Haematologica, Volume 108, Issue 11 by Haematologica

haematologica Journal of the Ferrata Storti Foundation

haematologica.org

VOL.

108 NOVEMBER 2023

ISSN 0390 - 6078

haematologica Editor-in-Chief Jacob M. Rowe (Jerusalem)

Deputy Editors Carlo Balduini (Pavia), Jerry Radich (Seattle)

Associate Editors Shai Izraeli (Tel Aviv), Steve Lane (Brisbane), Pier Mannuccio Mannucci (Milan), Pavan Reddy (Houston), David C. Rees (London), Paul G. Richardson (Boston), Francesco Rodeghiero (Vicenza), Gilles Salles (New York), Kerry Savage (Vancouver), Aaron Schimmer (Toronto), Richard F. Schlenk (Heidelberg), Sonali Smith (Chicago)

Statistical Consultant Catherine Klersy (Pavia)

Editorial Board Walter Ageno (Varese), Sarit Assouline (Montreal), Andrea Bacigalupo (Roma), Taman Bakchoul (Tübingen), Pablo Bartolucci (Créteil), Katherine Borden (Montreal), Marco Cattaneo (Milan), Corey Cutler (Boston), Kate Cwynarski (London), Ahmet Dogan (New York), Mary Eapen (Milwaukee), Francesca Gay (Torino), Ajay Gopal (Seattle), Alex Herrera (Duarte), Martin Kaiser (London), Marina Konopleva (Houston), Johanna A. Kremer Hovinga (Bern), Nicolaus Kröger (Hamburg), Austin Kulasekararaj (London), Shaji Kumar (Rochester), Ann LaCasce (Boston), Anthony R. Mato (New York), Matthew J. Mauer (Rochester) Neha Mehta-Shah (St. Louis), Moshe Mittelman (Tel Aviv), Alison Moskowitz (New York), Yishai Ofran (Haifa), Farhad Ravandi (Houston), John W. Semple (Lund), Liran Shlush (Toronto), Sarah K. Tasian (Philadelphia), Pieter van Vlieberghe (Ghent), Ofir Wolach (Haifa), Loic Ysebaert (Toulouse)

Managing Director Antonio Majocchi (Pavia)

Editorial Office Lorella Ripari (Office & Peer Review Manager), Simona Giri (Production & Marketing Manager), Paola Cariati (Graphic Designer), Giulia Carlini (Graphic Designer), Debora Moscatelli (Graphic Designer), Igor Poletti (Graphic Designer), Marta Fossati (Peer Review), Diana Serena Ravera (Peer Review), Laura Sterza (Account Administrator)

Assistant Editors Britta Dost (English Editor), Rachel Stenner (English Editor), Anne Freckleton (English Editor), Rosangela Invernizzi (Scientific Consultant), Marianna Rossi (Scientific Consultant), Massimo Senna (Information Technology), Luk Cox (Graphic Artist)

Haematologica | 108 - Ocotber 2023

Brief information on Haematologica Haematologica (print edition, pISSN 0390-6078, eISSN 1592-8721) publishes peer-reviewed papers on all areas of experimental and clinical hematology. The journal is owned by a non-profit organization, the Ferrata Storti Foundation, and serves the scientific community following the recommendations of the World Association of Medical Editors (www.wame.org) and the International Committee of Medical Journal Editors (www.icmje.org). Haematologica publishes Editorials, Original articles, Review articles, Perspective articles, Editorials, Guideline articles, Letters to the Editor, Case reports & Case series and Comments. Manuscripts should be prepared according to our guidelines (www.haematologica.org/information-for-authors), and the Uniform Requirements for Manuscripts Submitted to Biomedical Journals, prepared by the International Committee of Medical Journal Editors (www.icmje.org). Manuscripts should be submitted online at http://www.haematologica.org/. Conflict of interests. According to the International Committee of Medical Journal Editors (http://www.icmje.org/#conflicts), “Public trust in the peer review process and the credibility of published articles depend in part on how well conflict of interest is handled during writing, peer review, and editorial decision making”. The ad hoc journal’s policy is reported in detail at www.haematologica.org/content/policies. Transfer of Copyright and Permission to Reproduce Parts of Published Papers. Authors will grant copyright of their articles to the Ferrata Storti Foundation. No formal permission will be required to reproduce parts (tables or illustrations) of published papers, provided the source is quoted appropriately and reproduction has no commercial intent. Reproductions with commercial intent will require written permission and payment of royalties. Subscription. Detailed information about subscriptions is available at www.haematologica.org. Haematologica is an open access journal and access to the online journal is free. For subscriptions to the printed issue of the journal, please contact: Haematologica Office, via Giuseppe Belli 4, 27100 Pavia, Italy (phone +39.0382.27129, fax +39.0382.394705, E-mail: info@haematologica.org). Rates of the printed edition for the year 2022 are as following: Institutional: Euro 700 Personal: Euro 170 Advertisements. Contact the Advertising Manager, Haematologica Office, via Giuseppe Belli 4, 27100 Pavia, Italy (phone +39.0382.27129, fax +39.0382.394705, e-mail: marketing@haematologica.org). Disclaimer. Whilst every effort is made by the publishers and the editorial board to see that no inaccurate or misleading data, opinion or statement appears in this journal, they wish to make it clear that the data and opinions appearing in the articles or advertisements herein are the responsibility of the contributor or advisor concerned. Accordingly, the publisher, the editorial board and their respective employees, officers and agents accept no liability whatsoever for the consequences of any inaccurate or misleading data, opinion or statement. Whilst all due care is taken to ensure that drug doses and other quantities are presented accurately, readers are advised that new methods and techniques involving drug usage, and described within this journal, should only be followed in conjunction with the drug manufacturer’s own published literature.

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Associated with USPI, Unione Stampa Periodica Italiana. Premiato per l’alto valore culturale dal Ministero dei Beni Culturali ed Ambientali Haematologica | 108 - Ocotber 2023

Table of Contents Volume 108, Issue 11: November 2023

About the Cover Image taken from the editorial by Mauricio Nicolas Ferrao Blanco et al. in this issue.

Landmark Papers in Hematology 2878

The International Prognostic Index in aggressive B-cell lymphoma Matthew J. Maurer https://doi.org/10.3324/haematol.2023.284070

2880

Gene therapy for congenital marrow failure syndromes – no longer grasping at straws? Richard A. Voit and Seth J. Corey https://doi.org/10.3324/haematol.2023.283462

2883

Are DDX41 variants of unknown significance and pathogenic variants created equal? Zhuoer Xie and Daniel T. Starczynowski https://doi.org/10.3324/haematol.2023.283416

2886

Charting a course through the acute promyelocytic leukemia (APL)-like nebula: the enigmatic cousins of APL Alexandra Ghiaur and Gabriel Ghiaur https://doi.org/10.3324/haematol.2023.283232

2889

A novel approach to overcome drug resistance in acute myeloid leukemia Candice Mazewski and Leonidas C. Platanias https://doi.org/10.3324/haematol.2023.283099

2891

Leukemia suppressing normal bone marrow: how long does it last? Mauricio Nicolas Ferrao Blanco et al. https://doi.org/10.3324/haematol.2023.282955

Editorials

Review Articles 2894

Clinical perspectives on the optimal use of lenalidomide plus bortezomib and dexamethasone for the treatment of newly diagnosed multiple myeloma Paul G. Richardson et al. https://doi.org/10.3324/haematol.2022.282624

Spotlight Review Articles 2913

Asciminib in chronic myeloid leukemia: a STAMP for expedited delivery? Sandeep Padala and Jorge Cortes https://doi.org/10.3324/haematol.2022.282361 Haematologica | 108 - November 2023

2919

Momelotinib (JAK1/JAK2/ACVR1 inhibitor): mechanism of action, clinical trial reports, and therapeutic prospects beyond myelofibrosis Ayalew Tefferi, Animesh Pardanani and Naseema Gangat https://doi.org/10.3324/haematol.2022.282612

2933

Acute Myeloid Leukemia FLT4 as a marker for predicting prognostic risk of refractory acute myeloid leukemia Ji Yoon Lee et al. https://doi.org/10.3324/haematol.2022.282472

2946

Acute Myeloid Leukemia Molecular targeting of the UDP-glucuronosyltransferase enzymes in high-eukaryotic translation initiation factor 4E refractory/relapsed acute myeloid leukemia patients: a randomized phase II trial of vismodegib, ribavirin with or without decitabine Sarit Assouline et al. https://doi.org/10.3324/haematol.2023.282791

2959

Blood Transfusion Inhibition of GPIb-a-mediated apoptosis signaling enables cold storage of platelets Irene Marini et al. https://doi.org/10.3324/haematol.2022.282572

2972

Cell Therapy & Immunotherapy Salvage radiotherapy in relapsed/refractory large B-cell lymphoma after failure of CAR T-cell therapy Hazim S. Ababneh et al. https://doi.org/10.3324/haematol.2023.282804

2982

Cell Therapy & Immunotherapy Consolidative radiotherapy for residual fluorodeoxyglucose activity on day +30 post CAR T-cell therapy in non-Hodgkin lymphoma Omran Saifi et al. https://doi.org/10.3324/haematol.2023.283311

2993

Cell Therapy & Immunotherapy Intestinal IgA-positive plasma cells are highly sensitive indicators of alloreaction early after allogeneic transplantation and associate with both graft-versus-host disease and relapse-related mortality Lucia Scheidler et al. https://doi.org/10.3324/haematol.2022.282188

3001

Cell Therapy & Immunotherapy Azacitidine, lenalidomide and donor lymphocyte infusions for relapse of myelodysplastic syndrome, acute myeloid leukemia and chronic myelomonocytic leukemia after allogeneic transplant: the Azalena-Trial Thomas Schroeder et al. https://doi.org/10.3324/haematol.2022.282570

3011

Chronic Lymphocytic Leukemia Interleukin-27 potentiates CD8+ T-cell-mediated anti-tumor immunity in chronic lymphocytic leukemia Giulia Pagano et al. https://doi.org/10.3324/haematol.2022.282474

3025

Hodgkin Lymphoma Effect of cumulative dose of brentuximab vedotin maintenance in relapsed/refractory classical Hodgkin lymphoma after autologous stem cell transplant: an analysis of real-world outcomes Charlotte B. Wagner et al. https://doi.org/10.3324/haematol.2023.282780

Articles

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Myelodysplastic Syndromes Clinical and molecular correlates of somatic and germline DDX41 variants in patients and families with myeloid neoplasms Talha Badar et al. https://doi.org/10.3324/haematol.2023.282867

3044

Non-Hodgkin Lymphoma Characterization and clinical impact of the tumor microenvironment in post-transplant aggressive B-cell lymphomas Suvi-Katri Leivonen et al. https://doi.org/10.3324/haematol.2023.282831

3058

Non-Hodgkin Lymphoma Predictors of SARS-CoV-2 Omicron breakthrough infection after receipt of AZD7442 (tixagevimab-cilgavimab) for pre-exposure prophylaxis among hematologic malignancy patients Justin C. Laracy et al. https://doi.org/10.3324/haematol.2023.283015

3068

Red Cell Biology & its Disorders Characterization of genetic variants in the EGLN1/PHD2 gene identified in a European collection of patients with erythrocytosis Marine Delamare et al. https://doi.org/10.3324/haematol.2023.282913

3086

Red Cell Biology & its Disorders Increased retention of functional mitochondria in mature sickle red blood cells is associated with increased sickling tendency, hemolysis and oxidative stress Sofia Esperti et al. https://doi.org/10.3324/haematol.2023.282684

3095

Red Cell Biology & its Disorders Engineered human Diamond-Blackfan anemia disease model confirms therapeutic effects of clinically applicable lentiviral vector at single-cell resolution Yang Liu et al. https://doi.org/10.3324/haematol.2022.282068

3110

TRES, a validated three-factor comorbidity score, is associated with survival in older patients with mantle cell lymphoma Max J. Gordon et al. https://doi.org/10.3324/haematol.2023.283074

3115

Clinical and molecular features of CBL-mutated juvenile myelomonocytic leukemia Taro Yoshida et al. https://doi.org/10.3324/haematol.2022.282385

3120

Transcriptional features of acute leukemia with promyelocytic differentiation lacking retinoic acid receptor rearrangements Zhan Su et al. https://doi.org/10.3324/haematol.2022.282426

3125

Transcriptomic profiling does dot refine mastocytosis diagnosis Lars Buschhorn et al. https://doi.org/10.3324/haematol.2022.282617

3131

A proposed predictive mathematical model for efficient T-cell collection by leukapheresis for manufacturing chimeric antigen receptor T cells Xinxin Huang et al. https://doi.org/10.3324/haematol.2022.282350

Letters

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III

3135

TET2 mutational status affects myelodysplastic syndrome evolution to chronic myelomonocytic leukemia Violaine Tran Quang et al. https://doi.org/10.3324/haematol.2022.282528

3142

Clinical responses in pediatric patients with relapsed/refractory leukemia treated with azacitidine and venetoclax Lisa M. Niswander et al. https://doi.org/10.3324/haematol.2022.282637

3148

Examining the impact of age on the prognostic value of ELN-2017 and ELN-2022 acute myeloid leukemia risk stratifications: a report from the SWOG Cancer Research Network Christina M. Termini et al. https://doi.org/10.3324/haematol.2023.282733

3152

Risk of relapse after SARS-CoV-2 vaccine in the Milan cohort of thrombotic thrombocytopenic purpura patients Marco Capecchi et al. https://doi.org/10.3324/haematol.2022.282478

3156

High-risk additional cytogenetic aberrations in a Dutch chronic phase chronic myeloid leukemia patient population Camille C.B. Kockerols et al. https://doi.org/10.3324/haematol.2022.282447

3160

Chemotherapy in solitary bone plasmacytoma to prevent evolution to multiple myeloma Sophia Ascione et al. https://doi.org/10.3324/haematol.2022.282214

3165

The oncogenetic landscape and clinical impact of BCL11B alterations in adult and pediatric T-cell acute lymphoblastic leukemia Marie Emilie Dourthe et al. https://doi.org/10.3324/haematol.2022.282605

3170

Outcome of patients with acute myeloid leukemia following failure of frontline venetoclax plus hypomethylating agent therapy Naseema Gangat et al. https://doi.org/10.3324/haematol.2022.282677

3175

B-lineage acute lymphoblastic leukemia causes cell-autonomous defects in long-term hematopoietic stem cell function Christina T. Jensen et al. https://doi.org/10.3324/haematol.2022.282430

Case Reports & Case Series 3181

Novel FIP1L1::KIT fusion in a myeloid neoplasm with eosinophilia, T-lymphoblastic transformation, and dasatinib response Aseel Alsouqi et al. https://doi.org/10.3324/haematol.2022.282636

3186

Protracted viral infections in patients with multiple myeloma receiving bispecific T-cell engager therapy targeting B-cell maturation antigen Breanna Palmen et al. https://doi.org/10.3324/haematol.2023.283003

Haematologica | 108 - November 2023

LANDMARK PAPER IN HEMATOLOGY

M.J. Maurer

The International Prognostic Index in aggressive B-cell lymphoma Matthew J. Maurer Mayo Clinic, Rochester, MN, USA E-mail: maurer.matthew@mayo.edu https://doi.org/10.3324/haematol.2023.284070 ©2023 Ferrata Storti Foundation Published under a CC BY-NC license

TITLE

A predictive model for aggressive non-Hodgkin's lymphoma.

AUTHORS International Non-Hodgkin's Lymphoma Prognostic Factors Project. JOURNAL The New England Journal of Medicine. 1993;329(14):987-994. doi: 10.1056/NEJM199309303291402. used to develop the model received combination chemotherapy containing anthracycline on clinical trials between 1982 and 1987. In the mid 1980s, pathology subtypes were based on International Working Formulation, Kiel or Rappaport classifications, CD20 was a relatively recently discovered protein, and rituximab was still a decade away from being available for these patients. The computational tools available for modeling at the time the IPI was developed were also greatly limited compared to the smartphone/point-of-care apps, modern data visualization, and advanced modeling and data science techniques available to clinicians and researchers today. However, the productof-its-time simplicity of the IPI model has made it appealing and approachable for clinicians. A score of five dichotomous variables (age, stage, lactate dehydrogenase, Eastern Cooperative Oncology Group Performance Status, and number of involved extranodal sites) (Table 1), while inefficient from a statistical approach, can easily be computed in real-time without the need for electronic tools or reference charts. The variables in the model are part of Table 1. Variables used to calculate the International Prognostic standard workups and can be applied in most clinical setIndex score. tings. Efforts to modify the IPI have made marginal improvements in the prognostication of aggressive B-cell Category Number of points lymphoma and the IPI retains a strong prognostic capabiAge >60 years +1 lity in the rituximab era (Figure 1).2 As the classification Stage III/IV +1 and management of aggressive B-cell lymphoma continues to evolve and increase in complexity, a simple fiveECOG Performance Status ≥2 +1 variable model built from patients treated in the 1980s LDH >upper limit of normal +1 remains the standard for patient prognostication and de≥2 Extranodal sites +1 termination of clinical trial eligibility in 2023.

Prognostication and risk assessment are standard components of a clinical workup for a patient with a newly diagnosed hematologic malignancy. The information given by a prognostic tool provides guidance for treatment selection, helps to define clinical trial eligibility, and is useful for patient counseling. The development of an accurate measure of prognosis that can be broadly applied in a disease setting is a deceptively difficult endeavor. It requires assembly of a dataset that has a sufficiently large number of entries for statistical modeling, is representative of patients with the disease, and has adequate follow-up to assess clinically relevant outcomes. Furthermore, information in models may become clinically obsolete as new therapies are developed and/or the natural history of the disease evolves. The International Prognostic Index (IPI)1 was published in 1993 and has been a clinically relevant prognostic tool in aggressive B-cell lymphoma for 30 years. The fact that we are still using the model to define patient populations in this disease in 2023 is frankly remarkable. The patients

IPI score

Sum of Points

ECOG: Eastern Cooperative Oncology Group; LDH: lactate dehydrogenase; IPI: International Prognostic Index.

Disclosure No conflicts of interest to disclose.

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M.J. Maurer

Figure 1. Prognostic discrimination of the International Prognostic Index score with regard to overall survival. Figure modified with permission, from Ruppert et al.2 IPI: International Prognostic Index; KM est: estimated Kaplan-Meier; 95% CI: 95% confidence interval.

References 1. International Non-Hodgkin's Lymphoma Prognostic Factors Project. A predictive model for aggressive non-Hodgkin's lymphoma. N Engl J Med. 1993;329(14):987-994.

2. Ruppert AS, Dixon JG, Salles G, et al. International prognostic indices in diffuse large B-cell lymphoma: a comparison of IPI, RIPI, and NCCN-IPI. Blood. 2020;135(23):2041-2048.

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EDITORIAL

R.A. Voit and S.J. Corey

Gene therapy for congenital marrow failure syndromes – no longer grasping at straws? Richard A. Voit1,2 and Seth J. Corey3,4 1

Division of Hematology/Oncology, Boston Children’s Hospital, Harvard Medical School, Boston, MA; 2Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA; 3Departments of Pediatrics and Cancer Biology, Cleveland Clinic, Cleveland, OH and 4Case Comprehensive Cancer Center, Cleveland, OH, USA

Correspondence: S.J. Corey Coreys2@ccf.org Received: Accepted: Early view:

June 1, 2023. June 6, 2023. June 15, 2023.

The clinical potential that stems from the discovery of DNA’s double helix in 1953, and the subsequent genomic knowledge about health and disease, is now beginning to be realized as targeted corrections of genetic lesions are being translated into therapies. Hematopoietic disorders, arising from an accessible tissue that is amenable to ex vivo manipulation, provide a framework for the development of gene therapy cures. With a particular focus on the hemoglobinopathies, hematologists have been at the forefront of these efforts. In this issue of Haematologica, Liu et al. report on the application of a non-traditional CRISPR/Cas9 delivery method to establish a faithful model of Diamond-Blackfan anemia (DBA) in primary human hematopoietic stem and progenitor cells (HSPC) that can be rescued by lentiviral gene replacement.1 Newborn screening for hemoglobinopathies has been universally performed in the United States for several decades, and recent clinical trials using lentiviral delivery of a non-sickle hemoglobin gene2 or an inhibitor of hemoglobin switching3 are now showing promising results. Indeed, the need is great for individuals who suffer from sickle cell anemia or β-thalassemia major. And the global market is large. A much, much smaller market with a comparable need is found in the congenital bone marrow failure syndromes, which are also leukemia and cancer predisposition syndromes. This expanding list of monogenic disorders includes: Fanconi anemia, DBA, Shwachman-Diamond syndrome, dyskeratosis congenita, severe congenital neutropenia, congenital amegakaryocytic thrombocytopenia, GATA2 deficiency, and SAMD9/9L syndromes. This list continues to expand. Inherited conditions affecting hematopoiesis and resulting in myeloid neoplasms are now recognized in the adult population with germline pathogenic variants found in ANKRD26, RUNX1, CEBPA, and DDX41. More comprehensive neonatal screening for blood and non-blood disorders looms. The question will then be how to prevent disease manifestation or progression, and this will require faithful disease modeling in relevant primary

human cells. Hematologists will again lead this charge. But how? One intriguing hematologic disorder with suboptimal models is DBA. DBA is a rare inherited bone marrow failure syndrome that presents in infancy with pallor due to a profound hypoplastic macrocytic anemia. The mainstays of therapy are chronic red blood cell transfusions and judicious use of corticosteroids, while the only cure is allogeneic bone marrow transplantation.4 Chronic steroid therapy is effective in about one-third of patients, but it can confer long-term morbidity, affecting immune function, the adrenal axis, glucose utilization, and fat deposition. Chronic steroid use can lead to gastric ulcers, cataracts, osteopenia, delayed growth, and neuropsychologic impairment.5 One goal has been to find another effective drug with fewer side effects. Increased erythroid output has been demonstrated in preclinical models of DBA following treatment with leucine,6 trifluoperazine,7 and sotatercept,8 and efforts to translate these therapies to the clinic are ongoing, currently with mixed results at best.9 These drug-discovery efforts too require an accurate experimental model. The discovery of mutations in the ribosomal protein gene RPS19 in DBA10 confirmed its genetic basis, raising the possibility of definitive gene therapy-based cures. However, at least 20 genes have been identified to cause DBA, almost all of which encode ribosomal structural proteins.11 Others are related to ribosomal function or are specifically impacted by reduced ribosome numbers (e.g., HEATR3 or GATA1).12,13 That the disease can be due to a number of genes makes gene therapy somewhat cumbersome and complicates the development of a unified gene therapy cure using traditional approaches.11 However, approximately 25% of patients have RPS19 mutations, leading to efforts by the Karlsson group and others to develop an RPS19-directed gene replacement strategy to treat this largest subset of DBA patients. Two major challenges must be addressed in the preclinical development of novel gene therapy approaches: 1) a

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R.A. Voit and S.J. Corey

faithful ex vivo model for the disease must be established to enable evaluation of therapeutic efficacy; and 2) a safe, efficient means for the delivery of the genetic payload must be developed to achieve therapeutic benefit while minimizing short- and long-term toxicities. Early retroviral gene therapy trials were marred by the development of insertional mutagenesis, leading to acute lymphoblastic leukemia in some children who received gene therapy for severe combined immunodeficiency14 or Wiskott-Aldrich syndrome.15 Viral vectors were improved, relying on safer lentiviral backbones and alternative promoters, and the risk for leukemic transformation appears to be lessened, although clonal expansion remains a theoretical, if not an actual, risk. The accompanying paper from the Karlsson group extends their prior work16 developing a lentiviral gene replacement strategy to ameliorate the erythroid maturation defect that is the hallmark of DBA (Figure 1). Using their previously validated EF1α-driven RPS19 lentivirus as a gene replacement tool, Liu et al. set out to design a more faithful model of RPS19 haploinsufficiency that would allow for direct evaluation of this and future DBA-

directed therapies. Taking advantage of the efficiency of CRISPR/Cas9 editing of RPS19, the authors knocked in a GFP reporter to the RPS19 locus, enabling tracking of RPS19 disrupted clones by the presence of the GFP signal. They found significant cellular toxicity related to ribonucleoprotein delivery of CRISPR/Cas9 components, unlike what was recently described in the RPS19 CRISPR model by Bhoopalan et al.,17 perhaps owing to differences in electroporation conditions. Nonetheless, to avoid some of this toxicity, the authors optimized mRNA delivery of CRISPR components by nanostraws and demonstrated the efficacy of this approach in primary human cells for the first time. These proof-of-principle nanostraw CRISPR delivery approaches may one day extend beyond the hematopoietic system to allow for efficient disease modeling in other difficult-to-transfect tissue types, although the requirement for specialized equipment for nanostraw production and use may limit their widespread applicability. Nanostraw-enabled generation of RPS19 haploinsufficient erythroid progenitors allowed the authors to profile transcriptional changes associated with RPS19 loss and sub-

Figure 1. Generation and rescue of primary human hematopoietic stem and progenitor cell model of Diamond-Blackfan anemia. Steps of model generation and gene therapy treatment. 1. Nanostraw delivery of Cas9 mRNA and RPS19 sgRNA amelio-

rates toxicity associated with other delivery methods. 2. Delivery of a homology-directed repair template with a gene fluorescent protein (GFP) cassette flanked by arms of homology to the RPS19 locus. 3. Integration of a GFP cassette at RPS19 generates trackable clones with RPS19 haploinsufficiency. 4. Delivery of EF1α-RPS19 by lentivirus. 5. RPS19 gene replacement improves erythroid differentiation and reverses many of the transcriptional consequences of RPS19 haploinsufficiency. AAV: adeno-associated virus. Haematologica | 108 November 2023

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sequent treatment with their gene therapy vector in an otherwise isogenic background. As other DBA-directed treatments (both gene therapy and small molecules) emerge, it is essential to have a primary human cell system that will allow for sensitive profiling of direct cellular effects of treatment as the authors show here. Liu et al. establish such a model, which may mean that hematologists who are pursuing gene therapy cures for inherited

bone marrow failure syndromes are no longer grasping at straws. Disclosures No conflicts of interest to disclose. Contributions RAV and SJC wrote and edited the manuscript.

References 1. Liu Y, Schmiderer L, Hjort M, et al. Engineered human Diamond-Blackfan anemia disease model confirms therapeutic effects of clinically applicable lentiviral vector at single-cell resolution. Haematologica 2023;108(11):3095-3109. 2. Kanter J, Walters MC, Krishnamurti L, et al. Biologic and clinical efficacy of lentiglobin for sickle cell disease. N Engl J Med. 2022;386(7):617-628. 3. Esrick EB, Lehmann LE, Biffi A, et al. Post-transcriptional genetic silencing of BCL11A to treat sickle cell disease. N Engl J Med. 2021;384(3):205-215. 4. Da Costa L, Leblanc T, Mohandas N. Diamond-Blackfan anemia. Blood. 2020;136(11):1262-1273. 5. Sieff C. Diamond-Blackfan anemia. In: Adam MP, Mirzaa GM, Pagon RA, et al., eds. GeneReviews®. (WA): University of Washington, Seattle; 1993. 6. Payne EM, Virgilio M, Narla A, et al. L-Leucine improves the anemia and developmental defects associated with DiamondBlackfan anemia and del(5q) MDS by activating the mTOR pathway. Blood. 2012;120(11):2214-2224. 7. Taylor AM, Macari ER, Chan IT, et al. Calmodulin inhibitors improve erythropoiesis in Diamond-Blackfan anemia. Sci Transl Med. 2020;12(566):eabb5831. 8. Cappellini MD, Porter J, Origa R, et al. Sotatercept, a novel transforming growth factor beta ligand trap, improves anemia in beta-thalassemia: a phase II, open-label, dose-finding study. Haematologica. 2019;104(3):477-484. 9. Vlachos A, Atsidaftos E, Lababidi ML, et al. L-leucine improves anemia and growth in patients with transfusion-dependent Diamond-Blackfan anemia: results from a multicenter pilot

phase I/II study from the Diamond-Blackfan Anemia Registry. Pediatr Blood Cancer. 2020;67(12):e28748. 10. Draptchinskaia N, Gustavsson P, Andersson B, et al. The gene encoding ribosomal protein S19 is mutated in DiamondBlackfan anaemia. Nat Genet. 1999;21(2):169-175. 11. Liu YL, Shibuya A, Glader B, Wilkes MC, Barna M, Sakamoto KM. Animal models of Diamond-Blackfan anemia: updates and challenges. Haematologica. 2023;108(5):1222-1231. 12. O'Donohue MF, Da Costa L, Lezzerini M, et al. HEATR3 variants impair nuclear import of uL18 (RPL5) and drive DiamondBlackfan anemia. Blood. 2022;139(21):3111-3126. 13. Ludwig LS, Gazda HT, Eng JC, et al. Altered translation of GATA1 in Diamond-Blackfan anemia. Nat Med. 2014;20(7):748-753. 14. Hacein-Bey-Abina S, Hauer J, Lim A, et al. Efficacy of gene therapy for X-linked severe combined immunodeficiency. N Engl J Med. 2010;363(4):355-364. 15. Braun CJ, Boztug K, Paruzynski A, et al. Gene therapy for Wiskott-Aldrich syndrome--long-term efficacy and genotoxicity. Sci Transl Med. 2014;6(227):227ra33. 16. Liu Y, Dahl M, Debnath S, et al. Successful gene therapy of Diamond-Blackfan anemia in a mouse model and human CD34(+) cord blood hematopoietic stem cells using a clinically applicable lentiviral vector. Haematologica. 2022;107(2):446-456. 17. Bhoopalan SV, Yen JS, Mayuranathan T, et al. An RPS19-edited model for Diamond-Blackfan anemia reveals TP53-dependent impairment of hematopoietic stem cell activity. JCI Insight. 2023;8(1):e161810.

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Z. Xie and D.T. Starczynowski

Are DDX41 variants of unknown significance and pathogenic variants created equal? Zhuoer Xie1 and Daniel T. Starczynowski2 1

Malignant Hematology Department, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL and 2Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital, Departments of Cancer Biology and Pediatrics, University of Cincinnati, University of Cincinnati Cancer Center, Cincinnati, OH, USA.

Correspondence: Z. Xie zhuoer.xie@moffitt.org Received: Accepted: Early view:

May 25, 2023. June 6, 2023. June 15, 2023.

A variant of unknown significance (VUS) is a type of genetic mutation whose impact on an individual’s health remains undetermined. It falls between benign and pathogenic variants, and we can reclassify it as we gather more evidence. In this issue of Hematologica, Bader et al. describe a comprehensive study to uncover the clinical impact of VUS for DDX41 (DDX41VUS) and compared it to that of the pathogenic variants of DDX41 (DDX41path) and the associated cytopenia, myelodysplasia syndromes and acute myeloid leukemia.1 The DDX41 gene, also known as DEAD/H-box RNA helicase, is located on chromosome 5q35 and belongs to a class of tumor suppressor genes. It plays a crucial role in hematopoiesis by regulating the processing of small nucleolar RNA, assembling ribosomes, and synthesizing proteins.2,3 When the function of DDX41 is reduced, it can lead to defects in hematopoietic cells and increase the risk of developing hematologic malignancies, such as certain types of blood cancer. It is important to distinguish precisely between germline and somatic variants of DDX41, as well as between DDX41VUS and DDX41path variants, in order to make accurate prognoses and manage myeloid neoplasms associated with DDX41 mutations. However, despite the availability of advanced sequencing techniques for diagnosing inherited myeloid neoplasmas,4 we still face challenges in differentiating between causal variants and those classified as VUS. This difficulty arises from our incomplete understanding of the function of DDX41 protein. As a result, we have yet to fully define the landscape of causal DDX41 variants. DDX41path-associated familial myelodysplastic syndrome/ acute myeloid leukemia has distinctive features. It typically occurs later in life, at a median age of 65-70 years, and affects males more often than females (ratio of 3:1). There is also variability in the family history of hematologic malignancies.5-7 Many of these patients (46%) have pre-existing cytopenia and a long latency (5.2 years) before the diagnosis of the myeloid neoplasms.5 Patients with DDX41-mutant myeloid neoplasms typically present

with hypocellularity on the bone marrow sample and with normal cytogenetics.8 Fortunately, patients with DDX41pathassociated myeloid neoplasms generally respond well to treatment. In fact, a study demonstrated that they had a 100% overall response rate and nearly 90% overall survival at the 2-year mark when treated with intensive chemotherapy or hypomethylating-based agents.9 To investigate the potential pathogenicity of DDX41VUS, Bader et al. screened DDX41 mutations from 4,524 patients treated at the Mayo Clinic who underwent targeted sequencing for suspected or known myeloid neoplasms. They classified the mutations into DDX41 causal variants and VUS based on established guidelines from the American College of Medical Genetics and the Association for Molecular Pathology (ACMG/AMP). The researchers carefully categorized the DDX41 variants as either purely DDX41VUS (63 cases) or DDX41path (44 cases, with 11 having both pathogenic and VUS variants). The authors described the clinical features and outcomes of this cohort and found comparable features, including molecular profiles, with no differences between patients in variant allele fraction, co-mutation patterns, and cytogenetics. Family history of hematologic malignancies, and the outcomes, including time to initiate treatment, progression-free survival, and overall survival were also comparable between patients with DDX41vus and DDX41path (Figure 1). It is worth noting that some specific variants of DDX41VUS, such as p.P258L, p.G173R, and M155L, were associated with a remarkably high frequency of myeloid neoplasms. For instance, the occurrence of myeloid neoplasms was 86%, 75%, and 40% among patients with these variants, respectively. In addition, 28% (5/18) of patients had cytopenia. Similarly to a previous study led by Li et al.,7 only two patients with DDX41vus had concurrent somatic DDX41path (R5252H) mutations, which suggests that these DDX41VUS alone could be oncogenic. On the other hand, a study by Chlon et al. using mouse models demonstrated that a single DDX41 mutation, known as a monoallelic mutation, is associated with age-dependent hematopoietic

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Figure 1. Comparable features between DDX41vus and DDX41path variants. VAF: variant allele frequency; AML: acute myeloid

leukemia; MDS: myelodysplastic syndromes/myeloid neoplasms; DDX41vus: variants of DDX41 of unknown significance; DDX41path: pathogenic variants of DDX41.

defects.3 However, the acquisition of a "second-hit" mutation, often R525H, has a disease-modifying effect that accelerates hematopoietic defects and leads to hematologic malignancies.3 Clinical studies led by Li et al.7 and Duployez et al.8 also demonstrated that 70-80% of patients with DDX41path mutations develop acute myeloid leukemia after the acquisition of the “second-hit” DDX41 somatic mutation. The development of myeloid neoplasms without an additional somatic hit in these variants suggests that these DDX41vus might represent bona fide risk factors for the progression of myeloid neoplasms rather than trivial or inconsequential findings. Previous studies showed that the enrichment of somatic mutations in ASXL1, EZH2, and SRSF2 is associated with acute myeloid leukemia progression, and patients with DDX41 variants that result in truncation of the protein experience a rapid progression to acute myeloid leukemia compared to those with non-truncating variants.10 However, within the current study, the authors did not find significant differences in clinical features or outcomes when comparing patients with isolated DDX41 variants and those with co-mutations nor between patients with proteintruncating variants versus non-protein-truncating variants.

It is important to note that this lack of significance may be due to the small number of cases included in the analysis. Collectively, the high frequency of myeloid neoplasms observed in patients with DDX41VUS, along with the comparable clinical features and outcomes between those with DDX41VUS and DDX41path, suggest that these VUS might actually be oncogenic. This study underscores the importance of accurately classifying variants as VUS or pathogenic and understanding the causal landscape of DDX41 variants. It is crucial to establish a standardized classification specifically for DDX41 variants. For hematologists, the identification of DDX41VUS should prompt increased vigilance in terms of genetic counseling referrals, monitoring blood counts, and tailoring management accordingly. Future studies should focus on performing functional analyses of DDX41VUS to overcome the challenges associated with interpreting these variants of uncertain relevance. Disclosures DTS serves on the scientific advisory board at Kurome Therapeutics, is a consultant for and/or received funding from

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Kurome Therapeutics, Captor Therapeutics, Treeline Biosciences, and Tolero Therapeutics, and has equity in Kurome Therapeutics. ZX has received speaker bureau fees from Novatis.

Contributions ZX wrote the initial draft; both authors reviewed, provided edits to subsequent manuscript versions, and approved the final manuscript for submission.

References 1. Badar T, Nanaa A, Foran JM, et al. Clinical and molecular correlates of somatic and germline DDX41 variants in patients and families with myeloid neoplasms. Haematologica. 2023;108(11):3033-3043. 2. Polprasert C, Schulze I, Sekeres MA, et al. Inherited and somatic defects in DDX41 in myeloid neoplasms. Cancer Cell. 2015;27(5):658-670. 3. Chlon TM, Stepanchick E, Hershberger CE, et al. Germline DDX41 mutations cause ineffective hematopoiesis and myelodysplasia. Cell Stem Cell. 2021;28(11):1966-1981.e6. 4. Bannon SA, Routbort MJ, Montalban-Bravo G, et al. Nextgeneration sequencing of DDX41 in myeloid neoplasms leads to increased detection of germline alterations. Front Oncol. 2021;10:1. 5. Sébert M, Passet M, Raimbault A, et al. Germline DDX41 mutations define a significant entity within adult MDS/AML patients. Blood. 2019;134(17):1441-1444.

6. Quesada AE, Routbort MJ, DiNardo CD, et al. DDX41 mutations in myeloid neoplasms are associated with male gender, TP53 mutations and high-risk disease. Am J Hematol. 2019;94(7):757-766. 7. Li P, Brown S, Williams M, et al. The genetic landscape of germline DDX41 variants predisposing to myeloid neoplasms. Blood. 2022;140(7):716-755. 8. Duployez N, Largeaud L, Duchmann M, et al. Prognostic impact of DDX41 germline mutations in intensively treated acute myeloid leukemia patients: an ALFA-FILO study. Blood. 2022;140(7):756-768. 9. Alkhateeb HB, Nanaa A, Viswanatha D, et al. Genetic features and clinical outcomes of patients with isolated and comutated DDX41-mutated myeloid neoplasms. Blood Adv. 2022;6(2):528. 10. Makishima H, Saiki R, Nannya Y, et al. Germ line DDX41 mutations define a unique subtype of myeloid neoplasms. Blood. 2023;141(5):534-549.

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A. Ghiaur and G. Ghiaur

Charting a course through the acute promyelocytic leukemia (APL)-like nebula: the enigmatic cousins of APL Alexandra Ghiaur1 and Gabriel Ghiaur2

Correspondence: G. Ghiaur gghiaur1@jhmi.edu

Fundeni Clinical Institute, Bucharest, Romania and Johns Hopkins School of Medicine, Baltimore, MD, USA

Received: Accepted: Early view:

May 19, 2023. May 29, 2023. June 8, 2023.

Acute promyelocytic leukemia (APL) is characterized by a constellation of well-established elements, including distinctive morphology, flow cytometry, and clinical presentation. Nowadays, the definitive diagnosis of APL requires the presence of a unique genetic feature, namely the t(15;17)(q24;q21), and/or the presence of the PML-RARα fusion protein.1 The management of this special subtype of acute leukemia relies on a unique approach based on differentiation induction therapy using all-trans retinoic acid (ATRA) and arsenic trioxide (ATO).2 This dual-targeted therapy has completely revolutionized outcomes in APL. Consequently, relapse or refractory disease in patients treated with ATRA/ATO is extremely unlikely, and practically nonexistent. The success of the ATRA/ATO approach has launched a search for similar types of acute myeloid leukemia (AML) that may benefit from this treatment. Based on the distinctive morphological features of APL, this type of leukemia was categorized as M3 in the original French-American-British classification in 1976.3 It was only 1 year later that Rowley and colleagues reported that most patients with M3 APL share similar abnormalities of chromosome 17, later shown to be t(15;17)(q24;q21).4 For the last 45 years, the overlap between morphologically and genetically defined APL has been a topic of intense debate and scientific interest. The World Health Organization-mandated requirement for the presence of t(15;17)(q24;q21) or the PML-RARα fusion for the diagnosis and definition of APL paved the way for a disease entity called variant APL.5 Variant APL, sometimes called APLlike disease, has all the morphological features of APL but lacks both t(15;17)(q23;q21) and PML-RARα. In most cases, alternative translocations that involve either RARA or RARG can be identified (Figure 1). The first APL variant was described in 1993 and is defined by the presence of t(11;17)(q23;q21), resulting in a novel fusion gene, ZBTB16RARA.6 The encoded fusion protein is involved in MLL-induced leukemogenesis and is the most common form of non PML-RARα APL. Patients harboring ZBTB16-RARA have an unfavorable prognosis and are resistant to ATRA/ATO

therapy. In these cases, conventional AML chemotherapy with or without differentiation agents is the most appropriate management approach. Patients with variant APL are less likely to achieve complete remission and have a lower overall survival than those with typical APL.7 Multiple translocations involving various retinoic acid receptors (RAR) have been frequently reported in variant APL. An extensive list was published by Sanz et al. in 2019.2 The response of these variant APL to ATRA and/or ATO is variable and, to some extent, unpredictable based on the type of genetic event alone. Lately, gene expression techniques have been used to characterize morphologically defined cases of APL that lack rearrangements involving any of the RAR. Such leukemias were also termed APL-like leukemias but distinctively selected to lack any abnormalities affecting RAR. It is currently unknown whether they represent a unitary type of AML with common molecular pathology, clinical presentation, and, most importantly, response to ATRA/ATO therapy. To date, six case reports have described non-RAR molecular aberrations, with the MLL rearrangement being the most cited fusion gene involved.7 In the current issue of Haematologica, Su et al. report the gene expression profile of four cases of APL-like disease that lack the classical PML-RARα fusion protein as well as other RAR chimeric transcripts.8 Using sophisticated genetic tools, including transcriptome sequencing, they identified novel non-RAR chimeric transcripts such as KSR1-LGALS9, GPBP1L1-CCDC17, GLYCTK-DNAH1, NUP98HOXD8, and CFD-GNA15.8 These genetic events are relatively abundant and non-overlapping, which begs the question of whether they are actually "driver" or "passenger" mutations. Some of the described partners, such as NUP98 and HOXD8, have well-established roles in normal hematopoiesis, and it is conceptually possible that their dysregulation leads to abnormal differentiation and hematologic malignancies. Nevertheless, the impact of these molecular events on the pathogenesis of APL-like disease remains to be determined.

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Figure 1. Schematic representation of the genetic features and therapeutic implications of acute promyelocytic leukemia (APL) and APL-like disease. PML: promyelocytic leukemia; RARα: retinoic acid receptor alpha; RT-PCR: reverse transcriptase polymerase chain reaction; ATRA: all-trans retinoic acid; ATO: arsenic trioxide; NPM1: nucleophosmin 1; AML: acute myeloid leukemia.

In a previous investigation, five pediatric patients with non-RAR APL-like disease were managed with a combination of ATRA/ATO and standard chemotherapy, and the outcomes were favorable.9 In the investigation by Su et al., one pediatric patient and three adults were treated with ATRA/ATO plus chemotherapy, with a considerably wide range of survival from 5 weeks to 44 months.8 Thus, the utility of ATRA/ATO in the treatment of non-RARα APL variants remains unclear. While the current report by Su et al. is a step forward towards a better understanding of the molecular landscape of APL-like disease,8 further studies are warranted to establish the role of each fusion gene in variant APL development. Studies such as this one represent an opportunity for more precise stratification of APL-like AML and, at the same time, a more appropriate treatment approach. Gene expression signatures, rather than the presence of

discrete molecular events, are more likely to predict clinical behavior in acute leukemia. The best-known data come from the use of gene expression signatures in preB-cell acute lymphoblastic leukemia to identify a group of patients who have Philadelphia chromosome-like disease even though they lack the t(9;22).10 Similar approaches in AML may one day, not too distant, lead to the identification of an APL-like signature that predicts response to ATRA/ATO regardless of the genetic category of the disease. Until then, it is important to clearly define our goals and terminology as we chart our course through future studies of this disease entity. Disclosures No conflicts of interest to disclose. Contributions AG and GG wrote and edited the manuscript.

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References 1. Khoury JD, Solary E, Abla O, et al. The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: Myeloid and Histiocytic/Dendritic Neoplasms. Leukemia. 2022;36(7):1703-1719. 2. Sanz MA, Fenaux P, Tallman MS, et al. Management of acute promyelocytic leukemia: updated recommendations from an expert panel of the European LeukemiaNet. Blood. 2019;133(15):1630-1643. 3. Bennett JM, Catovsky D, Daniel MT, et al. Proposals for the classification of the acute leukaemias. French-American-British (FAB) co-operative group. Br J Haematol. 1976;33(4):451-458. 4. Rowley JD, Golomb HM, Dougherty C. 15/17 translocation, a consistent chromosomal change in acute promyelocytic leukaemia. Lancet. 1977;1(8010):549-550. 5. Geoffroy MC, de The H. Classic and variants APLs, as viewed from a therapy response. Cancers (Basel). 2020;12(4):967. 6. Chen SJ, Zelent A, Tong JH, et al. Rearrangements of the

retinoic acid receptor alpha and promyelocytic leukemia zinc finger genes resulting from t(11;17)(q23;q21) in a patient with acute promyelocytic leukemia. J Clin Invest. 1993;91(5):2260-2267. 7. Guarnera L, Ottone T, Fabiani E, et al. Atypical rearrangements in APL-like acute myeloid leukemias: molecular characterization and prognosis. Front Oncol. 2022;12:871590. 8. Su Z, Liu X, Zhang Y, et al. Transcriptional features of acute leukemia with promyelocytic differentiation lacking retinoic acid receptor rearrangements. Haematologica. 2023;108(11):3120-3124. 9. Zhao J, Liang JW, Xue HL, et al. The genetics and clinical characteristics of children morphologically diagnosed as acute promyelocytic leukemia. Leukemia. 2019;33(6):1387-1399. 10. Ribera JM. Philadelphia chromosome-like acute lymphoblastic leukemia. Still a pending matter. Haematologica. 2021;106(6):1514-1516.

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C. Mazewski and L.C. Platanias

A novel approach to overcome drug resistance in acute myeloid leukemia Candice Mazewski1,2 and Leonidas C. Platanias1,2,3 1

Robert H. Lurie Comprehensive Cancer Center of Northwestern University; 2Division of Hematology-Oncology, Feinberg School of Medicine, Northwestern University and 3 Department of Medicine, Jesse Brown Veterans Affairs Medical Center, Chicago, IL, USA

Correspondence: L.C. Platanias l-platanias@northwestern.edu Received: Accepted: Early view:

April 13, 2023. May 3, 2023. May 11, 2023.

The prognosis of acute myeloid leukemia (AML) remains poor for the majority of patients suffering from this disease. Undoubtedly, there is a desperate need for new treatments and therapeutic efficacy. In the current issue of Haematologica, Assouline et al. report on a phase II clinical trial (clinicaltrials.gov: identifier NCT02073838) conducted with vismodegib and ribavirin, with or without decitabine in AML patients.1 Most of the patients had at least two prior lines of therapy. The authors delve into two aspects of drug resistance, glucuronidation and drug transport into the cells, and help provide future strategies to tackle drug resistance in AML. Drug resistance continues to thwart the treatment success of therapeutics in AML as well as other cancers, leading to poor survival. Overall categories of resistance mechanisms include drug-resistant proteins, genetic alterations, miRNA alterations, and aberrant signaling activation.2 However, resistance mechanisms and targeting strategies can be specific to certain subtypes and mutations. A previous study from the same group explored ribavirin as a single agent in M4/M5 AML.3 In that clinical trial, some clinical responses associated with the reduction of eukaryotic translation initiation factor 4E (eIF4E) levels were seen. Ribavirin competitively inhibits the binding of eIF4E to the m7G RNA cap. The introduction of vismodegib, which targets glioma-associated protein 1 (GLI1) through the Smoothened receptor (Smo), was based on the novel discovery by this group that UDP-glucuronosyltransferase 1 A (UGT1A) and GLI1 have increased expression in resistant AML cells and induce glucuronidation of ribavirin and cytarabine.4 This glucuronidation is carried out by the UGT enzymes, which leads to inactivation of drug activity, increased water solubility, and elimination of the drug imparted by the conjugation.5 This group also demonstrated that GLI1-inducible glucuronidation by UGT1A was not limited to these two drugs but included approximately forty other drugs from a variety of families, including nucleosides, antifolates, and anthracyclines.6 This underscores the importance and potential of targeting UGT enzymes.

In the current study, 23 patients were enrolled, 15 in the vismodegib and ribavirin plus decitabine arm and 8 in the vismodegib and ribavirin arm. Inclusion criteria included elevated eIF4E levels compared to healthy volunteers and functional equilibrative nucleoside transporter 1 (ENT1). The overall response rate was 40% for the arm with decitabine, while there were no responders among the patients who received only vismodegib and ribavirin, although 3 had prolonged stable disease. Of the responders in the vismodegib and ribavirin plus decitabine arm, the majority of them had been previously treated with hypomethylating agents, demonstrating a possible re-sensitization with the vismodegib and ribavirin treatment. In addition to the exploration of drug resistance through glucuronidation, the authors also evaluated changes in ENT1 levels since many drugs enter cells through ENT1, and reduced levels can lead to resistance. Most patients in this study did have a reduction in ENT1 RNA levels post treatment, indicating a likely acquired mechanism of drug resistance. ENT1 regulation related to drug resistance is not fully understood. Researchers demonstrated that ENT1 removal from the cell surface was induced by bone marrow stroma cell-secreted factors, and this contributed to cytarabine resistance in AML cells.7 Another recent study found that acquired cytarabine resistance in relapsed AML was related to the downregulation of ENT1 through the inactivation of histone 3 lysine 27 demethylase 6A (KDM6A) which commonly has loss-of-function mutations in cancer.8 These studies emphasize that ENT1 is related to drug resistance specific to AML. However, although ENT1 has the potential to be used as a resistance predictor, strategies to avoid ENT1-related resistance are lacking and require further exploration. Since ribavirin is ultimately acting to reduce the oncogenic capacity of eIF4E in a specific way, this raises the possibility of simultaneous targeting of eIF4E by other means to minimize resistance. Targeting MAPK interacting kinases (MNK) in combination with glucuronidation inhibitors may provide such an approach. MNK phosphory-

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late eIF4E at serine 209 leading to its activation and promoting its transforming potential, and high nuclear phosphorylated eIF4E have been associated with higher tumor burden in AML patients.9 Previous studies have shown antineoplastic effects with a breadth of MNK inhibitors on AML cells in in vitro and in vivo models.10,11 One of the studies demonstrated that the combination of an MNK inhibitor (SEL201) with 5-azacytidine, a hypomethylating agent similar to decitabine, enhanced the reduction of viability and colony formation.10 It remains to be seen if specific MNK inhibitors are capable of therapeutic efficacy in patients. High importance has been placed on understanding mechanisms and progression of resistance in AML. In this work, the authors demonstrated baseline expression differences in these heavily pre-treated patients compared to healthy volunteers, as well as changes over the time of therapy in eIF4E and UGT1A levels. There was a reduction in both UGT1A and eIF4E levels at best molecular response compared to before-treatment measurements, with these increasing again to near baseline at relapse, providing a better understanding of how cells adapt in patients. Other groups have created resistance modeling in vitro, such as in a gilteritinib resistance study in FLT3-mutated AML where authors examined the metabolic programming in the progression from early to late resistance.12 They noted that the cells depended on Aurora kinase B (AURKB) in early resistance and suggested using AURKB inhibitors as

a strategy to re-sensitize to gilteritinib before the late resistance programming marked by pre-existing NRAS mutant subclones ensued.12 These studies highlight the importance of timing in treatment and the need to further elucidate the mechanisms of progressive resistance. Overall, the work by Assouline and colleagues provides proof of principle that it is possible to target glucuronidation, and specifically UGT1A levels, in patients without severe toxicity. As noted by the authors, because vismodegib does not target UGT1A levels directly, there are underlying opportunities for the cells to circumvent this pathway inhibition. The development of a more direct inhibitor may lead to more substantial and robust clinical effects. Another study previously found that selective compounds could inhibit UGT1A activity, and researchers are working to optimize these as potential glucuronidation inhibitors.13 The study by Assouline et al. is important and clinically relevant, as it provides a method to overcome one aspect of drug resistance. Using this as a basis, future studies should advance the field further by providing approaches to target glucuronidation, with the ultimate goal being lasting clinical responses. Disclosures No conflicts of interest to disclose. Contributions CM and LCP wrote the manuscript.

References 1. Assouline S, Gasiorek J, Bergeron J, et al. Molecular targeting of the UDP-glucuronosyltransferase enzymes in high-eukaryotic translation initiation factor 4E refractory/relapsed acute myeloid leukemia patients: a randomized phase II trial of vismodegib, ribavirin with or without decitabine. Haematologica. 2023;108(11):2946-2958. 2. Zhang J, Gu Y, Chen B. Mechanisms of drug resistance in acute myeloid leukemia. Onco Targets Ther. 2019;12:1937-1945. 3. Assouline S, Culjkovic B, Cocolakis E, et al. Molecular targeting of the oncogene eIF4E in acute myeloid leukemia (AML): a proof-of-principle clinical trial with ribavirin. Blood. 2009;114(2):257-260. 4. Zahreddine HA, Culjkovic-Kraljacic B, Assouline S, et al. The sonic hedgehog factor GLI1 imparts drug resistance through inducible glucuronidation. Nature. 2014;511(7507):90-93. 5. Allain EP, Rouleau M, Levesque E, Guillemette C. Emerging roles for UDP-glucuronosyltransferases in drug resistance and cancer progression. Br J Cancer. 2020;122(9):1277-1287. 6. Zahreddine HA, Culjkovic-Kraljacic B, Gasiorek J, Duchaine J, Borden KLB. GLI1-inducible glucuronidation targets a broad spectrum of drugs. ACS Chem Biol. 2019;14(3):348-355. 7. Macanas-Pirard P, Broekhuizen R, González A, et al. Resistance of leukemia cells to cytarabine chemotherapy is mediated by bone marrow stroma, involves cell-surface equilibrative

nucleoside transporter-1 removal and correlates with patient outcome. Oncotarget. 2017;8(14):23073-23086. 8. Stief SM, Hanneforth AL, Weser S, et al. Loss of KDM6A confers drug resistance in acute myeloid leukemia. Leukemia. 2020;34(1):50-62. 9. Zhou H, Jia X, Yang F. Elevated nuclear phospho-eIF4E body levels are associated with tumor progression and poor prognosis for acute myeloid leukemia. Biocell. 2021;45(3):711-722. 10. Kosciuczuk EM, Kar AK, Blyth GT, et al. Inhibitory effects of SEL201 in acute myeloid leukemia. Oncotarget. 2019;10(67):7112-7121. 11. Kosciuczuk EM, Saleiro D, Kroczynska B, et al. Merestinib blocks Mnk kinase activity in acute myeloid leukemia progenitors and exhibits antileukemic effects in vitro and in vivo. Blood. 2016;128(3):410-414. 12. Joshi SK, Nechiporuk T, Bottomly D, et al. The AML microenvironment catalyzes a stepwise evolution to gilteritinib resistance. Cancer Cell. 2021;39(7):999-1014.e8. 13. Osborne MJ, Coutinho de Oliveira L, Volpon L, Zahreddine HA, Borden KLB. Overcoming drug resistance through the development of selective inhibitors of UDPglucuronosyltransferase enzymes. J Mol Biol. 2019;431(2):258-272.

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M.N. Ferrao Blanco et al.

Leukemia suppressing normal bone marrow: how long does it last? Mauricio Nicolas Ferrao Blanco,1 Mirjam Belderbos,1 Hermann Josef Vormoor1,2 1

Princess Máxima Center for Pediatric Oncology and 2University Medical Center Utrecht, Utrecht, The Netherlands

Correspondence: H.J. Vormoor h.vormoor@prinsesmaximacentrum.nl Received: Accepted: Early view:

March 18, 2023. March 24, 2023. April 6, 2023.

Patients with newly diagnosed leukemia often present with signs and symptoms of bone marrow (BM) failure rather than with symptoms caused by proliferation of the leukemia itself: e.g., paleness, tiredness and lack of energy due to anemia; pro-longed or severe infections due to neutropenia; and petechial rash and nose bleeds due to thrombocytopenia. BM failure is a relatively late event during leukemia development. It is thought to be either a direct, cyto-/chemokine-mediated effect of the leukemic cells or an indirect effect via re-modeling of the BM environment. Either or both lead to suppression of normal BM function. Many studies have investigated the interaction of normal hematopoietic stem and leukemia cells with their BM niche. It has been shown that different types of leukemia affect differentiation and function of various cells in the BM, including bone progenitor cells, endothelial cells, nerve fibers and myofibroblasts,1 leading to a loss of support of normal hematopoiesis. In this issue of Haematologica, Jensen et al. investigated the effect of B-lineage acute lymphoblastic leukemia (BALL) on residual normal hematopoiesis.2 They use a spontaneous murine leukemia model with heterozygote deletions of Pax5 and Ebf1, and study the effect of the leukemia on normal hematopoiesis by transplanting leukemic blasts onto wild-type mice. Interestingly, and in contrast to what one might expect as a consequence of modulation of the BM niche, the most immature LinKIT+SCA1+ (LSK) and CD150+ LSK stem-like compartment persists, while more mature, lineage-restricted progenitors disappear from the leukemic marrow. A similar observation was made in 19 human patients with B-ALL in which the frequency of non-leukemic CD19CD34+CD38low/- putative stem-like cells was comparable to normal controls. Most intriguingly, the authors describe a lasting effect of the leukemia on the reconstitution potential of these murine stem cells after primary and secondary transplantation. Secondary mice transplanted with “leukemia-exposed” hematopoietic stem cells showed lower levels of reconstitution. The “leuke-

mia-exposed” CD150+ LSK cells displayed an expression profile suggestive of mitochondrial dysfunction, with fluorescent dye tracking confirming a reduced mitochondrial membrane potential in this residual stem cell population. Impaired mitochondrial function is a hallmark of cellular aging,3 and these data are, therefore, suggestive of premature aging of “leukemia-exposed” hematopoietic stem cells. There are two key questions that arise from this work. 1) What is the mechanism by which the leukemia imposes such a long-lasting effect on the repopulation potential of normal stem cells leading to mitochondrial dysfunction and premature aging of the hematopoietic compartment? Mitochondrial dysfunction is a common feature of many types of cancer, including leukemia. To meet their high energetic demands, leukemia cells rely on mitochondrial oxidative metabolism. Pharmacologic inhibition of oxidative metabolism has been demonstrated to inhibit leukemia cell growth and to increase sensitivity to chemotherapy, both in vitro and in vivo.4,5 Intriguingly, the damaging effects of chemotherapy can, at least in part, be rescued by transfer of mitochondria between leukemia cells and their surrounding BM stroma.6,7 Similarly, BM stromal cells were found to transfer mitochondria to healthy hematopoietic stem cells as a means to promote their capacity to respond to proliferative stress.8 Although direct exchange of mitochondria between leukemia cells and healthy hematopoietic stem/progenitor cells has not been demonstrated, it is interesting to think about whether or how mitochondrial transfer could account for the findings by Jensen et al. Alternatively, one could envision a tripartite exchange in which BM niche cells are the intermediate party, transferring mitochondria from leukemia cells towards the resident normal hematopoietic stem cells (and potentially, vice versa). Although a role for mitochondrial exchange remains speculative, if confirmed, it could provide the route to develop new therapeutic interventions to protect the healthy hemato-

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Figure 1. Leukemia-induced mitochondrial dysfunction in hematopoietic stem and progenitor cells limits their ability for long-term reconstitution, recapitulating aging. B-cell acute lymphoblastic leukemia (B-ALL)-exposed hematopoietic stem and progenitor cells (HSPC) produce reduced numbers of lineage-restricted progenitor cells, and exhibit mitochondrial dysfunction. Some of these effects may be indirectly mediated, by cross-talk of B-ALL blasts, bone marrow mesenchymal stromal cells (MSC) and the healthy HSPC compartment. Created with BioRender (BioRender.com).

poietic stem cell compartment from leukemia-mediated suppression. 2) Does this long-lasting effect of the leukemia on normal hematopoietic stem cells also occur in human patients? Or is the described effect on stem cells unique to this mouse model? Clonal hematopoiesis has been studied in long-term survivors of pediatric cancer.9 There is a significant increase in clonal hematopoiesis in childhood cancer survivors; however, this is mainly thought to be therapy-related rather than there being evidence for a leukemia-mediated accelerated aging of normal hematopoietic stem cells. Late BM failure is not usually regarded as a typical late

effect after ALL.10 However, data on clonal hematopoiesis in survivors of childhood ALL, particularly in very longterm survivors, are scarce. This interesting observation suggests that aging of the hematopoietic system in our patients warrants further attention, and future prospective studies are needed to look at BM function in longterm survivors. Disclosures No conflicts of interest to disclose. Contributions MF, MB and JV wrote the manuscript. MF designed the figure.

References 1. Pinho S, Frenette PS. Haematopoietic stem cell activity and interactions with the niche. Nat Rev Mol Cell Biol. 2019;20(5):303-320. 2. Jensen CT, Åhsberg J, Tingvall-Gustafsson J, et al. B-lineage acute lymphoblastic leukemia causes cell autonomous defects in long-term hematopoietic stem cell function. Haematologica. 2023;108(11):3175-3180.

3. Kauppila TES, Kauppila JHK, Larsson NG. Mammalian mitochondria and aging: an update. Cell Metab. 2017;25(1):57-71. 4. Saito K, Zhang Q, Yang H, et al. Exogenous mitochondrial transfer and endogenous mitochondrial fission facilitate AML resistance to OxPhos inhibition. Blood Adv. 2021;5(20):4233-4255. 5. Bosc C, Saland E, Bousard A, et al. Mitochondrial inhibitors

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circumvent adaptive resistance to venetoclax and cytarabine combination therapy in acute myeloid leukemia. Nat Cancer. 2021;2(11):1204-1223. 6. Griessinger E, Moschoi R, Biondani G, Peyron JF. Mitochondrial transfer in the leukemia microenvironment. Trends Cancer. 2017;3(12):828-839. 7. Burt R, Dey A, Aref S, et al. Activated stromal cells transfer mitochondria to rescue acute lymphoblastic leukemia cells from oxidative stress. Blood. 2019;134(17):1415-1429. 8. Mistry JJ, Marlein CR, Moore JA, et al. ROS-mediated PI3K

activation drives mitochondrial transfer from stromal cells to hematopoietic stem cells in response to infection. Proc Natl Acad Sci U S A. 2019;116(49):24610-24619. 9. Hagiwara K, Natarajan S, Wang Z, et al. Dynamics of age- versus therapy-related clonal hematopoiesis in long-term survivors of pediatric cancer. Cancer Discov. 2023;13(4):844-857. 10. Dixon SB, Chen Y, Yasui Y, et al. Reduced morbidity and mortality in survivors of childhood acute lymphoblastic leukemia: a report from the Childhood Cancer Survivor Study. J Clin Oncol. 2020;38(29):3418-3429.

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Clinical perspectives on the optimal use of lenalidomide plus bortezomib and dexamethasone for the treatment of newly diagnosed multiple myeloma Paul G. Richardson,1 Brian G. Durie,2 Laura Rosiñol,3 Maria-Victoria Mateos,4 Angela Dispenzieri,5 Philippe Moreau,6 Shaji Kumar,5 Noopur Raje,7 Nikhil Munshi,1 Jacob P. Laubach,1 Peter O’Gorman,8 Elizabeth O’Donnell,7 Peter Voorhees,9 Thierry Facon,10 Joan Bladé,3 Sagar Lonial,11 Aurore Perrot12 and Kenneth C. Anderson1 Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA; 2Cedars-Sinai Samuel Oschin Cancer Center, Los Angeles, CA, USA; 3Hospital Clínic de Barcelona, Institut d'Investigacions Biomèdiques August Pi i Sunyer, University of Barcelona, Barcelona, Spain; 4University Hospital of Salamanca, IBSAL, Institute of Cancer Molecular and Cellular Biology, Salamanca, Spain; 5Division of Hematology, Mayo Clinic Cancer Center, Rochester, MN, USA; 6 Hematology Department, University Hospital Hôtel-Dieu, Nantes, France; 7Center for Multiple Myeloma, Massachusetts General Hospital Cancer Center, Boston, MA, USA; 8 Department of Haematology, Mater Misericordiae University Hospital, University College Dublin, Dublin, Ireland; 9Department of Hematologic Oncology and Blood Disorders, Levine Cancer Institute, Atrium Health, Charlotte, NC, USA; 10University of Lille, Centre Hospitalier Universitaire Lille, Service des Maladies du Sang, Lille, France; 11Department of Hematology and Oncology, Winship Cancer Institute, Emory University, Atlanta, GA, USA and 12Institut Universitaire du Cancer de Toulouse-Oncopole, Toulouse, France 1

Correspondence: Paul G. Richardson Paul_Richardson@dfci.harvard.edu Received: Accepted: Early view:

March 29, 2023. July 24, 2023. August 24, 2023.

Abstract To improve the outcomes of patients with the otherwise incurable hematologic malignancy of multiple myeloma (MM), a key paradigm includes initial treatment to establish disease control rapidly followed by maintenance therapy to ensure durability of response with manageable toxicity. However, patients’ prognosis worsens after relapse, and the disease burden and drug toxicities are generally more challenging with subsequent lines of therapy. It is therefore particularly important that patients with newly diagnosed multiple myeloma (NDMM) receive optimal frontline therapy. The combination of lenalidomide, bortezomib, and dexamethasone (RVd) has consistently demonstrated a tolerable safety profile with significant and clinically relevant benefit, including deep and durable responses with improved survival in patients with NDMM regardless of their transplant eligibility. Furthermore, comparative studies evaluating this triplet regimen against both doublet and other triplet regimens have established RVd as a standard of care in this setting based upon its remarkable and concordant efficacy. Given the breadth of clinical data, physician familiarity, inclusion in treatment guidelines, and the emerging potential of RVd-containing quadruplet regimens, RVd will likely continue as a key cornerstone of the treatment of NDMM, and its role will therefore likely continue to grow as a therapeutic backbone in the initial treatment of MM.

Introduction Combinations of lenalidomide, bortezomib, and dexamethasone (RVd) are recommended for the treatment of newly diagnosed multiple myeloma (NDMM).1,2 Lenalidomide and bortezomib are approved for use in the USA for either transplant-ineligible (TNE) or transplant-eligible (TE) patients with NDMM. In 2019, RVd was approved in the European Union (EU) for patients with TNE NDMM3 and is used in Switzerland, Australia, and Brazil for NDMM regardless of transplant eligibility.

In NDMM trials, RVd has achieved deep, durable responses that are among the best reported with triplet regimens, which have been further improved with the introduction of monoclonal antibodies.4 The efficacy and tolerability of RVd have been demonstrated in TNE and TE populations across numerous studies and dose schedules. This review summarizes data supporting RVd as standard of care in NDMM when administered as induction therapy in settings of autologous stem cell transplant (ASCT) or in TNE patients, and as part of emerging quadruplet regimens.

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RVd: rationale and background MM remains incurable, despite therapeutic advances having led to substantially improved progression-free (PFS) and overall survival (OS).5,6 Disease burden, toxicities, and outcomes typically worsen with each subsequent line of therapy, confirming critical needs for effective frontline intervention.7 Achieving sustained deep responses (very good partial response [VGPR], complete response [CR], minimal residual disease [MRD] negativity) is a key treatment goal for NDMM and can improve survival.8-10 Frontline regimens should be highly effective and tolerable to attain successful induction and maintain continuous therapy. Avoiding agents that deplete stem cells and interfere with their collection is important in order to preserve the option of ASCT. Lenalidomide (an immunomodulatory agent) and bortezomib (a proteasome inhibitor) are backbones of pharmacotherapy for NDMM.1,5 Lenalidomide has pleiotropic mechanisms of action and can synergistically enhance antimyeloma effects of other drugs (e.g., dexamethasone).11-13 The efficacy and tolerability of lenalidomide + dexamethasone (Rd) in NDMM have been demonstrated in multiple trials, including the phase III FIRST study, which established continuous Rd treatment in TNE NDMM.14,15 Likewise, bortezomib enhances the antimyeloma activity of dexamethasone and other agents16-18 and has been evaluated extensively in NDMM.19 Rd has synergistic activity, as confirmed in relapsed/refractory MM (RRMM) phase I/II studies in which lenalidomide-exposed and bortezomib-exposed patients achieved durable responses, with favorable toxicity.20,21 Each drug individually, especially bortezomib, has positive effects on bone metabolism.22,23 Since the treatment landscape has shifted toward triplet regimens due to their improved efficacy over doublet regimens, clinical evaluations of RVd in NDMM have accelerated. With the success of RVd in NDMM, newer agents have been evaluated in this setting with Rd, including carfilzomib,24 ixazomib,25 daratumumab (DARA),26 and elotuzumab.27

RVd: phase II studies in newly diagnosed multiple myeloma The RVd regimen was initially evaluated with bortezomib administered intravenously (IV) (Table 1). In the first and seminal phase I/II trial, following RVd induction, patients achieving a partial response (PR) or better could undergo ASCT, and all responding patients could then receive tailored RVd maintenance after eight cycles.28 The randomized phase II EVOLUTION trial, which included lenalidomide and bortezomib with weekly dexamethasone treatment (the so-called VRd regimen) versus three other

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bortezomib-, dexamethasone-, and cyclophosphamidecontaining regimens, in patients with TE or TNE NDMM followed.29 This study demonstrated a combined efficacy and manageable toxicity profile (including the lowest rates of grade ≥3 hematologic and overall adverse events [AE] among the evaluated regimens), which warranted further investigation in phase III trials.29 The IFM 2008 trial evaluated RVd, administered in three 21-day cycles, in TE patients with NDMM.30 Patients then proceeded to ASCT, after which those who had not progressed received two 21-day cycles of RVd consolidation. This study demonstrated the favorable efficacy of the RVd regimen in TE patients. The second wave of phase II RVd trials (using subcutaneous [SC] bortezomib) provided supporting data, exploring different dosing strategies, such as the RVd Lite regimen designed to minimize toxicities in older TNE patients by using lower dose intensities.31-33 Notably, promising results from the phase II GRIFFIN trial evaluating quadruplet RVd-DARA versus RVd34,35 have led to implementation of RVd as a basis for quadruplet regimens, which are poised for inclusion in the NDMM treatment paradigm.34,36 Key characteristics of the study populations, outcomes, and selected safety findings of these phase II studies are shown in Table 1, and the phase II RVd dosing schedules are shown in Table 2.

RVd: phase III studies in newly diagnosed multiple myeloma Phase III studies have further supported the use of RVd in NDMM (Table 3). The pivotal SWOG S0777 trial demonstrated greater efficacy of RVd than Rd in patients not intended for immediate ASCT, supporting regulatory approval of RVd.3,37 Patients randomly assigned to RVd received eight 21-day cycles (Table 2), followed by Rd maintenance. After a median follow-up of 7 years, improved PFS (median, 41 vs. 29 months; P=0.003 ) (Figure 1A) and OS (median, not reached [NR] vs. 69 months; P=0.0114) (Figure 1B) were observed with RVd versus Rd.38 RVd also improved depth of response, with 75% of patients achieving ≥VGPR versus 53% with Rd. Rates of toxicities were generally similar between the treatment groups, but more grade ≥3 neurologic toxicities were observed with RVd than with Rd (34.6% vs. 11.3%, respectively), likely due to the use of IV bortezomib. A post-hoc analysis of this trial also evaluated RVd versus Rd in patients stratified by age.39 In patients <65 years of age (n=269), improved PFS (median, 55.4 vs. 36.6 months; hazard ratio [HR]=0.63, 95% confidence interval [95% CI]: 0.46-0.87) and OS (median, NR vs. 68.9 months; HR=0.61, 95% CI: 0.39-0.97) were observed with RVd versus Rd. Higher rates of grade ≥3 treatment-emergent adverse events (TEAE) (87 vs.

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Table 1. Phase II studies evaluating RVd in patients with newly diagnosed multiple myeloma.a

Study

Population

Response

PFS and OS

Selected safety findings Grade 3/4 AE

Richardson et al. (NCT00378105)

Regimen: 8 × 21-day cycles of RVd (optional ASCT after 4 cycles if ≥PR) → RVd maintenance after 8 cycles (if responding)b

Phase II (best response): ORR: 100% ≥VGPR: 74% N=66

18-mth PFS rate: 75% All patients (best response): ORR: 100% ≥VGPR: 67%

Phase II: N=35

18-mth OS rate: 97%

Lymphopenia: 14% Neutrophils: 9% Platelets: 6% Neuropathic pain: 3% Neuropathy, sensory: 2% Neuropathy, motor: 2% No grade 4 neuropathy

EVOLUTION29 (NCT00507442) Regimens: (8 × 21-day cycles of RVd vs. VDC vs. VDC-mod vs. VDCR) → BORT maintenance

Grade ≥3 AE

N=140

ORR (best response): 85%

RVd: N=42 (98% TE)

≥VGPR (best response): 51%

1-yr PFS rate: 83% 1-yr OS rate: 100%

ORR (end of consolidation): 97% IFM 200830 (NCT01206205) 3 × 21-day cycles of RVd → ASCT → 2 cycles of RVd consolidation → LEN maintenance (+ BORT if patient had high-risk features)

≥VGPR (end of consolidation): 87%

N=31

ORR (best response at any time): 100%

TE patients

Neutropenia: 10% Thrombocytopenia: 12% Neuropathy: 17%

Grade 3/4 AE (during RVd induction or consolidation)

3-yr PFS rate: 77% 3-yr OS rate: 100%

Neutropenia: 35% Thrombocytopenia: 13% Grade 3/4 AE (reported at any time) Neutropenia: 65% Thrombocytopenia: 19%

≥VGPR (best response at any time): 84%

No grade 3/4 neuropathy Grade ≥3 related AE

CTRIAL-IE (ICORG) 13-1733 (NCT02219178)

ORR (after 4 cycles of RVd): 92.5%c

N=42

4 × 21-day cycles of RVd → TE or TNE patients (ASCT or 4 more cycles of RVd) → LEN maintenance

≥VGPR (after 4 cycles of induction): 62.5%c

Not reported

Thrombocytopenia: 16.7% Fatigue: 11.9% Neutropenia: 9.5% PN: 4.8% No grade 4 PN

RVd Lite31 (NCT01782963)

Grade ≥3 TEAE N=50

9 × 35-day cycles of RVd Lite → 6 × 28-day cycles of LEN + BORT consolidation → LEN maintenance

TNE patients

FMG-MM0232 (NCT01790737) N=80 3 × 21-day cycles of RVd → (CY + FIL mobilization vs. FIL mobilization) → ASCT → LEN maintenance

TE patients

ORR (after 4 cycles of RVd Lite): 86% ≥VGPR (after 4 cycles of RVd Lite): 66%

ORR (best response at any time): 89% ≥VGPR (best response at any time): 68%

Median PFS: 35.1 mths Median OS: NR

1-/2-/3-yr PFS rates: 78%/67%/52% 1-/2-/3-yr OS rates: 96%/90%/83%

Hypophosphatemia: 34% Fatigue: 16% Neutropenia: 14% PN: 2% Thrombocytopenia: 2% Grade ≥3 AE in patients who received RVd induction (N=78) Neutropenia: 24% Infections: 23%d Febrile neutropenia: 22% Thrombocytopenia: 14% PN: 3% Continued on following page.

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Study

Population

Response

P.G. Richardson et al.

PFS and OS

Selected safety findings Grade 3/4 TEAE

GRIFFIN34, 35 (NCT02874742) (4 × 21-day cycles of RVd vs. RVd-DARA) → ASCT → (2 cycles of RVd vs. RVd-DARA consolidation) → (LEN vs. LEN + DARA maintenance)

N=207 RVd: N=104 RVd-DARA: N=103 TE patients

ORR (by end of induction) RVd: 91.8% RVd-DARA: 98.0% (by end of consolidation) RVd: 91.8% RVd-DARA: 99.0% sCR (by end of induction) RVd: 7.2% RVd-DARA: 12.1% (by end of consolidation) RVd: 32.0% RVd-DARA: 42.4% P=.068e

1-yr PFS rate RVd: 95.3% RVd-DARA: 96.9% 2-yr PFS rate RVd: 89.8% RVd-DARA: 95.8% 1-yr OS rate RVd: 97.9% RVd-DARA: 99.0% 2-yr OS rate RVd: 93.4% RVd-DARA: 95.8%

Neutropenia RVd: 22% RVd-DARA: 41% Lymphopenia RVd: 22% RVd-DARA: 23% Thrombocytopenia RVd: 9% RVd-DARA: 16% PNf RVd: 8% RVd-DARA: 7% Grade 3/4 TEAE

PLEIADES69 (NCT03412565)

≥VGPR rate (at primary analysis) RVd-DARA: 71.6%

N=199 RVd-DARA: 4 × 21-day cycles of RVd-DARA VMP-DARA: 9 × 42-day cycles → 28-day cycles until PD Rd-DARA: 28-day cycles until PD

RVd-DARA: N=67 VMP-DARA: N=67 Rd-DARA: N=65

ORR (at primary analysis) VMP-DARA: 88.1% Rd-DARA: 90.8%

Neutropenia RVd-DARA: 28.4% VMP-DARA: 37.3% Rd-DARA: 49.2% Not reported

Lymphopenia RVd-DARA: 16.4% VMP-DARA: 22.4% Rd-DARA: 10.8% Thrombocytopenia RVd-DARA: 14.9% VMP-DARA: 43.4% Rd-DARA: 13.8%

Due to differences in study design and procedures, cross-trial comparisons must be interpreted with caution. bRVd maintenance therapy comprised 21-day cycles of lenalidomide (dose tolerated at the end of cycle 8) on days 1-14, bortezomib (dose tolerated at the end of cycle 8) on days 1, 8, and dexamethasone 10 mg on days 1, 2, 8, 9. cOf 40 response-evaluable patients. dNot including febrile neutropenia. ePre-set one-sided α of 0.1. fGrouped term that includes peripheral neuropathy and peripheral sensory neuropathy. AE: adverse events; ASCT: autologous stem cell transplant; BORT: bortezomib; CY: cyclophosphamide; DARA: daratumumab; DEX: dexamethasone; FIL: filgrastim; IV: intravenously; LEN: lenalidomide; NR: not reached; ORR: overall response rate; OS: overall survival; PD: progressive disease; PFS: progression-free survival; PN: peripheral neuropathy; PR: partial response; RdDARA: lenalidomide, dexamethasone, and daratumumab; RVd: lenalidomide, bortezomib, and dexamethasone; RVd-DARA: lenalidomide, bortezomib, dexamethasone, and daratumumab; RVd Lite: modified lenalidomide, bortezomib, and dexamethasone; sCR: stringent complete response; TE: transplant eligible; TNE: transplant ineligible; TEAE: treatment-emergent adverse events; VDC: bortezomib, dexamethasone, and cyclophosphamide; VDC-mod: bortezomib, dexamethasone, and cyclophosphamide with an additional cyclophosphamide dose; VDCR: bortezomib, dexamethasone, cyclophosphamide, and lenalidomide; VGPR: very good partial response; VMP-DARA: bortezomib, melphalan, prednisone, and daratumumab; mth/mths: month/months; yr: year. a

79%) and treatment discontinuation due to toxicity (29 vs. 18%) were observed with RVd than with Rd. In patients ≥65 years of age (n=202), PFS (median, 33.1 vs. 25.8 months; HR=0.83, 95% CI: 0.60-1.16) and OS (62.9 vs. 53 months; HR=0.83, 95% CI: 0.55-1.23) were no longer statistically significant for RVd versus Rd. While rates of grade ≥3 TEAE were similar (93% for RVd vs. 89% for Rd), discontinuation due to toxicity was higher for RVd (47 vs. 26%). Interim results from the randomized phase III ENDURANCE (E1A11) trial, evaluating RVd versus carfilzomib, lenalidomide, and dexamethasone (KRd) in NDMM (regardless of

intent to undergo ASCT) demonstrated similar efficacy.40 In this trial, which enrolled standard-risk patients as well as those with fluorescence in situ hybridization (FISH)identified t(4;14) but excluded those with other high-risk cytogenetics, such as 17p deletion, PFS (censoring at SCT or alternative therapy) was 34.4 versus 34.6 months with RVd versus KRd, respectively (P=0.74), after a median follow-up of 15 months. The rate of ≥CR (14.8% vs. 18.3%; P=0.13) was also similar, although more patients achieved ≥VGPR with KRd (64.7% vs. 73.8%; P=0.0015). Notably, rates of grade ≥3 serious AE overall and cardiac, pulmonary, and

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REVIEW ARTICLE - Lenalidomide/bortezomib/DXM in newly diagnosed MM renal AE were lower with RVd than with KRd, while rates of grade 3/4 peripheral neuropathy (PN) were higher. The DSMM XIV study, evaluating induction with RVd (given as three 21-day cycles) compared with lenalidomide, doxorubicin, and dexamethasone (RAD), followed by response-adapted SCT (autologous or allogeneic) and lenalidomide maintenance, was designed to confirm the noninferiority of RAD for the induction phase primary endpoint of CR rate.41 In the RVd versus RAD arms, the rates of ≥CR were similar (13.0% vs. 11.8%), but there were nonsignificant trends toward higher rates of ≥VGPR (46.3% vs. 38.9%) and MRD negativity, as determined by next-generation sequencing (26.8% vs. 21.3%), with RVd. Rates of grade ≥3 neutropenia and thrombocytopenia were similar in the two arms, whereas grade ≥3 polyneuropathy/neuralgia was observed in 2.1% of patients treated with RVd and none treated with RAD. In an updated analysis (median follow-up, 40.2 months), median PFS from first randomization was longer with RVd than with RAD (53.7

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vs. 41.7 months; P=0.0439).42 In the IFM 2009 study, TE patients received three 21-day cycles of RVd induction (Table 2) followed by ASCT and two cycles of RVd consolidation (RVd + ASCT) or three cycles of RVd induction followed by five cycles of RVd consolidation (RVd); lenalidomide maintenance was then administered for 1 year.43,44 PFS was improved with RVd + ASCT versus RVd by approximately 12 months (median, 47.3 vs. 35.0 months; HR=0.70; 95% CI: 0.59-0.83; P<0.001).44 Remarkably, OS was similar (HR=1.03; 95% CI: 0.80-1.32; P=0.81); the median OS was NR in either arm.44 The rate of ≥VGPR was higher with RVd + ASCT than with RVd (88% vs. 77%; P=0.001).43 Grade 3/4 neutropenia, thrombocytopenia, and febrile neutropenia were more common with RVd + ASCT than with RVd; however, rates of grade 3/4 PN were similar. With a median follow-up of ≥43 months, the incidence of second primary malignancy per 100 patient-years did not differ significantly between patients treated with RVd + ASCT or RVd (1.5 vs. 1.1), nor

Table 2. RVd nomenclature and dosing schedules. Nomenclaturea RVd NDMM trials

Population

Cycle length

Lenalidomide

Bortezomibb

Dexamethasone

Richardson et al.28, c CTRIAL-IE (ICORG) 13-1733 FMG-MM0232 SWOG S077737 DSMM XIV41 IFM 200943, d ENDURANCE40, e RVD 100065 DETERMINATION45, f

TE or TE/TNE

21 days

25 mg days 1-14

1.3 mg/m2 days 1, 4, 8, 11

20 mg days 1, 2, 4, 5, 8, 9, 11, 12

Non-traditional RVdg

EVOLUTION29 IFM 200830

TE or TE/TNE

21 days

25 mg days 1-14

1.3 mg/m2 days 1, 4, 8, 11

40 mg days 1, 8, 15

Non-traditional RVdg

GRIFFIN34

21 days

25 mg days 1-14

1.3 mg/m2 days 1, 4, 8, 11

20 mg days 1, 2, 8, 9, 15, 16

GEM-RVdg

GEM 201249

28 days

25 mg days 1-21

1.3 mg/m2 days 1, 4, 8, 11

40 mg days 1-4, 9-12 20 mg ≤75 yr: days 1, 2, 8, 9, 15, 16, 22, 23 >75 yr: days 1, 8, 15, 22

RVd Classic

RVd Lite

15 mg days 1-21

1.3 mg/m2 days 1, 8, 15, 22

RVd LITE

TNE

35 days

RVd Ultra Lite

-h

TNE, frail

28-35 days

15 mg days 1-21

1.3 mg/m2 days 1, 8, 15

20 mg days 1, 2, 8, 9, 15, 16

RVd Premium Lite

-h

TNE

28 days

25 mg days 1-21

1.3-1.6 mg/m2 days 1, 8, 15, 22

20 mg days 1, 2, 8, 9, 15, 16, 22, 23

RVd regimen nomenclature is not yet standardized. bCTRIAL-IE (ICORG) 13-17, RVd LITE, FMG-MM02, DSMM XIV, GRIFFIN, and GEM2012 used SC bortezomib. ENDURANCE used SC or IV bortezomib. All other listed trials used IV bortezomib. SC bortezomib is generally used for the RVd Ultra Lite and RVd Premium Lite regimens, but IV bortezomib may be administered with IV normal saline for those patients who are not tolerant of SC bortezomib. cPhase II dosing for this trial. dDuring the consolidation phase of IFM 2009, RVd was administered with dexamethasone 10 mg in the transplant arm. eBortezomib on days 1 and 8 in cycles 9-12; dexamethasone reduced to 10 mg starting in cycle 5, and limited to days 1, 2, 8, and 9 during cycles 9-12. fIV or SC bortezomib; dexamethasone dose was 20 mg for cycles 1-3 and reduced to 10 mg starting in cycle 4. gThe RVd regimens used in these trials do not yet have widely accepted or proposed nomenclatures. hRegimen used in some clinics but not yet used in a published phase II or phase II clinical trial. IV: intravenous; NDMM: newly diagnosed multiple myeloma; RVd: lenalidomide, bortezomib, and dexamethasone; SC: subcutaneous; TE: transplant eligible; TNE: transplant ineligible; yr: year. a

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REVIEW ARTICLE - Lenalidomide/bortezomib/DXM in newly diagnosed MM did the frequency of acute myeloid leukemia (4 cases vs. 1 case) or myelodysplastic syndromes (1 case each), although follow-up remained short.43 The safety and efficacy of adding ASCT to RVd were also evaluated in the recently reported DETERMINATION study.45 Eligible patients received one cycle of RVd (Table 2) and were then randomly assigned 1:1 to receive two cycles of RVd with stem cell mobilization followed by either five cycles of RVd (RVd alone) or high-dose melphalan with ASCT and two subsequent cycles of RVd (RVd + ASCT). Both groups received maintenance therapy with daily lenalidomide until disease progression, unacceptable toxicity, or both. With a median follow-up of 76.0 months, PFS was significantly improved with RVd + ASCT versus RVd alone (median PFS, 67.5 vs. 46.2 months; HR=0.65; 95% CI: 0.52-0.81; P<0.001) (Figure 2A). However, no OS benefit with RVd + ASCT over RVd alone was observed (Figure 2B). The 5-year OS in patients with high-risk cytogenetics was greater with RVd + ASCT than with RVd alone (63.4% vs. 54.3%). Response rates were similar with RVd + ASCT and RVd alone (≥PR, 97.5% vs. 95.0%; ≥VGPR, 82.7% vs. 79.6%; ≥CR, 46.8% vs. 42.0%). A greater percentage of

P.G. Richardson et al.

patients achieved MRD negativity with RVd + ASCT than with RVd alone (54% vs. 40%; odds ratio=0.55; 95% CI: 0.30-1.01). In patients who were MRD positive, the median PFS was greater with RVd + ASCT than with RVd alone (50.6 vs. 33.4 months), but no difference in median PFS was seen in between the two arms for patients who were MRD negative. The absence of OS benefit is notable, especially given the use of ASCT in only 28% of patients in the delayed transplant arm to date.46 This is in contrast to the IFM/DFCI 2009 study in which salvage ASCT was used in almost 80% of patients.44 Moreover, while the overall rate of second primary malignancies was similar in both arms, ten cases of acute myeloid leukemia or myelodysplastic syndromes were seen in the transplant arm compared to no cases in the RVd-alone arm by the time of data cutoff (October 2021; P=0.002).47 Considering that four of the ten patients who developed acute myeloid leukemia or myelodysplastic syndromes had died by the time of data cutoff, careful monitoring is warranted. Finally, during ASCT, a significant and clinically meaningful decrease in quality of life occurred, which proved transient after several

Figure 1. Progression-free survival and overall survival for RVd vs Rd in the SWOG S0777 trial (median follow-up, 84 months) (A) Progression-free survival. (B) Overall survival. Figures reprinted from Durie BGM, et al. Blood Cancer J. 2020;10(5):53. Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).38 NR: not reached; OS: overall survival; PFS: progression-free survival; Rd: lenalidomide + dexamethasone; RVd: lenalidomide, bortezomib, and dexamethasone. Haematologica | 108 November 2023

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Table 3. Phase III studies evaluating RVd in patients with newly diagnosed multiple myeloma.a

Study

Population

Response

Selected safety findings

PFS and OS

Grade ≥3 AEc N=525 RVd: N=264 (N=215 for SWOG S077737,38 ORR; N=235 for PFS and (NCT00644228) OS) Rd: N=261 (N=207 for 8 × 21-day cycles of RVd vs. 6 ORR; N=225 for PFS and × 28-day cycles of Rd → Rd OS) b maintenance Patients not planned for immediate ASCT

ORR RVd: 90.2% Rd: 78.8% ≥VGPR RVd: 74.9% Rd: 53.2% CR RVd: 24.2% Rd: 12.1%

Blood or bone marrow RVd: 47.3% Rd: 46.0%

Median PFS RVd: 41 mths Rd: 29 mths P=0.003

Infectiond RVd: 14.5% Rd: 13.7%

Median OS RVd: NR Rd: 69 mths P=0.0114

Neurologicale RVd: 33.2% Rd: 11.1% Paine RVd: 12.0% Rd: 4.0% Grade ≥3 TEAE

DSMM XIV41,42 (NCT01685814) 3 × 21-day cycles of RVd vs. 3 × 28-day cycles of RAD → response-adapted SCT and LEN maintenance

476 patients randomized 469 received ≥1 dose of study drug RVd: N=237 RAD: N=232

≥CR (post-induction) RVd: 13.0% RAD: 11.8% P=0.697 ≥VGPR (post-induction) RVd: 46.3% RAD: 38.9% P=0.110

TE patients

Median PFS (from first randomization) RVd: 53.7 mths RAD: 41.7 mths P=0.0439

Neutropenia RVd: 5.5% RAD: 6.5% Thrombocytopenia RVd: 2.1% RAD: 2.6% PN/neuralgia RVd: 2.1% RAD: 0% Grade 3/4 AE

IFM 200943 (NCT01191060) 3 × 21-day cycles of RVd → 5 × 21-day cycles of RVd alonef vs. ASCT + 2 × 21-day cycles of RVdf → LEN maintenance

Neutropenia RVd alone: 47.4% RVd + ASCT: 92.0%

ORR (best response) RVd alone: 97% RVd + ASCT: 98% P=0.02g N=700 RVd alone: N=350 RVd + ASCT: N=350

≥VGPR (best response) RVd alone: 77% RVd + ASCT: 88% P=0.001

Median PFS RVd alone: 36 mths RVd + ASCT: 50 mths P<0.001

TE patients CR (best response) RVd alone: 48% RVd + ASCT: 59% P=0.03

Febrile neutropenia RVd alone: 3.4% RVd + ASCT: 14.9% Thrombocytopenia RVd alone: 14.3% RVd + ASCT: 83.1% Anemia RVd alone: 8.9% RVd + ASCT: 19.7% PN RVd alone: 12.0% RVd + ASCT: 12.9%

ORR (best response) RVd + ASCT: 97.5% RVd alone: 95.0%

DETERMINATION45 (NCT01208662) 3 × 21-day cycles of RVd → stem cell collection → 5 × 21day cycles of RVd alone vs. high-dose MEL + ASCT + 2 × 21-day cycles of RVd → LEN maintenance until PD

N=873 RVd + ASCT: N=365 RVd alone: N=357

≥VGPR (best response) RVd + ASCT: 82.7% RVd alone: 79.6% CR (best response) RVd + ASCT: 46.8% RVd alone: 42.0%

Grade ≥3 TEAE

Median PFS RVd + ASCT: 67.5 mths RVd alone: 46.2 mths P<0.0014

Neutropenia RVd + ASCT: 86.3% RVd alone: 42.6% Thrombocytopenia RVd + ASCT: 82.7% RVd alone: 19.9% Leukopenia RVd + ASCT: 39.7% RVd alone: 19.6%

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Study

Population

Response

P.G. Richardson et al. Selected safety findings

PFS and OS

ORR (after induction): 83.4% PETHEMA/ GEM201249 (NCT01916252) 6 × 28-day cycles of RVd → ASCT (IV busulfan + MEL) vs. ASCT (MEL) → 2 × 28-day cycles → RVd consolidation

≥VGPR (after induction): 66.6%

Grade 3/4 AE through induction

ORR (after ASCT): 81.2%

N=458

≥VGPR (after ASCT): 75.1%

TE patients

Median PFS: NR

ORR (after consolidation): 80.6%

Neutropenia: 12.9% Thrombocytopenia: 6.3% Infection: 9.2% PN: 3.9%h

≥VGPR (after consolidation): 75.5% ORR (after induction) RVd: 84.3% KRd: 86.7% P=0.13

N=1087 ENDURANCE (E1A11)40 (NCT01863550)

RVd: N=542 (N=527 for ORR, safety) 12 × 21-day cycles of RVd vs. 9 KRd: N=545 (N=526 for ORR, safety) × 28-day cycles of KRd → LEN maintenance × 2 years vs. LEN Patients not planned for maintenance until PD early ASCT

≥VGPR (after induction) RVd: 64.7% KRd: 73.8% P=0.0015 ≥CR (after induction) RVd: 14.8% KRd: 18.3% P=0.26

Median PFS RVd: 34.4 mths KRd: 34.6 mths P=0.74 3-yr OS RVd: 84% KRd: 86%

Grade ≥3 cardiac, pulmonary, and renal RVd: 4.7% KRd: 16.0% P<0.0001 Grade 3/4 PN RVd: 8.3% KRd: 0.8% Grade 3-5 SAE RVd: 22.0% KRd: 44.5% P<0.0001

Due to differences in study design and procedures, cross-trial comparisons must be interpreted with caution. bStem cell collection was allowed for patients considering future transplant. cAE considered to be unlikely related to treatment were stated to be excluded from reporting in the SWOG S0777 publication. dReported as a hematologic AE. eReported as a neurological AE. f During consolidation, patients received a reduced daily dose of DEX 10 mg. gP value for “best response during the study” overall. h PN was a grouped term including PN, neuralgia, polyneuropathy, and sensory loss. AE: adverse events; ASCT: autologous stem cell transplant; CR: complete response; DEX: dexamethasone; IV: intravenously; KRd: carfilzomib, lenalidomide, and dexamethasone; LEN: lenalidomide; MEL: melphalan; NR: not reached; ORR: overall response rate; OS: overall survival; PD: progressive disease; PFS: progression-free survival; PN: peripheral neuropathy; RAD: lenalidomide, doxorubicin, and dexamethasone; Rd: lenalidomide + dexamethasone; RVd: lenalidomide, bortezomib, and dexamethasone; SAE: serious adverse events; SCT: stem cell transplant; TE: transplant eligible; TEAE: treatment-emergent adverse events; VGPR: very good partial response; VMP; bortezomib, melphalan, and prednisone; mths: months. a

months and then improved over time.45 These findings corroborate similar results seen in the IFM/DFCI 2009 trial.48 In the PETHEMA/GEM2012 study, patients with TE NDMM received six 28-day cycles of RVd induction (Table 2) followed by ASCT with IV busulfan + melphalan versus melphalan and RVd consolidation (2 cycles).49 The RVd schedule was devised to increase lenalidomide and dexamethasone dose intensity in order to maximize response. At a median follow-up of 84.4 months, the median PFS was 80.8 months.50 An induction analysis of the pooled population showed that the rate of ≥VGPR was 66.6% and increased with more cycles during RVd induction, ranging from 55.6% to 70.4% for cycles 3-5 and post-induction, respectively. The rate of MRD negativity after induction was 28.8%. Common grade 3/4 AE were neutropenia, infection, and thrombocytopenia. The rate of grade 3/4 PN (including neuralgia, polyneuropathy, and sensory loss) was 3.9%.

The bortezomib, thalidomide, and dexamethasone (VTD) regimen has been used in NDMM outside of the USA,51 with no randomized controlled trials comparing RVd versus VTD conducted to date. Thus, findings from the PETHEMA/GEM2005, PETHEMA/GEM2012, IFM 2009, and IFM 2013-04 trials were used to conduct an integrated analysis52 evaluating RVd versus VTD in TE NDMM.43,49,53,54 In the GEM studies, the rate of ≥VGPR after induction was higher with RVd than with VTD (70.1% vs. 55.9% at cycle 6); findings from the IFM analyses (four 21-day–cycle regimens) showed noninferiority between RVd (57.1%) and VTD (56.5%). Safety findings were consistent with the individual toxicity profiles of the constituent agents.

RVd: adverse events and management AE reported with RVd are generally consistent with the

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REVIEW ARTICLE - Lenalidomide/bortezomib/DXM in newly diagnosed MM profiles of lenalidomide and bortezomib combined with dexamethasone, and include thrombocytopenia, neutropenia, infection, and PN.15,55 Multiple AE prevention strategies are relevant for RVd in clinical practice. RVd is likely to induce emesis in some patients (<30%); therefore, antiemetic prophylaxis can be given on bortezomib treatment days, as needed.56 Optimal use of granulocyte colony-stimulating factor prophylaxis in this population is dynamic, and it may be considered for neutropenia management. Antiviral prophylaxis is considered mandatory against varicella zoster, while antibacterial prophylaxis is recommended for some patients based on their individual risk factors. Thromboprophylaxis is mandatory unless contraindicated. Other strategies as supportive care may also ameliorate toxicities, including emollients and supplements for treatment-emergent PN or infusion of normal saline during bortezomib administration.57 Stem cell collection following RVd induction is an important consideration for patients who can later pursue ASCT. 28-30,34,49 Stem cell collection should be completed within four to six cycles of RVd induction therapy.58

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RVd: dose and schedule The posology of RVd is critical to optimize effectiveness. Reduced dose intensity via modifying cycle length and dosing frequency can attenuate the risk or severity of AE. Appropriate dose modifications (i.e., interruptions, reductions, or discontinuations) are key for AE management after onset.57 Importantly, the use of SC rather than IV bortezomib can reduce the intensity and frequency of PN without compromising efficacy.59 If discontinuation of bortezomib is warranted, Rd may be continued until progressive disease. RVd has been administered in varying posologies (Table 2), leading to nomenclature that reflects varying schedules of dose intensity which we will use throughout this review: • “RVd Classic”: 21-day cycle, lenalidomide 25 mg (days 114), bortezomib 1.3 mg/m2 (days 1, 4, 8, 11), dexamethasone 20 mg (days 1, 2, 4, 5, 8, 9, 11, 12) • “RVd Lite”: 35-day cycle, lenalidomide 15 mg (days 1-21), bortezomib 1.3 mg/m2 (days 1, 8, 15, 22), dexamethasone 20 mg (≤75 years of age: days 1, 2, 8, 9, 15, 16, 22, 23; >75 years of age: days 1, 8, 15, 22)

Figure 2. Progression-free survival and overall survival for RVd + autologous stem cell transplant versus RVd alone in the DETERMINATION trial (median follow-up, 76.0 months). (A) Progression-free survival. (B) Overall survival. Figures from the New England Journal of Medicine, Richardson PG, et al., “Triplet therapy, transplantation, and maintenance until progression in myeloma”.45 Copyright© (2022) Massachusetts Medical Society. Reprinted with permission. ASCT, autologous stem cell transplant; RVd, lenalidomide, bortezomib, and dexamethasone. Haematologica | 108 November 2023

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REVIEW ARTICLE - Lenalidomide/bortezomib/DXM in newly diagnosed MM • “RVd Ultralite”: 28- to 35-day cycle, lenalidomide 15 mg (days 1-21), bortezomib 1.3 mg/m2 (days 1, 8, 15), dexamethasone 20 mg (days 1, 2, 8, 9, 15, 16) • “RVd Premium Lite”: 28-day cycle, lenalidomide 25 mg (days 1-21), bortezomib 1.3-1.6 mg/m2 (days 1, 8, 15, 22), dexamethasone 20 mg (days 1, 2, 8, 9, 15, 16, 22, 23) • “GEM-RVd”: 28-day cycle, lenalidomide 25 mg (days 121), bortezomib 1.3 mg/m2 (days 1, 4, 8, 11), dexamethasone 40 mg (days 1-4, 9-12) The use of these names remains fluid. VRd, which is sometimes used as an alternate, describes the steroid-attenuated regimen first developed in the EVOLUTION trial. The distinctions otherwise center on cycle length and bortezomib frequency. Most commonly, RVd was administered in 21-day cycles, with lenalidomide 25 mg on days 1-14 and bortezomib 1.3 mg/m2 (IV or SC) on days 1, 4, 8, and 11; some studies used dexamethasone 40 mg once weekly,29,30 and others split the dose to 20 mg on days of and after bortezomib (“partnered dosing”, totaling 80 mg/week).32,33,37,41,43 Importantly, although weekly dexamethasone may be more convenient for some patients, the severity of bortezomibinduced PN may be mitigated by partnered dosing.60 PETHEMA/GEM2012 used 28-day cycles,49 which provide higher lenalidomide and dexamethasone dose intensities and a lower bortezomib dose intensity compared with 21day regimens, which may allow for increased efficacy and completion of planned induction. RVd Lite was developed specifically to maximize tolerability in older patients by extending cycle length, reducing the lenalidomide dose, administering bortezomib SC once weekly four times, and using an age-based schedule for dexamethasone.31 RVd Lite has the lowest lenalidomide intensity of all reviewed regimens, but offers long-term lenalidomide treatment (9 RVd induction cycles, 6 lenalidomide + bortezomib consolidation cycles, and optional lenalidomide maintenance) and demonstrates striking activity, less toxicity, and impressive clinical benefit.15,61 A recent observational, single-center study evaluated another modified version of RVd in TE NDMM, using fulldose lenalidomide and once-weekly bortezomib with the goal of minimizing PN risk.62 Patients received induction or salvage therapy with lenalidomide 25 mg on days 1-21; bortezomib 1.3 mg/m2 SC on days 1, 8, and 15; and dexamethasone 40 mg on days 1, 8, and 15 (28-day cycles). The overall response rate (ORR) was 87%, and 63% of patients achieved ≥VGPR. Of note, those who received RVd for induction had an ORR of 89% compared to 75% in those who received salvage, with no cases of grade ≥3 PN. To date, there are no head-to-head comparisons of the various RVd dosing regimens, and thus the advantages of any one regimen over another are not definitive. Although data from single-center studies support weekly bortezomib use,63 selection of this schedule should be individualized based on risks and benefits. Moreover, additional data

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reported across numerous studies with different patient populations allow clinicians considerable flexibility to factor in patient- and disease-specific factors when selecting the RVd dose and schedule, although most patients receive RVd Classic dosing regimens.64,65

RVd and quadruplet regimens Given its proven efficacy, RVd has been used as a foundation for quadruplet regimens for the treatment of NDMM. The first study of an RVd-based quadruplet was the previously discussed EVOLUTION trial, which included a VRd + cyclophosphamide (VRdC) arm.29 The efficacy of VRdC and VRd was similar (ORR, 88% vs. 85%; 1-year PFS rates, 86% and 83%). Hematologic toxicity rates were higher with VRdC than with VRd, especially grade 3/4 neutropenia (44% vs. 10%) and leukopenia (13% vs. 0%), with treatment-related mortality in the VRdC arm. Additionally, a phase I/II study investigated RVd + pegylated liposomal doxorubicin in eight 21-day cycles.66 Patients achieving ≥PR after four cycles could proceed to ASCT, and those achieving stable disease or better after eight cycles and not proceeding to ASCT could receive RVd maintenance. The phase II dose used RVd Classic, with dexamethasone 10 mg in cycles 5-8 and pegylated liposomal doxorubicin 30 mg/m2 on day 4: the ORR after four and eight cycles was 96% and 95%, with ≥VGPR in 57% and 65%, respectively. The median PFS was NR, but 18-month PFS was 81.6%, with grade 3/4 neutropenia and thrombocytopenia reported in 19% and 11% of patients, respectively. Results of these trials suggest that conventional chemotherapy may not be the ideal addition to RVd. Conversely, in the previously described phase II GRIFFIN study, patients with TE NDMM received either RVd or RVdDARA.34 The rates of grade 3/4 neutropenia and thrombocytopenia were higher with RVd-DARA than with RVd; however, rates of grade 3/4 lymphopenia and PN were similar. A final analysis of the safety run-in cohort of the GRIFFIN study found that 15 (93.8%) of the 16 patients receiving RVd-DARA achieved a stringent CR as best response at last follow-up.67 Additionally, the phase III COLUMBA RRMM trial has demonstrated non-inferiority of SC versus IV daratumumab, with an improved safety profile.68 The phase II PLEIADES study examined the addition of SC daratumumab to standard-of-care regimens and found that SC daratumumab had comparable efficacy to IV daratumumab (≥VGPR of 71.6% for RVd-DARA, ORR of 88.1% for VMP-DARA, ORR of 90.8% for Rd-DARA), with a median infusion duration of only 5 minutes and a low rate (≤9%) of infusion-related reactions.69 The ongoing phase III NDMM studies MMY3019 (NCT03652064) and PERSEUS (NCT03710603) are evaluating RVd-DARA versus RVd using SC daratumumab in patients not planned to undergo ASCT and in TE patients,

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REVIEW ARTICLE - Lenalidomide/bortezomib/DXM in newly diagnosed MM respectively. Of note, the PERSEUS study used a regimen of oral lenalidomide 25 mg on days 1-21 and oral dexamethasone 40 mg on days 1-4 and days 9-12 of each 28-day induction cycle. Additionally, the ongoing phase II MMY2040 study is evaluating multiple daratumumab-containing regimens (including RVd-DARA) using the SC formulation (NCT03412565). In the phase I portion of SWOG S1211, elotuzumab + RVd demonstrated limited additive toxicity to RVd alone.70 However, in the randomized phase II portion in high-risk NDMM, elotuzumab + RVd did not significantly improve patients’ outcomes compared to RVd alone (ORR=83% vs. 88%; median PFS, 31 vs. 34 months).71 This finding was supported in a follow-up analysis of RVd + elotuzumab versus RVd in SWOG-1211, with no improvement in median PFS (29 vs. 34 months) or OS (NR vs. 68 months) observed with a median follow-up of 6 years.71 Another phase II trial of patients with TE NDMM demonstrated an ORR (after 4 cycles) of 82.5%, with 55.0% of patients achieving ≥VGPR.72 However, 50% of patients experienced infections, including one grade 5 sepsis. In the phase III GMMG HD6 trial, four induction cycles of elotuzumab + RVd produced similar response outcomes as RVd alone (ORR, 82.4% vs. 85.6%; ≥VGPR, 58.3% vs. 54.0%).73 Isatuximab + RVd was well tolerated in a phase I study and extremely active,74 with phase I (NCT02513186), phase II/III (UK-MRA Myeloma XV RADAR [2019-001258-25]), and phase III (GMMG-HD7 [NCT03617731] and IMROZ [NCT03319667]) NDMM clinical trials ongoing.75 Notably, the combination of panobinostat + RVd demonstrated activity and tolerability in a phase Ib study of patients with RRMM76 and favorable efficacy in a phase Ib study of TE NDMM patients.77 In the TE NDMM study, patients who received panobinostat + RVd at the maximum tolerated dose (RVd Classic with SC bortezomib and panobinostat 10 mg on days 1, 3, 5, 8, 10, and 12) had an ORR of 96% after ≤4 cycles, including a ≥VGPR rate of 87%.77 The toxicity of panobinostat + RVd manifested as primarily low-grade gastrointestinal effects, which were usually manageable with supportive care. Finally, in a phase I trial of vorinostat + RVd in NDMM, vorinostat proved most tolerable at 200 mg given on days 1-14 of each 21-day cycle with the RVd Classic regimen.78 An objective response was observed in 96% of patients, with 48% of patients achieving complete remission.78 Gastrointestinal symptoms (87%), fatigue and PN (60%), and thrombocytopenia (33%) were the most common AE.78 In summary, RVd-based quadruplet regimens with monoclonal antibodies have exhibited promising activity and tolerability, although data from patients with high-risk MM remain limited, and demonstrate the clinical potential of RVd as a foundation for four-drug regimens. Importantly, toxicity profiles were not additive and proved manageable.

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Perspective Although multiple triplet regimens have been explored in the NDMM setting (Table 4), RVd is a particularly attractive option. RVd has been extensively evaluated in phase II and III trials, demonstrating impressive clinical activity, deep and durable responses in both TE and TNE NDMM populations, and a manageable safety profile. Moreover, the variety of dosage schedules investigated, including both high- and low-dose intensity modifications to the common 21-day cycles, facilitate unique customization for clinicians who may want to emphasize deep responses or tailored tolerability and treatment duration. Additionally, the improvement in median PFS observed with RVd + ASCT in frontline treatment, particularly in high-risk patients, demonstrates how RVd can be used as a platform to build patient-tailored treatments and reaffirms earlyline ASCT as a standard of care in selected patients.45 This benefit of RVd as a backbone regimen in high-risk patients was further supported by the results of the UK Optimum/MUKnine trial, which reported a 94% ORR at the end of induction and an 83% ORR at day 100 after ASCT in ultra-high-risk patients with NDMM.79 Thus, RVd has become a standard of care in NDMM. Global treatment guidelines (including those in the USA and EU) recommend RVd regardless of transplant eligibility.1,2 A post-hoc subgroup analysis of SWOG S0777 suggesting a smaller magnitude of benefit with RVd in older patients (≥65 years) is a consideration; however, further study in this population is needed. Recent approvals will likely increase the use of RVd in clinical practice, particularly in the EU. The availability of generic bortezomib and lenalidomide will also likely reduce the cost associated with induction therapy and contribute to increased use in real-world practice. Moreover, the excellent activity of RVd has been confirmed outside of the clinical trial setting.65 The RVD 1000 study, a database cohort study of 1,000 patients with NDMM who received RVd induction ± ASCT and risk-adapted maintenance, reported an ORR of 97.1% after induction (≥VGPR, 67.6%; ≥CR, 35.9%), a median PFS of 65.0 months, and a median OS of 126.6 months, demonstrating the substantial long-term benefit of RVd. The large size of the study enables subanalyses, including for standard- versus high-risk cytogenetics (median PFS, 76.5 vs. 40.3 months; median OS, NR vs. 78.2 months, respectively). In the context of the TE NDMM population, the recent results of DETERMINATION, with its relative maturity of follow-up, provide insights into the benefit of RVd in different populations and validate the tailoring of treatment in each individual patient, based upon the outcomes reported.45-47 The comparisons between DETERMINATION and IFM/DFCI 2009 further validate the importance of lenalidomide maintenance until progression after RVd-based induction

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as well as the benefit and competing risks of the use of steps in improving outcomes are well underway and inhigh-dose melphalan with its impact on OS.43-45 The next clude the integration of monoclonal antibodies and other

Table 4. Phase III induction data from select non-RVd-based triplet regimens for newly diagnosed multiple myeloma. Regimen

VMP

Study

VISTA88,89 (NCT00111319) 9 × 42-day cycles of VMP (IV BORT)

Population

N=344 (N=337, response; N=340, safety)

Response

Selected safety findings

PFS and OS

ORR: 74.5% ≥VGPR: 41.2% ≥CR: 32.9%

Median TTP: 24.0 mths

ORR: 81.0% ≥VGPR: 49.8% ≥CR: 24.1%

Median PFS: 24.8 mths

3-yr OS rate: 68.5%

TNE patients

VMP

GIMEMA-MM-03-0590,91 N=257 (NCT01063179) (N=253, response/safety) 9 × 42-day cycles of VMP (IV BORT) TNE patients UPFRONT92 (NCT00507416) 8 × 21-day cycles of VMP (IV BORT) → 5 × 35-day cycles of BORT maintenance

N=167 (N=145 response; N=163 safety) TNE patients

Induction response: ORR: 67.6% ≥VGPR: 36.6% ≥CR: 2.8%

Median OS: 60.6 mths

Median PFS: 17.3 mths Median OS: 53.1 mths

Grade 3/4 AE Neutropenia: 40.0% Thrombocytopenia: 37.1% Leukopenia: 22.6% Lymphopenia: 19.7% Anemia: 18.2% PN: 12.9% Grade 3/4 AE Neutropenia: 28.1% Thrombocytopenia: 19.8% PN: 5.1% Grade ≥3 AE PN: 19.6% Neutropenia: 19.0% Infection: 17.8% Thrombocytopenia: 14.7% Grade ≥2 PN: 35.0%

VMP

VTD

ALCYONE93,94 (NCT02195479) 9 × 42-day cycles of VMP (SC BORT)

CLARION95 (NCT01818752) 9 × 42-day cycles of VMP (IV or SC BORT)

GIMEMA MMY-300651,96 (NCT01134484) 3 × 21-day cycles of VTD (IV BORT) → tandem ASCT → consolidation with 2 × 35-day cycles of VTD → DEX maintenance IFM 2007-0297 (NCT00910897) 4 × 21-day cycles of VTD (reduced dose THAL/IV BORT) → ASCT (post-ASCT treatment at physician discretion) GEM200554 (NCT00461747) 6 × 28-day cycles of VTD (IV BORT) → ASCT → maintenance (IFN-α2b vs. VT)

N=356 TNE patients

N=477 (N=470, safety) TNE patients

N=236 TE patients

N=100 TE patients

N=130 TE patients

ORR: 73.9% ≥VGPR: 49.7% ≥CR: 25.3%

ORR: 78.8% ≥VGPR: 49.3% ≥CR: 23.1%

Induction response: ORR: 93.2% ≥VGPR: 61.9% ≥CR: 18.6%

Induction response: ORR: 88% ≥VGPR: 49% ≥CR: 13%

Induction response: ORR: ≈85% ≥VGPR: ≈60% ≥CR: 35%

Median PFS: 19.3 mths 36-mth OS rate: 67.9%

Median PFS: 22.1 mths

10-yr PFS rate: 34% 10-yr OS rate: 60%

Median PFS: 26 mths

Median PFS: 56.2 mths 4-yr OS rate: 74%

Grade 3/4 AE Neutropenia: 38.7% Thrombocytopenia: 37.6% Anemia: 19.8% Infections: 14.7% PN: 4.0% Grade ≥3 AE Neutropenia: 29.4% Thrombocytopenia: 21.1% Anemia: 13.6% Leukopenia: 12.8% PN: 7.9%

Grade 3/4 AE during induction Skin rash: 10.2% PN: 9.7%

Grade 3/4 AE during induction Infections: 10.0% PN: 3.0%

Grade 3/4 AE during induction Infection 20.8% PN: 13.1% DVT/PE: 11.5% Neutropenia: 10.0%

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Regimen

VTD

CyBorD

RAD

DRd

Study IFM 2013-0453 (NCT01564537) 4 × 21-day cycles of VTD (SC BORT) → ASCT (conditioning regimen, single vs. tandem ASCT, consolidation, maintenance at discretion of each center) CASSIOPEIA98 (NCT02541383) 4 × 28-day cycles of VTD (SC BORT) → ASCT → 2 × 28-day cycles of VTD → maintenance (DARA vs. observation) UPFRONT92 (NCT00507416) 8 × 21-day cycles of VTD (IV BORT) → 5 × 35-day cycles of BORT maintenance

GMMG-MM599 3 × 21-day cycles of CyBorD → ASCT (single or tandem) → 2 cycles LEN consolidation → LEN maintenancea

IFM2013-0453 (NCT01564537) 4 × 21-day cycles of CyBorD (SC BORT) → ASCT (conditioning regimen, single vs. tandem ASCT, consolidation, maintenance at discretion of each center) DSMM XIV41,42 (NCT01685814) 3 × 28-day cycles of RAD → response adapted SCT and LEN maintenance MAIA26,100 (NCT02252172) 28-day cycles of DRd until PD or unacceptable toxicity

Population

N=169 (ITT) TE patients

N=542 (N=538, safety) TE patients

N=167 (N=133 response; N=158 safety) TNE patients

N=251 (ITT); (N=250, safety) TE patients

N=169 (ITT) TE patients

N=232 TE patients

N=368, (N=364 for safety) TNE patients

KRd

ENDURANCE40 (E1A11) (NCT01863550) N=545 (N=526 for ORR, 9 × 28-day cycles of KRd → safety) LEN maintenance × 2 years Patients not planned for vs. LEN maintenance early ASCT until PD

Response

Induction response: ORR: 92.3% ≥VGPR: 66.3% ≥CR: 13.0%

Induction response: ORR: 89.9% ≥VGPR: 56.1% ≥CR: 8.9%

Induction response: ORR: 78.9% ≥VGPR: 48.9% ≥CR: 0.8%

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PFS and OS

Grade 3/4 AE during induction Neutropenia: 18.9%

Not evaluated

Grade 2-4 PN: 21.9%

18-mth PFS rate: 85%

Grade 3/4 AE Stomatitis: 16.4% Neutropenia: 14.7% PN: 8.6%

Median PFS: 15.4 mths

Grade ≥3 AE PN: 27.2% Infection: 15.8% Fatigue: 12.0%

Median OS: 51.5 mths

Grade ≥2 PN: 47.5%

Induction response: ORR: 78.1% ≥VGPR: 37.1%

Grade ≥3 AE during induction Leukocytopenia/neutropenia: 35.2% Not reported Grade ≥2 AE Infections and infestations: 22.4% Neuropathy: 8.4%

Induction response: ORR: 83.4% ≥VGPR: 56.2% ≥CR: 8.9%

Not evaluated

Grade 3/4 AE during induction Neutropenia: 33.1% Thrombocytopenia: 10.6% Infection: 10.1% Grade 2-4 PN: 12.9%

Induction response: ≥CR: 13.5% ≥VGPR: 40.6%

Median PFS (from first randomization) RAD: 41.7 mths

Grade ≥3 AE Neutropenia: 6.5% PN/neuralgia: 0%

48-mth PFS rate: 60%

Grade 3/4 AE Neutropenia: 53.3% Pneumonia: 18.4% Anemia: 16.2% Lymphopenia: 16.2% Infections: 40%

ORR: 92.9% ≥VGPR: 80.7% ≥CR: 51.1%

Induction response: ORR: 86.7% ≥VGPR: 73.8% ≥CR: 18.3%

Median PFS: 34.6 mths 3-year OS rate: 86%

Grade 3/4 AE Dyspnea: 7.2% Hyperglycemia: 6.5% PN: 0.8% Grade ≥3 cardiac, pulmonary, and renal AE: 16.0%

Data are provided for informational purposes only. No cross-trial comparisons should be made. Differences in patient population, study design (length of induction; use of transplant, consolidation, and/or maintenance), assessment criteria, and/or study conduct may have an impact on the results of each trial. aBortezomib was changed from intravenous to subcutaneous during the trial. AE: adverse effects; ASCT:autologous stem cell transplant; BORT: bortezomib; CR: complete response; CyBorD: cyclophosContinued on following page. Haematologica | 108 November 2023

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P.G. Richardson et al.

phamide, bortezomib, and dexamethasone; DARA: daratumumab; DEX: dexamethasone; DRd: daratumumab, lenalidomide, and dexamethasone; DVT: deep vein thrombosis; IFN: interferon; ITT: intent-to-treat; IV: intravenously; KRd: carfilzomib, lenalidomide, and dexamethasone; LEN: lenalidomide; ORR: overall response rate; OS: overall survival: PD: disease progression; PE: pulmonary embolism; PFS: progression-free survival; PN: peripheral neuropathy; RAD: lenalidomide, adriamycin, and dexamethasone; RVd: lenalidomide, bortezomib, and dexamethasone; SC: subcutaneously; TE: transplant eligible; THAL: thalidomide; TNE: transplant ineligible; TTP: time to progression; VCD: bortezomib, cyclophosphamide, and dexamethasone; VGPR: very good partial response; VMP: bortezomib, melphalan, and prednisone; VT: bortezomib and thalidomide; VTD: bortezomib, thalidomide, and dexamethasone; mth/mths: month/months; yr: year.

novel strategies to enhance the effectiveness of treatment paradigms in NDMM.46,47 In consideration of RVd, the potent activity of the secondgeneration proteasome inhibitor carfilzomib is fully acknowledged. Unlike RVd, carfilzomib + Rd (KRd) is approved only in the RRMM setting. In the USA, KRd is indicated for the treatment of patients with RRMM after one to three prior lines of therapy, and in the EU, KRd is approved for use in patients with RRMM after one or more prior lines of therapy. Regulatory approval of KRd for RRMM was based on findings from the phase III ASPIRE trial,80 which led to the clinical investigation of KRd in TE NDMM patients in the randomized phase II FORTE trial.81 Patients were randomized to either 12 cycles of KRd (KRd12); KRd followed by ASCT and KRd consolidation (KRd + ASCT); or carfilzomib, cyclophosphamide, and dexamethasone (KCd) followed by ASCT and KCd consolidation (KCd + ASCT). Both KRd regimens resulted in deep responses, with premaintenance ≥CR rates of 52% (KRd12) and 49% (KRd + ASCT), and MRD-negativity rates of 54% and 58%, respectively. KRd has also been evaluated as a basis for quadruplet regimens; KRd + daratumumab in the phase Ib MMY1001 trial in NDMM showed a tolerability profile consistent with KRd and very promising response rates.82 Although the depth of response conferred by KRd is profound, interim results of the phase III ENDURANCE (E1A11) NDMM study evaluating RVd versus KRd in patients with standard-risk MM demonstrated similar efficacy of the two regimens.40 Clinicians must also consider toxicity when evaluating proteasome inhibitors. In addition to thrombocytopenia,76 the most commonly reported AE are PN for bortezomib and cardiovascular, renal, thromboembolic, and pulmonary toxicities for carfilzomib. Bortezomib-associated PN can be debilitating and lead to the interruption or cessation of treatment, but the use of SC bortezomib as well as supportive measures and modified RVd regimens have alleviated much of this risk.31,59 Carfilzomib is associated with a much lower rate of PN than bortezomib, but confers an increased risk of potentially life-threatening cardiovascular toxicity.83 A systemic review and metaanalysis of carfilzomib-associated cardiovascular toxicity demonstrated an estimated cumulative incidence of 5% for grade ≥3 toxicity.84 Furthermore, concomitant use of carfilzomib and an immunomodulatory agent was found to significantly increase the risk of cardiovascular toxicity compared to that of carfilzomib without an immunomodu-

latory agent (6.45% vs. 4.34%; P=0.033). Notably, the incidence of cardiotoxicity was similar with high and standard doses of carfilzomib, although another systemic review and meta-analysis concluded that cardiovascular AE rates were higher with carfilzomib doses ≥45 mg/m2.83,84 In contrast, the bortezomib-associated risk of PN is known to be dosedependent, and can be ameliorated by dose reductions and schedule changes.31,59,60 Interim findings from the phase III ENDURANCE study provide crucial comparative safety data between the regimens: rates of grade ≥3 cardiac, pulmonary, and renal TEAE were lower with RVd than with KRd (P<0.0001), while rates of grade 3/4 PN were higher with RVd than with KRd.40 Rates of overall grade ≥3 serious AE were lower with RVd (P=0.038). Similar considerations when selecting front-line regimens for TE or TNE NDMM patients are overall toxicity and impact on quality of life relative to survival benefit. In DETERMINATION, a robust PFS benefit was noted with RVd + ASCT versus RVd, but patients treated with RVd + ASCT had greater rates of grade ≥3 treatment-related AE and transient but meaningful decreases in quality of life.45 In the absence of an OS benefit and the availability of multiple efficacious combination therapies for NDMM, patients and healthcare providers will need to consider the benefits of adding ASCT to regimens for TE NDMM patients relative to the risk of toxicity and impact on quality of life. This consideration provides a unique opportunity to tailor treatment regimens to a given patient’s characteristics, lifestyle, cytogenetic risk profile, and treatment goals while still inducing deep and durable responses.45 Personalized decision-making for patients regarding treatments is essential in the NDMM setting. Given the positive prognostic value of MRD status for patients’ outcomes, MRD status is increasing in clinical relevance. In a preliminary analysis of MRD status in DETERMINATION, a greater proportion of patients were MRD-negative with RVd + ASCT than with RVd alone, and patients with MRDnegative status had longer median PFS compared with patients with MRD-positive status.45 However, PFS was similar in patients receiving RVd or RVd + ASCT in whom no MRD was detected, suggesting that treatment adaptation based upon MRD status may be a potential alternative to the current paradigm of ASCT use, although further trials will be required to fully understand the interaction of MRD status with optimal duration of therapy.45 A final point of discussion is subsequent therapy following induction and relapse as a strategic consideration. Pa-

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REVIEW ARTICLE - Lenalidomide/bortezomib/DXM in newly diagnosed MM tients may benefit from purposeful preservation of highly effective agents until after initial progressive disease, since management of early relapse is another crucial juncture in a patient’s treatment journey.7 Frontline use of RVd (or an RVd-containing quadruplet regimen), bolstered by the well-established clinical profile and improved manageability of RVd, represents an attractive initial intervention. This facilitates longer-term treatment with RVd until progressive disease in the TNE NDMM population or completion of intended induction in TE NDMM patients. Using RVd in this setting also secures the option of using carfilzomib- and pomalidomide-containing regimens at early relapse. Either approach has been associated with impressively deep responses and broad efficacy in RRMM, which can be complemented by a monoclonal antibody, further improving long-term outcomes.45,85,86 The development of novel alkylating agents, such as melflufen, and novel cellular therapeutics, such as chimeric antigen receptor T cells, cereblon E3 ligase modulatory drugs, and bispecific T-cell engagers, may further help to tailor treatments and diversify options available for patients who are refractory to certain agents or drug classes.87 Given the robust body of data in clinical trials across multiple settings, increasing physician familiarity with RVd, and the evolving maturity of promising data regarding RVd-containing quadruplet regimens, RVd is established as a foundation of NDMM combination therapy with consistent clinical benefit now and into the future. Disclosures PGR reports serving on advisory committees for Karyopharm, Oncopeptides, Celgene, a Bristol-Myers Squibb Company, Takeda, Janssen, GSK, Regeneron, and AstraZeneca, and has received research funding from Oncopeptides, Celgene, a Bristol-Myers Squibb Company, Takeda, and Karyopharm. AD reports serving on advisory committees for Janssen and Alnylam Pfizer, Takeda, and Bristol Myers Squibb, and has received research funding from Bristol Myers Squibb. LR reports honoraria from Janssen, Celgene, a Bristol-Myers Squibb Company, Amgen, Takeda, Sanofi, GSK, and Karyopharm. EO reports serving on advisory boards for Bristol Myers Squibb, Takeda, Karyopharm, Adaptive, Oncopeptides, and Janssen. TF reports serving on advisory committees for Janssen, Bristol Myers Squibb, Takeda, Amgen, Roche, Oncopeptides, Karyopharm, and AbbVie. MVM has received honoraria and speakers bureau compensation from Janssen, Celgene, a Bristol-Myers Squibb Company, Takeda, Amgen, GSK, AbbVie, Pfizer, Regeneron, Adaptive, Sanofi, Oncopep-

P.G. Richardson et al.

tides, Seagen, Roche, and bluebird bio. PM has received honoraria from Amgen, Celgene, a Bristol-Myers Squibb Company, Janssen, AbbVie, Sanofi, Oncopeptides, and GSK. NR reports consultancy for Amgen, Bristol Myers Squibb, Janssen, and Takeda. NM reports consultancy for Adaptive Biotech, Amgen, Bristol Myers Squibb, Janssen, Karyopharm Therapeutics, Legend, OncoPep, and Takeda; and reports stock ownership with OncoPep. PV reports serving on advisory boards for Adaptive Biotechnologies, Bristol Myers Squibb, Janssen, and Teneobio; has served in a consultancy role for Novartis and Oncopeptides; and has received honoraria from GSK and Oncopeptides. JB has received honoraria from Celgene, a Bristol-Myers Squibb Company, Janssen, and Amgen. SL reports consultancy for AbbVie, Bristol Myers Squibb, Celgene, a Bristol-Myers Squibb Company, GSK, Janssen, Karyopharm, Novartis, and Takeda. AP has received research funding from Takeda and honoraria from AbbVie, Amgen, Bristol Myers Squibb, Celgene, a Bristol-Myers Squibb Company, Janssen, Sanofi, and Takeda. KCA reports serving on advisory boards for Celgene, a Bristol-Myers Squibb Company, Millennium, Janssen, Sanofi, Bristol Myers Squibb, Gilead Sciences, Precision Biosciences, and Tolero, as well as being the Scientific Founder of OncoPep and C4 Therapeutics. BGD, SK, JPL, and PO have no conflicts of interest to disclose. Contributions The authors met the criteria for authorship as recommended by the International Committee of Medical Journal Editors (ICMJE). The authors were fully responsible for all content and editorial decisions, were involved at all stages of manuscript development, and approved the final version that reflects the authors’ interpretations and conclusions. Acknowledgments The authors thank Shawn Vahabzadeh, PharmD, Peter J. Simon, PhD, and Martin Haschak, PhD, from The Nucleus Group Holdings, Inc. The authors also gratefully acknowledge the editorial support of Jack Sparacino, Ashley Ford, and Catherine O’Connor, funded in part by the RJ Corman Multiple Myeloma Research Fund. Funding Funding for medical writing assistance in the preparation of this manuscript was provided by a grant from Celgene, a Bristol Myers Squibb Company, with editorial support also funded by the RJ Corman Multiple Myeloma Research Fund.

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REVIEW ARTICLE - Lenalidomide/bortezomib/DXM in newly diagnosed MM Clin Oncol. 2020;38(17):1928-1937. 66. Jakubowiak AJ, Griffith KA, Reece DE, et al. Lenalidomide, bortezomib, pegylated liposomal doxorubicin, and dexamethasone in newly diagnosed multiple myeloma: a phase 1/2 Multiple Myeloma Research Consortium trial. Blood. 2011;118(3):535-543. 67. Voorhees PM, Rodriguez C, Reeves B, et al. Daratumumab plus RVd for newly diagnosed multiple myeloma: final analysis of the safety run-in cohort of GRIFFIN. Blood Adv. 2021;5(4):1092-1096. 68. Mateos MV, Nahi H, Legiec W, et al. Subcutaneous versus intravenous daratumumab in patients with relapsed or refractory multiple myeloma (COLUMBA): a multicentre, openlabel, non-inferiority, randomised, phase 3 trial. Lancet Haematol. 2020;7(5):e370-e380. 69. Chari A, Rodriguez-Otero P, McCarthy H, et al. Subcutaneous daratumumab plus standard treatment regimens in patients with multiple myeloma across lines of therapy (PLEIADES): an open-label phase II study. Br J Haematol. 2021;192(5):869-878. 70. Usmani SZ, Sexton R, Ailawadhi S, et al. Phase I safety data of lenalidomide, bortezomib, dexamethasone, and elotuzumab as induction therapy for newly diagnosed symptomatic multiple myeloma: SWOG S1211. Blood Cancer J. 2015;5(8):e334. 71. Usmani SZ, Ailawadhi S, Sexton R, et al. Primary analysis of the randomized phase II trial of bortezomib, lenalidomide, dexamethasone with/without elotuzumab for newly diagnosed, high-risk multiple myeloma (SWOG-1211). J Clin Oncol. 2020;38(15):8507. 72. Laubach J, Nooka AK, Cole C, et al. An open-label, single arm, phase IIa study of bortezomib, lenalidomide, dexamethasone, and elotuzumab in newly diagnosed multiple myeloma. J Clin Oncol. 2017;35(15):8002. 73. Goldschmidt H, Mai EK, Salwender H, et al. Bortezomib, lenalidomide and dexamethasone with or without elotuzumab as induction therapy for newly-diagnosed, transplant-eligible multiple myeloma. EHA Library. 2020:S203. 74. Ocio EM, Rodriguez Otero P, Bringhen S, et al. Preliminary results from a phase i study of isatuximab (ISA) in combination with bortezomib, lenalidomide, dexamethasone (VRd), and in patients with newly diagnosed multiple myeloma (NDMM) noneligible for transplant. Blood. 2018;132(Suppl 1):595. 75. Orlowski RZ, Goldschmidt H, Cavo M, et al. Phase III (IMROZ) study design: isatuximab plus bortezomib (V), lenalidomide (R), and dexamethasone (d) vs VRd in transplant-ineligible patients (pts) with newly diagnosed multiple myeloma (NDMM). J Clin Oncol. 2018;36(15 suppl):TPS8055. 76. Laubach JP, Tuchman SA, Rosenblatt JM, et al. Phase I, openlabel study of panobinostat, lenalidomide, bortezomib + dexamethasone in relapsed and relapsed/refractory multiple myeloma. EHA Library. 2020;EP953. 77. Manasanch EE, Shah JJ, Lee HC, et al. Bortezomib, lenalidomide, and dexamethasone with panobinostat for frontline treatment of patients with multiple myeloma who are eligible for transplantation: a phase 1 trial. Lancet Haematol. 2018;5(12):e628-e640. 78. Kaufman JL, Mina R, Shah JJ, et al. Phase 1 trial evaluating vorinostat plus bortezomib, lenalidomide, and dexamethasone in patients with newly diagnosed multiple myeloma. Clin Lymphoma Myeloma Leuk. 2020;20(12):797-803. 79. Kaiser MF, Hall A, Walker K, et al. Depth of response and minimal residual disease status in ultra high-risk multiple myeloma and plasma cell leukemia treated with daratumumab, bortezomib, lenalidomide, cyclophosphamide and dexamethasone (Dara-CVRd): results of the UK Optimum/MUKnine trial. J Clin Oncol. 2021;39(15_suppl):8001.

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80. Stewart AK, Rajkumar SV, Dimopoulos MA, et al. Carfilzomib, lenalidomide, and dexamethasone for relapsed multiple myeloma. N Engl J Med. 2015;372(2):142-152. 81. Gay F, Scalabrini DR, Belotti A, et al. Updated efficacy and MRD data according to risk-status in newly diagnosed myeloma patients treated with carfilzomib plus lenalidomide or cyclophosphamide: results from the FORTE trial. EHA Library. 2018;S109. 82. Jakubowiak A, Chari A, Lonial S, et al. Daratumumab (DARA) in combination with carfilzomib, lenalidomide, and dexamethasone (KRd) in patients (pts) with newly diagnosed multiple myeloma (MMY1001): an open-label, phase 1b study. J Clin Oncol. 2017;35(15_suppl):8000. 83. Waxman AJ, Clasen S, Hwang WT, et al. Carfilzomib-associated cardiovascular adverse events: a systematic review and metaanalysis. JAMA Oncol. 2018;4(3):e174519. 84. Shah C, Bishnoi R, Jain A, et al. Cardiotoxicity associated with carfilzomib: systematic review and meta-analysis. Leuk Lymphoma. 2018;59(11):2557-2569. 85. Shah JJ, Stadtmauer EA, Abonour R, et al. Carfilzomib, pomalidomide, and dexamethasone for relapsed or refractory myeloma. Blood. 2015;126(20):2284-2290. 86. Richardson PG, Oriol A, Beksac M, et al. Pomalidomide, bortezomib, and dexamethasone for patients with relapsed or refractory multiple myeloma previously treated with lenalidomide (OPTIMISMM): a randomised, open-label, phase 3 trial. Lancet Oncol. 2019;20(6):781-794. 87. Lakshman A, Kumar SK. Chimeric antigen receptor T-cells, bispecific antibodies, and antibody-drug conjugates for multiple myeloma: an update. Am J Hematol. 2022;97(1):99-118. 88. San Miguel JF, Schlag R, Khuageva NK, et al. Bortezomib plus melphalan and prednisone for initial treatment of multiple myeloma. N Engl J Med. 2008;359(9):906-917. 89. Mateos MV, Richardson PG, Schlag R, et al. Bortezomib plus melphalan and prednisone compared with melphalan and prednisone in previously untreated multiple myeloma: updated follow-up and impact of subsequent therapy in the phase III VISTA trial. J Clin Oncol. 2010;28(13):2259-2266. 90. Palumbo A, Bringhen S, Larocca A, et al. Bortezomib-melphalanprednisone-thalidomide followed by maintenance with bortezomib-thalidomide compared with bortezomib-melphalanprednisone for initial treatment of multiple myeloma: updated follow-up and improved survival. J Clin Oncol. 2014;32(7):634-640. 91. Palumbo A, Bringhen S, Rossi D, et al. Bortezomib-melphalanprednisone-thalidomide followed by maintenance with bortezomib-thalidomide compared with bortezomibmelphalan-prednisone for initial treatment of multiple myeloma: a randomized controlled trial. J Clin Oncol. 2010;28(34):5101-5109. 92. Niesvizky R, Flinn IW, Rifkin R, et al. Community-based phase IIIB trial of three UPFRONT bortezomib-based myeloma regimens. J Clin Oncol. 2015;33(33):3921-3929. 93. Mateos MV, Dimopoulos MA, Cavo M, et al. Daratumumab plus bortezomib, melphalan, and prednisone for untreated myeloma. N Engl J Med. 2018;378(6):518-528. 94. Mateos MV, Cavo M, Blade J, et al. Overall survival with daratumumab, bortezomib, melphalan, and prednisone in newly diagnosed multiple myeloma (ALCYONE): a randomised, openlabel, phase 3 trial. Lancet. 2020;395(10218):132-141. 95. Facon T, Lee JH, Moreau P, et al. Carfilzomib or bortezomib with melphalan-prednisone for transplant-ineligible patients with newly diagnosed multiple myeloma. Blood. 2019;133(18):1953-1963. 96. Tacchetti P, Pantani L, Patriarca F, et al. Bortezomib,

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REVIEW ARTICLE - Lenalidomide/bortezomib/DXM in newly diagnosed MM thalidomide, and dexamethasone followed by double autologous haematopoietic stem-cell transplantation for newly diagnosed multiple myeloma (GIMEMA-MMY-3006): long-term follow-up analysis of a randomised phase 3, open-label study. Lancet Haematol. 2020;7(12):e861-e873. 97. Moreau P, Avet-Loiseau H, Facon T, et al. Bortezomib plus dexamethasone versus reduced-dose bortezomib, thalidomide plus dexamethasone as induction treatment before autologous stem cell transplantation in newly diagnosed multiple myeloma. Blood. 2011;118(22):5752-5758. 98. Moreau P, Attal M, Hulin C, et al. Bortezomib, thalidomide, and dexamethasone with or without daratumumab before and after

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autologous stem-cell transplantation for newly diagnosed multiple myeloma (CASSIOPEIA): a randomised, open-label, phase 3 study. Lancet. 2019;394(10192):29-38. 99. Mai EK, Bertsch U, Durig J, et al. Phase III trial of bortezomib, cyclophosphamide and dexamethasone (VCD) versus bortezomib, doxorubicin and dexamethasone (PAd) in newly diagnosed myeloma. Leukemia. 2015;29(8):1721-1729. 100. Kumar SK, Facon T, Usmani SZ, et al. Updated analysis of daratumumab plus lenalidomide and dexamethasone (D-Rd) versus lenalidomide and dexamethasone (Rd) in patients with transplant-ineligible newly diagnosed multiple myeloma (NDMM): the phase 3 MAIA study. Blood. 2020;136(Suppl 1):24-26.

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Asciminib in chronic myeloid leukemia: a STAMP for expedited delivery? Sandeep Padala and Jorge Cortes

Correspondence: J. Cortes jorge.cortes@augusta.edu

Georgia Cancer Center at Augusta University, Augusta, GA, USA

Received: Accepted: Early view:

February 28, 2023. April 19, 2023. April 27, 2023.

Abstract Asciminib is a novel tyrosine kinase inhibitor (TKI) that specifically targets the myristoyl pocket. It has increased selectivity and potent activity against BCR-ABL1 and the mutants that most frequently prevent the activity of the ATPbinding competitive inhibitors. Results for clinical trials in patients with chronic myeloid leukemia that have received two or more TKI (randomized against bosutinib) or who have a T315I mutation (single arm study) have shown high levels of activity and a favorable toxicity profile. Its approval has offered new options for patients with these disease features. There are, however, a number of unanswered questions that remain to be defined, including the optimal dose, understanding the mechanisms of resistance, and, importantly, how it compares to ponatinib in these patient populations for whom we now have these two options available. Ultimately, a randomized trial is needed to answer questions to which we currently offer speculative informed guesses. The novelty of its mechanism of action and the exciting early data offer the potential for asciminib to address some of the remaining needs in the management of patients with chronic myeloid leukemia, including second-line therapy after resistance to a front-line second-generation TKI and improving successful treatment-free remission. Multiple studies are ongoing in these areas, and one can only hope that the desired randomized trial comparing asciminib to ponatinib will be conducted soon.

Essential thrombocythemia in the realm of myeloproliferative neoplasms It was a relatively short time in drug development terms from the initial description of the in vitro efficacy of a novel tyrosine kinase inhibitor (TKI), CGP5714S (now imatinib),1 to the initial clinical demonstration of its clinical activity in chronic myeloid leukemia (CML).2 Shortly thereafter, imatinib became standard therapy for patients with CML.3 Second-generation TKI (2G-TKI; dasatinib, nilotinib and bosutinib) were a new leap forward, providing new options for patients in whom imatinib had failed, and eventually in the front-line setting. Ponatinib was a later breakthrough providing a much needed option for patients with T315I mutation or with resistance to multiple prior TKI. Through these innovations, life expectancy for patients with CML has nearly reached that of the general population,4 and some patients may even do what was initially considered unimaginable, stop therapy. Despite all

this progress, only approximately 50% of patients treated with 2G-TKI achieve sustained MR4.5 by ten years, and approximately half the patients who stop therapy relapse.5 Upon failure, response rates and overall survival decrease as patients progress through subsequent TKI. Safety concerns have also evolved, with arterio-occlusive events (AOE) now recognized with most available TKI, particularly affecting the wider use of ponatinib.6 This has triggered continued development of new TKI. Asciminib (ABL001) is a first-in-class TKI that, unlike all other available TKI that inhibit ABL kinase activity in an ATP-competitive manner, binds to the myristoyl pocket of ABL1, inducing an inactive conformation of the kinase (Specifically Targeting the ABL Myristoyl Pocket or STAMP inhibitor).7 Asciminib offers several distinct features with potential clinical implications that makes it unique and valuable. Among them are its activity against T315I, and a distinct pattern of resistant mutations, different from that of the ATP-competitive agents. Myristoyl pockets are present in only a limited number of kinases, offering the potential for greater selectivity.7 The distinct binding site

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and complementary mechanism of resistance also offers in this patient population. The counter-argument is that, the possibility of combination therapy with traditional TKI at the time ASCEMBL was designed, there were major conwhich, in animal models, has led to complete, durable re- cerns about the risk of AOE with ponatinib in the pivotal missions.7 These properties made asciminib an exciting phase II trial (PACE), and a study to define the optimal ponew agent to bring to the clinic. The results have not dis- natinib dose was ongoing (OPTIC).12 This made ponatinib a apointed, but challenges remain and there are opportun- desirable but questionable control that could challenge ities for further development. completion of the study if these concerns dissuaded investigators and/or patients from enrolling. There were also imbalances in the two cohorts (e.g., a numerical trend for more patients that had received ponatinib and more TKI, The data or were resistant vs. intolerant in the bosutinib cohort). The phase I study of monotherapy asciminib suggested its Bosutinib is, among the 2G-TKI, the only one with prosefficacy in patients with resistance or intolerance to pective data in third-line,13,14 which made it a 'next best' multiple TKI. A dose range of from 10 to 200 mg, once (QD) alternative. This choice precluded the enrollment of paor twice (BID) daily was investigated. By 12 months, a tients with T315I and V299L. As a result, the data for T315I major cytogenetic response (MCyR) was achieved in 60% patients, although encouraging, remain limited (n=52 paof patients without T315I and 55% with T315I. Correspond- tients) and have not been controlled. The dose of bosutiing figures for major molecular response (MMR) were 36% nib used in ASCEMBL is the standard beyond first-line, and 24%.8 These encouraging results led to a pivotal ran- and, in this regard, it cannot be questioned. However, curdomized trial (ASCEMBL) for patients in chronic phase rent practice and recent studies have suggested that (CP)-CML with resistance or intolerance to ≥2 TKI without starting at a lower dose (e.g., 200-300 mg), and escalating T315I or V299L. Patients were randomized to asciminib (40 as tolerated and as needed, allows better tolerability.15,16 mg BID) or bosutinib (500 mg daily). The primary endpoint There was a very high rate of early treatment discontinuof MMR at 24 weeks was met: 25.5% with asciminib and ation (71.1%) after a median follow-up of only 14.9 months.9 13.2% with bosutinib.9 Additional follow-up shows MMR Bosutinib may have also underperformed (MMR in ASrates of 37.6% and 15.8%, respectively, at 96 weeks. Also CEMBL 19% by 48 weeks) compared to other series. In the important is the rate of BCR::ABL1 ≤1%, which for patients BYOND trial, the MMR rate with bosutinib by one year was with resistance and/or intolerance to multiple prior ther- 74.5% in third-line and 56.3% in fourth-line. With a median apies should be considered an adequate response. The follow-up of 30.4 months, 54.1%, and 49.0% of patients, rates at 96 weeks were 45.1% and 19.4%, respectively. The respectively, remained on therapy.14 Studies using a lower safety profile generally favored asciminib, with fewer pa- starting dose and escalation based on tolerance and effitients treated with asciminib discontinuing therapy due to cacy have also yielded far better tolerability, with excellent adverse events (AE) compared to bosutinib after a median responses even in older patients.15 Still, there is perhaps follow-up of 2.3 years (7.7% and 26.3%, respectively). little doubt that, for patients with resistance to ≥2 prior Overall, 45.9% of patients treated with asciminib discon- TKI, asciminib is a more effective agent than bosutinib. tinued therapy, most frequently due to lack of efficacy.10 The approval for patients with T315I is welcome and the Asciminib, at a dose of 200 mg BID, has also induced high outcomes have been excellent, but it is based on a yet response rates in patients with CP-CML with T315I. Among unpublished small cohort of patients. We can only hope 52 patients, 46.9% achieved MMR.11 The results of these that a randomized study against ponatinib will soon be studies constituted the basis for the approval of asciminib conducted to help us better understand the relative role for the treatment of patients with resistance or intoler- of these two valuable agents for these patients. ance to ≥2 TKI in many parts of the world; approval for patients with T315I has also been granted in some countries.

The dose The dose of asciminib in ASCEMBL was 40 mg BID; yet the dose approved in the US for patients treated with ≥2 prior ASCEMBL demonstrated the benefit of asciminib for pa- TKI includes 80 mg daily. In either case, no food should be tients with CP-CML with resistance or intolerance to ≥2 consumed at least two hours prior and one hour after adTKI. The design and the results, however, have not been ministration. For patients with T315I, the approved dose is without controversy. A central question has been the se- 200 mg BID, based on the fact that in the phase I study, 3 lection of bosutinib for the control arm. Undoubtedly, a of the 4 patients with T315I who responded received >150 direct comparison with ponatinib would have been ideal mg. In the phase I study, the MMR rates were numerically to better define the role of these two drugs, both effective higher with QD dosing compared to BID in patients without

The analysis

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T315I, both by 6 months (47% and 38%, respectively) and 12 months (69% and 53%). With smaller numbers, the opposite was seen in patients with T315I.8 No maximum tolerated dose was identified, but pancreatitis, although infrequent (3% of all patients) occurred only at doses >40 mg. A response by dose was not reported for non-T315I patients. It is thus not evident that the optimal dose has been identified. The higher dose required for patients with T315I is explained by a 10-fold lower anti-proliferative activity of asciminib in Ba/F3 cells expressing T315I compared to cells expressing the wild-type variant.17 Despite the encouraging clinical activity reported in ASCEMBL, one can speculate as to whether better outcomes could be achieved with higher doses and/or a QD schedule. With the safety reported in the T315I cohort similar to that with lower doses, it is reasonable to consider exploring higher doses in non-T315I patients to improve outcomes. A QD schedule is more practical for patients considering the need for fasting conditions. In contrast to nilotinib, which also requires fasting conditions, plasma concentration of asciminib decreases when administered with food, particularly if it is a high-fat meal.18 The dosing schedule for patients with T315I is also inconvenient because the formulations currently available (20 mg and 40 mg tablets) require ten tablets to administer the full dose. The financial implications of these higher dose schedules cannot been ignored. Identifying the optimal dose and improving the available formulations are important aspects of the optimal use of asciminib.

Safety The safety profile of asciminib has been consistent through the studies that have been reported. Not surprisingly for a heavily-treated patient population, myelosuppression is the most common AE, particularly thrombocytopenia (22.4% grade ≥3 in ASCEMBL). Among the non-hematologic AE, the only grade ≥3 event occurring in >5% was hypertension. Lipase elevation is also frequently observed, reported in 26.7% of patients in the phase I study (10% grade ≥3) and 5.1% in ASCEMBL (3.8% grade ≥3).8,10 Lipase elevation is a class effect AE, reported at similar rates with other TKI. In ASCEMBL, it occurred in 6.6% with bosutinib (5.3% grade ≥3). It clearly deserves attention when using asciminib (and other TKI), but it is seldom associated with clinical pancreatitis. So far, the favorable toxicity profile is in keeping with the selectivity of the binding to the myristoyl pocket. An AE category of special interest is AOE. AOE were reported with asciminib in 5.1% of patients in ASCEMBL. The overall incidence of AOE is influenced by the breadth of the search (i.e., what specific MedDRA terms are included; not described in ASCEMBL). It is also influenced by the duration of follow-up, as the incidence increases over time. For

example, with nilotinib in the front-line ENESTnd study, the reported cumulative incidence of such events was 7.5% by five years19 and 16.5% by ten years.5 The overall incidence reported with asciminib is low (5.1%), but still higher than with bosutinib (1.3%), even when adjusting for exposure (3.0 vs. 1.4 per 100 patient-years).10 At least two patients were reported to have died of such events.9 As is the case with other TKI, AOE occur predominantly among patients with risk features for such events and those with a higher Framingham score. It is thus important to consider the potential risk of AOE with asciminib, including assessment and management of co-morbidities, and risk factors at baseline and during therapy.

Resistance The response rate to asciminib has been encouragingly high, and responses have been durable: 97% maintained MMR and 95% maintained BCR::ABL1 ≤1% at the time of last report.10 However, at least half of the patients still experienced treatment failure.10 Being such a heavily-treated patient population this might be expected, but it still begs the question as to why patients may not respond to treatment with such an active drug with a novel mechanism of kinase inhibition. Furthermore, among patients who have had sequencing after failure of asciminib, 25.6% of patients treated with asciminib and 6.7% of those treated with bosutinib had newly emerging mutations.10 Remarkably, six of the ten newly emerging mutations were in the ATP-binding site, including M244V, E355G, F359V, and T315I; four others were in the myristoyl pocket. Six other patients had mutations at baseline that persisted at the end of treatment, including F317L (n=2), F359C/V (n=3), and Y253H (n=1). The growth inhibitory IC50 in cellular assays (Ba/F3 cells) of some of these emerging mutations (e.g., E355G, F359V ) are significantly higher than for the wild-type, being in the same range or higher than for T315I. Whether a dose escalation would overcome resistance in such instances deserves clinical investigation. It is also important to recognize that focusing on BCR::ABL1 mutations as the sole mechanism of resistance is an oversimplification of the complexity of the disease and the patients. Asciminib may be subject to ABCG2 efflux.20 We now also know that mutations in other cancer-related genes, such as ASXL1, and other gene fusions not associated with the Philadelphia translocation occur in a sizeable percentage of patients with CP-CML at the time of diagnosis and they confer a poor prognosis, with lower probability of achieving deep molecular response (DMR) and higher risk of progression.21 The frequency of these events among patients enrolled in asciminib trials has not been reported. These events, particularly those involving other genes, may not be responsive to ABL kinase inhibition and may require alternative approaches.

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The context: comparison with ponatinib

perior, would deny many of them of options that may offer efficacy or safety benefits for individual cases.

In the absence of a randomized trial of ponatinib and asciminib for patients with resistance or intolerance to ≥2 TKI and/or with T315I, an analysis of the results of the recent trials may shed some light on their value in this setting. This is important, as a physician is faced with the question of which drug to use for a given patient who meets the criteria for the use of either. A formal comparison of these separate trials is not possible or appropriate since, despite the similarities in the target population, not only are these independent trials, but many aspects of the trial design and selection of the patients differ or are not clearly described. The patients' characteristics and outcomes are also reported differently. A summary of these trials is presented in Table 1. Patients are younger in the OPTIC trial and more patients with resistance (vs. intolerance) are enrolled in the PACE and OPTIC trials. This summary shows good levels of response with both agents, but many patients have not responded to either drug. BCR::ABL1 levels of ≤1% are achieved in approximately 50% of patients with both ponatinib and asciminib. The follow-up is short in these studies, but the probability of response seems to plateau at around 48 weeks. For example, in ASCEMBL, the rate of BCR::ABL1 ≤1% was 50.6% by 48 weeks and 53.7% by 96 weeks.10 Still, some patients may achieve DMR. With asciminib, the rate of MR4 at 96 weeks was 17.2% and of MR4.5 10.8%.10 With ponatinib, with median follow-up of 56.8 months, they occur in 30% and 24%, respectively.22 Thus, unless the patient has an alternative option with a realistic expectation of a better outcome (e.g., stem cell transplantation), treatment can be continued in patients who achieve BCR::ABL1 ≤1%. Excellent survival rates are reported with both agents. The safety profile is generally adequate with both drugs, with some shared AE such as myelosuppression. A major safety concern are AOE; these are reported with both ponatinib and asciminib. With most TKI there seems to be a dose effect for AOE. In ENESTnd, for example, cardiovascular events occurred in 16.5% of patients with 300 mg BID and 23.5% with 400 mg BID.5 In OPTIC, the exposure-adjusted rates are 4.5 per 100 patient-years at 45 mg and 3.0 at 30 mg.12 The most salient message is the need to assess, monitor, and manage co-morbidities and risk factors for AOE in all patients treated with TKI. Ultimately, having the added option of asciminib for patients with resistance to ≥2 TKI or with T315I is a very welcome development. This allows the patient's and the disease characteristics to be carefully reviewed in the context of the available information for each drug and for carefully weighted decisions to be made as to the most appropriate treatment for each patient. Drawing general conclusions for all patients in these scenarios, with one drug or the other being regarded as su-

The future The high efficacy and favorable safety profile with asciminib in settings where poor outcomes have historically been observed have triggered interest in exploring its use in other areas. Perhaps the one with the greatest needs is second-line therapy after resistance to front-line 2GTKI. There is limited experience with prospective studies in this setting, but considering that the rate of complete cytogenetic response with 2G-TKI after resistance to only front-line imatinib is only 45-50%, treatment options with better outcomes are needed. Ongoing studies are exploring asciminib in this setting. There is also interest in combining asciminib in patients who have not reached a DMR. A recent analysis of the ASC4MORE trial reported, in small cohorts, a higher probability of achieving MR4.5 in this setting with adding asciminib at either 40 mg or 60 mg to imatinib compared to switching to nilotinib or continuing with imatinib.23 The non-clinical data suggesting synergy in preventing the emergence of resistant clones makes this approach attractive. It is also possible that switching to asciminib instead of adding it to imatinib could achieve similar results with less toxicity, cost and inconvenience. A cohort using this approach has been added to the study but results are not yet available. The hope is that this strategy may make successful treatment-free remission (TFR) achievable by more patients. The magnitude of any observed improvement will need to be balanced against safety, financial issues, convenience, and other implications to determine the ultimate value of this strategy. An intriguing possibility is to use asciminib as front-line therapy. Several studies are ongoing in this context. Early results of the first of these studies to report data show encouraging rates of early molecular response (92% BCR::ABL1 ≤10% at 3 months), although 9 of 63 patients had discontinued therapy for various reasons.24 The main benefit of this approach would be to increase the probability of TFR. Considering the generally favorable results achieved in most patients with current therapy, such an improvement would have to be sizeable to trigger a shift in treatment standards in a significant number of patients. Other intriguing possibilities would be to use asciminib in advanced phase CML, and the use of combinations, particularly for patients in blast phase or Philadelphia-chromosome positive acute lymphoblastic leukemia. In summary, asciminib is a new leap forward in the management of patients with CML, with a novel mechanism of action and increased selectivity. Its current indication addresses some ongoing needs, and its mechanism of action

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Table 1. Summary of the pivotal studies with ponatinib and asciminib.

Characteristic Patients’ characteristics N Median age, yrs (range) CV risk factors, %

Prior TKI, %

Resistance, % BCR-ABL1 mutation, % Best response last TKI, % Baseline BCR::ABL1, % Efficacy BCR::ABL1, % patients with response When assessed Last report, % patients with response (time) Median follow-up Median duration of exposure PFS, %l OS, % Safety AOE

HTN, % Lipase elevation, % Thrombocytopenia, %

Subcategory

PACE22,25

OPTICj12,26

ASCEMBL10,27

Asciminib T315I11

HTN Diabetes mellitus Hyperlipidemia BMI — kg/m2 1 2 ≥3 No mutation T315I ≥MCyR >10%

270 60 (18-94) 53 16 51 Obesity 24a 7 36 57 84 51 24 26g NR

94 46 (19-81) 28 5 20 27a 1 46 53 98 54 27 30k 79

157 52 (24-83) NR NR NR NR 0 52 48 61 87 0 NR 62

52 54 (26-86) NR NR NR NR 17b 31b 52b NR 0 100 NR 54

≤10% ≤1% -

60c 54c Median 57 mthsh

44 At 12 mths

43 At 48 wks

60 (by 36 mths) 34 (overall) 32 mths 72% >12 mths 79.99 (2-yrs) 91.28 (2-yrs)

54 (by 96 wks) 41 (by 96 wks) 33 (by 48 wks) 2.3 yrs 24 mths 94.4 (2-yrs) 97.3 (2-yrs)

47 (by 96 wks) 43 (by 48 wks) 68 wks -

31i 14.1i

10 4.5f

5d 3.0d

5.8e

14 13 35

9 11 30

6.4 3.8 22.4

5.7 15.4 17.3

≤1% ≤0.1% (total) ≤0.1% (12 mths) Overall, % Per 100 patient-years Grade ≥3 -

40 (overall) 37 (by 12 mths) 57 mths 32 mths 53 (5-yrs) 73 (5-yrs)

N: number; NR: not reported; yrs: years; mths: months; wks: weeks; CV: cardiovascular disease; HTN: hypertension; TKI: tyrosine kinase inhibitor; MCyR: major cytogenetic response; PFS: progression-free survival; OS: overall survival; AOE: arterio-occlusive events. aObesity: Body Mass Index (BMI) ≥30. Overweight: BMI 25-29.9. b60% prior ponatinib. cReflects MCyR and complete cytogenetic response (CCyR) by 12 mth. d 96-week report. eMedian duration of exposure 68.4 wk. fWith 45 mg (3.0 with 30 mg). gMost recent dasatinib or nilotinib treatment. hBy 12 mth, MCyR 56%, CCyR 46%, major molecular response 34%. i17% and 10.4% per 100 patient-years after adjudication.28 jPresented for 45 mg cohort. kBetter than complete hematologic response (CHR). lIn OPTIC, defined as the interval between the first dose and disease progression (progression to accelerated-phase chronic myeloid leukemia [CML] or blast-phase CML, loss of CHR or MCyR, or doubling of white blood cell count to 20x109/L on 2 occasions at least 4 weeks apart in patients without CHR); in ASCEMBL, no definition was provided in the manuscript.

and non-clinical data open new possibilities in areas where current treatment is adequate but not optimal. The quest for cure for most patients with CML continues, and new agents such as asciminib may get us closer to reaching this elusive goal for more patients.

has received research support (to his institution) from Novartis, Pfizer, Takeda, Incyte, Sun Pharma and Ascentage.

Contributions SP reviewed the literature. JC conceived the study and designed the outline of the manuscript, reviewed and analyzed the literature, and wrote the manuscript. Both authors Disclosures SP has no conflicts of interest to disclose. JC has been a edited the manuscript and approved the final version for consultant for Novartis, Pfizer, Takeda, Sun Pharma, and publication.

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S. Padala and J. Cortes

References 1. Druker BJ, Tamura S, Buchdunger E, et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med. 1996;2(5):561-566. 2. Druker BJ, Talpaz M, Resta DJ, et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med. 2001;344(14):1031-1037. 3. O'Brien SG, Guilhot F, Larson RA, et al. Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med. 2003;348(11):994-1004. 4. Sasaki K, Strom SS, O'Brien S, et al. Relative survival in patients with chronic-phase chronic myeloid leukaemia in the tyrosinekinase inhibitor era: analysis of patient data from six prospective clinical trials. Lancet Haematol. 2015;2(5):e186-193. 5. Kantarjian HM, Hughes TP, Larson RA, et al. Long-term outcomes with frontline nilotinib versus imatinib in newly diagnosed chronic myeloid leukemia in chronic phase: ENESTnd 10-year analysis. Leukemia. 2021;35(2):440-453. 6. Lipton JH, Brummendorf TH, Gambacorti-Passerini C, et al. Long-term safety review of tyrosine kinase inhibitors in chronic myeloid leukemia - What to look for when treatment-free remission is not an option. Blood Rev. 2022;56:100968. 7. Wylie AA, Schoepfer J, Jahnke W, et al. The allosteric inhibitor ABL001 enables dual targeting of BCR-ABL1. Nature. 2017;543(7647):733-737. 8. Hughes TP, Mauro MJ, Cortes JE, et al. Asciminib in chronic myeloid leukemia after ABL kinase inhibitor failure. N Engl J Med. 2019;381(24):2315-2326. 9. Rea D, Mauro MJ, Boquimpani C, et al. A phase 3, open-label, randomized study of asciminib, a STAMP inhibitor, vs bosutinib in CML after 2 or more prior TKIs. Blood. 2021;138(21):2031-2041. 10. Hochhaus A, Rea D, Boquimpani C, et al. Asciminib vs bosutinib in chronic-phase chronic myeloid leukemia previously treated with at least two tyrosine kinase inhibitors: longer-term followup of ASCEMBL. Leukemia. 2023;37(3):617-626. 11. Cortes J, Hughes T, Mauro M, et al. Asciminib, a first-in-class STAMP inhibitor, provides durable molecular response in patients (pts) with chronic myeloid leukemia (CML) harboring the T315I mutation: primary efficacy and safety results from a phase 1 trial. Blood. 2020;136(Suppl 1):47-50. 12. Cortes J, Apperley J, Lomaia E, et al. Ponatinib dose-ranging study in chronic-phase chronic myeloid leukemia: a randomized, open-label phase 2 clinical trial. Blood. 2021;138(21):2042-2050. 13. Cortes JE, Khoury HJ, Kantarjian HM, et al. Long-term bosutinib for chronic phase chronic myeloid leukemia after failure of imatinib plus dasatinib and/or nilotinib. Am J Hematol. 2016;91(12):1206-1214. 14. Hochhaus A, Gambacorti-Passerini C, Abboud C, et al. Bosutinib for pretreated patients with chronic phase chronic myeloid leukemia: primary results of the phase 4 BYOND study. Leukemia. 2020;34(8):2125-2137. 15. Castagnetti F, Bocchia M, Abruzzese E, et al. Bosutinib dose optimization in the second-line treatment of elderly CML

patients: extended 3-year follow-up and final results of the best study. Hemasphere. 2022;6:593-594. 16. Cortes JE, Apperley JF, DeAngelo DJ, et al. Management of adverse events associated with bosutinib treatment of chronicphase chronic myeloid leukemia: expert panel review. J Hematol Oncol. 2018;11(1):143. 17. Manley PW, Barys L, Cowan-Jacob SW. The specificity of asciminib, a potential treatment for chronic myeloid leukemia, as a myristate-pocket binding ABL inhibitor and analysis of its interactions with mutant forms of BCR-ABL1 kinase. Leuk Res. 2020;98:106458. 18. Hoch M, Zack J, Quinlan M, et al. Pharmacokinetics of asciminib when taken with imatinib or with food. Clin Pharmacol Drug Dev. 2022;11(2):207-219. 19. Hochhaus A, Saglio G, Hughes TP, et al. Long-term benefits and risks of frontline nilotinib vs imatinib for chronic myeloid leukemia in chronic phase: 5-year update of the randomized ENESTnd trial. Leukemia. 2016;30(5):1044-1054. 20. Qiang W, Antelope O, Zabriskie MS, et al. Mechanisms of resistance to the BCR-ABL1 allosteric inhibitor asciminib. Leukemia. 2017;31(12):2844-2847. 21. Branford S, Wang P, Yeung DT, et al. Integrative genomic analysis reveals cancer-associated mutations at diagnosis of CML in patients with high-risk disease. Blood. 2018;132(9):948-961. 22. Cortes JE, Kim DW, Pinilla-Ibarz J, et al. Ponatinib efficacy and safety in Philadelphia chromosome-positive leukemia: final 5year results of the phase 2 PACE trial. Blood. 2018;132(4):393-404. 23. Cortes J, Hughes T, Geissler J, et al. Efficacy and safety results from ASC4MORE, a randomized study of asciminib (ASC) add-on to imatinib (IMA), continued IMA, or switch to nilotinib (NIL) in patients (Pts) with chronic-phase chronic myeloid leukemia (CML-CP) not achieving deep molecular responses (DMRs) with ≥1 year of IMA. Blood. 2022;140(Suppl 1):195-197. 24. Yeung DT, Shanmuganathan N, Reynolds J, et al. Early and deep molecular responses achieved with frontline asciminib in chronic phase CML - interim results from ALLG CML13 AscendCML. Blood. 2022;140(Suppl 1):192-194. 25. Cortes JE, Kim DW, Pinilla-Ibarz J, et al. A phase 2 trial of ponatinib in Philadelphia chromosome-positive leukemias. N Engl J Med. 2013;369(19):1783-1796. 26. Cortes J, Deininger M, Lomaia E, et al. Three-year update from the Optic trial: a dose-optimization study of 3 starting doses of ponatinib. Blood. 2022;140(Suppl 1):1495-1497. 27. Mauro M, Minami Y, Rea D, et al. Efficacy and safety results from ASCEMBL, a multicenter, open-label, phase 3 study of asciminib, a first-in-class STAMP inhibitor, vs bosutinib in patients with chronic myeloid leukemia in chronic phase after ≥2 prior tyrosine kinase inhibitors: update after 48 weeks. Blood. 2021;138(Suppl 1):310. 28. Januzzi JL, Garasic JM, Kasner SE, et al. Retrospective analysis of arterial occlusive events in the PACE trial by an independent adjudication committee. J Hematol Oncol. 2022;15(1):1.

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Momelotinib (JAK1/JAK2/ACVR1 inhibitor): mechanism of action, clinical trial reports, and therapeutic prospects beyond myelofibrosis Ayalew Tefferi, Animesh Pardanani and Naseema Gangat

Correspondence: A. Tefferi

Division of Hematology, Department of Medicine, Mayo Clinic, Rochester, MN, USA

tefferi.ayalew@mayo.edu Received: Accepted: Early view:

January 2, 2023. February 20, 2023. March 2, 2023.

Abstract Janus kinase (JAK) 2 inhibitors are now part of the therapeutic armamentarium for primary and secondary myelofibrosis (MF). Patients with MF endure shortened survival and poor quality of life. Allogeneic stem cell transplantation (ASCT) is currently the only treatment modality in MF with the potential to cure the disease or prolong survival. By contrast, current drug therapy in MF targets quality of life and does not modify the natural history of the disease. The discovery of JAK2 and other JAK-STAT activating mutations (i.e., CALR and MPL) in myeloproliferative neoplasms, including MF, has facilitated the development of several JAK inhibitors that are not necessarily specific to the oncogenic mutations themselves but have proven effective in countering JAK-STAT signaling, resulting in suppression of inflammatory cytokines and myeloproliferation. This non-specific activity resulted in clinically favorable effects on constitutional symptoms and splenomegaly and, consequently, approval by the Food and Drug Administration (FDA) of three small molecule JAK inhibitors: ruxolitinib, fedratinib, and pacritinib. A fourth JAK inhibitor, momelotinib, is poised for FDA approval soon and has been shown to provide additional benefit in alleviating transfusion-dependent anemia in MF. The salutary effect of momelotinib on anemia has been attributed to inhibition of activin A receptor, type 1 (ACVR1) and recent information suggests a similar effect from pacritinib. ACRV1 mediates SMAD2/3 signaling which contributes to upregulation of hepcidin production and iron-restricted erythropoiesis. Targeting ACRV1 raises therapeutic prospects in other myeloid neoplasms associated with ineffective erythropoiesis, such as myelodysplastic syndromes with ring sideroblasts or SF3B1 mutation, especially those with co-expression of a JAK2 mutation and thrombocytosis.

Introduction Myelofibrosis (MF) is an operational terminology that refers to a primary form, a post-polycythemia vera form and postessential thrombocythemia MF.1 These three variants of MF are morphologically and molecularly inter-related myeloproliferative neoplasms (MPN) whose pathogenesis is centered around JAK-STAT activating JAK2, CALR or MPL mutations, with specific phenotypic expressions.2 Morphologically, all three MPN variants display variable degrees of trilineage myeloproliferation associated with a bone marrow stromal reaction that is most intense in MF, in which abnormal megakaryocyte proliferation is often accompanied by bone marrow fibrosis, ineffective erythropoiesis (clinically apparent as anemia), aberrant cytokine expression (clinically apparent as constitutional symptoms

and cachexia), and extramedullary hematopoiesis (clinically apparent as hepatosplenomegaly).3 The three MPN variants also differ in disease course, survival outcome, and risk of progression into blast-phase disease, with reported median survivals for primary MF, post-polycythemia vera MF and post-essential thrombocytopenia MF being 4.4, 15, and 18 years, respectively, with corresponding leukemic transformation rates of 9.3%, 3.9%, and 2.6%.4 Patients with MF are subject not only to premature death4 but also to poor quality of life.5 The latter is manifest as severe anemia (often requiring red blood cell transfusions), marked hepatosplenomegaly, constitutional symptoms (including fatigue, night sweats, and low-grade fever), progressive cachexia with loss of muscle mass, bone pain, splenic infarct, pruritus, non-hepatosplenic extramedullary hematopoiesis, thrombosis and bleeding.6 Consequences

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of hepatosplenic extramedullary hematopoiesis include portal hypertension, which might lead to variceal bleeding or ascites, while those of non-hepatosplenic extramedullary hematopoiesis include spinal cord compression, ascites, pleural effusion, pulmonary hypertension or extremity pain.6 The mechanism of anemia in MF involves multiple factors including ineffective erythropoiesis, bleeding, hemolysis, splenic sequestration of red cells, nutritional deficiency and the side effects of drugs. Ineffective erythropoiesis in MF might also contribute to extramedullary hematopoiesis and its underlying mechanisms might be similar to those seen in myelodysplastic syndromes (MDS) with ring sideroblasts (RS).7

Anemia and risk stratification in primary myelofibrosis Among 1,109 consecutive patients with primary MF, a hemoglobin level of below the lower limit of normal, adjusted for sex, was present in 950 (86%) patients and ranged in severity from mild (hemoglobin ≥10 g/dL but less than sexadjusted lower limit of normal) in 35%, to moderate (hemoglobin ≥8 and <10 g/dL) in 14%, to severe (hemoglobin <8 g/dL or transfusion-dependent) in 37%.8 In the particular study, U2AF1 mutations clustered with severe anemia and multivariable analysis confirmed prognostic relevance for all severity grades of anemia.8 Anemia is currently included in contemporary risk models for primary MF, including MIPSS709 and MIPSS70+ version 2.0 (MIPSSv2).10 MIPSS70 (Mutation-enhanced International Prognostic Scoring System for transplant-age patients) utilizes mutations and clinical variables9 while MIPSSv2 utilizes mutations, karyotype and clinical variables.10 MIPSSv2 scores very high-risk karyotype (4 points), unfavorable karyotype (3 points), ≥2 high molecular risk mutations (3 points), presence of one high molecular risk mutation (2 points), absence of type 1/like CALR mutation (2 points), constitutional symptoms (2 points), severe anemia (2 points), moderate anemia (1 point) and circulating blasts ≥2% (1 point).10 MIPSSv2 includes five risk categories: very high risk (≥9 points); high risk (5-8 points); intermediate risk (3-4 points); low risk (1-2 points); and very low risk (0 points) in patients aged 70 years or younger. The corresponding median survivals (10-year survival rates) were 1.8 years (<5%), 4.1 years (13%), 7.7 years (37%), 16.4 years (56%) and “median not reached” (92%).

Current treatment approaches Survival-directed treatment At present, allogeneic stem cell transplantation (ASCT) is the only treatment modality in MF with the potential to cure the disease or prolong survival.11 In a multicenter,

retrospective study of 4,142 patients with MF receiving ASCT and followed for a median of 48 months, 3-year survival, relapse, and non-relapse mortality rates were 58%, 22% and 29%, respectively.12 The study showed a significant trend in terms of older age distribution (median 59.3 years) and utilization of matched unrelated donors (45.2%) in more recent times.12 The study also showed decreasing rates of acute and chronic graft-versus-host disease, with recent rates of extensive chronic graft-versus-host disease at 23%. Observations from other studies were consistent regarding the value of ASCT in older patients13 and the possibility of using family mismatched/haplo donors.14 In a recent study of 556 transplanted patients with MF aged ≥65 years (median 67; range, 65-76), followed for a median of 3.4 years, 5-year survival, non-relapse mortality, and relapse rates were 40%, 37%, and 25%, respectively.13 The possibility of transplant-related mortality and morbidity dictates careful risk-benefit analysis in the individual patient with MF and a number of risk models assist in this regard: MIPSSv210 and the Myelofibrosis Transplant Scoring System (MTSS).15 Newer effective therapies for graft-versus-host disease (e.g. ruxolitinib) have contributed to recent improvements in post-transplant outcome in MF16-18 while the use of JAK inhibitors before and after ASCT is currently under investigation.19 Symptom-directed treatment: conventional non-JAK inhibitor drugs Unlike the case with ASCT, current drug therapy in MF is directed at improving quality of life through control of splenomegaly, constitutional symptoms, and anemia. Prior to the introduction of JAK inhibitors, the drugs used depended on specific treatment indications. Accordingly, drugs used for the treatment of anemia include androgen preparations, prednisone, immunomodulatory drugs (thalidomide, lenalidomide, pomalidomide), or danazol.6 Lenalidomide works best in the presence of del(5q31)20 while there is limited benefit from using erythropoiesis-stimulating agents21,22 or luspatercept.23,24 Anemia response rates to each one of the aforementioned drugs are less than 25% and responses are temporary, often lasting for less than 2 years. The aforementioned drugs used for combating anemia are often ineffective in controlling splenomegaly, which is typically treated with hydroxyurea.24 Patients not responding to hydroxyurea or who manifest constitutional symptoms are best served by treatment with JAK inhibitors (discussed below). Treatment options for drug-resistant splenomegaly include splenectomy and involved-field radiotherapy. The latter is most effective for symptomatic non-hepatosplenic extramedullary hematopoiesis or localized bone pain.

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Symptom-directed therapy: Food and Drug Administration-approved JAK2 inhibitors The discovery of JAK2V617F in 200525 opened Pandora’s box for the development of several JAK inhibitors, with the objective of targeting constitutive JAK-STAT activation resulting from gain-of-function mutations involving JAK2, CALR and MPL. Currently available JAK inhibitors are not specific to mutation-induced JAK-STAT activation26 but their non-specific inhibition of JAK2 produces broad suppression of inflammatory cytokines and myeloproliferation with resultant favorable effects on constitutional symptoms and splenomegaly.6,27 The demonstration of benefit in quality of life, by way of effective control of splenomegaly and constitutional symptoms, has allowed Food and Drug Administration (FDA) approval of ruxolitinib (2011), fedratinib (2019), and pacritinib (2022).6 None of these currently FDA-approved JAK inhibitors induces morphological or molecular remissions and their value is mostly palliative.6,26 Furthermore, ruxolitinib and fedratinib have not been recognized for their impact on transfusiondependent anemia in MF.28,29 The COMFORT clinical trials demonstrated the superiority of ruxolitinib over placebo (42% vs. <1%) or best available therapy (BAT; 28.5% vs. 13.9%) in reducing spleen size.30,31 Ruxolitinib treatment was also associated with alleviation of symptoms in approximately half of affected patients. Ruxolitinib-associated side effects, compared to placebo, included anemia (31% vs. 13.9%) and thrombocytopenia (34.2% vs. 9.3%). Fedratinib has also been compared to placebo, with spleen response rates of 36% versus 1% (JAKARTA-1).29 By contrast, spleen response rates for pacritinib were lower at 19% versus 5% (compared to BAT excluding JAK inhibitors; PERSIST-1)32 and 18% versus 3% (compared to BAT including JAK inhibitors; PERSIST-2).33 The latter study included patients with platelet counts <100x109/L. Fedratinib is currently approved for use in patients intolerant of or resistant to ruxolitinib, with a reported response rate of approximately 31% (JAKARTA-2),34 although this has not been validated in a real-world setting, in which spleen response rates were 0% in patients who were on ruxolitinib ≥20 mg BID dosing prior to the switch to fedratinib.35 Pacritinib is currently approved for patients with platelet count <50x109/L and recent observations suggest additional value in combating anemia through ACRV1 or IRAK1 inhibition.36 JAK inhibitors are immunosuppressive and can therefore be associated with serious opportunistic infections37-39 and poor response to COVID-19 vaccination.40 Long-term experience with ruxolitinib has also revealed high treatment discontinuation rates and the occurrence of “ruxolitinib withdrawal syndrome” with abrupt treatment discontinuation, characterized by a rapid relapse of symptoms, splenomegaly, worsening of cytopenias and occasional hemodynamic decompensation.41,42 Treatment-emergent side effects for fe-

dratinib included Wernicke encephalopathy, anemia, thrombocytopenia, gastrointestinal distress and elevations in serum liver function tests and pancreatic enzymes; and for pacritinib included cardiac events, severe diarrhea, nausea, thrombocytopenia, anemia and hemorrhage.

Momelotinib: mechanism(s) of action Momelotinib is an ATP-competitive small molecule that inhibits JAK1 (half maximal inhibitory concentration [IC50]=11 nM), JAK2 (IC50=18 nM), JAK3 (IC50=155 nM) and TYK2 (IC50=17 nM), among other kinases.43,44 The drug is orally administered in a tablet form and a 200 mg dose was shown to provide plasma exposure similar to that of a 300 mg capsule formulation, in healthy subjects; the effect of food or omeprazole was not considered clinically meaningful.45 Additional pharmacokinetic and safety studies have suggested that dose adjustment for momelotinib might not be necessary in patients with renal or mild to moderate hepatic impairment but dose reduction was advised for patients with severe hepatic impairment.46 In vitro, momelotinib has been shown to inhibit growth of Ba/F3-JAK2V617F and human erythroleukemia (HEL) cells (IC50=1,500 nM) and Ba/F3-MPLW515L cells (IC50=200 nM), but not BCR-ABL1-harboring K562 cells (IC50=58,000 nM).43 In addition, cell lines harboring mutated JAK2 were inhibited more potently than those harboring mutated JAK3 alleles, and STAT-5 phosphorylation was inhibited in HEL cells with an IC50 of 400 nM. Momelotinib selectively suppressed the in vitro growth of erythroid colonies harboring JAK2V617F from patients with polycythemia vera43 and induced growth suppression and apoptosis in JAK2-dependent hematopoietic cell lines. In a murine model of MPN, momelotinib normalized blood counts and spleen size, and suppressed the levels of inflammatory cytokines.44 Additional targets for momelotinib include CDK2/cyclin A, MAPK8 (JNK1), PRKCN (PKD3), PRKD1 (PKCμ), ROCK2, TBK1, FLT3-ITD, and ACVR1.44,47,48 Momelotinib’s inhibition of JAK2 is primarily responsible for its well-established palliative value in patients with MF, which includes reduction of spleen size and alleviation of constitutional symptoms. These effects are realized through inhibition of JAK-STAT-mediated activation of genes that are important for myeloid cell proliferation and survival, as well as suppression of cytokine-mediated inflammatory and constitutional symptoms (Figure 1). In addition, unlike the case with ruxolitinib and fedratinib, momelotinib and pacritinib also inhibit ACVR1, which is particularly appealing in the context of MF-associated anemia.36,48 ACVR1 (Activin A Receptor type 1 gene) is located on chromosome 2q24.1 and encodes ACVR1, which is a transmembrane serine/threonine kinase belonging to the

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Figure 1. Mechanism of action of momelotinib.

transforming growth factor-beta (TGF-β) receptor superfamily and is also known as Activin Receptor-Like Kinase 2 (ALK2).49 Signaling through ACVR1 is complex and involves other type 1 and type 2 receptors that engage various ligands, including activins and bone morphogenetic proteins (BMP) (Figure 1).50,51 These ligands are involved in multiple physiological and disease processes through distinct Smad (similar to the gene products of Drosophila mothers against decapentaplegic' and the C. elegans gene Sma) pathways; activins signal via Smad2/3 and BMP Smad1/5/8. Germline mutation of ACVR1 causes a rare heterotropic ossification disease, fibrodysplasia ossificans progressiva,52 and ACVR1 has also been implicated as a cancer-driver gene in childhood brainstem glioma (diffuse intrinsic pontine glioma). ACVR1 interacts with type II receptors to form heterotetrameric receptor complexes (two type I and two type II) that can bind various ligands, including activins and BMP (Figure 1). Ligand-receptor engage-

ment leads to canonical SMAD and non-canonical nonSMAD signaling, resulting in nuclear translocation and regulation of transcription.49 SMAD2/3 signaling has also been implicated in ineffective erythropoiesis and inhibition of terminal erythroid differentiation.53 The latter has led to the development of luspatercept, a recombinant activin receptor type IIB fusion protein that was designed to trap TGF-β superfamily ligands (including activin), for the treatment of anemia associated with transfusion-requiring β-thalassemia and low/intermediate-risk MDS-RS without thrombocytosis or with thrombocytosis (MDS-RS-T).23,54,55 Luspatercept is currently being investigated in a phase III study in transfusion-dependent patients with MF on JAK inhibitor therapy (ClinicalTrials.gov Identifier: NCT04717414). In a rat model of anemia of chronic disease, momelotinib treatment normalized hemoglobin concentration and red blood cell count, believed to have resulted from direct inhibition of ACVR1, and associated reduction of hepcidin

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production.48 Such activity was not apparent for another JAK1/2 inhibitor, ruxolitinib, and did not appear to be mediated by inhibition of JAK2-mediated ferroportin degradation.48 Momelotinib-induced inhibition of ACVR1 might therefore downregulate hepcidin expression and result in increased mobilization of cellular iron stores.48 Consistent with this supposition, clinical documentation of an improvement in anemia in a phase II study of MF patients treated with momelotinib was associated with reduction in blood hepcidin levels and increased markers of iron availability and erythropoiesis.56 The downregulation of hepcidin by momelotinib is particularly relevant in MF in which previous studies have shown increased circulating levels of hepcidin and inflammatory cytokines in patients with primary MF, compared to healthy controls;57,58 increased hepcidin levels in the particular study correlated with anemia, red cell transfusion need, and serum ferritin of >500 μg/L.57 In the same study, hepcidin and inflammatory cytokines were independently associated with inferior survival.57,58 In another recently published report of MF patients receiving momelotinib therapy, anemia response correlated with lower serum ferritin level59 whereas an earlier study had revealed increased plasma hepcidin levels in MF and their correlation with the degree of anemia and serum ferritin level.57 Taking these observations together, it is reasonable to consider that changes in hepcidin production, via ACVR1 inhibition, contribute to the salutary effect of momelotinib on anemia.48,56,60 However, it should be noted that active erythropoiesis, per se, might result in downregulation of hepcidin via erythroferrone and clarification of the precise mechanism of momelotinib-induced improvement in MFassociated anemia requires additional studies.61 Whether or not reported differences in transcriptional, proteomic, and phenotypic biomarker profiles, including disparately modulated inflammatory cytokine production and immune function, between momelotinib and other JAK inhibitors explain differences in their impact on response patterns and toxicity profile remains to be clarified.62,63

Momelotinib: published clinical reports Table 1 presents summaries of published clinical reports on momelotinib therapy in MF and includes the original Mayo Clinic-centered early phase and subsequent phase II and phase III studies. The original Mayo Clinic-centered phase I/II clinical trial The findings of the first-in-human, phase I/II study of momelotinib in MF (n=166; NCT00935987) were serially published in 201364 and 2018.65 Drug doses ranged between 100 and 400 mg once daily while the dose confirmation phase

utilized 150 or 300 mg once daily (Table 1). The study population included 143 JAK inhibitor-naïve cases. In the particular study, momelotinib therapy produced responses in anemia (54%), resolution of red cell transfusion need (68%), and clinically assessed reduction in spleen size (40%). Although not uniformly assessed, improvement in constitutional symptoms was clinically documented in the majority of the study patients. Adverse events included grade 3/4 thrombocytopenia (34%) and neutropenia (8%), grade 1/2 diarrhea (48%), nausea (39%), vomiting (24%), dizziness (40%), peripheral neuropathy (30%), and first-dose effects of flushing, hypotension, dizziness and nausea (11%); in addition, increases in liver function tests and pancreatic enzymes were documented in 15-18% and 11-13% of cases, respectively. In 2015, we reported additional observations from the original phase I/II study including treatment-emergent peripheral neuropathy in 44% of the 100 consecutive patients treated at the Mayo Clinic.66 Assessment of response in the first 60 patients on the original phase I/II study (NCT00935987), according to the 2013 revised International Working Group criteria included 0% complete remission, 2% partial remission, 57% clinical improvement, 45% anemia response (median response duration 13 months), 53% resolution of transfusion need (median response duration 12 months), and 42% spleen response (median response duration 10 months). In 2015, we published the initial analysis of genetic predictors of response and showed a correlation between spleen response and presence of CALR and absence of ASXL1 mutation; a smaller spleen size and absence of constitutional symptoms were also predictive of spleen response in univariate but not multivariable analysis.67 Subsequent publications of the above-described phase I/II momelotinib clinical trial (NCT00935987) provided more mature data in terms of overall and leukemia-free survival and predictors of treatment response.59,68,69 In 2018, we published the 7-year follow-up of the NCT00935987 study regarding the 100 Mayo Clinic participants, comprising 79 JAK inhibitornaïve patients and 21 patients previously exposed to ruxolitinib.69 At the time, protocol therapy was discontinued in 91% of the patients, after a median treatment duration of 1.4 years. In multivariable analysis, absence of CALR type 1/like and presence of ASXL1 or SRSF2 mutations adversely affected survival while SRSF2 mutations, very high-risk karyotype, and circulating blasts ≥2% predicted leukemic transformation. Post-momelotinib treatment survival (median 3.2 years) was not significantly different from that of a risk-matched MF cohort not receiving momelotinib.69 More recently, we reported the 12-year survival data on the 79 JAK inhibitor-naïve patients from the aforementioned NCT00935987 phase I/II study and compared the results with 50 patients treated with ruxolitinib in a separate clinical trial (NCT00509899).68 The median follow-up for living patients was 11.7 years for momelotinib and 14.2 years for ruxolitinib. Median survival periods from the initiation of

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treatment with the study drug were 3.5 years (10-year survival 20%) for momelotinib and 4.0 years (10-year survival 23%) for ruxolitinib (P=0.32). ‘Drug survival’ (i.e., treatment discontinuation-free survival) was superior for momelotinib, compared to ruxolitinib, with 3-year drug discontinuation

rates of 68% versus 88% (P<0.01). ASCT after failure of JAK inhibitor treatment had a favorable survival impact with a 10-year survival estimate of 68% versus 15% for non-transplanted patients (P<0.01).68 A separate publication regarding 183 Mayo Clinic patients with high/intermediate-risk MF en-

Table 1. Clinical trials with momelotinib for the treatment of myelofibrosis. Trial

Treatment arms dose & schedule

Phase I/II NCT00935987 Pardanani et al. Leukemia (2013)

Dose escalation 100-400 mg/day N=60 (JAKi-naïve and JAKi-exposed) N=49, JAKi-naïve

Phase I/II NCT00935987 Pardanani et al. Leukemia (2018)

Dose escalation 100-400 mg/day Dose confirmation 150 mg QD, 300 mg QD 150 mg bid N=166 (JAKi-naïve and JAKi-exposed)

40% by palpation

Phase I/II NCT01423058 Gupta et al. Haematologica (2017)

Momelotinib 200 mg bid (N=54) Momelotinib 250 mg bid (N=7) (JAKi-naïve and JAKi-exposed) N=53, JAKi-naïve

Week 24 Spleen volume reduction ≥35% 45.8% 72% by palpation

Resolution of transfusion need 51.7%

All grades Diarrhea (45.9%) Total symptom score Peripheral neuropathy reduction ≥ 50% (44.3%) 30.8% Thrombocytopenia (39.3%) Dizziness (36.1%) Hypotension (24.6%)

Phase II NCT02515630 Oh et al. Blood Adv (2020)

Momelotinib 200 mg QD N=41 Transfusion-dependent (JAKi-naïve and JAKi-exposed) N=36, JAKi-naïve

Week 24 Spleen volume reduction ≥35% 19%

Week 24/anytime Resolution of transfusion need 34%/41%

Total symptom score reduction ≥ 50% 29%

Phase III NCT01969838 SIMPLIFY-1 Mesa et al. JCO (2017)

Momelotinib 200 mg QD vs. Ruxolitinib 20 mg bid N=432 (JAKi-naïve)

Week 24 Spleen volume reduction ≥35% 26.5% vs. 29% (P=0.01)

Week 24 Resolution of transfusion need 66.5% vs. 49.3% (P<0.001)

Grade 3/4 (Momelotinib arm) Total symptom score Thrombocytopenia (7%) reduction ≥ 50% Anemia (5.6%) 28.4% vs. 42.2% Diarrhea (2.8%) (P=0.98)a Hypertension (2.8%) Neutropenia (2.8%)

Phase III NCT02101268 SIMPLIFY-2 Harrison et al. Lancet Haematol (2017)

Momelotinib 200 mg QD vs. Best available therapy including ruxolitinib N=156 (JAKi-exposed)

Week 24 Spleen volume reduction ≥35% 7% vs. 6% (P=0.90)

Week 24 Resolution of transfusion need 43% vs. 21% (P=0.0012)

Total symptom score Grade 3/4 reduction ≥ 50% (Momelotinib arm) 26% vs. 6% Anemia (14%) (P=0.0006) Thrombocytopenia (7%)

Phase III NCT04173494 MOMENTUM Mesa et al. JCO (2022)

Momelotinib 200 mg QD N=130 vs. Danazol 600 mg QD N=65 (JAKi-exposed)

Week 24 Spleen volume reduction ≥35% 23.1% vs. 3.1% (P=0.0006)

Week 24 Resolution of transfusion need 30.8% vs. 20% (P=0.0064)

Week 24 Grade 3/4 Total symptom score (Momelotinib arm) reduction ≥ 50% Thrombocytopenia (22%) 24.6% vs. 9.2% Infections (15%) (P=0.0095) Anemia (8%)

Spleen response

48% by palpation

Anemia response (IWG-MRT)

Symptom response

Adverse effects

Resolution of transfusion need 70%

3-month resolution Pruritus 75% Night sweats 79% Bone pain 63% Fever 100% Anorexia 40%

Grade 3/4 Thrombocytopenia (32%) ↑AST (3%) ↑ALT (3%) ↑Lipase (5%) Headache (3%)

Not uniformly assessed

Grade 3/4 Thrombocytopenia (33.7%) Anemia (40.4%) Neutropenia (7.8%) Grade 1/2 Peripheral neuropathy (30.1%)

Week 12 Resolution of transfusion need 68%

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Grade 3/4 Anemia (12%) Neutropenia (12%)

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rolled in consecutive phase I/II JAK inhibitor clinical trials included the aforementioned group of 79 momelotinib- and 50-ruxolitinib treated patients, as well as 23 cases treated with fedratinib and 31 treated with BMS-911543.70 The 10year survival rate for all 183 JAK inhibitor-treated patients was 16% and was not significantly different across the four drug cohorts (P=0.33). Multivariable analysis of pre-treatment variables identified age >65 years, absence of type 1/like CALR mutation, baseline transfusion need, and presence of ASXL1/SRSF2 mutation as risk factors for survival. In addition, spleen and anemia responses were independently associated with improved short-term survival while long-term survival was secured only by ASCT (10-year survival rate 45% vs. 19% in non-transplanted patients; P<0.01).70 In our most recent updated analysis of 72 Mayo Clinic patients who were JAK inhibitor-naïve and anemic (i.e., hemoglobin level below sex-adjusted normal range) prior to treatment with momelotinib,59 44% experienced an anemia response at any time during treatment (median response duration ⁓20 months; range, 3-81). In the particular study, spleen and symptom responses were documented in 45% and 44% of evaluable patients, respectively. In multivariable analysis, predictors of anemia response included post-essential thrombocytopenia MF (83% vs. 37%), serum ferritin level <55 μg/L (89% vs. 38%), and time from diagnosis to initiation of momelotinib therapy of <23 months (65% vs. 26%). Among 28 patients who were transfusiondependent at baseline, resolution of transfusion need was documented in 13 (46%) patients and the response lasted for a median of 20.3 months (range, 4-61.3); independent predictors of response in this group of patients included intermediate- versus high-risk disease (100% vs. 0%), serum ferritin level <833 μg/L (80% vs. 28%), and post-essential thrombocytopenia versus primary/post-polycythemia vera MF (80% vs. 39%).59 Among all 72 study patients, treatment was discontinued in 93% after a median treatment duration of 20 months. The median post-momelotinib survival was 3.2 years with 5- and 10-year survival rates of 31% and 19%, respectively. In multivariable analysis, survival was positively affected by anemia response (median 3.8 vs. 2.8 years), presence of type 1/like CALR mutation (median 11 vs. 3 years), and absence of ASXL1 or SRSF2 mutation (median 3.7 vs. 2.9 years). The favorable impact of anemia response on survival was also confirmed in transfusion-dependent patients (median 3.7 vs. 1.9 years: 10year survival 8% vs. 0%). Taken together, the above-elaborated series of analyses from the original NCT00935987 phase I/II study of patients treated with momelotinib suggested therapeutic value in terms of all three quality of life offenders in MF: anemia, splenomegaly, and constitutional symptoms. In addition to thrombocytopenia and peripheral neuropathy, adverse events included gastrointestinal disturbances and liver and

pancreas function test abnormalities. Analyses of mature data suggested short-term survival benefit associated with favorable genetic profile and anemia response, but longterm survival remained dismal without intervention with ASCT. Subsequent phase I/II clinical trials Several other phase I/II studies of momelotinib in both MF56,71 and essential thrombocytopenia and polycythemia vera72 were subsequently published. The most notable in this regard (NCT02515630) included 41 transfusion-dependent patients with MF among whom momelotinib-induced resolution of transfusion need was documented in 17 patients (41%).56 In the particular study, 21 (50%) patients experienced grade 3 or higher adverse events, similar in spectrum to those seen in the above-discussed phase I/II study. Laboratory correlative studies demonstrated a momelotinib treatment-associated decrease in circulating hepcidin levels and increased markers of iron availability and effective erythropoiesis. Predictors of anemia response included lower hepcidin level.56 Another phase I/II study included 61 patients with MF who received momelotinib at a dose of 200 mg twice daily;71 based on conventional response criteria, anemia response was documented in 45%, spleen response in 72% by palpation and 46% by imaging, and symptom response in the majority of patients. Adverse events in the particular study included diarrhea (45.9%), peripheral neuropathy (44.3%), thrombocytopenia (39.3%), and first-dose associated dizziness (36.1%). Laboratory correlative studies showed drug-induced suppression of inflammatory cytokines.71 Momelotinib was also evaluated at daily doses of 100 mg and 200 mg in 28 patients with polycythemia vera and 11 with essential thrombocytopenia; only two patients among all 39 cases showed a response, as per study response criteria; adverse events included peripheral neuropathy in seven (18%) patients.72 Taken together, the phase I/II studies after NCT00935987 confirmed the observations from the initial NCT00935987 study and, in addition, provided a mechanistic explanation for the erythropoietic effect of momelotinib in MF.56 Phase III studies The aforementioned observations from phase I/II studies were subsequently confirmed in three phase III studies, which ultimately led to acceptance of a New Drug Application (NDA) for momelotinib. In SIMPLIFY-1 (NCT01969838), 432 JAK inhibitor-naïve patients with high/intermediate-risk MF were assigned to receive either momelotinib (200 mg once daily; n=215 ) or ruxolitinib (20 mg twice daily; n=217).73 At week 24, spleen volume reduction of ≥35% was achieved at a similar rate (26.5% and 29%, respectively) while symptom reduction score was higher in the ruxolitinib arm (42.2% vs. 28.4%). Transfusion independence at week 24 was documented in 66.5% and

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49.3% of patients treated with momelotinib and ruxolitinib, respectively. Furthermore, achievement of transfusion-independence in patients receiving momelotinib was associated with a higher 3-year survival rate of 77.2% vs. 51.6%. Treatment-emergent myelosuppression was similar in the two treatment arms, with the exception of more anemia in the ruxolitinib arm and first-dose effects in the momelotinib arm. Peripheral neuropathy was reported in 10% and 5% of patients receiving momelotinib or ruxolitinib, respectively. In SIMPLIFY-2 (NCT02101268), 156 MF patients with either suboptimal response to or intolerance of ruxolitinib were randomly assigned to receive momelotinib 200 mg once daily (n=104) or BAT (which included ruxolitinib in 89% of the cases; n=52). Spleen volume response of ≥35% was reported in 7% of the momelotinib group and 6% of the BAT group. As was the case in SIMPLIFY-1, the rate of transfusion-independence at week 24 was higher in the momelotinib group than in the BAT group (49.3% vs. 21%).74 Peripheral neuropathy occurred in 11% of momelotinibtreated patients. In a recent updated analysis of the SIMPLIFY trials, 2-year overall and leukemia-free survival data for JAK inhibitor-naïve patients enrolled in SIMPLIFY-1 were similar in patients initially treated with momelotinib (81.6% and 80.7%, respectively) and those initially treated with ruxolitinib (80.6% and 79.3%, respectively). Results were similar in the context of previously ruxolitinib-exposed patients in SIMPLIFY-2 assigned to momelotinib or BAT. Baseline transfusion need in both SIMPLIFY trials was associated with inferior survival while momelotinib-induced transfusion-independence in SIMPLIFY-1 was associated with superior survival.75 The most recent phase III study included 195 JAK inhibitor-exposed patients with high/intermediate-risk MF with a hemoglobin <10 g/dL, a symptom score of ≥10, and a platelet count ≥25x109/L, assigned to either momelotinib (200 mg daily; n=130) or danazol (600 mg daily; n=65), both in conjunction with placebo pills, for 24 weeks, after which patients could receive open-label momelotinib.76 Transfusion-independence rates at baseline and at week 24 were 13% versus 31% for momelotinib and 15% versus 20% for danazol (P<0.05; met criteria for non-inferiority); rates of no transfusions to week 24 were 35% for momelotinib and 17% for danazol (met criteria for superiority). At week 24, spleen volume reduction of ≥35% occurred in 23% of patients treated with momelotinib versus 3% treated with danazol (met criterion for superiority); the corresponding symptom score response rates were 24.6% and 9.2% (met criteria for superiority). Grade ≥3 hematologic and nonhematologic side effects were similar in the momelotinib and danazol treatment groups.76 The follow-up period for the MOMENTUM study remains relatively short (approximately 9 months) and the crossover design of the study confounds estimation of comparative survival; regardless,

it is unlikely that momelotinib-treated patients in the MOMENTUM trial would behave differently from their counterparts in earlier phase II/III trials, in terms of survival or duration of treatment response.

Momelotinib: therapeutic prospects beyond myelofibrosis The somewhat unexpected discovery of ACVR1-SMAD pathway inhibition by momelotinib opens up new therapeutic avenues for the drug in other myeloid neoplasms and non-hematologic conditions associated with ineffective or iron-restricted erythropoiesis.61 The BMP-ACVR1-SMAD pathway is central to regulation of hepcidin transcription and also contributes to ineffective erythropoiesis driven by other pathogenic mechanisms.61,77 Inflammatory cytokines, such as interleukin-6, are markedly increased in MF and likely contribute to increased circulating levels of hepcidin.58,61 Similar mechanisms of hepcidin upregulation are considered in other myeloid neoplasms and non-hematologic conditions associated with iron-restricted erythropoiesis, including anemia of inflammation.61 In addition to MF, myeloid neoplasms associated with anemia include MDS with (MDS-RS) or without ring sideroblasts and with (MDS-SF3B1) or without SF3B1 mutation.78,79 The underlying mechanisms for anemia associated with MDS are complex but likely include ineffective erythropoiesis and aberrant SMAD signaling, which is now considered a legitimate target for the development of drugs, such as luspatercept (TGF-β ligand trap).54,55,80 Luspatercept is a recombinant activin receptor type IIB fusion protein that was designed to trap TGFβ superfamily ligands (including activin) and thus inhibit SMAD2/3 signaling, which is believed to inhibit terminal erythroid differentiation.53 Luspatercept is currently approved for use in adult patients with transfusion-requiring β-thalassemia and low/intermediate-risk MDS-RS and MDS/MPN-RS-T, based on controlled evidence of efficacy in alleviating anemia.23,54,55 Galunisertib (an ALK5 inhibitor) is another drug that targets SMAD signaling and has shown modest activity in ameliorating transfusion-dependent anemia in patients with low/intermediate-risk MDS.81 These observations suggest a similar activity as that of momelotinib in these myeloid neoplasms, especially in MDS-RS/MDS-SF3B1 in which a subset of patients display JAK2 mutations and thrombocytosis (MDS-RS-T). However, it is unlikely that the drug will be able to overcome other underlying contributors to disease-associated anemia, including intrinsic clonal defects, which explains the incomplete and non-durable anemia responses seen so far with momelotinib and luspatercept. We are also aware of emerging information on the drug’s potential as a FLT3-ITD inhibitor47 and ongoing clinical trials in solid tumors (clinicaltrials.gov).

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Table 2. Comparison of Food and Drug Administration-approved JAK2 inhibitors with momelotinib for treatment of myelofibrosis.

Mechanism of action

FDA-approved indication

FDA-approved dose & schedule

Ruxolitinib

Fedratinib

JAK1/JAK2 inhibition

JAK2/FLT3/RET inhibition

IPSS High/intermediate risk

Pacritinib JAK2/FLT3/ACVR1 IRAK1/CSF1R inhibition

DIPSS High/intermediate risk IPSS High/intermediate-2 risk First-line and second-line First-line and second-line for platelet count <50x109/L

20 mg twice daily (platelet count >200 x109/L) 200 mg twice daily 400 mg twice daily 15 mg twice daily 9 (platelet count ≥50 x10 /L) (Platelet count <50x109/L) (platelet count 150-200 x109/L) JAKARTA-1 (36%) JAKARTA-2 (55%)

PERSIST-1 (19%) PERSIST-2 (18%)

Not well defined

Resolution of transfusion need PERSIST-1 (25%) PERSIST-2 (37%/24%)μ

Symptom response†

COMFORT-1 (45.9%)

JAKARTA-1 (36%) JAKARTA-2 (26%)

PERSIST-1 (19%) PERSIST-2 (25%)

Adverse effects

Thrombocytopenia Anemia Bruising Dizziness Headache Withdrawal syndrome Opportunistic infections Poor response to COVID-19 vaccines

Monthly average wholesale price

$19,440

Spleen response*

Anemia response#

COMFORT-1 (41.9%) COMFORT-2 (28%)

Diarrhea Thrombocytopenia Anemia GI symptoms Thrombocytopenia Anemia GI symptoms ↑Liver function tests Peripheral edema ↑Amylase/lipase Pneumonia Wernicke encephalopathy Cardiac failure (boxed warning) Pyrexia Squamous cell skin cancer $27,520

$25,715

Momelotinib JAK1/JAK2/ACVR1 inhibition Approval pending MOMENTUM trial DIPSS High/Intermediate risk Anemia Palpable spleen ≥5 cm Symptoms Approval pending MOMENTUM trial 200 mg daily SIMPLIFY-1 (26.5%) SIMPLIFY-2 (7%) MOMENTUM (23.1%) Resolution of transfusion need SIMPLIFY-1 (66.5%) SIMPLIFY-2 (43%) MOMENTUM (30.8%) SIMPLIFY-1 (28.4%) SIMPLIFY-2 (26%) MOMENTUM (24.6%) Thrombocytopenia Neutropenia Anemia Infections ↑Liver function tests ↑Amylase/lipase Peripheral neuropathy First-dose effect€ Approval pending

COMFORT-1: ruxolitinib vs. placebo; COMFORT-2: ruxolitinib vs. best available therapy; JAKARTA-1: fedratinib vs. placebo; JAKARTA-2: fedratinib in patients previously treated with ruxolitinib; PERSIST-1: pacritinib vs. best available therapy excluding JAK inhibitors; PERSIST-2: pacritinib vs. best available therapy including ruxolitinib in patients with platelet count <100x109/L; SIMPLIFY-1: momelotinib vs. ruxolitinib; SIMPLIFY2: momelotinib vs. best available therapy including ruxolitinib; MOMENTUM: momelotinib vs. danazol. *Spleen response: spleen volume reduction ≥35% at week 24. #Anemia response: Gale criteria: absence of red blood cell transfusions for 12 weeks in the PERSIST-1 trial; IWG-MRT criteria: absence of red blood cell transfusions and hemoglobin ≥8 g/dL in the prior 12 weeks at week 24 in SIMPLIFY-1/2 and MOMENTUM trials; Gale criteria/IWG criteria in the PERSIST-2 trial. †Symptom response: total symptom score reduction ≥50% at week 24. €Hypotension, flushing, dizziness, nausea. FDA: Food and Drug Administration; IPSS: International Prognostic Scoring System; DIPSS: Dynamic International Prognostic Scoring System; COVID-19: coronavirus disease 2019; GI: gastrointestinal; IWG-MRT: International Working Group for Myelofibrosis Research and Treatment.

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Momelotinib-inclusive treatment algorithm and concluding remarks ASCT currently remains the only treatment option in MF that can secure long-term survival. The number of allogeneic transplants in MF has increased in recent years and it is encouraging to witness, over time, a higher number of patients who are older and less fit but are transplanted, increased utilization of matched unrelated donors, improvements in overall and relapse-free survival, decreased incidence of graft-versus-host disease and stable incidence of non-relapse mortality.12 In transplant-ineligible patients, optimal palliative care requires attention to all three quality-of-life offenders: anemia, splenomegaly, and

constitutional symptoms.6 In this regard, because of its salutary effect on anemia, as well as splenomegaly and constitutional symptoms, momelotinib might have an edge over currently FDA-approved JAK inhibitors. However, scientifically sound comparisons between different JAK inhibitors can only be accomplished through prospective controlled studies and should also consider other factors, including side effects (Table 2). Emerging information suggests similar erythropoietic benefit from pacritinib but it is not certain whether its activity against splenomegaly and constitutional symptoms would be as potent as that of momelotinib.36 Currently available JAK inhibitors, including momelotinib, are inherently immunosuppressive and carry multiple side

Figure 2. Our current risk-adapted treatment approach in primary myelofibrosis based on impending approval of momelotinib. Risk stratification is based on the Mutation-enhanced International Prognostic Scoring System, version 2.0. (MIPSSv2): very high risk karyotype = 4 points; unfavorable karyotype = 3 points; ≥2 high molecular risk mutations = 3 points; one high molecular risk mutation = 2 points; absence of a type 1 CALR mutation = 2 points; constitutional symptoms = 2 points; severe anemia = 2 points; moderate anemia = 1 point; ≥2% circulating blasts = 1 point. ESA: erythropoiesis-stimulating agents; JAKi: JAK inhibitors. Haematologica | 108 November 2023

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effects that necessitate due diligence in their use (Table 2). Current indications for JAK inhibitor therapy in MF include hydroxyurea-refractory splenomegaly and severe constitutional symptoms. The availability of momelotinib in the near future might expand the list of indications to include anemia. However, in the absence of symptomatic splenomegaly or constitutional symptoms, we prefer initial therapy with non-JAK inhibitor drugs (Figure 2). Similarly, we prefer initial treatment with hydroxyurea, for the treatment of splenomegaly, leukocytosis, or extreme thrombocytosis, in the absence of associated anemia or severe constitutional symptoms (Figure 2); such an approach considers the superior activity of hydroxyurea, compared to JAK inhibitors, in terms of controlling leukocytosis and thrombocytosis as well as the fact that the spleen effect of ruxolitinib or other JAK inhibitors is often not durable and the value of these inhibitors might be best reserved for those patients in whom treatment with hydroxyurea fails. Our second-line drug of choice in the latter instance is ruxolitinib, considering its comparatively better toxicity profile, compared to that of other JAK inhibitors (Table 2). The projected approval of momelotinib might result in modification of the current treatment algorithm in MF, including the possibility of its use as the first-line JAK inhibitor of choice in the presence of anemia (Figure 2). We prefer pacritinib as the first-line JAK inhibitor of choice in the presence of a platelet count <50x109/L. The more favorable toxicity profile of ruxolitinib, compared to that of all other JAK inhibitors, argues for its use as the first-line JAK inhibitor of choice, in the absence of anemia. In cases in which ruxolitinib fails, we prefer ruxolitinib dose modification first before switching treatment to other JAK inhibitors (Figure 2). Real-world experience suggests limited value of switching from ruxolitinib to fedratinib in MF patients already receiving adequate doses of ruxolitinib (≥20 mg twice daily).35,82 There is currently no evidence to support the value of JAK inhibitors in asymptomatic patients with MIPSSv2 low or

very low risk disease, whose expected 10-year survival rates were reported to be 50% and 86%, respectively.10 Furthermore, the risk-benefit balance for ASCT in such patients favors deferring the procedure until there is evidence of progressive disease.83 On the other hand, ASCT is the preferred treatment of choice for patients with MIPSSv2 high or very high risk disease, in whom 10-year expected survival rates, without transplantation, might be as low as 10% and <3%, respectively (Figure 2).10 ASCT might also be considered for carefully selected MIPSSv2 intermediate-risk patients in whom 10-year projected survival without a transplant is estimated to be 30%.10 In general, investigational therapy is preferred for transplant-ineligible patients with high/very high-risk or symptomatic lower-risk disease (Figure 2). The possibility of further enhancing benefit from momelotinib by changing the dose schedule (i.e., 100 mg twice daily), without increasing the total daily dose (i.e., 200 mg), warrants exploration, based on recently published data on jaktinib, a deuterated form of momelotinib,84 where a phase II multicenter study (NCT03886415) revealed higher rates of spleen and anemia response using the drug at a dose of 100 mg twice daily rather than 200 mg once daily.85 However, it should be noted that the twice-daily dosing schedule in the latter study (NCT03886415) was associated with a higher frequency of serious adverse events.85 Finally, we underscore that our proposed treatment algorithm outlined in Figure 2 assumes approval of momelotinib in the current calendar year and reflects our current preferences and practice, which are subject to change based on emerging new information. Disclosures The authors participated in the original phase I/II study of momelotinib, ruxolitinib, and fedratinib for myelofibrosis. They have no other conflicts of interest to disclose. Contributions AT wrote the paper. All authors participated in the concept and design of the study and approved the final manuscript.

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Leukemia. 2018;32(5):1254-1258. 9. Guglielmelli P, Lasho TL, Rotunno G, et al. MIPSS70: Mutationenhanced International Prognostic Score System for transplantation-age patients with primary myelofibrosis. J Clin Oncol. 2018;36(4):310-318. 10. Tefferi A, Guglielmelli P, Lasho TL, et al. MIPSS70+ version 2.0: Mutation and karyotype-enhanced International Prognostic Scoring System for primary myelofibrosis. J Clin Oncol. 2018;36(17):1769-1770. 11. Ali H, Bacigalupo A. 2021 update on allogeneic hematopoietic stem cell transplant for myelofibrosis: a review of current data and applications on risk stratification and management. Am J Hematol. 2021;96(11):1532-1538. 12. McLornan D, Eikema DJ, Czerw T, et al. Trends in allogeneic haematopoietic cell transplantation for myelofibrosis in Europe between 1995 and 2018: a CMWP of EBMT retrospective analysis. Bone Marrow Transplant. 2021;56(9):2160-2172. 13. Hernandez-Boluda JC, Pereira A, Kroger N, et al. Allogeneic hematopoietic cell transplantation in older myelofibrosis patients: a study of the Chronic Malignancies Working Party of EBMT and the Spanish Myelofibrosis Registry. Am J Hematol. 2021;96(10):1186-1194. 14. Raj K, Eikema DJ, McLornan DP, et al. Family mismatched allogeneic stem cell transplantation for myelofibrosis: report from the Chronic Malignancies Working Party of European Society for Blood and Marrow Transplantation. Biol Blood Marrow Transplant. 2019;25(3):522-528. 15. Hernandez-Boluda JC, Pereira A, Alvarez-Larran A, et al. Predicting survival after allogeneic hematopoietic cell transplantation in myelofibrosis: performance of the Myelofibrosis Transplant Scoring System (MTSS) and development of a new prognostic model. Biol Blood Marrow Transplant. 2020;26(12):2237-2244. 16. Zeiser R, Polverelli N, Ram R, et al. Ruxolitinib for glucocorticoid-refractory chronic graft-versus-host disease. N Engl J Med. 2021;385(3):228-238. 17. Zeiser R, von Bubnoff N, Butler J, et al. Ruxolitinib for glucocorticoid-refractory acute graft-versus-host disease. N Engl J Med. 2020;382(19):1800-1810. 18. Miklos D, Cutler CS, Arora M, et al. Ibrutinib for chronic graftversus-host disease after failure of prior therapy. Blood. 2017;130(21):2243-2250. 19. Kroger N, Sbianchi G, Sirait T, et al. Impact of prior JAK-inhibitor therapy with ruxolitinib on outcome after allogeneic hematopoietic stem cell transplantation for myelofibrosis: a study of the CMWP of EBMT. Leukemia. 2021;35(12):3551-3560. 20. Tefferi A, Lasho TL, Mesa RA, Pardanani A, Ketterling RP, Hanson CA. Lenalidomide therapy in del(5)(q31)-associated myelofibrosis: cytogenetic and JAK2V617F molecular remissions. Leukemia. 2007;21(8):1827-1828. 21. Huang J, Tefferi A. Erythropoiesis stimulating agents have limited therapeutic activity in transfusion-dependent patients with primary myelofibrosis regardless of serum erythropoietin level. Eur J Haematol. 2009;83(2):154-155. 22. Tefferi A, Silverstein MN. Recombinant human erythropoietin therapy in patients with myelofibrosis with myeloid metaplasia. Br J Haematol. 1994;86(4):893. 23. Tefferi A. New drugs for myeloid neoplasms with ring sideroblasts: luspatercept vs imetelstat. Am J Hematol. 2021;96(7):761-763. 24. Martinez-Trillos A, Gaya A, Maffioli M, et al. Efficacy and tolerability of hydroxyurea in the treatment of the hyperproliferative manifestations of myelofibrosis: results in 40 patients. Ann Hematol. 2010;89(12):1233-1237.

25. James C, Ugo V, Le Couedic JP, et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature. 2005;434(7037):1144-1148. 26. Tefferi A, Gangat N, Pardanani A, Crispino JD. Myelofibrosis: genetic characteristics and the emerging therapeutic landscape. Cancer Res. 2022;82(5):749-763. 27. Tefferi A, Barbui T. Polycythemia vera and essential thrombocythemia: 2021 update on diagnosis, risk-stratification and management. Am J Hematol. 2020;95(12):1599-1613. 28. Verstovsek S, Kantarjian H, Mesa RA, et al. Safety and efficacy of INCB018424, a JAK1 and JAK2 inhibitor, in myelofibrosis. N Engl J Med. 2010;363(12):1117-1127. 29. Pardanani A, Harrison C, Cortes JE, et al. Safety and efficacy of fedratinib in patients with primary or secondary myelofibrosis: a randomized clinical trial. JAMA Oncol. 2015;1(5):643-651. 30. Verstovsek S, Mesa RA, Gotlib J, et al. A double-blind, placebocontrolled trial of ruxolitinib for myelofibrosis. N Engl J Med. 2012;366(9):799-807. 31. Harrison C, Kiladjian JJ, Al-Ali HK, et al. JAK inhibition with ruxolitinib versus best available therapy for myelofibrosis. N Engl J Med. 2012;366(9):787-798. 32. Mesa RA, Vannucchi AM, Mead A, et al. Pacritinib versus best available therapy for the treatment of myelofibrosis irrespective of baseline cytopenias (PERSIST-1): an international, randomised, phase 3 trial. Lancet Haematol. 2017;4(5):e225-e236. 33. Mascarenhas J, Hoffman R, Talpaz M, et al. Pacritinib vs best available therapy, including ruxolitinib, in patients with myelofibrosis: a randomized clinical trial. JAMA Oncol. 2018;4(5):652-659. 34. Harrison CN, Schaap N, Vannucchi AM, et al. Fedratinib in patients with myelofibrosis previously treated with ruxolitinib: an updated analysis of the JAKARTA2 study using stringent criteria for ruxolitinib failure. Am J Hematol. 2020;95(6):594-603. 35. Gangat N, McCullough K, Al-Kali A, et al. Limited activity of fedratinib in myelofibrosis patients relapsed/refractory to ruxolitinib 20 mg twice daily or higher: a real-world experience. Br J Haematol. 2022;198(4):e54-e58. 36. Oh ST, Mesa R, Harrison C, et al. Pacritinib is a potent ACVR1 inhibitor with significant anemia benefit in patients with myelofibrosis. Blood. 2022;140(Suppl 1):1518-1521. 37. Heine A, Brossart P, Wolf D. Ruxolitinib is a potent immunosuppressive compound: is it time for anti-infective prophylaxis? Blood. 2013;122(23):3843-3844. 38. Tsukamoto Y, Kiyasu J, Tsuda M, et al. Fatal disseminated tuberculosis during treatment with ruxolitinib plus prednisolone in a patient with primary myelofibrosis: a case report and review of the literature. Intern Med. 2018;57(9):1297-1300. 39. Eyal O, Flaschner M, Ben Yehuda A, Rund D. Varicella-zoster virus meningoencephalitis in a patient treated with ruxolitinib. Am J Hematol. 2017;92(5):E74-E75. 40. Guglielmelli P, Mazzoni A, Maggi L, et al. Impaired response to first SARS-CoV-2 dose vaccination in myeloproliferative neoplasm patients receiving ruxolitinib. Am J Hematol. 2021;96(11):E408-E410. 41. Tefferi A. JAK inhibitors for myeloproliferative neoplasms: clarifying facts from myths. Blood. 2012;119(12):2721-2730. 42. Coltro G, Mannelli F, Guglielmelli P, Pacilli A, Bosi A, Vannucchi AM. A life-threatening ruxolitinib discontinuation syndrome. Am J Hematol. 2017;92(8):833-838. 43. Pardanani A, Lasho T, Smith G, Burns CJ, Fantino E, Tefferi A. CYT387, a selective JAK1/JAK2 inhibitor: in vitro assessment of kinase selectivity and preclinical studies using cell lines and

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primary cells from polycythemia vera patients. Leukemia. 2009;23(8):1441-1445. 44. Tyner JW, Bumm TG, Deininger J, et al. CYT387, a novel JAK2 inhibitor, induces hematologic responses and normalizes inflammatory cytokines in murine myeloproliferative neoplasms. Blood. 2010;115(25):5232-5240. 45. Xin Y, Shao L, Maltzman J, et al. The relative bioavailability, food effect, and drug interaction with omeprazole of momelotinib tablet formulation in healthy subjects. Clin Pharmacol Drug Dev. 2018;7(3):277-286. 46. Xin Y, Kawashima J, Weng W, Kwan E, Tarnowski T, Silverman JA. Pharmacokinetics and safety of momelotinib in subjects with hepatic or renal impairment. J Clin Pharmacol. 2018;58(4):522-532. 47. Azhar M, Kincaid Z, Kesarwani M, et al. Momelotinib is a highly potent inhibitor of FLT3-mutant AML. Blood Adv. 2022;6(4):1186-1192. 48. Asshoff M, Petzer V, Warr MR, et al. Momelotinib inhibits ACVR1/ALK2, decreases hepcidin production, and ameliorates anemia of chronic disease in rodents. Blood. 2017;129(13):1823-1830. 49. Valer JA, Sanchez-de-Diego C, Pimenta-Lopes C, Rosa JL, Ventura F. ACVR1 function in health and disease. Cells. 2019;8(11):1366. 50. Kaliya-Perumal A-K, Carney TJ, Ingham PW. Fibrodysplasia ossificans progressiva: current concepts from bench to bedside. Dis Model Mech. 2020;13(9):dmm046441. 51. Bousoik E, Montazeri Aliabadi H. “Do we know Jack” about JAK? A closer look at JAK/STAT signaling pathway. Front Oncol. 2018;8:287. 52. Shore EM, Xu M, Feldman GJ, et al. A recurrent mutation in the BMP type I receptor ACVR1 causes inherited and sporadic fibrodysplasia ossificans progressiva. Nat Genet. 2006;38(5):525-527. 53. Kubasch AS, Fenaux P, Platzbecker U. Development of luspatercept to treat ineffective erythropoiesis. Blood Adv. 2021;5(5):1565-1575. 54. Fenaux P, Platzbecker U, Mufti GJ, et al. Luspatercept in patients with lower-risk myelodysplastic syndromes. N Engl J Med. 2020;382(2):140-151. 55. Cappellini MD, Viprakasit V, Taher AT, et al. A phase 3 trial of luspatercept in patients with transfusion-dependent betathalassemia. N Engl J Med. 2020;382(13):1219-1231. 56. Oh ST, Talpaz M, Gerds AT, et al. ACVR1/JAK1/JAK2 inhibitor momelotinib reverses transfusion dependency and suppresses hepcidin in myelofibrosis phase 2 trial. Blood Adv. 2020;4(18):4282-4291. 57. Pardanani A, Finke C, Abdelrahman RA, Lasho TL, Tefferi A. Associations and prognostic interactions between circulating levels of hepcidin, ferritin and inflammatory cytokines in primary myelofibrosis. Am J Hematol. 2013;88(4):312-316. 58. Tefferi A, Vaidya R, Caramazza D, Finke C, Lasho T, Pardanani A. Circulating interleukin (IL)-8, IL-2R, IL-12, and IL-15 levels are independently prognostic in primary myelofibrosis: a comprehensive cytokine profiling study. J Clin Oncol. 2011;29(10):1356-1363. 59. Gangat N, Begna KH, Al-Kali A, et al. Predictors of anemia response to momelotinib therapy in myelofibrosis and impact on survival. Am J Hematol. 2022;98(2):282-289. 60. Truksa J, Lee P, Beutler E. Two BMP responsive elements, STAT, and bZIP/HNF4/COUP motifs of the hepcidin promoter are critical for BMP, SMAD1, and HJV responsiveness. Blood. 2009;113(3):688-695. 61. Nemeth E, Ganz T. Hepcidin and iron in health and disease.

Annu Rev Med. 2022;74:261-277. 62. Singer JW, Al-Fayoumi S, Taylor J, Velichko S, O'Mahony A. Comparative phenotypic profiling of the JAK2 inhibitors ruxolitinib, fedratinib, momelotinib, and pacritinib reveals distinct mechanistic signatures. PLoS One. 2019;14(9):e0222944. 63. Kong T, Yu L, Laranjeira AB, He F, Allen MJ, Oh ST. Comprehensive profiling of clinical JAK2 inhibitors in myeloproliferative neoplasms. Blood. 2022;140(Suppl 1):3951-3952. 64. Pardanani A, Laborde RR, Lasho TL, et al. Safety and efficacy of CYT387, a JAK1 and JAK2 inhibitor, in myelofibrosis. Leukemia. 2013;27(6):1322-1327. 65. Pardanani A, Gotlib J, Roberts AW, et al. Long-term efficacy and safety of momelotinib, a JAK1 and JAK2 inhibitor, for the treatment of myelofibrosis. Leukemia. 2018;32(4):1035-1038. 66. Abdelrahman RA, Begna KH, Al-Kali A, et al. Momelotinib treatment-emergent neuropathy: prevalence, risk factors and outcome in 100 patients with myelofibrosis. Br J Haematol. 2015;169(1):77-80. 67. Pardanani A, Abdelrahman RA, Finke C, et al. Genetic determinants of response and survival in momelotinib-treated patients with myelofibrosis. Leukemia. 2015;29(3):741-744. 68. Tefferi A, Pardanani A, Begna KH, et al. Momelotinib for myelofibrosis: 12-year survival data and retrospective comparison to ruxolitinib. Am J Hematol. 2022;97(12):E433-E435. 69. Tefferi A, Barraco D, Lasho TL, et al. Momelotinib therapy for myelofibrosis: a 7-year follow-up. Blood Cancer J. 2018;8(3):29. 70. Gangat N, Begna KH, Al-Kali A, et al. Determinants of survival and retrospective comparisons of 183 clinical trial patients with myelofibrosis treated with momelotinib, ruxolitinib, fedratinib or BMS- 911543 JAK2 inhibitor. Blood Cancer J. 2023;13(1):3. 71. Gupta V, Mesa RA, Deininger MW, et al. A phase 1/2, open-label study evaluating twice-daily administration of momelotinib in myelofibrosis. Haematologica. 2017;102(1):94-102. 72. Verstovsek S, Courby S, Griesshammer M, et al. A phase 2 study of momelotinib, a potent JAK1 and JAK2 inhibitor, in patients with polycythemia vera or essential thrombocythemia. Leuk Res. 2017;60:11-17. 73. Mesa RA, Kiladjian JJ, Catalano JV, et al. SIMPLIFY-1: a phase III randomized trial of momelotinib versus ruxolitinib in Janus kinase inhibitor-naive patients with myelofibrosis. J Clin Oncol. 2017;35(34):3844-3850. 74. Harrison CN, Vannucchi AM, Platzbecker U, et al. Momelotinib versus best available therapy in patients with myelofibrosis previously treated with ruxolitinib (SIMPLIFY 2): a randomised, open-label, phase 3 trial. Lancet Haematol. 2018;5(2):e73-e81. 75. Mesa R, Harrison C, Oh ST, et al. Overall survival in the SIMPLIFY-1 and SIMPLIFY-2 phase 3 trials of momelotinib in patients with myelofibrosis. Leukemia. 2022;36(9):2261-2268. 76. Verstovsek S, Gerds AT, Vannucchi AM, et al. Momelotinib versus danazol in symptomatic patients with anaemia and myelofibrosis (MOMENTUM): results from an international, double-blind, randomised, controlled, phase 3 study. Lancet. 2023;401(10373):269-280. 77. Qu X, Zhang S, Wang S, et al. TET2 deficiency leads to stem cell factor-dependent clonal expansion of dysfunctional erythroid progenitors. Blood. 2018;132(22):2406-2417. 78. Orazi A, Hasserjian RP, Cazzola M, Dohner H, Tefferi A, Arber DA. International Consensus Classification for myeloid neoplasms at-a-glance. Am J Hematol. 2023;98(1):6-10. 79. Khoury JD, Solary E, Abla O, et al. The 5th edition of the World Health Organization classification of haematolymphoid tumours:

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myeloid and histiocytic/dendritic neoplasms. Leukemia. 2022;36(7):1703-1719. 80. Patnaik MM, Santini V. Targeting ineffective hematopoiesis in myelodysplastic syndromes. Am J Hematol. 2022;97(2):171-173. 81. Santini V, Valcarcel D, Platzbecker U, et al. Phase II study of the ALK5 inhibitor galunisertib in very low-, low-, and intermediaterisk myelodysplastic syndromes. Clin Cancer Res. 2019;25(23):6976-6985. 82. Gupta V, Cerquozzi S, Foltz L, et al. Patterns of ruxolitinib therapy failure and its management in myelofibrosis: perspectives of the Canadian Myeloproliferative Neoplasm

Group. JCO Oncol Pract. 2020;16(7):351-359. 83. Gowin K, Ballen K, Ahn KW, et al. Survival following allogeneic transplant in patients with myelofibrosis. Blood Adv. 2020;4(9):1965-1973. 84. Tefferi A, Gangat N, Pardanani A. Jaktinib (JAK1/2 inhibitor): a momelotinib derivative with similar activity and optimized dosing schedule. Am J Hematol. 2022;97(12):1507-1509. 85. Zhang Y, Zhou H, Jiang Z, et al. Safety and efficacy of jaktinib in the treatment of Janus kinase inhibitor-naive patients with myelofibrosis: results of a phase II trial. Am J Hematol. 2022;97(12):1510-1519.

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ARTICLE - Acute Myeloid Leukemia

FLT4 as a marker for predicting prognostic risk of refractory acute myeloid leukemia Ji Yoon Lee,1,2 Sung-Eun Lee,3 A-Reum Han,1 Jongeun Lee,2 Young-sup Yoon2,4 and Hee-Je Kim1,3 1

Leukemia Research Institute, College of Medicine, the Catholic University of Korea, Seoul, Korea; 2Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Korea; 3Catholic Hematology Hospital, Seoul St. Mary’s Hospital, College of Medicine, the Catholic University of Korea, Seoul, Korea and 4Department of Medicine, Emory University, Atlanta, GA, USA

Correspondence: Y-s. Yoon yyoon5@emory.edu H-J. Kim cumckim@catholic.ac.kr Received: Accepted: Early view:

November 24, 2022. June 5, 2023. June 15, 2023.

Abstract Treating patients with refractory acute myeloid leukemia (AML) remains challenging. Currently there is no effective treatment for refractory AML. Increasing evidence has demonstrated that refractory/relapsed AML is associated with leukemic blasts which can confer resistance to anticancer drugs. We have previously reported that high expression of Fms-related tyrosine kinase 4 (FLT4) is associated with increased cancer activity in AML. However, the functional role of FLT4 in leukemic blasts remains unknown. Here, we explored the significance of FLT4 expression in leukemic blasts of refractory patients and mechanisms involved in the survival of AML blasts. Inhibition or absence of FLT4 in AML blasts suppressed homing to bone marrow of immunocompromised mice and blocked engraftment of AML blasts. Moreover, FLT4 inhibition by MAZ51, an antagonist, effectively reduced the number of leukemic cell-derived colony-forming units and increased apoptosis of blasts derived from refractory patients when it was co-treated with cytosine arabinoside under vascular endothelial growth factor C, its ligand. AML patients who expressed high cytosolic FLT4 were linked to an AML-refractory status by internalization mechanism. In conclusion, FLT4 has a biological function in leukemogenesis and refractoriness. This novel insight will be useful for targeted therapy and prognostic stratification of AML.

Introduction Acute myeloid leukemia (AML) is a lethal hematopoietic malignancy characterized by uncontrolled proliferation of blood precursor cells.1 Approximately, 10-40% of newly diagnosed-AML patients do not respond to chemotherapy, thereby failing to achieve complete remission (CR). This condition is defined as refractory AML and is one of the most significant challenges in AML treatment.2 Furthermore, two-thirds of patients in CR eventually relapse despite being given specific post-remission therapy.3,4 It has been suggested that this refractory and relapsed AML is caused by the existence of CD34+CD38- cells (marked AML blasts in the present study) on the surface; however, subsequent studies demonstrated considerable heterogeneity in surface phenotypes of AML blasts as well as leukemic stem cells (LSC).5 Targeted therapy for refractory AML has been challenging due to the lack of sophisticated markers that allow identification of blasts,

which are leukemogenic and confer refractoriness to treatment. Fms-related tyrosine kinase 4 (FLT4) is a surface receptor for vascular endothelial growth factor C (VEGF-C) and plays a crucial role in lymphangiogenesis.6,7 Under normal conditions, FLT4 is not expressed in CD45+ hematopoietic cells. However, FLT4 is expressed on certain circulating progenitor cells or support cells which maintain progenitor cells in some pathological conditions 8-12 and is also expressed on mononuclear cells (MNC) in cancer and inflammatory diseases.13,14 Studies reported that FLT4 and its ligand VEGF-C are expressed in leukemic blasts in AML and that the role of this signaling axis is closely related to blast survival.15,16 Dias et al. reported that VEGF-C and FLT4 are expressed in the bone marrow (BM) of AML and VEGF-C can protect AML blasts against chemotherapy by inducing their proliferation, showing the importance of VEGF-C signaling in blast maintenance.17 In fact, high levels of VEGF-C in peripheral blood (PB) and BM cells are

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associated with poor clinical outcome of AML.15,16,18 However, to date no studies explored whether FLT4 can play a significant role in leukemogenesis and refractoriness. In this study, we found that FLT4 can function as an important marker for AML blasts and is able to confer resistance to conventional chemotherapy to AML blasts. Inhibition of AML blasts with FLT4 suppressed homing of AML blasts to the BM in immunocompromised mice and specifically inhibited the engraftment of AML blasts. Moreover, FLT4 blockage by an antagonist, MAZ51, significantly reduced the number of leukemic cell-derived colony-forming units (CFU) and promoted apoptosis of AML blasts derived from refractory AML patients when cotreated with cytosine arabinoside (Ara-C). MAZ51 inhibits VEGF-C-induced activation of FLT4 tyrosine kinase selectively by blocking FLT4 phosphorylation at low concentration.19 MAZ51 was reported to directly inhibit survival and proliferation of lymphatic endothelial cells and tumor cells, which express FLT4.20 We also found that MAZ51 did not affect Ara-C-induced blast apoptosis in the absence of VEGF-C. However, in the presence of VEGF-C, MAZ51 induced blast apoptosis with Ara-C. Additionally, we also found that a high level of cytoplasmic FLT4 in the AML blasts derived from AML-BM at diagnosis was associated with refractory status after chemotherapy, whereas a low levels of cytosolic FLT4 in AML blasts was tied to complete remission later. We further demonstrated that this phenomenon is caused by internalization of FLT4 receptor in refractory cases, which activated cell survival. Collectively, this study suggests the therapeutic and prognostic implications of FLT4, particularly for refractory AML.

Methods Patients All experiments were performed with authorization from the Institutional Review Board for Human Research at the Catholic University of Korea (KC11TASI0526) and were performed in accordance with the Helsinki Declaration. Blood and BM samples were collected from 60 healthy donors, 103 patients with AML at diagnosis, 18 CR patients, nine AML patients who received stem cell transplantation (SCT), 12 refractory, and five relapsed AML patients, following the AML subtype classification designated by the World Health Organization (WHO). BM-derived mononuclear cells (BM-MNC) and peripheral blood-derived MNC (PB-MNC) were fractionated by density gradient centrifugation using Ficoll-Paque™ PLUS (171440-03; GE Healthcare Life Sciences, Piscataway, NJ, USA). The clinical characteristics and experimental information regarding patients with AML enrolled in the present study are listed in the Online Supplementary Table S1.

Humanized leukemic mouse model All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committees of the Catholic University of Korea (CU-2010-0189-03) and all animal procedures were performed in accordance with approved guidelines and regulations. NOD/ShiLtSz-scid/IL2Rgnull (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzL) or Nod scid γ (NSG) mice were purchased from the Jackson Laboratory and housed in ventilated micro-isolator cages in a high-barrier facility under specific pathogen-free conditions. Autoclaved water and irradiated food were provided ad libitum. All experiments were performed as previously described.21 For the leukemic xenograft model, 8-week-old mice were sublethally irradiated with 300 cGy total body irradiation 24 hours before intravenous injection of CD34+CD38- cells. For MAZ51 treatment (EMD-Calbiochem, San Diego, CA, USA), MNC cells from AML patients were isolated and cultured for 3 hours in DMEM containing either 1% fetal bovine serum or 5 μM MAZ51, a FLT4 antagonist, in dimethyl sulfoxide. CD34+ cells, either MAZ51 treated or untreated, were isolated and suspended in phoshate-buffered saline (PBS) at a final concentration of 5×105 cells per 200 μL of PBS and cells were injected into irradiated NSG mice via the tail vein. Mice were monitored daily for symptoms of disease such as ruffled coat, hunched back, weakness, and reduced motility. Once injected animals showed signs of distress, they were sacrificed. In the absence of these signs of stress, mice were analyzed over 15 weeks following transplantation. The time from transplantation to sacrifice varied from 8 to 15 weeks with an average of 10 weeks. Statistical analysis All results were presented as the mean ± standard error of the mean. Statistical analyses were performed with the Mann–Whitney U test for comparisons between two groups and the Kruskal-Wallis ANOVA test for >2 groups. Values of P<0.05 were considered to denote statistical significance. The GraphPad Prism version 4 software (GraphPad Software, La Jolla, CA, USA) was used for statistical analysis. In clinical correlation with FLT4 expression, the two-tailed Student t test was used to analyze continuous variables between two groups and one-way ANOVA followed by the Scheffé post hoc analysis was used to compare continuous variables among three groups.

Results FLT4 functions as an additional marker to define stem cell activities in acute myeloid leukemia blasts in acute myeloid leukemia patients We previously reported high expression of lymphatic endothelial genes including FLT4, PROX1, LYVE1 and PDPN in PM-MNC from AML patients (AML-MNC).22 In this study, we

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investigated whether these genes are also enriched in stem cells marked by CD34 in the BM of AML patients. Quantitative real-time polymerase chain reaction was then performed on CD34+ cells isolated from BM cells using the primer/probe sets presented in the Online Supplementary Table S2 and data showed that CD34+ cells showed higher expression of all four lymphatic endothelial genes, compared to CD34- cells (Figure 1A; P<0.05). We then per-

formed immunocytochemistry for FLT4 and CD34 on AMLMNC and found FLT4 expression in CD34+ cells (Figure 1B). We examined the expression of FLT4 in AML blasts via flow cytometry, comparing BM cells collected from normal donors, patients with AML, and AML with complete remission (CR). The number of AML blasts was significantly lower in AML-CR compared to AML (0.3±0.1% vs. 10.9±2.8%), which is similar to the number in normal donors (0.9±0.3%).

Figure 1. Expression of FLT4 in mononuclear cells derived from acute myeloid leukemia patients and its potential for stem/progenitor function. (A) Quantitative real-time polymerase chain reaction analysis for MACS-isolated CD34+ and CD34cells (N=5). Each with technical triplicates; *P<0.05. Mann–Whitney U test with two-sided P values. (B) Confocal microscopic imaging of acute myeloid leukemia mononuclear cells (AML-MNC), which were double-stained for Fms-related tyrosine kinase 4 (FLT4) (green) and CD34 (red) expression. DAPI: blue. Scale bar =20 μm. (C) Flow cytometric analysis of peripheral blood cells from normal donors, AML, and AML with complete remission (AML-CR) patients. Numbers in boxes are the percentages of CD34+CD38- and FLT4+ cells from the gated region. Normal (N=14), AML (N= 80), CR (N=12). *P<0.05; ***P<0.001 Kruskal-Wallis ANOVA test. FSC: forward scatter. Haematologica | 108 November 2023

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However, the proportion of FLT4+ cells in CD34+CD38- cells was similarly higher in both AML and CR patients compared to normal donors (AML, 26.7±2.3%; CR, 23.4±5.4%; normal, 4.7±1.0%) (Figure 1C; *P<0.05; ***P<0.001). Next, we performed leukemia CFU assays (L-CFU) with FLT4+AML blasts versus FLT4- AML blasts. L-CFU were classified as one of the following types: granulocyte, erythrocyte, monocyte, and megakaryocyte-L-CFU (L-CFU-GEMM); granulocyte and macrophage-L-CFU (L-CFU-GM); granulocyte-L-CFU (L-CFU-G); or macrophage-L-CFU (L-CFU-M), according to the cellular contents.23,24 The CFU assays showed that FLT4+AML blasts formed more L-CFU-GEMM colonies than FLT4-AML blasts (Online Supplementary Figure S1A). FLT4-AML blasts-derived CFU-colonies generally formed normal-looking colonies (Online Supplementary Figure S1B). We further determined the function of FLT4 in forming L-CFU by blocking FLT4 tyrosine kinase activity using MAZ51, a FLT4 antagonist.19 We cultured BM-MNC collected from AML patients in L-CFU culture medium for 14 days after pretreating them with MAZ51 for 2 hours (hrs). In these assays, L-CFU colonies were smaller with atypical morphology (Online Supplementary Figure S1C), compared to CFU colonies derived from FLT4-AML blasts (Online Supplementary Figure S1A). In MNC, the statistical analysis for the Online Supplementary Figure S1C were displayed in the Online Supplementary Figure S1D. These data implied that FLT4 expression in AML blasts can not only be a marker to represent leukemic blasts in refractory patients but also endow leukemic blasts activities. FLT4 expression in acute myeloid leukemia blasts increased the homing and engraftment in NSG mice We then determined the effects of FLT4 on homing and engraftment characteristics of AML blasts (Figure 2A). First, we investigated the role of FLT4 in the homing efficiency. After treating PB-MNC from AML patients with MAZ51, AML blasts were sorted by fluorescence-activated cell sorting and injected intravenously into NSG mice. Eighteen hrs later, the number of AML blasts was counted in the mouse BM, PB, and spleen. In flow cytometric analysis, the number of homed AML blasts in the BM and PB was significantly reduced in the MAZ51-treated group compared to the untreated groups (Figure 2B). Immunostaining confirmed the existence of human CD34+ cells in flushed BM cells of NSG mice (Online Supplementary Figure S2A) and brightly- and dimly-stained human CD45+ cells (Online Supplementary Figure S2B). These data indicate that inhibition of FLT4 significantly reduced homing efficiency of AML blasts to BM and PB. We further compared the homing efficiency of FLT4+AML blasts and FLT4-AML blasts. Again, the homing efficiency to the BM was significantly higher in the mice injected with FLT4+AML blasts than with FLT4-AML blasts (Online Supplementary Figure S2C). Next, we investigated the effects of FLT4 on the engraft-

ment potential of AML blasts. NSG mice were injected with either AML blasts or MAZ51-treated AML blasts and sacrificed 6 to 9 weeks later, and AML blasts were counted in BM and spleen of the humanized mice. The frequencies of AML blasts in BM and spleen were similar between the MAZ51 untreated and treated groups, indicating no effects of MAZ51 on the engraftment of CD34+CD38– cells (Online Supplementary Figure S2D). Engraftment of human CD45+ cells was further evaluated by flow cytometry. The results showed no significant differences in the number of human CD45+ cells in BM, PB and spleen between the two groups (Figure 2C). Additional engraftment experiments with FLT4+AML blasts and FLT4-AML blasts also showed no significant difference in the number of engrafted human CD45+ cells in the BM and spleen (Online Supplementary Figure S3). However, in detailed flow cytometric analyses of MAZ51 inhibition experiments, we found that there were two distinct populations of engrafted human CD45+ cells, having bright or dim intensities in flushed BM and PB cells (Figure 2C). Intriguingly, while the total number of human CD45+ cells in PB was not different between control and MAZ51-treated groups, in the control AML group, most human CD45+ cells were in the CD45dim fraction, which is regarded as leukemic blasts; however, in the MAZ51-treated group, a majority of CD45+ cells were in the CD45bright fraction (Figure 2C, middle panel). It is known that AML blasts have lower expression of CD45 (CD45dim) than normal hematopoietic cells (CD45bright) when MNC were gated.25 Taken together, these data suggest that FLT4 plays a significant role in the homing of AML blasts and engraftment of CD45dim blasts. Inhibition of FLT4 in acute myeloid leukemia blasts significantly reduced engraftment of CD45dimCD34+CD38cells, but not CD45brightCD34+CD38- cells In order to further elucidate the effects of FLT4 on the engraftment of AML blasts, we analyzed the flow cytometry data in detail. The frequency of AML blasts in PB-MNC was lower in the MAZ51-treated group than in the control AML group (control AML vs. MAZ51-treated, 11.0±10.4% vs. 4.3±4.2%) (Figure 3A; **P<0.01), suggesting that FLT4 inhibition reduced the frequency of overall engrafted AML blasts. We then further analyzed the frequency of engrafted AML blasts within CD45 bright and CD45dim populations in both MAZ51-treated and untreated groups (Figure 3B). In the CD45bright population, CD34+CD38- normal HSC displayed no significant difference between the control group and the MAZ51-treated group. However, in the CD45dim blast population, AML blasts were remarkably decreased in the MAZ51-treated group compared to the untreated control group (Figure 3C; **P<0.01). Together, these data demonstrate that FLT4 inhibition in AML blasts restrictively reduces engraftment of CD45dim blasts.

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Figure 2. Inhibition of homing and engraftment of acute myeloid leukemia CD34+CD38- cells in NSG mice by ex vivo MAZ51 treatment. (A) A schematic showing animal experiments of homing and engraftment of CD34+CD38- cells derived from acute myloid leukemia peripheral blood (AML-PB). (B) Homing of injected AML-derived CD34+CD38- cells in NSG mice. Homing efficiency in bone marrow (BM), PB, and spleen of mice B following ex vivo MAZ51 treatment (N=2-3 per group, each with technical triplicates). (C) Engraftment of injected AML-derived CD34+CD38- cells in NSG mice. Percentages of engrafted human CD45+ cells in the BM, PB, and spleen of NSG mice following treatment with or without ex vivo MAZ51 (N=2-6 per group, each with technical triplicates). *P<0.05; **P<0.01. Mann–Whitney U test with two-sided P values. Representative images of PB immunostained for human CD45 (hCD45).

FLT4 inhibition together with arabinoside in the presence of VEGF-C induced apoptosis of acute myeloid leukemia blasts derived from refractory patients In order to determine whether the effects of MAZ51 on the engraftment of leukemic MNC and AML blasts were attributed to direct toxicity of MAZ51, we performed an apoptotic assay using an Annexin V test after directly treating MNC and AML blasts with MAZ51. The results showed that there was no increase in apoptosis in either cell type regardless of MAZ51 treatment in different concentrations, indicating no direct toxicity of MAZ51 (Figure 4A; *P<0.05; **P<0.01). Next, we determined the effects of VEGF-C and FLT4 inhibition on apoptosis of leukemic MNC induced by

Ara-C. Treatment of Ara-C, but not MAZ51, to MNC culture induced apoptosis in the absence of VEGF-C (Figure 4B). Addition of VEGF-C to the culture significantly reduced apoptosis by Ara-C treatment. However, addition of MAZ51 to the MNC culture containing Ara-C and VEGF-C again increased apoptosis of MNC, suggesting FLT4 inhibition blocked the anti-apoptotic effects of VEGF-C on MNC. We then investigated the effects of FLT4 inhibition and VEGF-C on the apoptosis of CD45dim AML blasts induced by Ara-C. In cells derived from patients with non-refractory AML, MAZ51 did not have any additional effects on Ara-C-induced apoptosis regardless of the presence of VEGF-C in the culture (Online Supplementary Figure S4). However, in the cells derived

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from three of four different refractory AML patients, MAZ51 and Ara-C treatment relatively increased the apoptosis of the cells in the presence of VEGF-C (Figure 4C). These data indicate that Ara-C-induced blast apoptosis can be enhanced by FLT4 inhibition in the presence of VEGF-C in refractory AML patients.

marker to define the LSC properties of AML blasts. In order to prove the role of FLT4+ cells in AML leukemic blasts, we determined whether FLT4+ cells in BM could predict higher measurable residual disease (MRD), which is presumed to be caused by preserved CD34+CD38- LSC post chemotherapy. We evaluated MRD using BM-MNC of CR patients by counting the frequency of lineage aberrancy of CD7-ex+ + + Enrichment of CD7 CD34 cells in FLT4 acute myeloid pressing cells in CD34+CD45dim cells, called the leukemialeukemia blasts, and reconstruction of sinusoidal associated immunophenotype (LAIP). LAIP is a common endothelial cells and protection of the endosteal niche denominator for defining MRD when the frequency reaches in bone marrow by inhibition of FLT4 in acute myeloid over 0.01%.26,27 Flow cytometry demonstrated that the freleukemia blasts quency of LAIP in FLT4+ blasts in BM was 3.5±2.5% (range, Thus far, our data have shown that FLT4 can be an additional 1.0-9.3%), suggesting MRD while in CR status (Figure 5A).

Figure 3. Reduction of engrafted CD34+CD38- cells in mouse bone marrow after injection of peripheral blood mononuclear cells derived from acute myeloid leukemia into mice, which were pretreated with MAZ51. (A) Representative flow cytometry plots comparing expression of human CD34 (hCD34) and hCD38 in human peripheral blood (PB) obtained from acute myeloid leukemia (AML) patients. Numbers in boxes are the percentages of CD34+CD38- cells (N=16; **P<0.01). Mann–Whitney U test with twosided P values. (B) Flow cytometry for hCD45 and mouse CD45 (mCD45) of bone marrow mononucelar cells (BM-MNC) in mice, showing bright and dim fractions in human CD45 cells. (C) Flow cytometry for hCD34 and hCD38 with CD45dim (low) and CD45bright (upper) cells in the mouse BM with and without treatment of cells with MAZ51 (N=5 for CD45dim; N=6-7 for in CD45bright, each with technical triplicates). Each value is the average of at least 4 different PB cell samples obtained from AML patients (number of mice =3-5 per each PB). Mann–Whitney U test with two-sided P values. Haematologica | 108 November 2023

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Next, we investigated whether FLT4+ AML blasts occupy endosteal niche and affect sinusoidal endothelial cells in the BM when injected, and whether FLT4 inhibition in FLT4+ AML blasts can reverse these characteristics. As preliminary control experiments, we examined the changes of BM after irradiation. Among BM compartments, sinusoidal endothelial cells (SEC) can function as exits for circulating cells and for stem cell retention. When damage occurs by cancer or irradiation,28 SEC

rapidly expand with aberrant morphology.29 After partial irradiation, BM architecture was destroyed and FLT4 expression in SEC was not detected in the long bone area, suggesting impairment of SEC (Figure 5B).30,31 We then performed the main experiments in which MAZ51-treated and untreated FLT4+ AML blasts were injected into NSG mice, and 5 weeks later, BM tissues were harvested and subjected to immunostaining. We investigated two aspects: i) the localization of the FLT4+ AML blasts in the

Figure 4. MAZ51 induces apoptosis of CD34+CD38- cells collected from bone marrow of refractory/relapsed acute myeloid leukemia under VEGF-C exposure. (A) The percentage of apoptosis of mononucelar cells (MNC) and CD34+CD38- cells collected from acute myeloid leukemia (AML) patients’ peripheral blood (PB) treated with different concentrations of MAZ51 (N=8, KruskalWallis ANOVA test). (B) The percentages of apoptosis of AML PB-MNC cultured with or without cytosine arabinoside (Ara-C), MAZ51, or vascular endothelial growth factor C (VEGF-C) (N=11; *P<0.05, **P< 0.01; Kruskal-Wallis ANOVA test). (C) The percentage of apoptosis of CD45dimCD34+CD38- cells cultured with or without Ara-C, MAZ51, or VEGF-C. The number in the title represents the patient identification number (e.g., AML 69). Haematologica | 108 November 2023

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stem cell zone and the reconstitution of SEC in the BM. Hematoxylin and eosin staining showed that in the MAZ51-untreated group, leukemic blasts were massively infiltrated both endosteal and vascular regions of the BM. These data are consistent with prior reports that CD34+CD38- LSC are preferentially localized at the endosteal surface abutting osteoblasts for retention (Figure 5C, left panel).32 However, in the MAZ51-treated group, leukemic blasts were enriched in the vascular niche but not in the endosteal niche (Figure 5C, right panel). These data

support the identity of FLT4+ AML blasts as more specific CD34+CD38- LSC and the inhibitory effects of MAZ51. Immunostaining for FLT4 further showed that in the MAZ51untreated group, SEC appeared aberrant by infiltration of leukemic blasts. However, in the MAZ51-treated group, SEC were reconstituted and circulating blast cells were decreased (Figure 5D). Together, these data indicate that FLT4+ AML blasts can function as CD34+CD38- LSC in AML and FLT4 inhibition by MAZ51 treatment can recover the SEC in BM.

Figure 5. Enrichment of FLT4+ cells in measurable residual disease and restoration of the bone marrow niche with protective endosteal niche by FLT4 inhibition. (A) Flow cytometric analysis of Fms-related tyrosine kinase 4-positive (FLT4+) cells for CD45, CD34, and CD7. CD45dim-gated blasts represent high CD34+CD7+ cells. Samples were collected from acute myeloid leukemia with complete remission (AML-CR) patients (N=5). (B) Immunostaining of bone marrow (BM) after irradiation of wild-type mice, showing destroyed microenvironment. (C) Hematoxylin and eosin (H&E) staining of BM after intravenous injection of MAZ51untreated and MAZ51-treated FLT4+CD34+CD38- cells. Mouse BM injected with MAZ51-untreated FLT4+CD34+CD38- cells showed successful engraftment of injected cells in the endosteal niche except for a small normal mouse region (red region) (left panel). MAZ51-treated FLT4+CD34+CD38- failed to engraft into the endosteal niche (the red region indicates normal mouse BM cells). (D) In the MAZ51-untreated group, aberrant sinusoidal endothelial cells (SEC) by infiltration of leukemic blasts were shown (red arrows on the left) whereas in the MAZ51-treated group, SEC were restored (brown staining). Representative examples of N=3. Scale bar = 100 μM. Haematologica | 108 November 2023

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Role of FLT4 and VEGF-C in leukemic blasts derived from refractory acute myeloid leukemia patients Previous studies32-34 and our data (Figure 5) demonstrated that the BM niche is important to maintain CD34+CD38cells and the concentration of VEGF-C is high in the BM of leukemia-induced mouse29 and in AML patients.22 In this study, we measured the concentration of VEGF-C in the BM

plasma of patients with newly diagnosed (ND)-AML, refractory AML, and AML with CR, and normal donors and found that it was significantly higher in all the leukemia groups compared to normal donors (Figure 6A; **P<0.01). Next, we compared the percentages of FLT4+CD34+CD38- cells collected from the BM of the same groups. Flow cytometric analysis showed that the frequency of FLT4+CD34+CD38-

Figure 6. Relationship between FLT4/VEGF-C levels at diagnosis and the clinical outcomes of acute myeloid leukemia. (A) Comparison of vascular endothelial growth factor C (VEGF-C) concentration in bone marrow (BM) plasma of newly diagnosed acute myeloid leukemia (ND-AML), refractory AML (Ref-AML), AML with complete remission (CR), and normal donor groups (N=9 in each group; **P<0.01; the Kruskal-Wallis ANOVA test). (B) Flow cytometry data for FLT4+CD34+CD38- cells in the peripheral blood (PB) of ND-AML, Ref-AML, CR, and normal donor groups. A total of 64 patients were analyzed for clinical correlation (NDAML, N= 30; Ref-AML, N=9; CR, N=18; normal donors, N=7). One-way ANOVA followed by the Scheffé post hoc analysis. (C) In the bone marrow (BM), the frequency of cytosolic Fms-related tyrosine kinase 4-positive (FLT4+) cells expressing CD34+CD38- at the time of diagnosis who later became non-CR or CR increased. High cytosolic FLT4 represents non-CR in the BM 8 (N=8-11). VEGFC expression at the mRNA levels in BM at diagnosis (N=10-27, two-tailed Student t test). (D) The percentage of surface and cytosolic FLT4+ cells among BM CD34+CD38- cells from refractory AML patients with and without VEGF-C measured by flow cytometry (N=7; **P<0.01; Mann–Whitney U test with two-sided P values). (E) Confocal microscopic images showing enrichment of FLT4 in the cytosolic region of BM mononucelar cells collected from refractory patients by treatment with VEGF-C. Haematologica | 108 November 2023

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cells was higher in all leukemia groups compared to the normal donors (Figure 6B; *P<0.05; **P<0.01). These data demonstrate that higher concentrations of VEGF-C and higher numbers of FLT4+ AML blasts are correlated with AML status, regardless of treatment, compared to normal donors. Based upon this correlation between VEGF-C and FLT4, we evaluated the effects of FLT4 expression on AML blasts at the time of diagnosis on achievement of CR after chemotherapy. Using the BM taken at the time of diagnosis, the frequency of FLT4 in AML blasts was compared between the patients who later became CR or refractory following standard induction chemotherapy. Intriguingly, refractory patients showed higher expression of cytosolic FLT4 than CR patients. We then measured the VEGF-C mRNA levels in the BM-MNC, taken at the time of diagnosis. The mRNA expression was also higher in the refractory group compared to the CR group (Figure 6C; *P<0.05). Meanwhile, the level of VEGF-C protein showed inconsistent results, compared to the results of mRNA levels (data not shown). The discrepancy led us to consider the internalization of VEGF-C in refractory patients. In such a case, cells take up secreted VEGF-C, resulting in a low external protein concentration. In addition, together with the results of reduced FLT4 expression on the surface of AML blasts in the refractory patients, we further thought that VEGF-C may also internalize its receptor, FLT4, leading to simultaneous reduction of external VEGF-C and surface FLT4. If this is the case, cytosolic FLT4 should become high. In order to test this hypothesis in in vitro experiments, we next isolated CD34+CD38- cells from BM of patients who later became refractory, treated them with or without VEGF-C, and performed flow cytometry for FLT4 expression in the cytosol. The results showed that FLT4 expression in CD34+CD38- cells was increased in the cytosol in the presence of VEGF-C in the BM cells (Figure 6D; *P<0.05; **P<0.01). Also, confocal microscopy was conducted using BM–MNC from refractory patients. Without VEGF-C exposure, the density of FLT4 was higher on the cell membrane than in the cytosolic region. With VEGF-C exposures, the density of FLT4 became higher in the cytosol, indicating internalization of FLT4 (Figure 6E). Taken together, newly diagnosed AML patients showing a high percentage of cytosolic FLT4+AML cells in the BM are likely to be refractory after chemotherapy, suggesting FLT4 as a diagnostic marker for refractory status in AML patients. We then explored how this FLT4 internalization could increase survival of LSC by performing western blotting using FLT4-expressing Jurkat cells. The results showed that VEGF-C induced phosphorylation of FLT4 and its downstream molecules, PI3K and AKT (Online Supplementary Figure S5). These data suggest that activation of FLT4 signaling through the PI3K/AKT-pathway can protect leukemic blasts in the presence of VEGF-C in refractory patients.

Discussion Despite extensive research focused on identifying LSC based on a CD34+CD38- phenotype,35,36 neither an effective marker nor their clinical significance for LSC involving refractory status has yet been determined due to CD34+CD38- cells as a broad pan marker. Here we have made two important discoveries regarding the role of FLT4 in CD34+CD38- AML blasts. First, FLT4-expressing CD34+CD38- (FLT4+ AML) cells can function as AML-LSC and their function can be suppressed by inhibition of FLT4. These findings open up a new option for treating AML by targeting FLT4 on CD34+CD38- AML blasts, especially in refractory patients. Second, FLT4 internalization in AML blasts under VEGF-C in the BM plays a crucial role in drug resistance in refractory patients. High cytosolic FLT4 expression in AML blasts at the time of diagnosis is associated with refractory status in AML; whereas relatively high FLT4 surface expression with low cytosolic FLT4 expression in AML blasts is related to CR owing to blast destruction by Ara-C treatment. This internalization of FLT4 under VEGF-C can protect AML blasts by activating cell survival signaling. Such paradoxical FLT4 expression suggests prognostic implications for predicting refractory patients at the time of diagnosis. Our study provides evidence that FLT4+ AML blasts can represent more specific CD34+CD38- LSC. It has been known that the VEGF-C/FLT4 axis plays a pivotal role in the invasion and metastasis of solid tumor cells37 and in regenerating lymphatic vessels at the postnatal stage.38 Interestingly, in AML BM plasma, VEGF-C concentration is increased and the level is correlated with poor clinical outcomes.17,18 In addition, FLT4, a major receptor for VEGFC, is expressed in AML blasts.17,22 Dias et al. also demonstrated that VEGF-C promotes blast survival through FLT4 and KDR, through paracrine and autocrine mechanisms, respectively.17,39 However, the role of FLT4 expression in CD34+CD38- cells has been elusive. In normal BM, FLT4 is expressed on SEC, but not on hematopoietic cells.30 We found that high levels of FLT4 and VEGF-C were restricted in CD45dim blast cells, but not in normal HSC in the BM, suggesting the association of FLT4 with CD34+CD38- cells. Our results also showed that FLT4+ AML blasts are still present even in the CR patients, who were treated with Ara-C. In addition, LAIP in FLT4+ cells in the BM is high, suggesting MRD even in CR status (Figure 5A).26,27 In relation to this, the multi-drug resistance (MDR)-1 gene was more highly expressed in FLT4+ cells than FLT4- cells among AML blasts, indicating drug refractoriness of FLT4+ cells (data not shown). These data suggest that therapies targeting FLT4+ AML blasts would help reduce MRD in AML. Animal and in vitro experiments provide direct evidence that FLT4+ cells play a key role in generating leukemia. FLT4+ AML blasts homed and engrafted to mouse BM more

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efficiently than FLT4- AML blasts, and FLT4+ AML blasts generated leukemic colonies more robustly in cultures than FLT4- AML blasts. Lastly, the flow-MRD detection by FLT4+CD7+ aberrant cell population in CR patients with AML should be a very good approach in a real clinical setting, as shown in Figure 5A. Our study further proved that FLT4 is not only a marker but functions as a key regulator to endow refractory leukemic properties. in vitro, FLT4 inhibition in AML blasts derived from AML patients reduced leukemic colony-forming activities. Specific inhibition of FLT4 by MAZ51 in AML blasts led to low homing and engraftment efficiencies of CD34+CD38- cells in the CD45dim blast population. FLT4 inhibition in FLT4+ AML blasts suppressed activities of AML blasts. This further implies that inhibition of FLT4 by MAZ51 can interrupt leukemia induction without damage to normal hematopoiesis. We also demonstrated effects of MAZ51 with Ara-C on AML blasts apoptosis under VEGFC, suggesting that the survival of AML blasts involves FLT4 binding by VEGF-C. The identification of the role of FLT4+ AML blasts in leukemogenecity further led to discovery of the role of FLT4 internalization in conferring refractory status to conventional chemotherapy. We found that a relatively low surface and high cytosolic FLT4+ AML blast at the time of diagnosis was associated with later refractory status among AML patients. Although individual variations existed, this paradox was noted in refractory patients compared to CR patients. Most receptor tyrosine kinases such as FLT4 bind to their ligands on the cell membrane and the ligand-receptor complex undergoes internalization, leading to complex signaling and cellular activities. Studies have shown that internalization of VEGF-C/FLT4 is required for lymphatic endothelial cell survival and proliferation.40,41 FLT4 internalization activates the PI3K-AKT pathway, which is crucial for survival of many types of cells.40,42,43 Such internalization occurs in FLT4+ AML blasts in BM while maintaining higher levels of VEGF-C mRNA. This persistent internalization could be a hallmark for CD34+CD38- cells and a low ratio of surface to cytosol FLT4 could represent a prognostic marker for refractory patients. Moreover, this mechanistic insight supports the idea that new therapeutic approaches are required for targeting the VEGF-C/FLT4 pathway, particularly for refractory patients. We also examined prognosis between 84 patients with AML in terms of high and low expression of FLT4 at initial diagnosis. The preliminary analysis displayed lower overall survival rates in the high FLT4 expression group, compared to that of the low FLT4 expression group (data not shown). Taken together, these results implied the

potential of FLT4 as a prognostic indicator. However, it cannot definitively represent the ultimate outcome due to the diversity of individual variations in patients with AML. We will investigate the clinical correlation of prognosis based on a large number of patients by integrating various variables with FLT4 expression in patients with AML. There are some limitations of this study. It remains to be determined how internalization could be sustained in AML blasts. The cutoff value for the surface/cytosolic FLT4 ratio needs to be investigated before it can be used for prognostic stratification. This would further help to determine the candidate patients who need anti-VEGF-C or anti-FLT4 therapies. No doubt, such a therapeutic and prognostic strategy needs to be tested prospectively in the future. Nevertheless, this study demonstrated that the VEGFC/FLT4 axis plays a crucial role for defining refractoriness and has a biological function in leukemogenesis. This novel insight will be useful for prognostic stratification and targeted therapy for AML. Disclosures No conflicts of interest to disclose. Contributions JYL, SEK, ARH and JL performed experiments and analyzed data. JYL, Y-sY and HJK conceived, designed research, analyzed data, interpreted data, and wrote the manuscript. Acknowledgments We would like to thank Curt Donghyun for the initial steps of this project, Hee-Sun Hwang and Gi June Min for research assistance, and the Integrative Research Support Center of the Catholic University of Korea for technical assistance with this project. Funding This work was supported by a Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (grant numbers 2017R1D1A1B03031406 and 2015R1D1A1A01059819); National Research Foundation of Korea (NRF) funded by the Korea government (MSIT) (grant numbers 2020R1A2C3003784, 2020M3A9I4038454 and 2021R1A2C1004571); the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare (HI16C2211); and NHLBI (R01HL150887). Data-sharing statement Data generated during this study are available from the corresponding author upon reasonable request.

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References 1. Ferrara F, Schiffer CA. Acute myeloid leukaemia in adults. Lancet. 2013;381(9865):484-495. 2. Thol F, Schlenk RF, Heuser M, et al. How I treat refractory and early relapsed acute myeloid leukemia. Blood. 2015;126(3):319-327. 3. Yilmaz M, Wang F, Loghavi S, et al. Late relapse in acute myeloid leukemia (AML): clonal evolution or therapy-related leukemia? Blood Cancer J. 2019;9(2):7. 4. Jongen-Lavrencic M, Grob T, Hanekamp D, et al. Molecular minimal residual disease in acute myeloid leukemia. N Engl J Med. 2018;378(13):1189-1199. 5. Karantanos T, Jones RJ. Acute myeloid leukemia stem cell heterogeneity and its clinical relevance. Adv Exp Med Biol. 2019;1139:153-169. 6. Kaipainen A, Korhonen J, Mustonen T, et al. Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. Proc Natl Acad Sci U S A. 1995;92(8):3566-3570. 7. Kukk E, Lymboussaki A, Taira S, et al. VEGF-C receptor binding and pattern of expression with VEGFR-3 suggests a role in lymphatic vascular development. Development. 1996;122(12):3829-3837. 8. Kerjaschki D, Huttary N, Raab I, et al. Lymphatic endothelial progenitor cells contribute to de novo lymphangiogenesis in human renal transplants. Nat Med. 2006;12(2):230-234. 9. Salven P, Mustjoki S, Alitalo R, et al. VEGFR-3 and CD133 identify a population of CD34+ lymphatic/vascular endothelial precursor cells. Blood. 2003;101(1):168-172. 10. Thiele W, Krishnan J, Rothley M, et al. VEGFR-3 is expressed on megakaryocyte precursors in the murine bone marrow and plays a regulatory role in megakaryopoiesis. Blood. 2012;120(9):1899-1907. 11. Srinivasan RS, Escobedo N, Yang Y, et al. The Prox1-Vegfr3 feedback loop maintains the identity and the number of lymphatic endothelial cell progenitors. Genes Dev. 2014;28(19):2175-2187. 12. Han J, Calvo CF, Kang TH, et al. Vascular endothelial growth factor receptor 3 controls neural stem cell activation in mice and humans. Cell Rep. 2015;10(7):1158-1172. 13. Lee JY, Park C, Cho YP, et al. Podoplanin-expressing cells derived from bone marrow play a crucial role in postnatal lymphatic neovascularization. Circulation. 2010;122(14):1413-1425. 14. Schoppmann SF, Birner P, Stockl J, et al. Tumor-associated macrophages express lymphatic endothelial growth factors and are related to peritumoral lymphangiogenesis. Am J Pathol. 2002;161(3):947-956. 15. Fielder W, Graeven U, Ergun S, et al. Expression of FLT4 and its ligand VEGF-C in acute myeloid leukemia. Leukemia. 1997;11(8):1234-1237. 16. de Jonge HJ, Weidenaar AC, Ter Elst A, et al. Endogenous vascular endothelial growth factor-C expression is associated

with decreased drug responsiveness in childhood acute myeloid leukemia. Clin Cancer Res. 2008;14(3):924-930. 17. Dias S, Choy M, Alitalo K, et al. Vascular endothelial growth factor (VEGF)-C signaling through FLT-4 (VEGFR-3) mediates leukemic cell proliferation, survival, and resistance to chemotherapy. Blood. 2002;99(6):2179-2184. 18. de Jonge HJ, Valk PJ, Veeger NJ, et al. High VEGFC expression is associated with unique gene expression profiles and predicts adverse prognosis in pediatric and adult acute myeloid leukemia. Blood. 2010;116(10):1747-1754. 19. Kirkin V, Mazitschek R, Krishnan J, et al. Characterization of indolinones which preferentially inhibit VEGF-C- and VEGF-Dinduced activation of VEGFR-3 rather than VEGFR-2. Eur J Biochem. 2001;268(21):5530-5540. 20. Kirkin V, Thiele W, Baumann P, et al. MAZ51, an indolinone that inhibits endothelial cell and tumor cell growth in vitro, suppresses tumor growth in vivo. Int J Cancer. 2004;112(6):986-993. 21. Han AR, Lee JE, Lee MJ, et al. Distinct repopulation activity in Hu-mice between CB- and LPB-CD34(＋) cells by enrichment of transcription factors. Int J Stem Cells. 2021;14(2):203-211. 22. Lee JY, Park S, Kim DC, et al. A VEGFR-3 antagonist increases IFN-gamma expression on low functioning NK cells in acute myeloid leukemia. J Clin Immunol. 2013;33(4):826-837. 23. Matsushita H, Nakajima H, Nakamura Y, et al. C/EBPalpha and C/EBPvarepsilon induce the monocytic differentiation of myelomonocytic cells with the MLL-chimeric fusion gene. Oncogene. 2008;27(53):6749-6760. 24. Miyamoto T, Weissman IL, Akashi K. AML1/ETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 8;21 chromosomal translocation. Proc Natl Acad Sci U S A. 2000;97(13):7521-7526. 25. Lacombe F, Durrieu F, Briais A, et al. Flow cytometry CD45 gating for immunophenotyping of acute myeloid leukemia. Leukemia. 1997;11(11):1878-1886. 26. Feller N, van der Pol MA, van Stijn A, et al. MRD parameters using immunophenotypic detection methods are highly reliable in predicting survival in acute myeloid leukaemia. Leukemia. 2004;18(8):1380-1390. 27. Paietta E. Minimal residual disease in acute myeloid leukemia: coming of age. Hematology Am Soc Hematol Educ Program. 2012;2012:35-42. 28. Salter AB, Meadows SK, Muramoto GG, et al. Endothelial progenitor cell infusion induces hematopoietic stem cell reconstitution in vivo. Blood. 2009;113(9):2104-2107. 29. Lee JY, Park S, Min WS, et al. Restoration of natural killer cell cytotoxicity by VEGFR-3 inhibition in myelogenous leukemia. Cancer Lett. 2014;354(2):281-289. 30. Hooper AT, Butler JM, Nolan DJ, et al. Engraftment and reconstitution of hematopoiesis is dependent on VEGFR2mediated regeneration of sinusoidal endothelial cells. Cell Stem Cell. 2009;4(3):263-274.

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31. Kiel MJ, Yilmaz OH, Iwashita T, et al. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell. 2005;121(7):1109-1121. 32. Ishikawa F, Yoshida S, Saito Y, et al. Chemotherapy-resistant human AML stem cells home to and engraft within the bonemarrow endosteal region. Nat Biotechnol. 2007;25(11):1315-1321. 33. Krause DS, Fulzele K, Catic A, et al. Differential regulation of myeloid leukemias by the bone marrow microenvironment. Nat Med. 2013;19(11):1513-1517. 34. Morrison SJ, Scadden DT. The bone marrow niche for haematopoietic stem cells. Nature. 2014;505(7483):327-334. 35. Lapidot T, Sirard C, Vormoor J, et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature. 1994;367(6464):645-648. 36. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med. 1997;3(7):730-737. 37. Su JL, Yang PC, Shih JY, et al. The VEGF-C/Flt-4 axis promotes invasion and metastasis of cancer cells. Cancer Cell. 2006;9(3):209-223.

38. Karkkainen MJ, Haiko P, Sainio K, et al. Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins. Nat Immunol. 2004;5(1):74-80. 39. Dias S, Hattori K, Zhu Z, et al. Autocrine stimulation of VEGFR-2 activates human leukemic cell growth and migration. J Clin Invest. 2000;106(4):511-521. 40. Wang Y, Nakayama M, Pitulescu ME, et al. Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis. Nature. 2010;465(7297):483-486. 41. Nakayama M, Nakayama A, van Lessen M, et al. Spatial regulation of VEGF receptor endocytosis in angiogenesis. Nat Cell Biol. 2013;15(3):249-260. 42. Makinen T, Veikkola T, Mustjoki S, et al. Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3. EMBO J. 2001;20(17):4762-4773. 43. Kim SO, Trau HA, Duffy DM. Vascular endothelial growth factors C and D may promote angiogenesis in the primate ovulatory follicle. Biol Reprod. 2017;96(2):389-400.

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ARTICLE - Acute Myeloid Leukemia

Molecular targeting of the UDP-glucuronosyltransferase enzymes in high-eukaryotic translation initiation factor 4E refractory/relapsed acute myeloid leukemia patients: a randomized phase II trial of vismodegib, ribavirin with or without decitabine Sarit Assouline,1 Jadwiga Gasiorek,2 Julie Bergeron,3 Caroline Lambert,2 Biljana CuljkovicKraljacic,2 Eftihia Cocolakis,1 Chadi Zakaria,1 David Szlachtycz,1 Karen Yee4 and Katherine L.B. Borden2 Jewish General Hospital and McGill University, Montreal, Quebec; 2Institute for Research in Immunology and Cancer and Department of Pathology and Cell Biology, University of Montreal, Montreal, Quebec; 3CEMTL Installation Maisonneuve Rosemont, Montreal, Quebec and 4Princess Margaret Cancer Centre, Division of Medical Oncology and Hematology, Toronto, Ontario, Canada

Correspondence: S. Asssouline sarit.assouline@mcgill.ca K.Borden katherine.borden@umontreal.ca

Received: Accepted: Early view:

January 24, 2023. March 16, 2023. March 23, 2023.

Abstract Drug resistance underpins poor outcomes in many malignancies including refractory and relapsed acute myeloid leukemia (R/R AML). Glucuronidation is a common mechanism of drug inactivation impacting many AML therapies, e.g., cytarabine, decitabine, azacytidine and venetoclax. In AML cells, the capacity for glucuronidation arises from increased production of the UDP-glucuronosyltransferase 1A (UGT1A) enzymes. UGT1A elevation was first observed in AML patients who relapsed after response to ribavirin, a drug used to target the eukaryotic translation initiation factor eIF4E, and subsequently in patients who relapsed on cytarabine. UGT1A elevation resulted from increased expression of the sonic-hedgehog transcription factor GLI1. Vismodegib inhibited GLI1, decreased UGT1A levels, reduced glucuronidation of ribavirin and cytarabine, and re-sensitized cells to these drugs. Here, we examined if UGT1A protein levels, and thus glucuronidation activity, were targetable in humans and if this corresponded to clinical response. We conducted a phase II trial using vismodegib with ribavirin, with or without decitabine, in largely heavily pre-treated patients with high-eIF4E AML. Pre-therapy molecular assessment of patients’ blasts indicated highly elevated UGT1A levels relative to healthy volunteers. Among patients with partial response, blast response or prolonged stable disease, vismodegib reduced UGT1A levels, which corresponded to effective targeting of eIF4E by ribavirin. In all, our studies are the first to demonstrate that UGT1A protein, and thus glucuronidation, are targetable in humans. These studies pave the way for the development of therapies that impair glucuronidation, one of the most common drug deactivation modalities. Clinicaltrials.gov: NCT02073838.

Introduction Drug resistance remains a major challenge in the treatment of many malignancies and is responsible for reduced overall survival in settings such as refractory and relapsed (R/R) acute myeloid leukemia (AML). Clinical response following initial chemotherapy for most AML patients is about one year.1-4 After first relapse, outcomes are worse, with overall survival (OS) lasting approximately 4-5 months and at second relapse approximately two months with chemotherapy.5,6 The outlook is similarly dismal at first relapse

with venetoclax plus hypomethylating agents (HMA), with a median OS of about 2.4 months.4 By understanding the molecular bases of clinical resistance, it is possible to design new therapeutic strategies to overcome them. Drivers of clinical resistance include impaired drug entry into the cell, inactivation of drugs through chemical modification, genetic re-wiring, and/or enhanced efflux of drugs from cells.7 These events can elicit multi-drug resistance even to therapies for which there were no prior exposures, which thereby impacts outcomes of subsequent regimens. For example, the commonly used AML drugs cytarabine,

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ARTICLE - Targeting of UGT1A correlates with response in AML azacytidine and decitabine all employ the equilibrative nucleoside transporter 1 (ENT1) to enter AML cells, and reduced ENT1 levels elicit resistance to all these drugs8-11 (Figure 1). One of the most common forms of drug resistance in pharmacology is the covalent addition of glucuronic acid to drugs, which results in inactivation, thereby driving drug resistance. Glucuronidation occurs both in the liver and extrahepatically,12-15 and AML cells can develop the capacity to glucuronidate multiple drugs simultaneously via elevated levels of UDP-glucuronosyltransferase 1A (UGT1A) enzymes.8,16 These events occur independently of hepatic glucuronidation. Glucuronidation impacts approximately 50% of prescribed drugs including cytarabine, venetoclax, decitabine and azacytidine8,12,16-18 (Figure 1). These observations highlight the importance of developing inhibitors that overcome drug resistance to produce durable responses in R/R AML patients. This ability of AML blasts to glucuronidate drugs was first revealed during clinical studies targeting the eukaryotic translation initiation factor eIF4E with ribavirin.8,12,16,17 eIF4E is elevated in a subset of de novo and R/R AML patients, and its elevation alone is correlated with worse outcomes.19 High-eIF4E AML specimens are typically characterized by highly elevated and often nuclear-enriched eIF4E.20-22 Here, eIF4E drives the production of factors that support malignancy through its impacts on several nuclear RNA metabolism steps, including capping, splicing, and RNA export, as well as on translation in the cytoplasm

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through association with their m7G RNA cap.19,20,23-29 In this way, eIF4E serves as an exemplar for targeting RNA metabolism in malignancies. Ribavirin acts as a m7G RNA cap competitor, and thereby represses the biochemical and oncogenic activities of eIF4E.8,30-42 In a phase II ribavirin monotherapy and subsequent ribavirin plus low-dose cytarbine (LDAC) phase I/II trial, treatments reduced higheIF4E AML cell numbers and re-localized eIF4E from the nucleus to the cytoplasm, corresponding to repressed oncogenic capacity and objective clinical responses, including complete remissions.21,22,43 Ribavirin also targeted eIF4E in other cancers, including those of the prostate and head and neck.44,45 Despite robust responses, all patients relapsed on these trials. We noted at relapse the emergence of populations of high-UGT1A cells.8,16 This correlated with increased glucuronidation and subsequent deactivation of ribavirin and cytarabine in cells.8,16 Some specimens additionally had reduced ENT1 levels, and thus impaired uptake of ribavirin and cytarabine, indicative of the development of multiple forms of resistance simultaneously.8,21,22 High-UGT1A cell populations also emerged at relapse in patients treated with standard of care (cytarabine with an anthracycline), indicating that glucuronidation-driven resistance is not restricted to ribavirin-based therapies.8 This glucuronidation led to drug resistance to several AML drugs, including cytarabine, venetoclax, azacytidine and decitabine.8,16 Increased UGT1A protein levels arose due to elevation of the

Figure 1. Schematic of forms of drug resistance relevant to this trial. Glucuronidation of ribavirin (Rib) and decitabine (Dec) is indicated by red boxes; the ENT1 transporter is depicted as the purple channel. While not shown, drug resistance can also occur via the simultaneous loss of ENT1 and elevated UGT1A (and thus increased glucuronidation). Model created using Biorender. Haematologica | 108 November 2023

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ARTICLE - Targeting of UGT1A correlates with response in AML hedgehog transcription factor GLI1, but can likely occur by other means as well.8,16 GLI1-inducible glucuronidation was repressed by vismodegib which indirectly targets GLI1 via the Smoothened extracellular receptor (SMO).8,16 Vismodegib reduced UGT1A levels, decreased glucuronidation, and resensitized cell lines to these drugs.8,16 We sought to examine the capacity of vismodegib to lower UGT1A levels, and thereby reduce drug glucuronidation, in largely heavily pre-treated high-eIF4E AML patients in a phase II clinical trial. Given that vismodegib binds an extracellular receptor, it escapes cellular glucuronidation, providing an additional rationale for its use here. We also included decitabine in our combination, as ribavirin combined with HMA provided better inhibition of high-eIF4E AML patient specimen growth ex vivo than either agent alone.32 Finally, both ribavirin and decitabine employ the ENT1 drug transporter,8,11-13,46 and thus we ensured patients entered the trial with active ENT1. This also enabled us to study flux in ENT1 levels as a predictor of relapse in these patients in parallel with monitoring UGT1A levels. In all, we sought to develop strategies to target heavily pre-treated AML patients focusing on UGT1A. Our studies demonstrated for the first time that glucuronidation can be targeted in humans and that this is associated with clinical benefit.

Methods Study design This was a multi-center, open-label, randomized phase II study of ribavirin and vismodegib with or without decitabine in AML. The primary objective was to determine the efficacy of ribavirin, vismodegib with or without decitabine using overall response rate defined as the rate of complete remission (CR), complete remission with incomplete blood count recovery (CRi), partial remission (PR), morphologic leukemia-free state (MLFS) or blast response (BR). Correlative studies were included to assess relevant molecular targets. Patients randomized to the VRD arm (vismodegib/ribavirin/decitabine) were administered decitabine at 20 mg/m2 intravenously daily on days -7 to -3 for cycle 1 and days 1 to 5 on subsequent cycles. Ribavirin at 1400 mg orally was taken twice daily and vismodegib was taken orally at 150 mg once a day starting on day 1. Treatment cycles were 28 days long. Patients randomized to the VR arm (vismodegib/ribavirin) received ribavirin 1400 mg twice a day and vismodegib 150 mg once a day. Ribavirin and decitabine were donated by Pharmascience Inc., Montreal, Quebec, Canada; vismodegib was donated by Hoffmann-La Roche Ltd., Mississauga, Ontario, Canada. A run-in phase of three patients in each arm was performed to ensure both the safety of these combinations and adequate ribavirin plasma levels. Based on the respective

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toxicity profiles, no safety signal was observed and adequate plasma levels of ribavirin were found. The VR arm of the trial was terminated early due to futility as per the Simon two-stage design (see Online Supplementary Methods). Enrolment on the VRD arm closed prior to reaching 21 participants due to overall challenges in recruiting evaluable participants during the COVID pandemic and the understanding that the treatment landscape for AML was evolving. Patient selection Patients at least 18 years of age with AML were eligible to participate in this study. All patients must have failed primary therapy (defined as two induction chemotherapies), must have relapsed, or must not have been suitable candidates for intensive induction chemotherapy. All patients reviewed and signed an appropriate informed consent which had been approved by the institutional review boards (CIUSSS West-Central Montreal Research Review Office, approval 14-046, and University Health Network Research Ethics Board, approval 15-9586C) and Health Canada in accordance with the Declaration of Helsinki. Eligibility criteria are detailed in the Online Supplementary Methods. Acute myeloid leukemia primary specimens Blasts were isolated from peripheral blood or bone marrow by flow cytometry using forward and side scatter and CD45, as described previously.22 Cells were sorted on a BD FACSAria flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). For comparison, normal CD34+ cells were obtained from Lonza (Hayward, CA, USA) or ATCC (Manassas, VA, USA). Immunofluorescence Immunostaining of cells with eIF4E (BD Biosciences, Mississauga, ON, Canada) and UGT1A antibodies (Antibodiesonline, further purified in-house) is described in detail in the Online Supplementary Methods, along with a description of the purification of the commercial UGT1A antibody to yield a single band on western blot. Western blotting, quantitative polymerase chain reaction and RNA interference Detailed methodology for immunoblotting, RNAi and reverse transcription-quantitative polymerase chain reaction (RTqPCR), including antibodies, primers and RNAi sequences used, is presented in the Online Supplementary Methods.

Results Patients' characteristics and clinical results Between May 2015 and February 2021, 23 patients were enrolled onto the study. To ascertain eligibility, we per-

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ARTICLE - Targeting of UGT1A correlates with response in AML formed molecular screening on 47 patients, examining AML blasts isolated as described.22 For inclusion, patients’ blasts had to have ≥3-fold elevated eIF4E levels versus CD34+ cells from healthy volunteers as described,21,22 and functional ENT1 as assessed by 3H-ribavirin uptake equivalent to, or higher than, CD34+ cells from healthy volunteers. Fourteen patients failed molecular screening: seven due to impaired ribavirin uptake, two without elevated eIF4E, and five due to insufficient material to screen (Online Supplementary Figure S1). Baseline patients' characteristics for the enrolled patients are detailed in Table 1. Median age was 65 years (range 28-85), patients were

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heavily pre-treated with 43% of patients having received >3 lines of therapy and 52% of the patients having failed primary induction. Testing for FLT3-ITD and NPM1 mutations was performed in all patients and were found in 30% and 35% of the patients, respectively. Post-hoc next generation sequencing (NGS) performed on ten patient baseline samples, did not identify TP53 mutations (see Table 1 for list of mutations). The median duration of treatment was 1.6 months (range 0.4-10.4). The most common treatment-emergent adverse events regardless of causality were febrile neutropenia (65%; grade ≥3: 65%), nausea (61%; grade ≥3: 9%), diarrhea (52%; grade ≥3: 4%), vomiting

Table 1. Patients' baseline characteristics. Total

Decitabine+VR

65 (28-85)

61 (42-85)

67 (28-72)

Sex, N Female Male

6 17

5 10

1 7

ECOG Performance Status, N 0 1 2

5 15 3

3 9 3

2 6 0

WHO classification*, N Defining genetic abnormalities NPM1 NUP98 CEBPA Myelodysplasia-related Defining by differentiation

20 8 1 1 10 3

13 6 0 0 7 2

7 2 1 1 3 1

FLT3-ITD, N

Next generation sequencing (N=10) Myelodysplasia-related, N ASXL1 RUNX1 SRSF2 Other**, N

1 2 2 5

1 1 1 5

0 1 1 0

Response to primary therapy, N Primary induction failure Relapsed ≥ 6 mth post induction Not eligible for induction***

12 6 5

6 5 4

6 1 1

Prior treatment for AML, N No prior treatment for AML**** 1 prior line 2 prior lines ≥3 prior lines

3 4 6 10

2 3 4 6

1 1 2 4

Prior allotransplant, N

Prior HMA, N For AML For myeloproliferative disorder*****

11 9 3

9 6 3

3 3 0

N of patients Median age in years (range)

N: number; ECOG: Eastern Cooperative Oncology Group; WHO: World Health Organization; V: vismodegib; R: ribavirin; mth: months. *WHO classification was determined at relapse. **Other mutations found: CBL, IDH2, JAK2, PTPN11, SETBP1. ***Includes patients that were treated on a clinical trial, had no prior treatment for acute myeloid leukemia (AML) or received a hypomethylating (HMA) agent. ****2/3 patients were previously treated with a HMA for myelodysplastic syndromes (MDS). *****2/3 patients received a HMA for MDS and 1/3 received a HMA for myelofibrosis. Haematologica | 108 November 2023

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ARTICLE - Targeting of UGT1A correlates with response in AML (48%; grade ≥3: 0%), and fatigue (43%; grade ≥3: 13%) (Online Supplementary Table S1). Seventeen patients were evaluable for clinical response, 16 of whom were heavily pre-treated for AML and/or myelodysplastic syndromes with a median of three prior therapies for the VRD arm and two for the VR arm; 8/17 patients relapsed from prior HMA, 1/17 prior venetoclax plus HMA, and 15/17 prior cytarabine. Overall, 4/10 patients in the VRD arm achieved objective responses: one PR and three BR (treatment range 5-10 cycles); two durable SD (treatment range 4-6 cycles); two SD and two PD (Table 2). Median time to response was 2.2 months (range 1.7-3.6). In comparison to an earlier study with decitabine alone, median time to response was 157 days (5.2 months) and only untreated or patients receiving second-line therapy responded.47 Thus, decitabine alone is not likely to have driven the observed responses in this trial given the short time to response, but rather the HMAribavirin combination was associated with response, as observed in ex vivo studies.32 Among responding patients, 4/6 had relapsed while on HMA, suggesting re-sensitization to decitabine by vismodegib and/or added benefit by targeting eIF4E with ribavirin. Responses in the VR arm were 3/7 SD and 4/7 PD, and this arm was closed. Pharmacokinetic studies indicated that vismodegib and ribavirin were not affected by decitabine exposure (Online Supplementary Figure S2). Molecular assessment reveals correlation between UGT1A and eIF4E targeting and clinical response To study the impact of glucuronidation in both treated and treatment naïve patients, we examined UGT1A protein levels relative to healthy volunteers (Figure 2). For this purpose, we employed immunofluorescence and confocal laser microscopy (IFCLM). The commercially available panUGT1A antibodies available were found to have extraneous

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bands and thus were further affinity purified prior to use. The resulting antibody revealed a single band on the western blot, and RNAi reduction using a pan-UGT1A RNAi versus luciferase RNAi controls demonstrated reduced levels in the endogenous UGT1A band in liver HepG2 cells, thereby indicating the antibody is specific (Figure 2A). Actin indicates equal protein loading and is not impacted by the RNAi, as expected. Consistent with previous studies, we observed that UGT1A levels were elevated in high-eIF4E AML THP-1 cells over-expressing GLI1 versus vector controls using IFCLM and western blot, and that addition of vismodegib reduced both GLI1 and UGT1A, further validating this purified antibody8,16 (Figure 2B, C). For patient specimens, we observed that our heavily pretreated cohort was characterized by elevated UGT1A prior to exposure to study drugs relative to healthy volunteers (Figure 2D). For comparison, eIF4E staining is shown for some of the same specimens and demonstrates there is no correlation between eIF4E and UGT1A levels; it also confirms elevated eIF4E levels (Figure 2D). Interestingly, 3/4 treatment naïve AML patients (not participants in this trial) had elevated UGT1A at baseline, potentially predicting future treatment failure. Our previous studies demonstrated that the other major UGT family, UGT2B, were not elevated in the AML patients examined and thus were not monitored here.8 In all, patients in our trial were characterized by elevated UGT1A levels as well as elevated eIF4E levels relative to healthy volunteers. We next measured median intensity of UGT1A protein levels to ascertain whether levels decreased in patients upon treatment with vismodegib and if that corresponded to ribavirin targeting of eIF4E (Table 3). We note that only 16/17 evaluable patients had material available for analysis. Staining was quantified on a per cell basis using FIJI measuring more than 35 cells per condition. Median intensities ± Standard Error of Mean (SEM) at best molecular re-

Table 2. Efficacy in treated patients. Total

Decitabine+VR

1 3 7 6 6 23.5

1 3 4 2 5 40

0 0 3 4 1 0

Time to best response in months, median (min-max)

2.2 (1.7-3.6)

Overall survival in months, median (min-max)

4.6 (0.7-16.5)

3.6 (0.7-16.5)

6.6 (0.8-9.3)

Time on study in months, median (min-max)

1.9 (0.7-10.5)

2.5 (0.7-10.5)

1.2 (0.7-2.1)

Duration of treatment in months, median (min-max)

1.6 (0.4-10.4)

1.9 (0.4-10.4)

1.1 (0.5-1.8)

Best overall response PR, N BR, N SD, N PD, N NE, N ORR, %

V: vismodegib; R: ribavirin; PR: partial remission; BR: blast response; SD: stable disease; PD: progressive disease; NE: not evaluable; ORR: overall response rate; NA: not applicable. Haematologica | 108 November 2023

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Figure 2. Characterization of UGT1A levels in cells and primary acute myeloid leukemia specimens. (A) UGT1A antibody purification is described in the Online Supplementary Methods. The resulting antibody revealed a single band when monitoring endogenous UGT1A in liver HepG2 cells on the western blot with reduction for pan-UGT1A RNAi versus luciferase RNAi controls. Actin blot is provided for loading. Pan-UGT1A antibody purified in (A) recognized elevated UGT1A in THP-1 cells over-expressing GLI1 as assessed both by western blot (B) and confocal microscopy (C). In addition, UGT1A levels are reduced by vismodegib in these cells (B). (D) UGT1A are elevated in AML patients, including some treatment naïve patients, relative to healthy volunteers. UGT1A levels were observed to be elevated in all the AML patient specimens examined including 3/4 treatment naïve AML patients relative to healthy volunteers. Scale bar: 10 µm.

sponse (BMR) and end of treatment (EOT) (or last available sample [LAS] if EOT was not available) were relative to before treatment (BT) which was set to 1. For patients on trial for one cycle, the same value is provided as both BMR and EOT/LAS relative to BT. Importantly, neither vismodegib nor

HMA impact eIF4E activity or levels.8,32 We observed a median 3.1-fold reduction in UGT1A and median 3.8-fold reduction in eIF4E levels relative to BT in patients who achieved PR, BR or durable SD (patients B-001, A-008, C002, B-004, A-011, C-003) (Table 3, Figures 3 and 4). As an

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ARTICLE - Targeting of UGT1A correlates with response in AML example, patient B-004 bone marrow blasts had a 6.25fold and 10-fold reduction in eIF4E and UGT1A levels, respectively, at BMR relative to BT and this correlated with reduction of blast count to <10% (Table 3, Figure 3). During response, patients generally had re-localization of eIF4E to the cytoplasm, as we had observed in our previous ribavirin trials, whereby eIF4E entry into the nucleus is prevented by ribavirin interference with eIF4E interaction with its importin.21,22,43 At relapse, eIF4E and UGT1A levels were elevated, nearing BT levels, which corresponded with increased blasts, and increased eIF4E levels and its nuclear re-entry were evident, as observed in our previous trials (Table 3, Figures 3 and 4). In parallel, we measured ENT1 and adenosine kinase (ADK) since its loss is also associated with ribavirin resistance.8,48 ENT1 and ADK RNA levels were measured using RT-qPCR and the largest reduction is shown relative to BT set at 1 (Table 3, Online Supplementary Table 2). We observed that 2/6 of these patients (C-002 and

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C-003) had reduced ENT1 levels which likely contributes to drug resistance in parallel to elevation of UGT1A relative to BT. We note that ribavirin did not impact on UGT1A protein expression8,49 and, moreover, patients in both the VR and VRD arms achieved reduced UGT1A levels, indicating that this reduction was not driven by decitabine (Table 3). For patients with short SD, most achieved targeting of either UGT1A or eIF4E, or only very transient targeting of both (e.g., patients A-001 and A-010) (Table 3). For most PD patients, there was no targeting of UGT1A protein levels, eIF4E protein levels or eIF4E localization; furthermore, ENT1 or ADK were generally reduced relative to BT (Table 3, Figure 4B, Online Supplementary Table S2). For one SD and one PD patient, there was reduced UGT1A but no eIF4E targeting whereby resistance likely resulted from reduction in ENT1 relative to BT (Table 3). In all, simultaneous targeting of UGT1A and eIF4E correlated with objective clinical response or durable SD, while loss of eIF4E targeting cor-

Table 3. Changes in protein levels of eIF4E and UGT1A or RNA levels of ENT upon treatment.

Response Patient ID Treatment

Months active on study

Previous therapies

eIF4E BMR

eIF4E LAS or EOT

UGT1A BMR

UGT1A LAS or EOT

ENT largest reduction or EOT

A-011

VRD

4.6

N/A

1.22±0.03*

N/A

1.09±0.02*

1.05±0.27*

B-004

VRD

10.4

0.16±0.01

0.59±0.02

0.10±0.01

0.78±0.02

N/A

C-003

VRD

5.3

0.26±0.01

0.54±0.02*

0.32±0.01

0.69±0.02*

0.49±0.08*

B-001

VRD

3.4

0.24±0.01

0.24±0.01*

0.76±0.02*

3.89±1.07*

C-002

VRD

6.8

0+MDS19

0.40±0.02

1.21±0.13

0.64±0.02

1.66±0.03

0.53±0.20*

A-008

VRD

1+MDS18

0.49±0.01

1.01±0.04

0.22±0.005

1.44±0.06

1.09±0.31

A-001

1.8

0.54±0.03

0.37±0.02

0.60±0.01

N/A

A-002

1.8

0.21±0.02

0.67±0.01

1.09±0.02

0.46±0.13*

A-010

1.7

2.14±0.08**

0.24±0.004** 0.25±0.005**

0.19±0.07*

B-002

VRD

1.2

1.91±0.05

0.86±0.04

N/A

C-004

VRD

2.1

0.38±0.01

0.49±0.01

0.50±0.10

A-004

VRD

1.5

0.70±0.01

0.70±0.01*

0.98±0.01

0.98±0.01*

0.44±0.12*

A-005

VRD

1.6

1.16±0.02

1.16±0.02*

0.97±0.06

0.97±0.06*

1.13±0.21*

A-006

1.2

0.93±0.01

0.93±0.01*

0.49±0.01

0.49±0.01*

0.50±0.12*

A-009

0.9

0.86±0.02

0.42±0.01

0.35±0.08

C-001

0.9

1.06±0.08

1.61±0.04

N/A

The light blue background indicates molecular responders. eIF4E and UGT1A protein levels in isolated blasts were measured by immunofluorescence and confocal laser microscopy (IFCLM) and quantified using FIJI. The reported median intensities are ± Standard Error of Mean at best molecular response (BMR) and end of treatment (EOT) (or Last Available Sample [LAS] if EOT was not available), compared to before treatment (BT) set to 1. ENT1 RNA levels were measured using reverse transcription-quantitative polymerase chain reaction and the largest reduction ± Standard Deviation is shown relative to BT set at 1. V: vismodegib; R: ribavirin; D: decitabine; N/A: not assessable; SD: stable disease; BR: blast response; PD: progressive disease; PR: partial remission. *Not EOT. **Comparison made with Cycle1 Day15 sample because BT sample was not available. Haematologica | 108 November 2023

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ARTICLE - Targeting of UGT1A correlates with response in AML responded to resistance and/or relapse via increased UGT1A protein levels and/or decreased ENT1 levels.

Discussion While glucuronidation has been a well-established impediment to the development of therapeutics in humans for more than 70 years,15 there have been no modalities reported to overcome this deactivation mechanism in pa-

S. Assouline et al.

tients. This is despite the observation that an estimated >50% of FDA-approved drugs,18 including many of the drugs used in the treatment of AML, are deactivated in this manner.18 In more recent years, it has become clear that glucuronidation is not limited to the liver, but also occurs in other tissues, indicating that drugs that bypass liver metabolism will not a priori escape this modification.8,13-15,50 Indeed, recent studies indicate that cancer cells, including AML blasts, can develop the capacity to glucuronidate drugs through the elevation of UGT1A proteins to evade the

Figure 3. Molecular response of eIF4E and UGT1A for patient B-004 who achieved a blast response. (A) Samples for completed treatment cycles (CT) 1 to 10 (1CT-10CT) were collected at the end of each cycle; the patient had a 28-day treatment interruption after 9CT. Percentage of bone marrow blasts is shown (in black). Changes in protein expression of eIF4E (green) and UGT1A (red) in AML blasts isolated by FACS and stained as described in the Online Supplementary Appendix. Graph generated using GraphPad Prism 7. (B) Representative confocal micrographs of blasts used for quantification in (A). eIF4E (green), UGT1A (red) and DAPI (blue) in sorted bone marrow blasts are shown. Note also that a higher fraction of eIF4E is in the nucleus before treatment (BT) and at end of treatment (EOT) than during response, as observed previously in patients treated with ribavirin.21,22 Dapi provided as a nuclear marker. Scale bars: 10 µm. Haematologica | 108 November 2023

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ARTICLE - Targeting of UGT1A correlates with response in AML impacts of chemotherapies.8,13-15,50 We had previously identified vismodegib to target UGT1A levels in AML cells resistant to ribavirin and cytarabine, whereby vismodegib correlated with reduced drug glucuronidation as observed by mass spectrometry and restored drug sensitivity.8,16 Herein, we report on the first clinically feasible avenue to reduce glucuronidation and re-sensitize cells to drugs in humans and demonstrate, for the first time, that UGT1A protein levels could be targeted in patients. Indeed, vismodegib resulted in an up to 10-fold reduction in UGT1A

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protein levels in patients which persisted for months. Given the central role that glucuronidation plays in endogenous metabolite metabolism, we note that this reduction in UGT1A levels did not cause severe toxicity, a longstanding concern for the development of clinically useful glucuronidation inhibitors. In patients who had reduced UGT1A levels, we observed ribavirin targeting of eIF4E as demonstrated by nuclear to cytoplasmic re-localization of eIF4E and reduction in eIF4E protein levels up to 6-fold in AML cells. While targeting DNA hypomethylation by deci-

Figure 4. UGT1A and eIF4E levels in responding and non-responding patients A-008 and A-004. Representative micrographs used for quantifications in Table 3. eIF4E (green), UGT1A (red) and DAPI (blue) in sorted bone marrow blasts are shown. AML blasts isolated by FACS and stained as described in the Online Supplementary Appendix. Patient A-008 (A) achieved a 4-month stable disease (SD) and patient A-004 (B) a progressive disease (PD). BT: before treatment; CT: completed treatment cycle. Dapi provided as a nuclear marker. Scale bars: 10 µm. Haematologica | 108 November 2023

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ARTICLE - Targeting of UGT1A correlates with response in AML tabine could not be directly measured due to insufficient material, it could be inferred from our previous studies into decitabine glucuronidation in cells.16 In all, simultaneous reduction in UGT1A protein levels and eIF4E targeting correlated with objective clinical benefits including a partial remission, blast responses, and durable SD. At clinical relapse, we observed that both UGT1A and eIF4E protein levels became elevated and eIF4E re-localized to the nucleus, suggesting that acquired resistance to vismodegib provided conditions whereby ribavirin glucuronidation led to reduced ribavirin-targeting of eIF4E and, by inference, deactivation of decitabine (Table 3, Figure 3).8,16 Indeed, re-emergence of UGT1A levels is correlated with multi-drug resistance.8,12-16,50,51 There are several precedents for vismodegib resistance arising in cancer patients which could be at play here. For example, vismodegib failure occurs in basal cell carcinoma due to SMO mutations which arise in as little as two months of treatment.52 Thus, SMO mutations could be related to the loss of response here; this will be tested in future studies. In addition, FLT3-ITD drives GLI1 production independently of SMO thereby circumventing vismodegib action.53 In our study, 3/5 PD patients harbored FLT3-ITD mutations which could explain why UGT1A protein levels were not impacted by vismodegib in these patients; this is consistent with the FLT3-ITD mutations harbored by PD patients A-005 and C-001 who had no change in UGT1A, while additional reasons underlie patient A-009 who had FLT3-ITD mutations but still had reduced levels of UGT1A. Previous studies indicated that vismodegib monotherapy had modest effects in AML,54 suggesting that responses observed here are co-operative, i.e., impaired drug glucuronidation with vismodegib restored sensitivity to ribavirin and decitabine. The appearance of SMO mutations and/or alternative means to elevate UGT1A protein levels suggest it would be beneficial to develop therapeutics that directly target selected UGT1A proteins. In this way, the rapid adaptation of upstream signaling pathways like SMO-GLI1 can be evaded. Early-stage inhibitors which directly bind to UGT1A were identified, and these reduced glucuronidation and restored sensitivity to ribavirin and cytarabine in cell lines.49 This presents a promising future direction for next generation therapeutics to target glucuronidation for longer in patients. To date, ribavirin has been the only means to target eIF4E in cancer patients that has led to objective clinical responses.21,22,44,45 In contrast, the antisense oligonucleotide strategies to lower eIF4E levels did not effectively reduce eIF4E protein levels in patients and did not produce objective clinical responses.55 The phosphorylation status of eIF4E is often linked to its oncogenicity and this can be targeted with MNK1/2 inhibitors in AML cells.56 Notably MNK1/2 inhibitors also target phosphorylation of many other proteins, including hnRNPA1 and PSF,57 and thus could be an interesting combination with ribavirin to pro-

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duce more robust impacts on eIF4E and, through MNK, more multifaceted effects. Moreover, whether or not MNK1/2 inhibitors are targets of inducible glucuronidation in patients has yet to be studied, and thus like many other drugs, combining these with effective glucuronidation inhibitors could be clinically relevant. eIF4E has been targeted with the 4GI-1 inhibitor which allosterically reduces the interaction of eIF4E with eIF4G to reduce translation and this could also be considered to enhance eIF4E inhibition.58 Finally, the nuclear fraction of eIF4E has recently been found to play a role in splicing.19 Thus the combination of ribavirin with splicing inhibitors, which would be predicted to severely impair splicing in AML cells, could improve efficacy. Clinical trials will be required to assess the utility of these combinations. These and previous studies position UGT1A and glucuronidation as important factors to consider for the development of AML therapeutics. In support of this, many of the mainstays of AML treatment are deactivated through glucuronidation in cell-line models, including venetoclax and azacytidine, and in patients, for instance, cytarabine and ribavirin.8,16 Here, we demonstrated that both treatment naïve and heavily pre-treated AML patients had substantial elevation of UGT1A protein levels relative to healthy volunteers (Figure 2), suggesting glucuronidation is a widespread barrier to effective AML treatment. Quantifying the prevalence of UGT1A protein dysregulation in AML patients on a global level is warranted, particularly given that UGT1A mRNA and protein levels do not always correlate,8,16 and, thus, RNA levels will not always be an accurate surrogate for UGT1A protein levels. Aside from glucuronidation, we also monitored the loss of the ENT1 transporter as another contributor to multi-drug resistance and relapse in these patients. While all patients who entered the trial passed functional screens for active ENT1, we observed that several patients manifested substantial reductions in ENT1 RNA levels during treatment. ENT1 is required for cellular entry of ribavirin and decitabine as well as many other AML drugs, including cytarabine and azacytidine. Reduced ENT1 corresponded to a loss of eIF4E targeting by ribavirin and clinical resistance even in the face of reduced UGT1A protein levels. There appears to be substantial selection on this transporter which likely influences responses to subsequent salvage regimens. In all, clinical responders in the trial required targeting of both UGT1A and eIF4E, and functional ENT1. In this third trial using ribavirin in high-eIF4E AML, we continue to observe robust targeting of eIF4E in patients corresponding to objective responses supporting the further clinical development of ribavirin, and, in the future, next generation eIF4E inhibitors. In prior ribavirin monotherapy and ribavirin with LDAC clinical trials, we observed higher overall response rates than those observed here with 6/15 and 5/14, respectively, including complete responses in

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ARTICLE - Targeting of UGT1A correlates with response in AML both trials.21,22 In comparison to the current trial, these patients had had a median of one prior therapy; patients in our trial were much more heavily pre-treated, with a median of three prior therapies in the VRD group, likely contributing to the observed differences in responses. In conclusion, this study demonstrated that UGT1A protein levels can be decreased in patients, and this is associated with objective clinical benefit. We also showed that an up to 10-fold reduction in UGT1A levels can be achieved in patients without substantial toxicity over the course of months (Table 3). These observations have implications far beyond AML, ribavirin and decitabine. Given the large number of FDA-approved drugs that can be deactivated via glucuronidation,17,51 these findings pave the way for the development of pharmaceuticals targeting glucuronidation in patients, one of the most widespread and longstanding problems in therapeutic development. Disclosures Vismodegib was provided at no cost from Roche and Ribavirin and decitabine from Pharmascience. No other support was provided by these companies. KLBB, BCK, JG, CL, CZ, EC and DS received no commercial support. SA received research support. Novartis Canada Honoraria: BMS, Abbvie, Astra Zeneca, Novartis, Pfizer, Jazz, Roche/Genentech, Janssen, Palladin. JB is a consultant for AbbVie, Servier, Amgen, Astellas Pharma, BMS, Jazz Pharmaceuticals, Novartis, and Pfizer, and received travel support from Amgen and Novartis. KWLY was a consultant for Bristol Myers Squibb/Celgene, F. Hoffmann-La Roche, GSK, Jazz Pharmaceuticals, Novartis, Pfizer, Shattuck Labs, Taiho Oncology, and Takeda Pharmaceutical Company, received research funding from Astex Pharmaceuticals, Forma

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Therapeutics, F. Hoffmann-La Roche, Forma Therapeutics, Genentech, Geron Corporation, Gilead Sciences, Janssen Pharmaceuticals, Jazz Pharmaceuticals, Novartis, and Treadwell BMS Therapeutics, and received honoraria from AbbVie and Novartis. JG holds stocks of BMS and Sanofi. BCK and KLBB hold the following patents: Combination therapy using ribavirin as elF4E inhibitor, Inventors: KLBB, HZ and BC-K (targeting inducible drug glucuronidation); US10342817B2 GRANTED Translation dysfunction based therapeutics Inventors: GJ, KLBB, BC and AK: US8497292B2, GRANTED. No royalties received. Contributions SA, EC, CL and KLBB designed the study. SA, JB and KY carried out clinical work. JG and BCK performed molecular correlate data collection and analyses. DS performed sequencing. SA, JG, CL, BCK, EC, CZ, DS and KLBB performed data analysis. KLBB, JG, EC and SA wrote the manuscript. Acknowledgments We are grateful for the gifts of ribavirin and decitabine from Pharmascience and vismodegib from Roche. Funding This work was funded by the Leukemia and Lymphoma Society USA TRP (R6478) and the Jewish General Hospital Foundation. KLBB holds a Canada Research Chair in Molecular Biology of the Cell Nucleus. SA is a Chercheur Clinicien de Merite of the FRSQ. Data-sharing statement Data are available upon reasonable request to the corresponding authors.

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ARTICLE - Targeting of UGT1A correlates with response in AML Blood. 2016;128(22):3446. 48. Mori K, Hiraoka O, Ikeda M, et al. Adenosine kinase is a key determinant for the anti-HCV activity of ribavirin. Hepatology. 2013;58(4):1236-1244. 49. Osborne MJ, Coutinho de Oliveira L, Volpon L, Zahreddine HA, Borden KLB. Overcoming drug resistance through the development of selective inhibitors of UDPglucuronosyltransferase enzymes. J Mol Biol. 2019;431(2):258-272. 50. Dellinger RW, Matundan HH, Ahmed AS, Duong PH, Meyskens FL Jr. Anti-cancer drugs elicit re-expression of UDPglucuronosyltransferases in melanoma cells. PLoS One. 2012;7(10):e47696. 51. Guillemette C, Levesque E, Rouleau M. Pharmacogenomics of human uridine diphospho-glucuronosyltransferases and clinical implications. Clin Pharmacol Ther. 2014;96(3):324-339. 52. Pricl S, Cortelazzi B, Dal Col V, et al. Smoothened (SMO) receptor mutations dictate resistance to vismodegib in basal cell carcinoma. Mol Oncol. 2015;9(2):389-397. 53. Wellbrock J, Latuske E, Kohler J, et al. Expression of hedgehog

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pathway mediator GLI represents a negative prognostic marker in human acute myeloid leukemia and its inhibition exerts antileukemic effects. Clin Cancer Res. 2015;21(10):2388-2398. 54. Bixby D, Noppeney R, Lin TL, et al. Safety and efficacy of vismodegib in relapsed/refractory acute myeloid leukaemia: results of a phase Ib trial. Br J Haematol. 2019;185(3):595-598. 55. Hong DS, Kurzrock R, Oh Y, et al. A phase 1 dose escalation, pharmacokinetic, and pharmacodynamic evaluation of eIF-4E antisense oligonucleotide LY2275796 in patients with advanced cancer. Clin Cancer Res. 2011;17(20):6582-6591. 56. Suarez M, Blyth GT, Mina AA, et al. Inhibitory effects of tomivosertib in acute myeloid leukemia. Oncotarget. 2021;12(10):955-966. 57. Joshi S, Platanias LC. Mnk kinase pathway: cellular functions and biological outcomes. World J Biol Chem. 2014;5(3):321-333. 58. Papadopoulos E, Jenni S, Kabha E, et al. Structure of the eukaryotic translation initiation factor eIF4E in complex with 4EGI-1 reveals an allosteric mechanism for dissociating eIF4G. Proc Natl Acad Sci U S A. 2014;111(31):E3187-3195.

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ARTICLE - Blood Transfusion

Inhibition of GPIb-α-mediated apoptosis signaling enables cold storage of platelets Irene Marini,1,2 Lisann Pelzl,1,2 Yoko Tamamushi,1 Chiara-Tanita Maettler,1 Andreas Witzemann,1 Karina Althaus,1,2 Stefanie Nowak-Harnau,2 Erhard Seifried3 and Tamam Bakchoul1,2

Correspondence: T. Bakchoul tamam.bakchoul@med.uni-tuebingen.de

Institute for Clinical and Experimental Transfusion Medicine, Medical Faculty of Tübingen, Tübingen; 2Center for Clinical Transfusion Medicine Tübingen and 3Institute of Transfusion Medicine and Immunohematology, German Red Cross Blood Transfusion Service BadenWürttemberg-Hessen, Frankfurt, Germany 1

Received: Accepted: Early view:

December 14, 2022. June 15, 2023. June 22, 2023.

Abstract Cold storage of platelets has been suggested as an alternative approach to reduce the risk of bacterial contamination and to improve the cell quality as well as functionality compared to room temperature storage. However, cold-stored platelets (CSP) are rapidly cleared from the circulation. Among several possible mechanisms, apoptosis has been recently proposed to be responsible for the short half-life of refrigerated platelets. In the present study, we investigated the impact of apoptosis inhibition on the hemostatic functions and survival of CSP. We found that blocking the transduction of the apoptotic signal induced by glycoprotein Ib (GPIb)-α clustering or the activation of caspase 9 does not impair CSP functionality. In fact, the inhibition of GPIb-α clustering mediated-apoptotic signal by a RhoA inhibitor better conserved δ granule release, platelet aggregation, adhesion and the ability to form stable clots, compared to untreated CSP. In contrast, upregulation of the protein kinase A caused a drastic impairment of platelet functions and whole blood clot stability. More importantly, we observed a significant improvement of the half-life of CSP upon inhibition of the intracellular signal induced by GPIb-α clustering. In conclusion, our study provides novel insights on the in vitro hemostatic functions and half-life of CSP upon inhibition of the intracellular cold-induced apoptotic pathway. Our data suggest that the combination of cold storage and apoptosis inhibition might be a promising strategy to prolong the storage time without impairing hemostatic functions or survival of refrigerated platelets.

Introduction Transfusion of platelet concentrates (PC) is an essential medical approach to treat bleeding in thrombocytopenic patients.1 Currently, PC are stored at room temperature (RT, 22-24°C). At this storage condition PC might be associated with increased risk of bacterial contamination,2 post-transfusion septic reaction3,4 and platelet storage lesions (PSL).5 Therefore, the storage time of PC is restricted to 4-7 days, depending on the national guidelines.1 Nevertheless, the risk of transmission of bacterial infection still remains high.2,6 Moreover, recent clinical studies reported on detrimental effects in patients who received RT-stored PC.7,8 In this context, alternative storage conditions like cold storage have been considered. In fact, cold storage might reduce the risk of bacterial contaminations, protect cells from storage lesions and extend products’ shelf-life.9,10 However, clinical appli-

cations of cold-stored platelets (CSP), even if characterized by better functionality,9-14 have been abandoned due to concerns regarding poor recovery and survival compared to RT.15 In fact, as reported in a recent radiolabeling study, the survival of autologous CSP upon 5 days of storage was significantly lower (around 20%) compared to platelets stored for 7 days at RT (around 50%).16 However, this drawback of CSP might be compensated by better functionality. Several groups have investigated potential mechanisms underlying the observed short half-life of CSP. One of the first proposed mechanisms was the desialylation of glycoproteins (GP).17 However, a clinical study showed that reconstitution of sialylation does not prevent the accelerated clearance of CSP.18 This indicates that alternative mechanisms, which do not require desialylation, trigger the fast elimination of refrigerated platelets. Interestingly, we and others detected increased apoptosis levels in pla-

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ARTICLE - Cold-induced apoptosis inhibition in platelet concentrates telets upon cold storage compared to RT suggesting that the apoptotic pathway might contribute to the reduced half-life of CSP.9,16,19 In fact, cold storage was shown to trigger clustering of GPIb-α20 on the platelet surface leading to apoptosis.19,21 Interestingly, it has been reported that the presence of an apoptosis inhibitor (p38 inhibitor) reduces the expression of apoptotic markers, like Bax and Bak, in pathogen-inactivated PC stored at RT but it did not improve platelet survival in vivo.22 In the present work, we aimed to verify the efficacy of three compounds that have been shown to inhibit the apoptotic intracellular signal at different levels by targeting three key regulatory proteins (Figure 1; Online Supplementary Appendix). Our hypothesis was that all compounds could reduce cold-induced apoptosis. Nevertheless, since the proteins targeted by the inhibitors are involved in essential mechanisms of platelet functionality we thought to investigate potential desire/undesired effects of each inhibitor on different cell functions in order

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to screen them and select the most promising one to perform in vivo studies.

Methods Preparation of platelet concentrates PC were collected using the apheresis device TRIMAAccel 7.0 (TERUMO BCT, Munich, Germany). Briefly, 166 mL platelet-rich plasma (PRP) were collected and resuspended in 271 mL additive solution (PASIII, Machropharm, Germany). Next, three inhibitors were added to inhibit the apoptotic signals. The first compound (G04) targets the RhoA GTPase, which has been shown to play an essential role in the transduction of the outside/inside signal downstream of GPIb-α.23 Furthermore, we tested a compound (forskolin), which upregulates the protein kinase A (PKA), via adenylate cyclase (AC), that is known to control the apoptotic pathway by inhibiting the pro-apoptotic protein

Figure 1. Schematic illustration of the intrinsic apoptotic pathway and the corresponding targets of the apoptosis inhibitors used in the present study. It is presumed that cold storage of platelets induces clustering of the glycoprotein Ib-α (GPIb-α) leading to apoptosis which in turn triggers the reduction of the mitochondrial membrane potential (MMP) and phosphatidylserine externalization. In the present study we used the following apoptosis inhibitors: G04 which inhibits RhoA binding its guanine exchange factor domain. Forskolin that enhances the functionality of adenylyl cyclase (AC) inducing conformational change upon its binding and increasing the production of cyclic adenosine monophosphate (cAMP). The latter, triggers the activation of protein kinase A (PKA) which in turn inhibits the pro-apoptotic protein Bad by phosphorylation. Caspase-9 activation is blocked by a covalent irreversable binding with the Caspase-9 inhibitor which prevents the autocatalytic cleavage of the pro-caspase-9. Cyto C: cytochrome C. Haematologica | 108 November 2023

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ARTICLE - Cold-induced apoptosis inhibition in platelet concentrates Bad upon phosphorylation (Figure 1).24 The last tested compound (Z-LEHD-fmk) prevents the autocatalytic cleavage of caspase-9 that is necessary to begin the caspase cascade (Figure 1).25 The following final concentrations were used: 150 µM G04 (RhoA Inhibitor, Millipore Corp., Darmstadt, Germany), 0.75 µM forskolin (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) and 40 µM caspase-9 inhibitor26 (Z-LEHD-fmk, BD Biosciences, San Jose, USA). Apoptosis inhibitors were added to different apheresis bags and stored at 4°C under agitation. See the Online Supplementary Appendix for further details. Detection of platelet apoptosis In order to detect changes in the mitochondrial membrane potential (MMP), the tetramethylrhodamine ethyl ester (TMRE) assay kit (Abcam, Cambridge, UK) was used and the externalization of phosphatidylserine (PS) was determined using Annexin-V (Immunotools, Friesoyhte, Germany) staining. See the Online Supplementary Appendix for further information. Assessment of platelet function In order to analyze platelet activation, CSP were stimulated with thrombin receptor-activating peptide-6 (TRAP6, 10 µM, HART Biologicals, Hartlepool, UK) and incubated with CD62-P (CLB-Thromb/6, Beckman Coulter, Krefeld, Germany) or CD63 (CLB-Gran/12, Beckman Coulter) and measured by flow cytometry (FC) (Navios, BeckmanCoulter, Krefeld, Germany). Light transmission platelet aggregation assay was performed using a four-channel aggregometer (LABiTec, LAbor BioMedical Technologies, Ahrensburg, Germany) to investigate the aggregation ability in response to 20 µM TRAP-6, 1.0 mg/mL ristocetin (HART Biologicals, Hartlepool, UK) or NaCl (Braun, Melsungen, Germany). Next, the adhesion ability of CSP was assessed as previously described using coverslips (Corning, New York, USA) coated with 100 mg/mL fibrinogen (Sigma Aldrich, Munich, Germany) or 5% human serum albumin (Grifols, Berlin, Germany).9 CSP were allowed to seed on coverslips in the presence of 10 µM TRAP-6. Platelet clot retraction was assessed upon incubation with 7.4 µM CaCl2 (Sigma Aldrich, Darmstadt, Germany) and thrombin 10 U/mL (Roche, Mannheim, Germany). Thromboelastography was performed using the ClotPro analyzer (Haemonetics, Munich, Germany) according to the manufacturer’s instructions. See the Online Supplementary Appendix for further information concerning each assay. In vivo studies In order to determine the survival and recovery of human CSP, we used an NSG (NOD [non-obese diabetic] Scid Gamma) mouse model. The experiment procedure was performed as previously described with minor modifications.9 See the Online Supplementary Appendix and Online

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Supplementary Figures S5 and S6 for additional information. Ethics and statistical analysis All studies involving human subjects were approved by the Ethics Committees of the University Hospital of Tübingen. Animal studies were approved by the State Animal Ethics Committees of Baden-Württemberg. Statistical analyses were performed using GraphPad Prism 9.4.1 (GraphPad Software, La Jolla, USA). See the Online Supplementary Appendix for additional details.

Results Inhibition of cold-induced platelet apoptosis In order to verify the efficacy of the apoptosis inhibitors during cold storage, two of the key steps of the apoptotic pathway were analyzed, the reduction of MMP and the PS externalization. We found that CSP started showing apoptotic phenotype from storage day 4, as suggested by the significant decrease of MMP compared to day 1 (MMP mean fluorescence intensity [MFI] mean ± standard error of the mean [SEM]: buffer day 1 vs. day 4, 30.00±0.61 vs. 24.33±1.26, P=0.0076; Figure 2A-C, respectively). This effect was time-dependent (MMP MFI mean ± SEM: buffer day 4 vs. day 7, 24.33±1.26 vs. 9.74±1.05, P=0.0001; buffer day 7 vs. day 10, 9.74±1.05 vs. 3.76±0.29, P=0.0081; Figure 2A-C, respectively). The inhibition of RhoA (G04, Figure 2A, D), activation of PKA (forskolin, Figure 2B, E) and downregulation of the autocatalytic cleavage of caspase-9 (Figure 2C, F) induced a significant reduction of the apoptotic signal. The mitochondrial integrity was better maintained on day 7 and 10, as indicated by the significant higher levels of MMP, in comparison to untreated cells (MMP MFI mean ± SEM: G04 vs. buffer day 7, 17.65±1.64 vs. 9.74±1.05, P=0.0091; day 10, 6.81±0.53 vs. 3.76±0.29, P=0.0080; forskolin vs. buffer day 7, 12.92±1.23 vs. 9.73±1.05, P=0.0434; day 10, 4.81±0.37 vs. 3.76±0.29, P=0.0404; caspase-9 inhibitor vs. buffer day 7, 12.45±0.83 vs. 9.74±1.05, P=0.0384; day 10, 5.08±0.61 vs. 3.76±0.29, P=0.0477; Figure 2A-C, respectively). Similarly, treatment of CSP with apoptosis inhibitors revealed a significant reduction of the percentage of cells exposing PS starting from storage day 7 compared to cells stored in buffer (% apoptotic cells, mean ± SEM: G04 vs. buffer day 7, 9±3% vs. 26±6%, P=0.0218; day 10, 39±5% vs. 67±6%, P=0.0023; forskolin vs. buffer day 7, 7±1% vs. 26±6%, P=0.0084; day 10, 48±4% vs. 67±6%, P=0.0319; caspase-9 inhibitor vs. buffer day 7, 10±3% vs. 26±6%, P=0.0105; day 10, 48±5% vs. 67±6%, P=0.0371; Figure 2D-F, respectively). Of note, after 10 days of storage all treated CSP showed increased PS externalization, compared to storage day 7. Nevertheless, the percentage of cells ex-

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Figure 2. Apoptosis inhibition during cold storage of platelet concentrates. The mitochondrial membrane potential (MMP; A-C) and the percentage of apoptotic cells (% Annexin-positive cells; D-F) of platelet concentrates stored at 4°C in buffer (white symbols) or with G04 (RhoA inhibitor; blue symbols), forskolin (PKA activator; orange symbols) and caspase-9 inhibitor (green symbols) were detected by flow cytometry after 1, 4, 7 and 10 days of cold storage, respectively. MFI: mean fluorescence intensity. Data are shown as box and whiskers ± standard error of the mean. *P<0.05; **P<0.01; ns: not significant, N=4.

posing PS on day 10 was significantly lower in the presence of the inhibitors than cells stored in buffer (Figure 2D-F). These results indicate that pretreatment of PC with apoptosis inhibitors can prevent the cold-induced apoptosis. Inhibition of GPIb-α clustering signal maintains β granule release better and preserves platelet aggregation and agglutination in cold-stored platelets The impact of apoptosis inhibition on platelet activation and aggregation was analyzed using FC and aggregometry,

respectively. First, the release of α (CD62-P) and δ (CD63) granules content was determined upon TRAP-6 stimulation (Figure 3). The inhibition of either GPIb-α clustering signal, induced by G04 incubation, or caspase-9 showed unchanged α granule secretion compared to untreatedCSP (Figure 3A, C). On the contrary, the incubation with the PKA agonist induced a significant reduction of the α granule secretion, already after 1 day of cold storage (fold increase [FI] CD62-P mean ± SEM: forskolin vs. buffer day 1, 2.03±0.24 vs. 4.18±0.74, P=0.0487; day 4, 0.91±0.16 vs. 2.25±0.39, P=0.0229; Figure 3B).

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ARTICLE - Cold-induced apoptosis inhibition in platelet concentrates Upon apoptosis inhibition with G04 a significant increase in the release of δ granule content was detected, in a time-dependent manner, compared to untreated-CSP (FI CD63 mean ± SEM: G04 vs. buffer day 1, 4.54±0.79 vs. 2.19±0.22, P=0.0394; day 7, 2.70±0.46 vs. 1.53±0.04, P=0.0466; day 10, 1.90±0.20 vs. 1.19±0.17, P=0.0133; Figure 3D). Upregulation of PKA as well as caspase-9 inhibition significantly impaired the TRAP-6-induced δ granule se-

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cretion on storage day 4 and 7 (FI CD63 mean ± SEM: forskolin vs. buffer day 4, 1.38±0.20 vs. 2.04±0.18, P=0.0333; day 7, 1.04±0.01 vs. 1.53±0.04, P=0.0005; caspase-9 inhibitor vs. buffer day 7, 1.14±0.10 vs. 1.53±0.04, P=0.0058; Figure 3E and F, respectively). Furthermore, we found that the inhibition of apoptosis induced by both G04 and caspase-9 show similar aggregation and agglutination ability upon stimulation with

Figure 3. The impact of apoptosis inhibition on α (CD62-P) and δ (CD63) granules release from cold-stored platelets. The expression of CD62-P (A-C) and CD63 (D-F) on platelet concentrates stored at 4°C in buffer (white symbols) or with G04 (RhoA inhibitor; blue symbols), forskolin (PKA activator; orange symbols) and caspase-9 inhibitor (green symbols) was analyzed after 1, 4, 7 and 10 days of cold storage, respectively. The expression of both markers was detected by flow cytometry after stimulation with TRAP-6 (10 µM). Data are shown as box and whiskers ± standard error of the mean. *P<0.05; **P<0.01; ***P<0.001; ns: not significant, N=4. MFI: mean fluorescence intensity. Haematologica | 108 November 2023

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TRAP-6 and ristocetin compared to untreated cells (Figure 4A, D and 4C, F, respectively). Of note, enhanced aggregation and agglutination were detected after 10 days of cold storage only in the presence of the inhibitor G04 compared to cells stored in buffer (% maximal aggregation mean ± SEM: G04 vs. buffer day 10, after TRAP-6, 53±9% vs. 19±9%, P=0.0192; after ristocetin, 83±4% vs. 63±7%, P=0.0108; Figure 4A, D, respectively). Similarly to the activation results (Figure 3B, E), upregulation of PKA caused a drastic reduction of the aggregation ability in response to TRAP-6 in comparison to untreated-CSP, already after 1 day of storage, with a time-dependent trend (% maximal

aggregation mean ± SEM: forskolin vs. buffer, day 1, 90±3% vs. 98±2%, P=0.0311; day 4, 15±5% vs. 91±8%, P<0.0001; day 7, 13±1% vs. 54±13%, P=0.0275; Figure 4B, respectively). The same undesired effect was detected upon ristocetin stimulation but only on storage day 7 (% maximal agglutination mean ± SEM: forskolin vs. buffer, 58±6% vs. 86±5%, P=0.0045, Figure 4E). Taking together, these data indicate that inhibition of GPIb-α clustering-mediated signal, induced by G04 incubation, better maintains δ granule release of CSP without affecting the α granule secretion as well as the aggregation and agglutination abilities.

Figure 4. Aggregation and agglutination of cold-stored platelets upon apoptosis inhibition. The maximal aggregation (A-C) and agglutination (D-F) abilities of platelet concentrates stored at 4°C in buffer (white symbols) or with G04 (RhoA inhibitor; blue symbols), forskolin (PKA activator; orange symbols) and caspase-9 inhibitor (green symbols) were measured after stimulation with the inductors TRAP (20 µM) and ristocetin (1 mg/mL), respectively. Data are shown as box and whiskers ± standard error of the mean. *P<0.05; **P<0.01; ****P<0.0001; ns: not significant, N=4. Haematologica | 108 November 2023

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ARTICLE - Cold-induced apoptosis inhibition in platelet concentrates The impact of apoptosis inhibition on cold-stored platelet adhesion Analyzing the adhesion ability of CSP to fibrinogen we found that the total number of adherent cells detected upon inhibition of RhoA was significantly higher in comparison to untreated-CSP on storage day 4 (number of adherent cells/field mean ± SEM: G04 vs. buffer, day 4, 93±17 vs. 49±13, P=0.0493; Figure 5A). Similar adhesion ability was observed upon upregulation of PKA and inhibition of caspase-9 compared to cells stored in buffer, after 4 and 7 days of cold storage (Figure 5A, B). Platelet adhesion is a multistep process, which leads to

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a complex reorganization of the cytoskeleton resulting in the formation of filopodia/lamellipodia ending with complete spreading of the cells. Therefore, we analyzed the morphology of CSP by quantifying the percentage of the different spreading patterns (type 1: resting cells; type 2: cells with filopodia; type 3: cells with lamellipodia and type 4: fully spread platelets; Figure 5C, D). Surprisingly, the higher number of total adherent cells observed on storage day 4 after RhoA inhibition (Figure 5A) was not correlated with a higher percentage of activated cells (type 2, 3 and 4) compared to cells stored in buffer (Figure 5C); whereas, the upregulation of PKA

Figure 5. Cold-stored platelet adhesion to fibrinogen upon apoptosis inhibition. The adhesion ability of platelet concentrates stored at 4°C in buffer (white symbols) or with G04 (RhoA inhibitor; blue symbols), forskolin (PKA activator; orange symbols) and caspase-9 (Casp-9) inhibitor (green symbols) was measured after TRAP-6 stimulation on storage day 4 (A, C) and 7 (B, D), respectively. The number of adherent cells (A, C) and the percentage of the different platelet phenotypes (type 1: resting cells; type 2: cells with filopodia; type 3: cells with lamellipodia and type 4: fully spreaded cells; C, D) were quantified from 6 different microscopic fields per coverslips, respectively. Representative immunofluorescence images of adherent cells (A and B; scale bar: 20 µm) and of the platelet phenotypes (C and D). Green signal, glycoprotein IIb/IIIa (A-D). Data are shown as box and whiskers ± standard error of the mean. *P<0.05; ns: not significant. C and D, if not indicated the data were not significant. N=4. Haematologica | 108 November 2023

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ARTICLE - Cold-induced apoptosis inhibition in platelet concentrates induced a significant shift of cell morphology from type 2 to type 1 (resting platelets), in comparison to untreated-CSP on day 4 (% platelet phenotype/field mean ± SEM: forskolin vs. buffer type 1, 65±18% vs. 31±13%, P=0.0433 and type 2, 21±5% vs. 44±4%, P=0.0176; Figure 5C, respectively). Of note, the inhibition of caspase-9 showed similar spreading phenotypes compared to untreated-CSP on storage day 4 (Figure 5C). Finally, comparable total number of adherent cells as well as spreading patterns were observed in all treated-CSP in comparison to cells stored in buffer on storage day 7 (Figure 5B, D). These data suggest that inhibition of cold-induced GPIbα clustering signaling better conserved the adhesion ability of CSP, upon 4 days of storage. Effect of apoptosis inhibition on the kinetic of clot formation In order to deeper investigate the hemostatic functions of treated-CSP, we analyzed the platelet clot formation performing a clot retraction assay. We found that inhibition of either GPIb-α clustering or caspase-9 did not impair clot retraction in comparison to cells stored in buffer (Figure 6A, B). In contrast, the extent of platelet clot retraction was almost completely abolished in the presence of the PKA agonist (% clot retraction mean ± SEM: forskolin vs. buffer day 4, 1±0% vs. 88±1%, P<0.0001

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and day 7, 1±0% vs. 91±1%, P<0.0001; Figure 6A, B, respectively). Since clot retraction is a crucial mechanism to stabilize thrombi, we investigated the kinetics of whole blood clot formation performing an thromboelastography assay. We designed an experimental setting that mimics platelet transfusion in thrombocytopenic patients. Briefly, we produced platelet-depleted full blood samples from healthy donors and spiked these samples with CSP. We found that CSP treated with RhoA or caspase-9 inhibitor maintain similar ability to form clots, in terms of maximum clot firmness (MCF) and maximum lysis (ML) compared to cells stored in buffer at 4°C (Figure 7A, D and 7C, F, respectively). In contrast, a significant decrease of the MCF on day 4 (MCF mean ± SEM: forskolin vs. buffer day 4, 45.66.±2.93 vs. 58.02±1.12, P=0.0173; Figure 7B) and increase of ML were detected after incubation with the PKA activator indicating reduced stability of the clots (% ML mean ± SEM: forskolin vs. buffer day 1, 4±0.3% vs. 23±2%, P=0.0006; day 4, 2±1% vs. 24±4%, P=0.0076; day 7, 2±0.6% vs. 14±4%, P=0.0206; day 10, 2±0.6% vs. 19±4%, P=0.0169; Figure 7E, respectively). Taken together our data indicate that the inhibition of apoptosis by blocking cold-induced GPIb-α- or caspase9-mediated signals does not affect the contribution of CSP to clot formation or stability.

Figure 6. Clot retraction ability of platelet concentrates after cold-induced apoptosis inhibition. The percentage of clot retraction of platelet concentrates stored at 4°C in buffer (white symbols) or with G04 (RhoA inhibitor; blue symbols), forskolin (PKA activator; orange symbols) and caspase-9 (Casp-9) inhibitor (green symbols), was analyzed after TRAP-6 stimulation on storage day 4 (A) and 7 (B), respectively. The clot surfaces were calculated as percentage of retraction area compared to the total area. For forskolin a virtual value of 1% was reported in the graphics. Lower panel: representative pictures taken after 1 hour. Data are shown as box and whiskers ± standard error of the mean. ****P<0.0001; ns: not significant, N=4. Haematologica | 108 November 2023

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Figure 7. The impact of cold-induced apoptosis inhibition on the kinetic of clot formation. The maximum clot firmness (A-C) and the percentage of maximum lysis (D-F) of platelet concentrates stored at 4°C in buffer (white symbols) or with G04 (RhoA inhibitor; blue symbols), forskolin (PKA activator; orange symbols) and caspase-9 (Casp-9) inhibitor (green symbols) were measured performing thromboelastography assay (extrinsic test), respectively. Data are shown as box and whiskers ± standard error of the mean. *P<0.05; **P<0.01; ***P<0.001; ns: not significant, N=4.

Preventing the transduction of the cold-induced GPIb-α clustering signal reduces the fast clearance of coldstored platelets in vivo Based on the results of our in vitro analyses, which suggest the RhoA inhibitor G04 as the most promising compound, and in accordance with the principles of the 3R

(replacement, reduction and refinement) for animal research, we investigated whether the survival of CSP could be improved by preventing cold-induced GPIb-α clustering signal transduction. After 7 days of storage, CSP were administrated to the mice and the survival was analyzed 1, 2, 5 and 24 hours (h) post injection. As shown in Figure

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ARTICLE - Cold-induced apoptosis inhibition in platelet concentrates 8 significantly higher percentage of treated-CSPs was detected in the mouse circulation 2 and 5 hours (h) post injection in comparison to cells stored in buffer (% survival of human platelets mean ± SEM: G04 vs. buffer, 2 h, 47±10% vs. 26±6%, P=0.0387; 5 h, 40±7% vs. 18±3%, P=0.0355; Figure 8). Of note, comparable recovery after injection was observed between platelets stored in buffer and with the RhoA inhibitor (see the Online Supplementary Figure S6). These data indicate that the inhibition of cold-induced apoptosis by blocking the GPIb-α clustering signal improves the survival of CSP.

Discussion In the present study, we investigated the impact of apoptosis inhibition on the hemostatic functions and life span of CSP. Giving the specific target of each inhibitor (Figure 1; Online Supplementary Appendix), we found that G04 might be the most promising one, as this agent provided the best compromise between apoptosis inhibition and side effects on platelet functions. We presume that this is due to the fact that G04 blocks the cold-induced apoptosis at an early stage (outside/inside signal transduction). Therefore, it might better maintain the platelet integrity and functionality during cold storage, compared to the other tested inhibitors that target downstream proteins (Figure 1). Interestingly, we found that the inhibition of apoptosis induced by G04, which prevents the transduction of the cold-induced GPIb-α clustering signal, improves δ granule release and platelet response to agonists. Furthermore, the ability of platelets to adhere to fibrinogen and to form a stable clot upon exposure to thrombin was conserved. More importantly, we observed a significant improvement of the survival of CSP upon inhibition of the intracellular signal induced by GPIb-α clus-

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tering indicating promising potential for clinical use. The apoptotic phenotype of CSP was detectable in our study already after 4 days of storage. The blockade of cold-induced apoptosis signaling pathway at three different stages (GPIb-α clustering signal, PKA as well as caspase-9; Figure 1) showed a protective effect as determined by testing MMP and PS surface externalization. First, we inhibited the transduction of the outside/inside signal induced by GPIb-α a clustering by blocking the RhoA guanine exchange factor binding domain, which maintains the protein in its inactive form (RhoA-bound GDP).27 In the presence of the inhibitor, we detected better CSP activation, aggregation and adhesion as well as similar clot retraction and thrombus stability in comparison to untreated cells. Given the key role of RhoA in the cytoskeletal assembly events regulating platelet functionality,28,29 our findings might appear unexpected. This may be explained by the fact that other members of the Rho GTPase family, like Rac1, Cdc42, RhoB and RhoC, have redundant functions in the regulation of platelet functionality.27,30 Therefore, one or more of these proteins might compensate the inhibition of RhoA. This hypothesis is supported by a previous study, where the RhoA inhibitor does neither affect either Rac1 nor Cdc42 functions.27 Another possible explanation might be a protective effect regarding the accumulation of PSL. The inhibition of GPIbα signaling at a very early stage might decrease PSL preserving cell integrity and reducing the formation of exhausted platelets. Next, we used an agent (forskolin) which upregulates PKA via AC activation. One of the downstream targets of PKA is the proapoptotic protein Bad, which is sequestrated upon phosphorylation into to the cytoplasm.31 By this mechanism the translocation of Bad to the mitochondrial membrane is prevented and the apoptosis signaling is inhibited. Despite the well-recognized role of PKA in the intrinsic apoptosis pathway, the impact of the modulation

Figure 8. Survival of cold-stored platelets upon inhibition of cold-induced GPIb-α clustering signal transduction. Cold-stored platelets (CSP) stored for 7 days in buffer (full squares and dashed blue line) or with G04 (RhoA inhibitor; full tringles and continues blue line) were added to the NSG mouse circulation via the lateral tail vein. Survival of human platelets in the mouse circulation was analyzed by flow cytometry by collecting murine blood 1, 2, 5 and 24 hours post injection. Data are shown as mean ± standard error of the mean. *P<0.05; ns: not significant; N=4. Haematologica | 108 November 2023

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ARTICLE - Cold-induced apoptosis inhibition in platelet concentrates of its activity on platelet functions still remains controversial, likely due to the wide range of downstream targets regulated by PKA-phosphorylation. Several groups observed that enhanced PKA activity prevents apoptosis induction increasing platelet life span in vivo.24,32 One study showed, however, that PKA activation correlates with enhanced apoptosis and reduced thrombin-induced platelet activation.33 In the present work, CSP treated with the PKA agonist showed sufficient prevention of apoptosis after cold storage but caused a drastic impairment of cell functions. This is likely due to a higher phosphorylation of one of the PKA downstream targets, vasodilator-stimulated phosphoprotein (VASP), which is known to negatively regulate platelet functionality.34,35 Another important finding of our study is that the clot stability (clot retraction and resistance to fibrinolysis) was impaired in PKA agonist-treated CSP. This finding might indicate disadvantages for clinical applications of this compound. In the third part of our in vitro study, we blocked the coldinduced apoptotic signal at a late stage by inhibiting the autocatalytic cleavage of caspase-9 (Figure 1). We found comparable platelet responsiveness between caspase-9 inhibitor treated-platelets and cells stored in buffer at 4°C. These data are in line with a previous study that showed normal functionality of caspase-9-depleted murine platelets.36 Taking together our in vitro data indicate that apoptosis and activation signaling pathways in CSP might be better dissected if the signal could be blocked at an early stage. The better responsiveness in the presence of RhoA inhibitor (Online Supplementary Table S1) suggests additional protective effect against apoptosis-independent PSL. RhoA inhibitor has been recently tested in mice.37 In this study, RhoA inhibitor was administered daily intraperitoneally at 40 mg/kg for 7 days. All animals survived and no adverse events were observed indicating the safety of this compound. Despite this observation and considering the expected dilution effect after PC transfusion, the removal of G04 from CSP to reduce potential systemic toxic effects still remain a relevant question; in particularly in the absence of a clinical study on its safety. Our in vitro results showed better platelet functions and cell integrity upon G04 incubation, compared to untreated CSP or those that have been treated with forskolin or caspase-9 inhibitor (Online Supplementary Table S1). Therefore, we thought to perform an in vivo study and we observed a significantly higher number of circulating CSP 2 and 5 h after injection, when cells were treated with RhoA inhibitor compared to buffer. This finding shows that the presence of the inhibitor effectively extends the halflife of CSP. Our in vivo results are in line with a recent study that reported improved survival of CSP after 14 days of storage upon inhibition of p38MAPK,38 which is known to be involved in the apoptotic signal in PC.39

I. Marini et al.

Nevertheless, our study has some limitations. Although better functionality and survival of RhoA inhibitor-treated CSP were observed, we did not verify their in vivo functionality. Therefore, further investigations testing the platelet functions in vivo are needed to address this crucial question. Moreover, since the aim of the present study was to screen three apoptosis inhibitors for their efficacy and impact on platelet functions and half-life, we focused on the individual effect. Future studies should, however, investigate the effects of combination of inhibitors during cold storage. Keeping in mind that the final goal would be to add reagent/s to enhance CSP survival without affecting their functionality, the possibility to combine compounds might be a promising approach that should be addressed in future studies. Furthermore, we reported that the higher functionality of refrigerated platelets was even more pronounced upon inhibition of the signal transduction induced by GPIb-α clustering. It could be argued that increased risk of thrombosis might exist for patients receiving these products. Even if this consideration is correct and legitimate, it can be speculated that in some clinical cases like active bleeding upon injury in thrombocytopenic patients, transfusion of CSP with better functionality would be more efficient to treat bleeding compared to PC stored at RT. Although robust data from clinical trials with a significant number of patients are still missing, a recent small pilot study investigated the safety and feasibility of CSP in patients during cardiothoracic surgery. CSP have been shown to be still functional after 14 days of storage and no significant difference in clinical outcome was observed compared to standard products. These data indicate toward the feasibility of CSP application to treat perioperative bleeding.40 In conclusion, our study provides novel insights on the in vitro hemostatic functions and half-life of CSP upon inhibition of cold-induced apoptotic signaling pathways. Our findings indicate that the combination of cold storage and apoptosis inhibition might provide a promising strategy to prolong the storage time without affecting cell functionality or reducing platelet survival. Nevertheless, further analysis and clinical studies are still needed to evaluate whether the use of these products might also give better patient outcome. Disclosures TB has received research funding from CoaChrom Diagnostica GmbH, DFG, Robert Bosch GmbH, Stiftung Transfusionsmedizin und Immunhämatologie e.V.: Ergomed, DRK Blutspendedienst, Deutsche Herzstiftung, Ministerium für Wissenschaft, Forschung und Kunst Baden-Württemberg, has received lecture honoraria from Aspen Germany GmbH, Bayer Vital GmbH, Bristol-Myers Squibb GmbH & Co., Doctrina Med AG, Meet The Experts Academy UG, Schoechl medical edu-

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ARTICLE - Cold-induced apoptosis inhibition in platelet concentrates cation GmbH, Mattsee, Stago GmbH, Mitsubishi Tanabe Pharma GmbH, Novo Nordisk Pharma GmbH, has provided consulting services to Terumo, has provided expert witness testimony relating to heparin induced thrombocytopenia (HIT) and non‐HIT thrombocytopenic and coagulopathic disorders. All of these are outside the current work. TB and ES together with DRK Blutspendedienst Baden-WürttembergHessen have a pending patent application on the use of apoptosis inhibitors for cold storage of blood platelets. Other authors declare no competing financial interests. Contributions IM, KA, SN-H, ES and TB designed the study. IM, LP, YT, C-TM and AW performed the experiments. IM, LP and TB analyzed the data and interpreted the results. IM and TB wrote the manuscript. All authors read and approved the manuscript.

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Acknowledgments The authors would like to thank Karoline Weich, Julian Duerr and the team of the blood donation center of Tübingen for their excellent technical support. Funding This study was supported by a grant from the German Red Cross, Blutspendedienst Baden-Württemberg-Hessen, to I.M. (Forschungs- und Entwicklungsprojekt, Projekt Nr. F+E_2018_016) and a grant for young scientist from the University Hospital of Tübingen to IM (Juniorantrag, Fortuene-Antrag Nr. 2706-0-0). Data-sharing statement Data generated from this study are available from the corresponding author upon reasonable request.

References 1. Schiffer CA, Bohlke K, Delaney M, et al. Platelet transfusion for patients with cancer: American Society of Clinical Oncology Clinical Practice Guideline Update. J Clin Oncol. 2018;36(3):283-299. 2. Ketter PM, Kamucheka R, Arulanandam B, Akers K, Cap AP. Platelet enhancement of bacterial growth during room temperature storage: mitigation through refrigeration. Transfusion. 2019;59(S2):1479-1489. 3. Li JW, Brecher ME, Jacobson JL, et al. Addressing the risk of bacterial contamination in platelets: a hospital economic perspective. Transfusion. 2017;57(10):2321-2328. 4. Center for Biologics Evaluation and Research. U.S. Food and Drug Administration. Fatalities reported to FDA following blood collection and transfusion: annual summary for fiscal year 2017. www.fda.gov/media/124796. Accessed September 2017. 5. Cho J, Kim H, Song J, et al. Platelet storage induces accelerated desialylation of platelets and increases hepatic thrombopoietin production. J Transl Med. 2018;16(1):199. 6. Ramirez-Arcos S, DiFranco C, McIntyre T, Goldman M. Residual risk of bacterial contamination of platelets: six years of experience with sterility testing. Transfusion. 2017;57(9):2174-2181. 7. Baharoglu MI, Cordonnier C, Al-Shahi Salman R, et al. Platelet transfusion versus standard care after acute stroke due to spontaneous cerebral haemorrhage associated with antiplatelet therapy (PATCH): a randomised, open-label, phase 3 trial. Lancet. 2016;387(10038):2605-2613. 8. Curley A, Stanworth SJ, Willoughby K, et al. Randomized trial of platelet-transfusion thresholds in neonates. N Engl J Med. 2019;380(3):242-251. 9. Marini I, Aurich K, Jouni R, et al. Cold storage of platelets in additive solution: the impact of residual plasma in apheresis platelet concentrates. Haematologica. 2019;104(1):207-214. 10. Yang J, Yin W, Zhang Y, et al. Evaluation of the advantages of platelet concentrates stored at 4 degrees C versus 22 degrees C. Transfusion. 2018;58(3):736-747. 11. Koessler J, Klingler P, Niklaus M, et al. The impact of cold storage on adenosine diphosphate-mediated platelet responsiveness. TH Open. 2020;4(3):e163-e172. 12. Nair PM, Pandya SG, Dallo SF, et al. Platelets stored at 4

degrees C contribute to superior clot properties compared to current standard-of-care through fibrin-crosslinking. Br J Haematol. 2017;178(1):119-129. 13. Johnson L, Vekariya S, Wood B, Tan S, Roan C, Marks DC. Refrigeration of apheresis platelets in platelet additive solution (PAS-E) supports in vitro platelet quality to maximize the shelflife. Transfusion. 2021;61(Suppl 1):S58-S67. 14. Miles J, Bailey SL, Obenaus AM, et al. Storage temperature determines platelet GPVI levels and function in mice and humans. Blood Adv. 2021;5(19):3839-3849. 15. Murphy S, Gardner FH. Effect of storage temperature on maintenance of platelet viability - deleterious effect of refrigerated storage. N Engl J Med. 1969;280(20):1094-1098. 16. Stolla M, Bailey SL, Fang L, et al. Effects of storage time prolongation on in vivo and in vitro characteristics of 4 degrees C-stored platelets. Transfusion. 2020;60(3):613-621. 17. Jansen AJ, Josefsson EC, Rumjantseva V, et al. Desialylation accelerates platelet clearance after refrigeration and initiates GPIbalpha metalloproteinase-mediated cleavage in mice. Blood. 2012;119(5):1263-1273. 18. Wandall HH, Hoffmeister KM, Sorensen AL, et al. Galactosylation does not prevent the rapid clearance of long-term, 4 degrees C-stored platelets. Blood. 2008;111(6):3249-3256. 19. van der Wal DE, Du VX, Lo KS, Rasmussen JT, Verhoef S, Akkerman JW. Platelet apoptosis by cold-induced glycoprotein Ibalpha clustering. J Thromb Haemost. 2010;8(11):2554-2562. 20. Chen W, Druzak SA, Wang Y, et al. Refrigeration-induced binding of von Willebrand factor facilitates fast clearance of refrigerated platelets. Arterioscler Thromb Vasc Biol. 2017;37(12):2271-2279. 21. Gitz E, Koekman CA, van den Heuvel DJ, et al. Improved platelet survival after cold storage by prevention of glycoprotein Ibalpha clustering in lipid rafts. Haematologica. 2012;97(12):1873-1881. 22. Stivala S, Gobbato S, Infanti L, et al. Amotosalen/ultraviolet A pathogen inactivation technology reduces platelet activatability, induces apoptosis and accelerates clearance. Haematologica. 2017;102(10):1650-1660. 23. Dutting S, Gaits-Iacovoni F, Stegner D, et al. A Cdc42/RhoA regulatory circuit downstream of glycoprotein Ib guides transendothelial platelet biogenesis. Nat Commun. 2017;8:15838.

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ARTICLE - Cold-induced apoptosis inhibition in platelet concentrates 24. Zhao L, Liu J, He C, et al. Protein kinase A determines platelet life span and survival by regulating apoptosis. J Clin Invest. 2017;127(12):4338-4351. 25. Lebois M, Josefsson EC. Regulation of platelet lifespan by apoptosis. Platelets. 2016;27(6):497-504. 26. Lopez JJ, Salido GM, Gomez-Arteta E, Rosado JA, Pariente JA. Thrombin induces apoptotic events through the generation of reactive oxygen species in human platelets. J Thromb Haemost. 2007;5(6):1283-1291. 27. Shang X, Marchioni F, Sipes N, et al. Rational design of small molecule inhibitors targeting RhoA subfamily Rho GTPases. Chem Biol. 2012;19(6):699-710. 28. Flevaris P, Stojanovic A, Gong H, Chishti A, Welch E, Du X. A molecular switch that controls cell spreading and retraction. J Cell Biol. 2007;179(3):553-565. 29. Pleines I, Hagedorn I, Gupta S, et al. Megakaryocyte-specific RhoA deficiency causes macrothrombocytopenia and defective platelet activation in hemostasis and thrombosis. Blood. 2012;119(4):1054-1063. 30. Rowley JW, Oler AJ, Tolley ND, et al. Genome-wide RNA-seq analysis of human and mouse platelet transcriptomes. Blood. 2011;118(14):e101-111. 31. Nagy Z, Smolenski A. Cyclic nucleotide-dependent inhibitory signaling interweaves with activating pathways to determine platelet responses. Res Pract Thromb Haemost. 2018;2(3):558-571. 32. Xiao W, Zhou K, Yang M, et al. Carbamazepine induces platelet apoptosis and thrombocytopenia through protein kinase A. Front Pharmacol. 2021;12:749930. 33. Rukoyatkina N, Butt E, Subramanian H, et al. Protein kinase A

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activation by the anti-cancer drugs ABT-737 and thymoquinone is caspase-3-dependent and correlates with platelet inhibition and apoptosis. Cell Death Dis. 2017;8(6):e2898. 34. Benz PM, Laban H, Zink J, et al. Vasodilator-stimulated phosphoprotein (VASP)-dependent and -independent pathways regulate thrombin-induced activation of Rap1b in platelets. Cell Commun Signal. 2016;14(1):21. 35. Massberg S, Gruner S, Konrad I, et al. Enhanced in vivo platelet adhesion in vasodilator-stimulated phosphoprotein (VASP)deficient mice. Blood. 2004;103(1):136-142. 36. White MJ, Schoenwaelder SM, Josefsson EC, et al. Caspase-9 mediates the apoptotic death of megakaryocytes and platelets, but is dispensable for their generation and function. Blood. 2012;119(18):4283-4290. 37. Francis TC, Gaynor A, Chandra R, Fox ME, Lobo MK. The selective RhoA inhibitor Rhosin promotes stress resiliency through enhancing D1-medium spiny neuron plasticity and reducing hyperexcitability. Biol Psychiatry. 2019;85(12):1001-1010. 38. Skripchenko A, Gelderman MP, Vostal JG. P38 mitogen activated protein kinase inhibitor improves platelet in vitro parameters and in vivo survival in a SCID mouse model of transfusion for platelets stored at cold or temperature cycled conditions for 14 days. PLoS One. 2021;16(5):e0250120. 39. Chen Z, Schubert P, Culibrk B, Devine DV. p38MAPK is involved in apoptosis development in apheresis platelet concentrates after riboflavin and ultraviolet light treatment. Transfusion. 2015;55(4):848-857. 40. Strandenes G, Sivertsen J, Bjerkvig CK, et al. A pilot trial of platelets stored cold versus at room temperature for complex cardiothoracic surgery. Anesthesiology. 2020;133(6):1173-1183.

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ARTICLE - Cell Therapy & Immunotherapy

Salvage radiotherapy in relapsed/refractory large B-cell lymphoma after failure of CAR T-cell therapy Hazim S. Ababneh,1 Andrea K. Ng,2 Matthew J. Frigault,3 Jeremy S. Abramson,3 Patrick Connor Johnson,3 Caron A. Jacobson4# and Chirayu G. Patel1#

Correspondence: C. G. Patel cpatel@mgh.harvard.edu

Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School; 2Department of Radiation Oncology, Brigham and Women's Hospital; 3Division of Hematology and Oncology, Massachusetts General Hospital, Harvard Medical School and 4 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA 1

Received: Accepted: Early view:

January 22, 2023. June 5, 2023. June 15, 2023.

https://doi.org/10.3324/haematol.2023.282804

CAJ and CGP contributed equally as senior authors.

Abstract Despite the success of CD19-targeted chimeric antigen receptor (CAR T)-cell therapy in patients with relapsed/refractory large B-cell lymphoma (LBCL), there is a need for effective salvage strategies post-CAR T-cell therapy failure. We conducted a multi-institutional retrospective study of patients who relapsed following CAR T-cell therapy (axicabtagene ciloleucel [axi-cel] or tisagenlecleucel [tisa-cel]) and received salvage therapies (radiation therapy [RT] alone, systemic therapy alone, or combined modality therapy [CMT]). A total of 120 patients with post-CAR T relapsed LBCL received salvage therapies (RT alone, 25 patients; CMT, 15 patients; systemic therapy alone, 80 patients). The median follow-up from CAR T-cell infusion was 10.2 months (interquartile range, 5.2-20.9 months). Failure occurred in previously involved sites prior to CAR T-cell therapy in 78% of patients (n=93). A total of 93 sites were irradiated in 54 patients who received any salvage RT post-CAR T failure. The median dose/fractionation were 30 Gy (range, 4-50.4 Gy) and 10 fractions (range, 1-28 fractions). The 1-year local control rate for the 81 assessable sites was 84%. On univariate analysis, the median overall survival (OS) from the start date of RT was significantly higher among patients who received comprehensive RT versus focal RT (19.1 months vs. 3.0 months; P=<0.001). Twenty-three of 29 patients who received comprehensive RT had limited-stage disease. Among these, there was no difference in median OS among the patients who received RT alone versus those who received RT followed by additional therapies (log-rank P=0.2). On multivariate survival analysis, achieving PR or CR post-CAR T (hazard ratio =0.5; 95% confidence interval: 0.3-0.9; P=0.01) was independently associated with superior OS. Our findings suggest that RT can provide local control for LBCL relapsed post-CAR T-cell therapy, particularly in patients with limited-stage relapsed disease treated with comprehensive RT.

Introduction CD19-targeted chimeric antigen receptor (CAR T)-cell therapy has transformed the treatment of relapsed/refractory large B-cell lymphoma (rel/ref LBCL). The landmark CAR T-cell therapy trials in rel/ref LBCL have shown durable remissions in approximately 40%1–3 of patients who would otherwise have poor prognoses using conventional therapies. Despite these favorable outcomes, more than half of all patients undergoing CAR Tcell therapy will develop progressive disease, leaving these patients in need of additional therapies post-CAR T failure. Potential biologic rationales have been purported for CAR T-cell therapy failure, related to CAR T cells, lymphoma, or

microenvironment. These include downregulation of the tumor-associated antigen or loss of the target antigen,4–6 T-cell exhaustion, or senescence,7–9 intrinsic CAR T-cell dysfunction, inadequate persistence or expansion of the CAR T cells in vivo,10,11 inadequate memory phenotype achieved by the CAR T cells3,12,13 and/or microenvironmentinduced immune suppression.14–16 The optimal approach following failure of CAR T-cell therapy is unknown. Potential therapeutic options include systemic therapy including a targeted agent (such as polatuzumab vedotin, tafasitimab/lenalidomide, or loncastuximab tesirine), radiation therapy (RT), allogeneic hematopoietic stem cell transplantation (HSCT), second CAR T-cell therapy infusion, or any combinatorial treatment based on these modalities. Bispecific mono-

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clonal antibodies, such as epcoritamab-bysp, which recently received accelerated approval by the Food and Drug Administration (FDA), are also inducing responses in the post-CAR T-cell setting. An important goal for treatment post-CAR T-cell therapy is to modulate the immune system and exert synergistic activity with CAR T cells, thus overcoming resistance and leading to durable remissions.17–24 Of late, there is a burgeoning interest in exploring RT after CAR T-cell failure due to immunomodulatory and potentially synergistic effects that may interplay between radiation therapy and cellular immunotherapies. In addition to the role of RT as a local therapy, it is even more compelling that RT has the potential to prime and act in concert with CAR T cells to achieve long-lasting remissions.16,25 Preclinical mechanistic studies have previously highlighted the synergy between RT and CAR Tcell therapy using cell lines of solid tumors.26,27 It was shown that low-dose RT might radiosensitize tumor cells by upregulating specific cytokines, leading to the trafficking of modified T cells into the irradiated sites. Furthermore, it was revealed that RT could lead to enhanced T-cell receptor (TCR) repertoire expansion by inducing an abscopal-like effect outside the radiation field, as was described in a case of multiple myeloma.28 Data available pertaining to the optimal salvage strategy following CAR T-cell therapy failure is limited to a few case series published to date.17–19,22–24 In an effort to unravel the ambiguity concerning the therapeutic dilemmas in patients progressing after CAR T-cell therapy, we sought to describe our multi-institutional experience to compare the impact of RT with other systemic regimens in LBCL patients who progressed following CD19-targeted CAR T-cell therapy.

acteristics were collected from the date of transformation. Eligible patients had experienced CAR T-cell failure, defined as refractory disease or relapse after initial response following CAR T-cell therapy, and received additional lymphoma-directed therapies. These patients were identified and analyzed using descriptive and statistical analysis. Salvage regimens were categorized into three groups: (i) RT delivered as a single treatment; (ii) systemic therapy, including as chemotherapy, checkpoint inhibitors, other targeted therapies, second CAR T infusion, and allogeneic HSCT; and (iii) combined-modality therapy (CMT), which included only patients who had been planned for both RT and systemic therapy as a first salvage regimen, regardless of the response to either. All three categories were defined at the time of the first salvage therapy following CAR T-cell therapy failure. The median follow-up was analyzed at two separate time points: the date of CAR T-cell therapy infusion and the start date of salvage therapy post-CAR T failure. Overall survival (OS1) was defined as the time between the date of CAR Tcell therapy infusion and the time of the last follow-up or death from any cause. OS2 was defined as the time from the start date of salvage therapy until the time of the last follow-up or death from any cause. To account for the heterogeneity of the cohort and consider all possible combinations of patients, subgroup analyses were also performed in the OS2 analysis. These analyses focused on two groups: patients who initially received systemic therapy and subsequently had disease progression for which they required RT and patients who received comprehensive RT and then experienced disease progression for which they received systemic therapy. Event-free survival (EFS1) was defined as the time from CAR T-cell therapy infusion until the date of disease progression, relapse, start of a new line of lymphoma therapy, or death from any cause. EFS2 was defined as the time from the start date of the first salvage therapy post-CAR T failure until the date of disease progression, relapse, or start of a new line of lymphoma therapy, or death from any cause, whichever occurs earlier. Early response was generally assessed at post-CAR T day 30 (interquartile range [IQR], 28-31 days), while the best overall response was assessed at any time between postCAR T-cell therapy and additional salvage therapies when there was the lowest disease burden. The overall response rate (ORR) was defined as the percentage of patients who achieved complete response (CR) or partial response (PR). In-field response for salvage RT was evaluated using postRT imaging and/or clinical assessment and then was analyzed based on the total number of irradiated sites. The in-field PFS was defined as the time between the start date of RT and the date of in-field progression/relapse. A separate analysis was then performed for patients who were treated with comprehensive versus focal RT. Com-

Methods Following Institutional Review Board approval, a multi-institutional retrospective study was conducted at two tertiary care centers for consecutive LBCL patients who received either tisagenlecleucel (tisa-cel) or axicabtagene ciloleucel (axi-cel) CAR T-cell therapy between 2017 and 2021 as part of a database of 352 patients. Eligible patients had rel/ref LBCL including the following: de novo diffuse large-B cell lymphoma (DLBCL); transformed follicular lymphoma (TFL); DLBCL arising from other lowgrade lymphomas; primary mediastinal large B-cell lymphoma (PMBCL); high-grade BCL not otherwise specified/ with rearrangement of MYC with BCL2, or BCL6, or both; B-cell lymphoma unclassifiable with features intermediate between DLBCL and classic Hodgkin lymphoma; or high-grade BCL with features intermediate between DLBCL and Burkitt’s lymphoma. In cases of transformed low-grade lymphomas, demographics and patient char-

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ARTICLE - Salvage radiation for post CAR T failure in LBCL prehensive RT in this setting was defined as RT administered to include all lymphoma sites as per the scan obtained immediately prior to the start of RT; patients with relapse in only one site who received RT were considered to have had comprehensive RT. Only the first RT course was included in this analysis for patients receiving >1 salvage RT course. For comparability between different dose/fractionation regimens, the biologically effective dose (BED) was calculated using an α/β ratio of 10. Further information on methodology and details of the statistical analysis are provided in the Online Supplementary Appendix.

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Results

Failure after CAR T-cell infusion The median time interval between CAR T-cell infusion and treatment failure was 3.0 months (IQR, 1.1-5.1 months) for the entire cohort, with a median follow-up after CAR Tcell infusion of 10.2 months (IQR, 5.2-20.9 months). Analysis of patterns of failure revealed that the majority of patients (n=93, 78%) had a component of failure in previously involved sites pre-CAR T, wherein 35 patients had failed in these sites alone (29%), and 58 patients (48%) had concurrent local and de novo failures. The remaining 27 patients (23%) demonstrated de novo failures. Of the 67 evaluable patients for CD19 antigen expression status at the time of failure, 59 patients (88%) had CD19-positive disease, and only eight patients (12%) demonstrated CD19-negative disease.

Patient and treatment characteristics Online Supplementary Table S1 outlines patient characteristics prior to CAR T-cell therapy infusion. A total of 120 patients meeting eligibility progressed following CAR Tcell therapy and went on to receive salvage therapies: 25 patients received RT alone, 15 patients received CMT, and 80 patients received systemic therapy alone. There was no significant difference in patient characteristics at the time of receiving salvage therapy between the RT, CMT, and systemic therapy cohorts, except for high LDH level (0.006) and Eastern Cooperative Oncology Group (ECOG) performance status (<0.001) (Table 1). The best ORR was 70% (n=84), 43% (n=51) of which were in CR, with a median time to CR being 1.0 month (range, 0.9-5.3 months) post-CAR T infusion. For patients who responded to CAR T, the median duration of response was 3.9 months (IQR, 3.0-6.6 months).

Survival following post-CAR T salvage therapy The median number of lines of salvage therapy following CAR T-cell failure was 2 (range, 1-8), with a median follow-up after post-CAR T salvage therapy of 5.6 months (IQR, 1.9-12.1 months). The median duration from CAR Tcell infusion to the start date of salvage therapy was 3.4 months (IQR, 1.7-6.5 months). The median OS1 was 15.0 months (95% confidence interval [CI]: 11.7-24.4) and the median OS2 was 9.8 months (95% CI: 6.3-18.6). The estimated 12-month and 24-month OS1 rates were 58% and 39%, respectively. The estimated 12-month and 24-month OS2 rates were 45% and 33%, respectively. The median EFS1 was 3.0 months (95% CI: 2.4-3.2). Kaplan–Meier survival curves of OS1 and EFS1 are illustrated in Figure 1A, B. The median EFS2 was 2.6 months (95% CI: 1.7-4.3). After stratifying by salvage regimen, the median OS2 was not

Figure 1. Kaplan–Meier survival estimates of overall survival and event-free survival from the date of CAR T-cell therapy. (A) KM curve of overall survival (OS1) and (B) K-M curve of event-free survival (EFS1). OS1 was defined as the time between the date of CAR T-cell therapy infusion and the time of the last follow-up or death from any cause. EFS1 was defined as the time from CD19-targeted chimeric antigen receptor (CAR T)-cell therapy infusion until the date of disease progression, relapse, start of a new line of lymphoma therapy, or death from any cause. Haematologica | 108 November 2023

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ARTICLE - Salvage radiation for post CAR T failure in LBCL reached for the RT group, 7.3 months for the CMT group, 6.6 months for the systemic therapy group, 6.9 months for the systemic therapy then RT group, and 15.6 months

H. S. Ababneh et al. for the RT then systemic therapy group. There was no significant difference in OS2 between the five groups based on the type of salvage therapy (P=0.6) (Figure 2A). The

Table 1. Characteristics of patients following CAR T-cell therapy infusion.

Bridging therapy Yes No CAR T product Axi-cel Tisa-cel Best response post-CAR T CR PR DP SD CNS disease at time of salvage therapy Yes No Bulky disease at time of salvage therapy* ≥5 cm <5 cm Missing Number of disease sites at time of salvage therapy ≥2 <2 Extranodal disease at time of salvage therapy Yes No ECOG PS at time of salvage therapy 0 1 2 3 4 Stage at time of salvage therapy I II III IV Elevated LDH at time of salvage therapy Yes No IPI score at time of salvage therapy 1 2 3 4 Disease status at time of last follow-up CR PR DP Alive status Deceased Living

RT N (%) (N=25)

CMT N (%) (N=15)

ST N (%) (N=80)

Overall N (%) (N=120)

12 (48.0) 13 (52.0)

8 (53.3) 7 (46.7)

38 (47.5) 42 (52.5)

58 (48.3) 62 (51.7)

0.917

14 (56.0) 11 (44.0)

10 (66.7) 5 (33.3)

58 (72.5) 22 (27.5)

82 (68.3) 38 (31.7)

0.298

12 (48.0) 8 (32.0) 5 (20.0) 0 (0)

5 (33.3) 6 (40.0) 4 (26.7) 0 (0)

34 (42.5) 19 (23.8) 26 (32.5) 1 (1.3)

51 (42.5) 33 (27.5) 35 (29.2) 1 (0.8)

0.747

5 (20.0) 20 (80.0)

2 (13.3) 13 (86.7)

13 (16.3) 67 (83.8)

20 (16.7) 100 (83.3)

0.848

5 (25.0) 15 (75.0) 0 (0)

5 (38.5) 7 (53.8) 1 (7.7)

21 (31.3) 42 (62.7) 4 (6.0)

31 (31.0) 64 (64.0) 5 (5.0)

0.61

15 (60.0) 10 (40.0)

10 (66.7) 5 (33.3)

61 (76.3) 19 (23.8)

86 (71.7) 34 (28.3)

0.261

18 (72.0) 7 (28.0)

12 (80.0) 3 (20.0)

61 (76.3) 19 (23.8)

91 (75.8) 29 (24.2)

0.839

12 (48.0) 9 (36.0) 1 (4.0) 0 (0) 3 (12.0)

2 (13.3) 9 (60.0) 4 (26.7) 0 (0) 0 (0)

15 (18.8) 53 (66.3) 9 (11.3) 3 (3.8) 0 (0)

29 (24.2) 71 (59.2) 14 (11.7) 3 (2.5) 3 (2.5)

<0.001

9 (36.0) 7 (28.0) 1 (4.0) 8 (32.0)

4 (26.7) 2 (13.3) 0 (0) 9 (60.0)

11 (13.8) 16 (20.0) 9 (11.3) 44 (55.0)

24 (20.0) 25 (20.8) 10 (8.3) 61 (50.8)

0.0872

8 (32.0) 17 (68.0)

11 (73.3) 4 (26.7)

52 (65.0) 28 (35.0)

71 (59.2) 49 (40.8)

0.00671

12 (48.0) 5 (20.0) 4 (16.0) 4 (16.0)

2 (13.3) 5 (33.3) 4 (26.7) 4 (26.7)

14 (17.5) 23 (28.8) 24 (30.0) 19 (23.8)

28 (23.3) 33 (27.5) 32 (26.7) 27 (22.5)

0.085

6 (24.0) 2 (8.0) 17 (68.0)

3 (20.0) 0 (0) 12 (80.0)

27 (33.8) 1 (1.3) 52 (65.0)

36 (30.0) 3 (2.5) 81 (67.5)

0.24

12 (48.0) 13 (52.0)

9 (60.0) 6 (40.0)

43 (53.8) 37 (46.3)

64 (53.3) 56 (46.7)

0.756

χ2 test was used to compare categorical variables, and ANOVA test was used to compare the means between 3 groups. CAR T: CD19-targeted chimeric antigen receptor; RT: radiation therapy; CMT: combined modality therapy; ST: systemic therapy; CR: complete response; PR: partial response; DP: disease progression; SD: stable disease; CNS: central nervous system; ECOG: Eastern Cooperative Oncology Group; PS: performance status; LDH: lactate dehydrogenase; IPI: International Prognostic Index. Axi-cel: axicabtagene ciloleucel; Tisa-cel: tisagenlecleucel. *Patients with central nervous system lymphoma were excluded. Haematologica | 108 November 2023

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ARTICLE - Salvage radiation for post CAR T failure in LBCL median EFS2 was 3.5 months for the RT group, 3.3 months for the CMT group, and 1.9 months for the systemic therapy group. There was no significant difference in EFS2 between the three groups based on the type of salvage therapy (P=0.84) (Figure 2B). Salvage radiation therapy following CAR T-cell therapy failure Fifty-four patients received any salvage RT post-CAR T failure, with a total of 93 sites were irradiated; eight patients (15%) had previously received bridging RT, and among them, one patient received salvage RT to the same site. The median time from CAR T-cell therapy infusion to RT start was 7.7 months (IQR, 3.1-14.4 months). The median dose/fractionation were 30 Gy (range, 4-50.4 Gy) and 10 fractions (range, 1-28 fractions). Irradiated sites were treated via 3-dimensional conformal techniques (3DCRT) (n=51, 64%), intensity-modulated radiation therapy (IMRT) (n=20, 25%), both 3DCRT/IMRT techniques (n=6, 7%), or electron beam (n=3, 4%). Radiation details were incomplete for 13 sites that were administered RT at outside institutions. Sites of RT included: central nervous system (CNS) (n=22, 24%), extremities (n=20, 21.5%), head and neck (n=14,15%), pelvis (n=13, 14%), abdomen (n=10, 11%), chest (n=7, 7.5%), and paraspinal area (n=7, 7.5%). Of the 75 sites assessable per positron emission tomography/computed tomography (PET/CT), 30 sites (40%) were bulky (≥5 cm) at the time of RT. The other 18

H. S. Ababneh et al. sites were not assessable per PET/CT as they were CNS. The in-field responses of the 81 evaluable sites were as follows: CR (n=48, 59%), PR (n=19, 23%), stable disease (n=3, 4%), and in-field progression (n=11, 14%); the remaining 12 sites (13%) were not evaluable since those patients died shortly after receiving RT due to progressive lymphoma. The 1-year LC rate for the 81 assessable sites was 84% (Figure 3). For the 11 sites that experienced recurrence, the median time to in-field progression was 3.4 months (range, 0.6-14.8 months; IQR, 2.3-7.0 months). On univariate analysis, in-field PFS for bulky sites as compared to non-bulky sites was not statistically different (median in-field PFS: 14.8 months vs. not reached; logrank P= 0.6); bulky sites were not treated to higher BED10 (>30 Gy) as compared to non-bulky sites (P=0.7). Comparative subgroup analysis: comprehensive radiation therapy versus focal radiation therapy A total of 54 patients were treated to 62 sites with a median of one irradiated site (range, 1-2 sites) during their first course of RT and formed the cohort of the comprehensive versus focal RT analysis. Twenty-nine patients were treated with comprehensive RT field to 32 sites with a median dose of 36.7 Gy (range, 4-50.4 Gy) while 25 patients were treated with focal RT field to 30 sites with a median dose of 30 Gy (range, 4-41.4 Gy) (P< 0.001). Radiation details were incomplete for 12 sites that were administered RT at outside institutions. On univariate

Figure 2. Kaplan–Meier estimates of median overall survival and event-free survival of patients who received salvage therapies following CAR T-cell therapy failure based on the type of salvage therapy. (A) K-M curve of median overall survival (OS2) and (B) K-M curve of median event-free survival (EFS2). OS2 was defined as the time from the start date of salvage therapy until the time of the last follow-up or death from any cause. EFS2 was defined as the time from the start date of the first salvage therapy post- CD19-targeted chimeric antigen receptor (CAR T) failure until the date of disease progression, relapse, or start of a new line of lymphoma therapy, or death from any cause, whichever occurs earlier. RT: radiation therapy; CMT: combined modality therapy; ST: systemic therapy. Haematologica | 108 November 2023

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analysis, higher OS was observed among patients who received high-dose RT (BED10>30 Gy) as compared to patients who received low-dose RT (BED10≤30 Gy) (median OS: 10.9 months vs. 2.0 months; log-rank P=0.006); all but one patient treated comprehensively received high-dose RT. There was no statistically significant difference in BED10 in sites with local failure vs. sites that remained locally controlled. Patients who received focal RT were more likely to have an IPI of ≥3 (P=<0.001), advanced-stage disease (P=<0.001), ≥2 sites of disease (P=0.04), extranodal disease (P=0.003), and bulky disease as per PET/CT (P=0.02). No significant difference was detected among patients with elevated LDH (P=0.4) and poor ECOG PS (P=0.16). The infield responses of the 30 evaluable sites for patients who were treated with comprehensive RT were as follows: CR (n=17, 57%), PR (n=7, 23%), and in-field progression (n=6, 20%). The in-field responses of the 24 evaluable sites for patients who were treated with focal RT were as follows: CR (n=12, 50%), PR (n=8, 33%), stable disease (n=3, 13%), and in-field progression (n=1, 4%). The other sites were not assessable since those patients succumbed shortly following RT due to progressive disease. The median survival among patients who received comprehensive RT was 19.1 months and for focal RT was 3.0 months (P=<0.001) (Figure 4). In the comprehensive RT group, only three patients received RT to more than one site. On univariate analysis, there was no difference in median OS among the patients who received RT to only one site versus those who received RT to two sites (log-rank P=0.2). Twentythree of 29 patients who received comprehensive RT had limited-stage disease, while two patients who received

focal RT had limited-stage disease. Among patients who received comprehensive RT with limited-stage disease, there was no difference in median OS among the patients who received RT alone versus those who received RT followed by additional therapies (log-rank P=0.2). It is noteworthy that five patients received RT peri-allogeneic HSCT following CAR T-cell failure, including four patients who achieved a CR after RT and systemic therapy, enabling them to proceed with HSCT. The median time from salvage RT to transplant was 5.4 months (range, 3.98.9 months). One of the four patients also received additional RT 2 months post-HSCT. The fifth patient received consolidative RT 6 months post-HSCT. At the time of the last follow-up, four patients achieved CR and one patient had disease progression thereafter. All five patients are still alive at a median of 8.8 months (range, 3.3-33.6 months) following allo-HSCT.

Figure 3. Kaplan–Meier estimate of local control rate for the 81 assessable sites treated with salvage radiation therapy following CAR T-cell therapy failure. CAR T: CD19-targeted chimeric antigen receptor.

Univariate and multivariate analyses The next step of the analysis focused on factors associated with OS1, OS2, and EFS2 after any therapy following CAR T-cell failure. On univariate analysis, patients who experienced PR or CR post-CAR T-cell therapy infusion had superior OS1 compared to non-responders (median OS1 19.6 months vs. 8.3 months; P=0.01). Receipt of bridging therapy and type of CAR T product were not associated with OS1. On multivariate analysis, achieving PR or CR post-CAR T (HR=0.5; 95% CI: 0.3-0.9; P=0.01) was independently associated with a superior OS1. Online Supplementary Table S2 outlines the univariate analysis results for OS from the salvage therapy start date (OS2). Factors at the time of receiving salvage therapy that

Figure 4. Kaplan-Meier estimate of overall survival of patients who were treated with focal radiation therapy compared to patients treated with comprehensive radiation therapy. Only the first radiation therapy (RT) course was included in this analysis for patients receiving >1 salvage RT course.

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predicted for inferior OS2 included presence of bulky disease (≥5 cm) (median OS2 4.0 months vs. 12.0 months; P=0.03), presence of ≥2 sites of disease (median OS2 6.8 months vs. 19.1 months; P=0.01), poor performance status (median OS2 2.9 months vs. 10.5 months; P=0.01), advanced-stage disease (median OS2 4.3 months vs. 19.1 months; P=0.002), elevated lactate dehydrogenase (LDH) (median OS2 4.0 months vs. not reached; P=<0.001), and IPI≥3 (median OS2 4.0 months vs. 15.6 months; P=<0.001). On multivariate analysis, advanced-stage disease (HR=2.2; 95% CI: 1.3-3.8; P=0.004) and elevated LDH (HR=2.9; 95% CI: 1.7-5.3; P=<0.001) at the time of receiving salvage therapy were independent factors associated with a shorter OS2. Online Supplementary Table S3 outlines the univariate analysis results for EFS2. Factors at the time of receiving salvage therapy that predicted for inferior EFS2 included presence of ≥2 sites of disease (median EFS2 1.9 months vs. 5.5 months; P=0.04), extranodal disease (median EFS2 1.8 months vs. 5.1 months; P=0.04), poor performance status (median EFS2 1.2 months vs. 3.2 months; P=0.02), advanced-stage disease (median EFS2 1.7 months vs. 5.6 months; P=<0.001), elevated LDH (median EFS2=1.6 months vs. 4.4 months; P=0.005), and IPI ≥3 (median EFS2 1.7 months vs. 5.4 months; P=0.001). On multivariate analysis, advanced-stage disease (HR=2.3; 95% CI: 1.4-3.6; P=<0.001) and elevated LDH (HR=1.7; 95% CI: 1.1-2.7; P=0.01) at the time of receiving salvage therapy were independent factors associated with a shorter EFS2.

groups, which could be attributed to the favorable baseline risk factors at the time of salvage regimen receipt, particularly the higher likelihood of limited stage disease in patients selected to receive RT alone. Our findings demonstrate that RT can be an important treatment option following CAR T-cell therapy failure, aligning with previous series that support the use of RT for relapse after primary therapy of DLBCL.29,30 RT has been established as a fundamental modality in the management of LBCL. It has been shown that RT can improve patient outcomes for aggressive BCL that demonstrate resistance to systemic therapy.31–33 In the cellular therapy era, RT, in addition to its local control benefits, may enhance the efficacy of CAR T-cell therapy through its broad immunomodulatory roles and immunogenic effects on the immune system.16,25 Mechanistically, a plethora of evidence has supported the consideration of RT in the salvage setting following failure of CAR T-cell therapy. Potential roles of RT to circumvent barriers faced by CAR T-cell therapy and/or orchestrate CAR T-cell response include (i) overcoming the immunosuppressive cells in the tumor microenvironment,14,15 (ii) radiosensitizing tumor cells by upregulating specific chemokines, which appears to help trafficking of CAR T cells to infiltrate the tumor microenvironment,26,27 (iii) inducing various tumor-associated antigens expression such as major histocompatibility complex class I, resulting in eliciting antitumor responses,34–36 (iv) modulating CAR T cells to reestablish the appropriate memory T-cell phenotype,3,12,13 and/or (v) reinvigorating CAR T cells after T-cell exhaustion or senescence.7–9 Using allogeneic SCT as a consolidative strategy following CAR T-cell therapy infusion has been previously explored in patients with acute lymphoblastic leukemia.37–41 In lymphoma, RT has shown clear roles in the peri-transplant setting for patients with rel/ref LBCL. In patients with localized refractory LBCL, pretransplant RT can cytoreduce local residual fluorodeoxyglucose-avid disease thereby producing a complete metabolic response pre-autologous HSCT.42–44 In our study, four of five patients who received RT in the peri-transplant setting entered a CR with RT, allowing them to proceed with allogeneic HSCT. Similarly, Imber et al. presented three patients who had local relapses at time of CAR T-cell failure, received bridging RT prior to allogeneic HSCT, and had no evidence of disease at the time of the last follow-up. These early favorable outcomes highlight the need to continue to study this combination as a salvage strategy in selected patients with CAR T-cell treatment failure. Our patterns of failure analysis showed 97 of 124 (78%) patients progressed or relapsed in previously involved sites pre-CAR T. Imber et al.17 reported on 11 of 14 (79%) patients who received RT to previously fluorodeoxyglucose-avid sites pre-CAR T. Figura et al.’s18 subset analysis showed

Discussion CAR T-cell therapy has redefined the treatment paradigm for heavily pretreated relapsed/refractory LBCL patients. The pivotal CAR T trials, ZUMA-1,1 JULIET,2 and TRANSCEND,3 showed impressive outcomes with response rates ranging from 52% to 82% and 1-year OS rates ranging between 48% and 59%. While these trials led to the approval of CD19-targeted CAR T-cell therapy, questions remain regarding salvage strategies following CAR T-cell failure, which represents the majority of patients. Therefore, investigation of strategies to address CAR T failure is of paramount importance. To date, only a few studies have reported the real-world experience with using salvage therapies following CAR Tcell failure.17–19,22–24 To the best of our knowledge, we report the largest study thus far, which has explored the role of RT in comparison with other therapies in depth. We investigated the role of RT in treating local/distant recurrences post-CAR T and compared the impact of RT with other systemic regimens in the salvage setting. Our data showed that patients who received RT alone post-CAR T had superior median OS2 and EFS2 as compared to the other

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ARTICLE - Salvage radiation for post CAR T failure in LBCL that 31 of 36 progressions (86%) involve a component of local failure before CAR T-cell therapy infusion. Similarly, Saifi and colleagues45 revealed that 57 of 65 (88%) postCAR T failures occurred in sites of prior involvement. Together, these findings highlight the potential therapeutic significance of incorporating RT pre-and/or post-CAR Tcell therapy infusion to provide local control and optimize outcomes. Small number of local failures and selection bias likely makes it difficult to show a significant association between bulk and local control, but this warrants further study. While we found a dose-response relationship favoring higher doses for improved OS, only one comprehensively treated patient received low dose RT and only three comprehensively treated patients had >1 site of disease. As such, those receiving higher doses had lower burden of disease, which likely also led to more favorable outcomes. Early evidence from the pivotal CAR T trials and real-world studies suggested that patients with high tumor burden are more likely to experience inferior survival outcomes and lower durable remission rates following CAR T-cell therapy.1,2,10,46 Moreover, surrogate tumor biomarkers such as LDH pre-CART have been proven to be promising tools in predicting outcomes post-CAR T.46–50 However, prognostic factors at the time of CAR T-cell treatment failure have yet to be undefined. In our multicenter retrospective analysis of risk factors at the time of salvage therapy, we demonstrated that the presence of bulky disease (≥5 cm), presence of ≥2 sites of disease (nodal and/or extranodal), elevated LDH, stage 3-4 disease, and high IPI (≥3) status were identified to be prognostic markers for worse OS2 and EFS2; though elevated LDH, and advanced-stage disease portended the poorest OS2 and EFS2 in multivariate analyses. Hence, our data provide the rationale for the implementation of risk-stratification models by incorporating treatment biomarkers and baseline risk factors at the time of CAR T-cell treatment failure for predicting patients’ outcomes in future studies. There are several limitations of our study. The overwhelming majority in the salvage RT group received RT sequentially or concurrently with systemic regimens, precluding our ability to estimate the out-of-field recurrence rates post-RT on the post-RT imaging to ascertain if the out-field response was attributed to the effect of RT, systemic regimens, or both. Our findings are also limited by its retrospective nature and heterogeneity in RT dose/fractionation which was largely based on the extent of the disease.

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Conclusion CAR T-cell therapy holds great promise for rel/ref LBCL patients who would otherwise have poor outcomes, yet failure of CAR T-cell therapy is a pivotal challenge. Our data shows that disease burden and surrogate tumor biomarkers such as LDH at the time of CAR T-cell failure are associated with prognosis. RT is a feasible and promising therapeutic strategy that can provide local control, particularly in selected patients with limited-stage disease, and are able to receive comprehensive RT field. Small number of local failures and selection bias may have limited analysis regarding a dose-response relationship or an association with bulk. Disclosures HSA has no conflicts of interest to disclose. AKN reports honoraria from Elsevier and UpToDate; served on the Board of Trustee for the American Board of Radiology. MJF reports a consultancy role for Celgene, Novartis, Arcellx and Gilead/Kite; research funding from Novartis and Gilead/Kite. JSA reports consulting fees from Bristol Myers Squibb, AbbVie, Genentech, Epizyme, BeiGene, Kymera, Bluebird Bio, Incyte, Kite Pharma, Genmab, Ono Pharmaceutical, Mustang Bio, MorphoSys, Regeneron, Century, AstraZeneca, Lilly, and Janssen; and received research funding from Bristol Myers Squibb and Seattle Genetics. PCJ reports consulting fees from AstraZeneca. CAJ reports honoraria from Kite/Gilead, Novartis, Celgene, Bristol Myers Squibb, Nkarta, Precision Biosciences and Humanigen; reports consulting or advisory Role from Kite/Gilead, Precision Biosciences, Novartis, Celgene, Humanigen, Bristol Myers Squibb, Nkarta and Lonza; Speakers’ Bureau participation for Clinical Care Options, Axis Bioservices; reports research funding from Pfizer; and reports travel, accommodations, expenses from Kite/Gilead, Novartis and Precision Biosciences. Contributions HSA, CGP, and CAJ conceived and designed the study and wrote the manuscript. CGP, and CAJ provided supervision. All authors interpreted data, and contributed to revising the manuscript, and approved the submitted version. Data-sharing statement: The data generated in this study are not publicly available due to information that could compromise patient privacy or consent.

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H. S. Ababneh et al. autologous hematopoietic cell transplantation in relapsed and refractory DLBCL patients with minimal to no response to salvage chemotherapy. Bone Marrow Transplantation. 2022;57(6):1038-1041. 45. Saifi O, Breen WG, Lester SC, et al. Does bridging radiation therapy affect the pattern of failure after CAR T-cell therapy in non-Hodgkin lymphoma? Radiother Oncol. 2022;166:171-179. 46. Vercellino L, di Blasi R, Kanoun S, et al. Predictive factors of early progression after CAR T-cell therapy in relapsed/refractory diffuse large B-cell lymphoma. Blood Adv. 2020;4(22):5607-5615. 47. Jacobson CA, Hunter BD, Redd R, et al. Axicabtagene ciloleucel in the non-trial setting: Outcomes and correlates of response, resistance, and toxicity. J Clin Oncol. 2020;38(27):3095-3106. 48. Rabinovich E, Pradhan K, Sica RA, et al. Elevated LDH greater than 400 U/L portends poorer overall survival in diffuse large Bcell lymphoma patients treated with CD19 CAR-T cell therapy in a real world multi-ethnic cohort. Exp Hematol Oncol. 2021;10(1):55. 49. Nastoupil LJ, Jain MD, Feng L, et al. Standard-of-care axicabtagene ciloleucel for relapsed or refractory large B-cell lymphoma: results from the US Lymphoma CAR T Consortium. J Clin Oncol. 2020;38(27):3119-3128. 50. Bethge WA, Martus P, Schmitt M, et al. GLA/DRST real-world outcome analysis of CAR T-cell therapies for large B-cell lymphoma in Germany. Blood. 2022;140(4):349-358.

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Consolidative radiotherapy for residual fluorodeoxyglucose activity on day +30 post CAR T-cell therapy in non-Hodgkin lymphoma Omran Saifi,1 William G. Breen,2 Scott C. Lester,2 William G. Rule,3 Bradley J. Stish,2 Allison Rosenthal,4 Javier Munoz,4 Yi Lin,5,6 Radhika Bansal,5 Matthew A. Hathcock,5,7 Patrick B. Johnston,5 Stephen M. Ansell,5 Jonas Paludo,5 Arushi Khurana,5 Jose C Villasboas,5 Yucai Wang,5 Madiha Iqbal,8 Muhamad Alhaj Moustafa,8 Hemant S. Murthy,8 Mohamed A. KharfanDabaja,8 Jennifer L. Peterson1 and Bradford S. Hoppe1 Department of Radiation Oncology, Mayo Clinic Jacksonville, Jacksonville, FL; 2Department of Radiation Oncology, Mayo Clinic Rochester, Rochester, MN; 3Department of Radiation Oncology, Mayo Clinic Phoenix, Phoenix, AZ; 4Division of Hematology and Medical Oncology, Mayo Clinic Phoenix, Phoenix, AZ; 5Division of Hematology, Mayo Clinic Rochester, Rochester, MN; 6Division of Experimental Pathology, Mayo Clinic Rochester, Rochester, MN; 7 Department of Biostatistics, Mayo Clinic Rochester, Rochester, MN and 8Division of Hematology and Medical Oncology, Mayo Clinic Jacksonville, Jacksonville, FL, USA

Correspondence: B. S. Hoppe hoppe.bradford@mayo.edu Received: Accepted: Early view:

April 9, 2023. June 1, 2023. June 15, 2023.

Abstract Majority of non-Hodgkin lymphoma (NHL) patients who achieve partial response (PR) or stable disease (SD) to CAR T-cell therapy (CAR T) on day +30 progress and only 30% achieve spontaneous complete response (CR). This study is the first to evaluate the role of consolidative radiotherapy (cRT) for residual fluorodeoxyglucose (FDG) activity on day +30 postCAR T in NHL. We retrospectively reviewed 61 patients with NHL who received CAR T and achieved PR or SD on day +30. Progression-free survival (PFS), overall survival (OS), and local relapse-free survival (LRFS) were assessed from CAR T infusion. cRT was defined as comprehensive - treated all FDG-avid sites - or focal. Following day +30 positron emission tomography scan, 45 patients were observed and 16 received cRT. Fifteen (33%) observed patients achieved spontaneous CR, and 27 (60%) progressed with all relapses involving initial sites of residual FDG activity. Ten (63%) cRT patients achieved CR, and four (25%) progressed with no relapses in the irradiated sites. The 2-year LRFS was 100% in the cRT sites and 31% in the observed sites (P<0.001). The 2-year PFS was 73% and 37% (P=0.025) and the 2-year OS was 78% and 43% (P=0.12) in the cRT and observation groups, respectively. Patients receiving comprehensive cRT (n=13) had superior 2year PFS (83% vs. 37%; P=0.008) and 2-year OS (86% vs. 43%; P=0.047) compared to observed or focal cRT patients (n=48). NHL patients with residual FDG activity following CAR T are at high risk of local progression. cRT for residual FDG activity on day +30 post-CAR T appears to alter the pattern of relapse and improve LRFS and PFS.

Introduction Aggressive relapsed and/or refractory (r/r) B cell nonHodgkin lymphoma (NHL) remains a significant therapeutic challenge with poor outcomes. Anti-CD19-directed chimeric antigen receptor T-cell therapies (CAR T) are approved for treatment of r/r B-cell NHL, specifically large B-cell lymphoma, mantle cell lymphoma and follicular lymphoma. CAR T has demonstrated an objective response rate of 46% to 86%, and complete response rate of 28% to 66% for refractory large B-cell subtype.1-5 However, sustained CAR T efficacy is limited, with durable complete response (CR) rates of approximately 40%.1,6 Furthermore, up to 30% of NHL patients achieve a partial response (PR) or stable disease (SD) to CAR T on day +30,

with most patients experiencing disease progression, specifically those with Deauville score (DS) 4-5.7 Only 20% to 30% of PR/SD patients achieve spontaneous CR by day +90 without additional therapies.1 Patients who progress post-CAR T have poor overall survival (OS).8 Therefore, innovative consolidative strategies for this population are needed to augment disease control and prevent relapses. There is compelling rationale to consider consolidative radiotherapy (cRT) for post CAR T disease. Patients who receive CAR T usually have purported chemoresistant but radiosensitive disease.9 The predominant pattern of relapse of NHL following CAR T involves a local component.10,11 Consolidative RT to initial bulky sites, extranodal sites, and residual fluorodeoxyglucose (FDG)avid disease has long proven to improve outcomes in pa-

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ARTICLE - cRT for residual FDG activity on day +30 post-CAR T tients with aggressive NHL.12-14 Similarly, RT improves outcomes when offered as part of the peri-autologous stem cell transplant regimen, specifically for patients with bulky disease, limited sites of relapse, or partially responded disease.15-19 RT has promising outcomes in the peri-CAR T settings as a bridging strategy. It helps control the disease during the CAR T manufacturing period, improve rates of CAR T infusion, and possibly augment local control.10,20-23 In the salvage setting, RT has recently shown to improve survival outcomes for limited relapsed NHL post autologous stem cell transplant and CAR T.18,24,25 To date, no data exist on the optimal management of patients with residual FDG activity on day +30 post-CAR T. This study is the first to report on the role of consolidative RT for residual FDG activity (DS 4-5) on day +30 post-CAR T in B-cell NHL.

Methods Following Institutional Review Board approval, records of consecutive patients diagnosed with r/r B-cell NHL who received CAR T and achieved PR or SD on day +30 were retrospectively studied across three institutions between 2018 and 2022. This study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. Ann Arbor staging system was used to define the number of involved/FDG-avid lymph node sites. Residual activity (RA) corresponded to patients with PR or SD who had FDG-avid (DS 4-5) sites on day +30. Only patients with ≤4 RA sites on day +30 post-CAR T were included. This number is based on a previous publication that defined post-CAR T limited relapsed disease.18 Bridging therapy (systemic or radiation) was defined as treatment administered between leukapheresis and lymphodepleting chemotherapy. Remote radiotherapy was defined as RT administered >30 days from CAR T infusion. Consolidative systemic therapy was defined as chemo/immuno/transplant therapy administered following day +30 post-CAR T for RA. cRT was defined as RT delivered following day +30 post-CAR T for RA. Comprehensive cRT was defined as treating all residual FDG-avid sites identified on day +30. Focal cRT was defined in cases where not all residual FDG-avid sites were treated. RT was at the discretion of the treating physician and consisted of positron emission tomography (PET)-directed residual site radiotherapy (PD-RSRT) or involved site radiotherapy (ISRT). RT-related toxicities were graded prospectively per the Common Terminology Criteria for Adverse Events version 5 (CTCAE v.5). The RT equivalent 2 Gy dose was calculated based on an α/β ratio of 10. Following the management approach on day +30, response to treatment was assessed on day +90-100 with a

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PET/computed tomography (PET/CT) scan using the Lugano criteria.26 Subsequent imaging were performed for assessment of response or lack thereof. CR was defined as a DS of 1-3, PR/SD were defined as a DS of 4-5 with absence of disease progression, and progression of disease (PD) was defined as a DS of 4-5 following a CR/PR. Local response of individual RA sites was assessed based on careful review of the location of RA sites identified on day +30 PET/CT scan and their response on subsequent imaging. Statistical analysis was performed using IBM SPSS 28.0 software. Continuous data were reported as medians and ranges. Categorical data were reported as frequencies and percentages. Comparisons of different characteristics between the two groups were done using Χ2 and Fisher’s exact tests. The non-parametric independent samples median test was used to compare median values between two groups. Progression-free survival (PFS), overall survival (OS), and local relapse-free survival (LRFS) were estimated by Kaplan-Meier survival curves. Survival differences were assessed by the log-rank test. All reported P values were two-sided, and differences were considered statistically significant at P<0.05. The median follow-up period was calculated from the CAR T infusion date to the last documented follow-up visit. LRFS was calculated based on the total number of RA sites and was defined from CAR T infusion to local relapse (LR) in the RA site identified on day +30. PFS was defined from CAR T infusion to any local/distant disease progression. OS was defined from CART infusion to death.

Results Management approach on day +30 post-CAR T Sixty-eight patients were identified with B-cell NHL who achieved PR or SD to CAR T with residual FDG activity on day +30 between 2018 and 2022. Among those, 61 patients had ≤4 residual FDG-avid sites and met our inclusion criteria, and seven patients had ≥5 residual FDG-avid sites and were excluded. Only one patient had biopsy proven persistent disease and the rest were unbiopsied and classified as having RA based on radiological assessment. Following day +30 PET/CT scan, 45 patients with RA were observed and 16 received cRT. Only one patient received consolidative systemic therapy (polatuzumab vedotin combined with bendamustine and rituximab) and belonged to the cRT group. The median follow-up from CAR T infusion was 21 months for the entire cohort. Baseline characteristics The median age of the cohort was 62 (range, 18-85) years, and the maximum number of residual FDG-avid sites per patient identified on day +30 post-CART was three. There

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ARTICLE - cRT for residual FDG activity on day +30 post-CAR T

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Table 1. Pre-CAR T and day +30 post-CAR T baseline characteristics. Management on day +30 post-CAR T Observation (N=45)

Consolidative radiotherapy (N=16)

Total (N=61)

Baseline characteristics at time of CAR T infusion Age in years Median (range) Sex, N (%) Female Male Histology, N (%) DLBCL FL HGBCL MCL PMBCL Disease stage, N (%) Advanced Limited Extranodal involvement, N (%) No Yes

62 (18-82)

61 (33-85)

62 (18-85)

0.49

13 (28.9) 32 (71.1)

5 (31.3) 11 (68.7)

18 (29.5) 43 (70.5)

0.86

39 (86.7) 0 (0) 5 (11.1) 0 (0) 1 (2.2)

14 (87.5) 1 (6.3) 0 (0) 1 (6.3) 0 (0)

53 (86.9) 1 (1.6) 5 (8.2) 1 (1.6) 1 (1.6)

0.1

17 (37.8) 28 (62.2)

9 (56.3) 7 (43.8)

26 (42.6) 35 (57.4)

0.2

29 (64.4) 16 (35.6)

7 (43.8) 9 (56.3)

36 (59.0) 25 (41.0)

0.15

2 (1-4)

2 (1-7)

0.47

Biggest tumor size, cm Median (range)

7.3 (1.1-20)

4.5 (2-17.1)

6.8 (1.1-20)

0.21

Highest SUVmax Median (range)

17.6 (3.2-42.2

17.5 (2.7-38.1)

17.6 (2.7-42.2)

0.51

LDH level, mU/mL Median (range)

232 (127-669)

218 (150-470)

228 (127-669)

0.2

CRP level, mg/L Median (range)

11.6 (1-160.9)

12.55 (1-75.1)

11.6 (1-160.9)

0.95

Bridging chemotherapy, N (%) No Yes

30 (66.7) 15 (33.3)

12 (75) 4 (25)

42 (68.9) 19 (31.1)

0.54

Bridging radiotherapy, N (%) No Yes

36 (80) 9 (20)

12 (75) 4 (25)

48 (78.7) 13 (21.3)

0.68

Previous lines of therapy, N Median (range)

Baseline characteristics on day +30 post-CAR T # Residual FDG-avid sites, N Median (range) RA site size, cm Median (range)

1 (1-3)

2 (1-3)

1 (1-3)

0.007

2.6 (0.7-16)

1.8 (0.5-14)

2 (0.5-16)

0.054

5.1 (2.4-38.2)

5.2 (2.0-32.0)

5.1 (2.0-38.2)

0.77

49 (76.6) 15 (23.4)

20 (74.1) 7 (25.9)

69 (75.8) 22 (24.2)

0.8

RA site SUVmax Median (range) RA site SUVmax>10, N (%) No Yes Deauville score, N (%) 4 5 LDH level, mU/mL Median (range)

36 (80) 9 (20)

14 (87.5) 2 (12.5)

50 (82) 11 (18)

0.5

205 (141-623)

194.5 (144-436)

200 (141-623)

0.28

CRP level, mg/L Median (range)

2.9 (1-163.7)

1 (1-14.5)

1.95 (1-163.7)

0.58

CAR T: anti-CD19-directed chimeric antigen receptor T-cell therapy; DLBCL: diffuse large B-cell lymphoma; FL: follicular lymphoma; HGBCL: high grade B-cell lymphoma; MCL: mantle cell lymphoma; PMBCL: primary mediastinal B-cell lymphoma; SUVmax: maximum standardized uptake volume; LDH: lactate dehydrogenase; CRP: C-reactive protein; FDG: fluorodeoxyglucose; RA: residual activity.

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ARTICLE - cRT for residual FDG activity on day +30 post-CAR T were no significant differences in the pre-CAR T baseline characteristics between patients who received cRT and those who did not. Prior to CAR T, bridging RT was given to 13 patients (9 in observation group and 4 in cRT group) and bridging chemotherapy was given to 19 patients (15 in observation group and 4 in cRT group). The residual FDGavid sites on day +30 were radiation-naïve in 48 patients (34 in observation group and 14 in cRT group), received remote RT in four patients (3 in observation group and 1 in cRT group), and received bridging RT in nine patients (8 in observation group and 1 in cRT group). There were no significant differences in the day +30 baseline characteristics, including the median size and maximum standardized uptake volume (SUVmax) of the residual FDGavid sites, between the two groups. However, the median number of residual FDG-avid sites on day +30 was higher in the cRT group (2 vs. 1; P=0.007). Further baseline disease specific, patient specific, and treatment specific characteristics are listed in Table 1.

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neck (n=5), pelvis (n=4), skeleton (n=3) and mediastinum/thorax (n=2). The median size and SUVmax of the irradiated sites with residual activity were 2.5 (range, 0.5-14) cm, and 5.2 (range, 2-32), respectively. Six residual FDG-avid sites consolidated with cRT were extranodal (skeleton n=3, soft tissue/muscles n=1, colon n=1, lung n=1). The median time between CART infusion and cRT initiation was 57 (range, 31-118) days. cRT was delivered through 3-dimentional (n=6), IMRT (n=9), or proton therapy (n=1) techniques. Thirteen patients were treated with PD-RSRT and three with ISRT. Comprehensive cRT was given to 13 patients and focal cRT was given to three patients. The number of residual FDG-avid sites in the comprehensive cRT patients ranged between one and three with all sites receiving cRT to a median EQD2 dose of 39.1 Gy. The number of residual FDG-avid sites in the focal cRT patients ranged between two and three with only one site per patient receiving cRT. Focal cRT was delivered to a definitive dose of ≥40 Gy EQD2 in two patients and to a palliative dose of 12 Gy EQD2 in one patient. The unirradiated residual FDG-avid sites in the focal cRT patients did not receive prior remote or bridging RT. One patient received cRT (proton SBRT 39 Gy in 3 fractions) to a site with residual activity that was previously irradiated in the bridging setting (37.5 Gy in 10

Consolidative radiotherapy The median equivalent 2 Gy (EQD2) dose for cRT was 39.1 (interquartile range [IQR], 39.1-40) Gy, and the most common cRT regimen was 37.5 Gy in 15 fractions. The target sites included the abdomen (n=8), axilla (n=5), head and

Table 2. Baseline and radiation treatment characteristics of patients consolidated with radiotherapy. Patient Local Number Number Disease Number ultimate recurrenc RT Sites of of FDGPatient Type RT stage Day +30 of overall RT in-field e in the Progression dose response previous avid # of cRT irradiate fractions pre- response response recurrence residual (Gy) to cRT lines of sites on CAR T d sites to CAR T FDG-avid therapy day +30 and cRT site(s) 1

Advanced

Focal

Yes

Advanced

Focal

Yes

Advanced

Focal

Limited

Comp

Limited

Comp

Limited

Comp

Advanced

Comp

37.5

Advanced

Comp

Limited

Comp

Limited

Comp

Limited

Comp

Limited

Comp

Advanced

Comp

Advanced

Comp

37.5

Yes

Advanced

Comp

37.5

Yes

Advanced

Comp

CAR T: anti-CD19-directed chimeric antigen receptor T-cell therapy; PR: partial response; SD: stable disease; CR: complete response; PD: progression of disease; FDG: fluorodeoxyglucose; cRT: consolidative radiotherapy; comp: comprehensive; RT: radiotherapy; Gy: Gray. Haematologica | 108 November 2023

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ARTICLE - cRT for residual FDG activity on day +30 post-CAR T fractions). Nine patients had grade zero, three patients had grade 1 (fatigue, pharyngitis, xerostomia), and four patients had grade 2 (fatigue, pharyngitis/esophagitis, xerostomia, nausea, diarrhea, dermatitis) RT-related toxicities. There were no grade 3 or higher RT-related toxicities in this cohort. None of the patients developed a flare of cytokine release syndrome following cRT. Detailed description of the 16 cRT patients is provided in Table 2. Local response A total of 91 sites with RA were identified on day +30 post-CAR T; 64 were observed and 27 received cRT. Sustained CR was achieved in 14 (22%) observed sites with-

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out additional therapies compared to 24 (92%) sites consolidated with RT (P<0.001). Forty-two (66%) observed sites experienced LR, while no cRT sites relapsed (P<0.001). The 2-year LRFS was 100% in the cRT sites and 31% in the observed sites (P<0.001) (Figure 1). Fifteen disease sites (11 in observation group and 4 in cRT group) corresponding to 13 patients received bridging RT prior to CART. Only three (20%) bridged sites (3 in observation group and 0 in cRT group) corresponding to three patients experienced local relapse in the bridging radiation field. Among the aforementioned BRT sites, nine (8 in observation group and 1 in cRT group) were identified on day +30 with RA, and three (33%) experienced disease progression.

Figure 1. Local recurrence-free survival by receipt of consolidative radiation to individual sites. RA: residual activity; RT: radiotherapy.

Table 3. Disease progression stratified by the number of sites with residual fluorodeoxyglucose activity and management approach on day +30 post-CAR T. Number of sites with residual activity on Management approach on day +30 post-CAR T day +30 post-CAR T (N) (N)

Disease progression N (%)

One site with residual activity (38 patients)

Observation (33) RT (5)

17 (51.5) 0 (0)

0.031

Two sites with residual activity (13 patients)

Observation (6) RT (7)

5 (83) 2 (29)

0.048

Three sites with residual activity (10 patients)

Observation (6) RT (4)

5 (83) 2 (50)

0.26

CAR T: anti-CD19-directed chimeric antigen receptor T-cell therapy; RT: radiotherapy.

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ARTICLE - cRT for residual FDG activity on day +30 post-CAR T Patients’ outcomes and pattern of relapse Among the observed patients, 15 (33%) achieved spontaneous CR without additional therapies with two of 15 subsequently relapsing, five (11%) remained with PR/SD, and 27 (60%) experienced disease progression with all relapses involving the original sites of residual activity. Among patients who received cRT, ten (63%) achieved CR, two (12%) achieved a PR, and four (25%) had disease

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progression with no relapses in the irradiated sites. The relapses (n=2) in the comprehensive cRT patients were out-of-field and involved new sites that were not present on day +30. The relapses (n=2) in the focal cRT patients involved the original sites with RA that were present on day +30 but did not get irradiated. None of the ten cRT patients achieving CR relapsed or required subsequent therapies. The effect of cRT was most prominent in pa-

Figure 2. Progression-free survival by management approach on day +30. CAR T: anti-CD19-directed chimeric antigen receptor T-cell therapy; RT: radiotherapy.

Figure 3. Overall survival by management approach on day +30. CAR T: anti-CD19-directed chimeric antigen receptor T-cell therapy; RT: radiotherapy.

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ARTICLE - cRT for residual FDG activity on day +30 post-CAR T tients with ≤2 residual FDG-avid sites on day +30, especially in those with only one FDG-avid site post-CAR T (Table 3). The 2-year PFS was 73% in the cRT group and 37% in the observation group (P=0.025) (Figure 2). The 2-year OS was 78% in the cRT group and 43% in the observation

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group (P=0.12) (Figure 3). Patients consolidated with comprehensive cRT to all residual FDG-avid sites (n=13) had superior 2-year PFS (83% vs. 37%; P=0.008) (Figure 4) and 2-year OS (86% vs. 43%; P=0.047) (Figure 5) compared to those who were observed or received focal cRT to some but not all residual FDG-avid sites (n=48).

Figure 4. Progression-free survival by receipt of comprehensive consolidative radiotherapy. Comp: comprehensive; cRT: consolidative radiotherapy.

Figure 5. Overall survival by receipt of comprehensive consolidative radiotherapy. Comp: comprehensive; cRT: consolidative radiotherapy.

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ARTICLE - cRT for residual FDG activity on day +30 post-CAR T

Discussion This study is the first to report on the role of consolidative radiotherapy for residual FDG activity on day +30 postCART in B-cell NHL. Despite the small sample size and relatively short follow-up period, patients with residual activity who received cRT had significantly better outcomes with minimal added toxicity compared to those who were observed on day +30 post-CAR T. In our cohort, patients who achieved a PR or SD to CAR T on day +30 and were observed, had a high risk of disease progression with a predominant localized pattern of relapse. However, cRT following day +30 altered the pattern of relapse, as evident with the absence of relapses in the irradiated FDG-avid sites. Such alteration in the pattern of relapse resulted in a local recurrence-free survival benefit which translated to an improvement in PFS. Such PFS benefit is promising and may result in an encouraging OS when all residual FDG-avid sites are consolidated with radiotherapy (comprehensive cRT). The current most likely adopted management approach for patients with residual activity/disease on day +30 post-CAR T is observation. This is based on the ZUMA-1 trial findings that showed a 20% to 30% conversion rate of PR/SD to CR without additional therapies,1 which coincides with the spontaneous conversion rate (33%) seen in our cohort. Thus, the ZUMA-1 trial concluded that it would be reasonable to monitor PR/SD patients to allow for an opportunity for an improved response, since consolidation with allogenic stem cell transplantation comes with a high rate of treatment-related death and would ablate CAR T cells.1 Yet, this recommendation ignores the fact that 70% to 80% of PR/SD patients will likely progress and that cRT can be an effective and minimally toxic consolidative treatment option, as we have shown in this study. This is further supported by Kuhnl et al. who showed that patients with a DS of 4-5 on day +30 have a high risk of relapse and shorter duration of response which calls for risk-adaptive treatment approaches for these patients to prevent/delay progression.7 The utility of cRT can be further supported by understanding the pattern of relapse of NHL following CAR T. Previous studies have shown >85% of post-CAR T relapses involve a local component.10,11 Such pattern of local relapse is more evident in those who achieve a PR/SD to CAR T, as 100% of the relapses in our observed cohort involved the sites with RA that were originally present prior to CAR T. The local relapse component has also been significantly present but to a lesser extent (40-60%) in the frontline setting as evident by multiple seminal studies that established the standard of care for NHL.27-30 This might suggest an increase in localized chemorefractory disease in the r/r setting following multiple lines of therapies, where persistent gross disease, not ablated by initial therapies, may

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be less responsive to new systemic treatments and more likely to relapse. This calls for local therapies to augment local control in the r/r setting, and intriguingly, none of the radiated FDG-avid sites in our cohort relapsed. The role of local therapy becomes more noticeable and valuable as we move forward in understanding and defining oligometastatic lymphoma. This can be inferred from the improved outcomes seen with stereotactic body radiotherapy (SBRT) when offered as a consolidative local therapy for oligometastatic solid malignancies.31,32 Our study included only patients with ≤4 residual FDG-avid sites based on a previous publication,18 and not based on any evidence-base for the utility/benefit of RT. That study suggested comprehensive RT to be feasible for ≤4 lymphoma disease sites with improvement in survival metrics18, and thus formed the basis to our inclusion criteria. From an oligometastatic standpoint, the impact of cRT in our cohort was mostly evident in patients with ≤2 residual FDG-avid sites, especially in those with only one site on day +30 post-CAR T (Table 3). While acknowledging the limited number of patients with >2 FDG-avid sites (n=10) in our study, the tumor burden, and the decreased feasibility of comprehensive cRT with increasing number of FDG-avid sites, one might consider adding consolidative systemic therapy to cRT for patients who have multiple sites of RA on day +30 post-CAR T. Furthermore, it is important to acknowledge that patients with one residual FDG-avid site have less tumor burden and will fall under the comprehensive cRT subcohort by default if they receive cRT. This may limit comparisons between focal and comprehensive cRT subgroups if tumor burden is not accounted for. Moreover, it was noted in our study (Table 3) that cRT was more likely to be offered to patients with >1 RA site, as the perception for 1 RA site is perhaps to resolve spontaneously. Intriguingly, 51.5% of patients with 1 RA site who were observed relapsed. All relapses had a local component and involved the same RA site identified on day +30, rationalizing the use of local therapy, such as RT, that showed to be effective for these patients in our study. Radiation has long proven to be a successful and effective consolidative treatment for aggressive NHL in the frontline and r/r setting. As part of a combined modality therapy in the upfront setting, consolidative radiotherapy improves outcomes compared to chemotherapy alone.30,33 This is mostly evident in patients with bulky disease,12,34 extranodal involvement,14 and residual partially responded disease.35,36 Compellingly, patients with PR who receive cRT have comparable survival outcomes to those who achieve CR to systemic therapy.35-37 In the r/r setting, peri-transplant RT augments local control and improves outcomes when offered as a consolidative therapy pre- or post-stem cell transplant.17,38-45 For limited relapsed (1 site) posttransplant disease, salvage RT improves OS compared to

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ARTICLE - cRT for residual FDG activity on day +30 post-CAR T salvage chemotherapy.24 Studies supporting the utility of RT in the peri-CAR T setting are more limited but have promising outcomes. RT was mostly studied in the bridging setting prior to CAR T and has shown to be safe and effective in controlling the disease during the manufacturing period with favorable local control rates that can augment the efficacy of CAR T.10,18,19,21-23,46 More recently, it was shown that comprehensive salvage RT for limited (≤4 disease sites) post-CAR T relapsed disease improves survival,18,25 supporting the utility of RT for oligoprogressive post-CAR T lymphoma. However, no studies investigated the utility of RT as a consolidative therapy following CAR T for residual non-progressive disease. Given the aforementioned reasons, RT was adopted in the participating institutions as a consolidative management approach on day +30 post-CAR T, with very favorable outcomes as reported in the results section. Utilizing RT in the bridging pre-CAR T or consolidative post-CAR T setting remains inconclusive. Eleven (24%) patients in our observation group received prior RT (3 remote RT and eight bridging RT) which might have precluded them from getting additional consolidative radiation on day +30, contemplating the optimal timing of peri-CAR T radiotherapy. However, four (25%) patients in our cRT cohort received bridging RT prior to CAR T, suggesting the feasibility of administering RT pre- and post-CAR T if needed. This was more achievable if the residual FDG-avid sites on day +30 were not radiated in the bridging or remote setting. Some concerns arise on whether an early administration of RT on day +30 may have an impact on circulating CAR T cells. There are no data yet to support or refute this argument, however, the favorable outcomes of our cohort suggest the safety of cRT with no obvious detrimental effect on CART. Animal studies have shown radiation to increase the efficacy of CART and perhaps induce and abscopal-like effect.47-49 A recent study showed a plausible synergistic effect of RT with CAR T, and there may be an optimal timing to deliver RT during peri-CAR T.18 Plausibly, RT when administered approximately 30 days following CAR T, may sensitize and reactivate CAR T cells. Studies investigating the impact of RT on circulating CAR T cells and released cytokines are needed. This study is limited by its retrospective nature and the relatively small sample size, especially in the cRT cohort. RT was delivered at the discretion of the treating radiation oncologist with no standardized dosing and fractionation regimens. There were no clear indications for referring patients to receive cRT and the decision was at the discretion of the treating hematologist. Furthermore, the majority of patients (98%) did not get a biopsy to confirm the FDG-avidity is actually reflective of persistent lymphoma, and, therefore, it would not be possible to tell how many patients had true persistent disease versus in-

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flammatory changes in both cohorts. Some patients with RA may spontaneously convert to CR, either due to the resolution of FDG-avid inflammatory changes, or due to the continued activity of CAR T cells against residual lymphoma. In these particular scenarios, which are difficult to identify/quantify, the addition of radiation may seem to be unnecessary. Therefore, identifying patients with RA at risk of progression is important to select the right management approach, and this was mostly based on clinical and radiological assessment in our study. Studies suggested that patients with SUVmax>10 on day +30,50 and DS of 4-57 have higher risk of progression, and, therefore, may be used as parameters to identify patients who may benefit from consolidative treatments. Nonetheless, the detrimental outcome of post-CAR T relapses calls for maximal utility of upfront peri-CAR T treatments, and consolidative radiotherapy appeared to be highly effective and minimally toxic in our cohort. Also, it is important to acknowledge the short follow-up period in cRT cohort, as evident in the Kaplan-Meier curves, given the recent adoption of this management approach by the participating institutions. Longer follow-up is needed to confirm the response durability to cRT. Finally, despite the absence of significant differences in baseline characteristics between both cohorts, there might be other unaccounted confounding factors

Conclusion Patients with B-cell NHL who achieve PR or SD by PET to CAR T are at high risk of local progression. cRT for residual FDG activity on day +30 post-CAR T appears to alter the pattern of relapse and improve local recurrence-free survival and PFS. Comprehensive cRT further improves PFS and results in promising overall survival outcomes. An equivalent 2 Gy dose of 39-40 Gy seems sufficient to provide excellent local control with low toxicity. Longer follow-up and larger multicenter prospective studies are needed to confirm our findings. Disclosures JM consults for Pharmacyclics/Abbvie, Bayer, Gilead/Kite Pharma, Pfizer, Janssen, Juno/Celgene, BMS, Kyowa, Alexion, Beigene, Fosunkite, Innovent, Seattle Genetics, Debiopharm, Karyopharm, Genmab, ADC Therapeutics, Epizyme and Servier; discloses research funding Bayer, Gilead/Kite Pharma, Celgene, Merck, Portola, Incyte, Genentech, Pharmacyclics, Seattle Genetics, Janssen and Millennium; has received honoraria from Targeted Oncology, OncView, Curio, Kyowa, Physicians' Education Resource, and Seattle Genetics; is part of the Speaker’s bureau of Gilead/Kite Pharma, Kyowa, Bayer, Pharmacyclics/Janssen, Seattle Genetics, Acrotech/Aurobindo, Bei-

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ARTICLE - cRT for residual FDG activity on day +30 post-CAR T gene, Verastem, AstraZeneca, Celgene/BMS and Genentech/Roche. YL consults for Kite/Gilead, Celgene/BMS, Juno/BMS, BlueBird Bio, Janssen, Legend BioTech, Gamida Cells, Novartis, Iovance, Takeda, Fosun Kite and Pfizer; grant/research support for the highlighted; serves on the data safety and monitoring board for Sorrento; is on the data review committee of Pfizer; is on the scientific advisory committee of NexImmune. HS is an advisory board member of CRISPR Therapeutics, Senti Biosciences and Jazz pharmaceuticals. All other authors have no conflicts of interest to disclose. Contributions OS, JLP and BSH concveived and designed the study. OS,

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JLP, BSH, MAKD, YL and WGB provided study materials or patients. OS, WGB, RB, JLP, BSH and MAKD collected and assembled data. OS, JLP, BSH and MAKD analyzed and interpreted data. All authors wrote the manuscript, gave the final approval of the manuscript and are accountable for all aspects of the work. Funding There was no funding for this project. Data-sharing statement All relevant data are available in the article, Online Supplementary Appendix or from the corresponding author upon reasonable request.

References 1. Neelapu SS, Locke FL, Bartlett NL, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med. 2017;377(26):2531-2544. 2. Abramson JS, Palomba ML, Gordon LI, et al. Lisocabtagene maraleucel for patients with relapsed or refractory large B-cell lymphomas (TRANSCEND NHL 001): a multicentre seamless design study. Lancet. 2020;396(10254):839-852. 3. Schuster SJ, Tam CS, Borchmann P, et al. Long-term clinical outcomes of tisagenlecleucel in patients with relapsed or refractory aggressive B-cell lymphomas (JULIET): a multicentre, open-label, single-arm, phase 2 study. Lancet Oncol. 2021;22(10):1403-1415. 4. Locke FL, Miklos DB, Jacobson CA, et al. Axicabtagene ciloleucel as second-line therapy for large B-cell lymphoma. N Engl J Med. 2022;386(7):640-654. 5. Bishop MR, Dickinson M, Purtill D, et al. Second-line tisagenlecleucel or standard care in aggressive B-cell lymphoma. N Engl J Med. 2022;386(7):629-639. 6. Nair R, Neelapu SS. The promise of CAR T-cell therapy in aggressive B-cell lymphoma. Best Pract Res Clin Haematol. 2018;31(3):293-298. 7. Kuhnl A, Roddie C, Kirkwood AA, et al. Early FDG-PET response predicts CAR-T failure in large B-cell lymphoma. Blood Adv. 2022;6(1):321-326. 8. Chow VA, Gopal AK, Maloney DG, et al. Outcomes of patients with large B-cell lymphomas and progressive disease following CD19-specific CAR T-cell therapy. Am J Hematol. 2019;94(8):E209-E213. 9. Martens C, Hodgson DC, Wells WA, et al. Outcome of hyperfractionated radiotherapy in chemotherapy-resistant nonHodgkin's lymphoma. Int J Radiat Oncol Biol Phys. 2006;64(4):1183-1187. 10. Saifi O, Breen WG, Lester SC, et al. Does bridging radiation therapy affect the pattern of failure after CAR T-cell therapy in non-Hodgkin lymphoma? Radiother Oncol. 2022;166:171-179. 11. Figura NB, Robinson TJ, Sim AJ, et al. Patterns and predictors of failure in recurrent or refractory large B-cell lymphomas after chimeric antigen receptor T-cell therapy. Int J Radiat Oncol Biol Phys. 2021;111(5):1145-1154. 12. Held G, Murawski N, Ziepert M, et al. Role of radiotherapy to bulky disease in elderly patients with aggressive B-cell lymphoma. J Clin Oncol. 2014;32(11):1112-1118.

13. Poeschel V, Held G, Ziepert M, et al. Four versus six cycles of CHOP chemotherapy in combination with six applications of rituximab in patients with aggressive B-cell lymphoma with favourable prognosis (FLYER): a randomised, phase 3, noninferiority trial. Lancet. 2019;394(10216):2271-2281. 14. Held G, Zeynalova S, Murawski N, et al. Impact of rituximab and radiotherapy on outcome of patients with aggressive B-cell lymphoma and skeletal involvement. J Clin Oncol. 2013;31(32):4115-4122. 15. Philip T, Armitage JO, Spitzer G, et al. High-dose therapy and autologous bone marrow transplantation after failure of conventional chemotherapy in adults with intermediate-grade or high-grade non-Hodgkin's lymphoma. N Engl J Med. 1987;316(24):1493-1498. 16. Ng AK, Yahalom J, Goda JS, et al. Role of radiation therapy in patients with relapsed/refractory diffuse large B-cell lymphoma: guidelines from the international lymphoma radiation oncology group. Int J Radiat Oncol Biol Phys. 2018;100(3):652-669. 17. Hoppe BS, Moskowitz CH, Filippa DA, et al. Involved-field radiotherapy before high-dose therapy and autologous stemcell rescue in diffuse large-cell lymphoma: long-term disease control and toxicity. J Clin Oncol. 2008;26(11):1858-1864. 18. Saifi O, Breen WG, Lester SC, et al. Don't put the CART before the horse: the role of radiation therapy in peri-CAR T-cell therapy for aggressive B-cell non-Hodgkin lymphoma. Int J Radiat Oncol Biol Phys. 2023;116(5):999-1007. 19. Saifi O, Breen W, Lester SC, et al. The impact of radiation timing in peri-CAR T-cell therapy on local control for relapsed/refractory B-cell non-Hodgkin lymphoma. Int J Radiat Oncol Biol Phys. 2022;114(3):S85-S86. 20. Saifi O, Breen W, Lester S, et al. In-field recurrences in relapsed/refractory (R/R) B-cell non-Hodgkin lymphoma (NHL) bridged with radiation prior to CD19 chimeric antigen receptor Tcell therapy (CART). J Clin Oncol. 2022;40(Suppl 16):S7556-7556. 21. Sim AJ, Jain MD, Figura NB, et al. Radiation therapy as a bridging strategy for CAR T cell therapy with axicabtagene ciloleucel in diffuse large B-cell lymphoma. Int J Radiat Oncol Biol Phys. 2019;105(5):1012-1021. 22. Wright CM, LaRiviere MJ, Baron JA, et al. Bridging radiation therapy before commercial chimeric antigen receptor T-cell therapy for relapsed or refractory aggressive B-cell lymphoma.

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ARTICLE - cRT for residual FDG activity on day +30 post-CAR T Int J Radiat Oncol Biol Phys. 2020;108(1):178-188. 23. Pinnix CC, Gunther JR, Dabaja BS, et al. Bridging therapy prior to axicabtagene ciloleucel for relapsed/refractory large B-cell lymphoma. Blood Adv. 2020;4(13):2871-2883. 24. Ladbury C, Kambhampati S, Othman T, et al. Role of salvage radiation treatment of relapses in relapsed/refractory diffuse large B cell lymphoma post-autologous stem cell transplant. Int J Radiat Oncol Biol Phys. 2022;113(3):594-601. 25. Saifi O, Breen W, Lester SC, et al. Comprehensive salvage radiotherapy for limited relapsed B-cell non-Hodgkin lymphoma following CD19 chimeric antigen receptor T-cell therapy. Int J Radiat Oncol Biol Phys. 2022;114(3):S56. 26. Cheson BD, Fisher RI, Barrington SF, et al. Recommendations for initial evaluation, staging, and response assessment of Hodgkin and non-Hodgkin lymphoma: the Lugano classification. J Clin Oncol. 2014;32(27):3059-3068. 27. Reyes F, Lepage E, Ganem G, et al. ACVBP versus CHOP plus radiotherapy for localized aggressive lymphoma. N Engl J Med. 2005;352(12):1197-1205. 28. Bonnet C, Fillet G, Mounier N, et al. CHOP alone compared with CHOP plus radiotherapy for localized aggressive lymphoma in elderly patients: a study by the Groupe d'Etude des Lymphomes de l'Adulte. J Clin Oncol. 2007;25(7):787-792. 29. Lamy T, Damaj G, Soubeyran P, et al. R-CHOP 14 with or without radiotherapy in nonbulky limited-stage diffuse large B-cell lymphoma. Blood. 2018;131(2):174-181. 30. Horning SJ, Weller E, Kim K, et al. Chemotherapy with or without radiotherapy in limited-stage diffuse aggressive nonHodgkin's lymphoma: Eastern Cooperative Oncology Group study 1484. J Clin Oncol. 2004;22(15):3032-3038. 31. Gomez DR, Blumenschein GR, Jr., Lee JJ, et al. Local consolidative therapy versus maintenance therapy or observation for patients with oligometastatic non-small-cell lung cancer without progression after first-line systemic therapy: a multicentre, randomised, controlled, phase 2 study. Lancet Oncol. 2016;17(12):1672-1682. 32. Palma DA, Olson R, Harrow S, et al. Stereotactic ablative radiotherapy for the comprehensive treatment of oligometastatic cancers: long-term results of the SABR-COMET phase II randomized trial. J Clin Oncol. 2020;38(25):2830-2838. 33. Miller TP, Dahlberg S, Cassady JR, et al. Chemotherapy alone compared with chemotherapy plus radiotherapy for localized intermediate- and high-grade non-Hodgkin's lymphoma. N Engl J Med. 1998;339(1):21-26. 34. Pfreundschuh M, Murawski N, Ziepert M, et al. Radiotherapy (RT) to bulky (B) and extralymphatic (E) disease in combination with 6xR-CHOP-14 or R-CHOP-21 in young good-prognosis DLBCL patients: results of the 2x2 randomized UNFOLDER trial of the DSHNHL/GLA. J Clin Oncol. 2018;36(Suppl 15):S7574-7574. 35. Persky DO, Li H, Stephens DM, et al. Positron emission tomography-directed therapy for patients with limited-stage diffuse large B-cell lymphoma: results of Intergroup National Clinical Trials Network Study S1001. J Clin Oncol. 2020;38(26):3003-3011. 36. Freeman CL, Savage KJ, Villa DR, et al. Long-term results of PET-guided radiation in patients with advanced-stage diffuse large B-cell lymphoma treated with R-CHOP. Blood.

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2021;137(7):929-938. 37. Phan J, Mazloom A, Medeiros LJ, et al. Benefit of consolidative radiation therapy in patients with diffuse large B-cell lymphoma treated with R-CHOP chemotherapy. J Clin Oncol. 2010;28(27):4170-4176. 38. Philip T, Guglielmi C, Hagenbeek A, et al. Autologous bone marrow transplantation as compared with salvage chemotherapy in relapses of chemotherapy-sensitive nonHodgkin's lymphoma. N Engl J Med. 1995;333(23):1540-1545. 39. Mundt AJ, Williams SF, Hallahan D. High dose chemotherapy and stem cell rescue for aggressive non-Hodgkin's lymphoma: pattern of failure and implications for involved-field radiotherapy. Int J Radiat Oncol Biol Phys. 1997;39(3):617-625. 40. Vose JM, Zhang MJ, Rowlings PA, et al. Autologous transplantation for diffuse aggressive non-Hodgkin's lymphoma in patients never achieving remission: a report from the Autologous Blood and Marrow Transplant Registry. J Clin Oncol. 2001;19(2):406-413. 41. Friedberg JW, Neuberg D, Monson E, Jallow H, Nadler LM, Freedman AS. The impact of external beam radiation therapy prior to autologous bone marrow transplantation in patients with non-Hodgkin's lymphoma. Biol Blood Marrow Transplant. 2001;7(8):446-453. 42. Hoppe BS, Moskowitz CH, Zhang Z, et al. The role of FDG-PET imaging and involved field radiotherapy in relapsed or refractory diffuse large B-cell lymphoma. Bone Marrow Transplant. 2009;43(12):941-948. 43. Biswas T, Dhakal S, Chen R, et al. Involved field radiation after autologous stem cell transplant for diffuse large B-cell lymphoma in the rituximab era. Int J Radiat Oncol Biol Phys. 2010;77(1):79-85. 44. Wendland MM, Smith DC, Boucher KM, et al. The impact of involved field radiation therapy in the treatment of relapsed or refractory non-Hodgkin lymphoma with high-dose chemotherapy followed by hematopoietic progenitor cell transplant. Am J Clin Oncol. 2007;30(2):156-162. 45. Wilke C, Cao Q, Dusenbery KE, et al. Role of consolidative radiation therapy after autologous hematopoietic cell transplantation for the treatment of relapsed or refractory Hodgkin lymphoma. Int J Radiat Oncol Biol Phys. 2017;99(1):94-102. 46. Yu Q, Zhang X, Wang N, et al. Radiation prior to chimeric antigen receptor T-cell therapy is an optimizing bridging strategy in relapsed/refractory aggressive B-cell lymphoma. Radiother Oncol. 2022;177:53-60. 47. Smith EL, Mailankody S, Staehr M, et al. BCMA-targeted CAR Tcell therapy plus radiotherapy for the treatment of refractory myeloma reveals potential synergy. Cancer Immunol Res. 2019;7(7):1047-1053. 48. Kostopoulos N, Bedgi S, Krimitza E, et al. Radiation therapy for bridging and improving CAR-T cell therapy. Int J Radiat Oncol Biol Phys. 2022;114(3):S83-S84. 49. DeSelm C, Palomba ML, Yahalom J, et al. Low-dose radiation conditioning Eenables CAR T cells to mitigate antigen escape. Mol Ther. 2018;26(11):2542-2552. 50. Breen WG, Hathcock MA, Young JR, et al. Metabolic characteristics and prognostic differentiation of aggressive lymphoma using one-month post-CAR-T FDG PET/CT. J Hematol Oncol. 2022;15(1):36.

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ARTICLE - Cell Therapy & Immunotherapy

Intestinal IgA-positive plasma cells are highly sensitive indicators of alloreaction early after allogeneic transplantation and associate with both graft-versus-host disease and relapse-related mortality Lucia Scheidler,1* Katrin Hippe,2* Sakhila Ghimire,1 Daniela Weber,2 Markus Weber,3 Elisabeth Meedt,1 Petra Hoffmann,1,4 Petra Lehn,5 Ralph Burkhardt,5 Andreas Mamilos,2 Matthias Edinger,1,4 Daniel Wolff,1 Hendrik Poeck,1,4 Matthias Evert,2 Andre Gessner,6 Wolfgang Herr1 and Ernst Holler1 Department of Internal Medicine 3 (Hematology/Oncology), University Hospital; Department of Pathology, University of Regensburg; 3Department of Trauma, Orthopedics and Sports Surgery, Barmherzige Brueder Regensburg; 4Leibniz-Institute for Immunotherapy (LIT); 5Department of Clinical Chemistry and Laboratory Medicine, University Hospital; 6Department of Medical Microbiology and Hygiene, University Hospital, Regensburg, Germany 1

Correspondence: E. Holler ernst.holler@ukr.de Received: Accepted: Early view:

September 30, 2022. May 25, 2023. June 1, 2023.

LS and KH contributed equally as first authors.

Abstract Intestinal immunoglobulin A (IgA) is strongly involved in microbiota homeostasis. Since microbiota disruption is a major risk factor of acute graft-versus-host disease (GvHD), we addressed the kinetics of intestinal IgA-positive (IgA+) plasma cells by immunohistology in a series of 430 intestinal biopsies obtained at a median of 1,5 months after allogeneic stem cell transplantation (allo-SCT) from 115 patients (pts) at our center. IgA+ plasma cells were located in the subepithelial lamina propria and suppressed in the presence of histological aGvHD (GvHD Lerner stage 0: 131+/-8 IgA+ plasma cells/mm2; stage 1-2: 108+/-8 IgA+ plasma cells/mm2; stage 3-4: 89+/-16 IgA+ plasma cells/mm2; P=0.004). Overall, pts with IgA+ plasma cells below median had an increased treatment related mortality (P=0.04). Time courses suggested a gradual recovery of IgA+ plasma cells after day 100 in the absence but not in the presence of GvHD. Vice versa IgA+ plasma cells above median early after allo-SCT were predictive of relapse and relapse-related mortality (RRM): pts with low IgA+ cells had a 15% RRM at 2 and at 5 years, while pts with high IgA+ cells had a 31% RRM at 2 years and more than 46% at 5 years; multivariate analysis indicated high IgA+ plasma cells in biopsies (hazard ratio =2.7; 95% confidence interval: 1.04-7.00) as independent predictors of RRM, whereas Lerner stage and disease stage themselves did not affect RRM. In contrast, IgA serum levels at the time of biopsy were not predictive for RRM. In summary, our data indicate that IgA+ cells are highly sensitive indicators of alloreaction early after allo-SCT showing association with TRM but also allowing prediction of relapse independently from the presence of overt GvHD.

Introduction Graft-versus-host disease (GvHD) and graft-versus-leukemia reactions (GvL) are major determinants of outcome following allogeneic stem cell transplantation (allo-SCT) and show a broad overlap thus frequently preventing clear clinical separation of beneficial GvL and deleterious GvH effects.1 Acute GvHD strongly affects the gastro-intestinal (GI) tract, and microbiota colonizing the GI tract have been identified as major modifiers of both, normal and pathologic immune reactions such as GvHD.2-4 Numerous im-

mune cells including alloreactive T cells, regulatory T cells and innate lymphoid cells are involved in immune regulation of GvHD but also maintenance of immunological homeostasis in the GI tract.1,5 Likewise, intestinal plasma cells producing mainly IgA antibodies play a pivotal role in this context: they are strongly reduced in germ-free mice,6 induced in Peyers patches and secondary lymphoid organs and disseminate to the lamina propria of the whole GI tract. They are the main producers of secretory IgA which plays a central role in the defense against pathogens but also in maintaining co-existence with com-

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ARTICLE - Intestinal IgA-positive cells indicate GvH and GvL mensal bacteria, and flow cytometry of immunoglobulin A (IgA)-coated bacteria in the GI lumen revealed coating of a wide range of commensals.7-9 Intestinal IgA-positive (IgA+) plasma cells have not been in the focus of GvHD pathophysiology and so far, only a few early studies reported suppression of intestinal plasma cells in patients (pts) dying from GvHD.10 More is known about serum IgA levels and B-cell deficiency in general in relation to GvHD: delayed and disturbed B-cell recovery is a hallmark of acute (aGvHD) and chronic GvHD (cGvHD)11 and both, decrease and low IgA levels and B-cell deficiency associate with history of aGvHD and severity of cGvHD.12 Impaired reconstitution of IgA levels is among the most sensitive indicator of the presence of cGvHD,12 and immunoglobulin deficiency can persist up to 5 years and more in pts with cGvHD.13 Based on the potential role of IgA in microbiota regulation and the association of GvHD and microbiota damage we decided to perform a large retrospective analysis of intestinal IgA+ plasma cells in a series of biopsies obtained from pts after allo-SCT: our study revealed a prolonged suppression and deficiency of IgA+ plasma cells in pts with GvHD and revealed a so far unreported association of increased IgA+ plasma cells with a highly increased relapserelated mortality (RRM) suggesting plasma cells as a sensitive target of alloreaction involved in GvL even beyond overt GvHD.

Methods Patients Patient characteristics are given in Table 1 and represent a typical adult allo-SCT population. All pts gave informed consent to use urine and serum samples as well as biopsy sections for additional analysis including IgA serum titers and IgA staining of biopsies. The study was approved by the local Ethical Review Board of the University of Regensburg (approval number 09/059). Biopsies A total of 430 biopsies from 128 pts were analyzed. Thirtysix had two biopsies, 61 pts had three and more biopsies, from different sites of the GI tract and at different time points. Thirty-four biopsies from 13 pts were obtained prior to allo-SCT to rule out other GI diseases and served as pretransplant controls. The median time to biopsies was 1.5 months after allo-SCT (range, 0.4-67 months), 85% of biopsies were obtained within a time period of less than 6 months after transplantation. In order to avoid any bias by multiple biopsies in pts with more severe courses, we defined one most relevant (=master) biopsy per pt for survival and outcome analysis: in pts with only one biopsy this corresponded to the only

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Table 1. Patient and transplant characteristics. Characteristics Sex F/M, N (%)

45/70 (39/61)

Age in years, median (range)

56 (17-70)

Underlying disease, N (%) AML ALL MDS MPS (OMF, CML) Myeloma LgNHL HgNHL Non-malignant

60 (54) 4 (3) 13 (11) 13 (11) 9 (8) 3 (2) 9 (8) 4 (3)

Stage at treatment, N (%) Early Intermediate Advanced

28 (25) 52 (45) 35 (30)

Conditioning, N (%) Standard Reduced intensity

102 (89) 13 (11)

Number of SCT, N (%) 1 2 or 3

99 (86) 16 (14)

Donor, N (%) Sibling Unrelated Haploidentical

34 (29) 72 (63) 9 (8)

Stem cell source, N (%) PBSC Bone marrow

105 (91) 10 (9)

115 patients after allogeneic stem cell transplantation (allo-SCT) were analyzed, absolute numbers and %/range are shown. f: female; m: male; AML: acute myelogenous leukemia; ALL: acute lymphoblastic leukemia; MDS: myelodysplastic syndrome; MPS: myeloproliferative syndromes; OMF: osteomyelofibrosis; CML: chronic myelogenous leukemia; NHL: non-Hodgkin Lymphoma; Lg: low-grade; Hg: highgrade; PBSC: mobilized peripheral blood stem cells.

available one. For pts with multiple biopsy, the biopsy obtained at onset of GvHD was selected, and for pts without GvHD, the first biopsy after allo-SCT was used. Selection was independent of biopsy location and the number of allo-SCT. Outcome was always analyzed in relation to the biopsy of the respective transplant. Immunoglobulin A staining IgA+ plasma cells in the investigated biopsies were stained by immunohistochemistry (polyclonal rabbit anti-human IgA, code-number A 0262; Dako Denmark A/S, Glostrup, DK), supported by a software-controlled slide stainer (VENTANA BenchMark ULTRA; Ventana Medical Systems Inc., Tucson, USA). The detailed protocol is given in the Online Supplementary Appendix. Factors influencing immunglobulin A-positive plasma cells In order to analyze the impact of time after allo-SCT, bi-

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ARTICLE - Intestinal IgA-positive cells indicate GvH and GvL opsies were grouped according to the time period of biopsy into biopsies pretransplant, biopsies obtained until day 100 and biopsies obtained later than day 100 after allo-SCT. In order to assess the impact of microbiota, we used urinary indoxylsulfate levels (µmol/mmol creatinine) as previously described14 which were available within 1 week in relation to biopsies for 197 pts. For each biopsy, we recorded the concomitant treatment with corticosteroids >20 mg/day and the use of rituxan. Immunoglobulin A serum levels In 108 pts, serum samples had been drawn within +/-7 days of master biopsies thus allowing pairwise assessment of IgA serum levels and IgA+ plasma cells. Serum samples were stored at -80°C until analysis. IgA serum levels were quantified in a DIN ISO 15189 accredited clinical laboratory using an immunoturbidimetric assay (Roche Tina-quant IgA Gen.2) on an automated clinical chemistry analyzer (Roche cobas pro, Grenzach Whylen, Germany, for details see the Online Supplementary Appendix). Statistical analysis Clinical data as well as data from histopathological analyses including Lerner stage and IgA+ cells/mm2 were collected in a SPSS database (Version 26, IBM New York, USA). For comparisons of mean IgA+ plasma cells nonparametric Wilcoxon tests were used. For survival analysis using Kaplan Meier, Cox Regression and competing risk assessment, master biopsies were selected.

Results Morphology and distribution in the gastro-intestinal tract IgA+ plasma cells could be easily identified by immunoh-

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istochemistry in the lamina propria and were in close contact to the epithelial lines (Figure 1). When we compared the number of plasma cells in relation to the site of biopsies, there was no significant difference between the upper and the lower GI tract: upper GI tract n=153: 124.5, (standard error [SE] 9.4) IgA+ plasma cells/ mm2 versus lower GI tract n=244: 110.0 (SE 6.4) IgA+ plasma cells/mm2. Low intestinal immunoglobulin A-positive plasma cell numbers associate with acute graft-versus-host disease and treatment-related mortality Next, we addressed the impact of aGvHD on the presence of the IgA+ plasma cells: IgA+ plasma cell numbers were highest in 200 pts with Lerner stage 0, and gradually decreased with more severe GvHD (Table 2). For the whole set of biopsies obtained after allo-SCT, GvHD-dependent effects were observed both in the upper and in the lower GI tract (Online Supplementary Table S1). When we analyzed pts according to organ involvement and Lerner stage, suppression of IgA+ plasma cells was only mild in pts with exclusive skin or liver GvHD but highly pronounced and significant in pts with overt GI involvement. A total of 32 (27.8%) of pts died from treatment-related complications such as GvHD +/- infections. In line with a stronger suppression of plasma cells in pts with more severe GvHD, patients with IgA+ plasma cells below median after allo-SCT experienced an increased treatment-related mortality (TRM) (log-rank 0.04; Figure 2). In a multivariate cox regression analysis of TRM, higher Lerner stage (hazard ratio [HR] =3.8; 95% confidence interval [CI]: 1.69.6) predicted TRM and higher IgA+ cell numbers were protective (HR=0.34; 95% CI:0.14-0.83), whereas underlying disease, age, stage and donor type did not have significant impact (data not shown). Suppression of IgA+ plasma cells by GvHD is independent of microbiota damage, corticos-

Figure 1. Histopathological example of immunoglobulin A staining. Immunohistological staining of immunoglobulin A-positive plasma cells; positive cells (arrows) were detected in the lamina propria with close association to the epithelial line. Haematologica | 108 November 2023

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Table 2. Graft-versus-host disease-dependent suppression of immunoglobulin A-positive plasma cells. Histological GvHD

IgA+ plasma cells/mm2 (SE)

No GvHD at all

200

129.5 (7.7)

Lerner stage 1/2

143

103.7 (8.2)**

Lerner stage 3/4

91.8 (17.3)*

Differences between Lerner stage 0 and Lerner stage 1/2 were significant with P=0.009, differences between Lerner stage 1/2 and Lerner stage 3/4 were significant with P=0.02. GvHD: graft-versus-host disease; IgA+: immunoglobulin A-positive; SE: standard error; *P<0.05, **P< 0.01.

teroid use or use of B-cell depleting agents at the time of biopsy and from time after allo- SCT When we grouped biopsies according to the time after allo-SCT, 41 biopsies obtained prior to allo-SCT contained 139.0 (SE 23.7) IgA+ plasma cells/mm2 whereas biopsies obtained until day 100 after allo-SCT showed 107.6 (SE 7.2) IgA+ plasma cells (n=244; P=0.08 vs. pretransplant). In biopsies obtained beyond day 100 IgA+ plasma cells started to recover again (n=153; 125.7 [SE 7.9] IgA+ plasma cells; P=0.01 vs. before day 100). GvHD dependent suppression, however, was observed for both time intervals: In 233 biopsies obtained before day 100, IgA+ plasma cells/mm2 were 121.9 (SE 10.4) in biopsies without histological GvHD and 87.8. (SE 9.2) in biopsies with GvHD (P=0.001), in 163 biopsies obtained after day 100, mean IgA+ plasma cells/mm2 were 140.6 (SE 11.0) in the absence of GvHD and 110.9 (SE 11.9) in the presence of GvHD (P=0.03). Analysis of urinary indoxylsulfate (IS) levels (indicating presence of commensal microbiota in the gut), was available for 197 biopsies: IS levels were 97.6 (SE 10.9) µmol/mmol creatinine for 87 biopsies with IgA below median and 87.2 (SE 9.2) µmol/mmol creatinine for 107 biopsies with IgA above median (difference not significant [ns]). As corticosteroid dosage and use of B-cell depleting agents like rituximab might affect IgA+ plasma cells, we also analyzed prednisolone usage above 20 mg/day at the time of biopsy and did not observe significant differences (data not shown). Similarly, mean IgA plasma cells were 88.2+/-18.0 IgA+ plasma cells/mm2 in 14 pts receiving rituximab prior to SCT versus 95.7+/-9.3 IgA+ plasma cells/mm2 in pts not receiving rituximab (ns). After SCT, only four pts received rituximab prior to biopsies for treatment of high EpsteinBarr virus (EBV) serum copy numbers to prevent EBV lymphoma which had no impact on mean IgA plasma cells in the biopsies (104.8+/-58.0 IgA+ plasma cells/mm2 as compared to 93.9+/-8.0 IgA+ plasma cells/mm2 in the remaining pts (ns). Thus, in a multivariate binary logistic regression, only advanced Lerner stage (overall resposnse [OR]: 0.51; 95% CI: 0.26-0.93), but not prednisolone dose, microbiota damage, time interval after allo-SCT, age and site of biopsy were of significant impact (data not shown).

Intestinal immunoglobulin A-positive plasma cells associate with relapse even in the absence of graftversus-host disease The presence of high IgA+ plasma cell numbers in the GI tract is on the other side associated with RRM. Whereas only 15% of 57 pts with IgA+ plasma cells below median at the time of biopsy died from relapse in long-term followup, 31% of 58 pts with high IgA+ plasma cell content (above median) died at 2 years and 46% at 5 years after allo-SCT (Figure 3; P=0.01). When we included six additional patients with relapse but alive at the end of the observational period and assessed relapse incidence in general, we observed a comparable association: only 11.5% of patients with low IgA plasma cells relapsed in contrast to 41% of patients with high IgA plasma cells (P=0.001 in log-rank). These associations were predominantly observed in pts without evidence of major GvHD in their biopsies (Lerner 0-1; log-rank P=0.03). In multivariate Cox regression only IgA+ plasma cells, but not Lerner stage, disease stage at the time of transplant or age-predicted RRM, suggesting a prognostic significance of IgA+ plasma cell numbers independently from presence of overt GvHD (Table 3). In order to rule out an impact of underlying diseases we performed a separate analysis for myeloid versus lymphoid malignancies: in both subgroups IgA+ plasma cells predicted RRM. In myeloid malignancies, RRM at 5 years was 16% versus 49% in pts with low and high IgA+ plasma cell numbers, respectively, whereas in lymphoid malignancies RRM was 0% and 53%, respectively. Both results strongly support a disease-independent observation. Finally, we performed a competing risk analysis to rule out mutual overlap between GvHD and GvL effects. IgA+ plasma cells were independent predictors of both, TRM (low IgA+ plasma cell numbers) and RRM (high IgA+ plasma cell numbers) (Figure 4). Immunoglobulin A serum levels show weak correlation with intestinal immuglobulin A-positive plasma cells and only add to prediction of treatment-related mortality As serum IgA might allow more rapid assessment of prog-

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L. Scheidler et al. Figure 2. Cumulative treatment-related mortality and immunoglobulin A-positive plasma cells. Twenty-three of 62 patients (pts) with low plasma cells died from treatment-related mortality contrast to only 11 of 61 pts with high immunoglobulin A-positive (IgA+) plasma cells. Differences were significant (log-rank P=0.015) A total of 115 pts and first biopsies were analyzed, time (in days) from biopsy is given. Cum.NRM: cumulative non-relapserelated mortality.

Figure 3. Cumulative relapse-related mortality. Cumulative relapse-related mortality (Cum.RelapseRM) is significantly increased in (pts) with high plasma cells (log rank P=0.03). Only 6 of 63 pts with immunoglobulin A-positive (IgA+) plasma cells died from relapse but 22 of 62 pts with high IgA+ plasma cells. A total of 115 pts and first biopsies were analyzed. Cum.RelapseRM: Cumulative Relapse related mortality

nostic IgA+ plasma cell deficiency, we addressed whether IgA serum levels at the time of master biopsies reflected intestinal IgA+ plasma cell content. There was some correlation between IgA serum levels and the number of IgA+ plasma cells (r=0.332; P=0.000) in 108 available serum/biopsy pairs (Online Supplementary Figure S1). Patients with higher Lerner stages showed a tendency to have lower IgA serum levels, as pts with Lerner 0 (n=48) had 82.0 (standard error of the mean [SEM]=9.3), pts with Lerner 1,2 (n=44) had 84.7 (SEM=13.4) and pts with Lerner 3,4 (n=16) had 48.1 (SEM=7.1) g/L IgA (Lerner 3,4 vs. 0; P=0.03, all other ns). When we compared IgA+ plasma cells in the GI tract and IgA serum levels (above/below median) in Kaplan Meier analyses regarding TRM and RRM, low serum IgA levels

were weakly associated with higher TRM but showed no correlation with RRM as observed for IgA+ plasma cells (data not shown).

Discussion Our study describes for the first time the association of intestinal IgA+ plasma cells with outcome following alloSCT in a large series of pts. Although it is well known, that the majority of IgA-producing plasma cells reside in the intestinal tract and contribute substantially to control of microbial inflammation,6,15 only a few studies so far have addressed their changes in the setting of allo-SCT and GvHD. These papers addressed IgA-bearing cells in human

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Table 3. Cox regression analysis of factors associated with increased relapse-related mortality. Factor

95% CI

Lerner stage

1.59

0.4-5.9

Age in years at allo-SCT

1.98

0.7-6.0

Advanced stage

2.18

0.7-8.5

Underlying disease

0.66

0.2-2.4

IgA+ plasma cells above median

3.33

0.03

1.1-10.1

High immunoglobulin A-positive (IgA+) plasma cells is the only independent predictor. HR: hazard ratio; allo-SCT: allogeneic stem cell transplantation CI: confidence interval; NS: not significant.

Figure 4. Competing risk analysis. Competing risk analysis for treatment-related mortality (TRM) and relapse-related mortality (RRM). Effects on RRM and TRM are independent (TRM P=0.001; RRM P=0.002).

autopsies and showed that loss of these cells is part of a general immune cell loss during the course of aGvHD of the intestinal tract.10,16 In the same line, we recently described B cells in the bone marrow and spleen as being the most sensitive indicators of aGvHD and that their loss could be prevented or reversed by treatment with donorderived regulatory T cells.17 Prolonged secondary IgA deficiency in the serum is a hallmark of impaired systemic immunoreconstitution following allo-SCT and it is aggravated in pts with cGvHD.12,13,19 Although the majority of serum IgA is produced by plasma cells residing in the bone marrow, intestinal plasma cells can also significantly contribute to systemic IgA levels. Thus low serum IgA could indicate damage to plasma cells, both in the marrow and the intestinal tract,18 which is in line with the moderate correlation between serum IgA and intestinal

IgA+ plasma cells seen in our study. Our systematic analyses confirmed a clear suppression of IgA+ plasma cells in intestinal aGvHD and a subsequent association of this suppression with an increase in TRM. Suppression was most prominent in the first 100 days after allo-SCT. Thereafter plasma cell numbers started to recover in the absence of aGvHD, whereas a stronger suppression persisted in patients with severe aGvHD throughout the study. Besides time after allo-SCT, we tried to identify other confounding factors suppressing IgA+ plasma cells, but neither microbiota damage at the time of biopsy, as indicated by urinary IS levels, nor concomitant immunosuppression with high-dose corticosteroids or B-cell depleting agents like rituximab nor the site of biopsy affected the strong impact of histological GvHD. Interestingly, suppression of intestinal IgA+ plasma cells

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ARTICLE - Intestinal IgA-positive cells indicate GvH and GvL was minor if clinical GvHD-involved only organs such as skin and/or liver, suggesting local mechanisms being active in GI-GvHD and damaging plasma cells residing in that organ. A still unanswered question is the origin of intestinal IgA+ plasma cells at different time points following allo-SCT. Based on immunoglobulin recovery in general and halflife of specific antibodies (like anti-HbS),20 donor plasma cells are starting to take over immunoglobulin production 12 to 18 months after allo-SCT. However, reports on intestinal plasma cells, e.g., after small bowel transplantation, suggest that individual recipient-derived plasma cells may even persist for years.21 Onset of regeneration of plasma cells beyond day 100 may be in line with these kinetics, however, detailed studies on plasma cell chimerism both in patients and in experimental murine models are required in the future. This should also contribute to decipher the underlying mechanism of plasma cell damage. Whereas our study suggests direct elimination of intestinal IgA+ plasma cells by alloreactive T cells, actual and ongoing murine and human studies by our group indicate a more general and broader damage of the B-cell compartment in GvHD with an arrest of B-cell and plasma cell maturation which might indicate damage to the B-cell niches independent from the actual chimerism of B-cell effectors. Whether these mechanisms contribute to the suppression of plasma cells observed in our study needs to be analyzed in future studies. The differential recovery of IgA+ plasma cells already highlights the high sensitivity of plasma cells to allo-reaction in the setting of GvHD. The unexpected finding of increased plasma cells early after allo-SCT as a sensitive predictor of relapse and RRM, is in line with this sensitivity, as our observations suggest that decreased elimination of plasma cells may reflect an impaired alloreaction against recipient hematopoietic cells, which is active also in the absence of overt clinical GvHD. This GvHD-independent alloreaction is currently thought to be the most broadly active mechanism of GvL effects mediated by a graft-versus-host hematopoiesis reaction.22 Again, detailed analysis of chimerism of plasma cells will help to sharpen this hypothesis. So far, appropriate broad biomarkers predicting relapse are missing. Antigen-specific T cells have been reported for WT1 antigen-positive leukemia cells,23 and analysis of minimal residual disease where available is another example of specific biomarkers. Direct analysis of the extent of alloreaction is only possible on the level of chimerism,24 thus assessment of intestinal plasma cells if confirmed as a predictor of relapse might be a useful indicator and help to guide preemptive strategies such as donor lymphocyte infusions. As the easily accessible serum IgA levels have not been analyzed in this context

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so far, we compared the prognostic significance of serum IgA and intestinal IgA+ plasma cells. In spite of some positive correlation, analysis of intestinal plasma cells seemed to be a stronger indicator of alloreaction as compared to assessment of systemic IgA levels. Secretion of IgA, e.g., in the form of fecal IgA, might be an alternative approach to assess intestinal plasma cell activity but it has not yet been addressed in pts after allo-SCT.25 In summary, our study reports a strong association of intestinal IgA+ plasma cells with alloreaction in the setting of GvHD which has impact on both, GvHD and GvL. Disclosures EH is a scientific advisory board member of Novartis, Pharmabiome (Zürich), Maat Pharma (Lyon) and Medac; and he has received a research grant from Neovii. DW has received a research grant from Novartis; and he has received honoraria from Takeda, Gilead, Sanofi, Mallinckrodt and Pfizer; he is a member of the board of directors of Behring. All other authors have no conflicts of interest to disclose. Contributions LS and KH contributed equally as principal investigators, designed the study and performed immunohistology and data analysis. AM and MEv supervised pathology analyses and contributed to data discussion. SG performed data analysis and contributed to discussion. DW, MW and EM collected clinical data, performed clinical and survival data analysis and contributed to discussion. PL and RB performed serum IgA analysis. PH, ME, DW. HP, AG and WH discussed the data and the manuscript. EH designed the study, supervised data analysis, wrote the manuscript and served as a senior author. Acknowledgments The authors wish to acknowledge the excellent help of Doris Gaag in performing and the assessment of IgA immunostaining and Heike Bremm, Tatjana Schifferstein and Yvonne Schuman for collecting and the assessment of clinical samples. Funding This work was supported by the Wilhelm Sander Stiftung, grant 2017.020.02 (to EH and SG) and partially by the Jose Carreras Leukemia foundation (grant DJCLS 01/GvHD2020). EH, DW, EM, HP, ME, PH and WH are funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), project number 324392634, SFB TR221 GvH/GvL. EM received a clinician scientist grant from the Else Kroener Fresenius Stiftung. Data-sharing statement Data will be shared on the Zenodo platform.

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References 1. Ferrara JL, Levine JE, Reddy P, Holler E. Graft-versus-host disease. Lancet. 2009;373(9674):1550-1561. 2. Jenq RR, Ubeda C, Taur Y, et al. Regulation of intestinal inflammation by microbiota following allogeneic bone marrow transplantation. J Exp Med. 2012;209(5):903-911. 3. Holler E, Butzhammer P, Schmid K, et al. Metagenomic analysis of the stool microbiome in patients receiving allogeneic stem cell transplantation: loss of diversity is associated with use of systemic antibiotics and more pronounced in gastrointestinal graft-versus-host disease. Biol Blood Marrow Transplant. 2014;20(5):640-645. 4. Peled JU, Gomes AL, Devlin SM, et al. Microbiota as predictor of mortality in allogeneic hematopoietic-cell transplantation. N Engl J Med. 2020;382(9):822-834. 5. Zheng D, Liwinski T, Elinav E. Interaction between microbiota and immunity in health and disease. Cell Res. 2020;30(6):492-506. 6. Pabst O, Slack E. IgA and the intestinal microbiota: the importance of being specific. Mucosal Immunol. 2020;13(1):12-21. 7. Bunker JJ, Bendelac A. IgA responses to microbiota. Immunity. 2018;49(2):211-224. 8. Bunker JJ, Erickson SA, Flynn TM, et al. Natural polyreactive IgA antibodies coat the intestinal microbiota. Science. 2017;358(6361):eaan6619. 9. Sterlin D, Fadlallah J, Adams O, et al. Human IgA binds a diverse array of commensal bacteria. J Exp Med. 2020;217(3):e20181635. 10. Beschorner WE, Yardley JH, Tutschka PJ, Santos GW. Deficiency of intestinal immunity with graft-vs.-host disease in humans. J Infect Dis. 1981;144(1):38-46. 11. McManigle W, Youssef A, Sarantopoulos S. B cells in chronic graft-versus-host disease. Hum Immunol. 2019;80(6):393-399. 12. Serpenti F, Lorentino F, Marktel S, et al. Immune reconstitutionbased score for risk stratification of chronic graft-versus-host disease patients. Front Oncol. 2021;11:705568. 13. Witherspoon RP, Storb R, Ochs HD, et al. Recovery of antibody production in human allogeneic marrow graft recipients: influence of time posttransplantation, the presence or absence of chronic graft-versus-host disease, and antithymocyte globulin treatment. Blood. 1981;58(2):360-368. 14. Weber D, Oefner P, Hiergeist A, et al. Low urinary indoxylsulfate levels early after transplantation reflect a disruoted

microbiome and are associated with poor outcome. Blood. 2015;126(14):1723- 1728. 15. Gommerman JL, Rojas OL, Fritz JH. Re-thinking the functions of IgA(+) plasma cells. Gut Microbes. 2014;5(5):652-662. 16. Cousineau S, Belanger R, Perreault C. Characterization of plasma cell populations at autopsy after human allogeneic bone marrow transplantation. Am J Pathol. 1986;124(1):74-81. 17. Riegel C, Boeld TJ, Doser K, Huber E, Hoffmann P, Edinger M. Efficient treatment of murine acute GvHD by in vitro expanded donor regulatory T cells. Leukemia. 2020;34(3):895-908. 18. Wilmore JR, Allman D. Here, there, and anywhere? Arguments for and against the physical plasma cell survival niche. J Immunol. 2017;199(3):839-845. 19. Storek J, Viganego F, Dawson MA, et al. Factors affecting antibody levels after allogeneic hematopoietic cell transplantation. Blood. 2003;101(8):3319-3324. 20. Knöll A, Boehm S, Hahn J, Holler E, Jilg W. Long-term surveillance of haematopoietic stem cell recipients with resolved hepatitis B: high risk of viral reactivation even in a recipient with a vaccinated donor. J Viral Hepat. 2007;14(7):478-483. 21. Landsverk OJ, Snir O, Casado RB, et al. Antibody-secreting plasma cells persist for decades in human intestine. J Exp Med 2017;214(2):309-317. 22. Blazar BR, Hill GR, Murphy WJ. Dissecting the biology of allogeneic HSCT to enhance the GvT effect whilst minimizing GvHD. Nat Rev Clin Oncol. 2020;17(8):475-492. 23. Rezvani K, Yong AS, Savani BN, et al. Graft-versus-leukemia effects associated with detectable Wilms tumor-1 specific T lymphocytes after allogeneic stem-cell transplantation for acute lymphoblastic leukemia. Blood. 2007;110(6):1924-1932. 24. Gambacorta V, Parolini R, Xue E, et al. Quantitative PCR-based chimerism in bone marrow or peripheral blood to predict acute myeloid leukemia relapse in high-risk patients: results from the KIM- PB prospective study. Haematologica. 2020;106(5):1480-1483. 25. Lin R, Chen H, Shu W et al. Clinical significance of soluble immunoglobulins A and G and their coated bacteria in feces of patients with inflammatory bowel disease. J Transl Med. 2018;16(1):359.

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ARTICLE - Cell Therapy & Immunotherapy

Azacitidine, lenalidomide and donor lymphocyte infusions for relapse of myelodysplastic syndrome, acute myeloid leukemia and chronic myelomonocytic leukemia after allogeneic transplant: the Azalena-Trial Thomas Schroeder,1,2 Matthias Stelljes,3 Maximilian Christopeit,4 Eva Esseling,3 Christoph Scheid,5 Jan-Henrik Mikesch,3 Christina Rautenberg,1 Paul Jäger,2 Ron-Patrick Cadeddu,2 Nadja Drusenheimer,6 Udo Holtick,5 Stefan Klein,7 Rudolf Trenschel,1 Rainer Haas,2 Ulrich Germing,2 Nicolaus Kröger4 and Guido Kobbe2 Department of Hematology and Stem Cell Transplantation, West German Cancer Center Essen, University Hospital Essen, Essen; 2Department of Hematology, Oncology and Clinical Immunology, University Hospital Duesseldorf, Medical Faculty, Heinrich Heine University, Duesseldorf; 3Department of Medicine A, Hematology and Oncology, University of Münster, Münster; 4University Hospital Hamburg-Eppendorf, Clinic for Stem Cell Transplantation, Hamburg; 5Department I of Internal Medicine, Medical Faculty and University Hospital, University of Cologne, Cologne; 6Coordination Center for Clinical Trials, University Hospital Duesseldorf, Medical Faculty, Heinrich Heine University, Duesseldorf and 7Department of Hematology and Oncology, University Hospital Mannheim, Mannheim, Germany 1

Correspondence: T. Schroeder Thomas.Schroeder@uk-essen.de Received: Accepted: Early view:

December 28, 2022. May 23, 2023. June 1, 2023.

Abstract Azacitidine (Aza) combined with donor lymphocyte infusions (DLI) is an established treatment for relapse of myeloid malignancies after allogeneic transplantation. Based on its immunomodulatory and anti-leukemic properties we considered Lenalidomide (Lena) to act synergistically with Aza/DLI to improve outcome. We, therefore, prospectively investigated tolerability and efficacy of this combination as first salvage therapy for adults with post-transplant relapse of acute myeloid leukemia, myelodysplastic syndromes and chronic myelomonocytic leukemia. Patients were scheduled for eight cycles Aza (75 mg/m2 day 1-7), Lena (2.5 or 5 mg, days 1-21) and up to three DLI with increasing T-cell dosages (0.5×106-1.5×107 cells/kg). Primary endpoint was safety, while secondary endpoints included response, graft-versus-host disease (GvHD) and overall survival (OS). Fifty patients with molecular (52%) or hematological (48%) relapse of myelodysplastic syndromes (n=24), acute myeloid leukemia (n=23) or chronic myelomonocytic leukemia (n=3) received a median of seven (range, 1-8) cycles including 14 patients with 2.5 mg and 36 with 5 mg Lena daily dosage. Concomitantly, 34 patients (68%) received at least one DLI. Overall response rate was 56% and 25 patients (50%) achieved complete remission being durable in 80%. Median OS was 21 months and 1-year OS rate 65% with no impact of type of or time to relapse and Lena dosages. Treatment was well tolerated indicated by febrile neutropenia being the only grade ≥3 non-hematologic adverse event in >10% of patients and modest acute (grade 2-4 24%) and chronic (moderate/severe 28%) GvHD incidences. In summary, Lena can be safely added to Aza/DLI without excess of GvHD and toxicity. Its significant anti-leukemic activity suggests that this combination is a novel salvage option for post-transplant relapse (clinicaltrials gov. Identifier: NCT02472691).

Introduction The curative potential of allogeneic stem cell transplantation (allo-SCT) in patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) is substantially impeded by the risk of relapse. As the major cause of treatment failure relapse occurs in 30% to 80% of patients1 and only a minority of these patients achieve longterm survival with conventional treatment options such as chemotherapy, donor lymphocyte infusions (DLI) and

second transplantation.2–5 During the last years, these therapeutic options have been augmented by the hypomethylating agent Azacitidine (Aza).6–10 Following treatment with Aza, mostly in combination with DLI, response rates ranging from 10% to 41% and 2-year survival rates ranging from 12% to 38%7,11–13 have been reported, with patients treated at a stage of low disease burden and/or late relapse beyond 6 months after transplant having the greatest benefit.14 Although these data clearly indicate efficacy of Aza as post-transplant salvage therapy, they also

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underline the need to enhance the activity of Aza monotherapy in order to improve outcome. The immunomodulatory drug Lenalidomid (Lena) has antileukemic activity as single agent in patients who relapse after transplant.15 Initial results in the non-transplant setting suggested synergistic acitivity of Aza and Lena in patients with high-risk MDS.16,17 However, administration of Lena as maintenance therapy in patients, who were in remission after transplant, was associated with high rates of severe graft-versus-host disease (GvHD).18,19 By contrast, Aza accelerates reconstitution of regulatory T cells after transplant,20,21 which could explain the low incidence and severity of GvHD following the combination of Aza and DLI.6,7,10,14 In a first phase I/II trial reported by Craddock et al. the combination of Aza and Lena was able to induce complete remission (CR) in 6 of 29 patients with relapsed AML and MDS after allogeneic stem cell transplantation (alloSCT).22 In order to expand the evidence that Aza together with Lena delivers synergistic antileukemic and immunomodulatory activity without increasing the risk of severe GvHD, we tested this combined combination incorporating also DLI into this approach in a phase II trial.

Study design and treatment After inclusion, patients were scheduled to receive up to eight cycles Aza (Vidaza, Celgene Corporation, Summit, NJ, USA) at a dose of 75 mg/m2/day subcutaneously on days 1-7 repeated every 28 days. In the absence of active GvHD DLI were envisaged after cycle 4, 6 and 8 at a dose of 0.5-1x106 CD3+ cells/kg (1st DLI), 1-5x106 CD3+ cells/kg (2nd DLI) and 5-15x106 CD3+ cells/kg (3rd DLI). Additional DLI were permitted according to the individual decision of the treating physician, but only beyond cycle 4. Lena was given concomitantly starting from cycle 1 on days 1-21 followed by a 7-day break every 28 days for a maximum of eight cycles. Acknowledging the potential risk of GvHD induction, the study incorporated a dose escalating schedule for Lena and two safety interim analyses. The first interim analysis was planned, as soon as ten patients had been treated with Lena (2.5 mg/day) and the tenth patient had either completed four cycles or had discontinued treatment. If the criteria to stop or modify study treatment (= dose limiting toxicity [DLT], defined in the Online Supplementary Appendix) were observed in this cohort, the study would have been closed. If these criteria were not met, the next ten patients would have been treated with 5 mg/day Lena followed by a second interim analysis. In case DLT criteria occurred in these ten patients, the remaining patients would have been treated with 2.5 mg/day, while in the absence of DLT 5 mg/day would have been the dosage for the remaining patients (Online Supplementary Figure S1). Independent from dose level and DLT, Lena had to be stopped in case of acute GvHD grade ≥2. As Aza was given in-label, treatment was allowed to be continued beyond eight cycles, and additional DLI were allowed based on an individual decision of the treating physician, but only after cycle 4.

Methods Eligibility As a prospective, open-label, phase-II single-arm, multicenter study the AZALENA trial (EudraCT 2013-001153-27) aimed to assess the safety and efficacy of Lena (investigational medical product) in combination with Aza and DLI (standard of care) as first salvage therapy for relapsed MDS, chronic myelomonocytic leukemia (CMML) or AML after first allo-SCT. Fifty adult patients with first molecular or hematological relapse as defined in detail in the Online Supplementary Appendix were included. According to the license status of Aza, only AML patients with a bone marrow (BM) blast count ≤29% were initially eligible. After extended marketing authorization in 2017, inclusion was expanded to all FLT3 and IDH2 wild-type AML patients independent from blast count acknowledging the option for targeted therapies such as Gilteritinib or Enasidenib. The absence of active GvHD treated with systemic immunosuppression within 4 weeks before inclusion and availability of DLI (donor still contactable, no cord blood as stem cell source) were indispensable prerequisites. Exclusion criteria included any bridging therapy between diagnosis of relapse and start of study treatment, uncontrolled infections, as well as renal and hepatic impairment (for details see the Online Supplementary Appendix). The Heinrich Heine University of Duesseldorf was the sponsor of the study, which was approved by the Ethics Committees of the Heinrich Heine University (approval number: MC-LKP-738) and the five other sites.

Study endpoints Primary endpoint was safety defined by incidence and severity of AE, which were assessed according National Cancer Institute Common Terminology Criteria for AE v4.0. Secondary endpoints included response as defined by the European Leukemia Net 2017 and International Working Group criteria,23,24 time to and duration of response, restoration of complete donor chimerism (DC) as determined and classified by the local standard methodology and overall survival (OS) as calculated from treatment onset. Secondary safety parameters consisted of incidence, course and severity of acute and chronic GvHD reported according to established criteria25,26 as well as the number of hospitalizations. Patients were followed until death or data lock (April 12, 2021). Immune evaluation Peripheral blood (PB) lymphocyte subsets including acti-

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vation status and exhaustion markers were serially monitored in a subgroup of 11 patients as described in detail in the Online Supplementary Appendix.

(72%) were treated with a daily starting dosage of 5 mg Lena. There were no differences between the two dose levels in number of Lena cycles per patient and length of treatment cycles (data not shown). A total of 101 DLI were administered to 34 patients (68%) corresponding to a median of three DLI (range, 1-11 DLI) and a median of 6.75x106/kg CD3+ cells (range, 0.5- 336.7x106/kg) per patient. Reasons to omit DLI in the remaining 16 patients were disease progression (n=13), GvHD (n=2) and unavailability of the donor (n=1). In those 16 patients, who did not receive DLI, the median number of cycles of Aza + Lena was two (range, 1-8).

Statistical analyses Details on statistical analyses as well as are available in the Online Supplementary Appendix.

Results Patients characteristics Fifty patients with AML (n=23, 46%), MDS (n=24, 48%) or CMML (n=3, 6%), who relapsed in median 233 days (range, 61-2,659 days) after transplant were recruited between June 2015 and August 2018. Of these, 26 patients (52%) experienced molecular relapse (median BM blast count 3%; range, 0-4%), while 24 patients (48%) suffered from hematological relapse (median BM blast count 18%; range, 0-70%; P<0.0001) with no statistical difference regarding time to relapse (255 vs. 188 days; P=0.74) and BM chimerism (85% vs. 63%; P=0.149). In one MDS patient hematological relapse was diagnosed on the basis of recurrent sign of dysplasia and cytogenetic features accompanied by a drop of donor chimerism. Molecular relapse was detected by reoccurrence of disease-specific markers in 21 patients (molecular n=8, cytogenetic n=6, combined molecular and cytogenetic n=7) with associated decrease of DC in 20, while isolated loss of complete DC was indicative for molecular relapse in the remaining five patients. Median follow-up of all patients was 20 months (range, 1-23 months). Detailed information on patient, transplant and relapse characteristics are given in Tables 1 and 2. Treatment The combination of Aza, Len and DLI was commenced as first treatment of relapse in median 14 days (range, 1-52 days) after diagnosis of relapse. There was no dropout of patients occurring between study inclusion and envisaged start of study treatment. Overall, 275 treatment cycles were administered to the 50 patients corresponding to a median of seven cycles (range, 1-8 cycles) per patient (Online Supplementary Figure S2). Lena was given concomitantly with Aza in 246 treatment cycles (89%), corresponding to a median of five cycles (range, 1-8 cycles) per patient. In the remaining 29 treatment cycles (11%) Lena as study medication was omitted in five individual patients due to hematoxicity (n=1), non-hematologic AE (n=1) and acute (n=2) or chronic GvHD (n=1). According to the study design 14 patients (28%) received Lena at a daily starting dosage of 2.5 mg. Since no DLT requiring premature stop or dose modifications were observed in two interim analyses, the remaining 36 patients

Safety and toxicity At study entry, 42% of patients exhibited at least one cytopenia grade >2 with grade 3/4 neutropenia and thrombopenia being already present in 30% and 38% of patients respectively, while no patient had grade 3/4 anemia. During the study, 275 treatment cycles were administered. Table 3 indicates that grade 3/4 neutropenia, anemia and thrombopenia occurred during 76%, 15% and 46% of treatment cycles, while renal and liver dysfunctions were uncommon. A total of 305 non-hematological AE were considered to be drug-related and the only treatment-related non-hematologic toxicities occurring in more than 10% of patients at grade 3 or greater was febrile neutropenia (12%) (Table 4). Overall, 19 patients (38%) had to be hospitalized at least once during treatment. Three patients (6%) developed a second primary malignancy (squamous cell carcinoma, basal cell carcinoma and vulvar carcinoma) during (n=1) or after study treatment (n=2). Clinical response and overall survival During the 8-month treatment period, 25 patients (50%) achieved CR and three patients (6%) achieved partial remission, resulting in an overall response rate of 56%. CR was achieved in 14 patients treated at the stage of molecular relapse and 11 patients treated at the stage of hematological relapse, respectively. Achievement of CR was accompanied by restoration of complete donor chimerism in 21 patients (84%) and disappearance of molecular/cytogenetic markers in all but one patient with trackable markers (Online Supplementary Table S1). Median time to achievement of CR was 113 days (range, 50-295 days), corresponding to a median of four treatment cycles (range, 1-8 cycles). Twenty (80%) of the 25 patients achieving CR received DLI with a median of three DLI (range, 1-9) per patient. Of these, ten patients (50%) were already in CR before the first DLI. Of note, CR rates did not differ between patients treated at the stage of molecular relapse and those initiated at hematological relapse (56% vs. 44%; P=0.778), neither between those with early and late relapse nor between the two dosage levels (Table 5). Also the presence of a dele-

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Table 1. Patient demographics (N=50). Characteristic Age in years, median (range) Sex (%) female male ECOG at screening (%) 0 1 2 HCT-CI (N=49) (%) low intermediate high WHO 2016 diagnosis (%) AML MDS CMML IPSS at diagnosis (N=19) (%) low intermediate-1 intermediate-2 high IPSS-R at diagnosis (N=21) (%) intermediate high very high Karyotype (N=45) (%) normal abnormal complex non-complex Molecular/genetic risk* (N=45) (%) favorable intermediate adverse Disease status at Tx (%) remission no remission primary refractory no response relapse untreated Conditioning (%) standard-dose reduced-intensity Donor/HLA-match (%) related unrelated 10/10 9/10 In vivo T-cell depletion (%) yes no Graft source (%) PBSC BM

2.5 mg (N=14)

5 mg (N=36)

63 (30–75)

64.5 (43-73)

62.5 (30-75)

19 (38) 31 (62)

4 (29) 10 (71)

15 (42) 21 (58)

0.52

14 (28) 32 (64) 4 (8)

4 (29) 8 (57) 2 (14)

10 (28) 24 (67) 2 (6)

0.31

18 (37) 16 (33) 15 (30)

6 (43) 5 (36) 3 (21)

12 (34) 11 (31) 12 (34)

0.50

23 (46) 24 (48) 3 (6)

3 (21) 10 (71) 1 (7)

20 (56) 14 (39) 2 (6)

0.049

0 (0) 8 (42) 6 (32) 5 (26)

0 (0) 3 (33) 3 (33) 3 (33)

0 (0) 5 (50) 3 (30) 2 (20)

0.65

9 (43) 8 (38) 4 (19

4 (57) 1 (14) 2 (29)

5 (36) 7 (50) 2 (14)

0.40

20 (44) 25 (56) 12 (24) 13 (32)

6 (46) 7 (54) 3 (23) 4 (31)

14 (44) 18 (56) 9 (28) 9 (28)

0.99

15 (33) 14 (31) 16 (36)

5 (38) 5 (38) 3 (24)

10 (31) 9 (28) 13 (41)

0.32

14 (28) 36 (72) 9 (18) 8 (16) 5 (10) 14 (28)

4 (29) 10 (71) 0 (0) 4 (29) 0 (0) 6 (43)

10 (28) 26 (72) 9 (25) 4 (11) 5 (14) 8 (22)

>0.99

34 (68) 16 (32)

9 (64) 5 (36)

25 (69) 11 (31)

0.75

9 (18) 41 (82) 44 (88) 6 (12)

1 (7) 13 (93) 12 (86) 2 (14)

8 (22) 28 (78) 32 (89) 4 (11)

0.41

41 (82) 9 (18)

13 (93) 1 (7)

28 (78) 8 (22)

0.41

49 (98) 1 (2)

14 (100) 0 (0)

35 (97) 1 (3)

>0.99

0.99

0.17

*For acute myeloid leukemia (AML) patients we used the ELN AML 2017 Genetic Risk Stratification. For patients with myelodysplastic syndrome (MDS) we used the International Prognostic Scoring System - revised (IPSS-R) genetic risk categories summarizing very good and good as well as high and very high. For chronic myelomonocytic leukemia (CMML), we used the genetic risk categories of the CMML-specific prognostic scoring system (CPSS). Molecular/genetic risk confers to the results obtained at the time of primary diagnosis. Numbers in parentheses display patients with available information. Data are given for the entire cohort as well as for the 2 daily dosage levels (2.5 mg and 5 mg). BM: bone marrow; ECOG: Eastern Cooperative Oncology Group; ELN: European Leukemia Net; HCT-CI: hematopoietic cell transplantation - specific comorbidity index; HLA: human leukocyte antigen; PBSC: peripheral blood stem cells; Tx: transplantation. Haematologica | 108 November 2023

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tion 5q (n=7, including 6 with a complex karyotype) had no impact on the likelihood to achieve CR. Twenty of the 25 patients (80%) achieving CR in the 8month treatment period remained in ongoing remission for a median of 15 months (range, 6-21 months) without any additional antileukemic treatment at last follow-up,

while five patients (20%) relapsed again in median 12 months (range, 3-20 months) after achieving CR (Figure 1). Three of the former patients with ongoing remissions died due to non-relapse-related causes (ischemic stroke, organ failure and sepsis). In addition to that, another eight patients (16%), who did

Table 2. Relapse characteristic (N=50). N All (N=50)

2.5 mg (N=14)

5 mg (N=36)

233 (61-2,659)

193 (91-2,659)

243 (61-1,487)

0.83

26 (52) 24 (48)

9 (64) 5 (36)

17 (47) 19 (53)

0.35

WBC x109/L, median (range)

3.57 (1.2-20.3)

3.6 (1.69-6.9)

3.57 (1.2-20.3)

0.74

PB blasts %, median (range)

0 (0-32)

0 (0-19)

0.08

BM blasts %, median (range)

4 (0-70)

4 (0-60)

0.77

11.1 (8.1-16.5)

10.1 (8.1-13.2)

11.4 (8.1-16.5)

0.39

87 (8-824)

83 (8-468)

90 (9-824)

0.91

197 (128-501)

175 (153-363)

202 (128-501)

0.08

BM chimerism %, median (range)

76 (6-100)

77.5 (30-100)

71.5 (6-100)

0.98

PB chimerism %, median (range)

95.5 (23-100)

87 (35-100)

96 (23-100)

0.61

9 (18) 4 (8) 4 (8) 1 (2) 4 (8) 4 (8)

1 (7) 0 (0) 1 (7) 0 (0) 0 (0) 0 (0)

8 (22) 4 (11) 3 (8) 1 (3) 4 (11) 4 (11)

Characteristic Time to relapse in days, median (range) Type of relapse (%) molecular hematological

Hb g/dL, median (range) Platelets x109/L, median (range) LDH U/L, median (range)

GvHD before relapse (%) acute grade 1 grade 2 unknown grade chronic mild

0.08

Immunosuppression at study entry (%) yes no taper/stop*

0.99 15 (30) 35 (70) 11 (73)

5 (36%) 9 (64%) 5 (100%)

10 (28) 26 (72) 6 (60)

*Percentage refers to the number of patients, who received immunosuppression at study entry. Numbers in parentheses display patients with available information. The cohort of patients with molecular relapse included 5 individuals with isolated loss of complete donor chimerism (DC). Data are given for the entire cohort as well as for the 2 daily dosage levels (2.5 mg and 5 mg). Hb: hemoglobin; GvHD: graft-versus-host disease; Hb: hemoglobin; LDH: lactate dehydrogenase; PB: peripheral blood; BM: bone marrow; WBC: white blood cells.

Table 3. Hematotoxicity and laboratory findings during the study. Parameters Grade

Absolute neutrophil count

Platelets

Anemia

Bilirubin

Creatinine

N (%)

Pat.

N (%)

Pat.

N (%)

Pat.

N (%)

Pat.

N (%)

Pat.

No toxicity

22 (8)

20 (7)

239 (87)

162 (59)

8 (3)

86 (31)

107 (39)

25 (9)

94 (34)

36 (13)

42 (15)

107 (39)

6 (2)

19 (7)

62 (23)

49 (18)

41 (15)

4 (1)

0 (0)

144 (53)

76 (28)

0 (0)

1 (0)

0 (0)

Summary table indicates maximum common toxicity criteria (CTC) severity grades per patients (Pat.) and cycle. A total of 275 treatment cycles was administered. Number (N) depicts the affected treatment cycles, while patients indicates the number of affected patients according to the maximum adverse events CTC severity during treatment. Haematologica | 108 November 2023

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not respond and, therefore, prematurely terminated study treatment, achieved CR during follow-up. Median time to remission in these patients was 162 days (range, 73-554 days) calculated from treatment start. Two of these patients achieved CR after additional DLI, while the remaining six patients entered remission after salvage therapy with chemotherapy (n=3) or Decitabine/Venetoclax (n=2) followed by second transplantation (n=6). Of these eight patients, six were alive with ongoing remissions in four of them for a median of 8 months (range, 3-11 months). At data lock 28 patients (56%) were alive including 17 patients, who were free of disease. Median OS of the entire cohort was 21 months and estimated 1-year OS rate was 65%. The median OS in patients who achieved CR was not reached compared to 9.7 months in non-responders (P=0.0004; Figure 1). Similar to response, the OS rate was not influenced by diagnosis, genetic risk, Lena dosage, time to as well as type of relapse (Table 5). Twelve patients died during or after treatment due to disease progression, while ten patients, of which seven had active disease at the time of death, succumbed to due infections (n=6), cardiovascular complications (n=3) or liver failure (n=1).

Graft-versus-host disease During the interval between transplantation and relapse a total of nine patients (18%) had suffered from acute GvHD (overall grade 1 n=4, grade 2 n=4, missing n=1) and four patients from mild chronic GvHD (8%). However, at relapse only one patient still suffered from grade 1 acute GvHD not requiring systemic immunosuppression. Fifteen patients were still on systemic immunosuppressive prophylaxis, which could be tapered or directly stopped in 11 of them. Overall, 15 patients (30%) developed aGvHD (overall grade 1 n=3, grade 2 n=7, grade 3 n=2, grade 4 n=3) and 19 patients (38%) developed cGvHD (mild n=5, moderate n=10, severe n=4) in median 112 days (range, 5-810 days) after inclusion (Online Supplementary Tables S3 and S4). In 11 patients de novo onset of cGVHD was observed, while cGvHD developed fom aGvHD in the remaining eight patients. Frequencies of GvHD in the 2.5 mg and 5 mg dosing cohort were 43% (n=6 patients) and 55% (n=20; P=0.533), respectively. In 13 of the 26 patients that developed acute or chronic GvHD the first DLI was administered in median 203 days (range, 16-708 days) before GvHD onset, while seven patients received DLI after developing GvHD.

Table 4. Drug-related non-hematologic adverse events during the study. Events

Grade 1 N (%)

Grade 2 N (%)

Grade 3 N (%)

Grade 4 N (%)

Patients

Blood and lymphatic system disorders

2 (15)

1 (8)

9 (69)

1 (8)

Ear and labyrinth disorders

1 (50)

2 (100)

Eye disorders

1 (50)

0 (0)

1 (50)

Gastrointestinal disorders

40 (61)

23 (33)

4 (6)

General disorders and administration site conditions

26 (67)

7 (28)

2 (5)

Immune system disorders

8 (35)

12 (52)

3 (13)

Infections and infestations

4 (8)

39 (75)

9 (17)

2 (4)

Injury, poisoning and procedural complications

0 (0)

1 (100)

Investigations

25 (47)

15 (28)

12 (23)

1 (2)

Metabolism and nutrition disorders

6 (50)

5 (42)

1 (8)

Musculoskeletal and connective tissue disorders

3 (43)

1 (14)

Neoplasms, benign, malignant and unspecified

1 (100)

Nervous system disorders

6 (60)

1 (10)

3 (30)

Psychiatric disorders

1 (100)

Reproductive system and breast disorders

1 (100)

Respiratory, thoracic and mediastinal disorders

4 (57)

3 (43)

Skin and subcutaneous tissue disorders

17 (68)

8 (32)

Vascular disorders

3 (60)

1 (20)

Endocrine disorders

Summary table indicates numbers and percentages of drug-related, non-hematologic adverse events (N=305) according to common toxicity criteria grades. Patients indicates the number of affected patients according to the respective adverse events term. Haematologica | 108 November 2023

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Immune evaluation As exposure to Aza and Lena both can modulate T-cell activity and functionality, we monitored T-cell numbers and functionality during study treatment in a subgroup of 11 patients. Consistent with the hypothesis of T-cell exhaustion, we observed a significant higher frequency of CD3+/CD8+ T cells expressing PD1, CTLA4 and TIM3 in patients at relapse compared with healthy controls (P<0.05; Online Supplementary Figure S3), which was not modulated during therapy. Furthermore, during study treatment the frequency of CD3+/CD4+/CD25+/FoxP3+ regulatory T cells significantly increased in comparison to the baseline level (Online Supplementary Figure S3).

Discussion We here demonstrate that Lena can safely be added to the backbone of Aza and DLI as salvage therapy for relapse of myeloid malignancies after allo-SCT without excess of GvHD or other toxicities. The combination of immunodulatory drugs and cellular therapy induced a remarkable response rate of 56%, durable remissions in 20 patients, who have remained in remission for a median of 15 months, and a 1-year OS rate of 65%. Generally, the outcome of relapse of AML or MDS after allo-SCT is dismal reflected by 2-year OS rates of 13.9%

Figure 1. Overall survival and disease-free survival. Overall survival is displayed for all patients (A) and for patients separated according to response (B). Complete remission (CR) (n=25, green curve), no CR (n=25, red curve). (C) Disease-free survival (DFS) is displayed for the 25 CR patients. DFS was calculated as time from CR to relapse, death or last follow-up in those alive and still in remission.

Table 5. Predictors of response and survival – univariate analyses. Variable

CR rate (%)

1-year OS (%)

Lena dosage 2.5 mg 5 mg

43 53

0.75

75 61

0.22

Diagnosis AML MDS/CMML

48 52

>0.9999

55 72

0.09

Type of relapse molecular hematologic

56 44

0.78

69 59

0.55

Time to relapse in days <233 ≥233

48 52

>0.9999

54 75

0.19

Genetic risk favorable/intermediate adverse

56 44

>0.55

84 50

0.07

AML: acute myeloid leukemia; CMML: chronic myelomonocytic leukemia; CR: complete remission; Lena: lenalidomide; MDS: myelodysplastic syndrome; OS: overall survival.

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and 29.7%, respectively.2,3 In detail, CR rates following intensive chemotherapy or DLI alone range between 15% to 30% and translate into 2-year survival rates between 8% and 21%.2,3,27,28 Similarly, a 2-year OS rate of 25% following second transplantation in selected patients has been reported, while targeted approaches such as Sorafenib or Enasidenib exert clinical activity, but are restricted to specific genotype-defined subtypes.4,29,30 Following singleagent Aza with or without DLI, CR rates of 15% to 41% and 2-year survival rates ranging from 12% to 38% have been reported from one prospective and several retrospective studies.6,7,10–14 Compared to conventional approaches, which can mostly be administered in an in-patient setting only, Aza and DLI is an outpatient approach resulting in similar or even better response and survival rates and has, therefore, become standard of care. Although a direct comparison is not possible in the absence of a randomized trial and while the follow-up of this trial is still limited, the response and survival following Aza, Lena and DLI appears very promising. This is in accordance with the results of a recently published clinical trial investigating therapy of post-transplant relapse with a combination of Aza and Lena22 indicating synergistic antileukemic and immunologic effects of the two compounds. Furthermore, the high response rate in our trial might also be related to the fact that, in contrast to the reports on single agent Aza,6,7,10–14 a higher fraction of patients (52%) was treated at the stage of molecular relapse mirroring the currrent practice of preemptive, measurable-disease guided interventions. In addition, inclusion of patients merely based on decreasing chimerism as well as patients with MDS may also have contributed to the results observed in our trial. Disease burden and early relapse after transplant inversely correlate with response and survival following Aza monotherapy as reflected by a CR and 2-year OS rate of 29% and 27% in those with early hematological relapse.14 In contrast, type of and time to relapse were no longer associated with response and survival in our actual trial as also demonstrated by five of 12 patients (42%) with early (<6 months) hematologic relapse achieving CR. Despite the limited patient number and the lack of a multivariate analysis, our data including a notable CR rate of 40% in patients with frank hematologic relapse suggest that patients with early hematologic relapse, who otherwise have a limited chance to respond to Aza monotherapy,14 may benefit from the addition of Lena. The observed clinical synergism may be related to the additive antileukemic activity of the two drugs or alternatively to the pharmacologic manipulation of the graft-versus-leukemia (GvL) effect. While Lena directly enhances T-cell activity, Aza upregulates tumor antigen expression on leukemic cells and also induces CD8+ T-cell response.21 In order to further address this on the translational level, we investigated T cells with regard to subset

composition and exhaustion/activation markers. In line with previous findings22,31 we observed a higher frequency of CD8+ T cells with exhausted phenotype (PD-1+, CTLA-4+, TIM3+) at relapse, which was not reverted by the combined therapy with Aza and Lena (Online Supplementary Figure S3). Thus, the mechanism, how Lena synergizes with Aza remains elusive here and requires further investigations. Still, but alternatives to reverse T-cell exhaustion may be an additional option to prevent or treat relapse. Aiming to decipher the role of DLI we looked at the 25 patients achieving CR, of whom 20 received DLI. Ten of these were already in remission prior the first DLI underlining the synergistic, antileukemic activity of Aza and Lena. This is in line with the results of Craddock et al., who observed remissions following this pharmacological combination without the regular use of DLI.22 The ten remaining patients achieved CR after first DLI suggesting an immunologic effect. Here, Aza and Lena had offered disease control and probably enhanced DLI-driven immune effect. Only five CR patients had lost response at last follow-up including three with previous DLI. Overall, we believe from our previous data14 and reports from others,2 that Aza and Lena can induce remissions, but additional donor-cell based consolidation is definitively required to achieve CR persistence. Nevertheless, the exact contribution of the two pharmacological compounds and the cellular therapy for remission induction and long-term disease control can only be dissected within a randomized trial. Similar to the results reported by Craddock et al.,22 response was not counterbalanced by severe toxicity including acute and chronic GvHD enabling outpatient treatment in most patients and an acceptable hospitalization rate. Drug-related, non-hematologic adverse event were mainly grade 1 and 2. Neutropenia and thrombocytopenia grade 3/4 occurred in a relevant proportion of patients, but were manageable and did not lead to omission of Lena in the majority of treatment cycles. This may be one advantage of the relatively low Lena dosages we used to take advantage of its immunomodulatory properties compared to other similar trials, where higher dosages were envisaged to excert its full antileukemic activity.22 Based on results from previous studies investigating Lena as maintenance therapy following transplant,18,19 GvHD induction was a major concern when planning this trial and was addressed by two safety interim analyses, a dose escalating scheme and rather low dosages of Lena. We observed rates of aGvHD (30%) and cGvHD (38%), which are comparable to those after hypomethylating agent/DLI therapy and even slightly lower than aGvHD and cGvHD rates of 43% and 46% observed after DLI alone.28 Thus, in combination with Aza, dosages of 2.5 and 5 mg Lena do not lead to an excess of GvHD. This might be related to the previously observed Aza-mediated expansion of regulatory T cells,20,21 which we also found here in the trial population

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treated with Aza combined with Lena (Online Supplementary Figure S3). Since an acceptable GvHD rate was reported even after 25 mg Lena,22 one might speculate that an increase of the daily Lena dosage may further enhance the efficacy of this combined approach of Aza, Lena and DLI. However, hematologic toxicity may then become a concern. Hematologic toxicity and associated infections are the most serious side effects of combination therapy with Aza or Decitabine and Venetoclax, which has also become a treatment option for the relapse of myeloid malignancies after allo-SCT.32 In addition Venetoclax, while enhancing the cytotoxicity of Aza on the one hand, may hinder the development of GvL effects and thereby long-term remissions due to its lymphotoxic properties on the other.33 Taken together, our data demonstrate that 5 mg Lena can be safely added to the combination of Aza plus DLI and exerts significant antileukemic and immune-modulatory activity in patients with relapse after allo-SCT, including those with early hematologic relapse. Our results establish the combination of Aza, Lena and DLI as valuable treatment alternative among other current treatment modalities for patients relapsing after allo-SCT.

closes advisory boards, and research funding from Celgene GmbH Germany. UG discloses advisory boards, lecture fees, research funding from Celgene GmbH Germany. All other authors declare no conflicts of interest.

Disclosures TS discloses advisory boards, lecture fees, travel support, and research funding from Celgene GmbH Germany. JHM discloses advisory boards, lecture fees, and travel support from Celgene GmbH Germany, Pfizer, Novartis, JAZZ, Astellas and Daiichi Sankyo. UH discloses advisory boards, lecture fees, and consultancy from Celgene GmbH Germany. NK dis-

Contributions GK and TS developed the concept and design of the study and wrote the manuscript. TS, CR, PSJ, ND and GK collected and assembled data. TS, CR, PSJ, ND and GK analyzed and interpreted data. All authors provided patient data and approved the final version of the manuscript. Acknowledgments We would like to thank the staff of the transplant unit of the Department of Hematology, Oncology and Clinical Immunology for excellent patient care. The study was conducted in cooperation with the Coordination Center for Clinical Trials at Heinrich Heine University, Duesseldorf, Germany. This study was an investigator-initiated trial with the Heinrich Heine University Duesseldorf acting as a study sponsor. Celgene Corporation financially supported some of the logistics of the study and provided the study drug. Funding This work was supported by a restricted grant of Celgene GmbH Germany. Data-sharing statement Details on the data can be provided on personal request.

References 1. Craddock C, Versluis J, Labopin M, et al. Distinct factors determine the kinetics of disease relapse in adults transplanted for acute myeloid leukaemia. J Intern Med. 2018;283(4):371-379. 2. Schmid C, Labopin M, Nagler A, et al. Treatment, risk factors, and outcome of adults with relapsed AML after reduced intensity conditioning for allogeneic stem cell transplantation. Blood. 2012;119(6):1599-1606. 3. Schmid C, de Wreede LC, van Biezen A, et al. Outcome after relapse of myelodysplastic syndrome and secondary acute myeloid leukemia following allogeneic stem cell transplantation: a retrospective registry analysis on 698 patients by the Chronic Malignancies Working Party of the European Society of Blood and Marrow Transplantation. Haematologica. 2018;103(2):237-245. 4. Christopeit M, Kuss O, Finke J, et al. Second allograft for hematologic relapse of acute leukemia after first allogeneic stem-cell transplantation from related and unrelated donors: the role of donor change. J Clin Oncol. 2013;31(26):3259-3271. 5. Bazarbachi A, Schmid C, Labopin M, et al. Evaluation of trends and prognosis over time in patients with AML relapsing after allogeneic hematopoietic cell transplant reveals improved survival for young patients in recent years. Clin Cancer Res. 2020;26(24):6475-6482.

6. Craddock C, Labopin M, Robin M, et al. Clinical activity of azacitidine in patients who relapse after allogeneic stem cell transplantation for acute myeloid leukemia. Haematologica. 2016;101(7):879-883. 7. Schroeder T, Rachlis E, Bug G, et al. Treatment of acute myeloid leukemia or myelodysplastic syndrome relapse after allogeneic stem cell transplantation with azacitidine and donor lymphocyte infusions - a retrospective multicenter analysis from the German Cooperative Transplant Study Group. Biol Blood Marrow Transplant. 2015;21(4):653-660. 8. Platzbecker U, Middeke JM, Sockel K, et al. Measurable residual disease-guided treatment with azacitidine to prevent haematological relapse in patients with myelodysplastic syndrome and acute myeloid leukaemia (RELAZA2): an openlabel, multicentre, phase 2 trial. Lancet Oncol. 2018;19(12):1668-1679. 9. Platzbecker U, Wermke M, Radke J, et al. Azacitidine for treatment of imminent relapse in MDS or AML patients after allogeneic HSCT: results of the RELAZA trial. Leukemia. 2012;26(3):381-389. 10. Schroeder T, Czibere A, Platzbecker U, et al. Azacitidine and donor lymphocyte infusions as first salvage therapy for relapse of AML or MDS after allogeneic stem cell transplantation.

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Leukemia. 2013;27(6):1229-1235. 11. Steinmann J, Bertz H, Wäsch R, et al. 5-Azacytidine and DLI can induce long-term remissions in AML patients relapsed after allograft. Bone Marrow Transplant. 2015;50(5):690-695. 12. Czibere A, Bruns I, Kröger N, et al. 5-Azacytidine for the treatment of patients with acute myeloid leukemia or myelodysplastic syndrome who relapse after allo-SCT: a retrospective analysis. Bone Marrow Transplant. 2010;45(5):872-876. 13. Tessoulin B, Delaunay J, Chevallier P, et al. Azacitidine salvage therapy for relapse of myeloid malignancies following allogeneic hematopoietic SCT. Bone Marrow Transplant. 2014;49(4):567-571. 14. Rautenberg C, Bergmann A, Germing U, et al. Prediction of response and survival following treatment with azacitidine for relapse of acute myeloid leukemia and myelodysplastic syndromes after allogeneic hematopoietic stem cell transplantation. Cancers (Basel). 2020;12(8):E2255. 15. Blum W, Klisovic RB, Becker H, et al. Dose escalation of lenalidomide in relapsed or refractory acute leukemias. J Clin Oncol. 2010;28(33):4919-4925. 16. Sekeres MA, Tiu RV, Komrokji R, et al. Phase 2 study of the lenalidomide and azacitidine combination in patients with higher-risk myelodysplastic syndromes. Blood. 2012;120(25):4945-4951. 17. Sekeres MA, O’Keefe C, List AF, et al. Demonstration of additional benefit in adding lenalidomide to azacitidine in patients with higher-risk myelodysplastic syndromes. Am J Hematol. 2011;86(1):102-103. 18. Kneppers E, van der Holt B, Kersten M-J, et al. Lenalidomide maintenance after nonmyeloablative allogeneic stem cell transplantation in multiple myeloma is not feasible: results of the HOVON 76 Trial. Blood. 2011;118(9):2413-2419. 19. Sockel K, Bornhaeuser M, Mischak-Weissinger E, et al. Lenalidomide maintenance after allogeneic HSCT seems to trigger acute graft-versus-host disease in patients with highrisk myelodysplastic syndromes or acute myeloid leukemia and del(5q): results of the LENAMAINT trial. Haematologica. 2012;97(9):e34-35. 20. Schroeder T, Fröbel J, Cadeddu R-P, et al. Salvage therapy with azacitidine increases regulatory T cells in peripheral blood of patients with AML or MDS and early relapse after allogeneic blood stem cell transplantation. Leukemia. 2013;27(9):1910-1913. 21. Goodyear OC, Dennis M, Jilani NY, et al. Azacitidine augments expansion of regulatory T cells after allogeneic stem cell transplantation in patients with acute myeloid leukemia (AML). Blood. 2012;119(14):3361-3369. 22. Craddock C, Slade D, De Santo C, et al. Combination lenalidomide and azacitidine: a novel salvage therapy in

patients who relapse after allogeneic stem-cell transplantation for acute myeloid leukemia. J Clin Oncol. 2019;37(7):580-588. 23. Döhner H, Estey E, Grimwade D, et al. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood. 2017;129(4):424-447. 24. Cheson BD, Greenberg PL, Bennett JM, et al. Clinical application and proposal for modification of the International Working Group (IWG) response criteria in myelodysplasia. Blood. 2006;108(2):419-425. 25. Glucksberg H, Storb R, Fefer A, et al. Clinical manifestations of graft-versus-host disease in human recipients of marrow from HL-A-matched sibling donors. Transplantation. 1974;18(4):295-304. 26. Filipovich AH, Weisdorf D, Pavletic S, et al. National Institutes of Health Consensus Development Project on criteria for clinical trials in chronic graft-versus-host disease: I. Diagnosis and Staging Working Group Report. Biol Blood Marrow Transplant. 2005;11(12):945-956. 27. Motabi IH, Ghobadi A, Liu J, et al. Chemotherapy versus hypomethylating agents for the treatment of relapsed acute myeloid leukemia and myelodysplastic syndrome after allogeneic stem cell transplant. Biol Blood Marrow Transplant. 2016;22(7):1324-1329. 28. Schmid C, Labopin M, Nagler A, et al. Donor lymphocyte infusion in the treatment of first hematological relapse after allogeneic stem-cell transplantation in adults with acute myeloid leukemia: a retrospective risk factors analysis and comparison with other strategies by the EBMT Acute Leukemia Working Party. J Clin Oncol. 2007;25(31):4938-4945. 29. Mathew NR, Baumgartner F, Braun L, et al. Sorafenib promotes graft-versus-leukemia activity in mice and humans through IL15 production in FLT3-ITD-mutant leukemia cells. Nat Med. 2018;24(3):282-291. 30. Stein EM, DiNardo CD, Fathi AT, et al. Molecular remission and response patterns in patients with mutant-IDH2 acute myeloid leukemia treated with enasidenib. Blood. 2019;133(7):676-687. 31. Noviello M, Manfredi F, Ruggiero E, et al. Bone marrow central memory and memory stem T-cell exhaustion in AML patients relapsing after HSCT. Nat Commun. 2019;10(1):1065. 32. Schuler E, Wagner-Drouet E-M, Ajib S, et al. Treatment of myeloid malignancies relapsing after allogeneic hematopoietic stem cell transplantation with venetoclax and hypomethylating agents-a retrospective multicenter analysis on behalf of the German Cooperative Transplant Study Group. Ann Hematol. 2021;100(4):959-968. 33. Strobl J, Pandey RV, Krausgruber T, et al. Anti-apoptotic molecule BCL2 is a therapeutic target in steroid-refractory graft-versus-host disease. J Invest Dermatol. 2020;140(11):2188-2198.

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ARTICLE - Chronic Lymphocytic Leukemia

Interleukin-27 potentiates CD8+ T-cell-mediated antitumor immunity in chronic lymphocytic leukemia Giulia Pagano,1,2* Iria Fernandez Botana,1,2* Marina Wierz,1 Philipp M. Roessner,3 Nikolaos Ioannou,4 Xiangda Zhou,5 Gheed Al-Hity,4 Coralie Borne,1 Ernesto Gargiulo,1 Susanne Gonder,1,2 Bin Qu,5 Basile Stamatopoulos,6 Alan G. Ramsay,4 Martina Seiffert,3 Anne Largeot,1 Etienne Moussay1# and Jerome Paggetti1# Tumor Stroma Interactions, Department of Cancer Research, Luxembourg Institute of Health, Luxembourg, Luxembourg; 2Faculty of Science, Technology and Medicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg; 3Molecular Genetics, German Cancer Research Center (DKFZ), Heidelberg, Germany; 4School of Cancer and Pharmaceutical Sciences, Faculty of Life Sciences & Medicine, King's College London, London, UK; 5 Biophysics, Center for Integrative Physiology and Molecular Medicine, School of Medicine, Saarland University, Homburg, Germany and 6Clinical Cellular Therapy Research Laboratory, Jules Bordet Institute, Brussels, Belgium 1

Correspondence: J. Paggetti jerome.paggetti@lih.lu Received: Accepted: Early view:

November 22, 2022. June 15, 2023. June 22, 2023.

https://doi.org/10.3324/haematol.2022.282474 Published under a CC BY license

GP and IFB contributed equally as first authors. EM and JP contributed equally as senior authors.

Abstract Chronic lymphocytic leukemia (CLL) cells are highly dependent on interactions with the immunosuppressive tumor microenvironment (TME) for survival and proliferation. In the search for novel treatments, pro-inflammatory cytokines have emerged as candidates to reactivate the immune system. Among those, interleukin 27 (IL-27) has recently gained attention, but its effects differ among malignancies. Here, we utilized the Eμ-TCL1 and EBI3 knock-out mouse models as well as clinical samples from patients to investigate the role of IL-27 in CLL. Characterization of murine leukemic spleens revealed that the absence of IL-27 leads to enhanced CLL development and a more immunosuppressive TME in transgenic mice. Gene-profiling of T-cell subsets from EBI3 knock-out highlighted transcriptional changes in the CD8+ T-cell population associated with T-cell activation, proliferation, and cytotoxicity. We also observed an increased anti-tumor activity of CD8+ T cells in the presence of IL-27 ex vivo with murine and clinical samples. Notably, IL-27 treatment led to the reactivation of autologous T cells from CLL patients. Finally, we detected a decrease in IL-27 serum levels during CLL development in both pre-clinical and patient samples. Altogether, we demonstrated that IL-27 has a strong anti-tumorigenic role in CLL and postulate this cytokine as a promising treatment or adjuvant for this malignancy.

Introduction Chronic lymphocytic leukemia (CLL) is the most frequent type of adult leukemia in the USA and Europe, affecting mainly older adults.1 Clinically, CLL is defined as a B-cell hematological malignancy characterized by accumulation of abnormal, monoclonal, B lymphocytes in the peripheral blood (PB) and secondary lymphoid organs.2 Current treatments against CLL do not have a curative potential, and a significant percentage of patients do not respond or become resistant.3 Consequently, there is a pressing need for the development of novel therapies for advanced and aggressive CLL, which unfortunately remains an incurable disease. CLL cells are highly dependent on interactions with surrounding non-malignant cells for survival and proliferation.4,5 In fact, they spontaneously

undergo apoptosis in monoculture, but co-culture with accessory cells significantly extends their survival.6-8 Given the pivotal role of the tumor microenvironment (TME) in CLL, an increasing number of studies are focusing on the identification of micro-environmental signals that could play a role in the pathogenesis of this disease. Interleukin 27 (IL-27) is a heterodimeric cytokine composed of two non-covalently linked subunits: IL-27p28 (P28) and Epstein-Barr-virus-induced molecule 3 (EBI3).9 IL-27 is produced by activated antigen presenting cells (APC) and it signals through a heterodimeric receptor (IL-27R) that comprises the gp130 and WSX-1 subunits, both essential for efficient signaling.10 IL-27 was initially characterized as being pro-inflammatory, given its ability to promote Th1 immunity. Nevertheless, it was later reported that IL-27 exerts a potent inhibitory role during Th2, Th17 and regulatory T-cell

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(Treg) differentiation. Hence, IL-27 mediates a wide range of functions involved in T-cell-mediated immunity.11 Not surprisingly, IL-27 is known to have pleotropic functions in the setting of cancer development in relationship to the biological context and experimental models considered. Most existing evidence refers to the anti-tumor activities of this cytokine.12 IL-27 has been reported to hinder tumor development and progression mainly by modulating the immune landscape surrounding the malignant cells.13 While most evidence points towards the upregulation of Th1 and Cytotoxic T lymphocyte (CTL) responses as the main anti-tumor contribution of IL-27 to the TME,14-16 other reports suggest that IL-27 can also mediate natural killer (NK) cell responses and inhibit M2 macrophage polarization.17,18 On the other hand, a few studies indicate that IL-27 might also contribute to tumorigenesis in specific settings. For example, elevated IL-27 serum levels are associated with poor prognosis and disease progression in some malignancies such as gastroesophageal cancer, melanoma and adult acute myeloid leukemia (AML).19-21 Additionally, IL-27 was found to modulate transcriptional programs and induce the expression of immune-regulatory molecules such as PD-L1 and IDO in human ovarian cancer cells in vitro.22,23 Given the dynamic nature of cytokine biology, and consistent with a rather variable role in cancer biology, the role of IL-27 and its mechanism of action must be carefully investigated in each malignancy. With this background, we asked whether IL-27 has an effect in the development and progression of CLL. Here, we describe a strong anti-tumorigenic role of IL-27 in CLL in distinct preclinical mouse models and patient samples. First, we show that the genetic depletion of the IL-27 subunit Ebi3 leads to a strongly enhanced CLL development and a more immunosuppressive TME using two in vivo approaches. Secondly, we elucidate a mechanism by which IL-27 enhances CD8+ T-cell anti-tumor immunity in CLL. Moreover, we measured lower plasma levels of IL-27 concomitant with CLL development in mice and patients. We functionally validate the enhanced anti-tumor ability of CD8+ T cells in the presence of IL-27 both in murine and human samples. Finally, we show that IL-27 neutralization recapitulates the enhanced leukemic progression observed in the transgenic mouse models.

Methods Additional information regarding the materials and methods used in this publication can be found in the Online Supplementary Appendix. Patient samples All experiments involving the use of human samples were conducted in accordance with the declaration of Helsinki

and approved by the appropriate local Ethics Committee: the Jules Bordet Institute Ethics Committee (Belgium), The Comité National d'Ethique de Recherche (Luxembourg), and the University of Saarland (Germany). PB samples were obtained from CLL patients and from age-matched healthy donors following informed consent. PB mononuclear cells (PBMC) were isolated from whole blood by density gradient centrifugation using the LeucoSep™ Separation Medium (Greiner Bio-One) as described in the manufacturer’s protocol. Animal experiments All animal experiments were performed under specific pathogen-free conditions with the approval of the Luxembourg Ministry of Agriculture in accordance with the guidelines from the European Union. Ebi3-/- (RRID: IMSR_JAX:008701) and FoxP3YFP/Cre (RRID: IMSR_JAX:016959) mice were purchased from the Jackson Laboratory (Bar Harbour, ME); and C57BL/6 (MGI: 3028467, RRID: IMSR_JAX:000664) from Janvier Labs (France). Eµ-TCL1 mice (on C57BL/6 background; MGI: 3527221) were kindly provided by Pr. Carlo Croce and Pr. John Byrd (OSU, OH). The Eμ-TCL1 Ebi3-/- strain was generated in-house by crossing Ebi3-/- mice with Eμ-TCL1, together with FoxP3YFP/Cre Ebi3-/- mice, obtained by breeding FoxP3YFP/Cre and Ebi3-/mice for several generations. Rag2-/- mice were held at specific pathogen-free conditions at the central animal facility of the German Cancer Research Centre (DKFZ). Flow cytometry Single-cell suspensions were stained with cell-surface antibodies (30 minutes [min], 4 °C) and washed twice with FACS buffer. Dead cell discrimination was performed using Zombie dye (Biolegend), resuspended in PBS (20 min, 4 °C). For the staining of intracellular proteins, surface-stained cells were fixed (30 min, room temperature) with eBioscienceTM Foxp3/Transcription Factor Staining Buffer Set (ThermoFisher Scientific). After additional washing steps, cells were permeabilized using eBioscienceTM Permeabilization Buffer (ThermoFisher Scientific) and stained with the intracellular antibody mix (30 min, 4°C). Samples were stored at 4°C in dark conditions until acquisition. Antibodies used for flow cytometry are listed in the Online Supplementary Table S1. Statistical analyses Sample size was determined based on expected variance of read-out. No samples or animals were excluded from the analyses. Statistical analysis was performed using GraphPad Prism software (version 9.1.2; RRID: SCR_002798). Data are displayed as mean ± standard error of mean (SEM). For the percentage of CLL cells in PB over time, we performed two-way ANOVA followed by multiple comparison test. The unpaired t test was used for the rest of the

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figures. A P value lower than 0.05 was considered statistically significant. Significance displayed in each figure is explained in figure legends.

Results EBI3 depletion promotes leukemia development and induces an enhanced immunosuppressive tumor microenvironment. In order to investigate the role of IL-27 in CLL development, we used Ebi3-/- mice, defective in the production of the heterodimeric IL-27. First, we validated the specific deletion of the Ebi3 subunit in murine splenocytes via quantitative polymerase chain reaction (qPCR), and confirmed the suitability of the models for further analyses (Online Supplementary Figure S1A). We adoptively transferred (AT) Ebi3-/mice and Ebi3+/+ mice (wild-type [WT], used as controls) with two independent clones of CLL cells obtained from

the spleen of leukemic Eμ-TCL1 mice. We then followed up leukemia development in the PB of recipient mice using flow cytometry (FC), which led to the observation of a strikingly enhanced tumor development in the absence of Ebi3 (Figure 1A; Online Supplementary Figure S1B). Consistently, we observed an increased number of splenocytes and spleen weight in mice lacking Ebi3 (Figure 1B). Then, we sorted multiple immune cell subsets from leukemic mice and we identified dendritic cells and monocytes as the main producers of IL-27 in leukemic mice, as they expressed high levels of both subunits Ebi3EBI3 and p28 (Online Supplementary Figure S1C). After, we characterized the immune landscape in the splenic CLL TME, focusing on T cells as main mediators of the anti-tumor immune response in vivo.24 We found an increased number of CD8+ effector T cells and CD4+ conventional T cells (Tconv) in mice lacking EBI3, while the Treg number remained stable (Online Supplementary Figure S1D). Moreover, Ebi3-/- mice presented more effector (CD62L- CD44+) and memory (CD62L+

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Figure 1. EBI3 depletion promotes leukemia development and induces an enhanced immunosuppressive tumor microenvironment. (A) Two cohorts of recipient Ebi3-/- and control (Ctrl) mice were injected with splenocytes isolated from leukemic Eμ-TCL1 mice. Two weeks after adoptive transfer (AT), mice were bled weekly to monitor chronic lymphocytic leukemia (CLL) development in peripheral blood (PB). Percentages of neoplastic CD5+ CD19+ cells were detected in PB by flow cytometry (FC); (N=18 for control [Ctrl] and N=19 for Ebi3-/-, two-way ANOVA). (B-F). For the second cohort, splenic tumor microenvironment (TME) was characterized by FC (N=9 for Ctrl and N=10 for Ebi3-/-). (B) Number of CD5+ CD19+ CLL cells in the spleen (left), and spleen weight (right) in Ebi3-/- compared to Ctrl mice. (C) Frequency of naive (CD62L+ CD44-), effector (CD62L- CD44+) or memory (CD62L+ CD44+) CD8+ T cells. (D) Percentage of proliferative Ki-67+ cells among CD8+ T cells in the spleen of Ctrl and Ebi3-/- mice. (E) Frequency of the indicated populations in Ctrl and Ebi3-/- CD8+ T cells. (F) Representative flow cytometry plot (left) depicting the gating strategy used to discriminate activated regulatory T cells (aTregs, CD62Lhi CD44lo) from resting regulatory T cells (rTregs, CD62Llo CD44hi). Number of rTregs and aTregs in spleens (middle). Frequency of the indicated populations in Ctrl and Ebi3-/- Tregs (right). Unpaired t test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

CD44+) CD8+ T cells, concomitant to a decrease in the frequency of naive cells (CD62L+ CD44-) (Figure 1C). In addition, we observed an increased frequency in Ki-67+ CD8+ T cells in Ebi3-/- mice (Figure 1D). It is known that some immune checkpoints (IC) are expressed by activated CD8+ T cells, but only terminally exhausted cells co-express several IC.25 Here, we observed an increased percentage of TIGIT+ and PD1hi in CD8+ T cells lacking EBI3, while no differences were observed in the frequency of PD1int cells (Figure 1E). In order to analyze the functional status of CD8+ T cells, we performed hierarchical clustering of CD8+ T cells based on marker expression. Seven CD8+ T-cell clusters (C) were identified based on CD44, Ki-67, PD1, IFN-γ, TIGIT and CD62L (Online Supplementary Figure S1E). In the absence of Ebi3, there was a decrease in naïve CD8+ T cells (C1) together with an increase in memory T cells (Tmem, C3) and effector T cells (Teff) showing high IC and Ki-67 expression (Online Supplementary Figure S1F), suggesting an exhausted phenotype. Moreover, activated Tregs (aTregs, CD62L- CD44+) are more abundant in leukemic Ebi3-/- mice (Figure 1F, left panel) and display higher levels of TIGIT and KLRG1 along with increased Ki-67 positivity (Figure 1F, right panel), thus exhibiting enhanced immunosuppression and proliferation. As observed for CD8+ T cells, the proportion of antigen-experienced CD4+ Tconv was also increased in Ebi3-/- mice (Online Supplementary Figure S1G). Clustering of CD4+ T cells revealed the presence of nine clusters (Online Supplementary Figure S1H, I), showing that activated and immunosuppressive Tregs were expanded in absence of Ebi3. Altogether, these findings suggest an anti-tumor role of EBI3 during CLL development, as its depletion enhances tumor growth, promotes CD8+ T-cell exhaustion and increases Treg immunosuppressive phenotype. In parallel, we deeply characterized the immune landscape in Ebi3+/+ and Ebi3-/- mice to confirm that the aforementioned differences in CLL growth were not due to major intrinsic differences between the transgenic strains. We used Foxp3YFP/Cre mice to analyze T cells and Tregs. As expected, immunophenotyping of Foxp3YFP/Cre and Foxp3YFP/Cre Ebi3-/splenocytes did not reveal any major difference between Ebi3+/+ and Ebi3-/- mice. We found no statistical differences in the frequency of T cells, NK cells, NK-T cells or myeloid cells between groups (Online Supplementary Figure S2A).

Moreover, the analysis of the myeloid cell compartment showed no differences in the frequency of neutrophils, dendritic cells, monocytes nor macrophages (Online Supplementary Figure S2B). Only a slight increase/decrease in the frequencies of CD8+ T cells and CD4+ Tconv cells, respectively, was observed, with no differences for Tregs (Online Supplementary Figure S2C). In order to evaluate T-cell functionality between phenotypes, we isolated splenic CD4+ and CD8+ T cells and analyzed IC and cytokines expression following ex vivo stimulation for 4 hours (h). We neither observed any difference in the frequency of indicated IC and cytokines in CD8+ T cells, CD4+ Tconv cells, nor in Tregs (Online Supplementary Figure S2D-F). These results indicate that Ebi3 depletion does not drastically impact immune cells distribution, effector cytokine secretion and IC expression in T cells, pointing towards a fundamental role of IL-27 in the tumor context rather than in physiological conditions. EBI3 depletion in transgenic Eµ-TCL1 mice affects mice survival In order to better understand the role of EBI3 during CLL development, we crossed the Eμ-TCL1 mouse model (referred as T mice) with Ebi3-/- mice to generate the Eμ-TCL1 Ebi3-/- mouse model (referred as TE mice) that spontaneously develop CLL in the absence IL-27 production. As opposed to the TCL1 AT model, TE mice lack Ebi3 expression in all cell types, including CLL cells. We observed a shorter survival for TE mice (median survival of 302 vs. 351 days; Figure 2A) and an increased percentage and number of CLL cells in the PB at an earlier time point, although not statistically significant (Figure 2B; Online Supplementary Figure S3A). The analysis of the splenic TME following euthanasia indicated, despite a similar tumor load (Online Supplementary Figure S3B, C), an increased frequency of CD8+ T cells among total T cells in leukemic TE mice (Figure 2C left panel), as well as an increase in TIGIT+ CD8+ T cells (Figure 2C right panel). Clustering analysis identified six clusters based on CD44, Ki-67, PD1, KLRG1, TIGIT and CD62L expression in CD8+ T cells, as well as an enrichment in proliferative Ki67+ CD8+ T cells (C6) (Figure 2D; Online Supplementary Figure S3D, E). In addition, a general increase in CD8+ T-cell subpopulations expressing sev-

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eral IC was observed in TE mice compared with their Ebi3+/+ counterparts (clusters C1-C4) (Online Supplementary Figure S3E), pointing towards a more exhausted phenotype of

CD8+ T cells in TE mice. Additionally, we observed a decrease in CD4+ T-cell percentage in leukemic mice lacking Ebi3 compared to Ebi3+/+ controls (Online Supplementary

Figure 2. EBI3 depletion in transgenic Eµ-TCL1 mice affects mice survival. (A) Transgenic Eμ-TCL1 mice were crossed with Ebi3-/mice to obtain leukemic mice deficient in interleukin 27 (IL-27). Starting at 6 months after birth, mice were bled every month to evaluate peripheral disease development (T=Eμ-TCL1 mice; TE=Eμ-TCL1 Ebi3-/- mice). Mouse survival was compared between groups (survival curve analysis). (B) Percentages of neoplastic CD5+ CD19+ cells were detected by flow cytometry (FC) in peripheral blood (PB) (N=17 for T mice and N=14 for TE mice, two-way ANOVA test). (C-E) Leukemic T and TE mice were euthanized and their splenocytes were analyzed using FC. (C) Percentage of CD8+ T cells in control (Ctrl) and Ebi3-/- CD3+ T cells and frequency of indicated populations among T and TE CD8+ T cells (N=6 for T group and N=6 for TE group). (D) Percentages of CD8+ T-cell clusters distribution in T and TE mice. (E) Percentage of conventional T-cell (Tconv) and regulatory T-cell (Tregs) populations among CD4+ T cells (left) and frequency of indicated cell populations among Tregs (right). (F) Leukemic Eμ-TCL1 mice and Eμ-TCL1 Ebi3-/- mice were euthanized, and chronic lymphocytic leukemia (CLL) cells were isolated from splenocytes. Isolated CLL cells were adoptively transferred into wild-type (WT) mice. Recipient mice were bled weekly to evaluate peripheral disease development. Percentages of neoplastic CD5+ CD19+ cells were detected in PB by FC (N=12 mice divided in 6 groups. Each group of 2 animals received 1 independent CLL clone coming from diseased T or TE mice). Unpaired t test, *P<0.05, **P<0.01. Haematologica | 108 November 2023

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Figure S3F). More precisely, TE mice exhibited a decreased proportion of CD4+ Tconv together with increased frequency of Tregs (Figure 2E left panel), which were more immunosuppressive, as highlighted by higher expression of CD44, CD73 and CTLA4 in the absence of Ebi3 (Figure 2E right panel). Unsupervised clustering revealed eight CD4+ T-cell clusters (Online Supplementary Figure S3G, I). Of particular relevance, a cluster representing highly immunosuppressive Tregs (C1) was enriched in Ebi3-deficient leukemic mice. Immunophenotyping of T and TE CLL cells showed that the expression of immunosuppressive cytokines, major histocompatibility complex molecules, activation markers, and IC remained unchanged; suggesting that EBI3 depletion in CLL cells does not play a role in the observed phenotype (Online Supplementary Figure S3J). In order to investigate whether the effects of Ebi3 depletion observed in vivo were mediated by cells of the TME or by CLL cells, we depleted Ebi3 exclusively in CLL cells. For this purpose, we isolated CLL cells from spleens of diseased T and TE mice and injected them in recipient control Ebi3+/+ mice. In this experimental setting, we could not observe any difference in tumor growth in mice injected with Ebi3-/- leukemic cells (Figure 2F). Altogether our results indicate that EBI3 depletion in cells of the TME and not in CLL cells favors CLL progression by impacting T-cell-mediated immunity. Specific T-cell-EBI3 depletion promotes leukemia development As the AT of EBI3-depleted CLL cells did not impact tumor growth (Figure 2F) while substantial changes in T cells were observed upon EBI3 depletion in the AT model and Eμ-TCL1 model (Figures 1C, D and 2C), we proceeded to specifically investigate the impact of Ebi3-/- CD3+ T cells on CLL development. We used Rag2-/- mice, deficient in producing mature T and B cells. CD3+ T cells were isolated from the

spleen of Ebi3-/- and Ebi3+/+ mice and intravenously injected in recipient Rag2-/- mice. Recipient mice were subsequently adoptively transferred with Eμ-TCL1 leukemic cells (Figure 3A). Monitoring of leukemic growth in the PB revealed that Rag2-/- mice injected with Ebi3-/- T cells showed an enhanced CLL development compared to mice injected with the WT counterpart (Figure 3A). Importantly, the differences observed between the two groups were not due to a variation in the T-cell number throughout the experiment (Online Supplementary Figure S4A). Consistently, post-euthanasia analysis of the leukemic spleens showed an increase in the percentage of CLL cells in the Ebi3-/group (Figure 3B). Regarding T cells, we did not observe any difference in the frequency of subpopulations (Figure 3C). Nonetheless, unsupervised analysis identified nine clusters of CD8+ T cells (Online Supplementary Figure S4B), and revealed the expansion of activated/exhausted CD8+ T cells in the mice injected with Ebi3-/- CD3+ T cells (Figure 3D; Online Supplementary Figure S4C). In addition, we identified an increased frequency of Tregs within the CD4+ T-cell population (Figure 3E) and a higher percentage of CD25+ and KLRG1+ Tregs (Figure 3F), indicating an enhanced immunosuppressive ability of Tregs in absence of EBI3. Moreover, unsupervised clustering analysis of analyzing CD4+ T cells identified an accumulation of activated and immunosuppressive Tregs (C2) in recipient mice injected with Ebi3-/- T cells (Online Supplementary Figure S4D, E). CD8+ T cells from Ebi3-/- mice have altered expression of genes involved in activation and functionality As our data demonstrates that IL-27 controls CLL development in a T-cell-mediated mechanism in vivo, we proceeded to investigate the transcriptional differences between T cells from Ebi3+/+ and Ebi3-/- mice after ex vivo activation (Figure 4A; Online Supplementary Figure S5A). Among the upregulated genes in CD8+ T cells, we ident-

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Figure 3. Specific T-cell-EBI3 depletion promotes leukemia development. (A) Control (Ctrl) and Ebi3-/- mice were euthanized, and CD3+ T cells were isolated from the splenocytes. Recipient Rag2-/- mice were injected with either Ctrl (N=10) or Ebi3-/- CD3+ T cells (N=10) and subsequently injected with splenocytes derived from leukemic Eμ-TCL1 mice. Experimental mice were bled weekly to evaluate peripheral disease development. Number of circulating neoplastic CD5+ CD19+ cells is shown in the 2 groups (two-way ANOVA test). (B) Percentage of chronic lymphocytic leukemia (CLL) cells in the spleen of recipient Rag2-/- mice. (C) Percentages of CD8+, conventional T cells (Tconv), and regulatory T cells (Tregs) among the injected Ctrl and Ebi3-/- CD3+ T cells. (D) Percentages of CD8+ T cells in clusters identified in spleens of recipient Rag2-/- mice. (E, F) Frequency of Tregs and of CD25+ and KLRG1+ Tregs in the spleen of recipient Rag2-/- mice. Unpaired t test, *P<0.05, ****P<0.0001.

ified Profilin-1 (Pfn1) (Figure 4B), an actin-binding protein and a negative regulator of effector T-cell-mediated cytotoxicity.26 Amidst the downregulated genes, we found several transmembrane transporters known to mediate T-cell activation27 (Slc2a8 and Slc7a5; Figure 4C, D). An ontology analysis indicated a reprograming of crucial functions as translation initiation and rRNA processing (Online Supplementary Figure S5B). We also compared the gene expression profile of Ebi3-/- CD8+ T cells with transcriptional signatures of four CD8+ T-cell subsets showing different degrees of exhaustion and dysfunctionality previously published.28 We observed that Ebi3-/- CD8+ T cells present similarities with progenitor CD8+ exhausted T cells (TexProg) but also with more terminally exhausted cells (Texterm; Online Supplementary Figure S5C, D). Indeed, looking at the third component of the PCA, explaining 13% of the variability, indicated that Ebi3-/- T cells and Texterm present similar gene expression profiles (Online Supplementary Figure S5D right panel). Therefore, we selected genes described to mirror T-cell activation and exhaustion and we observed that Ebi3-/- T cells clustered between progenitor and terminally exhausted T cells (Figure 4E). The expression of these marker genes also showed differences between Ebi3+/+ and Ebi3-/- T cells, mostly for Tox, Tbet, Il7r, Ccr1 and Pfn1 (Figure 4F). Regarding CD4+ Tconv and Treg compartments, gene expression analysis revealed no

differences between groups (Online Supplementary Figure S5E-F), suggesting that, in absence of Ebi3, effector CD8+ T cells are the major T-cell population affected and appear less functional when activated in vitro. Ebi3-/- CD8+ T cells present impacted cytotoxic activity while IL-27 enhances their potential both in murine and human settings In order to functionally characterize Ebi3-/- and Ebi3+/+ CD8+ T cells and validate the gene expression data, we performed ex vivo activation of CD3+ T cells for 3 days and analyzed CD8+ T cells by FC and confocal microscopy (Figure 5A). In line with previous findings, after 3 days, we could observe a decrease in TOX abundance in Ebi3-/- CD8+ T cells (Figure 5B left panel). Very interestingly, we could not observe any difference in cytokine production between the two conditions after 3 days of activation (Online Supplementary Figure S6A), whereas after 6 days, Ebi3-/- CD8+ T cells presented decreased production of IL2 and IFN-γ compared to WT cells (Figure 5B right panel). Clustering analysis of CD8+ T cells co-expressing different cytokines (C3) appeared significantly reduced (Figure 5C; Online Supplementary Figure S6B-D), confirming that in the absence of Ebi3, CD8+ T cells are less polyfunctional. After 6 days of activation, we quantified Profilin1 protein level in CD8+ T cells by immunofluorescence. As expected,

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Figure 4. CD8+ T cells from Ebi3-/- mice have altered expression of genes involved in activation and functionality. (A) Foxp3YFP/Cre and Foxp3YFP/Cre Ebi3-/- mice (N=3 per group) were euthanized at 8 weeks of age and CD3+ T cells were isolated from the splenocytes. T cells were activated for 72 hours, sorted into CD8+ T cells, CD4+ conventional T cells (Tconv), and regulatory T cells (Tregs) (YFP+) by flow cytometry (FC), and subjected to RNA sequencing (RNAseq). (B) Volcano plot depicting differentially expressed genes (DEG) between control (Ctrl) and Ebi3-/- CD8+ T cells. (C, D) Heatmap and score of transmembrane transporters in Ctrl and Ebi3-/- CD8+ T cells. (E) Heatmap depicting the similarity of Ebi3-/- CD8+ T cells with the subset of proliferating progenitor exhausted T cells described in 43. (F) Expression profiles of single genes in CD8+ T cells from Ctrl and Ebi3-/- mice. Unpaired t test, *P<0.05, **P<0.01. Haematologica | 108 November 2023

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the analysis revealed an increase in Profilin1 in Ebi3-/- CD8+ T cells (Figure 5D). In order to assess if IL-27 could indeed affect the cytotoxic capabilities of CD8+ T cells, we performed a cytotoxic assay with WT or Ebi3-/- CD8+ T cells treated with IL-27 for 48 h (Figure 5E), and super antigen (sAg)-loaded Eμ-TCL1 CLL cells as target cells. We noted an increased cytotoxic ability of CD8+ T cells in the presence of IL-27 (Figure 5E, Online Supplementary Figure S6E). In order to inspect whether the effect of IL-27 on CD8+ T cells was conserved in human cells, first, we purified and activated T cells from PBMC of four healthy donors, and then cultured them in the presence or absence of IL-27 for 6 days (Online Supplementary Figure S6F). T cells were co-cultured with target cells for 30 h and the killing efficiency of CD8+ T cells during co-culture was quantified. In line with previous results, we observed a decrease in the percentage of live target cells, translated into an increased killing efficiency of human CD8+ T cells in the presence of IL-27 for all donors (Online Supplementary Figure S6G, H). Finally, we isolated T cells and CLL cells from CLL patients, and performed a killing assay in the absence or presence of IL-27. Again, we confirmed the positive effect of IL-27 on the cytotoxic activity of CLL patients’ T cells (Figure 5F). IL-27 level is reduced in the blood of leukemic mice and chronic lymphocytic leukemia patients and IL-27 neutralization enhances chronic lymphocytic leukemia development In order to better understand the role of IL-27 in CLL, we quantified IL-27 in the serum of leukemic mice and of CLL patients by ezyme-linked immunosorbant assay (Figure 6A, B). Leukemic mice presented a decreased level of IL-27 in serum during leukemia progression (Figure 6A). Similar findings were made in CLL patients, where IL-27 concentration was decreased compared to healthy control individuals (HC) (Figure 6B), in line with the anti-tumor role of IL-27 in CLL development. In addition, the analysis of several publicly available gene expression datasets revealed a lower EBI3 expression in different immune populations between CLL patients and HC (Online Supplementary Figure S7A) and between leukemic Eμ-TCL1 and WT mice (Online Supplementary Figure S7B). Since a number of studies indicate that IL-27 exerts a direct inhibitory effect on malignant cells,29 we assessed whether IL-27 can also have a direct anti-tumor effect on CLL cells. Thus, we treated both patient and mouse CLL samples ex vivo for 48 h with different concentrations of human and murine IL-27 respectively (25 and 50 ng/mL) (Online Supplementary Figure S7C, F). Assessment of viability and expression of key immunosuppressive markers indicated that neither the apoptosis rate nor the expression of markers were significantly altered, suggesting that Il-27 treatment does not impact

the viability and the phenotype of murine and human CLL cells. In order to support our results on Ebi3 genetic deletion and confirm the implication of IL-27 in CLL progression, we depleted IL-27 in vivo in C57BL/6 mice before injecting TCL1 cells (Figure 6C). Monitoring leukemic growth in the PB revealed an increase in CLL in mice depleted for IL-27, consistent with a higher number of CLL cells in the spleen following euthanasia (Figure 6D). Despite not observing differences in the frequency of CD8+ T cells between groups (Online Supplementary Figure S8A), conventional and clustering analyses confirmed the enrichment of proliferative and activated CD8+ T cells (Ki-67+ CD44hi) (Figure 6E; Online Supplementary Figure S8D-E), similarly to the adoptive transfer in Ebi3-/- mice (Figure 1D). Moreover, we observed a significant decrease in the percentage of CD4+ T cells (Online Supplementary Figure S8E), even though the frequencies of Tconv and Tregs within the CD4+ T-cell compartment were unaltered between the two groups (Online Supplementary Figure S8F). Analysis of CD4+ T cells confirmed the increased immunosuppressive and activated phenotype of Tregs in the absence of IL-27 in recipient mice (C2) (Online Supplementary Figure S8G, H). Importantly, Tregs appeared more suppressive and proliferative in IL-27-depleted mice, as demonstrated by the increased percentage of KLRG1 and Ki 67-expressing Tregs (Figure 6F).

Discussion Given the pleiotropic function of IL-27 in the tumorigenesis of multiple solid and hematologic malignancies, we investigated the role of this cytokine in the development and progression of CLL. Using a wide range of mouse models and patient samples, we demonstrated that IL27 has an anti-tumoral role in CLL pathobiology. Moreover, we explored the microenvironment, cellular, and transcriptional mediators responsible for this observation. A number of studies have previously assessed the role of IL-27 in CLL using a variety of cell lines and clinical samples.30-32 Nevertheless, the existing research remains contradictory and inconclusive. Here, using adoptive transfer of TCL1 leukemia cells in C57BL/6 as well as transgenic mouse models, we demonstrate for the first time that the lack of IL-27 results in a much faster CLL development and earlier death. Additionally, CD8+ T cells are known to be dysfunctional in CLL.33,34 Phenotypic characterization of the splenic immune subsets revealed an increasingly immunosuppressive TME in the absence of IL-27, suggesting that this cytokine has a role in modulating the anti-tumor response in CLL. In line with the varying effects of IL-27 reported in the lit-

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Figure 5. Ebi3-/- CD8+ T cells present impacted cytotoxic activity while IL-27 enhances their potential both in murine and human. (A) Control (Ctrl) and Ebi3-/- mice (N=3 per group) were euthanized at 8 weeks and CD3+ T cells were isolated from splenocytes. T cells were activated in vitro for 6 days and analyzed after 3 days by flow cytometry (FC) and after 6 days by FC and confocal microscopy. (B) Mean fluorescence intensity (MFI) of TOX after 3 days of activation and frequency of cells expressing the selected markers after 6 days of activation in Ctrl vs. Ebi3-/- CD8+ T cells. (C) Percentages of CD8+ T cells in clusters of wild-type (WT) vs. Ebi3-/- CD8+ T cells. (D) Detection and quantification of Profilin1 (red) between Ctrl vs. Ebi3-/- activated CD8+ T cells (green) after 6 days of activation (scale bar, 5μm). (E) In vitro cytotoxic assay. CD3+ T cells were isolated from Ctrl mice (N=6) and incubated with or without interleukin-27 (IL-27) for 48 hours (h) (left). TCL1-derived chronic lymphocytic leukemia (CLL) cells loaded with the super-antigen (sAg) were co-cultured with T cells for 4 h and stained to investigate cytotoxicity efficiency by FC (right). (F) Patient-derived CLL cells cytotoxicity assay (left panel). Peripheral blood mononucelar cells (PBMC) were isolated from CLL patient PB. One fraction was enriched in CD3+ T cells, and these cells were then activated in the presence or absence of IL-27. Antigen-pulsed CLL cells were co-cultured for 4 h with previously activated autologous CD3+ T cells. The cytotoxicity efficiency was measured by FC (right panel). Unpaired t test, *P<0.05, **P<0.01, ****P<0.0001.

erature, the serum levels of this cytokine have been found to be either significantly elevated or decreased in a wide range of malignancies,19,35,36 contributing to disease progression or control respectively. In consonance with our previous findings, our data revealed a significant and consistent decrease of IL-27 serum levels as CLL progresses in both murine and patient samples. This observation highlights both the involvement of this cytokine in CLL development as well as further supports its antitumor role in this malignancy. Even though the EBI3 subunit is shared by IL-35, EBI3-deficient C57BL/6 mice are often used to investigate the role of IL-27 as they reportedly result in a dominant IL-27-deficient phenotype due to the higher expression of IL-27 and the wider range of cells producing the cytokine. This is evidenced by the presence of functional Tregs expressing high levels of IL-10.11 Nevertheless, the interpretation of data obtained from knock-out mouse models of cytokine subunits or their receptors is often complicated due to the promiscuous usage of chains among the different members of each family. In order to overcome this source of variability, we further validated the role of IL-27 by neutralizing this cytokine in an in vivo leukemia model. Previous studies reported a direct anti-tumor role of IL27 in several tumors, including hematological malignancies such as pediatric AML and CLL.30,37 Nonetheless, our data indicated that there are no significant changes in the viability or phenotype of murine and patient CLL cells, both in vivo and following in vitro treatment with IL-27. Alternatively, we provided evidence that T cells are the main mediators in the observed enhanced CLL development in the absence of IL-27, as CD3+ T cells controlled CLL development more efficiently in the presence of this cytokine. The T-cell compartment has a critical function in antitumor immunity, and has been widely reported to show a significant functional impairment in CLL.38 Using gene expression analysis, we identified transcriptional changes in CD8+ T cells in the presence or absence of IL-27. Interestingly, we detected a decreased expression of transporters key in T-cell activation, proliferation, and cytotoxicity, most notably SLC7a5, which could poten-

tially contribute to the altered T-cell activity described in the IL-27-depleted environment. Moreover, the expression of profilin-1 was dramatically increased in this setting. This actin binding protein plays a pivotal role in cytoskeleton remodeling, and has been shown to severely hinder CTL-mediated cell killing by disrupting the formation of the T-cell cytolytic synapse.38 Altogether, these results suggest that the progressively decreasing levels of IL-27 in CLL patients leads to dysfunctional T-cell dynamics through different mechanisms, including the downregulation of important metabolic pathways and the inability to mount an appropriate immune response due to the lack of a functional lytic immune synapse. Further research must investigate the mechanisms behind the suggested mediators of T-cell dysfunction in the absence of IL-27. A number of studies suggest that the immune enhancing properties of IL-27 lead to a potent anti-tumor response through the upregulation of Th1 and CTL responses.39,40 Here, we show that IL-27 enhances the cytotoxicity of CD8+ T cells in mice and in humans. These observations are consistent with previous reports in the literature suggesting that IL-27 acts on CD8+ T cells through the activation of STAT1 and induces the T-bet and EOMES transcription factors, which are in turn critical for induction of effector molecules, and hence, the anti-tumor immune response.15,41,42 Of particular relevance, we demonstrate that IL-27 is able to partially restore the anti-tumor ability of CLL patientderived T cells against autologous leukemic cells, emphasizing the potential clinical prospects of this approach in the treatment of CLL. Here we identified IL-27 as a relevant mediator in CLL pathogenesis, highlighting its correlation with disease progression and suggesting a potential role as disease biomarker. Collectively, our data unveiled a novel mechanism by which IL-27 promotes anti-tumor immunity in CLL by enhancing CD8+ T-cell cytotoxicity, and established this cytokine as a promising immunotherapeutic agent in CLL. Further research should assess its efficacy in a pre-clinical setting in combination with other existing treatments, such as chemotherapy and immunotherapy agents, paving the path towards an affordable and effec-

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Figure 6. IL-27 level is reduced in the blood of leukemic mice and chronic lymphocytic leukemia patients and IL-27 neutralization enhanced chronic lymphocytic leukemia development as observed in Ebi3-/- mice. (A) (Left) Serum levels of interleukin 27 (IL27) in mice before and after chronic lymphocytic leukemia (CLL) development. (Right) Correlation between IL-27 serum levels and CLL development in mice measured by enzyme-linked immosorbant assay (ELISA). (B) Serum levels of IL-27 in CLL patients (N=53) and healthy controls (N=16) measured by ELISA. (C-F) (Right) A cohort of recipient Ctrl mice was intraperitoneally (i.p.) injected with α-IL-27 or isotype control antibodies and subsequently adoptively transferred with splenocytes from leukemic EμTCL1 mice. Mice were bled weekly to evaluate peripheral disease development. The injection of antibodies was maintained once per week until euthanasia. (C) (Left) Percentages of circulating leukemic CD5+ CD19+ cells in the 2 groups (N=7 for isotype group and N=8 for α-IL-27 group, two-way ANOVA test. (D-F) Mice were euthanized and their splenocytes were analyzed by flow cytometry. (D) Number of CLL cells in the spleen of both experimental conditions. (E) Distribution of cells in cluster C1 among CD8+ T cells from isotype- and α-IL-27-treated mice. (F) Percentage of KLRG1+ or Ki67+ Treg from isotype- and α-IL-27-treated mice. Unpaired t test, *P<0.05, **P<0.01, ****P<0.0001.

tive personalized medicine regimen for the treatment of CLL. Disclosures No conflicts of interest to disclose.

Contributions GP and IFB designed and performed experiments, analyzed results and wrote the manuscript. MW designed and performed experiments and analyzed results. PMR and MS performed the Rag2-/- experiments and analyzed results.

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NI, GAH, and AGR performed the cytotoxicity assays with murine and human cells and analyzed results. XZ and BQ performed the 3D killing assay on human T cells and analyzed results. EG, SG, CB, and AL provided experimental assistance and analyzed results. BS provided CLL patient samples, performed the ELISA assay and analyzed results. EM and JP designed and supervised the study, performed bioinformatics analyses, analyzed results, and wrote the final version of the manuscript. All authors revised the manuscript. Acknowledgments We thank Pr. Carlo Croce and Pr. John Byrd (Ohio State University, US) for the kind gift of Eµ-TCL1 mouse. We thank the National Cytometry Platform (LIH; Dr Antonio Cosma, Dr Céline Hoffmann, Thomas Cerutti, Fanny Hedin) for assistance in flow cytometry and confocal microscopy, and all the Animal Facility (LIH) staff, particularly Anais Oudin and Coralie Pulido. We thank Pr. Markus Hoth for inspiring discussion and continuous support, as well as for NALM6-pCasper cells (with Eva C. Schwarz). We thank

the LUXGEN platform LIH/LNS; Nathalie Nicot, Pol Hoffmann, Arnaud Muller and Dr Daniel Stieber) for RNA sequencing. We thank Dr Guy Berchem, Dr Jean-Hugues François, Dr Susan Cortez Clemente, Dr Vincent Schlesser, Dr Sigrid De Wilde and Dr Laurent Plawny from the Centre Hospitalier du Luxembourg for their help in sample collection. Funding This work was supported by grants from FNRS-Télévie to GP (7.4501.18, 7.6518.20), IFB (7.4529.19, 7.6603.21), MW (7.4508.16, 7.6504.18), SG (7.4502.19, 7.6604.21), CB (7.4577.22), and AL (7.4502.17, 7.4503.19), and from the Luxembourg National Research Fund (FNR) and Fondation Cancer to EG, EM and JP (PRIDE15/10675146/CANBIO, C20/BM/14582635, and C20/BM/14592342). Data-sharing statement The data set presented in this study is openly available in the Gene Expression Omnibus with the accession number GSE216131.

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ARTICLE - Hodgkin Lymphoma

Effect of cumulative dose of brentuximab vedotin maintenance in relapsed/refractory classical Hodgkin lymphoma after autologous stem cell transplant: an analysis of real-world outcomes Charlotte B. Wagner,1 Ken Boucher,1 Adrienne Nedved,2 Ivana N. Micallef,2 Sanjal Desai,3 Haris Hatic,4 Gaurav Goyal,4 Erin Zacholski,5 Amanda Fegley,5 Audrey M. Sigmund,6 David A. Bond,6 Courtney Samuels,7 Manali K. Kamdar,7 Sheeba Ba Aqeel,8 Pallawi Torka,8 Kira MacDougall,9 Azra Borogovac,9° Sridevi Rajeeve,10 Suchitra Sundaram,10 Kalub Fedak,11 Dipenkumar Modi,11 Elizabeth Travers,12 Sabarish Ayyappan,13 Nitin Chilakamarri,14 Elizabeth A. Brem,14 Daniel A. Ermann,1 Lindsey A. Fitzgerald,1 Boyu Hu,1 Deborah M. Stephens1 and Harsh Shah1

Correspondence: H. Shah harsh.shah@hci.utah.edu Received: Accepted: Early view:

January 19, 2023. April 19, 2023. April 27, 2023.

https://doi.org/10.3324/haematol.2023.282780 1

University of Utah Huntsman Cancer Institute, Salt Lake City, UT; Mayo Clinic, Rochester, MS; University of Minnesota, Twin Cities of Minneapolis and Saint Paul, MS; 4University of Alabama Medicine, Birmingham, AL; 5Virginia Commonwealth University Health, Richmond, VI; 6The Ohio State University, Columbus, OH; 7University of Colorado Cancer Center, Aurora, CO; 8Roswell Park Comprehensive Cancer Center, Buffalo, NY; 9University of Oklahoma Health Sciences Center, Oklahoma, OH; 10Tisch Cancer Institute at Mount Sinai, New York, NY; 11Karmanos Cancer Institute, Detroid, MI; 12University of Kentucky HealthCare, Lexington, KY; 13University of Iowa Hospitals and Clinics, Iowa City, IA and 14University of California, Irvine, CA, USA 3

°Current address: City of Hope Medical Center, Duarte, CA, USA

Abstract Sixteen cycles of Brentuximab vedotin (BV) after autologous stem cell transplant (ASCT) in high-risk relapsed/refractory classical Hodgkin lymphoma demonstrated an improved 2-year progression-free survival (PFS) over placebo. However, most patients are unable to complete all 16 cycles at full dose due to toxicity. This retrospective, multicenter study investigated the effect of cumulative maintenance BV dose on 2-year PFS. Data were collected from patients who received at least one cycle of BV maintenance after ASCT with one of the following high-risk features: primary refractory disease (PRD), extra-nodal disease (END), or relapse <12 months (RL<12) from the end of frontline therapy. Cohort 1 had patients with >75% of the planned total cumulative dose, cohort 2 with 51-75% of dose, and cohort 3 with ≤50% of dose. The primary outcome was 2-year PFS. A total of 118 patients were included. Fifty percent had PRD, 29% had RL<12, and 39% had END. Forty-four percent of patients had prior exposure to BV and 65% were in complete remission before ASCT. Only 14% of patients received the full planned BV dose. Sixty-one percent of patients discontinued maintenance early and majority of those (72%) were due to toxicity. The 2-year PFS for the entire population was 80.7%. The 2-year PFS was 89.2% for cohort 1 (n=39), 86.2% for cohort 2 (n=33), and 77.9% for cohort 3 (n=46) (P=0.70). These data are reassuring for patients who require dose reductions or discontinuation to manage toxicity.

Introduction In patients with relapsed/refractory classical Hodgkin lymphoma (r/r cHL) high-dose chemotherapy followed by autologous stem cell transplant (ASCT) leads to improved progression-free survival (PFS) over chemotherapy alone and is the accepted standard to achieve durable disease response.1 Historically, this treatment strategy cures approximately half of the patients with r/r cHL.1 Risk factors associated with poor outcomes after ASCT include

chemo-resistant disease, B symptoms at relapse, residual disease before ASCT, and extra-nodal involvement at relapse.2-4 Brentuximab vedotin (BV) is an antibody drug conjugate targeting CD30 that carries monomethyl auristatin E (MMAE) payload.5,6 The phase III AETHERA trial assessed whether early maintenance with BV after ASCT improved PFS compared to placebo in patients deemed high-risk for relapse after ASCT by the following risk factors primary refractory disease (PRD), extra-nodal disease (END) at re-

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ARTICLE - Cumulative dose BV maintenance real-world outcomes lapse <12 months (RL<12). Patients treated on the BV arm received 16 cycles at 1.8 mg/kg and had a significantly longer 2-year PFS compared to placebo, 63% compared to 51%, respectively.7 In an updated 5-year analysis, the PFS improvement was sustained, 59% in the BV arm compared to 41% in the placebo arm.8 The most common adverse events were peripheral neuropathy (PN) (56%), infections (60%) and neutropenia (35%); additionally, 53% of patients on the BV arm discontinued treatment early, with the main reason for discontinuation being adverse events (69%).7 In this study, PN was managed by dose delays or reductions. Those who required dose delay or reduction due to PN had a similar PFS to the entire population in a post hoc analysis.9 In the real world, patients are rarely able to tolerate all 16 cycles of BV maintenance at full dose, mainly due to toxicity. In a study conducted in Italy, early treatment discontinuations were reported at a rate of 44%, with the most common reason for discontinuation being toxicity.10 A real-world study assessing patients who received BV maintenance in Europe found that the median cycles of BV maintenance completed was 11.11 A similar study from Colombia also illustrated that the median number of cycles for BV maintenance was 12 and 18% of patients had grade 3 or higher PN.12 However, none of these studies examined cumulative dose of BV maintenance and its effect on PFS. Patients on AETHERA trial had no prior exposure to BV in front-line or in the pre-ASCT salvage setting. The landscape of frontline and salvage treatment of cHL has shifted dramatically since the publication of the AETHERA trial. The ECHELON-1 trial showed both improved PFS and OS of BVAVD compared to ABVD in frontline advanced cHL.13,14 Additionally, many phase II studies have showed excellent results with combination of BV and chemotherapy (such as ifosfamide, carboplatin, etoposide or bendamustine) during salvage before ASCT, resulting in high complete remission (CR) rates.15,16 Anti-programmed death receptor antibodies (anti-PD1) such as nivolumab or pembrolizumab have also been combined with either chemotherapy (ifosfamide, carboplatin, etoposide or gemcitabine, vinorelbine, liposomal doxorubicin) or BV for patients needing salvage therapy, also illustrating high CR rates in the range of 80-90%.17-19 The role of 16 cycles of BV maintenance in patients who might have had previous exposure to BV or anti-PD1 therapy before ASCT has not been determined. Due to physical and financial toxicity related to 16 cycles of BV maintenance, it is important to determine whether the full planned cumulative dose of BV maintenance is beneficial to patients in the era of earlier use of BV and anti-PD1 therapies. We set out to determine if dose reduction or treatment discontinuation, measured by cumulative dose of BV maintenance received, affects the 2-year PFS in patients with r/r cHL who received BV maintenance after ASCT.

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Methods Patients This is a retrospective, multicenter study of patients with r/r cHL who were administered at least one dose of BV maintenance after ASCT. We included patients ≥18 years of age who underwent ASCT from July 1, 2015 through June 30, 2019 from 13 institutions across the United States. Patients were required to have at least one high-risk disease feature as defined in AETHERA:7 PRD, END, or RL <12. We used an Excel ® (Microsoft ®, Redmond WA) database with built in data validation to capture variables uniformly across institutions. This project was exempt by the Institutional Review Board (IRB) at the University of Utah as the lead site. Each participating center obtained local IRB approval or exemption and a data use agreement. Cumulative dose of Brentuximab vedotin maintenance Patients were divided into three different cohorts based on total cumulative dose of BV maintenance (in mg/kg) received. Cohort 1 (C1) included those who received >75% of the planned total cumulative dose, cohort 2 (C2) included those who received 51-75% and cohort 3 (C3) included those who received ≤50%. In order to avoid bias from conditioning on future exposure, the total cumulative dose was treated as a time-varying exposure.20 For example, if a hypothetical patient was administered 100% of the total dose uniformly over exactly 1 year, then for the first 6 months the patient is in the “BV ≤50%” category. For the next 3 months the patient is in the “BV 51-75%” category, and for the remainder of follow-up time until censoring or progression the patient is in the “BV >75%” category. The other covariates in the analysis are fixed. We linearly interpolated when each dose was administered based on when the first and last dose of BV maintenance were administered. Patients were excluded if we did not have a date of first or last BV maintenance treatment. Study objectives The primary objective of the study was to determine the difference in 2-year PFS between the three cohorts of patients in each of the cumulative dosing cohorts. The secondary objectives of this study were to examine adverse events related to BV, treatment discontinuation rate and to examine the effect of prior chosen factors on the 2-year PFS. Statistical analysis Fisher’s exact tests were used to compare categorical variables and Wilcoxon rank-sum tests were used for continuous variables. Patients were censored at the date of last observation. PFS was defined as the time from the date of ASCT to the date of progression or death from any cause, whichever occurred first. Kaplan-Meier estimates were used

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ARTICLE - Cumulative dose BV maintenance real-world outcomes to summarize the distributions of PFS. Univariable Cox proportional hazards regression models were fit to assess associations between prior chosen predictors. Due to small sample size, P values are not reported for the multivariate analysis but the effect size for the odds ratio (OR) is reported assuming small, medium and large are 1.5, 2 and 3, respectively.21 Adverse events were defined using CTCAE v. 5 criteria. Response criteria was defined by Lugano criteria.22

Results Patient characteristics There were a total of 123 patients identified from 13 institutions. After excluding five patients who did not have a known date of first and/or last BV maintenance dose, 118 patient records were included for evaluation (Figure 1). There were 39 patients who received >75% of the total adjusted cumulative dose of BV maintenance (C1), 33 patients who received 51-75% of the total adjusted cumulative dose of BV maintenance (C2) and 46 patients who received ≤50% of the total adjusted cumulative dose of BV maintenance (C3). The median age of this population was 36 (range, 27-42) years at the time of ASCT, 53% were men, and the majority (95%) received ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine) as frontline therapy (Table 1). Sixty-nine percent of patients had advanced stage disease at diagno-

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sis. High-risk features at relapse were as follows: 50% of patients had PRD, 29% had RL<12 and 39% had END. Eighteen percent of patients had B symptoms at relapse. Twenty-three percent had more than one line of salvage therapy before ASCT (Table 2). The majority of patients achieved a CR (65%) prior to ASCT. Forty-four percent of patients had BV exposure prior to ASCT, mostly during salvage therapy (98%). Fifty-six percent of patients received a traditional chemotherapy only salvage regimen, while rest (44%) had exposure to a novel agent (anti-PD1 or BV) during salvage. Of the novel salvage patients, 27% patients had BVBendamustine, 60% had BV single agent, and 19% had any exposure to an anti-PD1 agent during salvage (Table 1). BEAM (etoposide, cytarabine and melphalan) was the most common conditioning regimen, reported in 94% of the patients. Table 1 describes the baseline characteristics of the patients by total cumulative dosing cohorts of BV maintenance and Table 2 describes the salvage treatment by dosing cohorts. There were no statistically significant differences in baseline characteristics or salvage treatment between the three cohorts. Toxicity and treatment discontinuation The median number of BV maintenance cycles completed was 12 (range, 1-25) with the majority (61%, 72/118) of patients discontinuing BV maintenance prior to completing all 16 cycles. Reasons for early discontinuation were as follows:

Figure 1. Consort diagram for eligibility of enrollment. BV: Brentuximab vedotin. Haematologica | 108 November 2023

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52 for toxicity (72%), ten for progression (14%), eight for patient preference (11%) and two for other reasons (3%). Fortyeight patients (41%) had a dose-reduction prior to discontinuation or completion of maintenance. Eighty-six percent of patients required either a dose reduction or discontinued BV maintenance early and only 14% of patients (16/118) received the full planned cumulative dose of BV (28.8 mg/kg). One patient continued maintenance past 16 cycles and received a total of 25 cycles. Any grade adverse events for peripheral neuropathy, neutropenia and infections were 79%, 20%, and 14% respectively and grade ≥3 adverse events were 17%, 7%, and 4% respectively. There was no difference in incidence of any grade neuropathy and grade ≥3 neuropathy in those who had prior BV exposure and those who did not have prior BV exposure (P=0.87). Additionally, there was no difference in the mean number of cycles between those who had prior BV exposure (10.64) and those who did not have prior BV exposure (10.97) (P=0.73).

(Figure 2A). Analyzing PFS according to cumulative BV dose cohorts in the time-variable analysis, the 2-year PFS was 89.2% (95% CI: 79.7-99.8) for C1, 86.2% (95% CI: 75.5-98.4) for C2, and 77.9% (95% CI: 67.2-90.2) for C3 (P=0.70) (Figure 2B). There was no statistically significant difference when comparing the 2-year PFS for C3 to C1 (P=0.68) or C2 to C1 (P=0.69) (Figure 2B). These findings were also supported in the time-independent analysis of 2-year PFS with no statistical difference between the three cohorts (Online Supplementary Figure S1). Only one death occurred in the entire population and, therefore, a difference in overall survival could not be estimated. In the multivariate analysis for PFS, ≤50% of adjusted total cumulative BV maintenance dose had OR of 1.57, predicting a trend towards worse PFS with a medium effect size. Prior BV exposure (OR=0.19) had large effect size for PFS and predicted for a trend towards improved PFS with post-ASCT BV maintenance. PRD (OR=5.91) and receiving ≥2 lines of salvage treatment (OR=9.34) had the largest effect size on PFS, Progression-free survival predicting a trend for worse PFS (Table 3). Prior BV exposure With a median follow up of 2.96 years 2-year PFS for all pa- and use of novel agents in salvage were almost perfectly tients was 80.7% (95% confidence interval [CI]: 73.8-88.2) correlated in the univariate analysis as there are only three

Table 1. Baseline characteristics by total cumulative dose of Brentuximab vedotin maintenance.

Baseline characteristics Total N Age in years, median (SD) Sex Male Female Stage at diagnosis Early favorable Early unfavorable Advanced Unknown Frontline therapy ABVD BV-AVD BEACOPP Other Unknown cHL status after frontline therapy PRD RL <12 months RL ≥12 months Unknown Extra-nodal disease B symptoms at relapse

Entire population N (%) 118 36 (12)

C1 N (%) 39 39 (12)

C2 N (%) 33 35 (12)

C3 N (%) 46 36 (12)

62 (53) 56 (47)

20 (51) 19 (49)

13 (39) 20 (61)

29 (63) 17 (37)

P 0.37 0.12 0.81

9 (8) 24 (20) 82 (70) 3 (2)

3 (8) 10 (26) 25 (64) 1 (2)

4 (12) 5 (15) 23 (70) 1 (3)

2 (4) 9 (20) 34 (74) 1(20)

110 (93) 2 (2) 3 (3) 1 (1) 2 (2)

37 (97) 1 (3) 0 (0) 0 (0) 1 (2)

30 (91) 0 (0) 2 (6) 0 (0) 1 (3)

43 (93) 1 (2) 1 (2) 1 (2) 0 (0)

0.68

0.18 59 (50) 34 (29) 24 (20) 1 (1) 46 (39) 21 (18)

17 (44) 12 (31) 10 (26) 0 (0) 21 (49) 5 (13)

21 (64) 5 (15) 7 (21) 0 (0) 9 (27) 8 (24)

21 (47) 17(38) 7 (16) 1 (2) 16 (40) 8 (18)

0.17 0.42

CI: cohort 1; C2: cohort 2; C3: cohort 3; SD: standard deviation; BV: Brentuximab vedotin; ABVD: doxorubicin, bleomycin, vinblastine and dacarbazine; BEACOPP: bleomycin, etoposide, adriamycine, cyclophosphamide, vincristine, procarbazine, and prednisone; cHL: classical Hodgkin lymphoma; PRD: primary refractory disease; RL: relapse. Haematologica | 108 November 2023

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ARTICLE - Cumulative dose BV maintenance real-world outcomes instances when these variables disagree. Therefore “novel agents” variable was omitted in the multivariate analysis as there is no information to separate the effect of these variables on PFS.

Discussion In this large, retrospective, multicenter study from 13 academic medical centers across the United States, the 2-year PFS was robust (80.7%) regardless of cumulative dose of BV maintenance and higher than the 63% reported in the AETHERA trial.7 The higher 2-year PFS observed in this study was most likely due to the recent incorporation of novel agents into salvage treatments prior to ASCT, which achieved a higher CR rate of 65% as compared to historical CR rates of 30-40% with chemotherapy-only salvage regimens. Furthermore, in the AETHERA trial,7 patients were excluded if they had previously received BV, while half of our population had BV exposure in their salvage regimens. Additionally, anti-PD-1 antibodies are being studied earlier in

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the treatment course for cHL, and we had 8% of total patients who had received anti-PD-1 antibodies prior to ASCT during salvage. The use of these novel therapies prior to ASCT could contribute to the increased CR rate of 65% prior to ASCT compared to the 37% in AETHERA.7 These results suggest achievement of a CR prior to ASCT remains an important treatment goal and that the use of novel agents in the salvage setting can help more readily achieve this CR. However, it should be noted that computed tomography (CT) scans were used to assess response criteria in the AETHERA trial while positron emission tomography/CT has become the standard which captures more instances of CR than CT alone. The cumulative dose of BV maintenance received did not appear to have a significant impact on 2-year PFS in this population, even when controlling for other variables in a multivariate analysis. This is reassuring as up to 72% of patients stopped treatment early due to toxicity. Seventy-nine percent of patients had neuropathy and 18% of total patients had grade ≥3, highlighting significant impact on quality of life and instrumental ADL. The data from this

Table 2. Salvage characteristics by total cumulative dose of Brentuximab vedotin maintenance.

Salvage characteristics Total N

Entire population N (%) 118

C1 N (%) 39

C2 N (%) 33

C3 N (%) 46

Salvage therapies before ASCT, N 1 >1 Unknown Best response to salvage therapy CR PR SD PD Prior BV exposure Type of salvage regimen Traditional Chemotherapy only Novel Agents in any salvage line BV Bendamustine BV monotherapy BV Nivolumab Pembrolizumab Nivolumab Median number of BV maintenance cycles* ASCT conditioning regimen BEAM CVP Unknown

P 0.60

90 (77) 27 (23) 1

30 (77) 8 (21) 1 (2)

27 (82) 6 (18) 0 (0)

33 (72) 13 (28) 0 (0) 0.45

77 (65) 39 (33) 1 (1) 1 (1) 53 (44)

23 (59) 15 (38) 1 (3) 0 (0) 19 (49)

25 (75) 8 (25) 0 (0) 0 (0) 15 (45)

29 (63) 16 (35) (0) 1 (2) 19 (41)

66 (56) 52 (44) 14 (26) 31 (60) 7 (12) 1 (1) 2 (1)

21 (54) 18 (46) 5 (27) 11 (61) 2 (12) 0 (0) 0 (0)

18 (55) 15 (45) 4 (27) 10 (67) 1 (6) 0 (0) 0 (0)

27 (59) 19 (41) 5 (26) 10 (52) 4 (20) 1 (1) 2 (1)

0.89

12 (2-25)

16 (13-25)

12 (8-16)

5 (1-10)

0.78

1.00 0.65 0.62 1.00 0.33

0.62 110 (93) 7 (6) 1 (1)

37 (95) 2 (5) 0 (0)

32 (97) 1 (3) 0 (0)

41 (91) 4 (9) 1 (0)

*Median and range. No P value because the difference in number of cycles is a design feature. CI: cohort 1; C2: cohort 2; C3: cohort 3; BV: Brentuximab vedotin; BEAM: etoposide, cytarabine and melphalan; CVP: cyclophosphamide, vincristine, and prednisone; ASCT: autologous stem cell transplantation; CR: complete remission; PR: partial response; SD: stable disease; PD: progressive disease. Haematologica | 108 November 2023

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ARTICLE - Cumulative dose BV maintenance real-world outcomes study suggest that receiving 51-75% of total planned dose had no impact on PFS (OR=0.94) when compared to >75% dose and receipt of ≤50% of the total planned cumulative dose of BV maintenance (equivalent to ≤8 cycles) had only a moderate effect size on PFS (OR=1.57) indicating that the 2-year PFS benefit is not significantly different from those who received a higher cumulative dose and was higher than reported in the AETHERA trial.

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Findings from our study highlight the difficulty in being able to deliver all 16 cycles of BV maintenance post-ASCT. In the AETHERA study, 47% of the patients received all 16 cycles of BV, however only 39% received all 16 cycles in the present real-world study; additionally, only 14% (16/118) received all 16 cycles at full cumulative dose of 28.8 mg/kg. A median number of 12 cycles were delivered to all patients, which is comparable to previously reported studies.10-12 Despite 44%

Figure 2. Two-year progression-free survival. (A) Progression-free survival of the entire study population. (B) Progression-free survival stratified by cohort, based on total planned cumulative dose, time-dependent analysis. Table 3. Univariate and multivariate analysis of progression-free survival.

Predictor Fraction of total BV dose% Type of salvage regimen

cHL Status Best response to salvage therapy Lines of prior therapy, N Prior BV?

Conditional logistic regression for progression Univariate Category OR (95% CI) BV >75% Reference BV 51-75% 1.20 (0.34-4.22) BV ≤50% 2.19 (0.74-6.55) Standard chemotherapy Reference Novel agent 0.54 (0.21-1.39) Relapse >12 months Reference Relapse <12 months 1.40 (0.25-7.96) Primary refractory disease 5.02 (1.09-23.1) CR Reference No CR 2.31 (0.97-5.49) 1 Reference 2+ 1.85 (0.70-4.90) No Reference Yes 0.51 (0.20-1.33)

Multivariate OR (95% CI) Reference 0.94 (0.19-4.62) 1.57 (0.43-5.69) Reference Reference 1.46 (0.22-9.95) 5.91 (0.99-35.1) Reference 1.88 (0.65-5.47) Reference 9.34 (1.57-55.4) Reference 0.19 (0.03-1.01)

Effect size 0.94 (none) 1.57 (small) 1.46 (small) 5.91 (large) 1.88 (small) 9.34 (large) 5.26 (large)

BV: Brentuximab vedotin; OR: overall response; cHL: classical Hodgkin lymphoma, CR: complete remission; CI: confidence interval. Haematologica | 108 November 2023

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ARTICLE - Cumulative dose BV maintenance real-world outcomes of patients having prior BV exposure, total mean number of BV cycles during maintenance was similar in the BV-exposed versus BV-naïve patients (10.6 vs. 11 cycles) which highlights that toxicity management and dose reductions were appropriate. Additionally, prior BV exposure had a large effect size on the 2-year PFS (OR=0.19) indicating a trend towards improved PFS compared to those without prior BV exposure. Seven patients had BV-refractory disease during salvage and yet still received BV maintenance therapy. Overall these findings suggest that prior response to BV during salvage may be an indicator of potential benefit of BV maintenance post-ASCT. During the time our study was conducted, multiple new treatment strategies including the use of novel therapies such as BV and anti-PD1 inhibitors have become incorporated into the treatment landscape of cHL. The replacement of bleomycin with BV in frontline therapy (BV + AVD) has demonstrated an overall survival benefit compared to ABVD in patients with late stage cHL.13 Additionally, both pembrolizumab and nivolumab have been studied in combination with chemotherapy in the front-line setting, and are being explored in the multipole prospective randomized phase III clinical trials such as clinicaltrials gov. Identifier: NCT03907488.23,24 There have also been studies indicating that adding an anti-PD-1 antibody to traditional chemotherapy salvage yields high complete response rates.18,19 These advances in both front-line and relapsed setting highlight the relevance of our data in a cohort with exposure to novel agents prior to ASCT, which leaves the role of BV maintenance less well defined within the modern treatment landscape. Many patients may have preexisting neuropathy due to exposure to BV as initial therapy or salvage, which may limit further use of BV maintenance. The data presented support that ongoing toxicity from BV can be managed with dose reduction and/or discontinuation without effect on long-term outcomes. Furthermore, post-ASCT maintenance with single agent anti-PD1 inhibitors and in combination with BV are being explored, which have demonstrated favorable safety and efficacy outcomes, which may supplant BV maintenance in the future.25 There are limitations to this study that impact the conclusions that we are able to draw. First, this study is retrospective in design, and, therefore, it should be viewed as hypothesis generating. We did not have data on previous cumulative dose of BV before maintenance, which could have some clinical implications, however we still highlighted impact of prior BV on PFS and toxicity. We also did not collect data on patients who may have qualified for BV maintenance, but did not receive it which could have further elucidated what role, if any, BV maintenance provides in the current treatment landscape. Additionally, a longer follow-up is necessary in order to validate the findings from our study.

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In summary, the 2-year PFS was high regardless of the cumulative BV maintenance dose received. This may be due to a shift in the treatment paradigm overall for cHL which is incorporating novel agents such as BV and anti-PD1 therapy earlier during salvage prior to ASCT, ultimately resulting in higher CR rate before ASCT. This study adds valuable contribution to the field of cHL that could have some important clinical implications. For example, for patients who have prior exposure to BV during salvage therapy with a response, there may still be a benefit of BV maintenance but reduced number of BV cycles (for example 8-12 cycles equivalent to 51-75% total cumulative dose of BV) could be considered to avoid unnecessary toxicity. For patients who are refractory to BV during frontline or salvage, maintenance BV has an unclear role as AETHERA study did not have such population and our study had had limited number of patients who were BV-refractory. Overall, our data is reassuring for clinicians who can feel comfortable dose reducing or discontinuing BV maintenance when facing toxicities that may impact quality of life in cHL patients. Disclosures CBW discloses funding from Abbvie Inc. MKK, TG Therapeutics, Novartis and Genentech; consultancy for AbbVie, AstraZeneca, Adaptive Biotechnologies, ADC Therapeutics, Beigene, ImpactBio, Celgene/BMS and Genentech; membership on the Board of Directors or advisory committees of Genentech and Celgene/BMS; is part of the Speakers Bureau of Seagen. PT discloses honoraria from Targeted Oncology, Physician Education Review; consultancy for Lilly USA, Epizyme, TG Therapeutics, ADC Therapeutics and Genentech. SM discloses membership on the Board of Directors or advisory committees of Seagen Inc. and MorphoSys; research funding from ADC Therapeutics, Karyopharm Therapeutics and Genentech; honoraria from AstraZeneca; is part of the Speakers Bureau of Beigene. SA discloses consultancy for TG Therapeutics, CSL, TG Therapeutics Behring, Genentech, Epizyme, Lilly, Celgene, AstraZeneca, AbbVie; Intellisphere and AstraZeneca; membership on the Board of Directors or advisory committees of Fate Therapeutics, TG Therapeutics, AbbVie, Intellisphere, AstraZeneca, Seattle Genetics and BeiGene; is part of the Speakers Bureau of Total CME. EB discloses membership on the Board of Directors or advisory committees of Morphosys/Incyte, Pharmacyclics/Janssen, Beigene, ADC Therapeutics, KiTE Pharma, Karyopharm and Bayer; is part of the Speakers Bureau of Pharmacyclics/Janssen, BeiGene and SeaGen. DMS discloses research funding from Novartis, Arqule, Acerta, JUNO, Karyopharm, Mingsight and Newave. BH discloses consultancy for ADC Therapeutics, Novartis and Bristol Meyers Squibb; research funding from Caribou Biosciences, Celgene, CRISPR Therapeutics, Genentech, Lymphoma Research Foundation, Morphosys AG and Repare Therapeutics. HS discloses research funding from ADCT,

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BeiGene, Astrazeneca, Seattle Genetics and Epizyme; data. CW drafted the manuscript. KB performed the statmembership on the Board of Directors or advisory commit- istical analysis. All authors reviewed and provided input on tees of AbbVie. the manuscript Contributions HS conceived the study question. CW and HS designed the data collection tool, cleaned and analyzed the data. CW, AN, HH, EZ, AS, CS, SB, KM, SR, KF, ET, SA and NC collected

Data-sharing statement The data supporting the findings of this study are available within the article. Additional data are available, upon request, from the corresponding author.

References 1. Schmitz N, Pfistner B, Sextro M, et al. Aggressive conventional chemotherapy compared with high-dose chemotherapy with autologous haemopoietic stem-cell transplantation for relapsed chemosensitive Hodgkin's disease: a randomised trial. Lancet. 2002;359(9323):2065-2071. 2. Sureda A, Constans M, Iriondo A, et al. Prognostic factors affecting long-term outcome after stem cell transplantation in Hodgkin's lymphoma autografted after a first relapse. Ann Oncol. 2005;16(4):625-633. 3. Majhail NS, Weisdorf DJ, Defor TE, et al. Long-term results of autologous stem cell transplantation for primary refractory or relapsed Hodgkin's lymphoma. Biol Blood Marrow Transplant. 2006;12(10):1065-1072. 4. Lazarus HM, Loberiza FR, Zhang MJ, et al. Autotransplants for Hodgkin's disease in first relapse or second remission: a report from the autologous blood and marrow transplant registry (ABMTR). Bone Marrow Transplant. 2001;27(4):387-396. 5. Sutherland MS, Sanderson RJ, Gordon KA, et al. Lysosomal trafficking and cysteine protease metabolism confer target-specific cytotoxicity by peptide-linked anti-CD30-auristatin conjugates. J Biol Chem. 2006;281(15):10540-10547. 6. Francisco JA, Cerveny CG, Meyer DL, et al. cAC10-vcMMAE, an antiCD30-monomethyl auristatin E conjugate with potent and selective antitumor activity. Blood. 2003;102(4):1458-1465. 7. Moskowitz CH, Nademanee A, Masszi T, et al. Brentuximab vedotin as consolidation therapy after autologous stem-cell transplantation in patients with Hodgkin's lymphoma at risk of relapse or progression (AETHERA): a randomised, double-blind, placebocontrolled, phase 3 trial. Lancet. 2015;385(9980):1853-1862. 8. Moskowitz CH, Walewski J, Nademanee A, et al. Five-year PFS from the AETHERA trial of brentuximab vedotin for Hodgkin lymphoma at high risk of progression or relapse. Blood. 2018;132(25):2639-2642. 9. Nademanee A, Sureda A, Stiff P, et al. Safety analysis of Brentuximab Vedotin from the phase III AETHERA trial in Hodgkin lymphoma in the post-transplant consolidation setting. Biol Blood Marrow Transplant. 2018;24(11):2354-2359. 10. Massaro F, Pavone V, Stefani PM, et al. Brentuximab vedotin consolidation after autologous stem cell transplantation for Hodgkin lymphoma: A Fondazione Italiana Linfomi real-life experience. Hematol Oncol. 2022;40(1):31-39. 11. Marouf A, Cottereau AS, Kanoun S, et al. Outcomes of refractory or relapsed Hodgkin lymphoma patients with post-autologous stem cell transplantation brentuximab vedotin maintenance: a French multicenter observational cohort study. Haematologica. 2022;107(7):1681-1686. 12. Patino B, Acon-Solano C, Pereira M, et al. Brentuximab vedotin as consolidation therapy post hematopoietic stem cell transplantation for patients with relapsed or refractory Hodgkin lymphoma: a real

world analysis of the Colombian experience. Blood. 2019;134(Suppl 1):S5275. 13. Ansell SM, Radford J, Connors JM, et al. Overall survival with Brentuximab vedotin in stage III or IV Hodgkin's lymphoma. N Engl J Med. 2022;387(4):310-320. 14. Connors JM, Jurczak W, Straus DJ, et al. Brentuximab vedotin with chemotherapy for stage III or IV Hodgkin's Lymphoma. N Engl J Med. 2018;378(4):331-344. 15. Lynch RC, Cassaday RD, Smith SD, et al. Dose-dense brentuximab vedotin plus ifosfamide, carboplatin, and etoposide for second-line treatment of relapsed or refractory classical Hodgkin lymphoma: a single centre, phase 1/2 study. Lancet Haematol. 2021;8(8):e562-e571. 16. LaCasce AS, Bociek RG, Sawas A, et al. Brentuximab vedotin plus bendamustine: a highly active first salvage regimen for relapsed or refractory Hodgkin lymphoma. Blood. 2018;132(1):40-48. 17. Advani RH, Moskowitz AJ, Bartlett NL, et al. Brentuximab vedotin in combination with nivolumab in relapsed or refractory Hodgkin lymphoma: 3-year study results. Blood. 2021;138(6):427-438. 18. Moskowitz AJ, Shah G, Schöder H, et al. Phase II trial of pembrolizumab plus gemcitabine, vinorelbine, and liposomal doxorubicin as second-line therapy for relapsed or refractory classical Hodgkin lymphoma. J Clin Oncol. 2021;39(28):3109-3117. 19. Mei MG, Lee HJ, Palmer JM, et al. Response-adapted anti-PD-1based salvage therapy for Hodgkin lymphoma with nivolumab alone or in combination with ICE. Blood. 2022;139(25):3605-3616. 20. Lund JL, Horváth-Puhó E, Komjáthiné Szépligeti S, et al. Conditioning on future exposure to define study cohorts can induce bias: the case of low-dose acetylsalicylic acid and risk of major bleeding. Clin Epidemiol. 2017;9:611-626. 21. Sullivan GM, Feinn R. Using effect size-or why the P value is not enough. J Grad Med Educ. 2012;4(3):279-282. 22. Cheson BD, Fisher RI, Barrington SF, et al. Recommendations for initial evaluation, staging, and response assessment of Hodgkin and non-Hodgkin lymphoma: the Lugano classification. J Clin Oncol. 2014;32(27):3059-3068. 23. Allen PB, Savas H, Evens AM, et al. Pembrolizumab followed by AVD in untreated early unfavorable and advanced-stage classical Hodgkin lymphoma. Blood. 2021;137(10):1318-1326. 24. Bröckelmann PJ, Goergen H, Keller U, et al. Efficacy of nivolumab and AVD in early-stage unfavorable classic Hodgkin lymphoma: the randomized phase 2 German Hodgkin Study Group NIVAHL Trial. JAMA Oncol. 2020;6(6):872-880. 25. Bachier C, Schade H, Zoghi B, Ramakrishnan A, Shah N. A phase II single arm study of nivolumab as maintenance therapy after autologous stem cell transplantation in patients with Hodgkin lymphoma at risk of relapse or progression. Blood. 2021;138(Suppl 1):S2455.

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ARTICLE - Myelodysplastic Syndromes

Clinical and molecular correlates of somatic and germline DDX41 variants in patients and families with myeloid neoplasms Talha Badar,1 Ahmad Nanaa,2,3 James M. Foran,1 David Viswanatha,4 Aref Al-Kali,5 Terra Lasho,5 Christy Finke,5 Hassan B. Alkhateeb,5 Rong He,4 Naseema Gangat,5 Mithun Shah,5 Ayalew Tefferi,5 Abhishek A. Mangaonkar,5 Mark R. Litzow,5 Laura J. Ongie,6 Timothy Chlon,7,8 Alejandro Ferrer4 and Mrinal M. Patnaik4 Division of Hematology-Oncology and Bone Marrow Transplant Program, Mayo Clinic, Jacksonville, FL; 2Division of Hematology, Mayo Clinic, Rochester, MN; 3John H. Stroger, Jr. Hospital of Cook County, Chicago, IL; 4Division of Hematopathology, Mayo Clinic, Rochester, MN; 5Division of Hematology, Mayo Clinic, Rochester, MN; 6Clinical Genomics, Mayo Clinic, Rochester, MN; 7Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH and 8Department of Pediatrics, College of Medicine, University of Cincinnati, Cincinnati, OH, USA 1

Correspondence: T. Badar badar.talha@mayo.edu M. S. Patnaik Patnaik.Mrinal@mayo.edu Received: Accepted: Early view:

February 1, 2023. May 10, 2023. May 18, 2023.

Abstract The diagnosis of germline predisposition to myeloid neoplasms (MN) secondary to DDX41 variants is currently hindered by the long latency period, variable family histories and the frequent occurrence of DDX41 variants of uncertain significance (VUS). We reviewed 4,524 consecutive patients who underwent targeted sequencing for suspected or known MN and analyzed the clinical impact and relevance of DDX41VUS in comparison to DDX41path variants. Among 107 patients (44 [0.9%] DDX41path and 63 DDX41VUS [1.4%; 11 patients with both DDX41path and DDX41VUS]), we identified 17 unique DDX41path and 45 DDX41VUS variants: 24 (23%) and 77 (72%) patients had proven and presumed germline DDX41 variants, respectively. The median age was similar between DDX41path and DDX41VUS (66 vs. 62 years; P=0.41). The median variant allele frequency (VAF) (47% vs. 48%; P=0.62), frequency of somatic myeloid co-mutations (34% vs 25%; P= 0.28), cytogenetic abnormalities (16% vs. 12%; P=>0.99) and family history of hematological malignancies (20% vs. 33%; P=0.59) were comparable between the two groups. Time to treatment in months (1.53 vs. 0.3; P=0.16) and proportion of patients progressing to acute myeloid leukemia (14% vs. 11%; P=0.68), were similar. The median overall survival in patients with high-risk myelodysplastic syndrome/acute myloid leukemia was 63.4 and 55.7 months in the context of DDX41path and DDX41VUS, respectively (P=0.93). Comparable molecular profiles and clinical outcomes among DDX41path and DDX41VUS patients highlights the need for a comprehensive DDX41 variant interrogation/classification system, to improve surveillance and management strategies in patients and families with germline DDX41 predisposition syndromes.

Introduction Germline pathogenic variants that predispose to familial cancers have been reported in several genes.1 In 2016 the World Health Organization recognized hematological malignancies associated with germline predisposition syndrome as a distinct sub-group with prognostic implications.2 DEAD/H-box helicase 41 gene (DDX41) is located on chromosome 5q35, is thought to be a tumor suppressor gene involved in the splicing of pre-mRNA and processing of ribosomal RNA. In experimental knockouts models, defects in DDX41 have been shown to contribute to the development of myeloid neoplasms (MN).3-6 Unlike traditional germline

predisposition syndromes, DDX41-associated germline predisposition syndrome has a late age of onset, commonly presents with myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML), and is associated with variable family histories of MN.7 The pro-leukemogenic properties of DDX41 pathogenic variants is also supported by lack of concurrent cytogenetic or molecular abnormalities that are usually seen in MDS/AML.8 Several studies including ours have demonstrated favorable prognostic outcomes associated with DDX41 mutations in MDS/AML.8-10 In spite of readily available genomic sequencing to assist with the diagnosis of MN, the diagnosis of DDX41 mutated MN is hindered by a long latency, variable family history and the frequent occur-

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ARTICLE - Molecular landscape of DDX41 in myeloid neoplasms rences of variants of uncertain significance (VUS), given inherent difficulties to characterize the DDX41 gene/protein function. The accurate recognition of germline and somatic DDX41 variants is vital for prognostication and the management of DDX41 variant-associated MDS and AML, including allogeneic stem cell transplant donor selection. In this large study we report the mutational profile of 107 patients with DDX41 variants (pathogenic and VUS) and their clinical outcomes. We also discuss challenges in interrogating and performing functional analysis for DDX41 VUS.

Methods We reviewed 4,524 consecutive patients who underwent next-generation sequencing (NGS; MC OncoHeme 42-gene panel) assessments between January 2018 and May 2022 for suspected or known myeloid disorders. The study was conducted at the Mayo Clinic and approved by the Mayo Clinic Institutional Review Board (IRB) (Figure 1). The clinical information was stored in a de-identified database. Germline testing in patients and families with suspected or known germline predisposition syndrome were conducted under IRB approved protocol (Mayo Clinic IRB# 16-004173; clinicaltrials gov. Identifier: NCT02958462). We identified 44 (0.9%) patients with DDX41 likely pathogenic/pathogenic (DDX41path) variants and 63 (1.3%) patients with DDX41 VUS (DDX41VUS) according to variant classification criteria from the American

T. Badar et al.

College of Medical Genetics/ the Association of Medical Genetics/ the Association for Molecular Pathology (ACMG/AMP) guidelines.11 We evaluated baseline characteristics, mutational profiles, and clinical outcomes of patients with DDX41path and DDX41VUS variants (Figure 1). For the purpose of this paper, we operationally defined DDX41 variants with a variant allele frequency (VAF) of ≥40% to be presumed germline, as previously reported.10,12 DDX41 variants with positive germline testing on DNA derived from skin fibroblasts or hair follicles were defined as confirmed germline. Patients with DDX41 variants who had negative germline testing defined as confirmed somatic. In the absence of direct germline testing, variants with VAF <40% were defined as presumed somatic (Figure 1). We defined clonal cytopenia(s) of undermined significance (CCUS) as unexplained cytopenia(s) associated with known somatic pathogenic variants (VAF ≥2%), with bone marrow dysplasia <10% and bone marrow blast% <5%. Patients having cytopenia(s) with an isolated DDX41VUS, without any somatic pathogenic variants, were classified as “cytopenia associated with DDX41VUS alone”, as the pathogenicity of DDX41VUS remain to be elucidated.13,14 Responses to therapy were assessed according to the International Working Group (IWG) MDS response criteria.15 Clinical NGS testing was performed on DNA extracted from fresh bone marrow aspirates (102/110 [93%]) or peripheral blood samples (8/110 [7%]). The Mayo Clinic NGS panel included 42 genes (Online Supplementary Appendix)

Figure 1. Flowchart of the study, illustrating germline versus somatic status, and proportion of patients with DDX41path and DDX41VUS. In this study, 107 patients with 132 DDX41 variants are identified from 4,524 adult patients screened. The yellow boxes indicate the number of patients, and the white boxes indicate the number of variants. We identified 44 patients with pathogenic DDX41, harboring 56 pathogenic and 11 variants of uncertain significance (VUS) variants. Sixty-three DDX41 VUS only, harboring 65 VUS variants. pts: patients; path: pathogenic; P/LP: pathogenic/likely pathogenic. Haematologica | 108 November 2023

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ARTICLE - Molecular landscape of DDX41 in myeloid neoplasms and has an accuracy of >99% and reproducibility of 100% for single-base substitutions and insertion/deletion events. The panel has a variant sensitivity of >5% VAF with a minimum depth coverage of 250x. All coding regions (exons 1-17) of DDX41 were covered by the panel. We manually reviewed the sequencing BAM files among patients with commonly occurring DDX41 pathogenic variants (M1I and D140fs) for the presence of DDX41 R525H somatic variants with low VAF (2-5%, below the report limit of the clinical laboratory). Germline testing was performed prospectively in our germline predisposition clinic, on DNA derived from skin fibroblasts or hair follicles as previously described.16 We also performed in depth curation assessments of frequently occurring DDX41VUS to analyze allelic diversity and pathogenicity. In order to make a pathogenic prediction, based on available literature, we considered in silico CADD (combined annotation dependent depletion) and REVEL (rare exome variant ensemble learner) scores >25 and >0.8 as being likely pathogenic, respectively.17,18 Statistical analysis Continuous variables were summarized as medians (range), while categorical variables were reported as frequencies (percentage). Unadjusted comparisons of patient characteristics and outcomes among the DDX41path and DDX41VUS variant groups were made using the Wilcoxon rank sum test (continuous variables) or Fisher’s exact test (categorical variables). The Kaplan-Meier method was used to estimate overall survival (OS). The median OS was calculated from the time of diagnosis to last follow-up or death. All tests were two-sided with P value <0.05 considered statistically significant. We have excluded asymptomatic carriers of DDX41 variants from survival analysis since they do not carry a diagnosis of myeloid neoplasm or cytopenia (s). At last, follow-up, none of these patients had developed cytopenia(s) or demonstrated progression to myeloid neoplasms.

Results Among 107 patients with DDX41 variants, we identified 17 unique DDX41path variants and 45 unique DDX41VUS (Figure 1). Eleven patients had both DDX41path variants and DDX41VUS. Twelve patients had two DDX41path variants. Among 24 (22%) DDX41-mutated patients that had germline testing, 13 (54%) had DDX41path, and 11 (46%) patients had DDX41VUS, respectively (Online Supplementary Table S1; Figure 2). The previously reported DDX41 pathogenic variants and VUS by our group are marked with (*) in the Online Supplementary Table S1. In addition to previously described germline and somatic variants in MN,10,12,19,20 novel germline variants identified in the current study (i.e., not previously reported in the literature or existing genetic databases) are summarized in the Online Supplementary Table S2.

T. Badar et al.

Germline DDX41VUS in patients with myeloid disorders We identified 74 patients with DDX41VUS, including 11 patients with both DDX41path and DDX41VUS. Among patients with DDX41VUS, frequently observed nucleotide/amino acid changes included c.773C>T; p.P258L (n=7 [9%]), c.517G>A; p.G173R (n=6 [8%]), c.465G>A; p.M155L (n=5 [7%]), c.992_994del; p. K331del (n=3 [4%]), c.1436G>A; p.R479Q (n=3 [4%]), c.1030G>T; p.D344Y (n=3 [4%]), c.1016G>A; p. R339H (n=2 [3%]), c.97T>C; p.Y33H (n=2 [3%]), c.571G>A; p.A191T (n=2 [3%]), c.616C>G; p.P206A (n=2 [3%]), c.653G>A; p.G218D (n=2 [3%]) c.740A>G; p.E247G (n=2 [3%]), c.1018T>A; p.Y340N (n=2 [3%]), c.1032C>G p.D344E (n=2 [3%]) and c.656G>A; p. R219H (n=2 [3%]). Among seven patients with the DDX41 c.773C>T; p.P258L variant, three (43%) patients had MDS, three (43%) patients had AML and one was an asymptomatic DDX41 carrier, identified after his family member with AML was found to have the same confirmed germline DDX41VUS. None of these patients had concurrent somatic mutations including involvement of the second DDX41 allele; all had VAF ≥40% (median 46.5%; range, 4550%) and three of seven (43%) patients required treatment for their MN. The second most common DDX41VUS seen was DDX41 c.517G>A; p.G173R (n=6). Of these six, four patients were diagnosed with MDS, one had clonal cytopenia of unknown significance (CCUS) with a somatic DNMT3A pathogenic variant (c.1233_1234insT; p. G412Wfs*9; VAF 7%) and one patient had pancytopenia. All six patients had VAF ≥40% (median 48%; range, 46-51%) and two (33%) of these six patients had concurrent somatic DDX41path (R525H) variants with low VAF (range, 5-7%). All six patients with this variant had a negative family history of hematological malignancies, with an indolent course only needing supportive care thus far. The DDX41 c.465G>A; p.M155LVUS, which has been reported previously as a germline variant,12 was also frequent in our cohort (n=5 [7%]) and was associated with pancytopenia (n=2), AML (n=1), CCUS (n=1) and essential thrombocythemia (ET, triple negative) (n=1). None of the patients with the c.465G>A; p.M155L variant had other comutations. Other DDX41VUS encountered and confirmed to be germline were c.992_994del; p.Lys331del (n=3 [4%]) c.740A>G; p.E247G (n=2 [3%]), c.1016G>A; p. R339H (n=2 [3%]), c.571G>A; p.A191T (n=2 [3%]), c.1018T>A; p.Y340N (n=2 [3%]), and c.959C>T; p.T320I (n=1 [1%]). Two patients with the E247G variant had a strong family history of MDS/AML. We performed in silico assessments of all the DDX41VUS in our cohort and compared their CADD/ REVEL scores with genomic curations carried out using ClinVar and the current ACMG classification in Online supplementary Table 2. Clinical demographics and mutational comparisons between DDX41path and DDX41VUS The median age in years at diagnosis of MN was comparable between DDX41path and DDX41VUS (66 vs. 62; P=0.41). Patients were predominantly male, and frequency was

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ARTICLE - Molecular landscape of DDX41 in myeloid neoplasms

T. Badar et al.

comparable between DDX41path and DDX41VUS (66% vs. 59%; P=0.64). A higher proportion of patients in the DDX41path group had MDS compared to DDX41VUS group, respectively (66% vs. 28%; P=<0.001). The proportion of patients with AML (20% vs. 24%; P=0.65), MPN (2% vs. 5%; P=0.54), CCUS (5% vs. 3%; P=0.64) or asymptomatic DDX41 variant carrier states (9% vs. 6%; P=0.78) were comparable between both groups. The median DDX41 VAF (47% vs. 48%; P=0.62), frequency of somatic myeloid co-mutations (34% vs. 25%; P=0.28), cytogenetic (CG) abnormalities (16% vs. 12%; P=>0.99) and family history of hematological malignancies (22% vs. 33%; P=0.59) were also comparable between the two groups (Table 1). The most frequently occurring somatic myeloid co-mutations in DDX41path and DDX41VUS groups were DNMT3A (15% vs. 5%; P=0.09), ASXL1 (9% vs. 5%; P=0.45), JAK2 (4% vs. 5%; P=>0.99) and EZH2 (4% vs. 3%; P=>0.99), respectively (Table 1). Time to treatment in months (1.53 vs. 0.3; P=0.16) and proportion of patients progressing to AML (14% vs. 11%; P=0.68), were not statistically significantly different between DDX41path and DDX41VUS, respectively. Higher proportion of DDX41path patients required treatment compared to DDX41VUS patients (73% vs. 48%; P=0.02). We did an additional subset analysis to look for differences in clinical characteristics and outcomes between DDX41path and DDX41VUS patients with one or more concurrent somatic mutations (Figure 3). We did not find statistically significant differences in age (P=>0.99), sex (P=>0.99), hemoglobin (g/dL) (P=0.36), white blood cell count (WBC) (x109/L) (P=0.36), platelet count (x109/L) (P=0.06), bone marrow blast percentage (P=0.14), cytogenetic abnormalities (P=>0.99), proportion of patients progressing to AML (P=0.31), time on observation (P=0.20) and median OS (P=0.69); as outlined in Online Supplementary Table S3. We did a subset analysis and assessed for risk of AML progression among truncating and non-truncating DDX41 variants. Among nine patients who progressed to AML, two (22%) and seven (88%) had truncating and nontruncating DDX41 variants (P=0.22), respectively. Asymptomatic individuals with germline DDX41 variants In our cohort, we identified eight asymptomatic individuals with germline DDX41 variants: four patients from three affected families. Among these four patients, three have DDX41path (c.3G>A: p. M1I) variants and one has DDX41VUS (c.773C>T: p.258L). Individuals with the DDX41 c.3G>A: p. M1I variant had a family history of advanced MDS and AML. The patient with DDX41vus c.773C>T: p.258L,

Figure 2. Distribution of DDX41 variants detected, positioned on the DDX41 protein and its functional domains with representation of germline and somatic status. NTD: N-terminal domain; ZFD: zinc finger domain; CTD: C-terminal domain; VUS: variants of uncertain significance. Haematologica | 108 November 2023

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Figure 3. Integrated matrix for DDX41path and DDX41VUS per case and observed concurrent mutations. *2: second pathogenic (path) DDX41 mutation. VUS: variants of uncertain significance.

ARTICLE - Molecular landscape of DDX41 in myeloid neoplasms

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T. Badar et al.

ARTICLE - Molecular landscape of DDX41 in myeloid neoplasms

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Table 1. Comparison of baseline characteristics, genomic profile, and clinical outcome between DDX41path and DDX41VUS variants. DDX41 pathogenica N=44

DDX41 VUS N=63

66 (30-84)

62 (23-83)

0.41

29 (66)

37 (59)

0.64

12 (0.01-45)

0 (0-95)

0.005

MDS overall , N (%)

28 (64)

18 (28)

<0.001

MDS as per IPSS-R classification, N (%) Very low risk Low risk Intermediate risk High risk Very high risk Non-evaluable

1 (3) 2 (6) 5 (15) 11 (33) 7 (21) 3 (9)

0 2 (11) 5 (28) 3 (17) 0 8 (44)

AML, N (%)

9 (20)

15 (24)

0.65

MPN, N (%)

1 (2)

3 (5)

0.54

DDX41 carrier, N (%)

4 (9)

4 (6)

0.78

CCUS, N (%)

2 (5)

2 (3)

0.64

20 (32)

<0.001

47 (5-52)

48 (14-91)

0.62

Variable Age in years, median (range) - at diagnosis Male sex, N (%) BM blasts %, median (range) b

<0.01

Cytopenia associated with DDX41VUS alone, N (%) DDX41 VAF (%) Most common DDX41 variants

Nucleotide/amino acid change (%) c.1574G>A; p. Arg525His (39)¥ ⁋

c.3G>A; p.M1I (36)

c.415_418dup; p. Asp140Glyfs*2 (14)

c.773C>T; p. Pro258Leu (11)⁋

⁋

c.517G>A; p. Gly173Arg (9.5) ⁋

c.465G>A p. Met155Ile (8)

⁋

c.1589G>A; p. p. Gly530Asp (7) ¥

c.992_994del p. Lys331del (5)⁋

c.931C>T; p. Arg311* (4.5)⁋

c.1436G>A p. Arg479Gln (5)⁋

⁋

c.1016G>A; p. Arg339His (3)

⁋

c.656G>A; p. Arg219His (3)⁋

Co-mutated, N (%)

15 (34)

16 (25)

0.28

Abnormal cytogenetics, N (%) NI Monosomal and or complex

7 (16) 0 2 (4)

4 (12) 36 1 (3)

>0.99

Family Hx of hematological malignancies, N (%) NI

9 (20) 0

9 (33) 36

0.59

Observation in mths in no Rx group, median (range)

22.7 (0.27-49.7)

8.38 (0.17-40)

0.24

Time in mths to treatment, median (range)

1.53 (0.03-92)

0.3 (0-25.4)

0.16

Progression to AML, N (%) NI

6 (14) 0

3 (11) 36

0.68

Proportion of pts received Rx, N (%) NI

32 (73) 0

13 (48) 36

0.02

HMA based therapy, N (%) NI

22 (50) 0

5 (18.5) 36

0.004

Intensive chemotherapy*, N (%) NI

7 (16) 0

1 (4) 36

0.20

Other treatment1, N (%) NI

3 (7) 0

7 (26) 36

0.61

Proportion of patient received allo-HCT NI

13 (32) 0

6 (24) 36

0.41

c.1030G>T. p. Asp344Tyr (5)

BM: bone marrow; MDS: myelodysplastic syndrome; IPSS-R: revised International Prognostic Scoring System; AML: acute myeloid leukemia; MPN: myeloproliferative neoplasms; CCUS: cytopenia of unknown significance; NI: no information; VAF: variant allele frequency; pts: patients; HMA: hypomethylating agent; Rx: treatment: Hx: history; mths: months. *Intensive chemotherapy includes 3+7, CPX-351, high dose cytarabine based treatment; 1other treatment includes growth factors support, erythropoietin stimulating agent, hydrea, other low intensity therapy; a11 patients had DDX41 pathogenic plus variants of uncertain significance (VUS); btotal MDS patients across all risk group as per IPSS-R; ⁋germline DDX41 variant; ¥somatic DDX41 variant.

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ARTICLE - Molecular landscape of DDX41 in myeloid neoplasms did have one affected family member with AML. Asymptomatic individuals with germline DDX41 variants are under active surveillance at the germline predisposition syndrome clinic at Mayo Clinic (clinicaltrial gov. Identifier: NCT02958462). Germline DDX41 variants in patients with myeloproliferative neoplasms DDX41 variants in patients with MPN have not been frequently reported. We identified four (4%) patients in this cohort; two with JAK2 V617F mutant polycythemia vera (c.138+5G>T, VAF 48% [DDX41path], one with JAK2 V617F mutant primary myelofibrosis (c.337del; p.E113Kfs*, VAF 48% [DDX41path]) and one with essential thrombocythemia to: ET (triple negative) (c.465G>A; p.M155I, VAF 48% [DDX41VUS]). One patient each with myelofibrosis and polycythemia vera, respectively, had additional somatic myeloid mutations (DNMT3A [c.2645G>A; p.R882H] and IDH2 [c.419G>A; p.R140Q]); while none of them had a family history of MN and only one patient with a CSF3R (c.1919 C>A; p. Thr640Asn) mutant chronic neutrophilic leukemia progressed to AML. Treatment and survival outcomes The proportion of patients requiring treatment in the DDX41path and DDX41VUS groups was 73% (n=32) and 48% (n=13), respectively (P=0.02). Decisions with regards to treatment were based on the presence of worsening cytopenia(s), high-risk disease features or overt progression to AML. Fifty percent (n=22) versus 18% (n=5) (P=0.004), 16% (n=7) versus 4% (n=1) (P=0.20) and 32% (n=13) versus 24% (n=6) (P=0.41) of patients received hypomethylating agent (HMA)-based, intensive chemotherapy and allogeneic stem cell transplantation in the DDX41path and DDX41VUS groups, respectively (Table 1). Among 21 of 39 (54%) evaluable MDS patients who received treatment, six (40%), three (14.5%), and 12 (57%) had complete remission (CR), hematological improvement (HI) and no response to treatment, respectively. Among eight of 11 evaluable AML patients who received leukemia directed therapy, seven (87.5%) achieved CR and one (12.5%) was refractory to treatment. Overall survival data was available on 63 of 107 (59%) patients. The median follow-up duration was 21.2 months (range, 1.5-158.0). We compared OS outcomes among MDS and AML patients in the DDX41path and DDX41VUS groups. The median OS from date of diagnosis till last follow up or death in patients with high-risk MDS or AML was 63.4 months and 55.7 months in the DDX41path (n=25) and DDX41VUS (n=6) groups, respectively (P=0.93; Figure 4A). At 4 years, median OS was 65% versus 60% in high-risk MDS or AML with DDX41path DDX41VUS, respectively. Similarly, median OS was not significantly different between patients with isolated (n=43) versus co-mutated (n=20) DDX41 variants (63.43 vs.

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158.03 months [at 4 years 78% vs. 59%]; P=0.63; Figure 4B). The median OS outcomes for patients with bone marrow (BM) blasts 10-19% (n=21) in comparison to BM blasts ≥20% (n=9) was not significantly different (136.7 vs. 63.4 months [at 4 years 70% vs. 64%]; P=0.90; Figure 4C). The median OS in patients with age <70 years (n=41) versus ≥70 years (n=22) (158 vs. 61.6 months [at 4 years 59% vs. 93%; P=0.93). Similarly, somatic (n=8) versus germline DDX41 variants (n=55) (158 vs. 63.4 months [at 4 years 69% vs. 68%]; P=0.29), and normal CG (n=53) versus abnormal CG (n=10) (136.7 vs. 55.73 months [at 4 years 75% vs. 58%]; P=0.81), were not significantly different (Figure 4D-F, respectively). Of note, asymptomatic DDX41 variants carriers were excluded from the survival analysis (refer to methods section for reason). We then looked at the survival difference between DDX41 truncating and non-truncating variants. Overall, 27 of 107 (25%) patients had truncating DDX41 variants, among them three of 27 were DDX41VUS (c.138+5G>T; p?, c.1732+4 A>G; p?, c.28-3C>T; p?). The median OS was 63.4 and 96.2 months (P=0.44) with DDX41 truncating and non-truncating variants, respectively.

Discussion In this large cohort of patients who underwent NGS for a known or suspected myeloid neoplasm, we identified 17 (16%) unique DDX41path variants and 45 (42%) unique DDX41VUS. Majority of the DDX41 variants occurred in isolation (n=76/107 [71%]) without any additional somatic variant or clonal cytogenetic abnormality. Our observations validate previous reports that germline DDX41 associated MN have a later age of onset (median age 65 years), are male predominant (61% males), usually present with indolent cytopenias (27% in DDX41path and 52% in DDX41VUS have not yet needed treatment thus far), with variable family histories of MN (26.5%).7,10,12,21,22 Importantly, we did not find significant differences in clinical and demographic factors between patients with DDX41path variants and DDX41VUS. We also describe the occurrence of MPN in patients with DDX41 variants. Recently, in a multicenter retrospective analysis by Li et al.12 from a cohort of 176 patients (DDX41path [n=116], DDX41VUS [n=60]), 15 patients with MPN harboring DDX41 variants were reported (11 with DDX41VUS), with 34% of these variants being somatic in nature. JAK2 and CALR mutations were not detected in patients with DDX41path and were reported in 72% of patients with DDX41VUS. We describe four patients with MPN, including two with JAK2 V617F mutant PV, and one each with triple-negative essential thrombocythemia and JAK2 V617F mutant primary myelofibrosis. In this cohort, three (75%) patients had presumed germline DDX41path (c.337del; p. E113Kfs*14,

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Figure 4. Kaplan Meier curves for overall survival. (A) Overall survival (OS) in high-risk myelodysplastic syndrome (MDS)/ acute myeloid leukemia (AML) patients with DDX41path and DDX41VUS, (B) isolated vs. co-mutated DDX41, (C) bone marrow (BM) blast 10%19% vs. ≥20% (D) age <70 years (yrs) vs. ≥70 yrs (E) germline vs. somatic DDX41, and (F) with or without cytogenetic abnormality. VUS: variants of uncertain significance; path: pathogenic. Haematologica | 108 November 2023

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ARTICLE - Molecular landscape of DDX41 in myeloid neoplasms c.465G>A; p.M155l and c.298+5G>A; p.?) and the remainder had presumed germline DDX41VUS (c.138+5G>T p?). We are not able to comment on the prognostic impact of DDX41 mutations in MPN patients given the small sample size. As previously reported, we did not find a significant impact of BM blast percentage (10-19% vs. ≥20%) on MDS/AML outcomes, in patients with DDX41 variants, germline or somatic.3,12,23 In contrast, DDX41-mutated MN with normal karyotypes had a trend towards a better OS compared to those with abnormal karyotypes, with the most common abnormal karyotype being del 20 (q11.2q13.1) (3/11 [27%]). In addition, survival outcomes for patients with DDX41-mutated MDS/AML were relatively favorable from historical cohort of high-risk MDS/AML and not significantly different between the DDX41path versus DDX41VUS groups. In a recent report by Makishima et al. on 346 patients with pathogenic/likely pathogenic germline DDX41 variants, MDS patients with truncating DDX41 variants had rapid progressions to AML in comparison to those with non-truncating variants, without any significant impact on survival.24 In our cohort, 25% (n=27) of patients had truncating DDX41 variants, among them three of 27 were DDX41VUS (c.138+5G>T; p?, c.1732+4 A>G; p?, c.28-3C>T; p?). One DDX41VUS was secondary to an inframe deletion (c.992_994del; p.Lys331del), making the adjudication as to whether or not this variant resulted in truncation of the protein challenging, hence it was excluded. We did not find significant differences in the rate of progression to AML between truncating and non-truncating DDX41 variants. The median OS was also not significantly different between DDX41 truncating and non-truncating variants. Further studies are needed to determine the clinical impact of truncating DDX41 variants in MDS and AML patients. Variant curation of DDX41 is challenging since many aspects of its protein function are incompletely understood and for which functional assays or strong heritability links are not yet described. Our molecular hematology laboratory uses standard ACMG criteria, with the help of genetic counselors for evaluating DDX41 germline variants. We tend to be conservative with our curation approach, to not assign potential risk allele and disease causality without sufficient evidence. Thus, several variants classified as VUS in this manuscript have been reported in ClinVar with different and sometimes conflicting ACMG classification.11 We are cautious about using ClinVar entry assertions when case counts are very limited. There are many inaccuracies in ClinVar and many entries with assertions provide little to no data for a designation of likely pathogenic variants. An example of this is the p.R339 site, which is now increasingly recognized as a recurrent event and a likely disease predisposing germline variant. It is also evident that these alterations have associations in SNP databases (e.g., gnomAD) with variable frequency among ethnic populations

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(overall frequency 0.003%). We feel this is a prudent approach given that data continues to emerge on the true effects of different DDX41 variants in relation to disease outcomes and currently there remains a large gap in our understanding due to a lack of functional data. Li et al.12 recently described a valuable DDX41-specific classification approach but included assumptions on certain ACMG criteria to enable a more dichotomous classification as causal/likely causal, versus uncertain. Specifically PM2 was applied in the situation that the polymorphic association of a new possible germline causal variant was less frequent in the “general population” than the most common two DDX41 germline variants (c.465G>A; p.M1I and c.415_418dup; p.D140fs). While the basis for this approach is reasonable, it may inadvertently segregate rare polymorphic variants of limited or no significance with potential disease-associated risk variants. We also apply this criterion (PM2) in our process, but not at its full value. Similarly, this group applied PM3 (typically used in recessive disorder assessment) in a modified fashion when considering occurrence of DDX41 somatic genetic variants. While these maneuvers are certainly logical, they are not necessarily definitive. Another recent paper by Duployez et al.25 applied ACMG/AMP criteria without apparent modification, although they did consider accompanying DDX41 somatic variants as strong evidence for a germline finding to be causal. Both papers identify many potential germline variants in patients with hematologic malignancies. In our cohort we find overlapping alterations, specifically with c.773C>T; p.P258L, c.1016G>A; p.R339H and c.992_994del; p.K331del variants, that have been described in several publications with a prevalence now exceeding that in ‘control’ patients with hematological malignancies. While the accumulating data supports the association of these rare alleles with disease risk, the standard ACMG classification would still render these as VUS calls. It is notable that in a more recent large international study Makishima et al.,24 the c.992_994del; p.K331del variant was not identified in our reading of the paper. While this may reflect an effect of different ethnic group distribution, this finding also supports a conservative approach to curation, along with appropriate interpretive commentary in our reporting. The pathogenicity of germline VUS associated with adult MN is difficult to determine through analysis of patient observational data alone, since these diseases often present with a complex array of co-mutations and cytogenetic abnormalities that may be epistatic to the effects of the DDX41VUS. Thus, experimental analysis of the effect of these variants on gene function is necessary, in conjunction with analysis of available patient data to confirm pathogenicity. Secondly, the effect of VUS on the tumor suppressive activities of DDX41 likely depends on the effect of each variant on the structure and function of the DDX41 protein. Most VUS are missense or cause deletion of a single

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ARTICLE - Molecular landscape of DDX41 in myeloid neoplasms amino acid and, thus their effect on tumor suppression activity is likely dependent on the role of the effected amino acid in the protein structure and function. The challenge in determining the effect of a variant on tumor suppression is that DDX41 has multiple functions and variants may not affect all functions equally. Experimental testing of the effect of each VUS on all known functions of DDX41 is required to resolve this question. However, this experimental analysis can be tedious because each variant must be analyzed separately. DDX41 is particularly difficult to examine experimentally since it is an essential gene, causing DDX41-knockout cell lines to grow inefficiently in culture and making it difficult to engineer cells where the VUS can be studied in isolation from wild-type DDX41. Furthermore, the precise function(s) of DDX41 that are responsible for its role as a tumor suppressor remain incompletely defined, adding additional complication to functional analysis of variants. Importantly, DDX41 is an essential gene for hematopoiesis and potentially other physiological processes relying on cell proliferation and it is unlikely that inhibition of DDX41 would provide clinical benefit as a therapeutic approach. However, since DDX41 mutant MDS/AML is known to have favorable treatment outcomes and slower progression rates than other adult MDS/AML subtypes, understanding the effect of each DDX41 variant on protein function would allow for further classification and risk stratification with individualized medicine approaches.5 In summary, we provide a comprehensive genomic landscape, including germline and somatic pathogenic variants and VUS in patients with DDX41 variant-associated hematological disorders. We report on the likely pathogenicity

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of several unique DDX41VUS and provide a detailed analysis on their clinical course and outcomes. We show that patients with DDX41path and DDX41VUS had similar clinical characteristics and clinical outcomes, underscoring the need for better variant interrogation and classification methods. Disclosures MS received research funding to institution from Astellas, Celgene, and Marker Therapeutics. MMP received research funding from Kura Oncology, Stem Line Pharmaceuticals and CTI Pharmaceuticals. Contributions TB developed the concept, cured data, and wrote and submitted the original draft. AN helped in collecting and analyzing. JMF, TL, CF, HBA, RH, DV, NG, AT, AAM and LJO contributed patients. AA, MS and MRL contributed patients and reviewed the manuscript. TC reviewed and edited the manuscript. AF reviewed genetic data and performed additional variant curation; he also reviewed the manuscript. MMP contributed patients, supervised the review, and edited the manuscript. Funding The study was funded by the The Henry J Predolin Leukemia foundation. We thank the Mayo Clinic Cancer Center Support Group (P30 CA015083) and the Mayo Clinic Center for Individualized Medicine for sponsoring the germline predisposition clinic. Data-sharing statement Original data can be provided on reasonable request.

References 1. Owen C, Barnett M, Fitzgibbon J. Familial myelodysplasia and acute myeloid leukaemia - a review. Br J Haematol. 2008;140(2):123-132. 2. Arber DA, Orazi A, Hasserjian R, et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood. 2016;127(20):2391-2405. 3. Maciejewski JP, Padgett RA, Brown AL, Müller-Tidow C. DDX41related myeloid neoplasia. Semin Hematol. 2017;54(2):94-97. 4. Omura H, Oikawa D, Nakane T, et al. Structural and functional analysis of DDX41: a bispecific immune receptor for DNA and cyclic dinucleotide. Sci Rep. 2016;6:34756. 5. Badar T, Chlon T. Germline and somatic defects in DDX41 and its impact on myeloid neoplasms. Curr Hematol Malig Rep. 2022;17(5):113-120. 6. Chlon TM, Stepanchick E, Hershberger CE, et al. Germline DDX41 mutations cause ineffective hematopoiesis and myelodysplasia. Cell Stem Cell. 2021;28(11):1966-1981. 7. Polprasert C, Schulze I, Sekeres MA, et al. Inherited and somatic defects in DDX41 in myeloid neoplasms. Cancer Cell. 2015;27(5):658-670. 8. Alkhateeb HB, Nanaa A, Viswanatha D, et al. Genetic features and clinical outcomes of patients with isolated and comutated

DDX41-mutated myeloid neoplasms. Blood Adv. 2022;6(2):528532. 9. Bernard E, Tuechler H, Greenberg PL, et al. Molecular International Prognostic Scoring System for myelodysplastic syndromes. NEJM Evid. 2022;1(7):EVIDoa2200008. 10. Sébert M, Passet M, Raimbault A, et al. Germline DDX41 mutations define a significant entity within adult MDS/AML patients. Blood. 2019;134(17):1441-1444. 11. Richards S, Aziz N, Bale S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17(5):405-424. 12. Li P, Brown S, Williams M, et al. The genetic landscape of germline DDX41 variants predisposing to myeloid neoplasms. Blood. 2022;140(7):716-755. 13. Arber DA, Orazi A, Hasserjian RP, et al. International Consensus Classification of myeloid neoplasms and acute leukemias: integrating morphologic, clinical, and genomic data. Blood. 2022;140(11):1200-1228. 14. Khoury JD, Solary E, Abla O, et al. The 5th edition of the World Health Organization classification of haematolymphoid

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ARTICLE - Molecular landscape of DDX41 in myeloid neoplasms tumours: myeloid and histiocytic/dendritic neoplasms. Leukemia. 2022;36(7):1703-1719. 15. Cheson BD, Greenberg PL, Bennett JM, et al. Clinical application and proposal for modification of the International Working Group (IWG) response criteria in myelodysplasia. Blood. 2006;108(2):419-425. 16. St Martin EC, Ferrer A, Wudhikarn K, et al. Clinical features and survival outcomes in patients with chronic myelomonocytic leukemia arising in the context of germline predisposition syndromes. Am J Hematol. 2021;96(9):E327-E330. 17. Ioannidis NM, Rothstein JH, Pejaver V, et al. REVEL: an ensemble method for predicting the pathogenicity of rare missense variants. Am J Hum Genet. 2016;99(4):877-885. 18. Rentzsch P, Schubach M, Shendure J, Kircher M. CADD-Spliceimproving genome-wide variant effect prediction using deep learning-derived splice scores. Genome Med. 2021;13(1):31. 19. Li P, White T, Xie W, et al. AML with germline DDX41 variants is a clinicopathologically distinct entity with an indolent clinical course and favorable outcome. Leukemia. 2021;36(3):664-674.

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20. Alkhateeb HB, Nanaa A, Viswanatha DS, et al. Genetic features and clinical outcomes of patients with isolated and comutated DDX41mutated myeloid neoplasms. Blood Adv. 2022;6(2):528-532. 21. Wan Z, Han B. Clinical features of DDX41 mutation-related diseases: a systematic review with individual patient data. Ther Adv Hematol. 2021;12:20406207211032433. 22. Lewinsohn M, Brown AL, Weinel LM, et al. Novel germ line DDX41 mutations define families with a lower age of MDS/AML onset and lymphoid malignancies. Blood. 2016;127(8):1017-1023. 23. Quesada AE, Routbort MJ, DiNardo CD, et al. DDX41 mutations in myeloid neoplasms are associated with male gender, TP53 mutations and high-risk disease. Am J Hematol. 2019;94(7):757-766. 24. Makishima H, Saiki R, Nannya Y, et al. Germline DDX41 mutations define a unique subtype of myeloid neoplasms. Blood. 2023;141(5):534-549. 25. Duployez N, Largeaud L, Duchmann M, et al. Prognostic impact of DDX41 germline mutations in intensively treated acute myeloid leukemia patients: an ALFA-FILO study. Blood. 2022;140(7):756-768.

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ARTICLE - Non-Hodgkin Lymphoma

Characterization and clinical impact of the tumor microenvironment in post-transplant aggressive B-cell lymphomas Suvi-Katri Leivonen,1,2,3 Terhi Friman,4 Matias Autio,1,2,3 Samuli Vaittinen,5 Andreas Wind Jensen,6 Francesco d’Amore,6 Stephen Jacques Hamilton-Dutoit,7 Harald Holte,8 Klaus Beiske,9 Panu E. Kovanen,10 Riikka Räty4 and Sirpa Leppä1,2,3 Applied Tumor Genomics Research Program, Medical Faculty, University of Helsinki, Helsinki, Finland; 2Department of Oncology, Helsinki University Hospital Comprehensive Cancer Center, Helsinki, Finland; 3iCAN Digital Precision Cancer Medicine Flagship, Helsinki, Finland; 4 Department of Hematology, Helsinki University Hospital Comprehensive Cancer Center and University of Helsinky, Helsinki, Finland; 5Department of Pathology, Turku University Hospital, University of Turku, Turku, Helsinki, Finland; 6Department of Hematology, Aarhus University Hospital, Aarhus, Denmark; 7Institute of Pathology, Aarhus University Hospital, Aarhus, Denmark; 8 Department of Oncology, Oslo University Hospital, Oslo, Norway; 9Department of Pathology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway and 10Department of Pathology, University of Helsinki, and HUSLAB, Helsinki University Hospital, Helsinki, Finland 1

Correspondence: S. Leppä sirpa.leppa@helsinki.fi Received: Accepted: Early view:

January 26, 2023. May 23, 2023. June 1, 2023.

https://doi.org/10.3324/haematol.2023.282831 Published under a CC BY license

Abstract Post-transplant lymphoproliferative disorders (PTLD) are iatrogenic immune deficiency-associated lymphoid/plasmacytic proliferations developing due to immunosuppression in solid organ or hematopoietic stem cell allograft patients. PTLD are characterized by abnormal proliferation of lymphoid cells and have a heterogeneous clinical behavior. We profiled expression of >700 tumor microenvironment (TME)-related genes in 75 post-transplant aggressive B-cell lymphomas (PTABCL). Epstein-Barr virus (EBV)-positive PT-ABCL clustered together and were enriched for type I interferon pathway and antiviral-response genes. Additionally, a cytotoxicity gene signature associated with EBV-positivity and favorable overall survival (OS) (hazard ratio =0.61; P=0.019). In silico immunophenotyping revealed two subgroups with distinct immune cell compositions. The inflamed subgroup with higher proportions of immune cells had better outcome compared to noninflamed subgroup (median OS >200.0 vs. 15.2 months; P=0.006). In multivariable analysis with EBV status, International Prognostic Index, and rituximab-containing treatment, inflamed TME remained as an independent predictor for favorable outcome. We also compared TME between post-transplant and immunocompetent host diffuse large B-cell lymphomas (n=75) and discovered that the proportions of T cells were lower in PT-diffuse large B-cell lymphomas. In conclusion, we provide a comprehensive phenotypic characterization of PT-ABCL, highlighting the importance of immune cell composition of TME in determining the clinical behavior and prognosis of PT-ABCL.

Introduction Post-transplant lymphoproliferative disorder (PTLD) is a rare complication and a leading cause of cancer-related mortality following solid organ or allogeneic hematopoietic stem cell transplantation.1 PTLD are characterized by abnormal proliferation of lymphoid cells associated with immunosuppression and are classified in distinct histological categories (non-destructive, polymorphic, monomorphic) with heterogeneous clinical behavior.2-4 The majority of monomorphic PTLD are aggressive B-cell lymphomas. The treatment of PTLD depends on the histological type and may include reduction of immunosuppressive therapy, usually combined with immunotherapy and chemotherapy.5,6

A substantial proportion (60-80%) of PTLD are associated with Epstein-Barr virus (EBV) positivity.7,8 As a result of immunosuppression, EBV can promote B-cell proliferation, and potentially transformation, in an unregulated fashion. Typically, EBV-positive PTLD arising in the setting of immunosuppression are associated with a latency type III viral gene expression program and express a wide range of EBV genes, including Epstein-Barr nuclear antigens (EBNA), EBV-encoded small RNA (EBER), and latent membrane proteins (LMP).5 In contrast, the pathogenesis of EBV-negative PTLD is less understood, and EBV-negative PTLD are considered to resemble lymphomas arising in immunocompetent hosts. EBV-positive and EBV-negative PTLD have been shown to have distinct gene expression

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profiles. EBV-positive PTLD is characterized by upregulation of antiviral immune response signaling and markers for immunotolerogenic microenvironment, such as programmed cell death receptor 1 ligand (PD-L1), indoleamine 2,3-dioxygenase 1 (IDO1), and M2-like macrophage marker CD163.9,10 The composition and function of the tumor microenvironment (TME) has been shown to have a major impact on development and prognosis of non-immunosuppressionassociated lymphomas.11 Data on the TME in PTLD are more limited, not least because of the rarity of the disease. In addition to the EBV-induced changes in the immune response, the TME in PTLD is markedly affected by the immunosuppressive therapy, which results in an impairment of T-cell immunity. The graft organ also causes chronic immune stimulation through chronic antigen presentation, and prolonged graft-mediated interaction between donor-derived host cells.12 In this study, we profiled the expression of over 700 TMErelated genes in 75 monomorphic post-transplant aggressive B-cell lymphomas (PT-ABCL) and correlated the findings with patient demographics and outcome. In addition, we compared the TME of post-transplant diffuse large B-cell lymphomas (PT-DLBCL) with that found in sporadic DLBCL. Our data support previous studies that have found differences between EBV-positive and EBV-negative PTABCL and provide evidence of a favorable impact of the Tcell-inflamed TME on the survival of patients with PT-ABCL.

Methods Patients and samples The study cohort included 75 patients with PT-ABCL diagnosed between 1997 and 2021 at the Helsinki (Finland), Turku (Finland), Oslo (Norway) and Aarhus (Denmark) University Hospitals (Table 1). All patients had received solid organ transplants and were >16 years old at the time of PTLD diagnosis. The histopathological diagnoses of all cases were reviewed and confirmed by expert hematopathologists and classified according to the World Health Organization classification of the lymphoid neoplasms (2016 revision).4 All study cases met the morphological and immunophenotypical criteria for the diagnosis of monomorphic PTLD. In particular, cases of non-destructive PTLD and polymorphic PTLD were excluded. Further details of the patients and their treatment are provided in the Online Supplementary Appendix. The study protocol, including biobanking, was approved in Finland by Helsinki Biobank permission (HUS/190/2018), the Ethics Committee of the Helsinki University Hospital (194/13/03/00/16 and 1916/2018§108), and by the National Institution for Health and Welfare (THL/1001/5.05.00/2016), in Norway by the Regional Committee for Medical and Health Research Ethics South-East

(191426) and in Denmark by the Aarhus University Hospital Ethics Committee (M-2021-45-21). The cohort of immunocompetent host DLBCL included 75 samples from patients with sporadic high-risk DLBCL not-otherwise-specified (NOS) treated in the Nordic LBC-05 and LBC-04 trials with biweekly R-CHOEP (ri-

Table 1. The Nordic post-transplant aggressive B-cell lymphoma cohort. Characteristics

N (%)

Patients

75 (100)

Sex Female Male

27 (36) 48 (64)

Age in years at PTLD diagnosis, median (range) <60 ≥60

56 (16-76) 47 (63) 28 (37)

Histology DLBCL, NOS GCB Non-GCB NA PCNSL Burkitt HGBL-triple hit

59 (79) 15 (26) 19 (32) 25 (42) 9 (12) 6 (8) 1 (1)

Stage Early (1-2) Advanced (3-4) NA

30 (40) 36 (48) 9 (12)

IPI Low (0–2) High (3–5) NA

32 (43) 34 (45) 9 (12)

EBER Positive Negative NA Time from transplantation to PTLD in years, median (range) <1 1-5 >5

42 (56) 26 (35) 7 (9) 9 (0.1–34) 14 (19) 16 (21) 45 (60)

Transplant Kidney Liver Heart Lung Kidney + Pancreas Lung + Heart NA

36 (48) 8 (12) 16 (22) 10 (13) 3 (4) 1 (1) 1 (1)

Rituximab-containing treatment Yes No NA

53 (71) 21 (28) 1 (1)

PTLD: post-transplant lymphoproliferative disorder; DLBCL, NOS: diffuse large B-cell lymphoma, not otherwise specified; GCB: germinal center B cell; PCNSL: primary central nervous system lymphoma; HGBL: high-grade B-cell lymphoma; NA: not assigned; IPI: International Prognostic Index; EBER: Epstein-Barr virus-encoded small RNA.

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tuximab, cyclophosphamide, doxorubicin, etoposide and prednisone) immunochemotherapy and systemic central nervous system (CNS) prophylaxis (high-dose methotrexate and cytarabine).13,14 The gene expression profiling results for these DLBCL patients have been previously described.15 Gene expression profiling RNA isolation and gene expression profiling are described in detail in the Online Supplementary Appendix. Briefly, RNA was extracted from formalin-fixed paraffin-embedded (FFPE) lymphoma blocks and subjected to gene expression analysis utilizing the Nanostring nCounter platform with the Human PanCancer Immunoprofiling Panel codeset (XT-CSO-HIP1-12, NanoString Technologies, Seattle, WA).

Immune cell phenotyping For in silico immune cell phenotyping, we applied the CIBERSORTx algorithm,16,17 which uses a set of reference gene expression values (an LM22 signature matrix of 547 genes) to infer the cell type proportions from gene expression data. In order to run CIBERSORTx, normalized gene expression data were uploaded to the CIBERSORTx web portal (http://cibersortx.stanford.edu/) and the algorithm run using the default LM22 signature matrix at 100 permutations. Details on the multiplex immunohistochemistry (mIHC) stainings are provided in the Online Supplementary Appendix. Statistical analysis All data analysis was performed using R version 4.0.2. Un-

Figure 1. Unsupervised clustering of post-transplant aggressive B-cell lymphoma samples. The heatmap visualizes unsupervised hierarchical clustering of the immune panel gene expression data from 75 post-transplant aggressive B-cell lymphoma (PT-ABCL) samples. Z-score transformed levels of gene expression are depicted according to the color scale shown. Rows represent genes and columns represent samples from PT-ABCL patients. NA: not assigned; IPI: International Prognostic Index; EBER: EpsteinBarr virus-encoded small RNA; pos: positive; neg: negative; HGBL: high-grade B-cell lymphoma; DLBCL: diffuse large B-cell lymphoma; CNS: central nervous system. Haematologica | 108 November 2023

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supervised clustering with Euclidean distance and ward. D linkage was carried out by the “pheatmap” package. The R package “limma” was utilized for differential gene expression analysis, whereas Wilcoxon rank sum test and Kruskall-Wallis tests were used for non-parametric comparisons between two or more groups, respectively. The Fisher’s exact test was used to assess whether differences in dichotomous clinical variables were significant between groups. P values were corrected for multiple testing using the Benjamin-Hochberg method. For survival analysis, Kaplan-Meier estimates with log-rank test, as well as Cox univariable and multivariable regression analysis were used.

ized by low expression of genes, which typically distinguish Burkitt lymphomas from DLBCL, such as NF-kB target gene and MHC class I signature18 (Online Supplementary Figure S2). On the contrary, post-transplant PCNSL with DLBCL histology shared the gene expression profile with a subset of PT-DLBCL (Figure 1; Online Supplementary Figure S3). This is in line with previous reports showing that according to their gene expression profile, PCNSL do not differ markedly from systemic DLBCL with regard to their gene expression profile but are instead distributed within the spectrum of DLBCL.19 However, since PCNSL are clinically considered to be a distinct entity with poor prognosis,20 we excluded these cases from further analyzes.

Results

Differential profiles between Epstein-Barr viruspositive and Epstein-Barr virus-negative posttransplant aggressive B-cell lymphomas EBV status plays a major role in the development of PTLD.21 Since unsupervised clustering analysis indicated that EBV-positive and EBV-negative cases have distinct gene expression profiles (Figure 1), we explored the differentially expressed genes based on the EBV status. EBVnegative cases were characterized by expression of the MHC class II genes (HLA-DMB, HLA-DMA, HLA-DOB) as well as B-cell development-associated genes, such as MS4A1, CD79B, and SYK, whereas in EBV-positive cases type I interferon signaling pathway and antivirus response genes, including IFITM1, ISG15, MX1, and IFI35, were enriched (Figure 2A, B; Online Supplementary Table S1). In addition, genes previously shown to associate with EBV positivity, IDO1 and granzymes (GZMB, GZMH, GZMA),9,22 were upregulated in EBV-positive cases. Gene expression-based in silico deconvolution of the proportions of distinct immune cells indicated that EBVnegative PT-ABCL had a higher proportion of B cells and follicular T helper (TFH) cells, whereas in EBV-positive PTABCL proportions of infiltrating T cells, especially memory CD4 T cells, and plasma cells were higher (Figure 2C). No significant differences in the proportions of macrophages were found (data not shown).

Baseline characteristics and patients outcome Baseline characteristics of the PT-ABCL patients (n=75) are presented in Table 1. Majority of the cases (n=59, 79%) had DLBCL histology. In addition, there were nine (12%) PCNSL, six (8%) Burkitt lymphomas, and one triple-hit high-grade B-cell lymphoma. In total, 56% of the PT-ABCL were EBV-positive, 35% were EBV-negative, and in 9% the EBV status was unknown. The time from transplantation to PTLD was highly dependent on the EBV status (linear regression P=4.86e-05) and all patients with early-onset disease (<1 year from transplantation, n=14, 19%) were EBV-positive. The median time from organ transplant to lymphoma diagnosis was 9.0 years (range, 0.1-34.0), and the median follow-up time was 10.7 years (interquartile range [IQR], 6.0-13.8). The median overall survival (OS) after lymphoma diagnosis was 4.3 years (IQR, 0.3-infinite) and median progressionfree survival (PFS) was 2.8 years (IQR, 0.3-infinite). Younger age (<60 years), low IPI (0-2) and early stage (1II) were associated with better OS and PFS (Online Supplementary Figure S1A, B), whereas the EBV status, type of the transplanted organ, and PTLD histology were not significantly associated with the outcome. Patients with earlier onset of the disease (<5 years) had a better outcome, but the result was not statistically significant (Online Supplementary Figure S1A, B). Gene expression profile of post-transplant aggressive B-cell lymphomas Gene expression profiling demonstrated a high degree of heterogeneity and variation in the immune response profile among PT-ABCL (Figure 1). Two major clusters were identified, and majority of the EBV-positive samples (71%, 30/42) clustered together, but the other clinical factors were not segregated into their own groups based on the gene expression. Most of the post-transplant Burkitt lymphomas formed their own subcluster character-

Cytotoxicity gene signature predicts better outcome Next, we focused on specific gene signatures in the Nanostring immune panel and extracted genes for the cytotoxic signature: HLA-A, HLA-B, HLA-C, GZMA, GZMB, GZMH, GZMK, GZMM, GNLY, and PRF1. Interestingly, we noticed that expression of the cytotoxic signature divided the patients into three distinct groups (Figure 3A), of which the high cytotoxicity group was associated with EBV positivity and earlier onset of the disease (Online Supplementary Table S2). The group with the highest cytotoxicity signature expression had significantly better OS compared with the low or intermediate groups (ha-

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zard ratio [HR] =0.61; 95% confidence interval [CI]: 0.400.92; P=0.019) (Figure 3B), suggesting that the inflammation status of the TME plays a role in the prognosis of

PTLD. In contrast, the cytotoxic signature was not associated with outcome in sporadic DLBCL (Online Supplementary Figure S4).

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Figure 2. Differential gene expression based on the Epstein-Barr virus status of post-transplant aggressive B-cell lymphoma. (A) Volcano plot showing differentially expressed genes by the Epstein-Barr virus (EBV) status. The genes with log2 fold change >0 have higher expression in EBV-positive post-transplant aggressive B-cell lymphoma samples (PT-ABCL), whereas the genes with log2 fold change <0 have higher expression in EBV-negative PT-ABCL. (B) Unsupervised hierarchical clustering of the differentially expressed genes with adj. P<0.05. (C) The proportions of 22 distinct immune cell types were estimated with the CIBERSORTx algorithm. The boxplots show the main cell types with significantly different proportions in EBV-positive vs. EBV-negative PT-ABCL (Mann-Whitney test). NA: not assigned; IPI: International Prognostic Index; EBER: Epstein-Barr virus-encoded small RNA; pos: positive; neg: negative; HGBL: high-grade B-cell lymphoma; DLBCL: diffuse large B-cell lymphoma.

Post-transplant aggressive B-cell lymphoma patients with T-cell-inflamed tumor microenvironment have better outcome In order to characterize the immune cell landscape of PTABCL in more detail, we performed unsupervised hierarchical clustering with CIBERSORTx deconvoluted immune cell types (Figure 4A). PT-ABCL clustered into two main groups: the smaller group (n=23, 35%) with inflamed TME was characterized by the presence of T cells, M1- and M2-like macrophages, and NK cells, but low B-cell proportions, whereas the non-inflamed group (n=43, 65%) had low proportions of T cells, higher macrophage/T-cell ratio, and high proportions of B cells. The inflamed subgroup was also enriched for the cytotoxic gene signature (Figure 4A). In silico-estimated cell proportions correlated well with multiplex immunohistochemistry stainings, which were available for a small subset of patients, corroborating the CIBERSORTx data (Figure 4B; Online Supplementary Figure S5). The inflamed subgroup was associated with EBV positivity (P=0.008) and with earlier onset of PTLD following transplantation (P=0.004) (Table 2). The type of transplant organ was not associated with the inflammation status of the TME (P=0.206), nor did the main immune cell types differ by the transplant organ (Online Supplementary Figure S6). In line with previous studies on DLBCL and other lymphomas,2327 inflamed PT-ABCL TME translated to better outcome (median OS >200.0 vs. 15.2 months; P=0.006; median PFS 89.0 vs. 14.4 months, P=0.049) (Figure 4C; Online Supplementary Figure S7). In multivariable analysis with EBV status, IPI and rituximab-containing treatment, the inflamed TME remained as an independent predictor for

favorable OS and PFS (Figure 4D). This highlights the importance of the TME also in PTLD despite the immunosuppressive treatment. Of the individual immune cells, the presence of resting dendritic cells (OS: HR=2.7; 95% CI: 1.3-5.5; P=0.008, and PFS: HR=2.5; 95% CI: 1.2-5.2; P=0.013) and activated mast cells (OS: HR=5.4; 95% CI: 1.8-16.0; P=0.009, and PFS: HR=4.8; 95% CI: 1.7-14, P=0.004) translated to unfavorable outcome, whereas regulatory T cells were associated with longer survival (OS: HR=0.05; 95% CI: 0.0-0.8; P=0.035, and PFS: HR=0.1; 95% CI: 0.1-1.0; P=0.046) (Online Supplementary Table S3A, B). In multivariable analysis with rituximab-containing treatment and IPI, activated mast cells remained as independent predictors for worse, and regulatory T cells (Tregs) for improved, OS and PFS (Online Supplementary Table S3C). Comparison of post-transplant diffuse large B-cell lymphomas with sporadic diffuse large B-cell lymphomas We next compared the PT-DLBCL (n=59) with DLBCLNOS of immunocompetent hosts (n=75). Regarding clinical characteristics of the two cohorts, the sporadic DLBCL cohort was enriched for high-risk, advancedstage patients, whereas neither age nor IPI were significantly different between the cohorts (Online Supplementary Table S4). Interestingly, principal component analysis and unsupervised clustering of the gene expression data indicated that PT-DLBCL did not form their own subgroup, but rather clustered closely with sporadic DLBCL (Figure 5A; Online Supplementary Figure

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Figure 3. Cytotoxicity gene signature predicts better outcome in post-transplant aggressive B-cell lymphoma. (A) Heatmap showing the unsupervised hierarchical clustering of genes associated with cytotoxicity. (B, C) Kaplan-Meier plots with the overall survival (OS) (B) and progression-free survival (PFS). (C) Estimates of high, intermediate, and low expression of the cytotoxic signature. HR: hazard ratio. NA: not assigned; IPI: International Prognostic Index; EBER: Epstein-Barr virus-encoded small RNA; pos: positive; neg: negative; HGBL: high-grade B-cell lymphoma; DLBCL: diffuse large B-cell lymphoma.

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Figure 4. Inflamed tumor microenvironment is associated with better survival in post-transplant aggressive B-cell lymphoma. (A) Heatmap visualizing the clustering of CIBERSORTx deconvoluted immune cell types. (B) Representative multiplex immunohistochemistry (mIHC) images of patients with non-inflamed and inflamed tumor microenvironment (TME). The inlets show magnified regions from the TMA cores, as indicated by the rectangles. Scale bar 75 µm in the images with the whole TMA cores, and 20 µm in the inlets. (C) Kaplan-Meier overall survival (OS) and progression-free survival (PFS) estimates of the inflamed and noninflamed TME subclusters. (D) Forest plots visualizing the Cox regression multivariable OS (left panel) and PFS (right panel) analysis of the inflamed TME subcluster with rituximab-containing treatment, International Prognostic Index (IPI) and the Epstein-Barr virus (EBV) status. NA: not assigned; EBER: Epstein-Barr virus-encoded small RNA; HGBL: high-grade B-cell lymphoma; DLBCL: diffuse large B-cell lymphoma; mo: months.

S8A). However, based on the first principal component, EBV-positive PT-DLBCL were more distinct from DLBCL compared with EBV-negative PT-DLBCL (Online Supplementary Figure S8B). Despite the similarities, a supervised analysis identified a gene signature, which could separate PT-DLBCL from sporadic DLBCL (Figure 5B, C). Genes having higher expression in sporadic DLBCL were enriched for Tcell signaling and T cell-related pathways (e.g., CD4, FOXP3, HLA genes), indicating that T-cell proportions are lower in PT-DLBCL (Online Supplementary Table S5). Indeed, this was verified by CIBERSORTx analysis, which confirmed that PTDLBCL have lower proportions of T cells, in particular TFH cells and γδ T cells, and resting mast cells (Figure 5D; Online Supplementary Figure S8C). In contrast, the proportions of NK cells, dendritic cells, plasma cells, and activated mast cells were higher in PT-DLBCL compared with sporadic DLBCL.

Discussion PTLD represent a heterogeneous spectrum of diseases ranging from early lesion and polymorphic lymphoproliferation to monomorphic lymphoma.2,3 PTLD also comprise various histologic subtypes, of which DLBCL is the most common. In this study, we focused on profiling the TME of 75 PT-ABCL utilizing digital gene expression profiling complemented with in silico phenotyping of TME-associated immune cells. To our knowledge, this is one of the largest gene expression profiling studies so far conducted on PTLD. Recently, a multidimensional characterization of PTLD TME and virome presented distinct immunogenomic features reflecting divergent PTLD biology.28 Here, we show that the PT-ABCL TME is heterogenous and can be classified as inflamed and non-inflamed according to the proportions of distinct immune cells. Although the PT-

ABCL TME is affected by the immunosuppressive therapy, a subset of patients displayed higher expression of cytotoxicity gene signature and an inflamed TME rich in T cells, translating to improved survival. Interestingly, the patient subgroup with low cytotoxicity signature expressed higher levels of B-cell receptor associated genes (PAX5, CD79A/B, CD19, MS4A1, SYK, BTK; data not shown), allowing speculation that these patients could benefit from targeted therapies, such as CD79B-targeting polatuzumab-vedotin,29-31 CD19-targeting tafasitamab in combination with lenalidomide,32-34 and SYK inhibitors.35,36 The subgroup with the inflamed TME was enriched for EBV-positive cases, which is in accordance with previous findings suggesting that T cells react to the EBV antigens and are attracted to the site of the lymphoma.22 In addition, EBV-positive and EBV-negative PTLD have been shown to form distinct entities with differences in the genomic and transcriptomic profiles.9,22,37 However, in line with other studies,38-40 EBV status itself was not prognostic in our cohort, suggesting that the favorable effect of the inflamed TME did not just reflect the EBV status. This was also supported by the multivariable analysis, which demonstrated that the inflamed TME was independent of IPI and EBV status in predicting favorable outcome. We also compared TME between PT-DLBCL and sporadic DLBCL. Our data indicate that PT-DLBCL and sporadic DLBCL do not cluster into distinct subgroups based on the immune response gene expression or TME characteristics. In general, sporadic DLBCL had higher proportions of T cells, and their gene expression profile was enriched for T-cell-related genes. The lack of T cells in the PTDLBCL was even more evident, when EBV-negative PTDLBCL were compared with sporadic DLBCL. This represents the main difference between PT-DLBCL and sporadic DLBCL and associates with the immunosuppressive treatment of PTLD. In previous studies, EBV-

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negative PT-DLBCL have been shown to resemble DLBCL genomically and transcriptionally, whereas EBV-positive PT-DLBCL have been considered more distinct; their transcriptomic profile substantially impacted by the EBV infection.8,9,22,37,41 Unfortunately, EBV status of the sporadic DLBCL in our study was not available for comparison. Another limitation of our study is that it is based solely on gene expression profiling. Therefore, the association of TME on distinct genomic alterations could not be addressed. Considering that stromal signatures associate

with distinct genomic abnormalities and outcome in DLBCL,42-44 it will be interesting to study the relationship between genomic abnormalities and microenvironmental signatures in PTLD as well. In the solid organ setting, therapy of PTLD consists of reduction of immunosuppression and is often followed by CD20 antibody rituximab in combination with chemotherapy, such as CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone). However, since the PTLD patients are clinically fragile, intensive chemotherapy is not the pre-

Table 2. Patient characteristics in the non-inflamed and inflamed groups. Characteristics

Non-inflamed, N (%)

Inflamed, N (%)

Patients

43 (100)

23 (100)

Age in years <60 ≥60

23 (53) 20 (47)

17 (74) 6 (26)

Stage Early (1-2) Advanced (3-4) NA

16 (4) 24 (56) 3 (7)

7 (30) 10 (43) 6 (26)

IPI Low (0-2) High (3-5) NA

16 (37) 24 (56) 3 (7)

9 (39) 8 (35) 6 (26)

EBER Negative Positive NA

21 (49) 15 (35) 7 (16)

5 (22) 18 (78) 0

Histology DLBCL Burkitt HGBL triple hit

38 (89) 4 (9) 1 (2)

21 (92) 2 (8) 0

0.122

1.000

0.397

0.008

1.000

Time from transplant in years to PTLD <1 1-5 5-9 ≥10

4 (9) 8 (19) 5 (12) 26 (60)

10 (43) 5 (22) 3 (13) 5 (22)

Transplant Kidney Heart Liver Lung Multiorgan NA

21 (49) 11 (25) 6 (14) 2 (5) 3 (7) 0

11 (48) 4 (17) 1 (4) 5 (22) 1(4) 1 (4)

Rituximab-containing treatment No Yes NA

9 (21) 33 (77) 1 (2)

5 (22) 18 (78) 0

Treatment response CR PR SD PD NA

0.004

0.169

1.000

0.082 22 (51) 4 (9) 2 (5) 6 (14) 9 (21)

18 (78) 0 2 (9) 0 3 (13)

NA: not assigned; IPI: International Prognostic Index; EBER: Epstein Barr-virus-encoded small RNA; DLBCL: diffuse large B-cell lymphoma; HGBL: high-grade B-cell lymphoma; PTLD: post-transplant lymphoproliferative disorder; CR: complete response; PR: partial response; SD: stable disease; PD: progressive disease. Haematologica | 108 November 2023

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ferred choice in the first-line therapy.45 In our cohort, the majority (71%) of the patients were treated with a rituximab-containing regimen, and over half of the patients also received chemotherapy. A limitation of our study was that information about the type of immunosuppressive therapy

used in the PTLD patients was not available for the analyzes. Immunosuppressive therapy can have a significant impact on the TME in PTLD by reducing the number and function of immune cells within the TME and thus leading to a less favorable TME, which promotes lymphoma growth

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Figure 5. Comparison of post-transplant diffuse large B-cell lymphoma with immunocompetent host diffuse large B-cell lymphoma. (A) Heatmap illustrating unsupervised hierarchical clustering of the immune panel gene expression in post-transplant lymphoproliferative disorder (PTLD) with diffuse large B-cell lymphoma (DLBCL) histology (N=59) and DLBCL from immunocompetent patients (N=75). (B) Volcano plot showing differentially expressed genes in post-transplant DLBCL (PT-DLBCL) compared to DLBCL. Genes denoted in red are significantly (adj. P<0.05) differentially expressed. Selected genes are annotated in the plot. (C) The heatmap visualizes clustering of PT-DLBCL and DLBCL based on the expression of the most significant (adj. P<0.0001) differentially expressed genes. (D) Immune cell proportions were deconvoluted by CIBERSORTx. Cell types that have differential levels in PT-DLBCL compared to DLBCL (P<0.05) are visualized in the heatmap. NA: not assigned; IPI: International Prognostic Index; EBER: Epstein-Barr virus-encoded small RNA; HGBL: high-grade B-cell lymphoma; NK: natural killer: GC: germinal center; NOS: not otherwise specified; COO: cell of origin. Haematologica | 108 November 2023

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and progression.12 In addition to its direct effects on immune cells, immunosuppressive therapy can impact the TME through its effects on the expression of cytokines and chemokines, such as interleukin-10 and interferon-γ, which play a critical role in regulating immune cell functions. In general, heart, lung, intestinal, and multi-organ transplant recipients are attributed to more aggressive immunosuppression due to severe consequences of graft failure owing to the rejection.46 In our study, the type of the transplant organ was, however, neither associated with the inflammation status of the PT-ABCL nor with the proportions of the distinct immune cells. In conclusion, we provide a comprehensive phenotypic characterization of PT-ABCL that highlights the importance of TME biology and in particular T-cell infiltration, in the clinical behavior and prognosis of PTLD.

and wrote the manuscript. TF designed the study, collected clinical data, and contributed to writing the manuscript. MA analysed mIHC data. SV, AWJ, FA, S-JH-D, HH, KB, and PEK collected samples and provided clinical data. RR designed the study and revised the manuscript. SL designed and supervised the study and revised the manuscript. All authors have read and accepted the final version of the manuscript.

Disclosures SL consults for Genmab, Gilead, Incyte, Novartis, Roche, Abbvie and Orion; she has received research funding from Genmab, Nordic Nanovector, Novartis, Roche, Bayer and Celgene Hutchmed all outside of the submitted work; she has received honoraria from Novartis and Gilead. Other other authors have no conflicts of interest to declare.

Funding This research was funded by the grants from the Academy of Finland (to SL), Finnish Cancer Organizations (to SL), Sigrid Juselius Foundation (to SL), University of Helsinki (to SL), Helsinki University Hospital (to SL).

Contributions S-KL designed and conceived the study, analyzed data,

Acknowledgements We thank the DNA Sequencing and Genomics Laboratory at the Institute of Biotechnology, University of Helsinki for the NanoString analyzes, Anne Aarnio for technical assistance, and Annabrita Schoonenberg, Teijo Pellinen and FIMM Digital Microscopy and Molecular Pathology Unit supported by HiLIFE and Biocenter Finland for the mIHC services.

Data-sharing statement The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request and Scandiatransplant (to TF).

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Predictors of SARS-CoV-2 Omicron breakthrough infection after receipt of AZD7442 (tixagevimab-cilgavimab) for pre-exposure prophylaxis among hematologic malignancy patients Justin C. Laracy,1-3 Judy Yan,1 Samantha N. Steiger,4 Carrie A. Tan,4 Nina Cohen,4 Elizabeth V. Robilotti,3,5 Jerome Fender,1,6 Sara Cohen,6 Neha Korde,3,7 Melissa Lee-Teh,4 Ariela Noy,3,8 Joseph H. Oved,9 Lindsey E. Roeker,3,10 Gunjan Shah,3, 11 N. Esther Babady,2,1 2 Mini Kamboj 1-3# and Susan K. Seo2,3# Infection Control, Memorial Sloan Kettering Cancer Center; 2Infectious Disease Service, Department of Medicine, Memorial Sloan Kettering Cancer Center; 3Department of Medicine, Weill Cornell Medical College; 4Department of Pharmacy, Memorial Sloan Kettering Cancer Center; 5Division of Infectious Diseases, Hospital for Special Surgery; 6Digital Informatics & Technology Solutions, Memorial Sloan Kettering Cancer Center; 7Myeloma Service, Department of Medicine, Memorial Sloan Kettering Cancer Center; 8Lymphoma Service, Department of Medicine, Memorial Sloan Kettering Cancer Center; 9Department of Pediatric Transplant and Cell Therapy, Memorial Sloan Kettering Cancer Center; 10Leukemia Service, Department of Medicine, Memorial Sloan Kettering Cancer Center; 11Adult Bone Marrow Transplant Service, Department of Medicine, Memorial Sloan Kettering Cancer Center and 12 Clinical Microbiology Service, Department of Pathology and Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA 1

Correspondence: J. Laracy laracyj@mskcc.org S. Seo seos@mskcc.org Received: Accepted: Early view:

March 6, 2023. June 15, 2023. June 22, 2023.

MK and SKS contributed equally as senior authors.

Abstract AZD7442 (tixagevimab-cilgavimab) is a combination of two human monoclonal antibodies for pre-exposure prophylaxis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection among high-risk patients who do not mount a reliable vaccine response. Foremost among these are hematologic malignancy patients with limited clinical trial or realworld experience to assess the effectiveness of this combination treatment since the emergence of Omicron and its subvariants. We performed a retrospective study of 892 high-risk hematologic malignancy patients who received AZD7442 at Memorial Sloan Kettering Cancer Center in New York City from January 1, 2022 to July 31, 2022. We evaluated demographic, clinical, and laboratory characteristics and performed regression analyses to evaluate risk factors for breakthrough infection. We also evaluated the impact of updated AZD7442 dosing regimens on the risk of breakthrough infection. Among 892 patients, 98 (10.9%) had a breakthrough infection during the study period. A majority received early outpatient treatment (82%) and eventually eight (8.2%) required hospitalization for management of Coronavirus Disease 2019 (COVID-19), with a single instance of severe COVID-19 and death. Patients who received a repeat dose or a higher firsttime dose of AZD7442 had a lower incidence of breakthrough infection. Univariate analyses did not reveal any significant predictors of breakthrough infection. While AZD7442 is effective at reducing SARS-CoV-2 breakthrough infection in patients with hematologic malignancies, no risk factors reliably predicted risk of infection. Patients who received updated dosing regimens as per Food and Drug Administration guidelines had better protection against breakthrough infection.

Introduction While vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has helped to reduce Coronavirus Disease 2019 (COVID-19)-related morbidity and mortality, individuals with underlying immune dys-

function remain at high risk for not achieving target immunological and clinical outcomes. For example, serological assessments after two or three primary mRNA vaccine doses demonstrated an abrogated seroconversion rate among cancer patients or early waning of immunity, especially among patients with hematologic malignancies.1-3

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ARTICLE - SARS-CoV-2 breakthrough infection after AZD7442 Subsequent studies found that vaccinated immunocompromised patients have higher odds of breakthrough infection and increased risk of severe disease leading to hospitalization and death compared to non-immunosuppressed patients.3,4 Furthermore, although data on variantspecific breakthrough among cancer patients are sparse, neutralizing activity of serum from vaccinated individuals is diminished against the Omicron subvariants.6-9 In December 2021, during the peak of the Omicron surge, the US Food and Drug Administration (FDA) granted emergency use authorization (EUA) to AZD7442, a combination of two human monoclonal antibodies for pre-exposure prophylaxis against COVID-19 in high-risk patients.10 The use of AZD7442 to prevent SARS-CoV-2 infection was the standard of care throughout 2022 for high-risk patients when regional variants retained susceptibility to this agent. AZD7442 was one of just a few medical innovations to be included in Times Magazine's "The Best Inventions of 2022".11 However, a vital gap in the trials leading to AZD7442’s EUA was the minimal representation of high-risk cancer patients.12 Shortly after its initial authorization, neutralization assays revealed decreased activity of AZD7442 against emerging Omicron subvariants.13,1 4 Subsequently, the FDA authorized revisions to the AZD7442 dosing regimen given concerns of reduced potency to certain Omicron subvariants. However, the EUA was rescinded on January 26, 2023, when the prevalence of susceptible variants in the US was less than 10%.15 The present study describes the incidence, predictors, and clinical outcomes among AZD7442-treated hematologic malignancy patients for the first 8 months after this drug received EUA for primary prevention of SARS-CoV-2 infection in high-risk patients.

Methods Study population From January 1, 2022, to July 31, 2022, all consecutive patients at Memorial Sloan Kettering Cancer Center (MSKCC) >=12 years of age who received AZD7442 were included in the study. A retrospective analysis of 892 AZD7442 recipients was conducted. Identification of case patients and their medical background and clinical course from COVID19 were extracted from the electronic medical record. The MSKCC Institutional Review Board granted a Health Insurance Portability and Accountability Act waiver of authorization to conduct this study. Laboratory methods SARS-CoV-2 RNA test Viral RNA was detected using nasopharyngeal swabs or saliva samples as previously described.16 Briefly, SARSCoV-2 RNA was tested for by real-time reverse transcrip-

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tion-polymerase chain reaction (RT-PCR) using several commercial assays. These included the Cobas® SARSCoV-2 test (Roche Molecular Diagnostics, Indianapolis, Indiana USA), the TaqPath™ COVID-19 Combo Kit (Thermo Fisher Scientific, Waltham, MA), the ePlex Respiratory Panel 2 (GenMark/Roche Molecular Diagnostics, Indianapolis, IN), and the BioFire Respiratory Panel 2.1 (BioMerieux, Salt Lake City, UT). Anti-SARS-CoV-2 spike IgG antibody assay was performed as previously described.17 SARS-CoV-2 whole genome sequencing Whole genome sequencing (WGS) was performed on samples with a cycle threshold (Ct) value <30 to increase the likelihood of successful sequencing. Samples from platforms that do not provide a Ct value (i.e., BioFire RP 2.1 and ePlex 2) underwent WGS without knowledge of Ct value. WGS was performed as previously described using the ARTIC protocol with version 4.1 primers (Integrated DNA Technologies [IDT (Integrated DNA Technologies)], Coralville, Iowa USA).18 Pangolin software (https://github.com/cov-lineages/pangolin) was used to assign lineages for each consensus sequence using the Pango nomenclature. Statistical analysis Baseline demographic and clinical characteristics for all study patients were reported as absolute frequency and percentage or median with interquartile range (IQR). Due to revised guidance from the FDA on AZD7442 dosing, study patients received varied dosing during the evaluation period. Therefore, we classified our study patients into four groups based on the dosage of AZD7442 they received: group 1 received one dose 150-150 mg, group 2 received two doses of 150-150 mg, group 3 received one dose of 150-150 mg and one dose 300-300 mg, and group 4 received one dose of 300-300 mg. Crude estimates of treatment effect were stratified by dose groups. The number of breakthrough infections, total number of person-days, and incidence rate per 1,000 person-days for each stratum were calculated. Follow-up person-days began from the initial date of AZD7442 administration until the SARS-CoV-2 breakthrough date, death, or the end of the study period. Incidence rate ratios (95% confidence interval [CI]) were calculated to assess the association between breakthrough infection by dose group. In order to examine the relationship between breakthrough infection and AZD7442 dosage, we performed extended Cox regression analyses using a counting process data structure to account for the varying dosages administered at different time periods. For each dose group, univariable Cox regression analyses were performed to evaluate potential risk factors for breakthrough infection. Variables that were significant at P<0.05 in the univariable analysis were considered for the multivariable cox regression model.

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ARTICLE - SARS-CoV-2 breakthrough infection after AZD7442

Results Baseline characteristics of AZD7442 recipients (study cohort) During the study period, 892 high-risk hematologic malignancy patients received AZD7442. The median age was 68 years (IQR, 59-75), 410 (46.0%) were female, and the underlying cancers were lymphoma (57.2%), leukemia/myelodysplastic syndrome (MDS) (28.8%), myeloma/amyloidosis

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(13.5%), and others (0.1%). One hundred ninety-six patients (21.9%) had received hematopoietic stem cell transplantation (HSCT) or chimeric antigen receptor (CAR) T-cell therapy within 1 year (Table 1). Almost 40% of patients had received anti-CD20 therapy within the previous 12 months. Dose groups 1, 2, 3, and 4 were comprised of 149 (16.7%), 292 (32.7%), 75 (8.4%), and 376 (42.2%) patients, respectively. Four hundred eighty-three (54.1%) patients were vaccinated. Patients were considered fully vaccinated ≥2 weeks

Table 1. Demographics and baseline characteristics of study patients. Characteristic

Total a, b N=892

Age in years, median (IQR)

68 (59-75)

Female sex, N (%)

410 (46.0)

Laboratory values, N (%) Creatinine ≥1.1 mg/dLc Creatinine <1.1 mg/dLc Lymphocyte ≥500 cell/mcLd Lymphocyte <500 cell/mcLd CD3+CD4+ (T4 helper) cells, blood ≥200 cell/mcLe CD3+CD4+ (T4 helper) cells, blood <200 cell/mcLe CD19 ≥50 cell/mcLe CD19 <50 cell/mcL Neutrophils ≥0.5 k/mcLf Neutrophils <0.5 k/mcLf Total IgG ≥500 mg/dL Total IgG <500 mg/dL

238 (26.9) 648 (73.1) 794 (89.3) 95 (10.7) 182 (61.3) 115 (38.7) 61 (20.9) 231 (79.1) 763 (98.5) 12 (1.5) 44 (53.1) 39 (47.0)

Anti-Spike SARS CoV-2 antibody levels (Abbot), N (%) Spike Ab ≥1,000 AU/mLg 81 (10.0) g Spike Ab <1,000 AU/mL 731 (90.0) COVID-19 vaccination status, N (%) ≥3 vaccine doses (Yes)h <3 vaccine doses (No)h

483 (54.1) 409 (45.9)

Comorbidities,i N (%) At least one co-morbidity No co-morbidity Atrial fibrillation (Yes) Atrial fibrillation (No) Chronic lung disease (Yes) Chronic lung disease (No) Heart failure (Yes) Heart failure (No) HIV (positive) HIV (negative) Hypertension (Yes) Hypertension (No) Diabetes (Yes) Diabetes (No) Renal failure (Yes) Renal failure (No)

503 (56.3) 389 (43.6) 107 (12.0) 785 (88.0) 47 (5.2) 845 (94.7) 42 (4.7) 850 (95.3) 8 (0.9) 884 (99.1) 378 (42.4) 514 (57.6) 91 (10.2) 801 (89.8) 167 (18.7) 725 (81.3)

Systemic steroids (Yes),j N (%)

199 (22.3)

Systemic steroids (No),j N (%)

693 (77.7)

Anti-CD20 therapy (Yes),k N (%)

349 (39.1)

Anti CD20 therapy (No),k N (%)

543 (60.9)

Characteristic

Total a, b N=892

Cancer types, N (%) Leukemia/myelodysplastic syndrome Myeloma/amyloidosis Other Lymphoma

257 (28.8) 120 (13.5) 1 (0.1) 510 (57.2)

Chemotherapy (Yes),l N (%)

549 (61.6)

Chemotherapy (No),l N (%)

343 (38.5)

Relapse/refractory disease (Yes), N (%)

179 (20.1)

Relapse/refractory disease (No), N (%)

713 (79.9)

BMT CAR T (Yes),m N (%)

196 (22.0)

BMT CAR T (No),m N (%)

696 (78.0)

Body mass index,n N (%) Underweight (< 18.5) Healthy weight (18.5-24.9) Overweight (25.0-29.9) Obese (≥30.0)

22 (2.5) 318 (35.7) 347 (39.0) 204 (22.9)

Ab: antibody; BMT: bone marrow transplant; CAR T: chimeric antigen receptor T-cell therapy; IgG: immunoglobulin G; HIV: human immunodeficiency virus; IQR: interquartile range. aClinical classifications were based on available data. bOdds ratios and P values were calculated from logistic regression applying Firth’s correction, where appropriate. cCreatinine, IgG: most recent values before first AZD7442 start date within the last 6 months. dLymphocytes: most recent 3 laboratory values within 12 months prior to first AZD7442 start date. All 3 counts must be <500 per microliter to be in the '<500' category. At least 1 count of value ≥500 per microliter is considered in the "≥500" category. One patient did not have 3 laboratory values prior to COVID-19 diagnosis. eCD4, CD19: most recent values before first AZD7442 start date within the last year. fNeutrophils: most recent values before first AZD7442 start date within 1 month. g Spike antibody: most recent test value before first AZD7442 start date within 1 year. hVaccination history (N): charts were manually reviewed for patients with no vaccine history. iChronic conditions (atrial fibrillation, chronic lung disease [chronic obstructive pulmonary disease, bronchiectasis, asthma], heart failure, hypertension, human immunodeficiency virus (HIV), diabetes, renal failure) were based on ICD10 diagnosis codes within 12 months of the study period. jSystemic steroids (dexamethasone, methylprednisolone, hydrocortisone, prednisone): given within 30 days prior to first AZD7442 start date. kAnti-CD-20 therapy (rituximab, obinutuzumab, tafasitamab, hyaluroinidase-rituximab, ofatumumab): given within previous 12 months of AZD7442. lChemotherapy during study period. m BMT and CAR T-cell therapies combined with service dates within 1 year of the study period. nBMI: most recent value during past 12 months.

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ARTICLE - SARS-CoV-2 breakthrough infection after AZD7442 after the final dose of a primary vaccination series.19 Five hundred and three (56.3%) patients had at least one additional comorbidity as outlined in Table 1. Incidence and clinical outcomes of breakthrough infection in AZD7442 recipients Ninety-eight (10.9%) unique patients had breakthrough infection from SARS-CoV-2 during the study period. Among these patients with breakthrough infection, the median age was 65 years (IQR, 53-74) and 37 (37.7%) were female. The underlying cancers included hematologic malignancy patients only: lymphoma (57.1%), leukemia/MDS (35.7%), and myeloma/amyloidosis (7.1%). Twenty-seven breakthrough patients had received HSCT or CAR T-cell therapy. The median time from the first AZD7442 administration to laboratory diagnosis of COVID-19 was 104 days (range, 4198; IQR, 67-140). Fifty-five (56.1%) patients were vaccinated, and 52 (53.0%) patients had at least one additional medical comorbidity. Dose groups 1 and 2 were each comprised of 35 (35.7%) patients while dose group 4 was comprised of 26 (26.5%) patients. Only 2 (2.0%) patients were in dose group 3. The incidence of breakthrough infection was highest in dose group 1 at 1.60 per 1,000 person days compared to 0.70, 0.15, 0.86 per 1,000 person days in dose groups 2, 3 and 4, respectively. Compared to dose group 1, the incidence rate ratio (IRR) was 0.43 (95% CI: 0.26-0.72), 0.09 (95% CI: 0.01-0.36), and 0.54 (95% CI: 0.32-0.92) for dose groups 2, 3, and 4, respectively (Table 2). Univariate analyses for patients in three of the four dosing groups did not show significant predictors for breakthrough infection. In particular, anti-CD20 therapy within the previous 12 months, a known risk factor for suboptimal binding and neutralizing antibody response, was not an independent predictor of infection in any dose group.20,21 Although neutrophil count was significant for patients in dose group 4, the low number of cases with ab-

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solute neutrophil count (ANC) <500/mcL and the small hazard ratio precluded meaningful statistical comparison (Table 3). Due to the limited number of significant predictors, multivariable analyses were not performed. Notable risk factors that did not predict breakthrough infection across all four dose groups included age, medical comorbidities, vaccination status, underlying cancer type, and prior HSCT or CAR T-cell therapy. Clinical outcomes of breakthrough infection and time to breakthrough infection by Omicron subvariants The clinical presentation and outcome of the 98 patients with breakthrough infection from SARS-CoV-2 are summarized in Table 4. Most received standard-of-care therapy with authorized monoclonal antibody therapy (n=47, 47.9%) or antivirals, most commonly nirmatrelvir-ritonavir (n=31, 31.6%). Eight patients (8.2%) were hospitalized for the management of COVID-19, of which two were hospitalized elsewhere without available records. The illness severity was mild to moderate for five of the six patients with available data. Treatments administered to the six hospitalized patients with available data included remdesivir for all six patients (4 patients at time of admission and 2 patients following admission when they developed an oxygen requirement) and dexamethasone for all three patients who developed an oxygen requirement. Of the three hospitalized patients who required supplemental oxygen, two patients recovered. The third patient, who had a prior history of lung cancer and Waldenstrom’s macroglobulinemia recently treated with anti-CD20 therapy, progressed to respiratory failure and eventually died due to COVID-19. This same patient had been fully vaccinated. Overall, six (6.1%) of the 98 breakthrough patients developed chronic COVID-19, resulting in two COVID-19related readmissions. As of March 31, 2023, a total of 15 (15.3%) AZD7442 recipients had experienced two breakthrough infections. The median time interval between the

Table 2. Comparison of AZD7442 dosing group as risk factors for SARS-CoV-2 breakthrough infection.

AZD7442 dosage

Total breakthrough cases

Total person-days

Median (IQR) Incidence rate days to COVID-19 per 1,000 diagnosis

Dose group 1: 1 dose 150-150 mg

149

21,834

92 (31-128)

1.60

ref.

Dose group 2: 2 doses 150-150 mg

292

50,222

138 (109-167)

0.70

0.43 (0.26-0.72)

Dose group 3: 1 dose 150-150 mg + 1 dose 300-300 mg

13,668

189.5 (181-198)

0.15

0.09 (0.01-0.36)

Dose group 4: 1 dose 300-300 mg

376

30,088

78 (43-97)

0.86

0.54 (0.32-0.92)

Incidence rate ratio (95% CI)

IQR: interquartile range; CI: confidence interval; COVID-19: coronavirus disease 2019; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. Haematologica | 108 November 2023

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Table 3. Cox regression analysis of risk factors for SARS-CoV-2 breakthrough infection among high-risk hematologic malignancy patients stratified by dose of AZD7442 for pre-exposure prophylaxis.

Characteristic

Crudea Hazard ratio (95% CI)

Group 1: 1 dose of 150-150 mg

Group 2: 2 doses of 150-150 mg

Group 4: 1 dose 300-300 mg

Age in years: ≥65 vs. <65 (ref.)

0.65 (0.34-1.26) 0.202

0.56 (0.27-1.08) 0.082

0.59 (0.27-1.28) 0.183

Sex: female vs. male (ref.)

0.79 (0.40-1.55) 0.484

0.64 (0.32-1.26) 0.195

0.67 (0.30-1.51) 0.334

Laboratory Creatinine: ≥1.1 mg/dL vs. <1.1 mg/dLc Lymphocyte: ≥500 cell/mcL vs. <500 cell/mcLd CD3+CD4+ (T4 helper) cells, blood: ≥200 cell/mcL vs. <200 cell/mcLe CD19: ≥50 cell/mcL vs. <50 cell/mcLe Neutrophils: ≥0.5 k/mcL vs.s <0.5 k/mcLf Total IgG: ≥500 mg/dL vs. <500 mg/dLc

0.73 (0.33-1.61) 0.44 1.41 (0.50-4.00) 0.515 0.70 (0.27-1.83) 0.464

0.54 (0.22-1.30) 0.167 3.42 (0.47- 25.0) 0.225 6.81 (0.89-52.1) 0.065

1.34 (0.60-3.00) 0.48 0.65 (0.20-2.18) 0.489 0.27 (0.06-1.13) 0.073

2.62 (0.94-7.31) 0.065 0.63 (0.04-10.8) 0.753 3.66 (0.71-18.94) 0.122

1.05 (0.29-3.82) 0.94 0.95 (0.06-16.27) 0.974 0.23 (0.01-9.39) 0.434

1.37 (0.28-6.81) 0.699 0.21 (0.05-0.88) 0.032 * 0.86 (0.02-35.9) 0.936

Anti-spike SARS CoV-2 antibody levels (Abbot) Spike Ab: ≥1,000 AU/mL vs. <1,000 AU/mLg

1.75 (0.53-5.76) 0.361

0.72 (0.14-3.78) 0.695

1.36 (0.46-3.96) 0.577

COVID-19 vaccination status ≥3 vaccine doses: Yes vs. Noh

1.03 (0.53-2.00) 0.928

1.14 (0.58-2.24) 0.706

1.20 (0.54-2.64) 0.655

0.81 (0.41-1.56) 1.29 (0.45-3.65) 2.39 (0.73-7.82) 1.04 (0.32-3.39) 1.70 (0.10-28.8) 0.95 (0.49-1.85) 0.64 (0.19-2.07) 1.00 (0.48-2.08)

0.81 (0.42-1.57) 0.77 (0.24-2.51) 0.29 (0.02-4.85) 0.57 (0.08-4.15) 1.59 (0.10-27.2) 0.65 (0.32-1.32) 0.11 (0.01-1.90) 0.78 (0.30-2.01)

0.523 0.663 0.387 0.577 0.748 0.232 0.130 0.605

0.76 (0.35-1.64) 0.488 0.13 (0.01-2.25) 0.161 0.56 (0.03-9.56) 0.693 0.84 (0.05-14.58) 0.903 1.44 (0.08-25.0) 0.802 1.15 (0.53-2.48) 0.731 0.22 (0.01-3.78) 0.295 1.38 (0.52-3.65) 0.522

Comorbidities At least one co-morbidity vs. no co-morbidity Atrial fibrillation: Yes vs. No Chronic lung disease: Yes vs. No Heart failure: Yes vs. No HIV: Yes vs. No Hypertension: Yes vs. No Diabetes: Yes vs. No Renal failure: Yes vs. No

0.532 0.635 0.149 0.953 0.714 0.879 0.452 0.998

Systemic steroids: Yes vs. Noj

1.28 (0.58-2.81) 0.547

0.54 (0.21-1.38) 0.196

1.27 (0.51-3.19) 0.608

Anti-CD20 therapy: Yes vs. Nok

0.68 (0.34-1.37) 0.277

1.67 (0.86-3.24)

0.13

0.51 (0.21-1.28) 0.153

Cancer types Lymphoma Leukemia/myelodysplastic syndrome Myeloma/amyloidosis Other

ref. N/A 1.64 (0.81-3.34) 0.173 0.72 (0.18-2.79) 0.63 1.86 (0.10-33.9) 0.677

ref. N/A 0.96 (0.48-1.93) 0.901 0.33 (0.06-1.82) 0.203 N/A N/A

ref. N/A 1.35 (0.56-3.25) 0.506 1.17 (0.39-3.50) 0.785 N/A N/A

Chemotherapy: Yes vs. Nol

0.74 (0.38-1.44) 0.374

0.82 (0.42-1.59) 0.549

0.81 (0.37-1.76) 0.589

Relapse/refractory disease: Yes vs. No

1.34 (0.64-2.79) 0.434

1.01 (0.44-2.32) 0.974

0.74 (0.26-2.16) 0.586

BMT CAR T: Yes vs. Nom

1.33 (0.68-2.61) 0.412

0.94 (0.39-2.26) 0.885

1.46 (0.59-3.64) 0.417

Body mass indexn Healthy weight (18.5-24.9) Underweight (<18.5) Overweight (25.0-29.9) Obese (≥30.0)

ref. N/A 0.29 (0.02-5.31) 0.403 0.96 (0.45-2.04) 0.905 0.71 (0.26-1.91) 0.5

ref. N/A 1.40 (0.18-11.04) 0.751 2.08 (0.94-4.61) 0.07 1.01 (0.36-2.84) 0.984

ref. N/A 2.10 (0.27-16.4) 0.481 0.86 (0.35-2.11) 0.734 0.93 (0.24-2.55) 0.884

Ab: antibody; BMT: bone marrow transplant; CAR T; chimeric antigen receptor T-cell therapy; IgG: immunoglobulin G; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2. *Cox model with AZD7442 dosage as time-varying covariate. Cox proportional hazards regression. Forward selection with inclusion and selection criterion of P<0.05. aClinical classifications were based on available data. bOdds ratios and P values were calculated from logistic regression applying Firth’s correction, where appropriate. cCreatinine, IgG: Most recent values before first AZD7442 start date within the last 6 months. dLymphocytes: Most recent 3 laboratory values within 12 months prior to first AZD7442 start date. All 3 counts must be <500 per microliter to be in the '<500' category. At least 1 count of value ≥500 per microliter is considered in the "≥500" category. One patient did not have 3 laboratory values prior to COVID-19 diagnosis. eCD4, CD19: most recent values before first AZD7442 start date within last year. fNeutrophils: most recent values before first AZD7442 start date within 1 month. gSpike antibody: most recent test value before first AZD7442 start date within 1 year. hFully vaccinated (N): charts were manually reviewed for patients with no vaccine history in patient database. iChronic conditions (atrial fibrillation, chronic lung disease [chronic obstructive pulmonary disease, bronchiectasis, asthma], heart failure, hypertension, human immunodeficiency virus (HIV), diabetes, renal failure) were based on ICD10 diagnosis codes within 12 months of the study period. jSystemic steroids includes patients who were on steroids within 30 days prior to first AZD7442 start date. Includes dexamethasone, methylprednisolone, hydrocortisone, prednisone. kAnti-CD-20 therapy (rituximab, obinutuzumab, tafasitamab, hyaluroinidase-rituximab, ofatumumab): given within the previous 12 months of AZD7442. lChemotherapy during the study period. m BMT and CAR T-cell therapies combined with service dates within 1 year of the study period. nBMI: most recent value during past 12 months. Haematologica | 108 November 2023

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ARTICLE - SARS-CoV-2 breakthrough infection after AZD7442 episodes was 157 days (range, 94-261 days). Notably, for 12 of 15 patients, the second infection occurred at a time when non-susceptible variants were dominant. Most patients with breakthrough infection tested positive for SARS-CoV-2 using home antigen tests, and clinical samples were not accessible for WGS. Of the 33 patients with available RT-PCR swabs performed at MSKCC, 27 subvariants were successfully identified using WGS. Figure 1 shows days to breathrough infection in dose groups 1, 2, and 4 by expected subvariant based on sequencing results when available or dominant circulating strain at the time of illness.

Discussion In our study cohort of highly vaccinated hematologic malignancy patients who received AZD7442 as pre-exposure prophylaxis against SARS-CoV-2 infection in the era of Omicron, 10.9% of recipients had breakthrough infection. Of those with breakthrough infection, the majority received early outpatient treatment (82%) and eventually eight (8.2%) required hospitalization for management of COVID-19, with a single instance of severe COVID-19 and death. Patients who received a repeat dose of AZD7442 or a higher first-time dose of AZD7442 had a lower incidence of breakthrough infection. Notably, no host factors or treatment-related factors predicted the risk of breakthrough infection in our study cohort. The critical PROVENT trial demonstrated an 83% relative risk reduction in developing symptomatic COVID-19 among vaccine-naïve patients who received AZD7442 for prevention compared to placebo at a median follow-up of 6 months.12 However, the study population only consisted of 7.2% cancer patients, and it only evaluated a single 150-mg dose each of tixagevimab and cilgavimab given as two consecutive intramuscular injections. The post-EUA experience marked by rapid evolution of Omicron subvariants with lower neutralizing activity raised concerns about decreased efficacy among immunocompromised patients. For example, an in vitro neutralization study by Boschi et al. demonstrated that the combination of AZD7442 was 233 times less active against B.1.1.529 than against the Delta variant.22 Stuver et al. found that AZD7442 failed to achieve meaningful neutralization of Omicron among 52 patients with hematologic malignancies who were treated with a single 150 mg dose each of tixagevimab and cilgavimab.23 Although the results were heterogenous, neutralization activity improved with either a second dose of 150 mg each of tixagevimab and cilgavimab or in those who received 300 mg each of tixagevimab and cilgavimab, supporting the revised FDA dosing regimen. There are likely multiple factors accounting for the vary-

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Table 4. Clinical characteristics and outcomes for 98 patients with breakthrough infection from SARS-CoV-2 after receiving AZD7442 for pre-exposure prophylaxis. Characteristic

N (%)a

Age in years, median (IQR)

65 (53-74)

Sex, male

61 (62.2)

Unvaccinated

6 (6.1)

Required hospitalization

8 (8.2)

Symptoms at hospitalization (N=6) Fever Dyspnea Cough Nasal congestion Poor oral intake Chest pain Fatigue

6 (100.0) 3 (50.0) 4 (66.7) 4 (66.7) 4 (66.7) 2 (33.3) 4 (66.7)

CT chest findings (N=2) Bilateral consolidative opacities Bilateral patchy ground-glass opacities Clear lungs

0 (0) 1 (50) 1 (50)

CXR findings (N=6) Unilateral opacities Bilateral opacities Clear lungs

1 (16.7) 1 (16.7) 4 (66.7)

Oxygen requirement (N=6) No oxygen requirement or <24 hours Nasal cannula >24 hours High flow oxygen Mechanical ventilation

3 (50.0) 2 (33.3) 1 (16.7) 0 (0)

COVID-19 severityb (N=6) Mild Moderate Severe/critical

3 (50.0) 2 (33.3) 1 (16.7)

COVID-19 treatment (N=98) Any COVID-19 treatment Nirmatrelvir-ritonavir Remdesivir Dexamethasone Bebtelovimab Sotrovimab

81 (82.6) 31 (31.6) 8 (8.2) 4 (4.1) 40 (40.8) 7 (7.1)

COVID-19 outcome (N=98) COVID-19-related deathc COVID-19-related readmission Chronic infectiond

1 (1.) 2 (2.0) 6 (6.1)

COVID-19: coronavirus disease 2019; CT: computed tomography; CXR: chest radiograph; IQR: interquartile range. aPatients can be in >1 category. bCOVID-19 severity based on maximum oxygen requirement through admission. Mild infections remain on room air or required nasal canula for <24 hours; moderate infections required nasal canula >24 hours; severe/critical infections required high flow oxygen or intubation. cCOVID-19-related deaths are based on review of electronic medical records and clinical judgement of patient presentation/illness in relation to patients’ primary disease. dChronic infection from COVID-19 is defined as patients who had progressive or recurrent COVID-19-related symptoms in the absence of an alternative explanation and with or without evidence of viral persistence.

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ARTICLE - SARS-CoV-2 breakthrough infection after AZD7442

J.C. Laracy et al. Figure 1. Days to breakthrough infection in dose groups 1, 2, and 4 by Omicron subvariant as predicted by date of positivity or whole genome sequencing, when available. (A) Dose group 1: single 150-150 mg dose of AZD7442. (B) Dose group 2: two 150150 mg doses of AZD7442. (C) Dose group 4: single 300-300 mg dose of AZD7442.

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ARTICLE - SARS-CoV-2 breakthrough infection after AZD7442 ing efficacy of AZD7442 observed across published studies. First, vaccination rates of AZD7442 recipients have varied significantly between studies. The critical PROVENT trial leading to the FDA’s EUA for AZD7442 was conducted in unvaccinated patients.12 However, postmarketing clinical studies such as those by Kertes et al. and Najjar-Debbiny et al., which demonstrated reduced efficacy of AZD7442 compared to PROVENT, had a majority of patients who received at least one dose of vaccine against SARS-CoV-2.24,25 A second major factor contributing to differences in AZD7442 efficacy between studies is the rapid evolution of SARS-CoV-2 variants with critical receptor binding domain mutations that reduce antibody binding. While PROVENT was conducted prior to the emergence of Omicron, subsequent studies have been conducted against different Omicron sublineages that have demonstrated wide variability in susceptibility to AZD7442.10 For example, in a large cohort study of 1,112 patients with heterogenous immunocompromising conditions, Nguyen et al. reported breakthrough infection in 4.4% of patients treated with 150-150 mg of AZD7442 during regional BA.1 and BA.2 predominance while Jondreville et al. observed that 22 of 161 (14%) of adult allogenic hematopoietic stem cell transplant recipients treated with 150-150 mg of AZD7442 developed breakthrough infection during the Omicron wave (sublineages not specified).26,27 The difference in infection rate between studies by Nguyen et al. and Jondreville et al. also highlights how differences in patient population may contribute to variability in AZD7442 efficacy. Another major factor contributing to differences in AZD7442 efficacy between studies is the variation in dosing regimens studied. In a study of 203 patients with hematologic malignancies, 97% of whom received the 300-300 mg dose of AZD7442 and nearly all of whom had received at least one dose of mRNA vaccine, Ocon et al. found that 19 (9.3%) patients developed breakthrough infection, a finding that is comparable to the breakthrough rate in our study.28 Multiple other studies have included patient cohorts treated with both 150-150 mg and/or 300-300 mg of AZD7442 with variability in observed efficacy of AZD7442 likely related to differences in patient population, vaccine history, dose, and circulating subvariants.29-33 There are several limitations to our study. First, and most importantly, the small sample sizes of SARS-CoV-2 breakthrough infections and missing laboratory parameters in each of the dose groups did not allow for robust analyses. Although we had a consistent mechanism for capturing self-administered rapid antigen tests, it is possible that we missed breakthrough infections and have underestimated the overall rate of mild infections. Second, the lack of a AZD7442 naïve comparator arm precludes more definitive discrimination of AZD7442 effectiveness. While our report is the most extensive co-

J.C. Laracy et al.

hort experience of high-risk hematologic malignancy patients, especially regarding measurement of dose-specific incidence of breakthrough infection, the findings do not extend to other immunocompromised patient populations. In contrast to published trials that were mostly conducted in those with elevated but non-cancer-related risk of SARS-CoV-2 infection, our study cohort overwhelmingly represents actively treated and vaccinated hematologic malignancy patients. Finally, the impact of AZD7442 alone on clinical outcomes (e.g., hospitalization, mortality) cannot be made at this time in the era of early effective anti-SARS-CoV-2 therapies. As newly circulating variants are non-susceptible to AZD7442, the results of our study cannot be generalized to the post-Omicron, mab-resistant era.34, 35 In summary, high-risk hematologic malignancy patients who received AZD7442 for pre-exposure prophylaxis against SARS-CoV-2 had a breakthrough infection rate of 11%, most infections were mild, with minimal risk of hospitalization and severe disease. In addition, our dose-specific analysis of breakthrough incidence rates shows that patients who received a second, or a higher initial dose of AZD7442, as per revised FDA guidance had better protection against breakthrough infection, providing the clinical evidence to support the FDA's dosing modification based on in vitro neutralization activity against circulating variants. Most importantly, our study's low incidence of severe outcomes underscores the swift and valuable therapeutic advancements that have been made for the prevention and early treatment of high-risk patients who cannot solely rely on vaccine-induced protection to reduce SARS CoV-2 related adverse outcomes. Disclosures EVR has received consulting fees from Replimune. NEB has received research grants from GenMark Diagnostics and ArcBio/Canta and is on the advisory board at Bio-Rad Molecular, Agena Diagnostics, and ArcBio/Canta. SKS has an investigator-initiated grant from Merck. MK has acted as a consultant for Regeneron and has received speaker fees for WebMD/Medscape. Contributions All authors critically reviewed and contributed to the writing of the manuscript, gave final approval of the version to be published, and agreed to be accountable for all aspects of the work. JL, JY, MK, and SKS were responsible for the conception and design of the study, interpretation of the data, and preparation of the manuscript. MK and SKS managed the study. JY, SNS, JL, JF, CAT, NC and SKS acquired the data. JY and NEB analyzed the data. JY performed statistical analysis. JY and JL produced the figures and tables. NEB analyzed laboratory samples; and all authors reviewed the final draft.

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ARTICLE - SARS-CoV-2 breakthrough infection after AZD7442 Acknowledgments The authors wish to thank all the healthcare workers within Memorial Sloan Kettering Cancer Center for their collective effort against the COVID-19 pandemic.

J.C. Laracy et al.

Data-sharing statement The authors are committed to the dissemination of data. However, the raw data are not available for sharing as no specific consent for this purpose was available.

References 1. Monin L, Laing AG, Muñoz-Ruiz M, et al. Safety and immunogenicity of one versus two doses of the COVID-19 vaccine BNT162b2 for patients with cancer: interim analysis of a prospective observational study. Lancet Oncol. 2021;22(6):765-778. 2. Fendler A, Shepherd STC, Au L, et al. Adaptive immunity and neutralizing antibodies against SARS-CoV-2 variants of concern following vaccination in patients with cancer: the CAPTURE study. Nat Cancer. 2021;2(12):1305-1320. 3. Lee LYW, Starkey T, Ionescu MC, et al. Vaccine effectiveness against COVID-19 breakthrough infections in patients with cancer (UKCCEP): a population-based test-negative casecontrol study. Lancet Oncol. 2022;23(6):748-757. 4. Wang W, Kaelber DC, Xu R, Berger NA. Breakthrough SARSCoV-2 infections, hospitalizations, and mortality in vaccinated patients with cancer in the US between December 2020 and November 2021. JAMA Oncol. 2022;8(7):1027-1034. 5. Yek C, Warner S, Wiltz JL, et al. Risk factors for severe COVID19 outcomes among persons aged ≥18 years who completed a primary COVID-19 vaccination series - 465 health care facilities, United States, December 2020–October 2021. MMWR Morb Mortal Wkly Rep. 2022;71(1):19-25. 6. Andrews N, Stowe J, Kirsebom F, et al. Covid-19 vaccine effectiveness against the omicron (B.1.1.529) variant. N Engl J Med 2022;386(16):1532-1546. 7. Higdon MM, Baidya A, Walter KK, et al. Duration of effectiveness of vaccination against COVID-19 caused by the omicron variant. Lancet Infect Dis. 2022;22(8):1114-1116. 8. Wang X, Ai J, Li X, et al. Neutralization of Omicron BA.4/BA.5 and BA.2.75 by booster vaccination or BA.2 breakthrough infection sera. Cell Discov. 2022 8:1. 2022;8(1):1-3. 9. Hachmann NP, Miller J, Collier A ris Y, et al. Neutralization escape by SARS-CoV-2 Omicron subvariants BA.2.12.1, BA.4, and BA.5. N Engl J Med. 2022;387(1):86-88. 10. COVID-19 Antibody Prevention | EVUSHELDTM (tixagevimab copackaged with cilgavimab). https://www.evusheld.com/en/patient Accessed November 28, 2022. 11. AstraZeneca Evusheld: The 200 best inventions of 2022 | TIME. https://time.com/collection/best-inventions2022/6229895/astrazeneca-evusheld/ Accessed November 28, 2022. 12. Levin MJ, Ustianowski A, Wit S de, et al. Intramuscular AZD7442 (tixagevimab–cilgavimab) for prevention of Covid-19. N Engl J Med. 2022;386(23):2188-2200. 13. Iketani S, Liu L, Guo Y, et al. Antibody evasion properties of SARS-CoV-2 omicron sublineages. Nature. 2022;604(7906):553-556. 14. Bruel T, Stéfic K, Nguyen Y, et al. Longitudinal analysis of serum neutralization of SARS-CoV-2 Omicron BA.2, BA.4, and BA.5 in patients receiving monoclonal antibodies. Cell Rep Med. 2022;3(12):100850. 15. FDA releases important information about risk of COVID-19 due to certain variants not neutralized by Evusheld | FDA. https://www.fda.gov/drugs/drug-safety-and-availability/fdareleases-important-information-about-risk-covid-19-due-certai n-variants-not-neutralized-evusheld Accessed January 10, 2023.

16. Babady NE, McMillen T, Jani K, et al. Performance of severe acute respiratory syndrome coronavirus 2 real-time RT-PCR tests on oral rinses and saliva samples. J Mol Diagn. 2021;23(1):3. 17. Tamari R, Politikos I, Knorr DA, et al. Predictors of humoral response to SARS-CoV-2 vaccination after hematopoietic cell transplantation and CAR AZD7442ell therapy. Blood Cancer Discov. 2021;2(6):577. 18. Chow K, Aslam A, McClure T, et al. Risk of healthcareassociated transmission of sever acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) in hospitalized cancer patients. Clin Infect Dis. 2022;74(9):1579-1585. 19. Adult immunization schedule by vaccine and age group | CDC. https://www.cdc.gov/vaccines/schedules/hcp/imz/adult.html Accessed January 10, 2023. 20. Mairhofer M, Kausche L, Kaltenbrunner S, et al. Humoral and cellular immune responses in SARS-CoV-2 mRNA-vaccinated patients with cancer. Cancer Cell. 2021;39(9):1171. 21. Liebers N, Speer C, Benning L, et al. Humoral and cellular responses after COVID-19 vaccination in antiCD20-treated lymphoma patients. Blood. 2022;139(1):142-147. 22. Boschi C, Colson P, Bancod A, Moal V, la Scola B. Omicron variant escapes therapeutic monoclonal antibodies (mAbs) including recently released Evusheld®, contrary to 8 prior main variant of concern (VOC). Clin Infect Dis. 2022;75(1):e534-e535. 23. Stuver R, Shah GL, Korde NS, et al. Activity of AZD7442 (tixagevimab-cilgavimab) against Omicron SARS-CoV-2 in patients with hematologic malignancies. Cancer Cell. 2022;40(6):590. 24. Najjar-Debbiny R, Gronich N, Weber G, Stein N, Saliba W. Effectiveness of Evusheld in immunocompromised patients: propensity score-matched analysis. Clin Infect Dis. 2023;76(6):1067-1073. 25. Kertes J, Shapiro S, David B, et al. Association between AZD7442 (tixagevimab-cilgavimab) administration and severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) infection, hospitalization, and mortality. Clin Infect Dis. 2023;76(3):e126-e132. 26. Nguyen Y, Flahault A, Chavarot N, et al. Pre-exposure prophylaxis with tixagevimab and cilgavimab (Evusheld) for COVID-19 among 1112 severely immunocompromised patients. Clin Microbiol Infect. 2022;28(12):1654. 27. Jondreville L, D’Aveni M, Labussière-Wallet H, et al. Preexposure prophylaxis with tixagevimab/cilgavimab (AZD7442) prevents severe SARS-CoV-2 infection in recipients of allogeneic hematopoietic stem cell transplantation during the Omicron wave: a multicentric retrospective study of SFGM-TC. J Hematol Oncol. 2022;15(1):169. 28. Ocon AJ, Ocon KE, Battaglia J, et al. Real-world effectiveness of tixagevimab and cilgavimab (Evusheld) in patients with hematological malignancies. J Hematol. 2022;11(6):210. 29. Calabrese C, Kirchner E, Villa-Forte A, et al. Early experience with tixagevimab/cilgavimab pre-exposure prophylaxis in

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ARTICLE - SARS-CoV-2 breakthrough infection after AZD7442 patients with immune-mediated inflammatory disease undergoing B cell depleting therapy and those with inborn errors of humoral immunity. RMD Open. 2022;8(2):e002557. 30. Bruel T, Hadjadj J, Maes P, et al. Serum neutralization of SARS-CoV-2 Omicron sublineages BA.1 and BA.2 in patients receiving monoclonal antibodies. Nat Med. 2022;28(6):1297-1302. 31. Aqeel F, Geetha D. Tixagevimab and Cilgavimab (Evusheld) in rituximab-treated antineutrophil cytoplasmic antibody vasculitis patients. Kidney Int Rep. 2022;7(11):2537-2538. 32. Al-Obaidi MM, Gungor AB, Kurtin SE, Mathias AE, Tanriover B, Zangeneh TT. The Prevention of COVID-19 in high-risk patients using tixagevimab-cilgavimab (Evusheld): real-world

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experience at a large academic center. Am J Med. 2023;136(1):96-99. 33. al Jurdi A, Morena L, Cote M, Bethea E, Azzi J, Riella LV. Tixagevimab/cilgavimab pre-exposure prophylaxis is associated with lower breakthrough infection risk in vaccinated solid organ transplant recipients during the omicron wave. Am J Transplant. 2022;22(12):3130-3136. 34. Imai M, Ito M, Kiso M, et al. Efficacy of antiviral agents against Omicron subvariants BQ.1.1 and XBB. N Engl J Med. 2023;388(1):89-91. 35. Wang Q, Iketani S, Li Z, et al. Alarming antibody evasion properties of rising SARS-CoV-2 BQ and XBB subvariants. Cell. 2023;186(2):279-286.

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ARTICLE - Red Cell Biology & its Disorders

Characterization of genetic variants in the EGLN1/PHD2 gene identified in a European collection of patients with erythrocytosis Marine Delamare,1,2* Amandine Le Roy,1,2* Mathilde Pacault,2,3* Loïc Schmitt,1,2* Céline Garrec,3 Nada Maaziz,4 Matti Myllykoski,5 Antoine Rimbert,2 Valéna Karaghiannis,1,2 Bernard Aral,4 Mark

Correspondence: B. Gardie

Catherwood,6 Fabrice Airaud,3 Lamisse Mansour-Hendili,7,8 David Hoogewijs,9,10 Edoardo Peroni,11,12

betty.gardie@inserm.fr

Salam Idriss, Valentine Lesieur, Amandine Caillaud, Karim Si-Tayeb, Caroline Chariau, Anne 13

14,15

Gaignerie, Minke Rab, 17

Torsten Haferlach, Manja Meggendorfer, Stéphane Bézieau, 18

19†

Benetti, Nicole Casadevall, Pierre Hirsch, Christian Rose, 7,21

Galacteros,

2,3

Andrea

Mathieu Wemeau, Frédéric

Bruno Cassinat, Beatriz Bellosillo, Celeste Bento,24 Richard van Wijk,14,15 Petro E.

6,26

Petrides, Maria Luigia Randi, Mary Frances McMullin, 4,27,28#

François Girodon,

‡

Peppi Koivunen, ECYT-3 consortium,

1,2,28#

and Betty Gardie

Ecole Pratique des Hautes Etudes, EPHE, PSL University, Paris, France; 2Université de Nantes,

CNRS, INSERM, l’Institut du Thorax, Nantes, France; 3Service de Génétique Médicale, CHU de Nantes, Nantes, France; 4Service d’Hématologie Biologique, Pôle Biologie, CHU de Dijon, Dijon, France; 5Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, Oulu Center for Cell-Matrix Research, University of Oulu, Oulu, Finland; 6Belfast Health and Social Care Trust, Belfast, North Ireland; 7Département de Biochimie-Biologie Moléculaire, Pharmacologie, Génétique Médicale, AP-HP, Hôpitaux Universitaires Henri Mondor, Créteil, France; 8Université Paris-Est Créteil, IMRB Equipe Pirenne, Laboratoire d’Excellence LABEX GRex, Créteil, France; 9

Section of Medicine, Department of Endocrinology, Metabolism and Cardiovascular System,

University of Fribourg, Fribourg, Switzerland; 10National Center of Competence in Research “Kidney.CH”, Zurich, Switzerland; 11Immunology and Molecular Oncology Unit, Veneto Institute of Oncology, IOV-IRCCS, Padova, Italy; 12Medical Genetics Unit, Mater Domini University Hospital, Catanzaro, Italy; 13Nantes Université, CHU Nantes, CNRS, Inserm, BioCore, Nantes, France; 14Central Diagnostic Laboratory - Research, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands; 15Department of Hematology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands; 16Munich Leukemia Laboratory, Munich, Germany; 17Department of Medicine-DIMED, University of Padua, Padua, Italy; 18Sorbonne Université, INSERM, Centre de Recherche Saint-Antoine, CRSA, AP-HP, SIRIC CURAMUS, Hôpital Saint-Antoine, Service d’Hématologie Biologique, Paris, France; 19Service d’Onco-Hématologie, Saint-Vincent de Paul Hospital, Université Catholique de Lille, Université Nord de France, Lille, France; 20Hematology Department, Claude Huriez Hospital, Lille Hospital, Lille, France; 21Red Cell Disease Referral Center-UMGGR, AP-HP, Hôpitaux Universitaires Henri Mondor, Créteil, France; 22Université Paris Cité, APHP, Hôpital Saint-Louis, Laboratoire de Biologie Cellulaire, Paris, France; 23Pathology Department, Hospital del Mar-IMIM, Barcelona, Spain; 24Hematology Department, Centro Hospitalar e Universitário de Coimbra; CIAS, University of Coimbra, Coimbra, Portugal; 25Hematology Oncology Center and Ludwig-Maximilians-University Munich Medical School, Munich, Germany; 26Queen’s University, Belfast, North Ireland; 27Inserm U1231, Université de Bourgogne, Dijon, France and 28Laboratoire d’Excellence GR-Ex, Paris, France. *

MD, ALR, MP and LS contributed equally as first authors.

FGi and BG contributed equally as senior authors.

†

deceased

‡

An appendix with all ECYT-3 consortium members can be found at the end of the manuscript.

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Received: Accepted: Early view:

February 10, 2023. June 6, 2023. June 15, 2023.

ARTICLE - Revisiting the classification of EGLN1 variants

M. Delamare et al.

Abstract Hereditary erythrocytosis is a rare hematologic disorder characterized by an excess of red blood cell production. Here we describe a European collaborative study involving a collection of 2,160 patients with erythrocytosis sequenced in ten different laboratories. We focused our study on the EGLN1 gene and identified 39 germline missense variants including one gene deletion in 47 probands. EGLN1 encodes the PHD2 prolyl 4-hydroxylase, a major inhibitor of hypoxia-inducible factor. We performed a comprehensive study to evaluate the causal role of the identified PHD2 variants: (i) in silico studies of localization, conservation, and deleterious effects; (ii) analysis of hematologic parameters of carriers identified in the UK Biobank; (iii) functional studies of the protein activity and stability; and (iv) a comprehensive study of PHD2 splicing. Altogether, these studies allowed the classification of 16 pathogenic or likely pathogenic mutants in a total of 48 patients and relatives. The in silico studies extended to the variants described in the literature showed that a minority of PHD2 variants can be classified as pathogenic (36/96), without any differences from the variants of unknown significance regarding the severity of the developed disease (hematologic parameters and complications). Here, we demonstrated the great value of federating laboratories working on such rare disorders in order to implement the criteria required for genetic classification, a strategy that should be extended to all hereditary hematologic diseases.

Introduction Red blood cell production is tightly regulated by the oxygen-sensing pathway, which controls the expression of erythropoietin (EPO), a glycoprotein hormone that stimulates the survival, proliferation, and differentiation of erythroid progenitors. The main enzymes that are capable of sensing oxygen are the dioxygenases whose enzymatic activity controls the hydroxylation of target proteins, utilizing oxygen and 2-oxoglutarate as co-substrates. This study focuses on the EGLN1 (Egl nine homolog 1) gene that encodes the dioxygenase prolyl hydroxylase domain-containing protein 2 (PHD2) (also called HIF prolyl 4-hydroxylase-2, HIF-P4H-2).1-3 In the presence of oxygen, PHD2 hydroxylates its main substrate, the α subunit of the hypoxia-inducible factor (HIF), which is a heterodimeric transcription factor (α/β) that plays a central role in oxygen homeostasis. The hydroxylated HIF-α (at proline residues 402 and 564 for HIF-1α and 405 and 531 for HIF-2α) then binds to von Hippel-Lindau (VHL) tumor suppressor protein, which directs HIF-α for proteasomal degradation through VHL E3 ubiquitin ligase activity. Thus, the orchestrated action of PHD and VHL proteins drives HIF degradation in the presence of oxygen. When oxygen concentration decreases, PHD enzymatic activity is diminished, which causes the stabilization of HIF-α. As a result, HIF-α accumulates in the nucleus, forms an active heterodimer complex with HIF-1β that binds hypoxia-responsive elements (HRE) and induces target gene expression. HIF regulates the transcription of more than 200 genes involved in many pathways,4 such as erythropoiesis (via the synthesis of EPO), iron regulation, angiogenesis, metabolism, cell proliferation and survival. EGLN1 is a HIF target gene and its expression contributes to a negative feedback mechanism that limits HIF-1 responses during reoxygenation.5-7 There are three isoforms of PHD (PHD1-3), with PHD2 showing the highest oxygen-dependent activity.8

Germline loss-of-function mutations in the EGLN1/PHD2 gene were first described in 2006 in a patient with hereditary erythrocytosis.9 Erythrocytoses are characterized by an elevated red cell mass reflected by increased hemoglobin and hematocrit10 levels. Primary hereditary erythrocytosis occurs when the mutations target the intrinsic mechanism of erythroid progenitors with overproliferation (i.e., EPOR), and a compensatory lowered EPO serum level. Secondary hereditary erythrocytosis occurs when a high level of EPO is produced, thus indirectly driving red blood cell overproduction. In this case, the circulating EPO levels are inappropriately normal or elevated. Patients with PHD2 mutations develop secondary hereditary erythrocytosis with high to normal EPO serum levels, a normal concentration being inappropriate in view of the high hematocrit. In contrast to patients with the Chuvash VHL-R200W mutation, erythroid progenitors do not exhibit hypersensitivity to EPO (a feature of primary erythrocytosis). However, mice targeted for Phd2 inactivation in hematopoietic precursors showed hypersensitivity to EPO.11 As discussed below, the impact of PHD2 mutations on erythroid progenitors is still under debate. Interestingly, genetic variants in the PHD2 and HIF2A genes have been associated with the adaptation of Tibetans to high altitude.12,13 The mechanisms involved appear to be complex,14,15 but studies have shown that particular variants of PHD2 (c.12C>G, p.Asp4Glu and c.380G>C, Cys127Ser in cis) result in a gain of function of the protein.15 Since the first described case, 64 different PHD2 genetic variants (in 72 families) have been described, including missense, frameshift and nonsense mutations (Online Supplementary Table S1, p.Asp4Glu and p.Cys127Ser not included).16-18 Patients with erythrocytosis due to a mutation in the EGLN1 gene are all heterozygous, with the exception of two siblings described as homozygous for p.Cys42Arg.19 The phenotypes of patients carrying these mutations are usually limited to erythrocytosis, but complications such as

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ARTICLE - Revisiting the classification of EGLN1 variants

M. Delamare et al.

thrombosis, hypertension, renal cysts, angiomas, and rarely paraganglioma20,21 or pheochromocytoma22 may arise.16,17 In vitro functional studies performed on PHD2 variants have not been able to explain the differences in clinical presentation.9,19-21,23-27 In particular, studies demonstrated that some PHD2 mutations induce a subtle loss of function, sometimes very close to the wild-type protein.26 This article describes a collaborative study of 34 novel genetic variants (in addition to 5 previously described16,19,28-33) identified in the EGLN1 gene using next-generation sequencing panels. The study includes data from sequencing of a total of 2,160 patients with hereditary or idiopathic erythrocytosis recruited in seven European countries. Forty-seven cases carrying genetic variants in PHD2 were identified. We performed comprehensive in silico and functional studies to decipher their potential causal role in the disease pathogenesis.

In vitro enzyme activity assays Flag-tagged PHD2 was expressed in insect cells and affinity-purified using anti-Flag, and the His-tagged HIF-2α oxygen-dependent degradation domain (ODDD) protein was expressed in E. coli and affinity-purified using NiNTA as described previously.36,33 A PHD2 enzymatic activity assay was performed to measure the radioactive CO2 produced during the decarboxylation of 2-oxo[1-14C]glutarate (Perkin-Elmer), which co-occurs with the substrate proline hydroxylation.37

Methods Sequencing All study participants signed written informed consent. Blood samples were collected for research purposes after receiving approval from the different local ethics committees. DNA was extracted and molecular screening was performed by next-generation sequencing with different technologies, depending on the sequencing center. In silico analyses The MobiDetails annotation platform34 was used for the interpretation of DNA variations (frequencies in the control population, prediction of the impact of missense variants and analysis of splicing with the Splicing Prediction Pipeline (SPiP)) and for the localization of the mutated amino acids on the three-dimensional protein structure (AlphaFold Protein Structure Database). The localization of the affected amino acids on the two-dimensional structure was analyzed using the MetaDome website.35 The UK Biobank resource was used to analyze the hematologic parameters of the carriers of EGLN1 genetic variants. Luciferase reporter assay End-point Luciferase assays were performed as previously described.20,26 Real-time Luciferase assays were performed on HEK 293T cells transfected with jetPRIME® (Ozyme Polyplus). Expression vectors pcDNA3-HA-PHD2 were co-transfected with pcDNA3-HA-HIF-2α, pGL3 3xHRE-luciferase reporter plasmid and pCMV-HA-empty vector. Luciferase activity was measured over 24 h using the bioluminometer WSL-1565 Kronos HT® (ATTO). Cells were harvested at different timepoints and lysed in passive lysis buffer (Invitrogen) for immunoblot detection with a mouse anti-HA antibody (clone 16B12, BioLegend).

Cycloheximide chase assay HEK cells were transfected with plasmids encoding pcDNA3-HA-PHD2 (100-800 ng) in addition to pCMV-HA-empty vector for a total amount of 800 ng of transfected DNA. Twenty-four hours after transfection, cells were treated with cycloheximide (Sigma-Aldrich) at a final concentration of 100 μg/mL. Cells were harvested at different timepoints. An equal volume of protein lysates was analyzed by western blot assay using a mouse anti-HA antibody, anti-actin (Sigma) and a goat anti-mouse secondary antibody (Jackson Immunoresearch). Splicing reporter assay Minigene constructs were prepared as described by Cooper and Gaildrat.38 Cells were transfected with 2 μg pCas2 plasmid containing the exon of interest and flanking intronic sequences surrounded by artificial exons (named A and B). Total RNA was extracted 24 h after transfection. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed with primers located in exons A and B. PCR products were resolved on a 2% agarose gel or quantified by using the Agilent TapeStation® system. Generation of human induced pluripotent stem cells and differentiation into hepatocyte-like cells Human induced pluripotent stem cells (hiPSC) were generated from peripheral blood mononuclear cells and characterized in the hiPSC Core Facility of Nantes University. hiPSC from passage numbers 21 to 25 were differentiated into hepatocyte-like cells, as described previously.39,40 After 22 days of differentiation, cells were cultured for 24 h in normoxia or at 1% O2 in an Invivo 400 Hypoxia Workstation (Baker Ruskinn).

Results Sequencing of patients with erythrocytosis Patients with erythrocytosis were recruited according to the following criteria: red cell mass >125% and/or elevated hematocrit (>52% in men, >47% in women) and hemoglobin (>18 g/dL in men and >16 g/dL in women) levels. Cases of polycythemia vera and secondary erythrocytosis, particularly related to cardiac or pulmonary insufficien-

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ARTICLE - Revisiting the classification of EGLN1 variants

M. Delamare et al.

cy, had been previously excluded. A flow chart detailing the steps that led to the molecular screening performed by high-throughput sequencing is presented in Figure 1. Next-generation sequencing enabled the investigation of large rearrangements (deletions and duplications) together with point mutations. Samples from a total of 2,160 patients

were sequenced in seven countries (10 diagnostic centers listed in Table 1). We selected variants with a gnomAD frequency ≤5x10-4. In total, we identified 39 genetic variants including one complete deletion in 47 families (Table 1). We identified, for the first time, a deletion of one copy of the entire EGLN1 gene by next-generation sequencing. The

Figure 1. Diagnostic flow chart for patients presenting with erythrocytosis. EPO: erythropoietin; EPOR: erythropoietin receptor, CO: carbon monoxide; Hb: hemoglobin; P50: partial pressure; O2: oxygen; MCHC: mean corpuscular hemoglobin concentration; RCM: red cell mass; NGS: next-generation sequencing.

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P #15

P#16

P #17

P #18

P #19

P #20

P #21

P #22

P #23

P #24

P #25

P #26

P #27

P #12

P #11

P #14

P #10

P #9

P #13

P #8

p.Gln134*

c.400C>T

P #7

p.Ala96Val

c.287C>T

P #6

p.Pro77Leu

c.230C>T

P #5

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L271fs

p.Ile269Thr p.Ile269Thr p.Ile269Thr p.Ile269Thr p.Ile269Thr

p.Leu271Argfs*15 p.Thr296Met

c.806T>C

c.808_811dup

c.887C>T

c.908A>G

c.891+1G>A

I269T

p.Thr268Ile

c.803C>T

NA Y303C

NA p.Tyr303Cys

T296M

I269T

T268I

G265V

p.Gly265Val

c.794_795delinsTT

K255N

p.Lys255Glu

Q239*

G233A

I222S

Q221*

V210Del

A190L

Y163*

Q157R

c.763A>G

p.Gln239*

c.715C>T

p.Gln221*

c.661C>T

p.Gly233Ala

p.Val210del

c.629_631delTGG

c.698G>C

p.Ala190Leu

c.568_569delinsTT

p.Ile222Ser

p.Tyr163*

c.489C>A

c.665T>G

p.Gln157Arg

c.470A>G

50/39

Q134*

A96V

P77L

D50H

p.Asp50His

c.148G>C

P #4

Y41C

p.Tyr41Cys

c.122A>G

P #3

R35H

c.104G>A

55.4

51.8

19.8

18.8

18.6 53.8

16.8 48.5

17.1 51.5

18.6

18.3 55.6

17.1 52.6

20.4 61.5

15.4 46.6

16.9

17.4

16.7

20.5

20.2 57.9

18.4

22.6 65.6

19.9 54.8

19.2

17.1 51.2

Age/ Hb Hct age at Dx Sex g/dL % in yrs

p.Arg35His

P #2

Letter code 73

c.-410G>T

5’UTR

P #1

Position protein NA

Position cDNA

Exon

7.1

5.58

6.48

5.87

6.11

6.23

5.7

5.09

6.83

5.07

6.1

6.97

6.17

5.71

6.05

11.9

5.8

17.9

3.5

6.6

10.4

9.2

3.2

6.03

6.3

RBC EPO Family 106/mm3 mIU/mL history

Table 1. List of genetic variants identified in the EGLN1 gene in patients with erythrocytosis.

None

Asthenia, HFE

None

Phleb/Asp

Phleb

Asp

Psychomotor retardation

Exertional dyspnea

PTE

Phleb/ Asp

MI, heavy smoker

Phleb

None

Asp

Ear pain, ulcerative colitis -

Asp

Portal vein thrombosis -

Asp

IFN-α

1, 5

Dx center

18, 20

10ψ

2, 4

Ref

Continued on following page.

VUS

Treatment Classification

Headaches

Hypereosinophilia

Other symptoms

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P #34

P #35

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1-5

P #40

P #41

P #42

P #43

P #44

P #45

P #46

P #47

p.Pro358Thr p.Phe366Leu p.Arg370Gly p.Gln377* p.Tyr384Tyr p.Trp389Arg p.Trp389Arg NA p.Asp419Glu p.Ser420Leu NA

c.1072C>A

c.1096T>C

c.1108C>G

c.1129C>T

c.1152C>T

c.1165T>C

c.1216+1G>T

c.1257T>G

c.1259C>T

Deletion of the entire gene

p.Gly349Cys

c.1045G>T p.Gly349Ser

p.Trp334Arg

c.1000T>C

c.1045G>A

p. Asn331*

p.Tyr329His

c.985T>C

c.990dup

p.Arg312His

Position protein

c.935G>A

Position cDNA

49 62

R312H R312H

S420L

D419E

W3389R

Y384Y

Q377*

R370G

F366L

P358T

G349S

G349C

W334R

N331*

50/34

R312H

Y329H

M 47

53.3

49.6

15.6 47.4

12.9

18.1

13.4 42.8

19.6 53.8

18.8 55.8

18.9 53.4

16.9 50.1

17.6 48-58

17.6 49.9

20.7

16.8 49.7

20.1 61.5

15.6

18.5

Age/ Hb Hct age at Dx Sex g/dL % in yrs

R312H

Letter code

5.33

5.91

6.08

5.87

6.66

5.87

6.22

5.75

6.54

5.85

5.95

9.7

24.1

2.4

8.07

16.5

Normal

6.2

45.5

RBC EPO Family 106/mm3 mIU/mL history

Thrombocytopenia

Asp

Phleb

Hydroxyurea

Phleb/Asp

TIA, myalgia, arthralgia -

Phleb

None

Phleb/ Asp

Phlebitis, RVO -

Asp

Phleb

VUS

Treatment Classification

None

CVA, OSAS

None

Other symptoms

2, 5

2, 1

Dx center

9ψ

Ref

ID: identification; Dx: diagnosis; Hb: hemoglobin (normal values are 13-18 g/dL for men and 12-16 g/dL for women); Hct: hematocrit (normal values are 40-52% for men and 37-47% for women); RBC: red blood cells (normal values are 4.2-5.7x106/mm3 for men and 4.2-5.2x106/mm3 for women); EPO: erythropoietin (normal values are 5-25 mIU/mL). It is not excluded that the hematologic values were measured after phlebotomies. Family history (+: erythrocytosis diagnosed in the family with the number of additional members indicated; No: no family history; Ad: adopted); Ref: references (detailed references are listed in the Online Supplementary Data); P #: patient number; UTR: untranslated region; NA: not applicable, -: data not available; M: male; F: female; *: stop codon, Fs: frameshift; MI: myocardial infarction; PTE: post-renal transplant erythrocytosis; HFE: hemochromatosis; CVA: cerebral vascular accident; OSAS: obstructive sleep apnea syndrome; RVO: retinal vein occlusion; TIA: transient ischemic attack; IFN-α: interferon-alpha; Asp: aspirin; Phleb: phlebotomies; VUS: variant of unknown significance; LB: likely benign; LP: likely pathogenic; P: pathogenic; Diagnosis Center: 1: Nantes (France), 2: Dijon (France), 3: Munich (Germany), 4: Belfast (UK), 5: Créteil (France), 6: Utrecht (Netherland), 7: Coimbra (Portugal), 8: Padova (Italy), 9: Barcelona (Spain), 10: Paris (France). ψ: same proband as already published but with updated information in the present paper.

P #39

P #33

P #38

P #32

P #31

P #37

P #30

P #29

P #36

Exon

P #28

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result was confirmed by quantitative PCR on DNA from additional biological samples. Remarkably, the patient with this deletion (P #47) did not present a more severe phenotype than that of patients carrying missense mutations. Of note, as usually described in hereditary erythrocytoses, the majority of probands were men (72.3%; 34 men vs. 13 women). Segregation studies of the identified variants were possible in a limited number of cases and allowed the identification of 34 additional carriers. Examples of pedigrees are shown in Figure 2A. One pedigree shows a large family with a history of erythrocytosis over three generations. The variant c.1000T>C, p.Trp334Arg was identified in proband III.216 and detected in four additional relatives. The other pedigree shows the family of patient P#42 carrying

the variant c.1165T>C, p.Trp389Arg, with segregation of the variant in family members with erythrocytosis, except for patient III.4, who was described as an asymptomatic carrier. After investigation, we found that this patient was a very assiduous blood donor, suggesting an incidental regulation of his hematologic parameters. In silico analyses of the variants First, we performed an in silico analysis of the variants identified to localize the missense variants in the protein structure. For this purpose, we first used the MetaDome server,35 which allows visualization of gene‐wide profiles of genetic tolerance on the two-dimensional protein structure. We localized the PHD2 variants described in the literature

Figure 2. Pedigrees of families carrying genetic variants in EGLN1 and localization of the mutated amino acids on the PHD2 protein. (A) Pedigrees of two families carrying genetic variants in EGLN1. (Left) The pedigree of patient #34 carrying the variant c.1000T>C, p.Trp334Arg. (Right) The pedigree of patient #42 carrying the variant c.1165T>C, p. Trp389Arg. +: carrier of the genetic variant. The arrow indicates the proband. (B) Localization of the targeted amino acids on the three-dimensional structural prediction of PHD2 obtained from the AlphaFold Protein Structure Database via the MobiDetails website. The regions of the structure modeled with high, medium, low, or very low confidence are colored blue, light blue, yellow, and orange, respectively. The prediction indicates that the relative positions of the two folded domains are not reliably modeled.

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(Online Supplementary Figure S1A, left panel) and the missense variants described in this study (right panel) on the Protein’s Tolerance Landscape. The variants were distributed throughout the protein, with no particular hotspot of mutations. The majority of the amino acids targeted by the genetic variations in the present study are expected to have a moderate impact (orange on the scale). Only three are expected to be highly intolerant (red on the scale): D50H, V210del, and Y329H (scores are detailed in Online Supplementary Table S3). The targeted amino acids on the three-dimensional structure were localized using the AlphaFold Protein Structure Database through the MobiDetails website (Figure 2B). PHD2 has two main domains: a N-terminal MYND-type zinc finger domain and a C-terminal prolyl hydroxylase domain with the double-stranded β-helix core fold supported by surrounding α-helices (in blue in Figure 2B). Most of the amino acids mutated in patients are located within the “core” domain, whereas R35, Y41 and D50 are located within the N-terminal zinc finger domain and P77L, A96, Q157, D419 and S420 are located outside the main domains. The following in silico analyses showed that the four variants located outside the structured domains are not pathogenic. Careful analysis of the N-terminal sequence of PHD2 revealed that the amino acids affected by the variations are located near to cysteines residues that play a major role in the function of the MYND-type zinc finger domain (Online Supplementary Figure S1B). In silico analysis was performed for the variant c.-410G>T, which is located upstream of the coding sequence. This variant is positioned precisely in the HRE-HIF binding consensus sequence responsible for the previously described regulation of PHD2 expression in hypoxia.6 Indeed, the variant targets the first G of the core HIF binding consensus sequence (ACGTG) and consequently may disrupt the regulation of PHD2 in hypoxia (Online Supplementary Figure S1C). The conservation of the PHD2 targeted amino acids was studied throughout a broad range of taxonomic classes covering primates, mammals, lower vertebrates as well as invertebrates (Online Supplementary Figure S2). A majority of them (63%, 17/27) are highly conserved through species and PHD isoforms (highlighted in black in the figure). We examined the likely functional impact of PHD2 missense variations using MobiDetails,34 a dedicated annotation platform for interpreting DNA variations. We used Radar graphs to represent the in silico prediction by the main software tools (Online Supplementary Figure S3). For each variant, the scores of individual and meta-predictors were plotted on a graph to allow their classification into three categories: probably damaging, possibly damaging/ tolerated, or benign (Figure 3A). These analyses showed that all identified variants had elevated scores, with the exception of variants P77L, A96V, Q157R, K255E and D419H (which are mainly located outside the main domains in the three-dimensional structure).

We analyzed the UK Biobank database, a large-scale biomedical database containing in-depth genetic and health information from half a million participants from the United Kingdom. We selected the PHD2 variants identified in the literature and this study (Figure 3B) and/or other variants associated with elevated hematocrit and hemoglobin levels (Online Supplementary Figure S4). We focused on men because, for this pathology, it may be difficult to interpret hematocrit and hemoglobin levels in women because of their menstrual cycles. For some variants (A96V, C127S, Q157H and Q157R, in green, Figure 3B), a significant number of carriers had hematocrit and hemoglobin levels distributed equally and comparably to those of wild-type individuals, strengthening the arguments in favor of a benign effect of the variants. Of note, we found two men carrying the Q221* and I269T variants (in red, Figure 3B) with hematocrit and hemoglobin values above the 99th percentile, a finding in favor of a deleterious effect of these variants. Some variants (R371H and T296M, light red) are associated with hematocrit and hemoglobin levels >90th to 99th percentile but their interpretation remains reserved. Additional variants have been associated with these elevated hematocrit and hemoglobin levels (Online Supplementary Figure S4), a list that could be very informative when identifying future variants. Functional studies of PHD2 variants using luciferase reporter tests The effect of the genetic variants on PHD2 activity was assessed using a reporter assay for HIF transcriptional activity. The HIF-2α subunit was co-expressed with a luciferase gene driven by HRE (Figure 4A). The activity of accumulated luciferase was quantified 24 h after transfection. The addition of wild-type PHD2 causes a dose-dependent suppression of HIF2-mediated induction of the reporter gene. When immunoblotted to monitor expression of the PHD2 protein, W334R and W389R proteins accumulated at levels below that of the wild-type protein for equivalent amounts of transfected plasmid (data not shown). To account for this effect, the amounts of transfected plasmid were adjusted to yield equivalent amounts of protein. We observed an inhibitory activity comparable to the wild-type for W334R, G349S and G349C variants, as well as for the previously published P200Q and R371H26 variants. Indeed, only the activity of the W389R mutant was impaired (Figure 4A). To test the hypothesis of a potential effect on the kinetics of HIF-2 inhibition by the different PHD2 variants, we performed a real-time luciferase reporter assay using the Kronos system, which measures the activity of expressed luciferase instantaneously. We detected a severe impairment for the control mutants D254H and P317R, as described elsewhere,9,26 and intermediate activity for the Y41C variant. All other PHD2 variants had a similar inhibitory effect to that of the wild-type protein (Figure 4B). Immunoblotting quantification showed that protein expression was reduced for two variants (I269T

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Figure 3. Scores from single and meta-predictors of studied variants and analysis of the UK Biobank data. (A) Representation of scores obtained by the in silico predictors analyzed by the MobiDetails annotation platform. Values are normalized (0-1), 0 being the least damaging and 1 the most for each predictor. The graph shows mean normalized scores obtained by single predictors (SIFT, Polyphen 2 HumDiv and HumVar) and meta-predictors (Fathmm, REVEL, ClinPred, Meta SVM, Meta LR, Mistic). For each variant, we analyzed the scores obtained with single and meta-predictors and classified the variants as benign when both scores were <0.4, and as deleterious when both scores were >0.6. (B) Analysis of hemoglobin and hematocrit levels in male carriers of PHD2 variants identified in the UK Biobank. The colored bars represent cases. The red dotted lines represent the 90th and 99th percentile values of the control population. *Variants present in the UK Biobank also identified in the present study. n: number of cases.

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and F366L), so the quantity of transfected plasmid was adjusted (Figure 4B and data not shown).

G349S, G349C, and R371H variants, expressed in insect cells and affinity-purified (Online Supplementary Figure S5), were determined by an enzymatic assay to detect the substrate hydroxylation-coupled decarboxylation of 2-oxglutarate compared with that of the wild-type PHD2. The results

Study of enzymatic activity The catalytic activity and kinetic properties of W334R,

Figure 4. Functional study of PHD2 mutants using luciferase reporter assays. (A) Functional study of PHD2 mutants using endpoint luciferase reporter assays. Cells were co-transfected with various amounts of PHD2 expression vectors (to enable the expression of the same amount of PHD2 proteins) in addition to HIF-2α expression vector, firefly luciferase reporter plasmid driven by hypoxia responsive elements and Renilla luciferase plasmid as a control of transfection efficiency. Luciferase activity was measured 24 h after transfection. Results are given as percentage of firefly luciferase activity normalized to Renilla luciferase activity. The amount of HA-PHD2 transfected (PHD2) was quantified by immunoblotting using anti-HA antibody. Results are mean values of experiments performed in triplicate. (B) Functional study of PHD2 mutants using real time luciferase reporter assays. Cells were co-transfected with various amounts of PHD2 expression vectors in addition to HIF-2α expression vector and firefly luciferase reporter plasmids driven by hypoxia-responsive elements. Cells were incubated for 24 h in the bioluminometer Kronos HT® (ATTO) and luciferase activity was measured during 10 sec every 30 min. Results are given in relative light units (counts/10 sec). The amount of HA-PHD2 transfected was quantified by immunoblotting using an anti-HA antibody. Results are mean values of experiments performed in triplicate. FL: firefly luciferase; RL: Renilla luciferase; R.L.U.: relative light unit; IB PHD2: immunoblot of transfected HA-PHD2 by using anti-HA antibody; WT: wild-type; ∅: cells transfected with an empty pcDNA3 vector.

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showed little difference between the variants and wildtype PHD2 through the Km values for 2-oxoglutarate or two peptide substrates; a short synthetic HIF-1α peptide or the recombinant HIF-2α ODDD (Table 2). The G349S and G349C variants had higher Km values for 2-oxo-glutarate compared with the wild-type protein, but these were probably compensated by their higher Vmax values (Table 2). The Vmax values of the R371H and W334R variants using either substrate were lower compared with those of the wild-type protein, but this could be, at least partially, compensated by their higher affinity (lower Km values). However, a higher Vmax value of the G349S variant was observed, especially with the HIF-1α substrate (Table 2).

We noticed decreased expression 10 h after cycloheximide treatment for the variants I269T, W334R, R371H, and W389R. Quantification of replicates confirmed the significant loss of stability of these variants (Figure 5; Online Supplementary Figure S6B).

Study of protein stability by a cycloheximide chase assay The stability of PHD2 variants was assessed by measuring the kinetics of PHD2 expression after treatment with cycloheximide, an inhibitor of protein biosynthesis due to its suppressive effect on translational elongation. Plasmids expressing the HA-PHD2 mutants were transfected 24 h before cycloheximide chase and expression was measured at different timepoints (Online Supplementary Figure S6A).

Study of the impact of variants on splicing using a minigene assay Since some of the genetic variants studied do not seem to have a major impact on the function or the stability of PHD2 proteins, we attempted to test their impact on splicing. The potential effects of some PHD2 variants on splicing were analyzed using Alamut Visual®, an exploration software application for genomic variation (Online Supplementary Figure S7A). These analyses showed a potential impact on splicing protein binding in all cases, except for the c.1165T>C, p.W389R, which was used as a control to study exon 4 splicing. In silico analysis using the SPiP site via MobiDetails, was more restrictive and showed only three deleterious splicing variants (c.891+1G>A, c.1152C>T, p.Y384Y, and c.1216+1G>T) (Online Supplementary Figure S7B). We first focused our study on variants located in exon 3 which are most likely to

Figure 5. Study of PHD2 protein stability by the cycloheximide chase assay. The graph shows the amount of transfected HA-PHD2 protein after treatment with cycloheximide, reported as a percentage of the initial HA-PHD2 protein level (100% at 0 h of cycloheximide treatment) normalized to the intensity of actin. Data are shown as mean ± standard error of the mean of three independent experiments. Two-way analysis of variance was used for statistics (****P≤0.0001). WT: wild-type.

Table 2. Km and Vmax values of erythrocytosis-associated PHD2 mutants for 2-oxoglutarate and HIF-1α and HIF-2α substrates. Parameter

Unit

Enzyme WT PHD2

W334R

G349S

G349C

R371H

Km of 2-oxoglutarate Vmax

μM % of WT PHD2

5.0 ± 1.5 100

10.5 ± 4.0 100

9.5 ± 2.5 170

8.8 ± 2.9 160

4.8 ± 1.0 60

Km of HIF-1α C-terminal peptide Vmax

μM % of WT PHD2

11.5 ± 7.7 100

5.3 ± 1.8 50

14.5 ± 13.1 200

10.7 ± 6.4 110

3.9 ± 1.1 35

Km of HIF-2α ODDD Vmax

μM % of WT PHD2

0.27 ± 0.18 100

0.14 ± 0.11 70

0.24 ± 0.07 120

0.29 ± 0.16 90

0.15 ± 0.11 60

Km and Vmax values were determined for 2-oxoglutarate, HIF-1α C-terminal peptide (19 residues with Pro564) and for HIF-2α-ODDD (250 residues, contains both hydroxylatable prolines). WT: wild-type; PHD2: prolyl hydroxylase domain-containing protein 2; ODD: oxygen-dependent degradation domain.

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be involved in the disease i.e., the same mutation described in two different families (c.1112 G>A, p.R371H)26,27 or the same nucleotide and amino acid targeted in two different families (c.1045G>A, p.G349C and c.1045G>T, p.G349S). The mutant c.1121A>G, p.H374R, which completely inhibits the activity of PHD2, was used as a control.20 The PHD2 exon 3 and intronic flanking sequences were between the SERPING1 exons (noted as A and B) of the splicing reporter plasmid pCAS2.38 Mutagenesis was then performed to obtain mutant constructs (Figure 6A). HEK293T cells were transfected with the different minigene constructs. Spliced transcripts were detected by RT-PCR using primers located in exons A and B and analyzed on agarose gel. Two bands were present (Figure 6B) corresponding to transcripts containing exons A and B spliced with or without PHD2 exon 3, demonstrating that wild-type PHD2 exon 3 is not fully spliced in this cell type. No significant difference was observed for the

different mutated minigene constructs, suggesting that the variants studied do not have an impact on splicing. We pursued the study with variants located in exon 4: c.1152C>T, p.Y384Y, and c.1216+1G>T in addition to c.1165T>C, p.W389R as a control. The results showed a deleterious effect of the c.1216+1G>T variant associated with a complete absence of PHD2-exon 4 inclusion in the three cell lines tested (Figure 6C). No impact on splicing was observed with c.1165T>C, p.W389R compared to the wild-type control, whereas the band corresponding to exon 4 skipping was slightly more highly expressed in the construct containing the synonymous variant c.1152C>T, p.Y384Y, in all the cell types tested (Figure 6C). Study of the impact of variants on splicing using patients’ cells To confirm the observed PHD2-exon 4 splicing defect, we next focused our study on a patient’s cells. RT-PCR using

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Figure 6. Functional studies of splicing variants. (A) Schematic representation of the splicing reporter assay (minigene experiment). The PHD2 exon of interest and flanking intronic sequences were cloned in a minigene pCAS2 plasmid between the SERPING1 exons (named A and B) flanked by short intronic sequences and conserved consensus splicing sequences. Mature and spliced mRNA were studied by reverse transcriptase polymerase chain reaction (RT-PCR) using primers located in exons A and B (schematized by arrows). (B) Characterization of PHD2-exon 3 splicing by a minigene experiment. RT-PCR was performed on mRNA obtained from HEK293T cells transfected with a minigene construct containing PHD2-exon 3 (wild-type or mutated) cloned in pCAS2. The pCAS2 plasmids were transfected, and the expression of the spliced chimeric transcripts was analyzed. Bands corresponding to exon A [A] and exon B [B] spliced together or with PHD2 exon 3 (Ex3) are indicated on the right (representative picture of agarose gel; N=3). (C) Characterization of PHD2-exon 4 splicing by the minigene experiments. RT-PCR was performed on mRNA obtained from cell lines (HEK293T, Hep3B or UT-7) transfected with a minigene construct containing PHD2-exon 4 (wildtype or mutated) flanked by intronic sequences cloned into pCas2 plasmids. The plasmids were transfected, and the expression of the spliced chimeric transcripts was analyzed. Bands corresponding to exon A and exon B spliced together or with PHD2-exon 4 (Ex4) are indicated on the left (representative picture of agarose gel, N=3). (D) Study of endogenous PHD2 splicing in a patient’s cells. RT-PCR using primers located in exons 3 and 5 of the PHD2 gene was performed on mRNA extracted from whole blood cells of the patient carrying the c.1152C>T, p.Y384Y variant collected into Paxgene® tubes. The lower band was purified and sequenced. The sequencing chromatogram is presented and shows the sequence of a transcript containing the exon 3 spliced with exon 5. (E) RT-PCR was performed on PHD2 mRNA (exons 3-5) extracted from peripheral blood mononuclear cells of the patient carrying the c.1216+1G>T variant. (F) RT-PCR was performed on PHD2 mRNA (exons 3-5) extracted from lymphoblastoid cell lines established from different patients. The cells were cultured in the absence (-) or presence (+) of puromycin, an inhibitor of nonsense-mediated mRNA decay mechanisms (representative picture of agarose gel, N=3). (G) Study of human induced pluripotent stem cells established from the patient carrying the c.1152C>T, p.Y384Y variant and differentiated into the hepatocyte-like cells. A representative gel of RT-PCR performed on PHD2 mRNA (exons 3-5) is shown on the left. The quantification of the percentage of PHD2-exon 4 skipping in all replicates was performed using the TapeStation® migration system and the results are shown on the right. Each column represents the mean ± standard error of the mean of independent experiments (see details in Online Supplementary Figure S8B). Two-way analysis of variance was used for statistics (**P≤0.01, ****P≤0.0001). M: molecular-weight size marker; WT: wild-type.

primers in exons 3 and 5 of the endogenous PHD2 mRNA was performed on whole blood cells obtained from the patient carrying the c.1152C>T, p.Y384Y variant; a barely detectable lower band was visible in the patient’s sample (Figure 6D). Purification, cloning, and sequencing identified the band as a PHD2 transcript containing exon 3 spliced with exon 5, thus confirming exon 4 skipping. We confirmed severe exon 4 skipping on peripheral blood mononuclear cells obtained from the patient carrying the heterozygous c.1216+1G>T mu-

tation (equal intensity of the two bands corresponding to the spliced and skipped exon 4) (Figure 6E). Theoretically, translation of these mis-spliced transcripts introduces, in frame with the PHD2 coding sequence, a premature stop codon at the beginning of exon 5 (Figure 6D, right panel). Although the premature stop codon is located in the last exon, we opted to assess whether these transcripts could be the target of nonsense-mediated mRNA decay. To this purpose, we established lymphoblastoid cell lines from

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blood cells of the two patients and cultured them in the absence or presence of puromycin, an inhibitor of nonsense-mediated mRNA decay. The results obtained with these cells confirmed a severe effect of the c.1216+1G>T mutant and a very weak effect of the c.1152C>T, p.Y384Y variant on splicing (Figure 6F). No effect of puromycin was observed, indicating that the mis-spliced transcripts were not degraded by nonsense-mediated mRNA decay. Because splicing is cell type-dependent and the effect of the c.1152C>T, p.Y384Y variant appeared to be more important, in minigene experiments, in the hepatocarcinoma cell line Hep3B (Figure 6C), we used a cellular model that mimics hepatocytes. We generated hiPSC from the patient’s peripheral blood mononuclear cells which were differentiated into hepatocyte-like cells (Online Supplementary Figure S8A). At the end of the differentiation (day 22), hepatocyte-like cells were cultured in 1% oxygen for 24 h. RT-PCR was performed to investigate PHD2-exon 4 skipping (Figure 6G, left panel; Online Supplementary Figure S8B). PCR products were quantified after their migration on TapeStation® (Figure 6G, right panel). Our results demonstrated a significant effect of the c.1152C>T, p.Y384Y variant on PHD2-exon 4 skipping in normoxia and hypoxia (at 1% oxygen). Remarkably, in control and mutant cells, we noted that hypoxia has a major impact on PHD2 splicing, with upregulation of the transcripts that do not retain exon 4.

with those of our patients (Online Supplementary Table S3). A total of 96 PHD2 variants were identified, of which 36 can be classified as pathogenic (or probably pathogenic), five as benign, and 55 are still of unknown significance. As in our study, no difference was observed between the pathogenic/not pathogenic categories regarding hematologic parameters (Online Supplementary Table S4).

Compiled analysis of data for variant classification We interpreted and classified the identified variants according to American College of Medical Genetics (ACMG) criteria41 and the recommendations of the French NGS group, based on genetic data (segregation in family, number of unrelated families, etc.), and the in silico and functional studies described here. In this study, 16 EGLN1 variants (in 24 patients) were classified as likely pathogenic or pathogenic, and 23 variants (in 23 patients) were classified as being of unknown significance (VUS) or likely benign. When comparing these two groups, there was no difference in the occurrence of thrombosis, either in the past history or after the diagnosis (4/24 [16.6%] vs. 2/24 [8.3%], respectively). Patients with a pathogenic variant were more frequently treated with phlebotomy (9/24 [37.5%] vs. 3/23 [13%]) and low-dose aspirin (10/24 [41.6%] vs. 2/23 8.7%]), whereas no significant difference was noted between these two groups of patients with respect to complete blood count values, including hemoglobin and hematocrit values (Online Supplementary Table S2). Surprisingly, two patients with a pathogenic EGLN1 variant were treated with cytoreductive drugs (interferon and hydroxyurea). A family history of erythrocytosis was found in a higher proportion of patients with a pathogenic variant (41.6%) compared to patients with a VUS (26%). We extended the in silico analysis to the genetic variants identified in the literature and compiled all these results

Discussion The development of next-generation sequencing panels for use in diagnostic laboratories allowed molecular screening of a larger number of patients and increased the number of VUS identified. Mutations in the EGLN1 gene are distributed throughout the protein with no hotspot, and no more than two families, of small size, per mutation have been described.31 In addition, mutations in hypoxia pathway genes associated with erythrocytosis may be hypomorphic.42 All these limitations make the classification of the identified variants difficult. The aim of this study was to bring together specialized genetic laboratories dedicated to genetic screening of mutations in EGLN1 and to propose a comprehensive approach to in silico and functional analyses in order to collect information and facilitate genetic diagnosis. Among the 2,160 patients sequenced by ten laboratories, we identified 39 genetic variants, including a complete heterozygous deletion of the gene, in 47 families. Our compiled in silico and functional studies allowed the classification of 16 variants as likely pathogenic/pathogenic mutations in 48 patients (24 probands and 24 relatives). The analysis extended to all variants in the literature showed that of the 96 PHD2 variants identified so far (including those in our study), 36 can be classified as pathogenic, five as benign and 55 are, in fact, of unknown significance (VUS). Although there are still many variants to be classified, the pooling of results from all our laboratories has been very beneficial. It has allowed the identification of families carrying similar mutations, which enabled the addition of ACMG classification criteria (such as PS4) and their classification into pathogenic mutations (I269T, R312H). In addition, the study of databases such as the UK Biobank has been very useful for the classification of benign variants (A96V, C127S, Q157H, Q157R). Examination of the clinical data showed that complications are rare in patients carrying EGLN1 mutations (in the present study, only 1 patient with portal vein thrombosis). Notably, clinical data from all the families described in the literature and in the present study (N=72 and N=47, respectively) showed that tumor development can be considered a very rare event (only 2 cases of paraganglioma20,21 and 1 case of pheochromocytoma42) and may, therefore, be associated with additional genetic events. However, given the demonstrated role of PHD2 in the pathogenesis of pseudo-hypoxic

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pheochromocytoma,43 it is essential to medically monitor the occurrence of a possible tumor. In this study there was no clear distinction in clinical or biological presentations between patients carrying PHD2 variants classified as pathogenic compared to those carrying VUS. This means that clinical parameters cannot help in the classification. Nevertheless, our data are retrospective, and in a certain number of cases, we were unable to obtain precise dates of the biological results, i.e., before any treatment, phlebotomies or during evolution, which are parameters that can modify the hematologic data. In addition, the number of variants classified as pathogenic according to ACMG criteria may be underestimated because a family history was found for a significant number of VUS (26%) but it was not possible to explore their segregation in the family. Further exploration in the families would allow the variants to be shifted to the pathogenic class or to open research into other causal genetic events. This study confirmed that erythrocytosis is more frequent in men than in women. In women, the disease could go undetected, mainly due to blood loss associated with menstruation. The example of the man carrying the W389R mutant (Family #42, P #III-4) who has no symptoms but is an assiduous blood donor supports this hypothesis (Figure 2A). The functional studies using endpoint and real-time luciferase assays showed loss of function for only two mutants, Y41C and W389R. Development of the luciferase assays under even more sensitive conditions (reducing the amount of vectors transfected, using PHD2 knock-out cells, etc.) did not improve the results (data not shown). We therefore used more sensitive in vitro enzymatic assays to evaluate the impact of some variants on PHD2 catalytic function. Unfortunately, these assays were not more informative. Measurement of Km and Vmax values did not demonstrate any significant loss of function of the tested variants. Indeed, the higher Km value (showing a lower affinity for the substrate or co-substrate) measured for the W334R, G349S, and G349C variants was compensated by a higher Vmax (speed of reaction) value. The same difficulty in demonstrating a loss of function was encountered with the R371H variant. The experiments showed a lower Vmax value compensated by a substrate with increased binding affinity (lower Km), confirming the results of previous studies.23,25 The examination of a loss of protein stability was more conclusive. The cycloheximide chase assay showed a decrease in the half-life of four variants: I269T, W334R, R371H, and W389R. Alteration of protein stability may theoretically have some impact on HIF activity, but was detected in our luciferase assays only for the W389R variant (for an equivalent amount of protein expressed). The absence of significant detectable PHD2 loss of function or protein stability suggests that other PHD2 partners need to be tested. Indeed, PHD2 binds a number of proteins, and the mutations could conceivably affect these interactions.44-46 For example, PHD2 binds FKPB38, which

plays a major role in PHD2 stability;47 LIMD1 is known to form a complex with PHD2 and VHL, creating an enzymatic niche that enables efficient degradation of HIF-1;48 and PHD2 binds p23, which allows recruitment of PHD2 to the heat shock protein 90 (HSP90) machinery to facilitate hydroxylation of HIF-1α.44 Of note, three variants in our series (R35H, Y41C and D50H), which are are located in the zinc finger domain, are involved in the stability of the PHD2/ p23/HSP90/HIF-α complex. Nonetheless, none of these variants targets a conserved cysteine that plays a major role in zinc chelating cysteine residues, and only the Y41C variant showed a decreased ability to downregulate HIF in our luciferase reporter assay. Interestingly, recent work has identified a new partner of the PHD2 zinc finger domain that binds to the ribosomal chaperone NACA, allowing PHD2 to co-translationally modify the nascent HIF-α polypeptide.49 More specific assays need to be developed in order to be able to investigate all these complex interactions. To test the hypothesis of a potential impact of variants on splicing, which has been already demonstrated for other hypoxia pathway genes involved in erythrocytosis,50 we performed splicing reporter assays. No impact was detected for the variants located in exon 3 (G349C, G349S, R371H), confirming the in silico prediction of the SPiP tool rather than that of the ALAMUT site which suggested a possible impact on the binding of the spliceosome proteins. Interesting results were obtained for exon 4 splicing. For the c.1216+1G>T variant targeting the splicing donor site, a deleterious effect with a high level of exon 4 skipping was detected, in all cells tested. Exon 4 skipping results in a transcript that contains an in-frame translation termination codon introduced by exon 5. We demonstrated that this transcript is not targeted by the nonsense-mediated decay machinery, which can, in consequence, lead to the expression of a truncated PHD2 protein (starting at amino acid 382). We have previously shown that a PHD2 protein truncated from amino acid 398 completely loses its function.26 Thus, we may conclude that exon 4 skipping is equivalent to loss of PHD2 function. A more subtle effect was detected for the synonymous c.1152C>T (Y384Y) located near the 5’ end of exon 4 of PHD2. Because splicing is cell type-specific, we developed a cellular model using hiPSC derived from the patient and differentiated into hepatocyte-like cells. All experiments showed a slight but readily reproducible impact of the variant on exon 4 skipping. Interestingly, our experiments performed in hiPSC cells confirmed that hypoxia upregulates the expression of PHD2, a described HIF target gene, but also showed for the first time that hypoxia increases exon 4 skipping. Since we know that the exon 4 skipping results in a non-functional PHD2, the described negative feedback loop of hypoxia-induced PHD2 might be attenuated because the expressed PHD2 is not fully effective. This result paves a new way to fine-tune the hypoxia pathway which regulates expression and splicing of its different players.

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In conclusion, our study provides an example of a comprehensive approach to classification of genetic variants that could be applied to many rare hematologic diseases. Here, we demonstrated the importance of collaborating and combining results from different diagnostic centers dedicated to rare diseases. In silico studies with analysis of databases such as the UK Biobank may also facilitate classification, especially of non-pathogenic variants. We have shown the importance of performing a wide range of functional analyses and of studying finer regulatory mechanisms of the gene, such as the splicing studied here, whose complexity remains to be explored. Finally, accurate classification is essential in order to make the most appropriate diagnosis in patients and thus ensure proper follow-up and treatment. In the absence of targeted or specific treatment, by analogy with polycythemia vera, phlebotomies and the use of low-dose aspirin, have been suggested,51 albeit still under debate.52 The recent encouraging results obtained with an HIF-2α inhibitor in VHL- and HIF-2α-associated diseases53,54 opens up new promising therapeutic perspectives in all disorders associated with the hypoxia pathway.

ed the study. All authors contributed to the research and approved the final version of the manuscript.

Disclosures No conflicts of interest to disclose.

Data-sharing statement Data and detailed information related to the study are available from the corresponding author upon request.

Contributions MD, ALR, MP, LS, MM, VK, DH, EP, VL, PK, and BG designed and/or performed in vitro and cellular functional experiments. MD, AC, KS-T, CC, and AG worked on hiPSC reprogramming and differentiation. AR performed the bioinformatics analyses. CG, NM, BA, MC, FA, LM, MR, TH, MM, SB, AB, NC, PH, CR, MW, FGa, BC, BB, CB, RvW, PP, MLR, MFMcM, FGi, and the ECYT3 consortium conducted the medical and genetic diagnostic studies. BG and FGi wrote the manuscript and designed the study. SI corrected the manuscript. BG direct-

Acknowledgments The present research has been conducted using the UK Biobank resource under application number 49823. We are most grateful to the Bioinformatics Core Facility of Nantes BiRD, member of Biogenouest, Institut Français de Bioinformatique (IFB) (ANR-11-INBS-0013) for the use of its resources and for its technical support. We thank Thomas Besnard for minigene expertise, Morgane Taligot for technical assistance and Dr Emmanuelle Verger for expertise in next-generation sequencing analysis. Funding This study was supported by grants from the Région des Pays de la Loire (project “EryCan”); the ANR (PRTS 2015 “GenRED” and AAPG 2020 “SplicHypoxia”); the labex GR-Ex, reference ANR-11-LABX-0051; Fonds Européen de Développement Régional (FEDER) Bourgogne Franche Comté; the VHL Alliance USA, the VHL France; the Génavie association and the Fondation Maladies Rares (FMR).

Appendix: ECYT-3 members Annalisa Andreoli, Emmanuel Bachy, Sarah Bonnet, Françoise Boyer, Serge Carillo, Brieuc Cherel, Florian Chevillon, Nataša Debeljak, Justine Decroocq, Roxana Dragan, Martine Escoffre, Jonathan Farhi, Arnaud Hot, Ludovic Karkowski, Catherine Humbrecht-Kraut, Philippe Joly, Jean-Jacques Kiladjian, Adrienne de Labarthe, Franck Lellouche, Guy Leverger, Emmanuel Raffoux, Dana Ranta, Benoit de Ranzis and Aline Schmidt.

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erythrocytosis due to EGLN1 mutation with review of the literature]. Rev Med Interne. 2020;41(3):196-199. 30. Bento C, Almeida H, Maia TM, et al. Molecular study of congenital erythrocytosis in 70 unrelated patients revealed a potential causal mutation in less than half of the cases (where is/are the missing gene(s)?). Eur J Haematol. 2013;91(4):361-368. 31. Barradas J, Rodrigues CD, Ferreira G, et al. Congenital erythrocytosis – discover of a new mutation in the EGLN1 gene. Clin Case Rep. 2018;6(6):1109-1111. 32. Al-Sheikh M, Moradkhani K, Lopez M, Wajcman H, Prehu C. Disturbance in the HIF-1alpha pathway associated with erythrocytosis: further evidences brought by frameshift and nonsense mutations in the prolyl hydroxylase domain protein 2 (PHD2) gene. Blood Cells Mol Dis. 2008;40(2):160-165. 33. Ansar S, Malcolmson J, Farncombe KM, Yee K, Kim RH, Sibai H. Clinical implementation of genetic testing in adults for hereditary hematologic malignancy syndromes. Genet Med. 2022;24(11):2367-2379. 34. Baux D, Van Goethem C, Ardouin O, et al. MobiDetails: online DNA variants interpretation. Eur J Hum Genet. 2021;29(2):356-360. 35. MetaDome: pathogenicity analysis of genetic variants through aggregation of homologous human protein domains - Wiel 2019 - Human Mutation - Wiley Online Library. https:// onlinelibrary-wiley-com.proxy.insermbiblio.inist.fr/doi/10.1002/ humu.23798 Accessed October 18, 2021. 36. Hirsilä M, Koivunen P, Xu L, Seeley T, Kivirikko KI, Myllyharju J. Effect of desferrioxamine and metals on the hydroxylases in the oxygen sensing pathway. FASEB J. 2005;19(10):1308-1310. 37. Koivunen P, Myllyharju J. Kinetic analysis of HIF prolyl hydroxylases. Methods Mol Biol. 2018;1742:15-25. 38. Gaildrat P, Killian A, Martins A, Tournier I, Frebourg T, Tosi M. Use of splicing reporter minigene assay to evaluate the effect on splicing of unclassified genetic variants. Methods Mol Biol. 2010;653:249-257. 39. DeLaForest A, Nagaoka M, Si-Tayeb K, et al. HNF4A is essential for specification of hepatic progenitors from human pluripotent stem cells. Development. 2011;138(19):4143-4153. 40. Si-Tayeb K, Noto FK, Nagaoka M, et al. Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology. 2010;51(1):297-305. 41. Richards S, Aziz N, Bale S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17(5):405-424. 42. Couvé S, Ladroue C, Laine E, et al. Genetic evidence of a precisely tuned dysregulation in the hypoxia signaling pathway during oncogenesis. Cancer Res. 2014;74(22):6554-6564. 43. Eckardt L, Prange-Barczynska M, Hodson EJ, et al. Developmental role of PHD2 in the pathogenesis of pseudohypoxic pheochromocytoma. Endocr Relat Cancer. 2021;28(12):757-772. 44. Song D, Li LS, Heaton-Johnson KJ, Arsenault PR, Master SR, Lee FS. Prolyl hydroxylase domain protein 2 (PHD2) binds a Pro-Xaa-Leu-Glu motif, linking it to the heat shock protein 90 pathway. J Biol Chem. 2013;288(14):9662-9674. 45. Vogel KS, Brannan CI, Jenkins NA, Copeland NG, Parada LF. Loss of neurofibromin results in neurotrophin-independent survival of embryonic sensory and sympathetic neurons. Cell. 1995;82(5):733-742. 46. Huo Z, Ye JC, Chen J, et al. Prolyl hydroxylase domain protein 2 regulates the intracellular cyclic AMP level in cardiomyocytes through its interaction with phosphodiesterase 4D. Biochem

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Biophys Res Commun. 2012;427(1):73-79. 47. Barth S, Nesper J, Hasgall PA, et al. The peptidyl prolyl cis/trans isomerase FKBP38 determines hypoxia-inducible transcription factor prolyl-4-hydroxylase PHD2 protein stability. Mol Cell Biol. 2007;27(10):3758-3768. 48. Foxler DE, Bridge KS, James V, et al. The LIMD1 protein bridges an association between the prolyl hydroxylases and VHL to repress HIF-1 activity. Nat Cell Biol. 2012;14(2):201-208. 49. Song D, Peng K, Palmer BE, Lee FS. The ribosomal chaperone NACA recruits PHD2 to cotranslationally modify HIF-α. EMBO J. 2022;41(22):e112059. 50. Lenglet M, Robriquet F, Schwarz K, et al. Identification of a new VHL exon and complex splicing alterations in familial erythrocytosis or von Hippel-Lindau disease. Blood. 2018;132(5):469-483.

51. McMullin MFF, Mead AJ, Ali S, et al. A guideline for the management of specific situations in polycythaemia vera and secondary erythrocytosis: a British Society for Haematology guideline. Br J Haematol. 2019;184(2):161-175. 52. Gordeuk VR, Miasnikova GY, Sergueeva AI, et al. Thrombotic risk in congenital erythrocytosis due to up-regulated hypoxia sensing is not associated with elevated hematocrit. Haematologica. 2020;105(3):e87-e90. 53. Kamihara J, Hamilton KV, Pollard JA, et al. Belzutifan, a potent HIF2α inhibitor, in the Pacak-Zhuang syndrome. N Engl J Med. 2021;385(22):2059-2065. 54. Yu Y, Yu Q, Zhang X. Allosteric inhibition of HIF-2α as a novel therapy for clear cell renal cell carcinoma. Drug Discov Today. 2019;24(12):2332-2340.

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Increased retention of functional mitochondria in mature sickle red blood cells is associated with increased sickling tendency, hemolysis and oxidative stress Sofia Esperti,1,2,3 Elie Nader,1,2+ Antoine Stier,4,5+ Camille Boisson,1,2 Romain Carin,1,2 Muriel Marano,6 Mélanie Robert,1,2,3 Marie Martin,1 Françoise Horand,3 Agnes Cibiel,3 Céline Renoux,1,2,7 Robin Van Bruggen,8 Colin Blans,8 Yesim Dargaud,6 Philippe Joly,1,2,7 Alexandra Gauthier,1,2,9 Solène Poutrel,1,2,10 Marc Romana,2,11 Damien Roussel4 and Philippe Connes1,2 Laboratoire interuniversitaire de Biologie de la Motricité (LIBM) EA7424, Team « Vascular Biology and Red Blood Cell », Université Claude Bernard Lyon 1, Université de Lyon, Lyon, France; 2Laboratoire d’Excellence du Globule Rouge (Labex GR-Ex), PRES Sorbonne, Paris, France; 3Erytech Pharma, Lyon, France; 4Laboratoire d’Ecologie des Hydrosystèmes Naturels et Anthropisés, CNRS, ENTPE, UMR 5023, Université Claude Bernard Lyon 1, Université de Lyon, Lyon, France; 5Université de Strasbourg, CNRS, Institut Pluridisciplinaire Hubert Curien, UMR7178, Strasbourg, France; 6UR4609, Hémostase & Thrombose, Université Claude Bernard Lyon 1, Université de Lyon, Lyon, France; 7Laboratoire de Biochimie et de Biologie Moléculaire, UF de Biochimie des Pathologies Erythrocytaires, Centre de Biologie et de Pathologie Est, Hospices Civils de Lyon, Lyon, France; 8Department of Molecular Hematology, Sanquin Research and Landsteiner Laboratory, University of Amsterdam, Amsterdam, the Netherlands; 9 Institut d’Hématologique et d’Oncologique Pédiatrique, Hospices Civils de Lyon, Lyon, France; 10 Service de Médecine Interne, Hôpital Edouard Herriot, Hospices Civils de Lyon, Lyon, France and 11Université de Paris, Université des Antilles, UMR S1134, BIGR, INSERM, Paris, France 1

Correspondence: P. Connes pconnes@yahoo.fr philippe.connes@univ-lyon1.fr Received: Accepted: Early view:

January 4, 2023. May 23, 2023. June 1, 2023.

EN and AS contributed equally.

Abstract Abnormal retention of mitochondria in mature red blood cells (RBC) has been recently reported in sickle cell anemia (SCA) but their functionality and their role in the pathophysiology of SCA remain unknown. The presence of mitochondria within RBC was determined by flow cytometry in 61 SCA patients and ten healthy donors. Patients were classified according to the percentage of mature RBC with mitochondria contained in the whole RBC population: low (0-4%), moderate (>4% and <8%), or high level (>8%). RBC rheological, hematological, senescence and oxidative stress markers were compared between the three groups. RBC senescence and oxidative stress markers were also compared between mature RBC containing mitochondria and those without. The functionality of residual mitochondria in sickle RBC was measured by high-resolution respirometry assay and showed detectable mitochondrial oxygen consumption in sickle mature RBC but not in healthy RBC. Increased levels of mitochondrial reactive oxygen species were observed in mature sickle RBC when incubated with Antimycin A versus without. In addition, mature RBC retaining mitochondria exhibited greater levels of reactive oxygen species compared to RBC without mitochondria, as well as greater Ca2+, lower CD47 and greater phosphatidylserine exposure. Hematocrit and RBC deformability were lower, and the propensity of RBC to sickle under deoxygenation was higher, in the SCA group with a high percentage of mitochondria retention in mature RBC. This study showed the presence of functional mitochondria in mature sickle RBC, which could favor RBC sickling and accelerate RBC senescence, leading to increased cellular fragility and hemolysis.

Introduction Sickle cell anemia (SCA) is an inherited hemoglobinopathy caused by a single point mutation in the β-globin gene which leads to the synthesis of an abnormal hemoglobin, called hemoglobin S (HbS). When deoxygenated, HbS polymerizes causing a mechanical distortion (i.e., sickling) of red

blood cells (RBC).1 Sickle RBC are less deformable and more fragile than normal RBC, resulting in chronic anemia2 and frequent vaso-occlusive crises.3 Moreover, the accumulation of free hemoglobin and heme in the plasma promotes oxidative stress, inflammation and endothelial dysfunction, which contribute to the pathophysiology of SCA and the development of chronic complications.4

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ARTICLE - Sickle cell disease and mitochondria

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Recent studies reported the presence of mitochondria in mature RBC in patients with SCA.5-7 Mitophagy is an important step for the survival of RBC during erythroid maturation.8 It has been demonstrated that the abnormal mitochondria retention in sickle RBC would be the result of a deficient mitophagy pathway throughout erythropoiesis.5 Jagadeeswaran et al.9 demonstrated that the use of the lysine-specific demethylase 1A (LSD1) inhibitor (RN-1), and the use of a mitophagy-inducing agent mammalian target of rapamycin (mTOR) inhibitor (sirolimus) increased RBC lifespan in a sickle cell mouse model. Of note, LSD1 inhibitor RN-1 could also improve RBC lifespan through its effects on HbF synthesis, as reported in non-human primates.10 In another study, it has been reported that SCA patients with a high percentage of mature RBC containing mitochondria exhibited higher levels of reticulocytes and total bilirubin, suggesting increased hemolysis in these patients compared to those with a low percentage of mature RBC with mitochondria.5 Indeed, although not formally proved, it has been hypothesized that the retention of mitochondria in mature RBC would favor hemolysis.9 It has been proposed that mitochondria retention in mature RBC could cause a shortening of their lifespan because of the accumulation of reactive oxygen species (ROS) generated by metabolically active mitochondria11 that would damage the cell.12 Promoting mitophagy in sickle cell mice was accompanied by a decrease in RBC ROS content.9 However, Martino et al.5 did not show any difference in the mitochondrial membrane potential using Mitrotracker orange between patients with mature RBC containing mitochondria compared to those without. Moreover, transmission electron microscopy experiments revealed that mitochondria in mature RBC were swollen, small and disorganized.5 It was concluded that mitochondria in mature RBC in SCA patients are not functional. However, another recent study supports the opposite.6 Proteomic, metabolomic and lipidomic analyses of mature RBC containing mitochondria and of mature RBC without mitochondria were performed, and higher levels of mitochondria metabolites were observed in the first population.6 Subsequent analyses in one patient with SCA showed detectable levels of oxidative phosphorylation activity and the presence of mitochondrial electron chain components in mature RBC containing mitochondria, supporting the idea that mitochondria would be functional. Indeed, the question of the functionality of mitochondria in mature RBC of SCA patients is still debated, and how mitochondria retention in mature RBC could be responsible for a rise in hemolysis in SCA remains unclear. The aim of this study was to test the associations between sickling tendency, RBC senescence, oxidative stress, hematological markers and the levels of mature RBC containing mitochondria in SCA patients and to investigate the functionality of these mitochondria.

Methods More details on the methods are given in the Online Supplementary Appendix. Blood samples from 61 SCA patients (HbSS genotype, 29 men and 32 women, at steady state, 23.2±14.2 years, non-transfused) and ten healthy racematched donors (AA) were collected in EDTA tubes. The study was conducted in accordance with the guidelines set by the Declaration of Helsinki, and all subjects gave informed written consent. The study was approved by the Regional Ethics Committees (L16-47, CPP Sud-Est IV, Hospices Civils de Lyon). Image Stream (Amnis, MK II) and Mitotracker Red CMXRos Dye (Invitrogen) were used to label active mitochondria. Flow cytometry (MACSQuant 16, Miltenyi), MitoTracker(R) Deep Red probe (MTKdr, Sigma-aldrich) and anti-CD71 antibody were used to determine the percentages of reticulocytes and mature RBC containing mitochondria. Since no criteria or threshold exist in the literature to decide which patient has high or low mitochondria retention in mature RBC, patients were divided into three groups using terciles according to the percentages of mature RBC containing mitochondria in the whole RBC population: “high”, “moderate” and “low” percentage of RBC containing mitochondria. Triple staining on blood samples from ten SCA patients was performed with anti-CD235a (Miltenyi) and anti-CD41 (Miltenyi) antibodies and Mitotracker Deep Red probe, to gate on the RBC+/platelet- population. Blood samples from eight patients and five AA were used for percoll gradient separation. The second layer containing mature RBC was collected and the presence of reticulocytes was assessed by flow cytometry by using an antiCD71 antibody (Miltenyi). Then, we investigated the functionality of mitochondria in mature RBC from SCA patients using a high-resolution respirometry protocol for intact blood cells.13,14 Endogenous O2 consumption was recorded before inhibiting ATP-dependent O2 consumption with Oligomycin (2.5 μM), an inhibitor of ATP synthase. The mitochondrial uncoupler FCCP (carbonyl cyanide-p-trifluoro-methoxyphenyl-hydrazone) was then titrated in 0.05 μM steps until the maximal uncoupled O2 consumption was reached. Finally, mitochondrial O2 consumption was fully inhibited by adding Antimycin A (2.5 μM). Flow cytometry was also used to determine the percentage of RBC exposing phosphatidylserine (PS) (Annexin-V-PE, Miltenyi 130-118-363), the anti-phagocytic CD47 antigen (antiCD47-PE antibody, Miltenyi) and intracellular ROS levels (2’,7’–dichlorofluorescin diacetate [DCFDA], Sigma-Aldrich) of the three groups categorized according to the percentages of mature sickle RBC containing mitochondria. Double staining for mitochondria retention (Mitotracker Deep Red) and i) intracellular Ca2+ (Fluo3 AM, ThermoFicher), ii) CD47, iii) PS or iv) intracellular ROS, was performed to compare these parameters between mature RBC containing mito-

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chondria and those without. Mitochondria superoxide production in sickle RBC was assessed using the MitoSOX Red mitochondrial superoxide indicator (Invitrogen, M36008) and flow cytometry, with and without Antimycin A, an inhibitor of the Respiratory Complex III. Reduced (GSH) and total (GSSG+GSH) intracellular glutathione were measured using the luminescence-based assay GSH-Glo glutathione Assay (Promega). Oxygen gradient ektacytometry was performed using the Oxygenscan protocol of the LORRCA Maxsis (Mechatronics) to measure RBC deformability in an oxygen gradient.15,16 The maximum RBC deformability (EImax) reached in normoxia and the oxygen pressure at which RBC start to sickle (point of sickling [PoS]) were determined.

Results Presence of functional mitochondria in mature sickle red blood cells Image Stream analysis showed the presence of mitochondria in mature RBC isolated from SCA patients, but not in RBC from healthy donors. Representative images of RBC

containing mitochondria from three SCA patients are shown in Figure 1A. Figure 1B shows the lack of mitochondria in RBC from three healthy donors. Flow cytometry analyses (gating strategy in Figure 1C) demonstrated no mitochondria in mature RBC from healthy individuals and highly variable percentages of mature RBC containing mitochondria in patients with SCA (Figure 1D). The mean percentages of mature RBC containing mitochondria in the three SCA groups categorized according to the percentages of mature RBC containing mitochondria in the whole RBC population were: low group 1.40±1.31%, moderate group 6.2±1.02% and high group 13.02±5.84%. The high-resolution respirometry assay showed active oxygen consumption being responsive to specific mitochondrial inhibitors (Figure 2A). Flow cytometry analysis of the isolated RBC fraction obtained after the Percoll separation gradient showed a very low percentage of residual reticulocytes (<0.6%) and platelets (<0.5%), indicating that the respirometry results were mainly attributable to sickle RBC. Specifically, oxygen consumption was observed in mature sickle RBC but not in healthy RBC (Figure 2A, interaction patient status *respiratory rate; P<0.001). As expected, respiration was undetectable and unaffected by

Figure 1. Presence of mitochondria in mature sickle red blood cells. (A) ImageStream and staining with Mitotracker Red CMRXRos dye of mitochondria in mature sickle red blood cells (RBC) of three sickle cell (SCA) patients. (B) ImageStream and staining with Mitotracker Red CMRXRos dye show no mitochondria in RBC from healthy donors. (C) Gating strategy for flow cytometry analysis to discriminate mature sickle RBC retaining mitochondria (CD71- Mitotracker+). (D) Mean percentages of mature RBC with mitochondria (mito) in 61 SCA (SS) patients and 10 healthy (AA) donors. Significant difference: ****P<0.0001.

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the mitochondrial inhibitors/uncoupler (Oligomycin, FCCP, and Antimycin A; all P>0.24) in healthy controls. In SCA patients, endogenous respiration was detectable and decreased after adding Oligomycin (Figure 2A; P<0.001), an inhibitor of ATP synthase (ATP-inhibited), showing that approximately half of the endogenous oxygen consumption was coupled to ATP synthesis in mature sickle RBC. Following the addition of the uncoupler FCCP thereby reducing the proton force in the inner mitochondrial membrane (i.e., uncoupled O2 consumption), the cellular respiration of mature sickle RBC increased back to the endogenous level (P<0.001). Finally, the use of Antimycin A, an inhibitor of Mitchondrial Complex III, caused a marked reduction of O2 consumption in mature sickle RBC (i.e., non-mitochondrial O2 consumption; P<0.001). Superoxide anion accumulation from mitochondria in mature RBC from 14 SCA patients was detected by the MitoSox Red probe with and without the use of Antimycin A. The percentages of mature sickle RBC accumulating mitochondrial superoxide anion increased after the incubation with Antimycin A (Figure 2B). Mitochondria retention in mature sickle red blood cells (RBC) is associated with increased RBC senescence markers and increased propensity of RBC to sickle under deoxygenation The comparisons of the three groups categorized ac-

cording to the percentages of mature sickle RBC retaining mitochondria contained in the whole RBC population showed no difference in intracellular ROS (Figure 3A), reduced (GSH, Figure 3B), oxidized (GSSG, Figure 3C) glutathione levels and the ratio between the two forms (Figure 3D). No difference in anti-phagocytic CD47 expression was observed between the three groups (Figure 3E). The percentages of RBC with externalized PS were greater in the groups with moderate and high percentages of mature RBC containing mitochondria (Figure 3F). The comparisons between mature sickle RBC with or without mitochondria showed higher Ca2+ levels, intracellular ROS, and lower CD47 expression in RBC retaining mitochondria (Figure 4A-C). Figure 4D shows greater percentages of RBC with externalized PS in mature sickle RBC with mitochondria compared to those without. No platelets contamination (CD41+) within the RBC population was observed. Rheological parameters of the total RBC population showed different tendencies according to the presence of mitochondria. EImax was lower in the two groups with the highest percentages of mature RBC containing mitochondria, with a further reduction in the third (high) group (Figure 5A). The PoS was greater in patients with a high percentage of mature RBC with mitochondria compared to the two other groups (Figure 5B).

Figure 2. Mitochondrial respiration in mature red blood cells from sickle cell disease patients. (A) Comparison of mitochondrial respiration between 5 healthy and 8 mature sickle red blood cells (RBC). (B) Effect of Antimycin A on superoxide anion generated by mitochondria in mature sickle RBC (N=13). Endogenous: basal mitochondrial O2 consumption; ATP-inhibited: mitochondrial O2 consumption after the use of ATP-synthase inhibitor (Oligomycin); uncoupled: maximal uncoupled O2 consumption obtained with the mitochondrial uncoupler (carbonyl cyanide-p-trifluoro-methoxyphenyl-hydrazone [FCCP]); non-mitochondrial: O2 consumption after adding the Antimycin A. Difference between healthy and mature sickle RBC: $P>0.05; $$$P<0.001. The addition of mitochondrial inhibitors or uncoupler had no effects on healthy RBC while changes were observed on mature sickle RBC: ***P<0.001. Difference between Mitosox and Mitosox+Antimycin A conditions: ***P<0.001. %RBC Mitosox+: percentages of RBC accumulating superoxide anion.

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Hematocrit was lower and hemolytic markers were greater in patients with high percentages of mature red blood cells containing mitochondria No difference in fetal hemoglobin (HbF) and mean corpuscular volume was observed between the three groups (Figures 6A, B). The mean corpuscular hemoglobin concentration was slightly but significantly increased in the group showing a higher percentage of mitochondrial retention compared to the group with a low percentage

(Figure 6C). Percentages of reticulocytes and bilirubin levels were greater, and hematocrit was lower, in the group of patients with a high percentage of mature RBC with mitochondria (Figure 6D-F). No association between mature sickle RBC mitochondria retention and previous history of clinical manifestations (i.e., vaso-occlusive crises and acute chest syndrome rates in the 3 preceding years, glomerulopathy, priapism, leg ulcers, osteonecrosis, stroke or pulmonary hypertension)

Figure 3. Comparisons of red blood cells oxidative stress and senescence markers between the three groups of sickle cell patients categorized according to the percentage of mature sickle red blood cell containing mitochondria (low group N=20, moderate group N=21, high group N=21) and healthy individuals (AA group N=10). (A-D) Red blood cell (RBC) oxidative stress markers, (E) CD47 expression and (F) percentages of RBC exposing phosphatidylserine (PS). ROS: reactive oxygen species; GSH: reduced glutathione; GSSG: oxidized glutathione; MFI: mean fluorescence intensity; ns: no significant difference. Significant difference: *P<0.05; **P<0.01; ***P<0.001; ****P< 0.0001. Haematologica | 108 November 2023

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was observed. Glucose-6-phosphate dehydrogenase deficiency frequency did not differ between the three groups (15%, 14% and 10% in the low, moderate, and high group, respectively; χ=0.39; P= 0.82).

The present study investigated the functionality of mitochondria in mature RBC from SCA patients and the associations between RBC mitochondria retention, the propensity of RBC to sickle under deoxygenation, hemolytic markers, RBC senescence and RBC oxidative stress. Our findings support the fact that mitochondria in ma-

ture RBC from SCA patients are still functional and able to produce ATP. In addition, we found that i) patients with a higher percentage of mitochondria retention in mature RBC were those exhibiting higher hemolytic rate, increased RBC senescence and greater propensity of RBC to sickle under deoxygenation, ii) oxidative stress and senescence markers were greater in mature sickle RBC containing mitochondria compared to those without. Martino et al.5 reported that the abnormal retention of mitochondria in sickle RBC was the result of a lower expression of mitophagy inducers PINK1, NIX and of a higher expression of HSP90 chaperone. The interest in studying mitochondria retention in sickle RBC is based

Discussion

Figure 4. Comparisons of oxidative stress and senescence markers between mature sickle red blood cells containing mitochondria and mature sickle red blood cells without. (A) Ca2+ levels, (B) intracellular reactive oxygen species (ROS), (C) red blood cells (RBC) exposing phosphatidylserine (PS) and (D) CD47 expression. MFI: mean fluorescence intensity. mito: mitochondria. Significant difference: *P<0.05; **P<0.01; ****P<0.0001.

Figure 5. Comparisons of red blood cell rheological parameters between the three groups of sickle cell patients categorized according to the percentage of mature sickle red blood cells containing mitochondria (low group N=20, moderate group N=21, high group N=21) and healthy individuals (AA group N=10). (A) EImax and (B) point of sickling (PoS). Elmax: red blood cell (RBC) deformability in normoxia; ns: no significant difference. Significant difference: *P<0.05; **P<0.01; ***P<0.001; ****<0.0001. Haematologica | 108 November 2023

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Figure 6. Influence of the presence of mitochondria in mature sickle red blood cells on hematological parameters. (A-D) Comparison of hematological parameters and (E-F) hemolytic markers between the 3 groups (low group N=20, moderate N=21, high N=21). HbF: percentage of fetal hemoglobin; MCV: mean corpuscular volume; MCHC: mean corpuscular hemoglobin concentration; ns: no significant difference. Significant difference: *P<0.05; **P<0.01.

on the fact that these organelles may be a source of ROS that could damage RBC and accelerate their death/removal from blood circulation. Our results did not show any difference in RBC oxidative stress markers between patients with high mitochondria retention in mature RBC and those with low retention. The lack of association between RBC oxidative stress level and the degree of mitochondria retention in mature RBC is in agreement with the study of Martino et al.5 However, the intracellular environment of sickle RBC is already characterized by a high level of oxidative stress caused by a high HbS auto-oxidation rate, increased NADPH oxidase activity and low antioxidant defense.17 In addition, the probe used to assess intracellular ROS in our study (i.e., DCFDA), although having several advantages, also has some limitations. DCFDA reacts with both nitric oxide and ROS18 and DCF formation is increased in the presence of heme-containing molecules, like peroxidase,

hematin or metal ions with redox action.19,20 Thus, even if the mitochondria in sickle RBC were functional, it is tempting to hypothesize that their contribution to the whole intracellular ROS production and RBC oxidative stress is too weak to be probed specifically by DCFDA. Nevertheless, the comparison of intracellular ROS levels between mature RBC containing mitochondria and mature RBC without, showed higher ROS content in the former RBC subpopulation. Indeed, the lack of intracellular ROS difference between the three groups could be due to the fact that the higher intracellular ROS content of the mature RBC containing mitochondria was diluted in the whole RBC population (i.e., mature RBC with and without mitochondria retention). The use of a more specific probe for mitochondrial superoxide anion detection (i.e., MitoSox Red) indicated the capability of these RBC mitochondria to produce superoxide anions, especially after the incubation with Antimycin A, a blocker of Mi-

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tochondrial Complex III, which resulted in increased mitochondrial ROS accumulation. Indeed, we suspect that mitochondria in mature sickle RBC could contribute to the production of ROS and thus explain why inducing mitophagy in a sickle mice model resulted in a decrease of RBC ROS levels and increased RBC survival.9 The functionality of mitochondria in mature RBC from sickle cell patients was confirmed by the respiration assay experiments. Indeed, we observed a mitochondria-dependent oxygen consumption in sickle RBC and the use of oligomycin demonstrated that about 48.7% of endogenous oxygen consumption was related to the production of ATP. Image Stream experiments also showed that active mitochondria are present in sickle RBC, further supporting mitochondrial functionality in mature sickle RBC. Although we were not able to measure individually the deformability of mature sickle RBC with mitochondria and of mature sickle RBC without mitochondria, we observed that patients with a high percentage of mitochondria retention in mature RBC had a lower mean RBC deformability and a greater propensity of RBC to sickle under deoxygenation compared to patients with a lower percentage of mature RBC containing mitochondria. Indeed, it is possible that a higher rate of oxygen consumption by mitochondria contained in mature RBC from SCA patients could facilitate sickling by decreasing the amount of oxygen available for HbS. The subsequent greater rheological alterations would then increase RBC fragility,2 which may explain the lower hematocrit and greater hemolytic rate found in the patients with high mitochondria retention in mature RBC, as previously reported.5 Moreover, the production of ROS by mitochondria could affect the RBC membrane and increase the risk of RBC membrane disruption. Alternatively, a high rate of hemolysis could induce abnormal erythropoiesis and altered RBC maturation process, which would lead in turn to a greater retention of mitochondria in mature RBC. Further studies are needed to investigate the mechanisms at the origin of the relationships between mitochondria retention in mature RBC and increased hemolysis in SCA patients. Patients with a high percentage of mature RBC containing mitochondria exhibited higher percentages of RBC with externalized PS, which could indicate an alteration of membrane asymmetry following scramblase stimulation and accelerated RBC senescence. The comparisons of mature RBC subpopulations according to the presence or not of mitochondria showed a higher percentage of RBC with externalized PS in mature RBC with mitochondria retention and that mature RBC with mitochondria had greater level of intracellular Ca2+ and lower membrane CD47 compared to mature RBC with no mitochondria. Of note, the evaluation of the percentages of RBC exposing PS was assessed using Annexin V, a protein with a high affinity for PS but that could also enter cells with membrane alterations and bind PS on the inner leaflet. This could have led to an overestimation

of external PS and the use of lactadherin should be considered in future studies. Increased cation permeability, and more particularly of Ca2+, has been reported in sickle RBC compared to normal RBC,21 notably in deoxygenated condition.22 Hanggi et al.23 reported increased Ca2+ conductance through NMDA receptors in sickle RBC and Wang et al.24 demonstrated increased Ca2+ accumulation in response to lysophosphatidic acid in sickle RBC compared to control RBC. Very recently, an important role of Piezo1 has been reported in SCA that may increase intracellular Ca2+ levels in sickle RBC.21 Intracellular accumulation of Ca2+ is known to stimulate scramblase, leading to a disruption of the membrane phospholipid asymmetry and PS externalization.25 The presence of mitochondria could also participate in the elevation of intracellular Ca2+ levels, as mitochondria have a remarkable ability to take up and store massive amounts of Ca2+,26 as well as in the release of it into the cytosol.27 Although mitochondria would provide RBC with ATP, the low residual mitochondria in sickle RBC are unable to maintain ATP levels high enough to ensure Ca2+ ATP-dependent extrusion. It is well known that reticulocytes still have the endoplasmic reticulum, which is a major source of Ca2+.28 Reticulocytes normally lose all their remaining organelles during maturation. The abnormal retention of mitochondria and the increased Ca2+ and PS exposure in mature RBC could point to a defect in the final maturation of reticulocytes.29 Further studies are needed to analyze the content in endoplasmic reticulum proteins29 in mature RBC in SCA. Furthermore, Ca2+ may cause the activation of the Gardos channel pathway, which mediates rapid K+ and Cl- efflux and water loss.30 The resulting cellular dehydration facilitates HbS polymerization and RBC sickling, which could explain why SCA patients with the greatest percentage of mature RBC containing mitochondria were also those with a lower deformability, a greater MCHC and a higher point of sickling, i.e., with a greater propensity of RBC to sickle under deoxygenation. In conclusion, this study showed the presence of functional mitochondria in mature sickle RBC, which could favor RBC sickling and accelerate RBC senescence, leading to increased cellular fragility and hemolysis. Disclosures No conflicts of interest to disclose. Contributions SE, EN, AS, RVB, CB, DR and PC designed the research. SE, EN, AS, CB, RC, MM, MR, CB, CR, PJ and DR performed the biological analyses. AG and SP included patients. SE, EN, AS and PC performed the statistical analyses. SE, EN, AS and PC wrote the first draft of the paper. CB, RC, MM, MR, FH, AC, CR, RVB, CB, YD, PJ, AG, SP, MR and DR read and approved the final version of the manuscript.

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Funding This study was supported by the European Framework Horizon 2020 under grant agreement number 860436 (EVIDENCE).

Data-sharing statement Data are available upon reasonable request to the corresponding author.

References 1. Kato GJ, Gladwin MT, Steinberg MH. Deconstructing sickle cell disease: reappraisal of the role of hemolysis in the development of clinical subphenotypes. Blood Rev. 2007;21(1):37-47. 2. Connes P, Lamarre Y, Waltz X, et al. Haemolysis and abnormal haemorheology in sickle cell anaemia. Br J Haematol. 2014;165(4):564-572. 3. Jang T, Poplawska M, Cimpeanu E, et al. Vaso-occlusive crisis in sickle cell disease: a vicious cycle of secondary events. J Transl Med. 2021;19(1):397. 4. Nader E, Romana M, Connes P. The red blood cell-inflammation vicious circle in sickle cell disease. Front Immunol. 2020;11:454. 5. Martino S, Arlet JB, Odievre MH, et al. Deficient mitophagy pathways in sickle cell disease. Br J Haematol. 2021;193(5):988-993. 6. Moriconi C, Dzieciatkowska M, Roy M, et al. Retention of functional mitochondria in mature red blood cells from patients with sickle cell disease. Br J Haematol. 2022;198(3):574-586. 7. Tumburu L, Ghosh-Choudhary S, Seifuddin FT, et al. Circulating mitochondrial DNA is a proinflammatory DAMP in sickle cell disease. Blood. 2021;137(22):3116-3126. 8. Mortensen M, Ferguson DJ, Edelmann M, et al. Loss of autophagy in erythroid cells leads to defective removal of mitochondria and severe anemia in vivo. Proc Natl Acad Sci U S A. 2010;107(2):832-837. 9. Jagadeeswaran R, Vazquez BA, Thiruppathi M, et al. Pharmacological inhibition of LSD1 and mTOR reduces mitochondrial retention and associated ROS levels in the red blood cells of sickle cell disease. Exp Hematol. 2017;50:46-52. 10. Rivers A, Jagadeeswaran R, Lavelle D. Potential role of LSD1 inhibitors in the treatment of sickle cell disease: a review of preclinical animal model data. Am J Physiol Regul Integr Comp Physiol. 2018;315(4):r840-r847. 11. Nohl H, Gille L, Staniek K. Intracellular generation of reactive oxygen species by mitochondria. Biochem Pharmacol. 2005;69(5):719-723. 12. Barodka VM, Nagababu E, Mohanty JG, et al. New insights provided by a comparison of impaired deformability with erythrocyte oxidative stress for sickle cell disease. Blood Cells Mol Dis. 2014;52(4):230-235. 13. Sjovall F, Ehinger JK, Marelsson SE, et al. Mitochondrial respiration in human viable platelets - methodology and influence of gender, age and storage. Mitochondrion. 2013;13(1):7-14. 14. Stier A, Romestaing C, Schull Q, et al. How to measure mitochondrial function in birds using red blood cells: a case study in the king penguin and perspectives in ecology and evolution. Meth Ecol Evol. 2017;8(10):1172-1182. 15. Boisson C, Rab MAE, Nader E, et al. Effects of genotypes and treatment on oxygenscan parameters in sickle cell disease. Cells. 2021;10(4):811.

16. Rab MAE, Kanne CK, Bos J, et al. Oxygen gradient ektacytometry-derived biomarkers are associated with vasoocclusive crises and correlate with treatment response in sickle cell disease. Am J Hematol. 2021;96(1):e29-e32. 17. Hebbel RP. Reconstructing sickle cell disease: a data-based analysis of the "hyperhemolysis paradigm" for pulmonary hypertension from the perspective of evidence-based medicine. Am J Hematol. 2011;86(2):123-154. 18. Aslan M, Thornley-Brown D, Freeman BA. Reactive species in sickle cell disease. Ann N Y Acad Sci. 2000;899:375-391. 19. Fuloria S, Subramaniyan V, Karupiah S, et al. Comprehensive review of methodology to detect reactive oxygen species (ROS) in mammalian species and establish its relationship with antioxidants and cancer. Antioxidants (Basel). 2021;10(1):128. 20. Tarpey MM, Wink DA, Grisham MB. Methods for detection of reactive metabolites of oxygen and nitrogen: in vitro and in vivo considerations. Am J Physiol Regul Integr Comp Physiol. 2004;286(3):R431-444. 21. Nader E, Conran N, Leonardo FC, et al. Piezo1 activation augments sickling propensity and the adhesive properties of sickle red blood cells in a calcium-dependent manner Br J Haematol. 2023 Apr 3. 2023;202(3):657-668. 22. Brugnara C. Sickle cell dehydration: pathophysiology and therapeutic applications. Clin Hemorheol Microcirc. 2018;68(2-3):187-204. 23. Hanggi P, Makhro A, Gassmann M, et al. Red blood cells of sickle cell disease patients exhibit abnormally high abundance of N-methyl D-aspartate receptors mediating excessive calcium uptake. Br J Haematol. 2014;167(2):252-264. 24. Wang J, Hertz L, Ruppenthal S, et al. Lysophosphatidic acidactivated calcium signaling is elevated in red cells from sickle cell disease patients. Cells. 2021;10(2):456. 25. Bevers EM, Williamson PL. Phospholipid scramblase: an update. FEBS Lett. 2010;584(13):2724-2730. 26. Strubbe-Rivera JO, Schrad JR, Pavlov EV, et al. The mitochondrial permeability transition phenomenon elucidated by cryo-EM reveals the genuine impact of calcium overload on mitochondrial structure and function. Sci Rep. 2021;11(1):1037. 27. Takeuchi A, Kim B, Matsuoka S. The destiny of Ca(2+) released by mitochondria. J Physiol Sci. 2015;65(1):11-24. 28. Moras M, Lefevre SD, Ostuni MA. From erythroblasts to mature red blood cells: organelle clearance in mammals. Front Physiol. 2017;8:1076. 29. Brusson M, Cochet S, Leduc M, et al. Enhanced calreticulin expression in red cells of polycythemia vera patients harboring the JAK2(V617F) mutation. Haematologica. 2017;102(7):e241-e244. 30. Maher AD, Kuchel PW. The Gardos channel: a review of the Ca2+-activated K+ channel in human erythrocytes. Int J Biochem Cell Biol. 2003;35(8):1182-1197.

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Engineered human Diamond-Blackfan anemia disease model confirms therapeutic effects of clinically applicable lentiviral vector at single-cell resolution Yang Liu,1,2* Ludwig Schmiderer,1* Martin Hjort,3,4,5 Stefan Lang,6 Tyra Bremborg,1 Anna Rydström,1 Axel Schambach,7,8 Jonas Larsson1 and Stefan Karlsson1

Correspondence: Yang Liu yang.liu@med.lu.se

Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden; 2Department of Medicine, Huddinge, Karolinska Institutet, Stockholm, Sweden; 3Chemical Biology and Therapeutics, Department of Experimental Medical Science, Lund University, Lund, Sweden; 4Navan Technologies, MBC Biolabs, San Carlos, CA, USA; 5 NanoLund, Lund University, Lund, Sweden; 6Division of Molecular Hematology and Stem Cell Center, Lund University, Lund, Sweden; 7Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany and 8Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA 1

Stefan Karlsson stefan.karlsson@med.lu.se Received: Accepted: Early view:

September 8, 2022. May 11, 2023. May 18, 2023.

*YL and LS contributed equally as first authors.

Abstract Diamond-Blackfan anemia is a rare genetic bone marrow failure disorder which is usually caused by mutations in ribosomal protein genes. In the present study, we generated a traceable RPS19-deficient cell model using CRISPR-Cas9 and homology-directed repair to investigate the therapeutic effects of a clinically applicable lentiviral vector at single-cell resolution. We developed a gentle nanostraw delivery platform to edit the RPS19 gene in primary human cord bloodderived CD34+ hematopoietic stem and progenitor cells. The edited cells showed expected impaired erythroid differentiation phenotype, and a specific erythroid progenitor with abnormal cell cycle status accompanied by enrichment of TNFα/NF-κB and p53 signaling pathways was identified by single-cell RNA sequencing analysis. The therapeutic vector could rescue the abnormal erythropoiesis by activating cell cycle-related signaling pathways and promoted red blood cell production. Overall, these results establish nanostraws as a gentle option for CRISPR-Cas9based gene editing in sensitive primary hematopoietic stem and progenitor cells, and provide support for future clinical investigations of the lentiviral gene therapy strategy.

Introduction Diamond-Blackfan anemia (DBA) is a rare genetic bone marrow failure disorder characterized by anemia and is associated with physical malformations and a predisposition to cancer. Around 75% of DBA cases are related to a heterozygous allelic variation in ribosomal protein genes of either small or large ribosomal subunit.1 The most frequently affected gene is ribosomal protein S19 (RPS19), which accounts for approximately 25% of the cases that occur in 6 out of 1 million live births.2 The defect has been defined functionally by a reduced ability to generate erythroid burst-forming unit (BFU-E) and erythroid colonyforming unit (CFU-E) colonies, and are associated with aberrant ribosome biogenesis and activation of p53-dependent apoptotic pathways.1,3,4 However, despite significant progress in understanding the molecular basis of DBA pathophysiology, up to now, there is no curable treat-

ment for patients except allogeneic bone marrow transplantation.1,2 Our group recently developed a self-inactivating lentiviral vector containing a codon-optimized RPS19 cDNA driven by the clinically applicable promoter, shortened version of human elongation factor 1α (EFS), and demonstrated the therapeutic effects in RPS19-deficient DBA mice.5 Due to the very limited availability of patient samples, creating a human cell model that faithfully mimics the naturally occurring loss of RPS19 would be critical for the development of a gene therapy approach that can eventually be used to treat patients. To achieve this, we aimed to generate a RPS19 haploinsufficient cell model using CRISPR-Cas9 (Cas9) and an adeno-associated virus (AAV)-based HDR template carrying a traceable marker gene via homologous recombination that can be used to label and identify successfully edited cells by their marker gene expression. Initially, we de-

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ARTICLE - scRNA-seq analysis of engineered human DBA model livered Cas9 editing cargo with electroporation, but found it difficult to recover sufficient numbers of viable edited cells. This is likely due to a combination of several factors. First, since homozygous loss of RPS19 is lethal, any biallelic knockout (KO) cells are not expected to be viable.2,6-8 Secondly, heterozygous RPS19 KO cells are expected to be viable, but highly sensitive to stress, since monoallelic loss of RPS19 generates nucleolar stress, p53 stabilization and activation of its targets.1 Last but not least, both electroporation and Cas9 activity have been shown to impair the function and viability of treated hematopoietic stem and progenitor cells (HSPC).9,10 The combined stress of these factors makes it challenging to generate an ideal RPS19-deficient CD34+ HSPC model. Novel non-viral transfection methodologies such as nanostraws have been shown to be a gentle and non-toxic alternative to electroporation.11-13 Nanostraws are hollow alumina nanotubes that can be used to inject mRNA and other biomolecules directly into the cytoplasm via the application of a gentle, pulsed electric field.11 Until now, however, they have only been used to successfully deliver smaller mRNA (e.g., gene fluorescent protein, GFP) to human HSPC. Efficient nanostraw-mediated Cas9 delivery to primary cells has so far not been demonstrated, even though this would be a major advancement for the generation of desired cell models in situations where the number of available cells is limited. In the present study, we demonstrate nanostraw-mediated Cas9 mRNA delivery that enables gentle gene editing in human CD34+ HSPC. Using this approach, we successfully generated a traceable RPS19-deficient cell model, and confirm the therapeutic effect of the therapeutic vector with erythroid differentiation assays and single-cell transcriptomics.

Methods Primary human samples, isolation and transduction of hematopoietic stem and progenitor cells Human cord blood samples were obtained from the maternity wards of Helsingborg General Hospital and Skåne University Hospital in Lund and Malmö, Sweden, after informed, written consent according to guidelines approved by the regional ethical committee. Mononuclear cells were separated through density-gradient centrifugation, as described previously.5 Cells were cultured in serum-free expansion medium, supplemented with stem cell factor, thrombopoietin, and FLT3-ligand at 100 ng/mL (all from Stem Cell Technologies). Transduction of the therapeutic lentiviral vector was performed at a multiplicity of infection (MOI) of 5, according to the published protocol.5

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Other experimental methods Descriptions of other methods used in the study are provided in the Online Supplementary Appendix.

Results Delivery of Cas9 and RPS19-targeting sgRNA with electroporation leads to loss of cell viability The primary aim of the study was to create a traceable RPS19-deficient cell model using Cas9 via homologous recombination in cord blood-derived human CD34+ HSPC.14 To this end, we selected a sgRNA with high editing efficiency and which causes an obviously impaired erythroid differention block phenotype (Online Supplementary Figure S1A, B). To make the edited cell traceable, we designed a complementary adeno-associated virus (AAV)-based homology-directed repair (HDR) template that allows GFP expression driven by PGK promoter (Figure 1A) (see Methods). With this system, cells with the expected RPS19-deficiency can be distinguished from unedited cells by their GFP expression. Moreover, by taking advantage of the lethal effects of homozygous loss of the RPS19 gene, the viable GFP+ cells are expected to be carrying heterozygous loss of RPS19.2,6-8 Initially, we used electroporation to deliver Cas9 RNP or mRNA into CD34+ HSPC, followed by AAV transduction at optimized MOI (Figure 1B). Almost no GFP signal can be detected when the MOI is equal or less than 5x104 vg/cell on day 4 in the AAV-only treated group (Online Supplementary Figure S2A-C). We decided to transduce with AAV at MOI of 5x104 vg/cell, which indicates that any GFP signals detected on day 4 or later should be primarily from successful HDR. Considering that the complete loss of RPS19 causes lethal effects, we included a control condition in which the cell surface marker CD45 was targeted, the KO of which is well tolerated by CD34+ HSPC.15 The cell viability was measured one day after editing. We found about 26% live cells (7-AADAnnexin V-) were recovered in the Cas9 RNP RPS19-edited group relative to the untreated group (Figure 1C, E) and up to 12% GFP+ cells (Figure 1C, F) on day 1 post electroporation. In the Cas9 RNP CD45-edited group, about 35% live cells were recovered relative to the untreated group (Online Supplementary Figure S3A, C). On day 4, however, less than 3% live cells in the Cas9 RNP RPS19-edited group and 28% live cells in the Cas9 RNP CD45-edited group were recovered relative to the untreated group (Figure 1D, E, Online Supplementary Figure S3B, C). Importantly, only very few cells carrying the desired edit and template insertion, i.e., being GFP positive, were recovered on day 4 in the RPS19edited group (Figure 1D, F). By counting the total number, only up to 150 live GFP+ cells can be obtained on day 4 from a starting cell number of 2x104 CD34+ HSPC, and the low efficiency therefore hinders further downstream analysis. Compared with this, the CD45 KO efficiency is about 63%

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ARTICLE - scRNA-seq analysis of engineered human DBA model in live cells in the Cas9 RNP CD45-edited group (Online Supplementary Figure S3B, D). This strongly indicates that the loss of RPS19 has a detrimental effect on the cells, which is possibly exacerbated by the stress from the electroporation. Since the negative effect of electroporation on HSPC function and viability is well documented,9 we decided to use a milder delivery method that might enable the recovery of higher numbers of successfully edited cells. Nanostraws enable gentle gene editing with improved cell viability Arguably the most gentle method of delivering nucleic acids to human HSPC is nanostraws.10 We previously showed that both the viability and function of CD34+ are fully maintained after nanostraw-mediated delivery of a

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transiently expressed mRNA (Figure 2A).10 To investigate if nanostraws could be used for stable gene editing via Cas9 mRNA delivery, we first attempted to KO CD45 (Figure 2B). As shown in Figure 2C-E, total live cell numbers at a rate of 75% (day 1) and 53% (day 4) relative to completely untreated cells could be recovered upon CD45targeting using Cas9 mRNA delivery. The efficiency of CD45 KO can reach up to 23% (Figure 2D, F). We also attempted to deliver Cas9 RNP instead of mRNA, but found that the efficiency was low (approx. 3.5% on average on day 4) (Figure 2D, F), which may be due to the differences in structure and charge between the protein and the GFP-mRNA previously used.10 Therefore, we decided to use Cas9 mRNA for the following experiments. We next investigated whether changing the delivery

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Figure 1. Genome editing via homologous recombination using electroporation leads to strongly decreased cell viability. (A) Schematic overview of RPS19 editing strategy. The RPS19 gene is targeted with Cas9, and gene fluorescent protein (GFP)-encoding adeno-associated virus (AAV) with homology arms flanking the cut site. Successful integration of the homology-directed repair (HDR) template leads to disruption of RPS19 expression, which allows traceable GFP expression. (B) Timeline of experiment. (C and D) Representative FACS plots of cell viability and GFP expression of cells on (C) day 1 and (D) day 4 after electroporation. (E) Percentage of live cell recovery relative to completely untreated cells on days 1 and 4 after electroporation (*P<0.05 by t-test, N=3). (F) Relative percentage of GFP+ cells compared to total recovered live cell numbers of the untreated condition, on days 1 and 4 after electroporation (*P<0.05, **P<0.01, ****P<0.0001 by one-way ANOVA test, N=3). LHA: left homology arm; RHA: right homology arm; HSPC: hemotopoietic stem and progenitor cells.

method from electroporation to nanostraws could improve the cell viability and recovery of RPS19-deficient cells. To this end, we delivered Cas9 mRNA and RPS19targeting sgRNA to CD34+ HSPC with nanostraws and added the AAV HDR-template at the optimized MOI to the cells (Figure 3A). In the RPS19-deficient group, we obtained similar numbers of viable cells (7-AAD-Annexin V-) compared to the untreated group on day 1, and 70% 7-AAD- cells relative to the untreated group on day 4 (Figure 3B, C). This was an enormous improvement over the electroporation results, where less than 3% viable cells compared to the untreated condition could be recovered on day 4. Importantly, we could obtain far more viable GFP+ cells on day 1 (12% on average) and day 4 (1% on average; Figure 3B, D) compared to electroporation (0.2% on average on day 4). On average, nanostraws en-

abled the recovery of 130-fold more live cells than electroporation, and about 7-fold more GFP+ cells than electroporation when treating the same initial number of cells (Online Supplementary Figure S4A). In addition, deep sequencing was performed on edited cells with no HDR template added. The frequency of insertions and deletions (indels) was on average 12% on day 1, but decreased to about 1% on day 4 (Online Supplementary Figure S4B). The decrease of the indels likely reflects the lethal effects of homozygous loss of the RPS19 gene. Consistent with this, we could generate around 1% live GFP+ cells on day 4. To detect the integration of the HDR template into the RPS19 locus and to assess the differentiation ability of edited cells, we sorted 7-AAD-GFP+ cells from the edited group and 7-AAD- cells from the mock group. PCR with

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ARTICLE - scRNA-seq analysis of engineered human DBA model primers flanking the homology arms confirmed that the HDR template integrated at the expected site (Online Supplementary Figure S5A, B). This was further confirmed by Sanger Sequencing of the PCR product (Online Supplementary Figure S5C). To investigate the differentiation ability, a CFU assay was performed. The number of colonies for BFU-E, CFU-G/M/GM, and CFU-GEMM in the edited group was significantly lower than in the mocktreated group (Figure 3E). In particular, GFP can be detected in individual colonies from the edited group (Online Supplementary Figure S6). To distinguish mono and bi-allelic integration into the RPS19 locus of edited cells, single colonies were picked and used to perform digital droplet PCR (ddPCR) (Online Supplementary Figure S7). The abundance of the edited allele compared to the alleles of a reference gene (APOE, on same chromosome) are in the range of 40-60% in the majority of colonies (85%), which indicated monoallelic integration in the GFP+ cells that successfully formed colonies (Figure 3F). This further confirms the generation of heterozygous RPS19 loss in our model. In particular, about 15% of all the colonies were found GFP-negative in the edited group on day 14, which may be due to the cytoplasmic non-integrated-AAV that also express GFP when we sorted the cells on day 4, and then gradually became GFP-negative after cell proliferation during culture. Overall, our results demonstrate a successful gentle genome editing strategy for generating RPS19-deficient CD34+ HSPC at levels where the edited cells are sufficient for further downstream analysis.

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EFS-RPS19 vector rescued impaired erythroid differentiation in RPS19-deficient hematopoietic stem and progenitor cells We next analyzed if the edited cells have the expected erythroid differentiation block phenotype, and whether this disease phenotype can be ameliorated by our therapeutic lentiviral vector, EFS-RPS19. Briefly, RPS19-deficient cells (RPS19-D) were generated using nanostraws after 2 days of culture (Figure 4A). A fraction of the RPS19D cells was transduced with the EFS-RPS19 (LV-RPS19) vector one day later. CD34+ HSPC without treatment (CD34), or Cas9-only treated cells (without sgRNA, named Cas9) were used as control groups. We sorted 7-AAD-GFP+ cells from the RPS19-D group on day 4, and these were cultured in erythroid differentiation medium. On the same day, the expression of endogenous RPS19 and the codonoptimized RPS19 (coRPS19) produced by EFS-RPS19 was measured by RT-qPCR. As expected, expression levels of endogenous RPS19 were strongly reduced in GFP+ cells, which further confirms the successful generation of RPS19-deficient CD34+ HSPC (Figure 4B). Interestingly, endogenous RPS19 expression was significantly reduced in cells transduced with the therapeutic vector, which might be caused by a compensatory mechanism triggered by the overexpression of the transgene RPS19 (Figure 4C). It is also possible that survival of potential homozygous RPS19-deficient cells that would not have survived otherwise was enabled by transduction with the EFS-RPS19 vector. The edited cells showed impaired erythroid differentiation

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Figure 2. Nanostraw-mediated Cas9 mRNA delivery enables CD45 knockout in human hematopoietic stem and progenitor cells. (A) Schematic overview and false-colored scanning electron microscope picture of the nanostraw delivery system. (B) Timeline of experiment. (C) Representative FACS plots of cell viability (7-AAD-/Annexin-) on day 1 (D1). (D) Representative FACS plots of edited cells on day 4. (E) Percentage of live cell recovery compared to completely untreated cells on days 1 and 4 upon Cas9 mRNA and RNP delivery to knockout CD45 (*P<0.05, **P<0.01, ***P<0.001 by one-way ANOVA test, N=3). (F) Efficiency of CD45 knockout in live (7-AAD-) cells on day 4 (***P<0.001, ****P<0.0001 by one-way ANOVA test, N=3). HSPC: hematopoietic stem and progenitor cells. Haematologica | 108 November 2023

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ARTICLE - scRNA-seq analysis of engineered human DBA model with increased numbers of progenitor cells (CD71-CD235) and BFU-E/CFU-E cells (CD71+CD235-) on day 10 (Figure 4D). This is consistent with clinical observations that differentiation of patient cells is inhibited at the initial stage (BFU-E and CFU-E).1 The erythroid differentiation block is consistently present throughout all measured timepoints

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(days 6, 8 and 10) during differentiation (Figure 4E). In contrast, cells treated with EFS-RPS19 could be rescued from the impaired erythroid differentiation, and showed significantly increased production of erythroblasts (CD71+CD235+) and mature red blood cells (CD71-CD235+) at all timepoints. The RPS19-D group produced a smaller

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Figure 3. Delivery of Cas9 mRNA with nanostraws enables the recovery of heterozygous GFP+ RPS19-deficient hematopoietic stem and progenitor cells with reduced cell viability. (A) Timeline of experiment. (B) Representative FACS plots of cell viability and GFP+ cells on (left) day 1 and (right) day 4. (C) Percentage of live cell recovery compared to completely untreated cells on days 1 (D1) and 4 upon using nanostraw to deliver Cas9 mRNA (*P<0.05 by t-test, N=3). (D) Relative percentage of GFP+ cells compared to total recovered live cell numbers of the untreated condition, using a nanostraw to deliver Cas9 mRNA on days 1 and 4 (**P<0.01, ***P<0.001 by t-test, N=3). (E) Number of colonies for BFU-E, CFU-G/M/GM and CFU-GEMM in each dish after culture with methylcellulose media for 14 days (####P<0.0001, #P<0.05 compared with the same colony category in the RPS19-deficient group by unpaired Mann-Whitney test, N=12 dishes in each group). (F) Ratio of edited allele (HDR-RPS19GFP) to reference gene (APOE) by ddPCR (a total of 20 colonies in the mock group, and 100 colonies in the RPS19-deficient group were analyzed). HSPC: hematopoietic stem and progenitor cells.

and fainter red pellet compared to the CD34 group, or the Cas9-only group, while the LV-RPS19 group produced larger pellets than the RPS19-D group on day 21 (Figure 4F). Overall, the results show that we successfully created a RPS19-deficient model that accurately mimics the impaired DBA erythroid differentiation phenotype, which could be rescued by our therapeutic EFS-RPS19 vector. Single-cell transcriptomic and differentiation trajectory during erythroid differentiation We next performed single-cell RNA sequencing (scRNAseq) to analyze the transcriptome of RPS19-D cells before and after treatment with EFS-RSP19 during the early stage of erythroid differentiation. Briefly, we cultured the sorted RPS19-deficient cells in erythroid differentiation medium for 6 days, followed by sorting GFP+ cells from the RPS19D and the LV-RPS19 group, and GFP-negative cells from the CD34 group for scRNA-seq analysis. The expression of GFP, endogenous RPS19 and EFS-RPS19-produced RPS19 (coRPS19) were checked in each group and were consistent with our previous results (Online Supplementary Figure S8). Unsupervised clustering by the Leiden method identified 11 distinct clusters from all the samples.16 We identified clusters based on the markers for hematopoietic progenitor compartments, megakaryocyte progenitors, and erythroid progenitors (EP) as described in the

Methods (Figure 5A, Online Supplementary Figure S9). For the identified EP-related clusters, the late EPs1 (LEPs1) showed high HBB expression and the LEPs2 showed high HBA1 and HBA2 expression (Figure 5A). The expression of GYPA increased gradually from the LEPs1 to the LEPs3, which may indicate the gradual erythroid differentiaton. This is further supported by the gradual increased expression of KLF1 (Figure 5A). On the contrary, both the 2 DBA EP showed lower HBB, HBA1 and HBA2 expression compared to the LEP. Specifically, we did not observe obvious differences on the expression of transcription factors of GATA1 and GATA2 among LEP clusters. In addition, cell cycle genes (MKI67 and AURKB) showed very low expression in the DBA EPs1, which is also the main LEP cluster in the RPS19-D group (38.9%, Figure 5B). The DBA EPs2 showed higher MKI67 and AURKB expression, but still lower expression of hemoglobin genes compared to the 3 LEP. Importantly, there was an almost 50% reduction of the DBA EPs1 cluster in the LV-RPS19 group (19.9%). Instead, the Rescued EP cluster became the main cluster (23.1%), with higher expression of hemoglobin (HBA1, HBA2) and cell cycle (MKI67 and AURKB) genes compared to the 2 DBA EP clusters. We next used a force-directed graph drawing algorithm, Force-Atlas2, to infer the differentiation trajectory of the cells during erythroid differentiation (Figure 5C).17 The ob-

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Figure 4. RPS19-deficient cells showed impaired erythroid differentiation ability which can be rescued by the lentiviral EFS-RPS19 vector. (A) Schematic overview of erythroid differentiation analysis of RPS19-deficient CD34+ cord blood hematopoietic stem and progenitor cells (HSPC). (B) Expression of endogenous RPS19 (*P <0.05, **P <0.01, ***P <0.001 by two-way ANOVA test, N=3). (C) Transgene RPS19 (coRPS19) expression (****P<0.0001 by two-way ANOVA test, N=3). (D) Representative FACS plots of GFP+ cells for erythroid differentiation on day (D) 10 in each group. (E) Statistical analysis of each population during erythroid differentiation from stage I (day 6) to stage II (day 10) (#P<0.001 compared to the CD34 and the Cas9 only groups; **P<0.01 compared to the RPS19-D group; ***P<0.001 compared to the RPS19-D group; ****P<0.0001 compared to the RPS19-D group, by two-way ANOVA test, N=3). (F) Formation of red blood cell pellets on day 21 in each group. EM: expansion medium; ED: erythroid differentiation. Haematologica | 108 November 2023

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ARTICLE - scRNA-seq analysis of engineered human DBA model tained global topology revealed high connections of the EEP with the LEP and Rescued EP, compared to the DBA EPs1, which were further supported by the partition-based approximate graph abstraction (PAGA) graph. The hemoglobin genes were highly expressed in the LEP and the Rescued EP (Online Supplementary Figure S10). These results indicate the abnormal erythroid differentiation of the DBA EPs1. Overall, specific clusters were identified in the RPS19-D and the LV-RPS19 group. The Rescued EP showed reversed erythroid marker gene expressions and higher correlations with the LEP in the CD34 group than the DBA EPs1 by the differentiation trajectory analysis. EFS-RPS19 helps to recover abnormal cell cycle in the erythroid progenitors of the RPS19-D group Since several LEP were observed, we next focused on the their transcriptomic differences. We compared genes related to erythroid progenitors and cell cycle according to a previous publication.3 There was an obvious reduction in the EP-related cell cycle genes in the DBA EPs1 (TUBA1B, TUBB, TUBB4B, etc.) (Online Supplementary Figure S11). On the contrary, the cell cycle-related genes were more

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highly expressed in the DBA Eps2, as mentioned above. We further analyzed the cell-cycle phase in all the LEP clusters. The DBA EPs1 showed a high percentage of genes in G1 phase (81.9%), while the DBA EPs2 showed a high percentage of genes in G2M (72.6%) and S phase (21.5%) compared to the other clusters (Figure 6A). Importantly, the Rescued EP showed similar cell cycle stage (G1: 54.4%; G2M: 25.9%; S: 19.7%) to the LEPs1 (G1: 55.1%; G2M: 25.9%; S: 19%). These results demonstrate the characteristics of the DBA EPs1 with impaired cell-cycle stage and lower hemoglobin gene expressions. However, treatment with the therapeutic vector could activate the cell cycle and improve hemoglobin gene expressions, as observed in the Rescued EPs. Since the cell-cycle stage in the Rescued EP reached a similar level to the LEPs 1, we applied the gene set enrichment analysis (GSEA) algorithm analysis among the LEPs1 (from the CD34 group), the DBA EPs1 (from the RPS19-D group), and the Rescued EP (from the LV-RPS19 group). TNFα, p53 and apoptosis signaling pathways were enriched in the DBA EPs1 compared to the LEPs1 and the Rescued EP (Figure 6B). On the contrary, cell cycle-related signaling pathways (e.g., MYC targets, G2M checkpoint and mitotic spindle signaling pathways)

Figure 5. Clustering analysis and differentiation trajectory during erythroid differentiation. (A) Heatmap of the mean expression value of manually selected marker genes for each cluster. CMP: common myeloid progenitors; GMP: granulocytemacrophage progenitors; MKP: megakaryocyte progenitors; EEP: early erythroid progenitors; LEP: late erythroid progenitors. (B) UMAP plot colored by (left) cluster and (middle) UMAP plot split by tissue. (Right) Frequency of each cluster in CD34, RPS19-D, and LV-RPS19 groups. (C) Partition-based approximate graph abstraction (PAGA) initialized embedding and PAGA graph of the differentiation trajectory. Size of dots is proportional to number of cells in the clusters.

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and heme metabolism were highly enriched in both the LEPs1 and the Rescued EP clusters (Online Supplementary Tables S1, S2). This further supports the conclusion that EFS-RPS19 could reverse the impaired erythroid differentiation of the RPS19-D cells by activating the cell-cycle status. The top 20 differentially expressed genes are

shown in Figure 6C. Specifically, EFS-RPS19 could reverse the expression of several cell cycle-related genes, such as HMGB1, HMGB2 and TUBA1B, which may contribute to the therapeutic effects. The recovery of the cell-cycle genes in the Rescued EP was also observed when we compared it to the LEPs2 (Online Supplementary Figure

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ARTICLE - scRNA-seq analysis of engineered human DBA model S12), and some heat shock proteins (HSP90AA1 and HSPD1) were also found to be highly expressed in the Rescued EP. Although the DBA EPs2 showed high expression of cellcycle genes, low hemoglobin genes were still observed in the cluster. By comparing it with the LEP, some long noncoding RNA (lncRNA), such as MALAT1 and XACT, were found highly expressed in the DBA EPs2 (Online Supplementary Figure S13A, B). Taken together, we identified specific cluster with abnormal cell-cycle status and enriched TNFα, p53 and apoptosis signaling pathways in the RPS19-D group. On the contrary, the EFS-RPS19-treated cells could recover cell-

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cycle status through activation of cell cycle-related signaling pathways, which are similar to the levels in the CD34 group.

Discussion Due to the very limited avaliablility of the patient samples, a traceable and precise DBA cell model would be useful to explore mechanisms and allow therapeutic investigations. We previously generated a RPS19 knockdown cell model using shRNA.5 However, modeling of haploinsufficient

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Figure 6. RPS19-deficient cells show abnormal cell cycle with activation of inflammatory signaling pathway that can be rescued by EFS-RPS19. (A) Donut plots showing percentages of cells in G1, S, and G2M phase in erythroid progenitor (EP) clusters. (B) Bubble plot showing abnormal significantly enriched signaling pathways (NOM: P<0.05) in the DBA EP 1 cluster from the RPS19-deficient group compared with the LEP 1 cluster from the CD34 group and Rescued EP cluster from the LV-RPS19 group. Pathways are from the Hallmark gene sets of the Molecular Signatures Database. (C) Dot plot showing the 20 top differentially expressed genes in the indicated clusters.

human diseases like DBA is challenging because the phenotype is highly dependent on the level of gene downregulation. Moreover, the shRNA-based knockdowns can be a useful approach to model the effect of disease-causing mutations, but it is difficult to precisely mimic the effect of heterozygous gene loss. Furthermore, shRNA-based knockdowns induce wide-ranging unintended off-target effects on unrelated genes. These issues make it more difficult to generate accurate disease models and to determine precise cause-effect relationships with shRNA.18 In this study, we set out to create a traceable RPS19-deficient model by using CRISPR-Cas9 to validate and generate the transcriptomic landscapes of our previously developed therapeutic lentiviral vector. Since DBA already presents early in infancy, we decided to use umbilical cord blood HSPC from healthy donors as the starting material for generating the cell model. However, working with these cells comes with multiple challenges. First, only limited numbers of cells can be obtained from each donor, which limits the scale of experiments that can be performed.19 Second, they are highly sensitive to stress, such as that induced by electroporation.20 Third, they are challenging to transfect.21 In our results, the conventional method of using electroporation for Cas9 delivery turned out to be highly detrimental for the cells when RPS19 was targeted, and did not allow sufficient recovery of edited cells. We speculate that several parts of the procedure likely contributed to the toxicity. Programmable nucleases, such as Cas9, cut DNA and induce double-stranded breaks (DSB).22

DSB can trigger apoptosis, differentiation or replicative arrest in HSPC, and limit their long-term engraftment capacity.9 It has also been shown that delivering Cas9 to HSPC via electroporation is toxic and activates p53, which leads to decreased cell viability and function.14,23,24 Furthermore, electroporation itself triggers a strong stress response in HSPC, even when a more benign cargo, such as GFP mRNA, is delivered. All of this, however, is not enough to explain the extreme toxicity that we observed when targeting RPS19 with electroporation-based Cas9 delivery, since targeting a different gene (CD45) with the same method was much less toxic and allowed for the recovery of a reasonable number of cells. Deficiencies in ribosomal genes such as RPS19 have been shown to induce the activation of p53, p21, and apoptosis.1,25 This might have compounded the negative effects of the electroporation and DSB, and caused the complete loss of viability that we observed. To overcome the toxicity issue, we used nanostraws to deliver Cas9 to HSPC. We adapted the nanostraws and delivery parameters to facilitate efficient disruption of RPS19 in HSPC enabling recovery of sufficient numbers of edited cells, which make it possible to perform downstream functional studies such as verifying the therapeutic effects of our clinically-applicable lentiviral vector. This approach has allowed us to establish a geno- and phenotypically correct DBA disease model using cord blood-derived CD34+ HSPC. This method of generating cell models will be useful for studying other disease-related genes that, until now, could not easily be knocked out in sensitive pri-

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ARTICLE - scRNA-seq analysis of engineered human DBA model mary cell types. The mechanistic basis for DBA pathophysiology is not fully understood, and this has limited the development of new therapeutic modalities. We observed a 75% recovery on the erythroid differentiation (increased CD235 expression) after treatment with EFS-RPS19. Interestingly, this is also consistent with our previously reported transduction efficiency of the vector at the same MOI.5 By applied scRNAseq analysis, our data indicate that the vector exerts its therapeutic effects by reversing the cell-cycle stage to promote cells to enter into cycling. Moreover, activated inflammatory signaling pathways were enriched in the RPS19-deficient group compared to the CD34 group and the vector-treated group. Consistent with our observation, recent studies demonstrated that elevated TNF-α can be detected in DBA bone marrow plasma,3 and inflammatory signature was shown in erythroblasts and red blood cells from DBA patients.26 Iskander et al. also demonstrated enriched TNF-α signaling via the NF-κB pathway in erythroid progenitors of DBA patients by RNA-sequencing.3 The role of inflammatory signaling pathway is worthy of future study. On the other hand, we also identified one cluster with activated cell-cycle status, but low hemoglobin gene expression in the RPS19-D group. The underlying mechanism remains unknown; however, lncRNA such as MALAT1 and XACT were found highly enriched the cluster. The MALAT1 has been shown to be regulated by p53 and plays a significant role in maintaining the proliferation potential of early-stage hematopoietic cells.27 This may indicate that abnormal epigenetic regulation also contributes to the impaired erythroid differentiation, which would be an interesting topic for future studies. Overall, we successfully generated a traceable RPS19-deficient CD34+ HSPC cell model by using nanostraws to deliver Cas9 mRNA and sgRNA. The nanostraw platform provides an optimal delivery option for targeting genes that sensitize cells to stress when knocked out, especially in sensitive primary stem cells. By using a clinically applicable lentiviral therapeutic vector EFS-RPS19, the impaired erythroid differentiation can be rescued with increased cell cycle to promote red blood cell production, which is also supported by the scRNA-seq results. Our results will encourage further investigation of the therapeutic effects of EFS-RPS19 in primary patient samples.

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Disclosures MH is a consultant for Navan Bio Inc., a startup commercializing nanostraw technology. LS, MH and JL are inventors on a patent application relating to nanostraws. Contributions YL, LS, MH and SK conceptualized the project and directed the research. YL, LS, MH, TDB and AR performed the experiments. YL and SL performed single cell RNA sequencing analysis. YL and LS analyzed the data. JL and AS provided materials and reagents. YL, LS, MH, JL and SK wrote the manuscript. Acknowledgments The authors thank Beata Lindqvist and Xiaojie Xian for lentivirus production, Jenny G Johansson for AAV production, Maria Björklund for digital droplet PCR technical assistance, and Zhi Ma for FACS technical assistance. We thank Kristijonas Žemaitis for help in characterizing colony-forming units and Veronika Žemaitė for the schematic demonstration of the nanostraw. We thank the staff in the Lund Nano Lab for assistance with tools needed for nanostraw fabrication. We thank the Center for Translational Genomics, Lund University and Clinical Genomics Lund, SciLifeLab for providing the sequencing service. Funding This work was supported by a Hemato-Linne grant from the Swedish Research Council Linnaeus, project grants from Swedish Research Council (to SK and MH), the Swedish Cancer Society and the Swedish Children’s Cancer Society (to SK), the Tobias Prize awarded by the Royal Swedish Academy of Sciences financed by the Tobias Foundation, a clinical research grant from Lund University Hospital (to SK), European Union project grants STEMEXPAND and PERSIST, a grant from The Royal Physiographic Society of Lund, Sweden (to YL and LS), and a grant from Stiftelsen Lars Hiertas Minne (to YL). Data-sharing statement All data needed to evaluate the conclusions in the paper are present in the paper and/or the Online Supplementary Appendix. The scRNA-sequencing count matrix are available in the GEO database of the NCBI under the GEO accession number GSE2232.

References 1. Da Costa L, Leblanc T, Mohandas N. Diamond-Blackfan anemia. Blood. 2020;136(11):1262-1273. 2. Ulirsch JC, Verboon JM, Kazerounian S, et al. The genetic landscape of Diamond-Blackfan anemia. Am J Hum Genet. 2018;103(6):930-947. 3. Iskander D, Wang G, Heuston EF, et al. Single-cell profiling of human bone marrow progenitors reveals mechanisms of failing

erythropoiesis in Diamond-Blackfan anemia. Sci Transl Med. 2021;13(610):eabf0113. 4. Khajuria RK, Munschauer M, Ulirsch JC, et al. Ribosome levels selectively regulate translation and lineage commitment in human hematopoiesis. Cell. 2018;173(1):90-103.e19. 5. Liu Y, Dahl M, Debnath S, et al. Successful gene therapy of Diamond-Blackfan anemia in a mouse model and human

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ARTICLE - scRNA-seq analysis of engineered human DBA model CD34(+) cord blood hematopoietic stem cells using a clinically applicable lentiviral vector. Haematologica. 2022;107(2):446-456. 6. Jaako P, Flygare J, Olsson K, et al. Mice with ribosomal protein S19 deficiency develop bone marrow failure and symptoms like patients with Diamond-Blackfan anemia. Blood. 2011;118(23):6087-6096. 7. An K, Zhou JB, Xiong Y, et al. Computational studies of the structural basis of human RPS19 mutations associated with Diamond-Blackfan anemia. Front Genet. 2021;12:650897. 8. Bhoopalan SV, Yet JS, Mayuranathan T, et al. A novel RPS19edited hematopoietic stem cell model of Diamond-Blackfan anemia for development of lentiviral vector gene therapy. Blood. 2021;138(Suppl 1):859. 9. Schiroli G, Conti A, Ferrari S, et al. Precise gene editing preserves hematopoietic stem cell function following transient p53-mediated DNA damage response. Cell Stem Cell. 2019;24(4):551-565. 10. Schmiderer L, Subramaniam A, Zemaitis K, et al. Efficient and nontoxic biomolecule delivery to primary human hematopoietic stem cells using nanostraws. Proc Natl Acad Sci U S A. 2020;117(35):21267-21273. 11. Xie X, Xu AM, Leal-Ortiz S, Cao Y, Garner CC, Melosh NA. Nanostraw-electroporation system for highly efficient intracellular delivery and transfection. ACS Nano. 2013;7(5):4351-4358. 12. Cao Y, Chen H, Qiu R, et al. Universal intracellular biomolecule delivery with precise dosage control. Sci Adv. 2018;4(10):eaat8131. 13. Chiappini C, Chen Y, Aslanoglou S, et al. Tutorial: using nanoneedles for intracellular delivery. Nat Protoc. 2021;16(10):4539-4563. 14. Bak RO, Dever DP, Porteus MH. CRISPR/Cas9 genome editing in human hematopoietic stem cells. Nat Protoc. 2018;13(2):358-376. 15. Yudovich D, Backstrom A, Schmiderer L, Zemaitis K, Subramaniam A, Larsson J. Combined lentiviral- and RNAmediated CRISPR/Cas9 delivery for efficient and traceable gene editing in human hematopoietic stem and progenitor cells. Sci Rep. 2020;10(1):22393. 16. Traag VA, Waltman L, van Eck NJ. From Louvain to Leiden:

Y. Liu et al.

guaranteeing well-connected communities. Sci Rep. 2019;9(1):5233. 17. Jacomy M, Venturini T, Heymann S, Bastian M. ForceAtlas2, a continuous graph layout algorithm for handy network visualization designed for the Gephi software. PLoS One. 2014;9(6):e98679. 18. Rao DD, Senzer N, Cleary MA, Nemunaitis J. Comparative assessment of siRNA and shRNA off target effects: what is slowing clinical development. Cancer Gene Ther. 2009;16(11):807-809. 19. Fares I, Chagraoui J, Gareau Y, et al. Cord blood expansion. Pyrimidoindole derivatives are agonists of human hematopoietic stem cell self-renewal. Science. 2014;345(6203):1509-1512. 20. Dever DP, Bak RO, Reinisch A, et al. CRISPR/Cas9 beta-globin gene targeting in human haematopoietic stem cells. Nature. 2016;539(7629):384-389. 21. Papapetrou EP, Zoumbos NC, Athanassiadou A. Genetic modification of hematopoietic stem cells with nonviral systems: past progress and future prospects. Gene Ther. 2005;12(Suppl 1):S118-130. 22. Kosicki M, Tomberg K, Bradley A. Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol. 2018;36(8):765-771. 23. Haapaniemi E, Botla S, Persson J, Schmierer B, Taipale J. CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response. Nat Med. 2018;24(7):927-930. 24. Enache OM, Rendo V, Abdusamad M, et al. Cas9 activates the p53 pathway and selects for p53-inactivating mutations. Nat Genet. 2020;52(7):662-668. 25. Vlachos A, Ball S, Dahl N, et al. Diagnosing and treating Diamond Blackfan anaemia: results of an international clinical consensus conference. Br J Haematol. 2008;142(6):859-876. 26. Kapralova K, Jahoda O, Koralkova P, et al. Oxidative DNA damage, inflammatory signature, and altered erythrocytes properties in Diamond-Blackfan anemia. Int J Mol Sci. 2020;21(24):9652. 27. Ma XY, Wang JH, Wang JL, Ma CX, Wang XC, Liu FS. Malat1 as an evolutionarily conserved lncRNA, plays a positive role in regulating proliferation and maintaining undifferentiated status of early-stage hematopoietic cells. BMC Genomics. 2015;16(1):676.

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TRES, a validated three-factor comorbidity score, is associated with survival in older patients with mantle cell lymphoma Mantle cell lymphoma (MCL) is a rare and aggressive nonHodgkin lymphoma.1 MCL prognosis is impacted by clinical, pathologic and molecular features.2-5 Age and comorbidities also impact survival in MCL.6,7 In contrast to well-defined clinical prognostic tools such as the Mantle Cell Lymphoma International Prognostic Index (MIPI),2 cellular proliferation rate measurements using Ki-67,5 and molecular features such as TP53 mutation/deletion and complex karyotype,8,9 there is no standardized measure of comorbidity in MCL. We previously developed and independently validated the chronic lymphocytic leukemia comorbidity index (CLL-CI), a three-category comorbidity scale which was associated with overall survival (OS) and event-free survival (EFS) in patients with CLL.10,11 We subsequently evaluated the CLLCI in older adults (>65 years) with non-Hodgkin lymphoma (NHL) using clinical information from the linked SEERMedicare databases. As this study included patients with multiple NHL subtypes as well as CLL, we renamed it the three-factor risk estimate scale (TRES).12 TRES is assigned in a manner identical to CLL-CI. The SEER-Medicare study included 40,486 patients with NHL with a median age of 77 years. TRES was independently associated with both OS and lymphoma-specific survival in multivariable models. TRES has not been evaluated in an independent dataset of patients with MCL. Additionally, SEER-Medicare does not include patients younger than 65 years nor does it include the clinical and pathologic granularity needed to utilize currently available risk assessment tools. The aim of the current study was to evaluate the association of TRES score with survival in an independent real-world cohort of patients with MCL including younger patients and in the context of established prognostic models. We conducted a multicenter retrospective study of patients with MCL from three US academic centers evaluated between 2007 and 2022. Patient demographic, diagnostic and therapeutic information were obtained from review of the electronic medical record. For the primary analysis cohort, we included all patients with newly diagnosed MCL who received frontline therapy and had at least 3 months of follow-up available from time of diagnosis. Comorbidity risk classification was retrospectively assigned using TRES at time of diagnosis. This was done by reviewing the patient’s medical record including provider notes, medication lists and laboratory results.10-12 TRES score is assessed by evaluating comorbidities in three categories: vascular, en-

docrine and upper gastrointestinal. If a comorbidity is present in each category one point is assigned, scores range from 0-3. A score of 0 is considered low-risk, 1 intermediate-risk, and 2-3 high-risk. We also stratified patients using the components of the MIPI score2 and the Ki-67 proliferation index (≥30% was considered high risk).5 OS was defined as time from diagnosis to death and EFS as time from treatment initiation to next line of therapy (excluding autologous stem cell transplant [ASCT] and rituximab maintenance), documented disease progression or death from any cause. Patient demographics were evaluated using descriptive statistics. Difference between groups was assessed by Fischer’s exact test and χ2. The Kaplan-Meier method was used to estimate OS and EFS with difference assessed by log-rank. Multivariable Cox regression models were utilized. Patients with missing data were removed from Cox models. A P value of <0.05 was considered significant for all. The study was approved by the City of Hope Institutional Review Board. The primary analysis cohort included 361 patients with MCL who received frontline therapy (Table 1). Median age was 63 years (range, 49-86). The majority were male and had advanced stage disease. Bendamustine and rituximab (BR) was the most common induction regimen (31.9%); 43.5% received ASCT consolidation. A total of 102 patients had vascular comorbidities (28.2%), 79 (21.9%) upper gastrointestinal and 76 (21.1%) endocrine comorbidities. The most commonly combined comorbidity categories were vascular and endocrine in 23 patients (6.3%) and 2.8% had comorbidities in all three categories. TRES score was low in 50.1%, intermediate in 31.3% and high in 18.6% of patients. High TRES score was numerically more frequent in patients >65 years (23.0%) compared with patients ≤65 years (15.9%). After a median follow-up of 61.2 months, the estimated 5year OS rate was 84.8%, 84.3% and 66.0% in low-, intermediate- and high-risk TRES groups, respectively (Figure 1A; P=0.002). Corresponding estimated 5-year EFS rates were 62.4%, 49.1% and 40.8% (Figure 1B; P=0.002). In multivariable Cox models including the components of the MIPI score (age, white blood cell count, lactate dehydrogenase [LDH, ratio of reported value to upper limit of normal], all considered as continuous variables, and Eastern Cooperative Oncology Group [ECOG] stratified 0-1 vs. 2-4) and Ki67, a high TRES score, compared to low and intermediate, remained independently associated with OS (hazard ratio

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LETTER TO THE EDITOR Table 1. Patient demographics. All patients N (%)

TRES low N (%)

TRES int. N (%)

TRES high N (%)

Cohort

361 (100)

181 (50.1)

113 (31.3)

67 (18.6)

Age in years Median (range) >65 ≤65

63 (49-86) 135 (37.4) 226 (62.6)

62 (37-86) 59 (43.7) 122 (54.0)

64 (41-86) 45 (33.3) 68 (30.1)

65 (37-86) 31(23.0) 36 (15.9)

0.12

Sex Female Male

91 (25.2) 270 (74.8)

44 (24.3) 137 (75.7)

32 (28.3) 81 (71.7)

15 (22.4) 52 (77.6)

0.63

ECOG 0-1 2-4

315 (87.5) 18 (12.5)

159 (87.7) 11 (6.1)

101 (89.4) 3 (2.7)

55 (82.1) 4 (6.0)

0.40

Stage 1-2 3-4

13 (3.6) 342 (94.7)

5 (2.8) 174 (96.1)

5 (4.4) 106 (93.8)

3 (4.5) 62 (92.5)

0.69

B symptoms Yes No

140 (38.8) 217 (60.1)

79 (43.6) 100 (55.2)

37 (32.7) 74 (65.5)

24 (35.8) 43 (64.2)

0.15

Ki-67 >30% Yes No Unknown

87 (24.1) 150 (41.6) 124 (34.3)

51 (28.2) 71 (39.2) 59 (32.6)

25 (22.1) 51 (45.1) 37 (32.7)

11 (16.4) 28 (41.8) 28 (41.8)

0.29

Treatment regimen BR HyperCVAD/Nordic/DHAP RCHOP Other

115 (31.9) 112 (31.0) 49 (13.6) 85 (23.5)

57 (31.5) 69 (38.1) 27 (14.9) 28 (15.5)

33 (29.2) 28 (24.8) 15 (13.2) 37 (32.7)

25 (37.3) 15 (22.4) 7 (10.4) 20 (29.9)

0.007

Autologous stem cell transplant Yes No

157 (43.5) 204 (56.5)

85 (47.0) 96 (53.0)

47 (41.6) 66 (58.4)

25 (37.3) 42 (62.7)

0.35

Maintenance rituximab Yes No

182 (50.4) 179 (49.6)

98 (54.1) 83 (45.9)

57 (50.4) 56 (49.6)

27 (40.3) 40 (59.7)

0.15

Simplified MIPI score 0-3 (low risk) 4-5 (intermediate risk) 6+ (high risk) Missing data

92 (25.5) 93 (25.5) 73 (20.2) 103 (28.5)

54 (29.8) 50 (27.6) 31 (17.1) 46 (25.4)

24 (21.1) 31 (27.4) 23 (20.4) 35 (31.0)

14 (20.9) 12 (17.9) 19 (28.4) 22 (32.8)

0.17

BR: bendamustine and rituximab; TRES: three-factor risk estimate scale; int.: intermediate; ECOG: Eastern Cooperative Oncology Group; HyperCVAD: hyper-fractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone, methotrexate, cytarabine; Nordic: rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone, cytarabine; DHAP: dexamethasone, cytarabine, cisplatin; RCHOP: rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone; MIPI: mantle cell lymphoma international prognostic index.

[HR] =1.95; 95% confidence interval [CI]: 1.07-3.57). Models of EFS showed a similar trend with high TRES score compared to low and intermediate (HR=1.34; 95% CI: 0.85-2.12). Because TRES had previously been studied only in older adults with MCL, we evaluated the association of TRES score with survival separately in older patients (>65 years) and younger patients (≤65 years). A total of 135 patients (37.4% of our cohort) were >65 years old, in whom BR was the most common induction regimen (41.4%). The esti-

mated 5-year OS rate in older adults was 74.3%, 84.0% and 40.1% in low-, intermediate- and high-risk TRES groups, respectively (Figure 1C; P=0.007). Corresponding to 5-year EFS rates of 55.4%, 55.0% and 26.5%, respectively (Figure 1D; P=0.051). In patients ≤65 years old, 66.8% received a cytarabine-containing induction regimen and 32.7% BR. OS was favorable independent of the TRES score with estimated 5-year rates of 90.2%, 84.7% and 86.4% in low, intermediate and high TRES, respectively (P=0.056).

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Figure 1. Survival by three-factor risk estimate scale comorbidity score risk group. Overall and event-free survival by threefactor risk estimate scale (TRES) score in all patients (A, B) and in those >65 years old (C, D). Low TRES score in blue, intermediate score in red and high score in green.

However, TRES remained significantly associated with EFS in younger patients with estimated 5-year rates of 66.1%, 45.3% and 52.2%, respectively (P=0.038). Notably, in multivariable models of the full study cohort which included TRES, age as a continuous variable and induction regimen (BR vs. cytarabine containing vs. rituximab, cyclophosphamide, doxorubicin, vincristine and oral prednisolone [RCHOP] vs. other), high TRES compared to low or intermediate TRES remained independently associated with OS (HR=2.18; 95% CI: 1.32-3.59) and EFS (HR=1.67; 95% CI: 1.152.43). In our previous work, we used propensity-matched models to demonstrate that TRES score was associated with OS in the MCL cohort derived from the SEER-Medicare database.12 In this report we validate the association of the TRES comorbidity score with survival in patients >65 years old using an independent cohort of patients with MCL treated at academic medical centers.12 A high TRES score, which was present in nearly one in five patients with MCL, was associated with significantly shorter OS and EFS.

When adjusted for MIPI a high TRES score remained independently associated with OS and was associated with nearly a two-fold increased risk of death. High-risk comorbidities were more common in patients >65 years old and were associated with a 5-year OS of only 40% in that population. In contrast to previous studies, we did not find a significant difference in survival between low- and intermediate-TRES risk patients. TRES was not significantly associated with OS in patients ≤65 years old, although it should be noted that OS events were rare in this population. TRES did remain associated with EFS in the younger patient cohort. Similar to prior reports,10,13 the impact of comorbidity appears greatest in older adults receiving frontline therapy. Whether the negative impact of comorbidity can be overcome by more effective, and/or better tolerated, treatments is an important area of future investigation. There are important limitations to studies which retrospectively assess comorbidity burden as only those conditions which are documented in the medical record or for

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LETTER TO THE EDITOR which patients are receiving active therapy can be identified. There are also inherent selection biases in treatment selection which may confound the stratification of patients based on treatment type and intensity. Additionally, information regarding TP53 mutation or deletion, complex karyotype and blastoid or pleomorphic histology were missing in a significant proportion of patients and, therefore, were excluded from all analyses. Bruton tyrosine kinase (BTK) inhibitors are increasingly used to treatment MCL and rare use of BTK inhibitors in this study cohort represents another potential limitation. We have previously shown that CLL-CI/TRES is predictive of outcomes among patients with CLL treated with ibrutinib.10 BTK inhibitors have demonstrated promising activity in MCL,14,15 whether they can improve outcomes for patients with comorbidities is an area for future investigation. TRES is a simple comorbidity score associated with survival in older patients with MCL. In addition to clinical and genetic prognostication, TRES could be a useful tool for designing prospective trials, including non-pharmaceutical interventions, targeting high-risk populations.

Early view: May 25, 2023. ©2023 Ferrata Storti Foundation Published under a CC BY-NC license Disclosures DAB has received consulting fees from SeaGen, Kite/Gilead, and Nurix and has received research funding from Novartis and Nurix. ASK has received consulting fees from AstraZeneca, Abbvie, BeiGene, Eli Lilly, Janssen, and Kite/Gilead and has ongoing research funding from Astra Zeneca. TJP has received consulting fees from Abbvie, AstraZeneca, ADC Therapeutics, Bayer, Beigene, BMS, Eli Lily, Epizyme, Genentech, Genmab, Gilead, Incyte, MEI, Pharmacyclics, Seattle Genetics, TG therapeutics and Xencor and has ongoing research funding from Bayer, BMS, Abbvie and Genentech. AVD has received consulting fees from Abbvie, AstraZeneca, BeiGene, Bristol Meyers Squibb, Genentech, GenMab, Incyte, Janssen, Lilly Oncology, MEI Pharma, Nurix, Oncovalent, Pharmacyclics and TG Therapeutics and has ongoing research funding from Abbvie, AstraZeneca, Bayer Oncology, Bristol Meyers Squibb, Cyclacel, Lilly Oncology, MEI Pharma, Nurix and Takeda Oncology. All other authors have no conflicts of interest to disclose.

Authors

Contributions MJG, DAB, AVD designed the research. NA, AS, SH, VN, GS and JBC collected the data. MJG and AVD analyzed the data. MJG, DAB, ASK,

Max J. Gordon, David A. Bond, Adam S. Kittai, Neda Amirmokhtari,

GS, JBC, TP and AVD wrote and edited the manuscript. All authors

Abigail Steinbrunner, Allison Huffman, Victor Orellana-Noia,

reviewed the manuscript and agree with presented format.

Geoffrey Shouse, Jonathon B. Cohen, Tycel Phillips and Alexey V. 4

Danilov4

Acknowledgments We thank the Lymphoma Database at The Ohio State University

University of Texas M.D. Anderson Cancer Center, Houston, TX;

Comprehensive Cancer Center, Columbus, OH.

Ohio State University, James Cancer Center, Columbus, OH; 3Emory

University Winship Cancer Institute, Atlanta, GA and 4City of Hope

Funding

National Medical Center, Duarte, CA, USA

This study was supported by the NCI 1R01CA244576 to AVD. AVD is a Leukemia and Lymphoma Society Scholar in Clinical Research

Correspondence:

(#2319-19). MJG is supported by a T32 training grant

A.V. DANILOV - adanilov@coh.org

(5T32CA009666-27). Research reported in this publication was also

M.J. GORDON - mjgordon@mdanderson.org

supported by The Ohio State University Comprehensive Cancer Center and the National Institutes of Health under grant number

https://doi.org/10.3324/haematol.2023.283074

P30 CA016058.

Received: March 13, 2023.

Data-sharing statement

Accepted: May 6, 2023.

Data will not be available.

References 1. Armitage JO, Longo DL. Mantle-cell lymphoma. N Engl J Med. 2022;386(26):2495-2506. 2. Hoster E, Dreyling M, Klapper W, et al. A new prognostic index (MIPI) for patients with advanced-stage mantle cell lymphoma. Blood. 2008;111(2):558-565. 3. Nabrinsky E, Danilov AV, Koller PB. High-risk mantle cell Llymphoma in the era of novel agents. Curr Hematol Malig Rep.

2021;16(1):8-18. 4. Yi S, Yan Y, Jin M, et al. Genomic and transcriptomic profiling reveals distinct molecular subsets associated with outcomes in mantle cell lymphoma. J Clin Invest. 2022;132(3):e153283. 5. Determann O, Hoster E, Ott G, et al. Ki-67 predicts outcome in advanced-stage mantle cell lymphoma patients treated with anti-CD20 immunochemotherapy: results from randomized

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LETTER TO THE EDITOR trials of the European MCL Network and the German Low Grade Lymphoma Study Group. Blood. 2008;111(4):2385-2387. 6. Glimelius I, Smedby KE, Eloranta S, Jerkeman M, Weibull CE. Comorbidities and sex differences in causes of death among mantle cell lymphoma patients - a nationwide populationbased cohort study. Br J Haematol. 2020;189(1):106-116. 7. Abrahamsson A, Albertsson-Lindblad A, Brown PN, et al. Real world data on primary treatment for mantle cell lymphoma: a Nordic Lymphoma Group observational study. Blood. 2014;124(8):1288-1295. 8. Greenwell IB, Staton AD, Lee MJ, et al. Complex karyotype in patients with mantle cell lymphoma predicts inferior survival and poor response to intensive induction therapy. Cancer. 2018;124(11):2306-2315. 9. Eskelund CW, Dahl C, Hansen JW, et al. TP53 mutations identify younger mantle cell lymphoma patients who do not benefit from intensive chemoimmunotherapy. Blood. 2017;130(17):1903-1910. 10. Gordon MJ, Kaempf A, Sitlinger A, et al. The Chronic Lymphocytic Leukemia Comorbidity Index (CLL-CI): a three-

factor comorbidity model. Clin Cancer Res. 2021;27(17):4814-4824. 11. Rotbain EC, Gordon MJ, Vainer N, et al. The CLL comorbidity index in a population-based cohort: a tool for clinical care and research. Blood Adv. 2022;6(8):2701-2706. 12. Gordon MJ, Duan Z, Zhao H, et al. A novel comorbidity score for older adults with non-Hodgkin lymphoma: the 3-factor risk estimate scale. Blood Adv. 2023;7(11):2632-2642. 13. Gordon MJ, Churnetski M, Alqahtani H, et al. Comorbidities predict inferior outcomes in chronic lymphocytic leukemia treated with ibrutinib. Cancer. 2018;124(15):3192-3200. 14. Jain P, Zhao S, Lee HJ, et al. Ibrutinib with rituximab in firstline treatment of older patients with mantle cell lymphoma. J Clin Oncol. 2022;40(2):202-212. 15. Dreyling M, Doorduijn JK, Gine E, et al. Efficacy and safety of ibrutinib combined with standard first-line treatment or as substitute for autologous stem cell transplantation in younger patients with mantle cell lymphoma: results from the Randomized Triangle Trial By the European MCL Network. Blood. 2022;140(Suppl 1):S1-3.

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LETTER TO THE EDITOR

Clinical and molecular features of CBL-mutated juvenile myelomonocytic leukemia Juvenile myelomonocytic leukemia (JMML) is characterized by excessive myelomonocytic cell proliferation and granulocyte–macrophage colony-stimulating factor hypersensitivity. Approximately 15% of children with JMML harbor homozygous CBL mutations.1,2 Niemeyer et al.1 identified germline CBL syndrome with developmental, tumorigenic, and functional consequences caused by hyperactive RAS/RAF/MEK/ERK signaling. Patients with CBLmutated JMML typically have a low-methylation profile and a less aggressive disease course compared to JMML patients with other RAS pathway mutations.3 In most cases, the disease resolves spontaneously,4 whereas vasculitides and other autoimmune disorders might develop in some patients.1 However, prior cohorts in the literature have been restricted to small numbers of patients, specifically those with somatic CBL mutations.2 Thus, this study aimed to retrospectively analyze a cohort along with a review of the literature.

This study retrospectively analyzed 25 children with CBLmutated JMML in Japan between September 1988 and November 2021 (Figure 1). JMML was diagnosed based on previously published internationally accepted diagnostic criteria.5 Our previous reports included 19 of 25 patients.6,7 Written informed consent was obtained from the guardians of all patients. This study was approved by the Ethics Committee of the Nagoya University Graduate School of Medicine. Bone marrow or peripheral blood samples were collected at the initial diagnosis. Ficoll–hypaque density gradient centrifugation was utilized to isolate mononuclear cells, which were cryopreserved until use. Genomic DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen, Chatsworth, CA). Whole-exome sequencing was used for the mutational analysis, as previously described.7 Canonical RAS pathway gene mutations, i.e., PTPN11, NRAS, KRAS, and CBL, were confirmed using Sanger sequencing.

Figure 1. Summary of the Japanese and literature review cohorts. (A) Japanese cohort and (B) literature review cohort. HCT: hematopoietic cell transplantation. Haematologica | 108 November 2023

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LETTER TO THE EDITOR Methylation analysis was performed using digital restriction enzyme analysis of methylation, as previously described.8 Of the 25 patients, 16 were analyzed with a 450k methylation array, as previously described.3,7 The Kaplan–Meier method was used to estimate overall survival (OS) and transplantation-free survival (TFS). Survival differences were evaluated using the log-rank test. OS was defined as the duration from the date of diagnosis to death, and TFS as the duration from the date of diagnosis to transplantation or all-cause death. All statistical analyses were performed using EZR software (version 1.36; Saitama Medical Center, Jichi Medical University, Saitama, Japan).9 P values were two-tailed in all analyses, and P values <0.05 were considered statistically significant. Literature review A systematic literature search was conducted using a combination of controlled vocabulary and keywords. PubMed (https://pubmed.ncbi.nlm.nih.gov) was searched for published articles from the date of its inception to February 2022. Searched terminologies included “JMML” and “CBL.” In total, 61 articles written in English were found. Of these, 26 abstracts, nine reviews, and seven articles reported from Japan that were identical or potentially identical to the study cases were excluded. The abstracts and text of the remaining 19 articles were carefully evaluated, and ten articles reporting 65 patients with CBL-mutated JMML from outside Japan were selected.1,2,10-17

Patient characteristics at diagnosis are presented in Figure 2; Online Supplementary Table S1 and Online Supplementary Table S2. Our cohort consisted of 25 patients, with a median age at diagnosis of 1 year (range, 1 month14 years). The median follow-up was 3.4 years (range, 0.5 months-23.6 years). Splenomegaly with ≥3 cm below the costal margin was determined in 19 patients. CBL mutations were homozygous (n=21) or heterozygous (n=4), with 18 missense mutations, four splice site mutations, and three deletions. Three patients with heterozygous gene deletions were identified as having no point mutation in the second allele by whole-exome sequencing. Moreover, 23 patients harbored germline CBL mutations, whereas two patients had somatic mutations only. One patient had a concomitant somatic PTPN11 mutation (UPN160). Methylation analysis classified all cases into the low-methylation (LM) group. Except for one patient who had trisomy 8 (UPN198), no patients had chromosomal abnormalities. In this cohort, three patients had moyamoya disease (Online Supplementary Figure S1), of whom one has neovascular glaucoma, whose clinical course was described in detail in a previous publication.6 Of the 25 patients in this cohort, six received allogeneic hematopoietic stem cell transplantation (HCT); four patients were diagnosed with JMML and had transplantation in the period before the CBL mutation was identified as the causative gene for JMML. The remaining two patients underwent HCT because of disease progression. Figure 3 shows an overview of all disease courses. The 5-

Figure 2. Clinical and genetic profiles of the Japanese and literature review cohorts. Each column indicates 1 patient. Methylation profiling data were available for 50 of 87 patients (22 in the Japanese cohort and 28 in the literature review cohort). Plt: platelet count; HbF: fetal hemoglobin; TFS: transplantation-free survival; OS: overall survival; HCT: hematopoietic cell transplantation. Haematologica | 108 November 2023

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LETTER TO THE EDITOR year OS and TFS rates were 70.3% (95% confidence interval [CI]: 47.4-84.6) and 52.7% (95% CI: 27.8-72.6), respectively. Spontaneous JMML resolution was experienced by 14 patients, without treatment (n=11) or with oral administration of 6-mercaptopurine (n=3). Four patients died of leukemia before transplantation, and six patients underwent allogeneic HCT after receiving different pretrans-

plant treatments. Among patients who underwent HCT, two went into remission, three died of JMML relapse, and one died of transplant-related complications. The literature review cohort consisted of 65 patients with a median age at diagnosis of 1 year (range, 1 month-25 years). The patient characteristics are provided in the Online Supplementary Table S2. Fifty-one patients had sple-

Figure 3. Swimmer plot and survival curves of the Japanese cohort. (A) Swimmer plot showing the clinical course of patients in the Japanese cohort. Each bar shows the clinical course of 1 patient. Symbols indicate the dates of hematopoietic cell transplantation (HCT), death, relapse, or spontaneous resolution. A color-coded arrow indicates the current status of the patient. Therapeutic agents received by the patient are shown on the left side using pattern-coded dots. Filled (if true) or empty (if false) boxes indicate the clinical features (sex, age, splenomegaly, moyamoya disease, Café au lait spot, platelet count [Plt], elevated fetal hemoglobin [HbF] for age, and abnormal karyotype). Dashed boxes are used to indicate unavailable data. 6-MP: 6-mercaptopurine; Ara-C: cytosine arabinoside. (B, C) Kaplan–Meier estimates of overall survival (OS) and transplantation-free survival (TFS). Five-year (yr) OS and TFS rate were 70.3% (95% confidence interval [CI]: 47.4-84.6) and 52.7% (95% CI: confidence interval: 27.8-72.6), respectively. Haematologica | 108 November 2023

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LETTER TO THE EDITOR nomegaly. CBL mutations were homozygous (n=24) or heterozygous (n=14) or with no available data about zygosity (n=28) with 53 missense mutations, two deletions, two frameshift, and eight splice site mutations. Three patients had chromosomal abnormalities, including chromosome 16 deletion (n=1), chromosome 8 derivation (n=1), and absence of detailed karyotype information (n=1). Methylation data were available for 28 cases, of which 27 were classified with an LM profile and one with an intermediate methylation profile. This literature review cohort identified three patients with vasculitides, including Takayasu arteritis and small vessel vasculitis. Information on transplantation was available for 47 of 65 patients. Of the 23 patients without transplantation, 18 (78%) survived and five (22%) died. Information on the cause of death of these five patients was unavailable. Of the 24 transplant recipients, 17 (71%) survived and seven (29%) died (transplant-related mortality, n=1; and no detailed information, n=6). We conducted a retrospective analysis of 25 cases of CBLmutated JMML diagnosed in Japan and identified an additional 65 cases by literature review. Studies1,4 have reported that most children with CBL-mutated JMML show a self-limiting clinical course with persistent clonal hematopoiesis, and observation is generally recommended, although splenomegaly and thrombocytopenia may require therapeutic intervention. However, of the 25 patients in the present study, four died of leukemia before HCT, and three died after HCT, resulting in a 5-year OS rate of 70.3%. Of the 65 patients with CBL-mutated JMML, 12 died (pre-transplant, n=5; post-transplant, n=7), indicating that CBL-mutated JMML is a heterogeneous population, and some patients experienced aggressive disease courses. A recent international collaborative study identified methylation classification as a potent prognostic factor in JMML, and the presence of CBL mutations is tightly associated with the LM subgroup.3 Therefore, of the 50 patients (Japanese cohort, n=22; literature review cohort, n=28) with evaluable methylation data, 49 were classified in the LM subgroup and one in the IM group. These data suggest that we need to develop additional biomarkers besides methylation profiling to understand the molecular pathogenesis and heterogeneity of CBL-mutated JMML. Moyamoya disease is a chronic cerebrovascular disease that is characterized by bilateral stenosis or artery occlusion around the progressive circle of Willis. CBL syndrome with germline CBL mutation shows a phenotype overlapping with Noonan syndrome, but it is associated with various vasculitides forms, including Takayasu disease, optic atrophy, hypertension, and acquired cardiomyopathy.1 Moyamoya disease was complicated in three patients with CBL syndrome without clinical JMML manifestations.18,19 In the present cohort, moyamoya disease was found in three of 25 patients with CBL-mutated JMML, including the pre-

viously reported case.6 Prospective screening for moyamoya disease and other vasculitides complications in patients with CBL-mutated JMML is recommended using imaging studies, including magnetic resonance angiography. This study has several limitations. First, the number of patients was insufficient to fully characterize the clinical features of CBL-mutated JMML associated with poor prognosis, although this is one of the largest CBL-mutated JMML studies so far. Second, there were only two cases with somatic CBL mutations and one case with secondary mutations in this cohort, making it difficult to evaluate the association between these genetic conditions and prognosis. Third, a significant proportion of the patients with CBL-mutated JMML in the literature review cohort lack basic genetic information such as CBL mutation zygosity and DNA methylation classification. In conclusion, patients with CBL-mutated JMML represent a heterogeneous patient population that includes cases requiring therapeutic interventions, such as HCT. Further international collaborative studies are needed to accurately assess the clinical profile of CBL-mutated JMML and identify clinical factors associated with a poor prognosis.

Authors Taro Yoshida,1 Hideki Muramatsu,1 Manabu Wakamatsu,1 Daichi Sajiki,1 Norihiro Murakami,1 Hironobu Kitazawa,1 Yasuhiro Okamoto,2 Rieko Taniguchi,1 Shinsuke Kataoka,1 Atsushi Narita,1 Asahito Hama,3 Yusuke Okuno4 and Yoshiyuki Takahashi1 Department of Pediatrics, Nagoya University Graduate School of

Medicine, Nagoya; 2Department of Pediatrics, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima; Department of Hematology and Oncology, Children's Medical

Center, Japanese Red Cross Aichi Medical Center Nagoya First Hospital, Nagoya and 4Department of Virology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan Correspondence: H. MURAMATSU - hideki-muramatsu@med.nagoya-u.ac.jp Y. TAKAHASHI - ytakaha@med.nagoya-u.ac.jp https://doi.org/10.3324/haematol.2022.282385 Received: November 10, 2022. Accepted: May 15, 2023. Early view: May 25, 2023. ©2023 Ferrata Storti Foundation Published under a CC BY-NC license

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LETTER TO THE EDITOR Disclosures

Chie Amahori for their valuable assistance and Takuro Nishikawa,

No conflicts of interest to disclose.

Katsuyoshi Hara, Atsushi Sato, Takeshi Taketani, Taichiro Tsuchimochi, Hideaki Ueki, Takashi Kaneko, Mariko Kakazu, Akihiro

Contributions

Iguchi, Mayuko Okuya, Junya Fujimura, Shinya Sasaki, Akira

TY gathered clinical information, designed the research, analyzed

Hayakawa, Masahiko Manabe, and Yuji Ishida for providing clinical

data, and wrote the paper. HM, YO and WM designed and performed

information.

the research, led the project, and wrote the paper. DS, NM and HK performed laboratory analyses. OY collected clinical samples and

Funding

information. RT, SK, AN, AH and YT cooperatively designed and

This study was supported by AMED under grant number

performed the research.

JP20ck0106611.

Acknowledgments

Data-sharing statement

We want to thank Yoshie Miura and Fumiyo Ando for their technical

Data used in this study will be provided to qualified researchers on

assistance. The authors would also like to thank Hiroko Ono and

reasonable request.

References 1. Niemeyer CM, Kang MW, Shin DH, et al. Germline CBL mutations cause developmental abnormalities and predispose to juvenile myelomonocytic leukemia. Nat Genet. 2010;42(9):794-800. 2. Hecht A, Meyer JA, Behnert A, et al. Molecular and phenotypic diversity of CBL-mutated juvenile myelomonocytic leukemia. Haematologica. 2022;107(1):178-186. 3. Schonung M, Meyer J, Nollke P, et al. International Consensus Definition of DNA Methylation Subgroups in Juvenile Myelomonocytic Leukemia. Clin Cancer Res. 2021;27(1):158-168. 4. Locatelli F, Niemeyer CM. How I treat juvenile myelomonocytic leukemia. Blood. 2015;125(7):1083-1090. 5. Chan RJ, Cooper T, Kratz CP, et al. Juvenile myelomonocytic leukemia: a report from the 2nd International JMML Symposium. Leuk Res. 2009;33(3):355-362. 6. Hyakuna N, Muramatsu H, Higa T, et al. Germline mutation of CBL is associated with moyamoya disease in a child with juvenile myelomonocytic leukemia and Noonan syndrome-like disorder. Pediatr Blood Cancer. 2015;62(3):542-544. 7. Murakami N, Okuno Y, Yoshida K, et al. Integrated molecular profiling of juvenile myelomonocytic leukemia. Blood. 2018;131(14):1576-1586. 8. Kitazawa H, Okuno Y, Muramatsu H, et al. Simple and robust methylation test for risk stratification of patients with juvenile myelomonocytic leukemia. Blood Adv. 2021;5(24):5507-5518. 9. Kanda Y. Investigation of the freely available easy-to-use software 'EZR' for medical statistics. Bone Marrow Transplant. 2013;48(3):452-458. 10. Perez B, Kosmider O, Cassinat B, et al. Genetic typing of CBL, ASXL1, RUNX1, TET2 and JAK2 in juvenile myelomonocytic leukaemia reveals a genetic profile distinct from chronic myelomonocytic leukaemia. Br J Haematol. 2010;151(5):460-468. 11. Park HD, Lee SH, Sung KW, et al. Gene mutations in the Ras pathway and the prognostic implication in Korean patients with

juvenile myelomonocytic leukemia. Ann Hematol. 2012;91(4):511-517. 12. Hanson HL, Wilson MJ, Short JP, et al. Germline CBL mutation associated with a noonan-like syndrome with primary lymphedema and teratoma associated with acquired uniparental isodisomy of chromosome 11q23. Am J Med Genet A. 2014;164A(4):1003-1009. 13. Bulow L, Lissewski C, Bressel R, et al. Hydrops, fetal pleural effusions and chylothorax in three patients with CBL mutations. Am J Med Genet A. 2015;167A(2):394-399. 14. Pathak A, Pemov A, McMaster ML, et al. Juvenile myelomonocytic leukemia due to a germline CBL Y371C mutation: 35-year follow-up of a large family. Hum Genet. 2015;134(7):775-787. 15. Coe RR, McKinnon ML, Tarailo-Graovac M, et al. A case of splenomegaly in CBL syndrome. Eur J Med Genet. 2017;60(7):374-379. 16. Lipka DB, Witte T, Toth R, et al. RAS-pathway mutation patterns define epigenetic subclasses in juvenile myelomonocytic leukemia. Nat Commun. 2017;8(1):2126. 17. Wang WH, Lu MY, Tsai CH, et al. A child with juvenile myelomonocytic leukemia possessing a concurrent germline CBL mutation and a NF1 variant of uncertain significance: a rare case with a common problem in the era of high-throughput sequencing. J Formos Med Assoc. 2021;120(4):1148-1152. 18. Guey S, Grangeon L, Brunelle F, et al. De novo mutations in CBL causing early-onset paediatric moyamoya angiopathy. J Med Genet. 2017;54(8):550-557. 19. Seaby EG, Gilbert RD, Andreoletti G, et al. Unexpected findings in a child with atypical hemolytic uremic syndrome: an example of how genomics is changing the clinical diagnostic paradigm. Front Pediatr. 2017;5:113.

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Transcriptional features of acute leukemia with promyelocytic differentiation lacking retinoic acid receptor rearrangements Acute promyelocytic leukemia (APL) is typically characterized by the rearrangement of RARA, the most common of which is the PML-RARA fusion gene. The emergence of all-trans retinoic acid and arsenic trioxide has led to a reversal of disease prognosis. However, there are a few cases of acute myeloid leukemia in which the cell morphology, immunology, and even clinical manifestations are similar to classical APL; however, no RARA fusion gene has been detected. This type of leukemia is also known as acute promyelocytic-like leukemia (APLL). Retinoic acid receptors (RAR) include three members: RARA, RARB, and RARG. They are evolutionarily highly conserved, and their sequences and functions are remarkably similar. Moreover, retinoic acid X receptors (RXR) are closely related to RAR and usually form heterodimers to perform functions together. The first fusion gene harboring RARG (NUP98-RARG) in APLL was discovered in 2011.1 Subsequently, PML-RARG, CPSF6-RARG, HNRNPC-RARG, and NPM1-RARG-NPM1 have been identified.2–7 Osumi et al. reported on five Japanese patients and identified the RARB-involved fusion gene.8 Nevertheless, there are other patients with promyelocytic differentiation unrelated to RAR rearrangements, suggesting the complexity of APLL genome abnormalities.9,10 Here, we performed transcriptome sequencing (RNA-seq) in four such patients, analyzed the characteristics of their expression profiles, identified novel fusion genes other than RAR, and focused on splicing alterations of RAR and RXR. Of the four patients (Online Supplementary Table S1), two were men and two were women. Their ages ranged from 8 to 71 years old. All patients had more than two blood morphology experts for the diagnosis of the cell morphology (Online Supplementary Figure S1). Karyotype analysis and reverse transcription polymerase chain reaction (RTPCR) of the fusion gene harboring RARA were performed, and there was no evidence of RARA rearrangement. Total RNA was extracted from the bone marrow or peripheral blood mononuclear cells using TRIzol and stored at -80℃. Illumina HiSeq 3000 and BGISEQ-500 sequencers were used for RNA-seq in paired-end mode. The HISAT software was employed to compare clean reads to the reference human genome hg19/GRCh37, with an average comparison rate of 90.64% for each sample. StringTie software was used to reconstruct the transcript of each sample, followed by Cuffcompare to compare the reconstructed transcript with reference annotation information to obtain

new transcripts. Chimeric transcripts from each sample were extracted using SOAPfuse. The RSEM package was used to calculate the expression levels of the transcripts. RT-PCR and Sanger sequencing were performed to verify the results. Furthermore, the Cancer Genome Atlas-Acute Myeloid Leukemia (TCGA-LAML) cohort was downloaded as a control group, whose expression profile was compared with that of three cases (with new transcripts of RAR or RXR). A total of six gene fusion events were detected by RNAseq in case 1, among which KSR1-LGALS9 and GPBP1L1CCDC17 were verified by RT-PCR and Sanger sequencing. The pattern diagram and Sanger sequencing of the KSR1LGALS9 fusion are shown in Figure 1A and the Online Supplementary Figure S2A, respectively. The break point was between exon 29 of KSR1 and exon 2 of LGALS9, and the reading frame was not shifted. The break point of GPBP1L1-CCDC17 was flanked by exon 15 of GPBP1L1 and exon 1 of CCDC17. The break point of GPBP1L1 was within the stop codon TAG, that is, the deletion of the sequence following the AG bases. The break point of CCDC17 is located in the 5'-UTR (untranslated region) of exon 1 (Figure 1B; Online Supplementary Figure S2B, F). Moreover, a large number of novel transcripts of numerous genes were identified, in which we detected a new transcript of the RXRA gene with 8.72 fragments per kilobase per million (FPKM). The variant consisted of 11 exons, and exon 1 was located in the intron region of the RXRA gene sequence released by NCBI and Ensembl genome databases, with a total of 63 bp. Its whole predicted sequence is shown in the Online Supplementary Document A. Figure 2A shows the Sanger sequencing at the junction of exons 1 and 2. In addition, we identified three new transcripts of the RARB gene (Online Supplementary Document A). The PCR verification was not performed since the FPKM value could not be measured. The three new RARB transcripts are composed of known exons. A total of 37 gene fusion events were detected in case 2. A novel fusion gene, GLYCTK-DNAH1, was validated. Exon 3 of GLYCTK was fused to exon 5 of DNAH1 in-frame (Figure 1C; Online Supplementary Figure S2C). Interestingly, GPBP1L1-CCDC17 was also found in case 2. No novel splicing variant was discovered in the RAR or RXR. In case 3, 16 gene fusion events were detected. Three novel NUP98-HOXD8 variants were identified, one of which was confirmed (Figure 1D). The break point was flanked

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LETTER TO THE EDITOR by exon 11 of NUP98 and exon 2 of HOXD8 in-frame (Online Supplementary Figure S2D). In addition, RNA-seq results showed that the exon 10 sequence of NUP98 was not consistent with the known sequences, and it was similar to the exon 10 sequence of the ENST00000397013.2 transcript, with two more bases (GT) than the latter at the 3'

end, which can be considered as an alternative 3' end. A novel RARB transcript was identified (0.63 FPKM). There were six exons in the variant, of which exon 6 was located in the intron region of the released RARB gene sequence, with a total of 412 bp (Figure 2B; Online Supplementary Document A).

Figure 1. Schematic of novel fusion genes, splicing variants and expression profile. (A-E) List of fusion genes. The small black square indicates that the display of some exons is omitted. Haematologica | 108 November 2023

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Figure 2. Sequences of retinoic acid receptor/retinoic acid X receptor novel transcripts and heat map of expression profiles. (A-D) Sanger sequencing of novel transcripts of RXRA, RARB, and RARA. In novel transcript of RARA, the sequence from exon 1 to exon 3 was used as a template for the polymerase chain reaction (PCR). Panel (C) is the junction sequence of exon 1 and exon 2, and panel (D) is that of exon 2 and exon 3. (E) Expression profile comparison. We downloaded TCGA-LAML cohort as a control group, which was compared to the expression profile of 3 cases in the experimental group (with new transcripts of the retinoic acid receptors [RAR] and retinoic acid X receptor [RXR]). The TCGA-LAML RPKM (reads per kilobase per million mapped reads) expression data was combined with the data of 3 cases, then they were clustered into 3 groups, namely experimental group (case), TCGA library classic promyelocytic leukemia group (TCGA_M3_Subtype), and TCGA library nonpromyelocytic leukemia group (TCGA_Other_Subtypes). Limma R package was employed to calculate the differential gene expression, and top 420 up- and downregulated genes were merged to define signatures.

LETTER TO THE EDITOR

LETTER TO THE EDITOR Two CFD-GNA15 variants were found in case 4, one of which was verified. Exon 4 of CFD was spliced with exon 7 of GNA15, which caused a reading frame shift in GNA15 (Figure 1E; Online Supplementary Figure S2E). Moreover, we detected a new transcript of RARA gene (0.49 FPKM). There were seven exons in the variant, all of which were known (Figure 2C, D; Online Supplementary Document A). Intriguingly, a new transcript of RARB (0.09 FPKM), identical to case 3, was also identified in case 4. As mentioned above, fusion genes involving RAR or RXR were absent in some APLL, whereas new fusions formed by other genes were found.9-11 Similarly, the results detected should fall into this category. Some of these chimeric transcripts are fused by adjacent genes located on the same chromosome, which can be observed in the cissplicing of adjacent genes. Whether these novel fusion genes directly or indirectly affect retinoic acid-related transcriptional regulation or block leukemic cell differentiation to the promyelocytic stage by other pathways needs to be further explored. We also detected new splicing variants of RXR and RAR members in three cases, including RXRA, RARA, and RARB. RXR can form homodimers or heterodimers with RAR, which are important transcriptional regulators. Activated by ligands (all-trans- or 9-cis-retinoic acid), they bind to target response elements to regulate gene expression in various biological processes. RXRA is the most abundant subtype of RXR in bone marrow cells and its expression varies according to the differentiation stage of the hematopoietic process. RARA is found in normal myeloid cells; however, RARB is rarely expressed in the bone marrow. Alternative splicing of mRNA is a common cellular process that leads to proteomic complexity in advanced eukaryotes and regulates gene expression patterns that dominate cell fate. Alternative splicing can occur in the UTR or coding region, resulting in corresponding functional alterations. In recent years, abnormal alternative splicing has been observed in various types of tumors. Abnormal splicing may be caused by gene mutations or epigenetic or spliceosome changes, and participates in the pathogenesis of multiple human diseases. Through comparison with the TCGA-LAML database, we described the expression profiles of three cases (cases 1, 3, and 4). Figure 2E shows a heat map of the expression profile. In addition, Online Supplementary Figure S2G, H shows gene ontology (GO) and KEGG enrichment analyses. The expression profiles of classic APL (M3) cases in TCGA cohort were consistent and significantly different from those of other AML cases (non-M3), which is likely due to its unique fusion gene. Additionally, the gene expression profiles of other RAR-rearranged APLL might be similar to those of classic APL, which has been confirmed by the discovery of the RARG-CPSF6 fusion gene.5 Non-M3 cases bear their characteristics, which

can be attributed to the diversity of types. Since the clinical data in the TCGA database were not detailed, we did not perform further groupings. Inconsistent with the RARG-CPSF6-positive APLL, the expression profiles of the experimental group were far from those of the M3 group. Moreover, the expression profiles of the experimental group were different from those of the non-M3 group and were unique. This may be explained by the absence of RAR-involved fusion genes in the experimental group. Hence, it can be deduced that the differentiation mechanism of APLL lacking RAR rearrangements is different from that of RAR-rearranged APL, which may be more complicated and involve distinctive biological functions and pathways. In summary, we described the transcriptome features of APLL cases lacking RAR rearrangements. In these cases, fusion genes other than RAR, as well as distinct variants of the RAR and RXR members, exist. Profiling suggests a complex molecular mechanism of the disease, which deserves further investigation.

Authors Zhan Su,1,2* Xin Liu,3* Yuanfeng Zhang,4 Wei Wang,2 Xuerong Li,5 Jie Yu,6 Xinru Wang7 and Jun Peng1 Department of Hematology, Qilu Hospital, Cheeloo College of

Medicine, Shandong University, Jinan; 2Department of Hematology, Affiliated Hospital of Qingdao University, Qingdao; 3Department of Stem Cell Transplantation, Blood Diseases Hospital & Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin; 4Department of Hematology, Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai; 5

Department of Pediatric Hematology, Affiliated Hospital of Qingdao

University, Qingdao; 6Department of Hematology, Weihai Municipal Hospital, Cheeloo College of Medicine, Shandong University, Weihai and 7Department of Hematology, Liaocheng People’s Hospital, Liaocheng, China *ZS and XL contributed equally as first authors. Correspondence: J. PENG - junpeng88@sina.com https://doi.org/10.3324/haematol.2022.282426 Received: December 6, 2022. Accepted: May 8, 2023. Early view: May 18, 2023. ©2023 Ferrata Storti Foundation Published under a CC BY-NC license

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LETTER TO THE EDITOR Disclosures

Funding

No conflicts of interest to disclose.

This work was supported by the Fundamental Research Funds for the Central Universities, 2022JC025.

Contributions JP, ZS and XL designed the studies and wrote the paper. YZ, WW

Data-sharing statement

and XL were involved in the management of the patients and

All data included in this study are available upon request by contact-

providing clinical data. ZS, JY and XW performed the molecular

ing the corresponding author.

studies. All authors read and approved the manuscript.

References 1. Such E, Cervera J, Valencia A, et al. A novel NUP98/RARG gene fusion in acute myeloid leukemia resembling acute promyelocytic leukemia. Blood. 2011;117(1):242-245. 2. Ha J-S, Do YR, Ki C-S, et al. Identification of a novel PML-RARG fusion in acute promyelocytic leukemia. Leukemia. 2017;31(9):1992-1995. 3. Liu T, Wen L, Yuan H, et al. Identification of novel recurrent CPSF6-RARG fusions in acute myeloid leukemia resembling acute promyelocytic leukemia. Blood. 2018;131(16):1870-1873. 4. Qin Y-Z, Huang X-J, Zhu H-H. Identification of a novel CPSF6RARG fusion transcript in acute myeloid leukemia resembling acute promyelocytic leukemia. Leukemia. 2018;32(10):2285-2287. 5. Miller CA, Tricarico C, Skidmore ZL, et al. A case of acute myeloid leukemia with promyelocytic features characterized by expression of a novel RARG-CPSF6 fusion. Blood Adv. 2018;2(11):1295-1299. 6. Su Z, Liu X, Xu Y, et al. Novel reciprocal fusion genes involving HNRNPC and RARG in acute promyelocytic leukemia lacking

RARA rearrangement. Haematologica. 2020;105(7):e376-e378. 7. Chen X, Wang F, Zhang Y, et al. A novel NPM1-RARG-NPM1 chimeric fusion in acute myeloid leukaemia resembling acute promyelocytic leukaemia but resistant to all-trans retinoic acid and arsenic trioxide. Br J Cancer. 2019;120(11):1023-1025. 8. Osumi T, Tsujimoto S-i, Tamura M, et al. Recurrent RARB translocations in acute promyelocytic leukemia lacking RARA translocation. Cancer Res. 2018;78(16):4452-4458. 9. Wang H-Y, McMahon C, Ali SM, et al. Novel FNDC3B and MECOM fusion and WT1 L378fs* 7 frameshift mutation in an acute myeloid leukaemia patient with cytomorphological and immunophenotypic features reminiscent of acute promyelocytic leukaemia. Br J Haematol. 2016;172(6):987-990. 10. Su Z, Liu X. Comment on Geoffroy, M.-C; de Thé, H. Classic and variants APLs, as viewed from a therapy response. Cancers 2020, 12, 967. Cancers (Basel). 2021;13(23):5883. 11. Cheng C-K, Chan H-Y, Yung Y-L, et al. A novel NUP98-JADE2 fusion in a patient with acute myeloid leukemia resembling acute promyelocytic leukemia. Blood Adv. 2022;6(2):410-415.

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Transcriptomic profiling does dot refine mastocytosis diagnosis The recent 5th edition of the World Health Organization (WHO) update on hematopoietic cancers includes relevant changes to the diagnostic criteria of mastocytosis.1 However, it remains entirely unclear whether transcriptional profiles of the bone marrow of patients with mastocytosis instruct diagnostic, prognostic or predictive information as it has been shown in many other cancer types.2 Here we show that transcriptional profiling of a large and clinically wellannotated dataset of systemic mastocytosis (SM) patients fails to achieve a further diagnostic refinement of SM above the level of genomic and clinical parameters. Nevertheless, transcriptional differences between clinical and genomic SM subgroups robustly link the expression of certain genes to the mutation-adjusted risk score (MARS) as a surrogate of prognosis. The advent of next-generation sequencing (NGS), with its increasing availability and reducing cost, has transformed cancer diagnosis and care. Especially in hematological malignancies, seminal breakthroughs showing the potential holistic nature of high throughput sequencing in the diagnosis of malignant disease1,3 have been achieved. Next to whole genome sequencing (WGS), transcriptomic profiling yields additional layers of complexity linking genetic lesions to signaling consequences and even guiding therapy.2 Furthermore, the combined usage of WGS and RNA sequencing may benefit the refinement of diagnosis as well as prognosis, bearing large untapped potential in the era of precision medicine. SM is a heterogeneous group of hematopoietic neoplasms characterized by amplified proliferation of aberrant mast cells in adult patients. Contrary to the predominantly pediatric cutaneous mastocytosis (CM), clonal mast cells in SM expand in the bone marrow and other organs, such as the liver, spleen, gut, and lymph nodes.4 The 2016 classification of the WHO5 further subdivides SM into indolent systemic mastocytosis (ISM), smoldering systemic mastocytosis (SSM), SM with associated hematologic neoplasm (SMAHN), aggressive systemic mastocytosis (ASM) and mast cell leukemia (MCL). SM-AHN, ASM, and MCL can be summarized as advanced SM (advSM). Pathognomonic for the disease are somatic gain-of-function mutations in KIT, which occur in >90% of all cases, with D816V accounting for >90% of all mutations.6,7 ISM patients experience mostly mediator-related symptoms such as nausea, flush, and pruritus, whereas advSM patients suffer primarily from symptoms caused by expansion of mast cells leading to bone marrow suppression and altered organ function, namely cytopenia, malabsorption, hepato-splenomegaly or

osteopenia.4 The severity of symptoms is additionally affected by often allergic comorbidities which can considerably impede oncological management. The clinical complexity translates to prognosis, reaching from near-normal life expectancy in indolent forms to a median survival of 2.9 years and 1.6 years in patients with SM-AHN and MCL, respectively.8,9 Baseline therapies aim to reduce mediator-related symptoms or serve as prophylaxis and comprise histamine receptor 1 and 2 antagonists, mast cell stabilizers, steroids, bisphosphonates, and vitamin D supplementation. In cases of advSM, systemic cytotoxic treatment with midostaurin,10 a tyrosine kinase inhibitor, or the recently approved avapritinib11 is considered the gold standard. However, polychemotherapy treatment analogous to de novo AML or cladribine-containing regimen remains the ultima ratio in cases of therapy-refractory or quickly progressive advSM.12 The majority of SM patients present with additional somatic mutations, predominantly in TET2, SRSF2, ASXL1, RUNX1, JAK2, CBL, N/KRAS, EZH2, IDH1/2, and SF3B1 which have a crucial impact on prognosis.13 A multivariate analysis of risk factors identified age >60 years, anemia (hemoglobin <10 g/dL), thrombocytopenia (platelets <100x109/L), presence of one mutation in SRSF2, ASXL1, and/or RUNX1 and presence of two or more mutations in respective genes as associated with overall survival time. The MARS integrates these parameters and was confirmed to be independent of WHO classifications.14 Despite the advances in genomic characterization, heterogeneity of the disease remains a challenge to both clinicians and scientists. Additional layers of information to feed the clinical workflow are crucial to improving diagnostics and patient stratification. Therefore, set out to investigate whether transcriptional portraits of different subtypes of SM, for which extensive clinical data were gathered for subsequent analysis, could aid this effort (Table 1; Online Supplementary Table S1). Bone marrow aspirate from 20 male and 10 female patients with SM was taken during routine diagnostic bone marrow punctures. The same material also underwent panel sequencing as part of routine genetic diagnostics (Figure 1A). Healthy control samples were obtained from femoral heads resected during hip joint replacement surgery from two male and two female donors. The cohort comprises cases with ISM (n=5), ASM (n=5), SM-chronic myelomonocytic leukemia (CMML) (n=5), SM-myelodysplastic syndromes/myeloproliferative neoplasm-unclassifiable (SM-MDS/MPNu) (n=6), SM-MDS (n=4), SM-chronic eosinophilic leukemia (SM-CEL) (n=2), SM-MPN (n=1), SM-

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LETTER TO THE EDITOR Table 1. Cohort characteristics and metadata. Characteristics Age in years, mean (SD)

60.87 (15.22)

Sex

Female

33.33

Male

66.67

WHO subtype

ISM

16.67

ASM

16.67

SM-AHN

66.67

SM-CMML

16.67

SM-MDS/MPNu

SM-MDS

13.33

SM-CEL

6.67

SM-MPN

3.33

SM-MGUS

3.33

SM-AML

3.33

range

mean (SD)

5-85

27.14 (16.06)

69.00

range

mean (SD)

Leukocytes/μL

2,580-35,600

10,506.21 (8,294.84)

Hemoglobin, g/dL

6.5-15.8

11.65 (2.43)

Thrombocytes x103/μL

12.0-515.0

196.80 (148.37)

Monocytes/μl

126.0-3,850.0

825.63 (945.56)

Eosinophils/μl

0.0-8,188.0

820.07 (1,624.05)

Tryptase, μg/L

23.2-850.0

187.89 (188.75)

Alkaline phosphatase, IU/L

3.0-629.0

217.10 (186.05)

Risk

range

mean (SD)

Mutation-adjusted risk score

0-5

1.76 (1.48)

Clinical parameters Mast cell infiltration in bone marrow (%) Splenomegaly Laboratory values

SD: standard deviation; WHO: world health organization; SM: systemic mastocytosis; ISM: indolent SM; ASM: aggressive SM; SM-AHN: SM with associated hematologic neoplasm; SM-MDS: SM-myelodysplastic syndromes; MPNu: myeloproliferative neoplasm unclassifiable; CEL: chronic eosinophilic leukemia; MPN: myeloproliferative neoplasm; MGUS: monoclonal gammopathy of undetermined significance; AML: acute myeloid leukemia.

monoclonal gammopathy of undetermined significance (SM-MGUS) (n=1) and SM-acute myeloid leukemia (SM-AML) (n=1). RNA was extracted and sequencing was performed using the prime sequencing protocol.15 Finally, extensive computational analyses were performed (Figure 1B). Principal component analysis (PCA) did not reveal clustering patterns (Figure 2A). UpSet plots were generated based on differential expression (DE) analysis (Online Supplementary Table S2) and gene set enrichment analysis (GSEA) was performed to visualize transcriptional inter- and intra-subtype similarities. Genes differentially expressed in SM of different subtypes are reported compared to healthy controls (Figure 2B). Notably, the most homogenous subgroups ASM and ISM show the highest numbers of (uniquely) differentially ex-

pressed genes. While various deregulated genes are shared between different subgroups, a subset of 26 genes is deregulated in all SM(-AHN) subgroups (Figure 2B; Online Supplementary Table S2). The overlap of hallmark gene sets enriched for highly expressed genes (false discovery rate [FDR] <=0.05) in SM subtypes compared to healthy controls was calculated (Figure 2C). Of note, 18 gene sets are enriched in ASM of which five are unique (Online Supplementary Table S2). None of the other subgroups show unique enrichment for any hallmark gene set, except for SM-MDS (Online Supplementary Table S2). Six gene sets were enriched across all SM subtypes; if SM-MDS is excluded, three gene sets are overlapping between ASM, ISM, SM-CMML and SM-MDS/MPNu (Online Supplementary Table S2). In

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LETTER TO THE EDITOR

Figure 1. Mutational profile of the analyzed cohort and sample processing workflow. (A) Oncoplot of patient for which panel sequencing was performed during routine diagnostics. No panel sequencing was performed for patient P01, P02 and P05. (B) Graphical illustration of the sample processing workflow. Ins: insertion; Del: deletion; BM: bone marrow; BMMNC: bone marrow mononuclear cells.

comparison, GSE of lowly expressed genes (FDR <=0.05) in SM subtypes versus healthy controls was calculated (Figure 2D). The highest number of enriched gene sets (14) is reported for SM-CMML, of which two are unique (Online Supplementary Table S2). Other uniquely enriched gene sets appeared in ISM and ASM. Three gene sets were enriched across all subtypes (Online Supplementary Table S2). In order to link gene expression and prognosis, differential expression of patient samples with a MARS of 5 compared to healthy controls was computed. Additionally, a correlation analysis was performed to extract genes correlating with MARS. In total, 226 genes are differentially expressed in samples with a MARS of 5, while ten genes show a high correlation (r>0.65 for both Pearson and Spearman) with MARS (Figure 2E). Overall, two genes are both significantly differentially expressed and correlate with MARS simultaneously (Figure 2E, panels 2-4, FLT3 rPearson=0.75, rSpearman=0.70, IGF2BP2 rPearson=0.68, rSpearman=0.72). Extensive differential expression analysis of WHO subgroups (advSM vs. ISM vs. healthy/SMall vs. healthy), pre- and post-treatment with midostaurin, all mutations detected by the NGS panel, presence/absence of splenomegaly, hemoglobin/thrombocytes below/above cut-offs (hemoglobin <10 g/dL, platelets <100x109/L), MARS risk level as well as correlation analyses for mast cell infiltration, levels of leukocytes, hemoglobin,

thrombocytes, monocytes, eosinophils, tryptase, alkaline phosphatase, and albumin were performed (Online Supplementary Table S3). Notably, the cytological parameters did not alter the above-mentioned results, despite the bulk material approach of this work. Additionally, we provide all read counts for the samples analyzed (Online Supplementary Table S3). Despite advances in the development of novel drugs for SM, targeted therapeutic approaches almost exclusively exploit the canonical KIT D816V mutation present in >90% of SM cases. Based on clinical phenotype and histopathological assessment, the new 2022 WHO classification of SM aims to subdivide the disease in a more granular manner but does not include more sophisticated biomarkers. Especially within the particularly heterogenous subgroup of advanced SM, extensive molecular profiling is of eminent importance to decipher the complexity of the disease to allow an optimized patient and subgroup stratification as this might ultimately lead to patient benefit. Although our transcriptomic analysis was able to point out individual cases (e.g., AML with stem-like signature, CEL with eosinophilic signature), the approach failed to reveal a distinct pattern in a collection of different SM subtypes. This highlights the potential of RNA sequencing to profile cases per se, but also stresses that broad profiling might not yield

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LETTER TO THE EDITOR

Figure 2. Multiple analyses aiming to refine systemic mastocytosis diagnosis. (A) Principal component analysis (PCA) of all patients who were sequenced within the analysis (P01-P30, see Online Supplementary Table S1). (B-D) UpSet plots showing the overlap of gene deregulation when comparing systemic mastocytosis (SM) with or without associated hematologic neoplasm (AHN) component to healthy samples. (B) Overlap of genes that were differentially expressed in SM subtypes compared to healthy. (C) Overlap of hallmark gene sets that had an enrichment of highly expressed genes in SM subtypes compared to healthy. (D) Overlap of hallmark gene sets that had an enrichment of lowly expressed genes in SM subtypes compared to healthy. (E) UpSet plot of genes differentially expressed between mutation-adjusted risk score (MARS) 5 and MARS 0 as well as genes correlating with MARS, with 2 genes (FLT3 and IGF2BP2) overlapping. Correlation of FLT3 and IGF2BP2 with MARS as well as separate expression levels (CPM) across MARS -1 (healthy) to MARS 5 shown separately. SM: indolent SM; ASM: aggressive SM; SM-AHN: SM with associated hematologic neoplasm; SM-MDS: SM-myelodysplastic syndromes; MPNu: myeloproliferative neoplasm unclassifiable; CEL: chronic eosinophilic leukemia; MPN: myeloproliferative neoplasm; MGUS: monoclonal gammopathy of undetermined significance; AML: acute myeloid leukemia.

distinct results due to the complexity of the disease, hence not allowing for diagnosis refinement based on expression data. The inability of RNA sequencing to refine the clinical classification of mastocytosis and segregate these into more granular signaling-specific subtypes opens up several questions. Physiologically, mast cells represent highly specialized and differentiated cell types that, after leaving the bone marrow, migrate to the periphery in order to exert their innate immune role. Mast cells are most prominently known

as cellular facilitators of allergic reactions after IgE crosslinking. However, the malignant expansion and thereby the occurrence of SM and MCL bears enormous clinical challenges. Current therapy protocols include the use of midostaurin but still display discouraging results. Unlike the malignant transformation process in leukemia, the transformation of mast cells is mostly associated with mutations in KIT. Moreover, mast cell disorder symptoms originate from a fully differentiated cell most likely already present in the periphery. We, thus hypothesize that unlike in AML,

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LETTER TO THE EDITOR aberrant signaling due to oncogenic hits would be less potent in these cells primed for tissue residency and terminal differentiation, which are processes most likely driven by strong inherent transcriptional signatures. Potential limitations of our study include the fact that in total a cohort of only 30 patients was investigated. Future studies with larger cohorts might yield more subtle transcriptional profiles which might refine diagnostic stratification, although the rarity of the disease poses a challenging hurdle. Altogether our data do not endorse transcriptomic approaches to refine molecular stratification of mast cell malignancies. Nevertheless, transcriptomic profiling revealed distinct signatures in individual patients and was able to link gene expression to surrogate risk markers.

Mannheim, Mannheim, Germany and 9Division of Clinical Oncology, Department of Internal Medicine, Medical University of Graz, Graz, Austria LB and DIO contributed equally as first authors.

AR, MJ and PJJ contributed equally as senior authors.

Correspondence: P.J. JOST - philipp.jost@medunigraz.at https://doi.org/10.3324/haematol.2022.282617 Received: January 20, 2023. Accepted: May 4, 2023. Early view: May 11, 2023. ©2023 Ferrata Storti Foundation

Authors

Published under a CC BY-NC license

Lars Buschhorn,1,2,3* Dorett I. Odoni,4,5* Johanna Geuder,6 Timo O.

Disclosures

Odinius, Celina V. Wagner, Stefanie Jilg,

No conflicts of interest to disclose.

3,7

Wahida,

Matthias Schlesner,

Jawhar

and Philipp J. Jost

1,2,3

4,5

Ulrike Höckendorf, Adam

Andreas Reiter,

Mohamad Contributions

3,9#

LB and DO performed the clinical and computational analysis, Division of Molecular Genetics, German Cancer Research Center

wrote the manuscript and designed the figures. JG performed RNA

(DKFZ), Heidelberg, Germany; 2Division of Gynecological Oncology,

sequencing. TOO, CW, SJ, UH and AW provided conceptual input. MS

National Center for Tumor Diseases (NCT), Heidelberg, Germany;

supervised the computational analysis. AR and MJ provided the

Department of Internal Medicine III, School of Medicine, Technical

analyzed material. PJJ conceived the analysis and supervised the

University of Munich, Munich, Germany; Bioinformatics and Omics

project. All authors provided critical feedback and helped shape the

Data Analytics, German Cancer Research Center, Heidelberg,

research, analysis and manuscript.

Germany; Biomedical Informatics, Data Mining and Data Analytics, 5

Augsburg University, Augsburg, Germany; 6Anthropology & Human

Data-sharing statement

Genomics, Faculty of Biology, Ludwig-Maximilians University,

The original data as well as protocols will be made available to other

Martinsried, Germany; 7Onkologie Erding, Erding, Germany; 8Medical

investigators without any restrictions. The data can be obtained upon

Department III for Hematology and Oncology, University Clinic

request via email to lars.buschhorn@med.uni-heidelberg.de.

References 1. Khoury JD, Solary E, Abla O, et al. The 5th edition of the World Health Organization classification of haematolymphoid tumours: myeloid and histiocytic/dendritic neoplasms. Leukemia. 2022;36(7):1703-1719. 2. Rodon J, Soria JC, Berger R, et al. Genomic and transcriptomic profiling expands precision cancer medicine: the WINTHER trial. Nat Med. 2019;25(5):751-758. 3. Duncavage EJ, Schroeder MC, O’Laughlin M, et al. Genome sequencing as an alternative to cytogenetic analysis in myeloid cancers. N Engl J Med. 2021;384(10):924-935. 4. Pardanani A. Systemic mastocytosis in adults: 2021 Update on diagnosis, risk stratification and management. Am J Hematol. 2021;96(4):508-525. 5. Valent P, Akin C, Metcalfe DD. Mastocytosis: 2016 updated WHO classification and novel emerging treatment concepts. Blood. 2017;129(11):1420-1427. 6. Lim KH, Tefferi A, Lasho TL, et al. Systemic mastocytosis in 342 consecutive adults: survival studies and prognostic factors.

Blood. 2009;113(23):5727-5736. 7. Garcia-Montero AC, Jara-Acevedo M, Teodosio C, et al. KIT mutation in mast cells and other bone marrow hematopoietic cell lineages in systemic mast cell disorders: a prospective study of the Spanish Network on Mastocytosis (REMA) in a series of 113 patients. Blood. 2006;108(7):2366-2372. 8. Sperr WR, Kundi M, Alvarez-Twose I, et al. International prognostic scoring system for mastocytosis (IPSM): a retrospective cohort study. Lancet Haematol. 2019;6(12):e638-e649. 9. Kennedy VE, Perkins C, Reiter A, et al. Mast cell leukemia: Clinical and molecular features and survival outcomes of patients in the ECNM registry. Blood Adv. 2023;7(9):1713-1724. 10. Gotlib J, Kluin-Nelemans HC, George TI, et al. Efficacy and safety of midostaurin in advanced systemic mastocytosis. N Engl J Med. 2016;374(26):2530-2541. 11. Gotlib J, Reiter A, Radia DH, et al. Efficacy and safety of avapritinib in advanced systemic mastocytosis: interim analysis

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LETTER TO THE EDITOR of the phase 2 PATHFINDER trial. Nat Med. 2021;27(12):2192-2199. 12. Gleixner KV, Valent P, Sperr WR. Treatment of patients with aggressive systemic mastocytosis, mast cell leukemia and mast cell carcoma: a single center experience. Blood. 2018;132(Suppl 1):S1769. 13. Muñoz-González JI, Jara-Acevedo M, Alvarez-Twose I, et al.

Impact of somatic and germline mutations on the outcome of systemic mastocytosis. Blood Adv. 2018;2(21):2814-2828. 14. Jawhar M, Schwaab J, Álvarez-Twose I, et al. MARS: mutationadjusted risk score for advanced systemic mastocytosis. J Clin Oncol. 2019;37(31):2846-2856. 15. Janjic A, Wange LE, Bagnoli JW, et al. Prime-seq, efficient and powerful bulk RNA sequencing. Genome Biol. 2022;23(1):88.

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LETTER TO THE EDITOR

A proposed predictive mathematical model for efficient Tcell collection by leukapheresis for manufacturing chimeric antigen receptor T cells Manufacturing chimeric antigen receptor T cells (CAR T cells) requires collection of CD3+ lymphocytes through mononuclear cell (MNC) leukapheresis. MNC leukapheresis for autologous CAR T cells manufacturing in patients with relapsed/refractory leukemia and lymphoma who have undergone multiple lines of chemotherapies creates various challenges. Firstly, patients’ leukopenia and lymphopenia make the red blood cell-white blood cell interface in the apheresis machine difficult to be established.1 Secondly, patients are subjected to long durations of leukaphereses with large volumes of blood processed in order to obtain sufficient CD3+ lymphocytes. Korell et al. advocated processing a minimal of 12-15 liters of blood in order to harvest sufficient number of CD3+ lymphocytes for CAR T-cell manufacturing.2 Patients often have suboptimal performance status and physical reserve to tolerate such long leukaphereses. Lastly, patients with relapsed/refractory leukemia and lymphoma usually have a small window period for successful leukapheresis when they are free of infections and their physical states are able to tolerate the leukapheresis. The current practices of MNC leukapheresis rely on processing a large volume of blood, which often leads to unnecessarily prolonged apheresis, wastage of manpower and hospital resources, and exposing the patients to additional risks associated with prolonged leukaphereses. Studies have reported various factors that impact the final CD3+ lymphocyte yield,3,4 and the preleukapheresis CD3+ count is the one common determining factor that has been repetitively mentioned. Preleukapheresis CD3+ count is difficult to be altered in patients with relapsed/refractory leukemia and lymphoma. Hence, patients with lower preleukapheresis CD3+ counts require larger volumes of blood processed during leukaphereses to meet a target yield and vice versa. A formula that can determine the required processed blood volume for patients based on their preleukapheresis CD3+ count to meet the target CD3+ lymphocyte yield will help to improve the efficiency of leukaphereses. In this study, we tried to understand the dynamics of the CD3+ lymphocyte collection through MNC leukapheresis and derive a predictive mathematical model using preleukapheresis CD3+ count to guide the required blood volume to be processed. We have included three sets of data in this study. The first set of data is from 12 MNC leukaphereses for CAR T-cell manufacturing at the Singapore General Hospital (SGH),

Department of Hematology from May 2020 to June 2021. The second set of data consists of five MNC leukaphereses performed at a different institute, the National University Hospital Singapore (NUH). This set of data was used to verify the consistency of the findings at a different institute. The third set of data consists of another six MNC leukaphereses for CAR T-cell manufacturing from June 2021 to April 2022 at SGH. This set of data was used to verify if the proposed mathematical equation derived from past data is applicable for future leukaphereses at the same institute. All patients had relapsed/refractory diffuse large B-cell lymphoma where majority had at least three lines of therapies. MNC leukaphereses were performed at least 1 month from the last cycle of chemotherapy. This study was approved by the Institutional Review Board and Ethics Committee of Singapore General Hospital. Consent were provided by all patients. All leukaphereses were performed using the “Terumo” Spectra Optia Apheresis system version 11.3 with continuous mononuclear cell collection (cMNC) protocol. Important parameters collected during the MNC leukaphereses were: preleukapheresis CD3+ lymphocyte count (denoted as Cpreleukapheresis, 109 cells/L), the total blood volume processed (denoted as VT, L), the total body blood volume for each patient (denoted as VB, L), the total amount of CD3+ lymphocyte yielded (denoted as T, 109 cells). The 12 leukaphereses at SGH between May 2020 and June 2021 were analyzed to derive the mathematical model. Table 1 summarizes the basic demographic, preleukapheresis laboratory data, and collection data of the 12 leukaphereses. Previous studies,5,6 described CD3+ lymphocytes collection efficiency (CE) as: CE=T/(Cpreleukapheresis*VT). The calculated CE of the 12 leukaphereses performed at SGH varied widely between 21.4-95.1% (mean 67.4%; standard deviation 20.5%). Similarly, CE of the five leukaphereses performed at NUH also varied widely between 26.3-75.0% (mean 53.7%; standard deviation 17.5%). Finding a representative CE for an institute may not be practical and may result in erroneous estimation of blood volume to be processed. The correlation between T and Cpreleukapheresis*VT in above equation only had an R2 of 0.75 for the 12 SGH leukaphereses. We think the reason for the wide variation of CE observed is due to the constant change of “real-time” circulating CD3+ lymphocytes concentration during leukapheresis, because CD3+ lymphocytes are constantly re-

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LETTER TO THE EDITOR Table 1. Summary of basic demographic, preleukapheresis laboratory data, and collection data for the 12 leukaphereses at the Singapore General Hospital from May 2020 to June 2021. Patients N=11 Leukaphereses N=12*

Mean

Range

Standard deviation

Female sex, N

10 (91%)

Age in years

19-73

Height, cm

160.7

148-179

8.3

Weight, kg

56.6

40-85.1

12.5

BMI

22.0

16.3-30.0

4.0

Hemoglobin level, g/dL

10.28

7.8-12.5

1.41

Hematocrit, %

30.63

24.3-35.2

3.60

Lymphocyte count x109/L

0.71

0.23-1.67

0.44

Prepheresis CD3+ lymphocyte count x109/L

0.57

0.20-1.49

0.37

Total body blood volume, L

3.57

2.80-5.45

0.68

Total blood volume processed, L

10.19

5.52-16.78

2.67

Total collection time, minute

319

117-410

Total mononuclear cell collected x109

12.25

5.75-19.65

4.90

Total CD3+ lymphocyte collected x109

3.57

0.67-7.66

1.97

Patient demographics

Preleukapheresis laboratory data

Leukapheresis data

*Note: the 12 leukaphereses were performed on 11 patients as 1 patient required a second collection. All patients had relapsed/refractory Bcell lymphoma and had previously been treated with at least 3 lines of systemic chemotherapies. BMI: body mass index.

moved from the peripheral blood during leukapheresis. The rate of CD3+ lymphocyte collection gradually slows down as more blood is being processed. Assuming a constant CE assumes a linear relationship between VT and T, i.e., doubling VT can result in doubling of T. This is unrealistic and will result in overestimation of the CD3+ lymphocyte yield. In order to account for the constant change in the “realtime” circulating CD3+ lymphocytes concentration, we made two assumptions: first the total amount of CD3+ lymphocytes circulating in the peripheral blood is not replenished from the extravascular space during MNC leukapheresis; secondly the apheresis machine removes a fraction (η) of the CD3+ lymphocyte from the blood that is fed to the machine (0< η <1). Figure 1 is a simplified illustration of the apheresis process, where t represents the duration of leukapheresis. The change of the amount of CD3+ lymphocytes in the patient’s body d(VB*C1) equals to the amount of CD3+ lymphocyte removed by the apheresis machine (C2-C1)*v*dt; therefore: d(VB*C1)/dt=(C2-C1)*v. By assumption 2, C2-C1=-η*C1. Solving the differential equation stated above results in: C1=e^constant*e^(η*v*t/VB). At t=0, C1 equals the preleukapheresis CD3+ lymphocyte concentration, Cpreleukapheresis; hence: C1=Cpreleuka-

*e^(-η*v*t/VB). v*t equals the total volume of blood processed, VT; hence: C1=Cpreleukapheresis*e^(-η*VT/VB). The total CD3+ lymphocyte collected can therefore be expressed as: T=(Cpreleukapheresis-C1)*VB=Cpreleukapheresis*VB*(1-e^(η*VT/VB)). This equation suggests that the maximum T from one leukapheresis is Cpreleukapheresis*VB, which is the total amount of CD3+ lymphocyte in the blood prior to leukapheresis. However, ten of the 12 leukaphereses performed at SGH between May 2020 and June 2021 were able to obtain more CD3+ lymphocytes than the total amount of CD3+ lymphocytes estimated in the blood prior to leukaphereses. Similar findings were observed in the five leukaphereses at NUH, where four of the five leukaphereses were able to obtain more CD3+ lymphocytes than estimated in the blood prior to leukaphereses. These findings suggest that CD3+ lymphocytes are possibly actively replenished from extravascular tissues during the leukapheresis, instead of the assumption that the total amount of CD3+ lymphocytes in the peripheral blood is not replenished during MNC leukapheresis. As discussed above, using a fixed collection efficiency assumes a linear relationship between VT and T (as shown in Figure 2 by a dotted line), which results in an overestimation; whereas assuming an MNC, leukapheresis is unpheresis

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LETTER TO THE EDITOR Figure 1. A simplified illustration of the leukapheresis process. C1: concentration of CD3+ lymphocytes in the patient’s peripheral blood at any time of leukapheresis; C2: concentration of CD3+ lymphocytes in the blood leaving the apheresis machine returning to the patient; VB: total body blood volume; v: apheresis machine blood flow rate; T: total number of CD3+ lymphocytes collected.

Figure 2. The visual representation of relationship between VT and T, where VT is the processed blood volume and T is the total CD3+ lymphocytes collected. The dotted line represents a linear relationship between VT and T, which results in overestimation; the long dash line represents a plateauing relationship between VT and T, which results in underestimation. The study postulated that the actual relationship is a curve shown in the solid line.

able to yield more than the amount of CD3+ lymphocytes in the blood prior to leukapheresis (as shown in Figure 2 by a long dashed line) which results in an underestimation of the actual CD3+ lymphocyte yield. Therefore, we postulate that the relationship is likely a curve as shown in Figure 2 by the solid line: the speed of CD3+ lymphocytes collected will gradually slow down, but the total number of CD3+ lymphocyte collected should continue to increase as leukapheresis continues. In order to describe the curve in a solid line, we used a logarithm equation to approximate it, where a and b are constants unique to each apheresis centre: T=a*ln(Cpreleukapheresis*VT)+b. Based on previous leukaphereses data, a and b can be obtained using regression line formula. Using the data from the 12 leukaphereses at SGH between May 2020 and June 2021, the equation for SGH was obtained: T=3.588*ln(Cpreleukapheresis*VT)-2.006 (R2: 0.90). The residual standard error for this equation was

0.66. The equation obtained was tested on the six subsequent MNC leukaphereses performed at SGH between June 2021 and April 2022 and it had an R2 value of 0.91. This showed that the equation obtained from previous data was still applicable for subsequent leukaphereses in the same center. This proposed mathematical model was also tested on data from a different institute, NUH. Constants a and b were calculated for NUH. The equation had an R2 value of 0.97. Previously published literature has described variables with predictive value for the CD3+ yield, including CD3+ count and hematocrit in one study3 and CD3+ count, hemoglobin level, and platelet count in the other.4 Despite some differences, the CD3+ count is consistently the most important variable that impacts the final CD3+ yield. What the current model adds to the existing ones is that it can be generalized. It requires each center to calculate their individual

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LETTER TO THE EDITOR constants a and b to fit the differences in the patient profiles, operator factors, and machine factors. In summary, this study demonstrates that the yield of CD3+ lymphocyte positively correlates to the preleukapheresis CD3+ lymphocyte count and the volume of blood processed. The equation T=a*ln(Cpreleukapheresis*VT)+b can be generalized to describe the CD3+ lymphocyte collection through MNC leukapheresis and helps provide an estimation of the minimum blood volume to be processed to meet the CD3+ lymphocyte target requirements.

Received: November 6, 2022. Accepted: May 3, 2023. Early view: May 11, 2023. ©2023 Ferrata Storti Foundation Published under a CC BY-NC license Disclosures No conflicts of interest to disclose. Contributions XH contributed to the design of the study, analysis of the data and

Authors

writing of the paper. GPLG, KKH and JJL contributed to the laboratory testing, data collection and drafting of the paper. SP, RS, JMLT and GX contributed to performing leukaphereses, data

Xinxin Huang, Gina Pei Ling Gan, Esther Hian Li Chan, Kee Khiang

collection and writing of the paper. EHLC, AYLH, WYKW, YCL, YC,

Heng,1 Susila Perumal,1 Rohani Salleh,1 Jessica Mei Ling Teo,1 Gaoge

JKSQ, HT and CN contributed to the recruitment and assessment

Xie,1 Jing Jing Lee,1 Aloysius Yew Leng Ho,1 William Ying Khee

of the patients, guidance in the study, drafting and review of the

Hwang, Yeh Ching Linn, Yunxin Chen, Jeffrey Kim Siang Quek, Hein

paper. FLWIL is the principle investigator. She contributed to the

Than, Chandramouli Nagarajan and Francesca Lorraine Wei Inng

conceptualisation and design of the study, guidance of the study,

Lim

writing and review of the paper.

Department of Haematology, Singapore General Hospital and

Acknowledgments

Department of Hematology, National University Hospital,

We would like to thank all participants and nurses for their support

Singapore

in this study.

Correspondence:

Data-sharing statement

F.L.W.I. LIM - francesca.lim.w.i@singhealth.com.sg

The data that support the findings of this study are available from

the corresponding author upon reasonable request. https://doi.org/10.3324/haematol.2022.282350

References 1. Fesnak A, Lin C, Siegel DL, Siegel DL, Maus MV. CAR-T cell therapies from the transfusion medicine perspective. Transfus Med Rev. 2016;30(3):139-145. 2. Korell F, Laier S, Sauer S, et al. Current challenges in providing good leukapheresis products for manufacturing of CAR-T cells for patients with relapsed/refractory NHL or ALL. Cells. 2020;9(5):1225. 3. O'Reilly MA, Malhi A, Cheok KPL, et al. A novel predictive algorithm to personalize autologous T-cell harvest for chimeric antigen receptor T-cell manufacture. Cytotherapy.

2023;25(3):323-329. 4. Jo T, Yoshihara S, Hada A, et al. A clinically applicable prediction model to improve T cell collection in chimeric antigen receptor T cell therapy. Transplant Cell Ther. 2022;28(7):365.e1-365.e7. 5. Allen ES, Stroncek DF, Ren J, et al. Autologous lymphapheresis for the production of chimeric antigen receptor T cells. Transfusion. 2017;57(5):1133-1141. 6. Hutt D, Bielorai B, Baturov B, et al. Feasibility of leukapheresis for CAR T-cell production in heavily pre-treated pediatric patients. Transfus Apher Sci. 2020;59(4):102769.

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LETTER TO THE EDITOR

TET2 mutational status affects myelodysplastic syndrome evolution to chronic myelomonocytic leukemia According to the 4th iteration of the World Health Organization (WHO) classification that came out in 2017, a diagnosis of chronic myelomonocytic leukemia (CMML) still required a persistent absolute (≥1x109/L) and relative (≥10% of white blood cell [WBC] count) monocytosis. Nevertheless, several reports suggested that up to 30% of myelodysplastic syndrome (MDS) evolved into genuine CMML,1 raising for many years the question of a “preCMML” state.2 An oligo-monocytic CMML entity was subsequently described in patients displaying a relative monocytosis with an absolute monocyte count between 0.5x109/L and 1x109/L,3,4 providing a rationale to revise CMML diagnosis criteria in the recent 5th edition of the WHO classification that considers a relative monocytosis ≥10% of WBC and an absolute monocyte count ≥0.5x109/L.5 An increased fraction of circulating classical monocytes (cMO, defined by flow cytometry as CD14++CD16- fraction) ≥94%, which was shown to distinguish CMML from reactive monocytosis,1,6 was included as a supportive criterion for CMML diagnosis. Notwithstanding these new diagnosis criteria, MDS with bone marrow (BM) monocyte infiltration could represent another group of pre-CMML. The present longitudinal study aims at identifying robust predictors of MDS evolution to “overt” CMML. Between 2018 and 2020, flow cytometry analysis of monocyte subset partition was performed in 44 patients with non-treated MDS and were included in a learning cohort together with 22 CMML patients (Table 1). In a control group of 19 patients with established, non-treated CMML according to the 2017 WHO classification, flow cytometry analysis of monocyte subsets mostly showed a relative accumulation of cMO ≥94% of total circulating monocytes.7 The same flow analysis in patients with an MDS diagnosis showed an increase in the fraction of cMO ≥94% in 18 of 44 (41%) patients, a proportion consistent with a previous report.1 These cases were defined as “CMML-like MDS”, while MDS cases without an increase in the fraction of cMO ≥94% were designated as “other MDS” (n=26) (Table 1; Figure 1A, B). Patients with a CMML-like MDS were older than other MDS patients (P=0.03, unpaired t test with Welch’s correction). They showed a significantly higher monocyte percentage in their peripheral blood (PB) (13.3±1.5%) and BM (7±1%) as compared with other MDS patients (PB: 8.8±1.1%, P=0.0226; BM: 3±0.2%, P=0.0098; unpaired t test with Welch’s correction) while the absolute monocyte count (AMC) and the Revised International Prognostic

Scoring System (R-IPSS) did not differ significantly between the two groups (Figure 1A; Table 1). Granulocyte-macrophage colony-stimulating factor (GMCSF) hypersensitivity was previously identified in approximetely 90% of CMML patients.8 In a standard clonogenic assay supplemented with stem cell factor (SCF), erythropoetin (EPO), interleukin 3 (IL-3), and GM-CSF, we observed a higher proportion of colony-forming unit granulocyte-macrophage (CFU-GM) as compared with burst-forming unit-erythroid (BFU-E) cells in CMML (78.9±5.9%; P=0.0009; Mann-Whitney test), CMML-like MDS (71.2±7.5%; P=0.0006, Mann-Whitney test) and in other MDS (63.7±7.2%; P=0.0159, Mann-Whitney test) (Figure 1C) showing a granulomonocytic differentiation bias. Without GM-CSF, growth factor-independent colony formation was barely detectable in MDS samples, with or without abnormal partition of monocyte subsets. Nevertheless, at low concentrations of GM-CSF, we detected an increased sensitivity of CMML-like MDS progenitor samples to GM-CSF as compared with those from other MDS, at both 0.1 ng/mL (P=0.048, Mann-Whitney test) and 1 ng/mL (P=0.0025; Mann-Whitney test) of GM-CSF (Figure 1D). The mutational profile was analyzed at diagnosis or during follow-up by next-generation sequencing (NGS) using a targeted panel of 38 genes recurrently mutated in myeloid malignancies as previously described.9 The mean number of mutations per sample in CMML-like MDS patients (3.5±0.2) was the same as that of CMML patients (3.5±0.3) and significantly higher than that of other MDS patients (1.7±0.3; P≤0.0001, unpaired t test with Welch’s correction). In the learning cohort, TET2 was the most frequently mutated gene in the three groups of patients, with a similar proportion of patients carrying at least one TET2 mutation in CMML (68.2%) and CMML-like MDS (66.7%) (P=0.92, Χ2 test) while this proportion was lower in other MDS (34.6%; P=0.036, Χ2 test) (Figure 1E). Sixty-eight TET2 mutations were identified in the learning cohort, including 52 (76.5%) truncating mutations, 14 (20.6%) missense mutations and two splice site mutations (2.9%). Truncating mutations were widely spread on the entire protein, while missense mutations were only located in the active domain of TET2: two in the Cys-N domain, two in the Cys-R domain, two in the low complexity insert, and eight in the double-stranded β-helix domain (DSBH) domain (Figure 2A). Among patients carrying TET2 mutations, multiple TET2 mutations were much more frequent among CMML (14/15, 93.3%) and CMML-like MDS

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LETTER TO THE EDITOR Table 1. Clinical and biological characteristics of the learning cohort. MDS

CMML

Total

CMML-like MDS

Other MDS

Age in years, mean (SD)

68.4 (2.5)

72.0 (1.8)

76.3 (2.5)

69.0 (2.5)

≤ 0.05

Male, N (%)

17 (77.2)

26 (55.3)

12 (66.7)

15 (53.8)

0.2339

Hemoglobin g/dL, mean (SD)

12.2 (0.5)

10.9 (0.3)

10.4 (0.6)

11.3 (0.4)

0.0589

Platelets x109/L, mean (SD)

167 (22)

178 (19)

178 (35)

177 (23)

0.7353

WBC x109/L, mean (SD)

10.5 (1.2)

4.7 (0.4)

4.5 (0.6)

4.6 (0.5)

≤ 0.001

ANC x109/L, mean (SD)

5.4 (0.9)

2.6 (0.3)

2.7 (0.5)

2.7 (0.4)

≤ 0.05

AMC x109/L, mean (SD)

2.5 (0.4)

0.5 (0.0)

0.5 (0.1)

0.4 (0.1)

≤ 0.001

Blood monocytes %, mean (SD)

24.9 (2.1)

10.6 (0.9)

13.3 (1.5)

8.8 (1.1)

≤ 0.001

Marrow monocytes %, mean (SD)

11 (1)

4 (1)

7 (1)

3 (0.2)

≤ 0.001

Marrow blasts %, mean (SD)

8 (1)

7 (1)

6 (1)

0.0889

Mean MO1 fraction %, (SD)

95.5 (0.7)

88.0 (1.5)

96.9 (0.3)

82.1 (1.7)

≤ 0.001

18 (40.9)

13 (59.1)

23 (52.3)

11 (61.1)

12 (46.2)

IPSS-R (%)

39 (88.6)

14 (77.8)

25 (96.2)

0.31

Very low IPSS-R, N (%)

5 (12.8)

2 (14.3)

3 (12.0)

Low IPSS-R, N (%)

18 (46.2)

4 (28.6)

14 (56.0)

Intermediate IPSS-R, N (%)

10 (25.6)

5 (35.7)

5 (20.0)

High IPSS-R, N (%)

4 (10.3)

2 (14.3)

2 (8.0)

Very high IPSS-R, N (%)

2 (5.1)

1 (7.1)

1 (4.0)

CPSS, N (%)

21 (95.5)

Low CPSS, N (%)

0 (0.0)

Intermediate-1 CPSS, N (%)

9 (42.8)

Intermediate-2 CPSS, N (%)

6 (28.6)

High CPSS, N (%)

6 (28.6)

Patients, N

Patients with cMo ≥94%, N (%) De novo diagnosis, N (%)

Myelodysplastic syndrome (MDS) patients have been classified into chronic myelomonocytic leukemia (CMML)-like MDS and other MDS according to the presence or absence of classical monocytes (cMO) accumulation ≥94%. Comparisons between CMML, CMML-like and other MDS patients were made using the Kruskal-Wallis test and Chi2 test. SD: standard deviation; WBC: white blood cell count; ANC: absolute neutrophil count; AMC: absolute monocyte count; IPSS-R: revised International Prognostic Scoring System; CPSS: CMML-specific prognostic scoring.

(10/12, 83.3%) compared to other MDS (2/9, 22.2%) patients (P=0.0007 and P=0.0092 respectively, Fisher’s exact test) (Figure 2B). The multiple mutations found in CMML and CMML-like MDS patients often showed very similar allelic ratio at around 0.5 and were eventually detected with several additional minor subclones. When CMML-like MDS patients carried a single mutation, the corresponding variant allele frequency (VAF) was above 50% (Figure 2C) indicative of a loss of heterozygosity (LOH). Overall, ten of 12 CMML-like MDS patients carrying TET2 mutations had,

therefore, mutation VAF indicative of a bi-allelic inactivation of TET2, which was observed in only one of nine MDS patients without accumulation of cMO (Figure 1E; Figure 2C; Online Supplementary Table S2). We previously showed that about 50% of MDS patients with cMO accumulation progressed to genuine CMML within 1 year.1 In the present study, among untreated MDS patients with at least 1 year of follow-up (n=21/44), we observed that 55.6% (n=5/9) of CMML-like MDS patients progressed to overt CMML (median follow-up 49 months).

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LETTER TO THE EDITOR Figure 1. A subgroup of myelodysplastic syndromes shares some common features with chronic myelomonocytic leukemia. (A) Comparison of classical monocyte (cMO) percentage in peripheral blood (PB) (upper left panel) including patients with a bulbous aspect associated to chronic myelomonocytic leukemia (CMML) with an inflammatory state (N=3, purple stars), monocyte percentage in PB (upper right panel), absolute PB monocyte count (AMC) (lower left panel) and monocyte percentage in bone marrow (lower right panel) among “other” myelodysplastic syndromes (MDS) (N=26, blue circles), “CMML-like” MDS (N= 18, green circles) and CMML patients (N=22, purple circles). (B) Examples of monocyte subset repartition in PB analyzed by flow cytometry in “other” MDS with no cMO accumulation (upper panel), while cMO >94% is displayed for CMML (middle panel) and “CMML-like” MDS (lower panel) patients. (C) Comparison of the proportion of erythroid colonies (BFU-E, solid circles) and granulo-monocytic colonies (CFU-GM, empty circles) obtained with colony-forming cell assay (CMML; P=0.0009; CMML-like MDS P=0.0006 and in other MDS P=0.0159, Mann-Whitney test). For each patient, 103 CD34+ enriched cells (N=17, CD34 Microbeads Kit, Miltenyi Biotec) or 7x104 bone marrow mononuclear cells (N=3) were seeded in duplicate in a methylcellulose based-medium with growth factors (Methocult H4434, STEMCELL Technologies) for 14 days at 37°C with 5% CO2. Results are expressed as the percentage of all colonies formed by at least 50 cells for “other” MDS (N=5), “CMML-like” MDS (N=7) and CMML (N=8) patients. (D) Colony-forming unit granulocyte-macrophages (CFU-GM) obtained after colony-forming cell assay with the same input cell numbers were seeded in methylcellulose without growth factors (Methocult H4230, STEMCELL Technologies) at various concentrations of granulocytemacrophage stem cell factor (GM-CSF) (Sandoz; 0 ng/mL, 0.1 ng/mL and 1 ng/mL). Results are expressed as the percentage of CFU-GM found compared to control cultures supplemented with growth factors (50 ng/mL SCF, 10 ng/mL IL-3, 3 U/mL erythropoietin [EPO], and GM-CSF 10 ng/mL, Methocult H4434, STEMCELL Technologies). The upper 95% confidence interval (CI) of the average percentage of normalized colonies derived from “other” MDS patients was calculated for each concentration of GM-CSF tested and used as a cut-off to classify CMML and CMML-like MDS progenitors as hypersensitive. (E) Mutational landscape of CMML, “CMML-like” MDS, and “other” MDS patients included in the learning cohort. Each column represents a patient. Vertical black bars show the total number of mutations detected. Horizontal black bars show the percentage of patients harboring at least one mutation for each gene out of all the patients included in the study. The first line depicts the absence (white square) or the presence of a relative accumulation of cMO ≥94% (green square); an abnormal distribution of monocyte subsets with a bulbous profile associated to CMML with an inflammatory state is represented by a green square with a white asterisk. One patient (represented as a green square with a black asterisk) had an uninterpretable flow cytometry profile due to paroxysmal nocturnal hemoglobinuria. Repartition of monocyte subsets was not available for two patients diagnosed in 2009. Normal karyotype, complex karyotype, and other cytogenetic profiles are shown as light blue square, dark blue square, and blue square, respectively. Revised International Prognostic Scoring System (IPSS-R) score for MDS patients and CMML-specific prognostic scoring (CPSS) score for CMML patients are depicted as low-risk profile (IPSS-R ≤3 and CPSS ≤1, light purple square) or highrisk profile (IPSS-R >3 and CPSS ≥2, dark purple square). In the mutation heatmap, the absence of mutation is shown by a light grey square, one mutation by a yellow square, and multiple mutations by a red square. Multiple mutations are defined by either several gene mutations or a single mutation with a variant allelic frequency (VAF) above 65%. Analysis not performed or uninterpretable results are shown as a dark grey square.

This rate progression to CMML was significantly higher compared with other MDS patients (1/12; 8.3%) (P=0.0393, log-rank test) (Figure 2D). Of note, CMML-like MDS patients overall survival was significantly reduced compared with other MDS patients (P=0.0483; log-rank test) (Figure 2E). This result would need further validation in larger independent cohorts. We next stratified MDS patients from the learning cohort according to the presence of multiple TET2 mutations (Figure 2F). MDS patients carrying multiple TET2 mutations had a significantly increased percentage of peripheral blood (15.8±1.8%; P=0.0025, unpaired t test with Welch’s correction) and BM (8±2%; P=0.0078, unpaired t test with Welch’s correction) monocytes compared with other MDS patients. They were significantly older (P=0.0269, unpaired t test with Welch’s correction) and also showed a reduced CMML progression-free survival (P=0.0015, log-rank test) (Figure 2G) with a median progression-free survival estimated at 3 months. Interestingly, the unique MDS patient without cMO accumulation who progressed to CMML had multiple TET2 mutations. This result was further confirmed with multicenter validation cohort of 39 untreated MDS patients (P=0.0016, log-rank test), including 14 patients with multiple TET2 mutations whose biological features were similar to those

from the learning cohort (Figure 2H; Online Supplementary Figure S1; Online Supplementary Table S1). Finally, among all MDS patients analyzed in the learning and validation cohorts, TET2I1873T (n=4) was significantly associated with evolution to CMML (P=0.008, LASSO penalized regression). Recent evolution of myeloid disease classification integrates so-called oligomonocytic CMML,3 which exhibits characteristic CMML features4 by decreasing the cut-off value that defines an absolute monocytosis to 0.5x109/L.5 The present study demonstrates that flow cytometry analysis of monocyte subsets in the PB, typically showing an accumulation of cMO ≥94% of total monocytes,6,7,10 together with typical combinations of somatic gene mutations, detects an additional number of CMML whose initial features are those of an MDS. Their genetic and epigenetic features already point to a CMML, while the characteristic increase in absolute and relative monocyte count will be observed several months or years later.1 While early identification of an overlap myelodysplastic/myeloproliferative neoplasm (MDS/MPN) may currently have a limited therapeutic impact, these entities deserve to be identified as they may indicate higher risk evolution with decreased survival.3 In the present study, the MDS to CMML evolution was evaluated in patients that didn’t receive hypomethyl-

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LETTER TO THE EDITOR Figure 2. TET2 mutational status and myelodysplastic syndrome evolution to chronic myelomonocytic leukemia. (A) Lollipop diagram showing mutation distribution across the TET2 protein in the learning cohort. Sixty-eight mutations from 36 patients are plotted (nonsense mutations are shown in black, frameshift mutations in pink, and missense mutations in green). (B) Patients harboring multiple mutations of TET2 gene in the 3 subgroups, expressed in percentage among “other” myelodysplastic syndromes (MDS) patients (N=26, blue), “chronic myelomonocytic leukemia (CMML)-like” patients (N=18, green), and CMML patients (N=22, purple). (C) Distribution of variant allelic ratios of TET2 mutations in the 3 subgroups. Each mutation is shown as a circle for each patient identified by their unique patient number (UPN). The dotted vertical line represents the 50% variant allele frequency (VAF) threshold. (D) Kaplan-Meier CMML progression-free survival (PFS) in “CMML-like” MDS (N=9, green line) and “other” MDS (N=12; blue line). (E) Kaplan-Meier overall survival (OS) in “CMML-like” MDS (N=9; green line) and “other” MDS (N=12; blue line). (F) Monocytes percentage (upper left panel) and AMC (upper right panel) in peripheral blood (PB), monocyte percentage in bone marrow (BM) (lower left panel), and percentage of circulating classical monocyte (cMO) (lower right panel) in the 4 subgroups defined according to their molecular profile: MDS patients carrying multiple TET2 mutations (solid red circle) or 1 mutation or no TET2 mutation (open black circle) and CMML patients carrying multiple TET2 mutations (solid purple circle) or 1 mutation or no TET2 mutation (open purple circle). (G) Kaplan-Meier CMML PFS of MDS patients carrying multiple TET2 mutations (N=6, red line) or one mutation or no TET2 mutation (N=15, black line) in the learning cohort. Multiple mutations are defined by several mutations affecting TET2 or a unique mutation with a VAF >65%. (H) Kaplan-Meier CMML PFS of MDS patients carrying multiple TET2 mutations (N=14, red line) or 1 mutation or no TET2 mutation (N=25, black line) in the validation cohort. In (D, E, G, and H) only patients with a minimal 12-month follow-up were included in the analysis. Patients who received hypomethylating agents after PB monocyte phenotyping were censured.

ating agents or hematopoietic stem cell transplantation. In the future, revisiting the boundaries between MDS and CMML may guide therapeutic approaches targeting the proliferative and highly inflammatory component of CMML to slow down its clinical progression. As proof of the proliferative component of the disease, CFU-GM hypersensitivity to GM-CSF was observed in both CMML-like MDS and CMML patients and comparable to those previously reported in juvenile myelomonocytic leukemia and CMML.8 Myeloid colony formation becomes independent of growth factors in a subset of highly proliferative CMML patients, which commonly correlates with clonal evolution including the acquisition of RAS pathway mutations,11 and is associated with an inferior outcome.12 In the present study, GM-CSF hypersensitivity was identified in patients who didn’t carry signaling mutations. The NGS panel covered most of the recurrently mutated signaling genes described in myeloid malignancies except NF1. Interestingly, a previous study showed efficient inhibition of CMML cell growth using an anti-GM-CSF antibody regardless of signaling gene mutations.8 Clonal architecture analysis showed that TET2 mutation is often the first hit in CMML,13 and bi-allelic TET2 inactivation may be a key event in driving the disease phenotype, either oligo-CMML or the full-blown disease.3,14 We noticed that the most frequent TET2 missense mutation TET2I1873T, was associated with bi-allelic alteration of TET2 and identified as a significantly independent factor of CMML evolution. TET2I1873T is located in a conserved region of the DSBH domain, and its functional impact is still only partially understood. In conclusion, a combination of phenotypic, genomic and functional features defines a subset of MDS patients who may eventually evolve into CMML, revisiting the boundaries between MDS and MDS/MPN and potentially defining a subgroup of patients with a poorest outcome.3 It may guide future therapeutic strategies targeting the prolifer-

ative and highly inflammatory component of CMML to slow down its clinical progression, such as lenzilumab, an anti-GM-CSF monoclonal antibody. These results also point to the usefulness of combining flow cytometry of peripheral blood monocyte subsets and analysis of their mutational status to better classify patients with dysplastic features.15

Authors Violaine Tran Quang,1,2 Benjamin Podvin,3+ Christophe Desterke,4+ Sihem Tarfi,1,2 Quentin Barathon,2 Bouchra Badaoui,2 Nicolas Freynet,2 Vincent Parinet,1,5 Mathieu Leclerc,1,5 Sébastien Maury,1,5 Eric Solary,6 Dorothée Selimoglu-Buet,6 Nicolas Duployez,4 Orianne Wagner-Ballon1,2# and Ivan Sloma1,2# Université Paris Est Créteil, INSERM, IMRB, Créteil; 2AP-HP, Hôpital

Henri Mondor, Département d’Hématologie et Immunologie, Créteil; Centre Hospitalier Régional Universitaire de Lille, Laboratoire

d’Hématologie, Lille; 4Université Paris-Sud, Faculté de Médecine Kremlin Bicêtre, INSERM UMS 33, Villejuif; 5AP-HP, Hôpital Henri Mondor, Département d'Hématologie, Créteil and 6INSERM Unité Mixte de Recherche (UMR) 1287, Faculté de Médecine, Université Paris-Sud, Gustave Roussy, Villejuif, France BP and CD contributed equally.

OW-B and IS contributed equally as senior authors.

Correspondence: I. SLOMA - ivan.sloma@aphp.fr https://doi.org/10.3324/haematol.2022.282528 Received: January 11, 2023. Accepted: April 14, 2023.

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LETTER TO THE EDITOR Early view: April 27, 2023.

and ND provided biological and clinical data. ES and DS-B critically reviewed the article. IS developed and validated the NGS panel

design, the bioinformatics pipeline and performed tertiary NGS

Published under a CC BY-NC license

analysis with help from VTQ. OW-B and IS designed the study and wrote the manuscript.

Disclosures ES, DS-B, and OW-B have a patent issued relevant to the work. The

Acknowledgments

remaining authors have no conflicts of interest to disclose.

We thank Alban Lermine and the Bioinformatics platform MOABI from Assistance Publique-Hôpitaux de Paris for bioinformatics

Contributions

pipeline implementation in routine clinical practice. VTQ received a

VTQ performed flow cytometry and cellular bone marrow analyses,

fellowship from la Fondation pour la Recherche Médicale.

progenitor assays, and helped to write the manuscript. CD performed the LASSO biostatistics analysis. ST, BB and NF also

Data-sharing statement

performed flow cytometry analyses. QB prepared the NGS library

The supporting data are available in the Online Supplementary Table

with help from VTQ. SM, VP and ML provided patient samples. BP

S2.

References 1. Selimoglu-Buet D, Badaoui B, Benayoun E, et al. Accumulation of classical monocytes defines a subgroup of MDS that frequently evolves into CMML. Blood. 2017;130(6):832-835. 2. Kasprzak A, Assadi C, Nachtkamp K, et al. Monocytosis at the time of diagnosis has a negative prognostic impact in myelodysplastic syndromes with less than 5% bone marrow blasts. Ann Hematol. 2023;102(1):99-106. 3. Calvo X, Garcia-Gisbert N, Parraga I, et al. Oligomonocytic and overt chronic myelomonocytic leukemia show similar clinical, genomic, and immunophenotypic features. Blood Adv. 2020;4(20):5285-5296. 4. Garcia-Gisbert N, Arenillas L, Roman-Bravo D, et al. Multi-hit TET2 mutations as a differential molecular signature of oligomonocytic and overt chronic myelomonocytic leukemia. Leukemia. 2022;36(12):2922-2926. 5. Khoury JD, Solary E, Abla O, et al. The 5th edition of the World Health Organization classification of haematolymphoid tumours: myeloid and histiocytic/dendritic neoplasms. Leukemia. 2022;36(7):1703-1719. 6. Talati C, Zhang L, Shaheen G, et al. Monocyte subset analysis accurately distinguishes CMML from MDS and is associated with a favorable MDS prognosis. Blood. 2017;129(13):1881-1883. 7. Selimoglu-Buet D, Wagner-Ballon O, Saada V, et al. Characteristic repartition of monocyte subsets as a diagnostic signature of chronic myelomonocytic leukemia. Blood. 2015;125(23):3618. 8. Padron E, Painter JS, Kunigal S, et al. GM-CSF-dependent pSTAT5 sensitivity is a feature with therapeutic potential in chronic myelomonocytic leukemia. Blood.

2013;121(25):5068-5077. 9. Gricourt G, Tran Quang V, Cayuela J-M, et al. Fusion gene detection and quantification by asymmetric capture sequencing (aCAP-Seq). J Mol Diagn. 2022;24(11):1113-1127. 10. Patnaik MM, Timm MM, Vallapureddy R, et al. Flow cytometry based monocyte subset analysis accurately distinguishes chronic myelomonocytic leukemia from myeloproliferative neoplasms with associated monocytosis. Blood Cancer J. 2017;7(7):e584-e584. 11. Geissler K, Jäger E, Barna A, et al. Chronic myelomonocytic leukemia patients with RAS pathway mutations show high in vitro myeloid colony formation in the absence of exogenous growth factors. Leukemia. 2016;30(11):2280-2281. 12. Geissler K, Jäger E, Barna A, et al. Correlation of RAS-pathway mutations and spontaneous myeloid colony growth with progression and transformation in chronic myelomonocytic leukemia - a retrospective analysis in 337 patients. Int J Mol Sci. 2020;21(8):3025. 13. Itzykson R, Kosmider O, Renneville A, et al. Clonal architecture of chronic myelomonocytic leukemias. Blood. 2013;121(12):2186-2198. 14. Awada H, Nagata Y, Goyal A, et al. Invariant phenotype and molecular association of biallelic TET2 mutant myeloid neoplasia. Blood Adv. 2019;3(3):339-349. 15. Solary E, Wagner-Ballon O, Selimoglu-Buet D. Incorporating flow cytometry and next-generation sequencing in the diagnosis of CMML. Are we ready for prime? Best Pract Res Clin Haematol. 2020;33(2):101134.

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LETTER TO THE EDITOR

Clinical responses in pediatric patients with relapsed/refractory leukemia treated with azacitidine and venetoclax Despite optimization and escalation of risk-based chemotherapy regimens, children with relapsed or chemotherapyrefractory acute leukemias, particularly acute myeloid leukemia (AML), remain difficult to cure.1,2 Traditional intensive cytotoxic chemotherapy salvage regimens require extended inpatient hospitalization due to infectious risk during severe myelosuppression and are accompanied by therapy-related morbidity and deleterious impact upon quality-of-life.3,4 The combination of the hypomethylating agent azacitidine with venetoclax, an oral selective BH3mimetic inhibitor of the anti-apoptotic protein B-cell lymphoma 2 (BCL-2), is effective at remission induction in elderly or intensive induction chemotherapy-ineligible adults with previously-untreated AML and is Food and Drug Administration-approved for this indication.5,6 The recent VENAML phase I clinical trial demonstrated safety and activity of venetoclax combined with idarubicin and/or highdose cytarabine in children and adolescents/young adults (AYAs) with relapsed refractory AML (clinicaltrials gov. Identifier: NCT03194932).7 However, clinical experience with the azacitidine/venetoclax regimen in children has been limited to small case series.8,9 Herein, we report clinical characteristics and outcomes of 37 pediatric patients with relapsed/refractory acute leukemias treated at our institution with commercially-available azacitidine/venetoclax therapy. We analyzed data from patients aged 0-21 years with multiply-relapsed/refractory acute lymphoblastic leukemia (ALL), AML, or mixed-phenotype acute leukemia (MPAL) treated with azacitidine/venetoclax without or with the CD33 antibody-drug conjugate gemtuzumab ozogamicin (GO) at the Children’s Hospital of Philadelphia from January 1st, 2018 to March 31st, 2022. Institutional Review Board exemption was obtained for retrospective chart review. Clinical data were abstracted from electronic medical records on baseline patient demographics, leukemia-associated immunophenotyping and genetic characteristics, therapy administration, clinical response, and post-azacitidine/venetoclax outcomes with clinical follow-up through June 30, 2022. Azacitidine 100 mg/m2 daily was given intravenously on days 1-5 of each 28-day cycle. Venetoclax was given once daily as oral tablets (swallowed intact or crushed) with 3day ‘ramp-up’ dosing during cycle 1 to minimize tumor lysis syndrome (TLS) risk with body surface area-adjusted adult exposure-equivalent dosing (AED) of 200 mg (day 1),

400 mg (day 2), and 800 mg (days 3-28).10 Subsequent cycles utilized venetoclax at full 800 mg AED for 28 days without ramp-up. Patients receiving concurrent moderate or strong CYP3A inhibitors, including azole-class anti-fungal mediations, received a 50% dose reduction of venetoclax.10 Some patients with AML treated with azacitidine/venetoclax also received GO 3 mg/m2/dose during cycle 1 usually given on days 4, 5, or 8. Of the 37 pediatric patients with relapsed/refractory acute leukemias treated with azacitidine/venetoclax-based therapy during the study period, 27 (73%) had AML, seven (19%) had B-ALL, one (3%) had T-ALL, and two (5%) had MPAL (Table 1). The majority of patients (n=29, 78%) had multiply-relapsed/refractory leukemia. Eight children (22%) had primary chemotherapy-refractory leukemia defined as never achieving bone marrow measurable residual disease (MRD) negativity by flow cytometry at thresholds of <0.01% for ALL and <0.1% for AML. Sex was evenly balanced between female and male patients, and the median age at initial leukemia diagnosis was 8 years. Many children had high-risk cytomolecular genetic alterations, including 11 patients with KMT2A-rearranged leukemia (n=3 B-ALL, n=7 AML, n=1 MPAL), four with CBFA2T3::GLIS2 acute megakaryoblastic leukemia (AMKL), one with NUP98::KDM5A AMKL, one with ETV6::EP300 AML, and one with FLT3-ITD AML (Table 2). Of note, four patients with refractory AML treated with azacitidine/venetoclax were initially diagnosed with B-ALL (n=3 KMT2A-rearranged, n=1 TCF3::ZNF384) and had relapsed with myeloid lineage switch following CD19-directed (n=3) or CD22-directed (n=1) chimeric antigen receptor T-cell immunotherapy (CAR T). Prior to initiation of azacitidine/venetoclax therapy, the median marrow MRD of patients who completed at least one 28-day cycle azacitidine/venetoclax was 10.5% (range, 0.01–91.5%). Three patients with AML and one with MPAL were in MRDnegative/low remission prior to starting azacitidine/venetoclax, which was administered as bridging therapy prior to allogeneic hematopoietic stem cell transplantation (HSCT). Patients received up to six cycles of azacitidine/venetoclax during the data collection window (median 2 cycles, range 0-6) and were followed for a median of 4.9 months (range, 0.1-32.6) after initiation of therapy (Table 1; Figure 1A). GO was given with azacitidine/venetoclax in nine of the 27 patients with AML. Thirty-one patients (84%) com-

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LETTER TO THE EDITOR pleted at least one full cycle of azacitidine/venetoclax, while six children (16%) received fewer than 28 days of therapy. Early discontinuation of azacitidine/venetoclax was due either to severe TLS (n=2) or rapidly progressive disease detected in peripheral blood (n=4). Treatment-free intervals (1 week, n=2; 2 weeks, n=4; ≥3 weeks, n=2) were required for seven patients with AML and one patient with B-ALL between cycles 1 and 2 to facilitate resolution of severe neutropenia or thrombocytopenia. Three patients with cycle 2 delay had concurrent infection (n=1 viral, n=1 bacteria, n=1 unknown etiology), and two others had persistent leukemia. Two patients proceeded to cycle 2 with continued cytopenias. During azacitidine/venetoclax therapy, six patients had acute bacteremia treated successfully with antibiotics, and two patients had biopsy-proven fungal infections. Response data at first post-azacitidine/venetoclax marrow assessment were available for 29 patients. Median MRD level was 0.5% (range, 0.01-90%), and 14 patients (n=12 AML, n=1 B-ALL, n=1 MPAL) achieved a complete response (CR) with MRD-negative remission (38%, 14 of 37 treated patients). Of the 25 patients with AML who completed cycle 1, 12 (48%) were in MRD-negative remission, including several children with high pretreatment disease burden (Table 2; Figure 1B). Eight of these patients remained in MRD-negative remission with further cycles of azacitidine/venetoclax, while three treated with palliative intent experienced disease progression after cycle 2 (n=2) or cycle 4 (n=1). Children with AML who received azacitidine/venetoclax with GO (n=9) had a similar CR rate (4/9, 44%) to those treated without GO. One patient with B-ALL and one with MPAL also achieved MRD-negative CR (Figure 1C). Of note, six of the 14 azacitidine/venetoclax-induced CR occurred in patients with primary chemorefractory leukemia (n=5 AML, n=1 B-ALL). Three patients with residual leukemia (n=2 AML, n=1 MPAL) after cycle 1 continued azacitidine/venetoclax therapy, and the one with MPAL achieved MRD-negative CR after cycle 2. Importantly, responses occurred in patients with high-risk leukemia genetics. Eight of 11 patients with relapsed/refractory KMT2A-rearranged leukemias (n=2 B-ALL, n=5 AML, n=1 MPAL) completed cycle 1 of azacitidine/venetoclax, and two patients achieved MRD-negative CR (n=1 infant B-ALL, n=1 AML). Patients with ALL-to-AML lineage switch relapse (n=4) had particularly aggressive leukemias with two children unable to complete cycle 1 due to disease progression and the other two experiencing persistent MRD after cycle 1. Strikingly, three of four children with CBFA2T3::GLIS2 AML achieved MRD-negative remission. Successful remission induction with azacitidine/venetoclax +/- GO was surprisingly HSCT-enabling for 11 patients with AML and one patient with MPAL. HSCT complications in these children included graftversus-host-disease of skin (n=3, grade 2-3 acute), liver (n=1, grade 1 acute), or lung (n=1, grade 3 chronic). Post-trans-

plant death occurred in five of 12 patients (n=3 relapse, n=2 infection in leukemia remission) at a median of 177 days. Two patients with persistent relapsed/refractory B-ALL received subsequent autologous CD19-directed CAR T, and one patient with refractory KMT2A-rearranged AML after infant B-ALL lineage switch relapse received allogeneic

Table 1. Demographics, clinical characteristics, and outcomes of the study cohort treated with azacitidine and venetoclax (N=37 patients). Demographics, clinical characteristics and outcomes Age at diagnosis (years) Median Range

8 0.04-21

Sex, N (%) Female Male

18 (49) 19 (51)

Leukemia subtype of patients who did not complete cycle 1 azacitidine/venetoclax (N=6), N (%) AML ALL (N=3 B-ALL, N=1 T-ALL)

2 (33) 4 (67)

Leukemia subtype of patients receiving ≥1 cycle (N=31), N (%) AML ALL (N=4 B-ALL) MPAL

25 (81) 4 (13) 2 (6)

Leukemia status at time of azacitidine/venetoclax, N (%) Primary chemorefractory Multiply-relapsed/refractory

8 (22) 29 (78)

Completed cycles of azacitidine/venetoclax, N (%) <1 ≥1

6 (16) 31 (84)

Number of cycles Median Range

2 0-6

Patients with AML treated with azacitidine/venetoclax + GO, N (%)

9 (36)

Chemotherapy held or delayed

18 (49)

First available response assessment, N (%) End of cycle 1 End of cycle 2 Flow cytometric MRD-negative CR achieved *

25 (68) 4 (11) 14 (38)

Duration of follow-up (months) Median Range

4.9 0.1-32.6

Outcome at last follow-up, N (%) Alive Deceased

12 (32) 25 (68)

Time to death after last azacitidine/venetoclax (months) Median Range

1.83 0.1-15.37

*0.1% for AML, <0.01% for ALL/MPAL. ALL: acute lymphoblastic leukemia; AML: acute myeloid leukemia; MPAL: mixed phenotype acute leukemia, GO: gemtuzumab ozogamicin; MRD: measurable residual disease.

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LETTER TO THE EDITOR Table 2. Patient characteristics, leukemia-associated cytomolecular genetic alterations, and treatment outcomes following azacitidine and venetoclax therapy in study cohort (N=37). Therapy cycles

≥1

Leukemia type

Age at diagnosis years

Sex

Genetic alterations

MRD^

B-ALL

0.5

KMT2A::MLLT1 fusion

B-ALL

0.5

KMT2A::MLLT10 fusion

B-ALL

ETV6::RUNX1 fusion, TP53 mutation

B-ALL

High hyperdiploidy

AML

DNMT3A and GATA2 mutations

AMKL

CBFA2TA::GLIS2 fusion

AML

NRAS mutation

AML

FLT3 TKD and RUNX1 mutations

AML

ELANE mutation

AML

FLT3-ITD, RUNX1, and WT1 mutations

AMKL

NUP98::KDM5A fusion

AMKL

0.9

CBFA2T3::GLIS2 fusion

AML

SET::NUP214 fusion

AML

WT1 mutation

AML

NRAS mutation

AML

Germline TP53 mutation

AML*

TCF3::ZNF384 fusion

AMKL

CBFA2T3::GLIS2 fusion

AML

RUNX1 mutation

AML

ETV6::EP300 fusion

AML

0.7

FLT3 TKD mutation

AMKL

CBFA2T3::GLIS2 fusion

AML

0.7

KMT2A::MLLT10 fusion, TP53 mutation

AML

FLT3-TKD and TP53 mutations

AML

KMT2A::MLLT1 fusion

AML

Monosomy 7, NRAS mutation

AML*

0.8

KMT2A::MLLT1 fusion

AML

KMT2A::MLLT6 fusion

AML

KMT2A-SEPT9 fusion, WT1 mutation

MPAL

KMT2A::AFF1 fusion

MPAL

GATA2::ERG fusion

B-ALL

High hyperdiploidy

B-ALL

KMT2A::MLLT3 fusion

B-ALL

Low hypodiploidy

T-ALL

NOTCH, PTPN11, and JAK3 mutations

AML*

0.1

KMT2A::AFF1 fusion, NRAS and KRAS mutations

AML*

KMT2A::AFF1 fusion

*Initial diagnosis of ALL with lineage switch to AML prior to receipt of azacitidine/venetoclax. ^Measurable residual disease (MRD) at first response assessment defined as <0.1% for AML and <0.01% for ALL/MPAL by flow cytometry analysis. ALL: acute lymphoblastic leukemia; AML: acute myeloid leukemia; MPAL: mixed phenotype acute leukemia; AMKL: acute megakaryoblastic leukemia; F: female; M: male; NA: not applicable or not assessed (due to disease progression). Haematologica | 108 November 2023

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LETTER TO THE EDITOR CD123-directed CAR T. At last follow-up, 12 patients (32%) were alive, all of whom received at least one cycle of azacitidine/venetoclax (Figure 1A). To our knowledge, we report the largest retrospective cohort of children and adolescents/young adults with multiply-relapsed/refractory acute leukemias treated with azacitidine/venetoclax to date. Within a heavily-pretreated and/or highly chemorefractory cohort of patients, we describe an encouraging 38% MRD-negative CR rate. Our observations are similar to those of Winters and colleagues, who described MRD-negative CR in three of six (50%) pediatric patients with relapsed/refractory AML treated with azacitidine/venetoclax and a lack of response in one

child with ALL-to-AML lineage switch relapse.8 Our data are also concordant with several retrospective case series of adults with relapsed/refractory AML in whom activity has been reported across numerous leukemia genetic backgrounds, including those with poor-risk cytogenetics.11-13 We observed remarkable rates of MRD-negative CR with azacitidine/venetoclax therapy in our cohort of children with multiply-relapsed/refractory leukemias, including many with high-risk cytomolecular genetic features such as CBFA2T3::GLIS2 AMKL. Importantly, our pediatric patients largely received azacitidine/venetoclax therapy in the outpatient oncology clinic with inpatient admission for tumor lysis or myelosuppression monitoring

Figure 1. Clinical course and responses of pediatric patients with multiply-relapsed/refractory acute leukemia treated with azacitidine and venetoclax. (A) Swimmer plot depicting the clinical course of each patient who received ≥1 complete cycle of azacitidine and venetoclax (aza/ven; N=31) is displayed. Each colored bar (grouped by leukemia subtype) represents the time after initiation of aza/ven for a single patient. Interior horizontal thick black bars represent aza/ven treatment duration. Squares represent patients with measurable residual disease (MRD)-negative remission at first evaluation (end of cycle 1 or cycle 2). Triangles represent treatment with chimeric antigen receptor T-cell immunotherapy (CAR T), including autologous CD19-directed CAR T (B-cell acute lymphoblastic leukemia [B-ALL]) or allogeneic CD123-directed CAR T (acute myeloid leukemia [AML]). Additional treatment modalities and outcomes are denoted by the symbols in the legend. (B) Clinical response assessment of pediatric patients with relapsed/refractory AML receiving ≥1 cycle of aza/ven. Prior to the initiation of aza/ven, patients had varying AML disease burden in the bone marrow as quantified by flow cytometric analysis of measurable residual disease (MRD). Lines depict response of individual patients from pretreatment (white circles) to first evaluation (blue circles, end of cycle 1 or 2). Many patients achieved MRD-negative remission at a threshold of <0.1% (dotted grey line) at first evaluation, including several patients with high pre-aza/ven treatment disease burden. (C) Clinical response assessment of pediatric patients with relapsed/refractory B-ALL (yellow squares) or mixed phenotype acute leukemia (MPAL) (green triangles) receiving ≥1 cycle of aza/ven as in (B). Lines depict flow cytometric analysis of bone marrow MRD for individual patients from pre-treatment (white symbols) to first evaluation (yellow or green symbols, end of cycle 1 or 2). One patient with B-ALL and 1 patient with MPAL achieved MRD-negative remission at a threshold of <0.01% (dotted grey line). HSCT: hematopoietic stem cell transplantation. Haematologica | 108 November 2023

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LETTER TO THE EDITOR not routinely required, comparable with experience in adult AML.14 As many children had spent significant time inpatient for receipt of prior frontline or salvage therapies, the ability to receive outpatient chemotherapy anecdotally provided significant quality of life benefit for patients and their families, an area that merits future formal study. Finally, our data suggest that clinical responses of children with relapsed/refractory leukemia, particularly AML, to the azacitidine/venetoclax regimen are rapid and binary with achieved MRD-negative CR typically occurring after cycle 1 therapy or not at all. Although our study is limited by its retrospective nature, a relatively small number of patients treated at a single institution, and short duration of follow-up, it highlights the robust potential of azacitidine/venetoclax as an effective salvage regimen for pediatric patients with multiply-relapsed and highly-chemorefractory acute leukemias. In our cohort, azacitidine/venetoclax was often initially administered with palliative intent, but was subsequently HSCT-enabling in many patients given their excellent response rates. Given the surprisingly high efficacy in a very chemorefractory population, favorable toxicity rate, and appreciable improvement in quality of life for patients, formal clinical trial investigation of azacitidine/venetoclax-based therapies in children with relapsed or refractory acute leukemias, ideally at an earlier stage of relapse, is warranted.

Early view: April 6, 2023. ©2023 Ferrata Storti Foundation Published under a CC BY-NC license Disclosures SKT receives/d research funding for unrelated studies from Beam Therapeutics, Incyte Corporation, and Kura Oncology, has consulted for bluebird bio, has received travel support from Amgen, and serves on scientific advisory boards of Aleta Biotherapeutics, Kura Oncology, and Syndax Pharmaceuticals. The remaining authors have no conflicts of interest to disclose. Contributions LMN contributed to study design, performed data collection, analyzed and interpreted data, and wrote the manuscript. PC performed data collection and analyzed and interpreted data. CD analyzed and interpreted data. SKT conceived and directed the study, interpreted data, and wrote and edited the manuscript. All authors reviewed, edited, and approved the final manuscript. Acknowledgments We are grateful to the physicians, advanced practice providers, and nurses of the Hematologic Malignancies Program at the Children’s Hospital of Philadelphia (CHOP) for their excellent clinical care of the patients whose data are described in this report. We also kindly acknowledge Drs Bianca Goemans and Uri Ilan at the Princess Máxima Centrum in Utrecht, the Netherlands for helpful discussion

Authors

and collaboration. Funding

Lisa M. Niswander, Perry Chung, Caroline Diorio, and Sarah K

These studies were supported by the National Institutes of Health

Tasian1,2,3

(NIH)/ National Institute of Child Health Development K12HD043245

1,2

(to LMN), NIH/National Cancer Institute 1U01CA232486 and Division of Oncology and Center for Childhood Cancer Research,

1U01CA243072 (to SKT), Precious Jules Foundation (to LMN),

Children’s Hospital of Philadelphia; Department of Pediatrics,

Canadian Institutes of Health Research (to CD), American Society of

University of Pennsylvania Perelman School of Medicine and

Clinical Oncology/Conquer Cancer Foundation (to CD), Department

Abramson Cancer Center, University of Pennsylvania Perelman

of Defense Translational Team Science Award CA180683P1 (to SKT),

School of Medicine, Philadelphia, PA, USA

and the V Foundation for Cancer Research (to SKT). LMN is a St.

Baldricks Foundation Fellow with support from Super Soph’s Correspondence:

Pediatric Cancer Research Fund and the Invictus Fund. SKT is a

S. K. TASIAN - tasians@chop.edu

Leukemia and Lymphoma Society Scholar and holds the Joshua Kahan Endowed Chair in Pediatric Leukemia Research at CHOP.

https://doi.org/10.3324/haematol.2022.282637 Data-sharing statement Received: December 23, 2022.

De-identified data without identifying patient health information are

Accepted: March 28, 2023.

available upon reasonable request from the corresponding author.

References 1. Hunger SP, Raetz EA. How I treat relapsed acute lymphoblastic leukemia in the pediatric population. Blood. 2020;136(16):1803-1812. 2. Zarnegar-Lumley S, Caldwell KJ, Rubnitz JE. Relapsed acute

myeloid leukemia in children and adolescents: current treatment options and future strategies. Leukemia. 2022;36(8):1951-1960. 3. Creutzig U, Zimmermann M, Reinhardt D, Dworzak M, Stary J,

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LETTER TO THE EDITOR Lehrnbecher T. Early deaths and treatment-related mortality in children undergoing therapy for acute myeloid leukemia: analysis of the multicenter clinical trials AML-BFM 93 and AMLBFM 98. J Clin Oncol. 2004;22(21):4384-4393. 4. Nagarajan R, Gerbing R, Alonzo T, et al. Quality of life in pediatric acute myeloid leukemia: report from the Children's Oncology Group. Cancer Med. 2019;8(9):4454-4464. 5. DiNardo CD, Jonas BA, Pullarkat V, et al. Azacitidine and venetoclax in previously untreated acute myeloid leukemia. N Engl J Med. 2020;383(7):617-629. 6. Cherry EM, Abbott D, Amaya M, et al. Venetoclax and azacitidine compared with induction chemotherapy for newly diagnosed patients with acute myeloid leukemia. Blood Adv. 2021;5(24):5565-5573. 7. Karol SE, Alexander TB, Budhraja A, et al. Venetoclax in combination with cytarabine with or without idarubicin in children with relapsed or refractory acute myeloid leukaemia: a phase 1, dose-escalation study. Lancet Oncol. 2020;21(4):551-560. 8. Winters AC, Maloney KW, Treece AL, Gore L, Franklin AK. Singlecenter pediatric experience with venetoclax and azacitidine as treatment for myelodysplastic syndrome and acute myeloid leukemia. Pediatr Blood Cancer. 2020;67(10):e28398.

9. Mishra AK, Mullanfiroze K, Chiesa R, Vora A. Azacitidine and venetoclax for post-transplant relapse in a case of CBFA2T3/GLIS2 childhood acute myeloid leukaemia. Pediatr Blood Cancer. 2021;68(11):e29221. 10. Place AE, Goldsmith K, Bourquin JP, et al. Accelerating drug development in pediatric cancer: a novel phase I study design of venetoclax in relapsed/refractory malignancies. Future Oncol. 2018;14(21):2115-2129. 11. Aldoss I, Yang D, Aribi A, et al. Efficacy of the combination of venetoclax and hypomethylating agents in relapsed/refractory acute myeloid leukemia. Haematologica. 2018;103(9):e404-e407. 12. Pollyea DA, Pratz KW, Wei AH, et al. Outcomes in patients with poor-risk cytogenetics with or without TP53 mutations treated with venetoclax and azacitidine. Clin Cancer Res. 2022;28(24):5272-5279. 13. Stahl M, Menghrajani K, Derkach A, et al. Clinical and molecular predictors of response and survival following venetoclax therapy in relapsed/refractory AML. Blood Adv. 2021;5(5):1552-1564. 14. Pelcovits A, Moore J, Bakow B, Niroula R, Egan P, Reagan JL. Tumor lysis syndrome risk in outpatient versus inpatient administration of venetoclax and hypomethlators for acute myeloid leukemia. Support Care Cancer. 2021;29(9):5323-5327.

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LETTER TO THE EDITOR

Examining the impact of age on the prognostic value of ELN-2017 and ELN-2022 acute myeloid leukemia risk stratifications: a report from the SWOG Cancer Research Network Recent revisions to the European LeukemiaNet (ELN) recommendations have redefined how acute myeloid leukemia (AML) is classified, monitored, treated, and risk-stratified.1,2 Some of the most significant risk stratification changes involve reclassification for some previously utilized mutations and the inclusion of additional mutations. The ELN-2022 guidelines have removed the FLT3-internal tandem duplication (ITD) ratio as a major risk classifier while promoting a single CEPBA mutation within the Zip domain as sufficient to convey a favorable risk. In the absence of favorable risk genomic alterations, the new ELN-2022 guidelines also recommend that mutations associated with myelodysplasia (i.e., myelodysplastic syndrome [MDS], or MDS-related) be considered adverse risk factors, even in patients without a history of MDS. The median age of AML patients at diagnosis is 68 years, highlighting that most AML patients are older,3 and these older patients frequently harbor MDS-related mutations despite not having documentation for antecedent MDS. It remains uncertain whether the “de novo” older patients with MDS-related mutations had an undiagnosed preceding MDS or not, but the MDS-related mutations in older AML patients are associated with an adverse risk.2 We and others have shown that age remains a major adverse risk factor, even after accounting for other age-related factors: type of therapy, performance status, cytogenetics, specific favorable-risk mutations, and even ELN-2017.4,5 Moreover, models incorporating age with ELN-2017 risk performed better than models with ELN-2017 risk alone.6 With the inclusion of MDS-related mutations into ELN-2022 guidelines, we hypothesized that ELN-2022 would outperform ELN-2017 - especially in older adults with AML, who tend to have a higher frequency of many of these MDS-related mutations. In order to examine this question, we compared the prognostic performance of the two versions of ELN guidelines. Since neither version incorporates age into its risk stratification, we evaluated whether a model with ELN2022 risk and age would improve the prognostic value of ELN-2022 as it does for ELN-2017. These models were evaluated in a well-defined cohort of patients treated with intensive chemotherapy as part of the SWOG Cancer Research Network clinical trials. Thus, we examined the molecular and clinical data from 351 patients previously enrolled in protocols SWOG-9031,

SWOG-9333, S0106, and S0112 and treated as previously described.6-10 Details of the patients and utilized specimens have been published and can be found in Online Supplementary Table S1.6-10 All participants provided written informed consent to participate in correlative research in compliance with the Declaration of Helsinki. All studies were conducted with the approval of Fred Hutch Cancer Center’s Institutional Review Board. ELN risk for patients was assigned based on previously described guidelines.1,2 Univariate and multivariable analyses of complete response (CR, logistic regression), overall survival (OS, Cox regression), and relapse-free survival (RFS, Cox regression) were used to evaluate the prognostic value of the ELN-2017 and ELN-2022 risk stratification. OS, CR, and RFS were defined as previously described.6 Multivariable analyses included age (modeled as a quantitative covariate) in addition to ELN risk. Note that there was no model or covariate selection performed in the analyses reported here. The objective was to describe how model performance changed by adding the covariate of age based on prior work. Therefore, we did not perform cross-validation. The area under the receiver operating characteristic curve (AUC) and C-statistics were calculated to assess model performance. Molecular mutation and cytogenetic profiles are specified in the Online Supplementary Table S2. Univariate analyses adjusting for ELN-2017 or ELN-2022 risk yielded similar statistical results for all outcomes: AUC of 0.7 for CR and C-statistics of 0.63 and 0.61 for OS and RFS, respectively (Table 1; Figures 1A, B; Online Supplementary Figure S1A, B). Specifically, 9% of all patients were reclassified based on their risk categorization when the ELN2022 guidelines were used instead of the ELN-2017 guidelines (Online Supplementary Table S1). Restricting the analyses to age of patients >55 years old, the models incorporating ELN-2022 or ELN-2017 risk had similar prognostic value as measured by C-statistics for OS (ELN-2022=0.60 vs. ELN-2017=0.58; Figure 1C, D) and RFS (ELN-2022=0.61 vs. ELN-2017=0.60; Online Supplementary Figure 1C, D). Restricting age of patients to ≤55 years old showed a similar prognostic value as measured by C-statistics for OS (ELN-2022=0.67 and ELN-2017=0.66; Figure 1E, F) and RFS (ELN-2022=0.60 and ELN-2017=0.60; Online Supplementary Figure 1E, F). As we previously described with ELN-2017, the ELN-2022 risk had greater prognostic

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LETTER TO THE EDITOR

Figure 1. Similar survival probability is seen in older patients analyzed using ELN-2017 and ELN-2022. Overall survival probability was analyzed for all patients classified into favorable, intermediate, adverse, or unknown risk groups based upon (A) ELN-2017 and (B) ELN2022 guidelines. Overall survival was analyzed for patients >55 years old classified into favorable, intermediate, adverse, or unknown risk groups based on (C) ELN-2017 and (D) ELN-2022 guidelines. Overall survival was analyzed for patients ≤ 55 years old classified into favorable, intermediate, adverse, or unknown risk groups based on (E) ELN-2017 and (F) ELN-2022 guidelines (N=351 total patients). Kaplan-Meier curves are shown for each group of patients; C-statistics are based on Cox proportional hazards models. Haematologica | 108 November 2023

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LETTER TO THE EDITOR Table 1. Age minimally changes the prognostic value of ELN-2017 and ELN-2022 guidelines. Comparison of age as a prognostic factor using ELN-2017 and ELN-2022 guidelines (N=351 total patients; N=272 patients with unknowns omitted). AUC or C-statistic

AUC or C-statistic (excluding unknowns)

Complete response (AUC) ELN-2017 ELN-2022 Age + ELN-2017 Age + ELN-2022

0.7 0.7 0.74 0.73

0.72 0.72 0.75 0.74

Overall survival (C-Statistic) ELN-2017 ELN-2022 Age + ELN-2017 Age + ELN-2022

0.63 0.63 0.71 0.72

0.65 0.65 0.71 0.72

Relapse-free survival (C-Statistic) ELN-2017 ELN-2022 Age + ELN-2017 Age + ELN-2022

0.61 0.61 0.67 0.67

0.61 0.62 0.67 0.68

Model

AUC: area under the curve; ELN: European LeukemiaNet.

value with respect to OS in younger patients (≤55) than in their older counterparts (Figure 1). We then examined the impact of incorporating age into the ELN-2022 risk model. Overall, incorporating age improved the prognostic value for CR, OS, and RFS – whether the model was based on ELN-2022 or ELN-2017. Moreover, the improvement was similar for ELN-2022 and ELN-2017 (Table 1), with the greatest improvement being for OS (Δ=0.08-0.09), followed by RFS (Δ=0.06), and then CR (Δ=0.03-0.04). Omitting patients with unknown risk group status from our analyses resulted in similar increases in the model performance when age was included for OS (Δ=0.06-0.07), RFS (Δ=0.06), and CR (Δ=0.03-0.02) (Table 1). We also performed similar analyses excluding those patients with FLT3-ITD mutations, given that examined patients did not receive an FLT3 inhibitor (Online Supplementary Table S3). When FLT3-ITD patients were removed from our analyses, we detected a slightly worse prognostic value of ELN-2022 compared to ELN-2017 for CR (Δ=0.02) and OS (Δ=0.01) when age was incorporated into the model, while RFS was unchanged (Online Supplementary Table S3). Taken together, these findings show a similar prognostic value of risk stratification for ELN-2022 and ELN-2017, which is consistent with only 9% of the patients in our cohort being reclassified for their risk stratification category under the ELN-2022 guidelines (Online Supplementary Table S1). Incorporating age into the ELN-2022 and ELN2017 models resulted in a similar magnitude of improved performance over the univariate models. Although the analyses included over 350 patients, we recognize that additional studies with even more patients will be required to examine the performance of the ELN-2022. However, it is

unlikely that the changes to ELN-2022 will dramatically improve risk stratification compared to ELN-2017. There are multiple reasons that likely contribute to our current lack of highly accurate prognostic and predictive biomarkers – with the lack of highly efficacious targeted therapy being just one. With the advent of more targeted therapies, investigators will hopefully be able to better refine and adapt risk models to incorporate more therapy-specific predictors, which will likely improve risk stratification and care.

Authors Christina M. Termini,1,2 Anna Moseley,3 Megan Othus,3 Frederick R. Appelbaum,4,5 Thomas R. Chauncey,4,6 Harry P. Erba,7 Min Fang,1,2 Stanley C. Lee,1,2 Jasmine Naru,1 Era L. Pogosova-Agadjanyan,1 Jerald P. Radich,1,4 Cheryl L. Willman,8 Feinan Wu,9 Soheil Meshinchi1,10 and Derek L. Stirewalt1,4 Translational Science and Therapeutics Division, Fred Hutch Cancer

Center, Seattle, WA; 2Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA; 3SWOG Statistical Center, Fred Hutch Cancer Center, Seattle, WA; 4Departments of Oncology and Hematology, University of Washington, Seattle, WA; Clinical Research Division, Fred Hutch Cancer Center, Seattle, WA;

VA Puget Sound Health Care System, Seattle, WA; 7Duke Cancer

Institute, Durham, NC; 8Department of Laboratory Medicine and Pathology, Mayo Clinic Comprehensive Cancer Center, Rochester, MN; 9Genomics and Bioinformatics Shared Resource, Fred Hutchinson Cancer Research Center, Seattle, WA and 10Department of Pediatrics, University of Washington, Seattle, WA, USA

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LETTER TO THE EDITOR Correspondence:

Acknowledgments

C.M. TERMINI - ctermini@fredhutch.org

The authors wish to gratefully acknowledge John E. Godwin and the late Dr. Stephen H. Petersdorf for their oversight of S9031 and S0106 trials, respectively. In addition, the authors would like to acknowledge

https://doi.org/10.3324/haematol.2023.282733

Patricia Arlauskas for her help coordinating the activities between the Received: January 12, 2023.

Stirewalt Lab and SWOG. The authors would also like to acknowledge

Accepted: March 24, 2023.

that much of the preliminary optimization for these studies utilized

Early view: April 6, 2023.

specimens obtained from the Fred Hutch Cancer Center/University of Washington Hematopoietic Diseases Repository.

Funding This work was funded by the following NIH grant awards: R01CA190661,

Disclosures

R01CA160872, U10CA180888, U10CA180819, U24CA196175, and

No conflicts of interest to disclose.

P30CA015704. In addition, parts of the studies were also funded through SWOG Hope Foundation.

Contributions CMT, AM, MO, and DLS developed the concept. CMT, AM, MO, FRA, TRC,

Data-sharing statement

HPE, JEG, MF, JN, ELPA, CLW, FW, SM and DLS provided resources.

The datasets generated and/or analyzed during the current study are

CMT, AM, MO, FRA, TRC, MF, JEG, JN, ELPA, CLW, FW and DLS cured

available in the dbGaP repository, dbGaP, under accession number

data. CMT, AM, MO, ELPA, FW and DLS performed formal analysis using

phs002805.v1.p1. Investigators can apply to access sequencing data

software. CMT, AM, MO, ELPA, FW and DLS performed formal analysis.

through standard dbGaP request procedures as described by NIH and

CMT, AM, MO, HPE, MF and DLS supervised the research. MO, SM and

found at dbgap_request_process.pdf (nih.gov). Additional data generated

DLS acquired funding. CMT, MO, ELPA, FW and DLS developed the

or analyzed during this study are included in the Online Supplementary

methodology. CMT, AM, MO, FRA, TRC, HPE, JEG, MF, SCL, JN, ELPA,

Appendix. Data and code to reproduce the analyses presented here are

JPR, CLW, FW, SM and DLS wrote, reviewed and edited the

available upon request from SWOG following SWOG’s data sharing policy

manuscript.

and process: https://www.swog.org/sites/default/files/docs/2019-12/Policy43_0.pdf

References 1. Döhner H, Estey E, Grimwade D, et al. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood. 2017;129(4):424-447. 2. Dohner H, Wei AH, Appelbaum FR, et al. Diagnosis and management of AML in adults: 2022 recommendations from an international expert panel on behalf of the ELN. Blood. 2022;140(12):1345-1377. 3. National Cancer Institute. Acute Myeloid Leukemia — Cancer Stat Facts [Internet]. Bethesda (MD) SEER; 2018 [updated 2020; accessed 2022 Sept 22] https://seer.cancer.gov/statfacts/html/amyl.html. 4. Appelbaum FR, Gundacker H, Head DR, et al. Age and acute myeloid leukemia. Blood. 2006;107(9):3481-3485. 5. Ostronoff F, Othus M, Lazenby M, et al. Prognostic significance of NPM1 mutations in the absence of FLT3-internal tandem duplication in older patients with acute myeloid leukemia: a SWOG and UK National Cancer Research Institute/Medical Research Council report. J Clin Oncol. 2015;33(10):1157-1164. 6. Pogosova-Agadjanyan EL, Moseley A, Othus M, et al. AML risk

stratification models utilizing ELN-2017 guidelines and additional prognostic factors: a SWOG report. Biomark Res. 2020;8:29. 7. Anderson JE, Kopecky KJ, Willman CL, et al. Outcome after induction chemotherapy for older patients with acute myeloid leukemia is not improved with mitoxantrone and etoposide compared to cytarabine and daunorubicin: a Southwest Oncology Group study. Blood. 2002;100(12):3869-3876. 8. Godwin JE, Kopecky KJ, Head DR, et al. A double-blind placebo-controlled trial of granulocyte colony-stimulating factor in elderly patients with previously untreated acute myeloid leukemia: a Southwest Oncology Group study (9031). Blood. 1998;91(10):3607-3615. 9. Hiddemann W, Kern W, Schoch C, et al. Management of acute myeloid leukemia in elderly patients. J Clin Oncol. 1999;17(11):3569-3576. 10. Petersdorf SH, Kopecky KJ, Slovak M, et al. A phase 3 study of gemtuzumab ozogamicin during induction and postconsolidation therapy in younger patients with acute myeloid leukemia. Blood. 2013;121(24):4854-4860.

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Risk of relapse after SARS-CoV-2 vaccine in the Milan cohort of thrombotic thrombocytopenic purpura patients Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy characterized by reduced levels of ADAMTS13 (<10%) secondary to the presence of antiADAMTS13 autoantibodies in the acquired immune form (iTTP) or to ADAMTS13 gene mutations in the congenital form (cTTP). Acute episodes of TTP may be triggered by pregnancy, drugs such as oral contraceptives, and infections. TTP has been occasionally described also after vaccination. In the pre-COVID19 era, six cases of TTP were reported after influenza, H1N1, pneumococcal and rabies vaccines, within 2 weeks. All cases but one were associated with vaccines against viral agents, and most of them (3) were associated with influenza vaccines, likely due to their wider availability.1 With the COVID-19 pandemic and the subsequent mass immunization program, safety concerns emerged about the possibility of TTP relapse after anti-SARS-CoV-2 vaccination. Since the availability of anti-SARS-CoV-2 vaccines, a total of 39 TTP cases (both first events or relapses) have been described, reporting a possible association between TTP onset and mRNA-based (Pfizer-BioNTech n=27, Moderna n=4), adenovirus vectors-based (AstraZeneca n=5, Janssen-Johnson & Johnson n=1) or inactivated whole-virus-based (Sinopharm n=1, CoronaVac n=1) vaccines. In this manuscript we report our single-center prospective cohort study aimed to evaluate the relapse rates in patients affected by TTP undergoing anti-SARS-CoV-2 vaccination. All consecutive adult TTP patients undergoing anti-SARSCoV-2 vaccination from March to May 2021 were enrolled and observed until 1 month after the second dose. Multiple blood samples were collected: 1 week before the first dose of vaccination (T0), at least 1 week after the first and before the second dose (T1), and at least 1 week after and within 1 month from the second dose (T2). Patients were observed from T0 to T2 for clinical or ADAMTS13 relapse (decrease in activity to <20%). Venous blood samples were tested for whole blood count, ADAMTS13 activity,2 anti-ADAMTS13 antibodies, prothrombotic markers (FVIII:C, VWF:Ag and Ddimer plasma levels), anticoagulant markers (protein C activity), anti-PF4 and anti-S antibodies. Data on demographics, type of vaccine and immunosuppression treatment were collected (Table 1). Categorical variables were expressed as counts and percentages and continuous variables as mean and standard deviation or median and interquartile range (IQR). Continuous variables at the different time points were compared by repeated measures ANOVA for normally distributed and KruskalWallis test for non-normally distributed variables. A total of 49 TTP patients were enrolled, 37 females and

12 males, in line with the reported 3:1 female prevalence of the disease with a median age of 50 years (IQR, 40-59 years). All patients were vaccinated with the Pfizer-BioNTech mRNA BNT162b2-Comirnaty vaccine. Forty-eight patients were affected by iTTP, while one had cTTP. The latter did not develop any clinical relapse and did not show any variation of the ADAMTS13 levels at the different time points. At baseline all iTTP patients were in clinical remission and the median plasma levels of ADAMTS13 were 62% (IQR, 34-87%). At T0 ADAMTS13 activity <20% was observed in five (10%) patients, two of which with an activity <10%, while nine (19%) patients had activity between 20% and 45%. Among patients with ADAMTS13 plasma levels below the lower limit of the normal range, only one had borderline anti-ADAMTS13 antibodies (15 IU/mL; normal range <12 IU/mL, borderline 12-15 IU/mL). Within 1 month from the second vaccine dose, no patients had a clinical TTP relapse and only one had an ADAMTS13 relapse with plasma levels <10%. Mean levels of ADAMTS13 activity were stable among the three time points (Figure 1). In only two patients a significant decrease of ADAMTS13 levels occurred after the first dose (from 28% to <3% and from 101% to 82%), and both remained stable after the sec-

Table 1. Demographic and clinical characteristics of acute thrombotic thrombocytopenic purpura patients. Characteristics

Values

Age in years, median (IQR)

50 (40-59)

Sex, N (%) Male Female

12 (25) 37 (77)

Number of TTP episodes, median (min-max)

1 (1-7)

Time from last TTP episode to first vaccine dose in years, median (IQR),

5 (3-9)

Immunosuppression therapy in the year before vaccination, N (%)* Rituximab Steroids Azathioprine Cyclosporine Hydroxycloroquine

13 (27) 8 (17) 7 (15) 1 (2) 3 (6)

Time from last rituximab to 1st vaccine dose in months, median (IQR)

6 (4-12)

Ongoing immunosuppression at 1st dose, N (%)

11 (23)

*Some patients were on concomitant treatment with more than one immunosuppressive agent. IQR: interquartile range; TTP: thrombotic thrombocytopenic purpura; N: number.

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LETTER TO THE EDITOR ond dose, with negative anti-ADAMTS13 antibodies. Notably, even though with undetectable ADAMTS13 plasma levels, the first patient received also the second vaccine dose, that didn’t elicit a clinical relapse. Due to a stable undetectable ADAMTS13 he was treated with 375 mg/m2 rituximab once weekly for 4 weeks showing a rapid ADAMTS13 response. Rituximab was started 1 month after the second dose to maximize the serological response to vaccination. One patient had positive basal anti-ADAMTS13 antibodies with a titer remaining stable after the two vaccine doses, while in another patient anti-ADAMTS13 antibodies became detectable after the first dose, with no corresponding drop in ADAMTS13 levels and a stable titer after the second dose. Among patients with iTTP, 21 were treated with an immunosuppressive drug over the last year before enrollment. Of those, most patients were treated with only one immunosuppressive drug, while four patients with two, and three patients with three different drugs. Thirteen patients had been treated with four to six infusions of rituximab with the standard schedule of 375 mg/mq weekly, eight patients with steroids (prednisone or metilprednisolone) at variable doses (from 1 mg/kg to low maintenance doses: 5 mg once-daily), three with hydroxychloroquine at the standard dose of 200 mg once-daily, six with azathioprine at a dose of 1.5-2.0 mg/kg once-daily, two with cyclosporine at a dose of 1.5-2 mg/kg/day. Nine of 13 patients had received the last dose of rituximab within 9 months of the first vaccine dose.3 Eleven patients were on immunosuppressive treatments at the time of the first vaccine dose. None of the patients at the time of the first vaccine dose were on more than 10 mg of prednisone equivalent dose, as previously recommended.4,5 Anti-PF4 antibodies were negative in all patients except one at T2. This patient was not exposed to heparin and did not show any other sign or symptom suggestive of vaccine-induced thrombotic thrombocytopenia (VITT). Indeed, no confirmed cases of VITT associated with mRNA vaccines have been reported in the literature.6 Six patients showed a positive titer of anti-spike antibodies before the first dose of vaccine. No systematically collected data on previous exposure to SARS-CoV-2 are available. After the first vaccine dose, 33 patients became positive, and nine more patients became positive after the second dose. A total of five (10%) patients did not show a serological response to the two doses of vaccine. Of those, two patients who had received the last dose of rituximab within 9 months from the first vaccine dose (2 and 4 months) and one patient was on continuous treatment with cyclosporine. For one patient who resulted negative after the first dose no serum sample was available after the second dose to evaluate the antibody response. A statistical analysis conducted with Student’s t-test showed no significant difference between the pa-

Figure 1. Plasma levels of ADAMTS13 in thrombotic thrombocytopenic purpura patients before (T0), 2 weeks after the first dose (T1) and 2 weeks after the second dose (T2) of anti-SARSCoV-2 vaccination. Horizontal bars represent mean and standard deviation.

tients that received immunosuppressive treatment in the year before the first vaccine dose and those off treatment in the levels of anti-spike antibodies titers. Concerning the procoagulant parameters FVIII:C, VWF:Ag and D-dimer, no statistically significant differences were found in plasma levels at the three time points. No difference was found for the natural anticoagulant protein C plasma levels, as well. No significant changes in white blood cells or platelet count at the three time points were observed (Table 2). Due to the inflammatory response induced by vaccines, a possible role of vaccines in the induction of autoimmune diseases has been proposed, via different mechanisms such as molecular mimicry and polyclonal immune response.7 Therefore vaccines may represent a trigger for TTP as well, being an autoimmune disease, even though only sporadic cases of acute TTP after vaccination have been reported in the literature so far. In our cohort no patient developed a clinical relapse and only one of 48 developed an ADAMTS13 relapse in the observation period for a rate of 1.36% per month, compared with the 2.6% clinical relapse rate reported in the literature8 and a 0.52 incidence rate observed/expected. Our results are in line with the results of two multi-center studies that showed an incidence rate of TTP relapse or new onset within 4 weeks after vaccination lower than expected in the vaccinated population.9,10 Conversely, we observed fewer relapses than another Italian monocentric study (overall 11 clinical/ADAMTS13 relapse 2% vs. 13% of cases). Of note, although the proportion of patients with baseline ADAMTS13 activity below normal was similar (29% vs. 31%), the proportion of patients with a baseline ADAMTS13 ac-

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LETTER TO THE EDITOR Table 2. Laboratory characteristics of acute thrombotic thrombocytopenic purpura patients. Characteristics

61 (33-81)

60 (37-80)

68 (43-81)

Anti-ADAMTS13 antibodies, N (%)

1 (2)

2 (4)

Anti-PF4 antibodies, N (%)

1 (2)

Anti-Spike antibodies, N (%)

6 (13)

33 (69)

35 (73)

ADAMTS13 %, median (IQR)

ported cases of TTP relapse after vaccination, it is of pivotal importance to carefully evaluate the platelet count and ADAMTS13 levels before and after the vaccination, with more strictly monitoring for patients with lower levels at baseline.

Authors Marco Capecchi,1,2 Pasqualina De Leo,1 Maria Abbattista,1 Ilaria Mancini,3 Pasquale Agosti,3 Marina Biganzoli,1 Chiara Suffritti,1

Platelet count x109/L, median (IQR)

240 259 260 (216-293) (226-310) (225-304)

FVIII:C %, median (IQR)

85 (66-105)

82 (67-100)

84 (73-104)

VWF:Ag %, median (IQR)

110 (90-138)

113 (85-132)

117 (91-138)

D-dimer FEU, median (IQR)

290 246 249 (157-387) (182-401) (169-376)

Barbara Ferrari,1 Anna Lecchi,1 Silvia La Marca,1 Lidia Padovan,1 Erica Scalambrino,1 Marigrazia Clerici,1 Armando Tripodi,1 Andrea Artoni,1 Roberta Gualtierotti1,3 and Flora Peyvandi1,3 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Angelo

Bianchi Bonomi Hemophilia and Thrombosis Center, Milan, Italy; Clinica Moncucco, Division of Hematology, Lugano, Switzerland and Università degli Studi di Milano, Department of Pathophysiology

Protein C %, median (IQR)

96 (84-109)

92 (82-108)

95 (82-115)

and Transplantation and Fondazione Luigi Villa, Milan, Italy

No statistically significant differences were observed between the 3 time points for ADAMTS13 and hemostatic parameters median levels. TO: before the first vaccination; T1: 2 weeks after the first dose of vaccination; T2: 2 weeks after the second dose; IQR: interquartile range; N: number; FVIII:C: factor VIII coagulant activity; VWF:Ag: von Willebrand factor antigen; FEU: fibrinogen equivalent units.

Correspondence: F. PEYVANDI - flora.peyvandi@unimi.it https://doi.org/10.3324/haematol.2022.282478 Received: November 30, 2022.

tivity of <20%, who are supposed to be at higher risk of TTP relapse after a trigger, was significantly lower in our study (10% vs. 22%), possibly explaining the observed differences. Overall, the analysis of the coagulation activation showed no increase of the procoagulant factors such as FVIII:C and VWF:Ag, suggesting that anti-SARS-CoV-2 vaccines do not induce an inflammatory response strong enough to determine a hypercoagulable state, in contrast to what is induced by the virus itself.12,13 In conclusion, the results of our study prospectively evaluating the effect of anti-SARS-CoV-2 vaccination on the risk of relapse in a large cohort of patients with TTP in Milan showed a lower than reported relapse rate (1.36% vs. 2.6%) with an observed/expected incidence rate ratio of 0.52, confirming the safety of mRNA-based anti-SARS-CoV-2 vaccination in TTP patients. Moreover, while the association of TTP relapse with any kind of mRNA vaccination is negligible, the association with infection, especially if characterized by a strong inflammatory response, is much higher (31% of TTP relapses in our historical cohort).14 Indeed, many reports on COVID-19-associated TTP have been reported since the pandemic onset. Based on our results, patients with TTP may safely receive the anti-SARS-CoV-2 vaccination. However, due to the re-

Accepted: March 14, 2023. Early view: March 23, 2023. ©2023 Ferrata Storti Foundation Published under a CC BY-NC license Disclosures IM received honoraria for participating as a speaker at educational meetings organized by Instrumentation Laboratory and Sanofi. AA received honoraria for participating as speakers at educational meetings organized by Sanofi. FP has received honoraria for participating as a speaker in education meetings organized by Grifols and Roche, and she is member of scientific advisory boards of Sanofi, Sobi, Takeda, Roche, Biomarin. The other authors do not have any conflicts of interests to disclose. Contributions MC, RG, AT and FP designed the study. MC, AA and BF enrolled the patients. MC, PDL and MA performed the statistical analysis. MC wrote the manuscript; all the other authors performed the laboratory tests. All authors critically revised and approved the last version of the manuscript. Data-sharing statement Original data will be made available upon request.

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References 1. Yavaşoğlu İ. Vaccination and thrombotic thrombocytopenic purpura. Turk J Haematol. 2020;37(3):218-219. 2. Cuker A, Cataland SR, Coppo P, et al. Redefining outcomes in immune TTP: an international working group consensus report. Blood. 2021;137(14):1855-1861. 3. Jyssum I, Kared H, Tran TT, et al. Humoral and cellular immune responses to two and three doses of SARS-CoV-2 vaccines in rituximab-treated patients with rheumatoid arthritis: a prospective, cohort study. Lancet Rheumatol. 2022;4(3):e177-e187. 4. Società Italiana di Reumatologia. A proposito della vaccinazione anti SARS-COV 2 nei pazienti reumatologici. https://www.reumatologia.it/obj/files/covid19/DocumentoSIRVaccin azioneCovid13febbraio2021. pdf. (Accessed on 19 December 2022). 5. European Alliance of Associations for Rheumatology. EULAR View-points on SARS-CoV-2 vaccination in patients with RMDs. https://www.eular.org/eular_sars_cov_2_vaccination_rmd_patien ts.cfm. (Accessed on 19 December 2022). 6. Schönborn L, Greinacher A. Longitudinal Aspects of VITT. Semin Hematol. 2022;59(2):108-114. 7. Segal Y, Shoenfeld Y. Vaccine-induced autoimmunity: the role of molecular mimicry and immune crossreaction. Cell Mol Immunol. 2018;15(6):586-594.

8. Falter T, Herold S, Weyer-Elberich V, et al. Relapse rate in survivors of acute autoimmune thrombotic thrombocytopenic purpura treated with or without rituximab. Thromb Haemost. 2018;118(10):1743-1751. 9. Picod A, Rebibou JM, Dossier A, et al. Immune-mediated thrombotic thrombocytopenic purpura following COVID-19 vaccination. Blood. 2022;139(16):2565-2569. 10. Shah H, Kim A, Sukumar S, et al. SARS-CoV-2 vaccination and immune thrombotic thrombocytopenic purpura. Blood. 2022;139(16):2570-2573. 11. Giuffrida G, Markovic U, Condorelli A, et al. Relapse of immunemediated thrombotic thrombocytopenic purpura following mRNA COVID-19 vaccination: a prospective cohort study. Haematologica. 2022;107(11):2661-2666. 12. Peyvandi F, Artoni A, Novembrino C, et al. Hemostatic alterations in COVID-19. Haematologica. 2021;106(5):1472-1475. 13. Mancini I, Baronciani L, Artoni A, et al. The ADAMTS13-von Willebrand factor axis in COVID-19 patients. J Thromb Haemost. 2021;19(2):513-521. 14. Mancini I, Pontiggia S, Palla R, et al. Clinical and laboratory features of patients with acquired thrombotic thrombocytopenic purpura: fourteen years of the Milan TTP Registry. Thromb Haemost. 2019;119(5):695-704.

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High-risk additional cytogenetic aberrations in a Dutch chronic phase chronic myeloid leukemia patient population Several chromosomal aberrations detected in addition to the pathognomonic Philadelphia chromosome (Ph) at diagnosis confer a poor prognosis in chronic myeloid leukemia (CML) chronic phase (CP) patients and herald earlier progression to accelerated phase (AP) or blast crisis (BC), and CML-related death.1-3 Their prognostic significance has been established both at diagnosis and when emerging in the course of the disease. Since not all clones have the same clinical relevance, several classifications have been proposed in the recent literature to define additional cytogenetic aberrations (ACA) presenting a higher risk of inferior outcomes.4-8 The conventional classification in “major” and “minor route” ACA was based on their prevalence and appeared to be too restricted to cover all “highrisk” ACA (HR-ACA). Besides four major-route ACA (trisomy 8, isochromosome 17q, additional Ph chromosome and trisomy 19 while excluding loss of Y), five other HR-ACA were identified (trisomy 21, 3q26.2 rearrangements, monosomy 7/7q-, 11q23 rearrangements, and complex karyotypes) in a recent study of CML-CP patients.8 In this study, their presence often preceded an increase in blast percentage and thereby anticipated progression. However, one study did not observe a prognostic impact of trisomy 8 or an additional Ph chromosome when occurring as a single ACA and only heralded inferior outcomes when in combination with other concurrent ACA.5 These discrepant results may be due to low observation numbers at diagnosis as HRACA remain relatively rare and are detected in less than 3% of de novo CML-CP patients.8-10 Consequently, the cohort sizes of previous studies of patients with HR-ACA have been relatively small and verification of findings is necessary. Here, we aim to assess the prevalence of ACA at diagnosis and their clinical impact in a Dutch nationwide patient cohort, with a focus on the recently proposed HRACA classification.8 In addition, we intend to assess the relation of HR-ACA to the EUTOS long-term survival (ELTS) score at diagnosis and to assess the impact of chromosomal aberrations on hematological toxicity (hemtox) of firstline tyrosine kinase inhibitor (TKI) treatment. Data were derived from a real-world population-based CML registry in the Netherlands (PHAROS-CML registry combined with HemoBase) covering a nationwide patient cohort diagnosed with CML between 2008 and 2014.11 We included all adult CML-CP patients with an evaluable cytogenetic assessment at diagnosis. HR-ACA were defined following Hehlmann et al. (+8, i(17q), +Ph, +19, +21, 3q26.2,

-7/7q-, 11q23.2 and complex karyotype; present in Ph-positive cells).8 Other ACA in Ph-positive cells were classified as low-risk ACA (LR-ACA). The emergence of chromosomal aberrations was also assessed during the first 24 months of TKI treatment, including clonal chromosomal aberrations in Ph-negative cells (CCA/Ph-). AP and BC were defined as described in the ELN recommendations.12 Hemtox was defined as de novo anemia, thrombocytopenia and/or leukopenia CTC grade 3 or higher, emerging during firstline TKI therapy. Survival analysis was performed with Kaplan-Meier estimates and the log-rank test was used to compare subgroups. Progression-free survival (PFS) was defined as the time from diagnosis until progression to AP/BC or death. Patients were censored at last follow-up visit. CML-related death was defined as death preceded by CML progression and was assessed using the cumulative incidence competing risk (CICR) method in which death of any other cause was considered as a competing event. Response milestones (complete hematological response [CHR], complete cytogenetic response [CCyR] and major molecular response [MMR]) were defined in accordance with the ELN recommendations.12 The achievement of CCyR, MR2.0 (BCR::ABL1 <1%IS) or MMR was assessed with the CICR method in which progression or death were considered as a competing event. A Cox proportional hazards model was used to assess different predictors for PFS including age, ELTS score (as a numeric variable) and the presence of HRACA at diagnosis. The Χ2 test was used to assess differences in hemtox across subgroups, only considering complete cases. The Medical Ethics Committee of the Erasmus Medical Center in Rotterdam approved this study and the exemption from informed consent. The study was conducted in accordance with the Declaration of Helsinki. A total of 398 CML-CP patients were included in this analysis. Thirty ACA (8%) were detected at diagnosis of which 15 were HR-ACA (4%) (Figure 1). The most frequent HR-ACA were trisomy 8 and an extra copy of Ph chromosome. Loss of the Y chromosome (-Y) as a solitary additional aberration in Ph-positive cells was observed in ten patients and was not designated as ACA since several studies did not report any clinical impact of this aberration.5,8 Patients with HR-ACA at diagnosis were younger than patients without HR-ACA, with a median age of 49 years (interquartile range [IQR], 34-61 years) versus 57 years ([IQR], 43-68 years) at diagnosis, respectively (P=0.198). Other

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LETTER TO THE EDITOR

Figure 1. Inclusion flowchart and prevalence of (additional) cytogenetic aberrations found in the Pharos-HemoBase chronic myloid leukemia patient population at diagnosis. AP/BC: accelerated phase or blast crisis; ACA: additional cytogenetic aberrations; HR: high-risk; LR: low-risk; kar: karyotype.

baseline characteristics were comparable between subgroups, including the ELTS score at diagnosis and the use of second generation TKI as first line treatment. There was no statistically significant association between ELTS categories and the presence of HR-ACA using the Χ2 test (P=0.168), nor was there a significant difference in the mean ELTS score in patients with or without HR-ACA using the Student's t-test (P=0.400). During the first 24 months of TKI treatment, one or more follow-up cytogenetic assessments were done in 257 patients. In these patients, four patients (2%) had newly emerging ACA in the context of disease progression, and 31 patients (12%) developed CCA/Ph-. Most frequent CCA/Ph- were –Y (n=12), +8 (n=11) and -7/7q- (n=4). Transition to myelodysplasia or acute myeloid leukemia was not observed during further follow-up of these patients. Five-year PFS for patients with HR-ACA, with LR-ACA or without ACA was 60% (95% confidence interval [CI]: 4091), 87% (95% CI: 71-100) and 85% (95% CI: 81-89), respectively, with a median follow-up duration of 5 years (IQR, 4-8 years) (Figure 2A). Of note, in patients with ACA, all events of progression or death occurred within 3 years from time of diagnosis. After further stratification based on HR-ACA and the ELTS score at diagnosis, an inferior PFS was noted in patients with HR-ACA in combination with an intermediate or high ELTS score (Figure 2B). In line with PFS results, the cumulative incidence of CML-related mortality was higher in patients with HR-ACA than patients without HRACA (13% vs. 3% at 5 years; P<0.032). No difference in PFS was observed for patients with solitary –Y or with emerg-

ing CCA/Ph- compared to patients without aberrations (Online Supplementary Figure S1). Again, when specifically assessing non –Y CCA/Ph-, no difference in PFS was noted (graph not shown; P=0.703). In a univariable Cox regression analysis, age, ELTS score and the presence of HR-ACA were predictive for PFS, with a hazard ratio (HR)=1.06, 95% CI: 1.04-1.08; HR=2.09, 95% CI: 1.39-3.15 and HR=2.81; 95% CI: 1.22-6.49, respectively (Online Supplementary Table S1). We fitted a multivariable model with ELTS score and HR-ACA, and excluded age since it is already part of the ELTS score calculation. The HR for PFS of HR-ACA and ELTS score were HR=3.13, 95% CI: 1.34-7.31 and HR=2.06, 95% CI: 1.37-3.11, respectively. Regarding the achievement of the ELN response milestones, CHR at 90 days was achieved in 80% versus 87% of patients with versus without HR-ACA, respectively (P=0.428). The cumulative incidence of CCyR or MR2.0 at 6 months was 10% (95% CI: 0-30) versus 38% (95% CI: 3243) in patients with versus without HR-ACA, respectively (P=0.261). The cumulative incidence of MMR at 12 months was 22% (95% CI: 0-51) versus 50% (95 % CI: 44-57) in patients with versus without HR-ACA, respectively (P=0.045). Of note, all HR-ACA patients who eventually presented with disease progression, failed to achieve the MR2.0 or MMR ELN milestone in time. In a final exploratory analysis, we assessed the occurrence of hemtox on first-line tyrosine kinase inhibitor (TKI) treatment. Patients with HR-ACA at diagnosis had significantly more hemtox than those without any ACA (39% vs. 16%; P=0.030), while this difference was not observed for pa-

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LETTER TO THE EDITOR

Figure 2. Progression-free survival Kaplan-Meier estimates. (A) Subgroups based on the presence of low-risk (LR) or high-risk (HR) additional cytogenetic aberrations (ACA). (B) Subgroups based on the presence of high-risk ACA and the EUTOS long-term survival (ELTS) score.

tients with LR-ACA (10% vs. 16%; P=0.607). Patients with CCA/Ph- emerging during the first 24 months of TKI treatment, also experienced more hemtox than patients without CCA/Ph- (32% vs. 16%; P=0.026). In CCA/Ph- patients, hemtox was mostly observed in case of +8 and/or -7/7q(7/15, 47%), and in lesser extent in case of –Y (2/12, 17%). In both groups (HR-ACA and CCA/Ph- patients) the difference in hemtox was mainly due to an increased incidence of thrombocytopenia, with or without concomitant anemia or leukopenia. In conclusion, our results support the recently proposed ACA risk classification.8 HR-ACA at diagnosis were associated with inferior responses, and a significantly higher probability of progression and (CML-related) death, while patients with LR-ACA had a PFS comparable to that of other CML-CP patients. Furthermore, HR-ACA at diagnosis remained independently predictive for PFS in a multivariable regression model including ELTS score, which is in line with a previous analysis.10 In contrast with HR-ACA, the emergence CCA/Ph- did not have an impact on PFS in our cohort. The prognostic significance of this entity remains controversial, more specifically for non –Y CCA/Ph-.13,14 Additionally, our data suggest that patients with HR-ACA at diagnosis or with CCA/Ph- emerging during TKI treatment, have a higher risk of TKI-related hemtox. CCA/Phmight interfere with normal (Ph-) hematopoiesis, predisposing to TKI-related hemtox. This is in line with previous studies showing an increased risk of development of myelodysplastic syndrome from a CCA/Ph- clone.15,16

Taken together, follow-up cytogenetic evaluation after diagnosis is warranted in case of failure to achieve molecular milestones in order to evaluate clonal progression,17 and also in case of hematological toxicity to evaluate emergence of CCA/Ph-, even when molecular response is optimal. Our results on their own should be interpreted with caution since the number of patients with HR-ACA and CCA/Ph- was low. However, our study contributes to the accumulating evidence that implies that patients with HRACA at diagnosis, particularly with a high ELTS, may benefit from a more aggressive treatment strategy with a secondgeneration TKI and an earlier switch to allogeneic stem cell transplantation if the response to TKI is unsatisfactory or results in significant hematological toxicity.

Authors Camille C.B. Kockerols,1 Inge G.P. Geelen,2 Mark-David Levin,1 Jeroen J.W.M. Janssen,3 H. Berna Beverloo,4 Avinash G. Dinmohamed,5,6,7 Mels Hoogendoorn,8 Jan J. Cornelissen2 and Peter E. Westerweel1 Department of Internal Medicine, Albert Schweitzer Hospital,

Dordrecht; 2Department of Hematology, Erasmus Medical Center, Rotterdam; 3Department of Hematology, Radboud University Medical Center, Nijmegen; 4Department of Clinical Genetics, Erasmus Medical Center, Rotterdam; 5Department of Research and Development, Netherlands Comprehensive Cancer Organisation

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LETTER TO THE EDITOR (IKNL), Utrecht; 6Department of Public Health, Erasmus University

Disclosures

Medical Center, Rotterdam; 7Department of Hematology, Amsterdam

The PHAROS-CML registry was financially supported by grants from

University Medical Center, location VUMC, Amsterdam and

Novartis and BMS to the Netherlands Comprehensive Cancer

Department of Hematology, Medical Center Leeuwarden,

Organisation (IKNL). The authors have no conflicts of interest to

Leeuwarden, the Netherlands.

disclose.

Correspondence:

Contributions

C.C.B. KOCKEROLS - c.c.b.kockerols@asz.nl

CK performed the main data-analysis and wrote the first draft of the manuscript. CK, PW and HB evaluated reported karyotypes and

https://doi.org/10.3324/haematol.2022.282447

cytogenetic aberrations. All authors revised and approved the final version of the manuscript.

Received: November 18, 2022. Accepted: March 14, 2023.

Data-sharing statement

Early view: March 23, 2023.

Data can be made available on request to other researchers, when in collaboration with the Dutch Cancer Registry, which is the owner of

the data.

Published under a CC BY-NC license

References 1. Anastasi J, Feng J, Le Beau MM, Larson RA, Rowley JD, Vardiman JW. The relationship between secondary chromosomal abnormalities and blast transformation in chronic myelogenous leukemia. Leukemia. 1995;9(4):628-633. 2. Marktel S, Marin D, Foot N, et al. Chronic myeloid leukemia in chronic phase responding to imatinib: the occurrence of additional cytogenetic abnormalities predicts disease progression. Haematologica. 2003;88(3):260-267. 3. Cortes JE, Talpaz M, Giles F, et al. Prognostic significance of cytogenetic clonal evolution in patients with chronic myelogenous leukemia on imatinib mesylate therapy. Blood. 2003;101(10):3794-3800. 4. Mitelman F, Levan G, Nilsson PG, Brandt L. Non-random karyotypic evolution in chronic myeloid leukemia. Int J Cancer. 1976;18(1):24-30. 5. Wang W, Cortes JE, Tang G, et al. Risk stratification of chromosomal abnormalities in chronic myelogenous leukemia in the era of tyrosine kinase inhibitor therapy. Blood. 2016;127(22):2742-2750. 6. Gong Z, Medeiros LJ, Cortes JE, et al. Cytogenetics-based risk prediction of blastic transformation of chronic myeloid leukemia in the era of TKI therapy. Blood Adv. 2017;1(26):2541-2552. 7. Alhuraiji A, Kantarjian H, Boddu P, et al. Prognostic significance of additional chromosomal abnormalities at the time of diagnosis in patients with chronic myeloid leukemia treated with frontline tyrosine kinase inhibitors. Am J Hematol. 2018;93(1):84-90. 8. Hehlmann R, Voskanyan A, Lauseker M, et al. High-risk additional chromosomal abnormalities at low blast counts herald death by CML. Leukemia. 2020;34(8):2074-2086. 9. Fabarius A, Leitner A, Hochhaus A, et al. Impact of additional cytogenetic aberrations at diagnosis on prognosis of CML: longterm observation of 1151 patients from the randomized CML

study IV. Blood. 2011;118(26):6760-6768. 10. Clark RE, Apperley JF, Copland M, Cicconi S. Additional chromosomal abnormalities at chronic myeloid leukemia diagnosis predict an increased risk of progression. Blood Adv. 2021;5(4):1102-1109. 11. Geelen IGP, Thielen N, Janssen JJWM, et al. Treatment outcome in a population-based, 'real-world' cohort of patients with chronic myeloid leukemia. Haematologica. 2017;102(11):1842-1849. 12. Hochhaus A, Baccarani M, Silver RT, et al. European LeukemiaNet 2020 recommendations for treating chronic myeloid leukemia. Leukemia. 2020;34(4):966-984. 13. Deininger M, Cortes J, Paquette R, et al. The prognosis for patients with chronic myeloid leukemia who have clonal cytogenetic abnormalities in Philadelphia chromosome-negative cells. Cancer. 2007;110(7):1509-1519. 14. Issa G, Kantarjian H, Gonzalez G, et al. Clonal chromosomal abnormalities appearing in Philadelphia chromosome-negative metaphases during CML treatment. Blood. 2017;130(19):2084-2091. 15. Bumm T, Müller C, Al-Ali H, et al. Emergence of clonal cytogenetic abnormalities in Ph-cells in some CML patients in cytogenetic remission to imatinib but restoration of polyclonal hematopoiesis in the majority. Blood. 2003;101(5):1941-1949. 16. Jabbour E, Kantarjian HM, Abruzzo LV, et al. Chromosomal abnormalities in Philadelphia chromosome negative metaphases appearing during imatinib mesylate therapy in patients with newly diagnosed chronic myeloid leukemia in chronic phase. Blood. 2007;110(8):2991-2995. 17. Geelen IGP, Thielen N, Janssen JJWM, et al. Omitting cytogenetic assessment from routine treatment response monitoring in chronic myeloid leukemia is safe. Eur J Haematol. 2018;100(4):367-371.

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Chemotherapy in solitary bone plasmacytoma to prevent evolution to multiple myeloma Solitary bone plasmacytoma (SBP) is a rare malignancy plasma cell disorder characterized by a proliferation of monoclonal (M) plasma cells localized within a single biopsy-proven lesion without evidence of overt multiple myeloma (MM).1 Standard of care relies on radiotherapy with a total dose of 40 to 50 grays.2 Surgery is sometimes combined, mainly in case of neurological compression. Although local disease control is frequent (about 90%), approximately 50% of patients will develop MM within 5 years of diagnosis.3,4 Some factors have been associated with survival outcomes in several retrospective studies, but these findings are sometimes divergent.3,5-9 Particularly, the impact of adjuvant chemotherapy has been highly debated, with controversial literature.3,5,10-13 However, the data mainly rely on small retrospective case series, conducted before the era of novel chemotherapeutic agents. The aim of this study was to describe the characteristics and outcome of patients treated for SBP, to identify factors associated with progression to MM and death, and to assess whether adjuvant chemotherapy improves outcome. Between 1992 and 2020, patients with histologically confirmed diagnosis of SBP according to the International Myeloma Working Group (IMWG) criteria,1 identified through a systematic search in the electronic database, were included. All Assistance Publique-Hôpitaux de Paris patients are informed that their clinical data can be used for research and give their consent for the use of their data unless they provide an opposition to it. None of the patients in this study objected to the use of their data. Therapeutic management (including radiotherapy, chemotherapy) was recorded and response to treatment was assessed after radiotherapy and then after chemotherapy. Treatment response was assessed retrospectively according to the IMWG criteria,14 (adapted to clinical practice, for patients with a detectable M-component on serum or urine immunofixation [IF]) and according to radiological response only for non-secretory SBP. Briefly, complete response (CR) for secretory SBP was defined as the disappearance of the M-component and no evidence of disease progression on imaging assessment when performed. For non-secretory SBP, CR was defined by the absence of an active lesion assessed by magnetic resonance imaging (MRI) or positron emission tomography/computed tomography (PET/CT). Progressive disease (PD) was defined by a significant increase in the size of the M-component or significant imaging progression. Otherwise, patients were considered partial responders (PR). Progression to MM was considered when bone marrow plasma-

cytosis (BMPC) was ≥10% or when BMPC reached <10% with multiple bone lesions. MM-free survival (MMFS) and overall survival (OS) were determined from the start of radiotherapy. Survival curves were computed using the Kaplan Meier method and compared with the log-ranktest. Cox proportional hazards models were used to identify possible independent predictive factors of MMFS and OS for SBP. All parameters associated with the outcome in the univariate analysis (P<0.2), and the variable of particular interest for this study (chemotherapy), were entered into a multivariate model. All analyses were performed with R software (version 1.2.1578 https://cran.rproject.org/). All P values were two-sided and P<0.05 was considered statistically significant. Seventy-seven patients with a diagnosis of SBP were included (Table 1). Overall, 21 (27.3%) patients had two bone marrow examinations at two different locations. Cytogenetics, available in 29 (37.7%) patients, detected a t(4;14) translocation in one patient (3.4%). Immunophenotyping of plasma cells, available in eight (10.4%) patients, revealed the presence of monoclonal plasma cells in the bone marrow in one case (12.5%). All patients received radiotherapy at a median dose of 45 grays (range, 30-55). The decision to combine chemotherapy and radiotherapy was left to the discretion of each referring hematologist. Chemotherapy was prescribed in 32 of 77 (41.6%) patients, concomitant to radiotherapy in eight of 32 (25%) patients or adjuvant in 24 of 32 (75%) patients. In these 24 patients, chemotherapy was initiated at a median of 4.3 months (range, 1.6-12.3) after the start of radiotherapy. Chemotherapy included mainly immunomodulatory drug combinations in 28 of 32 (87.5%) (Table 1). The median follow-up duration was 87.1 months (range, 1.6-306.8). MM occurred in 45 (58.4%) patients. Only one (1.3%) local recurrence of plasmacytoma occurred. Thirteen (16.9%) patients died: ten (76.9%) from MM, two (15.4%) from solid cancer and one (7.7%) of unknown cause. Five-year MMFS and OS were 47.9% and 86.8%, respectively (Online Supplementary Figure S1). In order to study the impact of adjuvant chemotherapy, the eight (10.4%) patients treated with concomitant chemotherapy were excluded from this analysis, since response to radiotherapy alone was not assessable and the indication of concomitant chemotherapy might reflect a more aggressive disease. Compared to patients not treated with chemotherapy, patients treated with adjuvant chemotherapy had more frequent detectable Mcomponent at diagnosis (95.8% vs. 73.3%, respectively; P=0.03; Online Supplementary Table S1). Absence of CR

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LETTER TO THE EDITOR Table 1. Characteristics of 77 patients with solitary bone plasmacytoma. Overall population N=77 Baseline demographic, clinical and biological characteristics Median age in years (range) Male sex, N (%) Site of plasmacytoma, N (%) Spine Limb Pelvis Rib Sternum Skull Symptoms at diagnosis (76 evaluated), N (%) Local pain Local swelling Incidental discovery Neurological compression syndrome Positive IF (serum and/or urine), N (%) Serum, N/N (%) IgG κ IgG λ IgA λ Light chain λ Light chain κ Urine (60 evaluated), N (%) Median serum M-spike, g/L (range) (74 evaluated) Median FLC ratio, (range) (51 evaluated) Abnormal FLC ratio (<0.26 or >1.65) (51 evaluated), N (%) Bone marrow involvement Median BMPC, % (range) BMPC ≥5%, N (%) Aberrant and/or immature PC on BM aspiration (72 evaluated), N (%) Clonal PC by immunophenotyping on BM aspiration (8 evaluated), N (%) Clonal PC infiltration by immunohistochemistry on BM biopsy (11 evaluated) Imaging procedure performed to exclude other location X ray, N (%) WB-CT, N (%) All spine and pelvis MRI or WB-CT, N (%) 18F-FDG PET/CT, N (%) Therapeutic management Median radiotherapy dose, grays (range) (72 evaluated) Radiotherapy alone, N (%) Radiotherapy + chemotherapy, N/N (%) Adjuvant chemotherapy Concomitant chemotherapy Regimen (32 evaluated), N (%) Edx-Dex TD RD VD MPT VTD VRD VCD VRD auto-SCT VRD Median duration of chemotherapy in months, N (range) Surgery, N (%)

59.0 (27.0-89.0) 47 (61.0) 42 (54.5) 13 (16.9) 8 (10.4) 4 (5.2) 2 (2.6) 8 (10.4) 66 (86.8) 3 (3.9) 7 (9.2) 12 (15.8) 62 (80.5) 57/77 (74.0) 33/57 (57.9) 18/57 (31.6) 4/57 (7.0) 1/57 (1.8) 1/57 (1.8) 12 (20.0) 4.3 (0-36.0) 1.5 (0-118.0) 30 (58.8) 2.0 (0-8.0) 12 (15.6) 25 (34.7) 1 (12.5) 0 40 (51.9) 5 (6.5) 50 (64.9) 63 (81.8) 45.0 (30.0-55.0) 45 (58.4) 32/77 (41.6) 24/32 (75.0) 8/32 (25.0) 1 (3.1) 16 (50.0) 3 (9.4) 2 (6.3) 1 (3.1) 3 (9.4) 4 (12.5) 1 (3.1) 1 (3.1) 7 (2.5-21.3) 22 (28.6)

auto-SCT: auto-stem cell transplantation; BM: bone marrow; Edx-Dex : cyclophosphamide + dexamethasone; FLC: serum free light chain; IF: immunofixation; Ig: immunoglobulin; M: monoclonal; MPT: melphalan + prednisone + thalidomide; PC: plasma cell; RD: lenalidomide + dexamethasone; TD: thalidomide + dexamethasone; VD: bortezomib + dexamethasone; VCD: bortezomib + cyclophosphamide + dexamethasone; VRD: lenalidomide + bortezomib + dexamethasone; VTD: thalidomide + bortezomib + dexamethasone; WB: whole body; X-ray: whole skeleton plain radiography; CT: computed tomography, PET: positron emssion tomography; MRI: magnetic resonance imaging. Haematologica | 108 November 2023

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LETTER TO THE EDITOR after radiotherapy was the main reason for prescribing chemotherapy, as none of the 24 patients treated with adjuvant chemotherapy were meeting CR criteria after radiotherapy, compared to 12 of 45 (26.7%) patients treated with radiotherapy alone (P=0.002; Online Supplementary Table S1). Nevertheless, 5-year MMFS was 62.9% for patients treated with adjuvant chemotherapy compared to 41.7% for patients treated with radiotherapy alone (P=0.2) (Figure 1A). In multivariate analysis, adjuvant chemotherapy was associated with a reduced risk of progression to MM (hazard ratio [HR]=0.30; 95% confidence interval [CI]: 0.14-0.64), as well as achieving CR after radiotherapy (HR=0.16; 95% CI: 0.05-0.56), while BMPC ≥5% at diagnosis was associated with an increased risk of developing MM (HR=2.81; 95% CI: 1.20-6.57) (Table 2). In sensitivity analyses performed in the entire study population, 5-year MMFS was significantly higher in patients achieving CR whatever treatment was (radiotherapy alone or combined with chemotherapy) (73.3% vs. 32.9%; P=0.0004). Five-year MMFS was 56% in patients treated with radiotherapy plus chemotherapy (adjuvant or concomitant) compared to 41.7% in patients treated with radiotherapy alone (P=0.2) (Figure 1B). In multivariate analysis, achieving CR compared to PR or PD was associated with a lower risk of progression to MM (HR=0.25; 95% CI: 0.11-0.59; Online Supplementary Table S2). In this model, chemotherapy (adjuvant or concomitant) and BMPC ≥5% were no longer associated with the risk of MM but were close to significance (HR=0.50; 95% CI: 0.25-1.00; and HR=2.40; 95% CI: 1.00-5.74, respectively; Online Supplementary Table S2). All deaths were observed in patients with detectable Mcomponent at baseline (P=0.04). Age was associated with the risk of death (HR=1.06; 95% CI: 1.00-1.11) whereas chemotherapy was not (P=0.45). Achieving CR after complete treatment was associated with improved survival (HR=0.22; 95% CI: 0.05-0.99) (Online Supplementary Table S2). In this retrospective study of 77 patients with SBP, we confirmed that about half of the patients develop MM within 5 years. Also, we found that chemotherapy, although frequently prescribed in patients with poorer prognosis, was associated with a lower risk of evolution to MM. Furthermore, we observed that response to the first line of treatment (radiotherapy +/- chemotherapy) was the main factor associated with the risk of MM and death. In the present study, the 5-year risk of developing MM was 52.1%, which is similar to previous data in the literature.3,4 However, this risk may differ depending on prognosis factors. Indeed, we observed that patients in CR after treatment had better outcomes in terms of MMFS and OS. Comparable results have been described in MM.15 In SBP, persistence of M-protein or abnormal serum free-light chain ratio after treatment was associated with an in-

creased risk of MM in several publications.7,9 Thus, it appears that the treatment goal should be to obtain a CR. However, response after radiotherapy alone is often incomplete leaving an important place for adjuvant chemotherapy in the therapeutic management of SBP. Many retrospective series did not find benefit of chemotherapy,3,5,13 but these series were mainly performed before the era of novel agents. Nevertheless, a series of 46 patients with solitary plasmacytoma showed improved outcomes for the concomitant lenalidomide-dexamethasone group compared to the radiation therapy-alone group (5-year disease-free-survival, 81.7% vs. 48.4%; P=0.047).10 Also, ad-

Figure 1. Comparison of the impact of treatment regimen on survival of multiple myeloma-free patients. Multiple myelomafree survival in patients treated with radiotherapy alone (RT) compared with (A) patients treated with radiotherapy and adjuvant chemotherapy (RT + adj.CT) and (B) patients treated with radiotherapy and chemotherapy (adjuvant or concomitant) (RT + CT).

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LETTER TO THE EDITOR Table 2. Factors associated with progression to multiple myeloma: univariate and multivariate analysis in 69 patients with solitary bone plasmacytoma treated with radiotherapy alone or combined with adjuvant chemotherapy. Parameter

Baseline characteristics Age in years Male sex Spine location Positive IF (serum and/or urine) Serum M-spike size, g/L BMPC, % BMPC ≥5% Aberrant and/or immature PC on BM aspiration Modern imaging for diagnosis (PET/CT or WB-MRI)

Univariate analysis

Multivariate analysis

HR (95% CI)

69 69 69 67 69 69 64 69 69

1.01 (0.98-1.03) 1.13 (0.60-2.13) 1.08 (0.58-2.01) 1.58 (0.68-3.68) 1.03 (0.99-1.07) 1.00 (0.86-1.17) 1.78 (0.87-3.65) 0.96 (0.50-1.85) 1.19 (0.50-2.85)

0.61 0.70 0.82 0.29 0.13 0.96 0.11 0.91 0.69

0.99 (0.95-1.04) 2.81 (1.20-6.57) -

0.83 0.02 -

Treatment regimen Radiotherapy dose, grays Radiotherapy dose ≥45 grays Surgery Adjuvant chemotherapy

64 64 69 69

1.03 (0.96-1.10) 1.15 (0.59-2.23) 0.93 (0.48-1.80) 0.65 (0.33-1.29)

0.46 0.68 0.84 0.22

0.30 (0.14-0.64)

0.002

Response after radiotherapy Positive IF Serum M-spike size, g/L Complete response

66 66 63

2.89 (1.35-6.15) 1.06 (1.02-1.11) 0.29 (0.10-0.83)

0.006 0.001 0.02

0.16 (0.05-0.56)

0.004

HR: hazard ratio; CI: confidence interval; BMPC: bone marrow plasma cell; IF: immunofixation (serum and/or urine); M: monoclonal; CT: computed tomography, PET: positron emission tomography; WB: whole body; MRI: magnetic resonance imaging.

juvant chemotherapy was associated with a decreased risk of progression in a retrospective study of 61 patients with SBP (HR=0.2; 95% CI: 0.04-0.97 for combined treatment vs. radiotherapy alone).12 A randomized therapeutic trial (IDRIS Trial, clinicaltrials gov. Identifier: NCT02544308) is on-going to evaluate whether adjuvant lenalidomide can improve progression-free survival compared to radiotherapy alone in patients with high-risk SBP. This study will definitively show the room of adjuvant therapy in SBP. Our study presents several limitations. Due to the rarity of SBP, inclusion time was very long and the number of patients was relatively small, but in line with existing large series. Thus, the treatment procedures were not standardized. Still, our results suggest a benefit of adjuvant chemotherapy used between 1992 and 2020 and it is likely that with the arrival of now available new drugs with even better efficacy, the positive impact of chemotherapy might be even higher. Finally, some data were missing and we could not rigorously use the IMWG response criteria which are, however, not totally adapted to SBP, particularly in non-secretory SBP. In conclusion, our results highlight the importance of achieving CR after treatment of SBP, which appears to be the main risk factor for progression to MM. They also suggest a benefit of adjuvant chemotherapy, especially for patients at high risk of progression, i.e., with persistent disease after radiotherapy. These observations need to be confirmed and justify a prospective trial.

Authors Sophia Ascione,1* Stéphanie Harel,2* Florent L. Besson,3,4 Rakiba Belkhir,1 Julien Henry,1 Bruno Royer,2 Bertrand Arnulf,2 Xavier Mariette1,5 and Raphaèle Seror1,5 Department of Rheumatology, AP-HP, Hôpital Bicêtre, Le Kremlin

Bicêtre; 2Department of Immunohematology, AP-HP, Hôpital SaintLouis, Paris; 3Department of Biophysics and Nuclear MedicineMolecular Imaging, AP-HP, Hôpital Bicêtre, Le Kremlin Bicêtre; 4

Université Paris-Saclay, CEA, CNRS, INSERM, BioMaps, Orsay and

Center of Immunology of Viral Infections and Auto-immune

Diseases (IMVA), INSERM UMR1184, Université Paris-Saclay, Le Kremlin Bicêtre, France *SA and SH contributed equally as co-first authors. Correspondence: R. SEROR - raphaele.seror@aphp.fr https://doi.org/10.3324/haematol.2022.282214 Received: October 10, 2022. Accepted: March 10, 2023. Early view: March 23, 2023. ©2023 Ferrata Storti Foundation

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LETTER TO THE EDITOR RS wrote the first version of the manuscript. All authors critically

Published under a CC BY-NC license

revised and approved the final version of the manuscript. Disclosures No conflicts of interest to disclose.

Acknowledgments The authors are indebted to all participants for their continued

Contributions

participation. The authors would like to thank Christophe

All authors contributed to the manuscript. SA, SH, FB, RB, JH, BR,

Hennequin for its help in patient’s inclusion process.

BA, XM, and RS were responsible for conception and design. SA, SH and RS were responsible for data collection and analysis. All

Data-sharing statement

authors were responsible for the interpretation of data. SA, SH and

Data are available on request by emailing the corresponding author.

References 1. Rajkumar SV, Dimopoulos MA, Palumbo A, et al. International Myeloma Working Group updated criteria for the diagnosis of multiple myeloma. Lancet Oncol. 2014;15(12):e538-548. 2. Caers J, Paiva B, Zamagni E, et al. Diagnosis, treatment, and response assessment in solitary plasmacytoma: updated recommendations from a European Expert Panel. J Hematol Oncol. 2018;11(1):10. 3. Knobel D, Zouhair A, Tsang RW, et al. Prognostic factors in solitary plasmacytoma of the bone: a multicenter Rare Cancer Network study. BMC Cancer. 2006;6:118. 4. Reed V, Shah J, Medeiros LJ, et al. Solitary plasmacytomas: outcome and prognostic factors after definitive radiation therapy. Cancer. 2011;117(19):4468-4474. 5. Katodritou E, Terpos E, Symeonidis AS, et al. Clinical features, outcome, and prognostic factors for survival and evolution to multiple myeloma of solitary plasmacytomas: a report of the Greek myeloma study group in 97 patients. Am J Hematol. 2014;89(8):803-808. 6. Tsang RW, Gospodarowicz MK, Pintilie M, et al. Solitary plasmacytoma treated with radiotherapy: impact of tumor size on outcome. Int J Radiat Oncol Biol Phys. 2001;50(1):113-120. 7. Dingli D, Kyle RA, Rajkumar SV, et al. Immunoglobulin free light chains and solitary plasmacytoma of bone. Blood. 2006;108(6):1979-1983. 8. Paiva B, Chandia M, Vidriales MB, et al. Multiparameter flow cytometry for staging of solitary bone plasmacytoma: new

criteria for risk of progression to myeloma. Blood. 2014;124(8):1300-1303. 9. Wilder RB, Ha CS, Cox JD, Weber D, Delasalle K, Alexanian R. Persistence of myeloma protein for more than one year after radiotherapy is an adverse prognostic factor in solitary plasmacytoma of bone. Cancer. 2002;94(5):1532-1537. 10. Mignot F, Schernberg A, Arsène-Henry A, Vignon M, Bouscary D, Kirova Y. Solitary Plasmacytoma treated by lenalidomidedexamethasone in combination with radiation therapy: clinical outcomes. Int J Radiat Oncol Biol Phys. 2020;106(3):589-596. 11. Avilés A, Huerta-Guzmán J, Delgado S, Fernández A, DíazMaqueo JC. Improved outcome in solitary bone plasmacytomata with combined therapy. Hematol Oncol. 1996;14(3):111-117. 12. Mheidly K, Lamy De La Chapelle T, Hunault M, et al. New insights in the treatment of patients with solitary bone plasmacytoma. Leuk Lymphoma. 2019;60(11):2810-2813. 13. Finsinger P, Grammatico S, Chisini M, Piciocchi A, Foà R, Petrucci MT. Clinical features and prognostic factors in solitary plasmacytoma. Br J Haematol. 2016;172(4):554-560. 14. Kumar S, Paiva B, Anderson KC, et al. International Myeloma Working Group consensus criteria for response and minimal residual disease assessment in multiple myeloma. Lancet Oncol. 2016;17(8):e328-346. 15. Harousseau JL, Attal M, Avet-Loiseau H. The role of complete response in multiple myeloma. Blood. 2009;114(15):3139-3146.

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The oncogenetic landscape and clinical impact of BCL11B alterations in adult and pediatric T-cell acute lymphoblastic leukemia T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive and heterogenous hematological cancer representing 15% of pediatric and 25% of adult ALL.1 It arises from the transformation of T-cell precursors arrested at specific stages of maturation. T-ALL are associated with a multitude of acquired genetic abnormalities that contribute to this developmental arrest and exacerbated proliferation, including the loss of major tumor suppressive pathways such as inactivating alterations of PTEN and CDKN2A/B and activation of oncogenic pathways (e.g., activating mutations in NOTCH1, IL7R/JAK/STAT).2 Most patients respond well to polychemotherapy, but at the cost of acute adverse effect and sequelae notably in the case of allogeneic stem cell transplantation (allo HSCT). Despite this initial favorable response, about 2030% of pediatric3 and 40% of adult T-ALL relapse, with 5year overall survival (OS) rates below 20%.4 Hence, identifying prognostic markers at diagnosis is a critical medical need to refine the treatment protocols and improve the patients' outcomes. BCL11B belongs to the Kruppel-like C2H2 type zinc finger transcription factor family that contains six C2H2 zinc fingers and proline-rich and acidic regions with 95% identity in their zinc finger domains. It encodes two different protein isoforms consisting of 823 and 894 aa in humans. These structures include DNA-binding and protein-interacting regions. BCL11B is critical in the development and maintenance of T-cell identity. It was initially discovered as a potential suppressor of radiation-induced T-cell lymphoma.5 The role of BCL11B in leukemogenesis was first pointed out through its cryptic translocation with TLX3, positioning this oncogene under the control of BCL11B regulatory elements.6 However, monoallelic BCL11B deletions or missense mutations were reported in 9% of T-ALL cases, showing that BCL11B is a haplo-insufficient tumor suppressor that could collaborate with additional oncogenic lesions during thymocyte development.7 Nevertheless, the incidence and clinical data on BCL11B alterations in large cohorts of patients are still lacking. Hence, we described the mutational landscape and clinical outcome related to BCL11B alterations in two cohorts of adult and pediatric TALL patients treated in GRAALL03/05 protocols or according to FRALLE2000T recommendations, respectively. We took advantage of an extensively annotated cohort of 476 patients included in the GRAALL03/05 French protocol for adults aged up to 60 years (n=215) or treated according

to FRALLE2000T recommendations for children (n=261). BCL11B alterations were identified in cases of 476 T-ALL cases by gene mutation screening and next-generation sequencing (NGS)-based analysis of copy number variation (CNV) as previously described. Array-comparative genomic hybridization (array-CGH) was also performed for 310 patients. All deletions identified in array-CGH were confirmed by NGS-based analysis of CNV. These alterations were more frequent in adults: 48 cases (22%) in GRAALL03/05 than in children: 38 cases (15%) in FRALLE2000T-treated patients (P=0.03) (Figure 1A). BCL11B mutations were the most frequent alterations: 73 patients (for a total of 99 mutations) (Figure 1C). BCL11B is a zinc finger transcription factor that binds DNA via its Cys2His2 zinc (C2H2 Zn) finger domains. Most mutations were missense (66%) within a mutational hotspot in exon 4 (83 mutations) affecting predominantly amino acids 452, 465, and 472 located in the C2H2 Zn finger domain (Figure 1C). Frameshift or non-sense mutations (34%) in exons 1-4 were also detected and predicted to produce truncated forms of the protein. BCL11B deletions were detected in 13 cases (3%) (Figure 1B). Only seven of 13 (54 %) presented focal intragenic deletions. Other cases harbored pan-genic deletions leading to haplo-insufficiency. The distribution of the alterations types was similar between adults and children, with a preponderance of mutations in both cohorts: 38 mutations (18%) and ten deletions (5%) in GRAALL03/05 and 35 mutations (13%) and three deletions (1%) in FRALLE2000T. The clinical and biological characteristics of the patients according to BCL11B status are described in Table 1. Patients carrying BCL11B alterations (BCL11Balt) are slightly older with a median age at diagnosis of 19.3 years (range, 1.8-57.0) compared to 14.8 years (range, 1.1-59.1) (P=0.045), with no difference in the sex ratio (Table 1; Online Supplementary Tables S1 and S2). BCL11Balt patients presented with reduced leukocytosis at diagnosis compared to the wild-type (wt) group (median white blood cell [WBC] count: 36.4x109/L vs. 67.6x109/L; P=0.004). No difference was observed in the central nervous system (CNS) involvement rate. BCL11Balt patients had a better good prednisone response (69% vs. 53%; P=0.008), a higher rate of early chemotherapy response (85% vs. 69%; P=0.005), and presented less detectable minimal residual disease (MRD) at the end of induction (10% vs. 42%; P<0.001) as compared to BCL11Bwt patients. Similar allo HSCT in the first complete remission were performed in both groups (17% vs. 23%).

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LETTER TO THE EDITOR Phenotypically, BCL11Balt T-ALL were less often described as ETP (8% vs. 21%; P=0.017) and mostly exhibited a cortical IMβ/Pre-αβ stage (76%) (Table 1; Online Supplementary Tables S1 and S2). Consequently, more TLX1/TLX3 deregulations (57%) were identified in this group. Conversely, TAL1 (5%) or HOXA9 (15%) overexpression were rare events in this subgroup, with no PICALM::MLLT10 rearrangement detected. While lesions of the NOTCH1 pathway or genetic deletions of CDKN2A/B are frequent oncogenetic traits of T-ALL (70% in our cohort), almost all BCL11Balt patients harbored these lesions (94% of NOTCH1 signaling alterations, 87% of CDKN2A/B deletions) (Online Supplementary Figure S1). Regarding JAK/STAT signaling, the gene mutation profile is more heterogeneous with more PTPN2 (16% vs. 7%) and DNM2 mutations (29% vs. 14%,) but less JAK3 mutations

(8% vs. 21%) in BCL11Balt, as expected due to more TLX1/3 deregulations. Epigenetic regulators have a specific profile with more KMT2D (9%) and PHF6 mutations (46%) and fewer SUZ12 alterations (5%). No significant difference was observed concerning PI3K signaling between BCL11Bwt and BCL11Balt. In consistency with these results, patients with BCL11Balt were fewer and scored a higher risk profile defined by the NOTCH1/FBXW7/RAS/PTEN (N/F/R/P) oncogenetic classifier: 28% versus 47% (P=0.001) (Table 1). In order to investigate the prognostic value of BCL11B alterations, survival analyses were performed in the entire cohort of 476 patients treated according to the GRAALL03/05 protocol and FRALLE2000T recommendations. Patients with BCL11Balt T-ALL have a favorable outcome in

Figure 1. BCL11B alterations and incidence in T-cell acute lymphoblastic leukemia. (A) Incidence of BCL11B alterations in GRAALL03/05 and FRALLE2000T treated patients. (B) BCL11B deletion mapping representation in chromosome 14. (C) Lollipop plot representing intragenic mutational patterns according to patients occurrence. (D) Kaplan-Meier curves according to BCL11B status in GRAALL03/05 and FRALLE2000T treated patients: overall survival (left), cumulative incidence of relapse (middle), event-free survival (right). Haematologica | 108 November 2023

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LETTER TO THE EDITOR terms of OS, cumulative incidence of relapse (CIR) and event-free survival (EFS) to BCL11BWT patients (Figure 1D). For BCL11Balt patients, the 5-year OS was 85.2% (95% confidence interval [CI]: 75.3-91.4) versus 67.9% (95% CI: 62.972.4) for BCL11BWT (hazard ratio [HR]=0.53; 95% CI: 0.35-0.80; P=0.01). The 5-year CIR was 21.4% (95% CI: 8.338.5) versus 30.7% (95% CI: 23.9-37.7) (HR=0.60; 95% CI: 0.39-0.92; P=0.047). The 5-year EFS was 73.5% (95% CI: 62.6-81.7) versus 59.4% (95% CI: 54.2-64.1) (HR=0.60; 95% CI: 0.44-0.90; P=0.025). A similar trend was observed in adults and children separately (Figure 2). However, in multivariate analysis, considering variables associated with OS in the univariate analysis as covariates (including oncogenetic classifier), the BCL11B status does not remain significantly associated with a better OS. In this large cohort of adult and pediatric homogeneously

treated T-ALL, BCL11B alterations were associated with an overall good response to treatment and a favorable longterm prognosis.8 These results have to be interpreted in the context of a favorable mutational landscape associated with this alteration.9 Indeed, BCL11B alterations were identified in a subgroup of T-ALL presenting a low-risk oncogenetic classifier with frequent NOTCH1 and rare NRAS or PTEN alterations. Recurrent cryptic t(5;14)(q35;q32) translocations juxtaposing BCL11B and TLX3 result in BCL11B gene regulatory elements driving the aberrant overexpression of TLX3. This translocation is responsible for the majority of TLX3 overexpression in T-ALL, thereby, inactivating one functional allele of BCL11B and leading to a haplo-insufficient phenotype in some cases. TLX3 alterations are observed in 20% of T-ALL in children and 13% in adults. The prognosis value

Table 1. Biological and clinical characteristics of patients according to BCL11B status. GRALL03/05 and FRALLE2000T

Overall N=476

BCL11Bwt N=390 (82%)

BCL11Balt N=86 (18%)

56 (18)

51 (21)

5 (8)

0.017

Biological characteristics ETP status2, N (%) Phenotypic classification,3 N (%) IM0/δ/γ IMβ/Pre-αβ TCR γδ TCR αβ

89 (21) 211 (50) 53 (13) 66 (16)

86 (26) 149 (44) 45 (13) 57 (17)

3 (4) 62 (76) 8 (10) 9 (11)

Molecular classification,4 N (%) PICALM::MLLT10 TLX1 TLX3 SIL-TAL1 HOXA9 overexpression Negative

13 (3) 54 (13) 72 (17) 57 (14) 79/336 (24) 219 (53)

13 (4) 22 (7) 57 (17) 53 (16) 69/270 (26) 188 (56)

0 (0) 32 (39) 15 (18) 4 (5) 10/66 (15) 31 (38)

Risk classifier, N (%) High Low

<0.001

0.001 209 (44) 267 (56)

185 (47) 205 (53)

24 (28) 62 (72)

15.33 (1.08-59.15)

14.8 (1.1-59.1)

19.3 (1.8-57.0)

357 (75) 119 (25)

295 (76) 95 (24)

62 (72) 24 (28)

WBC x109/L, median (range)

63.80 (0.30-980.00)

67.60 (0.30-965.00)

36.40 (4.25-980.00)

0.004

CNS Involvement, N/N (%)

51/474 (11)

38/388 (10)

13/86 (15)

0.18

Response to therapy, N/N (%) Good prednisone response Chemosensitivity MRD1 >10-4 Complete remission Allogeneic HSCT

259/467 (55) 337/467 (72) 123/340 (36) 440/476 (92) 101/456 (22)

202/384 (53) 266/383 (69) 117/280 (42) 357/390 (92) 87/372 (23)

57/83 (69) 71/84 (85) 6/60 (10) 83/86 (97) 14/84 (17)

0.008 0.005 <0.001 0.17 0.19

Clinical characteristics Age at Diagnosis in years, median (range) Sex, N (%) Male Female

0.045 0.49

Welch two-sample t-test; Fisher's exact test for count data; Fisher's exact test for count data with simulated P value. 2ETP status was available for 307 samples; 3phenotypic classification was available for 419 samples; 4molecular classification was available for 415 samples. CNS: central nervous system; ETP: early T-cell precursor; IM: immature; MRD1: minimal residual disease; SCT: stem cell transplantation; TCR: T-cell receptor; WBC: white blood cell count; wt: wild-type; alt: alterations. 1

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LETTER TO THE EDITOR

Figure 2. Kaplan-Meier curves according to BCL11B status in FRALLE2000T or GRAALL03/05 treated patients. (A, D) Kaplan Meier curves representing overall survival (OS) according to BCL11B status. (B, E) Kaplan Meier curves representing event-free survival (EFS) according to BCL11B status. (C, F) Kaplan Meier curves representing cumulative incidence of relapse (CIR) according to BCL11B status.

of TLX3 overexpression is either neutral or dismal in several adults and pediatric series.1,10–12 Previous work considered that BCL11B inactivation could be secondary to this translocation and lead to pathogenic consequences.7 However, our results showed that BCL11B alterations are associated with a favorable outcome, contrary to TLX3 overexpression, suggesting that TLX3 overexpression could mitigate the favorable profile of BCL11B alterations. In line with this, heterozygous loss of Bcl11b was reported to reduce lethal thymic lymphoma in ATM-/- mice by suppressing lymphoma progression, but not the initiation of the disease.13 During physiological hematopoiesis, BCL11B expression in T-cell precursors is maximal during the onset of the DN2 phase, then maintained throughout the subsequent maturation stages. In human T-ALL cell lines, BCL11B loss of function led to apoptosis.14 On the contrary, BCL11B overexpression has been reported to convey a chemo-protective effect. Interestingly, recent studies reported on the role of BCL11B in lineage ambiguous stem cell leukemia, T/M MPAL and ETP-ALL. Several mechanisms were described. The translocation t(2;14)(q22;q32) yields an inframe ZEB2-BCL11B fusion product that leads to the misexpression of BCL11B in early progenitor cells where the BCL11B enhancer is not normally active. Another re-

arrangement identified a transcriptional regulatory sequence hijacked by the BCL11B gene itself. All these rearrangements result in high expression of BCL11B.15–17 Altogether, these data supported multiple roles for BCL11B in the pathogenesis of acute leukemia according to maturation arrest: as a tumor suppressor in “typical” T-lineage ALL with loss of function mutation or deletion, or as a stage-specific oncogene in hematopoietic stem or early progenitors by existing or de novo super-enhancers maximally active in hematopoietic stem cell progenitors.

Authors Marie Emilie Dourthe,1,2,3 Guillaume P. Andrieu,1 Amandine Potier,1,4,5 Estelle Balducci,1 Julie Guerder,1 Mathieu Simonin,1,4 Lucien Courtois,1 Arnaud Petit,4 Elizabeth Macintyre,1 Nicolas Boissel,6,7 André Baruchel3,7 and Vahid Asnafi1 Université Paris Cité, Institut Necker-Enfants Malades (INEM),

Institut National de la Santé et de la Recherche Médicale, INSERM U1151; 2Laboratory of Onco-Hematology, Assistance PubliqueHôpitaux de Paris, Hôpital Necker Enfants-Malades; 3Université de

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LETTER TO THE EDITOR Paris Cité, Department of Pediatric Hematology and Immunology,

collected and assembled data. MED and GA performed statistical

Robert Debré University Hospital (AP-HP); 4Department of Pediatric

analysis. MED, GA, AP, JG, EB and VA analyzed and interpreted

Hematology and Oncology, Assistance Publique-Hôpitaux de Paris

data. MED, GA and VA wrote the manuscript. All authors approved

(AP-HP), GH HUEP, Armand Trousseau Hospital; Sorbonne

the manuscript.

Universités, UPMC Université de Paris 06, UMRS 938, CDR SaintAntoine, GRC n°07, GRC MyPAC; 6AP-HP, Hôpital Saint Louis, Unité

Acknowledgments

d’Hématologie Adolescents et Jeunes Adultes and Université Paris

The authors would like to thank all participants in the GRAALL-2003

Cité, Institut de Recherche Saint-Louis, EA-3518, Paris, France

and GRAALL-2005 study groups, the SFCE and the investigators of

the 16 SFCE centers involved in collection and provision of data and Correspondence:

patient samples, and V. Lheritier for collection of clinical data.

V. ASNAFI - vahid.asnafi@aphp.fr Funding https://doi.org/10.3324/haematol.2022.282605

The GRAALL was supported by grants P0200701 and P030425/AOM03081 from the Programme Hospitalier de Recherche

Received: December 20, 2022.

Clinique, Ministère de l’Emploi et de la Solidarité, France and the

Accepted: February 28, 2023.

Swiss Federal Government in Switzerland. Samples were collected

Early view: March 9, 2023.

and processed by the AP-HP “Direction de Recherche Clinique” Tumor Bank at Necker-Enfants Malades. MED was supported by

“CARPEM”. MS was supported by « Action Leucémie » and « Soutien

Published under a CC BY-NC license

pour la formation à la recherche translationnelle en cancérologie dans le cadre du Plan cancer 2009-2013 ». LC was supported by

Disclosures

“Fondation pour la Recherche Médicale”. GA was supported by

No conflicts of interest to disclose.

“Fondation de France”. This work was supported by grants to Necker laboratory from the “Association Laurette Fugain”, Institut National

Contributions

du Cancer PRT-K 18-071 and PLBIO2021-097-PL

MED, GA and VA conceived the study and oversaw the project. MED, MS, AP, NB, AB provided study materials or patients. MED

Data-sharing statement

and GA performed molecular analyses. MED, GA, AP, JG, EB and LC

No data will be shared.

References 1. Ferrando AA, Neuberg DS, Staunton J, et al. Gene expression signatures define novel oncogenic pathways in T cell acute lymphoblastic leukemia. Cancer Cell. 2002;1(1):75-87. 2. Belver L, Ferrando A. The genetics and mechanisms of T cell acute lymphoblastic leukaemia. Nat Rev Cancer. 2016;16(8):494-507. 3. Pui C-H, Yang JJ, Hunger SP, et al. Childhood acute lymphoblastic leukemia: progress through collaboration. J Clin Oncol. 2015;33(27):2938-2948. 4. Huguet F, Leguay T, Raffoux E, et al. Pediatric-inspired therapy in adults with Philadelphia chromosome–negative acute lymphoblastic leukemia: the GRAALL-2003 study. J Clin Oncol. 2009;27(6):911-918. 5. Rothenberg EV. Transcriptional drivers of the T-cell lineage program. Curr Opin Immunol. 2012;24(2):132-138. 6. Bernard OA, Busson-LeConiat M, Ballerini P, et al. A new recurrent and specific cryptic translocation, t(5;14)(q35;q32), is associated with expression of the Hox11L2 gene in T acute lymphoblastic leukemia. Leukemia. 2001;15(10):1495-504. 7. Gutierrez A, Kentsis A, Sanda T, et al. The BCL11B tumor suppressor is mutated across the major molecular subtypes of T-cell acute lymphoblastic leukemia. Blood. 2011;118(15):4169-4173. 8. Van Vlierberghe P, Ambesi-Impiombato A, De Keersmaecker K, et al. Prognostic relevance of integrated genetic profiling in adult Tcell acute lymphoblastic leukemia. Blood. 2013;122(1):74-82. 9. De Keersmaecker K, Real PJ, Gatta GD, et al. The TLX1 oncogene drives aneuploidy in T cell transformation. Nat Med. 2010;16(11):1321-1327.

10. Ballerini P, Blaise A, Busson-Le Coniat M, et al. HOX11L2 expression defines a clinical subtype of pediatric T-ALL associated with poor prognosis. Blood. 2002;100(3):991-997. 11. Bergeron J, Clappier E, Radford I, et al. Prognostic and oncogenic relevance of TLX1/HOX11 expression level in T-ALLs. Blood. 2007;110(7):2324-2330. 12. Attarbaschi A, Pisecker M, Inthal A, et al. Prognostic relevance of TLX3 (HOX11L2) expression in childhood T-cell acute lymphoblastic leukaemia treated with Berlin-Frankfurt-Münster (BFM) protocols containing early and late re-intensification elements. Br J Haematol. 2010;148(2):293-300. 13. Pinkney KA, Jiang W, Lee BJ, et al. Haploinsufficiency of Bcl11b suppresses the progression of ATM-deficient T cell lymphomas. J Hematol Oncol. 2015;8:94. 14. Karanam NK, Grabarczyk P, Hammer E, et al. Proteome analysis reveals new mechanisms of Bcl11b-loss driven apoptosis. J Proteome Res. 2010;9(8):3799-3811. 15. Montefiori LE, Bendig S, Gu Z, et al. Enhancer hijacking drives oncogenic BCL11B expression in lineage ambiguous stem cell leukemia. Cancer Discov. 2021;11(11):2846-2867. 16. Montefiori LE, Mullighan CG. Redefining the biological basis of lineage-ambiguous leukemia through genomics: BCL11B deregulation in acute leukemias of ambiguous lineage. Best Pract Res Clin Haematol. 2021;34(4):101329. 17. Di Giacomo D, La Starza R, Gorello P, et al. 14q32 rearrangements deregulating BCL11B mark a distinct subgroup of T and myeloid immature acute leukemia. Blood. 2021;138(9):773-784.

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LETTER TO THE EDITOR

Outcome of patients with acute myeloid leukemia following failure of frontline venetoclax plus hypomethylating agent therapy Venetoclax (Ven) in combination with hypomethylating agents (HMA) is Food and Drug Administration-approved for elderly/unfit acute myeloid leukemia (AML) patients. In the phase III VIALE-A study, complete remission (CR) with or without count recovery (CRi) was achieved in 66.4% of previously untreated patients with AML receiving Ven and azacitidine. CR/CRi was superior in the presence of IDH1/2, NPM1, FLT3, and TP53 mutations in the Ven and azacitidine arm compared to treatment with azacitidine alone.1 However, disease progression or relapse was documented in 42% of patients on Ven and azacitidine therapy.1 Similarly, in a separate study of 95 newly diagnosed AML patients treated on frontline Ven+HMA clinical trials, 41 (43%) of patients experienced relapsed or refractory disease; median overall survival after Ven+HMA failure was considerably inferior at 2.4 months, particularly in patients that did not receive salvage therapy (1.3 months).2 On the other hand, outcomes of AML patients following failure of upfront Ven+HMA therapy outside the context of clinical trials have not been well-studied. Accordingly, in the current study, our primary objective was to describe the clinical outcomes of patients with AML following failure of frontline Ven+HMA therapy in routine clinical practice and identify clinical and molecular predictors of survival after Ven+HMA failure. Patients with newly diagnosed AML treated with frontline Ven+HMA outside clinical trials at the Mayo Clinic between 2018 and 2020 were retrospectively recruited after Institutional Review Board approval. Follow-up was updated in November 2022. All patients received at least one cycle of either azacitidine 75 mg/m2 days 1-7 or decitabine 20 mg/m2 days 1-5 with Ven dose-adjusted based on azole antifungal prophylaxis.3 Bone marrow biopsy was obtained after either cycle 1 or 2 based on treating physician discretion with response assessed according to the 2017 European Leukemia Net (ELN) criteria.4 Treatment failure was defined as inability to achieve CR/CRi (refractory) or loss of CR/CRi (relapsed). Cytogenetic and molecular studies were performed at the time of AML diagnosis by conventional karyotype, and next-generation sequencing (42-gene panel), respectively in all patients, while a subset of patients underwent molecular testing at the time of relapsed/refractory disease. Survival was calculated from the time of treatment initiation and from onset of relapsed or refractory disease to last follow-up or death and survival curves prepared by the Kaplan-Meier method and

compared by the log-rank test. Cox proportional hazard model was used for multivariable analysis. JMP Pro 16.0.0 software package, SAS Institute, Cary, NC was used for statistical analysis. Outcomes from initial treatment with venetoclax in combination with hypomethylating agents Seventy-one of 103 (69%) patients with treatment-naïve AML (median age 74 years; range, 37-91; 70% males; 61% de novo, 23% secondary, 17% therapy-related) were either refractory (n=43, 61%) or relapsed (n=28, 39%) following Ven+HMA therapy. Initial treatment consisted of decitabine in 45 of 71 (63%) patients and the remainder received azacitidine with median Ven dose of 200 mg (range, 50-400 mg) for a median of three cycles (range, 1-16 cycles). In addition, 59 (83%) of patients received concomitant azole antifungal prophylaxis. Relapsed and refractory patients received a median of six cycles (range, 1-16 cycles) and two cycles (range, 1-19 cycles) of Ven+HMA, respectively. Median duration of CR (n=15) or CRi (n=13) was 5 months (range, 1-22 months); moreover, three of eight evaluable patients were measurable residual disease (MRD)-positive by multi-parametric flow cytometry testing. Treatmentrelated toxicities were noted in 51 (72%) of patients which included prolonged cytopenias (n=26), infections (n=20), major hemorrhage (n=3), and tumor lysis syndrome (n=1). Table 1 provides detailed clinical characteristics at time of initiation of Ven+HMA for AML patients that were relapsed or refractory to frontline Ven+HMA. ELN 2017 cytogenetic risk included intermediate (51%, n=36) or adverse (49%, n=35). Mutations involved TP53 in 23 patients (32%), ASXL1 in 12 (17%), IDH1/2 in 11 (16%), FLT3 in ten (14%), N/KRAS in nine (13%) (NRAS, n=5) and NPM1 in seven (10%). All TP53 mutations except one were classified as multi-hit based on the 2022 International Consensus classification5, with median variant allele frequency of 44.5% (range, 6-96%). A comparison of clinical characteristics of refractory versus relapsed patients revealed similarities in age (median age 73 vs. 76 years; P=0.6), secondary/therapy-related AML (44% vs. 32%; P=0.5), ELN adverse cytogenetics (53% vs. 43%; P=0.4), FLT3 (16% vs. 11%; P=0.5), NPM1 (9% vs. 11%; P=0.8), IDH1/2 (14% vs. 18%; P=0.7), and N/KRAS mutations (14% vs. 11%; P=0.7). On the other hand, patients that were refractory to Ven+HMA were more likely to harbor TP53 mutations (40% vs. 21%; P=0.1) while ASXL1 mutations were predominant in relapsed patients (32% vs. 6%; P=0.01). At

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LETTER TO THE EDITOR Table 1. Clinical characteristics at time of initiation of venetoclax plus hypomethylating agents including treatment details for 71 patients with acute myeloid leukemia relapsed or refractory to frontline therapy with venetoclax plus hypomethylating agents. Variables

All patients N=71

Patients with relapsed AML N=28

Patients with refractory AML N=43

Age in years, median (range)

74 (37-91)

76 (37-88)

73 (40-91)

0.6

Male, N (%)

50 (70)

20 (71)

30 (70)

0.9

AML type, N (%) De-novo AML Secondary AML t-AML

43 (61) 16 (23) 12 (17)

19 (68) 6 (21) 3 (11)

24 (55) 10 (23) 9 (21)

Mutations, N (%) FLT3 TP53 TET2 ASXL1 IDH1/2 RUNX1 SRSF2 K/NRAS NPM1

10 (14) 23 (32) 18 (25) 12 (17) 11 (16) 13 (18) 12 (17) 9 (13) 7 (10)

3 (11) 6 (21) 9 (32) 9 (32) 5 (18) 5 (18) 6 (20) 3 (11) 3 (11)

7 (16) 17 (40) 9 (21) 3 (6) 6 (14) 8 (19) 6 (14) 6 (14) 4 (9)

0.5 0.1 0.3 <0.01 0.7 0.9 0.4 0.7 0.8

ELN 2017 cytogenetic risk, N (%) Intermediate Adverse

36 (51) 35 (49)

16 (57) 12 (43)

20 (47) 23 (53)

0.4

Hemoglobin g/dL, median (range)

8.6 (5.1-14)

8.6 (5.2-14)

8.5 (5.1-12.9)

0.4

Leukocyte count x109/L, median (range)

4.4 (0.5-117)

6 (0.7-117)

4.3 (0.5-107)

0.3

Platelet count x109/L, median (range)

60 (7-444)

62 (10-444)

52 (7-239)

0.3

Circulating blasts %, median (range)

14 (0-92)

18 (1-86)

14 (0-92)

0.4

Bone marrow blasts %, median (range)

48 (20-95)

49 (20-95)

43 (20-91)

0.2

26 (36) 45 (63)

7 (25) 21 (75)

19 (44) 24 (55)

0.1 0.1

200 (50-400)

200 (100-400)

200 (50-400)

0.1

Azole antifungal, N (%)

59 (83)

26 (93)

33 (76)

0.1

Number of cycles of Ven+HMA, median (range)

3 (1-16)

6 (1-16)

2 (1-9)

<0.01

HMA therapy, N (%) Azacitidine Decitabine Venetoclax dose in mg, median (range)

0.5

AML: acute myeloid leukemia; t-AML: therapy-related AML: ELN: European Leukemia Net; HMA: hypomethylating agents; mg: miligram; Ven: venetoclax.

a median follow-up of 6 months (range, 1-40 months) from initial treatment with Ven+HMA, 67 (94%) deaths were recorded. Median overall survival was 5.9 months (95% confidence interval [CI]: 2.7-14.1), and was superior in patients that were relapsed versus those refractory to Ven+HMA (median overall survival 11.2 vs. 3.1 months; P<0.01). Online Supplementary Table S1 provides a description of 103 AML patients treated with frontline Ven+HMA including a comparison of clinical and laboratory characteristics of patients with or without relapsed/refractory disease following Ven+HMA. As expected, patients relapsed/refractory to Ven+HMA compared to responders were more likely

to harbor adverse cytogenetics (49% vs. 34%; P=0.15), TP53 (32% vs. 6%; P=0.002) and FLT3-internal tandem duplications (ITD) (14% vs. 0%; P=0.005) mutations. Outcomes from the time of relapsed or refractory disease Molecular testing at the time of relapsed/refractory disease was obtained in a subset of patients (n=18) which revealed persistence of TP53 in all six (100%) TP53-mutated patients and one of two (50%) NRAS-mutated patients that were tested. New emergent clones in the remainder of ten patients tested included mutations in NRAS, CEPBA and

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LETTER TO THE EDITOR BCOR in one patient each. Salvage therapy was pursued in 11 of 71 (15%) patients with gilteritinib (n=6), ivosidenib (n=2), Ven+gilteritinib (n=1), CPX-351 (n=1), and clinical trial (n=1) of which three patients (27%) achieved CR with one patient proceeding to allogeneic stem cell transplant. Notably, none of the patients received standard induction salvage chemotherapy which was not surprising given the median age of study patients was 74 years. On the other hand, in a recent single institution series of younger patients (median age 62 years) and predominantly ELN adverse risk disease (79%), 19 of 208 (9%) went on to receive intensive chemotherapy following failure of Ven+HMA.6 A total of 21 patients had FLT3-ITD (n=10) and IDH1/2 mutations (n=11), of which two patients were co-mutated for FLT3-ITD and IDH. Seven of ten FLT3-ITD-mutated patients received FLT3 inhibitors, three patients did not receive salvage therapy because of mortality from sepsis (n=2) and fungal pneumonia (n=1). On the other hand, two of 11 IDH1/2 mutated patients received IDH inhibitors, the remainder of patients passed away from sepsis (n=3), fungal pneumonia (n=1), intracranial hemorrhage (n=1) or transitioned to hospice due to poor performance status/medical comorbidities (n=4). However, it is to be noted that although survival was not significantly different in patients that received or did not receive targeted therapy (8.6 vs. 3.0 months; P=0.71), the observed difference was likely a reflection of the superior performance status/medical condition of patients that went on to receive salvage therapy. At a median follow up duration of 3 months from the time

of relapsed or refractory disease, median overall survival was similar for relapsed versus refractory patients (median survival 3.1 vs. 2.8 months; P=0.82). In univariate survival analysis, age <60 years (2.5 vs. 3.2 months; P=0.02), presence of TP53 mutation (2.2 vs. 3.6 months; P=0.01), presence of K/NRAS mutation (0.7 vs. 3.2 months; P<0.01), presence of ASXL1 mutation (2.2 vs. 3.2 months; P=0.1), and ELN adverse cytogenetics (2.7 vs. 3.4 months; P=0.04) predicted inferior survival following Ven+HMA failure (Table 2). In subsequent multivariable analysis, presence of TP53 mutation (hazard ratio [HR]=3.1; 95% CI: 1.7-5.7; P<0.01), K/NRAS mutation (HR=3.2; 95% CI: 1.5-6.8; P<0.01) and ASXL1 mutation (HR=2.2; 95% CI: 1.1-4.3; P=0.03) retained significance. Accordingly, TP53/RAS/ASXL1 mutational status predicted survival with median survival of 4.6 months (1-year survival 42%) in the absence of all three mutations (n=33), versus 2.2 months (1-year survival <1%) in the presence of one or more mutations (n=38) (Figure 1A). Similar results were obtained when survival was assessed in 103 AML patients from time of initiation of frontline Ven+HMA with median survival of 16 months versus 5.4 months in the absence versus in the presence of TP53/RAS/ASXL1 mutations (P<0.01) (Figure 1B). The current study confirms the grim prognosis of AML patients that are relapsed/refractory to upfront Ven+HMA therapy. Moreover, salvage therapy was pursued in a minority (15%) of patients and demonstrated limited ability to induce CR with one patient proceeding to allogeneic stem cell transplant. The Mayo clinic cohort comprises AML patients treated outside the context of clinical trials

Table 2. Predictors of inferior survival* following failure of venetoclax plus hypomethylating agents in 71 patients with acute myeloid leukemia relapsed or refractory to frontline therapy with venetoclax plus hypomethylating agents. Overall survival

Overall survival Univariate P

Multivariate P

Age <60 years

0.02

0.13

Sex

0.16

Secondary/therapy-related AML

0.60

Bone marrow blasts %

0.41

Complex or monosomal karyotype

0.11

ELN adverse karyotype

0.04

Presence of NPM1 mutation

0.59

Absence of IDH1/2 mutation

0.10

Presence of RAS mutation

0.03

<0.01

3.4 (1.5-7.5)

Presence of TP53 mutation

<0.01

0.01

2.5 (1.2-5.3)

Presence of ASXL1 mutation

0.10

0.02

2.2 (1.1-4.3)

Absence of FLT3-ITD mutation

0.07

Variables obtained at diagnosis

HR (95% CI)

0.37

*Survival was determined from time of relapsed/refractory disease. HR: hazard ratio; CI: confidence interval; AML: acute myeloid leukemia; ELN: European Leukemia Net; ITD: internal tandem duplication. Haematologica | 108 November 2023

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Figure 1. Overall survival of patients with acute myleoid leukemia (AML) following frontline venetoclax plus hypomethylating agent stratified by presence/absence of TP53, K/NRAS, ASXL1 mutations. (A) Overall survival of 71 patients with acute myleoid leukemia (AML), relapsed/refractory following frontline venetoclax (Ven) plus hypomethylating agents (HMA) stratified by presence/absence of TP53, K/NRAS, ASXL1 mutations. (B) Overall survival of 103 patients with AML treated with frontline Ven plus HMA stratified by presence/absence of TP53, K/NRAS, ASXL1 mutations.

and was enriched with secondary/therapy-related AML (40%) and adverse cytogenetics (49%). The corresponding figures for secondary AML and adverse cytogenetics in the VIALE-A study were lower at 25% and 36%, respectively.1 These differences explain the shorter CR duration and inferior median overall survival observed in our study in comparison to VIALE-A study.1,7 Our findings differ from an MD Anderson study which included 41 clinical trial patients, in which 24 of 41 (59%) of patients with AML that were relapsed/refractory disease following upfront Ven+HMA therapy received salvage therapy.2 However, akin to our study, only 21% of patients responded to salvage therapy and one patient underwent allogeneic stem cell transplant.2 Previous studies on the impact of mutations on response and survival in treatment-naïve and relapsed/refractory AML patients treated with Ven+HMA did not provide detailed information on survival following treatment failure. In our prior report on Ven+HMA-treated newly diagnosed AML patients, the presence of ASXL1 mutations and absence of FLT-ITD and TP53 mutations were associated with superior response; on the other hand, the presence of ASXL1 mutations, adverse karyotype and absence of CR/CRi predicted inferior survival.7 In contrast, in clinical trial patients with AML receiving frontline Ven+HMA or low-dose cytarabine, durable remissions and prolonged survival were reported with NPM1 and IDH2 mutations while TP53 and FLT3-ITD mutations were associated with adaptive resistance.8 Similarly, in relapsed/refractory AML patients treated with Ven combination therapy, responses were superior with NPM1 mutation while survival was shortened with TP53, K/NRAS and SF3B1

mutations.9 In addition, in a Mayo Clinic study of relapsed/refractory AML patients treated with Ven+HMA, the presence of ASXL1 mutation and absence of adverse karyotype predicted superior response, while survival was negatively impacted by the presence of TP53 mutations and absence of IDH1/2 mutations.10 In an exploratory analysis of AML patients treated on Ven+azacitidine clinical trials, stratified according to ELN 2017 risk, adverse risk patients with TP53 mutation had distinctly poor outcome with median overall survival of 5.42 months.11 The current study unveils the prognostic impact of TP53, RAS and ASXL1 mutations on survival in the setting of Ven+HMA failure in treatment-naïve patients with AML. Our observations provide a practically relevant survival prediction model based on the presence versus absence of TP53, RAS and ASXL1 mutations (1-year survival <1% vs. 42% in their presence vs. absence) which should be incorporated in patient counseling. However, whether these findings are specific to Ven+HMA therapy remains to be determined. Taken together, the current study underscores the prognostic relevance of TP53, RAS and ASXL1 mutations in treatment-naïve AML patients following failure of frontline Ven+HMA therapy.

Authors Naseema Gangat,* Rimal Ilyas,* Isla M. Johnson, Kristen McCullough, Aref Al-Kali, Hassan B. Alkhateeb, Kebede H. Begna, Abhishek Mangaonkar, Mark R Litzow, William Hogan, Mithun Shah, Mrinal M.

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Patnaik, Animesh Pardanani and Ayalew Tefferi

Published under a CC BY-NC license Division of Hematology, Mayo Clinic, Rochester, MN, USA

Disclosures MRL has received research funding from AbbVie. All other authors

*NG and RI contributed equally as first authors.

have no conflicts of interest to disclose.

Correspondence:

Contributions

N. GANGAT - gangat.naseema@mayo.edu.

NG, RI and AT designed the study, collected data, performed analysis and co-wrote the paper. IMJ, KM, AA, HA, KHB, AM, MRL, WH, MS,

https://doi.org/10.3324/haematol.2022.282677

MMP and AP contributed patients. All authors reviewed and approved the final draft of the paper.

Received: December 31, 2022. Accepted: February 22, 2023.

Data-sharing statement

Early view: March 2, 2023.

For original data please contact the corresponding author.

References 1. DiNardo CD, Jonas BA, Pullarkat V, et al. Azacitidine and venetoclax in previously untreated acute myeloid leukemia. N Engl J Med. 2020;383(7):617-629. 2. Maiti A, Rausch CR, Cortes JE, et al. Outcomes of relapsed or refractory acute myeloid leukemia after frontline hypomethylating agent and venetoclax regimens. Haematologica. 2021;106(3):894-898. 3. Agarwal SK, DiNardo CD, Potluri J, et al. Management of venetoclax-posaconazole interaction in acute myeloid leukemia patients: evaluation of dose adjustments. Clin Ther. 2017;39(2):359-367. 4. Dohner H, Estey E, Grimwade D, et al. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood. 2017;129(4):424-447. 5. Arber DA, Hasserjian RP, Orazi A, et al. Classification of myeloid neoplasms/acute leukemia: Global perspectives and the international consensus classification approach. Am J Hematol. 2022;97(5):514-518. 6. McMahon CM, Gil K, Amaya ML, et al. Response to intensive induction chemotherapy after failure of frontline azacitidine+venetoclax in acute myeloid leukemia. Blood. 2022;140(Suppl 1):S6185-6186.

7. Gangat N, Johnson I, McCullough K, et al. Molecular predictors of response to venetoclax plus hypomethylating agent in treatment-naïve acute myeloid leukemia. Haematologica. 2022;107(10):2501-2505. 8. DiNardo CD, Tiong IS, Quaglieri A, et al. Molecular patterns of response and treatment failure after frontline venetoclax combinations in older patients with AML. Blood. 2020;135(11):791-803. 9. Stahl M, Menghrajani K, Derkach A, et al. Clinical and molecular predictors of response and survival following venetoclax therapy in relapsed/refractory AML. Blood Adv. 2021;5(5):1552-1564. 10. McKerrow Johnson I, Ilyas R, McCullough K, et al. Molecular predictors of response and survival in patients with relapsed/refractory acute myeloid leukemia following venetoclax plus hypomethylating agent therapy. Blood. 2022;140(Suppl 1):S3233-3234. 11. Döhner H, Pratz KW, DiNardo CD, et al. ELN risk stratification is not predictive of outcomes for treatment-naïve patients with acute myeloid leukemia treated with venetoclax and azacitidine. Blood. 2022;140(Suppl 1):S1441-1444.

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B-lineage acute lymphoblastic leukemia causes cellautonomous defects in long-term hematopoietic stem cell function Many of the clinical manifestations of leukemia, including infections, anemia and hemorrhage, reflect a progressive disruption of normal blood cell development. While chronic lymphoid leukemia impairs the function of hematopoietic stem cells (HSC),1 acute myeloid leukemia2 and T-lineage acute lymphoblastic leukemia (T-ALL)3 primarily target lineage-restricted progenitor compartments. Data obtained from a xenograft model of B-lineage acute lymphoblastic leukemia (B-ALL) suggested that leukemia expansion results in a reduced expression of the chemokine (C-X-C motif) ligand 12 (CXCL12), causing defective HSC homing and reduction of CD34+ cell populations in the bone marrow (BM).4 However, single-cell RNA sequencing from a mouse B-ALL model indicated a substantial presence of cells with the RNA expression pattern of HSC in the BM even at advanced stages of leukemia.5 Thus, the impact of B-ALL on HSC function and residency in the BM remains unclear. We have developed a murine B-ALL model based on transplantation of primary tumor cells from mice carrying combined heterozygote mutations in the Pax5 and Ebf1 genes.6 Pax5 and Ebf1 encode two transcription factors frequently targeted in human B-ALL and a significant proportion of the trans-heterozygote mice develop monoclonal or oligoclonal B-ALL before 40 weeks of age.6 To determine the impact of B-ALL progression on hematopoiesis, we transplanted 150,000 primary mouse tumor cells from the lymph node of a leukemic Cd45.2+/+Ebf1+/–Pax5+/– mouse to wildtype CD45.1 mice by tail vein injection (Figure 1A). The transplants were performed without pre-conditioning allowing us to determine the impact of B-ALL on steadystate hematopoiesis (Figure 1B). Disease was manifested by palpable accessory axillary or subiliac lymph nodes 3-4 weeks after transplantation. At this time the tumor burden in the BM of the transplanted mice was over 90% (Figure 1C) and the absolute numbers of CD45.1 cells in the BM reduced (Online Supplementary Figure S1A). To determine the composition of the remaining BM population, the lineage negative (Lin–)CD45.1+KIT+ host populations were divided into lineage-restricted Lin–KIT+SCA1–, (LK) progenitors and Lin–KIT+SCA1+ (LSK) multipotent progenitors including HSC (Figure 1D). Sub-fractionation of the LK compartment7 (Figure 1D) revealed 10- to 100-fold reductions in both relative and absolute numbers of myeloid lineage-restricted progenitors in tumor transplanted mice as compared to the numbers in sham transplanted control mice (Figure 1E).

This included the pre-granulocyte/monocytes, granulocyte/monocyte progenitors, pre-megakaryocyte/erythrocytes, megakaryocyte progenitors, pre-colony-forming unit erythrocytes and colony-forming unit erythrocytes (Figure 1E). The multipotent LSK population was fractionated into CD150– short-term and CD150+ long-term HSC8 (Figure 1D, E). The frequency as well as absolute numbers of LSK were reduced in leukemic mice (Figure 1E). However, even though the absolute numbers of CD150+ HSC were reduced 1.6fold, the relative frequency of the most primitive progenitors was increased. Transplantation of two other independently generated pro-B-ALL tumors generated similar results as we detected reduced frequency of lineage restricted progenitors but no reduced frequency of CD150+LSK in mice transplanted with tumor (Online Supplementary Figure S1B-D). To further analyze the cellular composition of the leukemic BM we performed single-cell RNA-sequencing analysis of Lin–KIT+CD45.1+ cells in control and leukemic mice (GSE207819). The data were processed using Seurat whereby the control and leukemic samples were integrated using Harmony prior to clustering. The cells were assigned an identity using a previously generated data set from hematopoietic progenitors9 (Online Supplementary Figure S2A, B) employing SingleR. This revealed an 18-fold enrichment of HSC and a 5-fold reduction in megakaryocyte/erythroid progenitors in the BM from tumor-exposed mice as compared to the numbers in control mice (Online Supplementary Figure S2C). Even though the single-cell RNA-sequencing analysis did not quite recapitulate the dramatic loss of linage-restricted progenitors detected by fluorescence activated cell sorting analysis, the data support the idea that the HSC compartment is relatively well conserved while the frequencies of lineage-restricted progenitors are reduced in B-ALL. To determine the status of the progenitor compartments in B-ALL patients, we analyzed flow cytometry data from the BM of 19 patients diagnosed with B-ALL (median age, 6.5; range, 0-59 years).10 Cells were gated on high expression of CD34, a marker for early progenitor cells,11 and lack of CD19, a defining B-ALL marker (Figure 2A). The CD34+ population was gated negative for CD20 and CD66 (Online Supplementary Figure S3) and was highly heterogeneous for expression of CD38 (Figure 2A). This allowed us to investigate leukemia-associated changes in the ratios between CD38high cells, reported to be largely lineage-re-

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Figure 1. Leukemia affects the formation of lineage-restricted progenitor cells rather than the phenotypic stem cell compartment. (A) Schematic model of the leukemic cell transplants in which either phosphate-buffered saline (PBS) or 150,000 CD45.2+ leukemic lymph node cells were transplanted into non-irradiated CD45.1 recipients to determine the impact of leukemia on recipient hematopoietic progenitors. (B) Fluorescence activated cell sorting (FACS) overlay plot of the distribution of cells with expression of CD19 and CD45.2 in CD45.1 mice injected with tumor (TM, blue) or PBS (red). (C) Percentage tumor engraftment (CD45.2+CD19+) in the bone marrow of either PBS (PBS group) or leukemic lymph node (tumor, TM group)-injected CD45.1 mice 22 days after transplantation. ****P<0.0001 (Student t test), from a total of eight PBS and nine tumor (TM) samples per condition obtained from two experiments. (D) FACS profiles of whole bone marrow displaying CD45.1+ lineage-negative (CD11b/Mac1–Gr1– TER119–CD3–CD11c–NK1.1–CD19–)SCA1+KIT+ (LSK) cells stained with antibodies against CD16/32, CD105, CD150 and CD41 to identify progenitor populations. Propidium iodide was used as a marker of viability and gates for each indicated cell population were set according to fluorescence minus one (FMO) controls. (E) Percent of bone marrow progenitors and numbers of progenitors in the bone marrow of PBS- or TM-injected non-irradiated CD45.1 recipients. **P<0.01, ***P<0.001, ****P<0.0001 (Student t test), from a total of eight PBS and nine tumor samples per condition from two experiments. B-ALL: B-lineage acute lymphoblastic leukemia; BM: bone marrow; MkP: megakaryocyte progenitors; GMP: granulocyte/monocyte progenitors; CFU-E: colony-forming unit erythrocytes; pCFU-E: pre-colony-forming unit erythrocytes; pGM: pre-granulocyte/monocytes; pMeg-E: pre-megakaryocyte/erythrocytes.

stricted progenitors, and CD38low/– progenitors, enriched for multipotent HSC.11 While the relative frequency of CD34+ cells was reduced in the patients’ BM as compared to control samples (Figure 2B), the frequency of CD34+CD38low/– cells was not significantly different. Re-analysis of singlecell RNA-sequencing data from leukemia patients12 using

SingleR as above provided additional support for the fact that the frequency of HSC was not reduced in leukemia patients (Figure 2C). Analyzing BM samples from B-ALL patients at diagnosis and at days 15, 29 and 78-79 after initiation of treatment, according to the NOPHO2008 protocol,13 revealed a rapid reduction in the frequency of CD19+

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LETTER TO THE EDITOR cells (Figure 2D), and increased abundance of CD34+CD38+ lineage-restricted progenitors (Figure 2E). The frequency of CD34+CD38–/low cells did not change to a significant degree throughout the treatment (Figure 2F). These data reveal that the earliest progenitor compartments are well preserved even in advanced stages of disease and the rapid

recovery after initiation of treatment suggests that they retain a substantial functional capacity for blood cell production. To determine the functional capacity of leukemia-exposed HSC, we sorted CD150+LSK from leukemic and control CD45.1 mice and transplanted 250 cells into lethally irradi-

E F

Figure 2. Leukemia patients retain CD34+CD38– early progenitors while the frequency of CD34+CD38+ lineage-restricted progenitors is reduced. (A) Representative flow cytometry profiles. (B) Frequencies of bone marrow (BM) progenitors of total cells in diagnostics BM samples (PBM) from patients with B-lineage acute lymphoblastic leukemia (B-ALL) or control BM (CBM). ****P<0.0001 (Student t test), from a total of seven controls and 19 PBM. (C) Diagrams displaying a re-analysis of the human BALL dataset, GSE130116. Scanpy v1.8.2 was used to merge mtx files from GSE130116 into a single anndata object. The object was read into R using zellkonverter v1.4.0 readH5AD function. Scuttle version 1.4.0 was used to normalize the data, and SingleR version 1.8.1 was used to predict cell types using the HumanPrimaryCellAtlasData as the reference. The percentages of BM progenitor populations were calculated after cells assigned to B-cell classes were removed. The proportions of assigned hematopoietic stem cells (HSC), granulocyte/monocyte progenitors (GMP) and CD16– monocytes collected from a single-cell RNA dataset of BALL patients and controls are shown as fractions of non-B lineage BM cells. **P<0.01 (Student t test), from a total of four healthy controls and seven diagnostic B-ALL samples (GSE130116). (D-F) Frequencies of CD19+ B cells (D), CD34+CD38+ BM multipotent progenitors (E) and CD34+CD38–/low HSC and early progenitors (F) in B-ALL diagnostic BM samples (PBM) or control BM (CBM) at diagnosis (day 0) and throughout the course of treatment. **P<0.01, ****P<0.0001 (Student t test), from a total of seven CBM and 19 patients at day 0, 16 patients at day 15, 18 patients at day 29 and 14 patients at day 78-79. All patients with a verified BALL diagnosis and treatment response were included in the analysis. Haematologica | 108 November 2023

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LETTER TO THE EDITOR ated (9 Gy) CD45.2 mice together with 200,000 CD45.2 BM support cells (Figure 3A). Analysis of peripheral blood 4 and 8 weeks after transplantation identified a comparable fraction of CD45.1+ cells generated from both control and

leukemia-exposed HSC (Figure 3B). We were unable to detect any significant lineage bias as both groups of mice presented comparable ratios of CD19+, CD3+ and GR1/MAC1+ cells (Figure 3B). Seventeen weeks after transplantation,

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LETTER TO THE EDITOR Figure 3. Hematopoietic stem cells exposed to leukemia display a cell-autonomous defect in long-term reconstitution activity. (A) A schematic representation of the serial transplantation experiment. CD45.1 mice were injected with phosphate-buffered saline (PBS) or CD45.2+ leukemic lymph node (tumor, TM) cells followed by leukemia development over 19 days. At day 19, bone marrow (BM) was harvested, LSKCD150+ cells were sorted and 250 cells were transplanted into lethally irradiated (9 Gy) CD45.2+ mice together with 200,000 CD45.2+ whole BM support cells. Peripheral blood (PB) and BM were analyzed by fluorescence activated cell sorting (FACS) at the depicted time-points. At 17 weeks 10% of the extracted BM was serially transplanted into lethally irradiated CD45.2 mice followed by analysis of PB and BM as outlined. (B) Graphs display the contribution of transplanted LSKCD150+ cells to total CD45.1 and the contribution of B cells (CD19), T cells (CD3), and myeloid cells (CD19–CD3–GR1/MAC1+) within the CD45.1+ compartment in PB at 4, 8 and 17 weeks after transplantation. Data were collected from a total of ten PBSinjected and ten TM-injected mice per condition from two experiments. (C) Graphs display total CD45.1, and the contribution of B cells (CD19), T cells (CD3), and myeloid cells (CD19–CD3–GR1/MAC1+) within CD45.1+ compartment in PB at 4, 8 and 16 weeks after serial transplantation of BM from LSKCD150+ transplanted mice. Data were collected from a total of ten PBS-injected and ten TM-injected mice per condition from two independent experiments. (D) A graph displaying FACS data of the BM engraftment of CD45.1+ hematopoietic stem cells (HSC) and myelo-erythroid progenitors, as identified in Figure 1) at 16 weeks after serial transplantation of BM from LSKCD150+ transplanted mice as the percentage of total BM. **P<0.01 (Student t test), from a total of ten PBS-injected and nine tumor (TM)-injected mice per condition from two experiments. (E) FACS data displaying the mean florescent intensity of Mitotracker Green (MTG) in HSC populations (primary tumor transplanted cells as in Figure 1) of control (PBS) and leukemia (TM)-transplanted mice. Samples were stained with MTG (30 nM) and 50 nM verapamil for 30 min at 37°C before analysis. (F) Mitotracker CMXRos (MTR) staining of HSC from tumor-transplanted or control mice was performed with 25 nM MTR and incubation with verapamil as above. *P<0.05, ****P<0.0001 (Student t test), from a total of seven or eight PBS-injected mice and nine tumor TM-injected mice from two experiments. B-ALL: B-lineage acute lymphoblastic leukemia; pCFU-E: pre-colony-forming unit erythrocytes; CFU-E: colony-forming unit erythrocytes; pGM: pre-granulocyte/monocytes; pMeg-E: premegakaryocyte/erythrocytes; GMP: granulocyte/monocyte progenitors; MkP: megakaryocyte progenitors.

we noted a tendency towards a reduced level of reconstitution in mice transplanted with B-ALL-exposed HSC (Figure 3B). To determine whether the phenotypic stem cells retained long-term reconstitution potential, we performed serial transplantation of total BM from the mice reconstituted with control or leukemia-exposed HSC into lethally irradiated CD45.2 mice and followed the generation of CD45.1+ cells (Figure 3C). Even though the lineage distribution was comparable between the two groups of serially transplanted mice, the reconstitution level was significantly reduced, already 4 weeks after transplantation, in mice that obtained BM containing tumor-exposed hematopoietic progenitors (Figure 3C). This effect became even more prominent at 16 weeks after the secondary transplantation and upon analysis of CD45.1+ progenitors in the BM we detected a relative reduction in frequency of all progenitor populations, including the CD150+LSK compartment, in mice transplanted with BM containing leukemia-exposed HSC (Figure 3D). Hence, even though exposure to leukemia does not have a major impact on the presence of HSC in the BM, the remaining stem cells display a defective ability for long-term reconstitution. To identify B-ALL-induced changes in gene expression patterns, we compared RNA levels in cells defined as part of a given population in the SingleR analysis (Online Supplementary Figure S2) in mice injected with phosphate-buffered saline or leukemia (Figure 1A). These changes were mined for significantly enriched gene ontology terms using ToppCluster (https://toppcluster.cchmc.org/) and displayed in a heatmap. A major part of the gene ontology terms were related to mitochondrial function and electron transport (Online Supplementary Figure S2D, E). To determine the total mitochondria content in progenitor cells we stained HSC from control and leukemic mice (Figure 1A) with Mitotracker green. While CD150+LSK contained a slightly higher mitochondria mass in

leukemic as compared to control mice, no such difference was found in the CD150–LSK population (Figure 3E). To compare mitochondrial membrane potential in control and leukemia exposed cells, we stained the LSK cells with Mitrotracker CMXRos (MTR). The retention of MTR was reduced in both the CD150+ and CD150– cells from the leukemic BM as compared to control cells (Figure 3F) indicating that the B-ALL-exposed cells harbor a deficiency in mitochondria function. The impact of leukemia on normal blood cell development is reported to involve displacement as well as changes in the microenvironment causing disruptions in stem and progenitor cell function.1-4 Our data show that even though the phenotypic HSC are well preserved in the BM in B-ALL, the cells display a defect limiting their ability for long-term reconstitution. As we were unable to detect pathological expansion of CD45.2+CD19+ cells or symptoms of leukemia in the mice transplanted with stem cells, we believe that the functional defect is HSC autonomous and not a consequence of contaminating leukemia cells. Furthermore, in contrast to our observation in leukemic mice (Figure 1E), the stem cell-transplanted animals show a clear reduction in the frequency of HSC (Figure 3D). The functional impairment was associated with defective mitochondria function in a manner resembling that observed in aged HSC14 likely arising from cellular stress in the leukemic BM. Even if such a defect would not have an impact on the initial recovery of blood cell production after treatment of a leukemia patient, it could potentially contribute to late effects such as clonal hematopoiesis linked to primary hematologic malignancies.15 We believe that our findings open an interesting path for further studies of the underlying causes of secondary cancers and long-term medical conditions in leukemia patients.

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Authors

Contributions CJ, JÅ, AP, JTG, RS, JU, and MS designed, conducted and analyzed the experiments. SS, SL, and CJ performed bio-informatics analysis

Christina T. Jensen, Josefine Åhsberg, Johanna Tingvall-Gustafsson, 1

of data. All authors contributed to writing the manuscript.

Rajesh Somasundaram, Stefan Lang, Jonas Ungerbäck, Anna 2

Acknowledgments

Porwit,3 Shamit Soneji1 and Mikael Sigvardsson1,2

We thank Maria Malmberg, Liselotte Lenner and Linda Bergström, the Division of Molecular Hematology, Lund University, Lund;

CTG and the SCC FACS core facility at Lund University for expert

Department of Clinical and Experimental Medicine, Linköping

technical assistance. We also thank Dr David Bryder for his critical

University, Linköping; and 3Division of Pathology, Lund University,

reading of the manuscript.

Lund, Sweden. Funding Correspondence:

This work was supported by grants from the Swedish Childhood

M. SIGVARDSSON - mikael.sigvardsson@med.lu.se

Cancer Foundation, the Swedish Cancer Society, the Swedish Research Council, including a strategic grant to the Stem Therapy program at Lund University, a donation from Henry Hallberg and Lund

https://doi.org/10.3324/haematol.2022.282430

as well as Linköping Universities. Received: November 21, 2022. Accepted: February 22, 2023.

Data-sharing statement

Early view: March 2, 2023.

The RNA-sequencing data generated are deposited in GEO (GSE207819). Excel files with FACS data from mouse experiments will

be shared upon request. Detailed materials and methods will be

Published under a CC BY-NC license

provided upon request.

Disclosures No conflicts of interest to disclose.

References 1. Kikushige Y, Ishikawa F, Miyamoto T, et al. Self-renewing hematopoietic stem cell is the primary target in pathogenesis of human chronic lymphocytic leukemia. Cancer Cell. 2011;20(2):246-259. 2. Miraki-Moud F, Anjos-Afonso F, Hodby KA, et al. Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation. Proc Natl Acad Sci U S A. 2013;110(33):13576-135681. 3. Hu X, Shen H, Tian C, et al. Kinetics of normal hematopoietic stem and progenitor cells in a Notch1-induced leukemia model. Blood. 2009;114(18):3783-3792. 4. Colmone A, Amorim M, Pontier AL, Wang S, Jablonski E, Sipkins DA. Leukemic cells create bone marrow niches that disrupt the behavior of normal hematopoietic progenitor cells. Science. 2008;322(5909):1861-1865. 5. Anderson D, Skut P, Hughes AM, et al. The bone marrow microenvironment of pre-B acute lymphoblastic leukemia at single-cell resolution. Sci Rep. 2020;10(1):19173. 6. Prasad MA, Ungerback J, Ahsberg J, et al. Ebf1 heterozygosity results in increased DNA damage in pro-B cells and their synergistic transformation by Pax5 haploinsufficiency. Blood. 2015;125(26):4052-4059. 7. Pronk C, Rossi D, Månsson R, et al. Elucidation of the phenotype, functional, and molecular topography of a myeloerythroid progenitor cell hierarchy. Cell Stem Cell. 2007;1(4):428-442.

8. Kiel MJ, Yilmaz OH, Iwashita T, Yilmaz OH, Terhorst C, Morrison SJ. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell. 2005;121(7):1109-1121. 9. Nestorowa S, Hamey FK, Pijuan Sala B, et al. A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation. Blood. 2016;128(8):20-31. 10. Modvig S, Hallbook H, Madsen HO, et al. Value of flow cytometry for MRD-based relapse prediction in B-cell precursor ALL in a multicenter setting. Leukemia. 2021;35(7):1894-1906. 11. Notta F, Zandi S, Takayama N, et al. Distinct routes of lineage development reshape the human blood hierarchy across ontogeny. Science. 2016;351(6269):aab2116. 12. Witkowski MT, Dolgalev I, Evensen NA, et al. Extensive remodeling of the immune microenvironment in B cell acute lymphoblastic leukemia. Cancer Cell. 2020;37(6):867-882. 13. Toft N, Birgens H, Abrahamsson J, et al. Results of NOPHO ALL2008 treatment for patients aged 1-45 years with acute lymphoblastic leukemia. Leukemia. 2018;32(3):606-615. 14. Ho YH, Del Toro R, Rivera-Torres J, et al. Remodeling of bone marrow hematopoietic stem cell niches promotes myeloid cell expansion during premature or physiological aging. Cell Stem Cell. 2019;25(3):407-418. 15. Jaiswal S, Fontanillas P, Flannick J, et al. Age-related clonal hematopoiesis associated with adverse outcomes. N Engl J Med. 2014;371(26):2488-2498.

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Novel FIP1L1::KIT fusion in a myeloid neoplasm with eosinophilia, T-lymphoblastic transformation, and dasatinib response Myeloid/lymphoid neoplasms with eosinophilia (MLN-eo) with tyrosine kinase fusions are a family of hematolymphoid diseases with common shared features of blood eosinophilia and activating fusions involving tyrosine kinase genes.1,2 FIP1L1::PDGFRA fusion is the most common genetic lesion in this class and is caused by a cytogenetically cryptic interstitial deletion at 4q12 that includes the CHIC2 locus and that leads to fusion of the 5 FIP1L1 and 3 PDGFRA genes, with retention of the PDGFRA tyrosine kinase domain.3-6 Constitutive tyrosine kinase activation induces hematopoietic proliferation through stimulation of downstream targets involving the STAT5 pathway. MLNeo with FIP1L1::PDGFRA fusion most commonly manifests as a myeloid neoplasm morphologically resembling chronic eosinophilic leukemia, but blastic manifestations, including T-lymphoblastic leukemia/lymphoma sometimes occur.6,7 KIT, located 3 to PDGFRA at 4q12, is a member of the same class III receptor tyrosine kinase family as PDGFRA, but despite the physical proximity and similar protein structure, FIP1L1::KIT fusions have not been described. We report a novel FIP1L1::KIT fusion in a myeloid neoplasm with eosinophilia that underwent blastic transformation with a T-cell phenotype. A 79-year-old female was seen in consultation for leukocytosis. Laboratory work-up showed a white blood cell count (WBC) of 18.2×109/L, absolute neutrophil count of 12×109/L, absolute eosinophil count of 2.5×109/L, absolute monocyte and absolute lymphocyte count of 1.6×109/L each. She had normal hemoglobin and platelet counts. Bone marrow biopsy showed a hypercellular marrow (90%), left-shifted granulocytic hyperplasia, increased eosinophils, and otherwise morphologically unremarkable hematopoiesis. BCR::ABL1 RT-PCR, JAK2 V617F mutation testing, and karyotypic analyses were normal. Given the monocytosis, a presumptive diagnosis of chronic myelomonocytic leukemia (CMML) was rendered. Since the patient was asymptomatic, she was followed with observation. Three months later, routine testing revealed a WBC of 166×109/L, hemoglobin of 113 g/L and platelet count of 31×109/L. The peripheral blood showed leukocytosis with a myeloid left shift, eosinophilia, monocytosis and basophilia (Figure 1A). The bone marrow was hypercellular with myelomonocytic expansion, dysplasia in the myeloid series and no increase in blasts, consistent with the prior diagnosis of CMML (Figure 1B, C). Mast cells were mildly increased but were phenotypically normal, without aggregates and with only very rare spindled forms. Given

the eosinophilia, fluorescence in situ hybridization (FISH) studies to assess for eosinophilia-associated rearrangements were initiated. Meanwhile, the patient received hydroxyurea and decitabine initially. This was complicated by cytopenia, after which her counts recovered, and her absolute eosinophil count rose. The FISH studies were negative for PDGFRA, PDGFRB, FGFR1, and JAK2 rearrangements; however, the PDGFRA FISH assay demonstrated loss of both the CHIC2 and PDGFRA signals, with retention of the signal 5 of FIP1L1 (Figure 1D). The karyotype was normal, but whole-genome microarray analysis revealed a ~1.274 Mb loss in the 4q12 region with breakpoints within the FIP1L1 and KIT genes, suggesting a FIP1L1::KIT fusion (Figure 1E). These findings were confirmed by RNA-sequencing studies that demonstrated an in-frame, intraexonic fusion joining exon 16 of FIP1L1 and exon 11 of KIT, with retention of the KIT tyrosine kinase domain (NM_030917.4:r.–198_1372::NM_000222.3:r.1679_*2158) (Figure 1F). The fusion breakpoint was confirmed in the specimen’s genomic DNA by Sanger sequencing (Figure 1G). High-throughput DNA-sequencing studies identified a STAG2 nonsense mutation. Based on these findings, the treatment was changed to dasatinib 20 mg daily. The patient’s blood counts normalized within 1 month, consistent with a good response to treatment. After over 1 year of treatment, she suffered from intermittent gastrointestinal symptoms and infections resulting in prolonged interruptions of dasatinib. Consequently, the patient reported progressive night sweats and cervical lymphadenopathy. Her WBC increased to 20×109/L with 17% eosinophils. A repeat bone marrow biopsy showed a hypercellular marrow with myeloid predominant hematopoiesis and mild to moderate myelofibrosis. She could not tolerate higher doses of dasatinib and elected not to switch to an alternative agent. Four months later, the patient presented to the emergency department with shortness of breath. Her WBC was 3.8×109/L (WBC trend in Figure 2A). Imaging showed bilateral moderate pleural effusions, diffuse lymphadenopathy, and moderate splenomegaly. She underwent an axillary lymph node biopsy that showed sheets of blasts expressing the T-cell-associated markers CD3 (cytoplasmic), CD2, CD7, and CD5 (<75% of blasts), with expression of some myeloid- and immaturity-associated markers such as CD34 (partial), CD33, CD117, and TdT (partial). Staining for CD1a was negative (Figure 2B, D). FISH on the lymph node confirmed loss of 4q12. The bone marrow was hypercellular with increased

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Figure 1. Pathologic and molecular characteristics of the patient’s disease at diagnosis. (A) Representative image of the WrightGiemsa-stained blood smear at diagnosis, exhibiting granulocytic left shift, granulocytic dysplasia, monocytosis, and eosinophilia (original magnification ×1,000). (B) Wright-Giemsa-stained bone marrow aspirate demonstrating left-shifted granulocytic matuContinued on following page. Haematologica | 108 November 2023

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CASE REPORT ration, granulocytic dysplasia, and increased monocytic and eosinophilic precursors (original magnification ×1,000). (C) Hematoxylin and eosin-stained bone marrow core biopsy demonstrating marked hypercellularity (original magnification ×40). (D) Fluorescence in situ hybridization pattern showing a loss of both the red (CHIC2) and aqua (3 to PDGFRA) signals with retention of the green (5 to FIP1L1) signal on one chromosome, with a normal green-red-aqua fusion pattern on the other chromosome. (E) Array comparative genomic hybridization assay demonstrating an ~1.274 Mb deletion of chromosome 4q12, with loss of exons 1719 in FIP1L1 and exons 1-10 of KIT. (F) Illustration of RNA-sequencing findings that identified an in-frame, intra-exonic fusion of exon 16 of FIP1L1 with exon 11 of KIT. The green shading in FIP1L1 represents the FIP1 motif, while the blue shading in KIT represents the tyrosine kinase domain. (G) The breakpoints were confirmed in the genomic DNA by amplifying a product using primers targeting intron 15 of FIP1L1 and intron 11 of KIT (gel image). Sanger sequencing of this polymearse chain reaction product confirmed the fusion sequence identified by RNA sequencing (bottom panel). The dashed lines indicate the fusion site.

Figure 2. Response to therapy over time and subsequent relapse. (A) Plot showing the patient’s white blood cell count over time (plotted on a log scale) and response to tyrosine kinase inhibition. The dotted line indicates a value of 11 K/μL. (B) Hematoxylin and eosin-stained section of the patient’s left axillary lymph node, demonstrating sheets of mononuclear cells morphologically consistent with blasts (original magnification ×400). Immunohistochemical stains demonstrated that the blasts were strongly positive for CD3 (C) and partially expressed TdT (D) (original magnifications ×400). (E) Concurrent bone marrow aspirate demonstrated an increased blast population (arrowheads), along with maturing myeloid cells (original magnification ×1,000). (F) Immunohistochemical staining for CD3 in the bone marrow biopsy showed increased, morphologically atypical blastoid cells (original magnification ×1,000). Haematologica | 108 November 2023

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CASE REPORT blasts with a T-lineage phenotype as seen in the lymph node, along with maturing myeloid cells (Figure 2E, F). The findings were diagnostic of a blastic transformation of the myeloid neoplasm, and the blasts exhibited a phenotype compatible with an early T-precursor leukemia. Repeat genetic testing on the bone marrow demonstrated persistent evidence of FIP1L1::KIT fusion, as FISH and microarray studies showed persistent loss of the CHIC2 and PDGFRA loci at 4q12, along with karyotypic evolution: 46,XX,dic(7;20)(p11.2;q13.3),del(12)(p13p11.2)[6]/46XX[14]. DNA sequencing identified a persistent mutation of STAG2, with new mutations in RUNX1, DNMT3A, and WT1. Given the transformation to T-cell lymphoblastic leukemia, the patient received one cycle of cyclophosphamide, vincristine, dexamethasone (mini-HyperCVD), and intrathecal methotrexate, with minimal response. Lymphadenopathy progressed despite two cycles of nelarabine. Consequently, dasatinib was restarted at a dose of 100 mg daily, with significant improvement in cervical and axillary lymphadenopathy. We report a case of a myeloid neoplasm with monocytosis and eosinophilia harboring FIP1L1::KIT fusion that underwent a blastic transformation with an early T-precursor immunophenotype. KIT, a receptor tyrosine kinase that plays a key role in hematopoiesis, is recurrently mutated in myeloid neoplasms such as systemic mastocytosis and acute myeloid leukemia, but only very rare reports have described activating fusions involving KIT in myeloid neoplasms.8 To our knowledge, the FIP1L1::KIT fusion has not been previously described; however, the FIP1L1::KIT fusion structure resembles that of FIP1L1::PDGFRA, with the entire intracellular domain with tyrosine kinase activity present at the C-terminal end of the fusion gene. Imatinib is a tyrosine kinase inhibitor (TKI) that inhibits signaling mediated by KIT and PDGFRA.9,10 Imatinib results in clinical and molecular response when used in patients with MLN-eo with FIP1L1::PDGFRA.3,4,11-13 Dasatinib, a second-generation TKI, inhibits the growth of MLN-eo with FIP1L1::PDGFRA through several mechanisms, including the dephosphorylation of FIP1L1::PDGFRA.14 In in vitro studies, dasatinib had a 67-fold higher potency than imatinib in the inhibition of PDGFRA in animal and human cells.15 Dasatinib resulted in a clinical response in our patient both initially and after blastic transformation. Her initial relapse occurred following multiple dasatinib interruptions, similar to the reported cases of relapse in MLN-eo with FIP1L1::PDGFRA after imatinib discontinuation.13 Both the 5th edition of the World Health Organization Classification of Hematolymphoid Tumors and the International Consensus Classification of Myeloid Neoplasms and Acute Leukemias include a category of MLN-eo with tyrosine kinase gene fusions, and in each classification specific tyrosine kinase genes involved in the fusions in this category are listed.1,2 KIT fusions are not specifically mentioned in

either classification system, though the World Health Oraganization classification does include the possibility of rare fusions involving unspecified kinases as belonging to the category. Our findings, including the patient’s longterm response to single-agent dasatinib, support that KIT fusions are targetable genetic lesions in myeloid/lymphoid neoplasms and support the explicit inclusion of KIT fusions in this group of neoplasms. KIT fusions are presumably rare in myeloid neoplasms. The identification of the KIT fusion in this patient was fortuitous, as it was identified in the microarray and PDGFRAdirected FISH studies since it was caused by an interstitial deletion in a genomic region of known interest. It is likely that FIP1L1::KIT fusions are very rare, since many laboratories assess this region in cases with eosinophilia. Our results, however, provide a rationale to further work up unusual FISH signal patterns in this region, as they may indicate a variant, clinically important fusion. Furthermore, our routine testing would likely not identify other KIT fusion partners, and KIT fusions are not included in many commercially available fusion panels such as anchored multiplexed polyerase chain reaction platforms, presumably since KIT is not currently recognized as a fusion partner in MLN-eo. This case emphasizes the utility and clinical relevance of broad, unbiased genomic testing in myeloid neoplasms to identify unexpected druggable targets.

Authors Aseel Alsouqi,1 Jeffrey Kleinberger,2 Taylor S. Werner,3 Rashid Awan,4 Saurav Chopra,2 Bryan Rea,2 Nidhi Aggarwal,2 Svetlana A. Yatsenko,2 Rafic Farah5 and Nathanael G. Bailey2 Department of Medicine, Division of Hematology and Oncology,

University of Pittsburgh Medical Center, Pittsburgh; 2Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh; School of Pharmacy, University of Pittsburgh, Pittsburgh; 4University

of Pittsburgh Medical Center, Conemaugh Memorial Medical Center, Johnstown and 5University of Pittsburgh Medical Center, Hillman Cancer Center, Pittsburgh, PA, USA Correspondence: N. G. BAILEY - baileyng@upmc.edu https://doi.org/10.3324/haematol.2022.282636 Received: December 22, 2022. Accepted: April 17, 2023. Early view: April 27, 2023. ©2023 Ferrata Storti Foundation Published under a CC BY-NC license

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CASE REPORT Disclosures

Acknowledgments

No conflicts of interest to disclose.

The authors would like to thank Dr. Hagop Kantarijan for his clinical guidance in this case. We would also like to thank our patient and

Contributions

her family for giving us permission to share and describe her case in

JK, SC, BR, NA, SAY, and NGB performed pathologic review, diagnostic

this report.

testing and contributed to manuscript writing. RF and RA performed clinical, diagnostic and response evaluations, and provided patient

Data-sharing statement

care. AA, TSW, NGB, SAY and RF drafted the manuscript. All authors

For additional information please contact the corresponding author.

reviewed and approved the final manuscript.

References 1. Arber DA, Orazi A, Hasserjian RP, et al. International Consensus Classification of Myeloid Neoplasms and Acute Leukemias: integrating morphologic, clinical, and genomic data. Blood. 2022;140(11):1200-1228. 2. Khoury JD, Solary E, Abla O, et al. The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: myeloid and histiocytic/dendritic neoplasms. Leukemia. 2022;36(7):1703-1719. 3. Cools J, DeAngelo DJ, Gotlib J, et al. A tyrosine kinase created by fusion of the PDGFRA and FIP1L1 genes as a therapeutic target of imatinib in idiopathic hypereosinophilic syndrome. N Engl J Med. 2003;348(13):1201-1214. 4. Pardanani A, Brockman SR, Paternoster SF, et al. FIP1L1-PDGFRA fusion: prevalence and clinicopathologic correlates in 89 consecutive patients with moderate to severe eosinophilia. Blood. 2004;104(10):3038-3045. 5. Schwaab J, Umbach R, Metzgeroth G, et al. KIT D816V and JAK2 V617F mutations are seen recurrently in hypereosinophilia of unknown significance. Am J Hematol. 2015;90(9):774-777. 6. Pozdnyakova O, Orazi A, Kelemen K, et al. Myeloid/lymphoid neoplasms associated with eosinophilia and rearrangements of PDGFRA, PDGFRB, or FGFR1 or with PCM1-JAK2. Am J Clin Pathol. 2020;155(2):160-178. 7. Metzgeroth G, Walz C, Score J, et al. Recurrent finding of the FIP1L1-PDGFRA fusion gene in eosinophilia-associated acute myeloid leukemia and lymphoblastic T-cell lymphoma. Leukemia. 2007;21(6):1183-1188. 8. Grand FH, Waghorn K, Ernst T, Ohyashiki K, Cross NC. The

t(4;9)(q11;q33) fuses CEP110 to KIT in a case of acute myeloid leukemia. Leukemia. 2011;25(6):1049-1050. 9. Buchdunger E, Cioffi CL, Law N, et al. Abl protein-tyrosine kinase inhibitor STI571 inhibits in vitro signal transduction mediated by c-kit and platelet-derived growth factor receptors. J Pharmacol Exp Ther. 2000;295(1):139-145. 10. Carroll M, Ohno-Jones S, Tamura S, et al. CGP 57148, a tyrosine kinase inhibitor, inhibits the growth of cells expressing BCRABL, TEL-ABL, and TEL-PDGFR fusion proteins. Blood. 1997;90(12):4947-4952. 11. Klion AD, Noel P, Akin C, et al. Elevated serum tryptase levels identify a subset of patients with a myeloproliferative variant of idiopathic hypereosinophilic syndrome associated with tissue fibrosis, poor prognosis, and imatinib responsiveness. Blood. 2003;101(12):4660-4666. 12. Pardanani A, Ketterling RP, Brockman SR, et al. CHIC2 deletion, a surrogate for FIP1L1-PDGFRA fusion, occurs in systemic mastocytosis associated with eosinophilia and predicts response to imatinib mesylate therapy. Blood. 2003;102(9):3093-3096. 13. Jovanovic JV, Score J, Waghorn K, et al. Low-dose imatinib mesylate leads to rapid induction of major molecular responses and achievement of complete molecular remission in FIP1L1PDGFRA–positive chronic eosinophilic leukemia. Blood. 2007;109(11):4635-4640. 14. Baumgartner C, Gleixner KV, Peter B, et al. Dasatinib inhibits the growth and survival of neoplastic human eosinophils (EOL-1) through targeting of FIP1L1-PDGFRα. Exp Hematol. 2008;36(10):1244-1253.

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Protracted viral infections in patients with multiple myeloma receiving bispecific T-cell engager therapy targeting B-cell maturation antigen Recent expansion in therapeutic landscape in multiple myeloma (MM) has resulted in significant improvement in patient survival. Specifically, chimeric antigen receptor (CAR) T cells and bispecific T-cell engagers (BiTE) targeting B-cell maturation antigen (BCMA) have resulted in unprecedented response rates.1 While infections remain the leading cause of morbidity and mortality in patients with relapsed/refractory multiple myeloma (RRMM),2 the on-target-off-tumor toxicities associated with BCMA-targeting agents lead to prolonged B-cell aplasia, hypogammaglobulinemia, and increase the cumulative risk for infections.3-6 Currently, two CAR T cells and one BiTE product targeting BCMA are approved by the Food and Drug Administration (FDA) for the treatment of RRMM. Patients receiving these agents, either in clinical trials or commercially, need to have received several prior lines of treatment often including autologous hematopoietic cell transplant (autoHCT), monoclonal antibodies, and have prolonged cytopenia. This further intensifies the net state of immunosuppression, superimposed upon the immunoparesis associated with myeloma. Prior studies in BCMA CAR T highlighted an infection rate ranging between 23-63%.3,4,79 A single-center study examined infections up to 1 year post CAR T in 55 patients and showed that 53% of infections were viral, 40% bacterial, and 6% fungal.9 Another single-center study in 104 patients with RRMM and NHL undergoing BCMA and CD19-directed CAR T showed that BCMA CAR T-cell recipients had significantly more viral infections than CD19-directed CAR T recipients.10 While there are evolving data among BCMA CAR T cell recipients, evidence remains limited among BCMA BiTE recipients. In a single-center analysis of MM patients receiving BCMA CAR (n=26) and BiTE (n=36), CAR T recipients had higher baseline absolute lymphocyte counts (ALC) and were less heavily pretreated. The cumulative incidence and burden of infections was higher among BCMA BiTE compared to BCMA CAR T-cell recipients.8 However, bacterial infections were predominant in this small study. A larger pooled analysis of ten clinical trials of MM BiTE in 790 MM patients (73% of patients treated with an agent targeting BCMA) showed grade 2-4 neutropenia in 37% and grade 3-4 infections in 26%. Importantly, non-BCMA targeted BiTE were associated with lower grade 2-4 neutropenia (45.6% vs. 24.4%) and lower grade 3-4 infections (27.5% vs. 16.9%) when compared to BCMA BiTE.11 Since CAR T-cell therapy is currently a one-time infusion,

most patients may still achieve at least partial immune reconstitution with resolution of cytopenia and hypogammaglobulinemia.4 Contrastingly, BiTE therapy is given indefinitely until disease progression or treatment intolerance. This can lead to a double-edged sword effect with BiTE therapy. While staying in remission, patients develop persistent plasma cell suppression, hypogammaglobulinemia, and suffer from significant morbidity due to recurrent infections, hospitalizations, and treatment interruptions. Herein, we present three cases of BCMA BiTE recipients who developed uncommon protracted viral infections (Table 1). Case 1. A 73-year-old white male, with International Staging System (ISS) stage-3 IgA λ MM since March 2018 who had received six prior lines of treatment including autoHCT with melphalan 200 mg/m2, remained in remission with a BCMA BiTE but developed parvovirus B19 infection. Patient's prior anti-myeloma treatment included immunomodulators (IMiD), lenalidomide and pomalidomide, proteasome inhibitors (PI) including carfilzomib, monoclonal antibody (mAb) targeting CD38 (daratumamab) and SLAMF7 (elotuzamab), BCL-2 inhibitor (venetoclax), and most recently a BCMA BiTE on a clinical trial initiated 3 years after the initial diagnosis of MM. The patient developed grade 1 cytokine release syndrome (CRS) with his first cycle of BCMA BiTE which resolved with tocilizumab. Within 3 months of BiTE initiation, the patient developed symptomatic anemia with a drop in hemoglobin (Hb) to 5.9 g/dL. He did not exhibit occult signs of clinically bleeding and physical examination was unremarkable for jaundice, icterus, koilonychia, lymphadenopathy, or hepatosplenomegaly. Hematologic and gastrointestinal investigations were non-revealing except for hemolytic biochemical picture. Infectious disease (ID) work-up revealed parvovirus B19 infection (+ serum qualitative polymerase chain reaction [PCR]). The patient has been treated with monthly intravenous immunoglobulins (IVIG) and his Hb level remained above 10 g/dL consistently. His BCMA BiTE therapy was discontinued after 1.5 years due to recurrent infections which included chronic sinusitis and skin/soft tissue infections with resultant treatment intolerance. The patient continues to remain in clinical and biochemical remission of his myeloma to date, after being off treatment for 3 months. Immune correlates of disease and infection course are shown in Figure 1A.

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CASE SERIES Table 1. Patient, disease, and infection characteristics of bispecific T-cell engagers recipients. Characteristic

Case 1

Case 2

Case 3

Age in years at MM diagnosis

Age in years at BiTE initiation

Age in years at infection diagnosis

Sex

Male

Race

White

Type of MM

IgA λ

IgA κ

Prior lines of therapy

Prior autoHCT

Number of cycles of BiTE

Duration of BiTE therapy in years

1.5

Ongoing

BiTE discontinued in months

Yes (off for 3 )

Yes (off for 17 )

Current disease status

In remission

CRS at initiation of BiTE

Yes (grade 1)

Use of steroids/tocilizumab

Tocilizumab

Parvovirus B19 (chronic)

Parvovirus B19

Norovirus

Parvovirus B19 DNA (qualitative PCR)

Stool norovirus (NAAT)

IgG level at the time of infection (mg/dL)

<40

368

ALC level at the time of infection (x103 cells/uL)

3.59

0.33

0.25

Interval in months between infection onset and BiTE initiation

Monthly IVIG

Monthly IVIG; nitazoxanide; stem cell boost

Resolved

Chronic

Viral infection Diagnostic method

Treatment of infection Outcome (resolution vs. ongoing vs. chronic vs. resistant)

BiTE: bispecific T-cell engagers; MM: multiple myeloma; autoHCT: autologous hematopoietic cell transplant; CRS: cytokine release syndrome; DNA: deoxyribonucleic acid; PCR: polymerase chain reaction; NAAT: nucleic acid amplified test; IgA: immunoglobulin A; ALC: absolute lymphocyte counts; IVIG: intravenous immunoglobulins; NA: not analyzed.

Case 2. A 67-year-old white male was diagnosed with ISS stage 3A κ light chain MM in 2005 and received highdose therapy (thalidomide/dexamethasone) followed by autoHCT. He then developed RRMM and received seven prior lines of treatment including two autoHCT with melphalan 200 mg/m2 in 2005 and 2014. Prior MM treatment included IMiD: thalidomide, lenalidomide and pomalidomide, PI: bortezomib and carfilzomib, mAb elotuzamab and daratumamab. For the RRMM, he started BCMA BiTE in July 2020. In February 2021, the patient presented with a cough and symptomatic anemia (Hb 9.8 g/dL) in the setting of profound hypogammaglobinemia (IgG <40 mg/dL). Physical examination was unremarkable for occult signs of clinical

bleeding. Hematocrit was 23% with a reticulocyte percentage of 0.3%. A thorough ID workup revealed parvovirus B19 infection (serum qualitative PCR). The patient has been treated with monthly IVIG for persistent parvovirus B19. Despite persistent viremia, his Hb remains above 11 g/dL consistently and an IgG level of above 800 mg/dL. The patient has received 42 cycles of BiTE treatment over the last 2.5 years and maintains remission. The disease course is presented in Figure 1B. Case 3. A 71-year-old White male was initially diagnosed with IgA κ MM with complex high-risk cytogenetics in 2008. He developed RRMM and received 10 lines of prior therapy including two autoHCT with melphalan 200

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CASE SERIES mg/m2 in 2009 and 2012. Prior treatment regimens consisted of IMiD: lenalidomide, PI: bortezomib, oprozomib, and carfilzomib, and anti-CD38 mAb (daratumumab). For RRMM, he was enrolled in a clinical trial of BCMA BiTE in April 2020. The patient developed grade 1 CRS during initial infusion which resolved without further complications. Seventeen months after BiTE initiation, he presented with severe diarrhea and abdominal pain. He had up to 25 loose

non-bloody bowel movements daily. Physical exam was unremarkable for jaundice, hepatosplenomegaly, lymphadenopathy, or ascites. At the time of presentation, his IgG level was 368 mg/dL (Figure 1C). ID workup was negative for common enteric pathogen and colonoscopic biopsy pathological stains were negative for cytomegalovirus (CMV), herpes simplex virus (HSV), and adenovirus. Noroviral infection was diagnosed via stool PCR specimen, coupled with radiologic evidence of colitis. The patient was

Figure 1. Immune correlates of the disease course, protracted viral infections, and treatment. (A) shows immune correlates of parvovirus B19 infection with absolute lymphocyte counts (ALC), immunoglobulin G (IgG), and hemoglobin (Hb) levels and the duration of bispecific T-cell engagers (BiTE) therapy; (B) shows the course of parvovirus B19 infection with ALC, IgG, and Hb levels with ongoing BiTE therapy; (C) illustrates the protracted course of norovirus infection, treatment with nitazoxanide and autologous stem cell boost, frequency of diarrhea, and the duration of BiTE therapy. Haematologica | 108 November 2023

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CASE SERIES treated with 14-days of nitazoxanide, with transient improvement in symptoms. Diarrhea worsened significantly after nitazoxanide completion, and he persistently tested positive for norovirus. Monthly IVIG infusions were initiated for chronic hypogammaglobulinemia, but he continued to have diarrhea due to recalcitrant norovirus infection and discontinued BCMA BiTE infusion after 20 cycles (total duration, 1 year). In order to achieve immune reconstitution (including neutropenia), he received a stem cell boost using frozen stem cells collected during a prior remission. Upon last follow up, the patient remains in complete remission after discontinuing BCMA BiTE infusion for nearly 1.5 years. However, he continues to test positive for norovirus, although his diarrhea is slowly improving. BCMA BiTE recipients develop distinctive infections with intracellular pathogens that may have a protracted course with frequent recurrences. BCMA targeting with bispecific T-cell engagement leads to persistent T-cell mediated cytotoxicity against plasma cells and B cells resulting in profound hypogammaglobulinemia.6 Further, usage of immunosuppressive medications/premedications, CRS, impaired immune reconstitution, prolonged cytopenia, B-cell aplasia and potential redirection/activation of regulatory T cells contribute to a heightened infection risk in RRMM patients with pre-existing immune paresis.1,6 While it is difficult to discern the relative contribution of BiTE to the risk for infections as patients with RRMM are heavily pretreated with a profoundly and globally immunosuppressed state, severe and protracted infections could largely be attributed to anti-BCMA agents as these are associated with persistent B-cell and plasma cell suppression. Patients with RRMM receiving BiTE therapy are at a high risk for frequent viral infections/reactivations and transient viremia. These viral infections may include CMV, Epstein-Barr virus (EBV), parvovirus, HSV, BK polyomavirus viremia, and other visceral infections. Further, these patients are at a particularly high risk of chronic infections with SARS-CoV-2 and prolonged viral shedding.12 Additionally, such patients may develop parvovirus-induced red cell aplasia as in our study. Additional infections that should be considered include adenoviral hepatitis, EBV-related lymphomas, progressive multifocal leukoencephalopathy due to John Cunningham virus, and Guillain-Barré syndrome. As evident from the cases, use of prophylactic IVIG should be the standard of care. Comprehensive screening for viruses should be performed prior to initiation of BiTE and treatment postponed until complete eradication of any baseline active viral reactivation/infection. Active monitoring and surveillance for viruses such as CMV, EBV, SARSCoV-2, and other community respiratory viruses should be pursued. A low threshold should be maintained for testing for appropriate pathogens based on apt clinical presentations (parvovirus, norovirus, HHV6B, etc). Pre-emptive

antiviral therapy for CMV and EBV viremia with therapy interruption may be considered. Prophylactic antiviral therapy is essential. Since antibody synthesis is paralyzed in patients with RRMM, available strategies to provide passive immunity against ongoing viral infections such as against SARS-CoV-2 variants of concern should be considered and asymptomatic infections treated early to prevent progression. Evolving evidence further supports the use of additional (booster) vaccine doses.13,14 Given significant risk of infections with prolonged use, examining BiTE therapies for RRMM on protocols with fixed duration or intermittent dosing are urgently needed. Meanwhile, comprehensive infection prevention strategies are urgently needed, particularly in patients with durable remission on ongoing therapy.15

Authors Breanna Palmen,1 Parameswaran Hari,2 Anita D’Souza2 and Muhammad Bilal Abid2,3 Department of Medicine, Medical College of Wisconsin; 2Division of

Hematology and Oncology, Department of Medicine, Medical College of Wisconsin and 3Division of Infectious Diseases, Department of Medicine, Medical College of Wisconsin, Milwaukee, WI, USA Correspondence: M.B. ABID - Bilal_abid@hotmail.com https://doi.org/10.3324/haematol.2023.283003 Received: February 22, 2023. Accepted: April 3, 2023. Early view: April 13, 2023. ©2023 Ferrata Storti Foundation Published under a CC BY-NC license Disclosures No conflicts of interest to disclose. Contributions BP and MBA drafted the manuscript. MBA, PH, and AD provided patient care and critically revised the manuscript. All authors were involved in the critical analysis and final version of the manuscript. Acknowledgments The authors gratefully acknowledge patients for their agreement to participate in this study. Data-sharing statement The data that support the findings of this study are available from the corresponding author upon reasonable request.

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9. Kambhampati S, Sheng Y, Huang C-Y, et al. Infectious complications in patients with relapsed refractory multiple myeloma after BCMA CAR T-cell therapy. Blood Adv. 2022;6(7):2045-2054. 10. Kambhampati S, Fakhri B, Sheng Y, et al. Infectious complications of BCMA-targeted and CD19-targeted chimeric antigen receptor T-cell immunotherapy. Blood. 2020;136(Suppl):S4-5. 11. Mazahreh F, Mazahreh L, Schinke C, et al. Risk of infections associated with the use of bispecific antibodies in multiple myeloma: a pooled analysis. Blood. 2022;140(Suppl 1):S4378. 12. Abid MB, Mughal M, Abid MA. Coronavirus disease 2019 (COVID19) and immune-engaging cancer treatment. JAMA Oncol. 2020;6(10):1529-1530. 13. Abid MA, Abid MB. SARS-CoV-2 vaccine response in CAR T-cell therapy recipients: a systematic review and preliminary observations. Hematol Oncol. 2022;40(2):287-291. 14. Abid MB, Rubin M, Ledeboer N, et al. Efficacy of a third SARSCoV-2 mRNA vaccine dose among hematopoietic cell transplantation, CAR T cell, and BiTE recipients. Cancer Cell. 2022;40(4):340-342. 15. Haroon A, Muhsen IN, Abid MB, et al. Infectious complications and preventative strategies following chimeric antigen receptor T-cells (CAR-T cells) therapy for B-cell malignancies. Hematol Oncol Stem Cell Ther. 2022;15(3):153-158.

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