Haematologica, Volume 101, issue 2 by Haematologica

haematologica Journal of the European Hematology Association Published by the Ferrata Storti Foundation

Editor-in-Chief Jan Cools (Leuven)

Deputy Editor Luca Malcovati (Pavia)

Managing Director Antonio Majocchi (Pavia)

Associate Editors Hélène Cavé (Paris), Ross Levine (New York), Claire Harrison (London), Pavan Reddy (Ann Arbor), Andreas Rosenwald (Wuerzburg), Juerg Schwaller (Basel), Monika Engelhardt (Freiburg), Wyndham Wilson (Bethesda), Paul Kyrle (Vienna), Paolo Ghia (Milan), Swee Lay Thein (Bethesda), Pieter Sonneveld (Rotterdam)

Assistant Editors Anne Freckleton (English Editor), Cristiana Pascutto (Statistical Consultant), Rachel Stenner (English Editor), Kate O’Donohoe (English Editor)

Editorial Board Omar I. Abdel-Wahab (New York); Jeremy Abramson (Boston); Paolo Arosio (Brescia); Raphael Bejar (San Diego); Erik Berntorp (Malmö); Dominique Bonnet (London); Jean-Pierre Bourquin (Zurich); Suzanne Cannegieter (Leiden); Francisco Cervantes (Barcelona); Nicholas Chiorazzi (Manhasset); Oliver Cornely (Köln); Michel Delforge (Leuven); Ruud Delwel (Rotterdam); Meletios A. Dimopoulos (Athens); Inderjeet Dokal (London); Hervé Dombret (Paris); Peter Dreger (Hamburg); Martin Dreyling (München); Kieron Dunleavy (Bethesda); Dimitar Efremov (Rome); Sabine Eichinger (Vienna); Jean Feuillard (Limoges); Carlo Gambacorti-Passerini (Monza); Guillermo Garcia Manero (Houston); Christian Geisler (Copenhagen); Piero Giordano (Leiden); Christian Gisselbrecht (Paris); Andreas Greinacher (Greifswals); Hildegard Greinix (Vienna); Paolo Gresele (Perugia); Thomas M. Habermann (Rochester); Claudia Haferlach (München); Oliver Hantschel (Lausanne); Christine Harrison (Southampton); Brian Huntly (Cambridge); Ulrich Jaeger (Vienna); Elaine Jaffe (Bethesda); Arnon Kater (Amsterdam); Gregory Kato (Pittsburg); Christoph Klein (Munich); Steven Knapper (Cardiff); Seiji Kojima (Nagoya); John Koreth (Boston); Robert Kralovics (Vienna); Ralf Küppers (Essen); Ola Landgren (New York); Peter Lenting (Le Kremlin-Bicetre); Per Ljungman (Stockholm); Francesco Lo Coco (Rome); Henk M. Lokhorst (Utrecht); John Mascarenhas (New York); Maria-Victoria Mateos (Salamanca); Simon Mendez-Ferrer (Madrid); Giampaolo Merlini (Pavia); Anna Rita Migliaccio (New York); Mohamad Mohty (Nantes); Martina Muckenthaler (Heidelberg); Ann Mullally (Boston); Stephen Mulligan (Sydney); German Ott (Stuttgart); Jakob Passweg (Basel); Rob Pieters (Rotterdam); Stefano Pileri (Milan); Miguel Piris (Madrid); Andreas Reiter (Mannheim); Jose-Maria Ribera (Barcelona); Stefano Rivella (New York); Francesco Rodeghiero (Vicenza); Richard Rosenquist (Uppsala); Simon Rule (Plymouth); Claudia Scholl (Heidelberg); Martin Schrappe (Kiel); Radek C. Skoda (Basel); Gérard Socié (Paris); Kostas Stamatopoulos (Thessaloniki); David P. Steensma (Rochester); Martin H. Steinberg (Boston); Ali Taher (Beirut); Evangelos Terpos (Athens); Takanori Teshima (Sapporo); Pieter Van Vlierberghe (Gent); Alessandro M. Vannucchi (Firenze); George Vassiliou (Cambridge); Edo Vellenga (Groningen); Umberto Vitolo (Torino); Guenter Weiss (Innsbruck).

Editorial Office Simona Giri (Production & Marketing Manager), Lorella Ripari (Peer Review Manager), Paola Cariati (Senior Graphic Designer), Igor Ebuli Poletti (Senior Graphic Designer), Marta Fossati (Peer Review), Diana Serena Ravera (Peer Review)

Affiliated Scientific Societies SIE (Italian Society of Hematology, www.siematologia.it) SIES (Italian Society of Experimental Hematology, www.siesonline.it)

haematologica Journal of the European Hematology Association Published by the Ferrata Storti Foundation

Information for readers, authors and subscribers 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, research articles, review articles, guideline articles and letters. 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 online (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. Detailed information about subscriptions is available online at www.haematologica.org. Haematologica is an open access journal. Access to the online journal is free. Use of the Haematologica App (available on the App Store and on Google Play) 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 International edition for the year 2015 are as following: Print edition

Institutional Euro 500

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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. Direttore responsabile: Prof. Edoardo Ascari; Autorizzazione del Tribunale di Pavia n. 63 del 5 marzo 1955. Printing: Tipografia PI-ME, via Vigentina 136, Pavia, Italy. Printed in January 2016.

haematologica calendar of events

Journal of the European Hematology Association Published by the Ferrata Storti Foundation

The 9th Annual Congress of the European Association for Haemophilia and Allied Disorders 2016 Chairs: P de Moerloose, C Hermans, J Astermark February 3 – 5, 2016 Malmö, Sweden 6th Congress of Cellular Therapy and Regenerative Medicine Society of Cellular Therapy and Regenerative Medicine Chair: O Ilhan February 27-28, 2016 Mersin, Turkey ESH International Conference on Aging and Hematological Malignancies: Biology and Therapy European School of Haematology (ESH) Chairs: B Löwenberg, L Balducci, P Fenaux, M Hallek March 11-13, 2016 Athens, Greece 42nd EBMT Annual Meeting European Society for Bone and Marrow Transplantation Chairs: MS Sanz, A Urbano, JL Díez April 3-6, 2016 Valencia, Spain

21st Congress of the European Hematology Association European Hematology Association June 9-12, 2016 Copenhagen, Denmark Hematology Tutorial on managing complications in patients with hematologic malignancies in the era of new drugs Chairs: E Parovichnikova, I Poddubnaya, R Foà July 1-3, 2016 Moscow, Russian Federation EHA Scientific Conference on Bleeding Disorders Scientific Program Committee: C Balduini (Chair), A Falanga (Chair), F Rodeghiero, I Pabinger, M Makris September 14-17, 2016 Barcelona, Spain

Calendar of Events updated on January 6, 2016

haematologica Journal of the European Hematology Association Published by the Ferrata Storti Foundation

Table of Contents Volume 101, Issue 2: February 2016 Cover Figure Aspects of hematology research - accompanying the opinion article on page 115 (Image created by www.somersault1824.com)

Editorial 101

Blood disorders stepping into the limelight Ulrich JĂ¤ger, et al.

Review Article 104

The role of targeted treatment in mantle cell lymphoma: is transplant dead or alive? - Leaders in Hematology review series Martin Dreyling, et al.

Opinion Article EHA Roadmap for European Hematology Research: a consensus document

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The European Hematology Association Roadmap for European Hematology Research: a consensus document Andreas Engert, et al.

Articles Blood Transfusion

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Red blood cell alloimmunization is influenced by the delay between Toll-like receptor agonist injection and transfusion Rahma Elayeb, et al.

Coagulation & Iys Disorders

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Clinical, instrumental, serological and histological findings suggest that hemophilia B may be less severe than hemophilia A Daniela Melchiorre, et al.

Non-Hodgkin Lymphoma

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Rationale for optimal obinutuzumab/GA101 dosing regimen in B-cell non-Hodgkin lymphoma Guillaume Cartron, et al.

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The addition of rituximab to fludarabine and cyclophosphamide chemotherapy results in a significant improvement in overall survival in patients with newly diagnosed mantle cell lymphoma: results of a randomized UK National Cancer Research Institute trial Simon Rule, et al.

Stem Cell Transplantation

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Time-dependent effects of clinical predictors in unrelated hematopoietic stem cell transplantation Daniel Fuerst, et al.

Haematologica 2016; vol. 101 no. 2 - February 2016 http://www.haematologica.org/

haematologica Journal of the European Hematology Association Published by the Ferrata Storti Foundation

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Monosomal karyotype as an adverse prognostic factor in patients with acute myeloid leukemia treated with allogeneic hematopoietic stem-cell transplantation in first complete remission: a retrospective survey on behalf of the ALWP of the EBMT Angelique V.M. Brands-Nijenhuis, et al.

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Peripheral blood stem cell graft compared to bone marrow after reduced intensity conditioning regimens for acute leukemia: a report from the ALWP of the EBMT Bipin N. Savani, et al.

Letters to the Editor Letters are available online only at www.haematologica.org/content/101/2.toc

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Elevated transpulmonary gradient and cardiac magnetic resonance-derived right ventricular remodeling predict poor outcomes in sickle cell disease Kim-Lien Nguyen, et al http://www.haematologica.org/content/101/2/e40

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Spinal glial activation and oxidative stress are alleviated by treatment with curcumin or coenzyme Q in sickle mice Yessenia Valverde, et al. http://www.haematologica.org/content/101/2/e44

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Iron deficiency anemia in cyclic GMP kinase knockout mice Elisabeth Angermeier, et al. http://www.haematologica.org/content/101/2/e48

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Relevant role of von Willebrand factor in neutrophil recruitment in a mouse sepsis model involving cecal ligation and puncture Shogo Kasuda, et al. http://www.haematologica.org/content/101/2/e52

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Molecular subtypes of NPM1 mutations have different clinical profiles, specific patterns of accompanying molecular mutations and varying outcomes in intermediate risk acute myeloid leukemia Tamara Alpermann, et al. http://www.haematologica.org/content/101/2/e55

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Spleen tyrosine kinase inhibitors reduce CD40L-induced proliferation of chronic lymphocytic leukemia cells but not normal B cells Anna Parente-Ribes, et al. http://www.haematologica.org/content/101/2/e59

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UGT2B17 expression: a novel prognostic marker within IGHV-mutated chronic lymphocytic leukemia? Sujata Bhoi, et al. http://www.haematologica.org/content/101/2/e63

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Positron emission tomography response and minimal residual disease impact on progression-free survival in patients with follicular lymphoma. A subset analysis from the FOLL05 trial of the Fondazione Italiana Linfomi Stefano Luminari, et al. http://www.haematologica.org/content/101/2/e66

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Minimal residual disease following autologous stem cell transplant in myeloma: impact on outcome is independent of induction regimen Ruth M. de Tute, et al. http://www.haematologica.org/content/101/2/e69

Haematologica 2016; vol. 101 no. 2 - February 2016 http://www.haematologica.org/

haematologica Journal of the European Hematology Association Published by the Ferrata Storti Foundation

The origin of a name that reflects Europe’s cultural roots.

Ancient Greek

aÂma [haima] = blood a·matow [haimatos] = of blood lÒgow [logos]= reasoning

Scientific Latin

haematologicus (adjective) = related to blood

Scientific Latin

haematologica (adjective, plural and neuter, used as a noun) = hematological subjects

Modern English

The oldest hematology journal, publishing the newest research results. 2014 JCR impact factor = 5.814

Haematologica, as the journal of the European Hematology Association (EHA), aims not only to serve the scientific community, but also to promote European cultural identify.

EDITORIALS Blood disorders stepping into the limelight Ulrich Jäger,1 Christine Chomienne,2 Jan Cools,3,4 and Carin Smand5 1 Department of Hematology, Medical University of Vienna, Austria; 2Hôpital Saint Louis, Institut Universitaire d'Hématologie, Paris, France; 3Center for Human Genetics, KU Leuven, Belgium; 4Center for the Biology of Disease, VIB, Leuven, Belgium; and 5European Hematology Association, The Hague, The Netherlands

E-mail: c.smand@ehaweb.org

doi:10.3324/haematol.2016.142018

hat should we do when the promising treatment options for patients provided by hematology research cost more than the current treatments, when funding for hematology research is limited, and when the increasing cost of health care is such a major concern in many European Member states? This is the question that, under the guidance of the European Hematology Association (EHA), the hematology community in Europe has been addressing. The result is a consensus document: the European Hematology Research Roadmap. More than 300 leading European hematology experts have contributed to this important document, as well as patient organizations, national hematology societies, clinical trial groups, and many others. Typical of such a consensus document is that it needs to ‘come alive’ and this editorial is the first step towards that. However, more needs to be done. • Politicians need to be made aware of the economic burden of hematologic diseases and of the huge number of patients involved. But these diseases do not only involve patients but also have an impact on society as a whole, and politicians need to be made aware of the importance of reducing this. • Research funders need to pay more attention to creating better funding opportunities for hematology research. • The European hematology research community needs to overcome the problem of fragmentation by bringing together basic researchers, clinical trial networks and patient advocates in comprehensive study groups. The European Hematology Research Roadmap is instrumental to this and the EHA needs to play a co-ordinating role in achieving these goals.

These are contradictory times for patients with blood disorders and hematology researchers. On the one hand, patients and those involved in delivering healthcare are being told repeatedly that the continuing effects of the world economic crisis means that health budgets and resources must continue to be constrained at a time when demand for healthcare is increasing. In the words of Dr. Vytenis P Andriukaitis, European Commissioner for Health, “health systems across the European Union (EU) face similar challenges, including: pressure on resources; an ageing population; increasing expectations and possibilities for treatment; and workforce shortages”, which are heightening “the eternal problem of how to do ‘more with less’ ”.1 More recent research in blood disorders including blood cancers, coagulation and platelet disorders, and common diseases such as anemia, has resulted in breakthrough discoveries, new diagnostic methods and better treatments at a breathtaking pace. But research has to be carried out in a conservative funding environment. Announcing the details of a €16 billion investment in research and innovation for 2016-17 under the Horizon 2020 program (the EU’s research and innovation funding scheme), haematologica | 2016; 101(2)

Carlos Moedas, European Commissioner for Research, Science and Innovation said: “Research and innovation are the engines of Europe’s progress and vital to addressing today’s new pressing challenges like (…) healthy societies. Over the next two years, €16 billion from Horizon 2020 will support Europe’s top scientific efforts, making the difference to citizens’ lives.”2 There is substantial evidence to show that there is a pressing medical need for more hematology research. As stated in the EHA Roadmap Consensus Document,3 an estimated 80 million people are currently affected with blood disorders in the EU. Various types of anemia affect more than 50 million children and adults in the World Health Organization’s European region.4 Blood cancers, some of which mainly affect young people, contribute strongly to premature cancer-related mortality and lost productivity in Europe.5 Among cancers, blood cancers (leukemias, Hodgkin and non-Hodgkin lymphomas, and multiple myeloma) together rank third after lung cancer and colorectal cancer in terms of age-adjusted mortality in the European Economic Area.6 Inherited blood diseases, such as thalassemia, sickle cell disease, and glucose-6-phosphate dehydrogenase deficiency, also affect millions of people, and cause substantial morbidity and mortality. Rare forms of congenital blood disorders cause an immense burden on those affected. Many infectious diseases affect various types of blood or blood-forming cells, causing widespread diseases such as malaria and HIV/AIDS. Blood disorders also have immense economic consequences. In parallel with its Roadmap project, the EHA commissioned a study by the University of Oxford, UK, into the societal burden and cost of blood disorders in Europe (EHA, 2015, unpublished data). This study showed that the combined societal cost of hematologic diseases for the EU, Norway, Iceland, and Switzerland is estimated at €23 billion per year. And yet, unfortunately, on a European level, current public spending on hematology research does not match this vast medical need. Of the €6.1 billion that the EU allocated to health research under its 7th Framework Programme (20072013), only 2.2% (€137 million) was granted to hematology research. This amounts to less than 0.1% of the societal cost of blood disorders in Europe over that same period. The current program (Horizon 2020) has been spared major budget cuts, but raising the relative level of funding for hematology research remains a work in progress. Governments, politicians and other policy-makers are responsible for making wellinformed decisions on regulation and funding priorities for health research and medicinal product development. At the same time, the research community has a responsibility to provide policy-makers with the kind of information and evidence that they need to make those informed decisions. Research in hematology has fundamentally improved our biological understanding of disease and has led to innovative 101

Editorials

Table 1. Overarching topics and unmet needs in hematology. 1. 2. 3. 4. 5. 6. 7.

Developing novel targeted therapies based on genomic profiling and chemical biology Unleashing the power of cellular immunotherapy Eradicating minimal residual disease in hematologic malignancies Creating smarter combination treatments Developing better tolerated treatments for blood disorders with a special emphasis on elderly patients Using gene therapy to tackle blood disorders Maximizing the clinical application of hematopoietic stem cells for transfusion, immunomodulation, and repair.

discoveries being made. Many of these discoveries are potent examples of how carefully designed basic research can lead to new approaches that block or interact with key pathways in diseased cells, resulting in very impressive anti-tumor effects. European hematologists have pioneered important inventions and played leading roles in developing curative approaches for patients with malignant diseases, such as lymphomas and leukemias.7,8 It is within this context that, in 2014, the EHA, Europe’s largest non-profit membership organization in hematology, decided to launch the Roadmap project. In addition to the general goal of raising public awareness of blood disorders and highlighting major achievements in their diagnosis and treatment, the specific goals of the EHA Roadmap include: 1) better informing European policy-makers and other stakeholders on the urgent needs and priorities of patients with blood disorders and of the hematology field for the next 5-10 years, thus helping them to make betterinformed decisions on hematology research; 2) improving research funding opportunities for hematology; 3) helping the European hematology research community to reduce fragmentation by bringing together basic researchers, clinical trial networks and patient advocates in comprehensive study groups; 4) promoting a European consensus on medical and research priorities, thus promoting excellence and collaboration between academia and other researchers into blood disorders, such as the pharmaceutical industry. The Roadmap Task Force included EHA board members and other top experts from all fields of hematology. Hundreds of hematologists, clinical trial groups, national hematology societies, patients’ representatives and others were invited to provide input and advice. Draft texts and figures were discussed, developed, and reviewed, and the final draft was sent for consultation to stakeholders such as national hematology societies, patient organizations, hematology trial groups and other European organizations in, for example, overlapping disease areas. In all, some 300 hematologists contributed to the drafting of the document and the various stages of review, and its subtitle, “a consensus document”, reflects this process. The final product, the EHA Roadmap, distinguishes nine ‘sections’ in hematology: normal hematopoiesis, malignant lymphoid and myeloid diseases, anemias and related diseases, platelet disorders, blood coagulation and hemostatic disorders, transfusion medicine, infections in hematology, 102

and hematopoietic stem cell transplantation. These sections span 60 smaller study groups. The EHA Roadmap identifies the greatest unmet needs in hematology research and clinical science, and describes: 1) state-of-the-art hematologic research; 2) the research that needs to be carried out most urgently; and 3) the impact this research is anticipated to have. Some overarching topics and unmet needs can be recognized in nearly all of the nine EHA Roadmap sections (Table 1). Chronic myeloid leukemia (CML) is a good example of successful hematology research that has led to a drastic improvement in treatment options and significantly increased survival rates. CML was almost always fatal until 15 years ago, but the excellent results of BCR-ABL1 tyrosine kinase inhibitor treatment are raising the expectation that a considerable proportion of patients will achieve treatment-free remission. Prevalence of patients with CML treated with kinase inhibitors is expected to increase by about 10% per year, so CML is a challenge for healthcare systems worldwide. With average treatment costs in Europe of between €40,000 and €70,000 per patient per year, the challenge now is how to maximize patient benefit with an affordable allocation of resources. The main objective is to integrate the leading European national trial groups in CML to form a co-operative network for advancements in CML-related research and healthcare. Indepth molecular and cellular characterization of CML patients will facilitate personalized medicine with regard to diagnosis, prognostication, and therapeutic decisions. Overall, a rational advanced treatment design will have a major impact on lowering the disease burden, reducing the rate of complications, and prospectively will result in higher remission rates, longer survival and a higher proportion of patients for whom treatment can be permanently discontinued as an indicator of cure. The cost of novel technologies and treatments might be balanced by their more specific application, having a favorable impact on patients’ quality of life and lessening the burden for caregivers. This will translate into a more general positive impact on society by reducing the burden on available resources and improving individual work capabilities. Classical Hodgkin lymphoma (HL) has become a highly curable malignancy and is considered one of the major success stories in hematology. More than 90% of patients with both localized and advanced-stage disease are alive five years after diagnosis. The progression-free survival ranges from 70% to 93%. As HL is one of the most common haematologica | 2016; 101(2)

Editorials

malignancies in young adults, most patients will have a very long survival, which translates into substantial treatment and societal costs. During follow up, however, a significant proportion of patients experience serious long-term toxicities, such as secondary cancers, cardiovascular diseases, and infertility. Most of these late toxicities have been related to treatment. European clinical trials in HL have made an important contribution to reducing both radiation dose and field size, as well as the number of chemotherapy cycles. Since most HL patients will ultimately be cured from their disease and experience long-term survival, research should focus on better identifying patients who can be cured with less aggressive treatments. As discussed in the Roadmap document, research should also aim at further refining the use of new drugs and possible associations with the currently available treatment modalities as part of an individualized therapy strategy, thus meeting the urgent need of avoiding unnecessary toxicities for the vast majority of patients. The EHA Roadmap also points the way towards repeating the success of research into CML and HL with diseases that currently suffer from low survival rates, such as acute myeloid leukemia (AML), where new technologies and additional funding are needed. Overall, the EHA Roadmap highlights major past achievements in diagnosing and treating blood disorders, identifies unmet clinical and scientific needs in those same areas, and points to the anticipated benefits for patients of the proposed research areas. The authors trust that this will help decision-makers to focus their attention on how to

haematologica | 2016; 101(2)

provide better funding for more targeted European hematology research. Acknowledgment The authors would like to thank the referees for their insight, comments and suggestions and Charles Charalambous, medical writer, Brussels, Belgium, who assisted with the final editing of this editorial.

References 1. Speech to the European Patients Forum conference on the CrossBorder Healthcare Directive, 2 July 2015. Available from: https://ec.europa.eu/commission/2014-2019/andriukaitis/announcements/european-patients-forum-conference-cross-border-healthcaredirective_en. 2. European Commission. Commission invests €16 billion in funding for research and innovation over next two years. Available from: http://europa.eu/rapid/press-release_IP-15-5831_en.htm. 3. Engert A, Balduini C, Brand A, et al. The European Hematology Association Roadmap for European Hematology Research: a consensus document. Haematologica. 2016;101(2):115-208. 4. Hanly P, Soerjomataram I, Sharp L. Measuring the societal burden of cancer: the cost of lost productivity due to premature cancer-related mortality in Europe. Int J Cancer. 2015;136(4):e136-145. 5. De Benoist B, et al. (eds.) Worldwide prevalence of anemia 1993-2005. WHO Global Database on Anemia. Geneva, World Health Organization, 2008. 6. Eurostat. Luxembourg, Luxemburg. Causes of Death, 2015. Available from: http://ec.europa.eu/eurostat/web/health/causes-death/data/ database 7. Döhner H, Stilgenbauer S, Benner A, et al. Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med. 2000;343(26):1910-1916. 8. Engert A, Plütschow A, Eich HT, et al. Reduced treatment intensity in patients with early-stage Hodgkin’s Lymphoma. N Engl J Med. 2010;363(7):640-652.

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REVIEW ARTICLE EUROPEAN HEMATOLOGY ASSOCIATION

Ferrata Storti Foundation

Haematologica 2016 Volume 101(2):104-114

Leaders in Hematology review series

The role of targeted treatment in mantle cell lymphoma: is transplant dead or alive? Martin Dreyling1 and Simone Ferrero,2 on behalf of European Mantle Cell Lymphoma Network

Department of Medicine III, Hospital of the University LMU München, Germany; and 2Division of Hematology, Department of Molecular Biotechnologies and Health Sciences, University of Torino, Italy 1

ABSTRACT

Correspondence: martin.dreyling@med.uni-muenchen.de

Received: 13/7/2015. Accepted: 17/11/2015.

doi:10.3324/haematol.2014.119115

Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/101/2/104

©2016 Ferrata Storti Foundation Material published in Haematologica is covered by copyright. All rights reserved to Ferrata Storti Foundation. Copies of articles are allowed for personal or internal use. A permission in writing by the publisher is required for any other use.

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ased on the profound biological insights of the last years into the molecular pathogenesis of mantle cell lymphoma and the clinical introduction of new targeted drugs, with high efficacy and a good safety profile, the therapeutic scenario for this tumor has been shown to be thoroughly favourable. No longer characterized by a uniformly dismal prognosis, mantle cell lymphoma has been revealed as a spectrum of different diseases, ranging from very indolent cases to highly aggressive and refractory ones. Thus, there is an urgent need to adapt therapy to accommodate the diverse presentations of the disease. High-dose chemotherapy, followed by autologous stem cell transplantation is the current standard of care for younger patients, generally providing high responses and long survival rates, but hampered by acute and long-term toxicity. In addition, some patients may be overtreated, while others could benefit from targeted approaches, based on the new, molecular-directed compounds. Such a personalized treatment based on the specific characteristics of individual patients may be guided by validated prognostic tools, such as the Mantle Cell Lymphoma International Prognostic Index and the Ki-67 Proliferative Index, as well as by early predictors of treatment response, like minimal residual disease analysis. Moreover, mutation screening of distinctive genomic alterations may provide new, predictive biomarkers, with an additional impact on clinical practice. Only after tailoring treatment according to the clinical and biological heterogeneity of the disease the role of transplantation and modern therapeutic options will be redefined in mantle cell lymphoma.

Introduction Mantle cell lymphoma (MCL) is a relatively rare lymphoma subtype, constituting nearly 6-8% of all non-Hodgkin Lymphomas (NHL) in Europe and North America. MCL is typically diagnosed in elderly males, with a median age at diagnosis of 65 years and a male preponderance of 3 to 1.1 Since its worldwide recognition in 1994, it has been known to have a dismal prognosis (“the worst lymphoma to have”), with a median overall survival (OS) rate of 3 years only. Unfortunately, no curative therapy has been established so far.2,3 After many years without significant advance in the management of patients with MCL, recently the prognosis for younger patients has improved significantly due to the introduction of dose-intensified regimens containing cytarabine, some incorporating autologous stem cell transplantation (ASCT), and the introduction of anti-CD20 monoclonal antibody rituximab. However, these intensive regimens raise some concern regarding acute and late toxicities and are not suitable for elderly patients, who represent the majority of MCL patients.4 The superiority of sequential regimens with ASCT consolidation after high-dose cytarabine schemes versus some intensive schedules like Hyper-CVAD/MA are debated, especially between European and American clinical groups.5 More importantly, even among younger patients, a minority presents with clinically indolent features (“indolent MCL”) or with classical MCL but characterized by low tumor mass and low-risk haematologica | 2016; 101(2)

The role of targeted treatment in mantle cell lymphoma

according to the MIPI (MCL International Prognostic Index) and/or the Ki-67 Proliferative Index. These patients also represent a dilemma for the clinician as to whether to offer them a high-dose therapy or not, as intensive treatments, with or without ASCT, are hampered by shortand long-term toxicities, including secondary malignancies.6,7 Moreover, recently the scientific and therapeutic scenario for MCL patients has rapidly changed: new biological insights into the molecular pathogenesis of MCL have highlighted some crucial oncogenic signaling pathways, underlying the aggressiveness and chemorefractoriness of the disease. Such discoveries have paved the way for the concept of personalized medicine in MCL. On one hand, the availability of these new diagnostic tests offers a better and more rational biology-based prognostic stratification of patients at baseline, suggesting different treatment strategies for patients with various risk profiles. On the other hand, a deeper unveiling of the underlying mechanisms has led to the clinical development of many new small molecules acting towards specific molecular targets, with high anti-lymphoma activity in some cases.8,9 The current availability of effective, targeted drugs and the increasing clinical application of robust and predictive diagnostic tools have already started to change the therapeutic algorithms of MCL and will challenge the established role of ASCT. Therefore, our review will draw on the current landscape of evidence supporting ASCT in MCL, subsequently describing the most important new drugs available in clinical practice for this lymphoma and will finally debate the role of ASCT in the near future, proposing a new therapeutic algorithm for MCL in the era of personalized medicine.

The role of high-dose therapy and autologous transplantation Soon after the recognition of MCL as a distinct entity in the REAL (Revised European-American Lymphoma) Classification back in 1994, it became obvious that this lymphoma subtype has a more aggressive clinical course with rapid relapses and subsequent chemorefractoriness, as compared to indolent lymphoma. Initially, MCL typically showed slightly lower response rates to polychemotherapy and a short event-free survival (EFS) and OS of 8 and 28 months, respectively, in a German series of 45 patients.2,3 The combination of rituximab with fludarabine or CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone), though improving the response rate and the time to treatment failure (TTF), did not impact on OS, resulting in a median progression-free survival (PFS) of only slightly more than one year, with virtually unchanged long-term perspectives.10,11 On the contrary, more promising results were obtained in phase II studies implementing high-dose cytarabine, with or without ASCT. A sequential CHOP-DHAP (dexamethasone, high-dose cytarabine and cisplatin) regimen led to a CR rate of >80% in a series of 28 patients. Responding patients underwent intensified consolidation with total body irradiation (TBI), high-dose Ara-C, melphalan and ASCT, resulting in an impressive 3-year EFS rate of 83% and 3-year OS rate of 90%.12 Similarly, high response rates of more than 90% were demonstrated by the MD Anderson Cancer Center with a haematologica | 2016; 101(2)

dose-intensified approach. Twenty-five patients received an alternating regimen of hyper-CVAD (hyperfractionated cyclophosphamide, vincristine, doxorubicin and dexamethasone) with high-dose cytarabine and methotrexate (MA). However, in this elderly patient population the median TTF was only 15 months, and hematologic toxicity was significant.13 The role of consolidation by dose intensification and ASCT was supported by encouraging results obtained by different phase II studies, aiming at the elimination of residual lymphoma cells after conventional chemotherapy.14-16 In addition, the benefit of TBI as part of the conditioning regimen in MCL was suggested by a retrospective analysis (PFS after 4 years: 71% vs. 0%, P<0.0001; OS 89% vs. 60%, P=0.07).17 Thus, in 1996, the European MCL Network embarked on a randomized comparison of CHOP followed by myeloablative radiochemotherapy (high-dose cyclophosphamide + 12 Gy TBI) followed by ASCT versus IFNÎą maintenance in patients under 65 years of age in order to assess more precisely the impact of ASCT. Patients in the ASCT arm experienced a significantly longer PFS, even though the 3-year OS was not significantly superior (Table 1).18 However, in a subsequent analysis the median OS was also superior in the ASCT arm after extended median follow-up (63 months) (90 months versus 54 months, P=0.034).19 Therefore, chemotherapy dose intensification and ASCT support became the standard of care for younger MCL patients. However, the non-curative potential of this intensive approach was witnessed by the continuous relapsing pattern and lack of molecular remissions (MR) (determined by Bcl-1 or immunoglobulin rearrangement nested-PCR approach).20,21 Nonetheless, the subsequent integration of rituximab and high-dose cytarabine into ASCT programs led to unprecedented levels of cytoreduction, making MR an attainable target in MCL patients. In 2003 Massimo Gianni et al. reported MR in 19 out of 20 patients receiving a rituximab-supplemented high-dose sequence (R-HDS), along with very favorable 4-year EFS and OS rates (79% and 89%, respectively).22 Comparable results were reported for a multicenter phase II trial by the Nordic Lymphoma Group. One hundred and sixty MCL patients received an induction with R-maxi-CHOP alternating with R-high dose cytarabine, followed by a high-dose consolidation (BEAM) with ASCT. MR was achieved in 92% of the 79 evaluable patients, while overall and complete response was achieved in 96% and 54%, respectively. The 6-year EFS, PFS and OS were 56%, 66%, and 70%, respectively, with no relapses observed after 5 years.23 Moreover, achievement of MR, irrespective of high-dose therapy with ASCT or less intensive immuno-chemotherapy regimens, was an independent predictor of clinical outcome.24,25 Additional phase II studies, as well as a large retrospective population-based analysis showed similar favorable clinical results of high-dose cytarabine-containing schedules followed by ASCT, with overall response rates (ORR) ranging from 70% to 100% (CR: 64-96%), 5-year OS ranging from 64% to 75% and acceptable toxicity profiles (treatment-related mortality â&#x2030;¤ 5%), but a significant dropout rate (13%-30%).26-29 Similarly, the MD Anderson Hyper-CVAD/MA regimen with rituximab resulted in excellent results in a monocen105

M. Dreyling et al. Table 1. Published clinical studies investigating first-line dose-intensified therapy in MCL.

ASCT Based regimens

Author

Study Features

Evaluable patients

Therapeutic regimen

ORR% (CR%)

Median PFS (years)

Median OS (years)

Dropout rate

TRM

Secondary tumors rate

Dreyling et al., 2005 [18]

Phase III, randomized

122

R-CHOP + TBI + ASCT vs. R-CHOP + TBI + interferon-α

98 (81) vs. 99 (37)

3,3 vs. 1,4

NR (83% 3-y OS) vs. NR (77% 3-y OS)

13% vs. na

5% vs. 0%

Hermine et al., 2012 [34]

Phase III, randomized

455

Damon et al., 2009 [26] Van't Veer et al., 2009 [27] Geisler et al., 2012 [39]

Phase II

13%

Phase II

30%

Phase II

160

R-CHOP + TBI + ASCT 98 (63) 3,8 6,8 vs. vs. vs. vs. R-CHOP/R-DHAP + HD-araC + ASCT 99 (61) 7,3 NR R-CHOP + methotrexate + 88 (69) NR NR HD-araC/etoposide + ASCT (56% 5-y PFS) (64% 5-y OS) R-CHOP + HD-araC + 70 (64) NR NR ASCT (36% 4-y PFS) (66% 4-y OS) R-Maxi-CHOP + 96 (54) 7,4 NR HD-araC+ (64% 10-y OS) ASCT R-CHOP/R-DHAP + 100 (96) 6,9 NR HD-araC + ASCT (75% 5-y OS) Different ASCT-based 83 (77) NR NR schedules (67% 3-y PFS) (83% 3-y OS) R-Maxi-CHOP + 94 (82) NR (71% 4-y PFS) NR (78% 4-y OS) HD-araC+/- Zevalin + ASCT R-DHAP + ASCT +/- rituximab na (92) NR (74% 3-y PFS) NR (83% 3-y OS) maintenance R-CHOP+R-CTX+HD-araC+ASCT 86 (78) NR (78% 2-y PFS) NR (89% 2-y OS) +/- lenalidomide maintenance

18%

1,5%

18%

2,5%

14%

22%*

29%

63%

6,5%

1,5%

39%

Non-ASCT based regimens

Delarue et al., 2013 Phase II [28] Touzeau et al., 2013 Retrospective [29] Kolstad et al., 2014 Phase II [40] Le Gouill et al., 2014 Phase III, [42] randomized Cortelazzo et al., 2015* Phase III, [99] randomized

Romaguera et al., 2010 Phase II, [6] monocentric Merli et al., 2012 Phase II, [31] multicentric Bernstein et al., 2013 Phase II, [32] multicentric

60 396 160 299 260*

R-Hyper-CVAD

97 (87)

4,6

R-Hyper-CVAD

83 (72) NR (73% 5-y PFS)

R-Hyper-CVAD

86 (55)

4,8

NR (64% 10-y OS) NR (61% 5-y OS) 6,8

* the accrual is not yet completed. MCL: mantle cell lymphoma; ORR: overall response rate; CR: complete response; PFS: progression-free survival; OS: overall survival; R: rituximab; CHOP: cyclophosphamide, doxorubicin, vincristine and prednisone; TBI: total body irradiation; ASCT: autologous stem cell transplantation; DHAP: dexamethasone, cytarabine and cisplatin; HD-araC: high dose cytarabine; R-CTX: rituximab-high dose cyclophosphamide; Hyper-CVAD: hyperfractionated cyclophosphamide, vincristine, doxorubicin and dexamethasone + methotrexate-cytarabine; NR: not reached; na: not available; ne: not evaluable; y: years; vs.: versus.

tric series of 97 patients. Nonetheless, this dose-intensive regimen was not devoid of TRM and high dropout rates (8% and 29%, respectively)30 and its application in multicentric trials revealed a limited feasibility.31,32 Finally, a recent “real-life”, population-based observational study by the Nordic Lymphoma Group, demonstrated that rituximab (n=766; HR=0.66; P<0.001) and ASCT (n=273; HR=0.55; P<0.004) were independently associated with improved OS among patients receiving systemic treatment.33 Based on these promising data, in 2004 the European MCL Network launched the “MCL Younger” phase III trial, comparing a conventional R-CHOP induction to the “experimental” French one (alternating induction of 3 courses of R-CHOP and R-DHAP), both followed by myeloablative consolidation, TBI and ASCT. Preliminary results confirmed that the R-CHOP/R-DHAP arm achieved a significantly improved median TTF and OS (Table 1), with a comparable number of treatment-related deaths in both groups.34 The impact of cytarabine on the 106

TTF rate was closely linked to MR in the bone marrow, which was much more frequent in the R-CHOP/R-DHAP arm (68% vs. 24%, P<0.001).35 Therefore, ASCT is currently considered the standard first-line consolidation therapy for younger MCL patients (including “low-risk” cases), as stated by international guidelines,36,37 as well as a recent consensus of the European MCL Network and the Lymphoma Working Party of the European Society for Blood and Marrow Transplantation (EBMT).38 However, although the overall results of all these highdose cytarabine-containing regimens are excellent, with a median OS of more than 10 years in the updated Nordic Lymphoma Group experience, late relapses continue to occur, highlighting that even ASCT-based programs alone are not able to eradicate MCL.39 A recent trial by the Nordic Lymphoma Group failed to demonstrate an improved outcome after 90Yttrium-ibritumomab tiuxetan-BEAM conditioning before ASCT.40 On the other hand, more promising maintenance strategies are being implemented after ASCT, haematologica | 2016; 101(2)

The role of targeted treatment in mantle cell lymphoma

Table 2. Recent published clinical studies investigating targeted approaches in MCL (with more than 10 evaluable MCL patients).

Author

Study Features

Goy et al., 2009 [46] Ruan et al., 2011 [50] Robak et al., 2015 [51] Hess et al., 2009 [52]

Evaluable Patients

Therapeutic regimen

Phase II, relapse Phase II, upfront Phase III, randomized, upfront Phase III, randomized, relapse

ORR% (CR%)

141 bortezomib 33 (8) 36 R-CHOP + bortezomib 91 (72) 244 R-CHOP vs. VR-CAP 89 (42) vs. 92 (53) 54 temsirolimus 75mg/75mg 22 (2) 54 temsirolimus 75mg/25mg 6 (0) 53 investigator's choice 2 (2) Ansell et al., 2011 [53] Phase II, relapse 69 temsirolimus + rituximab 59 (19) Witzig et al., 2011 [56] Phase II, relapse 57 lenalidomide 35 (12) Eve et al., 2012 [57] Phase II, relapse 26 lenalidomide 31 (8) Goy et al., 2013 [59] Phase II, relapse 134 lenalidomide 28 (8) Wang et al., 2012 [60] Phase II, relapse 44 lenalidomide + rituximab 57 (36) Zaja et al., 2012 [58] Phase II, relapse 33 lenalidomide + dexamethasone 52 (24) Albertsson-Lindblad et al., 2015 [62]Phase II, upfront 51 lenalidomide + rituximab 91 (78) + bendamustine Zaja et al., 2015 [61] Phase II, relapse 52 lenalidomide + rituximab 79 (55) + bendamustine Wang et al., 2013 [63] Phase II, relapse 111 ibrutinib 68 (21) Kahl et al., 2014 [64] Phase I, relapse 40 Cal-101 40 (5) Morschhauser et al., 2013 [67] Phase II, relapse 40 (15 MCL) GA-101 27 (13) Lin et al., 2010 [71] Phase I** 10 flavopiridol + fludarabine 80 (70) + rituximab Evens et al., 2012 [75] Phase II 11 abexinostat 27 (na)

Median PFS (months)

Median OS (months)

6,7 (TTP) 44% (2-y PFS) 14,4 vs. 24,7 4,8 3,4 1,9 9,7 8,8 3,9 4 11,1 12 42

23,5 86% (2-y OS) 54% vs. 64% (4-y OS) 12,8 10 9,7 29,5 NR 10 19 24,3 20 53

51% (2-y PFS) 13,9 3,7 2,7* 21,9

66% (2-y OS) NR (1,5-y OS 58%) na na na

*Data derived from the overall population of the study, not exclusively from patients with MCL. **6 patients received the schema as first-line therapy, 4 patients after relapse. MCL: mantle cell lymphoma; ORR: overall response rate; CR: complete response; PFS: progression-free survival; OS: overall survival; TTP: time to progression; R-CHOP: rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone; VR-CAP: bortezomib, rituximab, cyclophosphamide, doxorubicin and prednisone; NR: not reached; na: not available; y: years; vs.: versus.

based on the results of rituximab maintenance in elderly MCL patients.41 The ongoing phase III Lyma trial (NCT00921414) investigated the role of rituximab maintenance after four courses of R-DHAP and ASCT. Besides confirming very favorable CR, PFS and OS rates (Table 1), the data from the interim analysis show a promising 2-year EFS of 93% in the rituximab maintenance arm versus 82% in the control arm (hazard ratio, HR = 2.1), suggesting that rituximab maintenance after ASCT may become a new standard of care in MCL.42 Currently, the randomized phase III trial FIL-MCL0208 (EudraCT Number 2009-012807-25) is exploring lenalidomide maintenance in young MCL patients after ASCT, and results are eagerly awaited.99 Despite high response rates and long-term survival advantages after the described high-dose schedules, the non-negligible toxicity profile of such an approach has to be disclosed. Dropout rates generally range between 10%30% in all the studies (Table 1), with the major adverse events being infectious (neutropenic fever, pneumonia) and gastrointestinal (10%-15% of patients), besides the need for red cell and platelet transfusions (10%-30% of cycles). TRM generally ranges between 2%-8%, mainly due to infectious and cardiac complications. Moreover, costs of hospitalization for high-dose therapy and ASCT have to be considered. Finally, all the reported intensive regimens displayed a significant rate of second tumor development, ranging from 4% up to 18% (Table 1). These results are in line with the long-term secondary neoplasia rates of a large retrospective study on more than 1000 lymphoma patients treated with high-dose therapy, haematologica | 2016; 101(2)

rituximab and ASCT (10-year rates of myelodysplasia/ acute leukemia, 4.5% and solid tumors, 6.8%).7 Table 1 describes the most important published clinical studies investigating first-line high-dose therapy in MCL.

The emerging role of new drugs During the last years, growing insights into the molecular biology of MCL have led to the systematic exploration of targeted approaches.8 Many new compounds are currently being tested within clinical trials, and some of them have already received approval both in the USA and Europe (Table 2), based on impressive activity in relapsed/refractory patients. Current trials are investigating the combinations with immunochemotherapy in earlier treatment lines, with the aim of enhanced efficacy, without adding further toxicity. In the USA the first new agent registered in relapsed MCL was bortezomib, a selective and reversible proteasome 26S inhibitor. Some phase II, single-agent data showed significant responses and favorable PFS and OS rates, with predictable toxic effects.43-46 Since then, many combinations with rituximab and chemotherapy were tested, mainly in a limited series of relapsed/refractory patients.47-50 More recently, the phase III trial LYM-3002 showed that the substitution of vincristine by bortezomib in front-line R-CHOP (â&#x20AC;&#x153;VR-CAPâ&#x20AC;? regimen) improved outcomes in elderly patients with MCL (Table 2); however, an increased hematologic toxicity was observed.51 107

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Temsirolimus, an intravenous mammalian target of rapamycin (mTOR) inhibitor, received the European Medicines Agency (EMA) approval in 2009, due to its single-agent activity. This approval was based on a randomized phase III trial, showing superiority to monochemotherapy (Table 2).52 The addition of rituximab showed even higher response rates in a phase II study.53 To further improve its efficacy, temsirolimus is currently being investigated in combination with BR: of note, all evaluable patients of the phase I part responded to this combination.54 The immunomodulatory compound lenalidomide showed high activity in relapsed/refractory MCL patients in many phase II trials, either as a single-agent or combined with dexamethasone.55-59 Subsequently, a chemofree lenalidomide-rituximab combination resulted in even higher response rates (Table 2) and impressive response duration of up to 19 months.60 Finally, preliminary results of a phase II trial in first relapse showed activity and the feasibility of a dose reduced rituximab, lenalidomide plus bendamustine combination, followed by lenalidomide maintenance.61 Nevertheless, a full dose combination in a front-line setting showed an excess of toxicity and secondary malignancies.62 Recently, highly promising data were reported for inhibitors of the B-cell receptor pathway. The covalent oral inhibitor of Bruton’s tyrosine kinase (BTK) ibrutinib showed durable single-agent efficacy in relapsed or refractory MCL.63 Based on an international phase II trial in heavily pre-treated MCL patients responses were achieved in the majority of patients paired with excellent tolerability (Table 2). Prior treatment with bortezomib had no effect on the response rate. The most common adverse events were mild or moderate diarrhoea, fatigue, and nausea. Grade 3 or higher hematologic events were infrequent and included neutropenia (16%), thrombocytopenia (11%), and anemia (10%). One phase III trial comparing ibrutinib versus temsirolimus monotherapies in relapsed patients (NCT0164021) has confirmed the superiority of the BTK inhibitor, and another trial assessing a BR schedule plus/minus ibrutinib in first-line therapy (NCT01776840) has completed accrual. Another antagonist of the BCR signal cascade, idelalisib, a specific inhibitor of phosphatidylinositol 3-kinase delta isoform, also achieved high response rates in MCL, but Table 3. Simplified MIPI calculation.

Points

Age (years) ECOG Performance LDH/ULN Status

0 1 2 3

<50 50-59 60-69 >69

0-1 2-4 -

<0.670 0.670-0.999 1.000-1.499 >1.499

Leukocytes (x109/L) 6700 6700-9999 10000-14999 >14999

For each prognostic factor, 0 to 3 points are given to each patient and points are summed up to define a category of risk

Risk stratification 0-3 points 4-5 points 6-11 points

low-risk intermediate-risk high-risk

MIPI: Mantle cell lymphoma International Prognostic Index; ECOG: Eastern Cooperative Oncology Group; LDH: lactate dehydrogenase; ULN: upper limit of normal; L: liter.

108

had a disappointing median duration of response of only 2.7 months.64 Finally, many other promising targeted drugs are also currently being tested in MCL. New anti-CD20 monoclonal antibodies (mAB), such as obinutuzumab and ofatumumab,65-67 bispecific anti-CD19/anti-CD3 mAB blinatumumab,68,69 the toxin-immunoconjugated mAB antiCD79b DCDS4501A,70 direct inhibitors of cyclin-dependent kinase 4 and 6 (flavopiridol and PD0332991),71-73 oral second generation BCL-2 inhibitors (venetoclax)74 and novel oral pan-histone deacetylase inhibitors (abexinostat).75 Overall, the above mentioned compounds showed activity in MCL. Nevertheless, additional studies on larger MCL patient cohorts are warranted to assess their specific role in this lymphoma subtype. A summary of the recently published clinical trials of targeted approaches in MCL is presented in Table 2.

Looking for a tailored treatment in MCL The well known biological and clinical heterogeneity of MCL, as well as the recent availability of highly active, but also expensive new compounds, urges the introduction of the concept of “personalized medicine” into the clinical practice of MCL. However, to effectively tailor the therapeutic approach according to the individual patient’s risk profile reliable prognostic tools applicable in clinical routine are mandatory. The ideal prognosticator should integrate clinical and biological features, taking into account the recent knowledge of molecular pathogenesis. Currently, the most widely applied tool is the prognostic MIPI score, encompassing simple clinical parameters such as age, performance status, LDH and the leukocyte count.76 Based on easy calculations available via internet, and validation in a “simplified” version76,77 (Table 3), the MIPI is able to stratify newly diagnosed patients into three risk classes with different 5-year OS rates: 83%, 63%, and 34% in MIPI low, intermediate, and high-risk groups, respectively.78 However, there are some important limitations: first of all, as the “age” is one of the most important variables, MIPI fails to correctly classify some younger “high-risk” patients; moreover, it is not able to precisely stratify the outcomes of “low” and “intermediate” risk groups among elderly patients.78 Therefore, the integration of a validated biomarker, such as the Ki-67 proliferative index, has been proposed (“biological-MIPI”, MIPI-b).76 A Ki-67 index ≥ 30% was associated with poor outcome in different patients series, after conventional or intensified chemotherapy plus rituximab.23,79,80 The Ki-67 integration into the MIPI-b was validated in a large series of patients from randomized trials carried out by the European MCL Network, identifying patients at higher relapse risk in both the younger and older age categories, but again not reliably stratifying between “low” and “intermediate” risk groups.78 This limitation has been overcome by a recently improved version of “combined” MIPI, MIPI-c, identifying four risk classes based on a 30% Ki-67 cut-off value (5year OS rates, 85%, 72%, 43% and 17%, respectively, P<0.0001)81 (Figure 1). However, an important limitation is the reproducibility of the Ki-67 evaluation in pathology labs, where the published guidelines may not be routinely followed.82 Moreover, a representative lymph node biopsy is required: thus, cases diagnosed only on BM histology are often not sufficiently evaluable. haematologica | 2016; 101(2)

The role of targeted treatment in mantle cell lymphoma

More recently, the integration of new biomarkers in the MIPI has been proposed. A study by the Nordic Lymphoma Group reported that microRNA (miR)-18b overexpression identifies MCL patients with poor outcome.83 Despite the intriguing biological rationale of this work, the wide application of this tool appears to be hampered by the missing availability of miR analysis in clinical routine. The only other validated prognosticator in MCL is the post-treatment evaluation of MRD by allele-specific oligonucleotide (ASO)-PCR. MRD analysis is able to detect very low levels (up to 1.00E-05) of residual lymphoma cells in patients achieving complete clinical response (CR) after treatment. This tool, currently applicable in 90% of MCL patients (with an available diagnostic specimen and BM or peripheral blood follow-up samples) is an effective early predictor of outcome, with an independent prognostic value in a large series of patients, which is even superior to CR achievement in multivariate analysis.25 Its value has been confirmed in various patients series through different treatments (both standard and high-dose chemotherapy, as well as maintenance therapy) in both young and elderly patients.24,25,35,39,84 Moreover, MRD prospective assessment is able to identify early those high-risk patients with molecular relapse only and thus prone to clinical relapse within the subsequent two years.84 This setting allows preemptive trials investigating non toxic treatments at MRD reappearances.85,86 The major limitations of this approach is the technical complexity of the MRD analysis with patient-specific primers, currently reliable only in specialized labs, applying standardized guidelines and performing regular quality control rounds (“EuroMRD group”).87 However, although such predictive tools effectively stratify patients into different risk classes, solid data on their application into personalized treatments are still lacking. To our knowledge, thus far only two trials designed by the Nordic Lymphoma Group have investigated tai-

lored therapy in MCL. The “MCL2” trial proposed a “preemptive” rituximab strategy for 26 patients experiencing MRD recurrence after ASCT:85 even though molecular reconversion rates and preliminary data on survival are promising, the limited patient number does not yet justify therapeutic approaches in clinical routine.39 Moreover, the attempt to improve the prognosis of “high-risk” MCL (according to MIPI and MIPI-b) by offering increased doses of cytarabine did not yield satisfying results (“MCL5”).88 Therefore, although broadly validated, neither MIPI nor Ki-67 nor MRD are currently routinely applied to guide treatment decisions in MCL.36 Thus, a practical application of these predictors in the next clinical trials is eagerly awaited, to finally investigate tailored therapies in MCL. In addition, in the last years, many new molecular pathways involved in tumor survival, aggressiveness and treatment refractoriness have been identified.8,89 Thus, numerous molecular markers (including SOX11 expression, p53 alterations and Notch-1 mutations)90-94 have been linked with outcome. However, a reliable translation of biological data into the context of clinical patient care is currently lacking, not yet allowing for a personalized strategy in the majority of MCL cases.

MCL: is transplant dead or alive? Given that a high-dose schedule containing cytarabine and rituximab, followed by an ASCT, is nowadays widely recognized as the standard of care for younger patients affected by MCL, some important considerations should be made that might change the therapeutic scenario in the upcoming years. First of all, the valuable results in terms of improved survival rates after high-dose therapy and ASCT consolidation are counterweighted by non-negligible toxicities, as previously described.7 Given that some patients do not

Figure 1. Alternative combination of Ki-67 index and MIPI: MIPIc. MIPI-c: combined mantle cell lymphoma international prognostic index.

haematologica | 2016; 101(2)

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M. Dreyling et al.

Figure 2. Schematic representation of the “Triangle” trial by the European MCL Network. MCL: mantle cell lymphoma; R: randomization; ASCT: autologous stem cell transplantation; RCHOP: rituximab, cyclophosphamide, doxorubicin, vincristine and prednison; RDHAP: rituximab, dexamethasone, cytarabine and cisplatin.

need immediate treatment (the clinically defined “indolent MCL”, potentially identified by the lack of SOX11 expression),95-97 and others benefit from very long remissions after ASCT, it is reasonable to challenge the value of intensified treatment in these patients in order to avoid unnecessary toxicities. In this context, the recent improvements in induction schemes,34,98 as well as the very promising data on rituximab maintenance,41 even after ASCT,42 strongly suggest a more sustained PFS, especially in younger patients. Moreover, the high activity of the targeted drugs as a single-agent in relapsed patients has prompted their investigation in combination with immunochemotherapy in first-line trials.51,62 Their impact on long-term survival will potentially result in new options for first-line treatment, and might challenge the current role of ASCT consolidation. This is precisely the concept of the upcoming phase III trial “Triangle” by the European MCL Network (EudraCT Number 2014-001363-12), assessing whether the implementation of a BTK-inhibitor in first-line treatment could eliminate the need for an ASCT consolidation in younger patients. In detail this trial offers a randomization between conventional induction R-CHOP/R-DHAP + ASCT, versus the identical scheme with the addition of ibrutinib, versus R-CHOP/R-DHAP + ibrutinib without ASCT (Figure 2). However, this trial does not implement personalized treatment according to the discussed prognosticators. Interestingly, some important data on the value of ASCT are emerging from MRD studies, supporting the concept of “dispensable therapy”. High-dose consolidation followed by ASCT demonstrated a high impact on tumor reduction in the pooled treatment arms (R-CHOP vs. R-CHOP/R-DHAP) of the European MCL Network 110

“Younger” trial, increasing the MR rate from 50% to 75% (P=0.0001). However, this improvement was prominent only in the R-CHOP arm (29% to 65%; P=0.0023), while detectable but not statistically significant after the more effective R-CHOP/R-DHAP arm (76% to 88%; P=0.18). Remarkably, MR after induction was associated with a significantly improved remission duration (89% vs. 74% at 24 months, P=0.002), and sustained MR during the first year after ASCT was also predictive for outcome.35 This observation underlines the important role of cytarabine in inducing sustained MR in MCL. Thus, it may be speculated that patients already achieving MR after high-dose cytarabine plus rituximab induction might do well without ASCT consolidation. Conversely, patients with persistent MRD positivity after a highly effective cytarabine induction might not benefit from ASCT, and thus may be potential candidates for experimental strategies. In accordance, such molecular results have been recently presented for the Italian “MCL0208” trial: preliminary MRD data demonstrate only a marginal improvement of the MR rate (from 67% to 73%) after ASCT.99 Of course, these MRD results have to be confirmed by subsequent PFS results: however, in our opinion, they should prompt the investigation of MRD-guided personalized treatment strategies. Finally, despite their high efficacy, ASCT based regimens do not lead to complete eradication of the disease. Actually, even among long-term responders, late relapses still continue to occur up to 10 years after the transplantation.39 In addition, MRD reappearance has shown to herald full-blown relapse (with a median time to clinical relapse of 18 months).84 Thus, even after ASCT, effective maintenance therapies have to be considered42 and MRDdriven pre-emptive treatments may be investigated in the context of maintenance trials. Finally, a problem which haematologica | 2016; 101(2)

The role of targeted treatment in mantle cell lymphoma

Figure 3. Suggested personalized treatment strategy according to risk stratification in mantle cell lymphoma (MCL). MIPI-c: combined MCL International Prognostic Index; MRD: minimal residual disease; ASCT: autologous stem cell transplantation; HD AraC: high-dose cytarabinebased regimen.

remains unresolved are the “very high-risk” patients, in whom the standard high-dose + ASCT approach does not result in long-term disease control.88 There is an urgent need to identify those patients at baseline, in order to investigate new front-line approaches, tailored on the high risk of early disease progression. Currently, some genomic alterations have been described, predicting high risk of treatment failure: in particular p53 and CDK2N mutations, as well as complex karyotype mutations.94,100 Those patients who are refractory to intensified therapies and ASCT are appropriate candidates to explore new, targeted approaches or to undergo immunological approaches, such as allogeneic transplantation or the recently described chimeric antigen receptor T cells.101 However, all of these therapeutic approaches have to be further investigated in the context of well-designed clinical trials, carefully weighing the pros and cons. In this regard, concerns are actually rising about the unsustainable costs of the unselected use of targeted drugs, as well as their long-term toxicity; very little is known about their role in inducing subsequent aggressive transformations of the disease.102 On the basis of all these considerations we propose a rational strategy of “personalized first-line treatment” for younger MCL patients, to be investigated in a clinical trial (Figure 3). In our model risk stratification is based on MIPIc and mutational analysis at baseline, and MRD evaluation during therapy: low-risk and MRD negative low-intermediate risk patients may not proceed to ASCT consolidahaematologica | 2016; 101(2)

tion, while high-intermediate and high-risk patients should receive a combined chemotherapy induction with biological agents; finally, consolidation and maintenance strategies may be carried out in all patients based on the post-treatment MRD result.

Conclusions The clinical scenario of MCL has completely changed during the last years due to the availability of highly effective targeted drugs, as well as reliable predictive tools determining the prognostic heterogeneity of such patients. Currently, a high-dose immunochemotherapeutic regimen based on cytarabine and rituximab, supported by ASCT, is the standard of care for younger patients, in spite of its non negligible toxicity. However, recent biological insights on MCL molecular pathogenesis are paving the way for both the development of new drugs and refined prognostication. Therefore, it is likely that in the near future the therapeutic approach in this disease will become more and more personalized, based on the individualized risk of relapse, and potentially ASCT will be reserved only for those cases who will really benefit from this effective, but toxic procedure. Acknowledgments The Authors would like to thank Antonella Fiorillo for the excellent secretarial support 111

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Funding SF was supported by Progetto di Rilevante Interesse Nazionale (PRIN 2009) from Ministero Italiano dell'Università e della Ricerca (MIUR), Roma, Italy (code: 7.07.02.60 AE01); Progetto di Ricerca Sanitaria Finalizzata 2009, head unit: Divisione di Ematologia S. Cortellazzo, A. O. S. Maurizio,

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Roadmap for European hematology research

The European Hematology Association Roadmap for European Hematology Research: a consensus document

OPINION ARTICLE EUROPEAN HEMATOLOGY ASSOCIATION

Ferrata Storti Foundation

Andreas Engert,1 Carlo Balduini,2 Anneke Brand,3 Bertrand Coiffier,4 Catherine Cordonnier,5 Hartmut Döhner,6 Thom Duyvené de Wit,7 Sabine Eichinger,8 Willem Fibbe,3 Tony Green,9 Fleur de Haas,7 Achille Iolascon,10 Thierry Jaffredo,11 Francesco Rodeghiero,12 Gilles Salles,13 Jan Jacob Schuringa,14 and the other authors of the EHA Roadmap for European Hematology Research

1 Universität zu Köln, Cologne, Germany; 2IRCCS Policlinico San Matteo Foundation, Pavia, Italy; 3Leids Universitair Medisch Centrum, Leiden, the Netherlands; 4Université Claude Bernard, Lyon, France; 5Hôpitaux Universitaires Henri Mondor, Créteil, France; 6 Universitätsklinikum Ulm, Germany; 7European Hematology Association, The Hague, the Netherlands; 8Medizinische Universität Wien, Vienna, Austria; 9Cambridge Institute for Medical Research, United Kingdom; 10Università Federico II di Napoli, Italy; 11Université Pierre et Marie Curie, Paris, France; 12Ospedale San Bortolo, Vicenza, Italy; 13Hospices Civils de Lyon/Université de Lyon, Pierre-Bénite, France; and 14Universitair Medisch Centrum Groningen, the Netherlands

Haematologica 2016 Volume 101(2):115-208

ABSTRACT

he European Hematology Association (EHA) Roadmap for European Hematology Research highlights major achievements in diagnosis and treatment of blood disorders and identifies the greatest unmet clinical and scientific needs in those areas to enable better funded, more focused European hematology research. Initiated by the EHA, around 300 experts contributed to the consensus document, which will help European policy makers, research funders, research organizations, researchers, and patient groups make better informed decisions on hematology research. It also aims to raise public awareness of the burden of blood disorders on European society, which purely in economic terms is estimated at €23 billion per year, a level of cost that is not matched in current European hematology research funding. In recent decades, hematology research has improved our fundamental understanding of the biology of blood disorders, and has improved diagnostics and treatments, sometimes in revolutionary ways. This progress highlights the potential of focused basic research programs such as this EHA Roadmap. The EHA Roadmap identifies nine ‘sections’ in hematology: normal hematopoiesis, malignant lymphoid and myeloid diseases, anemias and related diseases, platelet disorders, blood coagulation and hemostatic disorders, transfusion medicine, infections in hematology, and hematopoietic stem cell transplantation. These sections span 60 smaller groups of diseases or disorders. The EHA Roadmap identifies priorities and needs across the field of hematology, including those to develop targeted therapies based on genomic profiling and chemical biology, to eradicate minimal residual malignant disease, and to develop cellular immunotherapies, combination treatments, gene therapies, hematopoietic stem cell treatments, and treatments that are better tolerated by elderly patients.

haematologica | 2016; 101(2)

Correspondence: a.engert@uni-koeln.de

Received: 15/12/2015. Accepted: 27/01/2016. Pre-published: 27/01/2016. doi:10.3324/haematol.2015.136739

Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/101/2/115

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A. Engert et al.

Introduction Blood can be described as one of the human body’s largest organs. It is essentially a liquid tissue containing many different types of specialized cells needed for the normal functioning of the human body. When one or more of these cell types do not perform well, a wide variety of blood disorders can result, ranging from blood cancers and coagulation and platelet disorders to very common diseases such as anemia. Hematology is the medical discipline concerned with diagnosing and treating all of these diseases. In the European Union (EU) alone, an estimated 80 million people are currently affected with blood disorders. Various types of anemia affect more than 50 million children and adults in the World Health Organization’s European region.1 Blood cancers, some of which mainly affect young people, contribute strongly to premature cancer-related mortality and lost productivity in Europe.2 Among cancers, blood cancers [leukemia, Hodgkin and non-Hodgkin lymphomas (HLs and NHLs), and multiple myeloma] together rank third after lung cancer and colorectal cancer in terms of age-adjusted mortality in the European Economic Area.3 Inherited blood diseases, such as thalassemia, sickle cell disease, and glucose-6-phosphate dehydrogenase deficiency, also affect millions of people and cause substantial morbidity and mortality. Rarer forms of congenital blood disorders represent an immense burden on those affected. Many infectious diseases affect various types of blood or blood-forming cells, causing widespread diseases such as malaria and HIV/AIDS. In recent decades, enormous progress has been made in terms of diagnosis and treatment of these diseases. Unfortunately, many blood disorders remain incurable. Approximately 115,000 patients die each year.4 Blood disorders have immense economic consequences as well. The combined societal cost of hematologic diseases for the EU, Norway, Iceland, and Switzerland has been estimated at €23 billion per year. At a European level, current public spending on hematology research does not match this vast medical need. Of the €6.1 billion that the European Union allocated to health research under its 7th Framework Programme (2007-2013), only 2.2% (€137 million) was granted to hematology research. That amounts to less than 0.1% of the societal cost of blood disorders in Europe over that same period.

Milestones in hematology and the contribution from Europe Research in hematology has fundamentally improved our understanding of the biology of hematologic diseases and resulted in many innovative discoveries. Many of these discoveries are powerful examples of how carefully designed basic research can lead to new approaches that block or interact with key pathways in diseased cells, resulting in very impressive anti-tumor effects. European hematologists have pioneered important inventions and played leading roles in developing, for example, curative approaches for patients with malignant diseases, such as lymphomas and leukemias,5,6 which often affect young patients. 116

Key milestones included the characterization of hemoglobin (Hb),7 induced pluripotent stem cells (iPSCs),8 and somatic driver mutations.9 The discovery of the Philadelphia chromosome and the subsequent identification of the BCR-ABL1 tyrosine kinase and its role in chronic myeloid leukemia (CML)10 led to the successful development of potentially curative targeted treatment in this form of blood cancer.11 This was an unprecedented rate of success and it occurred in a malignancy that previously could only be treated by allogeneic transplant in a very select number of patients. Acute promyelocytic leukemia became one of the first malignancies that could be cured without conventional chemotherapy.12 Another key development in hematology was that of a wide range of monoclonal antibodies following the original invention by Köhler and Milstein in the UK.13 Humanized or fully human monoclonal antibodies are now used in hematology for both diagnostic and therapeutic purposes. The clinical breakthrough was a humanized monoclonal antibody targeting the CD20 antigen on B-cell lymphoma.14 Today, monoclonal antibodies or antibody-based conjugates are used successfully in most malignant lymphomas and leukemias. They can, however, also be effective in nonmalignant blood disorders such as paroxysmal nocturnal hemoglobinuria (PNH), a rare acquired clonal stem cell defect leading to increased fragility of hematopoietic cells and hemolytic anemia (HA), thrombosis, and bone marrow failure (BMF). Prognosis of patients with severe PNH used to be less than five years, but changed radically with the advent of an anti-complement monoclonal antibody that counteracts membrane fragility.15 Today, PNH patients treated with this antibody have a normal life expectancy. Severe hemophilia represents another story of unprecedented success. Patients used to be confined to wheelchairs or face the specter of death because of untreatable hemorrhage or blood-born infections such as HIV/AIDS. Today, new recombinant substitutive therapy is completely safe and effective in long-term prophylaxis. Hematology expects to further improve in this area, with innovative factor VIII or IX molecules that have increased activity and prolonged half-life. Gene therapy is becoming a reality for more and more blood diseases, while treatment of malignant and nonmalignant hematologic diseases is impossible without blood transfusions and blood-derived medicinal products. “Haemovigilance”, a European initiative that provides a surveillance registry of serious unwanted transfusion effects, is now up and running in most EU member states.

European research policy Governments, politicians and other policy makers carry the responsibility for making well informed decisions on regulation and funding priorities for health research and medicinal product development. The research community has a responsibility in providing policy makers with the kind of information and evidence that they need to make those informed decisions. With respect to research funding, the authors feel that hematology was underfunded in the EU’s 7th Framework Programme. The current Framework Programme (Horizon 2020) was spared major budget cuts, but raising the relative level of funding for hematology research needs to be improved. haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

With respect to regulation, a key issue on the table is the EU’s new regulation on clinical trials on medicinal products for human use, which will come into effect in 2016. Over the past years, the number of clinical trials in Europe has decreased. These trials are key to medical research. European research groups have been instrumental in setting up multicenter clinical trials to test important new products. However, the new regulation has the potential of making future trials in Europe too expensive and too complex to carry out, especially in terms of academic research, and, therefore, may lead to a further decrease in clinical trials. A drop in the number of trials and the number of participants would harm the interests of European patients and damage Europe’s knowledge infrastructure and future economy.

ogy societies, patients' organizations, hematology trial groups, and other European organizations in, for example, overlapping disease areas. All comments were discussed and integrated before submission of the manuscript to Haematologica. In all, around 300 European hematologists and top experts helped to create the Roadmap. At the request of the EHA board, the University of Oxford simultaneously carried out a study into the societal burden and cost of blood disorders in Europe. Outcomes from their analysis also informed various parts of this Roadmap.

The European Hematology Association Roadmap

1. developing novel targeted therapies based on genomic profiling and chemical biology; 2. unleashing the power of cellular immunotherapy; 3. eradicating minimal residual disease (MRD) in hematologic malignancies; 4. creating smarter combination treatments; 5. developing better tolerated treatments for blood disorders with a special emphasis on elderly patients; 6. using gene therapy to tackle blood disorders; 7. maximizing the clinical application of hematopoietic stem cells (HSCs) for transfusion, immunomodulation, and repair.

In 2014, at its 19th Annual Congress in Milan, Italy, the European Hematology Association (EHA), Europe’s largest non-profit membership organization in the field of hematology, decided to launch a Roadmap project. One of its goals was to better inform European policy makers and other stakeholders about the urgent needs and priorities of patients with blood diseases and the field of hematology. Another goal was to help the European hematology research community in harnessing resources by bringing basic researchers, clinical trial networks and patient advocates together in comprehensive study groups. A European consensus on medical and research priorities will also promote excellence and collaboration between academics and the pharmaceutical industry. The EHA Roadmap Task Force included EHA board members and other top experts from all fields of hematology. Hundreds of hematologists, clinical trial groups, drug makers, national hematology societies, patient representatives and others were invited to provide input and advice. Many contributed to the drafting of the document and the various stages of review. This Roadmap is the outcome of this project. It identifies the greatest unmet needs in hematology research and clinical science, describing: 1) state-of-the-art hematologic research; 2) the most urgent research priorities; and 3) the anticipated impact this research could have. The EHA Roadmap Task Force identified nine major ‘sections’ in hematology: normal hematopoiesis, malignant lymphoid and myeloid diseases, anemias and related diseases, platelet disorders, blood coagulation and hemostatic disorders, transfusion medicine, infections in hematology, and hematopoietic stem cell transplantation (HSCT). For each section, the Roadmap Task Force appointed one or two editors. Together, the Roadmap Task Force and section editors drafted and reviewed a more detailed framework of 60 ‘subsections’ of groups of diseases and conditions. Section editors selected experts from their various fields to contribute as subsection editors or authors. Each section and subsection adapted the same basic format. Draft texts and figures were discussed by the Roadmap Task Force and section editors during three meetings between October 2014 and March 2015. Sections were then reviewed by the Roadmap Task Force, the EHA board, and a selection of experts. The final draft was sent for consultation to stakeholders such as national hematolhaematologica | 2016; 101(2)

Some dominating topics and unmet needs can be recognized in nearly all of the nine EHA Roadmap sections. They include:

Taken together, this EHA Roadmap highlights major past achievements in the diagnostics and treatment of blood disorders, identifies unmet clinical and scientific needs in those same areas, and will enable better funded and more focused European hematology research. The EHA will pro-actively bring this Roadmap to the attention of all stakeholders involved in hematology, and calls upon those stakeholders to do the same.

Acknowledgments The authors wish to thank all who contributed to the creation of this document.

The EHA Roadmap for European Hematology Research Section 1. Normal hematopoiesis

Section editors: Jan Jacob Schuringa, Thierry Jaffredo. Hematopoiesis, the formation of blood, is initiated in our bone marrow by hematopoietic stem cells (HSCs), first identified by Till and McCulloch in the 1960s. After cell division, these HSCs can generate progenitor cells that gradually differentiate into all the erythroid, myeloid, and lymphoid lineages that reconstitute our blood. Via a process termed self-renewal, they are also able to generate new stem cells to ensure a lifelong reservoir of HSCs. In the past decades, excellent in vitro and in vivo model systems have been generated that have allowed us to obtain a thorough understanding of hematopoiesis at the molecular and cell biological level. HSCs were also the first stem cells that were used in a clinical setting through bone marrow transplantation (BMT). It is, therefore, not surprising that the hematopoi117

A. Engert et al. etic system has served as a paradigm for the study of many other stem cell types as well. We have learned much about growth factors and cytokines that regulate the fate of HSCs and their progenies. With the availability of genome-wide multiomics technologies, transcription factor (TF) networks and epigenetic landscapes of cells within the hematopoietic hierarchy are currently being characterized at a rapid pace. Step by step, we are now beginning to understand how these are interlinked and how they control the transcriptomes and proteomes of hematopoietic cells. We have learned a lot about the microenvironment within the bone marrow that keeps HSCs in their quiescent state and regulates their self-renewal. We have learned about how and where HSCs are formed during embryogenesis, and we are also beginning to better understand how HSCs age. Fundamental translational research has been critically important in getting us where we are today. But still many questions remain. Among many others, these include the question as to how (epi)genetic aberrations cause hematologic malignancies, and how we can use these insights to develop better therapeutic strategies. It is now being realized that there is a clonal heterogeneity in many hematologic cancers, and possibly even within the normal HSC compartment. But how does this affect disease development and current treatment options? In contrast to adult life, HSCs are rapidly expanding during embryogenesis. So can we unravel those mechanisms and apply them to in vitro HSC expansion protocols for clinical use? A thorough understanding of embryonic versus adult hematopoiesis might also help us to better understand the differences between childhood and adult hematologic malignancies. Reprogramming now allows patient-specific induced pluripotent stem cells (iPSCs) to be generated, but the generation of fully functional HSCs from these is still rather challenging. Can this be improved? We live in a continuously aging society, but how does HSC aging actually affect health and disease? Within the first section, we have brought together leading scientists and clinicians in the field of hematopoiesis. They provide an overview of the current status of the field and an outlook on where future research should be directed (Figure 1). We firmly believe that combining fundamental and translational research will result in not only a better understanding of the hematopoietic system, but also in the development of better therapeutic approaches for hematologic malignancies, many of which are still difficult to treat.

1.1. Erythropoiesis Sjaak Philipsen (Erasmus MC, Rotterdam, the Netherlands), Joan-Lluis Vives Corrons (Universitat de Barcelona, Barcelona, Spain), Lucia de Franceschi (Università degli Studi di Verona, Verona, Italy), Olivier Hermine (Université Paris Descartes, Paris, France), Douglas Higgs (University of Oxford, Oxford, United Kingdom), Marina Kleanthous (Cyprus School of Molecular Medicine, Nicosia, Cyprus).

Introduction The major cell type in our blood is the red blood cell (RBC) or erythrocyte. RBCs transport oxygen from the lungs to other parts of the body, and from there they carry carbon dioxide back to the lungs. An adult has approxi118

mately 5 liters of blood, containing 25x1012 RBCs. Because the lifespan of an RBC is approximately 120 days, a healthy person needs to produce 2.4x106 RBCs per second to maintain a constant number of RBC.16 The oxygen carrier hemoglobin (Hb), composed of two α-like and two b-like globin proteins, makes up approximately 90% of soluble protein in RBCs. RBCs and Hb form during a process called erythropoiesis, which includes the initial specification of HSCs from mesoderm during embryogenesis, the decision of these cells to self-renew or differentiate, the process of proliferation and erythroid specification, and, finally, their terminal differentiation and post-mitotic maturation. Terminally differentiating erythroid cells extrude their nucleus and shed their endoplasmic reticulum and mitochondria. The new cells enter the circulation as reticulocytes, which are still engaged in protein translation. Finally, the population of mature, biconcave RBCs with diameters of only 6-8 micrometers creates a large surface area for gas exchange, which, through RBC membrane deformability, extends from major blood vessels into the microcirculation. Abnormally low Hb levels cause anemia. Approximately one-third of the world’s population has some form of anemia, making this diverse group of disorders by far the most common clinical problem worldwide. Perturbation of erythropoiesis might be acquired and related to iron deficiency or to different systemic disorders associated with chronic inflammation (e.g. autoimmune diseases and cancers) or myelodysplasia. A multitude of different inherited anemias affect erythropoiesis by diverse mechanisms, such as thalassemias (by reduced or absent functional Hb), sickle cell disease (SCD) (by a pathological Hb variant), HAs (by defects in membrane proteins, metabolic enzymes, or pathological Hbs), Diamond Blackfan anemia (DBA) (by impaired ribosome biogenesis), Fanconi anemia (FA) (by DNA repair defects), and congenital dyserythropoietic anemia (CDA) (e.g. CDA type II by defects in protein trafficking). Polycythemia vera (PV), although not limited to erythropoiesis and also seen in myeloproliferative neoplasms (MPNs), is caused by activating JAK2 kinase mutations. The physiological and molecular mechanisms underlying these disorders are still not completely understood, while erythroid defects are also associated with many other, and often still unknown, genetic defects. Elucidation of normal erythropoiesis is, therefore, essential to develop new strategies for treating the wide variety of conditions affecting the erythroid system.

European research contributions Historically, research of the hematopoietic system has driven novel biological concepts and methods, owing to the accessibility and ready purification of hematopoietic progenitor cells (HPCs) for molecular and functional analyses. Early European contributions included the Nobel Prize winning discovery of the structure of Hb7 and understanding the etiology and epidemiology of inherited anemias, leading to implementation of pre-natal diagnostic programs.17 Other European contributions include determining the origin of hematopoietic stem cells (HSCs), the transcriptional circuitry underlying erythropoiesis, the molecular control of differentiation versus apoptosis, the role of iron metabolism, and DNA sequences driving high-level expression of Hb, which are now applied in gene therapy vectors. Other discoveries, such as the roles of serotonin and transferrin receptors, also heralded significant haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

progress in our understanding of normal erythropoiesis. Recently, purified cells have been characterized using â&#x20AC;&#x153;omicsâ&#x20AC;? techniques to determine their transcriptional profiles, epigenetic programs, and responses to cell signaling. A database dedicated to erythroid disorders has been established18 aiming to integrate data from fundamental and translational research with data from routine clinical care. Translational research has resulted in optimized BMT protocols, magnetic resonance imaging (MRI) monitoring of iron overload, improved iron chelation therapies, and targeted inhibition of signaling pathways mutated in (pre)leukemic conditions.19

Proposed research for the Roadmap Previous research has laid the foundation on which a comprehensive framework for understanding erythropoiesis can be built. Next generation sequencing (NGS) technologies have opened up exciting new avenues for qualitative and quantitative biology, with unprecedented sensitivity and specificity. For instance, mutation detec-

tion in single cells is now possible, and quantitative gene expression profiles can be generated from hundreds of individual cells in a single experiment. For the first time, this allows hierarchical relationships between cells of a single lineage to be determined and the impact of cell-cell interactions and signaling cascades on erythroid development to be unraveled. Although pioneering research will rely on the use of cellular and animal model systems, the protocols developed will be quickly translated to the study of erythropoiesis in human subjects, taking full advantage of single-cell omics analyses. Our goal is to apply this deeper understanding of erythropoiesis to improve diagnosis, prognosis, and treatment algorithms for patients with conditions affecting the erythroid system.

Anticipated impact of the research Understanding the basic physiological and molecular mechanisms of normal erythropoiesis will have a direct and long-lasting impact on the medical care of patients with hereditary and acquired anemias. Firstly, improved diagno-

Figure 1. An overview of the current â&#x20AC;&#x2DC;normal' hematopoiesis field and an outlook on where future research should be directed.

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A. Engert et al. sis will enable clinicians to predict disease progression for individual patients much more accurately, leading to betterinformed decisions on disease management by transfusion, iron chelation, cytokines or cytokine inhibitors, and splenectomy. Secondly, fundamental knowledge of normal erythropoiesis will likewise guide the development of safer and more effective curative treatments, such as those involving gene correction or gene therapy, and identification of new therapeutic targets to be exploited in public-private partnerships for the development of new treatments for erythroid disorders. Thirdly, an increased understanding of the microenvironment and cell signaling mechanisms will enable the development of clinical algorithms for management of patients with anemias. Fourthly, in vitro generation of fully functional human erythrocytes will ultimately bring completely defined and guaranteed disease-free human blood units to the clinic, with a major impact on transfusion medicine. Finally, the erythroid system represents a fascinating and tightly regulated process of proliferation, survival, and differentiation and has always served as a paradigm for other biological systems. New fundamental insights into erythropoiesis will, therefore, likely continue to facilitate discoveries in all fields of medicine, leading to an improved understanding of disease mechanisms and better clinical care for patients.

1.2. Myelopoiesis Kim Theilgaard-Mönch (Københavns Universitet, Copenhagen, Denmark), Niels Borregaard (Københavns Universitet, Copenhagen, Denmark), Jörg Cammenga (Linköpings Universitet, Linköping, Sweden), Ruud Delwel (Erasmus MC, Rotterdam, the Netherlands), Henk Stunnenberg (Radboud Universiteit, Nijmegen, the Netherlands), Ivo Touw (Erasmus MC, Rotterdam, the Netherlands).

Introduction Myeloid cells, including granulocytes, monocytes/ macrophages, and dendritic cells, are key effector cells of the innate immune defense against invading micro-organisms.20 Myeloid cells are continuously generated from hematopoietic stem cells (HSCs) in the bone marrow through a tightly regulated process referred to as myeloid differentiation or myelopoiesis. This complex process is regulated in part by growth factors and epigenetic and transcriptional regulators that in concert orchestrate cell survival, proliferation, and, most importantly, instruction of lineage-restricted differentiation of HSCs via a series of hematopoietic progenitor cells (HPCs) into all types of fully mature myeloid cells. For the past two decades, a plethora of studies have demonstrated the pathognomonic link of genetic aberrations in myeloid key regulators with several hematologic disease entities, such as acute myeloid leukemias (AMLs), myeloproliferative neoplasms (MPNs), and severe congenital, as well as cyclic neutropenia. Hence, characterization of myeloid regulators and their function during steady-state hematopoiesis at both the molecular and systems level is important to understand: 1) the biology of myeloid differentiation and innate immune defense; and 2) how genetic aberrations of myeloid regulators affect normal myeloid differentiation and cause myeloid disease.

European research contributions Lineage priming is an idea that was brought forward by scientists in Europe. The concept of lineage priming, 120

which postulates that lineage-specific transcription factors (TFs) are already present in uncommitted HSCs, is now widely accepted. European scientists have also made substantial contributions to the understanding of TF networks in hematopoiesis, as well as identification of the earliest cells in the hematopoietic hierarchy that can give rise to myeloid cells. Moreover, research groups in Germany and the Netherlands have identified mutations in the granulocyte colony-stimulating factor receptor as the cause of severe congenital neutropenia, and have shown that treatment with granulocyte colonystimulating factor can improve survival but also lead to AML.21 Later, HAX1 and JAGN1 mutations were identified by a German research group as another genetic cause for severe congenital neutropenia. Other European researchers have pioneered our understanding of neutrophil differentiation and antimicrobial granule proteins of neutrophils.22 The role of the TF CEBPA as a key regulator in normal and malignant myeloid differentiation was pioneered by European research groups. Specifically, these groups applied genetic models to uncover the important role of CEBPA for granulocytic lineage commitment and differentiation, as well as the causative role for mutant CEBPA in AML, thereby dissecting the complexity of how biallelic CEBPA mutations contribute to leukemogenesis.23,24

Proposed research for the Roadmap The advent of novel comprehensive omics technologies, such as RNA-seq/microarray analyses, miRNA array analysis, ChIP-seq analyses of transcriptional and epigenetic regulators, metabolomics, and finally proteomics and phosphoproteomics, allow us to define phenotypes of cellular states at the systems level. The current proposal is to apply a comprehensive omics strategy to improve our understanding of normal myeloid differentiation and innate immune defense at a systemic level and how genetic aberrations cause perturbations of normal cellular activities, resulting in myeloid disease phenotypes. Given this, the proposal will combine omics technologies and comprehensive cell sorting to generate a state-ofthe art reference omics data set of prospectively purified human bone marrow populations representing successive stages of myeloid differentiation (i.e. HSCs, myeloid progenitors, and mature myeloid cells) in healthy subjects. The resultant data set will improve our understanding of how dynamic regulatory networks control cell fate and function during normal myeloid differentiation and immune defense. Moreover, the research community will be able to match the resultant normal reference omics data set with omics data sets of sorted bone marrow populations from patients with myeloid diseases harboring defined genetic aberrations. This strategy will allow a standardized omics data set comparison of normal and disease states, which will unravel how specific genetic aberrations in patients promote aberrant cellular activities (e.g. signaling, proliferation, metabolism, apoptosis, etc.) underlying the phenotype of specific myeloid diseases. Significantly, comprehensive data mining of the normal “reference” and patient omics data sets will allow us to identify novel diagnostic markers, as well as targets for therapeutic interventions, and ultimately improve treathaematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

ment and clinical outcome of patients suffering from AML, MPNs, and other myeloid diseases. A co-ordinated European effort involving basic researchers, clinical researchers, and bioinformatic technicians is required to achieve the following aims of the proposal. 1. Establishment of a European expert group that will discuss and define a standard for cell sorting of myeloid cells, applied omics technology platforms, and the development of bioinformatic methodologies for integrated omics and clinical data analysis. 2. Establishment of core facilities/hospitals for standardized collection and biobanking of human bone marrow samples from healthy subjects for the project. 3. Establishment of core facilities/research teams for sorting of bone marrow populations according to the standard sorting strategy defined by the expert group (see point 1). 4. Establishment/identification of core facilities/research teams for omics analysis of sorted bone marrow populations according to the consensus omics standard platform defined by the expert group (see point 1). 5. Establishment of a European core bioinformatics group for concerted processing and analysis of omics data in order to generate a â&#x20AC;&#x153;referenceâ&#x20AC;? omics data set of normal myeloid differentiation. Ideally, the bioinformatics group will also assist European clinicians with standardized comparison of the obtained reference omics data set and omics data sets of patients enrolled in clinical trials. In addition, the bioinformatics group will develop an open-access web-based platform allowing researchers worldwide to download and match omics data from patients with myeloid diseases for comparison with the reference omics data set.

Anticipated impact of the research The proposed research program relies on a concerted multidisciplinary European effort to generate a comprehensive omics reference data set of myeloid differentiation. The resultant data set represents an extremely powerful tool for the research community, as it can be used in part as a reference of how expression and activity of genes, proteins, or signaling pathways change during normal myeloid differentiation and are perturbed by genetic aberrations in myeloid diseases. Significantly, the latter is important for pre-clinical and clinical research programs aiming at identifying: 1) novel diagnostic markers for improved prognostication; and 2) novel therapeutic targets for the development of more effective treatment modalities improving survival of patients with AML, MPNs, and other rare myeloid diseases.

in the blood shear of their marrow precursors, namely the megakaryocytes. A large part of the platelet production is regulated by the megakaryocyte size through polyploidization. The regulation of megakaryopoiesis is dependent on a cytokine/hormone called thrombopoietin (THPO), which signals through the MPL receptor. However, THPO is not directly involved in the last differentiation steps directly responsible for platelet production. In terms of development, megakaryopoiesis is extremely close to erythropoiesis, and the regulation of megakaryopoiesis and HSCs unexpectedly share many common features concerning gene transcription and regulation by THPO with the presence of megakaryocytebiased hematopoietic stem cells (HSCs). Megakaryopoiesis is affected by numerous acquired and hereditary disorders. Most of them target the THPO/MPL signaling or the actin and tubulin cytoskeletons, which play a central role in late stages of megakaryopoiesis.25-28 Platelet transfusion is the most common way to treat profound thrombocytopenia, but this increasing need in platelet transfusion is limited by a donor deficit, and thus, there is now a place for alternative approaches, including ex vivo platelet production and small molecules stimulating platelet production in vivo. All of these approaches require major progress to be made in basic research.

European research contributions Researchers from Europe have played a central role in understanding the regulation of megakaryopoiesis. 1. They have been pioneers in the identification of the MPL/THPO axis and the main transcription factors (TFs) (GATA1, FLI1, TAL1, and LYL1). 2. They have largely contributed to the mechanisms of polyploidization and proplatelet formation. 3. They have developed new investigational techniques, such as 2-D and 3-D cultures, videomicroscopy, and use of shear to produce platelets.29

Proposed research for the Roadmap The major topics that require intense research resources and efforts are listed here. Mechanisms of megakaryocyte commitment and differentiation from HSCs: defining these different cellular steps in terms of transcription factors, epigenetic regulators, and growth factors involved in this cellular process will be important to: 1) increase platelet production in vivo; 2) develop in vitro techniques for somatic cell reprogramming toward the megakaryocyte lineage; and 3) increasing the megakaryocyte potential of induced pluripotent stem cells (iPSCs).

1.3. Megakaryopoiesis William Vainchenker (Institut Gustave Roussy, Villejuif, France), Alessandra Balduini (UniversitĂ degli Studi di Pavia, Pavia, Italy), Cedric Ghevaert (University of Cambridge, Cambridge, United Kingdom).

Introduction Megakaryopoiesis is the differentiation process that leads to platelet production. This is a unique cell biology system, because platelets arise from the fragmentation haematologica | 2016; 101(2)

Further characterization of the THPO/MPL functions: MPL plays a central role in regulating megakaryopoiesis through direct signaling, as well as by clearing THPO. Studies of THPO synthesis, the precise MPL signaling pathways, including their consequences on gene regulation and MPL cell trafficking will be important for understanding the mechanisms of thrombocytopenia or thrombocytosis and for developing new molecules capable of positively or negatively regulating MPL. Determination of the precise mechanisms of polyploidization: 121

A. Engert et al. the processes of endomitosis and its regulation by both extrinsic and intrinsic mechanisms, including ontogenesis processes, are poorly known. A complete understanding will be important for developing in vitro cell systems able to produce highly polyploid megakaryocytes. In addition, this topic might be relevant to understanding the processes of polyploidization in malignant tumors. Regulation of platelet formation: in order to discover disease mechanisms and new therapeutic targets, it is fundamental to understand how megakaryocytes differentiate and form platelets. These mechanisms include all the bone marrow environment molecules that participate in the regulation of TFs and biochemical signaling through activation of specific receptors. New cellular techniques, including single-cell assays, should be developed, generating candidate regulators of this crucial step of platelet production. Intrinsic cellular determinants, such as the actin and tubulin cytoskeletons and their regulation, will have to be studied. It will be important to determine the precise mechanism of platelet abscission and the role of the shear. These approaches may also provide candidate molecules implicated in proplatelet formation and the migration of megakaryocytes in the marrow. The endothelium participates in the regulation of megakaryocyte function, and megakaryocytes have to remodel the basement membrane of the sinusoids in order to extend proplatelets through the vascular wall of the bone marrow sinusoids. It will be important to understand how proplatelets interact with the endothelium to reach the blood flow and be released, and how megakaryocytes and endothelial cells mutually regulate their behavior. New processes to be considered also include the role of calcium in megakaryocyte development and the importance of autophagy in megakaryocyte development within the bone marrow environment. Mutual regulation of megakaryocyte and bone marrow environment: it is known that megakaryocytes are able to express and release different molecules that may regulate bone marrow homeostasis in both steady-state, postinjury conditions, and in malignant and inflammatory disorders. Alteration of these processes may lead to pathological conditions or support diseases. On this basis, it is important to understand which molecules megakaryocytes actively express and release, and their role in the bone marrow regulation.

Anticipated impact of the research In recent decades, much progress has been made in our knowledge of megakaryopoiesis, but better understanding its regulation and the function of the increasing number of genes found to be mutated in pathology will require significant effort. The precise understanding of endomitosis and platelet formation may have important clinical consequences, particularly for developing new technologies for large platelet production in vitro and also new molecules capable of modifying platelet production in vivo. Europe has played a leading role in studies of megakaryopoiesis, but research has remained fragmented. Integrated European programs will provide the critical mass of resources and expertise needed to develop the large ambitious programs required to proceed from basic to clinical research. 122

1.4. Lymphopoiesis Isabelle Andre-Schmutz (Université Paris Descartes, Paris, France), Jean-Christophe Andrau (Institut de Génétique Moléculaire de Montpellier, Montpellier, France), Sophie Ezine (Université Paris Descartes, Paris, France), Francoise Pflumio (Institut de recherche en radiobiologie cellulaire et moléculaire (IRCM), Paris, France), João Pedro Taborda Barata (Universidade de Lisboa, Lisbon, Portugal), Tom Taghon (Universiteit Gent, Ghent, Belgium).

Introduction The immune system constitutes the body’s defense system against disease and foreign cells/micro-organisms. Immune cells are diverse, conventionally divided into innate and adaptive subsets. B and T lymphocytes are the main protagonists in adaptive immunity and are generated by a complex process referred to as lymphopoiesis, which involves numerous finely regulated steps, including the lymphoid-specific somatic rearrangement of genes encoding the immunoglobulins and T-cell receptors for antigens. Lymphocytes are generated within defined microenvironments that provide the growth factors and signals necessary for their commitment, survival, expansion, and education to self-/non-self-discrimination. A deep understanding of the steps that allow the production and amplification of lymphoid precursors and mature cells is crucial in order to obtain an efficient immune system, which is important for lymphoid-based therapies in humans.

European research contributions European groups have a strong research record in the field of lymphopoiesis and achieved the first successful gene therapy protocols for human genetic defects affecting lymphopoiesis.30 They contributed to the development of in vitro assays, allowing studies on T- and B-cell differentiation using bone marrow-derived stromal cell/progenitor and organotypic thymic cultures further complemented by humanized murine models in which the generation of human T and B cells could be studied.31 Such tools helped identify murine and human lymphoid progenitors, as well as thymus-seeding progenitors. Moreover, lymphoid progenitor expansion conditions for cell therapy purposes have been recently described. Pioneering European studies also characterized the thymic microenvironment, crosstalks between the epithelium and developing T cells, and the role of the Notch pathway and regulators in the molecular regulation of T-cell differentiation.32 Other groups focused on the early steps of B-cell development in mice and identified progenitor B-cell regulators that modulate pathological immune responses. Studies on human B-cell deficiencies emphasized species differences between mice and humans, underlining the need for the establishment of novel research strategies that help us understand speciesspecific peculiarities and the common features of B- and T-cell development. Several pathologies involving lymphoid deficiencies were found to be caused by defects in various steps of V(D)J recombination, implicating general and specific mechanisms of DNA repair in lymphopoiesis and telomere maintenance.33 Finally, innate lymphoid cell and natural killer (NK)-cell development were described,34 bringing robust cell role-players to develop new therapies. haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

Proposed research for the Roadmap Mature lymphocytes and their subsets concomitantly arise from very infrequent progenitor subsets that are not well characterized. These processes take place in complex “niches,” the settings of which are only just being unraveled. Dissecting the role of various non-hematopoietic and hematopoietic cell subsets of the bone marrow and thymus involved in lymphoid development/progenitor maintenance will favor identification of the major players in steady-state conditions and new molecular targets for anti-cancer and anti-viral immunotherapies. This knowledge will be important for modeling normal T/B/NK-cell development within their specific niches, using scaffolds as surrogate thymic and bone marrow niches to help uncover side effects and resistance mechanisms observed in chemotherapies. A precise description of the migratory pattern of developing T and B cells will be necessary to efficiently transplant newly generated, ex vivo lymphoid progenitors from different HSC sources, including induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs). Identification of drugs able to mobilize lymphoid progenitors for cell/gene therapy also needs to be investigated. Further studies are required to assess the impact of these and other drugs/small molecules on normal developmental processes, such as hematopoiesis. Such experiments will support the design of new assays for human lymphoid development. They should also stimulate drug research for acute lymphoblastic leukemias (ALLs). For realistic lymphoid-based therapies to become “routine” in the near future, one should learn from previous experiences with the successes achieved in gene therapy. These experiences emphasize the continuous need for modification of existing viral vectors to improve transduction of lymphoid progenitors, as well as the development of lymphoid-specific protocols of gene therapy. Improved modeling of human disease and cellular therapy of immune deficiencies is also essential and is required to support the development and expansion of lymphoid cells from iPSCs or ESCs, as well as to generate lymphoid progenitors ex vivo from isolated hematopoietic cell populations. Identification of human HSCs biased toward Tand B-cell production will facilitate understanding of the origin of lymphoid cells and their potent expansion prior to transplant into patients.

pathogens and neutralize autoimmunity and response to tumor cell growth. Additional knowledge about normal lymphopoiesis will undoubtedly benefit new drug discovery for leukemia/lymphoma/myeloma therapies.

1.5. Hematopoietic stem cells Gerald de Haan (Universitair Medisch Centrum Groningen, Groningen, the Netherlands), Dominique Bonnet (The Francis Crick Institute, London, United Kingdom), Hartmut Geiger (Universitätsklinikum Ulm, Ulm, Germany), Gerwin Huls (Universitair Medisch Centrum Groningen, Groningen, the Netherlands).

Introduction Hematopoietic stem cells (HSCs) were the first adult stem cells to find their way into the clinic. Indeed, each year more than 50,000 patients receive HSCs for various (benign and malignant) diseases. Therefore, HSCs are frequently regarded as a role model in adult stem cell biology. Notwithstanding their very successful and beneficial clinical applicability, many aspects of basic HSC biology remain unresolved, precluding additional rational approaches to further expand their use, or that of their more mature cellular derivatives, in the clinic. In this subsection, we will briefly discuss research topics that will need to be addressed to further expand and maximize the impact of the use of HSCs (and/or their progenies) in the clinic.

Proposed research for the Roadmap and anticipated impact of the research HSC heterogeneity and clonal diversity: it is still not known how many stem cells contribute to blood cell development in normal individuals, nor whether all contributing HSCs in fact contribute equally.35 Multiple hypotheses have been put forward over the past decades, yet we do not know how many HSCs there are in the human body, how many of these cells contribute to blood cell production, how long individual stem cells remain active, whether active stem cells can become dormant and whether this process is reversible, and to what extent active stem cells differ individually in their contribution to the various blood cell types.

Anticipated impact of the research

HSC aging and rejuvenation: most patients who develop a hematologic disease (including anemia, immune senescence, lymphoma, myelodysplasia, and chronic and acute leukemias) are older, typically 65 years and over. It has become apparent that aged HSCs suffer from multiple functional defects: they show reduced self-renewal, overall produce fewer mature cells per stem cell, and are impaired in terms of generating lymphocytes. The molecular causes of these defects have not been defined, but may involve DNA damage, telomere attrition, erosion of epigenetic marks, replication stress, or loss of cell polarity, or, indeed, may result from microenvironmental perturbations.36 Elucidation of the molecular cause(s) of stem cell aging will be required to assess whether, and how, it may be possible to reverse it. Reversion or prevention of stem cell aging will contribute to delaying age-related hematopoietic deficiencies.

The primary goals of such studies are lymphoid-based therapies, the maintenance of a healthy immune system in the elderly, and the engineering of personalized treatment. The tools developed should enable clinicians to reconstitute the lymphopoiesis of patients carrying invading

Generation of HSCs from non-stem cells: in the postYamanaka era, many labs are attempting to generate bona fide HSCs from induced pluripotent stem cells (iPSCs), or indeed to embark on direct conversion of non-HSCs to

At the molecular level, epigenetic changes and major regulatory signals (microRNA, long non-coding RNA, and chromatin modifications) need to be explored by developing robust genome-wide protocols on small cell subsets. This may help to: 1) define an “epigenetic and transcriptomic ID card” of aforementioned progenitors and B- and T-subsets in normal and pathological development; and 2) evaluate the consequences of infections, inflammations, irradiations, and hypoxia in this development. A major factor that compromises the immune system is aging, and future studies should be aimed at maintaining a fit, longlived lymphopoietic compartment.

haematologica | 2016; 101(2)

123

A. Engert et al. transplantable stem cells. Whereas these attempts initially proved to be very cumbersome, substantial progress has recently been made in this field. It has been proved possible, using an array of transcription factors (TFs), to induce hematopoietic (stem) cell activity, while the molecular mechanisms are still unknown.37 The generation of functional HSCs from non-stem cells will greatly expand the clinical use of stem cells and their differentiated progenies. HSC expansion, transplantation, and homing: HSCs are very rare cells, and many attempts have been made to amplify them in order to improve engraftment kinetics after transplant and to allow manipulation of these cells prior to transplantation. In the light of very significant clinical progress in the field of (hematopoietic) gene therapy, the ability to maintain, grow, and expand HSCs during gene transduction protocols becomes more important than ever. Classical protocols that used cytokines have not been successful in expanding stem cells. More recently, however, small molecule-based approaches have suggested that the massive expansion that occurs in vivo when few stem cells are transplanted to conditioned recipients can be recapitulated in vitro. Efforts to explore such expansion protocols are highly warranted and should include studies aimed at increasing the homing efficiency of transplanted stem cells to their proper niche in the bone marrow. Novel imaging tools have been developed to record, in real time, the lodging of transplanted cells to specific preferred sites.38 Such tools and the insight they provide are essential for developing methods to improve homing and to identify the microenvironmental cells to which normal and leukemic cells home in transplantation settings. HSCT will benefit from in vitro manipulation of the graft to expand more desired cells and decrease the number of less desired cells in certain conditions (i.e. facilitate the development of “designer grafts”). HSC transformation, cell of origin, and pre-leukemia: it has become clear that the identity of the hematopoietic stem or progenitor cell in which a leukemic event first arises plays a crucial role in the biology of the disease. It seems plausible that the epigenetic context in which a (pre)leukemic lesion first arises plays an important role in establishment and progression of the disease. Preleukemic lesions have been identified that appear to confer a proliferative advantage to not yet transformed primitive cells, the progenies of which then continue to accumulate additional mutations.39 Cataloging these preleukemic events and identification of the primitive cells in which they first arise will be crucial in order to embark on approaches that allow very early detection of aberrant hematopoiesis. Understanding the value of these preleukemic events in the predisposition of developing a fullblown leukemia will allow us to screen mutations in elderly patients’ blood and decrease the risk of leukemia development. In addition, despite our knowledge of the mutation landscape present in leukemic stem cells (LSCs), the importance of the order in which a mutation or mutations occurred might provide us with a better understanding of the co-operative effect of different mutations. Furthermore, understanding the biology of stem cells will provide insight into how leukemic (stem) cells hijack specific biological processes exploited by stem cells to remain undifferentiated. Consequently, this insight should result in better treatment strategies. 124

1.6. Developmental aspects of hematopoiesis Elaine Dzierzak (University of Edinburgh, Edinburgh, United Kingdom), Anna Bigas (Institut Hospital del Mar d'Investigacions Mèdiques, Barcelona, Spain), Charles Durand (Université Pierre et Marie Curie, Paris, France), Thierry Jaffredo (Université Pierre et Marie Curie, Paris, France), Alexander Medvinsky (University of Edinburgh, Edinburgh, United Kingdom), Roger Patient (University of Oxford, Oxford, United Kingdom), Irene Roberts (University of Oxford, Oxford, United Kingdom).

Introduction Throughout adult life, the hematopoietic system is a highly dynamic, self-renewing hierarchy of cells founded by robust hematopoietic stem cells (HSCs) that produce billions of mature blood cells daily. How these self-renewing HSCs in the bone marrow are first generated during embryonic life is only beginning to be understood. Already, the clinical use of umbilical cord stem cells suggests that ontogenetically early cells have a therapeutic advantage. Research into the mechanisms of hematopoietic fate determination, expansion, and homing during embryogenesis and in successive ontogenetic microenvironments will be instrumental to the production and amplification of HSCs from pluripotent stem cells (PSCs), two current issues in regenerative medicine, and this knowledge will have relevance in understanding/treating hematologic disease and childhood leukemias. Europe has been, and continues to be, the major leader in the field of developmental hematopoiesis. Among the key challenges are: 1. HSC generation: currently limited to the embryo and occurs through the transdifferentiation of endothelial cells; 2. extrinsic microenvironmental and cell-intrinsic factors that govern the HSC generative program; 3. by understanding and harnessing the program of hematopoietic cell ontogeny, it may be possible to reprogram somatic cells and produce HSCs for regenerative therapies and leukemic treatments.

European research contributions European developmental biologists spearheaded the search for the embryonic origins of the adult hematopoietic system.40,41 As demonstrated in chick and frog embryos, the adult blood system originates in an intraembryonic site encompassing the dorsal aorta, whereas the yolk sac contributes to transient embryonic hematopoiesis. Extensive analyses in these models and in the mouse and human intraembryonic aorta-gonadmesonephros region clearly demonstrate that the first adult-type HSCs are generated in that region in a process called endothelial-to-hematopoietic transition. This surprising natural cell transdifferentiation event was revealed by real-time imaging of the developing aorta in mouse and zebrafish embryos. The late embryonic development of these potent HSCs has raised the question of why there are many earlier, less potent hematopoietic progenitors in the embryonic hematopoietic tissues, such as the yolk sac, placenta, and fetal liver.40,41 Early hematopoietic cells (primitive erythrocytes, macrophages, etc.) support the growth of the embryo, may influence the generation of HSCs, and prepare the developing hematopoietic microenvironments to receive the cells of the adult haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

hematopoietic system. The unique complexity of HSCs arises through several maturational steps orchestrated by molecular regulators. Mouse deficiencies for hematopoietic transcription factors (TFs) (many of them involved in leukemic chromosomal translocations) and in vitro hematopoietic differentiation of embryonic stem cells (ESCs) have facilitated the identification of pivotal regulators.

Proposed research for the Roadmap The key factors initiating HSC generation and the adult hematopoietic stem cell program will be found by comparing gene expression profiles obtained from embryonic endothelial/HSC precursor cells and the first HSCs generated in the aorta.42 Computational methods and the comparative analysis of the transcriptomes of HSC subsets across different vertebrate models will also further define the molecular signature of HSCs through the identification of TF complexes and epigenetic regulators that each play a role in modulating the hematopoietic program during ontogeny. Mouse models and systems biology approaches are beginning to provide insight into the molecular differences between embryonic, fetal, and adult hematopoietic cells and their specific developmental microenvironments.43 Considerable advances have been made regarding the cellular complexity of the bone marrow HSC niche, but very little is known about specific cellular components and the molecular signatures of the cells within the HSC supportive niches of the developing embryo. Examination and comparison of the gene regulatory networks active in the aorta-gonad-mesonephros, placenta, and fetal liver hematopoietic supportive microenvironments will be instrumental for identifying the molecular pathways involved in specific processes, such as the emergence, amplification, and differentiation of hematopoietic progenitors and stem cells. This knowledge can then be interpreted in the context of childhood leukemias that resolve at later developmental stages. For example, Down syndrome trisomy 21 impacts fetal, neonatal, and childhood hematopoiesis, and expands HSCs and megakaryocyte-erythroid progenitors.44 Acquired GATA1 mutations in these cells lead to abnormal myelopoiesis. These initiating events are occurring in utero during embryonic and fetal stages when hematopoietic progenitors represent a major part of the hematopoietic system. The transient nature of these progenitors prevents their lifelong persistence and acquisition of additional mutations leading to leukemia. Understanding the cellular targets of particular leukemias during the different developmental stages and in the different microenvironmental compartments presents an important challenge for ongoing and future research. Induced pluripotent stem cells (iPSCs) from patients and animal models in which leukemia can be induced during development are among the options for such studies. The ex vivo expansion of HSCs for clinical transplantation has continued to be a major challenge in the field despite many years of research. Our knowledge of the TFs and other regulators that play a role in endothelial-tohematopoietic transition, together with the molecular programs of HSCs and their surrounding microenvironments, hold promise for unlimited production of such cells for therapeutic purposes. These data will also have an impact haematologica | 2016; 101(2)

on how to generate HSCs from PSCs. Yamanaka-style reprogramming may allow the de novo production of HSCs via gene transduction/factor stimulation of endothelial cells or other somatic cell types. In Europe, some success is currently being seen in the production of blood and platelet production from iPSCs.

Anticipated impact of the research Taken together, by understanding all the hematopoietic cell types, progenitors, and stem cells produced in embryonic, fetal, and neonatal stages, we will begin to establish how the hematopoietic microenvironment is shaped, what mechanisms co-operate in regulating the emergence and amplification of HSCs, how this relates to changes in HSC heterogeneity during ontogeny, and how leukemia is initiated at developmentally early stages. These findings are sure to have an impact on treatment regimens, especially during postnatal periods. Moreover, once we are able to directly establish HSCs by reprogramming somatic cells from the patient, graft rejection issues will become a thing of the past and will allow all patients to receive transplants in cases of hematopoietic malignancy and failure.

1.7. Mesenchymal and other stromal cells Simón Méndez-Ferrer (Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain), Rosa Bernardi (IRCCS San Raffaele Scientific Institute, Milan, Italy), Cristina Lo Celso (Imperial College London, London, United Kingdom), Pierre Charbord (Université Pierre et Marie Curie, Paris, France), Willem Fibbe (Leids Universitair Medisch Centrum, Leiden, the Netherlands), Daniela Krause (Georg Speyer Haus - Institute for Tumorbiology and Experimental Therapy, Frankfurt, Germany), Robert Oostendorp (Technische Universität München, Munich, Germany), Marc Raaijmakers (Erasmus MC, Rotterdam, the Netherlands).

Introduction Hemopoiesis is critically regulated by non-hematopoietic cells that are capable of controlling the production of blood and immune cells according to the demands of the organism. These stromal cells make up the so-called hematopoietic microenvironment. It is becoming increasingly clear that different stromal populations regulate distinct subsets of hematopoietic cells, and vice versa. The complexity of these networks is further increased by the recognition of heterogeneity among bone marrow stem cells and their progenies. Recent technological developments will allow this complexity to be addressed experimentally. This will be critical in fulfilling the enormous potential of stromal cells in immune modulation, tissue regeneration, and cancer treatment.

European research contributions Soon after Till and McCulloch demonstrated the existence of hematopoietic stem cells (HSCs), European researchers contributed to characterizing the hematopoietic microenvironment and extrapolating this knowledge to the clinical and technological arenas. Simultaneously with the discovery of mesenchymal stem cells (MSCs) in the bone marrow, Owen and Friedenstein dissected skeletal turnover at the cellular level and demonstrated the capacity of osteoprogenitor cells to give rise to both skeletal-forming and hematopoietic-supporting stroma. Dexter 125

A. Engert et al. devised protocols to maintain blood cell production in long-term culture and Schofield hypothesized that a specific niche in the bone marrow is essential to maintaining HSCs. The stem cell niche concept was later extrapolated to other organs. Remarkably, at variance from niches made of fully differentiated cells (e.g. Drosophila ovariole and mammalian intestine), some of the cells belonging to the niche are the immediate progenies of the MSCs, or the MSCs themselves.

Proposed research for the Roadmap Unraveling the physiology of mesenchymal-hematopoietic networks: HSCs and their microenvironment probably represent the best-characterized hierarchical stem cell system in vertebrates, paving the path to understanding how other organs function. Dissecting the regulation of HSCs and their progenies by their microenvironment, and vice versa, will be key to optimizing the use of HSCs and therapeutically applying the emerging role of the HSC microenvironment in hematologic disorders. Ongoing research by European groups has identified MSCs in vivo and characterized their functions in the HSC niche. Detailed characterization of the properties of MSCs will rely on the development of markers to isolate relevant subsets of human MSCs and understand their HSC-supporting and regulatory properties at the anatomical and functional levels.43 Understanding the role of mesenchymal elements in human disease: future work will uncover the complexity of HSCstroma reciprocal regulation in normal and pathological settings. Recent intravital microscopy studies showed altered physical interaction between HSCs and stroma during infection.45 Mesenchymal elements have recently been experimentally implicated in the initiation, progression, and drug resistance of a variety of hematopoietic neoplasms. Osteoblastic cells are altered in chronic myeloid leukemia (CML) and effectively support leukemic stem cells (LSC) maintenance while hampering normal hematopoiesis. Parathyroid hormone stimulation of osteoblastic cells attenuates CML progression, but enhances acute myeloid leukemia (AML).46 Another type of myeloproliferative neoplasms (MPN), polycythemia vera (PV), reduces nestin+ MSCs, and their rescue is associated with disease blockade.47 In parallel to a concept in which (pre)malignant hematopoietic elements alter their niche to promote disease progression, emerging research is showing that primary (genetic) abnormalities in mesenchymal cells can initiate malignant transformation in hematopoietic cells.48 Finally, stromal cells might promote leukemic cell survival and/or protect them from chemotherapy. A better understanding of the molecular mechanisms driving these pivotal contributions of mesenchymal cells to disease pathogenesis opens the way to not only more effective treatments, but also to novel strategies to prevent malignant evolution and the associated socio-economic burden. Mesenchymal cells in regeneration, immune modulation and systemic disease: bone marrow stromal cells do not only regulate HSCs, they are also instrumental in the (re)generation of other bone marrow cell types, including bone cells. Characterizing the mechanisms by which stromal cells maintain and regulate hematopoiesis and osteogenesis will be essential for optimizing recovery following injury or bone marrow transplantation (BMT), as well as 126

increasing the number of patients that can benefit from cell therapies. In addition, stromal cells also have a profound impact on different immune cells, having therapeutic benefits in sepsis, autoimmune disorders, and graft-versus-host disease (GvHD). More mature stromal cells, or cells isolated from other sources (e.g. adipose tissues), also seem to display some of these immunomodulatory properties. Bone marrow stromal cells are also responsive to factors mediating diabetes, obesity, and aging, which are likely to increase in Europe in the next ten years. Understanding how the hematopoietic niches respond to these physical conditions and how these changes affect the blood cell system will be essential. Research in this area holds promise for correcting some of the debilitating effects associated with these conditions.

Anticipated impact of the research An increase research in the hematopoietic microenvironment will, scientifically, feed into other (hierarchical) stem cell systems and, clinically, provide new ways to modulate and treat hematopoietic diseases, immune responses, and regenerative processes. Both pharmacological modulation and cellular therapy are expected to emanate from further efforts. As a result of the productive research, the European Union is already the world region with the second highest number of registered clinical trials using mesenchymal stem/stromal cells.

1.8. Transcriptional/epigenetic networks Berthold Gottgens (University of Cambridge, Cambridge, United Kingdom), Joost Martens (Radboud Universiteit, Nijmegen, the Netherlands), Carsten MĂźllerTidow (UniversitĂ¤tsklinikum Halle, Halle, Germany), Henk Stunnenberg (Radboud Universiteit, Nijmegen, the Netherlands).

Introduction Maintaining a balanced output of mature hematopoietic cell lineages is critically dependent on exquisite control of cell fate choices at the stem and progenitor level within the hematopoietic hierarchy.49-53 These cell fate choices are executed through the interplay of extracellular signaling pathways with the intracellular decision-making machinery. The latter is driven by networks of transcriptional regulators that interact in a combinatorial fashion and can form larger protein complexes with different functions dependent on composition and cellular context. These establish cell type-specific transcriptional programs and also mediate developmental transitions when cells differentiate down a particular hematopoietic lineage. Within these transcriptional regulatory networks, transcription factor (TF) proteins bind to specific DNA sequences, and therefore represent the primary stage of decoding regulatory information present in lineage-specific gene regulatory elements. Once bound, TF proteins recruit a number of accessory proteins with enzymatic activity, which cause either modifications to the DNA (e.g. DNA methylation) or covalent modification of histone proteins (e.g. histone methylation, acetylation, phosphorylation). These so-called epigenetic modifications in turn influence the accessibility of the DNA template for subsequent binding of further TFs, and therefore serve a critical function in the establishment of stable transcriptional programs. The epigenetic status of the chromatin haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

template can also influence additional aspects of transcriptional control, such as the assembly of RNA polymerase complexes on the promoter, and also the rate of transcriptional elongation. In addition, these TF-regulated networks are related to higher-order structure organization and nuclear localization of DNA elements.

on genomics, epigenomics, and metabolomics, integrative analysis of data is mandatory. Computational, conditional dependency models need to be established, for example by creating Bayesian networks, to formulate the relationship between the cell niche, environmental factors, TF binding, epigenetic alterations, and the presence of various hematologic disorders.

European research contributions European researchers have contributed significantly to the identification and characterization of TFs involved in normal and aberrant hematopoiesis. In addition, they have extended many of these analyses toward genome-wide mapping of normal as well as mutated TFs in primary hematopoietic cells from healthy and diseased individuals, thereby revealing multiple unanticipated functions of these proteins. European researchers have been working at the forefront of epigenetic research, a prime research interest of the European Union, which has supported a multitude of projects on this subject in the past decade (e.g. Epigenome NoE, HEROIC, EPITRON, and Epigenesys). Recently, these interests specifically focused on the hematopoietic lineage with the support of BLUEPRINT, a project that set out to generate epigenomic data of more than 100 blood cell types from healthy individuals and patients suffering from blood diseases. This project has already provided many new insights into the interplay of TFs with chromatin to establish epigenetic patterns that define cell type functionality, but also led to the realization that much can still be learned about how blood cell types develop and how we could modulate their activities to prevent or counteract disease. This will require a concerted effort to better understand regulatory networks in both normal hematopoiesis and disease, and will only be accomplished through close collaboration between experimentalists, computational biologists, statisticians, and clinicians.

Proposed research for the Roadmap Mutations in transcriptional and epigenetic regulators are some of the most common mutations in hematologic malignancies. Given that these proteins function as regulatory network components, it will be important to gain an understanding of the malignant state as a perturbation of wider regulatory networks. Research on the concerted actions as well as post-transcriptional regulation of TFs, and on how these regulate the local and global epigenetic environment, should be intensified. Although the main TFs involved in disease development have been identified, many components that drive the functional fine-tuning of the blood cell types are still not known. These are likely controlled by the niche in which the cells reside, the availability of metabolites, both endogenous (amino acids, sugars, and vitamins) and exogenous (drugs, food additives, and toxins), and other environmental factors. In addition, it has become clear that not only the adaptive immune system confers memory potential, but that also cells of the innate immune system can be trained and are functionally dependent on past and present behavior, offering another starting point to utilize cells of the hematopoietic system in disease prevention and control. Apart from creating additional comprehensive data sets haematologica | 2016; 101(2)

Anticipated impact of the research Intensifying the research on normal blood development provides an opportunity to better understand the intrinsic molecular systems that regulate normal hematopoiesis and provide knowledge of how these mechanisms can be shaped and regulated to the benefit of the individual. It will provide a framework to which similar analysis on hematologic diseases can be compared, allowing the identification of the primary mechanisms that are disrupted and providing starting points for the development of targeted interventions. Finally, the depth to which these networks can be studied in vitro and in vivo will be of great importance to deciphering transcriptional and epigenetic networks, not only in blood cell formation, but in multiple other organ systems as well.

1.9. Reprogramming/induced pluripotent stem cells/embryonic stem cells Valerie Kouskoff (University of Manchester, Manchester, United Kingdom), Lesley Forrester (University of Edinburgh, Edinburgh, United Kingdom), Thomas Graf (Center for Genomic Regulation, Barcelona, Spain), Hannes Klump (UniversitĂ¤tsklinikum Essen, Essen, Germany), Georges Lacaud (University of Manchester, Manchester, United Kingdom), Pablo Menendez (Universitat de Barcelona, Barcelona, Spain), Joanne Mountford (University of Glasgow, Glasgow, United Kingdom).

Introduction Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), represent a limitless source of cells for investigations ranging from developmental processes to drug discovery. Dissecting the molecular and cellular mechanisms underlying the in vitro differentiation of PSCs to blood progenitors has been instrumental in furthering our knowledge of the early steps of hematopoietic development. The use of human ESCs has proved particularly useful given the difficult and restricted access to human embryos. Furthermore, dissecting the in vitro differentiation of iPSCs derived from cells of patients with hematologic diseases is starting to provide invaluable insights into these pathological conditions. But one of the greatest promises of the stem cell field remains the in vitro derivation of cell populations that can be used in the clinic for therapeutic purposes. PSC-derived cells could be used to regenerate a damaged hematopoietic system or to modulate the repair of other tissues (e.g. macrophage in fibrosis) or the immune response [e.g. chimeric antigen receptor (CAR)-expressing T cells]. Finally, the fast-expanding field of direct reprogramming in which one somatic lineage is directly converted into another clinically useful cell type is gaining momentum and may complement the derivation of cell populations from PSCs.

European research contributions In the past two decades, European researchers have contributed significantly to the field of embryonic hematopoiesis, using PSCs as a model system to decipher 127

A. Engert et al. the early steps of mesoderm specification and blood commitment. These efforts have led to the identification of the hemogenic endothelium as a cornerstone in the in vitro generation of blood progenitors.54,55 Studies by several European laboratories have provided insights into the role of key transcription factors (TFs) (e.g. RUNX1, ETV2, TAL1, HOXB4, and HOXA9) and Notch signaling (e.g. DLL4 and CDCA7) in promoting hematopoiesis. Innovative studies exploring the derivation of in vivo engrafting blood progenitors from ESCs56 and investigations on the instructive role of leukemogenic fusion genes (e.g. MLL-AF4) in human ESCâ&#x20AC;&#x201C; derived blood cells have also been undertaken in European laboratories. Furthermore, the use of disease-specific iPSCs to unravel inherited hematologic disorders, such as Fanconi anemia (FA), chronic granulomatous disease, and pyruvate kinase deficiency, provided unique clues to understanding the mechanisms underlying these diseases. On the translational front, European teams have made seminal progress toward establishing protocols for the in vitro generation of platelets, macrophages, dendritic cells, and red blood cells (RBCs) and their manufacturing for use in the clinic.57 Alternative methods for the derivation of hematopoietic populations are now starting to emerge with direct reprogramming as a forerunner in this fast expanding area of research. Although still in its infancy, direct reprogramming explores the instructive conversion of one somatic lineage into another mediated by the ectopic expression of TFs. Pioneering work was performed in Europe to decipher the reprogramming of committed immune cells into macrophages mediated by CEBPA.58 More recently, the reprogramming of fibroblasts to hematopoietic progenitors was achieved upon ectopic expression of specific TFs.

Proposed research for the Roadmap Fundamental research focusing on developmental processes of the hematopoietic specification of PSCs needs to be maintained, because this area of investigation holds the key to establishing robust and efficient protocols for the production of clinically applicable blood and immune cells. These protocols would benefit from the reproducible derivation of long-term repopulating hematopoietic stem cells (HSCs) with adult characteristics. Deriving therapeutic cells from PSCs by modulating the differentiating conditions is a priority, but other approaches should be followed in parallel. One of the new avenues to be pursued is the direct reprogramming of human non-hematopoietic cells to blood cells/progenitors for therapeutic purposes. There is still much to be understood about the fundamental aspects of somatic cell reprogramming, and funding opportunities should be provided for European groups to intensify their efforts in this groundbreaking field. Both basic and translational areas need to be explored in order to remain competitive at an international level. Another important area that European research should focus on is further developing the use of iPSCs as a model system for hematologic disease in both fundamental and applied studies. This approach represents a unique opportunity to: 1) determine the contribution of the epigenome to disease initiation and maintenance; and 2) understand the developmental impact of leukemia-specific mutations and chromosomal translocations in blood specification and cell fate decisions. 128

Anticipated impact of the research Ultimately, harnessing the power of PSCs for regenerative medicine and the production of blood derivatives will profoundly benefit patients, families, and European society as a whole. To reach that goal, however, we will need to gain a deeper understanding of the differentiation processes leading to the generation of the desired hematopoietic subsets. We will also need to implement robust protocols for the large-scale production of desired blood products. iPSCs as model systems to understand mechanisms of disease emergence and maintenance will be instrumental in identifying and developing novel treatment options for the relevant disorders.

The EHA Roadmap for European Hematology Research Section 2. Malignant lymphoid diseases

Section editors: Bertrand Coiffier, Gilles Salles. Malignant lymphoid diseases represent the most frequent hematologic malignancies, with an age-adjusted estimated incidence of 24.5 per 100,000 inhabitants in Europe,59 and are associated with significant mortality60 and morbidity. This disease group is highly heterogeneous in terms of frequency, epidemiology, biology, genetic abnormalities, and outcome. While in a way all individual lymphoma subtypes could be seen as rare diseases, some of them are relatively common, e.g. multiple myeloma, chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphomas (DLBCLs), follicular lymphomas (FLs), and Hodgkin lymphomas (HLs). Others are less common e.g. mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), T-cell lymphoma, and mucosa-associated lymphoid tissue (MALT) lymphoma, or even very rare, e.g. some subsets of marginal zone lymphomas (MZLs) and HIV-associated lymphoma. After the progress made in the morphological classification of these tumors in the 1990s, the advent of large-scale genomic approaches enabled identification of multiple molecular subsets, which may further subdivide the different entities in multiple rare diseases.61-63 These achievements justify the need for European-based epidemiological studies and contributions to the InterLymph consortium64 to investigate the role of environmental and lifestyle factors, which, in the context of inherited genetic background, may favor the development of these malignancies. Significant progress was also made in unraveling key biological features of these diseases, including: 1) the more precise delineation of intrinsic genetic defects in tumor cells, delineation still ongoing with NGS approaches;63,65,66 2) the growing understanding of the complex interplays between malignant cells and their microenvironment, which is especially critical in these diseases arising in lymphoid organs;67 and 3) the emerging identification of constitutional genetic traits associated with an increased susceptibility to develop these malignancies.68,69 Finally, several recent developments have pointed toward the existence of â&#x20AC;&#x153;lymphoid cancer stem cells,â&#x20AC;? which may represent highly desirable targets to achieve a definitive cure in these malignancies.70-72 Although several European groups have already made outstanding contributions to this field, in part within large international consortia, further haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

achievements will only be possible if major investments can be realized. These should particularly focus on establishing new cellular and animal models (critically rare in the field of mature lymphoid malignancies) to better understand how these diseases develop and for pre-clinical assessment of new therapeutic agents. Despite important advances in the past few years,73 the survival of patients with lymphoid malignancies remains unsatisfactory. This is true for the most aggressive malignancies (e.g. ALLs, T-cell lymphomas, and some forms of DLBCL), which still are frequently fatal. In addition, the lack of cure in patients with multiple myeloma or indolent lymphoma is equally challenging. Furthermore, short- or long-term morbidities such as infertility, secondary malignancies, as well as cardiac, pulmonary, renal, or neurological dysfunction are associated with intensive treatment in HL or DLBCL. Chronic exposure to therapeutic agents such as in indolent lymphoma and CLL also represents a health burden for patients, as well as an increasingly relevant economic burden for the European Union.2,74 Attention to malignancies occurring in elderly patients should also be considered in this regard given the fact that life expectancies continue to grow. European co-operative groups have been leading clinical research in lymphoid malignancies in the past decades. Progress is being made in investigating the role of targeted agents in well-characterized molecular subsets. The number of new therapeutic agents under development in this field demands further academic research collaboration. For example, analyzing the medico-economic impacts of patient management should clarify costs and benefits of new therapeutic strategies, including those related to public health economics. These groups also need further support in their translational research activities, especially in their efforts to constitute and analyze large biobanks with high-quality clinical annotations. Efforts should also aim to eliminate the differences in outcome observed in different parts of Europe and to improve patients’ survival and quality of life.

malignancies in young adults, most patients will have a very long survival. During their follow up, however, a significant proportion of patients experience serious longterm toxicities, such as second malignancies, cardiovascular diseases, and infertility. Most of these late toxicities have been related to the treatments for HL. To reduce long-term, treatment-related toxicities, optimization of the balance between the risks and benefits of the different treatment strategies is still the subject of controversy and the main goal of most clinical trials.

European research contributions Two European risk models (proposed by the German Hodgkin Study Group and the European Organisation for Research and Treatment of Cancer/the Lymphoma Study Association) are commonly used and were established to separate HL into three different risk categories: early favorable, early unfavorable or intermediate, and advanced disease. Using these prognostic categories, European lymphoma groups have been major contributors in clinical trials that established ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine) as a reference chemotherapy for HL. Alteration of ABVD by omitting any drug including bleomycin was also recently reported as inferior in terms of disease control.75 However, as approximately 30% of patients with advanced disease relapse after ABVD, a more intensified chemotherapy, BEACOPPesc (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, and prednisone) was developed for these patients. It showed a better progression-free survival (PFS), although it is also associated with more toxicity.76 Since the late 1980s, children and adolescents with HL have been treated with chemotherapy designed to limit cumulative doses of anthracycline, alkylating agents, and bleomycin to limit long-term toxicity. Recently, OEPA (vincristine, etoposide, prednisone, and doxorubicin) associated with COPDAC (cyclophosphamide, vincristine, prednisone, and dacarbazine) was commonly used in European studies and demonstrated global efficacy comparable to treatment with regimens containing procarbazine with, therefore, an expected lower rate of impaired fertility in both men and women.

2.1. Hodgkin lymphoma Marc André (Université Catholique de Louvain, Yvoir, Belgium), Anke van den Berg (Universitair Medisch Centrum Groningen, Groningen, the Netherlands), Peter Borchmann (University Hospital Cologne International, Cologne, Germany), Massimo Federico (Università degli studi di Modena e Reggio Emilia, Modena, Italy), Judith Landman-Parker (Hôpital Armand Trousseau, Paris, France), Stephan Mathas (Charité Universitätsmedizin Berlin, Berlin, Germany), John Radford (University of Manchester, Manchester, United Kingdom).

Introduction Classical Hodgkin lymphoma (HL) is a highly curable disease and is considered one of the most successful stories in hematology. For both localized and advanced-stage disease, more than 90% of patients are alive five years after diagnosis, and the progression-free survival (PFS) is 85%-93% for localized disease and 70%-89% for patients with advanced disease. After a first relapse, the disease remains curable in nearly half of the cases when high-dose chemotherapy with autologous stem cell transplantation (ASCT) is feasible. As HL is one of the most common haematologica | 2016; 101(2)

Radiotherapy is a major contributor to the late toxicities, such as secondary cancers and cardiovascular diseases. European trials provided an important contribution to the reduction of radiotherapy for HL treatment. Indeed, several trials have shown that when given in combination with chemotherapy, the radiotherapy fields can be reduced from extended to involved. In recent years, even more restricted radiotherapy modalities, such as involved node and involved site radiotherapy, have also been proposed and evaluated in randomized clinical trials. Moreover, these trials have also demonstrated that the dose of radiotherapy can be safely reduced without compromising treatment efficacy. The dose of radiotherapy in the more favorable group of localized patients has been reduced to 20 Gy. In the vast majority of cases, radiotherapy in children and adolescents is delivered at 20 Gy and restricted to the involved site in order to limit growth abnormalities, and long-term vascular and heart toxicity. More recently, European trials in pediatric, adolescent, and adult patients have also tested the possibility of omitting radiotherapy in select patients. As it has been shown that patients with a fluorodeoxyglucose positron-emission tomography (PET) 129

A. Engert et al. negativity after two cycles of ABVD have an excellent outcome, it was suggested that those PET-negative patients could receive less intense therapy without any radiotherapy.77 This PET-driven treatment adaptation is still restricted to clinical trials and whether this select population of patients can be safely treated without radiotherapy remains controversial and a matter of debate. Importantly, HL trials led by several European co-operative groups allowed nuclear medicine physicians to establish criteria for a good and reproducible interpretation of PET-CT scans in lymphoma patients. This 5-point scale, referred as the Deauville scale, is now commonly adopted by the international community to properly evaluate interim and end-of-treatment PET-CT scan results in the field of lymphoma.78

with relapsed/refractory HL.79 More recently, when given as a consolidation therapy after ASCT, this drug was also shown to improve PFS of patients treated with high-dose chemotherapy for a first relapse. Finally, this drug is now being tested for both first- and second-line therapy. More recently, as pre-clinical studies suggested that Hodgkin/Reed-Sternberg cells exploit the programmed death-1 (PD-1) pathway to evade the immune system, PD1–blocking antibodies were evaluated in ASCT and CD30 antibody-drug conjugate refractory patients and showed substantial therapeutic activity with an acceptable safety profile. The place of these new drugs and possible associations with the currently available treatment modalities need to be further refined, especially with the aim of limiting long-term toxicities.

Apart from these clinical achievements, various European groups have a long history of research on HL pathogenesis, beginning with the identification of HL as a B-cell-derived malignancy. Most of the currently known molecular defects considered to be key alterations of HL were identified by European groups, including the deregulated NF-κB activity and genomic defects of components of the NF-κB and JAK/STAT signaling pathway.

As late toxicities and complications are a major concern in this situation, continuous and rigorous evaluation of long-term survivors also remains a major topic of interest for clinical research.

Proposed research for the Roadmap Research should be pursued into the etiology and epidemiology of HL with attention to genetic, immune-based and infectious (e.g. Epstein-Barr virus) determinants. Most patients with HL will ultimately be cured from their disease and experience long-term survival. We are now in a situation of trying to better identify patients who will be more readily cured from those who will need more intensified therapy (e.g. radiotherapy for localized patients and BEACOPPesc for advanced patients). This will allow us to meet the urgent need of avoiding unnecessary toxicities for the vast majority of patients who can be cured with less aggressive treatments. Special efforts towards optimizing treatment for elderly patients should also be made. PET-CT and biomarker-driven strategies are currently being explored with the hope of individualizing treatment decisions. However, pre-treatment prognostic markers (e.g. circulating biomarkers), genomic markers, or molecular markers have to be identified to stratify patients before starting treatment or early thereafter. To develop such tools, coupling international clinical trials with the establishment of a biobank including tumor tissue and blood samples is essential. Moreover, as the pathogenesis of HL is still largely unknown, establishment of a comprehensive picture of the role of the microenvironment, genetic alterations, and molecular pathways driving pathogenesis of the disease is required. This may also lead to the identification of novel targets for treatment that could potentially cause fewer undesired side effects. Interestingly, for the first time since the 1970s and the introduction of doxorubicin, new drugs are now becoming available in the field of HL and are suggested as treatments with a safe toxicity profile. A CD30 antibody-drug conjugate was recently approved for the treatment of relapses after high-dose chemotherapy with ASCT and for patients failing two previous lines of chemotherapy but ineligible for high-dose chemotherapy. This drug induced durable remissions and favorable long-term survival in patients 130

Anticipated impact of the research As HL is a disease of the third and fourth decade of life, and one of the most common cancers in young adults, cured patients are potential long-term survivors. Reducing their risk of late toxicities, while keeping their high cure rate, is of utmost importance for increasing their individual chances of becoming active members of society in the long term. The recent successful implementation of targeted treatment strategies shows the importance and clinical relevance of unraveling the pathogenesis of HL. Moreover, knowledge about disease relapse is lacking and intensive research is needed into this. This will only be possible within large European clinical trials combined with extensive biobanking and the development of tools that allow analysis of the scarce tumor cell population characteristic of HL. In addition, these clinical trials provide a solid basis to identify, if possible, pre-treatment prognostic biomarkers to select patients who will benefit from the respective treatment regimen. Finally, the definition of the place of already-developed new drugs in our armamentarium and individualized therapy strategies is one of the main goals of the next generation of clinical trials. Special attention should be given to disseminate knowledge and innovation to vulnerable populations in Europe and elsewhere.

2.2. Diffuse large B-cell lymphoma and Burkitt lymphoma in adults and children Hervé Tilly (Université de Rouen, Rouen, France), Igor Aurer (University of Zagreb, Zagreb, Croatia), Peter Johnson (University of Southampton, Southampton, United Kingdom), Georg Lenz (Universitätsklinikum Münster, Münster, Germany), Véronique Minard (Institut Gustave Roussy, Villejuif, France), Vincent Ribrag (Institut Gustave Roussy, Villejuif, France), Andreas Rosenwald (Universität Würzburg, Würzburg, Germany), Umberto Vitolo (Università degli Studi di Torino, Turin, Italy).

Introduction Diffuse large B-cell lymphoma (DLBCL) is the most common mature B-cell neoplasm, with an estimated incidence of 3.8 per 100,000. This indicates that approximately 30,000 cases occur in Europe each year, with some variations in geographic distribution.59 The median age at diagnosis is 60 years. The standard method of diagnosis is haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

a surgical excision biopsy with morphology and immunohistochemistry study. Gene expression profiling has identified three major subtypes of DLBCL according to the cell of origin of the malignant cells: germinal center B-cell, activated B cell-like, and primary mediastinal B-cell lymphoma. A large majority of DLBCL in adolescents belongs to the germinal center B-cell subtype, and the proportion of the activated B cell-like subtype increases with age. Next generation sequencing (NGS) studies demonstrated very heterogeneous genomic alterations among these subtypes, which could be related to a variable outcome and could indicate putative targets for therapeutic interventions. The combination of the anti-CD20 antibody rituximab with chemotherapy resulted in an important improvement in survival over the past decades.73,80 A proportion of 50%-90% of patients can be cured by immunochemotherapy depending on age and other clinical prognostic factors gathered in the International Prognostic Index.81 As salvage treatment is often disappointing, a successful first-line treatment is the key to longer survival. Burkitt lymphoma (BL) is an aggressive B-cell lymphoma characterized by the presence of a translocation that activates the oncogene MYC. The sporadic form found in Europe mostly affects children and young adults with a crude incidence of 0.22 per 100,000, accounting for 80% of B-cell lymphomas in these age groups. It is associated with immunosuppression, especially HIV infection, mostly among older patients. Intensive chemotherapy and supportive care can cure most young patients in highincome countries, but outcome is less favorable for other populations. MYC translocations can also occur in lymphoma with features intermediate between DLBCL and BL. This B-cell lymphoma unclassifiable has a more aggressive behavior and potentially needs a specific therapeutic approach.

European research contributions Large randomized studies conducted by European cooperative study groups have contributed to the establishment of a worldwide standard treatment. Fifteen years ago, the advantages of the combination of rituximab and standard chemotherapy CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) over chemotherapy alone were demonstrated by these study groups in young and older patients.80 Further studies have proposed optimal combinations varying according to prognostic factors, the exploration of salvage treatment, the evaluation of treatment by functional imaging, and the description of biological characteristics correlated to clinical outcome in patients treated with immunochemotherapy. Efforts in children and adolescents consisted of intensive chemotherapies to increase a high-rate cure, sometimes accompanied with immediate toxicity, but with the aim of reducing long-term toxicity. European hematologists have contributed to an international collaborative project aiming at defining the molecular definition of lymphoid malignancies. This consortium was at the origin of the identification of molecular subtypes of DLBCL and the distinction between BL and DLBCL with MYC translocations.82 These investigators explored the genomic and transcriptional mechanisms haematologica | 2016; 101(2)

implicated in lymphomagenesis and identified genetic alterations modifying major cellular pathways, influencing clinical outcome of patients with DLBCL and representing possible therapeutic targets.

Proposed research for the Roadmap An effort to further characterize the genomic, transcriptional, epigenetic, proteomic, and metabolomic landscape of each DLBCL subtype is a common goal in research into hematologic malignancies. Development of new cell lines and animal models representative of these subtypes are certainly needed to improve our understanding of the important biological mechanisms of the lymphoma cell, the interaction with the tumor microenvironment, and to explore the efficacy of new drugs. The most important challenge in DLBCL is to improve survival of those patients who have refractory disease or who relapse early in the course of the lymphoma. Molecular heterogeneity is, at least in part, responsible for this outcome. If whole-exome sequencing has redefined the genetic landscape of the disease, identification and characterization of genetic alterations in this population requires a large number of samples, important clinical data, and access to extensive sequencing and analysis possibilities. This approach should overcome the inherent complexity of these alterations, the low frequency of some of them, the tumor heterogeneity, and the mechanisms of resistance. New tools for children and adolescents will be developed to assess risk stratification, early response, and monitoring of the disease in order to tailor chemotherapy and strike a balance between acute toxicity and the risk of long-term toxicity. This will require further international collaboration, along with partnerships with adult lymphoma groups. Early access to targeted drugs will be conditional on the ability of the European centers to collect the tumor samples and establish a viable network of platforms to exchange data, define common protocols, and share quality-control processes. A large number of new agents targeting driver mutations involved in lymphomagenesis are awaiting clinical application. The selection of patients who are likely to respond to a single agent should be based on the identification of subtypes, the exploration of pathways, or the presence of genetic abnormalities. In this view, targeting MYC would be a highly desirable goal in both BL and DLBCL. Combining targeted drugs with standard first-line immunochemotherapy in order to increase its efficacy and decrease its toxicity will certainly be the major therapeutic path to be explored in the coming years. The next step could then be the advent of chemotherapy-free regimens containing a combination of targeted molecules or a combination of these molecules with monoclonal antibodies or other immune therapies. Access to platforms and development of these novel combinations will be of critical importance in elderly patients who represent the fastestgrowing group and the frailest population. New biomarkers of response and survival need to be explored. Functional imaging has been shown to be a useful tool for evaluating early response and correlating it to clinical outcome. Efforts to develop new markers of spe131

A. Engert et al. cific pathways or new evaluation modalities could help guide treatment. Circulating DNA could also be a powerful tool to help diagnosis, evaluate response to treatment, and predict relapse.

Anticipated impact of the research All of these research directions aim at a better understanding of the biology of DLBCL and BL. Future management of patients with these diseases will have to change from empiric administration of chemotherapy to a combination of precision therapy, thus leading to more personalized treatment approaches. This change will have a major impact on increasing treatment efficacy, decreasing the rate of treatment complications, and ultimately prolonging survival. A close collaboration between patients, academic laboratories, pharmaceutical and biotech companies, and co-operative study groups must work towards ensuring this translation in adults and children. Finally, a reduction of hospital costs and optimization of treatment strategies will allow this policy to be adopted in all parts of Europe.

2.3. Mantle cell lymphoma Marek Trneny (Univerzita Karlova, Prague, Czech Republic), Elias Campo (Universitat de Barcelona, Barcelona, Spain), Martin Dreyling (Ludwig-MaximiliansUniversität München, Munich, Germany), Steven LeGouill (Université de Nantes, Nantes, France), Simon Rule (Derriford Hospital, Plymouth, United Kingdom).

Introduction Mantle cell lymphoma (MCL) represents approximately 7% of all non-Hodgkin lymphomas (NHLs) and is genetically characterized by the translocation t(11;14)(q13;q32) and the overexpression of CCND1. Most cases have an aggressive course and require intensive treatment. The standard approach is based on immunochemotherapy, which consists of rituximab and CHOP-like and/or highdose, Ara-C–containing regimens followed by high-dose treatment (HDT) with autologous stem cell transplantation (ASCT). Elderly patients are usually treated with rituximab and CHOP (R-CHOP) or R-bendamustine chemotherapy, and rituximab maintenance is used for responding patients. The MCL International Prognostic Index (MIPI) can identify patients with a high risk, who still have a very poor outcome in spite of intensive treatment. Proliferation index evaluated by Ki-67 has been established as an important prognostic marker. There is growing evidence that patients who achieve minimal residual disease (MRD) negativity have a significantly better outcome than patients who remain MRD-positive.83 Patients who relapsed have a very poor outcome with median overall survival of approximately 18 months. A small subset of MCL biologically characterized by IGHV hypermutation and SOX11 negativity has a favorable outcome and usually does not require immediate treatment after diagnosis.

European research contributions The effort in Europe has been focused on several different priorities: 1) description of a clinically relevant prognostic index with the use of some biological parameters; 2) development of treatment strategies for the young as well as the elderly population; and 3) response criteria. The German Low Grade Lymphoma Study Group established an MIPI that consists of age, lactate dehydrogenase level, 132

white blood cell count, and performance status.84 This index has been accepted worldwide as a tool to discriminate between different outcomes. Evaluation of proliferation index by Ki-67 adds important prognostic information; the most common threshold used is 30% positive cells.84 High-dose chemotherapy (HDCT) and ASCT is regarded the standard of care for young MCL patients. The addition of rituximab and high-dose Ara-C has been explored by the Nordic Lymphoma Study Group and the Lymphoma Study Association.85 The European Mantle Cell Lymphoma Network showed that incorporating DHAP (dexamethasone, high-dose Ara-C, and cisplatin) led to a better outcome in these patients. Whether the improvement is due to high-dose Ara-C only or if combination high-dose Ara-C with platinum is important has not been tested; there is, however, some hint that combination (DHAP) can be better than high-dose Ara-C monotherapy. The issue of total body irradiation (TBI) as part of HDT has not been resolved; it might well be that the incorporation of TBI results in better outcomes. Maintenance treatment after stem cell transplant has been evaluated by the Lymphoma Study Association. Although significant improvements have been achieved, young patients with a high MIPI score still have a poor outcome.85 However, the majority of MCL patients are ineligible for intensive treatment with HDCT and ASCT. The treatment with RCHOP or R-bendamustin has been accepted as standard, and the European Mantle Cell Lymphoma Network study demonstrated that rituximab maintenance for two years after R-CHOP significantly prolongs progression-free survival (PFS).86 Although there is significant improvement in terms of survival, the majority of patients suffer from poor outcomes. This led to collaborative trials with new drugs including temsirolimus, lenalidomide, ibrutinib, bortezomib, and others.87 It has been clearly demonstrated that outcome depends on response. Several groups have worked to define the impact of MRD negativity. Pooled data from different trials under the umbrella of the European Mantle Cell Lymphoma Network demonstrated that MRD negativity is a more important prognostic factor than classical staging.

Proposed research for the Roadmap The general outcome of MCL patients has improved but is still worse than that of other lymphoma subtypes. The growing understanding of MCL biology and improvements in therapeutic strategies have led to a situation in which the MCL patient population is considered more heterogeneous than it was 15 years ago. Heterogeneity regarding MCL epidemiology in different parts of Europe should be investigated. On one side, there is a subgroup of MCL patients with an indolent course; these patients can be followed until treatment is required. There is, however, the need for a better biological description of this subgroup. For the other MCL patients, MIPI or Ki-67 is not yet used for treatment tailoring. All young patients outside clinical trials are treated similarly with HDCT and ASCT as standard management. There is, however, a significant survival difference according to prognostic subgroups and there is a need for individualized treatment. Patients with more aggressive MCL (blastoid variants, high Ki-67, p53 mutations or deletion, mutations in some related genes, such as NOTCH1 or NOTCH2, several chromatin modifiers, such as WHSK1, and others) still have a very poor outcome. haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

On the other hand, a description of patients with good prognosis, who could be treated without HDT, is also needed. Targeted therapy at relapse seems to improve outcome, but the median survival is still only approximately two years. Due to the low incidence of MCL, prospective trials based on international collaboration are warranted. These trials should test new classes of drugs combined with established immunochemotherapy, and translational research should be included. The important issue is to collect biological samples of patients who fail the treatment in order to understand the mechanism of resistance. Another task is to define how to use the information on MRD. Should it become a standard response criterion? Until now, this information has been used without affecting therapeutic decisions. The question of whether there is room for treatment intensification in MRD-positive patients or treatment reduction in MRD-negative patients should be answered in prospective clinical studies. It has been accepted that post-induction treatment with rituximab improves outcome in MCL. The question that should be explored is whether targeted treatments, such as ibrutinib, lenalidomide, or others, should be used as a maintenance approach in first-line treatment.

Anticipated impact of the research Mantle cell lymphoma is a rare lymphoma subtype with a genetically well-defined primary event and many secondary events. This disease has a very poor prognosis with some recent improvements but results are still far from satisfactory. The European collaborative effort could provide a fresh insight into the impact of different secondary genetic events in MCL and identify subgroups for individualized therapeutic approaches. The important issue is establishing tissue bank samples, not only from the time of diagnosis but also at the time of relapse. This can only be achieved through large international efforts, such as those initiated under the umbrella of the European Mantle Cell Lymphoma Network. Collaborative efforts should aim at further improvements in the outcome of this disease. Although MCL is a rare disease, it represents a paradigm for the exploration of innovative, targeted therapies.

2.4. Follicular lymphoma John Gribben (Queen Mary University, London, United Kingdom), Christian Buske (Universitätsklinikum Ulm, Ulm, Germany), Jude Fitzgibbon (Queen Mary University of London, London, United Kingdom), Peter Hoskin (Mount Vernon Hospital, Northwood, United Kingdom), Armando Lopez-Guillermo (Hospital Clínic de Barcelona, Barcelona, Spain), Bertrand Nadel (Université de la Méditerranée, Marseille, France).

Introduction Follicular lymphoma (FL) is the second most common lymphoma, comprising approximately 20% of all nonHodgkin lymphomas (NHLs), with an incidence in Europe of approximately 2.18 cases per 100,000 people per year. FL typically presents in middle age and in the elderly, and the median age at diagnosis is 60 years. FL arises from germinal center B cells and maintains the gene expression profile of this stage of B-cell differentiation. haematologica | 2016; 101(2)

Morphologically, the disease is composed of a mixture of centrocytes and centroblasts; an increased percentage of centroblasts is predictive of a poor outcome. A hallmark of the disease is the chromosomal translocation t(14.18), contributing to overexpression of the antiapoptotic protein BCL2. In addition, next generation sequencing (NGS) studies have identified several recurring mutational events targeting genes, highlighting the importance of epigenetic dysregulation in the pathogenesis of the disease and tumor microenvironment modulation through NF-κB and B-cell receptor signaling pathways, as well as defects in DNA repair and apoptosis, challenging the notion that t(14;18) is sufficient for tumor initiation and demonstrating the genetic heterogeneity of the disease.88 Guidelines for the diagnosis of indolent lymphomas were outlined by the European Society for Medical Oncology. The majority of affected individuals exhibit a characteristic indolent disease course with multiple relapses requiring repeated courses of treatment; others develop aggressive disease and histological transformation with shortened overall survival. The disease remains incurable in most cases.

European research contributions In the past decades, European scientists have made major contributions to the understanding of the molecular basis of the disease and the relationship between the tumor cells and their microenvironment. The conduct of large controlled randomized trials within highly organized lymphoma co-operative groups has changed the treatment of FL and improved outcome. In particular, European-led trials have paved the way to demonstrating the benefit of immunochemotherapy over chemotherapy and the advantage of maintenance anti-CD20 monoclonal antibody in first and subsequent remission, and have defined new approaches to optimize first-line treatment.89

Proposed research for the Roadmap Despite major progress in the management of FL, the biological basis is not fully understood, and there is currently no cure. To find an effective treatment, we need to be able to determine the molecular basis of the disease so that more targeted treatment approaches can be adopted. Because there does not appear to be a single target that can be applied to all cases, a combination of novel biomarkers (based on genomic, proteomic, transcriptomic, and metabolomic analyses of biopsies) will be required. It will be important to identify those molecular events involved in early development of the disease90,91 and those involved in progression and transformation.88 The prognosis and clinical course of FL appears to be highly dependent on the tumor microenvironment, and immunohistochemical methods are being assessed to address this.92 The validation of these techniques will require the integration of longitudinal standardized data, as well as uniform criteria for diagnosis and outcome that can be applied in the clinical setting. Better integration of basic and clinical research will also be crucial. Salient features of that integration should include the following: 1. Where possible, consent should be obtained for the procurement and storage of use of excess tissue from lymph node biopsies and normal tissue at the time of presentation and at each subsequent disease relapse for research purposes in order to investigate the molecular biology of these diseases. 133

A. Engert et al. 2. A biobank of biopsies linked to the clinical database should be available, with protocols for standardized sampling and storage procedures adapted to genomics and functional assays (including live cells), which would form the basis of correlative and biomarker studies. Of particular importance would be the banking of longitudinal samples from patients at diagnosis and at subsequent relapses and transformation to the nature of relapsing disease. 3. A database (a co-ordinated pan-European registry) that can be accessed by all research partners, containing the biological and clinical information collected for each participating patient, should be made available. 4. Uncovering the molecular mechanisms involved in FL, especially in its early stages90-92 and the processes involved in transformation, should be a common goal.88 A key issue would be performing both genetic and microenvironment analyses on longitudinal and/or paired FL biopsies to obtain an integrated view on bidirectional dependency. 5. Robust biomarkers (both prognostic and predictive) should be developed, reflecting the molecular biology of the disease and the impact of the immune microenvironment for disease development and treatment outcome.92 6. Novel animal models that recapitulate disease features and allow pre-clinical investigation could also be developed. No good animal models of this disease are currently available, limiting research and drug development. 7. Academic clinical research should also address issues related to the costs and benefits of different therapeutic options and optimal strategies in the elderly population.

Anticipated impact of the research The current lack of understanding of the molecular basis of the disease, the nature of the lymphoma “stem cell”, and the events involved in progression and transformation limit our ability to cure this disease. The research plans above hold the key to understanding the molecular pathogenesis of disease and identifying key targets for optimal therapeutic intervention. The characterization of the genetic, genomic, proteomic, transcriptomic, and metabolomic profile of individual patients and patient cohorts will allow the most appropriate treatment to be selected within clinical trials investigating novel targeted therapeutic agents. This will also allow identification of robust biomarkers for monitoring response to treatment in order to allow a precision therapeutic strategy to be applied to improve the survival and quality of life of patients with FL.

2.5. Marginal zone lymphoma: extranodal, nodal, and splenic forms Andrés Ferreri (IRCCS San Raffaele Scientific Institute, Milan, Italy), Ming-Qing Du (University of Cambridge, Cambridge, United Kindom), Carlos Montalbán (MD Anderson Cancer Center Madrid, Madrid, Spain), Kostas Stamatopoulos (Institute of Applied Biosciences, Thessaloniki, Greece), Alexandra Traverse-Glehen (Centre Hospitalier Lyon Sud, Lyon, France).

Introduction Marginal zone lymphomas (MZLs) are a diverse group of clinic-pathological entities, comprising extranodal (also called MALT lymphoma), nodal, and splenic forms. The 134

ontogeny of these lymphomas is in most cases related to autoimmune disorders and chronic infections. Indeed, Sjögren syndrome, systemic lupus erythematosus, rheumatoid arthritis, and Hashimoto thyroiditis, among the former, and hepatitis C virus, Helicobacter pylori, and Chlamydia psittaci, among the latter, have been linked to MZL development. Persistent (auto)antigenic stimulation by chronic infections or autoimmune disorders leads to lymphoid proliferation, susceptible to malignant transformation; the acquisition of genetic aberrations culminates in the activation of intracellular survival pathways and clonal outgrowth due to proliferation and resistance to apoptosis. However, the interactions between cell-extrinsic (environmental factors) and cell-intrinsic (genetic, molecular, and immunological abnormalities) factors that underlie disease pathogenesis are still not completely understood. From a clinical standpoint, MZLs are indolent disorders, often manageable with a “watch and wait” strategy, and exhibiting excellent survival when treated with conventional immunochemotherapy or radiotherapy. However, such approaches result in overtreatment of many patients affected by these indolent lymphomas. Accordingly, several investigators are exploring new active and less toxic therapies. In particular, monoclonal antibodies, immunomodulators, antibiotics, and other targeted therapies have been tested, sometimes with promising results. Nevertheless, efficacy rates are still lagging behind those achieved with conventional treatments.

European research contributions In the past 20 years, European researchers have made great progress in basic, translational, and clinical research into MZL. In particular, the establishment of pathogenic links with microorganisms has boosted understanding of the mechanisms underlying MALT lymphomagenesis and advanced therapeutic concepts.93 A seminal finding was that t(1;14)(p22;q32)/BCL10-IGH, t(14;18)(q32;21)/IGHMALT1, and t(11;18)(q21;q21)/API2-MALT1, recurrently seen in MALT lymphomas, activate the nuclear factor NFκB pathway. More recently, high-throughput studies have identified several genetic changes that are useful as biomarkers for disease diagnosis and refined classification (e.g. BRAF, MYD88, NOTCH2, and KLF2 and NOTCH2 mutations), and new targets to be translated into therapeutic interventions (e.g. BCR, TLR, Notch, NF-κB, and MAPK signaling pathways), especially in splenic MZL. Splenic MZL was also shown to display a remarkably skewed immunoglobulin gene repertoire because up to one-third of cases express clonotypic B-cell receptor immunoglobulin utilizing the IGHV1-2*04 gene, supporting antigen selection in splenic MZL ontogeny. The precise diagnosis of MZL remains challenging, but real progress has been achieved. The contribution of European researchers though the Splenic Lymphoma Working Group has been important, especially for the establishment of guidelines for the diagnosis, treatment, and monitoring of splenic MZL. Their studies have been instrumental in the characterization of a broad category of variably well-defined provisional entities, involving primarily the spleen, that do not fall into any of the other distinct types of splenic B-cell neoplasms (splenic B-cell lymphoma/leukemia unclassifiable), especially splenic diffuse haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

red pulp lymphoma and hairy cell leukemia variant, and their precise ontogenetic relationship with splenic MZL. Furthermore, positive diagnostic markers (e.g. MNDA and FCRL4) have been established, and gene expression studies have identified a specific gene expression profile that separates nodal MZL from other lymphoma types. The Splenic Lymphoma Working Group has also formulated standard criteria to initiate treatment and a simple but effective prognostic score for splenic MZL.94 Such advancements have complemented the therapeutic progress in MZLs achieved thanks to European trials, often performed within the International Extranodal Lymphoma Study Group. An International Extranodal Lymphoma Study Group trial and a nationwide Spanish trial have established a standard of care with immunochemotherapy for extranodal MZL.95,96 Moreover, the activities of drugs such as rituximab, everolimus, bortezomib, lenalidomide, and clarithromycin, among others, in MZLs have been addressed in European trials. Importantly, studies of the association between infectious agents and MZL resulted in the development of safe, costbenefit, and effective therapeutic strategies,93 as exemplified by the efficacy of antiviral therapy for hepatitis C virus-related MZL.97

Proposed research for the Roadmap A concerted use of high-throughput platforms available in several European research centers will significantly advance our knowledge of these lymphomas. The main objectives of future studies should be the following. 1. Characterize genetic aberrations and molecular mechanisms involved in the natural history of MZL, map recurrent mutations to molecular pathways, and investigate their correlation with gene expression signature and potential oncogenic co-operation among the altered molecular pathways. 2. Unravel the ontogenetic mechanisms of splenic MZL and other lymphomas of MZ origin, through genome (e.g. whole-exome sequencing), transcriptome (e.g. RNA-seq), epigenome (e.g. DNA methylome), and immunoglobulin repertoire analysis and comparison to normal B-cell subsets from human spleen and lymph nodes. 3. Analyze multi-stage lymphomagenesis and clonal evolution, including transformation, applying deep sequencing to longitudinal samples from different phases of the disease. 4. Define functional immune profiles of disease subgroups with particular clinical and/or biological features. 5. Identify other micro-organisms that could play a pathogenic role and serve as target for more specific therapies. 6. Identify and characterize the antigens and immune pathways that drive lymphoma development, thus paving the way for tailored treatment strategies applicable to each major immunogenetic subgroup. 7. Investigate the precise mechanisms associated with chronic inflammation involved in the development and evolution of nodal MZL arising in patients with autoimmunity. 8. Address the complex interactions between the neoplastic B cells and the surrounding microenvironment, haematologica | 2016; 101(2)

including both cellular and humoral components. 9. Develop prospective clinical trials on risk-adapted treatments that result in improved efficacy and reduced toxicity.

Anticipated impact of the research Advances in unraveling the molecular abnormalities and mechanisms of antigenic triggering combined with progress in genetic profiling will likely result in the identification of subjects with an increased risk of MZL and, consequently, the potential to implement prevention strategies. Knowledge of involved antigens, pathogenic mechanisms, altered molecular pathways, and the crosstalk between tumor cells and the microenvironment will promote personalized therapies, thus maximizing benefits while minimizing unnecessary toxicities and costs. Given the recent refinement of some entities and their relative rarity, pan-European co-operation is a prerequisite for real progress. This is especially important given the emerging trend of designing clinical trials for highly select subgroups of MZL patients, which is a challenge considering the rarity of these lymphomas. Patient selection will always be based on clinical criteria, but biological and molecular parameters will be progressively incorporated as selection criteria in important trials. This is necessary for testing new compounds, as well as for guiding patient choice among the armamentarium of personalized therapies.

2.6. T-cell and NK-cell lymphoma Philippe Gaulard (HĂ´pital Henri-Mondor, Creteil, France), Bertrand Coiffier (UniversitĂŠ Claude Bernard, Lyon, France).

Introduction T-cell and natural killer (NK)-cell lymphomas are rather heterogeneous and uncommon malignancies. They represent less than 15% of all non-Hodgkin lymphomas (NHLs) worldwide but their epidemiology shows important geographic variations, partly overlapping with the prevalence of certain viral infections [e.g. Epstein-Barr virus (EBV) and human T-cell leukemia virus type 1] and linked to the heterogeneous distribution of genetic backgrounds. The current WHO classification recognizes more than 20 entities grouped according to their predominant nodal, extranodal, cutaneous, or leukemic presentation; some of them indolent, but most of them aggressive or very aggressive.98 With some exceptions, such as ALK-positive anaplastic large cell lymphoma, survival is usually short. The longterm overall survival for all entities is less than 30%. Unfortunately, there is no real standard treatment for most T-cell/NK-cell lymphoma, except for NK/T-cell nasal type lymphoma where the role of L-asparaginase (alone or in combination) has been well demonstrated.99 Prognostic biomarkers are not well characterized for most groups or entities. In addition, most entities lack pre-clinical models. Leading researchers in the field feel that substantial and continuous efforts should be made while appreciating the challenges represented by the rarity of these lymphomas.

European research contributions In recent years, a better understanding of most entities of the different T-cell and NK-cell proliferations has been established.100,101 The different identities have become better defined, primarily through pathological classification. 135

A. Engert et al. Several European groups have long-lasting research activity and experience in the pathogenesis of T-cell lymphoma. In larger cohorts, genome-wide molecular profiling of available tumor material has provided new insights into the pathobiology of these diseases. This subsequently led to the identification of new markers with diagnostic, prognostic, and/or therapeutic implications. The identification of the follicular helper T-cell subset as the cell of origin of angioimmunoblastic T-cell lymphoma and a proportion of peripheral T-cell lymphoma (PTCL) represented an important step in defining markers with diagnostic and therapeutic implications. European groups described the currently known molecular signatures of most T-cell lymphomas. These findings also contributed to the discovery of several genetic aberrations and deregulated pathways, such as the involvement of mutations in epigenetic modifiers and dysregulation in important signaling pathways, including JAK-/STAT, PDGFRA, and NF-κB. Now there is a good chance that at least some of these pathways may serve as targets for the development of novel therapies. New drugs such as romidepsin, pralatrexate, and belinostat have shown clinical responses in up to 30%-35% of relapsing patients but the question as to their role in daily patient management remains unanswered. Promising advances include a targeted immunoconjugate against CD30-positive T-cell lymphoma (expressed on anaplastic lymphoma, but also on some others T-cell subtypes) and small molecules against the activity of the ALK kinase (e.g. crizotinib). However, no substantial improvement has been made in defining the best first-line treatment or the role of high-dose chemotherapy and transplant for these patients. Several phase III clinical trials have been launched that evaluate therapeutic options such as transplants in first remission and the addition of new drugs to CHOP, but results are still pending.

exome sequencing studies, the genetic landscape and potential driver alterations remain poorly characterized. A European effort to collect clinical and biological data of PTCL patients enrolled within clinical trials or registries is critical. It should aim to perform whole-exome and wholegenome sequencing analysis on a large number of clinically well-annotated PTCL samples of each entity to identify driver alterations and novel candidates for therapy. Early access to targeted drugs will be needed for European groups in order to collect tumor samples and establish a viable network of platforms to exchange data, define common protocols, and share quality-control processes. A number of novel compounds targeting driver mutations (e.g. demethylating agents and IDH2 inhibitors in PTCLs with mutations in epigenetic modifiers) are awaiting clinical application. The selection of patients who are likely to respond to a single agent should be based on the identification of the alterations, the exploration of pathways, or the presence of genetic abnormalities. Evaluation of targeted drug combinations should also be undertaken. Access to platforms and development of these novel combinations will be of critical importance in PTCL patients, a group of patients with a very poor outcome. In parallel, new response and outcome biomarkers need to be explored. Functional imaging has been shown to be a useful tool for evaluating early response and correlating it to clinical outcome. Within the populations of patients collected in Europe, in view of the rarity of the diseases, efforts to develop new markers of specific pathways or new evaluation modalities could help guide treatment. Circulating DNA could also be a powerful tool to help diagnosis, evaluate response to treatment, and predict relapse.

Anticipated impact of the research Proposed research for the Roadmap Insights into the molecular basis of T-cell and NK-cell lymphomas will probably help define future risk stratification, predict treatment response, and provide the basis for novel drug design. The emphasis is put on defining the best combination for first-line patients. They represent the best opportunity for finding curative treatments, as salvage treatments for these lymphomas clearly remain insufficient. Given the modest efficacy of most agents, improving outcome will likely rely on drug combinations with complementary mechanisms of action. Furthermore, we need to better identify patients who will respond to therapies, with correlative studies performed in parallel. Despite some improvement in recent years, there is a need to characterize the genomic, transcriptional, epigenetic, and metabolomic landscape of each PTCL subtype. Development of new cell lines and animal models representative of these subtypes are also needed to explore the efficacy of new drugs, and to improve our understanding of the important biological mechanisms of the lymphoma cell and the interaction with tumor microenvironment. The most important challenge in PTCL is to develop alternative therapies to the conventional CHOP or CHOP-like chemotherapy regimens in order to improve survival. Biologically oriented strategies with drugs targeting altered genes, pathways or surface molecules expressed in specific PTCL entities need to be developed. Despite recent whole136

All of these projects will help describe well-defined entities with their specific genetic modifications and will enable new targets for innovative drugs to be evaluated; these could provide more efficient and personalized treatment approaches for PTCL patients and prolong their survival. PTCLs are certainly the best example of lymphomas for which there is still a need for biologically oriented novel strategies. Randomized studies will help set new standards and enable better entity-specific treatment regimens to be tested. Close collaboration between patients, academic laboratories, pharmaceutical and biotech companies, and European co-operative lymphoma study groups with a long-standing tradition of working together offers the possibility of a rapid translation of biological studies into the clinic. Finally, reduction of hospital costs and optimization of treatment strategies will allow this policy to be adopted in all parts of Europe.

2.7. Lymphoma and immune deficiency (including AIDS, post-transplant, and drug-induced immunodeficiency) José Maria Ribera (Institut Catala d'Oncologia, Barcelona, Spain), Antonino Carbone (Centro di Riferimento Oncologico, Aviano, Italy), Jose-Tomas Navarro (Institut Catala d'Oncologia, Barcelona, Spain), Ralf Trappe (Charité-Universitätsmedizin Berlin, Berlin, Germany).

Introduction The incidence of Hodgkin lymphoma (HL), and espehaematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

cially non-Hodgkin lymphoma (NHL), in patients with immune deficiencies is higher than in the normal population. Although particularly evident in patients with HIV infection, this also occurs in other situations, such as after solid organ and hematopoietic transplants, and in patients receiving immunosuppressive therapies or with autoimmune diseases. Characteristically these lymphomas have high-grade malignancy and are in advanced stage, with frequent extranodal involvement. Since the introduction of combination antiretroviral therapy (cART), there have been a number of changes in the spectrum of cancer affecting HIV-infected individuals. Although the incidence and proportion of deaths related to non-AIDSâ&#x20AC;&#x201C;defining malignancies are increasing, lymphoma is still the most frequent neoplastic cause of death among HIV-infected individuals. The incidence of NHL initially fell in the cART era but has now stabilized. On the other hand, the incidence of diffuse large B-cell lymphoma (DLBCL) and primary central nervous system lymphomas has decreased, whereas that of Burkitt's lymphoma (BL) and HL has increased. Post-transplant lymphoproliferative disorders (PTLDs) include a range of diseases ranging from benign proliferations to malignant lymphomas. Risk factors for developing PTLD include Epstein-Barr virus (EBV) infection, recipient age, transplanted organs, type of immunosuppression, and genetics. Uncontrolled proliferation of EBV-infected B cells is implicated in EBV-positive PTLD, whereas the pathogenesis of EBV-negative PTLD may be similar to that of NHL arising in the general population. The management of lymphomas in immunosuppressed patients differs according to the cause of the immunosuppression. In HIV-infected patients, the extensive use of cART has allowed these patients to be treated with identical schedules of immunochemotherapy as those used in the general population (together with cART and adequate prophylaxis of opportunistic infections). In PTLD, the first step is the removal of immunosuppressive therapy, followed by anti-CD20 immunotherapy, moving quickly to standard immunochemotherapy schedules if response is not rapidly achieved. Sequential therapy using rituximab followed by chemotherapy has demonstrated promising results and may establish a standard of care. The remaining immunosuppression-associated lymphomas are managed like those arising in the normal population.102-106

come with the addition of rituximab to the different chemotherapy schedules in patients not severely immunosuppressed. A new prognostic score for HIV-related lymphomas in the rituximab era (AIDS-Related Lymphoma International Prognostic Index) has been developed, combining patients from Europe and the United States included in phase II or phase III trials of immunochemotherapy. This score includes the age-adjusted International Prognostic Index (IPI), the number of extranodal sites, and the HIV-score (composed of CD4 count, viral load, and prior history of AIDS). Twenty-eight percent of patients were defined as low risk by the AIDS-Related Lymphoma International Prognostic Index and had an estimated 5year overall survival of 78%, 52% as intermediate risk (5year overall survival of 60%), and 20% as high risk (5-year overall survival of 50%). Another European-US study has shown that the outcome of patients with AIDS-related lymphomas has improved in the past two decades, and effective HIV-directed therapies have reduced the impact of HIV-related prognostic factors on outcome, allowing curative anti-lymphoma therapy to be used in most patients with aggressive NHL. As far as HL is concerned, the results of chemotherapy schedules used in different European countries, such as BEACOPP (Germany) and VEBEP (Italy), have been comparable to those obtained with the classical ABVD regimen (Spain and other countries). Similarly to NHL, an international effort (Europe, the US, and South America) has been made to define the prognostic factors of HL patients treated with ABVD and cART, showing the negative impact of low CD4 lymphocyte counts on overall and progression-free survival. A comparative analysis of HIV-related lymphoma and a matched cohort of HIV-negative lymphoma patients from several European countries was conducted by the European Group for Blood and Marrow Transplantation (EBMT). Comparable survival between HIV-positive and HIV-negative NHL and HL patients undergoing autologous peripheral blood stem cell transplantation was observed, leading to the conclusion that, in the cART era, HIV-infected patients with lymphoma should be considered for autologous peripheral blood stem cell transplantation according to the same criteria adopted for HIV-negative lymphoma patients. Finally, a co-operative study from the European Group of AIDS and Tumors analyzed the autologous peripheral blood stem cell mobilization policies used in HIV-associated lymphomas, evaluated the failure rate, and identified factors influencing mobilization results.

European research contributions Most of the achievements made through collaborative efforts in Europe have been in HIV-related lymphomas. Several national groups from European Union countries have conducted phase II trials showing similar results of the treatment of HIV-related lymphomas in the cART era. The most frequent schedules used for DLBCLs are RCHOP, R-CDE (rituximab, cyclophosphamide, doxorubicin, and etoposide) and R-EPOCH (rituximab, etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin); for BLs the most frequent schedules are RCODOX-M/IVAC (rituximab, cyclophosphamide, vincristine, doxorubicin, and methotrexate/ifosfamide, etoposide, and cytarabine), LMB, NHL2002, Burkimab, and dose-adjusted R-EPOCH, among others. A general consensus has been reached on the improvement in outhaematologica | 2016; 101(2)

In summary, most of the co-operative European studies on lymphomas associated with immunosuppression have been focused on therapeutic strategies and the identification of prognostic factors. Pan-European trials have also established treatment standards for the management of post-transplant B-cell lymphomas, using sequential administration of rituximab followed by rituximabchemotherapy. However, rare post-transplant lymphoma entities remain challenging.

Proposed research for the Roadmap Basic aspects 1. To improve the knowledge of the mechanisms of lymphomagenesis in immunosuppression-related lymphomas, especially EBV-driven lymphomagenesis and 137

A. Engert et al. the relation with other viruses (e.g. other gammaherpesviruses, HIV, and hepatitis viruses). 2. To evaluate the potential value of plasma load of gammaherpesviruses as a surrogate marker of residual disease in lymphomas in immunosuppressed patients. 3. To develop early biological predictors of development of lymphomas in immunosuppressed patients (e.g. EBV viral load and serum-free light chains). 4. To study the dynamics of the T-cell and natural killer (NK)-cell repertoire in immunosuppressed patients and its relation with the development of lymphomas. Clinical aspects The key clinical research goals are as follows. 1. To develop pan-European clinical trials with new drugs, especially in the setting of relapsed/refractory patients with HL and NHL, since immunosuppressed patients (especially those who are HIV-infected) are usually excluded from current clinical trials. Given their low frequency, specific trials for these patients are required in a multicenter setting. 2. To compare the strategies based on R-CHOP with those based on infusional dose-adjusted chemotherapy (e.g. dose-adjusted-R-EPOCH) in the treatment of immunosuppression-related NHL. 3. To develop a joint effort to conduct specific clinical trials for the treatment of infrequent subtypes of lymphoma arising in immunosuppressed patients (e.g. plasmablastic, peripheral T-cell, and primary effusion lymphomas). 4. To conduct joint trials with therapies including antiviral agents, adoptive immunotherapy (e.g. genetically modified EBV-specific cytotoxic T cells), and monoclonal antibodies targeting cytokines for the prevention and treatment of PTLD.

Anticipated impact of the research With the extensive use of immunologically-based therapies to treat cancer and immunological diseases, the increased frequency of solid organ and stem cell transplants, and the increased life expectancy of patients with HIV infection, the number of lymphomas arising in immunosuppressed patients is expected to increase in the coming years. However, the frequency of these lymphomas is still lower than that of those involving the nonimmunosuppressed population, making it essential to initiate co-operative efforts in order to improve the knowledge of the mechanisms of lymphomagenesis and to develop more effective therapies. As the first-line therapy of HL and aggressive NHL in these patients has been reasonably well standardized in Europe, efforts must mainly be focused on relapse and refractory patients, for whom promising new drugs and immunologically-based therapies are in development. Progress in the knowledge of the mechanisms of lymphoma development in these patients will contribute to improving the treatment results, and will hopefully help in the prevention of these lymphomas.

2.8. Chronic lymphocytic leukemia and other chronic lymphoproliferative disorders Stephan Stilgenbauer (Universitätsklinikum Ulm, Ulm, Germany), Frederic Davi (Université Pierre et Marie Curie, Paris, France), Dimitar Efremov (International Centre for Genetic Engineering and Biotechnology, Trieste, Italy), Peter Hillmen (University of Leeds, Leeds, United 138

Kingdom), Emili Montserrat (Hospital Clínic de Barcelona, Barcelona, Spain), Tadeusz Robak (Uniwersitet Medyczny W Lodzi, Lodz, Poland).

Introduction Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults in Europe. The disease is characterized by a complex pathogenesis due to the interplay between genetic and microenvironmental factors and a variable clinical course, making it a paradigm for the understanding of other malignancies. Recent discoveries in biology, therapy, and the relationship between these two have led to groundbreaking advances, and underline the impact of this “translational” approach for cancer research in general.

European research contributions Research in Europe has been at the forefront regarding CLL biology and therapy. This goes back to the defining of the Binet staging system, the discovery of pathogenic mechanisms and prognostic parameters, the development of the current standard of care, and the latest innovations regarding novel therapies and resistance mechanisms. Details of each of these are outlined in the priorities of the proposal in the following section.

Proposed research for the Roadmap The cellular origin of CLL has been difficult to identify. The finding that the clinical behavior of CLL differs dramatically depending on the mutational load and rearrangements of their immunoglobulin genes has opened new perspectives. Although it was initially postulated that CLL with mutated IGHV corresponded to memory B cells while CLL with unmutated IGHV corresponded to naive B-lymphocytes, the current consensus is that both represent clonal expansions of antigen-experienced cells. Based on similar patterns of antigen recognition, both have been thought to derive either from marginal zone B cells or putative human B1 cells. Recent studies coupling gene expression profiling and IGHV repertoire analysis have led to the conclusion that both IGHV subtypes derive from subsets of CD5+ B lymphocytes. Surprisingly, recent data from xenotransplantation experiments and next generation sequencing (NGS) suggest that the disease may, in fact, originate within hematopoietic progenitor (CD34+) cells. Thus, although progress has been made, the precise cellular origin still needs to be elucidated. As indicated by the importance of the immunoglobulin genes and rearrangements and the clinical success of BCR pathway inhibitors (see below), the BCR and its downstream signaling elements play a crucial role in the pathogenesis of CLL. However, as BCR pathway inhibitors cannot cure the disease and the functional pathogenic mechanisms of BCR stimulation remain elusive, further research in this area is needed. Specific topics that need to be addressed in order to allow selective therapeutic targeting of disease subsets are related to the characterization of the stimuli and interactions that activate the BCR pathway and determine their impact and the downstream signaling molecules activated by different types of BCR interactions.107 Furthermore, it will be extremely important to: 1) develop rational therapeutic strategies with curative potential by combining BCR pathway inhibitors with drugs that target other essential pathways, such as apophaematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

tosis regulation; and 2) decipher mechanisms underlying treatment resistance. Genomic aberrations and gene mutations have been identified as major factors determining resistance to therapy and poor survival. 17p deletion and/or TP53 mutation remain the strongest prognostic markers in multivariable analyses despite the improvement in treatment with immunochemotherapy (see below), and NOTCH1 mutation may be a predictive marker indicating a decrease in benefit from the addition of CD20 antibody. Mutations of specific target structures, such as BTK, and downstream signaling molecules, such as PLCG2, have been identified as resistance mechanisms against targeted therapies, such as ibrutinib.108 Questions remain, however; for example, those related to resistance mechanisms and their impact on treatment decisions for novel treatments such as PI3K and BCL2 inhibitors and novel antibodies. Furthermore, the outcome of some disease subgroups (e.g. 17p-CLL) still appears inferior to other subgroups, and the transformation of CLL to more aggressive lymphoma (Richter transformation) is a frequent phenomenon of unclear etiology, leading to new and urgent research questions. The standard conventional treatment approach in CLL is now immunochemotherapy with FCR (fludarabine, cyclophosphamide, and rituximab) for young/fit patients109 and chlorambucil plus anti-CD20 antibodies (rituximab, obinutuzumab or ofatumumab) for elderly/unfit patients.110 Despite the dramatic improvements in efficacy with these regimens, a number of critical issues remain. The first regards when to initiate therapy in the light of novel developments; also important are the therapeutic objectives [symptom control/palliation vs. minimal residual disease (MRD) eradication/cure]. Moreover, there is a relentless pattern of relapse despite the often initially deep remissions, making cure unlikely with these regimens. Therefore, predictive factors allowing informed treatment choice (i.e. is any one treatment superior to another in a particular patient?) represent an urgent need for individualized treatment (“precision medicine”). The improved understanding of disease biology in CLL has led to the identification of targeted therapeutic approaches against BTK (e.g. ibrutinib), PI3K (e.g. idelalisib), BCL2 (e.g. venetoclax), and CD20 (e.g. obinutuzumab and ofatumumab). These agents have provided compelling evidence not only for dramatic efficacy but also for outstanding tolerability.111 Nevertheless, a number of critical questions have emerged, in particular, related to the choice and handling of these agents. With some novel agents, “benign” disease persistence (lymphocytosis) is a frequent phenomenon, whereas there is rapid disease eradication with other agents (e.g. immunochemotherapy, BCL2 antagonists), and the principles guiding treatment aims between disease control and eradication remain to be determined. New, rare adverse events (e.g. bleeding, atrial fibrillation, and colitis) have been identified in spite of the generally outstanding treatment tolerability, and the longterm (side) effects of novel treatments are unclear. Given that combination treatments have been beneficial on the one hand, whereas on the other hand the novel compounds have already shown great single agent activity, will the concept of a combination approach or a sequential haematologica | 2016; 101(2)

use of the novel agents lead to better long-term results? As differences are seen, on the one hand, between patients' and their disease characteristics and, on the other hand, between the specific features of each compound, will all patients be treated with the same approach, or how can subgroups be identified for the greatest individual benefit? Lastly, given the cost of indefinite treatment duration with the currently licensed novel agents, and the worldwide demand for efficacious cancer therapy, how will the issue of cost and equal access to these treatments be handled?

Anticipated impact of the research As is often the case with breakthroughs, the answers from the biological and clinical studies mentioned above open up new questions, and it appears that the magnitude of these questions is at least as great as the advances made through a new understanding of CLL biology and treatment. Clearly, to move beyond the understanding and success witnessed already, more well-designed clinical trials are needed with the ultimate goal of cure. Of equal importance, and underlined by the development of these targeted agents in CLL, is the advance of laboratory science. Taken together, CLL can serve as a valid model system for cancer in general by highlighting the dramatic progress that can be made within a short time frame when linking biology to therapy in a truly translational approach.

2.9. Acute lymphoblastic leukemia Monique den Boer (Erasmus MC, Rotterdam, the Netherlands), André Baruchel (Hôpital Universitaire Robert-Debré, Paris, France), Andrea Biondi (Università degli Studi di Milano-Bicocca, Monza, Italy), Nicola Gökbuget (Universitätsklinikum Frankfurt, Frankfurt, Germany), Elizabeth MacIntyre (Université Paris Descartes, Paris, France), Anita Rijneveld (Erasmus MC, Rotterdam, the Netherlands).

Introduction Acute lymphoblastic leukemia (ALL) is a life-threatening disease if not treated immediately. ALL occurs most frequently in children under 15 years of age and accounts for 25% of pediatric cancers and less than 1% of adult cancers. ALL arises from hematopoietic stem cells (HSCs) in the bone marrow that have acquired genomic lesions that result in the survival and a proliferative capacity of immature, non-functional malignant cells at the expense of normal, functionally differentiated white blood cells. This results in an impaired immune response against pathogens, resulting in fever or infections, anemia, and decreased wound healing capacity or bleeding. The type of genomic lesions differs somewhat between children and adults; for example, KMT2A gene fusions are predominantly found in infants (< 1 year of age), and the ETV6-RUNX1 gene fusion is mainly found in children, whereas the BCR-ABL1 gene fusion is most frequently found in adults. Treatment mainly consists of combination chemotherapy and allogeneic hematopoietic stem cell transplantation (HSCT), mainly limited to highrisk categories of ALL. Due to risk-stratified treatment, more optimized treatment protocols, and improved supportive care, the 5-year event-free survival on contemporary treatment protocols is more than 80% for children and is approaching 50% for adults.112 The short- and long-term side effects of therapy are considerable, however, particularly with respect to the quality of life (QoL) in at least some of the adult survivors of childhood cancer. 139

A. Engert et al. European research contributions The treatment of ALL is relatively well structured in Europe by the assembly of national and international study groups, such as the International-Berlin-FrankfurtMunster study group for childhood ALL and the European Working Group for adult ALL (EWALL). New prognostic subtypes of ALL were identified by screening large patient cohorts, facilitating structured diagnosis and treatment of ALL. The European collaborative studies under the umbrella of EuroMRD have been key in standardizing and developing monitoring minimal residual disease (MRD) and in building risk-adapted therapies that benefit ALL patient outcomes. The prognosis of adolescents and young adults has been significantly improved by pediatric protocols. In addition, pediatric-like therapies, including the use of L-asparaginase, have significantly improved outcome for adults with ALL. In addition to disease monitoring, major achievements have been obtained in molecularly redefining ALL. Initially, chromosomal lesions were identified, such as those affecting chromosomal copy number (e.g. high hyperdiploidy with more than 50 chromosomes, as well as good prognosis) and those leading to aberrant chromosomes [e.g. the Philadelphia chromosome t(9;22), which gives rise to the BCR-ABL1 gene fusion and is predictive of an unfavorable outcome]. The development of the molecular toolbox, mainly driven by the deciphering of the human genome in 2001, has accelerated the oncogenomics field in the past decade. Together with their US colleagues, European researchers have significantly contributed to the molecular characterization of ALL. Gene expression profiling has identified new subtypes of B-cell precursor and T-cell lineage ALL, and deepened our knowledge of mechanisms of resistance to frequentlyused chemotherapeutic drugs, such as prednisone and Lasparaginase.114 The BCR-ABL1–like ALL subtype, which was originally identified in children, has also been identified in adults with ALL, representing a relatively large new unfavorable prognostic subtype. Genomic screens and next generation sequencing (NGS) have revealed many new fusion genes, including more than 10 ABL1-class and more than 10 JAK-class fusion genes, which result in constitutively-activated gene products that can be targeted with precision medicines, such as ABL1 inhibitors like imatinib and JAK inhibitors like ruxolitinib. In addition to gene fusions, smaller genetic mutations have been identified, which often affect the survival and proliferative capacity of leukemic cells.

Proposed research for the Roadmap The molecular deciphering of ALL revealed the complexity of this disease; the heterogeneity among ALL patients, and between children and adults, is further complicated by the identification of mutated subclones, which can resist chemotherapy or be acquired during treatment, and which can give rise to (late) relapse.115 In addition, our knowledge about the supportive (and protective) role of the bone marrow microenvironment in the progression and treatment response of leukemia is still limited. Research will improve our knowledge of the pathobiology of ALL and its interaction with the microenvironment, which presumably also plays a role in the spread of ALL to extramedullary sites, such as the central nervous system, liver, and testicles, associated 140

with an adverse clinical outcome. Many new genomic/molecular lesions have been identified in past years, of which a few are prognostic. It is a huge mistake to only foster research dedicated to identifying and unraveling prognostic lesions because the improvements in clinical outcome will also limit the number of new prognostic lesions that can be identified, whereas the side effects of treatment will remain considerable. Research should increasingly focus on functional studies addressing the tumor dependency of new lesions (including those found in subclones) in relevant leukemia models. Last but not least, studies need to address how new precision medicines should be combined with (reduced) up-front chemotherapy to minimalize side effects without jeopardizing clinical outcome. Attention should also be paid to age-related differences in pharmacokinetics of novel and old drugs in order to individualize dosing and accelerate the implementation of new drugs in children. Support by regulatory authorities, such as the European Medicines Agency (EMA), the development of European laws to facilitate drug development in children (Pediatric Regulation, 2001/20/EC), and early access to new potential drugs for ex vivo (patients’ leukemic cells) and in vivo (ALL animal models) studies are essential for accomplishing better therapies with reduced side effects.116 Furthermore, harmonizing the backbone of chemotherapeutic protocols of different study groups will be highly beneficial for performing clinical trials with new drugs in rare subsets of patients and will accelerate early drug development programs together with pharmaceutical companies. Recently developed monoclonal antibodies (bispecific antibodies and immunoconjugates) in ALL need to be further evaluated and eventually integrated in the standard management of some patients, particularly in adult ALL. Early results using cellular therapy (adoptive modified T cells, such as CAR-T cells) are promising and point towards alternative innovative strategies that might be effective in ALL. Leukemias have always represented proof-of-concept cancers for solid tumors, whereby their understanding permeates to less easily accessible tumors in both adults and children. Reinforced funding for acute leukemia will, therefore, have an impact well beyond the small percentage of cancers they represent.

Anticipated impact of the research The treatment will change from disease-type to molecular target-type, and from risk-stratified treatment schedules to more personalized therapies with precision medicines. In addition, individualized drug dosing may prevent underdosing and hence may reduce the risk of relapse, while therapeutic drug monitoring may also prevent overdosing and associated toxic side effects. More specific therapies and new immunotherapy-based approaches are of the utmost importance, not only for improving the prognosis of high-risk patients but also for significantly reducing treatment-related morbidity for patients of all ages, and especially for long-term survivors of childhood cancer.

2.10. Multiple myeloma and other plasma cell neoplasms Meletios Dimopoulos (National and Kapodistrian University of Athens, Athens, Greece), Hervé Avet-Loiseau (Centre Hospitalier Universitaire de Toulouse, Toulouse, France), Monika Engelhardt (Universitätsklinikum Freiburg, haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

Freiburg, Germany), Hartmut Goldschmidt (UniversitĂ¤tsklinikum Heidelberg, Heidelberg, Germany), Antonio Palumbo (UniversitĂ degli Studi di Torino, Turin, Italy), Evangelos Terpos (National and Kapodistrian University of Athens, Athens, Greece).

Introduction Plasma cell disorders are common hematologic malignancies; multiple myeloma, the most common of these disorders, accounts for about 2% of all neoplasms and is the second most common hematologic malignancy. The median survival of patients with myeloma is 3-7 years; however, there is significant heterogeneity depending on the characteristics of the disease, of the patient and the therapy. The prevalence of the precursor monoclonal gammopathy of undetermined significance (MGUS) increases with age, and it is estimated that approximately 3%-5% of individuals over 65 years of age may have a MGUS. The risk of developing myeloma or other lymphoproliferative disorders with underlying MGUS is approximately 1% per year and remains lifelong. Myeloma is a disease of the elderly with a median age at diagnosis of around 65-70 years. In the past two decades, there has been a major increase in the number of patients over 75 years of age who are diagnosed and receive therapy for myeloma, and this has been largely attributed to the major demographic changes that have occurred in Europe. Thus, more patients of advanced age will require therapy, presenting a major challenge in terms of management and health care costs. Despite the recent improvements in the survival of patients with myeloma, there is a significant minority of patients who have a very poor prognosis even with the use of the most intensive therapies, including transplantation. This group includes patients who are refractory to both proteasome inhibitors and immunomodulatory drugs, as well as patients with high-risk cytogenetics [del17p, t(4;14), and add1q], plasma cell leukemia, extramedullary relapses, or myeloma of the central nervous system. New therapies and innovative strategies are urgently needed for the treatment of these patients. The recent advances made with the introduction of new drugs are also a challenge for the health care system due to the high cost of these therapies. Furthermore, these therapies are moving forward to front-line/early applications, they are combined very intensively, and many of them are given continuously until disease progression, resulting in substantial additional costs. Therefore, there must be a prudent allocation of health care resources in order to provide the best therapy for patients within a sustainable health care system.117-121

European research contributions The European Myeloma Network (EMN) was established in 2003 by integrating 27 research institutions and 14 trial groups with the intent of supporting development of novel diagnostics and therapies for multiple myeloma. Now, the EMN is a legal entity and has initiated co-operative clinical trials and laboratory research in different research fields in plasma cell dyscrasias. The close relationship between these research areas facilitated the exchange of information and experience, and has created a spirit of co-operation within the European area, prohaematologica | 2016; 101(2)

moting clinical and laboratory research in myeloma and related disorders. Different groups have initiated research programs and projects within the EMN and most of the researchers in Europe have been involved. As a result, several clinical trials have been conducted in myeloma, both multicenter phase III and phase I and II. The results of these clinical studies have framed the contemporary management of the disease in Europe. Importantly, through the collaborative network of the EMN it has been possible to conduct large clinical trials in rare diseases, such as WaldenstrĂśm macroglobulinemia and light chain systemic amyloidosis, which represent major advances in the field. Through the EMN, a network of laboratories is working on several projects and participates in European Framework Programmes. As a result, the EMN has regularly published recommendations and guidelines on the management and other aspects of myeloma and related disorders, and these provide guidance to European and other physicians who care for myeloma patients, thus advancing the quality of care of patients with plasma cell malignancies.

Proposed research for the Roadmap Genetic studies have revealed the complex nature of myeloma and other plasma cell disorders. Large-scale genetic studies will provide new insights into the pathogenesis of plasma cell malignancies but, importantly, will uncover mechanisms of development, resistance, and relapse. Biobanking will be crucial for collecting sufficient high-quality samples. Genetic studies require the development of additional tools for interpretation and application of the results of the genetic mapping. The EMN has set up a network for biobanking, and for providing guidance and facilities. Asymptomatic myeloma and MGUS are models for progression of the disease, and the integration of advanced genomics will provide the required knowledge of the evolution of plasma cell malignancies. Myeloma is characterized by inherent genomic instability and evolution of clonal disease has been shown for different pathways. Studies on disease evolution and genetic instability, integrating next generation technology and the detailed and prospective evaluation of the genetic evolution of the disease, will provide a framework for understanding mechanisms of resistance and escape. The role of the microenvironment is crucial in the development and evolution of the disease, and genetic and functional studies that will address the role of other cells in the microenvironment of the plasma cell can delineate the pathobiology of myeloma and provide new rational targets for therapy. Although mechanisms of resistance are crucial in order to build new therapies and combinations of existing drugs, it is also important to develop technologies and in vitro/ex vivo systems that can provide reasonable sensitivity and specificity and predictive tools for responses to various therapies in order to avoid toxicity and reduce health care costs. In particular, valid and reproducible animal models are needed in multiple myeloma. These require advanced technologies in order to become widely available and usable. In view of the deep responses that are now attained by a 141

A. Engert et al. significant fraction of patients with myeloma, the prognostic importance and the characteristics of minimal residual disease (MRD) are issues of intensive investigation. MRD may be considered a surrogate for cure or survival in clinical trial settings, but the different aspects of MRD need to be thoroughly and prospectively assessed. Technologies that involve multicolor flow cytometry, next generation sequencing (NGS), and single cell analysis will provide the data needed to build new regimens and modify therapeutic targets. In this setting, it will not be sufficient to monitor the disease by the traditional serum and urine electrophoresis, and new markers of disease for both diagnostic and monitoring procedures need to be developed and validated.

The EHA Roadmap for European Hematology Research

Imaging of the disease is another area of intensive research, which may provide crucial information on the extent, prognosis, and response of the disease. New technologies (PET/CT, PET/MRI, DWI-MRI) and advanced imaging software can identify disease foci with high sensitivity. Over the next few years, these advances will change the landscape of disease imaging and it is expected that evaluation of response will imply imaging criteria.

In recent years, European scientists have made major contributions to the understanding of the molecular basis of these disorders. Key disease-associated gene mutations have been discovered in myelodysplastic syndromes (MDSs), acute myeloid leukemias (AMLs), and myeloproliferative neoplasms (MPNs).9,122,123

Survival outcome (usually progression-free survival and, less often, overall survival) is the main end point of most clinical studies that aim to improve disease outcome. However, the availability of new therapies with different toxicity profiles and the changing demographics of the disease require an appraisal of quality of life (QoL) as a critical end point of clinical studies. Redefining outcome based not only on metrics of survival end points, but also on QoL, is an area of intense study, with major social and economic impact, which will become important for the choice of therapy, as well as for the approval and financial compensation of new drugs. Furthermore, well-designed clinical trials, including more investigatorinitiated efforts, are needed. Appropriate treatment options need to be harmonized within corporate groups to explore and answer questions such as: 1) when should treatment be initiated? 2) for how long should a regimen be given? 3) is cure rather than long-term disease control attainable, and in which patient cohort? and (4) what exactly benefits ultra-high-risk patients (i.e. patients with poor cytogenetics, RISS-3, plasma cell leukemia or extramedullary disease)?

Anticipated impact of the research The intensification of research in the field of plasma cell dyscrasias and related disorders is expected to improve outcome, according to all end points. This has been proved in the past decades where a major survival improvement has changed the landscape of these diseases as a result of new therapies, advanced technologies, and improved criteria for definition, diagnosis, and treatment initiation. These improvements reflect the major advances in our understanding of the disease biology, which have led to the improvement of therapies in terms not only of the availability of new drugs, but also by improving treatment strategies and delivery of therapy. It is also crucial that several aspects of this research result in the development and establishment of new technologies in genetics, single cell analysis, ex vivo predictive systems, and imaging. In addition, a better understanding of the disease and patientsâ&#x20AC;&#x2122; needs will allow a rational allocation of health care resources, with significant social impact. 142

Section 3. Malignant myeloid disease

Section editor: Hartmut DĂśhner. The malignant myeloid diseases that are discussed in this section are disorders of hematopoietic progenitor cells (HPCs) characterized by varying degrees of cell maturation defects and/or uncontrolled proliferation. These disorders commonly affect older patients and will, therefore, constitute an increasing burden for caregivers in an aging society.

The conduct of large controlled randomized trials within highly organized leukemia co-operative groups, in conjunction with the implementation of correlative science programs on well-annotated patient samples, has been one of the great assets of the European hematology community. The introduction of 1st- and 2nd-generation ABL1 TKIs in chronic myeloid leukemia (CML), characterized by the BCR-ABL1 gene fusion, has been the paradigm for the successful development of precision medicine in cancer. New strategies are now being studied that aim at curing this previously fatal disease. The discovery of the JAK2 mutation has been instrumental in rapidly bringing the first JAK1/JAK2 inhibitor into the clinic for the treatment of patients with MPNs.124,125 European investigators have also played a leading role in the development of hypomethylating agents for the treatment of MDS and AML.126,127 European co-operative groups have shown that around 95% of patients with acute promyelocytic leukemia can be cured by a chemotherapy-free combination therapy with the vitamin A derivative all-transretinoic acid and arsenic trioxide.128 For the great majority of patients with MDS and AML, however, progress has been very modest, and a high unmet medical need remains. The European research groups, under the umbrella of the European Hematology Association (EHA) and the European LeukemiaNet (ELN),129 and in collaboration with international investigators, have been instrumental in providing fundamental information to the scientific community by publishing recommendations and guidelines for clinical and laboratory practice of all the malignant myeloid diseases discussed here.130-133 Nevertheless, major challenges remain, as outlined in the following subsections. To further advance precision medicine for these disorders, a complete understanding of the disease biology will be needed. With the advent of novel technologies, comprehensive analyses of the genomes, epigenomes, transcriptomes, and metabolomes of these heterogeneous disorders will become possible. Robust bioinformatics tools need to be developed that are capable of processing such complex data and that can be applied on a clinical scale. Due to the demographic develhaematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

opment in Western countries, particular attention should be paid to the study of older patients in order to improve their outcome and, importantly, also their quality of life (QOL). A joint effort of clinicians and scientists, research consortia, and leukemia co-operative groups on an international level, in close collaboration with the biotech and pharmaceutical industry, will be essential for more rapid scientific achievements.

3.1. Myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms Mario Cazzola (Università degli Studi di Pavia, Pavia, Italy), Pierre Fenaux (Hôpital Saint Louis, Paris, France), Ulrich Germing (Universitätsklinikum Düsseldorf, Düsseldorf, Germany), Eva Hellström-Lindberg (Karolinska Institutet, Stockholm, Sweden).

Introduction Myelodysplasia is a term used to describe morphological abnormalities in one or more of the major myeloid cell lineages of hematopoiesis and is a typical feature of myelodysplastic syndromes (MDSs). MDSs are clonal disorders of hematopoiesis with a propensity to evolve into acute myeloid leukemia (AML), caused by somatic mutations that occur in hematopoietic stem cells (HSCs). These disorders include primary conditions as well as secondary and therapy-related forms. Primary MDSs occur mainly in older people as a result of stem cell aging, and their crude incidence rate is 4 per 100,000 people per year, indicating that more than 30,000 new cases are expected in Europe each year. Myelodysplasia is also found in other myeloid malignancies, in particular in the so-called myelodysplastic/myeloproliferative neoplasms (MPNs), which include chronic myelomonocytic leukemia and atypical chronic myeloid leukemia (CML). MDS and MDS/MPNs show marked clinical heterogeneity, ranging from conditions with an indolent clinical course and a near-normal standardized mortality ratio to entities with very poor prognosis. A risk-adapted treatment strategy is, therefore, mandatory for these disorders. Several treatments have been proposed for MDS, but only a few have met evidence-based criteria of efficacy. At present, the only treatment with a potentially curative effect is allogeneic hematopoietic stem cell transplantation (HSCT), but less than 20% of all MDS patients are eligible for such treatment and have a donor. Azacitidine can prolong survival in patients with high-risk MDS, while erythropoiesis-stimulating agents and lenalidomide improve anemia in patients with lowrisk MDS and the MDS associated with deletion 5q, respectively. Red cell transfusion remains the mainstay of therapy for many patients with MDS.

European research contributions In the past few years, the genetic basis of MDS has been revealed by means of massive parallel DNA sequencing, and seminal studies have been performed in Europe.134,135 Approximately 90% of MDS patients carry one or more oncogenic mutations, and two-thirds of them are found in individuals with a normal karyotype. Driver mutations have been identified in genes involved in RNA splicing, DNA methylation, chromatin modification, transcription regulation, DNA repair, and signal transduction. Only six genes are consistently mutated in 10% or more MDS haematologica | 2016; 101(2)

patients, while a long tail of more than 50 genes are mutated less frequently. Seminal contributions have also been made in pediatric hematology, for example, in elucidating the genetic predisposition to juvenile myelomonocytic leukemia.136 European hematologists have provided pivotal contributions to developing effective treatments for MDS, including erythropoietin and azacitidine.126,137 Recommendations for treatment of the individual patient with MDS have also been developed.130

Proposed research for the Roadmap Myeloid malignancies appear to be propagated by rare self-renewing mutant HSCs. However, the cellular and molecular mechanisms that regulate development, propagation, and therapy resistance of these myelodysplastic stem cells remain unknown. Studies are needed to: 1) delineate the stem and progenitor cell hierarchy in order to identify the cancer-propagating cells in MDS and MDS/MPN patients; 2) characterize the cellular and molecular mechanisms underlying disease development, progression, and therapy resistance; and 3) identify therapeutic targets suitable for efficient elimination of the MDS-propagating cells. In order to decipher the genetic complexity of MDSs, prospective studies of comprehensive mutational profiling of acquired gene mutations should be performed in large patient populations, ideally within clinical trials. The combined analysis of the genome and transcriptome may identify the impact of recurrent molecular abnormalities on gene expression. Particular focus should be given to spliceosome mutations, which occur in about half of all patients with MDS and are highly specific for this myeloid malignancy, suggesting an important role in disease pathogenesis. Gender and age significantly influence prognosis of MDS patients; in particular, age is an independent adverse prognostic factor. One or more comorbidities are found in more than half the patients at the time of diagnosis, and they have a significant impact on survival. Studies that analyze the relationships between genotypes, gender, age, and comorbidities are needed. The findings of these studies should be used to develop prognostic/predictive models. Outcome improvements in MDS patients remain modest. Identifying drugs that may further improve survival of patients with high-risk MDS and drugs that may inhibit ineffective erythropoiesis and improve anemia in those with low-risk MDS represents a priority. The importance of spliceosome and epigenetic mutations in MDS suggests that novel drugs targeting these pathways should be specifically investigated. Patient inclusion in clinical trials should be encouraged. The relationship between the genetic basis of MDS and the outcome of allogeneic HSCT should be explored, and more effective transplantation procedures should be developed.

Anticipated impact of the research The current lack of understanding of the molecular mechanisms that regulate MDS stem cell development and their escape from therapeutic targeting is limiting our ability to efficiently eliminate the cells required for MDS propagation. The research lines described above have the potential to decipher these mechanisms. 143

A. Engert et al. The current diagnostic approach to MDS and MDS/MPNs includes a complete blood count, peripheral blood and bone marrow morphology, and cytogenetics. Mutational profiling of peripheral blood has the potential to dramatically improve our approach to the diagnosis of myeloid malignancies, leading to a clinically relevant molecular classification of these disorders. The characterization of genomic and transcriptomic profiles of each individual patient with MDS or MDS/MPNs will allow the most appropriate treatment to be selected, patients to be enrolled in ad hoc clinical trials investigating new targeted therapeutic agents, and molecular biomarkers for monitoring response to treatment to be identified. This will eventually lead to precision medicine strategies.

3.2. Acute myeloid leukemia Gert Ossenkoppele (VU University Medical Center, Amsterdam, the Netherlands), Lars Bullinger (Universitätsklinik Ulm, Ulm, Germany), Robin Foà (Università degli Studi di Roma ‘La Sapienza’, Rome, Italy), Ralf Rambach (Deutsche Leukämie- und Lymphomhilfe (DLH), Bonn, Germany), Tadeusz Robak (Uniwersitet Medyczny W Lodzi, Lodz, Poland), Jorge Sierra (Hospital de la Santa Creu i de Sant Pau, Barcelona, Spain).

Introduction Acute myeloid leukemia (AML) is a clonal disorder arising from hematopoietic progenitor cells (HPCs) characterized by defects in their maturation program and by uncontrolled proliferation.138 AML is the most common form of acute leukemia with an estimated incidence of 3 per 100,000 individuals, resulting in 15,000 newly diagnosed patients each year in Europe. AML most commonly affects the elderly population, males more commonly than females, with a median age that has reached 70 years. The incidence of the disease will rapidly rise due to the proportional increase of the aging population. A further increase is expected from the rising incidence of therapy-related AML (i.e. myeloid neoplasms occurring in cancer survivors after successful treatment of a primary cancer). A particularly significant unmet medical need lies in the management of older patients with AML. Whereas in younger patients cure rates of 40%-50% can now be achieved, the outcome of older patients has remained very poor, in particular for those patients who are considered unsuitable for intensive chemotherapy. The backbone of treatment for AML, the combination of an anthracycline and cytarabine, has not changed in decades. This demonstrates the urgent need for the development of new agents, the mechanisms of action of which are based on a better understanding of the disease biology. Using novel genomics technologies, such as next generation sequencing (NGS) techniques, major progress has been made in deciphering the heterogeneity of the disease; however, translating this knowledge into clinical practice is lagging behind.

European LeukemiaNet (ELN) has facilitated the internationally embraced ELN recommendations on the diagnosis and management of AML as well as the consensus statement on allogeneic hematopoietic cell transplantation (HCT).131,139 European hematologists have played a leading role in the improved management of a particular form of AML, acute promyelocytic leukemia, by showing superiority of the chemotherapy-free combination of the vitamin A derivative all-trans-retinoic acid and arsenic trioxide over conventional treatment, with cure rates of approximately 95%, a first highly successful step toward precision medicine in AML.128 European investigators have made major contributions to our understanding of the molecular basis of AML, as exemplified by the discovery of the mutation in the nucleophosmin 1 gene (NPM1), one of the most important biomarkers currently used in the clinic.140 The identification of new biomarkers was paralleled by the introduction of the concept of minimal residual disease (MRD) detection either by quantitative polymerase chain reaction (PCR) or by flow cytometry, which is now implemented in many treatment algorithms. Recently, investigators have shown that clonal hematopoiesis with somatic mutations previously implicated in hematologic cancer (DNMT3A, ASXL1, and TET2) is increasingly common as people age, and it is associated with an increased risk of hematologic cancer. The data from this study are instrumental for the further understanding of the biology of AML in the aging population, one of the main challenges that we are now facing.141

Proposed research for the Roadmap Concerted efforts from basic, translational, and clinical hematologists will be required to make major advances in the forthcoming years. One important prerequisite to advance the field is the further understanding of the disease pathogenesis. This includes the identification and characterization of preleukemic cells and leukemic stem cells (LSCs), the analysis of the clonal architecture of genomic lesions, and their clonal evolution during the disease course, as well as the analysis of primary and acquired resistance mechanisms. To capture the entire complexity of leukemia biology, it will be instrumental to also analyze the transcriptional and epigenetic landscape of the leukemic cells. Integrated analysis of these complex omics data sets will require the parallel development of appropriate bioinformatics tools. These studies should be performed on well-annotated biosamples from patients treated in controlled clinical trials. A particular focus should be on the study of older patients, who in the past have been largely under-represented in both clinical trials and correlative science studies. Instruments need to be developed to better define patients who are considered fit for intensive therapy versus those who a priori should be considered for investigational treatment.

European research contributions In Europe there is a long-standing tradition of controlled randomized trials within well-organized leukemia co-operative groups and this has largely contributed to the scientific achievements of European hematology. The 144

A large number of new drugs targeting leukemic drivers or a multitude of deregulated pathways are awaiting clinical application. Careful pre-treatment selection by extensive molecular profiling will pave the way to a successful haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

outcome. Examples for targeting of defined subgroups are: AML with IDH1/IDH2 mutations (using small molecule IDH inhibitors), AML with FLT3 mutations [tyrosine kinase inhibitors (TKIs)], AML with KMT2A rearrangement (DOT1L or CDK6 inhibitors), and AML with DNMT3A mutations (hypomethylating agents). Another promising route of investigation is offered by the new avenues of immunotherapy, beyond the further development of the concept of allogeneic hematopoietic cell transplantation that will remain a mainstay in the management of AML patients. New immunotherapy approaches, such as vaccination, CAR T cells, natural killer (NK) cells, bispecific T-cell engagers, novel monoclonal antibodies, and immunoconjugates, hold great promise for treatment of bulk disease or for targeting residual LSCs. Finally, harmonization and standardization of complex diagnostic procedures, such as gene panel diagnostics and monitoring of MRD, need to be realized on an international level, because results from these diagnostic tests are expected to have a major impact on informing patient management.

Anticipated impact of the research The program aims at further understanding the complex molecular heterogeneity of the disease. Deciphering this enormous complexity will be essential for the development of personalized approaches to AML treatment. Given the current knowledge of the clonal architecture of the disease, no single drug is expected to cure it; rather, the combination of established therapies with novel agents that target disease-associated molecular lesions will be needed. Special attention must also be paid to the better management of older patients, given the more unfavorable biology and the still dismal outcome of the disease in these patients. Dissecting the molecular trajectories of the disease using well-annotated biosamples from patients treated in innovative clinical trials will be instrumental to achieve these goals. These research programs are expected to make a major contribution to improving outcome in patients with this fatal disease.

3.3. Chronic myeloid leukemia Andreas Hochhaus (Universitätsklinikum Jena, Jena, Germany), Francisco Cervantes (Universitat de Barcelona, Barcelona, Spain), Jan Geissler (CML Advocates Network, Bern, Switzerland), Francois Guilhot (Université de Poitiers, Poitiers, France), Guiseppe Saglio (Università di Torino, Turin, Italy).

Introduction Chronic myeloid leukemia (CML) is a malignant neoplastic disease of the hematopoietic stem cells (HSC). CML is typically linked with the Philadelphia chromosome, a shortened chromosome 22 as the result of a reciprocal translocation of chromosomes 9 and 22 leading to fusion of BCR and ABL1 genes. CML constitutes approximately 15% of all leukemia and occurs with an incidence of approximately 1.2 per 100,000 people. CML was almost always fatal until 15 years ago, but the excellent haematologica | 2016; 101(2)

results of BCR-ABL1 TKI treatment are raising the expectation that a considerable proportion of patients will achieve a treatment-free remission. The use of interferon (IFN) α in parallel with or after tyrosine kinase inhibitor (TKI) therapy is associated with the induction of an immune response against the leukemic clone with further improvement of the remission rate. An essential part of the management of CML patients is rigorous use of cytogenetic and molecular follow up with standardized methods to regularly assess the residual disease status.132 Prevalence of patients with CML treated with TKIs is expected to increase by approximately 10% per year so that CML is a challenge for health care systems worldwide. With average treatment costs in Europe of between €40,000 and €70,000 per patient per year, the challenge is how to maximize patient benefit with an affordable allocation of resources.

European research contributions European co-operative CML study groups were established 30-40 years ago and have continued to contribute to the optimization of management. The impact of interferon (IFN) has been investigated in a series of large studies. IFN as an immunomodulatory agent has activity in CML and has resulted in sustained cytogenetic remissions in an important minority of patients. Meta-analyses of conflicting studies revealed new prognostic factors for IFN response. In 1998, the EUROscore was presented to better discriminate patients with a favorable, intermediate, and unfavorable outcome. The place of stem cell transplantation in disease management had been gradually evolving, having been displaced as first-line treatment by 2002, and then moving to a 3rd-line option after the licensing of the 2nd-generation TKI in 2006. From 2001 on, European investigators have participated in the clinical development of five TKIs. National and supranational [the European LeukemiaNet (ELN)] networks of European CML investigators and clinicians have provided fundamental knowledge for clinical practice. National and multinational studies with imatinib, IFN α, Ara-C, nilotinib, and dasatinib have contributed to understanding the biology of TKI response, the impact and potential problems of combination therapies, and base-line and time-dependent prognostic factors. As a result of a study of the ELN involving more than 2000 patients, a new score predicting the chance of a complete cytogenetic response on imatinib therapy has been presented.142 The predictive value of early molecular response, deep molecular response, and the velocity of response has been established in Europe.143 ELN expert recommendations for CML management were published in 2006, 2009, and 2013, and have become a key reference for CML treatment worldwide.132 In basic and translational research, European investigators significantly contributed to the understanding of the mechanisms of TKI resistance and how to prevent and overcome it. Molecular monitoring of CML has been developed, optimized, and standardized in Europe, allowing accurate quantification of residual disease in a dynamic range of six orders of magnitude.144 Such contributions permitted the successful attempt to discontinue treatment after deep molecular response.145 Currently, the mechanisms that allow persistence of BCR-ABL1–positive stem cells are a major focus of research. Other research directions are the origin of CML, the clonal molecular evolu145

A. Engert et al. tion, and the characterization of the BCR-ABL1–negative hematopoiesis. Still, challenges remain, in particular in those patients who develop resistance mechanisms and eventually fail current treatment options.

Proposed research for the Roadmap Tyrosine kinase inhibitors have substantially improved survival of CML patients. There is reasonable expectancy to cure the disease in a significant proportion of patients. The main objective is to integrate the leading European national trial groups in CML to form a co-operative network for advancements in CML-related research and health care. A clinical trials platform was created to promote the performance of clinical trials with new drugs and/or treatment strategies. Standardization of diagnostic and therapeutic procedures allows outcome to be compared across Europe. The formation of an exemplary “European platform to cure CML” is proposed to consolidate and lead international efforts to improve CML therapy with harmonized methods of molecular monitoring, definition of prognostic factors, and assessment of quality of life (QOL), as well as to reveal the biology of CML stem cells in order to induce immune response or to target specific features. We aim to improve: 1) the rate of deep molecular response; and 2) the rate of patients in durable remission after stopping TKIs. New induction therapies, combination with immunotherapies or stem cell active drugs, and new approaches of stem cell transplantation after treatment failure are methods to improve treatment responses. Patients in durable deep molecular remission after withdrawal of TKIs are considered cured of the disease. The complexity of CML blast crisis pathophysiology, the failure of TKIs to eradicate CML at the stem cell level, and the observation of molecularly defined BCR-ABL1negative clones demand further research despite major improvements in the standard of care for CML. A better understanding of the events governing LSC behavior might lead to the biological cure of CML and effective treatment of blast crisis. Translational studies will contribute to early outcome prediction and treatment surveillance. Biostatisticians and patient advocacy groups co-operate with the study groups and with a European clinical trials platform that will support the co-ordination of the studies.

Anticipated impact of the research In-depth molecular and cellular characterization of CML patients will facilitate personalized medicine with regard to diagnosis, prognostication, and therapeutic decisions. Overall, this will have a major impact on lowering the disease burden, reducing the rate of complications, and, prospectively, prolonging survival. The cost of novel technologies and treatments might be balanced by their more specific application as well as a favorable impact on patients’ QoL and the lessening of the burden for caregivers; this will translate to a more general favorable impact for society by reducing use of resources and improving individual work capabilities. Standardization of diagnostics and therapeutic procedures will further strengthen integration. The resultant comparability of outcome will facilitate recognition of interstudy differences and rare subtypes. Recommendations, network meetings, training courses, and exchange of researchers will spread excellence and raise standards of research and patient care across Europe. The CML network and its activities provide the critical mass for added value and 146

European leadership. The impact on the future management of CML patients is expected to be considerable, because a rational advanced treatment design will likely lead to higher remission rates, longer survival, and a higher proportion of patients in whom treatment can be permanently discontinued as an indicator of cure.

3.4. Myeloproliferative neoplasms Alessandro Maria Vannucchi (Università degli Studi di Firenze, Florence, Italy), Martin Griesshammer (Mühlenkreiskliniken, Minden, Germany), Claire Harrison (Guy’s and St Thomas’, London, United Kingdom), Francesco Passamonti (Ospedale di Circolo e Fondazione Macchi, Varese, Italy).

Introduction The chronic Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) are disorders of hematopoietic stem cells (HSCs) and include polycythemia vera (PV), essential thrombocythemia (ET), and primary and post-PV/post-ET myelofibrosis. These are chronic disorders usually affecting individuals in middle to advanced age; the estimated overall incidence in Europe is 1-5 people per 100,000 a year. Life expectancy is close to normal in ET and modestly reduced in PV, whereas in primary myelofibrosis median survival is 5-6 years. There is no reliable estimate of the prevalence of MPNs; however, the prevalence is likely rising due to earlier diagnosis and prolonged survival. MPN patients suffer from major cardiovascular events and, less commonly, hemorrhages. Patients, and particularly those with myelofibrosis, may present with disabling constitutional symptoms, including marked cachexia, and suffer the consequences of abnormal cell proliferation with massive splenomegaly, hepatomegaly, and foci of extramedullary hematopoiesis. MPNs have an intrinsic tendency to transform to acute leukemia.

European research contributions European researchers have a long-standing record of achievements in this field regarding both basic/translational and clinical research. European researchers have discovered the JAK2V617F mutation, JAK2 exon 12 mutations, and CALR mutations that constitute major diagnostic criteria in the up-dated WHO classification.146-148 The characterization of the key pathogenetic role of activated JAK/STAT signaling has been instrumental to directing pharmaceutical research that culminated in the approval of the first JAK1 and JAK2 inhibitor, ruxolitinib. The pivotal COMFORT-II study in myelofibrosis patients has been co-ordinated and performed in Europe;124 the international pivotal RESPONSE study in PV has been co-ordinated and largely carried out in Europe as well.125 European researchers first discovered genetic haplotypes predisposing to MPNs and highlighted the prognostic impact of subclonal mutations in primary myelofibrosis. National and supranational [European LeukemiaNet (ELN)] networks of European investigators and clinicians specifically focusing on MPNs have produced fundamental knowledge for clinical practice, including the drawing up of clinical risk scores, definitions of drug intolerance and resistance, treatment response criteria, and clinical end points for novel drug studies. Major phase III studies that established standards of care for MPN patients were performed in Europe, such as: 1) the ECLAP study, which demonstrated the safety and haematologica | 2016; 101(2)

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efficacy of low-dose aspirin for thrombosis prophylaxis in PV; 2) the PT-1 and the ANAHYDRET study, which compared hydroxyurea versus anagrelide for high-risk ET patients; and 3) the CYTO-PV trial, which established the optimal hematocrit target in PV. European researchers have produced the largest prospective experience of stem cell transplantation in myelofibrosis.

Proposed research for the Roadmap In spite of the above achievements in molecular characterization of MPNs, additional genomic research is needed to identify novel phenotype driver mutation(s) in the approximately 20% of patients with ET and primary myelofibrosis who still lack a molecular marker; reaching this goal will certainly facilitate earlier and more accurate diagnosis. However, a more ambitious goal is the discovery of the initiating mutation for MPNs, because the currently known mutations are certainly required for disease manifestation but are not essential for its development. Disease progression to either secondary myelofibrosis or acute leukemia is a particularly important aspect that is poorly understood and has not been studied in adequate depth, notwithstanding its eventual occurrence in more than 25% of patients with ET and PV whose conditions are regarded as more indolent. A better understanding of the molecular framework of MPNs would be important for improving our ability to subcategorize patients according to their risk of disease progression and dying. This would also permit appropriate therapies to be better selected. For example, stem cell transplantation may be a curative option, but it carries considerable risk; conversely, the recently developed JAK2 inhibitors must be credited with an incredible efficacy for symptomatic disease management, possibly resulting in prolongation of survival, but are unable to cure the disease. Thus, research is also needed to better delineate the cell-intrinsic abnormalities that determine and/or accompany these diseases; such insights might enable development of novel and more efficacious therapeutic strategies. Lastly, better animal models are required as an integral part of proposed research plans in order to facilitate a full understanding of the functional consequences of mutations and to test novel therapies. Overall, these studies might be greatly facilitated by supporting and reinforcing the existing networks of European scientists and clinicians in order to share carefully annotated patient samples and take advantage of existing technological platforms and expertise.

Anticipated impact of the research Detailed molecular and cellular characterization of MPN patients would facilitate personalized medicine in the context of diagnosis, prognostication, and therapeutic decisions. Overall, this would have a major impact on lowering the disease burden, reducing the rate of complications, and, prospectively, prolonging survival. The cost of novel technologies and treatments might be balanced by their more specific application as well as a favorable impact on patientsâ&#x20AC;&#x2122; quality of life and the lessening of the burden for caregivers; this would translate into a favorable outcome for society in general by reducing use of resources and improving individual work capabilities. Scientific achievements frequently result in patent development, which, by strengthening the relationships between European academia and the industry, facilitates public and private investments. haematologica | 2016; 101(2)

The EHA Roadmap for European Hematology Research Section 4. Anemias and related diseases

Section editor: Achille Iolascon. Anemia affects 1.6 billion people worldwide1 and has a huge direct impact on human health and economic wellbeing, as well as being associated with worse prognosis and higher treatment costs because of the numerous comorbid diseases. Global anemia prevalence is approximately 47% in children under the age of five years, 42% in pregnant women, and 30% in non-pregnant fertile women.1 The consequences of morbidity associated with chronic anemia extend to loss of productivity due to impaired work capacity, cognitive impairment, increased susceptibility to infection, and in the elderly, a huge contribution to comorbidities, which places a substantial economic burden on health care systems.149 Nevertheless, anemia frequently goes unrecognized and untreated, causing high direct and indirect costs both to the individual and at a national level. The globalization of migration flows in recent decades has increased the multicultural diversity of our societies. According to the Organisation for Economic Co-operation and Development, the percentage of foreign-born populations within the European Union in 2008 ranged from 4% in Finland to 37% in Luxembourg, with an overall average of 8%.150 Health care services in these countries have to deal with increasingly culturally diverse populations. Due to the movement of immigrants in Europe, there is a new epidemiology of acquired and inherited anemias. It is important to know the exact distribution of the different forms of anemia in each country in order to plan healthcare interventions. To carry this out, it appears mandatory to have European guidelines for diagnosis and establish a common and more sustainable therapeutic approach. Moreover, clinical trials on new drugs and therapeutic procedures could ameliorate the quality of treatment of patients affected with these diseases, that are mainly inherited, enhance their quality of life, and extend their life expectancy. Co-ordinated efforts should be made to develop strategies for prevention of acquired and inherited anemias in individuals at risk in Europe. Detailed epidemiological studies in all countries, especially in Western Europe, are a prerequisite for the implementation of effective prevention programs. For a correct diagnosis, it is mandatory to share a common diagnostic flowchart for each of the main forms of anemias. Thus, new tools urgently need to be developed to reliably diagnose anemias and this fits well with the needs of personalized medicine. We expect that the development of such diagnostic tools will improve timely diagnosis throughout Europe, especially in those countries where it is difficult to gain access to â&#x20AC;&#x153;classicalâ&#x20AC;? diagnostic tests. In the past 15 years, hematology research has made a big leap forward. The emergence of sophisticated genetic 147

A. Engert et al. and molecular tools [e.g. next generation sequencing (NGS) techniques] have allowed spectacular progress to be made in our understanding of the structure and function of the blood cells in health and disease. Our general aim will be to solve several hematologic problems using these new approaches.151,152 Research on rare iron-related genetic diseases that are informative biological models may contribute to a further understanding of iron metabolism. Precise diagnosis of these diseases might help avoid unnecessary and costly diagnostic tests and possibly harmful treatments.

4.1. New technologies for anemia and related disorders Stefano Rivella (Weill Medical College, New York, United States of America), Nicholas Anagnou (University of Athens School of Medicine Athens, Greece), Celeste Bento (Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal), Achille Iolascon (Università Federico II di Napoli, Naples, Italy), Mayka Sanchez (Josep Carreras Leukaemia Research Institute, IJC, Badalana, Barcelona, Spain).

Introduction The production of the oxygen carrier red blood cells (RBCs) is called erythropoiesis, a process that begins with pluripotent hematopoietic stem cells (HSCs) and terminates with the production of RBCs. Anemia, defined as a decreased amount of hemoglobin (Hb), can result from blood loss as well as from decreased synthesis of Hb, decreased RBC production, or increased RBC destruction. Examples include hemolytic anemias (HAs), anemia of inflammation, chronic kidney disease (CKD), some forms of myelodysplastic syndromes (MDS), and bone marrow failure (BMF). In Europe, it is estimated that each year there are 8 new cases of MDS per every 100,000 people, 13% of people present clinical or biochemical evidence of CKD, and anemia of inflammation affects approximately 50% of patients with chronic inflammatory disease. Clinical management includes administration of anti-inflammatory molecules in anemia of inflammation, erythropoiesis-stimulating agents and iron in CKD, and blood transfusions, administration of hematopoietic growth factors, low-intensity chemotherapy, and bone marrow transplantation (BMT) in MDS. Hb disorders, such as b-thalassemia, α-thalassemia, and sickle cell anemia (SCA), are monogenic disorders characterized by reduced or altered synthesis of the b- or α-globin chain, components of the oxygen carrier Hb. Causes of morbidities and mortality associated with hemoglobinopathies are extramedullary hematopoiesis, iron overload, thrombosis, pain, bone defects, and liver and heart failure. Hemoglobinopathies represent the most frequent disorder worldwide, with at least 300,000 children born with these disorders every year. Clinical management has been focusing on supportive therapy, such as blood transfusions, iron chelation, and management of pain. BMT has also been utilized as a definitive curative option, although it is not without risks. Additional anemias are due to dietary limitations, such as folate, vitamin B12, and iron deficiency, while others are due to infections or exposure to toxic agents.

European research contributions Advances have been made in our understanding of molecules and pathways that could be targeted to improve the anemia or the secondary manifestations associated with 148

defective erythropoiesis, such as splenomegaly, bone abnormalities, iron overload, and pain. B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11A (BCL11A) and Krüppel-like factor 1 (KLF1), which modulate the production of fetal hemoglobin, are important modifiers of clinical severity in b-thalassemia and SCA.153 JAK2 and growth differentiation factor 11 (GDF11), modulators of erythropoiesis, have been negatively associated with anemia in bthalassemia, SCA, and MDS.154 Cell adhesion molecules, such as E-, L-, and P-selectin, are being investigated for their potential role in hemolysis, inflammation, pain, and thrombosis in SCA.155 Hepcidin (HAMP), the main regulator of iron absorption, has been associated with increased iron absorption in b-thalassemia and iron-restricted anemia in anemia of inflammation.154 Molecules that control dietary iron absorption in the gut and HAMP production in the liver, such as hypoxia-inducible factor 2α, divalent metal transporter 1, duodenal cytochrome B , ferroportin, transferrin, erythroferrone, and type II transmembrane serine protease, have also been investigated for their contribution to iron overload and anemia in b-thalassemia.154 Despite huge progress, however, there is still no radical treatment for b-thalassemia and SCA, besides allogeneic HSCT, which has limitations such as donor histocompatibility. Gene therapy and gene editing approaches have been introduced as realistic alternatives to treat b-thalassemia and SCA. The technology for viral-mediated gene addition of the b-globin chain gene reached the clinical trial stage with promising results in two ongoing clinical trials in the United States.154 This technology allows insertion of the curative gene by random integration in the genome. However, this might be associated with genotoxicity (i.e. undesirable gene disruption or oncogene activation). Gene editing technologies are focusing on the correction of mutations in the b-globin gene or in modifying genes that modulate fetal hemoglobin expression.156 The latter technology could be intrinsically safer, because it does not require random integration of a curative transgene.

Proposed research for the Roadmap All of these new potential targets and technologies may translate into clinical treatments with a positive impact on the management of these disorders. For instance, new agents that target GDF11 are now in phase I clinical trials for hemoglobinopathies and MDS.154 Drugs that limit iron absorption and improve anemia are showing very promising results in pre-clinical studies.154 As some of these drugs might target additional molecules, however, negative side effects following long-term treatments have not yet been excluded. Gene therapy trials for the cure of b-thalassemia show very promising results, but long-term effects are unknown. In Europe, there has been significant progress in generating appropriate gene therapy vectors, but this needs to be further supported in order to reach the clinic. Gene editing requires additional studies before it can be

Table 1. Key priorities in a European Anemia Research Roadmap. • Epidemiology of anemias in Europe • Common flowcharts for diagnosis • Pathogenesis studies of rare inherited anemias to have new therapeutic targets • Enhancement of clinical trials for new drugs • Use of new technologies for a personalized diagnosis and therapy

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EHA Roadmap for European Hematology Research

translated into mainstream clinical therapy.156 Genetic variability will likely influence the efficacy of these new therapeutics or therapies. For this reason, we recommend the use of next generation (omics) technologies to assess the role of genetic makeup of the patients, modifiers, and environment. This will help clinicians develop precision medicine (i.e. prevention and treatment strategies that take individual variability into account, leading to personalized treatment for each patient).157 Therefore, as many of these approaches are still under characterization or in an early phase of development, we propose investing in these new lines of investigations and technologies in order to validate their potential and transform them into effective and safe ways of treatment (Figure 2).

Anticipated impact of the research The clinical cost and socio-economic impact of these disorders is tremendous. Children affected by hemoglobinopathies require life-long transfusion for survival but, eventually, much morbidity develops, leading to a decreased life span. Anemia of inflammation, CKD, and MDS predominantly impact the adult population, representing a growing and pressing issue in Europe. Therefore, efforts to develop new scientific discoveries and therapeutics can have a major impact on the growing and aging population in Europe at many different levels. This new source of information and potential novel technologies might have a profound impact, not only on these disorders, but also on many other diseases that require management of RBC production, such as in cancer therapies or following BMT. Moreover, many additional incurable disorders could benefit from the development of gene therapy and gene editing technologies developed for b-thalassemia and SCA.

that iron may worsen infections, as shown in children living in malaria-endemic areas.159 Treatment of IDA is apparently simple, because several (oral and intravenous) iron preparations are available.

European research contributions European researchers have contributed identifying hepcidin, the key liver hormone regulating systemic iron homeostasis, defining its role in genetic iron-related disorders and clarifying how high hepcidin induces iron sequestration and iron-deficient erythropoiesis in ACD.160 Our advanced understanding of iron metabolism has allowed the recognition of rare genetic iron-related anemias that are challenging to diagnose. The e-rare JTC 2009 HMA-IRON project has helped raise awareness of these novel entities in Europe. Orphanet and the European Network for Rare and Congenital Anaemias (ENERCA) (www.enerca.org), a European network of expert centers, offer online tools useful for the diagnosis of these rare anemias. From genetic iron-refractory IDA, characterized by high hepcidin levels and iron refractoriness, the lesson is that hepcidin levels should be low/undetectable to allow oral (and pharmacological) iron absorption and that, when needed, intravenous iron should be preferentially employed in the presence of inflammation with high hepcidin. However, analytically and clinically validated hepcidin tests are available in only a few European centers.

4.2. Iron-deficiency anemia Clara Camaschella (San Raffaele Institute, Milan, Italy), Adlette Inati (Lebanese American University, Beirut, Lebanon), Mariane de Montalembert (Necker-Enfants Malades University Hospital, Paris, France), Dorine Swinkels (Radboud Universitair Medisch Centrum, Nijmegen, the Netherlands), Sule Unal (Hacettepe University, Ankara, Turkey).

Introduction Iron-deficiency anemia (IDA) is the most common form of anemia worldwide, affecting almost 1 billion people. There are strong differences in IDA prevalence between developing and high-income countries, and even among European countries.158 Individuals at risk are those with increased iron needs, such as pre-school children, adolescents, and young (especially pregnant) women. Pathological causes of IDA, such as malabsorption and chronic blood loss, are common and may be associated with cancer, especially in the elderly. IDA poses a major burden to society: it has been reported to cause cognitive defects in children, increased morbidity and mortality in pregnancy, reduced physical performance in workers, and is a common comorbidity in the elderly. Genetic causes of IDA are extremely rare, often remaining undiagnosed, though relevant in children. Also challenging is the diagnosis of IDA in the context of anemia of chronic disease (ACD), a condition frequently found in the elderly. Diet fortification is an effective preventive modality of IDA, although there are concerns haematologica | 2016; 101(2)

Figure 2. A roadmap for research into new technologies in anemias and related disorders.

149

A. Engert et al. The results of clinical trials comparing the efficacy of novel intravenous preparations with oral iron drugs are available or being processed. Intravenous preparations appear safe even at high doses (up to 1 g) administered in a single infusion. However, experience is limited, and criteria for using oral or intravenous iron are only partially defined. Long-term effects and cost-effectiveness also need to be evaluated.

Proposed research for the Roadmap Co-ordinated efforts should be made to develop strategies for the following. 1. Prevention and treatment of IDA in individuals at risk in Europe. From the available data, Eastern European countries show a higher IDA prevalence than countries in Western Europe.158 Detailed epidemiological studies in all countries, especially Western countries, are a prerequisite for the implementation of effective prevention programs. 2. A better understanding of the impact of iron-deficiency on physical performance and cognitive and physical development in children, even independently from anemia; understanding is now based only on epidemiological evidence, also necessitating molecular and biological studies. A flowchart should be developed and shared for differential diagnosis of IDA, IDA in ACD, and ACD, and for therapeutic criteria.161 3. Clinical trials focused on the new intravenous iron preparations should be designed to compare their efficacy and side effects. There is a need for evidencebased strategies for accurate and timely diagnosis and optimal treatment of genetic iron-related anemias. 4. Clarify the possible genetic propensity to develop iron deficiency and the relationship of iron with erythropoiesis efficiency. We have just started to understand how erythropoiesis adapts to iron deficiency. It is unclear why and how iron deficiency induces microcytosis and through which mechanisms it increases platelet production in severe cases. Iron deficiency is a positive modifier of ineffective erythropoiesis in preclinical murine models of b-thalassemia,162 but the mechanisms have not been explored and should be verified in patients. 5. The iron-related changes in the composition of gut microbiota, with a prevalence of pathogens over the beneficial lactobacilli, is an emerging problem in developing countries that needs to be further explored considering the relevance of microbiota for human health and the safety of oral iron supplementation in developing countries.

for acquired disorders of erythropoiesis in myeloproliferative disorders [e.g. polycythemia vera (PV)] and low-risk MDSs. The results of clinical trials may indicate when and how intravenous iron should be safely used. Targeted therapies are the results of increased knowledge. The discovery of hepcidin is fostering its therapeutic manipulation as a novel approach to control iron levels. Trials with hepcidin antagonists that aim at making the sequestered iron in inflammation available for erythropoiesis are ongoing in European centers and beyond. Epidemiological surveys, biological studies, and clinical observations suggest an important role for iron in common disorders, including heart failure, obesity, CKD, diabetes, and metabolic syndrome. Studies triggered by IDA have the potential to help establish the optimal levels of iron according to age and sex in these disorders.

4.3. Dyserythropoietic and hyporegenerative anemias Hannah Tamary (Schneider Children's Medical Center of Israel, Petach Tikva, Israel), Wilma Barcellini (Ospedale Maggiore Policlinico, Milan, Italy), Lydie da Costa (Hôpital R. Debré, Paris, France), Irma Dianzani (Università di Torino, Turin, Italy), Roberta Russo (Università Federico II di Napoli, Naples, Italy).

Introduction Congenital dyserythropoietic anemias (CDAs) and Diamond Blackfan anemia (DBA) are rare hereditary anemias caused by abnormal erythropoiesis that is ineffective in the former and hypo/aregenerative in the latter. The prevalence of the CDAs in Europe varies according to European region and is 0.1-3 cases per million live births, while that of DBA is 4-7 cases per million live births.163 The CDAs are characterized by moderate to severe anemia, distinct morphological features in bone marrow late erythroblasts, and development of secondary iron overload. The morphological classification initially proposed by Heimpel and Wendt (CDA I, II, and III) is still valid in clinical practice and is now supported by the identification of different genes mutated in each type.164 CDA I is caused mainly by mutations in CDAN1, but also in C15orf41. CDA II is a result of mutations in SEC23B and CDA III of KIF23. However, there are families that fulfill the general definition of the CDAs but do not conform to any of the three classical types (CDA variants). Moreover, mutations in erythroid-specific TFs genes GATA1 and KLF1 have been described in few such patients. The protein encoded by KIF23 is a mitotic kinesin crucial for cytokinesis; however, the possible role of the proteins encoded by CDAN1, C15orf41, and SEC23B in erythropoiesis is still unknown.

Anticipated impact of the research To correct IDA is simple and usually inexpensive. The underlying cause in some cases is more relevant than anemia itself. Society will benefit from programs aimed at controlling iron and Hb levels in all age groups. Research on rare genetic iron-related diseases that are informative biological models may contribute to further understanding iron metabolism and its regulation. Their precise diagnosis might lead to avoiding unnecessary and costly diagnostic tests and possibly harmful treatments. Elucidating the role of iron in ineffective and effective erythropoiesis would benefit patients with b-thalassemia and other inherited anemias and may have implications 150

Children with DBA classically present with severe macrocytic anemia in the first year of life. The bone marrow discloses a paucity of erythroid precursors. Approximately 30% of DBA patients also have physical anomalies (e.g. craniofacial, thumb, and cardiac malformations). The risk of solid tumors, myelodysplastic syndromes (MDS), or leukemia is elevated in DBA and was calculated to be 20% by the age of 46 years.165 Following the first year of life, the anemia is currently treated with corticosteroids. Infants in the first year of life or patients who do not respond to steroids or require high doses with unacceptable toxicities receive chronic red blood cell (RBC) transfuhaematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

sions.166 These patients often develop substantial iron overload and require careful monitoring to detect this, as well as iron chelation therapy. Stem cell transplantation is an alternative to chronic transfusions. DBA is the first ribosomopathy described resulting in haploinsufficiency of 16 genes encoding ribosomal proteins, RPS19, RPL5, RPS10, RPL11, RPL35A, RPS26, RPS24, RPS17, RPS7, RPL26, RPS27, RPS28, RPS29, RPL15, RPL27, and TRS2. Mutations in ribosomal genes account for 60%-70% of DBA cases. The effect of decreased ribosomal activity in vivo and in a tissuespecific manner is unknown; p53 activation has been observed in bone marrow from DBA patients, after depletion of ribosomal proteins. Recently, it has been shown that rare mutations in the GATA1 gene can cause DBA. Subsequently, an elegant study suggested that impaired translation of GATA1 mRNA (as a consequence of ribosomal protein haploinsufficiency) is an important factor in mediating the erythroid defect observed in DBA.

European research contributions Almost all the work carried out on CDAs, including the description of the clinical picture and identification of the genes involved, was done in Europe. Heimpel made an important contribution to the field, becoming the first to diagnose the disorder and to describe its morphological and clinical features. Iolascon did much of the work on CDA II, defining the molecular genetics and the genotypephenotype correlation of this disease. The genes mutated were mainly described in Europe. Although the major gene mutated in CDAI (CDAN1) was identified in Israel, the second gene causing CDA I was described in the UK. The CDA II gene identification was an Italian-German collaboration; that of the CDA III gene was performed in Sweden.

mutation is identified by targeted NGS, a wholeexome sequence will be performed to identify new causative genes. 3. Perform functional studies to define the role of proteins, encoded by known and any new genes in erythropoiesis. The proteins will be studied in relevant human erythroid progenitors grown in liquid medium CD34+ cells and also using patientsâ&#x20AC;&#x2122; induced pluripotent stem cells (iPSCs). 4. Investigate the role of new drugs that modulate erythropoiesis (anti-JAK2 and TGF-b ligand modifiers) using in vitro models of erythropoiesis. Anti-JAK2 has the potential to decrease ineffective erythropoiesis and thus ameliorate anemia in CDAs. TGF-b ligand modifiers correct anemia by promoting late-stage erythropoiesis and have recently been shown to decrease transfusion requirements and serum ferritin level with favorable safety profile in patients with bthalassemia.167 Because of the similarity in pathogenesis between thalassemia and CDAs, their study in CDAs is warranted. 5. Implement studies for CDAs/DBA. Gene therapy has also been proved to cure diseases that affect hematopoietic cells, such as severe combined immunodeficiency.

Anticipated impact of the research As CDAs/DBA are rare disorders, there is often misdiagnosis or a delay in diagnosis which may result in years of inappropriate therapy, including iron preparations. Patient registries and cutting-edge molecular technology will contribute to accurate diagnosis and optimal therapy for most patients. Defining the role of the proteins encoded by mutated genes in CDAs and DBA in erythropoiesis may eventually be exploited therapeutically. Gene therapy

The main DBA patient registries are kept in France, Germany, Italy, the UK, and the US. Israel also keeps a registry of bone marrow failure (BMF) syndromes that includes Diamond Blackfan anemia (DBA). Collaboration among European groups led to the identification of the first DBA gene, RPS19. Subsequently, many collaborative papers explored several aspects of genotype-phenotype correlation. A group from Lund has developed many cellular and mice models of DBA, and is involved in gene therapy and new drug research for the anemia. Educational activities train young doctors and researchers in their understanding of these disorders, and patients have organized themselves into national organizations throughout Europe.

Proposed research for the Roadmap We suggest programs to improve diagnosis and optimal clinical care for patients with these rare disorders, as well as basic research programs to better understand the role of the proteins encoded by mutated genes in erythropoiesis (Figure 3). 1. Improve CDA and DBA European registries by harmonization and collaboration among the existing national registries to create a unique European database. 2. Improve molecular diagnosis and identification of potential new genes by using next generation sequencing (NGS) methods. The first step in this proposal is to employ targeted NGS with a panel of known genes mutated in CDAs, DBA, and other rare anemias. If no haematologica | 2016; 101(2)

Figure 3. A roadmap for research into dyserythropoietic and hypogenerative anemias.

151

A. Engert et al. approaches and new drugs that modulate erythropoiesis have the potential to ameliorate the anemia and iron overload, and thus improve patients’ quality of life and survival.

intensive research by internationally recognized EU groups, aimed at elucidating the molecular bases of these diseases, as is the case for hereditary stomatocytosis and enzymopathies.168,169

4.4. Hemolytic anemias, including membrane and enzyme defects

A great step forward in the classification of these rare defects, as well as in the identification of expert centers for diagnosis, has been made in the past ten years by ENERCA. ENERCA is an EU project currently in its fourth phase (e-ENERCA). An important outcome is the ENERCA White Book containing the recommendations and the definition of the criteria that Centers of Expertise and local centers have to fulfill as health care providers.170

Alberto Zanella (Ospedale Maggiore Policlinico, Milan, Italy), Patricia Aguilar Martinez (Hôpital Saint-Eloi, Montpellier, France), Immacolata Andolfo (Università Federico II di Napoli, Naples, Italy), Paola Bianchi (Ospedale Maggiore Policlinico, Milan, Italy), Richard van Wijk (Universitair Medisch Centrum Utrecht, Utrecht, the Netherlands).

Introduction Hemolytic anemias (HAs) are a heterogeneous group of hereditary and acquired disorders. Among hereditary forms, the most common are defects of the red cell membrane and enzymopathies that disturb red cell metabolism. Except for glucose-6-phosphate dehydrogenase deficiency, which affects more than 400 million people, the most frequent congenital hemolytic disease in Europe is hereditary spherocytosis, a cytoskeletal defect with a prevalence of 1-5 cases per 10,000 individuals. Other hereditary HAs are rare or extremely rare (Figure 4). Hereditary HAs are characterized by anemias of variable degree, from fully compensated hemolysis to severe and transfusion-dependent anemia. Other manifestations of clinical significance include jaundice, splenomegaly, and iron overload. Hydrops fetalis has been reported in rare cases. In some enzymopathies (those involving genes with ubiquitous expression) and in rare conditions, non-hematologic symptoms, such as neurological/neuromuscular impairment, may also be present. Because the pathophysiology of around 30% of hereditary HAs is poorly understood, these disorders represent a substantial and heterogeneous group of diseases that still lack easy-to-apply tools for diagnosis, clinical management, and patient stratification. Moreover, epidemiological data in Europe are generally still incomplete, and the estimated prevalence of some defects varies widely among countries. This is likely due to the limited and incomplete availability of diagnostic tools.163 Although the general diagnosis of anemia is part of daily clinical practice, the differential diagnosis of HAs is often difficult, requiring specialized analyses available in only a few expert EU centers. In addition, the conclusive diagnosis is often delayed, thus increasing the overall costs of the health care systems and causing considerable degrees of distress for patients and their families. It has been calculated that the cost of diagnosis for one of these anemias is between €850 and €2500; that can triplicate or even quadruplicate if a conclusive diagnosis is not reached. Although the total number of affected individuals is substantial, the rarity and heterogeneity of HAs have resulted in limited interest from the pharmaceutical industry.

European research contributions In past 20 years, inherited HAs have been the object of 152

Due to the concerted efforts of several EU groups, a new edition of diagnostic guidelines for hereditary spherocytosis is about to be published.171 However, no specific guidelines are currently available for the rarer HAs.

Proposed research for the Roadmap Create an EU network and registry for rare HAs: through the work carried out by ENERCA, it has been possible over the past years to map EU expert centers for the referral of cases with RBC cytoskeletal membrane disorders, hereditary stomatocytosis, and RBC enzymopathies. This experience has shown that an officially recognized European Reference Network (ERN-RA), combining different areas of expertise and dedicated specialists, is needed to better define these disorders and share common diagnostic and therapeutic flowcharts. The creation of European registries for these rare disorders will also be of great help to increase knowledge about their prevalence and to collect a greater number of patients; this will improve clinical diagnosis, allow a better definition of complications, and facilitate possible therapeutic trials. Understand the pathophysiological mechanisms and identify new genes to develop new diagnostic tools: despite detailed, exhaustive hematologic and molecular investigations, approximately 10%-15% of HA patients remain undiagnosed. Moreover, the wide heterogeneity of their phenotypical expression has made it difficult in the past to develop easy-to-apply molecular diagnostic tools. The advent of next generation sequencing (NGS) technologies make these new approaches useful tools to investigate the genetic basis of undiagnosed cases and to identify new nosological entities. Moreover, the reduction in cost of these technologies may allow the development of NGSbased diagnostic tools (i.e. by creation of a panel of known genes) and their market development. Develop new therapeutic approaches (e.g. new drugs and gene therapy models): whereas new drugs and therapeutic approaches recently became available for acquired HA, no specific or curative treatments are available for congenital HAs except for hematopoietic stem cell transplantation (HSCT). Because most of these defects are monogenic, gene therapy may represent a therapeutic option. In this respect, a promising approach concerns the use of gammaretroviral vectors that has proved effective in correcting the disease in a pyruvate kinase-deficient mouse model as recently developed by EU groups. A therapeutic and clinically applicable lentiviral vector has recently received the orphan drug designation for the treatment of pyruvate kinase deficiency by the European Commission haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

(EU/3/14/1130). Finally, investigation of new drugs that could increase specific enzymatic activity and/or activate isoenzymes could pave the way for attractive therapeutic approaches to RBC enzyme disorders.

Anticipated impact of the research A correct diagnosis will have a major impact on patientsâ&#x20AC;&#x2122; quality of life (QoL) and survival, especially by the early detection of complications such as iron overload, and will allow for appropriate genetic counseling of patients and their families. A more timely diagnosis will also result in a significant reduction of the overall costs of the health care systems. The increased knowledge of pathophysiology of these disorders and the identification of new nosological entities will be of great help in improving the diagnosis and will offer the basis for the development of new therapeutic approaches for HAs. Finally, the creation of EU registries for rare HAs will improve awareness of these rare disorders and their prevalence.

4.5. Congenital bone marrow failure, aplastic anemias, and paroxysmal nocturnal hemoglobinuria Antonio Risitano (UniversitĂ Federico II di Napoli, Naples, Italy), Carlo Dufour (Istituto Giannina Gaslini, Genoa, Italy), Antonis Kattamis (Athens University, Athens, Greece ), Regis Peffault de Latour (National Institutes of Health, Bethesda, MD, United States of America), Irene Roberts (University of Oxford, Oxford, United Kingdom).

Introduction Bone marrow failure (BMF) syndromes are a heterogeneous group of diseases characterized by a quantitative deficiency in one or more blood cell lineages. Inherited BMFs include different entities, such as Fanconi anemia (FA) (which is due to impaired DNA repair and cytokine hypersensitivity),172,173 dyskeratosis congenita, Diamond Blackfan anemia (DBA), and Shwachman-Diamond syndrome (all associated with impaired ribosomal or telomere function). Inherited BMFs are rare disorders, the most common of which is FA (1-3 per 500,000 newborns).172 Indeed, the majority of BMFs are acquired forms, mostly idiopathic; the most typical form, idiopathic aplastic ane-

Figure 4. Red blood cell membrane disorders.

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A. Engert et al. mia (IAA), has an incidence of 1-5 people per million in Western countries. Another less common acquired BMF is paroxysmal nocturnal hemoglobinuria (PNH); here the underlying bone marrow disorder is associated with the expansion of an abnormal, non-malignant, blood cell population that is deficient in the expression of glycosylphosphatidylinositol-linked proteins due to a somatic PIGA mutation. As well as BMF, which is seen in only a proportion of PNH patients, the clinical phenotype of PNH is characterized by anemia due to complement-mediated intravascular hemolysis secondary to the lack of the glycosylphosphatidylinositol-linked complement regulators CD55 and CD59, along with an increased frequency of major thromboembolic events.

European research contributions The improved understanding of all BMFs has led to better patient management in Europe; the Working Party for Severe Aplastic Anemia (WPSAA) of the EBMT has contributed greatly to improved clinical outcome in this field. Milestones, where European hematologists have taken the lead, include the first observations on the use of antithymocyte globulin (ATG) as treatment for IAA and the first use of hematopoietic stem cell transplantation (HSCT) in patients with FA. There have also been improvements in diagnostic strategies in BMF, mostly in the differential diagnosis of inherited versus acquired forms of BMF. (The former are often cryptic and may appear even in adolescents or adults.) In terms of treatment, the most relevant improvements include: 1) intensive immunosuppressive treatment (mainly for acquired IAA); 2) anticomplement treatment (for hemolytic forms of PNH); 3) HSCT, for inherited BMFs, IAA, and, more rarely, PNH. These improvements are particularly relevant given that they were achieved in the setting of rare disorders where there are innate difficulties in making progress. The database of the WPSAA of the European Group for Blood and Marrow Transplant (EBMT) contains data on more than 11,000 patients with different subtypes of BMFs, thereby providing a unique opportunity for investigating many different critical aspects of these diseases. The WPSAA of the EBMT continues to run a multinational database to collect data from all European BMFs; the aim is to combine this retrospective work with prospective studies to address further improvements in the complex treatment of these disorders.

Proposed research for the Roadmap Patients suffering from BMFs continue to represent a challenge for the medical community because of their poor prognosis when the underlying disease is not controlled. Additional efforts are needed to offer the most appropriate treatment to all European patients and improve current standards of care. The WPSAA of the EBMT is dedicated to this goal through different research lines. 1. Improvement of non-transplant treatment for acquired IAA: current immunosuppressive treatment for acquired IAA is based on the combination of horse ATG and cyclosporine A. The recent withdrawal of the horse ATG preparation from the European market has had detrimental effects on outcome in European IAA patients, as demonstrated in several studies,174,175 leading the WPSAA to highlight the need for this ATG preparation for IAA patients.175,176 In addition, ongoing efforts are investigating the benefit in randomized, 154

controlled trials of the addition of newer agents (e.g. the THPO-mimetic agent eltrombopag) on the “scaffold” of standard immunosuppression (which may be different according to the severity of IAA). 2. Improvements in HSCT for inherited and acquired forms of BMF: HSCT remains a key treatment option for all BMF patients, with the current indication depending on the phase/severity of the disease and on the availability of alternative treatments. Possible improvements in HSCT procedures will exploit different (and possibly combined) strategies: a) identification of improved HSCT protocols (including pre-transplant conditioning and pre-, peri-, and post-transplant immunosuppression) to reduce posttransplant mortality and morbidity (e.g. comparing different immunosuppressive regimens); b) development of novel HSCT protocols in the setting of unrelated donor HSCT, aiming to neutralize the detrimental effect of a non-related donor, such that unrelated HSCT could be used earlier in the treatment algorithm of IAA; c) investigation of HSCT from alternative donors, such as human leukocyte antigen (HLA)–haploidentical donors and cord blood units, aiming to offer a HSCT option to all candidate patients; d) identification of novel HSCT procedures tailored to specific conditions (e.g. for PNH or for FA and other inherited BMFs) 3. Observational studies on PNH: the treatment of PNH has been revolutionized in the past decade by the introduction of the anticomplement agent eculizumab. The WPSAA is currently looking at the actual role (and most appropriate procedures) of HSCT in PNH in the eculizumab era, as well as evaluating possible unmet medical needs that may still remain during this treatment. 4. Observational studies on the natural history of BMFs: these studies aim to improve the diagnosis, classification, and definition of response categories of both acquired and inherited BMFs, with the goal of identifying burning clinical questions to be investigated by specific investigations.

Anticipated impact of the research The management of BMF represents an urgent medical need not only for individual patients but also for society as a whole. Many affected patients will become unproductive and require lifelong, expensive treatments. In the past four decades, BMFs have changed from inevitably fatal diseases into curable ones, with an overall survival rate approaching 70% at ten years. These outcome data can still be improved. It is only through the design and execution of stringent and well-focused studies that the scientific community will learn how to deliver better treatment options to these patients. Furthermore, improvement of the management of BMF will lead to a better use of increasingly restricted resources, which will also have a positive impact on society, also from a financial point of view.

4.6. Thalassemia and congenital hemoglobinopathies Maria Domenica Cappellini (Università degli Studi di Milano, Milan, Italy), Emanuele Angelucci (Ospedale A. Businco, Cagliari, Italy), Androulla Eleftheriou (Thalassaemia International Federation, Strovolos, haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

Cyprus), Antonio Piga (UniversitĂ di Torino, Turin, Italy), Ali Taher (American University of Beirut Medical Center, Beirut, Lebanon), Vip Viprakasit (Mahidol University, Bangkok, Thailand).

Introduction The thalassemia syndromes are a heterogeneous group of inherited hemolytic anemias (HAs) characterized by reduced or absent production of one or more of the globin chains of hemoglobin (Hb). This leads to imbalanced globin chain synthesis that is the hallmark of thalassemia syndromes. In hemoglobinopathies, globin chain synthesis is usually balanced but one chain is abnormal: HbS, HbC, and HbE are the most common and relevant. The thalassemias and hemoglobinopathies are the most common single gene disorders in the world population, with estimated carrier number of more than 270 million and an annual birth rate of more than 300,000. They are most frequent in southern Asian, Middle Eastern, and Mediterranean countries, and North and Central Africa. As a result of migration, however, these conditions are found all over Europe.177

European research contributions The natural history of these diseases has changed significantly in Europe during the past decades due to three main reasons: 1. carrier screening and prenatal diagnosis; 2. advances in diagnosis and conventional treatment; 3. HSCT. Carrier screenings and prenatal diagnosis: a couple identified at risk for a severe form of thalassemia or hemoglobinopathy can be offered prenatal diagnosis to avoid the birth of an affected child. Prenatal diagnosis of thalassemia was introduced in Europe in the late 1970s, initially performed through globin chain synthesis on cord blood, and then in the 1980s by DNA analysis. Since then, the birth rate of children with thalassemia in Cyprus and Italy has dropped almost to zero. However, the wide variability of the phenotype of many mutation combinations demands great experience and counseling. Prenatal diagnosis may be difficult for religious and cultural reasons in recent migrants.178 Improvement of conventional treatment: for thalassemias, the improved understanding of the pathophysiology and the availability of safe and high-quality blood in Europe has allowed an optimal suppression of ineffective erythropoiesis by appropriate transfusion therapy. Iron chelation has had a major impact on morbidity and mortality in Europe. The standard chelation therapy for more than 40 years was deferoxamine, given by continuous subcutaneous infusion 5-7 days per week. The long-term efficacy of deferoxamine has been extensively documented in large cohorts of patients. Unfortunately, long-term compliance with daily subcutaneous infusions is a serious limiting factor. This has led to identifying safe, effective oral iron chelators. At present, two oral iron chelators are available: deferiprone and deferasirox. Deferiprone was registered in Europe and only recently in the United States and Canada. Deferiprone may be more effective than deferoxamine in protecting the heart from the accumulation of haematologica | 2016; 101(2)

iron. Combined deferoxamine/deferiprone therapy is used in high-risk patients, such as those with heart iron or cardiac dysfunction. The more recent oral iron chelator, deferasirox, has been shown to be effective and safe in removing excess iron from different organs, including the heart. While the availability of oral iron chelators has improved patientsâ&#x20AC;&#x2122; compliance, the introduction of noninvasive techniques to quantify tissue iron, especially MRI T2* to measure myocardial iron, has significantly contributed to optimizing and intensifying iron chelation, reducing cardiac mobility and mortality.179 HSCT: allogeneic HSCT in thalassemia syndromes has been increasingly successful during the past three decades, mainly in b-thalassemia major. Predictors of transplant outcome established by the Pesaro group categorized patients into three risk classes. The probability of thalassemia-free survival for patients under 17 years of age who receive the allograft from an HLA-identical relative is above 85% in class 1 or 2 patients and is much lower in young patients in class 3. The progressive adjustment of conditioning therapy in class 3 patients and in adults (over 17 years of age) has also significantly improved outcome in this class. HSCT from unrelated donors has a higher risk of acute and chronic graft-versushost disease (GvHD), particularly in thalassemia. A recent study from Eurocord reported no mortality and better outcome in 33 patients with class 1 and 2 thalassemia who received cord blood HSCTs from HLA-identical siblings, suggesting that related cord blood HSCT is a safe procedure for thalassemia patients. The European experience in bone marrow transplantation (BMT) in thalassemia represents a milestone for thalassemia treatment in the world. HSCT in sickle cell disease (SCD) has also been developed in Europe, and recent recommendations have been proposed.180

Proposed research for the Roadmap Boost transplantation options: HSCT may be considered a definitive treatment for the major Hb disorders; however,

Figure 5. Proposed priorities for European research on hereditary hemolytic anemias.

155

A. Engert et al. most patients with thalassemia lack a compatible sibling donor and thus there is interest is using alternative donors. In this context, other approaches require further development: 1. matched unrelated donor; 2. matched unrelated cord blood; 3. mismatched related or haploidentical donors. Pharmacological intervention: although there is currently no definitive treatment (with the exception of HSCT), the potential of correcting the globin chain imbalance through pharmacological intervention is an approach that holds tremendous promise and could lead to widespread therapeutic options for patients. This includes identification of new and potent fetal hemoglobin inducers or new molecules that may potently modulate the ineffective erythropoiesis, such as agents that block the activity of certain TGF-b–family cytokines, as well as JAK2 kinase inhibitors.181 Gene therapy: gene therapy is the major investment for the future for the cure of thalassemias and SCD. Quality of life (QoL): more research work should be undertaken to investigate the impact of good social and psychiatric support in improving the QoL of this group of patients. Most thalassemia and sickle patients in Europe are immigrants who, in addition to their lifelong disease, are exposed to extra stress when they move to a new country with regulations and a social and cultural life that are different from that of their country of origin.

Anticipated impact of the research During the past three decades, the improvement in diagnosis and management has enabled patients to live normal lives but has increased the economic and social burden. The cure of thalassemias and hemoglobinopathies represents an urgent medical need, not only for individual patients, but also for society as a whole.

4.7. Iron overload disorders Yesim Aydinok (Ege Üniversitesi, Izmir, Turkey), Barbara Butzeck (European Federation of Associations of Patients with Haemochromatosis, Croissy sur Seine, France), Domenico Girelli (Università degli Studi di Verona, Verona, Italy), Martina Muckenthaler (Universitätsklinikum Heidelberg, Heidelberg, Germany), Jecko Thachil (Manchester Royal infirmary, Manchester, United Kingdom), Sophie Vaulont (Institut Cochin, Paris, France).

Introduction Iron overload represents a major health problem worldwide. Excess iron accumulates in vital organs of the human body and increases the risk for liver disease (cirrhosis or cancer), heart failure, diabetes mellitus, metabolic syndrome, osteoarthritis, and hypogonadism, and in some cases it causes premature death. Iron overload can be a consequence of inherited diseases, such as hereditary hemochromatosis, the most frequent genetic disorder in the Caucasian population (carrier frequency of 1:8). Similarly, patients with “iron-loading anemias” (e.g. with α-thalassemia) present with elevated iron levels. Iron accumulates dramatically in patients that require regular blood transfusions. Furthermore, mild to moderate elevation of 156

tissue iron levels exacerbates the pathologies of common acquired diseases, such as chronic liver disease, diabetes, atherosclerosis, and cardiovascular disease. Iron misdistribution in the brain hallmarks the main neurodegenerative disorders (i.e. Alzheimer and Parkinson diseases).

European research contributions Research into mechanisms that cause iron overload was fueled by the discovery of mutations in the HFE gene as the cause of hereditary hemochromatosis. Subsequently, European researchers identified more aggressive subtypes of hereditary hemochromatosis,182 as well as novel disease entities characterized by iron accumulation (e.g. ferroportin disease).183 The discovery by European researchers of the iron-regulatory hormone hepcidin and its target receptor ferroportin improved our understanding of how iron overload develops in hereditary hemochromatosis and provided new insights into mechanisms that underlie iron accumulation in blood diseases caused by insufficient or malfunctioning red blood cells (RBCs) [e.g. myelodysplastic disease syndromes (MDS) and thalassemias].160 These anemic patients frequently require blood transfusions, which exacerbates iron overload. (One unit of RBCs contains 200-250 mg iron.) Iron overload causes oxidative stress, and up till now, use of phlebotomy and chelation therapies has been common to prevent iron toxicity. European research groups established disease models for iron overload disorders to identify mechanisms that control iron balance.184 These important research findings not only gave an insight into the classical iron-related disorders, but also significantly improved our knowledge of how iron accumulates and contributes to the pathologies of acquired diseases, such as chronic liver disease, heart failure, and diabetes mellitus. Importantly, basic research into iron metabolism disorders was successfully translated into novel therapeutic opportunities. Together with European biotech companies, novel therapies were defined that are currently being tested in clinical trials.185 Educational activities train young doctors and researchers in their understanding of iron-related disorders throughout Europe. Patients have organized themselves in the European Federation of Associations of Patients with Haemochromatosis.

Proposed research for the Roadmap The past decades provided us with important insights into cellular and systemic iron metabolism that have improved understanding of the pathophysiology of iron overload disorders. Despite that, fundamental questions remain unanswered. 1. An improved understanding of the etiology and pathogenic mechanisms of iron overload in inherited disorders. We need to identify: a) the signals sent from the erythroid compartment to regulate systemic iron homeostasis and how these signals are controlled and sensed by different organs; b) how iron traffics inside cells; c) how iron causes toxicity; d) how different organs handle iron or heme that is released during hemolysis. 2. An improved understanding of clinical implications of iron overload in inherited disorders. We need to understand how iron influences the early stages of erythropoiesis and how iron overload damages eryhaematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research thropoiesis in conditions such as b-thalassemia, as well as how the kidneys handle iron, which undergoes glomerular filtration and reabsorption with important physiological and potentially therapeutic implications. Similarly, the nervous system poses an enormous challenge. Despite the fact that patients with high plasma and systemic iron levels are protected from iron accumulation in the brain, local alterations of iron metabolism contribute to neurodegeneration. We need to devise strategies to prevent or ameliorate its course. We still do not fully understand the role that iron overload plays in inflamed or infected tissues and how this affects the immune system. The link between mild to moderate iron overload and type 2 diabetes or other highly prevalent insulinresistant conditions (e.g. the metabolic syndrome) is increasingly recognized, but the molecular mechanisms underlying these associations are largely unknown. 3. An unanswered question remains as to how iron metabolism differs in the fetal, neonatal, and infant stages and how this compares with adulthood. Furthermore, there are uncertainties regarding when chelation therapy should be started and what the safe levels of body iron burden are. An improved understanding of basic cell biology principles of iron metabolism will allow further exploration of disease states and will help devise new therapeutic concepts. 4. Novel diagnostic means to identify patients with iron overload at risk of clinical progression and development of comorbidities. We need to identify modifier genes that affect the pathological course of iron overload disorders and that can be applied as diagnostic parameters. Epidemiological and prospective cohort studies are required to substantiate our knowledge of how perturbations of iron homeostasis are linked to disease progression and development of comorbid complications in other acquired disorders, such as cancer and cardiovascular, liver, kidney, or bone disease. 5. Clinical trials to evaluate the potential of targeted therapies in reducing systemic iron levels in widespread diseases, including atherosclerosis, diabetes, chronic liver disease, and neurodegenerative disorders. Integrated pre-clinical and clinical approaches are needed to continue the translation of novel targeted approaches that were designed based on the impressive progress in unraveling the regulation of hepcidin and ferroportin expression, in addition to commonly applied therapies, such as phlebotomy and iron chelation.

Anticipated impact of the research Continued research into iron metabolism will discover novel iron-related genes and regulatory mechanisms that maintain iron homeostasis. This will improve our understanding of the etiology, pathogenic mechanisms, and clinical implications of iron overload in inherited disorders, as well as in those diseases where iron accumulates secondary to primary disease pathology (e.g. iron-loading anemias, dysmetabolic iron overload syndrome, atherosclerosis, and chronic liver disease). This is expected to have a major impact on the treatment of hereditary and acquired iron overload disorders. The identification of “iron signatures” in iron overload diseases will be an important diagnostic means of identifying patients with iron overload at haematologica | 2016; 101(2)

risk of clinical progression and development of comorbidities or to differentiate true iron overload from other disorders characterized by high hyperferritinemia. Clinical trials will evaluate the potential of targeted therapies in reducing systemic iron levels in widespread diseases, including atherosclerosis, diabetes, chronic liver disease, and neurodegenerative disorders.

4.8. Anemia in the elderly Reinhard Stauder (Medizinische Universität Innsbruck, Innsbruck, Austria), Swee Lay Thein (King’s College London, London, United Kingdom), Joan-Lluis Vives Corrons (Universitat de Barcelona, Barcelona, Spain), Giovanna Graziadei (Università degli Studi di Milano, Milan, Italy), Gerwin Huls (Radboud Universitair Medisch Centrum, Nijmegen, the Netherlands).

Introduction Anemia is associated with an increased risk of adverse outcome in older adults, including hospitalization, impaired cognitive capacities, diminished quality of life (QoL), frailty, and higher mortality. Analyses have revealed a prevalence of anemia (WHO definition: Hb <130 g/L for men; <120 g/L for women) of 12% in adults over 65 years of age living in the community, 40% in those admitted to the hospital, and 47% in nursing home residents (Table 2). Based on an overall proportion of 17% in the general population,186,187 approximately 15 million elderly citizens (over 65 years of age) in the European Union are affected by anemia. Hence, anemia is a frequent condition in the elderly population, exceeding 40% in those aged 85 years or over. Anemia has been associated with increased morbidity, mortality, and hospital stays. Despite this clinical importance, anemia in the elderly is often neglected, and evidence-based guidelines for diagnostic workups and individualized therapeutic algorithms are lacking. Causes of anemia in the elderly include nutritional deficiency in approximately one-third (primarily iron deficiency), while one-third have chronic inflammation (ACD) or chronic kidney disease (CKD). While ACD is caused by cytokine- and hepcidin-mediated processes,188 the underlying mechanisms of anemia in approximately 30% of cases remain unexplained.189 Moreover, decreased testosterone levels have been considered as an underlying mechanism.

Table 2. Late-life anemia is frequent in the elderly. Numbers based on a cohort of 19,758 university hospital inpatients and outpatients.203

Age group (years) Men 64-69 70-74 75-79 80-84 85-89 >89 All

18% 21% 25% 34% 40% 47% 23.4%

Anemia prevalence* Women 13% 16% 20% 25% 29% 33% 19.3%

*As defined by WHO (Hb <12 g/dL in women and <13 g/dL in men).

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A. Engert et al. European research contributions The rational therapy of anemia is hampered by the difficulty of dissecting the underlying pathological mechanisms and the lack of evidence-based guidelines. Whereas a $16 million grant supports studies of unexplained anemia in the United States (Partnership for Anemia: Clinical and Translational Trials in the Elderly; www.agingportfolio.org/projects/project/5U01AG034661-02), very few European programs have addressed issues of anemia in the elderly. A network for the recognition, epidemiological surveillance, and medical education of rare anemias was established in Europe (ENERCA). The aim is to offer an improved public health service in rare anemias. Based on clinical studies, several drugs have so far achieved approval by the European Medicines Agency, including different iron formulations and erythropoiesisstimulating agents. Studies on promising drugs, including drugs directed at hepcidin,190 TGF-b superfamily ligand traps, and novel oral iron supplementations, are ongoing.

d) new drugs, such as TGF-b superfamily ligand traps. 5. Exclude congenital disorders of erythropoiesis, including thalassemias and congenital dyserythropoietic anemias (CDAs), because inherited anemias might represent an underlying cause of anemia. 6. Evaluate the effect of these tailored health care interventions on Hb levels, survival, and other health-related outcomes. Patient-reported outcomes, including scores to assess fatigue and quality of life (QoL) and objective functional parameters (e.g. gait speed), will be included and analyzed.

Anticipated impact of the research It is anticipated that a refined definition of the causes and pathological classification of anemias in the elderly will widen our understanding and achieve the following goals. 1.

Proposed research for the Roadmap We propose to study the epidemiology of anemia in the elderly and to raise awareness among health care providers. The most essential objective is to develop and perform evidence-based clinical treatment strategies and health care interventions, which are based on a refined pathological algorithm. The innovative combination of new anti-inflammatory drugs and novel iron formulations will improve health care interventions in end-of-life anemia.

Objectives of the research agenda include the following. 3. 1. Study Hb concentrations as well as the epidemiology and prognostic impact of anemia in distinct cohorts of elderly patients in select geographical regions throughout Europe. Structured evaluation of comorbidities and functional capacities will allow the estimation of the prevalence and relative contribution of different chronic diseases and frailty on Hb concentrations. 2. Raise awareness of the clinical relevance of a structured workup, diagnosis, and treatment. A European competence network on “anemia in the elderly” encompassing relevant stakeholders, such as physicians, researchers, health care providers, regulatory institutions, and patient groups, for the dissemination and utilization of up-todate, evidence-based recommendations will be established. The network will actively facilitate the diffusion of evidence-based guidelines among patient organizations and European anemia study groups. 3. Develop a new pathological classification of anemia, particularly addressing unexplained anemia, in different patient groups. This will be based on parameters such as hepcidin, erythroferrone, cytokine, and hormone levels in combination with established hematologic parameters such as serum ferritin, reticulocyte count, Hb concentration, and soluble transferrin receptors. 4. Infer individualized algorithms based on these refined laboratory analyses that will assist differential diagnosis and rational treatment for the principal therapeutic interventions in anemia: a) supplementation of oral or IV iron; b) erythropoiesis-stimulating agents; c) antihepcidin strategies; and 158

Develop and distribute new health care interventions in anemia: a) develop and validate simple and evidence-based guidelines for diagnosis and treatment of anemia in the elderly, with particular consideration of gender aspects; b) improve therapeutic outcome by clinical studies; c) reduce morbidity and mortality related to anemia in a group of vulnerable patients through tailored treatment. Develop an algorithm for intervention in cases that are likely to succeed. a) exclude inherited anemias; b) identify early patients in whom specific strategies are ineffective or even harmful; c) develop a predictive model to classify patient populations with high likelihood of treatment response. Assess and improve clinical outcome of anemia by generation of new core outcome sets: a) raise awareness of novel therapeutic options for anemia; b) generate clinical evidence even in non-fit elderly patients displaying comorbidities; c) comprehensively address patients’ needs and support treatment algorithms by using parameters that are relevant to the patient and society, including patient-reported outcomes, QoL, and functional capacities; d) generate new and innovative core outcome sets and add them to classical parameters; e) form the basis for cost-effective use of diagnostic and health care resources for diagnosis and treatment. European competitiveness in the field includes the following: a) generate data to form the basis for a rational design of clinical trials to apply for European Medicines Agency registration and marketing of novel therapeutic substances; b) generate a European competence network on “anemia in the elderly.”

4.9. Sickle cell disease Lucia De Franceschi (Università degli Studi di Verona, Verona, Italy), Jacques Elion (Université Paris Diderot, Paris, France), Frederic Galacteros (Hôpitaux Universitaires Henri Mondor, Créteil, France), Cornelis Harteveld (Leids Universitair Medisch Centrum, Leiden, the Netherlands), David Rees (King’s College Hospital, London, United Kingdom). haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

Introduction Sickle cell disease (SCD) is a rare hereditary red cell disorder caused by a point mutation in the b-globin gene that results in the synthesis of pathological hemoglobin S (HbS). The term SCD is used to indicate different genotypes that cause the characteristic clinical syndromes (SS, SC, bS). SCD is a chronic and disabling disorder, that is still associated with a high mortality. In the past two decades, due to immigration fluxes from areas endemic for SCD, SCD has spread throughout the European Union (EU). The prevalence of SCD newborns and SCD carriers in the EU is approximately 1-5 in 10,000 and one in 150, respectively. Epidemiological predictions suggest an increasing global burden of SCD between 2010 and 2050, making SCD an emerging public health problem with limited therapeutic options. In fact, the years lived with disability for hemoglobinopathies and SCD is estimated to be 10,197, which is a dramatic observation, because the years lived with disability for cardiovascular disorders is 21,985. In the past two decades, the survival of children with SCD has significantly improved due to the introduction of hydroxycarbamide therapy and the comprehensive care provided through pediatric age to adulthood. However, the shape of survival curves for SCD has not been affected by these therapeutic approaches and has been shifted to the second and third decade of life, showing a mortality rate that remains high for young adult SCD patients.191-195

European research contributions Historically, SCD was first described by James Herrick in 1910. The identification of the molecular defect and the characterization of the peculiar biochemical properties of pathological HbS that polymerize when deoxygenated have definitely opened a new era of research in the SCD field. Studies on red cell membrane physiology in SCD have allowed the identification of the key membrane ion systems involved in the generation of dense dehydrated red cells, which play an important role in the pathogenesis of acute and chronic clinical manifestation of SCD. Increasing fetal Hb levels was explored as a therapeutic strategy to delay HbS polymerization. This resulted in the introduction of hydroxycarbamide treatment for SCD patients with improvement of patientsâ&#x20AC;&#x2122; survival and ameliorations of clinical outcome, such as pain crisis rate and chronic complications with some impact also on their quality of life (QoL). European research contributions have included: 1) the long-term evaluation of hydroxycarbamide in treatment of pediatric and adult patients with SCD; 2) the development of comprehensive sickle cell centers for multidisciplinary care of SCD patients; and 3) the development of hematopoietic stem cell transplantation (HSCT) programs for pediatric patients with severe forms of SCD. In addition, EU research has contributed to the development of the first transgenic mouse model for SCD, which has improved knowledge of the disease, starting from identification of new mechanisms of disease to testing new therapeutic targets in SCD. Furthermore, the availability of animal models for SCD has allowed the European research community to open new high-risk and high-innovative therapeutic approaches to SCD through the development of different strategies for gene therapy in the disease. This has recently been moved from benchside to bedside in an ongoing trial in patients with various haematologica | 2016; 101(2)

hemoglobinopathies and also SCD. The European scientific community has also contributed to the definition of the biocomplexity of SCD, which involved chronic inflammation, vasculopathy, oxidative stress (i.e. free heme), and cytokine storm, as well as to the clinical definition and characterization of severe chronic organ complications related to SCD, such as pulmonary hypertension and cerebrovascular disease. In conclusion, European research has made an important contribution to state-ofthe-art advances in the field of SCD and to a widening of knowledge of the disease within the international scientific community.

Proposed research for the Roadmap Although significant progress has been made in our knowledge of SCD, treatment strategies remain unsatisfactory for both acute and chronic clinical complications. Thus, future research horizons in the disease should face up to that SCD biocomplexity which makes it a multiorgan disease. The recent development of high-throughput techniques for both molecular and protein analysis, integrated with a rigorous phenotype characterization of the large EU SCD cohort of patients, will allow us to identify new biomarkers of disease severity to generate subgroups of patients to be driven to personalized medicine (secondary outcomes: SCD EU registry, biobanking). These will also allow us to select new targets for future development of new therapeutic options. The presence of both in vitro and in vivo animal models for SCD within the EU research teams will ensure the exploitation of new therapeutic approaches will lead to clinical trials in the near future. These might involve SCD vasculopathy, chronic hemolysis, free heme, or novel fetal hemoglobin inducers. In addition, the novel imaging techniques developed for functional studies will help the scientific community learn more about the disease and the management of SCD with the generation of new diagnostic approaches to organ damage to facilitate early treatment. This will have an impact on the natural history of SCD and improve the survival of adult patients.

Anticipated impact of the research Future European research programs on SCD will widen our knowledge of SCD and will deliver: 1) new therapeutic molecules for clinical management of SCD; 2) new profiling of disease severity for personalized medicine; 3) optimization of SCD patient care; and 4) clinical trials addressing basic aspects of clinical care. These will positively affect: 1) patient health outcome and QoL; 2) national and European health systems by reducing hospitalization lengths and care costs; and 3) national and European welfare spending due to the reduction in disabilities of SCD patients and in the level of sick leave from work. In addition, the novel identified therapeutic molecules would have an impact on the management of SCD in other high-income countries, such as the United States, as well as in low- and middle-income countries, such as 159

A. Engert et al. Africa and India, where SCD is a significant health problem. These will also stimulate public-private partnerships for orphan drug development largely based on innovation, generating new opportunities in the context of international competition.

The EHA Roadmap for European Hematology Research Section 5. Platelet disorders

Section editor: Carlo Balduini. Knowledge about platelet disorders has greatly expanded in recent years, and this topic has become one of the most complex in the field of hematology. Many new diseases have been identified, and a better understanding of old diseases revealed their complexity in terms of etiology, pathogenesis, and clinical features.196-200 Moreover, we are now recognizing that the peculiar genetic background of each patient may modulate the clinical expression of both acquired and inherited platelet disorders,201 while even slight differences in the mutations affecting one gene may be the origin of quite different clinical pictures in subjects with inherited thrombocytopenias (ITs).202 Finally, the therapeutic armamentarium has been enriched with new, targeted drugs interfering specifically with the pathogenic mechanisms of diseases and promising to modify their natural history.199,203-205 Thus, platelet disorders have truly entered the era of personalized medicine, and European researchers have played a key role in achieving this goal. Despite the remarkable advances made, the increase in knowledge has not gone forward at the same rate for the different diseases. For instance, the number of welldefined inherited forms of thrombocytopenia has increased from 3 to more than 20 in the past 15 years,196 while that of inherited disorders of platelet function have changed little, and the majority of affected subjects still remain without a definite diagnosis because the nature of their illness has not yet been identified.206 Also, some forms of acquired platelet disorders (APDs), in spite of their high prevalence, remain poorly defined; this is the case of, for example, platelet dysfunction in chronic liver and kidney diseases, for which we do not have a clear understanding of clinical relevance, standardized diagnostic methods, or validated therapeutic approaches. Druginduced platelet dysfunction is even more important considering the impact it has on general health, especially with the increasing number of subjects receiving antiplatelet drugs for the prevention of thrombosis. In addition, for patients receiving drugs that interfere with platelet function, we have no evidence-based guidelines to help them deal with bleeding or hemostatic challenges. Another undefined issue of great clinical relevance is the differentiation between primary and secondary immune thrombocytopenia (ITP) and other forms of acquired thrombocytopenia, especially those associated with infection. Finally, new curative approaches for ITP based on restoring the immune dysregulation are required. Major outcomes of new therapeutic studies should be rooted in bleeding assessment and quality of life (QoL) more than on platelet count.207 Given the great expertise being applied to platelet studies, stimulating European 160

research on these neglected topics is expected to lead quickly to a better management of these conditions, to the benefit of patients and the health care system. Another major problem in the field of platelet disorders is the gap between what is done in everyday clinical practice and what should be done. Many diseases are rare or exceedingly rare, and awareness of these forms is not widespread in the medical community. As a consequence, affected patients often receive misdiagnoses and inappropriate treatments.208 Moreover, when the right diagnosis is suspected, its confirmation requires tests that are available in only a few specialized laboratories.209 The resulting diagnostic delay due to logistic difficulties can even put the lives of patients at risk because, as in thrombotic microangiopathies, a very early therapeutic intervention maximizes the chances of survival.210,211 Thus, creating a network of centers for the diagnosis of specific platelet disorders is expected to have a strong impact on the quality of care for affected subjects. Moreover, centralizing diagnosis may facilitate the creation of registries and conduct of collaborative clinical trials, which are essential for widening our knowledge and improving treatment of rare diseases (Figure 5).

5.1. Congenital platelet disorders: number and function Alessandro Pecci (Università degli Studi di Pavia, Pavia, Italy), Remi Favier (Hôpital d'Enfants ‘A. Trousseau’, Paris, France), Andrew Mumford (University of Bristol, Bristol, United Kingdom), Hana Raslova (Université Paris Sud, Villejuif, France), Barbara Zieger (Universitätsklinikum Freiburg, Freiburg, Germany).

Introduction Knowledge in the field of inherited thrombocytopenias (ITs) and inherited platelet function disorders (IPFDs) has greatly improved in the past 15 years. More than 20 new genes responsible for ITs and IPFDs have been identified, leading to the definition of novel nosographic entities and better characterization of some disease phenotypes. This has also provided novel information that widens our understanding of human platelet production and function. Despite impressive advances, many gaps still have to be filled. In a considerable proportion of patients, a definite diagnosis still cannot be made because their ITs and IPFDs are still unknown. For example, nearly 50% of patients with ITs are affected by disorders that have not yet been identified. Moreover, the clinical features of some disorders that have had their genetic defects identified remain poorly characterized, as the information currently available comes from studies conducted in select single families or small series, thus hampering a general representation of clinical features of patients and preventing a data-driven clinical management. Differential diagnosis of ITs and IPFDs is currently based on pre-genetic laboratory assays that are usually complex and only available in a few centers; in addition, the diagnostic significance of laboratory findings is often uncertain. Therapy of ITs and IPFDs also needs to be improved. For most patients, no evidencebased protocols are available for treatment of bleeding or management of the bleeding risk associated with hemostatic challenges. Hematopoietic stem cell transplantation (HSCT) is the treatment of choice for a few, very severe forms, and alternative treatments are needed for patients with these forms for whom HSCT is not possible. Finally, haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

validated alternative options to platelet transfusions for the treatment of major bleedings or prophylaxis of hemostatic challenges are needed.

European research contributions Researchers from Europe identified 15 novel genes responsible for ITs and seven genes causing IPFDs. Through the construction of patient databases, European groups have provided a thorough definition of the mutation spectrum, clinical features, and genotype/phenotype correlations of some disorders, including MYH9-related disease, Bernard-Soulier syndrome, Glanzmann thrombasthenia, Wiskott-Aldrich syndrome/X-linked thrombocytopenia, and thrombocytopenia 2. European researchers have provided effective tools to standardize the diagnosis of both ITs and IPFDs; European networks have developed methodologies that combine phenotyping of IPFDs and application of next generation sequencing (NGS) to unravel the genetic complexity of these diseases.196,212,213 By using in vitro models of megakaryopoiesis or animal models, European investigators identified the pathogenesis of several ITs and IPFDs, including congenital amegakaryocytic thrombocytopenia, disorders deriving from FLI1 or RUNX1 haploinsufficiency, and thrombocytopenia 2.214,215 Groups from Europe contributed to the improvement of HSCT in WiskottAldrich syndrome or congenital amegakaryocytic thrombocytopenia, performed the first clinical trials of gene therapy for Wiskott-Aldrich syndrome, and successfully tested a drug for increasing platelet production in one form of IT.

Proposed research for the Roadmap Identify new genes responsible for ITs and IPFDs: the first step toward this goal is the recruitment of large series of patients with ITs and IPFDs of unknown genetic origin through international co-operative efforts to collect biological samples and clinical data of patients. Application of NGS approaches (whole-exome or -genome or RNA sequencing) appears the most powerful tool for identifying candidate genes. The demonstration of pathogenicity of genetic variations requires functional studies that preferably use in vitro models of human platelet biogenesis or, alternatively, animal models (although these do not always reproduce the phenotypes observed in IT/IPFD patients). Establish national and international registries of patients with known genetic defects: registries should be aimed at two main objectives. 1. To define the clinical consequences of the mutations responsible for ITs or IPFDs by the systematic investigation of large series of consecutive patients. This concerns not only the disorders that will be defined by the recognition of new causative genes, but also some ITs and IPFDs with known molecular defects but a yet poorly characterized clinical picture. 2. To define evidence-based protocols for the management of bleeding risk. Optimize in vitro models of human platelet biogenesis (megakaryopoiesis and platelet formation) and platelet function for pathogenetic investigations and pre-clinical studies of novel therapies. We have identified three priorities. haematologica | 2016; 101(2)

1. To optimize in vitro models of platelet biogenesis obtained by peripheral blood of IT and IPFD patients. Patientderived models are those that more closely reproduce human diseases. Because marrow sampling is often not feasible for ethical reasons, improved in vitro models obtained by circulating progenitors or using induced pluripotent stem cells (iPSCs) derived from peripheral blood should be prioritized. The establishment of a patient-derived European bank of iPSCs would provide an unlimited source of cells for pathogenetic studies. 2. To develop models of platelet biogenesis that reproduce as close as possible the bone marrow microenvironments: the endosteal niche that regulates megakaryocyte differentiation/maturation and the vascular niche where platelets are released into flowing blood. 3. To improve models that reproduce the conditions of human circulation to investigate platelet function defects. Validate single-step sequencing approaches for diagnosis of ITs and IPFDs: the single-step sequencing of all known IT/IPFD genes (or whole-genome sequencing) as the first diagnostic approach for patients with ITs or IPFDs may prove more effective and cheaper than the complex and time-consuming sequence of traditional assays currently in use. Identify and/or develop novel therapeutic options: these include the following. 1. Defining the role of THPO mimetics and other drugs that stimulate megakaryopoiesis with different mechanism in the treatment of ITs. 2. Promoting studies of gene therapy for the most severe forms of IT and IPFD.

Anticipated impact of the research Identifying new genes responsible for ITs and IPFDs will increase the number of patients for whom it is possible to make a molecular diagnosis and will lead to the identification of new key players in platelet production and function. Validating single-step sequencing of all the causative genes as the first-line diagnostic approach for IT and IPFD patients will make it easier and more effective to reach a molecular diagnosis. The definition of the clinical phenotypes deriving from the different mutations is the basis for providing patients with a personalized, genotype-driven prognostic assessment, and, therefore, to set the appropriate follow up, choose the best treatments, and offer correct genetic counseling. Optimizing in vitro models of platelet biogenesis and function will provide powerful tools to validate the pathogenicity of the genetic variations identified by NGS, identify novel therapeutic approaches, and test them in pre-clinical studies.

5.2. Acquired non-immune thrombocytopenia and acquired disorders of platelet function Paolo Gresele (Università degli Studi di Perugia, Perugia, Italy), Christel van Geet (KU Leuven, Leuven, Belgium), Michael Makris (Royal Hallamshire Hospital, Sheffield, United Kingdom), A Koneti Rao (Temple University School of Medicine, Philadelphia, United States of America), Rüdiger E. Scharf (Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany). 161

A. Engert et al. Introduction The understanding of the regulation of platelet function and number has increased enormously during the past decade. However, most of the advancements come from the study of inherited platelet disorders or immune thrombocytopenia (ITP). In spite of this, qualitative and quantitative APDs, different from ITP, are very common but relatively little-studied. Several systemic disorders of large epidemiological impact, such as chronic kidney and liver disease, are associated with APD and a bleeding diathesis. Despite the large epidemiological burden, the clinical relevance of APD is still unclear, and no consensus is available on its assessment and treatment. Moreover, many drugs and foods transiently modify platelet function and may provoke, especially when taken before surgery, increased bleeding. Finally, nonimmune thrombocytopenia (NITP) is very frequent in acutely ill patients, especially in association with infection, but not well understood due to its multifactorial pathogenesis, difficult differentiation from ITP, heterogeneity of involved pathogens, comorbidities, and the potential confounder represented by antimicrobial drugs.216 Differentiation from ITP and other forms of acquired thrombocytopenia is a crucial issue because treatment is completely different.

European research contributions European researchers have strongly contributed to characterizing APD in chronic liver and kidney disease.217-219 European investigators clarified, in particular, the role of altered platelet formation versus platelet destruction in thrombocytopenia and of in vivo platelet activation, platelet â&#x20AC;&#x153;exhaustionâ&#x20AC;?, altered nitric oxide signaling, and enhanced nitric oxide formation in platelet dysfunction. Similarly, the role of uremic toxins and enhanced generation of nitric oxide in uremic platelet dysfunction have been shown by European investigators.218 The bleeding risk associated with drug-induced platelet dysfunction in patients undergoing surgery and its management has been the object of position statements from European experts.219 Finally, several studies have documented the strong and independent negative prognostic value of thrombocytopenia in critically ill patients.220 Mechanisms causing pathogen-induced thrombocytopenias, including decreased platelet production by megakaryocyte invasion and/or enhanced platelet consumption by direct interaction with platelet receptors or by antiplatelet antibodies or immune complexes, have been described by European researchers.220 European researchers have made an important contribution towards identifying the role of platelet toll-like receptors (TLRs) in the response to infection.220 TLR expression enables activated platelets to bind and kill bacteria during sepsis and stimulate neutrophils to extrude their DNA-forming neutrophil extracellular traps that capture bacteria and perpetuate platelet activation, promoting thromboinflammatory processes or disseminated intravascular coagulation.220

Proposed research for the Roadmap Clarification of the clinical relevance of APD in chronic liver and kidney disease: uncertainty remains about the clinical relevance of APD in the hemostatic abnormalities of these chronic conditions. A large international, collaborative, prospective study on the prognostic value of a clinical bleeding risk score, previously employed in congenital hemostatic disorders and ITP, may clarify the clinical rele162

vance of the mild mucocutaneous bleeding diathesis typical of liver or kidney failure patients. Moreover, studies employing new and sensitive techniques for the assessment of platelet/vessel wall interactions and correlating the results with clinical bleeding may conclusively unravel the significance of APD in chronic liver and kidney disease. In addition, a rational diagnostic algorithm for the identification of clinically relevant APD needs to be generated. Finally, the best management of impaired platelet function and number needs to be established by prospective, European collaborative studies. Another issue to be developed is the role of platelets in liver regeneration and fibrosis. Drug-induced platelet dysfunction in patients undergoing surgery: clinical relevance and treatment. The relevance of drug-induced platelet dysfunction in patients undergoing surgery and of its management may initially be evaluated by a large European retrospective survey assessing the relationship between pre-surgical drug intake and surgical bleeding. Previous experience from the EHA-SWG shows that surveys, although based retrospectively on clinical records, may provide clinically relevant information when the database obtained is sufficiently large. The survey will analyze different drug treatments and various types of surgery, classified as major or minor and by organs/systems. Another subject of future research is the possibility of guiding surgery and minimizing prophylactic platelet transfusions by pre-operative platelet function testing with pointof-care devices; appropriately designed, collaborative prospective studies will clarify this important issue. Infection-associated thrombocytopenia (IATP): mechanisms, diagnosis, and treatment. Identification of the mechanism(s) leading to IATP (decreased platelet production, enhanced platelet consumption, or a combination of both), its diagnosis, and treatment are the crucial objectives of future research. The questions to be addressed are whether the dynamics of IATPs differ from other NITPs; whether platelet count profiles differ depending on the invading pathogen; if the immature platelet fraction is a reliable index of thrombopoietic activity; what the bleeding risk is depending on the degree of thrombocytopenia (using the ISTH score); if THPO receptor agonists are safe and may minimize prophylactic platelet transfusions in IATP; what the frequency is of antiplatelet antibodies/immune complexes in IATPs; what immune mediators are expressed by platelets during infection (cytokines, CD40, CD154, TLR, and P-selectin); what the expression profile is of platelet-, monocyte-, and endothelial cellâ&#x20AC;&#x201C;derived microparticles and their receptors; and what the platelet-induced inflammatory responses are that can be protective or detrimental to the host. To answer these questions, an IATP registry and prospective multicenter studies complemented by murine sepsis models will have to be established. The differentiation between ITP and NITP, clinical and laboratory diagnosis: the rapid differentiation between ITP and acquired NITP is a crucial task of future research as the clinical management of the two conditions is very different. Better and more rapid tests for the confirmation of ITP are urgently required. In the area of pregnancy, a collaborative network for thrombocytopenia should be established to better document the natural history in mothers and fetuses. haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

Anticipated impact of the research The clarification of the clinical meaning of APD in disorders of large epidemiological impact will improve clinical knowledge in the complex hemostatic impairment of these diseases and provide a guide to their diagnosis and management. Moreover, understanding of the impact of drug-induced platelet dysfunction on surgical bleeding and its possible prediction by laboratory testing may greatly reduce surgical and cardiovascular morbidity and mortality. In addition, the proposed research will provide further insights into the mechanisms leading to thrombocytopenias associated with infections and improve their management. Finally, the differentiation between ITP and NITP will allow more appropriate and rapid treatment.

5.3. Primary and secondary immune thrombocytopenia and fetal neonatal alloimmune thrombocytopenia Thomas Kühne (Universitäts-Kinderspital Beider Basel, Basel, Switzerland), Bertrand Godeau (Hôpitaux Universitaires Henri Mondor, Créteil, France), Axel Matzdorff (Caritasclinic Saarbrücken, Saarbruecken, Germany), Irene Roberts (University of Oxford, Oxford, United Kingdom), John Semple (St. Michael's Hospital, Toronto, Canada).

Introduction Primary immune thrombocytopenia (ITP) is a bleeding disorder of unknown etiology characterized by low platelet count, immune-mediated platelet destruction, and reduced platelet production. As with many other autoimmune disorders, it is thought that environmental and genetic factors may incite the autoimmune response against some platelet and megakaryocyte membrane glycoproteins. Immune dysregulation manifests with a complex pathophysiological mechanism, including peculiar features of antigen-presenting cells and B and T cells. ITP occurs in all age groups, with half of patients older than 60 years of age and 20% older than 75. Data for estimation of frequency of this disorder are primarily from Europe and include an incidence of 1.9 to 6.4 per 100,000 children per year and 3.3 per 100,000 adults per year. In children, a minority of patients has severe bleeding and persistence of symptomatic thrombocytopenia, which is not predictable at an early stage of the disease. In adults, comorbidity and co-medication affect the bleeding phenotype, and there is an increasing number of patients (currently approx. 20%) with secondary forms of ITP, which have still not been well studied. Severe hemorrhages and fatal outcome increase with age and may become a cause of death in approximately 5%-10% of refractory cases. It has been realized that primary ITP is not a clinical-pathological entity, but rather has a heterogeneous background. Secondary ITP occurs in patients with malignancy, systemic autoimmune disease, chronic viral infection, primary immune deficiency, or drugs. Fetal and neonatal alloimmune thrombocytopenia (FNAIT) due to fetomaternal alloimmunization to paternal human platelet antigens (HPAs) is a rare disorder (incidence one in 1000 live births), but causes severe thrombocytopenia and a high bleeding risk in fetuses and neonates. Intracranial hemorrhage occurs in approximately one per 10,000 fetuses and neonates. Screening of all pregnant women for HPA alloimmunization has been suggested, because the risk of complications is very high and effective haematologica | 2016; 101(2)

intervention is available. The rarity of the disorder, and the fact that alloantibodies are not identified in many cases, makes FNAIT difficult to study except through large transnational collaborative groups.

European research contributions For both ITP and FNAIT, European researchers are playing a key role in epidemiology and clinical research. There has been a significant increase in knowledge of the natural history of ITP based on registries. It has been recognized that research activity should be co-ordinated for an effective use of resources. These international collaborative activities have resulted in the development of practice guidelines; standardizing of definitions, terminology, and outcome criteria; therapeutic outcome measures, such as assessing bleeding; and co-ordination of research activities.221 New innovative therapies have been successfully introduced and have questioned current therapeutic strategies and stimulated expert discussions.222,223 Despite the relative rarity of FNAIT, several important advances have been made in the past ten years. Firstly, new diagnostic methods, including fetal genotyping on maternal blood samples and antibody detection using aptamers or recombinant peptides, have been developed and are gradually being introduced into clinical practice. Secondly, improvements in the management of pregnant women with a positive history of FNAIT have resulted from systematic studies of the use of antenatal immunomodulation with IVIG in place of invasive treatment (in utero platelet transfusion).224,225 Finally, large screening studies have shown the potential benefit of testing in combination with planned delivery and prophylactic perinatal platelet transfusion.

Proposed research for the Roadmap The pathophysiological mechanisms of ITP and FNAIT are still not well understood and need to be studied in more detail in order to precisely diagnose the disease at an early stage, identify predictors of severe bleeding, and find novel treatments. In both ITP and FNAIT, the functional effects of putative or known auto- and alloantibodies on megakaryopoiesis and thrombopoiesis should be studied, as having a better understanding of this may help guide new treatment protocols with existing drugs as well as develop novel therapies. Pathophysiology of ITP: new findings of immune responses in autoimmune disorders include the identification of the role of antigen-presenting cells, T and B cells, and their interactions. Regulatory T cells maintaining self-tolerance are involved in modulating ITP pathogenesis. Study of the increased platelet mass due to various therapies, and its role in presenting platelet autoantigens to T cells and potential effects on activating and suppressing regulatory T cells, may elucidate novel pathomechanisms. Improving the knowledge of the pathogenesis of ITP is an essential starting point for identifying effective diagnostic tests for this form of thrombocytopenia, a very important goal that has not yet been reached. Pathophysiology and management of FNAIT: the assessment of platelet function in thrombocytopenic individuals is challenging and may be important for predicting bleeding risk. Thus, the development of functional assays for 163

A. Engert et al. diagnostic and management applications is needed. The application of high-throughput genomics to the identification of causal antibodies and/or antigens should be investigated. Effective therapies for antenatal and postnatal management should be developed and assessed. Some promising avenues include the development of modified HPA-1a recombinant antibodies and monoclonal antibodies to induce neonatal Fc receptor blockade. Studies in animal models and clinical trials are a priority for further development and testing of these strategies. Clinical proposals in children and adults with ITP: a diagnostic algorithm in children and adults with chronic primary ITP should be developed to adequately diagnose patients at acceptable costs. Clinical research activity in all age groups is required to assess treatment end point alternatives or additional to the platelet count, such as bleeding and health-related quality of life (QoL), and to develop innovative treatments. Criteria for defining both the bleeding and the thrombotic risk, which seems to be increased in some cases, are required to develop personalized treatment strategies in all age groups of patients, particularly in elderly patients. New treatments of patients with ITP: the introduction of innovative treatments, such as THPO receptor agonists, may change current therapeutic strategies, reducing the rate of splenectomies and reserving this intervention to the few patients who do not achieve a good QoL with medical treatments. The occasional observation that THPO receptor agonists may induce durable responses in a small subgroup of adult patients that have been weaned from therapy may reflect a tolerance-like activity of these drugs and should be studied in animal models. New curative approaches of ITP based on restoring the immune dysregulation are required. Outcomes of new therapeutic interventions should be rooted in bleeding assessment and QoL more than on platelet count measurements.

Anticipated impact of the research The systematic study of immune response mechanisms in health, autoimmune (ITP), and alloimmune (FNAIT) disorders will provide more insights into a highly complex system and may identify, not only new diagnostic tools with the potential to define patient prognosis (mild and moderate disease or more severe forms with life-threatening bleedings), but also better and more personalized therapeutic approaches.

5.4. Heparin-induced thrombocytopenia and other drug-dependent immune thrombocytopenias Andreas Greinacher (Universitätsmedizin Greifswald, Greifswald, Germany), Tamam Bakchoul (Universitätsmedizin Greifswald, Greifswald, Germany), Tadeja Dovcˇ Drnovšek (Zavod RS za transfuzijsko medicino, Ljubljana, Slovenia), Yves Gruel (Hôpital Trousseau, Tours, France), Volker Kiefel (Universitätsmedizin Rostock, Rostock, Germany).

Introduction Drug-induced immune thrombocytopenias (DITPs) result from drug-dependent antibodies destroying platelets in the presence of drugs. DITPs are a major safe164

ty concern for patients and also for approval of new drugs. DITPs are: 1) life-threatening and require rapid recognition to allow appropriate measures to be taken to avoid harm for the patient; 2) iatrogenic adverse effects, with medico-legal implications; 3) relevant for drug approval, with a major economic impact on the development of new compounds. As drug-induced immune reactions are infrequent, they are typically recognized at a late stage of clinical development, or even only after approval. When they then cause the drug to be withdrawn from the market, several hundreds of millions of euros have often already been spent; and 4) DITPs are models to understand mechanisms causing the human immune system to attack self-proteins. DITPs are based on different mechanisms: 1) the drug may alter the immune system that produces autoantibodies against several tissues; or 2) the drug or its metabolite(s) binds to platelets and thereby induces the formation of antibodies that cause platelet destruction. The most frequent DITP is heparin-induced thrombocytopenia (HIT).226 HIT is currently the underlying cause of more than 95% of all confirmed DITPs. Whereas most DITPs increase the risk for bleeding, HIT is prothrombotic and affects 0.5% of intensive care patients and approximately 1%-3% of cardiac surgery patients. By conservative assumption, the incidence of HIT is 1:10,000 in-hospital patients, making DITP a substantial health issue in Europe. Diagnosis of DITPs is based on clinical criteria followed by laboratory confirmation of drug-dependent antibodies. Clinical criteria for non-HIT DITPs: clinical criteria for non-HIT DITPs are the exposure to the candidate drug started approximately 1-2 weeks before the onset of thrombocytopenia and recovery from thrombocytopenia after discontinuing the candidate drug. Clinical criteria for HIT (e.g. 4Ts score): clinical criteria for HIT include: 1) decrease in platelet count by more than 50% from the highest platelet count; 2) decrease in platelet count between days 5 and 10 after start of heparin; 3) often associated with new thromboembolic complications; and 4) no other obvious cause of thrombocytopenia. These clinical criteria for DITPs are not very specific, and diagnosis requires confirmation of drug-dependent antibodies by laboratory tests. The currently available tests, however, have major limitations. Laboratory tests for non-HIT DITPs: laboratory tests for non-HIT DITPs show low sensitivity (but high specificity), are restricted to specialized laboratories, and are poorly standardized.227 In contrast, the widely available HIT laboratory tests228 are easy to perform, show high sensitivity but a low predictive value, and have unsatisfactory specificity, whereas the much more specific functional assays are technically demanding and not widely available. Open issues include the following. 1. There is a strong need for sensitive but also specific screening tests. 2. Access to appropriate testing is needed throughout Europe. haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

3. A better understanding of the pathogenesis to develop preventive measures is needed. 4. Current treatments of HIT and its clinical sequelae are a major cost burden for hospitals.

European research contributions Several European groups made major contributions to the pathogenesis of DITPs229 and developed test systems and treatment recommendations. Access of physicians and patients to appropriate laboratory testing is well developed in some European countries (e.g. France, Germany, Austria, Switzerland, the Netherlands, and the UK).

the underlying mechanisms will not only improve patient safety but will also strengthen the European biopharmaceutical industry. All DITPs are caused by antibodies reacting with endogenous (self) cells. Identifying the underlying mechanisms at the molecular level will help to understand mechanisms of autoimmunity.

5.5. Thrombotic thrombocytopenic purpura and other thrombotic microangiopathies Marie Scully (University College London Hospitals, London, United Kingdom), Paul Coppo (HĂ´pital SaintAntoine, Paris, France), Johanna Kremer Hovinga (UniversitĂ¤t Bern, Bern, Switzerland).

Proposed research for the Roadmap

Introduction

A better understanding of the pathogenesis of DITPs: understanding the pathogenesis of DITPs is important not only for drug development but also for gaining new insights into pathological states of the immune system.

Thrombotic thrombocytopenic purpura (TTP) is a form of thrombotic microangiopathy (TMA) characterized by microangiopathic hemolytic anemia (HA), thrombocytopenia, and organ failure of variable severity generated by microvascular aggregation of platelets causing ischemia in the brain, heart, kidneys, and other organs. TTP is distinct from other TMAs such as the hemolytic uremic syndrome (HUS), in which renal involvement due to fibrin-rich thrombi is the prominent feature, or the HELLP syndrome in pregnant women. TMAs are very rare: the incidence of TTP is between 6 and 10 per million of the population, and for atypical HUS (aHUS), approximately 0.5 per million of the population.

1. With regard to drug development, it is of major relevance that several polyanionic drugs, including DNA/RNA-based230 and polysaccharide-based drugs, can induce a HIT-like syndrome. In vitro assays able to predict potential immunogenicity of polyanionic compounds based on their interaction pattern with PF4 are needed for the development of polyanion-based drugs, such as RNA aptamers and antisense drugs, and the rapidly developing field of carbohydrate engineering, which allows screening of new drugs for their immunogenicity during pre-clinical development. 2. The immune response to PF4/polyanion complexes is so frequent that this immune reaction can be studied systematically in humans. This may be instrumental to better understanding which factors lead the immune system to develop antibodies against endogenous proteins other than PF4. 3. Recent data make it highly likely that PF4 is a protein involved in pathogen host defense. It acts as a danger signal for the immune system. Further understanding of these mechanisms could optimize antibacterial and antiviral treatments and even identify new strategies for anti-tumor treatment. 4. For the other drug-dependent thrombocytopenias, first evidence suggests that the immune reaction is caused by binding of the drug to the hypervariable region of a pre-formed IgG antibody, which thereby gains high affinity to an endogenous protein. A better understanding of this mechanism may become very important for understanding other autoimmune disorders. Improvement in diagnostic methods of DITPs: widely applicable assays for drug-dependent antibodies are needed. Especially for HIT, an easy-to-apply assay with a high positive predictive value is one of the main needs in most laboratories. Establishing networks in Europe providing rapid access to diagnostic assays for DITPs with locally available screening tests and a rapid turnaround time, followed by confirmatory tests with high specificity, will be a solution for a currently unmet need.

Anticipated impact of the research Adverse immune reactions are the biggest threat for the development of new biotherapeutic drugs. Understanding haematologica | 2016; 101(2)

Thrombotic thrombocytopenic purpura is usually idiopathic, although it can also occur in association with HIV infection, connective tissue disease, pregnancy, or cancer. TTP is most frequently an acquired disorder, but rarely it derives from inherited defects in ADAMTS13, which may manifest itself not only in childhood but also in adult life, sometimes in association with pregnancy. Early recognition and treatment of TMAs is essential, because they are most often fatal when left untreated. Moreover, an early differentiation among the different TMAs is crucial given the availability of targeted therapies for each form.

European research contributions In the past 15 years, advances have delineated the molecular mechanisms of most of the TMA syndromes, including TTP, aHUS, and the HELLP syndrome, providing evidence that they are caused by distinct molecular defects.231 In particular, complement dysfunction and immune-mediated ADAMTS13 deficiency are responsible for aHUS and TTP, respectively. The novel concepts and disease mechanisms identified in the laboratory were rapidly and successfully transferred into the clinic for the benefit of patients, and recent studies reporting on the use of monoclonal antibodies in the management of TTP and HUS provided convincing examples of translational medicine. Indeed, the B-cell depleting monoclonal antibody rituximab successfully treated refractory or relapsing acquired TTP.232 The results of two international studies involving multiple European teams clearly indicated that the complement blocker eculizumab represents a breakthrough in the management of aHUS by preventing the evolution to end-stage renal disease and allowing dialyzed patients to have a successful kidney transplant.233 Therefore, the rapid distinction between TTP and aHUS at the time of diagnosis is now mandatory. 165

A. Engert et al. From the research point of view, TTP represents a relevant model to better understand the interrelations between microbes, other environmental influences, the immune system, and the endothelium within a still uncharted specific genetic background. In this regard, three European groups reported independently that HLAs DRB1*11 and DQB1*03 were both susceptibility alleles for acquired TTP and confirmed the protective role of DRB1*04.234 Future large-scale studies should lead to the identification of additional genetic risk factors associated with acquired idiopathic TTP and in other forms of TMA. The ability to increase our knowledge and experience in the field of TMAs was challenged in the past by the low incidence of these diseases and their clinical heterogeneity. Several national groups have recently set up large registries that include hundreds of patients with various forms of TTP, however, and these reports have shed light on the epidemiology, clinical presentation, prognosis, and long-term outcome of the disease.235 This provides evidence that collaborations at the national and international level remain key to the continued advancement of the knowledge and treatment of rare diseases. Collaborative works have progressively led to the proposal of consensual treatment modalities and the definitions of treatment responses based on large series of patients. Though arbitrary and based only on clinical experience, these definitions are progressively and advantageously shared by different groups and may foster a common language that can allow fruitful meta-analyses in the future.

Proposed research for the Roadmap 1. Raise awareness of TMAs through multidisciplinary educational programs for general medicine physicians, emergency department physicians, and all other specialists possibly involved in the management of TTP. Create a network of laboratories to facilitate patients' access to specific tests (ADAMTS13 measurement and genetics and complement genetics) to prove the nature of their diseases and personalize treatment. 2. Identify tools for quickly distinguishing TTP from HUS and other TMAs in order to use early targeted therapies for each form of TMA. 3. Develop an international registry for TTP to identify: a) the genetic risk factors that, interacting with environmental factors, are responsible for the onset of TTP; b) relevant early prognostic factors to adapt treatment to the severity of the disease; c) parameters that identify patients at risk of relapse (e.g. decreasing ADAMTS13 levels in remission). 4. Organize international clinical trials to: a) identify the efficacy of early introduction of targeted therapies (e.g. B-cell depleting therapies for TTP and complement blockers for HUS); b) test the effect of innovative, promising compounds (e.g. recombinant ADAMTS13 and blockers of the von Willebrand factor-glycoprotein Ib/IX pathway); c) verify the efficacy of B-cell depleting therapies in the prevention of TTP relapses.

Anticipated impact of the research The improvement of knowledge of TTP and other TMAs at a multidisciplinary level will increase their diag166

nosis in emergency situations, and undoubtedly improve patientsâ&#x20AC;&#x2122; prognosis. The development of clinical trials at the international level (mandatory to achieve significant patient numbers given the rareness of TTP) will allow rapid evaluation of new strategies in the early management of TTP, and for the prevention of relapses. The understanding of genetic factors involved in the occurrence of autoimmune TTP, as well as the interaction between genetic and environmental factors will increase our knowledge about the initiation of the disease and, again, better prevention of its occurrence. The proposed initiatives are needed to improve the knowledge of clinicians about this disease and related TMAs that require a diagnosis and an adapted treatment in emergency situations. Moreover, through collaboration with industry, Europeâ&#x20AC;&#x2122;s role in the improvement of knowledge of TTP and its management will become more important, at a time when the field of rare diseases is becoming a major goal.

The EHA Roadmap for European Hematology Research Section 6. Blood coagulation and hemostatic disorsers

Section editor: Sabine Eichinger. Thrombotic and bleeding disorders are a global disease burden with considerable morbidity and a high mortality. Estimates for the European Union (EU) arrived at a death toll of 500,000 venous thrombosis (VT)-related deaths per year.236 About one in 300 people is affected by an inherited bleeding disorder. Uncontrolled bleeding is a major cause of death not only among these patients but also among those with an acquired bleeding disorder, including liver disease and severe trauma. Europe has a long-standing tradition in basic, translational, and clinical science in practically all areas of blood coagulation and hemostatic disorders.237 The identification of coagulation factors, their interplay within the clotting system, and the role of platelets in hemostasis are all seminal discoveries made in Europe.238-242 Owing to the dedication and scientific curiosity of physicians in many European countries, the clinical aspects and pathophysiological features of genetic and acquired coagulation disorders have been described for the first time. Based on this knowledge, coagulation assays were developed and the foundation for standardized nomenclature in thrombosis and hemostasis was laid. Drugs that saved or improved the lives of millions of people worldwide, such as antithrombotics or procoagulants, have been developed by researchers based in Europe. Notably, these research activities are not clustered in certain parts of the continent but are ongoing across the whole of Europe, from the far north to the south, from east to west. In the past decades, Europe has experienced exciting developments and dramatic breakthroughs that offer a myriad of possibilities to continue along the road of enlightenment in order to unravel still hidden secrets of thrombosis and hemostasis, thereby further improving the management of the diseases. Moreover, we saw changes and will see even more so in the upcoming years throughout the continent due to an expanding EU, an aging population, increasingly self-determined patients, intra- and intercontihaematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

nental migration, and a globalization that facilitates collaboration with research institutions also outside Europe. These developments pose substantial challenges to researchers, physicians, and health authorities alike, but at the same time pave the way for exploring novel tools and approaches in research and treatment options. Increasing demands of regulatory authorities and decreasing financial resources also weigh heavy on the shoulders of the academic research community in particular.243 The burden can be alleviated at least to some extent by defining needs and major goals, focusing on main research questions, streamlining resources, combining efforts, and intensifying collaborations. Scientists and physicians working in the field of blood coagulation and hemostatic disorders are privileged by the fact that bleeding and thrombotic disorders are of utmost relevance in almost all medical disciplines, which per se fosters collaboration and provides a fruitful and inspiring atmosphere for research. The following chapters give an overview of the developments and multi-disciplinary aspects of hemostasis and thrombosis. Although hematologists concern themselves with clinical, consultative, and research aspects of arterial thrombosis, including acute coronary syndromes and cerebral infarction, we will focus here on venous thromboembolic diseases and bleeding disorders. We describe major needs and cutting-edge questions in basic and clinical research, and provide a view not only of the future but also beyond.

6.1. Genomics in hemostasis Pieter Reitsma (Leids Universitair Medisch Centrum, Leiden, the Netherlands), Anne Goodeve (University of Sheffield, Sheffield, United Kingdom), Christine Mannhalter (Medizinische Universität Wien, Vienna, Austria), Pierre-Emmanuel Morange (Aix-Marseille Université, Marseille, France), Johannes Oldenburg (Universitätsklinikum Bonn, Bonn, Germany), José Manuel Soria (Hospital de la Santa Creu i Sant Pau, Barcelona, Spain).

Introduction Inherited disorders of the hemostatic system can be divided into those that increase the risk of bleeding and those that increase the risk of thrombosis. Common inherited bleeding disorders include the hemophilias and von Willebrand disease, but a multitude of rare inherited bleeding disorders involving blood coagulation factors or platelets also amount to a significant disease burden. Today, we have good evidence that the severity of the bleeding phenotype can be modified by multiple common genetic variations, such as the ABO blood group and the activation status of platelets, as well as other confounders. Severe inherited disorders that lead to thrombosis are quite rare and mostly limited to homozygosity or compound heterozygosity for loss of function mutations in genes encoding natural anticoagulants. Lifetime risk of thrombosis is influenced by inherited risk factors, such as factor V Leiden; PT20210A; genetic variations in platelet glycoproteins, such as P-selectin; and also the ABO blood group.

European research contributions The field of human genetics in Europe has a proud hishaematologica | 2016; 101(2)

tory of leadership in the study of inherited hemostatic disorders. Technological breakthroughs were always quickly incorporated into European laboratories and clinics. For example, in the early days of recombinant DNA technology, restriction fragment length polymorphism–based diagnosis was immediately implemented for genetic diagnosis and counseling in hemophilia A and B.244 Common risk factors for VT were among the first risk factors for complex diseases to be characterized in detail and applied for risk stratification, and most of the research is this area was led by European research groups.245

Proposed research for the Roadmap Bleeding: in hemophilia A, genetic imprinting studies and sequencing of the entire F8 gene have shown that, in rare cases, mutations in intronic regions of the F8 gene can cause a severe hemophilia phenotype. Furthermore, there are preliminary data to suggest that the severity of hemophilia is influenced by the number of circulating activated platelets present in the circulation. An answer to this important question can be obtained only if many hemophilia centers collaborate more actively. In addition, new knowledge has been obtained regarding the epigenetic modification and imprinting of the F8 gene in females. However, it is currently unclear whether certain mutations (e.g. the intron 22 inversion) are associated with skewed imprinting. To clarify this, large numbers of patients with and without the mutation will have to be tested again, underlining the need for collaboration. The diagnosis and study of rare bleeding disorders, including those involving blood platelets, will greatly benefit from the introduction of next generation sequencing (NGS) technology.246 To take full advantage of this, a stable and sustainable database of genetic variation in hemostatic genes is required. There is also a need for standardization of clinical reporting of genetic test results for hemostatic disorders to have long-term stability of reference sequences to avoid confusion about “versioning” of DNA sequences, and the locus reference genomic sequences aim to achieve this. VT: high-throughput genotyping technologies in the framework of genome-wide association studies have led to the identification of at least eight new loci associated with the risk of first VT.247 However, the currently known genetic factors explain only approximately 5% of VT heritability.248 An important question is how to discover the missing heritability. The following research strategies can be used to achieve this. 1. Increase sample sizes by pooling large collections of comparable data. Importantly, the number of variants discovered is strongly correlated with sample size. Thus, increasing the sample size will increase the number of discovered variants. 2. Risk factors for first and recurrent VT may not be identical. Identifying genetic risk factors specific for recurrence is important as it could influence treatment decision, in particular duration of anticoagulant treatment after a first VT event. To date, no genome-wide association study has been published on recurrent VT and there is a need for a collaborative effort to reach a sufficient sample size to approach this question. 3. Missing heritability for VT can be attributed to rare 167

A. Engert et al. variants (minor allele frequency less than 0.5%). Such variants are not sufficiently captured by current genome-wide association genotyping arrays. The best method for the detection of rare single nucleotide polymorphisms is sequencing (ideally whole-genome or alternatively exome) using NGS technology. These approaches should be scaled up both in families with a strong history of VT and in unselected individuals. 4. Missing heritability is not necessarily explained by simple Mendelian genetics. Epigenetic processes that influence gene expression may be highly important. Investigating these processes might provide new insights into the molecular mechanisms underlying VT, as recently suggested for other human diseases. For this purpose, specific high-throughput technologies are becoming available to quantify non-coding RNA expression and methylation profiles from cells, tissues, and blood. The second goal for VT is to translate the genetic discoveries into useful clinical applications that lower the burden of thrombotic disease. This process should primarily focus on the prevention of secondary thrombosis after a first event. Clinical trials need to be designed that stratify patients with a first event based on genetic and non-genetic risk factors. Patients with a high risk should be treated differently from patients with a low or intermediate risk, and the effect of such triaging on the number of thrombotic and bleeding events should be recorded. This precision medicine approach should lead to guidelines for thrombosis prevention that are tailored to the individual characteristics of the patient. Research groups in Europe have a strong track record and the infrastructure to carry out such trials.

Anticipated impact of the research With the advent of genome-wide association studies and NGS technologies, the introduction of genomic analysis in the clinical care of patients with inherited bleeding and thrombotic disorders is rapidly progressing, and there is a need to achieve the safe and appropriate introduction of this technology in clinical care delivery. In parallel, standardized and harmonized tools to collect clinical data should be developed, adopting an ontological approach, in order to attribute the right clinical significance to the specific genetic abnormality. This should lead to a lower disease burden and improved public health. The discovery of the missing heritability in VT may point to genes that operate outside the canonical coagulation system. This may yield drug candidates that decrease thrombotic risk without increasing bleeding risk, which would revolutionize treatment and prevention.

6.2. Novel mechanisms for coagulation activation Wolfram Ruf (Johannes Gutenberg-Universität Mainz, Mainz, Germany)

Introduction Activation of the plasmatic coagulation cascade generates the key enzyme thrombin that is pivotal for converting the soluble plasma protein fibrinogen to fibrin and the activation of blood platelets. Together, these form 168

hemostatic plugs to seal sites of vascular injury. Thrombin is essential not only for hemostasis (the prevention of bleeding) but also for thrombosis (the formation of blood clots in vessels). Since the first description of the waterfall model of co-factor-amplified, consecutive proteolytic activation of coagulant protease zymogens 50 years ago, a wealth of biochemical data has delineated the details of blood clotting in vitro. Coagulation initiates through the cellular enzyme-co-factor complex of coagulation factor VIIa and tissue factor or factor XIIa that is activated in the context of contact with exogenous or endogenous polyanions, including DNA, RNA, and polyphosphates (Figure 6). The main routes of coagulation initiation have been refined by description of amplification loops connecting these pathways (e.g. the direct activation of the anti-hemophilic factor IX by tissue factor–factor VIIa or the feedback activation of factor XI by thrombin). Thrombin generation is tightly controlled by plasma serine protease inhibitors that prevent intravascular clotting, and thus thrombosis. Important information on the physiological regulation of coagulation initiation has been further uncovered in patients with thrombophilia and validated by in vivo studies in model organisms. Key roles are played by the vascular antithrombotic mechanisms provided by the anticoagulant thrombomodulin-protein C-protein S pathway and plasma- or platelet-derived tissue factor pathway inhibitor. Although the principles of initiation and regulation of coagulation are well laid out, much remains to be learned about the fine-tuning of these responses to vascular injury and the implications for normal hemostasis and pathological thrombosis.

European research contributions European scientists were major contributors to recent conceptual advances in our understanding of molecular connections between innate host defense mechanisms and coagulation, and the relevance of these interactions to thromboembolic disease. The factor XII–dependent contact pathway has experienced a flurry of research activities after the realization that factor XII and factor IX deficiencies, while causing minimal hemostatic impairments, confer resistance to vascular thrombosis in a variety of animal models.249 Complementary research has identified pathophysiologically relevant activators of this pathway, including DNA and RNA released in the context of cell damage and polyphosphates derived from platelets and microbial pathogens.250 Initial clinical proof-of-concept studies emphasize the feasibility of therapeutic intervention in the contact pathway for antithrombotic benefit without impairment of hemostasis during surgery. In recent years, complex interactions of the coagulation cascade have been uncovered, not only with platelets but also with multiple intra- and extravascular cell types. These studies led to the realization that coagulation enzymes and their cognate receptors mediate crucial cell signaling events in angiogenesis, inflammation, and immunity.251 Conversely, effector mechanisms of innate immunity couple coagulation with inflammation. Activation of immune cells by injury signals are known to trigger a variety of acute and chronic inflammatory diseases, but the same cellular signaling mechanisms are increasingly recognized as directly responsible for the generation of procoagulant tissue factor–bearing thromboinhaematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

flammatory microparticles. In addition, the complement system serving host defense is connected through multiple reciprocal effects with the coagulation cascade. Complement activation not only enhances coagulation by perturbing membrane integrity, exposure of negatively charged phospholipids, and direct activation of coagulation factors, but also through thiol-disulfide exchangeâ&#x20AC;&#x201C; dependent and protein disulfide isomeraseâ&#x20AC;&#x201C;dependent activation of tissue factor on monocytic cells.252 These redox and innate immune reactions are emerging as important regulators of both coagulation and platelet activation, and should remain a priority for future studies. A key function of the coagulation system is to generate fibrin that serves as a transitional barrier at sites of injury. Fibrin formation also serves to wall off and protect the organism from invading pathogens. Novel surveillance functions of leukocytes have been defined that promote similar protective functions within the vascular bed. In these processes, termed immuno-thrombosis,253 neutrophil-derived proteases degrade tissue factor pathway inhibitor leading to localized tissue factorâ&#x20AC;&#x201C;dependent coagulation and fibrin formation. Alternatively, neutrophils can respond under certain conditions with coordinated chromatin remodeling and expulsion of nuclear DNA that entrap intravascular micro-organisms. The formed neutrophil extracellular traps also serve as a matrix for platelet deposition and both extrinsic and intrinsic coagulation activation. These primarily host-protective mechanisms are now recognized as crucial contributors to the development of thrombosis, in particular in the venous system. Important research into the multicellular interactions in low- and high-flow vascular beds remains to be pursued to understand the unique causes of coagulation initiation in the venous and arterial system.

Proposed research for the Roadmap Coagulation research has evolved novel technologies and in vivo and translational approaches to move from simple test-tube research to a detailed understanding of coagulation initiation in specific vascular and extravascular locations. This productive path should be continued with a special focus on understanding the common and discriminating regulatory mechanisms of coagulation activation in thrombosis versus hemostasis. The specific research areas with a high likelihood of return in resolving these fundamentally important questions are as follows. 1. Triggers, modulators, and regulators of intrinsic and extrinsic coagulation activation in the venous and arterial circulation. 2. Contributions of the coagulation system to the complex multicellular interactions of the blood and the vascular endothelium under physiological and pathological conditions, including cancer. 3. Thromboinflammatory circuits contributing to vascular dysfunction, thrombosis, or hematologic and immunological disorders. 4. Environmental, metabolic, age, and gender effects on the reciprocal interactions of the hematopoietic and the coagulation systems.

Anticipated impact of the research These areas of research are highly significant for the rapidly evolving landscape of antithrombotic therapy. The availability of diverse target selective anticoagulants has already changed the practice of hematology, and future advances in anticoagulant therapy are on the horizon. The proposed research areas will have an impact on these developments by defining new interactions and pathogenic roles of the coagulation system in hemato-oncological

Figure 6. Synergistic activation of coagulation by contact and tissue factor pathways.

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A. Engert et al. diseases and vascular medicine. New insights into coagulation-supported pathomechanisms can be used to tailor and individualize antithrombotic therapy with new anticoagulants and may indicate additional areas for therapeutic interventions with antithrombotic drugs in more complex thromboinflammatory diseases.

tively inhibit distinct coagulation factors (factor Xa or thrombin), thereby protecting patients with VTE from recurrence to the same extent as the vitamin K antagonists but at a lower risk of (major) bleeding and without the need of regular coagulation monitoring.

Proposed research for the Roadmap

6.3. Venous thromboembolism

European research contributions

Venous thromboembolism is a multicausal disease, and its development can be explained by the interaction of various genetic and environmental risk factors. It is the main task of future thrombosis research to find strategies for identifying individuals/patients at risk for a first/recurrent thrombotic event on the basis of their risk factor profile, to delineate methods of prevention and prove that such a personalized medicine approach is beneficial. To accomplish this ambitious task, large clinical studies are required that enable differentiation between individuals at a high or low risk for thrombosis. In these studies, clinical and biochemical characteristics have to be complemented with genetic determinates of VTE. It is important to recognize that the development of VTE is to a great extent genetically determined. It is very doubtful that the presence or absence of a few, so far unknown, strong risk factors determines the thrombosis risk. It is much more likely that, according to the common variantâ&#x20AC;&#x201C;common disease hypothesis, VTE development is driven by common variants in many genes, which occur with a high frequency in the general population, with each variant at each gene exerting a small additive or multiplicative effect on the disease phenotype.257

European scientists were responsible for many important breakthroughs in both basic and clinical thrombosis research. This subsection addresses only a few of them.

Specifically, future research on VTE should do the following.

Paul Kyrle (Medizinische UniversitĂ¤t Wien, Vienna, Austria).

Introduction Venous thromboembolism (VTE), a syndrome consisting of deep vein thrombosis and/or pulmonary embolism (PE), occurs in one to 2 per thousand people per year. VTE is one of the leading causes of death in Europe. It is estimated that in France, Germany, Italy, Spain, Sweden, and the UK together, VTE occurs in more than 750,000 people per year and that VTE-related death affects more than 370,000 individuals annually in these countries.236 Millions of VTE events or deaths per annum are expected in the European Union. Most importantly, VTE is one of the best examples of a preventable disease as antithrombotic treatment is highly effective in both primary and secondary prophylaxis. It is therefore of major importance to improve the diagnostic and therapeutic strategies in order to reduce the number of people affected by this frequent and potentially fatal disease.

The recognition of important pathomechanisms leading to VT or PE has an essentially European origin. The German pathologist Rudolf Virchow the etiology of VTE was already clarified back in 1865, postulating that thrombi occurring within the veins, particular in those of the extremities, become dislodged and migrate to the pulmonary vasculature. A few years later, the French internist Armand Trousseau described for the first time the association between cancer and vessel wall inflammation due to blood clots, which are recurrent and appear in different locations over time. In modern thrombosis research, European scientists have greatly capitalized on advances in genetics to identify important determinants of VT, such as the factor V Leiden mutation and the G20210A mutation in the prothrombin gene,254-256 which represent the two most frequent congenital risk factors of VT known today. A tremendous amount of pioneering work in the field of antithrombotic treatment has been carried out in Europe. Sir John Vane discovered the mechanism by which acetylsalicylic acid (aspirin) inhibits platelet function, thereby paving the way for large clinical trials that the effectiveness of aspirin in reducing the incidence of both arterial and venous thrombosis on firm ground. As for the treatment of acute PE, the first (and for ethical reasons last) placebo-controlled trial was carried out in England to show that patients greatly benefit from anticoagulant treatment with heparin in terms of both morbidity and mortality. Only recently, European researchers were the masterminds behind the development of the novel anticoagulant drugs rivaroxaban and dabigatran, which selec170

1. Explore the incidence of VTE in well-defined populations of patients or so far unaffected individuals by the use of prospective observational studies with large numbers of individuals included. 2. Identify single genes, networks of genes, and signaling pathways responsible for VTE development using genome-wide linkage association studies. 3. Investigate the relationship between VTE occurrence and hemostatic system activity by measuring (molecular) markers of platelet and coagulation activation. 4. Enable the construction of prediction tools that are capable of differentiating between high- and low-risk individuals on the basis of clinical, genetic, and molecular evidence. This should be achieved in all groups of patients with VTE including those with VTE provoked by surgery, pregnancy or trauma and also in patients with cancer. 5. Improve our knowledge on the association between hematologic malignant and non-malignant diseases and the risk of VTE. Upon completion of these studies, findings have to be validated in separate studies, and eventually management studies will be required to prove that such a personalized risk stratification strategy is helpful.

Anticipated impact of the research The large number of people suffering from VTE is a serious challenge to European health care systems now and will continue to be so in the years to come. The research proposed in this document will provide indications for haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

prevention in patients as well as in those up till now healthy individuals. It is a step-by-step plan that firstly consists of the definition of populations that might benefit, and subsequently of the estimation of risk based on clinical, molecular, and genetic features, eventually allowing a personalized prevention and treatment strategy. Only a concerted action that brings together basic scientists and clinically orientated researchers will eventually be successful at achieving such an ambitious goal.

6.4. Venous thrombosis in aging populations John-Bjarne Hansen (Universitetet i Tromsø, Tromsø, Norway), Sigrid Brækkan (Universitetet i Tromsø, Tromsø, Norway), Ellen Brodin (Universitetssykehuset Nord-Norge, Tromsø, Norway), Frits Rosendaal (Leids Universitair Medisch Centrum, Leiden, the Netherlands).

Introduction Advancing age is associated with a shift of the hemostatic balance in a prothrombotic direction. The incidence of VT [e.g. deep vein thrombosis and pulmonary embolism (PE)] increases sharply with age. The overall incidence of first symptomatic VT is one to 2 per 1000 person-years in the general population, increasing from one per 10,000 person-years in the age group 25 to 30 years to 5-8 per 1000 person-years in those above 75 years of age258 (Figure 7). Therefore, the risk of VT is 50- to 80-fold higher in the older population, leading to a high attributable risk for age-related factors. In the Scandinavian Thrombosis and Cancer study, a merged cohort including individual data from three large population-based cohorts in Scandinavia (the Tromsø Study, the HUNT Study, and the Danish Diet, Cancer and Health study), approximately 45% out of a total of 2444 subjects with a first VT were over 70 years of age. Based on these results, age accounted for 78% of the VT events occurring in this age group (attributable risk), whereas previous estimations of the population attributable risk were approximately 90%, indicating that 90% of total incidence of VTE could be explained by age-related factors.259

European research contributions Despite a striking increase in the incidence of VT with age, few studies have addressed etiology and prevention strategies in the elderly. Some studies have reported agerelated changes of the hemostatic system, such as increased plasma concentrations of procoagulant coagulation factors (fibrinogen, factor V, factor VII, factor VIII, factor IX, and von Willebrand factor) and inhibitors of the fibrinolytic system (PAI-1 and TAFI),260 but none have investigated the interactive effect between age and these hemostatic factors on VT risk. A major proportion of the VT events (45%-60%) occur during or shortly after hospitalizations. However, the thrombotic risk associated with hospitalization has not been studied in the elderly. Because the incidence of hospitalization doubles with age, the population attributable risk is expected to be highest in the elderly (10% in the young and approximately 40% in the elderly).259 Short-term bed rest in subjects over 65 years of age was associated with a 6-fold higher VT risk than in subjects without bed rest,261 suggesting that a strategy to either avoid bed rest or offer prophylaxis will substantially affect the VT incidence in elderly. The overall risk of VT in cancer patients is 5- to 7-fold higher than in individuals without cancer, and cancer-related VTs account for 20%-25% of all VTs in the general population. Due to the high incidence of cancer in the elderly, it has been estimated that the population attributable risk for VT in cancer patients is 15% in the young and 35% in the elderly. Recently, Blix et al. calculated age-specific population attributable risks of VT due to cancer based on incidences of cancer and VT in the Tromsø Study and found a smaller actual difference than postulated between the young (<50 years, population attributable risk 14%) and the elderly (>70 years, population attributable risk 18%).262 These findings show that malignancy does not explain a substantial proportion of the VT events in the elderly, and, most importantly, that studies focusing on risk factors in the elderly are urgently needed, because extrapolations do not suffice. A limited number of studies have investigated the impact of age-specific risk factor on VT risk, and prelimi-

Figure 7. Venous thrombosis (VT) incidence increases with age. Incidence rates per 100,000 person-years with 95% confidence intervals. Data are derived from the Tromsø Study (1994–2012), in which 26,853 individuals were followed for a median of 17.7 years, and a total of 710 first VTs occurred.

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A. Engert et al. nary data have shown that thickening of the venous valves occurs with age, which may contribute to the high incidence of thrombosis in the elderly.

Targeted prevention strategies are needed to reduce the incidence of VT in the elderly in order to reduce personal suffering and the health burden in Europe, and promote healthy aging.

Proposed research for the Roadmap The key question is: why does the incidence of VT increase with age? We believe that the answer is multifaceted and involves at least three fundamental aspects: 1) aging may be associated with an increased prevalence of conventional risk factors (e.g. immobility, malignancy, comorbidity, hemostatic factors, and genetic factors); 2) VT risk may be conferred by age-specific risk factors (e.g. reduced muscle strength, endothelial dysfunction, venous insufficiency, and frailty); 3) synergistic effects with age may occur for established risk factors, meaning that the impact of a risk factor on VT may differ between young, middle-aged, and elderly individuals. The following issues need to be addressed. 1. Investigate the prevalence of established risk factors (e.g. immobility, malignancy, comorbidity, surgery, hemostatic factors, and genetic factors) in the elderly and their effect on the thrombotic risk in different age groups. 2. Identify novel risk factors of VT in the elderly by applying state-of-the-art technologies in imaging, biochemistry, genomics, genome-wide methylation patterns, and proteomics. 3. Investigate joint effects between age and thrombotic risk factors on VT risk. 4. Develop risk prediction models for VT in high-risk situations (e.g. malignancy, hospitalizations for acute medical conditions, and surgery) specifically for the elderly. 5. Identify age-specific risk factors (e.g. muscle strength, endothelial dysfunction, venous insufficiency, and hemodynamic changes) and elucidate underlying mechanisms for VT risk. These issues should be addressed in observational studies with validated information on exposures, effect modifiers, and end points, and with no upper age limit. Risk prediction models should be developed and validated in prospective cohort studies.

Anticipated impact of the research The total suffering and economic burden caused by VT is tremendous, particularly among the elderly. VT is associated with short- and long-term complications, such as fatal or non-fatal recurrence (affecting 30% of the patients within 10 years), post-thrombotic syndrome (affecting 25%-50% of VT patients), and pulmonary hypertension, all of which severely impair mobility and quality of life (QoL). Extrapolated data from six EU countries estimated that a total of 680,000 deep vein thrombosis events, 430,000 PE events, 610,000 post-thrombotic events, and 540,000 VT-related deaths occur in the EU each year. As the European population is becoming older, the incidence of VT is expected to increase. Therefore, efforts are urgently needed to identify inherited and acquired risk factors and age-specific factors, and reveal their interactions with advanced age, on risk of VT. 172

6.5. Bleeding disorders Flora Peyvandi (Università degli Studi di Milano, Milan, Italy), Isabella Garagiola (Università degli Studi di Milano, Milan, Italy), Michael Makris (University of Sheffield, Sheffield, United Kingdom), Brian O’Mahony (European Haemophilia Consortium, Brussels, Belgium), Johannes Oldenburg (Universitätsklinikum Bonn, Bonn, Germany).

Introduction Inherited abnormalities of coagulation factors or platelets lead to lifelong bleeding. Hemophilia A and B due to deficiency or dysfunction of factor VIII and factor IX account for the majority of the severe disorders. Von Willebrand disease is mostly a milder but much more prevalent disorder of primary hemostasis. The remaining defects, such as factor II, V, VII, X, XI, and XIII, are very rare, with a prevalence of around one in 1 million. There are 106,000 people with an inherited bleeding disorder in Europe according to the World Federation of Hemophilia Survey of November 2014. Mild deficiencies may be asymptomatic until suitable challenges, such as surgery or trauma, and may go undiagnosed until adult life. Due to their low prevalence, current knowledge of the genetic, laboratory, and clinical characteristics of these disorders remains limited, making their diagnosis and management difficult. Their treatment involves replacement of the missing factor with plasma-derived or recombinant concentrates. Their management has recently improved with the development of new extended half-life drugs. Gene therapy is also emerging as a possible future treatment with recent reports of success in hemophilia B. The development of antibodies against the replaced factor (inhibitors) is a major issue, affecting up to 30% of patients with severe hemophilia. The rare coagulation disorders present significant difficulties in management. Affected individuals suffer increased morbidity and mortality as a result of recurrent, often spontaneous, bleeding, and there are wide variations in the availability and quality of specialized health care delivery in Europe.263-267

European research contributions In the past decade, European researchers have made major contributions to the field, with some large collaborative projects funded by the European Commission. The PedNet registry of European pediatricians has contributed to studies on severe hemophilia in terms of genotype, phenotype, role of prophylaxis, and risk of inhibitor development by endogenous and exogenous factors. Large collaborative efforts of UK and French researchers have also investigated inhibitor development. The Malmö International Brother Study identified genetic determinants of inhibitor development. The European Haemophilia Network project established the hemophilia center standards required for delivery of care, and the European Haemophilia Safety Surveillance project prospectively monitors the safety of hemophilia treatments across Europe. haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

The European Network of Rare Bleeding Disorders identified the critical levels in clotting factor deficiency leading to clinical problems and has established prospective studies to determine the natural history of the disorders. The multicenter European study Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease documented that von Willebrand factor mutations in type 1 disease are common and established the use of bleeding scores in the diagnosis of the disease.

Proposed research for the Roadmap We propose the following topics for future research in the area. 1. The use of new technologies, such as next generation DNA sequencing, to clarify the genetic determinants involved in the complex multifactorial process of inhibitor development in hemophilia. 2. The harmonization of standards for collecting and sharing data on the development of inhibitors in patients previously untreated and previously treated in Europe for both standard and novel engineered products (e.g. the longer-acting factor concentrates). 3. The establishment of an optimal data collection system through a European network to obtain more accurate data on safety and efficacy of novel bioengineered coagulation factors in the post-marketing investigation period. 4. The monitoring of the safety and efficacy of gene therapy clinical trials and ensuring collaboration in the consistent long-term collection and sharing of these data at a European level. 5. The collection of data on the prevalence and management of the most frequent age-related hemophilic comorbidities, in particular cardiovascular disease. 6. The prospective collection of data on the natural history of the very rare bleeding disorders, which can only be carried out by multinational, worldwide collaboration. 7. The investigation of the ascertainment, diagnosis, clinical implications, and natural history of mild bleeding disorders. 8. The improvement of bleeding risk prediction in subgroups such as fragile and elderly patients. 9. The collaboration between diagnostic manufacturers and clinical services to improve the quality, sensitivity, and economic value of laboratory assays.

Anticipated impact of the research Developing appropriate European networks based on real collaboration and appropriate data sharing and agreed methodologies will significantly enhance the power of the data collected, minimize bias, and prevent wastage of resources due to the current models of individual working. The aim is to improve the health and quality of lives of European citizens with these rare inherited bleeding disorders through improved awareness, diagnosis, and knowledge of the clinical manifestations and sequelae of these disorders.

6.6. Women, hemostasis, and thrombosis Rezan Kadir (The Royal Free Hospital, London, United Kingdom), Hannah Cohen (University College London, London, United Kingdom), Jacqueline Conard (HĂ´pital HĂ´tel-Dieu, Paris, France), Chris Gardiner (University of haematologica | 2016; 101(2)

Oxford, Oxford, United Kingdom), Debra Pollard (The Royal Free Hospital, London, United Kingdom).

Introduction Inherited bleeding disorders are lifelong conditions that remain largely undiagnosed in women and result in significant morbidity and impaired quality of life (QoL). A World Federation of Hemophilia global survey in 2013 showed that the majority of patients with von Willebrand disease, the most common bleeding disorder reported by 107 countries, are women in their reproductive years.268 Affected women suffer significant reproductive health problems, with heavy bleeding during menstruation, ovulation, and postpartum. There is an increased risk of pregnancy loss in women with severe deficiency of certain coagulation factors. Affected newborns are at an increased risk of intracranial hemorrhage during birth. The incidence of intracranial hemorrhage is unknown, however, without consensus on the optimal mode of delivery. Venous thromboembolism (VTE) is rare in women of childbearing age, but pulmonary embolism (PE) is a leading cause of maternal mortality. The optimal diagnostic approach for PE during pregnancy and postpartum has not been established, and lung imaging carries risks, including those of radiation exposure for both the mother and baby. Low molecular weight heparin is the standard treatment of VTE in pregnancy and postpartum, although the evidence for dosing regimens is limited. Thrombophilias, both acquired and heritable, increase the risk of VTE in relation to pregnancy and hormonal intake; increase the risk of recurrent pregnancy loss and placenta-mediated complications, such as pre-eclampsia, placental abruption, and fetal growth restriction, and may be associated with recurrent implantation failure in assisted conception.

European research contributions Research has highlighted increased menstrual and gynecological morbidity in women with bleeding disorders. Heavy menstrual bleeding is now recognized as a predictor for bleeding disorders in women. A systematic review of literature showed a 13% prevalence of von Willebrand disease in women with menorrhagia, with a higher prevalence of 18% in the European population.269 Analysis of cell-free fetal DNA in maternal plasma has been established as a non-invasive prenatal diagnosis method for assessment of fetal sex in pregnant carriers of hemophilia and can be used to detect fetal genotypes for hemophilia mutations in male fetuses.270 The role of multidisciplinary management of pregnancy has also been established for safe delivery, with regional block for pain relief and anesthesia during labor and reduced risk of postpartum hemorrhage. Postpartum hemorrhage remains a leading cause of maternal mortality and morbidity in Europe. Hypofibrinogenemia has been shown to be a good marker for progression to severe postpartum hemorrhage.271 However, how to assess the fibrinogen level and when to administer fibrinogen remains controversial. The risks of thrombosis and pregnancy morbidity associated with heritable thrombophilias have been established. VTE risk assessment scores have been proposed to guide VTE thromboprophylaxis and for exclusion of PE during pregnancy and postpartum. Low-dose aspirin plus 173

A. Engert et al. heparin have been demonstrated to lead to a significant increase in live births in women with antiphospholipid syndrome-related recurrent miscarriage. The TIPPS trial did not show benefit with antenatal low molecular weight heparin in women with heritable thrombophilia; however, the power calculations were based on an aggregate of adverse outcomes rather than individual obstetric complications.272 The role of heparin in women undergoing assisted conception is unclear.

Proposed research for the Roadmap Bleeding disorders 1. Studies in women with heavy menstrual bleeding on assessing underlying bleeding disorders, the predictive value for the menstrual pictorial blood assessment chart and bleeding assessment tool, and to validate the benefits of incorporating these tools into routine clinical practice. 2. Studies to assess gynecological problems and treatment options in women with bleeding disorders; development and validation of a disease-specific QoL tool, to develop optimal care services. 3. Assessment of the role of local hemostatic mechanisms within the endometrium as an underlying cause for gynecological pathologies, such as heavy menstrual bleeding and endometriosis, and also in implantation and early placental development, in particular in women with recurrent early pregnancy loss and recurrent implantation failure following in vitro fertilization. 4. Refinement of non-invasive prenatal diagnostic techniques for the prenatal diagnosis of severe and common mutations, such as intron 22 inversion; qualitative research to explore feelings that influences decision making about reproductive choices. 5. Pooling of European prospective data to establish the risk of intracranial hemorrhage with various modes of delivery, and the impact of neonatal intracranial hemorrhage on neurocognitive development. 6. For women with rare bleeding disorders, multicenter studies and/or a web-based international registry to provide more evidence for management. 7. Assessment of real-time hemostatic monitoring by point-of-care testing in postpartum hemorrhage, to guide a tailored approach to management of coagulopathy and administration of blood components/ products.

Thrombotic disorders 1. Prospective studies to establish the optimal strategies for management of VTE (stratification of risk factors and clinical prediction rules for suspected VTE, the potential use of higher D-dimer cut-off values, and appropriate low molecular weight heparin dosing) in pregnancy and postpartum. 2. Prospective studies to investigate the pathogenesis, diagnostic validity, management implications, and long-term outcome in women with thrombotic and obstetric complications, associated with antiphospholipid antibodies. 3. Research to define the optimal management of pregnancy in women with heritable thrombophilia and other thrombotic states, including hemoglobinopathies, MPNs, and thrombotic microangiopathies. 4. Assessment of combining cardiovascular measure174

ments with circulating biomarkers for early prediction of pre-eclampsia; development of diagnostic algorithms; definition of mechanisms for improved outcome with aspirin or other agents; definition of the role of complement. 5. Prospective studies on the effects of heparin in assisted conception.

Anticipated impact of the research Research will enable improved diagnosis and an evidence-based approach for minimizing obstetric and gynecological morbidity. This will improve quality of life (QoL) and, in turn, educational achievement and productivity at work. Definition of the diagnosis of mild bleeding disorders will enable assessment as to whether this improves reproductive health outcome and/or has a health economic benefit. Development of non-invasive prenatal diagnosis for hemophilia and information on the risk of intracranial hemorrhage during birth improve obstetric management, avoiding unnecessary intervention and medicalization of delivery. Development of strategies for the prevention and diagnosis of VTE and obstetric morbidity will reduce maternal and fetal/neonatal morbidity and mortality. A further understanding of the pathogenesis of pregnancy morbidity associated with thrombotic states and predictive biomarkers will inform optimal management. Appropriate management of obstetric antiphospholipid syndrome, as well as other obstetric morbidity associated with thrombotic states, will reduce long-term disability in the offspring, along with its health care and economic implications. Improvements in the understanding of mechanisms responsible for pre-eclampsia will achieve the long-term goal of curing this condition.

The EHA Roadmap for European Hematology Research Section 7. Transfusion medicine

Section editor: Anneke Brand. Transfusion therapy started a century ago with the aim of rescuing soldiers with large blood loss. Since then, transfusions reduced maternal mortality and enabled major surgery, leukemia treatment, and transplantation. Despite a lack of studies on when and how much a patient needs, transfusions became increasingly used. Recent studies lowering transfusion thresholds, conducted in surgical and intensive care patients, mostly favor a restrictive policy.273 However, for most indications, the balance between benefit and harm of transfusion is still unknown.274,275 Although in rich countries transfusions are safe, alertness for emerging infections remains, and for under-resourced countries and immunocompromised patients transfusion-transmitted infections still pose a considerable risk. Besides transfusion-transmitted infections, harm caused by transfusion results from transfusioninduced alloimmunization and transfusion-related immunomodulation, causing (transient) immune suppression.276 Transfusion represents a huge (economic) market, albeit with a wide range of use in Europe. Reported by 32 of 47 haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

(70%) member states, on average 37 units of red blood cells (RBCs) are transfused per 1000 inhabitants (range 8– 126 units), identifying three countries with a possibly insufficient blood supply (<20 units/1000). One-third of platelet transfusions are supplied by apheresis, and 8.5 L plasma/1000 inhabitants (range 0-49 L) is fractionated into medicinal products. In about half of the countries, all RBC and platelet transfusions are leukocyte-reduced. All countries have legally binding national regulations and 90% have a hemovigilance registry.277 Future blood supply needs are expected to reflect an increase in patients needing blood components because of age-related morbidity coinciding with less accrual of young donors and donors from ethnic minorities. Although for approximately half of the patients transfusion is a single lifetime event, patients with hereditary anemias from Europe’s former colonies and residents in Mediterranean and eastern Europe need lifelong RBC support, leading to iron overload. Overarching research proposals include the following (Figure 8). 1. Evidence-based indications of virtually all (individual, pooled/cellular, or plasma-derived) blood components demand new research methodology for clinical trials, as well as (bio)markers for indication and monitoring of effects.278,279 Improved disease-specific IVIG products require human research on the mechanisms of immunomodulation and optimal dose and timing of administration. Hemovigilance would include surveillance of thrombotic complications of blood products. 2. Technology is available to match donors and recipients for almost any antigen avoiding alloimmunization, but scarcity of matched donors makes this impossible for all transfusions.280,281 An immunovigilance registry of allo-(RBC, HLA, and HPA) immunized patients will assist selection of eligible patients (patient groups) for pre-emptive matching.282 Such a registry can also stimulate collaborative studies that aim to reverse antibody production283 and explore new treatment for severe complications of incompatible transfusions, transplants, or unborn children.284 3. For (emerging) transfusion-transmitted infections, the best future solution must be sought. Additional tests for immune-compromised patients and pathogenreduction methods that optimally preserve intended (also red) cell functions should be compared. In the long term, ex vivo culture of transfusion-transmitted infection-safe (and antigens defined) blood cells lie ahead.285 These three approaches need triage for feasibility, costs, and patient selection. 4. Good donor management, not solely considering them as resource material, must safeguard donors of blood, cells, tissues, and organs.286 Achievements of transfusion medicine (non-remunerated donors, good manufacturing practice, traceability, and hemovigilance) can support quality management of new cellular products for immunotherapy and repair treatment.287

7.1. Conventional blood products: indications and usage Simon Stanworth (John Radcliffe Hospital, Oxford, United Kingdom), Shubha Allard (Barts Health NHS Trust & NHS Blood and Transplant, London, United Kingdom), Milos Bohonek (Central Military Hospital, Prague, Czech haematologica | 2016; 101(2)

Republic), Enrico Lopriore (Leiden University Medical Centre, Leiden, the Netherlands), Miguel Lozano (Universitat de Barcelona, Barcelona, Spain), Cynthia SoOsman (Sanquin Research, Amsterdam, the Netherlands).

Introduction The most commonly transfused blood component remains red blood cells (RBCs), followed by platelets and plasma. Granulocyte transfusions are still considered an experimental product. Bleeding with shock and anemia is an undisputed indication for RBC transfusion. However, RBCs are commonly used to correct anemia in patients without bleeding to improve the oxygen-carrying capacity, for instance in critically unwell patients and bone marrow failure (BMF) disorders. Although the evidence base for RBC transfusion practice is incomplete, randomized studies in surgery and intensive care settings consistently support a more restrictive use of RBCs, with no evidence of benefit (and arguably harm by increasing post-operative infections and organ failure) for maintaining patients at higher Hb thresholds (liberal strategy).288 The past ten years have also seen an increasing scrutiny of RBC usage in medical patients (accounting in the UK for approx. twothirds of all RBC transfusions) compared with surgical indications. The degree to which the optimal Hb transfusion “trigger” should be modified for patients with specific risk factors (e.g. coronary disease, radiotherapy, and chemotherapy) remains unclear, and clinical dilemmas are clearly shown when considering the appropriate RBC transfusion threshold in the extremes: elderly, and premature neonates. The evidence for a prophylactic platelet transfusion threshold of 10x109 /L instead of higher triggers has come from randomized controlled trials in hematology patients with chemotherapy-associated thrombocytopenia.289 More recent studies even questioned the effectiveness of prophylactic platelet transfusions in all patients with (onco) hematologic diseases. There is a lack of evidence to guide use of platelet transfusions to cover invasive/surgical procedures in patients with platelet dysfunction and/or thrombocytopenia, and guidelines for these indications outside hematologic settings remain largely based only on expert opinion. Fresh frozen plasma contains pro- and anticoagulant factors and other proteins. Most guidelines describe the use of laboratory coagulation tests to guide administration of plasma, but there are uncertainties about the value of standard coagulation tests in predicting clinical bleeding risk and a lack of evidence of benefit for the prophylactic administration of fresh frozen plasma in non-bleeding patients.

European research contributions Major steps toward questioning the evidence base for use of blood were established in Europe, beginning with descriptive data by the Sanguis Study Group, which in 1994 reported large differences between hospitals and clinical teams in the use of RBC transfusions (and other blood products) for the same surgical procedures with no clear clinical explanation. Many subsequent activities (e.g. the European Society of Anaesthesiology Clinical Trial Network, established in 2010) have facilitated clinical research in anesthesia and intensive care. An ongoing study termed the European Transfusion Practice and 175

A. Engert et al. Outcome Survey has collected transfusion and outcome data from large numbers of patient transfusion episodes from many centers across Europe. According to reports from the European Blood Alliance, although average RBC use per 1000 inhabitants in Europe is substantially lower than in the US, large variations in usage rates are still reported between European countries, and audits indicate greater than 15% inappropriate use. Several leading clinical trials have been conducted in Europe for platelets, red cells, and granulocyte and fresh frozen plasma transfusions,289-292 but given the broad ranges of clinical setting where transfusions occur, many research gaps remain.

Proposed research for the Roadmap Clinical studies on appropriate use/linking to ongoing studies and patient input: the aforementioned European Transfusion Practice and Outcome Survey on blood usage for surgical interventions in Europe still has no counterpart for (onco) hematologic diseases. There is an opportunity to explore current hospital databases to characterize blood use in (onco) hematology and hemoglobinopathy, to generate key base-line information on blood usage practice. Incorporation of data collection for transfusion usage and bleeding outcome in hemato-oncology trials should be encouraged. Policies to enhance patient engagement, for instance in transfusion-dependent myelodysplasia, are required. This could be achieved by introduction of patient-reported outcomes related to physical activity, well-being, and quality of life (QoL) as part of post-transfusion follow up. Research is warranted to identify (predictive) risk factors for severe thrombocytopenic bleeding. A European registry is required that reports bleeding after commonly applied interventions in hematology (e.g. lumbar puncture and organ biopsies), in relation to platelet count, coagulation profile, and use of platelet transfusions and/or plasma/coagulation factors. Studies on transfusion management for pre-term infants and elderly patients undergoing cancer treatment are essentially lacking. Emphasis should also be given to medical and economic considerations of different approaches to transfusion or alternatives, including hematopoiesis-stimulating agents, iron, and prohemostatic drugs. Indications and patterns of use of granulocyte transfusions are unknown. Better biomarkers identifying transfusion needs and results: the hemoglobin (Hb) concentration is still used to define the need for red cell transfusions, calculate the dose of RBCs required, and monitor the response to transfusion or alternative treatment. However, Hb is a surrogate marker, and research should address better-targeted measures of oxygen requirements that can identify specific patient needs. Similarly, to identify patients with a high bleeding risk requiring prophylactic platelet or plasma transfusions, the safety of alternatives (near-patient point-of-care tests) and the value of biomarkers for endothelial damage preceding bleeding should be explored. Physician behavior and education: many interventions are undertaken to change transfusion practice based on suspected wrong practice or guidelines, but there are uncertainties about their effectiveness and durability. There is a need to define the determinants of transfusion behavior to deliver 176

optimal transfusion practice. Electronic blood ordering and better information technology support for prescribers may be of value. Prevention of hospital-acquired anemia is one key tenet of patient blood management, defined as a patient-centered, evidence-based approach of good clinical transfusion practice. Patients may lose significant volumes of blood for laboratory evaluation. In pre-term infants, this is even the major cause of transfusions. Non-invasive assays or microtesting would help minimize the need for transfusions.

Anticipated impact of the research Blood components are biological products and a costly resource. The uptake of patient blood management, which includes consideration of transfusion alternatives, remains highly variable. Studies in surgical and intensive care patients generally support a restrictive use of blood components. There are hardly any studies in (onco) hematologic diseases. The proposed research will contribute to patient blood management for hematologic patients.

7.2. Plasma-derived and recombinant human plasma proteins Srini Kaveri (Institut National de la Santé et de la Recherche Médicale, Paris, France), Sébastien LacroixDesmazes (Institut National de la Santé et de la Recherche Médicale, Paris, France), Stephan von Gunten (Universität Bern, Bern, Switzerland), Jagadeesh Bayry (Institut National de la Santé et de la Recherche Médicale, Paris, France).

Introduction Plasma products are produced by (non-profit) blood establishments or by the pharmaceutical industry. Plasma is recovered from whole blood donations or collected by (sometimes remunerated) plasmapheresis. Plasma is used as single-donor fresh frozen plasma units (quarantine, methylene-blue-treated) or as pooled plasma products requiring a pathogen-reduction treatment. Pooled plasma and fractionated plasma products (e.g. factor VIII, albumin, prothrombin concentrate, immunoglobulins, von Willebrand factor, and fibrinogen). as well as factors produced by recombinant techniques (e.g. factor VIIa, factor VIII, and factor IX), are pharmaceutical products. The huge variation in usage of various plasma-derived and recombinant products per 1000 inhabitants in the European member states is not explained by differences in plasma-product-dependent diseases. According to a 2010 report by the International Plasma Fractionation Association 2010, high-dose IVIG usage in European member states is, on average, 36.5 g per 1000 inhabitants (range 1.2–97 g), compared with an average of 120 g per 1000 US inhabitants. Indications for recombinant and plasma-derived coagulation factors comprise substitution in massive bleeding and prevention of bleeding in patients with congenital or acquired coagulation factor deficiencies. Patients with primary or acquired hypogammaglobulinemia are substituted with immunoglobulins. Anti-RhD immunoglobulin is applied for immunoprophylaxis of hemolytic disease of the newborn. IVIG is used for many (auto-)immunemediated disorders and to neutralize alloantibodies. IVIG exerts multiple immunoregulatory mechanisms, such as anti-idiotypic activity, inhibition of activation of B cells and antigen-presenting cells such as dendritic cells and macrophages, enhancement of IgG catabolism, and reciprocal regulation of regulatory T cells and pathogenic Th17 haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

and Th1 cells. The precise mechanisms underlying its beneficial effects in various diseases are still largely unknown. Many indications for IVIG are not evidencebased, and off-label use is frequent.

phases. Recent efforts aspire to generating products with longer half-lives, to improve patientsâ&#x20AC;&#x2122; quality of life (QoL), or to fully human products to ensure normal glycosylation and sulfation, aiming to reduce alloimmunization and neutralizing antibodies.

European research contributions The past decades have seen enormous progress in the development of therapeutic coagulation factors, greatly improving the treatment of bleeding disorders. Improvement in the very nature of the recombinant products has been constant, starting, in the case of procoagulant factor VIII, from albumin-stabilized products to 3rd-generation products that are devoid of contact with mammal proteins in both the production and purification

It was a European discovery by Imbach in 1981 that high-dose IVIG increased the platelet count in an autoimmune thrombocytopenia patient, and since then a large number of immune-mediated diseases are treated with IVIG. European researchers and physicians have made a considerable contribution toward understanding the mechanisms of action of IVIG and conducted randomized clinical trials with IVIG contributing to the evidence base for (new) indications.

Figure 8. Activation of the adaptive antigen-specific immune response. Potential targets for interventions to reduce Ab production or mitigate destruction. Potential interventions include high-dose IVIG and PDN (prednisolone).

haematologica | 2016; 101(2)

177

A. Engert et al. Proposed research for the Roadmap Immunogenicity of coagulation factors and gene therapy: replacement therapy with procoagulant products is complicated by the occurrence of neutralizing antidrug antibodies. Particularly in patients with hemophilia A, the prevalence of these antibodies may reach 30% and pose a major clinical concern. Several strategies are being investigated to reduce the immunogenicity of therapeutic factor VIII. These include alteration of factor VIII moieties implicated in its processing/recognition by immune effectors, control of the inflammatory status of the patients at the time of replacement therapy and induction of active immune tolerance, for example, upon oral or transplacental transfer of factor VIII,293 or using cell therapy for tolerance induction. Pre-clinical studies with recombinant adeno-associated viral vectors encoding variant human factor VIII294 are encouraging and may lead to a successful gene therapy strategy for hemophilia A similar to that achieved with hemophilia B. Prediction and treatment of massive bleeding: evidencebased assays to predict bleeding severity and monitor optimal substitution (bedside point-of-care and coagulation factor levels) are required. European consortia should continue to investigate the optimal mix of coagulation factor suppletion and drug treatment in massive bleeding of various origins (e.g. military and civil casualties, major surgery, postpartum hemorrhage, and congenital or acquired bleeding disorders) (see Section 6). Addressing these questions should proceed independently and free of any influence from producers. Thrombotic complications: for both antifibrinolytic treatment and coagulation factor substitution, the window of opportunity may be small, depending on the underlying condition, and late treatment may enhance thrombotic complications. Also, possible contamination of coagulation factors in IVIG products carries a risk of thrombotic events. A step has recently been made to remove coagulation factor impurities in IVIG. Besides hyperviscosity, thrombogenic activity of some products may play an additional role. Thrombotic complications should be monitored in plasma product surveillance systems, preferably European-wide. National hemovigilance registrations, which are in place in many European member states, can possibly facilitate this. Innovation of IVIG products and identification of biomarkers: the same IVIG products are used for substitution of immunodeficiency conditions and for immunomodulation in immune-mediated diseases. For substitution, research should aim for the conception of highly concentrated immunoglobulin products to reduce the volume of injection by subcutaneous route. For immunomodulation studies, identification of biomarkers to predict IVIG responders and appropriate dose are warranted. Although controversies regarding dependence of Fc-sialylation toward anti-inflammatory functions of IVIG exist,295 the necessity of CD209 for the IVIG-mediated expansion of regulatory T cells is established in both humans and mice.296,297 Sialylation-mediated structural modifications can be mimicked by specific amino acid modifications at position 241 (F→A) of the CH2 domain.297 This variant 178

IgG Fc molecule demonstrated anti-inflammatory effects similar to IVIG. Therefore, sialylation is not mandatory for anti-inflammatory effects of IVIG. The eventual clinical application of these innovative IVIG products needs to be further explored.

Anticipated impact of the research In well-resourced countries with access to products to combat bleeding after trauma, delivery, or surgery, validated assays to predict bleeding risk and monitor treatment can improve survival. Despite its complex clinical environment, European groups treating these patients and adjustments to (inter)national reporting systems will help to arrive at more evidence-based use of available treatments. Reducing the incidence of immune response to coagulant factors, such as anti–factor VIII, would reduce the cost associated with patients’ management. As IVIG is the driving force for plasma collection and represents a huge budget, appropriate dosage, treatment window, cellular and molecular mechanisms, and randomized clinical trials to confirm off-label usage may ultimately save costs. Similarly, clinical and basic research can ultimately lead to the conception of IVIG similars or IVIG-derived therapeutic molecules for treating specific pathological conditions.

7.3. Hemapheresis Hans Vrielink (Sanquin Research, Amsterdam, the Netherlands), Olivier Garraud (Université Jean Monnet, Saint-Etienne, France), Jörg Halter (Universitätsspital Basel, Basel, Switzerland), Luca Pierelli (Università degli Studi di Roma ‘La Sapienza’, Rome, Italy), Bernd Stegmayr (Umeå Universitet, Umeå, Sweden).

Introduction Hemapheresis encompasses a method of obtaining one or more blood components by using a special blood separation device processing whole blood, removing the component of interest, and returning the residual components of the blood to the donor/patient during or at the end of the process. Apheresis can be divided into apheresis to collect blood components from donors/patients and therapeutic apheresis. Component apheresis involves collection of plasma, red blood cells (RBCs), and platelets from blood donors for the purpose of direct cellular therapy or further modification. Both patients and donors donate peripheral hematopoietic stem cells (HSCs) and other mononuclear cells. Therapeutic apheresis is performed to remove large numbers of cells or plasma from patients for specific disease treatment and is applied for more than 75 different, mostly immune-mediated, rare diseases, varying from macular degeneration to sickle cell crisis.298 In 2007, the American Society for Apheresis started an initiative to classify the applied indications for therapeutic hemapheresis according to evidence base, and these recommendations are regularly up-dated. The latest update of 2013298 recommends hemapheresis for 25 diseases either as a first-line lifesaving treatment or a valid adjuvant to other therapies. Still, there are more than 50 benefit-of-doubt indications. Hemapheresis serves a multitude of medical disciplines (neurology, nephrology, dermatology, ophthalmology, dermatology, cardiology, and haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

hematology), requiring joint studies and multidisciplinary recommendations. Besides the most pivotal plasmaexchange indication (TTP), for hematology, the collection of cells for autologous and allogeneic use play an increasing dominant role. Of the more than 20,000 hematopoietic stem cell transplants (HSCTs) performed worldwide each year, more than 70% are obtained by blood stem cell apheresis.

of an EBMT board committee on donor follow up (2012) with one of the goals to set up a donor follow-up registry for all EBMT related and unrelated stem cell donors and to provide the possibility for systematically collecting adverse events and longer follow up for family donors. The safety of new stem cell mobilization drugs (e.g. biosimilars and CXCR4 agonists) requires European collaboration and uniform medico-ethical procedures.301

European research contributions

Plasma and platelet donation from unrelated donors: a donorrelated aspect is whether the repeated exposure to citrate (binding calcium) and protein removal is harmful and poses risks for osteoporosis and hypogammaglobulinemia in the long term, an important issue for recommendation on plasma- or apheresis-derived platelet products.302

The European Society for Hemapheresis collaborates with the national apheresis societies to establish standardized education and certification programs for specialized nurses for apheresis. There is also co-operation in contributing to evidence-based medicine indications (e.g. systemic lupus erythematosus, as well apheresis treatment in inflammatory bowel disease). The evolving use of blood stem cell, lymphocyte, and monocyte/dendritic cell collections resulted in dedicated working parties within the European Group for Blood and Marrow Transplantation (EBMT) and the International Society for Hematotherapy and Graft Engineering. Two Italian scientific societies for hematology and hemapheresis (SIDEM and GITMO) produced best practice recommendations for stem cell mobilization in children and adults.299 Extracorporeal photopheresis is a treatment in which blood mononuclear cells collected by apheresis are incubated with methoxsalen and exposed to UV light, and subsequently reinfused to the patient. Extracorporeal photopheresis is by UK and Italian scientific/clinical consensus groups recommended as 2nd-line treatment for graft-versus-host disease (GvHD) after allogeneic stem cell transplantation and for scleroderma and other autoimmune diseases.300 Because of the high costs, however, many patients cannot be treated with extracorporeal photopheresis. Stem cell donations by healthy donors pose special medico-ethical problems. Surveys in Europe and the US reveal that the care for the family donor differs from the strict guidelines from the World Marrow Donor Association aiming at optimal donor safety for unrelated donors. Collections of mononuclear cells and subsequently ex vivo preparation of the collected leukocytes for cellular therapies is currently being explored as a novel potent anticancer treatment or antiviral vaccination therapy. This requires a further commitment and poses a burden for the volunteer donor.

Proposed research for the Roadmap Therapeutic apheresis: there remains an ongoing need to contribute to evidence base indications for therapeutic apheresis. Given the orphan character of diseases this requires broad collaboration with all involved institutes in Europe and the establishment of European databases in conjunction with the scientific societies of various medical disciplines. Stem cell donation by autologous, related, and unrelated donors: for stem cell donation, the establishment of a global standardized system for related donor care comparable to unrelated volunteer donors is in progress. This initiative is a joint effort of the World Marrow Donor Association and EBMT, resulting from the establishment haematologica | 2016; 101(2)

Anticipated impact of the research Hemapheresis represents a unique medical (supportive) specialty for patient treatment and depends highly on industry-driven technology and drug development to obtain optimal efficacy. Moreover, a huge part of the worldwide allogeneic hematopoietic transplants, as well as the European blood supply of plasma and platelet products, depends on apheresis. Although short-term adverse effects of patient as well as donor apheresis are minimal, long-term effects of many procedures applied in healthy (volunteer, non-remunerated) donors are less well studied. The proposed research subjects can provide the European community with information about the risk and safety issues for donors, and promote convenient devices and drugs for their voluntary donations.

7.4. Immunological transfusion complications: alloimmunization/transfusion-related immunomodulation/ hemovigilance Andreas Greinacher (Ernst-Moritz-Arndt-UniversitĂ¤t, Greifswald, Germany), Anneke Brand (Leids Universitair Medisch Centrum, Leiden, the Netherlands), Frans Claas (Leids Universitair Medisch Centrum, Leiden, the Netherlands), France Noizat-Pirenne (Etablissement FranĂ§ais du Sang, CrĂŠteil, France), Martin Olsson (Lunds Universitet, Lund, Sweden), Sacha Zeerleder (Sanquin Research, Amsterdam, the Netherlands).

Introduction Currently, donor counseling, sensitive laboratory tests, good manufacturing practice, and hemovigilance have substantially increased transfusion safety with respect to transfusion-transmitted infections. However, blood transfusions also bear immunological risks, yet lack a surveillance system. Although alloantibodies can result from pregnancy, most are induced by transfusions. Red blood cell (RBC) antibodies can cause (sub)acute hemolytic transfusion reactions and hemolytic disease of the fetus/newborn. Leukocyte/HLA antibodies can cause transfusion-related acute lung injury and limit the chance of finding a compatible (organ/stem cell) graft. HLA and platelet antibodies destroy transfused blood platelets needed for leukemia treatment and stem cell transplantation. Many European countries apply universal leukoreduced transfusions, which reduces HLA immunization, but HLA antibodies still hamper optimal platelet transfusion and transplantation treatment. Except (Rh-K) matching of RBC transfusions for (pre)fertile women and for 179

A. Engert et al. patients with hemoglobinopathies, applied by some European countries, measures to avoid antibody formation are not yet possible. Although some countries have a national registry for RBC/HPA-immunized pregnant women, the overall prevalence of alloimmunization across Europe is unknown. However, immunization has a huge economic impact in the need to provide safety nets for immunized patients needing blood or transplants, or who are pregnant.

throughput RBC, HLA, and HPA typing platforms of donors, recipients, pregnant females, and fetuses. Immunovigilance would also serve as post-marketing surveillance of pathogen-reduced products to exclude immunogenic neoantigen formation. A registry of transfusion-dependent RBC diseases in Europe can identify the incidence and stimulate collaborative treatment studies for hyperhemolysis, a life-threatening transfusion complication.

Besides antigen-specific immunization, clinical studies suggest transfusion-related immunomodulation, an illdefined complication also affecting the innate (not antigen-specific) recipient immune system, transiently impairs resistance against infections, enhancing organ damage and resulting in unknown consequences for cancer immunosurveillance.

Prevention: prevention of alloimmunization requires better matching and the detection of potentially dangerous memory responses. Currently, about 300 RBC antigens are genetically and/or molecularly defined. Approximately 40 more residual (orphan) groups need to be unraveled, while new RBC groups are still discovered through unexpected antibodies. It is important to know their genetic backgrounds for inclusion in platforms for high-throughput donor and patient typing. The polymorphism of the HLA system with more than 10,000 identified alleles complicates relevant clinical matching. Every HLA allele poses a unique combination of antigenic epitopes, but many epitopes are shared with other HLA alleles.307 Consequently, the immune response to a foreign HLA antigen can be explained by a restricted number of epitopes. New strategies should focus on matching for the relevant immunogenic epitopes between donor and recipient rather than matching for the HLA alleles. The complex pathophysiology of high and low responder individuals to alloantigens needs studies of genetic and environmental factors for T- and B-cell activation, memory, and antibody persistence. Instead of studies in mice, pregnancy and transfusions in humans can enhance insight.

Research on the mechanism of alloimmunity has mostly focused on animal models, even though pregnancies and blood transfusions in humans provide unique models for systematic studies of in vivo reactions of the human immune system.

European research contributions In the past decade, European research groups have advanced our knowledge of the genetics, polymorphism, expression, function, and pathophysiology of the many blood groups expressed on RBCs, leukocytes, and platelets. Molecular genotyping of most antigens is now possible for donors and patients, and even fetal polymorphic antigens can be determined in maternal blood during pregnancy. Demonstration of the absence of (fetal) RhDpos DNA in Rh-Dneg gravidas avoids unnecessary immunoprophylaxis with human anti-D immunoglobulin and saves money.303 A European platform performed preparations for implementation of â&#x20AC;&#x153;RBC bloodmatch on a chip.â&#x20AC;?304 For stem cell and organ transplantation and donor selection for platelet transfusions, provided electronic matching programs can select HLA-compatible donors from large international registries. HLA-net aims at networking researchers in bone marrow transplantation (BMT), epidemiology, and population genetics to improve the molecular characterization of genetic HLA diversity of human populations with an impact on both public health and fundamental research (Allele Frequency Net Database). A European subgroup of the Allele Frequency Net Database (EUROSTAM/HLA-net) is setting up an HLA allele frequency database to enable renal transplantation in highly sensitized patients on the basis of acceptable HLA mismatches.305 The European platelet immunology working party contributed insight in fetal alloimmune thrombocytopenia.306 Unraveling the minor histocompatibility antigens aims at dissecting the T-cell immunity causing graft-versus-host disease (GvHD) and graft-versus-tumor effect (see Section 9).

Reversal of immunity: alloimmunization cannot be completely abolished. To reverse antibody production and memory immune cells is extremely complex. For this purpose, the effect of selected drugs/immune cells shall be explored on the behavior of B/plasma cells, regulatory mechanisms at the T-cell level, antibody affinity development, and the role of (post-pregnancy) chimerism. Mitigating severe alloimmune complications: antibodies can cause severe (lethal) complications, such as hyperhemolysis after transfusion, severe bleeding and lack of compatible platelet donors, graft rejection, and fetal morbidity and mortality. New drugs mitigating complement activation and other sequels (cytokine storm) of antigen-antibody reactions may save lives and safeguard transplanted organs. To reach these goals, translational research programs including bioinformatics analyzing big-data output304-307 should be combined with basic studies using the unique situation of the highly ethical exchange of alloantigens by blood transfusions.

Proposed research for the Roadmap Four broad research goals are proposed. Epidemiology: the current EU hemovigilance registries could be extended with immunovigilance. Registration of alloantibodies is important to define which phenotypes we should focus on for blood and stem cell donor recruitment (e.g. ethnic minorities), for composition of high180

Anticipated impact of the research For patients suffering from a wide range of (onco) hematologic diseases, transfusions, stem cell transplantation, and immunotherapy can be indispensable. Alloimmunization hampers optimal access and increases costs related to these treatments. The proposed research will result in the reduction of immunization and of damhaematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

age caused by immune incompatibility, and can reduce the current high cost of care for alloimmunized patients.

7.5. Blood-borne infections and hematologic patients: lines of research Hans Zaaijer (Sanquin Research, Amsterdam, the Netherlands), Dragoslav DomanovicÂ´ (European Center for Disease Prevention and Control, Stockholm, Sweden).

Introduction The donation of organs, tissues, and cells as well as the pooling of large numbers of donor plasma units for protein fractionating bears the risk of transmission of pathogens to recipients. For hepatitis B, hepatitis C, HIV, and variant Creutzfeldt-Jakob disease, donor counseling, testing, product processing, and traceability of donors and recipients are successfully applied to limit the risk of transmission by transfusion and transplantation. However, the increase in global travel, intensive animal farming, and climate change may cause a quick spread of emerging bacterial, viral (e.g. West Nile virus, chikungunya virus, and dengue virus), and parasitic infections.

European research contributions Safeguarding blood products and other substances of human origin (SoHO), intended for human treatment, is regulated in the European Union by several directives, launched from 2004 to 206 (2004/23/EC, 2006/17/EC, and 2006/86/EC). The European Commission leads the regulatory activities, co-ordinates the work of national competent authorities for SoHO, and runs Rapid Alert platforms monitoring serious adverse reactions and events caused by SoHO therapies and traceability from donor to patient and vice versa. The European Network of Competent Authorities for Tissues and Cells participates in the vigilance and surveillance of SoHO. For the blood supply, the European Blood Alliance emerging infectious disease monitoring group ensured quick monthly information with recommendations for donor/donation exclusion, in particular related to traveling. In 2005, the European Centre for Disease Prevention and Control was established in Stockholm. Its mission is to identify, assess, and divulge information about the current and emerging threats to human health posed by infectious diseases. The center communicates with Coordinating Competent Bodies in each member state to strengthen and develop continent-wide disease surveillance and early warning systems. In the field of SoHO safety, the center provides member states and the European Commission with the best advice available concerning infectious risks of donations and human use of SoHO. The center also develops risk assessments and tools related to infectious safety of SoHO and provides weekly maps of areas affected by West Nile virus infection, to support blood banks and pharmaceutical production plants to take preventive measures and help with validation of infectious disease tests.

transmission of these agents to one residual infection in 110 million transfusions. Nevertheless this risk is not zero, and for hematologic patients the risk is higher than for many other categories of recipients. Besides prolonged immune impairment in transplant recipients, it has been shown that patients treated with chemotherapy for hematologic cancer may also lose pre-existing immunity.308 It is obvious that the ongoing research in pathogen inactivation and pathogen removal is of great importance for blood recipients that are especially vulnerable to blood-transmitted infections (see Subsection 7.6; pathogen inactivation and removal). An important topic, especially relevant for blood disorders, is the question of whether blood and blood-derived products for immunosuppressed hematologic patients should be safer than for other categories of patients. At least three elements of this question are candidates for applied research. 1. Does severe (iatrogenic) immunodeficiency warrant additional safety measures for blood transfusions and blood-derived products [e.g. pooled plasma and hematopoietic stem cells (HSCs)] in hematologic patients, and if so, which agents and which safety measures must be considered and what are the triggers for their implementation? At the same time, it seems desirable to apply this question on a more practical level to at least two specific infectious agents. 2. In parts of the Western world, currently there is a high incidence of silent hepatitis E virus genotype 3 infection among blood donors. Transmission via blood and blood-derived products has been demonstrated. Hepatitis E virus is a non-enveloped virus and is not sufficiently inactivated by the current solvent-detergent procedures used for pathogen reduction (PR) of pooled plasma products, which are in large quantities administered to patients with thrombotic thrombocytopenic purpura (TTP). In immunosuppressed patients, hepatitis E virus genotype 3 may cause silent, chronic infection that rapidly leads to cirrhosis.309 Chronic hepatitis E is often interpreted as a druginduced liver injury or a sign of graft-versus-host disease (GvHD), which may lead to an increase in immunosuppressive treatment and thus may worsen the viral infection. From a technical point of view, donor screening for hepatitis E virus genotype 3 RNA is possible, but so far it has not been implemented. 3. Parvovirus B19 (B19V) infection has a seasonal and cyclic nature: each year in springtime blood donors may harbor acute, asymptomatic B19V infection. In addition, every four years, there is a marked increase of B19V infections. In immunosuppressed patients, exposure to B19V may cause severe and chronic anemia, which can be controlled only by repeated blood transfusions or monthly administration of IVIG. To prevent B19V infection of vulnerable patients, some blood transfusion services (e.g. in the Netherlands) provide â&#x20AC;&#x153;B19V-safeâ&#x20AC;? blood for specific categories of recipients. It is desirable to evaluate this policy in the EU member states.

Proposed research for the Roadmap In the Western world, the risk that blood transfusions and blood-derived products transmit blood-borne infections to patients is very small. For example, the introduction of sensitive nucleic acid amplification testing for HIV1/2, hepatitis B virus, and hepatitis C virus reduced haematologica | 2016; 101(2)

Anticipated impact of the research With the implementation of serological and molecular testing, at least in high-income countries, transfusiontransmitted infections of a limited number of specific pathogens (HIV, hepatitis B virus, hepatitis C virus, and 181

A. Engert et al. human T-cell leukemia virus) have become extremely rare. Full control of infectious disease transmission has not yet been achieved because many pathogens are not included in test protocols and new agents continue to emerge. The immune status of recipients has a considerable impact on the outcome of transfusion-transmitted infections. This particularly affects hematologic patients who received anti-lymphocytic agents or underwent stem cell transplantation. The results of the proposed research can be used to better assess patient risks, enhance the cost-effectiveness of treatments, and increase the safety of blood and blood-derived products for hemato-oncology patients across Europe.

7.6. Pathogen reduction of blood components Lello Zolla (University of Tuscia, Viterbo, Italy), Paolo Rebulla (Ospedale Maggiore, Milan, Italy), Sara Rinalducci (University of Tuscia, Viterbo, Italy), Peter Schlenke (Medical University Graz, Graz, Austria), Jean Daniel Tissot (University of Lausanne, Lausanne, Suisse).

Introduction Nowadays, thanks to the measures adopted to increase the safety of transfusions (including optimized donor selection programs and mandatory screening tests), the residual risk of suffering a transfusion-related pathogen infection is extremely low, especially with regard to viral infections. Transfusion medicine research has, therefore, moved toward the development of methods intended to reduce the risks posed by bacterial contamination, particularly in platelet components. Transfusion of bacteria-contaminated platelets can cause a septic reaction in the recipients (1:20,000 to 1:50,000). The fatality rate is expected to be around 10%; however, transfusion-associated bacterial infections may be underestimated due to the fact that platelet transfusions are frequently administered to patients suffering from hemato-oncology diseases where the use of antibiotics-based therapies may mask the symptoms of a septic transfusion reaction. To prevent platelet transfusion-related bacterial sepsis, several countries have implemented bacterial detection, but unfortunately, available bacterial screening methods (even the more sensitive ones) are not able to completely eliminate the cases of this infectious complication of transfusion. Moreover, one should not forget the issue of novel emerging pathogens, including not only undiscovered strains of bacteria, but also viruses with genomic mutations susceptible to immune escape mechanisms or known parasites/viruses that are continuously on the rise in nonendemic regions in view of globalization and climate changes (e.g. Plasmodium spp., Babesia spp., Trypanosoma cruzi and HIV, as well as dengue, West Nile, and chikungunya viruses).310,311 The arduous search for the zero-risk strategy to reach a completely safe blood transfusion will probably never end, although a complicated and intense debate about the implementation of pathogen reduction (PR) technologies continues. This is due to the paradox that innovation directly related to the safety of blood products is most frequently associated with impairment of a productâ&#x20AC;&#x2122;s intrinsic quality. Thus, scientists have to find the equilibrium between safety and clinical efficiency. This observation means that we urgently need basic as well as clinical research. Although in some European countries PR technologies for plasma and platelets fulfill the overall criteria of acceptability, there are currently no 182

acceptable PR methodologies for whole blood and red blood cells (RBCs).

European research contributions The variety of emerging pathogens and the costs of development of new counseling and detection assays were (besides more ideological purposes for underresourced countries) driving forces to develop techniques for PR of cellular blood components, often in close collaboration between research centers and pharmaceutical companies. For platelets, three PR techniques have been developed and approved in Europe.311 One is based on the addition of amotosalen and illumination with UVA light; a second combines the addition of riboflavin (vitamin B2) and illumination with 265 to 370 nm UV light; and the third applies only UVC (below 280 nm) under loose strong agitation. However, in France only A-L (Intercept) has been approved (by the National Agency for the Safety of Medicines and Health Products, in 2003). The first two methodologies are approved for clinical application or in phase III studies, while Theraflex UV is currently in phase I-II.311 In Europe, there is a wide range of use of PR-treated platelets for routine transfusion. Published hemovigilance data predominantly concern the A-L (Intercept) method with all reports confirming both the safety and efficacy of A-Lâ&#x20AC;&#x201C;treated platelets in a huge number of platelet transfusions.312 If on the one hand, PR technologies are designed to irreversibly disrupt nucleic acids of pathogens, on the other, they also cause collateral reactive oxidation-related damage to platelet proteins, thus enhancing the platelet storage lesion. Comparison of such damage by different PR techniques has been shown by a consortium represented by four (Switzerland, Italy, France, Germany) European research groups, along with a Canadian group.313 Studying platelet proteome changes revealed that each PR technique affects different protein pathways relevant for essential platelet functions.313 A great challenge is posed by PR of RBCs for which UV light cannot be applied. Currently, the S-303â&#x20AC;&#x201C;based technology (Cerus Corporation, Concord, USA) is the pre-eminent system for PR of whole blood and RBCs.314

Proposed research for the Roadmap The following are important issues to be examined: 1) to what extent PR of platelets affect the potential to prevent/treat bleeding; 2) immunogenicity of PR-treated cellular blood products; 3) clinical comparison of blood products treated by different PR techniques; 4) PR of RBCs; and 5) assessment of cost-effectiveness of PR interventions.

Anticipated impact of the research The proposed research will contribute to improving the quality of established commercial technologies for PR in platelets and plasma through the co-operation of academic expert centers in proteomic analysis and cellular biology. Moreover, experience gained in the studies performed in platelets will facilitate the development of novel technologies for PR in RBCs and whole blood. The achievement of a unique procedure carried out on whole blood will contribute to reducing the high costs of current PR procedures. Besides this important economical benefit, the development of a global PR reduction technology for whole blood will significantly reduce the frequency and severity of pathogen transmission with blood transfusions. haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

7.7. Toward large-scale production of blood cells for transfusion purposes Luc Douay (UniversitĂŠ Pierre et Marie Curie, Paris, France), Emile van den Akker (Sanquin Research, Amsterdam, the Netherlands), David Anstee (NHSBT Blood Centre, Filton, United Kingdom), Dominique Baruch (UniversitĂŠ Paris Descartes, Paris, France ), Johan Flygare (University of Lund, Lund, Sweden), Marieke von Lindern (Sanquin Research, Amsterdam, the Netherlands), Anna Rita Migliaccio (Mount Sinai Hospital, New York, United States of America), Kenichi Miharada (University of Lund, Lund, Sweden), Wassim el Nemer (INSERM UMR S1134, Paris, France), Ash Toye (University of Bristol, Clifton, United Kingdom).

Introduction Blood transfusions in Europe depend on the recurrent, non-remunerated contribution of blood by donors. Part of the donor population is genetically typed. In addition, banks store typed erythrocyte units in liquid nitrogen, available for patients with alloantibodies having no other option. It is, however, logistically impossible to provide all patients that require regular blood transfusions with matched blood products. A solution to this problem would be the availability of cultured red blood cells (cRBCs) and cultured platelets (cPLTs) that are perfectly matched for all blood groups. When successful, this will be a major clinical breakthrough providing transfusion-dependent patients with a non-immunizing safe transfusion product, likely diminishing iron overload in transfusion-dependent patients. 315-319

European research contributions Over the past decades, several major steps towards cultured human blood cells were established in Europe, paving the way to major advances in our understanding of the mechanisms that regulate erythropoiesis/megakaryopoiesis. The first key development for the ex vivo production of erythroid cells was made at the end of the last century when liquid culture systems replaced semisolid culture, allowing the expansion and differentiation of pure erythroid cultures in distinct medium conditions. This enabled large-scale culture of cRBC from hematopoietic stem cells (HSCs) of adult or umbilical cord blood origin, which was achieved with different cocktails of cytokines and additives. Improved culture conditions yielded high numbers of fully mature cRBCs and cPLTs, and the prospect of culturing blood cells for transfusion purposes became closer to a reality. Industrial production of both cRBCs/cPLTs has been tackled by simplification of protocols. Procedures without stroma or xenogenic components, using pharmaceutical-grade reagents, have been developed, allowing the production of cRBCs/cPLTs for transfusion from various stem cell sources. The first proof of concept (autologous mini-transfusion with cRBC from adult CD34+ cells) in a healthy volunteer was performed in Paris. Erythropoiesis and megakaryopoiesis should ideally be initiated from an unlimited source, such as induced pluripotent stem cells (iPSCs). The big advantage of iPSCs is that they can be selected for their phenotype of interest, or manipulated to generate specific phenotypes. Several EU teams can now produce cRBCs/cPLTs from various haematologica | 2016; 101(2)

sources (HSCs, human ESCs, iPSCs), but not yet at sufficient numbers or quality. An alternative may reside in immortalized cell lines, for which a Japanese team provided proof of concept by production of cRBCs and cPLTs. The major remaining challenge for in vitro blood production is the affordable cost. The development of bioreactor conditions for cost-effective production at a therapeutic scale, however, is still at an early phase. The production of cRBCs/cPLTs is optimized in standard liquid bioreactors under good manufacturing practice conditions to generate enough cells for mini-transfusions for further clinical trials. In addition, 3-D bioengineered scaffolds are being tested for more efficient stem cell expansion and cost reduction. This has brought our EU consortium to a lab-to-market stage. For cPLTs in particular, 3-D culture is required, which requires the development of flow devices that are based on microfluidics and engineered biomaterials mimicking the bone marrow environment, such as silk and hydrogels.

Proposed research for the Roadmap The next step is to optimize the production of cRBCs/cPLTs at the required scale for safe patient transfusion. EU blood supply centers and scientists must cooperate to: 1) fulfill regulatory compliance of cRBCs/cPLTs as new advanced therapy medicinal products (ATMPs), to define the release criteria and set functional standards for cRBCs/cPLTs; 2) prepare cRBCs/cPLTs for clinical studies; and 3) optimize culture conditions to reduce costs for the large scale required for clinical use. The key objectives for the next few years can be summarized in three steps. 1. Achieving regulatory compliance: the objective is to define standards for quality and safety regulations and release criteria that comply with the good manufacturing practice requirements for the production of ATMPs. These standards should be used by regulatory committees throughout Europe that have to decide on the use of cRBCs/cPLTs in clinical trials. European teams need to organize consensus approach and progress meetings with stakeholders that include experts in transfusion technology, immunohematology, clinical transfusion science, hematologists, representatives of patient organizations, and representatives of donor councils throughout the European research area. 2. Establishing proof of principle by large-scale transfusion of cRBCs/cPLTs: proof of principle by large-scale transfusion of cRBCs/cPLTs should be established by transfusion studies in healthy volunteers, giving insight into: 1) the stability and functionality of cRBCs/cPLTs after administration; and 2) the risk of immunization against neoantigens. The aim of the clinical trials will be to validate the release criteria set in vitro for their suitability to predict cellular function in vivo. 3. Therapeutic-scale production at reasonable cost: the aim is to generate cRBCs/cPLTs more efficiently without compromising the functional parameters and thresholds that define a safe cRBC/cPLT product. Modified culture conditions should result in a more efficient, automated, and economical process. There is a need to devise optimal bioreactor conditions to culture cells at high density in a low-cost medium. Immortalized 183

A. Engert et al. cells derived from universal donors that generate suitable, high-quality transfusion units for (almost) all patients, without adverse transfusion reactions, have to be designed. Efficient expansion and maturation of these cells may be supported by small molecular compounds. Chemical libraries can be tested to find substances that control or enhance expansion and maturation of erythroid/megakaryocytic cells, and to determine expression of appropriate types of Hbs for cRBCs.

Anticipated impact of the research cRBCs and cPLTs derived in vitro with rare or near universal phenotypes would revolutionize blood transfusion practices for future safe blood transfusions. Development of culture conditions to produce pharmaceutical quantities of blood involves a considerable amount of knowledge, innovation, and technology generation. To this end, EU teams should collaborate closely with industrial partners. In addition to the transfusion market, RBCs may be manipulated for drug delivery, promising a further revolution in blood transfusion practices.

The EHA Roadmap for European Hematology Research Section 8. Infections in hematology

Section editor: Catherine Cordonnier. Parallel to great progress made in the treatment of hematologic malignancies in the past 20 years, the research carried out by the hematology community has led to equally valuable progress in the incidence, causes, presentation, and mortality of infectious complications. These changes have usually been poorly anticipated, and sometimes not identified before several trials have assessed a new drug. Since tyrosine kinase inhibitors (TKIs) have been made available for chronic myeloid leukemia (CML), the indication for allogeneic hematopoietic stem cell transplantation (HSCT) has decreased considerably in this disease, and although TKIs impact on immunity, infection is now extremely rare in this setting before acute transformation of the disease. On the other hand, new monoclonal antibodies such as rituximab, which is highly efficient in many CD20-positive lymphoid malignant disorders, have created a risk of prolonged B-cell deficiency, increasing a natural risk of infection due to encapsulated pathogens and impairing the response to vaccines. In general, few new therapeutic approaches have been devoid of adverse infectious risks: the older age of HSCT candidates increases the risk of many infections after transplant; alemtuzumab treatment increases the risk for cytomegalovirus (CMV) infection and disease; eculizumab, a humanized anti-C5 monoclonal antibody used for treatment of paroxysmal nocturnal hemoglobinuria (PNH), results in susceptibility to meningococcal infection, requiring vaccination. However, many unmet needs remain and will continue to remain as long as new therapies are being developed. Understanding the mechanism of predisposition to certain pathogens is of crucial importance to develop strategies, as are exhaustive epidemiological data each time a 184

new treatment or strategy is assessed in hematology. This should be done not only in the classical high-risk patient populations such as neutropenic patients or HSCT recipients, but also in patients with lympho- or myeloproliferative chronic disorders who are increasingly being managed as outpatients and deserve specific considerations in terms of control of infection. Europe has been highly active in the field. The Infectious Diseases Group of the European Organisation for Research and Treatment of Cancer has been one of the very first in the world to prospectively study febrile neutropenia.320 Twenty-five years ago, the European Group for Blood and Marrow Transplantation (EBMT) created a dedicated working party for infectious complications after HSCT. The EBMT was able to show that death from infections significantly declined over time since the very early times of transplant, although it remains a major concern.321 The European Conference on Infections in Leukemia was initiated ten years ago to share practices and expert opinions, elaborate guidelines for the management of infectious complications in hematology patients, and define new areas of research. More recently, the European Society of Clinical Microbiology and Infectious Diseases created a group for immunocompromised patient populations. Pharmaceutical companies have also been very active in developing new anti-infective drugs for high-risk patients. However, the evolution of bacterial resistance worldwide, more aggressive therapies for some conditions, and an older age limit for performing HSCT mean these advances are associated with a greater risk of patients dying from infection, often before benefiting from the treatment of their underlying disease. This is especially the case in acute leukemia or high-risk lymphoma patients, allogeneic HSCT recipients, and primary immunodeficient children. Several issues concern all of these populations, and targeted studies should be encouraged. 1. We need to better anticipate the infectious risk and adopt a more preventative approach on an individual basis. This implies good epidemiological data in hematology patients, better identification of specific infectious risks, and the development of scoring systems322 to better target prophylaxis, which, for many reasons, including toxicity, resistance, and cost, cannot be universal. 2. We need new classes of antibacterials to overcome the inevitable increase of multi-drug resistance, which is a worldwide phenomenon and may soon constrain us to give up curative treatment in hematologic diseases. In parallel, we should explore how to limit the administration of the available antimicrobials as far as possible each time they are needed. To reach this goal, antimicrobial stewardship in the hematology ward is crucial, and yet more difficult than with other patient populations.323 This requires studies and resources. 3. In order to challenge the empirical anti-infectious approach widely used in many hematology patients, we need to develop sensitive, direct or indirect markers of bacterial, fungal, and viral infection and assess their clinical value in prospective trials. The main goal should be to restrict the administration of anti-infectives as much as possible, on the basis of infection markers. haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

4. Faced with the real lack of new antibacterials, we need to develop alternative strategies to prevent and treat infection. To this end, vaccination, monoclonal antibodies, and targeted cellular therapy should be properly assessed in well-designed prospective and controlled trials. Except after HSCT, where it has been possible to produce evidenced-based guidelines, vaccination has been poorly explored in hematology patients so that clinical practice is heterogeneous. Prospective trials on vaccination in specific diseases should be encouraged, taking into account the timing of the therapeutic program of the underlying disease.324 Although the role of the pharmaceutical industry is of the utmost importance to produce new tests and new drugs and support large, well-designed trials, more academic involvement is essential for translational research, epidemiology, and prospective randomized trials within the field. The unmet needs of infectious complications in hematology patients are mainly observed in four different settings. 1. HSCT recipients. 2. Neutropenic patients. 3. Non-neutropenic patients with acquired immune deficiencies. 4. Primary immune deficiencies. We also think that exploring genetic predisposition to infections in these patients will greatly help anticipate and manage the risks.

8.1. Infections in HSCT recipients Per Ljungman (Karolinska Institutet, Stockholm, Sweden), Simone Cesaro (Policlinico GB Rossi, Verona, Italy).

Introduction Infections have been major obstacles to the success of allogeneic stem cell transplantation from the beginning, more than 40 years ago. The immunosuppression needed for successful transplantation, including the conditioning regimen, prevention of, and treatment of graft-versushost disease (GvHD), all contribute to these risks. Transplant strategies are continuously evolving with the introduction of new conditioning regimens, an increased utilization of alternative donors (e.g. haploidentical donors), and single and double cord blood grafts. Many of these new techniques will affect the speed and degree of immune reconstitution and be related to the risk of infections. Haploidentical transplantation is performed in an increasing number of patients. However, the strategies used to perform allogeneic transplantations vary between centers. Strategies are likely to affect the immune reconstitution in different ways, and knowledge of the risks of specific infection in short, intermediate, and long-term perspectives are still limited.

pneumococci and influenza, and are also likely to have comorbidities that influence the risk for infections. Viral infections have been recognized as important for outcome, especially in allogeneic stem cell transplant recipients. Despite many advances in the field, these infections are still associated with morbidity and mortality. Several new antiviral drugs, as well as cytomegalovirus (CMV) vaccines are in late clinical development. Techniques allowing monitoring of CMV-specific immune responses in the individual patient are also being developed and might be valuable in the management algorithm. A challenge for the next few years will be to implement these new agents and techniques in the clinical management of stem cell transplant recipients, taking into consideration efficacy, toxicity, and cost aspects. New drugs are likely to be more expensive than the currently available agents, but might have significant advantages in terms of toxicity. In addition, vaccines and specific T-cell therapies are under development, and strategies for their implementation need to be developed addressing efficacy, safety, and costs.325 The importance of human herpesvirus 6 has been discussed for at least two decades, but the diagnostics and management of these infections have been controversial. Recent studies suggest that human herpesvirus 6 is an important pathogen mainly causing complications in the central nervous system in patients with poor or delayed T-cell reconstitution, such as cord blood transplant recipients.326 For the moment, the available antiviral drugs are not very effective in controlling this virus. A changing epidemiology of viral infections poses new challenges. Respiratory viruses such as respiratory syncytial virus and influenza are well recognized as important pathogens especially after allogeneic stem cell transplantation. During the past decade, at least 10 new respiratory viruses have been described, of which several have the potential to become relevant pathogens. Previously known viruses can change their epidemiological pattern and appear as important pathogens. This is illustrated by the recent emergence of severe infections caused by enterovirus D68 in outbreaks in both North America and Europe.327 Recent experience also includes the emergence of the West Nile virus, outbreaks of chikungunya virus, and the expanding areas of the world where patients are at risk of dengue virus infections. A recently recognized potentially important pathogen is the hepatitis E virus that has been associated with chronic hepatitis and possibly the rapid development of cirrhosis in small patient series.328 Several of these viruses can also be transmitted from stem cell donors, possibly requiring testing for new viruses. Vaccines are important for preventing infections in the general population. There are major gaps in our knowledge of how best to utilize vaccines in stem cell transplant.329

European research contributions Patient populations are changing, especially with more elderly patients undergoing allogeneic transplantation. Elderly patients are more vulnerable to infections such as haematologica | 2016; 101(2)

European centers and collaborative groups, such as the Infectious Diseases Working Party of the European Group for Blood and Marrow Transplantation (EBMT) and the 185

A. Engert et al. European Organisation for Research and Treatment of Cancer working groups, have been active in the field of infectious diseases in transplant recipients. Topics of particular interest for European investigators include the management of viral infections due to CMV, Epstein-Barr virus (EBV), respiratory syncytial virus, and polyomavirus (BK). Other topics include the development of vaccination strategies for HSCT recipients, randomized studies of antibacterial agents, and diagnosis and management of fungal infections. Many of these studies have changed practice, not only in Europe.

reduce the morbidity and mortality of infections, and to develop management strategies that quickly meet new challenges.

Proposed research for the Roadmap

Introduction

1. Immune reconstitution and infections with special emphasis on new transplant strategies. a) studies are needed in patients undergoing haploidentical transplantation; b) studies addressing the specific needs of elderly patients must also be developed. 2. Herpesviruses. a) CMV: strategies are needed for introduction of new prophylactic, monitoring, and therapeutic strategies in patient management; b) Human herpesvirus 6: new strategies must be developed through proper controlled clinical trials. 3. Respiratory viruses - new viruses. a) Careful surveillance and rapid recognition of respiratory viruses by sensitive and specific diagnostic techniques are necessary for the development of infection control and management strategies. New antiviral drugs against respiratory syncytial virus, parainfluenza viruses, and influenza are undergoing clinical tests, and well-designed studies in the HSCT patient population are important. 4. T-cell therapy for infections. a) Approaches aiming to improve the immune reconstitution with a minimal risk of uncontrollable GvHD, such as the use of suicide gene-expressing T cells, should be encouraged; b) Multi-specific T cells with activity against several infections are interesting options, especially for prevention of viral infections; c) T cells against aspergillosis might become important, because the development of new antifungals remains limited; d) CMV-specific T cells are commercially available and need to be tested in larger trials. 5. Vaccines. The following topics need to be addressed: a) whether different vaccine schedules should be used in patients having undergone different transplant procedures needs to be examined; b) new vaccines, such as inactivated varicella-zoster vaccines, CMV vaccines, and vaccines against human papillomaviruses, should be evaluated in welldesigned studies.

Infections are the leading cause of death in patients with hematologic malignancies undergoing myelosuppressive chemotherapy. The majority of infections are caused by bacteria, but invasive fungal or viral infections also occur frequently.

Anticipated impact of the research In past decades, major improvements have been achieved in infectious disease management in allogeneic HSCT recipients. Despite these advances, infections remain important causes of non-relapse mortality. New infections can quickly become severe threats, as shown by the 2009 influenza pandemic. Therefore, the impact of the proposed projects will be in two areas: to further 186

8.2. Infections in neutropenic patients Georg Maschmeyer (Ernst von Bergmann Klinikum, Potsdam, Germany), Peter Donnelly (Radboud Universitair Medisch Centrum, Nijmegen, the Netherlands), Inge Gyssens (Universiteit Hasselt, Hasselt, Belgium), Hans Hirsch (UniversitĂ¤t Basel, Basel, Switzerland), Marie von Lilienfeld-Toal (UniversitĂ¤tsklinikum Jena, Jena, Germany).

European research contributions A variety of European scientific associations and groups have played a leading role in advancing the diagnosis, prevention, and treatment of infectious diseases in patients with hematologic malignancies. In particular, Europeans have pioneered research in the field of invasive fungal diseases. Most of the diagnostic tools (e.g. galactomannan antigen and the detection of Aspergillus nucleic acid by PCR) have been developed and validated in Europe, leading to the globally adapted definitions of invasive fungal diseases by the European Organisation for Research and Treatment of Cancerâ&#x20AC;&#x201C;Mycoses Study Group in 2008 (www.eortc.org) and the European Aspergillus PCR Initiative (www.eapcri.eu) has developed a standard for detecting Aspergillus nucleic acid. Moreover, European clinical trials of antifungal treatment and prophylaxis have resulted in an overall survival benefit. Beyond this, European groups have led the field in clinical studies on antibiotic treatment and thus paved the way for internationally recognized clinical guidelines on antimicrobial strategies in patients with hematologic malignancies.

Proposed research for the Roadmap Despite this progress, there are medical and scientific needs that remain unmet in the field of infections in neutropenic patients. The most pressing topics include: 1) the need for antimicrobial stewardship in the face of emerging multi-drug-resistant bacteria given the lack of new antimicrobial agents; 2) the challenge of community-acquired respiratory viruses; 3) improvements in control of infection; and 4) establishing the role of the microbiome. Proposed research topics: Antimicrobial stewardship in prophylaxis and therapy: the current standard of care for neutropenic patients is a predefined form of antimicrobial prophylaxis and empiric antimicrobial therapy as soon as fever occurs. This has been a lifesaving approach for many years, but several issues remain: 1) improving the initial response to antimicrobial therapy; 2) establishing the minimum duration of systemic antimicrobial therapy after defervescence, as current approaches often result in unnecessarily prolonged use of antibiotics; and 3) in the era of multidrugresistant pathogens,330 the current recommendations for haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

antimicrobial prophylaxis and treatment, even after establishing an etiology, need to be reevaluated. Thus, we propose research regarding the following topics. 1. Use of therapeutic drug monitoring to personalize dosing and improve response to prophylaxis and initial empirical therapy. 2. Prospective comparison of the duration of antibiotic treatment, including in de-escalation (step-down) strategies, with or without guidance by inflammatory markers such as C-reactive protein, procalcitonin, interleukin-6, and markers of gut function (e.g. citrulline). 3. Prospective evaluation of different antimicrobial prophylaxis regimens utilizing commonly used fluoroquinolones, as well as "older" drugs such as cotrimoxazole, with prospective monitoring of inflammatory markers and citrulline. 4. Prospective evaluation of a systematic approach based on clinical and laboratory findings to guide pre-emptive compared with empiric antimicrobial therapy including antifungal therapy. 5. Clinical trials of novel antibacterial agents with new targets and mechanisms of action in neutropenic patients. Community respiratory viruses (CRV): a series of outbreaks have brought CRVs to the attention of clinicians and scientists.331 CRVs are regarded as an uncommon cause of fever during neutropenia, because they tend to occur seasonally and are difficult to diagnose, but they have not yet been studied systematically. However, the increasing number of reports of fatal CRV infections has illustrated the need for a better understanding of CRV epidemiology. Further research is warranted, and we suggest the following topics for clinical studies. 1. Studies on epidemiology, seasonality, and clinical relevance of CRVs in neutropenic patients using modern diagnostic methods. 2. Prospective evaluation of infection control measures, especially in asymptomatic patients shedding the virus. 3. Clinical development of novel antiviral agents with activity against relevant CRVs. Efficacy of infection control measures: infection control measures are adopted routinely to reduce the risk of hospital-acquired infections. These measures include protective isolation, use of gloves and gowns, rigorous disinfection measures, reduction of inhaled potentially infective particles by air filtration and masks worn by patients leaving their rooms or wards, and low-microbial hospital diets. However, there is no evidence of benefit from these measures in terms of reducing infection-related morbidity and mortality.332 In the light of an increasing threat from multi-drug-resistant bacteria and fungi among hematologic patients, a comprehensive review is long overdue. What is needed is a series of prospective, randomized clinical studies focused on the use of the following. 1. Low-microbial hospital diet. 2. High-effiency air particle filtration. 3. Disposable gowns, gloves, and masks for caregivers and visitors. haematologica | 2016; 101(2)

4. Well-fitting masks for patients outside their treatment rooms. Microbiome: in recent years, a rapidly evolving insight into the essential role of the intestinal and skin microbiome for the quality of immune responses in normal and immunocompromised individuals has been attained. This has been shown to have a significant impact on the clinical course of allogeneic stem cell transplant recipients, but potentially also on the general interaction between host and tumor cells.333,334 There is a need to better understand the pathogenesis and background of fever and infections in patients undergoing myelosuppressive chemotherapy, for distinguishing febrile immune reactions from infections due to microbial pathogens, and for designing more specific (“targeted”) antimicrobial or anti-inflammatory treatment in this severely immunocompromised patient cohort. Clinical and translational studies are needed in neutropenic patients to explore the role of the microbiome in the following. 1. 2. 3. 4.

The intestinal tract. The oral cavity and teeth. The respiratory system. The skin.

Anticipated impact of the research These projects will result in the following. 1. A significant improvement of survival rates of neutropenic patients. 2. An improvement of the quality of life of neutropenic patients through better prophylaxis of infectious events, deletion of unnecessary procedures, shortening of antimicrobial therapy duration, and subsequent reduction of collateral damages of antibacterials. 3. Reduction of unnecessary antimicrobial prophylaxis and therapy leading to reduced pathogen and commensal resistance rates. 4. Evidence-based update of the guidelines on the rational use of infection control measures for severely immunocompromised patients. 5. A fundamental revision of the current approach to fever and infections in neutropenic patients.

8.3. Infections in non-neutropenic patients other than hematopoietic stem cell transplantation recipients Claudio Viscoli (Università degli Studi di Genova, Genova, Italy), Malgorzata Mikulska (Università degli Studi di Genova, Genova, Italy).

Introduction There is a growing population of patients with hematologic malignancies who receive chemotherapeutic agents that do not cause neutropenia but result in other types of severe immunodeficiencies, such as T-cell defects and hypogammaglobulinemia. These patients are at risk of developing potentially life-threatening infections, which are different from those commonly seen during neutropenia, due to differences in the underlying immunodeficiency. There is little reliable and systematic information on infections in non-neutropenic hematologic patients, even though they significantly outnumber those with neutropenia.335 Studies on infectious complications in patients with 187

A. Engert et al. hematologic malignancies are paramount for determining the efficacy of chemotherapeutic agents. Indeed, even a drug with a 100% curative effect of the underlying hematologic disease cannot be used if it results in a 90% rate of severe infections, which cannot be prevented or treated. Thus, the lack of advances in infectious diseases (e.g. the shortage of efficacious antibiotics against multidrug-resistant gram-negative bacteria) may result in unacceptably high mortality, hampering advances in chemotherapy.336 Most of the infections developing in subjects undergoing different chemotherapeutic treatments are not unexpected, and they could be predicted and prevented if timely studies on the infectious complications were to be carried out. The monoclonal antibody eculizumab is a good example; it predisposes patients to meningococcal infection, and this could be foreseen based purely on the knowledge of its mechanism of action. Eculizumab mimics the C5 complement deficit, which can occur as an inherited immunodeficiency, and it is associated with repeated or relapsing meningococcal infections. Secondly, if registration trials were properly designed, the true rate of specific infections could have been established before the drug was marketed and proper preventive measures could be designed. Another example is alemtuzumab: here, no regular monitoring of cytomegalovirus (CMV) reactivation was performed in the first efficacy studies. Dedicated studies and appropriate recommendations on CMV management in this setting could only be made several years later. Last but not least, post-marketing monitoring of infectious complications would benefit from a standardized and dedicated approach so that real-life incidence of infectious complications can be properly assessed. Preventing infectious complications has always been the most appealing approach, yet the benefit of prophylaxis must be carefully weighed against its short- and long-term side effects, such as drug toxicity and a change in epidemiology or resistance patterns. The latter is of particular importance in our era of multidrug-resistant bacteria, yet it has rarely been evaluated in trials aimed at short-term benefits.337 Antibiotics are the only drugs in which improper use does not reflect on the patient being treated, but rather on other patients and generations of future patients. In addition, there is a global understanding that hospital stay is associated with several risks and substantial costs, such as colonization and infection with nosocomial pathogens, including resistant bacteria or Clostridium difficile. Along with a reduced risk of health careâ&#x20AC;&#x201C;associated infections, the undeniable benefits of outpatient therapy are better quality of life (QoL) for patients and reduced costs.338 Although some neutropenic patients are also cared for in their home environment, outpatient management is particularly suitable for non-neutropenic populations. In order to perform secure treatment in outpatient settings, all management procedures, such as diagnostic methods, infection control measures, and treatment, should be adapted to the outpatient condition. Therefore, rapid diagnosis, possibly based on a point-of-care approach, should be developed; oral or once-daily intravenous therapy should be preferred.

tious disease specialists, hematologists, microbiologists, and infection control specialists, are needed in the field of infectious complications in hematology patients, and several studies have been successfully carried out in Europe for more than 40 years.339 Scientific organizations and societies, such as the Infectious Diseases Group of the European Organisation for Research and Treatment of Cancer, the European Conference on Infections in Leukemia, the European Group for Blood and Marrow Transplantation (EBMT), the Immunocompromised Host Society, the European Conference on Infections in Leukemia and the European Society of Clinical Microbiology and Infectious Diseases, have significantly contributed with studies and guidelines on the management of infectious diseases in patients with hematologic malignancies.

Proposed research for the Roadmap Several initiatives at different levels, ranging from basic science to large epidemiological studies, should be undertaken to comprehensively address the problem of infections in non-neutropenic populations. Major areas of interest requiring research resources and efforts include the following. 1. Background and basic science studies aiming at understanding the intimate mechanism of action and the potential proinfectious effect of novel molecules or biological agents, with certain infectious problems possibly anticipated even before clinical trials. 2. Inclusion in clinical trials of new agents of an ad hoc section for the detection of infectious complications, designed and reviewed by experts in the field of infectious diseases. 3. Setting up pre- and post-marketing registers of infectious complications in patients receiving chemotherapy, in order to establish long-term safety of new products and to react immediately when unexpected problems arise, which would also allow full assessment of the risk profile of novel therapies. 4. Studies on the need for prophylaxis of bacterial, viral (including hepatitis B), and fungal (including Pneumocystosis pneumonia) infections, its efficacy, costeffectiveness, and short- and long-term benefits and risks. 5. Studies on the role of vaccination for preventable diseases (e.g. influenza and pneumococcus), and implementation of new efforts for new approaches. 6. Tuned-up revaccination strategies. 7. Studies in the non-neutropenic population on the performance of diagnostic assays based on antigen or DNA detection for bacterial and fungal diseases, and further development of immunological diagnosis by means of monitoring cellular immune responses to specific pathogens (e.g. ELISPOT). 8. Novel rapid diagnostic methods, to be used especially in the outpatient setting (e.g, point-of-care tests and molecular diagnostics). 9. Studies on effective and convenient outpatient therapy of infections, including oral antibiotics and antibiotics with prolonged half-lives.

Anticipated impact of the research European research contributions Multidisciplinary efforts, including efforts from infec188

Implementing the studies and initiatives described above, we should be able to obtain a more complete and haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

thorough approach to infectious complications in nonneutropenic patients. Thus, mortality and morbidity can be reduced, providing full benefit of novel therapies by minimizing the drawback of unexpected or uncontrolled infections. The individualization of the management of infectious risk and the development of outpatient infection control protocols and tools for diagnosis and treatment of infections should result in better quality of life and reduced cost.

8.4. Infections in primary immune deficiencies Andrew Cant (Great North Children’s Hospital, Newcastle upon Tyne, United Kingdom), Marieke Emonts (Great North Children’s Hospital, Newcastle upon Tyne, United Kingdom), Charlene Rodrigues (Great North Children’s Hospital, Newcastle upon Tyne, United Kingdom).

Introduction Although individually rare disorders, there are now more than 200 genetic primary immunodeficiencies (PIDs) described, with 1:2500 people having one PID or another. The lifetime burden of infection in this group is high and infections can be unusual and difficult to treat. The risk is increased by immunosuppression associated with hematopoietic stem cell transplantation (HSCT). Early diagnostics and effective treatment for bacterial, viral, and fungal infection is important, as is surveillance within the bone marrow transplantation (BMT) unit setting. The research is relevant for various patient populations. 1. Severe combined immunodeficiencies, including conditions such as X-linked severe combined immunodeficiency, adenosine deaminase deficiency, and Omenn syndrome. 2. Combined immunodeficiency syndromes, including conditions such as Wiskott-Aldrich syndrome, T-cell activation defects, MHC class II deficiency, and CD40 ligand deficiency. 3. Antibody deficiencies, including X-linked agammaglobulinemia, common variable immunodeficiency, and selective IgA/specific antibody deficiencies. 4. Diseases of immune dysregulation, including hemophagocytic lymphohistiocytosis and X-linked lymphoproliferative syndrome. 5. Congenital defects of phagocyte number, function, or both, including disorders such as chronic granulomatous disorder. 6. Defects in innate immunity, including conditions such as NEMO deficiency and complement deficiencies.

Proposed research for the Roadmap Viral infections: improved rapid methods for detection of viruses are now in clinical use. PCR methodology allows an accurate identification and quantification of viral load, as well as better planning of treatment strategies and monitoring of therapeutic effects; however, some caveats remain. 1. There is a need for better treatments; current approaches are associated with significant toxicities for the patient (e.g. foscarnet and cidofovir) and potentially also for the staff (e.g. nebulized ribavirin). The prolonged treatment required confers a risk of emerging resistance. Little is known about efficacy and toxicity of combination therapy in these complex cases. Particular viral treatment challenges include Epstein-Barr virus (EBV), adenovirus, and cytomegalovirus (CMV), where cellular (virus-specific cytotoxic lymphocytes) and antibody (monoclonal antibody, e.g. respiratory syncytial virus–specific palivizumab) therapies seem to be of limited effectiveness and not always easy to arrange and administer. 2. Improved prevention and treatment of respiratory infections are needed, especially for rhinovirus, respiratory syncytial virus, parainfluenza, influenza, and adenovirus.340 3. Human papillomaviruses cause severe massive skin warts in specific immunodeficient patients, and better therapeutic options are needed. 4. New live attenuated vaccines, such as the rotavirus vaccine, given prior to PID diagnosis, confer new challenges. Ideally, PIDs should be diagnosed prior to first vaccinations. Central line-associated bloodstream infections: this remains a significant issue in oncology and chronic diseases. The morbidity and mortality of central line–associated bloodstream infections mean that all children with fevers and central lines undergo blood culturing and empirical broad-spectrum antibiotic treatment, affecting antimicrobial resistance in a high-risk population.341 1. There remains a need for more timely diagnostics and better prevention of central line-associated bloodstream infections, including adjuncts or alternatives to antibiotic use (e.g. biofilm inhibitors). 2. A better antimicrobial stewardship is required to preserve antibiotics for now and the future in an era of increased antimicrobial resistance. Gastrointestinal infections: PID patients are affected by many bacterial and viral gut pathogens with an ongoing impact on nutrition and fluid management, a significant cause of morbidity and mortality.

European research contributions More than 200 PIDs have now been genetically defined and infectious complications and prognosis are more predictable. Viral PCR tests enable much more rapid accurate diagnosis. Furthermore, new antiviral and antifungal drugs improve outcome. Clearer guidelines for the management of febrile neutropenia and research networks for studying infections in the immunocompromised have been developed through learned societies such as the European Society for Paediatric Infectious Diseases and the European Society of Clinical Microbiology and Infectious Diseases. haematologica | 2016; 101(2)

1. Improved diagnostics and treatments for Cryptosporidium spp. are required.342 2. Improved treatment modalities for enteroviruses in the context of X-linked agammaglobulinemia, where paralysis and death can result from ongoing infection, are required. 3. A better understanding of the human gut microbiome and its impact on the immune system is needed. 4. Improved diagnostics to distinguish gut graft-versushost disease (GvHD) from infectious enteritis requiring distinct management are needed. 189

A. Engert et al. Nosocomial infections: PID patients who require prolonged or multiple hospital admissions are at a very high risk of nosocomial infections. These potentially increase the length of hospital stay, having not only clinical but also economic implications on health services. 1. Better estimation of the prevalence of nosocomial infections (e.g. norovirus and sapovirus) is needed. The morbidity and mortality associated with these infections require effective preventative strategies and new therapeutic interventions. 2. For those patients requiring ventilation, ventilatorassociated pneumonia is an important cause of morbidity. Better anti-infective and anti-inflammatory treatments are needed here. Infections associated with biologics: biological agents, particularly monoclonal antibodies to proinflammatory cytokines, are being widely used, increasing the risk of infections in a high-risk population:343 1. More information regarding specific infectious risks of this family of agents would be useful to allow appropriate prophylaxis to be instituted. 2. More information regarding how this family of agents affects immune response to vaccination is warranted. While aiming to prevent vaccine strain infections, whenever possible, appropriate protection with vaccination is needed. Timing of vaccination is likely to be important and should be considered prior to commencing immune-modulating therapy. Fungal diagnostic markers and treatment monitoring: morbidity and mortality due to invasive fungal infections are increasing in PID patients, especially those who are on immunosuppression.344 Candida spp. and Aspergillus spp. diagnostics are currently limited to culture and histopathology, with serum markers such as galactomannan showing limited reliability. 1. Surveillance throughout HSCT could be improved with better rapid diagnostic tests to allow more timely management and reduced mortality. 2. Call for standardized diagnostic tests to be established to allow universal diagnostic criteria to be set and commercial assays or protocols to set up services. 3. Large interindividual variabilities of antifungal drug levels are observed in children. Pharmacokinetic/pharmacodynamic data are limited in this group and are warranted for prompt informed adjustment of dosing, allowing optimal treatment with these potentially lethal infections. 4. Optimal duration of antifungal treatment is ill defined, as is subsequent secondary prophylaxis. Surveillance of infections post HSCT: PID patients preparing for HSCT are screened for blood, respiratory, and gut pathogens. Although reasonably controlled pre-HSCT, these pathogens can cause significant problems as the child progresses through HSCT with the conditioning and immunosuppressive regimens. A further understanding of control mechanisms and preventative strategies are needed for CMV, EBV, adenovirus, and cryptosporidium in the PID patient leading up to HSCT.

190

Anticipated impact of the research The impact of these proposed research areas will have a huge benefit on the management of PID patients. Their vulnerability to both common and opportunistic pathogens leaves them at a high risk even before they go through the HSCT process. We have a limited repertoire of prophylactic antimicrobials, immunoglobulins, and treatment modalities for these patients. Even those who are appropriate for and successfully transplanted are highly susceptible at various points. Therefore, in order to minimize the impact of their underlying diseases and HSCT, improved diagnostics, therapeutics, and monitoring are desperately needed.

8.5. Genetic predisposition factors for infection in hematology and hematopoietic stem cell transplantation patients Pierre-Yves Bochud (Centre Hospitalier Universitaire Vaudois/UniversitĂŠ de Lausanne, Lausanne, Switzerland), Agnieszka WĂłjtowicz (Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland).

Introduction Infectious complications represent a major challenge in patients undergoing intensive chemotherapy for the treatment of hemato-oncological diseases and/or hematopoietic stem cell transplantation (HSCT). The most common pathogens are gram-positive and gram-negative bacteria (usually causing bloodstream infections during the course of neutropenia); fungi such as Candida or Aspergillus species (causing invasive infections during the course of neutropenia or after HSCT); and viruses such as cytomegalovirus (CMV) (that can be reactivated and/or cause disease after HSCT). Although specific risk factors, such as patient age, comorbid conditions, and type and duration of immunosuppression, have been identified, it is still difficult to accurately predict which patient will develop which infection, and at which time. Because most infections are severe, prophylactic and empirical drugs are increasingly administered, exposing patients to potentially unnecessary side effects and the development of resistance.345 It is becoming clear that an individualized risk assessment may dramatically improve the appropriateness of antimicrobial use in such immunocompromised patients. Over the past 15 years, a number of studies have evaluated whether genetic factors influence susceptibility to infections in hematologic patients. These studies have been made possible through several concomitant events, including the availability of the full human genome in the early 2000s; the development of new, rapid, and inexpensive genotyping techniques; and major discoveries in the field of innate immunity. At the molecular level, the detection of pathogens is mediated by pattern recognition receptors located on the surface or within immune cells. Pattern recognition receptors detect specific molecular patterns from microorganisms and mediate immune response and cytokine production. Therefore, polymorphisms in gene-encoding cytokines and pattern recognition receptors, such as TLRs and C-type lectin receptors (e.g. CLEC7A), were extensively studied.

haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

However, such studies were often limited by several factors, including problematic design, lack of precise definitions for infections, inappropriate control selection, small sample size, increasing use of prophylactic regimen, failure to account for important covariates, and/or lack of correction for multiple testing.345 Another important limitation has been the lack of a definite role for some of the reported genes and/or their polymorphism(s) in the immune response mechanisms against the offending pathogen. So far, existing data have not supported the use of such polymorphisms for individual risk stratification in clinical practice. However, recent associations have been reported that are more promising, because they have been replicated in different studies and/or different populations at risk and/or are supported by strong functional evidence.

ic activities and reduced Aspergillus clearance. Altogether, these features currently make PTX3 rs3816527 the most promising marker for invasive fungal infections in hemato-oncological patients.345

Proposed research for the Roadmap Implement prospective cohorts of hemato-oncological patients: efficient exploration of host genetics in susceptibility to infections among hemato-oncological patients needs data from a much larger number of patients than most studies have been able to collect so far. This could be obtained by implementing a large multicenter cohort with efficient structures for prospective data collection (e.g. electronic case report forms, use of standard definitions for relevant phenotypes, and systematic quality checking) and biobanking (e.g. centralized storage and processing and genotyping) in accordance with existing regulations.

European research contributions Most genetic association studies in hemato-oncological patients focused on susceptibility to invasive fungal infections (n=30). Only a limited number of studies analyzed the risk of developing bacterial (n=8) and viral infections (n=4). Among studies reporting associations supported by replication and/or convincing functional evidence, all were performed on susceptibility to invasive fungal infections in relatively large cohorts of Caucasian patients undergoing HSCT,346-348 mostly in European centers.347,348 In a study of 702 HSCT recipients (336 included in a discovery and 366 in a replication cohort), rs4986790 and rs4986791 single nucleotide polymorphisms in TLR4 were associated with an increased risk for the development of invasive aspergillosis. The relevant polymorphisms were found in the donor, not the recipient, a specific feature that was observed in subsequent studies of HSCT patients. Although TLR4 is a detector for O-linked mannan, an important component of the fungal cell wall, the exact mechanism by which the polymorphisms influenced fungal pathogenesis has remained controversial. In another study of 205 HSCT patients, a stop codon polymorphism in CLEC7A (Y238X, found either in the donor and/or the recipient) was associated with susceptibility to invasive aspergillosis.347 CLEC7A is the detector of b-glucan, a key component of the fungal cell wall. The Y238X polymorphism leads to CLEC7A deficiency and was also associated with different forms of Candida infections.349 Although promising, associations with relatively infrequent polymorphisms, such as those in TLR4 (minor allele frequency ~0.05) and CLEC7A (minor allele frequency ~0.08), may be difficult to replicate and implement as risk predictors in clinical practice. A more frequent polymorphism in donor pentraxin 3 (PTX3, rs3816527, minor allele frequency ~0.43) was associated with susceptibility to invasive aspergillosis in a study of 268 HSCT recipients. The investigators replicated the association in an independent cohort of 330 HSCT patients. Furthermore, a similar association was observed in a cohort of 1101 solid organ transplant recipients.350 PTX3 is a pattern recognition receptor that can directly bind to Aspergillus conidia, thereby acting as an opsonizing factor for complement activation and phagocytosis, or can interact with CLEC7A or TLR4 to enhance immune recognition. PTX3 variants were associated with reduced PTX3 production in neutrophils with defective phagocythaematologica | 2016; 101(2)

Improving the quality of genetic association studies: there is an urgent need to improve the quality and reliability of genetic association studies. Future proposals need to follow stringent criteria for cases and control enrollment (e.g. standard definition of cases, controls exposed to the same risk as cases, and period of observation and/or use of time-dependent analyses), study design (e.g. complete publication of polymorphisms tested, rationale for gene/polymorphism selection, and replication for relevant associations), genotyping (e.g. standardization and quality controls), and statistics (e.g. power calculation, multiple testing correction, and multivariate analyses accounting for all factors influencing the phenotype).

Anticipated impact of the research Well-conducted studies based on large, multicenter cohorts are needed to further establish the role of host genetics on susceptibility to infections in high-risk hemato-oncological patients. Such factors may contribute to improved infectious risk stratification and individualized prevention strategies.

The EHA Roadmap for European Hematology Research Section 9. Hematopoietic stem cell transplantation and other cell-based therapies

Section editor: Willem Fibbe. More than 35,000 hematopoietic stem cell transplantations (HSCTs) were performed in Europe in 2011. Of these, 60% were autologous and 40% were allogeneic transplants. It is worthy of note that allogeneic HSCT remains the single potentially curative option for many patients with hematologic malignancies, despite recent progress with chemotherapeutic treatment modalities. Allogeneic stem cell transplantation largely relies on the principle that the immune system has the power to cure hematologic tumors, provided that tolerance is achieved for the immune cells that mediate these activities. With the development of the reduced intensity conditioning regimens aiming primarily at tolerance induction rather than at eradicating disease, allogeneic stem cell transplantation can now be applied in elderly patients and in patients with significant comorbidities. Issues that remain to be solved, and that require a better understanding, 191

A. Engert et al. include graft-versus-host disease (GvHD), promotion of immune reconstitution, and mechanisms underlying the graft-versus-tumor effect. In particular, following reduced intensity conditioning, the transplant procedure itself may not be sufficient to eradicate disease. Therefore, additional steps are required that include post-transplant immunotherapy, such as donor lymphocyte infusions or other forms of cellular immunotherapy. In this regard, significant scientific progress has been made in the past few years with the development of novel approaches in this setting. These include the targeting of extracellular antigens through chimeric antigen receptor (CAR) T cells, Tcell therapy with gene-modified T-cell receptors, and strategies aiming at amplification of in vivo immune responses. Through these “checkpoint-blocking agents”, potentially curative immune responses become apparent by blocking inhibitory signals to immune cells. The identification of antigens underlying these responses will be of crucial importance to further develop disease- or patient-specific forms of immunotherapy. The disadvantage of checkpoint blockers via inducing autoimmunity underscores the need for further research into this promising new approach. Expansion of hematopoietic stem and progenitor cells may aim at solving the problem of hematopoietic and immune reconstitution that is observed particularly following cord blood transplantation. A further understanding of the hematopoietic niche in regulating HSC expansion and differentiation is required to develop novel expansion technologies. This will also be important for the further development of genetically repaired hematopoietic stem cells (HSCs) through the use of artificial nucleases. Through these novel technologies, it may be possible to edit the genome of patients with congenital hematologic abnormalities, thereby bypassing the need for allogeneic stem cell transplantation (ASCT). A requirement to make this technology successful will be amplifying the corrected HSCs to the numbers that are required for functional engraftment. This will enable a novel form of ASCT. These new therapeutic strategies require substantial manipulation of cells, including expansion, differentiation, and gene transfer. Cells that have undergone this more than “minimal manipulation” are regarded as advanced therapy medicinal products (ATMPs) and are regulated as drugs. As such, approval is required of a national regulatory and ethical committee before clinical studies can be undertaken. In addition, ATMPs have to be produced under good manufacturing practice conditions and require an Investigational Medicinal Product Dossier and an Investigator’s Brochure. There is a clear need to make the regulatory procedure less complicated in order to allow an easier and more rapid development of these novel therapeutics.

9.1. Allogeneic stem cell transplantation Andrea Bacigalupo (Ospedale San Martino, Genova, Italy), Gerard Socie (Hôpital Saint Louis, Paris, France).

Introduction Prevention and control of graft-versus-host disease (GvHD): 192

during the past decade, our understanding of the pathophysiology of acute GvHD has greatly improved. Much has been learned from pre-clinical models and less from correlations with clinical observations or therapeutic interventions. Little progress has been made since the mid 1980s, and GvHD still develops in approximately 40%60% of recipients. Thus, there is a deep need to develop newer approaches to mitigate and effectively treat GvHD, which may facilitate the wider use of allogeneic hematopoietic stem cell transplantation (HSCT). Promoting immune reconstitution without increasing GvHD is still the main challenge, especially after umbilical cord blood or T-cell depleted haploidentical HSCT, which are associated with prolonged immunodeficiency. While a slow T-cell reconstitution is regarded as primarily responsible for deleterious infections, GvHD, and relapse, the importance of innate immune cells for disease and infection control is currently being re-evaluated. Relapse following allogeneic HSCT has remained unchanged throughout the past three decades, as recently reviewed in the second workshop convened by the National Institutes of Health.351

European research contributions Clinical trials mainly conducted in the European Union (EU) demonstrated that intensifying GvHD prophylaxis by T-cell depletion with antithymocyte globulin prevents GvHD. Up to 60% of patients who develop GvHD will have inadequate responses to corticosteroids, however, portending a dismal prognosis. Trials conducted have demonstrated that high doses are not more efficient than standard dose steroids, and that adding any new drug to steroids improves outcome.

Proposed research for the Roadmap Considering the increased utilization of hematopoietic cell transplantation, the morbidity and mortality associated with GvHD, and the limitations inherent to contemporary therapies, novel approaches are urgently needed. Two main priorities have been identified. 1. Developing translational research at the European level on human GvHD to study pathophysiology and develop biomarkers to assess disease severity. 2. Favoring the development of clinical trials with ancillary studies based on strong biological background, leading to rapid development to phase III trials through “pick-the-winner” phase II trials. A variety of approaches have been explored pre-clinically and clinically, including cytokines, keratinocyte growth factor, growth hormone, cytotoxic lymphocytes, and mesenchymal stromal cells (MSC) or blockade of sex hormones. Dissecting GvHD from graft-versus-leukemia has been the aim of several decades of unsuccessful pre-clinical and clinical studies. A recent study352 suggests that the combined use of regulatory and conventional T cells prior to selected CD34+ cell transplants may result in a surprisingly low rate of relapse, without increasing GvHD. In other words, we now have several studies suggesting that leukemia relapse can be diminished either by pre-transplant, peri-transplant, or post-transplant interventions. We also have new cellular tools. haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

Prospective trials with a control arm are badly needed to prove this is the case. Strategies to prevent or treatment post-HSCT relapse351 include the following. Prophylaxis pre-transplant: several agents can be given before HSCT in the attempt of reducing relapse; preHSCT azacytidine, other chemotherapy, and bispecific antibodies have been used in phase II trials. In particular, one study has reported interesting results in refractory or relapsed patients with acute myeloid leukemia (AML): a short course chemotherapy, given prior to the conditioning regimen, produced survival in excess of 50%, but only if patients had received one to two courses of induction therapy.353 If patients had received more than two courses of chemotherapy, results were very poor.353 Therefore, AML patients with primary induction failure should proceed to transplant immediately after the second course of failed chemotherapy, rather than attempting sequential intensive additional chemotherapy. The role of pre-transplant azacytidine in patients with refractory anemia with excess blasts is controversial; unfortunately there has been no prospective randomized trial, and a large retrospective study by the French transplant group has failed to show any advantage of pretransplant azacytidine on post-transplant relapse or survival.354 Prospective randomized trials are necessary to assess whether a short course of chemotherapy the week prior to conditioning in AML or azacytidine in refractory anemia with excess blast patients will reduce the risk of post-transplant relapse. Prophylaxis post transplant: prophylactic tyrosine kinase inhibitors (TKIs) have been tested prospectively post HSCT against controls and have been shown to reduce the risk of relapse in patients with Ph+ acute lymphoblastic leukemia (ALL). Recent data would suggest that prophylactic post-transplant panobinostat or azacytidine may be beneficial in patients with AML; these data warrant confirmation through controlled trials. Preemptive treatment of molecular relapse: markers for minimal residual disease (MRD) are required to implement pre-emptive treatment: in acute myeloblastic leukemia, treatment driven by WT1-based MRD, with donor lymphocyte infusions, has limited success. The use of bispecific antibodies and cellular therapy with CAR T cells355 may produce significantly better results and should be tested prospectively. Treatment of hematologic relapse: once hematologic relapse has occurred, outcome depends on the pace of the underlying disease; in chronic disorders, long-term survival is still possible.351 In patients with acute leukemia, the major predictor of outcome is the interval between the first transplant and relapse:351 patients relapsing within six months are at a very high risk of early death, whereas delayed relapses can be successfully treated in a proportion of cases with a second transplant or chemotherapy and donor lymphocyte infusions.351 The use of bispecific antibodies and CAR T cells may offer better results,355 and trials are under way to assess optimal protocols. haematologica | 2016; 101(2)

Anticipated impact of the research HSCT is increasingly performed in Europe (15,000 per year). Recent progress now allows transplantation in older patients (over 60 years of age). Prediction and improved treatment of early immunological complications remain an unmet medical need. Progresses in managing infectious complications have contributed to improved survival rates during the past ten years. Further improvement should incorporate recent knowledge about innate lymphoid cells and immune response to cancer to improve long-term outcome.

9.2. Immune-based therapies for hematologic malignancies Hermann Einsele (UniversitĂ¤tsklinikum WĂźrzburg, WĂźrzburg, Germany), Marcel van den Brink (Memorial Sloan Kettering Cancer Centre, New York, United States of America), Fred Falkenburg (Leids Universitair Medisch Centrum, Leiden, the Netherlands).

Introduction Targeted therapy in patients with hematologic malignancies aims at generating new agents, such as monoclonal antibodies, that are less toxic than conventional chemo- or radiotherapy. Monoclonal antibodies are effective in a number of hematologic malignancies. Most of the currently identified targets for monoclonal antibodies are also expressed on non-malignant cells. The ability to genetically engineer structure and function of these antibodies has significantly improved their effectiveness. T lymphocytes recognize antigens through a unique antigen-specific T-cell receptor (TCR), promoting the elimination of a given target and amplifying the attack through recruitment of other components of the immune response. T cells can target antigens derived from both intracellular and extracellular proteins, including peptides encoded by mutated genes. T lymphocytes can actively distribute themselves within tissues and in the tumor environment and have the potential for in vivo expansion and self-maintenance, as they can establish a memory compartment. Donor lymphocyte infusion is used to treat relapsed or residual tumor cells following allogeneic stem cell transplantation. In addition, adoptive transfer of antigen-specific cytotoxic T lymphocytes and CD4+ T-helper cells has been used to treat viral or fungal infections. Immunotherapy based on a personalized dendritic cell cancer vaccine represents an innovative approach for hematologic malignancies. In some cancer entities, dendritic cell/peptide vaccines have demonstrated clinical benefit in phase III trials. A clear shortcoming of cancer vaccines is the difficult standardization of the antigenic material. Therefore, most investigators favor mixing relevant immunogenic peptides to be used directly as peptide vaccines or loaded onto dendritic cells as professional antigen-presenting cells. Checkpoint blockade is a form of releasing the brakes on tumor-specific T cells, allowing them to persist and expand to attack malignant cells. Cancers can develop in part as a consequence of cancer-induced immunosuppression. In many individuals, immunosuppression is mediated by CTLA4 and PD-1, two immunomodulatory receptors 193

A. Engert et al. expressed on T cells. Monoclonal antibody–based therapies targeting CTLA4 or PD-1 have shown significant clinical effects in patients with hematologic malignancies, such as Hodgkin lymphoma (HL). Recent advances have allowed genetic modifications of T cells to provide robust, personalized lymphocytes that target specific tumor-associated antigens. Gene transfer in human T lymphocytes can be accomplished by several means. Here, long-term culture is required, significantly compromising their capacity to survive long term in vivo. Gene delivery can also be achieved through retroviral vectors. These vectors can be manufactured on a large-scale producing stable integration into the genome of the T cell and its progeny. Adverse consequences due to insertional mutagenesis in T cells have not yet been reported. Lentiviral vectors have also been used to engineer T cells. These vectors are particularly attractive when less differentiated T-lymphocyte subsets are targeted, as they have the unique ability to infect T cells even upon minimal activation, a property lacking in retroviral vectors. Novel nonviral systems (Sleeping Beauty and PiggyBac) allow larger fragments of DNA to be inserted than viral vectors permit. T cells redirected to specific surface antigens on malignant cells by engineered CARs are also emerging as powerful therapies for hematologic malignancies. More than 10 clinical trials using CAR T cells for treatment of hematologic malignancies have been reported. Dramatic responses were observed, especially in patients with B-lineage ALL, even in high-risk patients. Similar to T-cell engaging antibodies, clinical use of CAR T cells especially in patients with advanced disease was associated with significant but reversible toxicity.

European research contributions New bispecific antibodies also have the properties of selective antigen specificity and T-cell activation. Very high response rates and long-lasting remissions, especially after the application of the CD3/CD19–directed bispecific antibody blinatumomab, indicate that in some patients either the tumor is eradicated during T-cell activation and expansion or, more likely, a memory T-cell response directed against the tumor cells was induced or expanded. The development of bispecific antibodies for hematologic malignancies was mainly done in Europe.356,357 Also new targets for bispecific antibodies will be tested in pilot trials in Europe, including multiple myeloma and AML. Technologies of gene modification of T cells have been intensively developed in Europe.358 However, Europe is lacking behind the large number of clinical trials initiated in the US. Currently, European patients are queuing up to receive treatment with CAR T cells for hematologic malignancies in US centers due to the lack of clinical trials in Europe. European biotech companies are also moving to the US for a much more rapid transfer of CAR T approaches into the clinic. T-cell therapy has been developed for clinical use in Europe, as have novel strategies of cell selection.359,360 TCR gene transfer has been developed by groups in Europe, and pilot trials are ongoing.

Proposed research for the Roadmap The following questions and research priorities have been identified. 194

1. Which antigens expressed by both malignant and nonmalignant cells can be safely targeted? 2. Do all tumor cells express similar tumor-specific antigen(s)? 3. Can the tumor be eradicated if only some tumor cells are susceptible to immune-mediated targeting? 4. Are there tumor stem/initiating cell-specific antigens? 5. Can stroma-specific antigens be used to target the tumor? 6. How are long-lasting remissions following treatment with bispecific antibodies induced? 7. How can resistance be overcome, especially target loss variants? 8. How can the neurotoxicity of the constructs be explained? 9. How can the significant cytokine release syndrome following treatment with bispecific antibodies be reduced, especially in patients with more advanced disease? 10. How can specificity by combinatorial approaches targeting several tumor antigens on the tumor cell be increased? 11. Are trispecific antibodies a better strategy? Other topics include strategies to: reduce the side effects of CAR T-cell therapy; improve long-term persistence of CAR T cells; improve efficacy of CAR T cells by combination with other T-cell activation modifiers; overcome resistance to CAR T cells, especially target antigen loss on tumor cells; and develop combinatorial approaches to improve. Efficacy and tolerability of CAR T-cell approaches tackling tumors other than the CD19+ B-cell malignancies will have to be shown. In addition, the best and most specific T-cell epitopes and most suitable T-cell subsets for adoptive T-cell therapy must be identified, and strategies to improve in vivo expansion must be developed. We also need to maximize the surface expression of the introduced TCR, optimize the translation of the introduced TCR α and b chain, identify high avidity T cells, and increase TCR affinity to promote in vivo persistence. To optimize induction of tumor-specific T cells and response to vaccination, we need better adjuvants to improve the mode of antigen delivery. However, little is known about the identity of tumor antigens that represent the targets of activated T cells. Currently, genomic and bioinformatic approaches are applied to identify tumorspecific mutant proteins following anti-PD-1 and/or antiCTLA4 therapy. In addition, efficacy of checkpoint blockade is dependent on a sufficiently broad T-cell repertoire. Therefore, strategies to enhance T-cell repertoire could enhance strategies involving checkpoint blockade.

9.3. The hematopoietic stem cell niche: regulation of hematopoietic stem cell function and significance for hematopoietic stem cell expansion Cristina Lo Celso (Imperial College London, London, United Kingdom), Simón Méndez-Ferrer (University of Cambridge, Cambridge, United Kingdom), Michael Milsom (Deutsche Krebsforschungszentrum Im Neuenheimer Feld, Heidelberg, Germany), Timm Schroeder (ETH Zurich, Basel, Switzerland).

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Introduction Europe has a long-lasting track record of research focusing on the hematopoietic stem cell (HSC) niche. In fact, in 1978, Manchester-based Ray Schofield was the first to postulate that HSC function critically depends on their occupancy of specific niches located within the bone marrow. Since then, numerous cell types comprising the HSC bone marrow niche have been identified, and some of the molecular signals that they transmit to HSCs have been discovered. These signals trigger the cell-intrinsic molecular mechanisms known to regulate HSC fate. Here we provide a short summary of these studies, and discuss the new challenges and questions faced by the HSC niche field. A better understanding of the HSC niche is critical to develop both novel therapies for and preventative strategies against hematologic disease. Furthermore, delineating the molecular pathways by which the niche regulates HSC fate decision may contribute to solving the unmet clinical need for HSC ex vivo expansion and enhanced in vivo reconstitution activity for regenerative therapies.

European research contributions Both functional studies and direct observation of HSCs in vivo and ex vivo have been critical in developing our knowledge of the HSC niche and have highlighted how several stromal and hematopoietic cells must co-operate to ensure that the necessary number of hematopoietic cells are produced every day. European researchers have both contributed to and led these studies since their earliest days. Osteoblastic cells were the first to be identified as a component of the HSC niche and subsequently several perivascular mesenchymal cells, including nestin+ progenitor cells, PDGFR/NG2/LepR-expressing cells, Schwann cells, neuronal terminations, adipocytes, and, within the hematopoietic lineages, macrophages, regulatory T cells, and megakaryocytes, have all been shown to produce molecular signals directing HSC quiescence and proliferation, self-renewal, and differentiation. These signals act through direct cell-cell interactions, such as NotchDelta and integrin signaling; short-range gradients, such as Wnt, CXCL12, Ang1, and SCF; and long-range signals, such as cytokines and hormones. More recently, the chemical and physical composition of the bone marrow microenvironment has begun to be studied, including factors such as oxygen levels and matrix stiffness.

Proposed research for the Roadmap Given the complexity of the bone marrow microenvironment, the abundance of most niche components, and the rarity of HSCs themselves, a central question within the field is whether unique and rare combinations of niche cells are required to ensure correct stem cell function or whether the bone marrow space is, in fact, more fluid than postulated and stem cells are free to migrate between multiple niches to undergo different fates. To this end, intravital microscopy has shown that infection-exposed, highly engrafting HSCs are migratory and interact with larger niches than stem cells at steady state.45 Although there is a clear appreciation that bone marrow physiology alters with age, most dramatically seen in the age-associated accumulation of adipocytes, we are only just beginning to understand how this affects the HSC-supporting function of the niche.361,362 Clearly this is a phenomenon that is likely to contribute haematologica | 2016; 101(2)

to the decreased efficacy of bone marrow transplantation (BMT) as a therapeutic modality in elderly individuals and is, therefore, an area that requires further attention. Interestingly, not only stem but also progenitor cell function is influenced by their microenvironment, and progenitor niches need to receive further attention as their manipulation is likely to lead to better short- and longterm recovery of BMT patients. Moreover, the bone marrow microenvironment is deeply affected by the development of hematologic malignancies,47 and it is, therefore, critical to understand the impact of both leukemia and chemotherapy on HSC niches, and how to restore the bone marrow microenvironment to improve outcome for therapeutic BMT. In addition, to prevent the development of hematologic malignancies, it is critical to study and understand their initial steps, when the niche conditions that support expansion of specific, often mutant, clones of HSCs are established. A better understanding of the relationship between hematopoietic stem and progenitor cells and the bone marrow microenvironment is needed not only to improve current BMT-based therapies, but also to enable us to culture or even expand HSCs for therapeutic purposes. Currently, as few as 5% of banked cord blood samples contain a sufficient amount of stem cells to be deemed usable for transplantation. Achieving efficient in vitro expansion of HSCs has long been a Holy Grail of the experimental hematology community, as this would both increase the size of the stem cell pool transplanted into each patient and, therefore, ensure more rapid recovery, while dramatically expanding the pool of usable cord bloods, increasing by orders of magnitude the number of successful haplotype matches. In order to achieve this aim, and potentially revolutionize HSCT by opening up novel sources of donor cells, significant further research needs to be performed in order to ascertain how HSC-supportive signals provided by the niche can be provided artificially in vitro. Cytokines have so far been the main factors used to improve HSC culture conditions. Co-culture systems, including stroma and hematopoietic cells in two or three dimensions, are currently being developed and will likely provide much insight into what molecular, chemical, and physical factors need to be combined to achieve efficient, clinical-grade, feeder-free expansion of HSCs. When not only freshly isolated but also genome-edited HSCs are expanded and transplanted, the patient population that will benefit from these studies will be even greater.

Anticipated impact of the research The past 30 years have seen many exciting discoveries highlighting the dazzling complexity of the HSC niche and providing some clues to its plasticity in response to stress, de-regulation when hematologic disease occurs, and critical factors that may be combined to achieve HSC expansion for therapeutic purposes. However, our knowledge about the cellular and molecular components of the hematopoietic niches and spatial organization and dynamics remain extremely rudimentary, and strong research efforts are required to enable potential future therapeutic use. The clinical need for better niche regeneration and larger availability of HSCs remains critically 195

A. Engert et al. unmet, and further in vivo and in vitro studies relying on genetically engineered mouse strains, xenograft models, clinical samples, and refined culture conditions are critical to start making sense of the complexity of the HSC niches. Prolonged observation of stem cells in vivo and ex vivo363 will allow an understanding of how self-renewal is achieved at the single cell level and how pre-malignant clones develop into full disease, so that not only improved therapies but also effective preventive strategies will be developed. This research will affect all patients with hematologic disease, as well as all of those requiring BMT for other reasons, and ultimately the population as a whole by reducing the burden of these diseases and, most importantly, their incidence.

9.4. Human pluripotent stem cells: source of hematopoietic stem cells and hematopoietic progenitor cells Christine Mummery (Leids Universitair Medisch Centrum, Leiden, the Netherlands), Frank Staal (Leids Universitair Medisch Centrum, Leiden, the Netherlands).

Introduction The present availability of human pluripotent stem cells (PSCs) (see subsection 1.9) raises promise for a universal resource for cell-based therapies in regenerative medicine. Rapid progress has been made in generating human PSCs amenable for clinical applications, culminating in reprogramming of adult somatic cells to autologous human PSCs that can be indefinitely expanded in vitro. However, how to differentiate human PSCs to specific lineages efficiently and how to select for cells that will function normally upon transplantation in adults remain major challenges. The hematopoietic lineage has been particularly refractory, and derivation of adult-type hematopoietic stem cells (HSCs) has not yet been possible, even though various other blood lineages can be obtained.364 If HSCs could be generated from a patient's own human PSCs, genetic engineering could easily be used to correct genetic defects prior to differentiation into transplantable HSCs, which would overcome some caveats of conventional hematopoietic stem cell transplantation (HSCT) therapies. Skin fibroblasts were the first human somatic cells to be reprogrammed into human induced pluripotent stem cells (iPSCs), but blood cells are increasingly used because of the availability of banked (cord and peripheral) blood samples covering a range of diseases, ages, genders, and ethnic backgrounds. Complementing human iPSC generation are attempts at “direct reprogramming” using sets of lineagespecific transfer factors (TFs) that act as master regulators and convert cells to a new differentiation state without intermediate pluripotency. Generating human iPSCs from mature B cells requires silencing of lineage-specific factors, such as PAX5, so that direct reprogramming into blood cells where this would not be necessary may, therefore, be quite feasible.365 The first steps in directed differentiation of human PSCs are guided by morphogens (e.g. Wnt, TGF-b, activin, and BMP) important in development. Human PSCs progress through a primitive-streak–like stage before forming the three germ layers, endoderm, ectoderm, and mesoderm, 196

which is the layer giving rise to (hemogenic) endothelial cells and blood. While early embryonic stages are quite faithfully mimicked in culture, patterning of the mesoderm requires anatomical structure of the embryo, embryonic cell-to-cell interactions, expression of patterning genes (Cdx/Hox), certain cell non-autonomous effects, and exposure to physical stimuli (flow) that occur at specific stages of development. Human PSC derivatives thus remain immature, and to date, all human PSC–derived HSCs have been deficient in their developmental potential and ability to self-renew and engraft upon transplantation in mice, even though animal studies have shown that this is possible in the context of a developing embryo. PSCs do have the innate ability to differentiate into fully functional, definitive HSCs. Normal HSC development during embryogenesis occurs in several distinct temporal/spatial waves, each characterized by its own set of HPCs. Only those produced from the latter, or “definitive”, wave give rise to mature, functional HSCs, but during in vitro differentiation, it is believed that the HSC-like cells produced are from the more primitive waves. These give rise to HSC-like cells biased toward myeloid lineages at the expense of lymphoid potential. These are unable to selfrenew in culture and lack long-term engraftment capacity. The key to overcoming this HSC bottleneck will be understanding: 1) the ontogeny of human HSCs; 2) how they progress at the molecular level to become mature, adult HSCs; and 3) intrinsic and extrinsic factors that govern HSC behavior and function.

European research contributions Several prominent European hematologists are using genomic approaches (e.g. gene expression and microRNA profiles and TF and histone ChIP data) to examine the molecular mechanisms that regulate cell fate decisions (e.g. self-renewal versus differentiation and quiescence versus proliferation and apoptosis) by HSCs or HPCs and their more differentiated progenies. This focus on genetic modification of stem cells especially using gamma-retroviral and lentiviral vectors has brought Europe to the global forefront of genetically modified stem cell therapy for treatment of various types of severe combined immunodeficiencies. This has been in part through EU FP7-funded projects that included PERSIST, EuroStemCell, and NOVEXPAND, among others. This momentum is poised to combine HSC expansion with gene editing approaches that will move the field forward toward innovative therapies in the coming decade.

Proposed research for the Roadmap An exciting new development in the gene therapy field is the use of designer nucleases, most recently CRISPR/Cas9-based, that are proving exceptionally efficient in engineering the genome.366 Recent work has established the potential for site-specific gene editing to repair disease mutations in the human genome by targeting the integration of a corrective cDNA into the IL2RG locus of HSCs from a severe combined immunodeficiency–X1 patient.367 For homologous recombination–based gene editing approaches to be successful, HSCs need to proliferate and template DNA harboring the corrective sequences needs to be efficiently introduced into the target cells. This can be readily achieved in human iPSCs so that these cells are prime targets for gene editing techniques. As gene editing can also be done in primary HSCs, haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

however, it is crucial to find conditions that allow HSCs to proliferate without loss of phenotype. Therefore, expansion protocols (see subsection 9.1) are directly relevant for the gene repair described here. Niche signals, expansion, directed differentiation toward induced HSCs, and genetic modification of HSCs and their progenies are related research topics that need an integrated approach for optimal exploitation in the clinical context.

Anticipated impact of the research A major bottleneck to curing genetic diseases originating from mutations in HSCs is that even though efficient methods for gene targeting are now available, HSCs from neither adult nor human PSC sources can be expanded sufficiently for this to become routine practice. The combined projects in subsections 9.1 and 9.2 seek to address this and bring safe gene therapy to the clinic via HSCs.

9.5. Advanced therapy medicinal products and other cell therapies Chiara Bonini (Ospedale San Raffaele, Milan, Italy), Jaap Jan Zwaginga (Leids Universitair Medisch Centrum, Leiden, the Netherlands), Willem Fibbe (Leids Universitair Medisch Centrum, Leiden, the Netherlands).

Introduction Advanced therapy medicinal products (ATMPs) comprise somatic cell therapy medicines, gene therapy medicines, and tissue-engineered medicines or combinations. The first class of ATMPs includes mesenchymal stem cells (MSCs). These are adult, fibroblast-like multipotent cells characterized by their ability to differentiate into tissues of mesodermal origin, including adipocytes, chondrocytes, and osteocytes. MSCs have been initially isolated from bone marrow, but can also be expanded from a variety of other tissues including periosteum, muscle connective tissue, adipose tissue, umbilical cord (blood), amniotic fluid, and placenta. Isolation relies on their ability to adhere to plastic surfaces and they are able to expand significantly by consecutive passaging in vitro. The lack of uniform criteria to define MSCs has prevented efforts to compare results from different experimental and clinical studies, and therefore the International Society for Cellular Therapy formulated minimal criteria for defining MSCs. Mesenchymal stem cells have also been shown to possess broad immunoregulatory abilities, which can influence both adaptive and innate immune response in vitro and in vivo. The mechanisms through which MSCs exert these functions, either through cell-to-cell contact or by secretion of soluble factors, are still not completely understood. Recent findings indicate that MSCs are able to actively interact with cells of the innate immune system through which they may display both anti-inflammatory and proinflammatory effects. This ability to adopt a different phenotype in response to sensing an inflammatory environment is not yet captured in assays that are currently used to characterize these cells. The putative role of stromal cells in maintaining tissue homeostasis serves as the basis for their application in disorders resulting from auto- or alloimmune responses, including graft-versus-host disease (GvHD) and autoimmune disorders. These potential anti-inflammatory properties of MSCs are distinct from their stem cell characteristics.

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Cell-based gene therapy is an innovative therapeutic approach based on the possibility of permanently introducing a gene of interest in the genome of human cells, thus conferring a new function to the cells and their progenies. The first clinical trials of gene therapy, performed in the 1990s with gamma-retroviral vectors, have produced promising clinical results. The recent introduction of innovative gene transfer vectors, such as self-inactivating lentiviral vectors, have further increased the efficacy and safety profile of gene therapy. Recently, the introduction of artificial nucleases, molecules able to mediate DNA strand breaks in selected genomic regions, allow not only a gene of interest to be added in human cells but also a cellular gene and thus a cellular function to be precisely substituted, thus offering new hope for the cure of several diseases. 368-372

European research contributions Whereas the development of cellular therapies in the US is primarily driven by biotech, the role of academic developments in Europe is more prominent. A broad range of phase I and II studies have been performed. Following the first report on a pediatric patient suffering from grade IV refractory acute GvHD of the liver who was rescued by the infusion of bone marrow-derived MSCs, the cells have been studied in the context of hematopoietic stem cell transplantation (HSCT), as well as immunoregulating and regenerative therapies. These studies confirm the safety of MSC therapy and indicate significant response rates in steroid-resistant acute GvHD, solid organ transplantation, Crohn disease (fistulas), and other autoimmune disorders. Cell-based gene therapy is currently being tested in clinical trials for the cure of genetic and acquired diseases. In recent years, patients affected by genetic diseases, such as immunodefiencies, metabolic diseases, and thalassemias, have been enrolled in phase I-II clinical trials based on the infusion of autologous HSCs genetically modified with viral vectors, to express the correct gene and restore cellular functions. Initial results have been highly promising. Gene therapy has been also recently applied to treat patients affected by hematologic malignancies. In this setting, T lymphocytes have been modified by viral vectors to express genes selected to increase the safety and efficacy of cancer immunotherapy. Suicide genes have been successfully introduced in allogeneic lymphocytes, to promote anti-tumor responses and control side effects. Molecules conferring cancer specificity, such as TCRs and CARs, have been introduced in patientsâ&#x20AC;&#x2122; T cells, to mediate tumor regression. In initial phase I-II clinical trials with CAR-redirected T cells, striking complete remissions have been observed in patients affected by B-cell malignancies.

Proposed research for the Roadmap The clinical efficacy for the treatment of allo- (e.g. acute GvHD and solid organ transplantation) and autoimmune (e.g. Crohn disease and multiple sclerosis) disorders is currently being studied in several randomized phase II and III studies. In addition to demonstrating improved outcome, it will be crucial to gain further insight into the biology of response. Advanced immunological monitoring that will 197

A. Engert et al. take into account the inflammatory status in the host at the time of treatment in conjunction with extensive analysis of the products will be crucial for the development of a signature that is associated with outcome. This will be crucial for the development of next generation MSC products with enhanced therapeutic efficacy. Several hurdles need to be cleared before ATMP therapy can be fully exploited in Europe. Multicenter phase III studies across Europe, required to validate promising approaches tested in phase I-II studies, must be carried forward together with many national regulatory agencies, some of which interpret the European guidelines on ATMPs in different ways. This complex scenario largely explains why and how the successful clinical trials with CAR T cells have been performed mainly in the US. The scientific and medical community, and most importantly, patients and patients’ associations, urgently need the use of cell-based gene therapy to be promoted in Europe, and this can be achieved only through harmonization, simplification of the procedures required for trial approval, and dedicated funding. Finally, the full exploitation of T-cell-based cancer gene therapy to additional cancer types requires a co-ordinated and multidisciplinary effort to speed up the generation of new cancer-specific receptors, able to target a wide range of cancer subtypes. This important aim, that can now be reached, thanks to the most innovative technologies currently available, can be achieved only by multidisciplinary teams and dedicated funding.

Anticipated impact of the research The development of a biomarker signature will allow the design of MSC products with enhanced therapeutic efficacy that can be applied in a variety of auto- and alloimmune disorders.

Appendix The authors of the European Hematology Association Roadmap for European Hematology Research First author and editor Andreas Engert (Universität zu Köln, Cologne, Germany)

Second authors and editors (in alphabetical order) Carlo Balduini (IRCCS Policlinico San Matteo Foundation, Pavia, Italy), Anneke Brand (Leids Universitair Medisch Centrum, Leiden, the Netherlands), Bertrand Coiffier (Université Claude Bernard, Lyon, France), Catherine Cordonnier (Hôpitaux Universitaires Henri Mondor, Créteil, France), Hartmut Döhner (Universitätsklinikum Ulm, Ulm, Germany), Thom Duyvené de Wit (European Hematology Association, The Hague, the Netherlands), Sabine Eichinger (Medizinische Universität Wien, Vienna, Austria), Willem Fibbe (Leids Universitair Medisch Centrum, Leiden, the Netherlands), Tony Green (Cambridge Institute for Medical Research, Cambrige, United Kingdom), Fleur de Haas (European Hematology Association, The Hague, the Netherlands), Achille Iolascon (Università Federico II di Napoli, Naples, Italy), Thierry Jaffredo (Université Pierre et Marie Curie, Paris, France), Francesco Rodeghiero (Ospedale San Bortolo, Vicenza, Italy), Gilles Salles (Hospices Civils de Lyon/Université de Lyon, Pierre-Bénite, France), and Jan Jacob Schuringa (Cancer Research Center Groningen, Groningen, the Netherlands).

Lead authors (in alphabetical order) Marc André (Université Catholique de Louvain, Yvoir,

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Belgium, Belgium), Isabelle Andre-Schmutz (Université Paris Descartes, Paris, France), Andrea Bacigalupo (Ospedale San Martino, Genova, Italy), Pierre-Yves Bochud (Centre Hospitalier Universitaire Vaudois/Université de Lausanne, Lausanne, Switzerland), Monique den Boer (Erasmus MC, Rotterdam, the Netherlands), Chiara Bonini (Ospedale San Raffaele, Milan, Italy), Clara Camaschella (San Raffaele Institute, Milan, Italy), Andrew Cant (Great North Children’s Hospital, Newcastle upon Tyne, United Kingdom), Maria Domenica Cappellini (Università degli Studi di Milano, Milan, Italy), Mario Cazzola (Università degli studi di Pavia, Pavia, Italy), Cristina Lo Celso (Imperial College London, London, United Kingdom), Meletios Dimopoulos (National and Kapodistrian University of Athens, Athens, Greece), Luc Douay (Université Pierre et Marie Curie, Paris, France), Elaine Dzierzak (University of Edinburgh, Edinburgh, United Kingdom), Hermann Einsele (Universitätsklinikum Würzburg, Würzburg, Germany), Andrés Ferreri (IRCCS San Raffaele Scientific Institute, Milan, Italy), Lucia De Franceschi (Università degli Studi di Verona, Verona; Italy), Philippe Gaulard (Hôpital Henri-Mondor, Creteil, France), Berthold Gottgens (University of Cambridge, Cambridge, United Kingdom), Andreas Greinacher (Universitätsmedizin Greifswald, Greifswald, Germany), Andreas Greinacher (Ernst-Moritz-Arndt-Universität, Greifswald, Germany), Paolo Gresele (Università degli Studi di Perugia, Perugia, Italy), John Gribben (Queen Mary University, London, United Kingdom), Gerald de Haan (Universitair Medisch Centrum Groningen, Groningen, the Netherlands), John-Bjarne Hansen (Universitetet i Tromsø, Tromsø, Norway), Andreas Hochhaus (Universitätsklinikum Jena, Jena, Germany), Rezan Kadir (The Royal Free Hospital, London, United Kingdom), Srini Kaveri (Institut National de la Santé et de la Recherche Médicale, Paris, France), Valerie Kouskoff (University of Manchester, Manchester, United Kingdom), Thomas Kühne (Universitäts-Kinderspital beider Basel, Basel, Switzerland), Paul Kyrle (Medizinische Universität Wien, Vienna, Austria), Per Ljungman (Karolinska Institutet, Stockholm, Sweden), Georg Maschmeyer (Ernst von Bergmann Klinikum, Potsdam, Germany), Simón Méndez-Ferrer (University of Cambridge, Cambridge, United Kingdom), Michael Milsom (Deutsche Krebsforschungszentrum Im Neuenheimer Feld, Heidelberg, Germany), Christine Mummery (Leids Universitair Medisch Centrum, Leiden, the Netherlands), Gert Ossenkoppele (VUmc, Amsterdam, the Netherlands), Alessandro Pecci (Università degli studi di Pavia, Pavia, Italy), Flora Peyvandi (Università degli Studi di Milano, Milan, Italy), Sjaak Philipsen (Erasmus MC, Rotterdam, the Netherlands), Pieter Reitsma (Leids Universitair Medisch Centrum, Leiden, the Netherlands), José Maria Ribera (Institut Catala d'Oncologia, Barcelona, Spain), Antonio Risitano (Università Federico II di Napoli, Naples, Italy), Stefano Rivella (Weill Medical College, New York, United States of America), Wolfram Ruf (Johannes Gutenberg-Universität Mainz, Mainz, Germany), Timm Schroeder (ETH Zurich, Basel, Switzerland), Marie Scully (University College London Hospitals, London, United Kingdom), Gerard Socie (Hôpital Saint Louis, Paris, France), Frank Staal (Leids Universitair Medisch Centrum, Leiden, the Netherlands), Simon Stanworth (John Radcliffe Hospital, Oxford, United Kingdom), Reinhard Stauder (Medizinische Universität Innsbruck, Innsbruck, Austria), Stephan Stilgenbauer (Universitätsklinikum Ulm, Ulm, Germany), Hannah Tamary (Schneider Children's Medical Center of Israel, Petach Tikva, Israel), Kim Theilgaard-Mönch (Københavns Universitet, Copenhagen, Denmark), Swee Lay haematologica | 2016; 101(2)

EHA Roadmap for European Hematology Research

Thein (King’s College London, London, United Kingdom), Hervé Tilly (Université de Rouen, Rouen, France), Marek Trneny (Univerzita Karlova, Prague, Czech Republic), William Vainchenker (Institut Gustave Roussy, Villejuif, France), Alessandro Maria Vannucchi (Università degli Studi di Firenze, Florence, Italy), Claudio Viscoli (Università degli Studi di Genova, Genova, Italy), Hans Vrielink (Sanquin Research, Amsterdam, the Netherlands), Hans Zaaijer (Sanquin Research, Amsterdam, the Netherlands), Alberto Zanella (Ospedale Maggiore Policlinico, Milan, Italy), Lello Zolla (University of Tuscia, Viterbo, Italy), Jaap Jan Zwaginga (Leids Universitair Medisch Centrum, Leiden, the Netherlands).

Authors (in alphabetical order) Patricia Aguilar Martinez (Hôpital Saint-Eloi, Montpellier, France), Emile van den Akker (Sanquin Research, Amsterdam, the Netherlands), Shubha Allard (Barts Health NHS Trust & NHS Blood and Transplant, London, United Kingdom), Nicholas Anagnou (University of Athens School of Medicine Athens, Greece), Immacolata Andolfo (Università Federico II di Napoli, Naples, Italy), Jean-Christophe Andrau (Institut de Génétique Moléculaire de Montpellier, Montpellier, France), Emanuele Angelucci (Ospedale A. Businco, Cagliari, Italy), David Anstee (NHSBT Blood Centre, Filton, United Kingdom), Igor Aurer (University of Zagreb, Zagreb, Croatia), Hervé Avet-Loiseau (Centre Hospitalier Universitaire de Toulouse, Toulouse, France), Yesim Aydinok (Ege Üniversitesi, Izmir, Turkey), Tamam Bakchoul (Universitätsmedizin Greifswald, Greifswald, Germany), Alessandra Balduini (Carlo Balduini (IRCCS Policlinico San Matteo Foundation, Pavia, Italy), Wilma Barcellini (Ospedale Maggiore Policlinico, Milan, Italy), Dominique Baruch (Université Paris Descartes, Paris, France ), André Baruchel (Hôpital universitaire RobertDebré, Paris, France), Jagadeesh Bayry (Institut National de la Santé et de la Recherche Médicale, Paris, France), Celeste Bento (Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal), Anke van den Berg (Universitair Medisch Centrum Groningen, Groningen, the Netherlands), Rosa Bernardi (IRCCS San Raffaele Scientific Institute, Milan, Italy), Paola Bianchi (Ospedale Maggiore Policlinico, Milan, Italy), Anna Bigas (Institut Hospital del Mar d'Investigacions Mèdiques, Barcelona, Spain), Andrea Biondi (Università degli Studi di Milano-Bicocca, Monza, Italy), Milos Bohonek (Central Military Hospital, Prague, Czech Republic), Dominique Bonnet (The Francis Crick Institute, London, United Kingdom), Peter Borchmann (University Hospital Cologne International, Cologne, Germany), Niels Borregaard (Københavns Universitet, Copenhagen, Denmark), Sigrid Brækkan, (Universitetet i Tromsø, Tromsø, Norway), Marcel van den Brink (Memorial Sloan Kettering Cancer Centre, New York, United States of America), Ellen Brodin, (Universitetssykehuset Nord-Norge, Tromsø, Norway), Lars Bullinger (Universitätsklinik Ulm, Ulm, Germany), Christian Buske (Universitätsklinikum Ulm, Ulm, Germany), Barbara Butzeck (European Federation of Associations of Patients with Haemochromatosis, Croissy sur Seine, France), Jörg Cammenga (Linköpings Universitet, Linköping, Sweden), Elias Campo (Universitat de Barcelona, Barcelona, Spain), Antonino Carbone (Centro di Riferimento Oncologico, Aviano, Italy), Francisco Cervantes (Universitat de Barcelona, Barcelona, Spain), Simone Cesaro (Policlinico GB Rossi, Verona, Italy), Pierre Charbord (Université Pierre et Marie Curie, Paris, France), Frans Claas (Leids Universitair Medisch Centrum, Leiden, the Netherlands), Hannah Cohen haematologica | 2016; 101(2)

(University College London, London, United Kingdom ), Jacqueline Conard (Hôpital Hôtel-Dieu, Paris, France), Paul Coppo (Hôpital Saint-Antoine, Paris, France), Joan-Lluis Vives Corrons (Universitat de Barcelona, Barcelona, Spain), Lydie da Costa (Hôpital R. Debré, Paris, France), Frederic Davi (Université Pierre et Marie Curie, Paris, France), Ruud Delwel (Erasmus MC, Rotterdam, the Netherlands), Irma Dianzani (Università di Torino, Turin, Italy), Dragoslav Domanović (European Center for Disease Prevention and Control, Stockholm, Sweden), Peter Donnelly (Radboud Universitair Medisch Centrum, Nijmegen, the Netherlands), Tadeja Dovč Drnovšek (Zavod RS za transfuzijsko medicino, Ljubljana, Slovenia ), Martin Dreyling (Ludwig-Maximilians-Universität München, Munich, Germany), Ming-Qing Du (University of Cambridge, Cambridge, United Kindom), Carlo Dufour (Istituto Giannina Gaslini, Genova, Italy), Charles Durand (Université Pierre et Marie Curie, Paris, France), Dimitar Efremov (International Centre for Genetic Engineering and Biotechnology, Trieste, Italy), Androulla Eleftheriou (Thalassaemia International Federation, Strovolos, Cyprus), Jacques Elion (Université Paris Diderot, Paris, France), Marieke Emonts (Great North Children’s Hospital, Newcastle upon Tyne, United Kingdom), Monika Engelhardt (Universitätsklinikum Freiburg, Freiburg, Germany), Sophie Ezine (Université Paris Descartes, Paris, France), Fred Falkenburg (Leids Universitair Medisch Centrum, Leiden, the Netherlands), Remi Favier (Hôpital d'enfants ‘A. Trousseau’, Paris, France), Massimo Federico (Università degli studi di Modena e Reggio Emilia, Modena, Italy), Pierre Fenaux (Hôpital Saint Louis, Paris, France), Jude Fitzgibbon (Queen Mary University of London, London, United Kingdom), Johan Flygare (University of Lund, Lund, Sweden), Robin Foà (Università degli Studi di Roma ‘La Sapienza’, Rome, Italy), Lesley Forrester (University of Edinburgh, Edinburgh, United Kingdom), Frederic Galacteros (Hôpitaux Universitaires Henri Mondor, Créteil, France), Isabella Garagiola (Università degli Studi di Milano, Milan, Italy), Chris Gardiner (University of Oxford, Oxford, United Kingdom), Olivier Garraud (Université Jean Monnet, Saint-Etienne, France), Christel van Geet (KU Leuven, Leuven, Belgium), Hartmut Geiger (Universitätsklinikum Ulm, Ulm, Germany), Jan Geissler (CML Advocates Network, Bern, Switzerland), Ulrich Germing (Universitätsklinikum Düsseldorf, Düsseldorf, Germany), Cedric Ghevaert (University of Cambridge, Cambridge, United Kingdom), Domenico Girelli (Università degli Studi di Verona, Verona, Italy), Bertrand Godeau (Hôpitaux Universitaires Henri Mondor, Créteil, France), Nicola Gökbuget (Universitätsklinikum Frankfurt, Frankfurt, Germany), Hartmut Goldschmidt (Universitätsklinikum Heidelberg, Heidelberg, Germany), Anne Goodeve (University of Sheffield, Sheffield, United Kingdom ), Thomas Graf (Center for Genomic Regulation, Barcelona, Spain), Giovanna Graziadei (Università degli Studi di Milano, Milan, Italy), Martin Griesshammer (Mühlenkreiskliniken, Minden, Germany), Yves Gruel (Hôpital Trousseau, Tours, France), Francois Guilhot (Université de Poitiers, Poitiers, France), Stephan von Gunten (Universität Bern, Bern, Switzerland), Inge Gyssens (Universiteit Hasselt, Hasselt, Belgium), Jörg Halter (Universitätsspital Basel, Basel, Switzerland), Claire Harrison (Guy’s and St Thomas’, London, United Kingdom), Cornelis Harteveld (Leids Universitair Medisch Centrum, Leiden, the Netherlands), Eva Hellström-Lindberg (Karolinska Institutet, Stockholm, Sweden), Olivier Hermine (Université Paris Descartes, Paris, France), Douglas Higgs (University of Oxford, Oxford, United Kingdom), Peter Hillmen (University 199

A. Engert et al. of Leeds, Leeds, United Kingdom), Hans Hirsch (Universität Basel, Basel, Switzerland), Peter Hoskin (Mount Vernon Hospital, Northwood, United Kingdom), Gerwin Huls (Universitair Medisch Centrum Groningen, Groningen, the Netherlands), Adlette Inati (Lebanese American University, Beirut, Lebanon), Peter Johnson (University of Southampton, Southampton, United Kingdom), Antonis Kattamis (Athens University, Athens, Greece ), Volker Kiefel (Universitätsmedizin Rostock, Rostock, Germany), Marina Kleanthous (Cyprus School of Molecular Medicine, Nicosia, Cyprus), Hannes Klump (Universitätsklinikum Essen, Essen, Germany), Daniela Krause (Georg Speyer Haus - Institute for Tumorbiology and Experimental Therapy, Frankfurt, Germany), Johanna Kremer Hovinga (Universität Bern, Bern, Switzerland), Georges Lacaud (University of Manchester, Manchester, United Kingdom), Sébastien Lacroix-Desmazes (Institut National de la Santé et de la Recherche Médicale, Paris, France), Judith Landman-Parker (Hôpital Armand Trousseau, Paris, France), Steven LeGouill (Université de Nantes, Nantes, France), Georg Lenz (Universitätsklinikum Münster, Münster, Germany), Marie von Lilienfeld-Toal (Universitätsklinikum Jena, Jena, Germany), Marieke von Lindern (Sanquin Research, Amsterdam, the Netherlands), Armando Lopez-Guillermo (Hospital Clínic de Barcelona, Barcelona, Spain), Enrico Lopriore (Leiden University Medical Centre, Leiden, the Netherlands), Miguel Lozano (Universitat de Barcelona, Barcelona, Spain), Elizabeth MacIntyre (Université Paris Descartes, Paris, France), Michael Makris (Royal Hallamshire Hospital, Sheffield, United Kingdom), Michael Makris (University of Sheffield, Sheffield, United Kingdom), Christine Mannhalter (Medizinische Universität Wien, Vienna, Austria), Joost Martens (Radboud Universiteit, Nijmegen, the Netherlands), Stephan Mathas (Charité Universitätsmedizin Berlin, Berlin, Germany), Axel Matzdorff (Caritasclinic Saarbrücken, Saarbruecken, Germany), Alexander Medvinsky (University of Edinburgh, Edinburgh, United Kingdom), Pablo Menendez (Universitat de Barcelona, Barcelona, Spain), Anna Rita Migliaccio (Mount Sinai Hospital, New York, United States of America), Kenichi Miharada (University of Lund, Lund, Sweden), Malgorzata Mikulska (Università degli Studi di Genova, Genova, Italy), Véronique Minard (Institut Gustave Roussy, Villejuif, France), Carlos Montalbán (MD Anderson Cancer Center Madrid, Madrid, Spain), Mariane de Montalembert (Necker-Enfants Malades University Hospital, Paris, France), Emili Montserrat (Hospital Clínic de Barcelona, Barcelona, Spain), Pierre-Emmanuel Morange (Aix-Marseille Université, Marseille, France), Joanne Mountford (University of Glasgow, Glasgow, United Kingdom), Martina Muckenthaler (Universitätsklinikum Heidelberg, Heidelberg, Germany), Carsten Müller-Tidow (Universitätsklinikum Halle, Halle, Germany), Andrew Mumford (University of Bristol, Bristol, United Kingdom), Bertrand Nadel (Université de la Méditerranée, Marseille, France), Jose-Tomas Navarro (Institut Catala d'Oncologia, Barcelona, Spain), Wassim el Nemer (INSERM UMR S1134, Paris, France), France Noizat-Pirenne (Etablissement Français du Sang, Créteil, France), Brian O’Mahony (European Haemophilia Consortium, Brussels, Belgium), Johannes Oldenburg (Universitätsklinikum Bonn, Bonn, Germany), Martin Olsson (Lunds Universitet, Lund, Sweden), Robert Oostendorp (Technische Universität München, Munich, Germany), Antonio Palumbo (Università degli Studi di Torino, Turin, Italy), Francesco Passamonti (Ospedale di Circolo e

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Fondazione Macchi, Varese, Italy), Roger Patient (University of Oxford, Oxford, United Kingdom), Regis Peffault de Latour (National Institutes of Health, Bethesda, MD, United States of America), Francoise Pflumio (Institut de recherche en radiobiologie cellulaire et moléculaire (IRCM), Paris, France), Luca Pierelli (Università degli Studi di Roma ‘La Sapienza’, Rome, Italy), Antonio Piga (Università di Torino, Turin, Italy), Debra Pollard (The Royal Free Hospital, London, United Kingdom), Marc Raaijmakers (Erasmus MC, Rotterdam, the Netherlands), John Radford (University of Manchester, Manchester, United Kingdom), Ralf Rambach (Deutsche Leukämie- und Lymphomhilfe (DLH), Bonn, Germany), A Koneti Rao (Temple University School of Medicine, Philadelphia, United States of America), Hana Raslova (Université Paris Sud, Villejuif, France), Paolo Rebulla (Ospedale Maggiore, Milan, Italy), David Rees (King’s College Hospital, London, United Kingdom), Vincent Ribrag (Institut Gustave Roussy, Villejuif, France), Anita Rijneveld (Erasmus MC, Rotterdam, the Netherlands), Sara Rinalducci (University of Tuscia, Viterbo, Italy), Tadeusz Robak (Uniwersitet Medyczny W Lodzi, Lodz, Poland), Irene Roberts (University of Oxford, Oxford, United Kingdom), Charlene Rodrigues (Great North Children’s Hospital, Newcastle upon Tyne, United Kingdom), Frits Rosendaal, (Leids Universitair Medisch Centrum, Leiden, the Netherlands), Andreas Rosenwald (Universität Würzburg, Würzburg, Germany), Simon Rule (Derriford Hospital, Plymouth, United Kingdom), Roberta Russo (Università Federico II di Napoli, Naples, Italy), Guiseppe Saglio (Università di Torino, Turin, Italy), Mayka Sanchez (Carreras Leukaemia Research Institute, IJC, Badalana, Barcelona, Spain), Rüdiger E. Scharf (Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany), Peter Schlenke (Medical University Graz, Graz, Austria), John Semple (St. Michael's Hospital, Toronto, Canada), Jorge Sierra (Hospital de la Santa Creu i de Sant Pau, Barcelona, Spain), Cynthia So-Osman (Sanquin Research, Amsterdam, the Netherlands), José Manuel Soria (Hospital de la Santa Creu i Sant Pau, Barcelona, Spain), Kostas Stamatopoulos (Institute of Applied Biosciences, Thessaloniki, Greece), Bernd Stegmayr (Umeå Universitet, Umeå, Sweden), Henk Stunnenberg (Radboud Universitair Medisch Centrum, Nijmegen, the Netherlands), Dorine Swinkels (Radboud Universitair Medisch Centrum, Nijmegen, the Netherlands ), João Pedro Taborda Barata (Universidade de Lisboa, Lisbon, Portugal), Tom Taghon (Universiteit Gent, Ghent, Belgium), Ali Taher (American University of Beirut Medical Center, Beirut, Lebanon), Evangelos Terpos (National and Kapodistrian University of Athes, Athens, Greece), Jecko Thachil (Manchester Royal infirmary, Manchester, United Kingdom), Jean Daniel Tissot (University of Lausanne, Lausanne, Suisse), Ivo Touw (Erasmus MC, Rotterdam, the Netherlands), Ash Toye (University of Bristol, Clifton, United Kingdom), Ralf Trappe (Charité-Universitätsmedizin Berlin, Berlin, Germany), Alexandra Traverse-Glehen (Centre Hospitalier Lyon Sud, Lyon, France), Sule Unal (Hacettepe University, Ankara, Turkey), Sophie Vaulont (Institut Cochin, Paris, France), Vip Viprakasit (Mahidol University, Bangkok, Thailand), Umberto Vitolo (Università degli Studi di Torino, Turin, Italy), Richard van Wijk (Universitair Medisch Centrum Utrecht, Utrecht, the Netherlands), Agnieszka Wójtowicz (Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland), Sacha Zeerleder (Sanquin Research, Amsterdam, the Netherlands), Barbara Zieger (Universitätsklinikum Freiburg, Freiburg, Germany).

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ARTICLE

Blood Transfusion

Red blood cell alloimmunization is influenced by the delay between Toll-like receptor agonist injection and transfusion

EUROPEAN HEMATOLOGY ASSOCIATION

Ferrata Storti Foundation

Rahma Elayeb,1,2,4 Marie Tamagne,1,2,4 Philippe Bierling,1,2,3,4 France NoizatPirenne,1,2,3,4 and Benoît Vingert1,2,4

1 Établissement Français du Sang, Créteil; 2Institut Mondor de Recherche Biomédicale, lnserm U955, Equipe 2, Créteil; 3Université Paris Est, Faculté de Médecine, Créteil; and 4 Laboratory of Excellence GR-Ex, Paris, France

Haematologica 2016 Volume 101(2):209-218

ABSTRACT

urine models of red blood cell transfusion show that inflammation associated with viruses or methylated DNA promotes red blood cell alloimmunization. In vaccination studies, the intensity of antigen-specific responses depends on the delay between antigen and adjuvant administration, with a short delay limiting immune responses. In mouse models of alloimmunization, the delay between the injection of Tolllike receptor agonists and transfusion is usually short. In this study, we hypothesized that the timing of Toll-like receptor 3 agonist administration affects red blood cell alloimmunization. Poly(I:C), a Toll-like receptor 3 agonist, was administered to B10BR mice at various time points before the transfusion of HEL-expressing red blood cells. For each time point, we measured the activation of splenic HEL-presenting dendritic cells, HEL-specific CD4+ T cells and anti-HEL antibodies in serum. The phenotype of activated immune cells depended on the delay between transfusion and Tolllike receptor-dependent inflammation. The production of anti-HEL antibodies was highest when transfusion occurred 7 days after agonist injection. The proportion of HEL-presenting CD8α+ dendritic cells producing interleukin-12 was highest in mice injected with poly(I:C) 3 days before transfusion. Although the number of early-induced HEL-specific CD4+ T cells was similar between groups, a high proportion of these cells expressed CD134, CD40 and CD44 in mice injected with poly(I:C) 7 days before transfusion. This study clearly shows that the delay between transfusion and Toll-like receptor-induced inflammation influences the immune response to transfused red blood cells.

Correspondence: benoit.vingert@efs.sante.fr

Received: 22/7/2015. Accepted: 1/10/2015. Pre-published: 1/10/2015. doi:10.3324/haematol.2015.134171

Introduction Sickle cell disease (SCD) is a devastating condition which still relies on red blood cell (RBC) transfusion. The main immunological complication of transfusion in SCD patients is alloimmunization against RBC antigens, leading to life-threatening post-transfusion hemolysis. Alloimmunization is more frequent in SCD patients than in other patients and represents a major concern in transfusion medicine.1 The high incidence of alloimmunization in this population is partly explained by the large disparity of blood groups between European donors and recipients of African descent. However, some SCD patients never become immunized, and can be qualified as “low responders”. The immune mechanisms underlying red blood cell alloimmunization are poorly understood.2 In humans, several genotypes of class II major histocompatibility complex (MHC II) could be implicated in alloimmunization against specific antigens but controversy remains regarding this.3,4 Little is known about the role of CD4+ T cells in alloimmunization,5 except for Treg cells.6-8 Recently, we showed that the phenotype of CD4+ T cells from SCD patients differs according to whether haematologica | 2016; 101(2)

Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/101/2/209

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the patients have been alloimmunized or not.9 Most knowledge about the mechanisms of alloimmunization has been provided by mouse models. However, it has been shown that SCD does not increase the rate of alloimmunization in mice.10 Despite important differences in the immune system between mice and humans, mouse models enable the investigation of different parameters separately, and provide hypotheses that can be tested in humans. Murine models of post-transfusion alloimmunization have been developed, such as those expressing transgenic human antigens, e.g. glycophorin A, or nonhuman antigens, e.g. hen egg lysozyme (HEL), at the erythrocyte membrane.11 In mouse models, Toll-like receptor (TLR) stimulation promotes alloimmunization. Prior to transfusion, the injection of CpG, a TLR9 agonist, facilitates the production of alloantibodies.12 Moreover, the injection of poly(I:C), a TLR3 agonist, also promotes alloimmunization in mice that are transfused.13,14 TLR3 and TLR9 are implicated in immunity to dsRNA viruses and bacterial infections, respectively.15 Poly(I:C) stimulates splenic CD11c+ dendritic cells (DC) to consume transfused RBC, and modifies the expression of co-stimulatory molecules on these DC.16 However, no study has yet tried to identify the RBC antigen-presenting DC and to characterize their phenotype. In the absence of TLR agonists, splenic macrophages consume RBC, preventing the production of alloantibodies.14 Indeed, transfusion in the absence of inflammation can lead to tolerance to RBC antigens.17 However, in murine models of vaccination, the administration of TLR agonist enables the maturation of DC, leading to the establishment of immune responses rather than tolerance.18 Two main subsets of CD11c+ DC, CD8α+ and CD8α- DC, have been described in the spleen19 and are distinct in terms of function: the CD8α+ population produces interleukin (IL)12.20 IL12 directly affects CD4+ T-cell responses because it induces Th1 polarization, leading to the production of IL2 and interferon (IFN)γ.19,20 Poly(I:C) injection directly modulates the function of CD4+ T cells and stimulates cytokine production and lymphoproliferation.21,22 In a mouse model of transfusion, poly(I:C) was confirmed to promote the lymphoproliferation of HEL-specific CD4+ T cells following transfusion.14 Using this TLR3 agonist, Longhi et al.22 found that the antigen-specific immune response was weak if inflammation occurred during antigen capture and presentation. Indeed, Th1-type immune responses were weak when poly(I:C) injection preceded antigen administration by a period of 6 h.22 It is, therefore, likely that the delay between antigen and adjuvant administration affects antigen-specific responses. In currently used mouse models of alloimmunization, poly(I:C) is typically injected 4 h before transfusion.13,14,16,17,23 In human transfusions, the role of viral and bacterial infections in the induction of alloimmunization has not been documented. However, in SCD patients, a recent study showed that an inflammatory state at the time of transfusion, independently of direct TLR stimulation, can influence RBC alloimmunization.24 The underlying inflammatory state of the patient and TLR signaling may, therefore, be central to the process of RBC alloimmunization. Here, we hypothesized that RBC alloimmunization is influenced by the delay between transfusion and the administration of a TLR3 agonist. To test this, B10BR mice were transfused at various time points after the adminis210

tration of poly(I:C) with HEL-expressing RBC obtained from HOD mice. For each delay, the production of antiHEL antibodies was measured in the recipient mice and the function of splenic HEL-presenting DC and HEL-specific CD4+ T cells was analyzed. We report here that the time between transfusion and TLR3 stimulation influences the immune response to transfused RBC.

Methods Mice B10BR mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and have class II MHC IAk. HOD mice (transgenic RBC-specific expression of HEL, ovalbumin, and Duffy b anchoring HEL to the RBC membrane) are issued from the FVB background and have class II MHC IAb. Mice were housed and bred at the Institut Mondor de Recherche Biomédicale (IMRB) conventional animal facility, in pathogen-free conditions. Transfusionrecipient mice were used at 7 to 9 weeks of age, in homogeneous mixed-sex groups. All procedures were approved by the local ethics committee.

Transfusion and treatment of mice

Mice received a 100 mL intraperitoneal injection of phosphatebuffered saline or a TLR3 agonist, poly(I:C) (100 mg, Amersham Piscataway, NJ, USA), at various time points (4 h, 3 days, 7 days or 14 days before transfusion). The mice then received a 100 mL transfusion of fresh HOD RBC concentrate into the lateral tail vein. All mice were sacrificed 2 days or 28 days after transfusion, and the spleen was harvested. Blood was collected from the retroorbital vein before death. Serum was isolated by centrifugation and frozen at -20°C.

Flow cross-matching and enzme-linked immunosorbent assay for the detection of anti-HEL responses The presence of anti-HEL antibodies was evaluated by flow cross-matching. Serum from B10BR mice, harvested 28 days after transfusion, was diluted 1:10 and incubated with RBC expressing HEL (HOD) antigen or control RBC (B10BR). Anti-HEL antibodies were detected by flow cytometry, with allophycocyanin (APC)labeled antibodies against total Ig (BD Biosciences, Franklin Lakes, NJ, USA). Serum from HEL immunized mice with aluminum salts incubated with HEL-expressing RBC (HEL+ RBC) was used as a positive control. Adjusted mean fluorescence intensity (MFI) was calculated as follows: adjusted MFI = (MFI of the serum incubated with HEL+ RBC) – (MFI after incubation with HEL- RBC). Enzymelinked immunosorbent assay (ELISA) was performed in a HELcoated plate, to determine the total amount of IgG antibody against HEL in the serum. The alkaline-phosphatase-conjugated secondary antibody used for detection was purchased from Jackson ImmunoResearch (West Grove, PA, USA). Antibody binding was detected by reaction with the pNPP substrate (SigmaAldrich, St-Louis, MO, USA). Anti-HEL antibody (Raybiotech, GA, USA), with known amounts of the IgG isotype, was used as a standard.

Dendritic cell immunostaining and intracellular cytokine staining After DC enrichment, at least 2x104 cells were cultured overnight in complete medium supplemented with brefeldin A (0.5 mg/well) to determine the production of cytokines. Before staining, the Fc receptors were blocked with CD16/CD32 antibodies (0.5 mg, eBioscience, San Diego, CA, USA). Cultured DC were incubated (15 min, 4°C) with antibodies against the following haematologica | 2016; 101(2)

RBC alloimmunization depends on transfusion timing

membrane proteins, to determine their phenotype: CD8α-PECF594 (BD Biosciences), CD40-PE-Cy7, CD252-AF647 (Biolegend, San Diego, CA, USA), CD11c-APC-eF780, and CD70PerCP-eF710 (eBioscience). DC were fixed and permeabilized with a commercial kit (eBioscience) for intramembranous staining with CD283-PE (Biolegend, San Diego, CA, USA), IL12-FITC and IFNγ-AF700 (BD Biosciences). The HEL protein contains an immunodominant peptide, NR16 (HEL46-61: NTDGSTDYGILQINSR). NR16-presenting DC were studied using an AW3.18 antibody. This antibody recognizes the MHC II I-Ak-NR16 complex.25 The AW3.18 antibody was detected with a biotinylated anti-IgG1 antibody (Jackson ImmunoResearch) and streptavidin-BV421 (Biolegend). The proportions of CD8α+ and CD8α- DC among total CD11c+ cells were calculated as follows: (number of CD8α cells / total number of CD11c+ cells) x 100.

Ex vivo staining of NR16-specific CD4+ T cells by MHC II tetramers Splenocytes were incubated with APC-labeled class II MHC IAk tetramers (4 mg/mL, 90 min, 4°C) in phosphate-buffered saline

– bovine serum albumin (1%, Sigma-Aldrich) supplemented with azide (0.02% Santa Cruz Biotechnology, Santa Cruz, CA, USA). Antibodies against membrane proteins, CD134-BV421 (Biolegend), CD40-PE-Cy5, CD3-APC-eF780 (eBioscience), CD4PE-CF594 and CD44-PerCP-Cy5.5 (BD Biosciences), were added in the last 30 min of tetramer staining. A negative control was obtained by staining with a tetramer loaded with class II-associated invariant chain peptide (CLIP).

Results Anti-HEL antibody production and the timing of poly(I:C) delivery To determine how the interval between poly(I:C) delivery and transfusion influences the induction of alloimmunization, we injected B10BR mice with poly(I:C) at 4 h, 3 days, 7 days or 14 days before transfusion. As in previous studies relating to the induction of antiRBC antibodies as efficiently as possible in mouse

Figure 1. Anti-HEL response 28 days after transfusion in mice injected with poly(I:C) at various times before RBC transfusion. At 4 h, 3 days, 7 days or 14 days after poly(I:C) or phosphate-buffered saline (control represented at 4 h) intraperitoneal injection, mice were transfused with HEL RBC and sera were obtained 28 days later. (A) Histograms of representative experiments showing the detection of anti-HEL antibodies by flow cross-matching with sera incubated with HEL+ RBC (red line) or HEL– RBC (black line). Serum from HEL-immunized mice was used as a positive control. Serum from HEL-naïve B10BR mice was used as a negative control. Sera were tested for the presence of anti-HEL antibodies by (B) flow cross-matching or by (C) ELISA. Comparisons were performed using the Kruskal-Wallis test and Dunn post test. *P<0.05; **P<0.01; ***P<0.005. Representative data from two independent experiments in six mice each are shown (mean ± SD).

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models,12,13 we evaluated the production of anti-HEL antibodies by flow cross-matching and ELISA, 28 days after the transfusion of blood from donor HOD mice (Figure 1). The adjusted MFI was calculated as described above, to eliminate the background noise of each sample. The HEL antibody response was significantly higher in mice injected with poly(I:C) 7 days before transfusion than in those injected 4 h before transfusion (42.5±16.4 versus 15.2±5.3, respectively; P<0.05) (Figure 1B). The anti-HEL IgG response was assessed by ELISA (Figure 1C). The titer of these antibodies was also significantly higher in mice receiving the injection 7 days before transfusion than in those receiving the injection 4 h before transfusion (0.99±1.01 mg/mL versus 0.15±0.10 mg/mL, respectively; P<0.05).

Effect of poly(I:C) injection before transfusion on the proportion of NR16-presenting dendritic cells We evaluated the effect of the timing of poly(I:C) injection (4 h, 3 days, 7 days or 14 days before transfusion) on CD8α+ and CD8α- splenic CD11c+ DC 48 h after transfusion. These two subsets are the main subpopulations of DC in the spleen. We determined the proportion of NR16-

presenting DC in the CD11c+ and CD8α-/+ subsets, using the AW3.18 antibody (Figure 2A). NR16-presenting DC represented 33.9±13.5% of cells in the CD11c+ DC population in mice injected with poly(I:C) 7 days before transfusion, but only 14.6±9.1% in mice injected with poly(I:C) 4 h before transfusion (P<0.05, Figure 2B). Regarding the different subpopulations, the proportion of NR16-presenting cells in the CD8α- subpopulation was highest in mice injected with poly(I:C) 7 days before transfusion (31.1±6.0%, P<0.05). By contrast, the proportion of NR16presenting cells in the CD8α+ subpopulation was highest in mice injected only 4 h before (34.6±16.7%). The proportion of CD8α+ NR16-presenting cells decreased as the delay between poly(I:C) injection and transfusion increased, and reached its lowest value of 4.0±1.8% in mice injected with poly(I:C) 14 days before transfusion (Figure 2C).

The functions of NR16-presenting dendritic cells are influenced by the timing of poly(I:C) delivery IL12 expression by the total CD8α+/- DC subset was not affected by the timing of poly(I:C) delivery (data not shown). However, to examine the function of NR16-pre-

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Figure 2. Proportion of splenic NR16presenting cells in the CD11c+ DC and CD8α+/- subpopulations, after transfusion following poly(I:C) injection. At 4 h, 3 days, 7 days or 14 days after poly(I:C) intraperitoneal injection, mice were transfused with HEL RBC and spleens were harvested 48 h later. (A) The AW3.18 antibody was used to detect NR16-presenting CD11c+ DC in the CD8α- (top) and CD8α+ (bottom) subpopulations. NR16 is the immunodominant peptide of HEL. Gating is shown for mice injected with poly(I:C) 4 h before transfusion. AW3.18- cells isolated from mice that received intravenous PBS instead of transfusion were used as a negative control. (B) The proportions of NR16-presenting cells in the CD11c+ DC population are given. (C) The proportions of NR16-presenting cells in the CD8α- CD11c+ (left) and CD8α+ CD11c+ (right) DC populations are given. Comparisons were performed using the Kruskal Wallis test and Dunn post test. P<0.05; **P<0.01; ***P<0.005. Representative data from two independent experiments in five mice each are shown (mean ± SD).

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senting DC, we also measured IL12 production in both the CD8α+ and CD8α- subsets, by intracellular staining (Figure 3A). The proportion of NR16-presenting CD8α+ CD11c+ DC producing IL12 was significantly higher in mice that received poly(I:C) 3 days before transfusion than in those injected only 4 h before transfusion (8.3±5.5% versus 21.8±5.2%, respectively; P<0.05). As expected, the CD8αsubpopulation produced low amounts of IL12; however, its production was not influenced by the timing of poly(I:C) delivery (Figure 3B). We also investigated IFNγ production by the two subpopulations: IFNγ expression in the NR16-presenting CD8α- subpopulation was significantly weaker in mice injected with poly(I:C) 3 days or 7 days before transfusion than in those that received poly(I:C) 4 h before transfusion (data not shown, P<0.05 and P<0.01, respectively). IFNγ production by the CD8α+ subpopulation was not affected by the timing of poly(I:C) injection (data not shown).

The timing of poly(I:C) delivery affects the phenotype of NR16-presenting dendritic cells We also evaluated the effect of the timing of poly(I:C) injection on the two NR16-presenting DC subpopulations 48 h after transfusion, by measuring the expression of the co-stimulatory molecules CD252, CD70 and CD40. In both the CD8α- and CD8α+ subpopulations, CD252 expression decreased as the delay between poly(I:C) injection and transfusion increased (Figure 4A). CD252 expression on NR16-presenting CD8α- DC was highest in mice injected with poly(I:C) 4 h before transfusion and lowest in those injected 14 days before transfusion (45.0±5.6% to 2.6±1.0%, respectively; P<0.005). We observed a similar pattern for CD8α+ DC (77.1±8.2% in mice injected 4 h

and 9.7±2.7% in those injected 14 days before transfusion; P<0.005). In both the CD8α- and CD8α+ subpopulations, the timing of poly(I:C) delivery had little effect on CD70 expression (Figure 4B). The proportion of CD8α- NR16-presenting DC expressing CD70 was slightly higher in mice injected with poly(I:C) 7 days than in those injected 4 h before transfusion (75.3±24.3% versus 63.1±9.6%, respectively; P<0.05). The proportion of CD8α+ NR16-presenting cells expressing CD70 was significantly lower in mice that received poly(I:C) 3 or 7 days than in those that received it 4 h before transfusion (P<0.01 and P<0.005, respectively) (Figure 4B). CD40 expression followed the same pattern as CD70 expression. The proportion of cells in the CD8α- DC subpopulation expressing CD40 was slightly higher in mice injected with poly(I:C) 14 days before transfusion than in those injected 4 h prior to the transfusion (72.7±5.3% versus 60.8±11.4%; P<0.05). In the CD8α+ subset, the proportion of cells expressing CD40 was significantly lower in mice injected 7 days than in those injected 4 hours before transfusion (56.6±2.2% versus 81.0±10.3%; P<0.005) (Figure 4C). Furthermore, as expected, poly(I:C) played the same activating role in the total DC population and in NR16-presenting DC (a representative example for the 7day group is presented in Online Supplementary Figure S1). Indeed, the levels of CD252, CD70 and CD40 expression were equivalent on total DC at all four time points studied, and changes in these levels followed the same pattern as those observed in NR16-presenting DC (data not shown). We also measured the effect of poly(I:C) delivery time on the intracellular expression of TLR3 in the CD11c+ DC subpopulations (Figure 5A). In the total CD8α+ subpopula-

Figure 3. Effect of the timing of poly(I:C) delivery on IL12 production by splenic NR16presenting DC. At 4 h, 3 days, 7 days or 14 days after poly(I:C) intraperitoneal injection, mice were transfused with HEL RBC and spleens were harvested 48 h later. (A) The example shows IL12 production by CD8α– (black line) and CD8α+ (red line) NR16-presenting (AW3.18+) cells in mice injected with poly(I:C) 3 days before transfusion. (B) IL12 production was measured in both the NR16-presenting CD8α– (left) and CD8α+ (right) DC subpopulations identified with the AW3.18 antibody. Comparisons were performed using the Kruskal Wallis test and Dunn post test. *P<0.05; **P<0.01; ***P<0.005. Representative data from two independent experiments in five mice each are shown (mean ± SD).

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tion, TLR3 expression was higher in mice injected with poly(I:C) 3 or 7 days before transfusion than in those injected 4 h before (P<0.01) (Figure 5B). TLR3 expression in the NR16-presenting CD8α+ subpopulation was also higher in mice that received poly(I:C) 3 or 7 days before transfusion than in those that received it only 4 h before (P<0.01 and P<0.05, respectively) (Figure 5B). As expected, the CD8α- subpopulation expressed low amounts of TLR3 and its expression was not influenced by the timing of poly(I:C) delivery (Online Supplementary Figure S2). In the NR16-presenting CD8α- subpopulation, TLR3 expression was weakest in the mice that received poly(I:C) 14 days before transfusion (Online Supplementary Figure S2).

The timing of poly(I:C) injection influences the abundance and phenotype of early NR16-specific CD4+ T cells We harvested the spleen 48 h after transfusion to investigate CD4+ T-cell induction at an early stage of TCR

engagement. We used MHC class II tetramers (Tet) to study how the timing of poly(I:C) injection influences NR16-specific CD4+ T effector cells ex vivo (Figure 6A). NR16-Tet+ cells were present in all four sets of conditions, but there were no important differences between groups. Indeed, the percentage of NR16-Tet+ cells was similar in mice injected with poly(I:C) 7 or 14 days before transfusion and those injected 4 h before, and it was only slightly lower in those injected 3 days before (P<0.05 versus injection at 4 h) (Figure 6B). We, therefore, analyzed the phenotype of NR16-Tet+ cells by evaluating expression of the activation molecules CD134, CD40 and CD44. The expression of CD134, CD40 and CD44 was lower on total T CD4+ cells than on NR16-Tet+ cells (Figure 6C). The proportion of total cells expressing CD134, CD40 or CD44 was smaller than that of NR16-Tet+ cells for each of the injection time points studied (Online Supplementary Figure S3). The proportion of NR16-Tet+ cells expressing CD134 was highest in mice injected 7 days before transfusion and

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Figure 4. The timing of poly(I:C) delivery influences the phenotype of NR16-presenting DC. At 4 h, 3 days, 7 days or 14 days after poly(I:C) intraperitoneal injection, mice were transfused with HEL RBC and spleens were harvested 48 h later. The expression of (A) CD252, (B) CD70 and (C) CD40 was measured on NR16presenting CD11c+ CD8α– (left column) and CD8α+ (right column) DC. Comparisons were performed using the Kruskal Wallis test and Dunn post test. *P<0.05; **P<0.01; ***P<0.005. Representative data from two independent experiments in five mice each are shown (mean ± SD).

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lowest in those injected 4 h before (26.8±6.2% versus 4.4±1.6%, respectively; P<0.01) (Figure 6D). The proportion of NR16-Tet+ cells expressing CD40 also tended to be correlated with the timing of poly(I:C) injection, and was significantly higher in mice injected 14 days before transfusion than in those injected 4 h before (8.5±6.7% versus 0.7±1.1%, respectively; P<0.01) (Figure 6D). CD44 was expressed on NR16-Tet+ cells, but the proportion of cells expressing this molecule was not significantly influenced by the timing of poly(I:C) injection (Figure 6D).

Discussion The HEL antibody response was significantly higher in mice injected with poly(I:C) 7 days before transfusion than in those injected 4 h before transfusion. To understand why antibody production depends on the delay between transfusion and poly(I:C) injection, we first explored DC subpopulations and their activation phenotypes. Poly(I:C) is widely used to promote the maturation of both mouse and human DC.22,26,27 In mouse studies, poly(I:C) induces complete activation of DC after 12 to 24 h.22,28 In humans, poly(I:C) induces a mature surface phenotype, which can still be detected at 72 h.29 TLR3 signaling stimulates the expression of co-stimulatory molecules on antigen-presenting cells, potentially enhancing CD4+ Tcell responses.26 We, therefore, also investigated antigenspecific CD4+ T-cell responses. In murine models of transfusion, poly(I:C) promotes the consumption of RBC by CD11c+ DC in the spleen.16 However, no studies have examined whether these DC present RBC antigens. We, therefore, explored whether the phenotypic modifications of CD11c+ DC observed by Hendrickson et al.16 were also detectable in HEL-presenting CD11c+ DC. Specifically, we examined whether the delay between poly(I:C) injection and transfusion affected the phenotype and function of HEL-presenting DC. In vaccination studies, immune responses are weak if the antigen is administered within 6 h of agonist injection.22 We studied CD11c+ DC subpopulations in the spleen, which can be differentiated by their CD8α expression.20 We found that the timing of agonist injection influenced the ratio of CD8α+ to CD8α- NR16-presenting CD11c+ DC. Poly(I:C) and the CD8α+ DC subset play an important role in the induction of Th1 polarization.19,22 Our observation was, therefore, surprising because we expected to find a high proportion of NR16-presenting CD8α+ CD11c+ DC in mice that received poly(I:C) 7 days before transfusion, rather than CD8α- CD11c+ DC. Given that the CD8α- subset outnumbers the CD8α+ subset in these conditions, it is possible that the CD8α- subset is more efficient at antigen uptake and presentation. Alternatively, either only the CD8α+ subset is primordial, independently of its proportion, or both subsets are implicated in RBC alloimmunization. These two subsets of DC can be differentiated by their expression of TLR3, with the CD8α+ subset expressing high levels of TLR3.19 TLR3 expression in the NR16presenting CD8α+ subset was positively correlated with the delay between transfusion and poly(I:C) injection, suggesting a higher sensitivity of the CD8α- subset to TLR3 agonists. However, these two DC subsets are also distinct in terms of function, which may be important for alloimmunization. Indeed, upon activation, the CD8α+ subpopulahaematologica | 2016; 101(2)

tion produces high levels of IL12, whereas the CD8α- subpopulation does not.20 We found that the proportion of NR16-presenting CD8α+ DC expressing IL12 was significantly higher in mice injected with poly(I:C) 3 days before transfusion than in those injected 4 h before. This result is consistent with studies showing that poly(I:C) stimulates CD8α+ DC to produce IL12,19,27 which may contribute substantially to RBC alloimmunization. Indeed, activated DC produce IL12, which induces the differentiation of naïve CD4+ T cells into T follicular helper (Tfh) cells.30 Tfh cells are specialized T helper cells that regulate antibody production and the development of memory B cells.31 The production of anti-HEL antibodies in mice receiving blood

Figure 5. The effect of the timing of poly(I:C) delivery on TLR3 expression by splenic CD11c+ CD8α+ DC after transfusion. At 4 h, 3 days, 7 days or 14 days after poly(I:C) intraperitoneal injection, mice were transfused with HEL RBC and spleens were harvested 48 h later. (A) TLR3 expression on the CD8α– (black line) and CD8α+ (red line) CD11c+ subpopulations in mice injected with poly(I:C) 4 h before transfusion is shown. The expression of TLR3 was measured in (B) total and (C) NR16-presenting CD11c+ CD8α+ DC. Comparisons were performed using the Kruskal Wallis test and Dunn post test. *P<0.05; **P<0.01. Representative data from two independent experiments in five mice each are shown (mean ± SD).

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transfusions may reflect changes in the phenotype of NR16-presenting CD8α+ DC. However, we cannot exclude a role for CD8α- DC in alloimmunization, because this subset produces IL10, which is important for B-cell differentiation and immunoglobulin switching.32 In this study, due to differences in phenotype observed between the different time periods studied, we can conclude that DC are involved in the induction of alloimmunization, but that these cells may not affect the magnitude of this phenomenon. Poly(I:C) tightly regulates the expression of co-stimulatory molecules at the surface of DC.22 In transfused mice,

poly(I:C) induces the expression of CD70, CD252 and CD40 molecules on DC.16 The expression of CD252 on DC promotes CD4+ T-cell expansion about 48 h after antigen stimulation.33 However, in this study, the level of CD252 expression was low in both subpopulations 48 h after transfusion in mice that received poly(I:C) 3 or more days before transfusion. Given that this model differs from classical models used to study antigen stimulation, it is likely that the exosomes issued from RBC or platelets also modulate immune responses to RBC antigens.34-36 However, other tumor necrosis factor receptors, notably CD70 and CD40, are required for complete CD4+ T-cell

Figure 6. The effect of the timing of poly(I:C) injection on NR16-specific CD4+ T cells. Mice were transfused with HEL RBC 4 h, 3 days, 7 days or 14 days after poly(I:C) intraperitoneal injection and spleens were harvested 48 h later. (A) The abundance of NR16-specific CD4+ T cells was measured ex vivo by class II MHC tetramers of the immunodominant NR16-peptide from HEL. Plots corresponding to mice injected with poly(I:C) 4 h before transfusion are shown. Tetramers of class II-associated invariant chain peptide (CLIP) were used as a specificity control for NR16 tetramers. (B) NR16-specific CD4+ T cells (NR16+ tetramers) were detected using tetramers of class II MHC and are reported as a percentage of total CD4+ T cells. (C) The expression of CD134, CD40 and CD44 was measured ex vivo on NR16-specific CD4+ T (red dot plot) and total CD4+ T cells (black contour plot). Plots for mice injected with poly(I:C) 7 days before transfusion are shown. (D) The phenotype of NR16-specific CD4+ T cells (NR16) in mice injected with poly(I:C) at various time points before transfusion. Comparisons were performed using the Kruskal Wallis test and Dunn post test. *P<0.05; **P<0.01. Representative data from two independent experiments of five mice each are shown (mean ± SD).

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activation.37 Indeed, the injection of immunogenic protein and TLR3/9 agonists combined with an anti-CD40 antibody upregulates CD70 expression at the DC membrane.37 This combination enables the binding of the CD70 ligand, CD27, which induces the differentiation of naïve-CD4+ T cells and memory T lymphocytes.38-40 Although we observed minor variations between groups regarding the expression of CD70 and CD40 on both subsets of NR16-presenting DC, the proportion of cells expressing these molecules remained high in all conditions. The expression of these co-stimulatory molecules on DC is associated with antigen-specific CD4+ T-cell responses.26 Furthermore, poly(I:C) directly affects the lymphoproliferation of RBC-specific CD4+ T cells in a mouse model of transfusion.14 For these reasons, we used class II MHC tetramers to investigate antigen-specific CD4+ T-cell responses 48 h after transfusion. The timing of poly(I:C) delivery did not appear to affect the proportion of NR16-specific CD4+ T cells. However, the CD4+ T-cell population was analyzed 48 h after transfusion, which may explain the small variation observed for CD4+ T-cell numbers. Nevertheless, both the activation and differentiation of NR16-specific CD4+ T cells were affected by the timing of agonist injection. Co-stimulatory molecules expressed by DC and CD4+ T cells are important for the activation and differentiation of immune system cells.33,41 Among these co-stimulatory molecules, CD134 (OX40) is expressed soon after the stimulation of the TCR on CD4+ T cells and binds to its ligand, CD252 (OX40L), on the DC membrane. We found that the proportion of NR16-specific CD4+ T cells expressing CD134 was highest in mice injected with poly(I:C) 7 days before transfusion. CD40 expression was also positively associated with the duration of the delay between TLR-agonist injection and transfusion. Both CD134 and CD40 play a role in CD4+ T-cell responses and autoimmunity.33,42-44 This finding is consistent with the high frequency of autoantibodies against RBC antigens in alloimmunized SCD patients.45,46 Moreover, autoimmunity has also been described in SCD patients.47-49 The strong expression of these two molecules may affect alloimmunization, especially when poly(I:C) is injected 7 days before transfusion. Specifically, the injection of TLR3 agonists combined with CD40 stimulation facilitates the secretion of pro-inflammatory cytokines by CD4+ T cells by promoting the expression of CD134.40,50 Cross-linking

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between CD134 on CD4+ T cells and CD252 on activated B cells results in B-cell proliferation and the secretion of all Ig isotypes.51 This high level of expression of CD134 at 7 days and its involvement in B-cell antibody secretion suggest a direct link between the CD4+ T-cell response and the level of antibody production, which were concordant for the same time interval between agonist injection and transfusion. Finally, the relationship between CD4+ T cells and B cells might be stronger than that between CD4+ T cells and DC in RBC alloimmunization. In conclusion, our study confirms that the degree of alloimmunization depends on the delay between TLR3induced inflammation and exposure to RBC antigens, with the largest effect observed at 7 days, for CD4+ T cells. TLR3 and TLR9 have been shown to affect alloimmunization in mouse models.12,14 However, it remains unknown whether exposure to RNA viruses or bacterial infections in the week before a transfusion increases the risk of alloimmunization in humans. We need to evaluate this risk in SCD patients, who experience inflammation and infection more frequently than the general population of individuals undergoing transfusion. The role of vaccination should also be taken into account. Indeed, in addition to the conventional adjuvants used in vaccination (aluminum salts, oil-emulsion or liposomes), which encourage antigen presentation and the activation of antigen-presenting cells,52 TLR agonists have also been proposed as new adjuvants for vaccines in clinical trials.52,53 It is likely that these new vaccines need to be used with caution in polytransfused SCD patients to limit RBC alloimmunization. Funding This work was supported by the Établissement Français du Sang (EFS) and the Université Paris-Est. Acknowledgments The authors would like to thank Sylvie Manin and Sophia Ballustre from the animal facility of IMRB and Yves Levy (Institut de Recherche Vaccinale, Créteil, France) for the use of the LSR II cytometer. The authors would also like to thank the NIH Tetramer Facility (Emory Vaccine Center) for kindly providing the class II MHC IAk tetramers, Jeanne E. Hendrickson (Emory University, Atlanta, GA, USA) for kindly providing HOD mice and Emil R. Unanue (Washington University School of Medicine) and Gilles Dadaglio (Institut Pasteur, Paris, France) for kindly providing the AW3.18 antibody.

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ARTICLE

Coagulation & Its Disorders

Clinical, instrumental, serological and histological findings suggest that hemophilia B may be less severe than hemophilia A

EUROPEAN HEMATOLOGY ASSOCIATION

Ferrata Storti Foundation

Daniela Melchiorre,1 Silvia Linari,2 Mirko Manetti,3 Eloisa Romano,1 Francesco Sofi,4,5 Marco Matucci-Cerinic,1 Christian Carulli,6 Massimo Innocenti,6 Lidia Ibba-Manneschi,3* and Giancarlo Castaman2* Department of Experimental and Clinical Medicine, Section of Internal Medicine, University of Florence, Rheumatology Unit, Careggi University Hospital; 2Center for Bleeding Disorders, Careggi University Hospital, Florence; 3Department of Experimental and Clinical Medicine, Section of Anatomy and Histology, University of Florence; 4 Department of Experimental and Clinical Medicine, University of Florence; 5Don Carlo Gnocchi Foundation, Onlus IRCCS, Florence; and 6First Orthopedic Clinic, Careggi University Hospital, Florence, Italy 1

Haematologica 2016 Volume 101(2):219-225

*LI-M and GC contributed equally to this work.

ABSTRACT

ecent evidence suggests that patients with severe hemophilia B may have a less severe disease compared to severe hemophilia A. To investigate clinical, radiological, laboratory and histological differences in the arthropathy of severe hemophilia A and hemophilia B, 70 patients with hemophilia A and 35 with hemophilia B with at least one joint bleeding were consecutively enrolled. Joint bleedings (<10, 10-50, >50), regimen of treatment (prophylaxis/on demand), World Federation of Hemophilia, Pettersson and ultrasound scores, serum soluble RANK ligand and osteoprotegerin were assessed in all patients. RANK, RANK ligand and osteoprotegerin expression was evaluated in synovial tissue from 18 hemophilia A and 4 hemophilia B patients. The percentage of patients with either 10-50 or more than 50 hemarthrosis was greater in hemophilia A than in hemophilia B (P<0.001 and P=0.03, respectively), while that with less than 10 hemarthrosis was higher in hemophilia B (P<0.0001). World Federation of Hemophilia (36.6 vs. 20.2; P<0.0001) and ultrasound (10.9 vs. 4.3; P<0.0001) score mean values were significantly higher in hemophilia A patients. Serum osteoprotegerin and soluble RANK ligand were decreased in hemophilia A versus hemophilia B (P<0.0001 and P=0.006, respectively). Osteoprotegerin expression was markedly reduced in synovial tissue from hemophilia A patients. In conclusion, the reduced number of hemarthrosis, the lower World Federation of Hemophilia and ultrasound scores, and higher osteoprotegerin expression in serum and synovial tissue in hemophilia B suggest that hemophilia B is a less severe disease than hemophilia A. Osteoprotegerin reduction seems to play a pivotal role in the progression of arthropathy in hemophilia A.

Introduction Hemophilia A (HA) and hemophilia B (HB) are X-linked recessive bleeding disorders caused by mutations in the genes encoding coagulation factor VIII (FVIII) and factor IX (FIX), respectively. Subjects with factor plasma levels less than 1 IU/dL are classified as severe hemophiliacs, whereas those with factor levels between 1 and 5 IU/dL and more than 5 IU/dL are affected by moderate and mild hemophilia.1 Although the bleeding phenotype may be rather heterogeneous,2,3 this classification reflects closely the severity of clinical symptoms. haematologica | 2016; 101(2)

Correspondence: daniela.melchiorre@unifi.it

Received: 13/7/2015. Accepted: 15/10/2015. Pre-published: 22/10/2015. doi:10.3324/haematol.2015.133462

Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/101/2/219

ÂŠ2016 Ferrata Storti Foundation Material published in Haematologica is covered by copyright. All rights reserved to Ferrata Storti Foundation. Copies of articles are allowed for personal or internal use. A permission in writing by the publisher is required for any other use.

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Traditionally, HA and HB have been considered clinically indistinguishable, with recurrent musculoskeletal bleeding, particularly joint bleeding, as hallmark of severe disease. Some evidence, however, suggests that patients with severe HB may have a less severe bleeding phenotype, a lower bleeding frequency, and better long-term outcomes compared to severe HA patients.4,5 More than 50 years ago, prior to the availability of clotting factor concentrates, Quick et al. noticed that severe HB was less handicapping than HA.6 More recently, many studies demonstrated a higher use of continuous prophylaxis and greater factor consumption in severe HA patients compared with those with severe HB.7-10 Moreover, Tagariello et al., in a retrospective survey of joint arthroplasty in the frame of the Italian Hemophilia Center Association, showed that patients with HA had a 3-fold higher risk of undergoing orthopedic arthroplasty, that is an indirect expression of severity of arthropathy.11 Finally, Mannucci et al. suggested that HB is milder than HA also because of the different expression of the pathogenetic gene defects.5 Indeed, the type of gene mutation does affect the residual coagulant activity of FVIII or FIX, so that gene defects that totally prevent the synthesis of the protein (referred to as null mutations) are usually associated with undetectable factor activity, whereas non-null mutations account for variable factor levels in the plasma, even when below 1 IU/dL. Null mutations are prevalent in severe HA, whereas missense mutations are prevalent in HB.12,13 The fact that less severe gene mutations are more

frequent in severe HB supports the view that some FIX activity may be present in the plasma of these patients, thus attenuating bleeding severity and frequency. Recurrent joint bleeding leads to initially independent adverse changes in both the synovial tissue and the articular cartilage/subchondral bone which reciprocally influence each other. The synovial inflammatory changes enhance articular cartilage damage and vice versa, eventually resulting in arthropathy and disability.14,15 The introduction into clinical practice of the ultrasound (US) evaluation coupled with the US score16 allows frequent monitoring of the evolution of arthropathy in HA and HB.17 Another crucial parameter of bone biology is the molecular triad consisting of osteoprotegerin (OPG), receptor activator of nuclear factor-kB (RANK) and RANK ligand (RANKL), which tightly controls bone turnover and is involved in the severity of arthropathy, as demonstrated in HA.18,19 OPG is a member of the tumor necrosis factor receptor superfamily, acts as a decoy receptor for RANKL, and competes with RANK for binding to RANKL.20-22 By this mechanism, OPG down-regulates osteoclast differentiation, activity and survival both in vivo and in vitro.23,24 Instead, RANKL is expressed by fibroblast-like synoviocytes (type B synoviocytes) and by activated T cells, and may induce osteoclastogenesis through a mechanism enhanced by several cytokines (e.g. tumor necrosis factor-α, interleukin-1 and interleukin-17) that promote both inflammation and bone resorption.25 With this as background, the aim of the present study was to investigate the differences in the severity of arthropathy in HA and HB by assessing clinical, imaging and biochemical markers.

Table 1. Clinical characteristics of hemophilia A and hemophilia B patient groups.

Hemophilia A (n=70)

Hemophilia B (n=35)

Median age and range (years) 33.5 (3-69) Primary and secondary 10 (15%) prophylaxis treatment (n, %) Tertiary prophylaxis treatment (n, %) 24 (34%) On demand treatment (n, %) 36 (51%) Viral infections HCV (n, %) 49 (70%) HCV-HIV (n, %) 7 (10%) None (n, %) 21 (30%)

34.6 (2-69) 5 (14%) 8 (23%) 22 (63%) 19 (54%) 5 (14%) 16 (46%)

Table 2. Clinical and imaging findings of hemophilia A and hemophilia B patient groups.

Hemarthrosis, n (%) <10 10-50 >50 Pettersson score, mean±SD WFH score, mean±SD US score, mean±SD US score >5, n (%)

Hemophilia A (n=70)

Hemophilia B (n=35)

11 (15.7) 16 (22.8) 43 (61.4) 6.81±3.99 36.6±21.6 10.91±4.05 46 (65.7)

15 (42.9) 3 (8.5) 17 (48.6) 5.64±4.02 20.2±14.6 4.34±3.39 11 (31.4)

<0.0001 0.001 0.03 0.2 <0.0001 <0.0001 0.003

WFH: World Federation of Hemophilia; US: ultrasound.

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Methods Patients’ characteristics Seventy hemophilia A patients and 35 hemophilia B patients attending the Center for Bleeding Disorders of Careggi University Hospital in Florence, Italy, were consecutively enrolled in the study. At recruitment, all these patients had suffered from at least one joint bleeding. Clinical and demographic characteristics of the study population are shown in Table 1. All patients gave informed consent, and the study protocol was approved by the institutional medical ethics committees.

Hemophilia A group The median age of HA patients was 33.5 years (range 3-69 years). All patients (100%) had severe HA (FVIII:C <1 IU/dL). Thirty-six out of 70 HA patients (51%) were treated on demand, and 10 of 70 (15%) and 24 of 70 (34%) with primary and secondary prophylaxis, respectively. According to the last guidelines for management of hemophilia of the World Federation of Hemophilia (WFH),26 the prophylaxis is defined as the long-term continuous factor replacement therapy two or three times per week at dosage of 25 U/kg. Primary prophylaxis is when it starts in the absence of documented osteochondral joint disease, as determined by physical examination and/or imaging studies, and before the second clinically evident large joint bleeding and the age of three years. Secondary prophylaxis is when it starts after two or more bleedings into large joints and before the onset of joint disease documented by physical examination and imaging studies.26 The tertiary prophylaxis is when it starts in the presence of documented joint disease. Fortynine out of 70 (70%) patients were HCV positive: HCV viremia haematologica | 2016; 101(2)

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was present in 28 of 49 subjects (57%) and HCV-RNA was undetectable (<15 IU/mol) in the other 21 patients (43%); 25 of 29 patients who had received anti-HCV therapy were still HCV positive. Seven out of 70 (10%) patients were also HIV positive with undetectable viremia (HIV-RNA <20 cp/mL), and were all receiving antiretroviral therapy.

Hemophilia B group Median age of HB patients was 34.6 years (range 2-69 years). All patients (100%) had severe HB (FIX:C <1 IU/dL). Twenty-two of 35 HB patients (63%) were treated on demand, 5 of 35 (14%) and 8 of 35 (23%) with primary and secondary prophylaxis, respectively. Nineteen of 35 (54%) patients were HCV positive: HCV viremia was present in 9 of 35 subjects (26%) and HCV-RNA was undetectable (<15 IU/mol) in the other 10 patients (29%) for sustained virological response to anti-HCV treatment. Five of 35 (14%) patients were also HIV positive with undetectable viremia (HIVRNA <20 cp/mL), and were all receiving antiretroviral therapy.

Clinical and imaging score The severity of arthropathy was measured using the WFH orthopedic joint scale score consisting of a physical examination

and pain scale.27 Knee X-ray was performed in all subjects over 14 years of age, while US was carried out and scored in each patient. X-ray score (Pettersson score) evaluates osteoporosis, enlarged epiphysis, irregular subchondral bone surface, narrowing of the joint space, subchondral cyst formation, erosions of the joint margins, gross incongruence of articulating bone ends, and deformity (angulation and/or displacement between articulating bones).28 The joint score for a single joint varies between 0 (normal joint) and 13 (i.e. a totally destroyed joint). US was performed by an experienced sonographer (DM) blinded with regard to diagnosis using ESAOTE my LAB 70 (linear probe 134 MHz, Milan, Italy). For a single joint, US score with 9 items was applied: 1) joint effusion; 2) fibrotic septa; 3) synovial hyperthropy with flags on power Doppler US (pDUS) or hemarthrosis; 4) synovial hyperthropy without flags on pDUS; 5) hemosiderin deposition; 6) bone erosion; 7) osteophytes; 8) bone remodeling; and 9) cartilage modifications. US score is based on a range from 0-21 with a cut off less than 5.16 Indeed, pDUS may identify synovial blood flow, synovitis or muscle hematoma in the extremities. Patients were divided into three groups according to the total number of hemarthrosis in their life: 1) patients with less than 10 hemarthrosis (<10); 2) patients with hemarthrosis 10-50 (10-50); and 3) patients with hemarthrosis greater than 50 (>50).

Serum analysis of soluble RANKL and OPG Blood samples were collected from all HA and HB patients. Thirty healthy subjects (median age 36.5 years, range 18-73 years) were used as controls. Serum levels of soluble RANKL (sRANKL) and OPG were measured by enzyme-linked immunosorbent assay according to the manufacturer’s instructions (Ampli-

Table 3. US findings of hemophilia A and hemophilia B patient groups.

Hemophilia A (n=70)

Figure 1. Circulating levels of osteoprotegerin (OPG) and soluble receptor activator of nuclear factor-κB ligand (sRANKL). Serum concentrations of OPG (A) and sRANKL (B) were determined by enzyme-linked immunosorbent assay in 70 patients with hemophilia A, 35 patients with hemophilia B and 30 healthy controls. Boxes show 25thand 75th percentiles. Vertical lines below and above boxes show 10th and 90th percentiles. Lines inside the boxes represent the medians, circles the outliers and asterisks the extreme values. Significant differences between patients with hemophilia A and healthy controls, as well as between hemophilia A and hemophilia B are indicated.

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Effusion, n (%) Small 16 (22.5) Moderate 18 (25.4) Large 22 (31) Fibrotic septa, n (%) 1 (1.4) Hemarthrosis, n (%) 11 (15.5) (>3 flags on pDUS) Synovial hypertrophy (without flags on pDUS), n (%) <1.5 mm 4 (5.6) 1.5-2.5 mm 8 (11.3) >2.5 mm 24 (33.8) Hemosiderin deposition, n (%) Small 10 (14.1) Moderate 14 (19.7) Large 9 (12.7) Bone erosion, n (%) 6 (8.5) Osteophytes, n (%) 27 (38) Bone remodeling, n (%) 63 (88.7) Cartilage modifications, n (%) Hyperechogenicity 33 (46.5) Irregular profile 18 (25.4) Calcification 17 (23.9)

Hemophilia B (n=35) 13 (37.1) 8 (22.9) 2 (5.7)* 2 (5.7) 5 (14.3)

3 (8.6) 2 (5.7) 13 (37.1) 2 (5.7) 2 (5.7) 2 (5.7) 1 (2.9) 5 (14.3)¥ 22 (62.9)** 4 (11.4)* 2 (5.2) 3 (8.6)

pDUS: power Doppler ultrasound; US: ultrasound. *P<0.0001 versus hemophilia A large effusion and hyperechogenicity. **P=0.001 versus hemophilia A bone remodeling. ¥P=0.01 versus hemophilia A osteophytes.

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sRANKL, Biomedica Medizinprodukte GmbH & Co, Wien; Human OPG Instant ELISA, Bender MedSystems, Wien, Austria).

Synovial biopsy samples and immunohistochemistry Eighteen HA and 4 HB patients suffering from severe knee arthropathy underwent arthroplasty and samples of synovial tissue obtained during surgery at the First Orthopedic Clinic in Florence were analyzed as described elsewhere.19 Synovial samples from 16 osteoarthritis (OA) patients were included as controls. Each synovial specimen was cut into small pieces, fixed in 10% buffered formalin and, after standard processing, embedded in paraffin wax and used for light microscopy. Immunohistochemistry was performed using the following mouse monoclonal antibodies: anti-RANK (Abcam, Cambridge, UK), anti-RANKL (Abcam), anti-OPG (Santa Cruz Biotechnology, Santa Cruz, CA, USA), as described elsewhere.19

Statistical analysis Statistical analysis was performed using the SPSS (Statistical Package for Social Sciences Inc., Chicago, IL, USA) software for Macintosh (v. 19.0). Values are expressed as mean ± standard deviation (SD) or median and interquartile range (IQR), as appropriate. c2 test was used to compare proportions. Student’s t-test was used

to compare two independent groups for normally distributed parameters, while Mann-Whitney U-test was used to compare two independent groups for non-normally distributed parameters. Spearman’s rank correlation coefficient (r) was used to analyze the relationship between two continuous variables. P<0.05 was considered statistically significant.

Results Clinical and imaging findings The overall results of clinical and imaging findings are shown in Table 2. The percentage of patients with less than 10 hemarthrosis was significantly higher in the HB group compared with the HA group (P<0.0001). Conversely, the percentage of patients with either 10-50 or more than 50 hemarthrosis was significantly greater in HA group than HB group (P<0.001 and P=0.03, respectively). The mean WFH clinical score and US score were significantly worse for the HA group, while no difference was observed in terms of the Pettersson score. The main results of the US findings are summarized in Table 3. Large joint effusion and cartilage modifications were more frequent in HA patients (P<0.0001 vs. HB).

Figure 2. Expression of receptor activator of nuclear factor-κB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG) in synovial tissue from patients with hemophilia A, hemophilia B and osteoarthritis. Representative microphotographs of tissue sections subjected to immunoperoxidase staining for RANK, RANKL and OPG (brownish-red color) and counterstained with hematoxylin are shown. Arrows indicate OPG immunostaining in the synovial lining layer. Original magnification: x20. Scale bar: 100 mm.

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Bone remodeling and osteophytes were also more frequent in HA patients (P=0.01 and P<0.01 vs. HB, respectively). There was no significant difference in hemarthrosis (>3 flags on pDUS), synovial hypertrophy without flags on pDUS, fibrotic septa, hemosiderin deposition and bone erosions between the two groups of patients. Furthermore, we compared Pettersson, WFH and US scores between HA and HB patients according to the number of hemarthrosis. All mean score values were higher for all HA groups (i.e. <10, 10-50 and >50) compared to the respective HB groups. In particular, a remarkable significant increase in WFH score was observed in HA more than 50 hemarthrosis group versus HB more than 50 hemarthrosis group (P<0.0001). As far as the US score is concerned, significantly higher scores were found in the HA 10-50 group versus the HB 10-50 group (P<0.0001), as well as in the HA more than 50 group versus the HB more than 50 group (P=0.001) (Table 4).

Circulating levels of OPG and sRANKL Serum OPG was significantly decreased in HA patients (median 22.15 pg/mL, IQR 15.83-39.63 pg/mL) compared both to controls (median 44.36 pg/mL, IQR 40.36-123.53 pg/mL) and HB patients (median 42.74 pg/mL, IQR 24.5450.81 pg/mL) (P<0.0001 for both comparisons) (Figure 1A). In HB patients, circulating levels of OPG did not differ to those from healthy controls (Figure 1A). When HA patients were stratified according to the number of hemarthrosis, OPG levels in less than 10 group (median 52.94 pg/mL, IQR 44.97-75.30 pg/mL) were significantly higher than in 10-50 (median 19.59 pg/mL, IQR 4.05-29.02 pg/mL) and in more than 50 (median 17.55 pg/mL, IQR 10.44-29.82 pg/mL) groups (P=0.004 and P<0.0001, respectively). In HB patients, serum OPG levels were higher, although not significantly, in less than 10 group (median 42.27 pg/mL, IQR 22.65-87.81 pg/mL) compared with both 10-50 (median 32.12 pg/mL, IQR 24.54-52.09 pg/mL) and more than 50 (median 29.69 pg/mL, IQR 20.19-43.59 pg/mL) groups. Furthermore, we compared OPG levels between HA and HB patients according to the number of hemarthrosis. Interestingly, OPG levels were significantly higher in the HB more than 50 group compared with the HA more than 50 group (P=0.02). In HA patients, circulating levels of OPG correlated inversely with WFH score (r=-0.44, P<0.0001), Pettersson score (r=-0.26, P=0.04) and US score (r=-0.39, P=0.001). In

HB patients, a trend toward a significant inverse correlation between OPG levels and all three scores was observed, although not statistically significant. Circulating levels of sRANKL were similar between HB patients and healthy controls (median 0.20 pmol/L, IQR 0.14-0.36 pmol/L vs. 0.23 pmol/L, IQR 0.15-0.68 pmol/L), while they were significantly lower in HA patients (median 0.16 pmol/L, IQR 0.09-0.20 pmol/L) compared both to controls (P=0.005) and HB patients (P=0.006) (Figure 1B). sRANKL levels did not correlate significantly with the number of hemarthrosis and scores in HA or HB patients (data not shown).

Expression of RANK, RANKL and OPG in synovial tissue Both in HA and HB synovium, RANK was strongly expressed in the lining and sublining layers, especially in synoviocytes and vascular endothelium. In OA, RANK was less expressed in the lining layer, while a strong immunopositivity was observed in the inflammatory infiltrate of the sublining layer (Figure 2). The expression of RANKL in the lining and sublining layers of HA and HB synovium was similar to that observed in OA (Figure 2). In synovial tissue from HB patients, the expression of OPG was increased compared with HA patients, particularly in the lining layer and sublining vessels. In HA synovium, only a few cells of the sublining layer were positive for OPG. In OA tissue, OPG was strongly expressed in synovial lining cells, as well as in endothelial cells (Figure 2).

Discussion In this study, we show that the WFH score and the US score are significantly worse in the HA group compared to the HB patient group when matched for age, even with a similar frequency of hemarthrosis. The lower mean US score observed in the HB group compared to the HA group (4.3 vs. 10.9) represents an important result for the follow up of these patients. US findings show that joint involvement is more marked in HA than in HB patients. Mainly, fewer large joint effusion and cartilage modifications (hyperechogenicity) and less bone remodeling were detected in HB patients. Similarly, the lower value of WFH clinical score in the HB group (20.2 vs. 36.6) indicates that the arthropathy is less severe in HB than in HA patients.

Table 4. Clinical, serological and imaging findings of hemophilia A and hemophilia B patient groups according to the number of hemarthrosis.

HA Number of hemarthrosis Patients, n (%) Age, years (range) Pettersson score, mean ± SD WFH score, mean ± SD US score, mean ± SD US score >5, n (%) OPG, median (range)

<10 11 (15.7) 26.6 (4-57) 4.2±2.7 10±5.7 5.8±3.7 6 (54.5) 52.94 (44.97-75.30)

<10 10-50 15 (42.9) 16 (22.8) 28.7 (1-65) 34.62 (11-69) 2.6±1.9 5.9±3.8 9±5.8 21.5±13.8 3.6±3.2 6.9±2.7 3 (8.5) 9 (56.2) 42.27 19.59 (22.65-87.81) (4.05-29.02)

10-50 3 (8.5) 56.33 (49-60) 4.3±1.1 16.3±13.5 1.7±0.6* 0 32.12 (24.54-52.09)

>50 43 (61.4) 35.5 (14-66) 9.6±8.6 48.6±16.2 9.4±4.2 31 (72) 17.55 (10.44-29.82)

>50 17 (48.6) 35.9 (12-69) 7.5±3.6 22.6.±16.4¥ 5.3±3.5** 8 (47) 29.69¥¥ (20.19-43.59)

HA: hemophilia A; HB: hemophilia B; WFH: World Federation of Hemophilia; US: ultrasound; OPG: osteoprotegerin. ¥P<0.0001 versus HA hemarthrosis >50. *P<0.0001 versus HA hemarthrosis 10-50; **P=0.001 versus HA hemarthrosis >50; ¥¥P=0.02 versus HA hemarthrosis >50.

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The lesser severity of HB with respect to HA is mostly supported by the fact that US and WFH scores were lower in HB than in HA patients matched for the number of hemarthrosis. Instead, the mean Pettersson score was 5.6 points for the HB group and 6.8 points for the HA group. These data may be explained both by the young patient age, also in the HA group, and because radiographic examination can detect abnormalities only in advanced stage, as demonstrated in previous studies.16,29-31 Another very important aspect concerning the severity of hemophilia was the number of hemarthrosis as marker of arthropathy.16 It is worthy of note that the percentage of the joint bleedings was lower in the HB group with respect to the HA group, also when matched for age. These results confirm the lower risk of bleeding and consequent arthropathy in HB, as also supported by the significant different distribution of patients according to the number of hemarthrosis between HA and HB groups. Moreover, as expected, we observed a greater use of on demand treatment in HB patients (63%) with respect to HA patients (51%) and a different use of prophylaxis in the two groups (49% in HA patients, 37% in HB patients). Our clinical data are in agreement with previous studies.7-9 In a previous study, we provided evidence of a strong correlation between the severity of arthropathy in HA patients and the expression of the RANK/RANKL/OPG triad in synovial tissue, as well as circulating levels of sRANKL and OPG.19 Therefore, in the present work we investigated for the first time the possible differences in the RANK/RANKL/OPG triad between HA and HB patients. Assuming that these cytokines are involved in the progression of the arthropathy, the markedly reduced expression of OPG, which plays a protective role for the subchondral bone, in HA, confirms the more severe clinical outcome of these patients. As a further confirmation, RANK and RANKL, which play a pivotal role in osteoclast activation and bone erosions, were strongly expressed in the synovium of HA. On the contrary, a marked increase in OPG and sRANKL serum levels in the HB compared to the HA group was found. This behavior mirrored the OPG

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and sRANKL serum levels in healthy controls, thus strengthening the hypothesis that the arthropathy in HB may be less severe and exhibit different features compared to HA. This conclusion is further supported by the significantly higher serum levels of OPG found in the HB more than 50 hemarthrosis group compared with the HA patients of the same group. Furthermore, the histological analysis on synovial tissue of 4 HB patients underlined important differences in the expression of OPG compared with HA. Collectively, these data confirm that the arthropathy is less severe in HB patients, in keeping with the lower number of patients who went on to arthroplasty, and the increased expression of OPG compared to the HA group. It has been demonstrated that even a single or a few episodes of joint bleeding are sufficient to initiate the arthropathy, since even microhemorrhages into the joint may cause articular deterioration in HA.32 Furthermore, joint bleeding affects the synovial tissue, resulting in synovitis and subsequent articular cartilage damage, mainly caused by the excretion of tissue-destructive mediators, such as enzymes and cytokines.14,15,33-35 In conclusion, our results suggest that there are clinical differences between HB and HA and that the degree of arthropathy is more severe in HA patients, as supported by the higher number of hemarthrosis and lower levels of OPG both in serum and synovium. Our data suggest that the synovitis may play a crucial role in blood-induced arthropathy provoking an overreaction which subsequently becomes independent from bleeding, as postulated in other studies.36,37 In addition, on the basis of our findings, the reduction in OPG seems to play a pivotal role in the progression of arthropathy and could even serve in the future as a biomarker of disease severity. Thus, an early clinical, instrumental and serological screening of all hemophiliacs may be recommended. Further investigation of the mechanisms promoting and sustaining blood-induced synovial inflammation will be necessary to shed additional light on the pathogenesis of hemophilic arthropathy.

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expression of the RANK/RANKL/osteoprotegerin system in peripheral spondyloarthritis is partially disconnected from inflammation. Arthritis Rheum. 2008;58(3): 718-729. Srivastava A, Mahlangu JN, Brewer AK, et al. Guidelines for the management of hemophilia. Hemophilia. 2013;19(1):e1-e47. Gilbert MS. Prophylaxis: musculoskeletal evaluation. Semin Hematol. 1993;30(3 Suppl 2):3-6. Pettersson H, Ahlberg A, Nilsson IM. A radiologic classification of the haemophilic arthropathy. Clin Orthop Relat Res. 1980;149:153-159. Kulkarni MV, Drolshagen LF, Kaye JJ, et al. MR imaging of hemophiliac arthropathy. J Comput Assist Tomogr. 1986;10(3):445449. Kilcoyne RF, Nuss R. Radiological assessment of haemophilic arthropathy with emphasis on MRI findings. Haemophilia. 2003;9(Suppl 1):57-63. Doria AS, Lundin B, Kilcoyne RF, et al. Reliability of progressive and additive MRI scoring systems for evaluation of haemophilic artrhopathy in children: expert MRI Working Group of the International Prophylaxis Study Group.

Haemophilia. 2005;11(3):245-253. 32. Manco-Johnson MJ, Abshire TC, Shapiro AD, et al. Prophylaxis versus episodic treatment to prevent joint disease in boys with severe haemophilia. N Engl J Med. 2007;357(6):535-544. 33. Roosendaal G, van Rinsum AC, Vianen ME, et al. Haemophilic arthropathy resembles degenerative rather than inflammatory joint disease. Histopatology. 1999;34(2): 144-153. 34. Jansen NW, Roosendaal G, Bijlsma JW, Degroot J, Lafeber FP. Exposure of human cartilage tissue to low concentrations of blood for a short period of time leads to prolonged cartilage damage: an in vitro study. Arthritis Rheum. 2007;56(1):199-207. 35. Hoots WK, Rodriguez N, Boggio L, Valentino LA. Pathogenesis of haemophilic synovitis: clinical aspects. Haemophilia. 2007;13(Suppl. 3):4-9. 36. Jansen NW, Roosendaal G, Lafeber FP. Understanding haemophilic arthropathy: an exploration of current open issues. Br J Haematol. 2008;143(5):632-640. 37. Rodriguez-Merchan EC. Cartilage damage in the haemophilic joints: pathophysiology, diagnosis and management. Blood Coagul Fibrinolysis. 2012;23(3):179-183.

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ARTICLE EUROPEAN HEMATOLOGY ASSOCIATION

Non-Hodgkin Lymphoma

Ferrata Storti Foundation

Rationale for optimal obinutuzumab/GA101 dosing regimen in B-cell non-Hodgkin lymphoma

Guillaume Cartron,1 Florence Hourcade-Potelleret,2 Franck Morschhauser,3 Gilles Salles,4 Michael Wenger,5 Anna Truppel-Hartmann6 and David J. Carlile7

CHRU Montpellier, UMR-CNRS 5235, Université de Montpellier, France; F. Hoffmann-La Roche Ltd, Basel, Switzerland*; 3CHRU Lille, Unité GRITA, Université de Lille, France; 4Hospices Civils de Lyon, Université Claude Bernard, France; 5Genentech, South San Francisco, CA, USA; 6F. Hoffmann-La Roche Ltd, Basel, Switzerland; and 7Roche Pharmaceutical Research and Early Development, Roche Innovation Center Welwyn, UK† 1

Haematologica 2016 Volume 101(2):226-234

*Current affiliation: Novartis Pharma AG, Basel, Switzerland † Current affiliation: AstraZeneca UK, Melbourn Science Park, Melbourn, UK

ABSTRACT

Correspondence: g-cartron@chu-montpellier.fr

Received: 10/07/2015. Accepted: 3/12/2015. Pre-published: 11/12/2015. doi:10.3324/haematol.2015.133421

Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/101/2/226

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binutuzumab (GA101) is a type II, glycoengineered anti-CD20 monoclonal antibody for the treatment of hematologic malignancies. Obinutuzumab has mechanisms of action that are distinct from those of rituximab, potentially translating into improved clinical efficacy. We present the pharmacokinetic and clinical data from the phase I/II GAUGUIN and phase I GAUDI studies that were used to identify the obinutuzumab dose and regimen undergoing phase III assessment. In phase I (GAUGUIN and GAUDI), non-Hodgkin lymphoma patients received up to a maximum 9 fixed doses (obinutuzumab 50-2000 mg). In GAUGUIN phase II, patients received obinutuzumab 400/400 mg or 1600/800 mg [first dose day (D)1, D8, cycle (C) 1; second dose D1, C2-C8]. The influence of demographic factors on pharmacokinetics and drug exposure on tumor response and toxicity were analyzed using exploratory graphical analyses. Obinutuzumab serum concentrations with 1600/800 mg were compared with a 1000 mg fixed-dose regimen (D1, D8 and D15, C1; D1, C2-C8) using pharmacokinetic modeling simulations. Factors related to CD20-antigenic mass were more influential on obinutuzumab pharmacokinetics with 400/400 versus 1600/800 mg. Higher serum concentrations were observed with 1600/800 versus 400/400 mg, irrespective of CD20-antigenic mass. Tumor shrinkage was greater with 1600/800 versus 400/400 mg; there was no significant increase in adverse events. Fixed dose 1000 mg with an additional C1 infusion resulted in similar serum concentrations to 1600/800 mg in model-based analyses. The obinutuzumab 1000 mg fixed-dose regimen identified in this exploratory analysis was confirmed in a full covariate analysis of a larger dataset, and is undergoing phase III evaluation. GAUGUIN and GAUDI are registered at www.clinicaltrials.gov (clinicaltrials.gov identifier:00517530 and 00825149, respectively).

Introduction The chimeric anti-CD20 monoclonal antibody rituximab (IgG1k) has revolutionized the treatment of hematologic malignancies. Rituximab plus chemotherapy has become the standard-of-care treatment for follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL).1-6 Single-agent rituximab is also used as FL maintenance therapy.7,8 Rituximab was the first monoclonal antibody approved for use in malignant diseases. The dose and schedule of haematologica | 2016; 101(2)

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rituximab used were mostly developed empirically, without extensive studies to establish dose or dose scheduling, as the substantial clinical benefits achieved with rituximab led to its rapid incorporation into treatment regimens. In phase I studies, indolent non-Hodgkin lymphoma (iNHL) patients received rituximab doses ranging from 10–500 mg/m2.9,10 As no dose-limiting toxicity or clear dose– response relationship was found, the 375 mg/m2 dose was selected for further clinical evaluation.11 This dose was approved in Europe and the US for the treatment of several hematologic cancers.12,13 Pivotal studies have shown large inter-individual variability in rituximab serum concentrations.11,14 As rituximab concentrations correlate with clinical response and progression-free survival (PFS),14-16 an optimized dosing regimen should translate into improved clinical response. Yet personalized, pharmacokinetic (PK)-based maintenance dosing of rituximab 375 mg/m2, administered when serum concentrations decreased to less than 25 mg/mL, yielded no clinical advantage in iNHL.17 However, treatment responders had higher serum concentrations of rituximab than non-responders at one and four months post-treatment, although patient numbers were too small to draw firm conclusions and non-responders still had rituximab serum levels near the target range.17 It is likely that rituximab serum concentrations are the result of a complex interplay of several biological factors that influence antibody behavior (i.e. distribution, catabolism), including histology (CLL vs. NHL), CD20 expression levels on malignant B cells, and tumor burden.11,14,18,19 In vitro studies showed that FcγRIIIa-158VF polymorphisms impact the concentration-effect relationship of rituximab-induced antibody-dependent cell-mediated cytotoxicity (ADCC),20 a critical mechanism of anti-tumor activity.21As these biological parameters can vary between individuals, the PK of rituximab, and thus disease control, can differ between patients. To optimize dosing, there is a critical need to understand the factors affecting the PK of anti-CD20 monoclonal antibodies, particularly those under clinical development. Obinutuzumab is a novel, type II, glycoengineered, antiCD20 monoclonal antibody. Glycoengineering of obinutuzumab has enhanced its binding to the Fc portion of FcγRIIIa expressed by effector cells versus the non-glycoengineered rituximab. The glycoengineered region of obinutuzumab is also expected to reduce the influence of the FcγRIIIa-158VF polymorphism on the in vivo activity of obinutuzumab versus rituximab. Thus, obinutuzumab exhibits enhanced in vitro ADCC versus rituximab.22 As obinutuzumab recognizes a type II epitope on the CD20 antigen, its mechanisms of action are distinct from that of type I anti-CD20 antibodies, such as rituximab.22 For example, the binding of obinutuzumab to CD20 does not induce the translocation of antibody-CD20 complexes into lipid rafts, leading to a lack of complement-dependent cytotoxicity (CDC). Moreover, the binding of obinutuzumab to CD20 leads to increased direct cell death versus rituximab. Obinutuzumab-induced direct cell death is mediated via pathways distinct from classical apoptosis, and may involve actin reorganization and homotypic adhesion.23 Animal studies showed that these mechanistic differences translate into superior efficacy for obinutuzumab versus rituximab.22 Clinically, obinutuzumab monotherapy has exhibited encouraging activity in phase I and II studies in NHL and CLL.24-27 Given the relationship haematologica | 2016; 101(2)

between rituximab serum concentrations and patient outcomes and the mechanistic differences between obinutuzumab and rituximab, early clinical trials assessed the relationship between PK, safety and efficacy with tumor markers. These data were used to explore the relationship between obinutuzumab PK and response, and, in conjunction with PK modeling techniques, to identify an optimized dose and regimen for phase III studies in NHL.

Methods Patients and study designs Pharmacokinetic data for analyzing optimal obinutuzumab dose and schedule were derived from iNHL and aggressive NHL (aNHL) patients participating in the phase I/II GAUGUIN and phase Ib GAUDI studies. In GAUGUIN phase I (dose escalation), 21 heavily pre-treated patients with relapsed/refractory CD20positive iNHL received obinutuzumab monotherapy at a dose of 50/100, 100/200, 200/400, 400/800, 800/1200, 1200/2000, or 1600/800 mg (8 x 21-day cycles; n=3 per cohort). The former dose of each regimen was administered at first infusion (D1, C1) and the latter was infused for the remainder of the regimen (D8, C1; D1, C2–C8).26 The dose selection for GAUGUIN phase II was based on the safety, efficacy and PK results obtained during the phase I dose escalation. During phase II, iNHL (FL) or aNHL [DLBCL, mantle cell lymphoma (MCL)] patients were randomized to obinutuzumab 400/400 mg or 1600/800 mg (former dose of each regimen administered on D1 and D8, C1; latter dose administered on D1, C2–C8; 21-day cycles).24,25 The GAUDI study examined the safety and efficacy of obinutuzumab plus chemotherapy [CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or FC (fludarabine and cyclophosphamide)] in relapsed/refractory FL. Patients (n=56) were randomized to obinutuzumab 400/400 mg plus CHOP, obinutuzumab 400/400 mg plus FC, obinutuzumab 1600/800 mg plus CHOP, or obinutuzumab 1600/800 mg plus FC (n=14 per cohort). The number of cycles received was dictated by the chemotherapy backbone (CHOP: 8 x 21-day cycles; FC: 6 x 28-day cycles). In all cohorts, obinutuzumab was administered on D1 and D8, of C1, and D1 of subsequent cycles.28 In both GAUGUIN and GAUDI, patients had 1 or more bidimensionally measurable lesion (>1.5 cm by computerized tomography scan), life expectancy of more than 12 weeks, and an Eastern Cooperative Oncology Group performance status of 0-2. All patients provided informed consent. The GAUGUIN and GAUDI studies conformed to the Declaration of Helsinki and the International Conference on Harmonisation Guidelines for Good Clinical Practice, and the protocols were approved by local or institutional ethics committees. CONSORT diagrams for GAUGUIN and GAUDI have been published previously.24,25,28

PK assessments and bioanalytical methods Timings for blood sample collection, bioanalytical methods used and calculation of PK parameters are described in the Online Supplementary Methods.

Tumor response and toxicity assessments Tumor response was assessed according to International Workshop criteria for NHL.29 Tumor burden was estimated by the sum of product diameters of six specified target lesions.14 Incidences of the grade 3/4 adverse events (AEs), neutropenia and infection were calculated according to the National Cancer 227

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Institute Common Terminology Criteria for Adverse Events (v.3.0).

Graphical analyses Exploratory graphical analyses were conducted to assess the influence of base-line characteristics on obinutuzumab PK, the relationship between obinutuzumab exposure and tumor response, and the relationship between exposure and toxicity (Online Supplementary Methods).

and inter-individual variability was observed within each cohort (Figure 1A). As one patient each in the 50/100 mg, 100/200 mg, 200/400 mg, 800/1200 mg, and 1600/800 mg dose groups responded to treatment, no relationship between dose and efficacy was identified.26 These PK data indicated that serum concentrations of obinutuzumab increased with higher doses, and that a dose of at least 400/800 mg was required for CD20 target saturation.26 As a result, two doses of obinutuzumab were selected for

Pharmacokinetic modeling Pharmacokinetic model development, evaluation and simulations are described fully in the Online Supplementary Methods. In brief, PK model simulations compared obinutuzumab serum concentrations following the 1600/800 mg regimen (D1 and D8, C1; D1, C2–C8) with a new 1000 mg fixed-dose regimen (D1, D8, and D15, C1; D1, C2–C8; 21-day cycle).

Results Patients' characteristics Demographic and base-line disease characteristics of iNHL and aNHL patients participating in phase I and II studies of obinutuzumab have been published24-26,28 and are summarized in Online Supplementary Table S1. Median age ranged from 59.8 years in patients randomized to obinutuzumab plus CHOP in GAUDI to 71.0 years among aNHL patients participating in GAUGUIN phase II. In contrast to GAUGUIN phase I, there were more male than female participants in phase II (43% vs. 63%-68%). Patients in GAUGUIN phase I also had less advanced disease than those in phase II (62% stage III-IV vs. 76%90%). The recruitment criteria resulted in disease histology and rituximab-refractory status varying across trials. Almost all patients in GAUGUIN and GAUDI had received rituximab, with the median number of any previous anti-lymphoma treatments received by patients ranging from 1-5.

Pharmacokinetics of obinutuzumab monotherapy In GAUGUIN phase I, patients received up to nine infusions of obinutuzumab monotherapy at doses ranging from 50-2000 mg. Two infusions were administered during cycle 1 to quickly elevate serum concentrations of obinutuzumab. No dose-limiting toxicity was established,

Figure 1. Mean obinutuzumab concentrations. Mean obinutuzumab concentrations (± standard error of mean) observed in (A) phase I and (B) phase II of GAUGUIN.

Table 1. Obinutuzumab pharmacokinetic parameters of patients with non-Hodgkin lymphoma participating in phase II of the GAUGUIN study.

Geometric mean (SD) Cmax, mg/mL Ctrough, mg/mL AUC7d, mg*day/mL AUClast, mg*day/mL VD, L CL, mL/day t1/2, days

Cycle 1 (day 1) 400/400 mg 1600/800 mg n=38 n=41 130 (93.0) NC 554 (255) NC NC NC NC

502 (263) NC 2250 (561) NC NC NC NC

Cycle 1 (day 8) Cycle 4 (day 1) Cycle 8 (day 1) 400/400 mg 1600/800 mg 400/400 mg 1600/800 mg 400/400 mg 1600/800 mg n=37 n=40 n=24 n=31 n=22 n=25 182 (108) NC 879 (360) NC NC NC NC

710 (163) NC 3940 (1010) NC NC NC NC

NC 133 (108) NC NC NC NC NC

NC 334 (187) NC NC NC NC NC

339 (179) 645 (265) 172 (116) 368 (196) 2020 (1130) 3850 (1650) 14900 (16100) 27200 (21900) 4.34 (1.49) 6.99 (5.29) 103 (83.8) 92.1 (35.5) 26.3 (11.5) 29.3 (11.7)

AUC: area under the curve; AUC7d: AUC from time zero to day 7; AUClast: AUC from time zero to the last recorded measurement; CL: clearance; Cmax: maximum (peak) plasma drug concentration; Ctrough: minimum (trough) plasma drug concentration; NC: not calculated (data availability limited by intensive dosing schedule during cycle 1 and restricted sampling in cycle 4); NHL: non-Hodgkin lymphoma; SD: standard deviation; t1/2: half life; VD: volume of distribution.

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evaluation in GAUGUIN phase II: a low-dose cohort receiving 400/400 mg (patients received 400 mg throughout the study: D1 and D8, C1; D1, C2â&#x20AC;&#x201C;C8), and a highdose (1600/800 mg) cohort [patients received 1600 mg of obinutuzumab on D1 and D8, C1, and 800 mg for subsequent cycles (D1, C2â&#x20AC;&#x201C;C8)].25 In the current analysis, AUClast values at cycles 1 and 8 demonstrated that obinutuzumab exposure was higher throughout treatment for the 1600/800 mg versus 400/400 mg dose group (P<0.001 at cycles 1 and 8). Cmax for the 1600/800 mg group on D8 of C1 was comparable to that on D1 of C8, confirming the role of the significant loading dose in achieving steady-state concentrations as early as possible after treatment initiation (Table 1 and Figure 1B). There was clear evidence of a relationship between dose and exposure with obinutuzumab, with statistically significantly higher Cmax and AUC observed in the 1600/800 mg cohort versus the 400/400 mg cohort (both P<0.05) (Table 1).

Factors influencing the pharmacokinetic parameters of obinutuzumab Demographic and base-line parameters: to explore the influ-

ence of different clinical parameters on obinutuzumab PK variability, patients in GAUGUIN phase II were stratified by age (older or younger than the median for each patient cohort), sex, bodyweight, B-cell count before treatment, disease stage (I-II vs. III-IV), and tumor bulk (>5 cm or <5 cm) (Figures 2 and 3). In the low-dose group (400/400 mg), serum concentrations of obinutuzumab were affected by clinical parameters related to CD20-antigenic mass (tumor bulk, disease stage, B-cell count), and varied more for those patients with higher versus lower base-line lymphocyte count, stage, and tumor burden. In contrast, for the high-dose group (1600/800 mg), CD20-antigenic mass had little impact on PK. There was also high inter-individual variation in serum concentration with the low dose of obinutuzumab (400/400 mg) (Figure 2A). However, this inter-individual variation decreased with increasing dose, with an apparent decrease in the elimination process at the highest dose (1600/800 mg) (Figure 2A). This pattern suggests that the CD20 target sites became saturated at the highest dose. In addition, tumor shrinkage was greater among patients in the 1600/800 mg group versus the 400/400 mg group (Figure 2B), further demonstrating that the 1600/800 mg dosing regimen allowed patients to

Figure 2. Serum concentrations of obinutuzumab and tumor sizes. (A) Inter-individual serum concentrations of obinutuzumab by tumor burden and dose group in patients with iNHL participating in phase II of GAUGUIN. (B) Inter-individual change in tumor size by dose group in patients with iNHL.

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Figure 3. Time-course of obinutuzumab serum concentrations stratified by clinical parameters and dose group in patients with nonHodgkin lymphoma participating in phase II of GAUGUIN.

obtain optimal obinutuzumab serum concentrations, which facilitates clinical efficacy. With regard to demographics, age did not appear to alter obinutuzumab PK for patients aged 22â&#x20AC;&#x201C;85 years (Figure 3A). In the 400/400 mg group, there was a small increase in serum concentrations for women versus men (Figure 3B), for patients with high bodyweight (> median) versus those with low bodyweight (< median) (Figure 3C), for patients with stage I/II versus stage III/IV disease (Figure 3E), and for patients with non-bulky disease versus those with bulky disease (Figure 3F). These differences in serum concentration with sex, weight, disease stage and bulky disease were not observed in the 1600/800 mg group, showing that this dosing regimen conferred high exposure irrespective of patientsâ&#x20AC;&#x2122; initial demographic characteristics. Trough serum concentrations did not vary according to B-cell count for either dosing regimen (Figure 3D). NHL subtype: an exploratory graphical analysis demonstrated a high degree of variability in obinutuzumab serum concentrations across different NHL subtypes (Figure 4). MCL patients had lower serum concentrations 230

of obinutuzumab than iNHL or DLBCL patients, possibly due to treatment during the leukemic phase of MCL for some patients. However, it should also be noted that only 4 MCL patients were recruited to GAUGUIN phase II.24 In terms of clinical efficacy, higher tumor response rates were observed in iNHL versus aNHL patients (55% vs. 32%). Although the percentage of responders with MCL is similar to that observed among iNHL patients (50% vs. 55%), only the 4 GAUGUIN MCL patients were included, 2 of whom exhibited partial response (PR).24

Effect of obinutuzumab exposure on overall response rates and tumor response Clinical response rates for patients participating in GAUGUIN phase II have been published.24,25 In the cohort of aNHL patients, overall response rate (ORR), measured as end-of-treatment response (ETR), was similar between the two dosing groups: for 400/400 mg, ETR was 24% (80%CI: 12-40) and 2 patients (10%) had a complete response (CR); for 1600/800 mg, ETR was 32% (80%CI: 18-49) and no patient had a CR; difference haematologica | 2016; 101(2)

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between groups was not statistically tested.24 In iNHL patients, an apparent improvement in ORR was observed for the higher dose group versus the lower dose group: for 1600/800 mg, ETR was 55% (95%CI: 32-76) and 2 patients (9%) had a CR, whereas for 400/400 mg, ETR was 17% (95%CI: 4-41) and no patient had a CR.25 The difference in ETR between groups in the iNHL patients was 38% (95%CI: 7-68), which was statistically significant.25 An exploratory graphical analysis indicated that patients with higher mean obinutuzumab serum concentrations during the first 5 cycles of treatment may experience greater inhibition of tumor growth. At week 6, tumor size decreased by a median of 17% (95%CI: −81-32) in patients with the lowest mean serum concentrations of obinutuzumab (<160 mg/mL), 38% (95%CI: −72- −5) in patients with serum concentration 160–371 mg/mL and 48% (95%CI: −72- −16) in those with the highest mean concentrations (>371 mg/mL) (Figure 5).

was also supported by the stronger response in iNHL patients with the 1600/800 mg dose (55%) than with the 400/400 mg dose (17%), coupled with more marked decreases in tumor size at the higher dose and at higher obinutuzumab concentrations (Figures 2B and 5). However, a single dose for all infusions would be significantly more convenient and easier to administer than a bodyweight- or size-adjusted dose. A dose of 1000 mg with three infusions during cycle 1 was then proposed. Under such a regimen, patients would receive a cumulative obinutuzumab dose of 3000 mg in cycle 1, similar to the 3200 mg received during cycle 1 by patients randomized to the 1600/800 mg dose arm of GAUGUIN phase II. PK model-based simulations showed that this new regimen would result in trough serum concentrations of obinutuzumab similar to those observed with the 1600/800 mg dose (Figure 6). Therefore, obinutuzumab 1000 mg administered on D1, D8, and D15 of C1 (21-day cycle) and D1 of subsequent cycles has been selected as the dose and regimen for phase III clinical trial assessment.

Effect of obinutuzumab exposure on toxicity Previous studies showed that the addition of rituximab to chemotherapy increases the incidence of neutropenia, which is usually transient and manageable.1,3,7 Combined AE data from the GAUGUIN phase II iNHL study and GAUDI study showed that incidence of grade 3 or 4 neutropenia was similar for low- and high-dose groups, i.e. 13 of 46 (28%) and 15 of 50 (30%) for the 400/400 mg and 1600/800 mg groups, respectively. The obinutuzumab doses received by 3 patients in the GAUGUIN phase II aNHL and GAUGUIN phase I studies who had grade 3 or 4 neutropenia were not reported. Exploratory graphical analysis indicated that there was no difference in obinutuzumab exposure between patients who had grade 3/4 neutropenia and those who did not (Online Supplementary Figure S1). No grade 3/4 infections were observed in GAUGUIN phase I.26 In phase II, 4 iNHL patients (400/400 mg, n=1; 1600/800 mg, n=3) and no aNHL patients experienced grade 3/4 infections.24,25 In the GAUDI study, there were 4 patients with grade 3/4 infections in the 400/400 mg cohort (G-CHOP, n=3; G-FC, n=1) and 7 in the 1600/800 mg cohort (G-CHOP, n=3; G-FC, n=4).28 In total in the GAUGUIN phase II and GAUDI studies, 5 of 67 patients (7%) who received the 400/400 mg dose and 10 of 69 (14%) who received 1600/800 mg experienced grade 3/4 infections. Exploratory graphical analysis showed that there was no difference in obinutuzumab exposure between patients who had grade 3/4 infections and those who did not (Online Supplementary Figure S2).

Discussion Obinutuzumab is a humanized, glycoengineered, type II anti-CD20 monoclonal antibody, with demonstrated clinical impact in rituximab-refractory patients with DLBCL, MCL, iNHL and FL.24,25,28 In the randomized, phase III CLL11 study in patients with previously untreated CLL and comorbidities, PFS was significantly longer with obinutuzumab plus chlorambucil (G-Clb) than with rituximab plus chlorambucil (R-Clb).30 The standard rituximab dose and regimen was used (375 mg/m2, D1, C1; 500 mg/m2 D1, C2-C6), while obinutuzumab was administered at a fixed dose of 1000 mg, with additional infusions on D8 and D15 of C1. As a result, the median cumulative antibody dose was increased in the G-Clb arm (8000 mg) versus the R-Clb arm (5106 mg).30 Median cumulative chlorambucil dose was similar between arms (G-Clb: 366 mg; R-Clb: 396 mg). Therefore, the observed differences in efficacy between the study arms may have been influenced by differences in exposure and pharmacokinetics of the two antibodies. Given differences in the nature of CD20 binding, the

Pharmacokinetic model for obinutuzumab A basic structural model of obinutuzumab PK is shown in Online Supplementary Figure S3, and a summary of the final parameter estimates of the time-varying model is shown in Online Supplementary Table S2. The goodness-offit diagnostic plots and visual predictive checks did not indicate any model deficiencies (Online Supplementary Figures S4 and S5).

Pharmacokinetic predictions to select an optimal dosing regimen Based on PK data collected during phases I and II of GAUGUIN, the 1600/800 mg dose of obinutuzumab was identified as likely to produce the highest serum concentrations of obinutuzumab in NHL patients. This decision haematologica | 2016; 101(2)

Figure 4. Time course of obinutuzumab serum concentrations in the 1600/800 mg dose group stratified by disease type.

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biological pathways activated and the underlying mechanism of action, the dose and schedule of obinutuzumab could not be extrapolated from rituximab and the early clinical development program was designed to identify an optimal dose for this new molecule. Consequently, the early clinical studies were undertaken to optimize the dose and schedule of obinutuzumab to achieve the maximal clinical response possible without adversely affecting safety. In this current analysis, PK data from phases I and II clinical studies and a population PK model were used to identify the treatment regimen likely to be clinically effective for the majority of patients with CD20-positive NHL: obinutuzumab 1000 mg administered on D1, D8, and D15 of C1 (21-day cycle) and D1 of C2â&#x20AC;&#x201C;C8. With this regimen, target saturation should occur quickly after treatment initiation and serum concentrations of obinutuzumab should be maintained thereafter throughout the treatment course (i.e. steady state). This dose and schedule is anticipated to be clinically effective for the majority of patients with CD20-positive NHL. At the doses tested clinically, obinutuzumab, like rituximab,9,10 did not display dose-limiting toxicity. In patients treated with low-dose obinutuzumab (400/400 mg), PK data showed an increase in serum concentrations over the treatment duration consistent with the hypothesis that target saturation was not fully achieved in the low-dose cohort. Among patients administered high-dose obinutuzumab (1600/800 mg), steady state was reached quickly, by cycle 2, consistent with the hypothesis of a high degree of target saturation. Clinically, this may translate into improved tumor response rates among patients in the high-dose cohort versus the low-dose cohort. With regard to patient demographics, weight and sex had an apparent influence on PK, with increased exposure in women or individuals with lower bodyweight. This observation is in agreement with the PK characteristics of antibodies in which clearance usually increases with bodyweight. A fixed-dose strategy with obinutuzumab will, therefore, lead to moderate overexposure in patients

with low bodyweight. In a separate detailed PK analysis, bodyweight was shown to not have a clinically significant effect on the PK of obinutuzumab.31 The observation that women with NHL had higher obinutuzumab exposure than men is similar to previous observations with rituximab PK in elderly DLBCL patients.32,33 Elderly females with DLBCL who received rituximab plus CHOP therapy had slower rituximab clearance and improved PFS versus elderly males; however, these trends were not seen for younger patients. This gender difference appears to be more pronounced in elderly patients treated with rituximab plus chemotherapy versus chemotherapy alone.33-36 Therefore, rituximab clearance rates in women appear to decrease with age at a faster rate than in men, resulting in greater exposure to rituximab and subsequently improved outcomes. Results from the SMARTE-R-CHOP and DENSE-R-CHOP trials suggested that increasing the dosing density of rituximab may allow elderly male patients to achieve the same exposure and clinical outcomes as their female counterparts.36,37 In our analysis, female NHL patients receiving the obinutuzumab 400/400 mg dose had higher obinutuzumab exposure than men, indicating a gender difference in obinutuzumab PK. However, women and men had equivalent serum obinutuzumab concentrations at the higher dosing regimen of 1600/800 mg, indicating that optimal exposure is achieved for both genders with this dosing regimen. This observation is supported by findings from the recent PK analysis, where sex did not have a clinically significant effect on the PK of obinutuzumab.31 Our data revealed a relationship between serum concentrations of obinutuzumab and base-line tumor burden: in patients with high base-line tumor burden who received low-dose obinutuzumab (400/400 mg), antibody PK was highly variable. In contrast, the PK of the higher obinutuzumab dose was more homogeneous and independent of base-line tumor burden. Serum concentrations of obinutuzumab are a likely reflection of CD20 saturation, with target saturation yielding higher systemic antibody levels.

Figure 5. Change in tumor size by mean plasma concentration of obinutuzumab during the first six weeks of treatment in phase I and II of the GAUGUIN study.

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Rationale for obinutuzumab dosing in B-cell NHL

Figure 6. Predicted serum concentrations of obinutuzumab 1000/1000 mg (blue line) versus 1600/800 mg (red line).

We, therefore, postulate that CD20 target sites become saturated at increased doses of obinutuzumab. Stratifying patients by disease stage and B-cell count, exploratory graphical analyses indicated marked differences in trough serum concentrations of obinutuzumab infused at 400/400 mg. For example, trough serum concentrations of antibody were lower in patients with stage III/IV versus stage I/II disease. These differences were not apparent or were considerably less evident in patients receiving the 1600/800 mg dose. Yet PK differences did emerge for the higher dose in patient subgroups defined by disease histology, with higher serum concentrations observed for iNHL versus aNHL patients. Higher clinical response rates were also observed in iNHL versus aNHL patients (55% vs. 32%).24,25 These findings are supported by recent detailed obinutuzumab PK analyses where MCL patients were shown to have higher clearance rates than CLL patients, while those with DLBCL or BCL had the lowest clearance rates.31 These observations are likely due to differences in the levels of circulating target B cells in these diseases, and the relative expression of CD20 receptors on circulating B cells.38-40 Although nearly all of the patients in GAUDI and GAUGUIN had previously received rituximab treatment, this would have had little or no influence on obinutuzumab PK. When obinutuzumab treatment started, concentrations of rituximab in patients previously treated with this antibody would have been minimal. The only possible carry-over effect of rituximab would have been a marginal depletion of the CD20-cell population, which would have allowed a slightly faster saturation of CD20 by obinutuzumab to occur. In those patients who were refractory to rituximab, circulating CD20 cells might still have been depleted, but some CD20 production sites would have been active, leading to relapse. In rituximab-naïve patients, the intensive dosing schedule of obinutuzumab would be expected to rapidly saturate the target and deplete the CD20-cell population.

References 1. Coiffier B, Lepage E, Briere J, et al. CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma. N Engl J Med. 2002;346(4):235-242.

haematologica | 2016; 101(2)

Because of an intensive dosing schedule during cycle 1 and limited sampling in cycle 4, the data collected did not allow adequate characterization of obinutuzumab elimination using simple non-compartmental analysis (NCA), so PK parameters such as distribution volume and clearance could not be determined. A more intensive sampling schedule during cycle 8 did allow adequate characterization, and pharmacokinetic modeling was, therefore, undertaken as planned, enabling fuller characterization, from which firmer conclusions could be drawn. The clinical safety of obinutuzumab has been evaluated in a number of clinical studies, including GAUGUIN,24-29,30 and no dose-limiting toxicity has been observed, despite administration of doses of up to 2000 mg. The major safety events observed have been neutropenia, infections and infusion-related reactions, none of which are related to obinutuzumab dose or exposure.31 Therefore, in the context of a large therapeutic window for obinutuzumab, this fixed-dose strategy is an acceptable outcome.

Conclusion Although the high dose of obinutuzumab (1600/800 mg) has favorable PK properties and demonstrable clinical efficacy, a single fixed-dose regimen would be significantly more convenient in routine clinical practice. PK predictions have shown that a fixed dose of 1000 mg of obinutuzumab with three doses instead of two in cycle 1 would yield a PK profile similar to that measured for the 1600/800 mg dose regimen assessed in GAUGUIN phase II.41 This regimen reflects a loading dose paradigm for obinutuzumab 3000 mg in cycle 1, which is directly comparable to the 3200 mg dose regimen administered in the high-dose group (1600/800 mg) of GAUGUIN. This regimen offers many significant benefits, such as reduced preparation time, less waste, and minimization of errors. Based on in vivo and in silico analyses, obinutuzumab 1000 mg administered in a 21-day cycle on D1, D8, and D15 of C1 and D1 of C2–C8 is anticipated to rapidly saturate CD20 and optimize serum concentrations of antibody, irrespective of patient demographics and disease characteristics. This dose and schedule is now being taken forward for clinical assessment in three phase III studies of obinutuzumab plus chemotherapy in NHL patients. Trial registration The GAUGUIN and GAUDI studies are registered at www.clinicaltrails.gov with the trial identifiers NCT00517530 and NCT00825149, respectively. Acknowledgments The GAUGUIN and GAUDI studies were funded by F. Hoffmann-La Roche Ltd. Support for third-party medical writing assistance for this manuscript was provided by F. Hoffmann-La Roche Ltd.

2. Coiffier B, Thieblemont C, Van Den Neste E, et al. Long-term outcome of patients in the LNH-98.5 trial, the first randomized study comparing rituximab-CHOP to standard CHOP chemotherapy in DLBCL patients: a study by the Groupe d’Etudes des Lymphomes de l’Adulte. Blood.

2010;116(12):2040-2045. 3. Marcus R, Imrie K, Solal-Céligny P, et al. Phase III study of R-CVP compared with cyclophosphamide, vincristine, and prednisone alone in patients with previously untreated advanced follicular lymphoma. J Clin Oncol. 2008;26(28):4579-4586.

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G. Cartron et al. 4. Salles G, Mounier N, de Guibert S, et al. Rituximab combined with chemotherapy and interferon in follicular lymphoma patients: results of the GELA-GOELAMS FL2000 study. Blood. 2008;112(13):48244831. 5. Hallek M, Fischer K, Fingerle-Rowson G, et al; International Group of Investigators; German Chronic Lymphocytic Leukaemia Study Group. Addition of rituximab to fludarabine and cyclophosphamide in patients with chronic lymphocytic leukaemia: a randomised, open-label, phase 3 trial. Lancet. 2010;376(9747):1164-1174. 6. Robak T, Dmoszynska A, Solal-Céligny P, et al. Rituximab plus fludarabine and cyclophosphamide prolongs progressionfree survival compared with fludarabine and cyclophosphamide alone in previously treated chronic lymphocytic leukemia. J Clin Oncol. 2010;28(10):1756-1765. 7. van Oers MH, Van Glabbeke M, Giurgea L, et al. Rituximab maintenance treatment of relapsed/resistant follicular non-Hodgkin’s lymphoma: long-term outcome of the EORTC 20981 phase III randomized intergroup study. J Clin Oncol. 2010;28(17): 2853-2858. 8. Salles G, Seymour JF, Offner F, et al. Rituximab maintenance for 2 years in patients with high tumour burden follicular lymphoma responding to rituximab plus chemotherapy (PRIMA): a phase 3, randomised controlled trial. Lancet. 2011;377 (9759):42-51. 9. Maloney DG, Liles TM, Czerwinski DK, et al. Phase I clinical trial using escalating single-dose infusion of chimeric anti-CD20 monoclonal antibody (IDEC-C2B8) in patients with recurrent B-cell lymphoma. Blood. 1994;84(8):2457-2466. 10. Maloney DG, Grillo-López AJ, White CA, et al. IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin's lymphoma. Blood. 1997;90(6):2188-2195. 11. McLaughlin P, Grillo-López AJ, Link BK, et al. Rituximab chimeric anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: half of patients respond to a four-dose treatment program. J Clin Oncol. 1998;16(8):2825-2833. 12. Rituximab Summary of Product Characteristics, 9 July 2014. Available from: http://www.ema.europa.eu/ema/index.jsp ?curl=pages/medicines/human/medicines/000165/human_med_000897.jsp&mi d=WC0b01ac058001d124. 13. Rituximab Prescribing Information, revised February 2010. Available from: http://www.accessdata.fda.gov/drugsatfda_docs/label/2010/103705s5311lbl.pdf 14. Berinstein NL, Grillo-López AJ, White CA, et al. Association of serum rituximab (IDEC-C2B8) concentration and anti-tumor response in the treatment of recurrent lowgrade or follicular non-Hodgkin’s lymphoma. Ann Oncol. 1998;9(9):995-1001. 15. Igarashi T, Kobayashi Y, Ogura M, et al. IDEC-C2B8 Study Group in Japan. Factors affecting toxicity, response and progression-free survival in relapsed patients with indolent B-cell lymphoma and mantle cell lymphoma treated with rituximab: a Japanese phase II study. Ann Oncol. 2002;13(6):928-943.

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16. Tobinai K, Igarashi T, Itoh K, et al; IDECC2B8 Japan Study Group. Japanese multicenter phase II and pharmacokinetic study of rituximab in relapsed or refractory patients with aggressive B-cell lymphoma. Ann Oncol. 2004;15(5):821-830. 17. Gordan LN, Grow WB, Pusateri A, Douglas V, Mendenhall NP, Lynch JW. Phase II trial of individualized rituximab dosing for patients with CD20-positive lymphoproliferative disorders. J Clin Oncol. 2005;23(6): 1096-1102. 18. Cartron G, Blasco H, Paintaud G, Watier H, Le Guellec C. Pharmacokinetics of rituximab and its clinical use: thought for the best use? Crit Rev Oncol Hematol. 2007;62(1):43-52. 19. Daydé D, Ternant D, Ohresser M, et al. Tumor burden influences exposure and response to rituximab: pharmacokineticpharmacodynamic modeling using a syngeneic bioluminescent murine model expressing human CD20. Blood. 2009;113(16):3765-3772. 20. Dall’Ozzo S, Tartas S, Paintaud G, et al. Rituximab-dependent cytotoxicity by natural killer cells: influence of FCGR3A polymorphism on the concentration-effect relationship. Cancer Res. 2004;64(13):46644669. 21. Cartron G, Dacheux L, Salles G, et al. Therapeutic activity of humanized antiCD20 monoclonal antibody and polymorphism in IgG Fc receptor FcgammaRIIIa gene. Blood. 2002;99(3):754-758. 22. Mössner E, Brünker P, Moser S, et al. Increasing the efficacy of CD20 antibody therapy through the engineering of a new type II anti-CD20 antibody with enhanced direct and immune effector cell-mediated B-cell cytotoxicity. Blood. 2010;115(22): 4393-4402. 23. Alduaij W, Ivanov A, Honeychurch J, et al. Novel type II anti-CD20 monoclonal antibody (GA101) evokes homotypic adhesion and actin-dependent, lysosome-mediated cell death in B-cell malignancies. Blood. 2011;117(17):4519-4529. 24. Morschhauser F, Cartron G, Thieblemont C, et al. Obinutuzumab (GA101) monotherapy in relapsed/refractory diffuse large B-cell lymphoma or mantle cell lymphoma: results from the phase II GAUGUIN study. J Clin Oncol. 2013;31(23): 2912-2919. 25. Salles G, Morschhauser F, Solal-Céligny P, et al. Obinutuzumab (GA101) in patients with relapsed/refractory indolent nonHodgkin’s lymphoma: results of the phase II GAUGUIN study. J Clin Oncol. 2013;31(23):2920-2926. 26. Salles G, Morschhauser F, Lamy T, et al. Phase 1 study results of the type II glycoengineered humanized anti-CD20 monoclonal antibody obinutuzumab (GA101) in B-cell lymphoma patients. Blood. 2012;119(22):5126-5132. 27. Sehn LH, Assouline SE, Stewart DA, et al. A phase I study of obinutuzumab induction followed by two years of maintenance in patients with relapsed CD20-positive B-cell malignancies. Blood. 2012;119(22):51185125. 28. Radford J, Davies A, Cartron G, et al. Obinutuzumab (GA101) in combination with FC or CHOP in patients with relapsed

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or refractory follicular lymphoma: final results of the phase I GAUDI study (BO21000). Blood. 2013;122(7):1137-1143. Cheson BD, Horning SJ, Coiffier B, et al. Report of an international workshop to standardize response criteria for nonHodgkin's lymphomas. NCI Sponsored International Working Group. J Clin Oncol. 1999;17(4):1244-1253. Goede V, Fischer K, Busch R, et al. Obinutuzumab plus chlorambucil in patients with CLL and coexisting conditions. N Engl J Med. 2014;370(12):1101-1110. Gibiansky E, Gibiansky L, Carlile DJ, Jamois C, Buchheit V, Frey N. Population pharmacokinetic analyses of obinutuzumab (GA101) in chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphoma (NHL) and exposure-response analyses in CLL. CPT Pharmacometrics Syst Pharmacol. 2014;3:e144. Müller C, Murawski N, Wiesen MH, et al. The role of sex and weight on rituximab clearance and serum elimination half-life in elderly patients with DLBCL. Blood. 2012;119(14):3276-3284. Pfreundschuh M, Müller C, Zeynalova S, et al. Suboptimal dosing of rituximab in male and female patients with DLBCL. Blood. 2014;123(5):640-646. Ngo L, Hee SW, Lim LC, et al. Prognostic factors in patients with diffuse large B cell lymphoma: Before and after the introduction of rituximab. Leuk Lymphoma. 2008;49(3):462-469. Riihijärvi S, Taskinen M, Jerkeman M, Leppä S. Male gender is an adverse prognostic factor in B-cell lymphoma patients treated with immunochemotherapy. Eur J Haematol. 2011;86(2):124-128. Pfreundschuh M, Held G, Zeynalova S, et al. Improved outcome of elderly poor-prognosis DLBCL patients with 6xCHOP-14 and 8 applications of rituximab (R) given over an extended period: results of the SMARTE-R-CHOP-14 trial of the German High-Grade Non-Hodgkin Lymphoma Study Group (DSHNHL). Blood (ASH Annual Meeting Abstracts). 2011; 118(21):(Abstract 592). Murawski N, Pfreundschuh M, Zeynalova S, et al. Optimization of rituximab for the treatment of DLBCL (I) : Dose-dense rituximab in the DENSE-R-CHOP-14 trial of the DSHNHL. Ann Oncol. 2014;25(9):18001806. Ginaldi L, De Martinis M, Matutes E, Farahat N, Morilla R, Catovsky D. Levels of expression of CD19 and CD20 in chronic B cell leukaemias. J Clin Pathol. 1998;51(5): 364-369. Suková V, Klabusay M, Coupek P, Brychtová Y, Doubek M, Mayer J. Density expression of the CD20 antigen on population of tumor cells in patients with chronic B-lymphocyte lymphoproliferative diseases. Cas Leuk Cesk. 2006;145(9):712-716. Prevodnik VK, Lavren ak J, Horvat M, Novakovi BJ. The predictive significance of CD20 expression in B-cell lymphomas. Diagn Pathol. 2011;6:33. Morschhauser F, Salles G, Cartron G, et al. Dose selection for Phase III studies of the monoclonal anti-CD20 antibody obinutuzumab (GA101) – a rational approach. Haematologica. 2011;96(8):(Abstract 0935).

haematologica | 2016; 101(2)

ARTICLE

Non-Hodgkin Lymphoma

EUROPEAN HEMATOLOGY ASSOCIATION

Ferrata Storti Foundation

Simon Rule,1 Paul Smith,2 Peter W.M. Johnson,3 Simon Bolam,4 George Follows,5 Joanne Gambell,2 Peter Hillmen,6 Andrew Jack,7 Stephen Johnson,4 Amy A Kirkwood,2 Anton Kruger, 8 Christopher Pocock,9 John F. Seymour,10 Milena Toncheva,2 Jan Walewski11 and David Linch12

Derriford Hospital, Plymouth, UK; 2Cancer Reasearch UK and UCL Cancer Trials Centre, London, UK; 3University of Southampton, Southampton, UK; 4Musgrove Park Hospital, Taunton, UK; 5Addenbrookes Hospital, Cambridge, UK; 6St. Jamesâ&#x20AC;&#x2122;s University Hospital, Leeds, UK; 7HMDS, Leeds General Infirmary, UK; 8Royal Cornwall Hospital, Truro, UK; 9 Kent and Canterbury Hospital, Canterbury, UK; 10The Peter MacCallum Cancer Centre, Melbourne, Australia; 11Maria Sklodowska-Curie Institute and Oncology Centre, Gilwice, Poland; and 12UCL Cancer Institute, London, UK 1

Haematologica 2016 Volume 101(2):235-240

ABSTRACT

antle cell lymphoma is an incurable and generally aggressive lymphoma that is more common in elderly patients. Whilst a number of different chemotherapeutic regimens are active in this disease, there is no established gold standard therapy. Rituximab has been used widely to good effect in B-cell malignancies but there is no evidence that it improves outcomes when added to chemotherapy in this disease. We performed a randomized, open-label, multicenter study looking at the addition of rituximab to the standard chemotherapy regimen of fludarabine and cyclophosphamide in patients with newly diagnosed mantle cell lymphoma. A total of 370 patients were randomized. With a median follow up of six years, rituximab improved the median progression-free survival from 14.9 to 29.8 months (P<0.001) and overall survival from 37.0 to 44.5 months (P=0.005). This equates to absolute differences of 9.0% and 22.1% for overall and progression-free survival, respectively, at two years. Overall response rates were similar, but complete response rates were significantly higher in the rituximab arm: 52.7% vs. 39.9% (P=0.014). There was no clinically significant additional toxicity observed with the addition of rituximab. Overall, approximately 18% of patients died of non-lymphomatous causes, most commonly infections. The addition of rituximab to fludarabine and cyclophosphamide chemotherapy significantly improves outcomes in patients with mantle cell lymphoma. However, these regimens have significant late toxicity and should be used with caution. This trial has been registered (ISRCTN81133184 and clinicaltrials.gov identifier:00641095) and is supported by the UK National Cancer Research Network.

Introduction Mantle cell lymphoma (MCL) is an uncommon and usually aggressive form of non-Hodgkin lymphoma with an annual incidence of approximately 1 per 100,000 of the population. In younger patients, the treatment of choice includes a high-dose cytarabine-containing regimen usually followed by autologous stem cell transplantation.1,2 However, with a median age at presentation in the mid sixth decade, such therapy is not applicable to the majority of patients. There is no generally accepted haematologica | 2016; 101(2)

Correspondence: fengx2@nhlbi.nih.gov

Received: 9/4/2015. Accepted: 19/11/2015. Pre-published: 26/11/2015. doi:10.3324/haematol.2015.128710

Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/101/2/235

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standard of care for older patients and a variety of treatments have been widely used. Newer therapeutic approaches are clearly needed as, despite improvements in outcome for patients treated within trial cohorts,3 recent SEER data show there has been no improvement in outcome for this disease over the last 20 years.3 Mantle cell lymphoma expresses the pan-B-cell surface antigen CD20, and with the advent of specific monoclonal antibodies targeting this antigen, a new therapeutic option became available. Rituximab (Rituxan, Mabthera) is a chimeric anti-CD20 monoclonal antibody that is widely used in lymphoproliferative disorders. It has wide international regulatory approval for use in both diffuse large Bcell and follicular lymphoma. As a single agent, rituximab produces response rates of approximately 35% in MCL5,6 and when added to the standard chemotherapeutic regimen CHOP (cyclophosphamide, doxorubicin, vincristine and prednisolone) within a phase II single arm study the combination demonstrated a very high overall response rate.7 There have subsequently been three randomized trials involving the addition of rituximab to a standard chemotherapeutic regimen in which patients with MCL have been included. All of these trials included a variety of ‘low-grade’ lymphomas, including MCL. Two of these Table 1. Base-line characteristics.

F/C n=184 n (%)

F/C/R n=186 n (%)

studies considered the addition of rituximab as part of the initial therapy to CHOP8 and MCP9 (mitoxantrone, chlorambucil and prednisolone). In both trials, no difference in progression-free survival (PFS) or overall survival (OS) was demonstrable within the subset of patients with MCL; however, only 122 and 90 patients were randomized in these trials, respectively. A subsequent relapse study examined the addition of rituximab to FCM (fludarabine, cyclophosphamide and mitoxantrone).10 This study did demonstrate an improvement in OS in the rituximab containing arm; however, there were only 24 patients with MCL in each arm. A subsequent meta-analysis of all three studies suggested an OS benefit for the addition of rituximab.11 However, no individual phase III study has yet demonstrated such a benefit, and thus the true impact of rituximab is still unclear. The purine nucleoside analog class of drugs have demonstrable activity in the treatment of MCL.12-15 Fludarabine is the most widely used nucleoside analog and when combined with cyclophosphamide in patients with MCL high response rates are achieved.12 This combination has the attraction of not including an anthracycline, which can be associated with cardiac toxicity in elderly patient populations and can be delivered as an oral combination. Given this, in 2002, a UK-based randomized trial was initiated exploring the addition of rituximab to oral FC.

Methods Study design

Age at randomization Median (range) Gender Male Female ECOG1 0 Performance status 1 2 3 4 Missing B symptoms Absent Present Missing Stage I II III IV Missing Serum LDH level Normal Elevated Missing MIPI risk group Low Intermediate High Missing

66 (37 - 85)

66 (36 - 88)

146 (79.3) 38 (20.7)

137 (73.7) 49 (26.3)

87 (47.3)

93 (50.0)

64 (34.8) 15 (8.2) 5 (2.7) 1 (0.5) 12 (6.5)

62 (33.3) 17 (9.1) 0 (0.0) 0 (0.0) 14 (7.5)

106 (57.6) 74 (40.2) 4 (2.2)

97 (52.2) 81 (43.5) 8 (4.3)

2 (1.1) 11 (6.0) 32 (17.4) 134 (72.8) 5 (2.7)

4 (2.2) 15 (8.1) 25 (13.4) 134 (72.0) 8 (4.3)

99 (53.8) 80 (43.5) 5 (2.7)

96 (51.6) 77 (41.4) 13 (7.0)

45 (24.5) 63 (34.2) 60 (32.6) 16 (8.7)

37 (19.9) 75 (40.3) 55 (29.6) 19 (10.2)

Eastern Cooperative Oncology Group; 2lactate dehydrogenase; 3the Mantle Cell Lymphoma International Prognostic Index.

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The trial began as a randomized 2-stage phase II study with eligible patients given either the standard chemotherapeutic regimen of FC or same regimen with the addition of rituximab (FCR). A Simon’s design was used, with a target response rate of more than 60%, compared to less than 40%, with 90% power and 10% twosided significance level (target sample size: 82 patients). After meeting the target response (reviewed by an independent data monitoring committee), the trial was extended into phase III, powered to detect an improvement in the 3-year OS rate of 11% (55% FC vs. 66% FCR) with 80% power and 5% two-sided test of statistical significance (log rank test), requiring 370 patients in total. Secondary end points included progression-free survival (PFS), response and toxicity.

Ethics and study management The study complied with the Declaration of Helsinki and was conducted in accordance with Good Clinical Practice guidelines. The protocol was approved by an independent ethics committee and by local review boards at each participating institution.

Patient selection Patients aged over 18 years with previously untreated MCL were eligible. Central pathological confirmation of MCL diagnosis including cyclin D1 overexpression or evidence of t(11:14) was performed retrospectively, but was not required for inclusion in the study. Patients required adequate organ function and a life expectancy of at least three months.

Study treatment Patients received oral 40 mg/m2 fludarabine and 250 mg/m2 cyclophosphamide on days 1-3 of a 28-day cycle. Patients randomized to FCR received intravenous (iv) 375 mg/m2 rituximab on day 1 of each cycle. In patients intolerant of oral FC, treatment could be given intravenously: cyclophosphamide at the same dose haematologica | 2016; 101(2)

Rituximab with chemotherapy increases survival in MCL

Table 2. Subgroup analysis.

Subgroup HR(95% CI) B symptoms Absent Present Stage I/II III IV Serum LDH Normal Elevated Age <70 ≥70 MIPI risk group Low Intermediate High

OS P*

PFS Events/n

HR(95% CI)

116/203 116/155

0.42 (0.30, 0.59) 0.71 (0.50, 1.00)

14/32 37/57 180/268

0.42 (0.16, 1.08) 0.57 (0.31, 1.05) 0.54 (0.41, 0.71)

109/195 119/157

0.44 (0.31, 0.61) 0.70 (0.49, 0.99)

133/233 107/137

0.51 (0.37, 0.69) 0.53 (0.37, 0.77)

37/82 82/138 97/115

0.36 (0.21, 0.64) 0.57 (0.38, 0.84) 0.55 (0.37, 0.82)

P=0.06 0.54 (0.37, 0.79) 0.91 (0.63, 1.32)

P=0.05

0.99 0.64 (0.21, 1.97) 0.74 (0.38, 1.45) 0.74 (0.55, 0.99)

0.70

0.15 0.59 (0.40, 0.86) 0.87 (0.60, 1.25)

0.65

0.72 0.65 (0.46, 0.92) 0.72 (0.49, 1.05)

0.94

0.70 0.50 (0.25, 0.99) 0.73 (0.47, 1.13) 0.72 (0.48, 1.08)

0.55

*P-value for the interaction.

and 25 mg/m2 fludarabine.16 Supportive care was provided according to institutional practice but Pneumocystis jirovecci (PJP) prophylaxis was mandatory, as was the use of irradiated blood products. Patients received 4 cycles of therapy before re-staging. If they showed no response or had already progressed they were taken off study. Those patients with responsive disease were treated to maximal response or a maximum of 8 cycles of treatment. At the completion of therapy, patients were re-staged and followed up as according to institutional practice. Follow up scans did not follow a standardized schedule. Standard response criteria were adopted.17 PET scans were not performed. Adverse events were reported using the National Cancer Institute Common Terminology Criteria for Adverse Events (v.3.0). Following treatment, patients were not permitted to receive any form of maintenance or consolidation therapy.

Statistical analysis All time-to-event analyses were performed on an intention to treat basis; however, response and toxicity analyses were limited to patients who received at least one dose of treatment. OS was measured from the date of randomization until the date of death and PFS from the date of randomization until the date of progression or death. Patients who did not experience an event were censored at the date last seen. OS and PFS distributions were examined using Kaplan-Meier curves, and Cox proportional hazards models after confirming the assumption of proportional hazards. All analyses were performed using Stata software (v.12.1) (StataCorp, TX, USA).

Results Patients’ characteristics A total of 370 patients were randomized (n=156 phase II and n=214 phase III) between the 2nd of September 2002 and the 2nd of December 2010 from 96 centers in the UK, Poland and Australia. Patients’ characteristics were well balanced between arms (Table 1). Median age at randomization was 66 years with a male predominance of 3:1. The vast majority haematologica | 2016; 101(2)

of the patients had intermediate- or high-risk disease, as assessed by the Mantle Cell International Prognostic Index (MIPI).18 Diagnostic material of 297 patients was centrally reviewed. Of these patients, 19 did not have sufficient material to confirm a diagnosis. From the remaining 278 patients there were 11 patients (4%) with incorrect diagnoses: 4 marginal zone lymphomas, one diffuse large B-cell lymphoma, one chronic lymphocytic leukemia, 4 with no evidence of lymphoma (on the material centrally reviewed) and one patient diagnosed with MCL which did not express cyclin D1.

Compliance The addition of rituximab did not affect the tolerance of FC chemotherapy, with the number of patients receiving 4 cycles or more being higher in the FCR arm than the FC arm: 128 (70.3%) vs. 102 (55.7%) (P=0.004). The proportion of patients whose chemotherapy was delayed or dose-reduced was similar in the two arms; 16.3% (142 of 877) of FC cycles and 15.3% (149 of 971) of FCR cycles were delayed and less than 90% of one or more drugs was given in 18.5% (162 of 877) of cycles of FC and 23.5% (236 of 971) of FCR [20.9% (203 of 971) of FCR cycles if only FC dose reductions were considered].

Efficacy More patients in the FCR arm achieved an objective disease response (CR/CRu/PR) at the end of treatment than in the FC arm: 137 (73.7%) vs. 125 (68.3%). However, this was not statistically significant (P=0.26). The proportion of complete responses (CR and CRu) was significantly higher in the FCR arm: 98 (52.7%) vs. 73 (39.9%), (P=0.014). Fewer patients experienced progression of disease on therapy in the FCR arm [16 (8.6%) vs. 27 (14.8%)] although this did not reach statistical significance (P=0.066). Figure 1 shows Kaplan Meier curves for OS and PFS. The median OS was 44.5 months in the FCR arm and 37.0 months in the FC arm. The patients who received FCR had a 31% reduction in the risk of death: hazard ratio (HR) 0.69, 95%CI: 0.54-0.90. At two years, the survival proportions are 237

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59.8% (95%CI: 52.3-66.5) in the FC arm and 68.8% (95%CI: 61.6-74.9) in the FCR arm. The improvement in PFS was even greater (median PFS: 29.8 months with FCR vs. 14.9 with FC) with a reduction in the risk of death or progression of 47% for patients given FCR (HR 0.53, 95%CI: 0.42-0.67; P<0.001). This represents an absolute difference of 22.1% in PFS at two years. The proportional hazards assumption held for PFS (P=0.11). More FCR patients received 4 cycles or more [128 (70.3%) vs. 102 (55.7%)], so it is plausible that adding rituximab allowed more cycles to be delivered, which might account for the observed treatment benefit. However, there was no clear pattern between HR and number of cycles. The interaction P-value was driven by the large HR among patients receiving 2 cycles (5.86) and when these are excluded the interaction P-value becomes statistically non-significant (P=0.17). Therefore, the overall HR of 0.69 for FCR versus FC is unlikely to be due to the number of cycles. Overall survival and PFS results held when patients without a centrally confirmed MCL diagnosis were excluded. The PFS results also held when patients who were given further systemic treatment (n=20) before progression were censored. Table 2 shows the HRs for OS and PFS according to prespecified base-line factors. There was no strong evidence of a difference in treatment effect within any of the subgroups.

Toxicity The treatment-related mortality (TRM) was low and similar between the 2 arms. Five (2.7%) patients in the FC arm and 6 (3.2%) in the FCR arm were recorded as dying from treatment-related causes, predominantly sepsis. The incidence of grade III/IV toxicity was similar between the two treatment arms (P=0.12) (Table 3A), and although more patients experienced grade III/IV hematologic toxicities in the FCR arm [105 (57.4%) vs. 125 (67.2%)] this did not reach statistical significance (P=0.051). There was more grade III/IV thrombocytopenia [53 (28.5%) vs. 33 (18.0 %); P=0.017] in the FCR arm. However, this did not result in any clinically significant bleeding episodes. There was more leukopenia [(32 (17.2%) vs. 18 (9.8%); P=0.039)] in the FCR arm, although there was no increase in neutropenia or observed infections. There were more allergic reactions in the rituximab-containing regimen, in keeping with the known infusion-related toxicity of this agent, although grade III/IV events occurred in only 12 (6.5%) patients. Although toxicity rates were slightly higher in the FCR arm, this may, in part, be due to the fact that these patients received more cycles of therapy than in the FC arm. For those toxicities recorded in the first 4 cycles (Table 3B), there is no significant difference between the arms with 85 (46.5%) patients in the FC arm experiencing a grade 3/4 hematologic toxicity versus 95 (51.1%) in the FCR arm (P=0.37). The rates of non-hematologic toxicity were almost identical: 69 (37.7%) patients in the FC arm experiencing a toxicity versus 69 (37.1%) in the FCR arm (P=0.90).

(Mycobacterium tuberculosis). The majority of other deaths were either second malignancies (7 in each arm, comprising 2 cases of AML and 5 various solid tumors in both arms) or cardiac events (5 post FC and 7 post FCR).

Discussion With a median follow up of 6.02 years, this study has demonstrated that the addition of rituximab to FC chemotherapy leads to a significant improvement in both PFS and OS for patients with MCL. The addition of rituximab produces a modest increase in hematologic toxicity, but, importantly, no increase in neutropenia or infections, with no clinically significant difference in long-term toxicity. The median age of the study population was 66 years making this a trial of predominantly elderly patients. The toxicity associated with this regimen is observed in the dose adjustments required throughout. Just below 40% of patients in both arms received less than the planned dose of one or more drug during at least one cycle. Despite this, the TRM was low in both arms (approx. 3%) but this might account for the relatively high number of patients experiencing disease progression on therapy. The other finding of concern is the number of patients who died following therapy of causes other than lymphoma, principle amongst these being infection. The propensity for patients to be at risk from opportunistic infections following purine analog therapy is well known because of the lymphoid suppression that can result

Late toxicity At a median follow up of almost 6.02 years, a total of 240 patients have died, 132 (71.7%) in the FC arm and 108 (58.1%) in the FCR arm. The most common cause of death was lymphoma, accounting for 94 (71.2%) and 66 (61.1%) deaths in each arm. Thirty patients in the FC arm and 36 patients in the FCR arm died of other causes. Approximately one-third were secondary to infections (12 FC, 15 FCR) of which only one was classed as an opportunistic infection 238

Figure 1. Kaplan Meier curves. (A) Overall Survival: HR (95% CI) 0.72 (0.550.94); P=0.016. (B) Progression Free Survival: HR (95% CI) 0.54 (0.42-0.69); P<0.001.

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Rituximab with chemotherapy increases survival in MCL

from it.19 However, only one patient had a true opportunistic infection (TB) in this series. A recent randomized trial comparing FCR with R-CHOP in elderly patients with MCL showed a survival benefit in favor of R-CHOP.20 The findings in the FCR arm of that study are virtually identical to those obtained in the current study. They described greater hematologic toxicity in the FCR arm and more progressive disease on therapy: 14% to our 8.6%. But as we found, a significant number of patients died whilst in remission of their lymphoma, usually of infection. The addition of rituximab to FC has also been explored in a large randomized trial in chronic lymphocytic leukemia (CLL).21 This demonstrated a statistically significant increase in both PFS and OS in favor of the rituximab treatment arm. Toxicity problems were similar, with dose reductions secondary to neutropenia observed in over one-third of cycles

and significant late effects, with approximately 50% of patients dying from causes other than leukemia. The delayed toxicity following FC-based therapy impacts on the subsequent delivery of treatment at the time of relapse. Another CLL trial22 considered the outcome of patients who received 3 different chemotherapy regimens, one of which was FC. Following progression, this group of patients had the worst outcome. It seems plausible that this inability to re-treat patients after relapse following FC-based therapy explains the survival difference observed in the Kluin-Nelemans20 study in favor of R-CHOP. In that trial, the R-CHOP treated patients had a superior outcome despite a very similar time to treatment failure. Interestingly, in those patients progressing on FCR, the median survival was only five months post induction. Does a survival benefit in favor of rituximab with FC mean

Table 3A. Toxicity.

In cycles 1-4 Toxicity III/IV Hematologic Anemia Neutropenia Thrombocytopenia Leukopenia Any hematologic Non-hematologic Fatigue Allergy Infection Fever Constipation Nausea Vomiting Anorexia Diarrhea Stomatitis Hypotension Bronchospasm Cardiac Pulmonary Skin rash Flushing Headaches Joint pain Neurological Renal Febrile neutropenia Neutropenic sepsis Gastrointestinal Other Pancytopenia Any non-hematologic Any toxicity

At any point

FC n=183

FCR n=186

FC n=183

FCR n=186

20 (10.9) 74 (40.4) 23 (12.6) 11 (6.0) 85 (46.5)

19 (10.2) 84 (45.2) 26 (14.0) 19 (10.2) 95 (51.1)

25 (13.7) 88 (48.1) 33 (18.0) 18 (9.8) 105 (57.4)

25 (13.4) 105 (56.5) 53 (28.5) 32 (17.2) 125 (67.2)

14 (7.7) 1 (0.5) 21 (11.5) 6 (3.3) 1 (0.5) 9 (4.9) 11 (6.0) 5 (2.7) 6 (3.3) 1 (0.5) 3 (1.6) 0 5 (2.7) 10 (5.5) 9 (4.9) 1 (0.5) 3 (1.6) 1 (0.5) 2 (1.1) 0 8 (4.4) 2 (1.1) 2 (1.1) 17 (9.3) 1 (0.5) 69 (37.7) 111 (60.7)

11 (5.9) 12 (6.5) 22 (11.8) 5 (2.7) 0 4 (2.2) 6 (3.2) 5 (2.7) 3 (1.6) 0 7 (3.8) 2 (1.1) 4 (2.2) 10 (5.4) 6 (3.2) 0 1 (0.5) 2 (1.1) 1 (0.5) 1 (0.5) 6 (3.2) 5 (2.7) 2 (1.1) 13 (7.0) 3 (1.6) 69(37.1) 118 (63.4)

16 (8.7) 1 (0.5) 26 (14.2) 7 (3.8) 1 (0.5) 10 (5.5) 12 (6.6) 5 (2.7) 7 (3.8) 1 (0.5) 3 (1.6) 0 6 (3.3) 12 (6.6) 10 (5.5) 1 (0.5) 4 (2.2) 1 (0.5) 2 (1.1) 0 10 (5.5) 5 (2.7) 2 (1.1) 18 (9.8) 5 (2 .7) 81 (44.3) 132 (72.1)

13 (7.0) 12 (6.5) 30 (16.1) 7 (3.8) 0 4 (2.2) 7 (3.8) 5 (2.7) 4 (2.2) 0 7 (3.8) 2 (1.1) 5 (2.7) 12 (6.5) 7 (3.8) 0 2 (1.1) 2 (1.1) 3 (1.6) 1 (0.5) 10 (5.4) 6 (3.2) 2 (1.1) 18 (9.7) 7 (3.8) 89 (47.9) 147 (79.0)

Table 3B. Toxicity by cycle.

Any hematologic toxicity Any non-hematologic toxicity Any toxicity haematologica | 2016; 101(2)

In cycles 1-4 FCR

FC 85 (46.5) 69 (37.7) 111 (60.7)

95 (51.1) 69 (37.1) 118 (63.4)

0.37 0.90 0.58

At any point FCR

FC 105 (57.4) 81 (44.3) 132 (72.1)

125 (67.2) 89 (47.9) 147 (79.0)

0.051 0.49 0.12 239

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that the same benefit would be seen if added to other standard chemotherapy approaches? The evidence in follicular lymphoma, where the benefit is consistent across a range of chemotherapies, would suggest this may be the case.23-26 However, there have been two previous randomized trials8,9 of rituximab in combination with CHOP and MCP in MCL where no survival benefit was observed. This is almost certainly a reflection of the small size of these studies, which were not sufficiently powered to demonstrate a difference. As rituximab had been shown to improve survival in randomized studies involving more common forms of lymphoma, the drug has been used widely in the context of MCL. However, in health care systems where specific evidence of a benefit is required, usually in the form of randomized evidence before a drug can be made generally available, it is increasingly important to design and complete appropriately powered studies. This study was predominantly performed in the UK and demonstrates that it is possible to carry

References 9. 1. Khouri IF, Romaguera J, Kantarjian H, et al. Hyper-CVAD and high-dose methotrexate/cytarabinefollowed by stemcell transplantation: an active regimen for aggressivemantle-cell lymphoma. J Clin Oncol. 1998;16(12):3803-3809. 2. Geisler CH, Kolstad A, Laurell A, et al. Long-term progression-free survival of mantle cell lymphoma after intensive front-line immunochemotherapy with in vivo-purged stem cell rescue: a nonrandomized phase 2 multicenter study by the Nordic Lymphoma Group. Blood. 2008; 112(7):2687-2693. 3. Herrmann A, Hoster E, Zwingers T, et al. Improvement of overall survival in advanced stage mantle cell lymphoma. J Clin Oncol. 2009;27(4):511-518. 4. Chandran R, Gardiner SK, Simon M, Spurgeon SE. Survival trends in mantle cell lymphoma in the United States over 16 years 1992-2007. Leuk Lymphoma. 2012; 53(8):1488-1493. 5. Coiffier B, Haioun C, Ketterer N, et al. Rituximab (anti-CD20 monoclonal antibody) for the treatment of patients with relapsing or refractory aggressive lymphoma: a multicenter phase II study. Blood. 1998;92(6):19271932. 6. Foran JM, Rohatiner AZ, Cunningham D, et al. European phase II study of rituximab (chimeric anti-CD20 monoclonal antibody) for patients with newly diagnosed mantlecell lymphoma and previously treated mantle-cell lymphoma, immunocytoma, and small B-cell lymphocytic lymphoma. J Clin Oncol. 2000;18(2):317-324. 7. Howard OM, Gribben JG, Neuberg DS, et al. Rituximab and CHOP induction therapy for newly diagnosed mantle-cell lymphoma: molecular complete responses are not predictive of progression-free survival. J Clin Oncol. 2002;20(5):1288-1294. 8. Lenz G, Dreyling M, Hoster E, et al. Immunochemotherapy with rituximab and cyclophosphamide, doxorubicin, vincristine, and prednisone significantly improves response and time to treatment failure, but not long-term outcome in patients with previously untreated mantle cell lymphoma: results of a prospective randomized trial of the German Low Grade Lymphoma Study

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out randomized studies in rare diseases. In summary, the addition of rituximab to FC chemotherapy improves survival in patients with mantle cell lymphoma. However, the evidence would suggest that purine analog combinations should be used with caution in elderly patients. Acknowledgments We would like to thank all the patients, participating centers and staff, and to the members of the Trials Steering Committee and Independent Data Monitoring Committee.The authors would also like to thank Cancer Research UK for funding the trial and Roche who provided free rituximab. Funding ClinicalTrials.gov number NCT00641095 This trial was funded by CR UK (CRUK/06/013) and conducted by the CR UK and UCL Cancer Trials Centre.

Group (GLSG). J Clin Oncol. 2005;23(9):19841992. Herold M, Haas A, Doerken B, et al. Immunochemotherapy (R-MCP) in advanced mantle cell lymphoma is not superior to chemotherapy (MCP) alone - 50 months up date of the OSHP Phase III study (OSHO #39). Ann Oncol. 2008;19:iv86. (Abstract). Forstpointner R, Dreyling M, Repp R, et al. The addition of rituximab to a combination of fludarabine, cyclophosphamide, mitoxantrone (FCM) significantly increases the response rate and prolongs survival as compared with FCM alone in patients with relapsed and refractory follicular and mantle cell lymphomas: results of a prospective randomized study of the German Low-Grade Lymphoma Study Group. Blood. 2004;104(10):3064-3071. Schulz H, Bohlius J, Skoetz N, et al. Chemotherapy plus Rituximab versus chemotherapy alone for B-cell non-Hodgkin's lymphoma. Cochrane Database Syst Rev. 2007;(4):CD003805. Zinzani PL, Magagnoli M, Moretti L, et al. Randomised trial of fludarabine versus fludarabine and idarubicin as frontline treatment in patients with indolent or mantle cell lymphoma. J Clin Oncol. 2000;18(4):773-779. Rummel MJ, Chow KU, Jager E, et al. Treatment of mantle cell lymphomas with intermittent two-hour infusion of cladribine as first-line therapy or in first relapse. Ann Oncol. 1999;10(1):115-117. Cohen BJ, Hedrick E, Moskowitz C, et al. Cyclophosphamide/fludarabine (CF) is active in the treatment of mantle cell lymphoma. Leuk Lymphoma. 2001;42(5):1015-1022. Johnson SA. Use of fludarabine in the treatment of mantle cell lymphoma, Waldenström’s macroglobulinaemia and other uncommon B- and T-cell lymphoid malignancies. Hematol J. 2004;5 Suppl 1:S50-61. Foran JM, Oscier D, Orchard J, et al. Pharmacokinetic study of single doses of oral fludarabine phosphate in patients with “lowgrade” non-Hodgkin’s lymphoma and B-cell chronic lymphocytic leukaemia. J Clin Oncol. 1999;17(5):1574-1579. Cheson BD, Horning SJ, Coiffier B, et al. Report of an international workshop to standardize response criteria for non-Hodgkin's lymphomas. NCI Sponsored International Working Group. J Clin Oncol.

1999;17(4):1244-1253. 18. 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. 19. Potter M. New anti-cancer therapies, new opportunities for infection. Curr Opin Infect Dis. 1999;12(4):359-363. 20. Kluin-Nelemans HC, Hoster E, Hermine O, et al. Treatment of older patients with mantlecell lymphoma. N Engl J Med. 2012;367 (6):520-531. 21. Hallek M, Fischer K, Fingerle-Rowson G, et al. Addition of rituximab to fludarabine and cyclophosphamide in patients with chronic lymphocytic leukaemia: a randomised, openlabel, phase 3 trial. Lancet. 2010;376(9747): 1164-1174. 22. Catovsky D, Richards S, Matutes E, et al. Assessment of fludarabine plus cyclophosphamide for patients with chronic lymphocytic leukaemia (the LRF CLL4 Trial): a randomised controlled trial. Lancet. 2007;370(9583):230-239. 23. Marcus R, Imrie K, Solal-Celigny P, et al. Phase III study of R-CVP compared with cyclophosphamide, vincristine, and prednisone alone in patients with previously untreated advanced follicular lymphoma. J Clin Oncol. 2008;26(28):4579-4586. 24. Herold M, Haas A, Srock S, et al. Rituximab added to first-line mitoxantrone, chlorambucil, and prednisolone chemotherapy followed by interferon maintenance prolongs survival in patients with advanced follicular lymphoma: an East German Study Group Hematology and Oncology Study. J Clin Oncol. 2007;25(15):1986-1992. 25. Salles G, Mounier N, de Guibert S, et al. Rituximab combined with chemotherapy and interferon in follicular lymphoma patients: results of the GELA-GOELAMS FL2000 study. Blood. 2008;112(13):48244831. 26. Hiddemann W, Kneba M, Dreyling M, et al. Frontline therapy with rituximab added to the combination of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) significantly improves the outcome for patients with advanced-stage follicular lymphoma compared with therapy with CHOP alone: results of a prospective randomized study of the German Low-Grade Lymphoma Study Group. Blood. 2005;106(12):37253732.

haematologica | 2016; 101(2)

ARTICLE

Stem Cell Transplantation

Time-dependent effects of clinical predictors in unrelated hematopoietic stem cell transplantation

EUROPEAN HEMATOLOGY ASSOCIATION

Ferrata Storti Foundation

Daniel Fuerst,1,2 Carlheinz Mueller,3,4 Dietrich W Beelen,5,6 Christine Neuchel,1,2 Chrysanthi Tsamadou,1,2 Hubert Schrezenmeier,1,2 and Joannis Mytilineos1,2,3

Institute of Clinical Transfusion Medicine and Immunogenetics Ulm, German, Red Cross Blood Transfusion Service, Baden-Wuerttemberg – Hessen, Ulm; 2Institute of Transfusion Medicine, University of Ulm; 3DRST – German Registry for Stem Cell Transplantation, Ulm Office; 4Zentrales Knochenmarkspender-Register Deutschland (ZKRD), Ulm; 5DRST – German Registry for Stem Cell Transplantation, Essen Office; and 6Department of Bone Marrow Transplantation, University Hospital, University of Duisburg-Essen, Essen, Germany 1

Haematologica 2016 Volume 101(2):241-247

ABSTRACT

ematopoietic stem cell transplantation is a multifactorial process. Some of the predictors exhibit time-dependent effects. We present a systematic analysis and description of selected clinical predictors influencing outcome in a time-dependent manner based on an analysis of registry data from the German Registry for Stem Cell Transplantation. A total of 14,951 patients with acute myeloid leukemia, acute lymphocytic leukemia, myelodysplastic syndrome and non-Hodgkin lymphoma transplanted with peripheral blood stem cells or bone marrow grafts were included. Multivariate Cox regression models were tested for time-dependent effects within each diagnosis group. Predictors not satisfying the proportional hazards assumption were modeled in a time-dependent manner, extending the Cox regression models. Similar patterns occurred in all diagnosis groups. Patients with a poor Karnofsky performance score (<80) had a high risk for early mortality until day 139 following transplantation (HR 2.42, CI: 2.19-2.68; P<0.001) compared to patients with a good Karnofsky performance score (80-100). Afterwards the risk reduced to HR 1.43, CI: 1.25-1.63; P<0.001. A lower mortality risk was found for patients after conditioning treatment with reduced intensity until day 120 post transplant (HR: 0.81 CI: 0.75-0.88; P<0.001). After this, a slightly higher risk could be shown for these patients. Similarly, patients who had received a PBSC graft exhibited a significantly lower mortality risk until day 388 post transplantation (HR 0.79, CI: 0.73-0.85; P<0.001), reversing to a significantly higher risk afterwards (HR 1.23, CI: 1.08-1.40; P=0.002). Integrating time dependency in regression models allows a more accurate description and quantification of clinical predictors to be made, which may help in risk assessment and patient counseling.

Introduction Outcome after hematopoietic stem cell transplantation is influenced by different factors. These include disease stage1 or cytogenetic risk,2 but also pre-transplant treatment-related variables, such as conditioning toxicity and selection of therapeutic agents.3 Furthermore, donor or graft properties may affect outcome.4 Of these, for example HLA-matching, cytomegalovirus (CMV) status or graft source are usually considered, as they were found to impact transplant-related outcome.5 Finally, haematologica | 2016; 101(2)

Correspondence: d.fuerst@blutspende.de

Received: 11/05/2015. Accepted: 25/11/2015. Pre-published: 26/11/2015. doi:10.3324/haematol.2015.130401

Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/101/2/241

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post-transplant treatment in the form of graft-versus-host disease (GvHD) prophylaxis and therapy with growth factors or steroids may influence morbidity and mortality.6 Predictors may be categorized as modifiable, such as type and dosage of therapeutic agent, or as invariable, e.g. genetic factors. In survival analysis, different groups are compared and effects may vary in size, also in a time-dependent manner, as the outcomes are time-to-event end points. Some effects may change in intensity over time or may be present only for a limited period, e.g. therapeutic interventions. From a clinical perspective, intensity of conditioning treatment is likely to show time-dependent effects as toxicity resolves with time. In addition, graft source may show time dependency as the kinetics of immune reconstitution is quite different in bone marrow (BM) and in peripheral blood stem cell (PBSC) grafts.7 Karnofsky performance score (KPS) has been shown to associate with a high early mortality.8,9 Finally, disease stage might be of interest, as relapse-associated and transplantation-associated events may have a greater impact on advancing disease stage early after transplantation. One important tool to evaluate survival is Kaplan-Meier analysis,10 in which time-dependent effects may manifest as crossing or diverging/converging survival curves (Online Supplementary Figures S1 and S2). However, Kaplan-Meier analysis is a univariate method and does not allow examination of multiple effects in combination. For multivariate analysis, the standard methodology is Cox regression analysis,11 which is limited by the ”Proportional Hazards Assumption“ (PHA). This means that all effects in the model are assumed to remain constant over time. This is often not the case, and would lead to false regression estimates, and, therefore, false hazard ratios for such effects if ignored. In such situations, the Cox regression model has been extended to allow for adjustment of time-dependent effects.12 To visualize such effects over time, dynamic

regression modeling has been proposed.13 Models including time-dependent variables have been used in important clinical studies.14,15 However, such time-dependent effects have not been studied in a dedicated analysis of HSCT data before, as their interpretation is not as intuitive as that of relative risk estimates obtained from variables fulfilling the PHA. In this study, we investigated selected clinical predictors of unrelated stem cell transplantation (HSCT) for timevarying effects and aimed to identify, describe and quantify these effects in such a way as to facilitate their interpretation by the clinician. For this reason, we analyzed a large cohort of patients based on data from the German Registry for Stem Cell Transplantation (DRST). Patterns that were observed in a similar fashion across the different disease entities are reported.

Methods Patients The DRST database is a subset of the European Group for Blood and Marrow Transplantation (EBMT) ProMISe database and includes patients transplanted in Germany. Clinical data of all patients receiving a first allogeneic transplant for the disease entities acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), myelodysplastic syndrome (MDS) and lymphoma were retrieved. The category lymphoma included chronic lymphocytic leukemia (CLL) and was sub-classified in aggressive and indolent. No missing values were accepted for disease stage, HLA-match type, and source of stem cells. Only adult patients and patients transplanted with BM or PBSC were included in this analysis. A total of 14,951 patients were eligible with a median age of 48 years (range 18-78). Transplantations were performed between 1976 and 2013. Median follow up of surviving patients was 49 months. Patients’ characteristics are summarized in Table 1 and Online Supplementary Table S1.

Table 1. Patients’ characteristics.

Category, n (%) Karnofsky Performance Score

Conditioning Graft source Patient age Patient sex

Donor type

Year of Tx

80-100 <80 Missing data MAC RIC PBSC BM Median Range Female Male Unknown mREL mUNREL mmREL mmUNREL 1976-2000 2001-2005 2006-2013

AML

ALL

MDS

Lym aggressive

Lym indolent

Total

5702 (79.9) 455 (6.4) 976 (13.7) 4654 (65.2) 2479 (34.8) 5958 (83.5) 1175 (16.5) 48 18-77 3477 (48.7) 3652 (51.2) 4 (0.1) 2986 (41.9) 2547 (35.7) 378 (5.3) 1222 (17.1) 1472 (20.6) 1774 (24.9) 3887 (54.5)

2095 (77.7) 179 (6.6) 422 (15.7) 2416 (89.6) 280 (10.4) 2055 (76.2) 641 (23.8) 36 18-74 1022 (37.9) 1670 (61.9) 4 (0.1) 1028 (38.1) 1072 (39.8) 125 (4.6) 471 (17.5) 712 (26.4) 782 (29.0) 1202 (44.6)

2019 (84.8) 179 (7.5) 182 (7.6) 1156 (48.6) 1224 (51.4) 2099 (88.2) 281 (11.8) 57 18-78 955 (40.1) 1422 (59.7) 3 (0.1) 782 (32.9) 1030 (43.3) 67 (2.8) 501 (21.1) 311 (13.1) 499 (21.0) 1570 (66.0)

1018 (85.0) 111 (9.3) 68 (5.7) 776 (64.8) 421 (35.2) 1092 (91.2) 105 (8.8) 47 18-75 420 (35.1) 775 (64.7) 2 (0.2) 468 (39.1) 449 (37.5) 62 (5.2) 218 (18.2) 140 (11.7) 274 (22.9) 783 (65.4)

1410 (91.3) 94 (6.1) 41 (2.7) 682 (44.1) 863 (55.9) 1444 (93.5) 101 (6.5) 55 18-76 441 (28.5) 1101 (71.3) 3 (0.2) 531 (34.4) 674 (43.6) 44 (2.8) 296 (19.2) 92 (6.0) 384 (24.9) 1069 (69.2)

12,244 (81.9) 1018 (6.8) 1689 (11.3) 9684 (64.8) 5267 (35.2) 12,648 (84.6) 2303 (15.4) 48 18-78 6315 (42.2) 8620 (57.7) 16 (0.1) 5795 (38.8) 5772 (38.6) 676 (4.5) 2708 (18.1) 2727 (18.2) 3713 (24.8) 8511 (56.9)

AML: acute myeloid leukemia; ALL: acute lymphoblastic leukemia; Lym: lymphoma; PBSC: peripheral blood stem cells; BM: bone marrow; MAC: myeloablative conditioning; RIC: reduced intensity conditioning; mREL: matched related; mUNREL: matched unrelated; mmREL: mismatched related; mmUNREL: mismatched unrelated; Tx: transplantation.

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Time-dependent predictors of uHSCT

Definitions The term “time dependent” is used for covariates not satisfying the PHA, which may be caused by an effect that changes in intensity over time or an effect that is present only for a limited time period and modifies subsequent risk. Disease stage definitions were adopted from the EBMT study group defining the EBMT risk score.14 Early disease stage was defined as transplantation in first complete remission for acute leukemia and as untreated or in first complete remission for MDS and lymphoma. Intermediate disease stage grouped for acute leukemia transplantation in second complete remission, for MDS in second complete or partial remission, and for lymphoma in second complete remission, partial remission or stable disease. Stages other than early or intermediate were classified as advanced disease stage. Regarding conditioning regimen intensity, the terms myeloablative (MAC), non-myeloablative (NMA), and reduced intensity conditioning (RIC) have been introduced.16 However, the EBMT ProMISe format currently classifies non-myeloablative and reduced intensity together as RIC. From retrospective data, it is sometimes difficult to distinguish between NMA and RIC. Therefore, our study only distinguished between myeloablative and conditioning with less intensity, termed RIC. According to the EBMT standards, MAC conditioning is defined as total body irradiation (TBI) of 10 Gy or more combined with cyclophosphamide or etoposide, or busulfan 16 mg/kg combined with cyclophosphamide 120-200 mg/kg. For lymphomas, also BEAM and CBV polychemotherapy were considered as MAC conditioning. Regimens with lower dosages were considered as RIC (see EBMT MED-AB forms manual Appendix III; www.ebmt.org). Overall survival (OS) was defined as the fraction of surviving patients at any given time point after transplantation. Death from any cause was considered as an event. Patients alive at the last follow up were censored. Disease-free survival (DFS) was defined as the proportion of patients alive without evidence of disease at any given time point after transplantation. Death from any cause or recurrence of diseases, whichever occurred earlier, was considered as an event. Patients alive and free from disease at the last follow up were censored. Consent for scientific data analysis was obtained upon registration in the DRST. The study was approved by the ethical committee of the University of Ulm, Germany (n. 108/15).

made on the structure of the base-line hazard (the distribution of survival times). In contrast, the Cox-Aalen model includes a multiplicative as well as an additive component. The multiplicative part incorporates covariates that modify the excess risk similarly to the Cox model, while the additive part models the base-line hazard rate allowing for time-varying effects.17,18 For OS and DFS multivariate Cox regression models were fitted and PHA was examined by a test based on weighted Scheonfeld residuals according to the algorithm proposed by Grambsch and Therneau implemented in the “survival package” of the R statistical software (R-3.0.2).19 The test basically checks for linearity of Schoenfeld-residuals over time. Covariates not satisfying the PHA were further examined by fitting a Cox-Aalen model and plotting cumulative hazard curves.20 These curves show the change in hazard over time. A positive slope adds risk and therefore correlates to increased hazard rates (i.e. HR>1), a negative slope corresponds to reduced hazard rates (i.e. HR<1), and a slope 0 (a horizontal line) implies no effect on outcome. In time periods where the slope is constant or, in other words, a straight line, the PHA is satisfied for these time periods. Based on this information, it is possible to extend the Cox model by splitting the follow up for time-dependent covariates into

CNPH curve OS for KPS <80 vs. 80-100

Statistical analysis The Cox regression model is a multiplicative hazard model, which means that the effect of covariates is modeled on a multiplicative scale in relationship to the underlying base-line hazard. While the effects of covariates are assumed to be proportional over time, no assumptions are

Figure 1. Cumulative non-parametric hazard (CNPH) curve, overall survival (OS) for Karnofsky Performance Score (KPS) less than 80 compared to KPS 80-100. The vertical red line marks day 139 after transplantation.

Table 2. Overall survival. Karnofsky Performance Score <80 vs. 80-100 Reduced intensity conditioning vs. myeloablative conditioning PBSC vs. BM

Time period

PHA-test P

Before d139 After d139 Unadjusted Before d120 After d120 Unadjusted Before d388 After d388 Unadjusted

2.42 1.43 2.01 0.81 1.11 0.97 0.79 1.23 0.94

2.19-2.68 1.25-1.63 1.85-2.18 0.75-0.88 1.04-1.18 0.92-1.02 0.73-0.85 1.08-1.40 0.87-1.01

<0.001 <0.001 <0.001 <0.001 0.003 0.220 <0.001 0.002 0.069

0.255 0.794 <0.001 0.626 0.310 <0.001 0.161 0.436 <0.001

HR: hazard ratio; CI: confidence interval; PHA: proportional hazards assumption; PBSC: peripheral blood stem cells; BM: bone marrow.

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D. Fuerst et al. Table 3. Disease-free survival. Karnofsky Performance Score <80 vs. 80-100 Reduced intensity conditioning vs. myeloablative conditioning

PBSC vs. BM Intermediate disease stage vs. early disease stage Advanced disease stage vs. early disease stage

Time period

PHA-test P

Before d130 After d130 Unadjusted Before d140 After d140 Unadjusted Before d242 After d242 Unadjusted Before d309 After d309 Unadjusted Before d242 After d242 Unadjusted

2.26 1.30 1.91 0.88 1.12 0.98 0.86 1.14 0.97 1.82 1.52 1.73 2.43 1.79 2.23

2.05-2.49 1.13-1.50 1.76-2.07 0.82-0.95 1.04-1.21 0.93-1.03 0.79-0.93 1.01-1.29 0.90-1.04 1.69-1.95 1.36-1.70 1.62-1.84 2.27-2.60 1.63-1.98 2.10-2.36

<0.001 <0.001 <0.001 0.001 0.002 0.458 <0.001 0.039 0.382 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

0.071 0.910 <0.001 0.835 0.991 <0.001 0.994 0.317 0.001 0.231 0.748 0.004 0.214 0.473 <0.001

HR: hazard ratio; CI: confidence interval; PHA: proportional hazards assumption; PBSC: peripheral blood stem cells; BM: bone marrow.

observation periods and to estimate regression coefficients separately for these respective time intervals. Cut-off points for these time intervals were determined by fitting models from ranges of cut-off points. Selection of cut-off points was based on optimal log likelihood. Multivariate models included risk factors defined by the EBMT risk score: age, disease stage, time from diagnosis to transplant, donor type, and recipient-donor sex combination. In addition, year of transplantation, graft source and conditioning therapy and KPS (<80=poor vs. 80100=good) were included. Missing data for KPS (11.3%) were included in the models as separate category. Stratification was performed for diagnosis and donortype; a center effect was adjusted using a gamma frailty term. Final models were tested for PHA and after adjustment for time-dependent effects all models satisfied PHA. P=0.01 was considered statistically significant.

Results As predictors of primary interest, we defined disease stage, graft source, conditioning regimen intensity, and KPS. Cox regression modeling for OS and testing of PHA indicated a strong non-proportionality of KPS (<80 vs. 80100; P<0.001), conditioning regimen (RIC vs. base-line MAC; P<0.001), and graft source (PBSC vs. BM; P<0.001) (Table 2). The estimates for disease stage showed no timedependent effect in OS analysis. For DFS, KPS (P<0.001), RIC vs. MAC (P<0.001), and PBSC vs. BM (P=0.001), as well as both the estimates for intermediate disease stage (intermediate disease stage vs. base-line early disease stage; P=0.004) and advanced disease stage (advanced disease stage vs. base-line early disease stage; P<0.001) indicated strong time-dependent effects. Based on these results, cumulative hazard curves were plotted to visualize the change in hazards over time [OS: Figures 1-3; DFS: Online Supplementary Figures S3-S7]. After selection of optimal cut-off points, final multivariate models were adjusted in a time-dependent manner. For the estimates of all time periods, the PHA assumption held; the PHA test was not significant (Tables 2 and 3), which proved that the specified cut-off points were adequate for our dataset. For OS analysis, these time periods were before and after day 244

CNPH curve OS for RIC vs. MAC

Figure 2. Cumulative non-parametric hazard curve (CNPH) curve, overall survival (OS) for reduced intensity conditioning (RIC) compared to myeloablative conditioning (MAC). The vertical red line marks day 120 after transplantation.

139 for KPS, before and after day 120 for conditioning intensity, and before and after day 388 for graft source (Figures 1-3). For DFS, these time periods were before and after day 130 for KPS, before and after day 140 for conditioning intensity, before and after day 242 for graft source, before and after day 309 after transplantation for intermediate disease stage, and before and after day 242 for advanced disease stage (Online Supplementary Figures S3S7). Follow up of the cohort was split into observation intervals, and separate hazard ratios were obtained for time-dependent covariates within the respective time periods. For analysis of KPS, the estimates obtained were considerably higher in the early period after transplantation as compared to the period later on. Unadjusted values lay in between (Tables 2 and 3). For conditioning therapy, no significant effect on OS and DFS was found in the unadjusted model, but modeling of time-dependent effects showed opposing significant effects before and after the cut-off time points for OS and DFS. In the early post-transplant period, reduced intensity conditioning appears to have a protective effect, whereas later on a higher hazard ratio was found for patients who had haematologica | 2016; 101(2)

Time-dependent predictors of uHSCT

undergone conditioning with a reduced intensity. The observed effects correlate with lower early mortality and higher late mortality in this patient cohort. In the unadjusted models for OS and DFS, graft source did not correlate with significantly different outcomes. Interestingly, the effect of graft source could be better described by modeling separate estimates for the early and for the later period after transplantation. Similarly for OS and DFS, significant effects were found in the early period after transplantation, indicating lower probability for adverse events for patients transplanted with PBSC, while in the later period these patients seemed to do less well, as higher hazard ratios were observed (Tables 2 and 3). Other predictive covariates in the final model were: age (OS: HR 1.013, CI: 1.011-1.015, P<0.001; DFS: HR 1.011, CI: 1.01-1.013, P<0.001), year of transplantation 20012005 (OS: HR 0.89, CI: 0.82-0.96, P<0.001; DFS: HR 0.94, CI: 0.87-1.01, P=0.092), year of transplantation 20062013 (OS: HR 0.73, CI: 0.67-0.79, P<0.001, DFS: HR 0.81, CI: 0.75-0.88, P<0.001), and time to transplantation from initial diagnosis of more than 12 months (OS: HR 1.18, CI: 1.12-1.25, P<0.001; DFS: HR 1.21, CI: 1.16-1.29, P<0.001). A subset analysis was performed for transplantations using PBSC grafts performed between 2006 and 2013. For OS analysis, KPS and conditioning intensity showed time-dependent characteristics. For DFS analysis, KPS, conditioning intensity as well as disease stage (intermediate and advanced stage) could be confirmed as time-dependent variables. For both end points, similar risk estimates such as those seen in the analysis of the complete dataset were obtained in the subset analysis (Online Supplementary Tables S2 and S3).

Discussion We show in a large German patient cohort that important clinical predictors (KPS, disease stage, conditioning regimen and graft source) exhibit significant time-dependent effects. These effects may be quantified in a sense that regression coefficients and relative risk can be calculated for follow-up periods, which satisfy the PHA, by extending the Cox regression model. We describe patterns that were valid for all disease entities included in this analysis. Our data contain a large proportion of transplantations performed in recent years (2006-2013, 56.9%) and may, therefore, represent current approaches in conditioning treatment and supportive care. The selection of diagnoses aimed to include a large number of patients, while, on the other hand, restricting disease-associated heterogeneity to a limited number of disease entities. Considering time-dependent effects may reveal relationships that could remain undetected in standard Cox regression models. Such effects can be shown for KPS in analysis of OS and DFS. In the early phase after transplantation, the hazard ratio is substantially higher as compared to the later phase after day 139 (OS) and 130 (DFS), respectively. The biological explanation is that the higher risk in the early phase may be attributed to a substantially higher early mortality. This indicates that patients with a poor KPS tolerate transplantation-associated morbidity/toxicity less well. In the later phase, the effect of transplantation morbidity/toxicity disappears (according to our data around 139) and the higher risk in this group diminishes. It is, therefore, no coincidence that the estimated cut-off haematologica | 2016; 101(2)

CNPH curve OS for PBSC vs. BM

Figure 3. Cumulative non-parametric hazard curve (CNPH) curve, overall survival (OS) for peripheral blood stem cells (PBSC) compared to bone marrow (BM) as graft source. The vertical red line marks day 388 after transplantation.

time points are similar for KPS and conditioning treatment (between days 120-140 post transplantation) as they most likely both correlate to conditioning-associated toxicity. Perhaps even more interesting is the observation that the intensity of conditioning regimen, which did not show a statistically significant effect in standard regression modeling, showed clearly significant effects depending on the time interval after transplantations. Our data show that, in the early phase after transplantation, risk is significantly reduced for patients who had undergone a dose-reduced conditioning regimen when compared to patients who had received a standard myeloablative conditioning therapy. This observation was obtained consistently for OSand DFS-associated risk (OS: HR 0.81; DFS: HR: 0.88). However, later on and presumably after resolution of acute conditioning toxicity, the slope of the cumulative hazard curve changes from negative to positive and the risk is higher in the patient group with reduced intensity conditioning. It has been shown that non-relapse mortality is lower in patients undergoing RIC as compared to patients treated with MAC.21 As non-relapse mortality is dominated by treatment-associated mortality soon after transplantation, this effect reflects a potentially protective effect of RIC in the early phase. However, later on, and as conditioning toxicity resolves, a differential effect on relapse remains. Lower relapse rates are observed in patients treated with MAC compared to patients treated with RIC, which explains the adverse effect of RIC in the later time period after HSCT.22 The time-dependent effect of conditioning treatment may, therefore, be explained by opposing effects with different time kinetics on treatmentrelated mortality and relapse incidence. The cumulative hazard curves are very similar for OS and DFS (Figure 2 and Online Supplementary Figure S4). The differences in cutoff time points are probably a result of optimal model selection rather than a correlate of clearly different time kinetics. We could show an almost 20% risk reduction in early mortality for patients with RIC in the OS analysis, which is of clinical significance. The above-mentioned differences were less intense in the DFS analysis, which may be attributed to the fact that higher relapse rates observed 245

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in RIC patients tend to partly attenuate the beneficial effect of RIC on mortality for this end point. Without considering time dependency, these effects would not have been noticed. In a Kaplan-Meier analysis, such a relationship is shown as overlapping survival curves (Online Supplementary Figure S1). Peripheral blood stem cells are considered by many transplant physicians as graft source of choice for adult patients with malignant diseases of the hematopoietic system.23,24 One advantage of PBSCs is the faster engraftment compared to bone marrow, leading to a shorter aplasia time, which may reduce early post-transplant morbidity.25 Our data do not support this practice, as patients who were transplanted with allogeneic PBSCs showed similar hazard ratios compared to patients receiving BM grafts (OS: HR 0.94, CI: 0.87-1.01; P=0.069). However, regarding both end points, a significant time-dependent effect was detected, which, similarly to the analysis of conditioning regimen, allowed a distinction between a phase with risk reduction and a phase with increased risk. In the early phase (before day 388 in OS and before day 242 in DFS), PBSC had a protective effect, possibly because of the faster engraftment time, which reduces aplasia time and in turn has a beneficial influence on transplant-related mortality early after transplantation.26 In the later phase, a risk increase for PBSC was observed, which might be attributed to late complications, e.g. chronic GvHD.27-29 The earlier cut-off time point for DFS is caused by the addition of relapse events, which accumulate early after transplantation when compared to OS. For analysis of OS, a 21% risk reduction in the early phase after transplantation was seen, while the risk afterwards was increased by 23%. These differences are in the magnitude of a single HLAmismatch and are of clinical relevance. DFS analysis revealed weaker differences compared to those observed in OS analysis, underscoring the stronger influence of relapse events in the analysis of DFS. In general, PBSC might be preferred in cases where high early post-transplant mortality is expected, e.g. in elderly patients or in patients with relevant comorbidity. In DFS analysis, the variables “intermediate disease stage” and “advanced disease stage” showed time-dependent effects when compared with early disease stage, which was not seen in the OS analysis. The reason for this observation is that occurrence of relapse is counted as an event in DFS analysis, whereas for OS only death from relapse accounts for an event. Since relapse does not necessarily lead to death from relapse, and as many relapse events occur early after transplantation, such events accumulate for DFS in the early phase after transplantation, particularly for patients with more advanced disease stages. The difference in relative risk seen between advanced and early disease stage patients (HR 2.43 early after transplantation vs. HR 1.79 in the later phase after transplantation) highlights the problem of early morbidity/mortality in advanced disease stage patients, while on the other hand predicting a more favorable outcome for such patients who managed to survive these early complications.

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Other commonly used approaches for inclusion of timedependent variables are stratification on non-proportional variables as well as landmark analysis with refitting of separate models. Stratification, however, allows no quantification or comparison of the effects of various levels of the stratified variable. For the landmark approach, different models are fitted based on landmark time points with inclusion of case subsets with a survival time, which is at least as long as the landmark time points.30 It is often difficult to interpret the results of landmark analysis from a clinical perspective, as subsets of patients are analyzed in each landmark model and multiple models might be necessary. Generally speaking, our approach could be considered as a variant of a landmark approach using the cut-off time points we describe as landmarks. However, only one statistical model is necessary to include all time-dependent variables, which facilitates interpretation. Adjustment of variables for time dependency, in the way we describe here, does not affect the estimates for the other covariates satisfying the PHA in the multivariate Cox regression model. To address the problem of confounding by older transplantations and over-representation of BM grafts in this group, a subset analysis was performed for patients transplanted with PBSC grafts between 2006 and 2013. Very similar risk estimates to those seen in the analysis of the complete dataset were found (Online Supplementary Tables S2 and S3), indicating that our approach did not introduce a relevant bias, but instead allowed us to evaluate the effect of graft source in the context of the other predictors mentioned. A limitation of our analysis is that exact HLA-matching patterns could not be included, as definitions of HLAmatching changed over time, and detailed information about the number and resolution of HLA-mismatches is currently not available from the DRST database. Therefore, stratification had to be performed for the covariate “donor type”. In addition, the dataset included also historical transplantations, which leads to heterogeneity with regard to changes in transplant protocols, graft source, donor selection, or supportive care. This heterogeneity is probably only partly reflected by including the time period of transplantation as covariate. Another important clinical covariate is cytogenetic risk.31,32 However, cytogenetic information is currently not available in the majority of patients in the DRST database, which precluded inclusion in our analysis. Hematopoietic stem cell transplantation is a highly complex and multifactorial process. Understanding and evaluating time-dependent effects allows more sophisticated risk quantification in HSCT to be made, particularly when a predictor has differential impact on outcome, as is the case for conditioning regimen intensity and graft source. Such information may help clinicians choose treatment according to the individual patient. Acknowledgments The authors would like to thank the DRST Data administrators F. Strehle and H. Neidlinger for providing the clinical data for this analysis.

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Time-dependent predictors of uHSCT

References 13. 1. Finke J, Schmoor C, Bethge WA, et al. Prognostic factors affecting outcome after allogeneic transplantation for hematological malignancies from unrelated donors: results from a randomized trial. Biol Blood Marrow Transplant. 2012;18(11):17161726. 2. Grimwade D, Hills RK. Independent prognostic factors for AML outcome. Hematology Am Soc Hematol Educ Program. 2009;385-395. 3. Blaise D, Castagna L. Do different conditioning regimens really make a difference? Hematology Am Soc Hematol Educ Program. 2012;2012:237-245. 4. Spellman SR, Eapen M, Logan BR, et al. A perspective on the selection of unrelated donors and cord blood units for transplantation. Blood. 2012;120(2):259-265. 5. Lee SJ, Klein J, Haagenson M, et al. Highresolution donor-recipient HLA matching contributes to the success of unrelated donor marrow transplantation. Blood. 2007;110(13):4576-4583. 6. Perez L, Anasetti C, Pidala J. Have we improved in preventing and treating acute graft-versus-host disease? Curr Opin Hematol. 2011;18(6):408-413. 7. Holtick U, Albrecht M, Chemnitz JM, et al. Comparison of bone marrow versus peripheral blood allogeneic hematopoietic stem cell transplantation for hematological malignancies in adults - a systematic review and meta-analysis. Crit Rev Oncol Hematol. 2015;94(2):179-188. 8. Algara M, Valls A, Marrugat J, Granena A. Early mortality in bone marrow transplantation for acute lymphocytic leukaemia a multivariate analysis of risk factors. Eur J Med. 1993;2(7):386-392. 9. Valls A, Algara M, Marrugat J, Carreras E, Sierra J, Granena A. Risk factors for early mortality in allogeneic bone marrow transplantation. A multivariate analysis on 174 leukaemia patients. Eur J Cancer. 1993;29A(11):1523-1528. 10. Kaplan EL, Meier P. Nonparametric estimation from incomplete observations. J Am Stat Assoc. 1958;53(282):457-481. 11. Cox D. Regression Models and Life-Tables. J Am Stat Assoc. Series B. 1972;34(2):187220. 12. Therneau T, Grambsch P. Modeling

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Survival Data: Extending the Cox Model. Springer-Verlag; 2000. Martinussen T, Scheike TH. Dynamic Regression Models for Survival Data. Springer; 2006. Gratwohl A, Stern M, Brand R, et al. Risk score for outcome after allogeneic hematopoietic stem cell transplantation: a retrospective analysis. Cancer. 2009;115(20):4715-4726. Furst D, Muller C, Vucinic V, et al. High-resolution HLA matching in hematopoietic stem cell transplantation: a retrospective collaborative analysis. Blood. 2013; 122(18):3220-3229. Bacigalupo A, Ballen K, Rizzo D, et al. Defining the intensity of conditioning regimens: working definitions. Biol Blood Marrow Transplant. 2009;15(12):16281633. Scheike TH, Zhang MJ. Extensions and applications of the Cox-Aalen survival model. Biometrics. 2003;59(4):1036-1045. Zahl PH. Regression analysis with multiplicative and time-varying additive regression coefficients with examples from breast and colon cancer. Stat Med. 2003; 22(7):1113-1127. Grambsch P, Therneau T. Proportional Hazards Tests and Diagnostics Based on Weighted Residuals. Biometrika. 1994; 81(3):515-526. Cortese G, Scheike TH, Martinussen T. Flexible survival regression modelling. Stat Methods Med Res. 2010;19(1):5-28. Ringden O, Labopin M, Ehninger G, et al. Reduced intensity conditioning compared with myeloablative conditioning using unrelated donor transplants in patients with acute myeloid leukemia. J Clin Oncol. 2009;27(27):4570-4577. Mohty M, Labopin M, Volin L, et al. Reduced-intensity versus conventional myeloablative conditioning allogeneic stem cell transplantation for patients with acute lymphoblastic leukemia: a retrospective study from the European Group for Blood and Marrow Transplantation. Blood. 2010; 116(22):4439-4443. Stem Cell Trialists Collaborative Group. Allogeneic peripheral blood stem-cell compared with bone marrow transplantation in the management of hematologic malignancies: an individual patient data meta-analysis of nine randomized trials. J Clin Oncol. 2005;23(22):5074-5087.

24. Bertz H, Spyridonidis A, Ihorst G, et al. Marrow versus blood-derived stem cell grafts for allogeneic transplantation from unrelated donors in patients with active myeloid leukemia or myelodysplasia. Biol Blood Marrow Transplant. 2012;18(6):894902. 25. Ringden O, Remberger M, Runde V, et al. Faster engraftment of neutrophils and platelets with peripheral blood stem cells from unrelated donors: a comparison with marrow transplantation. Bone Marrow Transplant. 2000;25 Suppl 2:S6-S8. 26. Champlin RE, Schmitz N, Horowitz MM, et al. Blood stem cells compared with bone marrow as a source of hematopoietic cells for allogeneic transplantation. IBMTR Histocompatibility and Stem Cell Sources Working Committee and the European Group for Blood and Marrow Transplantation (EBMT). Blood. 2000; 95(12):3702-3709. 27. Mohty M, Kuentz M, Michallet M, et al. Chronic graft-versus-host disease after allogeneic blood stem cell transplantation: long-term results of a randomized study. Blood. 2002;100(9):3128-3134. 28. Schmitz N, Beksac M, Bacigalupo A, et al. Filgrastim-mobilized peripheral blood progenitor cells versus bone marrow transplantation for treating leukemia: 3-year results from the EBMT randomized trial. Haematologica. 2005;90(5):643-648. 29. Schmitz N, Eapen M, Horowitz MM, et al. Long-term outcome of patients given transplants of mobilized blood or bone marrow: A report from the International Bone Marrow Transplant Registry and the European Group for Blood and Marrow Transplantation. Blood. 2006;108(13):42884290. 30. Anderson JR, Cain KC, Gelber RD. Analysis of survival by tumor response and other comparisons of time-to-event by outcome variables. J Clin Oncol. 2008; 26(24):3913-3915. 31. Tallman MS, Dewald GW, Gandham S, et al. Impact of cytogenetics on outcome of matched unrelated donor hematopoietic stem cell transplantation for acute myeloid leukemia in first or second complete remission. Blood. 2007;110(1):409-417. 32. Armand P, Gibson CJ, Cutler C, et al. A disease risk index for patients undergoing allogeneic stem cell transplantation. Blood. 2012;120(4):905-913.

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ARTICLE EUROPEAN HEMATOLOGY ASSOCIATION

Stem Cell Transplantation

Ferrata Storti Foundation

Angelique V.M. Brands-Nijenhuis,1 Myriam Labopin,2 Harry C. Schouten,3 Liisa Volin,4 Gérard Socié,5 Jan J. Cornelissen,6 Anne Huynh,7 Per Ljungman,8 Florent Malard,9 Jordi Esteve,10* Arnon Nagler,11,2* and Mohamad Mohty9*

Catharina Ziekenhuis Eindhoven, Department of Internal Medicine, Division of Hematology, Eindhoven, the Netherlands; 2EBMT Data Office Paris, Hospital Saint Antoine, Department of Hematology, France; 3Maastricht University Medical Center, the Netherlands; 4Helsinki University Central Hospital, Department of Medicine, Finland; 5 Hospital St. Louis, Department of Hematology, Paris, France; 6Erasmus Medical Center Rotterdam, the Netherlands; 7Hôpital de Purpan CHU Department Hematologie Toulouse, France; 8Karolinska University Hospital, Department of Hematology, Stockholm, Sweden; 9Hospital Saint Antoine, Department of Hematology, Paris, France; 10 Hospital Clínic, Department of Hematology, IDIBAPS, Barcelona, Spain; and 11Chaim Sheba Medical Center, Tel-Hashomer, Israel *These authors contributed equally 1

Haematologica 2016 Volume 101(2):248-255

ABSTRACT

Correspondence: mohamad.mohty@inserm.fr

Received: 24/06/2015. Accepted: 13/11/2015. Pre-published: 20/11/2015. doi:10.3324/haematol.2015.132654

Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/101/2/248

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espite the overall benefit from allogeneic hematopoietic stem cell transplantation observed in patients with poor cytogenetic risk acute myeloid leukemia in first complete remission, the precise effect of this procedure for different poor-risk subtypes has not been fully analyzed. This retrospective analysis was performed to investigate whether allogeneic hematopoietic stem cell transplantation performed in first complete remission in patients with monosomal karyotype can overcome the adverse prognosis associated with these patients. Of the 4635 patients included in the study, 189 (4%) harbored a monosomal karyotype. The presence of a monosomal karyotype was associated with a worse outcome, with an inferior leukemia-free survival and overall survival (5-year leukemia-free survival and overall survival: 24±3% and 26±3% vs. 53±1% and 57±1% in monosomal-karyotype and non-monosomal-karyotype, respectively; P<0.0001) and higher relapse risk after transplantation (cumulative incidence of relapse at 5 years: 56±4% in monosomal-karyotype vs. 28±1% in nonmonosomal-karyotype; P<0.0001). The adverse negative impact of monosomal karyotype cytogenetics was confirmed in the entire cohort in a multivariate analysis [Hazard Ratio (HR): 1.88, 95% Confidence Interval (CI):1.292.73, P=0.001 for relapse incidence; HR:1.71, 95%CI:1.27-2.32, P<0.0001 for leukemia-free survival; HR:1.81, 95%CI:1.32-2.48, P=0.0002 for overall survival], and was independent of the presence of other poor-risk cytogenetic subtypes. In summary, monosomal karyotype arises as a strong negative prognostic feature in acute myeloid leukemia also in patients who undergo allogeneic hematopoietic stem cell transplantation in first complete remission, stressing the need to develop additional pre- and post-transplantation strategies aimed at improving overall results. Nonetheless, allogeneic hematopoietic stem cell transplantation in early phase is currently the best therapy for this very poor-risk acute myeloid leukemia subtype. haematologica | 2016; 101(2)

alloHSCT for monosomal karyotype AML

Introduction Acute myeloid leukemia (AML) is a disorder of hematopoietic progenitor cells with a great biological and clinical diversity, as a result of very different genetic alterations leading to an impaired differentiation capacity and increased proliferation ability of the leukemic population.1 It has long been recognized that several cytogenetic and molecular abnormalities are of prognostic importance, and the clinical relevance of these is reflected in the WHO 2008 classification of AML.2 The European LeukemiaNet defines three genetic subgroups to classify AML (excluding acute promyelocytic leukemia) according to the risk of relapse: favorable, intermediate and unfavorable.1 Between 15% and 20% of AML patients belong to the favorable group, characterized by t(8;21), inv(16) or t(16.16). The unfavorable group is a heterogeneous group of patients, with 25%-40% of AML patients with diverse cytogenetic abnormalities such as loss of long arm or monosomy of chromosomes 5 or 7, EVI1 rearrangement associated to translocation 3q26, different types of rearrangement involving the MLL gene, deletion of 17p region related to TP53 loss, and other cases carrying multiple clonal cytogenetic abnormalities known as complex karyotypes (CK, â&#x2030;Ľ3 abnormalities in the karyotype). Finally, the remaining AML patients with t(9;11)(q22;q23) and cytogenetic abnormalities not otherwise classified belong to the intermediate risk group. The benefit of allogeneic hematopoietic stem cell transplantation (alloHSCT) in first complete remission has been clearly confirmed in the unfavorable risk and intermediate risk groups, presumably depending on the underlying genotypes.3,4 However, the precise effect of this procedure for differ-

ent poor-risk subtypes has not been fully analyzed. Breems et al. identified a very poor prognostic cytogenetic subgroup named monosomal karyotype (MK),5 with an overall survival (OS) of only 3% at four years. This group of MK patients was defined by the presence of one autosomal monosomy together with at least one other structural chromosomal abnormality or the presence of at least two autosomal monosomies. Cornelissen et al. reported that post-consolidation therapy using alloHSCT in MK AML in CR1 is associated with a significant reduction in relapse and improvement of survival with a similar relative reduction in death and relapse to other cytogenetic risk categories.6 Similarly, Fang et al. and Oran et al. reported a negative prognostic impact on survival of MK also in patients who underwent alloHSCT.7,8 However, the number of transplanted patients with MK included in the studies mentioned was limited, making it difficult to accurately define the outcome of MK AML patients after alloHSCT, especially for patients in CR1. To address this issue, we performed a retrospective analysis using data from the European Group for Blood and Marrow Transplantation (EBMT) registry in patients with primary AML in first complete remission (CR1).

Methods Study population We searched the registry of the EBMT with the following criteria: patients aged 18 years or over, initial diagnosis of AML, in CR1, transplanted with allogeneic donors (related and unrelated; syngeneic excluded), transplant source of bone marrow and/or peripheral blood stem cells (cord blood excluded), transplanted

Table 1. Main characteristics of patients with and without monosomal karyotype. N. of patients (%) Sex (female:male), n (%) Age (years), median (range) AML subtype (FAB), n (%) M1 to M6 M0 M7 & other Cytogenetics at diagnosis, n (%) MK CK OP Any 7 abnormality Monosomy 5 or del5q 17p abnormality WBC at diagnosis (109/L), median (range) Patients requiring >1 induction course to achieve CR1, n (%) Interval diagnosis to CR1, days median (range) Donor type (MRD/MUD), n (%) Stem cell source (BM/PB), n (%) Intensity of conditioning regimen (MAC/RIC), n (%)

Non-MK AML

MK AML

4446 (96) 2213:2230 (50:50) 45 (18-76)

189 (4) 97:92 (51:49) 48.5 (18-68)

0.76 0.007

3691 (86) 296 (7) 298 (7)

125 (68) 23 (15) 36 (19)

<0.0001 <0.0001

0 182 (4) 706 (16) 222 (5) 91 (2) 16 (0.4) 12.9 (0.2-879) 1072 (31) 46 (15-110) 3174 (71)/1272(29) 1514 (34)/2932(66) 3023 (69)/1375(31)

189 (100) 130 (69) 151 (82) 119 (66) 70 (37) 28 (15) 4.6 (0.45-290) 61 (41) 53 (10-174) 110 (58)/79 (42) 60 (32)/129(68) 117 (64)/67(36)

<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.01 0.008 <0.0001 0.51 0.14

AML: acute myeloid leukemia; MK: monosomal karyotype; CK: complex karyotype; OP: other poor risk cytogenetics; any 7 abnormality: any abnormality involving chromosome 7; WBC: white blood cell count; MRD: matched related donor; MUD: matched unrelated donor; BM: bone marrow; PB: peripheral blood stem cells; MAC: myeloablative conditioning; RIC: reduced intensity conditioning.

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A.V.M. Brands-Nijenhuis et al. Table 2. Multivariable analysis among the entire cohort (n=4635).

Prognostic factors

Relapse incidence HR (95% CI)

Age > 45 years WBC at diagnosis (>12.3x109/L) Time to achieve CR (>45 days) Unrelated donor Conditioning intensity (RIC vs. MAC) Acute GvHD (grade 2-4 vs. 0-1) Chronic GvHD (present vs. absent) Monosomal karyotype (present vs. absent) Complex karyotype (present vs. absent) Other poor cytogenetics No (reference) Monosomy 7 Other AML subtype (FAB) (M0-M7 vs. other)

Non-relapse mortality HR (95% CI)

Leukemia-free survival HR (95% CI)

Survival HR (95% CI)

0.97 (0.82-1.14) 1.47 (1.26-1.71)

0.69 <0.0001

1.70 (1.41-2.05) 0.98 (0.83-1.17)

<0.0001 0.83

1.26 (1.12-1.42) 1.19 (1.07-1.33)

0.0002 0.001

1.30 (1.15-1.47) 1.20 (1.07-1.34)

<0.0001 0.001

1.56 (1.34-1.81)

<0.0001

1.26 (1.07-1.50)

0.007

1.40 (1.26-1.56)

<0.0001

1.45 (1.29-1.62)

<0.0001

1.02 (0.87-1.20) 1.28 (1.07-1.53)

0.81 0.006

1.31 (1.09-1.57) 0.70 (0.57-0.86)

0.005 0.001

1.16 (1.04-1.31) 1.08 (0.95-1.22)

0.01 0,26

1.19 (1.06-1.35) 1.04 (0.91-1.19)

0.004 0.53

0.75 (0.62-0.90)

0.002

2.74 (2.30-3.26)

<0.0001

1.39 (1.24-1.56)

<0.0001

1.55 (1.37-1.74)

<0.0001

0.77 (0.64-0.93)

0.007

2.50 (2.00-3.11)

<0.0001

1.20 (1.06-1.37)

0.005

0.96 (0.85-1.10)

0.58

1.88 (1.29-2.73)

0.001

1.30 (0.72-2.34)

0.39

1.71 (1.27-2.32)

<0.0001

1.81 (1.32-2.48)

0.0002

1.59 (1.20-2.11)

0.001

0.90 (0.59-1.37)

0.62

1.26 (1.01-1.58)

0.04

1.28 (1.01-1.61)

0.04

1 1.85 (1.28-2.67) 1.41 (1.17-1.70) 1.08 (0.88-1.32)

0.001 0.0003 0.46

1 1.13 (0.65-1.97) 0.87 (0.68-1.12) 1.04 (0.82-1.32)

0.67 0.28 0.76

1 1.43 (1.07-1.92) 1.17 (1.02-1.35) 1.16 (1.01-1.34)

0.02 0.03 0.05

1 1.32 (0.97-1.79) 1.08 (0.93-1.26) 1.15 (0.99-1.34)

0.08 0.30 0.07

HR: hazard ratio; CI: confidence interval; WBC: white blood cell count; CR: complete remission; RIC: reduced intensity conditioning; MAC: myeloablative conditioning; GvHD: graft- versus-host disease; FAB: French-American-British Classification.

between 1995 and 2010, and known cytogenetics. A total of 11,001 patients were identified. Data were provided and approved for this study by the Institutional Review Board of the Acute Leukemia Working Party (ALWP) of the EBMT group registry. This a voluntary working group of more than 500 transplant centers that are required to report all consecutive stem cell transplantations and follow-up visits once a year. Audits are routinely performed to determine the accuracy of the data. Since 1990, patients have been providing informed consent authorizing the use of their personal information for research purposes. The participating centers are listed on the Online Supplementary Appendix.

Cytogenetic categories Cytogenetics was based on local reports. All cytogenetic data were reviewed by 2 of the authors (AVMBN, JE) according to the European LeukemiaNet.1 Patients with two or more autosomal chromosome monosomies or a single autosomal monosomy in the presence of at least one other structural chromosomal abnormality, excluding those patients with unidentified marker chromosomes, were assigned to the MK group (as defined by Breems et al.5) while the remainder of patients were assigned to the non-MK group. Two additional cytogenetics categories were distinguished within the unfavorable risk group, namely AML with a complex karyotype (CK) and other poor-risk cytogenetic entities (OP). CK was defined as the presence of three or more structural chromosomal abnormalities in the absence of a recurrent cytogenetic abnormality.1,2 OP comprised the following cytogenetic abnormalities: t(3;3)(q21;q26) or inv(3q)/EVI1 rearrangement, t(6;9)(p23;q34)/DEK-NUP214, del5q/-5, del7q/-7, t(1;22)(p13;q13), MLL rearrangement and t(9;22)/BCR-ABL.1,9 Those cases with insufficient information to be cytogenetically assigned were 250

Figure 1. Distribution of autosomal monosomies within the study group.

excluded from the analysis and those not fulfilling criteria for MK, OP or CK were defined to have intermediate risk (IR). Overall, 4635 patients met the inclusion criteria and were included in the study. haematologica | 2016; 101(2)

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Statistical analysis The probabilities of overall survival (OS), leukemia-free survival (LFS), relapse incidence (RI), and non-relapse mortality (NRM) were the primary study end points. LFS was defined as time interval from alloHSCT until either relapse or death in months, and was calculated using the Kaplan-Meier estimate. NRM was defined as death in the absence of relapse. RI and NRM were calculated using cumulative incidence curves in a competing risks set-

ting, death in remission being treated as a competing event to relapse and relapse in the NRM estimation setting, respectively.10 Univariate analyses were performed using log rank test for OS and LFS while Gray’s test was applied for RI and NRM. Patient-, disease-, and transplant-related variables of both groups were compared, using the c2 test for categorical and the Mann-Whitney test for continuous variables. Variables considered were: recipient age, sex, disease characteristics [FAB classification, white blood count

Table 3. Multivariate analysis among patients with a monosomal karyotype acute myeloid leukemia (n=189). LFS Poor-risk cytogenetics No (reference) Monosomy 7 Other poor-risk Age <35 (years) [35-60] vs. <35 (years) ≥60 (years) vs. [35-60] Interval diagnosis-CR1>median (53d) Donor (UD vs. HLA-id sibling) Conditioning intensity (RIC vs. MAC) RI Poor-risk cytogenetics No (reference) Monosomy 7 Other poor-risk Age <35 (years) [35-60] vs. <35 (years) ≥60 (years) vs. [35-60] Interval diagnosis-CR1>median (53d) Donor (UD vs. HLA-id sibling) Conditioning intensity (RIC vs. MAC) NRM Poor-risk cytogenetics No (reference) Monosomy 7 Other poor-risk Age <35 (years) [35-60] vs. <35 (years) ≥60 (years) vs. [35-60] Interval diagnosis-CR1>median (53d) Donor (UD vs. HLA-id sibling) Conditioning intensity (RIC vs. MAC) OS Poor-risk cytogenetics No (reference) Monosomy 7 Other poor-risk Age <35 (years) [35-60] vs. < 35 (years) ≥60 (years) vs. [35-60] Interval diagnosis-CR1>median (53 d) Donor (UD vs. HLA-id sibling) Conditioning intensity (RIC vs. MAC)

0.04 0.07

1.00 1.85 1.69

1.02 0.95

3.38 2.98

34.3 (18.3-51.4) 18.2 (7.7-28.8) 20.1 (11.6-28.6)

0.005 0.12 0.71 0.55 0.13

2.16 1.55 1.07 1.10 0.71

1.26 0.89 0.74 0.77 0.45

3.69 2.67 1.55 1.62 1.1

46.2 [29.5-62.8] 19.8 [12.2-27.3] 10.3 [0-21.4] 18.3 (9.6-27) 23.3 (13.7-32.8) vs. 24.1 (15.7-32.5) 16.6 (6.9-26.2) vs. 27.9 (19.5-36.4)

0.06 0.07

1.00 2.11 1.97

0.98 0.95

4.54 4.09

46.6 (28.4-62.8) 62.2 (47.4-73.9) 59 (47.8-68.6)

0.05 0.33 0.62 0.64 0.36

1.89 1.4 0.95 1.11 0.78

1.0 0.71 0.61 0.71 0.45

3.58 2.79 1.49 1.75 1.34

45.5 [28.2-61.3] 57.6 [47.8-66.3] 65.5 [44.2-80.3] 54 (42.3-64.3) 52 (40.1-62.6) vs. 59.2 (48.9-68.1) 66.8 (52.9-77.4) vs. 51.1 (41.3-60)

0.89 0.98

1.00 1.08 1.01

0.36 0.36

3.25 2.84

18.6 [9.5-30] 19.6 [10.2-31.1] 20.9 [11.2-32.6]

0.02 0.57 0.15 0.20 0.16

5.4 1.39 1.73 1.60 0.52

1.24 0.45 0.82 0.78 0.21

23.4 4.31 3.64 3.29 1.29

8.3 [0.8-27.6] 22.6% [5.6-46.3] 24.1 [6.4-47.9] 27.7 [18.4-37.7] 24.7 [17-33.2] vs. 16.7 [10.4-24.4] 16.7 [10.5-24.1] vs. 21 [14-28.9]

0.10 0.33

1.00 1.65 1.33

0.9 0.75

3.03 2.37

34.3 (17.8-50.9) 22 (10.9-33.1) 22.1 (13.2-31.1)

0.007 0.53 0.34 0.35 0.05

2.1 1.74 1.2 1.19 0.63

1.23 0.99 0.83 0.82 0.39

3.6 3.05 1.74 1.73 0.99

48.7 [32-65.4] 22.7 [14.9-30.6] 10.3 [0-21.4] 20.3 (11.3-29.3) 23.9 (14.2-33.6) vs. 27.6 (18.9-36.3) 22.5 (12.4-32.7) vs. 28.2 (19.5-36.8)

95% CI

% at 5 years

RI: relapse incidence; NRM: non-relapse related mortality; LFS: leukemia-free survival; OS: overall survival; MAC: myeloablative conditioning; RIC: reduced intensity conditioning; CK: complex karyotype; OP: other poor-risk cytogenetics; ID sibling: HLA identical sibling; UD: unrelated donor.

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(WBC) at time of diagnosis, interval from diagnosis to CR1 in days], donor characteristics (age, sex) and transplant characteristics (including type of donor, conditioning, and source of stem cells). Factors differing in distribution between the two groups with P<0.15 were included in the final models. We also performed a separate analysis of MK patients to determine prognostic factors associated to patient, donor and transplant characteristics in this subgroup. For all prognostic analyses, continuous variables were first categorized into five categories according to the quintiles. If there was no substantial difference in relative event rates between two or more adjacent categories, these categories were grouped. Otherwise, the median was used as a cut-off point. Associations of MK with outcomes were evaluated in multivariate analyses, using Cox proportional hazards model including time-dependant variables. All tests were two-sided. The type I error rate was fixed at 0.05 for determination of factors associated with time to event outcomes. Statistical analyses were performed with SPSS 19 (SPSS Inc., Chicago, USA) and R (R Development Core Team, Vienna, Austria) software packages.

Results Patientsâ&#x20AC;&#x2122; characteristics From a total of 4635 patients 189 (4%) harbored a MK. Basic characteristics of this group are listed in Tables 1 and 2, compared to the remaining 4446 patients. Patients with MK were older (median age 45 years for non-MK vs. 48.5 years for MK; P=0.007), presented with a lower white blood cell count (WBC) at diagnosis (12.9x109/L for non-MK vs. 4.6x109/L for MK; P<0.0001), presented with a higher proportion of FAB AML subtype M0 (7% for nonMK (n=296) vs. 15% for MK (n=23); P<0.0001) and required a longer interval to achieve CR1 (46 days for nonMK vs. 53 days for MK; P=0.008), indicative of a higher proportion of patients with an MK AML who required more than one induction course to achieve CR1 (41% among MK vs. 31% among non-MK; P=0.01).

Cytogenetic features Two-thirds of patients with an MK also harbored a CK, and 82% of MK had a karyotype fulfilling criteria for OP

Figure 2. Outcome after alloHSCT in patients harboring a monosomal karyotype compared to other cytogenetic abnormalities. (A) Leukemia-free survival (LFS). (B) Relapse incidence (RI) after alloHSCT. (C) Non-relapse mortality (NRM). (D) Overall survival (OS) after alloHSCT.

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as described in the Methods section. On the contrary, 58% of the CK AML patients did not correspond to MK and MK only represented a minority (15%) of OP. The distribution of autosomal monosomies present in both MK and non-MK patients is shown in Figure 1. In MK patients, monosomy 7 was the most frequent (93 patients, 24.3%), followed by monosomy 5 (39 patients, 10.2%), 17 (24 patients, 6.3%), 13 (22 patients, 5.7%), and monosomies of chromosomes 18 and 20 (both 18 patients, 4.7%). In non-MK patients, monosomy 7 was also the most frequent (80 patients, 68.4%), followed by monosomy 5 (5 patients, 4.3%), monosomy 11 and 20 (both 4 patients, 3.4%), and monosomy 21 (3 patients, 2.6%).

Outcome after alloHSCT Median follow up of patients was 75 months (range 1.2235) and median year of transplant was 2005. Five-year probability of LFS in MK and non-MK patients was 24±3% and 53±1%, respectively (P<0.0001) (Figure 2A). Cumulative incidence of relapse at five years was markedly increased in MK, with 56±4% versus 28±1% in non-MK

(P<0.0001) (Figure 2B), whereas there was no difference in NRM between the two groups (5-year NRM: 20±3% for MK and 19±1% for non-MK; P=0.77) (Figure 2C). MK patients also experienced a shorter survival after alloHSCT, with OS at five years of 26±3% for MK versus 57±1% for non-MK (P<0.0001) (Figure 2D). In the subgroup of patients with CK, 5-year OS was significantly decreased in MK (n=130), with 27.1% versus 51.8% in patients without MK (n=182, 51.8%; P<0.0001). Similarly, in this subgroup of patients, LFS was significantly decreased in patients with MK, with 24.1% compared to 45% in patients without MK (P<0.0001). In order to analyze the impact of MK on outcome, a multivariate analysis was performed including the following covariates: age, FAB subtype, WBC at diagnosis, interval from diagnosis to CR, donor type, conditioning intensity, diagnosis, GvHD (acute and chronic) and cytogenetic categories (including separately MK, CK and other cytogenetics, which was categorized into 3 classes: non-OP, monosomy of chromosome 7, and OP subtypes). MK was an independent adverse prognostic factor in multivariate

Figure 3. Outcome after alloHSCT among patients with MK-AML according to age. (A) Leukemia-free survival (LFS). (B) Relapse incidence (RI) after alloHSCT. (C) Non-relapse mortality (NRM). (D) Overall survival (OS) after alloHSCT.

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analysis for LFS (HR: 1.71, 95%CI: 1.27-2.32; P<0.0001), RI (HR 1.88, 95%CI: 1.29-2.73; P=0.001) and OS (HR: 1.81, 95%CI: 1.32-2.48; P=0.0002). Remarkably, the adverse impact of MK was independent of the presence of a CK and OP. Details of other prognostic factors in multivariate analysis are shown in Table 2.

Prognostic factors among MK patients After confirming the negative prognostic impact of MK in the entire cohort, we performed a separate analysis of MK patients to determine the prognostic effect of additional variables. A total of 189 MK patients were identified in this cohort. The results of the multivariate analysis are summarized in Table 4. After adjustment, the only factor significantly associated with OS, NRM, RI and LFS was age, with a more favorable outcome among the subgroup of 37 patients under 35 years of age (Table 3 and Figure 3). A comparison of the main characteristics of MK patients under and over 35 years of age is indicated in Online Supplementary File 2.

Discussion This analysis confirms the negative prognostic impact on survival of the well-recognized high-risk cytogenetic subcategory of MK also in patients who undergo alloHSCT in CR1,7 mainly due to a high relapse incidence observed after transplant (over 50% at 5 years). The detrimental effect of MK on outcome was independent of other variables, including the presence of other known adverse cytogenetics features such as monosomy 7 and CK, supporting the recognition of this entity as a challenging subgroup of patients with distinct biological and clinical features. Therefore, the prognosis of patients with a CK was significantly worse in the subgroup of patients with both CK and MK compared to patients with CK alone. Nonetheless, alloHSCT in early phase still represents the best available option for this very high-risk group of patients, providing a long-term response for a significant subgroup, not observed with other approaches.6 Despite the overall unfavorable results associated with MK, the outcome of this cohort of patients transplanted in CR1 seems to be improved compared to those transplanted in a more advanced phase or who do not receive an alloHSCT, with a long-term LFS plateau of 24% possibly indicative of the curative potential of an alloHSCT in a fraction of these patients.11 Moreover, this observation emphasizes the importance of increasing the proportion of MK patients who achieve CR1 and who could benefit from an alloHSCT in this early phase. In this regard, a subanalysis of the results from the HOVON/SAKK trial addressing the effect of higher doses of cytarabine in induction and consolidation (200 mg/m2 for 6 days in induction and 1000 mg/m2 for 5 days in first consolidation vs. high-dose cytarabine, 1000 mg/m2 for 6 days in induction and 2000 mg/m2 for 4 days in first consolidation) showed a better outcome in the subgroup of younger MKAML patients who received higher cytarabine dose, with an increase in event-free survival and OS at five years from

References 1. Dohner H, Estey EH, Amadori S, et al. Diagnosis and management of acute myeloid leukemia in adults: recommenda-

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0% to 13% and 0% to 16%, respectively.12 In contrast, a similar study from the same group comparing escalated dose of daunorubicin (90 mg/m2 for 3 days vs. 45 mg/m2 for 3 days in induction) in patients over 60 years of age, was not associated with a statistically improved outcome in the MK subgroup.13 These findings support the design of specific studies aimed to identify that combination of induction chemotherapy associated with the highest initial CR rate for this subgroup of patients with MK that could increase the proportion of the patients who could benefit from an alloHSCT. Our analysis here, however, is based on a highly selected patient population of patients who have reached CR1 and who were suitable for an alloHSCT in the setting of a matched donor being available. Therefore, the incidence of MK identified in this large population of allografted patients (4%) is lower than that previously reported in unselected AML populations (16%-37% depending on age).4 Given the high relapse incidence in MK patients, and the higher number of induction courses necessary for reaching CR1 compared to other cytogenetic groups (41% of MK patients required more than one induction course to achieve CR), the majority of MK patients are obviously not able to reach alloHSCT or are transplanted in a more advanced phase. The outcome of alloHSCT for MK patients in more advanced phase was analyzed in a study by the AML Study Group which showed a very poor prognosis, with only 5 out of 46 patients undergoing alloHSCT not in CR being alive at two years after the procedure. These results emphasize the importance of improving the efficacy of pre-transplant therapy in this AML subtype to increase the proportion of patients who can ultimately benefit from an alloHSCT.14 Other measures that can be implemented to reduce the relapse risk are optimization of the conditioning regimen and development of post-transplant strategies aimed at preventing relapse. Myeloablative regimes are associated with a lower relapse risk, although this beneficial effect, coupled with a higher NRM among more intensive regimens, does not translate into a neat clinical benefit in more heterogeneous high-risk groups. Therefore, implementation of novel regimes maintaining the anti-leukemic effect of more intensive conditioning schemes but with reduced toxicity are warranted. Potential post-transplant intervention that could result in a reduced relapse incidence are the administration of prophylactic or pre-emptive donor lymphocyte infusion (DLI), or the use of azacitidine maintenance.15 In conclusion, the presence of a monosomal karyotype is associated with a worse outcome compared to other cytogenetic abnormalities in patients undergoing alloHSCT in CR1, with a high relapse risk after transplantation and a durable response only in approximately one-quarter of patients. Improvement of induction regimens, conditioning regimens and post-transplant treatment for prevention of relapse is warranted in this highrisk group of patients, in order to improve the number of patients that can benefit from alloHSCT and therefore improving outcome.

tions from an international expert panel, on behalf of the European LeukemiaNet. Blood. 2010;115(3):453-474. 2. Swerdlow SH, Campo E, Harris NL, et al, (ed.) WHO Classification of tumours of

haematopoietic and lymphoid tissues. 4th Ed, Lyon, France: IARC; 2008. 3. Koreth J, Schlenk R, Kopecky KJ, et al. Allogeneic stem cell transplantation for acute myeloid leukemia in first complete

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remission: systematic review and metaanalysis of prospective clinical trials. JAMA. 2009;301(22):2349-2361. Slovak ML, Kopecky KJ, Cassileth PA, et al. Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia: a Southwest Oncology Group/Eastern Cooperative Oncology Group Study. Blood. 2000; 96(13):4075-4083. Breems DA, Van Putten WL, De Greef GE, et al. Monosomal karyotype in acute myeloid leukemia: a better indicator of poor prognosis than a complex karyotype. J Clin Oncol. 2008;26(29):4791-4797. Cornelissen JJ, Breems D, van Putten WL, et al. Comparative analysis of the value of allogeneic hematopoietic stem-cell transplantation in acute myeloid leukemia with monosomal karyotype versus other cytogenetic risk categories. J Clin Oncol. 2012;30(17):2140-2146. Fang M, Storer B, Estey E, et al. Outcome of

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patients with acute myeloid leukemia with monosomal karyotype who undergo hematopoietic cell transplantation. Blood. 2011;118(6):1490-1494. Oran B, Dolan M, Cao Q, Brunstein C, Warlick E, Weisdorf D. Monosomal karyotype provides better prognostic prediction after allogeneic stem cell transplantation in patients with acute myelogenous leukemia. Biol Blood Marrow Transplant. 2011; 17(3):356-364. Estey E. High cytogenetic or molecular genetic risk acute myeloid leukemia. Hematology Am Soc Hematol Educ Program. 2010;2010:474-480. Gooley TA, Leisenring W, Crowley J, Storer BE. Estimation of failure probabilities in the presence of competing risks: new representations of old estimators. Stat Med. 1999; 18(6):695-706. Guo RJ, Atenafu EG, Craddock K, Chang H. Allogeneic hematopoietic cell transplantation may alleviate the negative prognostic

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impact of monosomal and complex karyotypes on patients with acute myeloid leukemia. Biol Blood Marrow Transplant. 2014;20(5):690-695. Lowenberg B, Pabst T, Vellenga E, et al. Cytarabine dose for acute myeloid leukemia. N Engl J Med. 2011; 364(11):1027-1036. Lowenberg B, Ossenkoppele GJ, van Putten W, et al. High-dose daunorubicin in older patients with acute myeloid leukemia. N Engl J Med. 2009;361(13):1235-1248. Kayser S, Zucknick M, Dohner K, et al. Monosomal karyotype in adult acute myeloid leukemia: prognostic impact and outcome after different treatment strategies. Blood. 2012;119(2):551-558. Bashir Q, William BM, Garcia-Manero G, de Lima M. Epigenetic therapy in allogeneic hematopoietic stem cell transplantation. Rev Bras Hematol Hemoter. 2013; 35(2):126-133.

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ARTICLE EUROPEAN HEMATOLOGY ASSOCIATION

Stem Cell Transplantation

Ferrata Storti Foundation

Peripheral blood stem cell graft compared to bone marrow after reduced intensity conditioning regimens for acute leukemia: a report from the ALWP of the EBMT

Bipin N. Savani,1 Myriam Labopin,2,3,4,5 Didier Blaise,6 Dietger Niederwieser,7 Fabio Ciceri,8 Arnold Ganser,9 Renate Arnold,10 Boris Afanasyev,11 Stephane Vigouroux,12 Noel Milpied,12 Michael Hallek,13 Jan J. Cornelissen,14 Rainer Schwerdtfeger,15 Emmanuelle Polge,2,3,4,5 Frédéric Baron,16 Jordi Esteve,17 Norbert C. Gorin,18 Christoph Schmid,19 Sebastian Giebel,20 Mohamad Mohty2,3,4,5 and Arnon Nagler2,21

Haematologica 2016 Volume 101(2):256-262

1 Vanderbilt University Medical Center, Nashville, TN, USA; 2EBMT Paris Study Office/CERESTTC, Paris, France; 3Department of Haematology, Saint Antoine Hospital, Paris, France; 4 INSERM UMR 938, Paris, France; 5Université Pierre et Marie Curie, Paris, France; 6 Département d'Hématologie - Centre de Recherche en Cancérologie de Marseille - AixMarseille University, Institut Paoli Calmettes, Marseille, France; 7University Hospital Leipzig, Hematology, Oncology and Hemostaseology Divisions, Germany; 8Hematology, IRCCS San Raffaele Scientific Institute, Milan, Italy; 9Hannover Medical School - Department of Haematology Hemostasis Oncology and Stem Cell Transplantation, Hannover, Germany; 10 Charité Universitätsmedizin Berlin, Campus Virchow Klinikum – Medizinische Klinik m. S. Hämatologie/Onkologie, Berlin, Germany; 11Saint Petersburg State Medical Pavlov University, Ratsa Gorbacheva Memorial Children’s Institute, Hematology and Transplantology, St. Petersburg, Russia; 12CHU Bordeaux - Hôpital Haut-leveque, Pessac, France; 13University of Cologne, Department of Medicine, Germany; 14ErasmusMC Cancer Institute, Erasmus University Medical Center, Rotterdam, the Netherlands; 15Department of Haematology, Oncology Helios-Klinikum Berlin-Buch, Germany; 16Department of Medicine, Division of Hematology, University of Liège, Belgium; 17 Department of Hematology, Hospital Clinic, Barcelona, Spain; 18Department of Hematology, Saint Antoine Hospital, APHP and University UPMS, Paris, France; 19Klinikum Augsburg, Department of Hematology and Oncology, University of Munich, Augsburg, Germany; 20Department of Bone Marrow Transplantation and Onco-Hematology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Poland; and 21Hematology Division, Chaim Sheba Medical Center, Tel Hashomer, Israel

Correspondence: Bipin.Savani@Vanderbilt.Edu

Received: 22/08/2015. Accepted: 10/11/2015. Pre-published: 12/11/2015. doi:10.3324/haematol.2015.135699

Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/101/2/256

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ABSTRACT

ncreasing numbers of patients are receiving reduced intensity conditioning regimen allogeneic hematopoietic stem cell transplantation. We hypothesized that the use of bone marrow graft might decrease the risk of graft-versus-host disease compared to peripheral blood after reduced intensity conditioning regimens without compromising graft-versus-leukemia effects. Patients who underwent reduced intensity conditioning regimen allogeneic hematopoietic stem cell transplantation from 2000 to 2012 for acute leukemia, and who were reported to the Acute Leukemia Working Party of the European Group for Blood and Marrow Transplantation were included in the study. Eight hundred and thirty-seven patients receiving bone marrow grafts were compared with 9011 peripheral blood transplant recipients after reduced intensity conditioning regimen. Median follow up of surviving patients was 27 months. Cumulative incidence of engraftment (neutrophil ≥0.5x109/L at day 60) was lower in bone marrow recipients: 88% versus 95% (P<0.0001). Grade II to IV acute graft-versus-host disease was lower in bone marrow recipients: 19% versus 24% for peripheral blood (P=0.005). In multivariate analysis, after adjusting for differences between both groups, overall survival [Hazard Ratio (HR) 0.90; P=0.05] and leukemia-free survival (HR 0.88; P=0.01) were higher in patients transplanted with peripheral blood compared to bone marrow grafts. Furthermore, peripheral blood graft was also associated with decreased risk of relapse (HR 0.78; P=0.0001). There was no significant difference in nonrelapse mortality between recipients of bone marrow and peripheral blood grafts, and chronic graft-versus-host disease was significantly higher after peripheral blood grafts (HR 1.38; P<0.0001). Despite the limitation of a retrospective registry-based study, we found that peripheral blood grafts after reduced intensity conditioning regimens had better overall and leukemia-free survival than bone marrow grafts. However, there is an increase in chronic graft-versus-host disease after peripheral blood grafts. Long-term follow up is needed to clarify whether chronic graft-versushost disease might increase the risk of late morbidity and mortality. haematologica | 2016; 101(2)

BMT or PBSCT after RIC regimen for acute leukemia

Introduction Indications for allogeneic hematopoietic stem cell transplantation (HCT) have changed in the past decade. The most common indication for HCT in 2013 was acute myeloid leukemia (AML), and currently nearly 50% of HCTs are being carried out for acute leukemia including AML and acute lymphoid leukemia (ALL).1,2 Furthermore, following the introduction of reduced intensity conditioning (RIC) regimens, individuals with co-morbidities and those 65 years of age and older are now eligible to undergo HCT.3-5 The use of peripheral blood stem cells (PB) has largely replaced bone marrow (BM) as the main stem cell source for HCT in adults with hematologic malignancies.1 However, limited data are available on the impact of the stem cell source (related or unrelated donor) in the setting of RIC HCT,6-10 including our small previous European Group for Blood and Marrow Transplantation (EBMT) series.9,10 In 8 of 9 published comparative randomized studies, all participants received a myeloablative HCT, using different regimens depending on the underlying disease.6 Only one randomized study by Anasetti et al.7 included RIC regimens in the comparative analysis of BMT versus PBSCT for a variety of hematologic malignancies. Participants predominantly received a myeloablative HCT, with only 20% of the BMT group receiving RIC, and very few were performed for acute leukemia. A study recently published by the Center for International Blood and Marrow Transplant Research (CIBMTR) reported comparative analysis of BMT versus PBSCT after RIC regimen.8 Similar to the report by Anasetti et al.,7 the study population included a variety of hematologic malignancies and the analysis included only 108 with AML patients in the BMT RIC group. Outcomes for unrelated donor allografting have improved over time. This is probably due to improved high-resolution HLA-typing and better matching, and the availability of intensive supportive care.11,12 Moreover, outcomes for unrelated donor HCT after RIC regimens appear comparable to those seen with sibling donor RIC HCT.3,13-15 Here we report on transplant outcomes after related or unrelated donor BMT (n=837) versus PBSCT (n=9011) in the setting of reduced intensity conditioning for the treatment of adults with acute leukemia using data reported to the ALWP of the EBMT from 2000 to 2012.

Methods Study design and data collection This was a retrospective multicenter registry analysis. Data were provided and approved for this study by the Acute Leukemia Working Party (ALWP) of the EBMT group registry. The EBMT is a non-profit, scientific society representing more than 600 transplant centers, mainly in Europe. The EBMT promotes all initiatives that aim to improve stem cell transplantation or cellular therapy, which includes registering all activities relating to stem cell transplants. Data are entered, managed, and maintained in a central database that includes all EBMT centers. There are no restrictions regarding centers that can report data except those legal requirements concerning patient consent, data confidentiality and accuracy. Quality control measures include several independent systems: confirmation of validity of the entered data by the reporting team, selective comparison of the survey data with MED-A data haematologica | 2016; 101(2)

sets in the EBMT registry database, cross-checking with the national registries, and regular in-house and external data audits. Since 1990, patients have provided informed consent authorizing the use of their personal information for research purposes. Eligibility criteria for this analysis included adult patients (age >18 years) with acute leukemia receiving HLA-matched or mis-

Table 1. Patientsâ&#x20AC;&#x2122; disease and transplant characteristics.

Patients N. of patients Recipient age at SCT, median (range)(years) Recipient gender, n (%) Male Female Missing Diagnosis AML ALL Median follow up (alive patients), months (IQR) Donor age (years, range) Donor gender, n (%) Male Female Missing Female donor to male recipient, n (%) Disease status at HCT, n (%) CR1 â&#x2030;ĽCR2 Active disease Karnofsky at HCT, <90%, n (%) Recipient positive CMV serology Donor positive CMV serology Human leukocyte antigen matching HLA-identical sibling URD (10/10 match) 1- locus mismatched-URD (9/10) Mismatched-related donor Conditioning regimen Bu-Flu Low-dose TBI Flu-Mel Treo-Bu Cy-Thiotepa Flu-Cy Other In vivo T-cell depletion, n (%) Anti-thymocyte globulin Anti-lymphocyte globulin Alemtuzumab GvHD prophylaxis Calcineurin inhibitors and either MTX or MMF or both Tacrolimus and sirolimus Other + missing

BMT

PBSCT

837 54 (18-77)

9011 57 (18-77)

<0.0001

459 (55%) 376 (45%) 2

4790 (53%) 4214 (47%) 7

702 (84%) 135 (16%) 29 (12-56) 43 (9-74)

8075 (90%) 936 (10%) 27 (11-54) 47 (13-80)

487 (59%) 338 (41%) 12 181 (22%)

5401 (61%) 3531 (39%) 79 1733 (19%)

475 (57%) 177 (21%) 185 (22%) 215 (37%) 508 (69%) 403 (54%)

5193 (57%) 1513 (17%) 2305 (26%) 2226 (31%) 5682 (67%) 4526 (53%)

388 (46%) 220 (26%) 107 (13%) 122 (15%)

4751 (53%) 2894 (32%) 950 (10%) 416 (5%)

314 210 149 30 66 29 39 394 (48%) 296 29 69

3240 2730 1845 307 155 384 350 5246 (61%) <0.0001 3808 215 1223 0.38 8471

0.33

0.27 0.005 0.42

0.08 0.002@

814 5 18

0.006 0.26 0.69 <0.0001

62 478

BMT: bone marrow transplantation; PBSCT: peripheral blood stem cell transplantation; AML: acute myeloid leukemia; CMV: cytomegalovirus; HCT: stem cell transplantation; Bu: busulfan; Cy: cyclophosphamide; Flu: fludarabine; Mel: melphalan; TBI: total body irradiation; MTX: methotrexate; Treo: treosulfan; @active disease versus complete remission (CR); P=0.03.

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matched related or unrelated donor BM or PB transplants after RIC regimens from 2000 to 2012. Two hundred and ninety-four transplant centers reported data on recipients of BM and PB grafts after related or unrelated donor transplantation. We do not have any information about why patients were allocated to a specific graft (BM vs. PB) in the registry, and it is difficult to distinguish between the role of the conditioning approach adopted and the role of a potential effect of the individual center (“center effect”); however, a center effect was not evident in the analysis. All unrelated donors were HLA (-A, -B, -C, DRB1, -DQB1) matched (10/10) or mismatched at one loci. Exclusion criteria included previous allogeneic or cord blood transplantation, and recipients of grafts that were either ex vivo T-cell depleted or CD34 selected. Data were collected on recipient and donor characteristics [age, gender, cytomegalovirus (CMV) serostatus], disease status at transplant, transplant-related factors including conditioning regimen, immunosuppression (in vivo T-cell depletion vs. none), stem cell source (BM or PB), graft-versus-host disease (GvHD) prophylaxis, and outcome variables [acute and chronic GvHD,16,17 relapse, nonrelapse mortality (NRM), leukemia-free survival (LFS), overall survival (OS), and causes of death]. Regimens were classified as RIC based on published criteria.18

Statistical analysis The primary end points of the study were OS and LFS. Secondary end points included relapse incidence (RI), NRM, engraftment, incidence and severity of acute and chronic GvHD. The starting point for time-to-event analysis was date of transplantation. OS was defined as the time to death from any cause. Surviving patients were censored at time of last follow up. LFS was defined as survival without relapse or progression. Patients surviving in continuous CR were censored at the time of last follow up. RI was defined as time to onset of leukemia recurrence. NRM was the competing risk, and patients surviving in continuous complete remission were censored at last contact. NRM was defined as death without relapse/progression (relapse was the competing risk). The two groups were compared according to the stem cell sources (BM vs. PB) using the c2 test for qualitative variables, whereas the Mann-Whitney test was applied for continuous

Table 2. Multivariate analysis adjusted outcomes after transplantation by graft source (peripheral blood vs. bone marrow).

Outcome variable Overall survival Leukemia-free survival Relapse Non-relapse mortality Acute GvHD (grade II-IV)* Chronic GvHD

HR (95% CI)

0.90 (0.81-1.00) 0.88 (0.79-0.97) 0.78 (0.69-0.88) 1.09 (0.91-1.31) 1.31 (1.07-1.60) 1.38 (1.17-1.60)

0.05 0.01 0.0001 0.34 0.008 0.0001

GvHD: graft-versus-host disease; *OR: odds ratio.

parameters. Univariate comparisons were made using the log rank test for OS, LFS, and the Gray test for RI, NRM and GvHD cumulative incidences. Multivariate analyses were performed using logistic regression for acute GvHD and Cox proportional hazards model for all other end points (variables tested are provided in Table 1). All factors known as potentially related to the outcome were included in the final model. First-order interactions between the main effect and the other variables were tested in multivariate models. All tests were two-sided. The type I error rate was fixed at 0.05 for determination of factors associated with time to event outcomes. Statistical analyses were performed with SPSS 22.0 (IBM Corp., Armonk, NY, USA) and R 3.1.1 software packages (R Development Core Team, Vienna, Austria).

Results Patients’, disease and transplant characteristics Details of patients’, disease and transplant characteristics are summarized in Table 1. A total of 9848 patients with AL were included in the study: 837 patients received BM and 9011 PB transplants performed between 2000 and 2012; 8777 (89.1%) patients had AML (BM=702, PB=8075) and 1071 (10.9%) ALL (BM=135, PB=936). PBSCT recipients were older with a median age of 57 years (range 18-77) in comparison to 54 years (range 18-77) for the BM group (P<0.0001). Median follow up of surviving patients in the BM group was 29 (IQR, 12-56) months, while that of the PB group was 27 (IQR, 11-54) months (P=0.27). Significantly higher numbers of patients had Karnofsky Performance Status (KPS) score less than 90% (37 vs. 31%; P=0.006) in the BM group. There were more patients with advanced disease in PB compared to the BM group (26 vs. 22%; P=0.002). There were no statistically significant differences in cytogenetic risk categories in AML or ALL groups between patients receiving BM or PB transplantations. The proportion of CMV seropositive recipients and donors were comparable in the BM and PB groups [69% vs. 67% (P=0.26) and 54% vs. 53% (P=0.69), respectively]. Details of transplant characteristics and conditioning regimen are summarized in Table 1. The majority of patients received chemotherapy-based RIC regimens (BM=75%, PB=70%; P=0.002). Among the BM recipients, 388 (46%) received a graft from HLA-identical sibling donor, 220 (26%) matched unrelated donor (MUD) (10/10), 107 (13%) mismatched unrelated donor (MMURD) (9/10), and 122 (15%) received mismatched-related donor HCT; corresponding numbers in the PB cohort were 4751 (53%), 2894 (32%), 950 (10%), and 416 (5%), respectively. The percentage of patients receiving in vivo T-cell depletion was higher in the PB group (61% vs. 48% in BM; P<0.0001). Patients receiving ex vivo T-cell depletion were

Table 3. The 2-year adjusted probabilities of transplant outcome after bone marrow versus peripheral blood grafts. RI NRM LFS OS BM 43% (39-47) 18% (16-21) 39% (35-42) 47% (44-51) PB 35% (34-37) 20% (18-23) 44% (43-45) 50% (49-52) P 0.0004 0.19 0.009 0.05

cGvHD 29% (26-32) 36% (35-37) 0.0003

BM: bone marrow; PB: peripheral blood; RI: relapse incidence; NRM: non-relapse mortality; LFS: leukemia-free survival; OS: overall survival; cGvHD: chronic graft-versus-host disease; BM: bone marrow; PB: peripheral blood.

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BMT or PBSCT after RIC regimen for acute leukemia

excluded from the analysis. The choice of conditioning, graft source and GvHD prophylaxis was dependent on the protocols of the individual centers and the strategies adopted for transplantation.

Engraftment and graft-versus-host disease

Median time to neutrophil recovery (PMN â&#x2030;Ľ0.5x109/L) was 20 days and 16 days after BM and PB HCT, respectively (P<0.0001). The corresponding day 60 probability of neutrophil recovery (PMN â&#x2030;Ľ0.5x109/L at day 60) was 88% (95%CI: 86-90) after BM versus 95% (95%CI: 95-96) in the PB group (P<0.0001). The rates of day 100 grade II-IV acute GvHD were 24% versus 19% (P=0.005) and 10% versus 6% (P=0.003) for grade III-IV GvHD in PB compared with BM transplant recipients. In multivariate analysis, the factors associated with increased risk of grade II-IV acute GvHD were PB versus BM (OR 1.31; 95%CI: 1.07-1.60; P=0.008; Table 2), active disease (OR 1.33; 95%CI: 1.18-1.51; P<0.0001), poor cytogenetic risk group (OR 1.30; 95%CI: 1.10-1.53; P=0.002), CMV donor seropositivity (OR 1.12; 95%CI: 1.01-1.25; P=0.05), female donor for male recipients (OR 1.19; 95%CI: 1.04-1.35; P=0.01), MUD (10/10) and MM-URD (9/10) allo-SCT compared with HLA-identical donor [OR 1.78; 95%CI: 1.57-2.02 (P<0.0001) and OR 2.30; 95%CI:

1.95-2.71 (P<0.0001), respectively], and absence of in vivo T-depletion (OR 1.46; 95%CI: 1.30-1.64; P<0.0001). The 2-year incidence of chronic GvHD was higher after PB grafts [36% (95%CI: 35-37) vs. 29% (95%CI: 26-32) in the BM group; P=0.0003] (Table 3 and Figure 1). The severity of chronic GvHD was graded as extensive in 1287 (16.6%) of PBSCT recipients compared with 69 (9.3%) in the BMT group (P<0.0001). Similarly, the GvHD-related mortality was higher after PBSCT compared to BMT (13% vs. 7.4%; P=0.001). In multivariate analysis, chronic GvHD was significantly higher after PB grafts (HR 1.38; 95%CI: 1.17-1.60; P<0.0001) (Table 2). Other factors determining the risk of cGvHD that were independent of graft source included active disease at transplant (HR 1.20, 95%CI: 1.09-1.34; P=0.0004), older age at allo-SCT (HR 1.02, 95%CI: 1.001.03; P=0.03), female donor for male recipients (HR 1.18, 95%CI: 1.08-1.30; P=0.0004), MUD (10/10) and MMURD (9/10) allo-SCT compared with HLA-identical donor (HR 1.12, 95%CI: 1.02-1.23; P=0.02, and HR 1.23, 95%CI: 1.07-1.41; P=0.003, respectively). There was no significant difference in risk of chronic GvHD between HLA-identical and related mismatched donors (HR 0.94, 95%CI: 0.761.15; P=0.54). In vivo T-cell depletion was associated with lower risk of chronic GvHD (HR 0.57, 95%CI: 0.53-0.63; P<0.0001) independent of graft type.

Figure 1. Long-term outcomes after transplantation by graft type. (A) RI: relapse incidence. (B) NRM: nonrelapse mortality. (C) LFS: leukemia-free survival. (D) OS: overall survival. (E) cGvHD: chronic graft-versushost disease.

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Non-relapse mortality There was no significant difference in 2-year NRM between the BM and PB groups in univariate analysis (18%, 95%CI: 16-21 after BM versus 20%, 95%CI: 18-23 after PB grafts; P=0.19) (Table 3 and Figure 1). In multivariate analysis, there was no significant difference in NRM between BM and PB grafts (HR 1.09, 95%CI: 0.91-1.31; P=0.34) (Table 2). Other factors associated with higher NRM independent of graft source were CR2 versus CR1 (HR 1.27, 95%CI: 1.11-1.45; P=0.0004), active disease (HR 1.85, 95%CI: 1.65-2.07; P<0.0001), secondary AML (HR 1.32, 95%CI: 1.17-1.48), older age at HCT (HR 1.08, 95%CI: 1.06-1.10; P<0.0001), CMV seropositive recipients (HR 1.34, 95%CI: 1.20-1.50; P<0.0001), CMV seropositive donors (HR 1.13, 95%CI: 1.02-1.25; P=0.02), female donor for a male recipient (HR 1.21, 95%CI: 1.071.36; P=0.002), matched-URD (10/10), MM-URD (9/10) and related mismatched donors compared with HLAidentical donor [HR 1.60, 95%CI: 1.42-1.80 (P<0.0001); HR 2.11, 95%CI: 1.81-2.45 (P<0.0001), and HR 2.34 95%CI: 1.94-2.83 (P<0.0001), respectively]. In vivo T-cell depletion was associated with lower NRM (HR 0.79, 95%CI: 0.71-0.87; P<0.0001) independent of graft type.

Relapse Two-year RI was lower after PB (35%, 95%CI: 34-37) compared to BM transplants (43%, 95%CI: 39-47) (P=0.0004) (Table 3 and Figure 1). In multivariate analysis, RI was significantly lower after PB grafts (HR 0.78, 95%CI: 0.69-0.88; P=0.0001) (Table 2). Factors associated with higher RI independent of graft source were: diagnosis of ALL versus AML (HR 1.38, 95%CI: 1.23-1.55; P<0.0001), CR2 versus CR1 (HR 1.26, 95%CI: 1.13-1.39), active disease (HR 2.45, 95%CI: 2.262.66; P<0.0001), poor cytogenetic risk group (HR 1.51, 95%CI: 1.36-1.68; P<0.0001), in vivo T-cell depletion (HR 1.10, 95% CI, 1.01-1.19; P=0.02), and low-dose total body irradiation (TBI)-based RIC regimens compared to chemotherapy-based regimens (fludarabine combined with busulfan or melphalan) (HR 1.12, 95%CI: 1.03-1.21; P=0.01). Relapse risk was lower for patients receiving MUD (10/10) or MM-URD (9/10) compared with an HLAidentical donor [HR 0.77, 95%CI: 0.71-0.85 (P<0.0001) and HR 0.82, 95%CI: 0.73-0.93 (P=0.002), respectively].

Leukemia-free survival Two-year LFS was higher after PB (44%, 95%CI: 43-45) compared with BM transplants (39%, 95%CI: 35-42) (P=0.009) (Table 3 and Figure 1). In multivariate analysis, the only factor associated with superior LFS was the use of PB grafts (HR 0.88, 95%CI: 0.79-0.97; P=0.01) (Table 2). Factors associated with inferior LFS independent of graft source were diagnosis of ALL versus AML (HR 1.42, 95%CI: 1.29-1.57; P<0.0001), CR2 versus CR1 (HR 1.26, 95%CI: 1.16-1.37), active disease (HR 2.22, 95%CI: 2.072.37; P<0.0001), poor cytogenetic risk group (HR 1.35, 95%CI: 1.23-1.47; P<0.0001), secondary AML (HR 1.16, 95%CI: 1.08-1.25), older age at HCT (HR 1.03, 95%CI: 1.02-1.04; P<0.0001), CMV-positive recipients (HR 1.07, 95%CI: 1.00-1.14; P=0.04), MM-URD (9/10) or mismatched-related donor compared with HLA-identical donor [HR 1.16, 95%CI: 1.05-1.27 (P=0.003) and HR 1.26, 95%CI: 1.12-1.43 (P=0.0002), respectively] and low-dose TBI-based RIC regimens compared with chemotherapy260

based regimens (fludarabine combined with busulfan or melphalan) (HR 1.08, 95%CI: 1.01-1.15; P=0.02).

Overall survival Two-year OS was higher after PB grafts (50%, 95%CI: 49-52) compared with BM transplants (47%, 95%CI: 4451) (P=0.05) (Table 3 and Figure 1). In multivariate analysis, factors associated with superior OS were PB grafts (HR 0.90, 95%CI: 0.81-1.00; P=0.05) (Table 2) and in vivo T-cell depletion (HR 0.93, 95%CI: 0.87-1.00; P=0.04). Factors associated with inferior OS independent of graft source included a diagnosis of ALL versus AML (HR 1.42, 95%CI: 1.28-1.57; P<0.0001), CR2 versus CR1 (HR 1.28, 95%CI: 1.17-1.39; P<0.0001), active disease at transplant (HR 2.21, 95%CI: 2.06-2.37; P<0.0001), poor cytogenetic risk group (HR 1.27, 95%CI: 1.15-1.39; P<0.0001), secondary AML (HR 1.17, 95%CI: 1.08-1.26; P=0.0001), older age at HCT (by 10 years) (HR 1.04, 95%CI: 1.03-1.06; P<0.0001), CMV-positive recipients (HR 1.12, 95%CI: 1.05-1.20; P=0.0001), female donor for male recipients (HR 1.09, 95%CI: 1.01-1.18; P=0.02), MUD (10/10), MMURD (9/10) donors and MM-related donor compared with HLA-identical donor [HR 1.09, 95%CI: 1.01-1.18 (P=0.02), HR 1.31, 95%CI: 1.18-1.45 (P<0.0001) and HR 1.44, 95%CI: 1.27-1.64 (P<0.0001), respectively] and low-dose TBI-based RIC regimens compared chemotherapy-based regimens (fludarabine combined with busulfan or melphalan) (HR 1.08, 95%CI: 1.01-1.16; P=0.03).

Impact of graft source on other variables The primary purpose of our analysis was to explore outcome differences between BM and PB transplantation in patients with acute leukemia after RIC regimens. The effect of stem cell source (BM vs. PB) on survival was independent of disease (interaction test P=0.22). There was no significant interaction between the graft source and disease status at transplant (interaction test P=0.57). Results were similar among HLA-mismatched pairs and recipients with active disease, although this study was not designed to detect potential differences within these subsets.

Discussion This large, multicenter, registry study shows that the use of PB grafts after RIC gives superior outcome in patients with acute leukemia. Moreover, PBSCT recipients had a lower risk of relapse, most probably due to a stronger graft-versus-leukemia (GvL) effect not just in patients in CR, but, more importantly, in patients with active disease pre-HCT. This finding is of major clinical significance as an increasing number of patients are undergoing RIC HCT for acute leukemia, a significant number of whom receive BM grafts;1 indeed, 8.5% (n=837) of 9848 patients transplanted from 2000 to 2012 at centers reporting to the EBMT received BM grafts. However, the increased risk of chronic GvHD observed in our series after PB grafts is alarming, and long-term follow up is awaited to clarify if excess risk of chronic GvHD among RIC PBSCT recipients translates into continued GvL effect or increased late morbidity and mortality.19 We investigated patient, disease, and transplantation factors affecting survival, LFS, relapse, NRM and GvHD in a well-characterized population of nearly 10,000 adult acute leukemia patients receiving either BMT or PBSCT. haematologica | 2016; 101(2)

BMT or PBSCT after RIC regimen for acute leukemia

Overall, nearly 50% of patients (Figure 1) transplanted after RIC HCT survived beyond two years, with higher survival for AML compared to ALL. Notably, the low-dose TBI-based regimen had inferior outcome (for both patients in CR1 and active diseases) compared to chemotherapybased RIC regimens. Results were similar among related or unrelated donor groups. Patients receiving in vivo T-cell depletion (either ATG or alemtuzumab) had a significantly lower risk of chronic GvHD and higher OS despite their higher relapse rate, which was offset by lower NRM. Anti-thymocyte globulin or alemtuzumab with standard GvHD prophylaxis (which has been shown to be effective in lowering GvHD rates as well as its severity) was used for 48% of BM and 61% of PB transplantations in our series. This study was not designed to analyze separately patients receiving lowdose ATG/alemtuzumab, which is commonly used today; however, previous studies have shown that low-dose ATG/alemtuzumab reduced the risk of chronic GvHD and NRM without compromising the GvL effect.20-25 Previous smaller studies demonstrated the non-inferiority of RIC PBSCT compared to RIC BMT for patients with acute leukemia.7-10 Our results differ from the findings in published studies comparing RIC outcomes with BM with PB grafts. This discrepancy may be due to differences in the study populations. Firstly, the improved survival, decreased relapse risk and higher rates of grade II to IV acute GvHD (also grade III-IV) after transplants with PB compared with BM grafts are in contrast to previous reports.6,8,26 The survival rates in the BM and PB groups in our study are comparable to a study from the CIBMTR by Eapen et al.,8 reporting a 3%-5% difference in OS and LFS between the two groups, which was not statistically significant. In our analysis, results were statistically significant, and this may reflect the difference in power between the two studies. Secondly, the cumulative incidence of engraftment and the risk of chronic GvHD were higher after PBSCT compared with BMT, in keeping with other reports.6,7,27,28 Chronic GvHD can impair quality of life and is associated with significant morbidity and mortality among HCT recipients. However, the costs involved, the economic burden and the use of resources to manage long-term complications associated with cGvHD have not been well described. Studies of transplantation costs are complex

References 1. Passweg JR, Baldomero H, Bader P, et al. Hematopoietic SCT in Europe 2013: recent trends in the use of alternative donors showing more haploidentical donors but fewer cord blood transplants. Bone Marrow Transplant. 2015;50(4):476-482. 2. Pasquini MC, Zhu, X. Current uses and outcomes of hematopoietic stem cell transplantation: 2014 CIBMTR Summary Slides. Available from: http://www.cibmtr.org 2014. 3. Reshef R, Porter DL. Reduced-intensity conditioned allogeneic SCT in adults with AML. Bone Marrow Transplant. 2015;50(6):759-769. 4. Bachanova V, Marks DI, Zhang MJ, et al. Ph+ ALL patients in first complete remission have similar survival after reduced

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and difficult to conduct because of the wide variation in transplant methods, conditioning approaches, GvHD prophylaxis regimens and supportive care practices. More research is needed to better understand the costs of cGvHD to patients, centers and the health care system, and to determine whether the lower incidence and severity of cGvHD with BM grafts leads to long-term savings of resources compared to PB grafts. Also, trials aimed at better GvHD prophylaxis that is either drug-mediated or through graft manipulation are needed to reduce chronic GvHD rates after RIC PBSCT. We acknowledge that there are differences in patientsâ&#x20AC;&#x2122;, disease, and transplantation characteristics between those who received BMT and PBSCT. We have addressed this by performing a carefully controlled well-adjusted analysis that considered patients', disease, and transplantation characteristics as well as any transplantation center effects in this multicenter registry analysis. In addition, there may be unmeasured and unknown factors that have not been considered, which is an inherent limitation of this type of analysis. However, we believe that the results of this analysis are very important in the absence of available prospective data. Only through the conduct of well-designed clinical trials will we be able to understand and appreciate the complexities of stem cell source choices and their outcome after RIC HCT. Unfortunately, there are no ongoing trials to compare outcomes after BMT with that after PBSCT following an RIC regimen for acute leukemia. Therefore, in the absence of any prospect of such comparative studies, our data support the use of PB (related or unrelated donor) grafts after RIC for adult patients with acute leukemia in remission or with advanced disease. Acknowledgments We thank all European Group for Blood and Marrow Transplantation (EBMT) centers and national registries for contributing patients to the study and data mangers for their superb work. Supplementary information is available at the EBMT Web site. The list of institutions reporting data included in this study is available in the Online Supplementary Appendix. BNS thanks Dr. Katy Rezvani, MD, PhD (Houston, TX, USA), Dr. Agnes Yong, MD, PhD (Adelaide, Australia) and Prof. John Greer (Nashville, TN, USA) for critical reading of the manuscript.

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