Romjoh 5 (4) 2014

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T H I S J O U R N A L I S I N T E R D I S C I P L I N A R Y A N D I S D E D I C A T E D T O A L L S P E C I A L I S T S I N T H E M E D I C A L F I E L D R E L A T E D T O O N C O LO GY A N D H E M A T O LO GY

O n c o l o g y & H e m at o l o g y

R o m a n i a n

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www.rom-joh.com Volume II I Issue 4 I 2014

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O n c o l o g y & H e m at o l o g y Issue 4 I Volume 2 I December 2014 PUBLISHED UNDER THE AUSPICES OF THE NATIONAL SOCIETY FOR MEDICAL ONCOLOGY IN ROMANIA; THE ROMANIAN HEMATOLOGICAL SOCIETY; THE ROMANIAN CANCER SOCIETY “VASILE PACURAR”; THE ROMANIAN ASSOCIATION FOR THE STUDY OF PAIN

Senior Editors Prof. Dr. Florin Bădulescu (Universitatea de Medicină şi Farmacie Craiova, Craiova, România) Prof. Dr. Anca Roxana Lupu (Universitatea de Medicină şi Farmacie "Carol Davila", Bucureşti, România) Şef Lucrări Dr. Lucia Stănculeanu (Universitatea de Medicină şi Farmacie "Carol Davila", Bucureşti, România) Şef Lucrări Dr. Simona Mihuţiu (Universitatea de Medicină şi Farmacie Oradea, Oradea, România) Cerc. St. Gr. I. Dr. Grigorescu Alexandru (Institutul oncologic Prof. Dr. Alexandru Trestiorean, Bucureşti, România)

Section Editor

Prevention & Screening Asist. Univ. Mircea O. D. Lupusoru (University of Medicine and Pharmacy “Carol Davila”; Director Medical - Spitalul de Psihiatrie Titan “Dr. C-tin Gorgos”)

Romanian Editorial Board Prof. Dr. Oltean Galafteon (Universitatea of Medicină şi Farmacie Târgu Mureș, Târgu Mureș, România) Prof. Dr. Ljubomir Petrov (Universitatea of Medicină şi Farmacie „Iuliu Haţieganu”, Cluj-Napoca, România) Prof. Dr. Ioniță Hortensia (Universitatea of Medicină şi Farmacie "Victor Babeș" , Timișoara, România) Prof. Dr. Cătălina Poiană (Universitatea of Medicină şi Farmacie "Carol Davila", Bucureşti, România) Conf. Dr. Monica Dragomir (Universitatea of Medicină şi Farmacie "Carol Davila", Bucureşti, România) Conf. Dr. Coriu Daniel (Universitatea of Medicină şi Farmacie "Carol Davila", Bucureşti, România) Conf. Dr. Adriana Coliță (Universitatea of Medicină şi Farmacie "Carol Davila", Bucureşti, România) Conf. Dr. Anca Coliţă (Universitatea of Medicină şi Farmacie “Carol Davila”, Bucureşti, România) Conf. Dr. Horia Bumbea (Universitatea of Medicină şi Farmacie "Carol Davila", București, România) Conf. Dr. Elena Copaciu (Universitatea of Medicină şi Farmacie “Carol Davila”, București, România) Șef Lucrări Dr. Laura Mazilu (Facultatea de Medicină, Universitatea Ovidius, Constanţa, România) Șef Lucrări Dr. Coliță Andrei (Universitatea of Medicină şi Farmacie "Carol Davila", Bucureşti, România) Şef Lucrări Dr. Cristian Silviu Voinea (Universitatea of Medicină şi Farmacie "Carol Davila", Bucureşti, România) Şef Lucrări Dr. Diana Paun (Universitatea of Medicină şi Farmacie “Carol Davila”, Bucureşti, România) Asist. Univ. Gabriela Elena Lupusoru (University of Medicine and Pharmacy “Carol Davila”) Asist. Univ. Dr. Carsote Mara (Universitatea of Medicină şi Farmacie "Carol Davila", Bucureşti, România) Asist. Univ. Dr. Victor Gabriel Clătici (Universitatea of Medicină şi Farmacie “Carol Davila”, Bucureşti, România Asist. univ. dr. Adina Alexandru (Universitate de Medicina si Farmacie "Carol Davila", Bucuresti, Romania) Asist. Univ. Dr. Trandafir Maria Silvia (Universitatea of Medicină şi Farmacie "Carol Davila", Bucureşti, România) Asist. Univ. Dr. Ioana Soare (Universitatea Titu Maiorescu, Facultatea de Medicină, București, România) Dr. Adrian Udrea (Medisprof, Cluj-Napoca, România) Dr. Radu Niculescu (Institutul Clinic Fundeni, Bucureşti, România) Dr. Ana Maria Boeru (Asociaţia Free of Pain, Bucureşti, România) Dr. Virgil Dincă (Asociația Română pentru Studiul Durerii, Bucureşti, România)

International Editorial Board Prof. Dr. med. Anca-L. Grosu (Klinik für Strahlenheilkunde, Universität Freiburg, Freiburg, Germany) Prof. Dr. Shibo Li (University of Oklahoma Health Sciences Center, Oklahoma City, USA) Prof. Dr. Mariusz Z. Ratajczak (University of Louisville, Louisville, USA) Prof. Dr. Arnold Ganser (Hannover Medical School, Hanover, Germany) Prof. Dr. Saverio Bettuzzi (University of Parma Via Volturno, Parma, Italy) Prof. Dr. Lodovico Balducci (Moffitt Cancer Center, Tampa, USA) Prof. Dr. Leonard Wartofsky (Georgetown University School of Medicine, Washington, USA) Prof. Dr. Robert Amato (Memorial Hermann Cancer Center, Texas, USA) Prof Dr. Kevin R. Loughlin (Harvard University, Cambridge, USA) Prof. Dr. Maureen Markman (Drexel University College of Medicine, Philadelphia, USA) Prof. Dr. Stephen P. Hunger (University of Colorado School of Medicine, Colorado, USA) Prof. Dr. M.W.M. van den Brekel (Academic Medical Center Amsterdam, Amsterdam, Netherlands) Prof. Dr. M Sitki Copur (University of Nebraska Medical Center, Nebraska, USA) Prof. Dr. Derek Raghavan (UNC School of Medicine, Levine Cancer Institute, Charlotte, NC, USA) Prof. Dr. Richard J. Ablin (University of Arizona, Arizona, USA) Prof. Dr. Florian Strasser (Cantonal Hospital St. Gallen, Switzerland) Prof. Dr. Michel Rigaud (Scientific advisor - IRST, Meldola, Italy) Associate Prof. Dr. Mishu Popa McKiver (Massachusetts General Hospital, Massachusetts, USA) Assistant Prof. Dr. Alina Mihai (Univ Pittsburgh School Medicine, Pittsburgh, USA) Assistant Prof. Dr. Doru Paul (Hofstra North Shore-LIJ School of Medicine, New York, USA) Assistant Prof. Dr. Bruno Vincenzi (University Campus Bio-Medico, Rome, Italy Assistant Prof. Dr. Elizabeta C. Popa (Weill Cornell Medical College, NY, USA) Assistant Prof. Dr. Gabriela Oprea (Emory University, Atlanta, USA) Dr. Wainer Zoli (Director of the Bioscience Laboratory, IRST, Meldola, Italy) Dr. Ciprian Enachescu (Centre Hospitalier Lyon Sud, Lyon, France) Dr. Javier Martín Broto (Son Espases Hospital, Palma de Mallorca Spain) Dr. David Gómez Almaguer (Universidad Autónoma de Nuevo León, Monterrey, México) Dr. Sankalp Yadav (General Duty Medical Officer-II, Chest Clinic Moti Nagar, North Delhi Municipal Corporation, New Delhi, India)

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EVENTS

Health Meeting pe tema leziunilor pre-neoplazice și neoplazice Eveniment interdisciplinar, marca Media Systems Communication

REVIEW

Neuroendocrine carcinoma of the breast: how, when, why Bacalbasa N., Gavril A.

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Events

Titlul articolului

Health Meeting pe tema leziunilor pre-neoplazice și neoplazice Eveniment interdisciplinar, marca Media Systems Communication 5 decembrie 2014 – București, Crystal Palace Ballrooms

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Compania Media Systems Communication (MSC) a organizat cea de-a doua ediție a evenimentelor interdisciplinare, intitulate Health Meeting, chiar în ajun de Moș Nicolae. Peste 200 de participanți din diverse specialități de la oncologie medicală, ginecologie, neonatologie, dermatologie, chirurgie până la medicină de familie și anatomie patologică, au venit pe 5 decembrie 2014 pentru a discuta despre "Leziunile pre-neoplazice și neoplazice".

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Conferința, cu durată de o zi, a avut loc la Crystal Palace Ballrooms, a fost creditată de Colegiul Medicilor din România cu 4 credite EMC, iar organizatorii le-au mai oferit participanților gratuit revista Romanian Journal of Oncology & Hematology, editată de MSC și creditată cu 5 puncte EMC. Lucrările științifice au fost prezentate în cele trei sesiuni ale evenimentului și au vizat subiecte de actualitate atât din perspectiva prevenției, depistării precoce, cât și din cea a tratării leziunilor pre-neoplazice și neoplazice. Și pentru a spori gradul de interactivitate și abordare interdisciplinară, la finalul fiecărei sesiuni a avut loc o dezbatere. Participanții au putut pune întrebări prin intermediul tabletelor, pe care le-au primit la venire și chiar le-au folosit cu încredere. Au fost însă și participanți care au preferat microfonul și au pus întrebări în direct.

Un element de noutate față de prima ediție a manifestării Health Meeting, care a avut loc tot în 2014, l-a constituit prezența reprezentanților a două asociații de pacienți cu boli oncologice respectiv Dorica Dan din partea Asociației Române de Cancere Rare (ARCRare) și Loredana Munteanu, din partea Asociației Casiopeea, tocmai pentru a arăta că din ecuația interdisciplinară nu poate lipsi pacientul. În loc de părerile noastre despre eveniment, preferăm să dăm cuvântul câtorva dintre speakerii prezenți la eveniment, care au văzut cum au stat lucrurile de pe podium: 1. Prof. Dr. Radu Vlădăreanu, Director de Departament Obstetrică-Ginecologie UMF Carol Davila Bucureşti, Şeful Clinicii de Obstetrică-Ginecologie al Spitalului Universitar de Urgenţă “Elias” Bucureşti.

,,Cancerul este o temă primordială, ori de câteori vorbim de medicină, iar asocierea cancerului cu sarcina este o încercare majoră pentru practician și pentru pacienta aflată în situația aceasta. De aceea, ni s-a părut că această asociere, nediscutată din câte știu eu până acum într-o manifestare de acest gen, merită să aibă locul ei. Eu cred că a suscitat interesul participanților prin prezența pe care au arătat-o la eveniment și prin întrebările foarte multe care au fost puse. Sunt convins că este un su-

biect care le-a atras atenția. Aceste situații, ca și altele, în medicină nu se pot face de către medicii dintr-o singură specialitate. Vin și colaborează extrem de puternic medicul curant, obstetricianul, neonatologul, oncologul, radioterapeutul și psihologul. Este o echipă întreagă care nu întotdeauna este rodată, mai ales atunci când sunt cazuri accidentale sau ocazionale. Eu personal, am avut cazuri, așa cum au avut și colegii mei.

Fiecare caz, care s-a rezolvat cu bine, reprezintă o satisfacție mult mai mare decât un caz obișnuit și pentru noi și bineînțeles pentru paciente și familiile acestora. Prin această manifestare, aș fi vrut să întărim faptul că e mai bine să previi cancerul decât să-l tratezi. Sunt convins că evenimentele organizate de MSC suscită un real interes prin faptul că reușesc să arate transnaționalitatea lor”.

2. Şef lucr. Dr. Simona Vlădăreanu, UMF Carol Davila Bucureşti, medic primar neonatologie, Şef Secţie Neonatologie Spitalul Universitar de Urgenţă “Elias” Bucureşti

,,Este extrem de dificil să le încurajezi și descurajezi pe aceste femei, mai ales dacă sunt la prima sarcină și află că au cancer, fiindcă de cele mai multe ori este o sarcină extrem de dorită. Trebuie să încurajezi o astfel de pacientă, dar să-i dai informații corecte. Lucrurile nu sunt chiar așa de ușoare din punct de vedere al sarcinii, mai intră în discuție tipul de cancer și tratamentul necesar și posibilitatea de prelungi perioada de supraviețuire a gravidei. Staff-ul medical care se ocupă de situații de genul acesta trebuie să cuprindă obstetricianul, oncologul și cel care recomandă terapia specifică bolii neoplazice, neonatologul și mai departe în urmărirea copilului pediatru. De mare ajutor este aportul adus de un psiholog deoarece situația e extrem de delicată. În acest context, găsesc că este extrem de interesantă abordarea acestei tematici, fiind foarte utilă societății noastre și cred că va trebui să continuăm cu astfel de dezbateri în care să fie implicați medici din toate specialitățile colaterale. Aportul fiecăruia e foarte bine venit. În clinica noastră, în ultimii ani am avut cazuistică nu foarte bogată de cancer diagnosticat în sarcină. Sigur există și situația în care femeia se știe cu o boală neoplazică și își dorește cu încăpățânare o sarcină. Lucruri le au mers bine în ceea ce privește copilul de cele mai multe ori, pe paciente uneori

Nume autori

For all those who would like more Enjoy more comfort on business trips in the new Premium Economy Class Enjoy restful and stress-free travel to your business meetings: in the new Premium Economy Class on long-haul routes you will experience signifi cantly more comfort and service in comparison to Economy Class as well as numerous extras on the ground and in the air.

More personal space – the Premium Economy Class seat with up to 50 per cent more room Especially comfortable seating: when you sit in the specially developed Premium Economy Class seat with its own armrest, adjustable backrest, footrest and generous legroom, you are never more than one seat away from the aisle. Enjoy more privacy and up to 50 per cent more space. You can reserve your seat in advance free of charge.

Enjoy a relaxed start to your journey With twice the free baggage allowance of Economy Class, you have plenty of room for work materials and everything that is essential for your business trip. You can also make sensible use of your time at the airport in the exclusive Lufthansa Business Lounge* – a perfect place to work undisturbed before your fl ight or simply to switch off and relax for a while.

Look forward to more extras on board On board, too, you can enjoy more peace and quiet and personal space. Here you can work undisturbed or relax. Thanks to the fold-out table, separate power socket and plenty of storage space, you can convert your spacious seat into a comfortable working area or simply stretch out in comfort and enjoy the fl ight. So you’ll arrive at your destination feeling on top form and ready to start your meetings.

You will be able to experience the new Premium Economy Class on selected routes from this December**. By late summer 2015, the entire Lufthansa long-haul fl eet will have been fi tted with the new seats.

Further information at LH.com/premium-economy

Benefi ts at a glance More personal space New seat with up to 50% more room More baggage Two bags each up to 23 kg free of charge*** More delight Meals served on china tableware More to look forward to A welcome drink to greet you on board More entertainment A larger 11- or 12-inch touchscreen More relaxation Bag with useful travel accessories More refreshment A separate bottle of water at your seat More exclusivity Access to selected Lufthansa lounges, for a fee****

* As a Premium Economy Class passenger you may use almost all Lufthansa Business Lounges and the Lufthansa Welcome Lounge at Frankfurt on payment of a fee of 25 or 50 euros respectively. ** As at July 2014. Subject to changes. *** HON Circle Members and Senators can check in three bags each up to 23 kg (approx. 50lb). **** For HON Circle Members, Senators and Frequent Travellers, lounge access is based on the respective Miles & More member status.

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le-a costat viața mai rapid decât în situația în care ar fi ales să nu ducă sarcina la capăt. Interesul a vizat mai mult sănătatea copilului decât a mamei, asta fiind dorința ei și a familiei, nu a echipei medicale. Dacă tratamentul nu se amâna șansele de supraviețuire erau mai mari. În cazul nostru, nou născuții au fost bine, nu au prezentat nici un fel de malformație, insuficiență cardiacă sau alte anomalii. Însă sunt foarte puține studii care au urmărit pe lungă durată, timp de 18-20 ani, acești copii și nu se știe exact incidența bolii neoplazice la un astfel de copil. Se discută în continuare de afectarea fertilității la acești copii și sunt necesare în continuare lămuriri sub acest aspect”.

3. Asist. Univ. Dr. Victor Clătici, UMF Carol Davila Bucureşti, Spitalul Universitar de Urgenţă “Elias” Bucureşti, medic primar dermatologie

,,Manifestarea a fost excepțională la fel ca și prima, pe tema HPV. S-a mers pe modelul de abordare integrat, multidisciplilnar, iar medicina este o specialitate multidisciplinară, de aceea trebuie să stimulăm colaborarea între diferitele ramuri: partea clinică, partea de laborator, imagistică, toate acestea își au rolul lor. O astfel de manifestare care adună sub același acoperiș medici din specialități diferite, sponsori care oferă servicii integrative își găsește locul în medicina actuală. În momentul de față medicina înseamnă colaborare, colaborare, colaborare. Melanomul malign din anumite puncte de vedere nu este o necunoscută, este o formă gravă de cancer de piele. Din alte puncte de vedere reprezintă o necunoscută inclusiv pentru noi medicii specialiști care ne ocu-

Titlul articolului

păm de el, pentru că sunt foarte mulți factori implicați în apariția melanomului, care influențează prognosticul și care influențează terapia. În privința terapiei vorbim de terapia personalizată, există modele de tratament care se bazează pe depistarea prezenței anumitor gene. Terapia per pacient, nu terapia pe boală, e scopul nostru.

Să găsim o ,,schemă” de tratament care să fie valabilă la mai mulți pacienți, dar nu la toți”.

4. Dr. Daciana Toma, Medic primar medicină de familie, Instructor formator în medicina de familie, secretar SNMF

,,E foarte important când vorbim de prevenție, să știm care sunt factorii de risc și să putem să facem o evaluare corectă, să alocăm timp, să avem cunoștințele necesare și să avem unde să trimitem mai departe pacienta. De exemplu, în cancerul de sân după o anumită vârstă se recomandă mamografia. În acest moment și vorbim de București nu știu de vreo variantă la care să nu existe coplată și destul de consistentă la mamografie, ori din start aceasta este o barieră. Este esențială abordarea multidisciplinară, pe de o parte acești pacienți au o evoluție sperăm pe perioadă îndelungată, pe de altă parte acești pacienți au și comorbidități și e normal ca eu, ca medic de familie, să știu ce i se întâmplă pe patologia oncologică atâta timp cât am nevoie să integrez cu celelalte patologii. Ce se întâmplă de fapt? Depinde de oameni, nu neapărat de sistem. Lucrurile sunt prevăzute de sistem, dar depinde de fiecare cât de bine vede locul echipei. Este foarte importantă prezența medicilor

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de familie la un astfel de eveniment pentru că nu poți să-ți faci o opinie corectă decât văzând opiniile tuturor. Au fost prezentări care în cea mai mare parte s-au adresat medicilor de alte specialități: ginecologie, oncologie și sincer nu mă așteptam la întrebări adresate medicului de familie. Nu am mai participat la altă ediție Health Meeting, organizată de MSC, așa că pentru mine a fost surprinzătoare deschiderea și interesul cu care oamenii au pus întrebări, nivelul de participare impresionant și ideea ca atare: actori din diferite poziții ale sistemului sunt împreună ceea ce nu poate să fie decât un beneficiu pentru fiecare dintre noi profesioniștii în sănătate. Recunosc că m-am consultat cu colegii mei din SNMF când am conceput lucrarea pe care urma s-o prezint, care sunt problemele pe care le întâlnim la pacientul oncologic și pe care vrem să le facem cunoscute din perspectiva medicului de familie și celorlalți colegi, colaboratorilor noștri”.

5. Dorica Dan, psiholog, președintele ARCrare

,,Am avut ocazia să particip la cea de-a 2-a ediţie a evenimentului ştiinţific interdisciplinar – Health Meeting și în acest context, am avut ocazia să vorbesc despre activitățile Asociației Române de Cancere Rare și despre obiectivele asumate în calitate de reprezentant al pacienților afectați de aceste boli. La conferință au fost prezenți lideri de opinie din diverse specialităţi implicate în diagnosticarea și managementul leziunilor pre-neoplazice şi neoplazice și am fost plăcut surprinsă să constat că există o preocupare majoră din partea specialiștilor către abordarea interdisciplinară a cazurilor, către intervenția personalizată și către îngrijirea integrată în vederea asigurării creșterii calității vieții pacienților”.

6. Conf. Dr. Alexandru Filipescu, UMF Carola Davila Bucureşti

,,Evenimentul foarte bine organizat de către dvs. a fost extrem de interesant tocmai datorită caracterului multidisciplinar de abordare a subiectelor puse în discuție.

Aștept cu drag invitația pentru următoarea conferință si mai ales ce subiecte veți aborda.”

7. Loredana Munteanu, kinetoterapeut independent, specialist Traumatologie-Ortopedie și Drenaj limfatic manual, reprezenant Asociaţia Casiopeea.

„Salut cu multă admirație inițiativa celor de la MSC de a organiza seria de Intruniri Medicale Interdisciplinare.

Tema „Leziuni pre-neoplazice si neoplazice” mi-a dat încă o data prilejul ca prin intermediul colegilor mei medici, să încurajez și să transmit pacientului importanța prevenției și tratamentului limfedemului post mastectomie. Existența unei echipe multidisciplinare ca și suport pacientului oncologic îi este mai mult decât vitală, iar întrunirile medicale interdisciplinare crează puntea de legătura în lucrul în echipă.’’

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Titlul articolului

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Review

Neuroendocrine carcinoma of the breast: how, when, why

NEUROENDOCRINE CARCINOMA OF THE BREAST: HOW, WHEN, WHY Bacalbasa N.1, Gavril A.2 1 University of Medicine and Pharmacy ‚Carol Davila’” Bucharest ; 2 University Emergency Hospital „Elias” Bucharest Corresponding author: Bacalbasa Nicolae Adress: Dimitrie Racoviță Street, no. 2, Bucharest, Romania E-mail: nicolae_bacalbasa@yahoo.ro Phone no: +40723540426

Open Access Article

Abstract Keywords: neuroendocrine carcinoma, endocrine carcinoma, invasive carcinoma, breast

Received: December 2014 Reviewed: December 2014 Accepted: December 2014

Background

Cite this article: Bacalbasa N., Gavril A. Neuroendocrine carcinoma of the breast: how, when, why.

Rom J Oncol Hematol.

2014; 5(4):182-188

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Neuroendocrine carcinoma (NEC) of the breast is a rare type of carcinoma that has not been well studied or characterized. Of the limited number of studies reported in the literature, most are case reports. A few small retrospective series studies have been reported.

We reviewed data recorded in the literature between 2003–2014 and evaluated disease incidence and patient age, sex, and race/ethnicity; clinicopathologic characteristics; and survival in comparison to invasive mammary carcinoma, not otherwise specified.

Review of the literature revealed that NEC is an aggressive variant of invasive mammary carcinoma. It generally occurred in older women (>60 years); present with larger tumor size (>20 mm), higher histologic grade, and higher clinical stage; and result in shorter overall survival and disease-specific survival than invasive mammary carcinoma. Overall survival and disease-specific survival were shorter in NEC at each stage than in NON-N EC of the same stage. Furthermore, when all NEC and NON-NEC cases were pooled together, neuroendocrine differentiation itself was an adverse prognostic factor independent of other known prognostic factors, including age, tumor size, nodal status, histologic grade, estrogen/progesterone receptor status, and therapy.

NEC is a rare but aggressive type of mammary carcinoma. Novel therapeutic approaches should be explored for this uniquely clinical entity.

Neuroendocrine carcinoma (NEC) of the breast is a very rare malignant tumor. Only a limited number of studies on NEC have been reported in the literature, most of them anecdotal case reports. Very few are series studies (1-11). Much of the current limited knowledge of this disease is based on these small retrospective series and thus is subject to selection/referral bias. Therefore, very little is known about the disease incidence, age and sex predilection, race/ethnicity distribution, clinicopathologic characteristics, and survival.

According to the most recent World Health Organization (WHO) classification of breast and gynecologic tumors, (4) their incidence is estimated, approximately, to 1% of all breast carcinomas.

To gain more insight into mammary NEC, we took advantage and evaluated the literature in the last decade. We evaluated the incidence and clinical course of mammary NEC in comparison to its more common counterpart, invasive mammary carcinoma, not otherwise specified (IMC-NOS).

Bacalbasa N., Gavril A.

Table 1. Reported cases of primary neuroendocrine carcinoma of breast

Features

Known facts – incidence, prevalence In the last 50 years histological and immuno­ histochemical studies have extended our know­ ledge about neuroendocrine tumors. Different diagnostic technologies allow us to distinguish pathological changes in endocrine cells. It is now possible to identify neuroendocrine features morphologically and biochemically within different histological types of invasive breast cancer. NECB was originally described in 1963 by Feyrter et al. when several cases of invasive breast cancer appeared to have a carcinoid growth pattern (1).

In 1977 Cubilla and Woodruff reported a case series of patients with breast tumors with the same features (2). Later on in 1982 a modified silver stain (grimelius) and electron microscopy were both routinely used to identify neurosecretory granules and if present within a tumor the patient was diagnosed with ‘argyrophilic breast carcinoma’, a term coined by Azzopardi et al.(5). Towards the end of the 1980s chromogranin and synaptophysin were found to be neuroendocrine differentiation markers and tumors that were once denominated ‘argylophilic breast carcinoma’ also tested positive for these markers (6). It was only until 2002 when Sapino et al.(7) first suggested a specific definition for NECB, which was subsequently adopted by the World Health Organization (WHO) in 2003 as

a means of endorsing it as a unique type of breast cancer (8). In the WHO classification neuroendocrine tumors have been defined as those in which one or more neuroendocrine markers, such as neuro specific enolase, chromogranin A, and/ or synaptophysin, are expressed in at least 50% of cancer cells (8). In addition to this, diagnosing primary NECB also requires fulfilling two other criteria: (1) other primary sites must be ruled out and (2) the tumor must show histological evidence of a breast in situ component (9).

The prevalence of primary NECB was once reported to be as high as 12% to 19.5%, but this was based on early diagnostic criteria, various sources of tissue, and IHC neuroendocrine markers (10-13). According to WHO diagnostic criteria the incidence is reported to actually range from 0.3% to 0.5% (14,15). The peer-reviewed literature reveals that >80 patients have been reported . A significant proportion (59.8%) of these cases was published after the WHO definition was established in 2003 allowing for consistency with respect to diagnostic criteria.

The majority of cases have been described in women and so far only two cases in men have been reported (16,17). The reported age of incidence ranges from 20 to 83 years with a higher incidence (60.2%) occurring in patients aged ≥50 years.

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Clinical features Patients with primary NECB do not have any distinctive clinical presentation. Indeed on clinical examination the findings are similar to those of any other type of invasive breast cancer. Nodule(s) size ranges from Tis to 18.0 cm and are not generally associated with axillary lymphadenopathy. The majority of tumors are <2.0 cm and patients are staged with early breast cancer. The radiological features may include a highly-dense mass with a spiculated or microlobulated margin on mamography and/or a homogenously hypoechoic massive lesion with normal sound transmission on ultrasonography (14). It is also possible for it to be mistaken for benign disease such as fibroadenomas or cysts because the tumor may have clear-cut, circumscribed borders (14). Distinguishing primary from metastatic NECB is not achieved with breast imaging.

Approach Before any further action is taken the NECB must be classified as ‘primary’ or ‘metastatic’. Physicians should primarily focus on ruling out breast metastasis from small cell carcinoma of the lung, the gastrointestinal tract, pancreas, and the cervix.

Metastatic neuroendocrine carcinoma from other organs cannot be distinguished from primary NECB because histologically they are similar . In this case workup with imaging is fundamental.

Differential diagnoses should also include Merkel cell carcinoma, lymphoma, carcinoid tumor, and melanoma . Combining clinical data, radiologic findings, and immunohistochemical stains specific to each tumor provides sufficient evidence to rule out metastatic sources of NECB and other primary tumors (11).

Tumors with neuroendocrine immunoreactivity below the 50% threshold should be considered to have focal neuroendocrine differentiation.

Generally these are IDC-not-otherwise specified (IDC-NOS) although lobular or medullary carcinomas can also present this feature. Breast cancers with focal neuroendocrine differentiation typically have 12% to 18% of IHC-positive neuroendocrine cells (12,13). These tumors tend to resemble common histologic types of breast cancer rather than NECB in terms of age of presentation, size, histologic grade, and lymph nodal status (10).

The histogenesis of NECB has not been fully clarified. An initial theory suggested that NECB cancer cells derived from argyrophilic cells of neural crest origin that migrated to the mammary ducts (2).

Another theory supports in situ development from neuroendocrine cells naturally found in the breast, but these cells have not been consistently found by other authors (4,). A novel hypothesis suggests that NECB results from an early divergent differentiation event in breast carcinogenesis in which neoplastic stem cells differentiate into both epithelial and endocrine lines (10,). Molecular analysis studies support this theory because studies have shown

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that the neuroendocrine component is clonally related to the intraductal component and that the whole tumor itself is of the luminal subtype.

In situ lesion Histologically describing the breast in situ component is important because this determines which adjuvant treatment regimens are chosen. Simultaneous presence of an intra-ductal component and the absence of other primary sites establish the breast as the primary organ of origin (11). Unlike the histogenesis of other common types of neuroendocrine carcinoma where there is evidence of benign neuroendocrine tumors these precursor lesions are extremely rare in the breast. Of the case reports published so far, only one has described a tumor with co-existing NECB and neuroendocrine cell hyperplasia. Kawasaki et al. has also described that neuroendocrine ductal carcinoma in situ (DCIS) could be considered a pre-invasive stage of NECB . Neuroendocrine DCIS are frequently underdiagnosed preoperatively because it resembles ductal hyperplasia and intraductal papilloma and sampling is quite difficult.

Primary NECB is not associated with any definitive gross pathological characteristics. The breast in situ component of primary NECB is usually found as an intraductal lesion co-existing with the neuroendocrine carcinoma component (19).

The breast in situ component consistently has histopathological features that include large or dilated ducts with the luminal spaces completely filled; distinctive cells with ovoid, polygonal, and spindle shapes; and a low- or moderate-grade of nuclear atypia (17). Additionally this specific component creates four pitfalls during diagnosis: (1) the invasive component of primary NECB can mimic DCIS; (2) non-specific glandular patterns within the tumor can lead to a diagnosis of IDCNOS; (3) cases of invasive lobular carcinoma or carcinoma with lobular features may not be recognized as having neuroendocrine dif­ ferentiation; and (4) the intraductal component of primary NECB may be mistaken for atypical intraductal hyperplasia or atypical papilloma In our case we initially overlooked this tumor because the IDC component prevailed on the core biopsy sample and final surgical pathology was necessary to obtain a definitive diagnosis. Over two-thirds of the cases in the literature report initial misdiagnosis later rectified after surgery.

Histological approach The diagnosis of NEBC relies on the presence of morphologic neuroendocrine features and neuroendocrine markers expressed in more than 50% of tumor cells. (4) The most sensitive and specific NE markers, are chromogranin A and B and synaptophysin. According to the above criteria, NEBCs constitute approximately 1% of all breast carcinomas. In 2012, the last edition of World Health Organization (WHO) classification of

Bacalbasa N., Gavril A.

Figure 1. Tumour cells arranged in diffuse sheets, clusters and at places trabecular pattern separated

by thin fibrous septae (H and E, ×100 and ×400) *Ann Med Health Sci Res. Nov 2013; 3(Suppl1): S35–S37 breast and gynecologic tumors, described 4 main histologic types: solid (usually of low to intermediate grade), small/oat cell and large cell, that are both poorly differentiated variants and lately added atypical carcinoid tumor. (11) In terms of prognostic and predictive factors, HER-2 is almost always absent in NEBCs, while the vast majority express estrogen and/ or progesterone receptors. The prognostic relevance of neuroendocrine differentiation is controversial, (12,13) though most studies report a relatively poor prognosis based on the extent of the neuroendocrine component and the degree of its differentiation.

WHO classified NEC of breast as solid NEC, atypical carcinoid tumor, small cell/oat cell carcinoma and large cell NEC. (2) Histopathologically the tumor cells are arranged in sheets, ribbons and cords. Individual cells are uniform with round to ovoid nucleus, salt and pepper chromatin and scant cytoplasm. (4) Immunohistochemistry shows more than 50% tumor cells positive for NE markers like chromogranin, synaptophysin and NSE. Aktar et al., reported that grimelius staining is specific and shows argyrophilia in NEC, on electron microscopy electron dense granules are demonstrated. Metastatic NEC to breast have also been reported hence CT of thorax, head and neck

Figure 2.

IHC stained section showing estrogen nuclear staining (×400) Ann Med Health Sci Res. Nov 2013; 3(Suppl1): S35–S37

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and abdomen should be done to rule out primary in the lung or other sites, (1,2,3,4) which was carried out in our case and found no abnormalities.

Hoang et al., (12) studied the molecular features of two cases of primary small cell carcinoma which revealed identical molecular alterations at multiple chromosomal regions including Breast Cancer gene 1, BRCA-2, p53 and retinoblastoma gene loci.

Weigelt et al. stated that out of six PNEC of breast, five NECs were of luminal A subtype, whereas one NE tumor was of luminal B phenotype. (13) Whereas Watrowski et al. studied a single case of PNEC of breast and classified as luminal B subtype also concluded that molecular classification helps in therapeutic interventions. (11) Outcome and treatment Patient outcome is not affected by the size of the neuroendocrine component (10); instead three histological parameters (histological grade, mucin production, and apocrine differentiation) have more significant impact. The most important histological factor is the histological grade, which is to some extent related to the histologic subtype.

For example, solid neuroendocrine carcinoma and atypical carcinoids, described as well-differentiated tumors, have a better prognosis than small cell and large cell NECB, which are poorly differentiated and have an unfavorable prognosis. Our case highlights the impact histological grade has on a patient’s outcome as we believe this may explain why the patient progressed to metastasis despite receiving optimal treatment. Mucin production is highly relevant because solid papillary carcinomas and mucinous carcinomas produce significantly longer survival times than other subtypes of NECB with no mucin content. Similarly the presence of apocrine differentiation has correlated with a better prognosis.

Specific recommendations regarding surgical management do not exist. Patients should be treated similarly to IDC whose choice of surgical procedure depends on the tumor’s location and clinical stage (12). Differentiating ‘primary’ from ‘metastatic’ NECB is crucial because the latter does not justify submitting a patient to mastectomy and axillary node dissection. Of the patients reported in the literature 48.3% have undergone mastectomy and 40% axillary lymph node dissection. Confirming negative surgical margins can be difficult, especially from intraoperative frozen sections, because NECB may have pagetoid involvement or a background of neuroendocrine cell hyperplasia can produce artifacts. To date there is limited information in the literature regarding safety of oncoplastic breast conservation and immediate breast reconstruction. Thus far only one study has reported the case of a patient that underwent mastectomy followed by deep inferior epigastric perforator flap reconstruction, however the authors failed to describe margin status and disease-free survival (15). Given the difficulty in assessing tumor margin status for this

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type of tumor, oncoplastic breast conservation and immediate breast reconstruction may not be beneficial to some patients with primary NECB. The real challenge with primary NECB lies in choosing the ideal type of cytotoxic therapy.

Currently there is no information that indicates what the most efficacious regimen is, but the general consensus is to treat it with chemotherapy regimens for common histologic types of breast cancer (11,15) and pulmonary small cell carcinoma neuroendocrine carcinoma (11). Of the cases that give detailed information on treatment (n=39), 28 received neo- and/or adjuvant chemotherapy.

Some examples of the chemotherapy regimens reported in the literature include fluorouracil/ epirubicin/cyclophosphamide followed by doce­ taxel; etoposimide and carboplatin or cisplatin; cisplatin/irinotecan; adriamycin and cytoxan or cisplatin; paclitaxel alone; and cyclophosphamide/ methotrexate/fluorouracil. Our patient was treated with cisplatin/etoposimide followed by paclitaxel/ carboplatinum; the former combination is the most widely used in primary NECB. Radiation therapy appears to be used to a lesser extent than chemotherapy; only 18 patients have received it alone or in combination with chemotherapy.

Because breast biomarkers have only recently become a standard in pathology reports many of the earlier case reports lack this information. Primary NECB tends to heterogeneously express ER, PR, and HER-2; this may explain why the overall outcome of these patients varies so much between different cohorts. Of the case reports with complete hormone receptor information available (n=18) 9 were triple negative, 7 were ER/PR positive and HER-2 negative, and the remaining 2 had other combinations. In this current case report the patient’s tumor was ER positive and PR and HER2 negative and hormone therapy was accordingly prescribed. The current recommendation is for patients with primary NECB to receive hormone therapy based on their hormonal receptor status .

The few studies that give insight into outcome have mixed results. A study by van Krimpen et al. suggested that the prognosis does not differ from that of the more common types of breast cancer (13). This is in accordance with Miremadi et al.’s study that reported that cases of primary NECB have the same mean age, size, histologic grade, nodal status, and prognosis as their non-primary NECB counterparts (10). It has also been suggested that patients with primary NECB of non-small cell type may even have a better prognosis than patients with IDC or invasive lobular carcinoma (15).

Alternatively other studies have described a worse outcome overall.

Several unsolved issues are frequently discussed in the literature. First of all case series have only analyzed their patients as a homogenous population without taking into consideration the different histologic subtypes (11). Second, standard prognostic parameters, specifically histologic grade, are not consistently taken into account

Bacalbasa N., Gavril A.

when comparing primary NECB cases with nonNECB (11). In addition to this randomized clinical trials comparing the different treatment regimens and their outcomes have not been carried out.

All these unsolved matters are obviously related to the rarity of this tumor. Interestingly enough many of the studies included in this review did not provide essential clinical, histologic, diagnostic, and therapeutic information in their case report, which makes comparing them difficult. In this respect if future cases of primary NECB could be prospectively and collectively registered in a single international database encompassing standard epidemiological information and stratifying patients according to histological and molecular subtype then physicians would have a valuable tool to truly understand this tumor.

Secondary disease Previous reports of literature mention that NEC breast shows aggressive behavior compared to ductal carcinomas with a higher propensity for local and distant recurrence. But more recent data suggest this tumor has more favorable prognosis.(1,7) Higher grade, increased tumor size and regional lymph node metastasis are associated with poor prognosis and decreased disease free survival.(3) Mucinous differentiation and Estrogen/ Progesterone receptors positivity are favorable prognostic factors. (7) Clinical outcomes reported in literature showed 15% of local recurrence by 5 years, with median recurrence free time of 177 months and 34% risk for distant recurrence within 5 years with median recurrence free time of 73 months among NEC patients. The common sites for distant metastasis are bone and liver, other sites include lungs, brain, pleura, mediastinal lymph nodes, adrenal glands, ovaries, fallopian tubes, colon, ileum and pancreas. (3) In the literature, there have been reports about NEBCs metastasizing to multiple sites, even years following treatment of the primary breast tumor and despite the administration of systemic therapy. (14–26) Although there are only case reports of NEBCs metastases, the diversity of metastatic sites is unremarkable , while there are scattered data regarding the treatment strategies.

Imamura,(14) has reported the curative resection of multiple hepatic metastases by means of a lateral segmentectomy of the liver. In other reports, an alternative approach included Transcatheter Arterial Chemoembolization (TACE) and octreo­ tide administration, along with resection of the breast primary was instituted. (16) The response to systemic therapy has been inconsistent with progression of the disease. (17–19,22–24) There is no widely accepted systemic therapy for NEBC for the moment. Close follow-up of patients is mandatory, since metastases can appear late and in diverse sites. Howlader et al. performed a review of the management of liver metastases from breast carcinoma and proposed a management guideline, based on the fact that prolonged survival can be obtained after hepatectomy. (27) Liver surgery is recommended, in absence of extrahepatic disease (except for bone metastases which are easily controlled by radiotherapy or isolated pulmonary metastasis) if an R0 resection is feasible with accepted mortality.

Conclusions In summary, this review showed that NEC is an aggressive mammary carcinoma subtype with significantly shorter overall survival than IMCNOS. It tends to present at greater age, with larger tumor size, higher histologic grade, and higher clinical stage. NEC also tends to be ER/PR positive, but positive ER status does not appear to confer a prognostic benefit as it does in other invasive mammary carcinomas. At the moment it is difficult to fully understand this rare tumor because issues such as histogenesis, optimal adjuvant therapy, and prognosis are still unknown. The limited number of patients in the literature have presented in different clinical stages and received different treatment combinations thus the data summarized in this review should be interpreted cautiously.

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International article

Clinical and biological impact of TET2 mutations and expression in younger adult AML patients treated within the EORTC/GIMEMA AML-12 clinical trial

CLINICAL AND BIOLOGICAL IMPACT OF TET2 MUTATIONS AND EXPRESSION IN YOUNGER ADULT AML PATIENTS TREATED WITHIN THE EORTC/GIMEMA AML-12 CLINICAL TRIAL First published in: Ann Hematol (2014) 93:1401–1412 Published online: 29 March 2014 #Springer-Verlag Berlin Heidelberg 2014 DOI 10.1007/s00277-0142055-7 The online version of this article (doi:10.1007/s00277014-2055-7) contains supplementary material, which is available to authorized users.

Mariam G. Aslanyan1, Leonie I. Kroeze1, Saskia M.C. Langemeijer1, Theresia N. Koorenhof-Scheele1, Marion Massop1, Patricia van Hoogen1, Ellen Stevens-Linders1, Louis T. van de Locht1, Evelyn Tönnissen1, Adrian van der Heijden1, Pedro da Silva-Coelho1,4, Daniela Cilloni5, Giuseppe Saglio5, Jean-Pierre Marie6, Ruoping Tang6, Boris Labar7, Sergio Amadori2, Petra Muus3, Roel Willemze4, Erik W. A. Marijt4, Theo de Witte8, Bert A. van der Reijden1, Stefan Suciu9, Joop H. Jansen1 1. Department of Laboratory Medicine, Laboratory of Hematology, Radboud University Nijmegen Medical Center, Geert Grooteplein zuid 8, 6525 GA Nijmegen, The Netherlands; 2. Department of Hematology, Tor Vergata University Hospital, Rome, Italy; 3. Department of Hematology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands; 4. Faculdade de Medicina da Universidade do Porto, Porto, Portugal; 5. Department of Hematology and Clinical & Biological Sciences, S Luigi Hospital, University of Turin, Turin, Italy; 6. Department of Hematology and Tumor Bank, Saint-Antoine Hospital, AP-HP and University Pierre and Marie Curie, Paris, France; 7. Department of Hematology and Clinical Institute of Laboratory Diagnosis, University School of Medicine, Zagreb, Croatia Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands; 8. Department of Tumor Immunology, Radboudumc, Nijmegen, The Netherlands; 9. European Organization for Research and Treatment of Cancer (EORTC) Headquarters, Brussels, Belgium Corresponding author: Joop H. Jansen E-mail: Joop.Jansen@Radboudumc.nl S. Suciu E-mail: stefan.suciu@eortc.be

Received: 25 February 2014

Open Access Article

Accepted: 6 March 2014

Abstract Keywords:

Cite this article: Mariam G. Aslanyan, Leonie I. Kroeze, Saskia M. C. Langemeijer, Theresia N. Koorenhof-Scheele, Marion Massop, Patricia van Hoogen, Ellen Stevens-Linders, Louis T. van de Locht, Evelyn Tönnissen, Adrian van der Heijden, Pedro da Silva-Coelho, Daniela Cilloni, Giuseppe Saglio, Jean-Pierre Marie, Ruoping Tang, Boris Labar, Sergio Amadori, Petra Muus, Roel Willemze, Erik W. A. Marijt, Theo de Witte, Bert A. van der Reijden, Stefan Suciu, Joop H. Jansen. Clinical and biological impact of TET2 mutations and expression in younger adult AML patients treated within the EORTC/ GIMEMA AML-12 clinical trial.

Rom J Oncol Hematol. 2014; 5(4):190-200

190

TET2 mutations, Acute myeloid leukemia, TET2 expression, Prognosis

We assessed the prognostic impact of TET2 muta- tions and mRNA expression in a prospective cohort of 357 adult AML patients< 60 years of age enrolled in the European Organization For Research and Treatment of Cancer (EORTC)/Gruppo Italiano Malattie Ematologiche dell’ Adulto (GIMEMA) AML-12 06991 clinical trial.

In addition the co-occurrence with other genetic defects and the function- al consequences of TET2 mutations were investigated. TET2 mutations occurred in 7.6 % of the patients and were an independent marker of poor prognosis (p =0.024). TET2 and IDH1/2 mutations strongly associated with aberrations in the DNA methyltransferase DNMT3A. Functional studies con- firmed previous work that neither nonsense truncations, nor missense TET2 mutations, induced 5-hydroxymethylcytosine formation. In addition, we now show that mutant TET2 forms did not act in a dominant negative manner when co-expressed with the wild-type protein. Finally, as loss-of-function TET2 mutations predicted poor outcome, we questioned whether low TET2 mRNA expression in cases of AML without TET2 mutations would affect overall survival. Notably, also AML patients with low TET2 mRNA expression levels showed inferior overall survival.

Mariam G. Aslanyan, Leonie I. Kroeze, Saskia M. C. Langemeijer, Theresia N. Koorenhof-Scheele, Marion Massop, Patricia van Hoogen, Ellen StevensLinders, Louis T. van de Locht, Evelyn Tönnissen, Adrian van der Heijden, Pedro da Silva-Coelho, Daniela Cilloni, Giuseppe Saglio, Jean-Pierre Marie, Ruoping Tang, Boris Labar, Sergio Amadori, Petra Muus, Roel Willemze, Erik W. A. Marijt, Theo de Witte, Bert A. van der Reijden, Stefan Suciu, Joop H. Jansen

Introduction Recently, a number of novel recurrent alterations have been described in adult AML, among which a group of epigenetic modifiers including TET2, ASXL1, BCOR, DNMT3A, IDH1 and IDH2 (1, 2). Verifying the relevance of these new genes as prognostic markers is crucial before they can be implemented into clinical practice to improve risk-adapted treatment. Un- derstanding the molecular biological consequences of genetic mutations allows the development and application of targeted forms of therapy, aiming at the impaired proteins or the biological pathways they are involved in. TET2 aberrations occur in various myeloid neoplasms.

Mutations were found in 20–25 % of myelodysplastic syndromes (3, 4), 10–15 % of myeloproliferative neoplasms (5) , and up to 50 % of chronic myelomonocytic leukemias (6). In AML, TET2 mutations affect 7–10 % of the adulthood cases (3–5) and 1.5–4 % of pediatric cases (7, 8). The impact of TET2 mutations on clinical outcome in AML is still controversial. In some stud- ies, TET2 mutations correlated with inferior survival, particularly in the subgroup of cytogenetically normal AML, whereas in other studies, no prognostic impact of TET2 mutations was observed (9–15). So far, few prospectively collected cohorts have been analyzed for the impact of TET2 mutations using multivariate analysis and including the currently established genetic and other prognostic parameters (10–12, 14).

Several studies based on mouse models have shown that inactivation of TET2 in myeloid cells induces a myeloprolif- erative syndrome, confirming the role of TET2 mutations in the pathogenesis of myeloid malignancies (16–18) . The TET protein family comprising TET1, TET2 and TET3, belongs to the group of 2 oxoglutarate (2OG)and Fe(II)-dependent dioxygenases, and is involved in a novel epigenetic modifi- cation, namely, the conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) (19). TET proteins further oxidize 5hmC to formylcytosine and carboxylcytosine (5fC and 5caC).

5fC and 5caC are recognized by the thymine- DNA glycosylase (TDG), and replaced by unmodified cytosines through the DNA repair machinery (20, 21). Mapping of TET protein binding sites and 5hmC on the genome has shown that they localize to the transcription start sites of genes, suggesting a role for the TETs in the regulation of gene expression through modification of chromatin at promoter regions (22–24).

In this study, we assessed the incidence and clinical impact of TET2 mutations in a prospective cohort of 357 AML patients who were treated uniformly according to the EORTC/GIMEMA AML-12 06991 clinical trial protocol. We determined the cooccurrence of TET2 mutations with other molecular markers. In addition, the function of different TET2 isoforms and TET2 mutants was tested in cell line models. Finally, mRNA levels of TET2 were measured and correlated to survival.

Materials and methods Patients Bone marrow samples from 357 patients included in the EORTC/GIMEMA 06991 AML-12 (25) clinical trial NCT0004128 (1999–2009) were obtained at diagnosis.

In- formed consent was obtained from all patients included in the study. To avoid bias, consecutive patients from the larger participating centers were included in the analysis. Median age was 46 years (range 15–60 years) and median follow-up was 5.63 years. The study was approved by the Institutional Review Board of the Radboud University Nijmegen Medical Center and all aspects of this study abided by the principles laid out in the declaration of Helsinki. All patients had un- treated newly diagnosed AML, ≥30 % blast cells in bone marrow smears, and represented all AML FAB subtypes, except AML-M3. Briefly, the AML-12 trial assessed the value of high vs. standard dose Ara-C during induction, as well as IL-2 after intensive consolidation/auto-SCT. Q-PCR and mutational analysis For Q-PCR analysis RNA was isolated from healthy donor- derived G-SCF-mobilized CD34+ cells, or patientderived bone marrow or peripheral blood mononuclear cells. mRNA levels of TET2 isoform 1 and isoform 2, TET1 and TET3, were determined as previously described (3,4).

DNA sequencing of TET2 isoform 1 (NM_001127208.2) was performed as de- scribed (3). cDNA sequencing primers are listed in Table S1. EVI1, IDH1 R132, IDH2, R140, and R172 detection was performed as described (26, 27). Direct DNA sequencing of DNMT3A (ENST00000264709) was performed on exons 7- 23 either by Sanger sequencing (primer sequences listed in Table S2) or 454-based next-generation sequencing (454 Life Sciences, Branford, CT, USA) (28). NGS data were analyzed using R453Plus1Toolbox, GS Variant Analyzer Software 2.6 (454 Life Sciences) and Sequence Pilot version 3.5.2 (JSI Medical Systems, Kippenheim, Germany)(29). NPM1 and FLT3 mutation analyses were developed previously (30, 31).

Statistical analysis Overall survival (OS) was computed from the date of registration in the EORTC 06991 study until death (whatever the cause) or until last follow-up (censored observation). Survival distribution of OS was estimated according to the Kaplan–Meier technique. The Cox Pro- portional Hazards Model was used to determine the prognostic importance of each factor analyzed and to obtain hazard ratio (HR) estimates as well as corre- sponding 95 % confidence intervals (95 % CI).

All statistical analyses were performed according to the intention-to-treat principle. Being a prognostic factor analysis, only eligible patients were included. For con- tingency tables, the significance of a relationship be- tween categorical two variables, the Fisher exact test was used. Statistical analyses were performed by using SAS® (Cary, USA).

Plasmids used for overexpression in cell line models N-terminally tagged eGFP-TET2 isoform 1 plasmid was kindly provided by Dr. O. Bernard.

TET2 isoform 1 (NM_001127208.2), TET2 isoform 2 (NM_017628.4), TET2 catalytic domain (a.a. 1104– a.a. 2002), and TET2 box 2 (a.a. 1789–a.a. 2002) sequences were cloned into pDONR201 using the Gateway cloning technology (Life Technologies, December 2014

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Carlsbad, CA, USA) according to the manufacturer’s instructions. The entry vectors were further transferred into a Gateway based pDest733 (32) destination vector to create N-terminal mRFP fusion proteins.

TET2 mutant sequences were generated using the QuikChange XL site-directed mutagenesis kit (Stratagene, Agilent Technologies, CA, USA) as recommended by the manufacturers and cloned into pDONR201.

The entry vectors were used for transfer into pDest733 and pDest501 Gateway-based expression plasmids (33) to create N-terminal mRFP and eCFP fusions, respectively.

Cell culture, transfection, and immunofluorescence COS-1 ce lls were grown i n I scove’s M odified Dulbecco’s Media (IMDM; Life Technologies) supplemented with 10 % fetal calf serum and penicillin and streptomycin. Cells were transfected with Lipofectamin 2000 reagent (Life Technologies).

Proteins were expressed for 20–24h. Subsequently, cells were fixed with 4 % PFA for 20 min, washed extensively with PBS and permeabilized with 0.5 % Triton X-100 in PBS for 5 min. To allow detection of 5hmC, cells were treated with 2 M HCl for 20–25 min.

HCl was neutralized with 100 mM Tris–HCl for 10 min (pH =8), followed by extensive washing with PBS. A blocking step was performed with 5 %BSA in PBS/ Tween for 30 min. Afterwards, samples were incubated with a primary antibody against 5hmC (Active Motif, CA, USA; 1:500 dilution) for 2 h at RT and subse- quently washed with PBS. To allow primary antibody detection, the following secondary antibodies were used: Alexa 488 (Life Technologies; 1:500 dilution) or Alexa 350 (Life technologies; 1:50 dilution). Samples were mounted with Vectashield with DAPI or Antifade Prolong Gold (in the case of Alexa 350). Samples were analyzed with a Leica DMBRE (type 301-371.011) microscope. Images were taken at 40× or 100× magnification using a DC350F (10447116) camera and Leica FW4000 acquisition software (Leica Microsystems, Wetzlar, Germany).

Results TET2 mutations correlate with poor overall survival in AML in univariate analysis Bidirectional Sanger sequencing of the entire TET2 cod- ing region, and the splice donor and acceptor sites, revealed mutations in 27/357 (7.6 %) of the patients (for detailed patient characteristics, see Table 1; localization of mutations is depicted in Fig. S1).

In 19 of the cases, mutations affected one allele, whereas in 8 pa- tients two TET2 aberrations coexisted, suggesting biallelic inactivation. In univariate analysis, the various cytogenetic subtypes showed the expected correlations with overall survival, with the t(8;21) and inv (16) translocations correlating with a good prognosis, and -5, -7, and complex karyotypes signifying poor outcome (Fig. S2a). NPM1 mutations did, however, not have prognostic impact (Fig. S2b). In line with published data, the presence of a FLT3- ITD mutation correlated with inferior clinical outcome (p <0.0001), whereas a FLT3-TKD mutation associated with intermediate or

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slightly better survival (Fig. S2c and d). Furthermore, EVI1 overexpression was strongly associated with inferior prognosis (Fig. S2e, p =0.0004). The presence of a TET2 mutation correlated with poor overall survival (Fig. 1a, p = 0.025). IDH2 muta- tions did not show a significant effect on overall sur- vival (Fig. 1c), whereas a trend toward poor prognosis was present in patients with an IDH1 mutation (Fig. 1d, p = 0.075).

Finally, DNMT3A mutations also predicted unfavorable outcome (Fig. 1b, p =0.04).

Association of TET2 mutations with other prognostic features including molecular and cytogenetic markers TET2 mutations correlated with older age, and were almost exclusively present in the group of cytogenetically normal AML. We found no association between TET2 mutations and performance status or WBC at diagnosis. There was, however, an association between TET2 mutations and FAB subtype (p=0.06).

TET2 mutations were less frequent in AML-M1, and enriched in AML-M5 patients. NPM1 and TET2 defects were significantly correlated (p=0.03), whereas FLT3-ITD and FLT3-TKD aberrations were often present together with TET2 mutations but their co-occurrence did not reach statistical significance.

TET2 and IDH mutations seldom co-existed in the same patient as may be expected since IDH mutations abrogate the activity of TET2 (34). Notably, both TET2 and IDH mutations strongly correlated with the presence of a DNMT3A mutation (p=0.02 and p= 0.004, respectively). Patients who carried DNMT3A aberra- tions together with either a TET2 or an IDH mutation, very frequently had an additional FLT3-ITD. TET2 mutations represent an independent marker of poor prognosis in AML Cox regression analysis was performed to determine whether TET2 mutations represent an independent marker of poor prognosis. In multivariate analysis, adjusting for age, cytogenetic/FLT3-ITD risk group, NPM1 muta- tion, AML type, institution, and stratifying by treatment arm, TET2 mutations remained an independent predictor of inferior survival (p = 0.01, HR = 1.86, 95% CI: 1.15–3.00. Within the poor risk group of patients carrying a FLT3-ITD and/or a high-risk cytogenetic profile (-5, -7, complex), which conferred the expected poor overall survival, the presence of an additional TET2 mutation defined a subgroup of patients with an even more dis- mal prognosis (Fig. 2a, p = 0.033). NPM1 mutations had only a minor effect toward favorable outcome in the cytogenetically normal subgroup without FLT3-ITD (Fig. S2b). Also within the NPM1 mutated group, TET2 mutations conferred a trend toward worse survival (Fig. 2b, p = 0.061). Finally, as DNMT3A and TET2 mutations frequently co-occurred, and DNMT3A mutations correlated with inferior survival (Fig 1b), we tested what the effect of TET2 mutations would be in the context of a concomitant DNMT3A mutation. Also in the DNMT3A mutated subgroup, the presence of a TET2 mutation further exacerbated the poor prognosis of patients (Fig. 2c, p = 0.066). Treatment with standard

Nume autori

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Figure 1. Univariate analysis assessing the effect of mutations in genes implicated in the DNA

(hydroxy)methylation pathway. TET2 (a), DNMT3A (b), IDH1 (c), and IDH2 (d) or high dose Ara-C did not alter theeffect of TET2 mutation, as these mutations remained a poor indicator of prognosis in both patient groups (Fig. S3).

Functionality of different TET2 splice and truncation variants Although studies so far have always focused on the longest isoform of TET2 (isoform 1), there are three major TET2 transcripts that may be expressed in cells, as we previously showed (3) (Fig. S4a). The shorter isoforms lack the catalytic domain and their function remains unknown. The expression levels of isoform 1 and of the shorter form, isoform 2, are comparable. Furthermore, isoform 2 is reminiscent of some of the truncation mutants found in patients (3). To investigate whether isoform 2 exhibits dominant negative features, we tested its effect on the capacity of isoform 1 to generate 5hmC. Several expression constructs were created that contained TET2 isoform 1, TET2 isoform 2, the catalytic domain (CD), and the second conserved region of the catalytic domain (box 2; Fig. S4b). To monitor expression, all proteins were N-terminally fused to monomeric red fluorescent protein (mRFP).

COS-1 cells were transiently transfected with each of the expression vectors and immunostaining was performed using an antibody specific for 5-hydroxymethylcytosine. Expression of isoform 1 and the isolated catalytic domain resulted in

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a clear formation of 5hmC. Isoform 2 and the isolated, conserved box 2 showed no enzymatic activity (Fig. S4c). Importantly, coexpression of isoform 2 with isoform 1 did not inhibit the generation of 5hmC (Fig. S4d), implying that isoform 2 does not exhibit dominant negative properties.

TET2 mutations result in loss of function and are not dominant negative Mutations in TET2 are scattered throughout the entire coding region of the gene and may result in the production of truncated proteins, as well as single amino acid substitutions. In vitro studies and mouse models have suggested that likely all TET2 mutations lead to loss of function. Moreover, in patients, despite the presence of a wild-type allele, DNA hydroxymethylation is strongly reduced (35). It remains, however, possible that the mutant protein inhibits the function of the wild-type allele in a dominant negative fashion. To test this, six mutant TET2 sequences were cloned into expression vectors, each N-terminally fused to mRFP. Two nonsense mutations were selected that either delete both conserved boxes (R506X)â&#x20AC;&#x201D;hence the whole catalytic domainâ&#x20AC;&#x201D;or yield a protein containing only the first conserved box (box 1), causing a partial deletion of the catalytic domain (S1758X). Additionally, four missense substitutions were generated, two in box 1 (R1214W and R1261L) and two in box 2 (I1873T and H1881Q; Fig. 3a). All six mutants,

Mariam G. Aslanyan, Leonie I. Kroeze, Saskia M. C. Langemeijer, Theresia N. Koorenhof-Scheele, Marion Massop, Patricia van Hoogen, Ellen StevensLinders, Louis T. van de Locht, Evelyn TĂśnnissen, Adrian van der Heijden, Pedro da Silva-Coelho, Daniela Cilloni, Giuseppe Saglio, Jean-Pierre Marie, Ruoping Tang, Boris Labar, Sergio Amadori, Petra Muus, Roel Willemze, Erik W. A. Marijt, Theo de Witte, Bert A. van der Reijden, Stefan Suciu, Joop H. Jansen

Figure 2. Kaplanâ&#x20AC;&#x201C;Meier survival curves of cytogenetic and genetic subgroups. An unfavorable effect

of TET2 mutationson overall survival was observed within the a cytogenetic high risk and/or FLT3ITD-positive patients, b NPM1-mutated patients, and c DNMT3A-mutated patients including the single amino acid substitutions, lost the capability to generate 5hmC (Fig. 3b). Furthermore, co-expression of GFP-tagged wild-type TET2 with each of the mutant proteins did not result in loss of hydroxymethylation, indicating that, with respect to the formation of 5hmC, the mutants are not dominant negative (Fig. 3c).

Effect of wild-type TET2 on survival of AML patients As diminished TET2 function appears to be the mechanism through which mutations in this gene contribute to oncogenesis, and patients who carry TET2 mutations show a significantly worse overall survival, we tested whether the expression levels of isoforms 1 and 2 of TET2 may impact clinical outcome in patients without a TET2 or IDH mutation. The mRNA levels of both isoforms varied between different patients. No significant differences in expres- sion were observed between patients with and without a TET2 mutation (Fig. S5a). When compared to mRNA levels of TET2 in CD34+ cells, AML patients showed expression levels that were distributed in a wider range (Fig. S5b).

The median levels of TET2 expression in patients were similar to those measured in healthy CD34+ control cells. There was no difference in overall survival when TET2 isoform 2 expression was considered (Fig. 4b). In contrast, overall survival analysis showed that low TET2 isoform 1 expressers had an inferior clinical outcome (Fig. 4a, p =0.05).

Discussion The prognostic significance of TET2 mutations in AML patients has been controversial. Initially, Abdel-Wahab et al. (9) showed an association with poor OS in a smaller, retrospective study, whereas Nibourel et al. (13) reported no correlation between TET2 mutational status and clinical outcome, albeit in a preselected group of de novo AML patients achieving complete remission.

Consequently, studies involving larger cohorts of patients were explored.

Shen et al. (14) found no prognostic significance for TET2 mutations in 605 de novo AML patients who had no other recognizable karyotypic abnormalities except 11q23, and Metzeler et al. (12) identified TET2 mutations as a predictor of inferior survival in favorable risk cytogenetically normal primary AML patients, as defined by the European LeukemiaNet (ELN) guidelines. Chou et al. (10) defined TET2 aberrations as a marker of poor prognosis in AML patients of intermediate risk, and Weissmann et al. (15) also showed a negative impact of TET2 mutations on sur- vival in favorable risk AML patients with normal cytogenetics.

Most recently, Gaidzik et al. (11) did not find a prognostic impact of TET2 mutations in 330 cytogenetically normal AML patients. In the current study, we show a negative prognostic impact of TET2 mutations in a sizeable, prospectively studied cohort December 2014

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Figure 3. TET2 mutant proteins show impaired catalytic activity, but do not exhibit dominant

negative features when co- expressed with wild-type protein. a Localization of TET2 mutations found in patients with myeloid malignancies used for functional testing. Two of the mutants result in truncated proteins lacking either both conserved boxes within the catalytic region, or only the second conserved box, whereas the rest of the mutants result in missense substitutions in the conserved boxes. All aberrations leading to amino acid changes have been confirmed either by us or other as true acquired somatic mutations. The H1881Q mutation targets an Fe(II)-binding site in the catalytic domain of TET2. b Mutant forms of TET2 found in patients are unable to produce 5hmC unlike wild-type protein. Constructs contain an N-terminal mRFP tag. Images were taken at 40Ă&#x2014; magnification. c mRFP-tagged mutant forms of TET2 do not act in a dominant negative manner over wild-type GFP-TET2. Images were taken at 100Ă&#x2014; magnification of adult AML patients < 60 years of age. The negative effect of TET2 mutation on overall survival was present in various risk groups. In accordance with previous reports, TET2 mutations were significantly associated with older age. In addition, TET2 alterations were reported in normal elderly individuals with clonal

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hematopoiesis (33), and TET2 mutations were shown to be present in a preleukemic stage of leukemia evolution (36). Further- more, TET2 mutations are found in many different subtypes of myeloid malignancies.

This suggests that cells carrying TET2 defects may clonally expand, but that additionally acquired

Vincenzi B, Santini D, Russo A, Spoto C, Venditti O, Gasparro S, Rizzo S, Beomonte B, Caricato M, Valeri S, Coppola R, Tonini G

Table 1. Baseline Characteristics of the Patients

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Figure 4.

Aberrant expression of the wild-type TET2 isoform 1 has a negative impact on overall survival (a), whereas the TET2 isoform 2 expression has no effect on survival (b)

mutations mostly determine the subtype and severity of the disease that eventually causes clinical problems.

It has previously been noted that TET2 and DNMT3A mutations co-occur in T-cell lymphomas (37). In AML, a correlation between IDH and DNMT3A has been ob- served in AML (14). In the present study, we show that both TET2 and IDH mutations were enriched in patients who also carried a DNMT3A defect. Although DNMT3A mutations conferred poor prognosis (in line with previous work (38)), the additional presence of TET2 alterations in these patients defined a subgroup with a particularly unfavorable outcome.

As established earlier, TET2 and IDH mutations were mutually exclusive since both groups of mutations perturb a common biological pathway (34,39,40). The high incidence of DNMT3A mutations in both of these groups is highly indicative of a cooperative

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mechanism through which mutations impairing DNA hydroxymethylation and DNA methylation contribute to leukemogenesis. Further functional characterization of this interaction in in vivo models is necessary and may provide important insights into targeted therapies aimed at the DNA (de) methylation pathway within patients with this particular genetic profile.

Testing of TET2 mutations in vitro (35) as well as in mouse models consistently points to a loss-of-function phenotype (16â&#x20AC;&#x201C;18, 41).

This is further supported in this manuscript. In addition, we show that TET2 mutants do not suppress the function of the wild-type protein and hence do not show dominant negative traits. As mRNA levels of TET2 in AML patients varied over a wide range compared to normal hematopoietic cells, this raised the possibility

Mariam G. Aslanyan, Leonie I. Kroeze, Saskia M. C. Langemeijer, Theresia N. Koorenhof-Scheele, Marion Massop, Patricia van Hoogen, Ellen StevensLinders, Louis T. van de Locht, Evelyn Tönnissen, Adrian van der Heijden, Pedro da Silva-Coelho, Daniela Cilloni, Giuseppe Saglio, Jean-Pierre Marie, Ruoping Tang, Boris Labar, Sergio Amadori, Petra Muus, Roel Willemze, Erik W. A. Marijt, Theo de Witte, Bert A. van der Reijden, Stefan Suciu, Joop H. Jansen

that the level of TET2 mRNA expression could be relevant in patients who do not carry loss-of-function mutations of the TET2 gene. Analysis of the effect of wild-type TET2 expression on clinical outcome in patients without TET2 or IDH mutations demonstrated that patients with low levels of TET2 expression had a worse overall survival. We conclude that both lossof-function mutations and low expression of TET2 are markers of poor prognosis in AML indicating that the development of therapies targeting the DNA hydroxymethylation pathway could be beneficial for these patients.

Acknowledgments This work was supported by grants from the EORTC (EGAM 2009 NOCI), the EORTC Charitable Trust, and the Netherlands Institute for Regenerative Medicine (NIRM).

Authors’ contributions MGA, LIK, SMCL, TNKS, MM, PvH, ESL, LvdL, ET, AvdH, PSC performed experiments, analyzed and interpreted data. DC, GS, JPM, RT, BL, SA, PM, RW, EWAM, TdW recruited patients, provided essential patient material and analyzed patient data. SS performed statistical analyses. MGA, BvdR, and JHJ designed the study. MGA and JHJ wrote the paper. All authors have read and approved the manuscript.

Conflicts of interest The authors declare no conflict of interest.

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Interventional treatment of the carcinoid syndrome

INTERVENTIONAL TREATMENT OF THE CARCINOID SYNDROME H. Ahlman a, O. Nilsson b, M. Olaussona Departments of a Surgery and b Pathology, Göteborg University, Sahlgrenska University Hospital, Göteborg, Sweden Corresponding author: Håkan Ahlman Department of Surgery, Sahlgrenska University Hospital, SE–413 45 Göteborg (Sweden) Tel. +46 31 342 1778, Fax +46 31 822 104, E-mail: hakan.ahlman@surgery.gu.se

Open Access Article

Abstract Keywords: carcinoids, tumour reduction, embolization, liver transplantation First published in: Neuroendocrinology 2004;80(suppl 1):67–73 Accessible online at: www. karger.com/nen

Liver metastases imply a major problem in patients with carcinoid tumours and hormone overproduction. Patients with distant metastases can undergo resection for potential cure or for symptom palliation. In patients with bilobar liver metastases other interventions are at hand, e.g. local ablation or hepatic arterial embolization. In selected cases liver transplantation can be a treatment alternative.

Prior to all interventions patients with midgut carcinoids are protected with somatostatin analogues to reduce hormone secretion. Patients with foregut carcinoids may present special problems with lifethreatening release of histamine during interventions.

DOI: 10.1159/000080745 Copyright © 2004 S. Karger AG, Basel

Cite this article: H. Ahlman, O. Nilsson, M. Olausson. Interventional treatment of the carcinoid syndrome. Rom J Oncol Hematol. 2014; 5(4):202-210

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Introduction

Preoperative considerations

Carcinoid tumours of the gastro-intestinal tract were initially divided according to embryological origin into foregut (stomach, duodenum, pan­ c reas), midgut (small bowel, appendix, ce­ c um) and hindgut carcinoids (colon, rectum). A revised classification took into account tumour location, histological grade and proliferative index (1). They can further be divided after degree of differentiation into well-differentiated (WDEC) or poorly differentiated (PDEC) endocrine car­ cinomas. The neuroendocrine (NE) phenotype is usually evaluated by antibodies against pro­ teins of the neuro secretory granules (chromo granin A, synaptophysin) or cytosolic markers (neuron-specific enolase, PGP9.5). WDEC are immunopositive for all markers in the majority of tumour cells, while PDEC often lack the granular markers with cytosolic markers retained. The proliferative activity can be assessed by mitotic rate or more reliably by the fraction of tumour cells that express the Ki67 antigen.

Prior to interventional treatment of NE tumours, the tumour type and hormone production must be assessed. Patients with midgut carcinoids are pretreated with long-acting somatostatin analogs, e.g. octreotide, to prevent carcinoid crises during surgery. In case of such a reaction (facial flush, bronchoconstriction and hypotension), adrenergic drugs should be avoided, since carcinoid tumour cells may express adrenoceptors and excessive release of tumour products, i.e. serotonin and tachykinins, can be elicited (fig. 1).

Instead the surgical manipulation should be interrupted, volume substituted according to haemodynamic parameters and octreotide in combination with cortisone given. Spinal anaesthesia with reduced arterial blood pressure and secondary release of catechol- amines from the adrenals, in turn activating serotonin release from the tumour, should be avoided.

For postoperative pain, epidural analgesia is preferred (2).

H. Ahlman, O. Nilsson, M. Olausson

Figure 1. The midgut carcinoid tumour cells express on its surface adrenoceptors, which mediate the

release of 5-HT, somatostatin receptors (SSTR), which inhibit the release of secretory products and receptors for interferon (IFN). Serotonin, or its precursor 5-HTP, can be taken up by the tumour cell via a membrane pump (inhibited by clomipramine) and incorporated into secretory granules via vesicular monoamine transporters (VMAT; inhibited by reserpine). Cytosolic 5-HT can be degraded within the tumour cell by monoamine oxidase (MAO) to 5-HIAA. Serotonin stored within granules is released by exocytosis together with chromogranin A (CgA). The tumour cell can also handle catecholamines or analogs (MIBG)

Foregut carcinoids with excess production of histamine can cause the life-threatening ‘atypical carcinoid syndrome’ (generalized flushing, broncho­­constriction, hypotension, lacrimation and oedema formation). Correct diagnosis relies on analyses of the main histamine metabolite, methylimidazole acetic acid, in urine and demon- stration of histamine, its synthesizing enzyme histidine decarboxylase or the vesicular monoamine transporter VMAT1 in tumour biopsies (3) (fig. 1). These patients have a decarboxylation deficit, i.e. they seldom have excess urinary excretion of the serotonin metabolite 5-HIAA. They are optimally pretreated with somatostatin analogs combined with a blockade of histamine (H1 and H2) receptors and cortisone. Histamine-releasing agents, e.g. morphine and tubocurarine, should be avoided (4).

Locoregional Disease Adequate treatment of locoregional disease is a prerequisite for subsequent curative liver surgery. There is a correlation between the

size of the primary tumour and the presence of metastases. In case of small-bowel carcinoids, tumours smaller than 1 cm present with lymph node metastases in 44%, 1- to 2-cm tumours in 77% and those larger than 2 cm in 85%, so second-look surgery is recommended for the latter two types (5). The tumours are often accompanied by retroperitoneal fibrosis or desmoplastic mesenteric reaction, which may lead to constriction of the bowel and adjacent structures and intestinal ischaemia. The primary surgical treatment should therefore include dissection and removal, or reduction, of the central mesenteric lymph nodes to mobilize the bowel for adequate resection and to prevent compression of the mesenteric vein (6).

Advanced Disease The primary surgical treatment of locoregional disease is not controversial, while the treatment of liver metastases is more demanding and may involve several interventional modalities.

There are only few randomized studies on medical treatment of advanced disease (7). For December 2014

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International article

Interventional treatment of the carcinoid syndrome

Table 1. Liver resection rates over two decades Galland & Blumgart 1986

n = 30

7%

McEntee et al. (Mayo 1970–1989)

n = 425

9% (1/2 curative)

Wängberg et al. (Göteborg 1985–1992) MC

n = 64

22% (all curative)

Carty et al. (NIH 1982–1991) EPT

n = 42

40% (2/3 curative)

Dousset et al. (Paris 1986–1994)

n = 34

50% (2/3 curative)

Norton et al. (San Francisco 1995–2003)

n = 16

50% (all curative)

Low mortality (! 5%) and complication rate (X 30%)

correct evaluation of such studies, a similar type of primary surgery is required, e.g. high mesenteric lymph node dissection around the mesenteric vessels. After surgery, correct staging of residual tumours is necessary. In case liver transplantation is planned as a second step, all extrahepatic tumours must be resected.

There is no general agreement when to start palliative interventions. In the Swedish MRC study on patients with the midgut carcinoid syndrome (bilobar liver disease), all patients had a similar type of primary surgery followed by embolization of each hepatic artery in two sessions. This treatment was initiated when the patients were symptomatic and had 5-HIAA levels exceeding 250 µmol/24 h (reference <50). Stratification of more severe disease was done and defined as patients with 5-HIAA levels exceeding 500 µmol and/or cardiac valvular disease. After completion of the interventional treatment, the patients were randomized to medical treatment with octreotide, alone or in combination with interferon-α, with no survival differences (7).

Interventional Treatment Modalities Liver Resection The type of resective liver surgery is delineated by the three principal hepatic veins (fig. 2). Left hepatectomy is the removal of segments II + III, right hepatectomy the removal of segments V–VIII and trisegmentectomy the removal of the three central segments (sometimes also

Figure 2.

Surgical anatomy of the liver

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including the caudate lobe segment I). Resection of indi- vidual segments requires identification and ligation of each portal pedicle, which can be difficult for the upper segments VII–VIII with impaired venous drainage from the residual lower segments. In some cases, extended right lobe resections, leaving only the two left segments, can be performed. A residual liver smaller than 20–25% of the original volume may lead to hepatic failure. In these cases, a portal embolization of the right tumour-bearing liver lobe can be performed, which leads to deviation of the portal blood flow to the intact left liver and subsequent hypertrophy over 4–6 weeks.

The planned resection can thereafter be safely performed (8).

So-called debulking, or cytoreductive, liver surgery can be performed according to similar principles in cases with slow tumour growth and severe hormonal symptoms. Palliative liver resections are generally performed in patients in whom more than 90% of the tumour volume can be safely excised (9, 10). The role of debulking surgery for enhanced survival has not yet been proven in randomized studies.

In selected series, there seemed to be a distinct 5-year survival advantage in those with combined chemotherapy and debulking vs. debulking alone (11). In a series from the Mayo Clinic, 74 patients with metastatic NE tumours were treated with liver resection (41 midgut carcinoids). Even though two thirds had palliative resection, the 5-year survival was about 70% with no difference between palliative and curative procedures (12).

In clinical series of patients with gastrointestinal NE tumours some 20 years ago, the liver resection rate with curative intent was low, about 10% (table 1). This was partly ascribed to diffuse spread in the liver at clinical presentation. In our own consecutive series of patients with the midgut carcinoid syndrome, 22% underwent potentially curative liver surgery. In the nineties, two series reported a resection rate of 40–50%; two thirds were

H. Ahlman, O. Nilsson, M. Olausson

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International article per- formed with intention to cure. Recently, in a consecutive series of 16 patients, curative resection has been done in half of the patients (13) . In all these series, the mortality rate was low (< 5%) and the complication rate below 30%.

Even with so-called curative resections, the tumour may recur. It is therefore important to have these patients under surveillance using tumour markers (chromogranin A in plasma and urinary 5-HIAA) in combination with octreotide scintigraphy + SPECT and radiological methods (spiral CT and MRT) in order to early detect subclinical disease and perform repeat surgery.

This strategy has been successful in patients with NE tumours treated by liver transplantation; in our experience, scintigraphy was superior to biochemical markers (14).

Vascular Interventions Markowitz (15) first proposed hepatic vascular inter- ventions as therapeutic procedures. The rationale for isch- aemic treatment of carcinoid tumours is their main blood and oxygen supply from the hepatic artery with maintained portal perfusion of the normal liver parenchyma.

Ischaemia can be established by several techniques directed against the hepatic artery: ligation, selective embolization, or temporary occlusion. The methods differ in completeness, distribution and duration of ischaemia (16).

Ligation has largely been abandoned; it is difficult to obtain adequate ischaemia due to rich collateral blood supply. The palliative effects are short-lasting and there is significant mortality in patients with advanced disease. The possibility for repeat vascular interventions is also restricted. Selective embolization causes complete, but temporary, ischaemia distal to the point of injection, where the arterial tree is filled with embolization material (gel foam powder or alcohol-Ivalon particles). The contraindications are tumour burden exceeding 50% of the liver volume, portal vein occlusion and hyperbilirubinaemia.

Relative contraindications are contrast allergy, coagulopathies, persistent elevation of liver enzymes, extrahepatic tumour dominance and poor performance status (16). In our experience, also patients with a large tumour volume can undergo embolization with superselective embolizations, well separated in time (2–4 weeks); this can also be done in patients with previous hemihepatectomy and recurrence in the residual liver.

Immediately prior to embolization, an arteriogram is performed to demonstrate arterial anatomy, tumour blood flow and patency of the portal vein. Upon reembolization, permanent occlusion in the proximal arterial can be achieved by steel coils.

Embolization is accompanied by pain, elevated transaminases, nausea and a late fever reaction (24–48 h). Serious complications may occur

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Interventional treatment of the carcinoid syndrome

in individual patients (gallbladder necrosis, pancreatitis, liver abscess, vascular damage, hepatorenal syndrome and carcinoid crises). In the major series, the mortality was < 5%, which serves as a quality standard (16). In our first series with 94 embolizations of 40 patients, the first 11 were done with conventional angiography catheters with complications like arterial occlusion (n = 3), hepatic arterial aneurysm (n = 1), hepatic abscess (n = 1), pancreatitis (n = 1) and fatal hepatorenal syndrome (n = 1).

After changing to a coaxial catheter system, 83 consecutive embolizations were per- formed with only 2 minor complications. In our second series of similar size, the complication rate has remained at this low level. To minimize adverse reactions, several precautions can be undertaken: experienced interventional radiologists, coaxial catheter system to reduce the risk of vascular damage, prophylactic use of somatostatin analogs, cholecystectomy at the time of primary surgery, intravenous hydration and haemodynamic monitoring to reduce the risk for hepatorenal syndrome (17).

Chemoembolization is embolization combined with intra-arterial administration of chemotherapy. Pro- nounced tumour regression has been reported with a modified procedure using iodized oil, doxorubicin and gelfoam particles (18).

The potential advantage of chemoembolization vs. bland embolization in advanced disease is currently studied. Radioembolization, i.e. microspheres loaded with ß-emitter, has been attempted. Several unsolved problems remain: arteriovenous shunting to the lung, exact dosimetry, non-homogenous distribution of microspheres and redistribution after radiation damage (19). Some centres have used temporary occlusion of the hepatic artery via external vessel loops (positioned during surgery) or via a port-operated tourniquet. A combination of peripheral embolization and temporary proximal occlusion can have additive ischaemic effects.

The results of ischaemic treatment depend on the stage of the disease. In the early series (n = 63) of Coupe et al. (20) in patients with advanced disease, half of the patients were embolized without survival advantage, while others have reported prolongation of expected survival with a few years (21).

A survival advantage for embolized pa­ tients was indicated in one small randomized study using surgery and interferon as common treatment (22). A non-randomized large patient series indicated that chemotherapy subsequent to embolization en­h anced the effectiveness of ischaemic treatment in patients with advanced endocrine pancreatic (EPT)/carcinoid tumours (23) .

In our initial series of 64 consecutive patients with the midgut carcinoid syndrome, we used

H. Ahlman, O. Nilsson, M. Olausson

Table 2. Treatment algorithm for the midgut carcinoid syndrome

the treatment algorithm shown in table 2: 14 patients had curative liver resection (5-year survival 100%), 40 had bilobar liver disease and underwent embolizations (5-year survival 63%) and 10 had other conditions (cirrhosis, synchronous malignancies, psychiatric disorders) and were only treated medically after primary surgery (5-year survival 0%).

The total survival was 69% at 5 years and seemed to stabilize around 60% at 10 years (6).

This figure compares favourably with previous series with 5-year survival rates between 19 and 40% (22â&#x20AC;&#x201C;27).

A 5-year survival between 40 and 60% was reached in the Swedish MRC multicentre trial on adjuvant medical treatment after embolization therapy (7). In our initial series (6), the embolized patients could be divided into two equally large groups based on radiological and biochemical responses; radiological responders (1 50% tumour reduction) had a pronounced reduction of 5-HIAA excretion (80%) and non-responders (<50% tumour reduction) had a moderate reduction of 5-HIAA levels (30%) with distinct survival advantage for responders over 5 years.

The M.D. Anderson experience (1992â&#x20AC;&#x201C;2000) with embolization (n = 50), or chemoembolization (n = 31), in patients with metastatic carcinoids has recently been reported (28). The radiological response could be assessed in a majority of patients; two thirds had major tumour reduction (650%) and palliation of hormonal symptoms.

The survival probability was 93% at 1 year, 62%

at 2 years and 24% at 5 years. The progressionfree survival was especially encouraging, since these patients had advanced disease, which was progressive during medical treatment.

Local Ablation In elderly patients, or patients with progressive tumours after previous treatment, percutaneous alcohol injections into isolated liver lesions have been attempted to destroy tumour tissue. Tumour volume is estimated ultrasonographically and the lesions injected with equal volumes of absolute alcohol. The tumours develop high echogenicity during injection (29). The tumours can also be destroyed by cryosurgical techniques or by interstitial laser. Cryo- probes can be equipped with intraoperative ultrasound and can destroy central liver tumours without damage to overlying normal parenchyma (30). Today the most com- monly used technique is radiofrequency ablation, which causes selective thermocoagulation of lesions up to 3â&#x20AC;&#x201C;4 cm in diameter. It can be used during surgery or percutaneously (31). Lesions adjacent to vessels can usually be treated, since the vessels are protected by cooling due to blood stream effects. Local ablative techniques are valuable complements to liver resection for treatment of localized tumours in the residual liver or for recurrent tumours.

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Interventional treatment of the carcinoid syndrome

Figure 3. Octreotide scintigraphy

and tumour markers in a patient with recurrent midgut carcinoid after liver transplantation, who was treated with 4 radiotherapies via somatostatin receptors.

Three years after radiotherapy, the patient has marked tumour regression, almost normal tumour markers and is symptom free (Cg = chromogranin)

hepatic arterial embolization is potentially dangerous with the risk for massive release of histamine. In individual cases, after adequate pharmacological blockade, we have delivered cytotoxic drugs (melphalan + cisplatin) to the liver by hyperthermic perfusion. During perfusion, the venous effluent from the liver (containing histamine) is shunted from the systemic circulation avoiding adverse effects.

The cytostatic perfusion of the isolated liver is made possible by a special perfusion catheter inserted in the caval vein.

The technique involves isolation of the hepatic artery, portal vein and caval vein together with a temporary portocaval shunt (allowing maintained blood flow rates in both the arterial and portal systems). Repeat perfusion can be difficult to perform due to intense fibrotic reaction of the vessels (32).

Liver Transplantation Transplantation may be considered in selected patients with NE tumours, if the tumours are not accessible for curative or cytoreductive surgery, are associated with lifethreatening hormone secretion (histamine), or are resistant to treatment given (14). Ten years ago, Bechstein and Neuhaus (33) reviewed the world literature on liver transplantation and NE tumours: the 1-year survival was only 52% with transplantation-associated death in half of the patients within 8 months.

Later the 5-year survival from Hannover was reported to be 80% (34) and similar figures were reported for carcinoid patients (lower for EPT patients) in a French multicentre study (35).

Relatively few multivisceral transplantations have been performed in patients with NE tumours, but seem to render a 5-year survival of about 60% (36). In a review of the literature 5 years

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ago, Lehnert (37) reported the first 103 patients transplanted for metastatic NE tumours. In this heterogenous survey, the 5-year survival was 47% and seemed to be favourably influenced by young age, the carcinoid phenotype and less extensive upper abdominal surgery prior to transplantation. Over 6 years, we have performed 14 transplantations for NE tumour disease. Also patients up to 60 years and those with a large tumour burden were included in our series. Nine patients with carcinoids, or taillocated EPT, first had histology-proven radical removal of the primary followed by careful work-up to exclude extrahepatic lesions prior to transplantation.

Patients with head-located EPT were treated with multivisceral transplantation . All our patients accepted for transplantation had WDEC tumours of relatively low proliferation (Ki67 <10%); 9 EPT and 5 carcinoid patients (14) . Two multivisceral transplantation patients died 4 months after surgery of posttransplant lymphoprolifera- tive disease and arteritis, respectively.

Only 1 patient with liver transplantation has died of tumour recurrence. A total of 3 patients developed recurrence long time after surgery; 2 could be resected again due to early diagnosis with octreotide scintigraphy/spiral CT and 1 was treated by 177Lu-DOTA-Tyr3-octreotate (7â&#x20AC;&#x201C;7.5 GBq i.v. x 4) in Rotterdam (fig. 3) and has now been observed for more than 3 years after radiotherapy.

This patient had a very long interval between primary surgery and liver transplantation (almost 10 years). Nine months after liver transplantation, he developed symptoms, elevated tumour markers and multiple lymph node metastases. He is currently symptomfree with marked tumour regression and

H. Ahlman, O. Nilsson, M. Olausson

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International article almost normal markers. We conclude that transplantation for metastatic WDEC tumours offers good relief of hormonal symptoms and long disease-free intervals. The initial survival of grafts and patients is comparable with liver transplantation for benign disease. The experience with multivisceral transplantation is still too limited. The possibility of completion radiotherapy via somatostatin receptors in case of recurrent and non-resectable tumours is most attractive.

Interventional treatment of the carcinoid syndrome

Acknowledgment This study was supported by grants from the Swedish RC, the Swedish Cancer Society and the Assar Gabrielsson Foundation.

This work is licensed under a Creative Commons Attribution 4 .0 Unported License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc/4.0/

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Immunotherapy resistance mechanisms in renal cell cancer

IMMUNOTHERAPY RESISTANCE MECHANISMS IN RENAL CELL CANCER Katarzyna Kaminska1, Gabriel Wcislo1, Anna M. Czarnecka1, Salem Chouaib2, Cezary Szczylik1 1. Department of Oncology with Laboratory of Molecular Oncology, Military Institute of Medicine, Warsaw, Poland; 2. Institut National de la Santé et de la Recherche Médicale (INSERM U753), Institut Gustave Roussy, Villejuif, France Corresponding author: Katarzyna Kaminska Laboratory of Molecular Oncology, Military Institute of Medicine, Warsaw, Szaserow 128, 04-141 Warsaw, Poland; Tel: +48 - 22 - 681 71 72; Fax: +48 - 22 - 610 30 98; E-mail: kkaminska@wim.mil.pl

Open Access Article

Abstract Keywords:

First published in: Current Signal Transduction Therapy, Vol. 8, No. 3 251-252 ©2013 Bentham Science Publishers

Renal cell carcinoma, immunotherapy resistance, renal cell cancer chemotherapy, renal cell cancer radiotherapy, IL-2 induced immunotherapy, IFN induced immunotherapy, TKI induced immunotherapy

Received: December 03, 2013 Revised: January 22, 2014

The successful treatment of renal cancer remains a therapeutic challenge. Clear Cell Renal Cell Carcinoma (ccRCC) is resistant to conventional radio and chemotherapy, but complete response has been observed after immunotherapy with high-dose interleukin-2 (IL-2) and interferon (IFN)-α. Nevertheless, immunotherapy strategies have shown response rates in the range of 5 to 10%. For the past 20 years, the mechanisms of treatment resistance have been studied, and immune escape of tumours in cancer development and spread has been a broadly investigated phenomenon. Multiple studies have revealed that genomic abnormalities of ccRCC promote the loss of major histocompatibility complex (MHC) molecules on the renal cancer cell surface, resulting in immune response resistance. Studies have shown that IFN-α-induced signalling pathways are deregulated in ccRCC cells and promote immune escape. Polymorphisms of multiple genes, including STAT3, have been shown to trigger immune-response deregulation. Investigation and understanding of the mechanisms of renal cell cancer immunotherapy resistance are extremely important for the design of rational combinatorial approaches and other novel therapies in the future. This mini-review focuses on immunotherapy resistance mechanisms in ccRCC.

Accepted: January 29, 2014

Renal cell carcinoma

Cite this article: Katarzyna Kaminska, Gabriel Wcislo, Anna M. Czarnecka, Salem Chouaib, Cezary Szczylik.

Immunotherapy resistance mechanisms in renal cell cancer. Rom J Oncol Hematol. 2014; 5(4):212-222

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The Renal Cell Carcinoma (RCC) is considered the most common urological tumour and accounts for almost 3% of human malignancies (1).

Several histological subtypes of this heterogeneous entity are associated with different molecular aberrations and clinical outcomes (2,3).

Clear-cell renal cell carcinoma (ccRCC) is the predominant subtype of RCC and affects up to 60,000 patients annually in the US; mortality was 13,500 in 2012 (Atlanta, GA: Cancer facts and

figures 2011; American Cancer Society 2012.).

Approximately 65,000 new cases of RCC were diagnosed, and more than 2500 deaths were reported in the European Union in 2012 (4). Within Europe, the highest incidence of RCC has been reported in the eastern region, primarily in the Czech Republic and Poland. The causes of regional differences in morbidity are not known (4) . CcRCC is characterized by high metastatic potential, with a prolonged asymptomatic course.

Metastatic disease develops in almost 50% of pa-

Katarzyna Kaminska, Gabriel Wcislo, Anna M. Czarnecka, Salem Chouaib, Cezary Szczylik

Table 1. Molecular mechanisms of tumour cells evading the immune recognition CTL T-cytotoxic lymphocytes; NK– natural killer cells Mechanisms

Ref.

Reduction or absence of the expression of major histocompatibility complex classes I and II

(18,19)

Loss of tumour antigens

(20,21)

Impairment of proteasome function or ATP-dependent transporter proteins TAP peptide

(22)

Lack of co-stimulatory signals, such as B7 or CD40 molecules, on the surface of tumour cells

(23,24)

Aberration in expression of adhesion molecule

(103)

Impaired expression of Fas receptor and/or Fas ligand, leading to apoptosis of T - cytotoxic lymphocytes (CTL) and/ or natural killer cells (NK)

(25-27)

Synthesis and secretion of immunosuppressive agents

(28,29)

Expression of TRIAL on tumour cells that leads to apoptosis of T lymphocytes

(30)

Clonal exhaustion of T cells or activation induced cell death

(31)

tients within five years of primary diagnosis (5). In 25–30% of patients with ccRCC, metastases are diagnosed synchronously with primary tumour.

Only 10% of patients diagnosed with metastatic ccRCC survive more than five years. In the population of patients with localized disease, up to 60% will survive five years (6,7).

Immune response resistance mechanisms Cancer immune surveillance is considered an important host-protection process to inhibit carcinogenesis and to maintain cellular homeostasis.

In the interaction of host and tumour cells, three essential phases have been proposed: elimination, equilibrium and escape (Burnet 1957). According to this theory, cancer cells develop more often than tumours. Cancer cells are constantly eliminated by the immune system. Innate immune responses, such as natural killer cells, can initially eliminate Nascent transformed cells. During tumour progression, though an adaptive immune response can be provoked by antigen-specific T cells, immune selection produces tumour cell variants that lose major histocompatibility complex class I and II antigens and decrease tumour antigens. Finally, tumour-derived soluble factors facilitate the escape from immune attack, allowing progression and metastasis (8). Even short-term disturbances in immune response may enable proliferating cancer cells to reach a high number.

Evidence for the existence of immune surveillance is supported by epidemiological studies, which have shown that patients with immuno-deficiencies develop lymphoblastic lymphoma and Kaposi’s sarcomas more often than the general popu-

lation (9,10). Further studies have revealed that the immunological deficiencies in patients result in a high rate of pathogen-related cancer development, including bacteria-induced tumours such as gastric cancer induced by Helicobacter pylori (11) ; virus-induced lymphoma by Epstein-Barr virus (12); Kaposi ‘s sarcoma, by human herpesvirus 8 (13); and cervical cancer, by HPV (14). Moreover, evidence for the immune system’s contribution to carcinogenesis is confirmed by the observations of immunocompromised transplant patients who develop multiple cancers (15,16).

Cancer immuno-editing theory states that all cancers in the early stages of development are immunogenic, causing the reaction of the immune system (17). Accumulating evidence indicating that tumours develop mechanisms allowing them to circumvent immuno-surveillance and escape immune system control is emerging (Table 1) (17). In the last two decades, a number of tumour “escape” mechanisms have been reported.

These include: 1. The reduction or absence of the expression of major histocompatibility complex classes I and II - (MHC I and II) (18, 19) 2. The loss of tumour antigen presentation (20, 21) 3. The incorrect processing of intracellular antigens (impairment of proteasome function or ATPdependent transporter proteins TAP peptide) (22) 4. The disruption of co-stimulatory signalling molecules, including B7 and CD40 receptors (23,24)

5. The impaired expression of the Fas receptor and/or Fas ligand on T-cytotoxic lymphocytes (CTL) Natural killer cell (NK) (25-27) December 2014

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International article 6. The synthesis and secretion of immunosuppressive agents, such as IL-10, TGF-β, PGE2, blocking the immune response (28,29) 7. The expression of TRIAL (TNF-related apoptosis-inducing ligand) on tumour cells, which leads to apoptosis of T lymphocytes (30) 8. The chronic stimulation of specific T cells leading to clonal exhaustion or death (activationinduced cell death – AICD) (31)

Renal cell cancer immunotherapy Poor efficacy of radio and chemotherapy in RCC has drawn attention to other treatment modalities such as immunotherapy. The first immunotherapy trial for renal cancer was recorded in 1914, when the New York surgeon WB Cooley published a study assessing the long-term effects of the application of a bacterial-toxin mixture to patients with renal tumours (Colley 1914). Immunotherapy utilizing IL-2 or IFN-α is now registered in RCC treatment in the US and EU (32-35). Two recombinant forms, rIFN-α-2a and rIFN-α-2b, are available for clinical use; Roferon (rIFN-α2a) is more immunogenic than Intron A (rIFN-α2b). Th1 cytokine production is induced by IFN-α, and promotes anti-tumour cytotoxicity (36). IL-2 is a growth/ differentiation factor inducing and maintaining the cytotoxicity of NK and T cells (37). Cytokine treatment, called nonspecific immunotherapy, induces nonspecific anti-tumour activity. IL-2 activates natural killer (NK) cells, and IFN-α promotes the maturation of antigen-presenting cells (APC).

IL-2 indirectly limits tumour escape mechanisms, such as defective tumour cell expression of Class I or II molecules or expansion of regulatory T cells.

Indirect effects of IL-2 on the tumour microenvironment are associated with T-cell infiltration during the global delayed-type hypersensitivity response. As a result, the use of IFN-α or IL-2 treatment regimens may results in a reduction of tumour burden by more than 50%. Unfortunately, the objective response rate (partial or complete) is in the range of 5 to 20% (38-41), and the median overall survival (OS) of responsive patients is about four months longer than nonresponders (Rini, Weinberg et al. 2004). Further subgroup analyses revealed that performance status (ECOG) is the most important predictor of IFN-α response (42). Moreover, the best outcomes were observed in patients with pulmonary or lymph-node metastases, while patients with liver or bone metastases showed a low percentage of objective response. During the IFN-α treatment, more that 30% of the patients developed antiIFN-α antibodies. The production of antibodies leads to neutralization of IFN and inhibits immune response and anti-tumour toxicity. The development of anti-IFN antibodies was associated with RCC relapse short response and survival (43,44).

Immunotherapies other than IFN were tested.

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Broad clinical studies demonstrated that IL-2 had a response rate of 15 to 30%, with approximately 5% complete responses; median survival was reported between 6 and 19 months (45 46).

However, use of high-dose IL-2 is limited by its toxicity, which can cause hypotension, cardiac arrhythmias, metabolic acidosis, renal failure, neurotoxicity and dermatologic complications. Between 1–5% mortality was observed (47). High-dose IL-2 may be administered in a facility that is prepared to provide blood pressure support, but treatment candidates have excellent organ function and a good or intermediate prognosis by Memorial Sloan-Kettering Cancer Center (MSKCC, New York, New York) criteria. Other trials have shown that patients receiving low dose IL-2 suffered significantly fewer side effects while maintaining the same OS. Although IL-2 was more clinically active at maximal doses, this did not produce an overall survival benefit (47, 48). Combined therapy of IL-2 and IFN-α exhibited longer median recurrencefree survival compared to single-agent treatment, but overall survival did not differ between treatment arms (46). All reported data confirms that both IL-2 and IFN-α are active agents in ccRCC, and that immunotherapy is still an option in RCC treatment (39,49).

To increase the efficacy of the immunotherapy, multiple agents were tested, including tumourinitiating lymphocytes, IL-4, IL-1β, IFN-γ, and lymphokine-activated killer cells that were coadministrated to enhance IL-2 therapeutic effect.

Trials did not prove clinical benefits of synergistic usage of other immune-stimulants in RCC treatment (46). IFN-α was administrated with IFN-β and chemotherapy (i.e., 5-Fu), but this did not benefit patients (42). Only reduced-dose monoclonal antibodies against VEGF (bevacizumab) increased IFN efficacy in RCC patients (50).

Recent insight into tumour-host interactions has also prompted new immunotherapeutic strategies for RCC, including the investigation of vaccinations in ccRCC. In particular, the TroVax vaccine targeting the 5T4 antigen, a protein found on 85% of solid tumours, was expected to boost the immune response (51). Researchers reported that this treatment resulted in longer PFS but no difference in OS (51). More recently, the importance of immune regulation by APC and regulatory Treg cells in ccRCC has been investigated. Primarily in mice models, researchers demonstrated that depletion of Tregs using anti-CD25 antibodies evoked effective anti-tumour immunity (52). In RCC patients, elimination of Tregs followed by vaccination with tumour RNA-transfected dendritic cells (DCs) led to improved stimulation of tumour-specific T cells (53).

The most advanced agent in RCC immunotherapy is nivolumab, which acts as an immuno-modulatory ligand, blocking activation of the receptor PD-L1. This therapy was developed due to high

Katarzyna Kaminska, Gabriel Wcislo, Anna M. Czarnecka, Salem Chouaib, Cezary Szczylik

levels of RCC cell production of the immunosuppressive-programmed death-ligand 1 (PD-L1).

This is a ligand for the T-cell checkpoint molecule PD-1 that inhibits activated T cells and down-regulates the anti-tumour response.

Mechanisms of immunotherapy resistance in ccRCC The limitations in immunotherapy efficacy are a result of multiple intrinsic and therapy-inducted resistance mechanisms. Wolf et al. (54) observed statistically significant enhanced gene expression of pathway B-cell receptor (BCR), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and T-cell receptor in ccRCC patients when compared to a healthy donor. All these pathways are wellknown inhibitors of immune response involved in the modulation of immune system functions with CTLA-4 (55). Observed discrepancies suggested a primordial defect of the immune system in patients who develop RCC and immunotherapy resistance. Down-regulation or lack of expression in MHC class I and II molecules is believed to be a predominant tumour-specific cell-mediated immune effector mechanism, which determines immune escape in ccRCC (18,19). Studies based on RCC tumour explants have revealed low expression of HLA class I on the RCC cell, which may represent an immune escape mechanism of RCC (56). Studies based on an in vitro RCC model revealed that CIITA (human gene encoding a protein called the class II major histocompatibility complex transactivator) transfection significantly delayed tumour growth when injected into BALB/c mice (57). However, compared to other investigated cancer cell lines, the renal cancer cell line is characterized by low stability of CIITA expression. Furthermore, ex vivo-isolated cells from CIITA tumour-bearing mice displayed retarded tumour growth, and showed an almost complete loss of MHC-II expression. Although CIITA-RENCA tumour cells (transfected renal cell line) could not be fully rejected, they grew in vivo with significantly reduced kinetics compared to parental cells (not transfected with CIITA) (57).

Kren et al. (58) evaluated the correlation between HLA-G and HLA-E expression and the prognostic significance in RCC patients. Authors demonstrated that up-regulation of HLA-G is connected with a worse prognosis, and increased expression of HLA-E carried a better prognosis (58) . The majority of RCC-infiltrating myeloid cells co-express MHC class II molecule (59) and variable levels of PD-L1, which play a major role in suppressing the immune system. RCC- infiltrating macrophages mediate immunosuppression also due to up-regulation of 15-lipoxygenase-2 (15-LOX2) (59). Inhibition of 15-LOX2 decreased the synthesis of the immunosuppressive cytokine IL-10 and monocyte chemoattractant protein

(CCL2), which provide enhanced recruitment of blood myelomonocytic cells. Targeting 15-LOX2 is a potentially valuable measure for limiting cancer-related inflammation mediated by CCL2, and for attenuating immuno-suppression mediated by tumour-associated macrophages (TAMs).

This measure thereby enhances anti-tumour immune response in patients with advanced RCC (59).

These findings underscore the relevance of IL10-producing TAMs to immunosuppression observed in patients with late-stage cancer. TAMs can promote the expression of forkhead box P3 (FOXP3) and cytotoxic T-lymphocyte associated antigen 4 (CTLA-4). CTLA-4 plays a key role in restraining the adaptive immune response of T cells toward a variety of antigens, including tumour-associated antigens (TAAs) (55) and FOXP3 function as a master regulator in the development and function of regulatory T cells (60). The direct and indirect influences of TAMs in promoting local immunosuppression and RCC tumour evasion has been demonstrated. The results suggest that the 15-LOX2/15(S)-HETE pathway is a key player in this mechanism. Hence, new therapeutic approaches should focus on the manipulation of the 15-LOX2 mediated in patients with advanced RCC (59).

Pre-incubation of peripheral blood mononuclear cells (PBMCs) with RCC cell line cells resulted in decreased cytotoxic potential of NK cells (61).

It was demonstrated that pre-incubated NK cells overexpress a CD94 surface marker, which recognizes MHC class I and further blocks cytotoxicity of NK cells (61). IL-15 has been identified as a key mediator in this mechanism. Researchers found that IL-15 enhances the cytotoxicity of NK cells both pre-incubated and co-cultured with RCC cells (61). The blocking of the CD94 receptor due to an increased level of IL-15 may enhance NK cytotoxicity in RCC patients and may augment the immune response against tumour cells (61). Inhibitors of DNA methyltransferase 1 may have clinical relevance for immune modulation by the augmentation of cytokine effects and/or expression of tumour-associated antigens, as demonstrated in an in vitro RCC model (62,63). Another surface marker, CD70, was proved to play an important role in immune escape in RCC (64). Co-culture of a T-cell line with RCC cells induced significant apoptosis of immune cells (64). The activation of the CD70 receptor on T cells resulted in apoptosis. Inhibition of the CD70 receptor in the presence of a recombinant soluble CD70 completely blocked this process (64). Since RCC cell lines express the CD70 ligand but not the CD70 receptor, and in co-culture RCC cells induce apoptosis in MOLT-4 T cells, it is highly possible that this mechanism is involved in complete ccRCC immune escape (64).

Further investigation revealed that other signalling pathways are involved in T-cell apoptosis.

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International article RCC cell lines secrete Fas ligand, which may protect tumour cells from immune-mediated elimination by Fas-expressing T cells, as FasL induces T-cell apoptosis (65). Blockage of the Fas pathway by anti-FasL Abs or by treating the T cells with an inhibitor of caspase-8, led to a 50% inhibition in T-cell apoptosis when incubated with RCC cells (65). FasL RCC cell lines also overexpress gangliosides, which can enhance apoptosis. Das et al. observed that the apoptosis of primary T cells occurs via a receptor-independent pathway by a mechanism that involves ganglio side-mediated initiation of the mitochondrial permeability (65). In summary, receptor-dependent and receptor-independent mechanisms act collectively to defeat the lymphocytes, which can promote the progressive growth of the RCC tumour (65).

Mechanisms of IFN-induced immunotherapy resistance in ccRCC Recent microarray analyses revealed that the observed immuno-resistance origins from transcription deregulation in renal cancer cells rather than is developed during immunotherapy (54).

No statistical differences were observed in gene expression of the peripheral blood lymphocyte population (PBL) collected from patients before and after treatment with IL-2, IFN-α and autologous dendritic cell (DC) tumour vaccination (54).

This result confirms that immunotherapy in metastatic ccRCC does not modulate the immune system through influence on lymphocytes to develop drug resistance. Modest changes were reported in levels of pro-inflammatory cytokines in peripheral blood, and it was hypothesized that the treatment influences cytokine secretion by cells other than immune cells. At the same time, responding patients showed up-regulation of the Hypoxiainducible factors α (HIFα) and class III receptor tyrosine kinase (FLT3) pathways. Cluster analysis of genes in these pathways allowed discrimination between “non-responder” and “responder” patients (66,67). Moreover, IFN-α promoted up-regulation of HLA-G surface expression on RCC cells (68) . HLA-G mediated suppression of T-cell and NK cell (69,70) HLA-G molecules, protected tumour cells from immune attack (68, 71) and reduced susceptibility to CTL, LAK and NK cell-mediated cytotoxicity (68, 71). Using an RCC in vitro model, researchers showed that IFN’s resistance has an unclear background, since the observed resistance is not due to structural aberrations of IFN-α receptor or failure to initiate the gene-inducing and anti-viral effect of IFN-α (72). Researchers investigated IFN-α biological action and determined that this agent is a therapeutic factor. Nevertheless, discrepancies in in vitro studies on RCC (73) cell lines and limited clinical benefit led to a question about the basis of observed heterogeneous effect of the same agent on the same type of cancer. One sugges-

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tion is that there are more pathways involved in apoptosis induced by IFN-α (73).

Up-regulation of the Fas pathway is involved in activation of the cell-death pathway in RCC cell lines (74), and interferons up-regulate Fas in RCC cell lines (75). Results obtained by Kelly et al. suggest that the IFN-α treatment Fas-dependent pathway as well as alternative is involved, resulting in apoptosis of RCC cells (73). Despite considerable efficacy, in vitro studies show that IFN-based treatment is not as successful. Recent evidence suggests that Fas receptor activation and apoptosis can be inhibited by Bcl-2 (76,77). Kelly et al. suggested that targeting Bcl-2 synergistically with IFN-α treatment might improve therapy. Studies from another groups revealed that the re-expression of the genes that are silenced due to methylation is needed to overcome the resistance (62,63).

The suppressor of cytokine signalling 3 (SOCS3), which negatively regulates the signal transducer and activator of transcription 3 (STAT3) (78) and IFN-α signalling (79), is another key factor in RCC resistance to IFN-α treatment (80). SOCS3 expression level was significantly increased after IFN-α stimulation and SOCS3 siRNA treatment resulted in IFN-α-mediated anti-proliferative effects in vitro and in vivo (80). The authors suggest that the silencing of SOCS3 expression is a possible strategy to restore sensitivity to IFN-α in resistant cells (80) . Other SNPs than the previously described SNP rs1905341 in STAT3-2 gene and low expression of SOCS3 could be responsible for the lack of response in RCC patients treated with IFN-α. Reconstitution of STAT2 expression in RCC cell lines restored the apoptotic effect of IFN-α (81). Shang et al. demonstrated that low expression of STAT1 is associated with the resistance of RCC to IFN-α, and the restoration of its expression may increase the susceptibility of RCC to IFN-α-based immunotherapy (82, 83).

Researchers demonstrated that STAT1, TRAILR1, IRF-7 and DAPK genes undergo epigenetic changes mediated by DNA methyltransferase 1 (DNMT1), which is essential for various actions of IFNs. Hypermethylation of these genes may confer growth advantage through resistance to endogenous IFNs (84-87). Epigenetic editing of gene expression by aberrant methylation of DNA helps RCC tumour cells escape attack from the innate and acquired immune systems. Resistance to anti-proliferative effects and apoptosis induction by IFNs was postulated to result from the silencing of IFN response genes by promoter hypermethylation; therefore, inhibitors of DNMT1 may have clinical relevance for immune modulation by augmentation of cytokine effects and/or expression of tumour-associated antigens (62). Rue et al. showed that the overexpression of XAF1 plays a significant role in apoptosis induction by IFNs (62).

Further analysis of cRNA array revealed another

Katarzyna Kaminska, Gabriel Wcislo, Anna M. Czarnecka, Salem Chouaib, Cezary Szczylik

Table 2. Molecular aberration resulting in RCC immune resistance or immune escape Gene

Factor

Abnormality in Resistance

Cell type

Abnormality/Resistance

CAIX

Catalyses the reversible hydration of carbon dioxide to carbonic acid

Up-regulation

Tumour cells

Better response on IL-2 based therapy

VHL

Tumour suppressor

Mutation

Tumour cells

IFN-α resistance

(91)

HLA-G

Immune auto-tolerance

Up-regulation after IFN-α treatment

Tumour cells

Protects tumour cells from immune attack; IFN-α resistance

(68,71)

HIF-1α; FLT3

HIF-1α: adaptation to low-oxygen conditions FLT3: cell survival, proliferation, and differentiation.

Up-regulation after immunotherapy

Peripheral blood lymphocytes

Better outcomes from IL-2; IFN-α treatment

(54)

BCR; CTLA4; TCR

Immune response

Up-regulation in RCC patients compared to healthy individuals

Peripheral blood lymphocytes

IL-2; IFN-α

(54)

CD94

Surface marker which recognizes MHC I

Up-regulation

Tumour cells

Blocking of CD90 increased cytotoxicity of NK cells

CD70

Immune activation

Constitutive expression

Tumour cells

Induction of CD70 receptor on T cells resulting in apoptosis

(64)

CD70

Immune activation

Constitutive expression

T cells

Immune escape

(64)

Fas ligand

Regulation of the immune system

Constitutive expression

Tumour cells

T-cell apoptosis

(65)

STAT3

Differentiation of the TH17 helper T cells, cell growth, apoptosis

Single nucleotide polymorphism

Tumour cells

Associated with better response to INF-α based therapy

(104)

SOCS3

Suppressor of cytokine signalling

Up-regulation

Tumour cells

IFN-α resistance

(80)

Down-regulation

Tumour cells

CIITA tumour-mediated CD4 T-cell priming is required for the generation of anti-tumour cytolytic T lymphocytes

(61)

Hypermethylation

Tumour cells

Inhibition of apoptosis induced by IFN

(62,63)

Down-regulation

Tumour cells

Immune escape

(56)

CIITA

Immune system functions

STAT1 – activation of IFN response; TRAILR1 – immuno-surveillance, IRF-7- regulator of IFNs, STAT1, XAF1 – apoptosis, TRAILR1, RASSF1A – cell proliferation, difIRF-7, XAF1, ferentiation, motility and apoptosis, RASSF1, DAPK DAPK –inhibition of cell adhesion/migration and promotion apoptosis HLA I

Control of the immune response

Ref.

(2,3)

CAIX carbonic anhydrase IX; VHL - von Hippel-Lindau; HLA-G - histocompatibility antigen, class I, G; HIF-1α - Hypoxiainducible factors α; FLT3 - class III receptor tyrosine kinase, BCR - B-cell receptor; TCR - T-cell receptor; CTLA-4 - cytotoxic T lymphocyte-associated antigen 4; CD94 - C-type lectin receptor; CD70 - tumour necrosis factor ligand; STAT1/3 - Suppressor of cytokine signalling 1/3; SOCS3 - cytokine signalling suppressor of cytokine signalling 3; CIITA - gene encoding an MHC class II transactivator, TRAILR1 – gene encoding tumour necrosis factor receptor superfamily member, IRF-7- virus-inducible cellular gene, XAF1 – gene encoding XIAP-associated factor 1, RASSF1 - gene encoding a protein associated to the RAS; DAPK - death-associated protein kinase gene; HLA I - histocompatibility antigen class I

19 genes with a possible effect on immune responses that were increased at least four-fold in DNMT1-depleted RCC cells (62). Studies revealed that the silencing of the RASSF1A gene abolishes the resistance to apoptosis induction by IFNs treatment (63). Other findings suggest that epigenetic changes play a crucial role in RCC treatment, allowing suppression to emerge as an important contributor to the development of clinical neoplasia. Finally, microarray studies of IFN-stimulated

gene expression patterns suggest that subtle differences in transcription profiles may contribute to differences in IFN responsiveness (88). The authors conclude that factors determining clinical response to IFN remain elusive, but resistance of RCC to IFN-alpha is associated with the lack of Jak1, Tyk2 and STAT1 expression and defective Jak-Stat activation, but not with deficiency of IFN receptors or suppressors of cytokine signalling induction (88).

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International article Mechanisms of IL-2-induced immunotherapy resistance in ccRCC In an IL-2 immunotherapy context, carbonic anhydrase IX (CAIX) has been identified as a potential response marker. High expression of CAIX in primary tumour has been correlated with response to IL-2, survival and pathologic risk categorization (2,3).

CAIX expression is mediated by the HIF-1 and is dependent on mutation in the von Hippel- Lindau (VHL) gene. Tumours with VHL mutation show higher CAIX expression than those without VHL mutations. VHL is mutated in almost in 80% of patients with RCC (4,89), and high CAIX expression has been identified in more than 85% of cases (4).

According to reports, CAIX high expression correlates with positive response to IL-2, therefore complete remissions should be observed in 85% of cases, not 5 to 10% (90). Nevertheless, it has been suggested that mutation of the VHL gene is involved in IFN-α resistance (91). As assessed by IFN-γ secretion, NK degranulation and cell lysis, the blocking of human leukocyte antigen (HLA)-Ispecific inhibitory NK receptor interactions pVHLtransfected cell lines produced a weaker activation of NK cells and substantially increased lysis of RCC-pVHL (91). The observed genetic aberration in RCC can predestinate this type of neoplastic change to be resistant to immunologic treatment.

Moreover, RCC with a papillary, no alveolar, type and/or more than 50% granular features responded poorly to IL-2, and should be considered for alternative treatments (10). Histological features are a consequence of genetic alterations. Table 2 summarizes known genes that may contribute to the phenomenon of resistance mechanisms in renal cancer. Their modification may have therapeutic significance in RCC immunotherapy.

Mechanismsof TKI-inducedimmunological resistance in ccRCC Targeted therapy agents affect neutrophil migration, T lymphocyte-dendritic cell cross talk, dendritic cell maturation, and immune cell metabolism and reactivity. The inhibiting vascular endothelial growth factor receptor VEGFR (sorafenib, sunitinib, pazopanib and axitinib) and the mammalian target of rapamycin (mTOR) (temsirolimus and everolimus), are particularly effective (92). Sorafenib, a multi-kinase inhibitor inter alia FLT3, is capable of modulating the immuno-biological activity of dendritic cells and a murine regulatory macrophage population (93,94) . Sorafenib (10 µm) inhibits proliferation of T cells and, at higher concentrations, T-cell apoptosis (95). The maximum concentration of sorafenib was more than 15 µm in the serum of patients, so the investigated concentrations are lower than those for clinical use (96). Further investigation showed that sorafenib (10 µm) significantly inhibits the macrophage viability (97). Moreover, sorafenib

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suppressed the CD80 expression and function of activated macrophages. Sorafenib-treated macrophages exhibited extensive development of cytoplasmic vacuoles, which suggests induction of autophagy (97). Thus, it should be kept in mind that a high dose of sorafenib leads to unrecoverable immunosuppression, and the dose-escalated strategy carries a risk for the long- term treatment of mRCC. The frightening conclusion from these findings is that sorafenib, and probably other TKI- based treatments, may induce immuno-resistance. Therefore, the blockade of one or few oncogenic pathways may lead to disease progression and increased tumour burden. Observed resistance may be the tumour’s attempt to overcome the blockade of the pathway that allows its development.

Sunitinib was not studied in such a context, but as these two tyrosine kinase inhibitors have similar modes of action, we hypothesized that the effect of sunitinib treatment could be similar.

Conclusion and future directions The immune system plays a significant role in the control of tumours. Growing knowledge has allowed the development of new immunotherapies and the use of multiple tumour antigens with the potential to induce a greater immune response.

IL-2 and interferon-α were the only effective therapies against ccRCC before the appearance of therapeutic options such as the novel agents that target the VEGFR and mTOR. IL-2 remains the only treatment capable of curing advanced RCC, albeit in few patients. Randomized phase II and III trials have proven that high-dose, intravenous bolus IL-2 elicits a superior response rate compared to regimens that involve either low-dose IL-2 and IFN-α, intermediate or low-dose IL-2 alone or lowdose IFN-α alone (98). Despite recent advances, there are still unmet needs among patients in the adjuvant setting, those with poor prognostic factors and those who have progressed on prior targeted therapies and/or immunotherapy. Improved understanding of host-tumour immune interactions has led to the recent development of novel immunotherapeutic agents, including various vaccines and antibodies against immune checkpoint proteins (PD-1 and CTLA-4) (99). Phase I and II trials of vaccination with allogeneic dendritic cell/tumour fusions in patients with metastatic ccRCC have also demonstrated immunological and clinical responses. T-cell modulating agents (e.g., PD-1 and CTLA-4 or soluble lymphocyte activation gene-3) and dendritic cell-activating tolllike receptor agonists have also shown encouraging evidence of efficacy.

All recent studies suggest that new immunotherapy may be an effective approach for patients with ccRCC. A number of new strategies are currently under investigation, including adoptive cell transfer (ACT) with T cells modified to target

Katarzyna Kaminska, Gabriel Wcislo, Anna M. Czarnecka, Salem Chouaib, Cezary Szczylik

proteins expressed by renal tumour cells such as MAGE- A3/12, DR4 and TRAIL. ACT with autologous natural killer cells is also under investigation (100). TAMs (tumour-associated macrophages) represent a promising and effective target for cancer therapy in RCC. Several strategies have been proposed to suppress TAMs recruitment, to deplete their number, to switch M2 TAMs into the anti-tumour M1 phenotype and to inhibit TAM-associated molecules. TAMs infiltration in RCC microenvironment contributes to cancer progression and metastasis by stimulating angiogenesis, tumour growth, cellular migration and invasion and epithelial-mesenchymal transition of RCC cancer cells in the development of tumour resistance to targeted agents (101). Advances in the understanding of the nature of tumour antigens and their optimal presentation, and progress in the understanding of regulatory mechanisms that govern the immune system, should provide multiple novel ccRCC immunotherapy intervention strategies with increased specificity and fewer side effects (102).

Conflict of interest The authors indicate no potential conflict of interest.

Acknowledgements CS, AMC and KK are supported by the Military Institute of Medicine statutory founding 1/1744 (101), National Science Centre projects 2011/01/B/NZ5/02822 and 2011/01/B/ NZ4/01602 and the Foundation for Polish Science TEAM project TEAM/2010-6/8. AMC is supported by the Ministry of Science and Higher Education “Juventus” grant CRU/WIM/275/2012. The authors acknowledge the support of the Scribendi, Inc. for professional editing and proofreading of this manuscript.

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