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

Editor-in-Chief Jan Cools (Leuven)

Deputy Editor Luca Malcovati (Pavia)

Managing Director Antonio Majocchi (Pavia)

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

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

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

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

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


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

Information for readers, authors and subscribers Haematologica (print edition, pISSN 0390-6078, eISSN 1592-8721) publishes peer-reviewed papers on all areas of experimental and clinical hematology. The journal is owned by a non-profit organization, the Ferrata Storti Foundation, and serves the scientific community following the recommendations of the World Association of Medical Editors (www.wame.org) and the International Committee of Medical Journal Editors (www.icmje.org). Haematologica publishes editorials, research articles, review articles, guideline articles and letters. Manuscripts should be prepared according to our guidelines (www.haematologica.org/information-for-authors), and the Uniform Requirements for Manuscripts Submitted to Biomedical Journals, prepared by the International Committee of Medical Journal Editors (www.icmje.org). Manuscripts should be submitted online at http://www.haematologica.org/. Conflict of interests. According to the International Committee of Medical Journal Editors (http://www.icmje.org/#conflicts), “Public trust in the peer review process and the credibility of published articles depend in part on how well conflict of interest is handled during writing, peer review, and editorial decision making”. The ad hoc journal’s policy is reported in detail online (www.haematologica.org/content/policies). Transfer of Copyright and Permission to Reproduce Parts of Published Papers. Authors will grant copyright of their articles to the Ferrata Storti Foundation. No formal permission will be required to reproduce parts (tables or illustrations) of published papers, provided the source is quoted appropriately and reproduction has no commercial intent. Reproductions with commercial intent will require written permission and payment of royalties. Detailed information about subscriptions is available online at www.haematologica.org. Haematologica is an open access journal. Access to the online journal is free. Use of the Haematologica App (available on the App Store and on Google Play) is free. For subscriptions to the printed issue of the journal, please contact: Haematologica Office, via Giuseppe Belli 4, 27100 Pavia, Italy (phone +39.0382.27129, fax +39.0382.394705, E-mail: info@haematologica.org). Rates of the International edition for the year 2016 are as following: Print edition

Institutional Euro 500

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Advertisements. Contact the Advertising Manager, Haematologica Office, via Giuseppe Belli 4, 27100 Pavia, Italy (phone +39.0382.27129, fax +39.0382.394705, e-mail: marketing@haematologica.org). Disclaimer. Whilst every effort is made by the publishers and the editorial board to see that no inaccurate or misleading data, opinion or statement appears in this journal, they wish to make it clear that the data and opinions appearing in the articles or advertisements herein are the responsibility of the contributor or advisor concerned. Accordingly, the publisher, the editorial board and their respective employees, officers and agents accept no liability whatsoever for the consequences of any inaccurate or misleading data, opinion or statement. Whilst all due care is taken to ensure that drug doses and other quantities are presented accurately, readers are advised that new methods and techniques involving drug usage, and described within this journal, should only be followed in conjunction with the drug manufacturer’s own published literature. Direttore responsabile: Prof. Edoardo Ascari; Autorizzazione del Tribunale di Pavia n. 63 del 5 marzo 1955. Printing: Tipografia PI-ME, via Vigentina 136, Pavia, Italy. Printed in May 2016.


haematologica calendar of events

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

21st Congress of the European Hematology Association European Hematology Association June 9-12, 2016 Copenhagen, Denmark

Anemia Diagnosis and Treatment in the Omics Era Chair: A Iolascon February 2-4, 2017 Barcelona, Spain

Hematology Tutorial on managing complications in patients with hematologic malignancies in the era of new drugs EHA-ROHS-RSH Chairs: E Parovichnikova, I Poddubnaya, R Foà July 1-3, 2016 Moscow, Russian Federation

EHA Hematology Tutorial on Lymphoid malignancies , Multiple myeloma and Bone Marrow Failure February 23-24, 2017 Colombo, Sri Lanka

Summer School of Personalised Medicine for Health Care Professionals European Alliance for Personalised Medicine (EAPM) July 4-7, 2016 Cascais, Portugal

EHA-SAH Hematology Tutorial on Thrombosis, Hemostasis & Myeloid Malignancies Chairs: R Foà, J Korin, G Kusminsky August 27-28, 2016 Buenos Aires, Argentina

EHA Scientific Conference on Bleeding Disorders Scientific Program Committee: C Balduini (Chair), A Falanga (Chair), F Rodeghiero, I Pabinger, M Makris September 14-17, 2016 Barcelona, Spain

2nd International Conference on New Concepts in B-Cell Malignancies European School of Haematology (ESH) Chairs: M Hallek, L Staudt, S Stilgenbauer, A Thomas-Tikhonenko September 9-11, 2016 Estoril, Portugal

12th Edition of the Educational Course of the EBMT Lymphoma Working Party on Treatment of Malignant Lymphoma: State-of-the Art and the Role of Stem Cell Transplantation European Group for Blood and Marrow Transplantation (EBMT) Chairs: S Montoto, P Dreger, A Sureda, E Vandenberghe September 21-23, 2016 Dublin, Ireland

10th Hodgkin Symposium University hospital of Cologne Chairs: A Engert, B von Treskow, B Böll October 22-25, 2016 Cologne, Germany

EHA Hematology Tutorial on EHA Hematology Tutorial on Lymphoid Malignancies March 17-18, 2017 Warsaw, Poland

Advances in Biology and Treatment of B Cell Malignancies, with a Focus on Rare Lymphoma Subtypes Chairs: MJ Kersten and M Dreyling March 10-12, 2017 Barcelona, Spain

Aging and Hematology Chair: D Bron May 4-6, 2017 Barcelona, Spain

22nd Congress of the European Hematology Association European Hematology Association June 22 - 25, 2017 Madrid, Spain

Challenges in the Diagnosis and Management of Myeloproliferative Neoplasms Chairs: JJ Kiladjian and C Harrison October 12-14, 2017 Location: TBC

Shaping the Future of Mesenchymal Stromal Cells Therapy Chair: W Fibbe November 23-24, 2017 Location: TBC

Calendar of Events updated on May 2, 2016


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

Table of Contents Volume 101, Issue 7: July 2016 Cover Figure Escaping detection by the immune system in Hodgkin lymphoma – accompanying the review article by Vardhana and Younes (Image created by www.somersault1824.com)

Editorials 789

Old and new faces of neutropenia in children Carlo Dufour, et al.

792

Patient-centered research and practice in the era of genomics: a novel approach Sam Salek, et al.

Review Articles 794

The immune microenvironment in Hodgkin lymphoma: T cells, B cells, and immune checkpoints - Leaders in Hematology review series Santosha Vardhana and Anas Younes

803

Management of Epstein-Barr Virus infections and post-transplant lymphoproliferative disorders in patients after allogeneic hematopoietic stem cell transplantation: Sixth European Conference on Infections in Leukemia (ECIL-6) guidelines Jan Styczynski, et al.

Articles Hematopoiesis

812

Human thrombopoiesis depends on Protein kinase Cδ/protein kinase Cε functional couple Cecilia Carubbi, et al.

Myeloproliferative Disorders

821

Ruxolitinib versus best available therapy in patients with polycythemia vera: 80-week follow-up from the RESPONSE trial Srdan Verstovsek, et al.

Chronic Myeloid Leukemia

830

Ultra-deep sequencing leads to earlier and more sensitive detection of the tyrosine kinase inhibitor resistance mutation T315I in chronic myeloid leukemia Constance Baer, et al

Acute Myeloid Leukemia

839

Salvage therapy with high-dose cytarabine and mitoxantrone in combination with all-trans retinoic acid and gemtuzumab ozogamicin in acute myeloid leukemia refractory to first induction therapy Marie-Luise Hütter-Krönke, et al.

846

Whole exome sequencing reveals a C-terminal germline variant in CEBPA-associated acute myeloid leukemia: 45-year follow up of a large family Anand Pathak, et al.

Haematologica 2016; vol. 101 no. 7 - July 2016 http://www.haematologica.org/


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

853

Whole exome sequencing in families at high risk for Hodgkin lymphoma: identification of a predisposing mutation in the KDR gene Melissa Rotunno, et al.

Non-Hodgkin Lymphoma

861

N-terminally truncated FOXP1 protein expression and alternate internal FOXP1 promoter usage in normal and malignant B cells Philip J. Brown, et al.

Plasma Cell Disorders

872

Analysis of renal impairment in MM-003, a phase III study of pomalidomide + low dose dexamethasone versus high-dose dexamethasone in refractory or relapsed and refractory multiple myeloma Katja C. Weisel, et al.

Stem Cell Transplantation

879

Clinical activity of azacitidine in patients who relapse after allogeneic stem cell transplantation for acute myeloid leukemia Charles Craddock, et al.

884

Unrelated alternative donor transplantation for severe acquired aplastic anemia: a study from the French Society of Bone Marrow Transplantation and Cell Therapies and the EBMT Severe Aplastic Anemia Working Party Raynier Devillier, et al.

Letters to the Editor Letters are available online only at www.haematologica.org/content/101/7.toc

e272

Tumor suppressors BTG1 and BTG2 regulate early mouse B-cell development Esther Tijchon, et al. http://www.haematologica.org/content/101/7/e272

e277

Histones stimulate von Willebrand factor release in vitro and in vivo Fong W. Lam, et al. http://www.haematologica.org/content/101/7/e277

e280

Are somatic mutations predictive of response to erythropoiesis stimulating agents in lower risk myelodysplastic syndromes? Olivier Kosmider, et al. http://www.haematologica.org/content/101/7/e280

e284

Relationship between event-free survival and overall survival in acute myeloid leukemia: a report from SWOG, HOVON/SAKK, and MRC/NCRI Megan Othus, et al. http://www.haematologica.org/content/101/7/e284

e287

A novel mechanism of NPM1 cytoplasmic localization in acute myeloid leukemia: the recurrent gene fusion NPM1–HAUS1 Paulo Vidal Campregher, et al. http://www.haematologica.org/content/101/7/e287

e291

The association of aberrant folylpolyglutamate synthetase splicing with ex vivo methotrexate resistance and clinical outcome in childhood acute lymphoblastic leukemia Anna Wojtuszkiewicz, et al. http://www.haematologica.org/content/101/7/e291

Haematologica 2016; vol. 101 no. 7 - July 2016 http://www.haematologica.org/


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

e295

Phase I study of single-agent CC-292, a highly selective Bruton’s tyrosine kinase inhibitor, in relapsed/refractory chronic lymphocytic leukemia Jennifer R. Brown, et al. http://www.haematologica.org/content/101/7/e295

e299

Additional trisomies amongst patients with chronic lymphocytic leukemia carrying trisomy 12: the accompanying chromosome makes a difference Panagiotis Baliakas, et al. http://www.haematologica.org/content/101/7/e299

e303

Associations between B-cell non-Hodgkin lymphoma and exposure, persistence and immune response to hepatitis B Geffen Kleinstern, et al. http://www.haematologica.org/content/101/7/e303

e307

Newly established myeloma-derived stromal cell line MSP-1 supports multiple myeloma proliferation, migration, and adhesion and induces drug resistance more than normal-derived stroma Pilar de la Puente, et al. http://www.haematologica.org/content/101/7/e307

e312

APRIL levels are associated with disease activity in human chronic graft-versus-host disease François Chasset, et al. http://www.haematologica.org/content/101/7/e312

e316

Multimodal intervention integrated into the clinical management of acute leukemia improves physical function and quality of life during consolidation chemotherapy: a randomized trial ‘PACE-AL’ Mary Jarden, et al. http://www.haematologica.org/content/101/7/e316

Haematologica 2016; vol. 101 no. 7 - July 2016 http://www.haematologica.org/


EDITORIALS Old and new faces of neutropenia in children Carlo Dufour,1 Maurizio Miano2 and Francesca Fioredda3 1

Haematology Unit. I.R.C.C.S. G. Gaslini Children's Hospital,Genova, Italy. The Scientific Working Group on Granulocytes and Monocyte disorders of the EHA; 2Haematology Unit. I.R.C.C.S. G. Gaslini Children's Hospital, Genova, Italy; and 3Haematology Unit. I.R.C.C.S. G. Gaslini Children's Hospital, Genova, Italy. The Scientific Working Group on Granulocytes and Monocyte disorders of the EHA

E-mail: carlodufour@gaslini.org doi:10.3324/haematol.2016.142760

I

n children, the term neutropenia usually identifies a group of inherited and acquired diseases characterized by a reduced number of mature circulating neutrophils and by an increased susceptibility to infections. Neutropenia is defined as severe when the absolute neutrophil count (ANC) is below 0.5x109/L.1 This article will provide an overview, with special focus on the most recent findings, on the two main forms occurring in children: severe congenital neutropenia (SCN) and primary autoimmune neutropenia (AIN).

Severe Congenital Neutropenia SCN encompasses a heterogeneous group of often inherited disorders appearing early in infancy with a variable clinical phenotype.2 In many cases neutropenia represents the sole disease, but sometimes it is associated to a multisystem involvement (neurological, endocrine, immune systems and other somatic districts).3 Mutations in different genes with various inheritance can cause SCN. In 1956 Rolf Kostman first described a cluster of neutropenia patients in a northern Swedish family in whom the disease was fatal within the first year of life because of infections. Later on this neutropenia was attributed to mutations of the HAX1 gene that is transmitted in an autosomal recessive fashion, and is often associated to neurological symptoms.4 The term Kostmann syndrome has sometimes been inappropriately used for neutropenia due to mutations of the ELANE gene, which is responsible for more than half of European and North American SCN patients.5 The ELANE gene encodes for neutrophil elastase. More than 120 distinct ELANE mutations, either transmitted in an autosomal dominant mode or sporadically, have been described so far.6 Some of them are shared in both cyclic and severe congenital neutropenia without a clear explanation of how a given genetic lesion may be associated to different phenotypes.6 Mutations in other genes like GFI1, WASP, G6PC3 and VPS45 are also the cause of SCN, although less frequently.7-9 JAG1 and TCIRG1 are the latest discovered genes resulting in isolated neutropenia.10-11 Mutations of COH (Cohen syndrome), BTK (X linked form), CD40L (Hyper IgM) and CXCR4 (WHIM syndrome) genes generate neutropenia in the context of immunodeficiencies. Recently, mutations of the CLBP gene were reported as a cause of SCN associated to cataracts, neurological impairment and increased urinary excretion of 3-Methylglutaconic acid (3-MGA) within the framework of the autosomal recessive metabolic disorder MEGCANN. The CLBP gene encodes for a mitochondria protein that is widely expressed in human tissues including granulocytes and- to a larger extent- the brain, and interacts with other proteins like HAX1 which has a critical role in the maintenance of mitochondria transmembrane potential, thus preventing excessive cell apoptosis.12 It is, however, worth noting that in spite of the untiring haematologica | 2016; 101(7)

research activity in the genetic neutropenia field, more than one third of SCN patients are still gene orphans thus far. In many cases the lack of neutrophil production is due to a marrow maturation block at the promyelocyte stage, as occurs in ELANE, HAX1, G6PC3, WAS and JAGN-1 gene mutations. In these cases, myeloid precursors beyond promyelocytes are not produced because of increased apoptosis13 occurring through different mechanisms like unfolded protein response (ELANE and G6PC3) or deranged mitochondria transmembrane potential (HAX1 and CLBP). Apoptosis may affect cells other than marrow myeloid precursors, like neurons, urinary tract cells, lymphocytes and natural killer cells, thus accounting for the multisystem phenotype observed in some forms of SCN (Kostman, glycogen storage disease 1b, GATA2 and MEGCANN diseases).14-15 In other circumstances the pathogenic mechanism resides in the lack of/scarce sensitivity to endogenous G-CSF due to the dysfunctionality of the extracellular portion of the G-CSF receptor (GCSF3R) or to the defective mobilization of bone marrow neutrophils (WHIM syndrome).16-17 The common denominators of the clinical phenotype of SCN are the infections and the risk of transformation into MDS/AML.18 After the introduction in the 1990’s of G-CSF in clinical practice, infections have become generally manageable.19-20 Conversely, the prolonged life duration achieved with G-CSF incremented the evolution towards MDS/AL whose cumulative incidence, according to the Severe Chronic Neutropenia International Registry (SCNIR) and the Severe Neutropenia French Registry (SNFR), is estimated at 22% and 10.8%, respectively, after 15 years from the start of G-CSF therapy. The risk of transformation has been correlated to the dose and the duration of G-CSF exposure, with amounts higher than 8 mg/kg/day being associated to increased risks.21-22 The neoplastic transformation is in part due to factors intrinsic to neutropenia and in part to pro-cancer elements acquired over time. Some neutropenia diseases are constitutively more prone to transformation. This is the case of Shwachman-Diamond syndrome, whose cumulative incidence of MDS/AL is 18–36% over a timespan of 20–30 years, according to the North American Shwachman-Diamond Syndrome Registry and the SNFR.22-23 Some specific ELANE mutations (i.e. G214R or C151Y) are more frequently associated to transformation.6 Another factor significantly associated with the development of MDS/AML in SCN is the acquisition of a truncating mutation of CSF3R genes that were found in 78% of SCN/AML cases, whereas they were present in only 34% of SCN non-leukemic patients.24 “Per se”,the presence of a CSF3R mutation does not automatically herald the advent of leukemia since distinct mutated clones may co-exist, and sometimes rise and disappear in a stochastic model.25-26 Additional “cooperative events” are required, throughout the 789


Editorials

course of transformation, after the first hit.27 In this respect a study of SCN patients on G-CSF who underwent leukemic transformation showed, in sequential analyses, that RUNX1 mutations appeared after CSF3R mutations in the transforming cells,28 thus pointing to this sequence of genetic events as a novel leukemogenic pathway in SCN. Other later events like monosomy 7, trisomy 8 and/or trisomy 21 may appear just before the MDS/AL onset and can be considered as a further event in the leukemogenic evolution.28 The mechanisms by which the genetic events contribute to clonal transformation have not yet been fully elucidated, but it is hypothesized that mutations of the external part of CSF3R induces a sustained activation of STAT5 that leads to ROS production with consequent intracellular DNAdamage.27 Other evoked mechanisms include the formation of RAS oncogene activating mutations and CSF3R gene mutations conferring proliferative advantage to transforming cells.29 Cyclic neutropenia (CyN), a disease characterized by oscillating neutrophil counts with a periodicity of 21-28 days,30 has long been considered a “relatively” benign disorder based on the absence of reports of clonal evolution in the international registries.21-22 Interestingly, a patient with CyN due to ELANE mutations, formerly detected in SCN but not in CyN subjects, was recently shown to evolve to AML. A CSF3R p.Gln741X mutation was found in leukemic cells and RUNX1 mutation p.Asp171Asn was present in the patient’s marrow cells.31 These findings suggest that CyN subjects may also undergo clonal evolution and that this may occur through a molecular mechanism similar to that seen in other “less benign” forms of SCN. These findings might potentially change the monitoring policy in CyN patients, who will have to be more tightly monitored for clonal escape than before. Overall, the above findings suggest a careful surveillance of the blood count and bone marrow of SCN patients with monitoring focused on CSF3R and RUNX1 mutations, particularly in those subjects treated with high cumulative doses of G-CSF. The Marrow Failure Study Group of the AIEOP (Associazione Italiana Emato-Oncologia Pediatrica) recommends an extensive bone marrow study with cytogenetics and G-CSFR mutation analysis every year, to be moved to every 6 months or earlier in the case that new mutations or abnormal clones appear.32 In patients poorly/non responding to G-CSF (i.e., requiring>10 mg/kg/day and > 20 mg/kg/day, respectively) or already transformed to MDS/AL, stem cell transplantation (SCT) is an indicated treatment option.33-34 The outcome of SCT in 136 SCN patients was analyzed in a collaborative study from EBMT and SCETIDE.34 The three year probability of OS and EFS were 82% and 71%, respectively. In multivariate analysis, factors associated to a better outcome were transplant before age 10 years from an HLA-matched, related or unrelated, donor. Late post-SCT tumors were not reported. Transplant related mortality was 17%, thus suggesting that this procedure should not be offered to patients who can be effectively and easily managed with standard doses of G-CSF (5 mg /kg/day or less).34 Research on new therapies are limited by the lack of ani790

mal models recapitulating the human severe neutropenia phenotype. Recently, induced pluripotent stem cell (iPSC) lines from SCN subjects have been generated and might become a helpful tool to screen possible new drugs.35

Autoimmune Neutropenia The most clinically relevant form of acquired neutropenia in children is primary autoimmune neutropenia (AIN), a disease typically appearing in early infancy due to the production of specific antibodies against human neutrophil antigens, that is regarded as due to the immaturity of the immune-suppression system allowing the autoantibody production as a consequence of a “surveillance escape event”. The clinical characteristics and outcome of AIN have been recently described in a large cohort of 157 patients.36 The median ANC and age at diagnosis were 0.45x109/L and 1.06 years, respectively, and the median time of resolution was 2.14 years. Severe infections occurred in 9.6% of cases, and recovery from neutropenia in about 89.9% within 5 years from diagnosis. No antibiotic prophylaxis was used, and G-CSF was administered in 7.1% of patients only during severe infections. In multivariate analysis, factors associated with a favorable outcome were early age at onset and lack of monocytosis. Although primary AIN generally appears as a substantially benign and self-limiting condition, some subcategories of patients, such as those with later onset or with disease persisting for many years or who develop involvement of further cell lineage need to be thoroughly assessed for secondary forms. Indeed, a multilineage immune-mediated cytopenia including neutropenia might be the manifestation of a wider autoimmune disorder or an epiphenomenon of an immune dysregulation,37 like autoimmune lymphoproliferative syndrome (ALPS) which is caused by defective FAS-mediated apoptosis signalling.38 Autoimmune cytopenias may also be secondary to common variable immune deficiency (CVID) or to “ALPS-like syndrome”, a clinical phenotype close to ALPS but with a still undefined genetic background.37 Of note, patients formerly diagnosed with one of the above diseases were recently found to have an abnormal T-cell hyperproliferation due to pathogenic mutations upregulating mTor (mammalian target of rapamycin),39 as in the case of the defective function of phosphoinositide 3kinase δ (PI3Kδ).40 Other mutations may either generate deficiency of the suppressing T-cell molecule CTLA-4,41 or impair its exposure on the cell membrane because of the deficiency of another anchor protein named LRBA (LPSResponsive beige-like anchor protein deficiency).42 These immune dysregulation diseases have a heterogeneous clinical phenotype which may include neutropenia. For these reasons children with atypical AIN, due to late onset, late recovery and involvement of additional cell lineage over time, require extensive immunological workup, including the evaluation of lymphocyte subsets with double negative T-cells and immunoglobulin serum level measurement. Clinical surveillance focused on organs which are potentially the target of immune dysregulation like the lungs, bowels and joints is also recommended. Finally, the identification of these non pure AIN patients has some important clinical implications. First, they have an increased risk of lymphomas, that in the case of ALPS is estimated to be 14- and 50-fold higher for non Hodgkin haematologica | 2016; 101(7)


Editorials

and Hodgkin disease, respectively, that requires close monitoring. Second, patients resistant to first-line therapies like steroids and intravenous immunoglobulins, may respond to immunosuppressive drugs such as MMF and sirolimus,43 normally not indicated in classical AIN. In conclusion, future efforts should concentrate on the definition of pathogenic and leukemogenic mechanisms and to further refine treatment to minimize infection and clonal evolution risks. Children with neutropenia should undergo a comprehensive diagnostic and monitoring program in specialized pediatric hematology centers.

References 1. Dinauer MC. The phagocyte system and disorders of granulopoiesis and granulocyte function. In: Nathan DG, OrkinSH,editors. Nathan and Oshi’s hematology of infancy and childhood. Philadelphia: WBS saunders Company; 2003. pp. 923-1010. 2. Welte K, Zeidler C, Dale DC. Severe congenital neutropenia. Semin Hematol. 2006;43(3):189-195. 3. Donadieu J, Beaupain B, Mahlaoui N, Bellanné-Chantelot C. Epidemiology of congenital neutropenia. Hematol Oncol Clin North Am. 2013;27(1):1-17. 4. Klein C, Grudzien M, Appaswamy G, et al. HAX1 deficiency causes autosomal recessive severe congenital neutropenia (Kostmann disease). Nat Genet. 2007;39(1):86-92. 5. Xia J, Bolyard AA, Rodger E, et al. Prevalence of mutations in ELANE, GFI1, HAX1, SBDS, WAS and G6PC3 in patients with severe congenital neutropenia. Br J Haematol. 2009;147(4):535-542. 6. Makaryan V, Zeidler C, Bolyard AA, et al. The diversity of mutations and clinical outcomes for ELANE-associated neutropenia. Curr Opin Hematol. 2015;22(1):3-11. 7. Person RE, Li FQ, Duan Z, et al. Mutations in proto-oncogene GFI1 cause human neutropenia and target ELA2. Nat Genet. 2003; 34(3):308312. 8. Boztug K, Appaswamy G, Ashikov A, et al. A syndrome with congenital neutropenia and mutations in G6PC3. N Engl J Med. 2009;360(1): 32-43. 9. Devriendt K, Kim AS, Mathijs G, et al Constitutively activating mutation in WASP causes X-linked severe congenital neutropenia. Nat Genet. 2001;27(3):313-317. 10. Makaryan V, Rosenthal EA, Bolyard AA, et al. TCIRG1-associated congenital neutropenia. Hum Mutat. 2014;35(7):824-827. 11. Boztug K ,Järvinen PM , Salzer E, et al. JAGN1 deficiency causes aberrant myeloid cell homeostasis and congenital neutropenia. Nat Genet. 2014;46(9):1021-1027. 12. Kiykim A, Garncarz W, Karakoc-Aydiner E, et al. Novel CLPB mutation in a patient with 3-methylglutaconic aciduria causing severe neurological involvement and congenital neutropenia. Clin Immunol. 2016;165:1-3. 13. Grenda DS, Murakami M, Ghatak J, et al. Mutations of the ELA2 gene found in patients with severe congenital neutropenia induce the unfolded protein response and cellular apoptosis. Blood. 2007;110(13): 4179-4187. 14. Donadieu J, Fenneteau O, Beaupain B, Mahlaoui N, Bellanné Chantelot C. Congenital neutropenia: diagnosis, molecular bases and patient management. Orphanet J Rare Dis. 2011;6:26. 15. Pasquet M, Bellanné-Chantelot C, Tavitian S, et al. High frequency of GATA2 mutations in patients with mild chronic neutropenia evolving to MonoMac syndrome, myelodysplasia, and acute myeloid leukemia. Blood. 2013;121(5):822-829. 16. Tricot A, Järvinen PM, Arostegui JI, et al. Inherited bi allelic CSFR3 mutations in Severe Congenital neutropenia. Blood. 2014;123(24): 3811-3817. 17. Hernandez PA, Gorlin RJ, Lukens JN, et al. Mutations in the chemokine receptor gene CXCR4 are associated with WHIM syndrome, a combined immunodeficiency disease. Nat Genet. 2003;34(1):70-74. 18. Dale DC, Cottle TE, Fier CJ, et al. Severe chronic neutropenia: treatment and follow-up of patients in the Severe Chronic Neutropenia International Registry. Am J Hematol. 2003;72(2):82-93. 19. Dale DC, Bonilla MA, Davis MW, et al. A randomized controlled phase III trial of recombinant human granulocyte colony-stimulating factor (filgrastim) for treatment of severe chronic neutropenia. Blood.

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1993;81(10):2496-2502. 20. Lehrnbecher T, Welte K. Haematopoietic growth factors in children with neutropenia. Br J Haematol. 2002;116(1):28-56. 21. Rosenberg PS, Zeidler C, Bolyard AA, et al. Stable long-term risk of leukaemia in patients with severe congenital neutropenia maintained on G-CSF therapy. Brit J Haematol. 2010;150(2):196-199. 22. Donadieu J, Leblanc T, Bader Meunier B, et al. Analysis of risk factors for myelodysplasias, leukemias and death from infection among patients with congenital neutropenia. Experience of the French Severe Chronic Neutropenia Study Group. Haematologica. 2005;90 (1):45-53. 23. Myers KC, Davies SM, Shimamura A. Clinical and Molecular Pathophysiology of Shwachman–Diamond Syndrome: An Update. Hematol Oncol Clin North Am. 2013;27(1):117-128. 24. Germeshausen M, Ballmaier M, Welte K. Incidence of CSF3R mutations in severe congenital neutropenia and relevance for leukemogenesis: Results of a long-term survey. Blood. 2007;109(1):93-99. 25. Link DC, Kunter G, Kasai Y, et al. Distinct patterns of mutations occurring in de novo AML versus AML arising in the setting of severe congenital neutropenia. Blood. 2007;110(5):1648-1655. 26. Kimmel M, Corey S. Stochastic Hypothesis of Transition from Inborn Neutropenia to AML: Interactions of Cell Population Dynamics and Population Genetics. Front Oncol. 2013;3:89. 27. Touw IP. Games of clones the genetic evolution of severe congenital neutropenia. Hematology Am Soc Hematol Educ Program. 2015; 2015:1-7. 28. Skokowa J, Steinemann D, Katsman-Kuipers JE. Cooperativity of RUNX1 and CSF3R mutations in severe congenital neutropenia: a unique pathway in myeloid leukemogenesis. Blood. 2014;123(14): 2229-2237. 29. Freedman MH, Alter BP. Risk of myelodysplastic syndrome and acute myeloid leukemia in congenital neutropenias. Semin Hematol. 2002;39(2):128-133. 30. Horwitz M, Benson KF, Person RE, Aprikyan AG, Dale DC. Mutations in ELA2, encoding neutrophil elastase, define a 21-day biological clock in cyclic haematopoiesis. Nat Genet. 1999;23(4):433–436. 31. Klimiankou M, Klimenokova O, Kanz L, Zeidler C, Welte K, Skokowa J. Time course of acquisition of a CSF3R Mutation and subsequent development of AML in a patient with cyclic Neutropenia. Blood. 2015;126(23):885 abstr. 32. Fioredda F, Calvillo M, Bonanomi S, Coliva T, Tucci F, Farruggia P. Congenital and acquired neutropenias consensus guidelines on therapy and follow-up in childhood from the Neutropenia Committee of the Marrow Failure Syndrome Group of the AIEOP (Associazione Italiana Emato-Oncologia Pediatrica). Am J Hematol. 2012;87(2):238-243. 33. Peffault de Latour R, Peters C, Gibson B, et al. Recommendations on hematopoietic stem cell transplantation for inherited bone marrow failure syndromes. Bone Marrow Transplant. 2015;50(9):1168-1172. 34. Fioredda F, Iacobelli S, van Biezen A, et al. Stem cell transplantation in severe congenital neutropenia: an analysis from the European Society for Blood and Marrow Transplantation. Blood. 2015;126(16):18851892. 35. Morishima T, Watanabe K, Niwa A, et al. Genetic correction of HAX1 in induced pluripotent stem cells from a patient with severe congenital neutropenia improves defective granulopoiesis. Haematologica. 2014;99(1):19-27. 36. Farruggia P, Fioredda F, Puccio G, et al. Autoimmune neutropenia of infancy: Data from the Italian neutropenia registry. Am J Hematol. 2015;90(12):E221-222. 37. Miano M. How I manage Evans Syndrome and AIHA cases in children. Br J Haematol. 2016;172(4):524-534. 38. Teachey DT, Manno CS, Axsom KM, et al. Unmasking Evans syndrome: T-cell phenotype and apoptotic response reveal autoimmune lymphoproliferative syndrome (ALPS). Blood. 2005;105(6):2443-2448. 39. Seidel MG. Autoimmune and other cytopenias in primary immunodeficiencies: pathomechanisms, novel differential diagnoses, and treatment. Blood. 2014;124(15):2337-2344. 40. Elgizouli M. Activating PI3k∂ mutation in a cohort of 669 patients with primary immunodeficiency. Clin Exp Immunol. 2016;183(2):221-229. 41. Kuehn HS, Ouyang W, Lo B, et al. Immune dysregulation in human subjects with heterozygous germline mutations in CTLA4. Science. 2014;345(6204):1623-1627. 42. Gámez-Díaz L, August D, Stepensky P, et al. The extended phenotype of LPS-responsive beige-like anchor protein (LRBA) deficiency. J Allergy Clin Immunol. 2016;137(1):223-230. 43. Miano M, Scalzone M, Perri K, et al. Mycophenolate mofetil and Sirolimus as second or further line treatment in children with chronic refractory Primitive or Secondary Autoimmune Cytopenias: a single centre experience. Br J Haematol. 2015 Jun 8. [Epub ahead of print].

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Editorials

Patient-centered research and practice in the era of genomics: a novel approach Sam Salek,1 Esther Oliva,2 and Tatyana Ionova3 The School of Life and Medical Sciences, University of Hertfordshire, Hatfield, UK; 2Hematology Unit, Azienda Ospedaliera, Reggio Calabria, Italy; and 3National Medical Surgical Centre and Multinational Center for Quality of Life Research, St Petersburg, Russia.

1

E-mail: mssalek@gmail.com doi:10.3324/haematol.2016.142844

I

n recent decades, therapeutic approach has been shifting from "clinician-centered", in which the clinicians assume responsibility as the sole competent person to look after patients’ interests and make decisions without the participation of the patients themselves, to “patientcentered”. Patient-centered care is defined as respectful of and responsive to individual patient preferences, needs and values, and ensures that patient values contribute to shared decision-making.1,2 The simple and reliable way to identify patient preferences, needs and values is to assess the patient’s quality of life and other patient-reported outcomes (PRO). The past decade has been characterized by increased attention to PRO assessment from the hematological community. A number of trials focusing on the assessment of quality of life, symptoms and other patient-reported outcomes in patients with hematological malignancies have been performed. Several instruments have been used to evaluate health-related quality of life and symptoms of patients with hematological malignancies, mostly in clinical trials.3 However, it cannot be established that the evaluation of PRO is widely accepted in the clinical practice of hematology. The assessment of the impact of illness on physical, mental and social functioning is an essential element of clinical diagnosis, a major determinant of therapeutic choices and efficacy, and a guide to longer-term care. The traditional approach to medical history taking and physical examination obtained by the clinician may not be sufficient for assessing the full range of health and/or treatment-related problems of patients with hematological malignancies. Clinicians vary widely in their ability to elicit relevant information from their patients, and patients vary in their ability to articulate their problems and concerns.4-7 Furthermore, hematologists frequently underestimate the patient’s level of psychosocial functioning, depression and the severity of important symptoms, while overestimating other aspects of the disease such as clinical parameters.8,9 It is clear that the formal diagnosis describes only the disease, and one cannot get any particular information on the patient’s individual characteristics from this formal diagnosis. It is known that the information on PROs received in clinical practice may influence various changes in intervention; the endpoints of individual quality of life measurement are not those associated with the evaluation of the efficacy of a single given treatment in a clinical trial. Thus, the implementation of PRO measures in routine clinical practice in patients with hematological malignancies is greatly needed and there is much demand from hematologists. However, there is no PRO measure that has been developed specially for use in routine hematological practice. The European Hematology Association Scientific Working Group for “Quality of Life and Symptoms” (EHA 792

SWG QoL & Symptoms) aims to facilitate patient-clinician communication through the development of a new instrument applicable in routine clinical practice. In this quest, The EHA SWG QoL & Symptoms has adopted a novel approach, placing the concept of “patient-centeredness” at the heart of such an initiative by involving a patient with a hematological malignancy to join the core research team as a “patient research partner”. Patient Engagement (PE), or Patient and Public Involvement (PPI), is increasingly viewed as a cornerstone of health-related research activities, practice and policy making. Effective patient engagement can profoundly change how patientcentered research and practice is conceptualised and conducted, resulting in better patient-centered care, management and measurement.10 With respect to the values that may underpin the process of PE in shared decision-making, the overarching principle is the importance of effective collaborative relationships underpinned by the importance of mutual respect for differing values and skills, greater transparency and the need for clarity in purpose and process (Table 1). “Trust” is something that grows as the patient-clinician relationship develops; trust is more of an outcome – it is important to build an environment where patients can trust. One does not need to agree with the patient, but needs to debate and discuss, and partnership negotiation depends on the nature of invlovement. Consequently, the impact of PE will be first on the quality, relevance and credibility of the outcome of the research or shared decision; and second, on the challenges and importance of developing an evidence base for PE practice. Developing Table 1. Values Underpinning Patient Engagement (PE)/Patient and Public Involvement (PPI). Improving the quality, relevance and credibility of research. – Transparency – Clarity in purpose and process – New / unique insights: experiential knowledge of patients – More explicit research – Asking the ‘right’ questions – Enhanced validity: improved relevance and credibility of research to patients’needs Improve the dissemination and impact of research Different approaches to PPI: – What works for whom, when and in what context – What level of representativeness is meaningful and appropriate – ‘Not just the posh articulate’ Importance of developing the evidence base – ‘How to do effective PPI?’ – Challenges Developing a genuine relationship between all stakeholders – A collaborative, respectful, deliberative and transparent relationship based on trust, reciprocity, co-learning and mutual respect haematologica | 2016; 101(7)


Editorials

effective relationships between the patient and all other stakeholders is central to both of these sets of values. “Effectiveness” is a shared value that would require knowledge and effort on the part of all participants. The fundamental right of the patient to have a say and to be empowered in their contribution to the research or therapeutic decision process should be widely valued. However, it is recognised that this requires the establishment of a genuine relationship between the patient and other partners, underpinned by mutual respect, clarity in the roles to be undertaken, and the valuing of different views and perspectives. An awareness of the different approaches to patient-centred practice or patient engagement – and what works, for whom, when and in what context is considered essential to enabling effective involvement, and requires the development of a strong evidence base with which to inform good practice guidance. A patient’s job is not to tell the clinician their story, rather it is to bring a reflective voice to the table. While the voice of patients is gaining power, and effective patient engagement in research and practice has become a reality, in parallel, hematologists are witnessing an evolution in the diagnosis and prognostication of patients through genetic and epigenetic discovery. To stay up-to-date, the WHO has classified tumors of hematopoietic and lymphoid tissues twice - in 2002 and 2008 - and is now in the process of reclassification. Hematologists have several tools to better define (score) prognosis in individual patients. Molecular targeted therapy is following the process with newly approved drugs, which include lenalidomide for myelodysplastic syndrome with deletion 5q chromosomal abnormality; imatinib and other more novel tyrosine kinase inhibitors for chronic myeloid leukemia; ibrutinib, a covalent inhibitor of the enzyme Bruton's tyrosine kinase (BTK), for chronic lymphocytic leukemia; monoclonal antibodies, such as rituximab, which targets CD20 of B cells in non-Hodgkin lymphomas, and elotuzumab, a SLAMF7-directed immunostimulatory antibody for multiple myeloma; and many others. Modern technology facilitates the determination of diagnosis and prognosis and challenges the role of patients when it comes to the choice of treatment offered, since the latter is becoming ever more “tailored”. Certainly, clinicians will tend to emphasize the value of therapeutic approaches, whether marketed or experimental, according to knowledge and experience and the patient will always make the final decision, but, in this fast era of genomics, probably the true modern approach to communication is equality between both patients and clinicians, “bringing different knowledge, needs, concerns, and gravitational

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pull but neither claiming a position of centrality”.11 This is further supported by the five values which have been defined by the European Patients Forum to underpin meaningful patient involvement: 1) Appropriate representation of patients; 2) Building on diversity and pooling knowledge to achieve more than can be achieved by each partner alone; 3) Equality, providing an empowering environment for patients; 4) Commitment to patient involvement being a positive experience that adds value to the project; and 5) Respect for patients as equal partners. Thus, the implementation of PRO measures in clinical practice will be of value to help clinicians and patients with hematological conditions to make more informed shared treatment decisions, and might facilitate patientphysician communication and ensure patient-centeredness. Such partnerships require new skills and sustained efforts from all parties: understanding the values that different stakeholders aspire to provide an essential foundation for effective patient engagement. This approach is an ambitious goal of health advocacy. In conclusion, patientcentered research and practice in hematology based on real patient-clinician partnership will help to provide risk adaptive treatment and enhance the quality of care that patients with hematological malignancies receive.

References 1. Institute on Medicine. "Crossing the Quality Chasm: A New Health System for the 21st Century". By the Committee on Quality of Health Care in America of the Institute of Medicine. 337 pp. Washington, D.C., National Academy Press, 2001. 2. Bardes CL. Defining "patient-centered medicine". N Engl J Med. 2012;366(9):782-783. 3. Guidelines. Patient-reported outcomes in hematology. Editors: Novik A, Salek S, Ionova T. Genoa: Forum service editore. 2012. 4. Beckman HB, Frankel RM. The effect of physician behaviour on the collection of data. Ann Intern Med. 1984;101(5):692-696. 5. Clarke DM, Miniad IH, Stuart GW. The prevalence of psychiatric morbidity in general hospital inpatients. Aust N Z J Psychiatry. 1991;25(3):322-329. 6. Ley P. Satisfaction, compliance and communication. Br J Clin Psychol. 1982;21(Pt 4):241-254. 7. Magurie P, Faulkner A, Booth K, et al. Helping cancer patients disclose their concerns. Eur J Cancer. 1996;32A(1):78-81. 8. Finlay AY, Khan GK. Dermatology life quality index (DLQI): a simple practical measure for routine clinical use. Clin Exp Dermatol. 1994;19(3):210-216. 9. Fitzpatrick R, Fletcher A, Gore S, Jones D, Spiegelhalter D, Cox D. Quality of life measures in health care: applications and issues in assessment. BMJ. 1992;305(6861):1074-1077. 10. Haywood K, Brett J, SALEK S, et al. Patient and public engagement in health-related quality of life and patient-reported outcomes research: what is important and why should we care? Findings from the first ISOQOL patient engagement symposium. Qual Life Res. 2015;24(5):1069-1076. 11. (European Patients Forum (20XX) The Value+ Toolkit: http://www.eupatient.eu/globalassets/projects/valueplus/value-toolkit.pdf).

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REVIEW ARTICLE EUROPEAN HEMATOLOGY ASSOCIATION

Ferrata Storti Foundation

Leaders in Hematology review series

The immune microenvironment in Hodgkin lymphoma: T cells, B cells, and immune checkpoints Santosha Vardhana and Anas Younes

Haematologica 2016 Volume 101(7):794-802

Lymphoma Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA

ABSTRACT

C

lassical Hodgkin lymphoma is curable in the majority of cases with chemotherapy and/or radiation. However, 15-20% of patients ultimately relapse and succumb to their disease. Pathologically, classical Hodgkin lymphoma is characterized by rare tumor-initiating Reed-Sternberg cells surrounded by a dense immune microenvironment. However, the role of the immune microenvironment, particularly T and B cells, in either promoting or restricting Classical Hodgkin lymphoma growth remains undefined. Recent dramatic clinical responses seen using monoclonal antibodies against PD-1, a cell surface receptor whose primary function is to restrict T cell activation, have reignited questions regarding the function of the adaptive immune system in classical Hodgkin lymphoma. This review summarizes what is known regarding T cells, B cells, and immune checkpoints in classical Hodgkin lymphoma.

Correspondence:

Introduction

younesa@mskcc.org

Received: January 13, 2016. Accepted: April 13, 2016. Pre-published: no publication. doi:10.3324/haematol.2015.132761

Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/101/7/794

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

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Nearly two hundred years after Thomas Hodgkin’s initial description of “morbid experiences of the absorbent glands and spleen”,1 the underlying pathophysiology of this eponymous disease remains highly enigmatic. While it has been established that the malignant Reed-Sternberg (RS) cells of classical Hodgkin lymphoma (CHL) are of B cell origin,2,3 these cells comprise only a small percentage of CHL tumor bulk while the remaining tumor microenvironment is rich in T cells, non-malignant B cells, granulocytes, eosinophils, and stromal cells. The contribution of the immune microenvironment to CHL pathogenesis remains incompletely defined; however, the recent success of novel treatments aimed at amplifying anti-tumor T cell responses suggests a potential therapeutic role for the immune system in this disease.4,5 This review will highlight both the relative contribution of non-malignant T and B cells to the pathogenesis and prognosis of CHL as well as the role of negative regulatory immune checkpoints in CHL pathophysiology and therapeutic potential.

T cells in CHL: friends or foes? The role of non-malignant T cells in CHL pathogenesis and treatment remains poorly understood. T cells are thought to suppress the development and growth of lymphomas; the increased incidence of lymphomas in patients receiving long-term immunosuppressants as well as immunodeficient mice supports this hypothesis.6-8 The presence of multiple tumor-infiltrating T cells “rosetting,” but failing to eliminate, malignant RS cells has been well-described in CHL and is highly suggestive of an ineffectual T cell response in this disease.9,10 This has been complemented by the demonstration of impaired proliferative responses to mitogenic stimuli in peripheral blood lymphocytes isolated from CHL patients.11 What explains the impaired T cell responses seen in CHL? First, the T cells that accumulate within the CHL microenvironment are largely skewed towards differentiation into either Th2 cells or regulatory T cells (Tregs).12-15 This accumulation is haematologica | 2016; 101(7)


The immune microenvironment in Hodgkin lymphoma

driven by a combination of selective recruitment as well as intratumoral functional reprogramming.16 RS cells produce a variety of Th2 and Treg-selective chemoattractants, including CCL17/TARC,17 CCL22,18 CCL5,19,20 IL-4, IL-5, IL-10, and IL-13.15,21,22 Production of these chemoattractants is associated with inferior responses to therapy.23,24 Additionally, RS cells secrete factors known to induce functional reprogramming of tumor-infiltrating T cells into Th2 cells and Tregs, such as galectin-1,25-28 macrophage migration inhibitory factor29 and IL-7.30 Stromal cells within the CHL microenvironment also recruit immunosuppressive myeloid-derived suppressor cells and Tregs by secreting factors such as indoleamine 2,3 dioxygenase (IDO)31 (Figure 1A). Second, effector T cells in CHL display features of chronic ineffectual antigen encounter, a phenomenon known as T cell “exhaustion” characterized by the upregulation of negative regulatory receptors such as the immunoglobulin superfamily member Programmed Death 1 (PD-1; CD279). PD-1 upregulation was initially characterized in models of chronic viral infection32,33 but is also seen in multiple lymphomas, including diffuse large B-cell lymphoma and follicular lymphoma.34,35 In CHL, the expression of PD-1 on T cells is likely driven by constitutive upregulation of its ligands, PD-L1 and PD-L2, on RS cells36 (Figure 1B). Accordingly, the presence of PD-1+ T cells, both in the microenvironment and in the peripheral blood, is a negative prognostic factor in CHL.37,38 Finally, impaired anti-tumor immunity in CHL may be due to an inability of T cells to recognize RS cells. RS cells

frequently lack expression of MHC-I and MHC-II, which are required for antigen recognition by CD8+ and CD4+ T cells, respectively. This can occur secondary to mutations, such as in the β2M39 and CIITA40,41 genes, or via epigenetic mechanisms at the CIITA promoter leading to decreased transcription.42 While T cells in CHL are rendered incapable of mediating anti-tumor responses, there is some evidence to suggest that they may actually support RS cell growth and survival. CHL has been noted to develop during the immune response to active viral infections, such as acute Epstein-Barr virus mediated mononucleosis,43 and during immune reconstitution following the initiation of antiretroviral therapy in HIV+ patients.44 Mechanistically, T cells in CHL can promote RS cell survival and proliferation via CD40/CD40 ligand-mediated alternative activation of NF-kB;45 this growth signal may be particularly important for the survival of RS cells, which have lost the ability to activate NF-kB through conventional B cell receptor-driven signals.46-48 The multiple mechanisms by which RS cells and the CHL microenvironment suppress immune responses are summarized in Figure 1; therapies aimed at breaking this pathological cycle of T cell fueled growth and immune evasion, primarily via checkpoint blockade, are discussed below.

B cells: innocent bystanders or active participants? Less is known regarding the role of non-malignant B cells in CHL pathogenesis and response to therapy as compared to T cells. Non-malignant B cells are prevalent in lymphocyte-predominant Hodgkin lymphoma (LP-HL), a biologically distinct disease in which the tumor-initiating cells also express CD20; this form of Hodgkin lymphoma

Figure 1. Suppression of anti-tumor T cell responses by the CHL microenvironment. (A) RS cells and stromal cells secrete cytokines, chemokines, and other soluble immunomodulatory factors, such as IL-10, CCL17/TARC, galectin-1, and indoleamine 2,3-dioxygenase (IDO) which both recruit Th2 and regulatory CD4+ T cells and favor the differentiation of tumor-infiltrating T cells into regulatory and Th2 cells via the induction of lineage specific transcription factors Gata3 (Th2) and FoxP3 (Treg). (B) RS cells evade recognition by CD8+ and CD4+ T cells by downregulating expression of MHC-I and MHC-II in the majority of cases. They also express ligands that activate negative regulatory receptors present on T cells, such as PD-1. Conversely, RS cells are able to derive growth signals from CD40L, which is present on the majority of T cells within the microenvironment and activates CD40 on RS cells, driving NF-kB signaling and RS cell proliferation.

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is frequently monitored and, when requiring therapy, can be successfully treated with radiation alone or single agent rituximab.49,50 In CHL, non-malignant B cells are also generally present in the microenvironment, likely due to the normal predominance of B cells within a non-malignant lymph node. However, their role in facilitating CHL growth is less established. Non-malignant B cells can easily be distinguished from RS cells, which lose expression of classical B cell antigens including CD20, CD79a, and PAX-5 due to mutations and/or epigenetic silencing.51 The effect of B cells within the CHL microenvironment is also not well established; B cell production of IL-10 may suppress anti-tumor T cell responses;52,53 on the other hand, non-malignant B cells may compete with RS cells for T cell-derived survival signals such as CD40L, and in this way suppress RS cell growth. In support of the latter hypothesis, gene expression signatures consistent with non-malignant B cells are associated with improved outcomes in CHL, although this may simply reflect low CHL tumor burden within an otherwise healthy LN.54-56 Targeting B cells within the tumor microenvironment with rituximab has shown some clinical activity, with an overall response rate of 22% as a single agent regardless of RS cell CD20 expression.57 In a phase 2 study of rituximab plus ABVD (adriamycin, bleomycin, vinblastine, and dacarbazine) in newly diagnosed CHL, five-year eventfree and overall survival rates of 83% and 96% compared favorably with historical controls treated with ABVD therapy alone.58 The reasons for rituximab efficacy in CHL are likely to be multifactorial. It has demonstrated benefit in a subset of patients whose RS cells express CD20.59 In the majority of CHL cases, which lack CD20 expression on RS cells, rituximab may deplete CHL precursor cells, which have a memory-like B cell phenotype and express CD20.60 In a phase 2 study of rituximab plus ABVD (R-ABVD) in untreated, advanced stage CHL, circulating CD20+ clonal B cells were found in 21 out of 25 assayed patients, and clearance of these precursor cells following treatment with R-ABVD was associated with a reduced risk of relapse as compared to patients in whom clonal CD20+ cells persisted.61 Ultimately, randomized controlled trials currently underway evaluating R-ABVD versus ABVD in unselected CHL patients with early stage (clinicaltrials.gov identifier: 00992030) and advanced stage (clinicaltrials.gov identifier: 00654732) disease will provide insight into the value of depleting CD20+ malignant and non-malignant B cells in CHL.

Immune checkpoints: breaks in the action Broadly speaking, immune checkpoints are a diverse group of proteins whose function is to restrict physiologic immune cell responses in order to limit damage to host tissues. These include members of the immunoglobulin superfamily such as CTLA-4, PD-1, and LAG-3.62 The essential role for negative regulators of the immune response was first established by the diffuse systemic immune hyperactivation and multisystem organ failure seen in mice lacking CTLA-4.63,64 Increasingly, malignant co-opting of immune checkpoints has emerged as a mechanism by which tumor cells can subvert immune surveillance and anti-tumor immunity. Targeting of immune checkpoints, particularly with the anti-PD-1 antibodies nivolumab and pembrolizumab, has 796

resulted in dramatic clinical responses in CHL,4,5 although the mechanisms by which these drugs induce an antitumor effect remain somewhat enigmatic. Furthermore, PD-1 represents only one of multiple immune checkpoints, all of which can promote immune evasion in CHL and might be amenable to therapeutic blockade. The specifics of individual immune checkpoints and their potential for therapeutic intervention are discussed below.

PD-1 PD-1, a costimulatory molecule within the immunoglobulin superfamily of receptors, was first established as a negative regulator of T cell activation based on the presence of a cytoplasmic inhibitory tyrosine-based ITIM motif, as well as the development of a lupus-like autoimmune disease in PD-1 knockout mice.65 Subsequently, PD-1 was found to be present on many tumor-infiltrating lymphocytes (TILs),66 and its ligand is upregulated in a variety of human cancers.67 Checkpointmediated immune evasion was established as a hallmark of CHL pathogenesis with the identification of amplifications of the 9p24 locus resulting in constitutive expression of PD-L1 and PD-L2 in more than 85% of CHL patients.36 Even in patients without genetic amplifications of PD-L1 or PD-L2, physiologic upregulation of these ligands likely occurs downstream of JAK/STAT signaling, IFNg production or, in EBV-associated cases of CHL, expression of the viral-associated protein LMP1. 67,68 In solid tumors, PD-1 blockade acts by promoting T cell activation via a variety of mechanisms. PD-1 blockade reverses SHP-2-mediated dephosphorylation of the proximal T cell receptor-associated kinase ZAP-70, leading to increased T cell activation.69 Furthermore, PD-1 blockade increases the dwell time of T cells on antigen-presenting and target cells, increasing the opportunity for a T cell to encounter its cognate antigen and successfully initiate an anti-tumor response.70 Indeed, the blockade of PD-1 increases the sensitivity of T cells to foreign antigens and increases effector function and cytokine production of both CD4+ and CD8+ T cells in models of both tumor and virally mediated chronic T cell exhaustion.71,72 PD-1 is thought to tune T cells during the effector, rather than priming, phase of T cell antigen encounter. This likely underlies the lower incidence of off-target, autoimmunelike adverse events associated with anti-PD1 as compared to anti-CTLA-4 therapy. Indeed, PD-1 knockout mice have a relatively mild, organ-specific autoimmune phenotype,65 and clinical PD-1 blockade does not induce the activation of peripheral blood T cells.73 Clinically in CHL, the reversal of PD-1 mediated T cell suppression using blocking monoclonal antibodies has resulted in impressive and durable remissions in patients with highly refractory disease. Nivolumab, a human IgG4 monoclonal antibody, elicited an overall response rate (ORR) of 87% and complete response (CR) rate of 17% in 23 patients with relapsed and refractory CHL whose disease had progressed after or were ineligible for autologous stem cell transplant.4 Pembrolizumab, also an IgG4 monoclonal antibody to PD-1, had an ORR of 65% with 16% complete remissions in 31 patients, all of whom had progressed or were ineligible for autologous stem cell transplant and had progressed on brentuximab vedotin.5 The median duration of response was not reached during the haematologica | 2016; 101(7)


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short follow-up time of less than one year in either study; however, recent data suggests that the majority of remissions have been durable for longer than one year.74 Objective biomarkers correlating with PD-1 response in CHL, however, have remained elusive. In some solid tumors, PD-L1 expression correlates with response to therapy,75-77 but this has not yet been demonstrated in CHL. Similarly, somatic mutation and neoantigen burden have been shown to correlate with anti-PD-1 response to therapy,78 but the mutational burden of CHL remains uncharacterized. The mechanism by which anti-PD-1 therapy promotes responses in CHL is likely to have implications in other types of lymphoma such as diffuse large B-cell lymphoma (DLBCL), in which PD-L1 expression on tumor cells was recently demonstrated to portend an adverse clinical outcome.79 Single agent studies of nivolumab and pembrolizumab in patients with relapsed/refractory disease (clinicaltrials.gov identifier: 02453594), in comparison with brentuximab vedotin (clinicaltrials.gov identifier: 02684292), as maintenance following autologous transplant (clinicaltrials.gov identifier: 02362997), and in relapsed patients following allogeneic transplant (clinicaltrials.gov identifier: 01822509) are currently underway. Single agent studies of antibodies targeting PD-L1 are also accuring patients (clinicaltrials.gov identifier: 01452334, clinicaltrials.gov identifier: 02603419). Finally, multiple trials combining PD-1 blockade with other checkpoint inhibitors, targeted agents, and chemotherapy are underway (Table 1). Currently, anti-PD-1 therapy has only been studied in highly refractory patients and has not yet been FDA approved for this indication. Furthermore, the role of anti-PD-1 therapy in untreated patients or those curable with autologous stem cell transplant (in which it is likely to be combined with chemotherapy) remains to be defined.

LAG-3 LAG-3 was discovered in 1990 and was initially reported to be a ligand for MHC-II.87,88 Subsequently it was determined that LAG-3, like PD-1, is upregulated on T cells during chronic antigen stimulation.89 LAG-3 suppresses CD4+ T cell expansion in response to antigen,90 and LAG-3 was found to be synergistic with CTLA-4 and PD-1 in mediating T cell suppression during chronic antigenic stimulation.91,92 Additionally, LAG-3 is important in promoting the function of regulatory T cells.93 As a result, antibodies to LAG-3 augment CD4+ T cell expansion94 and CD8+ T cell function95 while blocking peripheral Treg differentiation and function.96,97 In CHL, CD4+ T cells from patients with active disease were found to express significantly higher levels of LAG-3 as compared to patients in long-term remission, and expression of LAG-3 was associated with impaired T cell responses to EBV-associated viral antigens LMP1 and LMP2.12 Intriguingly, LAG-3 is also expressed on natural killer (NK) cells.98 Thus, LAG-3 upregulation may suppress antitumor immunity through effects on T cells, Tregs, and NK cells, and is an intriguing candidate for therapeutic targeting. Monoclonal antibodies to LAG-3 are currently in clinical development, with early phase studies demonstrating that a LAG-3 monoclonal antibody is well tolerated with objective responses both as a single agent and in combination with chemotherapy in solid tumors.99,100 Given the established synergy between LAG-3 and PD-1, both in double knockout mice101 and with dual blockade in mouse models,62 this may be an attractive target for combination therapy. A phase I study of the anti-LAG-3 antibody BMS-986016 is currently accruing patients (clinicaltrials.gov identifier: 02061761).

CTLA-4

Checkpoint blockade in CHL: a mechanistic conundrum

CTLA-4 was initially discovered as an additional member of the immunoglobulin superfamily involved in cellcell interactions in 1987.80 Subsequently, CTLA-4 was shown to be a critical negative regulator of T cell activation based both on in vitro studies81,82 and in fatal lymphoproliferative disorders seen in mice lacking CTLA-4.64 The repression of immune responses by CTLA-4 occurs via a number of mechanisms. In effector T cells, CTLA-4 competes strongly with CD28 for effective costimulation by CD80/86, leading to impaired T cell costimulation and functional inactivation. CTLA-4 also impairs the “stop signal� initiated by T cells upon antigen encounter leading to impaired T cell activation.83 Finally, CTLA-4 induces transendocytosis of the costimulatory ligands CD80 and CD86, restricting opportunities for further T cell activation.84

While it is clear that checkpoint blockade produces clinical responses in the majority of CHL patients, the mechanism by which this occurs has not been fully characterized. As described above, checkpoint blockade enhances T cell activation by eliminating negative regulation of either T cell receptor signaling or positive costimulatory signals. In solid tumors, checkpoint blockade primarily augments CD8+ T cell responses to tumor antigens pre-

Pre-clinical rationale for targeting CTLA-4 in CHL was seen shortly after CTLA-4 was characterized with histopathologic demonstrations of CTLA-4+ T cells infiltrating CHL tumors.85 The best evidence to support clinical activity of CTLA-4 blockade comes from a phase I trial of patients with malignancies progressing after allogeneic stem cell transplantation.86 Two complete remissions were seen out of 14 CHL patients treated in the study. A clinical trial of ipilimumab, nivolumab, or both in combination with brentuximab vedotin in patients with relapsed or refractory CHL is currently accruing patients (clinicaltrials.gov identifier: 01896999). haematologica | 2016; 101(7)

Table 1. Clinical trials investigating combination strategies with checkpoint blockade in CHL.

PD-1 Antibody Nivolumab Nivolumab Nivolumab Nivolumab Nivolumab Pembrolizumab Pembrolizumab Pembrolizumab

Combination Agent

Combination Target

Ipilimumab Lirilumab Brentuximab +/- Ipilimumab AVD* Brentuximab Epacadostat AFM13 Brentuximab ICE*** ACP-196

CTLA-4 KIR CD30 CTLA-4 Chemotherapy CD30 IDO1** CD30/CD16a CD30 Chemotherapy Btk

Identifier NCT01592370 NCT01592370 NCT01896999 NCT02181738 NCT02572167 NCT02327078 NCT02665650 NCT02408042 NCT02408042 NCT02362035

*Adriamycin, Vinblastine, and Dacarbazine; ** Indoleamine 2,3 dioxygenase 1;*** Ifosamide, Carboplatin, and Etoposide

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sented by MHC class I molecules on tumor cells. Correspondingly, anti-PD-1 activity correlates with the presence of CD8+ TILs at the invasive margin of the tumor.77 In the setting of checkpoint blockade, CD8+ T cells can recognize tumor antigens, including self-antigens for which T cell tolerance is incomplete, including those with restricted tissue expression, or tumor “neoantigens� produced by somatic mutations within tumor cells.102,103 Recent reports suggest that the somatic mutational and consequent neoantigen burden correlates with response to anti-CTLA-4 and anti-PD-1 therapy in mouse models78 as well as in patients with melanoma and non-small cell lung cancer,104,105 in which neoantigen-specific CD8+ T cell clonal expansion could be detected in the peripheral blood. In CHL, however, there are multiple barriers to CD8+ T cell recognition of tumor antigens in the setting of checkpoint blockade. First, it is unclear whether the CHL somatic mutational burden generates sufficient neoantigens to drive anti-tumor responses. Median somatic mutational burdens vary widely across cancers,106 and correlate strongly with neoantigen burden. The mutational burden in CHL is not well established as sequencing efforts have thus far been hampered by the paucity of RS cells within CHL tumors, although this can be overcome by either flow cytometry or microdissection-based cell enrichment.39,107 Another intriguing option for assessment of mutation burden is via assessment of cell-free DNA, which can be detected in the serum of the majority of CHL patients,108 although it is not yet clear whether cellfree or circulating tumor DNA can be used for comprehensive whole exome sequencing. More importantly, the majority of CHL samples demonstrate a loss of beta-2 microglobulin, leading to an absence of MHC-I expression

on RS cells.39 As CD8+ T cells require antigen presentation on MHC-I molecules for their effector function, they are highly unlikely to be the primary mediators of the antiPD-1 response (Figure 2A). It remains possible that CD4+ T cells could be major contributors to the anti-PD-1-mediated anti-tumor response in CHL. CD4+ T cells are able to mediate tumor rejection, both through the production of pro-inflammatory cytokines and via the recruitment and activation of innate effector cells, such as macrophages and NK cells. Both reversal of Th1 anergy and an increased IFNgresponse signature are seen in in vitro models38 as well as in patients in response to anti-PD-1 therapy, suggesting that the amplification of effector CD4+ T cell responses may be important to the anti-PD-1 response. Whether CD4+ T cells exert anti-tumor immunity directly or through recruitment of innate effector cells has yet to be established. Arguing against a role of CD4+ T cells in mediating the anti-PD-1 response is the loss of MHC-II on RS cells in at least 40% of patients, and likely higher in patients with relapsed disease.40 In a minority of cases this likely results from gene fusions involving CIITA, a transactivator required for MHC-II synthesis.41 However, unlike CD8+ T cell function, which requires class I antigen presentation on tumor cells, CD4+ T cells could be primed in CHL by APCs in the microenvironment or draining lymph node, and so loss of MHC-II does not preclude a CD4+ T cell mediated effect in anti-PD-1 treated patients (Figure 2B). Furthermore, both class I and class II restricted neoantigens have been described with associated expansion of neoantigen-specific CD4+ as well as CD8+ T cells,109-111 suggesting that a neoantigen-specific CD4+ T cell response may be possible in CHL.

Figure 2. A model for anti-tumor immunity in the setting of checkpoint blockade. (A) In solid tumors, anti-tumor immunity is mediated primarily by CD8+ T cell responses that are amplified in the setting of PD-1 blockade. However, in CHL this is mitigated by downregulation of MHC-I in the majority of cases. (B) This may predispose RS cells to killing by NK cells, which also express PD-1. Similarly, RS cell downregulation of MHC-II may limit CD4+ T cell responses following checkpoint blockade, but CD4+ T cells can also be primed by other APCs within the CHL microenvironment that do express MHC-II. Additionally, checkpoint blockade may impair the immunosuppressive function of infiltrating regulatory T cells, increasing productive T cell activation.

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Checkpoint blockade may also induce anti-tumor responses in CHL in an effector T cell-independent fashion. PD-1 is expressed on NK cells as well as T cells,67,112 and PD-1 is upregulated on NK cells in models of chronic infection.113 PD-1 blockade may thus promote anti-tumor immunity by facilitating NK cell recognition of MHC-I deficient RS cells, and this effect has been seen in primary hematopoietic cancer cells114 (Figure 2A). Meanwhile, Tregs are actually activated by PD-1 ligand binding,115,116 suggesting that the suppression of Treg function may be another potential immunomodulatory effect of anti-PD-1 therapy (Figure 2B). Finally, blockade of the PD-1/PD-L1 interaction may have cell autonomous effects on tumor growth, as suggested by a recent study demonstrating that blockade of PD-L1 reduces glucose consumption by tumors. This blockade simultaneously inhibits tumor cell growth and increases extracellular glucose availability permitting T cell activation, proliferation, and cytokine production.117 The lack of a defined mechanism of action for checkpoint blockade in Hodgkin lymphoma has resulted in the lack of biomarkers predicting response to therapy. Expression of PD-L1 is unlikely to predict response, as it is amplified in the overwhelming majority of patients treated with checkpoint inhibitors. A recent analysis of peripheral blood from patients treated with the anti-PD-1 antibody pembrolizumab demonstrated an increase in the absolute number of CD4+, CD8+, and NK cells with parallel gene expression profiles demonstrating an increased IFNg response signature,118 but whether these changes correlate with treatment response has not been established. Future investigations into the mechanism of response to checkpoint blockade should focus both on evaluating the extent to which known immunosuppressive features of RS cells and the CHL microenvironment affect response to checkpoint blockade, as well as identifying the effector cells responsible for mediating this response. These studies would include assessment of tumor mutational and neoantigen burden, MHC-I and MHC-II expression, intratumoral effector and regulatory T cells, and development of clonal CD4+ and CD8+ T cell responses in response to therapy (Table 2).

Towards rational combination strategies in Hodgkin lymphoma Despite the encouraging clinical responses seen with checkpoint blockade, and particularly with anti-PD-1 therapy, complete remissions to immunotherapy remain rare, with only 15-20% of patients achieving a complete remis-

sion to PD-1 blockade.5,74 This may be due to a variety of factors, both on RS cells and within the tumor microenvironment. Effective anti-tumor immune responses may not be feasible in the setting of restricted antigen expression, either due to epigenetic silencing or downregulation of antigen presentation machinery. Additionally, tumorinfiltrating Tregs and immunosuppressive tumor-associated macrophages may effectively negate anti-tumor responses even in the presence of checkpoint blockade. Rational combination strategies may help to overcome these limitations and provide sustained remissions. Combinations of checkpoint inhibitors, including PD-1 and CTLA-4 blockade, are part of ongoing active clinical trials (clinicaltrials.gov identifier: 01896999, clinicaltrials.gov identifier: 01592370, clinicaltrials.gov identifier: 01592370). Combining checkpoint blockade with agonist antibodies against costimulatory molecules present on T cells, such as OX40 and 4-1BB, represents an intriguing strategy to overcome multiple mechanisms of immunosuppression known to be present within the CHL microenvironment, and agonist antibodies against OX40 and 4-1BB are currently being investigated in active clinical trials (clinicaltrials.gov identifier: 02205333, clinicaltrials.gov identifier: 01644968, clinicaltrials.gov identifier: 02253992, clinicaltrials.gov identifier: 01775631).119 An additional candidate for combination therapy with checkpoint blockade is the family of chromatin-modifying agents, including hypomethylating agents and histone deacetylase (HDAC) inhibitors. These agents mediate direct apoptosis of CHL cell lines in in vitro studies but have additional effects that may cooperate with checkpoint blockade to increase antitumor immunity. Hypomethylating agents may increase tumor antigen expression, leading to more diverse antigen-specific responses that can prevent immune escape.120 HDAC inhibition also suppresses RS production of multiple cytokines and chemokines favoring Th2 cell recruitment and differentiation. For example, the treatment of CHL cell lines with vorinostat was shown to reduce STAT-mediated production of Th2 polarizing cytokines IL-5, IL-10 and IL-13 as well as the Th2 recruiting chemokine TARC.121 These findings were paralleled in phase 2 studies of mocetinostat and panobinostat, in which treatment-induced decreases in TARC correlated with reductions in tumor burden and progression-free survival.122,123 HDAC inhibition can also reinvigorate exhausted T cells in CHL by upregulating OX40 on RS cells124 and by downregulating PD-1 expression on CD4+ and CD8+ T cells.125 Finally, HDAC inhibition may selectively deplete Tregs by suppressing FoxP3

Table 2. Potential biomarkers under investigation to predict response to checkpoint blockade in CHL.

Potential Biomarker

Assay

Tumor mutational and neoantigen burden Clonal T cell responses Effector:Regulatory T cell ratio

Whole exome sequencing of flow-sorted or laser capture microdissected RS cells High throughput TCR sequencing Flow cytometry-based quantitation of naive and memory CD4+, CD8+ effector T cells and regulatory T cells IHC or flow cytometry-based evaluation of MHC-I, MHC-II, β2M, and CIITA IHC or flow cytometry-based evaluation of PD-L1/PD-L2, OX40/OX40L, CTLA-4, 4-1BB/4-1BBL, TIM3, LAG3

Loss of antigen presentation Expression of immune checkpoints

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expression and depleting intratumoral accumulation of myeloid-derived suppressor cells.126,127 The multiple pleiotropic effects of HDAC inhibition may collectively tip the balance towards deeper responses to checkpoint blockade.

Future directions CHL remains an enigmatic disease in which components of the microenvironment, including T and B cells, may help

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haematologica | 2016; 101(7)


REVIEW ARTICLE

Management of Epstein-Barr Virus infections and post-transplant lymphoproliferative disorders in patients after allogeneic hematopoietic stem cell transplantation: Sixth European Conference on Infections in Leukemia (ECIL-6) guidelines

Jan Styczynski,1 Walter van der Velden,2 Christopher P. Fox,3 Dan Engelhard,4 Rafael de la Camara,5 Catherine Cordonnier,6 and Per Ljungman7 on behalf of the Sixth European Conference on Infections in Leukemia, a joint venture of the Infectious Diseases Working Party of the European Society of Blood and Marrow Transplantation (EBMT-IDWP), the Infectious Diseases Group of the European Organization for Research and Treatment of Cancer (EORTC-IDG), the International Immunocompromised Host Society (ICHS) and the European Leukemia Net (ELN)

EUROPEAN HEMATOLOGY ASSOCIATION

Ferrata Storti Foundation

Haematologica 2016 Volume 101(7):803-811

Department of Pediatric Hematology and Oncology, Collegium Medicum, Nicolaus Copernicus University Torun, Jurasz University Hospital, Bydgoszcz, Poland; 2 Department of Hematology, Radboud University Medical Centre, Nijmegen, The Netherlands; 3Center for Clinical Hematology, Nottingham University Hospitals NHS Trust, Nottingham, UK; 4Department of Pediatrics, Hadassah-Hebrew University Medical Center, Jerusalem, Israel; 5Hospital de la Princesa, Department of Hematology, Madrid, Spain; 6Department of Hematology, Henri Mondor Hospital, Assistance PubliqueHôpitaux de Paris, and University Paris-Est-Créteil, Créteil, France; 7Karolinska University Hospital, Departments of Hematology and Allogeneic Stem Cell Transplantation, Karolinska Institutet, Division of Hematology, Department of Medicine, Huddinge, Stockholm, Sweden 1

Correspondence: jstyczynski@cm.umk.pl ABSTRACT

E

pstein-Barr virus-related post-transplant lymphoproliferative disorders are recognized as a significant cause of morbidity and mortality in patients undergoing hematopoietic stem cell transplantation. To better define current understanding of post-transplant lymphoproliferative disorders in stem cell transplant patients, and to improve its diagnosis and management, a working group of the Sixth European Conference on Infections in Leukemia 2015 reviewed the literature, graded the available quality of evidence, and developed evidence-based recommendations for diagnosis, prevention, prophylaxis and therapy of post-transplant lymphoproliferative disorders exclusively in the stem cell transplant setting. The key elements in diagnosis include non-invasive and invasive methods. The former are based on quantitative viral load measurement and imaging with positron emission tomography; the latter with tissue biopsy for histopathology and detection of Epstein-Barr virus. The diagnosis of post-transplant lymphoproliferative disorder can be established on a proven or probable level. Therapeutic strategies include prophylaxis, preemptive therapy and targeted therapy. Rituximab, reduction of immunosuppression and Epstein-Barr virusspecific cytotoxic T-cell therapy are recommended as first-line therapy, whilst unselected donor lymphocyte infusions or chemotherapy are options as second-line therapy; other methods including antiviral drugs are discouraged. haematologica | 2016; 101(7)

Received: February 13, 2016. Accepted: April 21, 2016. Pre-published: no prepublication. doi:10.3324/haematol.2016.144428

Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/101/7/803

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

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J. Styczynski et al.

Introduction Post-transplant lymphoproliferative disorders (PTLD) are a heterogeneous group of diseases occurring in the setting of transplantation of either hematopoietic stem cells (HSCT) or solid organs (SOT). PTLD results from the uncontrolled neoplastic proliferation of lymphoid or plasmacytic cells. It can occur at any age and after all types of transplant; recipients of allogeneic HSCT are at a particular risk for developing PTLD.1,2 In contrast to the SOT setting, post-HSCT PTLD are almost exclusively EBV-related, although rare cases of non-EBV-PTLD also exist in this setting. PTLD is one of the most severe complications associated with transplantation. Before 2000, an attributable mortality for PTLD of 84.6% after HSCT was reported.1 With the introduction of new approaches for EBV disease/PTLD, including the use of monitoring for EBV by PCR, pre-emptive therapy and timely treatment with rituximab, considerable improvements in outcome have been achieved. However, mortality remains high; approximately one-third of diagnosed patients.3 Recently, guidelines for management of PTLD in the SOT setting were published.4-6 The first recommendations for management of EBV infections in patients undergoing HSCT or therapy for hematological malignancies were produced following the Second European Conference on Infections in Leukemia (ECIL-2) in 2007.7 The goal of this paper is to present updated recommendations based on analysis of recent data.

Methods The main task of ECIL is to develop evidence-based guidelines for management of infectious complications in subjects with leukemia including HSCT. An EBV-PTLD Working Group was hence created. The group defined the relevant issues, questions and outcomes to be addressed, and evaluated these issues and questions prior to the consensus conference through a systematic literature review.8 PubMed was searched using each of the following terms: lymphoproliferative disorder, PTLD, Epstein-Barr virus, EBV, together with leukemia, hematopoietic transplantation, HSCT, bone marrow transplantation, or cord blood. Relevant studies were reviewed up to August 2015. Recommendations were elaborated within the group and graded for quality of evidence (I–III) and strength of recommendation (A–D) using the ESCMID/EFISG grading system (Table 1).9 The ECIL-6 conference (September 11-12, 2015) was attended by 55 experts from 25 countries, including 16 European countries. Experts in hematology, microbiology, and infectious diseases were mostly selected for their active participation in the host organizations. The group presented its literature review and guideline proposals in plenary session. After panel debate, the recommendations were revised as necessary until reaching a final consensus.

Definitions and diagnostic criteria Primary EBV infection is defined when EBV is detected (nucleic acid or serologically) in an EBV-naïve individual (most often asymptomatic acquisition, or occasionally presenting as infectious mononucleosis). Recurrent EBV DNA-emia is diagnosed by detection of EBV DNA in the blood of a previously infected individual, as defined by 804

detection of EBV-specific IgG-antibodies. EBV-associated disease following transplantation can be categorized as EBV-PTLD or other EBV-associated post-transplant manifestations; also referred to as EBV end-organ disease. EBV-PTLD can be diagnosed as probable or proven. Probable EBV disease: significant lymphadenopathy, hepatosplenomegaly or other end-organ manifestations (without tissue biopsy, but in the absence of other documented cause), together with significant EBV DNA-emia. Proven EBV disease: detection of EBV nucleic acids or EBV-encoded proteins in a tissue specimen, together with symptoms and/or signs from the affected organ. The diagnosis of EBV-PTLD should be based on at least two of the following histological features: (i) disruption of underlying cellular architecture by a lymphoproliferative process, (ii) presence of monoclonal or oligoclonal cell populations as revealed by cellular and/or viral markers, (iii) evidence of EBV infection in many of the cells i.e. DNA, RNA or protein. Detection of EBV nucleic acid in blood is not, eo ipso, sufficient for the diagnosis of EBVPTLD. The recommended method for histological specimens, conferring high sensitivity and specificity, is the detection of EBV-encoded RNA by in situ hybridization (EBER-ISH). Immunohistochemistry for viral proteins have good specificity but lower sensitivity; these proteins are variably expressed in PTLD biopsies. Detection of EBV DNA by PCR of histological extracts is not an appropriate method for PTLD diagnosis given the very high sensitivity but low positive predictive value (PPV) (Table 2).10-15 The histopathologic criteria of PTLD were defined by Swerdlow and Greig.16 The WHO classification is most commonly used, with four types of morphological lesions being recognized: polyclonal early lesions, polymorphic, monomorphic (B-cell or T/NK-cell) and classical Hodgkin lymphoma-type PTLD.17

Epidemiology The incidence of EBV DNA-emia and EBV-PTLD varies between transplant centers. The reported incidence of EBV DNA-emia ranging between 0.1-63% is largely dependent on the type of transplant, assay sensitivity, defined level of DNA-emia, use of systematic screening and its timing.18-27 In a recent EBMT study, the overall incidence of PTLD after allogeneic HSCT was 3.2%, varying from 1.2% in matched family donor (MFD) to 2.8% in mismatched family donor (haploidentical/MMFD), 4.0% in matched unrelated donor (MUD), and 11.2% in mismatched unrelated donor (MMUD) recipients.3 In recipients of unrelated cord blood (CBT), the incidence of EBV-PTLD was 2.63.3% for myeloablative transplants, and 7-12.9% in nonmyeloablative transplants.24,28 Interestingly, data from haplo-HSCT incorporating post-transplant cyclophosphamide (haplo-PTCy-HSCT) indicate a very low EBVPTLD incidence.23 The median time to development of EBV-PTLD after HSCT is 2-4 months.3,29 Only 4% of cases develop later than 12 months after HSCT, and cases occurring >5 years after HSCT are extremely rare.3 PTLD after autologous-HSCT is very rare.30-32

Risk factors for EBV-PTLD Risk factors for developing EBV-PTLD can be considered as existing pre-20,24,33-35 or developing post-transplant7,34-37 (Table 3). Importantly, assessing the risk of EBV-PTLD is haematologica | 2016; 101(7)


ECIL-6 guidelines for EBV-PTLD after HSCT

Table 1. ECIL-6 scoring system.

Strength of Recommendation (SoR)*

Definition

Grade A Grade B Grade C Grade D

ECIL strongly supports a recommendation for use ECIL moderately supports a recommendation for use ECIL marginally supports a recommendation for use ECIL supports a recommendation against use

Quality of Evidence (QoE)

Definition

Level I

Evidence from at least 1 properly designed, randomized, controlled trial (orientated on the primary endpoint of the trial) Evidence from at least 1 well-designed clinical trial (including secondary endpoints), without randomization; from cohort or case-controlled analytic studies (preferably from > 1 center; from multiple time series; or from dramatic results of uncontrolled experiments Evidence from opinions of respected authorities, based on clinical experience, descriptive case studies, or reports of expert committees

Level II

Level III

Added Index

Source of Level II Evidence

r t

Meta-analysis or systematic review of RCT Transferred evidence: data from different patient cohorts with comparable clinical features and/or immune function Comparator group: historical control Uncontrolled trials Published abstract presented at an international symposium or meeting

h u a

*poor quality of design, inconsistency of results, indirectness of evidence etc. would lower the SoR.

dependent on the HSCT context with potentially complex interactions between the primary hematological malignancy, HSCT procedure, source, and other factors. Given that the risk of EBV-PTLD is predominantly related to the degree of T-cell depletion or impairment, this should be regarded as the principal risk factor (AIIu). Strategies that deplete T cells from the graft increase the risk of EBVPTLD.38 CBT confers an intrinsic risk for EBV-PTLD because of T-cell naĂŻvety related to the HSC source. A high incidence of EBV-PTLD in both pediatric and adult patients after CBT, following reduced intensity conditioning regimens using anti-thymocyte globulin (ATG) or alemtuzumab (anti-CD52), has also been reported.28,39 This likely reflects both the delayed recovery of EBV-specific CTLs after such transplants, alongside the persistence of recipient-derived B cells. The use of alemtuzumab during conditioning in other types of HSCT can also be regarded as a risk factor for EBV-PTLD.27,29 There appears to be a dose-dependent risk with the in vivo use of ATG in children,40 which is probable also in adults. Current data do not suggest any significant differences between children and adults with respect to epidemiology and risk factors. Patients undergoing HSCT can be classified for the risk for EBV-PTLD as low risk (auto-HSCT), standard risk (MFD allo-HSCT without risk factors, haplo-PTCyHSCT), and high risk (MFD with at least one risk factor, MUD/MMUD, alternative donors including CBT).

plantation (Table 4). Since EBV sero-mismatch is a risk factor for PTLD,34,35 the selection of an EBV matched donor, if possible, might be beneficial. As EBV-PTLD after HSCT is usually of donor origin and EBV might be transmitted with the graft, the risk of EBV-PTLD is higher when the donor is seropositive. Neither in vivo/ex vivo CD34-positive selection nor CD3/CD19 depletion prevents EBVPTLD.11,31 Allo-HSCT recipients should be closely monitored clinically, together with prospective monitoring for EBV DNA in peripheral blood. Importantly, monitoring and intervention strategies might be individualized, informed by a holistic assessment of EBV-PTLD risk.

ECIL recommendations for prevention of EBV diseases including PTLD

ECIL recommendations for diagnosis of EBV-disease/PTLD

ECIL recommends that all allo-HSCT patients and donors should be tested for EBV antibodies before trans-

Fever and lymphadenopathy are the most common symptoms and signs of EBV-PTLD and are, if not treated,

haematologica | 2016; 101(7)

ECIL recommendations for diagnosis and monitoring of EBV DNA-emia Prospective monitoring of EBV DNA performed by quantitative PCR is recommended. There are no data to support a preference for whole blood, plasma or serum; all are appropriate specimens for monitoring EBV DNAemia.7,41-43 Screening for EBV DNA-emia should start within the first month after allo-HSCT. However, the incidence of EBVPTLD during the first month after HSCT is estimated to be below 0.2%.3 Monitoring should continue for at least 4 months after HSCT, with a frequency of at least once a week. As the calculated doubling time for EBV might be as short as 56 hours,44 more frequent sampling in patients with rising EBV DNA-emia may be warranted (Table 5).

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J. Styczynski et al. Table 2. Relative merits of EBV assays.

Material

Value

Recommendation

Reference

Whole blood, plasma, serum Tissue specimen Tissue specimen Tissue specimen

high sensitivity and specificity, low PPV very high sensitivity but low PPV high sensitivity and specificity high specificity but lower sensitivity; variably expressed in PTLD biopsies

AIIu

10-12

DIIu AIIu CIII

13

Assay DNA by PCR

EBER ISH Viral proteins (e.g. LMP1 and EBNA1)

14 15

Table 3. Risk factors for EBV-PTLD after HSCT.

Pre-transplant risk factors • T-cell depletion (either in vivo or ex vivo) • EBV serology donor/recipient mismatch • Cord blood transplantation (CBT) • HLA mismatch • Splenectomy • Second HSCT

Post-transplant risk factors • Severe acute (especially steroid-refractory) or chronic GvHD requiring intensive immunosuppressive therapy • High or rising EBV viral load • Treatment with mesenchymal stem cells

frequently associated with rapidly progressive multi-organ failure and death.45 The diagnostic approach to EBV-PTLD should, preferably, be based on biopsies of enlarged lymph nodes and other sites of suspected EBV disease (Table 5). However, if this is impossible due to the clinical status of the patient, a non-invasive approach, encompassing quantitative EBV DNA-emia combined with PET-CT/CT imaging, can be considered.29,46,47 The diagnostic work-up of EBV-PTLD includes: (a) physical examination, including an examination for fever, tonsillitis, adenopathy and organomegaly; (b) PET-CT/CT imaging; (c) endoscopy in case of gastro-intestinal symptoms; (d) tissue biopsy with histological examination, including EBER ISH and/or immunohistochemistry for viral antigens, and/or flow cytometry; (e) peripheral blood EBV viral load by PCR. The clinical staging of EBV-PTLD includes: nodal vs. extranodal, limited (unifocal) vs. advanced (multifocal) disease.3 The Ann Arbor classification, established for staging of lymphoma, can also be recommended. As PTLD is an FDG-avid malignancy, EBV-PTLD can be staged according to the Lugano classification by PET-CT, both in children and adults.47-50

clonal antibodies), reduction of immunosuppression (RI), EBV-CTL, donor lymphocyte infusion (DLI) and chemotherapy. RI is defined as a sustained decrease of at least 20% of the daily dose of immunosuppressive drugs with the exception of low-dose corticosteroid therapy.21 Pooling results from published studies in HSCT recipients suggest that administration of rituximab results in a positive outcome for approximately 90% patients treated pre-emptively, and 65% with EBV-PTLD.2,3,11,12,19,20,24,27,51-62 Recent data demonstrate that RI, when applied in combination with rituximab, appears to improve the outcome by over 80%.3 RI used alone as preemptive therapy resulted in a 68% success rate.21,51 The use of EBV-CTLs leads to a positive outcome for >90% of patients treated pre-emptively, and approximately 75% in therapy of EBVPTLD.51,63-68 There are no studies directly comparing efficacy of rituximab±RI vs. EBV-CTL in either prophylaxis, preemptive or targeted therapy. Thus, there is insufficient evidence to support a recommendation for one treatment modality over another as a first line approach for centers with access to both therapies.

Management strategies

Rituximab. B-cell depletion by prophylactic use of rituximab before or shortly after allo-HSCT might reduce the risk of EBV DNA-emia and PTLD (Table 6).20,23,69,70 In a large retrospective analysis, prophylactic post-transplant rituximab significantly reduced the risk of EBV DNAemia; however, no statistically significant impact on PTLD incidence, treatment-related mortality, and overall survival in comparison to a pre-emptive approach was demonstrable.69 Low risk of EBV-PTLD was observed also after the use of post-transplant high-dose cyclophosphamide,23 or sirolimus as GvHD prophylaxis.20 Since rituximab treatment after allo-HSCT has been related to an increased risk of life-threatening cytopenias71 and bacterial infections,72

There are three approaches for EBV infection, EBV disease and EBV-PTLD after HSCT: prophylaxis, pre-emptive therapy and treatment of EBV disease/PTLD. Prophylaxis of EBV disease includes any intervention (e.g. drug or cellular therapy) given to an asymptomatic EBV-seropositive patient to prevent EBV DNA-emia. Pre-emptive therapy includes any intervention given to a patient with EBV DNA-emia to prevent EBV disease. Treatment of EBV disease includes therapeutic interventions for patients with probable or proven EBV disease. Prophylaxis and treatment approaches of EBV-PTLD include: administration of rituximab (anti-CD20 mono806

ECIL recommendations for prophylaxis of EBV DNA-emia

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ECIL-6 guidelines for EBV-PTLD after HSCT

Table 4. Recommendations for prevention of EBV disease after HSCT.

Allo-HSCT patients • All allo-HSCT patients and donors should be tested before transplantation for EBV antibodies (AIIu). • For an EBV-seronegative patient, an EBV-seronegative donor is preferred (BIIu). • For an EBV-seropositive recipient, an EBV-seropositive donor might be beneficial, due to the presence of EBV-positive CTLs (CIII). • Patients at high risk for EBV-PTLD after allo-HSCT should be closely monitored for symptoms or signs attributable to PTLD or other end-organ EBV disease (AIIu). • After high-risk allo-HSCT, prospective monitoring of EBV DNA-emia is recommended (AIIu). • The risk in HLA-identical family transplant recipients not receiving T-cell depletion and without GvHD is low and no routine screening for EBV is recommended (DIIu).

Auto-HSCT or conventional chemotherapy patients • It is not recommended that auto-HSCT patients be routinely monitored for EBV before and after HSCT (DIII). • It is not recommended that conventional chemotherapy patients be routinely monitored for EBV before and during treatment (DIII).

Table 5. Recommendations for diagnosis of EBV DNA-emia and EBV-disease/PTLD.

Recommendations for diagnosis of EBV DNA-emia • Prospective screening of EBV DNA-emia by quantitative PCR is recommended after allo-HSCT at high-risk for EBV-PTLD (AIIu). • Whole blood, plasma and serum are all appropriate biological specimens for monitoring EBV DNA-emia (BIIu). • Beginning of screening: no later than 4 weeks after the day of HSCT; in patients with several risk factors earlier screening might be considered (AIIu). • Frequency of screening: testing for EBV DNA is recommended once a week in high-risk EBV PCR-negative patients (BIIu); in patients with rising EBV DNA-emia more frequent sampling might be considered (BIIu). • End of screening: at least 4 months after HSCT in high risk patients (BIIu). • Longer monitoring is recommended in patients considered to have poor T-cell reconstitution: on treatment for severe acute/chronic GvHD, after haplo HSCT, with the use of TCD, after conditioning with ATG/alemtuzumab, or in those having experienced an early EBV reactivation (BIIu).

Recommendations for diagnosis of EBV-disease/PTLD • The diagnosis of EBV-PTLD must be based on symptoms and/or signs consistent with PTLD together with detection of EBV by an appropriate method applied to a specimen from the involved tissue (AIIu). • Non-invasive methods: quantitative EBV DNA-emia (in blood, plasma or serum) (AIIu), and PET-CT/CT (BIIt). PET-CT is preferred to CT in extranodal disease (BIII). • Invasive methods: biopsy of lymph node and/or other sites suspected for EBV disease (AIIu). • Diagnosis of proven EBV-PTLD requires biopsy and histological examination with EBV detection (AIIu). • EBV detection requires in situ hybridization for the EBER transcripts or detection of viral antigens (AIIu).

the use of rituximab should be restricted to patients at highest risk of EBV-PTLD and, following its use, accompanied by close monitoring for hypogammaglobulinemia with consideration of Ig replacement and other strategies to limit infectious-related mortality. EBV-CTLs. High efficacy of prophylaxis has been shown with the use of EBV-CTLs in a high-risk group in one study.63 Current use of EBV-CTLs is, however, limited as it is available only in selected centers. Antiviral drugs. Although aciclovir, ganciclovir, foscarnet, and cidofovir show some in vitro activity against replicating EBV,73 antiviral treatment of latent EBV has been unsuccessful74 since latently infected B cells do not express the EBV thymidine kinase enzyme transcript or protein. There is no evidence to recommend any anti-EBV antiviral prophylaxis in patients with hematological malignancies in non-allo-HSCT setting (DIII).

ECIL recommendations for preemptive therapy against EBV disease Indications. The indication for preemptive therapy is significant EBV DNA-emia without clinical symptoms/disease in patients with high risk for EBV-PTLD (Table 7). The goal of preemptive therapy is to obtain a negative EBV PCR or EBV DNA-emia below the initial threshold without relapse. Implications of EBV DNA-emia. EBV DNA-emia mostly haematologica | 2016; 101(7)

occurs prior to the onset of clinical symptoms but data are somewhat conflicting.7,75-78 Currently available data do not allow elucidation of an EBV-DNA threshold for the development of EBV disease. Indeed, probable/proven PTLD has been described in a significant proportion of patients with EBV DNA levels below commonly adopted intervention thresholds.29 Threshold value. In the absence of universal standards for Nucleic Acid Test assays, ECIL cannot recommend a specific threshold value of EBV DNA-emia for giving preemptive therapy. Some authors employ a threshold of 1,000 EBV copies/mL,10,11,20 10,000 EBV copies/mL,2,12,34,37 or 40,000 EBV copies/mL19,27 when determined in whole blood, plasma, serum; or 1,000 copies as determined per 105 PBMC69 to initiate pre-emptive therapy. The rate of increase of EBV copy number is likely to be clinically significant given that increases in EBV DNA-emia are due to the expansion of EBV-infected memory B cells in the peripheral blood. Local experience based on correlation of clinical and laboratory data might be a rationale for center-specific cut-off value. Rituximab. The primary method for preemptive therapy is rituximab, dose 375 mg/m2, once weekly until EBV DNA-emia negativity. The number of doses should be assessed locally on the basis of changes in EBV DNA-emia and an assessment of the patient’s immune function. Typically, 1-4 doses are sufficient. Reduction of immunosuppression. Rituximab should be 807


J. Styczynski et al. Table 6. Recommendations for prophylaxis against EBV disease.

Recommendations for prophylaxis against EBV disease • B-cell depletion with prophylactic rituximab might reduce the risk of EBV DNA-emia (CIIu). • Prophylactic use of EBV-CTLs should be considered as first line prophylactic treatment whenever possible (CIIu). • There are no data to support any positive impact of antiviral drugs on the development of EBV-PTLD. Antiviral drugs are not recommended for EBV prophylaxis (DIIu). • Interferon and IVIG are not recommended for EBV prophylaxis (DIII). Table 7. Recommendations for preemptive therapy of EBV disease.

Recommendations for preemptive therapy of EBV disease • • • • • •

Significant EBV DNA-emia without clinical symptoms of EBV disease is an indication for preemptive therapy with rituximab (BIIu). No specific threshold of EBV DNA-emia can currently be recommended for initiation of preemptive therapy. Rituximab once weekly (1-4 doses) is recommended until EBV DNA-emia negativity (AIIu). Rituximab should be combined with reduction of immunosuppression, if possible (AIIu). Donor or third party EBV-specific cytotoxic T lymphocytes (CTL) should be considered, if available (CIIu). Antiviral drugs are not recommended for preemptive therapy (DIIh).

Table 8. Recommendations for therapy of EBV-PTLD.

First line therapy in EBV-PTLD 1. Rituximab, 375 mg/m2, once weekly (AIIu). 2. Reduction of immunosuppressive therapy combined with rituximab should always be considered, if possible (AIIu). 3. Cellular therapy as adoptive immunotherapy with in vitro generated donor or third-party EBV-specific CTL, if available (CIIu).

Second line therapy in EBV-PTLD 1. Cellular therapy (EBV specific-CTLs or DLI) (BIII). 2. Chemotherapy±rituximab is a potential option after failure of other methods (CIIh). 3. Surgery, IVIG, interferon and antiviral agents are not recommended for therapy of PTLD (DIII) CNS EBV disease.

CNS EBV disease • Therapeutic options in EBV-PTLD in central nervous system include: rituximab ± chemotherapy (BIIh), rituximab systemic or intrathecal monotherapy (CIII), anti-EBV T-cell therapy (CIII) or radiotherapy (CIII).

combined with RI, if possible, except in patients with uncontrolled severe acute or chronic GvHD. Other options. Donor or third party EBV-specific cytotoxic T lymphocytes (CTL) are highly efficacious; however, this approach is not widely available. Antiviral drugs are not effective against EBV.

ECIL recommendations for treatment of EBV-PTLD First line therapy. In case of proven or probable EBVPTLD, therapy should be started as soon as practicable due to the risk of a rapidly growing high-grade lymphoid tumor, together with the risk of multi-organ impairment. Rituximab monotherapy is the treatment of choice for EBV-PTLD (Table 8) with positive outcome reported in almost 70% of patients. Rituximab is usually administered once weekly for up to 4 doses while monitoring EBV viral load. Additional doses might result in down-regulation of CD20 expression and thereby possibly decreased efficacy. Reduction of immunosuppression (RI) is rarely successful as the sole intervention in PTLD following HSCT,21,79 and may increase the risk of rejection or GvHD.77 It should be combined with rituximab administration.3 Additionally, rituximab may reduce the risk of acute/chronic GvHD.80,81 Central nervous system (CNS) EBV disease. CNS localisation of EBV-PTLD warrants special consideration due to the risk of neurocognitive dysfunction, notwithstanding the successful eradication of EBV-infected cells from the 808

CNS. To date, no standard therapy has been accepted. Possible therapeutic options include: (i) chemotherapy±rituximab in line with primary CNS lymphoma protocols based on high dose methotrexate and/or cytarabine82 or hydroxyurea;83 (ii) monotherapy with rituximab, either systemic3,84 or intrathecal;85 (iii) T-cell therapy with EBVspecific CTLs;63,68 (iv) radiotherapy. Response to therapy. The treatment goal is resolution of all signs and symptoms of PTLD, including a negative viral load. Response to rituximab therapy can be identified by a decrease in EBV DNA-emia of at least 1 log10 in the first week of treatment (BIIh). Younger age is a favourable factor predicting outcome to rituximab-based therapy. Positive prognostic factors for outcome to rituximab therapy include: age below 30 years, underlying non-malignant disease, no acute GvHD, RI at EBV-PTLD diagnosis, and decrease of EBV DNA-emia after initial therapy.3 Second line therapy. In the setting of rituximab failure, second-line therapy options include cellular therapy (DLI or CTLs) or chemotherapy±rituximab. Unselected DLI from an EBV-positive donor are employed to restore broad T-cell reactivity, including EBV-specific responses; unselected DLI, however, can be associated with severe GvHD.86,87 Previous GvHD is usually a contraindication to DLI. ECIL’s preferred approach is specific cellular therapy; however, EBV-specific CTLs are not readily available in all centers. Apart from donor-derived CTLs, the haematologica | 2016; 101(7)


ECIL-6 guidelines for EBV-PTLD after HSCT

novel development of 3rd party EBV-CTLs may represent a promising option for the recipients of cord blood transplant, or those who have EBV-negative donors and/or donors who are unable to provide further donation for cellular therapy.64,66-68 Data on efficacy of DLI or chemotherapy in EBV-PTLD are limited. Chemotherapy for EBV-PTLD after HSCT is not recommended as firstline therapy due to poor tolerability in HSCT patients and the risk of inducing neutropenia and graft failure.51 Chemotherapy for EBV-PTLD is therefore restricted for refractory/relapsing cases.88

ECIL recommendations for treatment of EBV-negative and/or T-PTLD A growing number of cases of EBV-negative B-PTLD have been reported, presenting late (>5 years) after transplant. These cases should be regarded as malignant lymphoma, not PTLD, and treated with appropriate chemotherapy protocols. T-PTLD after HSCT are extremely rare, and also should be regarded as malignant lymphoma and treated with appropriate chemotherapy protocols.

Possible future developments The possible future anti-EBV prophylaxis and/or therapies include cellular therapy, new monoclonal antibodies and new antivirals. Active immunization against EBV is not available. Ex vivo-generated EBV-CTL have proved to be an effective prophylactic measure, pre-emptive therapy, or treatment for PTLD post-HSCT. EBV-CTL can be isolated and expanded ex vivo from EBV-seropositive stem cell or third-party donors. Considering the recent success and safety profile of obinutuzumab in CD20-positive malignancies,89,90 novel anti-CD20 monoclonal antibodies are possible candidates for future use in EBV-PTLD. The possibility of new and experimental therapies for EBVPTLD has also recently emerged in the transplant setting, including brentuximab vedotin, anti-CD30 antibodies. Brincidofovir, a new, currently unlicensed antiviral agent, has excellent antiviral activity against EBV in vitro.91 Further

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study, however, is needed in order to establish whether prophylaxis with this drug will be able to reduce the risk of EBV replication and possibly EBV-PTLD. Acknowledgments The authors would like to thank the participants of the ECIL-6 meeting: Manuel Abecassis, Portugal; Murat Akova, Turkey; Mahmoud Aljurf, Saudi Arabia; Dina Averbuch, Israel; Rose Mary Barnes, UK; Ola Blennow, Sweden; Pierre Yves Bochud, Switzerland; Emilio Bouza, Spain; Stephane Bretagne, France; Roger BrĂźggemann, The Netherlands; Thierry Calandra, Switzerland; Jordi Carratala, Spain; Simone Cesaro, Italy; Catherine Cordonnier, France; Oliver Cornely, Germany; Tina Dalianis, Sweden; Rafael De La Camara, Spain; Peter Donnelly, The Netherlands; Lubos Drgona, Slovakia; Rafael Duarte, Spain; Hermann Einsele, Germany; Dan Engelhard, Israel; Christopher Fox, UK; Corrado Girmenia, Italy; Andreas Groll, Germany; Dag Heldal, Norway; Jannick Helweg-Larsen, Denmark; Raoul Herbrecht, France; Hans Hirsch, Switzerland; Elisabeth Johnson, UK; Galina Klyasova, Russia; Minna Koskuenvo, Finland; Katrien Lagrou, Belgium; Russel Lewis, Italy; Per Ljungman, Sweden; Johan Maertens, Belgium; Georg Maschmeyer, Germany; Malgorzata Mikulska, Italy; Marcio Nucci, Brazil; Christophe Padoin, France; Livio Pagano, Italy; Antonio Pagliuca, UK; Zdenek Racil, Czech Republic; Patricia Ribaud, France; Christine Rinaldo, Norway; ValĂŠrie Rizzi-Puechal (Pfizer), France; Emmanuel Roilides, Greece; Christine Robin, France; Montserrat Rovira, Spain; Markus Rupp (MSD), Germany; Sonia Sanchez (Gilead Sciences), UK; Peter Schellongovski, Austria; Peter Sedlacek, Czech Republic; Janos Sinko, Hungary; Monica Slavin, Australia; Isabelina Sousa Ferreira, Portugal; Jan Styczynski, Poland; Frederic Tissot, Switzerland; Andrew Ullman, Germany; Marie von Lilienfeld-Toal, Germany; Claudio Viscoli, Italy; Katherine Ward, UK; Anne-Therese Witschi (Basilea), Switzerland. The authors thank the group GL-Events, Lyon, France, for the organization of the meeting. Funding The ECIL-6 meeting has been supported by unrestricted educational grants from Basilea, Gilead Sciences, Merck and Pfizer.

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2007;45(7):2151-2155. 42. Baldanti F, Gatti M, Furione M, et al. Kinetics of Epstein-Barr virus DNA load in different blood compartments of pediatric recipients of T-cell-depleted HLA-haploidentical stem cell transplantation. J Clin Microbiol. 2008;46(11):3672-3677. 43. Ruf S, Behnke-Hall K, Gruhn B, et al. Comparison of six different specimen types for Epstein-Barr viral load quantification in peripheral blood of pediatric patients after heart transplantation or after allogeneic hematopoietic stem cell transplantation. J Clin Virol. 2012;53(3):186-194. 44. Stevens SJ, Verschuuren EA, Pronk I, et al. Frequent monitoring of Epstein-Barr virus DNA load in unfractionated whole blood is essential for early detection of posttransplant lymphoproliferative disease in highrisk patients. Blood. 2001;97(5):1165-1171. 45. Xuan L, Jiang X, Sun J, et al. Spectrum of Epstein-Barr virus-associated diseases in recipients of allogeneic hematopoietic stem cell transplantation. Transplantation. 2013;96(6):560-566. 46. Dierickx D, Tousseyn T, Requile A, et al. The accuracy of positron emission tomography in the detection of posttransplant lymphoproliferative disorder. Haematologica. 2013;98(5):771-775. 47. Cheson BD, Fisher RI, Barrington SF, et al. Recommendations for initial evaluation, staging, and response assessment of Hodgkin and non-Hodgkin lymphoma: the Lugano classification. J Clin Oncol. 2014;32(27):3059-3068. 48. Barrington SF, Mikhaeel NG, Kostakoglu L, et al. Role of imaging in the staging and response assessment of lymphoma: consensus of the International Conference on Malignant Lymphomas Imaging Working Group. J Clin Oncol. 2014;32(27):3048-3058. 49. Sandlund JT, Guillerman RP, Perkins SL, et al. International Pediatric Non-Hodgkin Lymphoma Response Criteria. J Clin Oncol. 2015;33(18):2106-2111. 50. Rosolen A, Perkins SL, Pinkerton CR, et al. Revised International Pediatric NonHodgkin Lymphoma Staging System. J Clin Oncol. 2015;33(18):2112-2118. 51. Styczynski J, Einsele H, Gil L, Ljungman P. Outcome of treatment of Epstein-Barr virusrelated post-transplant lymphoproliferative disorder in hematopoietic stem cell recipients: a comprehensive review of reported cases. Transpl Infect Dis. 2009;11(5):383392. 52. Blaes AH, Cao Q, Wagner JE, Young JA, Weisdorf DJ, Brunstein CG. Monitoring and preemptive rituximab therapy for EpsteinBarr virus reactivation after antithymocyte globulin containing nonmyeloablative conditioning for umbilical cord blood transplantation. Biol Blood Marrow Transplant. 2010;16(2):287-291. 53. Coppoletta S, Tedone E, Galano B, et al. Rituximab treatment for Epstein-Barr virus DNAemia after alternative-donor hematopoietic stem cell transplantation. Biol Blood Marrow Transplant. 2011;17(6):901907. 54. D'Aveni M, Aissi-Rothe L, Venard V, et al. The clinical value of concomitant Epstein Barr virus (EBV)-DNA load and specific immune reconstitution monitoring after allogeneic hematopoietic stem cell transplantation. Transpl Immunol. 2011; 24(4):224-232. 55. Muramatsu H, Takahashi Y, Shimoyama Y, et al. CD20-negative Epstein-Barr virus-associated post-transplant lymphoproliferative

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ECIL-6 guidelines for EBV-PTLD after HSCT

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disease refractory to rituximab in a patient with severe aplastic anemia. Int J Hematol. 2011;93(6):779-781. Bordon V, Padalko E, Benoit Y, Dhooge C, Laureys G. Incidence, kinetics, and risk factors of Epstein-Barr virus viremia in pediatric patients after allogeneic stem cell transplantation. Pediatr Transplant. 2012;16(2):144150. Pinana JL, Sanz J, Esquirol A, et al. Umbilical cord blood transplantation in adults with advanced hodgkin's disease: high incidence of post-transplant lymphoproliferative disease. Eur J Haematol. 2016;96(2):128-135. Kuriyama T, Kawano N, Yamashita K, Ueda A. Successful treatment of Rituximab-resistant Epstein-Barr virus-associated post-transplant lymphoproliferative disorder using RCHOP. J Clin Exp Hematop. 2014;54(2):149153. Meyer SC, Medinger M, Halter JP, et al. Heterogeneity in clinical course of EBV-associated lymphoproliferative disorder after allogeneic stem cell transplantation. Hematology. 2014;19(5):280-285. Han SB, Bae EY, Lee JW, et al. Features of Epstein-Barr virus reactivation after allogeneic hematopoietic cell transplantation in Korean children living in an area of high seroprevalence against Epstein-Barr virus. Int J Hematol. 2014;100(2):188-199. Helgestad J, Rosthoj S, Pedersen MH, et al. Very late relapse of PTLD 10 yr after allogeneic HSCT and nine yr after stopping immunosuppressive therapy. Pediatr Transplant. 2014;18(1):E35-39. Weber T, Wickenhauser C, Monecke A, et al. Treatment of rare co-occurrence of Epstein-Barr virus-driven post-transplant lymphoproliferative disorder and hemophagocytic lymphohistiocytosis after allogeneic stem cell transplantation. Transpl Infect Dis. 2014;16(6):988-992. Heslop HE, Slobod KS, Pule MA, et al. Longterm outcome of EBV-specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients. Blood. 2010;115(5):925-935. Barker JN, Doubrovina E, Sauter C, et al. Successful treatment of EBV-associated posttransplantation lymphoma after cord blood transplantation using third-party EBV-specific cytotoxic T lymphocytes. Blood. 2010;116(23):5045-5049. Moosmann A, Bigalke I, Tischer J, et al. Effective and long-term control of EBV PTLD after transfer of peptide-selected T cells. Blood. 2010;115(14):2960-2970. Doubrovina E, Oflaz-Sozmen B, Prockop SE, et al. Adoptive immunotherapy with unselected or EBV-specific T cells for biopsyproven EBV+ lymphomas after allogeneic hematopoietic cell transplantation. Blood. 2012;119(11):2644-2656. Leen AM, Bollard CM, Mendizabal AM, et al. Multicenter study of banked third-party virus-specific T cells to treat severe viral infections after hematopoietic stem cell transplantation. Blood. 2013;121(26):5113-5123.

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68. Vickers MA, Wilkie GM, Robinson N, et al. Establishment and operation of a Good Manufacturing Practice-compliant allogeneic Epstein-Barr virus (EBV)-specific cytotoxic cell bank for the treatment of EBV-associated lymphoproliferative disease. Br J Haematol. 2014;167(3):402-410. 69. Dominietto A, Tedone E, Soracco M, et al. In vivo B-cell depletion with rituximab for alternative donor hemopoietic SCT. Bone Marrow Transplant. 2012;47(1):101-106. 70. Liu D, Tammik C, Zou JZ, et al. Effect of combined T- and B-cell depletion of allogeneic HLA-mismatched bone marrow graft on the magnitude and kinetics of EpsteinBarr virus load in the peripheral blood of bone marrow transplant recipients. Clin Transplant. 2004;18(5):518-524. 71. McIver Z, Stephens N, Grim A, Barrett AJ. Rituximab administration within 6 months of T cell-depleted allogeneic SCT is associated with prolonged life-threatening cytopenias. Biol Blood Marrow Transplant. 2010;16(11):1549-1556. 72. Petropoulou AD, Porcher R, Peffault de Latour R, et al. Increased infection rate after preemptive rituximab treatment for EpsteinBarr virus reactivation after allogeneic hematopoietic stem-cell transplantation. Transplantation. 2012;94(8):879-883. 73. Williams-Aziz SL, Hartline CB, Harden EA, et al. Comparative activities of lipid esters of cidofovir and cyclic cidofovir against replication of herpesviruses in vitro. Antimicrob Agents Chemother. 2005; 49(9):3724-3733. 74. Perrine SP, Hermine O, Small T, et al. A phase 1/2 trial of arginine butyrate and ganciclovir in patients with Epstein-Barr virusassociated lymphoid malignancies. Blood. 2007;109(6):2571-2578. 75. Gartner BC, Schafer H, Marggraff K, et al. Evaluation of use of Epstein-Barr viral load in patients after allogeneic stem cell transplantation to diagnose and monitor posttransplant lymphoproliferative disease. J Clin Microbiol. 2002;40(2):351-358. 76. Aimoto M, Yamane T, Inoue A, et al. [Epstein-Barr virus-associated post-transplant lymphoproliferative disorder diagnosed by the episode of intestinal perforation following allogeneic hematopoietic stem cell transplantation]. Rinsho Ketsueki. 2010;51(12):1775-1780. 77. Gross TG. Treatment for Epstein-Barr virusassociated PTLD. Herpes. 2009; 15(3):64-67. 78. Weinstock DM, Ambrossi GG, Brennan C, Kiehn TE, Jakubowski A. Preemptive diagnosis and treatment of Epstein-Barr virusassociated post transplant lymphoproliferative disorder after hematopoietic stem cell transplant: an approach in development. Bone Marrow Transplant. 2006;37(6):539546. 79. Cesaro S, Murrone A, Mengoli C, et al. The real-time polymerase chain reaction-guided modulation of immunosuppression enables the pre-emptive management of EpsteinBarr virus reactivation after allogeneic haematopoietic stem cell transplantation. Br

J Haematol. 2005;128(2):224-233. 80. Ratanatharathorn V, Ayash L, Reynolds C, et al. Treatment of chronic graft-versus-host disease with anti-CD20 chimeric monoclonal antibody. Biol Blood Marrow Transplant. 2003;9(8):505-511. 81. Ratanatharathorn V, Logan B, Wang D, et al. Prior rituximab correlates with less acute graft-versus-host disease and better survival in B-cell lymphoma patients who received allogeneic peripheral blood stem cell transplantation. Br J Haematol. 2009;145(6):816824. 82. Mahapatra S, Chin CC, Iagaru A, HeeremaMcKenney A, Twist CJ. Successful treatment of systemic and central nervous system post-transplant lymphoproliferative disorder without the use of high-dose methotrexate or radiation. Pediatr Blood Cancer. 2014;61(11):2107-2109. 83. Pakakasama S, Eames GM, Morriss MC, et al. Treatment of Epstein-Barr virus lymphoproliferative disease after hematopoietic stem-cell transplantation with hydroxyurea cytotoxic T-cell lymphocytes. and Transplantation. 2004;78(5):755-757. 84. Wroblewska M, Gil LA, Komarnicki MA. Successful treatment of Epstein-Barr virusrelated post-transplant lymphoproliferative disease with central nervous system involvement following allogeneic haematopoietic stem cell transplantation - a case study. Cent Eur J Immunol. 2015;40(1):122-125. 85. Czyzewski K, Styczynski J, Krenska A, et al. Intrathecal therapy with rituximab in central nervous system involvement of post-transplant lymphoproliferative disorder. Leuk Lymphoma. 2013;54(3):503-506. 86. Lucas KG, Burton RL, Zimmerman SE, et al. Semiquantitative Epstein-Barr virus (EBV) polymerase chain reaction for the determination of patients at risk for EBVinduced lymphoproliferative disease after stem cell transplantation. Blood. 1998;91 (10):3654-3661. 87. Papadopoulos EB, Ladanyi M, Emanuel D, et al. Infusions of donor leukocytes to treat Epstein-Barr virus-associated lymphoproliferative disorders after allogeneic bone marrow transplantation. N Engl J Med. 1994;330(17):1185-1191. 88. Knight JS, Tsodikov A, Cibrik DM, Ross CW, Kaminski MS, Blayney DW. Lymphoma after solid organ transplantation: risk, response to therapy, and survival at a transplantation center. J Clin Oncol. 2009;27(20):3354-3362. 89. Goede V, Fischer K, Busch R, et al. Obinutuzumab plus chlorambucil in patients with CLL and coexisting conditions. N Engl J Med. 2014;370(12):1101-1110. 90. Illidge T, Klein C, Sehn LH, Davies A, Salles G, Cartron G. Obinutuzumab in hematologic malignancies: lessons learned to date. Cancer Treat Rev. 2015;41(9):784-792. 91. Hostetler KY. Synthesis and early development of hexadecyloxypropylcidofovir: an oral antipoxvirus nucleoside phosphonate. Viruses. 2010;2(10):2213-2225.

811


ARTICLE EUROPEAN HEMATOLOGY ASSOCIATION

Hematopoiesis

Ferrata Storti Foundation

Human thrombopoiesis depends on Protein kinase Cδ/protein kinase Cε functional couple

Cecilia Carubbi,1 Elena Masselli,1 Silvia Martini,1 Daniela Galli,1 Franco Aversa,2 Prisco Mirandola,1 Joseph E. Italiano Jr,3 Giuliana Gobbi,1 and Marco Vitale1

Department of Biomedical, Biotechnological and Translational Sciences (SBiBiT), University of Parma, Italy; 2Department of Clinical and Experimental Medicine, University of Parma, Italy; and 3Hematology Division, Department of Medicine, Brigham and Women’s Hospital, and Harvard Medical School, Boston, MA, USA. 1

Haematologica 2016 Volume 101(7):812-820

ABSTRACT

A

Correspondence: marco.vitale@unipr.it

Received: October 16, 2015. Accepted: April 12, 2016. Pre-published: April 14, 2016. doi:10.3324/haematol.2015.137984

Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/101/7/812

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

812

deeper understanding of the molecular events driving megakaryocytopoiesis and thrombopoiesis is essential to regulate in vitro and in vivo platelet production for clinical applications. We previously documented the crucial role of PKCε in the regulation of human and mouse megakaryocyte maturation and platelet release. However, since several data show that different PKC isoforms fulfill complementary functions, we targeted PKCε and PKCδ, which show functional and phenotypical reciprocity, at the same time as boosting platelet production in vitro. Results show that PKCδ, contrary to PKCε, is persistently expressed during megakaryocytic differentiation, and a forced PKCδ down-modulation impairs megakaryocyte maturation and platelet production. PKCδ and PKCε work as a functional couple with opposite roles on thrombopoiesis, and the modulation of their balance strongly impacts platelet production. Indeed, we show an imbalance of PKCδ/PKCε ratio both in primary myelofibrosis and essential thrombocythemia, featured by impaired megakaryocyte differentiation and increased platelet production, respectively. Finally, we demonstrate that concurrent molecular targeting of both PKCδ and PKCε represents a strategy for in vitro platelet factories.

Introduction Platelets are circulating anucleate elements derived from megakaryocytes (MK), with major roles in hemostasis, thrombosis and inflammation.1,2 Appropriate numbers and function of circulating platelets are essential in hemostasis. Indeed, uncontrolled platelet release and activation is associated with thrombotic risk.3,4 On the contrary, low levels of platelets, as well as their functional defects, might compromise the healing of wounds, resulting in bleeding.5 The therapeutic strategy to prevent severe bleeding is platelet transfusion; however, the use of platelet units derived from human donors has several limitations.6,7 Consequently, both scientific and technological efforts are currently active for generating large platelet supplies, including in vitro platelet producing systems and pharmacological treatments able to modulate in vivo thrombopoiesis and platelet production.8,9 A deeper understanding of the molecular regulation of thrombopoiesis clearly plays a key role in this context. Thrombopoiesis is a complex process resulting in the generation of thousands of platelets from a single megakaryocyte which, following polyploidization, forms elongated cellular processes called proplatelets (proPLT).10 Several molecules, including transcription factors and their intermediates, have been found to be involved in the regulation of this process and the perturbation of the expression and activity of these proteins leads to alterations in platelet number, morphology or function.11-15 Protein kinase C (PKC) is a family of serine-threonine kinase involved in many cellular functions, including cell death, proliferation, migration and differentiation.16,17 Protein Kinase C epsilon (PKCε) and Protein Kinase C delta (PKCδ) are both members of the novel sub-family of PKCs, which can be considered as “yin and haematologica | 2016; 101(7)


PKCdelta in human thrombopoiesis

yang” because of their antithetical roles in several cellular functions.18 PKCε is largely considered as an oncogene because of its anti-apoptotic and pro-proliferative functions,19 whereas PKCδ generally slows down proliferation and induces cell cycle arrest and apoptosis.20,21 In the heart, they are among the most widely expressed PKC isoforms, playing an opposite role in ischemic-reperfusion preconditioning.22,23 In the hematopoietic system it has been demonstrated that protein expression levels of ε and δ isoforms are opposite during erythroid differentiation.24-26 Moreover, while PKCε down-regulation sensitizes primary acute myeloid leukemia (AML) blasts to the apoptogenic and pro-differentiative effects of TRAIL,27 PKCδ activation mediates pro-differentiative and antileukemic effects of statin and INF-a in AML blasts, including acute promyelocytic leukemia cells.28-30 We previously demonstrated that PKCε has a key role in human megakaryocytopoiesis in vitro27,31 and platelet function in vivo,3 as well as in proPLT production in the murine model.32 Specifically, PKCε levels increase in the early phase of in vitro human megakaryocytic (MK) differentiation and decrease in the late phase before platelets release,31 and a forced PKCε overexpression prevents MK full maturation,31 while its down-regulation increases MK differentiation.33 Additionally, more recently we have demonstrated that primary MK from myelofibrotic (PMF) patients express higher levels of PKCε than those from healthy donors (HD), and that PKCε inhibition in PMF restores a bona fide normal MK differentiation.33 Although in murine models it has been demonstrated that PKCδ deficiency enhances megakaryopoiesis,34 its role in human

megakaryocytopoiesis still remains unexplored. The few data available from the literature shows increased levels of the delta isoform in K562 and HEL cell lines when committed to megakaryocytic differentiation.35-37 On these bases we hypothesized that PKCε and PKCδ may have an antithetical role in human MK differentiation and platelet formation. Therefore we investigated herein the role of PKCδ during in vitro human normal and malignant megakaryocytopoiesis and the effects of PKCε and PKCδ modulation on platelet release, in the translational perspective of clinical applications.

Methods CD34+ cell isolation and cell culture Primary CD34+ cells were isolated from peripheral blood of healthy donors, primary myelofibrosis (PMF) patients, and essential thrombocythemia (ET) patients. Samples were collected following written informed consent and approval by the Ethical Committee of Parma University Hospital, Italy. Clinical and laboratory characteristics of PMF and ET patients are reported in Table 1. Cells were cultured for up to 14 days in serum free X-Vivo medium supplemented with recombinant human thrombopoietin, recombinant human stem cell factor and recombinant human interleukin-3. For details see the Online Supplementary Material.

shRNA cell infection For shRNA-based gene silencing we used a pLKO.1 lentiviral vector encoding short hairpin RNAs (shRNA) against human PKCδ and, as control, a MISSION pLKO.1-puro Non-Target

Table 1. Clinical and laboratory characteristics of PMF and ET patients.

PMF patients PLT Blast mm3 %

Code

Sex, age

JAK2 V617F

WBC mm3

Hb g/dL

PMF1 PMF2 PMF3 PMF4 PMF5 PMF6 PMF7 PMF8 PMF9

F, 65 M, 35 M, 64 M, 33 M,55 M,66 F, 68 M, 65 M, 59

pos pos pos pos neg pos nd pos pos

10,130 30,510 66,200 27,260 5,650 5,000 8,740 25,100 6,120

11.6 11.2 8.2 10.9 13.6 7.6 10.5 10.8 15.6

Code

Sex, age

JAK2

WBC

Hb

ET patients Ht PLT

ET1 ET2 ET3 ET4 ET5 ET6 ET7

F, 12 F, 77 F, 55 F, 66 F, 73 F, 47 F, 85

V617F nd pos pos pos neg pos pos

mm3 11,390 7,920 8,200 13,380 6,860 11,210 10,120

g/dl 14.4 14 13.8 15.4 13.9 13.8 15.3

(%) 43.2 43.4 41 45.7 41.3 41.4 46.6

360,000 257,000 525,000 210,000 784,000 107,000 1,080,000 843,000 950,000

0 3 1 2 0 8 0 1 0

mm3 661,000 633,000 689,000 951,000 642,000 565,000 736,000

Spleen Ø cm

IPSS*

DIPSS#

Constitutional symptoms

18,5 28,5 22 23.5 9 12 8 14 12.6

High Low

Int-1 High High High Int-1 -

yes yes yes yes no yes no no no

Int-1 Low

Spleen Ø

IPSET

thrombosis

cm 11.5 10.5 9.6 8 8.5 9 12

int high low high int int int

no yes no no no no no

*IPSS (International Prognostic Scoring System) has been calculated for patients with newly diagnosed disease at time of sample collection. #DIPSS (Dynamic International Prognostic Scoring System) has been calculated for patients with ongoing disease at time of sample collection. IPSET (International Prognostic Score for ET), has been calculated for patients with newly diagnosed disease at time of sample collection. Int-1: intermediate-1; Int: intermediate; neg: negative; nd: not determined; pos: positive.

haematologica | 2016; 101(7)

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C. Carubbi et al.

Pharmacological inhibition and activation of PKCδ and PKCε activity

declining in the final steps of this process. On the contrary, herein we find that human PKCδ levels rise at the beginning of megakaryocytopoiesis, remaining high throughout the entire maturation process (Figure 1A,B). On the basis of the previous results obtained by our31,33 and other groups,41 we proceeded to assess the level of expression of Bcl-xL and Bax, involved in both normal and neoplastic megakaryocytic differentiation and known as downstream mediators of PKCε anti-apoptotic and PKCδ pro-apoptotic effects.42 We found that both Bcl-xL and Bax expressions are significantly modulated in differentiating MKs, with a kinetic similar to PKCε and PKCδ, respectively (Figure 1A,C).

Morphological evaluation of MK differentiation

PKCδ down-regulation reverses the normal expression of Bcl-xL and Bax

shRNA Control Plasmid, containing an shRNA insert that does not target any known genes from any species. Cells were infected at Day 8 of TPO-culture, selected according to puromycin-resistance cells and cultured for up to 14 days. For details see the Online Supplementary Material.

PKCδ and PKCε activities were inhibited by δV1-1 (SFNSYELGSL) and by εV1-2 (CEAVSLKPT) peptides, respectively, whereas PKCδ or PKCε activities were enhanced by using ψδRACK (MRAAEDPM) or ψεRACK (CHDAPIGYD) peptides.23,38 For details see the Online Supplementary Material.

At 14 days of culture, cells were analyzed using a phase contrast microscope (40X/0.5NA). The percentage of megakaryocytes extending proPLT and cell diameter were determined using ImageJ software analyzing a minimum of 100 cells for each treatment from at least 4 independent experiments. For details see the Online Supplementary Material.

Flow cytometric analysis Flow cytometry analyses were performed at day 14 of culture. Cell culture viability was assessed by FITC conjugate Annexin V (ACTIPLATE; Valter Occhiena, Torino, Italy) in Ca2+ and PI staining buffer, following manufacturer's protocol. For ploidy analysis, cells were permealized with 70% ethanol overnight and incubated in PBS containing PI 80*10-6 mmol/L and RNAse-A 7*10-3 mmol/L for 15 minutes before flow cytometry analysis. Platelets produced in culture were quantified by staining with anti-CD41-RPE and Calcein AM and adding a fixed volume of calibration beads at known concentration, as previously described.27,39,40 Analysis of the samples was performed by a FC500 flow cytometer and the Expo ADC software (Beckman Coulter). For details see the Online Supplementary Material.

Western blot Cultured cells were collected on days 0, 3, 6, 9 and 14 for healthy donors and on day 14 for PMF and ET patients. Cells were lysed and 25 μg of proteins from each sample were run on SDSacrylamide gels, blotted onto nitrocellulose membranes and incubated with specific primary antibodies. Specifically, we used mouse monoclonal anti-PKCδ antibody, rabbit polyclonal antiPKCε antibody, rabbit polyclonal anti-Bax antibody, rabbit polyclonal anti-Bcl-xL antibody and monoclonal anti-GAPDH antibody, and secondary antibody peroxidase-conjugated anti-rabbit or peroxidase-conjugated anti-mouse IgG. Proteins were resolved by a chemiluminescence detection method and densitometric analyses were performed by using the ImageJ software system. Statistical analysis was performed using a t-test or analysis of variance (ANOVA) and Tukey’s test, when applicable. For details see the Online Supplementary Material.

Results PKCδ/PKCε and Bax/Bcl-xL expression levels are differently modulated during MK differentiation In agreement with our previous studies in human megakaryocyte cultures,31 PKCε protein expression increases during the early phases of MK differentiation, 814

We previously demonstrated that during the late phases of MK differentiation the forced expression of PKCε induces Bcl-xL up-regulation.31 Taking advantage of PKCδspecific shRNA, we sought to determine whether PKCδ expression was necessary to keep Bcl-xL and Bax expression at the levels required for a successful megakaryocytopoiesis. Therefore we used recombinant lentiviral vectors to introduce and stably express shRNA that specifically target PKCδ into MK differentiating cells at day 8 of culture. Analysis of puromicyn-selected megakaryocyte cultures at day 14 (day 5 post-infection) revealed that abrogation of PKCδ was specific, not modifying the expression of PKCε (Figure 2A,B). However, the selective down-regulation of PKCδ dramatically reduces Bax while, to the contrary, boosting Bcl-xL expression (Figure 2A,B). The densitometric analysis (Figure 2B) of Western blot assays clearly shows the significant modulation of the tested proteins only in the presence of PKCδ-specific shRNA (shPKCδ), as compared to the samples infected with control shRNA (shCT), which are similar to uninfected controls.

PKCδ down-regulation impairs MK differentiation and platelet formation We previously demonstrated that in mouse MK differentiation the PKCε down-regulation impairs proplatelet production.32 Furthermore, Kostyak and colleagues have shown that, in a mouse model, PKCδ down-regulation reinforces MK differentiation and platelet production.34 Since it is well documented that PKCε and PKCδ have opposite expression and function in mouse versus human platelets,43 we hypothesized that high levels of PKCδ are necessary for adequate human MK differentiation and platelet release. Indeed, analysis of puromycin-selected human MK cultures at day 5 post-infection revealed that abrogation of PKCδ impaired MK differentiation (Figure 3). We showed that PKCδ-specific shRNA (shPKCδ) infected cells resulted more viable (Figure 3A), smaller (Figure 3B) and less polyploid (Figure 3C,D), as compared to controls (Uninfected and shCT). Moreover, although few residual branched protrusions could still be observed, proPLTs generated by shPKCδinfected cells were characterized by few abortive branches (Figure 4A). On the contrary, shCT proPLTs, as well as uninfected samples, were characterized by the presence of proPLTs formation (Figure 4A). Platelet release in the culture medium is the terminal step of MK differentiation and, in our system, we haematologica | 2016; 101(7)


PKCdelta in human thrombopoiesis

observed a reduction of greater than 50% in platelet numbers in PKCδ knockout cultures (Figure 4B).

The PKCδ and PKCε balance is altered in human pathological megakaryocytopoiesis. In our model, PKCε and PKCδ have opposite expression levels at the end (day 14) of MK differentiation (Figure 1). We hypothesized that the proper expression of both PKC isoforms could be critical for terminal megakaryocytopoiesis and platelet production. In order to test our speculation, CD34+ cells were isolated from the peripheral blood of both patients affected by PMF and ET, which are hematologic neoplasms characterized by abnormal MK differentiation and platelet production. Specifically, MKs

generated in vitro from PMF CD34+ cells show an impaired differentiation and proplatelet formation; conversely, an increase in proplatelet formation is normally observed in ET CD34+ cell cultures.33,44 PMF, ET and HD isolated CD34+ cells were therefore cultured up to day 14 in the presence of TPO, in order to induce MK differentiation, and then collected for Western blot analysis (Figure 5). As compared to HD, PKCε and Bcl-xL expression was significantly higher in PMF (in agreement with published data33,45), and significantly lower in ET (Figure 5A). On the contrary, PKCδ and Bax showed an opposite modulation, being significantly increased in ET and almost halved in PMF, (Figure 5A), hinting again at an antithetical role of PKCε and δ on thrombopoiesis.

A

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Figure 1. PKCδ/PKCε and Bax/Bcl-xL expression levels are differently modulated during MK differentiation. (A) Western blot detection of PKCδ, PKCε, Bax, Bcl-xL protein expression in CD34+-derived MK cultures. GAPDH was monitored for protein loading. (B) Relative PKCδ and PKCε protein expression during megakaryocytic differentiation of human CD34+ cells normalized for GAPDH expression levels. Densitometric measurements of Western blots from 3 replicates were performed by ImageJ software (means ± SD; *P<0.05 ANOVA and Tukey's Tests), Error Bar = SD. (C) Relative Bax and Bcl-xL protein expression during megakaryocytic differentiation of human CD34+ cells normalized for GAPDH expression levels. Densitometric measurements of Western blots from 3 replicates were performed by ImageJ software (means ± SD; * P<0.05 ANOVA and Tukey's Tests), Error Bar = SD.

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Figure 2. PKCδ down-regulation reverses the normal expression of Bcl-xL and Bax. (A) Western blot detection of PKCδ, PKCε, Bax and Bcl-xL in uninfected CD34+-derived MK cultures (Uninfected) and in puromycinselected CD34+-derived MK cultures infected with PKCδ-specific shRNA (shPKCδ), and with Non-Target shRNA control (shCT) at day 5 post-infection. GAPDH was monitored for protein loading. (B) Densitometric analyses of proteins expression, normalized for GAPDH expression levels and expressed as fold increase of shCT, were performed using ImageJ software. Densitometric measurements of Western blots from 4 replicates (means ± one SD; *P<0.05 ANOVA and Tukey's Tests), Error Bar = SD.

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In summary, at day 14 of culture decreased PKCδ/PKCε and Bax/Bcl-xL ratios typify diseases –like PMF- characterized by impaired MK differentiation and proplatelet formation. On the contrary, these ratio values increase in MK culture characterized by enhanced megakaryocytopoiesis and increased proplatelet and platelet formation, like in ET (Figure 5B).

The amount of platelet production can be modified by modulating PKCε/PKCδ function Given these results, we asked whether the pharmacological modulation of PKCε and PKCδ activity might impact platelet formation in normal and pathologic conditions. MK differentiating cells were treated with specific activatory and/or inhibitory peptides at day 8 and then cultured for a further 5 days. In MK precursors, both the concomitant inhibition of PKCε and activation of PKCδ, or activation of PKCε and inhibition of PKCδ activity affect thrombopoiesis (Figure 6). Indeed, in normal MK precursors, the simultaneous inhibition of PKCδ and activation of PKCε (δV11/ψεRACK) halves the percentage of MKs producing proplatelets (Figure 6A) and the number of platelets released in culture (Figure 6B). Conversely, the concurrent PKCδ activation and PKCε inhibition (ψδRACK /εV1-2) significantly increase both the percentage of MKs producing proplatelets and platelets release (Figure 6 C,D). We then tested whether PKCε and PKCδ pharmacological modulation could affect the expression levels of the

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downstream effectors Bcl-xL and Bax. As expected, the combination of peptides that reduces platelet output, (δV1-1/ψεRACK), is also capable of reducing Bax and increasing Bcl-xL expression levels, while the combination of peptides that increase platelet production, (ψδRACK /εV1-2), has the opposite effect on Bax and BclxL (Figure 6E). This data further reinforces the PKCδ/PKCε and Bax/Bcl-xL axis in the context of thrombopoiesis. Finally, we investigated whether pharmacological modulation of the two studied novel PKC isoforms could impact on in vitro platelet production in PMF and ET malignant megakaryocytopoiesis. We found that the combination of PKCε inhibition and PKCδ activation was capable of increasing platelet release from PMF-derived MK and, conversely, PKCε activation combined with PKCδ inhibition was able to reduce platelet output from ET-derived MK (Figure 6F). Collectively, this data shows that platelet production can be modulated in vitro by tuning PKCε/PKCδ activity, likely via Bax and Bcl-xL.

Discussion Megakaryocytopoiesis is the process by which hematopoietic stem cells differentiate into megakaryocytes, eventually capable of releasing mature platelets into the bloodstream through a process called throm-

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Figure 3. PKCδ down-regulation impairs MK differentiation. (A) Cell viability analysis of uninfected CD34+-derived MK cultures (Uninfected) and puromycin-selected CD34+-derived MK cultures infected with PKCδ-specific shRNA (shPKCδ), and with Non-Target shRNA control (shCT) at day 5 post-infection. Percentage of Annexin V/Propidium Iodide- cells from 3 replicates (cells data expressed as percentage of shCT; means ± one SD; *P<0.05 ANOVA and Tukey's test), Error Bar = SD. (B) Analysis of size distribution within uninfected CD34+-derived MK cultures (Uninfected) and puromycin-selected CD34+-derived MK cultures infected with PKCδ-specific shRNA (shPKCδ), and with Non-Target shRNA control (shCT) at day 5 post-infection. Percentage of cells with a given diameter range of 3 replicates. (mean ± one SD; *P<0.05 shPKCδ vs. Uninfected, #P<0.05 shPKCδ vs. shCT, ANOVA and Tukey’s test), Error Bar = SD. (C) Representative histogram of cell ploidy analysis of uninfected CD34+-derived MK cultures (Uninfected) and puromycin-selected CD34+-derived MK cultures infected with PKCδ-specific shRNA (shPKCδ), and with Non-Target shRNA control (shCT) at day 5 post-infection. (D) Percentage of cells with a DNA content >4N of uninfected CD34+-derived MK cultures (Uninfected) and puromycin-selected CD34+-derived MK cultures infected with PKCδ-specific shRNA (shPKCδ), and with Non-Target shRNA control (shCT) at day 5 post-infection. Cells data were obtained from 3 replicates and expressed as an arbitrary unit of shCT (means ± one SD; *P<0.05 ANOVA and Tukey's test), Error Bar = SD.

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independent supply of platelets for clinical applications.8,9 Solid data emerged from our and other groups in recent years showing that PKCε has a specific role in the regulation of human megakaryocytopoiesis.47 Nevertheless, sparse data, particularly in non-human hematopoietic and other systems, also show that PKC functions may not be necessarily confined to one specific isoform, whereas they can also be surrogated by members of the same protein family. For instance, human mature platelets do not express PKCε but do express PKCδ, whereas mouse platelets do exactly the opposite.48 Such characteristics complicate the translation of basic science discoveries in

bopoiesis. The entire process is characterized by a progressive increase of cellular dimensions, DNA content and, finally, proplatelet formation and fragmentation.46 A deeper understanding of the molecular events driving megakaryocytopoiesis and thrombopoiesis is essential: i) to develop new drugs able to overcome the cellular metabolic key nodes of MK maturation and platelet production that characterize primary thrombocytopenias, thrombocytoses, or accompany different hematopoietic disorders; ii) to achieve massive platelet production in vitro. Indeed, ex vivo MK cultures and in vivo MK infusion are being developed as strategies to obtain an unlimited, donor-

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Figure 4. PKCδ down-regulation impairs proplatelets formation. (A) representative images of proplatelet forming MK of uninfected CD34+-derived MK cultures (Uninfected) and puromycin- selected CD34+-derived MK cultures infected with PKCδspecific shRNA (shPKCδ), and with Non-Target shRNA control (shCT) at day 5 postinfection. Cells were analysed using a Leica DM IL phase contrast microscope (40X/0.5NA) and images were obtained with a Leica ICC50 HD camera (Leica Microsystems, Wetzlar, Germany) and analyzed using ImageJ software. (B) Analysis of platelet production of uninfected CD34+-derived MK cultures (Uninfected) and puromycin-selected CD34+-derived MK cultures infected with PKCδ-specific shRNA (shPKCδ), and with Non-Target shRNA control (shCT) at day 5 post-infection. Data were obtained from 5 replicates and were normalized for shCT (means ± one SD; *P<0.05 ANOVA and Tukey's test), Error Bar = SD.

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Figure 5. The PKCδ and PKCε balance is impaired in human pathological megakaryocytopoiesis. (A) Densitometric analysis of PKCδ, PKCε, Bax and Bcl-xL in human CD34+-derived MK at day 14 of differentiation of healthy donor (HD), PMF and ET patients. Protein expression is normalized for GAPDH expression levels, and expressed as fold increase of HD. Densitometric measurements of Western blots from 4 replicates is performed using ImageJ software (means ± one SD; *P<0.05 vs. HD t-test; **P<0.01 vs. HD t-test; #P<0.05 vs. PMF t-test; ##P<0.01 vs. PMF t-test), Error Bar = SD. (B) Densitometric analysis of PKCδ, PKCε, Bax and Bcl-xL in human CD34+-derived MK at day 14 of differentiation of of HD, PMF and ET patients. Protein expression is normalized for GAPDH expression levels, and expressed as the ratio between PKCδ and PKCε, and between Bax and Bcl-xL. Densitometric measurements of Western blots from at least 4 replicates is performed using ImageJ software (means ± one SD; *P<0.05 vs. HD t-test; #P<0.05 vs. PMF t-test; ##P<0.01 vs. PMF t-test), Error Bar = SD.

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Figure 6. The amount of platelet production can be revised by modulating PKCε/PKCδ function. (A) Analysis of proplatelet producing megakaryocytes of untreated (UNTR), treated with TAT47-57 peptide (TAT47-57), PKCδ inhibitor peptide, PKCε activator peptide alone (δV1-1 and ψεRACK, respectively) or in combination (δV1-1/ ψεRACK) cultures, at day 5 post-treatment. Data are expressed as percentage of megakaryocyte extending proplatelet analysing 100 cells for each treatment from at least 3 independent experiments. (means ± one SD; *P<0.05, ANOVA and Tukey's Test), Error Bar = SD. (B) Analysis of platelet production of untreated (UNTR), treated with TAT47-57 peptide (TAT47-57), PKCδ inhibitor peptide, PKCε activator peptide alone (δV1-1 and ψεRACK, respectively) or in combination (δV1-1/ ψεRACK) cultures, at day 5 post-treatment. Data are expressed as an arbitrary unit of control (TAT47-57) from at least 3 independent experiments. (means ± one SD; *P<0.05, ANOVA and Tukey's Test), Error Bar = SD. (C) Analysis of proplatelet producing megakaryocytes of untreated (UNTR), treated with TAT47-57 peptide (TAT47-57), PKCδ activator peptide, PKCε inhibitor peptide alone (ψδRACK and εV1-2, respectively) or in combination (ψδRACK / εV1-2 ) cultures, at day 5 post-treatment. Data are expressed as percentage of megakaryocyte extending proplatelet analysing 100 cells for each treatment from at least 3 independent experiments. (means ± one SD; *P<0.05, ANOVA and Tukey's Test), Error Bar = SD. (D) Analysis of platelet production of untreated (UNTR), treated with TAT47-57 peptide (TAT47-57), PKCδ activator peptide, PKCε inhibitor peptide alone (ψδRACK and εV1-2, respectively) or in combination (ψδRACK / εV1-2 ) cultures, at day 5 post-treatment. Data are expressed as an arbitrary unit of control (TAT47-57) from at least 3 independent experiments. (means ± one SD; *P<0.05, ANOVA and Tukey’s Test), Error Bar = SD. (E) Densitometric analysis of Bax and Bcl-xL in human CD34+-derived MK at day 14 of differentiation of healthy donor untreated (UNTR), treated with TAT47-57 peptide (TAT47-57), with PKCδ inhibitor- and PKCε activator peptide (δV1-1/ ψεRACK), with PKCδ activator- and PKCε inhibitor peptide (ψδRACK / εV1-2 ). Protein expression is normalized for GAPDH expression levels, and expressed as fold increase of TAT47-57. Densitometric measurements of Western blots from 4 replicates were performed by ImageJ software (means ± SD; ANOVA and Tukey’s Tests, statistical significant variations are indicated by the horizontal lines), Error Bar = SD. (F) Analysis of platelet production of human CD34+-derived MK at day 14 of differentiation in ET and PMF patients. ET patients were treated with control peptide (TAT47-57) or with PKCδ inhibitor- and PKCε activator peptide (δV1-1/ ψεRACK), PMF patients were treated with control peptide (TAT47-57) or with PKCδ activator- and PKCε inhibitor peptide (ψδRACK / εV1-2 ). Data are expressed as an arbitrary unit of control (TAT47-57) from 3 independent experiments. (means ± one SD; *P<0.05 vs. TAT47-57, t-test), Error Bar = SD.

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this field to the therapy, and might be the theoretical reason for the limited success of the related clinical trials.23 Starting from the above mentioned observation of functional and phenotypical “reciprocity” of PKCε and PKCδ in the megakaryocytopoietic systems of mouse and man, we therefore changed our methodological approach and started thinking in terms of “PKC couples” playing a role in a specific cellular pathway of maturation, which, in the case of megakaryocytopoiesis, could most likely be represented by PKCε and PKCδ. We already know that: i) PKCε increases in the early phases of MK differentiation and then decreases to undetectable levels in the late phases; ii) forced expression of PKCε reduces MK maturation and platelet release.31 Since PKCε and PKCδ have antithetical roles in many cellular systems,18 we hypothesized that they may also mediate opposing effects on MK differentiation, concurring, however, to the final success of the process. Our results show that PKCδ has opposite kinetics and functional roles in megakaryocytopoiesis when compared to PKCε. In fact it is: i) constantly expressed during MK differentiation; ii) high levels of PKCδ are required in the final steps of megakaryocytopoiesis to allow full MK maturation and PLT production. Indeed, PKCδ down-modulation during the later phase of differentiation impairs MK maturation reducing cell dimensions, polyploidization and platelet production: exactly the same alterations induced by PKCε overexpression, and previously described.31 To summarize, successful human megakaryocytopoiesis requires both late PKCε down-regulation in the presence of persistently high levels of PKCδ. Of course, our subsequent question was about their downstream effectors. Consistent with our previous data24,31 and with our theoretical expectations, the experiments with megakaryocytes in vitro showed that the downstream effectors of PKCε and PKCδ are represented by two Bcl2family members, Bcl-xL and Bax. Given the well documented role of apoptosis in MK differentiation,41,49,50 this result was nicely predictable: proapototic Bax lays downstream PKCδ and is up-regulated in the late phases of megakaryocytopoiesis, whereas antiapoptotic Bcl-xL, that lays downstream PKCε, is down-regulated. Interestingly however, forced PKCδ down-modulation in MKs not only down-regulates Bax but also up-regulates Bcl-xL, further confirming that the two upstream PKCs work as a functional couple. To both reinforce our hypothesis and give a translational perspective to our findings, we then took advantage of two human haematological disorders characterized by thrombocytopenia or thrombocytosis, where we would expect to find an imbalance between PKCδ and PKCε expression during MK differentiation. We very recently demonstrated that primary myelofibrosis (PMF)-derived megakaryocytes express higher levels of PKCε as compared to healthy subjects, and that its forced down-modulation or inhibition restores a normal MK maturation and platelet formation.33 On this basis, we herein studied the

References 1. Ware J, Corken A, Khetpal R. Platelet function beyond hemostasis and thrombosis. Curr Opin Hematol. 2013;20(5):451-456.

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expression levels of PKCδ, PKCε, Bax and Bcl-xL in human primary myelofibrosis (PMF) (characterized by a platelet count reduction), and in essential thrombocythemia (ET) (characterized by an enhanced MK maturation and platelet production). As expected, at day 14 of culture we found that both the PMF and the ET CD34+- derived megakaryocytes expressed altered levels of all these target proteins. Indeed, PKCδ/PKCε and Bax/Bcl-xL ratio values were significantly decreased in PMF while, to the contrary, significantly increased in ET, as compared to healthy subjects. Eventually, we tested the possibility to modulate platelet production both in normal and pathologic MK differentiation, by using PKCδ and PKCε specific, commercially available, activatory and inhibitory peptides already in use for clinical trials.23 In MKs derived from healthy subjects, the combined inhibition of PKCδ and activation of PKCε significantly reduced platelet production in vitro, reducing Bax levels and increasing Bcl-xL levels; conversely, the concurrent activation of PKCδ and inhibition of PKCε boosted platelet production, via up-regulation of Bax and down-regulation of Bcl-xL levels. Additionally, in disease models of abnormal MK differentiations (i.e., PMF and ET), the simultaneous modulation of these two PKC isoforms was capable of reverting, in vitro, the altered thrombopoiesis. In fact, the combined inhibition of PKCδ and activation of PKCε significantly reduced platelet production in ET patients; conversely, the concurrent activation of PKCδ and inhibition of PKCε boosted platelet output in PMF patients, proving that a fine pharmacological tuning of both kinases can revert the thrombocytotic phenotype in ET and the thrombocytopenic phenotype in PMF. Collectively this data show that: i) during human megakaryocytopoiesis PKCδ has an opposite kinetic expression compared to PKCε and their balance is critical for adequate MK maturation and PLT production; ii) PKCδ and PKCε work as a functional couple with opposite roles on thrombopoiesis, and the modulation of their balance strongly impacts platelet production, likely via the pathway of Bax and Bcl-xL; iii) as far as we can now say, ex vivo both thrombocytopenia and thrombocytosis can be corrected acting on the PKCε/PKCδ system both in normal and pathologic conditions. On this basis, we also suggest that the modulation of both PKCδ and PKCε expression and function might represent a strategy for platelet factories under the proper conditions. Acknowledgements The authors would like to thank Luciana Cerasuolo, Vincenzo Palermo, Domenico Manfredi and Davide Dallatana, University of Parma, Italy, for technical support. This work was supported by Regione Emilia-Romagna Area 1 - Strategic Program 2010-2012, and FIRB-accordi di programma 2010 (IT-Ministry for Universities and Scientific and Technological Research/Ministry of Education, Universities and Research, MIUR), RBAP10KCNS_002.

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ARTICLE

Myeloproliferative Disorders

Ruxolitinib versus best available therapy in patients with polycythemia vera: 80-week follow-up from the RESPONSE trial

Srdan Verstovsek,1 Alessandro M. Vannucchi,2 Martin Griesshammer,3 Tamas Masszi,4 Simon Durrant,5 Francesco Passamonti,6 Claire N. Harrison,7 Fabrizio Pane,8 Pierre Zachee,9 Keita Kirito,10 Carlos Besses,11 Masayuki Hino,12 Beatriz Moiraghi,13 Carole B. Miller,14 Mario Cazzola,15 Vittorio Rosti,16 Igor Blau,17 Ruben Mesa,18 Mark M. Jones,19 Huiling Zhen,19 Jingjin Li,20 Nathalie Francillard,21 Dany Habr,20 and Jean-Jacques Kiladjian22

EUROPEAN HEMATOLOGY ASSOCIATION

Ferrata Storti Foundation

Haematologica 2016 Volume 101(7):821-829

1 The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 2Center for Research and Innovation of Myeloproliferative Neoplasms, AOU Careggi, University of Florence, Italy; 3Johannes Wesling Clinic, Minden, Germany; 4St. István and St. László Hospital, Semmelweis University 3rd Department of Internal Medicine, Budapest, Hungary; 5 Royal Brisbane & Women’s Hospital, Brisbane, QLD, Australia; 6Department of Clinical and Experimental Medicine, University of Insubria, Varese, Italy; 7Guy’s and St. Thomas’ NHS Foundation Trust, London, UK; 8University of Naples Federico II, Italy; 9ZNA Stuivenberg, Antwerp, Belgium; 10Department of Hematology and Oncology, University of Yamanshi, Chuo-shi, Japan; 11Hematology Department, Hospital del Mar, Barcelona, Spain; 12 Department of Clinical Hematology and Diagnostics, Osaka City University Graduate School of Medicine, Japan; 13Hospital Jose Maria Ramos Mejia, Buenos Aires, Argentina; 14 Saint Agnes Cancer Institute, Baltimore, MD, USA; 15Department of Hematology, University of Pavia, Italy; 16Center for the Study of Myelofibrosis, IRCCS Policlinico San Matteo Foundation, Pavia, Italy; 17Medical Department, Division of Hematology, Oncology, and Tumor Immunology, Charité Universitätsmedizin Berlin, Germany; 18Department of Hematology/Oncology, Mayo Clinic Cancer Center, Scottsdale, AZ, USA; 19Incyte Corporation, Wilmington, DE, USA; 20Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA; 21Novartis Pharma S.A.S, Rueil Malmaison, France; and 22Centre d’Investigations Cliniques (INSERM CIC 1427), Hôpital Saint-Louis and Université Paris Diderot, Paris, France

ABSTRACT

R

ESPONSE is an open-label phase 3 study evaluating the Janus kinase 1/Janus kinase 2 inhibitor ruxolitinib versus best available therapy for efficacy/safety in hydroxyurea-resistant or intolerant patients with polycythemia vera. This preplanned analysis occurred when all patients completed the Week 80 visit or discontinued. Objectives included evaluating the durability of the primary response (Week 32 phlebotomy-independent hematocrit control plus ≥35% spleen volume reduction), its components, and that of complete hematologic remission; and long-term safety. Median exposure was 111 weeks; 91/110 (82.7%) patients randomized to ruxolitinib remained on treatment. No patients continued best available therapy (98/112 [87.5%] crossed over to ruxolitinib, most at/soon after Week 32). At Week 32, primary response was achieved by 22.7% vs. 0.9% of patients randomized to ruxolitinib and best available therapy, respectively (hematocrit control, 60.0% vs. 18.8%; spleen response, 40.0% vs. 0.9%). The probability of maintaining primary and hemat-ocrit responses for ≥80 weeks was 92% and 89%, respectively; 43/44 spleen responses were maintained until Week 80. Complete hematologic remission at Week 32 was achieved in 23.6% of ruxolitinib-randomized patients; the probability of maintaining complete hematologic remission for ≥80 weeks was 69%. Among ruxolitinib crossover patients, 79.2% were not phlebotomized, and 18.8% achieved a ≥35% reduction from baseline in spleen volume after 32 weeks of treatment. New or worsening hematologic laboratory abnormalities in ruxolitinib-treated patients were primarily grade 1/2 decreases in hemoglobin, lymphocytes, and platelets. The thromboembolic event rate per 100 patient-years was 1.8 with randomized ruxolitinib treatment vs. 8.2 with best available therapy. These data support ruxolitinib as an effective long-term treatment option for hydroxyurea-resistant or intolerant patients with polycythemia vera. This trial was registered at clinicaltrials.gov identifier: 01243944. haematologica | 2016; 101(7)

Correspondence: sverstov@mdanderson.org

Received: February 2, 2016. Accepted: April 15, 2016. Pre-published: April 21, 2016. doi:10.3324/haematol.2016.143644

Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/101/7/821

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

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Introduction Polycythemia vera (PV) is a myeloproliferative neoplasm primarily characterized by erythrocytosis, although increased white blood cell and platelet counts are also common.1 Patients with PV have increased risks of morbidity and mortality relative to comparable subjects in the general population (eg, same sex/age),2,3 often resulting from thromboembolic events or progression to myelofibrosis (MF) or acute myeloid leukemia (AML).2 Treatment for PV aims to reduce the risk of thromboembolic events, relieve symptom burden, and minimize the risk of disease transformation to MF or AML.4,5 Some patients obtain clinical benefit from cytoreductive treatment, often hydroxyurea;6-8 however, approximately 25% of patients become resistant to or intolerant of hydroxyurea.9 Ruxolitinib is a Janus kinase (JAK)1/JAK2 inhibitor approved by the US Food and Drug Administration (FDA) for patients with PV who have an inadequate response to or are intolerant of hydroxyurea,10 and by the European Medicines Agency (EMA) for adult patients with PV who are resistant to or intolerant of hydroxyurea.11 The ongoing RESPONSE trial is a global, multicenter, phase 3 study comparing ruxolitinib with best available therapy in patients with PV who were resistant to or intolerant of hydroxyurea, per modified European LeukemiaNet (ELN) criteria.4,12 In the primary analysis, a significantly greater proportion of patients treated with ruxolitinib achieved hematocrit control without phlebotomy along with a ≥35% reduction in spleen volume from baseline at Week 32 (the primary study endpoint) compared with patients treated with best available therapy.12 This was a second preplanned analysis of the RESPONSE trial assessing durability of efficacy and the long-term safety of ruxolitinib treatment after all patients completed the Week 80 visit or discontinued the study.

Methods

Declaration of Helsinki; all patients provided written informed consent.

Endpoints The primary analysis occurred when all patients completed the Week 48 visit or discontinued; the current preplanned analysis occurred when all patients completed the Week 80 visit or discontinued. The primary endpoint was the proportion of patients achieving both (1) hematocrit control without phlebotomy (defined as no phlebotomy eligibility between Weeks 8 and 32 with ≤1 phlebotomy eligibility from randomization to Week 8; phlebotomy eligibility was defined as hematocrit >45% and ≥3 percentage points higher than baseline or >48%, whichever were lower) and (2) ≥35% reduction from baseline in spleen volume (as measured by magnetic resonance imaging [MRI]) at Week 32. Complete hematologic remission (CHR; defined as hematocrit control, platelet count ≤400×109/L, and white blood cell count ≤10×109/L) was a key secondary endpoint. Because most patients randomized to best available therapy crossed over to receive ruxolitinib at or immediately after Week 32, long-term comparisons between study treatment arms were no longer appropriate. Therefore, this analysis evaluated the durability of efficacy in patients originally randomized to the ruxolitinib arm and in those who received ruxolitinib after crossover, including durability of the primary response, hematocrit control, spleen volume reduction, and CHR. Patient-reported outcomes were not collected after Week 32, with the exception of the endof-study visit for patients who discontinued; therefore, these data are not summarized. Adverse events are reported regardless of causality and not limited to those considered to be related to treatment; serious adverse events and deaths are also reported. Adverse event data from the 80-week analysis are reported for patients originally randomized to the ruxolitinib arm, patients who received ruxolitinib after crossover (i.e. all randomized to the best available therapy arm and received ≥1 dose of ruxolitinib after crossover), and patients who received best available therapy. Please see the Online Supplementary Section for details concerning exploratory and statistical analyses included in this report.

Study Design RESPONSE is an international, randomized, open-label, phase 3 study. The study design and primary analysis results have been described previously.12 Briefly, eligible patients were randomized 1:1 to receive ruxolitinib (10 mg twice daily) or best available therapy (single-agent therapy deemed most appropriate by treating physician). Treatment options for best available therapy included hydroxyurea, interferon or pegylated interferon, pipobroman, anagrelide, immunomodulators (e.g. lenalidomide, thalidomide), or observation without pharmacologic treatment (except aspirin). Dose adjustments were permitted for safety and efficacy in patients receiving ruxolitinib; modifications could be made to best available therapy regimens for lack of response or side effects requiring treatment discontinuation. Low-dose aspirin was administered to all patients unless contraindicated. Phlebotomies as monotherapy or in combination with study treatment were mandatory for patients with a confirmed hematocrit >45% that was ≥3 percentage points higher than baseline or a confirmed hematocrit >48%, whichever were lower. Patients randomized to best available therapy were permitted to cross over to ruxolitinib at Week 32 if the primary endpoint was not met or after Week 32 following signs of disease progression (i.e. phlebotomy eligibility or splenomegaly progression). The study was approved by the central ethics committee or institutional review board at each participating institution and was conducted in accordance with the 822

Results Patients In total, 222 patients were randomized to ruxolitinib (n=110) or best available therapy (n=112); patient enrollment and demographics were previously reported. 12 Median age in the ruxolitinib and best available therapy arms (62.0 and 60.0 years, respectively), median time since PV diagnosis (8.2 and 9.3 years), median duration of previous hydroxyurea therapy (3.1 and 2.8 years), mean JAK2V617F allele burden (76.2% and 75.0%), and median spleen volume (1195 and 1322 cm3) at baseline were similar between treatment arms. Additionally, 60.0% of patients treated with ruxolitinib and 71.4% of patients treated with best available therapy were men. At the time of data cutoff for the 80-week analysis, 91 patients (82.7%) randomized to receive ruxolitinib were still being treated (Figure 1), and the median exposure was 111 weeks. No patients were actively receiving best available therapy (median exposure, 34 weeks); 98 patients (87.5%) had crossed over to ruxolitinib, 81 (82.7%) of whom continued to receive ruxolitinib at data cutoff (median exposure, 75.6 weeks). Mean (SD) ruxolitinib dose was 26.7 mg/d (10.8 mg/d) at Week 32 and 28.4 mg/d haematologica | 2016; 101(7)


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Figure 1. Patient disposition. *One patient withdrew consent and was not treated on study; initial best available therapy included hydroxyurea (n=66), interferon/pegylated interferon (n=13), anagrelide (n=8), immunomodulators (n=5), pipobroman (n=2), and observation (n=17). †2 patients who discontinued because of an adverse event died during follow-up.

(11.1 mg/d) at Week 80 (Online Supplementary Figure S1). The distribution of ruxolitinib dosing was similar at Week 32 and Week 80; the most common ruxolitinib dose was 20 mg/d at both time points (36.1% and 33.0%, respectively; Online Supplementary Figure S2). Among patients originally randomized to ruxolitinib, the most common reasons for discontinuation of study drug included disease progression (5.5%), patient decision (5.5%), and adverse events (4.5%) (Figure 1).

Efficacy The primary endpoint was previously reported to have been achieved by 23 patients (20.9%) originally randomized to ruxolitinib, and 1 patient (0.9%) receiving best available therapy at Week 32 (P<0.001).12 During MRI data review for the current 80-week analysis, 2 additional patients randomized to ruxolitinib were identified as primary responders, bringing the total number of primary responders to 25 (22.7%). No additional responders were identified in the best available therapy arm. The probability of maintaining the primary response among patients originally randomized to ruxolitinib for ≥80 weeks from time of response was 92% (Figure 2). The primary analysis previously reported that 60.0% of patients originally randomized to ruxolitinib achieved hematocrit control without phlebotomy by Week 32 compared with 19.6% of patients randomized to best available therapy;12 however, analysis of data from the 80-week data cutoff revealed an additional patient in the best available therapy arm who had a phlebotomy at Week 8, bringing the proportion of patients with hematocrit control at Week 32 down to 18.8%. Among patients originally randomized to ruxolitinib, the probability of haematologica | 2016; 101(7)

maintaining hematocrit control up to Week 80 from time of response was 89% (Figure 3). Of the 98 patients still receiving ruxolitinib at Week 32, 88 (89.8%) had no phlebotomy procedures between Weeks 32 and 80. Of the 109 patients randomized to best available therapy who did not discontinue before Week 8, 68 (62.4%) had ≥1 phlebotomy and 22 (20.2%) had ≥3 phlebotomies between Weeks 8 and 32.12 A higher proportion of patients originally randomized to ruxolitinib achieved ≥35% reduction in spleen volume at Week 32 compared with the best available therapy arm (40.0% vs. 0.9%); none of these patients lost their response at Week 80. Additionally, mean reductions in spleen volume increased over time in the ruxolitinib arm (Online Supplementary Figure S3). The primary analysis previously reported that a CHR at Week 32 was achieved by 26 patients (23.6%) originally randomized to ruxolitinib compared with 10 patients (8.9%) randomized to best available therapy (P=0.003);12 however, after correcting for the patient in the best available therapy arm who had a phlebotomy at Week 8, only 9 patients achieved CHR at Week 32 (8.0%; unadjusted, P=0.0016; with adjustment for baseline white blood cell and platelet status, P=0.0013 ). For patients originally randomized to ruxolitinib, the probability of maintaining their CHR for at least 80 weeks was 69%. Blood cell counts improved over time in patients originally randomized to ruxolitinib (Figure 4). Among patients with elevated white blood cell counts (>10×109/L) at baseline, improvements to ≤10×109/L were achieved in 31.0% (27/87) of patients at Week 32, and 47.1% (41/87) at Week 80. Among patients with elevated baseline platelet counts (>400×109/L), improvements to ≤400×109/L were achieved 823


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Figure 2. Durability of primary response with ruxolitinib treatment.

by 44.4% (24/54) of patients at Week 32 and 59.3% (32/54) at Week 80.

ruxolitinib was also observed in patients treated with ruxolitinib after crossover (Figure 4).

Exploratory analyses

JAK2V617F Allele Burden The mean (median) percentage change from baseline in JAK2V617F allele burden at Week 32 was –12.2% (– 10.1%) and +1.2% (0.0%) in patients originally randomized to ruxolitinib and in those receiving best available therapy, respectively.12 At Week 80, the mean (median) percentage change from baseline in JAK2V617F allele burden among patients originally randomized to ruxolitinib was –22.0% (–18.4%). Among patients who received ruxolitinib after crossover, the mean (median) percentage change from crossover baseline in JAK2V617F allele burden was –6.7% (–5.5%) 48 weeks after crossover.

Efficacy with ruxolitinib after crossover Phlebotomy requirement was similar between patients treated with ruxolitinib after crossover from best available therapy and patients originally randomized to the ruxolitinib arm. Among patients treated for up to 32 weeks, phlebotomy was not required by 73.6% (81/110) of patients originally randomized to the ruxolitinib arm, 79.2% (76/96) receiving ruxolitinib after crossover, and 25.0% (28/112) during treatment with best available therapy. Median (range) time to first phlebotomy was 131 (30– 568) days for patients originally randomized to ruxolitinib, 144 (6–483) days for those receiving ruxolitinib after crossover, and 113 (31–337) days for those receiving treatment with best available therapy. The phlebotomy rate per 100 patient-years of exposure was lower among patients originally randomized to ruxolitinib (34.1) and receiving ruxolitinib after crossover (38.5) compared with patients receiving best available therapy (196.8). Ruxolitinib after crossover was associated with reductions in spleen volume after 16 weeks of treatment (Online Supplementary Figure S4). After 32 weeks of treatment, a greater proportion of patients achieved a ≥35% reduction from baseline in spleen volume in patients originally randomized to ruxolitinib (40.0% [44/110]) and those receiving ruxolitinib after crossover (18.8% [18/96]) compared with patients receiving best available therapy (0.9% [1/112]; Online Supplementary Table S1). After 32 weeks of treatment, the mean percentage change from original baseline in spleen volume was –27.7% in patients originally randomized to ruxolitinib, –14.2% in those receiving ruxolitinib after crossover, and +4.5% in those receiving best available therapy. The positive trend toward improved (i.e. reduced) blood cell counts observed in patients originally randomized to 824

Safety The most common nonhematologic adverse events in the originally randomized ruxolitinib arm were headache, diarrhea, pruritus, and fatigue (Table 1); most events were grade 1 or 2. Relatively few new adverse events were observed after the primary analysis; the number of patients with any given adverse event from the 48-week to the 80-week analysis increased by no more than 4, and by 1 or 2 for most individual events. New or worsening hematologic laboratory abnormalities in the originally randomized ruxolitinib arm through Week 80 were primarily grade 1 or 2 decreases in hemoglobin, lymphocytes, and platelets (Table 2). Patients receiving treatment with ruxolitinib after crossover had a higher rate of decreased hemoglobin compared with those originally randomized to ruxolitinib; the rates of other hematologic adverse events (Table 2) and nonhematologic adverse events (Table 1) were generally consistent with those observed in patients originally randomized to ruxolitinib. The rates of all grade and grade 3/4 thromboembolic events per 100 patient-years of exposure were 1.8 and 0.9, respectively, among patients originally randomized to ruxhaematologica | 2016; 101(7)


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olitinib vs. 4.1 and 2.7 in those receiving ruxolitinib after crossover, and 8.2 and 2.7 in those receiving best available therapy (Table 3). In the originally randomized ruxolitinib arm, thromboembolic events included portal vein thrombosis, cerebral infarction, ischemic stroke, and retinal vascular thrombosis; thromboembolic events in the best available therapy arm included deep vein thrombosis, myocardial infarction, pulmonary embolism, splenic infarction, thrombophlebitis, and thrombosis. Other adverse events of interest are shown in Table 4. Rates of herpes zoster infection were higher in patients receiving ruxolitinib (per 100 patient-years of exposure: originally randomized to ruxolitinib, 5.3; with ruxolitinib after

crossover, 5.4; with best available therapy, none). Rates of nonmelanoma skin cancer per 100 patient-years of exposure were 4.4 in those originally randomized to ruxolitinib, 2.0 with ruxolitinib after crossover, and 2.7 with best available therapy. Among patients with a history of nonmelanoma skin cancer (originally randomized to ruxolitinib, n=12; with ruxolitinib after crossover, n=6; with best available therapy, n=7), rates of nonmelanoma skin cancer were similar between randomized treatments (24.2, 10.6, 22.3 per 100 patient-years of exposure, respectively). Among patients without a history of nonmelanoma skin cancer (originally randomized to ruxolitinib, n=98; with ruxolitinib after crossover, n=92; with best available ther-

Table 1. Nonhematologic adverse events in the 80-week and 48-week analyses adjusted for exposure.

80-Week Analysis Ruxolitinib Crossover (n=98) 147.6

Ruxolitinib (n=110) Exposure, Patient-Years Rate per 100 Patient-Years of Exposure† Headache Diarrhea Pruritus Fatigue Muscle spasms Dizziness Increased weight Dyspnea Abdominal pain Arthralgia Back pain Cough Nasopharyngitis Constipation Herpes zoster Pyrexia

227.7

Best Available Therapy (n=111*) 73.6

All Grades

Grade 3 or 4

All Grades

Grade 3 or 4

All Grades

Grade 3 or 4

10.5 9.7 9.7 8.3 7.9 7.5 7.5 7.0 6.6 6.1 5.7 5.7 5.7 5.3 5.3 5.3

0.9 0 0.4 0.4 0.4 0 0.4 1.3 0.9 0 0.4 0 0 0.4 0.9 0

8.8 5.4 8.8 6.8 3.4 7.5 6.8 2.7 4.7 4.7 5.4 5.4 6.1 6.8 5.4 5.4

0 0 0 0 0 0 0 0 0 0 0.7 0 0 0 0.7 0.7

28.5 12.2 32.6 23.1 9.5 14.9 1.4 2.7 17.7 10.9 6.8 8.2 12.2 4.1 0 6.8

1.4 1.4 5.4 4.1 0 0 0 0 0 1.4 0 0 0 0 0 0

48-Week Analysis Ruxolitinib Best Available (n=110) Therapy (n=111*) 170.0 72.8 All Grade Grades 3 or 4 13.5 12.4 11.2 11.2 8.8 8.8 7.6 8.8 7.1 7.6 5.9 7.6 7.6 7.1 6.5 5.9

1.2 0 0.6 0 0.6 0 0 1.8 1.2 0 0.6 0 0 0.6 1.2 0

All Grades

Grade 3 or 4

28.8 12.4 34.3 23.4 6.9 15.1 1.4 2.7 17.9 11.0 6.9 8.2 12.4 4.1 0 6.9

1.4 1.4 5.5 4.1 0 0 0 0 0 1.4 0 0 0 0 0 0

*1 patient was randomized to best available therapy but did not receive study treatment. †All grades adverse events occurring at a rate of ≥5 per 100 patient-years of exposure in the ruxolitinib arm in the 80 week analysis.

Table 2. New or worsening decrease in hematologic laboratory values in the 80-week analysis adjusted for exposure.

Ruxolitinib (n=110) Exposure, Patient-Years Rate per 100 Patient-Years of Exposure Hemoglobin Lymphocytes Platelets Leukocytes Neutrophils

227.7

80-Week Analysis Ruxolitinib Crossover (n=98) 147.6

Best Available Therapy (n=111*) 73.6

48-Week Analysis Ruxolitinib Best Available (n=110) Therapy (n=111*) 170.0 72.8

All Grades

Grade 3 or 4

All Grades

Grade 3 or 4

All Grades

Grade 3 or 4

All Grades

Grade 3 or 4

All Grades

27.2 27.2 14.9 6.6 2.2

0.9 9.7 2.6 0.9 0.4

40.0 29.8 16.9 6.8 1.4

2.7 6.8 0.7 0.7 0.7

47.6 78.8 29.9 19.0 12.2

0 27.2 5.4 2.7 1.4

34.7 32.9 19.4 8.2 2.9

1.2 11.8 3.5 1.2 1.2

48.1 78.3 30.2 19.2 12.4

Grade 3 or 4 0 27.5 5.5 2.7 1.4

*1 patient was randomized to best available therapy but did not receive study treatment.

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apy, n=104), rates of nonmelanoma skin cancer were 2.0, 1.4, and 1.4 per 100 patient-years of exposure, respectively. Rates of transformation to MF and AML in patients originally randomized to ruxolitinib were 1.3 and 0.4 per 100 patient-years of exposure, respectively. One patient in the best available therapy arm had transformation to MF before crossover to ruxolitinib (rate of transformation, 1.4 per 100 patient-years of exposure); no patients in the best available therapy arm had transformation to AML before crossover. Among patients treated with ruxolitinib after crossover, 3 patients had transformation to MF (rate of transformation, 2.0 per 100 patient-years of exposure), 1 of whom developed AML (rate of transformation, 0.7 per 100 patient-years of exposure). Serious adverse events occurred at a rate of 12.7 per 100 patient-years of exposure in patients originally randomized to ruxolitinib, and 19.0 with ruxolitinib after crossover at the 80-week analysis; the only serious adverse events reported by ≥2 patients in those originally

randomized to ruxolitinib were basal cell carcinoma (1.3 per 100 patient-years of exposure), chest pain (0.9), and pneumonia (0.9). At the 48-week analysis, 2 patients in the best available therapy arm had died after crossing over to ruxolitinib; 1 due to central nervous system hemorrhage, and 1 due to multiorgan failure and hypovolemic shock. In the patient who died from central nervous system hemorrhage (a 66-year-old white woman), platelet counts were 1174×109/L during screening and 351×109/L at the Week 64 visit (137 days after crossover and 18 days before death), there was no history of hemorrhage, and the patient was receiving treatment with aspirin 81 mg once daily. The patient had grade 3 hypertension at randomization and intermittently throughout study treatment, which the investigator considered as a possible cause for the central nervous system hemorrhage. The patient who died from multiorgan failure and hypovolemic shock (a 50-year-old Asian woman) discontinued ruxolitinib on Day 645 because of grade 3 anemia. Fourteen days later,

Table 3. Thromboembolic events in the 80-week analysis adjusted for exposure.

Exposure, Patient-Years Events, Rate per 100 PatientYears of Exposure

Ruxolitinib (n=110) 227.7 All Grades

Grade 3 or 4

1.8 0.4 0.4 0.4 0.4 0 0 0 0 0 0 0 0 0

0.9 0.4 0.4 0 0 0 0 0 0 0 0 0 0 0

All thromboembolic events Portal vein thrombosis Cerebral infarction Ischemic stroke Retinal vascular thrombosis Myocardial infarction Bone infarction Coronary artery occlusion Disseminated intravascular coagulation Thrombosis Deep vein thrombosis Pulmonary embolism Splenic infarction Thrombophlebitis

Ruxolitinib Crossover (n=98) 147.6 All Grade Grades 3 or 4 4.1 0 0 1.4 0 1.4 0.7 0.7 0.7 0.7 0 0 0 0

2.7 0 0 1.4 0 0.7 0 0 0.7 0 0 0 0 0

Best Available Therapy (n=111*) 73.6 All Grade Grades 3 or 4 8.2 0 0 0 0 1.4 0 0 0 1.4 2.7 1.4 1.4 1.4

2.7 0 0 0 0 1.4 0 0 0 0 1.4 1.4 0 0

*1 patient was randomized to best available therapy but did not receive study treatment.

Table 4. Adverse events of interest in the 80-week analysis adjusted for exposure.

Exposure, Patient-Years Events, n (Rate per 100 Patient-Years of Exposure) All infections Grade 3 or 4 Herpes zoster infection Grade 3 or 4 Nonmelanoma skin cancer Disease progression† Myelofibrosis Acute myeloid leukemia

Ruxolitinib (n=110) 227.7

Ruxolitinib Crossover (n=98) 147.6

Best Available Therapy (n=111*) 73.6

67 (29.4) 9 (4.0) 12 (5.3) 2 (0.9) 10 (4.4)

41 (27.8) 8 (5.4) 8 (5.4) 1 (0.7) 3 (2.0)

43 (58.4) 3 (4.1) 0 0 2 (2.7)

3 (1.3) 1 (0.4)

3 (2.0) 1 (0.7)

1 (1.4) 0

*1 patient was randomized to best available therapy but did not receive study treatment. †There was 1 additional report of myelofibrosis in the ruxolitinib arm, but this was not confirmed on bone marrow biopsy.

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the patient developed grade 4 disseminated intravascular coagulation, grade 3 acidosis, grade 1 pyrexia, and respiratory distress; was moved into the intensive care unit; and received a blood transfusion for anemia. The next day (16 days after the last dose of ruxolitinib), the patient died because of shock related to multiorgan failure, with the following ongoing events: anemia, cardiac failure, disseminated intravascular coagulation, dyspnea, peripheral edema, nephrotic syndrome, and pulmonary hypertension. The investigator managing this patient’s care suspected an association between ruxolitinib treatment and the pulmonary hypertension event, but not other events (i.e. disseminated intravascular coagulation, hypovolemic shock, multiorgan failure, or nephrotic syndrome). These deaths were not considered treatment related. No new deaths were reported at the 80-week analysis data cutoff.

Discussion Long-term follow-up of the phase 3 RESPONSE trial demonstrates the durability of ruxolitinib efficacy in patients with PV who were resistant to or intolerant of hydroxyurea. For patients who achieved the composite primary response, Kaplan-Meier estimates predicted that most would maintain that response (92%) or the hematocrit control component (89%) up to Week 80. Furthermore, no patients who achieved spleen response at Week 32 (i.e. the primary analysis time point) lost the response at Week 80. Among all patients originally randomized to ruxolitinib treatment, mean hematocrit levels were approximately 40% at Week 32, where they remained through Week 80. Between Weeks 32 and 80, mean white blood cell counts decreased from 12.0×109/L to 10.7×109/L. During this time frame, mean reduction in spleen volume from baseline improved from −27.7% to −38.6%. Patients who were treated with ruxolitinib after

crossover from best available therapy achieved similar benefits in hematocrit control, reduction in spleen volume, and normalization of blood cell counts as patients originally randomized to ruxolitinib. Furthermore, although patient-reported symptom severity was not assessed after Week 32, fatigue and pruritus were recorded as adverse events throughout the study. These events continued to occur at lower rates in patients who were randomized to or crossed over to ruxolitinib compared with those receiving best available therapy. Control of blood cell counts is an important treatment goal for patients with PV. A large-scale randomized controlled trial testing the intensity of cytoreductive therapy in PV (CYTO-PV) demonstrated that high hematocrit levels (45%–50% vs. <45%) and high white blood cell counts (≥11×109/L vs. <7.0×109/L) are associated with increased risk of cardiovascular/thromboembolic events.6,13 However, maintaining a therapeutic hematocrit level with phlebotomy and/or hydroxyurea may be challenging for some patients. In the CYTO-PV study, approximately 25% of patients had hematocrit levels that were outside the target range 6 months after randomization.14 Treatment with ruxolitinib in RESPONSE was associated with durable improvements in hematocrit levels, as well as reductions in white blood cell counts. Furthermore, although the study was not designed to evaluate thromboembolic event rates, the originally randomized ruxolitinib arm of RESPONSE was associated with a lower rate of thromboembolic events compared with the best available therapy arm. At Week 32, before crossover to ruxolitinib, there were 6 thromboembolic events in the best available therapy arm compared with 1 event in the ruxolitinib arm.12 Although treatment with ruxolitinib after crossover was associated with rapid normalization of blood cell counts, thromboembolic event rates remained higher than in patients originally randomized to ruxolitinib. These data emphasize the importance of imple-

Figure 3. Duration of hematocrit control with ruxolitinib treatment. *Duration of the absence of phlebotomy eligibility is defined as the time from first occurrence of absence of phlebotomy eligibility until the date of the first documented progression.

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menting early treatment changes to control blood counts, especially hematocrit, in patients with hydroxyurea-resistant or intolerant PV to minimize the risk of thromboembolic events. Ruxolitinib continued to be tolerated by most patients during long-term treatment following the primary analysis, with 83% of patients still receiving treatment at a median exposure of 111 weeks. In agreement with the pri-

mary analysis,12 most adverse events were grade 1 or 2, with relatively few new adverse events observed in the 80-week analysis. The rate of herpes zoster continued to be higher in the originally randomized ruxolitinib arm, as reported in the primary analysis.12 Most herpes zoster infections were grade 1 or 2 and were resolved without sequelae. Nonmelanoma skin cancers were observed in the originally randomized ruxolitinib arm, mainly in

A

B

C

Figure 4. Mean hematocrit levels (A), white blood cell counts (B), and platelet counts (C) over time. Includes all data points with >5 patients. For patients in the ruxolitinib crossover group, baseline represents the date of crossover to ruxolitinib. Ruxolitinib and best available therapy arm data are from the 80-week data cutoff; ruxolitinib crossover data are from the 48-week data cutoff. WBC: white blood cell.

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patients with a history of nonmelanoma skin cancer or precancer; however, exposure-adjusted rates at the time of this analysis were generally similar between the originally randomized ruxolitinib and best available therapy arms. Rates of transformation to MF and AML were consistent with published rates for similar patient populations with PV.9,15,16 These safety and tolerability data are important because many patients require long-term therapy to manage their PV. Hydroxyurea is often prescribed for patients who require cytoreductive treatment. Although many patients receive clinical benefits from hydroxyurea,6-8 a considerable proportion will not tolerate therapy or may become resistant.9 Patients who develop resistance are at increased risk of fibrotic/leukemic disease transformation and mortality,9 with few second-line treatment options available. Ruxolitinib represents a new treatment option for this hydroxyurea-resistant or intolerant patient population that has durable responses and long-term tolerability based on the 111-week follow-up of RESPONSE. This study had several limitations that should be considered. The design of the RESPONSE trial permitted crossover from best available therapy to ruxolitinib for patients who did not achieve the primary endpoint. Most patients crossed over to ruxolitinib shortly after they became eligible at Week 32, precluding long-term comparisons between ruxolitinib treatment and best available therapy. Many patients in the best available therapy arm received hydroxyurea (58.9%), despite established resistance or intolerance.12 While this scenario is perhaps coun-

References 1. Tefferi A, Vardiman JW. Classification and diagnosis of myeloproliferative neoplasms: the 2008 World Health Organization criteria and point-of-care diagnostic algorithms. Leukemia. 2008;22(1):14-22. 2. Stein BL, Moliterno AR, Tiu RV. Polycythemia vera disease burden: contributing factors, impact on quality of life, and emerging treatment options. Ann Hematol. 2014;93(12):1965-1976. 3. Hultcrantz M, Kristinsson SY, Andersson TM, et al. Patterns of survival among patients with myeloproliferative neoplasms diagnosed in Sweden from 1973 to 2008: a population-based study. J Clin Oncol. 2012;30(24):2995-3001. 4. Barbui T, Barosi G, Birgegard G, et al. Philadelphia-negative classical myeloproliferative neoplasms: critical concepts and management recommendations from European LeukemiaNet. J Clin Oncol. 2011;29(6):761-770. 5. Barosi G, Mesa R, Finazzi G, et al. Revised response criteria for polycythemia vera and essential thrombocythemia: an ELN and IWG-MRT consensus project. Blood. 2013;121(23):4778-4781. 6. Marchioli R, Finazzi G, Specchia G, et al.

haematologica | 2016; 101(7)

7.

8.

9.

10. 11. 12.

terintuitive, it is not uncommon in real-world clinical practice where limited treatment options were available before the approval of ruxolitinib for patients with PV who have an inadequate response to or are intolerant of hydroxyurea.10 In addition, although ELN criteria for hydroxyurea resistance and intolerance17 are important for defining patient populations in clinical trials, they may not be as useful in clinical practice. Finally, because patients received hydroxyurea and other traditional treatment options before randomization to ruxolitinib, the causal relationship between ruxolitinib and adverse events such as nonmelanoma skin cancer are difficult to determine. In conclusion, patients treated with ruxolitinib who achieved protocol-defined treatment responses for hematocrit control, spleen volume reduction, and CHR at the primary analysis12 were likely to maintain their responses during the 111-week follow-up period of this study. Long-term follow-up with ruxolitinib did not identify new safety signs or progressively worsening toxicity. The observed adverse events were expected, and most were manageable with standard clinical monitoring and care. Taken together, these data support ruxolitinib as an effective long-term treatment option for patients with PV who are resistant to or intolerant of hydroxyurea. Acknowledgments Writing assistance was provided by Cory Pfeiffenberger, PhD (Complete Healthcare Communications, LLC, an ICON plc company), whose work was funded by Incyte Corporation.

Cardiovascular events and intensity of treatment in polycythemia vera. N Engl J Med. 2013;368(1):22-33. Fruchtman SM, Mack K, Kaplan ME, Peterson P, Berk PD, Wasserman LR. From efficacy to safety: a Polycythemia Vera Study Group report on hydroxyurea in patients with polycythemia vera. Semin Hematol. 1997;34(1):17-23. Kiladjian JJ, Chevret S, Dosquet C, Chomienne C, Rain JD. Treatment of polycythemia vera with hydroxyurea and pipobroman: final results of a randomized trial initiated in 1980. J Clin Oncol. 2011;29(29):3907-3913. Alvarez-Larran A, Pereira A, Cervantes F, et al. Assessment and prognostic value of the European LeukemiaNet criteria for clinicohematologic response, resistance, and intolerance to hydroxyurea in polycythemia vera. Blood. 2012;119(6):1363-1369. JAKAFIÂŽ (ruxolitinib) [package insert]. Wilmington, DE: Incyte Corporation. December 2014. JAKAVIÂŽ (ruxolitinib) [package insert]. Horsham, West Sussex, United Kingdom: Novartis. April 2015. Vannucchi AM, Kiladjian JJ, Griesshammer M, et al. Ruxolitinib versus standard therapy for the treatment of polycythemia vera. N Engl J Med. 2015;372(5):426-435.

13. Barbui T, Masciulli A, Marfisi MR, et al. White blood cell counts and thrombosis in polycythemia vera: a subanalysis of the CYTO-PV study. Blood. 2015;126(4):560561. 14. Marchioli R, Finazzi G, Specchia G, Masciulli A, Mennitto MR, Barbui T. The CYTO-PV: a large-scale trial testing the intensity of CYTOreductive therapy to prevent cardiovascular events in patients with polycythemia vera. Thrombosis. 2011;2011:794240. 15. Finazzi G, Caruso V, Marchioli R, et al. Acute leukemia in polycythemia vera: an analysis of 1638 patients enrolled in a prospective observational study. Blood. 2005;105(7):2664-2670. 16. Passamonti F, Rumi E, Pietra D, et al. A prospective study of 338 patients with polycythemia vera: the impact of JAK2 (V617F) allele burden and leukocytosis on fibrotic or leukemic disease transformation and vascular complications. Leukemia. 2010;24(9):1574-1579. 17. Barosi G, Birgegard G, Finazzi G, et al. A unified definition of clinical resistance and intolerance to hydroxycarbamide in polycythaemia vera and primary myelofibrosis: results of a European LeukemiaNet (ELN) consensus process. Br J Haematol. 2010; 148(6):961-963.

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ARTICLE EUROPEAN HEMATOLOGY ASSOCIATION

Chronic Myeloid Leukemia

Ferrata Storti Foundation

Haematologica 2016 Volume 101(7):830-838

Ultra-deep sequencing leads to earlier and more sensitive detection of the tyrosine kinase inhibitor resistance mutation T315I in chronic myeloid leukemia Constance Baer, Wolfgang Kern, Sarah Koch, Niroshan Nadarajah, Sonja Schindela, Manja Meggendorfer, Claudia Haferlach and Torsten Haferlach MLL Munich Leukemia Laboratory, Germany

ABSTRACT

C

Correspondence: torsten.haferlach@mll.com

Received: March 10, 2016. Accepted: April 19, 2016. Pre-published: April 21, 2016.

hronic myeloid leukemia cells acquire resistance to tyrosine kinase inhibitors through mutations in the ABL1 kinase domain. The T315I mutation mediates resistance to imatinib, dasatinib, nilotinib and bosutinib, whereas sensitivity to ponatinib remains. Mutation detection by conventional Sanger sequencing requires 10%-20% expansion of the mutated subclone. We studied the T315I mutation development by ultra-deep sequencing on the 454 XL+ platform (Roche) in comparison to Sanger sequencing. By ultra-deep sequencing, mutations were detected at loads of 1%-2%. We selected 40 patients who had failed first-line to third-line treatment (imatinib, dasatinib, nilotinib) and had high loads of the T315I mutation detected by Sanger sequencing. We confirmed T315I mutations by ultra-deep sequencing and investigated the mutation dynamics by backtracking earlier samples. In 20 of 40 patients, we identified the T315I three months (median) before Sanger sequencing detection limits were reached. To exclude sporadic low percentage mutation development without subsequent mutation outgrowth, we selected 42 patients without resistance mutations detected by Sanger sequencing but loss of major molecular response. Here, no mutation was detected by ultradeep sequencing. Additional non-T315I resistance mutations were found in 20 of 40 patients. Only 15% had two mutations per cell; the other cases showed multiple independently mutated clones and the T315I clone demonstrated a rapid outgrowth. In conclusion, T315I mutations could be detected earlier by ultra-deep sequencing compared to Sanger sequencing in a selected group of cases. Earlier mutation detection by ultra-deep sequencing might allow treatment to be changed before clonal increase of cells with the T315I mutation.

doi:10.3324/haematol.2016.145888

Introduction Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/101/7/830

Š2016 Ferrata Storti Foundation Material published in Haematologica is covered by copyright. All rights reserved to the Ferrata Storti Foundation. Copies of articles are allowed for personal or internal use. Permission in writing from the publisher is required for any other use.

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The use of BCR-ABL1 tyrosine kinase inhibitors (TKI) has revolutionized the treatment of chronic myeloid leukemia (CML) by achieving long remission periods.1 However, lack of efficacy, progression or adverse events can require treatment discontinuation. For imatinib, the discontinuation rate for first-line treatment was 34% within the first six years.2 Therapy with the 2nd-generation inhibitors dasatinib or nilotinib for newly diagnosed CML patients had lower discontinuation rates, but still some patients progressed or failed therapy.3-5 Mutations in the ABL1 tyrosine kinase domain (TKD) are the best studied mechanism of acquired TKI resistance.6-8 Two-thirds of patients who fail imatinib treatment have acquired at least one mutation in the TKD, and at least one-third of resistant patients on dasatinib or nilotinib have developed mutations.9 Over 100 point mutations leading to amino acid exchanges were described in different TKI resistant CML patients.10,11 Well characterized resistance profiles of each TKI have been described and this allows the TKIs to be changed and offer the possibility of overcoming resistance.8 Only the threonine-to-isoleucine exchange at amino acid position 315 (T315I) caused by the single base substitution act>att results in resisthaematologica | 2016; 101(7)


Early T315I detection by ultra-deep sequencing

ance to most TKIs, and sensitivity remains only to ponatinib.12 Although the mutation itself does not increase the kinase activity,13 the resistance to imatinib, dasatinib, nilotinib and bosutinib causes patients with T315I to show a rapid increase in malignant cell burden and to progress to blast crisis.14 An early detection of the T315I mutation may be advantageous and allow treatment intervention before disease progression. Low-level mutations (â&#x2030;¤1%) are selected on treatment in patients receiving a TKI for which the mutation causes resistance, and these lead to lower response rates.15 However, the sensitivity of conventional Sanger sequencing to detect mutations is generally 10%-20%. Consequently, reliable detection of T315I mutated CMLs by Sanger sequencing requires strong expansion of the mutated clone or the entire CML cell mass to bear the mutation. In contrast, ultra-deep sequencing (UDS) can overcome the sensitivity limits of conventional sequencing studies.16,17 In comparison to highly sensitive but mutation-specific PCRs (e.g. digital PCR assays18 or allele specific PCRs), UDS assays can be designed to identify all mutations in the TKD. This is essential in order to discover novel mutations and to diagnose patients who have gained more than one resistance mutation. The evolution of two or more mutations in the same clone (compound mutations) can occur rapidly under treatment with nonsensitive inhibitors. It changes the TKI resistance profile within months16 and results in an extremely resistant CML.19 Therefore, these cases need to be distinguished from those with multiple individual clones carrying one resistance mutation each. In previous studies, individual PCR products were cloned before Sanger sequencing to determine the clonal architecture.20 These elaborate cloning steps can be avoided with UDS because individual sequencing reads are the equivalent to cloned PCR products. CML cells with the T315I in combination with other key position mutations (e.g. G250E, Y253H or E255V) are resistant in vitro to currently available TKIs, including ponatinib.19 For those patients, therapy options without TKIs, such as allogeneic stem cell transplantation (alloSCT), urgently need to be considered. Here we used UDS in comparison to conventional Sanger sequencing8 to study the appearance and evolution of the T315I. By backtracking CML patients with a T315I mutation level, which was already detectable by Sanger sequencing, we describe the early dynamics and the progression of the mutational landscape.

Methods CML patient cohort Samples were received at our laboratory between June 2006 and October 2014 for routine diagnostic assessment. Patients gave written informed consent in accordance with the Declaration of Helsinki. The study was approved by the Munich Leukemia Laboratory Institutional Review Board. Cohort 1 included 40 CML patients (2-5 time points per patient) with a known T315I mutation detected by Sanger sequencing. At the end of the monitoring period, patients had a median age of 61 years (19-85 years) and had received treatment by 1-3 TKIs and alloSCT. We included patients and time points from chronic phase to blast crisis (Online Supplementary Table S1). Cohort 2 consisted of 42 patients who had achieved but subsequently lost major molecular response (MMR). In these patients, no resistance mutations were identified haematologica | 2016; 101(7)

by Sanger sequencing for routine diagnostic purposes. The samples sequenced in this part of the study were selected after MMR loss. Patients had a median age of 58 years (26-79 years). Cytogenetic aberrations in addition to the Philadelphia chromosome are given in Online Supplementary Table S2.

RNA isolation, cDNA synthesis RNA was isolated after red blood cell lysis from peripheral blood or mononucleated bone marrow cells. For isolation and cDNA synthesis, routine protocols were used.21

BCR-ABL1 fusion transcript quantification The BCR-ABL1/ABL1 ratio was determined according to previously published protocols22,23 and normalized to %IS (international scale) by the conversion factor 0.989.24

BCR-ABL1 TKD sequencing Sanger sequencing was performed as previously described.25 For UDS, PCRs were performed with FastStart High Fidelity PCR System (Roche Applied Science, Penzberg, Germany). BCR-ABL1 fusion transcript was amplified (first PCR), sequencing amplicons of the TKD were generated (second PCR) with Assay-on-Demand (AoD) oligonucleotide primer plates including multiplex identifiers (MIDs) 1-26. AoD plates were part of the IRON-III study (Roche Diagnostics, Penzberg, Germany). Primer sequences and PCR conditions are given in the Online Supplementary Tables S3 and S4. PCR products were tested on the Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Products were purified by Agencourt AMPureXP beads (Beckman Coulter, Krefeld, Germany) and pooled (1st/2nd amplicon ratio: 4/5). Pools were quantified using Quant-iT PicoGreen dsDNA kit (Invitrogen, Carlsbad, CA, USA). Sequencing with the XL+ kit for extended read length on GS Junior (Roche/454, Branford, CT, USA) was performed according to the manufacturerâ&#x20AC;&#x2122;s instructions.

Cytogenetic and molecular genetic analyses Cytomorphology and chromosome banding analysis combined with interphase fluorescence in situ hybridization were performed as previously described.26

Data analysis Sequencing raw data was processed with the long amplicon pipeline 1 and analyzed by GS Amplicon Variant Analyzer 3.0 (Roche/454) and Sequencing Pilot Module SeqNext 4.1.1 (JSI Medical Systems, Kippenheim, Germany). Sanger sequencing was analyzed by Mutation Surveyor 4.0 (SoftGenetics, State College, PA, USA) or Sequencing Pilot Module SeqPatient 4.1.2 (JSI). We calculated the percentage of mutated BCR-ABL1 transcripts relative to all BCR-ABL1 transcripts.16,17 To distinguish compound from multiclonal mutations, individual reads were investigated for the presence of more than one mutation using the GS Amplicon Variant Analyzer 3.0. In cases with ambiguous read distribution, the divergent progression of mutations during disease course was used to identify multiclonal disease development.

Results Sensitivity of ultra-deep XL+ sequencing We sequenced the TKD of ABL1 with high sensitivity on the 454 XL+ platform. Extended read length of the XL+ assay allowed covering the TKD (exons 4-10) by two sequencing amplicons (555 bp, 575 bp). To sequence only ABL1 from the BCR-ABL1 fusion gene, we partially amplified the fusion gene from cDNA (first PCR) serving as tem831


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plate for the sequencing amplicons in a second (nested) PCR (Figure 1). The percentage of mutated BCR-ABL1 relative to all BCR-ABL1 transcripts (in %) was calculated. Equal distribution of forward and reverse reads and a median coverage of 2257X (range 1012-4558X) per base were reached. Dilution tests were performed to prove quantitative and sensitive mutation detection. cDNAs from 2 samples with high and well defined mutation loads (identified by 454 XL+ sequencing) were mixed in different ratios to simulate specific or low percentage mutation loads (Online Supplementary Table S5). We obtained a high correlation of the calculated mutation load and the sequenced result for each single nucleotide variant including 1% and 2% mutation loads (Figure 2A; correlation coefficient calculated by linear regression: R2=0.998). Next, we performed independent technical replicates (n=5) of patient samples with mutations at levels below the Sanger sequencing detection limit. Replicates were performed from one cDNA with independent PCR amplifications and independent sequencing reactions. Different MIDs were used. Mutated reads were detectable for each mutation and in each individual test (Figure 2B), confirming the sensitivity of 1%2%, which is required for early mutation detection.

T315I mutation detection by Sanger sequencing and UDS We selected 40 CML patients (cohort 1), who had received treatment with at least one TKI (Table 1), and in whom we had previously identified the T315I mutation by conventional Sanger sequencing for routine diagnostic purposes: 36 of 40 had a BCR-ABL1/ABL1 (%IS) â&#x2030;Ľ1 (median 46.139; range 1.572-89.727) and prior TKI treatment of six months or longer (Online Supplementary Table S1). Two patients were in the ELN warning categories1 [BCRABL1/ABL1 (%IS) of 40.200 and treatment with imatinib for four months; BCR-ABL1/ABL1 (%IS) of 0.812 after 19 months of second-line dasatinib treatment] and 2 patients were sequenced after only two months of TKI treatment due to the cytomorphological detection of a blast crises and BCR-ABL1/ABL1 (%IS) of more than 10.

Early T315I mutation detection First, we performed UDS in comparison to Sanger sequencing and obtained a high degree of correlation of the

mutation loads (R2=0.948) (Figure 2C). Next, we sequenced earlier samples of all 40 patients with sensitive UDS. Resistance mutations with less than 5% mutation load were confirmed in an independent sequencing run if they occurred only once per patient during disease course. Sequencing analysis of samples with BCR-ABL1/ABL1 (%IS) 1 or less than 1 were confirmed in independent runs. If possible, we continued the backtracking until we reached a sample without any detectable T315I mutation with 454 XL+ sensitivity (n=37). For 3 patients, we completed the backtracking until we reached a sample with a T315I mutation load below the Sanger sequencing detection limit. We used cDNA from routine BCR-ABL1/ABL1 ratio analysis with a median interval between the sampling time points of three months. For patients who carried a T315I above the Sanger sequencing limit (15%) in more than one sample, we concluded that also by conventional Sanger sequencing the mutation would have been detected but was not because no Sanger sequencing was performed. For those samples, the earliest time point with a mutation load of more than 15% was defined as the end point of our monitoring interval (Online Supplementary Figure S1). The resulting monitoring intervals contained 2-5 samples per patient and covered a median period of five months (2-27 months) (Table 1 and Online Supplementary Table S1). At the beginning of our monitoring interval, patients had a median BCRABL1/ABL1 (%IS) of 29.739 (interval 0.046-74.007) and 61% (14 of 23) of patients were already in blast phase. During our monitoring interval, 13% (3 of 23) progressed to blast phase in parallel with the T315I mutation outgrowth. An increase in BCR-ABL1/ABL1 ratio in parallel with the T315I increase was observed for 15% of patients (6 of 39). Only 26% (6 of 23) stayed in complete hematologic response or chronic phase during the entire monitoring interval (Online Supplementary Table S1). In 20 of 40 patients, backtracking by UDS uncovered an early sample with a T315I mutation level below 15% (Patients #1 to #20). In the other 20 of 40 patients (Patients #21 to #40), no low percentage T315I mutation was detected by backtracking. In these patients, the earliest measureable T315I load was 27% or more (range 27%100%, median 60%), and was, therefore, detectable by both Sanger sequencing and UDS (Figure 3A). Differences between the samples with and without a low level T315I cannot be attributed to different sampling intervals (medi-

Figure 1. Amplicon design. The fusion transcript was amplified from cDNA (first PCR). The first PCR served as template for amplification of two amplicons for 454 XL+ sequencing (nested PCR). The position of the T315I is marked by an arrow. Key motive structures of the ABL1 tyrosine kinase domain (TKD, light green) are shown: Ploop (phosphate binding loop, yellow), SH2 contact region (dark green), SH3 contact region (blue), A-loop (activating loop, brown). The b3a2 (e14a2) fusion transcript with the exons of the BCR gene in light and the exons of the ABL1 gene is shown in dark gray.

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Early T315I detection by ultra-deep sequencing

an interval 3 months for both subsets; range 1-13 months for Patients #1 to #20 vs. 1-14 months for Patients #21 to #40). In addition, there was no difference in the time from initial CML diagnosis to T315I onset between the patient subsets with and without a low percentage T315I mutation. The T315I mutation occurred in 21 of 40 patients before or within the first year of disease (11 patients with and 10 patients without detectable low level T315I) (Online Supplementary Table S1). T315I could develop up to 14 years after CML was diagnosed. Patients with late T315I development were mainly on second- or third-line TKI therapy (Online Supplementary Table S1). For 21

patients, molecular and/or cytogenetic response data was available from initial diagnosis to T315I development. Only 2 patients (#15, #37) had achieved major treatment milestones before mutation development (CCyR/MMR) and lost the response status in parallel with T315I mutation development (Online Supplementary Table S1). The other 19 patients did not reach molecular or complete cytogenetic response before T315I mutation development [median period between initial diagnosis and T315I detection: 7 months (interval 2-60 months)]. Importantly, 5 of 19 patients were under TKI treatment for 12 months or longer, a time point at which MMR achievement would be required to define optimal response according to ELN guidelines.1

A

T315I mutation evolution Next, we used the patients with low level mutations to uncover the dynamics and time frames until the T315I mutation loads reached 15% or higher. It required a median period of three months (range 1-27 months) (Figure 3B) for the T315I to expand to more than 15%. Patient #17 (indicated by * in Figure 3B) did not show the rapid clonal expansion observed in the other patients. This patient received hydroxyurea alternating with imatinib every four weeks during the period analyzed. For the other samples, usually at the next monitoring time point, the T315I mutation load had greatly expanded. No low percentage T315I mutation was observed that disappeared during our monitoring period. However, our initial cohort included only patients with at least one Sanger sequencing positive sample (â&#x2030;Ľ15%). According to this study design, we cannot exclude the possibility that low level mutations may arise but disappear before being

B

Table 1. Patientsâ&#x20AC;&#x2122; characteristics.

Cohort 1: Cohort 2: longitudinal T315I monitoring control Total cohort Sex (male/female) Median monitoring interval, months (range) Median number of samples/patient (range)

C

n=40 27/13 5 (2-27) 2 (2-5)

n=42 23/19 1

End of monitoring period Median age, years (range)

61 (19-85)

58 (26-79)

12 (3-379) 164 (20-352) 11 (8-15)

6.5 (3-53) 219 (107-1192) 13 (9-16)

12 2 1 22 3 4

32 0 1 7 1 1

Peripheral blood counts1 Median WBC, x109/L (range) Median platelets, x 109/L (range) Median hemoglobin, g/dL (range)

Therapy2

Figure 2. Quality controls for 454 XL+ sequencing of the BCR-ABL1 kinase domain. (A) Mutation loads (%) are shown as obtained through calculation of the mixing ratio of mutated BCR-ABL1 from different patients (black line) and as sequenced by the 454 XL+ protocol (red crosses). (B) Sequencing was performed in replicates (n=5) for known resistance mutations. Bar charts show mean (+/-standard deviation). Bubbles display individual sequencing analysis, areas represent read numbers (white: all; red: mutated; example bubbles on the left side: 1000 total and 50 mutated reads). (C) The T315I mutation load is shown as obtained by Sanger sequencing and by 454 XL+ sequencing. Correlation coefficient R2 was calculated by linear regression.

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Imatinib only Dasatinib only Nilotinib only Two TKIs Three TKIs AlloSCT in addition to TKI

WBC: white blood cells; alloSCT: allogeneic hematopoietic stem cell transplantation; TKI: tyrosine kinase inhibitors. 1Available for 28 patients of cohort 1 and 25 of cohort 2. 2Available for all patients of cohort 1 and 41 of cohort 2; 13 patients of cohort 1 and 10 patients of cohort 2 received antineoplastic agents in addition to TKI: hydroxyurea, interferon alpha, cytarabine, FLAGIda [idarubicin, fludarabine, cytarabine, granulocyte colony-stimulating factor (G-CSF)].

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detected by Sanger sequencing in a context of routine diagnostics. Therefore, we selected a second control cohort of 42 CML patients (cohort 2) (Table 1). Patients of cohort 2 had lost the MMR status while treated with TKIs, but were negative for BCR-ABL1 resistance mutations by Sanger sequencing. We performed UDS for all patients, but could neither identify a low percentage T315I mutation nor any other resistance mutation (data not shown).

Additional TKI resistance mutations In cohort 1, sequencing the entire BCR-ABL1 TKD allowed identifying 20 cases without and 20 with resistance mutations in addition to T315I. For reasons of comparability, we limited our analysis of non-T315I mutations to aberrations detectable in the sample of the first T315I detection. This excluded aberrations, which were completely eradicated by previous therapeutic interventions before the T315I developed. Patients had acquired up to seven different non-T315I mutations. In total, 18 different mutation types were identified to occur with the T315I. The total number of additional mutations was 52. All mutations resulted in amino acid exchanges with known effects on TKI resistance.8,16 In our T315I mutated cohort, additional mutations were most frequently found at E255 (E255K: n=8 patients; E255V: n=7 patients). Importantly, only 18 of 52 non-T315I mutations had a mutation load above the Sanger sequencing detection limit and 34 of 52 were detectable by UDS only (Figure 4). Next, we determined the clonal architecture of the CMLs with more than one mutation. Of all additional mutations, 49 of 52 (94%) were located in the first sequencing amplicon together with the T315I mutation. Sequencing reads from multiclonal CMLs should carry only one mutation per read, whereas for compound mutated CMLs the aberrations should be seen on the same sequencing read. However PCR-mediated recombination artifacts may cause sequence variants from different CML clones to be seen on the same read.27 CMLs can, therefore, be falsely classified as compound mutated.28 We also observed cases with a read distribution, which could be derived from either a multiclonal or a compound mutated case. To determine the extent of false compound mutations in our sequencing assay, we mixed cDNA from patients with only one mutation (Y253H+E255K; Y253H+T315I; E255K+T315I). The recombination rate (calculated as previously described by Deininger et al.29) was on average 17% per 100 bp (data not shown). In critical cases, we used the longitudinal design of our study as additional information to eliminate misclassification. Patients could be classified as multiclonal, despite reads with a chimeric mutation pattern, if one mutation decreased or disappeared, while the other increased or stayed unaltered during the course of the disease. By applying this additional criterion, we could classify 12 of the 20 patients with additional mutations as multiclonal and 3 as compound mutated (Figure 4). Five patients could not be unequivocally categorized. This was due to technical limitations for 4 patients (mutations in two amplicons or potentially strong PCR-mediated recombination). For Patient #21, the clonal architecture could not be resolved because of an acquired Philadelphia chromosome duplication (Online Supplementary Table S2). Here, mutations from different reads could be derived from either different clones or different Philadelphia chromosomes. Figure 5 shows the detailed clonal development of 4 834

patients with additional non-T315I resistance mutations. We extended the backtracking to the diagnosis sample. In the multiclonal Patients #15 and #16, the T315I arose and overgrew all otherwise mutated clones within less than one year (Figure 5A and B). In Patient #37, the E255V clone gained a T315I mutation under dasatinib treatment and expanded to 100% within five months (Figure 5C). In Patient #33, the Y253H mutated clone further diversified into three compound mutated subclones, of which the Y253H+T315I clone arose to 76% within 79 days (Figure 5D and Online Supplementary Figure S2).

Discussion By backtracking patients with a high T315I mutation load detected by Sanger sequencing (cohort 1), we identified UDS as a sensitive approach for earlier mutation identification. We excluded the possibility that low level TKD mutations arise sporadically and frequently without subsequent expansion in a second cohort. Cohort 2 was selected to contain CML patients from the European LeukemiaNet (ELN) warning or failure category, in whom sequencing is usually performed. In contrast to cohort 1, UDS in cohort 2 did not reveal any low percentage mutation.

A

B

Figure 3. Detection of the T315I by ultra-deep sequencing and development. The percentage of T315I mutated BCR-ABL1 of all BCR-ABL1 transcripts is shown as detected by 454 XL+ sequencing. Sanger sequencing detection limit is defined as 15% (red line). (A) The T315I load (%) at the earliest detectable time point is given (UPI: unique patient identifier). (B) In patients for whom a low T315I mutation level (<15%) was detected, subsequent time points are shown. Patient #17 (*) was treated with hydroxyurea or imatinib changing every four weeks for 31 weeks.

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Early T315I detection by ultra-deep sequencing

In the T315I positive cohort 1, we sequenced two to five longitudinal time points from patients treated with imatinib, dasatinib and/or nilotinib, which allowed a retrospective description of the mutation evolution. We found a rapid and ever increasing nature of the T315I mutation. For the majority of patients under ineffective TKIs, a 1%2% mutation load expanded to Sanger sequencing detection limits in between two routine analysis time points corresponding to three months. As a consequence of the rapid T315I subclonal outgrowths, each sequenced sample is only a snapshot of the mutational landscape at one time point and despite narrow monitoring intervals, the low percentage mutation onset can be missed. In fact, we only identified the early onset of the T315I mutation in every second patient. For patients without any detectable low percentage T315I mutation, we had sequenced at least one earlier time point and could not detect any mutation with at least a 1% load. The rapid T315I outgrowths demonstrated in our study supports the quest for introduction of UDS in routine clinical procedures16,30 and the relevance of mutation monitoring requested by ELN guidelines1 for patients with suboptimal response (“failure category” and “warning category”). After the first year, patients with BCR-ABL1/ABL1 (%IS) below 1 fall into the “warning category.” Therefore, absolute BCR-ABL1 transcript numbers are low, despite good RNA quality. Mutations in these patients can only be detected if the mutation load is high. Mutations detected at those time points are very informative; however, the number of false negative sequencing results is probably considerable, and negative results should be interpreted with caution. A one-time negative sequencing result cannot exclude the contribution of a mutation in the course of resistance. Patients with rapid clonal evolution might benefit from BCR-ABL1 sequencing several times during disease evolution. Although all low percentage (<15%) T315I mutations were detectable by Sanger sequencing at subsequent routine sampling time points, results from UDS would have allowed an earlier therapy intervention and earlier consideration of alloSCT, as recommended by the current ELN guidelines for patients with T315I mutations. This would increase the time frame needed to prepare an alloSCT, e.g. initiation of HLA typing of patient’s siblings or the search for unrelated stem cell donors.1 Given the retrospective nature of our study, we can

only speculate as to the benefit of changing TKI earlier. Firstly, for some patients, the T315I mutation outgrowths was parallel by an increase in the BCR-ABL1/ABL1 ratio and progress to blast crisis. Support for a change in TKI before progression comes from the phase II trial on ponatinib in Philadelphia chromosome-positive leukemias (PACE). Among chronic phase patients, 66% achieved complete cytogenetic response (CCyR) under ponatinib; however, among patients with accelerated phase and blast phase CML, only 33% and 21% reached CCyR, respectively.31 Secondly, early eradication of mutated cells could prevent the malignant clone from further diversification, e.g. the gain of cytogenetic aberrations or additional resistance mutations. We observed 50% of patients with other TKD mutations in addition to the T315I mutation and found that more than half of them were also present only at low levels. This showed that not only for T315I, but also for all other resistance mutations, conventional Sanger sequencing would have identified only a small subset of mutations. Importantly, other highly sensitive assays for T315I (e.g. digital PCR or mass spectrometry) can detect the mutation even below 1% (e.g. 0.05%15 or less than 10 mutated copies18), but are limited to the detection of the T315I mutation. These assays do not reveal any of the additional mutations. The high number of additional mutations could explain why we did not see a correlation between the increase in the T315I mutation load and the overall malignant cell burden (BCR-ABL1/ABL1 ratio) in every patient. The dynamics of total CML cell expansion are likely to be the sum of all resistance mechanisms and depend on the administered TKIs.32 In line with other previously published BCR-ABL1 sequencing studies, we also observed that the variety of detected mutations reflected the selective pressure of previously administered TKIs.16,20,33 In our study, a strong dominant outgrowth of the T315I clone over all other mutated clones was observed. This demonstrated the strong resistance of the mutated cells against first- and second-line TKIs and the extraordinary importance of considering a T315I mutation before changing TKI. In patients with the T315I mutation and other mutations in one clone, the compound mutated clone strongly expanded and the detected mutation loads of both mutations remained high during the monitoring period. Treatment for patients with compound mutations is chal-

Figure 4. Additional tyrosine kinase inhibitor resistance mutations. The number and type of additional mutations per patient is shown. Only mutations present at the time point of initial T315I detection are included. Mutation load is color-coded (green: <15%; orange: 15-49%; dark red: >50%). Cases can have multiclonal (multi) or compound (comp) mutations and 5 cases could not be definitely defined. Tyrosine kinase inhibitor treatment up to the time point of initial T315I mutation detection is displayed (im: imatinib; da: dasatinib; ni: nilotinib). Patient #5 had three mutations resulting in a F317L amino acid change: ttc>ctc, 12%; ttc>tta, 27%; ttc>ttg>12%.

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lenging. In vitro data suggested that many combinations of mutations (including those with T315I) render cells resistant to available TKIs including ponatinib,19,34 However, recent data from the PACE trial encourages the use of ponatinib in heavily pre-treated patients with compound mutations.29 Compound mutated CML cases can be distinguished from cases with multiple independent clones by

their read distribution using the longer amplicons design of 454 XL+ sequencing. However, a study by Parker et al. suggested that technical artifacts cause a false classification of multiclonal cases as compound mutated.28 PCR mediated recombination generates one PCR product, which is derived from two original molecules, but presents as one double mutated read.27 To clarify the clonal

Figure 5. Examples of clonal evolution. Patient #15 and #16 are examples of multiclonal chronic myeloid leukemia (CML) patients (A and B) and patient #37 and #33 (C and D) have compound mutated clones. We extended the backtracking analysis to the initial CML diagnosis sample. Sampling dates are shown on the x-axis and the mutation load on the y-axis. Tyrosine kinase inhibitor treatment by imatinib (im) and dasatinib (da) is indicated by an arrow. (A and B) Each CML subclone is shown as individual area. The T315I is displayed in red; the other resistance mutations are displayed in gray, green and orange. (C) The E255V mutated clone (gray) gained a T315I+E255V double mutated subfraction (red). (D) Of the Y253H mutated BCR-ABL1 transcripts (gray), 98% gained one additional mutation: 7% the T317L (orange), 15% the T317I (green) and 76% the T315I (red) mutation (Online Supplementary Figure S2).

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Early T315I detection by ultra-deep sequencing

architecture of cases with mixed reads, we used the longitudinal design of our study. Mutations derived from the same clone cannot diverge during disease course. However, we cannot exclude the possibility that, in rare cases, a nucleotide exchange at one position would occur independently several times in one patient but in independent clones. In future studies, the generation of chimeric reads could be reduced by modified sequencing strategies (single molecule consensus sequencing)28 or could be mathematically corrected.29 However, the aforementioned advances are not sufficient to resolve the clonal evolution of cases with cytogenetic aberrations in addition to the initial Philadelphia chromosome, e.g. duplication of the Philadelphia chromosome.35 We observed additional cytogenetic aberrations in one-third of our cohort with the T315I mutation (cohort 1). We were unable to identify the clonal development of some patients because mutated reads could be assigned to either of the two Philadelphia chromosomes or to different CML clones; only single cell sequencing would clarify their clonal architecture.36 Importantly, additional cytoge-

References 10. 1. Baccarani M, Deininger MW, Rosti G, et al. European LeukemiaNet recommendations for the management of chronic myeloid leukemia: 2013. Blood. 2013;122(6):872-884. 2. Hochhaus A, O'Brien SG, Guilhot F, et al. Six-year follow-up of patients receiving imatinib for the first-line treatment of chronic myeloid leukemia. Leukemia. 2009;23(6): 1054-1061. 3. Kantarjian HM, Shah NP, Cortes JE, et al. Dasatinib or imatinib in newly diagnosed chronic-phase chronic myeloid leukemia: 2year follow-up from a randomized phase 3 trial (DASISION). Blood. 2012;119(5):11231129. 4. Kantarjian HM, Hochhaus A, Saglio G, et al. Nilotinib versus imatinib for the treatment of patients with newly diagnosed chronic phase, Philadelphia chromosome-positive, chronic myeloid leukaemia: 24-month minimum follow-up of the phase 3 randomised ENESTnd trial. Lancet Oncol. 2011;12(9): 841-851. 5. Hochhaus A, Rosti G, Cross NC, et al. Frontline nilotinib in patients with chronic myeloid leukemia in chronic phase: results from the European ENEST1st study. Leukemia. 2016;30(1):57-64. 6. Shah NP, Nicoll JM, Nagar B, et al. Multiple BCR-ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia. Cancer Cell. 2002;2(2):117-125. 7. Hochhaus A, La RosĂŠe P. Imatinib therapy in chronic myelogenous leukemia: strategies to avoid and overcome resistance. Leukemia. 2004;18(8):1321-1331. 8. Soverini S, Hochhaus A, Nicolini FE, et al. BCR-ABL kinase domain mutation analysis in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors: recommendations from an expert panel on behalf of European LeukemiaNet. Blood. 2011;118(5): 1208-1215. 9. Soverini S, Branford S, Nicolini FE, et al. Implications of BCR-ABL1 kinase domain-

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mediated resistance in chronic myeloid leukemia. Leuk Res. 2014;38(1):10-20. Ernst T, Hoffmann J, Erben P, et al. ABL single nucleotide polymorphisms may masquerade as BCR-ABL mutations associated with resistance to tyrosine kinase inhibitors in patients with chronic myeloid leukemia. Haematologica. 2008;93(9):1389-1393. Apperley JF. Part II: management of resistance to imatinib in chronic myeloid leukaemia. Lancet Oncol. 2007;8(12):1116-1128. Cortes JE, Kantarjian HM, Shah NP, et al. Ponatinib in refractory Philadelphia chromosome-positive leukemias. N Engl J Med. 2012;367(22):2075-2088. Corbin AS, Buchdunger E, Pascal F, Druker BJ. Analysis of the structural basis of specificity of inhibition of the Abl kinase by STI571. J Biol Chem. 2002;277(35):3221432219. Nicolini FE, Ibrahim AR, Soverini S, et al. The BCR-ABLT315I mutation compromises survival in chronic phase chronic myelogenous leukemia patients resistant to tyrosine kinase inhibitors, in a matched pair analysis. Haematologica. 2013;98(10):1510-1516. Parker WT, Lawrence RM, Ho M, et al. Sensitive detection of BCR-ABL1 mutations in patients with chronic myeloid leukemia after imatinib resistance is predictive of outcome during subsequent therapy. J Clin Oncol. 2011;29(32):4250-4259. Soverini S, De Benedittis C, Machova Polakova K, et al. Unraveling the complexity of tyrosine kinase inhibitor-resistant populations by ultra-deep sequencing of the BCRABL kinase domain. Blood. 2013;122(9): 1634-1648. Kastner R, Zopf A, Preuner S, et al. Rapid identification of compound mutations in patients with Philadelphia-positive leukaemias by long-range next generation sequencing. Eur J Cancer. 2014;50(4):793800. Oehler VG, Qin J, Ramakrishnan R, et al. Absolute quantitative detection of ABL tyrosine kinase domain point mutations in chronic myeloid leukemia using a novel nanofluidic platform and mutation-specific PCR. Leukemia. 2009;23(2):396-399.

19. Zabriskie MS, Eide CA, Tantravahi SK, et al. BCR-ABL1 Compound Mutations Combining Key Kinase Domain Positions Confer Clinical Resistance to Ponatinib in Ph Chromosome-Positive Leukemia. Cancer Cell. 2014;26(3):428-442. 20. Khorashad JS, Kelley TW, Szankasi P, et al. BCR-ABL1 compound mutations in tyrosine kinase inhibitor-resistant CML: frequency and clonal relationships. Blood. 2013;121 (3):489-498. 21. Meggendorfer M, Haferlach T, Alpermann T, et al. Specific molecular mutation patterns delineate chronic neutrophilic leukemia, atypical chronic myeloid leukemia, and chronic myelomonocytic leukemia. Haematologica. 2014;99(12):e244-e246. 22. Emig M, Saussele S, Wittor H, et al. Accurate and rapid analysis of residual disease in patients with CML using specific fluorescent hybridization probes for real time quantitative RT-PCR. Leukemia. 1999;13(11):18251832. 23. Cross NC, White HE, Colomer D, et al. Laboratory recommendations for scoring deep molecular responses following treatment for chronic myeloid leukemia. Leukemia. 2015;29(5):999-1003. 24. Hughes T, Deininger MW, Hochhaus A, et al. Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for harmonizing current methodology for detecting BCRABL transcripts and kinase domain mutations and for expressing results. Blood. 2006;108(1):28-37. 25. Hochhaus A, Kreil S, Corbin AS, et al. Molecular and chromosomal mechanisms of resistance to imatinib (STI571) therapy. Leukemia. 2002;16(11):2190-2196. 26. Schoch C, Schnittger S, Bursch S, et al. Comparison of chromosome banding analysis, interphase- and hypermetaphase-FISH, qualitative and quantitative PCR for diagnosis and for follow-up in chronic myeloid leukemia: a study on 350 cases. Leukemia. 2002;16(1):53-59. 27. Parker WT, Phillis SR, Yeung DT, et al. Many BCR-ABL1 compound mutations reported in chronic myeloid leukemia patients may

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Genes Chromosomes Cancer. 2010;49(10): 910-918. Shapiro E, Biezuner T, Linnarsson S. Singlecell sequencing-based technologies will revolutionize whole-organism science. Nat Rev Genet. 2013;14(9):618-630. Schnittger S, Haferlach C, Nadarajah N, et al. CML Patients with Resistance to Tyrosine Kinase Inhibitors and without BCR-ABL1 Resistance Mutation Frequently Carry Other Gene Mutations. Blood. 2014;124(21):4516. Soverini S, de Benedittis C, Mancini M, Martinelli G. Mutations in the BCR-ABL1 Kinase Domain and Elsewhere in Chronic Myeloid Leukemia. Clin Lymphoma Myeloma Leuk. 2015;(15 Suppl):S120-128. Bixby D, Talpaz M. Mechanisms of resistance to tyrosine kinase inhibitors in chronic myeloid leukemia and recent therapeutic strategies to overcome resistance. Hematology Am Soc Hematol Educ Program. 2009:461-476.

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ARTICLE

Acute Myeloid Leukemia

Salvage therapy with high-dose cytarabine and mitoxantrone in combination with all-trans retinoic acid and gemtuzumab ozogamicin in acute myeloid leukemia refractory to first induction therapy

Marie-Luise Hütter-Krönke,1 Axel Benner,2 Konstanze Döhner,1 Jürgen Krauter,3,4 Daniela Weber,1 Margit Moessner,1 Claus-Henning Köhne,5 Heinz A. Horst,6 Ingo G.H. Schmidt-Wolf,7 Mathias Rummel,8 Katharina Götze,9 Elisabeth Koller,10 Andreas L. Petzer,11 Hans Salwender,12 Walter Fiedler,13 Heinz Kirchen,14 Detlef Haase,15 Stephan Kremers,16 Matthias Theobald,17 Axel C. Matzdorff,18 Arnold Ganser,4 Hartmut Döhner,1 and Richard F. Schlenk1

EUROPEAN HEMATOLOGY ASSOCIATION

Ferrata Storti Foundation

Haematologica 2016 Volume 101(7):839-845

Department of Internal Medicine III, University Hospital Ulm, Germany; 2Division of Biostatistics, German Cancer Research Center, Heidelberg, Germany; 3Department of Oncology and Hematology, Klinikum Braunschweig, Germany; 4Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Germany; 5 Department of Oncology and Hematology, Klinikum Oldenburg, Germany; 6Department of Internal Medicine II, University Hospital Schleswig-Holstein Campus Kiel, Germany; 7 Department of Internal Medicine III, CIO, University Hospital of Bonn, Germany; 8 Department of Hematology/Oncology, University-hospital Giessen, Germany; 9Department of Internal Medicine III, Technical University of Munich, Germany; 10Department of Hematology/Oncology, Hanuschkrankenhaus, Wien, Austria; 11Department of Medical Oncology and Hematology, Krankenhaus der Barmherzigen Schwestern, Linz, Austria; 12 Department of Hematology/Oncology, Asklepios Klinik Altona, Hamburg, Germany; 13 Department of Internal Medicine II, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 14Department of Hematology/Oncology, Krankenhaus der Barmherzigen Brüder, Trier, Germany; 15Department of Hematology and Oncology, GeorgAugust-University Hospital of Göttingen, Germany; 16Department of Hematology/Oncology, Caritas-Krankenhaus, Lebach, Germany; 17Department of Medicine III, Johannes Gutenberg-University Mainz, Germany; 18Department of Hematology/Oncology, CaritasKrankenhaus, Saarbrücken, Germany 1

Correspondence: ABSTRACT

O

utcome of patients with primary refractory acute myeloid leukemia remains unsatisfactory. We conducted a prospective phase II clinical trial with gemtuzumab ozogamicin (3 mg/m² intravenously on day 1), all-trans retinoic acid (45 mg/m² orally on days 46 and 15 mg/m² orally on days 7-28), high-dose cytarabine (3 g/m²/12 h intravenously on days 1-3) and mitoxantrone (12 mg/m² intravenously on days 2-3) in 93 patients aged 18-60 years refractory to one cycle of induction therapy. Primary end point of the study was response to therapy; secondary end points included evaluation of toxicities, in particular, rate of sinusoidal obstruction syndrome after allogeneic hematopoietic cell transplantation. Complete remission or complete remission with incomplete blood count recovery was achieved in 47 (51%) and partial remission in 10 (11%) patients resulting in an overall response rate of 61.5%; 33 (35.5%) patients had refractory disease and 3 patients (3%) died. Allogeneic hematopoietic cell transplantation was performed in 71 (76%) patients; 6 of the 71 (8.5%) patients developed moderate or severe sinusoidal obstruction syndrome after transplantation. Four-year overall survival rate was 32% (95% confidence interval 24%-43%). Patients responding to salvage therapy and undergoing allogeneic hematopoietic cell transplantation (n=51) had a 4-year survival rate of 49% (95% confidence intervaI 37%-64%). Patients with fms-like tyrosine kinase internal tandem duplication positive acute myeloid leukemia had a poor outcome despite transplantation. In conclusion, the described regimen is an effective and tolerable salvage therapy for patients who are primary refractory to one cycle of conventional intensive induction therapy. (clinicaltrials.gov identifier: 00143975) haematologica | 2016; 101(7)

richard.schlenk@uniklinik-ulm.de

Received: December 27, 2015. Accepted: March 24, 2016. Pre-published: April 25, 2016. doi:10.3324/haematol.2015.141622

Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/101/7/839

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

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Introduction The prognosis of patients with acute myeloid leukemia (AML) refractory to first induction chemotherapy is poor. About 15%-20% of younger patients (<61 years) are primary refractory to one cycle of standard 7+3 induction chemotherapy.1-3 Substantial long-term survival in this patient population has only been observed if allogeneic hematopoietic cell transplantation (HCT) was performed, resulting in overall survival (OS) rates of 10%-31% measured from date of HCT.4-9 Best outcome after allogeneic HCT is achieved when transplantation is performed in complete remission (CR) or partial remission (PR) after salvage therapy.6,8,9 Therefore, better response rates to salvage therapy are crucial to improve OS in these patients. Different high-dose cytarabine-based salvage regimens, often used in combination with anthracyclins and epipodophyllotoxins, resulted in CR rates between 11% and 70%.3 In order to increase CR rates, high- or intermediate-dose cytarabine has been combined with a wide spectrum of different drugs such as idarubicin and fludarabine (FLAG-Ida), clofarabine, gemtuzumab ozogamicin Table 1. Clinical and genetic characteristics at diagnosis and study entry.

Characteristics Age, years Median (range) Sex, n. (%) Male Female Type of AML, n. (%) De novo s-AML t-AML Cytogenetics risk, n. (%) CBF-AML Intermediate Adverse* Missing Mutated-NPM1, n. (%) Missing FLT3-ITD, n. (%) Missing WBC, x 109/L Median (range) Missing Platelets, x109/L Median (range) Missing Hemoglobin, g/dL Median (range) Missing Bone marrow blasts, % Median (range) Missing Peripheral blood blasts, % Median (range) Missing, n. CD33 expression Cut off at 20%, n. (%) Positive Missing, n.

At diagnosis

At study entry

48 (22-62)

Methods Patients Patients 18-60 years of age with AML defined by the 2001 World Health Organization Classification of Tumours21 who did not achieve a CR, CR with incomplete blood count recovery (CRi) or partial remission (PR) after one cycle of standard chemotherapy, and who had adequate organ function, were eligible for entry into the trial. Patients with acute promyelocytic leukemia and patients with a concomitant uncontrolled infection were not eligible. Written informed consent was obtained from all patients at study entry according to the Declaration of Helsinki. The study was approved by the local Ethics Review Committee and registered at clinicaltrials.gov identifier: 00143975.

45 (48) 48 (52) 77 (83) 4 (4) 12 (13) 3 (3.5) 48 (58) 32 (38.5) 10 12 (15) 14 18 (22) 10

Study design

16.0 (0.6-243.6) 0

1.41 (0.1-7.1) 3

74 (5-585) 0

33.5 (2-223) 3

9.1 (5.0-13.3) 0

9.35 (6.80-12,5) 3

70 (7-100) 5

60 (5.5-100) 4

47 (0-100) 3

4.5 (0-46) 19

The trial was a single-arm multi-center phase II trial. All patients received one cycle of GO-A-HAM consisting of GO 3 mg/m² intravenously (IV max. 5 mg absolute) over two hours on day 1; cytarabine 3 g/m² every 12 hours IV on days 1-3; mitoxantrone 12 mg/m² IV days 2 and 3; oral all-trans retinoic acid 45 mg/m² on days 4-6 and 15 mg/m² on days 7-28. In all patients, allogeneic HCT from a matched related or matched unrelated or from a haploidentical family donor was intended irrespective of the remission status after GO-A-HAM.

Table 2. Overall treatment response.

Parameter

70 (86%) 12

s-AML: secondary AML after preceding MDS; t-AML: therapy-induced AML; CBF: core binding factor; NPM1: nucleophosmin; FLT3-ITD: FMS-like tyrosine kinase 3 gene internal tandem duplication; WBC: white blood count. *According to European LeukemiaNet recommendations.1

840

(GO), and all-trans retinoic acid (ATRA).10-13 The GermanAustrian AML Study Group (AMLSG) evaluated the conventional (HAM) and a sequential (S-HAM) HAM regimen in patients with refractory disease. No beneficial effect could be shown with the dose-intense S-HAM regimen.14 In the subsequent trial, AML HD98A, ATRA was added to the HAM regimen (A-HAM) based on promising in vitro15,16 and in vivo data.17,18 The sequential administration of ATRA after HAM led to an overall response rate of 47% and was thus remarkably better than HAM alone.9 In line with our data, Montillo et al. reported a CR rate of 70% induced by a salvage therapy combining ATRA with fludarabine, cytarabine, idarubicin, and granulocyte-colony stimulating factor (G-CSF).10 Several phase I-II clinical trials evaluating GO in relapsed/refractory AML showed response rates up to 33% when used as single agent, and 12%-68% in combination with chemotherapy.13,19,20 Based on these promising results, we combined GO with our previously established A-HAM regimen (GO-A-HAM) in patients refractory to one cycle of 3+7-based induction therapy. The main objectives of the study were to assess GO-A-HAM with regard to response rate, toxicity [including sinusoidal obstruction syndrome (SOS)] after allogeneic HCT, and survival.

Complete remission Complete remission with incomplete blood count recovery Partial remission Overall response rate Refractory disease Early death

Patients (n=93) 28 19

(30%) (20%)

10 57 33 3

(11%) (61%) (35%) (3%)

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Impact of GO in primary refractory AML

Statistical analyses, efficacy and safety end points The primary end point of the study was achievement of CR or CRi at a maximum of 30 days after start of therapy with GO-AHAM defined by standard criteria.22 Beyond CR/CRi, partial remission (PR) defined according to standard criteria22 was documented and evaluated. A continuous safety assessment was performed during the study. Toxicities reported during therapy were evaluated according to the National Cancer Institute Common Toxicity Criteria (NCI-CTC), v.3.0. The safety end points with corresponding maximally tolerated rates were: i) NCI-CTC grade 4+5 liver toxicity â&#x2030;¤ 10%; ii) rate of deaths within 30 days after start of GO-A-HAM 25% or under; and iii) rate of severe SOS after allogeneic HCT or under 20%. SOS was defined according to the Baltimore criteria23 and graded as described by Bearman.24 Management of SOS followed local standard operating procedures of the respective transplantation centers. Univariable and multivariable logistic regression models were used to test the influence of covariates on response to induction therapy. The Kaplan-Meier method was used to estimate the distribution of OS. Survival distributions were compared using the log rank test. To address the time dependence of the variable allogeneic HCT, a multivariable analysis based on an extended Cox regression model was used according to the method of Andersen and Gill.25 Missing data were replaced by 50 imputations using multivariate imputations by chained equations applying predictive mean matching.26 Backward selection applying a stopping rule based on P-values was used in multivariable regression models to exclude redundant or unnecessary variables.26 For all analyses, P<0.05 was considered statistically significant. All eligible patients who started with GO-A-HAM were included in the analysis. Statistical analyses were performed with the statistical software environment R v.3.2.1 using the R package cmprsk, survival, rms and Hmisc.27

Results Patientsâ&#x20AC;&#x2122; characteristics From July 2004 to June 2007, 95 patients from 23 institutions fulfilled the eligibility criteria and were enrolled in the study. Two patients withdrew their consent before initiation of treatment; thus, a total of 93 patients are reported. Prior treatments of the patients were as follows: 29 patients received standard induction therapy with idarubicin, cytarabine, and etoposide (ICE), 26 patients ICE in combination with ATRA (A-ICE), 17 patients with valproic acid (V-ICE), and 12 patients with both ATRA and valproic acid (VA-ICE), according to the initial randomization of the AMLSG 07-04 protocol (clinicaltrials.gov identifier: 00151242);28 9 patients were treated in the German AML Intergroup trial29 and received standard induction therapy with daunorubicin and cytarabine (DA). Median age was 48 years (range 22-62 years); further demographics and baseline characteristics of the 93 patients are shown in Table 1. Eighteen of 83 (22%) patients had an fms-like tyrosine kinase internal tandem duplication (FLT3-ITD) mutation and 12 of 79 (15%) patients a nucleophosmin (NPM1) mutation at time of initial diagnosis. The surface marker CD33 was expressed in 87% of the patients with a 20% expression cut-off level.

Response rate and treatment outcome Twenty-eight patients (30%) achieved CR, 19 patients (20%) CRi, and 10 patients PR (11%), resulting in an overall response rate of 61%; 33 patients (35%) were refractory and 3 patients (3%) died within 30 days (Table 2). Multivariable analysis on the end point CR/CRi or overall response revealed no prognostic influence of the following variables assessed at diagnosis: age, sex, cytogenet-

Figure 1. Flow chart on study conduct. Flow-chart showing subsequent treatment and outcome according to response to GO-A-HAM. A: all-trans retinoic acid; AlloHCT: allogeneic hematopoietic cell transplantation; CR: complete remission; CRi: CR with incomplete hematologic recovery; FLAG-I/M: fludarabin, cytarabine, G-CSF+ idarubicin or mitoxantrone; GO: gemtuzumab ozogamicin; HA high-dose cytarabine; HAM: high-dose cytarabine and mitoxantrone; LD-ARAC: low-dose cytarabine; MTCG: mitoxantrone, topotecan, cytarabine, imatinib; PR: partial remission; RD: refractory disease.

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M.-L. Hütter-Krönke et al. Table 3. Infectious complications.

Infection site Septicemia Pulmonary Gastrointestinal Skin/soft tissue ENT Esophagus Liver Urogenital Other Grading according CTC NCI version 3

Grade 1/2 1 (1%) 2 (2%) 1 (1%) 2 (2%)

1 (1%)

Grade ≥3 43 (46%) 20 (22%) 11 (12%) 5 (5%) 3 (3%) 1 (1%) 1 (1%) 1 (1%) 1 (1%)

Grading according to the National Cancer Institute's Common Terminology Criteria for Adverse Effects v.3. ENT: ear/nose/throat.

Table 4. Non-hematologic adverse events (excluding infection).

Adverse event Figure 2. Survival after allogeneic hematopoietic cell transplantation according to response to GO-A-HAM salvage therapy.

ics (according to European LeukemiaNet criteria),1 white cell count (WBC), bone marrow blast percentage, type of AML [de novo versus secondary AML evolving from myelodysplastic syndrome (AML) versus therapy-related AML (t-AML)], CD33 expression, mutated NPM1, FLT3-ITD and prior treatment with ATRA. Of note, patients with t-AML had a nearly equal CR/CRi rate of 50% compared to de novo AML with 53%, whereas none of the 4 patients with s-AML responded to GO-A-HAM.

Gastrointestinal Neurological Cardiac Pulmonary Hemorrhage Thrombosis Renal Liver Pain Endocrine Skin General condition

Grade 1/2 1 (1%)

1 (1%)

2 (2%) 2 (2%) 2 (2%)

Grade ≥3 14 (15%) 10 (11%) 5 (5%) 4 (4%) 4 (4%) 3 (3%) 1 (1%) 2 (2%) 2 (2%) 1 (1%)

Grading according to the National Cancer Institute's Common Terminology Criteria for Adverse Effects v.3.

Toxicity Hematologic toxicity Median times of WBC (>1x109/L), neutrophil (>0.5x109/L) and platelet (>20x109/L) recovery were 22, 25 and 21 days, respectively.

Non-hematologic toxicity In 60 (65%) of the 93 patients, a total of 86 infections with a CTC grade 3 or over occurred. The most frequent infections were septicemia (n=43; 46%), pneumonia (n=20; 22%), and infections of the gastrointestinal tract (n=11; 12%). Other infection sites included skin and soft tissue (n=5; 5%), ear-nose-throat (n=3; 2%), urogenital tract (n=1; 1%), liver (n=1; 1%), and esophagus (n=1; 1%) (Table 3). Five patients died of severe infection, including 3 patients who died within 30 days after start of GO-AHAM. Other non-hematologic toxicities were seen in 30 patients, and 42 adverse events with a CTC grade 3 or more were reported (Table 4). The most common events were gastrointestinal toxicities (n=14) including diarrhea, nausea and mucositis, and neurological symptoms (n=10) with polyneuropathy, ataxia and hallucination. Cardiac events [tachyarrhythmia absoluta, left ventricular failure (n=2), pericardial effusion and pericarditis] occurred in 5 patients. A total of 4 episodes of hemorrhage CTC grade 3 were noted during treatment, two central nervous system bleedings, one vaginal bleeding, and one bleeding 842

after central venous system installation. Four patients developed respiratory insufficiency. An additional 3 events of thrombosis were reported, two of them located in the internal jugular vein. One patient experienced renal failure. SOS was not observed during or after GO-AHAM. Median duration of hospitalization was 27.5 days (range 8-96 days).

Subsequent treatment Consolidation therapy with allogeneic HCT was performed in 71 of 93 patients (76%); 51 patients achieved CR, CRi or PR after GO-A-HAM, and 20 patients had persistent refractory disease. Reasons for not proceeding to an allogeneic HCT were death after GO-A-HAM (n=3), comorbidities and bad performance status (n=12), no compatible donor (n=3) and patients’ wish (n=4). Of the 71 patients who received allogeneic HCT, 50 patients proceeded immediately to allogeneic HCT, 19 patients received one or two additional cycles of intensive chemotherapy [A-HAM, n=5; HAM/high-dose cytarabine, n=9; GO-A-HAM, n=3; high-dose cytarabine with mitoxantrone and topotecan (clinicaltrials.gov identifier: 00744081), n=1; fludarabine, cytarabine, G-CSF (FLAG),±idarubicin/mitoxantrone, n=1], one patient received GO as single agent, and one received low-dose cytarabine (Figure 1). Median time from start of therapy with GO-A-HAM to allogeneic HCT was 70 days; 16 haematologica | 2016; 101(7)


Impact of GO in primary refractory AML

patients were transplanted from matched related donors, 50 patients from matched unrelated donors, and 5 patients from haplo-identical related donors. Myeloablative conditioning regimens (n=34) included cyclophosphamide (Cy) and total body irradiation (TBI-Cy) or busulfan and Cy (Bu-Cy) (n=26), Bu-Cy with radioimmunotherapy (RIT) (n=6),30 and fludarabine, melphalan and thiotepa (n=2). Dose-reduced conditioning regimens (n=37) included FLAMSA-based regimens (n=24),31 fludarabine plus total body irradiation (n=6), fludarabine plus busulfan (n=2), fludarabine plus melphalan+/-BCNU (n=3), and fludarabine plus or threosulfan (n=2). OS after allogeneic HCT with myeloablative and dose-reduced conditioning was comparable (P=0.54). Of 19 patients not proceeding to allogeneic HCT, 7 received additional cycles of intensive therapy (HAM/high-dose cytarabine n=2, single agent GO, n=1; FLAGÂąidarubicin/mitoxantrone, n=4) followed by autologous HCT in 2 patients. Twelve patients received no further intensive treatment (Figure 1).

ic HCT (n=71), 4-year OS after transplant was 39% (95%CI: 29%-52%), with a significantly better OS (P=0.0006) (Figure 2) since the timepoint of allogeneic HCT in patients (n=51) responding to GO-A-HAM (49%, 95%CI: 37%-64%) compared to those (n=20) not responding (11%, 95%CI: 3%-41%); there was no difference in outcome in responding patients according to type of response (i.e. CR, CRi, PR; P=0.48). An Andersen Gill regression model (Table 5) on OS after GO-A-HAM with allogeneic HCT as a time-dependent co-variable after limited backward selection revealed allogeneic HCT (P=0.04) and response to GO-A-HAM (P<0.0001) as prognostic favorable variables, whereas adverse cytogenetics according to European LeukemiaNet1 criteria (P=0.09), older age (P=0.04), s-AML/t-AML (P=0.02) and FLT3-ITD (P=0.04) were unfavorable parameters. All 18 patients with FLT3-ITD positive AML proceeded to allogeneic HCT (n=13 responded to GO-A-HAM), but outcome was poor despite allogeneic HCT (Figure 3).

Discussion Sinusoidal obstruction syndrome after allogeneic HCT and safety analysis according to predefined safety end points Nine patients developed SOS after allogeneic HCT; in 3 patients SOS was classified as mild, in 5 patients as moderate, and one patient died of severe SOS, leading to a rate of moderate/severe SOS of 8.5% (95%CI: 3.9%-17.2%). In 7 of 34 patients, SOS occurred after myeloablative conditioning, including the patient who died from SOS, whereas only 2 of 37 patients developed SOS after dosereduced conditioning (P=0.08). Grade 4/5 liver toxicity was not observed. The rate of early and hypoplastic death within 30 days after start of GO-A-HAM was 3%. All rates were below the maximally tolerated death-rate predefined in the protocol.

Survival analysis

Here we report on the prospective phase II study evaluating the GO-A-HAM regimen consisting of gemtuzumab ozogamicin in combination with all-trans retinoic acid, high-dose cytarabine and mitoxantrone in patients with primary refractory disease. Primary refractory disease in this study was defined as AML not responding to one cycle of standard 3+7-based induction therapy with either CR, CRi or PR. Half of the patients achieved CR or CRi, and another 11% of patients achieved PR, leading to an overall response rate of 61% that compares favorably to those in previous AMLSG protocols which used the HAM and S-HAM14 as well as A-HAM regimens9 in this patient population. Chevallier et al. reported on a similar salvage regimen combining intermediate-dose cytarabine with mitoxantrone and GO at a dosage of 9 mg/m2 given on day 4 in refractory or relapsed AML patients.13 Although a direct

Median follow up for survival was 48.8 months. In total, 62 of the 93 patients died; median OS was 16.0 months and the 4-year OS rate 32% (95%CI: 24%-43%) for the whole cohort. OS at four years after start of treatment was poor (7%, 95%CI: 1%-42%) in patients not proceeding to allogeneic HCT (n=22). In patients proceeding to allogene-

Table 5. Anderson Gill regression model on overall survival including allogeneic HCT as time-dependent co-variable. Allogeneic HSCT Response (CR, CRi, PR) to GO-A-HAM Age in years, 10-year difference s/t-AML Adverse cytogenetics1 FLT3-ITD

HR

P

0.47 0.24 1.35 2.16 1.64 2.13

0.04 <0.0001 0.04 0.02 0.09 0.04

Parameters withdrawn by limited backward selection; WBC, CD33 expression, sex, NPM1 mutational status, prior treatment with ATRA. Results of univariable Cox-regression analyses: response to GO-A-HAM (HR 0.27; P<0.0001). Age in years (HR for a 10year difference 1.46; P=0.004), s/t-AML (HR 3.22; P=0.0001), adverse cytogenetics1 (HR 2.05; P=0.01), FLT3-ITD (HR 1.66; P=0.11); log10(WBC) (HR 1.12; P=0.53), CD33 expression with 20% cut off (HR 0.93; P=0.86), male sex (HR 1.12; P=0.65), mutated NPM1 (HR 0.98; P=0.96). HCT: hematopoietic cell transplantation; s/t-AML: secondary AML after preceding MDS/therapy-induced AML; FLT3-ITD: FMS-like tyrosine kinase 3 gene internal tandem duplication; WBC: white blood count; NPM1: nucleophosmin.

haematologica | 2016; 101(7)

Time (years) Figure 3. Kaplan-Meier plot illustrating prognostic impact of FLT3-ITD status in patients receiving an allogeneic hematopoietic cell transplantation measured from the date of transplantation.

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comparison is difficult, the authors reported a CR rate of 39% in the refractory patient cohort. Whether the somewhat superior CR/CRi rate in our trial was due to the addition of ATRA remains elusive. Of note, in a large randomized trial in refractory/relapsed AML conducted by the Medical Research Council, no significant impact of ATRA as adjunct to intensive chemotherapy could be shown.32 However, ATRA in this study was initiated at day 1 of chemotherapy raising the important issue of which is the best schedule with significant beneficial effects of ATRA when given after chemotherapy,33 as in the GO-A-HAM regimen. Several clinical trials20,34,35 and a meta-analysis36 showed that GO given as adjunct to intensive induction chemotherapy improved several survival end points, in particular in patients with favorable or intermediate cytogenetic risk AML. Of note, GO was only effective if given early in the treatment course, i.e. in first induction therapy; however, there was no impact on the response rate.36 Interestingly, the addition of GO to induction therapy was particularly effective in AML with FLT3-ITD based on a subset analysis.34 However, this beneficial effect was based on a very small sample size and has not been confirmed by others.35 In our study, we were not able to identify prognostic factors for the response to GO-A-HAM. Compared to our historical controls,9,14 GO had a major impact in improving CR rates in primary refractory patients mainly with adverse or intermediate cytogenetic risk profile. In fact, in our multivariable analyses on OS, adverse cytogenetics represented the only trend associated with an inferior outcome, with a much weaker impact compared to type of AML, age, and presence of FLT3-ITD (Table 5). Thus, our results do not support a beneficial effect of GO in AML with FLT3-ITD, despite the fact that all patients in our study of this subgroup proceeded to allogeneic HCT (Figure 3). To improve outcome of patients with FLT3-ITD positive AML, the incorporation of FLT3 inhibitors before and after transplantation is currently being evaluated in clinical trials (clinicaltrials.gov identifers: 01477606, 01468467, and 02298166, and EudraCT 2010018539-16). The majority (76%) of enrolled patients could proceed to allogeneic HCT. Four-year OS since start of treatment of patients responding to GO-A-HAM and receiving allogeneic HCT was 49% compared to only 11% in patients who were refractory to salvage therapy. These results again underline the strong impact of the disease status at the time of transplantation on long-term survival, as has been previously reported.5-7,9 Thus, the two major prerequisites for long-term survival in primary refractory

References 1. Döhner H, Estey EH, Amadori S, et al. Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood. 2010;115(3):453-474. 2. Schlenk RF, Döhner H. Genomic applications in the clinic: use in treatment paradigm of acute myeloid leukemia. Hematology Am Soc Hematol Educ Program. 2013;2013:324-330. 3. Thol F, Schlenk RF, Heuser M, Ganser A. How I treat refractory and early relapsed

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patients are chemo-sensitivity to salvage therapy followed by allogeneic HCT. An important point to consider when GO is added to intensive chemotherapy is the dosage, especially if allogeneic HCT is planned. In most regimens conducted in Europe, doses lower than 9 mg/m2 (the dose originally approved by the US Food and Drug Administration in 2000 for single agent therapy) were applied. In our trial, a dosage of 3 mg/m2 had only a modest impact on toxicity, especially with regard to the development of SOS directly after salvage therapy as well as after transplantation, with a rate of moderate/severe SOS after transplantation of 8.5%, which is expected in this patient population.30,31,37,38 Thus, in contrast to previous data showing an enormously increased incidence of SOS (64%) by pre-treatment with GO (6 mg/m2 or 9 mg/m2) in a 3.5-month period prior to transplant,39 we were able to show that salvage therapy including GO in a dosage of 3 mg/m2 is safe and can be followed by allogeneic HCT without increasing the rate of SOS. Whether an increment of the GO dosage by using fractionated administration,40,41 comparable to successful attempts in first-line therapy,35 is safe in terms of SOS and even more effective compared to our GO-A-HAM regimens remains elusive. In summary, the addition of GO in a dosage of 3 mg/m2 and of ATRA to intensive chemotherapy in patients with AML refractory to one cycle of induction therapy resulted in a high response rate and a high proportion of patients proceeding to allogeneic HCT with very limited additional toxicity. However, primary refractory AML with FLT3-ITD still had a very poor outcome despite allogeneic HCT. Funding This work was supported by grants 01GI9981 [Network of Competence Acute and Chronic Leukemias], and 01KG0605 [IPD-Meta-Analysis: A model-based hierarchical prognostic system for adult patients with acute myeloid leukemia (AML)] from the German Bundesministerium für Bildung und Forschung (BMBF), the German Research Foundation (DFG FI405/5-1, BU 1339/3-1 and BU 1339/5-1, SFB 1074 B3), the Deutsche José Carreras Leukämie-Stiftung (DJCLS H 05/02), and an unrestricted grant from Wyeth/Pfizer. Acknowledgments We are also grateful to all members of the German-Austrian AML Study Group (AMLSG) for providing leukemia specimens and clinical data; a list of AMLSG institutions and investigators participating in this study appears in the Online Supplementary Appendix.

acute myeloid leukemia. Blood. 2015;26(3): 319-327. 4. Biggs JC, Horowitz MM, Gale RP, et al. Bone marrow transplants may cure patients with acute leukemia never achieving remission with chemotherapy. Blood. 1992;80(4):1090-1093. 5. Craddock C, Labopin M, Pillai S, et al. Factors predicting outcome after unrelated donor stem cell transplantation in primary refractory acute myeloid leukaemia. Leukemia. 2011;5(5):808-813. 6. Michallet M, Thomas X, Vernant JP, et al. Long-term outcome after allogeneic hematopoietic stem cell transplantation for

advanced stage acute myeloblastic leukemia: a retrospective study of 379 patients reported to the Société Française de Greffe de Moelle (SFGM). Bone Marrow Transplant. 2000;26(11):1157-1163. 7. Duval M, Klein JP, He W, et al. Hematopoietic stem-cell transplantation for acute leukemia in relapse or primary induction failure. J Clin Oncol. 2010;28(23):37303738. 8. Aoudjhane M, Labopin M, Gorin NC, et al. Comparative outcome of reduced intensity and myeloablative conditioning regimen in HLA identical sibling allogeneic haematopoietic stem cell transplantation

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intermediate-risk cytogenetics acute myeloid leukemia (AML) in first relapse with gemtuzumab and cytarabine versus cytarabine: results of a retrospective comparative study. Cancer. 2011;117(5):974-981. Thol F, Schlenk RF. Gemtuzumab ozogamicin in acute myeloid leukemia revisited. Expert Opin Biol Ther. 2014;14(8):11851195. Jaffe ES, Harris NL, Stein H, Vardiman JW (eds). World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France: IARC Press; 2001. Cheson BD, Bennett JM, Kopecky KJ, et al. Revised recommendations of the international working group for diagnosis, standardization of response criteria, treatment outcomes, and reporting standards for therapeutic trials in acute myeloid leukemia. J Clin Oncol. 2003;21(24):4642-4649 Jones RJ, Lee KS, Beschorner WE. Venoocclusive disease of the liver following bone marrow transplantation. Transplantation. 1987;44(6):778-783. Bearman SI. The syndrome of hepatic veno-occlusive disease after marrow transplantation. Blood. 1995;85(11):3005-3020. Andersen P, Gill RD. Cox’s regression model for counting processes: A large sample study. Ann Stat. 1982;10:1100-1120. Harrell FE. Regression Modeling Strategies: With Applications to Linear Models, Logistic Regression, and Survival Analysis. New York, NY: Springer; 2001. R Development Core Team. R. A language and environment for statistical computing. R Foundation for Statistical Computing: 2007; Vienna, Austria. Schlenk RF, Döhner K, Krauter J, et al. Alltrans retinoic acid improves outcome in younger adult patients with nucleophosmin-1 mutated acute myeloid leukemia: results of the AMLSG 07-04 randomized treatment trial. Blood. 2011;118(21):80a Büchner T, Schlenk RF, Schaich M, et al. Acute Myeloid Leukemia (AML): different treatment strategies versus a common standard arm--combined prospective analysis by the German AML Intergroup. J Clin Oncol. 2012;30(29):3604-3610. Bunjes D, Buchmann I, Duncker C, et al. Rhenium 188-labeled anti-CD66 (a, b, c, e) monoclonal antibody to intensify the conditioning regimen prior to stem cell transplantation for patients with high-risk acute myeloid leukemia or myelodysplastic syndrome: results of a phase I-II study. Blood. 2001;98(3):565-572. Schmid C, Schleuning M, Schwerdtfeger R, et al. Long-term survival in refractory acute myeloid leukemia after sequential treatment with chemotherapy and reducedintensity conditioning for allogeneic stem cell transplantation. Blood. 2006; 108(3):1092-1099.

32. Milligan DW, Wheatley K, Littlewood T, Craig JIO, Burnett AK. Fludarabine and cytosine are less effective than standard ADE chemotherapy in high-risk acute myeloid leukemia, and addition of G-CSF and ATRA are not beneficial: results of the MRC AML-HR randomized trial. Blood. 2006;107(12):4614-4622. 33. Schlenk RF, Fröhling S, Hartmann F, et al. Phase III study of all-trans retinoic acid in previously untreated patients 61 years or older with acute myeloid leukemia. Leukemia. 2004;18(11):1798-1803. 34. Castaigne S, Pautas C, Terré C, et al. Acute Leukemia French Association. Effect of gemtuzumab ozogamicin on survival of adult patients with de-novo acute myeloid leukaemia (ALFA-0701): a randomised, open-label, phase 3 study. Lancet. 2012; 379(9825):1508-1516. 35. Burnett AK, Hills RK, Milligan D, et al. Identification of patients with acute myeloblastic leukemia who benefit from the addition of gemtuzumab ozogamicin: results of the MRC AML15 trial. J Clin Oncol. 2011;29(4):369-377. 36. Hills RK, Castaigne S, Appelbaum FR, et al. Addition of gemtuzumab ozogamicin to induction chemotherapy in adult patients with acute myeloid leukaemia: a metaanalysis of individual patient data from randomised controlled trials. Lancet Oncol. 2014;15(9):986-996. 37. Tsirigotis PD, Resnick IB, Avni B, et al. Incidence and risk factors for moderate-tosevere veno-occlusive disease of the liver after allogeneic stem cell transplantation using a reduced intensity conditioning regimen. Bone Marrow Transplant. 2014; 49(11):1389-1392. 38. Carreras E, Díaz-Beyá M, Rosiñol L, Martínez C, Fernández-Avilés F, Rovira M. The incidence of veno-occlusive disease following allogeneic hematopoietic stem cell transplantation has diminished and the outcome improved over the last decade. Biol Blood Marrow Transplant. 2011; 17(11):1713-1720. 39. Wadleigh M, Richardson PG, Zahrieh D, et al. Prior gemtuzumab ozogamicin exposure significantly increases the risk of venoocclusive disease in patients who undergo myeloablative allogeneic stem cell transplantation. Blood. 2003;102(5):1578-1582. 40. Pilorge S, Rigaudeau S, Rabian F, et al. Fractionated gemtuzumab ozogamicin and standard dose cytarabine produced prolonged second remissions in patients over the age of 55 years with acute myeloid leukemia in late first relapse. Am J Hematol. 2014;89(4):399-403. 41. Walter RB, Medeiros BC, Gardner KM, et al. Gemtuzumab ozogamicin in combination with vorinostat and azacitidine in older patients with relapsed or refractory acute myeloid leukemia: a phase I/II study. Haematologica. 2014;99(1):54-59.

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ARTICLE EUROPEAN HEMATOLOGY ASSOCIATION

Acute Myeloid Leukemia

Ferrata Storti Foundation

Haematologica 2016 Volume 101(7):846-852

Whole exome sequencing reveals a C-terminal germline variant in CEBPA-associated acute myeloid leukemia: 45-year follow up of a large family Anand Pathak,1 Katja Seipel,2 Alexander Pemov,1 Ramita Dewan,1 Christina Brown,3 Sarangan Ravichandran,4 Brian T. Luke,4 Michael Malasky,5 Shalabh Suman,5 Meredith Yeager,5 NCI DCEG Cancer Genomics Research Laboratory, NCI DCEG Cancer Sequencing Working Group, Richard A. Gatti,3,6 Neil E. Caporaso,7 John J. Mulvihill,8 Lynn R. Goldin,7 Thomas Pabst,2 Mary L. McMaster,7* and Douglas R. Stewart1*

Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA; 2 Departments of Medical Oncology and Clinical Research, University Hospital and University of Berne, Switzerland; 3Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA; 4Advanced Biomedical Computing Center, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, USA; 5Cancer Genomics Research Laboratory, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Rockville, MD, USA; 6Department of Human Genetics, David Geffen UCLA School of Medicine, Los Angeles, CA, USA; 7Genetic Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA; and 8Department of Pediatrics, Section of Genetics, The University of Oklahoma College of Medicine, OK, USA 1

*MLMcM and DRS contributed equally.

ABSTRACT

Correspondence: drstewart@mail.nih.gov

Received: May 29, 2015. Accepted: December 29, 2015. Pre-published: December 31, 2015. doi:10.3324/haematol.2015.130799

Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/101/7/846

Š2016 Ferrata Storti Foundation Material published in Haematologica is covered by copyright. All rights are reserved to the Ferrata Storti Foundation. Copies of articles are allowed for personal or internal use. Permission in writing from the publisher is required for any other use.

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F

amilial acute myeloid leukemia is rare and linked to germline mutations in RUNX1, GATA2 or CCAAT/enhancer binding protein-a (CEBPA). We re-evaluated a large family with acute myeloid leukemia originally seen at NIH in 1969. We used whole exome sequencing to study this family, and conducted in silico bioinformatics analysis, protein structural modeling and laboratory experiments to assess the impact of the identified CEBPA Q311P mutation. Unlike most previously identified germline mutations in CEBPA, which were N-terminal frameshift mutations, we identified a novel Q311P variant that was located in the C-terminal bZip domain of C/EBPa. Protein structural modeling suggested that the Q311P mutation alters the ability of the CEBPA dimer to bind DNA. Electrophoretic mobility shift assays showed that the Q311P mu-tant had attenuated binding to DNA, as predicted by the protein modeling. Consistent with these findings, we found that the Q311P mutation has reduced transactivation, consistent with a loss-of-function mutation. From 45 years of follow up, we observed incomplete penetrance (46%) of CEBPA Q311P. This study of a large multi-generational pedigree reveals that a germline mutation in the C-terminal bZip domain can alter the ability of C/EBP-a to bind DNA and reduces transactivation, leading to acute myeloid leukemia. Introduction Familial acute myeloid leukemia (AML) is a rare disease and is linked to mutations in RUNX1,1 GATA22,3 and CCAAT/enhancer binding protein-a (CEBPA).4 CEBPA is located on 19q13.1 and is a single exon gene.4 Its protein product, C/EBP-a, is a granulocyte differentiation factor. The protein consists of two N-terminal transactivating domains, a basic DNA binding domain and a C-terminal bZIP leucine-zipper dimerization domain. C/EBP-a is a critical protein in myeloid differentiation and regulates the expression of granulocyte specific genes.5 CEBPA knock-out mice develop a phenotype similar to AML with a block in granulocyte maturation.6 haematologica | 2016; 101(7)


Familial AML due to a germline CEBPA Q311P variant

Germline CEBPA mutations segregate in familial AML in an autosomal dominant manner. Smith et al. reported the first germline mutation in CEBPA in a family.4 The causative mutation was an N-terminal (212delC) mutation that abrogated the production of the wild-type 42-kDa transcript, leading to the production of a 30-kDa product from an alternative start site. This mutation resulted in the loss of a transactivation domain. Five additional pedigrees have been identified with similar germline N-terminal frameshift insertions and deletions, leading to the production of the alternate dominant-negative 30-kDa product (Online Supplementary Table S1).7-10 These germline mutations have also been frequently associated with somatic C-terminal insertions or deletions. This evidence suggests that the pathogenesis of AML caused by CEBPA may require bi-allelic inactivation of the gene. In addition, a previous study has uncovered germline C-terminal mutations in CEBPA associated with sporadic AML.11 Somatic CEBPA mutations also occur in sporadic AML with a frequency of between 5%-14%.12 Fasan et al. analyzed 2296 sporadic AML cases and found that 244 patients (10.6%) carried CEBPA mutations: 140 were CEBPA single mutant cases, 104 were CEBPA double mutant cases.13 Patients with the CEBPA double mutation had a more favorable prognosis than the CEBPA single mutation.13 In 1970, Snyder et al. reported a kindred of 6 family members with AML in three generations evaluated at the National Cancer Institute.14 A follow-up report on the family in 1977 revealed that a previously healthy sister had also developed AML.15 Cells from 2 affected sisters and their mother all had increased cellular transformation on exposure to the SV40 virus.15 This family was also studied for sister chromatid exchange, along with other familial leukemia families.16 We have continued to follow up both the nuclear and extended family since their original description up to the present time. This large pedigree has 10 AML patients as well as 4 obligate carriers (Figure 1). Since there are multiple genes presumed to be associated with AML, we applied whole exome sequencing to 3 AML patients as well as to 2 obligate carriers to search for mutations in known and novel genes.

Methods Detailed clinical descriptions of the patients are presented in the Online Supplementary Appendix. Patients from this family were enrolled into NIH protocol “Clinical, Laboratory and Epidemiologic Characterization of Individuals and Families at High Risk of Cancer” (#78-C-0039; NCT-00001163), which was subsequently merged into a specific familial leukemia/lymphoma protocol “Clinical, Laboratory and Epidemiologic Characterization of Individuals and Families at High Risk of Hematologic Cancer” (#02C-0210; NCT-00039676). The study was approved by the NCI Institutional Review Board and written informed consent was obtained from all participants. Briefly, in 1970, 2 brothers had died of AML at 4 (IV.2) and 8 (IV.3) years of age and a sister (age 12 years, IV.4) was affected (Figure 1). Twin brothers (IV.5 and IV.6) were healthy, though a healthy sister (IV.1) developed AML in 1976.15 Through the mother (III.2), an obligate carrier, an uncle (II.5), an aunt (II.8) and a cousin (III.7) were also affected; they had died at 63, 53 and 38 years of age, respectively. The mother (III.2) had a history of cervical cancer in 1963 that had been treated with radiotherapy; she had no history of leukemia. Three additional relatives have haematologica | 2016; 101(7)

developed AML (III.8, IV.7 and V2). Cytogenetic studies on peripheral bone marrow from IV.1, IV.4 and V.2 were all normal. A high percentage of peripheral blasts were observed in patients III.7 (68%) and V.2 (77%), as is typical of CEBPA mutated AML.

Discovery phase Exome sequencing and annotation: whole exome sequencing on the AML family was performed at the National Cancer Institute Division of Cancer Epidemiology and Genetics Cancer Genomics Research Laboratory (NCI DCEG CGR). The procedure used has been described previously.17,18 Briefly, 1.1 mg of genomic DNA was extracted by standard methods from low-passage cultured fibroblasts (IV.1, IV.4 and III.2) or from lymphocytes (III.5 and III.8). Pretreatment leukemic cells were not available for any of the tested AML patients; all testing was performed on healthy tissue and diseased cells were not available for testing. Variant filtering: the variants were filtered by the following criteria: 1) present in all 5 individuals in the pedigree, 3 AML affected patients (III.8, IV.1 and IV.4) and 2 obligate carriers (III.2 and III.5); 2) appearing in less than or equal to 10 individuals in an in-house internal control group (“NCI CGR Out Group”) of 1170 exomes in individuals from families with cancers other than hematologic cancers; 3) appearing in less than or equal to 1.0% in the NHLBI Exome Sequencing Project (ESP) European-American population (the ethnic background of this family); and 4) not present in areas of repeats or segmental duplications. In silico analysis: we utilized SIFT,19 PolyPhen-2,20 Mutation Taster,21 Mutation Assessor,22 FATHMM,23 and the Combined Annotation Dependent Depletion-scaled C-score,24 for assessment of deleteriousness of mutations. For information on the degree of conservation at the site, we used GERP25 and PhyloP.26 Full details of the materials and methods are provided in the Online Supplementary Methods.

Results Whole exome sequencing and Sanger validation identifies a CEBPA Y11525.1:c.932A>C mutation (p.Gln311Pro, Q311P) in the NCI AML family Exome sequence data were generated for the 3 affected members (IV.1, IV.4 and III.8) of the pedigree and the 2 obligate carriers (III.2 and III.5). There were a total of 479,900 variants (both synonymous and non-synonymous) identified in this family. We restricted analysis to non-synonymous mutations (missense, nonsense, splice site mutations) and frame-shift mutations, resulting in 31,448 variants. After filtering for genetic segregation in all 5 members of the family, and rarity (in-house NCI CGR outgroup database ≤10 and NHLBI ESP EA ≤1.0%), and removal of variants in regions of repeats or segmental duplication, there were only 3 variants remaining in the following genes: ANGEL1, CEBPA and COL4A6. Even with a more permissive filtering strategy (eliminating the NCI CGR Outgroup filter), there were no mutations in FLT3, KRAS, NRAS, NPM1, RUNX1, MLL, KIT, PTPN11, TP53, GATA2, IDH or DNMT3A detected, as segregating with this pedigree. Based on in silico bioinformatic predictions (Online Supplementary Table S2) and literature review, a rare missense variant in the CEBPA gene (CEBPA; g.19.33792389T>G; p.Gln311Pro, NM_004364:c.A932C) was deemed to be the most likely causative mutation of the familial AML clustering. This variant has not been previously reported in 1000 Genomes (2577 exomes), the NHLBI ESP database (6503 exomes) or the Broad Institute Exome Aggregation Consortium (61,486 exomes). In 847


A. Pathak et al. Table 1. Sanger sequencing of the extended NCI AML family.

Pedigree ID Age at Dec 2013 (years) II.5 II.8 III.2 III.5 III.6 III.7 III.8 III.10 IV.1 IV.2 IV.3 IV.4 IV.5 IV.6 IV.7 IV.9 IV.10 IV.11 IV.12 IV.13 V.1 V.2

Deceased 1970, age 63 Deceased 1956, age 53 Deceased 1980, age 44 88 Deceased 2008, age 68 Deceased 1961, age 38 Deceased 2012, age 69 Deceased 2013, age 80 Deceased 1978, age 22 Deceased 1956, age 4 Deceased 1961, age 8 Deceased 1970, age 12 54 54 Deceased 1996, age 43 64 52 60 51 47 27 24

Cause of death

Cell/tissue type

NM_004364: c.932A>C (CEBPA Q311P)

AML

Age at AML diagnosis (years)

AML AML Adenocarcinoma of the rectum N/A Renal cancer AML AML Myocardial infarction Sepsis Gastrointestinal hemorrhage AML Anaphylaxis N/A N/A AML N/A N/A N/A N/A N/A N/A N/A

N/A N/A Fibroblasts Lymphocytes Lymphocytes N/A Lymphocytes Lymphocytes Fibroblasts N/A N/A Fibroblasts Lymphocytes Lymphocytes N/A Lymphocytes Lymphocytes Lymphocytes Lymphocytes Lymphocytes Lymphocytes Lymphocytes

N/A N/A (obligate carrier) Yes (heterozygous) Yes (heterozygous) No N/A Yes (heterozygous) Yes (heterozygous) Yes (heterozygous) N/A N/A Yes (heterozygous) Yes (heterozygous) Yes (heterozygous) N/A (obligate carrier) No No Yes (heterozygous) No No Yes (heterozygous) Yes (heterozygous)

Yes Yes No No No Yes Yes No Yes Yes Yes Yes No No Yes No No No No No No Yes

62 53 N/A N/A N/A 36 58 N/A 20 3 7 11 N/A N/A 41 N/A N/A N/A N/A N/A N/A 22

N/A: not applicable.

addition, this variant was not present in our in-house NCI CGR cancer exome database. This mutation was present in all 4 family members with AML for whom we had a DNA sample. We used bi-directional Sanger sequencing to validate the CEBPA variant initially detected by exome sequencing. Online Supplementary Figure S1 shows that patient IV.1 had a c.932A>C heterozygous mutation, resulting in one allele coding for proline (codon CCG), instead of glutamine (codon CAG). Sanger validation was performed on fibroblasts from IV.1, IV.4 and III.2 and on lymphocytes on III.5 and III.8. We sequenced lymphocytes from 11 additional blood relatives from the family (Table 1); we were able to detect the CEBPA Q311P in the only other AML patient (V.2) in this group. While AML patients III.8, IV.1, IV.4, and V.2 all exhibited the mutation, subjects III.2, III.5, III.10, IV.5, IV.6, IV.11, and V.1 carried the mutation and did not develop AML.

CEBPA Q311 is highly conserved We investigated the conservation of the CEBPA Q311P residue using GERP and Phylo-P. We found that Q311P is conserved by both GERP (score=4.7) and Phylo-P (score=4.5). Alignment of CEBPA orthologs (Online Supplementary Figure S2, top panel) shows absolute conservation of the glutamine 311 residue. In addition, the residue was conserved in four out of five CEBPA paralogs (Online Supplementary Figure S2, bottom panel).

Molecular modeling predicts reduced transcriptional activity of CEBPA Q311P To regulate transcription, two protein chains of C/EBP-a dimerize in a crisscross orientation at its 848

“leucine zipper” domain (residues 317-345); the DNA strand is held using the basic motif (residues 286-313) in a “scissors-grip”27-29 (Online Supplementary Figure S3A). The mutated residue, Q311, is located at the interface between the two C/EBP-a chains and is in an optimal position to form hydrogen bonds with residue T310 from the opposing chain (Online Supplementary Figure S3B) as previously suggested.29 The Q311 mutation to proline, a rigid amino acid and a protein “helix-breaker”, is thus predicted to perturb the leucine zipper dimer and its complex with DNA. Configurations of the wild-type and mutant homo- and heterodimers are presented in Online Supplementary Figure S4. The computed binding energies of wild-type and mutant dimers were estimated using molecular dynamics simulations. Using the average of the energies from two simulations (Online Supplementary Table S3), the C/EBP-a dimerization energy and DNA binding energies were calculated (Online Supplementary Table S4A and B, respectively). These binding energies represent the amount of energy that would be required to separate the dimer into the two component monomers, and to remove the DNA from the C/EBP-a dimer, respectively. The binding energy of the heterodimer (Q:P) was 3.9 kcal/mol greater than for the wild-type dimer (Q:Q) and the binding energy of two mutated C/EBP-a (P:P) was 13.2 kcal/mol greater than the wild-type dimer (Q:Q), suggesting that the P:P dimer is the most stable and the wild-type Q:Q dimer is the least stable. This means that a mutated C/EBP-a monomer would rather bind to another mutated C/EBP-a. Modeling suggests that DNA was bound more tightly to the wild-type dimer than to the mutant haematologica | 2016; 101(7)


Familial AML due to a germline CEBPA Q311P variant

Figure 1. Pedigree of NCI AML family. CEBPA Q311P genotyping was performed on all individuals with available DNA. Filled symbols: AML cases; symbol with a dot in the center: obligate carrier. Boxed symbols: exome-sequenced samples; Q311 is the wild-type allele and Q311P is the variant at this position. Arrow: proband.

dimer (Figure 2 and Online Supplementary Table S4B). This DNA binding energy was 14.8 kcal/mol larger than that of the mutant dimer. Given that the dimerization and DNA binding are reversible processes, the energy differences were used to calculate the Boltzmann probabilities of occurrence (Online Supplementary Table S3). These probabilities represent the relative abundance of the different complexes when the cell has achieved a steady-state equilibrium. These results show that the great majority of mutated C/EBP-a monomers are more likely to bind to another mutated C/EBP-a than to the wild-type protein. Therefore, in C/EBP-a-haploinsufficient cells, the predominant mutant dimer species is P:P. The wild-type monomers will have to form Q:Q dimers, since there will be very few mutated monomers available for wildtype dimerization. The modeling predicts that since the P:P dimer does not bind DNA nearly as tightly as the wild-type Q:Q dimer, we expect that its ability to initiate transcription will also be greatly reduced (Figure 3). Therefore, in a cell with a single Q311P mutation, the overall transcription activity should be reduced by about a factor of 2 since there are only half as many dimers Q:Q available for binding.

Experimental evidence that CEBPA Q311P is a loss-of-function allele Molecular modeling predicts that Q311P, located in the hinge region between the basic DNA binding region and the leucine zipper region of the C/EBP-a protein, interferes with DNA binding and dimerization. The Q311P point mutation was introduced into the CEBPA expression haematologica | 2016; 101(7)

plasmid by site-directed mutagenesis and was tested for transcriptional activator function in MOLM-16 AML cells (Figure 4A). As predicted, CEBPA Q311P was a loss-offunction allele since it was unable to activate reporter gene expression by itself (pcDNA/Q311 vs. pcDNA/Q311P; P=0.0002), despite comparable levels of wild-type and mutant protein (Figure 4B). In the heterozygous state (Q311+Q311P), the reporter gene induction was higher than in the hemizygous wild-type (pcDNA+Q311) (P=0.0011). If the Q311P was a dominant-negative, we would expect the heterozygous (Q311+Q311P) induction to be lower than the hemizygous wild-type (pcDNA+Q311). These data indicate that the Q311P mutation has a loss of transcriptional activator function, but has not acquired a dominant negative function, similar to other germline N-terminal frameshift mutations that are associated with familial AML. The homozygous WT state (Q311+Q311) exhibited significantly more transactivation than the heterozygous state (Q311+Q311P) (P=0.0343). There were no apparent differences in the C/EBP-a protein-DNA complexes of C/EBP-a wild-type and Q311P mutant in vitro on a high affinity CEBP bind-ing site (Figure 3A). On a lower affinity site, however, only the wild-type C/EBP-a was able to bind, indicating that Q311P exhibits comparatively reduced DNA binding (Figure 3B).

Discussion Members of this family met the diagnostic criteria for â&#x20AC;&#x153;acute myeloid leukemia with mutated CEBPAâ&#x20AC;?, accord849


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Figure 2. Comparison of the wild-type C/EBP-α DNA fragment (PDB: 1NWQ; left) complex to the final conformation from the simulated double Q311P mutant (right). Residue 311 is shown in “ball-and-stick style”. The mutant C/EBPa is predicted to bind to DNA with less avidity than the wild-type, affecting transcriptional regulation.

ing to the WHO Classification of Tumors of the Hematopoietic and Lymphoid Tissues.30 AML with mutated CEBPA has higher peripheral blood blast cell counts than CEBPA non-mutated AML and a more favorable prognosis. Germline mutations in CEBPA are associated with familial AML.4,7-10 To date, the majority of reported variants have been N-terminal frameshift mutations that create a premature stop codon and thus trigger transcription from an alternate start site; this results in the production of a truncated 30-kDa isoform, which lacks a transactivation domain. In addition, a study by Taskesen et al. has also identified germline C-terminal mutations associated with AML.11 In a large, multi-generational pedigree with AML and 45 years of follow up, we report a C-terminal germline missense mutation (Q311P) in the C-terminal bZip domain of CEBPA, distinct from the previously reported causative variants. The majority of reported CEBPA somatic mutations in familial AML are in the Cterminus, located in close proximity to glutamine 311, the amino acid affected in this family. In addition, in the COSMIC database,31 there are 13 missense/frameshift mutations at residue Q311, although Q311P was not observed. Given the location of Q311P, we hypothesized that C/EBP-a dimerization and DNA binding ability would be affected. Protein molecular modeling pointed to preferential P:P (mutant:mutant) dimerization and a decreased ability of the Q311P dimer to bind DNA compared to the wild-type dimer. Experimental evidence showed that the ability of the Q311P mutant to transactivate a CEBP luciferase construct was significantly reduced, indicating that it is a loss-of-function allele. In the heterozygous state, with both the normal Q311 and mutant Q311P expressed, transactivation was no further reduced than the pcDNA and normal Q311, indicating that this mutant allele is not a dominant negative, as has been observed with other CEBPA mutations.32 In accordance with the protein molecular modeling prediction, an electrophoretic mobility shift assay showed comparatively reduced binding of the Q311P mutant to DNA versus the wild-type Q311. This explains how the Q311P mutant loses its ability to activate transcription from a CEBP promoter. 850

Notably, a somatic in-frame duplication of amino acids 312-317 and a deletion of the lysine 312 in C/EBP-a also lead to the loss of DNA binding and reduced transactivation.32 We have 603 person-years of follow up in this AML family. The age of onset of AML ranged from three years to 62 years. The mean age of onset of AML in this family was 31.3 years (median 29 years). Unlike the generally good prognosis observed in CEBPA mutation carriers, the overall survival of the majority of our patients was only 12 years. This may be due to the diagnosis and treatment of the majority of the family members in the 1950s-1970s, when the survival after AML diagnosis was extremely poor. Family members more recently affected with AML survived longer. One patient, diagnosed at 57 years of age in 2001, survived 11 years (III.8) with chemotherapy, and another member recently diagnosed at 22 years of age (V.2) is doing well two years after a double umbilical cord transplant. It is possible that a C-terminal bZip mutation such as Q311P may have a worse prognosis than the Nterminal frameshift mutation, a pattern that has been previously reported.4 In familial AML, it is well documented that a second somatic mutation in CEBPA may be required for leukemia development;4,7-10 however, we did not have any appropriate tumor tissue available to test for this and only tested healthy tissue in this study. Thus, we cannot speculate whether the prognosis of our patients is associated with a single mutation or a double mutation.13 We observed a penetrance for AML of 46% [6 of 13 confirmed mutation carriers (III.8, IV.1, IV.4 and V.2) or obligate carriers (II.8 and IV.7)]; this rate is 59% (10 of 17) if we include family members (e.g. II.5, III.7, IV.2, IV.3) who died of AML but before DNA was collected and who presumably had the Q311P mutation. This is lower than the almost complete penetrance reported in the literature for the N-terminal frameshift mutations,4,7-10 although the sample sizes are limited in these studies. The mother (III.2) was diagnosed with cervical cancer at 27 years of age and received radium and cobalt therapy of uncertain dose and intensity in 1963. Pathological materials from this diagnosis were not available to for review. She developed poorly differentiated adenocarcinoma of haematologica | 2016; 101(7)


Familial AML due to a germline CEBPA Q311P variant

the rectum 16 years later (pathology materials reviewed by NCI Laboratory of Pathology) at 43 years of age and died of its complications at 44 years of age. There are 7 family members with the Q311P mutation who to date have not developed AML, other leukemia or lymphoma (III.5, IV.5, IV.6, IV.11, V.1; age range 27-88 years, mean 57 years). In summary, we report an incompletely penetrant

CEBPA Q311P mutation in all tested, affected members of the largest multi-generational AML pedigree reported to date with 45 years of longitudinal follow up. The Q311P mutation was predicted to be highly deleterious by in silico algorithms and the Q311 position was highly conserved among CEBPA orthologs and paralogs. Protein structural modeling suggested that the Q311P mutation alters the ability of the C/EBP-a dimer to bind DNA. The

A

B Figure 3. Transactivation potential of CEBPA Q311P versus wild-type. (A) CEBP promoter transactivation assays in MOLM16 cells for pcDNA, CEBPA Q311 (WT) and CEBPA Q311P (and all combinations), show a loss-of-function of CEBPA Q311P (experiments were performed in triplicate). P-value paired t-test values: pcDNA/Q311 vs. pcDNA/Q311P, P=0.0002; pcDNA/Q311 vs. Q311/Q311P, P=0.0011; Q311/Q311 vs. Q311P/Q311P, P= 0.0021; Q311/Q311 vs. Q311/Q311P, P=0.0343; Q311P/Q311P vs. Q311/Q311P, P=0.0013. (B) anti-CEBPA and anti-GAPDH Western blot of total extracts of transiently transfected MOLM16 cells.

A

B

Figure 4. DNA binding potential of CEBPA Q311P versus CEBPA Q311 (wild-type). Electrophoretic mobility shift assay performed with nuclear extracts of H1299 cells transiently transfected with equal amounts of pcDNA, CEBPA Q311 (wild-type) and CEBPA Q311P expression plasmids. (A) Binding to high affinity site and (B) binding to low affinity site.

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transactivation potential of C/EBP-a Q311P was reduced, suggesting it is a loss-of-function mutation. Furthermore, EMSA studies showed that the Q311P mutant had attenuated binding to DNA, providing a mechanism for its lossof-function. Clinically, the CEBPA Q311P mutation was incompletely penetrant for AML; 46% of the carriers developed the disease. Thus, we conclude that a germline mutation in the C-terminal bZip domain, distinct from previously reported mutations, can alter the ability of C/EBPa to bind DNA and reduces transactivation, leading to AML, though with lower penetrance than the canonical risk variants. Acknowledgments NCI DCEG Cancer Sequencing Working Group: Bari Ballew, Stephen J. Chanock, Mark H. Greene, Alisa M. Goldstein, Allan Hildesheim, Nan Hu, Maria Teresa Landi, Jennifer Loud, Phuong L. Mai, Lisa Mirabello, Lindsay Morton,

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Melissa Rotunno, Sharon A. Savage, Philip R. Taylor, Geoffrey S. Tobias, Margaret A. Tucker, Jeannette Wong, Xiaohong R. Yang, Guoqin Yu NCI DCEG Cancer Genomics Research Laboratory: Sara Bass, Joseph Boland, Aaron J. Bouk, Maria Brown, Laurie Burdett, Salma Chowdhury, Michael Cullen, Casey Dagnall, Belynda Hicks, Herbert Higson, Amy A. Hutchinson, Kristine Jones, Sally Larson, Kerrie Lashley, Hyo Jung Lee, Wen Luo, Michelle Manning, Jason Mitchell, David Roberson, Aurelie Vogt, Mingyi Wang, Kathleen Wyatt, Xijun Zhang, Bin Zhu Funding This project has been funded in part with funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. This work was supported by the Division of Cancer Epidemiology and Genetics of the Intramural Research Program of the National Cancer Institute.

11. Taskesen E, Bullinger L, Corbacioglu A, et al. Prognostic impact, concurrent genetic mutations, and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients: further evidence for CEBPA double mutant AML as a distinctive disease entity. Blood. 2011;117(8):2469-2475. 12. Pabst T, Eyholzer M, Fos J, Mueller BU. Heterogeneity within AML with CEBPA mutations; only CEBPA double mutations, but not single CEBPA mutations are associated with favourable prognosis. Br J Cancer. 2009;100(8):1343-1346. 13. Fasan A, Haferlach C, Alpermann T, et al. The role of different genetic subtypes of CEBPA mutated AML. Leukemia. 2014;28 (4):794-803. 14. Snyder AL, Henderson ES, Li FP, Todaro GJ. Possible inherited leukaemogenic factors in familial acute myelogenous leukaemia. Lancet. 1970;1(7647):586-589. 15. McKeen EA, Miller RW, Mulvihill JJ, Blattner WA, Levine AS. Familial leukaemia and SV40 transformation. Lancet. 1977; 2(8032):310. 16. Cheng WS, Mulvihill JJ, Greene MH, Pickle LW, Tsai S, Whang-Peng J. Sister chromatid exchanges and chromosomes in chronic myelogenous leukemia and cancer families. Int J Cancer. 1979;23(1):8-13. 17. Stewart DR, Pemov A, Johnston JJ, et al. Dubowitz syndrome is a complex comprised of multiple, genetically distinct and phenotypically overlapping disorders. PLoS ONE. 2014;9(6):e98686. 18. Shi J, Yang XR, Ballew B, et al. Rare missense variants in POT1 predispose to familial cutaneous malignant melanoma. Nat Genet. 2014;46(5):482-486. 19. Kumar P, Henikoff S, Ng PC. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc. 2009;4(7):1073-1081. 20. Adzhubei IA, Schmidt S, Peshkin L, et al. A method and server for predicting damaging missense mutations. Nat Methods. 2010;7 (4):248-249. 21. Schwarz JM, Rodelsperger C, Schuelke M, Seelow D. MutationTaster evaluates disease-causing potential of sequence alterations. Nat Methods. 2010;7(8):575-576. 22. Reva B, Antipin Y, Sander C. Predicting the functional impact of protein mutations:

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ARTICLE

Hodgkin Lymphoma

Whole exome sequencing in families at high risk for Hodgkin lymphoma: identification of a predisposing mutation in the KDR gene

EUROPEAN HEMATOLOGY ASSOCIATION

Ferrata Storti Foundation

Melissa Rotunno,1 Mary L. McMaster,1 Joseph Boland,2 Sara Bass,2 Xijun Zhang,2 Laurie Burdett,2 Belynda Hicks,2 Sarangan Ravichandran,3 Brian T. Luke,3 Meredith Yeager,2 Laura Fontaine,4 Paula L. Hyland,1 Alisa M. Goldstein,1 NCI DCEG Cancer Sequencing Working Group, NCI DCEG Cancer Genomics Research Laboratory, Stephen J. Chanock,5 Neil E. Caporaso,1 Margaret A. Tucker,6 and Lynn R. Goldin1

Genetic Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Bethesda, MD; 2Cancer Genomics Research Laboratory, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Bethesda, MD; 3 Advanced Biomedical Computing Center, Leidos Biomedical Research Inc.; Frederick National Laboratory for Cancer Research, Frederick, MD; 4Westat, Inc., Rockville MD; 5 Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Bethesda, MD; and 6Human Genetics Program, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Bethesda, MD, USA 1

Haematologica 2016 Volume 101(7):853-860

ABSTRACT

H

odgkin lymphoma shows strong familial aggregation but no major susceptibility genes have been identified to date. The goal of this study was to identify high-penetrance variants using whole exome sequencing in 17 Hodgkin lymphoma prone families with three or more affected cases or obligate carriers (69 individuals), followed by targeted sequencing in an additional 48 smaller HL families (80 individuals). Alignment and variant calling were performed using standard methods. Dominantly segregating, rare, coding or potentially functional variants were further prioritized based on predicted deleteriousness, conservation, and potential importance in lymphoid malignancy pathways. We selected 23 genes for targeted sequencing. Only the p.A1065T variant in KDR (kinase insert domain receptor) also known as VEGFR2 (vascular endothelial growth factor receptor 2) was replicated in two independent Hodgkin lymphoma families. KDR is a type III receptor tyrosine kinase, the main mediator of vascular endothelial growth factor induced proliferation, survival, and migration. Its activity is associated with several diseases including lymphoma. Functional experiments have shown that p.A1065T, located in the activation loop, can promote constitutive autophosphorylation on tyrosine in the absence of vascular endothelial growth factor and that the kinase activity was abrogated after exposure to kinase inhibitors. A few other promising mutations were identified but appear to be “private”. In conclusion, in the largest sequenced cohort of Hodgkin lymphoma families to date, we identified a causal mutation in the KDR gene. While independent validation is needed, this mutation may increase downstream tumor cell proliferation activity and might be a candidate for targeted therapy. Introduction Classical Hodgkin lymphoma (HL) is a lymphoproliferative malignancy of B-cell origin with an age-adjusted incidence in 2011 in the United States of 2.8/100000.1 The number of new HL cases in the United States diagnosed in 2014 was estimated to be 9190 with 1180 estimated deaths. Etiologic clues about HL have been suggested by several observations including: 1) the bimodal age distribution with one peak occurring in the third decade of life and a second peak after 50 years of age; 2) an elevated risk in males; 3) an elevated risk in individuals with higher socioeconomic haematologica | 2016; 101(7)

Correspondence: goldinl@mail.nih.gov

Received: August 19, 2015. Accepted: January 7, 2016. Pre-published: June 13, 2016. doi:10.3324/haematol.2015.135475

Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/101/7/853

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

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status and in smaller families; 4) the occurrence of EpsteinBarr virus (EBV) in HL tumor cells and 5) a strong familial risk.2 Specifically, there is strong evidence for genetic factors based on evidence from multiply affected families from case series, twin, case-control, and population-based registry studies.3-9 Furthermore, our group analyzed data from registries in Scandinavia and found significant familial aggregation of HL (RR = 3.1) and other lymphoproliferative tumors. Relative risks were higher in men compared with women, in siblings of cases compared with parents and offspring, and in relatives of patients with diagnosis under the age of 40.4 In addition, we showed that nonHodgkin lymphoma (NHL) also aggregates in HL families. The human leukocyte antigen (HLA) region on chromosome 6 has been associated with HL in several studies.10-12 Other non-HLA susceptibility loci have been identified through GWAS.13-15 The identification of genes with major susceptibility effects has been more difficult. A study of a family with a reciprocal translocation between chromosomes 2 and 3 segregating with HL led to the identification of a disruption in the gene KLDHC8B, which codes for a midbody kelch protein, hypothesized to disrupt cytokinesis thereby promoting tumorigenesis. Additional familial patients were found to carry an uncommon 5’ UTR variant in this gene.16 Recently, a mutation in the NPAT gene was identified in an extended pedigree segregating for the less common nodular lymphocytic predominant subtype of HL.17 A homozygous deletion in the ACAN gene was identified in 3 siblings with classical HL.18 We conducted a genome-wide linkage study in 44 HL families that revealed several regions of the genome possibly linked to HL,19 but no susceptibility genes were subsequently identified. In this study, we have attempted to identify high risk genes causing susceptibility to HL using whole exome sequencing and follow-up targeted sequencing in families segregating HL.

Methods Description of Families and Study Design The 65 HL-prone families included in this study are participants in an IRB approved NCI study (NCI-02-C-0210) who were accrued through healthcare professionals or self-referral19 and had at least 2 confirmed HL patients. The sequencing study design is shown in Table 1. In the first phase, 63 affected (including 2 NHL patients) or obligate carrier individuals and 6 unaffected spouses from 17 ‘discovery families’ (required to have at least 3 affected members or obligate carriers with available DNA) were whole exome sequenced to identify candidate HL susceptibility variants. In the second phase, selected candidate genes from Phase 1 were (a) re-sequenced in the original 17 families to validate the variants and to determine the mutation status of 27 additional unaffected members first-degree relatives of cases and (b) tested for replication in an additional 48 ‘replication families’ corresponding to 80 sequenced individuals (64 from 32 HL siblings or child-parent pairs, 16 from multiplex families with only one patient with DNA available for sequencing). The distribution of sequenced individuals among all families is shown in Online Supplementary Table S1.

Sequencing and Analyses of Sequence Data Whole exome sequencing (WES) was performed at the Cancer Genomics Research Laboratory, National Cancer Institute (CGR, NCI), as previously described.20 Details of the bioinformatics pipeline for variant alignment and calling used in this study appear in the Online Supplementary Methods. 854

Annotation of each variant locus was made via a custom software pipeline based on public data sources described in the Online Supplementary Methods. Filtering of WES variants called in discovery families was based on the following criteria: 1) present in all affected (HL and NHL) or obligate carrier individuals in the pedigree and absent in unaffected married-in individuals; 2) present in ≤ 1% of families from an in-house database (700 families), in ≤ 1% of the NHLBI Exome Sequencing Project (ESP) European American population (4300 individuals) and in ≤ 1% of the phase 1 1000 Genomes Project European population (379 individuals) and 3) occurring in exonic or UTR regions or in locations linked to epigenetic findings from ENCODE. Prioritization of the filtered-in variants was based on mutation type, low minor allele frequency and functional predictions using the programs described in the Online Supplementary Methods. We also considered the genes’ links to cancer or immune-related processes from the literature and whether the variant or gene was present in multiple HL families. Validation and replication of a set of genes selected was based on the above prioritized list and on amplicon length to optimize available resources. Validation was performed in all available families using Ion Torrent (see Online Supplementary Methods). We checked for technical validation in whole exome sequenced samples, for segregation in additional family members, and for replication in additional families. Re-sequencing the whole genes allowed us to test the hypothesis that different variants from the same gene might occur in different families. In silico analysis was performed for the variants that replicated in multiple families (see Online Supplementary Methods).

Results As noted previously, and consistent with other studies of familial HL, patients in the families we studied are skewed toward younger age at diagnosis. The average age at diagnosis of 169 patients in these families is 27.4, with more than 90% being less than 45 years old at diagnosis. The ratio of male to female patients in the families is 1.25, which is consistent with population incidence rates. As expected from the age distribution of patients, among 141 HL patients with subtype information, 82% had nodular sclerosis and 15% had mixed cell. Based on our filtering criteria between 9 and 406 variants were shared by all cases or obligate carriers within each discovery family, making a total of 2699 variants (listed in the Online Supplementary Table S2) whose characteristics are described in Table 2. The 2699 variants and the corresponding 2383 genes were prioritized based on mutation types, low minor allele frequency, functional prediction, possible role in epigenetic regulation, literature linked to cancer or immunerelated processes, and presence in multiple HL families. In general, the highest priority was given to the 650 non-synonymous variants and, among those, to the 180 variants identified in families with 4 or 5 affected members. These genes were further prioritized based on the information available in literature. Based on these criteria, 23 genes were selected to be followed up through Ion Torrent targeted sequencing (see Table 3). Additional genes could have been included based on our criteria but we were limited by the number and size of genes that we could investigate. As shown in the Online Supplementary Table S3, WES haematologica | 2016; 101(7)


A germline mutation in KDR in familial Hodgkin lymphoma

Table 1. Summary of study design.

Family pedigrees Hodgkin Lymphoma Non-Hodgkin Lymphoma Obligate Carrier Spouse unaffected Related unaffected Total subjects Results

Discovery of variants 17 (â&#x2030;Ľ3 affected or obligate carriers sequenced each)

Validation of 23 genes Same 17 discovery families

Replication of 23 genes 48 new families (1 or 2 affected sequenced each)

44 2 17 6 0 69 2699 variants, 2383 genes

45 2 14 3 27 91 All variants validated

80 0 0 0 0 80 One variant replicated

results were technically validated by Ion Torrent for all tested variants. Targeted sequencing in the additional families produced none or little additional evidence in support of the variants and genes investigated. Specifically, the variants generally did not segregate with HL in the new families. However, of the 23 genes selected for follow-up in the discovery set, only one variant was found in two additional HL patients in one family in our replication set, therefore co-segregating with HL/NHL in two independent families (indicated as F6 and F30 in Figure 1 and Online Supplementary Tables S1 and S4). This is a C-to-T missense mutation located at chr4:55955969 (rs56302315) in the Kinase Insert Domain Receptor (KDR) gene, a type III receptor tyrosine kinase. KDR produces a 1356 amino acid protein transcript (NP_002244.1) called VEGFR2. The identified variant results in a single amino acid residue change (UniProt: P35968; p.A1065T) of the wild-type VEGFR2, has an allele frequency of 7.8x10-4 in the ExAC European (non-Finnish) database and is highly conserved. Consistent with this low frequency, we found it to be present in only one individual among the 1700 in approximately 700 non-lymphoid cancer families sequenced in our lab. Because the same KDR variant was identified in two families, it was further investigated through additional indepth functional in silico analysis. The KDR gene contains 30 exons and the missense variant NM_002253.2:c.3193C>T occurs in the first nucleotide position of exon-24. The mutated residue is part of the kinase domain activation loop (kinase domain: 806-1171 AA; activation loop: 1046-1075 AA),21 that undergoes major conformational changes upon phosphorylation to allow for phosphate transfer. The fully activated kinase serves as the signaling center for further downstream events such as cell proliferation and growth. The wildtype residue, alanine, is non-polar and hydrophobic, whereas the modified residue, threonine, is polar and can form up to three hydrogen bonds.22 Since the VEGFR2 variant p.A1065T yields a polar residue with an acquired ability to get phosphorylated, this modification could make VEGFR2 prone to take more open and solvent accessible conformations, similar to those observed during activation. Structure-based impact assessment analysis was based on forty-seven partial VEGFR2 structures available (27 May 2015) from RCSB PDB. All the structures exhibited inactive-like folds and were co-crystallized with different inhibitors. Ten of these PDB structures were selected for our structure-based impact assessment analysis based haematologica | 2016; 101(7)

on the structure availability of kinase domain, the structure quality (e.g., resolution and R-value) and the presence of kinase activation loop residues (D1046-E1075).21 A sequence alignment based structure overlay was carried out in a Discovery Studio Visualizer using the PDB structure ID 3VO323 as the template. The template (see Figure 2) was selected based on both structure release date and quality (i.e., R-value), and the alignment resulted in two unique inactive fold groups, represented in our study by PDB IDs 3VO3 (group 1) and 2OH424 (group 2). Structurally aligned conformations of group representative structures are shown in the Online Supplementary Figure S1. Conformational analysis using the ten selected PDB structures of VEGFR2 showed that the activation loop containing p.A1065 had definite mobility (Online Supplementary Figure S1). Using the representative group structures we were also able to identify the group affiliations of all the remaining VEGFR2 structures. Group 2 (e.g., PDB ID 2OH4 and 1VR2) structure analysis showed that the p.A1065 containing segment is closer to the catalytic residues p.D1028 and p.R1032, and the neighboring residues R1066-P1068 in 1VR2 adopt an inhibitory conformation that can impact the substrate binding.21 With the p.A1065T modification (data not shown in Figure 2), the inhibitory conformation could be weakened, therefore promoting a more open, active-like conformation and possibly leading to a constitutively active state. Most impact assessment software predicted the p.1065T variant to be damaging or deleterious to the VEGFR2 function (see Online Supplementary Table S5). Other promising variants confirmed in single large (i.e., with more than 3 affected or carrier members) families were missense T-to-C changes at chr20:54956620 in AURKA, and at chr6:133073884 in VNN2, and missense C-to-T changes at chr13:49281386 in CYSLTR2, at chr20:39989937 in EMILIN3, and at chr16:82033722 in SDR42E1.

Discussion The most important finding based on the sequencing of 65 HL families (17 discovery and 48 replication families) is the presence of a rare non-synonymous c.3193G>A mutation in the KDR gene shared by patients and obligate carriers in two families. It appears that NHL in these families also shares the same genetic susceptibility, which is consistent with our previous familial aggregation data based 855


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on Scandinavian registries that showed significant co-segregation of HL and NHL.4 The first family (F6) is particularly informative as it contains HL patients who are first cousins. One of the obligate carriers in this family was not available for direct testing but did have a diagnosis of diffuse large B-cell lymphoma (DLBCL) based on a slide review. The second family (F30) is consistent with the mutation causing susceptibility, but the family structure is somewhat more complex. The father and daughter with confirmed HL both have the mutation. However, two unaffected siblings of the daughter, who were tested at a relatively young age, also have the variant. This is not surprising because of the siblings’ young age and the expected incomplete penetrance of mutations predisposing to familial HL based on the observed proportion of patients in high-risk HL families. In addition, either parent of the father in this second family, F30, (II.3 and II.2, Figure 1) could be a gene carrier and thus the genetic information is limited. KDR (also known as VEGFR2) is a tyrosine-protein kinase that acts as a receptor for VEGFA, VEGFC, and VEGFD. It is an important gene in tumor angiogenesis, but is also involved in cell proliferation and survival. Our structural analyses of the p.A1065T mutation show that it is located in the activation loop, close to key active site residues, and able to impact protein function by interrupting the inactive conformation of VEGFR2. The codon for the A1065 amino acid residue is also in a splice region at the border of intron-exon 24, and based on the known donor/acceptor patterns25 the c.3193C>T change might cause a splice variant. However the acceptor site is unlikely altered, since alignments of homologous VEGFR sequences show that the VEGFR1 protein (FLT1 gene) contains a naturally occurring threonine residue, p.T1059, also found in exon 24, and has the same sequence context in the splicing region. Moreover, the p.A1065T germline mutation found in the HL families has also been studied somatically in an angiosarcoma tumor and found to be associated with high mRNA and protein KDR expression. Transfection of the mutant into COS-7 cells showed that p.A1065T could be responsible for ligand-independent activation of the kinase.26 This same mutation was also identified as germline in a study that explored the somatic mutation patterns of kinase genes in cancer genomes.27 Based on the experimental information and our sequencestructure analysis, we believe that the p.A1065T variant could impact the VEGFR2 function via structural (conformational) effects. Thus, we have good evidence that the (p.A1065T) mutation observed in the two families is functionally significant. There is also evidence supporting the potential functional significance of KDR and VEGF in lymphomas, including HL.28 VEGF and constitutively active KDR have been found to be expressed in Hodgkin ReedSternberg (RS) cell lines and in RS cells from patients, as well as in serum from patients.29,30 KDR also has high expression in DLBCL tumors and is associated with poor treatment response.31 EBV is involved in the pathogenesis of both HL and NHL tumors. In one study, EBV positivity in NHL tumors was associated with VEGFA expression.32 VEGF inhibitors are used in cancer treatment, including lymphomas, and have shown some efficacy in a small study of HL patients and in a HL preclinical model.33 KDR mRNA was not expressed in the peripheral blood samples from our two HL families with the p.A1065T mutation (data not shown). Based on publicly available data from the 856

Table 2. Characteristics of 2699 variants identified in 17 HL discovery families after filtering.

Characteristic Reference/Variant A/T & T/A C/T & T/C G/T & T/G A/C & C/A G/C & C/G A/G & G/A Indels Family size (affected) 3 4 5 Number of non LPD* families 0 1,2,3 4,5,6,7 ESP EA 0 (0-0.001] (0.001-0.005] (0.005-0.01] Genomic region exonic intronic UTR3 UTR5 downstream upstream intergenic splicing missing 1000 Genome 0 (0-0.001] (0.001-0.005] (0.005-0.01] GERP ≥2 <2 FATHMM (SCORE) <-0.75 ≥-0.75 MUTATIONTASTER (PRED) D or A N or P Mutation type non-exonic non-synonymous synonymous frameshift non-frameshift MUTATIONASSESSOR (PRED) high medium low neutral SIFT (SCORE) ≤ 0.05 > 0.05 POLYPHEN2 HVAR (PRED) D P B Epigenetic Finding (ENCODE) Yes No

No Variants 124 867 192 203 253 887 173 1991 425 283 992 1021 686 1890 442 319 48 1183 868 376 119 60 49 21 5 18 2467 139 86 7 422 238 123 432 222 325 1671 650 337 31 10 21 158 191 202 217 391 139 106 329 1527 1172

*LPD indicates Lymphoproliferative diseases.

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A germline mutation in KDR in familial Hodgkin lymphoma

NIH Roadmap Epigenomics projects,34 KDR mRNA is not expressed in blood cells, but it is expressed in the epithelial tissues of HL target organs, such as the liver and the spleen. We could not study the effect of the mutation directly in HL tumor cells from carrier patients since we did not have fresh-frozen tumor available. We speculate that an early prerequisite epigenetic event is needed in order for p.A1065T carriers to develop anomalous KDR expression that then promotes enhanced proliferation. We have identified a few other potential susceptibility variants for Hodgkin lymphoma in the families that we have surveyed. However, these additional potential candidate mutations appear to be private, at least in this cohort. In particular, the largest HL family (F4, 4 HL cases and 1 obligate carrier), in addition to having changes in 2 genes from our follow-up study (GPT and SMTNL2), presented with an A to G missense change at chr20:54956620 corresponding to the N192D amino acid change in Aurora Kinase A (AURKA). This mutation, which is highly conserved and predicted to be deleterious, is located in the kinase domain of the AURKA gene and absent in the ESP, 1000 Genome, and ExAC databases. AURKA is a kinase regulating mitosis, aberrantly expressed in mouse and human lymphoma cells,35 HL cell lines, and lymph nodes.36 AURKA inhibitors are used in cancer treatment, including lymphomas.37 Interestingly, we did not find shared variants in the three genes (KLHDC8B, NPAT, and ACAN) previously identified in high-risk HL families,16-18 while we observed

the POT1 rs202187871 variant also reported in a high-risk melanoma family.20 Given the large total number of genes (2383) with family-shared variants that we identified, we also conducted pathway analyses using two independent tools, Ingenuity Variant Analysis (IVA)38 and the program, GoMiner39 (Online Supplementary Tables S6A-6F). As seen in Online Supplementary Table S6A, the most enriched IVA pathways included Macrophages, T cell and B Cell Signaling in Rheumatoid Arthritis, Hematopoiesis, Allograft Rejection Signaling, OX40 Signaling, Innate and Adaptive Immune Cells, IL-10 Signaling, and Autoimmune Thyroid Disease Signaling. The most enriched GoMiner gene ontologies involved cell adhesion biological processes (Online Supplementary Table S6E) and protein binding molecular functions (Online Supplementary Table S6F). We did not observe significant enrichment in kinase activities or in cell division processes or in Kelch gene families (Online Supplementary Table S6B). It has been suggested that, based on increased sex concordance of HL sibling pairs seen in some studies,40,41 there could be a susceptibility gene in the pseudoautosomal region (PAR). Our overall sample of HL families do not show increased sex concordance, although our families were selected for greater density of cases occurring in multiple generations. Still, there could be rare families with susceptibility due to this mechanism. We do not see an enrichment of gene variants that may be in the PAR among our families (Online Supplementary Table S2). However, we do have one 3-generation family with 3 HL

Figure 1. Family pedigrees in the KDR mutation segregating families.

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Figure 2. Inhibitor bound VEGFR-2 structure (PDB ID: 3VO3). The wildtype protein structure display is set to solid ribbon and colored using N(blue)-to-C(red) terminal coloring style. The active site residue locations are colored in green and the activation loop segment in cyan. The inhibitor molecule is shown in balland-stick style and highlighted with a transparent closed surface. The amino acid residue A1065 is shown in CPK (solid spheres) style.

Table 3. Results in genes selected for Ion Torrent validation and follow-up study.

Count 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 858

Gene symbol

Chromosome

Position

Reference Allele

Sample Allele

Gene Region

Translation Impact

Protein Variant

AURKA BCAR1 CD33 COX7A2L CYSLTR2 EMILIN3 FAM86A FSTL5 GPT HLTF IL17RA ITGB2 KDR KRT82 MYL6 NKD2 PPP1R32 RHOG SDR42E1 SMTNL2 SUDS3 VNN2 ZGPAT; LIME1

20 16 19 2 13 20 16 4 8 3 22 21 4 12 12 5 11 11 16 17 12 6 20

54956620 75268923 51742917 42578483 49281386 39989937 5140298 162841700 145730071 148757909 17589509 46308800 55955969 52789643 56553920 1033498 61249376 3849350 82033735 4500575 118821819 133073884 62367256

T G G G C C T A C A T C C C C G A C G A T T G

C A A A T T A G T G C T T T T A G A C G C C T

Exonic Exonic Exonic Exonic Exonic Exonic Exonic Exonic Exonic Exonic Exonic Exonic Exonic ncRNA Exonic Exonic Exonic Exonic Exonic Exonic Exonic Exonic Exonic

missense missense missense missense missense missense stop gain missense missense missense missense missense missense ncRNA missense missense missense missense missense missense missense missense missense

p.N192D p.P671L p.E230K; p.E357K p.P74L p.R145W p.E758K p.K143*; p.K177* p.C89R; p.C88R p.R83C p.I804T p.L467P p.E630K p.A1065T none p.L113F p.A72T p.Y32C p.V7L p.Q55E p.T406A; p.T262A p.M72T p.H181R; p.H128R p.K507N; p.K498

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patients and 1 obligate carrier that are all male. Future deep sequencing of the Y chromosome or PAR genes would be appropriate in this family. The strength of our study is the ability to search comprehensively for coding variants causing susceptibility to HL in a number of highly informative families. Nonetheless, there are limitations in applying WES to find germline susceptibility genes. First, it is possible that the key susceptibility loci are outside of exonic regions and we would therefore not be able to detect them. Some genes and some regions within genes are not well covered by exome targets. Our filtering may be too stringent and thus we could have missed important mutations. It is also possible that not all top potential candidate genes were selected for the replication study. Alternatively, heritable epigenetic marks, rather than specific gene variants, may cause disease susceptibility. The underlying genetics of HL may be more complex than assumed in this study, even in high-risk families. There could be extensive genetic heterogeneity so that some causative mutations are â&#x20AC;&#x153;privateâ&#x20AC;?. It is also possible that more than one susceptibility gene is involved, even in a single family. These factors generally make it more difficult to detect critical loci and ultimately large consortia may be required to combine the sequencing of an even larger number of families. Future studies could include whole genome sequencing as well as integrated epigenetic and RNA-Seq expression

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studies in order to narrow down critical loci. Although our finding in the KDR gene is promising, this must be verified in independent studies. The identified germline mutation in the KDR gene has been shown to encode a constitutively active form of KDR that may have the capacity to enhance cell proliferation activity in the absence of ligand. Importantly, the phosphorylation was decreased and the kinase activity was abrogated after exposure to sunitinib and sorafenib drugs,26 suggesting that the p.A1065T KDR mutation could be targeted for therapy in HL and NHL patients carrying this mutation. In addition, our study is the largest sequenced cohort of HL families to date and provides the scientific community with potentially important variants readily available for replication in independent family studies. Funding This work was supported by the Intramural Research Program of the U.S National Institutes of Health (NIH), National Cancer Institute (NCI), Division of Cancer Epidemiology and Genetics. This work was supported in part by funds from the National Cancer Institute (NCI) /National Institutes of Health (NIH) contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views of policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government.

9. Westergaard T, Melbye M, Pedersen JB, Frisch M, Olsen JH, Andersen PK. Birth order, sibship size and risk of Hodgkin's disease in children and young adults: a population-based study of 31 million person-years. Int J Cancer. 1997;72(6):977-981. 10. Diepstra A, Niens M, Vellenga E, et al. Association with HLA class I in EpsteinBarr-virus-positive and with HLA class III in Epstein-Barr-virus-negative Hodgkin's lymphoma. Lancet. 2005;365(9478):2216-2224. 11. Harty LC, Lin AY, Goldstein AM, et al. HLADR, HLA-DQ, and TAP genes in familial Hodgkin disease. Blood. 2002; 99(2):690693. 12. Niens M, van den Berg A, Diepstra A, et al. The human leukocyte antigen class I region is associated with EBV-positive Hodgkin's lymphoma: HLA-A and HLA complex group 9 are putative candidate genes. Cancer Epidemiol Biomarkers Prev. 2006;15(11):2280-2284. 13. Cozen W, Timofeeva MN, Li D, et al. A meta-analysis of Hodgkin lymphoma reveals 19p13.3 TCF3 as a novel susceptibility locus. Nat Commun. 2014; 12:(5):3856. 14. Frampton M, da Silva Filho MI, Broderick P, et al. Variation at 3p24.1 and 6q23.3 influences the risk of Hodgkin's lymphoma. Nat Commun. 2013;4:2549. 15. Kushekhar K, van den Berg A, Nolte I, Hepkema B, Visser L, Diepstra A. Genetic associations in classical hodgkin lymphoma: a systematic review and insights into susceptibility mechanisms. Cancer Epidemiol Biomarkers Prev. 2014; 23(12):2737-2747. 16. Salipante SJ, Mealiffe ME, Wechsler J, et al. Mutations in a gene encoding a midbody kelch protein in familial and sporadic classical Hodgkin lymphoma lead to binucleated cells. Proc Natl Acad Sci USA. 2009;

106(35):14920-14925. 17. Saarinen S, Aavikko M, Aittomaki K, et al. Exome sequencing reveals germline NPAT mutation as a candidate risk factor for Hodgkin lymphoma. Blood. 2011; 118(3):493-498. 18. Ristolainen H, Kilpivaara O, Kamper P, et al. Identification of homozygous deletion in ACAN and other candidate variants in familial classical Hodgkin lymphoma by exome sequencing. Br J Haematol. 2015; 170(3):428-431. 19. Goldin LR, McMaster ML, Ter-Minassian M, et al. A genome screen of families at high risk for Hodgkin lymphoma: evidence for a susceptibility gene on chromosome 4. J Med Genet. 2005;42(7):595-601. 20. Shi J, Yang XR, Ballew B, et al. Rare missense variants in POT1 predispose to familial cutaneous malignant melanoma. Nat Genet. 2014;46(5):482-486. 21. McTigue MA, Wickersham JA, Pinko C, et al. Crystal structure of the kinase domain of human vascular endothelial growth factor receptor 2: a key enzyme in angiogenesis. Structure. 1999;7(3):319-330. 22. Cooper PS, Lipshultz D, Matten WT, et al. Education resources of the National Center for Biotechnology Information. Brief Bioinform. 2010;11(6):563-569. 23. Miyamoto N, Sakai N, Hirayama T, et al. Discovery of N-[5-({2-[(cyclopropylcarbonyl)amino]imidazo[1,2-b]pyridazin-6yl}oxy)-2-methylph enyl]-1,3-dimethyl-1Hpyrazole-5-carboxamide (TAK-593), a highly potent VEGFR2 kinase inhibitor. Bioorg Med Chem. 2013;21(8):2333-2345. 24. Hasegawa M, Nishigaki N, Washio Y, et al. Discovery of novel Benzimidazoles as potent inhibitors of TIE-2 and VEGFR-2 tyrosine kinase receptors. J Med Chem.

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M. Rotunno et al. 2007;50(18):4453-70. 25. Bacolla A, Temiz NA, Yi M, et al. Guanine holes are prominent targets for mutation in cancer and inherited disease. PLoS Genet. 2013;9(9):e1003816. 26. Antonescu CR, Yoshida A, Guo T, et al. KDR activating mutations in human angiosarcomas are sensitive to specific kinase inhibitors. Cancer research. 2009;69(18):7175-7179. 27. Greenman C, Stephens P, Smith R, et al. Patterns of somatic mutation in human cancer genomes. Nature. 2007; 446(7132):153158. 28. Marinaccio C, Nico B, Maiorano E, Specchia G, Ribatti D. Insights in Hodgkin Lymphoma angiogenesis. Leuk Res. 2014;38 (8):857-861. 29. Agarwal B, Naresh KN. Re: DoussisAnagnostopoulou et al. Vascular endothelial growth factor (VEGF) is expressed by neoplastic Hodgkin-Reed-Sternberg cells in Hodgkin's disease. 2003;201(2):334-335. 30. Doussis-Anagnostopoulou IA, Talks KL, Turley H, et al. Vascular endothelial growth factor (VEGF) is expressed by neoplastic

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Hodgkin-Reed-Sternberg cells in Hodgkin's disease. J Pathol. 2002;197(5):677-683. Jorgensen JM, Sorensen FB, Bendix K, et al. Expression level, tissue distribution pattern, and prognostic impact of vascular endothelial growth factors VEGF and VEGF-C and their receptors Flt-1, KDR, and Flt-4 in different subtypes of nonHodgkin lymphomas. Leuk lymphoma. 2009; 50(10):1647-1660. Paydas S, Ergin M, Erdogan S, Seydaoglu G. Prognostic significance of EBV-LMP1 and VEGF-A expressions in non-Hodgkin's lymphomas. Leuk Res. 2008; 32(9):1424-1430. Reiners KS, Gossmann A, von Strandmann EP, Boll B, Engert A, Borchmann P. Effects of the anti-VEGF monoclonal antibody bevacizumab in a preclinical model and in patients with refractory and multiple relapsed Hodgkin lymphoma. J Immunother. 2009;32(5):508-512. http://www.roadmapepigenomics.org/ [last accessed 20 Nov 2015]. den Hollander J, Rimpi S, Doherty JR, et al. Aurora kinases A and B are up-regulated by Myc and are essential for maintenance of

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the malignant state. Blood. 2010;116(9): 1498-1505. Mori N, Ishikawa C, Senba M, Kimura M, Okano Y. Effects of AZD1152, a selective Aurora B kinase inhibitor, on Burkitt's and Hodgkin's lymphomas. Biochem Pharmacol. 2011;81(9):1106-1115. Friedberg JW, Mahadevan D, Cebula E, et al. Phase II study of alisertib, a selective Aurora A kinase inhibitor, in relapsed and refractory aggressive B- and T-cell non-Hodgkin lymphomas. J Clin Oncol. 2014; 32(1):44-50. www.qiagen.com/ingenuity [last accessed 20 Nov 2015]. Zeeberg BR, Feng W, Wang G, et al. GoMiner: a resource for biological interpretation of genomic and proteomic data. Genome biology. 2003;4(4):R28. Altieri A, Hemminki K. The familial risk of Hodgkin's lymphoma ranks among the highest in the Swedish Family-Cancer Database. Leukemia. 2006;20(11):20622063. Horwitz M, Wiernik PH. Pseudoautosomal linkage of Hodgkin disease. Am J Hum Genet. 1999;65(5):1413-1422

haematologica | 2016; 101(7)


ARTICLE

Non-Hodgkin Lymphoma

N-terminally truncated FOXP1 protein expression and alternate internal FOXP1 promoter usage in normal and malignant B cells

EUROPEAN HEMATOLOGY ASSOCIATION

Ferrata Storti Foundation

Philip J. Brown,1*† Duncan M. Gascoyne,1* Linden Lyne,1 Hayley Spearman,1 Suet Ling Felce,1 Nora McFadden,2 Probir Chakravarty,3 Sharon Barrans,4 Steven Lynham,5 Dinis P. Calado,2,6 Malcolm Ward,5 and Alison H. Banham1

Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, Oxford University; 2Immunity and Cancer Laboratory, The Francis Crick Institute, Lincoln's Inn Fields Laboratory, Lincoln's Inn Fields, London 3Computational Biology Laboratory, The Francis Crick Institute, Lincoln’s Inn Fields Laboratory, Lincoln’s Inn Fields, London; 4Leeds Teaching Hospitals NHS Trust, HMDS, Leeds Cancer Centre; 5 Centre of Excellence for Mass Spectrometry, Institute of Psychiatry, Psychology and Neuroscience, Kings College London; and 6Peter Gorer Department of Immunobiology, Kings College London, UK 1

*PJB and DMG contributed equally to this work. †Deceased, 2nd March 2014

Haematologica 2016 Volume 101(7):861-871

ABSTRACT

S

trong FOXP1 protein expression is a poor risk factor in diffuse large B-cell lymphoma and has been linked to an activated B-cell-like subtype, which preferentially expresses short FOXP1 (FOXP1S) proteins. However, both short isoform generation and function are incompletely understood. Here we prove by mass spectrometry and Nterminal antibody staining that FOXP1S proteins in activated B-cell-like diffuse large B-cell lymphoma are N-terminally truncated. Furthermore, a rare strongly FOXP1-expressing population of normal germinal center B cells lacking the N-terminus of the regular long protein (FOXP1L) was identified. Exon-targeted silencing and transcript analyses identified three alternate 5ʹ non-coding exons [FOXP1-Ex6b(s), FOXP1-Ex7b and FOXP1-Ex7c], downstream of at least two predicted promoters, giving rise to FOXP1S proteins. These were differentially controlled by B-cell activation and methylation, conserved in murine lymphoma cells, and significantly correlated with FOXP1S protein expression in primary diffuse large B-cell lymphoma samples. Alternatively spliced isoforms lacking exon 9 (e.g. isoform 3) did not encode FOXP1S, and an alternate long human FOXP1 protein (FOXP1AL) likely generated from a FOXP1Ex6b(L) transcript was detected. The ratio of FOXP1L:FOXP1S isoforms correlated with differential expression of plasmacytic differentiation markers in U-2932 subpopulations, and altering this ratio was sufficient to modulate CD19 expression in diffuse large B-cell lymphoma cell lines. Thus, the activity of multiple alternate FOXP1 promoters to produce multiple protein isoforms is likely to regulate B-cell maturation.

Correspondence: alison.banham@ndcls.ox.ac.uk

Received: January 15, 2016. Accepted: April 4, 2016. Pre-published: April 7, 2016. doi:10.3324/haematol.2016.142141

Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/101/7/861

Introduction Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease entity originating from germinal center (GC) or post-GC B cells such as plasmablasts.1-4 The majority of DLBCL can be classified according to cell-of-origin gene expression profile, as either germinal center (GC-DLBCL) or activated B-cell (ABC-DLBCL) subtype.5-9 While addition of rituximab to CHOP chemotherapy has improved DLBCL patients’ survival significantly,10 new therapies are needed for non-responding or relapsed patients (reviewed by Sehn and Gascoyne).11 Novel molecularly-targeted therapies are being sought particularly for the poorer prognosis ABC-DLBCL subtype following identification of key biological pathways contributing to disease pathogenesis, such as NF-kB pathway mutations and activation,12-15 B-cell receptor (BCR) signaling,16 MALT1 activity,17 and BLIMP1 mutahaematologica | 2016; 101(7)

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

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P.J. Brown et al. tions.18 Maintenance of BCR signaling and prevention of plasma cell maturation to disrupt normal maturation/differentiation pathways is a common paradigm. High FOXP1 expression correlates with the ABCDLBCL subtype4 and poor clinical outcome in both the pre- and post-rituximab eras.19-22 FOXP1 amplification and trisomy have been described in ABC-DLBCL,23 and translocations involving the immunoglobulin heavy chain (IGH) locus24 drive expression of a long ~75kDa FOXP1 protein (FOXP1L) that may contribute to GC-DLBCL tumor growth by potentiating Wnt/β-catenin signaling.25 Also, we have described abundant expression of short ~65kDa activation-induced FOXP1 proteins (FOXP1S) in ABC-DLBCL.26 Oncogenic activity of N-terminally truncated FOXP1 has been proposed following its truncation by an oncogenic virus27 and non-IGH translocations targeting the FOXP1 coding region in lymphoma.24,28,29 Studies manipulating Foxp1 expression have established biological roles in early B-cell development30,31 and in mature B cells.32 Direct FOXP1 target genes, including PAX5, PRDM1, and POU2F1, support a functional role in the GC reaction.32 Recent studies have provided insight into FOXP1 function in DLBCL cells, indicating contributions to proliferation,33 inhibition of apoptosis to compliment NF-kB-dependent regulation of proliferation,34 and Wnt signaling.25 However, further understanding of FOXP1 isoform expression and functionality is required to pursue emerging evidence indicating that FOXP1L and FOXP1S proteins have distinct functions.22,31 Here we demonstrate that FOXP1S proteins in DLBCL are encoded by transcripts with alternate 5ʹ non-coding exons, not internal exon skipping.26 We identify a novel alternate long FOXP1 protein (designated FOXP1AL) and a GC B-cell population lacking the FOXP1L N-terminus. Isoform-biased FOXP1 depletion altering FOXP1L:FOXP1S stoichiometry regulates expression of CD19 in DLBCL cell lines, providing further evidence for isoforms having distinct functional roles in B-cell biology.

Gene expression analyses Random-primed cDNA was prepared from total RNA according to the manufacturer’s protocols (Life Technologies), and real-time PCR analyses performed by Chromo4 (BioRad, Herts., UK) using Express qPCR supermix or SYBR GreenER supermix (Life Technologies), and primers/probes as detailed in Online Supplementary Table S1. Standard RT-PCR to discriminate FOXP16b(L/S) transcripts used forward Ex6b(L)#1, Ex6b(L)#2, Ex6b(S), or control forward primers Ex6 or Ex8, all paired with reverse primer Ex10 (Online Supplementary Table S1). RNAseq analysis was performed on NCBI Gene Expression Omnibus (GEO) lymphoma dataset GSE50721.35

Immunoblot and immunoprecipitation Whole cell extracts were prepared in RIPA, or nuclear and cytoplasmic extracts by commercial reagent (Affymetrix, High Wycombe, UK). Immunoprecipitations from 50 mg nuclear lysate were performed in IP buffer (1% Triton, 150 mM NaCl, 10 mM Tris, 1 mM EDTA) and complexes purified using mMACS Protein G microbeads (Miltenyi Biotec, Surrey, UK). Primary antibody details are provided in Online Supplementary Table S1.

Mass spectrometry JC12 immunoprecipitates were subjected to parallel SDS-PAGE analyses for immunoblotting and silver staining. Stained bands corresponding to ~65kDa and ~75kDa FOXP1 proteins were excised, subjected to reduction, alkylation and trypsin digestion, and resulting peptides identified by mass spectrometry as previously described36 with formic acid modified to 0.1% in the gradient.

Immunohistochemistry After formalin fixation, paraffin embedding and sectioning, slides were dewaxed and antigen retrieved by microwaving in 50 mM Tris, 2 mM EDTA pH9.0. Immunostaining with primary antibodies (Online Supplementary Table S1) was followed by secondary antibody and detection (Envision-Dako, Ely, UK).

Flow cytometry Methods Cell culture Diffuse large B-cell lymphoma and myeloma lines were sourced and cultured as described previously.26 COS-1, 293T and NIH-3T3 (ATCC) were cultured in DMEM containing 10% fetal bovine serum (Life Technologies, Paisley, UK), and murine B-cell lines 5TGM1-GFP (a gift of Claire Edwards, Oxford, UK) and A20 (ATCC) in RPMI containing 10% serum, 100 mM non-essential amino acids, 1 mM sodium pyruvate, 50 mM 2-mercaptoethanol.

Primary human samples Diffuse large B-cell lymphoma patient samples were collected with informed consent in accordance with the Declaration of Helsinki, and the study was performed under local ethics committee approval from the Leeds West Regional Ethics Committee, Leeds, UK. RNA was isolated from DLBCL samples obtained prior to therapy as described.26 Primary B cells from blood buffy-coat preparations (National Blood Service, Bristol, UK) were activated with 50 mg/mL anti-IgM or 1:20,000 SAC plus 5 ng/mL recombinant human IL-2 (Sigma, St Louis, USA). Reactive tonsils were obtained with informed consent from John Radcliffe Hospital, Oxford, UK, and studies were conducted under ethical approval from NRES Committee South Central – Oxford B (C02.162). 862

Cells were labeled in PBS containing 0.5% bovine serum albumin, 2 mM EDTA with primary antibodies (Online Supplementary Table S1) and/or isotype controls, and/or secondary antibody (streptavidin APC, eBioscience) and data obtained by FACSCalibur (Becton Dickinson, San Jose, USA).

Transfection For overexpression, cells were harvested 48 h after transfection with pcDNA4-HisMax expression vectors encoding human FOXP1-4 proteins using Lipofectamine (Life Technologies). For knockdown, cells were electroporated in the presence of 1 mM Stealth siRNA duplexes (Life Technologies) (Online Supplementary Table S1) using Amaxa Nucleofector (Lonza, Slough, UK) generally using Solution L, program X-001.

Results Short FOXP1 proteins (FOXP1S) in ABC-DLBCL lack the N-terminus We used mass spectrometry to characterize FOXP1S and FOXP1L proteins immunoprecipitated from GC- and ABC-DLBCL cell lines (Figure 1A and B). Peptides translated from most exons encoding the FOXP1L protein were identified, while peptides from the N-terminal coding exons (Ex), Ex6 and Ex7, were absent from FOXP1S prohaematologica | 2016; 101(7)


N-terminal FOXP1 truncation in DLBCL

teins in ABC-DLBCL cell lines RIVA and OCI-Ly3 (Figure 1B). This is consistent with FOXP1S proteins deriving from transcripts where translation initiates in Ex8 (e.g. isoform 9)26 but inconsistent with internal deletion of Ex8 and/or Ex9 and/or Ex10 identified in FOXP1 isoforms 3, 5 and 8, which retain Ex6 and Ex7.26 To confirm N-terminal truncation of FOXP1 proteins in DLBCL, we validated a commercially available FOXP1 polyclonal antibody against an Ex7-encoded peptide. This antibody detected recombinant FOXP1L protein (Ex6Ex21), but not FOXP1S protein (Ex8-Ex21), using both immunohistochemistry and Western blotting. However, there was cross-reactivity with both the related FOXP2 protein and an unknown, FOXP1 siRNA resistant, ~70kDa cytoplasmic protein in DLBCL cell lines (Online Supplementary Figure S1A-D). Thus, this reagent was used to study only nuclear expression in cell lines and tissues with known FOXP2 status. In DLBCL nuclear extracts, this N-terminal antibody recognized only FOXP1L, while our anti-C-terminal JC12 antibody detected both FOXP1S and FOXP1L proteins (Figure 1C). Thus FOXP1S proteins in multiple ABC-DLBCL cell lines lack the N-terminal epitopes encoded by Ex7.26 The N-terminal antibody was ineffective at routinely distinguishing ABC- versus GCBDLBCL cell lines by immunohistochemistry (Online Supplementary Figure S2A), reflecting FOXP1L co-expression, non-specific cytoplasmic staining, and FOXP2 expression in RIVA. Interestingly, immuno-precipitation of FOXP1L using the N-terminal antibody co-immunoprecip-

A

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itated FOXP1S, providing evidence for a physical FOXP1LFOXP1S interaction in ABC-DLBCL (Online Supplementary Figure S1E).

A rare subset of germinal center B cells lack the FOXP1L N-terminus

The ABC-DLBCL cell-of-origin is proposed to be a plasmablastic B cell poised to exit the GC.1-4 Tonsillar B-cell follicles (FOXP2-negative) consistently exhibited comparable intensity and pattern of mantle zone staining when immunolabeled with N- and C-terminal FOXP1 antibodies (Figure 1D). However, rare GCs in some tonsils contained a small subpopulation of strongly FOXP1+CD20+ B cells (Figure 1D; inset) that were not effectively labeled using the N-terminal antibody. Thus, a small population of GC B cells may share the abundant FOXP1S protein expression observed in ABC-DLBCL. However, we cannot exclude an alternate FOXP1L protein (FOXP1AL), which we have identified in ABC-DLBCL.

Transcripts encoding FOXP1S proteins in ABC-DLBCL have variable 5’ non-coding exons and share coding exons 3’ from Ex8 N-terminally truncated FOXP1S proteins in ABC-DLBCL might derive from post-translational cleavage of the normal FOXP1L protein or alternate promoter usage. To distinguish these possibilities, we performed exon-targeted siRNA across the FOXP1 locus (Figure 2A), thus identifying transcripts producing FOXP1 proteins in ABC-DLBCL

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Figure 1. Short FOXP1 proteins (FOXP1S) in ABC-diffuse large B-cell lymphoma (ABC-DLBCL) and rare germinal center B cells lack the FOXP1 N-terminus. (A) JC12 anti-FOXP1 immunoblot of immunoprecipitates from three DLBCL cell lines after JC12 (a-FOXP1) or isotype (control) immunoprecipitation. ‘Input’ represents 5%; ‘flow’ represents flow-through to show efficient precipitation; both FOXP1L (L) and FOXP1S (S) isoforms are indicated together with antibody heavy chain (asterisk); long and short FOXP1 proteins as indicated left were excised from silver-stained SDS-PAGE gels run in parallel to those in (A) and mass spectrometry performed. (B) Schematic to show presence (+) or absence (–) of FOXP1 peptides from each coding exon. (C) Representative FOXP1 immunoblot analysis of DLBCL cell line nuclear extracts using JC12 or FOXP1 N-terminal-specific antibodies. α-TBP blotting controlled for loading and transfer; (D) FOXP1 immunohistochemistry of serial sections through a secondary follicle (with strongly JC12-positive germinal center cells) from reactive human tonsil using both JC12 and N-terminal antibodies. Note similar mantle zone but less germinal center immunoreactivity with the N-term pAb. Inset shows CD20 surface expression on the majority of strongly JC12-positive germinal center cells by dual color immunohistochemistry for FOXP1 (JC12, brown nuclei) and CD20 (blue membranous).

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cell lines (RIVA and OCI-Ly3, and as a control the GCDLBCL cell line DB) (Figure 2). FOXP1 coding exon targeting generally reduced FOXP1L levels, although this was sometimes difficult to detect in OCI-Ly3 due to low FOXP1L expression (Figure 2B). Consistent with siRNA targeting of the 5ʹ coding region being inefficient for some genes, Ex1-2 siRNA did not work at all, and Ex5 and Ex6 siRNAs targeted poorly. In contrast, targeting of FOXP1 Ex8 onwards silenced FOXP1 protein expression effectively, confirming coding function of the 3ʹ exons and the absence of FOXP1S coding transcripts with internal Ex8-9 deletions. Ex5 and Ex6 targeting had no effect on FOXP1S expression, suggesting that FOXP1S proteins were not post-translationally processed from FOXP1L. Interestingly, two independent siRNAs targeting Ex7 that effectively silenced FOXP1L also partially depleted FOXP1S in both ABC-DLBCL cell lines (Figure 2B and C).

As no Ex7-encoded peptides were identified in FOXP1S by mass spectrometry, a proportion of FOXP1S-coding transcripts may contain a non-coding Ex7. Indeed one such transcript, with transcription starting in the 3ʹ end of alternative Ex6b(S) is described (Figure 3). Thus FOXP1S-coding transcripts in ABC-DLBCL share common 3ʹ exons (from exon 8 onwards), have variable 5ʹ non-coding exons, and are not encoded by previously reported splice variants26 lacking exons 8, 9 and/or 10.

DLBCL cell lines expressing FOXP1S protein transcribe multiple 5ʹ alternate exon-containing FOXP1 mRNA species To explore the relationship between FOXP1 proteins and transcripts, panels of GC- and ABC-DLBCL lines were ranked by increasing FOXP1S:FOXP1L protein expression ratio (Online Supplementary Figure S2B). Based on our data,

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C Figure 2. Transcripts encoding FOXP1S proteins in activated B-cell like-diffuse large B-cell lymphoma (ABC-DLBCL) share coding exons from Ex8 onwards with FOXP1L. (A) Schematic illustration of human FOXP1 exons to show location of siRNA target sequences. (B) Immunoblot analysis of whole cell extracts from DLBCL cells harvested 48 h after transfection with FOXP1-targeting (+) or matched control (–) siRNA duplexes, representative of two or more experiments. (C) Schematic summary of immunoblotting data in (B). + indicates successful siRNA targeting; - indicates the FOXP1 isoform was unaffected; +/- indicates suboptimal/partial targeting.

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N-terminal FOXP1 truncation in DLBCL

additional database searching, and published FOXP1 RNA-sequencing data from primary DLBCL biopsies,24 we assembled a list of FOXP1 transcripts with the potential to encode FOXP1L and FOXP1S proteins in ABC-DLBCL (Figure 3A). There appear to be two transcriptional start sites within Ex6b, with the 5ʹ longer Ex6b transcript (Ex6b(L)) predicted to encode a long FOXP1 protein with an alternate N-terminus (FOXP1AL), while the shorter (Ex6b(S)) initiates translation from Ex8, as do transcripts containing other alternate 5ʹ non-coding exons Ex7b and Ex7c. Real-time PCR analysis of common 3ʹ exons (Ex20-21) demonstrated increased FOXP1 expression in most ABCDLBCL cell lines, while expression of 5ʹ Ex6-7 was vari-

able (Figure 3B). Alternate exons Ex7b and Ex7c were preferentially transcribed in ABC-DLBCL cell lines, while Ex6b (encoding FOXP1L or FOXP1S proteins) was only slightly more abundant in ABC-DLBCL lines (Figure 3C). RT-PCR analysis demonstrated that, in contrast to Ex6b(S), Ex6b(L) was abundant only in the ABC-DLBCL cell line HBL-1 (Online Supplementary Figure S3A). Interestingly, reduced recognition of long FOXP1 by the N-terminal antibody in HBL-1 (Figure 1C) is consistent with expression of an additional long FOXP1 protein containing an alternate N-terminus, FOXP1AL. Transfection confirmed that Ex6b(L) encodes only the FOXP1AL protein (Online Supplementary Figure S1F). Increased expression of alternate 5ʹ Ex6b and Ex7b tran-

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C Figure 3. Diffuse large B-cell lymphoma (DLBCL) cells expressing FOXP1S protein transcribe multiple 5ʹ exon-containing FOXP1 mRNA species. (A) Schematic illustration of human FOXP1 transcripts containing alternative 5ʹ exons (purple), non-coding exons (light blue), coding exons (yellow), exons containing initiating methionine (green), and termination codons (red). Note 6b(L) exon is an alternative exon colored green not purple due to presence of an initiating methionine. (B and C) Real-time PCR analyses of human FOXP1 transcript expression in DLBCL cell lines ordered as in Online Supplementary Figure S2B (according to FOXP1S to FOXP1L protein ratio); n=3±SD.

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scripts in ABC-DLBCL versus GC-DLBCL lines was also detectable by RNA-sequencing (Figure 4 and Online Supplementary Table S2), although only the Ex6b(L) increase was significant. Ex7c was not detected by this method, reflecting its relatively low abundance. In contrast to previously reported RT-PCR data,26 Ex4a was not reliably increased in ABC-DLBCL (Figure 4 and Online Supplementary Figure S3B). Thus, no single alternate transcript was specifically associated with increased FOXP1S expression, and almost all ABC-DLBCL cell lines expressed multiple alternate transcripts.

FOXP1 transcripts with alternate exons 6 and 7 correlate with FOXP1S protein expression in primary human DLBCL The expression of FOXP1 total transcripts and those with alternate 5’ exons was further validated in a panel of primary DLBCL cases with known FOXP1L and FOXP1S protein expression, determined previously by immunoblotting.26 Expression of FOXP1 transcripts containing Ex6b or Ex7c was significantly increased in tumors where FOXP1S protein levels were equivalent to or exceeded FOXP1L, while there was no relationship with total FOXP1 transcripts (Figure 5A). Expression of Ex6b, Ex7b, Ex7c and total FOXP1 was significantly higher in non-GC-DLBCL (Figure 5B). Combining expression profiles of all three FOXP1S-encoding transcripts did not improve the accuracy of predicting GC or non-GC status (data not shown); some non-GC-DLBCL exhibited low expression of them all. Expression of Ex7b and Ex7c transcripts was significantly related to the intensity of tumoral nuclear FOXP1 protein positivity determined by immunohistochemistry (Figure 5C). Thus, despite lower relative abundance in DLBCL cell lines, only Ex7c-containing transcript levels correlate significantly with both FOXP1 protein expression levels, DLBCL cell-of-origin subtype, and predominant FOXP1S protein expression in primary tumors. Ex4a expression was not significantly related to FOXP1 protein or GC/non-GC status in primary DLBCL (Online Supplementary Figure S3C).

Figure 4. RNA-sequencing analysis detects increased FOXP1 alternate 5ʹ promoter usage in ABC- versus germinal center (GC)-diffuse large B-cell lymphoma (DLBCL) cells. Analysis of FOXP1-annotated transcripts was performed in dataset GSE50721 that includes ABC-DLBCL cell lines OCI-LY3, OCI-LY10, SUDHL2, U-2932, and GCB-DLBCL cell lines OCI-LY7, SU-DHL4, SU-DHL6, SUDHL10, and OCI-LY19.35 FASTQ files were downloaded, decrypted and aligned to hg19 genome using RSEM (bowtie2 mapping tool). Output gene and transcript count data were filtered to remove genes with 10 or fewer reads, then input into EdgeR (Bioconductor) and normalized to take into account the number of mapped reads and a normalization factor using a trimmed mean of M values (TMM). Output was log2 transformed normalized expression data. Expression of FOXP1 transcripts containing Ex6bS, Ex7b or Ex6bL was higher in ABC cell lines (red data points, gray boxes) versus GCB-DLBCL cell lines (blue data points, black boxes), while Exon7c expression was not detected by this method. Differently-expressed transcripts are highlighted (triangle).

Malignant mature murine B cells express Foxp1S protein and N-terminally truncated Foxp1 transcripts partially conserved with mature human B cells Cross-species conservation supports biological significance, and a murine Foxp1S isoform termed Foxp1D (initiating exon originally designated mEx2b,37 revised to mEx5b)38 has been described.37 Multiple transcripts with alternate non-coding 5ʹ exons encoding murine Foxp1S protein in a murine B-cell lymphoma model, A20, that expresses both Foxp1L and Foxp1S proteins were investigated (Online Supplementary Figure S4A). Comparisons of the murine Foxp1 genomic sequence with alternate 5ʹ exons in human FOXP1 transcripts identified potential murine exons with homology to human Ex6b (mEx4b), Ex7b (mEx5b) and Ex7c (mEx5c) (Online Supplementary Figure S4B and C). However, the revised mEx2b alternate 5ʹ exon (predicted to encode Foxp1L or Foxp1A)37 had only low similarity to human Ex4a sequences. Importantly, expression of Foxp1S -coding murine transcripts containing alternate 5ʹ exon mEx4b or mEx5b was detectable in the malignant mature B-cell lines A20 and 5TGM1 and associated with reduced expression of Foxp1L Ex2-4 transcripts (Online Supplementary Figure S4D). Despite the significance of Ex7c in human DLBCL, transcripts containing its poten866

tial murine equivalent (mEx5c) could not be reliably detected. In summary, multiple transcripts with alternate 5ʹ exons contribute to FOXP1S protein expression in both murine and human lymphoma.

FOXP1 alternate 5’ exon usage is differentially induced by activation/maturation stimuli The redundancy of multiple alternate 5ʹ exons giving rise to FOXP1S may enable several pathways (potentially different cell types or developmental stages) to fine-tune FOXP1S protein expression. Supporting this hypothesis, activation of primary human naïve B cells from 2 individuals with multiple stimuli, previously shown to induce FOXP1S protein,26 increased expression of Ex6b(L) Ex7b and Ex7c, but had little effect on total Ex6b transcripts (Figure 6A). The latter indicates relatively high expression of Ex6b(S) versus Ex6b(L), as in DLBCL cell lines (Online Supplementary Figure S3A). Activation of B-cell-derived cell lines induced Ex7c but not total Ex6b or reliably Ex7b expression, while Ex6b transcripts were induced only in the myeloid cell line HL-60, despite successful activation haematologica | 2016; 101(7)


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Figure 5. FOXP1 transcripts with alternate exons Ex6b and Ex7b/c correlate with FOXP1S protein expression in primary human diffuse large B-cell lymphoma (DLBCL) samples. Real-time PCR analysis of multiple human FOXP1 transcript expression in 79 primary DLBCL biopsies. Expression relative to RIVA cell line, normalized to 18S. Dots represent means of triplicate determinations per sample. Samples grouped according to (A) predominant FOXP1L (long: FOXP1L > FOXP1S) or FOXP1S (short; FOXP1L ≤ FOXP1S) protein expression as determined previously by western blot. (B) DLBCL subtype with germinal center (GC) or non-GC, as previously described,26 or (C) intensity of tumoral FOXP1 positivity by immunohistochemistry, as previously described.26 Horizontal bars within each scatterplot represent mean of each group. *P≤0.05, **P≤0.01.

of other myeloid lines (Figure 6B and Online Supplementary Figure S5A and B). Lack of Ex6b(L) transcripts (Figure 6B; inset) indicates this Ex6b to be FOXP1Scoding. Thus, multiple conserved FOXP1 transcripts with alternate 5ʹ exons are induced by activation/maturation events in a cell-type specific manner to produce FOXP1S protein. Treatment of several GC-DLBCL cell lines with 5-azacytidine increased Ex7b and Ex7c but not Ex6b expression (Online Supplementary Figure S5C), implicating methylation as another mechanism differentially regulating alternate Foxp1 promoter activity between DLBCL subtypes.

FOXP1S:FOXP1L ratio controls CD19 expression in human DLBCL As GC- and ABC-DLBCL differ in their putative cell-oforigin, the relationship between FOXP1S:FOXP1L ratio and expression of B-cell activation/maturation cell surface markers was investigated further across DLBCL cell lines. Expression of the B-cell marker CD20 was uniformly robust, and that of the plasma cell marker CD138 uniformly weak or absent (Figure 7A). In contrast, surface expression of CD19, CD45 (B220 isoform), and CD27 generally decreased as the FOXP1S to FOXP1L expression haematologica | 2016; 101(7)

ratio increased (Figure 7A), with relatively few exceptions (e.g. CD19 in DB and CD45 in Karpas-422). Cell lines such as SU-DHL-4 and U-2932 exhibited both intermediate FOXP1S:FOXP1L ratios and intermediate expression of the immaturity/memory markers CD19, B220 and/or CD27. Importantly, in these ‘intermediate’ DLBCL cell lines, transfection of siRNAs targeting Ex7 to increase the FOXP1S:FOXP1L ratio (Figure 2), but not targeting of total FOXP1, significantly reduced CD19 expression (Figure 7B and C). The ABC-DLBCL cell line U-2932 has two subpopulations ‘R1’ and ‘R2’ (Figure 8A) both present as ‘clones’ in the original patient (CD20hiCD38hi and CD20loCD38lo, respectively), which can be maintained stably and display both common and unique genetic aberrations.39 Purified R1 and R2 populations exhibited clumped versus singlecellular growth habits, respectively (Figure 8B), and R1 showed higher FOXP1S protein expression and increased expression of FOXP1 Ex6b, Ex7b and Ex7c transcripts (Figure 8C and D). Furthermore, R1 has elevated expression of plasmablastic markers (IRF4, XBP1) and reduced expression of the FOXP1S-repressed target gene HIP1R22 (Figure 8D), again demonstrating a positive association between FOXP1S and B-cell maturity. 867


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Discussion Characterization of FOXP1 isoform complexity in both human and murine lymphoma cells is important in order to understand their roles in lymphoma biology and clinical relevance. We have proven our original hypothesis26 that FOXP1S proteins in ABC-DLBCL lack the N-terminus using both mass spectrometry and a commercially available polyclonal N-terminal antibody. An improved N-terminal antibody, lacking the non-FOXP1 cytoplasmic cross-reactivity and nuclear FOXP2 cross-reactivity seen with the current antibody, might routinely identify strongly JC12+ (FOXP1 C-terminal epitope) DLBCL that lack the N-terminus. Despite its limitations, the current polyclonal antibody may be of interest within the research community, e.g. to validate expression of C-terminally truncated FOXP1 proteins, such as the FOXP1w isoform up-regulated by SF3B1 mutation in poor prognosis B-cell chronic lymphocytic leukemia. Western blotting data have demonstrated that normal lymphoid tissue and the microenvironment surrounding FOXP1-negative tumors express predominantly FOXP1L protein(s).26 Thus, our identification of a rare GC B-cell population with high-level expression of FOXP1 protein(s) lacking Ex7-encoded epitopes is particularly interesting. Additional reagents (e.g. antibodies to novel epitopes in the N-terminus of FOXP1AL) will help to definitively characterize this strongly JC12+ population and examine whether FOXP1AL or FOXP1S isoforms predominate. Such studies may help to define a normal counterpart for ABCDLBCL. FOXP1 exon-targeted siRNA and expression studies have identified at least three distinct FOXP1 proteins in DLBCL; FOXP1L, FOXP1AL (long forms) and FOXP1S, the latter two being primarily expressed in ABC-DLBCL. FOXP1AL detection in the HBL-1 ABC-DLBCL cell line is consistent with published transcript data describing potential expression of this isoform (FOXP1-011, ENST00000491238) in primary DLBCL.24,26 Since all alternate 5ʹ exons were targeted inefficiently by siRNA in DLBCL cells, methods such as FOXP1 locus editing could help to determine particular transcript contributions. Importantly, transcript conservation in murine lymphoma cells (e.g. human Ex6b and murine equivalent mEx4b being most abundant) should simplify Foxp1S functional studies. While no individual 5ʹ non-coding exon defines FOXP1S protein expression in primary DLBCL, Ex7c remains the best single transcript predictor and could be a useful addition to prognostic/diagnostic gene panels for DLBCL. Our data suggest that the FOXP1S/L ratio may also help define the stage of developmental block in DLBCL and improve stratification of DLBCL subgroups. Although FOXP1 proteins expressed in malignant and normal B cells are similar, the control of FOXP1 expression appears distinct. Genetic abnormalities truncating FOXP1 are infrequent,24,28,29 and our data indicate the majority of N-terminally-deleted FOXP1 expression in lymphoma is generated by alternate internal 5ʹ promoter usage (Ex6b being particularly abundant). Thus, it will be important to understand the likely oncogenic mechanisms controlling these transcriptional events. Significantly, BLIMP1 has been shown to bind the FOXP1 locus,40 and thus BLIMP1 mutation18 may drive ABC-DLBCL lymphomagenesis at least in part by mis-regulating FOXP1 isoform expression. Historically, many clinical studies have not evaluated 868

A

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Figure 6. FOXP1 alternate 5’ exon usage is differentially induced by activation/maturation stimuli. (A) Real-time PCR analysis of multiple human FOXP1 transcripts as indicated in primary naïve B cells purified from peripheral blood, cultured for 48 h without (Naïve) or with (anti-IgM or IL-2 + SAC) stimuli. Expression relative to the RIVA cell line, normalized to 18S. Mean of data from 2 individuals±S.E (B) Real-time PCR analysis of multiple human FOXP1 transcripts as indicated in B lineage and myeloid cell lines cultured for 48 h with either carrier alone (u/t) or phorbol ester (PMA), expression relative to RIVA cell line, normalized to 18S, representative of at least two experiments. (B) (Inset) Standard RT-PCR amplification to detect human FOXP1 exon expression (top) in HL-60 cells treated with PMA identified Ex6b(S) but not Ex6b(L) transcripts.

heterogeneity of marker expression across areas of a tumor. However, studies of clonal evolution have highlighted the importance of heterogeneity in a variety of malignancies, particularly in developing treatment resistance. We previously noted in a lymphoma biopsy with FOXP1 translocation that the localized area of tumor containing the translocation had stronger FOXP1 protein expression ((PJ Brown et al, unpublished data, 2014). Here we demonstrate that subpopulations from a single DLBCL patient (present in U-2932) exhibit distinct FOXP1 isoform patterns alongside different immunophenotypes and expression of transcription factors involved in B-cell differentiation. Greater understanding of FOXP1 isoform heterogeneity may help to identify therapy-resistant DLBCL clones, as elevated FOXP1 is linked to resistance in response to both CHOP-R and rituximab monotherapy in lymphoma.19,21,22,41 Abundant Ex6bS transcript expression in GC-DLBCL cell lines lacking FOXP1S protein expression suggests posttranscriptional regulatory mechanisms may also regulate FOXP1S protein expression in DLBCL. Alternate exon-specific miRNA activity, in addition to the previously reporthaematologica | 2016; 101(7)


N-terminal FOXP1 truncation in DLBCL

A

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C

Figure 7. FOXP1S to FOXP1L expression ratio controls developmental status of human DLBCL cells. (A) Flow cytometric analysis of CD19, CD138, CD45, CD20, CD27 and CD30 surface expression on DLBCL cell lines with increasing FOXP1S:FOXP1L protein ratio, as indicated. Note reasonably constant expression of CD20 and weak/absent CD138 and CD30 expression, while CD19, CD45 and CD27 show generally decreasing expression with increasing FOXP1S to FOXP1L ratio. (B) Representative flow cytometric analysis of CD19 surface expression on SUDHL-4 (GC-DLBCL) and U-2932 (ABC-DLBCL) cell lines 48 h after introduction of control siRNA or siRNA targeting FOXP1 Exon 7 to increase the FOXP1S to FOXP1L ratio. (C) Mean fluorescence intensityÂąS.D. of surface CD19 determined by flow cytometric analysis of cells treated as in (B), both FOXP1 Ex7 targeting siRNAs significantly reduced CD19 expression (P<0.05) [n=6 (SU-DHL-4) or n=3 (U-2932)] while Ex13 targeting siRNA (total FOXP1si) did not; n=3 for both lines.

ed global miRNA targeting of FOXP1,31,33 could be one regulatory mechanism, but there are many possibilities. Our finding that the first methionine codon in hEx6b(L) enabling translation of FOXP1AL is not conserved in mouse must be considered in studies of murine lymphoma, and may also prove significant outside of lymphoma and B-cell biology. Particularly the established neuronal roles of FOXP1,42 emerging evidence for FOXP1 alterations in human behavioral disorders,43 and the proposed contribution of related FOXP2 sequence evolution to language acquisition44 indicate that neuronal studies of human-specific FOXP1AL may be warranted. Total FOXP1 depletion studies indicate a contribution to cell viability and regulation of developmental target genes such as PAX5 and PRDM1 in lymphoma cells.25,32,33,45 In addition, Dekker et al. have recently shown that total FOXP1 activates nearly all BCR-dependent genes; however, as FOXP1 silencing did not decrease BCR clustering, they concluded that it contributed to chronic B-cell receptor signaling (CABS) but only minimally to CABS-directed NF-kB activation.46 Our findings, haematologica | 2016; 101(7)

showing no decrease in CD19 expression upon total FOXP1 silencing, are consistent with these data. Interestingly, an siRNA that preferentially silenced FOXP1L was recently reported to be less effective at inducing cell death than total FOXP1 targeting in multiple DLBCL cell lines.45 While the same study generally observed stronger regulation of gene expression on silencing all FOXP1 isoforms, there were some exceptions.47 Our isoform-biased depletion studies presented here indicate that, in accordance with our previous findings (where the FOXP1 target gene HIP1R was preferentially regulated in ABC-DLBCL),22 and complimentary to some common functions,47 FOXP1L and FOXP1S have at least some distinct functional properties, including CD19 regulation. CD19 is a critical regulator of BCR signaling48 via both BCR-dependent and independent mechanisms, thus FOXP1 isoforms (rather than total expression levels) may contribute to surface BCR activity. Distinct long and short FOXP1 functions are highly likely to derive from altered protein-protein interactions at the N-terminus. Given that induction of FOXP1S expression, for example 869


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D

Figure 8. U-2932 DLBCL subpopulations display distinct FOXP1 expression patterns and maturation status. (A) Flow cytometric analysis of CD20 and CD38 expression in U-2932 parental (pre-sort sample) and subpopulations R1 and R2 sorted using gates as described previously.39 Phenotypes were stable while samples were harvested over a 2-week period. (B) Representative phase-contrast light microscopy of sorted cultures to show clumping behavior of R1 on the left, as exhibited by multiple other ABC-DLBCL lines (data not shown). (C) Representative JC12 immunoblot analysis of FOXP1 in nuclear extracts. (C) Real-time PCR analysis as indicated, expression relative to RIVA cell line, normalized to 18S; n=5ÂąS.D.

during B-cell activation, is associated with many other changes, the cellular environment is likely to play a crucial role in defining long and short FOXP1 functions. Overall, we favor a model in which temporally- or spatially-distinct FOXP1L and FOXP1S expression have some conserved functions but mediate also distinct isoform-specific functions during mature B-cell development and in lymphoma pathogenesis. CD19 regulation may provide a potential mechanism for the reported FOXP1 regulation of BCR signaling,46,49 and modulating FOXP1 could, therefore, have clinical benefit for therapies targeting CD19 in DLBCL, e.g. anti-CD19 chimeric antigen receptors.50 Studying expression, regulation and function of FOXP1 isoforms in relation to developmental blocks in DLBCL may also lead to the identification of novel therapeutic differentiation strategies for this disease.

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Acknowledgments The authors would like to acknowledge the important contributions from Dr Philip Brown, who sadly died before the completion of this study and drafting of the manuscript. We thank Dr Helen Ferry for cell sorting and Aengus Stewart at The Francis Crick Institute for analysis of HiSeq data. Funding PJB, DMG, LL, SLF and HS were supported by Specialist Program Grants (Ref:10044, 13047) to AHB from Bloodwise and the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre program. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. NM and DPC were supported by core funding from The Francis Crick Institute, and a MRC career development award MR/J008060/1 to DPC.

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Sci Signal. 2015;8(362):ra12. 26. Brown PJ, Ashe SL, Leich E, et al. Potentially oncogenic B-cell activationinduced smaller isoforms of FOXP1 are highly expressed in the activated B cell-like subtype of DLBCL. Blood. 2008; 111(5):2816-2824. 27. Pajer P, Pecenka V, Kralova J, et al. Identification of potential human oncogenes by mapping the common viral integration sites in avian nephroblastoma. Cancer Res. 2006;66(1):78-86. 28. Goatly A, Bacon CM, Nakamura S, et al. FOXP1 abnormalities in lymphoma: translocation breakpoint mapping reveals insights into deregulated transcriptional control. Mod Pathol. 2008;21(7):902-911. 29. Wlodarska I, Veyt E, De Paepe P, et al. FOXP1, a gene highly expressed in a subset of diffuse large B-cell lymphoma, is recurrently targeted by genomic aberrations. Leukemia. 2005;19(8):1299-1305. 30. Hu H, Wang B, Borde M, et al. Foxp1 is an essential transcriptional regulator of B cell development. Nat Immunol. 2006;7(8):819826. 31. Rao DS, O'Connell RM, Chaudhuri AA, et al. MicroRNA-34a perturbs B lymphocyte development by repressing the forkhead box transcription factor Foxp1. Immunity. 2010;33(1):48-59. 32. Sagardoy A, Martinez-Ferrandis JI, Roa S, et al. Downregulation of FOXP1 is required during germinal center B-cell function. Blood. 2013;121(21):4311-4320. 33. Craig VJ, Cogliatti SB, Imig J, et al. Mycmediated repression of microRNA-34a promotes high-grade transformation of B-cell lymphoma by dysregulation of FoxP1. Blood. 2011;117(23):6227-6236. 34. van Keimpema M, Gruneberg LJ, Mokry M, et al. FOXP1 directly represses transcription of proapoptotic genes and cooperates with NF-kappaB to promote survival of human B cells. Blood. 2014; 124(23):34313440. 35. Hardee J, Ouyang Z, Zhang Y, et al. STAT3 targets suggest mechanisms of aggressive tumorigenesis in diffuse large B-cell lymphoma. G3 (Bethesda). 2013;3(12):21732185. 36. Hooper C, Sainz-Fuertes R, Lynham S, et al. Wnt3a induces exosome secretion from primary cultured rat microglia. BMC Neurosci. 2012;13:144. 37. Wang B, Lin D, Li C, Tucker P. Multiple domains define the expression and regulatory properties of Foxp1 forkhead transcriptional repressors. J Biol Chem. 2003; 278(27):24259-24268. 38. Gabut M, Samavarchi-Tehrani P, Wang X,

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871


ARTICLE EUROPEAN HEMATOLOGY ASSOCIATION

Plasma Cell Disorders

Ferrata Storti Foundation

Analysis of renal impairment in MM-003, a phase III study of pomalidomide + low‐dose dexamethasone versus high‐dose dexamethasone in refractory or relapsed and refractory multiple myeloma

Katja C. Weisel,1 Meletios A. Dimopoulos,2 Philippe Moreau,3 Martha Q. Lacy,4 Kevin W. Song,5 Michel Delforge,6 Lionel Karlin,7 Hartmut Goldschmidt,8 Anne Banos,9 Albert Oriol,10 Adrian Alegre,11 Christine Chen,12 Michele Cavo,13 Laurent Garderet,14 Valentina Ivanova,15 Joaquin Martinez-Lopez,16 Stefan Knop,17 Xin Yu,18 Kevin Hong,18 Lars Sternas,18 Christian Jacques,18 Mohamed H. Zaki,18 and Jesus San Miguel19

Hematology and Oncology, Department of Medicine, University Hospital Tübingen, Germany; 2National and Kapodistrian University of Athens, Greece; 3Hematology, University Hospital Hôtel-Dieu, Nantes, France; 4Division of Hematology, Mayo Clinic, Rochester, MN, USA; 5Vancouver General Hospital, BC, Canada; 6Department of Hematology, University Hospitals Leuven, Belgium; 7Centre Hospitalier Lyon Sud/Hospices Civils de Lyon, Pierre-Bénite, France; 8Universitäts Klinikum Heidelberg, Germany; 9Hematology, Centre Hospitalier de la Côte Basque, Bayonne, France; 10Institut Catala d'Oncologia, Hospital Germans Trias I Pujol, Barcelona, Spain; 11Hospital Universitario La Princesa, Madrid, Spain; 12Princess Margaret Hospital, Toronto, ON, Canada; 13Bologna University School of Medicine, Institute of Hematology and Medical Oncology, Bologna, Italy; 14Hôpital Saint-Antoine, Paris, France; 15GUZ Moscow City Clinical Hospital S. P. Botkin, Moscow, Russia; 16Hospital Universitario 12 de Octubre, Madrid, Spain; 17Hematology and Oncology, Würzburg University Medical Center, Würzburg, Germany; 18Celgene Corporation, Summit, NJ, USA; and 19Clínica Universidad de Navarra, CIMA, IDISNA, Pamplona, Spain 1

Haematologica 2016 Volume 101(7):872-878

ABSTRACT

Correspondence: katja.weisel@med.uni-tuebingen.de

Received: December 15, 2015. Accepted: April 12, 2016. Pre-published: April 14, 2016. doi:10.3324/haematol.2015.137083

Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/101/7/872

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

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P

omalidomide + low-dose dexamethasone is effective and well tolerated for refractory or relapsed and refractory multiple myeloma after bortezomib and lenalidomide failure. The phase III trial MM-003 compared pomalidomide + low-dose dexamethasone with high-dose dexamethasone. This subanalysis grouped patients by baseline creatinine clearance ≥ 30 - < 60 mL/min (n=93, pomalidomide + low-dose dexamethasone; n=56, high-dose dexamethasone) or ≥ 60 mL/min (n=205, pomalidomide + low-dose dexamethasone; n=93, high-dose dexamethasone). Median progression-free survival was similar for both subgroups and favored pomalidomide + low-dose dexamethasone versus high-dose dexamethasone: 4.0 versus 1.9 months in the group with baseline creatinine clearance ≥ 30 - < 60 mL/min (P<0.001) and 4.0 versus 2.0 months in the group with baseline creatinine clearance ≥ 60 mL/min (P<0.001). Median overall survival for pomalidomide + low-dose dexamethasone versus high-dose dexamethasone was 10.4 versus 4.9 months (P=0.030) and 15.5 versus 9.2 months (P=0.133), respectively. Improved renal function, defined as an increase in creatinine clearance from < 60 to ≥ 60 mL/min, was similar in pomalidomide + low-dose dexamethasone and high-dose dexamethasone patients (42% and 47%, respectively). Improvement in progression-free and overall survival in these patients was comparable with that in patients without renal impairment. There was no increase in discontinuations of therapy, dose modifications, and adverse events in patients with moderate renal impairment. Pomalidomide at a starting dose of 4 mg + low-dose dexamethasone is well tolerated in patients with refractory or relapsed and refractory multiple myeloma, and of comparable efficacy if moderate renal impairment is present. This trial was registered with clinicaltrials.gov identifier 01311687 and EudraCT identifier 2010-019820-30. haematologica | 2016; 101(7)


Renal impairment in the phase III MM-003 study

Introduction

Methods

Renal impairment is a common presenting feature for patients with multiple myeloma (MM). Approximately 20% to 40% of patients with newly diagnosed MM, who are primarily aged over 65 years, have renal impairment at diagnosis, and this rate increases during the course of disease.1-4 Renal impairment as a complication of MM often leads to cast nephropathy, potentially resulting in renal tube atrophy and tubulointerstitial fibrosis.5,6 In addition, impaired renal function may be a result of aging and can also be caused by comorbidities unrelated to MM, such as diabetes, hypertension, vascular disease, or prior nonmyeloma therapies.3 Renal impairment in patients with MM is associated with shortened survival, but recovery of renal function during treatment may improve survival in these patients and even achieve similar outcomes in patients with a history of normal renal function.3,4,7 The past several decades have seen advances in the treatment of MM, and outcomes have improved for patients with varying degrees of renal impairment;3,8 despite this, most patients will ultimately relapse.9,10 Control of MM via effective therapy has been shown to improve renal function in a large proportion of patients.11,12 However, patients who are refractory to bortezomib and have relapsed following treatment with an immunomodulatory agent, or patients who are refractory to or ineligible to receive an immunomodulatory agent, have a poor prognosis (median survival 9 months).13 Physicians may perceive that treatment options in these patients may be further limited by the presence of renal impairment, as some novel agents rely on metabolism/excretion by the kidneys.7 Pomalidomide (POM) acts via direct antimyeloma, stromal-support inhibitory, and immunomodulatory effects.14,15 POM in combination with low-dose dexamethasone (LoDEX) was evaluated for the treatment of relapsed and refractory MM in patients after lenalidomide and bortezomib failure in the MM-002 and MM-003 studies. The phase I component of MM-002 established the maximum tolerated dose for POM at 4 mg daily with or without LoDEX (40 mg weekly).16 This dose was moved forward into an open-label, randomized, phase II component, which found POM + LoDEX to be more efficacious than POM alone [overall response rate (ORR) 33% vs. 18%; median progression-free survival (PFS) 4.2 vs. 2.7 months; median overall survival (OS) 16.5 vs. 13.6 months]. The pivotal, multicenter, open-label, randomized, phase III study MM-003 verified the safety and efficacy of POM + LoDEX (n=302) compared with high-dose dexamethasone (HiDEX; n=153) in patients with refractory or relapsed and refractory MM previously treated with (and 74% refractory to) lenalidomide and bortezomib.17 Overall trial results included significant improvements in ORR (31% vs. 10%; P<0.0001), PFS (4.0 vs. 1.9 months; P<0.0001), and OS (12.7 vs. 8.1 months; P=0.0285).17 Preliminary pharmacokinetic data suggest that renal impairment probably does not impact on POM exposure.18 The aim of the current analysis was to examine efficacy and safety of POM + LoDEX versus that of HiDEX in patient subgroups enrolled in MM-003 by renal function at baseline [creatinine clearance (CrCl) ≥ 30 - < 60 mL/min vs. ≥ 60 mL/min]. In addition, we examined median PFS and OS in patients who had improvement of renal function from CrCl ≥ 30 - < 60 mL/min at baseline to ≥ 60 mL/min at any point during study treatment.

MM-003 was an open-label, randomized, phase III trial conducted in 93 centers in Europe, Russia, Australia, Canada, and the United States (clinicaltrials.gov identifier: 01311687; EudraCT 2010019820-30). Full details have been described previously.17 All authors and the study sponsor were involved in data collection and analysis, review and interpretation of results, and the writing of the report.

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Patients Eligible patients were aged 18 years or over with refractory or relapsed and refractory MM, had measurable serum or urine M protein, and were refractory to their last prior treatment [documented progressive disease (PD) during or within 60 days of last therapy]. Prior bortezomib and lenalidomide treatment (≥ 2 consecutive cycles, alone or in combination) must have failed [i.e. refractory (never experienced a response), PD within six months after at least a partial response (PR), or intolerant (to bortezomib only)]. Adequate prior alkylator therapy was required. Exclusion criteria included: absolute neutrophil count < 1x109/L; platelets < 75x109/L (< 30x109/L if ≥ 50% of bone marrow nucleated cells were plasma cells); CrCl < 45 mL/min; peripheral neuropathy grade ≥ 2; prior exposure to POM; hypersensitivity to thalidomide, lenalidomide, or DEX; or resistance to DEX.

Study design and treatment Patients were randomized 2:1 to POM + LoDEX (28-day cycles; oral POM: 4 mg/day, days 1-21; LoDEX: 40 mg/day, days 1, 8, 15, and 22) or HiDEX (40 mg/day, days 1-4, 9-12, and 17-20). For patients aged over 75 years, DEX dose was reduced to 20 mg/day in both treatment arms. Patients continued treatment until PD or unacceptable toxicity. Thromboprophylaxis was required for all patients receiving POM and any patient at high risk of developing thrombosis. Patients provided written informed consent. The study received institutional review board or independent ethics committee approval at all participating centers according to the Declaration of Helsinki and the International Conference on Harmonisation Guidelines on Good Clinical Practice.

Subgroup analyses In this retrospective analysis, renal impairment cohorts included patients with moderate renal impairment (baseline CrCl ≥ 30 - < 60 mL/min) or without renal impairment (baseline CrCl ≥ 60 mL/min), based on the Cockroft-Gault formula.19,20 CrCl data were collected prospectively and were estimated at the start of each treatment cycle and upon discontinuation.

Assessments The primary end point was investigator-assessed PFS. Survival distribution functions for each treatment group were estimated with the Kaplan-Meier product-limit method and compared with the log-rank test. Key secondary end points included OS, ORR [≥ PR by International Myeloma Working Group (IMWG) criteria], safety [adverse events (AEs) graded according to National Cancer Institute Common Terminology Criteria for Adverse Events v.4.0], and improvement of renal function from CrCl ≥ 30 - < 60 mL/min at baseline to ≥ 60 mL/min at any point during study treatment (only assessed in patients with baseline and post-baseline CrCl data). As an additional retrospective analysis, renal response was assessed according to IMWG criteria.5 Efficacy was assessed in the intent-to-treat population (all patients randomized to treatment) using IMWG criteria, and tolerability was assessed in the safety population (all patients who received ≥ 1 dose of study drug). PFS 873


K.C. Weisel et al. Table 1. Baseline patients’ characteristics and prior therapies.

Patients’ characteristics

Median age (range), years Male sex, % Median time from initial diagnosis, years ECOG status 0/1/2/3, %a ISS stage at study entry I/II/III, %a Median prior treatments (range), n Prior LEN/BORT/DEX, % Prior SCT, % LEN refractory, % BORT refractory, % LEN and BORT refractory, % Presence of del(17p)/t(4;14), %

POM + LoDEX (n=93)

Baseline CrCl ≥ 30 - < 60 mL/min HiDEX (n=56)

POM + LoDEX (n=205)

Baseline CrCl ≥ 60 mL/min HiDEX (n=93)

69 (41-84) 46 5.5 39/38/23/0 11/38/51 5 (2-12) 100/100/99 60 99 77 76 30

69 (36-87) 45 5.8 16/59/21/4 11/28/61 5 (2-17) 100/100/100 57 89 84 79 34

61 (35-80) 67 5.3 36/50/14/0 36/42/22 5 (2-14) 100/100/97 76 93 80 74 23

61 (35-80) 65 6.2 30/56/13/1 33/45/22 5 (2-16) 100/100/99 79 94 76 71 17

BORT: bortezomib; CrCl: creatinine clearance; DEX: dexamethasone; ECOG: Eastern Cooperative Oncology Group; HiDEX: high-dose dexamethasone; ISS: International Staging System; LEN: lenalidomide; LoDEX: low-dose dexamethasone; POM: pomalidomide; SCT: stem cell transplant. aPercentages based on number of patients with data available.

and OS for patients with renal improvement were calculated according to European Medicines Evaluation Agency criteria. Statistical analyses were performed using SAS software v.9.2.

Results Patients' characteristics This analysis was performed using a data cut off of September 1st, 2013, corresponding to a median follow up of 15.4 months. A total of 302 patients received POM + LoDEX, and 153 patients received HiDEX (Online Supplementary Figure S1). Patients were heavily pre-treated, with a median of 5 prior lines of treatment in all groups. For patients with baseline CrCl ≥ 30 - < 60 mL/min, data were available for 93 POM + LoDEX and 56 HiDEX patients. For those with baseline CrCl ≥ 60 mL/min, data were available for 205 POM + LoDEX and 93 HiDEX patients. Eight patients were not included in the analysis: 3 patients had missing baseline CrCl values (2 in the POMLoDEX arm and 1 in the HiDEX arm), and 5 patients had baseline CrCl levels below 30 mL/min (2 in the POMLoDEX arm and 3 in the HiDEX arm) (Online Supplementary Figure S1). Patients with baseline CrCl ≥ 30 - < 60 mL/min were more likely to be older and less likely to have undergone prior SCT compared with patients with baseline CrCl ≥ 60 mL/min (Table 1). The moderate renal impairment cohort also exhibited higher rates of International Staging System stage II-III disease, Eastern Cooperative Oncology Group status of 3, and high-risk cytogenetics.

Efficacy Median PFS with POM + LoDEX versus HiDEX was consistent in patients with baseline CrCl ≥ 30 - < 60 mL/min (4.0 vs. 1.9 months; P<0.001) (Figure 1A) and ≥ 60 mL/min (4.0 vs. 2.0 months; P<0.001) (Figure 1B). POM + LoDEX also improved median OS compared with HiDEX in patients with baseline CrCl ≥ 30 - < 60 mL/min (10.4 vs. 4.9 months; P=0.030) (Figure 2A) and ≥ 60 mL/min (15.5 vs. 9.2 months; P=0.133) (Figure 2B). The OS advantage of POM + LoDEX over HiDEX was achieved despite a substantial pro874

portion of HiDEX patients receiving subsequent POM (50% of HiDEX patients with baseline CrCl ≥ 30 - < 60 mL/min and 60% of HiDEX patients with baseline CrCl ≥ 60 mL/min crossed over to POM + LoDEX). POM + LoDEX significantly improved ORR versus HiDEX regardless of baseline renal function (Table 2). Duration of response (for patients achieving ≥ PR) was consistently longer for POM + LoDEX versus HiDEX in both groups.

Improvement in renal function A total of 273 patients in the POM + LoDEX arm and 128 patients in the HiDEX arm had CrCl data for baseline and ≥ 1 post-baseline assessment and were thus evaluable for change in renal function (for patients with > 1 postbaseline assessment, the best value during the first 6 cycles was used). Renal improvement to CrCl ≥ 60 mL/min was noted in 42% of patients (33 of 79) treated with POM + LoDEX who had renal impairment (CrCl < 60 mL/min) at baseline (Table 3). Renal improvement was seen in 47% (20 of 43) of HiDEX-treated patients. The median time to improvement was similar for each treatment arm (POM + LoDEX: 1.0 month; HiDEX: 0.9 months). In these POM + LoDEX and HiDEX-treated patients with renal improvement, median PFS was 6.5 (95%CI: 4.6, 8.4) and 3.2 (95%CI: 2.1, 5.5) months, respectively; median OS was 12.6 (95%CI: 7.6, 25.7) and 10.1 (95%CI: 5.7, 17.7) months, respectively. According to IMWG criteria for renal response,5 32% (14 of 44) of POM + LoDEX-treated patients with estimated glomerular filtration rate (eGFR) < 50 mL/min/1.73 m2 achieved complete response of CrCl ≥ 60 mL/min (Table 3). In a similar analysis of the HiDEX-treated patients, 43% (9 of 21) achieved a complete response. Few or no patients in either treatment arm were eligible for partial or minimal response by having baseline eGFR < 30 mL/min/1.73 m2.

Safety The AE profiles for POM + LoDEX and HiDEX were similar across both renal function subgroups (Table 4). The most common grade 3/4 AEs for the POM + LoDEX treatment arm were neutropenia (48% for baseline CrCl ≥ 30 haematologica | 2016; 101(7)


Renal impairment in the phase III MM-003 study < 60 mL/min and 49% for baseline CrCl ≥ 60 mL/min), anemia (40% and 30%, respectively), and infections (31% and 34%, respectively). With mandatory thromboprophylaxis, incidence of grade 3/4 deep vein thrombosis/pulmonary embolism was low (≤ 2% in both groups). POM discontinuations and dose modifications due to AEs were similar regardless of moderate renal impairment (Table 5). Median duration of POM treatment was similar in patients with baseline CrCl ≥ 30 - < 60 mL/min (3.7 months) and ≥ 60 mL/min (4.6 months). Renal function did not affect frequency of dose reductions and interruptions. Median relative dose intensity was consistent at 90% for both renal function subgroups.

Discussion POM + LoDEX was efficacious and well tolerated in patients with refractory or relapsed and refractory MM and moderate renal impairment. POM + LoDEX significantly extended PFS versus HiDEX for all patients regardless of renal impairment (baseline CrCl ≥ 30 - < 60 mL/min, 4.0 vs. 1.9 months; baseline CrCl > 60 mL/min, 4.0 vs. 2.0 months; P<0.001 for both groups), similar to the benefits observed in the general study population (4.0 vs. 1.9 months; P<0.001).17 OS results showed a similar 5- to 6-month benefit for POM + LoDEX in both renal function subgroups (baseline CrCl ≥ 30 - < 60 mL/min, 10.4 vs. 4.9

Proportion of Subjects

A

Proportion of Subjects

B

Figure 1. Progression-free survival for patients with baseline creatinine clearance ≥ 30 - < 60 mL/min (A) or ≥60 mL/min (B).

Table 2. Response to treatment (IMWG criteria).

Variable

ORR (≥ PR), % sCR CR VGPR PR SD, % Median DOR, monthsa

POM + LoDEX (n=93)

Baseline CrCl ≥ 30 - < 60 mL/min HiDEX (n=56)

POM + LoDEX (n=205)

Baseline CrCl ≥ 60 mL/min HiDEX (n=93)

28 0 0 7 22 52 6.6

11 0 0 0 11 48 4.4

34 1 2 5 26 50 7.5

12 0 0 1 11 50 5.1

CR: complete response; CrCl: creatinine clearance; DOR: duration of response; HiDEX: high-dose dexamethasone; IMWG: International Myeloma Working Group; LoDEX: low-dose dexamethasone; ORR: overall response rate; POM: pomalidomide; PR: partial response; sCR: stringent complete response; SD: stable disease;VGPR: very good partial response. aFor patients with ≥ PR.

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months; baseline CrCl > 60 mL/min, 15.5 vs. 9.2 months) compared with those of the overall patient population (13.1 vs. 8.2 months),17 although these results were only statistically significant for patients with reduced renal function (baseline CrCl ≥ 30 - < 60 mL/min, P=0.030; baseline CrCl > 60 mL/min, P=0.133). This finding is likely to

be confounded by the high number (56% overall) of HiDEX patients crossing over to receive POM after progression, as per protocol. A substantial number of renally impaired patients treated with POM + LoDEX had improved renal function (42% per protocol criteria and 32% per IMWG criteria), which

A

B

Figure 2. Overall survival for patients with baseline creatinine clearance ≥ 30 - < 60 mL/min (A) or ≥ 60 mL/min (B).

Table 3. Improvement in renal function.

Category Per-protocol improvement IMWG CRrenal IMWG PRrenal IMWG MRrenal

Baseline renal function CrCl < 60 mL/min eGFR < 50 mL/min/1.73 m2 eGFR < 15 mL/min/1.73 m2 eGFR < 15 mL/min/1.73 m2 eGFR 15-29 mL/min/1.73 m2

Best renal function

CrCl ≥ 60 mL/min CrCl ≥ 60 mL/min CrCl 30-59 mL/min CrCl 15-29 mL/min CrCl 30-59 mL/min

POM + LoDEX, n/N (%)a

HiDEX, n/N (%)a

33/79 (42) 14/44 (32) 0/3 0/0

20/43 (47) 9/21 (43) 0/1 0/0

CrCl: creatinine clearance; CRrenal: complete renal response; eGFR: estimated glomerular filtration rate; HiDEX: high-dose dexamethasone; IMWG: International Myeloma Working Group; LoDEX: low-dose dexamethasone; MRrenal: minimal renal response; POM: pomalidomide; PRrenal: partial renal response. aPercentages represent patients who improved from baseline to most extreme post-baseline value, divided by the total number of patients with evaluable baseline and post-baseline data.

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Renal impairment in the phase III MM-003 study

was similar to the improvement rate noted in the HiDEX arm. HiDEX treatment can rapidly suppress M-protein and light-chain excretion leading to recovery of renal function and can be used for acute myeloma treatment.12,21,22 In the present study, however, response rates in renally impaired patients greatly favored the POM+LoDEX arm compared with the HiDEX arm: 28% versus 11%, respectively. In patients treated with POM + LoDEX whose kidney function improved from moderate impairment to normal, PFS reached 6.5 months, which exceeded the results of patients with normal baseline kidney function (4.0 months). Despite a similar proportion of patients with renal improvement noted in the HiDEX arm, their PFS was only 3.2 months. The OS improvement observed in these patients was in the same range as that in patients with normal renal function. Tolerability profiles were consistent regardless of baseline renal function. Rates of discontinuation due to AEs were similar in both subgroups, indicating that patients with moderate renal impairment did not experience increased toxicity. Slightly increased incidences of pneumonia were observed in patients with baseline CrCl ≥ 30 - < 60 mL/min. Duration of treatment and dose intensity were not affected by baseline renal function. These findings demonstrate that up-front dose modification is not required in patients with moderate renal impairment, and

that 4 mg is a safe and appropriate starting dose of POM in combination with LoDEX for these patients. The efficacy results of POM + LoDEX in renal function subgroups of MM-003 confirm those found previously. In the phase II component of MM-002, patients without renal impairment (baseline CrCl > 60 mL/min) had an ORR of 34%, median PFS of 5.4 months, and median OS of 16.9 months, and patients with moderate renal impairment (baseline CrCl ≥ 45 to ≤ 60 mL/min) had an ORR of 43%, median PFS of 4.7 months, and median OS of 19.5 months. It should be noted, however, that there were only 14 patients in this subgroup in that study.23 Safety profiles and relative dose intensities for these subgroups were consistent between the phase II study and the one presented here.23 This cumulative body of evidence regarding POM + LoDEX further supports use of a 4-mg starting dose for patients with mild or moderate renal impairment without up-front dose reduction. The finding that POM does not require dose adjustment in patients with moderate renal impairment versus patients with normal renal function is related to its metabolism and excretion. In contrast to lenalidomide, which is excreted primarily via the kidneys,24 POM is extensively metabolized (with only 2.2% excreted as the parent drug in urine) and, therefore, does not require dose reductions in patients with impaired renal function;18,25 the same obser-

Table 4. Safety profile.

Event

Baseline CrCl ≥ 30 - < 60 mL/min POM + LoDEX HiDEXa (n=93) (n=56)

Grade 3/4 hematologic AEs in ≥ 10% of pts, % Neutropenia Anemia Thrombocytopenia Febrile neutropenia Grade 3/4 non-hematologic AEs in ≥ 10% of pts, % Infections Pneumonia Grade 3/4 AEs of interest, % DVT/PE Peripheral neuropathyb Discontinuation due to AEs, %

Baseline CrCl ≥ 60 mL/min POM + LoDEX HiDEXa (n=203) (n=90)

48 40 22 5

21 46 38 0

49 30 23 11

16 34 20 0

31 19

27 9

34 11

24 8

1 1 13

0 2 11

2 2 8

0 1 10

AE: adverse event; CrCl: creatinine clearance; DVT: deep vein thrombosis; HiDEX: high-dose dexamethasone; LoDEX: low-dose dexamethasone; PE: pulmonary embolism; POM: pomalidomide; pt: patient. aPatients may have received POM + LoDEX after crossover. bPeripheral neuropathy includes the preferred terms “hyperesthesia,”“neuropathy peripheral,” “peripheral sensory neuropathy,”“paresthesia,”“hypoesthesia,” and “polyneuropathy.”

Table 5. Pomalidomide dose modification due to adverse events and dose intensity.

Variable

POM dose modifications due to AEs, % Interruption Reduction Discontinuation POM dose intensity Planned POM dose/day, mg Median relative dose intensity (range)a Median duration of treatment (range), months

Baseline CrCl ≥ 30 - < 60 mL/min (n=93)

Baseline CrCl ≥ 60 mL/min (n=203)

69 28 12

67 28 7

4 0.9 (0.4-1.3) 3.7 (0.1-21.4)

4 0.9 (0.3-1.2) 4.6 (0.1-25.6)

AEs: adverse event; CrCl: creatinine clearance; POM: pomalidomide. aRelative dose intensity = dose intensity/planned dose intensity. May be more than 1 due to patient discontinuation prior to end of 28-day cycle.

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K.C. Weisel et al. vation applies to thalidomide.26,27 The safety profile of POM + LoDEX as assessed in the study presented here demonstrated no difference in frequency of dose reductions in patients with moderate renal impairment and only slightly higher rates of anemia and pneumonia. However, this analysis was limited by the fact that it concerns only patients with normal or moderately impaired renal function, as the study excluded patients with CrCl < 45 mL/min at the time of screening [although 28 (9%) POM + LoDEX and 15 (10%) HiDEX patients had baseline CrCl below this cut off due to the time that had elapsed between screening and the first treatment cycle]. To address this, 2 trials are in progress to assess the use of POM + LoDEX in patients with severe renal impairment, including those requiring dialysis: MM-008 in the United States (clinicaltrials.gov identifier 01575925) and MM-013 in the European Union (clinicaltrials.gov identifier 02045017). Newer treatment options, including lenalidomide, thalidomide, and bortezomib, have improved outcomes and survival for many patients with MM in recent years.28,29 In addition, although newer agents can improve

References 1. Alexanian R, Barlogie B, Dixon D. Renal failure in multiple myeloma. Pathogenesis and prognostic implications. Arch Intern Med. 1990;150(8):1693-1695. 2. Kyle RA, Gertz MA, Witzig TE, et al. Review of 1027 patients with newly diagnosed multiple myeloma. Mayo Clin Proc. 2003;78(1):21-33. 3. Dimopoulos MA, Delimpasi S, Katodritou E, et al. Significant improvement in the survival of patients with multiple myeloma presenting with severe renal impairment after the introduction of novel agents. Ann Oncol. 2014;25(1):195-200. 4. Knudsen LM, Hjorth M, Hippe E. Renal failure in multiple myeloma: reversibility and impact on the prognosis. Nordic Myeloma Study Group. Eur J Haematol. 2000;65(3): 175-181. 5. Dimopoulos MA, Terpos E, Chanan-Khan A, et al. Renal impairment in patients with multiple myeloma: a consensus statement on behalf of the International Myeloma Working Group. J Clin Oncol. 2010; 28(33):4976-4984. 6. Suzuki K. Diagnosis and treatment of multiple myeloma and AL amyloidosis with focus on improvement of renal lesion. Clin Exp Nephrol. 2012;16(5):659-671. 7. Chanan-Khan AA, San Miguel JF, Jagannath S, Ludwig H, Dimopoulos MA. Novel therapeutic agents for the management of patients with multiple myeloma and renal impairment. Clin Cancer Res. 2012; 18(8):2145-2163. 8. Uttervall K, Duru AD, Lund J, et al. The use of novel drugs can effectively improve response, delay relapse and enhance overall survival in multiple myeloma patients with renal impairment. PLoS One. 2014; 9(7):e101819. 9. Attal M, Harousseau JL, Leyvraz S, et al. Maintenance therapy with thalidomide improves survival in patients with multiple myeloma. Blood. 2006;108(10):3289-3294. 10. Kumar SK, Rajkumar SV, Dispenzieri A, et al. Improved survival in multiple myeloma and the impact of novel therapies. Blood.

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outcomes, including renal function, for many patients with MM with renal impairment,5,7 it remains a significant risk factor for early death in MM.3,30 Thus, the efficacy of POM + LoDEX in renally impaired relapsed/refractory MM populations is of particular importance. This analysis has demonstrated that a starting dose of POM 4 mg may be used safely regardless of moderate renal impairment, with no unexpected toxicities observed and no additional dose modifications or discontinuations required when compared with the overall trial population. PFS and OS benefits achieved by patients with moderate renal impairment were also consistent with the overall MM-003 trial population. Acknowledgments The authors would like to thank the patients who took part in the trial, clinical staff at the participating study sites, and representatives of the sponsors who were involved in data gathering and analyses. We also thank Peter Simon, PhD, and Nicola Hanson, PhD, of MediTech Media for writing assistance, which was funded by Celgene Corporation.

2008;111(5):2516-2520. 11. Bladé J, Rosiñol L. Renal, hematologic and infectious complications in multiple myeloma. Best Pract Res Clin Haematol. 2005; 18(4):635-652. 12. Kastritis E, Anagnostopoulos A, Roussou M, et al. Reversibility of renal failure in newly diagnosed multiple myeloma patients treated with high dose dexamethasone-containing regimens and the impact of novel agents. Haematologica. 2007; 92(4):546-549. 13. Kumar SK, Lee JH, Lahuerta JJ, et al. Risk of progression and survival in multiple myeloma relapsing after therapy with IMiDs and bortezomib: a multicenter International Myeloma Working Group study. Leukemia. 2012;26(1):149-157. 14. Quach H, Ritchie D, Stewart AK, et al. Mechanism of action of immunomodulatory drugs (IMiDs) in multiple myeloma. Leukemia. 2010;24(1):22-32. 15. Mark TM, Coleman M, Niesvizky R. Preclinical and clinical results with pomalidomide in the treatment of relapsed/refractory multiple myeloma. Leuk Res. 2014;38(5):517-524. 16. Richardson PG, Siegel D, Baz R, et al. Phase 1 study of pomalidomide MTD, safety, and efficacy in patients with refractory multiple myeloma who have received lenalidomide and bortezomib. Blood. 2013;121(11):19611967. 17. San Miguel J, Weisel K, Moreau P, et al. Pomalidomide plus low-dose dexamethasone versus high-dose dexamethasone alone for patients with relapsed and refractory multiple myeloma (MM-003): a randomised, open-label, phase 3 trial. Lancet Oncol. 2013;14(11):1055-1066. 18. Hoffmann M, Kasserra C, Reyes J, et al. Absorption, metabolism and excretion of [14C]pomalidomide in humans following oral administration. Cancer Chemother Pharmacol. 2013;71(2):489-501. 19. Cockcroft DW, Gault MH. Prediction of creatinine clearance from serum creatinine. Nephron. 1976;16(1):31-41. 20. Luke DR, Halstenson CE, Opsahl JA, Matzke GR. Validity of creatinine clearance estimates in the assessment of renal function. Clin

Pharmacol Ther. 1990;48(5):503-508. 21. Bayraktar UD, Warsch S, Pereira D. Highdose glucocorticoids improve renal failure reversibility in patients with newly diagnosed multiple myeloma. Am J Hematol. 2011;86(2):224-227. 22. Ishikawa H, Tanaka H, Iwato K, et al. Effect of glucocorticoids on the biologic activities of myeloma cells: inhibition of interleukin-1 beta osteoclast activating factor-induced bone resorption. Blood. 1990;75(3):715-720. 23. Siegel DS, Richardson PG, Baz R, Chen M, Zaki M, Anderson KC. Pomalidomide (POM) with low-dose dexamethasone (LoDEX) in patients with relapsed and refractory multiple myeloma (RRMM): impact of renal function on patient outcomes. Blood. 2012;120(21):4072. 24. Chen N, Lau H, Kong L, et al. Pharmacokinetics of lenalidomide in subjects with various degrees of renal impairment and in subjects on hemodialysis. J Clin Pharmacol. 2007;47(12):1466-1475. 25. Pomalyst® (pomalidomide) [package insert]. Summit, NJ: Celgene Corporation; 2015. 26. Thalomid® (thalidomide) [package insert]. Summit, NJ: Celgene Corporation; 2013. 27. Eriksson T, Hoglund P, Turesson I, et al. Pharmacokinetics of thalidomide in patients with impaired renal function and while on and off dialysis. J Pharm Pharmacol. 2003;55(12):1701-1706. 28. Liwing J, Uttervall K, Lund J, et al. Improved survival in myeloma patients: starting to close in on the gap between elderly patients and a matched normal population. Br J Haematol. 2014;164(5):684-693. 29. Pulte D, Gondos A, Brenner H. Improvement in survival of older adults with multiple myeloma: results of an updated period analysis of SEER data. Oncologist. 2011;16(11):1600-1603. 30. Augustson BM, Begum G, Dunn JA, et al. Early mortality after diagnosis of multiple myeloma: analysis of patients entered onto the United Kingdom Medical Research Council trials between 1980 and 2002: Medical Research Council Adult Leukaemia Working Party. J Clin Oncol. 2005;23(36): 9219-9226.

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ARTICLE

Stem Cell Transplantation

Clinical activity of azacitidine in patients who relapse after allogeneic stem cell transplantation for acute myeloid leukemia

EUROPEAN HEMATOLOGY ASSOCIATION

Ferrata Storti Foundation

Charles Craddock,1 Myriam Labopin,2 Marie Robin,3 Juergen Finke,4 Patrice Chevallier,5 Ibrahim Yakoub-Agha,6 Jean Henri Bourhis,7 Henrik Sengelov,8 Didier Blaise,9 Thomas Luft,10 Michael Hallek,11 Nicolaus Kröger,12 Arnon Nagler,*13 and Mohamad Mohty*14

1 Centre for Clinical Haematology, Queen Elizabeth Hospital, Birmingham, UK; 2Faculté de Médecine Saint Antoine Hospital, Paris, France; 3Hôpital Saint-Louis, Assistance Publique Hôpitaux de Paris, France; 4Freiburg University Medical Centre, Germany; 5 Centre Hospitalier et Universitaire, Nantes, France; 6Institut de Cancerologie Gustave Roussy, Villejuif, France; 7Division of Hematology, Institut Gustav Roussy, Villejuif, France; 8Department of Hematology, National University Hospital, Rigshospitalet, Copenhagen, Denmark; 9Hematology Department, Paoli Calmettes Institute, Marseille, France; 10Department Medicine V, University of Heidelberg, Germany; 11Department I of University Medicine, University of Cologne, Germany; 12Department of Bone Marrow Transplantation, University Hospital of Hamburg-Eppendorf, Hamburg, Germany; 13 Chaim Sheba, Medical Centre, Tel Hasomer, Israel; and 14Faculté de Médecine Saint Antoine Hospital, Paris, France

*AN and MM contributed equally to this work

Haematologica 2016 Volume 101(7):879-883

ABSTRACT

D

isease relapse is the most common cause of treatment failure after allogeneic stem cell transplantation for acute myeloid leukemia and myelodysplastic syndromes, yet treatment options for such patients remain extremely limited. Azacitidine is an important new therapy in high-risk myelodysplastic syndromes and acute myeloid leukemia but its role in patients who relapse post allograft has not been defined. We studied the tolerability and activity of azacitidine in 181 patients who relapsed after an allograft for acute myeloid leukemia (n=116) or myelodysplastic syndromes (n=65). Sixtynine patients received additional donor lymphocyte infusions. Forty-six of 157 (25%) assessable patients responded to azacitidine therapy: 24 (15%) achieved a complete remission and 22 a partial remission. Response rates were higher in patients transplanted in complete remission (P=0.04) and those transplanted for myelodysplastic syndromes (P=0.023). In patients who achieved a complete remission, the 2-year overall survival was 48% versus 12% for the whole population. Overall survival was determined by time to relapse post transplant more than six months (P=0.001) and percentage of blasts in the bone marrow at time of relapse (P=0.01). The concurrent administration of donor lymphocyte infusion did not improve either response rates or overall survival in patients treated with azacitidine. An azacitidine relapse prognostic score was developed which predicted 2-year overall survival ranging from 3%-37% (P=0.00001). We conclude that azacitidine represents an important new therapy in selected patients with acute myeloid leukemia/myelodysplastic syndromes who relapse after allogeneic stem cell transplantation. Prospective studies to confirm optimal treatment options in this challenging patient population are required. Introduction Allogeneic stem cell transplantation (SCT) is an important curative option in patients with high-risk acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). The major cause of treatment failure remains disease relapse, which occurs in 40%-70% of patients.1,2 Little progress has been made to date in haematologica | 2016; 101(7)

Correspondence: charles.craddock@uhb.nhs.uk

Received: January 7, 2016. Accepted: April 12, 2016. Pre-published: April 14, 2016. doi:10.3324/haematol.2015.140996

Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/101/7/879

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

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developing effective treatment options for patients with recurrent disease after allogeneic SCT, and the great majority remain destined to die of resistant disease.3 Although a small number of patients with disease recurrence can survive long term after a second transplant or donor lymphocyte infusion (DLI), the success of both these treatment modalities is contingent on the prior acquisition of morphological remission with salvage therapy.4,5 At present, the only established salvage option for patients who relapse post allograft is intensive chemotherapy which is variably effective, with reported complete response (CR) rates of 15%-30%, and is often poorly tolerated in this heavily pre-treated population patients.3-5 The low rates of response to myelosuppressive chemotherapy, coupled with its substantial toxicity and requirement for lengthy hospitalization, makes the identification of more effective and better tolerated re-induction therapies for patients relapsing after allogeneic SCT a significant unmet clinical need. Azacitidine (AZA) is a DNA methyltransferase inhibitor (DNMTI) which demonstrates significant clinical activity in patients with AML and high-risk MDS.6,7 The mechanism of the anti-tumor activity of AZA has not been determined but may be due to its ability to reverse epigenetically silenced pro-apoptotic pathways. AZA also has the capacity to up-regulate the expression of epigenetically silenced tumor antigens, and can induce a CD8+ T-cell response to tumor antigens post transplant, raising the possibility that it may have the potential to augment a graft-versus-leukemia (GvL) response.8,9 A number of small series have reported that AZA can induce remissions in patients who relapse after an allogeneic transplant, raising the possibility that this agent represents a potential new treatment strategy in this challenging patient population.1012 Furthermore, it has been suggested in single arm studies that AZA may augment the anti-tumor activity of DLI in patients who relapse after an allograft.13 However, importantly, so far there has been no systematic analysis of the clinical activity of AZA in patients who have relapsed after an allograft for AML or MDS, or of whether its activity is increased by the concurrent administration of DLI.

Table 1. Patients' characteristics. Diagnosis Patient sex Donor sex Female to male Donor Number of transplant before relapse TBI Conditioning Source of SC CMV patient

CMV donor CMV donor/patient

Cytogenetics in AML

Cytogenetics in MDS

AML MDS Male Female Male Female No Yes Identical sibling Matched unrelated Mismatched relative First Second allograft No Yes MAC RIC BM PB Negative Positive Missing Negative Positive Missing Neg to neg Pos to neg Neg to pos Pos to pos Missing Good Intermediate Adverse N/A Good Intermediate Adverse NA

N

%

116 65 111 70 119 59 142 36 72 105 4 161 20 142 39 46 135 18 163 71 109 1 91 89 1 50 20 41 68 2 6 58 39 13 22 25 13 5

64.1 35.1 61.3 33.1 66.9 34.9 79.8 20.2 39.8 58.0 2.2 89.0 11.0 78.5 21.5 25.4 74.6 9.9 90.1 39.4 60.6 50.6 49.4 27.9 11.2 22.9 38.0 5.2 50.0 33.6 36.7 41.7 21.7

AML: acute meyloid leukemia; MDS: myelodysplastic syndromes; TBI: total body irradiation; SC: stem cells; CMV: cytomegalovirus; MAC: myeloablative conditioning; RIC: reduced intensity conditioning; BM: bone marrow; PB: peripheral blood; NA: not applicable.

Methods Patients The study cohort was made up of 181 patients from the EBMT database who had received AZA for the treatment of morphological disease relapse between 2006 and 2010 after an allogeneic stem cell transplantation for AML or MDS. Only patients who received AZA within one month of disease recurrence were included. Patients who had received prophylactic or pre-emptive post-transplant AZA were specifically excluded from this analysis. Study patients' characteristics are summarized in Table 1; 116 patients had undergone transplantation for AML and 65 for MDS. Median time from transplant to relapse was eight months (range 1-71 months); 72 patients relapsed within six months of transplant, 52 between six and 12 months post transplant, and 57 more than 12 months post transplant. In patients who had been transplanted as treatment for AML, presentation cytogenetics were classified as either good, intermediate or adverse risk according to the Medical Research Council (MRC) criteria14 and in patients transplanted for MDS utilizing the International Prognostic Scoring System.15 Seventy-two patients were transplanted using a matched sibling donor and 109 from an adult unrelated donor. 880

Forty-six patients received a myeloablative conditioning (MAC) regimen and 135 patients received a reduced intensity (RIC) regimen according to EBMT criteria.16

Azacitidine therapy Azacitidine was administered at a dose of 75 mg/m2 for 5-7 consecutive days every month. Median time from relapse to commencement of AZA was 11 days (range 1-30 days). The median duration of AZA treatment was 53 days (2-1196 days) and the median total AZA dose delivered to the study population was 1050 mg/m2 (75-10,500 mg/m2). Sixty-nine patients received DLI in addition to AZA treatment, 39 of whom received DLI within two months of commencing AZA salvage and in the absence of a clinical response. Thirty-five patients proceeded to a second allogeneic transplant after AZA salvage therapy at a median of 119 days (range 12-1183 days) after the commencement of salvage AZA. Twenty-four patients were allografted before an assessment of response to AZA salvage was made (median 82 days), 6 of whom were transplanted after acquisition of a major response [complete response/partial response (CR/PR)] following AZA therapy and 5 after loss of a major response to AZA. haematologica | 2016; 101(7)


Azacitidine in relapsed AML post transplant

Non-hematologic toxicities were graded using the National Cancer Institute's Common Terminology Criteria for Adverse Events (http://ctep.cancer.gov/protocoldevelopment/electronic_applications/ctc.htm). Hematologic toxicity was not assessable in the presence of active leukemia.

Response criteria Response to AZA was assessed by conventional morphological criteria and evaluated in patients who had received AZA alone or AZA in combination with DLI. CR was defined as acquisition of less than 5% blasts on bone marrow assessment. PR was defined using the criteria defined by Cheson et al.17

Statistical analysis The primary end point of the study was overall survival (OS). Secondary end points were response rate (CR or PR). Outcome parameters were measured from the date of commencement of AZA. Cumulative incidence curves were used to estimate CR and response rate, as death was a competing event.18 The probability of OS was calculated using the Kaplan-Meier method. Two Cox proportional hazards models were developed, including or not DLI within two months as a time-dependent variable. Fixed variables were diagnosis (AML or MDS), cytogenetics at time of diagnosis, time to relapse, the percentage of blasts in bone marrow at time of relapse, chronic GvHD before AZA, DLI before AZA, and year of transplantation. A stepwise procedure was then used for selection of variables with a P value of 0.05. The two remaining significant fixed factors, time interval from transplant to relapse and percentage of blasts in bone marrow at time of relapse, were used to develop a risk score in an additive way. Twenty-four patients who received AZA and proceeded to a second transplant were excluded from the prognostic factor analysis.

Results Tolerability of AZA in patients who have relapsed post allograft Fifty-two patients developed grade 3-4 non-hematologic toxicity during the course of AZA administration. Eighteen patients developed Grade 3-4 sepsis/infectiousrelated complications, 15 patients Grade 3-4 pneumonia, and 7 patients Grade 3-4 liver/gastrointestinal toxicity. It is likely that disease-related neutropenia made a substantial contribution to infection-related toxicities. Thirteen patients developed Grade 2-4 GvHD after AZA therapy, of whom 7 had received prior DLI. Five of the 112 patients who did not receive DLI developed Grade 2-4 GvHD. One patient developed GvHD after DLI followed by a second transplant.

Overall survival after AZA salvage therapy The median follow up after commencement of AZA therapy was 24 months (range 2-72 months). At the time of latest follow up, 163 patients had died: 130 of disease relapse, 25 of infection-related complications, 5 of GvHD, and 3 of miscellaneous transplant-related complications. Eighteen patients are still alive. The 2-year OS for the whole group was 12.4%. The 2year OS in patients achieving a CR after AZA salvage was 48.4%, and 28.7% in patients with a major response. In multivariable analysis, the following factors were predictive of 2-year OS at the time of relapse (Table 3): time to relapse 6-12 months versus less than six months [HR 0.51: (0.35-0.76); P=0.001] or more than 12 months [HR 0.29 (0.19-0.44); P=<10-4), respectively, and blasts in bone marrow greater than median (20%) at time of relapse [HR 1.5 (1.1-2.13); P=0.012].

Table 2. Multivariable analysis of factors determining acquisition of a major clinical response in patients relapsing after an allogeneic transplant for acute myeloid leukemia or myelodysplastic syndrome who were treated with azacitidine.

P Diagnosis AML vs. MDS CR at transplant

0.023 0.04

HR 0.48 1.92

95% CI inf

sup

0.26 1.03

0.90 3.58

AML: acute myeloid leukemia; MDS: myelodysplastic syndrome; CR: complete response.

Table 3. Multivariable analysis of factors determining 2-year overall survival after azacitidine treatment.

P Interval SCT-relapse < 6 mo (Ref) 6-12 mo vs. <6 mo 0.001 >12 mo vs. <6 mo <10-4 Blasts in BM at relapse 0.012 >median

HR

0.51 0.29 1.53

inf

95% CI sup

1.00 0.35 0.19 1.10

0.76 0.44 2.14

SCT: stem cell transplantation; mo: months; BM: bone marrow; Ref: reference.

Clinical response to AZA salvage therapy Forty-six of 157 (29.3%) assessable patients treated with AZA or AZA and DLI in combination demonstrated a major response (CR/PR) to AZA salvage therapy. Twenty-four (15.3%) patients achieved a CR and 22 (14%) a PR. Median time to achieve a CR after commencement of AZA was 108 days. In multivariate analysis, transplantation for MDS as opposed to AML was associated with a higher probability of achieving a major response [HR 0.48 (0.26-0.90); P=0.023] and transplantation in CR [HR 1.92 (1.03-3.58); P=0.04] (Table 2). The additional administration of DLI within two months of AZA administration was analyzed as a time-dependent variable and had no impact on OS [HR=1.04 (95%CI: 0.67-1.61); P=0.86]. haematologica | 2016; 101(7)

Figure 1. Two-year overall survival after azacitidine therapy in patients who relapsed after an allogeneic transplant for acute myeloid leukemia or myelodysplastic syndrome according to the AZA Relapse Prognostic Score.

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In a time-dependent multivariable analysis (Table 4), the following factors were predictive of 2-year OS: time to relapse post allograft 6-12 months versus less than six months [HR 0.5 (0.33-0.77); P=0.001] and more than 12 months, respectively [HR 0.29 (0.19-0.44); P=<10-5] and blasts in bone marrow greater than median (20%) at time of relapse [HR1.52 (1.08-2.15); P=0.02]. The administration of DLI within two months of commencement of AZA salvage had no impact on either the probability of achieving a major response or 2-year OS.

The development of the AZA Relapse Prognostic Score Using factors previously identified to determine survival after AZA therapy, it was possible to create a scoring system based on interval from transplant to relapse and blast percentage in the bone marrow at the time of relapse. In the AZA Relapse Prognostic Score (ARPS), the interval from transplant to relapse was assigned 2 points if less than six months, 1 if 6-12 months, and 0 if greater than 12 months. A blast percentage greater than the median (20%) at relapse was assigned 1 point. Utilizing the ARPS predicted the likelihood of achieving both a CR and major response as well as 2-year OS after AZA salvage therapy (Table 5 and Figure 1).

Table 4. Time-dependant multivariable analysis of factors determining 2-year overall survival after azacitibine treatment.

Blast in BM at relapse>median Interval SCT-relapse <6 mo (Ref) 6-12 mo vs. <6 mo >12 mo vs. <6 mo

P

HR

95% CI sup inf

0.02

1.52

1.08

2.15

0.001 <10-5

1.00 0.50 0.29

0.33 0.19

0.77 0.44

BM: bone marrow; SCT: stem cell transplantation; mo: months; Ref: reference.

Table 5. Two-year overall survival of 181 patients who relapsed after an allogeneic transplant for acute myeloid leukemia or myelodysplastic syndrome treated with azacitibine according to the AZA Relapse Prognostic Score.

Risk score

2-year OS after AZA

Major response (CR/PR after AZA)

Response (CR after AZA)

0 (n=33) 1 (n=51) 2-3 (n=97)

37.2% [19.4-55] 15% [4.8-25.3] 3.1% [0-6.5] P=<0.00001

48.5% [30.3-64.5] 27.9% [16.2-40.9] 16.5% [9.9-24.6] P=0.0017

33.9% [18.1-50.3] 14% [6-25.2] 8.2% [3.9-14.9] P=0.0019

AZA: azacitibine; CR: complete response; PR: partial response; mo: months. Scoring based on time interval of transplant to relapse (< 6 mo. = 2 points, 6-12 mo. = 1 point, >12 mo. = 0 points) and blasts in BM at relapse >median = 1 point.

Discussion The ability of AZA to produce major clinical responses in a proportion of patients who relapse after an allograft for AML or MDS conclusively identifies this agent as a new treatment option in this challenging clinical setting. Furthermore, our study represents the first systematic analysis of activity of AZA in patients relapsing post transplant and it defines factors predicting the likelihood of response, which will assist its logical deployment. A second transplant or administration of DLI represent the only treatment modalities with the capacity to deliver longterm survival in patients with recurrent disease after an allogeneic transplant, but their utility is almost entirely dependent on the prior acquisition of a morphological remission.3 Currently the only established therapy in this patient population is intensive chemotherapy which has been reported to result in 2nd CR rates in the region of 15%-30% but is associated with significant toxicity and prolonged hospitalization.3-5 Our data permit, for the first time, the identification of patients with a significant chance of responding to AZA in whom DLI or a second transplant can be delivered with potential curative effect. The retrospective nature of these data, in common with previous reports of outcome after intensive chemotherapy, introduces significant potential selection bias. We have deliberately only studied patients who received AZA within a month of relapse in order to minimize this bias. It will be important, however, for future studies addressing this important clinical challenge to be performed prospectively, either in a registration study or as a randomized comparison of AZA and intensive chemotherapy. For patients who have experienced the rigors of a previous allograft, considerations of both treatment toxicity and patient disposition are important. In general, AZA reinduction was well tolerated. Although approximately 30% of patients experienced Grade 3-4 non-hematologic toxicities, these were principally due to infection and likely to be consequent upon the cytopenias associated with 882

disease relapse rather than being directly attributable to AZA. There was a notably low incidence of GvHD observed in this study, which is surprising given the likelihood that many patients had undergone a rapid immunosuppression taper. Although this observation requires prospective validation, it is consistent with the demonstration that AZA has the capacity to expand regulatory T cells post transplant, which may result in a reduced risk of GvHD.19,20 An additional, potentially valuable benefit of the use of AZA compared with intensive chemotherapy in this patient population is the opportunity to deliver salvage therapy as an out-patient. The other pressing therapeutic challenge in the management of patients with relapsed disease is to maximize the curative potential of a second transplant in patients who have responded to salvage therapy. Transplant toxicity remains substantial in this setting and it is possible that AZA results in less organ toxicity than conventional chemotherapy. In the light of the significant number of patients who do not respond to AZA therapy, an important question raised by our data is whether it is possible to increase the response rate to AZA in this patient population. One proposed approach has been to combine AZA with DLI.21 This is the first study to study the impact of concurrent DLI on AZA response, and we failed to demonstrate any benefit associated with the co-administration of DLI. Coadministration of a histone deacetylase inhibitor, such as sodium valproate or vorinostat, may increase both the overall response rate and its speed in patients with AML and MDS,22-24 and it would be interesting to study such an approach in patients who relapse after an allograft. AZA has previously been shown to up-regulate the expression of epigenetically silenced tumor antigens, and one of its mechanisms of action in patients who have relapsed after an allogeneic transplant is the augmentation of a GvL effect. Consequently, combined administration of AZA haematologica | 2016; 101(7)


Azacitidine in relapsed AML post transplant

with lenalidomide, an immunomodulatory drug with the capacity to activate CD8+ T cells, which also has the capacity to salvage patients with relapsed myeloid malignancies who relapse post transplant, would be of interest.25 In conclusion, our data demonstrate a potentially important role for AZA in the management of selected patients with relapsed AML or MDS after an allograft. Given its acceptable toxicity and ease of administration, these results emphasize a role for AZA as a novel treatment strategy in patients with recurrent disease. The develop-

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ment of cellular or pharmacological strategies with the capacity to increase response rates is a priority. Acknowledgments The hard work of data mangers at the BMT centers returning data for this study is gratefully acknowledged. Funding A component of the data analysis presented in this manuscript was supported by an educational grant from Celgene.

allo-SCT: the good without the bad? Blood. 2012;119(14):3199-3200. Platzbecker U, Wermke M, Radke J, et al. Azacitidine for treatment of imminent relapse in MDS or AML patients after allogeneic HSCT: results of the RELAZA trial. Leukemia. 2012;26(3):381-389. Antar A, Otrock ZK, Kharfan-Dabaja M, et al. Azacitidine in the treatment of extramedullary relapse of AML after allogeneic hematopoietic cell transplantation. Bone Marrow Transplant. 2013;48(7):994995. Tessoulin B, Delaunay J, Chevallier P, et al. Azacitidine salvage therapy for relapse of myeloid malignancies following allogeneic hematopoietic SCT. Bone Marrow Transplant. 2014;49(4):567-571. Schroeder T, Rachlis E, Bug G, et al. Treatment of acute myeloid leukemia or myelodysplastic syndrome relapse after allogeneic stem cell transplantation with azacitidine and donor lymphocyte infusions--a retrospective multicenter analysis from the German Cooperative Transplant Study Group. Biol Blood Marrow Transplant. 2015;21(4):653-660. Grimwade D, Hills RK, Moorman AV, et al. Refinement of cytogenetic classification in acute myeloid leukemia: determination of prognostic significance of rare recurring chromosomal abnormalities among 5876 younger adult patients treated in the United Kingdom Medical Research Council trials. Blood. 2010;116(3):354-365. Onida F, Brand R, van Biezen A, et al. Impact of the International Prognostic Scoring System cytogenetic risk groups on the outcome of patients with primary myelodysplastic syndromes undergoing allogeneic stem cell transplantation from human leukocyte antigen-identical siblings: a retrospective analysis of the European Society for Blood and Marrow Transplantation-Chronic Malignancies Working Party. Haematologica. 2014; 99(10):1582-1590. Bacigalupo A, Ballen K, Rizzo D, et al. Defining the intensity of conditioning regimens: working definitions. Biol Blood Marrow Transplant. 2009;15(12):1628-1633. Cheson BD, Bennett JM, Kopecky KJ, et al. Revised recommendations of the International Working Group for Diagnosis, Standardization of Response Criteria,

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Treatment Outcomes, and Reporting Standards for Therapeutic Trials in Acute Myeloid Leukemia. J Clin Oncol. 2003; 21(24):4642-4649. Fine JP. Regression modeling of competing crude failure probabilities. Biostatistics. 2001;2(1):85-97. Goodyear OC, Dennis M, Jilani NY, et al. Azacitidine augments expansion of regulatory T cells after allogeneic stem cell transplantation in patients with acute myeloid leukemia (AML). Blood. 2012; 119(14):33613369. Schroeder T, Frobel J, Cadeddu RP, et al. Salvage therapy with azacitidine increases regulatory T cells in peripheral blood of patients with AML or MDS and early relapse after allogeneic blood stem cell transplantation. Leukemia. 2013; 27(9):19101913. Schroeder T, Czibere A, Platzbecker U, et al. Azacitidine and donor lymphocyte infusions as first salvage therapy for relapse of AML or MDS after allogeneic stem cell transplantation. Leukemia. 2013;27(6): 1229-1235. Craddock C, Quek L, Goardon N, et al. Azacitidine fails to eradicate leukemic stem/progenitor cell populations in patients with acute myeloid leukemia and myelodysplasia. Leukemia. 2013; 27(5): 1028-1036. Silverman LR, Verma A, Odchimar-Reissig R, et al. A Phase I Trial of the Epigenetic Modulators Vorinostat, in Combination with Azacitidine (azaC) in Patients with the Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML): A Study of the New York Cancer Consortium. ASH Annual Meeting Abstract. 2008;3656. Garcia-Manero G, Yang H, Bueso-Ramos C, et al. Phase 1 study of the histone deacetylase inhibitor vorinostat (suberoylanilide hydroxamic acid [SAHA]) in patients with advanced leukemias and myelodysplastic syndromes. Blood. 2008; 111(3):1060-1066. Sockel K, Bornhaeuser M, MischakWeissinger E, et al. Lenalidomide maintenance after allogeneic HSCT seems to trigger acute graft-versus-host disease in patients with high-risk myelodysplastic syndromes or acute myeloid leukemia and del(5q): results of the LENAMAINT trial. Haematologica. 2012;97(9):e34-35.

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ARTICLE EUROPEAN HEMATOLOGY ASSOCIATION

Stem Cell Transplantation

Ferrata Storti Foundation

Haematologica 2016 Volume 101(7):884-890

Unrelated alternative donor transplantation for severe acquired aplastic anemia: a study from the French Society of Bone Marrow Transplantation and Cell Therapies and the EBMT Severe Aplastic Anemia Working Party

Raynier Devillier,1 Jean-Hugues Dalle,2 Austin Kulasekararaj,3 Maud D'aveni,4 Laurence Clément,4,5 Alicja Chybicka,6 Stéphane Vigouroux,5 Patrice Chevallier,7 Mickey Koh,8 Yves Bertrand,9 Mauricette Michallet,10 Marco Zecca,11 Ibrahim Yakoub-Agha,12 Jean-Yves Cahn,13 Per Ljungman,14 Marc Bernard,15 Pascale Loiseau,16,17 Valérie Dubois,18 Sébastien Maury,19 Gérard Socié,20 Carlo Dufour,21* and Regis Peffault de Latour20*

1 Hematology Department, Institut Paoli Calmettes, Marseille, France; 2Pediatric Hematology Unit, Robert Debré University Hospital, Paris, France; 3Hematology Department, King’s College Hospital, London, UK; 4Pediatric Hematology Unit, University Hospital, Nancy, France; 5Hematology Department, Haut-Leveque Hospital and Bordeaux University Hospital, Pessac, France; 6Hematology Department, Wroclaw Medical University, Poland; 7Hematology Department, University Hospital, Nantes, France; 8Hematology Department, St. George’s Hospital, London, UK; 9Pediatric Hematology Unit, University Hospital, Lyon, France; 10Hematology Department, University Hospital, Lyon, France; 11Hematology Department, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; 12Hematology Department, University Hospital, Lille, France; 13 Hematology Department, University Hospital, Grenoble, France; 14Hematology Department, Karolinska University Hospital, Stockholm, Sweden; 15Hematology Department, University Hospital, Rennes, France; 16HLA Laboratory, Saint-Louis University Hospital, Paris, France; 17French Society of Histocompatibility and Immunogenetics (SFHI), France; 18HLA Laboratory, Etablissement Français du Sang, Lyon, France; 19Hematology Department, Henri Mondor Hospital, Créteil, France; 20BMT Department, Saint-Louis Hospital, Diderot Paris 7 University, Paris, France; and 21Clinical and Experimental Haematology Unit, Giannina Gaslini Children’s Hospital, Genova, Italy

Correspondence: regis.peffaultdelatour@aphp.fr

*CD and RPdL contributed equally to this work.

ABSTRACT

Received: October 29, 2015. Accepted: April 5, 2016. Pre-published: April 7, 2016. doi:10.3324/haematol.2015.138727

Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/101/7/884

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

884

U

nrelated allogeneic transplantation for severe aplastic anemia is a treatment option after immunosuppressive treatment failure in the absence of a matched sibling donor. Age, delay between disease diagnosis and transplantation, and HLA matching are the key factors in transplantation decisions, but their combined impact on patient outcomes remains unclear. Using the French Society of Bone Marrow Transplantation and Cell Therapies registry, we analyzed all consecutive patients (n=139) who underwent a first allogeneic transplantation for idiopathic severe aplastic anemia from an unrelated donor between 2000 and 2012. In an adjusted multivariate model, age over 30 years (Hazard Ratio=2.39; P=0.011), time from diagnosis to transplantation over 12 months (Hazard Ratio=2.18; P=0.027) and the use of a 9/10 mismatched unrelated donor (Hazard Ratio=2.14; P=0.036) were independent risk factors that significantly worsened overall survival. Accordingly, we built a predictive score using these three parameters, considering patients at low (zero or one risk factors, n=94) or high (two or three risk factors, n=45) risk. High-risk patients had significantly shorter survival (Hazard Ratio=3.04; P<0.001). The score was then confirmed on an independent cohort from the European Group for Blood and Marrow Transplantation database of 296 patients, with shorter survival in patients with at least 2 risk factors (Hazard Ratio=2.13; P=0.005) In conclusion, a simple score using age, transplantation timing and HLA matching would appear useful to help physicians in the daily care of patients with severe aplastic anemia.

haematologica | 2016; 101(7)


Unrelated transplantation for severe aplastic anemia

Introduction In the absence of a matched sibling donor for patients with severe aplastic anemia (SAA), allogeneic hematopoietic stem cell transplantation (allo-HSCT) from an unrelated donor (UD) is considered to be the standard treatment after immune suppressive therapy (IST) failure. The graftversus-host disease (GvHD), morbidity, and mortality after UD allo-HSCT observed in early reports partly explain this strategy.1,2 Although IST provides high long-term overall survival and reasonable response rates, patients are still exposed to clonal evolution toward paroxysmal nocturnal hemoglobinuria, myelodysplastic syndrome and acute leukemias, whereas the latter complications are rarely observed following allo-HSCT. In the last two decades, optimized HLA typing, along with better defined conditioning regimens and GvHD prophylaxis approaches have drastically improved outcomes after UD allo-HSCT.3-6 Recently, the Severe Aplastic Anemia Working Party (SAAWP) of the European Group for Blood and Marrow Transplantation (EBMT) reported 1500 allo-HSCTs for SAA performed between 2005 and 2009, with no differences between matched sibling and UD allo-HSCT when stratified by independent risk factors such as time from diagnosis to allo-HSCT, age at the time of allo-HSCT, and the use of peripheral blood stem cells as graft source.7 Moreover, excellent UD allo-HSCT results were reported in children receiving in vivo T-cell depletion as part of their conditioning regimen, in the setting of both IST failure8 and up-front treatment.9 Together, these recent results suggest considering early UD allo-HSCT as a preferred option for younger patients who lack HLA-identical siblings, provided the graft be performed early after diagnosis. However, although the age limit for this procedure, the timing of UD allo-HSCT and the impact of HLA mismatch have been tested as singular factors, the combined effect of these variables during the natural history of the disease remains unclear.3,5,10 We, therefore, analyzed all patients who received an initial allo-HSCT for idiopathic SAA in France from a UD between 2000 and 2012, with the particular aim of evaluating the predictive value of the combination of age, timing of allo-HSCT and HLA matching on overall survival (OS) after UD allo-HSCT.

Methods Design and selection criteria The study population was made up of all patients who received a first allo-HSCT for idiopathic SAA from a UD in France between 2000 and 2012. Clinical data were prospectively collected using ProMISe (Project Manager Internet Server), an internet-based data registry system shared by all centers of the French Society of Bone Marrow Transplantation and Cell Therapies (SFGM-TC). Data concerning HLA typing were collected and cross-validated using the French Society of Histocompatibility and Immunogenetics (SFHI) and the French Biomedical Agency (ABM). Patients were separated into two groups (10/10 and 9/10) by HLA matching at 10 loci at high-level resolution (HLA-A, -B, -C, -DRB1 and -DQB1). Patients with more than one mismatch at these 10 loci, as well as those for whom allele-level HLA typing was unavailable, were excluded from the study (n=5). All conditioning regimens and GvHD prophylaxes were considered for analysis. All graft sources were accepted, with the exception of cord blood. haematologica | 2016; 101(7)

We analyzed a different cohort of patients used as a validation set. Clinical data of these patients were collected from the SAAWP of EBMT using the standard minimal required forms. One hundred and eleven EBMT centers participated in this study (a complete list of participating centers is available in the Online Supplementary Appendix). Inclusion criteria were: 1) first alloHSCT for idiopathic SAA between 2000 and 2012; 2) patients transplanted in France were excluded; 3) HLA matched or mismatched unrelated donor (10 loci HLA-A, -B, -C, -DRB1 and DQB1, high resolution). The study was approved by the scientific committees of both the SFGM-TC and the Severe Aplastic Anemia Working Party of EBMT, and was conducted in accordance with the Declaration of Helsinki for clinical research. All patients provided written signed informed consent for clinical data collection (entered in ProMISe database) and participation in retrospective database analysis.

Statistical analyses Overall survival was the primary end point, estimated from the date of allo-HSCT using the Kaplan-Meier method, and univariate comparisons were made using the log rank test.11 GvHD was assessed as previously described.12,13 The cumulative incidence of chronic GvHD (cGvHD) was calculated considering death before GvHD as a competing event, and univariate comparisons were made using the Gray test.14 In the study population, we analyzed the impact of our 3 main parameters: age at the time of allo-HSCT (≤ 30 vs. > 30 years), the time between diagnosis and allo-HSCT (≤ 12 vs. > 12 months) and allele-level HLA matching (9/10 vs. 10/10) on outcome. We tested the impact on outcome of following potential confounding factors: transplantation period (2000-05 vs. 2006-12), donor age at the time of allo-HSCT (≤ 35 vs. > 35 years), cytomegalovirus (CMV) serostatus (donor and recipient: negative vs. other combinations), graft source [bone marrow vs. peripheral blood stem cells, (PBSC)], conditioning regimen [without total body irradiation (TBI) vs. with TBI], GvHD prophylaxis [cyclosporine A (CSA) + methotrexate (MTX) vs. other] and the use of in vivo T-cell depletion as part of the conditioning regimen (no vs. yes). Among them, those with P<0.150 were selected to adjust the impact of the 3 main variables of interest (age, timing and HLA matching) in a multivariate Cox model.15 Subsequently, a score was calculated based on the three variables of interest (age, timing of alloHSCT and HLA matching) in which each of them carried a weight defined by Cox model hazard ratios. The impact of this score was then evaluated in univariate and multivariate analyses (the latter adjusted for the same co-variables). Finally, the score was tested on the validation independent set of patients in both univariate and multivariate analyses.

Results Characteristics of patients of the training set (SFGM-TC database) One hundred and thirty-nine consecutive patients (64 male; 46%) met the selection criteria. Median age was 23 years (range 1-66) (Table 1). Median follow up was 51 months (range 2-140), and 124 patients (89%) had a minimal follow up of 24 months. All patients were in failure of first-line of treatment by IST. Forty-six and 93 patients were transplanted in the periods 2000-05 and 2006-12, respectively. In the more recent period, patients were significantly older (median age 2000-05 vs. 2006 12: 18 years vs. 25 years, respectively; P=0.030), more frequently 885


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received in vivo T-cell depletion (2000-05 vs. 2006-12: 57% vs. 91%; P<0.001, respectively) and were more prone to receive PBSC as graft source (2000-05 vs. 200612: 7% vs. 19%, respectively; P=0.074). There was no difference in interval between diagnosis and allo-HSCT (as continuous variable: Mann-Whitney test: P=0.312; as categorical variables ≤12 vs. >12 months: P=0.855), HLA matching and GvHD prophylaxis between the two transplantation periods.

Transplantation-related events Ten patients (7%) experienced primary graft failure. Forty-eight patients developed grade II-IV acute GvHD, leading to a day-100 cumulative incidence of 35% (95CI: 26-42) (grade II 32 of 48, 66%; grade III 10 of 48, 21%; grade IV 6 of 48, 13%). The use of PBSC was associated with a higher incidence of grade II-IV acute GvHD (PBSC vs. bone marrow: 52% vs. 31%; P=0.044) (Table 2) but not with higher incidence of chronic GvHD. The cumulative incidence of chronic GvHD at four years was 24% (95CI: 17-31) (extensive GvHD 8%). Chronic GvHD occurred at a median time of six months after allo-HSCT (range 3-24). No specific risk factor was identified (Table 2).

Overall survival With a median follow up of 51 months (range 2-140), 4year OS was 66% (95CI: 58-75). Median time from alloHSCT to death was four months (range 1-46). Forty-one of the 45 deaths (91%) occurred within the first two years after allo-HSCT. Causes of death were GvHD (n=17, 37%), severe infections without GvHD (n=14, 31%), graft failure (n=8, 18%), secondary malignancy (n=2, 4%), and other causes (n=4, 9%). Univariate analyses of OS by age at the time of alloHSCT, interval from diagnosis of SAA to allo-HSCT, and HLA matching (10 loci) are illustrated in Table 3. To select factors for adjustment in the multivariate model, we tested the impact of these confounding factors in univariate analyses and found that the transplantation period significantly influenced OS (2000-05 vs. 2006-12: 52% vs. 74%; P=0.018). Moreover, CMV sero status (D-/R- vs. other: 71% vs. 61%; P=0.125) and the use of in vivo T-cell depletion (yes vs. no: 67% vs. 55%; P=0.145) were selected for the adjusted model, having P values <0.150. The remaining factors (donor age, graft source, conditioning regimen and GvHD prophylaxis) did not significantly influence outcome and were not considered to adjust the multivariate model (Online Supplementary Table S1). In multivariate analyses adjusted for the transplantation period, CMV sero status and the use of in vivo T-cell depletion, we found that age over 30 years [HR=2.39 (1.23-4.66); P=0.011], time from diagnosis to allo-HSCT over 12 months [HR=2.18 (1.09-4.37); P=0.027] and the use of a 9/10 mismatched UD [HR=2.14 (1.05-4.38) P=0.036] were independent risk factors that significantly influenced OS (Table 3).

Predictive score for overall survival Using a number of risk factors [age (> 30 years), time from diagnosis to allo-HSCT (> 12 months) and HLA matching (9/10)] we created a score to predict OS. We attributed the same weight to these three variables since the hazard ratios produced by the Cox model were similar (i.e. close to 2) (Table 3). No risk factors were seen in 35 patients (25%), one risk factor was seen in 59 patients 886

(42%), two risk factors in 41 patients (30%), and three risk factors in 4 patients (3%). We stratified patients into a low-risk group (zero or one risk factors, based on the fact that we found no significant difference in OS between patients with zero and one risk factor (Online Supplementary Table S2) and a high-risk group (two or three risk factors, because the number of patients with 3 risk factors is too low for a separate analysis). Four-year OS was 74% in the low-risk group and 49% in the highrisk group (P<0.001) (Table 3 and Figure 1A). After adjustment for the transplantation period in a multivariate Cox model, patients in the high-risk group had significantly shorter survival [HR=3.04 (1.64-5.62); P<0.001] (Table 3). In both low-risk and high-risk groups, main causes of death were GvHD (39% and 36%, respectively), infections (39% and 23%, respectively) and graft failure (10% and 27%, respectively).

Independent validation set (EBMT database) Two-hundred and ninety patients matched inclusion criteria for the validation set. Validation cohort characteristics are shown in Online Supplementary Table S3. Age was over 30 years in 63 patients (21%), 175 patients (59%) were transplanted later than 12 months after diagnosis of SAA, and 44 patients (15%) received grafts from a mismatched unrelated donor (MUD). Based on these 3 factors, 232 (78%) and 64 (22%) were categorized as lowrisk (zero or one risk factor) and high-risk (2 or 3 risk fac-

Table 1. Patients’, disease and transplantation characteristics in the study population.

All patients (N = 139) N. % Median age (years) Time from diagnosis to allo-HSCT (months) ≤ 12 months > 12 months CMV serostatus D-/ROthers Unknown Conditioning regimen Cy +/- Flu Bu-Cy +/- Flu Other Use of TBI In vivo T-cell depletion GvHD prophylaxis CSA CSA + MTX CSA + MMF Others Unrelated donor 10/10 MUD 9/10 MUD Median donor age (years) Graft source BM PBSC

23

[1-66]

58 81

42% 58%

53 83 3

39% 61%

100 24 15 64 112

72% 17% 11% 46% 81%

11 94 17 17

8% 68% 12% 12%

113 26 35

81% 19% [3-60]

118 21

85% 15%

BM: bone marrow; Bu: busulfan; CSA: cyclosporine A; Cy: cyclophosphamide; D-/R-: seronegative donor and recipient; Flu: fludarabine; GvHD: graft-versus-host disease; MMF: mycophenolate mofetil; MTX: methotrexate; MUD: matched unrelated donor; PBSC: peripheral blood stem cell; TBI: total body irradiation.

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tors), respectively. Low-risk patients had significantly better 4-year OS compared to patients of the high-risk group (low-risk vs. high-risk: 76% vs. 60%; P=0.003) (Figure 1B). In adjusted multivariate analyses, we confirmed the prognostic impact of the score, with a shorter OS in the highrisk group [HR=2.13 (1.26-3.59); P=0.005].

Discussion This paper provides details of the French experience of UD allo-HSCT from 2000 to 2012 for idiopathic SAA, with a reasonable follow-up period (89% of patients for at least 24 months). Four-year OS was 66%, and importantly, results continued to improve over time, with the best results from 2006 onwards. Online Supplementary Table S4 reviews major series of UD allo-HSCT for SAA, showing overall improvement of OS over the last 20

years.3-5,16-18 Due to an improvement in transplantation procedures, recent survival after matched UD allo-HSCT has approached that of post HLA-identical sibling donor allo-HSCT. This is supported by the recent analysis of our group showing that, once stratified using risk factors, OS was roughly similar using a related or matched unrelated donor.7 In this paper, we focused on the impact of age, timing between diagnosis and allo-HSCT as well as HLA matching (excluded from the recent EBMT analysis7) on outcome after transplantation for patients with refractory aplastic anemia. In addition, in order to provide physicians with a more complete tool to make therapeutic decisions when patients are considered for UD alloHSCT, we tested the combined effect of these factors to predict outcomes in our cohort. We acknowledge that other variables may influence outcome. However, most of them are related to transplantation procedure (e.g. conditioning regimen, in vivo T-cell depletion, stem cell source

Table 2. Cumulative incidences of acute and chronic graft-versus-host disease in the study population.

Day-100 aGvHD

(95%CI)

35%

(26-42)

44% 30%

(27-56) (20-39)

44% 32%

All patients (n = 139) Transplantation period 2000-05 (n = 46) 2006-12 (n = 93) In vivo T-cell depletion No (n = 27) Yes (n =112) Age at allo-HSCT ≤ 30 years (n = 93) > 30 years (n = 46) Time from diagnosis to allo-HSCT ≤ 12 months (n = 58) > 12 months (n = 81) Graft source BM (n = 118) PBSC (n = 21) HLA matching 10/10 MUD (n = 113) 9/10 MUD (n = 26)

P

P

4-years cGvHD

(95%CI)

24%

(17-31)

0.073

22% 26%

(9-33) (16-34)

0.704

(22-60) (23-40)

0.137

22% 25%

(5-36) (16-33)

0.789

38% 28%

(27-47) (14-40)

0.330

25% 22%

(16-34) (9-34)

0.813

33% 36%

(20-44) (25-45)

0.575

26% 23%

(14-37) (13-31)

0.609

31% 52%

(23-39) (25-70)

0.044

24% 24%

(16-32) (3-40)

0.946

35% 35%

(14-51) (25-43)

0.949

25% 23%

(16-32) (5-38)

0.816

95%CI: 95% confidence interval; aGvHD: acute graft-versus-host disease; BM: bone marrow; cGvHD: chronic graft-versus-host disease; HSCT: allogeneic hematopoietic stem cell transplantation; MUD: matched unrelated donor; PBSC: peripheral blood stem cell.

Table 3. Univariate and multivariate analyses of overall survival in the study population.

Age at allo-HSCT ≤ 30 years (n = 93) > 30 years (n = 46) Time from diagnosis to allo-HSCT ≤ 12 months (n = 58) > 12 months (n = 81) HLA matching 10/10 (n = 113) 9/10 (n = 26) Predictive score 0 or 1 risk factor (n = 94) 2 or 3 risk factors (n = 45)

4-y

Univariate analysis [95%CI]

P

HR

Multivariate analysis* [95%CI]

P

70% 57%

[61-81] [44-74]

0.037

1 2.39

[1.23-4.66]

0.011

77% 58%

[65-90] [48-70]

0.017

1 2.18

[1.09-4.37]

0.027

68% 57%

[59-78] [40-80]

0.196

1 2.14

[1.05-4.38]

0.036

74% 49%

[65-84] [36-67]

< 0.001

1 3.04

[1.64-5.62]

<0.001

*Adjustment with cytomegalovirus serostatus, in vivo T-cell depletion and transplantation period; 4-y: 4-year; HR: Hazard ratio; 95%CI: 95% confidence interval.

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or GvHD prophylaxis) and have been optimized over the years to continuously improve overall outcome. Although these parameters are determining factors in how to proceed, they are not usually key factors in deciding whether or not to transplant. Variables not available at the time of decision (e.g. the number of CD34 cells) were purposely excluded from the analysis. Finally, we validated the impact of our key factors as a composite score in an independent population provided by the EBMT SAAWP (Severe Aplastic Anemia Working Party), including patients with idiopathic SAA who underwent first allo-HSCT from a UD during the same period of time. Patients over 30 years of age experienced shorter survival (57%) compared with younger patients (70%). The finding of younger age as associated with a better outcome is in line with two recent large EBMT studies that showed OS rates of 83% and of 78%, respectively, in children and adolescents undergoing UD HSCT after failed IST.19,20 Moreover, it is of interest that previous reports identified a significant age cut off of 20 years for UD allo-HSCT.16,21 In the EBMT series,3 Bacigalupo et al. showed that, in the context of global improvement of transplantation procedures and outcomes, an age cut off of 27 years could not predict OS, underlining the need to reassess the age limit in this context. We, therefore, suggest that UD allo-HSCT should be safely considered up to 30 years of age, approaching the clinically relevant age cut off of 40 years in the setting of HLA-identical sibling alloHSCT.22,23 For older patients, UD allo-HSCT should be performed with caution in highly selected patients, especially in the presence of additional poor risk factors such as comorbidities.5 Regarding the timing of allo-HSCT, we found better OS when allo-HSCT was performed earlier, within the first year following diagnosis. This is in line with previous reports showing better results with early allo-HSCT for SAA in different clinical settings.3,22,24,25 Because of the absence of a non-transplanted control group, we were not able to directly assess the optimal timing for UD allo-HSCT after first IST failure. However, we suggest that patients should be transplanted as early as possible in this situation, which implies starting the search for an unrelated donor soon after diagnosis in younger patients without a sibling donor. Lastly, we confirmed the unfavorable impact of HLA mismatches, which underlines the importance of HLA matching in a cohort exclusively comprising allele-level HLA-matched patients, which is in agreement with previous reports suggesting better outcomes with matched UD.4,17,18 Accordingly, and in order to provide a more comprehensive evaluation tool, we built a prognostic OS score taking into account age (â&#x2030;¤ 30 vs. > 30 years), timing of allo-HSCT (â&#x2030;¤ 12 vs. > 12 months) and HLA matching (9/10 vs. 10/10). In the same low-risk group, we decided to combine patients with zero and one risk factor because we found no statistical difference in OS between these 2 groups in the study population or in the validation cohort (Online Supplementary Table S2). However, we cannot exclude the possiblity that OS might be better for patients who present zero risk factors in comparison with patients with one risk factor (hazard ratio of 1.41 and 1.87, respectively, in the study population and validation cohort). That said, a dramatic impairment of survival was observed in patients with more than one risk factor (Online Supplementary Table S2). Therefore, we separated patients into a low-risk (0 or 1 risk factor) and a high-risk 888

(2 or 3 risk factors) group. We found that patients with more than one of these risk factors experienced lower survival (49%), while those with zero or one risk factor obtained a 4-year OS of 74%. As some of these patients were transplanted in the early 2000s, we might expect lower mortality rates in more recent times following the systematic introduction of procedures that have since been described to provide better results (in vivo T-cell depletion, bone marrow as graft source, fludarabine-based conditioning regimens).3 Moreover, supportive care has improved over time, partly contributing to the lower mortality rate in the most recent period.26 We then evaluated the impact of our prognostic score on the independent validation set and confirmed that it is easy to use and reproducible. Indeed, it turned out to have strongly predictive value in both univariate and multivariate analyses, although the high number of participating centers leads to a high heterogeneity in the baseline characteristics of patients and transplantation

A

B

Figure 1. Overall survival by the presence of selected risk factors: age > 30 years; time from diagnosis to allogeneic hematopoietic stem cell transplantation (allo-HSCT) > 12 months; presence of an HLA mismatch. (A) Study population. (B) Validation cohort.

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procedures. Stratifying risk in this manner may prove to be an easy way to select low-risk patients for UD allo-HSCT, while care should be exercised when considering those with more than one risk factor, and for whom an additional course of IST could be debated. As recently published, there has been a significant decrease in invasive fungal infections, infection-related mortality, and overall mortality in patients with SAA unresponsive to initial IST, with a 5-year OS among non-responders of approximately 57%, similar to that expected in patients with more than one risk factor in our study.26 Our work has both strengths and limitations. Its strengths include the large enrollment of all consecutive patients with idiopathic SAA undergoing UD allo-HSCT in France over the most recent 12-year period. This provides not only a clear picture of our current practice in this setting, but also shows how transplantation strategies have improved over time. Moreover, our analyses focus on patients with available allele-level HLA matching at 10 loci. This allows for a clear assessment of the impact of HLA matching, which is already known to have contributed to some degree to the overall improvement in transplantation strategies. Moreover, the presence of a validation set of patients represents a major strength to evaluate the power and the reproducibility of a new prognostic score. Our study also has a number of limitations that are mostly due to its retrospective nature. It is likely that only the healthiest patients over 30 years of age were considered for UD allo-HSCT, especially in the presence of an HLA mismatch. We do not have data for similar patients who were referred for a UD allo-HSCT but were considered unfit. It would have been interesting to evaluate their outcomes after further non-allogeneic treatment in comparison with our cohort. Data concerning previous

References 6. 1. Kernan NA, Bartsch G, Ash RC, et al. Analysis of 462 transplantations from unrelated donors facilitated by the National Marrow Donor Program. N Engl J Med. 1993;328(9):593-602. 2. Kojima S, Inaba J, Yoshimi A, et al. Unrelated donor marrow transplantation in children with severe aplastic anaemia using cyclophosphamide, anti-thymocyte globulin and total body irradiation. Br J Haematol. 2001;114(3):706-711. 3. Bacigalupo A, Socie’ G, Lanino E, et al. Fludarabine, cyclophosphamide, antithymocyte globulin, with or without low dose total body irradiation, for alternative donor transplants, in acquired severe aplastic anemia: a retrospective study from the EBMTSAA Working Party. Haematologica. 2010;95(6):976-982. 4. Maury S, Balère-Appert M-L, Chir Z, et al. Unrelated stem cell transplantation for severe acquired aplastic anemia: improved outcome in the era of high-resolution HLA matching between donor and recipient. Haematologica. 2007;92(5):589-596. 5. Marsh JC, Gupta V, Lim Z, et al. Alemtuzumab with fludarabine and cyclophosphamide reduces chronic graftversus-host disease after allogeneic stem cell

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7.

8.

9.

10.

treatment were also lacking for a significant number of patients. We acknowledge that these data would have been helpful in order to make an accurate evaluation of the disease risk at the time of allo-HSCT. As a surrogate marker, we used the time from diagnosis to allo-HSCT, but the number, type and response duration to previous IST would have been more informative to better identify the optimal timing of UD allo-HSCT in the treatment strategy of SAA. Despite these obvious limitations, our results have led us to consider UD Allo-HSCT soon after IST failure and to start the search for an UD at time of diagnosis in younger patients, although age and HLA matching should be key factors in the decision-making process. Integrating these 3 simple parameters in our proposed prognostic score would appear useful in selecting patients for UD allo-HSCT. Although it seems to be reproducible in an independent cohort, a prospective evaluation is needed to better define the overall treatment strategy of idiopathic SAA in IST failure. Acknowledgments We would like to thank all the participating centers of the Société Française de Greffe de Moelle et Thérapie Cellulaire (SFGM-TC) (see Online Supplementary Appendix), the Société Francophone d’Histocompatibilité et d’Immunogénétique (SFHI) and all the participating centers of the Severe Aplastic Anemia Working Party (SAAWP) of the European Society of Blood and Marrow Transplantation (EBMT) (see Online Supplementary Appendix). We would also like to thank Dr. Raphael Porcher (Centre d’Epidémiologie Hôtel-Dieu, Paris, France; INSEMR U1153, Paris, France) for his critical review of the statistical analyses.

transplantation for acquired aplastic anemia. Blood. 2011;118(8):2351-2357. Lee SJ, Klein J, Haagenson M, et al. High-resolution donor-recipient HLA matching contributes to the success of unrelated donor marrow transplantation. Blood. 2007;110 (13):4576-4583. Bacigalupo A, Socié G, Hamladji RM, et al. Current outcome of HLA identical sibling versus unrelated donor transplants in severe aplastic anemia: an EBMT analysis. Haematologica. 2015;100(5):696-702. Samarasinghe S, Steward C, Hiwarkar P, et al. Excellent outcome of matched unrelated donor transplantation in paediatric aplastic anaemia following failure with immunosuppressive therapy: a United Kingdom multicentre retrospective experience. Br J Haematol. 2012;157(3):339-346. Dufour C, Veys P, Carraro E, et al. Similar outcome of upfront-unrelated and matched sibling stem cell transplantation in idiopathic paediatric aplastic anaemia. A study on behalf of the UK Paediatric BMT Working Party, Paediatric Diseases Working Party and Severe Aplastic Anaemia Working Party of EBMT. Br J Haematol. 2015;171(4):585-594. Yagasaki H, Kojima S, Yabe H, et al. Acceptable HLA-mismatching in unrelated donor bone marrow transplantation for patients with acquired severe aplastic anemia. Blood. 2011;118(11):3186-3190.

11. Kaplan E, Meier P. Nonparametric estimation from incomplete observations. J Amer Statist Assoc. 1958;53(282):457-481. 12. Glucksberg H, Storb R, Fefer A, et al. Clinical manifestations of graft-versus-host disease in human recipients of marrow from HL-Amatched sibling donors. Transplantation. 1974;18(4):295-304. 13. Shulman HM, Sullivan KM, Weiden PL, et al. Chronic graft-versus-host syndrome in man. A long-term clinicopathologic study of 20 Seattle patients. Am J Med. 1980;69 (2):204-217. 14. Fine J, Gray R. A proportional hazards model for the subdistribution of a competing risk. J Amer Statist Assoc. 1999;94(446): 496-509. 15. Cox D. Regression models and life tables. J R Stat Soc Series B Stat Methodol. 1972;34 (2):187-220. 16. Deeg HJ, Amylon ID, Harris RE, et al. Marrow transplants from unrelated donors for patients with aplastic anemia: minimum effective dose of total body irradiation. Biol Blood Marrow Transplant. 2001;7(4):208215. 17. Kojima S, Matsuyama T, Kato S, et al. Outcome of 154 patients with severe aplastic anemia who received transplants from unrelated donors: the Japan Marrow Donor Program. Blood. 2002;100(3):799-803. 18. Viollier R, Socié G, Tichelli A, et al. Recent

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R. Devillier et al. improvement in outcome of unrelated donor transplantation for aplastic anemia. Bone Marrow Transplant. 2008;41(1):45-50. 19. Dufour C, Pillon M, Sociè G, et al. Outcome of aplastic anaemia in children. A study by the severe aplastic anaemia and paediatric disease working parties of the European group blood and bone marrow transplant. Br J Haematol. 2015;169(4):565-573. 20. Dufour C, Pillon M, Passweg J, et al. Outcome of aplastic anemia in adolescence: a survey of the Severe Aplastic Anemia Working Party of the European Group for Blood and Marrow Transplantation. Haematologica. 2014;99(10):1574-1581. 21. Bacigalupo A, Locatelli F, Lanino E, et al. Fludarabine, cyclophosphamide and antithymocyte globulin for alternative donor

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with conventional conditioning regimen. Haematologica. 2009;94(9):1312-1315. 24. Peffault de Latour R, Porcher R, Dalle J-H, et al. Allogeneic hematopoietic stem cell transplantation in Fanconi anemia: the European Group for Blood and Marrow experience. Blood. Transplantation 2013;122(26):4279-4286. 25. Peffault de Latour R, Schrezenmeier H, Bacigalupo A, et al. Allogeneic stem cell transplantation in paroxysmal nocturnal hemoglobinuria. Haematologica. 2012;97 (11):1666-1673. 26. Valdez JM, Scheinberg P, Nunez O, et al. Decreased infection-related mortality and improved survival in severe aplastic anemia in the past two decades. Clin Infect Dis. 2011;52(6):726-735.

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