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A pilot study of the Histone-Deacetylase inhibitor Givinostat in patients with JAK2V617F positive chronic myeloproliferative neoplasms

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A pilot study of the Histone-Deacetylase inhibitor Givinostat in patients with JAK2V617F positive chronic myeloproliferative neoplasms
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  A pilot study of the Histone-Deacetylase inhibitor Givinostatin patients with JAK2V617F positive chronic myeloproliferativeneoplasms Histone-Deacetylases (HDACs) are enzymes involved in theremodelling of chromatin, and have a key role in the epigeneticregulation of gene expression. In addition, the activity of non-histone proteins can be regulated through HDAC-mediatedhypoacetylation. In recent years, inhibition of HDACs hasemerged as a potential strategy to reverse aberrant epigeneticchanges associated with cancer, and several classes of HDACinhibitors have been found to have potent and specificanticancer activities. Givinostat (ITF2357; Italfarmaco S.p.A.,Cinisello Balsamo, Italy) is a new synthetic class I and II HDACinhibitor(Leoni  et al  , 2005) with a potent anti-proliferativeand pro-apoptotic activity against several haematologicalmalignancies, including acute myeloid leukaemia (AML)(Barb-etti  et al  , 2008) and multiple myeloma (MM) cells(Golay  et al  , 2007) , (Todoerti  et al  , 2010). In addition to these anti-tumour features, preclinical studies of Givinostat also showedthat it could inhibit the production/release of severalcytokines, including tumour necrosis factor- a  (TNF a ), inter-leukin (IL)-1 b , IL-6, IL-12,  c -interferon (IFN c ) and vascularendothelial growth factor (VEGF) by neoplastic as well asendothelial and mesenchymal cells (Carta  et al  , 2006; Golay  et al  , 2007).In advanced cancer patients, some evidence of clinicalactivity has been observed in MM (Galli  et al  , 2010) andHodgkin disease(Carlo-Stella  et al  , 2008). In all these studies,Givinostat, when used up to the dose of 200 mg/d, proved tobe well tolerated with non-specific gastrointestinal symptomsand reduction in platelets being the most frequent adverseevents.Moreover, we have recently shown that Givinostat inhibitsthe cell growth and clonogenic potential of the JAK2V617F-mutated HEL cell line as well as the autonomous proliferation Alessandro Rambaldi, 1 Chiara MariaDellacasa, 1 Guido Finazzi, 1 AlessandraCarobbio, 1 Maria Luisa Ferrari, 1 PaolaGuglielmelli, 2 Elisabetta Gattoni, 3 SilviaSalmoiraghi, 1 Maria Chiara Finazzi, 1 Silvia Di Tollo, 4 Carmine D’Urzo, 4 Alessandro M. Vannucchi, 2 GiovanniBarosi 3 and Tiziano Barbui 1 1 Haematology, Ospedali Riuniti Di Bergamo,Largo Barozzi, Bergamo,  2 Haematology,University of Firenze, Viale Morgagni, Firenze, 3 Clinical Epidemiology and Centre for the Study of Myelofibrosis, Fondazione Istituto Di Ricovero eCura a Carattere Scientifico Policlinico S. Matteo,Viale Golgi, Pavia, and   4 Clinical R&DDepartment, Italfarmaco SpA, Via dei Lavoratori,Cinisello Balsamo (MI), Italy  Received 5 March 2010; accepted for publication29 April 2010Correspondence: Alessandro Rambaldi, MD,U.S.C Ematologia, Ospedali Riuniti di Bergamo,Largo Barozzi 1, 24128 Bergamo, Italy.E-mail: arambaldi@ospedaliriuniti.bergamo.it Summary A phase II A study was conducted to evaluate the safety and efficacy of Givinostat, a novel Histone-Deacetylases inhibitor, in patients withPolycythaemia Vera (PV,  n  = 12), Essential Thrombocythaemia (ET, n  = 1) and Myelofibrosis ( n  = 16), bearing the JAK2V617F mutation. Thestudy was approved by the local ethics committees and all humanparticipants gave written informed consent. Givinostat was given orally for24 weeks at a starting dose of 50 mg twice daily. The median treatmentduration was 20 weeks. Reasons for treatment discontinuation were diseaseprogression( n  = 6),grade2thrombocytopenia( n  = 1),psychiatricsymptoms( n  = 1) and withdrawn consent ( n  = 2). A dose reduction was applied in 10patients while a temporary interruption occurred in 15. Among 13 PV/ETpatients, 1 complete, 6 partial and 4 no responses were documented atstudy end while 2 patients went off-study, prematurely. Three majorresponses were registered among 16 MF patients. Pruritus disappeared inmost patients and reduction of splenomegaly was observed in 75% of PV/ETand 38% of MF patients. Reverse transcription polymerase chain reactionidentified a trend to reduction of the  JAK2 V617F allele burden. Givinostatwas well tolerated and could induce haematological response in most PV andsome MF patients. Keywords :  Polycythaemia Vera, Essential Thrombocythaemia, Myelofibrosis,HDAC inhibitors, Givinostat. research paper First published online 15 June 2010doi:10.1111/j.1365-2141.2010.08266.x  ª  2010 Blackwell Publishing Ltd,  British Journal of Haematology  ,  150 , 446–455  of haematopoietic cells from patients with Polycythaemia Vera(PV) and Essential Thrombocythaemia (ET). Givinostatinduced a specific down modulation of the phosphorylatedJAK2V617F protein and inhibition of its downstream signal-ling while it did not affect the wild-type JAK2 or STATproteins in cells lacking the JAK2V617F mutation(Guerini et al  , 2008).Based on these properties we planned to evaluate the safety and efficacy of Givinostat in the treatment of JAK2V617F-positive PV and ET as well as in Myelofibrosis, both primary (PMF) and secondary to a previous PV or ET (post-PV/ETMF). Patients and methods Study design and eligibility  This was a phase II A, pilot, multi-centre, open-label, non-randomized study (Study No.: DSC/07/2357/28; EudraCT No.:2007-004480-21, ClinicalTrials.Gov Identifier: NCT00606307).This study enrolled a population of patients with a confirmeddiagnosis of JAK2V617F-positive PV, ET, PMF or post-PV/ETMF according to the revised World Health Organizationcriteria(Tefferi  et al  , 2007). To be eligible for study entry patients had to be in need of cytoreductive therapy beingintolerant or refractory to hydroxycarbamide according to thedefinition given by the LeukemiaNet European Collaborationfor ET (Barosi  et al  , 2007) and PV/MF (Barosi  et al  , 2009a). Inaddition, younger patients who had refused hydroxycarbamidedue to concerns regarding its potential leukemogenic effectwere also eligible.After providing informed written consent before undertak-ing any protocol-related procedure, a unique number wasassigned by the sponsor to identify each patient throughouthis/her participation to the study.Patients were excluded in case of platelet count <100  ·  10 9 /l,neutrophil count <1 Æ 2  ·  10 9 /l, blast cells in peripheral blood>10%, treatment with IFN a  or hydroxycarbamide within the14 d prior to enrolment, treatment with anagrelide within the7 d prior to enrolment and any other investigational drugwithin the 28 d prior to enrolment. Moreover, exclusioncriteria also included Eastern Cooperative Oncology Groupperformance status  ‡ 3, or the concomitant presence of acutecoronary syndrome, uncontrolled hypertension, congestiveheart failure, any cardiac arrhythmia or baseline prolongationof QT/QTc interval (e.g. repeated demonstration of a QTcinterval > 450 ms, according to Bazett’s correction formula)abnormal kidney or liver function, any active bacterial viral orfungal infection, pregnancy or lactation. Treatment schedule Givinostat was supplied as 50 mg hard gelatine capsules fororal administration. All recruited patients received an initialdose of 50 mg b.i.d. that could subsequently be escalated to50 mg t.i.d. in case of lack of toxicity. Treatment wasadministered on an outpatient basis and lasted up to amaximum of 24 cumulative weeks. Criteria for treatment continuation/discontinuation and dose reduction To continue the treatment with Givinostat all the followingcriteria had to be satisfied at each check point foreseen in theprotocol: neutrophil count  ‡ 1 Æ 0  ·  10 9 /l, platelet count >100  ·  10 9 /l, normal heart, kidney and liver function, all othertoxicities grade  £ 2. If any worse toxicity was identified,treatment with Givinostat had to be discontinued and patientremained untreated until recovery of the observed toxicities tothe level identified as mandatory for treatment continuation.Upon recovery to admitted toxicity level, treatment had to berestarted at the reduced dose of 50 mg o.d. If no dose-limitingtoxicity was observed, this same dose (50 mg o.d.) wasmaintained throughout the remaining part of study untilcompletion. If new toxicities were observed at 50 mg o.d., thepatient had to leave the study. In any event, if recovery fromprevious toxicities required 4 weeks or more, treatment withGivinostat was not restarted. Concomitant therapy  Patients were not allowed to receive any other investigationalor chemotherapeutic drug while on this study protocol.Patients were not allowed to receive any drug generally accepted to have a risk of causing Torsade de Pointes (TdP)(http://www.torsades.org). Hydroxycarbamide was permittedto control the platelet count in case of new ischaemicsymptoms at any platelet level and at any time during thestudy period. Treatment with allopurinol (150–300 mg/o.d.)and aspirin (100 mg/o.d.) was allowed. Other allowed con-comitant medications were recorded in the clinical recordforms (CRFs). Efficacy and safety assessment  The efficacy evaluation included all patients and was per-formed on an intention to treat basis. Assessment of responsestatus of the patients was performed after 12 and 24 weeks of treatment, and reviewed and cross-checked independently by the investigators. For PV/ET patients the clinical response wasevaluated according to the recently published EuropeanLeukemiaNet criteria(Barosi  et al  , 2009b). In MF, the responseevaluation was planned by protocol according to the EuropeanMyelofibrosis Network (EUMNET) criteria(Barosi  et al  , 2005).When the study was completed, the clinical response was alsoevaluated according to the International Working Group(IWG) criteria (Tefferi  et al  , 2006), which are most commonly used in the USA.Toxicity was evaluated on the reported adverse events,physical examination, and changes in laboratory results, Givinostat Treatment for CMNs ª  2010 Blackwell Publishing Ltd,  British Journal of Haematology  ,  150 , 446–455  447  according to the  nci-ctcae  criteria version 3.0 (http://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/ctcaev3.pdf). Statistical methods, ethics and good clinical practice Drug activity was evaluated based upon a one-stage Flemingstudy design for determination of response rates based on asingle treatment group. A sample size of 27 evaluable patientswas estimated using exact method (binomial).The primary endpoint was the number of objectiveresponses and the number of patients experiencing an adverseevent; the secondary endpoint was the reduction of JAK2V617F mutated allele burden.This study was carried out in accordance to Good ClinicalPractice, as described in the International Conference onHarmonization Tripartite Guidelines for Good ClinicalPractice 1996, the European Economic Community (EEC)Directive 91/507/EEC, the Rules Governing Medicinal Prod-ucts in the European Community and the US Code of FederalRegulations dealing with clinical studies.Equality of matched pairs of observations was tested usingthe Wilcoxon matched-pairs signed-ranks test. Nonparametrictest for trend across ordered groups were performed fordifferences in JAK2V617F allele burden distribution. Quantitative analysis of JAK2V617F tumour allele burden The mutational state of JAK2V617F was determined on DNAfrom peripheral-blood granulocytes as described by Guerini et al   (2008). The burden of mutated allele was measured by aquantitative Real-Time reverse transcription polymerase chainreaction assay (qRT-PCR), as recently published(Guglielmelli et al  , 2009). Single colony genotyping was performed asdescribed (Guerini  et al  , 2008). Results Patient characteristics Between December 2007 and June 2008, 29 patients (15 males/14 females) with PV ( n  = 12), ET ( n  = 1) or MF ( n  = 16) wereenrolled into this study. Among these latter MF patients, six had primary disease (PMF), while five had a previous ET(post-ET MF) and five a previous PV (post-PV MF). Themedian disease duration for all patients was 9 Æ 2 (range 1 Æ 4–26 Æ 2) years. Prior therapies included cytotoxic agents, such asHydroxycarbamide, Busulfan, Pipobroman, and Bortezomib,and non-cytotoxic agents, such as Aspirin, Anagrelide, Dana-zol, Thalidomide, Erythropoietin, IFN a , and Pentofillin. Fivepatients (4 PV/ET and 1 MF) were previously untreated andwere offered this study because they specifically asked for anexperimental, potentially non-leukemogenic treatment. TwoMF patients were transfusion-dependent and 3 had beensplenectomized. Fourteen patients (11 PV and 3 post-PV MF)were phlebotomy-dependent. All patients harboured theJAK2V617F mutation (homozygous in 17 patients). Mostpatients had a palpable splenomegaly (76%) and pruritus wasclinically relevant in 52%. Safety   Adverse events.  Safety was evaluated on all 29 enrolledpatients (Table I). Overall, Givinostat was usually welltolerated and no grade IV toxicity was observed. The mostfrequent drug-related adverse events were: (i) gastrointestinaldisorders including diarrhoea (62% of patients, but grade III inonly one case), nausea and gastric pain (10% and 7%,respectively, but all below grade III toxicity); (ii) anaemia(21%, but grade III in only one case) and thrombocytopenia(10%, but all below grade III toxicity); (iii) fatigue (17%, butall below grade III toxicity) and fever (7%, all grade I); (iv)elongation of QTc and weight loss (17% and 7%, respectively,but all below grade III toxicity); (v) anorexia (7%, all grade I);(vi) rash (7%, all grade I). There were no life-threateningadverse events. Serious adverse events.  Four serious adverse events (SAEs)were documented among MF patients during the clinicalstudy, but all were considered unrelated to Givinostattreatment. One MF patient experienced two SAEs, both dueto infection of foot soft tissue (cellulites). Another MF patientwas hospitalized because of disease progression. Finally, oneMF patient was hospitalized due to bleeding from gastric ulcer.No SAEs were registered in PV patients. Reasons for temporary or definitive Givinostat interruption. Drug-related adverse events led to at least one dose levelreduction in 10 patients (34%), for a median of 30 d (range, 5–49 d). Fifteen patients experienced at least one temporary interruption. Reasons for dose reduction or transientsuspension included grade 1–3 diarrhoea, gastric pain, liverenzymes elevation, fatigue, transient QTc elongation and on-target side effects (grade 2 thrombocytopenia and grade 3anaemia in one patient each).Primary reasons for definitive treatment discontinuationwere disease progression ( n  = 6, 21%), drug-related adverseevent ( n  = 1, 4%, grade 2 thrombocytopenia at week 9), notdrug-related adverse event ( n  = 1, 4%, psychiatric symptomsat week 10), withdrawn Informed Consent ( n  = 2, 8%), andbaseline QTc out of normal ( n  = 1, 4%). Grade 3 psychiatricsymptoms (the patient was disoriented), considered unrelatedto study drug, were observed in one PV patient who went off study after 10 weeks of treatment. The QTc prolongation wasobserved in one MF patient who had a borderline QTc >450 ms (QTc = 457 ms) before the experimental treatmentwas started and experienced a transient further QTc elonga-tion (grade 2 toxicity). The patient was immediately droppedfrom the study and treatment stopped after 9 d from study entry. A. Rambaldi  et al  448  ª  2010 Blackwell Publishing Ltd,  British Journal of Haematology  ,  150 , 446–455  Efficacy  Efficacy end-points are presented on an intention-to-treat(ITT) basis, by which all 29 patients were considered. One PVpatient (Patient 13, Table II) and 1 MF patient (Patient 22,Table III) did not receive at least 14 doses of Givinostattreatment without interruption; whereas one MF patient(Patient 26, Table III) was inadvertently recruited in violationof the protocol having a borderline QTc > 450 ms(QTc = 457 ms) before Givinostat was started. The medianduration of therapy was 20 weeks (range, 6–24). Clinical response in PV/ET patients.  Eleven out of 13 PV/ETpatients completed the 24 scheduled weeks of treatment(Table II). One patient (Patient 12) left the study after10 weeks of Givinostat treatment because of psychiatricsymptoms (disorientation). In one patient (Patient 1),complete haematological response was demonstrated after12 weeks and was maintained until the final evaluation atweek 24. Notably, this patient was included in the study because of intolerance to hydroxycarbamide (leg ulcers) andpruritus. Six additional patients achieved a rapid andsustained response qantified as partial at the end of treatment. Phlebotomy requirement was abolished in 7/10patients (70%) and the median platelet count significantly decreased from 865 (range, 347–1458)  · 10 9 /l at study entry to 497 (range, 233–1602)  · 10 9 /l at the end of treatment( P   = 0 Æ 0128). Similarly, the median leucocyte countsignificantly decreased, from 16 (range, 5–45 Æ 2)  · 10 9 /l atstudy entry to 11 Æ 7 (range, 3 Æ 7–35 Æ 8)  · 10 9 /l at the end of treatment ( P   = 0 Æ 0076).Among eight patients presenting with palpable splenomeg-aly at baseline, five (Patients 3, 4, 5, 7 and 8) achieved acomplete response (spleen no more palpable) and one patient(Patient 9) had a partial response after 12 weeks of Givinostattreatment. One patient (Patient 8) lost the clinical completeresponse after 24 weeks due to reappearance of a palpablespleen. A short course of concomitant hydroxycarbamide was Table I.  Safety assessment . Systemic organ class Preferred termCTCAE grade Total Total1 2 3 N % N %Patients evaluable for AEs – – – – – – 29 100Patients with AEs – – – – – – 28 97Gastrointestinal disorders Diarrhoea 10 7 1 18 62 27 91Nausea 2 1 0 3 10Gastric pain 0 2 0 2 7Abdominal pain 0 1 0 1 3Dyspepsia 1 0 0 1 3Flatulence 1 0 0 1 3Gingival bleeding 1 0 0 1 3Blood and lymphatic system disorders Anaemia 5 0 1 6 21 11 37Thrombocytopenia 2 1 0 3 10Leucopenia 0 1 0 1 3Neutropenia 0 1 0 1 3General disorders and administration siteconditionsFatigue 1 4 0 5 17 7 24Fever 2 0 0 2 7Investigations Electrocardiogram QT prolonged 3 2 0 5 17 7 24Weight decreased 1 1 0 2 7Metabolism and nutrition disorders Anorexia 2 0 0 2 7 3 10Hypertriglyceridaemia 0 1 0 1 3Skin and subcutaneous tissue disorders Rash 2 0 0 2 7 4 13Erythema 0 0 1 1 3Pruritus 1 0 0 1 3Nervous system disorders Paraesthesia 1 0 0 1 3 2 6Psychiatric symptoms 0 0 1 1 3Hepatobiliary disorders Hypertransaminasaemia 0 0 1 1 3 1 3Reproductive system and breast disorders Breast discomfort 1 0 0 1 3 1 3Surgical and medical procedures Constipation 1 0 0 1 3 1 3Note that: (a) each patient is counted once in each row; (b) in case of more than one occurrence of the same Adverse Event (AE) per patient the onewith the worst grade is used.Adverse Events assessed as possibly related to Givinostat were evaluated on all 29 patients and scored according to the MedDRA dictionary version11.0 (http://www.meddramsso.com). The percentage of adverse events was determined on all 29 patients. Givinostat Treatment for CMNs ª  2010 Blackwell Publishing Ltd,  British Journal of Haematology  ,  150 , 446–455  449  Table II.  Baseline and post-treatment parameters of 13 PV/ET patients . PtAge(years)/Sex HCpre-baselinetreatmentBaseline Week 12 Week 24HCtreatmentduringthe Study ResponseHCT(%) PhlebPlateletcount(10 9 /l)WBCcount(10 9 /l)Spleensize(cm) Pruritus  JAK2 V617F MutationalStatus (%)HCT(%) PhlebPlateletcount(10 9 /l)WBCcount(10 9 /l)Spleensize(cm) Pruritus  JAK2 V617F MutationalStatus (%)HCT(%) PhlebPlateletcount(10 9 /l)WBCcount(10 9 /l)Spleensize(cm) Pruritus  JAK2 V617F MutationalStatus (%)1 47/F Yes 42 Yes 347 5 Æ 0 0 Grade 2 39 35 No 279 4 Æ 0 0 No 31 34 No 287 3 Æ 7 0 No 18 Yes CR 2 48/M No 48 Yes 704 13 Æ 4 0 Grade 2 39 43 Yes 325 7 Æ 4 0 No 41 46 No 406 7 Æ 1 0 No 35 No PR 3 42/F Yes 48 Yes 1174 16 Æ 9 2 Grade 1 41 30 Yes 641 10 Æ 4 0 No 37 42 No 768 11 Æ 7 0 No 37 No PR 4 57/M No 44 Yes 1039 16 Æ 0 2 Grade 2 45 36 No 427 6 Æ 7 0 No 33 37 No 461 6 Æ 8 0 No 30 No PR 5 60/F Yes 41 Yes 865 11 Æ 6 1 Grade 2 71 46 Yes 695 20 Æ 3 0 Grade 1 68 45 No 497 14 Æ 3 0 No 56 Yes PR 6 36/F No 45 Yes 447 10 Æ 9 14 Grade 2 23 49 No 328 9 Æ 6 10 No 19 46 No 233 8 Æ 8 12 No 15 Yes PR 7 76/F Yes 43 No 987 38 Æ 9 3 Grade 2 100 42 No 675 32 Æ 8 0 No 91 40 No 705 35 Æ 8 0 No 96 No PR 8 42/M No 42 Yes 1022 20 Æ 1 12 Grade 2 67 49 No 379 12 Æ 6 8 Grade 1 75 48 No 453 13 Æ 3 8 Grade 1 58 No No9 66/F Yes 45 Yes 1273 23 Æ 1 19 No 71 44 Yes 628 14 Æ 3 0 No 61 52 Yes 586 7 Æ 0 3 No 50 No No10 40/M No 46 Yes 613 21 Æ 9 0 No 37 46 No 517 11 Æ 4 0 No 31 46 Yes 676 16 Æ 5 0 No 25 No No11* 55/M Yes 41 No 1458 45 Æ 2 0 No 29 37 No 1071 17 Æ 6 0 No 33 41 No 1602 29 Æ 5 0 No 32 Yes No12 71/F Yes 43 No 620 11 Æ 6 5 Grade 2 100 – – – – – – – – – – – – – – No Out of study atweek 10  13   66/F Yes 50 Yes 728 9 Æ 6 0 Grade 1 38 – – – – – – – – – – – – – – No Out of study atweek 11§*ET patient.  Not drug related AE.  Patient not evaluable for efficacy.§Withdrawn consent.Pruritus was defined and graded according to the common toxicity criteria: grade 1 if mild and localized, grade 2 if intense or widespread, grade 3 if intense or widespread and interfering with quality of life.HC, hydroxycarbamide; WBC, white blood cell. A .R  a m b   a l     d  i      e  t   a l     4   5   0    ª 2  0 1  0 B l     a  c k  w e l    l    P   u b  l    i     s h  i    n  gL   t    d   , B r  i     t  i     s h   J    o ur  n a l     of   H  a  e m a  t   ol     o  g  y   , 1   5   0    ,4 4  6 –4  5  5 
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