Page 79 - Haematologica3

Pegylated interferon-α in myelofibrosis
interferon therapy may provide a survival benefit for patients with intermediate- or high-risk Lille and dynamic International Prognostic Scoring System scores. It also reduced the JAK2V617F allele burden in most patients. These results further support the use of pegylated interferon in selected patients with myelofibrosis. (Clinicaltrials.gov #NCT02910258 and #NCT02897297).
Introduction
Primary myelofibrosis is a Philadelphia chromosome- negative myeloproliferative neoplasm characterized by splenomegaly, constitutional symptoms and cytopenia and/or proliferative features in peripheral blood. Secondary myelofibrosis may develop from either poly- cythemia vera or essential thrombocythemia.1,2 The main causes of death of patients with myelofibrosis include dis- ease progression leading to cachexia or infection, and acceleration and transformation of their disease into acute myeloid leukemia.3 The JAK2V617F mutation can be found in about half of myelofibrosis patients, while 20-30% carry a calreticulin (CALR) mutation and 5-10% a mutation in the thrombopoietin receptor gene MPL. These three driver mutations influence the clinical presentation and out- come: for example, CALR-mutated myelofibrosis patients are predominantly male, have higher platelet and lower leukocyte and red cell counts, and longer survival than those with the JAK2V617F mutation.4,5
In addition to these three driver mutations, other muta- tions are frequently found in myelofibrosis patients, main- ly in genes involved in epigenetic regulation or the splicing machinery. Some of these mutations have been associated with poorer survival and Vannucchi et al. have defined five “high molecular risk” genes: ASXL1, EZH2, SRSF2, and IDH1/2. Mutations in any of these genes dramatically decreased overall and event-free survival of the affected patients and the presence of more than one additional mutation conferred an even worse outcome.6,7
Several strategies have been used to alleviate the prolif- erative aspects of myelofibrosis (e.g., hydroxyurea, pipo- broman, 6-mercaptopurine) or the cytopenic ones (e.g., thalidomide and its derivatives, androgens, recombinant erythropoietin), as well as to manage splenomegaly (e.g., hydroxyurea, radiotherapy, splenectomy). The efficacy of these approaches is generally modest, especially with regards to cytopenia and does not clearly modify disease evolution.2
Ruxolitinib, a non-specific JAK1/JAK2 inhibitor approved for the treatment of symptomatic myelofibrosis patients, was recently shown to be very effective in reduc- ing the inflammatory component of these diseases with significant improvement of pruritus, fever, weight loss and splenomegaly. Although still debated, results of the COM- FORT I and II studies also suggest that ruxolitinib may increase overall survival of high-risk myelofibrosis patients compared to that of patients treated in the place- bo or “best available therapy” arms.8-12 To date, however, allogeneic stem cell transplantation remains the only cura- tive option for these patients.13-16
We have previously reported on the feasibility and hematologic results of myelofibrosis treatment with pegy- lated interferon-α2a in a prospective observational study conducted by the French Intergroup of Myeloproliferative neoplasms (FIM).17,18 Herein, we report the long-term out- comes of this large cohort of patients, focusing on survival
and incidence of acute leukemia. In addition, next-genera- tion sequencing data enabled us to assess the impact of interferon treatment on the prognosis associated with mutational patterns, and with the presence of non-driver mutations.
Methods
Patients’ recruitment
haematologica | 2018; 103(3)
Between December 2006 and April 2011 we prospectively recruited 62 patients treated with pegylated interferon-α2a in 17 centers affiliated to the FIM group. The inclusion criteria and methodology have been described elsewhere.18 This study was approved by the local Institutional Review Board and registered in ClinicalTrials.gov (NCT02910258). All participants gave written informed consent. The patients treated in the CHRU of Brest were also registered in the OBENE observatory (NCT02897297).
Pegylated interferon-α2a was initiated by physicians in accor- dance with local and national guidelines. During the period of this study, ruxolitinib was only available through clinical trials (approval for use in myelofibrosis in France was obtained in August 2012).
Molecular analyses
Samples from all the patients were characterized for the three driver mutations. Genomic DNA was extracted from blood neu- trophils or total leukocytes using the Flexigene DNA kit (Qiagen, Germany) according to the manufacturer’s recommendation.
JAK2V617F was quantified by real-time quantitative polymerase chain reaction analysis according to previously described meth- ods.19 MPLW515K/L mutations were screened for using the MPLW515L/K MutaScreen Kit (Qiagen) according to manufacturer’s instructions. Quantitative polymerase chain reactions were performed on ABI7500 instruments (Applied Biosystems). CALR exon 9 muta- tions were screened for by fragment analysis according to pub- lished methods.4 Polymerase chain reaction products were ana- lyzed on an ABI3130 instrument (Applied Biosystems).
Next-generation sequencing
Targeted next-generation sequencing was performed in 49 samples collected at the time of starting pegylated interferon-α2a treatment (34 JAK2V617F-positive, 12 CALR-positive, 3 triple-nega- tive). The next-generation sequencing panel included 26 genes (ASXL1, BCOR, CBL, CSF3R, DNMT3A, ETNK1, ETV6, EZH2, IDH1, IDH2, JAK2, KRAS, MPL, NRAS, PDGFRA, RUNX1, SETBP1, SF3B1, SH2B3, SRSF2, STAG2, TET2, TP53, U2AF1, ULK1, and ZRSR2) and the sequencing was performed using AmpliseqTM (Thermo Fisher Scientific, Foster City, CA, USA) cus- tom design. Library preparation and sequencing using PGMTM (Thermo Fisher Scientific) were performed according to the man- ufacturer’s instructions.
Mutations were detected using the Variant Caller v4.2 plugin from Torrent Suite Software and IonReporter v5.2 (Life Technologies). For mutation calling, arbitrary filters were fixed with variant allele frequencies >2% and depth >50X. False posi- tive variants were dropped after BAM analysis on Alamut®
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