Molecular responses and chromosomal aberrations in patients with polycythemia vera treated with peg-proline-interferon alpha-2b

Fifty-one polycythemia vera (PV) patients were enrolled in the phase I/II clinical study PEGINVERA to receive a new formulation of pegylated interferon alpha (peg-proline-IFNα-2b, AOP2014/P1101). Peg-proline-IFNα-2b treatment led to high response rates on both hematologic and molecular levels. Hematologic and molecular responses were achieved for 46 and 18 patients (90 and 35% of the whole cohort), respectively. Although interferon alpha (IFNα) is known to be an effective antineoplastic therapy for a long time, it is currently not well understood which genetic alterations influence therapeutic outcomes. Apart from somatic changes in specific genes, large chromosomal aberrations could impact responses to IFNα. Therefore, we evaluated the interplay of cytogenetic changes and IFNα responses in the PEGINVERA cohort. We performed high-resolution SNP microarrays to analyze chromosomal aberrations prior and during peg-proline-IFNα-2b therapy. Similar numbers and types of chromosomal aberrations in responding and non-responding patients were observed, suggesting that peg-proline-IFNα-2b responses are achieved independently of chromosomal aberrations. Furthermore, complete cytogenetic remissions were accomplished in three patients, of which two showed more than one chromosomal aberration. These results imply that peg-proline-IFNα-2b therapy is an effective drug for PV patients, possibly including patients with complex cytogenetic changes. Am. J. Hematol. 90:288–294, 2015. © 2014 The Authors. American Journal of Hematology published by Wiley Periodicals, Inc.


Introduction
Polycythemia vera (PV) represents a frequent type of BCR-ABL negative myeloproliferative neoplasm (MPN) and is characterized by an elevated erythrocyte mass, as well as a variable presence of thrombocytosis and leukocytosis [1,2]. PV patients have a risk of developing thrombosis, bleeding and disease transformation to secondary myelofibrosis as well as secondary acute myeloid leukemia. In the vast majority (95%) of PV patients the JAK2-V617F mutation is found and in around 3% of PV patients the less common JAK2 exon 12 mutations [3][4][5][6][7][8][9]. The functional consequences of these mutations are a constitutively active JAK2 kinase signaling and cytokine hypersensitivity of myeloid cells [5,8,[10][11][12].
Present treatment options for PV patients include phlebotomy, low-dose aspirin, hydroxyurea and interferon alpha (IFNa) [2]. Already in the 1980s therapeutic efficacy of recombinant IFNa for MPN patients was reported [13][14][15]. IFNa not only induced hematologic and molecular responses in most MPN patients but was also described to be nonleukemogenic [16]. However, the use of recombinant IFNa was limited by frequent drugrelated toxicities and subsequent treatment discontinuations. The chemical linkage of polyethylene glycol (peg) to IFNa increased the plasma half-life and improved the toxicity profile of peg-IFNa considerably [17]. To date, the cellular and molecular mechanisms involved in the myelosuppressive Additional Supporting Information may be found in the online version of this article. The copyright line for this article was changed on 20 August 2015 after original online publication. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Interne 1 Hemato-Oncology, Krankenhaus Der Elisabethinen Linz, Linz, Austria; 5 Department of Internal Medicine IV, Wels-Grieskirchen Hospital, Wels, Austria; 6 effect of IFNa remain as unclear as the influence of constitutive and acquired genetic changes on the magnitude of IFNa responses. To evaluate the impact of acquired chromosomal aberrations we analyzed chromosomal lesions by high-resolution SNP microarrays in PV patients treated with a new formulation of peg-IFNa (AOP2014/P1101, peg-proline-IFNa-2b) in a phase I/II clinical trial.

Methods
Patient samples. Patients were enrolled in the phase I/II clinical study PEGIN-VERA (identifier: NCT01193699; www.clinicaltrials.gov) [18,19]. The study was approved by the Institutional Review Board of the Medical University of Vienna and the Austrian Drug Agency (AGES) and all patients provided written consent. Granulocytes were isolated from peripheral blood by standard gradient centrifugation using Histopaque 1077 (Sigma-Aldrich, St. Louis, MO). Genomic DNA was isolated from granulocytes and whole blood samples using the Wizard Genomic DNA Purification Kit and the Maxwell 16 Tissue DNA Purification Kit (Promega, Madison, WI).
Hematologic response criteria. Hematologic response was defined similar to the European LeukemiaNet (ELN) criteria [20]. Complete hematologic response (CHR) required hematocrit <45%, platelet count 400 3 10 9 /L, white blood cell count 10 3 10 9 /L, normal spleen size, freedom of phlebotomy in the past 2 months and absence of thromboembolic events. In case of concomitant hydroxyurea (HU), at least 2 weeks after the last HU administration had to pass to qualify for diagnosing CHR. Partial hematologic response (PHR) was defined by hematocrit <45% without phlebotomy with persistent splenomegaly or elevated platelet count (>400 3 10 9 /L) or at least a 50% reduction of phlebotomy requirements. No hematologic response (NHR) was any response that did not fulfill CHR or PHR criteria. Best individual hematologic response was defined as the best response observed for a given patient. Detailed clinical outcomes were reported recently [18,19].
Molecular response criteria. To evaluate molecular response, JAK2-V617F burden was determined by allele specific PCR and JAK2 exon 12 mutations by a fragment analysis-based assay, as previously reported (each with a detection limit of 1%) [21][22][23]. Molecular response was defined similar to the revised ELN criteria [24]. Patients required a JAK2 mutant allele burden of 20% at baseline for response assessment and latest follow-up samples were used for response definition. Complete molecular response (CMR) was defined by an undetectable residual JAK2 mutant allele burden. Partial molecular response (PMR) required 50% decrease in JAK2 mutant allele burden from baseline value and no molecular response (NMR) was a <50% reduction in JAK2 mutant allele burden from baseline value.
SNP microarray analysis. DNA samples were processed and hybridized to Genome Wide Human SNP 6.0 arrays (Affymetrix, Santa Clara, CA) according to the manufacturer's protocol. Raw data was analyzed for quality, copy number variation and loss of heterozygosity using Genotyping Console version 4.1.1 software (Affymetrix). Chromosomal aberrations (deletions, gains and uniparental disomies-UPDs) were annotated manually. UPDs were annotated if they had a terminal location (end of chromosomal arm) and size of 1 Mb. Terminal UPDs in patients with extensive (>10 Mb) interstitial runs of homozygosity and all aberrations that mapped to known copy number variation loci according to the Database of Genomic Variants (DGV version 10, human reference genome assembly hg19) were excluded.
Statistical analysis. To test for significant differences Fisher's exact test was used for categorical data and Mann-Whitney test for continuous data. Wilcoxon signed rank test was used to compare JAK2 mutant allele burden at baseline and on pegproline-IFNa-2b treatment. All tests were two-tailed and P values lower than 0.05 were considered significant. For data management and analysis Microsoft Office Excel 2007 (Microsoft, Redmond, WA) and GraphPad Prism version 6.01 for Windows (GraphPad Software, La Jolla, CA) were used.

Patient characteristics
Fifty-one PV patients were included in the phase I/II clinical trial PEGINVERA to receive a new formulation of peg-IFNa (AOP2014/ P1101, peg-proline-IFNa-2b). Patient characteristics are outlined in Table I and Supporting Information Table I. The cohort included 31 male patients (61%) and the median age of patients was 56 years. The median time from PV diagnosis to study entry was 511 days and 17 patients (33%) received hydroxyurea before study entry. For 11 patients (22%) PV related events (thrombosis and bleeding) were observed before investigational treatment. Patients received a median dose of 244 lg peg-proline-IFNa-2b and the median follow-up time of the cohort was 80 weeks. The median JAK2 mutant allele burden at treatment start was 41% and JAK2 mutant allele burden changes over time for the whole cohort are displayed in Supporting Information Fig. 1.

Influence of chromosomal aberrations on peg-proline-IFNa-2b response
To analyze chromosomal aberrations for patients treated with pegproline-IFNa-2b, we performed high-resolution SNP microarrays for 51 baseline samples (at start of peg-proline-IFNa-2b treatment) and 46 follow-up samples (latest follow-up samples that were available at time of array analysis) (Supporting Information Table II and Fig.  1A). Typical aberrations for MPN were observed, such as chromosome 9p uniparental disomies (UPDs), 14q UPDs, trisomies of chromosome 8 and 9. Over 70% of the baseline samples showed at least one chromosomal aberration, of which chromosome 9p UPDs were the most prevalent ones.
To evaluate if cytogenetic lesions influence the response to pegproline-IFNa-2b we compared the number of chromosomal aberrations in the different response groups. Similar numbers of chromosomal aberrations were observed in the diverse molecular response groups (Fig. 1B). Also in hematologic response groups no differences were found (Fig. 1D). Furthermore, we assessed if specific, recurrent (present in at least two patients) cytogenetic changes might explain the diverse response outcomes. Lesions that were present in more than one patient were distributed over all three molecular response groups with no significant differences in their frequencies (Fig. 1C). Comparing hematologic response groups revealed similar results with no associations of recurrent aberrations to specific response groups and both chromosome 14q UPDs were observed in complete hematologic response (CHR) patients (Fig. 1E). Overall this indicates that responses on molecular and hematologic levels are achieved independently of chromosomal aberrations.

Peg-proline-IFNa-2b induces hematologic and molecular response
From the 51 included patients 47 were evaluable for hematologic responses with a median follow-up time of 74 weeks (range, 10-146 weeks). A partial hematologic response (PHR) was present in 45% of the patients (Fig. 2A). Only one patient (2%) did not show any response (NHR) and 53% of the patients reached a CHR. The median time to CHR was 18 weeks (range, 10-122 weeks).
Thirty-five patients were assessed for molecular responses as they had a JAK2 mutant allele burden of 20% or higher at treatment start and at least one follow-up sample available (Supporting Information Table III). The median follow-up time for molecular response assessment was 584 days (range, 61-1,127 days). A molecular response was generally accomplished gradually over time and the median time to response was 433 days (range, 133-633 days). Molecular responses were achieved in 51% of the patients and in 26% a residual JAK2  mutant allele burden of 5% or lower was accomplished (Fig. 2B). In two patients (6%) the JAK2-V617F mutation was undetectable in the latest follow-up sample, indicating a complete molecular response (CMR). A partial molecular response (PMR) required a decrease of 50% or more of the JAK2 mutant allele burden. This was accomplished in 46% of the patients (Fig. 2B). As PMR patients showed a continuous reduction of the JAK2 mutant allele burden, we suspect that the remission depth will further improve over time on continuous therapy (Supporting Information Table III). Moreover, the overall JAK2 mutant allele burden of the 35 patients analyzed for molecular response decreased significantly on peg-proline-IFNa-2b treatment (P < 0.0001; Fig. 2C). The JAK2 mutant allele burden changed from a median of 48% at treatment start to 21.5% at the current median follow-up time of 19 month. We have not observed patients that initially showed a molecular response and relapsed molecularly during therapy to no molecular response (NMR).
Next we compared the hematologic and molecular responses in 33 patients that had both response data available (Fig. 2D). Patients achieving CHR showed significantly better molecular responses than PHR patients (P 5 0.0373). Notably, in some cases a CHR or PHR was achieved without a molecular response. In conclusion, pegproline-IFNa-2b leads to a sound clinical and molecular response in the majority of PV patients.

Factors affecting the response to peg-proline-IFNa-2b
Because the number and type of chromosomal aberrations had no significant influence on the outcome of the therapy, we next assessed which other parameters could have affected response rates. The first factor examined was the JAK2 mutant allele burden at the start of therapy. Both clinical and molecular responses were achieved independently of the initial JAK2 mutant allele burden (Fig. 2E,F). Furthermore, we divided patients into two groups according to their JAK2 mutant allele burden at baseline (low, 20-59%; high 60%). We observed similar median times to molecular response in both groups (442 and 423 days, respectively). Thus, JAK2 mutant allele burden at treatment start seems to have no impact on peg-proline-IFNa-2b treatment outcomes, at least in our cohort. To exclude observation time biases we focused our analysis on patients with a follow-up time of more than 400 days (mean duration to achieve a molecular response). No significant differences between molecular responding (CMR and PMR) and nonresponding (NMR) patients were seen for factors such as sex, age or previous hydroxyurea therapy, besides a trend for older patients responding worse to the therapy (Table  II). Overall these results suggest that other factors (such as germline variants) might influence the response to peg-proline-IFNa-2b.

Cytogenetic changes during peg-proline-IFNa-2b therapy
Chromosomal aberrations that were not present at peg-proline-IFNa-2b treatment start and only found in the follow-up sample may indicate clonal evolution during therapy. These emerging clones might cause acquired peg-proline-IFNa-2b resistance and/or accelerated disease progression. Acquired cytogenetic lesions were detected in three patients. Patient 5007 with PMR showed a small clone with a deletion of chromosome Y, patient 1004 with NMR had a single gene deletion on chromosome 10p (USP6NL) and patient 3003 (baseline JAK2 mutant allele burden lower than 20%) showed a single gene gain on chromosome 3q (FXR1) and a single gene deletion on chromosome 7p (NXPH1) (Supporting Information Table II).
Chromosomal aberrations that are lost on peg-proline-IFNa-2b treatment indicate complete cytogenetic remission, thus a molecular response. In three patients such a complete cytogenetic response was achieved, all three of them showing chromosome 9p UPDs at treatment start (Fig. 3). One patient had in addition to the 9p UPD trisomies of chromosome 8 and 9 and another one a chromosome 14q UPD. This indicates that peg-proline-IFNa-2b is able to target clones with lesions in addition to mutated JAK2.

Discussion
In a previous study we reported that chromosomal aberrations emerged at the time of IFNa resistance in a patient with primary myelofibrosis [25]. Therefore, we hypothesized that either specific types or the number of cytogenetic lesions might impact IFNa treatment outcome. In this study we performed a detailed analysis of molecular responses to peg-proline-IFNa-2b in a large cohort of PV patients in which high-resolution SNP array karyotypes were obtained. We observed similar numbers and types of chromosomal aberrations in the different response groups (Fig. 1B-E and Table II). Thus, responses to peg-proline-IFNa-2b are independent of chromosomal lesions in patients with PV. Patients with complex karyotypes may perhaps equally benefit from peg-proline-IFNa-2b therapy as patients with no detectable chromosomal aberration.
Peg-proline-IFNa-2b treatment led to high response rates on both molecular and clinical levels and as previously reported, molecular responses were generally preceded by hematologic responses ( Fig.  2A-C) [26,27]. CHR was accompanied by significantly better responses on molecular levels compared to PHR (Fig. 2D). Similar results were reported for MPN patients that were treated with hydroxyurea, where the main factor for completing a major molecular response was a major hematologic response [28]. In contrast, another study reported that responses on clinical and molecular levels were not associated with each other [29]. We observed as well absence of molecular response for some CHR or PHR cases, but at a much lower rate.
No difference in the JAK2 mutant allele burden was observed in the diverse response groups at treatment start, suggesting that the outcome of peg-proline-IFNa-2b therapy is likely independent of baseline JAK2 mutant allele burden (Fig. 2E,F and Table II). Similar observations were reported for peg-IFNa-2a treated PV patients, where the JAK2-V617F reduction was not influenced by the initial JAK2-V617F allele burden [26]. Moreover, PV patients with a fast molecular response to peg-IFNa-2a showed no significant difference in  their initial JAK2-V617F allele burden compared to slow molecular responders [30]. Factors such as age and hydroxyurea pretreatment did not significantly influence the outcome to peg-proline-IFNa-2b therapy (Table II). Complete cytogenetic remission on peg-proline-IFNa-2b treatment was observed in three patients, showing that clones with more lesions than JAK2 mutation are also sensitive to the therapy (Fig. 3). Because we did not find evidence for chromosomal lesions being responsible for an absence of IFNa response, the question on the mechanisms underlying IFNa resistance remains open. Recent studies report inconclusive findings whether clones with mutated TET2 can persist IFNa therapy and potentially be involved in IFNa response outcomes [31][32][33]. Also the frequency of MPN associated mutations in addition to JAK2 did not significantly influence IFNa responses [33,34].
We did not observe any relapses to NMR, indicating that a resistance to IFNa was present from the start of therapy. This intrinsic resistance might be caused by somatic changes already present at treatment start or constitutive germline variants, such as in the case of hepatitis C therapy where IL28B was significantly associated with the response to IFNa and ribavirin [35][36][37]. Because we and others could not obtain sufficient evidence for the involvement of somatic aberrations in IFNa resistance, we believe that germline variants may determine the outcome to IFNa in most cases. The observed high response rates, that are independent of chromosomal aberrations, makes peg-proline-IFNa-2b a promising therapeutic for PV. Future studies should help to reveal factors that affect IFNa therapy and ultimately identify resistance mechanisms as well as patients that benefit from IFNa therapy.