Reduction In JAK2V617F Allele Burden Research Articles

Effective Management of Polycythemia Vera With Ropeginterferon Alfa-2b Treatment.

Polycythemia vera (PV) is a myeloproliferative neoplasm. Ropeginterferon alfa-2b is a new-generation polyethylene glycol-conjugated proline-interferon. It is approved for the treatment of PV at a starting dose of 100 µg (50 µg for patients receiving hydroxyurea (HU)) and dose titrations up to 500 µg by 50 µg increments. The study was aimed at assessing its efficacy and safety at a higher starting dose and simpler intra-patient dose escalation. Forty-nine patients with PV having HU intolerance from major hospitals in China were treated biweekly with an initial dose of 250 µg, followed by 350 µg and 500 µg thereafter if tolerated. Complete hematological response (CHR) was assessed every 12 weeks based on the European LeukemiaNet criteria. The primary endpoint was the CHR rate at week 24. The secondary endpoints included CHR rates at weeks 12, 36 and 52, changes of JAK2V617F allelic burden, time to first CHR, and safety assessments. The CHR rates were 61.2%, 69.4% and 71.4% at weeks 24, 36, and 52, respectively. Mean allele burden of the driver mutation JAK2V617F declined from 58.5% at baseline to 30.1% at 52 weeks. Both CHR and JAK2V617F allele burden reduction showed consistent increases over the 52 weeks of the treatment. Twenty-nine patients (63.0%) achieved partial molecular response (PMR) and two achieved complete molecular response (CMR). The time to CHR was rapid and median time was 5.6 months according to central lab results. The CHRs were durable and median CHR duration time was not reached at week 52. Mean spleen index reduced from 55.6 cm2 at baseline to 50.2 cm2 at week 52. Adverse events (AEs) were mostly mild or moderate. Most common AEs were reversible alanine aminotransferase and aspartate aminotransferase increases, which were not associated with significant elevations in bilirubin levels or jaundice. There were no grade 4 or 5 AEs. Grade 3 AEs were reversible and manageable. Only one AE led to discontinuation. No incidence of thromboembolic events was observed. The 250-350-500 µg dosing regimen was well tolerated and effectively induced CHR and MR and managed spleen size increase. Our findings demonstrate that ropeginterferon alfa-2b at this dosing regimen can provide an effective management of PV and support using this dosing regimen as a treatment option.

Ruxolitinib Treatment in Polycythemia Vera Results in Reduction in JAK2 Allele Burden in Addition to Improvement in Hematocrit Control and Symptom Burden

Introduction:In the pivotal RESPONSE and RESPONSE 2 trials, the Janus kinase (JAK)1/JAK2 inhibitor ruxolitinib was superior to best available therapy (BAT) at providing hematocrit control and complete hematologic response, as well as in improving disease-related symptoms, in patients with polycythemia vera (PV). Here we report the results of a post hoc pooled efficacy analysis using data from the RESPONSE studies. Methods:RESPONSE and RESPONSE 2 were randomized, open-label, multicenter phase 3 trials that assessed efficacy and safety of ruxolitinib in adults with PV who had resistance or intolerance to hydroxyurea. Splenomegaly was required for enrollment into RESPONSE but not for RESPONSE 2; patients in both studies were randomized 1:1 to ruxolitinib (starting dose, 10 mg twice daily) or BAT (most commonly hydroxyurea [59%, 49%] or interferon formulations [12%, 13%] in RESPONSE and RESPONSE 2, respectively). Patients in either trial who were randomized to BAT could cross over to ruxolitinib at the time of primary analysis (Week 32 in RESPONSE; Week 28 in RESPONSE 2) or later for efficacy or safety reasons. In this pooled analysis, hematocrit control was assessed in the pooled population at Weeks 28 and 80 and was defined as hematocrit <45% that was maintained since Week 16 with ≤1 phlebotomy occurring postrandomization and before Week 4. Symptom control (using the Myeloproliferative Neoplasm Symptom Assessment Form Total Symptom Score [MPN-SAF TSS]) and changes over time in JAK2V617F allele burden were also assessed. Response rate 95% CIs were calculated using the Clopper-Pearson exact method, and the odds ratio (OR) and 95% CI for comparisons of the proportion of patients achieving ≥50% reduction from baseline in MPN-SAF TSS were calculated by the Mantel-Haenszel method. Results:Overall, 371 patients were randomized in RESPONSE and RESPONSE 2 (ruxolitinib, n=184; BAT, n=187). Patient baseline characteristics were similar across trials and treatment groups and have been previously published. Mean (SD) age in the overall pooled analysis population was 61.8 (11.0) years; most patients were male (62.5%) and White (88.1%). At Week 28, 62.0% (95% CI, 54.5%-69.0%) of ruxolitinib patients achieved hematocrit control compared with 18.2% (12.9%-24.5%) of BAT patients (Figure 1). Durable hematocrit control was maintained to Week 80 by 47.3% (95% CI, 39.9%-54.8%) of patients randomized to ruxolitinib (nearly all patients in the BAT arm crossed over to ruxolitinib). The percentage of patients achieving ≥50% reduction from baseline in MPN-SAF TSS at Week 16 (the only common timepoint in both studies) was higher for ruxolitinib vs BAT (48.7% [95% CI, 40.7%-56.8%] vs 18.0% [95% CI, 12.5%-24.6%]; OR, 4.3 [95% CI, 2.6-7.2]). Mean (SD) change from baseline to Week 16 in MPN-SAF TSS was −4.4 (10.0) for ruxolitinib patients and 0.6 (6.9) for BAT patients. JAK2V617F allele burden decreased consistently from baseline to Week 208 in patients randomized to ruxolitinib. Mean JAK2V617F allele burden decreased from 66.1% to 41.4% at 4 years. Best change from baseline in JAK2V617F allele burden is shown in Figure 2, with a decrease of ≥25% in 57.1% of patients randomized to ruxolitinib and ≥50% decrease in 38.9%. Similar reductions in JAK2V617F allele burden were observed following crossover from BAT to ruxolitinib (Figure 2), with a best decrease of ≥25% and ≥50% in 48.7% and 35.3% of patients, respectively. Analysis of the BAT group is limited by small patient numbers after Week 32, as most patients crossed over to ruxolitinib, with a best decrease of ≥25% and ≥50% in 7.2% and 2.6% of patients respectively. Conclusions:In this pooled analysis of RESPONSE and RESPONSE 2, patients with PV treated with ruxolitinib achieved durable hematocrit control through Week 80 and had better symptom control at Week 16 than those who received BAT. Reductions in JAK2V617F allele burden were consistently observed through Week 208 in patients treated with ruxolitinib, including those who crossed over from BAT. Taken together, these results provide further evidence of the patient benefit of ruxolitinib in patients with PV with or without splenomegaly.

Ropeginterferon-alfa2b resolves angina pectoris and reduces JAK2V617F in a patient with clonal hematopoiesis of indeterminate potential: A case report

The JAK2V617F mutation is an acquired somatic mutation, which is prevalent in patients with the Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs). In these diseases the mutation gives rise to constitutive JAK-STAT signaling with increased blood cell counts and in vivo activation of neutrophils and platelets as well, which altogether contribute to a chronic inflammatory and thrombogenic state with a 12-fold increased risk of coronary disease. Treatment with recombinant interferon-alpha2 (rIFN) reduces the JAK2V617F allelic burden in a large number of MPN-patients. Long-term treatment with rIFN associates with low-burden JAK2V617F in a subset of patients and a decreased thrombosis risk as well. In the general population the JAK2V617F mutation has been shown to associate with ischemic heart disease and thrombosis. Based upon the above observations we herein report the first patient with CHIP-JAK2V617F, in whom treatment with rIFN resolved severe angina pectoris. During a short period off rIFN the symptoms reappeared to resolve in concert with reduction of JAK2V617F allele burden, when rIFN was reinstituted. The JAK2V617F mutation may be a novel therapeutic target to prohibit the development of cardiovascular diseases using rIFN either as monotherapy or in combination with potent anti-inflammatory agents.

Treatment of Ropeginterferon Alpha-2b Achieves Hematologic Remission and Molecular Response in Patients with Hydroxyurea- and/or Anagrelide-Resistant/Intolerant Myeloproliferative Neoplasms

▪Background:Ropeginterferon alpha-2b (Ropeg) is a novel pegylated interferon-alpha approved for the treatment of polycythemia vera (PV) in Europe and Taiwan. Prior to its approval in Taiwan, the major options for patients with myeloproliferative neoplasms (MPNs) included hydroxyurea (HU) and/or anagrelide. Patients who are HU/anagrelide resistant/intolerant have limited options, as ruxolitinib is not subsidized by the national health insurance in Taiwan for PV. In 2017, the manufacturer provided a compassionate use program (CUP) for patients who were HU and/or anagrelide resistant/intolerant. Herein, we assessed the efficacy and safety of Ropeg in 20 MPN patients.Methods:To be eligible, patients must be resistant or intolerant to currently available therapies for MPN in Taiwan, mainly HU and anagrelide. Patients with autoimmune disorders, psychiatric illness, and acute/chronic infections were excluded. An accelerated dosing regimen was used, starting from 250 µg, and increased by 100 µg every two weeks until it reached the target dose of 500 µg, if no self-reported discomforts or abnormalities in biochemical or hematological profiles were observed. Efficacy assessments included hematologic parameters, phlebotomy need, and JAK2V617F allele burden. Hematologic remission was defined as platelets ≤400 x 10^9/L and white blood cells <9.5 x 10^9/L for essential thrombocythemia (ET), and platelets ≤400 x 10^9/L, white blood cells <10 x 10^9/L, and hematocrit <45% with no phlebotomy in the past 3 months for PV. Molecular response was defined as a reduction in JAK2V617F allele burden of at least 50% from baseline if baseline value was less than 50%, and a reduction of at least 25% from baseline if the baseline level was at least 50%. Each case was independently reviewed and approved for the use of Ropeg by both Institutional Review Board and the Ministry of Health and Welfare in Taiwan.Results:A total of 20 patients received treatment, which included 14 PV, 4 ET, 1 post-ET myelofibrosis (MF), and 1 pre-fibrotic primary MF (Table 1). There were 12 female and 8 male patients with a median age of 56.1 years old. Of these 20 patients, 18 had JAK2V617F mutation and 5 had a history of thrombosis. Of the 18 ET and PV patients, 13 achieved hematologic remission. The ET patients seemed to achieve hematologic remission faster than PV patients (19.3 vs. 33.2 weeks). Of the 18 patients with JAK2V617F mutation, 7 PV patients and 1 post-ET MF patient achieved molecular response, which took a median of 46 weeks after Ropeg treatment. Reduction in JAK2V617F allele burden was observed in 12 patients. One MF patient discontinued treatment due to disease progression. Another PV patient discontinued treatment due to acute myeloid leukemia transformation, although after treatment, the patient returned to PV state and continued Ropeg treatment. Overall, the drug was well tolerated, as most of the treatment-related adverse events (AEs) were mild to moderate. The AE profile was consistent with those from the phase 3 PROUD/CONTI-PV study. There were no unbearable side effects that led to treatment discontinuation.Conclusion:Our study provided evidence in the efficacy and safety of Ropeg for the treatment of HU-/anagrelide-resistant/intolerant MPNs. Hematologic remission was observed in ET and PV patients, whereas molecular response was observed in only PV patients, possibly due to the small sample size of ET patients. Our experience with Ropeg suggests it to be a promising option for the treatment of MPNs with drug-resistance/intolerance. [Display omitted] DisclosuresChen: PharmaEssentia: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen, Celgene, Novartis, and Panco Healthcare: Honoraria. Shih: PharmaEssentia Co: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene Ltd: Research Funding; Ltd: Research Funding; Novartis: Research Funding. OffLabel Disclosure:Ropeginterferon alfa-2b is a novel interferon alpha indicated for the treatment of polycythemia vera in Europe and in Taiwan. This abstract describes the use of this agent for the treatment of myeloproliferative neoplasm patients with hydroxyurea/anagrelide resistance/intolerance.

A pharmacokinetic evaluation of ropeginterferon alfa-2b in the treatment of polycythemia vera

ABSTRACT Introduction Polycythemia vera (PV) is a Philadelphia chromosome-negative chronic myeloproliferative neoplasm (MPN). A newly developed PV treatment option, ropeginterferon alfa-2b, contains recombinant human alfa monoisomer as an active ingredient, resulting in a novel pharmacologic profile and improved tolerability. Efficacy studies conclude remarkable long-term hematological response and sustained JAK2V617F allele burden reduction. Ropeginterferon alfa-2b compound has been approved for the treatment of polycythemia vera without symptomatic splenomegaly. Areas covered Current clinical trials are investigating the role of ropeginterferon alfa-2b in the first-line setting of treatment for PV. The safety and efficacy results of completed trials are summarized in this review. Metabolic, pharmacokinetic issues are also discussed of ropeginterferon alfa-2b. Expert opinion Ropeginterferon alfa-2b is a targeted therapeutic option in the treatment of PV, representing a significant improvement compared to conventional cytoreductive therapies. The single isomer entity of the recombinant human interferon alfa-2b and the mono-pegylation method imparts favorable properties to the compound. The use of ropeginterferon alfa-2b allows extended dosing interval, reduces side effects, and may increase the overall survival of PV patients by reducing the risk of progression to myelofibrosis or acute leukemia. Clinical data suggests that the compound may provide a disease-modifying option for PV patients with asymptomatic splenomegaly.

Ruxolitinib for Patients (Pts) with Polycythemia Vera: Responders Vs Non-Responders As Defined in the Response Trial

Introduction: The RESPONSE trial (NCT01243944) compared ruxolitinib (Rux) and best available therapy (BAT) in pts with polycythemia vera (PV) who were intolerant of or resistant to hydroxyurea (HU) according to modified European LeukemiaNET criteria. In the primary analysis, at week (Wk) 32, 60% of (pts) randomized to Rux achieved HCT control (HCT &lt;45%). The present analysis evaluated the effect of baseline characteristics on HCT response at Wk 32, and aimed to determine the long-term clinical efficacy of Rux in pts who did and did not achieve the protocol-defined HCT control (i.e., HCT control responders and non-responders at Wk 32) in RESPONSE at Wk 256. Methods: Adult pts with phlebotomy-dependent PV with splenomegaly, and resistant to or intolerant of HU were enrolled. Pts were randomized to receive Rux (at a starting dose of 10 mg BID) or single-agent BAT (1:1). HCT control was defined as lack of phlebotomy eligibility between Wks 8−32 with no more than 1 phlebotomy eligibility between randomization and Wk 8. Phlebotomy eligibility was based on protocol-defined HCT values (HCT &gt; 45% and ≥ 3 percentage points higher than baseline or &gt; 48%, whichever was lower; regardless of receipt of phlebotomy), and pts with missing data or assessments outside of protocol-defined time windows were considered non-responders. In this analysis, a logistic regression model was fitted to identify the significant baseline factors to predict HCT control response at Wk 32. Time to phlebotomy eligibility in the HCT control responders and time from the first phlebotomy eligibility to the second phlebotomy eligibility in the HCT control non-responders were plotted, and the changes in hematological parameters (HCT, WBC and platelet count), spleen volume and allele burden over time, up to Wk 256, were studied in HCT control responders and non-responders who were randomized to Rux treatment arm in RESPONSE. Results: A total of 222 pts were randomized to receive either Rux (n = 110) or BAT (n = 112). Baseline WBC (P=0.0198) and baseline JAK2 V617F allele burden (P=0.0159), were found to be predictors of the HCT response within Rux treated pt group (n = 110). In the HCT responder subgroup of the Rux arm, 23% (15/66) pts needed their first phlebotomy by Wk 256. In the HCT non-responder subgroup of the Rux arm, out of 28 patients who experienced their first phlebotomy between Wk 8 and Wk 32, 64% (18/28) of pts required subsequent phlebotomy by Wk 256, with a median duration of 28.4 Wks (12.7, NA). Pts receiving Rux demonstrated controlled hematologic parameters (HCT, WBC, and platelets) over the course of study, regardless of whether they were HCT control responders and HCT control non-responders at Wk 32. From Wk 48 to Wk 80, 97% HCT control responder pts and 84% HCT control non-responder pts of the Rux treatment arm required no phlebotomies. From Wk 80 to Wk 256, 91% and 68% of the evaluable pts in the Rux treatment arm remained phlebotomy-free for HCT control responders and non-responders, respectively. By Wk 256, spleen volume on an average was reduced from baseline by approximately 35% and 50% for HCT control responders and non-responders, respectively. In pts with available assessments, allele burden on an average was reduced approximately from 80% at baseline to 55% at Wk 256 in the HCT control responders, and approximately from 70% at baseline to 40% at Wk 256 in the HCT control non-responders. Conclusions: The results from present analysis demonstrated that the benefits of the Rux treatment were not limited to pts who achieved HCT control at Wk 32. Patients treated with Rux were able to maintain hematological parameters, spleen volume reduction, and JAK2 V617F allele burden reduction for a longer duration (up to 5 years), regardless of whether they were HCT control responders or non-responders at Wk 32. Disclosures Verstovsek: Constellation: Consultancy; Pragmatist: Consultancy; Incyte: Research Funding; Roche: Research Funding; NS Pharma: Research Funding; Celgene: Consultancy, Research Funding; Gilead: Research Funding; Promedior: Research Funding; CTI BioPharma Corp: Research Funding; Genetech: Research Funding; Blueprint Medicines Corp: Research Funding; Novartis: Consultancy, Research Funding; Sierra Oncology: Research Funding; Pharma Essentia: Research Funding; Astrazeneca: Research Funding; Ital Pharma: Research Funding; Protaganist Therapeutics: Research Funding. Kiladjian:Novartis: Honoraria, Research Funding; Celgene: Consultancy; AOP Orphan: Honoraria, Research Funding. Wroclawska:Novartis Pharma AG: Employment. Dong:Novartis: Employment. Vannucchi:Celgene: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Italfarmaco: Membership on an entity's Board of Directors or advisory committees.

Evaluation of Bone Marrowmmorphology in Addition to JAK2 Allele Burden Is Essential for Assessing Disease Status in Recombinant Interferon Alpha-Treated Polycythemia Vera Patients

IntroductionPolycythemia vera (PV) is a myeloproliferative neoplasm (MPN) associated with the JAK2 V617F mutation. Recombinant interferon alfa-2b (rIFNα-2b) or pegylated rIFN2α (pegrIFNα-2a) treatment in PV is effective in inducing hematologic and molecular responses. Reduction in JAK2V617Fallele burden (%V617F) has been used increasingly as a surrogate marker of disease response to treatment and as a unique criterion for discontinuation of therapy. To date, no studies have evaluated the relationship between changes in %V617F and established indicators of disease activity such as bone marrow cellularity and degree of fibrosis. Having observed persistent marrow hypercellularity or progressive fibrosis in PV patients despite a significant reduction in %V617F, we therefore systematically examined the correlation of %V617F with marrow morphology. We also assessed immunohistochemical expression of pSTAT5 as a marker of JAK2 activation.MethodsThe diagnosis of PV was based on the WHO criteria which include presence of erythrocytosis and/or an increased Cr51 red cell mass, JAK2V617F mutation and typical bone marrow findings such as hypercellularity due to panmyelosis and megakaryocyte morphology consistent with PV. Patients with two marrow examinations and quantitative JAK2 levels measured at a median of 1.2 months from the biopsy date were eligible for inclusion. The JAK2V617Fallele burden in peripheral blood samples was determined by pyrosequencing. Clinical, hematologic and molecular responses were graded according to ELN criteria. Marrow fibrosis was scored according to the WHO three-tiered semi quantitative grading system. Immunohistochemical staining for p-STAT5 was performed on clot sections in a subset of patients.ResultsWe identified 15 patients for inclusion. The median %V617F at the time of the initial biopsy was 62.2% (18.2-100%), which decreased to a median of 33% (0-93.7%) on subsequent evaluation (p=0.02). Patients were initially treated with peg rIFNα-2a 45-90 mcgm weekly (n=13) or rIFNα-2b (n=2), 1x106units thrice weekly, and gradually increased based upon response and tolerance. The median duration of treatment between biopsy was 4.2 years (0.8 - 6.6). All patients achieved either a complete (CHR) (n=8) or a partial (PHR) (n=7) hematologic response. Of these 15 patients, 3 achieved a complete molecular response (CMR), 4 a partial molecular response (PMR), and 8 patients no molecular response (NMR). We observed no correlation between clinical and molecular response: of the 8 patients who achieved CHR, 2 achieved a CMR and 6 did not. There was no correlation between clinical response and fibrosis or cellularity; of 8 patients who achieved CHR, 5 showed persistent/increased hypercellularity and 6 had persistent/increased fibrosis.We also found no correlation between marrow morphologic changes and molecular response. Of the 3 patients with CMR, all had persistent hypercellularity (range: 60-90%) and increased/persistent fibrosis. Among the 4 patients who achieved a PMR, 2 had increased cellularity and 2 decreased cellularity. All had increased or persistent fibrosis. Among the 8 patients with NMR, 2 had an increase in marrow cellularity, 2 no change, and 4 an insignificant decrease. Fibrosis increased in 2, was unchanged in 3, and decreased in 3. Thus, there were no significant correlations between changes in %V617F and cellularity (κ=0.02) and fibrosis (κ=0.04). Immunohistochemical staining for p-STAT5 expression in megakaryocyte nuclei showed no correlation between degree of positivity and %V617F.ConclusionsAlthough we observed a statistically significant decrease in %V617F after treatment with rIFNα, there were no parallel changes in marrow morphology. In all patients with CMR, we observed persistent or worsening marrow disease, suggesting to us a need for continued therapy. In patients who achieved either CHR or PHR, we observed persistent hypercellularity and persistent/progressive fibrosis. Our results question the use of JAK2V617F allele burden as the sole criterion for discontinuation of rIFNα therapy. The prognostic significance of failing to attain a morphologic response in patients who have achieved a hematologic and/or molecular response remains unresolved. Resolution of these issues with longer follow-up is important, as premature discontinuation of rIFNα may allow the pathogenic mechanisms of the disease to progress unfettered. DisclosuresOrazi:Novartis: Honoraria.

A phase II study of panobinostat in patients with primary myelofibrosis (PMF) and post-polycythemia vera/essential thrombocythemia myelofibrosis (post-PV/ET MF)

Myelofibrosis is a chronic and progressive myeloproliferative neoplasm characterized by anemia, splenomegaly, debilitating symptoms and leukemic transformation. Ruxolitinib, an oral JAK1/2 inhibitor, is highly effective in ameliorating systemic symptoms and reducing splenomegaly. Current clinical research is focused on the evaluation of agents based on pre-clinical rationale that can result in disease course modification. Panobinostat is a pan-histone deacetylase inhibitor that has demonstrated clinical activity as a single agent in early phase trials of myelofibrosis. We previously conducted a phase I trial of panobinostat monotherapy in patients with myelofibrosis and determined 25mg thrice weekly as the recommended phase II dose. We then completed an investigator initiated, Simon 2-stage, phase II trial of 22 myelofibrosis patients at our single institution. After 6 cycles of therapy, the overall response rate by IWG-MRT criteria was 36% (8/22; 95% CI: 16–56%). The median percent reduction in spleen volume was 34% (range, 1.6%–73%) in eight evaluable patients. The average reduction in JAK2V617F allele burden was 6.8% (Range; −4.0% to 20.2%) and one patient obtained a complete molecular response. Six patients remained on therapy in the extension phase for a median of 18 months (range, 7–44). Treatment discontinuation was frequent due to patient/physician perception of therapy ineffectiveness. The optimal dosing of panobinostat for the treatment of MF remains somewhat ill-defined but appears to be most effective and better tolerated when administered at lower doses over a prolonged duration of therapy.

Impact of Underlying Mutational Profile, and Changes during Treatment, in MPN Patients Treated with JAK Inhibitors

Background. Most patients (pts) with MPN derive clinical benefits from treatment with JAK inhibitors (JAKi) but predictors of response or loss of response have not been clearly identified.Aims. To address the impact of underlying mutational profile of driver mutations (JAK2V617F, CALR, MPL), including other genes of JAK/STAT pathway, and "subclonal" (epigenetic, spliceosome) mutations, and their modifications under treatment, on the response to JAKi.Methods. 100 patients with WHO2008-diagnosis of MPN, 39 PV, 14 ET, 64 MF (37 PMF, 27 PPV/PET-MF), were included. 25 (12PV, 13 MF) had received hydroxyurea (HU), 75 (20 PV, 7 ET, 48 MF) a JAKi (67 ruxolitinib, 8 fedratinib). Response criteria were as by IWG-MRT (Blood 2013; 121:4778; Blood 2013; 122:1395). Mutations in 22 genes (JAK1, JAK2, JAK3, EZH2, ASXL1, TET2, IDH1, IDH2, CBL, SRSF2, DNMT3A, NFE2, SOCS1, SOCS2, SOCS3, SH2B3, STAT1, STAT3, STAT5A, STAT5B, SF3B1, U2AF1) were analyzed in blood DNA at baseline (bl) and at the latest available sample by deep sequencing with Ion Torrent-PGM. CALR mutations were analyzed by capillary electrophoresis; JAK2 V617F allele burden was measured by RT-qPCR assays. The nonparametric Wilcoxon rank-sum test, Kaplan-Meier method and Cox regression, were used for statistical analysis.Results. A. Impact of baseline mutational profile. PV and ET:At bl, 17/40 (42.5%) pts had subclonal mutations. Spleen volume reduction (SVR) by IWG-MRT criteria was obtained in 100% of pts without subclonal mutations compared to 66% of those with >1 mutations (P=0.04). Presence of NFE2 mutations uniformly predicted for lack of SVR (P=0.03). Loss of SVR was predicted by ASXL1 mut (67% vs 12% in un-mutated; P<0.05). No correlation of mutations with symptomatic improvement, normalization of leukocyte and platelet count, and control of hematocrit to <45% in PV pts was noticed. A trend to lower pruritus responses were seen in PV pts harboring >1 subclonal mutations (P=0.06). JAK2 V617F homozygous pts were more prone to have platelet count control (81%) compared to heterozygous (30%; P<0.04). MF:At bl, 33/64 (51.5%) pts had subclonal mutations. No correlation of mutations with symptomatic improvement, normalization of leukocyte and platelet count was found. SVR was not predicted by a high molecular risk status (Leukemia 2013;27:1861; Blood 2014;123:2157); however, harboring >1 of the 22 mutations was negatively associated with SVR (P=0.02) (HR 1.9, 95% CI 1.0-4.6). No correlation of JAK2 V617F or CALR mutations, or the allele burden, on SVR or loss of SVR was discovered.B. Changes during treatment. PV and ET:follow-up mutational analysis was performed at median treatment duration of 2.9y HU, 4.7y ruxolitinib, 1.7y fedratinib in 50 pts for driver mutation and 40 pts for subclonal genes. Of HU pts (n=12), 33% each had JAK2 V617F allele burden stable, increased and reduced by >10%. Median reduction of allele burden was -18.3%. 1 pt acquired 2 novel subclonal mutations (ASXL1, EZH2) and 2 showed increased allele burden of ASXL1 and TET2 by >10%. Among JAKi pts (n=30), 20 pts (66.7%) had JAK2 V617F allele burden reduction by a median of -33.3% (-13.6 to -100%), stable in 9. Degree of allele burden reduction was positively correlated with length of JAKi treatment (P=0.04). 1 pt acquired novel ASXL1, 2 pts had increase (ASXL1, EZH2) and 6 pts reduction of >10% of allele burden (4 TET2, 1 DNMT3A, 1 SH2B3). No correlation was seen with clinical response over time. MF: follow-up mutational analysis was performed at median treatment duration of 2.4y HU, 1.9y ruxolitinib, 1.4y fedratinib. HU pts (n=13): of the 7 JAK2 V617F mut, 4 increased and 1 decreased the allele burden, and 1 of 3 CALR mutated reduced allele burden, by >10%. 1 pt acquired CBL mutation, other mutations (n=7) were stable. Among JAKi treated pts, of the 41 JAK2 V617F mutated, 51% showed a median allele burden reduction of -14.8% (-10.5% to -53.9%). Of 10 pts with subclonal mutations at baseline, 4 clones (3 ASXL1 and 1 NFE2) increased and 2 (EZH2, ASXL1) decreased by >10%. Only 1 pt acquired a novel mutation in EZH2. No correlation was seen with clinical response over time.Discussion. These data suggest that only minimal influence on clinical response is provided by driver mutations and their allele burden, or subclonal mutations, in MPN patients receiving JAKi. The clinical relevance of different clone fluctuations over treatment with conventional therapy and JAKi remains to be addressed. DisclosuresVannucchi:Baxalta: Membership on an entity's Board of Directors or advisory committees; Shire: Speakers Bureau; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Mutational Profile of Patients with Polycythemia Vera Treated with Ruxolitinib in the Phase III Controlled Response Study

Background. The JAK1/2 inhibitor ruxolitinib (RUX) demonstrated clinical benefit compared with best available therapy (BAT) in patients (pts) with polycythemia vera (PV) in a phase 3 study (RESPONSE TRIAL) (NEJM 2015; 372:426). A reduction of JAK2 V617F allele burden of 12.1% from baseline at week (w)32 was observed in pts receiving ruxolitinib compared to BAT (1.2%). Conversely, no information was available about other mutations that may occur in some PV pts.Aim. To analyze the molecular landscape of PV pts enrolled in the RESPONSE trial specifically as regarded subclonal mutations.Methods: In the RESPONSE study, PV pts with intolerance or refractoriness to hydroxyurea, showing an enlarged spleen volume (SV) >450 ml and phlebotomy requirement, were randomized (1:1) to receive RUX (n = 110) or BAT (n = 112). After institutional approval and informed written consent, samples for genotyping were available in 150 cases (67.5% of total, 75 each RUX and BAT). Mutations in 22 genes (JAK1, JAK2, JAK3, EZH2, ASXL1, TET2, IDH1, IDH2, CBL, SRSF2, DNMT3A, NFE2, SOCS1, SOCS2, SOCS3, SH2B3, STAT1, STAT3, STAT5A, STAT5B, SF3B1, U2AF1) were analyzed in blood DNA at baseline and at the latest available sample by deep sequencing with Ion Torrent-PGM. CALR mutations were analyzed by capillary electrophoresis. JAK2 V617F allele burden was confirmed by RT-qPCR assays.Results. 149/150 patients had informative sequencing results (RUX, n = 74 and BAT, n = 75). Mutation frequency at baseline in RUX pts was: JAK2 V617F 97.3% (mean allele burden=83.7±20.0%); JAK2Exon12 1.3%; other mutations in JAK2 5.4%; JAK3 1.3%; ASXL1 4.0%; TET2 20.3%; EZH2 4.0%; CBL 1.3%; SH2B3 2.0%; SOCS1 2.7%; NFE2 2.7%; STAT5A 1.3%. No somatic variants were detected in CALR, JAK1, SRSF2, IDH1, IDH2, SOCS2, SOCS3, DNMT3, STAT1, STAT5B, SF3B1, U2AF1. These frequencies were comparable in BAT arm. One patient was un-mutated in all assessed genes. 28.4% and 8.1% of RUX and 32.0% and 8.0% in BAT pts had 1 and >2 subclonal mutations, respectively. The proportion of PV pts harboring at least 1 mutation in either ASXL1, EZH2, SRSF2, IDH1/2, was significantly lower (8.1% in RUX and 10.6% in BAT) compared to reference series of primary myelofibrosis pts (31%) as it was for those having >2 HMR mutations (0.0 in RESPONSE vs 7.4% in PMF) (Leukemia 2014;28:1804)The median duration of treatment corresponding to the latest available sample for analysis was 82.8w; at that time, 43 pts (58.1%) randomized to RUX achieved a JAK2 V617F allele burden reduction ≥10%, of which 15 (20.3%) had >50% reduction. Among the latter pts, the median allele burden was 83.7% at baseline, 84.9%, 55.1% and 44.3% at 1, 2, and 3 years. Three patients attained an allele burden below 5% (from 65.1%, 17.3% and 83.7% at baseline to 3.2%, 0.5% and 1.4%, respectively, at latest follow up).Of the 27 pts harboring subclonal mutations at baseline, 12 (44.4%) presented a reduction of mutational allele burden ≥10%: 4 in JAK2 (other than JAK2 V617F/exon 12 mutations), 4 in TET2, 3 in ASXL1 and 1 each in JAK3, EZH2 and SH2B3. In 10 of the 12 pts, comparable decreases in JAK2 V617F allele burden were observed, suggesting reduction of a single clone expressing both mutations. Conversely, in 5 pts (18.5%) the allele burden of a baseline TET2 clone at increased by ≥10% (range: 10-37%); of these, one had concurrent reduction of JAK2 V617F burden from 17.3 to 0.5, thereby suggesting two independent clones.Eight pts (29.6%) acquired new mutations: 3 in TET2, 3 in U2AF1, 1 in DNMT3A and 1 in IDH1. Among these, 4 pts had achieved a reduction ≥10% of JAK2 V617F allele burden (18.4%, 25.4%, 30.5% and 39.4%, respectively). Three pts (4.0%) progressed during treatment (2 myelofibrosis,1 acute leukemia); no novel acquired mutation in the 22 genes was observed in these pts. All 3 pts were homozygous for V617F (92.3%, 72.7% and 59.0%) and did not show appreciable changes of allele burden during treatment.Conclusions. The current study identifies mutations and mutational combinations at baseline and during follow up in a representative cohort of pts enrolled in RESPONSE trial and treated with Ruxolitinib. We observed progressive reduction of JAK2 V617F allele burden that in some cases was associated with concurrent reduction of subclonal mutations. Conversely, emergency of novel clones as observed in some pts, whose significance might be clarified by ongoing analysis of hydroxyurea and phlebotomy treated patients that will be presented at the meeting. DisclosuresMahtab:Novartis Pharma AG: Employment. Rodriguez:Novartis Pharma: Employment. Vannucchi:Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Shire: Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees.

Busulfan for the Treatment of Myeloproliferative Neoplasms: The Mayo Clinic Experience

Background: The cytoreductive treatment options for myeloproliferative neoplasms, in those who have indications, may be limited to hydroxyurea and interferon. Busulfan has been reported to have some activity, but used less frequently in the management of BCR/ABL negative MPNs (Haanen CM et al Br J Cancer 1981; Brodsky American J of clinical oncology 1998). One study has shown significant JAK2V617F allele burden reduction with busulfan (Kuriakose ET et al; Haematologica 2013). Ruxolitinib was approved by the FDA for intermediate and high risk myelofibrosis; and as second line therapy for polycythemia vera in those who are intolerant or develop side effect to hydroxyurea.Objective: To assess the utility of busulfan, an old drug, in patients with myeloproliferative neoplasms who are intolerant to other forms of therapy.Methods: The mayo clinic data base from 1970 to 2014 was interrogated using the terms myeloproliferative disorder, polycythemia vera (PV), essential thrombocythemia (ET), myelofibrosis (MF) and busulfan. Patients' follow up information was collected till July 2015.Results: Seventy five patients with full demographic, diagnostic and therapeutic information were identified, and those included 37 patients with ET, 22 with PV, 12 with MF, and 4 with myeloproliferative neoplasms unclassifiable. The median age was 64 (range 31-91) years. After a median follow up of 17 years, 40 patients (53 %) died, and leukemic transformation was documented in 4 (5%). The median time for leukemic transformation was 86 months (12-229).1. Essential Thrombocytopenia (n=37): Twenty nine (79%) were females, and the median (range) age was 67 (33-90) years. At diagnosis the median (range) hemoglobin (Hgb) (gm/dL), white blood cell count (WBC) (X109/L), and platelet count (X109/L) were 13.6 (9.8-16.9), 10.2 (5-231), 1113 (593-2062) respectively. After a median follow up time of 230 months, 15 patients (41%) died, and leukemic transformation was documented in 1 patient who was also treated with radioactive phosphorous (P32). Leukemic transformation was documented 230 months from date of diagnosis. Follow up complete blood count was available in 20 patients and the median (range) Hgb, WBC and platelet count was 12 (9.9-16), 7.4 (3.1-25), and 267 (126-573) respectively.2. Polycythemia Vera (n=22): Fourteen (61%) were females, and the median (range) age was 64 (46-91) years. At diagnosis the median (range) Hgb, WBC, and platelet count were 17.5 (15.1-20.8), 11.5 (1.2-26.6), and 669 (185-2370) respectively. After a median follow up time of 188 months 13 (57%) patients died, and leukemic transformation was documented in 2 patients and one of them was given P32. Follow up complete blood count was available in 21 patients and their median (range) Hgb, WBC and platelet count was 12.9 (10-15.2), 7.2 (2.8-20), and 303 (124-833) respectively.3. Myelofibrosis (Primary and Post-PV and Post-ET myelofibrosis) (n=12): The median age was 52 (31-75) and 5 were females. The median Hgb, WBC, and platelet count were 13.6, 14.5, and 472. Six (50 %) patients did have splenomegaly (and 5 of them have splenic size reduction after busulfan), 3 underwent splenectomy, and 3 have no palpable spleen. At a median follow up of 208 months, and 39 months (range 78-401) from the start of busulfan, 10 (84%) patients died and no leukemic transformation was documented.Conclusion: Busulfan should be considered as alternative therapy in myeloproliferative neoplasms especially in ET and PV that are intolerant to other forms of cytoreductive therapy. Busulfan may be given as pulse therapy in controlling cell counts. DisclosuresOff Label Use: Busulfan as alternative therapy in myeloproliferative neoplasms.

Imetelstat, a Telomerase Inhibitor, Therapy for Myelofibrosis: A Pilot Study

Background: Current drugs in myelofibrosis (MF), including JAK inhibitors, do not induce complete or partial remissions. Imetelstat is a 13-mer lipid-conjugated oligonucleotide that targets the RNA template of human telomerase reverse transcriptase. A phase-2 study in essential thrombocythemia showed platelet-lowering activity of imetelstat accompanied by reduction in JAK2V617F allele burden, thus providing the main rationale for conducting the current study.Methods: The current study (NCT01731951) is investigator-driven and IRB approved. Drug and research funding were provided by Geron Corporation (Menlo Park, CA, USA). The objective was to obtain preliminary information on therapeutic activity and safety of imetelstat in DIPSS-plus high or intermediate-2 risk MF. Adverse events (AEs) were monitored by Common Terminology Criteria for Adverse Events (Version 4.03) and responses by the International Working Group criteria (Blood2013). Laboratory correlative studies included measurement of plasma cytokines and analysis of mutations.Results: 33 patients were accrued and met eligibility criteria (median age 67 years; 67% males); type of MF was primary 55% and post-PV/ET 45%; risk distribution was high 48% and intermediate-2 52%. Karyotype was abnormal in 16 (48.5%) patients. 79% of patients had received prior therapy including JAK inhibitors in 33%.Protocol treatment: Imetelstat was administered by a 2-hour intravenous infusion (9.4 mg/kg) every three weeks (cohort A; n=19) or weekly x 3 followed by every three weeks (cohort B; n=16). As of 7/24/2014, 11 (33.3%) patients remain on active treatment (median number of cycles completed for all patients = 7; range 1-21). Three patients have died post-registration: intracranial hemorrhage (related), upper gastrointestinal hemorrhage (unrelated) and disease progression. Median follow-up for living patients was 11.0 months (range, 3.6 – 18.4). 22 (66%) patients have discontinued treatment, mainly because of suboptimal response (n=15) or disease progression (n=3).Toxicity: Treatment-related grade 4 neutropenia was seen in 6 (18%) patients and grade 4 thrombocytopenia in 7 (21%). Grade 3 anemia was seen in 9 (27%) patients. Two patients experienced grade 4+ non-hematologic AE: grade 5 intracranial hemorrhage (related), grade 5 upper gastrointestinal hemorrhage (not related); other grade 3+ non-hematologic AEs were seen in only one patient. Regardless of attribution, treatment-emergent grade 1 or 2 liver function test abnormalities affected bilirubin 46%, ALP 52%, AST 55% and ALT 24%. There were 3 instances of grade 3 ALP and one of bilirubin elevations.Efficacy: There were 7 (21.2%) complete (CR; n=4) or partial (PR; n=3) remissions, occurring at a median of 5 cycles of treatment (range 1-9). All 4 CR patients experienced reversal of BM fibrosis and 3 of them a complete molecular response. Six of the 7 CR/PR patients remain in remission (median 9.9 months, range 4.8-14.7). Other responses included anemia response in 4 (31%) of 13 transfusion-dependent patients, >50% reduction in palpable spleen size in 9 (39%) of 23 evaluable patients, ≥50% reduction in leukocyte count in 8 (80%) of 10 patients with marked leukocytosis (WBC >25 x 10(9)/L), resolution of leukoerythroblastosis in the majority of patients, and normalization of platelet count in 9 (75%) of 12 patients with thrombocytosis.Laboratory correlative studies: Mutational frequencies were 79% for JAK2, 18% for CALR, 3% for MPL, 33% for ASXL1, 9% for IDH1/2 and 33% for spliceosome component mutations. CR/PR rates were 27% in JAK2-mutated vs 0% unmutated (p=0.3) and 32% in ASXL1-unmutated vs 0% mutated (p=0.07). CR rate was 38% in SF3B1/U2AF1-mutated vs 4% unmutated (p=0.036). Grade 3+ neutropenia or thrombocytopenia was more likely to occur in JAK2-unmutated (p=0.02) and ASXL1-mutated cases (p=0.049). Multi-cytokine panel screening showed significant differences between baseline and post-treatment samples involving several cytokines, including IL-1b, IL-5, IL-7, IL-17, VEGF, IL-8 and TNF-α (p<0.001 for all). CR/PR did not correlate with baseline cytokine levels.Conclusions: The current study identifies imetelstat as an active drug in patients with MF, but also reveals its potential to cause significant myelosuppression. The association between CR/PR and specific mutations suggests potential targeted activity that might be exploited for patient and disease selection. DisclosuresNo relevant conflicts of interest to declare.

JAK2V617F Complete Molecular Remission in Long-Term Follow-up of Patients with Polycythemia Vera and Essential Thrombocythemia Treated with Ruxolitinib

Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms (MPN) characterized by the presence of JAK2V617F mutation in >95% and 60% of patients (pts), respectively. This mutation usually affects one allele in ET while most PV pts are homozygous due to mitotic recombination. Acquisition of the JAK2V617F mutation is strongly associated with the germline 46/1 predisposition haplotype. Ruxolitinib is a JAK1/JAK2 inhibitor recently approved for myelofibrosis (MF) and under investigation in PV and ET pts intolerant or resistant to hydroxyurea. We enrolled 24 pts, 11 with PV and 13 with ET, in the phase II INCB18424-256 trial that overall included 34 PV and 39 ET pts. 21/24 pts were still on treatment at 5 years (yr), of which 19 JAK2V617F mutated. Results of the PV cohort have been reported recently (Verstovsek et al. Cancer, 2014): with a median follow up of 35 months (mo), the JAK2V617F allele burden decreased by a mean of 8%, 14%, and 22%, respectively, after 12, 24 and 36 mo. The proportion of pts who achieved a reduction ≥50 % at any time during the 1st yr, 2nd yr, and 3rd yr were 5.9%, 14.7%, and 23.5%, respectively, but no patients achieved a complete remission. In our series of pts we evaluated the JAK2V617F allele burden by two RTQ-PCR methods, according to Lippert (sensitivity, 0.8%) and to Larsen (sensitivity, 0.08%) method. We also analysed by next generation sequencing (NGS; Ion Torrent platform) a series of MPN-associated mutations including TET2, ASXL1, IDH1/2, LNK, CBL, SRSF2, EZH2 and MPL at baseline and at 5 yr of treatment in ruxolitinib treated pts who achieved a >25% JAK2V617F allele burden reduction at 5 yr (n=13/19). JAK2V617F allele burden decreased by a mean of 7%, 11%, and 19% at 12, 24 and 36 mo, and decreased further by a mean of 28% after 60 mo. Three (1 PV, 2 ET) of 19 pts (16%) achieved a 50% or greater allele burden reduction after 2 yr; no additional pts achieved this degree of allele burden reduction even in prolonged follow up. These 3 pts further improved their molecular response to a complete molecular response (CMR) after 5 yr of treatment. Their mean JAK2V617F allele burden was 46.6% at baseline, 28.3%, 16.3%, 8.7% and 0% after 1 yr, 2 yr, 3 yr and 5 yr, respectively. The JAK2 CMR was confirmed in at least one independent sample at 3 mo after first discovery. At this last timepoint, the PV pt was in complete haematological remission according to ELN criteria, the 2 ET pts were in partial remission due to platelet count still >400x109/L: 422x109/L and 812x109/L, respectively. BM histopathology in the 2 ET pts at 5 yr, while they were in CMR, showed still evidence of megakaryocyte hyperplasia. In the PV pt, histopathology at 5 yr is pending; evaluation at 3 yr, a time when she was in complete hematologic remission and JAK allele burden had decreased from 69 to 8%, showed normalization of cellularity, megakaryocyte and myeloid lineage compared to baseline but still slight erythroid hyperplasia. All 3 pts had normal karyotype at baseline that remained unchanged thereafter. CMR for JAK2V617F was confirmed by NGS. The 2 ET pts achieving CMR did not show any additional mutations, while the PV pts presented a TET2 Y867H mutation with an allele burden of 48.9% and 52%, respectively at baseline and 5 yr. No recurrent mutations in genes other than JAK2 were found in all other examined cases at baseline or at 5 yr. In 3 informative pts, we also analysed the proportion of JAK2V617F homozygous, heterozygous and wild type clones by the method of Hasan et al (Leukemia 2013) based on allelic discrimination of 46/1 haplotype and JAK2. We found that JAK2V617F/V617F clones were reduced by a mean of 95.5%, JAK2V617F/WT showed an uneven trend with a mean reduction of 45.54% while JAK2WT/WT conversely increased (mean 61.43%) at 5 yr, suggesting that in a subset a patients who present significant reduction of VF allele burden ruxolitinib may preferentially target the homozygous clones. Until now, complete molecular remission in PV pts has been described only in patients treated with interferon. Our data suggest that a subset of pts who present a rapid and sustained reduction of the JAK2V617F allele burden under ruxolitinib may eventually reach a condition of CMR with prolonged treatment. However, similar to findings with interferon, mutations establishing clonality, such as in TET2, may still persist in patients who eventually show the disappearance of JAK2V617F mutated subclones. DisclosuresVerstovsek:Incyte: Research Funding. Vannucchi:Novartis: Membership on an entity’s Board of Directors or advisory committees, Research Funding.

Abstract 3684: The combination of JAK inhibitor, ruxolitinib, pan-PIM inhibitor, LGH447, and CDK4/6 inhibitor, LEE011, in a preclinical mouse model of myeloproliferative neoplasia

Abstract The JAK/STAT axis is a critical component downstream of multiple cytokine and growth factor receptor signaling pathways. The genetic aberration of JAK2V617F and the associated activation of STAT in myeloproliferative neoplasia (MPN) is one example of the involvement of this pathway in human cancer. Activated JAKs phosphorylate STAT proteins, which then up-regulate the transcription of STAT target genes such as PIM1. Pim kinases are involved in the regulation of cell cycle and proliferation. The inhibition of JAK1/2 by the JAK inhibitor, ruxolitinib (RUX), results in the suppression of the JAK-STAT pathway and promotes significant clinical benefit in patients with myelofibrosis. Our previous preclinical studies in a Ba/F3-JAK2V617F-driven MPN model demonstrate that the combination of RUX and the pan-PIM inhibitor, LGH447, exhibits greater inhibition of spleen weight and some reduction of JAK2V617F allele burden, compared with the RUX monotherapy. Mutant JAK2V617F has been shown to increase CDC25A transcription through activated STAT5. It also regulates p27 at both the gene expression and phosphorylation levels. The activation of CDC25A and the modulation of p27 have been postulated to trigger the activation of cyclin dependent kinases (CDK), such as CDK4, to initiate cell cycle progression. Additionally, activated STATs and Pims have been shown to activate D cyclins that are upstream of CDK4/6. Here, we explored the hypothesis that CDK4/6 inhibition, in conjunction with JAK and PIM inhibition, would enhance therapeutic efficacy against MPN. LEE011 is a potent and selective inhibitor of CDK4/6. The combination of RUX and LEE011 was tested in an MPN model with Ba/F3 cells harboring EPOR-JAK2V617F. While RUX monotherapy reduced spleen weight and total tumor burden by more than 50%, it had marginal effect on JAK2V617F allele burden in this model. The combination of RUX and LEE011 resulted in an additional 2 to 3 fold reduction in spleen weight and total tumor burden. Yet, no clear modulation of JAK2V617F allele burden was observed. To further enhance the anti-tumor activity, we tested the triple combination of RUX, LGH447 and LEE011 in this MPN model. This triple combination resulted in &amp;gt;99% reduction of total tumor burden and a ∼96% reduction of spleen weight. Furthermore, the triple combination of RUX, LGH447 and LEE011 significantly down-modulated JAK2V617F allele burden by &amp;gt; 80%. Our preclinical data indicate that the triple combination of RUX, CDK4/6 inhibitor, LEE011, and pan-PIM inhibitor, LGH447, may preferentially impact the JAK2V617F mutant MPN clones. This combination also achieves greater reductions in tumor burden and spleen weight in our preclinical MPN model. Therefore, potentially greater therapeutic benefit in subgroups of MPN patients may be achieved with the triple combination of RUX, LEE011 and LGH447. Citation Format: Maria Pinzon-Ortiz, Xianhui Rong, Abdel Saci, Robert Schlegel, Gary Vanasse, Giordano Caponigro, Z. Alexander Cao. The combination of JAK inhibitor, ruxolitinib, pan-PIM inhibitor, LGH447, and CDK4/6 inhibitor, LEE011, in a preclinical mouse model of myeloproliferative neoplasia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3684. doi:10.1158/1538-7445.AM2014-3684

Combination therapy with interferon and JAK1-2 inhibitor is feasible: Proof of concept with rapid reduction in JAK2V617F-allele burden in polycythemia vera

We report a 55 year old woman with post-ET PV for 12 years, who experienced resolution of severe constitutional symptoms within 3 days, a marked reduction in splenomegaly and a rapid decline in the JAK2V617F allele burden during combination therapy with interferon-alpha2a and ruxolitinib. Within 4 weeks the patient achieved complete hematological remission with normalization of peripheral blood counts and within 10 months the JAK2V617F-allele burden was reduced from 90% to 28%. Such a rapid decline in the JAK2V617F allele burden is highly unusual in PV-patients during low-dose IFN-alpha2 monotherapy and this finding warrants a prospective study with combination therapy.

Molecular analysis of patients with polycythemia vera or essential thrombocythemia receiving pegylated interferon α-2a

AbstractPegylated interferon α-2a (PEG-IFN-α-2a) has previously been shown to induce hematologic and molecular responses in patients with polycythemia vera (PV) or essential thrombocythemia (ET). Here we present a follow-up of a phase 2 trial with PEG-IFN-α-2a treatment in 43 PV and 40 ET patients with detailed molecular analysis. After a median follow-up of 42 months, complete hematologic response was achieved in 76% of patients with PV and 77% of those with ET. This was accompanied by complete molecular response (CMR) (ie, undetectable JAK2V617F) in 18% and 17%, of PV and ET patients, respectively. Serial sequencing of TET2, ASXL1, EZH2, DNMT3A, and IDH1/2 revealed that patients failing to achieve CMR had a higher frequency of mutations outside the Janus kinase–signal transducer and activator of transcription pathway and were more likely to acquire new mutations during therapy. Patients with both JAK2V617F and TET2 mutations at therapy onset had a higher JAK2V617F mutant allele burden and a less significant reduction in JAK2V617F allele burden compared with JAK2 mutant/TET2 wild-type patients. These data demonstrate that PEG-IFN-α-2a induces sustained CMR in a subset of PV or ET patients, and that genotypic context may influence clinical and molecular response to PEG-IFN-α-2a.

Reductions in JAK2V617F allele burden with ruxolitinib treatment in COMFORT-II, a phase III study comparing the safety and efficacy of ruxolitinib to best available therapy (BAT).

6514^ Background: Ruxolitinib is a potent and selective JAK1/2 inhibitor approved for the treatment of myelofibrosis (MF) based on results of the phase 3 COMFORT studies. Ruxolitinib demonstrated rapid and durable reductions in splenomegaly and improved MF-related symptoms and quality of life of patients (pts) with MF. Since one measure of efficacy is molecular response, this analysis correlates changes in mutant allele burden (%V617F) with spleen size reduction in COMFORT-II. Methods: COMFORT-II is a randomized, open-label, phase 3 study comparing ruxolitinib 15 or 20 mg twice daily (BID) with BAT. The primary endpoint was a ≥ 35% reduction in spleen volume from baseline (BL) at week 48. Change in %V617F was measured by allele specific qPCR. Pts were stratified by reduction in %V617F (&lt; 10%, 10-20%, &gt; 20%) and results were correlated with achievement of the primary endpoint. Results: More pts in the ruxolitinib arm had ≥ 10% V617F reductions compared with BAT (41% vs 5%; P = .01; Table). The majority of reductions &gt; 20% were gradual and progressive over the course of the study; 2 pts had rapid reductions from 48% to 1% and 45% to 9% over 48 weeks. In the ruxolitinib arm, significantly more pts with a &gt; 20% V617F reduction achieved the primary endpoint compared with pts with a &lt; 10% reduction (79% vs 30%; P = .004); in each group, gender did not affect spleen response. For pts with &lt; 10% reductions (15 mg BID, n = 16; 20 mg BID, n = 24), the average total daily dose (TDD) was ruxolitinib 29.6 mg; pts with &gt; 20% reductions (15 mg BID, n = 3; 20 mg BID, n = 11) had a TDD of 35.3 mg. Conclusions: Pts who received ruxolitinib had larger reductions in JAK2V617F allele burden compared with BAT. %V617F reductions were gradual over the course of the 48-week study; longer follow-up is needed to determine the extent of allele burden reduction. [Table: see text]

Dynamics of Mutant Alleles in Patients with Advanced Essential Thrombocythemia (ET) or Polycythemia (PV) During Pegylated Interferon-Alfa-2A (PEG-IFN-α-2A; Pegasys) Therapy

Abstract▪281▪This icon denotes a clinically relevant abstract Background:PEG-IFN-α-2A induces complete hematologic response (CHR) in 76% and 80% of pts with advanced polycythemia vera (PV) or essential thrombocythemia (ET), respectively, which is accompanied by a reduction of at least 50% in the JAK2V617F mutant allele burden in 52% of pts with PV (undetectable in 18%) and in 29% of those with ET (undetectable in 18%). These data suggest selective targeting of JAK2V617F–positive clones. The impact of PEG-IFN-α-2A on other mutations frequently found in patients with PV or ET is unknown. Objectives:To investigate the dynamics of mutant JAK2, TET2, MPL, ASXL1 DNMT3a, EZH2, and IDH1/2 alleles in 71 pts with high-risk PV or ET treated with PEG-IFN-α-2A in a phase II study.Patients and Methods: We performed high-throughput mutational analysis of the regions of known mutations in JAK2, MPL, IDH1, IDH2, and all coding regions of TET2, ASXL1, EZH2, and DNMT3a in pretreatment samples from 71 patients from this trial and in 21 of them obtained serially during treatment or post-treatment. We then analyzed the mutational data to determine (a) distribution of mutations amongst response groups and (b) whether genetic aberrations in addition to the JAK2 mutational burden pre- or post-treatment correlated with response to therapy. Results:71 pts were analyzed: 12 good responders (at least partial molecular response [≥50% reduction in JAK2V617F allele burden]), 50 intermediate responders (only complete hematologic response), and 9 worst responders (no response or only a partial hematologic response). Median age was 50 years (range, 18–79), time from diagnosis to PEG-IFN-α-2a 51 months (range, 0–355), and number of prior therapies was 1 (range, 0–6). The median JAK2V617F allele burden at study entry was 20% (range, 4.8%-93.6%). The median follow-up was 40 months (range, 8–62). Mutations were found in the following genes in patients from this cohort as follows (using mutations found at any point in treatment): 39.4% JAK2, 14.1% TET2, 14.1% DNMT3a, 5.6% ASXL1, 1.4% EZH2, 1.4% IDH2. No significant differences were observed regarding the number of mutations in genes other than JAK2 between the worst responders and the best responders. The mutational frequency according to response to PEG-IFN-α-2A is shown in Table 1.The proportion of patients carrying 2 or more mutations was 8.3% among the good responders, 0% among the intermediate, and 11.1% among the worst responders. Samples during PEG-IFN-α-2a therapy were obtained in 21 pts (13 good, 4 intermediate, and 4 worst responders). Of note, 50% of the best responders and 22.2% of the worst responders had a mutation in addition to JAK2 prior to starting PEG-IFN-α-2a therapy. Interestingly, high-throughput sequencing done in sequential samples from 11/12 best responders and in 8/9 worst responders revealed that 16.7% of the former and 44.4% of the latter had acquired a new mutation in a gene other than JAK2 during therapy (p=0.04). High-throughput sequencing of individual clones retrieved from clonogenic cultures of bone marrow samples obtained during PEG-IFN-α-2a therapy will be presented.Table 1Mutational frequency by response groupJAK2TET2ASXL1DNMT3aEZH2IDH1/2Best (n=12)83.3%25%16.7%25%8.3%0%Intermediate (n=50)24%6%2%6%0%0%Worst (n=9)77.8%44.4%22.2%44.4%0%11.1%P value0.3850.2210.5810.1860.2750.129 Conclusion:High throughput sequencing analysis of a large number of patients with PV or ET receiving PEG-IFN-α-2a revealed important molecular heterogeneity. The fact that more patients in the worst responder group acquired mutations during therapy compared with the best responder group, may reflect either a possible effect of additional mutations on resistance to therapy, or it may be a consequence of genetic evolution of the malignant clone resistant to PEG-IFN-α-2a-induced eradication in the worst responders. Disclosures:No relevant conflicts of interest to declare.

High Rates of Molecular Response After Long-Term Follow-up of Patients with Advanced Essential Thrombocythemia (ET) or Polycythemia Vera (PV) Treated with Pegylated Interferon-ALFA-2A (PEG-IFN-α-2A; PEGASYS)

AbstractAbstract 461 Background:The use of IFN-α in polycythemia vera (PV) and essential thrombocythemia (ET) has been hampered by poor tolerance and inconvenient dosing schedules. The covalent attachment of polyethylene glycol to IFN-α renders a molecule with prolonged serum half-life, which can be administered weekly. Objectives:We conducted a phase II study of subcutaneous PEG-IFN-α-2a (Pegasys) in 84 patients (pts) with high-risk PV (n=44) or ET (n=40). We performed high throughput mutational analysis of JAK2, MPL, TET2, and ASXL1 in all pts. Patients and Therapy:Median age was 51 years (range, 18–79), time from diagnosis to PEG-IFN-α-2a 51 months (range, 0–355), and number of prior therapies was 1 (range, 0–6), including hydroxyurea (HU; n=47), anagrelide (AG; n=26), IFN-α (n=12: 5 oral and 7 sc), imatinib (n=7), and dasatinib (n=1). PEG-IFN-α-2a was initial therapy in 16 (19%) pts (7 PV) that refused HU. JAK2V617F was detected in 19/40 (48%) ET and in 42/44 (95%) PV pts. Nine (11%) pts had abnormal cytogenetics. Initial PEG-IFN-α-2a starting dose was 450 mcg/wk, but that was modified to the current starting dose of 90 mcg/wk. Results:After a median follow-up of 40 months (range, 8–62), 66/83 (80%) assessable pts have responded. Median time to response was 4 weeks (range, 0.5–26). Complete response (CR) was achieved by 62 (75%) pts (for ET: platelets <440×109/L, in the absence of thromboembolic events; for PV: Hb <15 g/dL, no phlebotomy, disappearance of splenomegaly) whereas 4 (5%) pts (2 PV, 2 ET) had a partial response ([PR]; no phlebotomy, off HU and AG, still palpable spleen). Of 5 pts with abnormal karyotype at study entry who were evaluable for response, 2 reverted to diploid cytogenetics.JAK2V617F to total JAK2 ratio was determined by quantitative pyrosequencing assay in all 84 pts prior to PEG-IFN-α-2a. Sixty-one (73%) pts carried the JAK2V617F mutation, which was quantitated at least once on therapy in 54 (64%). Overall, 29 (54%) had >20% reduction in JAK2V617F allele burden, including 10 (19%) in whom the mutation became undetectable (complete molecular response [CMR]) and 15 (28%) who had a >50% reduction (partial molecular response). Molecular responses have not yet reached a plateau among pts with PV.We also analyzed pts for mutations in exon 12 of JAK2, MPL, and the tumor suppressors TET2 and ASXL1 to determine their impact on response to PEG-IFN-α-2a. No pts carried JAK2 exon 12 mutations. One JAK2V617F−negative pt with ET had a MPLW515L mutation, achieved CHR but did not achieve a molecular response. Full length resequencing of all exons of TET2 and ASXL1 genes identified somatic TET2 mutations in 9/71 (13%) and somatic ASXL1 mutations in 3/71 (4%) pts; we identified TET2 (3 JAK2V617F− ET, 2 JAK2V617F+ ET, 3 JAK2V617F+ PV, 1 JAK2V617F− PV) and ASXL1 (1 pt with ET JAK2V617F+, ET JAK2V617F−, and PV JAK2V617F+) mutations in PV and ET pts who were JAK2V617F–positive and negative. TET2 or ASXL1 mutational status did not impact the likelihood of achievement of JAK2 molecular responses, and there was no difference in JAK2V617F allele burden with PEG-IFN-α-2a according to TET2 or ASXL1 mutational status. One pt with baseline mutations in JAK2, TET2, and ASXL1 became JAK2V617F–negative on PEG-IFN-α-2a.Most pts had grade 1–2 toxicities but at doses ≤90 mcg/wk, grade 3–4 toxicity was infrequent. Twenty-five (30%) patients were taken off study after a median of 9 months (range, 3–36) but only 13 (15%) of them due to therapy-related toxicity: g3 neutropenia, anorexia, depression (n=3), ischemic retinopathy, g2 fatigue (n=5), dyspnea, g2 neuropathy. The remaining 59 pts are currently receiving 450 mcg/wk (n=1), 360 mcg/wk (n=1), 240 mcg/wk (n=1), 180 mcg/wk (n=2), 135 mcg/wk (n=3), 90 mcg every 1 (n=8), 2 (n=12), 3 (n=2), or 4 wks (n=1), 45 mcg every 1 (n=9), 2 (n=5), 3 (n=6), or 4 wks (n=8). Conclusion:PEG-IFN-α-2a is remarkably active and acceptably safe in advanced, previously treated PV and ET. Clinical responses are frequently accompanied by significant reduction of JAK2V617F allele burden, which becomes undetectable in a proportion of them suggesting selective targeting of the JAK2V617F clones. Quantitative analysis of ASXL1 and TET2 mutational allele burden during PEG-IFN-α-2a therapy to determine clonal evolution, and methylcellulose-based clonogenic assays in pts who achieved CMR to assess for the presence of erythropoietin independent colony formation are ongoing and will be presented. Disclosures:No relevant conflicts of interest to declare.

A Phase I Evaluation of TG101348, a Selective JAK2 Inhibitor, in Myelofibrosis: Clinical Response Is Accompanied by Significant Reduction in JAK2V617F Allele Burden.

Abstract 755 Background:TG101348 is a potent, orally bioavailable, JAK2-selective small molecule inhibitor, that is currently being evaluated in a Phase I study for the treatment of myelofibrosis. Data from the dose escalation cohort (n=28; 30-800mg administered as a single daily dose) showed dose-linear plasma exposure, with mean elimination T1/2 at steady state ranging from 20 to 52 hours. The dose-limiting toxicity was asymptomatic grade 3 or 4 amylasemia/lipasemia that was reversible, and the maximum tolerated dose (MTD) was 680mg. The most frequent non-hematological toxicities were mild nausea, vomiting, and/or diarrhea that were easily controlled or resolved spontaneously. Grade 3/4 neutropenia and thrombocytopenia were observed in 14% and 25% of patients, respectively. TG101348 had activity in reducing spleen size, leukocyte count, and JAK2V617F (VF) allele burden. Here, we present updated results with a focus on data from the dose confirmation cohort who initiated treatment at a dose of 680mg/day. Results:Fifty nine patients (median age=66 years; range 43-86) have been treated – 28 in the dose escalation phase, and 31 in the dose confirmation phase. Overall, 44 patients had PMF, 12 post-PV MF, and 3 post-ET MF; 86% were VF-positive. Median palpable spleen size was 18cm and 22 patients were RBC transfusion-requiring at study enrollment. After a median follow-up of 12 weeks (range <1-76), 18 (31%) patients have discontinued treatment due to toxicity (n=7; thrombocytopenia=3, neutropenia=1), comorbidities (n=5), withdrawal of consent (n=4), or non-compliance/lack of response (1 each). The remaining 41 patients are currently at the following dose levels: 680mg (n=14), 520-600mg (n=16), 360-440mg (n=10), and 240mg (n=1). The cumulative drug exposure to date is 362 patient-months; exposure at or above MTD (≥680mg) is 154 patient-months. Forty patients (68%) started treatment at ≥680mg. Toxicity:TG101348 is well tolerated. Of the patients who started at ≥680mg, Gr3/4 neutropenia was observed in 15/0% and Gr3/4 thrombocytopenia in 20/10%. Twenty four (60%) patients did not require RBC transfusions at baseline (median Hgb=9.6g/dL; range 7.4-13.1); of these, 42% and 8% of patients developed Gr3 and Gr4 anemia, respectively. The majority of patients who started at ≥680mg developed mild nausea (1 Gr3), vomiting (1 Gr3), and/or diarrhea (3 Gr3) that were self-limited or easily controlled. Other non-hematological toxicities included Gr1/2 transaminitis (38%), Gr1/2 serum creatinine elevation (38%), and asymptomatic hyperlipasemia (33%). Efficacy:Thirty three patients who started at ≥680mg have completed at least 3 cycles of treatment; at 3 months, reduction in palpable spleen size (baseline median=18cms; range 6-32) was at least 50% in 22 (67%) patients; the spleen became non-palpable in 9 (27%) patients. All 21 patients with leukocytosis at baseline (WBC range 11 to 203 ×109/L) who started at ≥680mg have experienced a marked reduction in their WBC count (range 4 to 90); 70% had a normal WBC count at their last follow-up visit. Overall, 48 of the 51 VF-positive patients completed at least 1 cycle and were evaluable for response in VF allele burden; at last available follow-up, the median decrease in granulocyte mutant allele burden was 48%; 21 (44%) patients have had a ≥50% reduction, and in the group who started treatment at ≥680mg, 48% have had a ≥50% reduction. Of those evaluable, there was clinically significant benefit or resolution of constitutional symptoms, including early satiety, fatigue, cough, pruritus, and night sweats. Conclusions:TG101348 continues to be well tolerated in patients with myelofibrosis. Spleen and leukocyte responses are frequent, observed early, and produce substantial clinical benefit for patients. These responses are associated with significant decrease in VF allele burden and point to activity of TG101348 against the malignant clone in myelofibrosis. Disclosures:Pardanani:TargeGen: Research Funding; Cytopia: Research Funding. Off Label Use: Data from ongoing clinical trial will be presented. Gotlib:TargeGen: Research Funding. Jamieson:Merck: Research Funding; Pfizer: Research Funding; Wintherix: Consultancy; TargeGen: Research Funding; Celgene: Research Funding. Cortes:Targegen: Research Funding. Stone:Cephalon: ad hoc advisory board. Silverman:TargeGen: Consultancy. Shorr:TargeGen: Employment, Equity Ownership. Gilliland:Merck: Employment. Tefferi:TargeGen: Research Funding.