Tefferi, Ayalew

Abstract Background : Current diagnosis of polycythemia vera (PV) is based on a set of clinical and laboratory criteria that were adopted more by consensus rather than because of support from systematic evidence. Accordingly, one major criterion for the diagnosis of PV requires the demonstration of increased red cell mass (RCM) as measured by radionuclide dilution methods. In order to adjust for the influence of obesity on RCM expressed in mL/kg, an expert radionuclide panel of the International Committee for Standardization in Haematology (ICHS) has recommended that the results be expressed in reference to body surface area and specific formulae for the prediction of normal values as well as guidelines for the interpretation of measured values have been proposed (J Nuclear Med1980;21:793, BJH1995;89:748). Nevertheless, there is limited data on either the performance or added value of RCM measurement, following these revised recommendations, for the diagnosis of PV in current clinical practice. Methods : The current study looks at a single institution experience with RCM measurement over the last 10 years involving patients in whom the test was performed to consider the diagnosis of PV. The study excluded patients that were previously treated with either phlebotomy or cytoreduction. Designation of diagnostic categories was based on both a retrospective and prospective analysis of clinical data, bone marrow histology, and other laboratory parameters including leukocyte count, platelet count, serum erythropoietin (EPO) level, serum B12 level, and leukocyte alkaline phospatase (LAP) score. A diagnosis of secondary polycythemia (SP) required the presence of a condition known to be associated with SP. Apparent polycythemia (AP) was represented by patients in whom the diagnosis of either PV or SP could not be made and the stability of hematocrit values was documented by serial measurements. Measurement and interpretation of RCM values were according to the aforementioned published criteria and separate analyses were performed for males and females. Results : i) Evaluation of test performance : The study cohort consisted of 105 patients (60 males; median age 62 years, range 16–89) including 25 with PV, 35 with SP, 38 with AP, and 7 with essential thrombocythemia (ET). Table 1 outlines the percentage of patients, in each disease category, whose measured values exceeded the 98–99% limits of the reference range (i.e. ±25% of the normal predicted mean for an individual patient). Table 1 Diagnosis % with increased RCM (m2) % with normal RCM (m2) % with decreased plasma volume % with increased plasma volume PV (n=25) 80 20 0 20 ET (n=7) 57.1 42.9 0 29 SP (n=35) 20 80 2.9 5.7 AP (n=38) 21.6 78.4 5.4 5.4 The results reveal that RCM measurement was neither adequately sensitive nor specific in distinguishing PV from the other disease categories. In addition, based on the aforementioned ICHS criteria, chronically contracted plasma volume appears to be an infrequent phenomenon in AP. ii) Evaluation of added value for the diagnosis of PV : Among the 19 PV patients with elevated RCM, serum EPO was measured in 17 and the results showed decreased levels in 16 (94%). Bone marrow biopsy was available for review in 9 patients and the results were consistent with PV in all instances (100%). LAP score was performed in 12 patients and 11 had LAP scores above 130 (92%). In none (0%) of the 19 patients was RCM measurement found to be vital for the diagnosis of PV. Conclusion : In the current retropsective study, RCM measurement was found to be neither diagnostically accurate nor essential for the diagnosis of PV.

Abstract Background : Neutrophil PRV-1 over-expression has been strongly associated with polycythemia vera (PV) and its quantitative measurement might complement the diagnostic distinction between PV and secondary polycythemia (Klippel et al. Blood2003;102:3569). Furthermore, the PV-specificity of the particular abnormality had been suggested by studies that demonstrated increased PRV-1 expression in post-polycythemic but not agnogenic myeloid metaplasia (Tefferi et al. Blood2003;103:3547) as well as by other studies that suggested the inevitable progression into PV of essential thrombocythemia (ET) associated with PRV-1 over-expression (Griesshammer et al. Ann Hematol2004;83:364). Still others have suggested the possibile utility of the particular assay in differentiating clonal from reactive myeloproliferation (Cilloni et al. Blood2004;103:2428). Methods : The purpose of the current study was two-fold; i) to further evaluate the specificity of neutrophil PRV-1 over expression to either PV or clonal myeloproliferation by testing the assay in atypical myeloproliferative disorders (AMPD) and ii) to evaluate eosinophil PRV-1 expression in AMPD associated with eosinophilia. In a prospective study, 13 patients with AMPD and 2 with reactive eosinophilia were evaluated for neutrophil PRV-1 expression (table 1). PRV-1 transcript quantitation was performed according to published methods (Klippel et al. Blood2003;102:3569). PRV-1 gene expression was considered elevated in the presence of a PRV-1/GAPDH ratio of 1.17 or lower. Table 1 Patient Diagnosis Neutrophil PRV-1/GAPDH ratio Neutrophil PRV-1 expression 1 Chronic myelomonocytic leukemia associated with myelofibrosis 1.05 Increased 2 FIP1L1/PDGFRA-positive eosinophilic disorder 1.39 Normal 3 FIP1L1/PDGFRA-positive eosinophilic disorder 1.19 Normal 4 FIP1L1/PDGFRA-positive eosinophilic disorder 1.10 Increased 5 Biphenotypic acute leukemia with prominent blood eosinophilia 0.99 Increased 6 Hypereosinophilic syndrome with cardiac involvement 1.15 Increased 7 Hypereosinophilic syndrome with cardiac involvement 1.13 Increased 8 Hypereosinophilic syndrome with sinus involvement 1.29 Normal 9 Reactive eosinophilia associated with atopic dermatitis 1.09 Increased 10 Reactive eosinophilia associated with T cell skin lymphoma 1.22 Normal 11 Aggressive systemic mastocytosis associated with chronic myelomonocytic leukemia 1.08 Increased 12 Aggressive systemic mastocytosis associated with myelofibrosis 1.09 Increased 13 Aggressive systemic mastocytosis with circulating mast cells 1.29 Normal 14 Indolent systemic mastocytosis 1.20 Normal 15 Indolent systemic mastocytosis 1.18 Normal In addition, eosinophil PRV-1 expression was measured in 2 patients from the above cohort (patients 3 and 8) and 3 additional patients with hypereosinophilic syndrome (HES). Results : Table 1 outlines specific diagnoses as well as the corresponding neutrophil PRV-1 expression levels in all 15 patients tested (median age 54 years, range 20–74; 11 males). Eosinophil PRV-1 expression was normal in all 5 patients tested (range of PRV-1/GAPDH ratio between 1.20–1.39). Conclusion : The current study suggests that neutrophil PRV-1 over-expression is a non-specific marker of clonal myeloproliferation that is seen in a broad spectrum of both typical and atypical myeloid disorders. As has been the case in ET, only a subset of AMPD patients display the specific abnormality, thus undermining its diagnostic utility. The preliminary information from the current study does not suggest increased eosinophil PRV-1 expression in either HES or clonal eosinophilia.

Abstract Introduction : Anecdotal reports of “idiopathic” cardiomyopathy (ICM) in patients receiving anagrelide therapy have surfaced but not formally studied. We undertook a tertiary-center comprehensive study of ICM that was diagnosed in the setting of either polycythemia vera (PV) or essential thrombocythemia (ET). Methods : Standard criteria were used for the diagnosis of both ET and PV. A diagnosis of ICM required echocardiogram documentation of a reduced left ventricular systolic ejection fraction (EF < 50%) as well as the absence of both coronary artery disease and other causes of cardiomyopathy. In order to avoid specific drug selection bias, we queried the entire Mayo Clinic Rochester database first for the diagnosis of ‘polycythemia’, ‘thrombocytosis’, or ‘thrombocythemia’ and subsequently cross-referenced the results with the diagnosis of either ‘cardiomyopathy’ or ‘heart failure’. Results : Initial data inquiry yielded a total of 434 patients whose medical histories were thoroughly reviewed for the presence of an echocardiogram-documented ICM that post-dated a diagnosis of either ET or PV. Nine such patients (7 females; age range 46–78 years) were identified. Anagrelide therapy, at standard doses (median 2 mg/day), was temporally associated with ICM in 4 of the 9 patients while treatment in the remaining 5 patients consisted of either phlebotomy alone (3 patients) or other cytoreductive agents (2 patients). A similar database inquiry at Mayo Clinic Jacksonville identified 2 more patients with ICM that was temporally associated with anagrelide treatment. A normal echocardiogram was documented in 3 of the 6 patients with anagrelide-associated ICM at 2, 15, and 39 months prior to treatment (table 1). Interval (months) Ejection fraction (%) Sex Diagnosis Diagnosis to ICM Anagrelide treatment to ICM Pre-treatment At ICM diagnosis Off-anagrelide therapy for 0.2–16 months F PV 66 60 69 30 and then 18 with continued anagrelide therapy 28 F ET 8 6 N/A 30 N/A F PV 120 36 N/A 20 66 F PV 156 13 73 35 55 F ET 108 10 N/A 35 44 F ET 108 11 58 10 34 Anagrelide therapy was immediately discontinued when ICM was diagnosed in 5 of the 6 patients and after further worsening of EF on continued anagrelide therapy in the sixth patient (first patient in table 1). All 6 patients with anagrelide-associated ICM experienced symptomatic improvement after drug discontinuation but the corresponding improvement in EF, after a period of 0.2–16 months, was often suboptimal (table 1). In contrast, ICM not associated with anagrelide therapy remained unchanged both in terms of symptoms and EF in all 5 cases. Conclusion : Anagrelide-induced cardiomyopathy might occur in some patients with either PV or ET. Prompt recognition and drug discontinuation has the potential to adequately reverse the situation in some but not all patients.

Abstract Background : Polycythemia Vera (PV) is associated with JAK2 mutations. In essential thrombocythemia (ET), ̴ 85% of patients harbor one of 3 "driver" mutations, with frequencies of ̴ 58%, 23% and 4%, for JAK2, CALR and MPL, respectively; ̴ 15% are wild type for all three mutations and are referred to as "triple negative". We applied next-generation sequencing (NGS) with a 27-gene panel of myeloid malignancy-relevant genes, in order to describe the prevalence of "non-driver" mutations and their prognostic relevance in PV and ET. Methods: Targeted capture assays were carried out on bone marrow or whole blood DNA for the following genes: TET2, DNMT3A, IDH1, IDH2, ASXL1, EZH2, SUZ12, SRSF2, SF3B1, ZRSR2, U2AF1, PTPN11, Tp53, SH2B3, RUNX1, CBL, NRAS, JAK2, CSF3R, FLT3, KIT, CALR, MPL, NPM1, CEBPA, IKZF, and SETBP1.Paired-end indexed libraries were prepared using the NEBNext Ultra Library prep protocol (NEB, Ipswich, MA /Agilent Technologies Inc, Santa Clara, CA). Capture libraries were assembled according to Nimblegen standard library protocol (Roche Nimblegen, Inc, Basel, Switzerland). Base-calling was performed using Illumina's RTA version 1.17.21.3. Genesifter® software (PerkinElmer, Danvers, Massachusetts) was utilized to analyze targeted sequence data. Nucleotide variants were called using the Genome Analysis Toolkit (GATK -Broad Institute, Cambridge, MA). Specific variants were deemed as mutations if they are associated with a hematologic malignancy (COSMIC database), or if they have not been associated with a dbSNP. Results: 314 patients with PV (n =133; median age 64 years, 53% females) or ET (n =181; median age 58 years, 59% females) were evaluated. Median follow-up was 9.8 years for PV and 9.2 years for ET. During this time 59 (44%) deaths, 15 (11%) fibrotic and 5 (4%) leukemic transformations were documented for PV and the corresponding percentages for ET were approximately 33%, 8% and 2%. Driver mutation distribution was 98% JAK2 for PV and 52% JAK2, 24% CALR, 20% triple-negative and 4% MPL for ET. Polycythemia Vera Mutations other than JAK2, CALR or MPL were seen in 58 (44%) patients; 29% harbored one, 14% two and one 3 mutations. None of 3 JAK2 -unmutated cases expressed non-driver mutations. Mutational frequencies were 18% TET2, 11% ASXL1, 5% SH2B3, 3% SF3B1 and ≤2% SETBP1, IDH2, DNMT3A, CEBPA, CSF3R and SUZ12, SRSF2, ZRSR2, TP53, CBL, NRAS, RUNX1, KIT, PTPN11 and FLT3. "Number of mutations" significantly affected overall (OS; Figure 1) and myelofibrosis-free (MFS) survival; respective HR (95% CI) were 2.6 (1.3-5.2) and 1.7 (0.97-3.1) and 13.7 (2.9-63.4) and 5.1 (1.5-17.6) for ≥2 mutations and one mutation, respectively. OS was also adversely affected by SRSF2 (p=0.006) and RUNX1 (p=0.04) and borderline affected by TET2 (p=0.06), IDH2 (p=0.08), ASXL1 (p=0.19) and SF3B1 (p=0.19) mutations. In multivariable analysis, SRSF2 and RUNX1 retained significance whereas the others remained borderline significant. In both univariate and multivariable analyses, leukemia-free survival (LFS) was adversely affected by IDH2 and RUNX1 mutations. In univariate analysis, ASXL1, IDH2, RUNX1 and KIT mutations predicted fibrotic progression, whereas SETBP1 was of borderline significance (p=0.07); all, including SETBP1, were significant during multivariable analysis. Essential thrombocythemia Mutations other than JAK2, CALR or MPL were seen in 83 (46%) patients; 35% harbored one, 7% two and 4% three mutations; prevalence in JAK2, CALR, MPL mutated and triple-negative cases was 52%, 43%, 43% and 35%, respectively (p=0.52). Mutational frequencies were: 13% TET2, 11% ASXL1, 6% DNMT3A, 5% SF3B1, 4% CEBPA, 2% TP53, SH2B3, EZH2 and CSF3R and <2% for SETBP1, IDH2, SRSF2, ZRSR2, CBL, NRAS, RUNX1, U2AF1, KIT, PTPN11 and FLT3. "Number of mutations" significantly affected OS (Figure 2) but not MFS or LFS; HR (95% CI) for OS were 6.6 (2.5-17.8) for 3 mutations and 2.2 (1.3-3.9) for one or two mutations. In univariate analysis, survival was adversely affected by CBL, EZH2, SF3B1, SRSF2 and IDH2 mutations. In multivariable analysis, EZH2 and SF3B1 remained significant. Univariate analysis identified SETBP1 and SF3B1 mutations as risk factors for fibrotic progression and EZH2, TP53 and CSF3R mutations for leukemic transformation. Conclusions: "Non-driver" mutations occur in more than 40% of patients with PV or ET; the number of such mutations and presence of certain specific ones likely predict OS, MFS or LFS. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Pardanani: Stemline: Research Funding.

Abstract Background : Thirty to 50% of patients with myelofibrosis with myeloid metaplasia display detectable bone marrow cytogenetic abnormalities at diagnosis with some(Demory et al. Blood1988;72:855, Dupriez et al. Blood1996;88:1013, Reilly et al. BJH1997;98:96) but not other studies (Cervantes et al. BJH1998;102:684, Okamura et al. Int J Hematol2001;73:194) suggesting a detrimental effect on survival. Methods : The current prospective study was conducted as a follow-up of the observations from an antecedent retrospective study that identified sole abnormalities of either 13q- or 20q- as ‘favorable’ to survival (Tefferi et al. BJH2001;113:763). All clinical and laboratory measurements, including bone marrow examination and karyotype analysis, were performed concurrently in consecutive cases of histologically confirmed MMM regardless of either duration of disease or presence of active therapy. Survival, leukemic transformation rate, and clinical progression were determined from the time of cytogenetic analysis. Results : Karyotypic analysis was successfully performed in a total of 88 patients (median age 61 years; 31 females) and the results were normal in 49 (56%) and abnormal in the remaining 39 patients with 9 (10%) displaying ‘favorable’, and 30 (34%) ‘other’ clonal abnormalities. At the time of study entry, 57 patients were not recieving any form of therapy, 18 were newly diagnosed, and the Dupriez prognostic score distributions were ‘0’ in 33 patients, ‘1’ in 35 and ‘2’ in 20 (Dupriez et al. Blood1996;88:1013). All patients were followed for a minimum of 3 years and a median of 42 months. The 3 cytogenetic categories were similar in age and gender distribution. Survival in the favorable cytogenetic group was similar to that of the normal group and significantly better than the group with other cytogenetic abnormalities (p=0.02). Figure Figure Similarly, no patient (0%) in the ‘favorable’ group transformed into acute leukemia while 6 of 30 (20%) in the ‘other’ and 5 of 49 (10%) in the normal groups did (p=0.21). In a Cox regression analysis, cytogenetic lesions other than the ‘favorable’ kind, hemoglobin below 10 g/dL, leukocytosis, hypercatabolic symptoms, increased blood blast %, blood CD 34 count above 100 x 106/L were individually associated with shortened survival. In a multivariate analysis, only hemoglobin level and leukocyte count retained their significance. Conclusion : Single chromosomal abnormalities of either 13q- or 20q- should be considered as ‘favorable’ cytogenetic lesions in MMM while other abnormalities are associated with an adverse prognosis. The current observation explains the discrepancy in the literature regarding the prognostic relevance of cytogenetic findings in MMM and warrants additional studies to determine the value of incorporating the specific information in prognostic scoring systems.

Abstract Background : CC-5013 (Revlimid™) is an immunomodulatory analog of thalidomide that is substantially more potent than the parent drug in terms of both anti-angiogenic and anti-TNF-α activity. CC-5013 has shown therapeutic activity in thalidomide-responsive hematological malignancies including relapsed multiple myeloma (Blood2002;100:3063) and myelodysplastic syndrome (Blood2002;100:96a). Myelofibrosis with myeloid metaplasia (MMM) has also been shown to respond to thalidomide (BJH2002;117:288). Furthermore, both angiogenesis and altered TNF-α activity have been pathogenetically implicated in MMM. Methods : The current report considers the first 15 patients in an ongoing phase II treatment trial of single agent CC-5013 in MMM based on a minimum of 3 month follow-up from day 1 of protocol treatment. Study eligibility criteria included no concomitant MMM-directed therapy, a hemoglobin (Hgb) level of < 10 g/dL, an absolute neutrophil count (ANC) of ≥ 1 x 109/L, and a platelet count of ≥ 100 x 109/L. Previous thalidomide therapy in the absence of high-grade skin toxicity was allowed. All patients were started with daily oral CC-5013 (10 mg/day) to be taken for three 28-day treatment cycles. Three additional treament cycles were planned in case of anemia response. Results : Median age was 64 years (range, 40–75) and 10 pts were males. Twelve of the 15 study pts (80%) were red cell transfusion-dependent, 14 (93%) were previously treated including 12 with cytoreductive and 4 with thalidomide therapy. The spleen was palpably enlarged in 13 pts (median 18 cm, range 7–23). Eight of the 15 pts (53%) successfully completed 3 months of protocol treatment. Treatment was discontinued in the remaining 7 pts because of drug toxicity (2 pts), disease progression (1 pt), other co-morbid condition (3 pts), or patient discretion (1 pt). Treatment response data : Clinically relevant responses were documented in 4 patients (27%) and consisted of measurable improvements in anemia (2 pts), splenomegaly (2 pts), and/or constitutional symptoms (3 pts). One pt experienced an increase in Hgb level from 8.3 to 13.4 g/dL, a decrease in leukocyte count from 66.2 to 7.7 x 109/L, and complete resolution of myelophthisis including disapperance of circulating blasts that measured 9% before treatment. Another pt became transfusion-independent (Hgb level increased to > 10 g/dL) as well as enjoyed a profound improvement in constitutional symptoms. Interestingly, serum lactate dehydrogenase (LDH) level decreased in 11 pts (73%) to either within the normal ramge (5 pts) or by more than 30 % (6 pts). Treatment toxicity data : Grade 3 or 4 adverse events that were possibly, probably, or definitely attributed to CC-5013 included rash (2 pts), neutropenia (5 pts), thrombocytopenia (3 pts), and fatigue (2 pts). In addition, 3 patients experienced drug-associated extreme thrombocytosis while a histologically-proven disseminated extramedullary hematopoiesis was documented in one patient after one month of CC-5013 treatment. Conclusion : The current preliminary report suggests a substantial biological activity of CC-5013 in MMM with a therapeutic potential for a subset of patients. Additional studies with a longer duration of therapy, different dose schedules, and combinations of CC-5013 and corticosteroids are warranted.

Abstract Background: Myelofibrosis with myeloid metaplasia (MMM) is a progressive and fatal myeloproliferative disorder with limited therapeutic options. R115777 is a non-peptidomimetic enzyme-specific inhibitor of farnesyl protein transferase (FT) which we recently reported has in vitro activity against aberrant myeloid progenitors from MMM patients (Leukemia2003;17:849). This observation, coupled with the clinical activity demonstrated in high risk acute myeloid leukemia (Blood2001;97:3361) and chronic myeloid disorders (Blood2003;101:1692) led us to perform a Phase II trial in patients (pts) with MMM. Methods: Eligible pts had histologically confirmed MMM and were symptomatic, defined by anemia (hemoglobin<10g/dL or transfusion dependent) or palpable hepatosplenomegaly. Pts received 300 mg of R115777 orally twice daily for the first 21 days of a 28 day cycle. Dose escalations (up to 1200mg/day) and reductions (down to 100mg/day) were allowed based on activity and toxicity. The primary endpoint was response as defined by improvement in either anemia or organomegaly. Results: 34 pts (median age 65 yrs, range 55–80; 71% male) were enrolled in the trial. All pts displayed symptomatic disease at enrollment, with 10 who were erythrocyte transfusion dependent; and 19 (56%) had a Lille MMM prognostic score ≥1; no pts were significantly thrombocytopenic at entry by enrollment criteria. Median pre-study spleen and liver sizes were 12 cm (range: 0–35) and 2 cm (range: 0–15) below the left and right costal margin, respectively. Median time to discontinuation of therapy was 5.5 months (95% CI:2.9–6.4). Reasons for early termination were progression (7), adverse reactions (5), co-morbidities (2), refusal of further treatment (2), and death on study (1). One pt died of grade 5 pneumonitis two weeks after study enrollment, which was felt to be unrelated to study treatment. Significant toxicities (≥ grade 3) were seen for myelosuppression (n=13), neuropathy (n=2), fatigue (n=1), rash (n=1) and hyponatremia (n=1). Overall, R115777 resulted in little improvement in anemia, with the exception of one anemia-based partial response. However, the myelosuppressive aspects of the agent may have masked improvements in cytopenias. In contrast, R115777 did result in clinically relevant decreases in organomegaly in 11 pts (33%) (splenomegaly in 3 pts, hepatomegaly in 5 pts, and both in an additional 3 pts.) Across all pts who had some reduction in splenomegaly, the median maximal reduction was 42.5% (of palpable component; range: 8–100%). Likewise, all patients with baseline hepatomegaly had resolution of this problem at some point during therapy. Four pts remain on the agent (from 14 to 22 months) for continued and sustained improvement in organomegaly. Responses observed did not significantly correlate with reductions in bone marrow fibrosis, osteosclerosis, neoangiogenesis, or resolution of baseline karyotypic abnormalities. Conclusions: R115777 has demonstrated preliminary activity against MMM associated organomegaly. Whether this latter benefit is independent of R115777 induced myelosuppression is unclear. The myelosuppression observed was generally mild and without clinical consequence, although it was difficult to differentiate between myelosuppression versus disease progression. Pts continue to be followed to evaluate durability of observed responses, long term effects on anemia and intramedullary manifestations of the disease.

Abstract Background : Momelotinib (a JAK1 and JAK2 inhibitor) induces both anemia and spleen response in patients with myelofibrosis (MF) (Leukemia 2013;27:1322). The favorable effect on anemia was a distinguishing feature for momelotinib, compared to other JAK inhibitors, but the drug was also associated with treatment-emergent peripheral neuropathy (TE-PN). We examined the natural history and risk factors of momelotinib-associated peripheral neuropathy. Methods : The study patients constitute part of a larger phase-1/2 clinical trial (CCL09101; NCT00935987) using momelotinib for the treatment MF. Toxicity grading was according to the Common Terminology Criteria for Adverse Events (CTCAE) v.4.03. Momelotinib capsules (as opposed to the newer tablet formulation used in the currently ongoing phase-3 study) were used in the current study, which represents a sponsor-independent analysis. Results : 100 patients with MF (median age 66 years; 58% males) were treated at the Mayo Clinic between 11/20/09 and 11/10/10. DIPSS-plus (JCO. 2011;29:392) risk distribution was 63% high, 36% intermediate-2 and 1% intermediate-1. 75% of the patients harbored JAK2V617F and 50% abnormal karyotype. Previous treatment included other JAK inhibitors in 21 patients, thalidomide/lenalidomide/pomalidomide in 31 and hydroxyurea or other cytoreductive agents in 55. History of diabetes was documented in 7% of the patients at baseline and 14 (14%) of the patients had symptoms of PN before starting treatment with momelotinib, all of which were grade-1. Momelotinib was initiated at 100 mg/day in 3 patients, 150 mg/day in 21, 150 mg twice-daily in 20, 200 mg/day in 3, 300 mg/day in 47 and 400 mg/day in 6. The maximum momelotinib dose was 150 mg/day in 6 patients, 150 mg twice-daily in 20, 300 mg/day in 68 and 400 mg/day in 6. Treatment duration was <6 months in 12 patients, 6-12 months in 22, 12-24 months in 19 and >24 months in 47. Median follow-up after the institution of protocol drug therapy was 35 months (range 1-49). During this period, TE-PN was documented in 44 (44%) patients; of these, 42 were in patients without baseline neuropathy. TE-PN involved the feet only in 28 patients, hands only in one patient and both feet and hands in 15 patients. Median (range) for the time of onset of TE-PN was 32 weeks (1-132) and duration of TE-PN 11 months (0-40). All 42 newly-emergent PN were grade-1 sensory while the two patients with grade-1 baseline PN progressed to grade-2. Formal neurological evaluation, which was performed in 9 patients, mostly showed length-dependent sensory-motor large and small fiber neuropathy with axonal features. TE-PN resulted in drug discontinuation in 7 patients and reduction in drug dose in 28 patients. Improvement after drug dose reduction was documented in only one patient and in none of those with drug discontinuation. Progression to grade-2 PN was documented in 3 patients. TE- PN was significantly associated with clinical improvement (CI) with 54% incidence in responders vs 30% in non-responders (p=0.02); however, longer treatment duration was also associated with higher prevalence of TE-PN (p=0.03). In univariate analysis, patients with TE-PN lived longer than those without TE-PN (p=0.048); however, significance was lost during multivariable analysis that included either achievement of CI or treatment duration. Neither the initial (p=0.53) nor maximum (p=0.37) momelotinib dose was correlated with TE-PN, which was also not influenced by JAK2V617F (p>0.99), karyotype (p=0.26), history of diabetes (p=0.39) or previous treatment with JAK inhibitor therapy (p=0.7), thalidomide (p=0.24), lenalidomide (p=0.59), pomalidomide (p=0.63) or hydroxyurea (p=0.56). There were no correlations between TE-PN and age (p=0.37), sex (p=0.37), type of MF (p=0.98), DIPSS-plus score (p=0.07), baseline transfusion dependency (p=0.15), constitutional symptoms (p=0.83) or spleen size (p=0.53). TE-PN did not correlate with hematologic (p=0.14) or extramedullary toxicity other than PN (p=0.41) or lipase/amylase abnormalities (p=0.35). Conclusions : Momelotinib-associated neuropathy in MF might not be reversible in the majority of cases but also does not appear to be progressive. We were not able to identify risk factors for TE-PN, other than duration of treatment, which also explains its apparent association with response/survival. We are currently looking into possible correlations with serum thiamine levels. Disclosures No relevant conflicts of interest to declare.

Abstract Background : In primary myelofibrosis (PMF), ̴ 88% of patients harbor one of three "driver" mutations, with mutational frequencies of approximately 60%, 22% and 6% for JAK2, CALR and MPL, respectively. Other "non-driver" mutations have also been described in PMF and some of them and their number have been associated with inferior survival (Leukemia. 2014;28:1804). We applied next generation sequencing (NGS) with a broader panel of MPN-relevant genes, in order to identify additional mutations of prognostic relevance as well as obtain additional information regarding the prognostic value of 'number of mutations'. Methods: Targeted capture assays were carried out on bone marrow or whole blood DNA specimens obtained at time of referral for the following genes: TET2, DNMT3A, IDH1, IDH2, ASXL1, EZH2, SUZ12, SRSF2, SF3B1, ZRSR2, U2AF1, PTPN11, Tp53, SH2B3, RUNX1, CBL, NRAS, JAK2, CSF3R, FLT3, KIT, CALR, MPL, NPM1, CEBPA, IKZF, and SETBP1. Paired-end indexed libraries were prepared from individual patient DNA using the NEB Next Ultra Library prep protocol on the Agilent Bravo liquid handler (NEB, Ipswich, MA/Agilent Technologies Inc, Santa Clara, CA). Capture libraries were assembled according to Nimblegen standard library protocol (Roche Nimblegen, Inc, Basel, Switzerland). Base-calling was performed using Illumina's RTA version 1.17.21.3. Genesifter® software was utilized (PerkinElmer, Danvers, Massachusetts) to analyze targeted sequence data. Nucleotide variants were called using the Genome Analysis Toolkit (GATK-Broad Institute, Cambridge, MA). Specific variants were deemed as mutations if they are associated with a hematologic malignancy (as identified by COSMIC database), or if they have not been associated with a dbSNP. Results: 180 PMF patients were evaluated (median age 63 years; 65% males). DIPSS-plus risk distribution was 32% high, 38% intermediate-2, 17% intermediate-1 and 13% low. Driver mutation distribution was 62% JAK2, 22% CALR, 9% triple-negative and 7% MPL. Karyotype was abnormal in 41% of patients and unfavorable in 12%. Mutations other than JAK2, CALR or MPL (i.e. "non-driver" mutations) were seen in 150 (83%) patients including 88% of "triple-negative" cases. 62 (34%) patients harbored one, 55 (31%) two, 16 (9%) three and 17 (10%) four or more. Mutational frequencies were: ASXL1 36%, TET2 18%, SRSF2 17%, U2AF1 17%, ZRSR2 11%, SF3B1 10%, DNMT3A 9%, CEBPA (9%), Tp53 7%, SETBP1 6%, CBL 5%, IDH1/2 5%, SH2B3 4%, CSF3R 4%, NRAS 4%, RUNX1 3% and ≤2% for SUZ12, KIT, PTPN11, NPM1 and EZH2. DIPSS-plus high/intermediate-2 risk patients displayed higher number of mutations (p=0.0004) and higher mutational frequencies for ASXL1 (p=0.02), SRSF2 (p=0.004) and CBL (p=0.02). Associations noted included JAK2 with U2AF1 (p=0.03), unfavorable karyotype with CBL (p=0.01) and normal karyotype with ZRSR2 mutations (p=0.04). At a median follow-up of 4 years, 111 (62%) deaths were documented. For examination of impact on survival, we considered 'number of mutations' and specific mutations with >2% frequency. Accordingly, in univariate analysis, survival was adversely affected by 'number of mutations' (Figure 1) and presence of ASXL1, SRSF2, IDH1/2, U2AF1, RUNX1 and CBL mutations. For multivariable analysis, we considered three categories (zero, 1-3 and ≥4) for number of mutations based on the results from univariate analysis (Figure 1); the results showed ≥4 mutations, 1-3 mutations, RUNX1, CBL, ASXL1 and SRSF2 mutations were independently associated with shortened survival; the respective HR (95% CI) were 4 (1.4-11.1), 3 (1.3-6.8), 2.9 (1.1-8.1), 2.8 (1.3-6.3), 1.8 (1.2-2.7) AND 1.7 (1.03-2.7). When the multivariable analysis was repeated including only the 150 patients with at least one non-driver mutation, the 'number of mutations' was no longer significant (p=0.35) but ASXL1, CBL, RUNX1 and SRSF2 mutations retained their significance. The prognostic relevance of ASXL1 and CBL continued to be apparent even after the addition of DIPSS-plus and driver mutation profile to the multivariable model. Conclusions: Mutations other than JAK2, CALR or MPL occur in more than 80% of patients with PMF, including those with "triple-negative" driver mutational status. The absence of such mutations is independently favorable for survival while the prognostic effect of their presence is influenced by ASXL1, CBL, RUNX1 and SRSF2 mutations. Figure 1. Figure 1. Disclosures Pardanani: Stemline: Research Funding.

Abstract Background Current risk stratification in Polycythemia Vera (PV), is designed to estimate the likelihood of thrombotic complications. In ECLAP large prospective study of 1638 PV cases diagnosed by Polycythemia Vera Study Group (PVSG) criteria, risk factors were age over 65 years and prior thrombotic events. After the JAK2V617F discovery, diagnosis has undergone substantial revision by the World Health Organization (WHO). Consequently, current risk factors determination should be described in patients diagnosed by the most recent recommendations and requires a large sample size with reliable and sufficiently long clinical course documentation. Accordingly, the current study includes 1545 patients with strictly WHO-defined PV diagnosed and followed-up in seven centers from Italy, Austria and the United States, belonging to the International Working Group for Myeloproliferative Neoplasm (MPN) Research and Treatment (IWG-MRT). Objective and design 1) Describe the rate of thrombosis in this population; 2) Report the sites and frequency of recurrences after a first arterial or venous event; 3) Identify separately risk factors for arterial versus venous thrombosis and develop practically useful prognostic models. Results 1. Presenting features The characteristics of the cohort recruited reflected well the heterogeneity of epidemiologic and clinical features that are found in the routine clinical practice of PV. Arterial and venous thrombosis history before or at diagnosis was documented in 246 (16%) and 114 (7.4%) patients, respectively. The frequencies of arterial and venous thrombosis were lower than those recorded in the ECLAP4 (27% and 11% respectively) but comparable to those reported in the recent Cyto-PV2 (arterial 17%, venous 12%) studies. The discrepancy might be partly related to earlier diagnosis in the post-ECLAP JAK2mutation era. 2. Thrombotic events after diagnosis Median follow-up was 6.9 years and treatment included aspirin (84%) and cytoreductive agents (73%), in addition to phlebotomy. Post-diagnosis total major thrombosis rate was 2.62% pt/yr, lower than reported in the ECLAP trial (4.4% pt/yr) but comparable to the Cyto-PV study (2.7% pt/yr) where management of cardiovascular risk factors was more intensive than in ECLAP. Arterial or venous thrombosis occurred in 184 (12%, rate: 1.59 % pts/yr) and 137 (9%, rate: 1.05 % pts/yr) patients, respectively. In addition, prior arterial events were associated in 75% of cases with subsequent arterial events and in the remaining 25% with venous thrombosis including splanchnic vein events. Moreover, patients with prior venous events had recurrences in venous district in 61% of cases and in the arterial district in 39%. 3. Risk factors for arterial versus venous thrombosis and prognostic models In multivariable analysis, arterial thrombosis was predicted by previous arterial event (HR 1.7, 95% CI 1.2-2.4), and hypertension (HR 1.6, 95% CI 1.2-2.2) while predictors of venous events were previous venous thrombosis (HR 2.6, 95% CI 1.5-4.4), and age ≥ 65 years (HR 1.7, 95% CI 1.2-2.5). We used the above-mentioned risk factors in order to develop prognostic models allowing to identify patients at different probability to develop arterial or venous thrombosis. For arterial thrombosis, the absence of previous arterial thrombosis and hypertension defined lower-risk (rate 1.27 % pts/yr) while the presence of one or both identified higher-risk patients (rate 2.04 % pts/yr). Previous venous event and/or age ≥ 65 years identified patients at higher risk of venous (rate 1.62 % pts/yr) thrombosis and, if absent, patients at lower risk (rate 0.78 % pts/yr). Conclusion The overall rate of thrombosis was half than in ECLAP and should be considered in future clinical trials. Of note, patients with a prior venous thrombosis had a more frequent subsequent recurrence in the venous district but a proportion of them (40%) had also arterial events; conversely, recurrences after previous arterial event were also registered in the leg and non-leg veins in 30 % of cases, indicating that in PV, venous and arterial thrombosis may share some common pathogenetic mechanisms. These findings may have practical implications and suggests antiplatelet therapy and anticoagulant therapy as the favorite choice to prevent arterial and venous thrombosis, respectively, but also suggests studies of antithrombotic drug combination. Disclosures No relevant conflicts of interest to declare.