Lasho, Terra L.

Abstract Background: We sought to retrospectively analyze the clinical differences, at diagnosis and throughout the disease course, between patients with primary myeloifbrosis whom undergo blastic transformation (PMF-BP) and those whom expire from PMF complications without undergoing transformation. Methods: An international collaborative database of patients with PMF which progressed to PMF BP, and a control group of individuals with PMF who expired secondary to PMF was created. Data regarding clinical course, bone marrow, karyotypic, quantitative JAK2V617F analysis (when able), laboratory values, and therapy at the diagnosis of PMF, PMF BP and up to 4 return visits (R1-4) in between these milestones was abstracted. Results: COMPARISON AT DIAGNOSIS OF PMF: 136 cases of PMF who eventually underwent PMF-BP were included, with a control group of 42 PMF patients, were analyzed. Both groups of patients had similar demographic (median age at diagnosis 59 years (range 23–83)/ 59.1 (15–93); and disease parameters (median hemoglobin 10 g/dL (range 2–15.7) /9.8 (4.9–14.6); median platelet count 181 x 109/L (range 7–1400)/168 (16–1916)), and Lille PMF prognostic scores (p = n.s.) for PMF-BP vs. PMF control group respectively. Additionally, blasts (peripheral blood /marrow) were a median of 0% (range 0–18%)/ 2% (range 0–16%) for the study group versus 0.25% (range 0–5%)/ 3% (range 0–4%) (p = n.s. for both) for the control group, respectively. Both groups had similar rates of requiring therapy (86%; 93%) and survival from diagnosis (median 34 months (range 2–441); 29 months (1–236)) (p = n.s.). However, lactate dehydrogenase (LDH) (although high in both groups) was higher at diagnosis (median 802 IU/L (range 83–10353) vs. 416 IU/L (124–2197)) (p=0.04), as well as an abnormal marrow karyotype (85% vs. 25%; p<0.001) amongst the PMF BP group. SUBSEQUENT CLINICAL COURSE: Analyzing the clinical evolution between groups demonstrated persistently higher LDH and worsening thrombocytopenia in the PMF-BP cohort (see figure). Decreasing Platelets Approaching PMF BP Decreasing Platelets Approaching PMF BP Peripheral blood blasts increased in both groups, but values above 10% were unique to the PMF BP group. Additionally marrow karyotype displayed clonal evolution developed in 56% of the PMF-BP patients as opposed to 14% of controls (p<0.001). Initial and serial quantitative JAK2V617F mutation analysis (available from 20 /19 patients from the PMF-BP and control group at diagnosis, with 11 / 12 serial samples) showed no difference between the groups, nor a pattern of mutation burden elevation with transformation to leukemia in the study group. Conclusions: Increasing LDH, peripheral blood blast percentages >10%, and clonal evolution are features commonly seen in PMF patients whom evolve to blast phase and may be a harbinger of movement towards PMF BP.

Abstract Background: Most patients with chronic myelomonocytic leukemia (CMML) are elderly and age >65 years is an adverse prognostic factor (JCO 2013;31:2428). Our objective in the current study was to examine risk factors for survival and treatment outcome in younger patients (age ≤65 years), with emphasis on molecular and cytogenetic markers. Methods: DNA analysis for SRSF2, SF3B1, U2AF1, ASXL1 and SETBP1 mutations was carried out on specimens obtained at time of diagnosis. We used both conventional and revised (see accompanying ASH 2014 abstract) cytogenetic risk models to query their application in the current study population. Treatment data included outcome of allogeneic stem cell transplant (HCT) and treatment with hypomethylating agents (HMA), including azacitidine (AZA) and decitabine (DAC). Results: Survival: 259 patients (median age 59 years, range 18-65; 75% males) with WHO-defined CMML and age≤ 65 years were included in the study: 108 from the French consortium, 81 Mayo Clinic, 47 Moffitt Cancer Center and 23 University of Milan; 217 (84%) patients had CMML-1 and the remainder CMML-2. At a median follow-up of 26 months, 124 (48%) deaths and 79 (31 %) leukemic transformations were documented. Mutational frequencies were 34% (61/178) for ASXL1, 45% (74/165) for SRSF2, and 4% (7/168) for SETBP1. Cytogenetic data was available in 248 of 259 study patients; 56 (23%) patients displayed abnormal karyotype and the revised cytogenetic risk designations included 196 (80%) low, 29 (12%) intermediate, and 23 (9%) high. In univariate analysis, lower hemoglobin (p<0.0001), higher WBC (p=0.005), higher monocyte count (AMC) (P=0.004), lower platelet count (p=0.004), presence of circulating immature myeloid cells (IMC) (p=0.004), presence of circulating blasts (p<0.0001), bone marrow (BM) blasts ≥10% (p=0.005), WHO morphological subtype (p=0.0003), Spanish cytogenetic risk stratification (p=0.0002), revised Mayo cytogenetic risk stratification (p<0.0001), SRSF2 (p=0.03) and ASXL1 (p=0.03) mutational status predicted shortened survival. In multivariable analysis, lower hemoglobin (p<0.0002), presence of circulating blasts (p<0.0001), SRSF2 (p=0.03) and ASXL1mutational status (p=0.04) retained their negative prognostic impact. A similar analysis identified circulating IMC (p<0.0001), presence of circulating blasts (p<0.0001), and BM blast % (p=0.0002) as predictors of blast transformation (BT). Response to hypomethylating agents: Seventy-two (28%) patients received HMA (AZA-46, DAC-26, both-6). In the AZA group, median number of cycles was 5 (range; 1-44), median duration of therapy 5 months (range;1-59) with 4 (6%) receiving DAC on progression and 26 (42%) proceeding with HCT. Over-all response rate (ORR) was 25% (6/24) and included complete response (CR) in 2 (8%) patients, partial response (PR) in 2 (8%) and hematological improvement in 2 (8%). In the DAC group, median number of cycles was 6 (2-25), median duration of therapy 5 months (range;1-49), with 2 (8%) receiving AZA on progression; ORR was 50% (7/14) and included CR in 4 (29%) patients, PR in 1 (7%) and HI in 2 (14%). ASXL1 (p=0.4), SRSF2 (p=0.7) mutational status or karyotype (p=0.5 for Spanish and 0.3 for revised Mayo) did not correlate with response to HMA. Allogeneic stem cell transplant: 50 patients underwent HCT. Conditioning regimen was reported in 41 patients and included myeloablative (MA) in 14 (34%) (median age 51 years, 27-62) and reduced intensity (RIC) in 27 (66%) (median age 48 years, 18-65). Donor source was documented in 40 patients and included matched related in 19 (48%), matched unrelated in 12 (30%), mismatched unrelated in 4, haploidentical in 2 and umbilical cord transplant in one. 27 (63%) patients had chronic GVHD (50% in MA group and 80% in the RIC group). Among 40 evaluable patients, 23 (57%) achieved CR, 8 (21%) relapsed and 8 (21%) died from transplant-related complications. ASXL1 (p=0.9), SRSF2 (p=0.5) mutational status or karyotype (p=0.2 for Spanish and 0.3 for revised Mayo) did not affect transplant outcome. Conclusions: Survival in young CMML patients is poor and even worse in the presence of SRSF2 or ASXL1 mutations, anemia or circulating blasts. Treatment outcome was more impressive with transplant than it was with HMA and neither was influenced by karyotype or ASXL1/SRSF2 mutational status. Disclosures No relevant conflicts of interest to declare.

Abstract Background : DNMT3A mutations result in epigenetic dysregulation and impart a negative prognostic impact in acute myeloid leukemia and myelodysplastic syndromes. In chronic myelomonocytic leukemia (CMML), DNMT3A mutations are seen in 2-5% of patients. In a large Groupe Français des Myélodysplasies (GFM) study (n=312), DNMT3A mutations were seen in 2% and were not included in further survival analyses (Itzykson JCO 2013). In a prior Mayo Clinic study (n=175), DNMT3A mutations were seen in 5% (n=9) and on univariate, but not multivariate analysis (Patnaik Blood C J 2016), were associated with shortened over-all survival (OS). We carried out this study on a larger CMML cohort (n=261), with more (n=15) informative cases to assess the impact of DNMT3A mutations. Methods : 261 patients with World Health Organization (WHO)-defined CMML were included in the study. All patients had bone marrow (BM) biopsies and cytogenetics performed at diagnosis. Targeted capture assays were carried out on BM DNA specimens obtained at diagnosis for the following genes; TET2, DNMT3A, IDH1, IDH2, ASXL1, EZH2, SUZ12, SRSF2, SF3B1, ZRSR2, U2AF1, PTPN11, Tp53, SH2B3, RUNX1, CBL, NRAS, KRAS, JAK2, CSF3R, FLT3, KIT, CALR, MPL, NPM1, CEBPA, IKZF, and SETBP1. The 2016 WHO diagnostic criteria were used. Results: Among the 261 study patients, 65% were males and median age was 70 years (range, 28-91). 154 (59%), 64 (25%) and 43 (16%) patients were classified as CMML-0, 1 and 2, respectively. At a median follow-up of 23 months, 174 (67%) deaths and 37 (14%) leukemic transformations (LT) were documented. Mutational frequencies ≥4% were encountered in; TET2 45%, ASXL1 45%, SRSF2 40%, NRAS 14%, SETBP1 13%, CBL 10%, JAK2 7%, RUNX1 6%, DNMT3A 6%, U2AF1 6%, SF3B1 5%, ZRSR2 4%, Tp53 4%, and IDH2 4%. i) DNTM3A mutated CMML: phenotypic and molecular correlates DNMT3A mutations were seen in 15 (6%) patients; 64% male with a median age of 64 years. DNMT3A amino acid substitutions included; R882H 50%, R882C 29%, R910P 7%, R598* 7% and R320* 7%. The median variant allele frequency burden was 45%. Concurrent gene mutations were detected in; TET2 43%, ASXL1 21%, SF3B1 21%, U2AF1 14%, RUNX1 14%, SETBP1 14%, NRAS 14%, SRSF2 7%, JAK2 7% and Tp53 7%. There was no difference between DNMT3A mutated and wild-type patients in terms of age and gender distribution, hemoglobin level, leukocyte, monocyte (AMC), and platelet counts, peripheral blood (PB) or BM blast content. Concurrent gene mutations were equally distributed with the exception for a higher prevalence of SF3B1 (p=0.003) and a lower prevalence of SRSF2 (p=0.004) mutations in DNMT3A mutated CMML. Four (29%) patients underwent leukemic transformation. ii) Impact on OS and leukemia-free survival (LFS): Median survival for the entire cohort (n=261) was 24 months. In univariate analysis, survival was shorter in DNMT3A mutated (median 8 months) versus wild-type (median 27 months) patients (p=0.0007; HR 2.9, 95% CI 1.5-5.7; Figure 1A). Other variables of significance, in univariate analysis, included lower hemoglobin (p=0.002), higher leukocyte count (p=0.0009), higher AMC (p=0.0012), PB blast % (p=0.001), circulating immature myeloid cells (IMC, p=0.01), BM blast % (p=0.045), abnormal karyotype (p=0.02), and ASXL1 (p=0.01) mutations. Survival was also adversely affected by the presence of either (n=133) or both (n=3) ASXL1/DNMT3A mutations (0=0.007, Figure 1B). In multivariable analysis (MVA) excluding ASXL1 and DNMT3A mutations, hemoglobin (p=0.03), IMC (p=0.013) and AMC (p=0.02) retained significance. When ASXL1 mutations were added to the MVA, ASXL1 (p=0.01) mutations, AMC (p=0.012) and IMC (p=0.03) retained significance. Similarly, when only DNMT3A mutations were added to the MVA, DNMT3A (p=0.003) mutations, IMC (p=0.01) and AMC (p=0.02) retained significance. When both DNMT3A and ASXL1 mutations were added to the MVA, only DNMT3A (p<0.0001) and ASXL1 (p=0.004) mutations remained significant. DNMT3A mutations predicted shortened OS, independent of the ASXL1 inclusive GFM model (p<0.0001) and Mayo Molecular Model (p=0.002). DNMT3A mutations (p=0.0018), along with low hemoglobin levels (p=0.003) independently predicted for a shorter LFS. Conclusions: DNMT3A mutations are seen in ~5% of patients with CMML and impart a negative prognostic impact on both OS and LFS. This finding warrants inclusion of DNMT3A mutations in molecularly integrated CMML prognostic models. Disclosures No relevant conflicts of interest to declare.

Abstract Background : An increased incidence of second skin and visceral malignancies in comparison to the general population has been reported in patients with hematologic neoplasms such as, chronic lymhocytic leukemia (CLL) (~11-36%) (Tsimberidou, JCO, 2009),chronic myeloid leukemia (CML) (4-6%) (Miranda, Leukemia 2016)and multiple myeloma (MM) (3-4.3%) (Palumbo, Lancet Oncol 2014).Diverse etiologies exist for this association. We carried out this study to describe the spectrum of second malignancies in patients with chronic myelomonocytic leukemia (CMML). Methods : Patients with World Health Organization defined CMML were included in the study. All patients had bone marrow (BM) biopsies performed at diagnosis. We only included malignancies that were diagnosed at the time of CMML diagnosis or subsequent to it. Patients that had concomitant systemic mastocytosis (n=18), therapy related CMML (n=19) and those that underwent allogeneic stem cell transplantation (n=23) were excluded. Standard cancer specific diagnostic and staging methods were utilized. Results: The study included 378 patients (median age 71 years; 66% males). Seventy-one concomitant and/or subsequent second malignancies were identified in thirty-two (8%) patients. Age group distribution was; 50-59 years 1 (1%), 60-69 years 21 (30%), 70-79 years 30 (42%) and ≥ 80 years 19 (27%) second malignancies. 22 (69%) patients had 1 second malignancy, 4 (13%) had 2, 2 (6%) had 4 (skin neoplasms), while 4 (13%) patients had ≥ 5 second malignancies (Table one). At a median follow-up of 18 months, 284 (75%) deaths and 54 (14%) leukemic transformations were documented. i) Second hematological malignancies: Thirteen (18%) concomitant (n=7, 54%) and subsequent hematological malignancies (n=6, 46%) were observed in 13 (3.5%) patients. In addition, 10 (3%) pre malignant states including, 7 patients with MGUS and 3 with monoclonal B-cell lymphocytosis (MBL) were seen. In one case each, the MGUS and MBL evolved into multiple myeloma (after 16 months) and CLL (after 30 months), respectively. Concomitant malignancies included: polycythemia vera (n=2, 15%), MM (n=2, 15%), marginal zone B-cell lymphoma (n=1, 8%), splenic lymphoma (n=1, 8%), ALK-rearranged T-cell lymphoma (n=1, 8%), while subsequent malignancies included: MM (n=1, 8%), diffuse large B-cell lymphoma (n=1, 8%), Waldenström's macroglobulinemia (n=1, 8%), CLL (n=2, 15%) and acute lymphoblastic leukemia (n=1, 8%). Four (31%) patients needed chemotherapy, with outcomes including: complete remission in one case (multiple myeloma), progression of disease in 2 (multiple myeloma and diffuse large B cell-lymphoma) and partial response in 1 case (ALK rearranged T cell lymphoma). CMML related cytopenias did not preclude patients from getting chemotherapy. Three patients died from causes related to their second malignancies. ii) Cutaneous malignancies: Fifty-one (71%) concomitant (n=12, 24%) and subsequent cutaneous malignancies (n=39, 76%) were seen in 13 (3%) patients. The concomitant malignancies included: 7 (14%) basal cell carcinoma (BCC), 5 (10%) squamous cell carcinoma (SCC), while subsequent cancers included: 3 (6%) malignant melanoma, 7 (14%) BCC and 29 (57%) SCC. Four cases (8%) were locally advanced and four patients (8%) had recurrent lesions. A concomitant diagnosis of BCC and SCC was made in 8 patients (16%). All patients were treated surgically or with electrodesiccation and curettage. No deaths or metastases were reported. iii) Visceral malignancies: Seven (10%) subsequent visceral malignancies were seen in 6 (1%) patients. These included; colonic adenocarcinoma (n=2, 29%), bladder adenocarcinoma (n=1, 14%), endometrial adenocarcinoma (n=1, 14%), prostate adenocarcinoma (n=1, 14%), clear cell renal carcinoma (n=1, 14%) and papillary thyroid carcinoma (n=1, 14%). All the cases were localized and surgically resected, with two patients (colonic and endometrial adenocarcinoma) needing adjuvant chemo-radiotherapy. All patients achieved complete remission with no mortality related to second malignancies. Conclusions: Approximately 8% of CMML patients have second cutaneous (3.5%), hematological (3.5%) and visceral (1%) malignancies, with most of these occurring in and beyond the seventh decade (~70%) of life. Mortality related to second cancers was low (2%), with most patients receiving appropriate surgical and adjuvant chemo-radiation therapy. Disclosures Al-Kali: Celgene: Research Funding; Onconova Therapeutics, Inc.: Research Funding.

Abstract Background JAK inhibitors (JAKi) currently represent a frontline treatment modality in patients with symptomatic intermediate- or high-risk myelofibrosis (MF). While the efficacy of JAKi in decreasing spleen size and improving disease-related symptoms is well documented, the effect of this drug class on overall survival and risk of leukemic transformation in MF patients is less well understood, primarily due to short follow up in individual patient cohorts. Furthermore, there is scant information on baseline predictors of overall and leukemia-free survival, and also spleen and/or anemia responses, at the onset of JAKi therapy. We conducted a sponsor-independent single-center retrospective review of patients treated with JAKi with long follow up to address the aforementioned issues. Here, we report our findings regarding baseline predictors of clinical outcome and treatment response in MF patients treated with JAKi. Methods Clinical, cytogenetic and molecular data were retrospectively collected on patients treated with one or more JAKi on a clinical trial at the Mayo Clinic, Rochester, MN. Baseline clinical data pertained to the onset of treatment with the first JAKi. Karyotype information was available for all patients. Screening for JAK2V617F, MPLW515, ASXL1, and SRSF2 mutations was performed as previously described (Leukemia. 2013 Apr 26. doi: 10.1038/leu.2013.119). Spleen size was measured by palpation by clinical investigators. Anemia and spleen responses were adjudicated as per IWG criteria (Tefferi, Blood, 2006). Information on survival and leukemic transformation was updated in July 2013. Results A total of 157 MF patients were studied (60% male; median age 65 years, range 34-89 years). Ninety seven patients (62%) had primary MF, 42 (27%) post-polycythemia vera MF, and 18 (11%) post-essential thrombocythemia MF. The DIPSS-plus distribution was: Low/Int-1 20 (13%), Int-2 66 (42%), and High 71 (45%). Other characteristics were: JAK2V617F positive 78%, unfavorable karyotype 17%, hemoglobin <10 g/dL 58%, leukocyte count >25 x 109/L 31%, platelet count <100 x 109/L 15%, peripheral blasts ≥1% 69%, red blood cell transfusion-dependent 37%, and constitutional symptoms 56%. Forty one patients (38%) were ASXL1 mutated (n=108) and 20 (18%) SRSF2 mutated (n=110). One hundred and thirty nine patients (89%) were evaluable for spleen response (median spleen size by palpation 19 cm, range 6-32 cm) and 91 (58%) for anemia response by IWG criteria. The first JAKi was momelotinib (CYT387) in 79 patients (50%), ruxolitinib in 51 (33%) and fedratinib (SAR302503) in 27 (17%). Median follow up from first JAKi treatment was 30 months (range 1-67 months); during this period, 72 deaths (46%) and 14 (9%) leukemic transformations have been documented. The median overall survival of the entire cohort was 41 months. Multivariable analysis identified the following DIPSS-plus independent predictors for survival: palpable spleen size >20 cm (HR 2.5), ASXL1 mutated status (HR 3.1) and SRSF2 mutated status (HR 3.8) (all p<0.01) (Figure). In contrast, there was no difference in survival with regards to the first JAKi used for MF treatment (p=0.3). Multivariable analysis identified the following predictors for leukemia-free survival: SRSF2 mutated (HR 9.7), unfavorable karyotype (HR 8.7), and palpable spleen size >20 cm (HR 9.1) (all p<0.01). Predictors of spleen response included baseline spleen size (≤20 cm 56%, >20 cm 34%; p=0.01), with borderline significance for ASXL1 mutation status (unmutated 51%, mutated 31%; p=0.06). Predictors of anemia response included RBC transfusion status at baseline (transfusion dependent 38%, transfusion independent 12%; p<0.01) and JAK2V617F status (unmutated 46%, mutated 22%; p=0.02). Conclusions Larger baseline spleen size and ASXL1 and SRSF2 mutated status predicted for inferior overall survival, independent of DIPSS-plus, at the onset of first JAKi treatment in MF patients. Similarly, multivariable analysis identified larger baseline spleen size and presence of unfavorable karyotype and SRSF2 mutations as being predictive for inferior leukemia-free survival. Spleen response rates were significantly superior in patients with smaller baseline spleen size and possibly ASXL1 unmutated status. Anemia response rates were significantly higher in patients who were transfusion-dependent at baseline and had JAK2V617F unmutated status. Disclosures: Pardanani: Bristol Myers Squibb: Clinical trial support Other; Sanofi: Clinical trial support, Publication support services, Clinical trial support, Publication support services Other; PharmaMar: Clinical trial support, Clinical trial support Other; JW Pharmaceutical Corp.: Clinical trial support, Clinical trial support Other. Off Label Use: Use of Ruxolitinib, Momelotinib, Fedratinib and Pomalidomide for treatment of Myelofibrosis in Clinical Trial setting.

Abstract Background Spliceosome pathway gene mutations are recurrent in myeloid malignancies with the highest frequencies reported for myelodysplastic syndromes associated with ring sideroblasts (MDS-RS; 85%), MDS without RS (44%) and chronic myelomonocytic leukemia (CMML; 55%) (Nature 2011;478:64). SF3B1 mutations were the most frequent (75%) in MDS-RS and SRSF2 mutations in CMML (28%); U2AF1 mutational frequencies were 12% in MDS without RS and 8% in CMML. We have previously described SRSF2 (Blood. 2012;120:4168) and SF3B1 (Leukemia. 2012;26:1135) mutations in 17% and 6.5% of patients with primary myelofibrosis (PMF); prognostic relevance was shown for the former but not the latter. Objectives The objectives of the current study were to i) describe the incidence of U2AF1 mutations in PMF and their correlation with clinical features, karyotype and other mutations and ii) examine the prognostic significance of U2AF1 mutations in PMF, in the context of both conventional prognostic models and other prognostically-relevant mutations. Methods Information on clinical and laboratory parameters and karyotype was available in all study patients, at time of referral, which coincided with time of sample collection for mutation screening. Risk stratification was according to the Dynamic International Prognostic Scoring System (DIPSS)-plus system. U2AF1 and other mutations were analyzed using standard PCR techniques and bidirectional sequencing; for U2AF1, two hot spots that included residues S34 and Q157 were amplified. Results A total of 251 PMF patients (median age 63 years; 160 males) were studied. DIPSS-plus risk distribution was high in 32%, intermediate-2 in 38%, intermediate-1 in 17% and low in 13% of the patients. The frequency of each DIPSS-plus adverse feature was as follows: age >65 years (42%), transfusion need (32%), hemoglobin <10 g/dL (47%), leukocyte count >25 x 10(9)/L, (16%), platelet count <100 x 10(9)/L (22%), ≥1% blasts (57%), constitutional symptoms (35%), and unfavorable karyotype (9%). Karyotype was normal in 156 (63%) patients. At a median follow-up of 48 months, 158 (63%) deaths and 27 (11%) leukemic transformations were recorded. Mutational frequencies Forty-one (16.3%) patients harbored U2AF1 mutations: 16 (39%) Q157P, 10 (24%) Q157R, 8 (20%) S34F, 4 (10%) S34Y and one each for Q157P/E159A, Q157R/S34Y and Q157-Y158insYE. Frequencies for other mutations were 11% for SRSF2, 7.3% for SF3B1, 31% for ASXL1, 5.5% for EZH2, 5% for IDH1/2 and 58% for JAK2V617F. U2AF1 mutations were usually, but not always, exclusive of other spliceosomal mutations: one patient expressed all three spliceosomal mutations. The frequency of any one of the three spliceosomal mutations was 34%. Clinical correlates U2AF1 mutations were significantly associated with older age (p=0.02), JAK2V617F (p=0.002), mutant ASXL1 (p=0.04), transfusion need (p<0.0001), hemoglobin <10 g/dL (p<0.0001), platelets <100 x 10(9)/L (p<0.0001) and normal karyotype (p=0.006). The associations with anemia, thrombocytopenia, JAK2V617F and normal karyotype were inter-independent; the frequency of U2AF1 mutations in the presence of anemia was 29%, transfusion need 36%, thrombocytopenia 35%, JAK2V617F 23% and normal karyotype 21%. Prognostic interactions U2AF1 mutations were associated with inferior overall (p=0.004) but not leukemia-free (p=0.6) survival. However, the survival association was fully accounted for by the above-mentioned clustering of U2AF1 mutations with anemia and thrombocytopenia. Similarly, although multivariable analysis of U2AF1, SRSF2, ASXL1, EZH2 and IDH mutations identified the first three as being independently predictive of poor survival, only ASXL1 and SRSF2 remained significant when either anemia or thrombocytopenia was included as a co-variate. Conclusions U2AF1 mutations are the most frequent spliceosome pathway mutations in PMF, cluster with normal karyotype and JAK2V617F, and are strongly associated with anemia and thrombocytopenia; the latter suggests a pathogenetic contribution to ineffective hematopoiesis in PMF. The current study also suggests that more than one third of patients with PMF carry a spliceosome mutation. Disclosures: No relevant conflicts of interest to declare.

Abstract Activating mutations in colony stimulating factor 3 receptor gene (CSF3R) have recently been identified as specific driver mutations in chronic neutrophilic leukemia (CNL)(NEJM 2013;368:1781). We sought to outline the clinical and laboratory characteristics and natural history of this molecularly defined entity. Methods : An IRB- approved retrospective analysis of all cases of CSF3R-mutated WHO-defined CNL was undertaken. Methods of mutation analysis have been previously published. (Leukemia 2013; 27: 1870) Results : From 10/1995 to 3/2013, fourteen patients (median age 67 years; 57% male) with CSF3R-mutated CNL were evaluated at the Mayo Clinic. Most of the CSF3R mutations identified occurred in isolation: T618I (n=11), M696T (n=2) and I598I SYN (n=1), although one T618I mutant patient also carried a cytoplasmic domain mutation (2341_2342insC ). All patients were BCR/ABL negative. Five carried a SETBP1 mutation: G870D (n=2), D868N (n=2) and G872R (n=1), all of whom carried the T618I mutation. Only one of 10 tested carried a CALR mutation (E398D) and none of 13 the JAK2 V617F mutation. Apart from fatigue, most patients were asymptomatic at diagnosis and leukocytosis had been mostly an incidental laboratory finding. Ten (71%) patients had preceding chronic leukocytosis, median duration 12.5 months (range 5-84), 5 reported weight loss, which was accompanied by fevers/ sweats in 3. Five (36%) patients had prior exposures to adjuvant chest wall irradiation for breast cancer (n=2), radioiodine for thyroid disease (n=2) and methotrexate/cyclophosphamide for autoimmune disease (n=2). Five (36%) patients had palpable splenomegaly. Peripheral blood (PB) parameters, median (range) at diagnosis were: Hb 10.8 g/dl (8.5-16.0), WBC: 54 x 10(9)/L (21.7-176.2), platelets: 201 x 10(9)/L (25-476), neutrophils: 85% (78-94), immature cells (myelocytes + metamyelocytes): 6% (0-11) and monocytes: 3% (0-10). Neutrophils had toxic granulation and Dohle Bodies in 71% of cases. LDH was elevated in all patients: 354 U/L (225-496). None had a monoclonal protein. The bone marrow (BM) was hypercellular in all patients: 95% (80-100%) and granulocytic precursors were markedly increased with left-shifted (n=11) or normal maturation. BM blasts were < 5%. Reticulin fibrosis was absent (n=5), 1+ (n=5) or 1-2+ (n=1) in those with staining. Cytogenetics were normal in all but one at diagnosis. One patient was lost to follow up after diagnosis. Hydroxyurea (HU) was the most frequently used therapy (initial therapy in 10 patients) and was ultimately received by all. Although initially effective at controlling leukocytosis, clinical course of most was defined by progressive refractory mature neutrophilia with the development of red cell (n=6) and platelet transfusion (n=3) dependancy. Other therapies included IFN or PEG/IFN (n=4), hypomethylating agents (n=4), ruxolitinib (n=2), thalidomide (n=2), cladribine (n=2) and imatinib (n=1). None of these additional drugs, induced remission and they provided primarily palliative and transient benefit. One patient transformed into AML at 17 months, one received induction chemotherapy for suspected AML (PB and BM blasts at 11-13%) at 19 months and two evolved into WHO-defined CMML, at 20 and 27 months, respectively. Both patients with documented/suspected AML were both T618I and SETBP1 positive. Of the cases progressing to CMML (one with T618I, one with M696T) neither was SETBP1 positive. At last follow up, eight patients had died and median survival was 22.5 months. Progressive refractory neutrophilic leukocytosis often preceded and contributed to death. Four (29%) patients are currently alive after a median follow-up of 61 months (14.3- 69.4). Conclusion : CSF3R-mutated CNL is an aggressive disease (median survival <2 years) characterized by progressive neutrophilic leukocytosis which is only partially controlled with current drugs, including the putative targeted therapy ruxolitinib (both ruxolitinib-treated patients progressed on treatment). Patients with CNL could progress into AML or CMML; however, most succumb from complications associated with progressive treatment-refractory leukocytosis. The observation that both patients with documented/suspected AML were SETBP1-mutation positive suggests potential pathogenetic link. Disclosures Off Label Use: Ruxolitinib, cladribine, thalidomideand inteferon. These drugs were used in a historical account of CNL and I report on these in a retrospective chart review only..

Abstract Background : Controversy persists regarding diagnostic accuracy as well as added value for quantitative assessment of neutrophil PRV-1 expression in polycythemia vera (Klippel et al. Blood2003;102:3569; Tefferi et al. Blood2004;103:3547). This is partly because of limited data regarding the relationship between neutrophil PRV-1 transcript level and other laboratory parameters in polycythemia vera (PV). More information is also needed on the PRV-1 expression profile in other myeloproliferative disorders (MPD), including essential thrombocythemia (ET) and agnogenic (AMM), post-polycythemic (PPMM), and post-thrombocythemic (PTMM) myeloid metaplasia. Methods : The current prospective study involves 141 subjects, including 49 with PV, 40 with secondary or spurious polycythemia (SP), 23 with ET, 11 with AMM, 4 with PPMM, 3 with PTMM, and 11 normal controls. Peripheral blood neutrophil PRV-1 expression was quantified by real-time PCR according to published methods (Klippel et al. Blood 2003). The results were first compared among the different disease categories and controls. Subsequently, within the specific disease categories of both PV and ET, PRV-1 expression was correlated with several laboratory [leukocyte count, absolute neutrophil count (ANC), leukocyte alkaline phosphatase score (LAP) score, CD34 count, platelet count, hematocrit, serum EPO level, MCV, serum ferritin, reticulin fibrosis] and clinical (age, sex, disease duration, spleen size, thrombosis history, and presence of treatment with cytoreductive agent, aspirin, or phlebotomy) parameters. Results : The incidence of increased neutrophil PRV-1 expression was the highest in PV and PPMM (p<0.0001) while the results in the other disease categories (ET, SP, AMM, PTMM) were similar among each other and controls ( p=0.89). Table 1 Parameters Cont. (n=11) SP (n=40) PV ( n=49) ET (n=23) AMM (n=11) PPMM (n=4) PTMM (n=3) Age in years median NA 51 58 46 60 63 47 range NA 19–78 18–83 18–81 41–80 56–71 47–72 Females NA 20% 55% 65% 18% 25% 33% PRV-1/GAPDH median 1.25 1.25 1.01 1.24 1.26 1.04 1.25 range 1.14–1.43 0.99–1.41 0.83–1.28 0.9–1.44 1.08–1.43 0.93–1.11 1.23–1.27 PRV-1 ↑ 9% 15% 76% 17% 18% 100% 0% In both PV and ET, neutrophil PRV-1 expression strongly correlated with LAP score (p=0.003 and 0.04, respectively). In addition, in PV, univariate analysis revealed significant correlations between PRV-1 expression and leukocyte count, ANC, and reticulin fibrosis. On multivariate analysis, only LAP score retained its significance. Figure Figure Conclusion :The current study confirms the previously recognized close association between increased neutrophil PRV-1 expression and PV as well as suggests a similarly strong association with PPMM but not with ET, MMM, or PTMM. The study also underscores the suboptimal diagnostic accuracy of the neutophil PRV-1 assay in distinguishing PV from SP. Most importantly, the demonstrated tight correlation between PRV-1 transcript level and LAP score raises the issue of added value to currently available diagnostic tests.

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 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.