Lasho, Terra L.

Abstract Background : Chronic myelomonocytic leukemia (CMML), a myeloid neoplasm with overlapping features of myelodysplastic syndromes and myeloproliferative neoplasms (MPN), is characterized by peripheral blood (PB) monocytosis and a risk for leukemic transformation (LT). Gene mutations commonly seen in CMML include, TET2 (~60%), SRSF2 (~50%) and ASXL1 (~40%). MPN associated-driver mutations such as JAK2V617F (~10%), MPL (<1%) and CALR (<1%) are uncommon, and in fact, the 2016 World Health Organization (WHO) guidelines state that the presence of these mutations tends to support a diagnosis of MPN with monocytosis. We carried out this study to assess the i) clinical correlates and ii) survival outcomes of MPN associated-driver mutations in CMML. Methods : 323 molecularly annotated patients with 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 30 myeloid relevant genes, by previously described methods. Results: Among the 323 study patients, 67% were males and median age was 71 years (range, 18-95). Twenty five (8%) patients had CMML with MPN associated-driver mutations; 24 (96%) with JAK2V617F and 1(4%) with a MPLW515L mutation. There were no patients with CALR or JAK2 exon 12 mutations. Additional signal-pathway mutations included NRAS (15%), KRAS (4%), CBL (14%), PTPN11 (3%), CSF3R (1%), and SH2B3 (<1%). i) Phenotypic correlates: The median age of CMML patients with MPN-associated driver mutations was 71 years and 66% were male. The WHO morphological subtypes included CMML-0 60%, CMML-1 24% and CMML-2 16%; while 91% of patients had a normal karyotype. The distribution of mutations included TET2 75%, SRSF2 54%, ASXL1 48%, RUNX1 20%, NRAS and EZH2 12% each, SETBP1 8%, SF3B1, U2AF1, CBL, PTPN11, FLT3-TKD, & Tp53 4% each, respectively. Risk stratification by the Mayo Molecular Model included high 16%, intermediate-2 32%, intermediate-1 36% and low risk 16%, respectively. In comparison to CMML patients without MPN associated-driver mutations, those with, had a higher hemoglobin (HB, p=0.001) and hematocrit (p=0.001), were more likely to have leukocytosis (p=0.02) and elevated LDH levels (p=0.0002), less likely to have thrombocytopenia (p=0.003), more likely to have a "proliferative" CMML phenotype (p=0.01) with palpable splenomegaly (p=0.0007), and more likely to have mutations involving TET2 (p=0.02). Six (2%) thrombotic events were documented in the cohort; 2 (8%) in patients with MPN-driver mutations and 4 (1%) in those without (p=0.07). There were no differences between the two groups with regards to the degree of monocytosis, PB and BM blasts, BM cellularity, BM megakaryocytic atypia, and BM fibrosis. ii) Survival outcomes: At last follow up 219 (68%) deaths and 55 (17%) LT were documented, of which 14 (56%) deaths and 2 (8%) LT occurred in the MPN-driver mutation group. The median OS for the entire cohort was 28 months (22-32); 31 months for CMML patients with MPN associated-driver mutations and 24 months for those without (p=0.4). On a univariate analysis, survival was adversely impacted by low HB (p<0.0001), high white blood count (p=0.0009) and absolute monocyte count (p=0.0004), circulating immature myeloid cells (p=0.02) and blasts (p=0.005), BM blasts (p=0.02), palpable splenomegaly (p=0.03), abnormal karyotype (p=0.001), presence of DNMT3A (p=0.006), ASXL1 (p=0.009), EZH2 (p=0.03) and Tp53 (p=0.02) mutations and the absence of TET2 (p=0.0009) mutations. Survival was not affected by the presence of JAK2V617F (p=0.4) or MPL (p=0.46) mutations. On a multivariable analysis that included the aforementioned significant variables, only HB <10 gm/dl (p=0.0016), presence of PB blasts (p=0.01), presence of DNMT3A (p=0.003) and the absence of TET2 mutations (p=0.02) retained significance. The presence of MPN associated-driver mutations did not impact LFS (p=0.36). Conclusions: The occurrence of MPN associated-driver mutations in CMML is infrequent (~8%) and is largely restricted to JAK2V617F. CMML patients with MPN-associated driver mutations have a higher HB/hematocrit, are more likely to have proliferative features and palpable splenomegaly, and are less likely to be thrombocytopenic. There was a trend towards an increased thrombotic risk, with no impact of these mutations on overall and leukemia free survival. Disclosures No relevant conflicts of interest to declare.

Abstract Background: Current prognostic models in primary myelofibrosis (PMF) target overall survival (OS) and utilize MIPSS70 (mutation-enhanced international prognostic scoring system for transplant-age patients), MIPSS70+ version 2.0 (karyotype-enhanced MIPSS70) and GIPSS (genetically-inspired prognostic scoring system, which is based on mutations and karyotype) (JCO 2018;36:310; JCO doi: 10.1200/JCO.2018.78.9867; Leukemia. 2018;doi:10.1038/s41375-018-0107). In the current study, we used logistic regression statistics to identify risk factors for leukemic transformation (LT) within 5 years of diagnosis/referral (i.e. early events) and also performed Cox regression analysis of overall leukemia-free survival (LFS). Methods : Study patients were recruited from the Mayo Clinic, Rochester, MN, USA. Diagnoses of LT and chronic phase PMF were confirmed by both clinical and bone marrow examinations, in line with the 2016 World Health Organization criteria (Blood. 2016;127:2391); specifically, LT required presence of ≥20% blasts in the peripheral blood (PB) or bone marrow (BM) (Blood 2016;127:2391). Statistical analyses considered clinical and laboratory data collected at the time of initial PMF diagnosis or Mayo Clinic referral point. Logistic regression statistics was used to identify predictors of LT at 5 years from initial diagnosis/referral; in the particular method, patients with documented LT within 5 years were "uncensored" while those followed up for at least 5 years, without developing LT, were "censored"; the analysis excluded patients without LT and not followed for at least 5 years. In addition, Cox regression analysis was performed to identify risk factors for overall LFS. The JMP® Pro 13.0.0 software from SAS Institute, Cary, NC, USA, was used for all calculations. Results: 1,306 patients with PMF (median age 65 years; 63% males) were included in the current study; MIPSS70+ version 2.0 risk distribution was 20% very high risk, 41% high risk, 19% intermediate risk, 16% low risk and 4% very low risk. 149 (11%) patients were documented to experience LT, and compared to the remaining patients (n=1157), they were more likely to be males (p=0.02) and mutated for ASXL1 (p=0.01), SRSF2 (0.001) and IDH1 (0.02) and present with higher risk MIPSS70+ version 2.0 (p=0.02). Multivariable logistic regression identified the following as predictors of LT in the first 5 years of disease: IDH1 mutation (odds ratio; OR 78.4), very high risk (VHR) karyotype (OR 57.6), ASXL1 mutation (OR 15.1), age >70 years (OR 13.3), SRSF2 mutation (OR 8.5), male sex (OR 6.9), PB blasts ≥3% (OR 5.4), presence of moderate or severe anemia, adjusted for sex (OR 3.6) and constitutional symptoms (OR 3.1). On Cox regression analysis, the following were associated with inferior LFS: IDH1 mutation (HR 4.3), PB blasts ≥3% (HR 3.3), SRSF2 mutation (HR 3.0), age >70 years (HR 2.1), ASXL1 mutation (HR 2.0) and presence of moderate or severe anemia, adjusted for sex (HR 1.9). Subsequently, HR-based risk point allocation resulted in highly discriminating LT predictive model with HR (95% CI) of 39.4 (10.8-114) for high risk and 4.1 (2.4-7.3) for intermediate risk (Figure 1). Conclusions: The current study identifies IDH1 mutation as a main predictor of LT in PMF. Our study also implicates SRSF2 and ASXL1 mutations and VHR karyotype as other genetic markers of early LT. Other independent contributors of early LT and inferior LFS, overall, included PB blasts ≥3%, moderate to severe anemia and older age. We provide LT prediction model, based on these variables, with leukemia risk ranging from 8% to 57%. Disclosures No relevant conflicts of interest to declare.

Abstract Background: The 2016 World Health Organization (WHO) system lists five morphological categories of systemic mastocytosis (SM): indolent (ISM), smoldering (SSM), SM with an associated hematological neoplasm (SM-AHN), aggressive (ASM) and mast cell leukemia (MCL) (Blood. 2016;127:2391). In the current study, we capitalized on the large number of accumulated cases of SM seen at the Mayo Clinic, between 1968 and 2015 (n=580), in order to devise two separate but complementary prognostic models that are based on either clinically-derived variables, or a combination of both clinical and molecular information. Methods : Study patients (n=580) were recruited from the Mayo Clinic, Rochester, MN, USA. Diagnoses of SM and its morphological subcategories were confirmed by both clinical and bone marrow examinations, in line with the 2016 WHO criteria (Blood. 2016;127:2391). Next-generation sequencing (NGS) was performed in a subset of the study population (n=150). Statistical analyses considered clinical and laboratory data collected at the time of initial diagnosis at the Mayo Clinic. Conventional statistics was used for calculation and of overall survival and determination of inter-independent risk factors. Receiver operating characteristic (ROC) curves were used to determine the prognostically most discriminative platelet threshold. The JMP® Pro 13.0.0 software from SAS Institute, Cary, NC, USA, was used for all calculations. Results: 580 patients with SM (median age 55 years; range 18-88 years; 52% males) were included in the current study; 291 (50%) constituted indolent and 289 (50%) advanced variants; the latter included 85 (15%) ASM, 199 (34%) SM-AHN and 5 MCL cases. At presentation, anemia defined by hemoglobin level below the lower limit of the sex-adjusted reference range, was present in 41% of the patients, hemoglobin <10 g/dl in 16%, red cell transfusion-dependency in 9%, platelet count below the ROC-determined limit of 150 x 109/l in 26%, serum albumin below the lower normal limit (LNL) of the reference range in 22%, serum alkaline phosphatase (ALP) above the upper normal limit (UNL) of the reference range in 54%, urticaria pigmentosa in 41%, mast cell mediator symptoms in 46%, palpable hepatomegaly in 21%, palpable splenomegaly in 31%, "C" findings in 23% and "B" findings in 42%. Median follow-up was 34 months with 239 (41%) deaths and 9 (1.5%) leukemic transformations documented. Cytogenetic information was available in 342 cases, including 51 (15%) with abnormal karyotype and 27 (8%) with unfavorable karyotype. NGS-derived mutation information was available in 150 cases and most frequent mutations were KIT (75%), TET2 (29%), ASXL1 (17%) and CBL (11%). Age-adjusted multivariable analysis of clinical variables, which did not include mutation or cytogenetic information, identified age >60 years (HR 2.5, 95% CI 1.8-3.4), advanced vs ISM (HR 2.3, 95% CI 1.5-3.7), thrombocytopenia <150 x 109/l (HR 2.4, 95% CI 1.8-3.4), anemia (HR 2.1, 95% CI 1.5-3.0), ALP above UNL (HR 2.1, 95% CI 1.5-3.1) and albumin below LNL (HR 1.4, 95% CI 1.0-1.9). A similar age-adjusted multivariable analysis for mutations identified ASXL1 (HR 3.3, 95% CI 1.9-5.5), RUNX1 (HR 4.1, 95% CI 1.2-10.2) and NRAS (HR 5.7, 95% CI 1.7-14.9) as independent molecular risk factors; these mutations were henceforth labelled as "adverse". The addition of "adverse" mutations (HR 2.7, 95% CI 1.6-4.7) to the list of the aforementioned clinical risk factors resulted in loss of significance for age and anemia while platelets below 150 x 109/l (HR 2.9, 95% CI 1.8-4.8), ALP above UNL (HR 3.2, 95% CI 1.8-6.1), albumin below LNL (HR 2.5, 95% CI 1.6-4.0) and advanced vs ISM (HR4.2, 95% CI 1.9-10.7) remained significant. Subsequently, based on the number of risk factors present, a "clinical" (figure 1a; n=380 informative cases) and "clinical-molecular" (figure 1b; n=129 informative cases) risk models were prepared; both risk models performed effectively for advanced and indolent SM, when analyzed separately (data not shown). Conclusions: The current study advances two separate but complimentary risk models for SM and illustrates the additional prognostic contribution of mutations and underlines the importance of thrombocytopenia and serum ALP and albumin levels in both risk models. Furthermore, we show effective application of both risk models in advanced and indolent SM. Disclosures No relevant conflicts of interest to declare.

Abstract Background Recent studies have suggested that increased circulating free light chains, inflammatory cytokines, hepcidin, ferritin, and ASXL1 mutated status have independent prognostic value for survival in primary myelofibrosis (PMF). In the current retrospective study, we studied all the aforementioned variables in the context of DIPSS-plus to examine their relative prognostic contributions for overall and leukemia-free survival. Methods The diagnosis of PMF was according to WHO criteria. The study population included all consecutive referrals to the Mayo Clinic, Rochester, MN where complete information on clinical and laboratory parameters and bone marrow karyotype was available. Inclusion to the study required availability of a research blood sample collected at referral. Levels of circulating free light chains (FLC) (J Clin Oncol. 2012 Apr 1;30(10):1087), inflammatory cytokines (J Clin Oncol. 2011 Apr 1;29(10):1356), hepcidin and ferritin (Am J Hematol. 2013 Apr;88(4):312) were determined as previously described. Disease-relevant mutations (ASXL1, SRSF2, EZH2, IDH-1/2, SF3B1, JAK2V617F and MPLW515) were identified as previously described (Leukemia. 2013 Apr 26. doi: 10.1038/leu.2013.119). Results A total of 224 PMF patients were studied (66% male; median age 63 years, range 18-83 years). The DIPSS-plus distribution was: Low 23 (10%), Int-1 26 (12%), Int-2 98 (44%), and High 77 (34%). Clinical characteristics were: unfavorable karyotype 13%, hemoglobin <10 g/dL 58%, leukocyte count >25 x 109/L 13%, platelet count <100 x 109/L 20%, peripheral blasts ≥1% 62%, red blood cell transfusion-dependent 38%, and constitutional symptoms 36%. Mutation frequencies were: JAK2V617F 61% (n=220), MPLW515 6% (n=204), ASXL1 34% (n=154), SRSF2 16% (n=189), IDH-1/2 3% (n=209), EZH2 1.5% (n=134), SF3B1 5% (n=91) and SETBP1 4% (n=114). Distribution of circulating biomarkers was: FLC >receiver operating characteristic (ROC) cutoff of 3.78 mg/dL 37% (n=222), interleukin (IL)-2R and/or IL-8 >3 standard deviations above normal mean (SD) 75% (n=197), ferritin ≥1000 mcg/L 18% (n=146), ferritin ≥500 30% (n=146), and hepcidin >3SD 72% (n=203). Median follow up from the time of referral was 45 months (range 1-317 months); during this period, 139 deaths (62%) and 27 (12%) leukemic transformations have been documented. The median overall survival of the entire cohort was 61 months. The following variables had DIPSS-plus independent predictive value for inferior survival: ferritin ≥1000 mcg/L (HR 1.9), ASXL1 mutated status (HR 1.7) and FLC >ROC cutoff (HR 2.0) and IL-2R and/or IL-8 >3SD (HR 1.6) (all p<0.05). When the aforementioned variables were added to the multivariate model (sequentially or altogether), only serum ferritin >1000 mcg/L retained its significance for overall survival independent of DIPSS-plus (HR 2.1; p=0.03). The predictive significance for increased ferritin level was maintained in RBC transfusion-dependent (ferritin ≥1000 mcg/L; n=26/54; median survival 21 versus 59 months; p=0.03) and -independent (ferritin >500 mcg/L; n=5/92; median survival 20 versus 80 months; p=0.0002) patients. Multivariable analysis identified the following predictors for inferior leukemia-free survival: SRSF2 mutated status (HR 4.8), unfavorable karyotype (HR 4.0), and circulating blasts ≥1% (HR 3.0) (p<0.05). Conclusions When considering composite risk-assessment in PMF where input variables were disease-relevant mutations and circulating biomarkers in addition to traditional clinical parameters, only increased serum ferritin levels at time-of-referral retained adverse predictive value for overall survival independent of DIPSS-plus. The prognostic value of serum ferritin was independent of anemia and RBC transfusion need. It appears to more fully capture the severity of the underlying erythropoietic defect in PMF by also likely reflecting (as an acute-phase reactant) the marrow-suppressive effect of disease-related inflammation. The shortened survival in patients with increased ferritin levels was unrelated to iron overload since none of the deaths, where information was available, were caused by cardiac siderosis. Serum ferritin levels were not associated with leukemic transformation risk. In the latter analysis, only SRSF2 mutations, unfavorable karyotype and presence of circulating blasts were independently associated with inferior leukemia-free survival. Disclosures: No relevant conflicts of interest to declare.

Abstract Myeloproliferative disorders (MPD) are a group of heterogeneous diseases that include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). They are characterized by increased hematopoiesis leading to elevated numbers of non-lymphoid cells and/or platelets in the peripheral blood. Beside thrombotic and hemorrhagic complications, MPD may evolve into secondary acute myeloid leukemia (sAML). Recently characterized markers suggest an opportunity to diagnose and identify subpopulations of MPD patients. In particular, altered expression and point mutations of PRV-1, MPL and JAK2 were commonly found in MPD, as well as deletions on chromosome 20q (del20q). Acquired uniparental disomy (UPD) on chromosome 1p (1pUPD) and 9p (9pUPD) leading to copy-neutral loss of heterozygosity (LOH) is a further mechanism found in MPD which often leads to homozygous activated mutations of MPL and JAK2, respectively. However, the molecular mechanisms involved in the transformation process to sAML remains unclear. Using standard metaphase cytogenetics (MC), chromosomal abnormalities are found in only a proportion of patients with MPD. We hypothesized that with new precise methods more genomic lesions can be uncovered that may be associated with leukemic transformation. To address this issue, we used 250K single nucleotide polymorphisms (SNP) Chip arrays to study chromosomal lesions in 40 sAML samples from patients who evolved from MPD; 7 had preexisting PV, 25 PMF, and 8 ET. Moreover, 43 additional samples of MPD (10 PV, 17 ET, and 16 PMF) were included in this study. SNP-chip analysis showed major chromosomal changes in almost all the sAML samples including monosomy 16, deletions of 1q-, 3p-, 6p-, 5q-, 7q-, 9q-, 12p-, 6q-, 3q-, 17p-, 19q-, and 20q- as well as trisomy 2, 3, 8, 9, 12, 15, 19, 21, and 22. We validated these data by MC. However, numerous new genomic alterations which contained potentially interesting genes that might contribute to leukemic transformation were detected by SNP Chip Array in patient samples with normal karyotype. Moreover, UPD was very frequent: 44% (19/43) of MPD and 53 % (21/40) sAML samples. 1pUPD occurred in 5 patients with MPD (1PV, 4 PMF; 12 %) compared to 5 patients with sAML (1 PV, 4 PMF; 13 %). 9pUPD was found in 16 MPD patients (8 PV, 7 PMF, 1 ET; 37%) and 6 sAML patients (3 PV, 2 PMF, 1ET; 15 %). All patients with 9pUPD proved to be positive for the JAK2 V617F mutation seen by allele specific PCR. Interestingly, the MPD samples only had UPD on 1p, 9p, and 12q. In contrast, sAML samples showed additional UPD regions on 7q, 11q, 12q, 16p, 17p, 19q, and 21q. Beside the evaluation of the non-matching groups of patients with MPD and sAML, we also evaluated 4 patients during their PMF and sAML stages by SNP Chip. The sAML samples acquired additional genomic changes including trisomy 8, 10, 14, 19, duplication on 3q and 6p, and heterozygote deletion on 18q. In contrast, 1pUPD, 9pUPD, and 12qUPD were detected in both MPD and sAML matching samples, suggesting that these changes do not play an immediate role in causing transformation. In conclusion, we detected chromosomal regions possessing genes which may be involved in the leukemic transformation of MPD patients.

Abstract Background : Thrombosis is a characteristic feature in essential thrombocythemia (ET) and polycythemia vera (PV). Previous studies had focused on thrombotic events occurring after diagnosis and did not always distinguish between arterial and venous thrombosis. Arterial thrombosis in ET has been associated with age >60 years, JAK2 mutations, leukocyte count >11 x 109/l, history of thrombosis and cardiovascular risk factors, and venous thrombosis with male sex (Blood 2011;117:5857). In the current two-enter study, we looked for associations between both driver and other mutations, with first incidences of arterial and venous thrombosis in ET and PV, analyzed together and separately. Methods : Study patients were recruited from the Mayo Clinic, Rochester, MN, USA and the University of Florence, Florence, Italy, based on availability of next-generation sequencing-derived mutation information. Diagnoses were according to the 2016 World Health Organization criteria (Blood. 2016;127:2391). Conventional statistics was employed to examine significance of associations, with separate analysis of arterial vs venous thrombosis, occurring at any time before or after formal diagnosis of ET or PV. For the purposes of the current study, only first events were considered. Results: 906 molecularly-annotated patients (416 from Mayo and 490 from Florence), including 502 ET and 404 PV cases, were included in the current study. The Mayo/Florence cohorts included 270/232 ET (median age 57/54 years, 60%/59% females) and 146/258 PV (median age 63/58 years, 52%/44% females) patients; median follow-up was 9.9/12.9 years for ET and 10.7/12.4 years for PV. Driver mutation distribution in ET was 55% JAK2, 27% CALR, 5% MPL and 13% triple-negative. Most frequent mutations, other than JAK2/CALR/MPL, were TET2 (14%; 11% in ET and 18% in PV) and ASXL1 (13%; 13% in ET and 13% in PV). 301 (33%) patients experienced first time thrombosis, before or after diagnosis, including 152 (30%) in ET and 149 (37%) in PV (p=0.03); arterial events occurred in 174 (19%) patients, including 96 (19%) in ET and 78 (19%) in PV (p=0.9); venous events occurred in 177 (20%) patients, including 82 (16%) in ET and 95 (24%) in PV (p=0.007). The number of vascular events after diagnosis was 174 (19%) for arterial and 154 (17%) for venous thrombosis, with no significant difference between ET and PV. Associations with arterial thrombosis: In multivariable logistic regression analysis that included both ET and PV (n=906), age >60 years (23% vs 16%; OR, 1.6, 95% CI 1.1-2.2) and absence of ASXL1 and RUNX1 mutations (21% vs 9%; OR 2.6, 95% CI 1.4-4.8) were associated with arterial thrombosis; these associations remained significant (or near significant) when ET and PV were analyzed separately; in addition, JAK2 mutations in ET displayed borderline significance (p=0.05). Overall incidence of arterial events in ET was estimated at 27% in the presence of all three risk factors (i.e. older age, presence of JAK2 mutations and absence of ASXL1/RUNX1 mutations), 21% with two risk factors, 13% with one risk factor and 9% in the absence of all three risk factors (p=0.01). Associations with venous thrombosis: In multivariable analysis, including both ET and PV (n=906), JAK2 mutations (p<0.001), higher leukocyte count (p=0.03) and younger age (p=0.04) were identified as being significant. Analysis of ET patients only (n=502) identified JAK2 mutations (p<0.001; OR 2.4, 95% CI 1.4-4.1), absence of SRSF2 mutations (p=0.03) and younger age (p=0.04) as independent risk factors and in PV patients (n=404), higher leukocyte count (p=0.06). Prognostic interaction between JAK2 mutations and age: In ET, the overall frequency of venous events was 27% for young patients with JAK2 mutations vs 13% for older JAK2 mutated cases vs 10% for JAK2 unmutated young patients vs 13% for JAK2 unmutated older patients (p<0.001). The corresponding percentages for arterial events were 20%, 24%, 14% and 19%, respectively (p=0.1). Conclusions: JAK2 mutations contribute to the risk of both venous (younger patients) and arterial (young and older patients) thrombosis, in an age-dependent manner; the association with venous thrombosis might explain the higher incidence of venous thrombosis in PV, compared to ET. Additional studies are required to confirm the observed lower risk of thrombosis in patients with ASXL1, RUNX1 and SRSF2 mutations and provide insight into the underlying biological explanation. Disclosures No relevant conflicts of interest to declare.

Abstract Background: While several population-based studies have reported on adverse outcomes and life expectancy in myeloproliferative neoplasms (MPN) (JCO. 2015;33:2288; AJH. 1999;61:10; Leukemia. 2013;27:1874; JCO. 2012;30:2995), large-scale studies reporting mature data have been rare and important questions remain unanswered. The current study documents the Mayo Clinic (MC) decades-long experience with over 3,000 consecutive MPN patients including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) with the majority of patients followed until death. Herein, we provide mature, and importantly, risk-stratified survival data and disease complication estimates. Methods: Study patients were recruited from MC, Rochester, MN, USA between 10/27/1967 and 12/29/2017. All MPN diagnoses and documentation of fibrotic/leukemic transformations were in accordance with the 2016 World Health Organization criteria (Blood. 2016;127:2391). Clinical and laboratory data were abstracted from medical records. Risk-stratification used conventional risk models considering age, leukocytes, and venous thrombosis for PV (Leukemia. 2013;27:1874), international prognostic score for ET (Blood. 2012;120:1197), and dynamic international prognostic scoring system for PMF (Blood. 2010;115:1703). Statistical analyses were based on parameters obtained at the time of referral to MC which, in the majority of cases, coincided with or was within 1 year of diagnosis. All patients were followed from diagnosis until death or date of last follow-up/contact. Recipients of allogeneic stem cell transplants were censored at the time of transplant. Standard statistical methods and time-to-event curves prepared using the Kaplan-Meier method were used to compare MPN subgroups. Survival data for low-risk ET/PV were compared with age- and sex-matched controls from Olmstead County, MN, USA. JMP® Pro 13.0.0 software (SAS Institute, Cary, NC, USA) was used for all analyses. Results: 3,023 consecutive patients (median age 62 years, range 18-96; 51% males) were considered, including 89% diagnosed within the year: 665 PV, 1076 ET and 1282 PMF. Conventional risk stratification in 2925 evaluable patients revealed low, intermediate, and high-risk status in 26%, 29%, and 45% of PV and 30%, 42%, and 28% of ET patients, respectively, while PMF cases were attributed low (14%), intermediate-1 (38%) -2 (40%) and high (8%) risk (Table 1). After a median follow-up of 8.2 years for PV (range 0-39), 9.9 for ET (range 0-47), and 3.2 for PMF (range 0-31), 1631 (54%) deaths, 183 (6%) leukemic transformations, 244 (14%) fibrotic progressions, and 516 (17%) thrombotic events were recorded (Table 1). Median overall survival (OS) was 18 years for ET, 15 for PV and 4.4 for PMF (p<0.05 for all inter-group comparisons) (Figure 1A). Leukemia-free survival was similar for ET and PV (p=0.22) and significantly worse for PMF (p<0.001) (Figure 1B). PV, compared to ET, was associated with higher risk of fibrotic progression (p<0.001) (Figure 1C). Thrombosis risk after diagnosis was highest in PV and lowest in PMF (p=0.002 for PV vs ET; 0.56 for ET vs PMF; and 0.001 for PV vs PMF) (Figure 1D). Following risk stratification, median OS in low-risk ET (28 years) and low-risk PV (27 years) were superimposed (p=0.89) (Figure 2). Similarly, intermediate or high-risk PV and ET patients had comparable OS within each risk strata (p=0.23 and 0.11, respectively). Low-risk PMF survival was analogous to that of intermediate-risk PV (p=0.06). All other risk-stratified categories disclosed significantly different inter- and intra-group survival patterns (p<0.001) (Figure 2). Despite their categorization as favorable-risk disease, both low-risk ET and low-risk PV displayed excess mortality relative to age- and sex-matched controls with median survival of 26.7 and 28.1 years respectively, compared to the expected 37.5 and 39.2 years (p<0.001) (Figure 3). Conclusions: The current study provides large-scale and uniquely mature survival and outcomes data in MPN and highlights MPN subgroup risk categorization as cardinal in appraising disease natural history. Interestingly, OS was only marginally better in ET, compared to PV, while the latter clearly displayed a higher risk of thrombosis and fibrotic progression. Moreover, data disclosed shortened life expectancy relative to matched controls even in MPN with favorable risk attribution. Disclosures Busque: BMS: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Paladin: Consultancy.

Abstract Background High frequency mutations in both the colony-stimulating factor 3 receptor (CSF3R) and SET binding protein-1 (SETBP1) have recently been described in World Health Organization (WHO)-defined chronic neutrophilic leukemia (CNL) (NEJM 2013;368:1781; Leukemia. 2013. Prepublished on 2013/04/23). Response to treatment with ruxolitinib (10-15 mg twice-daily) was also described in one patient with CSF3RT618I mutation (NEJM 2013;368:1781). Primary cells from this patient were reportedly sensitive to inhibition by ruxolitinib (IC50, 127 nM). In a report of 12 patients with WHO-defined CNL, CSF3RT618I was seen in 10 (83%) patients, four (40%) of whom also expressed SETBP1 mutations (Leukemia. 2013. Prepublished on 2013/04/23). In the current study, we investigated the clonal distribution of mutant CSF3R and SETBP1in a CNL patient expressing both mutations and describe our observations regarding the effect of JAK inhibitors, both in vitro and in vivo. Methods Under institutional review board approved protocol, peripheral blood and buccal cells were collected and density gradient centrifugation was applied to enrich for mononuclear and granulocyte cell fractions. Antibody-labeled magnetic bead separation was used to obtain CD3+ and CD34+ cell fractions. DNA sequencing was used to screen for CSF3R and SETBP1 mutations. Single colonies were obtained by plating mononuclear cells in duplicate with cytokine enriched methylcellulose with and without the addition of fedratinib (a JAK2 inhibitor) or a commercially available JAK1 inhibitor. On day 11, erythroid and granulocyte individual colonies were counted and collected at each concentration. Single colonies were screened for both SETBP1 and CSF3Rmutations. Results The study patient was a 66-year-old lady with history of radiation therapy after lumpectomy for breast cancer in 1997. In October, 2012 the discovery of three synchronous lesions in the left breast necessitated mastectomy. White blood cell count (WBC) was approximately 13 x 10(9)/L at the time. Subsequently, her WBC gradually increased to 180 x 10(9)/L. Bone marrow examination on March 15, 2013 showed predominantly granulocytic proliferation with over 95% cellularity and no dysplastic features or monocytosis. Cytogenetic studies and mutation screening for JAK2V617F and BCR-ABL1were negative. A diagnosis of CNL was made and the patient was placed on hydroxyurea therapy. i) In vitro studies The patient harbored both CSF3RT618I and SETBP1D868N mutations in granulocytes, mononuclear cells and CD34+ myeloid progenitors; neither mutation was present in buccal or CD3+ cells. Colony forming unit (CFU) assay-derived 30 single colonies (15 erythroid and 15 granulocyte) were analyzed for the presence of CSF3R and SETBP1 mutations and all (100%) harbored heterozygous SETBP1D868N and 27 (90%) heterozygous CSF3RT618I; two granulocyte colonies were wild-type and one homozygous for CSF3RT618I. Fedratinib and the JAK1 inhibitor revealed activity in suppressing colony formation by >50% at a drug concentration of ≥600 nM. Mutation analysis in post-treatment residual single colonies revealed persistence of both mutations, even under conditions of higher ambient drug concentrations. However, CSF3R unmutated colonies were more likely to emerge than SETBP1unmutated colonies after in vitro drug exposure, which resulted in the appearance of occasional colonies that were negative for both mutations. ii) Treatment with ruxolitinib Ruxolitinib treatment at 10 mg twice-daily was started on 6/10/13, in addition to 500 mg/day of hydroxyurea. At the time, WBC was 86.5 x 10(9)/L, Hgb 11.8 g/dL and platelets 122 x 10(9)/L. On 6/17/13, WBC decreased to 43.9 and hydroxyurea was held. On 6/25/13, WBC increased to 89.6 x 10(9)/L and ruxolitinib was increased to 15 mg twice-daily. On 7/15/13, WBC had further increased to 190 x 10(9)/L and hydroxyurea was added to the treatment regimen at 500 mg/day. On 8/2/13, WBC was recorded at 91.3 x 10(9)/L. Conclusions In a double mutated CNL patient, we found CSF3R and SETBP1 mutations to be myeloid cell-restricted and co-expressed in erythroid and granulocytic cells; the latter antedated the former in order of acquisition. In vitro JAK inhibition had non-selective activity in suppressing myeloid colony formation. Treatment of the study patient with single agent ruxolitinib was ineffective. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 5158 Background: Vitamin D insufficiency is commonly observed in clinical practice. Observational studies have suggested an association of vitamin D insufficiency with increased risk of cancer development. Further, vitamin D insufficiency has been associated with poorer prognosis in solid tumors as well as hematologic malignancies, including non-Hodgkin lymphoma and chronic lymphocytic leukemia. In this study, we examined the clinical and prognostic correlates of low plasma levels of 25-hydroxyvitamin D (25[OH]D), which reflects whole-body vitamin D stores, in a cohort of myeloproliferative neoplasm (MPN) and myelodysplastic syndrome (MDS) patients. Methods: We studied a total of 409 patients: 247 (60%) had primary myelofibrosis (PMF), 74 (18%) de novo MDS, 63 (15%) polycythemia vera (PV) and 25 (6%) essential thrombocythemia (ET). Inclusion criteria required availability of archived plasma, bone marrow aspirate and biopsy and cytogenetic information at the time of first referral to our institution. Vitamin D measurements were performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). For the purposes of this study, plasma 25(OH)D levels <10 ng/mL indicated severe deficiency, <25 ng/mL insufficiency, and 25–80 ng/mL optimal levels. Results: Patient characteristics were as follows: (i) PMF: median age=63 years, >65 years=41%, males=68% and serum creatinine above the upper normal limit (UNL)=15%; (ii) MDS: median age=72 years, >65 years=77%, males=69% and creatinine >UNL=12%; (iii) PV median age=61 years, >65 years=37% and males=50%; and (iv) ET: median age=48 years, >65 years=10% and males=28%. Median (range) plasma 25(OH)D levels (ng/mL) were as follows: PMF 25 (0–60), PV 27 (6.8–51), ET 31 (6–50) and MDS 30 (7.7–72) (p=0.005). The proportion of patients with 25(OH)D insufficiency were: PMF 48%, PV 43%, ET 28% and MDS 28% (p=0.01). Similarly, the proportion of patients with severe 25(OH)D deficiency were: PMF 9%, PV 3%, ET 12% and MDS 1% (p=0.05). In PMF and MDS, 25(OH)D insufficiency was not significantly correlated with age (≤65 vs. >65 years), gender, red cell transfusion need, DIPSS-plus/IPSS category, hemoglobin (<10 vs. ≥10 g/dL), thrombocytopenia (<100 vs. ≥100 × 109/L), circulating blasts, unfavorable karyotype, serum creatinine >UNL, or JAK2V617F (PMF only) (p>0.05). 25(OH)D insufficiency was not predictive of inferior overall survival (OS) in PMF, MDS, PV, or ET (p>0.05). Similarly, in PMF and MDS, 25(OH)D insufficiency was not predictive of inferior leukemia-free survival (LFS) (p>0.05). Severe 25(OH)D deficiency was infrequently observed in MDS or PV patients; in PMF or ET patients, there was no prognostic impact of severe 25(OH)D deficiency on OS or LFS (p>0.05). Conclusions: Vitamin D insufficiency is relatively common in PMF, PV, ET and de novo MDS however this finding had no prognostic value for OS in the aforementioned patients. In PMF and MDS patients, low 25(OH)D levels had no clearly identifiable clinical correlates and further did not have prognostic value for LFS. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 971 Unlike the case in acute myeloid leukemia, there is limited information on the prognostic impact of IDH mutations in myelodysplastic syndromes (MDS). In the current study of 277 patients with MDS, IDH mutations were detected in 34 (12%) cases: 26 IDH2 (all R140Q) and 8 IDH1 (six R132S and two R132C). Mutational frequency was 4% (2 of 56) in refractory anemia with ring sideroblasts (RARS), 12% (16 of 130) in refractory cytopenia with multilineage dysplasia, 14% (2 of 14) in MDS-unclassifiable, 14% (6 of 42) in refractory anemia with excess blasts (RAEB)-1, and 23% (8 of 35) in RAEB-2. Normal karyotype was noted in all but one IDH1-mutated cases and 13 IDH2-mutated cases. Multivariable analysis identified presence of mutant IDH1 (p=0.0004; HR 4.0, 95% CI 1.9–8.8), revised International Prognostic Scoring System (IPSS-R) risk category (p<0.0001), and red cell transfusion need (p=0.002) as independent predictors of inferior survival (Figure 1). In a similar multivariable analysis, mutant IDH1 was the only variable associated with shortened leukemia-free survival (p=0.001; HR 7.0, 95% CI 2.3–20.8). The presence of IDH2 R140Q did not affect overall (p=0.54) or leukemia-free (p=0.81) survival (Figure 2). The current study suggests a powerful adverse prognostic effect for mutant IDH1 in MDS. Disclosures: No relevant conflicts of interest to declare.