Hanson, Curtis A.

Abstract Background: We have long introduced the concept of host genetic variations in the phenotypic diversity of myeloproliferative neoplasms (MPN) (Blood 2008;111:2785). Previous studies have established an association between JAK2 mutations in myeloproliferative neoplasms (MPN) and the germline GGCC (46/1) haplotype, which constitutes a string of single nucleotide polymorphisms (SNPs) near the JAK2 gene that are inherited together on chromosome 9p (reviewed recently;Int J Mol Sci. 2018; 19: 1152). In 2010, we reported an association between shortened survival in primary myelofibrosis (PMF) and nullizygosity for the JAK2 46/1 haplotype (Leukemia 2010; 24:105), although our findings were not confirmed in another study (Leukemia 2010; 24:1533). Others have reported an association with splanchnic vein thrombosis, that was not accounted for by JAK2 mutations (Ann Hematol 2014;93:1845). In the current study, we have increased the number of informative cases to 414 (from 130 reported in 2010), in order to revisit with the phenotypic and prognostic relevance of the JAK2 46/1 haplotype in PMF. Methods : Study patients were recruited from the Mayo Clinic, Rochester, MN, USA. Diagnoses PMF and its leukemic transformation were confirmed by both clinical and bone marrow examinations, in line with the 2016 World Health Organization criteria (Blood. 2016;127:2391). Screening for the JAK2 46/1 haplotype included rs12343867 SNP genotyping, as previously detailed (Leukemia 2010; 24:105), and using a commercially available TaqMan SNP genotyping assay (Applied Biosystems Inc., Foster City, CA, USA). Statistical analyses considered clinical and laboratory data collected at the time of initial PMF diagnosis or Mayo Clinic referral point. Conventional statistics was used for confirming phenotypic associations and calculation of overall (OS) and leukemia-free (LFS) survival. The JMP® Pro 13.0.0 software from SAS Institute, Cary, NC, USA, was used for all calculations. Results: 414 patients with PMF (median age 63 years; 63% males) were included in the current study; among 324 evaluable cases, MIPSS70+ version 2.0 risk distribution was 18% very high risk, 41% high risk, 19% intermediate risk, 18% low risk and 4% very low risk. Driver mutation distribution was 63% JAK2, 17% type 1-like CALR, 3% type 2-like CALR, 7% MPL and 10% triple-negative. JAK2 46/1 haplotype was documented in 69% of the study patients, including 25% in homozygous and 44% in heterozygous state. Driver mutation frequency in patients homozygous/heterozygous/nullizygous for the 46/1 haplotype was 78%/60%/56% JAK2, 10%/20%/18% type 1-like CALR, 3%/2%/5% type 2-like CALR, 4%/8%/7% MPL and 6%/10%/14% triple-negative (p=0.02). The three 46/1 haplotype groups were phenotypically mostly similar, with the exception of platelet count (p=0.02) and leukocyte count (p=0.003), which were both higher with homozygous 46/1 haplotype. In univariate analysis, nullizygosity for the JAK2 46/1 haplotype was associated with inferior overall survival (HR 1.5, 95% CI 1.1-1.9; figure 1a); this survival effect was most pronounced in JAK2 mutated cases (figure 1b; p<0.001), as opposed to CALR/MPL mutated cases (figure 1c; p=0.48) or triple-negative cases (figure 1d; p=0.27). Multivariable analysis that included age and other genetic risk factors, including karyotype, driver mutational status and presence of high molecular risk mutations, such as ASXL1 and SRSF2, confirmed the independent prognostic contribution of nullizygosity for the 46/1 haplotype (p=0.02; HR 1.4, 95% CI 1.1-1.8). Nullizygosity for 46/1 also remained significant in the context of the recently unveiled genetics-based prognostic model, GIPSS (genetically-inspired prognostic scoring system) (p=0.04) (Leukemia.2018 doi: 10.1038/s41375-018-0107-z), but not in the context of MIPSS70+ version 2.0 (karyotype and mutation-enhanced international prognostic scoring system for transplant-age patients) (p=0.4). (JClinOncol.2018 doi: 10.1200/JCO.2018.78.9867). Leukemia-free survival was not affected by the 46/1 haplotype (p=0.6). Conclusions: The current study confirms the association of nullizygosity for the JAK2 GGCC (46/1) haplotype with inferior survival in PMF, primarily in JAK2-mutated cases; the observed survival effect was independent of currently acknowledged genetic risk factors, including karyotype and high molecular risk mutations. Disclosures No relevant conflicts of interest to declare.

Abstract Background: Current drug therapy in primary myelofibrosis (PMF) offers palliative value only, including alleviation of splenomegaly, constitutional symptoms and anemia; hydroxyurea (HU), JAK2 inhibitors and interferon (IFN)-α are used for splenomegaly; JAK2 inhibitors for constitutional symptoms; and immunomodulatory drugs (IMiDs), erythropoiesis-stimulating agents (ESAs) and androgens for anemia. The current study constitutes a retrospective evaluation of specific drug response in anemia or splenomegaly in PMF. Methods : Study patients were recruited from the Mayo Clinic, Rochester, MN, USA. Diagnoses were according to the 2016 World Health Organization criteria (Blood. 2016;127:2391). For the purposes of the current study, conventional response criteria were modified to reflect clinical benefit assessment without strict adherence to criteria designed for clinical trials; accordingly, spleen response was evaluated only in patients with palpable splenomegaly and was defined as a minimum 50% reduction in palpable spleen size, regardless of response duration; anemia response was evaluated only in patients with hemoglobin level <10 g/dl and was defined as achieving transfusion-indpendence lasting for at least one month or an increase in hemoglobin of 2 g/dl, regardless of response duration. The JMP® Pro 13.0.0 software from SAS Institute, Cary, NC, USA, was used for all statistical calculations. Results: 432 cytogenetically- and molecularly-annotated patients with PMF were accessed in order to identify 333 patients who received first-line treatment with HU (n=97), JAK2 inhibitors (n=41), IFN-α (n=22), IMiDs (n=58), androgens (n=19), ESAs (n=54) and various other drugs (n=42), and were evaluable for response; driver mutational status was JAK2 59%, CALR type 1/like 20%, CALR type 2/like 4%, MPL 7% and triple-negative 10%; karyotype included very high risk (VHR) 6%, unfavorable 18% and favorable 76%; 60% of the patients harbored high molecular risk (HMR) mutations including ASXL1 (45%), SRSF2 (18%), U2AF1Q157 (10%), EZH2 (4%), IDH2 (4%) and IDH1 (2%). MIPSS70+ version 2.0 risk distribution was very high 17%, high 46%, intermediate 21%, low 14% and very low 2%. Overall, 249 patients were evaluable for spleen response, including 218 that were treated with the specific drugs analyzed in the current study. Anemia response was evaluated in 222 patients, including 194 (105 transfusion-dependent) that were treated with the specific drugs analyzed in the current study. Predictors of response to HU: Spleen response to HU was more likely in the presence of HMR mutations (37% vs 11%; p=0.02); however, responses were mixed and limited to presence of ASXL1 (40% vs 17%; p=0.06) or SRSF2 (47% vs 22%; p=0.1) mutations while none of 8 patients with either U2AF1Q157 or IDH2 mutations responded. None of 5 patients with VHR karyotype and only 1 (9%) of 11 patients with platelet count <100 x 109/l responded. Anemia responses to HU were infrequent. Overall response rate to HU was predicted by the absence of U2AF1Q157 mutations (64% vs 0%; p=0.007). Predictors of response to JAK2 inhibitors: Spleen response to JAK2 inhibitors was more likely in female patients (87% vs 50%; p=0.01) and in the presence of CALR type 1/like mutations (90% vs 57%; p=0.06). Anemia responses to JAK2 inhibitors were largely unpredictable. In order to further verify the aforementioned observed associations, we accessed data from a previous formal clinical trial of momelotinib (JAK2 inhibitor); among 91 evaluable patients, spleen response was higher in the presence of CALR mutations (73% vs 37%; p=0.009) and female sex (49% vs 39%, p=NS). Predictors of response to other agents: Spleen response to IFN-α was unlikely in the presence of ≥2% circulating blasts (0/6 responded) or presence of ASXL1 mutations (0/7 responded). IFN-α was ineffective for the treatment of anemia. Spleen responses to treatment with IMiDs, androgens or ESAs were unusual while anemia response to all three agents was not predicted by either genetic or clinical markers. Conclusions: Our observations, which require additional examination in a prospective setting, suggest limited value of genetic and clinical markers in predicting response to currently available drugs for PMF; this is consistent with the non-specific mechanism of action for these drugs. CALR (JAK inhibitors) and U2AF1Q157 (HU) mutations respectively predict favorable or unfavorable spleen response. Disclosures No relevant conflicts of interest to declare.

Abstract Background: Type 1 calreticulin (CALR) variants comprise ~70% of all CALR mutations in primary myelofibrosis (PMF) and form a distinct phenotypic and prognostic disease subset (Leukemia. 2014;28:1568). Determinants of long-term outcome have not, however, been systematically appraised in this population. The current study documents the natural history, molecular correlates, and independent predictors of overall (OS), leukemia-free (LFS), and thrombosis-free (TFS) survival in CALR type 1/like-mutated myelofibrosis. Methods: Patients were recruited from the Mayo Clinic, Rochester, MN, USA. Diagnoses were consistent with World Health Organization (PMF, fibrotic/leukemic transformations) (Blood. 2016;127:2391) and International Working Group for Myeloproliferative Neoplasms Research and Treatment criteria (post-essential thrombocythemia (ET) MF) (Leukemia. 2008;22:437). Laboratory and clinical data were retrospectively abstracted corresponding to time of referral (PMF) or myelofibrotic transformation (post-ET MF). Conventional prognostic scoring was as previously outlined (Blood. 2010;115:1703; J Clin Oncol. 2018;36:1769). Recipients of allogeneic stem cell transplant were censored at the time of transplant. Standard statistical methods were used for all analyses using the JMP® Pro 13.0.0 software package (SAS Institute, Cary, NC, USA). Results: A total of 162 consecutive patients with CALR type 1/like-mutated myelofibrosis were identified: 139 (86%) with PMF and 23 (14%) with post-ET MF with median age 55 years (range 23-85), 62% male. The PMF and post-ET MF cohorts displayed similar phenotypic features with the exception of higher platelet counts (median 443 vs 340 x 109/l; P=0.02) in post-ET MF (Table 1). The most frequent co-existing mutations were ASXL1 (n=47; 37%) and SRSF2 (n=4; 3%), with ASXL1 seen more frequently in PMF (39% vs 11% post-ET MF; P=0.07). Over a median follow-up of 6 years (range 0-25 years), a total of 20 (12%) leukemic transformations and 66 (41%) deaths were recorded, with no significant differences between the PMF and post-ET MF cohorts (P=0.16 and 0.11, respectively). Kaplan-Meier survival estimates revealed comparably favorable median OS in both CALR type 1/like-mutated variants: not yet reached and 13 years in post-ET MF vs PMF, respectively (P=0.7) (Figure 1A). Multivariable analysis disclosed moderate to severe sex-adjusted anemia (P<0.001), ≥2% circulating blasts (P<0.001), very high risk (VHR) karyotype (P<0.001), age >70 years (P=0.006), and constitutional symptoms (P=0.008) to be independent predictors of inferior OS in CALR type 1/like-mutated PMF (Table 2). LFS was significantly shortened in the presence of IDH1 mutations (P=0.01) and platelets <100 x 109/l (P=0.02) while IDH2 mutations (P=0.02), leukocytosis ≥11 x 109/l (P=0.03) and history of arterial thrombosis (P=0.04) were independent predictors of shortened TFS. Importantly, myelofibrosis variant (primary vs post-ET) did not influence survival (P=0.7) or complication rates (P=0.8 for LFS, P=0.09 for TFS) (Table 2). The karyotype- and mutation-enhanced international prognostic scoring system (MIPSS70+ version 2.0), was effective in risk stratifying type 1/like CALR-mutated PMF (P-values 0.01 to <0.0001), with the exception of low vs intermediate (P=0.22) and intermediate vs high risk (P=0.49) (Figure 1C). The detrimental influences of unfavorable/VHR karyotype and ASXL1 mutations were confirmed (Figure 2A-B) while borderline adverse and prognostically neutral effects were seen on OS for U2AF1 (n=116; P=0.05) and SRSF2 (n=120; P=0.98) respectively (Figure 2C-D). Conclusions: The current study documents analogous disease patterns in primary and post-ET CALR type 1/like-mutated MF and provides information on determinants of long-term 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 While unfavorable karyotype at referral is associated with significantly inferior overall and leukemia-free survival in myelofibrosis (MF) patients (Blood. 2011 Oct 27;118(17):4595), the incidence of clonal evolution during JAK inhibitor (JAKi) treatment and its clinical significance are not currently known. We conducted a sponsor-independent, single-center retrospective analysis examining the phenotypic correlates and prognostic impact of clonal evolution during JAKi treatment. Methods Clinical, cytogenetic and molecular data were collected on MF patients treated with one or more JAKi on a clinical trial at the Mayo Clinic, Rochester, MN. Baseline data pertained to the onset of treatment with first JAKi. Karyotype risk categories were as previously defined (Blood. 2011 Oct 27;118(17):4595). Serial bone marrow biopsies were obtained at varying frequencies based on individual JAKi protocol requirements. Mutation screening was performed as previously described (Leukemia. 2013 Apr 26. doi: 10.1038/leu.2013.119). 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). The DIPSS-plus distribution was: Low/Int-1 20 (13%), Int-2 66 (42%), and High 71 (45%). 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 were documented. Eighty nine patients (57%) had a median total 3 (range 2-8) bone marrow biopsies with karyotype analysis during JAKi treatment. Of these, 21 patients (24%) showed clonal evolution (ie, acquisition or loss of one or more chromosome abnormalities when comparing last observed karyotype during JAKi treatment to baseline). Overall, 13 patients (15%) had upstaging of karyotype risk category (eg, normal to unfavorable), 9 (10%) had downstaging (eg, favorable to normal) and 67 (75%) had no change. Acquisition of worse-than-baseline karyotype was significantly higher in DIPSS-plus high-risk patients (26%) versus non-high risk patients (6%) (p=0.03). There was no association between acquisition of a worse karyotype category and baseline karyotype risk category or ASXL1 or SRSF2 mutation status. There was borderline association with RBC transfusion-dependence and constitutional symptoms at baseline (p=0.06). There was no relationship between clonal evolution and achievement of spleen or anemia response during JAKi treatment (p>0.05). The median overall survival of the entire cohort was 41 months. On univariate analysis, overall survival was significantly worse in patients who acquired a worse-than-baseline karyotype risk category during JAKi treatment (n=13, 77% deaths, median survival=31 months) versus those who did not (n=76, 43% deaths, median survival=52 months) (p=0.01) (Figure). On multivariate analysis, the prognostic significance of acquiring a worse karyotype risk category was maintained against all individual DIPSS-plus prognostic variables, including baseline karyotype risk category, except age >65 years. On univariate analysis, leukemia-free survival was significantly worse in patients who acquired a worse-than-baseline karyotype risk category during JAKi treatment (n=13, 31% leukemic transformation, median leukemia-free survival=32 months) versus those who did not (n=76, 8% leukemic transformation, median leukemia-free survival not reached) (p=0.0006) (Figure). On multivariate analysis against known predictors of leukemic risk in PMF patients (ie, baseline unfavorable karyotype, SRSF1 mutated status and platelet count <100 x 109/L), only acquisition of a worse-than-baseline karyotype risk category maintained its prognostic value (HR 5.3, p=0.03) Conclusions This is the first study to examine the incidence of clonal evolution during JAKi treatment in MF patients. Approximately 25% of patients exhibited clonal evolution; acquisition of a worse-than-baseline karyotype risk category had a strong and independent association with inferior leukemia-free survival. A similar association was also observed for inferior overall survival, albeit not independent of older age. In contrast, there was no association with spleen or anemia response during JAKi treatment. Disclosures: Pardanani: Bristol Myers Squibb: Clinical trial support Other; Sanofi: Clinical trial support. Publication support service., Clinical trial support. Publication support service. Other; PharmaMar: Clinical trial support, Clinical trial support Other; JW Pharmaceutical Corp.: Clinical trial support, Clinical trial support Other.

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 Background : Pre-treatment determinants of survival in adult acute myeloid leukemia (AML) include performance status, age, karyotype, the distinction between primary and non-primary AML and FLT3-ITD/NPM1 mutational status. Current literature on AML natural history and risk factors is often based on information derived from stringent protocol studies. In the current series of 1,123 adult patients seen at the Mayo Clinic between 2004 and 2017, we examined long-term survival specified by a number of primarily established risk factors. Methods: Diagnosis of AML and its sub-classification was according to World Health Organization criteria. Study subjects were recruited from the Mayo Clinic AML database. Conventional response criteria were used for CR and CRi assignment; the latter met all criteria for CR with the exception of platelet count <100 x 109/l or absolute neutrophil cunt <1 x 109/l. Standard statistical methods were used for analysis of overall survival, calculated from the initial diagnosis of AML to date of death or last follow-up; analyses were performed both in the absence and in the presence of censoring of survival at time of allogenic stem cell transplant (ASCT). The JMP® Pro 13.0.0 software from SAS Institute, Cary, NC, USA, was used for all calculations. Results: 1,123 adult patients (median age 65 years, range 18-94; 61% males) with non-APL AML were considered; 61% were age >60 years. 351 (31%) patients were seen between 2004 and 2009 and 772 (69%) between 2010 and 2017. AML subcategories were primary (56%), post-myelodysplastic syndromes (post-MDS; 24%), therapy-related (10%), post-myeloproliferative neoplasm (post-MPN; 8%) and post-MDS/MPN (3%). Cytogenetic risk distribution, according to the European LeukemiaNet classification, was favorable in 47 (4%), intermediate in 650 (58%) and adverse risk in 426 (38%) patients. FLT3, NPM1 and CEBPA mutation information was available in 462 (24% mutated), 393 (27% mutated) and 228 (11% mutated) patients, respectively. Treatment included intensive chemotherapy (IC) in 766 (68%) patients, less aggressive chemotherapy, including the use of hypomethylating agents in 144 (13%) and supportive care alone in 213 (19%) patients. ASCT was utilized in 258 (23%) cases, almost all performed after achieving CR/CRi. Complete remission (CR) and CR with incomplete count recovery (CRi) were listed in 333 (30%) and 248 (22%) patients, respectively; the corresponding rates in IC-treated patients were 43% and 32%. Almost all cases of CR/CRi occurred in IC-treated patients; 2 (1.4%) CRs were listed for less aggressive chemotherapy. After a median follow-up of 11 months, 798 (71%) deaths were recorded. Median survival for all 1,123 patients was 14 months with 1-, 3- and 5-year survival rates of 54%, 29% and 23%, respectively; a slight but significant (p=0.01) improvement in survival was apparent in more recent years (Figure 1a). Figures 1b, 1c and 1d depict analysis stratified by AML subcategories, treatment received and response obtained, respectively. Similarly, figures 2a, 2b and 2c depict analysis stratified by karyotype, use of ASCT and age. Multivariable analysis of pre-treatment parameters evaluable in all 1,123 patients identified age >60 years (HR 2.2, 1.9-2.6), adverse karyotype (HR 2.9, 1.9-4.9), intermediate-risk karyotype (HR 1.6, 1.02-2.6), post-MPN AML (HR 1.9, 1.5-2.4) and other secondary AML (HR 1.3 (1.1-1.6), as risk factors for shortened survival; the inclusion in the model of FLT3/NPM1 mutational status in 392 informative cases confirmed the adverse prognostic effect of age >60 years (HR 1.8, 1.4-2.5), adverse karyotype (HR 3.7, 1.4-15.3), post-MPN AML (HR 2.8, 1.6-4.6), other secondary AML (HR 1.4, 1.0-1.9) and FLT3+NPM1- (HR 2.8, 1.6-4.9) and FLT3-NPM1- (HR 1.7, 1.2-2.7) profile. Results were unchanged when survival was censored at time of ASCT. Additional prognostic interaction with treatment strategy and ASCT is further elaborated in an accompanying abstract to be presented. Conclusions: The current study, the largest coming from a single institution, provides practically useful information that should assist with patient consultation on AML prognosis and treatment. The study confirms the primary prognostic importance of age, karyotype and FLT3/NPM1 mutational status, in real-life practice. Novel observations included comparable value of CRi vs CR and the particularly worse prognosis associated with post-MPN AML. Disclosures Al-Kali: Novartis: Research Funding.

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.