Ketterling, Rhett P.

Abstract Background: Young patients with myeloproliferative neoplasms (MPN), including essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF), constitute a distinct, steadily growing subpopulation (Eur J Haematol. 2017;98:85). Most available data, including current prognostic models, are excerpted from older populations and may not accurately mirror the unique phenotypic and prognostic patterns seen in younger patients. The objective of the current study was to comprehensively assess the phenotypic features and long-term outcomes in MPN patients age ≤ 40 years. Methods: The study population was recruited from a master database of MPN patients seen at our institution between 1967 and 2017. Diagnoses were in accordance with 2016 World Health Organization criteria (Blood. 2016;127:2391). Data was abstracted from the time of referral, which coincided in the majority with the time/within one year of diagnosis. Additional molecular data was obtained based on availability of next-generation sequencing (NGS) information. Conventional statistics was utilized for group comparisons and calculation of survival data. Results: The study population was recruited from a total of 3,023 MPN patients: 665 PV, 1076 ET, and 1282 PMF. Of these, 361 (12%) were ≤ 40 years of age at the time of initial diagnosis; the corresponding incidences were 12% in PV (n=79), 20% in ET (n=219) and 5% in PMF (n= 63). Young PV patients (n=79): Median age (range) was 32 years (18-40) and males constituted 54% (Table 1). Over a median follow-up of 11.3 years, fibrotic transformation rates were 22% vs 25% and 10% in those 41-60 and >60 years of age, respectively (P<0.001). Leukemic conversion rates were balanced across age categories (4% vs 5% vs 3% for successively older cohorts) (P=0.4). Interestingly, young patients had lower rates of arterial (P<0.001) and higher of venous thrombosis (P=0.01) prior to/at diagnosis vs older cohorts while events post-diagnosis were comparable across age groups (P=0.6). Young ET patients (n=219): Median age (range) was 32 years (18-40) with a minority (31%) constituted of males. During a median follow-up of 13 years, fibrotic progressions rates were 16% vs 16% and 9% in those aged 41-60 and >60 years, respectively (P<0.001). The leukemic conversion rate (2%) was analogous to that of older cohorts (5% and 3%, respectively) (P=0.1). Young patients also experienced significantly fewer arterial thrombotic events prior to/at diagnosis (6% vs 14% and 18%) and after diagnosis (8% vs 16% and 22%) compared to older cohorts (P<0.0001). Young PMF patients (n=63): Median age (range) was 37 years (19-40) with a preponderance of males (59%). High risk karyotype was not seen in young patients vs in 5% and 8% of those 41-60 and > 60 years of age, respectively (P=0.004). During median follow-up of 7.1 years, leukemic conversions were recorded in 10% of young patients vs 10% and 9% of respectively older cohorts (P=0.9). Young subjects also experienced fewer arterial thrombotic events prior to/at diagnosis (2% vs 6% and 13%) and lower rates of venous thrombosis after diagnosis (2% vs 10% and 4%) compared to cohorts 41-60 and > 60 years of age, respectively (P<0.0001). NGS data disclosed significant clustering of high risk molecular mutations with successively older subgroups (≤ 40 vs 41-60 vs > 60 years); ASXL1: 19 vs 40 vs 45% (P=0.005), SRSF2: 6 vs 8 vs 18% (P=0.0004), and U2AF1: 3 vs 11 vs 19% (P=0.004) (Table 1). Survival data Kaplan-Meier survival curves stratified by age bracket (≤ 40 vs 41-60 vs > 60 years) disclosed median OS in successively older age groups to be 37, 22, and 10 years for PV (Figure 1A), 35, 22, and 11 years for ET (1B), and 20, 8, and 3 years for PMF (1C); exposing significantly longer median survival estimates in younger patients, systematically across all disease subtypes (P<0.001). When appraised as a function of MPN subtype, survival data confirmed the significantly reduced OS in PMF relative to PV and ET (P<0.001), though PMF patients aged ≤ 40 years were still projected to have a reasonably favorable median life expectancy of 20 years (Figure 2). Conclusions: Young MPN patients comprise a unique disease subset defined by an attenuated-risk cytogenetic and mutational backdrop compared to their older counterparts. Caution must be exercised in counseling and managing this population as historical 'one-size-fits all' data fails to reflect the true natural history and disease biology in the young. Disclosures No relevant conflicts of interest to declare.

Abstract Background: Subnormal serum erythropoietin (EPO) level is widely accepted as a minor diagnostic criterion for polycythemia vera (PV) and has retained its value in the revised 2016 World Health Organization (WHO) classification scheme of myeloid neoplasms (Blood. 2016;127:2391). While over 80% of PV patients exhibit subnormal EPO values (Haematologica. 2004;89:1194), very little is known about the incidence, phenotypic connotation, or prognostic significance of serum EPO levels in essential thrombocythemia (ET). Methods: Study patients were recruited from the Mayo Clinic, Rochester, MN, USA. Diagnosis of ET was in strict accordance with the 2016 WHO criteria (Blood. 2016;127:2391) and only patients with EPO measurements at diagnosis or during their chronic phase were considered under the following provisions: i) no evidence of myelofibrotic or leukemic transformation; ii) no treatment with cytoreductive agents at the time of sample collection (and for those with EPO sampling at diagnosis, no prior exposure to cytoreductive therapy); iii) no hemoglobin levels below sex-adjusted values for anemia; and iv) systematic exclusion of all cases of masked PV. Serum erythropoietin levels were measured using Mayo-verified standard immune-enzymatic assay consistent with CLIA requirements with reference range between 4-21 mIU/mL. Standard statistical methods and time-to-event curves prepared using the Kaplan-Meier method were used to compare EPO-stratified groups. The JMP® Pro 13.0.0 software package was used for all analyses (SAS Institute, Cary, NC, USA). Results: Among 177 informative ET patients (median age 51 years, range 18-88; 71% females), median serum EPO concentrations were 5.2 mIU/mL (range <1-25) overall and 2.6 mIU/mL (range <1-3.9) and 7.2 mIU/mL (range 4-25) in those with subnormal versus normal/above normal EPO levels, respectively (p<0.0001). The majority of EPO measurements were at the time or within one year of diagnosis (67%), with the remainder obtained at referral or during the chronic clinical course. Risk stratification disclosed low, intermediate, and high risk status in 45%, 33% and 23% of patients, respectively, with no appreciable differences between EPO-stratified subsets. Subnormal serum EPO levels clustered clinically with JAK2 mutations (95% vs 60%; p<0.0001), higher hemoglobin (median 14.7 vs 13.8 g/dl, range: 12.1-17.8 vs 12-17.1; p<0.0001) and leukocyte counts (median 9.7 vs 8.2 x 109/l, range: 4-28.1 vs 3.9-22; p=0.0001), and arterial thrombosis after diagnosis (13% vs 5%; p=0.04). In addition, all three documented conversions to PV (p=0.01) and none of the six fibrotic progressions (p=0.02) occurred in patients with subnormal serum EPO levels (Table 1). While serum EPO level had no bearing on overall survival data (p=0.41) (Figure 1A), subnormal circulating EPO concentrations were significantly associated with superior myelofibrosis-free survival (p=0.02) (Figure 1B) but conversely, inferior polycythemia-free survival (p=0.03) (Figure 1C). Conclusions: We conclude that subnormal serum EPO levels are relatively frequent in ET, cluster with disease features that are reminiscent of polycythemia vera, and might be associated with a lower risk of fibrotic progression. Disclosures No relevant conflicts of interest to declare.

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 : 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 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 Therapy-related myeloid neoplasm (tMN) includes t-myelodysplasia (tMDS) and t-acute myeloid leukemia (tAML) and are serious late effects of the treatment of cancer. Prognosis of tMN is poor, related to the increased frequency of adverse cytogenetics and other clinical features which predict poor response to conventional treatment. Over the last years, azacitidine (AZA) has become the standard of treatment for high risk MDS (Silverman, JCO 2002; Fenaux, Lancet Oncol 2009) and has shown efficacy in AML. AZA represents an interesting option for patients with tMN considering its safety profile and its efficacy in poor prognosis subgroups of apparently de novo MDS patients including those with monosomy 7. Most prospective trials of AZA have excluded patients with tMN. Most tMN data are retrospective or registry studies (Bally, Leuk Res 2013). This abstract presents the results of 47 t-MN patients prospectively enrolled as a specific cohort in the E1905 study. Methods E1905 study is a randomized phase 2 study from the North American Leukemia Intergroup (NCT00313586, Prebet , ASH 2010) testing 10 days of AZA (50mg/m2/d s.c.) vs. 10 days of AZA + the histone deacetylase inhibitor entinostat (4 mg/m2/d PO days 3 and day 10). 6 cycles of treatment were planned; responding patients could receive up to 24 cycles. MDS, CMML, and AML with myelodysplasia-related changes were included. Patients with tMN were subsequently accrued as a separate cohort after amendment of the protocol. Response was assessed using IWG 2000 criteria (Cheson et al, Blood 2000); the primary endpoint of the overall trial was achievement of a normal hemogram in 25% of treated patients in either treatment arm. Results A total of 47 patients were included. Median age was 70 years (39y-83y), 45% male, and 94% of patients had ECOG PS 0-1. 29 patients could be subclassified as t-MDS and 18 as t-AML. At inclusion, median peripheral blood counts were: neutrophils 1.0 G/l, Platelets 35 G/L, Hemoglobin 9.2 g/l, peripheral blood blasts 0%, marrow blasts 14.0%. 68% of patients were RBC transfusion dependent and 40% platelet transfusion dependent. As expected, the cytogenetic evaluation showed a high frequency of unfavorable risk cytogenetics (74%) as compared to normal or intermediate risk cytogenetics (26%). Baseline characteristics were not statistically different between the 2 arms. 24 patients were treated with AZA monotherapy and 23 with AZA+entinostat. The median number of administered cycles was 4 and was significantly higher in patients treated with AZA monotherapy (6 cycles vs.3 cycles, p=0.008). 8 patients in the combination arm and 1 patient in the AZA monotherapy arm died of infection or hemorrhage before cycle 3. In an intent to treat analysis, overall response rates (CR, PR, or trilineage Hematologic improvement) were 11/24 (46%) in AZA monotherapy (95% CI 26 – 67%) and 4/23 (17%) in the combination arm (95% CI 5 – 39%, p=0.06 comparing the two arms). Median overall survival in the two arms were 12.8 months and 5.7 months (p=0.008). Conclusions In this group of very high risk patients with therapy-related myeloid neoplasms, the use of the novel 50 * 10 schedule of azacitidine monotherapy appears effective, with response rates comparable to those for patients with de novo MDS/AML treated on the same protocol (74 pts, 40% with unfavorable cytogenetics, ORR=32%, median OS=18 months). Because there are no prospective data examining the approved dose schedule of AZA (75 mg/m2/day * 7 days), it is not clear if this surprisingly high response rate derives from the current extended schedule of lower dose AZA, or would be true with standard dose AZA as well. This high response may enable many of these patients to proceed to allogeneic stem cell transplant. Entinostat combined with azacitidine at the dose and schedule applied in E1905 does not appear to be effective and tolerable as compared to azacitidine alone. Disclosures: Prebet: CELGENE: Honoraria. Off Label Use: Use of azacitidine in acute myeloid leukemia. Gore:CELGENE: Equity Ownership, Research Funding.