Lasho, Terra L.

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

Abstract Background : Prognosis in chronic myelomonocytic leukemia (CMML) is dire and blast transformation (BT) is a main cause of death. Current risk factors and prognostic models are mostly designed to predict overall (OS) and not leukemia-free (LFS) survival. Established risk factors for OS in CMML include anemia, thrombocytopenia, leukocytosis, monocytosis, presence of circulating immature myeloid cells (IMC), peripheral blood (PB) and bone marrow (BM) blasts, lymphocytosis, ASXL1 mutations, high risk abnormal karyotype and advanced age. In the current study, we examined risk factors for BT in CMML and survival outcome after BT. Methods : All CMML patients seen at our institution and with complete presentation data were considered. Clinical and laboratory variables, including cytogenetic information, at time of CMML diagnosis and also at time of BT were collected. DNA analysis for SRSF2, SF3B1, U2AF1, ASXL1 and SETBP1 mutations was carried out on specimens obtained at time of CMML diagnosis. We used both conventional and revised (see accompanying ASH 2014 abstract) cytogenetic risk models to query their influence in the current study population. Results: Clinical and laboratory features at time of CMML diagnosis: 277 consecutive patients (median age 71 years, range 18-91; 67% males) with WHO-defined CMML were considered. 236 (85%) patients had CMML-1 and the remainder CMML-2. According to the molecular Mayo risk model, 86 (33%) were high, 77 (29%) were intermediate-2, 77 (29%) were intermediate-1 and 24 (9%) were low risk. Mutational frequencies were 40% for ASXL1 (n evaluable =264), 44% for SRSF2 (n evaluable =263), 5% for SETBP1 (n evaluable =263). According to the revised Mayo-French consortium cytogenetic risk model, karyotype was high risk in 5%, intermediate in 21% and low risk in 74%. Risk factors for blast transformation (BT) : At a median follow-up of 16.2 months for all 277 patients from time of CMML diagnosis, 204 (74%) deaths and 38 (14%) BTs were documented. Median time from CMML diagnosis to BT was 13.3 months. In univariate analysis, female sex, younger age, lower hemoglobin, higher leukocyte count, higher absolute monocyte count, higher lymphocyte count, presence of circulating immature myeloid cells (IMC), increased peripheral blood (PB) blast %, increased bone marrow (BM) blast %, BM blasts ≥10%, CMML-2, abnormal karyotype, high risk karyotype per the Spanish or Mayo-French consortium, and higher risk disease per Mayo, MDACC or Dusseldorf models were all associated with higher risk of BT. In contrast, ASXL1 (p=0.84), SRSF2 (p=0.28) or SETBP1 (p=0.97) mutations were not found to be significant. In multivariable analysis, only PB blast % (p<0.0001), presence of PB IMC (p=0.004) and younger age (p=0.03) remained significant. Outcome of blast transformation : At time of BT, karyotype was abnormal in 58% and clonal evolution from time of CMML diagnosis was documented in 13 (36%) of 36 evaluable patients. Among the 38 patients with BT, induction chemotherapy was documented in 12 patients and allogeneic stem cell transplant (ASCT) in 6. At a median follow-up of 4.2 months from time of BT, 34 (89%) of the 38 patients with BT had died. Among the four patients who were alive at the time of this writing, 3 had received induction chemotherapy and two ASCT. Median survival of patients with BT, from time of disease transformation, was 4.5 months with 1, 2 and 3 year survival rates of approximately 30%, 16% and 3%, respectively. In univariate analysis, higher leukocyte count (p=0.01) and not receiving induction chemotherapy (p=0.03) were associated with shortened survival. Neither ASXL1 nor SRSF2 mutations affected survival after BT. Conclusions : Blast transformation in CMML occurs in approximately 14% of patients followed for a median of less than 1.5 years. Presence of circulating immature cells and percentage of peripheral blood blasts are the major determinants of BT in CMML. Genetic risk factors for overall survival did not appear to influence leukemia-free survival. Prognosis of post-CMML BT is dismal with median survival of less than 6 months and a 3-year survival of less than 5%. Outcome was better in patients receiving induction chemotherapy followed by ASCT. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 430 Background: The Dynamic International Prognostic Scoring System (DIPSS-plus) uses eight risk factors to predict overall survival (OS) in primary myelofibrosis (PMF): unfavorable karyotype, peripheral blood (PB) blast count ≥1%, platelet count <100 × 109/L, white blood cell count (WBC) >25 × 109/L, hemoglobin level <10 g/dL, red blood cell transfusion need, constitutional symptoms, and age >65 years (JCO 2011;29:392). Among these risk factors, karyotype holds a dominant role in predicting both overall (OS) and leukemia-free (LFS) survival (Blood 2011;118:4595). More recently, certain PMF-associated somatic mutations (IDH, EZH2, ASXL1, SRSF2) have been reported (in print or at recent meetings) to carry prognostic relevance for OS or LFS in PMF while other mutations (JAK2, MPL, TET2, SF3B1) did not. The purpose of the current study was to examine the individual and combined prognostic relevance of the former in the context of cytogenetic risk stratification and DIPSS-plus. Methods: An updated Mayo Clinic database of 1008 karyotypically- and DIPSS-plus-annotated patients with PMF was used to identify study patients in whom DNA at time of referral was available for mutation analysis. OS and LFS were calculated from the time of referral, which was also the time of study sample collection. Results: In an ongoing project, a total of 397 PMF patients have so far been studied for IDH1/2 (n=367), SRSF2 (n=355), ASXL1 (n=260) and EZH2 (n=43) mutations. The corresponding mutational frequencies were 5.4% (20/367), 14.4% (51/355), 31.2% (81/260) and 7% (3/43). These mutations were not exclusive of either each other or JAK2 or MPL mutations and a significant clustering was noted between SRSF2 and IDH mutations; IDH mutational frequency was 21% (10/48) in SRSF2-mutated versus 3% (9/279) in unmutated cases (P<0.0001). Mutational hotspots were P95 in exon 1 for SRSF2 and exons 1, 2 and 5 for ASXL1. OS and LFS effect have so far been examined for ASXL1, SRSF2 and IDH 1/2 mutations; in univariate analysis, all three mutations significantly affected OS (ASXL1 RR 1.9, 95% CI 1.4–2.7; SRSF2 RR 1.7, 95% CI 1.2–2.5; IDH 1/2 RR 1.8, 95% CI 1.1–3.1) whereas only IDH 1/2 (RR 5.5, 95% CI 2.5–11.8) and SRSF2 (RR 3.5, 95% CI 1.7–7.0) but not ASXL1 (p=0.29) mutations affected LFS. The observed prognostic relevance for OS was sustained during multivariable analysis that included ASXL1 with either SRSF2 or IDH mutations, as co-variates, whereas both IDH and SRSF2 mutations remained significant for LFS. Based on the above observations, study patients were classified into two groups: i) unmutated for ASXL1, SRSF2 and IDH1/2 (all unmutated; n=146) and ii) mutated for at least one of the three mutations (one or more mutated; n=130); the latter group constituted 62%, 52%, 18% and 27% of DIPSS-plus high, intermediate-2, intermediate-1and low risk patients, respectively (p<0.0001). The corresponding rates for patients with favorable and unfavorable karyotype were 47% and 43% (p=0.7) and for normal vs. abnormal karyotype were 53% and 36% (p=0.008). The “all unmutated group”, compared to the “one or more mutated group” enjoyed a significantly superior OS (RR 0.5, 95% CI 0.4–0.7) and LFS (RR 0.3, 95% CI 0.2–0.7) that was DIPSS-plus-independent for OS (p=0.03) and karyotype-independent for LFS (p=0.004). The prognostic value of distinguishing “all unmutated” from “one or more mutated” patient groups was most evident for DIPSS-plus intermediate-2 or lower risk groups (p=0.002) and its added value for high-risk group was limited (p=0.23) (Figure). Conclusions: A concomitant analysis strategy reveals that SRSF2, ASXL1 and IDH mutations carry inter-independent prognostic relevance for overall or leukemia-free survival in PMF. Any one of these three mutations is more likely to occur in patients with normal karyotype and such an event predicts shortened survival that is not accounted for by DIPSS-plus and a higher risk of leukemic transformation that is independent of karyotype. The current study suggests that such information might be useful in treatment decisions involving intermediate-2 or lower risk patients. Disclosures: No relevant conflicts of interest to declare.

AbstractHypomethylating agents (HMA) are currently the only FDA approved therapy for patients with chronic myelomonocytic leukemia (CMML). In the current retrospective study, we assessed response rates as adjudicated by the IWG (International Working Group) MDS (myelodysplastic syndrome) and MDS/MPN myeloproliferative neoplasm overlap syndrome response criteria, in 121 CMML patients treated with Azacitidine (AZA, n = 56) and Decitabine (DAC, n = 65). The overall response rates were 41% by the IWG MDS (AZA‐ 45%, DAC‐39%), and 56% by the IWG MDS/MPN (AZA‐56%, DAC‐58%) response criteria, with CR (complete remission) rates of <20% for both agents, by both criteria. There were no significant differences in response rates between proliferative and dysplastic CMML. Moreover, 29% of CMML patients in a CR with HMA progressed to AML (blast transformation), underscoring the limited impact of these agents on disease biology. Progression after HMA response was associated with a median overall‐survival (OS) of 8 months, while median OS in patients with primary HMA failure was 4 months. Lower serum LDH levels (<250 Units/L) were associated with HMA responses by both criteria; while ASXL1 and TET2 mutational status had no impact. HMA treated patients had a longer median OS (31 vs 18 months; P = .01), in comparison to those treated with conventional care regimens (excluding observation only patients), without any differences between AZA vs DAC (P = .37). In conclusion, this study highlights the inadequacies of HMA therapy in CMML, retrospectively validates the IWG MDS/MPN response criteria and underscores the need for newer, rationally derived therapies.

Abstract JAK2 mutations in myeloproliferative neoplasms (MPNs) are associated with the germline GGCC (46/1) haplotype. In 2010, we reported an association between shortened survival in primary myelofibrosis (PMF) and nullizygosity for the JAK2 46/1 haplotype. In the current study, we have increased the number of informative cases from 130 to 414 (median age 63 years; 63% males), in order to revisit with the phenotypic and prognostic relevance of the JAK2 46/1 haplotype in PMF. 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 = .02). 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), most pronounced in JAK2 (P <.001), as opposed to CALR/MPL mutated (P = .48) or triple‐negative cases (P = .27). Multivariable analysis that included karyotype, driver mutational status and high‐molecular risk mutations confirmed the independent prognostic contribution of nullizygosity for the 46/1 haplotype (P = .02; HR 1.4, 95% CI 1.1‐1.8). Nullizygosity for 46/1 also remained significant in the context of the genetically‐inspired GIPSS risk model (P = .04), but not in the context of the integrated genetics‐clinical MIPSS70+ version 2.0 model (P = .4). Leukemia‐free survival was not affected by the 46/1 haplotype (P = .6). The current study confirms the association of nullizygosity for the JAK2 GGCC (46/1) haplotype with inferior survival in JAK2‐mutated PMF.

AbstractIn the last decade, several prognostic models for primary myelofibrosis (PMF) have been introduced and shown to be effective in predicting overall survival. The main objective for this study was to identify clinical and genetic markers of very long (20+ years) survival in PMF. A total of 1282 patients with PMF were considered (median age 65 years, range 19‐92; 63% males); 26 (2%) patients (median age 51 years, range 28‐71; 38% males) survived their disease for at least 20 years (long‐lived patients) and 626 (49%) patients (median age 68 years, range 27‐92; 66% males) died within 5 years of their diagnosis (short‐lived patients). Multivariable logistic regression analysis identified 7 variables that were associated with survival beyond 20 years: age ≤ 70 years (P = .002); female sex (P = .03); hemoglobin level ≥ 10 g/dL for women and ≥ 11 g/dL for men (P = .03), leukocyte count ≤25 × 109/L (P = .009), platelet count ≥100 × 109/L (P = .002), circulating blasts <2% (P = .03) and absence of constitutional symptoms (P = .04). Five‐year mortality was independently predicted by high‐molecular risk mutations (P < .001); unfavorable or very high risk karyotype (P < .001); absence of type 1/like CALR mutation (P < .001); age > 70 years (P < .001); constitutional symptoms (P < .001); hemoglobin level < 10 g/dL for women and < 11 g/dL for men (P < .001); leukocyte count >25 × 109/L (P = .004); and circulating blasts ≥2% (P = .001). This study suggests that genetic risk factors in PMF are associated with early mortality while survival beyond 20 years could be predicted by easily accessible clinical variables, including age, sex, blood counts, and symptoms.

Abstract Systemic mastocytosis (SM) is a clinically heterogeneous disease with prognosis chiefly assigned based on World Health Organization (WHO) morphologic subclassification. We assessed the feasibility of developing contemporary risk models for SM based on clinical and integrated clinical-genetics information. Diagnosis of SM was per WHO criteria, and karyotype and next-generation sequencing data were available in a subset of the total 580 patients (median age, 55 years; range, 18-88 years) seen at the Mayo Clinic between 1968 and 2015. Morphologic subcategories were indolent/smoldering in 291 (50%) and “advanced” in 289 (50%): SM with an associated hematological neoplasm in 199, aggressive SM in 85, and mast cell leukemia in 5. Multivariable analysis of clinical variables identified age &gt;60 years, advanced SM, thrombocytopenia &lt;150 × 109/L, anemia below sex-adjusted normal, and increased alkaline phosphatase (ALP) as independent risk factors for survival; respective hazard ratios (HRs) 95% confidence intervals (95% CIs) were 2.5 (1.9-3.4), 2.7 (1.8-4.0), 2.5 (1.9-3.4), 2.2 (1.6-3.1), and 2.1 (1.5-3.0). In addition, ASXL1 (HR, 4.5; 95% CI, 2.6-7.6), RUNX1 (HR, 4.3; 95% CI, 1.3-10.8), and NRAS (HR, 5.0, 95% CI, 1.5-13.2) mutations were independently associated with inferior survival. Combined clinical, cytogenetic, and molecular risk factor analysis confirmed the independent prognostic contribution of adverse mutations (2.6, 1.6-4.4), advanced SM (4.0, 1.8-10.0), thrombocytopenia (2.8, 1.7-4.5), increased ALP (2.1, 1.2-4.0), and age &gt;60 years (2.2, 1.3-3.6). These data were subsequently used to develop clinical and hybrid clinical-molecular risk models. The current study advances 2 complementary risk models for SM and highlights the independent prognostic contribution of mutations.

Key PointsSerum albumin and cholesterol levels predict survival in primary myelofibrosis, independent of each other and contemporary risk models. The cachexia index, determined by serum albumin and cholesterol levels, might further refine current prognostic models in myelofibrosis.

AbstractBetween 1967 and 2017, 361 patients with myeloproliferative neoplasms (MPN), age ≤ 40 years, were seen at our institution, constituting 12% of all MPN patients (n = 3023) seen during the same time period; disease‐specific incidences were 12% in polycythemia vera (PV; n = 79), 20% in essential thrombocythemia (ET; n = 219) and 5% in primary myelofibrosis (PMF; n = 63). Compared to their older counterparts, younger patients were more likely to present with low risk disease (P < .001) and display female preponderance in ET (P = .04), lower incidence of arterial events overall (P < .001), and higher incidence of venous thrombosis in PV (P = .01). Younger patients were also more likely to express CALR mutations, in ET and PMF, normal karyotype, in PV and PMF, and lower incidence of high molecular risk mutations in PMF (P significant in all instances). Over median follow‐up of 11.3, 13, and 7.1 years for PV, ET, and PMF, leukemic transformations were respectively documented in 4%, 2%, and 10% (P values 0.1‐0.9) while incidences of fibrotic progression in PV (22%) and ET (16%) were expectedly higher in young patients, because of their longer survival (P < .001). Median survival in young patients was 37 years for PV, 35 for ET and 20 for PMF; the corresponding values were 22, 22, and 8 years for ages 41‐60 years and 10, 11, and 3 years for ages >60 years (P < .001). Young MPN patients comprise a unique disease subset defined by an attenuated‐risk cytogenetic and mutational backdrop and conspicuously longer survival compared to their older counterparts, which requires assertion during patient counseling.