Finke, Christy M.

Introduction Clonal cytopenias of undetermined significance (CCUS) is defined by the presence of somatic driver mutations/copy number variations in hematopoietic cells in patients with low blood counts, in the absence of morphological evidence for myeloid neoplasms. Patients with CCUS are often symptomatic and can be transfusion dependent (TD), with high rates of progression to myeloid neoplasms, especially myelodysplastic syndromes (MDS). In addition, similar to MDS, there are reports of CCUS patients responding to hypomethylating agents. Due to the lack of a formal diagnosis/morphological dysplasia, CCUS patients are often denied therapies or clinical trial enrollment. We carried out this study to validate our hypothesis that CCUS shares similar clinical and survival characteristics with lower risk MDS (LR-MDS). Methods CCUS patients were prospectively identified from the clonal hematopoiesis clinic, whereas LR-MDS patients were retrospectively identified from our institutional database. LR-MDS was defined as very low, low and intermediate risk MDS based on the R-IPSS categorization. Baseline demographics, blood counts, bone marrow (BM) morphology, cytogenetics, and NGS results were abstracted. Transfusion dependency was defined as requiring at least one unit of red cell or platelets every 4 weeks. The Mann-Whitney-U and Fischer's exact test were used to compare quantitative and qualitative data in subgroups. Kaplan-Meier overall survival (OS) estimates were used for survival analysis and compared using the log-rank test. Results 186 patients were included in the study; 74 (40%) with CCUS and 112 (60%) with LR-MDS, median age 66 years, with 63% being male (Table 1). In the CCUS group, 93% had one or more mutations (67% >1 mutation) detected by NGS, while 7% had clonal cytogenetic abnormalities. Common mutations in CCUS included TET2 (30%), SRSF2 (20%), DNMT3A (13%) and ASXL1 (11%); with 28% of patients being red cell TD and 15% being platelet TD. On application of the R-IPSS stratification, 40%, 45%, 12% and 3% were in the very low, low, intermediate and high risk categories, respectively. LR-MDS subtypes included MDS-RS (50%), MDS-MLD (21%), MDS-EB (10%), MDS del5q (7%), and MDS-U (12%). SF3B1 mutations were seen in 67% (95% of MDS-RS), while TET2 and DNMT3A mutations were seen in 29% and 21%, respectively. 60% of LR-MDS patients had >1 mutation. 44% were red cell TD, while 10% were platelet TD. In comparison to patients with CCUS, LR-MDS patients were more likely to have higher white blood cell counts (p=0.002), higher neutrophil counts (p=0.009), higher platelet counts (p<0.001), be red cell TD (p=0.031), have BM ring sideroblasts (p<0.001), have higher BM blast% (p=0.004) and carry SF3B1 mutations (p<0.001); whereas CCUS patients were more likely to have higher monocyte counts (p=0.009) and more likely to carry IDH1 (p=0.005), ZRSR2 (p=0.005) and ATM (p=0.012) mutations. Importantly there were no differences with regards to R-IPSS cytogenetic risk groups, R-IPSS prognostic categories, platelet TD, and mutations involving TET2, DNMT3A, SRSF2 and ASXL1 and TP53. Notably signal pathway mutations and mutations involving tumor suppressor genes were infrequent (<5%) in both groups. The LR-MDS patients had a longer median follow up (53 vs 15 months) and at last follow up 72 (64%) and 11 (15%) deaths had been documented in the LR-MDS and CCUS groups respectively. 13 (18%) CCUS patients progressed to MDS (10) and AML (3) over a median of 15 months, while 9 (8%) LR-MDS patients progressed to higher grade MDS (1) and AML (8), respectively (median follow up 53 months). There was no difference in the median survival between CCUS (median OS not reached) versus LR-MDS (median OS 8.3 years) (p=0.372, Figure 1). Conclusion In spite of subtle phenotypic/molecular differences between LR-MDS and CCUS, both entities had similar prognostication, distribution of high risk mutations and survival outcomes. Within a short follow-up, 18% of CCUS patients progressed to MDS/AML, indicating that regardless of the absence of dysplasia these entities are a continuum of myeloid neoplasms. In order to improve access to treatments and clinical trial opportunities, we recommend that close consideration be given to consider CCUS as a MDS subtype. Disclosures Shah: Dren Bio: Consultancy.

Introduction: Truncating mutations in Additional Sex Combs-Like 1 (ASXL1) are associated with a high-risk disease phenotype in myeloid malignancies. In chronic myelomonocytic leukemia (CMML), truncating ASXL1 mutations are known to increase transcriptional activity of leukemic driver genes and have been associated with gene body hypermethylation. We interrogated the transcriptome and methylome of patients with ASXL1-mutant (MT) and -wildtype (WT) CMML using a multi-omics approach to test the hypothesis that gene expression is mediated through gene body methylation. Methods: Bone marrow mononuclear cells from patients with ASXL1 WT (n=8) and MT (n=8) CMML were subjected to targeted NGS of DNA, whole transcriptome shotgun sequencing (RNA-seq), and immunoprecipitation of DNA methyl residues (DIP-seq). After quality control all samples were sequenced on an Illumina HiSeq 4000 before further processing and data analysis. Differential gene expression analysis was performed to identify genes up-regulated in MT CMML. The samples in the two groups were treated as biological replicates and subjected to a consensus peak calling strategy requiring an overlap of at least 30% between samples and an adjusted p-value < 5x10-5 for a methylation peak to be considered statistically significant. For validation purposes methylation analysis was performed on 3 ASXL1 MT and 3 WT CMML patients using Illumina Infinium MethylationEPIC microarrays and differentially methylated regions were identified using a bump hunting strategy. Gene body methylation was defined as methylation in gene bodies (outside the promoter region, i.e. transcription start site ±2kb). Gene body methylation was compared between WT and MT CMML for the up-regulated genes and correlated with expression of all genes in MT CMML. Results: Sixteen WHO-defined CMML patients were included, median age 69 years (48-77), 63% male, 50% had truncating ASXL1 frame shift mutations. Abnormal karyotypes were observed in the same number of patients and the burden of co-mutations was similar between the two groups (median number per group 3 vs. 3, p=0.508). This included several modulators of DNA methylation including TET2, DNMT3A, and IDH2 (median number per group 1 vs. 1, p=0.699). There was a predominant up-regulation of gene expression in MT CMML: 707 genes up- and 124 down-regulated (FDR<0.050) without evidence of differential methylation in promoter regions of the differentially expressed genes. There was no difference in the number of genes with consensus methylation peaks in the gene body or the extent of gene body methylation (area under the consensus peaks) between MT and WT CMML for the differentially expressed genes (Figure 1a). We further validated these results using methylation microarrays (Figure 1b). Among the MT CMML patients, there was a curvilinear relationship between gene body methylation and gene expression across all genes with intermediate gene body methylation being associated with the highest gene expression (Figure 1c). As an alternative unbiased approach, we identified 1595 differentially methylated regions (DMR, regions with FDR<0.05) in the validation data set. We mapped all hypermethylated regions to gene bodies requiring that there was no concurrent hypermethylation of the promoter region of the same gene. With this approach we identified one of the 707 up-regulated genes (0.14%) with evidence of isolated gene body hypermethylation (HBZ, log2-fold change in gene expression 4.25, FDR=0.0008, DMR area=1.61, DMR FDR=0.018). Representative examples of up-regulated mitotic kinases without evidence of differential gene body methylation are shown in Figure 1d (pie charts represent the mean β-values of microarray probes, p-values represent the comparisons of mean β-values between MT and WT CMML per gene). Conclusions: Gene body methylation was positively associated with gene expression in MT CMML. However, the lack of differential gene body methylation between WT and MT CMML for the up-regulated genes make it an unlikely explanation for the observed increase in transcriptional activity among patients with MT CMML. Figure 1 Disclosures Ordog: Millipore Sigma: Patents & Royalties.

Introduction: Additional Sex Combs-Like 1 (ASXL1) is a chromatin modifier frequently affected by truncating mutations in myeloid malignancies. In chronic myelomonocytic leukemia (CMML), truncating ASXL1 mutations are associated with the overexpression of leukemogenic driver genes, however the molecular mechanisms underlying this transcriptional activation remain controversial. We performed single-cell chromatin accessibility studies of ASXL1-mutant (MT) and -wildtype (WT) CMML to identify cis-regulatory elements (CREs) such as distal enhancer elements that may explain the observed transcriptional activity in MT CMML. Methods: Bone marrow mononuclear cells from a patient with WT and a patient with MT CMML were subjected to single-cell DNA transposase accessibility studies (scATAC-seq). The cryopreserved cells were thawed and resuspended and approximately 100000 viable mononuclear cells per sample were subjected to transposase assays before proceeding to single-cell partitioning into gel beads in emulsion, barcoding, library construction, and sequencing following an established 10X Genomics workflow. The target cell recovery was approximately 2000 cells per sample. Genomic libraries were sequenced on an Illumina HiSeq 4000 before demultiplexing, alignment to the reference genome, and post-alignment quality control. The 10X Genomics Cell Ranger ATAC software was used for demultiplexing, alignment of the reads to the GRCh38 reference genome, filtering and quality control, counting of barcodes and unique molecular identifiers, identification of transposase cut sites, detection of accessible chromatin peaks, count matrix generation for peaks and transcription factors, clustering, and differential accessibility analysis. The two-sample Kolmogorov-Smirnov test for equality of distribution functions was used to compare the distributions of single-cell entropies. Single-cell cis-co-accessibility scores were estimated using Cicero. Results: To mitigate potential effects on the analysis introduced by unequal cell recovery due to experimental conditions, we randomly sampled 1500 single cells from each genotype. Visualizing the first two principal components of the chromatin accessibility data revealed the differences in chromatin conformation between genotypes and also the increased heterogeneity within the MT sample (Figure 1a). To quantify this increase in intratumoral heterogeneity of chromatin accessibility in MT CMML, we calculated the single-cell entropies for both genotypes. There was an increase in the dispersion of single-cell entropy values (mean 0.793±0.026 versus 0.811±0.012, p=6.19x10-151) in MT compared to WT disease (Figure 1b). To leverage the power of the single-cell chromatin accessibility data, we estimated cis-co-accessibility scores (two-way chromatin interactions). There were 631 genes involved in two-way chromatin interactions that were either specific to MT CMML or were shared between MT and WT CMML and showed an increase in co-accessibility in MT CMML. Co-accessibility was higher in MT compared to WT CMML, regardless whether the chromatin interaction was MT-specific or shared between the genotypes (Figure 1c). Gene ontology analysis revealed increased chromatin accessibility in genes involved in the regulation of cell cycle, nuclear division, chromosome organization, cell cycle phase transition, DNA conformation change, and DNA replication (FDR<1.00x10-5). The construction of cis-co-accessibility networks demonstrated a remodeling of the chromatin landscape with MT-specific chromatin interactions involving key mitotic kinases such as MAP2K3 (Figure 1d). Conclusions: MT CMML is characterized by an increase in intratumoral heterogeneity and increased chromatin accessibility. The increased chromatin co-accessibility involved key mitotic kinases and may serve as a plausible explanation for the increased transcriptional activity observed in MT CMML. Figure 1 Disclosures Ordog: Millipore Sigma: Patents & Royalties.

Abstract Introduction: Primary myelofibrosis (MF) is a chronic clonal malignancy which is compounded by the presence of inflammation, bone marrow fibrosis/scarring and risk of transformation to acute myeloid leukemia (AML). Ruxolitinib (Rux), a JAK2 inhibitor, is beneficial for reduction of symptoms and splenomegaly, however, significant myelosuppression is also a toxic side effect of this treatment, limiting optimal drug dosage in many patients. 9-ING-41 is a small molecule GSK-3β inhibitor which has significant clinical activity in patients with advanced malignancies and causes no myelosuppression. We thus assessed the effect of 9-ING-41in MF alone or in combination with Rux. We performed ex-vivo colony assays with primary cells from cases with untreated MF along with normal bone marrow (BM). The experiments were designed to assess the effect of drug on the ability of MF stem/progenitor cells to proliferate/differentiate by evaluating the number, size, and the morphology of colonies, both with and without treatment. Methods: Primary peripheral blood mononuclear cells from MF patients and BM from controls were plated in methylcellulose in duplicate, containing cytokines promoting hematopoietic growth, in the presence of either no drug, Rux only (0.05µM), 9-ING-41 only (range: 0.125µM - 10µM), or a combination of 9-ING-41 (range: 0.125µM - 10µM) and Rux (0.05uM). Colonies were counted ten days later, and colony growth frequency, distribution, and morphology were calculated. Results: We observed an increasing presence of large primitive granulocytic/erythroid/macrophage/monocyte (GEMM) colonies proportional to no drug treatment and linear to increasing concentrations of 9-ING-41 in both MF cases relative to other colony types and normal BM, suggestive of a selective primitive proliferative and/or differentiation effect of GSK-3β inhibition on MF hematopoietic progenitor growth. We did not observe this effect in the combinatory treatments (9-ING-41 + Rux), and there was no significant difference on MF colony number or size in colonies grown without treatment or with Rux alone (Figure 1 A/C/E). However, the morphology of the colonies within different treatments was remarkable. In both MF cases, either without treatment or addition of Rux only (0.05µM), the colonies appeared similar to each other -irregular and disorganized relative to normal bone marrow (Figure 1, B/D/F). In the presence of 9-ING-41 alone, we observed morphologically very large, fully differentiated primitive colonies in both MF cases. However, the most striking observation was the comparison of colonies after combination of both inhibitors. These colonies appeared normal and healthy-looking -discreet, round size and similar in morphology to normal bone marrow colonies. Additional studies are currently underway to look at the signaling consequences, including proliferation, differentiation, and cell cycle effect. Our preliminary results suggest this same mechanism might be occurring in MF cases and could explain the selective presence of large differentiated primitive colonies in MF vs. normal BM in the presence of 9-ING-41. Combining 9-ING-41 with Rux may reduce the dose needed for optimal therapeutic response and/or reverse myelosuppression. A Phase 2 study of 9-ING-41 in patients with advanced MF will be conducted. Citation Format: Terra L. Lasho, Francis Giles, Christy Finke, Naseema Gangat, Ayalew Tefferi. 9-ING-41, a GSK-3β-selective small molecule inhibitor, in combination with ruxolitinib in JAK2617F primary myelofibrosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5217.

Abstract Background: We recently reported on MPLW515L/K mutations in 1182 patients with myeloid disorders (Blood, First Edition July 25, 2006) and found low frequency occurrence in in agnogenic myeloid metaplasia (AMM) (~5%) and essential thrombocythemia (ET) (~1%), but not polycythemia vera (PV). Of the 20 MPLW515L/K-positive patients in that series, 6 (30%) also carried a low burden of the JAK2V617F mutation. Studies of mutation prevalence in individuals with clonal granulopoiesis as well as familial myeloproliferative disorders (MPD) studies suggest that these mutations (JAK2V617F) may be acquired as secondary genetic events. Aim: to gain additional insight regarding the aforementioned two mutations and MPD pathogenesis by determining (i) concordance between mutation expression and clonal hematopoiesis, (ii) the instructive role of each mutation in determining lineage fate, iii) differences in pattern and lineage restriction of these mutations between various MPD, and (iv) chronology of clonal emergence and clonal dominance for the 2 mutations. Methods: Sixteen MPD patients were studied (13 JAK2V617F+ and 3 MPLW515L/K+); 9 had PV (all JAK2V617F+), 2 ET (both JAK2V617F+), 1 CMML (JAK2V617F+), and 4 AMM (2 MPLW515K+, and one each with MPLW515L and JAK2V617F). CD34+ progenitor cells were plated in methylcellulose and resulting hematopoietic colonies were scored on day 10–12. Colonies were obtained with (all patients) and without (10 patients; 5 with PV, 1 ET, and 4 AMM) cytokine support. Individual colonies were screened for presence of either mutation by DNA sequencing. In addition, MPLW515L/K analysis was performed in T and B lymphocytes in 3 informative cases. Finally, serially stored bone marrow was examined in 3 patients with co-expression of MPL515 and JAK2V617F mutations. Results: A total of 552 hematopoietic colonies from 16 MPD patients were genotyped. We found cohabitation of both mutation-positive and mutation-negative endogenous colonies in PV as well as other MPD. For JAK2V617F+ patients, the majority (75–100%) of erythropoietin-independent erythroid colonies (EEC) but not endogenous granulocyte colonies (20–70%) were mutation-positive. In contrast, no EEC were obtained from progenitors from the 3 MPLW515L/K+ patients. For 2 of the 3 patients, the endogenous myeloid colonies were predominantly mutation-positive (~90%). A mixed allele pattern (homozygous and/or heterozygous) was seen with both mutations regardless of MPD phenotype, but the overall mutant colony burden was higher with MPLW515L/K mutations relative to JAK2V617F. When such testing was possible, MPL515 mutations were detected in T-lymphocytes (2 of 3 patients) as well as B-lymphocytes (2 of 2 patients). Finally, among 3 patients carrying both mutations, serial marrow studies (25 time points over 4–8 years) revealed the occurrence of both mutations at diagnosis and persistent dominance of MPLW515L/K over JAK2V617F over time. Conclusions: Our observations provide direct evidence for clonal hematopoiesis that antedates acquisition of either MPLW515L/K or JAK2V617F that bias towards myeloid and erythroid fates, respectively. Furthermore, both mutations appear to occur early in the disease course and lineage studies suggest a hierarchically earlier stem cell origin for MPLW515L/K as compared to JAK2V617F.

Abstract Introduction: The Multiple Myeloma Research Consortium (MMRC) has established a tissue bank for the deposition of bone marrow (BM) samples from patients with multiple myeloma (MM) to be mailed and processed under good laboratory practices (GLP). To date over 300 samples have been collected. The ability for multiple sites to utilize the same GLP protocols for isolation and storage of BM for batch testing can be cost prohibitive and can introduce derived variability. Currently, limited information is available on shipped BM aspirates in regards to cell viability, cell yield, cell purity, and subsequent RNA yield and quality. Materials and methods: To test these determinants we performed a pilot study on behalf of the MMRC where samples were drawn at Mayo Clinic Rochester (MCR) pooled and split into 2 equal aliquots. One-half of each sample was processed following the provided GLP compliant standard operating procedures (SOP’s), immediately after sample procurement, at MCR. The CD138+ cells were stored in TRIZOLTM and the RNA was isolated and analyzed in a single batch. The other half of the aspirate was sent overnight to Mayo Clinic Scottsdale (MCS) where they were processed using an identical protocol. At both locations samples were tested for the following quality determinants; cell yield, RNA yield and integrity, and viability using a 3 color flow cytometric method (CD45, CD38 and 7ADD). Cell counts were performed on the CD138+ fraction to determine plasma cell recovery and a slide based immunofluorescent assay used to determine purity. RNA recovery and integrity were assessed using the Agilent BioanalyzerTM. Lastly, gene expression profiles was compared to determine the “signature” emanating from the shipment of samples. Results: In aggregate, all quality determinants showed similar values when the two sets of samples were compared. Cell viability was similar in both sets of samples as was our ability to collect a highly enriched plasma cell population. The cell yield was very similar (r2=0.52) but slightly lower in the shipped samples (median 71% of locally processed, range 40–140%), probably due to some shipment-associated apoptosis with subsequent loss of cell surface CD138 antigen. The purity of the shipped samples was very similar to that of locally processed (median 94%). Subjective qualitative analysis of the RNA was similar between both groups (shipped yield being 80% of local) with no evidence of degradation in the shipped samples. Details regarding the shipment signatures using gene expression profiling will be presented at the meeting. Conclusion: Here we show that the shipment of samples is feasible with no appreciable loss in cell yield or quality of derived products. % APOPTOTIC+ DEAD PC YIELD PURITY (%) RNA Recovered MCR MCS MCR MCS MCR MCS MCR MCS 8.9 ND 15.2 6.8 93 90 11.4 12.8 6.2 ND 2 2.8 95 99 8 17.2 17.4 8 1.4 1.4 75 88 2.2 0 2.1 18.7 2.8 1.6 96 68 7.2 2 18.1 9.5 1.2 .8 100 94 3.4 .4 5 5.0 8.5 7.2 98 91 12.6 17.8 3.7 5.1 .4 .4 88 87 ND ND 19.5 10.3 1.6 1.2 88 79 .4 3.6 29.1 ND 9.2 1.2 100 56 30.2 1.4 5.6 ND 9.2 7.6 92 88 5.2 16.8 20.3 62.8 2.4 .8 84 61 4.6 0.2 4.6 3.7 10.4 2.4 100 94 29.4 8.4 26.5 13.5 5.8 2.8 ND 97 16.8 5.8 4.5 6.2 2.2 2 100 99 10 4.4 17.5 ND 19.2 12 ND ND 16.4 14

Abstract Background: Conventional cytogenetic studies in myeloproliferative neoplasms (MPNs) have provided pathogenetic, diagnostic and prognostic information and in certain instances guide therapeutic decisions. It is therefore reasonable to hypothesize that a higher resolution examination of chromosomes might reveal additional changes that are scientifically as well as clinically relevant. Methods: Granulocyte DNA from 70 patients (median age 56 years, range 18–84; 43 males) with classic BCR-ABL-negative MPNs were studied: 27 with primary myelofibrosis (PMF), 22 with polycythemia vera (PV), 12 with essential thrombocythemia (ET), 6 with post-ET MF and 3 with post-PV MF. Twelve healthy volunteers were used as controls. Cytogenetic information, collected at variable times before the current study, was available in 57 patients. Samples were analyzed by comparative genomic hybridization (CGH) with a gender matched normal control using oligoarrays with either 44,000 or 105,000 oligonucleotides. Data were analyzed with CGH Analytics (Agilent). Copy number changes were considered significant (CNCs) if they were defined by 4 or more adjacent oligonucleotides spanning at least 150,000 base pairs, contained at least one gene, and were not identified in the Database of Genomic Variants. Results: Array CGH results were equivocal in 6 cases, which were therefore excluded from further analysis. Among the 12 normal controls, granulocyte DNA displayed no CNCs. The percentage of patients with CNCs was 52% for PMF, 32% for PV and 17% for ET. In addition, 2 of 6 patients with post-ET MF and 1 of 2 patients with post-PV MF displayed CNCs. There was no correlation between the occurrence of CNCs and JAK2V617F mutational status in PMF and ET. Array CGH revealed CNCs in 16 of 49 patients (33%) with normal and 7 of 8 patients (88%) with abnormal cytogenetic findings; one patient with t(2;12)(p13;q14) in 19 of 20 metaphases detected at 9 months prior to sample collection for the array CGH study displayed no CNCs. In patients with abnormal cytogenetic findings, array CGH also detected imbalances at chromosomal translocations, including t(1;6), t(9;11), and t(1;18), as well as additional abnormalities that were not detected by cytogenetic analysis. A total of 23 single occurrence CNCs were documented and included all the ET [del(2)(q31.3q32.1), del(10)(q11.21q11.22)] and post-ET MF [del(2p12), del(Xq12)] associated CNCs. In contrast, several specific abnormalities were shared between PV and PMF including del(6p22) and dup(15q11.2). As expected, del(13q) and del(20q) abnormalities were recurrent in PMF. Conclusions: Conventional cytogenetic analysis underestimates the extent of genomic gains and losses in BCR-ABL-negative MPNs. The current study suggests that oligonucleotide array CGH is more sensitive to detect small genomic alterations and we are currently studying higher resolution arrays in a larger group of patients to detect additional genomic alterations as well as assess overall clinical utility. The current data also suggest that chromosomal translocations are commonly associated with imbalances.

Abstract Background: Systemic mastocytosis (SM) has a varied presentation ranging from indolent forms with limited morbidity over many years to the rapidly fatal mast cell leukemia (MCL). This heterogeneity complicates clinical decision making regarding choice and timing of therapy. The aim of this study was to evaluate the prognostic value of the 2001 World Health Organization (WHO) classification of SM, and to refine risk stratification within SM sub-groups to facilitate clinical decision making. Information on KITD816V and JAK2V617F mutation analysis and clinical correlates is also provided. Methods: Patient records in the institutional electronic database from January 1976 to October 2007 were reviewed and SM patients 18 years of age or older identified; the date of diagnosis ranged from July 1964 to October 2007. Only those patients where diagnosis was confirmed by bone marrow (BM) histology were included in the analysis. KITD816V mutation analysis was performed by DNA sequencing. JAK2V617F was screened by allele-specific quantitative PCR analysis. Results: i. Clinical characteristics at presentation: A total of 342 SM patients were identified (154 female; median age at diagnosis=57 years; range 19 to 87 years). Per the 2001 WHO classification, 159 had indolent SM (ISM; median age=49 years), 41 aggressive SM (ASM; median age=65 years), 138 SM with associated clonal hematological non-mast cell lineage disease (SM-AHNMD; median age=65 years), and 4 MCL (median age=55 years) (p-value for age <0.0001). 140 (41%) patients had urticaria pigmentosa (UP) (63% with ISM; p<0.0001), 160 (47%) mast cell (MC) mediator release symptoms (69% with ISM; p<0.0001), 107 (31%) skeletal symptoms, and 142 (42%) constitutional symptoms (61% with ASM/SM-AHNMD versus 19% with ISM; p<0.0001). 123 patients (36%) had palpable splenomegaly (57% with SM-AHNMD; p<0.0001). 56 patients (16%) exhibited prominent eosinophilia (absolute eosinophil count >1500/mcl) – 31% and 22% with SM-AHNMD and ASM, respectively, versus 3% with ISM (p<0.0001); 12 of 23 patients (52%) with eosinophilia who were tested carried the FIP1L1-PDGFRA mutation. Serum tryptase (normal <11.5 ng/mL) was measured in 160 patients (47%) and almost all (96%) had an elevated level (median=64 ng/mL; range=4 to 2000 ng/mL; 33 patients had >200 ng/mL). ii. Disease course and prognostic factors After a median follow-up of 20.7 months (range=0–417), 153 deaths were recorded. 17 patients (5%) transformed to acute myeloid leukemia (AML; n=14) or MCL (n=3), most frequently with SM-AHNMD (11% versus 0% for ISM; p<0.0001). The median overall survival was 63 months (ISM=198 months, ASM=41 months; SM-AHNMD=24 months, and MCL=2 months; p<0.0001). Multivariate analysis of parameters at the time of diagnosis showed a significant and independent association between inferior survival and WHO sub-type (p<0.0001), age at diagnosis (p<0.0001), history of weight loss (p=0.01), anemia (p=0.007), thrombocytopenia (p=0.0008), hypoalbuminemia (p=0.0008), and excess BM blasts (>5%; p=0.004). A similar analysis in ISM identified anemia (p=0.04), hypoalbuminemia (P=0.002), and BM MC ≥ 30% (p=0.03) as independent predictors of inferior survival; the corresponding parameters in ASM were age at diagnosis (p=0.002) and history of weight loss (p=0.003). iii)KITD816V andJAK2V617F analysis Archived bone marrow was available in 165 patients for KITD816V and JAK2V617F analysis. By DNA sequencing, overall KITD816V detection rate was 28%: ISM 20%, ASM 29%, SM-AHNMD 33% (p=0.4). In patients with ASM (p=0.003) and SM-AHNMD (p=0.001), detection of KITD816V was associated with inferior survival. Greater than 1% (range 1–57) JAK2V617F allele burden was detected in 6 patients (4%); all belonged to SM-AHNMD including 4 with non-MC lineage myeloproliferative neoplasm. Conclusions: The current study confirms the prognostic value of the WHO subclassification of SM and identifies advanced age, weight loss, anemia, thrombocytopenia, hypoalbuminemia, and excess BM blasts as additional adverse prognostic factors. In ISM, presence of anemia, hypoalbuminemia, or BM MC ≥ 30% is associated with inferior survival. In ASM and SM-AHNMD, BM KITD816V allele burden might influence survival. JAK2V617F is rare in SM and when found, it is almost always in the context of SM-AHNMD.