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Abstract Background and Aims A platelet count ≤100,000/µL is regarded as an independent prognostic marker for the development of leukemia and is associated with inferior survival in myelofibrosis (MF) (Huang et al. 2008). The approved JAK2 inhibitor, ruxolitinib, is associated with treatment-emergent thrombocytopenia, and patients with platelets ≤100,000/µL were excluded from the phase III COMFORT trials. Efficacy and safety data are limited in patients with MF and platelets ≤100,000/µL treated with ruxolitinib. Pacritinib (SB1518), an oral JAK2/FLT3 inhibitor, did not appear to be associated with clinically significant treatment emergent anemia or thrombocytopenia during early phase studies in patients with myeloid and lymphoid malignancies. This integrated analysis was performed to further evaluate the safety and efficacy of pacritinib in MF patients with baseline platelet counts of ≤100,000/µL. Methods The databases from 2 phase I/II clinical trials of pacritinib in advanced myeloid malignancies and MF were integrated to evaluate the rate of adverse events (AEs) and favorable spleen responses in patients with MF and thrombocytopenia. Results One hundred twenty-nine patients with advanced myeloid malignancies and MF were treated with pacritinib in phase I and II clinical trials. Sixty-five patients with MF were treated with 400 mg orally once daily in 2 phase II studies. Of these, 28 patients with MF [21 primary MF (75%), 5 post-polycythemia vera-MF, 2 post-essential thrombocytosis-MF] had baseline platelet counts of ≤100,000/µL (median: 58,500/µL, range: 15,000/µL – 97,000/µL). The median age was 69 years. Twenty three (82%) patients had the JAK2V617F mutation. During the phase 2 studies, spleen volume responses were assessed by MRI and are shown for all patients (Figure 1) and for patients with platelets ≤100,000/µL (Figure 2). Thirty-seven percent and 43% of all evaluable patients and patients with ≤100,000/µL platelets, respectively, had ≥35% reduction in spleen volume from baseline (Table). Among the 21 informative patients with allelic JAK2V617F quantification at more than 1 study visit, 3, including 2 patients with homozygous mutations, had ≥35% reduction in spleen volume and substantial reductions in allelic burden from baseline (mean reduction 64%, range 44 to 88%). In the 11 (17%) patients with MF and baseline platelet counts ≤50,000/μL, overall spleen volume reduction was 38% (3/8) in evaluable patients and the median decline in platelet count observed at study end was 6,000/µL. Integrated safety analysis for all 129 patients with advanced myeloid malignancies showed the most common adverse events (AEs) were gastrointestinal (GI), predominantly diarrhea, and most of these were grade 1 (43%) and grade 2 (26%). The time to onset of diarrhea was ≤30 days in the majority of those affected, but only 2% had drug discontinuation as a consequence. Among the 129 patients in the phase 1 and 2 trials, 57 patients had ≤100,000 platelets; of these 26 (46%) had no CTC grade change in hemoglobin (Hb) and platelet counts from baseline values. Hb and platelet counts improved by one grade in 16% and 18% of patients, respectively. One grade worsening was seen in 28% and 30% in Hb and platelet counts, respectively. Conclusions Pacritinib was well-tolerated in patients with MF, regardless of their baseline platelet counts. Grade 1 or 2 GI toxicities, particularly diarrhea, were the most common AEs. Spleen responses were similar in patients regardless of baseline platelet counts (≥100,000/µL vs. ≤100,000/µL). Notably, even patients with initial platelet counts ≤50,000/µL tolerated therapy and the majority maintained stable hemoglobin and platelet counts. A phase III trial will evaluate pacritinib versus best available therapies in patients with MF and platelet counts ≤100,000/µL. Disclosures: Dean: Cell Therapeutics, Inc: Employment. Cernohous:Cell Therapeutics, Inc: Employment. Mesa:Incyte, CTI, Gilead, Celgene, Genetech, NS Pharma, Lilly: Research Funding. Campbell:Cell Therapeutics, Inc: Employment. Caldwell:Cell Therapeutics, Inc: Employment. Wang:Cell Therapeutics, Inc.: Employment. Myint:Cell Therapeutics, Inc: Employment.

Abstract Both genome wide association studies (GWAS) of common variation and exome wide association studies (EXWAS) of rare variation have successfully identified disease susceptibility variants for a variety of diseases. One GWAS of inherited susceptibility to Acute Myeloid Leukemia (AML) has been conducted, but no EXWAS have been performed to measure risk of AML attributable to low-frequency constitutional genetic variation. We performed the first EXWAS of risk of AML as a nested case-control study in the DISCOVeRY-BMT (Determining the Influence of Susceptibility Conveying Variants Related to one-Year mortality after BMT) cohorts. The DISCOVeRY-BMT parent study examined transplant-related mortality in leukemia patients undergoing unrelated donor allogeneic BMT. To identify low frequency variants and genes contributing to increased susceptibility to AML we used genotype data from the Illumina HumanExome BeadChip typed in the DISCOVeRY-BMT cohorts; the HumanExome BeadChip contains 242,901 variants, which are mainly protein-coding variants. The optimal sequence kernel association test (SKAT-O) was used to analyze gene-level associations with risk of AML. These gene-based tests evaluate the cumulative effects of multiple single gene variants on risk of AML. Analyses were performed in all European American AML cases and two subtypes: 1) de novo AML, 2) de novo AML with normal cytogenetics. Models were adjusted for age at transplant and principal components to control for population stratification. For gene-based tests at least 2 variants with minor allele frequency (MAF) ≤ 5%, were required to be present in the gene. This yielded a total of 13,687 genes tested, and a Bonferroni corrected significance level of P<3.65 x 10-6. Association tests were performed in 1,189 AML cases reported to CIBMTR 2000-08 (Cohort 1) and 327 AML cases reported to CIBMTR from 2009-11 (Cohort 2). Controls in Cohorts 1 (n=1,986) and 2 (n= 515) were 10/10 HLA-matched unrelated donors who passed a comprehensive medical exam and deemed healthy. We used metaSKAT to combine Cohorts 1 and 2 and obtain p-values of association with AML. We present the results of gene-level tests significant in both cohorts. The likely pathogenicity of these variants was determined in silico using SIFT, PolyPhen and MutationTaster. Patient characteristics are in Table 1. DNMT3A, on chromosome 2, was associated in the gene-based test with risk of AML (Pmeta=1.70x10-9, Table 2). Three missense variants at MAF <1% comprise both overall AML and de novo AML gene-based association: exm177559 (Asn->Ser), exm177507 (Arg->His), and exm177543 (Arg->Trp). Normal cytogenetics de novo AML gene-based assocations consisted of only 2 of these variants: exm177559 and exm177507 (Table 2). While prevalence of exm177507 is <1% for all AML cases, in de novo AML with normal cytogenetics the MAF was higher at 3%. The other 2 variants had a MAF<1% irrespective of subtype. Somatically, DNMT3A is most frequently mutated in hematologic malignancies, with >30% of de novo AML cases with a normal karyotype and >10% of MDS patients having DNMT3A mutations. Although these are germline gene associations all three of the variants found have been reported somatically in hematologic malignancies. In 200 AML cases from The Cancer Genome Atlas (TCGA) p.R882H (represented as exm177507 on the exome chip) was a frequent somatic mutation (25%). Exm177543 (p.R635W) and exm177559 (p.N501S) are reported in the Catalogue of Somatic Mutations in Cancer (COSMIC) as somatic mutations involved in hematopoietic and lymphoid tissue in both cell lines and humans. Exm177507 and exm177543 show evidence of pathogenicity in all three in silico tools, while exm177559 was reported as deleterious and disease causing by Sift and MutationTaster, respectively. Our results show that multiple potentially pathogenic missense germline variants in DNMT3A comprise the gene-based association with AML, specifically de novo AML with normal cytogenetics. Given the functional nature of these variants it is possible germline risk stratification could be informative in determining AML risk, and subsequently development of AML harboring DNMT3A mutations. Confirmation of these findings in additional cohorts could have implications for individualized risk screening, prediction and prognosis. Additional cytogenetic subgroup analyses, including treatment-related AML, are underway. Disclosures Hahn: Novartis: Equity Ownership; NIH: Research Funding. McCarthy:Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; The Binding Site: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gamida Cell: Honoraria, Membership on an entity's Board of Directors or advisory committees. Sucheston-Campbell:NIH/NCI: Research Funding.

Abstract Abstract 2922 The myelodysplastic syndromes (MDS) are a heterogeneous group of hematological malignancies characterized by ineffective hematopoiesis and a highly variable clinical course, for which novel treatments are beginning to emerge. Conventional cytogenetic studies (CCS) of bone marrow (BM) are routinely used in clinical practice to detect abnormal clones in proliferating (metaphase) cells, identifying clonal aberrations in ∼50% of de novo MDS cases. Cytogenetics is also one of the key International Prognostic Scoring System (IPSS) components used to estimate overall survival and leukemia-free survival in MDS. Chromosome abnormalities may be quantified at presentation and during treatment by the use of fluorescence in situ hybridization (FISH) with DNA probes for specific chromosome loci (e.g., chromosomes 5, 7, 8 and 20) in non-proliferating (interphase) nuclei. However, it is not clear whether or not peripheral blood CCS will yield the same diagnostic and prognostic data as bone marrow CCS at disease presentation or whether patients without apparent chromosome abnormalities by CCS have “hidden” abnormalities that can be identified by interphase FISH. To answer these questions, 15 members of the International Working Group on MDS Cytogenetics agreed to perform CCS and FISH in parallel on both peripheral blood and bone marrow samples collected from MDS patients. To be certain that all participating sites scored and interpreted their individual FISH data in a similar fashion, a quality assurance (QA) study was completed with each site studying two identical test (proficiency) samples. Concordance among sites was very good to excellent allowing for the establishment of a standardized protocol with clear scoring criteria before patient samples were processed. In the second phase of the study, a total of 77 MDS patients were accrued to the study with 61% showing an abnormal karyotype. A FISH panel consisting of eight probe sets [-5/5q-, -7/7q-/der(1;7), +8/8q-, -11/+11/11q-/add(11q), 12p-/+21/t(12;21), -13/13q-, 17p- and 20q-/i(20q)/i(20p), Abbott Molecular, Inc.] was performed on both specimen types. While CCS was frequently unsuccessful (57.5%) in the PB specimens, FISH was informative (concordant with BM/PB CCS) in 92% of cases, with 49% of PB FISH demonstrating an abnormal clone. FISH was discordant in 4 of 77 BM and PB samples (5%), while CCS and FISH on BM and PB were discordant in 6 of 77 BM specimens (8%) and 6 of 73 PB specimens (8%). The data suggest that evaluation of interphase nuclei from PB on follow-up (non-diagnostic) FISH studies on MDS patients will be equally informative (and less costly and stressful) as a BM sample. Disclosures: Slovak: PerkinElmer: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding. Ohyashiki:Nippon Shinyaku Co., Ltd.: Research Funding.

Abstract Mutation status of the immunoglobulin heavy chain variable region (IgVH) in B cell chronic lymphocytic leukemia (B-CLL) is a critical prognostic tool. Although patients with unmutated (UM) IgVH genes exhibit an overall shorter survival than those with mutated (M) IgVH genes, considerable heterogeneity in clinical progression exists among UM B-CLL patients. The goal of this study was to evaluate UM CLL patients (n=215) in a large B-CLL cohort for Ig V, D, and J gene usage and relevant clinical parameters to identify Ig molecular features in addition to UM vs. M status that have prognostic value. Consistent with the literature, the most commonly expressed IgVH gene in our UM B-CLL cohort was VH 1–69 (69/215). We first evaluated D and J usage in VH 1-69 vs. non-VH 1–69 UM patients. The factors that were significantly different between VH1–69 vs. non-VH 1–69 cohorts were JH6 usage (p=0.0014), D3–3 usage (p=0.0025), and the combination of JH6 and D3–3 usage (p=0.0002). We then examined potential associations between patient time to treatment (TTT) and specific IgVH molecular features. Although there was a trend that VH 1–69 patients exhibited a shorter TTT than non-VH 1–69 patients, the association did not reach statistical significance (p=0.06). When all UM patients were instead grouped on the basis of D and J usage, JH6 usage was not significantly associated with TTT, but D3–3 usage, irrespective of VH or JH usage, significantly correlated with shorter TTT (p=0.005). Of interest, when JH6 patients were excluded from the analysis, differences in TTT between those with and without D3–3 usage were particularly pronounced (p=0.011). We next explored whether a specific D3–3 reading frame (RF) is associated with TTT. Within the group of D3–3 patients, we evaluated differences in TTT between those with RF 2 (n=38) vs. RF 3 (n=19) but did not study RF1 patients due to small numbers (n=6). Comparison of D3–3/RF 3 patients (n=19) with all other UM patients (n=190), did not reveal a significant difference in TTT, however, there was a significant difference (p=0.012) in TTT between D3–3/RF 2 patients (n=38) and all other UM patients (n=171). Rai risk was still the best overall prognostic factor, and was the only significant factor (p<0.0001) in the multivariable (MV) setting for TTT using the entire UM cohort. However, when we categorized patients by both Rai risk (low vs. intermediate/high) and D3–3 usage, the most pronounced differences for TTT were observed based on D3–3 usage in the low Rai risk = 0 patients. Thus, the D3–3 cohort within the Rai 0 risk group displayed a significantly shorter TTT than did the non-D3–3 Rai 0 risk cohort (p=.0006). In model-building approaches in the MV setting using both the stepwise and score statistic methods for Rai 0 patients, D3–3 usage was the only significant factor for TTT (p=0.0004) even when RF 2 usage, JH6 usage, and absolute mutation percentage were also considered. These data illustrate for the first time the clinical importance of D gene usage and D gene RF in disease progression in early stage UM B-CLL. Moreover, this study adds further support to the notion that there is selection for specific Ig receptors in B-CLL and that the antigenic specificity of certain receptors may be associated with aggressive disease. Finally, these data demonstrate that the prognostic utility of IgVH mutation testing goes beyond simply determining categorical IgVH mutation status.

Context.— Biomedical terminologies such as Logical Observation Identifiers, Names, and Codes (LOINC) were developed to enable interoperability of health care data between disparate health information systems to improve patient outcomes, public health, and research activities. Objective.— To ascertain the utilization rate and accuracy of LOINC terminology mapping to 10 commonly ordered tests by participants of the College of American Pathologists (CAP) Proficiency Testing program. Design.— Questionnaires were sent to 1916 US and Canadian laboratories participating in the 2018 CAP coagulation (CGL) and/or cardiac markers (CRT) surveys requesting information on practice setting, instrument(s) and test method(s), and LOINC code selection and usage in the laboratory and electronic health records. Results.— Ninety of 1916 CGL and/or CRT participants (4.7%) responded to the questionnaire. Of the 275 LOINC codes reported, 54 (19.6%) were incorrect: 2 codes (5934-2 and 12345-1) (0.7%) did not exist in the LOINC database and the highest error rates were observed in the property (27 of 275, 9.8%), system (27 of 275, 9.8%), and component (22 of 275, 8.0%) LOINC axes. Errors in LOINC code selection included selection of the incorrect component (eg, activated clotting time instead of activated partial thromboplastin time); selection of panels that can never be used to obtain an individual analyte (eg, prothrombin time panel instead of international normalized ratio); and selection of an incorrect specimen type. Conclusions.— These findings of real-world LOINC code implementation across a spectrum of laboratory settings should raise concern about the reliability and utility of using LOINC for clinical research or to aggregate data.

Abstract #the authors contributed equally to this work While survival outcomes after HLA-matched unrelated donor (URD) blood and marrow transplant (BMT) have significantly improved over the last two decades, about 40% of patients die before 1-year post URD BMT. Previously we performed a genome-wide association study (GWAS) named DISCOVeRY-BMT (Determining the Influence of Susceptibility COnveying Variants Related to one-Year mortality after BMT) to investigate the contributions of common genetic variants on survival outcomes. To address the specific contributions of low frequency (≤ 1%) exonic variants on survival outcomes, we used the DISCOVeRY-BMT cohorts typed with the Illumina HumanExome BeadChip containing 242,901 variants, which are mainly protein-coding variants. We used a gene-based test (the optimal sequence kernel association test (SKAT-O)) to evaluate the cumulative effects of multiple genetic variants within a gene on overall survival (OS), transplant-related mortality (TRM), and disease-related mortality (DRM) 1 year after URD BMT. SKAT-O measures the overall significance of the gene and allows the variants to have different directions and magnitude of effects. We used a 2-stage study design: SKAT-O analysis was carried out in Cohort 1 consisting of 1,972 AML, ALL or MDS patients reported to the Center for International Blood and Marrow Transplant Research from 2000 to 2008 and their 2,006 10/10 HLA-matched URD. Genes with a suggestive association with each outcome (P < 1×10-3) entered the final meta-analysis including Cohort 1 and an independent cohort (Cohort 2), consisting of 503 recipients and 520 10/10 HLA-matched URD from 2009 to 2011. The analysis included recipients and URD of European descent due to the low frequency of other populations. All association models included age at BMT, diagnosis (AML, ALL, MDS), disease status at BMT, cell source (peripheral blood, marrow), year of BMT, and principal components from EIGENSTRAT to control for population stratification. Only coding variants (missense and nonsense) with minor allele frequency ≤ 1% were included in our analysis, which resulted in > 12,000 genes tested in our gene-based analysis in recipients and donors. Exome-wide significance was set at Pmeta< 4.10×10-6 after Bonferroni correction for the total number of genes tested. The likely pathogenicity of these variants was determined in silico using SIFT and PolyPhen. In recipients, we identified OR51D1, a member of the olfactory receptor gene family, as significantly associated with both OS and TRM. The OS association with ORD51D1 is driven by TRM as the same six missense variants contribute to both TRM and OS, but not to DRM. In donors, four (ALPP, EMID1, SLC44A5, LRP1), one (HHAT), and two (LYZL4, NT5E) genes were significantly associated with OS, TRM, and DRM, respectively (Table). ALPP encodes an alkaline phosphatase. SLC44A5 is a member of the solute carrier gene family. LRP1 (low density lipoprotein receptor-related protein 1) encodes an endocytic receptor involved in several cellular processes, including intracellular signaling, lipid homeostasis, and clearance of apoptotic cells. HHAT encodes an enzyme that acts within the hedgehog secretory pathway, which is involved in hematologic malignancy. LYZL4 is a member of a family of lysozyme-like genes, which have a protective role in host defense. The encoded protein of NT5E, CD270, is used as a determinant of lymphocyte differentiation. The likely pathogenicity of several of the functional variants in all significant genes identified was predicted to be both (possibly or probably) damaging and deleterious (Table), thus indicating that the amino acid substitution of the variant may not be well tolerated. One damaging variant in LRP1 has been identified as a somatic mutation in hematopoietic and lymphoid tissue. Our study is the first analysis of the low-frequency coding variant contribution to URD BMT survival outcomes. We found that the missense variants contributing to many of these significant gene associations shows evidence of pathogenicity and thus it is biologically plausible these variants are contributing to survival outcomes. Further confirmation of these findings, and studies of the functional consequences of protein-coding changes in these genes, may provide more individualized risk prediction and prognosis as well as alternative donor selection strategies. Table. Table. Disclosures McCarthy: Gamida Cell: Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; The Binding Site: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hahn:Novartis: Equity Ownership; NIH: Research Funding. Sucheston-Campbell:NIH/NCI: Research Funding.

Abstract Over the past decade, there have been numerous published candidate gene association studies of non-HLA variants, genes and/or pathways in relation to BMT survival outcomes. Most of these studies investigated a small number of candidate single nucleotide polymorphisms (SNPs), chosen based on known gene function, in a limited number of cases without replication. Here we test the association of previously reported SNPs with survival outcomes (overall survival (OS), progression-free survival (PFS), death due to disease (DD) or transplant-related mortality (TRM)) after URD BMT using data from the DISCOVeRY-BMT (Determining the Influence of Susceptibility Conveying Variants Related to one-Year mortality after BMT) study which included 2,052 European descent recipient-unrelated donor pairs (Cohort 1) and 763 recipient-unrelated donor pairs (Cohort 1) reported to the Center for International Blood and Marrow Transplant Research (CIBMTR) between 2000-2009 and 2009-2011, respectively. DISCOVeRY-BMT is a Genome Wide Association Study (GWAS) that includes over 9 million typed or imputed SNPs. This represents the largest study to date and is well powered to test both SNP and gene-based association hypotheses. We conducted an extensive literature review of PubMed using MESH terms related to transplant and hematologic disease which yielded 76 candidate gene association studies that investigated a total of 46 genes with any survival outcome. Of the 46 genes, 11 genes (CTLA4, CYP2B6, CYP2C19, GSTM1, GSTT1, IL23R, IL6, MTHFR, NOD2/CARD15, TNFSRF1B, and VDR) were studied in at least 2 publications. A total of 16 SNPs in 8 of these genes reported a significant (P< 0.05) association with at least one survival outcome (OS, PFS, DD or TRM). We tested these 16 SNPs for an association with the survival outcome originally reported in each article (Table 1). All association models included age at BMT, diagnosis (AML, ALL, MDS), disease status at BMT, cell source (blood, bone marrow) and year of BMT. P-values from Cohorts 1 and 2 were combined using a fixed-effects model in METAL software. Since the candidate gene studies have a gene-based hypothesis, we also performed gene-level testing using the VErsatile Gene-Based Association Study (VEGAS2) software to measure the aggregate effect of all available typed and imputed SNPs from our GWAS data in these 8 genes. The candidate gene association studies included heterogeneous patient populations of autologous and/or allogeneic (related or unrelated donors) BMT for a variety of hematologic diseases. Most candidate gene studies performed genotyping on both donors and recipients, and SNPs were tested either independently for donor or recipient genomes, or considering the total number of variant alleles the recipient-donor pair had at a given locus (0-4) (Table 1). The 16 SNPs from the previous candidate gene association studies were not significantly associated at a nominal P<0.05 to either the survival outcome reported in the original published paper, or to any survival outcome (Table 1). Likewise, the VEGAS2 gene-based analysis did not reveal any statistically significant associations for any of the 8 genes at P<0.05. Our study also corroborated prior reports of SNPs and genes that were not associated with survival outcomes. Candidate gene association studies use a priori biological knowledge to select genes that are likely associated with outcome and subsequently SNPs within the gene thought likely to affect function based on knowledge at the time of the investigation. Our findings suggest that neither the genetic variation tested in these candidate genes, nor other individual SNPs in the gene independently or collectively are not associated with survival outcomes in recipients of URD BMT. Our failure to replicate the prior candidate gene studies may be due to these SNPs being dependent on the underlying disease (ie. AML, ALL, etc), the donor relation (related, unrelated, autologous), or that there is no true association of these SNPs with the outcomes studied. #These authors contributed equally Table 1. Table 1. Disclosures McCarthy: Gamida Cell: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; The Binding Site: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hahn:Novartis: Equity Ownership; NIH: Research Funding. Sucheston-Campbell:NIH/NCI: Research Funding.

Abstract Background: We sought to retrospectively analyze the clinical differences, at diagnosis and throughout the disease course, between patients with primary myeloifbrosis whom undergo blastic transformation (PMF-BP) and those whom expire from PMF complications without undergoing transformation. Methods: An international collaborative database of patients with PMF which progressed to PMF BP, and a control group of individuals with PMF who expired secondary to PMF was created. Data regarding clinical course, bone marrow, karyotypic, quantitative JAK2V617F analysis (when able), laboratory values, and therapy at the diagnosis of PMF, PMF BP and up to 4 return visits (R1-4) in between these milestones was abstracted. Results: COMPARISON AT DIAGNOSIS OF PMF: 136 cases of PMF who eventually underwent PMF-BP were included, with a control group of 42 PMF patients, were analyzed. Both groups of patients had similar demographic (median age at diagnosis 59 years (range 23–83)/ 59.1 (15–93); and disease parameters (median hemoglobin 10 g/dL (range 2–15.7) /9.8 (4.9–14.6); median platelet count 181 x 109/L (range 7–1400)/168 (16–1916)), and Lille PMF prognostic scores (p = n.s.) for PMF-BP vs. PMF control group respectively. Additionally, blasts (peripheral blood /marrow) were a median of 0% (range 0–18%)/ 2% (range 0–16%) for the study group versus 0.25% (range 0–5%)/ 3% (range 0–4%) (p = n.s. for both) for the control group, respectively. Both groups had similar rates of requiring therapy (86%; 93%) and survival from diagnosis (median 34 months (range 2–441); 29 months (1–236)) (p = n.s.). However, lactate dehydrogenase (LDH) (although high in both groups) was higher at diagnosis (median 802 IU/L (range 83–10353) vs. 416 IU/L (124–2197)) (p=0.04), as well as an abnormal marrow karyotype (85% vs. 25%; p<0.001) amongst the PMF BP group. SUBSEQUENT CLINICAL COURSE: Analyzing the clinical evolution between groups demonstrated persistently higher LDH and worsening thrombocytopenia in the PMF-BP cohort (see figure). Decreasing Platelets Approaching PMF BP Decreasing Platelets Approaching PMF BP Peripheral blood blasts increased in both groups, but values above 10% were unique to the PMF BP group. Additionally marrow karyotype displayed clonal evolution developed in 56% of the PMF-BP patients as opposed to 14% of controls (p<0.001). Initial and serial quantitative JAK2V617F mutation analysis (available from 20 /19 patients from the PMF-BP and control group at diagnosis, with 11 / 12 serial samples) showed no difference between the groups, nor a pattern of mutation burden elevation with transformation to leukemia in the study group. Conclusions: Increasing LDH, peripheral blood blast percentages >10%, and clonal evolution are features commonly seen in PMF patients whom evolve to blast phase and may be a harbinger of movement towards PMF BP.

Abstract Histiocytic neoplasms are clonal, hematopoietic disorders characterized by an accumulation of abnormal monocyte-derived dendritic cells or macrophages in Langerhans Cell (LCH) and non-Langerhans (non-LCH) histiocytoses, respectively. The discovery of the BRAF V600E mutation in ~50% of patients with LCH and the non-LCH Erdheim-Chester Disease (ECD) provided the first molecular target in these patients and novel insights into the pathogenesis of these disorders. However, recurrent mutations in the majority of the ~50% of BRAF V600E-wild type patients with non-LCH are unknown. Moreover, recurrent mutations outside of the MAP kinase pathway are undefined throughout histiocytic neoplasms. To address these issues, we performed whole exome sequencing (WES) of frozen biopsies from 24 patients with LCH (n=10) or ECD (n=14) paired with peripheral blood mononuclear cells. 13/24 patients also underwent RNA sequencing (RNA-seq). All mutations in activating kinases were validated by droplet-digital PCR, while targeted-capture next-generation sequencing validated all others. Both adult (n=18; n=2 with LCH) and pediatric cases (n=9; n=8 with LCH) were included. Using combined WES/RNA-seq, activating kinase alterations were identified in 100% of patients. In LCH, 60% and 40% had BRAF V600E and MAP2K1 mutations, respectively. In non-LCH 51%, 14%, 14%, and 7% were BRAFV600E, ARAF, MAP2K1, and NRAS mutant (Fig1A). Overall, a mean of 7 non-synonymous mutations per adult patient was identified (range 1-22) compared with 5 mutations per pediatric patient (range 4-9; p =ns). Mutations affecting diverse cellular processes were found to co-exist with kinase mutations including mutations in epigenetic modifiers and the p38/MAPK pathway. In addition to kinase point mutations, RNA-seq identified recurrent, in-frame kinase fusions-a first for these disorders. All identified fusions were validated using FISH and RT-PCR. This includes novel fusions in BRAF (RNF11-BRAF and CLIP2-BRAF), as well as therapeutically important fusions in ALK (2 separate KIF5B-ALK fusions) and NTRK1 (LMNA-NTRK1;Fig1B). Expression of each fusion in Ba/F3 cells conferred cytokine-independent growth. Importantly, the BRAF fusions were found to be sensitive to MEK inhibition but resistant to vemurafenib while the ALK fusions conferred sensitivity to the ALK inhibitors crizotinib or alectinib. We next interrogated a validation cohort of 37 BRAF V600E-wild type, non-LCH, formalin-fixed, paraffin-embedded tissue samples using targeted mutational profiling for MAP2K1, ARAF, NRAS, KRAS, and PIK3CA. This revealed activating mutations in MAP2K1 (32%; n=12), NRAS (16%; n=6), KRAS (11%; n=4), PIK3CA (8%; n=3), and ARAF (3%; n=1). Three of the investigated non-LCH patients with refractory disease and progressive organ dysfunction were treated with targeted therapies based on the discovery of novel kinase alterations described above. Treatment of 2 refractory MAP2K1- mutant, non-LCH patients with MEK inhibitors (trametinib or cobimetinib) resulted in dramatic clinical improvement (Fig1C). Both patients have been maintained on MEK inhibitor single-agent therapy with a sustained clinical response for >100 days. Further evidence of effective targeted inhibition was found in a refractory ECD patient carrying an ARAF S214A mutation. This patient failed to respond to 3 lines of prior therapies and suffered near blindness due to disease infiltration in the retina and optic nerves. Given a recent report of complete response to sorafenib in a lung cancer patient with an ARAF S214C mutation, we initiated sorafenib. Within 12 weeks, there was improvement in the patientÕs eyesight and decreased infiltrative disease, coinciding with >50% decrease in mutant ARAF DNA in plasma cell-free DNA. Whole exome and transcriptome sequencing identified activating kinase mutations or translocations in all patients with the common downstream effect of activating the MAPK pathway. The preliminary, dramatic, clinical efficacy observed with use of MEK and RAF inhibitors in MAP2K1 - and ARAF-mutated, non-LCH patients further supports the central role of targeting the MAPK pathway in these tumors. The discovery of the discussed mutations and fusions in diverse kinases provides critical new insights into the genetic events central to a spectrum of adult and pediatric histiocytic neoplasms. Figure 1. Figure 1. Disclosures Off Label Use: This abstract describes use of MEK inhibitors (both tremetinib and cobimetinib) as well as sorafenib for MEK1 and ARAF mutant histiocytosis. . Stephens:Foundation Medicine, Inc.: Employment, Equity Ownership. Miller:Foundation Medicine, Inc.: Employment, Equity Ownership. Ross:Foundation Medicine Inc.: Employment. Ali:Foundation Medicine Inc.: Employment. Hyman:Chugai Pharma: Consultancy; Biotherapeutics: Consultancy; Atara: Consultancy, Honoraria.

Key PointsIKZF1 associations with high-risk B-ALL may differ by age and sex. A novel variant on chromosome 14, rs189434316, is associated with over a 3.5-fold risk of normal cytogenetic B-ALL.