Lasho, Terra L.

Introduction: Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic neoplasm characterized by sustained peripheral blood (PB) monocytosis and an inherent risk for leukemic transformation. Clonal origins of the disease can be detected in hematopoietic progenitor cells (CD34+/CD38-), while the complete spectrum of mutational evolution can be seen in circulating monocytes (CD14+). Cell sorting strategies have been employed to select cells in CMML, and while there are adequate monocyte numbers in the PB, there are very few circulating progenitor cells. In addition, attrition related to the selection process significantly depletes primary cells available for biological experiments and multiomics studies such as RNA-seq, ChIP-seq, ATAC-seq, and DIP-seq. While single-cell methods may be able to overcome this challenge, bulk sequencing methods remain a robust and cost-effective approach. We hypothesized that, secondary to the stem cell origin of this disease and significant myeloproliferation, PB mononuclear cells (MNC) would provide comparable results with regards to transcriptomic analysis, in comparison to cell selection procedures. Methods: Peripheral blood obtained from 15 molecularly annotated patients with WHO-defined CMML was ACK-lysed and subjected to a Ficoll procedure for collection of MNC. MNC were left unsorted (n=5) or further selected for CD34+/CD38- (n=5) and CD14+ (n=5) using a fully automated RoboSep-S (StemCell Technologies) protocol. All samples were then subjected to bulk whole transcriptome shotgun sequencing (using Illumina TruSeq and an Illumina HiSeq 4000). After data quality control, counts of detectable transcripts were log2-normalized and Pearson's product-moment correlation coefficients were calculated to evaluate the correlation between the two cell-sorting strategies and unsorted cells in terms of detectable transcripts. To visualize sample differences log2-normalized transcripts counts were centered and scaled per gene for a select number of genes relevant to myeloid biology as well as a number of housekeeping genes. Results: Fifteen patients with WHO-defined CMML, median age 69 years (55-73 years), 66% male, were included. Next generation sequencing for somatic mutations was performed on PB MNC obtained at CMML diagnosis (Figure 1, top heatmap). Considering the small sample size, mutations were evenly distributed among groups with the exception of ASXL1 (higher frequency in CD14+ and CD34+/CD38- cells), ZRSR2 (higher frequency in unsorted cells), and TET2 (lower frequency in CD14+ cells). The three groups were also well matched with regards to other CMML-related variables such as WHO and FAB morphological subtypes, cytogenetic abnormalities, and risk stratification by the Mayo Molecular Model. Transcriptomic analysis revealed a strong positive correlation between the median number of log2-normalized detectable transcripts in unsorted cells and CD34+/CD38- cells (ρ = 0.96, p < 0.001, top scatterplot). Likewise, there was a strong positive correlation between the median number of log2-normalized detectable transcripts in unsorted cells and CD14+ cells (ρ = 0.91, p < 0.001, bottom scatterplot). The latter correlation was marginally lower, which was explained by increased global gene expression in 3 of the 5 CD14+ samples (bottom heatmap). Increased gene expression in these 3 samples involved key myeloid genes and housekeeping genes known to have stable expression across human tissues alike. In comparison to PB MNC, both cell sorting strategies resulted in significant depletion of primary cells required for other experiments, and for procedures such as ChIP-seq, DIP-seq and ATAC-seq (CD34+/CD38- had greater depletion than CD14+). Additional experiments to assess this strategy for the above mentioned epigenetic studies are currently being planned. Conclusions: Accounting for sample differences, different cell sorting strategies (unsorted, CD34+/CD38- selection, and CD14+ selection) yielded similar results when performing bulk transcriptomic assessments on PB MNC from patients with CMML. For the purpose of gene expression profiling there was no clear advantage with CD34+/CD38- or CD14+ selection. These results support the use of unsorted cells for bulk transcriptomic analysis in CMML. Figure 1 Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Background: Myelodysplastic syndrome (MDS)/myeloproliferative neoplasm (MPN) with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T) was formally included as a unique WHO-defined entity in the 2016, iteration of the classification of hematological neoplasms (under MDS/MPN overlap syndromes). Patients with MDS/MPN-RS-T have features of MDS-RS-single lineage dysplasia (SLD), persistent thrombocytosis (platelet count ≥450x109/L), with proliferation of large atypical megakaryocytes. Anemia, including red blood cell transfusion dependency (RBC-TD) is a major problem. While extensive studies have documented erythroid response rates (ERR) to erythropoiesis stimulating agents (ESA) in MDS and MDS-RS-SLD, the data in MDS/MPN-RS-T is less clear. We carried out this study to asses ERR in ESA treated patients with MDS/MPN-RS-T. Methods: After approval by the IRB, patients with 2016, WHO-defined MDS/MPN-RS-T were included in the study. The bone marrow (BM) morphology, cytogenetics and BM RS% were retrospectively reviewed. All study patients underwent next generation sequencing for 29 myeloid-relevant genes, obtained on BM mononuclear cells, at diagnosis, or at first referral, by previously described methods (Patnaik et al., BCJ 2016). Treatment details, including the type of ESA used, dose administered, side effects and reason for discontinuation were retrospectively abstracted. Erythroid responses were assessed using the 2006, international working group (IWG) MDS and the 2015, IWG MDS/MPN response criteria. Results: Forty seven patients with MDS/MPN-RS-T were identified, of which 37 (79%) patients received ESA treatment at any time point during their disease course; median age 73 years (range, 52-93), 46% male.The hemoglobin at diagnosis (HB) and baseline erythropoietin (EPO) levels were 9.1 gm/dL (range, 6.6-12.1) and 39 IU/L (range, 8-500), respectively. Ten (29%) patients were RBC-TD at baseline. None of the 37, ESA-treated patients were on concomitant cyto-reductive therapy for thrombocytosis. Twenty (54%) patients were treated with erythropoietin-a, 13 (35%) with darbepoetin, and 4 (11%) with both sequentially. Median doses were 40,000 IU weekly (range, 20,000-80,000) for erythropoietin-a and 200 mcg every 2 weeks (range, 100-500), for darbepoetin. Median treatment duration was 14 months (range, 1-173) and causes for discontinuation included; ESA failure/loss of response in 20 (54%), sustained HB rise in 1 (3%) and other causes, including adverse events in 4 (11%) patients. With regards to safety, the following events were considered potentially ESA related; treatment emergent hypertension in 2 (5%), venous thromboembolism, ischemic stroke and splenic infarction in 1 (3%) patient each, respectively. All patients with thrombotic/ischemic events had been on low dose aspirin prophylaxis. Erythroid responses by the IWG MDS and MDS/MPN response criteria were assessable in 35 (95%) patients: with 16 (46%) meeting criteria for an erythroid response, by both assessment systems. The median duration of ER was 20 months (range, 2-172). In comparison to ESA non-responders, those that responded were more likely to have baseline EPO levels ≤44 IU/L (ROC analysis; median, 28 vs 112 IU/L; p=0.0080), while there was a trend for inferior responses associated with RBC-TD (p=0.0857). Age, gender, baseline white blood cell and platelet counts, type of ESA used, BM RS %, JAK2V617F, SF3B1, DNMT3A, TET2 and ASXL1 mutational status did not impact ESA response (Table 1). On a univariate survival analysis (OS), survival was not significantly different between ESA responders vs non-responder (median OS, 76 vs 45 months; p=0.2929). After ESA failure, 7 (19%) patients received hypomethylating agents (HMA), while 5 (14%) received lenalidomide. There were no erythroid responses to HMA, while 1 of 3 (33%) assessable lenalidomide treated patients achieved a morphological complete response. Conclusion: Erythropoiesis stimulating agents are effective first line therapies for the management of anemia in patients with MDS/MPN-RS-T, with 46% achieving an erythroid response (median duration of response of 20 months). Low baseline endogenous EPO level (<44 IU/L) was the best predictor of response, with a trend towards an inferior response in the presence of RBC-TD. In the context of clonal thrombocytosis, potentially related thrombotic/ischemic events were documented in 8% of patients. Disclosures Al-Kali: Astex Pharmaceuticals, Inc.: Research Funding. Patnaik:Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Introduction: Myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN) overlap syndromes consist of 5 distinct WHO-defined entities; namely chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia, BCR/ABL1- (aCML), juvenile myelomonocytic leukemia (JMML), MDS/MPN with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T), and MDS/MPN, unclassifiable (MDS/MPN-U) (Arber et al., Blood 2016). With the notable exception of JMML, a bona fide RASopathy, the other entities are characterized by clinical heterogeneity and molecular diversity. Loss of function TET2 mutations (TET2MT) are common in myeloid neoplasms, especially CMML (60%), and are known leukemogenic drivers. We carried out this study to assess the TET2 mutational landscape and phenotypic correlates in patients with MDS/MPN overlap syndromes. Methods: After approval by the institutional review board, adult patients with WHO defined MDS/MPN overlap syndromes were included; with the exception of JMML. The BM morphology, cytogenetics and 2016, WHO-diagnoses were retrospectively reviewed and all patients underwent targeted next generation sequencing for 29 myeloid-relevant genes, obtained on BM mononuclear cells, at diagnosis, or at first referral, by previously described methods (Patnaik et al., BCJ 2016). Results: Five hundred and four patients were included in the study; including 387 (77%) with CMML, 48 (10%) with MDS/MPN-RS-T, 17 (3%) with aCML and 52 (10%) with MDS/MPN-U. The median age at diagnosis was 71 (range, 18-99) years, and 333 (66%) were male. TET2MT were seen in 212 (42%) patients, with the frequency of other mutations being: ASXL1 45%, SRSF2 40%, NRAS 15%, SF3B1 13%, CBL, RUNX1 and SETBP1 12% each, and JAK2 V617F 11% (Figure B). Among the MDS/MPN overlap syndromes, TET2 was more frequently mutated in CMML (49%) and aCML (47%) compared to MDS/MPN-RS-T (10%) and MDS/MPN-U (15%). The prevalence of patients with TET2MT increased with age, a finding consistent across all MDS/MPN subtypes (Figure C). Overall, 341 TET2MT were identified in 212 patients (mean 1.6 variants/patient, range 0-5): 120 (24%) had >1 TET2MT, while 113 (22%), 5 (1%) and 2 (0.4%) had 2, 3 and 5 mutations, respectively. CMML and aCML patients were more likely to have an age-independent increase in multiple TET2MT (28% and 24%), in comparison to MDS/MPN-RS-T (4%) and MDS/MPN-U (8%). TET2 MT spanned the entire coding sequence and were mostly truncating (78%, Figure A): 59 (17%) were missense, 14 (4%) involved the splice-donor/acceptor sites, 2 (0.5%) were in-frame deletions, 129 (38%) were nonsense, and 137 (40%) were frameshift mutations. Overall, the distribution of TET2MT was superimposable across CMML, aCML, and MDS/MPN-U; the only exception being the absence of splice site mutations in the latter two. One hundred and eighty-seven (55%) TET2MT were secondary to pathogenic single nucleotide variants (SNV), while the remainders were secondary to deletions (25%) and insertions (15%). Transitions comprised the most frequent type of SNV (65%), with the C:G>T:A being the most common (56%). Patients with MDS/MPN overlap syndrome and TET2MT were more likely to have additional gene mutations compared to wild type patients (mean mutation number 3.1 vs 2.1, p<0.0001), with common co-mutations being SRSF2 (51%), ASXL1 (42%), and CBL (17%). The median overall survival (OS) of the entire cohort was 29 (range, 0-170) months; 29 months for CMML, 63 months for MDS/MPN-RS-T, 14 months for aCML, and 25 months for MDS/MPN-U. On univariate analysis, OS was superior in CMML patients with TET2MT (35 months) compared to wild type cases (21 months, p<0.0001, Figure D), and in CMML patients with >1 TET2MT (41 months) in comparison to wild type (21 months, p<0.0001) and single TET2MT (29 months, p=0.0476) cases (Figure E). These observations were not seen in patients with aCML, MDS/MPN-RS-T, and MDS/MPN-U. Conclusion: Our study demonstrates that TET2MT are among the most frequent mutations in patients with MDS/MPN overlap syndromes (42%), especially CMML (49%), with an age-dependent increase in the frequency and number of TET2MT. Mutations in TET2 were found to span the entire coding sequence, with truncating mutations being more common (78%). Importantly, in CMML, TET2MT, including number of TET2MT, were associated with favorable survival outcomes. Figure Disclosures Al-Kali: Astex Pharmaceuticals, Inc.: Research Funding. Patnaik:Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Introduction: Chronic myelomonocytic leukemia (CMML), an aggressive myeloid malignancy, can be categorized into two subtypes, proliferative CMML (pCMML) and dysplastic (dCMML), based on a white blood cell (WBC) count of ≥ 13 x 109/L for the former (Arber et al. Blood 2016). While this WBC cut off is somewhat arbitrary, patients with pCMML have unique phenotypic features and a shorter survival. We carried out this study to assess the genomic, transcriptomic and epigenetic landscapes of these two CMML subtypes. Methods: Peripheral blood (PB) and bone marrow (BM) mononuclear cells (MNC) were obtained from WHO-defined CMML patients. Next generation sequencing (NGS) using a 36-gene panel was performed on 350 patients with Illumina HiSeq4000 platform with median read depth of 400X. RNA sequencing (RNA-seq) was performed on 25 patients by bulk whole transcriptome sequencing using Illumina TruSeq. DNA immunoprecipitation sequencing (DIP-seq) was performed on 18 patients using 5-methylcystocine (5mC), 5-hydroxymethylcytosine (5hmC) and bridging monoclonal antibodies with subsequent paired-end sequencing using HiSeq4000. Chromatin immunoprecipitation sequencing (ChIP-seq) was performed on 30 patients with Illumina HiSeq2500 to a depth of 25 million for histone 3 lysine 4 monomethylation (H3K4me1) and histone 3 lysine 4 trimethylation (H3K4me3) and 50 million reads for histone 3 lysine 27 trimethylation (H3K27me3) and Input per sample. Results: Five hundred and seventy-three patients with WHO defined CMML were included; median age 71 years (range 18-95 years), 67% males. 282 patients had pCMML (49%), while 291 (51%) had dCMML. As pre-defined, patients with pCMML were more likely to have higher absolute monocyte counts (p<0.0001), circulating immature myeloid cells (p<0.0001), PB blasts (p<0.0001), and higher lactate dehydrogenase levels (p=0.03). At last follow up 234 (41%) deaths and 70 (20%) leukemic transformations were documented. The median OS for pCMML vs dCMML in this cohort was 19 months vs 30 months (p<0.0001, Figure 1A) and validated in an independent Austrian cohort (p=0.02). Genomic profiling: NGS performed on 350 patients (BM MNC) revealed a higher frequency of NRAS (35 vs 17%, p=0.004), cumulative RAS pathway (NRAS, KRAS, CBL and PTPN11) (73 vs 47%, p=0.001), ASXL1 (p=0.003) and JAK2V617F (p=0.04) mutations in pCMML relative to dCMML (Figure 1B); while dCMML had a higher frequency of SF3B1 mutations (p=0.02). There were no differences in distribution of TET2 and SRSF2Transcriptomic analysis: RNA-seq was performed on PB MNC from RAS pathway mutant pCMML patients (n=12) and RAS pathway wildtype dCMML patients (n=13). Unsupervised clustering analysis resulted in appropriate segregation revealing distinct expression profiles between disease subtypes (Figure 1C). Compared to dCMML, 3729 genes were significantly differentially upregulated and 2658 genes were differentially downregulated in pCMML. Among genes most highly upregulated were mitotic checkpoint kinases including AURBK, PLK1, PLK2, PLK4 andEpigenetic profiling: ChIP-seq of PB and BM MNC from pCMML (n=18) and dCMML (n=12) patients and healthy, age-matched controls (n=10) revealed a global increase in H3K4me1, without significant differences in H3K4me3 or H3K27me3 occupancies (regardless of stratification by ASXL1 mutational status; 40% ASXL1mt in pCMML, 30% dCMML) in pCMML vs dCMML (Figure 1D). H3K4me1 occupancy was also increased in a sequence-specific manner at the transcription start sites of the aforementioned mitotic kinases (PLK1 and WEE1). DIP-seq was performed on PB MNC to assess global differences in 5-mC and 5-hmC levels, between pCMML (n=9) and dCMML (n=9), with no differences seen between the two subtypes (regardless of TET2 mutational status, 40% TET2mt in each subtype) (Figure 1E). Conclusions: Despite the somewhat arbitrary WBC distinction between pCMML and dCMML, clear phenotypic, genetic, transcriptomic, epigenetic and survival differences exist between the two subtypes, providing strong biological rationale for this distinction. pCMML patients have a higher frequency of oncogenic RAS pathway mutations, a unique transcriptomic profile enriched in mitotic check point kinases and a unique chromatin configuration with global and sequence specific enrichment in H3K4me1, with no significant global differences in 5mC, 5hmC, or H3K4me3 and H3K27me3 occupancy. Figure 1 Disclosures Geissler: AOP: Honoraria; Pfizer: Honoraria; AstraZeneca: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Roche: Honoraria; Abbvie: Honoraria; Ratiopharm: Honoraria; Amgen: Honoraria. Al-Kali:Astex Pharmaceuticals, Inc.: Research Funding. Patnaik:Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Introduction: Additional Sex Combs-Like 1 (ASXL1) is a chromatin modifier frequently affected by truncating mutations in myeloid malignancies. These mutations are associated with poor survival outcomes and increased rates of acute leukemic transformation. In chronic myelomonocytic leukemia (CMML), ASXL1 mutations are thought to affect transcriptional activity mainly by modifying histone marks, however additional epigenomic mechanisms have not been fully explored. We interrogated the epigenome of patients with ASXL1-mutant (MT) and -wildtype (WT) CMML using a multiomics approach to define cis-regulatory elements (CREs) such as distal enhancers (DEs). Methods: Bone marrow mononuclear cells from patients with CMML were subjected to targeted NGS of DNA, whole transcriptome shotgun sequencing (RNA-seq), immunoprecipitation (IP) of DNA (hydroxy-)methyl residues (DIP-seq), IP of the histone modifications H3K4me1, H3K4me3, and H3K27me3 (ChIP-seq), and DNA transposase accessibility studies (ATAC-seq). After quality control all samples were sequenced on an Illumina HiSeq 4000 before further processing and data analysis. Global assessments of DNA (hydroxy-)methylation, DNA accessibility, and histone modifications between ASXL1 MT and WT CMML were performed. 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 signal peak to be considered statistically significant. Differential gene expression was estimated to define the up-regulated genes in ASXL1 MT CMML. Potential CREs were defined as sites with statistically significant signal peaks overlapping in at least two of the three epigenomic marks: H3K4me1, 5hmC, and ATAC. Potential DEs were defined as CREs in non-coding regions outside promoter regions (defined as transcription start site ±3kb) that were annotated in GeneHancer. Annotated DEs only present in ASXL1 MT but not WT CMML (specific DEs) were intersected with the list of up-regulated genes and the ReMap atlas. Results: Sixteen WHO-defined CMML patients were included, median age 69 years (48 - 77), 63% male; of which 8 patients (50%, all truncating frame shift mutations) were ASXL1 MT and 8 (50%) WT. The burden and spectrum of co-mutations was similar between ASXL1 WT and MT CMML (21 versus 23 per group; p = 0.684; heatmap). There was a predominant up-regulation of gene expression in ASXL1 MT CMML: 707 genes up- and 124 down-regulated (volcano plot, FDR < 0.050 for all genes). There were 64336 potential CREs, the vast majority (97%) being present in both ASXL1 MT and WT CMML (left Venn diagram). These CREs were most commonly located in introns, promoter regions, and distal non-coding regions (bar graph and pie chart). There were 1303 CREs unique to ASXL1 MT CMML (specific DEs), 1161 (90%) of which were annotated in GeneHancer (left Euler diagram). Of these 1161 annotated specific DEs 859 (74%) were located outside promoter regions and 34 (4%) of them were known to be associated with genes up-regulated in ASXL1 MT CMML (Euler diagrams). These specific DEs were characterized by an increase in H3K4me1 occupancy and DNA accessibility (average signal tracks, purple bars indicating annotated DEs, thin bars below peaks indicating statistical significance). We previously observed epigenomic modification of promoter regions in 519 of the 707 up-regulated genes (73%) facilitating transcriptional activity in ASXL1 MT CMML. For 13 of the up-regulated genes (right Venn diagram, blue genes in volcano plot) the specific DEs were the sole identified mechanism, while for the other 21 genes there were additional mechanisms noted in the promoter region. The top five transcription factor candidates binding the 34 specific DEs included JMJD1C, MYC, KDM5B, RCOR1, and HDAC2 (-log10(E) > 40 for all candidates). Conclusions: Using a multiomics approach based on H3K4me1, 5hmC, and ATAC data we identified potential CREs in ASXL1 MT CMML and characterized potential DEs using publicly available annotation data. Specific DEs were associated with up-regulated genes serving as a possible explanation for the observed transcriptional activity, shedding further light on the adverse prognostic impact associated with ASXL1 mutations. Figure 1 Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Introduction: Isocitrate dehydrogenase 1 and 2 (IDH1/2) are metabolic enzymes in the citric acid cycle, producing alpha-ketoglutarate (αKG). Mutations in specific regions of these genes have been characterized in gliomas, AML and chronic myeloid neoplasms such as MDS and MPN. These mutations produce an oncometabolite 2-hydroxyglutarate (2HG) that disrupts the epigenetic phenotype of myeloid cells promoting oncogenesis. While this process has been characterized for arginine hot spot (R132/R140) mutations, there exist variants of unclear significance (IDHVUS), with potential pathogenic predictions using in silico assessment models. With the availability of mutant IDH inhibitors in practice, it has become important to characterize these VUS. We carried out this study to functionally interrogate potentially pathogenic IDHVUS in MDS and MPN. Methods: After institutional approval, bone marrow (BM) DNA of patients with MDS and MPN, including overlap syndromes were subjected to next generation sequencing (29-gene, including the entire coding region of IDH1/2) by previously described methods (Patnaik et al., BCJ 2016). A sub-set of patients with available plasma [IDH wild type (IDHWT), bona fide IDH mutations (IDHMT) and IDHVUS with predicted pathogenicity (≤0.01% minor allele frequency, and predicted damaging in silico scores)] were submitted for mass spectrometry (LC-MS) based detection of IDH-related metabolites; D and L isomers of 2HG, αKG, and total 2HG. The impact of variations on protein secondary structure was assessed by the GorIV prediction algorithm. Potential alterations within the tertiary structure of the proteins was evaluated by in silico mutagenesis. Results: One hundred and seventeen patients were included in the study; median age 68 years (range, 39-94), 68% male. Of these, 40 (34%) had MDS, 42 (36%) MPN, and 35 (30%) MDS/MPN overlap syndromes. Eighty six (74%) had IDHMT, including n=28 (24%) with IDH1MT (R132C/G/H/S) (11 MDS, 8 MPN, 9 MDS/MPN) and n=59 (50%) with IDH2MT (R140G/Q/W) (22 MDS, 20 MPN, 17 MDS/MPN). IDHVUS were detected in 32 (27%) patients, including 1 IDH1MT patient, and involved IDH1 in 14 (12%) cases and IDH2 in 18 (15%) cases. All IDHVUS were missense, except for a 2 amino acid insertion in IDH2, with the most frequent VUS being IDH1Y183C (7%) and IDH2T352P (7%). While there were no phenotypic differences between IDH1/2MUT and IDH1/2VUS, in comparison to patients with IDH1/2MT, those with IDH1VUS were more likely to have higher WBC counts (median 8.1 vs 3.6 x109/L, p=0.0322), higher neutrophil counts (median 3.3 vs 1.3 x109/L, p=0.0203), and lower BM blast % (median 1 vs 3%, p=0.02061). Compared to IDH1/2MT patients, those with sole IDH1/2VUS more frequently had mutations in SF3B1 (p=0.0167), while they were associated with a lower frequency of SRSF2 mutations (p=0.0002). Interestingly, TET2 mutations were detected in only 5% of patients with IDHMT and 16% of IDHVUS patients, although without reaching statistical significance. LC-MS analysis on 75 plasma samples (n=43 IDHMT, n=9 IDHVUS, n=20 IDHWT, 3 normal) revealed a significant increase in total 2HG levels in IDHMT vs IDHVUS (p=0.0089), and IDHWT (p=0.0020) cases (Figure 2A-D). While there were no significant differences in αKG levels between the groups, the D-2HG enantiomer levels were higher in IDHMT samples vs IDHVUS (p=0.0199) and IDHWT (p=0.0055). On examination of the crystal structures of the IDH genes using PDB, while we observed a direct interaction between R132/R140/R172 and the catalytic core, the IDHVUS were located away from this active center and were hence thought to be allosteric in nature (Figure 2F). These VUS however, did demonstrate predictive steric clashes in the tertiary structure context of IDH1 using the PyMol artificial mutagenesis tool. The absence of wild-type crystal structure for IDH2precluded a similar analysis for IDH2. Median overall survival (OS) of the entire cohort (Figure 1) was 36 (range, 0-190) months, with OS being inferior in IDH1/2MT patients (34 months) vs those with IDH1/2VUS (50 months, p=0.0255). Conclusion: Our study demonstrates that in spite of predictive pathogenicity of IDH1/2VUS using in silico predictive tools and crystal studies, the lack of involvement of the arginine hot spot precludes IDH neomorphic activity, as shown by normal 2HG levels. In addition, in comparison to IDHMT patients, those with IDHVUS had fewer BM blasts and a better survival. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Introduction: Germline RUNX1 mutations/deletions result in a Familial Platelet Disorder with propensity to Myeloid Malignancy (FPDMM); an autosomal dominant condition characterized by thrombocytopenia, qualitative platelet defects, and myeloid clonal evolution (MDS and AML). The advent of next generation sequencing (NGS) has allowed detection of clonal cytopenias of unclear significance (CCUS) prior to bone marrow (BM) morphological changes. Somatic RUNX1 mutations (RUNX1MT) can be seen in myeloid malignances, including MDS/MPN overlap syndromes, with an indeterminate prognostic impact. We carried out this study to define the landscape of clonal evolution in FPDMM patients and to compare germline RUNX1MT with somatic mutations. Methods After IRB approval, families with RUNX1MT FPDMM (detected by germline sequencing and deletion/duplication assays) and patients with 2016, WHO-defined MDS/MPN overlap syndromes (except JMML) were included in the study. Index patients with FPDMM underwent BM biopsies and in select cases NGS at diagnosis and then as indicated for follow up, or at disease progression. All MDS/MPN overlap syndrome patients underwent a 29-gene panel NGS assay on BM samples obtained at diagnosis by previously described methods (Patnaik et al., BCJ 2016). Results Eight index patients with FPDMM were included; median age at diagnosis 58 (range, 0-71) years, 3 (38%) males (Figure A). Four (50%) had intragenic deletions involving multiple exons in 3 cases and 2 nucleotides in 1 case, 1 (13%) each had missense, frameshift and splice site mutations, while 1 (13%) patient had a whole gene deletion (Figure C). Notably, in 4 (100%) of 4 patients with large deletions, NGS testing was negative for RUNX1 abnormalities. Four (50%) index patients presented with a myeloid neoplasm; JAK2, SRSF2, ASXL1 mutant CMML, MDS/MPN-U with trisomy 8, AML with del7q and normal karyotype AML (negative NGS). Three (38%) index patients had CCUS at diagnosis with TET2 (67%) being the most common acquired mutation, and others including SRSF2, EZH2, and SF3B1. Cumulatively, myeloid clonal evolution was seen in 7 (88%) patients. Three (38%) index patients (AML-2, CMML-1) underwent allogeneic HCT, and at last follow up were in remission. Median age at myeloid clonal evolution was 61 (range, 4-71) years; median overall survival (OS) from date of myeloid clonal evolution was 12 (range, 7-53) months. For the comparative analysis, 499 patients with MDS/MPN overlap syndromes were included; 382 (77%) with CMML, 48 (10%) with MDS/MPN-RS-T (MDS/MPN with ring sideroblasts and thrombocytosis), 17 (3%) with aCML (atypical CML), and 52 (10%) with MDS-MPN-U (unclassifiable). Median age at diagnosis was 71 (range, 18-99) years, and 329 (66%) were male. RUNX1MT were detected in 57 (11%) patients; 43 (11%) with CMML, 5 (30%) with aCML, and 9 (17%) with MDS-MPN-U. No RUNX1MT were found in MDS-MPN-RS-T. Overall, 62 RUNX1MT were identified in 57 patients, with 52 (39 CMML, 4 aCML, 9 MDS-MPN-U) and 5 (4 CMML, 1 aCML) patients harboring 1 and 2 RUNX1MT, respectively. RUNX1MT spanned the entire coding sequence without specific hotspots (Figure B): 27 (44%) were missense, 2 (3%) involved the splicing sites, 5 (8%) were nonsense and 28 (45%) were frameshift. There was only one RUNX1MT (R273K), seen in both germline and somatic patients (Figures B and C). Compared to wild type patients, RUNX1MT patients with MDS/MPN overlap syndromes had significantly lower platelet counts (median 72 vs 117 x109/L, p<0.0001), without any other phenotypic differences. In the somatic RUNX1MT cohort, co-occurring mutations included ASXL1 (54%), SRSF2 (54%), TET2 (49%), and NRAS (28%), with NRAS (28% vs 13%, p=0.0028), and SRSF2 (54% vs 39%, p=0.0218) mutations clustering with RUNX1MT. Median OS of the entire cohort was 29 (range, 0-170) months. On univariate analysis, while there was a trend for an inferior OS in RUNX1MTvs wild type patients (20 vs 29 months, p=0.0523), the leukemia-free survival was clearly inferior (p=0.0175, Figure D). Conclusion RUNX1 MT FPDMM is a germline predisposition syndrome with a high rate of myeloid clonal evolution (88%), including clonal cytopenias (38%). Germline RUNX1MT have minimal overlap with somatic mutations seen in myeloid malignancies, with NGS testing often missing RUNX1 deletions. Somatic RUNX1MT in myeloid malignancies are significantly associated with thrombocytopenia and an inferior leukemia-free survival. Figure Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Abstract Background: Current prognostication in primary myelofibrosis (PMF) utilizes 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). These contemporary models are inter-complimentary and highly efficient in predicting overall survival. However, specific risk factors for very long survival (e.g. 20+ years) and short-term mortality (i.e. death within 5 years of diagnosis) have not been systematically looked into. 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). Logistic regression statistics was used to identify predictors of 20-year survival and 5-year mortality. Variables evaluated in the logistic model included those that are currently listed in MIPSS70, MIPSS70+ version 2.0 and GIPSS, as well as age (≤70 vs >70 years) and sex. Receiver operating characteristic (ROC) curves and area under the curve (AUC) were used to estimate predictive accuracy. The JMP® Pro 13.0.0 software from SAS Institute, Cary, NC, USA, was used for all calculations. Results: A total of 1,282 consecutive patients with PMF (median age 65 years, range 19-92; 63% males) were used to identify 26 (2%) patients (median age 51 years, range 28-71; 38% males) who survived their disease for at least 20 years (very long-lived patients) and 626 (49%) patients (median age 68 years, range 27-92; 66% males) who died within 5 years of their diagnosis. Clinically-derived information, including the clinical variables used in MIPSS70 and MIPSS70+ version 2.0, with the exception of fibrosis grade (information available in 60%), was available in 99% of the study population; cytogenetic information was available in 94%, driver mutational status in 71% and the full profile of high molecular risk (HMR) mutations, including ASXL1, SRSF2, U2AF1 Q157, EZH2, IDH1 and IDH2, in 42%. MIPSS70+ version 2.0 risk distribution for the entire study population (n=1,282) was 4% very low risk, 16% low risk, 19% intermediate risk, 40% high risk and 21% very high risk; the corresponding percentages for the 26 very long-lived patients were 14%, 29%, 14%, 43% and 0% and for the 626 short-term survivors were 0%, 4%, 10%, 47% and 39% (p<0.001). Cytogenetic, driver mutation and HMR mutation information was available in 100%, 58% and 27% of the 26 long-term survivors, respectively, and 98%, 63% and 39% of the 626 short-term survivors. Multivariable logistic regression analysis of 20-year survival identified the following seven variables as being favorable: age ≤70 years (p=0.002); female sex (p=0.03); hemoglobin level ≥10 g/dl for women and ≥11 g/dl for men (p=0.03), leukocyte count ≤25 x 109/l (p=0.009), platelet count ≥100 x 109/l (p=0.002), circulating blasts ≤1% (p=0.03) and absence of constitutional symptoms (p=0.04). The particular phenotypic profile exhibited a high degree of predictive accuracy with an estimated AUC of 0.90 (Figure 1a); karyotype and mutations did not retain significance in this particular analysis; in fact, 5 (71%) of the 7 long-term survivors who were informative harbored at least one HMR mutation. A similar analysis for 5-year mortality identified the following risk factors: high molecular risk mutations (p<0.001); unfavorable or very high risk karyotype (p<0.001); absence of type 1/like CALR mutations (p<0.001); age >70 years (p<0.001); constitutional symptoms (p<0.001); hemoglobin level <10 g/dl for women and <11 g/dl for men (p<0.001); leukocyte count >25 x 109/l (p=0.004); and circulating blasts >1% (p=0.001); predictive accuracy was estimated at AUC 0.87 (Figure 1b). Conclusions: The phenotypic profile of long-lived patients in PMF includes young age (≤70 years old), female sex, asymptomatic disease (i.e. no constitutional symptoms) and absence of moderate to severe anemia, thrombocytopenia or leukocytosis and ≤1% circulating blasts; each one of these parameters, with the exception of female sex, were present in ≥80% of long-term survivors. Karyotype and mutations were more relevant in predicting short-term mortality. Disclosures No relevant conflicts of interest to declare.

Abstract Background : Cytogenetic abnormalities occur in approximately a third of patients with primary myelofibrosis (PMF) and 5-20% of those with essential thrombocythemia (ET) or polycythemia vera (PV). Abnormal karyotype in PV and specific cytogenetic abnormalities in PMF have been shown to adversely affect survival (Leukemia. 2018;32:1189; Br J Haematol. 2017 Jun 9. doi: 10.1111/bjh.14798). In the current retrospective study, we considered 650 Mayo Clinic patients with myeloproliferative neoplasms (MPN), including ET, PV and PMF, with at least two documented bone marrow (BM) biopsies, in order to examine the incidence and pattern of changes in karyotype and their clinical correlates. Methods : Study patients were recruited from the Mayo Clinic, Rochester, MN, USA, based on documentation of at least two serial BM biopsies. Diagnoses were according to the 2016 World Health Organization criteria (Blood. 2016;127:2391). BM biopsy #1 referred to the baseline karyotype at time of initial diagnosis/referral and BM biopsy #2 to the first repeat BM biopsy after diagnosis. In addition to documenting the presence or absence of changes in karyotype, a notation was made regarding disease phase, at the time of the repeat biopsy, in order to allow accurate interpretation of the data and evaluation of survival impact. Results: 650 patients with MPN, including 153 ET, 105 PV and 392 PMF, were included in the current study and had undergone at least two BM biopsies; 227 patients had three, 108 four, 48 five, 19 six, and 4 seven repeat BM biopsies during their clinical course. Cytogenetic clonal evolution in patients with normal karyotype at baseline : Baseline karyotype was normal in 466 (72%) patients, including 139 (91%) ET, 91 (87%) PV and 236 (60%) PMF. Clonal evolution, which constituted a change from "normal" to "abnormal" karyotype, was documented in 16 (12%) patients with ET, 22 (24%) with PV and 75 (33%) with PMF; the latter included favorable karyotype in 11% of the cases, unfavorable in 12% and very high risk (VHR) in 10%. 11 (69%) of the 16 documented cases of clonal evolution in ET, 10 (45%) of 22 in PV and 11 (15%) of 75 in PMF were accompanied by clinically evident progression to blast or fibrotic phase disease, at the time of the repeat BM biopsy. Cytogenetic clonal evolution in patients with abnormal karyotype at baseline : Baseline karyotype was abnormal in 187 (28%) patients, including 17 (9%) ET, 14 (13%) PV and 156 (40%) PMF. Additional changes in karyotype, during serial BM biopsies, were documented in 6 (35%) ET, 5 (36%) PV and 71 (46%) PMF patients. In PMF, the pattern of changes in karyotype included "favorable" to "favorable" in 7%; "favorable" to "unfavorable" in 13%; "favorable" to "VHR" in 5%; "unfavorable" to "unfavorable" in 13%; "unfavorable" to "VHR" in 3%; and "VHR" to "VHR" in 6%. All 11 (100%) patients with ET or PV who underwent clonal evolution displayed clinically overt disease transformation into leukemic or fibrotic phase disease, at the time of the repeat BM biopsy. Leukemic transformation at the time of the repeat BM biopsy was evident in 13 (18%) of the 71 PMF cases with clonal evolution. Survival impact of cytogenetic clonal evolution without change in disease phase : The survival impact of cytogenetic clonal evolution was examined under the following provisions: i) analysis was limited to events occurring at BM biopsy #2 with survival calculated from the date of BM biopsy #2; ii) patients with leukemic or fibrotic transformation at time of BM biopsy #2 were excluded from analysis; and iii) clonal evolution for the purposes of survival analysis constituted changes from "normal" to "abnormal", for ET and PV, while the type of changes were taken under consideration for PMF. Under these stipulations, clonal evolution appeared to affect survival in general, although statistical significance was apparent for only ET and PMF (Figures 1a, 1b and 1c). Conclusions: Cytogenetic clonal evolution in ET and PV is infrequent, in the absence of clinically overt disease transformation, and is more likely to occur in PMF. Such events, when documented in chronic phase disease, appeared to predict shortened survival in all three MPNs, which was most apparent in ET and with emergence of VHR or unfavorable abnormalities in PMF. These observations warrant prospective studies that account for indication bias. Disclosures No relevant conflicts of interest to declare.