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The spectrum of somatic alterations in hematologic malignancies includes substitutions, insertions/deletions (indels), copy number alterations (CNAs), and a wide range of gene fusions; no current clinically available single assay captures the different types of alterations. We developed a novel next-generation sequencing-based assay to identify all classes of genomic alterations using archived formalin-fixed paraffin-embedded blood and bone marrow samples with high accuracy in a clinically relevant time frame, which is performed in our Clinical Laboratory Improvement Amendments-certified College of American Pathologists-accredited laboratory. Targeted capture of DNA/RNA and next-generation sequencing reliably identifies substitutions, indels, CNAs, and gene fusions, with similar accuracy to lower-throughput assays that focus on specific genes and types of genomic alterations. Profiling of 3696 samples identified recurrent somatic alterations that impact diagnosis, prognosis, and therapy selection. This comprehensive genomic profiling approach has proved effective in detecting all types of genomic alterations, including fusion transcripts, which increases the ability to identify clinically relevant genomic alterations with therapeutic relevance.

Introduction: The Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders, which include polycythemia vera, essential thrombocythemia and myelofibrosis. These disorders are characterized by activation of the JAK-STAT pathway via somatic mutations in JAK2, MPL, and CALR. However, a number of recurrent somatic mutations outside the JAK-STAT pathway have been described. Many of these mutations, such as ASXL1 and EZH2, are know to confer risk for disease progression. Other mutations, such as TET2 mutations, appear to alter the biology of disease in preclinical and clinical studies. Thus, further investigation of co-occurring mutation events is important to further the understanding of disease biology. Recurrent activating mutations in the RAS signaling pathway have been described in patients with MPNs. We have sought to determine the clinical impact of RAS mutations in patients with MPN, and to assess the pattern of co-mutational events in patients with RAS mutations. Methods : A targeted 28-gene, amplicon based next-generation sequencing assay was used to sequenced bone marrow aspirate or peripheral blood samples from 211 pts with Philadelphia negative MPN including: ET=61, PV=35, PMF=54, Post ET MF=16, Post PV MF=17, Post MPN AML=20, MPN unclassified=5 and systemic mastocytosis=3. Pts with MDS/MPN overlap syndromes were excluded. NRAS and KRAS mutations were grouped together. FisherÕs exact test and Wilcoxon rank-sum test were used to compare categorical and continuous variables, respectively. Results: N/KRAS mutations occurred at a frequency of 6 % in this cohort (Fig 1a). The median variant allele frequency (VAF) of RAS mutations was 15.6%. This was lower than the VAF of JAK2 V617F (44.6%) (p RAS mutations were associated with mutations in TET2 with 4/32 (13%) with mut-TET2 vs 6/152 (4%) wild type TET2 having RAS mutations, though this result did not reach statistical significance (p=0.074) (Fig 1d). No other gene mutation was positively associated with mutations in RAS. 3/6 pts with TET2/RAS co-mutation had post MPN AML, while the remaining 3 had PMF or post ET MF (Fig 1c). The VAF of mut-RAS was always lower than mut-TET2 in patients where these were co-mutated, a pattern not seen for RAS VAF relative to JAK2 V617F VAF. This suggests that RAS mutations were acquired after TET2 in all cases assessed. Patients with RAS/TET2 co-mutation had the highest incidence of post-MPN AML. In those with wild type TET2 and mutant RAS proliferative disease features were noted including high WBC, monocyte count and splenomegaly. Finally, patients with myelofibrosis and RAS mutations were significantly more likely to have high DIPSS scores. Conclusions: RAS mutations occur in 6% of pts with Philadelphia negative MPN. Patients with co-occurring JAK-STAT pathway activating mutations and RAS mutations had more proliferative disease and a higher incidence of post MPN AML. RAS mutations were more frequent in patients with mutant TET2 and in this context they were associated with post MPN AML. The RAS VAF was always lower relative to TET2 and other MPN driver mutations suggesting they are acquired later in disease evolution. These data suggest the presence of RAS mutations may alter the disease biology of MPNs, and raise the question of the possible clinical efficacy of utilizing therapeutic agents, such as MEK inhibitors, in MPN patients with RAS mutations. Further preclinical and clinical evaluation of the role of this pathway in MPN pathobiology is warranted. Disclosures Levine: Qiagen: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.

Subsets of pediatric acute leukemias are refractory to frontline therapy or relapse with standard treatment. Pediatric leukemias are associated with distinct molecular and genomic alterations. Clinical genomic profiling has been used effectively to improve the diagnosis of adult leukemias. However, its use and utility for pediatric leukemias are not well defined. Here, we used FoundationOne Heme, a clinical grade, high-throughput, hybridization capture-based next-generation sequencing (NGS) assay for targeted sequencing of all exons of 405 genes as well as RNA sequencing of 265 genes. We analyzed the results of samples from 71 patients sequenced on the FoundationOne® Heme assay from children and young adults with acute lymphoblastic leukemia (ALL) (n= 34); early T cell precursor ALL (ETP) (n=3); acute myelogenous leukemia (AML) (n= 19); ambiguous lineage leukemia (n=3); and myelodysplastic syndrome (MDS) (n=12) who were treated at Memorial Sloan Kettering Cancer Center in the Department of Pediatrics over a 26 month period (January 2014-March2016). Age of patients ranged from 1-24 years of age (median =10 years). 42 samples were from patients who had yet to receive cytotoxic therapy (newly diagnosed) and 29 samples were from patients who had relapsed (n=24) or had refractory disease (n=5). The current average turnaround time for FoundationOne® Heme is 12-14 days. Overall 99% (70/71) of specimens were successfully profiled. We identified 230 genomic alterations, including 59.1% (136/230) missense or nonsense mutations of genes, 15.6% (36/230) copy number alterations, and 25.6% (59/230) gene fusions or rearrangements. Molecular aberrations were detected in 81% (9/11) of patients with normal karyotypes; 55% (5/9) of these were cytogenetically cryptic genomic rearrangements including NUP98-NSD1 (n=2) and 1 MLL-PTD (n=1). We identified one novel translocation involving SATB1-PDGFRB in a sample from a patient with pre B ALL. Another patient with pre B ALL was found to have a unique combination of genomic alterations including an MLL-PTD and SSBP2-JAK2. Two patients with MDS were identified as harboring germline GATA2 mutations, indicating the likely benefit of an allogeneic hematopoietic stem cell transplantation. Three patients had therapy modifications based on findings with the addition of dasatinib (ZMIZ1-ABL1), ruxolitinib (PCM1-JAK2) and trametinib (PTPN11mutation), and two patients had risk stratification altered based on MLL rearrangements which were not identifiable by conventional cytogenetics. Overall, the use of comprehensive genomic profiling led to improved accuracy of diagnosis or alteration of therapy in 10% (7/71) cases, including molecular based therapy selection (n=3), escalation of conventional chemotherapy due to high-risk features (n=2), and inclusion of stem cell transplantation (n=2). Thus, clinical genomic profiling of pediatric acute leukemia led to discovery of new pathobiology, improved accuracy of clinical diagnosis, and inclusion of targeted molecular therapies. Figure Figure. Disclosures Kobos: Janssen: Employment. He:Foundation Medicine, Inc: Employment, Equity Ownership. Roshal:BD Biosciences: Consultancy. Zhong:foundation medicine: Employment. Balasubramanian:Foundation Medicine, Inc: Employment, Equity Ownership. Nahas:Foundation medicine: Employment. Vergilio:Foundation Medicine: Employment. Ross:Foundation Medicine, Inc: Employment. Stephens:Foundation Medicine, Inc: Employment, Equity Ownership. Mughal:Foundation Medicine: Employment, Equity Ownership. Miller:Foundation Medicine: Employment, Equity Ownership. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.

Background: Acute myeloid leukemia (AML) is an aggressive malignancy with poor prognosis, especially in the elderly for whom hypomethylating agent therapy may be the only standard treatment option. Data from The Cancer Genome Atlas Research Network indicates 44% of patients with de novo AML carry mutations in genes that regulate DNA modifications, such as IDH1/2, DNMT3A, and TET2. Small retrospective series have reported differential response to hypomethylating agents (HMAs) in patients with mutations in the DNA modification pathway. For example, Itzykson et al. reported an increased response rate with azacitidine in MDS and AML patients with TET2 mutations compared with those without (82% vs 45%, p = 0.007). Next, Metzeler et al. reported an increase in CR rate with decitabine therapy in patients with DNMT3A mutations as compared with those without (75% vs 34% CR, p = 0.05). Emadi et al. noted a higher response rate in AML patients with IDH1/2 mutations compared to those without (71% vs 23%, p = 0.01) in patients receiving either decitabine or azacitidine. In contrast, Dinardo et al. did not find an association between responses to HMAs in patients with IDH1/2 and DNMT3A mutations in a retrospective analysis of 68 elderly AML patients. Recent evidence demonstrates that WT1 mutations lead to loss of TET2 function, suggesting thatAML pts with WT1 mutations may also be sensitive to HMAs. These findings suggest that mutations in the TET/IDH/WT1/DNMT3A pathway might confer increased sensitivity to HMAs. We aimed to determine if there was an association between response and mutations in the DNA methylation pathway in AML patients treated with HMAs at our institution. Methods: This is a single institution, retrospective study. Of a cohort of 288 molecularly characterized AML patients, 76 patients were treated with HMAs; either alone (n= 59) or in combination with another agent (n= 17). In our cohort, 39 patients were treated with HMA in the frontline setting and 37 were treated in the relapsed/refractory setting. DNA was obtained from peripheral blood or bone marrow aspiration samples. Testing was performed using a targeted enrichment NGS assay designed with the RainDance DeepSeq system, covering clinically relevant regions in 30 genes known to be mutated in myeloid malignancies. Samples were subjected to microdroplet emulsion PCR and sequenced on an Illumina MiSeq to an average depth of at least 500X. 51.3% of patients had mutational profiling at time of diagnosis, and the remainder had mutational profiling at time of relapse/refractory disease. Genes affecting DNA modification pathway (DNMT3A, IDH1/2, TET2, WT1) were analyzed as a composite (present/absent). Results: Among patients receiving HMA for AML, the mean age was 67.1 years and 68% of patients were male. Using the NCCN cytogenetic/molecular risk categories, 7/76 had "better" risk, 35/76 had "intermediate" risk, 28/76 had "poor" risk and 6/76 patients' risk status could not be determined. Four of the 76 patients had incomplete molecular data available and were excluded from analysis; therefore, the final cohort under study was 72 patients. 34/39 of the frontline HMA patients were evaluable for response and 32/37 of the relapsed/refractory patients were evaluable for response. Among the 34 patients treated with HMAs in the frontline setting, 16 had mutations affecting DNA methylation and 18 patients did not. The CR rate (CR + CRi) in patients with DNA pathway mutations was 8/18 (44%) vs. 3/16 (19%) in patients without mutations (p = 0.15). Among the patients treated with HMAs in the relapsed/refractory setting, responses were rare with only 3 patients (9.4%) achieving CR. Responses were too few to demonstrate statistical relationships. Conclusions: We noted a trend toward an improved CR rate in patients with DNA modification mutations. However, the difference was not statistically significant in this small study cohort. Given the rarity of mutations and size of published cohorts, including ours, it has been difficult to demonstrate statistically significant associations between mutations and response to HMAs. A meta-analysis of existing data may overcome barriers of small numbers in individual studies, though retrospective nature of studies and publication bias may influence these results. A prospective trial with comprehensive molecular profiling of all patients is warranted to determine true associations. Disclosures Levine: Foundation Medicine: Consultancy; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees.

BACKGROUND: Cardiovascular (CV) events are a known complication to proteasome inhibitor therapy in myeloma. Underlying mechanisms are unknown. We recently completed an investigator initiated, single institution Phase II study of high dose carfilzomib (56mg/m2) in patients with relapsed/refractory MM (NCT01351623). Among 42 response evaluable patients, 11 patients (25%) developed treatment-emergent heart failure of any grade, and 5 patients (11%) developed severe heart failure requiring mechanical ventilation. We undertook a study to identify potential biomarkers that may point to underlying mechanisms of CV events among multiple myeloma patients treated with carfilzomib therapy. METHODS: We performed a nested case-control study with 7 patients who experienced a CV event on our high dose carfilzomib study and had pre-treatment (baseline) plasma stored and 19 case matched controls treated on the same study who did not have a CV event. We screened for 90 proteins known to be associated with CV disease using O-linked glycosylation. We used the Proseek Multiplex CVD I 96x96 platform which is based on the Proximity Extension Assay (PEA) technique. PEA is a 96-plex immunoassay that allows high throughput detection of protein biomarkers in liquid samples. For each biomarker, a matched pair of antibodies linked to unique oligonucleotides (proximity probes) binds to the respective protein target. Upon binding, the unique proximity probes can hybridize to each other and subsequently be detected and quantified by real-time PCR. Mean biomarker levels were compared using a t-test. False discovery rate (FDR) was used for multiple comparisons adjustment. RESULTS: Using samples collected prior to initiation of carfilzomib therapy, in an agnostic statistical model we identified the following four proteins to have altered levels in myeloma patients who developed CV events (p=0.002-0.004, unadjusted; p=0.089, after FDR correction): matrix metalloproteinase-1 (MMP-1, heparin-binding EGF-like growth factor (HB-EGF), TNF-related apoptosis-inducing ligand (TRAIL), and myoglobin (MB). Myeloma patients who developed CV events had 37% lower MMP-1, 15% lower MB, and 4% lower HB-EGF, while TRAIL was 7% higher in patients who developed CV events. Matrix metalloproteinases are a family of proteolytic enzymes responsible, among other functions, for myocardial extracellular protein degradation. Interestingly, several MMP species, including MMP-1, have been identified within the human myocardium and are thought to be dysregulated in congestive heart failure. HB-EGF is a mitogenic and chemotactic glycoprotein that is essential for maintaining normal cardiac function and is known to play an important role in myocardial remodeling. CONCLUSIONS: We found that there was a trend towards lower MMP-1, HB-EGF, and MB levels and higher TRAIL levels in patients with CV events while receiving proteasome therapy. MMP-1 appears to be the most promising potential biomarker based on our data. Our study supports further investigation of these proteins as potential biomarkers for patients at risk of CV events when treated with carfilzomib. Table 1. CV event No CV event N=7 N=19 CKD Proteins1 Mean (SD) Mean(SD) Unadjusted P-value Adjusted P-value MMP_1 1.7 (0.5) 2.7 (0.9) 0.002 0.089 HB_EGF 6.9 (0.2) 7.2 (0.3) 0.004 0.089 TRAIL 8 (0.3) 7.5 (0.5) 0.004 0.089 MB 5 (0.5) 5.9 (0.8) 0.004 0.089 HSP_27 2.2 (0.3) 2.7 (0.8) 0.032 0.528 PDGF_subunit_B 4 (0.7) 5 (1.5) 0.036 0.528 CD40_L 3.4 (0.6) 4.2 (1.2) 0.042 0.533 EGF 3.7 (0.9) 4.7 (1.4) 0.053 0.592 CX3CL1 5.9 (0.2) 5.6 (0.6) 0.092 0.895 TRAIL_R2 4.2 (0.4) 4.6 (0.6) 0.101 0.895 1. Proteins are listed based on the p-value associated with the difference between patients who did and did not have CV events, with lowest p-value on the top. The top 10 biomarkers are shown. Disclosures Ekman: Olink Bioscience: Employment. Grundberg:Olink Bioscience: Employment. Hassoun:Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Takeda: Research Funding; Novartis: Consultancy. Lesokhin:Aduro: Consultancy; Efranat: Consultancy; Genentech: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Janssen: Consultancy, Research Funding. Landau:Janssen: Consultancy; Prothena: Consultancy, Honoraria; Janssen: Consultancy; Spectrum Pharmaceuticals: Honoraria; Onyx: Honoraria, Research Funding; Takeda: Research Funding. Giralt:TAKEDA: Consultancy, Honoraria, Research Funding; JAZZ: Consultancy, Honoraria, Research Funding, Speakers Bureau; AMGEN: Consultancy, Research Funding; SANOFI: Consultancy, Honoraria, Research Funding; CELGENE: Consultancy, Honoraria, Research Funding. Landgren:Onyx: Honoraria; Celgene: Honoraria; BMJ Publishing: Consultancy; International Myeloma Foundation: Research Funding; Bristol-Myers Squibb: Honoraria; Onyx: Research Funding; Medscape: Consultancy; Medscape: Honoraria; BMJ Publishing: Honoraria; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Onyx: Consultancy.

The Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs) Essential Thrombocytosis (ET), Polycythemia Vera (PV), and Myelofibrosis (MF) are characterized by mutations, which drive JAK-STAT pathway activation. Several studies have demonstrated the presence of recurrent somatic mutations outside of the JAK-STAT pathway, which accumulate over time, and may impact disease phenotype and outcome. We sought to determine the influence of somatic mutations on clinical phenotype and prognosis. We sequenced a total of 30 genes recurrently mutated in myeloid malignancies in a cohort of 162 MPN patients (pts) using a next generation sequencing platform. The cohort included 49 pts with ET, 26 PV, 38 Primary Myelofibrosis (MF), 11 Post ET MF, 14 Post PV MF, 12 with leukemic transformations of MPN (LT), 7 with MPN-unclassified (MPN-U) and 5 others. Median age was 59 years and 79 were men. A Total of 288 gene mutations were identified with the most commonly mutated genes being JAK2 (n=121, 74%), TET2 (n=31, 19%), DNMT3A (n=18, 11%), ASXL1 (n=16, 10%), IDH2 (n=10, 6%), RAS (n=12, 7%), TYK2 (n=8, 5%) and TP53 (n=7, 4%). We did not find any mutations in NPM1, CBL, SRSF2 and no FLT3 -ITD. CALR was not assessed in 20 pts and these were excluded from mutation number analysis. Importantly, we identified a relationship between the absolute number of mutations found per pt, disease phenotype, and age (table 1). Pts with/without prior chemotherapy or radiotherapy exposure did not have a difference in mutation number (1.5 vs. 1.9). Cases of ET or PV with fibrotic transformation had more mutations in ASXL1, RAS, EZH2, PHF6 and MPL than pre fibrotic ET or PV suggesting these may be relevant in disease progression and development of fibrosis. Mutations in TET2, RAS and PHF6 were more frequent in cases with LT compared to those with chronic phase MPN. Pts over 40 were more likely to have mutations in TET2 (p=0.026) and JAK2 (p=0.019) and ASXL1 mutations were more common in pts with abnormal cytogenetics than in those with normal cytogenetics (p=0.003). Thrombotic events, which are an important cause of morbidity in MPN patients, negatively correlated with mutations in ASXL1 (p=0.044). Prognosis as measured by DIPPS and DIPSS-Plus scores appeared to correlate with the average number of mutations found in MF patients (table 2). We examined several cases for which serial samples were available, and noted the acquisition of new mutational events despite ongoing therapy. We noted that the most commonly acquired mutations occurred in epigenetic modifying (DNMT3A, TET, IDH, ASXL1) and in growth signaling pathway (RAS, CBL) genes. These occurred despite active therapy and often without an overt change in clinical phenotype. Further details of these serial samples will be presented. We conclude that the number and spectrum of somatic mutations correlate with disease phenotype of MPN. Younger pts have fewer mutations, as do pts with normal cytogenetics. JAK2 and TET2 mutations were more common in older pts. We show that a subset of pts acquire mutations in epigenetic modifiers and in genes involved in growth signaling pathways during disease course, and that mutations in TET2, RAS and PHF6 were enriched at the time of leukemic transformation. Taken together, these results indicate that mutations outside the JAK-STAT pathway influence disease phenotype, and that the acquisition of mutations over time may predict for disease progression. Serial evaluation of mutational burden over time therefore warrants exploration in the clinical setting. Table 1. Average number of mutations appeared to correlate with disease phenotype, age and abnormal cytogenetics. Average Number of Mutations N Mean (SD) P-value Age < 40 years 13 1.4 (0.9) 0.026 Age > 40 years 12 2 (1) No Thrombosis 113 2 (1) 0.712 Thrombosis 28 1.9 (1) Normal Cytogenetics 64 1.8 (0.9) 0.016 Abnormal Cytogenetics 40 2.3 (1.2) ET/PV/PMF 99 1.8 (0.8) 0.029 LT 10 3 (1.5) ET/PV 66 1.6 (0.7) 0.01 Post ET/PV MF 22 2.3 (1.1) ET 44 1.6 (0.7) < 0.001 PV 22 1.5 (0.9) PMF 33 2.2 (0.9) Post ET/PV MF 22 2.3 (1.1) LT 10 3 (1.5) Table 2. Disease prognostic scores in MF appear to correlate with the average number of mutations found per patient. Risk category N Average number mutations DIPSS Low 8 1.5 Intermediate-1 19 2.4 Intermediate-2 9 1.8 High 1 5 DIPSS-Plus Low 6 1.5 Intermediate-1 12 2 Intermediate-2 14 2.2 High 1 5 Figure 1. Comutation map of genomic alterations. Each hash mark on x-axis represents an individual patient. Figure 1. Comutation map of genomic alterations. Each hash mark on x-axis represents an individual patient. Disclosures Levine: CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy.

Background: The clinical utility of mutation profiling in hematologic malignancies requires robust detection of all classes of genomic alterations in a single analysis across different tumor types. We developed a novel, hybrid capture-based NGS assay (FoundationOne® Heme) designed to comprehensively assess the genomic landscape of hematologic malignancies from archived FFPE, blood and bone marrow aspirate samples, sequencing both DNA and RNA to improve sensitivity for driver fusion events. We have analyzed the genomic alterations including substitutions, indels, copy number alterations and genomic rearrangements of 795 samples with various hematolymphoid malignancies including lymphoma, leukemia and multiple myeloma to demonstrate the diagnostic, therapeutic and prognostic utility of this test in routine clinical practice. Method: Genomic profiles of 746 out of 795 (94%) consecutive samples received in a CLIA-certified laboratory (Foundation Medicine) were successfully characterized by the FoundationOne® Heme test including 527 from bone marrow aspirates, 176 FFPE samples and 92 samples derived from blood. The clinical cohort is composed of 375 multiple myeloma cases, 185 leukemia cases, 150 lymphoma cases, 75 MDS/MPN cases, and 15 unknown hematologic disorder cases (Figure A). Adaptor-ligated sequencing libraries were captured by solution hybridization using a custom bait-set targeting 405 blood cancer-related genes and 31 frequently rearranged genes by DNA-seq, and 265 frequently-rearranged genes by RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging 498x for DNA and ~7M on-target unique pairs for RNA, to enable the sensitive and specific detection of substitutions, indels, copy number alteration and gene rearrangements. Results: 705/746 (95%) patients had at least one alteration reported as a somatic driver mutation, 68% of cases harbored at least one alteration linked to a targeted therapy or would inform enrollment in a clinical trial consistent with therapeutic relevance. These potentially actionable alterations included mutations in KRAS (14% of cases), NRAS (13%), CDKN2A (8%), DNMT3A (6%), IDH1/2 (5%), BRAF (4%) and FLT3 (3%) (Figure B). In addition, 64% of cases harbored at least one alteration that have been shown to predict outcome and have prognostic relevance, including TP53 (14% of cases), TET2 (7%), ASXL1 (7%), CDKN2B (5%), CREBBP (5%), MLL (5%) and NPM1 (3%)(Figure B). We also detected 344 genomic rearrangements in 280/746 (38%) patients. Of the 344 reported rearrangements, 166 were known fusions in various disease types and 178 were novel rearrangements involving known oncogenes and tumor suppressor genes, including a novel RCSD1-ABL2 fusion in a patient with B-cell ALL who will derive benefit from kinase inhibitor therapy as part of their anti-leukemic regimen. Genomic rearrangements (detected in 38% of cases) can lead to various functional consequences: 260 (76%) rearrangements detected in this cohort are predicted to create activating fusions; 19 (6%) rearrangements are predicted to be activating intragenic rearrangements in MLL, FLT3 and CARD11; and 60 (17%) rearrangements are likely truncating events in tumor suppressors. Conclusions: We demonstrated that hybrid capture-based targeted DNA and RNA sequencing can be used to identify all classes of genomic alterations in genes known to be therapeutic targets or prognostic predictors in a broad spectrum of hematologic malignancies. Moreover, our data suggests that the FoundationOne® Heme comprehensive genomic profiling test can alter therapeutic strategy in routine clinical practice. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures He: Foundation Medicine: Employment, Equity Ownership. Nahas:Foundation Medicine, Inc: Employment. Wang:Foundation Medicine, Inc.: Employment, Equity Ownership. Young:Foundation Medicine: Employment, Equity Ownership. Brennan:Foundation Medicine, Inc: Employment. Donahue:Foundation Medicine: Employment. Young:Foundation Medicine, Inc: Employment. Ross:Foundation Medicine, Inc.: Employment, Equity Ownership. Morosini:Foundation Medicine, Inc. : Employment, Equity Ownership. van den Brink:Foundation Medicine, Inc: Consultancy. Intlekofer:Foundation Medicine, Inc: Consultancy. Dogan:Foundation Medicine, Inc: Consultancy. Armstrong:Epizyme, Inc: Consultancy. Yelensky:Foundation Medicine, Inc.: Employment, Equity Ownership. Otto:Foundation Medicine, Inc.: Employment. Lipson:Foundation Medicine, Inc.: Employment, Equity Ownership. Stephens:Foundation Medicine, Inc.: Employment, Equity Ownership. Miller:Foundation Medicine, Inc: Employment. Levine:Foundation Medicine, Inc: Consultancy.

Xenotransplantation of primary AML samples into immunodeficient mice (PDX models) represents a unique opportunity for pre-clinical testing on a group of primary human samples that possess defined genomic lesions. However, given recent recognition that multiple genomically distinct sub-clones can exist in AML, there is a risk that there may be selection for sub-clones from the transplanted sample that might not fully represent the patient’s disease. We transplanted 160 (70 T-ALL 56 AML, 32 B-ALL, 2 MLL) patient samples of which 120 engrafted into at least 1 irradiated NSG mouse. 45 AML samples engrafted with a median latency 107+/-41 days. Transplantation of 6 PDX AML samples resulted in immunophenotypically identical disease within 87+/-35 days. 2 MLL samples engrafted in 100% of mice with a median latency of 103+/-13 days. 25 B-ALL samples engrafted with a median latency of 95+/-44 days. Secondary transplantation of 3 PDX B-ALL samples resulted in engraftment of leukemia cells with an identical immunophenotype in 100% of transplanted mice within 52+/-3 days. 48 T-ALL samples engrafted in at least one mouse within 50 days. Secondary transplantation of a single T-ALL PDX sample resulted in 100% engraftment within 31+/-10 days. Genomic DNA and total RNA were isolated from 150 (AML: 16Pt+33PDX; MLL 2Pt+6PDX; B-ALL 17Pt+38PDX; T-ALL 19Pt+19PDX) samples. Adaptor ligated sequencing libraries were captured by solution hybridization using baitsets for 405 cancer-related genes and selected introns for 31 genes frequently rearranged for DNA-seq, and 405 cancer-related and 265 genes frequently rearranged for RNA-seq. All libraries were sequenced averaging >500x for DNA and >6M total pairs for RNA (HiSeq). We detected on average 23+/-12 including a mean 5+/-4 known pathogenic variants such as CDKN2A/B deletion (20/13); FLT3 (SNV & -ITD) and NOTCH (11 ea); WT1 and TP53 (10 ea); NRAS (9); PTPN11 (7); NPM1c, PTEN, and KRAS (6) DNMT3A, IDH1/2, and ASXL1 (5 ea); FBXW7, CEBPA, and TET2 (4 ea); PHF6 and NF1 (3 ea); IKZF1, ATM, and JAK2 (2 ea). Analyses of fusion RNA molecules detected known fusions: MLL-AF4 (4); MLL-AF9 (2), CRLF2-P2RY8, ETV6-RUNX1 or TEL-AML1, PBX1-TCF3 (2 ea); MLL-AF10, MLL-ELL, MLL-EP300, MLL-PTD, BCR-ABL, BCL2-IGK, MYH11-CBFB, along with novel fusions: TCF3-OAZ1, RB1-RCBTB2, PAX5-FLI1, and PAX5-MSI2. The mutations found in the 54 patient samples were consistently identified in the 96 PDX, however some cases showing variation in allele frequency between diagnostic and engrafted samples. Collectively, all 1420 and 288 disease relevant variant allele frequency (VAF) correlated significantly between patient and PDX samples (R2=0.55, R2=0.43), respectively. We then assessed VAF changes from diagnostic to PDX sample as a measure of clonal concordance. Diagnostic and PDX sample were considered discordant if at least one disease relevant VAF demonstrated significant variation between these samples, accounted for small variability of infrequent variances considering SD of sequencing detection. 31 samples were scored as concordant and 23 as discordant which were similarly distributed between disease lineages and did not correlate with diseases status, future relapse or overall survival. Using the same rules we further accessed concordance only between PDX samples in 23 cases when patient samples were transplanted into multiple mice. All 10 groups of PDX samples that were concordant with patient samples were also concordant within the groups. 5 groups of PDX samples that were discordant with patient samples were concordant within groups. 8 groups of PDX samples that were discordant with patient samples were also discordant within their groups. Overall 15 samples produced concordant engraftment in mice and 8 samples produced discordant engraftment. We hypothesized that specific genomic lesions in the 8 groups might underline this discordance. Mutations of FLT3, RAS, TP53, PTPN11 and NOTCH1 correlated with clonal discordance. These findings show that the leukemias that are engrafted in mice mirror the genomic diversity of primary leukemia samples, and that the majority of PDX samples have a genotype similar to that observed in the clinical isolate. More importantly, our data demonstrate the feasibility of developing a large, genetically annotated bank of PDX leukemia models that can be used to test and credential novel therapeutics that target driver mutations in different leukemia subsets. Disclosures Stein: Seattle Genetics, Inc.: Research Funding; Janssen Pharmaceuticals: Consultancy. Wang:Foundation Medicine Inc: Employment. Miller:Foundation Medicine: Employment. Armstrong:Epizyme: Consultancy.

Background: A variety of next-generation sequencing methods have been used to investigate the genomic landscape of primary lymphoma patient samples, including whole-genome, whole-exome, and RNA sequencing. In diffuse large B cell lymphoma (DLBCL), sequencing studies identified numerous genomic alterations (GAs) of potential clinical relevance, but the distribution and frequency of GAs have not been precisely determined. Discrepancies in existing data likely arise from variability in types of specimens examined, sequencing technologies employed, and depth of coverage utilized to identify GAs. In this study, we performed comprehensive DNA/RNA sequencing of genes known to be important across the spectrum of hematologic malignancies in order to determine the nature and prevalence of GAs with potential diagnostic, prognostic, or therapeutic implications in a cohort of 112 well-annotated clinical DLBCL cases. Methods: We performed hybridization capture of 405 cancer-related genes and 31 genes commonly rearranged in cancer (FoundationOne Heme) and 265 frequently rearranged genes for RNA-seq applied to ³50ng of DNA and sequenced to high, uniform coverage. Genomic alterations, including short variants (small indels and base substitutions), rearrangements, and copy number alterations, were determined. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging >500x for DNA and >20,000,000 total pairs for RNA, to enable the sensitive and specific detection of GAs. Significant non-synonomous variants were identified as mutations from the COSMIC database, amplifications of established oncogenes, or homozygous deletions and/or clear loss-of-function mutations of known tumor suppressors. Results: GAs with diagnostic, prognostic, or therapeutic relevance were identified in 96% of cases, with a median of 5 (range 0 to 12) alterations per sample. Figure 1 shows the frequencies of different GAs. De novo DLBCL tended to exhibit fewer GAs (median 4) than relapsed/refractory (median 6) or transformed disease (median 6) (p=0.179; Wilcoxon rank sum). Compared with reported frequencies ranging from 5 to 44%, we detected alterations in CREBBP/EP300 in 21% of cases, with CREBBP mutations preferentially found in germinal-center B cell-like (GCB) compared to non-GCB DLBCL (26% vs. 8%; p=0.02; Pearson's chi-squared). Although previously described only in Burkitt lymphoma, we identified mutations in ID3 (L70P, P98R, Q100P) and TCF3 (N551K) in GCB-phenotype DLBCL cases with aggressive pathologic features and no MYC expression by IHC. Additional novel findings included alterations of genes involved in cellular metabolism in 18% of cases, including one IDH2 R172M mutation and two cases with SDHA L649fs* truncating mutations. We also identified several mutations more commonly found in solid tumors and leukemias, including BRAF V600E and KRAS G13D. With respect to genomic rearrangements, combined DNA/RNA capture and sequencing detected translocations/fusions in MYC, BCL2, and BCL6, which were concordant with cytogenetics/FISH analysis. We also identified rearrangements involving TBXL1XR1-P63, NOTCH2, SOCS1, and ETV6. Copy number alterations were detected in 26 different genes, including amplifications of CD274/PDCD1LG2 (PD-L1/PD-L2) (n=4) that were restricted to non-GCB cases. Alterations in TP53 were more frequently observed in transformed (42%) and relapsed/refractory (26%) compared to de novo DLBCL (12%; p=0.007; Pearson's chi-squared). TP53 mutations predicted for lack of response to chemotherapy, with chemo-refractory disease occurring in 48% of TP53-mutant patients compared to 10% of patients with intact TP53 (p=0.0004; Fisher's exact). Truncating mutations or deletions of RB1, albeit rare (5% of cases), were the most significant negative prognostic factor and were associated with therapeutic resistance and poor overall survival. Conclusions: Our results demonstrate the utility of comprehensive combined DNA/RNA next-generation sequencing as a promising method to identify clinically relevant GAs in clinical lymphoma specimens. This streamlined approach has the potential to combine multiple molecular and cytogenetic tests into a single platform. Future efforts will be directed at incorporating this approach both retrospectively and prospectively into clinical trials to identify predictive biomarkers to guide therapeutic decisions. Figure 1 Figure 1. Disclosures Intlekofer: Foundation Medicine, Inc: Consultancy. Levine:Foundation Medicine, Inc: Consultancy. Zelenetz:Foundation Medicine, Inc: Consultancy. Dogan:Foundation Medicine, Inc: Consultancy. Palomba:Foundation Medicine, Inc: Consultancy. van den Brink:Foundation Medicine, Inc: Consultancy. Brennan:Foundation Medicine, Inc: Employment. Young:Foundation Medicine, Inc: Employment. He:Foundation Medicine: Employment. Nahas:Foundation Medicine, Inc: Employment. Yelensky:Foundation Medicine, Inc: Employment. Otto:Foundation Medicine, Inc: Employment. Lipson:Foundation Medicine, Inc: Employment. Stephens:Foundation Medicine, Inc: Employment. Miller:Foundation Medicine, Inc: Employment. Younes:Foundation Medicine, Inc: Consultancy.

Genome and exome sequencing have provided important insights into the biology of hematologic malignancies, and have led to the identification of prognostically relevant mutations and therapeutically targetable genomic alterations. However, the utility of genomic profiling in routine clinical practice remains an area of active investigation, and there is a need for evidence-based approaches to application of these data. Although most adults with acute leukemia relapse after initial therapy, only 25% of acute leukemia patients are enrolled onto clinical trials. Reasons for limited enrollment are multifold, but include a limited understanding by clinicians of genomic alterations, and lack of access to broad-based analytically validated clinical genomic profiling, both hampering the ability of clinicians to enroll patients on genotype-specific, mechanism-based clinical trials. As such, the development of high throughput, state-of-the-art genomic profiling assays for clinical practice, such as FoundationOneHeme, provide an unprecedented opportunity. Here we analyzed a consecutive series of 62 leukemia patients at Memorial Sloan Kettering Cancer Center for whom FoundationOneHeme was performed to determine the incidence and applicability of genomic findings on clinical care. The clinical sample cohort included 17 patients with myelodysplastic syndromes (MDS), 14 patients with acute myeloid leukemia (AML), 14 patients with acute lymphoblastic leukemia (ALL), 6 patients with myeloproliferative neoplasms (MPN), 5 patients with chronic lymphocytic leukemia (CLL), 4 patients with aplastic anemia (AA), and 2 patients with chronic myeloid leukemias (CML). DNA and RNA were successfully extracted from 58/62 samples (94%). Adaptor ligated sequencing libraries were captured by solution hybridization using a custom bait-set targeting 405 cancer-related genes and 31 frequently rearranged genes by DNA-seq, and 265 frequently-rearranged genes by RNA-seq. Samples were sequenced to high depth (Illumina HiSeq) in a CLIA-certified CAP-accredited laboratory (Foundation Medicine), averaging 590x for DNA and >20M total pairs for RNA, to enable the sensitive and specific detection of short variants (substitutions and indels), CNAs and gene fusions. A genomic alteration was characterized as “therapeutic” if it has been shown to determine response or resistance to an available therapy, or mechanism-based trial and “prognostic” if it has been shown to be predictive of outcome by individual disease states. Of the 58 patients with informative genomic data, 84% had at least one pathogenic variant identified. A total of 154 such alterations were identified (2.7 alterations per sample), including 81 base substitutions, 39 indels, 6 splice mutations, 10 CNAs and 18 fusions/rearrangements. 69% of patients had variants identified that were classified as clinically significant. 53% of patients had potential therapeutically relevant genomic alterations, and 43% of patients had prognostically relevant alterations identified. 36% of patients had both therapeutic and prognostic alterations identified. The most common alterations identified in our cohort were TP53 (n= 8), NRAS (n= 6), KMT2A, KRAS, RUNX1 (n= 5), CDKN2A, CDKN2B, SF3B1, TET2 (n= 4). In sum, 54 prognostic and 47 therapeutic genomic alterations were identified. 7 MDS cases, 4 AA cases, 2 AML cases, 2 ALL cases, 1 CML case, and 1 CLL case did not have a pathogenic or actionable allele, demonstrating that almost all patients in our cohort had mutations with biologic and clinical relevance. We also noted a set of variants of unknown significance, including 37 deletions and 26 insertions, which are under investigation in regard to prognostic and therapeutic significance. However, given that at least one genomic variant was identified in the majority of patients, our data demonstrate the utility of this approach in the identification of somatic genomic alterations for prognostic and therapeutic value, and to identify clonal markers which can be used to track molecular response during anti-leukemic therapy. In summary, we analyzed the mutational profiles of leukemia patients using an analytic and clinically validated assay, which allowed us to identify both prognostic and therapeutically relevant mutations in the majority of patients, confirming the utility of comprehensive next-generation sequencing in clinical practice. Disclosures He: Foundation Medicine: Employment. Lipson:Foundation Medicine, Inc.: Employment. Otto:Foundation Medicine, Inc.: Employment. Miller:Foundation Medicine, Inc: Employment. van den Brink:Foundation Medicine, Inc.: Consultancy. Armstrong:Foundation Medicine, Inc.: Consultancy. Stephens:Foundation Medicine: Employment, Equity Ownership. Levine:Foundation Medicine, Inc.: Consultancy.

Although DNA methyltransferase Inhibitor (DNMTI) therapy demonstrates efficacy in patients (pts) with Myelodysplastic Syndromes (MDS), to date there are no clinical or genomic markers to select patients most likely to benefit from DNMTI therapy. Two recent retrospective studies have suggested TET2 mutations (muts) are associated with a higher response rate to 5-azacytidine (5-aza) and decitabine (DAC). We investigated the efficacy of DAC (20 mg/m2/day x 5 days) plus tretinoin (ATRA) given on days 10-19 of a 28-day schedule in MDS pts in a phase I/II study. We comprehensively analyzed pretreatment samples from 23 pts (15M,8F; median age 67, range, 44-78) treated on this study for alterations in known oncogenes to identify potential correlations between mut profile and clinical outcomes. The median follow-up was 41 mos (range, 10-66). IPSS cytogenetics (CG) risk categories included Good (n=8), Intermediate (n=5), and High risk (n=8) pts (2 karyotype failures). The overall IPSS risk categories were Int-1 (n=3), Int-2 (n=16), and High (n=4). Of 23 pts, 16 responded, including 2 CRs, 9 mCR+/-HI, 1 PR, and 4 HI. Genomic DNA and total RNA were isolated from all pt samples. Adaptor ligated sequencing libraries were captured by solution hybridization using two custom baitsets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 258 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging >584X for DNA and >20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. The median number of muts detected was 2 (range, 0-6), with 22/23 (96%) pts having at least 1 mut. The number of muts detected for 14 PB samples with ≤ 1% blasts was 2 (range, 0-6) compared to 4 muts (range, 1-5) for 8 pts with > 1% PB or BM blasts (unknown source for 1 sample). The most common mut identified in this cohort was TP53 (35%). Previous studies have shown that complex CGs and mut TP53 are strong poor prognostic indicators. Six pts with complex CGs and either mut or deleted TP53 had a median of 0 (range 0-2) additional muts, whereas pts with non-complex CGs who were wild-type (WT) TP53 (n=12) had a median of 3 (range 1-6) muts in other disease alleles (p=0.006). We identified mutually exclusive recurrent muts in RNA splicing factors including SRSF2 (26%), SF3B1 (17%), and ZRSR2 (9%), and recurrent muts in RUNX1 (22%), IDH1/2 (22%), TET2 (17%), ASXL1 (17%), PTPN11 (13%), CEBPA (13%), STAG2 (9%), and KRAS (9%). Thirteen additional muts were detected in single pts, and we identified novel candidate muts in DNA repair pathways (ATM, ATR, FANCA BRCA2), and in the recently described tumor suppressor FAT3. We also hypothesized that prognostication of pts with normal karyotype (NK) MDS may be aided by molecular stratification, as we and others have shown in AML. Of note, all 5 pts with IDH1/2 muts occurred in pts with NK, and in 4/5 cases they were associated with concomitant SRSF2 muts that were previously shown to be associated with adverse outcomes. We identified TET2 muts in 4 pts who were also ASXL1 WT. Notably, all 4 TET2 mutated/ASXL1 WT pts responded to DAC/ATRA therapy. The other 12 responders included 7 pts with TP53 mut/loss +/- other muts. No other muts were associated with a differential response to DAC/ATRA in this trial cohort. The strong association between TET2 muts and DAC/ATRA response, and more detailed mutational profiling of the 12 TET2 WT pts who responded to DAC/ATRA therapy is being investigated in additional pt samples and will be presented in more detail. Pts with TP53 muts showed a trend toward a worse survival which was not statistically significant; however, no other individual mut was associated with a differential survival in this small cohort. Conclusions The use of comprehensive mutational profiling employed in this study allowed us to identify muts in 25 genes in MDS pts, and allowed us to show that the overall mut frequency was higher in pts with a NK and without TP53 muts, thereby providing evidence for alternate disease initiation events and pathogenesis in these two genomically defined MDS subsets. All pts with mutated TET2 responded to DAC/ATRA, suggesting that pts with muts in TET2 are the best candidates for novel therapeutic trials which include DNMTI therapy. Disclosures: Wang: Foundation Medicine, Inc: Employment. Sanford:Foundation Medicine, Inc: Employment. Brennan:Foundation Medicine, Inc: Employment. Nahas:Foundation Medicine, Inc.: Employment. Otto:Foundation Medicine, Inc: Employment. Lipson:Foundation Medicine, Inc: Employment. Stephens:Foundation Medicine, Inc: Employment. Levine:Foundation Medicine, Inc: Consultancy.

Recent whole-genome sequencing, whole-exome sequencing, and copy number analysis studies in small sets of patients with chronic lymphocytic leukemia (CLL) at presentation versus progression have demonstrated that clonal evolution has clinical and biologic relevance in CLL. Although the time and cost required to perform whole genome and exome sequencing are improving, challenges still exist in implementing these genomic strategies in real-time clinical practice. Here we performed DNA and RNA sequencing of an extensive panel of all genes known to be recurrently mutated in lymphoid, myeloid, and solid tumor malignancies, at high sequencing depth in patients with CLL at clinical presentation and progression. This allowed us to obtain integrated mutation, copy number, and gene fusion events in CLL and to use this data to obtain new insights into clonal evolution of CLL. Genomic DNA and total RNA was isolated from 59 CLL samples (including 29 CLL paired patients sampled at the time of initial presentation when no clinical indication for therapy was met and at a later time point of disease progression requiring therapy). The median time between samples was 2.07 years (range 0.08-4.95 years)). Adaptor ligated sequencing libraries were captured by solution hybridization using two custom baitsets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 272 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging >590X for DNA and >20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. All clinical data as well as biomarkers previously validated for clinical use in CLL (IGHV mutational status, CD38 expression, metaphase cytogenetics and FISH) were also obtained for all samples at each time point. This targeted sequencing approach detected at least one clonal somatic mutation in 95% of CLL patients including 144 individual mutations, 3 functional rearrangements, and 1 homozygous deletion. Consistent with prior reports, mutations in NOTCH1 (25%), SF3B1 (19%), and TP53 (14%) were amongst the most commonly mutated genes in this cohort. Of note, activating mutations in KRAS (15%) and BRAF (10%) were also amongst the most commonly mutated genes targeted by somatic mutations in CLL (Figure). Taken together, mutations resulting in constitutive MAP kinase signaling occurred in 36% of all patients, second only to mutations activating Notch signaling which occurred in 40% of patients. The two most commonly mutated genes in the Notch signaling pathway were mutations in NOTCH1 followed by loss of function mutations in SPEN (7%). SPEN is a nuclear receptor transcription factor which blocks the differentiation of precursor B cells into marginal zone B cells through interactions with RBP-J. Also identified as recurrent were mutations in FAT3, an inhibitor of b-catenin signaling. In addition, sequencing identified a novel fusion event in BIRC3 fusing BIRC3 to LRRC40 as well as a truncating mutation in one patient in BRCA2 at disease progression. We then performed paired mutational analysis of samples at initial sample acquisition and at disease progression requiring therapy (Figure). Mutational analysis at the time of clonal evolution identified mutations in DNMT3A, EED, IDH2, IRF4, VHL, and RB1 only at the time of disease evolution. Mutations in NOTCH1, KRAS, TP53, NRAS, and BCOR were more common at disease progression than initial presentation. In contrast, mutations in SF3B1 and XPO1 were always retained at disease progression if found at earlier disease states. These data demonstrate the utility of clinical grade, high throughput targeted sequencing in CLL to identify targetable genomic alterations, discover novel mutations not previously reported, and identify important markers of disease evolution which may represent incipient biomarkers for clinical disease progression. This includes a much more frequent occurrence of targetable mutations in the MAP kinase pathway than previously described in CLL and recurrent loss of function mutations in SPEN and FAT3 in CLL. Disclosures: Levine: Foundation Medicine, Inc: Consultancy. Heaney:Novartis: Research Funding; Sanofi-Aventis: Consultancy, Research Funding; Onconova: Research Funding; Incyte: Consultancy, Research Funding. Abdel-Wahab:Foundation Medicine, Inc: Consultancy.

Disease relapse remains the greatest cause of mortality following allogeneic stem cell transplantation (SCT). Improved predictive markers are needed to identify patients most likely to benefit from SCT. Several mutations reported recently in MDS and AML have potential prognostic importance, however the relevance of these mutations to clinical outcome after SCT is poorly understood. In order to evaluate mutations present in transplant patients and provide an initial assessment of their clinical significance, we performed next-generation sequencing of myeloid malignancies from 55 patients (23 MDS, 32 AML) treated with SCT at MSKCC from 2010-2013. Median recipient age was 56 (20-69); 22/55 patients were transplanted in remission. Stem cell sources were CD34-selected (36) or unmanipulated (14) peripheral blood, unmanipulated marrow (2), or cord blood (3). 40/55 allografts were HLA matched (20 related, 20 unrelated). Sequencing was performed on peripheral blood or marrow aspirates in patients with >20% blasts (AML) or >20% dysplastic cells (MDS). Adaptor ligated sequencing libraries were captured with two custom baitsets targeting 374 cancer-related genes and 24 genes often rearranged for DNA-seq, and 272 genes often rearranged for RNA-seq. Captured libraries were sequenced to high depth (Illumina HiSeq), averaging >590X for DNA and >20,000,000 total pairs for RNA. Statistics included cumulative incidence functions for relapse, Kaplan-Meier estimates for relapse-free survival (RFS), and outcome comparison with a permutation-based logrank test. Only mutations observed in at least 5 patients were analyzed. No adjustments were made for multiple comparisons. Median follow-up of survivors was 16.2 months (5.5-32.8). We detected genetic variants in each patient, suggesting the utility of this approach for identifying somatic mutations to track minimal residual disease (MRD) post-SCT. Six patients had known FLT3 mutations detected by a CLIA-certified test; all 6 of these mutations were identified by the sequencing platform. In addition, 3 FLT3 mutations and 1 FLT3 amplification were identified in patients who previously tested FLT3 negative. We identified 13 patients with IDH mutations (5 IDH1, 8 IDH2), eight with NPM1 mutations (all in AML), and 10 with DNMT3A mutations. We identified MAP kinase pathway mutations in 12 patients, including NRAS (7), KRAS (5), and NF1 (4), and we identified mutations in novel genes previously implicated in MDS/AML, including STAT4, CASP8, APC, and ALK. We next evaluated if specific mutations were associated with SCT outcome. Patients with IDH mutations (all of whom had normal karyotype) demonstrated significantly less relapse than patients with wild-type (WT) IDH (1 yr incidence: 0% vs 29%, p=.027, Fig 1). This translated into improved RFS (p=.037) in patients with IDH mutant AML (Fig 2). Treatment-related mortality (TRM) was similar with and without IDH mutations, suggesting the improved outcome was due to reduced relapse. For FLT3, 5/10 patients with FLT3 abnormalities relapsed. All 5 that relapsed were IDH WT. In contrast, IDH mutations were present in 4/5 FLT3+ AMLs that did not relapse, suggesting that IDH mutations may predict for improved SCT outcomes in patients with intermediate cytogenetic risk AML and in patients with FLT3 mutations. Mutant KRAS correlated with reduced overall survival in AML (p=.008), but the significance of this was unclear due to the absence of relapses and high TRM, including infection and GVHD, in this group. We also evaluated disease progression in 2 AML patients who relapsed post-transplant with archived samples collected pre-SCT and at relapse. In both patients we observed a distinct mutational profile pre and post-transplant consistent with clonal evolution. Of note, 1 patient gained a NF1 mutation post-SCT, while the other patient lost a NF-1 mutation, although when detected, both mutations were present at a frequency less than 10%. In summary, we performed mutational profiling in SCT patients using a novel high throughput platform, which allowed us to identify clinically relevant mutations, including some not detected by clinical laboratory testing. Notably, we found that IDH mutations may predict for favorable outcome after SCT, even in FLT3 mutant AML. These data suggest that mutational profiling can identify clinically relevant biomarkers pre-SCT and identify mutations for tracking MRD. Disclosures: Miller: Foundation Medicine, Inc: Employment. Lipson:Foundation Medicine, Inc: Employment. Stephens:Foundation Medicine, Inc: Employment. Otto:Foundation Medicine, Inc: Employment. Yelensky:Foundation Medicine, Inc: Employment. Nahas:Foundation Medicine, Inc: Employment. Wang:Foundation Medicine, Inc: Employment. Levine:Foundation Medicine, Inc: Consultancy.

Background A subset of patients with the Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs) transform to AML, and the prognosis of post-MPN AML patients is very poor. As such, new genomic insights are needed to define the mechanisms that govern transformation from MPN to AML, and to identify new therapeutic targets for clinical intervention in this poor-risk myeloid neoplasm. Importantly, patients with JAK2V617F mutant chronic-phase disease transform to JAK2 wildtype AMLs in approximately half of cases, indicating that diverse genomic paths lead to transformation. Aims To characterize the genomic alterations, including point mutations, short indels, translocations, and copy number alterations, in 33 post-MPN AML samples. Methods Genomic DNA and total RNA was isolated from formalin fixed paraffin embedded (FFPE) tissue, blood and bone marrow aspirates. Adaptor ligated sequencing libraries were captured by solution hybridization using two custom baitsets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 272 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging >590X for DNA and >20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. We achieved a mean coverage depth of 511x (range 405-645). Results The most common genomic alterations identified in post-MPN AML samples were mutations in JAK2 (51.5%), ASXL1 (48.5%), and IDH2 (30.3%). Mutually exclusive mutations in the genes of spliceosome components SRSF2, U2AF1, and SF3B1 were identified in 39% of patients in this cohort, suggesting that somatic mutations in splicing factors are a common genomic event in transformation from MPN to AML. These data were in contrast to de-novo AML, in which mutations in FLT3, NPM1, and DNMT3A are the most common disease alleles (NEJM 2013; 368: 2059), suggesting that the spectrum of genomic alteration in post-MPN AML differs from that in de novo AML. Within our cohort of post–MPN AML samples, 52% of the patients had JAK2V617F positive AML and 48% of our cohort presented with JAK2 wildtype AML. Notably, the two subsets of post-MPN AML had distinct mutational patterns. In the JAK2V617F mutant subgroup, we identified frequent mutations in ASXL1 (41.2%), TP53 (41.2%), and IDH2 (41.2%). In JAK2-wildtype AML subgroup we identified frequent ASXL1 mutations (56.3%) and NRAS point mutations (37.5%). NRAS mutations were exclusive of JAK2 mutations in the entire cohort, consistent with alternate disease alleles which activate signaling in JAK2V617F positive AML and in JAK2 wildtype AML. SETBP1 mutations were found in 19% of patients with JAK2 wildtype post-MPN AML but not in any patients with JAK2V617F-positive post-MPN AML. Several alterations not previously described in post-MPN AML, to the best of our knowledge, were identified in this cohort including MLL-PTD, which was observed in both JAK2V617F and wildtype JAK2 patients. Copy number analysis of our high-depth sequencing data allowed us to identify homozygous deletions of TET2 and ETV6 and MYC amplifications in post-MPN AML. Univariate analysis demonstrated TP53 mutations were associated with significantly impaired overall survival (p Conclusion The spectrum of genomic alterations in post-MPN AML is strikingly distinct from de novo AML. Furthermore, capture-based sequencing allowed us to show there are important differences in the mutational profile of JAK2V617F and JAK2 wildtype post-MPN AML. NRAS mutations may represent driver mutations in JAK2V617F negative post-MPN AML and further biologic evaluation of these mutations is needed. Most importantly, the identification of NRAS, IDH2, and MLL-PTD mutations in post-MPN AML suggest specific therapies including MAPK inhibition, IDH2 inhibition and dot1L/BRD4 inhibition may be of value in genotypically defined subsets of post-MPN AML. These finding help to further define the biology of leukemic transformation, as well as offer new therapeutic avenues for the treatment of post-MPN AML. Disclosures: Rampal: Foundation Medicine: Consultancy. Verstovsek:Incyte Corporation: Research Funding.

Xenotransplantation of primary AML samples into immunodeficient mice (PDX models) represents a unique opportunity for pre-clinical testing on a group of primary human samples that possess defined genetic lesions. However, given our recent recognition that multiple genetically distinct subclones can exist in AML, there is a risk that there may be selection for sub-clones from the xenotransplanted sample that might not fully represent the patient’s disease. We sought to establish a collection of genetically defined AML samples capable of engraftment in immunodeficient mice. We transplanted 30 AML patient samples; within 150 days (median 91 days) post transplantation 12 samples produced human CD45+ CD33+ CD19- CD3- engraftment in one or multiple NSG mice. Median patient sample amplification in 25 mice was 21 fold. Genomic DNA and total RNA was isolated from 7 AML patient samples (3 diagnostic samples from patients who remain in remission; 2 diagnostic samples from patients who later relapsed, 2 diagnostic samples from patients with refractory disease) and 14 matched xenotransplanted samples (2 mice per patient sample). Adaptor ligated sequencing libraries were captured by solution hybridization using two custom baitsets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 272 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging >499x for DNA and >20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. The mutations found in the 7 diagnostic samples were consistently identified in the 14 engrafted AML samples, but with some cases showing variation in allele frequency between diagnostic and engrafted samples. This finding shows that the human disease that engrafted in mice mimics the genetic makeup of the disease found in patients. We then assessed for allele frequency (AF) changes from diagnostic to xenografted sample as a measure of clonal progression. Clonal progression was defined as emergence of a clone carrying a novel genetic variant in the xenografted sample as compared to the diagnostic patient sample. Five patient samples (from 10 mice) did not show emergence of novel genetic lesions. In this group 2 patients had refractory disease and 3 patients remain in remission. Two patient samples (from 4 mice) demonstrated apparent emergence of novel genetic lesions not detected in diagnostic patient samples. Both of these patients have relapsed since the diagnostic samples were acquired. In the first case, both xenotransplanted mice engrafted with disease carrying NRAS N12S mutation (AF 0.05 and 0.09), which subsequent evaluation revealed to be present below the limit-of-detection (AF 0.004) in the clinical isolate obtained from patient presentation. We are currently conducting the same analysis on the relapsed sample from this patient. In the second case, both mice engrafted with disease carrying PTPN11 E76V (AF 0.03 and 0.0016) while the patient diagnostic sample did not contain any evidence of the alteration at 718x unique sequence coverage. Of note, one xenografted sample had an IDH1 R132C and another had IDH2 R140Q mutation, both of which have previously been shown to play a role in AML pathogenesis. Available AML cell lines do not carry IDH1/2 mutations, making it challenging to test IDH1/2 inhibitors in pre-clinical settings. These xenografted samples offer an opportunity to test such inhibitors. Overall we conclude that the xenotransplanted samples possess the diversity of genetic abnormalities found in diagnostic AML samples and thus can be used to assess efficacy of novel targeted therapies. We would like to further investigate a model in which the absence of clonal progression in xenografted samples would predict a better patient outcome, while emergence of novel clones might indicate an increased potential for relapse. We are currently expanding the study to include more diagnostic, xenotransplanted and relapsed samples to assess the associations between the ability of a sample to engraft in mice with clinical outcome and genetic/epigenetic lesions. Disclosures: Armstrong: Epizyme Inc.: Has consulted for Epizyme Inc. Other.

Background The Myeloproliferative Neoplasms (MPNs), including Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Primary Myelofibrosis (PMF) are clonal hematopoietic disorders. JAK2V617F mutations are observed in approximately 90-95% of PV cases, but only 40-50% of ET and PMF cases. Although JAK2 exon 12 and LNK mutations are observed in the majority of JAK2V617F-negative PV patients, candidate gene and exome sequencing studies to date have not identified activating oncogenes in the majority of JAK2V617F-negative ET/PMF patients. Thus, further genetic investigations are needed to define the mutational architecture of these JAK2 wildtype MPNs in order to gain insight into the biology of these diseases, the clinical implications of genetic events that do occur, and the elucidation of potential therapeutic targets. Aims To characterize the spectrum of genetic alterations in JAK2 wildtype chronic-phase myeloproliferative neoplasms. Methods We identified 32 patients with a confirmed diagnosis of an MPN (per 2008 WHO criteria), including MF, PV and ET, who were negative for JAK2V617F using a CLIA-certified allele specific assay for the JAK2 disease allele. Genomic DNA and total RNA was isolated from formalin fixed paraffin embedded (FFPE) tissue, blood and bone marrow aspirates. Adaptor ligated sequencing libraries were captured by solution hybridization using two custom baitsets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 258 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging >590X for DNA and >20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. Results High coverage sequencing allowed us to identify JAK2V617F mutations in two patients (allele burden 3-5%) that were below the limit of detection of the CLIA assay. The most common mutations observed in JAK2V617F-negative MPN were in ASXL1 (22% of patients) and in TET2 (9%). Taken together, mutations in known epigenetic modifiers (ASXL1, TET2, DNMT3A, EZH2, MLL) were observed in 43% of samples, including a MLL-PTD mutation in one patient with PMF. We identified mutations in spliceosome components (SRSF2, U2AF1), in a subset of patients, consistent with previous reports, and in each case mutations in spliceosome components were mutually exclusive. We identified mutations in the JAK-STAT pathway (MPL, TYK2) and the RAS pathway components (KRAS, NF1) in 9% of this patient cohort, suggesting that there are alternate disease alleles that activate signaling in JAK2V617F-negative MPN. RNA-sequencing identified a ETV6-ABL1 fusion in one patient, and we identified amplification of PIK3CA in one patient in our cohort; these data suggest fusion genes and amplifications activate signaling in a subset of patients with JAK2V617F-negative MPN. We also identified novel mutations in MPN patients which have not been reported to date, including mutations in DNA repair genes (ATM and BRCA) in 25% of cases and mutations in the Notch signaling pathway (NOTCH1-4) in 31% of cases. The functional implications of these novel mutations remain to be elucidated. In univariate analysis, ASXL1 mutations were found to associate with impaired overall survival (Figure 1, p=0.049). These findings are consistent with data demonstrating an impaired survival in patients with MDS and PMF, and suggest that ASXL1 mutations represent an important biomarker for adverse outcome in JAK2V617F-negative MPN. Conclusions These data demonstrate that the mutational spectra of JAK2V617F-negative MPN includes genes implicated in epigenetic regulation, novel mutations which activate gene signaling, and fusion genes/copy number alterations which provide a novel mechanism of oncogenic activation not previously reported in MPN. ASXL1 mutations occur frequently in JAK2 wildtype Philadelphia-Chromosome negative MPNs, and are associated with impaired overall survival. Collectively, these findings support the importance of ASXL1 mutations in predicting outcome in JAK2V617F-negative MPN, demonstrate that mutations in signaling effectors and in epigenetic regulators are common in MPN, and illustrate the genetic heterogeneity of JAK2V617F-negative MPN. Disclosures: Rampal: Foundation Medicine: Consultancy. Levine:Foundation Medicine, Inc: Consultancy.

Background Rapid advancements in cancer genomics and in the development of targeted therapies provide expanding opportunities to use genomic profiling to improve patient outcomes. However, most patients do not have access to clinical genomic profiling platforms, and currently available assays capture a small set of known mutations or translocations tailored to specific tumor types. The spectrum of somatic alterations in leukemia, lymphoma, and myeloma includes substitutions, insertions/deletions (indels), copy number alterations (CNAs) and gene fusions; no current assay captures the different types of alterations in a single clinical genomic test. We developed a novel, CLIA-certified next-generation sequencing-based assay designed to provide targeted assessment of the genomic landscape of hematologic malignancies, including identification of all classes of genomic alterations using archived FFPE, blood and bone marrow aspirate samples with high accuracy in a clinically relevant timeframe. Methods DNA and RNA were successfully extracted from 350/362 (96%) specimens from 319 patients, including 57 FFPE samples, 150 blood samples and 142 bone marrow aspirates. The initial sample cohort included 20 ALL, 83 AML, 53 CLL, 57 DLBCL, 48 MDS, 32 MPN and 57 multiple myeloma samples. Adaptor ligated sequencing libraries were captured by solution hybridization using a custom bait-set targeting 374 cancer-related genes and 24 frequently rearranged genes by DNA-seq, and 258 frequently-rearranged genes by RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq) in a CLIA-certified laboratory (Foundation Medicine), averaging 590x for DNA and >20M total pairs for RNA, to enable the sensitive and specific detection of substitutions, indels, CNAs and gene fusions. Results Sufficient tumor content (≥20%) was present in 317/350 (91%) of the samples (289/319 patients), and a total of 885 alterations were identified (3.1 alterations per sample), including 555 base substitutions, 213 indels, 36 splice mutations, 51 CNAs and 36 fusions/rearrangements. The most frequent alterations across all hematologic malignancies included mutations in TP53 (9%), ASXL1, KRAS, NRAS, IDH2, TET2, SF3B1, JAK2, MLL2, DNMT3A, RUNX1, and SRSF2 (2-5% each); FLT3 ITDs (2%); MLL PTDs (1%); homozygous loss of CDKN2A/B (3%); and focal amplification of REL (1%). Rearrangements in BCL2/6, MYC, MLL, MLL2, NOTCH2, ABL1 and ETV6 were identified using DNA and RNA targeted sequencing, demonstrating the ability of this platform to reliably identify gene fusions with immediate clinical relevance. Overall high accuracy of the assay for substitutions, indels and CNAs was previously demonstrated by extensive validation studies achieving 95-99% across alteration types with high specificity (PPV>99%) [Frampton et al, Nat Biotech, in press]. Comparison of detected alterations to previous molecular testing for JAK2, NPM1, IDH2, FLT3 and CEBPA in MPN/AML samples demonstrated 97% sensitivity (33/34) in our ability to identify known mutations in these clinical samples. We identified additional clinically relevant mutations that were not detected using standard clinical assays, including alterations in JAK2, FLT3 and IDH2, which can inform therapeutic decisions. The use of our content rich sequencing platform allowed us to identify clinically actionable mutations in hematologic malignancies, including IDH1/2 mutations in a spectrum of myeloid/lymphoid malignancies, recurrent BRAF mutations in refractory CLL and myeloma, and mutations in the JAK-STAT signaling pathway in diffuse-large B cell lymphoma. These results demonstrate that a targeted sequencing platform which includes a large set of known disease alleles/therapeutic targets can identify mutations with therapeutic relevance in disease contexts where gene-specific assays are not currently performed in the clinical setting. Conclusions We have developed a sensitive, high throughput assay to detect somatic alterations in hundreds of genes known to be deregulated in hematologic malignancies, which can be used for clinical sequencing of frozen/paraffin samples. We demonstrate that targeted DNA and RNA sequencing can be used to identify all classes of genomic alterations in genes known to be therapeutic targets in a broad spectrum of hematologic malignancies. Disclosures: Lipson: Foundation Medicine, Inc: Employment, Equity Ownership. Nahas:Foundation Medicine, Inc: Employment, Equity Ownership. Otto:Foundation Medicine, Inc: Employment, Equity Ownership. Yelensky:Foundation Medicine, Inc: Employment, Equity Ownership. Wang:Foundation Medicine, Inc: Employment, Equity Ownership. He:Foundation Medicine, Inc: Employment, Equity Ownership. Rampal:Foundation Medicine: Consultancy. Brennan:Foundation Medicine, Inc: Employment, Equity Ownership. Brennan:Foundation Medicine, Inc: Employment, Equity Ownership. Young:Foundation Medicine, Inc: Employment, Equity Ownership. Donahue:Foundation Medicine, Inc: Employment, Equity Ownership. Sanford:Foundation Medicine, Inc: Employment, Equity Ownership. Greenbowe:Foundation Medicine, Inc: Employment, Equity Ownership. Frampton:Foundation Medicine, Inc: Employment, Equity Ownership. Fichtenholtz:Foundation Medicine, Inc: Employment, Equity Ownership. Young:Foundation Medicine, Inc: Employment, Equity Ownership. Erlich:Foundation Medicine, Inc: Employment, Equity Ownership. Parker:Foundation Medicine, Inc: Employment, Equity Ownership. Ross:Foundation Medicine, Inc: Employment, Equity Ownership. Stephens:Foundation Medicine, Inc: Employment, Equity Ownership. Miller:Foundation Medicine, Inc: Employment, Equity Ownership. Levine:Foundation Medicine, Inc: Consultancy.

Whole genome and exome sequencing studies have identified numerous genomic alterations in DLBCL, but these methods have limited applicability for the clinical care of lymphoma patients due to cost, specific tissue requirements, and laborious bioinformatic analysis. FoundationOne-Heme (FOH) is a novel next-generation sequencing platform designed to provide targeted assessment of the genomic landscape of hematologic malignancies, including identification of mutations within specific genes, copy number changes, and translocations. FOH can be performed on small quantities of formalin-fixed paraffin-embedded (FFPE) tissue, detect rare variants due to extensive depth of sequencing coverage, and rapidly provide results via streamlined bioinformatic interpretation. Here we report the first experience using this novel platform to evaluate the genetic landscape of DLBCL. Genomic DNA and total RNA were isolated from FFPE tissue on a cohort of 53 cases of DLBCL, including de novo (n=30), relapsed/refractory (n=12), and large cell transformation from low-grade lymphoma (n=11). The cohort included 25 cases with combined MYC and BCL2 overexpression by IHC (criteria for positivity: >40% MYC, >70% BCL2), of which only one had a known translocation involving MYC. Adaptor ligated sequencing libraries were captured by solution hybridization using two custom bait sets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 258 frequently-rearranged genes for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging >658x for DNA and >20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. Significant non-synonomous variants were identified as mutations from the COSMIC database, amplifications of established oncogenes, or homozygous deletions and/or clear loss-of-function mutations of known tumor suppressors. The DNA sequencing component of FOH detected translocations in BCL2, BCL6, and MYC, while the RNA sequencing component detected fusion transcripts involving BLC6 and MYC, in agreement with independent cytogenetic analysis via karyotype and FISH where available. The assay detected copy number alterations of 44 different genes, most commonly amplification of REL (15%) or loss of CDKN2A/CDKN2B (17%). The most frequent alterations of known significance are detailed in Figure 1. The most commonly altered gene was CDKN2A, exhibiting either homozygous deletion or loss of function mutation in 28% of cases. Chromatin modifying factors (e.g. MLL2, CREBBP, EZH2) represented the most frequently altered biologic category with alterations occurring in >50% of cases. Recurrent alterations in components of the Notch pathway (NOTCH1/2/4, FBXW7, SPEN), each predicted to activate the pathway, were identified in 23% of cases. Cell-of-origin was determined as per the Hans model using IHC for CD10, BCL6, and IRF4/MUM1; CD79B mutations were detected exclusively in non-GCB and EZH2 mutations were found exclusively in GCB-phenotype cases. Furthermore, IHC MYC+/BCL2+ de novo DLBCL cases (n=11) exhibited more frequent hypermutation of PIM1 (46%) compared with the 19 cases of IHC MYC-/BCL2- de novo DLBCL (11%). When comparing the various clinical categories, we found that mutations in tumor suppressors were significantly more common in relapsed/refractory than de novo DLBCL (47% vs 75%, p=0.02). Alterations in TP53 were most frequently observed in transformed lymphoma (55%). Our results demonstrate the feasibility of using a targeted next-generation sequencing platform on FFPE clinical specimens from patients with DLBCL as a means of providing an integrated analysis of gene mutations, copy number alterations, and translocations. This streamlined approach combines multiple molecular and cytogenetic tests into a single platform and uses a small amount of tissue to perform a multifaceted assessment of genomic alterations with potential diagnostic, prognostic, and therapeutic implications. Future efforts will be directed at analyzing additional cases of DLBCL to better establish the biologic and clinical significance of the observed genetic alterations, and to prospectively incorporate this novel platform to select patients for mechanism-based targeted therapy. Disclosures: Intlekofer: Foundation Medicine, Inc: Consultancy. Levine:Foundation Medicine, Inc: Consultancy. Zelenetz:Foundation Medicine, Inc: Consultancy. Palomba:Foundation Medicine, Inc: Consultancy. van den Brink:Foundation Medicine, Inc: Consultancy. Brennan:Foundation Medicine, Inc: Employment. Young:Foundation Medicine, Inc: Employment. He:Foundation Medicine, Inc: Employment. Nahas:Foundation Medicine, Inc: Employment. Yelensky:Foundation Medicine, Inc: Employment. Otto:Foundation Medicine, Inc: Employment. Lipson:Foundation Medicine, Inc: Employment. Stephens:Foundation Medicine, Inc: Employment. Miller:Foundation Medicine, Inc: Employment. Younes:Foundation Medicine, Inc: Consultancy.

Abstract 2482 Despite the advent of prognostic factors such as cytogenetics and IGH mutational status, the natural history of chronic lymphocytic leukemia (CLL) remains heterogenous and difficult to predict in individual patients. We used the carbocyanine dye, JC-1, to investigate mitochondrial membrane potential in patients with untreated, predominantly early stage CLL. We found that JC-1 staining separated CD5+/CD19+ CLL cells into two discrete populations, Low Red (R)/Green (G) and High R/G, which mainly differed with regard to JC-1 green fluorescence (530 nm) in all patients (Fig. 1). In evaluating CLL patients, we found substantial variation in the proportion of cells in the two populations, with a range of 1.1% to 96.7% (median=79.9%) in the High R/G population. CD19+ B-cells from normal individuals typically had few cells with high JC-1 red fluorescence and had a JC-1 staining pattern that was distinct from CLL patients. Electron microscopy revealed disorganized mitochondrial christae, particularly in patients with elevated High R/G cell populations, compared with mononuclear cells from normal donors. Consistent with the morphologic disorganization, we found that the two populations of CLL cells had differential survival in culture with pyruvate and glucose as primary energy sources; cells in the High R/G population had inferior survival (p=0.016), while cells in the Low R/G population had equal survival in pyruvate compared with glucose (Fig. 2). This result indicates that the CLL clonal cell population can be subdivided into cells able to use oxidative phosphorylation and those dependent on aerobic glycolysis fro metabolic energy needs. Interestingly, patient samples with High R/G cell populations >90% had better cell survival in glucose-containing medium than samples derived from patients with < 75% High R/G cells (p=0.0085). We found that the mononuclear cells from patients with CLL expressed the M2 isoform of pyruvate kinase, which has been linked to the regulated utilization of aerobic glycolysis. We assessed the outcome of 100 patients with untreated CLL with more than 1 month of follow up (median 36 months) after measuring the proportion of cells in the mononuclear fraction of the peripheral blood with in the High R/G population. The clinical characteristics of the patients at the time of JC-1 staining are characteristic of patients with early stage, untreated CLL. The population was divided into approximate quartiles based on the percentage of mononuclear cells in the High R/G population, and progression-free survival (PFS) was estimated within each quartile. Patients with 50% or fewer cells in the High R/G gate appeared to have an indolent natural history; an estimated 88% (95% CI: 73%, 99%) of the patients had no evidence of significant clinical progression at two years. In contrast, patients with more than 90% of cells in the gate with High R/G cells had rapid progression with an estimated PFS of 50% (95% CI: 31%–82%) at two years (Fig. 3). Patients with 50–90% cells in the High R/G gate had an intermediate disease course. There was strong evidence of a difference in survival distributions across the four JC-1 categories (log-rank p-value < 0.001). The percentage of cells in the High R/G gate provided prognostic information that was independent of good risk or poor risk cytogenetic changes. In summary, we found that the clonal, neoplastic cells in patients with CLL comprise discrete populations separated on their metabolic dependence on aerobic glycolysis and aerobic glycolysis. We also show that the percentage of cells utilizing glycolysis has a significant adverse impact on the natural history of patients with CLL. Aerobic glycolysis, the so-called Warburg effect, is a common and incompletely understood aspect of cancer biology. These data tie the acquisition of aerobic glycolysis to more aggressive cancer biology, identify an important new prognostic measure for CLL and could lead to the development of therapies that target metabolic differences in cancer cells. Disclosures: No relevant conflicts of interest to declare.

Abstract 1992 The myeloproliferative neoplasms (MPN) can have a variable natural history. Polycythemia vera and essential thrombocythemia, in particular, are conditions that can extend over decades, but some patients have clinical progression to myelofibrosis or acute myeloid leukemia. As first articulated by Warburg, cancers are metabolically distinguished from normal tissues by the use of glycolysis under aerobic conditions. To metabolically characterize the blood cells of patients with myeloproliferative neoplasms, we measured the mitochondrial membrane potential using the cyanine dye, JC-1. In examining cells derived from the blood and/or marrow of 159 patients with primary myelofibrosis, polycythemia vera and essential thrombocythemia, we found that the mitochondrial membrane potential (FL2/FL1=electrochemical potential/mitochondrial mass) was elevated compared to the blood cells of normal individuals. Thirty five percent of patients with polycythemia vera and essential thrombocythemia had normal MMP. In contrast, 97% of patients with primary myelofibrosis, post-polycythemia myelofibrosis, post-essential thrombocythemia myelofibrosis and acute myeloid leukemia following an MPN had evidence of cell populations with higher mitochondrial membrane potential. Cells with distinctly higher mitochondrial membrane potential could be indentified in platelets and polymorphonuclear leukocytes; however the MMP of lymphocytes was normal, indicating that the alteration in metabolic state likely occurred in a multipotential myeloid stem cell. Cell populations were confirmed by co-staining with anti-CD19, -CD45, -GlycophorinA and -β3-integrin antibodies. Sequential analysis of patient samples found that the acquisition of higher mitochondrial membrane potential was stable and persistent over 2 years or more of follow up and that elevated membrane potential predisposed patients to disease progression. The balance of patients (65%) with ET had evidence of increased MMP suggesting the possibility of disease in an early state of evolution to a more aggressive condition. The increased MMP did not correlate with the presence of mutation in JAK2. These results indicate that clinically advanced MPN can be characterized by changes in mitochondrial physiology that might be identified non-invasively by flow cytometric staining with JC-1. In addition, the early nature of these changes may help to identify therapeutic targets. Disclosures: No relevant conflicts of interest to declare.