Melnick, Ari

Abstract Studying gene expression and clinical outcome data from 136 clinical trials for patients with cancer (~21,000 patients with 26 cancer types), we found CD25 as one of the strongest predictors of poor clinical outcome in patients with B-cell malignancies, but not in other cancer types. This was unexpected because CD25 is known as one of three chains of the IL2 receptor on T-cells and NK-cells. Interleukin-2 (IL2) functions as essential T-cell growth factor. IL2 signals through β- and γ-, but not α-chains (CD25) of its heterotrimeric receptor. CD25-deficiency causes lymphoproliferation and autoimmunity, however, its mechanistic role is unclear. Our experiments based on genetic mouse models and engineered patient-derived B-cell leukemia and lymphoma xenografts revealed that CD25 expressed on B-cells is not an IL2 receptor chain, but in fact binds the B-cell receptor (BCR) to regulate its activity. Suggesting IL2-independent functions, defects in CD25-/-B-cells were not replicated in IL2-deficient mice. CD25 bound the BCR but not IL2Rβ- and IL2Rγ-chains. IL2Rβ- and IL2Rγ-chains can pair with other chains to form receptors for different cytokine-ligands. However, CD25 represents the first example of a cytokine receptor chain that binds to the BCR for negative feedback regulation. Likewise, in T-cells, CD25 had a bifunctional role and either functioned as IL2 receptor chain or as negative feedback regulator of T-cell receptor signaling. CD25-function was regulated by cell-membrane translocation, which required phosphorylation of its cytoplasmic tail at S268 (see schematic, left). In a family with monogenic autoimmunity, a mutation immediately preceding S268 compromised CD25-surface translocation, which was restored by homology-directed repair of the S268 motif. CD25-interactome analyses identified PKCd as critical effector molecule downstream of CD25 to mediate B-cell selection during normal B-cell development and calibrate oncogenic BCR signaling in B-cell tumors. In B-cell malignancies, BCR-dependent survival and proliferation signals are often substituted by oncogenic BCR-mimics (e.g. BCR-ABL1, JAK2, BRAFV600E, LMP2A, CD79B mutations; see schematic, right). Accordingly, we identified CD25 surface-expression as biomarker of oncogenic BCR-signaling and predictor of poor clinical outcomes. CD25-/-B-cell leukemia failed to initiate fatal disease in transplant recipients. Owing to imbalances of oncogenic BCR-signaling and p53-checkpoint activation, CD25-/- B-cell leukemia failed to initiate fatal disease in transplant recipients. In patient-derived xenograft models of drug-resistant B-cell malignancies, treatment with a CD25-specific antibody drug-conjugate (ADCT-301) extended survival of transplant recipients or eradicated disease. These findings identified CD25 as previously unrecognized feedback regulator of oncogenic BCR-signaling and provide a rationale for therapeutic targeting of CD25 in refractory B-cell malignancies. Figure. Figure. Disclosures Forman: Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding. Weinstock:Genentech/Roche, Monsanto: Consultancy; Novartis: Consultancy, Research Funding; Novartis, Astra Zeneca, Abbvie, Aileron, Surface Oncology, Daiichi Sankyo: Research Funding; Novartis, Dragonfly, Travera, DxTerity, Travera: Consultancy; Travera: Equity Ownership; Astra Zeneca, JAX, Samumed, Regeneron, Sun Pharma, Prescient: Patents & Royalties. Uzel:Novartis: Research Funding.

Abstract Abstract 87 Background: The International Prognostic Index (IPI) remains the most powerful predictor of clinical outcome in DLBCL in the R-CHOP era, serving as a useful surrogate for the biology we are just beginning to understand. With a follow-up of more than 9 years, its uniformly staged and treated patients and its prospectively collected unstained slides for correlative studies, the clinical data set from E4494, the US Intergroup trial comparing CHOP and RCHOP in patients >60 years with DLBCL, stands as a valuable resource for investigating prognosis in DLBCL. Based on immunohistochemistry, we previously showed that rituximab modulates the prognostic significance of some biomarkers in DLBCL. With these same specimens, the quantitative nuclease protection assay (qNPA), a methodology for measuring mRNA levels in FFPET, was used to develop separate prognostic signatures for the CHOP and RCHOP arms that can be simply applied using stored unstained slides. Methods: Five micron unstained FFPET sections from 183 eligible and evaluable cases enrolled on E4494 and submitted for prospective immunohistochemical correlative studies more than ten years ago were used for this analysis. Tissue was scraped from slides and a multiplexed qNPA was performed in triplicate using a customized Array Plate assay (HTG, Inc) for 43 genes of interest. TBP served as a housekeeping gene. Association between standardized log gene expression and patient failure-free survival (FFS) and overall survival (OS) was obtained using the Cox proportional hazards model. A weighted analysis was used to eliminate the confounding effect of maintenance rituximab. Genes that showed at least marginal significance in the univariate analysis were used to perform LASSO (penalized method to select best subset) to select a final list of genes in the multivariate analysis. Using the predictive model for either CHOP or R-CHOP induction, individual risk scores were calculated based on the multivariable model, and cases were dichotomized into low and high risk groups based on the median risk score. The model was then validated using the Lenz dataset (NEJM, 2008). Results: In six cases, tissue from slides prepared >10 years ago was compared to freshly cut sections from corresponding blocks, and showed excellent concordance (corr=0.86). On-study characteristics for the 176 cases with analyzable data were representative of the greater E4494 patient population. Six gene predictors were developed for each arm of the trial: RCHOP: FN1, LMO2, AKT1, HIF1a, AKT3, BCL2; and CHOP: PDCD4, HLADRB1, COL3A1, LMO2, ROBO4, TP53. Both signatures proved powerful predictors of FFS and OS among CHOP (FFS: p=0.0031; OS: p=0.0013) and RCHOP (FFS: p=0.001; OS=p=0.0015) treated patients. When adjusted for the clinically-based IPI, the gene-risk score retained its significance (CHOP: FFS p=0.0007; OS p=0.0011; RCHOP: FFS p=0.003; OS, p=0.001) while the IPI became only marginally significant (CHOP: FFS: p=0.06, OS p=0.06; R-CHOP: FFS p=0.09, OS p=0.02), suggesting that the gene predictor accounted for much of the predictive power of the IPI. The predictive model was then validated using the Lenz dataset for OS. The predictor models for CHOP and R-CHOP-treated patients effectively dichotomized patients into prognostic subgroups (CHOP: p<0.0001; RCHOP: p=0.0014; see figure below) and this difference was maintained when the subset over age 60 was analyzed (CHOP: p=0.0008; RCHOP: p=0.017). When adjusted for the IPI, the molecular predictor developed for CHOP treated patients remained robust (OS: P=.0001; HR 2.39), while the molecular predictor for RCHOP predicted OS marginally (p=.06; HR 1.82), with shorter followup than the CHOP cohort. Conclusions: Unstained slides from FFPET stored for many years may be used to investigate gene expression in lymphoma biopsy specimens for which there is mature followup. Gene risk scores based on the expression of a limited number of genes are powerful predictors of clinical outcome. Disclosures: Pollock: HTG, Inc.: Employment, Equity Ownership. Botros:HTG, Inc.: Employment, Equity Ownership. Horning:Genentech: Employment, Equity Ownership.

Abstract Chronic idiopathic myelofibrosis (MF) is a clonal hematopoietic disorder that leads to progressive marrow fibrosis and peripheral cytopenias. Very little is known about the role of aberrant DNA methylation in the pathobiology of this disease. Whole genome methylation was analyzed by a recently described novel method, the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation specific restriction digestion by HpaII and MspI followed by amplification, two color labeling and cohybridization to quantitatively determine individual promoter methylation. A whole genome human promoter array (Nimblegen) was used to determine the level of methylation of 25626 gene promoters by calculating HpaII/MspI cut fragment intensity ratio. Peripheral blood leucocytes from 9 patients with IMF were compared to 9 age-matched normal and anemic controls. Gene expression analysis was performed using 37K oligo maskless arrays using cDNA from the same samples. Methylation analysis showed that myelofibrosis samples clustered separately from normal and anemic controls when grouped by unsupervised clustering based on Pearson’s correlation coefficient. On the other hand, global gene expression demonstrated no clear cut differences between myelofibrosis and control samples suggesting that methylation profiling has greater discriminatory power when compared to conventional gene expression profiling. A high correlation (r=0.88–0.96) was observed between whole genome methylation profiles of matched sets of bone marrow and peripheral blood leucocyte samples from selected patients demonstrating that peripheral blood leucocytes can act as a valid surrogates for epigenomic analysis. Further analysis showed that genes aberrantly methylated in all myelofibrosis samples included v-myc, histone 2A, TNF, TNF Receptor1, FGF14 and others. Functional pathway analysis by Ingenuity showed that pathways involved in Inflammation and Cell signaling were the most affected by epigenetic silencing. Most interestingly, a large proportion of gene promoters were also aberrantly hypomethylated. These included genes for chemokine CXCL13, APC, IL-3, STAT2 and others. The pathways most activated by hypomethylation were involved in hematopoiesis and cell growth and proliferation demonstrating the biological validity of our analysis. Thus, our data demonstrates that myelofibrosis is characterized by distinct epigenetic aberrations that are preserved in peripheral blood leucocytes. These can be the basis of future studies on pathogenesis and diagnosis for this disease and lead to translational studies with agents targeting DNA methylation.

Abstract Abstract 852 Adult acute lymphoblastic leukemia (ALL) is an aggressive disease with <30% long-term survival. This relatively poor outcome can be explained in part by an increased frequency of high-risk molecular subtypes compared to childhood ALL, such as BCR/ABL (20%-40% in adults vs 2%-5% in children). We hypothesized that aberrant epigenetic gene regulation contributes to the pathogenesis and clinical features of adult ALL. We therefore performed genome-wide DNA methylation and gene expression microarray studies of 215 adult patients with B-lineage ALL enrolled in the ECOG E2993 phase III trial. Patients had a median follow-up 4.75 years (3.5 months to 13 years) and median age 39 years (17 to 63 years). BCR/ABL(+) cases (n=83) had a worse overall survival (OS, p=0.08) than BCR/ABL(-) cases (n=132). The smaller difference in survival in this series between BCR/ABL(+) and (-) cases is likely due to the use of imatinib in some of these patients. The HELP microarray assay was used to measure DNA methylation at 50,000 CpGs annotated to ∼22,000 RefSeq promoters. The accuracy of HELP was confirmed by extensive quantitative single locus validation studies. Supervised analysis revealed the presence of a markedly aberrant DNA methylation signature (166 genes) in BCR/ABL(+) ALL with the cutoff values of p<0.001 (FDR<0.002) and log fold change>1, and a differential gene expression profile of 416 genes at p<0.001 (FDR<0.004) and log fold change>1. Integrative analysis of expression and DNA methylation indicated that many of these genes are functionally connected within a gene network centered around IL8, which was over expressed and hypomethylated in BCR/ABL(+) cases, along with IL2RA(CD25), CEBPB, ABL1, ID1, IL15, BCL2L13, CD69, NOV, S100A8 and S100A9. KEGG and BioCarta pathway analysis also showed enrichment for IL8 signaling, NF-kB Activation, B Cell Development and Antigen Presentation pathways, suggesting these cytokine networks might play central and distinct roles in BCR/ABL(+) ALL. To identify a core set of functionally relevant genes, we explored the overlap of the DNA methylation and gene expression signatures. The overlap consisted of 13 genes, among which 11 showed inverse correlation, including CD200, GAB1, HLA-DQA1, HLA-DQB1, IL2RA (CD25), LST1, LTB, NOV, ROB04, S100A9, and CD38. Consistent with previous ECOG findings, CD25 positivity was a more dominant predictor than BCR/ABL, and could further stratify BCR/ABL(+) patients into a favorable (CD25-) and a poor (CD25+) outcome group (OS, p=0.07). When comparing CD25(+) and CD25(-) groups among BCR/ABL(+) cases, we found RhoH and UBE2J1 as the top differentially methylated and IL2RA(CD25) as the top differentially expressed genes, and all three genes showed concordant corresponding changes in expression and methylation. The aberrant DNA methylation signature of MLL/AF4 cases was even more dramatic, with 469 identified as differentially methylated (p<0.001 (FDR<0.015), log fold change>1) and 1108 genes differentially expressed (p<0.001 (FDR<0.009), log fold change>1). Integrated analysis of DNA methylation and expression implicated gene pathways centered around TNFA and MYC, respectively. A core set of 44 genes were identified overlapping between the methylation and gene expression signatures, among which 34 showed inverse correlation, including ANXA5, BRE, CAPG, CEBPA, FAIM, FLT3, FUT4, IGFBP7, IL1R2, ITGA7, ITGAE, MAP1A, MAP7, MRPL33, PARP8, RBKS, SLITRK4 and TFR2 with overexpression and hypomethylation, and BTBD3, CCR6, FYN, GAB1, GYPC, HPS4, IL2RA, KCNK3, LCK, LST1, LTB, PRKCH, QPCT, S100A13, SGPL1 and ZAP70 with underexpression and hypermethylation in MLL/AF4(+) ALL. All signatures were independent of B-ALL differentiation stage. Using univariate Cox Hazard Regression model adjusted by age, WBC, CD25 and BCR/ABL status, we furthermore identified 259 expression and 115 methylation markers which were significantly correlated with patient OS risk (p<0.01). Molecularly or immunophenotypically defined poor prognosis adult B- ALLs thus feature specific aberrant DNA methylation profiles with associated inversely correlated gene expression. These gene sets delineate specific biological functions that may contribute to disease phenotype and offer an opportunity for development of targeted therapy. Aberrantly methylated genes in adult B-ALL correlate with clinical risk independent of other clinical or molecular risk factors. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 851 Clinical studies have demonstrated that acute myeloid leukemia (AML) is heterogeneous with respect to presentation and to clinical outcome, and many studies have shown that cytogenetics can be used to improve prognostication. More recently, it has been shown that FLT3, NPM1, and CEBPA mutations can be used to further risk stratify AML patients. Although recent studies have identified novel, recurrent somatic mutations in AML in genes including TET2, ASXL1, IDH1, IDH2, and PHF6, their prognostic value has not yet been evaluated among patients treated on a large phase III clinical trial. Here we report full-length DNA resequencing of FLT3, NPM1, CEBPA, H/K/NRAS, KIT, WT1, TET2, ASXL1, IDH1/2, TP53, RUNX1, PTEN, and PHF6 in pre-treatment genomic DNA from 398 patients with de novo AML younger than 60 years of age enrolled in the ECOG E1900 protocol. We could identify a clonal alteration, defined as a somatic mutation or cytogenetic abnormality, in 91.2% of all patients; 42% had 1 somatic alteration, 36.4% had 2 alterations, 11.3% had 3 alterations and 1.5% had 4 or more somatic alterations. Mutational Analysis: We identified somatic mutations in FLT3 (37% total; 30% ITD, 7% TKD), NPM1 (14%), CEBPA (10%), TET2 (10%), NRAS (10%), WT1 (10%), KIT (9%), IDH2 (8%), IDH1 (6%), RUNX1 (6%), ASXL1 (4%), PHF6 (3%), KRAS (2.5%), TP53 (2%), and PTEN (1.5%) (Figure 1A). We then performed correlation analysis to determine the frequency of different mutational combinations in AML patients. In addition to previously identified mutational correlations (FLT3 and NPM1, KIT and core binding factor leukemia), we found that FLT3 and ASXL1 mutations were mutually exclusive in this large cohort (p =0.0008). In addition, we found that IDH1/IDH2 mutations were mutually exclusive of both TET2 (p=0.02), and WT1 (p=0.01) mutations consistent with overlapping roles in AML pathogenesis (Figure 1B). Integration of mutational data with response data and outcome: Mutations in ASXL1 (CR rate 33.3% versus 66.5% for ASXL1-WT patients; p= 0.009) were enriched in patients who did not respond to induction chemotherapy. We found that mutations in FLT3 (p=0.0005), ASXL1 (p=0.005; Figure 1C), and PHF6 (p=0.02) were associated with reduced OS. Notably, the adverse effects of ASXL1 and PHF6 mutations on outcome were restricted to patients treated with standard dose daunorubicin (p<0.01 for each mutational cohort); ASXL1 and PHF6 mutational status did not affect outcome in patients treated with high-dose daunorubicin, demonstrating that increased intensity induction chemotherapy can improve outcomes in genetically defined poor risk AML subsets. We also found that mutations in CEBPA (p= 0.04) and in IDH2 (p=0.003) were associated with improved OS; the favorable impact of IDH2 mutations on outcome was exclusive to patients with IDH2 R140Q mutations (Figure 1D). Within the subset of patients with cytogenetically defined intermediate risk AML, we found, as expected, that FLT3-ITD mutations were associated with reduced OS and CEBPA mutations were associated with improved OS. Importantly, we found that IDH2 R140Q mutations were associated with improved OS (p=0.003) in patients with intermediate risk AML regardless of FLT3 mutational status. In contrast, TET2 mutations (P=0.0002) and PHF6 (p<0.0001) were associated with reduced OS in intermediate risk, FLT3-WT, AML. Summary: These data provide important clinical implications of genetic alterations in AML and provides insight into the multistep pathogenesis of adult AML. We identified novel mutations that impact response to induction chemotherapy and predict overall outcome, including ASXL1, PHF6, TET2 and IDH2, suggesting these mutations can be used to improve AML risk stratification and therapeutic decisions in AML patients 18–60 years of age. Most importantly, we identify TET2, IDH1/2, and WT1 mutations as mutually exclusive somatic alterations that form a novel mutational complementation group that contributes to AML pathogenesis. Figure 1. A) Circos diagram depicting relative frequency and pairwise co-occurrence of mutations in de novo AML patients enrolled in the ECOG protocol E1900. B) Circos diagram showing mutational events in IDH1/2, TET2, and WT1 mutant AML patient. C) Overall survival in AML patients with and without ASXL1 mutations (p=0.005). D) Overall survival in AML patients with and without IDH2 R172 and R140 mutations (p=0.003). Figure 1. A) Circos diagram depicting relative frequency and pairwise co-occurrence of mutations in de novo AML patients enrolled in the ECOG protocol E1900. B) Circos diagram showing mutational events in IDH1/2, TET2, and WT1 mutant AML patient. C) Overall survival in AML patients with and without ASXL1 mutations (p=0.005). D) Overall survival in AML patients with and without IDH2 R172 and R140 mutations (p=0.003). Disclosures: No relevant conflicts of interest to declare.

Abstract Background: Induction therapy with daunorubicin (Dauno) & cytarabine (Ara-C) [DA] has been the standard of care for eligible older adults (age ≥ 60 years) with newly diagnosed acute myeloid leukemia (AML) for over 2 decades. Single agent Clofarabine (CLO) induction & consolidation (Consol.) therapy has demonstrated important clinical activity in this age group in large phase II studies. Lower induction mortality (IM) & similar reported complete remission rate (CR) & overall survival (OS), as well as notable activity in those with higher risk disease features [including unfavorable cytogenetics, therapy-related AML (t-AML) & prior antecedent hematologic disorder (AHD)] raises the possibility that a non-Ara-C-based regimen could achieve similar or superior OS with lower toxicity. Methods: We performed a randomized United States Intergroup Phase III trial of single agent CLO [30mg/m2 x 5 days induction; 20 mg/m2 re-induction (if indicated) & 2 cycles Consol.] vs. standard DA therapy [Dauno 60mg/m2 D1-3 & Ara-C 100mg/m2 D1-7 induction x 1-2 cycles; 2 cycles Consol. with Ara-C (1.5g/m2 Q12hrs D1-6 age 60-69; once daily if age 70+)] in patients (pts) age ≥ 60 yrs with newly diagnosed AML. Patients with serum creatinine >1.0 (or GFR <60 mL/min) and those with AML-M3 and ECOG performance status >3 (PS>2 if age 70+ yrs) were excluded. Randomization was stratified by age (60-69 vs. 70+), t-AML, & AHD. Pts with HLA-matched donor were eligible for allogeneic transplantation (AlloHCT) after induction, and those completing Consol. were eligible for randomization #2 (R#2) to maintenance decitabine [20mg/m2 x 3D, monthly x 1 year] versus observation. With a target accrual of 747, E2906 was powered to determine non-inferiority [and possible superiority] of CLO vs. standard DA, and primary endpoint was OS. A weighted statistical analysis was performed to account for confounding impact of R#2. AlloHCT patients were censored at transplant in this analysis. Responses & cytogenetics were confirmed centrally and OS & CR rates were monitored by an independent Data Safety Monitoring Committee (DSMC) at pre-specified time points. Results: As of Feb 23, 2015, 727 pts were randomized. Median age was 68 years (range 60-86); 57% were male, and 38% were age ≥70 yrs. Treatment arms are well balanced for all baselineclinical & AML characteristics, & 30% had unfavorable cytogenetics. Of 659 with complete treatment information reported, 30.4% on DA vs. 40.1% on CLO received 2 cycles of induction (p=0.006). Median follow-up of surviving patients is 7.6 months. Table 1. shows early treatment results (CR, toxicity) for the 686 pts randomized as of Dec 23, 2014 (2 months prior to study end, & excluding 90 with ongoing response evaluation). DA CLO p-value CR/CRi 43.8% 42.8% p=0.87 30-day mortality 8.5% 7.9% p=0.89 60-day mortality 14.9% 13.1% p=0.58 Gr 4-5 Non-Heme Tox.Induction 27% 19% p=0.02 Gr 4-5 Non-Heme Tox.Consol. 20% 7% p=0.001 374 pts have died (174, DA; 200, CLO) & significantly inferior OS was observed for CLO vs. DA [Hazard Ratio (HR) 1.41 (95% CI 1.12-1.78)] (Fig. 1). Planned subgroup analyses were performed (Table 2) demonstrating significant differences in OS after CLO for patients age 60-69 yrs, without AHD, & with intermediate risk cytogenetics; but not for those with Unfav. Cytogen. (Fig. 2) or t-AML. Based on the primary weighted analysis, DSMC recommended suspension of new accrual to E2906 on Feb 23, 2015 & all active patients on CLO were transitioned to DA Arm. Table 2.NHR CLO/Standard (95% CI)*Weighted OS7271.41 (1.12-1.78)Unweighted OS7271.23 (1.00-1.50)Age 60-694491.48 (1.10-1.99)Age 70+2781.34 (0.93-1.93)Intermed. Risk Cytogen.3781.77 (1.27-2.47)Unfav. Risk Cytogen.2160.96 (0.65-1.43)No AHD6041.46 (1.13-1.89)AHD1231.22 (0.74-2.00)De novo AML6271.52 (1.18-1.96)Therapy-related AML1000.94 (0.54-1.61) Conclusions: Despite similar CR & IM, OS after single agent CLO is inferior to standard DA therapy for pts age ≥60 years with newly diagnosed AML who are fit for intensive therapy, and DA remains the standard of care. However no difference in OS was observed after CLO in some pre-specified high risk AML subgroups. R#2 & AlloHCT arms continue in E2906 for pts already enrolled. Embedded prospective minimal residual disease study at CR is being performed to identify pts at higher risk after CLO & DA. Figure 1. Weighted Kaplan-Meier Curves for OS Figure 1. Weighted Kaplan-Meier Curves for OS Figure 2. Unfavorable Cytogenetics OS by Therapy Figure 2. Unfavorable Cytogenetics OS by Therapy Disclosures Off Label Use: Use of clofarabine in AML, and maintenance therapy with decitabine in AML. Claxton:Medimmune: Research Funding; BMS: Consultancy; Astellas: Research Funding; Cyclacel: Research Funding; Merck: Research Funding; Ambit: Research Funding. Levine:Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy. Altman:Seattle Genetics: Consultancy; BMS: Consultancy; Spectrum: Consultancy; Astellas: Consultancy; Ariad: Consultancy; Novartis: Consultancy. Al-Kali:Novartis: Research Funding.

Abstract Acute Lymphoblastic Leukemia (ALL) is the most common malignancy observed in the pediatric population. Recent genomic studies have made considerable strides in improving our understanding of the molecular pathogenesis of pediatric B-cell ALL. This includes the discovery of recurrent somatic alterations targeting B-cell transcription factors, mutations in epigenetic regulators, and oncogenic mutations which activate signaling effectors, including in Ph-like B-ALL. However, the molecular pathogenesis of adult B-cell ALL has been less well studied, and there is a need to identify novel therapeutic targets in adult B-ALL. We therefore performed detailed genomic profiling in adults with B-cell ALL. We performed targeted DNA/RNA capture and sequencing of 189 patients with B-cell ALL enrolled on the ECOG E2993 clinical trial. All samples were taken at the time of diagnosis before patients were treated with multi-agent chemotherapy and followed for clinical outcomes including overall survival and remission/relapse duration and kinetics. We used the FoundationOne® Heme assay for our genomics study. This platform allows us to identify short events (point substitutions, short insertions/deletions) copy number and fusion events present in DNA and/or RNA. Of the 203 samples profiled, 189 had sufficient quality genomic data with a median coverage of 435 on DNA sequencing and 7.85 million on target distinct reads per case on RNA sequencing. In all, we identified cancer-related mutations in 108 genes, 55 of these genes being mutated in more than one sample. We identified a median of 2.98 genomic alterations per sample (standard deviation of 1.78) in the target gene set, which included all genes known to be altered in B-ALL and in other hematologic malignancies. The most common genomic events were the BCR-ABL1 fusion (69 samples, 36.5%), followed by CDKN2A loss (57 samples, 30%), CDKN2B loss (35 samples, 18%), NRAS and KRAS point mutations (32 samples, 16.9%), and MLL fusion genes with a spectrum of partners (25 samples, 13.2%). Notably, we found that BCR-ABL1 fusion are mutually exclusive with both RAS mutations (p<10^-7) and MLL mutations (p<5*10^-4). We next investigated the spectrum and frequency of alterations observed in different classes of oncogenes/tumor suppressors. We identified mutations which result in activation of oncogenic signaling pathways in 130 cases or 68.7% of our cohort. This included known mutations/fusions, such as BCR-ABL, RAS-MAPK pathway mutations, and JAK-STAT pathway alterations, and included missense alleles, insertion/deletion events, and fusion genes targeting a spectrum of genes including ABL, PDGFRB, and CRLF2. In aggregate, 33.8% of the cohort had mutations which activate signaling exclusive of BCR-ABL, demonstrating the high prevalence of the Ph-like B-ALL subtype in adult B-ALL. We identified mutations affecting key transcription factors, including PAX5 and IKZF1, in 31.2% of our cohort including missense, nonsense and deletion events. To confirm our ability to detect small deletions of PAX5 and IKZF1, we performed array CGH of 50 cases and compared copy number calls using high-throughput sequencing and CGH. Of note, all deletions targeting PAX5 and IKZF1 identified by NGS were confirmed by CGH. We also identified mutations in epigenetic regulators in 30.6% of cases, including MLL fusions, mutations in the DNA methylation/hydroxymethylation the pathway (DNMT3A, TET1, TET2), and mutations targeted chromatin modulators. Detailed analyses of the mutational spectra and their relationship to clinical outcome will be presented at the meeting. Taken together, these data show we can use a clinically validated sequencing assay to identify known and novel somatic mutations in adult B-ALL, including sequence alterations, rearrangements and copy number aberrations. These data demonstrate the feasibility of combined DNA and RNA study to identify a complex landscape of fusions and short mutations with prognostic and therapeutic relevance, and inform the use of genomic profiling in B-ALL in the clinical context. Figure 1. Figure 1. Disclosures He: Foundation Medicine, Inc.: Employment, Equity Ownership. Brennan:Foundation Medicine, Inc.: Employment, Equity Ownership. Nahas:Foundation Medicine, Inc.: Employment, Equity Ownership. Balassubramanian:Foundation Medicine, Inc.: Employment, Equity Ownership. Otto:Foundation Medicine, Inc.: Employment, Equity Ownership. Lipson:Foundation Medicine, Inc.: Employment, Equity Ownership. Stephens:Foundation Medicine, Inc.: Employment, Equity Ownership. Miller:Foundation Medicine, Inc.: Employment, Equity Ownership. van den Brink:Boehringer Ingelheim: Consultancy, Other: Advisory board attendee; Merck: Honoraria; Regeneron: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tobira Therapeutics: Other: Advisory board attendee. Rowe:Amgen: Consultancy; BioSight Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx Ltd.: Consultancy. 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.

Abstract Background: The results of the primary endpoint of the E2906 North American Intergroup phase 3 trial - demonstrating significantly inferior OS after single agent clofarabine versus standard intensive daunorubicin & cytarabine induction and consolidation - strongly support the use of intensive therapy for patients age ≥60 years with newly-diagnosed AML who are fit for treatment. This result was achieved despite similar CR rates (including CRi - CR with incomplete blood count recovery) and similar early (30- & 60-day) induction mortality between study arms, raising the question as to the importance of achieving CR as an endpoint. Therefore, we performed a follow-up analysis to address the factors that contribute to early mortality, the necessity for 2nd induction cycle to achieve CR, and to assess the survival impact of the quality of remission. Methods: E2906 design, study population and results for the primary objective were reported previously (ASH 2015, abstr. #217a). We performed this updated analysis to evaluate clinical risk factors for early mortality and re-induction, and to assess the impact of response quality on OS. Univariate comparisons were made with Fisher's exact test (category) and t-test (continuous variable), and logistic regression models were used to examine risk factors associated with early mortality, and with requiring a 2nd cycle of induction therapy. We performed a landmark analysis (beginning Day 60 after initiation of therapy) to determine the impact of response quality (CR/CRi, morphologic leukemia-free-state (MLFS), and treatment failure - i.e. residual/refractory AML) on OS. Survival comparisons were performed with log-rank test. Results: A total of n=727 patients were registered to E2906, the median age is 68 years (range 60-86) and the median follow-up of surviving patients is now 18.3 months. Adjusting for treatment, cytogenetic risk group, FAB subtype, WBC count, and secondary AML, increasing age (p=0.06) and performance status (PS) >1 (p=0.05) were associated with 30-day mortality and only PS >1 (p=0.06) with 60-day mortality. Treatment arm (p=0.003), adverse cytogenetic risk group (p=0.02), increasing age (p=0.03) and baseline WBC <10K (p=0.03) were each independently associated with higher risk of receiving 2nd cycle induction, but there was no difference in CR/CRi (44.2% vs. 42.2%, p=0.58) or in median OS (13.8 versus 12.0 months, p=0.11) for those receiving 1 vs. 2 induction cycles, respectively. We next performed a landmark analysis (day 60, n=608 evaluable patients) to evaluate OS in relation to the quality of response (Table). We observed an advantage in achieving CR and CR/CRi, although the benefit of CRi diminished greatly after 12 months, and OS after MLFS appeared more similar to treatment failure (Figure, p<0.001). The relative adverse impact of CRi and MLFS (vs. CR) was most pronounced for patients treated with clofarabine, where median OS was shorter for those achieving CRi (12.2m vs. 22.6m standard therapy, p=0.007) and MLFS (6.6m vs. 13.4m standard therapy, p=0.15). Conclusion: This follow-up analysis confirms the importance of CR regardless of the number of cycles of induction therapy required to achieve remission, and identifies risk factors associated with early mortality and with requiring re-induction. At longer follow-up the OS after CRi appears to be inferior to true CR, particularly after clofarabine. We hypothesize that this may be due to higher rates of minimal residual disease in CRi, and studies to determine this are being performed. MLFS is not associated with a better overall survival than frank treatment failure (i.e. residual disease) and therefore its value as a response category is uncertain. These results highlight the role of standard intensive remission induction therapy with the intent of achieving CR, which remains the standard of care for fit older adults. Table. Table. Figure 1. Figure 1. Disclosures Foran: karyopharm: Honoraria; novartis: Honoraria; medscape: Honoraria; Millennium Pharmaceuticals, Inc.: Research Funding; pfizer: Honoraria; boehringer ingelheim: Research Funding; agios: Research Funding; Cellerant: Research Funding. Melnick:Janssen: Research Funding. Altman:Novartis: Honoraria; BMS: Honoraria; Janssen: Honoraria; Syros: Honoraria. Al-Kali:Novartis: Research Funding; Celgene: Research Funding.

Neutrophils are vital for antimicrobial defense; however, their role during viral infection is less clear. Furthermore, the molecular regulation of neutrophil fate and function at the viral infected sites is largely elusive. Here we report that BCL6 deficiency in myeloid cells exhibited drastically enhanced host resistance to severe influenza A virus (IAV) infection. In contrast to the notion that BCL6 functions to suppress innate inflammation, we find that myeloid BCL6 deficiency diminished lung inflammation without affecting viral loads. Using a series of Cre-transgenic, reporter, and knockout mouse lines, we demonstrate that BCL6 deficiency in neutrophils, but not in monocytes or lung macrophages, attenuated host inflammation and morbidity following IAV infection. Mechanistically, BCL6 bound to the neutrophil gene loci involved in cellular apoptosis in cells specifically at the site of infection. As such, BCL6 disruption resulted in increased expression of apoptotic genes in neutrophils in the respiratory tract, but not in the circulation or bone marrow. Consequently, BCL6 deficiency promoted tissue neutrophil apoptosis. Partial neutrophil depletion led to diminished pulmonary inflammation and decreased host morbidity. Our results reveal a previously unappreciated role of BCL6 in modulating neutrophil apoptosis at the site of infection for the regulation of host disease development following viral infection. Furthermore, our studies indicate that tissue-specific regulation of neutrophil survival modulates host inflammation and tissue immunopathology during acute respiratory viral infection.

BACKGROUND. Variable response to chemotherapy in acute myeloid leukemia (AML) represents a major treatment challenge. Clinical and genetic features incompletely predict outcome. The value of clinical epigenetic assays for risk classification has not been extensively explored. We assess the prognostic implications of a clinical assay for multilocus DNA methylation on adult patients with de novo AML. METHODS. We performed multilocus DNA methylation assessment using xMELP on samples and calculated a methylation statistic (M-score) for 166 patients from UPENN with de novo AML who received induction chemotherapy. The association of M-score with complete remission (CR) and overall survival (OS) was evaluated. The optimal M-score cut-point for identifying groups with differing survival was used to define a binary M-score classifier. This classifier was validated in an independent cohort of 383 patients from the Eastern Cooperative Oncology Group Trial 1900 (E1900; NCT00049517). RESULTS. A higher mean M-score was associated with death and failure to achieve CR. Multivariable analysis confirmed that a higher M-score was associated with death ( P = 0.011) and failure to achieve CR ( P = 0.034). Median survival was 26.6 months versus 10.6 months for low and high M-score groups. The ability of the M-score to perform as a classifier was confirmed in patients ≤ 60 years with intermediate cytogenetics and patients who achieved CR, as well as in the E1900 validation cohort. CONCLUSION. The M-score represents a valid binary prognostic classifier for patients with de novo AML. The xMELP assay and associated M-score can be used for prognosis and should be further investigated for clinical decision making in AML patients.