ALBERT

Description: Drugs that reverse epigenetic silencing, such as the DNA methyltransferase inhibitor (DNMTi) 5-azacytidine (AZA), have profound effects on transcription and tumor cell survival. AZA is an approved drug for myelodysplastic syndromes and acute myeloid leukemia, and is under investigation for different solid malignant tumors. AZA treatment generates self, double-stranded RNA (dsRNA), transcribed from hypomethylated repetitive elements. Self dsRNA accumulation in DNMTi-treated cells leads to type I IFN production and IFN-stimulated gene expression. Here we report that cell death in response to AZA treatment occurs through the 2′,5′-oligoadenylate synthetase (OAS)-RNase L pathway. OASs are IFN-induced enzymes that synthesize the RNase L activator 2-5A in response to dsRNA. Cells deficient in RNase L or OAS1 to 3 are highly resistant to AZA, as are wild-type cells treated with a small-molecule inhibitor of RNase L. A small-molecule inhibitor of c-Jun NH2-terminal kinases (JNKs) also antagonizes RNase L-dependent cell death in response to AZA, consistent with a role for JNK in RNase L-induced apoptosis. In contrast, the rates of AZA-induced and RNase L-dependent cell death were increased by transfection of 2-5A, by deficiencies in ADAR1 (which edits and destabilizes dsRNA), PDE12 or AKAP7 (which degrade 2-5A), or by ionizing radiation (which induces IFN-dependent signaling). Finally, OAS1 expression correlates with AZA sensitivity in the NCI-60 set of tumor cell lines, suggesting that the level of OAS1 can be a biomarker for predicting AZA sensitivity of tumor cells. These studies may eventually lead to pharmacologic strategies for regulating the antitumor activity and toxicity of AZA and related drugs.

Description: Myelodysplastic syndromes (MDS) are a heterogeneous group of blood cancers characterized by bone marrow (BM) failure, peripheral blood cytopenias, dysplasia, chromosomal abnormalities and an increased risk for transformation to acute myeloid leukemia (AML). Patients (pts) with higher risk disease are primarily treated with pharmacologic treatments like hypomethylating therapy (HMT) (5-azacytidine and decitabine). 5-azacytidine (AZA) and decitabine (DAC) can result in overall response rates of 36% with a median duration of response of 15 months and 17-21% with a median duration of response of 10 months, respectively. Pts refractory to HMT have poor outcomes with a median overall survival of ∼4 months. Spliceosome gene mutations are frequently found in certain subtypes of MDS specifically SF3B1 (∼28%), U2AF1 (6-12%) and SRSF2 (6-12%). The prognostic value of spliceosome mutations in different MDS subtypes has been largely investigated while the impact of these mutations on treatment response is still unknown. We aim to investigate the frequency of three commonly mutated spliceosome genes (SF3B1, U2AF1, and SRSF2) in pts who failed HMT in order to define mutational frequency and evaluate the feasibility of targeted therapy with next generation spliceosome inhibitors. We screened a cohort of 120 pts (MDS, 70; MDS/MPN, 33; MDS/sAML, 17; median age: 69; male/female: 85/35) that underwent HMT (AZA: 58; DAC: 21; AZA/DAC: 7; AZA/REV: 25; DAC/REV: 4; AZA/DAC/REV: 5). Forty-eight percent of pts failed HMT therapy as refractory or relapse. We performed Sanger sequencing on BM/peripheral blood DNA for known pathways involved in MDS pathogenesis including methylation (TET2, DNMT3A, IDH1/2), histone (ASXL1, UTX, EZH2), signaling (CBL, N/KRAS), transcription (RUNX1, TP53, JAK2), and RNA splicing (SF3B1, U2AF1, SRSF2). Data analysis was available for 90 pts. We detected a total of 131 mutations in different pathways. In total, spliceosome mutations were observed in 28/90 (31%) of pts. When we analyzed the presence of the mutations in relation to the rate of response, we found that pts who failed HMT have frequent spliceosome mutations: 17/58 (29%). We have reported that molecular mutations in TET2 and DNMT3A can predict response to treatment to HMT (Traina F, Blood (ASH Annual Meeting Abstracts), Nov 2011; 118: 461). Indeed, the frequency of mutations in methylation genes was lower in the group of pts who failed HMT (11/58; 18.9%) compared to pts who achieved hematological response (11/32; 34%). Spliceosome inhibitors have been proposed for targetted therapy in MDS. The presence of spliceosome mutations in pts who failed HMT can open a new era of investigation leading to the possibility of using spliceosome inhibitors in pts who fail conventional therapy. We performed RNA-sequencing analysis on BM cells of pts who failed HMT compared to pts who achieved hematological response (n=2 vs 2) in order to define any specific gene signature explaining the differences in response to HMT. We performed differential gene expression testing on 11,459 expressed genes. In total, 158 genes were differentially expressed at FDR 〈 .2 in responders compared to not responders. We identified several interesting genes involved in tumorigenesis and epigenetic regulation such as YPEL3, and ST14, which were up-regulated in responders vs not responders (FC: 4 and 7.5; P

Description: Abstract 461 Aberrant DNA methylation is a hallmark of myelodysplastic syndromes (MDS), MDS/myeloproliferative neoplasms (MDS/MPN) and secondary acute myeloid leukemia (sAML). It provides a rationale for treating these malignancies with hypomethylating agents like 5-azacitidine (AZA) and decitabine (DAC). However, treatment outcomes remain limited and heavily weighed on morphologic/cytogenetic results. The discovery of novel mutations has provided important insight into the pathogenesis of MDS and related disorders. Genes implicated in epigenetic regulation, including DNMT3A, TET2, IDH1/IDH2, EZH2, ASXL1 and UTX have been found mutated in MDS, while others have also been implicated in MDS pathogenesis. There is limited data on the predictive value of these genetic defects for treatment response and disease outcome. We hypothesized that these defects are important biomarkers predictive of response to hypomethylating agents. We studied 88 patients with MDS (RCUD=2, RARS=6, RCMD=11, MDS-U=3, RAEB-1/2=29, CMML1/2=16, MDS/MPN-U=5, RARS-T=5, AML from MDS=11) who received hypomethylating agents (AZA=53, DAC=24, both=11). The median number of cycles was 7 [range 1–35], median age was 69 years (range 42–82) and median follow-up was 18 months (range 0–76). Responses were scored according to IWG criteria. DNMT3A, TET2, IDH1/2, EZH2, ASXL1, UTX, KRAS, NRAS, CBL, RUNX1, TP53 and SF3B1 were sequenced using standard techniques. Categorical variables were analyzed using Chi-square statistics. Overall survival (OS) was analyzed using Kaplan-Meier; p-values ≤ 0.05 were considered statistically significant. Mutated patients were older than wild type (WT) cases (72 vs. 68 years, p=.01) but were well matched for marrow blast %, cytogenetic risk group and cycles of hypomethylating agents received. We found mutations in 40/88 (45%) patients. Mutations were most frequent in SF3B1 (6/11; 55%), ASXL1 (13/50; 26%), TET2 (18/88; 20%), KRAS (3/34; 9%), and DNMT3A (7/88; 8%). Less common were mutations in EZH2 (2/43; 5%), TP53 (1/23; 4%), IDH1 (4/88; 5%), IDH2 (3/88; 3%), and UTX (1/36;3%). No mutations were found in CBL, NRAS or RUNX1. Based on single mutations, overall response rate (ORR) was higher in mutated vs WT patients for DNMT3A (6/7 [86%] vs 33/81 [41%]; p=.02), ASXL1 (11/13 [85%] vs 14/37 [38%]; p=.003), and TET2 (12/18 [67%] vs. 27/70 [39%]; p=.03). All heterozygous DNMT3A mutants responded to hypomethylating agents. Differences remained significant when stratified to AZA treatment alone for DNMT3A (6/7 [86%] vs 21/56 [38%]; p=.01) and ASXL1 (9/11 [82%] vs 12/29 [41%]; p=.02) but not TET2 (6/10 [60%] vs 21/53 [40%]; p=0.22). The predictive value of combined mutations were analyzed for DNMT3A, TET2 and/or IDH1/2, showing better response to hypomethylating therapy in patients who had a mutation; ORR (mutated: 18/28 (64%) vs WT: 21/60 (35%); p=.01). This difference remained significant in patients receiving only AZA (n=53); ORR was 11/18 (61%) in mutant and 11/35 (31%) in WT patients (p=.03). No differences in ORR were noted for KRAS, EZH2 and IDH1/2 mutant and WT patients. No SF3B1 mutants responded to treatment while both patients with UTX and TP53 mutations responded. The frequency of AML evolution was also analyzed and showed no difference between mutant and WT cases for TET2 (7/18 [39%] vs 22/70 [31%];p=.52), ASXL1 (4/10 [40%] vs 11/35 [31%]; p=.61), and DNMT3A (3/7 [43%] vs 26/81 [32%];p=.56). No differences in OS and progression free survival (PFS) were noted between responders and non-responders to hypomethylating therapy (28 vs 17 mos, p=.25; 16 vs 8 mos, p=.54). Comparison of survival outcomes for mutant and WT patients showed no significant difference for DNMT3A (OS: 30 vs 21 mos, p=0.43; PFS: 20 vs 11, p=.53), ASXL1 (OS: 28 vs 22, p=.68; PFS: 16 vs 10, p=.88), and TET2 (OS: 30 vs 20 mos, p=.30). PFS was better in TET2 mutants compared to WT (19 vs 9, p=.03). No survival differences were noted between mutant and WT cases who responded to hypomethylating agents for DNMT3A (OS: 25 vs 28,p=.84; PFS: 14 vs 16, p=.78), ASXL1 (OS: 10 vs 18, p=.48; PFS: 10 vs 6, p=.76) TET2 (OS: 27 vs 16, p=.79; PFS: 18 vs10, p=.19). In conclusion, DNMT3A, ASXL1 and TET2 mutations were independently associated with a better response to hypomethylating drugs. Moreover, combined mutations in DNMT3A/TET2/IDH1/IDH2 may influence the response to hypomethylating agents, especially AZA supporting its role as a predictive biomarker in MDS treatment. Disclosures: Maciejewski: Celgene and Eisai, NIH, AA&MDS Foundation: Research Funding. Tiu:MDS Foundation Young Investigator Award: Research Funding.

Description: Abstract 2015 Allogeneic hematopoietic stem cell transplantation (AlloSCT) remains the only curative option for MDS. Several retrospective studies evaluated the impact of various prognostic factors (i.e. cytogenetic risk group, WHO classification, ferritin level etc.) on post-transplant outcomes of pts with MDS, however comprehensive analyses including a cytogenetic abnormalities detected by SNP array (SNP-A) karyotyping method have not been performed. We have analyzed prognostic factors of post-AlloSCT outcomes among 74 pts with MDS (2000–2010) including the predictive value of SNP-A abnormalities. Cox proportional hazards analysis was used to identify univariable prognostic factors for acute GVHD (aGVHD), chronic GVHD (cGVHD), disease relapse, relapse free (RFS) and overall survival (OS). Multivariable prognostic factors were identified by stepwise Cox proportional hazards analysis. The median time from MDS diagnosis to transplant for all pts was 6 mos (range, 0.2– 141 mos). The median age at transplant was 51 yrs; 32% of the pts had a hematopoietic cell transplant co-morbidity index (HCT-CI) score ≥ 3; 69% had ≥1 prior chemotherapies; and only 30% were in remission prior to their transplant. 27 pts (37%) had RAEB-2, 11 (15%) had RAEB-1, and 9 (12%) had treatment-related MDS. 42 pts (58%) belonged to an intermediate-2 or higher IPSS risk category. 23 pts (31%) had adverse karyotype (complex or monosomy 7) detected by metaphase cytogenetics (MC). SNP abnormalities were identified in 58% of patients; 79% of all patients with SNP abnormalities had lesions not previously detected by traditional cytogenetic techniques. Median pre-transplant ferritin level was 1127 (range, 9–5201). 73% of the pts received myeloablative conditioning. In 61% of cases stem cells were harvested from the bone marrow. Matched related donors accounted for half of the cases. Twelve pts (16%) died within 100 days of transplant and 39 pts (53%) within the median follow up of 36 mos (range, 5–114). MDS relapse occurred in 22 pts (30%). The rates of grade II-IV aGVHD and extensive cGVHD were 49% and 24% respectively. Disease relapse was the most common cause of death (31%) followed by aGVHD (18%) and cGVHD (13%). In univariate analysis, aGVHD was associated with myeloablative (p

Description: Abstract 1012 Poster Board I-34 Background: Disparities in survival between black and white patients (pts) exist for many malignancies, including AML. Potential causes include differential access to care; variable aggressiveness of therapy; and biological heterogeneity. Black men with AML have lower complete remission (CR) and overall survival (OS) rates than black women and whites, as shown in a previous Cancer and Leukemia Group B (CALGB) study in which induction therapies were defined, but subsequent treatment compliance and intensity was unknown. We investigated whether differences in post-remission therapy (PRT) might explain disparity in outcome. Methods: All pts with newly diagnosed AML treated with cytarabine-based induction therapy between 1997 and 2008 were included. PRT was defined as either cytarabine-based chemotherapy or bone marrow transplant (autologous or allogeneic) administered to pts achieving a CR and prior to relapse or in the setting of no relapse. PRT was coded according to intensity, in cumulative mg/m2 of cytarabine or mitoxantrone received, with BMT assigned the highest intensity of cytarabine. Data on known prognostic factors (age, white blood cell count (WBC) at diagnosis, pathologic subtype, AML etiology, cytogenetic risk groups (as defined by CALGB 8461)) were collected and controlled for in multivariable analyses. Time to PRT was measured in days from date of discharge after induction chemotherapy to date of PRT initiation, excluding patients with time to PRT 〉150 days, considered too long to be true consolidation. Comparisons between black and white pts were performed using linear, logistic, and proportional hazard regressions, exploring intensity of and time to PRT, and number of PRT cycles, along with potential interaction terms, controlling for known prognostic factors for outcome. Results: Of 460 pts, 421 had adequate data on PRT. Of these, 379 (90%) were white, 32 (8%) black, 10 (2%) other, and 46% were female. Similar to CALGB data, and compared to whites, blacks were younger (mean age 53.5 vs. 57.5 years, p=.11), had a higher proportion of favorable (18.8% vs. 12.7%) and poor risk (34.4% vs. 24.5%) cytogenetics (p=.28), and were less likely to attain a complete remission (CR, 66% vs. 73%, p=.39), though differences did not reach significance due to sample size. Other baseline characteristics, including reinduction rates, were similar. The 236 pts who received PRT included 18 blacks (56%) and 218 whites (58%, p=.89). In univariate analyses, for blacks vs. whites, median time to PRT initiation was 31 vs. 23 days (p=.33); cytarabine intensity was 66,654 mg/m2 vs. 50,630 mg/m2 (p=.26); and number of cycles was 2.4 vs. 2.1 (p=.41), respectively. Median time to PRT among all men was 24 days, compared to 21 days for all women (p=.09), and for pts =60 years (p=.43). Median survival was 0.72 years in both black and white patients (p=.57). Among those receiving PRT, median survival was 3 years in blacks and 1.4 years in whites (p =.22); for pts not receiving PRT, median survival was.22 years in blacks and.32 years in whites (p =.25). In multivariate analyses, time to PRT was shorter for whites (HR=.25, p=.016), particularly when white men were compared to black men (HR=.12, p=.012), whereas no differences were found for women. Survival differences did not reach significance; nor was there an interaction for male and black race. Conclusions: Time to PRT is shorter for whites compared to blacks with AML, though cycle dose intensity and number are similar. Despite this, overall survival was not different between blacks and whites receiving PRT. In the post-remission setting, blacks and whites appear to receive similar chemotherapy management, and thus differential treatment aggressiveness and compliance are not explanations for varying outcome between races. Disclosures: No relevant conflicts of interest to declare.

Description: Isochromosome 17q [i(17q)], a poor prognostic cytogenetic abnormality is a product of the breakage or inappropriate division of the pericentromere leading to the duplication of the long and loss of the short arm of chromosome 17. The region of the breakpoints maps at 17p11, a region encompassing a key tumor suppressor gene: TP53. I(17q) are detected in myelodysplastic/ myeloproliferative neoplasms (MDS/ MPN), chronic myeloid leukemia (CML), and acute myeloid leukemia (AML). This abnormality can occur as a sole structural abnormality or in combination with other chromosomal defects. The presence of i(17q) is associated with poor therapeutic response, disease progression, and an unfavorable clinical outcome. Elucidation of the molecular architecture of patients (pts) carrying i(17q) may lead to better understanding of disease biology and development of novel compounds that can target this disease. We selected 11 pts with i(17q) to characterize their genomic differences. We applied whole exome sequencing (WES) in order to define latent molecular defects explaining the clinical phenotype of this disease. The index case was a male MDS/MPN pt with isolated i(17q), 27% RS, hypercellular bone marrow (BM), mild splenomegaly, and atypical megakaryocytes. The pt developed 7% BM blasts without clinical response to growth factors. Molecularly this pt was a wild type SF3B1, a gene frequently mutated in RARS-T and associated with lower transformation rate to leukemia, better survival, and good/intermediate risk cytogenetic abnormalities. WES was performed on 2 ug of total DNA extracted from BM cells. Non-clonal CD3+ cells were used as source of germ-line control. Twenty-millions reads were run on an Illumina HiSeq2000 sequencer. Using a stringent bio-informatic algorithm developed in house, all variants were filtered based on a variation score (〉=30) and a coverage (30X) and the tumor nucleotide variation analysis was performed for each pair (tumor vs. germ-line), where only the variants unique to the tumor were retained. Variants were ultimately filtered in order to exclude SNPs by an in-house annotation and importing the hg19 SNP135. We detected 65 unique candidate genes. Four genes were confirmed to be somatic: 3 were novel: ZFP42 (4q35.2), P4HTM (3p21.31), and VPRBP (3p21.2) and 1 includes the newly discovered SETBP1 (18q12.3) gene. Three variants detected on the chromosome 17 had a wild type configuration. The subsequently genotyped all the pts (MDS/MPN/-U 3; AML 4; RCMD 1; CML 1; RAEB-1 2; mean age: 68 years; male/female: 8/3; i(17q)/other abnormalities:3/8) for the above genes and for a panel of genes known to be mutated in MDS/MPN and other diseases in order to find any genetic association explaining the disase phenotype. We applied Sanger sequencing to DNA derived from BM/peripheral blood cells (BM/PB:7/4) for the following genes and respective exons: TP53 (all exons), SF3B1 (13-16), SRSF2 (1-2), U2AF1 (2 and 6), TET2 (all exons), DNMT3A (18-23), IDH1/2 (4), CBL (8-9), N/KRAS (1-2), ASXL1 (12), JAK2 (12 and 14), EZH2 (16, 18 and 19), MPL (exon 10), BCAS3 (12, 15 and 16), FLT3 (11 and 17), and CSF3R (13,14, and 17). In total, we found 16 heterozygous missense mutations and 1 tandem duplication. We found somatic mutations in ZFP42, P4HTM, and VPRBP in 1 pt. The index case reported a mutation in SETBP1 and SRSF2. SF3B1 was detected as a sole abnormality in 1 patient. Of note, the patient with SF3B1 mutation (K700E) had 50% RS and achieved a complete hematologic remission after decitabine therapy. The most frequent mutations were found in SETBP1 and SRSF2. SETBP1 was found to be mutated in 4/11 (36.3%) pts (D868N, I871T, and G870S was common in 2 pts) while SRSF2 mutations (P95H/R) were found in 3/11 (27.2%) pts. Three pts showed concomitant SRSF2 and SETBP1 mutations. NRAS (G12D) was mutated in 1 pt and associated with SRSF2 and SETBP1 mutations. One pt showed mutations in TET2, JAK2, and TP53. Of note, this pt did not respond to treatment. One pt with MDS/MPN showed a mutation in CSF3R (Q741X), a novel gene discovered in chronic neutrophilic leukemia and atypical CML. The pt also has monosomy 7 and i(17q) abnormality. FLT3-ITD was found in 1 pt. As of last follow-up, only 2 pts remain alive. In sum, we found that poor risk molecular mutations in SRSF2 and SETBP1 are frequently found in i(17q) myeloid malignancies and may be the drivers of poor outcomes in this disease. Disclosures: No relevant conflicts of interest to declare.