Citations

Abstract The International ALL trial, conducted jointly by the MRC in the UK and ECOG in the US (UKALL XII/E2993), recruited 1929 patients between 1993 and 2006. All patients aged 16 to 64 years with newly diagnosed ALL, received the identical two phases of induction therapy. Patients with an HLA-identical sibling were assigned to a sibling allogeneic transplant and those who were Ph-positive could also get an unrelated-donor transplant. Patients who did not have a donor were to be randomized between a single autologous transplant, after conditioning with etoposide/total body irradiation, versus consolidation/maintenance therapy for 2.5 years. Following randomization, but prior to receiving the assigned or randomized therapy, all patients received intensification with 3 cycles of high-dose methotrexate. After excluding patients assigned to an allogeneic transplant, 1028 patients were eligible for randomization but, as in other major transplant studies, only 457 were randomized. The rationale underlying the randomization was based on the fact that protracted consolidation/maintenance therapy in adults, extrapolated from the pediatric experience, had never been prospectively evaluated in the era of intensive chemotherapy. Given that the mortality from autotransplant is not higher than chemotherapy (Goldstone et al. Blood. 2008), it was postulated that if a single autologous transplant is at least as good as standard protracted chemotherapy that may make it the preferable option, except possibly in females in whom fertility may be an issue. The overall survival and the event-free survival were significantly superior among the chemotherapy patients (p = .05) as previously reported (Goldstone et al. Blood. 2008). Because the literature contains reports suggesting a trend in favor of autologous transplantation in some patients with ALL (Dhédiu et al. Leukemia, 2006), an analysis was performed to see if any subgroup of patients could be identified in whom: 1. chemotherapy may not be superior to autologous transplant and 2. autologous transplant may be superior. The table lists a detailed analysis of overall survival looking at various age groups, B- versus T-lineage, the rapidity with which a complete remission was achieved –after phase I or only after phase II of induction. Detailed analysis was also performed by cytogenetics looking at those with standard risk versus those with high risk abnormalities [ t(9;22), t(4;11), t(8;14)] and low hypodiploidy/near triploidy or a complex karyotype (Moorman et al. Blood. 2007)]. In all groups, the chemotherapy group was at least as efficacious as the autograft group, and in some was even significantly superior to autologous transplantation. There is no evidence that other risk factors, such as relapse rate or treatment-related mortality, influenced these overall survival data. In addition, the tests for heterogeneity were not significant across any group. In conclusion, the overall survival was longer in patients randomized to chemotherapy, although the superiority was not statistically significant in most of the groups. There is no clinical indication for autologous transplantation at any age and particularly this should not be considered for patients over age 50, those with high-risk cytogenetics or T-lineage, and late remitters, for whom it might appear most attractive. Overall Survival (OS) at 5 years in 457 Randomized Patients Chemo Auto Subgroups n OS n OS P (log rank) Age (years) < 20 50 58% 43 46% > 0.1 20–29 61 52% 70 43% > 0.1 30–39 46 39% 46 32% > 0.1 40–49 38 31% 35 31% > 0.1 50 + 33 38% 35 31% > 0.1 B-lineage 169 46% 158 35% 0.03 T-lineage 45 54% 54 49% > 0.1 Time to CR phase I 193 48% 190 41% 0.1 phase II 26 36% 29 19% 0.08 Cytogenetics standard-risk 109 51% 122 44% > 0.1 high-risk 29 23% 27 7% 0.02

Abstract Abstract 4377 Introduction MicroRNA (miRNA) profiling in CLL has revealed a leukemia specific gene signature in CLL patients. Our earlier work found a unique signature of 13 deregulated microRNAs from 190 previously untreated CLL patients. This signature included some of the most frequently deregulated miRNAs in different types of hematological malignancies (miRNA-15-a/16-1, miRNA-29 family, miRNA-155, miRNA-221) and correlation with CLL prognostic factors (ZAP-70 expression and immunoglobulin heavy chain variable region (IGVH) mutations). Since these miRNAs have both diagnostic and prognostic implications, we wished to extend our observations to a cohort of relapsed/refractory CLL patients entered on the ECOG trial E2903. This trial is a chemoimmunotherapy protocol that uses 6 cycles of pentostatin, cyclophosphamide and rituximab to induce responses followed by consolidation with Alemtuzumab. Methods To assess miRNA status, we used our previously described method which employs a bead-based profiling that interrogates 649 microRNA genes. We used the Trizol extracted RNA from the CLL B-cells of 33 patients entered onto the E2903 clinical trial. All patients were also characterized by determining their CD38, ZAP-70, fluorescence in situ hybridization (FISH) and IGVH status. All microRNA and prognostic parameters were done on baseline CLL B-cells obtained from the patients prior to going on study. Results The demographics of this CLL patient population included 28 males with median age of 61 (range; 47-76), 1 Rai stage 3 and 24 Rai stage 4. The median number of prior treatments was 2 (range;1-5). To date with PCR treatment only assessed, there were 15 partial responses (PR), 15 stable disease, and 3 unevaluable patients. Because of a batch effect, we restricted our analysis in this report to only 24 patients. We decided to initially compare the microRNA profiles for CLL patients segregated on the following; responders (CR/PR) vs. non responders (SD or less), ZAP-70+ vs. ZAP-70- (20 % cutoff), CD38+ vs. CD38– (30% cutoff), Low-risk FISH (normal, 13q- or trisomy 12) vs. High-Risk FISH (17p- or 11q- or complex) and age '65 vs. ≥ 65. In addition, we did combine some of the prognostic parameters or responses together for comparison of microRNA status (ZAP-70 with either response, IGVH status, or CD38 status). This analysis revealed a set of 10 microRNA genes with a median false discovery rate of 20% that were aberrantly expressed when we compared the ZAP-70- responders to the ZAP-70+ non-responders. In addition, we found 15 differentially expressed miRNAs when we compared ZAP-70- to ZAP-70+ cohorts. We then evaluated the association of the 15 genes found in the ZAP-70 comparison with the overall survival and progression free survival (PFS). We found that miR-625* was significantly associated with PFS (p=0.01). The expression level of miR-625* was more than 2-fold increased in ZAP-70- compared to ZAP-70+. miR-625*, located at 14q23.3, has more than 60 putative target genes, including nuclear and cell cycle, and cell adhesion targets. Conclusion Interrogation of the genes that are impacted by the deregulated miRNAs uncovered in our analysis of this previously treated CLL cohort may provide valuable information regarding the mechanism of resistance commonly seen in this cohort of heavily pretreated CLL patients. Disclosures: Kay: Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding; Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees.

The critical importance of specific proteins including Bcl-2, Mcl-1, XIAP, Bax, Caspase-3, Traf-1, and ZAP70 in predicting disease progression, prognosis, and response to therapy in CLL have been extensively studied through in vitro experiments. We now report molecular data obtained from a large prospective clinical trial involving 278 previously untreated patients with CLL. This ECOG-led intergroup trial involved participation from CALGB and SWOG, and currently provides quantitative information regarding protein levels on 217 cases assessed immediately prior to treatment. NCI criteria for therapy in CLL were met before patients were randomized to receive either Fludarabine or Fludarabine plus Cytoxan. On-study analyses included: interphase cytogenetics by Fluorescence In Situ Hybridization (FISH), p53 mutational analysis, immunoglobulin variable heavy chain (IgVH) gene rearrangement, and quantitative analysis of the aforementioned specific proteins. Peripheral blood mononuclear cells were obtained from each patient at registration before starting therapy. Lysates were prepared from the patient cells and separated by SDS-PAGE. Proteins were detected by immunoblot analysis using the multiple antigen detection method previously developed by our group. Detection was performed by an enhanced chemiluminescence method and quantified by densitometry. Expression levels were calculated relative to expression in the Jurkat T cell line (for ZAP70) or the RS11846 lymphoblastoid cell line (for apoptosis-related proteins). Using a two-sided Wilcoxon rank sum test, molecular data were compared with FISH and IgVH mutational status. Patients with either p53 mutations or del(17p) had significantly lower levels of XIAP (p=0.022). Patients with del(11q) had significantly higher Bcl-2/Bax ratios (p=0.052). Patients with trisomy 12 had lower levels of Bcl-2 (p=0.066); higher Mcl-1 (p=0.026); higher Bax (p=0.09); and higher Caspase-3 (p=0.019) levels. Interestingly, these specimens also had lower ratios of Bcl-2/Bax (p=0.0016). Patients with del(13q) had significantly higher levels of Bcl-2 (p=0.0059) lower levels of Caspase-3 (p=0.047), and higher Bcl-2/Bax ratios (p=0.0569). IgVH mutated patients had significantly lower levels of Caspase-3 using either a 98% cut-off (p=0.0008) or the 97% cut-off (p<0.0001). Finally, patients with higher ZAP70 protein expression had significantly higher levels of Caspase-3 (p=0.0099) and Traf-1 (p=0.016). In 163 patients evaluable for both response and the molecular studies, either elevation of Bcl-2 protein (p= 0.01) or an abnormality of p53 (either del 17p or a p53 mutation) (p=0.08) correlated significantly with attainment of a complete remission. Potential opportunities to predict response to therapy in CLL will hopefully identify those patients likely to benefit from therapy. Based upon our previous work, these molecular parameters can be modulated both in vitro and in vivo. Therefore, understanding the relationship of these important proteins to therapeutic response can facilitate strategic plans to circumvent drug resistance to chemotherapy and immunotherapy in CLL.

Several prognostic factors including un-mutated VH mutational status, select interphase cytogenetic abnormalities [del(11q22.3), del(17p13.1)] and p53 mutations have been associated with shorter interval time from diagnosis to symptomatic disease requiring treatment, shortened progression-free survival (PFS) and overall survival (OS). Limited prospective data exists relative to the relevance of these biologic markers on PFS following treatment with modern therapies for CLL. E2997 is a randomized phase III trial of 278 previously untreated, symptomatic CLL patients who received fludarabine and cyclophosphamide (FC) versus fludarabine (F). We analyzed the response rates and PFS of the patients for whom interphase cytogenetics, VH mutational status, and p53 mutational status studies have been completed. FC therapy had a higher complete response (CR) (23% versus 5%, p<0.002), overall response [OR] (73% versus 50%, p<0.002), and median PFS (33.5 months versus 15 months, p<.0001) as compared to F. The CR and OR was not different based upon interphase cytogenetics or VH mutational status for the entire group of patients or when analyzed by arm of treatment. However, the OR for pts with del(17) or p53 mutations was lower in both the FC arm (69%, versus 76% in patients with normal p53) and F arm (27%, versus 54% in normal p53). Using the Dohner hierarchical classification [del(17p)>del(11q)>del(6q)>tri12>normal> del(13q14)], the relationship of interphase cytogenetics, VH mutational status and p53 mutational status with PFS is summarized below: Median PFS on FC (months) Median PFS on F (months) NR = median not reached; p53 mut+ = mutation present del(17p) 11.9 8.2 del(11q) 30.6 12.8 del(6q) 26.9 26.7 tri 12 NR 20.3 normal NR 11.2 del(13q) NR 21.2 del(17p)/p53 mut+ 11.9 8.9 no del(17p)/p53 mut+ NR 17.8 VH<98% NR 21.2 VH>98% 21.4 13.4 Using the interphase cytogenetic groups, there was a significant difference in PFS for both the FC (p=0.04) and F (p=0.01) treatment arms. Similarly, the presence of a p53 mutation or deletion [del(17p)] predicted for shorter PFS for both FC (p=0.005) and F (p=0.02) therapies. PFS did not statistically differ among VH mutational groups for either arm. We next investigated a Cox proportional hazards analysis to assess the impact of multiple molecular features and treatment arm on PFS. Treatment with F (hazard ratio 3.14, p<0.0001), presence of del(17p) or p53 mutation (hazard ratio 3.13, p=0.0001), and presence of del(11q) (hazard ratio 2.067, p=0.019) were highly significant in the model. Finally, VH unmutated status and del(11q) were highly associated. In conclusion, our data demonstrate that high risk genomic features including p53 mutations, del(17p), and del(11q) are highly predictive of shortened PFS. Our data provide support for a risk-stratified therapy approach for the treatment of CLL.

Abstract Deletions or mutations of the tumor suppressor p53 have been associated with more aggressive disease in many types of cancer including CLL, and are more commonly found in relapsed or refractory patients. We studied symptomatic patients (pts) enrolled on ECOG 2997, a randomized phase III trial of fludarabine monotherapy (F) versus fludarabine plus cyclophosphamide (CF) for previously untreated CLL. A total of 235 pretreatment samples were analyzed for p53 mutations in exons 5–9 using denaturing gradient gel electrophoresis (DGGE), followed by confirmation of mutations using automated sequencing. Cytogenetic analysis was also performed on 243 patient samples by Fluorescence In Situ Hybridization (FISH) using a probe to detect deletions in 17p13.1, the chromosomal location of the p53 gene. In the analysis by DGGE, a total of 25 pts (10.6%) were found to have p53 mutations. In 22 cases there was a single mutation, and in three cases there were two mutations. Mutations in exon 6 were the most common, and in each of the cases with two mutations, one of these was in exon 6. Results by exon are as follows: Exon 5 (1 pt, 0.4%), Exon 6 (15 pts, 6.4%), Exon 7 (5 pts, 2.1%), Exon 8 (3 pts, 1.3%), Exon 9 (4 pts, 1.7%). The interphase cytogenetic analysis included 24 of the 25 pts with a p53 mutation by DGGE. Of the 19 (7.8%) pts with del(17p13.1) as the leading cytogenetic anomaly, seven were cases that also had mutations in p53 by DGGE. The remaining 17 pts with mutations in p53 by DGGE had as leading cytogenetic anomaly: del(11q) (2 pts); trisomy 12 (3 pts), normal cytogenetics (2 pts), and del(13q) (10 pts). Results from both the DGGE and FISH analyses were combined to identify a group of 37 pts with either del(17p) or p53 mutation. This constituted 16% of the 230 pts in which either FISH or p53 mutational analysis was performed. Immunocytochemistry staining for p53 was performed on all pts, and no correlation was found between over-expression of p53 protein with mutation and/or deletion of this gene. The pts were then assessed by treatment arm for response and progression-free survival (PFS), relative to patients without p53 abnormality either by DGGE or FISH analysis. In the FC group: 13 pts had either a p53 mutation or del(17p), nine (69%) of whom achieved a CR or a PR, versus 56/74 (76%) of those with normal p53 (p=0.73). In the F group: 15 pts had either a p53 mutation or del(17p), of whom four (27%) achieved a CR or a PR, versus 38/70 (54%) of those with normal p53 (p=0.09). Pts with a detectable abnormality in p53 had a significantly reduced PFS, regardless of whether they received fludarabine alone (p=0.02) or fludarabine plus cyclophosphamide (p=0.005). This study suggests that routine immunocytochemistry staining should not be substituted for p53 mutational studies. While some short responses were observed in patients with p53 mutation and/or deletion receiving fludarabine and/or fludarabine cyclophosphamide, the time to progression was significantly shorter, emphasizing the prognostic significance of p53 abnormalities for this disease.

Abstract Purine nucleoside combination therapy, with and without immunotherapy has become the standard treatment approach to CLL. Past approaches, other than stem cell transplantation, have not been successful in curing the disease. Inherent resistance of CLL precursors, impaired immune surveillance (T-cells, NK cells) and microenvironmental abnormalities may all play a role in the ability to effectively eradicate leukemia. PCR has been successfully employed for previously treated (Lamanna 2006) and previously untreated (Kay 2007) patients with CLL. In addition, CA has been added to FCR (Wierda 2006) to treat relapsed/refractory CLL disease. We designed a study to evaluate PCR followed by CA for relapsed/refractory CLL. This ECOG study includes, in addition to response criteria, molecular correlates (FISH anomalies and CpG karyotype), risk parameters analysis, including CD38, ZAP-70 and IgVH mutational status. We assessed the baseline and sequential changes in the angiogenic status of patients by measuring plasma VEGF, bFGF, thrombospondin and their immune status via T-cell repertoire. From September 2004 to September 2006, 32 patients with relapsed/refractory CLL were enrolled in the protocol. The protocol combined 2 regimens (PCR and CA). Step 1, PCR, was planned for 6 monthly cycles. Steps 2 and 3 consisted of short term CA (4 weeks) or standard CA (18 weeks) depending upon Step 1 response. There were 3 ineligible, 1 not evaluated, and 28 evaluable patients. FISH anomalies revealed that 8 patients had a homozygous or heterozygous 13q-defect, 9 had complex FISH defects (more than one FISH anomaly), 5 patients had FISH anomalies that included del(17p13.1), 4 patients had FISH anomalies that included 6q-and 3 had FISH anomalies that included 11q-. CD38 was positive in 10 patients, 11 were ZAP-70 positive and 12 of 19 patients tested had unmutated type clones. The planned 6 cycles of Step 1 (PCR) was administered to 14/32 patients (44%), the others receiving 0–5 cycles. All responses were considered partial responses (11/29; 37.9%), with 2 patients (6.9%) demonstrating progressive disease. However, patients receiving the planned 6 cycles of PCR, achieved a partial response of 71% (10/14) with 2 cases of tumor lysis syndrome. Toxicity (Step 1-PCR) was predominately myelosuppressive (Grade 1–4). A case of chronic idiopathic polyneuropathy and a case of “paraneoplastic pemphigus” were reported. There were 4 deaths. CMV reactivation was diagnosed in 2 patients, neither of which was fatal. During Step 2 (CA), short-term administration in one patient led to grade 3 skin toxicity, which limited administration. During long-term administration of CA (PR, PD, SD) (n=7), infectious complications occurred in 6 patients all of whom developed grade 4 lymphopenia. The median survival for the entire group was 15 months. In conclusion, a significant number of heavily pre-treated and poor prognosis patients with CLL will respond to PCR. Further analysis will determine whether a significant number of patients will achieve a higher level of response and longer survival after CA administration. Studies of association of outcome with the risk parameters are ongoing and will be presented with the clinical response data.

Abstract Abstract 2568 Background: Obesity is associated with an increased risk of development of adult AML (Relative Risk approx. 2-fold) (Ross J, Cancer Epidemiol Biomarkers Prev 2004;13:1810) and significantly higher mortality in pediatric AML (Lange B, JAMA 2005;293:203). We therefore examined the association of obesity with presenting characteristics in 2 recent large ECOG phase III adult AML trials to determine whether there was a unique clinical and genetic phenotype, and also the impact of obesity on clinical outcomes (complete remission - CR; early death - ED; overall survival - OS) in association with other known risk factors. The impact of obesity on receiving full-dose daunorubicin (D) was also evaluated in these studies. We included patients (pts) from E1900 receiving standard infusional cytarabine with intensified D (45 vs. 90mg/m2) in adults age ≤60 yrs (NEJM 2009;361:1249), and from E3999 receiving conventional intensive chemotherapy +/− the MDR-inhibitor zosuquidar in adults age >60 yrs (Blood 2010;116:4077). Obesity was defined as Body Mass Index (BMI) >30kg/m2. Methods: Pt baseline characteristics were compared using Fisher's exact (categories variables) or Wilcoxon rank sum tests (continuous variables). Logistic regression models were used to examine the association between obesity and CR or ED; Cox models stratified on induction treatment arms were used to examine the association between obesity and OS. Multivariate models to examine the effect of Obesity controlled for known prognostic factors (including cytogenetics & secondary AML - sAML). Results: A total of 1089 pts (E1900, n=651; E3999, n=438) with available BMI data were included, the median follow-up of living pts is 4.7 yrs. Obesity was prevalent, with n=359 (33%) of pts with a BMI>30 at presentation. For all patients, median BMI was 27.1 (range 16.7–93.0); for non-obese patients median BMI was 25.0 (range 16.7–29.96) while for obese patients the median BMI was 34.0 (range 30.0–93.0). Obese pts were older (p=0.06), and significantly more likely female gender (p=0.05), ECOG PS>0 (p=0.01), & Intermediate Risk cytogenetics (p=0.006) but not sAML (14% overall, p=ns). Genotyping for FLT3 & MLL was performed in E1900 pts (age ≤60yrs): there was no difference in incidence of FLT3-ITD, but lower incidence of MLL-PTD was observed (p=0.08) in obese pts (not performed in E3999) (See Table). Overall, only 3.7% of pts received <90% of intended D dose. However obesity was significantly associated with receiving <90% intended D dose: in E1900, 11% of obese pts received <90% intended D (45mg/m2 cohort, p<0.001; 90mg/m2 cohort p=0.002), while in E3999 4% obese pts received <90% intended D (45mg/m2, p=0.07). There was no clear difference in toxicity/adverse events during initial induction therapy reported in obese vs. non-obese pts. On multivariate analysis obesity was not significantly associated with achieving CR (p=0.09), and had no direct independent association with ED (p=0.38). Kaplan-Meier plots of OS are shown in Figure. On multivariate analysis, age (p<0.001); ECOG PS (p<0.001); cytogenetic risk group (p<0.001); sAML (p=0.03); gender (p=0.05); and peripheral blasts (p=0.003) but not obesity were significantly associated with OS. Receiving <90% intended D dose was marginally associated with increased hazard ratio for OS (HR 1.50, 95%CI 0.98–2.28, p=0.06). Conclusion: Obesity is associated with specific presenting clinical characteristics in adult AML (female, older, PS>0, Int. risk cytogenetics, less frequent MLL-PTD), but has no direct impact on CR, ED or OS in the setting of a controlled therapeutic trial. Obese pts are at risk of receiving <90% intended D dose, which may impact OS. This analysis suggests that factors that predispose to adult AML may present with a unique clinical phenotype. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: Eastern Cooperative Oncology Group trial E1900 (E1900) showed that induction therapy with a high daily dose of daunorubicin (90 mg/m2) (DNR 90) improves survival in younger patients (pts) (<50 yrs) and intermediate (int) cytogenetic risk AML, but at 2 years of follow-up no benefit was seen in older pts (50-60 yrs), or in those with unfavorable cytogenetic risk or FLT3-ITD mutant AML (Fernandez et al. N Engl J Med 2009). Here we update results of E1900 with longer follow-up, focusing on the benefit of DNR 90 in cytogenetic and common molecular subgroups. Methods: Overall survival (OS) was measured from randomization for induction therapy to death from any cause (censored at last contact). Hazard ratios (HR) for death were computed using univariate and multivariable Cox proportional hazards models; multivariable Cox models were adjusted for sex, age, hemoglobin level, leukocyte count, platelet count, and cytogenetic profile. All conclusions regarding the impact of DNR 90, unless noted, are similar based on univariate and multivariable analysis. Results: Overall, 657 pts were enrolled with a median follow-up of 80.1 months. The HR for death in the DNR 90 group as compared with the standard-dose daunorubicin (45 mg/m2) (DNR 45) group was 0.74 (p=0.001). Pts <50 yrs benefited from DNR 90 (p = 0.002) while those >=50 yrs were not proven to benefit (p = 0.12). Pts with favorable (fav) and int. cytogenetic risk benefited from DNR 90 (p = 0.03 and p = 0.02, respectively). A benefit for pts with unfavorable cytogenetic risk was seen on multivariable analysis (p = 0.04). Impact of DNR 90 by mutation status: The 3 most common mutations were FLT3-ITD (24%), NPM1 (26%), and DNMT3A (24%). AML pts with any of these 3 mutations benefited from DNR 90 (p = 0.009, p = 0.002, and p = 0.02, respectively). FLT3-ITD pts who received DNR 90 had a 4-yr OS of 31%. Benefit was seen in pts age 50-60 with FLT3-ITD or NPM1 mutation (p = 0.02 and p = 0.04, respectively). No benefit of DNR 90 was seen in a small cohort of pts with MLL-PTD (p = 0.06). Benefit of DNR 90 in FLT3-ITD, NPM1, and DNMT3A mutant AML was confirmed in the int. cytogenetic risk group. Impact of DNR 90 on prognostic impact of NPM1: The presence of an NPM1 mutation conferred an improvement in OS in the DNR 90 group which was not seen in the DNR 45 group (p = 0.01 vs p = 0.3). This finding was confirmed in the int. cytogenetic risk group. Conclusion: With median follow-up of over 6 years on E1900, we confirm that DNR 90 improves outcome in pts with fav/int cytogenetic risk, DNMT3A or NPM1 mutant AML, or age < 50 (Patel et al. N Engl J Med 2012). Additionally, we now demonstrate that DNR 90 additionally benefits pts with FLT3-ITD AML, and pts with unfavorable cytogenetic risk, regardless of age. Moreover, we show that the favorable prognostic impact of the NPM1 mutation is only present when pts receive DNR 90. Given the benefit of DNR 90 across all cytogenetic risk groups as well as common molecularly defined subgroups of AML, DNR 90 should be the standard for all pts up to age 60 who are candidates for induction chemotherapy. Table HR for death by AML cohort. Subgroup N Univariate Model DNR 45 DNR 90 HR (DNR 90/DNR 45) & 95% CI Wald P All patients (n=657) Overall 330 327 0.74 (0.61, 0.89) 0.001 Age < 50 yrs³ 50 yrs 188 142 172 155 0.66 (0.50, 0.85) 0.81 (0.62, 1.06) 0.002 0.12 Cytogenetic Favorable Intermediate Unfavorable 38 232 59 51 212 63 0.51 (0.28, 0.93) 0.76 (0.61, 0.96) 0.79 (0.54, 1.16) 0.03 0.02 0.22 FLT3-ITD WT MUT 215 83 241 64 0.74 (0.59, 0.92) 0.61 (0.42, 0.89) 0.008 0.009 MLL-PTD WT MUT 290 16 296 15 0.70 (0.58, 0.86) 0.46 (0.21, 1.04) 0.0004 0.06 NPM1* WT MUT 180 65 192 65 0.70 (0.55, 0.89) 0.50 (0.32, 0.78) 0.003 0.002 DNMT3A WT MUT 177 61 194 58 0.66 (0.52, 0.85) 0.62 (0.41, 0.94) 0.001 0.02 * Statistically significant test of interaction (p<0.2) Figure 1A: Overall Survival by NPM1 Mutation Status and Treatment Arm Figure 1A:. Overall Survival by NPM1 Mutation Status and Treatment Arm Figure 1B: Overall Survival of FLT-ITD Mutant AML Patients by Treatment Arm Figure 1B:. Overall Survival of FLT-ITD Mutant AML Patients by Treatment Arm Disclosures No relevant conflicts of interest to declare.

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 Introduction: The therapy of relapsed/refractory AML remains poor and new treatment options are sorely needed. The Leukemia Committee of ECOG devised a randomized phase II trial of 3 novel regimens to determine overall response rates could be improved. Methods: Patients between the ages of 18 and 70 years with a prior diagnosis of AML who relapsed <1 year after initial achievement of complete remission (CR) or who were refractory to initial induction therapy (<2 courses) or to first reinduction after a relapse (<1 course) were eligible for this trial. Eligible patients were randomized equally to one of three regimens described in Table 1 below. The primary endpoint was achievement of a morphologic complete remission (CR) or a CR with incomplete blood count recovery (CRi). A 2 stage statistical design was planned with 18 patients (assuming 16 eligible) to be accrued in the first stage. If at least 3 CR+CRi were seen among the 16 eligible patients, an additional 19 patients (assuming 17 eligible) would be entered in the second stage. If 8 or more CR+CRi were seen in the 33 eligible patients, this would be considered evidence of a promising response rate. Results: This study activated October 16, 2008 and closed August 2, 2013 with a total of 92 patients accrued (91 eligible). The eligibility criteria were amended during the study to lower the upper age limit from 70 to 65 years because 6 treatment related deaths occurred in the initial 27 patients accrued to the FLAM regimen. All of these patients died of septic shock or multi-organ failure and 5 of the 6 died within 1 month of the start of therapy. Most of these patients were over age 65 ( 53, 62, 67, 68, 69 and 69 years). At the first stage analysis, of the first 16 eligible patients accrued to each arm there were 2 CR and 2 CRi in the CT arm, 2 CR and 2 CRi in the FLAM arm and 2 CR in the sirolimus-MEC arm. Thus, the sirolimus-MEC arm was closed to accrual with a total of 20 eligible patients and second stage accrual continued for the CT and FLAM arms. Among the 35, 36 and 20 eligible patients accrued to arms A, B and C, respectively, there were 2 CR and 3 CRi in the CT arm, 6 CR and 4 CRi in the FLAM arm, and 2 CR and 1 CRi in the sirolimus-MEC arm. All 10 CR or CRi of the FLAM arm were among the first 33 eligible patients, therefore FLAM was the only regimen to achieve our target. The overall response rates for arms A, B and C were 14% (90% CI 7.1% - 34.7%), 28% (90% CI 16.1% - 43.0%) and 15% (90% CI 4.2% - 34.4%), respectively. Among the 92 patients accrued, there were 5, 10 and 2 grade 5 events within 30 days of the end of therapy in arms A, B and C, respectively. The vast majority of these deaths were attributed to infection and multi-organ failure except two which were secondary to disease. Conclusion: In this high risk group of AML patients the FLAM regimen showed an encouraging response rate. A randomized phase II trial has also recently been completed exploring its use in the front-line setting with newly diagnosed high-risk AML. While the FLAM regimen was excessively toxic in elderly patients with relapsed or refractory disease, it is a suitable option for younger patients in this setting. Arm A: CT Carboplatin 150 mg/m2/day over 24 hours by continuous infusion IV for 5 days (days 1-5) Topotecan 1.6 mg/m2/day over 24 hours by continuous infusion IV for 5 days (days 1-5) Arm B: FLAM Flavopiridol 30 mg/m2 for ½ hour followed by 60 mg/m2 over 4 hours IV per day for 3 days Cytarabine 667 mg/m2 over 24 hours by continuous infusion IV for 3 days (days 6-8) Mitoxantrone 40 mg/m2 IV over 1-2 hours (day 9) Arm C: Sirolimus-MEC Sirolimus 12 mg by mouth on day 1, followed by 4 mg by mouth daily for 8 days (days 2-9) Mitoxantrone 8 mg/m2/day over 15 minutes IV for 5 days (days 4-8) Etoposide 100 mg/m2/day over 1 hour IV for 5 days (days 4-8) Cytarabine 1000 mg/m2/day over 3 hours IV for 5 days (days 4-8) Table 2. Responses Table 1. Chemotherapy Regimens Regimen n Median age (range), years Responses (%) Deaths (first 30 days) (%) CT 35 55 (23-67) 5 (14%) 5 (14%) FLAM 36 62 (19-69) 10 (28%) 10 (28%) Sirolimus-MEC 20 52 (22-68) 3 (15%) 2 (10%) Disclosures Off Label Use: Sirolimus-potentiate chemotherapy Carboplatin-chemotherapy for AML treatment Topotecan-chemotherapy for AML treatment.