Paietta, Elisabeth

Abstract Background: Despite advances, the majority of patients (pts) with acute myeloid leukemia (AML) will die of their disease. Current genetics-based risk classification schemes inadequately predict outcome. We sought to determine if a novel methylation-based biomarker could enhance risk stratification in AML. Methods: Using a novel methylation assay (xMELP) validated for the clinical laboratory, we performed DNA methylation assessment at 17 previously identified prognostic loci to calculate a summary methylation statistic (M-score, Wertheim et al. Clin Chem 2015) for 166 pts with de novo AML treated at the University of Pennsylvania (UPENN). Targeted next-generation sequencing (NGS) of 33 hematologic malignancy-associated genes was performed for a 136 pt subset. The association of M-score with other prognostic variables and outcome [complete remission (CR) and overall survival (OS)] was evaluated. Median follow-up was 68.1 mos (range 1.4-150.2) among 38 survivors and 10.5 mos (range 0.1-95.2) among those (n=128) deceased. The optimal M-score for identifying groups with differing OS in the total UPENN cohort defined a binary classifier. The classifier was validated in UPENN subgroups and in a separate ECOG ACRIN E1900 trial cohort. Results: The mean and median M-score for the UPENN cohort was 92.3 (95% confidence interval [CI], 87.4-97.2) and 91.4 (range, 30.8-97.3), respectively; the M-score was not significantly associated with age, gender, specimen type, blast percent, NPM1, or FLT3-ITD. Patients with favorable cytogenetics had a lower mean M-score than those with non-favorable cytogenetics; there was no difference between intermediate and unfavorable cytogenetic groups. Univariate analyses demonstrated that a 10-unit increase in M-score was associated with a 10% increase in the hazard of death (HR 1.1; P<.0001) and a 20% increase in the odds of failing to achieve CR (OR 1.2, P<.0001). In multivariable Cox analysis, higher M-score (HR 1.1, P=.011) and older age (P=.001) were significantly associated with increased hazard of death, while NPM1+/FLT3-ITD-status (P=.031) was associated with decreased hazard of death. In a multivariable logistic analysis, higher M-score (HR, 1.1, P=.034) and older age (P=.007) were associated with increased odds of failing to achieve CR, while favorable cytogenetics (P=.030) was associated with achievement of CR. Based on the maximization of the log-rank statistic, the optimal M-score cutpoint was 86, which defined a binary risk classifier (hazard of death for high vs low M-score: unadjusted HR 2.5, P<.0001; adjusted HR 1.9, P=.003). Median OS was 26.6 vs. 10.6 mos for low and high M-score groups (Figure). The CR rates for low and high M-score groups were 84% and 61%, respectively. The performance of the M-score classifier was confirmed in pts ≤ 60 yrs with intermediate cytogenetics (log-rank P=.001, OS in high vs low M-score groups: 36.4 vs 14.0 mos) and among pts who achieved CR with initial therapy (log-rank P<.00001, OS 43.9 vs 17.2 mos). The M-score classifier identified groups with different outcome regardless of allogeneic transplant. The M-score classifier was validated in the E1900 cohort (n=383). The mean and median M-scores were similar to the UPENN cohort and M-score was significantly associated with OS on multivariable analysis (P<0.0001). The M-score classifier identified E1900 subgroups with different OS (log-rank P<.00001; OS 29.5 vs 12.6 mos), a finding confirmed in the intermediate cytogenetics group, in those aged <50 and ≥50 yrs, and among pts assigned to both standard and high dose daunorubicin. Notably, high dose daunorubicin benefited patients with high M-scores (P=.001), but not those with low M-scores (P=.328). We also evaluated the prognostic value of the M-score in the context of an extended molecular profile. Random forest analysis of the UPENN cohort showed that M-score and age are the most robust predictors of OS, while a subset of recurrent mutations (FLT3-ITD, NPM1, IDH2, TET2, TP53, NRAS, CEBPA) also contribute to prognosis, but to a lesser degree. Conclusion: The M-score and associated classifier represent promising tools for clinical decision-making in AML and deserve further investigation. The prognostic value of the M-score may be superior to mutational analysis. Optimal methods for integration of M-score, clinical and genetic information are being defined. Figure 1. Figure 1. Disclosures Loren: Merck: Research Funding. 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 Abstract 4013 Background: In recent years, azacytidine (AZA) has become the standard of treatment for high risk myelodysplasia (MDS). The approved schedule of AZA uses a 75mg/m2/d s.c. regimen for 7 days based on the CALGB-9221 (Silverman, JCO 2002) and the AZA-001 studies (Fenaux, Lancet Oncol 2009). The clinical response rates after AZA have been extensively presented but there is only limited data on the rates of cytogenetic response (CyR). Based on the review of the literature, there are no specific cytogenetic data published on prospective trials. Methods: We based our analysis on a randomized phase 2 study from the US Leukemia Intergroup (E1905 study, NCT00313586) testing 10 days of AZA (50mg/m2/d s.c.) vs 10 days of AZA+ the histone deacetylase inhibitor entinostat (4 mg/m2/d PO days 3 and day 10). MDS, CMML, and AML with myelodysplasia-related changes were included. This analysis includes all patients with cytogenetic abnormalities (at baseline or acquired following treatment) with available cytogenetic follow-up (cycle 6). Of 150 patients, 70 demonstrated baseline cytogenetic abnormalities. To date, forty patients (27 MDS and 13 AML) were evaluable for both time points. Karyotypes were performed at local laboratories, and reviewed centrally (RPK and GH). Cytogenetic response was assessed using IWG 2000 (Cheson et al, Blood 2000) criteria. Results: The clinical response rate (CR+PR+ trilineage HI) according to IPSS cytogenetic risk stratification were of 20%, 33%, and 35% for favorable, intermediate and poor cytogenetic risk groups respectively (p=NS). Patients with Chr 7 abnormalities (i.e. -7 or -7q, n=18) had a response rate of 28% including 17% CR. Of patients with complete cytogenetic data, the rate of overall CyR was 52% (n=21): 22% (n=9) complete CyR, 30% (n=12) partial CyR. This represents a complete CyR of 13% and a partial CyR of 23% as a proportion of all treated patients with initial cytogenetic abnormalities (including those who did not receive six cycles of therapy). To date, confirmatory FISH analyses were available for 4 patients with CyR (2 CCyR and 2 PCyR). All four had complete clearance of their cytogenetic clone. Among the cytogenetic responders, 15 had MDS and 6 had AML (p=NS). CyR did not differ between the two treatment arms. CyR and clinical response were highly correlated (p<0.001): all patients with complete CyR and 7/10 partial CyR had a clinical CR. Only 3 patients (1 CCyR and 2 PCyR) had a CyR without associated clinical response. Among 8 patients with cytogenetic progression, 3 pts showed increased frequency of a known aberration and 5 showed new or additional aberration including 2 cases involving Chr7. 65 AZA-treated patients from AZA001 had comparable cytogenetic results available. Only 3 patients experienced complete CyR 4.6% with 33.8% partial CyR (n=22). In contrast, the reported rate of clinical response was similar in the two studies. Complete CyR were more frequent in the E1905 10 days AZA schedule compared to the 7 days AZA schedule (p=0.007). There was no difference in overall CyR between the two studies. Conclusions: Both the standard AZA schedule and the 10 day schedule administered on E1905 lead to high rates of cytogenetic response. The novel schedule explored in E1905 demonstrated a particularly high rate of complete CyR (22%) with complete clearance of abnormal clone by FISH in 4 tested patients. Further prospective comparison of different AZA schedules will be needed to confirm this higher rate of complete CyR and determine its clinical significance. Disclosures: Beach: Celgene: Employment. Gore:Celgene: Consultancy, Research Funding, stock options; Syndax: Consultancy.

Abstract Abstract 524 The International UKALLXII/E2993 trial enrolled 2091 pts with newly diagnosed adult ALL between 1993 and 2006. Patients received 2 months of combination induction chemotherapy followed by an intensification course of high dose methotrexate and L-asparaginase after which pts were randomized to autologous hematopoietic stem cell transplant (HSCT) or consolidation and maintenance chemotherapy. Patients <55 years of age with a matched sibling donor were assigned myeloablative allogeneic HSCT (Goldstone, et al, Blood 2008; 111:1827). Here we report a detailed analysis of 1,229 pts from this trial with B-ALL, excluding Philadelphia-chromosome-positive patients. The median age was 30 years (range 14–65) and 59% were male. The median WBC was 9.2×109/L (range 0.1–930) and 73% of pts had a WBC <30 × 109/L. CNS involvement was present in 4% of pts at diagnosis. Comprehensive centralized immunophenotyping was performed in 372 of the 409 (91%) ECOG pts enrolled and initial analysis demonstrated pro-B/pre-pre-B in 17%, early pre-B in 52%, pre-B in 14%, either early pre-B or pre-B in 6%, mature B in 2% and B-lineage, not otherwise specified, in 9%. Outcomes were similar between patients with immunophenotyping data and those without. The complete remission (CR) rate was 93% and there were 4% induction deaths. The overall survival (OS), event-free survival (EFS), relapse risk (RR) and non-relapse mortality (NRM) at 5 years for the overall group is shown in the table. There were no significant differences in CR rate, OS, EFS, RR or NRM for the immunophenotypic subgroups. There was better OS and EFS with less relapse and similar risk of NRM for pts randomized to chemotherapy as compared with autologous HSCT (Figure 1). In the matched sibling donor-no donor comparison, OS and EFS were improved, RR was less and NRM was higher in the donor group (Figure 2). Cytogenetic analysis confirmed that patients with t(4;11) (n=77, 8% total), t(8;14) (n=19, 2% total), a complex karyotype (≥5 abnormalities in the absence of an established chromosomal abnormality) (n=38, 5% total), or low hypoploidy/near triploidy (n=41, 5% total) had an inferior OS and patients with high hyperdiploidy had a superior OS when compared to other patients. In conclusion, this is the largest series of B-ALL in uniformly treated adults ever reported and confirms the superior outcome of patients randomized to chemotherapy as compared with autologous HSCT and shows that patients with a matched sibling donor have an improved survival over patients without a donor. No difference in CR rates or outcome was noted for patients with different immunophenotypic subtypes of B-ALL. Future improvements in outcome of pts with B-ALL will rely on the addition of new agents such as B cell monoclonal antibodies and improvements in allogeneic HCT.Table.Outcomes at 5 years of follow-upnCR rate (%)OSEFSRRNRMOverall12299342375021Pro-B/pre-pre-B639236345421Early pre-B/pre-B2689340345421Age ≤ 35 years746 (61%)9653454516Chi square p<0.0001<0.0001<0.0001<0.0001<0.0001Age >35 years483 (39%)8932265930Randomized to autologous HSCT15735316610Log rank p0.030.010.01NSRandomized to chemotherapy1694742558Matched sib donor34452483032Chi square p0.050.03<0.001<0.001No Donor47445415510Values are % except n and p values. CR=complete remission, OS=overall survival, EFS=event-free survival, RR=relapse risk, NRM=non-relapse mortality NS=not significant. The p values represent the difference between age groups for the first comparison, randomized to autologous HSCT versus chemotherapy for the second comparison and between matched sibling donor and no donor for the third comparison. Overall survival by randomized allocation at 10 years Overall survival by donor availability at 10 years Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 579 Background: In AML, age and cytogenetics remain among the major prognostic factors. Age above 55 years and a complex karyotype (CK; ≥ 3 abnormal clones) or the presence of monosomy 7 (M7) are universally associated with unfavorable prognosis. Recent data from HOVON-SAKK (J Clin Onc 26: 4791) suggest that in patients under the age of 60 years, subgroups with poor risk versus very poor risk can be delineated based on the presence of a MK. MK was defined as either the presence of at least two autosomal monosomies or one monosomy plus at least one structural abnormality. Limited data exists regarding the impact of MK in AML patients over the age of 55 years. Methods: Data from 935 AML patients above age 55 enrolled on three prospective ECOG therapeutic clinical trials (E1490, E3993, E3999) between 1990 and 2005 were retrospectively reviewed to identify those with MK with single and/or multiple monosomies, MK in the presence or absence of a CK, CK with/without MK, MK with/without M7, and normal karyotype (CN). CK was defined as ≥ 3 clonal abnormalities for the purposes of this study. Complete remission (CR), disease-free survival (DFS), and overall survival (OS) were assessed. Results: The median age of evaluable patients was 68 years (range 56 – 93) with a male/female ratio of 391/299. Cytogenetic data were evaluable in 690 (74%) subjects. Clonal cytogenetic abnormalities occurred in 399 (58%). Core-binding factor (CBF) abnormalities accounted for 16/399 (4%) whereas CK was detected in 181/399 (45%). MK was present in 172 (25%) evaluable cases, predominantly in the setting of CK (140/172, 81%). Overall CR was 44%. CR was achieved in 49% MK−, 29% MK+, 31% CK+, and 49% of CK−. Data for the combinations of MK and CK are presented in Table 1. Median follow-up for survivors was 5.0 years. OS at 4-years was 9% for all evaluable patients and 4y-DFS was 12% in those achieving CR. Median OS of MK+/CK+ (0.3y) was significantly worse (p<0.001) than MK+/CK−(0.6y), MK−/CK+ (0.8y), and MK−/CK−(0.9y). Median DFS of MK+/CK+ (0.3y) was also significantly poorer (p<0.001) than MK+/CK− (0.9y), MK−/CK+ (0.8y), and MK−/CK− (0.9y). M7 was present in 82 (13%) enrolled patients with 98% (80/82) of M7 occurring within a MK. CR was achieved in 31% of MK+/M7+ and 27% of MK+/M7−. Neither median OS nor DFS were significantly impacted by M7 status as noted in Table 2. Conclusions: AML patients above the age of 55 years have an unfavorable prognosis with the majority of patients harboring adverse clonal cytogenetic abnormalities and failing to achieve a CR. Fewer than 10% survive 4-years from diagnosis. Results from the ECOG experience demonstrate the prognostic predictive value of MK in older AML patients. In contrast to the data presented by the HOVAN-SAKK group, the prognostic value of MK in patients over 55 years is limited to those with MK in combination with CK. These results also demonstrate the failure of monosomy 7 to provide prognostic value in the setting of MK. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2739 Background: Internal tandem duplication mutations of the FMS-like tyrosine kinase 3 receptor (FLT3-ITD) are among the most frequent mutations in de novo acute myeloblastic leukemia (AML) and result in constitutive activation of the receptor tyrosine kinase. Based on data showing that these mutations negatively affect outcome in AML, FLT3 receptor kinase activity is currently being targeted in the clinic. To date, only FLT3 receptor mutant AML patients have been selected for trials involving FLT3 receptor kinase inhibitors, with surprisingly variable clinical responses. Downstream targets of FLT3 receptor activation, whether by receptor mutation or FLT3 ligand binding, involve Stat5, PI3-kinase/Akt and the Ras/Raf/Erk kinase signal transduction pathways which ultimately affect cell survival and proliferation. Functional characterization of those signaling pathways in mutated versus non-mutated FLT3 receptor (FLT3 -WT) myeloblasts has significant potential to reveal heterogeneity among these genetically defined subgroups and to predict for response to kinase inhibition, independent of FLT3 receptor mutation status. Methods: Modulated single cell network profiling (SCNP) was used to evaluate the activation state of intracellular signaling molecules (i.e. nodes), including phosphorylated (p)-Akt, p-Erk, p-S6, p-Stat5 and cleaved-PARP, at baseline and after treatment with specific modulators [including cytokines (such as IL-27) growth factors (such as FLT3 ligand) and drugs (such as cytosine arabinoside)] in 7 healthy bone marrow mononuclear blasts (BMMb) and leukemic myeloblasts, characterized for FLT3 receptor mutation status, from 44 AML patients (38 FLT-WT and 6 FLT3-ITD), aged >60 years (ECOG trial E3999). A total of 64 node-metrics were analyzed. Results: Signaling profiles differed significantly in FLT3-ITD vs. FLT3-WT AML, and in FLT3-WT vs. BMMb (shown in Figure 1 for a representative node, FLT3 ligand induced p-S6). Specifically, compared to BMMb, FLT3-ITD blasts uniformly showed increased basal p-Stat5 levels, decreased FLT3 ligand-induced activation of P13K and Raf/Ras/Erk pathways, minimal IL-27 induced activation of the Jak/Stat pathway, and higher apoptotic responses to DNA-damaging agents. Two AML samples harboring a low FLT3-ITD mutant burden, however, exhibited a signaling pattern similar to FLT3-WT AML samples. By contrast, FLT3-WT samples displayed heterogeneous signaling profiles, overlapping both with those of FLT3-ITD and BMMb samples, suggesting that a fraction of FLT3-WT AML samples exhibit FLT3 receptor pathway deregulation even without FLT3-ITD. Conclusions This study showed that SCNP, which provides a detailed view of intracellular signaling networks at the single-cell level, subclassified patients with AML beyond their molecularly determined FLT3 mutation status. In particular, a fraction of FLT3-WT AML samples signaled as if containing a FLT3 receptor length mutation while FLT3-ITD samples with low mutational load signaled like FLT3-WT AML samples. The clinical relevance of this observation, both for disease prognosis and response to kinase inhibitors, will be revealed only if AML patients are accrued to kinase inhibition trials irrespective of FLT3 receptor mutation status. The wide range of signaling responses observed in FLT3-WT AML suggests that disease across FLT3-WT patients is heterogeneous, likely promoted through distinct mutations and alterations, giving rise to distinct signaling profiles in individual patients Our data also provide evidence for the co-existence of differentially signaling blast populations in individual patients. The potential impact of signaling heterogeneity on clinical response needs to be assessed and may require an individualized combination of treatment modalities. Disclosure: Rosen: Nodality Inc.: Employment, Equity Ownership. Putta:Nodality Inc.: Employment, Equity Ownership. Gayko:Nodality Inc.: Employment, Equity Ownership. Cesano:Nodality Inc.: Employment, Equity Ownership.

Abstract Abstract 3405 Poster Board III-293 Background: Autologous hematopoietic cell transplantation (AutoHCT) as consolidation has been an alternative to standard intensive chemotherapy and/or allogeneic HCT for younger patients with acute myeloid leukemia (AML) in first complete remission (CR). However concerns about toxicity and true benefits in long-term outcome have hampered its use. Methods: We present the follow-up of younger patients with de novo AML treated on ECOG study E1900, on an intention-to-treat (ITT) and per protocol (PP) analysis. DFS and OS were measured from the time of registration to consolidation therapy. Results: 657 patients younger than 61 years were initially randomized to standard-dose cytarabine and either standard- or high-dose daunorubicin in a 7+3 approach. Of the 352 patients who entered consolidation phase, 307 patients, 68 with favorable-risk cytogenetics (FRC), 128 with intermediate-risk cytogenetics (IRC), 90 with indeterminate cytogenetics (IDT) and 21 with unfavorable-risk cytogenetics (URC), were allocated to AutoHCT. Patients in these cohorts were treated with high-dose cytarabine 3g/m2 x 6 doses in 5 days, for two cycles. Peripheral blood cells were collected after the second cycle. Initially patients were randomized to receive gemtuzumab ozogamicin (GO) or not; however, no benefit was derived from GO therapy and the randomization was closed. 156 patients did not receive the planned AutoHCT due to disease progression, alternative therapy, failure to collect, insurance denial, toxicity and other reasons. 151 patients (33 FRC, 64 IRC, 47 IDT and 7 URC) were conditioned with IV busulfan 0.8 mg/kg daily x 16 doses (without dose adjustment) and cyclophosphamide 60 mg/kg x 2 days. Autologous cells were infused on day 0 and patients were supported with sargramostim until ANC recovery > 500/μL. Veno-occlusive disease occurred in three patients. Treatment-related mortality (TRM) of AutoHCT was 1.3% (2/151) at 30 days. Median follow-up was 31.8 months. In the ITT analysis, median disease-free survival (DFS) was 21.1 months and overall survival (OS) was 31.8 months. For patients treated PP, median DFS and OS were 29.8 months and 39.7 months, respectively. Median DFS by cytogenetics was, 20.5 months for IRC and 22.4 months for IDT; not reached for the FRC group (p=0.10) and median OS was 32.8 months for IRC and 34.4 months for IDT; not reached for the FRC group (p=0.06). For FRC patients treated with high dose daunorubicin induction and AutoHCT the DFS and OS was ≥ 80% at 4 years based on K-M estimates. Patients who were FLT3-positive (n=27) demonstrated a trend towards worse DFS (16.2 vs. 30.3 months) and OS (20.0 vs.39.7 months) than those who were FLT3-negative (n=113) (p=0.12 and 0.10, respectively). Conclusions: Auto HCT is a safe consolidation approach with a low TRM in younger patients with de novo AML in first remission. Results are excellent in respect to DFS and OS particularly for the FRC patients on the high-dose daunorubicin arm. Due to small numbers, we could not demonstrate a negative outcome in FLT3-positive patients receiving AutoHCT. Further study, however, with a larger number of patients is warranted. Disclosures: Fernandez: Otsuka: Honoraria. Off Label Use: IV busulfan in the treatment of acute myeliod leukemia .

Abstract Abstract 2695 Background: Age and cytogenetics are major determinants of outcome in older patients with AML. Complete response (CR) to induction chemotherapy is observed in less than half of de novo AML patients (excluding APL) older than 60 years. Because unfavorable treatment results are often obtained despite intensive chemotherapy, which carries the potential for significant treatment-induced morbidity and mortality, it would be valuable to be able to predict with high accuracy at the individual patient level who will achieve a CR. Prospective identification of patients unlikely to benefit from anthracycline/cytarabine-based chemotherapy could spare patients from unnecessary treatment-related toxicities and allow consideration for alternative therapeutic strategies. Single Cell Network Profiling (SCNP) is used to measure simultaneously the effects of multiple modulators (including drugs) on signaling cascades at the single cell level. Using SCNP technology we have developed a set of classifiers that predict for likelihood of response to anthracycline/cytarabine-based induction therapy in older patients with AML. Methods: SCNP assays were performed on paired, bone marrow (BM) and peripheral blood (PB), samples from 44 AML patients (de novo, evolved from an antecedent MDS or MPN or treatment related), >60 years old, enrolled on ECOG trial E3999. Based on two previous training studies, 38 combinations of modulators and intra-cellular proteins (signaling nodes along the phosphoinositide 3-kinase (P13K ), the Janus Kinases (Jak) signal transducers and activators of transcription (Stat) and the DNA damage response and apoptosis pathways) were investigated. Basal and modulated protein levels and the effect of modulation on proteins levels in the leukemic blast cells were expressed using a variety of metrics. A total of 64 node/metric combinations (dimensions) were used to build multi-parametric classifiers (ranging from 2 to 10 nodes/metrics) using different modeling methodologies (including random forest, boosting, lasso and a bootstrapped best subsets logistic modeling approach that shrinks regression coefficients (BBLRS)) able to predict the likelihood of response to induction therapy. The performance characteristics of the classifiers built on the BM samples were then evaluated independently on the paired PB samples. Results: Several promising models with high area under the operator/receiver curve (AUROC) values (indicating strong agreement between actual clinical responses and responses as predicted by the model) were developed based on SCNP proteomic read outs for BM samples. The observed and predicted values from the current best BBLRS model are shown in Figure 1. The unadjusted AUROC of this model is 0.98 and the expected AUROC for the model when applied to an independent (validation) sample is 0.84. Five signaling nodes are represented in this model; they include nodes belonging to growth factor- induced survival pathways (PI3K, RAS/MAPK) as well as DNA damage response and apoptosis pathways. When the predictive accuracy of the lead SCNP classifier was compared to that of a model based on traditional clinical/molecular predictors (i.e. the combination of age, therapy-related AML, and karyotype) the adjusted AUROC of the SCNP classifier far surpassed that of the clinical predictors (adjusted AUROC=0.61 for clinical/molecular predictors vs. adjusted AUROC= 0.84 for the SCNP classifier). Finally, when the nodes in the best BBLRS model developed on data from BM samples were used to model read outs from the paired PB samples, the adjusted AUROC of the resulting BBLRS model was comparable to that of the model fit to BM samples. Conclusion: This training set data show the value of performing quantitative SCNP under modulated conditions as the basis for developing highly predictive tests for response to induction chemotherapy. Most importantly, the predictions made by the SCNP classifier are independent of established prognostic factors, such as age and cytogenetics The ability of one set of nodes to accurately predict response in paired BM or PB samples from individual patients suggests that the predictive power of the SCNP assay is independent of sample source, further improving the practicality of the test. Independent validation studies are ongoing. Disclosures: Cesano: Nodality Inc.: Employment, Equity Ownership. Putta:Nodality Inc.: Employment, Equity Ownership. Gayko:Nodality Inc.: Employment, Equity Ownership. Hackett:Nodality Inc.: Consultancy. Rosen:Nodality Inc.: Employment, Equity Ownership.

Abstract Abstract 2693 Background: Single Cell Network Profiling (SCNP) is used to measure simultaneously the effects of multiple modulators (including drugs) on signaling cascades at the single cell level. Using this technology, ECOG in collaboration with Nodality is developing several novel biomarker assays with the aim to find blast functional signaling profiles predictive of response to induction therapy and risk of relapse in AML patients. To date, such assays utilized patient bone marrow (BM) as the sample source of blasts. However, in about 65% of patients with AML, circulating peripheral blasts are detected and peripheral blood (PB) sampling is easier and less invasive for patients than BM sampling. Objectives: The objective of this study was to compare by SCNP the functional effects of a panel of compounds simultaneously on different signaling pathways (such as the phosphoinositide 3-kinase (P13K )and the Janus Kinases (Jak) signal transducers and activators of transcription (Stat) pathway) relevant both to the biology of the disease and the development of new therapeutics, in paired, diagnostic, cryopreserved PB mononuclear cells (PBMC) and BMMC samples from 44 AML patients. A paired sample was defined as a BMMC and PBMC specimen collected from the same patient on the same day. Methods: Modulated SCNP using a multiparametric flow cytometry platform was used to evaluate the activation state of intracellular signaling molecules in leukemic blasts under basal conditions and after treatment with specific modulators (Table 1). The SCNP phosphoflow assay was performed on 88 BMMC/PBMC pairs from ECOG trial, E3999. The relationship between readouts of modulated intracellular proteins (“nodes”) between BMMC and PBMC was assessed using linear regression, Bland-Altman method or Lin's concordance correlation coefficient. Results: Table 1 shows the goodness of fit (R2) values from the linear regression analysis for both the basal levels and the modulated levels of intracellular signaling proteins. Most of the signaling nodes show strong correlations (R2 >0.64) with several of the exceptions belonging to nodes with weak response to modulation (e.g. SCF -> p-Akt) or antibodies with dim fluorphores (i.e. Alexa 647). The lack of response is however, consistent between the tissue types for the weak response nodes. Using a rank based metric that is less sensitive to the absolute intensity levels seem to perform better for the antibodies with dim fluorophores. Results from other methods; Bland Altman and Lin's Concordance also show good concordance between the tissue types. Conclusions: The data presented here demonstrate: 1) Specimen source (BM or PB) does not affect proteomic signaling in patients with AML and circulating blasts. 2) PB myeloblasts can be used as a sample source for Nodality SCNP assays to identify functionally distinct leukemic blats cell populations with distinct sensitivities to therapy. 3) The ability to use PB as a sample source will greatly improve the utility of these assays. In particular, our results will facilitate the monitoring of cellular signaling effects following the administration of targeted therapies, e.g., kinase inhibitors, at time-points when BM aspirates are not clinically justifiable. Disclosures: Cesano: Nodality Inc.: Employment, Equity Ownership. Rosen:Nodality Inc.: Employment, Equity Ownership. Putta:Nodality Inc.: Employment, Equity Ownership. Gayko:Nodality Inc.: Employment, Equity Ownership.

Abstract This study was coordinated by the ECOG-ACRIN Cancer Research Group (Robert L. Comis, MD and Mitchell D. Schnall, MD, PhD, Group Co-Chairs) and supported in part by Public Health Service Grants CA180794, CA180820, CA180795, CA180791, CA189859, CA180790, CA180853, and from the National Cancer Institute, National Institutes of Health and the Department of Health and Human Services. Its content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute. Background: Relapse after achieving a complete remission (CR) in AML portends for a poor prognosis and allogeneic transplant after achieving a second remission is the only chance for cure. Patients who undergo a transplant have a 30-40% chance of long term survival. This is a follow-up of a report published 10 years ago (Rowe JM, ASH 2005, abstract 546) and includes contemporary studies and longer follow-up. The study examines the long term overall survival (OS) of AML patients who relapsed after achieving first CR in 9 successive ECOG-ACRIN trials for newly-diagnosed AML patients (E3483, E3489, PC486, E3993, E4995, E1490, E3997, E1900 and E3999) from March 1984 to November 2008. Methods: OS was defined as time from first relapse to death from any cause. Kaplan-Meier estimates were used to estimate the event-time distributions for OS. Multivariate model stratified on protocol and treatment were used to examine whether the following factors are prognostic for OS from relapse: age, gender, cytogenetic risk, ECOG performance status, WBC, platelets, hemoglobin, marrow blasts, peripheral blasts, and duration of CR. All P values were based on 2-sided tests. Results: A total of 3160 patients were enrolled in the 9 studies. The median follow-up on patients still alive was 10.0 years. Among those 3160 patients enrolled, 1864 (58.9%) achieved first CR of which 1086 (58.2%) had documented relapse. The median age at diagnosis of the relapsed patients was 50 (range: 16-84) and 50.6% were males. Fifty percent of the patients had reliable cytogenetic results. Of those, 13.5% had favorable cytogenetics, 55.8% - intermediate and 30.6% had unfavorable baseline results. The median OS from relapse was 0.5 years. The 2- and 5-year OS were 16(±1)% and 10(±1)%, respectively. This is true in even the most contemporary studies (E1900 and E3999) with median OS of 0.6 and 0.4 years, respectively. By age stratification (< or ≥ 55), 5-year OS was 13(±1)% and 5(±1)%, respectively (figure 1). Among patients<55, those with unfavorable cytogenetics had the poorest prognosis (median OS of 0.4 years and 5-year OS of 6(±3)%. Those with favorable and intermediate cytogenetics had similar OS with 0.7 and 0.6 years median OS and 5-year OS of 16(±5)% and 17(±3)%, respectively (figure 2). Multivariate analysis was perfomed on 517 patients who had enough baseline information, including cytogenetics. Factors that were significantly associated with OS from relapse included: age (p<0.001), ECOG performance status (p=0.04), hemoglobin (p=0.03), cytogenetics (p=0.045) at baseline and duration of CR (p<0.001). Conclusions: The short- and long-term OS of AML patients post-relapse is dismal (<10%). Although age<55 and favorable cytogenetics at diagnosis are relatively good prognostic factors, the general survival of even these patients is very poor. Disappointingly, these data are also applicable to the most contemporary studies. Long-term survival was possible only in the minority of patients who survived the relapse, achieved a second CR and then successfully underwent an allogeneic transplant. These data are crucial when considering post-remission strategy, and suggest that offering a therapy most likely to lead to cure in CR1 is the preferred option. Figure 1. Probability of OS from relapse by age group Figure 1. Probability of OS from relapse by age group Figure 2. Probability of OS from relapse by cytogenetic risk for patients age <55 Figure 2. Probability of OS from relapse by cytogenetic risk for patients age <55 Disclosures Douer: Gilead: Consultancy. Rowe:BioSight Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx Ltd.: Consultancy; Amgen: Consultancy.

Abstract The ECOG-ACRIN Cancer Research Group Led Trial, E2902, was a randomized phase III North American Leukemia Intergroup trial of the Farnesyl Transferase Inhibitor , R115777(Tipifarnib), as maintenance therapy for acute myeloid leukemia (AML). Patients (pts) with AML in remission after requiring salvage therapy (primary induction failure or second or subsequent remission) or over the age of 60 in first remission were eligible. Pts were eligible if they were in a complete remission (CR) or complete remission without platelet recovery (CRp) as confirmed by bone marrow examination within 2 weeks prior to randomization. Consolidation or post remission therapy prior to enrollment was allowed but not required. Pts who had received an allogeneic transplant in this remission were ineligible. Adequate blood counts (absolute neutrophil count >= 1000/mm3 and platelet count >=50,000/mm3), and normal renal, and hepatic function were required within 2 weeks of randomization. Pts were randomized to either receive tipifarnib twice daily (BID) or to observation. Pts were stratified by remission ( CR1 vs > CR1) and whether or not they received consolidation therapy in the current remission. The main objective of this study was to compare disease free survival(DFS) in the two groups. Between August 2004 and December 2009, 144 pts were accrued to the study. The median age of the pts was 69 (range 28-86). Seventy-three pts (51%) were male and 135 pts (94%) were white. The majority of pts enrolled on the study (70%) were in first CR, and had post remission chemotherapy (56%) prior to randomization. Seventy-three pts were enrolled onto the treatment arm (Arm A) and 71 pts were enrolled onto the observation arm (Arm B). When the study was initially opened, pts on Arm A were treated with tipifarnib at a dose of 400 mg BID with dose reductions and interruptions defined per protocol for toxicity. After the first planned interim analysis, based on toxicity data, the ECOG Data Monitoring Committee recommended that the starting dose be decreased to 300 mg BID. While non-hematologic toxicity was acceptable, significant hematologic toxicity was seen in pts treated at an initial dose of either 400 mg or 300 mg BID. Of note, hematologic toxicity was also seen on the observation arm although less frequently than with both treatment doses. The primary endpoint of the study was analysis of DFS on an intent to treat basis. The median DFS for arms A and B are 8.87 months and 5.26 months respectively ( p=0.058, HR 0.760). When restricted to eligible patients only(n=134 ), the DFS for arms A and B are 10.81 vs 5.26 months, respectively ( p=0.019, HR 0.690). An unplanned subgroup analysis was performed by gender. The median DFS for male pts (n=73) is 5.36 vs 5.91 months for arms A and B respectively (p=0.868, HR 1.320) and 12.09 vs 3.91 months for female pts (n=71)(p=0.0002, HR=0.408) In a multivariate Cox proportional hazards model to adjust for baseline clinical variables, there is a significant interaction between treatment and gender. (Table 1) Similar results were noted for OS (Figure 1). ECOG ACRIN Trial E2902 evaluated the potential role of Tipifarnib ( R115777) in patients with AML in remission with a high risk of relapse. While the primary endpoint for improved DFS was not reached when evaluating all randomized pts, when restricted to eligible patients only, there is a statistically significant improvement in DFS seen. Moreover, an unplanned subgroup analysis by gender demonstrates an improvement in DFS and OS for females who were enrolled on the study which persists on multivariate analysis. Possible explanations include the use of flat dosing rather than BSA based dosing on this study. However futher evaluation of patient and disease characteristics, dosing and toxicity profiles of responders and nonresponders is needed. Given what appears to be a potentially clinically relevant benefit, further evaluation of this agent is warranted. Table 1. Multivariate Cox Proportional Hazards Model for DFS: All Randomized Patients Hazard Ratio (95% CI) P value Arm B vs A 2.559(1.547,4.236) 0.00025 Male vs Female 2.005(1.228,3.274) 0.00544 CR1 vs >CR1 0.447 (0.305,0.656) 0.00004 Figure 1. (A) DFS (B) OS in Female Patients Figure 1. (A) DFS (B) OS in Female Patients Disclosures Rowe: BioSight Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; BioLineRx Ltd.: Consultancy. Larson:Astellas: Consultancy, Research Funding.