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Purpose To determine the frequency of TET2 mutations, their associations with clinical and molecular characteristics and outcome, and the associated gene- and microRNA-expression signatures in patients with primary cytogenetically normal acute myeloid leukemia (CN-AML). Patients and Methods Four-hundred twenty-seven patients with CN-AML were analyzed for TET2 mutations by polymerase chain reaction and direct sequencing and for established prognostic gene mutations. Gene- and microRNA-expression profiles were derived using microarrays. Results TET2 mutations, found in 23% of patients, were associated with older age (P < .001) and higher pretreatment WBC (P = .04) compared with wild-type TET2 (TET2-wt). In the European LeukemiaNet (ELN) favorable-risk group (patients with CN-AML who have mutated CEBPA and/or mutated NPM1 without FLT3 internal tandem duplication [FLT3-ITD]), TET2-mutated patients had shorter event-free survival (EFS; P < .001) because of a lower complete remission (CR) rate (P = .007), and shorter disease-free survival (DFS; P = .003), and also had shorter overall survival (P = .001) compared with TET2-wt patients. TET2 mutations were not associated with outcomes in the ELN intermediate-I–risk group (CN-AML with wild-type CEBPA and wild-type NPM1 and/or FLT3-ITD). In multivariable models, TET2 mutations were associated with shorter EFS (P = .004), lower CR rate (P = .03), and shorter DFS (P = .05) only among favorable-risk CN-AML patients. We identified a TET2 mutation-associated gene-expression signature in favorable-risk but not in intermediate-I–risk patients and found distinct mutation-associated microRNA signatures in both ELN groups. Conclusion TET2 mutations improve the ELN molecular-risk classification in primary CN-AML because of their adverse prognostic impact in an otherwise favorable-risk patient subset. Our data suggest that these patients may be candidates for alternative therapies.

Purpose To evaluate the prognostic significance of expression levels of a single microRNA, miR-181a, in the context of established molecular markers in cytogenetically normal acute myeloid leukemia (CN-AML), and to gain insight into the leukemogenic role of miR-181a. Patients and Methods miR-181a expression was measured in pretreatment marrow using Ohio State University Comprehensive Cancer Center version 3.0 arrays in 187 younger (< 60 years) adults with CN-AML. Presence of other molecular prognosticators was assessed centrally. A gene-expression profile associated with miR-181a expression was derived using microarrays and evaluated by Gene-Ontology analysis. Results Higher miR-181a expression associated with a higher complete remission (CR) rate (P = .04), longer overall survival (OS; P = .01) and a trend for longer disease-free survival (DFS; P = .09). The impact of miR-181a was most striking in poor molecular risk patients with FLT3-internal tandem duplication (FLT3-ITD) and/or NPM1 wild-type, where higher miR-181a expression associated with a higher CR rate (P = .009), and longer DFS (P < .001) and OS (P < .001). In multivariable analyses, higher miR-181a expression was significantly associated with better outcome, both in the whole patient cohort and in patients with FLT3-ITD and/or NPM1 wild-type. These results were also validated in an independent set of older (≥ 60 years) patients with CN-AML. A miR-181a-associated gene-expression profile was characterized by enrichment of genes usually involved in innate immunity. Conclusion To our knowledge, we provide the first evidence that the expression of a single microRNA, miR-181a, is associated with clinical outcome of patients with CN-AML and may refine their molecular risk classification. Targeted treatments that increase endogenous levels of miR-181a might represent novel therapeutic strategies.

The clinical impact of FLT3-internal tandem duplications (ITDs), an adverse prognostic marker in adults aged < 60 years with primary cytogenetically normal acute myeloid leukemia (CN-AML), requires further investigation in older patients. In CN-AML patients aged ≥ 60 years treated on Cancer and Leukemia Group B frontline trials, we found that FLT3-ITD remained associ-ated with shorter disease-free survival (P < .001; hazard ratio = 2.10) and overall survival (P < .001; hazard ratio = 1.97) in multivariable analyses. This impact on shorter disease-free survival and overall survival was in patients aged 60-69 (P < .001, each) rather than in those aged ≥ 70 years. An FLT3-ITD–associated gene-expression signature revealed overexpression of FLT3, homeobox genes (MEIS1, PBX3, HOXB3), and immunotherapeutic tar-gets (WT1, CD33) and underexpression of leukemia-associated (MLLT3, TAL1) and erythropoiesis-associated (GATA3, EPOR, ANK1, HEMGN) genes. An FLT3-ITD–associated microRNA-expression signature included overexpressed miR-155 and underexpressed miR-144 and miR-451. FLT3-ITD identifies older CN-AML patients with molecular high risk and is associated with gene- and microRNA-expression signatures that provide biologic insights for novel therapeutic approaches.

BAALC and ERG expression levels are prognostic markers in younger (< 60 years) cytogenetically normal acute myeloid leukemia (CN-AML) adults; their prognostic impact in older (≥ 60 years) patients requires further investigation. We evaluated pretreatment expression of BAALC and ERG in 158 de novo patients treated on cytarabine/daunorubicin-based protocols. The patients were also characterized for other established molecular prognosticators. Low BAALC and ERG expression levels were associated with better outcome in univariable and multivariable analyses. Expression levels of both BAALC and ERG were the only factors significantly associated with overall survival upon multivariable analysis. To gain biological insights, we derived gene expression signatures associated with BAALC and ERG expression in older CN-AML patients. Furthermore, we derived the first microRNA expression signatures associated with the expression of these 2 genes. In low BAALC expressers, genes associated with undifferentiated hematopoietic precursors and unfavorable outcome predictors were down-regulated, whereas HOX genes and HOX-gene–embedded microRNAs were up-regulated. Low ERG expressers presented with down-regulation of genes involved in the DNA-methylation machinery, and up-regulation of miR-148a, which targets DNMT3B. We conclude that in older CN-AML patients, low BAALC and ERG expression associates with better outcome and distinct gene and microRNA expression signatures that could aid in identifying new targets and novel therapeutic strategies for older patients.

We previously reported the adverse prognostic impact of Wilms tumor 1 gene (WT1) mutations in younger adult cytogenetically normal acute myeloid leukemia (CN-AML). Here, we investigated 243 older (≥ 60 years) primary CN-AML patients. WT1 mutated (WT1mut) patients (7%) had FLT3-ITD more frequently (P < .001), lower hemoglobin (P = .01), higher white blood cell count (P = .03) and percentage blood blasts (P = .03), and a shorter overall survival (P = .08) than WT1 wild-type (WT1wt) patients. Comparing older and younger WT1mut patients, they had similar pretreatment characteristics and outcome. By contrast, among WT1wt CN-AML, younger patients had a significantly better outcome. A WT1 mutation-associated gene-expression signature, reported here for the first time, included CD96, a leukemia stem cell-specific marker, and genes involved in gene regulation (eg, MLL, PML, and SNRPN) and in proliferative and metabolic processes (eg, INSR, IRS2, and PRKAA1), supporting the role of mutated WT1 in deregulating multiple homeostatic processes. Our results indicate that WT1mut CN-AML represents a distinct entity with poor treatment response across age groups. This study has been registered at www.clinicaltrials.gov as #NCT00900224.

Purpose To analyze the frequency and associations with prognostic markers and outcome of mutations in IDH genes encoding isocitrate dehydrogenases in adult de novo cytogenetically normal acute myeloid leukemia (CN-AML). Patients and Methods Diagnostic bone marrow or blood samples from 358 patients were analyzed for IDH1 and IDH2 mutations by DNA polymerase chain reaction amplification/sequencing. FLT3, NPM1, CEBPA, WT1, and MLL mutational analyses and gene- and microRNA-expression profiling were performed centrally. Results IDH mutations were found in 33% of the patients. IDH1 mutations were detected in 49 patients (14%; 47 with R132). IDH2 mutations, previously unreported in AML, were detected in 69 patients (19%; 13 with R172 and 56 with R140). R172 IDH2 mutations were mutually exclusive with all other prognostic mutations analyzed. Younger age (< 60 years), molecular low-risk (NPM1-mutated/FLT3-internal tandem duplication–negative) IDH1-mutated patients had shorter disease-free survival than molecular low-risk IDH1/IDH2-wild-type (wt) patients (P = .046). R172 IDH2-mutated patients had lower complete remission rates than IDH1/IDH2wt patients (P = .007). Distinctive microarray gene- and microRNA-expression profiles accurately predicted R172 IDH2 mutations. The highest expressed gene and microRNAs in R172 IDH2-mutated patients compared with the IDH1/IDH2wt patients were APP (previously associated with complex karyotype AML) and miR-1 and miR-133 (involved in embryonal stem-cell differentiation), respectively. Conclusion IDH1 and IDH2 mutations are recurrent in CN-AML and have an unfavorable impact on outcome. The R172 IDH2 mutations, previously unreported in AML, characterize a novel subset of CN-AML patients lacking other prognostic mutations and associate with unique gene- and microRNA-expression profiles that may lead to the discovery of novel, therapeutically targetable leukemogenic mechanisms.

Purpose To determine the prognostic importance of the meningioma 1 (MN1) gene expression levels in the context of other predictive molecular markers, and to derive MN1 associated gene– and microRNA–expression profiles in cytogenetically normal acute myeloid leukemia (CN-AML). Patients and Methods MN1 expression was measured in 119 untreated primary CN-AML adults younger than 60 years by real-time reverse-transcriptase polymerase chain reaction. Patients were also tested for FLT3, NPM1, CEBPA, and WT1 mutations, MLL partial tandem duplications, and BAALC and ERG expression. Gene- and microRNA-expression profiles were attained by performing genome-wide microarray assays. Patients were intensively treated on two first-line Cancer and Leukemia Group B clinical trials. Results Higher MN1 expression associated with NPM1 wild-type (P < .001), increased BAALC expression (P = .004), and less extramedullary involvement (P = .01). In multivariable analyses, higher MN1 expression associated with a lower complete remission rate (P = .005) after adjustment for WBC; shorter disease-free survival (P = .01) after adjustment for WT1 mutations, FLT3 internal tandem duplications (FLT3-ITD), and high ERG expression; and shorter survival (P = .04) after adjustment for WT1 and NPM1 mutations, FLT3-ITD, and WBC. Gene- and microRNA-expression profiles suggested that high MN1 expressers share features with high BAALC expressers and patients with wild-type NPM1. Higher MN1 expression also appears to be associated with genes and microRNAs that are active in aberrant macrophage/monocytoid function and differentiation. Conclusion MN1 expression independently predicts outcome in CN-AML patients. The MN1 gene- and microRNA-expression signatures suggest biologic features that could be exploited as therapeutic targets.

Abstract 98 Mutations in the TET2 gene were recently identified in a variety of myeloid neoplasms including AML. However, the frequency and clinical relevance of TET2 mutations in CN-AML, the largest cytogenetic subgroup of adult AML, have not been well defined. We report here the frequency and spectrum of TET2 mutations, and their associations with clinical and molecular characteristics, treatment outcomes and genome-wide gene- and microRNA (miR)-expression signatures in a relatively large cohort of 427 patients (pts) with primary CN-AML. The pts, aged 18–83 years, were intensively treated on CALGB frontline protocols, and were analyzed centrally for TET2 mutations by PCR and direct sequencing, and for other prognostic gene mutations (FLT3 internal tandem duplications [ITD] and tyrosine kinase domain mutations, MLL partial tandem duplications, and mutations in NPM1, CEBPA, WT1 and IDH1&IDH2). If available, buccal swabs or remission marrow samples were used to determine TET2 germline status. Gene- and miR-expression profiles were derived using microarrays (Affymetrix HG-U133 plus 2.0 and OSUCCC custom miR array v4.0). At least 1 sequence variation in TET2 was found in 104 pts. Frameshift (n=59) and nonsense (n=34) variations were distributed throughout all coding exons, while missense changes (n=37) clustered mainly (28/37) in 2 evolutionarily conserved domains of TET2. The remaining missense variations in 9 pts were located outside the conserved domains, and analysis of available buccal swabs or remission samples showed that these sequence changes were present in the germline. Since it is unclear whether they represent innocent polymorphisms or disease-relevant mutations, these 9 pts were excluded from further analyses. TET2-mutated (TET2-mut) pts were older (P Disclosures: No relevant conflicts of interest to declare.

Abstract 577 Trisomy 8 (+8), the most frequent numerical aberration in AML, occurs in approximately 10% of pts. In one-third of them, +8 is the sole chromosome (chr) anomaly; it is considered to confer an intermediate/adverse prognosis but further studies are required to define the clinical and biologic significance of this cytogenetic abnormality. We assessed the associations of sole +8 with prognostic gene mutations, outcome, and gene and miR expression profiles by comparing a relatively large cohort of sole +8 de novo AML pts (n=80; median, 63 years [y]; range, 18–84 y) with cytogenetically normal (CN) de novo AML pts (n=483; median, 60 y; range, 18–83 y). CN pts constitute the largest cytogenetic group in AML, with an overall intermediate prognosis modified by molecular markers. Markers analyzed in the present sole +8 cohort included mutations in NPM1, FLT3 (FLT3 internal tandem duplication [ITD], FLT3 tyrosine kinase domain), CEBPA, WT1, IDH1/2, N/KRAS and RUNX1. All pts were enrolled on frontline cytarabine/daunorubicin based CALGB protocols. No pt included in outcome analyses received allogeneic stem cell transplant in 1st complete remission (CR). Median follow-up was 7.1 y for pts alive. Compared with CN pts, sole +8 pts had lower platelet counts (P Disclosures: No relevant conflicts of interest to declare.

In younger (60 y) requires further evaluation. Pre-therapy bone marrow samples from 140 older de novo CN-AML pts (median age 68 y; range 60 – 81 y) enrolled on cytarabine/daunorubicin-based protocols were studied centrally by RT-PCR. Using the median expression value for MN1, as a cutoff pts were grouped as high & low MN1 expressers. Analysis of the mutation status for FLT3-ITD, FLT3-TKD, NPM1, CEBPA & WT1 & gene & microRNA (miR) expression profiling using, respectively, Affymetrix U133 plus 2.0 & OSU CCC v4.0 arrays, were performed centrally. At diagnosis, higher MN1 associated with wild-type NPM1 (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2717.

The Cancer and Leukemia Group B has performed central review of karyotypes submitted by institutional cytogenetics laboratories from patients with acute myeloid (AML) and acute lymphoblastic (ALL) leukemia since 1986. We assessed the role of central karyotype review in maintaining accurate, high quality cytogenetic data for clinical and translational studies using two criteria: the proportion of karyotypes rejected (i.e. inadequate), and, among accepted (i.e. adequate) cases, the proportion of karyotypes whose interpretation was changed on central karyotype review. We compared the first four years during which central karyotype review was performed with a recent 4-year period and found that the proportion of rejected samples decreased significantly for both AML and ALL. However, during the latter period, central karyotype reviews still found 8% of AML and 16% of ALL karyotypes inadequate. Among adequate cases, the karyotype was revised in 26% of both AML and ALL samples. Some revisions resulted in changing the patients' assignment to particular World Health Organization diagnostic categories and/or moving patients from one prognostic group to another. Overall, when both data on rejection rates and data on karyotype revisions made in accepted cases were considered together, 32% of AML and 38% of ALL samples submitted were either rejected or revised on central karyotype review during the recent 4-year period. These data underscore the necessity of continued central karyotype review in multi-institutional cooperative group studies.

Abstract 1616 Poster Board I-642 CD74 is a type II integral membrane protein receptor that binds its ligand MIF to induce phosphorylation of the extracellular signal-regulated kinase-1/2 (ERK-1/2) and drive cellular proliferation via nuclear factor-kappa B (NF-kB) activation. CD74 expression has been identified in human solid tumors, and its expression is associated with adverse prognosis in advanced pancreatic cancer. As CD74 is expressed and NF-kB constitutively activated in myeloblasts, we hypothesized that CD74 expression might also be associated with adverse outcome in AML. To investigate the prognostic impact of CD74 expression in the context of other predictive molecular markers in CN-AML, we assessed CD74 expression levels by Affymetrix HG-U133 Plus 2.0 microarray in 102 younger [ Figure 1 Disease free survival Figure 1. Disease free survival Figure 2 Overall survival Figure 2. Overall survival Disclosures No relevant conflicts of interest to declare.

Abstract 2602 Poster Board II-578 One of the major revisions in the 2008 WHO classification of “AML and Related Precursor Neoplasms” is the use of certain cytogenetic abnormalities (abns) as a criterion for inclusion in the “AML with myelodysplasia-related changes” category. These abns include 9 specific reciprocal translocations, 8 unbalanced abns, and complex karyotypes (CK), ie, ≥3 unrelated abns. The clinical features and outcome of patients (pts) with these abns require further study to confirm the appropriateness of their inclusion in this WHO category. Therefore, we evaluated 2,724 consecutive untreated adults meeting criteria for possible inclusion in this WHO category (ie, non-therapy-related AML and not part of the first WHO AML category) in the CALGB cytogenetics database; 516 (19%) pts harbored ≥1 myelodysplasia-related abn and/or CK, and had outcome data available. Their median age was 62 years (y; range, 15–88 y). The 9 reciprocal translocations were very rare, with t(3;5)(q25;q34) found in 6 pts, t(3;21)(q26.2;q22.1) in 2, t(1;3)(p36.3;q21.1) and t(2;11)(p21;q23) in 1 pt each and the remaining 5 translocations not detected. Their rarity precluded further analyses. With the exception of idic(X)(q13), found in 2 pts, the remaining 7 unbalanced abns were more common (Table). Importantly, the abns were not mutually exclusive, ie, ≥2 different abns could co-exist in the same karyotype and/or be part of a CK. Among 453 pts with ≥1 specific unbalanced abn (Table), 62% had CK; the highest % of CK, 89–95%, were seen in the i(17q) or t(17p), −13 or del(13q) and −5 or del(5q) groups and the lowest, only 24%, in del(9q) pts. As a group, non-CK pts had a higher complete remission (CR) rate (P=.002) and longer overall survival (OS; P Disclosures: No relevant conflicts of interest to declare.

214 BAALC & ERG are aberrantly expressed in younger (1 (

Abstract 1579 Poster Board I-605 While the prognostic role of FLT3 ITD has been validated in younger CN-AML adults (2-fold higher miR-155 expression in FLT3 ITD pts, was associated with shorter DFS and OS in the 60-69 y pt subgroup (P=.001, each) but not in the ≥70 y subgroup (P=.26 and P=.89, respectively), suggesting an age-associated prognostic role of the miRs. In summary, our data show FLT3 ITD is an independent marker for poor outcome in CN-AML pts aged 60-69 y but not in those aged ≥70 y. Although the ≥70 y pts with FLT3 ITD had a seemingly better prognosis than the corresponding 60-69 y pts, the outcome for both groups is poor and novel treatment approaches are needed in older pts. Table 1 Outcomes in older CN-AML pts with and without FLT3ITD Overall 60-69 y Pts ≥70 y Pts FLT3 ITD (n=72) FLT3 WT (n=147) P FLT3 ITD (n=41) FLT3 WT (n=78) P FLT3 ITD (n=31) FLT3 WT (n=69) P % achieving CR 67% 70% .64 71% 75% .67 61% 65% .82 DFS % disease-free at 3 y 10% 18% .007 7% 19% 1) indicate lower (higher) risk for an event for the first category listed for the dichotomous variables and for the higher values of the continuous variables. Variables considered in the models were those significant at á=0.20 in univariable analyses. † Variable did not meet the proportional hazards assumption, a covariate was used to account for time dependence. Disclosures Stone: Cephalon: ad hoc consultancy; Novartis: Research Funding, ad hoc consultancy.