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Salivary duct carcinomas (SDC) and Her2/Neu3-overexpressing invasive breast carcinomas (HNPIBC/IBC) are histologically indistinguishable. We investigated whether common histopathologic and immunophenotypic features of SDC and IBC are mirrored by a similar microRNA (miRNA) profile. MiRNA profiling of 5 SDCs, 6 IBCs Her2/Neu3+, and 5 high-grade ductal breast carcinoma in situ (DCIS) was performed by NanoString platform. Selected miRNAs and HOXA1 gene were validated by RT-PCR. We observed similar miRNA expression profiles between IBC and SDC with the exception of 2 miRNAs, miR-10a and miR-142-3p, which were higher in IBC tumors. DCIS tumors displayed increased expression of miR-10a, miR-99a, miR-331-3p and miR-335, and decreased expression of miR-15a, miR-16 and miR-19b compared to SDC. The normal salivary gland and breast tissues also showed similar expression profiles. Interestingly, miR-10a was selectively increased in both IBC and normal breast tissue compared to SDC and normal salivary gland tissue. Moreover, our NanoString and RT-PCR data confirmed that miR-10a was upregulated in IBC and DCIS compared to SDC. Finally, we show downregulation of HOXA1, a miR-10 target, in IBC tumors compared to normal breast tissue. Taken together, our data demonstrates that, based on miRNA profiling, SDC is closely related to HNPIBC. Our results also suggest that miR-10a is differentially expressed in IBC compared to SDC and may have potential utility as a diagnostic biomarker in synchronous or metachronous malignant epithelial malignancies involving both organs. In addition, miR-10a could be playing an important role as a mammary-specific oncogene, involved in breast cancer initiation (DCIS) and progression (IBC), through mechanisms that include modulation of HOXA1 gene expression. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12105-018-0971-x) contains supplementary material, which is available to authorized users.

Receptor-interacting protein kinase-3 (RIP3 or RIPK3) is an essential part of the cellular machinery that executes "programmed" or "regulated" necrosis. Here we show that programmed necrosis is activated in response to many chemotherapeutic agents and contributes to chemotherapy-induced cell death. However, we show that RIP3 expression is often silenced in cancer cells due to genomic methylation near its transcriptional start site, thus RIP3-dependent activation of MLKL and downstream programmed necrosis during chemotherapeutic death is largely repressed. Nevertheless, treatment with hypomethylating agents restores RIP3 expression, and thereby promotes sensitivity to chemotherapeutics in a RIP3-dependent manner. RIP3 expression is reduced in tumors compared to normal tissue in 85% of breast cancer patients, suggesting that RIP3 deficiency is positively selected during tumor growth/development. Since hypomethylating agents are reasonably well-tolerated in patients, we propose that RIP3-deficient cancer patients may benefit from receiving hypomethylating agents to induce RIP3 expression prior to treatment with conventional chemotherapeutics.

Emerging data demonstrate important roles for the TYRO3/AXL/MERTK receptor tyrosine kinase (TAM RTK) family in diverse cancers. We investigated the prognostic relevance of GAS6 expression, encoding the common TAM RTK ligand, in 270 adults (n=71 aged

Recently, we showed that increased miR-181a expression was associated with improved outcomes in cytogenetically normal acute myeloid leukemia (CN-AML). Interestingly, miR-181a expression was increased in CN-AML patients harboring CEBPA mutations, which are usually biallelic and associate with better prognosis. CEBPA encodes the C/EBPα transcription factor. We demonstrate here that the presence of N-terminal CEBPA mutations and miR-181a expression are linked. Indeed, the truncated C/EBPα-p30 isoform, which is produced from the N-terminal mutant CEBPA gene or from the differential translation of wild-type CEBPA mRNA and is commonly believed to have no transactivation activity, binds to the miR-181a-1 promoter and up-regulates the microRNA expression. Furthermore, we show that lenalidomide, a drug approved for myelodysplastic syndromes and multiple myeloma, enhances translation of the C/EBPα-p30 isoform, resulting in higher miR-181a levels. In xenograft mouse models, ectopic miR-181a expression inhibits tumor growth. Similarly, lenalidomide exhibits antitumorigenic activity paralleled by increased miR-181a expression. This regulatory pathway may explain an increased sensitivity to apoptosis-inducing chemotherapy in subsets of AML patients. Altogether, our data provide a potential explanation for the improved clinical outcomes observed in CEBPA-mutated CN-AML patients, and suggest that lenalidomide treatment enhancing the C/EBPα-p30 protein levels and in turn miR-181a may sensitize AML blasts to chemotherapy.

High BAALC expression levels are associated with poor outcome in cytogenetically normal acute myeloid leukemia (CN-AML) patients. Recently, miR-3151 was discovered in intron 1 of BAALC. To evaluate the prognostic significance of miR-3151 expression levels and to gain insight into the biologic and prognostic interplay between miR-3151 and its host, miR-3151 and BAALC expression were measured in pretreatment blood of 179 CN-AML patients. Gene-expression profiling and miRNA-expression profiling were performed using microarrays. High miR-3151 expression was associated with shorter disease-free and overall survival, whereas high BAALC expression predicted failure of complete remission and shorter overall survival. Patients exhibiting high expression of both miR-3151 and BAALC had worse outcome than patients expressing low levels of either gene or both genes. In gene-expression profiling, high miR-3151 expressers showed down-regulation of genes involved in transcriptional regulation, posttranslational modification, and cancer pathways. Two genes, FBXL20 and USP40, were validated as direct miR-3151 targets. The results of the present study show that high expression of miR-3151 is an independent prognosticator for poor outcome in CN-AML and affects different outcome end points than its host gene, BAALC. The combination of both markers identified a patient subset with the poorest outcome. This interplay between an intronic miR and its host may have important biologic implications.

Increased expression levels of miR-181 family members have been shown to be associated with favorable outcome in patients with cytogenetically normal acute myeloid leukemia. Here we show that increased expression of miR-181a and miR-181b is also significantly (P < .05; Cox regression) associated with favorable overall survival in cytogenetically abnormal AML (CA-AML) patients. We further show that up-regulation of a gene signature composed of 4 potential miR-181 targets (including HOXA7, HOXA9, HOXA11, and PBX3), associated with down-regulation of miR-181 family members, is an independent predictor of adverse overall survival on multivariable testing in analysis of 183 CA-AML patients. The independent prognostic impact of this 4-homeobox-gene signature was confirmed in a validation set of 271 CA-AML patients. Furthermore, our in vitro and in vivo studies indicated that ectopic expression of miR-181b significantly promoted apoptosis and inhibited viability/proliferation of leukemic cells and delayed leukemogenesis; such effects could be reversed by forced expression of PBX3. Thus, the up-regulation of the 4 homeobox genes resulting from the down-regulation of miR-181 family members probably contribute to the poor prognosis of patients with nonfavorable CA-AML. Restoring expression of miR-181b and/or targeting the HOXA/PBX3 pathways may provide new strategies to improve survival substantially.

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.

Purpose To evaluate the prognostic significance of CEBPA mutations in the context of established molecular markers in cytogenetically normal (CN) acute myeloid leukemia (AML) and gain biologic insights into leukemogenesis of the CN-AML molecular high-risk subset (FLT3 internal tandem duplication [ITD] positive and/or NPM1 wild type) that has a significantly higher incidence of CEBPA mutations than the molecular low-risk subset (FLT3-ITD negative and NPM1 mutated). Patients and Methods One hundred seventy-five adults age less than 60 years with untreated primary CN-AML were screened before treatment for CEBPA, FLT3, MLL, WT1, and NPM1 mutations and BAALC and ERG expression levels. Gene and microRNA (miRNA) expression profiles were obtained for the CN-AML molecular high-risk patients. Results CEBPA mutations predicted better event-free (P = .007), disease-free (P = .014), and overall survival (P < .001) independently of other molecular and clinical prognosticators. Among patients with CEBPA mutations, 91% were in the CN-AML molecular high-risk group. Within this group, CEBPA mutations predicted better event-free (P < .001), disease-free (P = .004), and overall survival (P = .009) independently of other molecular and clinical characteristics and were associated with unique gene and miRNA expression profiles. The major features of these profiles were upregulation of genes (eg, GATA1, ZFPM1, EPOR, and GFI1B) and miRNAs (ie, the miR-181 family) involved in erythroid differentiation and downregulation of homeobox genes. Conclusion Pretreatment testing for CEBPA mutations identifies CN-AML patients with different outcomes, particularly in the molecular high-risk group, thus improving molecular risk-based classification of this large cytogenetic subset of AML. The gene and miRNA expression profiling provided insights into leukemogenesis of the CN-AML molecular high-risk group, indicating that CEBPA mutations are associated with partial erythroid differentiation.

Posttranslationally modified histones and DNA hypermethylation frequently interplay to deregulate gene expression in cancer. We report that acute myeloid leukemia (AML) with an aberrant histone methyltransferase, the mixed lineage leukemia partial tandem duplication (MLL-PTD), exhibits increased global DNA methylation versus AML with MLL-wildtype (MLL-WT; P = .02). Among the differentially methylated genes, the SLC5A8 tumor suppressor gene (TSG) was more frequently hypermethylated (P = .003). In MLL-PTD+ cell lines having SLC5A8 promoter hypermethylation, incubation with decitabine activated SLC5A8 expression. Ectopic SLC5A8 expression enhanced histones H3 and H4 acetylation in response to the histone deacetylase inhibitor, valproate, consistent with the encoded protein—SMCT1—short-chain fatty acid transport function. In addition, enhanced cell death was observed in SMCT1-expressing MLL-PTD+ AML cells treated with valproate. Within the majority of MLL-PTD AML is a mechanism in which DNA hypermethylation silences a TSG that, together with MLL-PTD, can contribute further to aberrant chromatin remodeling and altered gene expression.

MLL (ALL-1) chimeric fusions and MLL partial tandem duplications (PTD) may have mechanistically distinct contributions to leukemogenesis. Acute myeloid leukemia (AML) blasts with the t(9;11)(p22; q23) express MLL-AF9 and MLL wild-type (WT) transcripts, while normal karyotype AML blasts with the MLLPTD/WT genotype express MLL PTD but not the MLL WT. Silencing of MLL WT in MLLPTD/WT blasts was reversed by DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors, and MLL WT induction was associated with selective sensitivity to cell death. Reduction of MLL PTD expression induced MLL WT and reduced blast colony-forming units, supporting opposing functions for MLL PTD and MLL WT whereby the MLL PTD contributes to the leukemic phenotype via a recessive gain-of-function. The coincident suppression of the MLL WT allele with the expression of the MLL PTD allele, along with the functional data presented here, supports the hypothesis that loss of WT MLL function via monoallelic repression contributes to the leukemic phenotype by the remaining mutant allele. These data from primary AML and the pharmacologic reversal of MLL WT silencing associated with a favorable alteration in the threshold for apoptosis suggest that these patients with poor prognosis may benefit from demethylating or histone deacetylase inhibitor therapy, or both.

Current treatments for acute myeloid leukemia (AML) are designed to target rapidly dividing blast populations with limited success in eradicating the functionally distinct leukemia stem cell (LSC) population, which is postulated to be responsible for disease resistance and relapse. We have previously reported high miR-126 expression levels to be associated with a LSC-gene expression profile. Therefore, we hypothesized that miR-126 contributes to “stemness” and is a viable target for eliminating the LSC in AML. Here we first validate the clinical relevance of miR-126 expression in AML by showing that higher expression of this microRNA (miR) is associated with worse outcome in a large cohort of older (≥60 years) cytogenetically normal AML patients treated with conventional chemotherapy. We then show that miR-126 overexpression characterizes AML LSC-enriched cell subpopulations and contributes to LSC long-term maintenance and self-renewal. Finally, we demonstrate the feasibility of therapeutic targeting of miR-126 in LSCs with novel targeting nanoparticles (NP) containing antagomiR-126 resulting in in vivo reduction of LSCs likely by depletion of the quiescent cell subpopulation. Our findings suggest that by targeting a single miR, i.e., miR-126, it is possible to interfere with LSC activity, thereby opening potentially novel therapeutic approaches to treat AML patients.

Background The purpose of this study is to determine whether microRNA for pluripotent stem cells are also expressed in breast cancer and are associated with metastasis and outcome. Methods We studied global microRNA profiles during differentiation of human embryonic stem cells (n =26) and in breast cancer patients (n = 33) and human cell lines (n = 35). Using in situ hybridization, we then investigated MIR302 expression in 318 untreated breast cancer patients (test cohort, n = 22 and validation cohort, n = 296). In parallel, using next-generation sequencing data from breast cancer patients (n = 684), we assessed microRNA association with stem cell markers. All statistical tests were two-sided. Results In healthy tissues, the MIR302 (high)/MIR203 (low) asymmetry was exclusive for pluripotent stem cells. MIR302 was expressed in a small population of cancer cells within invasive ductal carcinoma, but not in normal breast (P < .001). Furthermore, MIR302 was expressed in the tumor cells together with stem cell markers, such as CD44 and BMI1. Conversely, MIR203 expression in 684 breast tumors negatively correlated with CD44 (Spearman correlation, Rho = -0.08, P = .04) and BMI1 (Rho = -0.11, P = .004), but positively correlated with differentiation marker CD24 (Rho = 0.15, P < .001). Primary tumors with lymph node metastasis had cancer cells showing scattered expression of MIR302 and widespread repression of MIR203. Finally, overall survival was statistically significantly shorter in patients with MIR302-positive cancer cells (P = .03). Conclusions In healthy tissues the MIR302(high)/MIR203(low) asymmetry was characteristic of embryonic and induced pluripotency. In invasive ductal carcinoma, the MIR302/MIR203 asymmetry was associated with stem cell markers, metastasis, and shorter survival.

Purpose Molecular risk stratification of acute myeloid leukemia (AML) is largely based on genetic markers. However, epigenetic changes, including DNA methylation, deregulate gene expression and may also have prognostic impact. We evaluated the clinical relevance of integrating DNA methylation and genetic information in AML. Methods Next-generation sequencing analysis of methylated DNA identified differentially methylated regions (DMRs) associated with prognostic mutations in older (≥ 60 years) cytogenetically normal (CN) patients with AML (n = 134). Genes with promoter DMRs and expression levels significantly associated with outcome were used to compute a prognostic gene expression weighted summary score that was tested and validated in four independent patient sets (n = 355). Results In the training set, we identified seven genes (CD34, RHOC, SCRN1, F2RL1, FAM92A1, MIR155HG, and VWA8) with promoter DMRs and expression associated with overall survival (OS; P ≤ .001). Each gene had high DMR methylation and lower expression, which were associated with better outcome. A weighted summary expression score of the seven gene expression levels was computed. A low score was associated with a higher complete remission (CR) rate and longer disease-free survival and OS (P < .001 for all end points). This was validated in multivariable models and in two younger (< 60 years) and two older independent sets of patients with CN-AML. Considering the seven genes individually, the fewer the genes with high expression, the better the outcome. Younger and older patients with no genes or one gene with high expression had the best outcomes (CR rate, 94% and 87%, respectively; 3-year OS, 80% and 42%, respectively). Conclusion A seven-gene score encompassing epigenetic and genetic prognostic information identifies novel AML subsets that are meaningful for treatment guidance.

Prognostic gene mutations and distinct gene- and microRNA-expression signatures in acute myeloid leukemia with a sole trisomy 8

The inv(16)(p13q22)/t(16;16)(p13;q22) in acute myeloid leukemia results in multiple CBFB-MYH11 fusion transcripts, with type A being most frequent. The biologic and prognostic implications of different fusions are unclear. We analyzed CBFB-MYH11 fusion types in 208 inv(16)/t(16;16) patients with de novo disease, and compared clinical and cytogenetic features and the KIT mutation status between type A (n = 182; 87%) and non–type A (n = 26; 13%) patients. At diagnosis, non–type A patients had lower white blood counts (P = .007), and more often trisomies of chromosomes 8 (P = .01) and 21 (P < .001) and less often trisomy 22 (P = .02). No patient with non–type A fusion carried a KIT mutation, whereas 27% of type A patients did (P = .002). Among the latter, KIT mutations conferred adverse prognosis; clinical outcomes of non–type A and type A patients with wild-type KIT were similar. We also derived a fusion-type–associated global gene-expression profile. Gene Ontology analysis of the differentially expressed genes revealed—among others—an enrichment of up-regulated genes involved in activation of caspase activity, cell differentiation and cell cycle control in non–type A patients. We conclude that non–type A fusions associate with distinctclinical and genetic features, including lack of KIT mutations, and a unique gene-expression profile.

The associations of mutations in the enhancer of trithorax and polycomb family gene ASXL1 with pretreatment patient characteristics, outcomes, and gene-/microRNA-expression profiles in primary cytogenetically normal acute myeloid leukemia (CN-AML) are unknown. We analyzed 423 adult patients for ASXL1 mutations, other prognostic gene mutations, and gene-/microRNA-expression profiles. ASXL1 mutations were 5 times more common in older (≥ 60 years) patients (16.2%) than those younger than 60 years (3.2%; P < .001). Among older patients, ASXL1 mutations associated with wild-type NPM1 (P < .001), absence of FLT3-internal tandem duplications (P = .002), mutated CEBPA (P = .01), and with inferior complete remission (CR) rate (P = .04), disease-free survival (DFS; P = .03), overall survival (OS; P = .006), and event-free survival (EFS; P = .002). Within the European LeukemiaNet (ELN) genetic categories of older CN-AML, ASXL1 mutations associated with inferior CR rate (P = .02), OS (P < .001), and EFS (P < .001) among ELN Favorable, but not among ELN Intermediate-I patients. Multivariable analyses confirmed associations of ASXL1 mutations with unfavorable CR rate (P = .03), DFS (P < .001), OS (P < .001), and EFS (P < .001) among ELN Favorable patients. We identified an ASXL1 mutation-associated gene-expression signature, but no microRNA-expression signature. This first study of ASXL1 mutations in primary CN-AML demonstrates that ASXL1mutated older patients, particularly within the ELN Favorable group, have unfavorable outcomes and may be candidates for experimental treatment approaches.

Low MN1 expression bestows favorable prognosis in younger adults with cytogenetically normal acute myeloid leukemia (CN-AML), but its prognostic significance in older patients is unknown. We analyzed pretherapy MN1 expression in 140 older (≥ 60 years) de novo CN-AML patients treated on cytarabine/daunorubicin-based protocols. Low MN1 expressers had higher complete remission (CR) rates (P = .001), and longer overall survival (P = .03) and event-free survival (EFS; P = .004). In multivariable models, low MN1 expression was associated with better CR rates and EFS. The impact of MN1 expression on overall survival and EFS was predominantly in patients 70 years of age or older, with low MN1 expressers with mutated NPM1 having the best outcome. The impact of MN1 expression was also observed in the Intermediate-I, but not the Favorable group of the European LeukemiaNet classification, where low MN1 expressers had CR rates and EFS similar to those of Favorable group patients. MN1 expresser-status-associated gene- and microRNA-expression signatures revealed underexpression of drug resistance and adverse outcome predictors, and overexpression of HOX genes and HOX-gene–embedded microRNAs in low MN1 expressers. We conclude that low MN1 expression confers better prognosis in older CN-AML patients and may refine the European LeukemiaNet classification. Biologic features associated with MN1 expression may help identify new treatment targets.

The alleles of the Wilms tumor 1 (WT1) polymorphism rs16754 harbor adenine (A) or guanine (G). Recently, rs16754 has been reported to affect the outcome of patients with cytogenetically normal acute myeloid leukemia. To validate this finding, we investigated pretreatment features and outcome associated with rs16754 in a large cohort of patients with cytogenetically normal acute myeloid leukemia.Four-hundred and thirty-three intensively treated and molecularly characterized cytogenetically normal patients with de novo acute myeloid leukemia (18-83 years old) were analyzed for rs16754. To gain biological insights, we studied the gene- and microRNA-expression profiles for associations with rs16754.Three-hundred and nine (71%) patients were homozygous for A (WT1(AA)), 112 (26%) were heterozygous (WT1(AG)) and 12 (3%) were homozygous for G (WT1(GG)). For comparison with previous studies, we grouped WT1(AG) and WT1(GG) patients and compared them with WT1(AA) patients divided into younger (60 years) and older (≥60 years) adults. We found no independent prognostic impact of WT1(AA). However, WT1(GG) patients, who were less often Caucasian than WT1(AG) (P=0.001) or WT1(AA) (P=0.008) patients, and had TET2 mutations more often than WT1(AG) (P=0.02) patients, had, among patients with FLT3-internal tandem duplication and/or NPM1 wild-type, better disease-free (P=0.02) and overall survival (P=0.04) than WT1(AA) and WT1(AG) patients combined. Unsupervised and supervised analyses of the gene- and microRNA-expression profiles suggested that there were no distinct expression patterns associated with any rs16754 genotype.We did not observe the previously reported adverse impact of WT1(AA) but found favorable outcomes associated with the homozygous WT1(GG). Considering its low frequency, confirmatory studies are necessary. The biological significance of rs16754 remains questionable as no distinct expression profiles were associated with the genotypes.

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.

Item does not contain fulltext The GFI1 gene encodes a transcriptional repressor, which regulates myeloid differentiation. In the mouse, Gfi1 deficiency causes neutropenia and an accumulation of granulomonocytic precursor cells that is reminiscent of a myelodysplastic syndrome. We report here that a variant allele of GFI1 (GFI1(36N)) is associated with acute myeloid leukemia (AML) in white subjects with an odds ratio of 1.6 (P < 8 x 10(-5)). The GFI1(36N) variant occurred in 1806 AML patients with an allele frequency of 0.055 compared with 0.035 in 1691 healthy control patients in 2 independent cohorts. We observed that both GFI1 variants maintain the same activity as transcriptional repressors but differ in their regulation by the AML1/ETO (RUNX1/RUNX1T1) fusion protein produced in AML patients with a t(8;21) translocation. AML1/ETO interacts and colocalizes with the more common GFI1(36S) form in the nucleus and inhibits its repressor activity. However, the variant GFI1(36N) protein has a different subnuclear localization than GFI1(36S). As a consequence, AML1/ETO does not colocalize with GFI1(36N) and is unable to inhibit its repressor activity. We conclude that both variants of GFI1 differ in their ability to be regulated by interacting proteins and that the GFI1(36N) variant form exhibits distinct biochemical features that may confer a predisposition to AML.

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.

A role of microRNAs in cancer has recently been recognized. However, little is known about the role of microRNAs in acute myeloid leukemia (AML).Using microRNA expression profiling, we studied samples of leukemia cells from adults under the age of 60 years who had cytogenetically normal AML and high-risk molecular features--that is, an internal tandem duplication in the fms-related tyrosine kinase 3 gene (FLT3-ITD), a wild-type nucleophosmin (NPM1), or both. A microRNA signature that was associated with event-free survival was derived from a training group of 64 patients and tested in a validation group of 55 patients. For the latter, a microRNA compound covariate predictor (called a microRNA summary value) was computed on the basis of weighted levels of the microRNAs forming the outcome signature.Of 305 microRNA probes, 12 (including 5 representing microRNA-181 family members) were associated with event-free survival in the training group (P0.005). In the validation group, the microRNA summary value was inversely associated with event-free survival (P=0.03). In multivariable analysis, the microRNA summary value remained associated with event-free survival (P=0.04) after adjustment for the allelic ratio of FLT3-ITD to wild-type FLT3 and for the white-cell count. Using results of gene-expression microarray analysis, we found that expression levels of the microRNA-181 family were inversely correlated with expression levels of predicted target genes encoding proteins involved in pathways of innate immunity mediated by toll-like receptors and interleukin-1beta.A microRNA signature in molecularly defined, high-risk, cytogenetically normal AML is associated with the clinical outcome and with target genes encoding proteins involved in specific innate-immunity pathways.

In CN-AML, mutations of specific genes have prognostic significance and are used for treatment guidance. However, these mutations are in general not mutually exclusive and CN-AML patients (pts) frequently harbor mutational combinations with unclear prognostic significance because of the concurrent presence of mutations with favorable (CEBPA, NPM1) and unfavorable (ASXL1, DNMT3A, IDH1, IDH2, FLT3-ITD, MLL-PTD, RUNX1, TET2, WT1) impact on outcome. Here, we report the frequency and clinical significance of isolated mutations and their combinations in 364 CN-AML pts. Younger [ Disclosures: Mrózek: AMERICAN SOCIETY OF HEMATOLOGY: I will review abstracls submitted for presentation at the 55th ASH Annual Meeting in the 611 category Other. Eisfeld:AMERICAN SOCIETY OF HEMATOLOGY: I will review abstracls submitted for presentation at the 55th ASH Annual Meeting in the 608 category Other.

1387 [][1] miR-155 is upregulated in aggressive subtypes of solid tumors and leukemia. In AML, higher miR-155 expression is associated with FLT3 -ITD. However, whether miR-155 upregulation impacts on clinical outcome independently from FLT3 -ITD and other prognosticators is unknown. We evaluated the prognostic impact of miR-155 in 363 CN-AML pts (153 age ( (

Abstract 1293 Receptor tyrosine kinases (RTKs) constitutively activated by gene mutation, overexpression and/or autocrine activation via ligand expression have been shown to negatively impact on outcomes of AML patients (pts). AXL, a member of the TAM (TYRO3, AXL, MERTK) RTK gene family was reported to be overexpressed and associated with poor survival in AML (Rochlitz, et al, Leukemia, 1999: 13:1352–8). No AXL mutations have been described, suggesting its activation may occur via aberrant expression in leukemic blasts of a TAM RTK ligand, GAS6. GAS6 was shown to be overexpressed in AML (Dirks, et al Leuk. Res. 23:643–51); yet its prognostic relevance is unknown. We report clinical and molecular associations and prognostic impact of aberrant GAS6 expression, in the context of TAM RTKs and known prognostic markers in de novo CN-AML pts (n=270; aged 18–83 y) treated with cytarabine/anthracycline-based therapies. Sixty-nine (26%) pts expressed GAS6 (>background signal; derived from microarray gene expression profiles of AML samples). TYRO3 expression status [positive (+) vs negative (–)] was similar in GAS6+ and GAS6– pts (P=.74), while AXL+ (P Table. MVA models Variable CR DFS OS EFS P OR (95% CI) P HR (95% CI) P HR (95% CI) P HR (95% CI) GAS6 expression, + v – .02 0.46 (0.24, 0.88) .03* 1.78 (1.07, 2.96) .05* 1.59 (1.00, 2.52) .03* 1.59 (1.06, 2.41) NPM1, mut v wt .001 2.97 (1.53, 5.77) – – – – .006 0.64 (0.46, 0.88) FLT3-ITD, present v absent – – .003 1.66 (1.19, 2.33) – – .005 1.55 (1.14, 2.11) WT1, mut v wt – – – – ORs > ( ( *There are interactions between GAS6 and TYRO3/AXL dual receptor status for DFS (P=.16), OS (P=.06) and EFS (P=.12). The P-values, HRs and CIs are for comparisons of GAS6+ v GAS6– pts within the TYRO3–/AXL– subset. Disclosures: No relevant conflicts of interest to declare.

Abstract 773 Altered expression of SPARC, [secreted protein, acidic, cysteine-rich (osteonectin)] a gene encoding a protein that modulates cell-matrix interactions, has been reported in cancer and leukemia, but its precise contribution to malignant transformation is unknown. We previously reported that SPARC was among the most upregulated genes in the gene-expression profile (GEP) associated with IDH2-R172 mutations (mut; Marcucci et al. JCO 2010;28:2348) and among the most downregulated genes associated with NPM1 mut (Becker et al. JCO 2010;28:596) in cytogenetically normal (CN) AML. Both mutations have strong but opposite impact (IDH2-R172 mut, adverse; NPM1 mut, favorable) on clinical outcome. Thus, we hypothesized that SPARC may act as an oncogene and contribute to AML aggressiveness. To determine functional and mechanistic activities of SPARC in AML, we conducted gain- and loss-of-function experiments in cell lines and primary blasts collected from patients under the OSU Leukemia Tissue Bank protocol. In vitro, ectopic expression and knockdown of SPARC in THP-1 and Kasumi-1 AML cells led to increase (2.5-fold P=0.03) and decrease (45%, P=0.03) in colony forming ability, respectively. Clonogenic assays in SPARC- vs empty vector (EV)-infected AML blasts also showed 2-fold increase in colony-formation (P=.01) supporting a role of SPARC overexpression in promoting leukemia growth. The leukemogenic activity of the SPARC protein was likely mediated by its binding to αvβ3 integrin and in turn by the activation of the kinase ILK. This induced GSK3β phosphorylation (p) and in turn activation of β-catenin, previously reported to contribute to AML. Indeed, while ILK knockdown abolished SPARC-induced p-GSK3β, ectopic SPARC expression led to a 4-fold increased p-GSK3β, measured by p-GSK3β (Ser-9) immunoblot assay, and to stabilization of β-catenin, measured by p-β-catenin(Ser-552) and p-β-catenin(Ser-675) immunoblot assays in AML blasts. As a result, we observed increase in translocation of β-catenin to the nucleus in SPARC overexpressing cells by confocal microscopy. Nuclear β-catenin led to increased activity of the TCF/LEF transcription factors as shown by a TCF/LEF luciferase reporter assay (5.8-fold, P The results in the preclinical models prompted us to evaluate whether higher SPARC expression associated with aggressive human disease. Thus, SPARC expression levels were measured by NanoString nCounter assay in a cohort of CN-AML patients (pts; n=362; age, 18–83 years) comprehensively characterized for mutations in the NPM1, FLT3, CEBPA, WT1, TET2, MLL, IDH1, IDH2, RUNX1, ASXL1 and DNMT3A genes, and treated with cytarabine-anthracycline based regimens. Higher SPARC expressers were more likely classified in the European Leukemia Net (ELN) Intermediate-I (CEBPA wt, NPM1 mut with FLT3-ITD, or NPM1 wt) than the ELN Favorable Genetic Group (CEBPA mut and/or NPM1 mut without FLT3-ITD; P60 years); shorter DFS (P=.07), once adjusted for FLT3-ITD, RUNX1, WBC and age group; and shorter OS (P In conclusion, high SPARC expression contributes to aggressive AML growth via β-catenin activation-mediated mechanisms. SPARC overexpression independently predicted worse outcome in CN-AML pts, and may represent a potentially novel prognostic marker and therapeutic target in AML. Disclosures: No relevant conflicts of interest to declare.

Introduction: Risk-adapted therapy of AML patients (pts) is presently based on cytogenetic and molecular findings. However, the outcome remains poor for the majority of pts. Overexpression of BAALC is implicated in leukemogenesis and poor survival of CN-AML pts, but the underlying mechanisms are unknown. We hypothesized that the elevated expression of BAALC might result from a genetic variant in or close to the gene (in cis). Methods and Results: BAALC and RUNX1 expression were measured in pre-therapy samples from a test set of 253 CN-AML pts (Cancer And Leukemia Group B) using Affymetrix U133 plus 2.0 arrays. Pts were grouped as high or low BAALC and high or low RUNX1 expressers using the median expression value as the cut-off and screened for single nucleotide polymorphisms (SNPs) by direct sequencing of the BAALC gene. Nine informative SNPs were identified. By genotyping, marker rs62527607, a non-coding SNP in the promoter region correlated with high BAALC expression (rs62527607G>T: genotypes TT/GT vs. GG: P=2.01E-04). Luciferase reporter constructs (pGL4.11) spanning the respective alleles demonstrated that the T allele (“risk allele”) of rs62527607 increased luciferase activity compared to the G allele (P=1.0E-02). The T allele of rs62527607 is predicted to create a binding site for the transcription factor RUNX1. Indeed, RUNX1-cotransfection showed an increase in luciferase activity for the T allele of rs62527607 (P=2.0E-02) but not for the G allele (P=2.7E-01). An Electrophoretic Mobility Shift Assay comparing the binding activity of the two alleles showed highly increased binding activity of RUNX1 to the T allele vs. the G allele. Testing of the pt samples for a potential correlation of RUNX1 and BAALC expression levels revealed a positive correlation in pts with genotypes TT/GT but not GG when restricting the analysis to the high RUNX1 expressing group (P=1.18E-04). For validation of our findings, a set of 105 CN-AML pts comprising 52 high and 53 low BAALC expressers (AML Study Group [AMLSG], quantitative Real-Time PCR [qPCR]) was genotyped for rs62527607, determined for RUNX1 expresser status (qPCR) and analyzed alone and in combination with the test set. A non-significant association of TT/GT vs. GG with high BAALC expression (31% vs. 19%; P=1.6E-01) was seen. Analyzing both sets combined resulted in a P-value of 9.3E-05 (Odds Ratio [OR]=2.59 [1.62, 4.22; 95%CI]). Combining the results of the CALGB & AMLSG series showed a highly significant association between RUNX1 and BAALC expression in pts with genotypes TT/GT belonging to the high RUNX1 expressing group (P=7.97E-05, OR=4.02 [2.05, 8.21; 95%CI]). Conclusion: We show that the risk allele of rs62527607 creates a RUNX1 binding site, thereby leading to increased transcription of BAALC. The observation that a heritable trait is involved in the leukemogenesis process might initiate a new era of personalized medicine in AML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1307. doi:1538-7445.AM2012-1307

Abstract 3454 RUNX1 encodes the α subunit of core binding factor, a heterodimeric transcription factor required for normal hematopoiesis. Acquired RUNX1 mutations (muts) have been associated with poor clinical outcome in AML; however, prior studies analyzed pts heterogeneous for cytogenetics, age, AML type (primary or secondary), and treatment received [including allogeneic stem cell transplant (alloSCT) in 1st complete remission (CR1)] and contained limited data regarding the potential molecular drivers of the worse outcome. We report a relatively large study testing the prognostic impact of RUNX1 muts in primary CN-AML pts (n=392) treated similarly with intensive cytarabine/anthracycline-based 1st-line therapy and without alloSCT in CR1. This cohort comprised both younger [1 corresponds to a higher risk for higher values of continuous variables and the 1st level listed of a dichotomous variable. To gain biological insight, RUNX1 mut-associated gene and miR expression signatures were derived in CN-AML for the first time. Older, NPM1-wt pts were analyzed since RUNX1 muts are more common in this age group and are nearly exclusive from NPM1 muts, which have their own characteristic gene-expression signature. This yielded 484 probe sets representing 278 named genes differentially expressed between RUNX1-mut (n=31) and RUNX1-wt (n=45) pts (P In summary, RUNX1 muts are twice as common in older CN-AML pts than younger. They negatively impact on outcome in both younger and older pts not receiving alloSCT in CR1. RUNX1-mut blasts have molecular features of normal/malignant stem cells and B cells, which may explain their chemoresistance and guide novel therapeutic approaches. Disclosures: No relevant conflicts of interest to declare.

Abstract 2514 Approximately 8% of de novo AML pts have inv(16)(p13q22) or t(16;16)(p13;q22) [inv(16)] and this cytogenetic group is associated with high complete remission (CR) rates and a relatively favorable outcome after cytarabine/daunorubicin (ara-c/dnr)-based induction and high-dose ara-c (HiDAC) consolidation therapy. However, ∼40% of these pts still relapse. The molecular mechanisms that impact on the prognosis of inv(16) pts remain to be fully elucidated. We and others reported that presence of the mutated KIT (mutKIT) gene is associated with worse outcome, but other contributing factors may play a role. Molecularly, inv(16) results in disruption of the MYH11 gene at 16p13 and CBFB gene at 16q22, creating a CBFB-MYH11 fusion gene. Since the genomic breakpoints within CBFB and MYH11 are variable and the fusion transcripts depend on the exons fused, at least 11 different sized CBFB-MYH11 fusion transcript variants have been found. The frequency of each fusion varies, with ∼85% being type A and ∼5% each types D and E; types B, C, and F-K were reported in single cases (hereafter we refer to non-type A fusions as “rare”). To our knowledge, only one study (Schnittger et al. Leukemia 2007;21:725) has examined the biological and clinical significance of rare CBFB-MYH11 fusions in AML. Fusion type was not found to be prognostic, but the cohort included pts with therapy-related and secondary AML. Thus, further studies are warranted. Accordingly, we analyzed 149 de novo CBFB-MYH11 positive AML pts aged Disclosures: No relevant conflicts of interest to declare.

Abstract 1462 Despite progress made in understanding the biology and risk-adapted treatment of AML, prognosis of older pts [age ≥60 years (y)] remains poor. We have shown that high expression of the BAALC gene bestows worse outcome in older CN-AML pts. Yet, the mechanism(s) by which BAALC affects response to therapy remain unknown. MicroRNAs (miRs) are small noncoding RNAs that hybridize to target mRNAs and inhibit their translation or promote degradation, resulting in downregulation of the corresponding proteins. Recently, miR-3151 was discovered embedded in intron 1 of BAALC. We thus hypothesized that miR-3151 might be co-expressed with BAALC and contributes alone or in concert with its host to poor prognostic impact in older CN-AML. MiR-3151 and BAALC expression were measured by quantitative real time (RT) PCR in pretreatment blood of 179 older CN-AML pts enrolled on Cancer and Leukemia Group B (CALGB) cytarabine/daunorubicin-based protocols. The expression values of miR-3151 and BAALC were normalized to the internal controls SNORD44 and RN18S1, respectively. MiR-3151 expression correlated only weakly with the expression of BAALC (Spearman correlation r= 0.3). For clinical and prognostic analyses pts were dichotomized into high and low miR-3151 and BAALC expressers at the median miR and gene transcript expression values and analyzed for other molecular prognosticators (FLT3 -ITD, FLT3 -TKD, CEBPA, IDH1, IDH2, NPM1, TET2, WT1 mutations and ERG expression). Gene (GEP) and microRNA (MEP) expression profiles associated with miR-3151 expression were derived, using Affymetrix U133 plus 2.0 & OSU CCC v4.0 arrays, respectively. At diagnosis, higher miR-3151 expression was associated with lower % blood blasts (P =.02), FAB types M4 and M5 (P =.05), and wild-type NPM1 (P Disclosures: No relevant conflicts of interest to declare.

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.

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 1599 Poster Board I-625 The greatest obstacle to routine clinical testing of gene expression levels has been the lack of reproducibility of currently used methodologies, such as quantitative reverse transcriptase PCR (qRT-PCR) and microarray expression profiling. While these assays are useful for retrospective analyses of batched samples, they cannot be used for upfront evaluation of individual patients (pts) for molecular risk and treatment assignment. To overcome this barrier, we tested a recently developed, high throughput, PCR-independent, digital quantification technology, the nCounter system (Nanostring® Technologies). This system counts individual mRNA molecules, rather than measuring non-linear fluorescence generated by PCR-amplified targets (eg qRT-PCR). Using 72 AML samples and spike-in controls, we and collaborators demonstrated that the nCounter system is highly reproducible, sensitive, and accurate to femtomolar concentrations (Payton, J, et al. JCI 119:1714-26; Geiss, G, et al. Nat Biotech 26:317-25). Here we validated this technology using an independent set of 101 pts with a diagnosis of de novo cytogenetically normal AML. At diagnosis, pts presented with FAB subtypes M0, M1, M2, M4, M5(A, B), had a median age of 43 years (range 19-59), median white blood count of 28.5× 103/μL (range 1.4-273.0), median of 69% BM blasts (range 22-95) and median of 65% PB blasts (range 0-97). Paired BM and PB specimens were available for 27 pts; blast percentages were ≥ 20% for all paired specimens. We used the nCounter system to measure mRNA abundance (‘counts‘) of 27 genes whose expression correlates with clinical and/or pathological criteria, including 3 genes associated with prognosis (BAALC, ERG, MN1), and control/housekeeping genes (GAPDH, ABL, Actin). Briefly, mononuclear cells from pretreatment BM or PB were enriched on Ficoll-Hypaque gradients and RNA was isolated using Trizol reagent; 100ng of total RNA was assayed in triplicate by nCounter according to the manufacturer's protocols. The nCounter results demonstrated substantial reproducibility, with a median CV [coefficient of variation, (standard deviation/mean *100)] 1.9 and RQI scores >9, to relatively low, with 260:280 ratios Disclosures No relevant conflicts of interest to declare.

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.

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

The WT1 gene on chromosome 11p13 encodes a zinc-finger protein regulating gene transcription. A recent study conducted on 70 CN-AML patients (pts) found that 7 (10%) harbored WT1 mutations (WT1mut) and suggested that WT1mut are associated with failure to achieve complete remission (CR) (Summers et al., Leukemia2007;21:550). However, the prognostic impact of WT1mut remains to be elucidated in a larger set of homogeneously treated adults with de novo CN-AML and in the context of established prognostic molecular markers. We studied the prevalence and prognostic impact of WT1mut in 196 younger [ Figure Figure

Although CEBPA mutations (CEBPA+) have been reported to predict favorable outcome in CN-AML, their prognostic value has not been evaluated in the context of such established prognostic molecular markers in CN-AML as the combination of FLT3-ITD and NPM1 mutational status and BAALC and ERG expression. 169 adults aged

Abstract 417 Mutations in the additional sex combs like-1 (ASXL1) gene have been identified in patients (pts) with myelodysplastic syndromes, myeloproliferative neoplasms, and acute myeloid leukemia (AML). We present here the first study on ASXL1 mutations in adult primary cytogenetically normal (CN-)AML, and report their associations with clinical and molecular characteristics, treatment outcomes, and gene- and microRNA- (miR-)expression profiles. We studied 423 primary CN-AML pts, aged 18–83 years (y) and treated on cytarabine/daunorubicin-based frontline protocols, for ASXL1 exon 12 mutations (frame shift and nonsense changes) and other prognostic gene mutations [FLT3-internal tandem duplications (ITD) and tyrosine kinase domain mutations, MLL partial tandem duplications, and mutations in NPM1, CEBPA, WT1, IDH1, IDH2 and TET2]. ASXL1mutations were 5 times more common in older (≥60y) than in younger ( Among older primary CN-AML pts, those with mutated ASXL1 had a lower complete remission (CR) rate (53% vs 71%; P=.04) and shorter disease-free survival (DFS; P=.03; 3y rates, 10% vs 19%), overall survival (OS; P=.006; 3y rates, 5% vs 23%) and event-free survival (EFS; P=.002; 3y rates, 5% vs 14%; Fig. A) than ASXL1-wt pts. Due to the strong associations of ASXL1 mutations with NPM1-wt, absent FLT3-ITD and mutated CEBPA, we studied their prognostic impact within the genetic categories defined in the European LeukemiaNet (ELN) guidelines [ELN Favorable (Fav): CN-AML with mutated CEBPA and/or mutated NPM1 without FLT3-ITD; ELN Intermediate-I: all remaining CN-AML pts]. ELN Fav/ASXL1-mut pts had a lower CR rate (50%) compared with ELN Fav/ASXL1-wt pts (82%; P=.04). All 6 ELN Fav/ASXL1-mut pts who achieved CR relapsed within 13 months, while 27% of ELN Fav/ASXL1-wt pts were alive and disease-free at 3y. All ELN Fav/ASXL1-mut pts died within 18 months after enrollment, whereas 34% of ELN Fav/ASXL1-wt pts were alive at 3y (OS, P Gene- and miR-expression profiles were derived using Affymetrix HG-U133 plus 2.0 and custom-made miR microarrays. We identified an ASXL1 mutation-associated gene-expression signature comprising 67 differentially expressed genes (92 probe-sets), including upregulation of WNT pathway co-receptor LRP6, cytochrome P450 enzyme CYP1B1, and GJA1 (connexin 43, mediating stem cell-stroma interactions in the bone marrow). No significant signature of differentially expressed miRs was found. In conclusion, in this first study of ASXL1 mutations focusing on primary CN-AML, we demonstrate that they associate with inferior outcomes in older pts, particularly within the ELN Fav genetic group. We also report the first ASXL1-mutation associated gene-expression signature in CN-AML that may provide useful insight into the biology of ASXL1-mut AML, and help design novel treatment approaches for this high-risk group of older pts.FigureFigure. Disclosures: No relevant conflicts of interest to declare.