Claudia D. Baldus

MicroRNA Expression in Cytogenetically Normal Acute Myeloid Leukemia

BACKGROUND A role of microRNAs in cancer has recently been recognized. However, little is known about the role of microRNAs in acute myeloid leukemia (AML). METHODS 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. RESULTS Of 305 microRNA probes, 12 (including 5 representing microRNA-181 family members) were associated with event-free survival in the training group (P<0.005). 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. CONCLUSIONS 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.

Prognostic Significance of, and Gene and MicroRNA Expression Signatures Associated With, CEBPA Mutations in Cytogenetically Normal Acute Myeloid Leukemia With High-Risk Molecular Features: A Cancer and Leukemia Group B Study

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.

Overexpression of the ETS-Related Gene, ERG, Predicts a Worse Outcome in Acute Myeloid Leukemia With Normal Karyotype: A Cancer and Leukemia Group B Study

PURPOSE To test the prognostic significance of ETS-related gene (ERG) expression in cytogenetically normal primary acute myeloid leukemia (AML). PATIENTS AND METHODS Pretreatment blood samples from 84 cytogenetically normal AML patients aged less than 60 years, who were characterized for BAALC expression, FLT3 internal tandem duplication (ITD), and MLL partial tandem duplication (PTD) and uniformly treated on Cancer and Leukemia Group B 9621 protocol, were analyzed for ERG expression by real-time reverse transcriptase polymerase chain reaction. Patients were divided into quartiles according to ERG levels and were compared for clinical outcome. High-density oligonucleotide arrays were used to identify genes differentially expressed between high and low ERG expressers. RESULTS With a median follow-up of 5.7 years, patients with the upper 25% of ERG expression values had a worse cumulative incidence of relapse (CIR; P < .001) and overall survival (OS; P = .011) than the remaining patients. In a multivariable analysis, high ERG expression (P < .001) and the presence of MLL PTD (P = .027) predicted worse CIR. With regard to OS, an interaction was observed between expression of ERG and BAALC (P = .013), with ERG overexpression predicting shorter survival only in low BAALC expressers (P = .002). ERG overexpression was an independent prognostic factor even when the unfavorable group of FLT3 ITD patients lacking an FLT3 wild-type allele was included. High ERG expression was associated with upregulation of 112 expressed-sequenced tags and named genes, many of which are involved in cell proliferation, differentiation, and apoptosis. CONCLUSION ERG overexpression in AML patients with normal cytogenetics predicts an adverse clinical outcome and seems to be associated with a specific molecular signature.

High Expression Levels of the ETS-Related Gene, ERG, Predict Adverse Outcome and Improve Molecular Risk-Based Classification of Cytogenetically Normal Acute Myeloid Leukemia: A Cancer and Leukemia Group B Study

PURPOSE To validate ERG overexpression as an adverse predictor and assess its prognostic value in the context of other molecular markers in cytogenetically normal (CN) -acute myeloid leukemia (AML). PATIENTS AND METHODS Seventy-six adult patients with primary CN-AML, younger than 60 years and treated on Cancer and Leukemia Group B (CALGB) trial 19808, were evaluated for ERG expression by quantitative reverse transcriptase polymerase chain reaction. They were then combined with 72 patients enrolled onto CALGB 9621 for analyses that included other molecular markers. RESULTS Similar to patients enrolled onto CALGB 9621, high ERG expressers on CALGB 19808 had fewer complete remissions (CRs; P = .03) and worse event-free survival (EFS; P = .016) than low ERG expressers. In the combined set, high expressers (n = 55) had fewer CRs (P = .004) and shorter EFS (P < .001) than low expressers (n = 93). High ERG predicted failure to achieve CR (P = .004) after adjusting for BAALC expression (P = .04) and age (P = .008), and EFS (P = .004) after adjusting for FLT3 internal tandem duplication (ITD; P < .001). Among patients without FLT3-ITD (FLT3-ITD negative), only high ERG predicted shorter EFS (P = .001). Among NPM1-mutated (NPM1 positive) patients, high ERG predicted shorter EFS (P = .003), after adjusting for FLT3-ITD (P < .001). When all three markers were considered together, in the favorable FLT3-ITD-negative/NPM1-positive subset, high ERG was the only factor predicting shorter EFS (P = .002). CONCLUSION We validated ERG overexpression as an adverse predictor in CN-AML. Moreover, by using ERG expression levels, we improved the previously proposed molecular-risk classification of CN-AML based on the presence or absence of FLT3-ITD and NPM1 mutations, given that we identified subsets with different outcome among FLT3-ITD-negative, NPM1-positive, and FLT3-ITD-negative/NPM1-positive patients.

High BAALC expression associates with other molecular prognostic markers, poor outcome, and a distinct gene-expression signature in cytogenetically normal patients younger than 60 years with acute myeloid leukemia: A Cancer and Leukemia Group B (CALGB) study

BAALC expression is considered an independent prognostic factor in cytogenetically normal acute myeloid leukemia (CN-AML), but has yet to be investigated together with multiple other established prognostic molecular markers in CN-AML. We analyzed BAALC expression in 172 primary CN-AML patients younger than 60 years of age, treated similarly on CALGB protocols. High BAALC expression was associated with FLT3-ITD (P = .04), wild-type NPM1 (P < .001), mutated CEBPA (P = .003), MLL-PTD (P = .009), absent FLT3-TKD (P = .005), and high ERG expression (P = .05). In multivariable analysis, high BAALC expression independently predicted lower complete remission rates (P = .04) when adjusting for ERG expression and age, and shorter survival (P = .04) when adjusting for FLT3-ITD, NPM1, CEBPA, and white blood cell count. A gene-expression signature of 312 probe sets differentiating high from low BAALC expressers was identified. High BAALC expression was associated with overexpression of genes involved in drug resistance (MDR1) and stem cell markers (CD133, CD34, KIT). Global microRNA-expression analysis did not reveal significant differences between BAALC expression groups. However, an analysis of microRNAs that putatively target BAALC revealed a potentially interesting inverse association between expression of miR-148a and BAALC. We conclude that high BAALC expression is an independent adverse prognostic factor and is associated with a specific gene-expression profile.

BAALC Expression and FLT3 Internal Tandem Duplication Mutations in Acute Myeloid Leukemia Patients With Normal Cytogenetics: Prognostic Implications

PURPOSE Evaluate the impact of BAALC (brain and acute leukemia, cytoplasmic), a gene whose expression has been associated with adverse outcome in acute myeloid leukemia (AML) with normal cytogenetics, and FLT3 internal tandem duplication (ITD) mutations as independent prognostic factors in a larger study. PATIENTS AND METHODS BAALC expression was determined by real-time reverse transcriptase polymerase chain reaction in pretreatment blood samples of 307 adults <or= 60 years of age with AML with normal cytogenetics. Patients were dichotomized at BAALCs median expression into low and high expressers. The FLT3 ITD mutant:wild-type ratio was determined by fragment analysis. RESULTS Compared with low-BAALC patients, high-BAALC patients had a higher rate of primary resistant leukemia (16% v 6%; P = .006). High BAALC expression was associated with a higher cumulative incidence of relapse (CIR; P = .018) and an inferior overall survival (OS; 3-year OS, 36% v 54%; P = .001). On multivariable analysis, high BAALC was independently predictive of resistant disease (P = .019), and high BAALC as well as a high FLT3 mutant:wild-type ratio were confirmed as the only factors predicting a high CIR (BAALC, P = .03; FLT3, P = .01) and inferior OS (BAALC, P = .001; FLT3, P = .012). CONCLUSION This study strengthens BAALC expression as one of the most important prognostic factors in AML patients with normal cytogenetics. BAALC expression and FLT3 mutation status should assist in tailoring induction and postremission therapies.

Prognostic Significance of Expression of a Single MicroRNA, miR-181a, in Cytogenetically Normal Acute Myeloid Leukemia: A Cancer and Leukemia Group B Study

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.

High activity of sorafenib in FLT3-ITD-positive acute myeloid leukemia synergizes with allo-immune effects to induce sustained responses

Preliminary evidence suggests that the multikinase inhibitor sorafenib has clinical activity in FLT3-ITD-positive (FLT3-ITD) acute myeloid leukemia (AML). However, the quality and sustainability of achievable remissions and clinical variables that influence the outcome of sorafenib monotherapy are largely undefined. To address these questions, we evaluated sorafenib monotherapy in 65 FLT3-ITD AML patients treated at 23 centers. All but two patients had relapsed or were chemotherapy-refractory after a median of three prior chemotherapy cycles. Twenty-nine patients (45%) had undergone prior allogeneic stem cell transplantation (allo-SCT). The documented best responses were: hematological remission in 24 patients (37%), bone marrow remission in 5 patients (8%), complete remission (with and without normalization of peripheral blood counts) in 15 patients (23%) and molecular remission with undetectable FLT3-ITD mRNA in 10 patients (15%), respectively. Seventeen of the patients without prior allo-SCT (47%) developed sorafenib resistance after a median treatment duration of 136 days (range, 56–270 days). In contrast, allo-SCT patients developed sorafenib resistance less frequently (38%) and significantly later (197 days, range 38–225 days; P=0.03). Sustained remissions were seen exclusively in the allo-SCT cohort. Thus, sorafenib monotherapy has significant activity in FLT3-ITD AML and may synergize with allogeneic immune effects to induce durable remissions.

Prognostic Importance of MN1 Transcript Levels, and Biologic Insights From MN1-Associated Gene and MicroRNA Expression Signatures in Cytogenetically Normal Acute Myeloid Leukemia: A Cancer and Leukemia Group B Study

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.

High Expression of the ETS Transcription Factor ERG Predicts Adverse Outcome in Acute T-Lymphoblastic Leukemia in Adults

PURPOSE In adult T-lymphoblastic leukemia (T-ALL) disease-free survival remains limited to 32% to 46%. The adverse prognosis in T-ALL has not been attributed to cytogenetic or molecular aberrations. We have determined the prognostic impact of the oncogenic transcription factor ERG in T-ALL. PATIENTS AND METHODS ERG expression was analyzed by real-time polymerase chain reaction (PCR) in 105 adults with newly diagnosed T-ALL treated on the German ALL protocols. Patients were dichotomized at ERGs median expression into low (n = 52) and high (n = 53) expressers. Homeobox (HOX) 11 and HOX11L2 expression was determined by real-time PCR. RESULTS High ERG expressers compared with low ERG expressers had an inferior overall survival (OS, P = .02; 5-year OS: high ERG 26% v low ERG 58%) and relapse-free survival (RFS, P = .003; 5-year RFS: high ERG 34% v low ERG 72%). On multivariable analysis high ERG expression (P = .005), immunophenotypic subgroups (early v mature v thymic T-ALL; overall P = .04), HOX11L2 positivity (P = .055), and absence of HOX11 (P = .017) were independent adverse risk factors predicting RFS. Patients with high ERG expression had a hazard ratio (HR) for relapse of 3.2. Within the good prognostic subgroup of thymic T-ALL (n = 57), high ERG (HR, 4.1; P = .02) and presence of HOX11L2 (HR, 6.6; P = .008) were independent adverse factors for RFS. CONCLUSION High expression of ERG is an adverse risk factor in adult T-ALL. Within thymic T-ALL, otherwise classified as standard-risk, high ERG expression-identified patients that were four times more likely to fail long-term RFS. The prognostic impact of ERG may assist treatment stratification and suggest the need of alternative regimens.