A standardized microarray assay for the independent gene expression markers in AML: EVI1 and BAALC.
ABSTRACT: High levels of BAALC, ERG, EVI1 and MN1 expression have been associated with shorter overall survival in AML but standardized and clinically validated assays are lacking. We have therefore developed and optimized an assay for standardized detection of these prognostic genes for patients with intermediate cytogenetic risk AML. In a training set of 147 intermediate cytogenetic risk cases we performed cross validations at 5 percentile steps of expression level and observed a bimodal significance profile for BAALC expression level and unimodal significance profiles for ERG and MN1 levels with no statistically significant cutoff points near the median expression level of BAALC, ERG or MN1. Of the possible cutoff points for expression levels of BAALC, ERG and MN1, just the 30th and 75th percentile of BAALC expression level and the 30th percentile of MN1 expression level cutoff points showed clinical significance. Of these only the 30th percentile of BAALC expression level reproduced in an independent verification (extended training) data set of 242 cytogenetically normal AML cases and successfully validated in an external cohort of 215 intermediate cytogenetic risk AML cases. Finally, we show independent prognostic value for high EVI1 and low BAALC in multivariate analysis with other clinically relevant molecular AML markers. We have developed a highly standardized molecular assay for the independent gene expression markers EVI1 and BAALC.
Project description: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.
Project description:Acute myeloid leukemia (AML) is a heterogenous disorder that results from a block in the differentiation of hematopoietic progenitor cells along with uncontrolled proliferation. In approximately 60% of cases, specific recurrent chromosomal aberrations can be identified by modern cytogenetic techniques. This cytogenetic information is the single most important tool to classify patients at their initial diagnosis into three prognostic categories: favorable, intermediate, and poor risk. Currently, favorable risk AML patients are usually treated with contemporary chemotherapy while poor risk AML patients receive allogeneic stem cell transplantation if suitable stem cell donors exist. The largest subgroup of AML patients (aproximately 40%) have no identifiable cytogenetic abnormalities and are classified as intermediate risk. The optimal therapeutic strategies for these patients are still largely unclear. Recently, it is becoming increasingly evident that it is possible to identify a subgroup of poorer risk patients among those with normal cytogenic AML (NC-AML). Molecular risk stratification for NC-AML patients may be possible due to mutations of NPM1, FLT3, MLL, and CEBPalpha as well as alterations in expression levels of BAALC, MN1, ERG, and AF1q. Further prospective studies are needed to confirm if poorer risk NC-AML patients have improved clinical outcomes after more aggressive therapy.
Project description:For most acute myeloid leukemia (AML) patients, an allogeneic hematopoietic stem cell transplantation (HSCT) offers the highest chance of sustained remissions and long-term survival. At diagnosis, high expression of the AML-associated genes BAALC (brain and acute leukemia, cytoplasmic) and MN1 (meningioma-1) were repeatedly linked to inferior outcomes in patients consolidated with chemotherapy while data for patients receiving HSCT remain limited. Using clinically applicable digital droplet PCR assays, we analyzed the diagnostic BAALC/ABL1 and MN1/ABL1 copy numbers in 302 AML patients. High BAALC/ABL1 and MN1/ABL1 copy numbers associated with common adverse prognostic factors at diagnosis. However, while high diagnostic copy numbers of both genes associated with shorter event free survival (EFS) and overall survival (OS) in patients receiving chemotherapy, there was no prognostic impact in patients undergoing HSCT. Our data suggests that the adverse prognostic impact of high BAALC and MN1 expression are mitigated by allogeneic HSCT. But preHSCT BAALC/ABL1 and MN1/ABL1 assessed in remission prior to HSCT remained prognosticators for EFS and OS independent of the diagnostic expression status. Whether allogeneic HSCT may improve survival for AML patients with high diagnostic BAALC or MN1 expression should be investigated prospectively and may improve informed decisions towards individualized consolidation options in AML.
Project description:Hematological malignancies possess a distinctive immunologic microenvironment compared with solid tumors. Here, using an established computational algorithm (CIBERSORT), we systematically analyzed the overall distribution of 22 tumor-infiltrating leukocyte (TIL) populations in more than 2000 bone marrow (BM) samples from 5 major hematological malignancies and healthy controls. Focusing on significantly altered TILs in acute myeloid leukemia (AML), we found that patients with AML exhibited increased frequencies of M2 macrophages, compared to either healthy controls or the other four malignancies. High infiltration of M2 macrophages was associated with poor outcome in AML. Further analysis revealed that CD206, a M2 marker gene, could faithfully reflect variation in M2 fractions and was more highly expressed in AML than normal controls. High CD206 expression predicted inferior overall survival (OS) and event-free survival (EFS) in two independent AML cohorts. Among 175 patients with intermediate-risk cytogenetics, the survival still differed greatly between low and high CD206 expressers (OS; P < .0001; 3-year rates, 56% v 32%; EFS; P < .001; 3-year rates, 47% v 25%). When analyzed in a meta-analysis, CD206 as a continuous variable showed superior predictive performance than classical prognosticators in AML (BAALC, ERG, EVI1, MN1, and WT1). In summary, M2 macrophages are preferentially enriched in AML. The M2 marker CD206 may serve as a new prognostic marker in AML.
Project description:BACKGROUND:Elevated protein expressions of CD markers such as IL2RA/CD25, CXCR4/CD184, CD34 and CD56 are associated with adverse prognosis in acute myeloid leukemia (AML). However, the prognostic value of mRNA expressions of these CD markers in AML remains unclear. Through our pilot evaluation, IL2RA mRNA expression appeared to be the best candidate as a prognostic biomarker. Therefore, the aim of this study is to characterize the prognostic value of IL2RA mRNA expression and evaluate its potential to refine prognostification in AML. METHODS:In a cohort of 239 newly diagnosed AML patients, IL2RA mRNA expression were measured by TaqMan realtime quantitative PCR. Morphological, cytogenetics and mutational analyses were also performed. In an intermediate-risk AML cohort with 66 patients, the mRNA expression of prognostic biomarkers (BAALC, CDKN1B, ERG, MECOM/EVI1, FLT3, ID1, IL2RA, MN1 and WT1) were quantified by NanoString technology. A TCGA cohort was analyzed to validate the prognostic value of IL2RA. For statistical analysis, Mann-Whitney U test, Fisher exact test, logistic regression, Kaplan-Meier and Cox regression analyses were used. RESULTS:In AML cohort of 239 patients, high IL2RA mRNA expression independently predicted shorter relapse free survival (RFS, p < 0.001) and overall survival (OS, p < 0.001) irrespective of age, cytogenetics, FLT3-ITD or c-KIT D816V mutational status. In core binding factor (CBF) AML, high IL2RA mRNA expression correlated with FLT3-ITD status (p = 0.023). Multivariable analyses revealed that high IL2RA expression (p = 0.002), along with c-KIT D816V status (p = 0.013) significantly predicted shorter RFS, whereas only high IL2RA mRNA expression (p = 0.014) significantly predicted shorter OS in CBF AML. In intermediate-risk AML in which multiple gene expression markers were tested by NanoString, IL2RA significantly correlated with ID1 (p = 0.006), FLT3 (p = 0.007), CDKN1B (p = 0.033) and ERG (p = 0.030) expressions. IL2RA (p < 0.001) and FLT3 (p = 0.008) expressions remained significant in predicting shorter RFS, whereas ERG (p = 0.008) and IL2RA (p = 0.044) remained significant in predicting shorter OS. Similar analyses in TCGA intermediate-risk AML showed the independent prognostic role of IL2RA in predicting event free survival (p < 0.001) and OS (p < 0.001). CONCLUSIONS:High IL2RA mRNA expression is an independent and adverse prognostic factor in AML and specifically stratifies patients to worse prognosis in both CBF and intermediate-risk AML.
Project description: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.
Project description:Cytogenetic aberrations and gene mutations have long been regarded as independent prognostic markers in AML, both of which can lead to misexpression of some key genes related to hematopoiesis. It is believed that the expression level of the key genes is associated with the treatment outcome of AML.In this study, we analyzed the clinical features and molecular aberrations of 560 newly diagnosed non-M3 AML patients, including mutational status of CEBPA, NPM1, FLT3, C-KIT, NRAS, WT1, DNMT3A, MLL-PTD and IDH1/2, as well as expression levels of MECOM, ERG, GATA2, WT1, BAALC, MEIS1 and SPI1.Certain gene expression levels were associated with the cytogenetic aberration of the disease, especially for MECOM, MEIS1 and BAALC. FLT3, C-KIT and NRAS mutations contained conversed expression profile regarding MEIS1, WT1, GATA2 and BAALC expression, respectively. FLT3, DNMT3A, NPM1 and biallelic CEBPA represented the mutations associated with the prognosis of AML in our group. Higher MECOM and MEIS1 gene expression levels showed a significant impact on complete remission (CR) rate, disease free survival (DFS) and overall survival (OS) both in univariate and multivariate analysis, respectively; and an additive effect could be observed. By systematically integrating gene mutational status results and gene expression profile, we could establish a more refined system to precisely subdivide AML patients into distinct prognostic groups.Gene expression abnormalities contained important biological and clinical informations, and could be integrated into current AML stratification system.
Project description: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.
Project description:Numerous studies have investigated the prognostic role of brain and acute leukemia, cytoplasmic (BAALC) gene expression in adult patients with acute myeloid leukemia (AML); however, the results are inconclusive. A meta-analysis was conducted to provide a comprehensive evaluation of the prognostic role of BAALC gene expression in AML. Eligible studies were searched through PubMed, Embase, the Cochrane Library, the China National Knowledge Infrastructure and the China Biology Medicine Disc. Correlations between the BAALC gene expression and clinicopathological features and prognosis were analyzed. A total of 15 studies were examined. The pooled results suggest that high BAALC expression had an unfavorable outcome in AML. The combined hazard ratio (HR) for overall survival (OS) was 1.53 and the summary HR for the disease-free survival rate was 1.64. In addition, subgroup analyses considering cytogenetic and survival analysis were also conducted. High BAALC gene expression appeared to be an adverse prognostic indicator in patients with cytogenetically normal AML (HR for OS, 1.43) and in subgroups of survival analysis with multivariate analysis (HR for OS, 2.35). These results indicate that high BAALC gene expression served as an independent poor prognostic indicator in adult patients with AML.
Project description:High expression of the leukemia-associated gene meningioma-1 (MN1) is frequently found at diagnosis of acute myeloid leukemia (AML) and associates with adverse outcomes. The presence of measurable residual disease (MRD) in complete remission (CR) indicates high risk of relapse and worse outcome in AML patients. However, the prognostic impact of MN1 expression levels as MRD marker has not been evaluated. Digital droplet polymerase chain reaction (ddPCR) is a novel technique allowing sensitive and specific absolute gene expression quantification. We retrospectively analyzed 124 AML patients who received allogeneic hematopoietic stem cell transplantation (HSCT) in CR or CR with incomplete peripheral recovery. Absolute MN1 copy numbers in peripheral blood were assessed prior to HSCT (median 7; range 0-29 days) using ddPCR. High pre-HSCT MN1/Abelson murine leukemia viral oncogene homolog 1 gene (ABL1) copy numbers associated with a higher cumulative incidence of relapse after HSCT and-in relapsing patients-shorter time to relapse. In multivariable analysis, high pre-HSCT MN1/ABL1 copy numbers remained an independent prognosticator for relapse after HSCT. Patients with the highest pre-HSCT MN1/ABL1 copy numbers also had the highest risk of relapse. MN1 copy number assessment also added prognostic information to nucleophosmin 1 gene (NPM1) mutation- and brain and acute leukemia, cytoplasmic (BAALC) and Wilm's tumor gene 1 (WT1) expression-based MRD evaluation. Our study demonstrates the feasibility of the novel ddPCR technique for MN1/ABL1 copy number assessment as a marker for MRD. Evaluation of MN1/ABL1 copy numbers allows the identification of patients at high risk of relapse, independently of other diagnostic risk factors and MRD markers.