MicroRNAs in Intermediate Risk Cytogenetic Acute Myeloid Leukemia Add Relevant Prognostic Information to Molecular Categorization

Abstract 235

The prognosis of AML patients within the intermediate cytogenetics category is mainly determined by the mutational status of some relevant genes, such as NPM1 mutations (NPMmut), or biallelic CEBPA mutations (CEBPAmut), associated with a favorable outcome, and with the presence of FLT3 internal tandem duplication (FLT3-ITD), which correlates with an adverse prognosis. Nonetheless, additional biological features such as microRNA (miRNA) expression pattern might contribute to refine prognosis and guide therapy in this setting.

The aim of the present study is to investigate whether miRNA expression is associated with molecular characteristics and clinical outcome in intermediate-risk AML patients (IR-AML).

We have analyzed samples from 85 IR-AML patients (median age, 52 [range, 18–71]; 52% males) who received intensive therapy from 1994 to 2009. Forty-three patients (51%) harbored NPMmut, 37 (44%) harbored FLT3-ITD (including 23 with NPMmut), and 11 (13%) harbored CEBPAmut, including 7 with biallelic mutations. The expression of 670 mature miRNAs was analyzed by multiplex Real Time PCR using TaqMan Human MicroRNA Arrays (Applied Biosystems). All PCR reactions were performed using an ABI 7900 HT sequence detection system. miRNA expression data was analyzed by the 2^−DDCt method, using RNU48 as endogenous control. Statistical analysis was performed with BRB Array Tools, SPSS version 15.0.1 and R software version 2.9.0.

Supervised analysis by means of t-test based on multiplex permutations (class comparisons analysis, p<0.001) revealed a distinctive miRNA signature in patients with NPMmut, with overexpression of miR-10a, miR-10a*, miR-10b and miR-196b, and downregulation of miR-126, miR126*, miR-424, miR-424* and miR-335, as well as patients with biallelic CEBPAmut, characterized by downregulation of miR-196b and upregulation of miR-181a. Response rate in this series of patients was 84%, with 5-year survival of 43±11% and relapse incidence (RI) of 55±14%. Multivariate analysis for overall survival(OS) including NPM status, FLT3-ITD status, age, WBC, and Log Rank OS significant miRNAs (miR-632, miR-23b, miR-409-3p, let-7a*, miR-565 and miR-196b) identified age, absence of NPMmut, and FLT3-ITD as unfavorable variables together with low expression of miR-409-3p (p<0.001; HR=3.3, 95% CI: 1.7–6.4), and increased level of let-7a* (p=0.026; HR=5.1, 95% CI: 1.21–21.5) and miR-196b (p=0.056; HR=7.27, CI: 0.95–55.6). Concerning risk of relapse (RR), multivariate analysis including NPM status, age, FLT3-ITD, WBC, and Log Rank RR significant miRNAs (miR-632, miR-155*, miR-135a, miR-409-3p, miR-150, miR-23a* and miR-363) the absence of NPMmut, FLT3-ITD and increasing leukocyte count were associated with a higher RI. Remarkably, decreased miR-409-3p expression (p=0.011; HR=3.3, 95% CI: 1.3–8.2) and miR-135a (p=0.02; HR=4.2, 95% CI: 1.2–14.2), together with higher levels of miR-23a* (p<0.001; HR=6.2, 95% CI: 2.61–14.7) were independently associated with a higher relapse risk. Of note, a decreased miR-409-3p level retained its adverse prognosis value in the subgroup of patients without favorable molecular markers (i.e., wild-type NPM1 and CEBPA and/or FLT3-ITD;p=0.001) together with low miR-361-3p (p=0.013, HR= 2.4, CI: 1.2–5.1). On the contrary, let-7a* levels segregated subgroups of patients in the category of favorable genotype (i.e., mutated NPM1 without FLT3-ITD p=0.027).

In this series of patients of intermediate-risk cytogenetic AML, measurement of expression levels of several miRNAs such as miR-409-3p, miR-135a, let-7a* or miR-23a* showed independent prognostic value, and contribute to predict the outcome within specific molecular subgroups. Nonetheless, confirmation of the prognostic impact of these miRNAs and investigation of possible underlying mechanisms account for this effect require future studies.