Screening for Prognostic microRNAs Associated with Treatment Failure in Diffuse Large B Cell Lymphoma

Abstract

Diffuse large B cell lymphoma (DLBCL) treatment with R-CHOP regimen produces 5-year progression-free survival and overall survival of around 60-70%. Our objective was to discover prognostic biomarkers allowing early detection of the remaining 30-40% with poor long-term outcome. For this purpose, we applied a novel strategy: from a cohort of DLBCL patients, treated with standard therapy, a discovery group of 12 patients with poor prognosis (advanced stage III-IV, R-IPI > 2) was formed, consisting of six chemoresistant (refractory/early relapse < 12 months) and six chemosensitive (complete remission > 3 years) subjects. By using microarray assays, the most differentially expressed miRNAs were defined as an initial set of prognostic miRNA candidates. Their expression was then analyzed in a validation cohort of 68 patients and the three miRNAs with the most significant impact on event-free and overall survival were selected. In the DLBCL cell line U-2932 the transfection with miR-1244 and miR-193b-5p, but not miR-1231, blocked the effect of CHOP on cell viability. A subsequent gene set enrichment analysis in patients revealed the implication of the first two miRNAs in cell cycle control and chemoresistance-related pathways, whereas the last one was involved in immunological processes. In conclusion, this novel strategy identified three promising prognostic markers for DLBCL patients at high risk of failure with standard therapy.

Keywords: chemoresistance; diffuse large B cell lymphoma; epigenetic markers; microRNA; prognosis.

Figure 1

Heatmap of unsupervised hierarchical clustering to visualize the miRNAs expression profiles. Selected miRNAs that were used for further analysis in the validation cohort are highlighted with an asterisk (*).

Figure 2

High levels of miR-1244, miR-193b-5p, or miR-1231 were related to worse event-free survival (EFS) and overall survival (OS). The expression of miR-1244 (A,B), miR-93b-5p (C,D), and miR-1231 (E,F) was studied for their impact on EFS and OS, respectively, in our series of patients with diffuse large B-cell lymphoma (DLBCL). EFS and OS were compared using the Kaplan–Meier curves and log rank test between the high (red line) and low (blue line) expression groups determined by each miRNA-specific threshold. p value indicates statistical significance.

Figure 3

Inhibition, but not expression, of miR-1244, miR-193b-5p or miR-1231 reduced cell viability in human diffuse large B-cell lymphoma (DLBCL) cells. U-2932 cells were reverse transfected separately with 100 nM of miR-1244, miR-193b-5p, or miR-1231 miRNA mimics (A) or inhibitors (B) for 48 h. In parallel, another set of cells were reverse transfected with miRNA negative control #1 (miR control (−)) or left entirely untreated. Cell viability was determined as detailed in Materials and Methods. Each column represents mean ± SEM of 3 independent experiments performed in triplicate, and normalized to cells transfected with miR control (−) (taken as 100%). * p < 0.05 and ** p < 0.001 versus cells transfected with miR control (−).

Figure 4

Expression of miR-1244 and miR-193b-5p, but not miR-1231, blocked the antitumoral effect of CHOP in human diffuse large B-cell lymphoma (DLBCL) cells. U-2932 cells were reverse transfected separately with 100 nM of miR-1244 (A), miR-193b-5p (B), or miR-1231 (C) mimics or with miRNA negative control #1 (miR control (−)). 24 h after transfection, cells were treated with vehicle (1% DMSO) or with increasing concentrations (0.3, 1, and 3 µg/mL) of CHOP. Cell viability was determined as detailed in Materials and Methods. Each column represents mean ± SEM of 4 independent experiments performed in triplicate, and normalized to cells transfected with miR control (−) (taken as 100%). ** p < 0.01 and *** p < 0.001 versus cells transfected with miR control (−) and treated with vehicle. ## p < 0.01 and ### p < 0.001 versus their respective cells transfected with miR control (−).

Figure 5

Gene set enrichment analysis with Gene Ontology terms was performed according to the expression profile between CR and RR patients using clusterProfiler package from Bioconductor. Only those pathways with genes for which a consistent up- or downregulation was detected in both patients and U-2932 cells were considered. Gene Ontology categories altered by miR-1244 (A), miR-193-5p (B), and miR-1231 (C) including their corresponding NES (Normalized Enrichment Score) values are indicated in the respective panels. The higher the enrichment score the stronger is the displacement of genes belonging to certain GO categories towards either end of the ranked list with positive values representing up- and negative values downregulation.