Micro-RNA Expression Profile Reveals MiR-222 As a Potential Biomarker For EBV-Positive Diffuse Large B-Cell Lymphoma

EBV+ diffuse large B-cell lymphoma of the elderly (EBV+DLBCLe) is considered a provisional entity in the latest World Health Organization classification. It affects individuals older than 50 years without prior documented immunodeficiency.This disorder has unfavorable clinical course even after the advent of immunotherapy associated to anthracycline-based chemotherapy. It is linked to Epstein-Barr virus and the physiopathology is related to the presence of the virus itself, senescence and immunological deterioration. Currently there is not a characteristic pattern of expression of microRNAs in EBV+DLBCLe.

To characterize a signature profile for this new entity and to explore microRNAs as biomarkers and potential alternative therapeutic targets for EBV+DLBCLe.

124 cases of patients of DLBCL were treated at Hospital Sao Paulo UNIFESP/EPM between 2000 to 2010 and had paraffin blocks available for immunohistochemical and molecular analyses. Seventy-one of 124 patients were more than 50 years and were potential candidates to be considered EBV+DLBCLe (pilot study). In situ hybridization was used for EBV detection (EBER1, Invitrogen) in a tissue microarray slide. Total RNA was obtained from tumor slides using Recover All Total Nucleic Acid Isolation kit (Applied Biosystems). We obtained cDNAs using Megaplex Pools for microRNA Expression (Applied Biosystems). The cDNA was inserted into two platforms containing 384 human microRNA each (Taqman Low Density Arrays) on 7900 Real Time PCR Systems (Applied Biosystems). Data analyses were made in mathematical-statistical environment “R”. The normalization method 2-deltaCt was performed using the endogenous RNU48, and it was identified as the most stable among samples by software Normfinder. It was also used RNU6 recommended by the manufacturer, in a comparative way. MicroRNAs differentially expressed in EBV+ group compared to EBV negative were identified by means of nonparametric tests rank products (RankProd) and Wilcoxon rank-sum (R-Stats). We considered differentially expressed microRNAs which average fold change above or below 1.5.After, real-time quantitative PCR was performed through 7500 Real time PCR Systems (Applied Biosystems) using TaqMan Small RNA kit assays and normalized with RNU48.

8.5% of cases of DLBCL were considered EBV+DLBCLe after ISH for EBV. 53.1% of the pilot study were considered GCB and 43.9% non-GCB according to Hans et al. algorithm (2004); 73.7% were classified as worse prognosis (groups 3 and 4) according to Salles et. al(2011) model combining IPI and immunohistochemical markers (bcl-2 and Ki67).We selected four of EBV+ and four of EBV negative samples matched by age, gender, stage and IPI to be analyzed in the PCR platforms. We found 10 deregulated microRNAs among the two groups. However, only seven microRNAs achieved statistically significant differences and would be the start point of a microRNA signature profile proposal to be validated in a larger multicentric cohort (total of 29 EBV+DLBCLe versus 65 DLBCL). Among them let-7g, miR-126, miR-146a, miR-146b, miR-150 and miR-155 were overexpressed in EBV+DLBCLe comparing to EBV-negative DLBCL whereas miR-151 was underexpressed. After validation in 29 EBV+DLBCLe (including 23 new cases) and 65 EBV negative cases we confirmed overexpression of miR-126 in 75.8% (median 2.14 vs 0.14,p< 0.0001), miR-146a in 62% (median 1.94 vs 0.49, p = 0.0035) ,miR-146b in 51.7% (median 1.51 vs 0.11 , p< 0.0001),miR-150 in 96.5 % (median 20.54 vs 2.56,p< 0.0001) and miR-222 in 23,8% of cases (median 0.67 vs 0.08,p< 0.0001,Mann-Whitney) and also confirmed underexpression of miR-151 in 96% of EBV+DLBCL cases. Although miR-222 was overexpressed in ¼ of the cases, it showed high speficity (98%) and positive predictive value (83%), Area Under the Curve= 0.87774, when EBV+ when compared to EBV negative cases.

The merit of the present study is to propose a microRNA signature for a recently described disease and to highlight miR-222 as a possible biomarker and therapeutic target for EBV+DLBCLe. The main routes deregulated are NF-KappaB and PI3K-AKT pathway, being PTEN a target of the overexpressed miR-222. Thus, the findings suggest that antagomiRs for miR-222,that are being tested in some types of cancer, could be also used as adjuvant therapy to R-CHOP in EBV+DLBCL. (Supported by FAPESP 2010/17668-6).

No relevant conflicts of interest to declare.