miR-18b overexpression identifies mantle cell lymphoma patients with poor outcome and improves the MIPI-B prognosticator

Recent studies show that mantle cell lymphoma (MCL) express aberrant miRNA profiles, however, the clinical effect of miRNA expression has not previously been examined and validated in prospective, large, homogenously treated cohorts. We analyzed diagnostic MCL samples from the Nordic MCL2 and MCL3 clinical trials, in which all patients had received Rituximab-high-dose cytarabin alternating with Rituximab-maxiCHOP, followed by BEAM and autologous stem cell support. We performed genome-wide miRNA microarray profiling of 74 diagnostic MCL samples from the MCL2 trial (screening cohort). Differentially expressed miRNAs were re-analyzed by qRT-PCR. Prognostic miRNAs were validated by qRT-PCR in diagnostic MCL samples from 94 patients of the independent MCL3 trial (validation cohort). Three miRNAs (miR-18b, miR-92a, miR-378d) were significantly differentially expressed in patients who died from MCL in both the screening- and the validation cohort. MiR-18b was superior to miR-92a and miR-378d in predicting high risk. Thus, we generated a new MIPI-B-miR prognosticator, combining expression-levels of miR-18b with MIPI-B data. This prognosticator improved identification of high risk patients compared to MIPI-B with regard to cause-specific survival (P=0.015), overall survival (P=0.006) and progression-free survival (P<0.001). Transfection of two MCL cell lines with miR-18b decreased their proliferation rate without inducing apoptosis, suggesting miR-18b may render MCL cells resistant to chemotherapy by decelerating cell proliferation. Thus, we conclude that overexpression of miR-18b identifies patients with poor prognosis in two large prospective MCL cohorts and adds prognostic information to MIPI-B. MiR-18b may reduce the proliferation rate of MCL cells as a mechanism of chemoresistance. For miRNA microarray profiling, 500 ng of total RNA from samples (AM6000) was labeled with Hy3 fluorescent dyes, using the miRCURY LNA array power labeling kit (Exiqon A/S, Vedbæk, Denmark). Samples with RNA concentrations lower than 160 ng/µl were vacuum-centrifuged (speedvac) to obtain higher concentrations. All samples were labeled the same day with the same master mix to minimize technical variation. The labeled samples were hybridized to miRCURY LNA 7th generation arrays (Manual version 2.2; Exiqon A/S, Vedbæk, Denmark), containing capture probes targeting all human miRNAs registered in miRBASE release 18.0 (1896 human miRNAs). The hybridization was performed overnight at 56°C according to the manufacturer’s specifications using an HS4800 hybridization station (Tecan, Männendorf, Switzerland). Since it was not possible to hybridize all arrays at the same time point, samples were randomly split into six batches to minimize day-to-day variation in the hybridization process, and analyzed over six consecutive days. After hybridization, the microarray slides were scanned in an ozone free environment in order to prevent potential bleaching of the fluorescent Hy5 dye. Scanning was done using a G2565BA Microarray Scanner system (Agilent Technologies, Santa Clara, CA, USA) at 10-µm resolution, and the resulting TIFF images were analyzed using the GenePix 6.1 software on standard settings.