P-401: miRNA-seq and clinical validation of miR-221/222 cluster in multiple myeloma

Introduction: Despite the substantive advancements that occurred in multiple myeloma (MM) therapy over the past years, the highly heterogenous treatment response hinders reliable prognosis and tailored therapeutics, highlighting the urgent need for the delineation of innovative prognostic markers to be incorporated into clinical care. In this context, high-throughput techniques have unveiled microRNAs (miRNAs), as potent post-transcriptional regulators of gene expression with critical implications in cancer onset and progression. The emergence of these techniques revealed that miRNAs are deregulated in all human tumors and highlighted their utility as potential prognostic indicators. Herein, using miRNA-seq we have investigated deregulated miRNAs between R-ISS III vs R-ISS I/II MM patients, aiming to reveal potential MM-related miRNAs able to improve patients’ risk stratification and prognosis.

Methods: Bone marrow aspiration (BMA) samples were collected from 121 MM patients at diagnosis. Mononuclear cells were isolated using Ficoll-Paque, while CD138+ plasma cells were positively selected using magnetic beads with anti-CD138 mAbs. Next, miRNA-seq was performed in CD138+ plasma cells from 24 MM patients (8 R-ISS I, 8 R-ISS II and 8 R-ISS III). Based on miRNA-seq, the prognostic impact of both miR-221-3p and miR-222-3p was further evaluated, for the first time, in our MM screening cohort. Subsequently, RNA extraction, 3’-end polyadenylation and reverse transcription, miR-221-3p and miR-222-3p levels were evaluated using in-house developed qPCR assays. Finally, patients’ mortality and disease progression were assessed as clinical endpoints for survival analysis.

Results: miRNA-seq revealed both miR-221 and miR-222 to be concurrently downregulated in R-ISS III vs R-ISS I/II patients (miR-221: Fold Change;FC=0.356; log2(FC)=-1.48; miR-222: FC=0.357; log2(FC)=-1.48). In our screening cohort, miR-221 loss was associated with a significantly higher risk of short-term disease progression (p=0.002) and poor overall survival following treatment (p=0.040), while univariate Cox regression analysis confirmed the adverse prognostic value of miR-221 loss in patients’ overall survival (HR=2.158; p=0.047) and disease progression (HR=2.386; p=0.004). Along the same lines, miR-222 loss was linked with poor overall survival (p=0.012) and higher risk of short-term disease progression (p=0.008), while univariate Cox regression analysis confirmed miR-222 loss as an unfavorable prognostic indicator of patients’ overall survival (HR=2.569; p=0.016) and short-term disease progression (HR=2.143; p=0.011) in MM patients.

Conclusions: Ultimately, we identified the loss of miR-221/222 in CD138+ plasma cells as a powerful indicator of adverse disease outcome, ameliorating tailored patient’s management.