Prognostic Value of Circulating Exosomal microRNAs in 112 Patients with Multiple Myeloma
Authors: Manier Salomon , Avet-Loiseau Herve , Campigotto Federico , Glavey Siobhan , Salem Karma , Huynh Daisy , Boswell Erica N , Sacco Antonio , Mishima Yuji , Moschetta Michele , Reagan Michaela R. , Kawano Yawara , Anderson Kenneth C. , Roccaro Aldo M , Moreau Philippe , Facon Thierry , Leleu Xavier , Weller Edie A. , Ghobrial Irene M. 
Published: October 2019

Abstract
Introduction.Exosomes are small vesicles (50-100 nm) of endocytic origin, which are released in the extra-cellular milieu by several cell types. Exosomes play a role in tumor progression since they have been shown to carry and transfer microRNAs (miRNAs) to recipient cells. In this study, we sought to characterize circulating exosomes in Multiple Myeloma (MM) patients and to assess the prognostic value of circulating exosomal miRNAs in a cohort of 112 newly diagnosed MM patients.
Methods. Exosomes were isolated from peripheral blood (serum samples) using an ultracentrifugation protocol and Exoquick® solution. They were characterized using electron microscopy with immunogold labeling for the detection of CD63 and CD81, as well as for size using Nanosight® analysis. MiRNAs were isolated using miRNeasy micro kit (Qiagen®) and profiled using nCounter miRNA expression assay (Nanostring® Technologies) in 5 healthy donors (HD) and 10 MM patients. We next studied a cohort of 112 newly diagnosed MM patients uniformly treated with Bortezomib-Dexamethasone (4 cycles) followed by high dose Melphalan and autotransplant on the IFM 2005-01 Phase III Trial. After exosome isolation and miRNA extraction, we performed a low density qRT-PCR miRNA array using Taqman Array Micro Fluidic Cards for a panel of 23 specific miRNAs. Data were normalized by robust median global normalization and conditional inference tree was used to select the cut-point for miRNA levels. Kaplan-Meier estimation, Log-Rank test and Cox regression were used to assess progression-free survival (PFS).
Results. Circulating exosomes were first characterized from 5 HD and 10 MM patients. CD63 and CD81 expression was demonstrated by immunogold labeling and electron microscopy. The size (approx. 100nm) and concentration of peripheral blood exosomes did not differ between HD and MM patients using Nanosight® analysis. We next performed a miRNA array (Nanostring) and identified differentially expressed miRNAs in circulating exosomes from MM patients compared to HD. Notably, the miR-17/92, -106b/25, -106a/363 clusters were more highly expressed and the cluster miR-15a/16 and let-7 family members were down regulated in circulating exosomes from MM patients as compared to HD. We then designed a panel of 23 specific miRNAs from circulating exosomes in MM and performed a Taqman Low Density Array on a cohort of 112 patients who were newly diagnosed and therapy-naive in the IFM 2005-01 Phase III Trial or treated as such. The mean patient’s age was 61 years, 36% of patients had a 13q deletion, 7% a t(4;14) and 4% a 17p deletion. ISS stage I, II and III distribution was 37%, 39% and 21% respectively. The median follow up was 5.4 years. Among tumor suppressor miRNAs, we found that patients with lower let-7e levels before treatment had significantly shorter PFS compared to patients with higher levels of let-7e (P<0.001). The median PFS was 2.46 years and 4.06 years respectively in low and high let-7e level groups (Figure). A multivariate Cox proportional hazards model was fitted and included ISS (III vs. I), cytogenetics (17p deletion and t(4;14) vs. no abnormality) and let-7e expression (high vs. low). This indicated that decreased let-7e expression is associated with decreased PFS [hazard ratio (HR) = 0.4; P=.0004] after adjusting for the effects of ISS stage (HR=1.4; P=.261) and adverse cytogenetic status (HR=1.8; P=.096).
Conclusion.These findings indicate that circulating exosomes differ between normal and MM patients in terms of miRNA content. In this study, we report for the first time the prognostic impact of exosomal miRNA content in MM. The data presented here indicate that circulating exosomal levels of let7-e are an independent predictor of PFS in patients with MM who are therapy-naive.
Figure 1 Kaplan-Meier PFS curves by let-7e exosome levels in 112 patients with MM Figure 1. Kaplan-Meier PFS curves by let-7e exosome levels in 112 patients with MM

Disclosures
Anderson: Celgene: Consultancy; Sanofi-Aventis: Consultancy; Onyx: Consultancy; Acetylon: Scientific Founder, Scientific Founder Other; Oncoprep: Scientific Founder Other; Gilead Sciences: Consultancy. Ghobrial:Sanofi: Research Funding; Noxxon: Research Funding; BMS: Advisory board, Advisory board Other, Research Funding; Onyx: Advisory board Other.