Circulating Exosomal microRNAs Are Prognostic Markers in Multiple Myeloma
Authors: Manier Salomon , Avet-Loiseau Herve , Campigotto Federico , Salem Karma , Huynh Daisy , Glavey Siobhan , Sacco Antonio , Roccaro Aldo M , Minvielle Stephane , Moreau Philippe , Facon Thierry , Leleu Xavier , Weller Edie A. , Ghobrial Irene M. 
Published: October 2019

Abstract
Background Exosomes are secreted by several cell types including cancer cells and can be isolated from peripheral blood. They contain proteins and nucleic acids and promote tumorigenesis in many types of cancer. We aimed to establish the prognostic significance of circulating exosomal microRNAs (miRNAs) in multiple myeloma (MM).
Methods We first analyzed the miRNAs content of circulating exosomes in MM by small RNA sequencing of 10 samples from MM patients and 5 healthy controls. We then analyzed 156 serum samples from newly diagnosed patients with MM, uniformly treated with a Bortezomib and Dexamethasone based regimen. Using a quantitative RT-PCR array for 23 miRNAs, we assessed the associations between exosomal miRNAs and progression-free survival.
Findings By next generation sequencing, we identified 158 differentially expressed miRNAs in MM compared to normal healthy controls, notably including let-7 family members, miR-17/92 or miR-99b/125a clusters. We further identified a three-miRNA signature based on 156 MM samples (combining miR-106b, miR-18a and let-7e) and calculated a risk score to classify patients as high risk or low risk. Compared to low risk score, patients with a high risk score had a shorter PFS in the training set (hazard ratio [HR] 1·8, 95% CI 1·0-3·0; p=0·0375) and the validation set (HR 2·6, 1·5-4·4; p=0·0005). To further validate this signature, we generated 500 randomly computed re-sampling of the data sets. The three-miRNA signature was consistently significant with a p-value < 0·05 in more than 78% and < 0·10 in 86% of the 500 randomizations. The circulating exosomal miRNA signature was an independent prognostic marker after adjusting for cytogenetics and ISS. In a receiver operating characteristic (ROC) analysis, a combination of this signature together with International Staging System (ISS) and cytogenetics had a better prognostic value than ISS and cytogenetics alone in the training set (2 years area under the ROC curve 0·64 [95% CI 0·56-0·72] vs. 0·60 [95% CI 0·52-0·69]) and the validation set (0·67 [0·59-0·75] vs. 0·58 [0·50-0·66]).
Interpretation This study demonstrates unprecedented evidence of the prognostic significance of exosomal miRNAs in patients with MM. We identified a three-miRNA signature in circulating exosomes that adds prognostic value to ISS and cytogenetic status and helps improve prognostic identification of newly diagnosed MM patients.

Disclosures
Avet-Loiseau: jansen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; jansen: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Moreau:Celgene, Janssen, Takeda, Novartis, Amgen: Membership on an entity's Board of Directors or advisory committees. Facon:Onyx: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Pierre Fabre: Membership on an entity's Board of Directors or advisory committees.