Multiple myeloma patients-derived exosomes as a potential new clinical tool

Abstract

Introduction: Multiple Myeloma (MM) is a highly heterogeneous neoplasia, usually preceded by a monoclonal-gammopathy-of-undetermined-significance (MGUS). MM has a diverse clinical presentation, variable response to treatment and outcome. Poor response to novel drugs or extramedullary-disease (EMD) are some of the parameters associated to poor prognosis. Exosomes (EXO) are extracellular vesicles implicated, among others, in promotion of supportive tumor microenvironments [1]. EXO in MM may act as biomarkers of disease aggressiveness [2] and potentially be used as “liquid biopsies” for better stratification of disease.

Materials and methods: EXO were isolated from peripheral blood (PB) and bone marrow (BM) patient samples by sequential ultracentrifugation. Patient (MM, MGUS) and healthy donors (HD) were compared regarding EXO concentration (EXO/ml) and average protein cargo/EXO (µg/EXO), in PB and BM. Informed consent of the subjects and acceptance of the study protocol by a local ethics committee has been obtained. µg/EXO was determined by the ratio between total quantity of EXO proteins, measured by protein assay using bicinchoninic acid, and the total number of exosomes, measured by Nanosight. Statistical analysis was done with GraphPad.

Results: There was no difference in EXO/ml in MM vs MGUS vs HD. However, a positive correlation in EXO/ml between PB and BM in MM and MGUS (Pearson R = 0.80; p < 0.0001) was found. MM patients (n = 10) had 2,8x more µg/EXO in PB, when compared to MGUS patients (n = 5) (p = 0.058); MM median 1.2 (0.50–3.33) µg/EXO versus MGUS median 0.43 (0.10–0.69) µg/EXO. In BM, µg/EXO was also 2,6x higher in MM than in MGUS: 0.92 (0.15–2.13) µg/EXO versus 0.36 (0.06–0.45) µg/EXO, respectively. Analyzing patients by clinical subgroups, we found that those with EMD had higher µg/EXO. Comparing µg/EXO pre-treatment (T1) and at response evaluation (T2), we found that patients with progressive disease had a lower decrease (48%) when compared to patients achieving response to treatment (90%): median T1 1.24 (1.12–3.45) µg/EXO and median T2 0.65 (0.5–0.72) µg/EXO vs median T1 3.29 (1.17–4.15) µg/EXO and median T2 0.35 (0.32–0.5) µg/EXO, respectively.

Discussion and conclusions: 1-The positive correlation of the number of EXO in PB and BM samples in MM and MGUS patients may support the use of EXO as liquid biopsies for biomarkers of BM disease, as a less invasive method. 2-MM patients have higher total exosomal protein cargo in PB when compared to MGUS patients and HD and this finding is particularly striking in patients with extramedullary disease and more aggressive forms of MM. The longitudinal evaluation in 2 different time-points (T1 and T2) suggests a correlation between EXO protein load and response to treatment, to be confirmed in a larger cohort. Our work will further evaluate the profile of the proteins included in these EXO and its roles on disease progression, response to treatment and micro environment modification.