Measurable Residual Disease in Extracellular Vesicles from Bone Marrow and Peripheral Blood of Patients with Multiple Myeloma: A Proof-of-Concept Study
Authors: Bergantim Rui , Barbosa Mélanie A.G. , Silva Sara Peixoto da , Polónia Bárbara , Caires Hugo R. , Guimarães José E. , Vasconcelos M. Helena 
Published: November 2021

Abstract
BACKGROUND: Multiple myeloma (MM) treatment improved substantially in the last years, with unprecedented survival outcomes. However, even when achieving complete remission, patients ultimately relapse. Therefore, monitoring measurable residual disease (MRD) is crucial to assess treatment response and define the depth of patients' remission status. However, this currently still requires invasive bone marrow (BM) aspirates, which severely hinders real-time monitoring of the disease. Therefore, the identification of biomarkers of MRD in the peripheral blood (PB) of patients would allow a more frequent and minimally invasive monitoring of MRD. Extracellular Vesicles (EVs) are small particles (30-1000nm) shed by all cells, which are found in all biofluids including the BM and PB. These particles carry a specific cargo from their cell of origin, including proteins, enclosed by a lipidic layer. Therefore, they have been described as a possible source of cancer biomarkers, with potential to monitor MRD. AIMS: This study aimed to implement a protocol for the isolation of EVs from the BM and PB of MM patients at distinct stages of the disease (diagnosis and remission), in order to detect and compare the levels of known MRD biomarkers in their cargo. METHODS: The study was previously approved by the Ethical Committee of CHSJ and patient's consent was obtained. EVs from BM and PB Platelet-Poor Plasma (PPP) were isolated by size-exclusion chromatography (SEC), and further concentrated by ultrafiltration (UF). Then, the EVs were characterized according to their size and concentration (by Nanoparticle Tracking Analysis), morphology (by Transmission Electron Microscopy), protein concentration (Lowry protein assay) and presence of EV-associated protein markers (Western Blot - WB). In addition, 16 known MRD and MM biomarkers were analyzed by WB in the isolated EVs from PB and BM of seven patients, at two main stages of the disease - diagnosis versus response after autologous stem cell transplant (ASCT). Clinical features regarding cytogenetics and immunophenotypic markers using multi-parameter flow cytometry (MFC) were analyzed and compared. RESULTS: The two-step protocol described allowed the isolation of size-resolved EVs from both PB and BM of MM patients. The EVs isolated (both from PB and BM) presented a size-range from 50 to 500nm and presented EV-associated protein markers, such as CD81 and CD63. Moreover, several MM MRD biomarkers (e.g. CD56, CD45, CD38 and light chain) were detected in the cargo of the EVs from BM and PB at diagnosis and complete remission. The biomarkers of MM and MRD detected in the cargo of PB EVs were mainly the same as the ones detected in the cargo of BM EVs. The complete remission after ASCT was mostly associated with a decrease in the expression of EV-associated MM markers in both the BM and the PB; however, in some patients a few of the markers persisted at this stage when compared to diagnosis. In fact, the expression of CD45 and HLA-DR persisted at the remission stage in 3 and 2, respectively, out of 5 patients presenting these markers at diagnosis. Moreover, an increased expression of CD56 was also detected at remission in 3 out of 7 patients. By correlating these data with patient's routine work-up it was found that patients with persistent CD45 didn't reach 10^-5 MRD negative by flow cytometry. CONCLUSIONS: Taken together, this work suggests that it is possible to detect MM markers in EVs from either BM or PB of MM patients and compare their expression at different stages of the disease (diagnosis and remission after ASCT). Importantly, our results demonstrate the importance and potential of analyzing EVs cargo from PB, suggesting the possibility of using them for minimally invasive monitoring of MRD in MM patients.
ACKNOWLEDGEMENTS: The authors acknowledge Celgene/BMS for providing funding to this work (Project Looker - Grant_138800). The authors acknowledge Cytogenetics Laboratory, Department of Clinical Hematology, Centro Hospitalar e Universitário São João and Flow Cytometry Laboratory, Department of Clinical Pathology, Centro Hospitalar e Universitário São João.

Disclosures
Bergantim: Amgen: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; BMS: Consultancy, Research Funding, Speakers Bureau; Takeda: Consultancy, Speakers Bureau. Barbosa: BMS: Research Funding. Silva: BMS: Research Funding. Polónia: BMS: Research Funding. Caires: BMS: Research Funding. Guimarães: BMS: Research Funding; Amgen: Research Funding. Vasconcelos: BMS: Research Funding; Amgen: Research Funding.