A High-Throughput Workflow for the Detection, Isolation and Genomic Analysis of Single Circulating Multiple Myeloma Cells
Authors: Raspadori Andrea , Forcato Claudio , Edoardo Petrini , Papadopulos Francesca Marzia , Ferrarini Alberto , Del Monaco Valentina , Terracciano Mario , Morano Carrie , Gross Steven , Bolognesi Chiara , Buson Genny , Fontana Francesca , Connelly Mark , Simonelli Cecilia , Medoro Gianni , Manaresi Nicolò 
Published: October 2019

Abstract
Introduction: Multiple myeloma (MM) is a malignancy of terminally differentiated plasma cells. The high heterogeneity of MM cells is one of the major cause of disease relapse. Detection of circulating MM cells (CMMC) from peripheral blood is a useful procedure to investigate tumor heterogeneity and provides a painless alternative to the classic bone marrow biopsy to monitor disease progression. Here we demonstrate that the synergy between CellSearch® (CS) and DEPArray™ (DA) technologies can be used to identify, isolate and characterize at the genetic level single and pure CMMCs .
Methods: 4.0 ml of peripheral blood samples were obtained from 3 patients with MM. Putative CMMCs were enriched with CS using anti-CD138 or anti-CD138/CD38 as positive selection marker and subsequently stained with CD38-PE, CD19/CD45-APC immunofluorescent probes. Cells detection and enumeration was performed based on the co-localization of nuclei DAPI staining and CD38-PE. Single CMMCs (CD38+/CD19- and CD45-/DAPI+) and White Blood Cells (WBCs: CD38-/CD19+ or CD45+/DAPI+) were then isolated using the DA NxT system. Single cells genomic DNA was amplified using Ampli1™ Whole Genome Amplification (WGA) kit and Illumina®-compatible libraries were obtained using Ampli1™ LowPass kit and a high-throughput, customized automated protocol using Hamilton STARLet Liquid handler. Highly-multiplexed, genome-wide single-cell Low-Pass Copy Number Alteration (LPCNA) analysis was performed using HiSeq 2500 Illumina® platform.
Results: CS and DA workflow* enabled the isolation of 215 single CMMC, selected for LPCNA analysis. 42 single WBCs were also included as normal controls. Copy-number profiles of single CMMCs showed relevant gains and losses of chromosomal segments, as result of a high-level genomic instability. Notably, intra-patient CMMCs revealed overall conserved CNA patterns with subclonal alterations, suggesting a certain level of branched tumor evolution. Conversely, a higher degree of heterogeneity in CMMCs CNA profiles was observed among different patients. Interestingly, CNAs detected in all patients are located in regions containing genes involved in cell cycle regulation (MAPK, NOTCH pathways) and cell signaling (IL6R), which might be involved in proliferative processes and immuno-surveillance escape.
Conclusion: The combination of CS and DA workflow* with a streamlined automated protocol allowed to obtain hundreds of genomic libraries from pure single CMMCs. The presented workflow constitutes a non-invasive, rapid and high-throughput approach for characterizing MM tumor heterogeneity and progression, suggesting a possible future implementation in clinical applications.
*For Research Use Only. Not for use in diagnostic procedures.

Disclosures
Raspadori: Menarini Silicon Biosystems: Employment. Forcato:Menarini Silicon Biosystems: Employment. Edoardo:Menarini Silicon Biosystems: Employment. Papadopulos:Menarini Silicon Biosystems: Employment. Ferrarini:Menarini Silicon Biosystems: Employment. Del Monaco:Menarini Silicon Biosystems: Employment. Terracciano:Menarini Silicon Biosystems: Employment. Morano:Menarini Silicon Biosystems: Employment. Gross:Menarini Silicon Biosystems: Employment. Bolognesi:Menarini Silicon Biosystems: Employment. Buson:Menarini Silicon Biosystems: Employment. Fontana:Menarini Silicon Biosystems: Employment. Connelly:Menarini Silicon Biosystems, Inc.: Employment, Other: Chief R&D Officer, USA. Simonelli:Menarini Silicon Biosystems: Employment. Medoro:Menarini Silicon Biosystems: Employment. Manaresi:Menarini Silicon Biosystems: Employment.