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Background: Multiple myeloma (MM) is a bone marrow derived cancer of plasma cells, which remains an incurable disease. Because of the invasive and painful nature of bone biopsy, an alternate tumor monitoring strategy is needed. We have previously shown that Circulating Multiple Myeloma Cells (CMMCs) isolated by CellSearch® (CS) are prognostic and indicative of disease burden through remission and relapse. Here we report, for the first time, the molecular characterization of pure single CMMCs isolated from a multiple myeloma patient, by integrating CS and DEPArray™ (DA) NxT systems, providing access to copy-number alteration (CNA) profiling. Methods: 4.0ml of peripheral blood from a patient with multiple myeloma was tested. On CS, target CMMCs were enriched using anti-CD138 for cell capture, and stained immunofluorescently with CD38-PE, CD19 and CD45-APC. Nuclei were stained with DAPI and detected target cells counted. The enriched sample was then analyzed using the DA NxT system: single CMMCs (CD38+/CD19- and CD45-/DAPI+), along with some single White Blood Cells (WBCs: CD38-/CD19+ or CD45+/DAPI+), were isolated. The DNA of each single cell was amplified using the Ampli1™ WGA kit and used for highly-multiplexed, genome-wide single-cell CNA analysis using a Ampli1™ LowPass kit (LPCNA) on Illumina® MiSeq. Results: CS identified 128 CMMCs. From DA NxT we harvested 20 individual CMMCs for LPCNA analysis. Copy-number profiles of CMMCs confirmed their tumor origin, showing a high-level of genome instability with several gains and few losses of chromosomal segments. Moreover, an unsupervised hierarchical cluster analysis highlighted a conserved pattern of alterations, enabling the separation between CMMCs and WBCs groups. A pattern of copy-number gains shared by all CMMCs, coupled with gains and losses shared by a subset of CMMCs, suggests a branched evolution of different tumor subclones. WBC profiles were flat as expected. Conclusions: Cell enrichment by CS followed by individual cell sorting using DA NxT, enabled the isolation of single CMMCs with 100% purity. Ampli1™ single-cell analysis demonstrated CMMC molecular heterogeneity suggestive of tumor subclones presence. This platform combination provides a reliable and non-invasive method for MM characterization enabling translational research and future clinical application. Citation Format: Mario Terracciano, Claudio Forcato, Edoardo Petrini, Alberto Ferrarini, Valentina del Monaco, Andrea Raspadori, Carrie Morano, Steven Gross, Chiara Bolognesi, Genny Buson, Thai Bui, Francesca Fontana, Gianni Medoro, Mark Connelly, Nicolo Manaresi. An integrated workflow for liquid biopsy of circulating multiple myeloma cells (CMMCs) with single cell resolution reveals tumor heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2198.

cancer research

Abstract 2198: An integrated workflow for liquid biopsy of circulating multiple myeloma cells (CMMCs) with single cell resolution reveals tumor heterogeneity