Abstract
Abstract Multiple Myeloma is bone marrow tumor that frequently results in the aberrant release of neoplastic plasma cells into circulation. The CELLSEARCH® system (Janssen Diagnostics, Raritan, NJ) has been used to capture, enumerate, and perform FISH analysis on circulating multiple myeloma cells (CMMC) from patients with active multiple myeloma, smoldering myeloma, and MGUS (Gross, et.al. Poster #1825, ASH 2011). This method utilizes a blood fixative and subsequently permeabilizes the cells, which confounds the ability to do mRNA analysis on these samples. Here we present a novel method to isolate pure circulating myeloma cells that is compatible with downstream genetic analysis. Blood was obtained through a commercial vendor (Conversant Bio) from multiple myeloma patients. Samples were processed in parallel using the existing method for enumeration and a novel method for genetic profiling. For CMMC enumeration, blood was collected in CellSave tubes (Janssen Diagnostics) and CMMC were enriched using paramagnetic ferrofluid recognizing CD138 and CS1 antigens. Cells were permeabilized and stained on the CELLSEARCH® AUTOPREP with CD38 (PE), CD45 (APC), CD19 (APC) and DAPI. CMMC were enumerated using the CELLTRACKS ANALYZER II® scanning platform. CMMC were defined as DAPI +, CD138+, CD38+, CD45-, CD19-. For genetic profiling, 4 ml EDTA blood was enriched using CD138 and CS1 ferrofluids on the AUTOPREP with no subsequent permeabilization. CMMC were stained offline using CD38 (Texas Red), CD45 (FITC), CD19 (FITC), CD3 (FITC) and NucBlue. Samples were transferred to glass a bottomed petri dish and observed through an inverted microscope using a Texas Red/FITC dual filter, individual FITC and Texas red filters, and a DAPI filter. CMMC were defined as NucBlue+, CD138+, CD38+, CD45-, CD19-, and CD3-. Pools of individual CMMC were picked from the sample using an Eppendorf Transferman micromanipulator. For comparison, contaminating leukocytes were also collected for analysis. RNA from picked cells was amplified using the SMARTer Ultra Low Input Kit (Clontech) and qPCR was performed for myeloma and leukocyte specific markers. Detection of CMMC using the novel method for genetic profiling was found to be concordant with the existing method for enumeration. We found this approach to provide a deep and unbiased characterization of gene pathways activated in CMMC by RT qPCR and RNASeq. This method will have applications in longitudinal studies of active myeloma patients as a way to gather information about relapse and minimal residual disease between bone marrow draws. Citation Format: Greg Brittingham, Chandra Rao, Peter Vulfson, Vipul Bhargava, Denis Smirnov, Brad Foulk. A novel method for isolation and genetic analysis of pure populations of circulating plasma cells from multiple myeloma patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1597. doi:10.1158/1538-7445.AM2015-1597
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