Abstract 1559: Detection and retrieval of multiple myeloma cells with single-cell molecular confirmation from peripheral blood by multiparameter immunofluorescence, automated scanning, and image analysis

Abstract

Abstract Background: The detection of multiple myeloma (MM) cells is currently performed by analysis of bone marrow aspirates. Detection of MM cells from the peripheral blood would allow for less invasive and more frequent assessments to monitor residual disease. Using the RareCyte platform, we developed a 5-parameter assay to visualize and retrieve MM cells from peripheral blood and applied it to MM patient samples under an IRB-approved protocol. We also used this to identify plasma cells from the peripheral blood of healthy volunteer donors. Materials and Methods: Healthy volunteer or MM patient peripheral blood was collected into RareCyte blood collection tubes. RPMI8226 cells spiked into normal donor blood samples were used as a model for MM cell assay development. Buffy coats isolated from 7.5mL of blood by AccuCyte® separation were spread onto slides. Slides were stained with a 5-marker panel that included antibodies to CD138, CD38, CD56, CD45, and a nuclear dye on the Leica Bond Rx auto-stainer. Slides were scanned with CyteFinder® and MM cells identified by positive CD138 and CD56 staining and negative CD45 staining. Identified MM cells and WBC control cells were mechanically retrieved with the integrated CytePicker module, whole genome amplified, and Sanger sequencing was performed for specific mutations in four genes (IDH1, NRAS, PHLDA1, RPH3AL) identified by genomic analysis of a bone marrow sample. Plasma cells from normal donors were identified by co-expression of CD138 and CD38. Results: In a pretreatment MM sample, the 5-parameter assay identified 55 MM cells per mL of whole blood. In isolated single cells with at least one successful sequencing reaction, 0 of 13 WBCs contained mutations in at least one of the genes. Mutations were found in 9 of 15 MM cells (defined as CD138+, CD56+, CD38-, and CD45-). In healthy volunteer samples, 5 plasma cells (defined as CD138+, CD38+, and CD45 dim/negative) were identified per mL of whole blood. Conclusions: The 5-parameter assay successfully detected rare cells with an MM phenotype from the peripheral blood of an MM patient. Individually retrieved MM cells were demonstrated to contain mutations specific to the patient's tumor, confirming that the cells identified were MM cells. This approach may allow for less invasive assessment of MM and monitoring of residual disease. Citation Format: Lance U'Ren, Nolan Ericson, Daniel Campton, Arturo Ramirez, C. Anthony Blau, Eric Kaldljian. Detection and retrieval of multiple myeloma cells with single-cell molecular confirmation from peripheral blood by multiparameter immunofluorescence, automated scanning, and image analysis [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 1559.