Abstract 513: Simultaneous monitoring of immune cell subsets and minimal residual disease in bone marrow aspirate using full-spectrum flow cytometry
Authors: van der Touw William , Stout Bhavani , McCauley James , Mahlakõiv Tanel , Hariri Robert , Zhang Xiaokui 
Published: July 2021

Abstract
Background: Treatment options for multiple myeloma (MM) patients have expanded and improved outcomes. Autologous stem cell transplantation, immunomodulatory drugs (IMiDs), monoclonal antibodies, and CAR-T cell therapy have direct and indirect immune modulating effects that favor patient responses. However, the majority of patients still relapse due to minimal residual disease (MRD). Current flow cytometry methods that monitor MRD and bone marrow immune cell populations require large cell numbers to achieve assay sensitivity. New approaches are needed to better capture high-resolution data given the limited recovery of bone marrow specimens. Here, we describe the simultaneous monitoring of immune cell subsets in the bone marrow and MRD using a single antibody panel and the Cytek Aurora full-spectrum flow cytometry platform. In a Phase 1/2 study to evaluate an off-the-shelf, allogeneic human placental-derived NK cell product in newly diagnosed MM, this method enables correlative analysis of MRD state with immune profiling.
Methods: Bone marrow aspirate (BMA) from 5 patients with newly diagnosed MM following induction therapy were collected for MRD assessment in parallel using next-generation sequencing (NGS) and MRD evaluation by flow cytometry. BMA was lysed of red blood cells and 2x107 cells were stained using TCRγδ, CD16, CD8, CD33, CD117, CD11c, CD3, CD19, CD1c, CD45, CD38, CD14, HLA-DR, CD138, CD141, CD34, CD25, CRTH2, CD56, CD303, CD4, and CD127. Cells were acquired on a Cytek Aurora 4 laser system followed by data analysis using FlowJo software. MRD threshold of 10-5 was used for positivity for both modalities of testing.
Results: The 22-parameter panel resolved the majority of immune cell subpopulations present in the bone marrow and peripheral blood including granulocytes, B cells, plasma cells, monocytes, MDSCs, plasmacytoid and classical dendritic cells, NK cells, and T cells (CD4, CD8, NKT, γδT, Treg). Total plasma cells were identified based upon CD38+CD138+ co-staining. Myeloma cells could be identified based on aberrant expression of CD45, CD19, CD56, and CD117. Of the five patients evaluated, one patient was MRD negative by NGS and flow cytometry with no detectable cells. One patient was MRD negative by NGS (0.7x10-5) but positive by flow cytometry (1.53x10-5). The three remaining patients were positive by NGS (6.32x10-4, 7.51x10-4, and 4.78x10-4) and flow cytometry (2.46x10-4, 5.37x10-4, and 9.74x10-4).
Conclusion: Utilizing full-spectrum flow cytometry provides streamlined monitoring of MRD while capturing BMA-associated immune cell populations. Compared to validated measures of MRD testing by NGS, our flow cytometry approach provides comparable results while profiling myeloid and lymphoid cell subpopulations, which enables correlative analyses of underlying pathophysiological activities.
Citation Format: William van der Touw, Bhavani Stout, James McCauley, Tanel Mahlakõiv, Robert Hariri, Xiaokui Zhang. Simultaneous monitoring of immune cell subsets and minimal residual disease in bone marrow aspirate using full-spectrum flow cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 513.