Abstract 4118: Molecular cytometry for immunotherapy trials

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The ability to simultaneously query both protein and mRNA expression on tens of thousands of single cells has emerged only recently, with the development of CITE-seq, REAP-seq, and Ab-seq platforms. Each technology relies on antibodies conjugated to oligonucleotide tags, followed by capture of antibody-stained cells for single cell RNA-sequencing. The technologies have important advantages over flow cytometry, chiefly in that they allow study of a limitless number of parameters, and single cell RNA sequencing, which cannot identify cell populations with as much verity as protein/antibody-based analysis. We characterized the newest molecular cytometry technique (Ab-seq, BD Biosciences) by titrating nearly 100 antibodies (in a single staining experiment and sequencing run), and demonstrated that oligonucleotide antibodies exhibit the same staining characteristics and patterns as flow cytometry antibodies. We also comprehensively examined cells discordant for protein and mRNA expression, in order to discriminate true features of immune cell biology from artifacts caused by drop-out in sequencing assays. Importantly, we also found that mRNA expression could guide the discrimination of cells expressing a protein versus those lacking expression, and define a remarkably low limit of detection for the technology: 1-5 molecules. This remarkable sensitivity will allow detection of cells expressing even the lowest levels of immune exhaustion and checkpoint molecules in cancer immunotherapy trials, and could enable better biomarker discovery than existing technologies. Moreover, this approach allows more detailed characterization of the tumor microenvironment and periphery than ever before. Indeed, in a lymphoma patient, we show that cells expressing both PD1 and CTLA4 carry a unique and specific signature that includes markers never revealed by flow cytometry or other technologies, which might represent new targets for immunotherapy. In sum, this presentation will characterize this new technology and demonstrate the power and promise of new molecular cytometry tools.Citation Format: Woodrow E. Lomas, Aidan F. Winters, Jason Alexandre, Jody Martin, Nidhanjali Bansal, Margaret Nakamoto, Brent Gaylord, Suraj Saksena, Pratip K. Chattopadhyay. Molecular cytometry for immunotherapy trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4118.

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