Abstract 594: Image-based identification of single circulating tumor cells in ovarian cancer patients for genomic analysis
Authors: Salmon Carolin , Levermann Janina , Buderath Paul , Kuhlmann Jan Dominik , Wimberger Pauline , Kimmig Rainer , Kasimir-Bauer Sabine 
Published: July 2021

Abstract
Background: In ovarian cancer (OC), one of the leading causes of death among gynecological tumors, predictive biomarkers for personalized treatment are lacking. Combining immunomagnetic enrichment and molecular analysis in circulating tumor cell (CTC) fractions of OC patients, excision repair cross-complementing group 1 (ERCC1) endonuclease-positive CTCs independently predicted survival and clinical platinum resistance, which further selected for CTCs in epithelial-mesenchymal transition (EMT). However, these analyses could not reflect single CTC (sCTC) heterogeneity. We here present a workflow for the detection and analysis of sCTCs using negative immunomagnetic enrichment and immunofluorescence imaging for cytokeratin (CK), ERCC1 and CD11b, a white blood cell adhesion molecule also identified on OC tumors, to provide insights into CTC heterogeneity in OC.
Patients and methods: 10 ml blood of 15 high-grade serous OC patients at the time of primary diagnosis was analyzed for sCTCs using density-gradient centrifugation to further apply MACS® Technology (Miltenyi Biotec, Germany) using CD45 (leucocyte reduction) and CD235a (erythrocyte reduction) antibodies coupled to MACS® MicroBeads. After two consecutive separations, image-based sCTC sorting was performed with the DEPArray® System, applying an immunofluorescence protocol staining for Hoechst, CK, ERCC1, CD11b, and CD45. After volume reduction, the recovered sCTCs were stored at -80°C for later whole genome amplification, comprising CTC cell lysis, DNA digestion, adaptor ligation and an amplification PCR for subsequent copy number variation (CNV) analysis.
Results: We were able to identify three cell populations of interest with different expression patterns. In total, 31 (0-5) Hoechstpos/CKpos/ERCC1pos/CD11bpos/CD45neg cells were identified in 13/15 patients, probably displaying epithelial sCTCs expressing CK. Interestingly, CK-positivity was frequently linked to CD11b-positivity, thereby, suggesting CD11b as a negative mesenchymal maker. Furthermore, single Hoechstpos/ERCC1pos/CD11bpos/CD45neg as well as Hoechstpos/ERCC1pos/CD11bneg/CD45neg cells, both lacking CK-positivity, were detected. Ongoing CNV analysis will clarify whether a) the latter subgroup of cells represents a more mesenchymal-like CTC subpopulation and b) whether CD11b sensitivity in Hoechstpos/ERCC1pos/CD11bpos/CD45neg is utilizable for the extended detection of epithelial-like CTCs or whether this expression pattern is related to immune cells that might influence the course of the disease. CNV analyses are ongoing and we expect the first sequencing data to be available at the meeting.
Conclusions: This ongoing study will characterize sCTCs in the follow-up of the disease and CNV analysis will provide further information on CTC heterogeneity and their malignant character to tailor treatment of OC in the future.
Citation Format: Carolin Salmon, Janina Levermann, Paul Buderath, Jan Dominik Kuhlmann, Pauline Wimberger, Rainer Kimmig, Sabine Kasimir-Bauer. Image-based identification of single circulating tumor cells in ovarian cancer patients for genomic analysis [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 594.