Abstract 6561: Phenotypic and genotypic characterization of single circulating tumor cells in the follow-up of high-grade serous ovarian cancer
Authors: Kasimir-Bauer Sabine , Stoecklein Nikolas H. , Neves Rui P. , Liffers Sven T. , Kuhlmann Jan D. , Wimberger Pauline , Buderath Paul , Kimmig Rainer , Salmon Carolin 
Published: March 2024

Abstract
Background: Ovarian Cancer (OC) is one of the leading causes of death among gynecological tumors with an insufficient understanding of OC disease evolution. Since tumor tissue is not always available, blood allows the investigation of single circulating tumor cells (sCTC) over time. We combined immunomagnetic enrichment and image-based sCTC sorting for phenotypic and genotypic analysis of sCTCs in the follow-up of OC to provide insights into sCTC heterogeneity and their malignant character.
Patients and Methods: 10 ml blood of high-grade serous OC patients (pts) at the time of primary diagnosis (n=42), after chemotherapy (n=23) and after Avastin (n=11) was analyzed for sCTCs using density-gradient centrifugation to further apply MACS® using CD45 and CD235a antibodies coupled to MACS® MicroBeads. For image-based sCTC sorting (DEPArray®), an immunofluorescence protocol staining for Hoechst, cytokeratin (CK), the folate receptor alpha (FRa), Vimentin (Vim) and CD45 was applied. Whole genome amplification (WGA) was performed in recovered single cells, comprising cell lysis, DNA digestion, adaptor ligation and an amplification PCR for subsequent copy number alteration (CNA) analysis. Ampli1TM low-pass analysis was used to analyze single cell WGA products. 21 primary tumors (PT) were assessed for whole-genome CNA and the Ampli 1TM OncoSeek panel was used for deep sequencing of sCTCs.
Results: Phenotypic assessment revealed FRa-positive cells in 28/42 (67%) pts, CK-positive cells in 32/42 (76%) pts and CK-FRa-double-positive cells in 15/42 (36%) pts, respectively, with a significant (p= 0.0205) reduction of cells after chemotherapy only documented for FRa-positive cells. Interestingly, cells with a high nuclear staining but no expression of target antigens were found in 37/42 pts (88%) and significantly (p=0.002) expanded during treatment. Genotyping revealed that only 7% (19/289) of single cells deriving from 13 different pts were aberrant. Whole genome CNA analysis in all pre-treatment sCTCs revealed enriched CNAs in chromosomes 2, 7 and 12. In post-treatment sCTCs, CNA dynamics highlighted the adaptive potential of the homologous recombination pathway and suggested further investigation of MAPK signaling in resistant OC. Low-pass CNA comparison between sCTCs and the PT was feasible in nine pts, 2/9 with persistent PT characteristics in sCTCs at different sampling times. TP53 variants were the most frequently detected ones, persistent CNA in the CDK4 oncogene and emerging CNA in the ALK oncogene were found in sCTCs from two pts.
Conclusion: We identified sCTCs in the follow-up of OC and demonstrated interpatient as well as intrapatient heterogeneity of sCTCs. Matched sCTCs and PT data showed similar CNAs to a certain amount. Our low percentage of aberrant cells suggests that higher patient numbers are needed to confirm our findings to understand disease progression.
Citation Format: Sabine Kasimir-Bauer, Nikolas H. Stoecklein, Rui P. Neves, Sven T. Liffers, Jan D. Kuhlmann, Pauline Wimberger, Paul Buderath, Rainer Kimmig, Carolin Salmon. Phenotypic and genotypic characterization of single circulating tumor cells in the follow-up of high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6561.