Genetic analysis of heterogeneous subsets of circulating tumour cells from high grade serous ovarian carcinoma patients

Abstract

Circulating tumour cells (CTCs) are heterogenous and contain genetic information from the tumour of origin. They bear specific intra- and extra-cellular protein markers aiding in their detection. However, since these markers may be shared with other rare cells in the blood, only genetic testing can confirm their malignancy. Herein, we analyse different CTC subsets using single cell whole genome DNA sequencing to validate their malignant origin. We randomly selected putative CTCs identified by immunostaining that were isolated from 4 patients with high grade serous ovarian cancer (HGSOC) and one with benign cystadenoma. We specifically targeted CTCs positive for epithelial (CK/EpCAM^pos), mesenchymal (vimentin^pos), and pseudoendothelial (CK/EpCAM^pos plus CD31^pos) markers. We isolated these cells and performed whole genome amplification (WGA) and low-pass whole-genome sequencing (LP-WGS) for analysis of copy number alterations (CNA). Of the CK/EpCAM^pos cells analysed from the HGSOC patients, 2 of 3 cells showed diverse chromosomal CNAs. However, the 4 pseudoendothelial cells (CK/EpCAM^pos plus CD31^pos) observed in the HGSOC cases did not carry any CNA. Lastly, two of the clusters of vimentin positive cells sequenced from those found in the benign cystadenoma case had CNA. Despite the low number of cells analysed, our results underscore the importance of genetic analysis of putative CTCs to confirm their neoplastic origin. In particular, it highlights the presence of a population of CK/EpCAM^pos cells that are not tumour cells in patients with HGSOC, which otherwise would be counted as CTCs.

Figure 1

Representative fluorescence images of CTCs and their corresponding CNV profiles. Scale bar (bottom right) represents 10 μm. (A) Cells were stained with antibodies for cytokeratin and EpCAM (FITC, green), CD45/CD16 (PE, red), PD-L1 (AF647, cyan) and nuclei staining in blue (Hoechst), in the first panel. It was followed by fluorescence quenching and re-staining with antibodies for PAX8 (FITC, green), CD31 (PE, red) and vimentin (AF647, cyan). (B) CNA profiles obtained from low-pass whole-genome sequencing are shown to the right. Blue dots indicate neutral CNA, red indicate gains, and green indicate losses.

Figure 2

Representative fluorescence images of CTCs and their corresponding CNV profiles from the case with a benign tumour. Scale bar (bottom right) represents 10 μm. (A) T Photomicrograph of the tumour from the benign case, which was histologically diagnosed to be serous cystadenoma. Isolated cells (B and C) from blood were stained with antibodies for cytokeratin and EpCAM and cytokeratin (FITC, green), CD45/CD16 (PE, red), PDL1 (AF647, cyan) and nuclei staining in blue (Hoechst), in the first panel. It was followed by fluorescence quenching and re-staining with antibodies for PAX8 (FITC, green), CD31 (PE, red) and vimentin (AF647, cyan). Detected cells were mainly vimentin positive clusters. (C) CNA profiles obtained from low-pass whole-genome sequencing are shown at the far right from randomly selected clusters. Blue dots indicate neutral CNA, red indicate gains, and green indicate losses.