Description

Mutation Analysis of Single Circulating Tumour Cells and Cell-Free DNA in Metastatic Breast Cancer Patients

Hills A*, Guttery DS², Fernandez-Garcia D², Page K², Rosales BM¹, Goddard KS¹, Hastings RK³, Luo J³, Ogle O¹, Woodley L¹, Ali S¹, Stebbing J¹, Shaw JA², Coombes RC¹

*Corresponding author

¹Cancer Research UK Imperial Centre, Imperial College London, London, UK; ²Department of Cancer Studies, University of Leicester, Leicester, UK; ³Cancer Research UK Leicester Centre, University of Leicester, Leicester, UK

Background: Currently, there is a limited clinical ability to monitor breast cancer patients for signs of drug resistance at the molecular level. Development of minimally invasive blood-based assays, with a focus on Circulating Tumour Cells (CTCs) and circulating, cell-free DNA (cfDNA), offers opportunities for real-time monitoring and treatment stratification that could significantly impact on patient outcome. CfDNA is potentially easier to obtain than CTCs and so it is important to determine which, if either, is the more suitable candidate for clinical use. The purpose of this study was to directly compare the mutational profiles of multiple, single CTCs and cfDNA isolated from blood samples taken from women with metastatic breast cancer (MBC).

Methods: MBC patient blood samples were enriched for EpCAM-positive CTCs by CellSearch® and a CTC count obtained for each. In 5 patients with ³100 CTCs/7.5 ml blood, multiple, single CTCs were isolated by the DEPArray System. Next generation sequencing (NGS) of ~2200 mutations in 50 known cancer genes was performed on isolated single CTCs, cfDNA from the same blood sample, and patient-matched primary tumour tissue.

Results: NGS analysis of 40 individual CTCs revealed mutational heterogeneity in PIK3CA, TP53, ESR1 and KRAS genes between individual CTCs in 4/5 patients. In all 4 patients, matched cfDNA profiles provided an accurate reflection of mutations observed in individual CTCs, although more mutations were detected in cfDNA than in CTCs. One patient had no mutations in cfDNA or CTCs. ESR1 and KRAS gene mutations were detected in cfDNA but absent from primary tumour tissue, and therefore likely reflect a minor sub-clonal mutation.

Conclusions: Our results demonstrate that single CTC analysis can be used to elucidate the heterogeneous cancer cell populations that exist within an individual patient. CfDNA reflects EpCAM-positive CTCs in patients with high CTC counts and may therefore enable monitoring of the metastatic burden at the molecular level, and aid in clinical decision-making.

1.Shaw, J. A. et al. Mutation Analysis of Cell-Free DNA and Single Circulating Tumor Cells in Metastatic Breast Cancer Patients with High Circulating Tumor Cell Counts. Clin. Cancer Res. 23, (2017).