Mutational and copy number analysis of numerous sorted circulating and disseminated tumour cells to interrogate subclonal evolution in advanced breast cancer.

Abstract Background. Liquid biopsies have been studied as an accessible method to capture spatial tumour heterogeneity and repeatedly evaluate targetable aberrations in cancer. Here, we analysed pure single and pools of circulating and disseminated tumour cells (CTC and DTC) to study genetic heterogeneity. Methods. From, three patients with metastatic breast cancer, CTC and DTC (bone marrow, pleural fluid, and cerebrospinal fluid) were CellSearch® enriched and DEPArray® sorted into 136 samples of singles and pools up to 150 cells. Sorted cells, primary tumour, circulating free (cf)DNA, and enriched CTC/DTC fractions were analysed for mutations and copy number alterations (CNA). Results. Two patients harboured a driver PIK3CA mutation in all samples. Variant allele frequencies matched the ploidy status calculated from copy number data. The majority of CNA were homogeneously present in most samples of each patient, including breast cancer subtype specific CNA. One patient with rapidly progressive disease harboured additional CNA in the CTC/DTC compartment, reflecting this aggressive course and a possible subtype switch. These clones emerged at distinct timepoints in the different compartments. Furthermore, various mutations were present in unique samples. Conclusion. Repeated tumour analysis with liquid biopsies unveils changing molecular characteristics over time, and even a difference between simultaneous primary and metastatic disease. We show that all major subclones can be captured by combined sequencing of enriched CTC and cfDNA at disease progression.