Patients with metastatic hormone receptor-positive breast cancer express PIK3CA oncogene mutational heterogeneity in circulating tumor cells

Abstract

We present a single-cell application to determine PIK3CA mutations in CTCs, which uncovered the degree of intra-patient heterogeneity in patients with metastatic hormone receptor-positive breast cancer (HR+ MBC) and high CTC count (>10 CTCs/7.5mL). Using CellSearch and DEPArray we isolated circulating tumor cells (CTCs) and white blood cells (WBCs) from peripheral blood and sequenced PIK3CA exons 9 and 20 by targeted amplicon sequencing. Comparative analysis between the primary tumor (PT, n=27 patients), circulating cell-free DNA (cfDNA, n=31 patients), single (n=146 CTCs) and pools (n=70 CTC suspensions, ranging 5-120 cells/suspension) of CTCs from 26 patients and metastases/DTCs (n=11 patients) was performed. Mutations were frequent in PT (15/27 (55.5%)) and showed slight and substantial agreement with cfDNA (n=21; kappa=0.14) and CTCs (n=22; kappa=0.6733), respectively. A wild-type genotype in WBCs indicates a high specificity. Inter-compartmental concordance was observed in 13/18 (72.2%) patients and temporal heterogeneity in 4/18 patients (22.2%). CTC analysis reveals both mutational homo-and heterogeneity with cases showing the presence of different mutant and wild-type CTC subpopulations. Additionally, unique double-mutated CTCs were detected in 8/26 patients (30.7%). The developed liquid biopsy provides an insight into inter and intra-patient PIK3CA mutational heterogeneity in patients with HR+ MBC, paving the way towards the application of a more personalized medicine.