Detection of Clonal and Subclonal Copy-Number Variants in Cell-Free DNA from Patients with Breast Cancer Using a Massively Multiplexed PCR Methodology

We demonstrate proof-of-concept for the use of massively multiplexed PCR and next-generation sequencing (mmPCR-NGS) to identify both clonal and subclonal copy-number variants (CNVs) in circulating tumor DNA. This is the first report of a targeted methodology for detection of CNVs in plasma. Using an in vitro model of cell-free DNA, we show that mmPCR-NGS can accurately detect CNVs with average allelic imbalances as low as 0.5%, an improvement over previously reported whole-genome sequencing approaches. Our method revealed differences in the spectrum of CNVs detected in tumor tissue subsections and matching plasma samples from 11 patients with stage II breast cancer. Moreover, we showed that liquid biopsies are able to detect subclonal mutations that may be missed in tumor tissue biopsies. We anticipate that this mmPCR-NGS methodology will have broad applicability for the characterization, diagnosis, and therapeutic monitoring of CNV-enriched cancers, such as breast, ovarian, and lung cancer.