Identification of mutations for targeted therapy in circulating tumor cells via a multiplex PCR-based next generation sequencing-panel

Targeted therapy has become the preferred approach to treat most cancers, including metastatic breast cancer. Using liquid biopsies, which can act as a dynamic diagnostic tool, is an appealing concept to identify effective therapies.

In order to identify mutations from circulating tumor cells (CTCs) on single cell level, we developed a multiplex PCR-based next generation sequencing-panel. The CTCs were enriched using the CellSearch system based on their expression of the EpCAM protein and isolated by micromanipulation followed by whole genome amplification of their DNA. Afterwards, mutation hotspot regions in the PIK3CA, the ESR1, the AKT1, and the ERBB2 genes were amplified and barcoded. Mutations in those genes have a high clinical utility and are often undetectable or not yet acquired in primary tumors. Sequencing was performed on a MiSeq system. The assay was validated with cells from various cell lines displaying the expected mutations. Mutations that provide the basis for clinically approved targeted therapies were detected in 10 out of 13 patients in all analyzed genes. In four patients, mutations in more than one gene were observed - either in the same cell or in different cells, suggesting the presence of different tumor cell clones, which might be targeted with combination therapies.

This assay is a time- and cost-effective tool to investigate the most relevant genomic positions indicative for targeted therapies in metastatic breast cancer. It supports therapy decision to improve the treatment of cancer patients.

Publication History

Article published online:
01 June 2021