Comparative Performance of a Targeted Next-Generation Sequencing Assay Against Multiplexed Digital PCR Assays in Detecting ERBB2, ESR1, and PIK3CA Mutations in Plasma Circulating Cell-Free DNA from Liquid Biopsies

Abstract

Analyzing somatic mutations in liquid biopsies poses a real challenge in treating patients with breast cancer. Because of the high sensitivity required to detect circulating tumor DNA, which may be present at low levels, digital PCR analysis seems highly appropriate. However, new targeted next-generation sequencing solutions are now available, enabling highly sensitive multigene analysis that could benefit patients. This study compared the analytical performance of multiplex digital PCR and targeted next-generation sequencing in detecting somatic erb-b2 receptor tyrosine kinase 2 (ERBB2), estrogen receptor 1 (ESR1), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA) mutations in a series of 32 plasma samples from patients with metastatic breast cancer. Forty-four mutations were detected, with an overall concordance of 95% (90/95) and a high degree of correlation (R² = 0.9786). Interestingly, two ESR1 mutations detected in multiplex drop-off digital PCR were also detected by targeted next-generation sequencing with comparable mutant allele frequencies, enabling the identification of these specific variants (p.D538N and p.536LYD>P). Moreover, an additional PIK3CA mutation (p.P539R) was first detected using targeted next-generation sequencing and later confirmed with a newly designed digital PCR assay. Although more expensive than multiplex digital PCR, these new types of small targeted next-generation sequencing gene panels could provide a rapid answer to a specific clinical question with a ready-to-use solution, which could benefit patients.