Development of multiplex digital PCR–based <i>ESR1</i> and <i>PIK3CA</i> mutation assays for therapeutic decision-making in HR+/HER2− breast cancer.

Abstract

e13008 Background: Mutations in ESR1 and PIK3CA are clinically important molecular alterations associated with treatment response and therapeutic decision-making in HR+/HER2− breast cancer. Accurate detection of these mutations is essential for guiding targeted therapy and supporting companion diagnostic (CDx) strategies. While next-generation sequencing (NGS) is widely used, its routine clinical implementation may be limited by cost, long turnaround time (TAT), and workflow complexity. Multiplex digital PCR (dPCR) provides a simplified and cost-efficient alternative, enabling highly sensitive and specific mutation detection with low DNA input. Based on these advantages, we developed multiplex dPCR–based assays for rapid detection of ESR1 and PIK3CA mutations. Methods: The DigiPlex ESR1 Mutation Test and DigiPlex PIK3CA Mutation Test were developed as multiplex dPCR assays targeting clinically actionable mutations in HR+/HER2− breast cancer. The ESR1 assay detects key hotspot mutations associated with acquired endocrine resistance, while the PIK3CA assay detects 25 mutations across exons 1, 4, 7, 9, and 20. Both assays were optimized for use on the Roche Digital LightCycler Analyzer with multi-channel fluorescence–based detection and low DNA input. Analytical performance was evaluated using reference materials and contrived samples with predefined mutant allele frequencies, including DNA derived from FFPE tissue and plasma, to assess sensitivity, specificity, limit of detection (LoD), and precision. Results: The DigiPlex ESR1 and PIK3CA Mutation Tests demonstrated robust multiplex detection performance on a dPCR platform. Reliable mutation detection was achieved using low DNA input (5–10 ng) from both FFPE tissue and plasma-derived DNA. All targeted mutations were consistently detected at the evaluated LoD, demonstrating high analytical sensitivity. No false-positive or false-negative results were observed, indicating high analytical specificity. Clear fluorescence signal separation and reproducible quantitative performance were maintained across independent runs on the Digital LightCycler Analyzer. Conclusions: Multiplex digital PCR–based DigiPlex assays for ESR1 and PIK3CA mutation detection were successfully developed and analytically validated on the Roche Digital LightCycler Analyzer. These assays combine high analytical sensitivity and specificity with low DNA input and a streamlined workflow, representing a practical alternative to sequencing-based approaches for molecular testing in HR+/HER2− breast cancer. Accurate detection of ESR1 and PIK3CA mutations using multiplex dPCR may support individualized molecular diagnosis and CDx-driven therapeutic strategies. Clinical validation studies are ongoing.