High-Sensitivity Detection of KRAS G12C by Digital PCR: Analytical Performance and Concordance with NGS in FFPE Tumor Samples

Introduction: The KRAS c.34G>T (p.G12C) mutation represents a clinically actionable alteration in non-small cell lung cancer (NSCLC) and colorectal cancer (CRC), conferring eligibility for selective KRAS G12C inhibitors. Accurate detection of this mutation is essential for precision oncology, particularly in tumor samples with low mutant allele frequency (MAF). This study evaluated the analytical performance of digital PCR (dPCR) for KRAS G12C detection in formalin-fixed paraffin-embedded (FFPE) tumor samples and assessed its concordance with next-generation sequencing (NGS) and quantitative real-time PCR (qPCR).

Materials and Methods: A total of 47 FFPE tumor tissue samples were included in this study. These samples previously characterized by NGS were analyzed using a commercially available allele-specific dPCR assay. Analytical validation included assessment of accuracy, sensitivity, reproducibility, and specificity. Results obtained by dPCR were compared with those generated by NGS and qPCR.

Results: dPCR demonstrated complete qualitative concordance with NGS (Cohen’s kappa = 1.00) and showed a high degree of quantitative correlation across a broad range of MAF values. The limit of detection for dPCR was 0.5% MAF, enabling reliable detection of low-frequency KRAS G12C variants. qPCR also showed full qualitative concordance with NGS but detected the mutation only in samples with higher MAF values (≥6.13%).

Conclusions: dPCR provides accurate, reproducible, and highly sensitive detection of KRAS G12C in FFPE tumor samples. While NGS remains essential for comprehensive genomic profiling, dPCR represents a valuable complementary method for targeted detection of clinically actionable mutations, particularly in samples with limited DNA input or low tumor cellularity. Its high analytical sensitivity also supports its use as a tool for longitudinal molecular monitoring of patients undergoing targeted therapies, contributing to precision oncology strategies through the early detection of molecular changes associated with treatment response or disease progression.