Establishment of a potential reference measurement procedure and reference material for PIK3CA H1047R mutation detection.

PIK3CA gene mutation is a promising predictor for many different human cancers, especially for breast cancer. Increasing numbers of non-invasive methods have been developed for the detection of PIK3CA mutations. However, a lack of reference measurement procedures (RMPs) and reference materials (RMs) impedes the consistency and comparability of test results in clinical practice. In this study, a potential RMP based on digital PCR (dPCR) and a genomic reference material (RM) for PIK3CA H1047R mutation have been established. The reference method showed good linearity (slope = 1.0078, R² = 0.9999) between the measured and prepared mutant values across the variant allele frequency range from 40% to 0.2%. The limit of detection (LoD) for H1047R was 2.9 copies per μL, corresponding to a fractional abundance of 0.38%. The genomic RM was developed, and its homogeneity and stability assessment indicated that the RM was homogeneous and stable for 16 months at -80 °C. The RM for PIK3CA H1047R was characterized using the established RMP, with the reference values of the copy number concentration for the H1047R mutant and wild-type and fractional abundance being (7.02 ± 0.37) × 10³ copies per μL, (7.09 ± 0.37) × 10³ copies per μL, and 49.75% ± 1.69%, respectively. Three test kits were assessed by the RM, and one of the kits displayed inconsistency with the claimed LoD. This indicates that the gDNA RM can provide a useful solution for improving the detection performance and quality control of PIK3CA H1047R assays.