Performance Evaluation of a Colorectal Cancer-Associated MassARRAY-Based Hotspot Genotyping Assay for KRAS, NRAS, BRAF, and PIK3CA Using Solid Tumor Specimens.
BACKGROUND Accurate identification of actionable somatic variants is essential for therapeutic stratification in colorectal cancer (CRC). While next-generation sequencing (NGS) enables comprehensive genomic profiling, targeted approaches may provide faster and more practical alternatives for routine diagnostics. METHODS This study evaluated the analytical performance of the Agena Bioscience iPLEX® High Sensitivity (HS) Colon Panel using MassARRAY MALDI-TOF technology for detection of hotspot variants in KRAS, NRAS, BRAF, and PIK3CA from formalin-fixed paraffin-embedded (FFPE) specimens. A total of 60 unique clinical and reference samples were analyzed, targeting 26 single-nucleotide variants and compared with an orthogonal targeted NGS assay. Limit of detection (LOD) was assessed using serial dilutions of reference materials, and intra- and inter-run reproducibility was evaluated across multiple runs. Analytical performance metrics including positive and negative percent agreement, predictive values, and error rates were calculated. RESULTS The assay demonstrated complete concordance with NGS across all evaluated variants, requiring only 20 ng of DNA input, compared to 80-120 ng for a successful NGS run. LOD studies showed reliable detection of multiple variants at approximately 5% variant allele frequency, with BRAF p.V600E detectable near 1%. Intra- and inter-run analyses achieved 100% concordance, confirming assay reproducibility. Aggregated performance metrics demonstrated high sensitivity and specificity across a heterogeneous sample set. CONCLUSIONS These findings establish the iPLEX® HS Colon Panel as a reliable platform for rapid detection of clinically actionable hotspot mutations. This study represents analytical validation using mixed FFPE tumor specimens; evaluation in larger colorectal cancer cohort, is warranted.