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Published on: September 25, 2011
Performance Evaluation of a Colorectal Cancer-Associated MassARRAY-Based Hotspot Genotyping Assay for KRAS, NRAS,
Vishakha Vashisht1, Ashutosh Vashisht1, Ashis K Mondal1
1Department of Pathology, Augusta University, Augusta, GA 30909, USA.
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.
