A Novel Fully Automated Molecular Diagnostic System (AMDS) for Colorectal Cancer Mutation Detection

Background: KRAS, BRAF and PIK3CA mutations are frequently observed in colorectal cancer (CRC). In particular, KRAS mutations are strong predictors for clinical outcomes of EGFR-targeted treatments such as cetuximab and panitumumab in metastatic colorectal cancer (mCRC). For mutation analysis, the current methods are time-consuming, and not readily available to all oncologists and pathologists. We have developed a novel, simple, sensitive and fully automated molecular diagnostic system (AMDS) for point of care testing (POCT). Here we report the results of a comparison study between AMDS and direct sequencing (DS) in the detection of KRAS, BRAF and PI3KCA somatic mutations. Methodology/Principal Finding: DNA was extracted from a slice of either frozen (n = 89) or formalin-fixed and paraffin-embedded (FFPE) CRC tissue (n = 70), and then used for mutation analysis by AMDS and DS. All mutations (n = 41 among frozen and 27 among FFPE samples) detected by DS were also successfully (100%) detected by the AMDS. However, 8 frozen and 6 FFPE samples detected as wild-type in the DS analysis were shown as mutants in the AMDS analysis. By cloning-sequencing assays, these discordant samples were confirmed as true mutants. One sample had simultaneous ‘‘hot spot’ ’ mutations of KRAS and PIK3CA, and cloning assay comfirmed that E542K and E545K were not on the same allele. Genotyping call rates for DS were 100.0 % (89/89) and 74.3 % (52/70) in frozen and FFPE samples, respectively, for the first attempt; whereas that of AMDS was 100.0 % for both sample sets. For automated DNA extraction and mutation detection b