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  • Title: A novel fully automated molecular diagnostic system (AMDS) for colorectal cancer mutation detection.
    Author: Kitano S, Myers J, Nakamura J, Yamane A, Yamashita M, Nakayama M, Tsukahara Y, Ushida H, Liu W, Ratain MJ, Amano M.
    Journal: PLoS One; 2013; 8(5):e62989. PubMed ID: 23671647.
    Abstract:
    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 by AMDS, frozen tissues (n = 41) were successfully detected all mutations within 70 minutes. CONCLUSIONS/SIGNIFICANCE: AMDS has superior sensitivity and accuracy over DS, and is much easier to execute than conventional labor intensive manual mutation analysis. AMDS has great potential for POCT equipment for mutation analysis.
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