title: A novel fully automated molecular diagnostic system (AMDS) for colorectal cancer mutation detection.
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.
  
  
  RESULTS
  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
  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.
authors.affiliation: Technical Research Institute, Toppan Printing Co., Ltd., Chiba, Japan. shiro.kitano@toppan.co.jp
authors.forename: Shiro
authors.initials: S
authors.lastname: Kitano
authors.forename: Jamie
authors.initials: J
authors.lastname: Myers
authors.forename: Junko
authors.initials: J
authors.lastname: Nakamura
authors.forename: Akio
authors.initials: A
authors.lastname: Yamane
authors.forename: Mami
authors.initials: M
authors.lastname: Yamashita
authors.forename: Masato
authors.initials: M
authors.lastname: Nakayama
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Tsukahara
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ushida
authors.forename: Wanqing
authors.initials: W
authors.lastname: Liu
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Ratain
authors.forename: Masahiko
authors.initials: M
authors.lastname: Amano
mesh_terms: D058534:Class I Phosphatidylinositol 3-Kinases; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods; D005557:Formaldehyde / Q000737:chemistry; D005629:Frozen Sections; D006801:Humans; D009154:Mutation; D016612:Paraffin Embedding / Q000379:methods; D057089:Pathology, Molecular / Q000295:instrumentation / Q000379:methods*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics; D016133:Polymerase Chain Reaction; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016707:Tissue Fixation / Q000379:methods; D018631:ras Proteins / Q000235:genetics
chemical_list: D005557:Formaldehyde; D019869:Phosphatidylinositol 3-Kinases; D058534:Class I Phosphatidylinositol 3-Kinases; C484760:PIK3CA protein, human; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D018631:ras Proteins