title: [Comparative analysis of real-time quantitative PCR-Sanger sequencing method and TaqMan probe method for detection of KRAS/BRAF mutation in colorectal carcinomas].
abstract: OBJECTIVE
  To compare the application values of real-time quantitative PCR-Sanger sequencing and TaqMan probe method in the detection of KRAS and BRAF mutations, and to correlate KRAS/BRAF mutations with the clinicopathological characteristics in colorectal carcinomas.
  
  
  METHODS
  Genomic DNA of the tumor cells was extracted from formalin fixed paraffin embedded (FFPE) tissue samples of 344 colorectal carcinomas by microdissection. Real-time quantitative PCR-Sanger sequencing and TaqMan probe method were performed to detect the KRAS/BRAF mutations. The frequency and types of KRAS/BRAF mutations, clinicopathological characteristics and survival time were analyzed.
  
  
  RESULTS
  KRAS mutations were detected in 39.8% (137/344) and 38.7% (133/344) of 344 colorectal carcinomas by using real-time quantitative PCR-Sanger sequencing and TaqMan probe method, respectively. BRAF mutation was detected in 4.7% (16/344) and 4.1% (14/344), respectively. There was no significant correlation between the two methods. The frequency of the KRAS mutation in female was higher than that in male (P < 0.05). The frequency of the BRAF mutation in colon was higher than that in rectum. The frequency of the BRAF mutation in stage III-IV cases was higher than that in stageI-II cases. The frequency of the BRAF mutation in signet ring cell carcinoma was higher than that in mucinous carcinoma and nonspecific adenocarcinoma had the lowest mutation rate. The frequency of the BRAF mutation in grade III cases was higher than that in grade II cases (P < 0.05). The overall concordance for the two methods of KRAS/BRAF mutation detection was 98.8% (kappa = 0.976). There was statistic significance between BRAF and KRAS mutations for the survival time of colorectal carcinomas (P = 0.039). There were no statistic significance between BRAF mutation type and BRAF/KRAS wild type (P = 0.058).
  
  
  CONCLUSIONS
  (1) Compared with real-time quantitative PCR-Sanger sequencing, TaqMan probe method is better with regard to handling time, efficiency, repeatability, cost and equipment. (2) The frequency of the KRAS mutation is correlated with gender. BRAF mutation is correlated with primary tumor site, TNM stage, histological types and histological grades.(3) BRAF gene mutation is an independent prognostic marker for colorectal carcinomas.
authors.affiliation: Department of Pathology, Shanxi Cancer Hospital, Shanxi Medical University, Taiyuan 030001, China.
authors.forename: Xun
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Pathology, Shanxi Cancer Hospital, Shanxi Medical University, Taiyuan 030001, China.
authors.forename: Yuehua
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Pathology, Shanxi Cancer Hospital, Shanxi Medical University, Taiyuan 030001, China.
authors.forename: Ning
authors.initials: N
authors.lastname: Gao
authors.affiliation: Department of Pathology, Shanxi Cancer Hospital, Shanxi Medical University, Taiyuan 030001, China. E-mail: jfenwang@qq.com.
authors.forename: Jinfen
authors.initials: J
authors.lastname: Wang
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics* / Q000473:pathology; D002288:Adenocarcinoma, Mucinous / Q000235:genetics / Q000473:pathology; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D018279:Carcinoma, Signet Ring Cell / Q000235:genetics / Q000473:pathology; D003110:Colonic Neoplasms / Q000235:genetics / Q000473:pathology; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D004252:DNA Mutational Analysis; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D060787:Neoplasm Grading; D009367:Neoplasm Staging; D015345:Oligonucleotide Probes; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D060888:Real-Time Polymerase Chain Reaction; D012004:Rectal Neoplasms / Q000235:genetics / Q000473:pathology; D017422:Sequence Analysis, DNA; D012737:Sex Factors; D055815:Young Adult; D018631:ras Proteins / Q000235:genetics*
chemical_list: C117307:KRAS protein, human; D015345:Oligonucleotide Probes; D011518:Proto-Oncogene Proteins; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins