Application of pyrosequencing technique for improved detection of K-Ras mutation in formalin-fixed and paraffin-embedded prostate carcinoma tissues in Chinese patients.
BACKGROUND
The purpose of this study was to comparatively evaluate the efficacy of pyrosequencing (PS) and Sanger sequencing (SS) methods for detecting K-Ras codon 12 and 13 mutations in formalin-fixed paraffin-embedded (FFPE) prostate cancer (PCa) samples from Chinese patients.


METHODS
The cancer cell lines, including the LS174T G12D mutant (GGT to GAT) and the COLO320 wild-type, were tested to determine the limitation of detection and reproducibility of the PS method. In addition, 101 PCa patient samples, 13 benign prostatic hyperplasia (BPH) and 12 normal adjacent tissue samples, were assayed by PS and SS to evaluate their detection abilities for K-Ras mutations in codons 12 and 13.


RESULTS
The PS assay was able to reproducibly detect 5% mutant alleles and had an intra-assay variability of 4.21% and inter-assay variability of 11.37%. The PS assay detected a higher number of K-Ras mutations in PCa samples than the SS assay (8.91% vs. 3.96%). Correlation and stratification analyses of the PCa samples and K-Ras mutation status revealed no associations between age, serum prostate specific antigen (PSA), depth of invasion (pT category), or Gleason score.


CONCLUSIONS
We demonstrated that the PS method detected more K-Ras mutations in codons 12 and 13 of FFPE prostate cancer samples from Chinese patients than the traditional SS method. In addition, the K-Ras mutation was more frequent in Chinese population than in Western populations but was similar to that of other Eastern populations, suggesting that these K-Ras mutations may contribute to the pathogenesis of prostate carcinoma in Asian patients.
Department of Urology, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, PR China.
Jianjun
J
Sha
Guoqing
G
Liang
Jiahua
J
Pan
Hanqing
H
Xuan
Ping
P
Ping
Dong
D
Li
Juanjie
J
Bo
Dongming
D
Liu
Wei
W
Shen
Weiwei
W
Liu
Yiran
Y
Huang
D000368:Aged; D044466:Asian People / Q000235:genetics*; D002277:Carcinoma / Q000175:diagnosis / Q000235:genetics*; D016022:Case-Control Studies; D045744:Cell Line, Tumor; D003062:Codon; D005404:Fixatives; D005557:Formaldehyde; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D016612:Paraffin Embedding; D011470:Prostatic Hyperplasia / Q000175:diagnosis / Q000235:genetics; D011471:Prostatic Neoplasms / Q000175:diagnosis / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*
D003062:Codon; D005404:Fixatives; D005557:Formaldehyde; D016283:Proto-Oncogene Proteins p21(ras)