title: Frequency of KRAS, BRAF, and PIK3CA mutations in advanced colorectal cancers: Comparison of peptide nucleic acid-mediated PCR clamping and direct sequencing in formalin-fixed, paraffin-embedded tissue.
abstract: KRAS, BRAF, and PIK3CA mutation testing before administration of anti-epidermal growth factor receptor therapy of metastatic colorectal cancer (CRC) has become important. However, considerable uncertainty exists regarding which detection method can be applied in a reproducible, sensitive, and simple manner in the routine diagnostic setting. We compared the detection rates of KRAS, BRAF, and PIK3CA mutations in 92 routine formalin-fixed, paraffin-embedded CRC specimens by 2 discrete methods: direct sequencing and peptide nucleic acid (PNA)-mediated PCR. The detection rates for KRAS, BRAF, and PIK3CA mutations by direct sequencing were 20.7%, 3.3%, and 1.1%, respectively. PNA-mediated PCR clamping significantly increased the percentages of KRAS, BRAF, and PIK3CA mutations by up to 7.6%, 1.2%, and 5.4%, respectively, compared to the detection rate of regular PCR followed by direct sequencing (p=0.039, p=0.250, and p=0.031, respectively). The tumor volume of discordant cases was not significantly different from concordant cases (56.2±28.7% vs. 67.6±17.9%, p=0.41), which implies that there is a minor population of mutant alleles in the heterogeneous tumor population. The PNA-mediated PCR clamping method is highly sensitive and is efficiently applicable to the detection of KRAS, BRAF, and PIK3CA mutations in a clinical setting.
authors: Department of Pathology, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, Anyang, Republic of Korea.
authors: Mi Jung
authors: MJ
authors: Kwon
authors: Seung Eun
authors: SE
authors: Lee
authors: So Young
authors: SY
authors: Kang
authors: Yoon-La
authors: YL
authors: Choi
mesh_terms: D000328:Adult; D000368:Aged; D015179:Colorectal Neoplasms / Q000175:diagnosis / Q000235:genetics*; D004252:DNA Mutational Analysis; D005260:Female; D005404:Fixatives; D005557:Formaldehyde; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics*; D016612:Paraffin Embedding; D020135:Peptide Nucleic Acids / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D014157:Transcription Factors / Q000235:genetics*; D018631:ras Proteins / Q000235:genetics*
chemical_list: D005404:Fixatives; C117307:KRAS protein, human; D009687:Nuclear Proteins; C503859:PI3KCA protein, human; D020135:Peptide Nucleic Acids; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors; D005557:Formaldehyde; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins