Molecular detection and characterization of KRAS mutations among patients with colorectal adenocarcinoma in a tertiary hospital in Metro Manila

• Main Author: Nuylan, Michelle L.
• Format: text
• Language: English
• Published: Animo Repository 2009
• Subjects: Adenocarcinoma; Colon (Anatomy)—Cancer; Rectum—Cancer; Cancer; Mutation (Biology); Biology
• Online Access: https://animorepository.dlsu.edu.ph/etd_masteral/6455; https://animorepository.dlsu.edu.ph/context/etd_masteral/article/13134/viewcontent/CDTG004784_P.pdf
• Institution: De La Salle University

Activation of KRAS oncogene is responsible for the transition from intermediate adenoma to carcinoma in the colon. The gene is composed of 5 exons and encodes a 21kDa protein that acts as an intermediate molecule in signal transduction pathway by transferring information from the membrane to the nucleus consequently regulating cell proliferation and differentiation. Approximately 30% to 60% of mutations in colorectal cancer occur in the hotspot codons 12 and 13 of exon 1 and codon 61 of exon 2. The aim of this study is to characterize mutation in the entire coding region of the KRAS gene among Filipinos with colorectal cancer and correlate mutations with the patients’ clinicopathological profile. Paired frozen normal and tumor tissues from the 35 previously identified CRC patients who underwent surgical resection were included in the study. DNA was extracted from these frozen colon tissues and genomic DNA was amplified by PCR using set primers. PCR amplicons (except of exon 1) were initially screened for mutation using denaturing high performance liquid chromatography (DHPLC). Product size of exon 1 is only 89 bp and is below the size range (100 to 500 bp) required by DHPLC analysis. Seventeen of the 35 samples showed aberrant DHPLC chromatogram profile. Five of these mutations were in exon 2, one in exon 3, two in exon 4A and nine in exon 4B. To characterize these mutations PCR products were further analyzed by DNA sequencing. Likewise, all purified PCR products for exon 1 were also sequenced. Eighteen of the 35 samples were found to carry mutations. Ten of these mutations were in exon 1, 3 were in exon 2 and five in exon 4B. Four mutations in exon 1 were missense, five were silent and one was frameshift. All genetic alterations in exon 2 were missense mutation and those in exon 4B were all silent type. Nine of the 18 mutations could be considered pathogenic because these occur within the conserved region of the gene and are believed to contribute to its oncogenic potential. Eight of these nine mutations are found outside the hotspot region of KRAS gene. Mutations were significantly associated with tumor stage (p= 0.007) and these are found mostly in patients with stage III cancer. Result of this study may suggest a different mutation profile of Filipino patients with colorectal adenocarcinoma: a high frequency of mutations outside the hotspot