p53 and Ki-ras mutations in colorectal tumor tissue from the EPIC-Norfolk study Free

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Colorectal cancer is one of the most common cancers in the Western World and attributes to over 9% of all cancer deaths worldwide (1). There is evidence to suggest that the progression of cancer is linked to the accumulation of mutations in several different genes (2). Mutations in the p53 tumor suppressor gene are found in 50% of all human cancers and are a key step in the progression of colorectal cancer. However, studies of twins, migrants, and secular trends show that environmental factors, including diet, are important in influencing risk (1).

The aim of this research is to identify p53 and Ki-ras somatic gene mutations in genomic DNA extracted from colorectal tumor tissue, fixed in formalin and embedded in paraffin blocks, from the Norfolk arm of the EPIC study (European Prospective Investigation into Cancer and Nutrition). Specific base substitutions in p53 and Ki-ras will be related back to the detailed diet and lifestyle questionnaires that were collected from healthy individuals at recruitment.

Areas of p53 mutational “hotspots” were amplified from matched EPIC normal and tumor DNA via PCR and sequenced and then compared with the human p53 reference sequence using GAP4 (Genome Assembly Programme). The protocol for extracting genomic DNA from tumor tissue was optimised in both snap frozen and matched formalin fixed paraffin embedded samples from 10 patients, with no differences in DNA sequence. p53 somatic mutations have been identified in approximately one third of the EPIC-Norfolk colorectal tumour cases analysed so far.

Ki-ras somatic mutations occur almost exclusively in codons 12 and 13 (in the 1st or 2nd amino acid in codon 12 and in the 2nd amino acid in codon 13). Due to this specificity Ki-ras mutations can be analysed by pyrosequencing, a more sensitive sequencing assay than dideoxy sequencing (3). Preliminary results from a subset of colorectal tumor cases have detected a single mutation in Ki-ras at either codon 12 or 13 in approximately one quarter of cases.

1. International Agency for Research on Cancer. World Cancer Report (eds Stewart, B. W. & Kleihues, P.) (IARC Press, Lyon, 2003).

2. Vogelstein, B., Fearon, E.R., Hamilton, S.R., Kern, S.E., Preisinger, A.C., Leppert, M., Nakamura, Y., White, R., Smits, A.M. and Bos, J.L. (1988) Genetic alterations during colorectal-tumor development. N Engl J Med, 319, 525-532.

3. Ogino, S., Kawasaki, T., Brahmandam, M., Yan, L., Cantor, M., Namgyal, C., Mino-Kenudson, M., Lauwers, G.Y., Loda, M. and Fuchs, C.S. (2005) Sensitive sequencing method for KRAS mutation detection by Pyrosequencing. J Mol Diagn, 7, 413-421.