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Concomitant mutations and splice variants in KRAS and BRAF demonstrate complex perturbation of the Ras/Raf signalling pathway in Colorectal Cancer
  1. Simon Crook (simon.crook{at}nottingham.ac.uk)
  1. University of Nottingham, United Kingdom
    1. Rashmi Seth (rashmi.seth{at}nottingham.ac.uk)
    1. University of Nottingham, United Kingdom
      1. Darryl Jackson (darryl.jackson{at}nottingham.ac.uk)
      1. University of Nottingham, United Kingdom
        1. Mohammad Ilyas (mohammad.ilyas{at}nottingham.ac.uk)
        1. University of Nottingham, United Kingdom

          Abstract

          Background: KRAS and BRAF mutations occur in colorectal cancers (CRC) and are considered mutually exclusive methods of activating the RAS/RAF/MEK/ERK pathway. This pathway is a therapeutic target and KRAS mutation may predict tumour responsiveness.

          Aims: To investigate the relationship between KRAS and BRAF mutations in 24 CRC cell lines.

          Methods: KRAS and BRAF mutations were detected using High Resolution Melting and sequencing. Expression of mutations was confirmed by RT-PCR and sequencing. CpG island methylator phenotype (CIMP) was tested by methylation-specific PCR.

          Results: KRAS or BRAF mutation occurred in 19/24 (79%) cell lines and all mutations were expressed. KRAS mutations occurred in 13/24 (54%) cell lines and were mainly in codons 12/13 although 5/13 (38%) mutations occurred outside these codons. Four cell lines had homozygous mutation. Heterozygous BRAF mutation occurred in 7/24 (29%) cell lines. The V600E mutation occurred most commonly and was associated with CIMP+ status (p=0.005). Mutations at codons 529 and 581 were also found and, in one case, BRAF and KRAS mutation co-occurred. Unexpectedly, BRAF splice variants (with a predicted kinase dead protein) were found in 5/24 (21%) cell lines.

          Conclusions: Disrupted Ras/Raf signalling is common in CRC. The occurrence of homozygous KRAS mutations and concomitant KRAS/BRAF mutations shows that this pathway may be gene dosage dependent. The significance of BRAF splice variants is uncertain but it may represent another layer of complexity. Finally, if KRAS mutation is to be used for predictive testing, then the whole gene needs to be screened as mutations occur outside codons 12/13.

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