title: Pyrosequencing-based methods reveal marked inter-individual differences in oncogene mutation burden in human colorectal tumours.
abstract: BACKGROUND
  The epidermal growth factor receptor-targeted monoclonal antibody cetuximab (Erbitux) was recently introduced for the treatment of metastatic colorectal cancer. Treatment response is dependent on Kirsten-Ras (K-Ras) mutation status, in which the majority of patients with tumour-specific K-Ras mutations fail to respond to treatment. Mutations in the oncogenes B-Raf and PIK3CA (phosphoinositide-3-kinase) may also influence cetuximab response, highlighting the need for a sensitive, accurate and quantitative assessment of tumour mutation burden.
  
  
  METHODS
  Mutations in K-Ras, B-Raf and PIK3CA were identified by both dideoxy and quantitative pyrosequencing-based methods in a cohort of unselected colorectal tumours (n=102), and pyrosequencing-based mutation calls correlated with various clinico-pathological parameters.
  
  
  RESULTS
  The use of quantitative pyrosequencing-based methods allowed us to report a 13.7% increase in mutation burden, and to identify low-frequency (<30% mutation burden) mutations not routinely detected by dideoxy sequencing. K-Ras and B-Raf mutations were mutually exclusive and independently associated with a more advanced tumour phenotype.
  
  
  CONCLUSIONS
  Pyrosequencing-based methods facilitate the identification of low-frequency tumour mutations and allow more accurate assessment of tumour mutation burden. Quantitative assessment of mutation burden may permit a more detailed evaluation of the role of specific tumour mutations in the pathogenesis and progression of colorectal cancer and may improve future patient selection for targeted drug therapies.
authors.affiliation: Biomedical Research Institute, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK.
authors.forename: S
authors.initials: S
authors.lastname: Weidlich
authors.forename: K
authors.initials: K
authors.lastname: Walsh
authors.forename: D
authors.initials: D
authors.lastname: Crowther
authors.forename: M E
authors.initials: ME
authors.lastname: Burczynski
authors.forename: G
authors.initials: G
authors.lastname: Feuerstein
authors.forename: F A
authors.initials: FA
authors.lastname: Carey
authors.forename: R J C
authors.initials: RJ
authors.lastname: Steele
authors.forename: C R
authors.initials: CR
authors.lastname: Wolf
authors.forename: G
authors.initials: G
authors.lastname: Miele
authors.forename: G
authors.initials: G
authors.lastname: Smith
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002277:Carcinoma / Q000235:genetics* / Q000473:pathology / Q000601:surgery; D015331:Cohort Studies; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology / Q000601:surgery; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D005787:Gene Frequency; D020022:Genetic Predisposition to Disease; D006801:Humans; D007206:Individuality; D008297:Male; D008875:Middle Aged; D009154:Mutation / Q000502:physiology; D009857:Oncogenes / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods