A comprehensive procedural approach to genotyping KRAS and BRAF from paraffin embedded tissues for diagnostic purposes

Raffaele Palmirotta, Giorgia Ludovici, Maria Laura De Marchis, Barbara Leone, Vincenzo Formica, Giuseppe Maria Ettorre, Francesco Cavaliere, David Della Morte, Patrizia Ferroni, Mario Roselli, Fiorella Guadagni

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

Background: Mutations in the Kirsten Ras 1 (KRAS) and V-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF) genes may be predictive of response to drugs directly linked to the Epidermal Growth Factor Receptor (EGFR) signaling pathway. Materials and Methods: A total of 230 samples from patients with metastatic colorectal cancer were analyzed for KRAS exon 1 and 2 and for BRAF exon 15 mutations. DNA from paraffinembedded tumor sections was analyzed using microdissection, direct sequencing analysis and allelic separation by cloning. Results: KRAS mutations were present in 44.3% of the tumor samples. The mutation frequency at hot-spot codons of exon 1 was 84.2%, whereas noncanonical variants had a frequency of 11.8%. Approximately 4% of the cases exhibited concomitant variations. BRAF mutations were present in 3.9% of the tumor samples. Conclusion: Our experience suggests that sequential microdissection, direct sequencing and allelic separation by cloning may improve the approach to mutational analysis of KRAS and BRAF in patients with colorectal cancer.

Original languageEnglish
Pages (from-to)537-548
Number of pages12
JournalIn Vivo
Volume26
Issue number4
StatePublished - Jul 1 2012

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Keywords

  • BRAF
  • Colorectal cancer
  • Concurrent mutation
  • Genotyping
  • KRAS
  • Mutational analysis

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Pharmacology

Cite this

Palmirotta, R., Ludovici, G., De Marchis, M. L., Leone, B., Formica, V., Ettorre, G. M., Cavaliere, F., Della Morte, D., Ferroni, P., Roselli, M., & Guadagni, F. (2012). A comprehensive procedural approach to genotyping KRAS and BRAF from paraffin embedded tissues for diagnostic purposes. In Vivo, 26(4), 537-548.