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 paraffin-embedded 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 non-canonical 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.

A Comprehensive Procedural Approach to Genotyping KRAS and BRAF from Paraffin Embedded Tissues for Diagnostic Purposes

Ferroni P;Guadagni F
2012

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 paraffin-embedded 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 non-canonical 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.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.12078/1861
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