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Updated: May 20, 2026
Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines
Published on: October 8, 2015
A comprehensive procedural approach to genotyping KRAS and BRAF from paraffin embedded tissues for diagnostic
Raffaele Palmirotta1, Giorgia Ludovici, Maria Laura De Marchis
1Department of Laboratory Medicine and Advanced Biotechnologies, IRCCS San Raffaele Pisana, via della Pisana 235, 00163 Rome, Italy. raffaele.palmirotta@sanraffaele.it
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.
Insights
Mutations in Kirsten Ras 1 (KRAS) and V-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF) genes are common in metastatic colorectal cancer. Analyzing these mutations may guide EGFR-targeted therapy selection.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Mutations in Kirsten Ras 1 (KRAS) and V-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF) genes are implicated in cancer development.
- These genetic alterations may predict patient response to Epidermal Growth Factor Receptor (EGFR) signaling pathway targeted therapies.
Purpose of the Study:
- To investigate the frequency and types of KRAS and BRAF mutations in metastatic colorectal cancer (mCRC).
- To evaluate the utility of specific molecular techniques for accurate mutation detection.
Main Methods:
- Analysis of 230 mCRC patient tumor samples.
- Utilized microdissection, direct sequencing, and allelic separation by cloning for KRAS (exons 1-2) and BRAF (exon 15) mutation analysis.
Main Results:
- KRAS mutations detected in 44.3% of samples, with high frequency at exon 1 hot-spot codons.
- BRAF mutations identified in 3.9% of samples.
- Concomitant KRAS and BRAF variations observed in approximately 4% of cases.
Conclusions:
- Sequential microdissection, direct sequencing, and cloning offer an improved approach for KRAS and BRAF mutational analysis in colorectal cancer.
- Accurate mutational profiling of KRAS and BRAF is crucial for personalized treatment strategies in mCRC.
