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Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions
Published on: September 25, 2018
Clinical pharmacogenomic testing of KRAS, BRAF and EGFR mutations by high resolution melting analysis and ultra-deep
Emma Borràs1, Ismael Jurado, Imma Hernan
1Hospital de Terrassa, Ctra, Torrebonica, Terrassa, Spain.
Background:
Epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices.
Methods:
We describe a high resolution melting (HRM) assay for mutation detection in EGFR exons 19-21, KRAS codon 12/13 and BRAF V600 using formalin-fixed paraffin-embedded samples. Somatic variation of KRAS exon 2 was also analysed by massively parallel pyrosequencing of amplicons with the GS Junior 454 platform.
Results:
We tested 120 routine diagnostic specimens from patients with colorectal or lung cancer. Mutations in KRAS, BRAF and EGFR were observed in 41.9%, 13.0% and 11.1% of the overall samples, respectively, being mutually exclusive. For KRAS, six types of substitutions were detected (17 G12D, 9 G13D, 7 G12C, 2 G12A, 2 G12V, 2 G12S), while V600E accounted for all the BRAF activating mutations. Regarding EGFR, two cases showed exon 19 deletions (delE746-A750 and delE746-T751insA) and another two substitutions in exon 21 (one showed L858R with the resistance mutation T590M in exon 20, and the other had P848L mutation). Consistent with earlier reports, our results show that KRAS and BRAF mutation frequencies in colorectal cancer were 44.3% and 13.0%, respectively, while EGFR mutations were detected in 11.1% of the lung cancer specimens. Ultra-deep amplicon pyrosequencing successfully validated the HRM results and allowed detection and quantitation of KRAS somatic mutations.
Conclusions:
HRM is a rapid and sensitive method for moderate-throughput cost-effective screening of oncogene mutations in clinical samples. Rather than Sanger sequence validation, next-generation sequencing technology results in more accurate quantitative results in somatic variation and can be achieved at a higher throughput scale.
Insights
This study presents a rapid, cost-effective high-resolution melting (HRM) assay for detecting cancer-driving mutations in EGFR, KRAS, and BRAF genes. The assay enables precise mutation screening in clinical samples, aiding in targeted therapy selection.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Mutations in EGFR, KRAS, and BRAF influence cancer treatment response.
- EGFR inhibitors are affected by EGFR kinase domain mutations, while KRAS and BRAF mutations confer resistance to cetuximab in colorectal cancer.
- Systematic mutation screening methods are crucial for personalized cancer therapy.
Purpose of the Study:
- To develop and evaluate a high-resolution melting (HRM) assay for detecting mutations in EGFR, KRAS, and BRAF.
- To assess the utility of HRM and next-generation sequencing (NGS) for analyzing somatic mutations in clinical cancer specimens.
Main Methods:
- A high-resolution melting (HRM) assay was developed for EGFR exons 19-21, KRAS codon 12/13, and BRAF V600.
- Somatic variation in KRAS exon 2 was analyzed using massively parallel pyrosequencing (GS Junior 454 platform).
- The HRM assay was applied to 120 formalin-fixed paraffin-embedded tumor samples from colorectal and lung cancer patients.
Main Results:
- Mutations in KRAS, BRAF, and EGFR were detected in 41.9%, 13.0%, and 11.1% of samples, respectively, and were mutually exclusive.
- Specific KRAS substitutions (G12D, G13D, G12C, etc.) and BRAF V600E were identified.
- EGFR mutations included exon 19 deletions and exon 21 substitutions (L858R, P848L), with one case showing a resistance mutation.
- Pyrosequencing validated HRM results and provided quantitative data for KRAS mutations.
Conclusions:
- HRM is a rapid, sensitive, and cost-effective method for moderate-throughput screening of oncogene mutations in clinical settings.
- Next-generation sequencing offers more accurate quantitative results and higher throughput for somatic variation analysis compared to Sanger sequencing.
- These methods facilitate appropriate therapeutic choices for cancer patients based on specific genetic profiles.
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