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High-resolution melting analysis as a sensitive prescreening diagnostic tool to detect KRAS , BRAF , PIK3CA , and
Jasmin Teresa Ney1, Stefanie Froehner, Angelika Roesler
1Institute of Pathology, University of Bonn, Germany.
Context:
As the availability of targeted therapies for several tumor types increases, the need for rapid and sensitive mutation screening is growing. KRAS mutations constitutively activate the RAS/RAF/mitogen-activated protein kinase (MAPK) pathway and therefore play an important role in anti-epidermal growth factor receptor therapy for patients with colorectal cancers. Mutationally activated PIK3CA and AKT1 genes are promising therapeutic targets in breast cancer. In 60% to 70% of malignant melanomas, a mutation in BRAF can be found. Thus, the blocking of the oncogenic signaling induced by this mutation is now used as treatment approach.
Objective:
To establish high-resolution melting assays for routinely used predictive analyses of KRAS , AKT1 , PIK3CA , and BRAF mutations.
Design:
High-resolution melting assays were developed by using specifically designed primers and genomic DNA isolated either from cell lines or formalin-fixed paraffin-embedded tissues, oligonucleotides, or plasmids. Melting curve analyses were performed on the LightCyler platform and mutation analyses were additionally confirmed by Sanger sequencing.
Results:
We developed high-resolution melting assays by using genomic DNA containing the desired mutation, which enabled us to detect percentages of mutated DNA (3.1% to 12.5%) mixed in a wild-type background. Assays were evaluated by hybridization probes and/or Sanger sequencing to exclude pseudogene amplification. The high-resolution melting assays were validated with genomic DNA from different tumor entities. The concordance between Sanger sequencing and high-resolution melting was 99% for KRAS exon 2 and PIK3CA exon 20 and 100% for the remaining assays.
Conclusions:
High-resolution melting provides a valid and powerful tool for detecting genomic mutations efficiently.
Insights
High-resolution melting assays efficiently detect KRAS, AKT1, PIK3CA, and BRAF mutations. This method is valuable for targeted cancer therapies, offering sensitive and rapid screening of genomic alterations.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Targeted therapies are increasing, driving the need for rapid mutation screening.
- KRAS, PIK3CA, AKT1, and BRAF mutations are crucial in colorectal cancer, breast cancer, and melanoma.
- These mutations activate oncogenic signaling pathways, making them targets for cancer treatment.
Purpose of the Study:
- To establish high-resolution melting (HRM) assays for predictive analysis of KRAS, AKT1, PIK3CA, and BRAF mutations.
- To provide a sensitive and efficient method for routine mutation screening.
Main Methods:
- Development of HRM assays using specifically designed primers.
- Utilized genomic DNA from cell lines, FFPE tissues, oligonucleotides, and plasmids.
- Melting curve analysis on the LightCycler platform, confirmed by Sanger sequencing.
Main Results:
- HRM assays detected low percentages (3.1%–12.5%) of mutated DNA in a wild-type background.
- Assays were validated with genomic DNA from various tumor types.
- High concordance (99%–100%) was observed between HRM and Sanger sequencing.
Conclusions:
- High-resolution melting is a valid and powerful tool for efficient genomic mutation detection.
- HRM assays are suitable for routine predictive analyses in oncology.
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