Related Experiment Video
Updated: Mar 3, 2026
Detection of Targetable Alterations in Non-small Cell Lung Cancer using Next-generation Sequencing
Published on: October 10, 2025
Detection of EGFR mutations in patients with non-small cell lung cancer by high resolution melting. Comparison with
Background:
The discovery of mutations in the epidermal growth factor receptor gene (EGFR) related to the clinical response to tyrosine kinase inhibitors, has transformed the management of non-small cell lung cancer (NSCLC). Several methods have been developed for determination of mutations in EGFR, with different sensitivity and potential ability to detect a different number of mutations.
Methods:
We developed a screening method by high resolution melting (HRM) to detect EGFR mutations, and compared the results of 123 fixed in formalin and paraffin embedded (FFPE) tumor tissue samples with the detection of mutations by allele-specific PCR. In samples with discordant results, Sanger and massive parallel sequencing (MPS) were additionally performed.
Results:
Eight samples showed discordant results between both methods. Three samples with negative results by allele specific PCR and positive by HRM were confirmed by Sanger sequencing (p.S768I+p.V769L, T751_I759del and p.E709K+p.G719A; patients 1, 3 and 4, respectively). One sample with a negative result by HRM, and positive by allele specific PCR (p.T790M; patient 2), was confirmed by Sanger sequencing. Additionally, two positive samples for a deletion in exon 19 by allele-specific PCR, were negative by Sanger sequencing and HRM (patients 2 and 5) and finally, two samples were negative by allele-specific PCR and positive by HRM and Sanger sequencing due to synonymous variants in exon 21.
Conclusions:
HRM is a good method for mutational screening in EGFR. It is able to detect any change in the sequence of exons 18-21, providing high cost/effectiveness, but samples with low tumor burden may produce false negatives results.
Insights
High-resolution melting (HRM) effectively screens for epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC). While cost-effective, low tumor burden may lead to false negatives.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Epidermal growth factor receptor (EGFR) mutations are key predictors of tyrosine kinase inhibitor response in non-small cell lung cancer (NSCLC).
- Accurate detection of EGFR mutations is crucial for personalized cancer therapy.
- Various methods exist for EGFR mutation detection, differing in sensitivity and scope.
Purpose of the Study:
- To develop and evaluate a high-resolution melting (HRM) screening method for epidermal growth factor receptor (EGFR) mutations.
- To compare the performance of HRM against allele-specific PCR, Sanger sequencing, and massive parallel sequencing (MPS).
Main Methods:
- Developed a high-resolution melting (HRM) assay for EGFR mutation screening.
- Analyzed 123 formalin-fixed, paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC) tumor samples.
- Compared HRM results with allele-specific PCR, Sanger sequencing, and MPS for discordant cases.
Main Results:
- Eight samples showed discordant results between HRM and allele-specific PCR.
- HRM identified specific mutations (p.S768I+p.V769L, T751_I759del, p.E709K+p.G719A, p.T790M) missed by allele-specific PCR in some cases.
- HRM and allele-specific PCR showed discrepancies for exon 19 deletions and synonymous variants in exon 21, requiring confirmation by Sanger sequencing.
Conclusions:
- High-resolution melting (HRM) is a cost-effective screening tool for detecting alterations in EGFR exons 18-21.
- HRM demonstrates good sensitivity for broad mutation screening.
- Low tumor cellularity in FFPE samples may result in false-negative HRM findings.
