Related Experiment Video
Updated: Jul 2, 2026
Ultra-Fast Amplicon-Based Next-Generation Sequencing in Non-Squamous Non-Small Cell Lung Cancer
Published on: September 8, 2023
PCR-based testing for therapy-related EGFR mutations in patients with non-small cell lung cancer
Regine Dahse1, Alexander Berndt, Hartwig Kosmehl
1HELIOS Clinic Erfurt, Institute of Pathology, Erfurt, Germany. rdahse@erfurt.helios-kliniken.de
Background:
In patients with non-small cell lung cancer (NSCLC), mutations in the epidermal growth factor receptor gene (EGFR) have been associated with improved response to tyrosine kinase inhibitors. Two hotspot mutations located in exon 19 and exon 21 account for about 90% of all EGFR mutations.
Materials And Methods:
We designed a Bi-PASA (bidirectional PCR amplification of specific alleles) assay to detect the most common exon 19 deletion (codons 746-750) and an allele-specific PCR for the L858R mutation in exon 21. To validate the assays for use in clinical diagnostics, 35 lung adenocarcinoma samples were analyzed.
Results:
Both assays provided the predicted amplification pattern for normal and mutant genotypes with high specificity and sensitivity. In serial dilution experiments, the mutant alleles were detectable in mixed samples with an at least 6-fold excess of normal DNA. Three exon 19 deletions were identified in the tumor samples.
Conclusion:
Both assays are fast and easy to perform in any routine PCR laboratory with no special equipment other than thermocyclers. They provide sensitive and cost effective initial EGFR testing for identifying lung cancer patients who might clinically benefit from tyrosine kinase inhibitors.
Insights
New PCR assays accurately detect common EGFR mutations in non-small cell lung cancer (NSCLC). These rapid, cost-effective tests identify patients who may benefit from targeted therapies, improving lung cancer treatment.
Area of Science:
- Molecular diagnostics
- Oncology
- Genetics
Background:
- Epidermal growth factor receptor (EGFR) mutations are key drivers in non-small cell lung cancer (NSCLC).
- Specific EGFR mutations (exon 19 deletions and L858R) predict response to tyrosine kinase inhibitors.
- Accurate detection of these mutations is crucial for personalized NSCLC treatment.
Purpose of the Study:
- To develop and validate novel assays for detecting common EGFR mutations in NSCLC.
- To establish sensitive and specific molecular diagnostic tools for clinical use.
- To facilitate the identification of NSCLC patients eligible for targeted therapies.
Main Methods:
- Development of a Bi-PASA (bidirectional PCR amplification of specific alleles) assay for exon 19 deletions.
- Development of an allele-specific PCR assay for the exon 21 L858R mutation.
- Validation using 35 lung adenocarcinoma samples and serial dilution experiments.
Main Results:
- Both developed assays demonstrated high specificity and sensitivity in detecting EGFR mutations.
- Mutant alleles were detectable even with a significant excess of wild-type DNA.
- Three cases of exon 19 deletions were successfully identified in the analyzed tumor samples.
Conclusions:
- The Bi-PASA and allele-specific PCR assays are rapid, cost-effective, and suitable for routine laboratories.
- These assays provide sensitive and reliable EGFR mutation testing for NSCLC patients.
- The developed methods can aid in selecting patients for tyrosine kinase inhibitor therapy.