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Published on: August 14, 2018
Single-Tube Mutation Scanning of The Epidermal Growth Factor Receptor Gene Using Multiplex LATE-PCR and
Shana M Tetrault1, John E Rice1, Lawrence J Wangh1
1Department of Biology, Brandeis University, 415 South St., Waltham, MA, USA.
Background:
Numerous mutations in exons 18-21 of the epidermal growth factor receptor (EGFR) gene determine the response of many patients with non-small cell lung carcinoma (NSCLC) to anti-EGFR tyrosine kinase inhibitors (TKIs). This paper describes a single closed-tube assay for simultaneous mutational scanning of EGFR exons 18-21.
Methods:
The assay first co-amplifies all four exons as separate single-stranded DNA products using Linear-After-The-Exponential (LATE)-PCR. The amplicons are then interrogated at endpoint along their length using sets of Lights-On/Lights-Off probes of a different color for each exon. The four resulting fluorescent signatures are unique for each underlying DNA sequence. Every mutation in a target potentially alters its unique fluorescent signature thereby revealing the presence of the mutation.
Results:
The assay readily detects mutations which cause sensitivity or resistance to TKIs and can distinguish these clinically important genetic changes from silent mutations which have no impact on protein function. The assay identifies as little as 5% mutant sequences in mixtures of normal DNA and mutant DNA prepared from cancer cell lines. Proof-of-principle experiments demonstrate mutation identification in formalin-fixed, paraffin-embedded NSCLC biopsies.
Conclusion:
The LATE-PCR EGFR assay described here represents a new type of highly informative, single-tube diagnostic test for mutational scanning of multiple gene coding regions and/or multiple gene targets for personalized cancer therapies.
Insights
A new assay simultaneously scans epidermal growth factor receptor (EGFR) exons 18-21 for mutations. This single-tube test aids in predicting non-small cell lung carcinoma (NSCLC) patient response to targeted therapies.
Area of Science:
- Molecular diagnostics
- Oncology
- Genetics
Background:
- Mutations in epidermal growth factor receptor (EGFR) exons 18-21 are crucial for predicting non-small cell lung carcinoma (NSCLC) patient response to tyrosine kinase inhibitors (TKIs).
- Accurate and efficient detection of these mutations is essential for personalized cancer therapy.
Purpose of the Study:
- To develop and validate a single, closed-tube assay for simultaneous mutational scanning of EGFR exons 18-21.
- To provide a highly informative diagnostic tool for identifying clinically relevant EGFR mutations.
Main Methods:
- Utilizes Linear-After-The-Exponential (LATE)-PCR for co-amplification of all four EGFR exons (18-21) into separate single-stranded DNA products.
- Employs Lights-On/Lights-Off fluorescent probes for endpoint interrogation of amplicons, generating unique fluorescent signatures for each DNA sequence.
- The assay's sensitivity allows for detection of mutations down to 5% in mixed DNA samples.
Main Results:
- The assay successfully detects clinically significant mutations affecting TKI sensitivity or resistance.
- It effectively distinguishes actionable mutations from silent mutations.
- Demonstrated proof-of-principle for mutation detection in formalin-fixed, paraffin-embedded NSCLC biopsies.
Conclusions:
- The developed Linear-After-The-Exponential PCR (LATE-PCR) EGFR assay is a novel, single-tube diagnostic test.
- It offers a highly informative approach for mutational scanning of multiple gene regions.
- This assay supports personalized cancer therapies by enabling precise identification of genetic targets.
