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Detection of epidermal growth factor receptor T790M mutation by allele-specific loop mediated isothermal
Srividya Arjuna1, Gunimala Chakraborty1, Rajesh Venkataram2
1Division of Molecular Genetics and Cancer, Nitte University Centre for Science Education and Research, Nitte (Deemed to be University), Mangaluru, Karnataka, India.
Introduction:
Targeted therapy using specific inhibitors against tyrosine kinases (TKs) is a paradigm in non-small-cell lung cancer management. However, the success of TK inhibitor (TKI) therapy depends on certain activating or acquired mutations, which render sensitivity or resistance to TKIs in the patients. The acquisition of epidermal growth factor receptor (EGFR) T790M point mutation is the most common mechanism of resistance to TKI in non-small cell lung cancer. A number of molecular strategies are now available for molecular testing of non-small cell lung cancers. However, almost all of them are cost-intensive and laborious and require high-end advanced equipment. Thus, assays that are rapid, simple, and cost-effective, yet sensitive, are most ideal in clinical settings for screening such therapeutically relevant mutations.
Materials And Methods:
Allele-specific loop-mediated isothermal amplification assay (AS-LAMP), which is a variant of the original LAMP assay, is a promising diagnostic technique for screening single-nucleotide polymorphisms. Using commercially available plasmid constructs as template DNA, AS-LAMP assay for EGFR T790M mutation was optimized with six different sets of reaction mixture containing varying concentrations of buffer and primers. The results of AS-LAMP assay were further validated by ultrasensitive droplet digital polymerase chain reaction.
Results:
Only one of the six sets of reaction mixture could accurately distinguish between wild type and mutated DNA, indicating that the primers and buffer are the two most critical components that determine the accuracy of AS-LAMP. The optimized AS-LAMP assay was further used to screen germ line and somatic T790M mutations in non-small cell lung cancer using blood and tissue samples collected from patients.
Conclusion:
Development of an accurate and rapid diagnostic assay that can detect resistant mutations without the need for sequencing is highly useful for clinicians in deciding on the eligibility of patients for TKI therapy. Considering its several inherent advantages, AS-LAMP assay could become an effective molecular tool for screening baseline or acquired EGFR T790M mutations in non-small cell lung cancer patients.
Insights
A new allele-specific loop-mediated isothermal amplification (AS-LAMP) assay accurately detects EGFR T790M mutations, a key resistance mechanism in non-small cell lung cancer. This rapid, cost-effective method aids in selecting targeted therapies for patients.
Area of Science:
- Molecular Biology
- Oncology
- Diagnostic Assays
Background:
- Targeted therapy with tyrosine kinase inhibitors (TKIs) is crucial for non-small cell lung cancer (NSCLC).
- EGFR T790M mutations confer resistance to TKIs, necessitating accurate detection.
- Current molecular testing methods are often expensive and complex.
Purpose of the Study:
- To develop a rapid, simple, and cost-effective diagnostic assay for detecting EGFR T790M mutations in NSCLC.
- To optimize and validate the allele-specific loop-mediated isothermal amplification (AS-LAMP) assay for clinical use.
Main Methods:
- Optimization of AS-LAMP assay using varying buffer and primer concentrations with plasmid DNA.
- Validation of AS-LAMP results against ultrasensitive droplet digital polymerase chain reaction (ddPCR).
- Screening of germline and somatic T790M mutations in NSCLC patient blood and tissue samples.
Main Results:
- Identified critical buffer and primer concentrations for accurate AS-LAMP detection of wild-type and mutant DNA.
- The optimized AS-LAMP assay demonstrated high accuracy in identifying EGFR T790M mutations.
- Successful screening of T790M mutations in clinical samples from NSCLC patients.
Conclusions:
- AS-LAMP assay provides a rapid, accurate, and cost-effective method for detecting EGFR T790M resistance mutations.
- This assay can assist clinicians in determining patient eligibility for TKI therapy.
- AS-LAMP is a promising molecular tool for screening EGFR T790M mutations in NSCLC.
