Non-invasive detection of EGFR mutations by cell-free loop-mediated isothermal amplification (CF-LAMP)
Srividya Arjuna1, Rajesh Venkataram2, Pandyanda Nanjappa Dechamma1
1Division of Molecular Genetics and Cancer, Nitte University Centre for Science Education and Research (NUCSER), Nitte (Deemed to be University), Kotekar-Beeri Road, Deralakatte, Mangaluru, 575018, India.
Abstract:
Targeting epidermal growth factor receptor (EGFR) through tyrosine kinase inhibitors (TKI) is a successful therapeutic strategy in non-small cell lung cancer. However, the response to TKI therapy depends on specific activating and acquired mutations in the tyrosine kinase domain of the EGFR gene. Therefore, confirming the EGFR status of patients is crucial, not only for determining the eligibility, but also for monitoring the emergence of mutations in patients under TKI therapy. In this study, our aim was to develop a cost effective, yet sensitive, technique that allows the detection of therapeutically-relevant EGFR hotspot mutations at isothermal conditions in a non-invasive manner. Previously, we developed an allele-specific loop-mediated isothermal amplification (AS-LAMP) assay for screening germline and somatic de novo T790M EGFR mutation in lung cancer patients. In this study, we used cell free DNA as a template in AS-LAMP assay (CF-LAMP) for non-invasive detection of two hotspot EGFR mutations (T790M, and L858R) and compared its efficiency with ultrasensitive droplet digital PCR (ddPCR) assay. The results of CF-LAMP assay were consistent with those obtained in ddPCR assay, indicating the robustness of the method. CF-LAMP may serve as a valuable and cost-effective alternative for liquid biopsy techniques used in molecular diagnosis of non-small cell lung cancer.
Insights
A new non-invasive method, cell-free DNA loop-mediated isothermal amplification (CF-LAMP), accurately detects epidermal growth factor receptor (EGFR) mutations in lung cancer patients. This cost-effective technique aids in monitoring treatment response and guiding therapy.
Area of Science:
- Oncology
- Molecular Diagnostics
- Genetics
Background:
- Targeting epidermal growth factor receptor (EGFR) with tyrosine kinase inhibitors (TKI) is a key strategy for non-small cell lung cancer (NSCLC).
- Patient response to EGFR-TKI therapy is dictated by specific mutations within the EGFR gene.
- Accurate EGFR mutation detection is vital for patient eligibility, treatment selection, and monitoring acquired resistance mutations.
Purpose of the Study:
- To develop a cost-effective and sensitive method for detecting therapeutically relevant EGFR hotspot mutations.
- To establish a non-invasive technique for EGFR mutation analysis using cell-free DNA (cfDNA).
- To evaluate the performance of the developed cell-free DNA loop-mediated isothermal amplification (CF-LAMP) assay against droplet digital PCR (ddPCR).
Main Methods:
- Development of an allele-specific loop-mediated isothermal amplification (AS-LAMP) assay utilizing cfDNA as the template (CF-LAMP).
- Detection of two common EGFR hotspot mutations: T790M and L858R.
- Comparative analysis of CF-LAMP assay results with ultrasensitive droplet digital PCR (ddPCR).
Main Results:
- The CF-LAMP assay demonstrated high consistency with the ddPCR assay for detecting EGFR mutations.
- The method proved robust and reliable for analyzing cfDNA.
- Successful non-invasive detection of T790M and L858R EGFR mutations was achieved.
Conclusions:
- CF-LAMP offers a sensitive and cost-effective approach for non-invasive EGFR mutation detection in NSCLC.
- This method can serve as a valuable alternative to existing liquid biopsy techniques.
- CF-LAMP facilitates molecular diagnosis and monitoring of NSCLC patients undergoing TKI therapy.
