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Sensitive genotyping of somatic mutations in the EGFR, KRAS, PIK3CA, BRAF genes from NSCLC patients using hydrogel
Marina Emelyanova1, Ksenia Arkhipova, Natalia Mazurenko
1*Laboratory for Biological Microchips, Engelhardt Institute of Molecular Biology of Russian Academy of Sciences †Laboratory for Clinical Oncogenetics, Clinical Oncology Research Institute ‡Laboratory for Cellular and Viral Oncogene Regulation, Carcinogenesis Institute §Oncogenomics Laboratory, Carcinogenesis Institute, N.N. Blokhin Russian Cancer Research Center of Russian Academy of Medical Sciences ∥Laboratory of Molecular Biology, Moscow City Oncology Hospital #62 of Moscow Healthcare Department, Moscow area, Russian Federation.
Abstract:
Targeted inhibitors of the epidermal growth factor receptor (EGFR) are used for the treatment of non-small cell lung cancer (NSCLC). Somatic mutations in the EGFR gene and key effectors of the EGFR-signaling pathway (KRAS, BRAF, PIK3CA) are associated with sensitivity to these drugs. We developed a highly sensitive LUNG CANCER (LC)-biochip approach for the detection of the most common EGFR, KRAS, PIK3CA, and BRAF gene mutations. The locked nucleic acid clamp PCR technique was used to increase the sensitivity of the assay, then allele-specific hybridization of a fluorescently labeled target on a biochip was performed. To prove the feasibility of the approach, clinical samples from 112 patients with NSCLC were analyzed. A total of 14 EGFR (12.5%) mutations, 21 (18.8%) KRAS mutations, 12 (10.7%) PIK3CA mutations, and 1 BRAF mutation (0.9%) were found. We compared the results with those from direct sequencing. We detected 50 different mutations by the LC-biochip assay and only 33 of them were found by direct sequencing. To demonstrate that the LC-biochip assay did not give false-positive results, the 17 specimens with discordant results were subjected to locked nucleic acid clamp PCR followed by sequencing. The results of this analysis were identical to the results obtained by the LC-biochip assay indicating that the biochip-based assay was both accurate and reliable. This approach was able to detect approximately 0.5% of mutated alleles in wild-type DNA background. The biochip-based assay is a reliable and inexpensive method for the identification of NSCLC patients, who may respond to a specific targeted therapy.
Insights
A novel biochip assay accurately detects common gene mutations in non-small cell lung cancer (NSCLC). This reliable method identifies patients who may benefit from targeted therapies, improving treatment selection for lung cancer.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Targeted therapies for non-small cell lung cancer (NSCLC) rely on identifying specific gene mutations.
- Epidermal growth factor receptor (EGFR) mutations and related pathway mutations (KRAS, BRAF, PIK3CA) predict response to EGFR inhibitors.
- Accurate and sensitive detection of these mutations is crucial for effective NSCLC treatment.
Purpose of the Study:
- To develop and validate a highly sensitive biochip assay for detecting common EGFR, KRAS, PIK3CA, and BRAF mutations in NSCLC.
- To compare the performance of the novel biochip assay against direct sequencing.
- To establish the reliability and accuracy of the biochip assay for clinical application.
Main Methods:
- Development of a lung cancer (LC)-biochip assay utilizing locked nucleic acid (LNA) clamp PCR for enhanced sensitivity.
- Allele-specific hybridization of fluorescently labeled targets on the biochip.
- Analysis of 112 NSCLC patient samples and comparison with direct sequencing results.
Main Results:
- The LC-biochip assay detected a higher number of mutations (50) compared to direct sequencing (33).
- Identified mutation frequencies: EGFR (12.5%), KRAS (18.8%), PIK3CA (10.7%), and BRAF (0.9%).
- Validation confirmed the assay's accuracy, reliability, and ability to detect low levels (0.5%) of mutated alleles.
Conclusions:
- The developed LNA clamp-based biochip assay is a sensitive, accurate, and reliable method for detecting key NSCLC mutations.
- This cost-effective approach facilitates the identification of NSCLC patients eligible for targeted therapies.
- The biochip assay offers a significant improvement over direct sequencing for mutation detection in NSCLC.
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