title: Sensitive genotyping of somatic mutations in the EGFR, KRAS, PIK3CA, BRAF genes from NSCLC patients using hydrogel biochips.
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.
authors.affiliation: *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.
authors.forename: Marina
authors.initials: M
authors.lastname: Emelyanova
authors.forename: Ksenia
authors.initials: K
authors.lastname: Arkhipova
authors.forename: Natalia
authors.initials: N
authors.lastname: Mazurenko
authors.forename: Alexander
authors.initials: A
authors.lastname: Chudinov
authors.forename: Irina
authors.initials: I
authors.lastname: Demidova
authors.forename: Irina
authors.initials: I
authors.lastname: Zborovskaya
authors.forename: Lyudmila
authors.initials: L
authors.lastname: Lyubchenko
authors.forename: Alexander
authors.initials: A
authors.lastname: Zasedatelev
authors.forename: Tatiana
authors.initials: T
authors.lastname: Nasedkina
mesh_terms: D000368:Aged; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics*; D058534:Class I Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors / Q000235:genetics*; D005260:Female; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D020100:Hydrogels; D008175:Lung Neoplasms / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D012680:Sensitivity and Specificity; D018631:ras Proteins / Q000235:genetics*
chemical_list: D020100:Hydrogels; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D019869:Phosphatidylinositol 3-Kinases; D058534:Class I Phosphatidylinositol 3-Kinases; C484760:PIK3CA protein, human; C512478:EGFR protein, human; D066246:ErbB Receptors; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins