Custom-Designed Probes for the Accurate Determination of Epidermal Growth Factor Receptor Mutations and Their Allelic Configuration

The identification of single nucleotide polymorphisms (SNPs) is of paramount importance for disease diagnosis and clinical prognostication. In the context of nonsmall cell lung cancer (NSCLC), the emergence of resistance mutations, exemplified by the epidermal growth factor receptor (EGFR) T790 M and C797S, is intricately linked to the therapeutic efficacy of EGFR tyrosine kinase inhibitors (EGFR-TKIs). Herein, a highly efficient and specific SNP detection platform for T790 M and C797S mutations has been engineered through the integration of an asymmetric polymerase chain reaction (PCR) and an ingeniously tailored four-way junction (4WJ) probe. Notably, a molecular beacon (MB) probe was judiciously designed to discern the allelic configuration of these mutations. The administration of first- and third-generation EGFR-TKIs demonstrates therapeutic efficacy solely when the mutations are in the trans configuration, characterized by a low fluorescence signal. In contrast, significant fluorescence by the MB probe is indicative of the C797S mutation being in a cis arrangement with T790M, thereby rendering the cells refractory to the therapeutic interventions of both first- and third-generation EGFR-TKIs. The assay is capable of concurrently detecting two point-mutations and ascertaining their allelic positions in a single test within 1.5 h, enhancing both efficiency and simplicity. It also exhibits high accuracy in the identification of clinical samples, offering promising implications for therapeutic guidelines. By enabling tailored treatment plans based on specific genetic profiles, our approach not only advances the precision of NSCLC treatment strategies but also marks a significant contribution to personalized medicine.