Endonuclease-Assisted Selective Exponential Amplification for Ultrasensitive Enrichment and Detection of Low-Abundance Mutant Alleles in Lung Cancer.

Lung cancer is one of the most prevalent and lethal malignancies worldwide. Despite recent advancements in precision medicine, early detection and therapeutic monitoring of lung cancer remain challenging. Herein, an endonuclease-assisted selective exponential amplification (ESEA) platform is presented that selectively depletes wild-type alleles through programmable endonuclease digestion (CRISPR-Cas or restriction enzymes) while simultaneously amplifying mutant alleles, enabling robust and cost-effective detection at mutant allele frequencies as low as 0.00625%. To address the protospacer adjacent motif-site limitations inherent in CRISPR-based enrichment strategies, a primer-directed restriction site programming approach that expands the theoretical coverage to more than 94% of mutations listed in COSMIC (Catalogue of Somatic Mutations in Cancer) is introduced. The ESEA system offers high sensitivity and cost-effectiveness compared with conventional methods, and enables multiplexed detection of key lung cancer hotspot mutations, including EGFR L858R, EGFR exon 19 deletions, EGFR T790M, BRAF V600E, as well as hotspots in KRAS and PIK3CA. In a small-sample-size test, the ESEA system achieved 100% sensitivity and specificity in five pleural effusion samples and a 100% circulating tumor DNA detection rate in six patients with extracranial lesions and disease progression. These results highlight its potential as a cost-effective, highly sensitive, and robust platform for dynamic, real-time companion diagnostics, as well as noninvasive monitoring of treatment response and tumor evolution.