A novel method for enrichment and detection of the six major subtypes of EGFR exon 19 deletions simultaneously by capturing mutant-alleles.

Abstract

e15539 Background: EGFR exon 19 deletions (19DELs) are important precision therapy targets for NSCLC patients. Due to over twenty subtypes of 19DEL mutations, it is usually challenging for detecting different deletion forms in one assay using limited DNA preparation from plasma sample. Methods: Previously we had developed a high sensitivity detection method, namely PEAC, to detect single somatic mutation in one assay. The method used locked¨Cnucleic acid modified short DNA probe to capture mutant fragment under an optimized temperature. In the present study, we have established a multiplexing PEAC approach for simultaneous enrichment and high sensitivity detection of six major subtypes of EGFR 19DELs.The six subtypes overall account for >86% EGFR exon 19 deletions in the patient population. Results: Genomic DNA of EGFR wild-type was mixed with DNA samples of known EGFR 19DEL mutations. The mutation allele fractions of 5%, 1%, and 0.1% for the six 19del subtypes were constructed, which included c.2235-2249del15 p.E746_A750del, c.2236-2250del15 p.E746_A750del, c.2237-2255delinsT p.E746_A752delinsV, c.2235-2249delinsC p.L747_A750delinsP, c.2240-2254del15 p.L747_T751del, and c.2240-2257del18 p.L747_P753delinsS. The mixed DNA samples were sonicated to mimic the fragment distribution of circulating-free DNA (cfDNA) and PCR amplified for exon 19 regions. Multiplexing enrichment using the six specific probes targeted to the 19DEL subtypes were performed to the amplified DNA fragments under the optimal binding conditions. Sanger sequencing all showed detectable variants in the enriched samples starting with 5%, 1%, or 0.1% mutants, which indicated the capacity and high sensitivity of multiplexing PEAC technology. To explore the potential of multiplexing with other EGFR mutants, the 0.1% mixed mutants of 19DEL were PCR amplified for EGFR exon 20 and exon 21 in addition to exon 19, and followed with enrichment and detection of the six 19DEL variants. Sanger sequencing can detect all the six subtypes of 19DELs. Our method is superior to ddPCR in some aspects such as high throughput and low starting DNA amounts. Conclusions: Multiplexing PEAC technology can simultaneously enrich and detect the six major subtypes of EGFR 19 deletions with high sensitivity. It also has potential to multiplex with other EGFR driver mutations such as in exons 18-21. The technology may serve as an attractive method for detecting mutations in liquid biopsies in clinical practice.