P1.02-026 Detection of Low-Abundant EGFR Somatic Mutations by PNA Clamping-Assisted Fluorescence Melting Curve Analysis

Abstract

Detecting mutations is becoming important for both predicting disease progression and drug responses to treatment of cancer patients. Current mutation detection methods for cancer diagnosis are mainly based on the invasive sampling technique such as a tissue biopsy, but some patients may not available for this invasive procedure. Therefore, circulating tumor DNA (ctDNA) would be a good alternative for those patients. However, testing methods for tissue biopsy sample are not applicable to ctDNA samples due to relatively lower sensitivity. A highly sensitive assay method is required for detecting mutations in liquid biopsy samples. We have developed a highly sensitive and simple method to detect somatic mutation from ctDNA in patient’s plasma. This new real-time PCR-based testing method (PANAMutyper™) has maximized unique properties of peptide nucleic acid (PNA). It contains a PNA clamp and PNA detection probes in the each reaction tubes. A optimized PNA clamp can tightly bind to only wild-type DNA sequences, and then suppress amplification during the PCR reaction. Meanwhile, a PNA detection probe that conjugated with a fluorescent dye and a quencher, can detect a specific target mutant-type DNA and each mutation can be genotyped by melting peak analysis. PANAMutyper™ is able to detect 47 different mutations in exon 18, 19, 20 and 21 of EGFR gene with detection limits as low as 0.01%. In order to confirm the validity in real condition, we conducted spiking test. Ten of mutant clones DNA were used as standard materials. (G719A, G719S, G719S, S768I, Exon19 deletion, two Exon20 insertion, T790M, L858R, L861Q). Mix the each mutant clone DNA and human plasma to 1, 10, 100, 1000 copies per microliter concentration. And then we extracted ctDNA from prepared sample. As a result, all of ten mutant clone DNA was detected 1 copy/㎕. This result suggest that PANAMutyper™ is applicable to ctDNA derived from patient’s plasma sample. The high concordance, specificity, and sensitivity of this test have demonstrated that EGFR mutation status can be accurately assessed by PANAMutyper™ using ctDNA. Therefore, PANAMutyperTM can be used in various clinical areas including companion diagnostics and monitoring acquired mutations.