Abstract

Genotyping epidermal growth factor receptor (EGFR) is an essential process to indicate lung adenocarcinoma patients for the most appropriate treatment. Liquid biopsy using circulating tumor DNA (ctDNA) potentially complements the use of tumor tissue biopsy for identifying genotype-specific mutations in cancer cells. We assessed the performance of a high-fidelity sequencing method that uses molecular barcodes called the nonoverlapping integrated read sequencing system (NOIR-SS) for detecting EGFR L858R-mutated alleles in 33 advanced or recurrent patients with L858R mutation-positive lung adenocarcinoma revealed by matched tissue biopsy. We compared NOIR-SS with site-specific droplet digital PCR (ddPCR), which was taken as the reference, in terms of sensitivity and ability to quantify L858R variant allele fractions (VAFs). NOIR-SS and ddPCR had sensitivities of 87.9% (29/33) and 78.8% (26/33) for detecting L858R alleles, respectively. The VAFs measured by each assay were strongly correlated. Notably, one specimen was positive with a VAF of 30.12% for NOIR-SS but marginally positive with that of 0.05% for ddPCR because of a previously poorly recognized mechanism: two-base substitution-induced L858R (c.2573_2574delinsGA). These results indicate that NOIR-SS is a useful method for detecting ctDNA, potentially overcoming a limitation of ddPCR which highly depends on the binding ability of primers to specific targeting sequences.

Highlights

  • Epidermal growth factor receptor (EGFR)-activating mutations [exon 19 deletion mutation and exon 21 L858R substitution mutation (L858R)] are the most and second most frequently identified driver alterations of lung adenocarcinoma in East Asian and Western countries, ­respectively[1,2,3]

  • All patients received epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) as the first-line treatment, except for two patients, including one who received an EGFR-TKI as the second-line treatment after cisplatin and docetaxel and the other who received an EGFR-TKI as the third-line treatment

  • The present study assessed the detection sensitivity of L858R circulating tumor DNA (ctDNA) using nonoverlapping integrated read sequencing system (NOIR-SS) in patients with advanced lung adenocarcinoma whose tumors had been genotyped as L858R-positive while using orthogonal Droplet digital polymerase chain reaction (ddPCR) as a reference

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Summary

Introduction

Epidermal growth factor receptor (EGFR)-activating mutations [exon 19 deletion mutation and exon 21 L858R substitution mutation (L858R)] are the most and second most frequently identified driver alterations of lung adenocarcinoma in East Asian and Western countries, ­respectively[1,2,3]. The isolation and analysis of circulating tumor DNA (ctDNA) from plasma cell-free DNA (cfDNA) provides a noninvasive tool that potentially complements or could, in some cases, replace tumor tissue biopsy for identifying genotypespecific mutations in cancer ­cells[13,14]. Droplet digital polymerase chain reaction (ddPCR) is an approach based on digital ­PCR20 that allows the independent amplification and fluorescence reading of tens of thousands of individual droplets in one well It enables the highly sensitive genotyping and absolute quantification of mutant genes. NOIR-SS enables more accurate quantification of molecules and measurement of allele fractions than conventional barcode sequencing by removing erroneous barcode tags during data analysis It remains unclear whether the NOIR-SS has equivalent sensitivity and accuracy in detecting mutant EGFR ctDNA in patients with advanced lung adenocarcinoma as that of ddPCR. We assessed the sensitivity of a ctDNA analysis targeting L858R assessed by NOIR-SS and ddPCR in patients with lung adenocarcinoma in whom tissue-derived DNA analyses had identified L858R

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