Quantitative identification of mutant alleles derived from lung cancer in plasma cell-free DNA via anomaly detection using deep sequencing data.

Junji Uchida,Toshie Niki,Takako Okuyama,Fumio Imamura,Kazuya Taniguchi,Toru Kumagai,Kazumi Nishino,Yoji Kukita,Shigeyuki Oba,Kikuya Kato

doi:10.1200/jco.2013.31.15_suppl.e19153

Abstract

e19153 Background: Selection of lung cancer patients to be treated with EGFR-TKI (gefitinib and erlotinib) is currently based on the presence of activating mutations in the EGFR kinase domain. The diagnosis of the mutations is based on biopsy samples; the procedure is invasive, and often difficult to perform. A non-invasive diagnostic procedure is desirable. Methods: We established a non-invasive diagnostic system to detect somatic mutations including three activating mutations (exon 19 deletion, L858R, L861Q) and one resistant mutation (T790M) with single-pass deep sequencing of EGFR gene fragments amplified from patients’ plasma DNA using Ion Torrent PGM. In principle, substitutions or deletions significantly higher (p=0.00002) than the error rates determined with the normal samples were judged as mutations. Read errors of the system were determined with deep sequencing (>100,000 reads) of EGFR fragments. The detection limits were less than 0.01% for the exon 19 deletion and L858R, 0.01% for L861Q, and 0.05% for T790M. Results: We examined 155 samples, mostly plasma cell-free DNA, with mutation data of primary lesions. Exon 19 deletion, L858R, and L861Q were detected in 44 out of 72 samples (61.1%), 32 out of 56 (57.1%), and four out six, respectively. T790M was found in 26 samples, five of which did not accompany activating mutations. Considerable numbers of samples were collected from the same patients at different time points in the disease course. A temporal analysis revealed rapid disappearance of mutations by the EGFR-TKI therapy before radiological changes. Conclusions: Deep sequencing of amplified EGFR fragments from plasma DNA could detect mutations in lung cancer tissues. Results of the temporal analysis suggested potential applications for disease management and for therapeutic decision making to select EGFR-TKI. Clinical trial information: UMIN000006762.

Highlights

For some molecular targeted drugs against cancer, the examination of genomic changes in target genes has become a diagnostic routine and is indispensable for treatment decisions
When the frequency of a base change at a target locus is higher than a predetermined read error rate (RER), we may judge the change to be due to the presence of a mutant sequence
Rare mutation detection of target loci through the deep sequencing of plasma cell-free DNA has a comparable sensitivity to BEAMing

Summary

Introduction

For some molecular targeted drugs against cancer, the examination of genomic changes in target genes has become a diagnostic routine and is indispensable for treatment decisions. The identification of the mutations is based on biopsy samples; the procedure is invasive and often difficult to perform. Cell-free DNA in the blood consists of DNA derived from cancer tissues and has been studied for non-invasive diagnostic procedures [3]. This DNA, termed circulating tumor DNA (ctDNA), is rare in blood, and its detection is a technical challenge. BEAMing (beads, emulsion, amplification and magnetics) [4] is most likely the most sensitive method. In BEAMing, PCR products amplified from a single molecule are fixed to a single magnetic bead using emulsion PCR. In spite of its high sensitivity and quantification ability, BEAMing has not gained in popularity because it is a laborious technology and requires oligonucleotides for each mutation position

Methods

Results

Conclusion