[Background] Mutational analysis is essential for recent personalized therapeutic approaches of non-small cell lung cancer (NSCLC) patients. Circulating cell-free DNA (cfDNA) from plasma is an emerging topic as a tool for liquid biopsy and possibly enable repetitive genetic diagnosis or monitoring of cancer status. However, detection of cfDNA from the circulation still remains challenging because of its highly fragmented and low concentration nature. In this study, we analyzed cfDNA samples by targeted deep sequencing at high coverage.

[Materials and Methods] Twenty matched DNA samples were collected from metastatic NSCLC patients with EGFR mutation, which was detected by a clinical EGFR mutant enriched PCR assay using PNA-LNA PCR clamp method. These samples were extracted from tissue samples of fresh tumor tissue except one formalin fixed paraffin embedded (FFPE) sample (tDNA), and plasma samples (cfDNA). Mutation hotspots were enriched by multiplex PCR using the GeneRead DNAseq Targeted Panels V2 Human Tumor Actionable Mutations panel (QIAGEN, Hilden, Germany), followed by library construction, and sequenced on the MiSeq sequencer. Resulting data was analyzed using CLC Genomics Workbench and Genomics Server and mapped to the full human genome.

[Results and discussion] The mean read depth in target region was 59,000 reads. Matched EGFR driver mutations between cfDNA and tDNA were detected with an overall concordance of 25% (5 of 20 samples). The concordance rate of exon21 L858R point mutation, exon19 deletions and exon20 T790M point mutation were 32.5% (3 of 8 samples), 16.7% (2 of 12 samples), and 66.7% (2 of 3 samples), respectively. The mutant allele frequencies in cfDNA ranged from 0.1% to 44.5% (median, 0.3%). While further improvement is mandatory, these results suggest that targeted deep sequencing of cfDNA at high coverage may have a potential utility as a non-invasive mutational analysis. In addition, there seemed to be a tendency of better detection rate for point mutations (45.5%) than deletions (16.7%), while the difference didn't reach statistical significance (p = 0.19), which were corresponding to previous reports.

Citation Format: Ken Suzawa, Shuta Tomida, Takahiro Matsubara, Kadoaki Ohashi, Yuho Maki, Hiromasa Yamamoto, Mizuki Morita, Junichi Soh, Hiroaki Asano, Kazunori Tsukuda, Katuyuki Kiura, Shinichiro Miyoshi, Shinichi Toyooka. Detection of EGFR mutations in circulating cell-free DNA of non-small cell lung cancer patients by next-generation sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2249.