Objectives: Next-generation sequencing technologies have revolutionized cancer genomics research by providing a comprehensive method of detecting somatic cancer genome alterations. Platforms for genomic DNA alterations are more common in clinical practice and include whole genome/exome sequencing analysis. These tests are still very expensive, although the costs are coming down substantially. Here, we aimed to determine the efficacy and advantages of targeted exome sequencing of known cancer-related genes in human cancers using amplicon sequencing.

Methods: DNA was extracted from 61 human cancer specimens and their corresponding non-cancerous tissues, including oral squamous cell carcinomas (OSCCs) and multiple myelomas (MMs). Forty nanograms of DNA were used for multiplex PCR amplification with an Ion Ampliseq Comprehensive Cancer Panel that offers targeted coverage of all exons in 409 tumor suppressor genes and oncogenes frequently cited and frequently mutated in human cancers. This platform was designed to be amplification based capture with 15,992 regions (1.6 megabases in total size). Purified DNA libraries were sequenced with 6-8 samples on Ion Proton P1 chip. Sequence reads of tumor and normal samples were aligned to the hg19 genome, and generated BAM files were used to detect somatic mutations (point mutation, insertion and deletion) and copy number variations.

Results: Each sample underwent on mean 8.4 million sequencing reads after quality filtering. The mean base coverage depth was 530, and >95% of targeted bases were represented by at least 20 reads. The number of non-synonymous somatic mutations in 47 patients with OSCC ranged from 1 to 21 with a mean of 7.5 (6.4/Mb). The most frequent mutations in OSCC were in TP53 (63.8%), NOTCH1 (25.5%), CDKN2A (19.2%), TAF1L (17.0%), SYNE1 (14.9%) and PIK3CA (8.5%). We also detected a mean of 6.1 (range 3-11) non-synonymous mutations per MM patient. Somatic mutations were found in known MM-associated genes, including TP53 and NRAS. Pathway assessment has shown that somatic aberrations within MM genomes are mainly involved in several important pathways, including cell cycle regulation, RTK-MAPK-PI3K and NF-kB. We found several genetic alterations that may have been associated with the poor prognosis and poor response to chemotherapy of MM patients.

Conclusions: This study demonstrates the utility of using a semiconductor-based sequencing to efficiently identify somatic genetic alterations in human cancers. The targeted next-generation sequencing using low amounts of FFPE DNA is a valuable tool for rapid (5 days) and high-throughput genetic testing in research and clinical settings.

Citation Format: Yasushi Sasaki, Takafumi Nakagaki, Miyuki Tamura, Hisayo Fukushima, Hiroshi Ikeda, Ryota Koyama, Masashi Idogawa, Takashi Tokino. Targeted exome sequences of cancer-related genes in human cancers using amplicon sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 387. doi:10.1158/1538-7445.AM2017-387