Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing

May Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing Kazuko Sakai 0 1 Junji Tsurutani 0 1 Takeharu Yamanaka 0 1 Azusa Yoneshige 0 1 Akihiko Ito 0 1 Yosuke Togashi 0 1 Marco A. De Velasco 0 1 Masato Terashima 0 1 Yoshihiko Fujita 0 1 Shuta Tomida 0 1 Takao Tamura 0 1 Kazuhiko Nakagawa 0 1 Kazuto Nishio 0 1 0 1 Department of Genome Biology, Kinki University Faculty of Medicine , Osaka-Sayama, Osaka , Japan , 2 Department of Medical Oncology, Kinki University Faculty of Medicine , Osaka-Sayama, Osaka , Japan , 3 Department of Biostatistics, Yokohama City University , Yokohama, Kanagawa , Japan , 4 Department of Pathology, Kinki University Faculty of Medicine , Osaka-Sayama, Osaka , Japan 1 Academic Editor: Ralf Krahe, University of Texas MD Anderson Cancer Center , UNITED STATES Somatic mutations in KRAS, NRAS, and BRAF genes are related to resistance to antiEGFR antibodies in colorectal cancer. We have established an extended RAS and BRAF mutation assay using a next-generation sequencer to analyze these mutations. Multiplexed deep sequencing was performed to detect somatic mutations within KRAS, NRAS, and BRAF, including minor mutated components. We first validated the technical performance of the multiplexed deep sequencing using 10 normal DNA and 20 formalin-fixed, paraffinembedded (FFPE) tumor samples. To demonstrate the potential clinical utility of our assay, we profiled 100 FFPE tumor samples and 15 plasma samples obtained from colorectal cancer patients. We used a variant calling approach based on a Poisson distribution. The distribution of the mutation-positive population was hypothesized to follow a Poisson distribution, and a mutation-positive status was defined as a value greater than the significance level of the error rate ( = 2 x 10-5). The cut-off value was determined to be the average error rate plus 7 standard deviations. Mutation analysis of 100 clinical FFPE tumor specimens was performed without any invalid cases. Mutations were detected at a frequency of 59% (59/ 100). KRAS mutation concordance between this assay and Scorpion-ARMS was 92% (92/ 100). DNA obtained from 15 plasma samples was also analyzed. KRAS and BRAF mutations were identified in both the plasma and tissue samples of 6 patients. The genetic screening assay using next-generation sequencer was validated for the detection of clinically relevant RAS and BRAF mutations using FFPE and liquid samples. - Funding: This work was supported by Grant-in-Aid for Young Scientists (B) (Subject No.29830098 to K. S.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and Applied Research for Innovative Treatment of Cancer (Subject No.14525177 to K.N.) from the Ministry of Health, Labour and Welfare of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction Colorectal cancer is one of the leading causes of deaths due to cancer. As improved understanding of the molecular pathology of colorectal cancer, a number of targeted agents have been developed which have demonstrated improved outcome in colorectal cancer patients [16]. The signaling pathway of epidermal growth factor receptor (EGFR) plays a central role Competing Interests: The authors have declared that no competing interests exist. for the biology of colorectal cancer because two monoclonal antibodies directed to EGFR (cetuximab and panitumumab) have become important tools in the management of advanced disease [2, 3, 5]. KRAS is GTP-binding protein that is activated by GTP binding caused by upstream signals, such as EGFR. Single base mutations in the KRAS gene decrease GTPase activity, resulting in the constitutive activation of KRAS. The most common KRAS mutations are found in codons 12 and 13. Mutations in codons 12 and 13 of KRAS occur in ~40% of metastatic colorectal cancers [7, 8]. The OPUS and CRYSTAL studies of colorectal cancer patients receiving the FOLFOX plus cetuximab and FOLFIRI plus cetuximab regimens, respectively, have demonstrated that mutations in codons 12 and 13 are negative predictive factor for the response to the antiEGFR antibody cetuximab [9, 10]. The addition of cetuximab improves survival only in patients with KARS wild-type colorectal cancer. Other studies have also shown that the KRAS mutation status was negatively correlated with the response to the treatment with anti-EGFR antibodies such as cetuximab and panitumumab [1113]. NRAS is closely related to KRAS, having 85% amino acid sequence identity [14]. Recent studies showed that other activating mutations in KRAS (exons 3 and 4) or NRAS (exons 2, 3 and 4), in addition to KRAS mutation in exon 2 are also associated with poor prognosis or resistance to anti-EGFR antibody in metastatic colorectal cancer. The PRIME study showed that not only KRAS, but also NRAS mutations, will predict a lack of response to panitumumab [13]. Similar results were presented for extended RAS analyzes in the CRYSTAL and OPUS trials [15]. BRAF, a member of the Raf family of serine/threonine kinases, is a direct downstream effector of KRAS. BRAF mutation is constitutive active and leads to constitutive activation of mitogen-activated protein kinases (MAPK) pathway. The most common BRAF mutation is a T to A transversion resulting in a valine to glutamic acid substitution (V600E), present in ~10% of metastatic colorectal cancer patients [16]. KRAS and BRAF mutations are considered to be mutually exclusive [17]. In a retrospective analysis from the CRYSTAL and OPUS trials, the patients who had BRAF mutations had a poor prognosis, regardless of receiving chemotherapy or chemotherapy plus cetuximab [15]. These prospective-retrospective analyses have concluded that genetic testing for KRAS, NRAS and BRAF gene mutations beyond the routine analysis of KRAS exon 2 will be important for the selection of anti-EGFR therapy. Nevertheless, an efficient assay system for analyzing extended RAS and BRAF mutations in addition to those of exon 2 of KRAS (codons 12 and 13) is yet to be established. Many experimental methods can be used to detect rare mutations, such as mutation-specific PCR-based assays and high-resolution melting analysis. One disadvantage of these assays is that they consist of a single-plex reaction. Next-generation sequencing (NGS) is a widely used technology for gene mutation analysis. NGS technologies have the potential to analyze mutations in several genes and from multiple patient samples simultaneously. However, the ability of NGS technology to detect low-level mutations has limited its clinical use. In this study, we established and validated a gene mutation assay for the detection of KRAS, NRAS, and BRAF mutations in FFPE and plasma samples using an Ion Torrent PGM semiconductor sequencer (Life Technologies, Carlsbad, CA). Materials and Methods Ethics statement This study was approved by the ethics committee of Kinki University Fa (...truncated)