According to several studies hotspots of RAS mutations have

According to several studies, hotspots of RAS mutations have been identified in codons 12, 13, 59, 61, 117, and 146, which are considered to induce conformational changes into constitutively active forms (Prior et al., 2012; Smith et al., 2010). In addition, these gene mutations tend to occur in a mutually exclusive manner. These findings increase the medical need for kits that can detect all RAS mutations occurring in these codons. During the last decade, various techniques for RAS testing (i.e., direct sequencing [DS], SURVEYOR®-WAVE method, pyrosequencing, allele-specific PCR, MALDI-TOF mass array, and BEAMing [bead, emulsion, amplification and magnetics] assay) have been developed (Diehl et al., 2008; Douillard et al., 2013; Heinemann et al., 2014; Maughan et al., 2011; Parsons et al., 2011; Parsons and Myers, 2013). Some of these methods require microdissected formalin-fixed paraffin-embedded (FFPE) tissues to enrich tumor cells for obtaining enough amount of tumor-derived DNA. Although real-time PCR is one of the common techniques for RAS testing, it requires a lot of wells and a large amount of DNA to detect all RAS mutations. Next-generation sequencing technologies are innovative, comprehensive and high throughput methods for RAS testing, which are larger-sized for RAS testing in the present clinical practice. On the other hand, Luminex®-xMAP® technology can provide multiplex molecular testing in a single well, and only requires a small amount of tumor-derived DNA, as previously reported (Bando et al., 2013; Fukushima et al., 2011), so that the MEBGENTM RASKET KIT (RASKET KIT) could be considered as more cost-effective for RAS testing. In this study, we evaluated the RASKET KIT to detect forty-eight kinds of RAS amino rivastigmine tartrate mutations in CRC patients. This study is also performed as a registration trial for regulatory approval.
Methods
Results
Discussion This study is the first to demonstrate a clinical usefulness of RASKET KIT, a CE-marked and approved by the Ministry of Health, Labour and Welfare of Japan in vitro diagnostics (IVD) kit for determination of all RAS mutation status in FFPE tissues of CRC patients. For the purpose, we prospectively compared the RASKET KIT to TheraScreen Kit and DS with MMD which are the gold standard for RAS testing (Massarelli et al., 2007). Our data revealed that the overall concordance rate (96.7%, 297/307) was satisfied with the predefined criterion (>90%). As a useful kit detecting all RAS mutations, the capability of detecting minor RAS mutations other than KRAS exon 2 is required. Our study also showed a high concordance rate of 98.4% (188/191) between mutational status obtained with the RASKET KIT and DS with MMD, in WT KRAS exon 2 population (n=191). Similar to the overall concordance rate, the positive and negative concordance rates were no less than 95%. The frequency of RAS mutations detected with the RASKET KIT in our study agreed with those reported in the several past studies (Bokemeyer et al., 2009; Douillard et al., 2010, 2013; Heinemann et al., 2014; Watanabe et al., 2013). For example, 37.1% (114/307) in exon 2 detected in this study corresponded with the 37.6% reported in a large-scaled Japanese study (Watanabe et al., 2013). KRAS testing to determine exon 2 status prior to anti-EGFR treatment for CRC patients has been widely used (Normanno et al., 2009). Many KRAS exon 2 mutation detection kits such as TheraScreen Kit are approved as IVD in Japan, USA, and Europe. Recent studies suggested that additional RAS mutation as well as KRAS exon 2 could predict an efficacy of anti-EGFR treatment, and consequently the indication of anti-EGFR therapeutic antibodies has been revised to direct the treatment of patients with wild-type RAS (both KRAS and NRAS) metastatic CRC (Douillard et al., 2013; Heinemann et al., 2014; Schwartzberg et al., 2014). Thus, expanded RAS testing using an approved IVD kit is recommended. We had several inconsistent results between the RASKET KIT and the reference assays. Six specimens were determined as positive with RASKET KIT and negative with the reference assays. According to the confirmation study using TaqMan Mutation Detection Assays, all of the six specimens were RAS mutation positive. Such discrepancy may be caused by a smaller amount of mutant DNA. Indeed, as shown in Table 3, the ratios of mutant RAS DNA to WT RAS DNA were 0.1–1% in two specimens and 1–5% in three out of the six specimens. Another conflicting data was found in a sample with over 5% of mutant RAS DNA. In this study, although FFPE sections were equally distributed to three reference laboratories, it could not be completely denied a possibility of intratumoral heterogeneity. Bando et al. reported that the results by TheraScreen Kit whose sensitivity seems to be 1–5% were correlated with anti-EGFR therapeutic efficacy more than those by DS (Bando et al., 2011). On the other hand, a recent data suggested that patients with low-frequency (<1%) KRAS mutations may benefit of targeted anti-EGFR therapies (Laurent-Puig et al., 2014). The detection sensitivity of the RASKET KIT would also be at least 1–5%, which means that the detection of each RAS mutation can be thoroughly secured in samples with 5% RAS mutant allele in wild-type RAS genes and some of those alleles could be detected even in case of 1% (Table S4a and S4b). Thus, this kit can provide clinically appropriate detection of RAS mutations, although not being able to quantify the mutations can be a limitation in using this kit to screen the KRAS mutations for metastatic CRC patients. Further investigation will be needed to clarify the most appropriate detection sensitivity of RAS mutation as a companion diagnostics prior to anti-EGFR therapies.