The frozen samples which harbored BRAF V600E mutation were not included in this study due to the limited amount of DNA. Statistical Analysis Statistical analysis was carried out for statistical power and �� coefficient tests [26] which were used to compare the occurrence selleck and degree of concordance in the detection of KRAS, BRAF and PIK3CA between DS and AMDS. Results Comparing Mutation Detection Sensitivity between AMDS and DS in the Plasmid DNA Titration Study To evaluate the sensitivity of the AMDS, we used serially diluted plasmid DNAs containing different ratios of mutant and wild type of KRAS, BRAF and PIK3CA genes. When the sample contained less than 25% of mutant DNA, the mutation peak in the DS electropherogram was not able to be distinguished from the background (Figure 2C).

In contrast, AMDS clearly detected KRAS G13D mutations at a 5% level (maximum 0.5%) as shown Figure 2D. Comparing Sensitivity of Mutation Detection between AMDS and DS in the Clinical Performance Study The mutation detection performance was compared between AMDS and DS with two sets of samples; fresh frozen tissues (n=89) and FFPE samples (n=70) from patients (n=153) with CRC. Paired frozen and FFPE samples were available for six patients, and the rest of the samples were from independent patients. The comparison was performed in a double-blinded manner. As shown in Table 1�C3, all KRAS, BRAF and PI3KCA mutations detected by DS in either frozen (total number of mutation, n=41, 46.0%) or FFPE (n=27, 38.5%) samples were also successfully (100%) detected by AMDS.

There were no samples which were determined as mutants in DS while detected as wild-type in AMDS. In the samples detected as wild-types by DS, however, AMDS was able to detect additional mutants in the frozen (n=8, 9.0%) and FFPE (n=6, 8.6%) samples. As shown in Table 4, mutations in both KRAS and PIK3CA were detected by AMDS in 6 patients (6/153, 3.9%). Notably, among these coexisting mutations, all PI3KCA mutations were specifically E545K, while KRAS mutations varied. Table 1 2��2 comparison of KRAS mutations frequency in frozen and FFPE tissue sections (n=159). Table 3 2��2 comparison of PIK3CA mutations frequency in frozen and FFPE tissue sections (n=159). Table 4 Multiple mutations (Frozen and FFPE tissue sections). Table 2 2��2 comparison of BRAF mutations frequency in frozen and FFPE tissue sections (n=159).

Results of DS and AMDS for a discordant example are shown in Figure 2E and 2F. This sample was plausibly determined as a mutant according to DS only with Cilengitide forward primer analysis. Due to the poor sequencing signal in reverse primer analysis, this sample was considered as a wild-type. In contrast, AMDS had a clear mutation signal. All other discordant results (8 in frozen tissue, 6 in FFPE tissues) had very similar patterns (data not shown).