Fumagalli et al. BMC Cancer 2010, 10:101 http://www.biomedcentral.com/1471-2407/10/101 Page 8 of 14 Figure 5 Quantification of the sensitivity with a cell line mixing experiment. Spectra of MCF-7 cells (mutant) alone or mixed with SKBR3 cells (WT) are shown. Percents are based on the ng amounts of DNA. This assay detects an E545K mutation in PIK3CA. that FFPET samples can be interrogated with the tech- nology described here and yield accurate data. While most of the specific amino acid mutations mir- ror what is seen on the COSMIC database, some unique colon cancer gene mutations were found, which include ABL1-F359V, AKT1-E17K, MET-R970C, and MET- T992I. Other amino acid changes that were not in the COSMIC database were amino acid changes R88Q, H701P, and C420R in PIK3CA, BRAF-594V/G, and KRAS-Q61R, and several in NRAS, including G12C, G12D, G13R, G13V, Q61H and Q61K (Table 2). MET mutations were found in C0-7 and amplified in sometumors MET mutations were found in 3.3% of C-07 samples. Interestingly, these mutations were not only unexpected in their appearance within the colon cancer population but also the frequency within the samples was unex- pected. In four of the eight samples with MET muta- tions, the mutant alleles were present at 58-70%, suggesting an amplification of the mutant allele or a loss of the wt gene (Fig. 6). Amplification may represent the best explanation, in that amplification of the MET geno- mic region, 7q31, has been observed in the Progenetix CGH Database in 23% of colorectal cancers [22] Sequenom data was reproducible Most of the assays in the OncoCarta panel did not detect mutations or the frequency of mutations was very low (below 1%) in our colon cancer samples. OncoCarta assays interrogate mutations in these 19 genes listed in Table 1. To reduce the cost, time and the amount of DNA required for profiling, only 24 assays, which detected mutations at a frequency of 1% or greater in C-07, were selected, resorted in 6 pools and included in a new panel, termed ColoCarta (Table 3). Mutation profiles of 32 mutant samples with 41 mutations were repeated with the ColoCarta. The mutations detected by the 2 panels (OncoCarta and ColoCarta) were identical, demonstrating the reprodu- cibility of the methodology. Multiple mutation frequencies suggest an order to the acquisition of different mutations A majority of the tumors (64%) contained at least one or more mutations in the following genes: BRAF, KRAS, NRAS, MET, or PIK3CA, and 18% had 2 or more mutations. The most common double mutation was in KRAS and PIK3CA, followed by PIK3CA and BRAF (Table 4). Most samples with PIK3CA mutations (80%) also had mutations in other genes, the most