Fumagalli et al. BMC Cancer 2010, 10:101 http://www.biomedcentral.com/1471-2407/10/101 Page 10 of 14 Figure 6 MET mutation is amplified. The proportion and position (blue dashed lines) of mutant and wt alleles are shown. The MET_1 assay detects R970C mutations in MET. Table 3 ColoCarta panel Sequenom’s Assay Name BRAF_15 &16 BRAF_9 HRAS_6* KRAS_1 & 2 KRAS_4 KRAS_5 KRAS_7 KRAS_8 MET_1 MET_2 NRAS_1 NRAS_2 NRAS_3 NRAS_4 NRAS_7 NRAS_8 PIK3CA_1 PIK3CA_3 PIK3CA_5 PIK3CA_6 PIK3CA_7 PIK3CA_8 PIK3CA_9 Amino Acid Change V600E/K/R/L BRAF-D594V HRAS-Q61L G12V/A/D/C/S/R/F KRAS-G13D KRAS-A59T KRAS-Q61L Q61H/Q61H MET-R970C MET-T992I NRAS-G12V NRAS-G12C NRAS-G13V NRAS-G13C NRAS-Q61H NRAS-Q61E PIK3CA-R88Q PIK3CA-C420R PIK3CA-E542K PIK3CA-E545K PIK3CA-Q546K PIK3CA-H701P PIK3CA-H1047R *HRAS_6 was included in panel but occurred in < 0.1% of samples. Primary tumors with KRAS and PIK3CA mutations vary with respect to the frequency of these mutant alleles In the samples with co-occurring mutations, the ratios of KRAS mutation ratio (KRAS mutation peak area/total peak area) to the PIK3CA mutation ratio (PIK3CA mutation peak area/total peak area) was determined. Twenty-two out of 31 samples (71%) had KRAS/PIK3CA ratios above 1.25 (Table 5). PIK3CA mutations were more prevalent in only 2 out of 31 samples. These dif- ferences demonstrate that in a majority of primary tumors with double mutations in KRAS and PIK3CA, the KRAS mutations are more prevalent than the PIK3CA. This unequal distribution of mutant alleles within a tumor may be due to the fact that a majority of the tumor cells have only the KRAS mutation, and cells with a PIK3CA mutation are in the minority, or it could be due to copy number variations in the KRAS and PIK3CA loci. BRAF mutations were correlated with poorly differentiated tumors and with mucinous tumors The frequency of mutations for KRAS, PIK3CA, and BRAF were tested for correlation to the degree of differ- entiation and to the prevalence of mucin in the tumor. BRAF mutations were found in 26.2% of the poorly dif- ferentiated tumors and in 8.2% of the moderate and well differentiated. These frequencies were significantly dif- ferent by Chi square test (p value = 0.001). BRAF muta- tions were also associated with mucinous tumors: BRAF mutations occurred in 28% of grade 3 mucinous tumors (>50% mucinous tumor cells) but in only 9.4% of the non-mucinous tumors (grade 1 and 2). This was signifi- cant by the Chi square test at p value = 0.006. Similar data have been reported previously [25,26]. KRAS and PIK3CA mutations did not correlate with either the degree of differentiation or with prevalence of mucinous cells. Mutation profiling demonstrated a majority of primary and lymph node samples were concordant but differences were detected Lymph node metastases were not routinely collected in C-07 but as a pilot study to determine the feasibility of using lymph nodes for mutation profiling was conducted. We isolated DNA from 39 lymph nodes containing tumor cells and their corresponding primary tumors. These primary and lymph nodes samples were profiled with the entire OncoCarta panel. The majority of lymph nodes and their corresponding primary tumors (89.7%) were concordant. A total of 26 mutations were detected in lymph nodes, including KRAS, BRAF, PIK3CA, and NRAS. Thirty-five out of 39 lymph nodes had identical mutation profiles, but in 4 cases mutations in the primary