Fumagalli et al. BMC Cancer 2010, 10:101 http://www.biomedcentral.com/1471-2407/10/101 Table 4 Single and double mutations in C-07 Page 11 of 14 Single 43.70% 20.10% 11.80% 3.30% 3.80% 60.20% Actual 10.40% 0 1.67% 0 KRAS Double Mutation Frequencies PIK3CA Expected 8.70% 5.10% 1.44% 1.66% Actual 10.40% 1.80% 0 0.42% Expected 8.70% 2.40% 0.66% 0.40% Actual 14.60% 15.50% 2.50% 1.67% 1.30% All other Expected 7.21% 8.06% 5.71% 2% 2.14% KRAS PIK3CA BRAF MET NRAS All Mutations No of Samples 22 7 2 Average Median Table 5 KRAS/PIK3CA ratio mutation frequencies within primary tumors tumors were not found in the corresponding lymph nodes (BRAF [2], PIK3CA [1] and KRAS [1]). Mutation profiles demonstrate that tumor cell populations may be different in lymph nodes and in the primary tumors Peak area evaluation of tumors that had 2 mutations and for which a metastatic lymph node was available demonstrated differences between the primary and lymph node samples. Table 6 details the frequency of mutant and wt alleles based on the peak areas for 5 such samples. KRAS to PIK3CA ratios demonstrated that there were more KRAS mutations than PIK3CA mutations in 4 of 4 primary samples, and in 3 of the 4 lymph node samples. However, it is also notable that the ratio of KRAS/ PIK3A was lower in the lymph node compared to their Sample C07-0388 C07-0717 C07-0940 C07-2244 C07-1837 Mutations M1. KRAS-G12D M2. PIK3CA-H1047R M1. KRAS-G13D M2. PIK3CA-H1047R M1. KRAS-G12D M2. PIK3CA-E542K M1. KRAS-G12C M2. PIK3CA-H1047R M1. BRAF-V600E M2. PIK3CA-H1047R KRAS/PIK3CA 1.25-3.22 0.93-1.13 0.81-.42 1.67 1.6 0.45 0.24 0.08 0.07 0.42 0.22 0.37 0.21 0.2 0.12 Table 6 Allele frequencies for primary tumors with two mutations and metastatic lymph nodes Mutant Allele Frequency Primary Lymph Node Mutation Ratios Primary M1/M2 Lymph node M1/M2 primary tumor in 3 out of 4 samples. In sample 0940, the KRAS/PIK3CA mutation decreased by almost 1/2 in the lymph node tumor compared to the primary. Thus, in these samples there is either a loss of KRAS muta- tions or an accumulation of PIK3CA mutations, suggest- ing that PIK3CA mutations may impart a selective advantage in the lymph node. In contrast, two other samples have a less frequent occurrence of their PIK3CA mutation in the lymph node than in the primary tumor. In sample 2244, the PIK3CA mutation was undetectable in the lymph node (Table 6). In fact, if there was a selection for both mutations in the lymph node, then the PIK3CA mutation frequency would have been the same as that of the KRAS mutation (0.15). On the other hand, if the PIK3CA/KRAS ratio were the same in the primary and lymph node tumor, then the PIK3CA mutation frequency would have been .08, which is still detectable with this technology (Fig. 3). Thus, in sample 2244 there were fewer PIK3CA mutant alleles in the lymph node than in the primary tumor. In sample 1837, mutations in both BRAF and PIK3CA were detected and the BRAF/PIK3CA ratio was 1.67, but increased to 4.4 in the metastatic lymph node. Discussion The Sequenom platform provides a superior technology for the screening of many hot spot mutations in cancer 0.44 0.27 0.09 0.1 0.34 0.31 0.15 0 0.22 0.05 1.88 1.14 1.91 1.76 1.67 1.63 0.90 1.10 _ 4.40