Real-time PCR were performed on Stratagene Mx3000P PCR machine with the following settings: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The mutant and wild-type alleles were amplified separately, and the levels of each mutation in the sample were calculated by normalizing to standard curves. The mutation ratio was defined as [mutation ratio % = level of mutants/(level of

mutants + level of wild type allele) × 100%]. Statistical analysis Statistical analysis was carried out using SPSS version 16.0 software (SPSS Inc., Chicago, IL, US). Fisher’s exact test was used to analyze whether the different categories had Z-VAD-FMK price different positive rates. Kappa test was used to analyze whether the two sampling regions had consistent p53 activator outcomes. Wilcoxon matched pairs test was used to compare the mutation ratios from the two regions. Two-sided p < 0.05 was considered statistically significant. Results EGFR mutations in primary tumors and metastases Of the 50 cases of NSCLC that had EGFR HKI-272 cell line mutations in primary tumors, exon 19 mutations (in-frame deletions only) were present

in 28 cases (56%), and exon 21 (L858R point mutations only) mutations were detected in 22 cases (44%). Mutations in exon 19 and 21 were mutually exclusive and no multiple mutations were found. Of the metastases samples, 47 were positive for EGFR mutation (94% concordance with the detection in primary tumors), and exon 19 and exon 21 mutations were detected in 26 cases (55%, 93% concordance) and 21 cases (45%, 95% concordance), respectively. Notably, all cases presented the same mutation type in the matching primary and metastatic tumors. EGFR mutation detection and the clinical characteristics were listed in Table 1. Among the 50 subjects, only 3 (6%) had different test results for EGFR mutations in primary tumor and metastases, however, the difference

was RAS p21 protein activator 1 insignificant (P = 0.242) as analyzed by Fisher’s exact test. EGFR mutations at different sites of primary tumors of the same patient We performed quantitative measurement of EGFR mutations at different sites of primary tumors (Table 2). The median mutation deviation for different primary sites (see footnote of Table 2 for the formula of calculation) was 18.3% (with a range of 0.0% ~ 54.3%), indicating that the results of the quantitative measurement of EGFR mutations in different sites of primary tumor in the same patient have a high level of concordance. Table 2 Quantitative measurement of EGFR mutation ratios in 3 primary tumor sites and one metastases of the same patient ID Mutation ratio (%) in different primary tumor sites Mutation ratio (%) of metastases 1 2 3 Median Deviation (%)* E001 85.9 91.1 80.1 85.9 12.8 <10 E002 39.1 25.9 44 39.1 49.8 41 E003 <10 <10 <10 <10 0.0 <10 E004 82.