Fumagalli et al. BMC Cancer 2010, 10:101 http://www.biomedcentral.com/1471-2407/10/101 resistance to EGFR antibody based therapies [7-11]. Publications have reported the same phenomenon with BRAF and PIK3CA mutations, although these observa- tions are still not well established [12,13]. The published study suggesting that BRAF mutant tumors were resis- tant to EGFR therapies was a small study [13]. Predic- tive value of PIK3CA mutations remains controversial in that other publications have shown that these mutations have no predictive value [14,15]. These inconsistencies, together with two other factors, have limited the impact of mutation profiling for prognosis and prediction in standard care of colon cancer. A large sample size is required to establish that a gene mutation has a signifi- cant impact for patient prediction or prognosis. Another limitation is that until recently conducting such large studies with the standard sequencing technologies was too time consuming and too expensive to be practical for clinical studies. Moreover, while the high frequency of KRAS, BRAF and PIK3CA mutations in colon cancer is well docu- mented, other potentially important mutations have not been profiled with a large number of clinical samples. Whole genome sequencing of a small number of colon samples demonstrated that somatic cancer mutations consist of a few genes that occur frequently and many more mutations that occur very infrequently in many different genes [16,17]. Mutations in these infrequently mutated genes could have a similar effect or synergize with mutations in KRAS, PIK3CA, and BRAF. Given these considerations, our goal was to find a cost-effective and high throughput methodology that would detect frequent and infrequent cancer mutations genes in a large number of samples. Furthermore, it was essential that the methodology would work with degraded DNAs isolated from FFPET. The mass spectrometric SNP genotyping technology based on the Sequenom platform provided an ideal choice for mutation profiling to address these several criteria. It has been shown to work with small amounts of degraded DNAs (5 ng), and the high multiplexing capacity minimizes the use of irreplaceable clinical sam- ples. In addition, a variety of studies have demonstrated that the sensitivity of mass spectrometric methods exceeds that of traditional Sanger sequencing and is highly concordant with Sanger sequencing, Pyrosequen- cing, and allele-specific PCR [16,18,19]. Furthermore, Sequenom has recently developed the OncoCarta Panel, an oncogene panel that offers a rapid and parallel analy- sis of 238 simple and complex cancer mutations across 19 genes. The OncoCarta panel includes assays for most colon cancer mutations in the clinically relevant genes, BRAF (99%), KRAS (98%), and PIK3CA (78%), and in addition contains assays for other cancer mutations in genes that Page 2 of 14 intersect with the same pathways as that of KRAS, BRAF, and PIK3CA, such as AKT1, EGFR, HRAS, NRAS, MET and others. The frequency of KRAS, PIK3CA, and BRAF mutations in the National Surgical Adjuvant Breast and Bowel Project (NSABP) trial C-07 were simi- lar to the frequencies for colon samples listed in the COSMIC data base. This observation provides evidence that the mutation data obtained with the Sequenom platform is accurate. Our results also demonstrate that a majority of colon cancer samples have aberrant PIK(3) - RAS/RAF network; similar results have been seen pre- viously [6]. Mutations in ABL1 and MET, not previously identified in colon cancer, were identified, and 13 other genes were screened and found not to be mutated in hot spot locations. Furthermore, this study identified the most frequent colon cancer mutations from OncoCarta, pro- viding the necessary information to reduce the number of assays from 187 to 24, creating a smaller, more speci- fic and economical panel, requiring less DNA, and thus conserving precious clinical samples. Methods Clinical samples and histological evaluation Samples used in this study were from NSABP clinical trial C-07. This trial enrolled patients between 02/2000 and 11/2002 to compare oxaliplatin and bolus 5-FU/LV to bolus 5FU/LV alone for resected stage II and III colon cancer [20]. Tissue samples were obtained at sur- gery before the patients had received any treatment and were routinely processed with FFPE. C-07 was approved by an institutional review board, and informed consent was obtained from each subject. A pathologist categor- ized tumors into poor, moderate, well differentiated and signet cells carcinoma, according to the World Health Organization (WHO) criteria. Samples were graded for mucinous character, based on the amount of mucin retained within the tumor (1 = no mucin, 2 = < 50% mucinous volume/total tumor volume, 3 = >50% muci- nous volume/total tumor volume). Only grade 3 tumors were considered mucinous tumors carcinoma, which is in accordance with WHO criteria. DNA isolation DNA was isolated from FFPE tumor blocks collected from patients participating in NSABP clinical trial C-07. FFPE tumor blocks were cut, and sections of the slide containing the most tumor cells were defined by a pathologist and isolated by macrodissection. Genomic DNA was extracted from 4 five μm unstained sections. After attempting several extraction procedures from a variety of manufacturers (Machery Negel, Qiagen, Ambion), it was determined that the Ambion Recover- All™ Total Nucleic Acid isolation kit (Applied Biosystem,