Fumagalli et al. BMC Cancer 2010, 10:101 http://www.biomedcentral.com/1471-2407/10/101 added to the PCR was reduced to 15 ng or less. DNAs were amplified using the OncoCarta PCR primer pools, unincorporated nucleotides were inactivated by shrimp alkaline phosphatase (SAP), and a single base extension reaction was performed using extension primers that hybridize immediately adjacent to the mutations and a custom mixture of nucleotides. Salts were removed by the addition of a cation exchange resin. Multiplexed reactions were spotted onto the SpectroChipII, and mutations, if present, were resolved by MALDI-TOF on the Compact Mass Spectrometer (Sequenom, San Diego, CA). The OncoCarta™ Panel v1.0 (Sequenom, San Diego, CA) consists of 24 pools of primer pairs and 24 pools of extension primers, and has the capacity to detect 238 mutations in 19 genes, listed in Table 1. Each pool con- sists of 5-9 primer pairs in the PCR reaction. Two types of assays have been designed in the OncoCarta panel, referred to as simple and complex. The simple assays are those in which a single assay is able to detect the amino acid changes at that codon. The complex assays are those that require more than one assay to identify codon changes or deletions and insertion, and thus are able to detect multiple different amino acid substitutions or deletions. An example of a complex assay is KRAS_1 and KRAS_2, which interrogate 2 different nucleotide positions within codon 12 and together identify all codon 12 amino acid changes. Much more complex assays are included in OncoCarta, which interrogate insertions and deletions within the EGFR gene. Data analysis Data analysis was performed using MassArray Typer Ana- lyzer software 4.0.4.20 (Sequenom), which facilitates visua- lization of data patterns as well as the raw spectra. Mutations were identified in two different ways. Typer automates the identification of mutants by comparing ratios of the wild type peak to that of all suspected mutants and generates an Onco Mutation report detailing specific mutations and the ratios of wild type and muta- tion peaks. In addition, raw data was exported to Excel and an in-house macro was used to duplicate the analysis. The area under the peaks allows for quantification for each allele, giving a direct evaluation of the proportion of mutated and wildtype (wt) allele in the sample [18]. All mutations from both the Onco mutation report and the in-house Excel report were reviewed manually by 3 investigators (DF, PGG, KPG). Manual review of mutations was necessary to identify “real” mutant peaks from salt peaks or other background peaks. Selected reviewed mutations from the Onco Mutation Report and from the in-house macro were compared and were concordant. Page 4 of 14 Results Mutations were detected in control DNAs from intact and FFPETsamples Previously described mutations in control cell lines were detected. BRAF_V600E, HRAS_G12D, NRAS_Q61L, PIK3CA_E545K, KRAS_G13D, NRAS_Q61K, EGFR1_S125L, and PIK3CA_H1047R were detected in the appropriate cell lines (A2058, HS578T, HL60, MCF7, MDAMB231, NCI-H1299, NCI-H1395, and UACC-893, respectively). The appropriate mutation was found in MCF-7 (PIK3CA_E545K) from both intact DNA and DNA isolated from FFPET. DNAs from clini- cal samples, control cell lines, and cell lines formalin- fixed, paraffin-embedded cell lines showed the same rates of primer extension and performance on mass spectrometer. The proportion of the mutated alleles in each cell line, as observed from the area under the mutant peak on the spectra, ranged from 0.4-0.6, as expected for a pure clonal population with a heterozygote mutation. Spectra for cell line UACC-893 had equal fractions of mutant and wt alleles (Fig. 2A). One exception to this distribu- tion among cell lines was seen in A2058, which showed spectra consistent with 2 copies of the WT allele and one mutant BRAF mutant allele (Fig. 2B). The 3 alleles of BRAF in A2058 are consistent with the observation that there are 3 copies of chromosome 7 in this cell line (COSMIC in the SNP Array Based LOH and Copy Number Analysis data base) [21]. The Sequenom platform was sensitive and quantitative Pilot studies demonstrated that the assays worked with as little as 1 ng of DNA (Fig. 3). The fraction of unex- tended primer was .09 even when the input DNA was between 1-3 ng, When concentrations of the amount of DNA was between 3-14 ng, the fraction of unextended primer was similar, .07. Thus, the assays worked well even when only 1 ng of DNA was used. In clinical samples with some assays it was possible to detect mutations that only represented 5% of the total 2 peak areas. The spectra in Fig. 4 show a small but clear peak at the expected size for a PIK3CA 1047R mutation in a lymph node. We also were able to demonstrate the sensitivity of the platform by performing a cell mixing experiment. Mutation analysis was done using MCF-7 cell line DNA alone or mixed with SKBR3 at various percentages. MCF-7 cells contain a PIK3CA mutation, and SKBR3 cells do not. Fig. 5 demonstrates that the mutation was detectable even when the MCF-7 cells represented only 5 to 10% of the total DNA and only 5 to 2.5% of the alleles. This sensitivity is important for mutation detection in clinical cancer samples, which usually contain some amount of normal tissue, which