81 (57%) of the cases and120 samples were wild-type (43%) We the

81 (57%) of the cases and120 samples were wild-type (43%). We then we performed a second independent mutation analysis on all samples selleck products and observed that the results of the BRAF/KRAS and PIK3CA/NRAS assays were completely reproducible. We found 130 KRAS, 32 PIK3CA, 13 BRAF and 6 NRAS mutations. Details of the mutations are given in table 1. Of the 32 PIK3CA mutations 12 were single mutations, 19 occurred together with a KRAS mutation and 1 with a BRAF mutation. The mutation assays were performed in a blinded fashion and the results were compared afterwards with the results of the sequence analysis. There were 14 samples with discrepant results between sequence analysis for KRAS exon 2 (covering codons 12 and 13) and the BRAF/KRAS mutation assay.

As the latter had already been confirmed by an independent assay, the 14 samples were resequenced. In 9 cases, the sequence outcome now appeared identical to the mutation assay result and in 4 cases sequencing was unsuccessful due to poor DNA quality. In the 1 remaining sample (no. 289) sequencing suggested wild-type whereas the mutation assay detected a G12V mutation with the mutant peak being 6% of the wild type peak, suggesting that the tumor was heterogeneous. Figure 3 depicts a concise overview of sequence and mutation assay results. A detailed overview of the results of sequencing and mutation assays is given in Supplementary Table S3. In this table we also included the relative peak height of the mutant peaks compared to the wild type peak as observed in the mutation assays.

Note that this is at best semi-quantitative because of the different absorbances of the fluorescent labels, however, it gives an indication of the relative proportions of mutant and wild-type genes. Wild-type peaks in the assays have a height between 2000�C8000. Peaks (mutant) with a peak height of 100 are always visible. Based on this we estimate that sensitivity is between 1�C5%. This correlates well with the sensitivity calculated from dilution experiments in a similar assay as published previously [13]. Note that in some samples the mutant KRAS peaks were much higher than the peaks representing the wild type allele. We presume that this indicates loss of the wild-type allele. Figure 3 Overview of the resultsobtained by the mutation assays (A) and by sequence analysis (B) in 294 tumor DNA samples from patients with advanced colorectal cancer.

GSK-3 Table 1 Mutations identified with the BRAF/KRAS and PIK3CA/NRAS assays. Costs Comparison Next we compared the costs of both approaches. We observed mutations in all 4 genes, hence we assume that future mutation analysis, be it by sequencing or any other technique, will include the PIK3CA, NRAS and BRAF genes. We calculated the costs for all materials and reagents including those associated with the running of samples on the ABI 3130 XL sequencer. These amounted to � 7.03 for the two mutation assays together and � 59.54 for bi-directional sequencing of the 7 exons. Details are given in Sup

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