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Solid tumor genotyping has become standard of care for the characterization of proto-oncogene mutational status, which has traditionally been accomplished with Sanger sequencing. However, companion diagnostic assays and comparable laboratory-developed tests are becoming increasingly popular, such as the cobas 4800 BRAF V600 Mutation Test and the INFINITI KRAS-BRAF assay, respectively. This study evaluates and validates the analytical performance of the INFINITI KRAS-BRAF assay and compares concordance of BRAFstatus with two reference assays, the cobas test and Sanger sequencing. DNA extraction from FFPE tissue specimens was performed followed by multiplex PCR amplification and fluorescent label incorporation using allele-specific primer extension. Hybridization to a microarray, signal detection, and analysis were then performed. The limits of detection were determined by testing dilutions of mutant BRAFalleles within wild-type background DNA, and accuracy was calculated based on these results. The INFINITI KRAS-BRAF assay produced 100% concordance with the cobas test and Sanger sequencing and had sensitivity equivalent to the cobas assay. The INFINITI assay is repeatable with at least 95% accuracy in the detection of mutant and wild-type BRAFalleles. These results confirm that the INFINITI KRAS-BRAF assay is comparable to traditional sequencing and the Food and Drug Administration–approved companion diagnostic assay for the detection of BRAFmutations.

Background-A variety of tumor-related DNA has been detected in the plasma and serum of cancer patients, including RAS, the BCL2-IgH transgene, and p53. It is not known whether the APCgene, which is frequently mutated in colorectal cancer (CRC), can be identified in the plasma of CRC patients. Similarly, it is unknown whether another tumor suppressor gene altered in CRC, p53, is detectable in people with precursor lesions to CRC. Materials and Methods-The plasma of 240 colonoscopy patients was tested for mutations at two frequently altered sites (codons 175 and 248). A restriction enzyme-mediated enrichment assay was used to detect these mutants. We also tested tumor tissue from 8 patients with CRC for mutations in the mutation cluster region of the APCgene using direct DNA sequencing. Corresponding plasma from any positive patient was then similarly sequenced. Results-Three plasma p53mutations were identified. Two of these patients had adenomas at biopsy, and 1 had a hyperplastic polyp. All were tested for the same p53mutations, and 1 of the adenomas was positive. One of the 8 CRC patients had a 5-base-pair deletion in the cancer and the same deletion was detectable in that patient's plasma, although at an amount that we estimate at 1-5 of the total APCDNA present. Conclusions-APCmutations are detectable in the plasma of CRC patients. p53mutants are detectable in the plasma of colorectal adenoma patients. These may have important implications for cancer screening and diagnosis in the large intestine and suggest that all malignant and premalignant DNA alterations from the colon are identifiable in the blood.