BRAF Mutation Testing (malignant melanoma, colorectal cancer)

About 50% of melanomas harbor activating mutations in BRAF. Over 90% of BRAF mutations result in a nucleotide 1799 T>A change leading to a valine-to-glutamic acid substitution at position 600 (V600E). The BRAF V600E mutation causes uncontrolled signaling of the MAPK pathway leading to excessive cell growth and proliferation. Agents targeting activated mutant BRAF (BRAF-inhibitors) have proven to be successful in patients with metastatic melanoma harboring this mutation.

Apart from its predictive value in melanoma, the BRAF V600E mutation also has prognostic value in colorectal cancer.

The BRAF V600E mutation is found in about 10% of metastatic colorectal carcinoma and has been associated with the presence of microsatellite instability. Overall, patients with BRAF mutant CRC have low response rates to conventional therapies and adverse prognosis. While BRAF V600-mutated melanomas are sensitive to BRAF-inhibitors, BRAF V600-mutated CRCs may not be as sensitive. Activation of EGFR in colorectal cancer could explain why colorectal cancers generally have a lower response to BRAF inhibitors. Therefore, it is advised to initiate combination therapy with EGFR- and BRAF-inhibitors.

The Cobas® 4800 BRAF V600 Mutation Test is based on two major processes:

• Manual extraction to obtain genomic DNA, starting from one or two 5 µm thick sections of FFPE tissue containing at least 25% tumor cells.
• PCR amplification of target DNA, using a complementary primer pair and two oligonucleotide probes labeled with different fluorescent dyes. Target DNA is detected by automated real-time PCR technology on the Cobas z 480 Analyzer.

Acceptable specimens for the assay are FFPE melanoma and colorectal carcinoma tissue specimens with a fixation time of 6-48 hours.

One representative paraffin block is preferred. Alternatively, 3 unstained 5 µm thick tissue sections are accepted.

Maintain and ship specimens at ambient temperature.

Insufficient tumor content may not allow the detection of BRAF mutations: 10% of tumor cells is required. Tumor content is evaluated prior to analysis and macrodissection is performed. Fixatives other than formalin or prolonged fixation time may give rise to inadequate results.

5 to 7 business days for respectively slides and paraffin blocks.