BRAF Mutation Testing (Malignant Melanoma, Colorectal Cancer and Lung carcinoma (NSCLC))

Methodology

Cobas® 4800 BRAF V600 Mutation Test (Roche Diagnostics)

The Cobas® 4800 BRAF V600 Mutation Test is a real-time polymerase chain reaction (PCR)based assay designed to detect the presence of the BRAF V600E mutation in formalin-fixed, paraffin-embedded (FFPE) melanoma tissue. The test has been validated at HistoGeneX to detect BRAF V600 mutations in melanoma, colorectal carcinoma and lung carcinoma (NSCLC) tissue.

The test is based on two major processes: (1) manual extraction to obtain genomic DNA, starting from one or two 5 µm thick sections of FFPE tissue containing at least 25% tumor cells; (2) 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.

The test can detect the BRAF V600E mutation at a 5% mutation level using the input of 5 ng/µl DNA. Cross-reactivity is possible with other, non-V600E mutants V600D, V600E2 and V600K.

Clinical Implication

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 (1-3).

Apart from its predictive value in melanoma, the BRAF V600E mutation also has prognostic value in colorectal cancer and in lung cancer (NSCLC) (4,5,7).

The BRAF V600E mutation is found in about 10% of metastatic colorectal carcinoma and has been associated with the presence of microsatellite instability (6). 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 BRAF V600E mutation is also found in 3-5% of all lung cancers (7). While BRAF-inhibitors have proven to be successful in V600E mutation-positive NSCLC patients, the FDA has approved use of the combination therapy with BRAF- and MEK-inhibitor for NSCLC patients with a V600E mutation based on the results of an international, multicenter, three-cohort, non-randomized, non-comparative, open-label, trial in patients with locally confirmed BRAF V600E mutation-positive metastatic NSCLC.

Specimen Requirements

Acceptable specimens for the assay are formalin-fixed, paraffin-embedded melanoma and colorectal carcinoma tissue specimens with a fixation time of 6-48 hours.

Volume

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

Storage and shipment instructions

Maintain and ship specimens at ambient temperature.

Limitations

Insufficient tumor content may not allow the detection of BRAF mutations (< 25%); 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.

Special requirements

None.

Turn-around-time

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

References

  1. Flaherty KT, Puzanov I, Kim KB et al. Inhibition of mutated, activated BRAF in metastatic NEJM 2010;363(9):809-819.
  2. Rubinstein JC, Sznol M, Pavlick AC et al. Incidence of the V600K mutation among melanoma patients with BRAF mutations, and potential therapeutic response to the specific BRAF inhibitor PLX4032. J Transl Med 2010; 8:67
  3. Chapman PB, Hauschild A, Robert C et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. NEJM 2011;364(26):2507-2516.
  4. Van Cutsem E, Köhne CH, Lang I et al. Cetuximab plus irinotecan, fluorouracil, and leucovorin as first-line treatment for metastatic colorectal cancer: updated analysis of overall survival according to tumor KRAS and BRAF mutation status. J Clin Oncol 2011;29(15):2011-2019.
  5. Douillard JY, Oliner KS, Siena S et al. Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer. NEJM 2013;369(11):1023-1034.
  6. Chen D, Huang JF, Liu K et al. BRAFV600E mutation and its association with clinicopathological features of colorectal cancer: a systematic review and meta-analysis. PloS One 2014;9(3):e90607.
  7. Marchetti A, Felicioni L. et al. Clinical features and outcome of patients with non-small-cell lung cancer harboring BRAF mutations. Journal of clinical oncology 2011 (29): 3574-3579.

Updated on 23 May 2019