BRAF sequencing

The BRAF gene provides instructions for making a protein, called B-Raf, that helps transmit chemical signals from outside the cell to the cell's nucleus. This protein is part of a signaling pathway known as the RAS/MAPK pathway, which controls several important cell functions. Specifically, the RAS/MAPK pathway regulates the growth and division (proliferation) of cells, the process by which cells mature to carry out specific functions (differentiation), cell movement (migration), and the self-destruction of cells (apoptosis). Chemical signaling through this pathway is essential for normal development before birth.

The BRAF gene belongs to a class of genes known as oncogenes. When mutated, oncogenes have the potential to cause normal cells to become cancerous.

Indications of Test

Tests Available:

  • BRAF V600E by real-time PCR
  • BRAF V600E mutation only by Sanger sequencing
  • BRAF full gene sequence analysis

Clinical Use:

Determine mutations in the BRAF gene.

Clinical Background

BRAF is a member of the mitogen-activated protein/extracellular signal-regulated (MAP/ERK) kinase pathway, which plays a role in cell proliferation and differentiation. Deregulation of this pathway is a key factor in tumor progression. Targeted therapies directed to components of this pathway have demonstrated some success with increases both in progression-free and overall survival in patients with certain tumors. Effectiveness of these therapies, however, depends in part on the mutation status of the pathway components.

Malignant melanoma, one of the most aggressive forms of skin cancer, has a high frequency of BRAF mutation. Approximately 44% to 70% of melanoma cases have a BRAF mutation, and of those, approximately 50% to 90% are the V600E mutation. Current data suggest that the efficacy of BRAF-targeted therapies in melanoma is confined to patients with tumors with activating BRAF mutations, such as V600E, which leads to increased activation of the kinase pathway. While this test was designed to evaluate for the V600E alteration, cross-reactivity with other alterations at the V600 codon have been described. BRAF V600 mutations have also a clinical utility in that it is a predictor of response to anti-mutant BRAF therapy. Inhibitors of B-Raf, and downstream MEK kinase inhibitors, have been developed as increasingly more effective combination cancer therapeutics.

Currently, BRAF mutation status is the only biomarker that predicts a therapeutic response in advanced melanoma.

If the patient has positive result: The mutations that activate BRAF can predict response to treatment, such as selective inhibitors of BRAF (e.g. vemurafenib and dabrafenib) and inhibitors of the downstream MEK kinase (e.g. trametinib and cobimetinib).

Testing whether BRAF mutation exists in melanoma is of critical therapeutic importance. Full sequencing of BRAF may be indicated as a follow-up test in patients with malignant melanoma and normal BRAF V600E test results, in order to identify additional BRAF mutations that may respond to specific therapy. Mutations in other genes including CTNNB1, GNA11, KIT, MAP2K and NRAS, as well as TERT promoter are also non-inherited causes of malignant melanoma, and additional genetic testing may be warranted in order to optimize treatment or modify prognosis.

Molecular testing for BRAF mutations in patients with advanced melanoma is currently a recommended standard of care for determining the course of therapy. Testing is recommended by the current National Comprehensive Cancer Network (NCCN)1) and European Society for Medical Oncology guidelines for melanoma2).

Compared to BRAF wild-type (WT) melanoma, BRAF-mutated melanoma tends to be more aggressive, occur in younger patients, are more likely to metastasize, and are historically linked to shorter overall survival. BRAF mutation occurs in ~40–60% of advanced melanoma cases, of which the V600 mutations account for more than 99%.

Papillary Thyroid cancer: The BRAF V600E mutation is present in 18-87% of papillary thyroid cancers. It is associated with an aggressive tumor phenotype and predicts a poorer disease outcome.

Metastatic colorectal cancer: The BRAF V600E mutation is found in approximately 14% of metastatic colorectal cancer (mCRC). At least one-half of BRAF mutations associated with mCRC are V600E. The presence of a BRAF mutation in mCRC has been reported to render the tumor resistant to anti-EGFR therapy. Full sequencing of BRAF may be indicated as a follow-up test in patients with mCRC and normal BRAF V600 E test results, in order to rule out other BRAF mutations that could result in drug resistance.

BRAF mutations have been found in many other cancer types, including lung cancer, sarcoma, glioma, ependymoma, non-Hodgkin lymphoma, acute lymphoblastic leukemia, liver cancer, stomach cancer, breast and ovarian cancer, and esophageal cancer, although at a low frequency. The clinical implication and utility of BRAF gene mutation testing in these types of cancers remain to be determined.

Specimen Requirements

Requisition form.

Specimen and Volume: Whole blood- Collect 0.3-5 cc of blood in EDTA or ACD BD Vacutainer tubes guide. Embedded Tissue. Other types of tissue may be accepted (please call 818-789-1033 to verify before sending). Dry blood spot DBS, feces (please call 818-789-1033 to verify before sending), or corporal fluid.

Contact your doctor's office or FirmaLab for buccal swab kits. Buccal Swab Kit Instructions

Temperature: Specimen can be at room temperature and needs to be at the laboratory no more than 72hrs after collection. Do not freeze whole blood .

Turn around time: 5-7 days

Policy: General rejection criteria for specimens received at the laboratory may be developed. Additionally, each test may have specific requirements and shall have specific rejection criteria under the Specimen Requirements of the NCCLS formatted description of the test.

Procedure: Below are general rejection criteria. If the specimen must be rejected based on the following criteria, or specific criteria for the requested test:

  1. The test requisition form has missing information. Requisition must contain the required information including:
    • Requesting party and referring authority, usually a healthcare professional.
    • Patient identifying information - Name or alias if anonymous;
    • Test requested, Clinical indication for testing;
    • Date and time of specimen collection;
    • Type of sample (e.g. whole blood or epithelial cells obtained by buccal swab);
    • All other identifying information (e.g. social security number, telephone number, hospital ID, sex … etc.) is optional, but useful for accurate differentiating between patients with similar names.
  2. Label is unclear;
  3. Blood specimen is from a recipient of bone marrow transplant;
  4. Specimen is inappropriate for test requested;
  5. Blood sample is clotted or hemolyzed;
  6. Incorrect container or Vacutainer used;


  1. Anderson S, Bloom KJ, Vallera DU, et al: Multisite analytic performance studies of a real-time polymerase chain reaction assay for the detection of BRAF V600E mutations in formalin-fixed paraffin-embedded tissue specimens of malignant melanoma. Arch Pathol Lab Med 2012 Feb;136:1-7.
  2. Flaherty KT, Puzanov I, Kim KB, et al: Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med 2010 Aug;363(9):809-819.
  3. Dadu, R., K. Shah, et al. (2014) “Efficacy and Tolerability of Vemurafenib in Patients with BRAF(V600E) -Positive Papillary Thyroid Cancer: M.D. Anderson Cancer Center Off Label Experience.” The Journal of Clinical Endocrinology and Metabolism: jc20142246.
  4. Lade-Keller, J., K.M. Romer, et al. (2013) “Evaluation of BRAF Mutation Testing Methodologies in Formalin-Fixed, Paraffin-Embedded Cutaneous Melanomas.” The Journal of Molecular Diagnostics: JMD 15(1): 70–80.
  5. Satoh, T., A. Smith, et al. (2012) “B-RAF Mutant Alleles Associated with Langerhans Cell Histiocytosis, a Granulomatous Pediatric Disease.” (A.Y.W. Chang, ed.)PLoS ONE 7(4):e33891.
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