BRAF V600 MUTATION ANALYSIS
Label Name: BRAF
Lab Discipline: Molecular Diagnostics
Institution:  Duke University Health System 
EAP ID:  LAB6125 
Last Review:  3/16/2017 2:47:57 PM
Specimen Type
  Tissue
Container & Volume
  Age Group   Container   Volume  
  0  - 18 Years CHECK WITH LABORATORY 1  ML
Collection Notes
  Adult:
  • Formalin Fixed Paraffin Embedded Tissue: The laboratory can receive either a paraffin embedded tissue block or four freshly cut (within one week)5uM thick unstained slides containing 3 to 20 square mm of tissue. Unstained slides should be accompanied by an H&E stained slide for histologic evaluation.

    Fine Needle Aspirate (FNA) of thyroid: After cytological examination, the residual sample in preservcyt solution (Cytyc corporation, Boxborough, MA) can be sent to the molecular diagnostics laboratory.
 
Transport
  Formalin fixed paraffin embedded tissue blocks, slides and FNA specimen in preservcyt solution can be send to the lab at ambient temperature.

Turn Around Time -  Routine: 10 days   Stat: N/A
Reference Values
BRAF
No BRAF V600E mutation detected (normal allele only).
Methodology
  This assay uses a taqman real-time PCR based technique to detect the presence of the p.V600E (c.1799T>A) mutation and the p.V600K (c.1798_1799GT>AA) mutation in the BRAF gene. An H&E stained slide for each case is first evaluated to identify the regions of greatest tumor content. These regions are then macro-dissected from adjacent unstained formalin-fixed paraffin-embedded sections and used to prepare genomic DNA. A two real-time PCR reactions are performed using a oligonucleotide primer pairs that amplifies both the mutant and wild-type BRAF alleles and allele specific Taqman probes that are fluorescently labeled with VIC-(wild-type BRAF), FAM-(V600E mutant BRAF) or NED- (V600K mutant BRAF). A threshold cycle value (Ct) is measured for each Taqman probe. This represents the PCR cycle at which amplified product is first detected for wild-type BRAF and BRAF V600E or V600K. For each sample, a delta Ct value is calculated as the difference in Ct value between wild-type BRAF and mutant BRAF. 5% BRAF V600E and V600K sensitivity controls, a ~50% BRAF mutant control, and a wild type (normal) BRAF control are included in each assay. Results are interpreted based on the patient delta Ct value relative to the controls. If a BRAF mutation is detected at a level above the 5% control, the sample is interpreted as positive. If a BRAF mutation is detected at a level below the 5% control, the sample is interpreted as indeterminate. If no BRAF mutation is detected, the sample is interpreted as negative. This test is performed using an ABI Prism 7500 Sequence Detection System.

This test was developed and its performance characteristics determined by the DUHS Clinical Molecular Diagnostics Laboratory. It has not been cleared or approved by the U.S. Food and Drug Administration. This test is used for clinical purposes. It should not be regarded as investigational or for research. This laboratory is certified under the Clinical Laboratory Improvement Amendments of 1988 ("CLIA") as qualified to perform high complexity clinical testing.
   
   
Clinical Significance and Interpretive Data
    BACKGROUND:

The BRAF gene on the long arm of chromosome 7 (7q34) is composed of 18 exons. This gene encodes a serine/threonine protein kinase and is part of the RAS-RAF-MAPK pathway which is involved in the transduction of mitogenic signals from the cell membrane to the nucleus. Somatic BRAF gene mutations have been reported in many cancer types. Greater than 95% of reported mutations are a thymine to adenosine transversion at nucleotide 1799 in exon 15, resulting in a valine to glutamic acid change at amino acid 600 (c.1799T>A; p.V600E) . The remaining mutations, which are less well described, are found mostly in exon 15. Rare mutations have been found in exon 11. The BRAF V600E mutation is an activating mutation that results in a constitutively active BRAF serine/threonine kinase activity leading to activation of the MAPK pathway and subsequent regulation of cell proliferation, differentiation and apoptosis. BRAF mutations have been described in the same cancer types as KRAS mutations, however, they are usually mutually exclusive.

BRAF AND COLORECTAL CANCER:

Colorectal cancer (CRC) is one of the leading causes of death worldwide. Approximately 15% of colorectal cancers have high levels of microsatellite instability (MSI). Familial germline mutations in DNA mismatch repair genes (MLH1, MSH2, MSH6 or PMS2) account for 20% of cases with MSI. These inherited mutations cause the tumor syndrome called hereditary non-polyposis colon cancer (HNPCC or Lynch syndrome). In the remaining 80% of cases, MSI is caused by acquired mutations that lead to somatic hyper-methylation of the promoter region in the MLH1 gene. The BRAF V600E missense mutation is extremely rare in colorectal carcinoma arising from germline mutations of the DNA mismatch repair genes (MMR). In addition, BRAF V600E is tightly associated with defective MMR due to hMLH1 promoter hypermethylation. Thus, the presence of BRAF V600E, suggests a sporadic origin of disease, making HNPCC very unlikely. Distinguishing the inherited tumor syndrome cases from the acquired mutation cases is critically important for appropriate follow-up testing, better disease management and counseling of patients

The presence of BRAF mutations can also be used to determine the appropriate therapy for CRC patients. Cetuximab and panitumumab are drugs that are commonly used to treat metastatic colorectal cancer. Cetuximab and panitumumab are monoclonal antibodies that bind to the extra-cellular domain of EGFR (epidermal growth factor receptor) to prevent EGF initiated signaling through the MAPK pathway. Mutations in BRAF bypass this EGFR-initiated signaling cascade, leading to constitutive activity of the signaling pathway. As would be predicted, the presence of BRAF mutations is associated with primary resistance to EGFR-targeted therapies. This has been shown in several retrospective studies and this data is summarized in Mao et al. and Di Nicolantonio et al. Thus, detection of BRAF V600E indicates that therapies which use these monoclonal antibodies to target the EGF receptor may not be sufficient or effective.

BRAF AND PAPILLARY THYROID CANCER (PTC):

Thyroid cancers have a low frequency in the general population, however papillary thyroid cancers account for 75-80% of diagnosed thyroid cancers each year. The BRAF V600E mutation is highly specific to PTC in the context of thyroid lesions. Benign and follicular neoplasms do not exhibit BRAF mutations. BRAF mutations are found in 29-83% of PTC cases. Papillary thyroid cancers can be subdivided into tall cell PTC (77% are BRAF+), conventional PTC (60% are BRAF+) and follicular variant PTC (12% are BRAF+). PTC presents as painless, palpable, solitary thyroid nodules. Thyroid nodules are found in 4-7% of U.S. adults, with 5% of these nodules being malignant. Fine needle aspirates (FNA) are used to evaluate thyroid nodules cytologically: 10-30% of these nodules are deemed indeterminate, and approximately 7% of the indeterminate cases are found malignant. Approximately 300,000 thyroid nodules are detected annually in the U.S. and cytologically 90,000 cases would be called indeterminate. BRAF molecular testing would provide a definitive positive result in 7,200 of these indeterminate cases. Thus, BRAF mutation testing can be used as a highly specific test to determine which indeterminate cases could be brought with confidence directly to surgical resection, and which cases might benefit from an additional diagnostic procedure prior to partial or total thyroidectomy.

BRAF AND MELANOMA:

For melanoma patients, The FDA approved cobas® 4800 BRAF p.V600 Mutation Assay is used. This assay utilizes real-time PCR and the cobas 480z analyzer to detect the presence of p.V600 mutations in the BRAF gene. An H&E stained slide for each case is first evaluated to identify the regions of greatest tumor content. These regions are then macro-dissected from adjacent unstained formalin-fixed paraffin-embedded sections and used to prepare genomic DNA. A single real time PCR reaction is performed using an oligonucleotide primer pair that amplifies both the mutant and wild-type BRAF alleles and allele specific probes that are fluorescently labeled with HEX- or FAM-. If a BRAF p.V600 mutation is detected, the result is interpreted as positive. If no BRAF p.V600 mutation is detected, the result is interpreted as negative.

The BRAF p.V600E (c.1799T>A) AND p.V600K (c.1798_1799GT>AA) mutations are acquired activating mutations that results in constitutively active BRAF serine/threonine kinase activity leading to activation of the MAPK pathway and subsequent regulation of cellular proliferation, differentiation and apoptosis. Approximately 50-60% of melanoma cases contain a mutation in the BRAF gene. The V600E (c.1799T>A) missense mutation and the V600K (c.1798_1799GT>AA) mutation account for 80-90% and 6-16% of these mutations, respectively.

A recent phase 3 clinical trial has demonstrated that patients with metastatic melanoma that harbor the V600E BRAF mutation show better overall survival and better progression free survival when treated with vemurafenib a BRAF inhibitor when compared to similar patients treated with the alkylating agent dacarbazine. Even though there is only scarce data available, melanoma patients with the BRAF V600K mutation may respond to these inhibitors in a way that is similar to patients harboring V600E BRAF mutation. Thus, BRAF mutation analysis may be useful in determining which patients with metastatic melanoma may benefit from BRAF targeted therapies. Multiple factors including clinical findings and the results of other laboratory tests contribute to utility, efficacy and appropriateness of specific chemotherapeutic agents in the treating melanoma patients. Thus, this test is intended for use as an aid in making individualized patient treatment decisions and is not a substitute for a physician's judgment and clinical experience.

REFERENCES:

Atlas of Genetics and Cytogenetics in Oncology and Haematology. URL http://AtlasGeneticsOncology.org.

Benlloch S, Payá A, Alenda C, Bessa X, Andreu M, Jover R, Castells A, Llor X, Aranda I and Massutí B. Detection of BRAF V600E Mutation in Colorectal Cancer. Journal of Molecular Diagnostics. November. 2006;8(5):540-3.

Bollag G et al., Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma. Nature. 2010 Sep 30;467(7315):596-9.

Chapman PB et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med. 2011; 364(26): 2507-2516.

Davies H, Bignell G, Cox C, Stephens P et al. Mutations of the BRAF Gene in Human Cancer. Nature. 2002; 417:949-954.

Di Nicolantonio F, Martini M, Molinari F, Sartore-Bianchi A, Arena S, Saletti P, De Dosso S, Mazzucchelli L, Frattini M, Siena S and Bardelli A. Wild Type BRAF is Required for Response to Panitumumab or Cetuximab in Metastatic Colorectal Cancer. Journal of Clinical Oncology. 2008; 26:1-10.

Ellerhorst JA et al. Clinical correlates of NRAS and BRAF mutations in primary human melanoma. Clin Cancer Res. 2011; 17(2):229-235.

Flaherty KT et al., Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med. 2010 Aug 26;363(9):809-19.

Jin L, Sebo T, Nakamur N et al. BRAF Mutation Analysis in Fine Needle Aspiration (FNA) Cytology of the Thyroid. Diagn Mol Pathol. 2006; 15:136-143.

Kimura E, Nikiforova M, Zhu Z, Knauf J, Nikiforov Y and Fagin J. High Prevalence of BRAF Mutations in Thyroid Cancer: Genetic Evidence for Constitutive Activation of the RET/PTC-RAS-RAF Signaling Pathway in Papillary Thyroid Carcinoma. Cancer Research. 2003; 63:1454-1457.

Long GV et al. Prognostic and clinicopathologic associations of oncogenic BRAF in metastatic melanoma. J Clin Oncol. 2011 Apr 1; 29(10):1239-46. Epub 2011 Feb 22.

Mao C, Liao RY, Chen Q. BRAF mutation predicts resistance to anti-EGFR monoclonal antibodies in wild-type KRAS metastatic colorectal cancer. J Cancer Res Clin Oncol. 2010 Aug;136(8):1293-4.

Minoo P, Moyer M and Jass J. Role of BRAF V600E in the Serrated Pathway of Colorectal Tumourigenesis. Journal of Pathology. 2007; 212:124-133.

Pollock P and Meltzer P. Lucky Draw in the Gene Raffle. Nature. 2002; 417:906-907.

Preto A, Figueiredo J, Velho S, Ribeiro A, Soares P, Oliveira C and Seruca R. BRAF Provides Proliferation and Survival Signals in MSI Colorectal Carcinoma Cells Displaying BRAF V600E but not KRAS Mutations. Journal of Pathology. 2008; 214:320-327.

Rajagopalan H, Bardelli A, Lengauer C, Kinzler K, Vogelstein B and Velculescu. RAF/RAS Oncogenes and Mismatch-Repair Status. Nature 2002; 418: 934.

Ribas A and Flaherty KT. BRAF targeted therapy changes the treatment paradigm in melanoma. Nat Rev Clin Oncol. 2011; 8(7): 426-433.

Rowe L, Bentz B and Bentz J. Utility of BRAF V600E Mutation Detection in Cytologically Indeterminate Thyroid NoduLes. CytoJournal. 2006; 3:10.

Rubinstein JC 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 Jul 14; 8:67.

Troncone G, Russo M, Malapelle U, Accardo M, Ferraro A, Cozzolino I and Palombini L. Cytological and MolecμLar Diagnosis of Solid Variant of Papillary Thyroid Cancinoma: A Case Report. Cytojournal. 2008 5:2.

Vandrovcova J, Lagerstedt-Robinsson K, Pĺhlman L and Lindblom A. Somatic BRAF V600E Mutations in Familial Colorectal Cancer. Cancer Epidemiology, Biomarkers and Prevention. 2006; 15:2270-2273.

Xing M, Westra W, Tufano R, Cohen Y et al. BRAF Mutation Predicts a Poorer Clinical Prognosis for Papillary Thyroid Cancer. Journal of Clinical Endocrinology and Metabolism. 2005; 90(12):6373-6379.

Xing M. BRAF Mutation in Thyroid Cancer. Endocrine-related Cancer. 2005; 12:245-262.

Xing, Mingzhao. BRAF Mutation in Papillary Thyroid Cancer: Pathogenic Role, MolecuLar Bases, and Clinical Implications. Endocrine Reviews 2007; 28(7):742-762.

   
Indications
    Colorectal cancer:

1. Retrospective studies in mCRC patients have shown that EGFR-based antibody therapies (cetuximab and panitumumab) have little to no efficacy if the tumor harbors the BRAF V600E mutation.

2. The BRAF V600E missense mutation is very rare in colorectal carcinoma arising from germline mutations in genes involved DNA mismatch repair genes (MMR). Thus, the presence of BRAF V600E in a tumor with a mismatch repair defect (microsatellite instability), suggests a sporadic origin of colon cancer, rather than inherited (heridatry non-polyposis colon cancer). Distinguishing the inherited tumor syndrome cases from the acquired mutation cases is critically important for appropriate follow-up testing (MMR gene sequencing), better disease management and counseling of patients

Thyroid Cancer:

In the context of thyroid lesions, the BRAF V600E mutation is highly specific to papillary carcinoma of the thyroid (PTC). Benign and follicular neoplasms do not exhibit BRAF mutations. On the other hand, BRAF mutations are found in 29-83% of PTC cases. Thus, BRAF mutation testing can be used as a highly specific test to determine which indeterminate cases could be brought with confidence directly to surgical resection, and which cases might benefit from an additional diagnostic procedure prior to partial or total thyroidectomy.

Melanoma:

Patient's with activating mutations in BRAF have improved survival with the targetted kinase inhibitor vemurafenib when compared to traditional chemotherapeutic treatment approaches.
   
Limitations
    This test detects only the BRAF p.V600E (c.1799T>A) AND p.V600K (c.1798_1799GT>AA) mutations. Other mutations that may occur in the BRAF gene would not be detected using this assay. Mutations or polymorphisms within the BRAF gene other than V600E and V600K may interfere with PCR primer and/or probe binding and subsequent detection of BRAF V600E or V600K. Although rare, this could lead to a false negative result. The analytical sensitivity of this assay is 5%. If cells harboring the BRAF V600E or V600K mutations make up less than 10% in this specimen, they may not be detected by this assay. Poor DNA quality, insufficient DNA quantity or the presence of PCR inhibitors can result in uninterpretable or (rarely) inaccurate results.

This assay is intended for use as an aid in determining patient specific prognosis or diagnosis and is not a substitute for a complete pathologic and clinical evaluation, or physician's judgment and clinical experience.
   
Related Tests
    KRAS TARGETED MUTATION ANALYSIS
    MICROSATELLITE INSTABILITY (MSI) BY PCR
   
Molecular Diagnostics Laboratory
(MDX)

Medical Director:
 Michael Datto, M.D., Ph.D.
 Phone: 919-684-6965
 Email: michael.datto@duke.edu
Lab Director:
 Catherine Rehder Ph.D, FACMG
 Phone: 919-613-8434
 Email: catherine.rehder@duke.edu
Lab Director:
 Siby Sebastian Ph.D., DABMG
 Phone: 919-613-8432
 Email: siby.s@duke.edu

Address: 
 Wadsworth Bldg, Cytogenetics, Rm 0220
 2351 Erwin Rd
 Durham,  NC  27705
 Phone: 919-684-2698
 FAX: 919-668-5424

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