Melanoma BRAF Testing

We are pleased to announce that we now offer BRAF testing to aid in the molecular sub classification of malignant melanoma.  The test is offered through our molecular pathology division, Delta MDx  (www.DeltaMDx.com).

Background
B-Raf is a member of the Raf kinase family of serine/threonine-specific protein kinases. This protein plays a role in regulating the RAF-ERK signaling pathway, which affects cell division, differentiation, and secretion (1).  The gene encoding this protein is known as BRAF.
Acquired mutations in the BRAF gene are the most frequent mutation observed in human melanoma and have been found in 50-80% of nevi and melanomas (2,3,4,5,8).  While BRAF analysis is not diagnostically useful for the distinction between benign and malignant melanocytic lesions, it does identify a subset of malignant melanomas that may show response to specific inhibitors of mutated or overactive B-raf protein.  Mutated BRAF is also associated with a worse prognosis in metastatic disease.

BRAF mutations have also been found in other malignancies including non-Hodgkin lymphoma, colorectal cancer, papillary thyroid carcinoma, and non-small cell lung carcinoma.  Although many different mutations may occur, in 90% of the cases, a Glu for Val substitution at exon 15 or residue 599 (now referred to as V600E) in the activation segment has been found in human cancers (6).  Oncogenic (mutated) BRAF results in an overactive protein that may lead to increased and uncontrolled cell proliferation and resistance to apoptosis (7).  Thus, oncogenic BRAF or the B-raf protein are potential therapeutic targets.

Testing

The test is performed on routinely processed formalin fixed paraffin-embedded tissue and utilizes a multiplex polymerase chain reaction (PCR) approach to targeting the major mutations in exon 15.  Using fluorescent label incorporation and allele specific primer extension (ASPE), this microarray assay has increased sensitivity and specificity (detectable mutations as low as 1%).    All tests are interpreted by experienced molecular pathology technologists under the direction of board certified dermatopathologists and our Director of Molecular Pathology, Mary Lowery Nordberg, PhD.

Clinical Implications

Oncologic pharmaceutical treatment regimens may include specific inhibitors of mutated B-raf protein (8).  One of the first drug candidates to show promising clinical activity is PLX4032 (also known as RG7204 or vemurafenib).  Recent data from phase II and III clinical trials indicate that PLX4032 causes tumor regression and stabilized disease in >50% of advanced melanoma patients harboring BRAF V600E tumors (9,10).  On August 17, 2011 the FDA approved Zelboraf (vemurafenib) (Roche) for the treatment of late-stage metastatic or unresectable melanoma showing the BRAF V600E mutation (11).

Similar to other diagnostic tests, the test is typically reimbursed by government and third party payers.  Thus, your patients should not realize any additional out of pocket costs, apart from the usual co-pays and deductibles, as part of the total pathology charge.  To order the test please call Delta at 318-364-2000.

References:

1.  Wan PT, Garnett MJ, Roe SM, et al. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell. 2004;116:855-867.
2.  Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer. Nature. 2002;417:949-954.
3.  Brose MS, Volpe P, Feldman M, et al. BRAF and RAS mutations in human lung cancer and melanoma. Cancer Res. 2002;62:6997-7000.
4.  Namba H, Nakashima M, Hayashi T, Hayashida N, Maeda S, Rogounovitch TI, Ohtsuru A, Saenko VA, Kanematsu T, Yamashita S. “Clinical implication of hot spot BRAF mutation, V599E, in papillary thyroid cancers”. J. Clin. Endocrinol. Metab. 2003;88 (9): 4393–7.
5.  Pollock PM, Harper UL, Hansen KS, et al. High frequency of BRAF mutations in nevi. Nat Genet. 2003;33:19-20.
6.  Puxeddu E, Moretti S, Elisei R, Romei C, Pascucci R, Martinelli M, Marino C, Avenia N, Rossi ED, Fadda G, Cavaliere A, Ribacchi R, Falorni A, Pontecorvi A, Pacini F, Pinchera A, Santeusanio F. “BRAF(V599E) mutation is the leading genetic event in adult sporadic papillary thyroid carcinomas”. J. Clin. Endocrinol. Metab. 2004;89 (5): 2414–20.
7.  McCubrey JA, Steelman LS, Abrams SL, et al. Roles of the RAF/MEK/ERK and PI3K/PTEN/AKT pathways in malignant transformation and drug resistance. Adv Enzyme Regul. 2006;46:249-279.
8.  King AJ, Patrick DR, Batorsky RS, Ho ML, Do HT, Zhang SY, Kumar R, Rusnak DW, Takle AK, Wilson DM, Hugger E, Wang L, Karreth F, Lougheed JC, Lee J, Chau D, Stout TJ, May EW, Rominger CM, Schaber MD, Luo L, Lakdawala AS, Adams JL, Contractor RG, Smalley KS, Herlyn M, Morrissey MM, Tuveson DA, Huang PS. “Demonstration of a genetic therapeutic index for tumors expressing oncogenic BRAF by the kinase inhibitor SB-590885”. Cancer Res. 2006;66 (23): 11100–5.
9.  Vultur A, Villaneuva J, Herlyn M. Targeting BRAF in advanced melanoma: a first step toward manageable disease.  Clin Cancer Res. 2011;17:1658.
10.  Chapman PB, Hauschild A, Robert C et al. Phase III randomized, open-label, multicenter trial (BRIM3) comparing BRAF inhibitor vemurafenib with dacarbazine (DTIC) in patients with V600E BRAF-mutated melanoma. Paper presented at: 2011 Annual Meeting of the American Society of Clinical Oncology; June 3-7, 2011; Chicago, IL. Abstract LBA4.
11.  FDA News Release, August 17, 2011.