BRAF Gene Mutation Tests

BRAF gene mutation testing has emerged as an important tool for diagnosis, prognosis, treatment, and predicting patient outcome in response to targeted therapy for multiple cancer types.

The BRAF gene mutation test result is positive (ie, a mutation is present) if V600E is found in the BRAF gene. V600E is the most common gene mutation for the BRAF gene and is the most common mutation tested for in clinical laboratories.

BRAF and melanoma

Approximately 40-60% of cutaneous melanomas carry mutations in the BRAF gene. About 90% of these mutations are found to be V600E. ^[1]

Vemurafenib (PLX4032) is a highly selective and potent inhibitor of BRAF V600E. It has marked antitumor effects against melanoma cell lines with the BRAF V600E mutation but not against cells with wild-type (non-mutated) BRAF. Evidence exists that vemurafenib improves overall and progression-free survival in patients with advanced melanoma with the V600E mutation. ^[2] Thus, establishing whether BRAF mutations exist in melanoma is now of critical therapeutic importance. ^[3]

BRAF and colorectal cancer

BRAF mutation testing will likely be increasingly used in the management of colorectal cancer, as more evidence emerges regarding its usefulness. ^{[4, 5]} Some indication exists that BRAF mutation testing may be used to evaluate the likelihood of having hereditary nonpolyposis colorectal carcinoma (HNPCC), or Lynch syndrome, versus sporadic colorectal cancer. Distinguishing HNPCC from sporadic colon cancer with MSI-H is important because patients with HNPCC and their family members can be offered genetic counseling and provided with opportunities to prevent such second cancers through close surveillance and prophylactic surgery. ^[6]

The presence of microsatellite instability (MSI) is one of the major mechanisms of disease in HNPCC; however, MSI-high (MSI-H) is also found in approximately 12% of sporadic cases of colorectal cancer. ^[7] Full gene sequencing, albeit expensive, can detect all of the defective mismatch repair genes in HNPCC. Pembrolizumab, a PD-1 inhibitor, gained accelerated approval from the FDA in May 2017 for unresectable or metastatic colon cancer that has tested positive for microsatellite instability-high (MSI-H) or deficient mismatch repair (dMMR), and has progressed following treatment with a fluoropyrimidine, oxaliplatin, and irinotecan. It is also approved for any solid tumor that has tested positive for MSI-H or MMR-deficiency in patients who have had prior treatment and have no satisfactory alternative treatment options. ^[8]

Preliminary tests, such as BRAF mutation testing, may be useful in determining which patients are likely to benefit from full gene sequencing. ^[6] Approximately 91% of sporadic colorectal cancers harbor BRAF mutation, whereas BRAF is almost never mutated in colorectal cancers that arise as a consequence of Lynch syndrome. ^[9] Thus, persons with colorectal cancers with the BRAF mutation may be unlikely to have HNPCC, and further testing with full gene sequencing is probably unwarranted.

Some evidence indicates that the presence of BRAF mutation may render colorectal cancer resistant to epidermal growth factor (EGFR)-inhibitors, such as cetuximab and panitumumab. K-ras, which lies upstream of BRAF, is mutated in 30-50% of colorectal cancersand is predictive of unresponsiveness to EGFR-inhibitor targeted therapy. Although not yet standard of practice, there is some suggestion that BRAF mutation testing may be a useful adjunct to k-ras testing, since the presence of mutated BRAF likewise confers resistance to anti-EGFR treatment ^[10]

In addition, the presence of BRAF mutation may possibly serve as a useful prognostic marker, as one study reported its significant association with decreased progression-free and overall survival in colorectal cancer ^[11]

A study by Morris et al found that for the minority of those patients with BRAF V600E–mutated metastatic colorectal cancer who achieve long-term survival, multiple factors seem to play a role in the outcome. The investigators reported, for example, that individuals in the study who survived the longest had a greater prevalence of tumors with MSI-H than did those with the shortest survival (36% vs 4%, respectively), with tumors also tending to have a mucinous histology (44% vs 12%, respectively). Moreover, the prevalence of prior tobacco exposure was greater in the longest-surviving patients (44% vs 16%, respectively). The longest-surviving individuals also tended to be older when metastatic disease was diagnosed (60.2 years vs 55.7 years, respectively). ^[12]

BRAF and thyroid cancer

The V600E BRAF mutation is the most common genetic alteration involved in the most prevalent type of thyroid cancer, papillary thyroid cancer (PTC), with an overall prevalence of 45% ^{[13, 14]}

Some studies indicate that the presence of the BRAF gene mutation may potentially be a putative prognostic marker of papillary thyroid cancer ^{[11, 15]} A systematic review and meta-analysis reported that BRAF mutation was significantly associated with recurrence, lymph node metastases, extrathyroidal extension, and advanced stage in papillary thyroid cancer ^[15]

BRAF gene mutation testing has also been reported as a specific marker for papillary thyroid carcinoma when used in conjunction with fine-needle aspiration biopsy (FNAB). Mutated BRAF has been demonstrated to occur exclusively in papillary thyroid cancer and PTC-derived anaplastic thyroid cancer ^[16] and is virtually absent in follicular, Hurthle cell, medullary carcinomas, and in benign thyroid tumors. ^[11]

BRAF and other cancers

The BRAF mutation may be a potential diagnostic tool to distinguish hairy-cell leukemia (HCL) from other B-cell lymphomas with similar clinical and morphologic features, such as the HCL-variant and splenic marginal zone lymphoma. A study by Tiacci et al demonstrated that BRAF mutation was present in all patients with HCL but not in other peripheral B-cell lymphomas or leukemia, which implicates the potential usefulness of BRAF mutation testing in diagnosis of HCL.

As mentioned, BRAF mutations are found to occur in many other cancer types, such as lung cancer, ^[17] glioma, ependymoma, non-Hodgkin lymphoma, acute lymphoblastic leukemia, liver cancer, stomach cancer, and esophageal cancer, although at a low frequency (1-3%). However, the clinical implication and utility of BRAF gene mutation testing in these types of cancers have yet to be determined.

The test for BRAF mutation can be applied to fresh, frozen or, more commonly, formalin-fixed, paraffin embedded (FFPE) tumor. ^[6] The V600E mutation is the primary mutation tested for commercially. The most commonly used technique to identify BRAF mutation is real-time polymerase chain reaction (PCR).

A more comprehensive evaluation of BRAF genotypes can be accomplished using DNA sequencing methods, including traditional Sanger sequencing or newer methods, such as pyrosequencing. ^[6] Although sequencing is considered the criterion standard, real-time PCR can perform as well at identifying the most common BRAF mutation, V600E, with a reported sensitivity and specificity of 100% in one study. ^[18] Moreover, real-time PCR has the advantages of reduced labor, faster turnaround, and lower cost. ^[18]

Description

The BRAF gene is located on chromosome arm 7q34. It encodes B-raf, a serine-threonine kinase that is part of the Ras-Raf-Mek-Erk-MAPK (mitogen-activated protein kinase) signaling cascade (see the illustration below). ^[19] Activation of this pathway has been implicated in promoting growth, proliferation, and differentiation of cells.

Three functional RAF proteins exist in humans, ARAF, BRAF, and CRAF. ^[3] Among them, BRAF has the highest basal kinase activity, and is the most potent activator of the MAPK pathway. ^[20]

The BRAF gene is composed of 18 exons, and the most common activating mutation is found in exon 15 at nucleotide position 1799, involving a transversion of thymine to adenine. ^[6] This results in a substitution of glutamic acid for valine at position 600, which is designated as V600E. This mutation occurs within the kinase domain of the B-raf protein and is reported to have 10 times more kinase activity than its normal counterpart. ^[1] It therefore acts as an oncogene, promoting cell growth, differentiation, and survival.

More than 30 different BRAF mutations have been identified. ^[21] However, V600E accounts for about 90% of all the BRAF mutations, and is the mutation commonly tested for in clinical laboratories. ^[20]

The BRAF gene mutation is found in many different cancers, with various frequencies reported. The highest incidence is found in malignant melanoma (27–70%), papillary thyroid cancer (36–53%), colorectal cancer (5–22%) and serous ovarian cancer (30%). However, it can also occur at a low frequency (1-3%) in other cancers, including lung cancer, glioma, ependymoma, non-Hodgkin's lymphoma, acute lymphoblastic leukemia, liver cancer, stomach cancer, and esophageal cancer. ^[22]

Indications/Applications

BRAF gene mutation testing has emerged as an important tool for diagnosis, prognosis, treatment, and predicting patient outcome in response to targeted therapy. ^[23]

Evidence indicates that BRAF gene mutation testing has a role in the following:

• Differentiating papillary thyroid cancer from other types of thyroid cancer and benign thyroid nodules

• Differentiating sporadic colorectal cancer with microsatellite instability-high (MSI-H) from hereditary nonpolyposis colorectal carcinoma (HNPCC) or Lynch syndrome

• Differentiating hairy cell leukemia from other B-cell lymphomas with similar clinical and morphologic features

• Determining prognosis, as cancers with BRAF mutation are found to be more aggressive than their counterparts without the mutation, particularly in papillary thyroid carcinoma and colorectal carcinoma

• In treatment decisions, because in patients with advanced colorectal cancer, BRAF mutation testing has been suggested since its presence confers resistance to epidermal growth factor inhibitors (cetuximab and panitumumab), when k-ras is not mutated

• In therapeutic targeting, because the presence of mutated BRAF has been a valuable therapeutic target, particularly in metastatic melanoma

Considerations

Studies continue to be published regarding the usefulness of detecting BRAF in various tumors in clinical practice. However, at present, BRAF mutation testing is only widely accepted for use in targeted therapy for advanced malignant melanoma. As more evidence emerges, BRAF mutation testing may become standard of care in the diagnosis and management of other cancers.