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BRAF V600E mutation-specific antibody: A review
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Available online at www.sciencedirect.com
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BRAF V600E mutation-specific antibody: A review Lauren L. Ritterhouse, MD, PhD, Justine A. Barletta, MDn Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St, Boston, Massachusetts 02115
article info
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Keywords:
The significance of BRAF mutations in neoplasia was first recognized in 2002 when mutations
BRAF V600E
were discovered in a broad range of cancers. Numerous subsequent studies expanded our
Immunohistochemistry
understanding of BRAF V600E as a critical diagnostic, prognostic, and predictive biomarker in
Melanoma
many cancers. Additionally, the advent of small-molecule inhibitors of BRAF V600E rendered
Thyroid carcinoma
assessment of BRAF mutation status essential in tumors such as melanoma. In clinical
Colorectal carcinoma
practice, evaluation of BRAF mutation status has routinely been performed by DNA-based assays utilizing polymerase chain reaction (PCR). However, molecular testing is not available at many hospitals since it is time-consuming, expensive, and requires expertise in molecular techniques. The first BRAF V600E-specific antibody was reported in 2011 (clone VE1). A purified version of this antibody as well as a second monoclonal antibody targeted to BRAF V600E is now commercially available. In this review, clinicopathologic characteristics associated with BRAF-mutant tumors will be highlighted, and the prognostic and predictive implications of a BRAF V600E mutation will be discussed with a focus on melanoma, thyroid carcinoma and colorectal carcinoma. Additionally, we will review the correlation between immunohistochemistry and molecular results and deliberate how BRAF immunohistochemistry might be utilized in the evaluation of these tumors. & 2015 Published by Elsevier Inc.
Introduction BRAF (v-raf murine sarcoma viral oncogene homolog B1) is a RAS-regulated serine–threonine kinase and activator of the MAPK signaling cascade. Extracellular signals act via this pathway to regulate cellular proliferation, differentiation, and survival. The importance of BRAF mutations in neoplasia was elucidated in 2002 when mutations were discovered in a broad range of cancers.1 The vast majority of mutations were found to occur in exon 15 with a thymine-to-adenine transversion at nucleotide 1799 (originally designated as nucleotide 1796), leading to a valine to glutamic acid substitution at codon 600 (V600E) (originally designated as V599E), a mutation thought to mimic phosphorylation of the activation site. Heterozygous n
Corresponding author. E-mail address: [email protected] (J.A. Barletta).
http://dx.doi.org/10.1053/j.semdp.2015.02.010 0740-2570/& 2015 Published by Elsevier Inc.
mutations in this codon were shown to significantly increase kinase activity in a RAS-independent manner and have transforming activity. Since 2002, mutations in BRAF have been described in a wide array of benign and malignant neoplasms, including melanoma,1 benign nevi,2 colon adenocarcinoma,3 serrated polyps,4 thyroid carcinoma,5 borderline serous ovarian tumors and low-grade serous carcinoma,6 Langerhans cell histiocytosis,7 several low-grade glioma subtypes as well as papillary craniopharyngioma,8,9 hairy cell leukemia,10 ameloblastoma,11 and metanephric adenoma,12 as well as rare nonsmall cell lung carcinomas,13 biliary tract carcinomas,14 head and neck squamous cell carcinomas,15 gastrointestinal stromal tumors,16 and plasma cell myelomas,17 among others. BRAF V600E and RAS mutations have been shown to be mutually
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exclusive in many of these tumors, suggesting that they have a similar tumorigenic effect.1,3,5,6 In clinical practice, evaluation of BRAF mutation status has routinely been performed by DNA-based assays utilizing polymerase chain reaction (PCR). Direct (Sanger) sequencing was the initial gold standard for BRAF mutation detection. However, a variety of additional DNA-based assays with higher sensitivity have subsequently been employed, including allele-specific PCR, pyrosequencing, and high resolution melting (HRM) analysis.18,19 Genetic analysis can fail as a result of poor DNA quality due to DNA fragmentation that can occur with tissue processing. It can also yield erroneous or artifactual results due to low tumor content.20 Additionally, these molecular methodologies all require DNA extraction, expensive equipment, expertise in molecular techniques, and strict quality control measures. As a result, molecular assays are not available at many hospitals, necessitating the test to be sent out which leads to a delay in results. The recognition of BRAF V600E as a critical diagnostic, prognostic, and predictive biomarker in many cancers, along with the advent of small-molecule inhibitors of BRAF V600E, prompted interest in developing immunohistochemistry (IHC) as a potentially faster, less expensive, more widely available methodology to detect the BRAF V600E protein in formalin-fixed and paraffin-embedded (FFPE) tissue. The first BRAF V600E-specific antibody was reported by Capper et al.21 in 2011. They developed hybridomas by immunizing mice with an 11 amino acid synthetic peptide representing the BRAF V600E-mutated amino acid sequence and fusing lymph node cells from the immunized mice with mouse myeloma SP2/O cells. Over 2000 clones were screened and only one (clone VE1) demonstrated a specific reaction with BRAF V600E by immunofluorescence using BRAF V600E-transiently expressing HEK 293T cells, immunoblotting, and IHC of FFPE samples of human melanoma and papillary thyroid carcinoma samples. IHC demonstrated moderate to strong cytoplasmic staining, consistent with the cytoplasmic localization of the tumor. Many of the IHC studies reviewed herein use either diluted or undiluted hybridoma supernatant. Additionally, a purified version of this antibody is now commercially available from Spring Bioscience and Ventana. A second monoclonal antibody directed against BRAF V600E (clone V600E, anti-B-Raf) is also commercially available from NewEast Biosciences. While an exhaustive cataloging of the implications of BRAF V600E in every cancer type is beyond the scope of this review, various facets will be highlighted including diagnostic potential, clinicopathologic characteristics, prognostic implications, and predictive value of BRAF V600E mutation with a focus on melanoma, thyroid carcinoma and colorectal carcinoma along with a brief summary in other tumor types. Additionally, we will review the correlation between IHC and molecular methodologies for the detection of the mutation and discuss how BRAF IHC might be utilized in the evaluation of these tumors.
Melanoma Mutations in BRAF are present in 40–60% of primary malignant melanomas and roughly half of metastatic melanomas.19,22,23 BRAF mutations are inversely correlated with age, more frequent at locations such as the trunk and lower extremities, and
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less frequently associated with solar elastosis,24 consistent with the finding that BRAF mutations arise in melanomas from skin that is intermittently exposed to sun and correlated with a lower ultraviolet dose.23 Interestingly, the frequency of BRAF mutations in melanomas arising in sites nearly completely protected from sun (i.e., acral and mucosal melanomas) is low.23,24 In melanoma, BRAF mutation seems to be an early event since many nevi harbor BRAF mutations.2 While BRAF mutations in primary melanomas have not been found to impact disease-free or overall survival, there is some indication that BRAF mutations in metastatic melanoma impart a decreased survival rate.25,26 The BRAF V600E mutation is responsible for 80–90% of BRAF mutations in melanoma.19 BRAF V600K is the second most common mutation (seen in 10–15%), and other BRAF mutations are rare.19 Given the dismal prognosis of patients with unresectable or metastatic melanoma and the limited benefit of standard chemotherapy, the advent of smallmolecule BRAF inhibitors revolutionized melanoma treatment. It also rendered BRAF mutation status in metastatic melanoma integral to treatment decisions. The Food and Drug administration (FDA) has approved two BRAF inhibitors, vemurafenib and dabrafenib, for the treatment of unresectable or metastatic melanoma.27 Both specifically inhibit BRAF V600E. Vemurafenib is approved for patients with V600E-mutant melanoma, while dabrafenib is approved in patients with melanomas with either a V600E or V600K mutation. Both of these drugs have an impressive response rate of approximately 50%.28,29 Although the development of resistance has been a limitation, the combination of a BRAF inhibitor along with a MEK inhibitor has recently been shown not only to increase response rate (to nearly 70%) but also to increase progression-free survival.30 The correlation between BRAF V600E IHC and BRAF mutation status assessed by molecular assays has been fairly robust in melanoma (Table).18,21,22,31–40 An example of a BRAF-mutant melanoma stained for BRAF V600E is shown in Fig. A. Reported sensitivities of the VE1 clone range from 85% to 100% and specificities range from 93% to 100%. The V600E clone has also demonstrated fairly good results with melanoma including a sensitivity ranging from 90% to 100% and specificities ranging from 81% to 97%. Several observations are worth noting. In studies with more than one interpreter of IHC, concordance of reviewers was near-perfect.22,36,37 In some cases with initial discordant IHC and molecular results, additional molecular testing confirmed the presence of a BRAF V600E mutation as was originally suggested by IHC.37 Many of the cohorts included non-BRAF V600E mutations (such as V600K), and, with rare exceptions, these tumors were negative for BRAF V600E by IHC.18,41 Tumors in general were shown to lack intratumoral heterogeneity; however, rare cases were described with heterogeneous staining, and rare discordant results between primary and metastatic tumors were reported as well.22,31 Finally, BRAF V600E IHC showed some variation in staining due to tissue characteristics, such as focal negativity in areas of cautery effect and necrosis and reduced staining in tissue first submitted for frozen section analysis or when IHC was performed on older tissue sections.21 Finally, rare cases were reported to demonstrate equivocal IHC results. Colomba et al.33 reported a perfect sensitivity and specificity for BRAF V600E IHC (V600E clone). On the basis of these findings, the authors suggest that IHC could be used as the initial test with
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Table – Correlation between BRAF V600E immunohistochemistry and BRAF mutation status assessed by molecular testing. Study
Tumor
# of cases
Antibody
Sensitivity
Specificity
Boursault et al.22 Busam et al.31 Capper et al.21 Chen et al.32 Colomba et al.33 Feller et al.34 Hofman et al.35 Ihle et al.18 Lade-Keller et al.36 Long et al.37 Pearlstein et al.38 Routhier et al.39
Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma
230 51 47 38 111 35 98 63 28 100 76 31
Skorokhod et al.40 Bullock et al.56 Capper et al.21 Crescenzi et al.57 Fisher et al., 201458 Ghossein et al.46 Ilie et al.59 Kim et al.60 Koperek et al.61 McKelvie et al.62 Routhier et al.39
Melanoma Thyroid Thyroid Thyroid Thyroid Thyroid Thyroid Thyroid Thyroid Thyroid Thyroid
29 96 21 21 41 91 194 91 39 71 23
Zagzag et al.63 Zimmermann et al.64 Adackapara et al.75 Affolter et al.76 Bledsoe et al.77 Capper et al.78 Day et al.65 Ilie et al.59 Kuan et al.80 Lasota et al.81 Routhier et al.39
Thyroid Thyroid Colon Colon Colon Colon Colon Colon Colon Colon Colon
37 48 52 31 204 91 505 310 128 113 32
Rössle et al.82 Sajanti et al.83 Sinicrope et al.84 Thiel et al.85 Toon et al.86
Colon Colon Colon Colon Colon
265 147 75 137 216
VE1 (Spring Bio) VE1 hybridoma VE1 hybridoma V600E (NewEast VE1 hybridoma V600E (NewEast VE1 hybridoma VE1 (Spring Bio) VE1 (Spring Bio) VE1 hybridoma VE1 (Spring Bio) VE1 (Spring Bio) V600E (NewEast VE1 hybridoma VE1 hybridoma VE1 hybridoma VE1 (Spring Bio) VE1 (Spring Bio) VE1 (Spring Bio) VE1 hybridoma VE1 (Spring Bio) VE1 hybridoma VE1 (Spring Bio) VE1 (Spring Bio) V600E (NewEast VE1 (Spring Bio) VE1 (Spring Bio) VE1 (Spring Bio) VE1 (Spring Bio) VE1 (Spring Bio) VE1 (Spring Bio) VE1 hybridoma VE1 (Spring Bio) VE1 (Spring Bio) VE1 (Spring Bio) VE1 (Spring Bio) V600E (NewEast VE1 (Spring Bio) VE1 (Spring Bio) VE1 (Spring Bio) VE1 (hybridoma VE1 hybridoma
97% 100% 100% 100% 100% 100% 96% 100% 93% 97% 85% 90% 90% 86% 100% 100% 100% 100% 100% 99% n/aa 100%b 100% 100% 100% 89% 94% 71% 100% 96% 100% 100% 94% 100% 85% 100% 88% 100% 100% 100% 100% 99%
100% 100% 100% 93% 100% 97% 100% 98% 100% 98% 100% 95% 81% 100% 90% 100% 100% 62% 91% 100% n/aa 100%b 94% 100% 70% 100% 94% 74% 100% 99% 99% 100% 100% 94% 68% 100% 93% 95% 99% 100% 100% 100%
Bio) Bio)
Bio)
Bio)
Bio)
and Spring Bio)
This table includes only studies in which assessment of BRAF immunohistochemistry (IHC) was a main aim of the project and studies in which IHC results could be correlated with molecular results in specific tumor types. a The scoring method in this study precluded calculation of sensitivity and specificity. b These percentages are excluding cases that were considered equivocal on IHC.
pyrosequencing employed on cases that are equivocal or negative by IHC. Based on the very high specificity of BRAF V600E IHC in the studies reviewed, utilizing IHC as a first-line test seems reasonable and would reduce molecular testing by roughly 50%. Testing of negative cases is not only important due to the magnitude of the clinical consequences of missing a tumor harboring a V600E mutation, but also needed to identify tumors with other BRAF mutations such as V600K.
Thyroid carcinoma An activating BRAF mutation is the most common genetic event in thyroid carcinoma, found in roughly 45% of papillary
thyroid carcinomas (PTCs).42,43 The BRAF V600E mutation accounts for the vast majority of these BRAF mutations, with other mutations such as K601E found in only 1–2% of these tumors.44 The rate of BRAF V600E mutation depends on the subtype of PTC. The tall cell variant has the highest mutation frequency (over 90%), the follicular variant of PTC (FVPTC) has the lowest frequency (5–10%), and the mutation rate for classical type PTC falls in the middle, at roughly 55–75%.42,43 Interestingly, the rate of BRAF V600E mutation has increased nearly 30% in classical type PTCs in the last three decades.43 BRAF V600E is also present in rare poorly differentiated thyroid carcinomas and a subset of anaplastic thyroid carcinomas (ATCs) that likely arose from PTC.45,46 BRAF V600E mutations are not present in follicular or medullary thyroid
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Fig – Immunohistochemistry for BRAF V600E (VE1, Spring Biosciences) in BRAF V600E-mutant tumors: (A) Melanoma, (B) papillary thyroid carcinoma, and (C) colon adenocarcinoma.
carcinomas or in benign thyroid tumors.45 Thus, the detection of a BRAF V600E mutation is diagnostic of malignancy in this context. The majority of studies evaluating the clinicopathologic significance of BRAF V600E mutation have shown
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that it is associated with high-risk histopathologic features, such as an increased frequency of extrathyroidal extension, lymph nodes metastases, and higher clinical stage.47 Tumors found to have a BRAF mutation preoperatively have been shown to be associated with central neck lymph node metastases with a positive predictive value of 47% and a negative predictive value of 91%, indicating that knowledge of BRAF status preoperatively could potentially help guide whether a prophylactic central neck dissection should be performed.48,49 BRAF mutations have also been linked to an increased rate of disease recurrence with an odds ratio of roughly 3–5, a positive predictive value of 30%, and a negative predictive value of 90%.50 In a retrospective study of 1849 patients with PTC, Xing51 also found that the presence of a BRAF V600E mutation was significantly associated with increased cancer-related mortality among patients with PTC. Although they found that this association lost significance when other tumor features such as extrathyroidal extension, lymph node metastases, and distant metastases were factored into the analysis, they observed that each of these factors alone has a modest associated mortality, which was significantly increased by a coexisting BRAF mutation. Moreover, they found that conventionally defined low-risk patients with tumors lacking a BRAF V600E mutation had a negligible mortality rate. These findings are significant since they suggests that low-risk patients with tumors that are wild-type for BRAF might be spared post-thyroidectomy radioactive iodine treatment and might require less vigilant surveillance as compared to patients with tumors harboring a BRAF mutation.49 Non-infiltrative follicular variant of papillary thyroid carcinoma has been reported by some groups to lack of the BRAF V600E mutation.52,53 Though the evidence at this point is limited, this finding could potentially be used to support lobectomy only for this subset of tumors. BRAF V600E mutations have also been linked to loss of radioactive iodine uptake and subsequent resistance to radioactive iodine treatment.47 The presence of V600E in aggressive radioactive iodine refractory (RAIR) disease extends a treatment option with a BRAF inhibitor such as vemurafenib. There is some indication that vemurafenib produces a clinical response in patients with BRAF V600E-mutant metastatic PTC and could even have efficacy in the setting of anaplastic thyroid carcinoma harboring a BRAF mutation.54,55 IHC with the VE1 antibody has demonstrated a high concordance rate with molecular methods with sensitivities ranging from 89% to 100% and specificities from 61.5% to 100% in FFPE tissue from PTC specimens (Table).21,39,46,56–64 An example of a PTC harboring a BRAF V600E mutation stained for BRAF V600E is shown in Fig. B. The wide range in specificity is the result of one study by Fisher et al.58 that included cases of medullary thyroid carcinoma and follicular thyroid carcinoma in addition to PTC, as well as a handful of fine-needle aspiration (FNA) cell block specimens. Excluding this study, the specificity ranged from 90% to 100%. While the underlying explanation of the false–positive cases reported by Fisher et al. may be in part the result of their scoring tumors as positive by IHC when as little as 10% of the tumor demonstrated moderate to strong staining, they indicated that several of their false–positive cases demonstrated 480% positivity. Therefore, the explanation in these cases is
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unclear since they report a high tumor volume in specimens submitted for molecular analysis and repeat negative results with additional molecular testing by pyrosequencing. Bullock et al.56 noted that some of their “false–positive” IHC cases were likely secondary to low tumor content yielding a spurious negative result by molecular testing. Additionally, Sanger sequencing (an assay known to have lower sensitivity than other assays such as pyrosequencing and allele-specific PCR) was utilized in several studies and may also have produced spurious false–positive results on IHC, which in turn would lead to an inaccurately low estimation of specificity.59 There are a few additional points to highlight. Similar to the findings in melanoma, interobserver reproducibility was excellent (495%). Also, the vast majority of tumors demonstrated homogenous staining indicating that BRAF V600E mutation is a clonal event in thyroid carcinoma.46 Some cases were reported to show slight variation in staining; however, this appeared attributable to tissue fixation, since negative or weak staining was observed in the center of the tissue section.56 Significantly, Zimmermann et al.64 demonstrated that BRAF V600E IHC performed well on cell blocks from FNA specimens, with IHC demonstrating a 93.8% sensitivity and specificity. And finally, staining must be interpreted with caution. In the study by Ghossein et al.,46 the authors showed that IHC could be used to identify BRAF V600E mutations in anaplastic and poorly differentiated thyroid carcinoma as well as PTC; however, they found that in anaplastic cases with high background staining, moderate staining did not correlate with an underlying BRAF V600E mutation. Kim et al.60 also found that correlation was weaker with an intermediate staining result. These findings indicate that if the staining cannot be readily categorized as negative/ weak or moderate/strong in the majority of a tumor with no background staining (excluding the weak non-specific staining of colloid that was reported in several studies and did not influence results), the stain should be considered equivocal and molecular testing should be performed.
Colon adenocarcinoma Mutations in BRAF occur in approximately 10–15% of colorectal carcinomas (CRCs).65,66 BRAF V600E mutation is the main BRAF mutation that occurs in CRC.66 It is an early mutation (found in serrated polyps) and appears to promote methylation of CpG islands found within promoter regions of many genes, which in turn silences the methylated gene.67 Tumors that have high levels of CpG island methylation are categorized as CIMP-H. Thus, BRAF mutation is strongly associated with a CIMP-H status (though not all CIMP-H tumors harbor a BRAF mutation).67,68 In many BRAF-mutant, CIMP-H cases, the mismatch repair gene MLH1 is silenced by promoter methylation, leading to defective mismatch repair and subsequent microsatellite instability (MSI-H).67 Thus, BRAF mutations are also strongly associated with MSI-H status.66 BRAF mutations virtually never occur in the setting of an MSI-H tumor with an underlying germline mutation in a mismatch repair gene (i.e., Lynch syndrome). This is highly clinically significant, since it means that the identification of a BRAF mutation can be used to exclude Lynch syndrome as
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the underlying cause of mismatch repair protein deficiency.69 BRAF-mutant CRCs are associated with older age, a female predominance, and localization to the right colon.70 Identification of a BRAF mutation in CRC also has prognostic implications. Studies have demonstrated that BRAF-mutant metastatic CRCs are associated with a worse overall survival.65,66,71 In a cohort of metastatic CRCs, Tran et al.66 reported a medial overall survival of 10.4 months for BRAFmutant tumors compared with 34.7 months for tumors wildtype for BRAF. Moreover, they found that BRAF mutation was an independent poor prognostic factor in multivariate analysis with a HR of roughly 10.6. BRAF mutation may also be a predictive biomarker in CRC. BRAF is downstream of the epidermal growth factor receptor (EGFR). Anti-EGFR therapy with agents such as cetuximab has become standard treatment for patients with advanced CRC.72 While results are conflicting, there is some indication that BRAF mutation may correlate with resistance to the anti-EGFR therapy.73 Thus, identification of a BRAF mutation is an established clinical tool to exclude Lynch syndrome, has prognostic value in metastatic CRCs, and may be used to guide treatment decisions. Finally, while BRAF-mutant metastatic CRC appears to be unresponsive to treatment with a singleagent BRAF V600E inhibitor, the lack of response has been shown to be due to feedback activation of EGFR, suggesting that combination therapy with a BRAF V600E inhibitor and an EGFR or MEK inhibitor may yield better results.74 While the concordance between BRAF V600E IHC and molecular detection of a BRAF V600E mutation demonstrates some variability between studies, the overall concordance is strong given the number of cases tested (Table).39,65,75–86 An example of a BRAF-mutant colon adenocarcinoma stained for BRAF V600E is shown in Fig. C. Investigations using the VE1 clone have demonstrated sensitivities ranging from 71% to 100% and specificities ranging from 68% to 100%. There are several points to highlight from these studies. Bledsoe et al.,77 showed that IHC correlated with mutation status even in the setting or prior chemotherapy, radiation, and/or targeted therapy, while Ilie et al.79 demonstrated that IHC was sensitive and specific even with limited tumor in biopsy specimens of pulmonary metastases of CRC. In the one study comparing clones VE1 and V600E in CRC, VE1 performed slightly better.39 In general more BRAF-mutant CRC cases have been reported to show weaker staining as compared to BRAF-mutant thyroid carcinomas and melanomas. Cases with signet-ring-cell morphology were reported to produce both false–positive and negative IHC results.77,82 Also, background staining was reported in nuclei of normal colonic mucosa, smooth muscle, mucin, and nerve bundles.77,79 All of these findings indicate that careful IHC interpretation is required, though interobserver reproducibility was reported in one study as virtually perfect.86 Additionally, the disparate results between studies suggest that methodological differences such as the antigenretrieval protocol, antibody incubation conditions, and automated versus manual staining likely influences results especially in the setting of CRC samples. As Kuan et al.80 indicated, these findings demonstrate that rigorous antibody optimization is required. Moreover, extensive “in-house” validation needs to be performed before BRAF V600E staining is put into clinical practice, and any cases with somewhat
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Other tumors Several primary CNS neoplasms have been found to harbor a BRAF V600E mutation. A BRAF V600E is seen in several pediatric low-grade gliomas.87 For example, a BRAF V600E mutation has been reported in approximately 60% of pleomorphic xanthoastrocytomas (PXA), 20–60% of gangliogliomas (GG), and roughly 10% of pilocytic astrocytomas (predominantly those with an extra-cerebellar location).8,9 Thus, BRAF V600E may serve as an ancillary diagnostic tool when evaluating these tumors. For example, the histologic differential of PXA includes glioblastoma (GBM). Because the vast majority of standard GBMs lack a BRAF V600E mutation, the presence of this mutation could be used to help exclude GBM.8,9 Moreover, with trials with BRAF V600E inhibitors currently underway in patients with these tumors, identification of a BRAF V600E mutation is essential. Recently, Brastianos et al.88 demonstrated that the presence of a BRAF V600E mutation distinguishes papillary craniopharyngioma from adamantinomatous craniopharyngioma. Papillary tumors have a BRAF V600E mutation frequency of 95%, while adamantinomatous tumors lack BRAF mutations but instead virtually always harbor a CTNNB1 mutation. Again, this finding indicates that BRAF V600E IHC would be valuable diagnostically and suggests BRAF inhibitors as a rational treatment option for papillary craniopharyngiomas. High concordance rates between BRAF V600E IHC and mutation status have been reported in primary CNS neoplasms. For example, Ida et al.89 demonstrated that BRAF V600E IHC (VE1 clone) showed perfect sensitivity and specificity in PXAs, and Brastianos et al.88 showed the same result (again with VE1) in craniopharyngiomas. Among hematologic malignancies, a BRAF V600E mutation is present in virtually all cases of hairy cell leukemia (HCL) and approximately 60% of cases of Langerhans cell histiocytosis (LCH).7,10 The identification of a BRAF V600E mutation is essentially diagnostic of HCL, since it is extraordinarily rare in other lymphoproliferative disorders (4% of plasma cell myelomas harbor a BRAF V600E mutation), and the presence of a V600E mutation suggests that BRAF inhibitors may hold promise for patients with HCL.10 Andrulis et al.90 demonstrated that detection of BRAF V600E by IHC (VE1 was used) showed 100% sensitivity and specificity in HCL. Of note, their cohort of 30 HCL included many bone marrow specimens that were subjected to decalcification in EDTA. While some of the cases showed only weak staining when the Ventana ultraView detection system was used, staining was stronger and background was reduced when the Ventana OptiView detection system was employed (a system that is optimized for the detection of low-expressing antigens), again highlighting the importance of optimization of IHC staining protocols. BRAF mutations are increasingly being reported in a variety of other tumor types, with many of those tested by IHC showing robust concordance with molecular methods of detection. IHC for BRAF V600E has been shown to have a close to perfect concordance in ameloblastoma, a rare benign
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but locally aggressive odontogenic neoplasm with a high frequency of BRAF V600E mutation (approximately 60%).11,91 Metanephric adenomas (MA) are rare benign renal tumors that can mimic renal malignancies such as papillary renal cell carcinoma and have a BRAF V600E mutation frequency of 90%.12 IHC for BRAF V600E (clone VE1) has been shown to correlate perfectly with BRAF mutation status; moreover, the presence of BRAF V600E expression along with histologic impression is virtually diagnostic of metanephric adenoma.92 BRAF V600E IHC has also been shown to strongly correlate with molecular results in serous ovarian tumors.93 The BRAF V600E mutation is present in borderline serous ovarian tumors and low-grade serous ovarian carcinoma, demonstrating that these tumors are genetically distinct from high grade serous tumors which virtually never have BRAF mutations.6 Ilie et al.13 reported the presence of BRAF mutations in 9% of lung adenocarcinomas that were wild-type for EGFR, KRAS, PI3KA, and HER2 and lacked EML4–ALK: 4% were BRAF V600E and 5% non-BRAF V600E mutations. IHC for BRAF V600E was positive in 90% of BRAF V600E-mutant tumors and negative in all tumors harboring non-BRAF V600E mutations. Finally, while the frequency of BRAF mutations in biliary tract cancers is controversial, a recent study demonstrated 100% concordance between BRAF V600E IHC (clone VE1) and direct Sanger sequencing, although only 1% of biliary tract cancers (all intrahepatic cholangiocarcinomas) were positive for BRAF V600E.14
Conclusion The discovery of the BRAF V600E mutation has significantly increased our understanding of the carcinogenesis of many tumor types and has broad clinical implications. BRAF V600E IHC has already been performed on thousands of tumor specimens; likely, this is just the beginning.
re fe r en ces
1. Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer. Nature. 2002;417(6892):949–954. 2. Pollock PM, Harper UL, Hansen KS, et al. High frequency of BRAF mutations in nevi. Nat Genet. 2003;33(1):19–20. 3. Rajagopalan H, Bardelli A, Lengauer C, et al. Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status. Nature. 2002;418(6901):934. 4. Chan TL, Zhao W, Leung SY, et al. BRAF and KRAS mutations in colorectal hyperplastic polyps and serrated adenomas. Cancer Res. 2003;63(16):4878–4881. 5. Kimura ET, Nikiforova MN, Zhu Z, et al. High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC–RAS–BRAF signaling pathway in papillary thyroid carcinoma. Cancer Res. 2003; 63(7):1454–1457. 6. Singer G, Oldt R, Cohen Y, et al. Mutations in BRAF and KRAS characterize the development of low-grade ovarian serous carcinoma. J Natl Cancer Inst. 2003;95(6):484–486. 7. Badalian-Very G, Vergilio JA, Degar BA, et al. Recurrent BRAF mutations in Langerhans cell histiocytosis. Blood. 2010;116 (11):1919–1923. 8. Dias-Santagata D, Lam Q, Vernovsky K, et al. BRAF V600E mutations are common in pleomorphic xanthoastrocytoma:
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diagnostic and therapeutic implications. PLoS One. 2011;6(3): e17948. Schindler G, Capper D, Meyer J, et al. Analysis of BRAF V600E mutation in 1,320 nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma, ganglioglioma and extra-cerebellar pilocytic astrocytoma. Acta Neuropathol. 2011;121(3):397–405. Tiacci E, Trifonov V, Schiavoni G, et al. BRAF mutations in hairy-cell leukemia. N Engl J Med. 2011;364(24):2305–2315. Kurppa KJ, Catón J, Morgan PR, et al. High frequency of BRAF V600E mutations in ameloblastoma. J Pathol. 2014;232(5):492–498. Choueiri TK, Cheville J, Palescandolo E, et al. BRAF mutations in metanephric adenoma of the kidney. Eur Urol. 2012;62(5): 917–922. Ilie M, Long E, Hofman V, et al. Diagnostic value of immunohistochemistry for the detection of the BRAFV600E mutation in primary lung adenocarcinoma Caucasian patients. Ann Oncol. 2013;24(3):742–748. Goeppert B, Frauenschuh L, Renner M, et al. BRAF V600Especific immunohistochemistry reveals low mutation rates in biliary tract cancer and restriction to intrahepatic cholangiocarcinoma. Mod Pathol. 2014;27(7):1028–1034. Weber A, Langhanki L, Sommerer F, et al. Mutations of the BRAF gene in squamous cell carcinoma of the head and neck. Oncogene. 2003;22(30):4757–4759. Agaram NP, Wong GC, Guo T, et al. Novel V600E BRAF mutations in imatinib-naive and imatinib-resistant gastrointestinal stromal tumors. Genes Chromosomes Cancer. 2008;47 (10):853–859. Chapman MA, Lawrence MS, Keats JJ, et al. Initial genome sequencing and analysis of multiple myeloma. Nature. 2011; 471(7339):467–472. Ihle MA, Fassunke J, König K, et al. Comparison of high resolution melting analysis, pyrosequencing, next generation sequencing and immunohistochemistry to conventional Sanger sequencing for the detection of p.V600E and non-p. V600E BRAF mutations. BMC Cancer. 2014;14(13):13. 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 formalinfixed, paraffin-embedded tissue specimens of malignant melanoma. Arch Pathol Lab Med. 2012;136(11):1385–1391. Andersen JB, Spee B, Blechacz BR, et al. Genomic and genetic characterization of cholangiocarcinoma identifies therapeutic targets for tyrosine kinase inhibitors. Gastroenterology. 2012; 142(4):1021–1031; [e1015]. Capper D, Preusser M, Habel A, et al. Assessment of BRAF V600E mutation status by immunohistochemistry with a mutation-specific monoclonal antibody. Acta Neuropathol. 2011;122(1):11–19. Boursault L, Haddad V, Vergier B, et al. Tumor homogeneity between primary and metastatic sites for BRAF status in metastatic melanoma determined by immunohistochemical and molecular testing. PLoS One. 2013;8(8):e70826. Curtin JA, Fridlyand J, Kageshita T, et al. Distinct sets of genetic alterations in melanoma. N Engl J Med. 2005;353(20): 2135–2147. Bauer J, Buttner P, Murali R, et al. BRAF mutations in cutaneous melanoma are independently associated with age, anatomic site of the primary tumor, and the degree of solar elastosis at the primary tumor site. Pigment Cell Melanoma Res. 2011;24(2):345–351. Long GV, Menzies AM, Nagrial AM, et al. Prognostic and clinicopathologic associations of oncogenic BRAF in metastatic melanoma. J Clin Oncol. 2011;29(10):1239–1246. Shinozaki M, Fujimoto A, Morton DL, et al. Incidence of BRAF oncogene mutation and clinical relevance for primary cutaneous melanomas. Clin Cancer Res. 2004;10(5):1753–1757.
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27. Kim G, McKee AE, Ning YM, et al. FDA approval summary: vemurafenib for treatment of unresectable or metastatic melanoma with the BRAF V600E mutation. Clin Cancer Res. 2014;20(19):4994–5000. 28. Chapman PB, Hauschild A, Robert C, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med. 2011;364(26):2507–2516. 29. Hauschild A, Grob JJ, Demidov LV, et al. Dabrafenib in BRAFmutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial. Lancet. 2012;380 (9839):358–365. 30. Long GV, Stroyakovskiy D, Gogas H, et al. Combined BRAF and MEK inhibition versus BRAF inhibition alone in melanoma. N Engl J Med. 2014;371(20):1877–1888. 31. Busam KJ, Hedvat C, Pulitzer M, et al. Immunohistochemical analysis of BRAF(V600E) expression of primary and metastatic melanoma and comparison with mutation status and melanocyte differentiation antigens of metastatic lesions. Am J Surg Pathol. 2013;37(3):413–420. 32. Chen Q, Xia C, Deng Y, et al. Immunohistochemistry as a quick screening method for clinical detection of BRAF(V600E) mutation in melanoma patients. Tumour Biol. 2014;35(6): 5727–5733. 33. Colomba E, Hélias-Rodzewicz Z, Von Deimling A, et al. Detection of BRAF p.V600E mutations in melanomas: comparison of four methods argues for sequential use of immunohistochemistry and pyrosequencing. J Mol Diagn. 2013;15(1):94–100. 34. Feller JK, Yang S, Mahalingam M. Immunohistochemistry with a mutation-specific monoclonal antibody as a screening tool for the BRAFV600E mutational status in primary cutaneous malignant melanoma. Mod Pathol. 2013;26(3):414–420. 35. Hofman V, Ilie M, Long-Mira E, et al. Usefulness of immunocytochemistry for the detection of the BRAF(V600E) mutation in circulating tumor cells from metastatic melanoma patients. J Invest Dermatol. 2013;133(5):1378–1381. 36. Lade-Keller J, Kristensen LS, Riber-Hansen R, et al. A role for immunohistochemical detection of BRAF V600E prior to BRAF-inhibitor treatment of malignant melanoma? J Clin Pathol. 2013;66(8):723–725. 37. Long GV, Wilmott JS, Capper D, et al. Immunohistochemistry is highly sensitive and specific for the detection of V600E BRAF mutation in melanoma. Am J Surg Pathol. 2013;37 (1):61–65. 38. Pearlstein MV, Zedek DC, Ollila DW, et al. Validation of the VE1 immunostain for the BRAF V600E mutation in melanoma. J Cutan Pathol. 2014;41(9):724–732. 39. Routhier CA, Mochel MC, Lynch K, et al. Comparison of 2 monoclonal antibodies for immunohistochemical detection of BRAF V600E mutation in malignant melanoma, pulmonary carcinoma, gastrointestinal carcinoma, thyroid carcinoma, and gliomas. Hum Pathol. 2013;44(11):2563–2570. 40. Skorokhod A, Capper D, von Deimling A, et al. Detection of BRAF V600E mutations in skin metastases of malignant melanoma by monoclonal antibody VE1. J Am Acad Dermatol. 2012;67(3):488–491. 41. Heinzerling L, Kühnapfel S, Meckbach D, et al. Rare BRAF mutations in melanoma patients: implications for molecular testing in clinical practice. Br J Cancer. 2013;108(10):2164–2171. 42. Adeniran AJ, Zhu Z, Gandhi M, et al. Correlation between genetic alterations and microscopic features, clinical manifestations, and prognostic characteristics of thyroid papillary carcinomas. Am J Surg Pathol. 2006;30(2):216–222. 43. Jung CK, Little MP, Lubin JH, et al. The increase in thyroid cancer incidence during the last four decades is accompanied by a high frequency of BRAF mutations and a sharp increase in RAS mutations. J Clin Endocrinol Metab. 2014;99(2):E276–E285. 44. Nikiforov YE. Molecular diagnostics of thyroid tumors. Arch Pathol Lab Med. 2011;135(5):569–577.
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45. Nikiforova MN, Kimura ET, Gandhi M, et al. BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas. J Clin Endocrinol Metab. 2003;88(11): 5399–5404. 46. Ghossein RA, Katabi N, Fagin JA. Immunohistochemical detection of mutated BRAF V600E supports the clonal origin of BRAF-induced thyroid cancers along the spectrum of disease progression. J Clin Endocrinol Metab. 2013;98(8):E1414– E1421. 47. Xing M, Westra WH, Tufano RP, et al. BRAF mutation predicts a poorer clinical prognosis for papillary thyroid cancer. J Clin Endocrinol Metab. 2005;90(12):6373–6379. 48. Howell GM, Nikiforova MN, Carty SE, et al. BRAF V600E mutation independently predicts central compartment lymph node metastasis in patients with papillary thyroid cancer. Ann Surg Oncol. 2013;20(1):47–52. 49. Xing M, Haugen BR, Schlumberger M. Progress in molecularbased management of differentiated thyroid cancer. Lancet. 2013;381(9871):1058–1069. 50. Xing M. Prognostic utility of BRAF mutation in papillary thyroid cancer. Mol Cell Endocrinol. 2010;321(1):86–93. 51. Xing M. BRAF V600E mutation and papillary thyroid cancer. J Am Med Assoc. 2013;310(5):535. 52. Howitt BE, Jia Y, Sholl LM, Barletta JA, et al. Molecular alterations in partially-encapsulated or well-circumscribed follicular variant of papillary thyroid carcinoma. Thyroid. 2013;23(10):1256–1262. 53. Rivera M, Ricarte-Filho J, Patel S, et al. Encapsulated thyroid tumors of follicular cell origin with high grade features (high mitotic rate/tumor necrosis): a clinicopathologic and molecular study. Hum Pathol. 2010;41(2):172–180. 54. Kim KB, Cabanillas ME, Lazar AJ, et al. Clinical responses to vemurafenib in patients with metastatic papillary thyroid cancer harboring BRAF(V600E) mutation. Thyroid. 2013;23(10): 1277–1283. 55. Rosove MH, Peddi PF, Glaspy JA. BRAF V600E inhibition in anaplastic thyroid cancer. N Engl J Med. 2013;368(7):684–685. 56. Bullock M, O’Neill C, Chou A, et al. Utilization of a MAB for BRAF(V600E) detection in papillary thyroid carcinoma. Endocr Relat Cancer. 2012;19(6):779–784. 57. Crescenzi A, Guidobaldi L, Nasrollah N, et al. Immunohistochemistry for BRAF(V600E) antibody VE1 performed in core needle biopsy samples identifies mutated papillary thyroid cancers. Horm Metab Res. 2014;46(5):370–374. 58. Fisher KE, Neill SG, Ehsani L, et al. Immunohistochemical investigation of BRAF p.V600E mutations in thyroid carcinoma using 2 separate BRAF antibodies. Appl Immunohistochem Mol Morphol. 2014;22(8):562–567. 59. Ilie MI, Lassalle S, Long-Mira E, et al. Diagnostic value of immunohistochemistry for the detection of the BRAF(V600E) mutation in papillary thyroid carcinoma: comparative analysis with three DNA-based assays. Thyroid. 2014;24(5):858–866. 60. Kim YH, Choi SE, Yoon SO, et al. A testing algorithm for detection of the B-type Raf kinase V600E mutation in papillary thyroid carcinoma. Hum Pathol. 2014;45(7):1483–1488. 61. Koperek O, Kornauth C, Capper D, et al. Immunohistochemical detection of the BRAF V600E-mutated protein in papillary thyroid carcinoma. Am J Surg Pathol. 2012;36(6):844–850. 62. McKelvie PA, Chan F, Yu Y, et al. The prognostic significance of the BRAF V600E mutation in papillary thyroid carcinoma detected by mutation-specific immunohistochemistry. Pathology. 2013;45(7):637–644. 63. Zagzag J, Pollack A, Dultz L, et al. Clinical utility of immunohistochemistry for the detection of the BRAF v600e mutation in papillary thyroid carcinoma. Surgery. 2013;154(6):1199–1204; [discussion 1204–1205].
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64. Zimmermann AK, Camenisch U, Rechsteiner MP, et al. Value of immunohistochemistry in the detection of BRAF(V600E) mutations in fine-needle aspiration biopsies of papillary thyroid carcinoma. Cancer Cytopathol. 2014;122(1):48–58. 65. Day F, Muranyi A, Singh S, et al. A mutant BRAF V600Especific immunohistochemical assay: correlation with molecular mutation status and clinical outcome in colorectal cancer. Target Oncol. 2014, PMID: 24859797, [in press]. 66. Tran B, Kopetz S, Tie J, et al. Impact of BRAF mutation and microsatellite instability on the pattern of metastatic spread and prognosis in metastatic colorectal cancer. Cancer. 2011; 117(20):4623–4632. 67. Huang CS, Farraye FA, Yang S, et al. The clinical significance of serrated polyps. Am J Gastroenterol. 2011;106(2):229–240; [quiz 241]. 68. Ogino S, Nosho K, Kirkner GJ, et al. CpG island methylator phenotype, microsatellite instability, BRAF mutation and clinical outcome in colon cancer. Gut. 2009;58(1):90–96. 69. Gibson J, Lacy J, Matloff E, Robert M. Microsatellite instability testing in colorectal carcinoma: a practical guide. Clin Gastroenterol Hepatol. 2014;12(2):171–176; [e1]. 70. Lin CC, Lin JK, Lin TC, et al. The prognostic role of microsatellite instability, codon-specific KRAS, and BRAF mutations in colon cancer. J Surg Oncol. 2014;110(4):451–457. 71. Tol J, Nagtegaal ID, Punt CJ. BRAF mutation in metastatic colorectal cancer. N Engl J Med. 2009;361(1):98–99. 72. Jonker DJ, O’Callaghan CJ, Karapetis CS, et al. Cetuximab for the treatment of colorectal cancer. N Engl J Med. 2007;357(20): 2040–2048. 73. Di Nicolantonio F, Martini M, Molinari F, et al. Wild-type BRAF is required for response to panitumumab or cetuximab in metastatic colorectal cancer. J Clin Oncol. 2008;26(35):5705– 5712. 74. Prahallad A, Sun C, Huang S, et al. Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR. Nature. 2012;483(7387):100–103. 75. Adackapara CA, Sholl LM, Barletta JA, et al. Immunohistochemistry using the BRAF V600E mutation-specific monoclonal antibody VE1 is not a useful surrogate for genotyping in colorectal adenocarcinoma. Histopathology. 2013;63(2):187–193. 76. Affolter K, Samowitz W, Tripp S, et al. BRAF V600E mutation detection by immunohistochemistry in colorectal carcinoma. Genes Chromosomes Cancer. 2013;52(8):748–752. 77. Bledsoe JR, Kamionek M, Mino-Kenudson M. BRAF V600E immunohistochemistry is reliable in primary and metastatic colorectal carcinoma regardless of treatment status and shows high intratumoral homogeneity. Am J Surg Pathol. 2014;38(10):1418–1428. 78. Capper D, Voigt A, Bozukova G, et al. BRAF V600E-specific immunohistochemistry for the exclusion of Lynch syndrome in MSI-H colorectal cancer. Int J Cancer. 2013;133(7):1624–1630. 79. Ilie MI, Long-Mira E, Hofman V, et al. BRAFV600E mutation analysis by immunohistochemistry in patients with thoracic metastases from colorectal cancer. Pathology. 2014;46(4):311–315. 80. Kuan SF, Navina S, Cressman KL, et al. Immunohistochemical detection of BRAF V600E mutant protein using the VE1 antibody in colorectal carcinoma is highly concordant with molecular testing but requires rigorous antibody optimization. Hum Pathol. 2014;45(3):464–472. 81. Lasota J, Kowalik A, Wasag B, et al. Detection of the BRAF V600E mutation in colon carcinoma: critical evaluation of the imunohistochemical approach. Am J Surg Pathol. 2014;38(9): 1235–1241. 82. Rössle M, Sigg M, Rüschoff JH, et al. Ultra-deep sequencing confirms immunohistochemistry as a highly sensitive and specific method for detecting BRAF V600E mutations in colorectal carcinoma. Virchows Arch. 2013;463(5):623–631.
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Available online at www.sciencedirect.com
www.elsevier.com/locate/semdp
BRAF V600E mutation-specific antibody: A review Lauren L. Ritterhouse, MD, PhD, Justine A. Barletta, MDn Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St, Boston, Massachusetts 02115
article info
abstra ct
Keywords:
The significance of BRAF mutations in neoplasia was first recognized in 2002 when mutations
BRAF V600E
were discovered in a broad range of cancers. Numerous subsequent studies expanded our
Immunohistochemistry
understanding of BRAF V600E as a critical diagnostic, prognostic, and predictive biomarker in
Melanoma
many cancers. Additionally, the advent of small-molecule inhibitors of BRAF V600E rendered
Thyroid carcinoma
assessment of BRAF mutation status essential in tumors such as melanoma. In clinical
Colorectal carcinoma
practice, evaluation of BRAF mutation status has routinely been performed by DNA-based assays utilizing polymerase chain reaction (PCR). However, molecular testing is not available at many hospitals since it is time-consuming, expensive, and requires expertise in molecular techniques. The first BRAF V600E-specific antibody was reported in 2011 (clone VE1). A purified version of this antibody as well as a second monoclonal antibody targeted to BRAF V600E is now commercially available. In this review, clinicopathologic characteristics associated with BRAF-mutant tumors will be highlighted, and the prognostic and predictive implications of a BRAF V600E mutation will be discussed with a focus on melanoma, thyroid carcinoma and colorectal carcinoma. Additionally, we will review the correlation between immunohistochemistry and molecular results and deliberate how BRAF immunohistochemistry might be utilized in the evaluation of these tumors. & 2015 Published by Elsevier Inc.
Introduction BRAF (v-raf murine sarcoma viral oncogene homolog B1) is a RAS-regulated serine–threonine kinase and activator of the MAPK signaling cascade. Extracellular signals act via this pathway to regulate cellular proliferation, differentiation, and survival. The importance of BRAF mutations in neoplasia was elucidated in 2002 when mutations were discovered in a broad range of cancers.1 The vast majority of mutations were found to occur in exon 15 with a thymine-to-adenine transversion at nucleotide 1799 (originally designated as nucleotide 1796), leading to a valine to glutamic acid substitution at codon 600 (V600E) (originally designated as V599E), a mutation thought to mimic phosphorylation of the activation site. Heterozygous n
Corresponding author. E-mail address: [email protected] (J.A. Barletta).
http://dx.doi.org/10.1053/j.semdp.2015.02.010 0740-2570/& 2015 Published by Elsevier Inc.
mutations in this codon were shown to significantly increase kinase activity in a RAS-independent manner and have transforming activity. Since 2002, mutations in BRAF have been described in a wide array of benign and malignant neoplasms, including melanoma,1 benign nevi,2 colon adenocarcinoma,3 serrated polyps,4 thyroid carcinoma,5 borderline serous ovarian tumors and low-grade serous carcinoma,6 Langerhans cell histiocytosis,7 several low-grade glioma subtypes as well as papillary craniopharyngioma,8,9 hairy cell leukemia,10 ameloblastoma,11 and metanephric adenoma,12 as well as rare nonsmall cell lung carcinomas,13 biliary tract carcinomas,14 head and neck squamous cell carcinomas,15 gastrointestinal stromal tumors,16 and plasma cell myelomas,17 among others. BRAF V600E and RAS mutations have been shown to be mutually
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exclusive in many of these tumors, suggesting that they have a similar tumorigenic effect.1,3,5,6 In clinical practice, evaluation of BRAF mutation status has routinely been performed by DNA-based assays utilizing polymerase chain reaction (PCR). Direct (Sanger) sequencing was the initial gold standard for BRAF mutation detection. However, a variety of additional DNA-based assays with higher sensitivity have subsequently been employed, including allele-specific PCR, pyrosequencing, and high resolution melting (HRM) analysis.18,19 Genetic analysis can fail as a result of poor DNA quality due to DNA fragmentation that can occur with tissue processing. It can also yield erroneous or artifactual results due to low tumor content.20 Additionally, these molecular methodologies all require DNA extraction, expensive equipment, expertise in molecular techniques, and strict quality control measures. As a result, molecular assays are not available at many hospitals, necessitating the test to be sent out which leads to a delay in results. The recognition of BRAF V600E as a critical diagnostic, prognostic, and predictive biomarker in many cancers, along with the advent of small-molecule inhibitors of BRAF V600E, prompted interest in developing immunohistochemistry (IHC) as a potentially faster, less expensive, more widely available methodology to detect the BRAF V600E protein in formalin-fixed and paraffin-embedded (FFPE) tissue. The first BRAF V600E-specific antibody was reported by Capper et al.21 in 2011. They developed hybridomas by immunizing mice with an 11 amino acid synthetic peptide representing the BRAF V600E-mutated amino acid sequence and fusing lymph node cells from the immunized mice with mouse myeloma SP2/O cells. Over 2000 clones were screened and only one (clone VE1) demonstrated a specific reaction with BRAF V600E by immunofluorescence using BRAF V600E-transiently expressing HEK 293T cells, immunoblotting, and IHC of FFPE samples of human melanoma and papillary thyroid carcinoma samples. IHC demonstrated moderate to strong cytoplasmic staining, consistent with the cytoplasmic localization of the tumor. Many of the IHC studies reviewed herein use either diluted or undiluted hybridoma supernatant. Additionally, a purified version of this antibody is now commercially available from Spring Bioscience and Ventana. A second monoclonal antibody directed against BRAF V600E (clone V600E, anti-B-Raf) is also commercially available from NewEast Biosciences. While an exhaustive cataloging of the implications of BRAF V600E in every cancer type is beyond the scope of this review, various facets will be highlighted including diagnostic potential, clinicopathologic characteristics, prognostic implications, and predictive value of BRAF V600E mutation with a focus on melanoma, thyroid carcinoma and colorectal carcinoma along with a brief summary in other tumor types. Additionally, we will review the correlation between IHC and molecular methodologies for the detection of the mutation and discuss how BRAF IHC might be utilized in the evaluation of these tumors.
Melanoma Mutations in BRAF are present in 40–60% of primary malignant melanomas and roughly half of metastatic melanomas.19,22,23 BRAF mutations are inversely correlated with age, more frequent at locations such as the trunk and lower extremities, and
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less frequently associated with solar elastosis,24 consistent with the finding that BRAF mutations arise in melanomas from skin that is intermittently exposed to sun and correlated with a lower ultraviolet dose.23 Interestingly, the frequency of BRAF mutations in melanomas arising in sites nearly completely protected from sun (i.e., acral and mucosal melanomas) is low.23,24 In melanoma, BRAF mutation seems to be an early event since many nevi harbor BRAF mutations.2 While BRAF mutations in primary melanomas have not been found to impact disease-free or overall survival, there is some indication that BRAF mutations in metastatic melanoma impart a decreased survival rate.25,26 The BRAF V600E mutation is responsible for 80–90% of BRAF mutations in melanoma.19 BRAF V600K is the second most common mutation (seen in 10–15%), and other BRAF mutations are rare.19 Given the dismal prognosis of patients with unresectable or metastatic melanoma and the limited benefit of standard chemotherapy, the advent of smallmolecule BRAF inhibitors revolutionized melanoma treatment. It also rendered BRAF mutation status in metastatic melanoma integral to treatment decisions. The Food and Drug administration (FDA) has approved two BRAF inhibitors, vemurafenib and dabrafenib, for the treatment of unresectable or metastatic melanoma.27 Both specifically inhibit BRAF V600E. Vemurafenib is approved for patients with V600E-mutant melanoma, while dabrafenib is approved in patients with melanomas with either a V600E or V600K mutation. Both of these drugs have an impressive response rate of approximately 50%.28,29 Although the development of resistance has been a limitation, the combination of a BRAF inhibitor along with a MEK inhibitor has recently been shown not only to increase response rate (to nearly 70%) but also to increase progression-free survival.30 The correlation between BRAF V600E IHC and BRAF mutation status assessed by molecular assays has been fairly robust in melanoma (Table).18,21,22,31–40 An example of a BRAF-mutant melanoma stained for BRAF V600E is shown in Fig. A. Reported sensitivities of the VE1 clone range from 85% to 100% and specificities range from 93% to 100%. The V600E clone has also demonstrated fairly good results with melanoma including a sensitivity ranging from 90% to 100% and specificities ranging from 81% to 97%. Several observations are worth noting. In studies with more than one interpreter of IHC, concordance of reviewers was near-perfect.22,36,37 In some cases with initial discordant IHC and molecular results, additional molecular testing confirmed the presence of a BRAF V600E mutation as was originally suggested by IHC.37 Many of the cohorts included non-BRAF V600E mutations (such as V600K), and, with rare exceptions, these tumors were negative for BRAF V600E by IHC.18,41 Tumors in general were shown to lack intratumoral heterogeneity; however, rare cases were described with heterogeneous staining, and rare discordant results between primary and metastatic tumors were reported as well.22,31 Finally, BRAF V600E IHC showed some variation in staining due to tissue characteristics, such as focal negativity in areas of cautery effect and necrosis and reduced staining in tissue first submitted for frozen section analysis or when IHC was performed on older tissue sections.21 Finally, rare cases were reported to demonstrate equivocal IHC results. Colomba et al.33 reported a perfect sensitivity and specificity for BRAF V600E IHC (V600E clone). On the basis of these findings, the authors suggest that IHC could be used as the initial test with
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Table – Correlation between BRAF V600E immunohistochemistry and BRAF mutation status assessed by molecular testing. Study
Tumor
# of cases
Antibody
Sensitivity
Specificity
Boursault et al.22 Busam et al.31 Capper et al.21 Chen et al.32 Colomba et al.33 Feller et al.34 Hofman et al.35 Ihle et al.18 Lade-Keller et al.36 Long et al.37 Pearlstein et al.38 Routhier et al.39
Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma Melanoma
230 51 47 38 111 35 98 63 28 100 76 31
Skorokhod et al.40 Bullock et al.56 Capper et al.21 Crescenzi et al.57 Fisher et al., 201458 Ghossein et al.46 Ilie et al.59 Kim et al.60 Koperek et al.61 McKelvie et al.62 Routhier et al.39
Melanoma Thyroid Thyroid Thyroid Thyroid Thyroid Thyroid Thyroid Thyroid Thyroid Thyroid
29 96 21 21 41 91 194 91 39 71 23
Zagzag et al.63 Zimmermann et al.64 Adackapara et al.75 Affolter et al.76 Bledsoe et al.77 Capper et al.78 Day et al.65 Ilie et al.59 Kuan et al.80 Lasota et al.81 Routhier et al.39
Thyroid Thyroid Colon Colon Colon Colon Colon Colon Colon Colon Colon
37 48 52 31 204 91 505 310 128 113 32
Rössle et al.82 Sajanti et al.83 Sinicrope et al.84 Thiel et al.85 Toon et al.86
Colon Colon Colon Colon Colon
265 147 75 137 216
VE1 (Spring Bio) VE1 hybridoma VE1 hybridoma V600E (NewEast VE1 hybridoma V600E (NewEast VE1 hybridoma VE1 (Spring Bio) VE1 (Spring Bio) VE1 hybridoma VE1 (Spring Bio) VE1 (Spring Bio) V600E (NewEast VE1 hybridoma VE1 hybridoma VE1 hybridoma VE1 (Spring Bio) VE1 (Spring Bio) VE1 (Spring Bio) VE1 hybridoma VE1 (Spring Bio) VE1 hybridoma VE1 (Spring Bio) VE1 (Spring Bio) V600E (NewEast VE1 (Spring Bio) VE1 (Spring Bio) VE1 (Spring Bio) VE1 (Spring Bio) VE1 (Spring Bio) VE1 (Spring Bio) VE1 hybridoma VE1 (Spring Bio) VE1 (Spring Bio) VE1 (Spring Bio) VE1 (Spring Bio) V600E (NewEast VE1 (Spring Bio) VE1 (Spring Bio) VE1 (Spring Bio) VE1 (hybridoma VE1 hybridoma
97% 100% 100% 100% 100% 100% 96% 100% 93% 97% 85% 90% 90% 86% 100% 100% 100% 100% 100% 99% n/aa 100%b 100% 100% 100% 89% 94% 71% 100% 96% 100% 100% 94% 100% 85% 100% 88% 100% 100% 100% 100% 99%
100% 100% 100% 93% 100% 97% 100% 98% 100% 98% 100% 95% 81% 100% 90% 100% 100% 62% 91% 100% n/aa 100%b 94% 100% 70% 100% 94% 74% 100% 99% 99% 100% 100% 94% 68% 100% 93% 95% 99% 100% 100% 100%
Bio) Bio)
Bio)
Bio)
Bio)
and Spring Bio)
This table includes only studies in which assessment of BRAF immunohistochemistry (IHC) was a main aim of the project and studies in which IHC results could be correlated with molecular results in specific tumor types. a The scoring method in this study precluded calculation of sensitivity and specificity. b These percentages are excluding cases that were considered equivocal on IHC.
pyrosequencing employed on cases that are equivocal or negative by IHC. Based on the very high specificity of BRAF V600E IHC in the studies reviewed, utilizing IHC as a first-line test seems reasonable and would reduce molecular testing by roughly 50%. Testing of negative cases is not only important due to the magnitude of the clinical consequences of missing a tumor harboring a V600E mutation, but also needed to identify tumors with other BRAF mutations such as V600K.
Thyroid carcinoma An activating BRAF mutation is the most common genetic event in thyroid carcinoma, found in roughly 45% of papillary
thyroid carcinomas (PTCs).42,43 The BRAF V600E mutation accounts for the vast majority of these BRAF mutations, with other mutations such as K601E found in only 1–2% of these tumors.44 The rate of BRAF V600E mutation depends on the subtype of PTC. The tall cell variant has the highest mutation frequency (over 90%), the follicular variant of PTC (FVPTC) has the lowest frequency (5–10%), and the mutation rate for classical type PTC falls in the middle, at roughly 55–75%.42,43 Interestingly, the rate of BRAF V600E mutation has increased nearly 30% in classical type PTCs in the last three decades.43 BRAF V600E is also present in rare poorly differentiated thyroid carcinomas and a subset of anaplastic thyroid carcinomas (ATCs) that likely arose from PTC.45,46 BRAF V600E mutations are not present in follicular or medullary thyroid
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Fig – Immunohistochemistry for BRAF V600E (VE1, Spring Biosciences) in BRAF V600E-mutant tumors: (A) Melanoma, (B) papillary thyroid carcinoma, and (C) colon adenocarcinoma.
carcinomas or in benign thyroid tumors.45 Thus, the detection of a BRAF V600E mutation is diagnostic of malignancy in this context. The majority of studies evaluating the clinicopathologic significance of BRAF V600E mutation have shown
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that it is associated with high-risk histopathologic features, such as an increased frequency of extrathyroidal extension, lymph nodes metastases, and higher clinical stage.47 Tumors found to have a BRAF mutation preoperatively have been shown to be associated with central neck lymph node metastases with a positive predictive value of 47% and a negative predictive value of 91%, indicating that knowledge of BRAF status preoperatively could potentially help guide whether a prophylactic central neck dissection should be performed.48,49 BRAF mutations have also been linked to an increased rate of disease recurrence with an odds ratio of roughly 3–5, a positive predictive value of 30%, and a negative predictive value of 90%.50 In a retrospective study of 1849 patients with PTC, Xing51 also found that the presence of a BRAF V600E mutation was significantly associated with increased cancer-related mortality among patients with PTC. Although they found that this association lost significance when other tumor features such as extrathyroidal extension, lymph node metastases, and distant metastases were factored into the analysis, they observed that each of these factors alone has a modest associated mortality, which was significantly increased by a coexisting BRAF mutation. Moreover, they found that conventionally defined low-risk patients with tumors lacking a BRAF V600E mutation had a negligible mortality rate. These findings are significant since they suggests that low-risk patients with tumors that are wild-type for BRAF might be spared post-thyroidectomy radioactive iodine treatment and might require less vigilant surveillance as compared to patients with tumors harboring a BRAF mutation.49 Non-infiltrative follicular variant of papillary thyroid carcinoma has been reported by some groups to lack of the BRAF V600E mutation.52,53 Though the evidence at this point is limited, this finding could potentially be used to support lobectomy only for this subset of tumors. BRAF V600E mutations have also been linked to loss of radioactive iodine uptake and subsequent resistance to radioactive iodine treatment.47 The presence of V600E in aggressive radioactive iodine refractory (RAIR) disease extends a treatment option with a BRAF inhibitor such as vemurafenib. There is some indication that vemurafenib produces a clinical response in patients with BRAF V600E-mutant metastatic PTC and could even have efficacy in the setting of anaplastic thyroid carcinoma harboring a BRAF mutation.54,55 IHC with the VE1 antibody has demonstrated a high concordance rate with molecular methods with sensitivities ranging from 89% to 100% and specificities from 61.5% to 100% in FFPE tissue from PTC specimens (Table).21,39,46,56–64 An example of a PTC harboring a BRAF V600E mutation stained for BRAF V600E is shown in Fig. B. The wide range in specificity is the result of one study by Fisher et al.58 that included cases of medullary thyroid carcinoma and follicular thyroid carcinoma in addition to PTC, as well as a handful of fine-needle aspiration (FNA) cell block specimens. Excluding this study, the specificity ranged from 90% to 100%. While the underlying explanation of the false–positive cases reported by Fisher et al. may be in part the result of their scoring tumors as positive by IHC when as little as 10% of the tumor demonstrated moderate to strong staining, they indicated that several of their false–positive cases demonstrated 480% positivity. Therefore, the explanation in these cases is
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unclear since they report a high tumor volume in specimens submitted for molecular analysis and repeat negative results with additional molecular testing by pyrosequencing. Bullock et al.56 noted that some of their “false–positive” IHC cases were likely secondary to low tumor content yielding a spurious negative result by molecular testing. Additionally, Sanger sequencing (an assay known to have lower sensitivity than other assays such as pyrosequencing and allele-specific PCR) was utilized in several studies and may also have produced spurious false–positive results on IHC, which in turn would lead to an inaccurately low estimation of specificity.59 There are a few additional points to highlight. Similar to the findings in melanoma, interobserver reproducibility was excellent (495%). Also, the vast majority of tumors demonstrated homogenous staining indicating that BRAF V600E mutation is a clonal event in thyroid carcinoma.46 Some cases were reported to show slight variation in staining; however, this appeared attributable to tissue fixation, since negative or weak staining was observed in the center of the tissue section.56 Significantly, Zimmermann et al.64 demonstrated that BRAF V600E IHC performed well on cell blocks from FNA specimens, with IHC demonstrating a 93.8% sensitivity and specificity. And finally, staining must be interpreted with caution. In the study by Ghossein et al.,46 the authors showed that IHC could be used to identify BRAF V600E mutations in anaplastic and poorly differentiated thyroid carcinoma as well as PTC; however, they found that in anaplastic cases with high background staining, moderate staining did not correlate with an underlying BRAF V600E mutation. Kim et al.60 also found that correlation was weaker with an intermediate staining result. These findings indicate that if the staining cannot be readily categorized as negative/ weak or moderate/strong in the majority of a tumor with no background staining (excluding the weak non-specific staining of colloid that was reported in several studies and did not influence results), the stain should be considered equivocal and molecular testing should be performed.
Colon adenocarcinoma Mutations in BRAF occur in approximately 10–15% of colorectal carcinomas (CRCs).65,66 BRAF V600E mutation is the main BRAF mutation that occurs in CRC.66 It is an early mutation (found in serrated polyps) and appears to promote methylation of CpG islands found within promoter regions of many genes, which in turn silences the methylated gene.67 Tumors that have high levels of CpG island methylation are categorized as CIMP-H. Thus, BRAF mutation is strongly associated with a CIMP-H status (though not all CIMP-H tumors harbor a BRAF mutation).67,68 In many BRAF-mutant, CIMP-H cases, the mismatch repair gene MLH1 is silenced by promoter methylation, leading to defective mismatch repair and subsequent microsatellite instability (MSI-H).67 Thus, BRAF mutations are also strongly associated with MSI-H status.66 BRAF mutations virtually never occur in the setting of an MSI-H tumor with an underlying germline mutation in a mismatch repair gene (i.e., Lynch syndrome). This is highly clinically significant, since it means that the identification of a BRAF mutation can be used to exclude Lynch syndrome as
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the underlying cause of mismatch repair protein deficiency.69 BRAF-mutant CRCs are associated with older age, a female predominance, and localization to the right colon.70 Identification of a BRAF mutation in CRC also has prognostic implications. Studies have demonstrated that BRAF-mutant metastatic CRCs are associated with a worse overall survival.65,66,71 In a cohort of metastatic CRCs, Tran et al.66 reported a medial overall survival of 10.4 months for BRAFmutant tumors compared with 34.7 months for tumors wildtype for BRAF. Moreover, they found that BRAF mutation was an independent poor prognostic factor in multivariate analysis with a HR of roughly 10.6. BRAF mutation may also be a predictive biomarker in CRC. BRAF is downstream of the epidermal growth factor receptor (EGFR). Anti-EGFR therapy with agents such as cetuximab has become standard treatment for patients with advanced CRC.72 While results are conflicting, there is some indication that BRAF mutation may correlate with resistance to the anti-EGFR therapy.73 Thus, identification of a BRAF mutation is an established clinical tool to exclude Lynch syndrome, has prognostic value in metastatic CRCs, and may be used to guide treatment decisions. Finally, while BRAF-mutant metastatic CRC appears to be unresponsive to treatment with a singleagent BRAF V600E inhibitor, the lack of response has been shown to be due to feedback activation of EGFR, suggesting that combination therapy with a BRAF V600E inhibitor and an EGFR or MEK inhibitor may yield better results.74 While the concordance between BRAF V600E IHC and molecular detection of a BRAF V600E mutation demonstrates some variability between studies, the overall concordance is strong given the number of cases tested (Table).39,65,75–86 An example of a BRAF-mutant colon adenocarcinoma stained for BRAF V600E is shown in Fig. C. Investigations using the VE1 clone have demonstrated sensitivities ranging from 71% to 100% and specificities ranging from 68% to 100%. There are several points to highlight from these studies. Bledsoe et al.,77 showed that IHC correlated with mutation status even in the setting or prior chemotherapy, radiation, and/or targeted therapy, while Ilie et al.79 demonstrated that IHC was sensitive and specific even with limited tumor in biopsy specimens of pulmonary metastases of CRC. In the one study comparing clones VE1 and V600E in CRC, VE1 performed slightly better.39 In general more BRAF-mutant CRC cases have been reported to show weaker staining as compared to BRAF-mutant thyroid carcinomas and melanomas. Cases with signet-ring-cell morphology were reported to produce both false–positive and negative IHC results.77,82 Also, background staining was reported in nuclei of normal colonic mucosa, smooth muscle, mucin, and nerve bundles.77,79 All of these findings indicate that careful IHC interpretation is required, though interobserver reproducibility was reported in one study as virtually perfect.86 Additionally, the disparate results between studies suggest that methodological differences such as the antigenretrieval protocol, antibody incubation conditions, and automated versus manual staining likely influences results especially in the setting of CRC samples. As Kuan et al.80 indicated, these findings demonstrate that rigorous antibody optimization is required. Moreover, extensive “in-house” validation needs to be performed before BRAF V600E staining is put into clinical practice, and any cases with somewhat
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Other tumors Several primary CNS neoplasms have been found to harbor a BRAF V600E mutation. A BRAF V600E is seen in several pediatric low-grade gliomas.87 For example, a BRAF V600E mutation has been reported in approximately 60% of pleomorphic xanthoastrocytomas (PXA), 20–60% of gangliogliomas (GG), and roughly 10% of pilocytic astrocytomas (predominantly those with an extra-cerebellar location).8,9 Thus, BRAF V600E may serve as an ancillary diagnostic tool when evaluating these tumors. For example, the histologic differential of PXA includes glioblastoma (GBM). Because the vast majority of standard GBMs lack a BRAF V600E mutation, the presence of this mutation could be used to help exclude GBM.8,9 Moreover, with trials with BRAF V600E inhibitors currently underway in patients with these tumors, identification of a BRAF V600E mutation is essential. Recently, Brastianos et al.88 demonstrated that the presence of a BRAF V600E mutation distinguishes papillary craniopharyngioma from adamantinomatous craniopharyngioma. Papillary tumors have a BRAF V600E mutation frequency of 95%, while adamantinomatous tumors lack BRAF mutations but instead virtually always harbor a CTNNB1 mutation. Again, this finding indicates that BRAF V600E IHC would be valuable diagnostically and suggests BRAF inhibitors as a rational treatment option for papillary craniopharyngiomas. High concordance rates between BRAF V600E IHC and mutation status have been reported in primary CNS neoplasms. For example, Ida et al.89 demonstrated that BRAF V600E IHC (VE1 clone) showed perfect sensitivity and specificity in PXAs, and Brastianos et al.88 showed the same result (again with VE1) in craniopharyngiomas. Among hematologic malignancies, a BRAF V600E mutation is present in virtually all cases of hairy cell leukemia (HCL) and approximately 60% of cases of Langerhans cell histiocytosis (LCH).7,10 The identification of a BRAF V600E mutation is essentially diagnostic of HCL, since it is extraordinarily rare in other lymphoproliferative disorders (4% of plasma cell myelomas harbor a BRAF V600E mutation), and the presence of a V600E mutation suggests that BRAF inhibitors may hold promise for patients with HCL.10 Andrulis et al.90 demonstrated that detection of BRAF V600E by IHC (VE1 was used) showed 100% sensitivity and specificity in HCL. Of note, their cohort of 30 HCL included many bone marrow specimens that were subjected to decalcification in EDTA. While some of the cases showed only weak staining when the Ventana ultraView detection system was used, staining was stronger and background was reduced when the Ventana OptiView detection system was employed (a system that is optimized for the detection of low-expressing antigens), again highlighting the importance of optimization of IHC staining protocols. BRAF mutations are increasingly being reported in a variety of other tumor types, with many of those tested by IHC showing robust concordance with molecular methods of detection. IHC for BRAF V600E has been shown to have a close to perfect concordance in ameloblastoma, a rare benign
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but locally aggressive odontogenic neoplasm with a high frequency of BRAF V600E mutation (approximately 60%).11,91 Metanephric adenomas (MA) are rare benign renal tumors that can mimic renal malignancies such as papillary renal cell carcinoma and have a BRAF V600E mutation frequency of 90%.12 IHC for BRAF V600E (clone VE1) has been shown to correlate perfectly with BRAF mutation status; moreover, the presence of BRAF V600E expression along with histologic impression is virtually diagnostic of metanephric adenoma.92 BRAF V600E IHC has also been shown to strongly correlate with molecular results in serous ovarian tumors.93 The BRAF V600E mutation is present in borderline serous ovarian tumors and low-grade serous ovarian carcinoma, demonstrating that these tumors are genetically distinct from high grade serous tumors which virtually never have BRAF mutations.6 Ilie et al.13 reported the presence of BRAF mutations in 9% of lung adenocarcinomas that were wild-type for EGFR, KRAS, PI3KA, and HER2 and lacked EML4–ALK: 4% were BRAF V600E and 5% non-BRAF V600E mutations. IHC for BRAF V600E was positive in 90% of BRAF V600E-mutant tumors and negative in all tumors harboring non-BRAF V600E mutations. Finally, while the frequency of BRAF mutations in biliary tract cancers is controversial, a recent study demonstrated 100% concordance between BRAF V600E IHC (clone VE1) and direct Sanger sequencing, although only 1% of biliary tract cancers (all intrahepatic cholangiocarcinomas) were positive for BRAF V600E.14
Conclusion The discovery of the BRAF V600E mutation has significantly increased our understanding of the carcinogenesis of many tumor types and has broad clinical implications. BRAF V600E IHC has already been performed on thousands of tumor specimens; likely, this is just the beginning.
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