BRAF V600E Mutation-specific Antibody Research Articles

Association of BRAF V600E immunoexpression with clinicopathological variant groups in papillary thyroid carcinoma

The detection of BRAF V600E immunoexpression in papillary thyroid carcinoma (PTC) is extremely important because of its association with poor prognosis. Is poor prognosis really associated with all histological variants of thyroid papillary carcinoma? To contribute to this elucidation, using the BRAF V600E mutation-specific antibody, the present study was performed to determine the association between BRAF V600E immunoexpression and clinicopathological variant groups of PTC. Tumour tissue samples collected from 95 patients with papillary thyroid cancer and tumour size <3.5 cm in diameter were processed by routine microscopic technique and using Ventana Benchmark XT automated stainer. BRAF V600E immunoexpression in the group of the aggressive clinicopathological group is directly proportional to lymph node and distal metastasis (p=0.005) in contrast to the favourable clinicopathological group. Finally, the lymph nodal and distal metastasis (poor prognosis) of the aggressive clinicopathological group of PTC can be better predicted when combining BRAF V600E immunostaining with conventional histopathological technique, conversely, these results could not be achieved in the favourable clinicopathological group. These findings suggest that BRAF V600E immunoexpression is a potential candidate for the prognosis of the aggressive clinicopathological group.

Negative reactions of BRAF mutation-specific immunohistochemistry to non-V600E mutations of BRAF.

BRAF mutations are rare driver mutations in non-small cell lung cancer (NSCLC), accounting for 1%-2% of the driver mutations, and the mutation spectrum has a wide range in contrast to other tumors. While V600E is a dominant mutation in melanoma, more than half of the mutations in NSCLCs are non-V600E. However, treatment with dabrafenib plus trametinib targets the BRAF V600E mutation exclusively. Therefore, distinguishing between V600E and non-V600E mutations is crucial for biomarker testing in NSCLC in order to determine treatment of choice. Immunohistochemistry (IHC) using the BRAF V600E mutation-specific antibody is clinically used in melanoma patients, but little is known about its application in NSCLC, particularly with regard to the assay performance for non-V600E mutations. In the present study, we examined 117 tumors with BRAF mutations, including 30 with non-V600E mutations, using BRAF mutation-specific IHC. None of the tumors with non-V600E mutations, including two compound mutations, showed a positive reaction. Furthermore, all V600E mutations were positive except for one case with combined BRAF V600E and K601_W604 deletion. Our findings confirmed that the BRAF V600E mutation-specific IHC is specific without any cross-reactions to non-V600E mutations, suggesting that this assay can be a useful screening tool in clinical practice.

High frequency of BRAF V600E mutation in Iranian population ameloblastomas.

Background Ameloblastoma is a common locally invasive but slow-growing neoplasm of the jaws with an odontogenic origin. Association between BRAF V600E mutation and clinicopathologic features and behavior of ameloblastoma remains controversial. This study aimed to evaluate BRAF V600E gene mutation and expression of its related proteins with clinicopathologic parameters in conventional ameloblastoma. Material and Methods 50 Formalin-fixed paraffin-embedded blocks were included in this study. Immunohistochemistry was done using rabbit monoclonal BRAF V600E mutation-specific antibody VE1. Quantitative real-time polymerase chain reaction assay was used for evaluating of BRAF V600E mutation. Results Expression of BRAF V600E antibody was Positive in 42 out of 50 cases (84%). 46 (92%) out of 50 specimens showed BRAF V600E mutation. There were 13 cases of recurrence (26%). 3 out of 4 cases with negative mutations did not show recurrence. Conclusions We report the highest frequency (92%) of BRAF V600E mutation in ameloblastomas in the Iranian population. Although there was not a significant association between BRAF V600E‑positive immunoexpression and recurrence and clinicopathologic parameters, its high frequency could emphasize its role as a therapeutic marker in the future. Key words:Conventional ameloblastoma, BRAF V600E, recurrence.

Microfluidic-Based Immunohistochemistry Combined With Next-Generation Sequencing on Diagnostic Tissue Sections for Detection of Tumoral BRAF V600E Mutation.

Tailored diagnostics requires immunohistochemistry (IHC) and next generation sequencing (NGS). Here we combined on a single paraffin-embedded slide microfluidic-based IHC (micro-IHC) and NGS for BRAF V600E mutation detection in BRAFomas. For micro-IHC, we performed the primary antibody incubation step of conventional chromogenic IHC in a LabSat device (Lunaphore Technologies SA). Tumor areas immunoreactive for pan-cytokeratin, pan-melanoma, and BRAF V600E mutation-specific antibody were H-scored, microdissected, and analyzed by NGS. After 2 minutes, pan-cytokeratin and BRAF micro-IHC increased exponentially (half-time values: 1.7 and 3.2 minutes). Pan-melanoma displayed a higher half-time value of 15 minutes. There was no significant difference in H-score and staining quality, respectively, between conventional and micro-IHC. BRAF V600E mutation was detected in all pan-cytokeratin and pan-melanoma stained samples without amplification but in only 40% of BRAF V600E stained samples with amplification. Micro-IHC enables short antibody incubation times and subsequent NGS. Preprocessing is critical for preservation of DNA quality.

BRAF V600E Mutation Across Multiple Tumor Types: Correlation Between DNA-based Sequencing and Mutation-specific Immunohistochemistry.

The B-Raf proto-oncogene (BRAF) encodes a cytoplasmic serine/threonine kinase with a key role in regulating the mitogen-activated protein kinase signal transduction pathway. An activating missense mutation in codon 600 of exon 15 (V600E) of BRAF gene has been identified in multiple neoplasms including melanoma, colorectal carcinoma, papillary thyroid carcinoma, hairy cell leukemia, and Langerhans cell histiocytosis. Patients with BRAF V600E-mutated melanoma respond to FDA-approved BRAF inhibitors. In addition, subsets of other BRAF V600E-mutated tumors may also benefit from BRAF inhibitor therapy. Currently, clinical laboratories typically use molecular-based methods for mutation analysis. However, recently a BRAF V600E mutation-specific antibody has become available as a cost-effective alternative method to DNA-based molecular testing. We analyzed multiple tumor types including melanoma, colorectal carcinoma, papillary thyroid cancer, hairy cell leukemia, and Langerhans cell histiocytosis using both DNA-based sequencing and the BRAF V600E mutation-specific antibody. Our results show a high degree of concordance between the 2 methods. However, the high concordance seems to be limited only to the V600E mutation since variant V600 mutations are missed by V600E mutation-specific immunohistochemistry.

Rapid BRAF mutation detection in melanoma patients by immunohistochemistry

The V600E mutation is the most common (~90%) activating mutation of the BRAF gene. BRAF mutations have been frequently investigated in melanoma, colorectal cancer and papillary thyroid carcinoma. The importance of the detection of BRAF mutations has been rising by the routine use of Braf inhibitor therapy. We evaluated the usefulness of the BRAF V600E mutation-specific monoclonal antibody (VE1) in metastatic melanoma patients. To confirm the results of immunohistochemistry (IHC), we used COBAS 4800 BRAF V600 mutation test and PCR amplification followed by Sanger sequencing.36 of 105 patients have wild-type BRAF gene, 64 have V600E mutation and 5 of 105 have V600K mutation. Predicting the mutation only by IHC using VE1 antibody, all 58 positively scored specimen were V600E mutant. The V600K, the wild-type patients and 7 patients from the V600E mutant group scored as negative. Thus the specificity is 100% and the positive predictive value is 1 of the IHC method. After processing our data we could establish a cheaper diagnostic algorithm for rapid detection ofBRAF mutation.

Abstract 1138: Prognostic impact of BRAF-V600E expression and correlation with mutation status in cutaneous melanoma

Abstract Around 50% of primary cutaneous melanomas harbour BRAF mutations, but their prognostic impact has not been clear. Recently, a BRAF-V600E mutation-specific antibody has become available for immunohistochemistry (IHC). Here, we investigated for the first time the prognostic impact of BRAF-V600E protein expression in primary melanoma. In a patient series of 248 nodular melanomas, BRAF-V600E and total BRAF expression were assessed by IHC using tissue microarray sections of paraffin-embedded archival tissue. Mutation status was assessed by real-time PCR. Positive BRAF-V600E expression was present in 86 (35%) of the cases and was significantly associated with increased tumor thickness, presence of tumor ulceration and reduced survival. Further, BRAF-V600E expression was an independent prognostic factor by multivariate survival analysis. There was 88% concordance between BRAF-V600E expression and mutation status. Our findings indicate that BRAF-V600E expression is a novel prognostic marker in primary melanoma. Citation Format: Emilia Hugdahl, May Britt Kalvenes, Hanne Puntervill, Rita Ladstein, Lars A. Akslen. Prognostic impact of BRAF-V600E expression and correlation with mutation status in cutaneous melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1138.

Concomitant KIT/BRAF and PDGFRA/BRAF mutations are rare events in gastrointestinal stromal tumors.

AIMThe BRAF mutation is a rare pathogenetic alternative to KIT/PDGFRA mutation in GIST and causes Imatinib resistance. A recent description of KIT and BRAF mutations co-occurring in an untreated GIST has challenged the concept of their being mutually exclusive and may account for ab initio resistance to Imatinib, even in the presence of Imatinib-sensitive KIT mutations. BRAF sequencing is generally limited to KIT/PDGFRA wild-type cases. Hence, the frequency of concomitant mutations may be underestimated.METHODSWe screened for KIT (exon 9, 11, 13, 17), PDGFRA (exon 12,14, 18) and BRAF (exon 15) mutations a series of 407 GIST. Additionally, we evaluated the BRAF V600E mutation-specific antibody, VE1, as a surrogate for V600E mutation, on a series of 313 GIST (24 on whole sections, 288 cases on tissue array), including 6 cases molecularly ascertained to carry the BRAF V600E mutation.RESULTSNo concomitant KIT/BRAF or PDGFRA/BRAF mutations were detected. BRAF mutation was detected only in one case, wild-type for KIT/PDGFRA. All the 6 BRAF-mutant cases stained positive with the VE1 antibody. A weak VE1 expression was observed in 14/287 (4.9%) BRAF wild-type cases, as observed also in 2/6 BRAF-mutant cases. Overall in our series, sensitivity and specificity of the VE1 antobody were 100% and 95.1%, respectively.CONCLUSIONThe concomitance of BRAF mutation with either KIT or PDGFRA mutation is rare in GIST. In these tumors, moderate/strong VE1 immunoreactivity is a valuable surrogate for molecular analysis. Instead, genotyping is warranted in the presence of weak VE1 staining.

BRAF-V600E expression in primary nodular melanoma is associated with aggressive tumour features and reduced survival.

Background:Around 50% of primary melanomas harbour BRAF mutations, but their prognostic impact has not been clear. Recently, a BRAF-V600E mutation-specific antibody has become available for immunohistochemistry. Here, we investigated for the first time the prognostic impact of BRAF-V600E protein expression in primary melanoma.Methods:In a patient series of 248 nodular melanomas, BRAF-V600E and total BRAF expression were assessed by immunohistochemistry using tissue microarray sections of paraffin-embedded archival tissue. Mutation status was assessed by real-time PCR in cases with sufficient tumour tissue (n=191).Results:Positive BRAF-V600E expression was present in 86 (35%) of the cases, and was significantly associated with increased tumour thickness, presence of tumour ulceration and reduced survival. Further, BRAF-V600E expression was an independent prognostic factor by multivariate analysis, whereas BRAF mutation status was not significant. There was 88% concordance between BRAF-V600E expression and mutation status.Conclusions:Our findings indicate that BRAF-V600E expression is a novel prognostic marker in primary melanoma.

Validation of a Manual Protocol for BRAF V600E Mutation-specific Immunohistochemistry.

Detection of BRAF V600E has diagnostic, prognostic, and therapeutic relevance. The recently developed BRAF V600E mutation-specific antibody has evolved into a feasible alternative to DNA analysis. The plethora of immunohistochemical protocols makes implementation tedious and, here we tested a set of manual and automated protocols and compared test performance with sequencing results. For assays, we employed formalin-fixed, in part decalcified, and paraffin-embedded tissue samples. Empiric testing of manual protocols included 10 variables in 17 protocols. Automated immunohistochemical staining and BRAF pyrosequencing served as independent test methods. Test performance measures were compared without considering 1 method as a standard. Four well-fixed samples (2WT/2Mut) were used for testing of all protocols and indicated 2 correctly classifying procedures. Practical performance assessment employed 33 independent tissue samples, composed of 27 leukemias (by pyrosequencing: 8 wild-type; 18 mutated; 1 noninformative) and 6 melanomas (V600E; V600K; wild-type, 2 each). Manual V600E staining was positive in 20 cases (19 of 20 V600E-containing samples plus the 1 sample that was noninformative), whereas all wild-type and V600K cases were immunonegative. Manual or automated staining as well as pyrosequencing would have missed an equal number of V600E-mutated cases and the correlation coefficient for these methods was 0.75 to 0.93 (substantial to almost perfect); the Youden index was 0.95. Detection of V600E-mutated BRAF at the protein level in routine and decalcified tissue samples is possible, and the presented manual protocols should expedite implementation in routine diagnostic practice. Our results indicate that both molecular techniques should be considered complementary.

Prospective immunohistochemical analysis of BRAF V600E mutation in melanoma.

The v-raf murine sarcoma viral oncogene homolog B1 (BRAF) V600E mutation is the most common activating genetic alteration of this oncogene and a predictive marker for the therapeutic use of BRAF inhibitors in melanoma. Our aim was to evaluate the performance of BRAF V600E mutation-specific monoclonal antibody (VE1) in a prospective diagnostic setting of melanoma patients (n = 102). All 41 cases (40.2%) that showed a V600E mutation in the cyclic minisequencing analysis of the DNA were also initially scored immunopositive. Two cases that were scored as BRAF V600E mutation positive by immunohistochemistry were negative in the DNA-based mutation analysis and determined to be immunonegative in a repeated staining with more representative specimens. Thus, BRAF V600E mutation detection using immunohistochemistry was 100% sensitive and 96.8% specific, when compared with the analysis of the DNA. None of the BRAF V600K mutations was detected by the VE1 antibody (n = 7). However, the VE1 antibody detected a rare V600E2 mutation. We also studied the role of BRAF V600E mutation in a set of melanoma patients who had been investigated for sentinel node metastasis. Melanoma lymph node metastases were diagnosed in 21.8% (12/55) of the sentinel nodes, and BRAF V600E immunopositivity was detected in 34.5% (19/55) of the cases. BRAF V600E mutation status did not correlate with any clinicopathological parameters. In conclusion, analysis of BRAF V600E mutation in melanoma by immunohistochemistry is a sensitive and specific method, which can be used to identify BRAF inhibitor-sensitive melanoma patients as a first-line method due to its rapid and affordable nature.

A testing algorithm for detection of the B-type Raf kinase V600E mutation in papillary thyroid carcinoma.

The B-type Raf kinase (BRAF) V600E mutation is a useful diagnostic marker for papillary thyroid carcinoma (PTC). We developed a testing algorithm for the BRAF mutation using BRAF immunohistochemistry (IHC) in PTC. Formalin-fixed, paraffin-embedded PTC tissues from 91 patients were immunostained with a BRAF V600E mutation-specific antibody. The immunostaining results were interpreted semiquantitatively by adding the scores of the proportion of positive cells (scored 0 to 5) and staining intensity (score 0 to 3). To validate BRAF IHC, 4 molecular methods were used: direct sequencing, pyrosequencing, peptide nucleic acid clamping polymerase chain reaction, and real-time polymerase chain reaction. The cut-off score of BRAF IHC for predicting the BRAF mutation was 5.5. In 68 cases, where the BRAF IHC score was 6 or more, 89.7% were positive for the BRAF mutation by 2 or more molecular methods and 97.1% by one or more methods. In 13 cases, where the IHC score was between 4 or more and less than 6, 61.5% were positive for the BRAF mutation by 2 or more methods and 69.2% by at least one method. In 10 cases, where the IHC score was less than 4, BRAF mutation was noted in one case only by one molecular method. BRAF V600E IHC scores of less than 4 or 6 or more should not require confirmation by additional tests. However, BRAF V600E IHC scores of 4 or 5 may require confirmation of the BRAF V600E mutation. In addition, BRAF IHC scores 6 or more were more sensitive than any single molecular method; therefore, BRAF IHC may overcome the limitations of using a single type of molecular method.

Improved molecular classification of serrated lesions of the colon by immunohistochemical detection of BRAF V600E

BRAF V600E mutation in serrated lesions of the colon has been implicated as an important mutation and as a specific marker for the serrated carcinogenic pathway. Recent findings point to microvesicular hyperplastic polyps that have similar histologic and molecular features to sessile serrated adenomas/polyps, as potential colorectal carcinoma precursors. The aim of this study was to evaluate BRAF V600E mutation status by immunohistochemistry in serrated lesions of the colon with regard to histomorphology. We investigated 194 serrated lesions of the colon, comprising 42 sessile serrated adenomas/polyps, 16 traditional serrated adenomas, 136 hyperplastic polyps and 20 tubular/tubulovillous adenomas (conventional adenomas) with the novel BRAF V600E mutation-specific antibody VE1. In addition, BRAF exon 15 and KRAS exon 2 status was investigated by capillary sequencing in selected cases. All sessile serrated adenomas/polyps (42/42, 100%), 15/16 (94%) traditional serrated adenomas and 84/136 (62%) hyperplastic polyps were VE1+. None of the VE1− serrated lesions showed BRAF V600E mutation. Forty out of 42 (95%) sessile serrated adenomas/polyps displayed areas with microvesicular hyperplastic polyp-like features. In microvesicular hyperplastic polyps, VE1 positivity was significantly associated with nuclear atypia (P=0.003); however, nuclear atypia was also present in VE1− cases. Immunostaining with VE1 allows not only the detection of BRAF V600E mutation but also the correlation with histomorphology on a cellular level in serrated lesions. VE1 enables a subclassification of microvesicular hyperplastic polyps according to the mutation status. This improved classification of serrated lesions including immunohistochemical evaluation of BRAF V600E mutation may be the key to identify lesions with higher potential to progression into sessile serrated adenoma/polyp, and further to BRAF V600E-mutated colorectal cancer.

BRAFV600E mutation in sporadic and neurofibromatosis type 1-related malignant peripheral nerve sheath tumors

Malignant peripheral nerve sheath tumors (MPNSTs) are rare, but deadly, soft tissue sarcomas that occur more commonly and at a younger age in individuals affected with the neurofibromatosis type 1 (NF1) inherited cancer predisposition syndrome. Currently, the only curative treatment for MPNSTs is surgery, and treatment of advanced disease is limited to cytotoxic chemotherapy with generally little effect on overall survival. Moreover, there is a pressing need for targeted therapeutic options for MPNSTs arising in individuals with NF1 and in the general population. Mutations in the NF1 gene have been reported in both NF1-related and sporadic MPNSTs,1 such that loss of NF1 protein (neurofibromin) function leads to increased Ras signaling.2–4 Given that the BRAF kinase molecule is also a known downstream Ras effector and that BRAF mutations are found in numerous nervous system cancers,5,6 we hypothesized that BRAF mutations may identify a molecularly-distinct subset of MPNST. To explore this hypothesis, we employed immunohistochemistry using a BRAFV600E mutation-specific antibody on formalin-fixed/paraffin-embedded samples representing 62 MPNSTs and reviewed the associated clinical data to determine NF1 status, overall survival, and time to metastasis. Representative images are shown in Fig. 1 (left panels). Confirmatory BRAFV600E sequencing was not performed on these tumors, based on our previous experience revealing complete concordance between sequencing and immunohistochemistry results in ganglioglioma7 as well as in other laboratories using different tumor types.8,9 Fig. 1. Left. BRAFV600E immunostaining in (a) human papillary thyroid cancer, positive control; (b) normal human brain, negative control; (c) one representative BRAFV600E-immunopositive MPNST; and (d) one representative BRAFV600E-immunonegative MPNST. Magnification, ... First, we found that individuals with NF1 developed MPNSTs ∼10–15 years earlier than their sporadic counterparts (P < .003; Fig. 1; right panel), which was similar to previous reports.10–12 Second, BRAFV600E mutation was observed in 20% (5/25) of sporadic MPNSTs. In these BRAFV600E-positive MPNSTs, 90%–100% of the tumor cells were BRAFV600E-immunoreactive, suggesting that this mutation could be a primary driver of malignancy and not merely a mutation found in a subset of tumor cells. The prevalence of BRAFV600E mutation in our series of 62 MPNSTs was slightly higher than previously reported in a smaller series of tumors (1 of 13 sporadic MPNSTs13) and was likewise higher than observed in sporadic glioblastoma.14 In this regard, analysis of available The Cancer Genome Atlas (TCGA) data using the C-BIO website application (http://cbio.mskcc.org) revealed that BRAFV600E mutations predominated in thyroid cancer (182/323 tumors; ∼56%) and melanoma (81/228 tumors; ∼36%) but were far less common in all other cancers examined (<9%). Interestingly, melanocytes and Schwann cells have neural crest origins, suggesting a susceptible cell of origin for BRAF mutation in the genesis of these cancers. Third, BRAFV600E mutation was not observed in benign neurofibromas (0/11 tumors), implicating BRAF mutation in malignant progression rather than in neurofibroma tumorigenesis. Fourth, only one NF1-associated MPNST harbored a BRAFV600E mutation (1/37; 2.7%). There was a statistically significant difference in the percentage of BRAFV600E mutations in NF1-associated MPNSTs relative to their sporadic counterparts (P = .035). While these molecular events might be considered mutually exclusive, BRAF molecular alterations have also been reported in another NF1-associated nervous system tumor (pilocytic astrocytoma).15 Fifth, BRAFV600E mutation does not appear to confer any statistically significant differences in overall patient survival or time to metastasis when patients were censored by either date of death or date of last follow-up. This may reflect the small sample size, the rarity of these tumors, and the fact that nearly one-third of the patients were lost to follow-up (and assumed to be living as they could not be found in the Social Security Death Index). While the prognostic value has yet to be established, this study identifies BRAF mutation as a significant molecular alteration in MPNST, potentially offering another therapeutic target for a deadly cancer with no currently efficacious treatment options.

Clinical utility of immunohistochemistry for the detection of the BRAF v600e mutation in papillary thyroid carcinoma

BRAF V600E mutation is the most common genetic alteration in papillary thyroid cancer (PTC). We used a mutation-specific antibody for immunohistochemical (IHC) detection of the BRAF V600E mutation and correlated expression with clinicopathologic features. The study was designed to validate the accuracy and determine the clinical importance of IHC detection of the BRAF V600E mutation in PTC. Direct sequencing and IHC for BRAF V600E mutation was performed in 37 consecutive patients with PTCs. IHC was scored on an intensity proportion scale. IHC positive tumors were stratified into intensity categories. The categories were assessed for clinicopathologic variables, including age, extrathyroidal extension, lymphovascular invasion, and lymph node metastases. A total of 25 PTCs were BRAF V600E-positive and 12 were BRAF mutation-negative on IHC. The BRAF V600E mutation-specific antibody had a sensitivity of 89% and specificity of 100% for detecting the mutation. Tumors with high-intensity staining were more likely to have extrathyroidal extension. IHC is an accurate method for the detection of the BRAF V600E mutation in PTC, and its ability to quantify the mutation expression may serve as a better predictor of tumor behavior than molecular sequencing. It provides a potentially rapid, easily applicable, and economic alternative to current techniques.

Immunohistochemistry is highly sensitive and specific for detection of BRAF V600E mutation in pleomorphic xanthoastrocytoma

BackgroundHigh frequencies of the BRAF V600E mutation have been reported in pleomorphic xanthoastrocytoma (PXA). Recently, a BRAF V600E mutation-specific antibody has been developed and validated. We evaluated the immunohistochemical (IHC) detection of BRAF V600E mutation in PXA by comparing to gold standard molecular analysis and investigating the interobserver variability of the IHC scoring. We performed BRAF V600E IHC in 46 cases, of which 37 (80%) cases had sufficient tumor tissue for molecular analysis. IHC detection was performed using monoclonal mouse antibody VE1 (Spring Bioscience). IHC slides were scored independently by four reviewers blind to molecular data, including a primary (gold standard) and three additional reviewers. BRAF V600E mutation status was assessed by allele-specific polymerase chain reaction (PCR) with fragment analysis.ResultsAll 46 cases showed interpretable BRAF V600E IHC results: 27 (59%) were positive (strong cytoplasmic staining), 19 (41%) were negative (6 of these cases with focal/diffuse weak cytoplasmic staining, interpreted as nonspecific by the primary reviewer). By molecular analysis, all 37 cases that could be tested had evaluable results: 22 (59%) cases were positive for BRAF V600E mutation and were scored as “IHC-positive”, and 15 (41%) were negative (including 11 cases scored as “IHC-negative” and 4 cases scored as negative with minimal nonspecific staining). IHC detection of BRAF V600E mutant protein was congruent in all 37 cases that were successfully evaluated by molecular testing (sensitivity and specificity of 100%). Agreement for IHC scoring among the 4 reviewers was almost perfect (kappa 0.92) when cases were scored as “positive/negative” and substantial (kappa 0.78) when minimal nonspecific staining was taken into account.ConclusionsWe conclude that detection of BRAF V600E mutation by immunohistochemistry is highly sensitive and specific. BRAF V600E IHC interpretation is usually straightforward, but awareness of possible nonspecific staining is necessary and training is recommended. It is a practical rapid method that may avoid the need of labor-intensive molecular testing and may be most valuable in small biopsies unsuitable for molecular analysis.

Application of a BRAF V600E Mutation-specific Antibody for the Diagnosis of Hairy Cell Leukemia

In recent times BRAF V600E mutations have emerged as a genetic hallmark of hairy cell leukemia (HCL). This specific point mutation is present in virtually all cases of HCL but is exceedingly rare in other peripheral B-cell neoplasms. In this study we investigated the application of a BRAF V600E mutation-specific antibody (clone VE1) to differentiate HCL from HCL mimics, such as HCL variant and splenic marginal zone lymphoma. A total of 52 routinely processed formalin-fixed paraffin-embedded tissue specimens were investigated (bone marrow, n=46; spleen, n=6) for expression of V600E-mutated BRAF protein. All 32 cases of HCL were scored positive, and all non-HCL cases were scored negative. In 28 of 30 HCL cases the presence of a BRAF V600E mutation could be confirmed by direct sequencing, whereas no BRAF mutations were detected among 20 HCL mimics. We further screened 228 mature B-cell neoplasms with VE1 and detected 1 positive case of chronic lymphocytic leukemia. Sequencing confirmed the presence of a BRAF V600E mutation. In conclusion, we demonstrate that VE1 immunohistochemistry can be used to reliably differentiate HCL from HCL-mimicking entities. This on-slide technique might be particularly helpful in interpreting challenging biopsies with low tumor content.

Assessment of BRAF V600E mutation status by immunohistochemistry with a mutation-specific monoclonal antibody

Activating mutations of the serine threonine kinase v-RAF murine sarcoma viral oncogene homolog B1 (BRAF) are frequent in benign and malignant human tumors and are emerging as an important biomarker. Over 95% of BRAF mutations are of the V600E type and specific small molecular inhibitors are currently under pre-clinical or clinical investigation. BRAF mutation status is determined by DNA-based methods, most commonly by sequencing. Here we describe the development of a monoclonal BRAF V600E mutation-specific antibody that can differentiate BRAF V600E and wild type protein in routinely processed formalin-fixed and paraffin-embedded tissue. A total of 47 intracerebral melanoma metastases and 21 primary papillary thyroid carcinomas were evaluated by direct sequencing of BRAF and by immunohistochemistry using the BRAF V600E mutation-specific antibody clone VE1. Correlation of VE1 immunohistochemistry and BRAF sequencing revealed a perfect match for both papillary thyroid carcinomas and melanoma metastases. The staining intensity in BRAF V600E mutated tumor samples ranged from weak to strong. The generally homogenous VE1 staining patterns argue against a clonal heterogeneity of the tumors investigated. Caution is essential when only poorly preserved tissue is available for VE1 immunohistochemical analysis or when tissues with only little total BRAF protein are analyzed. Immunohistochemistry using antibody VE1 may substantially facilitate molecular analysis of BRAF V600E status for diagnostic, prognostic, and predictive purposes.