VE1 Staining Research Articles

Concurrent enhancer of Zeste homolog 2 (EZH2) over-expression with BRAFV600E mutation predicts a poor outcome in patients with Langerhans cell histiocytosis (LCH): A pilot study.

e18536 Background: Prior studies demonstrated upregulated EZH2 protein expression and p-ERK1/2 cascade signaling in histiocytic and dendritic cell neoplasms. BRAFV600E is the most common driver mutation reported in LCH patients (34-64%). In melanoma studies, a signaling pathway with BRAFV600E activating mutation resulted in upregulation of EZH2. The frequency of EZH2 co-expression with BRAFV600E is unknown in LCH. Given the limited durable responses after discontinuation of treatment with BRAF inhibitors in histiocytic disorders, we investigated the prognostic role of EZH2 overexpression and BRAFV600E mutation in LCH. Methods: We analyzed a cohort of 27 consecutive patients (pts) diagnosed with LCH at Moffitt Cancer Center and Tampa General Hospital between 2010-2022. Immunohistochemical (IHC) staining using antibodies against EZH2 (D2C9 clone, Cell Signaling) and BRAFV600E (VE1 clone, Ventana medical systems) were performed. Expression was assessed by an IHC score of stain intensity (0, 1+, 2+ and 3+) multiplied by percent positive cells (H score). H score ≥ 30 was considered as positive. Pyrosequencing was performed to detect BRAFV600E mutations and correlated with VE1 staining results. A positive outcome from either IHC or PCR was deemed indicative of mutation positivity, given the robust presence of mutation representation in melanoma studies through IHC. Results: Entire cohort were grouped for pts who had both EZH2 overexpression and BRAFV600E mutation (EZH2/BRAFV600E) versus those who did not. Out of 27 pts, 25.9% (n=7) had both EZH2/BRAFV600E. EZH2/BRAFV600E group differed from control group by age ( p=0.046). BRAFV600E mutation was identified in 37.03% (10) pts, and 40.7% (11) showed expression of VE1 by IHC study. Discrepancies between PCR and IHC study were observed in 4 pts and potentially attributable to the restricted availability of tissue samples. 63% and 48.1% of pts were positive for EZH2 and BRAFV600E respectively. 6 pts were observed, 8 underwent surgery with or without radiation, 12 received induction chemotherapy, and only 1 received a BRAF inhibitor. After 18.6 months of median follow up, 92.6% pts were alive. Overall response rate (ORR) to the 1st line therapy was 55.6%. In univariate analysis of OS, H score and BRAFV600E mutation trended towards worse OS but were not statistically significant owing to small sample size. However, BRAFV600E mutation and EZH2 over expression demonstrated a poor overall survival (OS) ( p=0.012). Conclusions: Our study indicates that concurrent presence of BRAFV600E mutation and EZH2 overexpression correlates with poor OS in LCH pts. We hypothesize that in a subset of LCH pts combination therapy targeting both EZH2 and BRAFV600E may improve ORR or overcome resistance to BRAF inhibitor monotherapy. However, the results of our pilot study need to be validated on a larger sample set.

Metanephric adenoma with BRAF V600K mutation and a doubtful radiological imaging: pitfalls in the diagnostic process

Metanephric adenoma (MA) is an uncommon benign renal tumor whose histomorphological aspect resembles that of Wilms’ tumor and papillary renal cell carcinoma. From a diagnostic and therapeutic perspective, recognition of this entity is important as it has a more favorable clinical outcome compared with Wilms’ tumor and papillary renal cell carcinoma. MA should not be treated with nephrectomy if the tumor size is small, opting for a conservative treatment. However, the preoperative diagnosis of this disease is extremely challenging. The present study describes a case of this rare disease, showing an ambiguous radiological imaging and that only after a percutaneous biopsy, was defined as a MA and treated with partial nephrectomy. Moreover, the histological diagnosis of this case was partially complicated by the equivocal immunohistochemical analysis showing negativity for BRAF VE1 staining. Only the mutational analysis demonstrated the presence of the BRAF V600K mutation (for the first time described in a case of metanephric adenoma), highlighting the necessity of sequencing in case of MA with negativity for BRAF VE1 clone.

Intra- and Inter-Tumor BRAF Heterogeneity in Acral Melanoma: An Immunohistochemical Analysis.

The current development of BRAF inhibitors has revolutionized the treatment of unresectable melanoma. As the potential heterogeneity of BRAF mutations in melanoma has been reported, accurate detection of BRAF mutations are important. However, the genetic heterogeneity of acral melanoma—a distinct type of melanoma with a unique genetic background—has not fully been investigated. We conducted a retrospective review of our acral melanoma patients. Of the 196 patients with acral melanoma, we retrieved 31 pairs of primary and matched metastatic melanomas. We immunostained the 31 pairs with VE1, a BRAFV600E-mutation-specific monoclonal antibody. Immunohistochemistry with VE1 showed a high degree of sensitivity and specificity for detecting BRAFV600E mutations compared with the real-time polymerase chain reaction method. A total of nine primary (29.0%) and eight metastatic (25.8%) acral melanomas were positive for VE1. In three patients (9.7%), we observed a discordance of VE1 staining between the primary and metastatic lesions. Of note, VE1 immunohistochemical staining revealed a remarkable degree of intra-tumor genetic heterogeneity in acral melanoma. Our study reveals that VE1 immunostaining is a useful ancillary method for detecting BRAFV600E mutations in acral melanoma and allows for a clear visualization of intra- and inter-tumor BRAF heterogeneity.

High-resolution melting and immunohistochemical analysis efficiently detects mutually exclusive genetic alterations of adamantinomatous and papillary craniopharyngiomas.

Craniopharyngioma consists of adamantinomatous and papillary subtypes. Recent genetic analysis has demonstrated that the two subtypes are different, not only in clinicopathological features, but also in molecular oncogenesis. Papillary craniopharyngioma (pCP) is characterized by a BRAF mutation, the V600E (Val 600 Glu) mutation. Adamantinomatous craniopharyngioma (aCP) can be distinguished by frequent β-catenin gene (CTNNB1) mutations. Although these genetic alterations can be a diagnostic molecular marker, the precise frequency of these mutations in clinical specimens remains unknown. In this study, we first evaluated BRAF V600E and CTNNB1 mutations in four and 14 cases of pCP and aCP, respectively, using high-resolution melting analysis followed by Sanger sequencing. The results showed that 100% (4/4) of pCP cases had BRAF V600E mutations, while 78% (11/14) of the aCP cases had CTNNB1 mutations, with these genetic alterations being subtype-specific and mutually exclusive. Second, we evaluated BRAF V600E and CTNNB1 mutations by immunohistochemical analysis (IHC). All pCP cases showed positive cytoplasmic staining with the BRAF V600E-mutant antibody (VE-1), whereas 86% (12/14) of aCP cases showed positive cytoplasmic and nuclear staining for CTNNB1, suggesting a CTNNB1 mutation. Only one case of wild-type CTNNB1 on the DNA analysis showed immunopositivity on IHC. We did not detect a coexistence of BRAF V600E and CTNNB1 mutations in any single tumor, which indicated that these genetic alterations were mutually exclusive. We also report our modified IHC protocol for VE-1 staining, and present the possibility that BRAF V600E mutations can be used as a diagnostic marker of pCP in the differentiation of Rathke cleft cyst with squamous metaplasia.

The use of BRAF V600E mutation-specific immunohistochemistry in pediatric Langerhans cell histiocytosis.

BRAF p.V600E mutations are detected in greater than 50% of pediatric Langerhans cell histiocytosis (LCH) lesions. However, the use of mutation-specific BRAF V600E immunohistochemistry (IHC) as a surrogate for molecular testing in pediatric LCH is unknown. We tested the mutation-specific BRAF V600E monoclonal antibody (clone VE1) in formalin-fixed, paraffin-embedded LCH samples from 26 pediatric patients (14 males and 12 females, ages 7mo-17y) using allele-specific real-time polymerase chain reaction (PCR) with a limit of detection of 0.5% as the comparative gold standard. BRAF VE1 staining was scored for both intensity (0-3+) and percentage of immunoreactive tumor cells (0%-100%). BRAF VE1 immunoreactivity was determined using both lenient (≥1+, ≥1%) and stringent (≥2+, ≥10%) scoring criteria. Using lenient-scoring criteria, we found that the sensitivity and specificity of IHC compared with allele-specific real-time PCR were 100.0% and 18.2%, respectively. The poor specificity of lenient IHC analysis was attributable to weak, 1+ staining in both BRAF-mutated and wild-type LCH. Using stringent-scoring criteria, we found that specificity improved to 100.0% at the expense of sensitivity that decreased to 80.0%. Stringent scoring generated 3 false-negative results, but in all cases, neoplastic tissue comprised less than 5% of the stained section and/or the specimen was decalcified. In conclusion, highly sensitive molecular assays remain the gold standard for BRAF mutation analysis in LCH paraffin-embedded lesions. To avoid false-positive results, unequivocal VE1 staining of 2+ intensity in greater than or equal to 10% neoplastic histiocytes is required. However, negative VE1 results require additional studies to exclude false-negatives, and stringent-scoring criteria may not be optimal for scant or decalcified specimens.

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.

Highly Concordant Results Between Immunohistochemistry and Molecular Testing of Mutated V600E BRAF in Primary and Metastatic Melanoma.

This study tested the sensitivity and specificity of VE1 antibody raised against BRAFV600E protein, on 189 melanoma samples, compared with molecular testing. In addition, the therapeutic response to BRAF inhibitors was analysed in 27 patients, according to staining intensity (scored from weak to strong) and pattern (homogeneous or heterogeneous). BRAFV600E status during melanoma progression was evaluated in a cohort of 54 patients with at least paired-samples. High sensitivity (98.6%) and specificity (97.7%) of VE1 were confirmed. During melanoma progression different samples showed concordant phenotypes. Heterogeneous VE1 staining was observed in 28.5% of cases, and progression-free survival was higher in patients with tumour samples displaying such staining. These findings suggest that only VE1-negative tumours would be genotyped to detect other BRAFV600 mutations, and that either primary melanoma or metastasis can be tested using immunohistochemistry, according to the material available.

Assessment of BRAF V600E Status in Colorectal Carcinoma: Tissue-Specific Discordances between Immunohistochemistry and Sequencing

Although sequencing provides the gold standard for identifying colorectal carcinoma with BRAF V600E mutation, immunohistochemistry (IHC) with the recently developed mouse monoclonal antibody VE1 for BRAF V600E protein has shown promise as a more widely available and rapid method. However, we identified anecdotal discordance between VE1 IHC and sequencing results and therefore analyzed VE1 staining by two different IHC methods (Leica Bond and Ventana BenchMark) in whole tissue sections from 480 colorectal carcinomas (323 BRAF wild-type, 142 BRAF V600E mutation, and 15 BRAF non-V600E mutation). We also compared the results with melanomas and papillary thyroid carcinomas (PTC). With the Bond method, among 142 BRAF V600E-mutated colorectal carcinomas, 77 (54%) had diffuse VE1 staining and 48 (33%) had heterogeneous staining, but 17 (12%) were negative. Among 323 BRAF wild-type colorectal carcinomas, 196 (61%) were negative, but 127 (39%) had staining, including 7 with diffuse staining. When positivity was defined as staining in ≥ 20% of tumor cells, VE1 IHC had sensitivity of 75% and specificity of 93% for BRAF V600E mutation. With the Ventana method, among 57 BRAF V600E-mutated colorectal carcinomas, 36 (63%) had diffuse VE1 staining, whereas 6 (11%) had no or weak (<20% of tumor cells) staining. Among 33 BRAF wild-type colorectal carcinomas, 16 (48%) had no or weak staining, whereas 15 (45%) had heterogeneous staining. In contrast with colorectal carcinoma, Bond and Ventana VE1 IHC in melanoma and PTC were highly concordant with sequencing results. We conclude that VE1 IHC produces suboptimal results in colorectal carcinoma and should not be used to guide patient management.

VE1 immunohistochemistry predicts BRAF V600E mutation status and clinical outcome in colorectal cancer.

VE1 is a monoclonal antibody detecting mutant BRAFV(600E) protein by immunohistochemistry. Here we aim to determine the inter-observer agreement and concordance of VE1 with mutational status, investigate heterogeneity in colorectal cancers and metastases and determine the prognostic effect of VE1 in colorectal cancer patients. Concordance of VE1 with mutational status and inter-observer agreement were tested on a pilot cohort of colorectal cancers (n = 34), melanomas (n = 23) and thyroid cancers (n = 8). Two prognostic cohorts were evaluated (n = 259, Cohort 1 and n = 226, Cohort 2) by multiple-punch tissue microarrays. VE1 staining on preoperative biopsies (n = 118 patients) was compared to expression in resections. Primary tumors and metastases from 13 patients were tested for VE1 heterogeneity using a tissue microarray generated from all available blocks (n = 100 blocks). Inter-observer agreement was 100% (kappa = 1.0). Concordance between VE1 and V600E mutation was 98.5%. Cohort 1: VE1 positivity (seen in 13.5%) was associated with older age (p = 0.0175) and MLH1 deficiency (p < 0.0001). Cohort 2: VE1 positivity (seen in 12.8%) was associated with female gender (p = 0.0016), right-sided tumor location (p < 0.0001), higher tumor grade (p < 0.0001) and mismatch repair (MMR)-deficiency (p < 0.0001). In survival analysis, MMR status and postoperative therapy were identified as possible confounding factors. Adjusting for these features, VE1 was an unfavorable prognostic factor. Preoperative biopsy staining matched resections in all cases except one. No heterogeneity was found across any primary/metastatic tumor blocks. VE1 is highly concordant for V600E and homogeneously expressed suggesting staining can be analysed on resection specimens, preoperative biopsies, metastatic lesions and tissue microarrays.

BRAFV600E protein expression in primary cutaneous malignant melanomas and paired metastases.

BRAFV600E mutations are present in approximately 50% of cutaneous malignant melanomas (CMMs). The use of BRAFV600E mutation-specific monoclonal antibody VE1 immunohistochemical analysis may facilitate rapid detection of BRAFV600E mutations in CMMs and demonstrate heterogeneity among tumors. To characterize the pattern of BRAFV600E protein expression in primary CMMs with matched metastases and to analyze the use of VE1 immunohistochemical analysis in clinical practice using formalin-fixed, paraffin-embedded tumor tissue. In this retrospective cohort study performed at Karolinska University Hospital from September 2012 to September 2013, we examined CMMs (124 primary tumors and 76 metastases) with VE1 immunohistochemical analysis, and results were compared with DNA mutation analyses. Determination of intratumoral and intertumoral heterogeneity as well as the sensitivity and specificity of VE1 immunohistochemical analysis. Positive staining results with the VE1 antibody were detected in 94 of 200 tumors (47.0%). In general, VE1 staining was homogeneous. However, VE1 staining intensity varied among the primary tumors and corresponding metastases in 63 of 135 tumors (46.7%), but a change of mutational status based on DNA analysis was found in only 4 matched tumors (3.0%). Discordant findings between DNA mutation analysis and immunohistochemical analysis were observed in 12 tumors. The overall sensitivity and specificity of VE1 immunohistochemical analysis were 96.7% and 94.5%, respectively. A comparable sensitivity was obtained for primary and metastatic CMMs. The specificity was lower among primary CMMs (92.4%) compared with metastases (98.0%). We found VE1 immunohistochemical analysis to be a useful and rapid assay for BRAFV600E mutations that may contribute to the detection of intratumoral and intertumoral heterogenetic subclones. Tumors with positive results, including strong staining, should be expedited for confirmatory BRAF mutation testing. If this test result is negative, a false-negative result of the mutation analysis should be considered. Validation of VE1 immunohistochemical analysis in clinical practice is needed.

VE1 antibody immunoreactivity in normal anterior pituitary and adrenal cortex without detectable BRAF V600E mutations.

The VE1 monoclonal antibody was developed to recognize the V600E mutation in BRAF, which is found in various tumors. We report that the VE1 antibody stains normal anterior pituitary gland and adrenal cortex, which lack detectable BRAF V600E mutations. Staining with the VE1 antibody was seen in the adenohypophysis and correlated well with adrenocorticotropic hormone (ACTH)-positive cells. ACTH-positive cells were typically most concentrated in the central mucoid wedge and pars intermedia, and VE1 staining was strong in these regions. Moreover, VE1 staining was seen in ACTH-expressing pituitary adenomas without detectable BRAF mutations. VE1 staining of the adrenal cortex was also significant, with the strongest staining seen in the inner segment of the zona fasciculata. Parathyroid glands, pancreatic islets, or parafollicular C cells in the thyroid showed no VE1 staining. Overall, VE1 staining of endocrine tissues strongly suggests limitations on the use of this antibody for the detection of BRAF mutations.

Intrapatient Homogeneity of BRAFV600E Expression in Melanoma

Concern regarding the presence of intertumoral heterogeneity of BRAF mutation status in patients with metastatic melanoma has led to uncertainty surrounding which specimens should preferentially undergo BRAF testing. We sought to examine the extent of intrapatient heterogeneity of BRAF(V600E) protein expression in patients with multiple tumors. Sixty-four patients with 171 tumors at various stages of disease progression had tumor BRAF(V600E) protein expression immunohistochemically (IHC) assessed using the BRAF(V600E) mutant-specific antibody VE1. Melanoma sections were examined for staining intensity (score 0 to 3), the presence of intratumoral heterogeneity, and concordance with molecular BRAF genotype. Intrapatient, intertumoral heterogeneity of BRAF(V600E) expression was also assessed by comparing VE1 staining on different tumors within the same patient. All specimens from 64 patients displayed complete intertumoral homogeneity of BRAF(V600E) expression status, and all tumors had concordant molecular and IHC BRAF status. Only 1 patient demonstrated >1 level of staining intensity heterogeneity between specimens. Intratumoral heterogeneity of staining intensity was not observed in any specimen. IHC-measured BRAF(V600E) protein expression displays complete intertumoral homogeneity, minimal intertumoral intensity heterogeneity, and no intratumoral heterogeneity in metastatic melanoma patients in various stages of disease progression. Our results suggest that, provided there is adequate quantity of viable tumor cells and minimal admixture of nontumor cells, testing any melanoma sample from a patient with metastatic disease will accurately determine BRAF status for treatment planning.

Mutational analysis of BRAF and KRAS in ovarian serous borderline (atypical proliferative) tumours and associated peritoneal implants

There is debate as to whether peritoneal implants associated with serous borderline tumours/atypical proliferative serous tumours (SBT/APSTs) of the ovary are derived from the primary ovarian tumour or arise independently in the peritoneum. We analysed 57 SBT/APSTs from 45 patients with advanced-stage disease identified from a nation-wide tumour registry in Denmark. Mutational analysis for hotspots in KRAS and BRAF was successful in 55 APSTs and demonstrated KRAS mutations in 34 (61.8%) and BRAF mutations in eight (14.5%). Mutational analysis was successful in 56 peritoneal implants and revealed KRAS mutations in 34 (60.7%) and BRAF mutations in seven (12.5%). Mutational analysis could not be performed in two primary tumours and in nine implants, either because DNA amplification failed or because there was insufficient tissue for mutational analysis. For these specimens we performed VE1 immunohistochemistry, which was shown to be a specific and sensitive surrogate marker for a V600E BRAF mutation. VE1 staining was positive in one of two APSTs and seven of nine implants. Thus, among 63 implants for which mutation status was known (either by direct mutational analysis or by VE1 immunohistochemistry), 34 (53.9%) had KRAS mutations and 14 (22%) had BRAF mutations, of which identical KRAS mutations were found in 34 (91%) of 37 SBT/APST-implant pairs and identical BRAF mutations in 14 (100%) of 14 SBT/APST-implant pairs. Wild-type KRAS and BRAF (at the loci investigated) were found in 11 (100%) of 11 SBT/APST-implant pairs. Overall concordance of KRAS and BRAF mutations was 95% in 59 of 62 SBT/APST-implant (non-invasive and invasive) pairs (p < 0.00001). This study provides cogent evidence that the vast majority of peritoneal implants, non-invasive and invasive, harbour the identical KRAS or BRAF mutations that are present in the associated SBT/APST, supporting the view that peritoneal implants are derived from the primary ovarian tumour.

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

The BRAF V600E mutation occurs in 15% of colorectal carcinomas (CRCs) and has important genetic, prognostic, and therapeutic implications. A monoclonal antibody (VE1) targeting the BRAF V600E mutant protein has become available with variable efficacy in literature reports. We investigated the utility of the VE1 antibody in detecting BRAF V600E mutant protein in two cohorts: (1) a retrospectively accrued series of 103 resected CRCs with (N = 57) and without (N = 46) known BRAF V600E mutation status by PCR and (2) a prospective series of 25 CRCs requiring BRAF analysis during routine screening for Lynch syndrome. All 74 cases with positive BRAF V600E mutation demonstrated cytoplasmic positivity with the VE1 antibody with most tumors (70/74, 95%) demonstrating moderate to strong staining. Of the 54 BRAF V600E-negative cases, 51/54 CRCs (94%) were negative with the VE1 antibody while 3 CRCs (6%) demonstrated weak cytoplasmic staining. The sensitivity and specificity of VE1 was 100% and 94%, respectively. Ten BRAF V600E-mutated CRCs had adjacent precursor lesions including 7 sessile serrated adenomas associated with CRCs with high-level microsatellite instability (MSI-H). All 10 precursor adenomas were positive for VE1 staining with the 7 sessile serrated adenomas maintaining preserved MLH1 expression. Our results indicate that VE1 immunohistochemistry is a useful surrogate for the detection of the BRAF V600E mutation in CRC, although weak staining must be evaluated by BRAF PCR analysis to exclude a false positive result. In addition, the BRAF V600E mutation appears to be an early event before the divergent development into MSS and MSI-H pathways.

BRAF V600E‐specific immunohistochemistry for the exclusion of Lynch syndrome in MSI‐H colorectal cancer

The differentiation between hereditary and sporadic microsatellite-unstable (MSI-H) colorectal cancer is a crucial step in Lynch syndrome diagnostics. Within MSI-H colorectal cancers, the BRAF V600E mutation is strongly associated with sporadic origin. Here, we asked whether BRAF V600E-specific immunohistochemistry (clone VE1) is helpful in separating sporadic from Lynch syndrome-associated MSI-H colorectal cancers. To that end, we performed VE1 immunohistochemistry and BRAF sequencing in a series of 91 MSI-H colorectal cancer specimens from patients tested for Lynch syndrome. Concordance of VE1 immunohistochemistry and molecular BRAF mutation status was observed in 90 of 91 (98.9%) MSI-H samples. All 11 tumors classified as BRAF V600E mutation-positive by Sanger sequencing were immunopositive, and 79 (98.8%) of 80 tumors classified as BRAF wild type showed negative staining. All VE1-positive tumors were MLH1- and PMS2-negative by immunohistochemistry. None of the tumors from mismatch repair (MMR) gene germline mutation carriers (n = 28) displayed positive VE1 staining, indicating that BRAF V600E mutation-specific immunostaining has a low risk of excluding Lynch syndrome patients from germline mutation analysis. In conclusion, implementation of VE1 immunohistochemistry was able to detect BRAF-mutated MSI-H colorectal cancers with a sensitivity of 100% and a specificity of 98.8%. Among MLH1-negative colorectal cancers, the rate of VE1-positive lesions was 21%, offering the exclusion of these patients from MMR germline testing. Therefore, we suggest the integration of VE1 immunohistochemistry into the diagnostic panel of Lynch syndrome.

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.