BRAF V600E Mutation of Non-Small Cell Lung Cancer in Korean Patients.

Background and Objectives: BRAF mutational status in resected non-small cell lung cancer (NSCLC) in the Korean population is poorly understood. We explored BRAF (particularly BRAF V600E) mutational status among Korean patients with NSCLC. Materials and Methods: This study included 378 patients with resected primary NSCLC who were enrolled from January 2015 to December 2017. The authors obtained formalin-fixed paraffin-embedded (FFPE) tissue blocks and performed peptide nucleic acid (PNA)-clamping polymerase chain reaction (PCR) for detecting BRAF V600, real-time PCR for detecting BRAF V600E, and immunohistochemical analyses using the mutation-specific Ventana VE1 monoclonal antibody. For positive cases in any methods mentioned above, direct Sanger sequencing was additionally performed. Results: The PNA-clamping method revealed the BRAF V600 mutation in 5 (1.3%) of the 378 patients. Among these five patients, real-time PCR, direct Sanger sequencing detected BRAF V600E mutations in three (0.8%) patients. Thus, two cases showed differences in their PNA-clamping and the others. Direct Sanger sequencing of PNA-clamping PCR product was performed for two cases showing negative results on direct Sanger sequencing; both contained BRAF mutations other than V600E. All patients harboring BRAF mutations had adenocarcinomas, and all patients with V600E mutation exhibited minor micropapillary components. Conclusions: Despite the low incidence of the BRAF mutation among Korean patients with NSCLC, lung adenocarcinoma patients with micropapillary components should be prioritized in terms of BRAF mutation testing. Immunohistochemical staining using Ventana VE1 antibody may serve as a screening examination for BRAF V600E.

Expression and Clinical Correlation of PD-1/PD-L1 and VE1(BRAFp.V600E) in Pediatric Langerhans Cell Histiocytosis.

Background And Objectives: Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasm with a wide spectrum of clinical presentations. Programmed Cell Death-1 (PD-1) receptor and its ligand (PD-L1) are overexpressed in LCH, but their clinical significance is unknown. We performed a clinical correlation study of PD-1/PD-L1 and VE1(BRAFp.V600E) expression in 131 children with LCH. Methods: A total of 111 samples were tested for PD-1/PD-L1 and 109 for VE1(BRAFp.V600E) mutant protein by immunohistochemistry. Results: PD-1, PD-L1 and VE1(BRAFp.V600E) positivity was observed in 40.5%, 31.53% and 55%, respectively. PD-1/ PD-L1 expression showed no significant effect on the rate of disease reactivations, early response to therapy or late sequelae. The 5-year EFS was not statistically different between patients with PD-1 positive compared to those with PD-1 negative tumours (47.7% vs.58.8%, p=0.17). Similar 5-year EFS rates were also seen in those who were PD-L1 positive compared to PD-L1 negative cases (50.5% vs.55.5%, p=0.61). VE1(BRAFp.V600E) positivity was associated with a significantly higher frequency of risk-organ involvement (p=0.0053), but no significant effect on early response to therapy or rates of reactivations or late sequelae. Conclusions: Our study showed no significant correlation between VE1(BRAFp.V600E) expression, PD-1 and PD-L1 and clinical outcome in pediatric LCH.

Assessment of BRAF V600E (VE1) immunochemistry for the detection of BRAF V600E mutation in non-small cell lung carcinoma cytology specimens.

BACKGROUND: Non-small cell lung carcinoma (NSCLC) patients with BRAF V600E-mutated tumors respond to targeted therapy. Testing for BRAF V600E is commonly performed with molecular methods; however, a mutation-specific VE1 antibody clone can provide an alternative testing option using immunohistochemistry (IHC) for practices using single-gene testing and in situations when the specimen is inadequate for molecular testing. This study evaluates the usefulness of VE1 IHC in screening for BRAF V600E mutations in NSCLC cytology specimens. METHODS: The authors retrospectively identified cytology cases with a diagnosis of NSCLC that had BRAF V600E IHC performed on cell block sections with the monoclonal VE1 antibody clone. The BRAF V600E IHC results were compared with those of molecular testing performed with an amplicon-based next-generation sequencing assay. RESULTS: There were 201 NSCLC cases evaluated. The VE1 IHC was positive in seven of seven BRAF V600E-mutated tumors (100%) and was negative in 158 of 158 nonmutated BRAF V600E tumors (100%). Thirty cases did not undergo molecular testing, primarily because of insufficient tissue or because molecular testing was performed on an alternative specimen. Six cases showed equivocal weak/focal staining: Two cases demonstrated BRAF V600E mutations, and four cases were negative by molecular testing. CONCLUSIONS: This study suggests that BRAF V600E IHC can be used reliably to screen NSCLC cytology specimens, and negative results strongly indicate the absence of a BRAF V600E mutation. Having a low threshold for equivocal staining is recommended with molecular confirmation of BRAF V600E for any cases demonstrating weak and/or focal cytoplasmic staining.

The BRAFV600E Mutation Is Not a Risk Factor for More Aggressive Tumor Behavior in Radiogenic and Sporadic Papillary Thyroid Carcinoma at a Young Age.

Histopathological changes in the fusion oncogene-driven papillary thyroid carcinomas (PTCs) from children and adolescents exposed to Chernobyl fallout have been extensively studied. However, characteristics of the radiogenic BRAFV600E-positive PTCs, whose proportion is growing with time, are not well described yet. We analyzed the relationship between the BRAFV600E status (determined immunohistochemically with the VE1 antibody) and the clinicopathological features of 247 radiogenic and 138 sporadic PTCs from young Ukrainian patients aged ≤28 years. The frequency of BRAFV600E was increasing with patient age, consistently remaining lower in radiogenic PTCs. In both etiopathogenic groups, the BRAFV600E-positive PTCs more frequently had a dominant papillary growth pattern, smaller tumor size, higher Ki67 labeling index, and a frequency of the major indicators of tumor invasiveness that is lower than or equal to that of the BRAFV600E-negative tumors. Comparison of the BRAFV600E-positive PTCs across the groups found a virtual absence of differences. In contrast, the BRAFV600E-negative radiogenic PTCs displayed less frequent dominant papillary and more frequent solid growth patterns, lower Ki67 labeling index, and higher invasiveness than the BRAFV600E-negative sporadic tumors. Thus, BRAFV600E is not associated with a more aggressive course of PTC in young patients regardless of etiology. The major clinicopathological differences between the radiogenic and sporadic PTCs are observed among the BRAFV600E-negative tumors.

Discrepancy between immunohistochemistry and sequencing for BRAF V600E in odontogenic tumours: Comparative analysis of two VE1 antibodies.

BACKGROUND: Although immunohistochemistry (IHC) along with molecular tests has been investigated in ameloblastoma for BRAF V600E detection, VE1 IHC has not been studied in odontogenic carcinomas (OCs) and benign mixed epithelial and mesenchymal odontogenic tumours (BMOTs). Here, we performed BRAF V600E mutation analysis, examined the expression pattern of VE1 IHC, and comparatively evaluated the performance of two VE1 antibodies in ameloblastomas, OCs and BMOTs. METHODS: BRAF V600E detection was performed using Sanger sequencing in a total of 47 odontogenic tumours: 28 ameloblastomas, 6 OCs and 13 BMOTs. VE1 IHC was conducted using two different antibodies (IHC-A and IHC-V), and their performance was analysed by calculating the sensitivity and specificity compared with sequencing. RESULTS: BRAF V600E mutations were identified in 24/28 (85.7%) ameloblastomas, 2/5 (40.0%) ameloblastic carcinomas (ACs), 3/7 (42.9%) ameloblastic fibromas and 1/2 (50.0%) ameloblastic fibro-odontomas. In the presence of the mutation, VE1 showed diffuse cytoplasmic staining in ameloblastomas and ACs, whereas all BMOTs were negative for VE1. IHC-A and IHC-V yielded a sensitivity of 76.7% and 60.0%, respectively, although both antibodies showed 100% specificity. CONCLUSION: OCs and BMOTs have BRAF V600E mutations in common at lower frequencies than ameloblastoma. Diffuse VE1 cytoplasmic staining in AC suggests the utility of MAPK-targeted therapy as selectively applied in ameloblastoma, and consistent VE1 false-negative expression in BMOTs requires further investigation. Considering the high specificity but low sensitivity of VE1 IHC, molecular tests should be performed to determine the presence of BRAF V600E mutations in odontogenic tumours.

VE1 immunohistochemistry is an adjunct tool for detection of BRAFV600E mutation: Validation in thyroid cancer patients.

Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy among other endocrine tumors, and BRAFV600E is a frequent genetic mutation occurring in the disease. Although different molecular techniques, most importantly sequencing has been widely recognized as a gold standard but molecular diagnosis remains an expensive, laborious, and time-intensive process. Recently, immunohistochemistry (IHC) with anti-BRAF V600E (VE1) antibody has increased practical utility and implemented clinically for the detection of BRAFV600E mutation. Therefore, the study aimed to evaluate diagnostic accuracy of VE1 IHC for detecting the BRAFV600E mutation frequency and clinical implementation in diagnostic laboratories. In this study, 72 formalin fixed paraffin-embedded tissues (FFPE) were used to determine the BRAFV600E mutation status using IHC and Sanger sequencing. The mutation was found in 29% and 28% cases using IHC and Sanger sequencing, respectively. Furthermore, the results showed 100% sensitivity, 98.07% specificity, 95.2% positive predictive value, and 100% negative predictive value. Notably, significant associations were found between BRAFV600E status and tumor stage, tumor focality, and extrathyroidal extensions, respectively. VE1 IHC was found to be a highly sensitive, specific, and diagnostically accurate method in this cohort. Therefore, BRAFV600E detection through IHC has been considered as the best tailored technique for routine pathology laboratories.

Analysis of 24 cases of epithelioid glioblastoma: Experience from a tertiary centre of North India.

BACKGROUND: Epithelioid glioblastoma (eGB) is a recently recognized and a rare variant of glioblastoma. This study aimed to describe the clinical, histological and immunohistochemical spectrum and outcome of eGB from a tertiary care hospital in north India. MATERIALS AND METHODS: Twenty four cases of eGB diagnosed over past 10 years were reviewed with detailed morphological and immunohistochemical analysis (GFAP, EMA, Vimentin, Myogenin, INI-1, Cytokeratin, Synaptophysin, CD99, S100, MelanA, IDH1, ATRX, p16, EZH2, Ki-67, and BRAF V600E mutant antibody). RESULT: The mean age was 29.9 years (3-54 years), with equal male and female patients. All had supratentorial tumor. All cases showed epithelioid cells in sheets; however, focal spindling (7 cases, 29.2%), grouping/nesting (6 cases, 25%) and papillary configuration (5 cases, 20.8%) were also noted. All showed microvascular proliferation (MVP) and all except one demonstrated areas of necrosis. INI1 was retained in all cases, while 2 showed patchy loss. EZH2 overexpression (>25%) was observed in 4 cases, while 5 cases showed loss of p16 expression. BRAF V600E mutant protein expression was seen in 12/23 (52.2%) cases. Outcome was available in 8 cases, out of which 6 (75%) experienced recurrence. The median survival was 25.5 months. Cases with tumor infiltrating lymphocytes had a better outcome. CONCLUSION: eGB is a distinct variant of glioblastoma which has predilection towards younger age group. It shows high percentage of BRAF V600E mutation and a subset of it shows longer survival. Cases with presence of tumor infiltrating lymphocytes are associated with better outcome.

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.

VE1 Immunohistochemistry Improves the Limit of Genotyping for Detecting BRAFV600E Mutation in Papillary Thyroid Cancer.

Detection of BRAFV600E is useful for making diagnosis and risk stratification of papillary thyroid carcinoma (PTC). Molecular testing, however, is not always available for routine clinical use. To assess the clinical utility and reliability of VE1 immunohistochemistry (IHC) for detecting BRAFV600E mutation in PTC, VE1 IHC was performed on the tissue microarrays of 514 patients with PTC and was compared with Sanger sequencing results. Of 514 PTC cases, 433 (84.2%) were positive for VE1 expression. Among 6 discordant cases between VE1 IHC and Sanger sequencing, 3 initial VE1-false negative cases turned out to be true false negative on repeat testing, and 3 VE1-false positive cases showed BRAFV600E mutation using digital PCR analysis. PTCs with low variant allele fraction were positive for VE1 IHC but were not detected using sequencing. VE1 IHC showed 99.3% sensitivity, 100% specificity, 100% positive predictive value, and 96.4% negative predictive value. The BRAFV600E mutation was significantly associated with older age, multifocality, extrathyroidal extension, lymph node metastasis, and advanced tumor stage. In conclusion, VE1 IHC is a reliable method for detecting BRAFV600E mutation in PTC specimens.

Immunohistochemical BRAF V600E Expression and Intratumor BRAF V600E Heterogeneity in Acral Melanoma: Implication in Melanoma-Specific Survival.

Acral melanoma, a distinct form of cutaneous melanoma originating in the glabrous skin of the palms, soles, and nail beds, has a different genetic background from other subtypes of cutaneous melanoma. The roles of oncogenic BRAF mutations of acral melanoma in pathogenesis and patient outcomes have not been fully elucidated. We retrieved a total of 112 patients with primary acral melanoma and checked their BRAF V600E status using immunohistochemical staining of VE1 antibody. Among these cases, 21 acral melanoma samples (18.8%) showed positive BRAF V600E staining, and of those, 11 samples (9.8%) showed a heterogeneous staining pattern, with a mixture of VE1-positive and VE1-negative cells. BRAF V600E positivity was significantly associated with thicker melanoma (p = 0.0015). There was no significant difference in clinicopathological factors between homogeneous and heterogeneous VE1-positive acral melanoma. Both patients with BRAF V600E-positive acral melanoma and those with heterogeneous BRAF V600E had significantly shorter melanoma-specific survival than those with BRAF V600E-negative melanoma in Kaplan-Meier analysis (p = 0.0283 and p = 0.0065, respectively). These findings provide novel insights into the pathobiology of acral melanoma.

Pediatric Langerhans cell histiocytosis: the impact of mutational profile on clinical progression and late sequelae.

Langerhans cell histiocytosis (LCH) is a clonal histiocytic disorder with recurrent mutations of BRAF and MAP2K1, but data on the impact of genetic features on progression and long-term sequelae are sparse. Cases of pediatric LCH with long-term follow-up from our institution were analyzed for mutations in BRAFV600 and MAP2K1 exons 2 and 3 by immunostaining with mutation-specific VE1 antibody, as well as allele-specific PCR and sequencing, respectively. Clinical and follow-up data were obtained from our files and a questionnaire sent to all former patients. Sixteen of 37 (43%) evaluable cases showed BRAFV600E, one case a BRAFV600D and eleven (30%) a MAP2K1 mutation. Nine cases were unmutated for both genes. All cases with risk organ involvement showed either BRAFV600 or MAP2K1 mutation. Patients with BRAFV600 mutation excluding Hashimoto-Pritzker cases had a significantly higher risk for relapses (p = 0.02). Long-term sequelae were present in 19/46 (41%) patients (median follow-up 12.5 years, range 1.0 to 30.8) with a trend for higher rates in mutated cases (mutated = 9/17, 53% versus non-BRAFV600/MAP2K1 mutated = 2/7, 29%). In addition, 8/9 cases with skin involvement including all Hashimoto-Pritzker cases (n = 3) were positive for BRAFV600E. Infants below 2 years more frequently had BRAFV600 mutations (p = 0.013). Despite favorable prognosis, pediatric LCH shows a high frequency of relapses and long-term medical sequelae.

The immunocytochemical expression of VE-1 (BRAF V600E-related) antibody identifies the aggressive variants of papillary thyroid carcinoma on liquid-based cytology.

BACKGROUND: The recently introduced monoclonal V600E antibody (clone VE1) is likely to be an alternative strategy for detecting this mutation in thyroid lesions. Although VE1 immunostaining and molecular methods used to assess papillary thyroid carcinoma in surgical specimens are in good agreement, evaluation of VE1 in cytology and cell block samples is rarely performed, and its diagnostic value in cytology has not been well established. In this study, we sought to determine if VE1 is suitable for fine needle aspiration (FNA) and cell block methods. METHODS: A total of 86 patients who had BRAF V600E mutations were investigated with molecular and immunocytochemical (ICC) analysis in 45 FNA and 41 cell blocks. In total, 83 (96.5%) patients underwent surgical treatment. Assessment of BRAF V600E mutation status was performed in 72 (83.7%) cases. RESULTS: Among the 72 cases analysed, 54 cases agreed (ICC+/BRAF+ or ICC-/BRAF-), seven cases were false positive (ICC+/BRAF-) and 11 cases were false negative (ICC-/BRAF+). False negative cases were not detected in the cell block method. The statistical analysis showed that sensitivity and specificity of ICC for detecting the BRAF V600E mutation were 61% and 77% in FNA samples and 100% and 73% in cell block. CONCLUSION: The use of antibody VE-1 is a reliable method and a negative result of VE1 immunostaining might help to save time and money, restricting the molecular test to antibody-positive cases only. The identification of the aggressive variants of papillary carcinoma might be enabled by the expression of the antibody in neoplastic cells with tall cell features.

Immunohistochemistry is a feasible method to screen BRAF V600E mutation in colorectal and papillary thyroid carcinoma.

AIMS: To investigate whether immunohistochemistry (IHC) could be used to screen BRAF mutation status in formalin-fixed paraffin-embedded (FFPE) colorectal carcinoma (CRC) and papillary thyroid carcinoma (PTC) samples. METHODS: Eight surgical resected samples, including 2 CRCs with mutated BRAF V600E, 2 PTCs with mutated BRAF V600E, 2 CRCs with wild-type BRAF and 2 PTCs with wild-type BRAF, were selected to explore the optimized IHC conditions for BRAF V600E (VE1) immunostaining using BenchmarkXT automated immunostainer VENTANA Medical System. BRAF V600E status was tested by optimized IHC and ARMS-PCR methods in 255 samples (123 CRCs and 132 PTCs). Sanger sequencing was performed to validate the BRAF V600E status if discordant results were found between optimized IHC and ARMS-PCR methods. RESULTS: Antigen retrieval time in 32 min and 64 min showed satisfactory intensity and homogeneity of BRAF V600E staining in CRC and PTC samples, respectively. The concordance between IHC and ARMS/Sanger sequencing was 99.2% (122/123) in CRCs and 96.2% (127/132) in PTCs. In CRCs, the sensitivity of IHC staining for BRAF V600E was 100% (3/3) and the specificity was 99.1% (119/120). In PTCs, the sensitivity was 100% (106/106) and specificity was 80.8% (21/26). The overall concordance across all cases was 97.6% (249/255). CONCLUSION: The appropriate antigen retrieval protocol is critical for accurate analysis of BRAF V600E status by Benchmark XT automated immunostainer. IHC is a suitable method to screen BRAF V600E mutation in FFPE samples of CRCs and PTCs.

A Graft Mimic Strategy for Verticillium Resistance in Tomato.

Grafting vegetables for disease resistance has increased greatly in popularity over the past 10 years. Verticillium wilt of tomato is commonly controlled through grafting of commercial varieties on resistant rootstocks expressing the Ve1 R-gene. To mimic the grafted plant, proteomic analyses in tomato were used to identify a suitable root-specific promoter (TMVi), which was used to express the Ve1-allele in susceptible Craigella (Cs) tomato plants. The results indicate that when infected with Verticillim dahliae, race 1, the transformed plants are comparable to resistant cultivars (Cr) or grafted plants.

BRAF V600E mutation and BRAF VE1 immunoexpression profiles in different types of glioblastoma.

Abnormalities in proto-oncogene B-Raf (BRAF) are typical in several subgroups of gliomas, including pilocytic astrocytomas, optic nerve gliomas, pleomorphic xanthoastrocytomas (PXA), anaplastic PXAs and gangliogliomas. However, they are rarely reported in adult gliomas. BRAF alterations are frequent in a distinct variant of glioblastomas (GBMs) known as epithelioid GBMs (E-GBMs). There are limited studies on whether immunohistochemistry (IHC) can be used to determine the presence of BRAF VE1 mutations in these tumors. The aim of the current study was to examine BRAF V600E mutations in 20 GBMs, including GBMs with epithelioid features, giant cell GBMs and conventional GBMs. V600 mutations were detected using the Cobas 4800 BRAF V600 Mutation Test, and IHC analysis was also performed. Of the 6 cases of GBM with epithelioid features, 1 exhibited a BRAF V600E mutation, while the other cases did not. IHC staining was positive in 3 out of the 8 conventional GBMs. Vemurafenib is a targeted therapy that has mainly been used for the treatment of melanoma patients for several years, and as a possible alternative treatment for cases of GBM harboring BRAF mutations, its existence may make testing for BRAF status important.

Defence cascade in Verticillium-infected grafted tomato.

In tomato the Ve1-gene provides resistance to the vascular pathogen, Verticillium dahliae, race 1; ve1 plants are susceptible. Reciprocal grafts of resistant and susceptible tomato near-isolines were used to examine proteomic changes and, in particular, the effect of the Ve1-gene on the defence/stress protein cascade induced during Verticillium wilt. Based on label-free LC-MS, the results indicate that this defence response is cell-specific, correlates with overall fungal colonization and is mitigated by Ve1 function. The influence of the Ve1-gene in resistant tissues, however, is not actually transferred to susceptible tissues in the grafted plant.

Desmoplastic non-infantile astrocytoma/ganglioglioma: rare low-grade tumor with frequent BRAF V600E mutation.

Desmoplastic infantile astrocytoma/ganglioglioma (DIA/DIG) are rare primary glioneuronal tumors that comprise 0.5% to 1.0% of all intracranial tumors. While BRAF mutation is found in up to 50% of pediatric gangliogliomas, data for DIA/DIGs is limited. This study was carried out to evaluate the frequency of BRAF V600E mutation in DIA/DIG. All cases of DIA/DIGs diagnosed over 7 years (2010-2016) were reviewed retrospectively. The clinical, radiological and histopathological characteristics of these patients were evaluated along with immunohistochemical analysis for glial and neuronal markers. We evaluated presence of BRAF V600E mutation by Sanger sequencing and immunohistochemistry using monoclonal antibody against clone VE1. Eight cases of desmoplastic infantile astrocytoma/ganglioglioma (6 DIGs and 2 DIAs) were evaluated. Four cases were of infantile type (age 10 months-2 years) and 4 cases of non-infantile type (age 10-14 years). BRAF VE1 immunohistochemistry (IHC) was positive in four cases (two DIG and two DIA). All these four cases also showed heterozygous BRAF V600E mutation (T replaced by A at nucleotide position 1799). Sequencing didn't detect BRAF mutation in any additional case. All four cases harboring this mutation were non-infantile (10-14 years). Cases with infantile presentation didn't carry this mutation. None of the cases showed recurrence on follow-up. Thus BRAF V600E mutation is common in desmoplastic non-infantile astrocytoma/ganglioglioma, but does not affect the prognosis. Identification of BRAF status opens the possibility of targeted therapies for the subset of cases that clinically progress post-resection.

BRAFV600E mutations and immunohistochemical expression of VE1 protein in low-grade serous neoplasms of the ovary.

AIMS: The most common BRAF mutation in ovarian low-grade serous neoplasms (LGSNs) involves substitution of valine by glutamic acid at position 600 (V600E). Small studies have demonstrated high specificity of immunohistochemistry with mutation-specific monoclonal antibody VE1. We sought to investigate the expression of VE1 protein in LGSNs and its correlation with BRAF mutation-associated histological features and BRAF mutation status. METHODS AND RESULTS: We reviewed pathology reports and available slides from ovarian serous borderline tumours (SBTs) and low-grade serous carcinomas (LGSCs) diagnosed between 2000 and 2012. VE1 immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue sections. Tumours with ≥50% positive cells were considered positive. Of 121 LGSNs, there were 73 SBTs, eight SBTs with micropapillary features (mpSBT) and 40 LGSCs (22 primary, 18 metastatic). VE1 was positive in 52% (38 of 73) of SBTs and 9% (two of 22) of primary LGSCs, and in none of the mpSBTs and metastatic LGSCs (P < 0.0001). Of 76 tumours with known mutation status, 42 (55%) harboured mutations, including BRAFV600E (26, 34%), KRASG12D (eight, 11%), and KRASG12V (eight, 11%). BRAFV600E mutations were present in 48% (25 of 52) of SBTs and 5% (one of 22) of LGSCs (P < 0.0001). VE1 was positive in 96% (25 of 26) of BRAFV600E -mutated tumours and correlated with BRAF mutation-associated histological features (P < 0.0001). CONCLUSIONS: BRAFV600E mutations are significantly more common in SBTs than in LGSCs. Immunohistochemical expression of VE1 protein is associated strongly with BRAFV600E mutation and BRAF mutation-associated histological features. VE1 immunohistochemistry is a reliable method for the detection of BRAFV600E mutations.

Sensitivity and Usefulness of VE1 Immunohistochemical Staining in Acral Melanomas with BRAF Mutation.

BACKGROUND: Acral melanomas are known to have a low frequency of BRAF mutation, in contrary to higher KIT mutation. Recently, VE1 immunostaining was reported to have a good correlation with BRAF mutation status. OBJECTIVE: We aimed to evaluate the clinicopathological features of BRAF-mutated acral melanomas and validate the correlation of the VE1 immunohistochemical stains in those cases. METHODS: The clinical features (age, sex, anatomical site), and histopathological characteristics of 41 patients with acral melanoma were evaluated. We performed a next-generation sequencing to detect BRAF mutation status. We also determined the correlation of VE1 immunohistochemical staining with BRAF mutation status. RESULTS: Among 19 acral melanomas with BRAF mutation, common histopathological subtype was acral lentiginous melanoma (8/19, 42%) and nodular melanoma (8/19, 42%) and superficial spreading melanoma (3/19, 16%) followed. VE1 immunostaining results were positive in all 15 cases with BRAF V600E mutation (sensitivity 100%), and negative in 4 cases of BRAF non-V600E mutation. However, VE1 immunostaining was negative in all 22 patients with BRAF wild-type. CONCLUSION: VE1 immunostaining had a good correlation with BRAF V600E mutation status.

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 7 mo-17 y) 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.