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      Reflectance confocal microscopy features of BRAF V600E mutated thin melanomas detected by immunohistochemistry

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          Abstract

          The classification of melanoma into four histological subtypes has been questioned regarding its clinical validity in providing relevant information for treatment for metastatic tumors. Specific genetic alterations are associated with particular clinical and histopathological features, suggesting that these could be helpful in refining existing melanoma classification schemes. We analyzed BRAF V600E mutated melanomas to explore the Reflectance confocal microscopy (RCM) utility as a screening aid in the evaluation of the most appropriate patients for genetic testing. Thus, 32 melanomas were assessed regarding their BRAF V600E mutational status. Experts blinded to dermoscopic images and V600E immunohistochemistry results evaluated RCM images regarding previously described melanoma features. BRAF positive melanomas were related to younger age (p = 0.035), invasive melanomas (p = 0.03) and to the presence of hiporreflective cells (p = 0.02), epidermal nests (p = 0.02), dermal-epidermal junction nests (p = 0.05), edged papillae (p = 0.05), and bright dots (p = 0.05), and to absence of junctional thickening due to isolated cells (p = 0.01) and meshwork (p = 0.02). This study can not characterize other mutations in the BRAF, because the immunohistochemistry is specific to the type V600E. The findings should encourage the genetic evaluation of BRAF mutation. This study highlights the potential of RCM as a supplementary tool in the screening of BRAF-mutated melanomas.

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          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.
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            The impact of in vivo reflectance confocal microscopy for the diagnostic accuracy of melanoma and equivocal melanocytic lesions.

            In vivo confocal reflectance microscopy recently showed promising results for melanoma (MM) diagnosis on a limited series. The aim of the study was to evaluate the sensitivity and specificity of confocal features for the diagnosis of MM 351 equivocal melanocytic lesions (136 MMs and 215 nevi) were evaluated for 37 confocal features by two blinded expert observers. Chi2 test, multivariate discriminant analysis and binary logistic regression were performed for the identification of the significant features and for testing newly created diagnostic models. Melanomas were mostly characterized by epidermal disarray and pagetoid cells in the epidermis, non-edged papillae, and cellular atypia at the junction, and atypical nests and bright nucleated cells in the upper dermis. On the other hand, regular dermal-epidermal architecture, and absence of pagetoid infiltration and atypical cells were suggestive of benign lesions. Five out of 136 melanomas, with mildly atypical melanocytes and occasional pagetoid cells at histopathology, were not diagnosed by confocal microscopy. Nevertheless, new diagnostic models showed no significant improvement compared with the previously proposed confocal microscopy algorithm. Owing to the visualization of cellular aspects, confocal microscopy seems useful for second level examination of clinically and dermoscopically equivocal lesions.
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              Immunohistochemistry is highly sensitive and specific for the detection of V600E BRAF mutation in melanoma.

              This study investigated the sensitivity and specificity of immunohistochemical (IHC) analysis using an anti-BRAF antibody to detect the presence of the BRAF V600E mutation in patients with metastatic melanoma. A total of 100 patients with American Joint Committee on Cancer stage IIIC unresectable or stage IV melanoma and who underwent tumor DNA BRAF mutation testing were selected. Paraffin-embedded, formalin-fixed melanoma biopsies were analyzed for the BRAF mutation status by independent, blinded observers using both conventional DNA molecular techniques and IHC with the novel BRAF V600E mutant-specific antibody, VE1. The antibody had a sensitivity of 97% (37/38) and a specificity of 98% (58/59) for detecting the presence of a BRAF V600E mutation. Of the BRAF-mutated cases, none of the non-V600E cases (including V600K) stained positive with the antibody (0/11). There were 5 cases with discordant BRAF mutation results. Additional molecular analysis confirmed the immunohistochemically obtained BRAF result in 3 cases, suggesting that the initial molecular testing results were incorrect. Two of these patients would not have received a BRAF inhibitor on the basis of the initial false-negative mutation testing result. Two cases remained discordant. The reported IHC method is an accurate, rapid, and cost-effective method for detecting V600E BRAF mutations in melanoma patients. Clinical use of the V600E BRAF antibody should be a valuable supplement to conventional mutation testing and allow V600E mutant metastatic melanoma patients to be triaged rapidly into appropriate treatment pathways.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ValidationRole: Visualization
                Role: ConceptualizationRole: InvestigationRole: Project administrationRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Formal analysis
                Role: Project administrationRole: Validation
                Role: InvestigationRole: Project administrationRole: ResourcesRole: Validation
                Role: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: MethodologyRole: Resources
                Role: ConceptualizationRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                29 June 2017
                2017
                : 12
                : 6
                : e0179745
                Affiliations
                [1 ]Cutaneous Oncology Department, AC Camargo Cancer Center, São Paulo, Brazil
                [2 ]Dermaimage Medical Associates, São Paulo, Brazil
                [3 ]Barretos Cancer Hospital, Teaching and Research Institute, Barretos, São Paulo, Brazil
                [4 ]Anatomy Pathology Department, AC Camargo Cancer Center, São Paulo, Brazil
                [5 ]Dermatology Service, Memorial Sloan Kettering Skin Cancer Center, New York, United States of America
                University of Queensland Diamantina Institute, AUSTRALIA
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                [¤a]

                Current address: Cutaneous Oncology Department, AC Camargo Cancer Center, São Paulo, Brazil.

                [¤b]

                Current address: Dermaimage Medical Associates, São Paulo, Brazil.

                ‡ These authors also contributed equally to this work.

                Author information
                http://orcid.org/0000-0002-8987-8156
                Article
                PONE-D-17-07514
                10.1371/journal.pone.0179745
                5491027
                28662062
                659253a5-0e83-4550-bf96-173d6e8550f4
                © 2017 Urvanegia et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 24 February 2017
                : 2 June 2017
                Page count
                Figures: 4, Tables: 7, Pages: 14
                Funding
                Funded by: Fapesp (São Paulo Research Foundation/ Fundação de Amparo à Pesquisa do Estado de São Paulo)
                Award ID: 2012 /13090-5
                Award Recipient :
                The research was funded by Grants from FAPESP (São Paulo Research Foundation/ Fundação de Amparo à Pesquisa do Estado de São Paulo) process number 2012 /13090-5, URL: http://www.fapesp.br/, to JPD.
                Categories
                Research Article
                Medicine and Health Sciences
                Oncology
                Cancers and Neoplasms
                Melanomas
                Biology and Life Sciences
                Anatomy
                Histology
                Medicine and Health Sciences
                Anatomy
                Histology
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Anatomical Pathology
                Histopathology
                Research and Analysis Methods
                Specimen Preparation and Treatment
                Staining
                Negative Staining
                Research and Analysis Methods
                Histochemistry and Cytochemistry Techniques
                Immunohistochemistry Techniques
                Research and Analysis Methods
                Immunologic Techniques
                Immunohistochemistry Techniques
                Research and Analysis Methods
                Specimen Preparation and Treatment
                Staining
                Immunostaining
                Research and Analysis Methods
                Specimen Preparation and Treatment
                Staining
                Cell Staining
                Research and Analysis Methods
                Microscopy
                Light Microscopy
                Confocal Microscopy
                Custom metadata
                All relevant data have been uploaded to Dryad at: http://doi.org/10.5061/dryad.s4f13.

                Uncategorized
                Uncategorized

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