RESUMO
Ocular melanoma, including uveal melanoma (UM) and conjunctival melanoma (CM), is the most common ocular cancer among adults with a high rate of recurrence and poor prognosis. Loss of epigenetic homeostasis disturbed gene expression patterns, resulting in oncogenesis. Herein, we comprehensively analyzed the DNA methylation, transcriptome profiles, and corresponding clinical information of UM patients through multiple machine-learning algorithms, finding that a methylation-driven gene RBMS1 was correlated with poor clinical outcomes of UM patients. RNA-seq and single-cell RNA-seq analyses revealed that RBMS1 reflected diverse tumor microenvironments, where high RBMS1 expression marked an immune active TME. Furthermore, we found that tumor cells were identified to have the higher communication probability in RBMS1+ state. The functional enrichment analysis revealed that RBMS1 was associated with pigment granule and melanosome, participating in cell proliferation as well as apoptotic signaling pathway. Biological experiments were performed and demonstrated that the silencing of RBMS1 inhibited ocular melanoma proliferation and promoted apoptosis. Our study highlighted that RBMS1 reflects a distinct microenvironment and promotes tumor progression in ocular melanoma, contributing to the therapeutic customization and clinical decision-making.
Assuntos
Proliferação de Células , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Melanoma , Microambiente Tumoral , Neoplasias Uveais , Humanos , Melanoma/patologia , Melanoma/genética , Melanoma/metabolismo , Neoplasias Uveais/genética , Neoplasias Uveais/patologia , Neoplasias Uveais/metabolismo , Apoptose/genética , Metilação de DNA , Neoplasias da Túnica Conjuntiva/genética , Neoplasias da Túnica Conjuntiva/patologia , Neoplasias da Túnica Conjuntiva/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Masculino , Feminino , Linhagem Celular TumoralRESUMO
PURPOSE: To identify high-risk histopathologic and molecular features of local recurrence, nodal metastasis, distant metastasis (DM) and disease-specific death (DSD) in conjunctival melanoma (CoM). METHODS: Ninety patients with pathologically diagnosed CoM between 2008 and 2023 were enrolled. Immunohistochemistry staining of BRAFV600E, NRASQ61R, CD117, PD-1 and PD-L1 was performed in 65 and 45 patients, respectively. Cox regression and Kaplan-Meier survival analysis were conducted to identify risk factors for local recurrence, nodal metastasis, DM and DSD. RESULTS: Pathologically, ulceration (hazard ratio [HR]: 3.170; 95% CI: 1.312-7.659; p = 0.01) and regression (HR: 3.196; 95% CI: 1.094-9.335; p = 0.034) were risk factors for DM. Tumour thickness ≥ 4 mm (HR: 4.889; 95% CI: 1.846-12.946; p = 0.001) and regression (HR: 4.011; 95% CI: 1.464-10.991; p = 0.007) were risk factors for DSD. For patients with tumour thickness < 4 mm, the presence of ulceration indicated a higher risk of nodal metastasis (log-rank p = 0.0011), DM (log-rank p = 0.00051) and DSD (log-rank p = 0.02). Patients with regression (+)/tumour-infiltrating lymphocytes (TILs) (+) had a higher risk for DM (log-rank p = 0.011) and DSD (log-rank p = 0.0032). Molecularly, the positive rate of BRAFV600E, NRASQ61R, CD117, PD-1 and PD-L1 was 40.00% (26/65), 43.08% (28/65), 70.77% (46/65), 46.67% (21/45) and 28.89% (13/45), respectively. Positive BRAFV600E was identified as an independent risk factor for DM (HR: 2.533; 95% CI: 1.046-6.136, p = 0.039). The expression level of BRAFV600E was positively correlated with vascular invasion (p = 0.01), as well as the expression levels of PD-1 (p = 0.038) and PD-L1 (p = 0.049). CONCLUSIONS: Tumour thickness ≥ 4 mm, ulceration, the coexistence of regression and TILs, and positive BRAFV600E were risk factors for poor prognosis of CoM patients. Besides, expression level of BRAFV600E was positively correlated with the expression levels of PD-1 and PD-L1.
Assuntos
Neoplasias da Túnica Conjuntiva , Melanoma , Humanos , Melanoma/genética , Melanoma/diagnóstico , Melanoma/patologia , Melanoma/metabolismo , Neoplasias da Túnica Conjuntiva/genética , Neoplasias da Túnica Conjuntiva/patologia , Neoplasias da Túnica Conjuntiva/metabolismo , Neoplasias da Túnica Conjuntiva/diagnóstico , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Estudos Retrospectivos , Fatores de Risco , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Adulto , Recidiva Local de Neoplasia , Metástase Linfática , Imuno-Histoquímica , Proteínas Proto-Oncogênicas B-raf/genética , Idoso de 80 Anos ou mais , Seguimentos , Taxa de Sobrevida/tendências , Estadiamento de Neoplasias , PrognósticoRESUMO
This study characterizes the transcriptional profile and the cellular tumor microenvironment of conjunctival extranodal marginal zone lymphoma (EMZL) and identifies prognostically relevant biomarkers. Ten formalin-fixed and paraffin-embedded conjunctival EMZL and eight healthy conjunctival specimens were analyzed by Massive Analysis of cDNA Ends (MACE) RNA sequencing. The 3417 upregulated genes in conjunctival EMZL were involved in processes such as B cell proliferation and Rac protein signaling, whereas the 1188 downregulated genes contributed most significantly to oxidative phosphorylation and UV protection. The tumor microenvironment, as determined by deconvolution analysis, was mainly composed of multiple B cell subtypes which reflects the tumor's B cell lineage. However, several T cell types, including T helper 2 cells and regulatory T cells, as well as innate immune cell types, such as anti-inflammatory macrophages and plasmacytoid dendritic cells, were also strongly enriched in conjunctival EMZL. A 13-biomarker prognostic panel, including S100A8 and S100A9, classified ocular and extraocular tumor recurrence, exceeded prognostic accuracy of Ann Arbor and American Joint Committee on Cancer (AJCC) staging, and demonstrated prognostic value for patient survival in 21 different cancer types in a database of 12,332 tumor patients. These findings may lead to new options of targeted therapy and may improve prognostic prediction for conjunctival EMZL.
Assuntos
Neoplasias da Túnica Conjuntiva , Linfoma de Zona Marginal Tipo Células B , Humanos , Neoplasias da Túnica Conjuntiva/genética , Neoplasias da Túnica Conjuntiva/metabolismo , Neoplasias da Túnica Conjuntiva/patologia , Prognóstico , Linfoma de Zona Marginal Tipo Células B/genética , Linfoma de Zona Marginal Tipo Células B/patologia , Linfoma de Zona Marginal Tipo Células B/terapia , Microambiente Tumoral/genéticaRESUMO
OBJECTIVE: The aim of this study was to evaluate the expression of endoglin and its correlation with histopathological and clinical findings in conjunctival nevi. METHODS: The study included archival formalin-fixed, paraffin-embedded tissue sections of 44 patients with conjunctival nevi. Immunohistochemical staining for CD105 had been performed with monoclonal mouse antihuman CD105 antibodies. The intratumoral microvessel density for quantification of tumoral vascularization had been determined by this marker. RESULTS: The expression of CD105 was positive in 30 (68.2%) cases. There was a statistically significant difference in the level of CD105 expression regarding the histological type of nevus (p=0.03) and intralesional cysts status (p=0.02). Spearman's rho (ρ -0.316) revealed a significant negative correlation between the expression of endoglin and the histological type of nevus (p=0.03) and between the expression of endoglin and the presence of intralesional cysts (ρ -0.380, p=0.01). CONCLUSION: This study suggests that endoglin could be a useful diagnostic and prognostic marker in differentiating between benign and malignant melanocytic ocular lesions.
Assuntos
Neoplasias da Túnica Conjuntiva , Cistos , Endoglina , Nevo Pigmentado , Neoplasias Cutâneas , Animais , Biomarcadores Tumorais/metabolismo , Neoplasias da Túnica Conjuntiva/metabolismo , Neoplasias da Túnica Conjuntiva/patologia , Cistos/metabolismo , Endoglina/biossíntese , Humanos , Camundongos , Nevo Pigmentado/metabolismo , Nevo Pigmentado/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologiaRESUMO
PURPOSE: Sunlight-induced p53 mutations are known to contribute towards increased risk of ocular surface squamous neoplasia (OSSN). Stratifin (14-3-3σ)/HEM (human epithelial marker) is a p53-mediated inhibitor of cell cycle progression and has been shown to be a target of epigenetic deregulation in various carcinomas. In the present study, Stratifin expression, its promoter methylation status as well as expression of mutant p53 in early and advanced AJCC stages (8th edition) of OSSN, was evaluated. METHODS: Sixty-four OSSN [20 conjunctival intraepithelial neoplasia (CIN) and 44 squamous cell carcinoma (SCC)] patients were registered for this study, and they were followed up for 36-58 months (mean 48 ± 3.6). Immunoexpression of Stratifin and mutant p53 protein, mRNA expression of Stratifin by reverse transcription polymerase chain reaction (PCR) and methylation status of Stratifin by methylation-specific PCR, was undertaken. RESULTS: Hypermethylation of Stratifin promoter in 63% (40/64), loss of Stratifin expression in 75% (48/64) and downregulation of Stratifin mRNA in 61% (39/64) were observed. Stratifin hypermethylation was significantly associated with reduced disease-free survival in both early and advanced T stage SCC cases. Expression of mutant p53 expression was seen in 48% (31/64) OSSN cases. Of the 31 patients with mutant p53 expression, 87% (27/31) also demonstrated loss of Stratifin immunoexpression. A significant association was seen between mutant p53 expression and Stratifin loss (p = 0.01) in advanced T stage SCC cases. CONCLUSIONS: Hypermethylation of Stratifin gene and its reduced mRNA expression both are potential biomarkers for identifying high-risk OSSN patients. Aberrant methylation of Stratifin and simultaneous mutant p53 expression implicates involvement of p53-Stratifin mediated signalling pathway in the pathogenesis of OSSN.
Assuntos
Proteínas 14-3-3/genética , Carcinoma de Células Escamosas/genética , Neoplasias da Túnica Conjuntiva/genética , Exorribonucleases/genética , Regulação da Expressão Gênica , Mutação , Estadiamento de Neoplasias , Proteína Supressora de Tumor p53/genética , Proteínas 14-3-3/biossíntese , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/metabolismo , Criança , Neoplasias da Túnica Conjuntiva/diagnóstico , Neoplasias da Túnica Conjuntiva/metabolismo , Análise Mutacional de DNA , DNA de Neoplasias/genética , Exorribonucleases/biossíntese , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Supressora de Tumor p53/biossíntese , Adulto JovemRESUMO
Uveal melanoma (UM) and conjunctival melanoma (CM) are ocular malignancies that give rise to life-threatening metastases. Although local disease can often be treated successfully, it is often associated with significant vision impairment and treatments are often not effective against metastatic disease. Novel treatment modalities that preserve vision may enable elimination of small tumors and may prevent subsequent metastatic spread. Very few mouse models of metastatic CM and UM are available for research and for development of novel therapies. One of the challenges is to follow tumor growth in-vivo and to determine the right size for treatment, mainly of the posterior, choroidal melanoma. Hence, the purpose of this study was to establish a simple, noninvasive imaging tool that will simplify visualization and tumor follow-up in mouse models of CM and UM. Tumors were induced by inoculation of murine B16LS9 cells into the sub-conjunctival or the choroidal space of a C57BL/6 mouse eye under a surgical microscope. Five to ten days following injection, tumor size was assessed by Phoenix MicronIV™ image-guided Optical Coherence Tomography (OCT) imaging, which included a real-time camera view and OCT scan of the conjunctiva and the retina. In addition, tumor size was evaluated by ultrasound and histopathological examination of eye sections. Tumor growth was observed 5-9 days following sub-conjunctival or sub-retinal injection of seven-thousand or seventy-thousand cells, respectively. A clear tumor mass was detected at these regions using the MicronIV™ imaging system camera and OCT scans. Histology of eye sections confirmed the presence of tumor tissue. OCT allowed an accurate measurement of tumor size in the UM model and a qualitative assessment of tumor size in the CM model. Moreover, OCT enabled assessing the success rate of the choroidal tumor induction and importantly, predicted final tumor size already on the day of cell inoculation. In conclusion, by using a simple, non-invasive imaging tool, we were able to follow intraocular tumor growth of both CM and UM, and to define, already at the time of cell inoculation, a grading scale to evaluate tumor size. This tool may be utilized for evaluation of new mouse models for CM and UM, as well as for testing new therapies for these diseases.
Assuntos
Neoplasias da Túnica Conjuntiva/diagnóstico por imagem , Modelos Animais de Doenças , Melanoma/diagnóstico por imagem , Tomografia de Coerência Óptica , Ultrassonografia , Neoplasias Uveais/diagnóstico por imagem , Animais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Neoplasias da Túnica Conjuntiva/metabolismo , Neoplasias da Túnica Conjuntiva/patologia , Imuno-Histoquímica , Antígeno MART-1/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Antígenos Específicos de Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monofenol Mono-Oxigenase/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Uveais/metabolismo , Neoplasias Uveais/patologiaRESUMO
PURPOSE: To determine the usefulness of melan-A, SOX10, HMB45, and p16 immunohistochemical stains in the distinction between the low-grade and high-grade conjunctival melanocytic intraepithelial lesions, either independently or as components of an immunohistochemical panel. DESIGN: Retrospective observational case series. METHODS: Institutional pathology records between 2014 and 2018 were searched for all patients with conjunctival melanocytic intraepithelial lesions. Biopsies without supporting clinical history or tissue available for review and immunohistochemical analysis were excluded. Clinical, histopathologic, and immunohistochemical (p16, SOX10, HMB45, and Ki-67) findings were recorded. RESULTS: Thirty-one patients underwent 47 biopsies for conjunctival melanocytic lesions between 2014 and 2018. Pathologic diagnoses were low-grade conjunctival melanocytic intraepithelial lesion (n = 18, 38%) and high-grade conjunctival melanocytic intraepithelial lesion/melanoma in situ (n = 29, 62%). The addition of melan-A and SOX10 immunohistochemical stains resulted in an upgrade of conjunctival melanocytic intraepithelial lesion from low-grade to high-grade in 2 (4%) of 47 cases. The addition of melan-A and SOX10 immunohistochemical stains did not downgrade any of the histomorphologically high-grade lesions. In a clinical-pathologic multivariable model, the parameters most predictive of high-grade melanocytic intraepithelial lesion/melanoma in situ were involvement of the caruncle (odds ratio [OR] = 19, confidence interval [CI] 1.6-212; P = .02] and p16 cytoplasmic H-score >30 (OR = 81, CI 2.7 to >999; P = .01) CONCLUSION: Although the stains for melanocytic markers melan-A and SOX10 facilitate assessment of melanocytic intraepithelial lesions, the current immunohistochemical panels have limited value in distinction between the low-grade and high-grade intraepithelial melanocytic proliferations and need to be used judiciously.
Assuntos
Neoplasias da Túnica Conjuntiva/diagnóstico , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Imuno-Histoquímica/métodos , Antígenos Específicos de Melanoma/metabolismo , Nevo Pigmentado/diagnóstico , Fatores de Transcrição SOXE/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Neoplasias da Túnica Conjuntiva/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nevo Pigmentado/metabolismo , Estudos Retrospectivos , Adulto Jovem , Antígeno gp100 de MelanomaAssuntos
Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/transmissão , Neoplasias da Túnica Conjuntiva/metabolismo , SARS-CoV-2/fisiologia , Serina Endopeptidases/metabolismo , Carcinoma/metabolismo , Carcinoma/patologia , Carcinoma/virologia , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/virologia , Neoplasias da Túnica Conjuntiva/patologia , Neoplasias da Túnica Conjuntiva/virologia , Humanos , Melanoma/metabolismo , Melanoma/patologia , Melanoma/virologia , Nevo/metabolismo , Nevo/patologia , Nevo/virologiaRESUMO
Recent advances in the field of biomedical research allow for elucidation of the transcriptional signature of rare tumors such as conjunctival squamous cell carcinoma (SCC). In this study we compare its expression profile to conjunctival papilloma (Pap) and healthy conjunctival tissue (Ctrl) and develop a classification tool to differentiate these entities. Seven conjunctival SCC, seven Pap and ten Ctrl were formalin-fixed and paraffin-embedded (FFPE) and analyzed using Massive Analysis of cDNA Ends (MACE) RNA sequencing. Differentially expressed genes (DEG) and gene ontology (GO) clusters were explored and the abundance of involved cell types was quantified by xCell. Finally, a classification model was developed to distinguish SCC from Pap and Ctrl. Among the most prominent DEG in SCC a plethora of keratins were upregulated when compared to Pap and Ctrl. xCell analysis revealed an enrichment of immune cells, including activated dendritic cells and T-helper type 1 cells (Th1), in SCC when compared to Ctrl. The generated classification model could reliably discriminate between the three entities according to the expression pattern of 30 factors. This study provides a transcriptome-wide gene expression profile of rare conjunctival SCC. The analysis identifies distinct keratins, as well as dendritic and Th1 cells as important mediators in SCC. Finally, the provided gene expression classifier may become an aid to the conventional histological classification of conjunctival tumors in uncertain cases.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias da Túnica Conjuntiva/metabolismo , Papiloma/metabolismo , Transcriptoma , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/diagnóstico , Estudos de Casos e Controles , Neoplasias da Túnica Conjuntiva/classificação , Neoplasias da Túnica Conjuntiva/diagnóstico , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Papiloma/diagnóstico , Análise de Sequência de RNA , Adulto JovemRESUMO
Conjunctival melanoma (CJM) is a rare but potentially lethal and highly-recurrent cancer of the eye. Similar to cutaneous melanoma (CM), it originates from melanocytes. Unlike CM, however, CJM is relatively poorly characterized from a genomic point of view. To fill this knowledge gap and gain insight into the genomic nature of CJM, we performed whole-exome (WES) or whole-genome sequencing (WGS) of tumor-normal tissue pairs in 14 affected individuals, as well as RNA sequencing in a subset of 11 tumor tissues. Our results show that, similarly to CM, CJM is also characterized by a very high mutation load, composed of approximately 500 somatic mutations in exonic regions. This, as well as the presence of a UV light-induced mutational signature, are clear signs of the role of sunlight in CJM tumorigenesis. In addition, the genomic classification of CM proposed by TCGA seems to be well-applicable to CJM, with the presence of four typical subclasses defined on the basis of the most frequently mutated genes: BRAF, NF1, RAS, and triple wild-type. In line with these results, transcriptomic analyses revealed similarities with CM as well, namely the presence of a transcriptomic subtype enriched for immune genes and a subtype enriched for genes associated with keratins and epithelial functions. Finally, in seven tumors we detected somatic mutations in ACSS3, a possible new candidate oncogene. Transfected conjunctival melanoma cells overexpressing mutant ACSS3 showed higher proliferative activity, supporting the direct involvement of this gene in the tumorigenesis of CJM. Altogether, our results provide the first unbiased and complete genomic and transcriptomic classification of CJM.
Assuntos
Neoplasias da Túnica Conjuntiva/genética , Variações do Número de Cópias de DNA , Melanoma/genética , Mutação , Transcriptoma , Linhagem Celular Tumoral , Neoplasias da Túnica Conjuntiva/metabolismo , Feminino , Humanos , Masculino , Melanoma/metabolismo , Pessoa de Meia-Idade , Neurofibromina 1/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas ras/genéticaRESUMO
Ocular melanoma is classified under the category of noncutaneous melanomas. Noncutaneous melanomas are relatively rare. Ocular melanoma commonly arises from choroid. Conjunctival melanoma is a rare but potentially lethal form of ocular melanoma. It can invade locally. Systemic spread is seen in up to 25% of cases, often associated with lymph node involvement. Metastatic sites include the lungs, liver, gastrointestinal tract, and the central nervous system. F-18 fluorodeoxyglucose positron emission tomography/computed tomography (F-18 FDG PET-CT) scanning is indicated for staging cutaneous melanoma patients. However, few studies have evaluated its role in the management of conjunctival melanoma. This case highlights the use of F-18 FDG PET/CT for imaging, preoperative staging, and evaluation for metastasis in conjunctival melanoma.
Assuntos
Neoplasias da Túnica Conjuntiva/patologia , Fluordesoxiglucose F18/metabolismo , Melanoma/patologia , Recidiva Local de Neoplasia/patologia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Neoplasias da Túnica Conjuntiva/diagnóstico por imagem , Neoplasias da Túnica Conjuntiva/metabolismo , Humanos , Masculino , Melanoma/diagnóstico por imagem , Melanoma/metabolismo , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/diagnóstico por imagem , Recidiva Local de Neoplasia/metabolismo , Prognóstico , Compostos Radiofarmacêuticos/metabolismoRESUMO
This study characterizes the transcriptome and the cellular tumor microenvironment (TME) of conjunctival melanoma (CM) and identifies prognostically relevant biomarkers. 12 formalin-fixed and paraffin-embedded CM were analyzed by MACE RNA sequencing, including six cases each with good or poor clinical outcome, the latter being defined by local recurrence and/or systemic metastases. Eight healthy conjunctival specimens served as controls. The TME of CM, as determined by bioinformatic cell type enrichment analysis, was characterized by the enrichment of melanocytes, pericytes and especially various immune cell types, such as plasmacytoid dendritic cells, natural killer T cells, B cells and mast cells. Differentially expressed genes between CM and control were mainly involved in inhibition of apoptosis, proteolysis and response to growth factors. POU3F3, BIRC5 and 7 were among the top expressed genes associated with inhibition of apoptosis. 20 genes, among them CENPK, INHA, USP33, CASP3, SNORA73B, AAR2, SNRNP48 and GPN1, were identified as prognostically relevant factors reaching high classification accuracy (area under the curve: 1.0). The present study provides new insights into the TME and the transcriptional profile of CM and additionally identifies new prognostic biomarkers. These results add new diagnostic tools and may lead to new options of targeted therapy for CM.
Assuntos
Neoplasias da Túnica Conjuntiva/genética , Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Recidiva Local de Neoplasia/genética , Microambiente Tumoral/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Neoplasias da Túnica Conjuntiva/metabolismo , Neoplasias da Túnica Conjuntiva/patologia , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Melanoma/metabolismo , Melanoma/patologia , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Prognóstico , TranscriptomaRESUMO
PURPOSE: Conjunctival squamous cell carcinoma (SCC) is primarily treated with surgical resection. SCC has various stages, and local recurrence is common. The purpose of this study was to determine molecular localization of epidermal growth factor receptor (EGFR) and the possibility of EGFR as a biomarker for the management of conjunctival SCC. METHODS: In this retrospective study, we performed immunohistochemistry to evaluate EGFR expression and localization in tumor cells, EGFR mutation-specific expression (E746-A750del and L858R), and human papillomavirus expression in a series of 29 conjunctival SCCs. RESULTS: All 29 tumors in our cohort were EGFR positive (100%). Twenty-one of 29 tumors (72%) showed focal EGFR staining, and seven (28%) showed diffuse EGFR staining. In addition, we calculated the percentages of the two most important mutations in EGFR (exon 19 746-A750del (8/29, 27.5%), exon 21 (L858R mutant (2/29, 6.8%)) in conjunctival SCCs. We observed that the translocation of EGFR from the membrane into the cytoplasm was related to clinical prognosis, as we detected correlations between EGFR cytoplasmic staining and final orbital exenteration and between decreased EGFR membrane staining and progression-free survival. CONCLUSIONS: EGFR is important in the pathology of ocular surface squamous neoplasia including SCC and is a prognostic factor. Increased understanding of EGFR mutations may have important implications for future treatment options.
Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias da Túnica Conjuntiva/genética , Adenocarcinoma/patologia , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/metabolismo , Estudos de Coortes , Neoplasias da Túnica Conjuntiva/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Neoplasias de Cabeça e Pescoço/genética , Humanos , Imuno-Histoquímica/métodos , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Mutação , Recidiva Local de Neoplasia/genética , Prognóstico , Estudos RetrospectivosRESUMO
Purpose: Previously, we demonstrated that Krüppel-like factor 4 (KLF4) promotes corneal epithelial (CE) homeostasis by suppressing epithelial-mesenchymal transition (EMT) and TGF-ß signaling. As TGF-ß affects epithelial apicobasal polarity (ABP) and plane of division, we investigated the role of KLF4 in these processes. Methods: Klf4 was ablated in adult ternary transgenic Klf4Δ/ΔCE (Klf4LoxP/LoxP/Krt12rtTA/rtTA/Tet-O-Cre) mouse CE using doxycycline chow. ABP and plane of division markers' expression in Klf4Δ/ΔCE and human ocular surface squamous neoplasia (OSSN) tissues relative to controls was evaluated by quantitative PCR, immunoblots, and/or immunofluorescent staining. Results: Klf4Δ/ΔCE CE cells displayed downregulation of apical Pals1 and Crumbs1, apicolateral Par3, and basolateral Scribble, as well as upregulation of Rho family GTPase Cdc42, suggesting disruption of ABP. Phalloidin staining revealed that the Klf4Δ/ΔCE CE actin cytoskeleton is disrupted. Klf4Δ/ΔCE cells favored vertical plane of division within 67.5° to 90° of the CE basement membrane (39% and 47% of the dividing cells relative to 23% and 26% in the control based on phospho-histone-H3 and survivin, respectively), resulting in more dividing cells within the Klf4Δ/ΔCE CE as reported previously. KLF4 was downregulated in human OSSN tissues that displayed EMT and downregulation of PAR3, PALS1, and SCRIB, consistent with a protective role for KLF4. Conclusions: By demonstrating that Klf4 ablation affects CE expression of ABP markers and Cdc42, cytoskeletal actin organization, and the plane of cell division and that KLF4 is downregulated in OSSN tissues that display EMT and lack ABP, these results elucidate the key integrative role of KLF4 in coordinating CE cell polarity and plane of division, loss of which results in OSSN.
Assuntos
Divisão Celular , Polaridade Celular , Epitélio Corneano/citologia , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proteínas de Ciclo Celular/metabolismo , Neoplasias da Túnica Conjuntiva/metabolismo , Neoplasias da Túnica Conjuntiva/patologia , Regulação para Baixo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fator 4 Semelhante a Kruppel , Proteínas de Membrana/metabolismo , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Núcleosídeo-Fosfato Quinase/metabolismo , Regulação para Cima , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
PURPOSE: The aim of this study was to investigate HIF-1α, HIF-2α, and ProExC expression in conjunctival intraepithelial neoplasia (CIN), to differentiate between metaplasia and dysplasia, and to access their value as diagnostic and prognostic immunohistochemical markers. Recurrence and progression into SCC (squamous cell carcinoma) were defined as endpoints. METHODS: Forty-three specimens including CIN I (2), CIN II (9), CIN III (29), with and without metaplasia, and metaplasia alone (3), as well as 21 conjunctival control specimens, were stained with antibodies against HIF-1α, HIF-2α, and ProExC. The percentage of positively stained cells were calculated and used for further analysis. RESULTS: The mean percentages of HIF-1α and HIF-2α were not increased in CIN. In comparison, the expressions of these markers were even significantly elevated in control specimens (p < 0.001). Upper epithelial cells in CIN were more often ProExC-positive compared with normal conjunctiva or metaplasia (p = 0.06 and p = 0.07). Cox proportional-hazards analysis was performed for characterization of factors influencing the combined endpoint and showed a significant elevated hazard ratio for staining with ProExC (p = 0.04) compared with HIF-1α (p = 0.26) and HIF-2α (p = 0.49). CONCLUSION: Our study shows that HIF-1α and HIF-2α do not serve as diagnostic or prognostic markers in CIN. ProExC seems to be a potential indicator for CIN, but not a reliable diagnostic marker. However, control specimens occasionally also display a high percentage of ProExC-positive cells and staining over the entire epithelial layer.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma de Células Escamosas/metabolismo , Neoplasias da Túnica Conjuntiva/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/patologia , Neoplasias da Túnica Conjuntiva/diagnóstico , Neoplasias da Túnica Conjuntiva/patologia , Feminino , Seguimentos , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos RetrospectivosRESUMO
BACKGROUND: Deep penetrating nevus (DPN) is not a widely recognised lesion on the conjunctiva and only a few cases consistent with combined DPN have been reported. METHODS: A review of all excised and histopathologically diagnosed conjunctival melanocytic lesions between 2003 and 2018 was performed in order to identify melanocytic nevi morphologically consistent with DPN. RESULTS: Thirty-four DPN were identified among 361 histopathologically examined conjunctival nevi (9.4%), including 33 (97%) combined with a common nevus and 1 (3%) pure DPN. The patients' age ranged from 7 to 51 years (median, 22 years). Clinically, 21 of 29 (72%) lesions with available data were darkly pigmented, and an increase in size and/or pigmentation was noted in 13 of 18 (72%) lesions with known history. All 24 lesions in which an immunohistochemical analysis was possible were diffusely positive for BRAFV600E (in DPN and common nevus components) and showed a diffuse nuclear positivity for beta catenin and cyclin D1 in the DPN component. None of the 21 lesions with available follow-up data recurred during a follow-up period from 0.3 to 16.3 years (median, 7.5 years). CONCLUSIONS: DPN of the conjunctiva is a relatively common lesion and usually presents as a combined nevus. Genetically, DPN of the conjunctiva are characterised by a combination of BRAFV600E mutation and activation of the beta catenin pathway. Recognition of DPN of the conjunctiva is important in order not to overdiagnose it as a melanoma, and to explain its potential atypical clinical features. DPN of the conjunctiva seems to be a benign lesion.
Assuntos
Neoplasias da Túnica Conjuntiva/patologia , Mutação , Nevo Pigmentado/patologia , Proteínas Proto-Oncogênicas B-raf/metabolismo , beta Catenina/metabolismo , Adolescente , Adulto , Criança , Neoplasias da Túnica Conjuntiva/metabolismo , Ciclina D1/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Nevo Pigmentado/metabolismo , Proteínas Proto-Oncogênicas B-raf/genéticaRESUMO
OBJECTIVE: To implement a double-staining technique to identify the most sensitive and specific combinations of melanoma antigen recognized by T cells (Melan-A), microphthalmia-associated transcription factor (MITF), human melanoma black 45 (HMB45), and Ki67 aiming to assist in the diagnosis of atypical melanocytic conjunctival lesions that are more prone to malignant progression. METHODS: Eight specimens of conjunctival melanoma and of primary acquired melanosis with moderate to severe atypia were double-immunostained with a combination of a cytoplasmic marker (anti-Melan-A or anti-HMB45), and a nuclear marker (anti-MITF or anti-Ki67). Eight specimens of normal conjunctiva and of conjunctival nevi served as controls. The specimens were processed using 3,3-diaminobenzidine substrate for nuclear stains and the fast-red substrate for cytoplasmic stains. Each slide was analyzed by light microscopy and provided a percent scale and a 0 to 4+ score for each nuclear and cytoplasmic component. RESULTS: Melan-A and MITF were strongly positive markers for all melanocytic cells, whereas Ki67 and HMB45 provided a variable response for identifying potentially proliferative or aggressive cells. HMB45 and MITF proved to be the best combination for differentiating between atypical and benign lesions on a percent scale and a 0 to 4+ scale (pâ¯=â¯0.0004), with the 3 other combinations providing mainly confirmatory diagnostic information (p < 0.05). CONCLUSIONS: Our study used an immunohistochemical double-staining approach to differentiate between atypical and benign melanocytic lesions of the conjunctiva. Our findings should aid in a more complete immunohistopathological diagnosis of conjunctival melanocytic lesions, particularly in diagnostically difficult cases.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Túnica Conjuntiva/diagnóstico , Melanoma/diagnóstico , Melanose/diagnóstico , Nevo Pigmentado/diagnóstico , Neoplasias da Túnica Conjuntiva/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Antígeno Ki-67/metabolismo , Antígeno MART-1/metabolismo , Masculino , Melanoma/metabolismo , Antígenos Específicos de Melanoma/metabolismo , Melanose/metabolismo , Fator de Transcrição Associado à Microftalmia/metabolismo , Pessoa de Meia-Idade , Nevo Pigmentado/metabolismo , Estudos Retrospectivos , Coloração e Rotulagem , Antígeno gp100 de MelanomaRESUMO
Purpose: Chemokines play a role in the progression and metastatic spread of both cutaneous and uveal melanomas. The aim of this study was to examine the prognostic value of expression of chemokine receptors CCR7, CXCR4, and CCR10 in conjunctival melanocytic lesions. Methods: In total, 44 conjunctival nevi, 21 cases of primary acquired melanosis (PAM) with atypia and 35 conjunctival melanomas, were included. After immunohistochemical staining for CCR7, CXCR4, and CCR10 the immunoreactive score (IRS) was determined. The findings were correlated for association with melanoma and development of metastasis. For mechanistic evaluation, we used a mouse melanoma metastasis model using two human conjunctival melanoma cell lines, CM2005.1 and CRMM1. Results: All tested chemokines showed a significantly higher expression in conjunctival melanoma than conjunctival nevi. There was a statistically significant difference between the IRS in nevi and PAM with atypia for nuclear IRS in CCR10 (P = 0.03) and both nuclear and cytoplasmic IRS in CXCR4 (P < 0.01 and P = 0.03, respectively); this was also true evaluating the groups PAM with atypia and melanoma all together (P < 0.01). Furthermore, a trend for lower IRS was seen in cases of melanoma without metastasis, with a suggestive pattern of a higher IRS in cases that did develop metastases, supported for CXCR4 using the mouse melanoma metastasis model. Conclusions: Expression of specific chemokines changes during the progression and metastatic spread of conjunctival melanocytic lesions. Differential chemokine profiles may hold prognostic value for patients with conjunctival melanomas and might be considered as a therapeutic target.