Study of the immunohistochemical expression of Plunc family proteins in mucoepidermoid carcinomas of the salivary glands

Orientadores: Márcio Ajudarte Lopes, Alan Roger dos Santos SilvaDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: Tumores de glândulas salivares são raros e geralmente apresentam características histopatológicas complexas que podem dificultar seu diagnóstico. O carcinoma mucoepidermóide (CME) é a neoplasia maligna mais frequente das glândulas salivares e é classificado histologicamente como de baixo grau, grau intermediário ou de alto grau. O uso de técnicas histoquímicas como o PAS (Periodic Acid-Schiff) ou o mucicarmin é de grande utilidade para identificar mucina nos tumores e permitir o diagnóstico do CME. Recentemente, descreveu-se que alguns tumores de glândulas salivares como o cistoadenocarcinoma papilar e o CME expressam proteínas da família PLUNC. O objetivo deste estudo foi avaliar a expressão imunoistoquímica das proteínas da família PLUNC em 30 casos de CMEs de glândulas salivares maiores e menores e testar a hipótese de que estas proteínas poderiam ser úteis no diagnóstico do CME. Os tumores foram revisados e classificados de acordo com o grau de malignidade e estudados por meio de reações histoquímicas (sendo todos os casos positivos para PAS e mucicarmin) e imunoistoquímicas para as proteínas SPLUNC1, LPLUNC1, SPLUNC2A, SPLUNC2B e LPLUNC2. A média de idade dos pacientes diagnosticados com CME foi de 44,07 anos. A maioria dos tumores (19) foi classificada como de baixo grau, 4 tumores foram classificados como grau intermediário e 7 como alto grau. A marcação imunoistoquímica foi classificada de acordo com a quantidade e intensidade de células marcadas no tumor. A maioria dos CMEs estudados foi positiva para SPLUNC1 (90%) e LPLUNC1 (93,33%), sendo identificada principalmente em células mucosas, plugs de mucina e células intermediárias. SPLUNC2A, SPLUNC2B e LPLUNC2 foram negativos na maioria dos tumores. LPLUNC2 apresentou positividade em células semelhantes a mastócitos em 83,33% dos tumores, expressão parece não ter sido relatada previamente na literatura. A expressão de SPLUNC1 e LPLUNC1 apresentou um padrão de marcação semelhante ao identificado com PAS e mucicarmin. A positividade para SPLUNC1 e LPLUNC1 nas células intermediárias, sugere que estas duas proteínas poderiam ser úteis no diagnóstico de casos de CME de alto grau de malignidadeAbstract: Salivary gland tumours are uncommon and generally present complex histopathologic features that can difficult the diagnosis. Mucoepidermoid carcinomas (MECs) are the most frequent malignant neoplasia of the salivary glands and are histologically classified as low, intermediate and high-grade. Histochemical stains such as PAS (Periodic Acid-Schiff) or mucicarmine are very useful to indentify mucine in tumors and to make the diagnosis of MEC. Recently, the expression of PLUNC family proteins has been described in salivary glands tumors, such as papillary cystoadenocarcinoma and MEC, suggesting that these proteins can be useful for the diagnosis in some difficult cases. The aim of this study was to evaluate the expression of PLUNC family proteins in 30 cases of MEC of the salivary glands and test the hypothesis that these proteins could be useful in the diagnosis of MEC. The tumors were reviewed and classified according to the grade of malignancy. Histochemical stains (PAS and mucicarmine) and immunohistochemical reactions for SPLUNC1, LPLUNC1, SPLUNC2A, SPLUNC2B and LPLUNC2 were performed. Patient's mean age was 44.07 years old. The majority of tumors (19) was histologically classified as low grade, 4 tumors were intermediate grade and 7 were high grade. All cases were positive for PAS and mucicarmine. Immunohistochemical stain was classified according to the quantity and intensity of stained cells in the tumor. Most of the MECs were positive for SPLUNC1 (90%) and LPLUNC1 (93.33%), particularly in mucous cells, mucin plugs and intermediary cells. SPLUNC2A, SPLUNC2B and LPLUNC2 did not present significant expression in the tumors. LPLUNC2 presented positivity for cells that remember mast cells in 83.33% of the tumors, expression that seems not to be previously reported in the literature. The expression pattern of SPLUNC1 and LPLUNC1 was similar to PAS and mucicarmine. The expression in intermediary cells for SPLUNC1 and LPLUNC1, suggest that these proteins could be useful in the diagnosis of high grade MECsMestradoEstomatologiaMestre em Estomatopatologi

Expressão de SPLUNC1 (BPIFA1) e SPLUNC2A (BPIFA2A) na saliva de pacientes submetidos à radioterapia

Orientador: Márcio Ajudarte LopesTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: A radioterapia causa alterações na composição salivar e as proteínas PLUNC participam na resposta imune inata da cavidade oral. O objetivo desse estudo foi verificar se a radioterapia é capaz de modificar a expressão de PLUNC salivar e se essas proteínas estão associadas com os efeitos colaterais. MATERIAIS E MÉTODOS: Foi coletada saliva não estimulada de 65 voluntários (45 pacientes com câncer e 20 controles). No grupo de estudo a coleta foi realizada uma semana antes do início da radioterapia, no meio do tratamento e uma semana após o término. A expressão de SPLUNC1 e SPLUNC2A foi detectada por western blot, e foi analisada com os dados clínico-patológicos e efeitos colaterais. RESULTADOS: Foi notada uma redução do fluxo salivar durante e após o término da radioterapia, sendo mais acentuada nos pacientes que foram submetidos a radioterapia envolvendo a região facial. O campo de radiação facial foi correlacionado com os efeitos colaterais, principalmente com a presença (p=0,0110) e intensidade (p=0,0143) de mucosite. SPLUNC1 e SPLUNC2A foram detectadas na saliva dos pacientes sem tratamento em concentrações variáveis. O grupo de estudo mostrou níveis de SPLUNC2A significantemente maiores que o grupo controle, enquanto SPLUNC1 não mostrou diferenças. A concentração de PLUNC foi modificada pela radioterapia, observando diminuição dos níveis de SPLUNC2A na sua forma glicosilada (p<,0001) e aumento dos níveis de SPLUNC1 (p=0,0081) na segunda e terceira coletas. A única associação entre efeitos colaterais da radioterapia e PLUNC foi a presença (p=0,0363) e intensidade (p=0,0500) da mucosite com maiores níveis SPLUNC1. CONCLUSÕES: O presente estudo reportou que os níveis de SPLUNC1 e SPLUNC2A glicosilada são afetados pela radioterapia, sugerindo que essas proteínas podem ter importância no microambiente oral dos pacientes irradiados na cabeça e pescoçoAbstract: Radiotherapy causes alteration in saliva composition and PLUNC proteins participate in innate immunity of the oral cavity. The aim of this study was to verify if radiotherapy is able to modify the salivary PLUNC expression and if they are associated with radiotherapy side-effects. MATERIALS AND METHODS: Unstimulated whole-mouth saliva of 65 voluntaries (45 cancer patients and 20 controls) was collected. In the study group the collection was performed one week before the beginning of radiotherapy, in the middle of the treatment and one week after finishing. SPLUNC1 and SPLUNC2A expression were detected by western blotting and was analyzed with clinicopathological data and radiotherapy side-effects. RESULTS: Reduction of salivary flow rates was observed during and after conclusion of radiotherapy, being more accentuated in patients who underwent radiotherapy involving the facial region. Facial radiation field was correlated with collateral effects, mainly with the presence (p=0.0110) and severity (p=0.0143) of mucositis. SPLUNC1 and SPLUNC2A were detected in saliva of patients without treatment in variable concentrations. The study group showed levels of SPLUNC2A significantly higher than the control group, while SPLUNC1 did not show differences. Concentration of PLUNC was modified by radiotherapy, observing decreasing of glycosilated form of SPLUNC2A levels (p<.0001) and increasing of SPLUNC1 levels (p=0.0081) in the second and third collections. The only association between collateral effects of radiotherapy and PLUNC was the presence of mucositis (p=0.0363) and its severity (p=0.0500) with higher levels of SPLUNC1. CONCLUSIONS: The present study reported that levels of SPLUNC1 and glycosilated SPLUNC2A are affected by the radiotherapy, suggesting that these proteins may have importance in the oral microenvironment of irradiated head and neck cancer patientsDoutoradoEstomatologiaDoutor em Estomatopatologi

Trophoblast cell surface antigen 2 expression predicts outcome in oral squamous cell carcinomas

Background Trophoblast cell surface antigen 2 (TROP2) has unclear clinical role in oral squamous cell carcinomas (OSCC). Here, we investigated the association of TROP2 immunoexpression with clinicopathological parameters and survival of OSCC patients. Subjects and Methods Cancer-specific survival (CSS) and disease-free survival (DFS) were assessed in a cohort composed of 266 OSCC. An independent cohort with 88 OSCC samples matched with the normal oral tissue, as well as 17 metastatic lymph nodes, was used for validation. Results Multivariate analysis showed TROP2 as an independent marker of favorable prognosis for both CSS (HR: 0.60, 95% CI: 0.40-0.90, p = .01) and DFS (HR: 0.57, 95% CI: 0.36-0.89, p = .01). Furthermore, TROP2 protein expression was significantly higher in morphologically normal tissues compared to primary tumors (p <.0001) and lymph node metastases (p = .001), and it was significantly associated with CSS (HR: 0.26, 95% CI: 0.09-0.74, p = .008) in the validation cohort. A pooled mRNA analysis performed on the Oncomine (TM) database confirmed the underexpression in OSCC compared with normal tissues (p = .014). Conclusions In summary, our results point to a favorable prognostic significance of TROP2 overexpression in a large cohort of oral cancer patients, suggesting it as an attractive clinical marker.Peer reviewe

Stress induced phosphoprotein 1 overexpression controls proliferation, migration and invasion and is associated with poor survival in oral squamous cell carcinoma

Objective: Although there have been remarkable achievements in the molecular landscape of oral squamous cell carcinoma (OSCC) in recent years, bringing advances in the understanding of its pathogenesis, development and progression, little has been applied in the prognosis and choosing the optimal treatment. In this study, we explored the influence of the stress induced phosphoprotein 1 (STIP1), which is frequently reported to be highly expressed in many cancers, in OSCCs. Methods: STIP1 expression was assessed in the TCGA database and in two independent cohorts by immunohistochemistry. Knockdown strategy was applied in OSCC cell lines to determine the impact of STIP1 on viability, proliferation, migration and invasion. The zebrafish model was applied for studying tumor formation and metastasis in vivo. The association of STIP1 and miR-218-5p was explored by bioinformatics and mimics transfection. Results: STIP1 was highly expressed in OSCCs and significantly associated with shortened survival and higher risk of recurrence. STIP1 down-regulation decreased proliferation, migration and invasion of tumor cells, and reduced the number of metastases in the Zebrafish model. STIP1 and miR-218-5p were inversely expressed, and the transfection of miR-218-5p mimics into OSCC cells decreased STIP1 levels as well as proliferation, migration and invasion. Conclusion: Our findings show that STIP1 overexpression, which is inversely associated with miR-218-5p levels, contributes to OSCC aggressiveness by controlling proliferation, migration and invasion and is a determinant of poor prognosis.Peer reviewe

Diagnostic approach to intramasseteric nodules

Intramuscular nodules can be the clinical presentation of several groups of lesions, such as reactive disorders and benign and malignant tumors. Here, we present three cases with similar clinical features and image aspects on Doppler ultrasonography. Two of the lesions were diagnosed as intramasseteric hemangioma and the third as intramasseteric metastasis from high-grade pleomorphic sarcoma of the thigh. The diagnosis of intramasseteric nodules is challenging, and various differential diagnoses must be considered. Clinical features, evolution time, and information from complementary examinations, such as Doppler ultrasonography and fine-needle aspiration cytology, are useful in making a precise diagnosis and providing appropriate treatment.Intramuscular nodules can be the clinical presentation of several groups of lesions, such as reactive disorders and benign and malignant tumors. Here, we present three cases with similar clinical features and image aspects on Doppler ultrasonography. Two of the lesions were diagnosed as intramasseteric hemangioma and the third as intramasseteric metastasis from high-grade pleomorphic sarcoma of the thigh. The diagnosis of intramasseteric nodules is challenging, and various differential diagnoses must be considered. Clinical features, evolution time, and information from complementary examinations, such as Doppler ultrasonography and fine-needle aspiration cytology, are useful in making a precise diagnosis and providing appropriate treatmentElsevier1231E16E21New York, N

Eukaryotic translation elongation factor 1 delta, N-terminal propeptide of type I collagen and cancer-associated fibroblasts are prognostic markers of oral squamous cell carcinoma patients

Objective. Identifying markers that influence oral squamous cell carcinoma (OSCC) prognosis is a fundamental strategy to improve the overall survival of patients. Markers such as eukaryotic translation elongation factor 1 delta (EEF1D), fascin, N-terminal propeptide of type I collagen (PINP), and cancer-associated fibroblasts (CAFs) have been noticed in OSCCs and their levels are closely related to the prognosis of tumors. Our aim was to confirm the role of those markers in OSCC prognosis. Study Design. Immunohistochemistry was performed in 90 OSCC specimens. The associations between clinicopathologic features and expression of markers were assessed by chi(2) test. Kaplan-Meier curves and univariate and multivariate Cox regression models were used for survival analysis. Markers were analyzed individually and in combination. Results. High expression of EEF1D (P =.017) and PINP (P =.02) and abundant density of CAFs in tumor stroma (P =.005) predicted significantly poor survival in OSCC patients. Multivariate analysis revealed that all 3 parameters are individually independent prognostic factors of OSCC patients, and their combination improved the discrimination of patients at high risk for poor survival. Conclusions. Our results suggested that the expression of EEF1D and PINP and the density of CAFs might influence the survival of patients with OSCC.Peer reviewe

Eukaryotic translation elongation factor 1 delta, N-terminal propeptide of type I collagen and cancer-associated fibroblasts are prognostic markers of oral squamous cell carcinoma patients

Objective. Identifying markers that influence oral squamous cell carcinoma (OSCC) prognosis is a fundamental strategy to improve the overall survival of patients. Markers such as eukaryotic translation elongation factor 1 delta (EEF1D), fascin, N-terminal propeptide of type I collagen (PINP), and cancer-associated fibroblasts (CAFs) have been noticed in OSCCs and their levels are closely related to the prognosis of tumors. Our aim was to confirm the role of those markers in OSCC prognosis. Study Design. Immunohistochemistry was performed in 90 OSCC specimens. The associations between clinicopathologic features and expression of markers were assessed by chi(2) test. Kaplan-Meier curves and univariate and multivariate Cox regression models were used for survival analysis. Markers were analyzed individually and in combination. Results. High expression of EEF1D (P =.017) and PINP (P =.02) and abundant density of CAFs in tumor stroma (P =.005) predicted significantly poor survival in OSCC patients. Multivariate analysis revealed that all 3 parameters are individually independent prognostic factors of OSCC patients, and their combination improved the discrimination of patients at high risk for poor survival. Conclusions. Our results suggested that the expression of EEF1D and PINP and the density of CAFs might influence the survival of patients with OSCC.Peer reviewe

Overexpression of heat-shock protein 47 impacts survival of patients with oral squamous cell carcinoma

Background: The expression of heat-shock protein 47 (HSP47) has been linked to collagen synthesis control and implicated in fibrotic disorders, but more recent studies have demonstrated its role in solid tumors. In this study, we explored the prognostic impact of HSP47 in oral squamous cell carcinomas (OSCC) and determined the in vitro effects of its loss-of-function on viability, proliferation, migration, invasion, and resistance to cisplatin of OSCC cells.Methods: The HSP47 expression in tumor samples was assessed by immunohistochemistry in two independent cohorts totaling 339 patients with OSCC, and protein levels were associated with clinicopathological features and survival outcomes. The OSCC cell lines HSC3 and SCC9 were transduced with lentivirus expressing short hairpin RNA to stably silence HSP47 and used in assays to measure cellular viability, proliferation, migration, and invasion.Results: HSP47 was overexpressed in OSCC samples, and its overexpression was significantly and independently associated with poor disease-specific survival and shortened disease-free survival in both OSCC cohorts. The knockdown of HSP47 showed no effects on cell viability or cisplatin sensitivity, but impaired significantly proliferation, migration, and invasion of OSCC cells, with stronger effects on SCC9 cells.Conclusion: Our results show a significant prognostic impact of HSP47 overexpression in OSCC and reveal that HSP47 inhibition impairs the proliferation, migration, and invasion of OSCC cells. HSP47 may represent a potential therapeutic target for OSCC.Peer reviewe

Combining discovery and targeted proteomics reveals a prognostic signature in oral cancer

Different regions of oral squamous cell carcinoma (OSCC) have particular histopathological and molecular characteristics limiting the standard tumor-node-metastasis prognosis classification. Therefore, defining biological signatures that allow assessing the prognostic outcomes for OSCC patients would be of great clinical significance. Using histopathology-guided discovery proteomics, we analyze neoplastic islands and stroma from the invasive tumor front (ITF) and inner tumor to identify differentially expressed proteins. Potential signature proteins are prioritized and further investigated by immunohistochemistry (IHC) and targeted proteomics. IHC indicates low expression of cystatin-B in neoplastic islands from the ITF as an independent marker for local recurrence. Targeted proteomics analysis of the prioritized proteins in saliva, combined with machine-learning methods, highlights a peptide-based signature as the most powerful predictor to distinguish patients with and without lymph node metastasis. In summary, we identify a robust signature, which may enhance prognostic decisions in OSCC and better guide treatment to reduce tumor recurrence or lymph node metastasis.Peer reviewe