Evaluation of polymorphisms and frequency of dental anomalies in patients with nonsyndromic cleft lip and/or cleft palate

Orientador: Ricardo Della ColettaTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: Fissuras do lábio e/ou palato (FL/P) representam a anomalia congênita mais comum em humanos. FL/P podem estar associadas a outras malformações, caracterizando uma síndrome, ou apresentar-se de forma isolada, recebendo a denominação não-sindrômica (FL/PNS). A etiologia das FL/PNS é complexa e envolve a participação de inúmeros genes, os quais estão sobre a influência de fatores ambientais. Alguns estudos demonstraram uma frequência elevada de anomalias dentais em pacientes com FL/PNS, sugerindo um defeito genético único para essas duas alterações. Os objetivos deste estudo foram: 1) verificar se polimorfismos nos genes responsáveis por síndromes que apresentam FL/P em seu espectro clínico, incluindo TP63, MID1, PVRL1, RUNX2 e TBX1, podem contribuir para a etiologia das FL/PNS, e 2) determinar a frequência de anomalias dentais em pacientes com FL/PNS. Sete regiões polimórficas foram genotipadas por PCR-RFLP (reação em cadeia da polimerase associada à análise de polimorfismo de fragmentos de restrição enzimática) em amostras de DNA provenientes de 367 pacientes afetados por FL/PNS (grupo experimental) e de 413 indivíduos clinicamente normais (grupo controle). Para determinar a frequência de anomalias dentais em pacientes com FL/PNS, 296 pacientes com idade entre 12 e 30 anos, sem história de extração dental e tratamento ortodôntico prévio e que apresentavam documentação radiográfica foram avaliados. Dos 7 prováveis polimorfismos, apenas 3 foram confirmados em nossa população. Após correção para múltiplas comparações pelo teste de Bonferroni, apenas o polimorfismo rs28649236 do gene TBX1 demonstrou diferença significante entre os grupos. O alelo G e os genótipos AG e GG ocorreram em uma frequência significantemente maior no grupo controle que no grupo experimental, e na presença do alelo G um efeito protetor para o desenvolvimento de FL/PNS foi observado (p=0,0002; OR: 0,41; IC 95%: 0,25-0,67). Anomalias dentais foram identificadas em 57,7% (n=296) dos pacientes com FL/PNS e pacientes com FL foram significantemente menos acometidos por anomalias dentais comparando-se com pacientes com FP ou FLP (p=0,004). Agenesia dental foi a mais frequentemente identificada entre os tipos de fissura, principalmente em pacientes com FLP e FP que em pacientes com FL (p=0,03). O presente estudo demonstrou que o polimorfismo rs28649236 do gene TBX1 apresenta um efeito protetor para o desenvolvimento de FL/PNS em uma população brasileira e que o risco de pacientes com FLP ou FP apresentar anomalias dentais é significantemente maior que o de pacientes com FL. Em adição, este estudo corrobora com evidências prévias que demonstraram a influência de genes responsáveis por síndromes contendo FL/P na etiopatogenia das FL/PNS.Abstract: Cleft lip and/or cleft palate (CL/P) is the most common congenital anomaly in humans. CL/P may be associated with other malformations, featuring a syndrome, or present itself in an isolated form, denominated nonsyndromic (NSCL/P). NSCL/P etiology is complex and involves the participation of numerous genes, which are under the influence of environmental factors. Some recent studies have demonstrated a high frequency of dental anomalies in patients with NSCL/P, suggesting a common genetic defect for these two alterations. The objectives of this study were: 1) to verify whether polymorphic variants of genes responsible for syndromes that have CL/P in their clinical spectrum, including TP63, MID1, PVRL1, TBX1 and RUNX2, may contribute to the etiology of NSCL/P, and 2) to determine the frequency of dental anomalies in patients with NSCL/P. Seven polymorphic regions were genotyped by PCR-RFLP (restriction fragment length polymorphism-polymerase chain reaction) in DNA samples from 367 patients affected by NSCL/P (experimental group) and 413 clinically normal subjects (control group). To determine the frequency of dental anomalies in patients with NSCL/P, 296 patients aged between 12 and 30 years without history of tooth extraction and orthodontic treatment and with radiographic documentation were evaluated. Out of 7 probable polymorphisms, only 3 were confirmed in our population. After correction for multiple comparisons by Bonferroni test, only rs28649236 TBX1 polymorphism showed significant difference between groups. The allele G and the AG and GG genotypes occurred in a significantly higher frequency in controls than in the experimental group, and in the presence of the G allele a protective effect against NSCL/P was observed (p=0.0002; OR: 0.41; 95% CI: 0.25-0.67). Dental anomalies were identified in 57.7% (n= 296) of patients with NSCL/P and patients with CL were significantly less affected by dental anomalies compared with patients with CP or CLP (p=0.004). Dental agenesis was the most frequently identified among the cleft types, especially in patients with CLP and ICP than in patients with FL (p=0.03). The present study demonstrated that rs2864923 TBX1 polymorphism has a protective effect in NSCL/P development in a Brazilian population, and patients with CLP or CP are frequently more affected by dental anomalies than patients with CL. In addition, this study corroborates with previous evidences demonstrating the influence of genes related to syndromes containing CL/P on etiopathogenesis of the NSCL/P.DoutoradoPatologiaDoutor em Estomatopatologi

Prognostication for oral carcinomas based on two histological scoring systems (BD and iBD models)

Peer reviewe

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

Stanniocalcin 2 contributes to aggressiveness and is a prognostic marker for oral squamous cell carcinoma

Stanniocalcin 2 (STC2), a glycoprotein that regulates calcium and phosphate homeostasis during mineral metabolism, appears to display multiple roles in tumorigenesis and cancer progression. This study aimed to access the prognostic value of STC2 in oral squamous cell carcinoma (OSCC) and its implications in oral tumorigenesis. STC2 expression was examined in 2 independent cohorts of OSCC tissues by immunohistochemistry. A loss-of-function strategy using shRNA targeting STC2 was employed to investigate STC2 in vitro effects on proliferation, apoptosis, migration, invasion, epithelial-mesenchymal transition (EMT) and possible activation of signaling pathways. Moreover, STC2 effects were assessed in vivo in a xenograft mouse cancer model. High expression of STC2 was significantly associated with poor disease-specific survival (HR: 2.67, 95% CI: 1.37-5.21, p = 0.001) and high rate of recurrence with a hazard ratio of 2.80 (95% CI: 1.07-5.71, p = 0.03). In vitro downregulation of STC2 expression in OSCC cells attenuated proliferation, migration and invasiveness while increased apoptotic rates. In addition, the STC2 downregulation controlled EMT phenotype of OSCC cells, with regulation on E-cadherin, vimentin, Snaill, Twist and Zeb2. The reactivation of STC2 was observed in the STC2 knockdown cells in the in vivo xenograft model, and no influence on tumor growth was observed. Modulation of STC2 expression levels did not alter consistently the phosphorylation status of CREB, ERK, JNK, p38, p70 S6K, STAT3, STAT5A/B and AKT. Our findings suggest that STC2 overexpression is an independent marker of OSCC outcome and may contribute to tumor progression via regulation of proliferation, survival and invasiveness of OSCC cells.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

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

Oral manifestation of lymphomatoid granulomatosis

Lymphomatoid granulomatosis (LYG) is a rare B-cell lymphoproliferative disorder driven by Esptein-Barr virus (EBV) that most commonly affects the lungs, although extra pulmonary sites like the central nervous system, skin, liver and kidney can also be involved. It is microscopically characterized by an angiocentric and angiodestructive growth pattern, predominantly composed by small T-cells, although a smaller population of atypical large B-cells is considered the true neoplastic component. Oral cavity involvement of LYG has rarely been described and the diagnosis of this neoplasm is very difficult. The aim of this report is to present a rare case of LYG affecting an 86-year-old female patient that was diagnosed due to an extensive, ulcerated and painful oral lesion affecting the hard palate. Detailed microscopic evaluation together with a large immunohistochemical study were necessary to achieve the correct diagnosis of LYG132270276FAPESP – Fundação de Amparo à Pesquisa Do Estado De São Paulo2017/14880-

Variable expressivity and novel PTEN mutations in Cowden syndrome

Cowden syndrome (CS) is a phosphatase and tensin homolog gene (PTEN) -associated condition characterized by multiple mucocutaneous hamartomas and an increased risk of malignancies. We reported an isolated case and another of several individuals in one family affected by CS. The isolated case showed typical features, including fibrocystic breast disease, benign thyroid nodules, and multiple papillomatous lesions in the face and oral cavity, and the cause was a novel nonsense mutation-guanine (G) to thymine (T) transition at position 940 (c. 940 G>T)-in PTEN. In the family, the proband showed erythema nodosum, duodenal ulcer, intestinal polyps, cervical lipoma, renal cysts, and glaucoma, whereas multiple members of her family were found to have intestinal polyps, and a sister had been diagnosed with breast cancer at early age. An intronic mutation-T>G transition at the +32 position of intron 8 (c.1026+32 T>G)-was found in this family, with in silico analysis revealing the creation of a new donor splice site. This study confirmed the involvement of PTEN in CS and the variable clinical expressivity of disease127115561FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2016/02667-