Oral squamous cell carcinoma (SCC) is a lethal, deforming disease of global significance and rising incidence. In 2011, 6,767 new cases and 2,056 deaths occurred in the UK. Cancers are preceded by oral potentially malignant disorders (PMD), recognizable mucosal diseases harbouring significantly increased risk of carcinoma. These manifestations offer clinicians a ‘therapeutic window’ to intervene during carcinogenesis, although contemporary practice remains unable to predict individual lesion behaviour or quantify risk for malignant transformation. No clear management guidelines exist and available scientific literature is unable to answer the fundamental question whether intervention prevents cancer. This MD thesis includes 7 published papers on oral pre-cancer and 4 observational studies from a specialist PMD service coordinated by the author in Newcastle upon Tyne in Northern England. PMD management involves a complex interaction between patients, clinicians’ views on diagnosis and treatment and histopathological assessment of mucosal biopsy specimens. Uncertainty regarding the ‘potentially malignant state’ remains a pernicious influence throughout. Newcastle PMD patients were profiled as predominantly male, with a median age of 60yrs, and regular users of both tobacco and alcohol. Most presented with single-site disease, primarily leukoplakia on the floor of mouth and ventro-lateral tongue, with over 80% exhibiting epithelial dysplasia on histopathological examination. Approximately 70% underwent interventional therapy using CO2 laser surgery. 840 laser treatments were performed between 1996 and 2015 and the efficacy of laser intervention was examined by reviewing clinico-pathological details and clinical outcome for 590 PMD patients followed for a mean of 7.3yrs. Histopathology required ‘up-grading’ in 36% following definitive examination of laser excision specimens. 74.2% of patients were disease free, primarily younger patients with ‘low-grade’ dysplasia, 9% exhibited persistent disease and were older with gingival lesions often proliferative verrucous leukoplakia (PVL). In 12%, unexpected SCC was identified on excision, whilst 4.8% transformed to malignancy. Interventional laser surgery facilitates definitive diagnosis and efficacious treatment, allows early diagnosis and treatment of SCC, identifies patients at risk of progressive disease and defines clinical outcome categories. Evidence is lacking, however, that intervention halts the progress of carcinogenesis. Multi-centre, prospective, randomized, controlled trials are needed to confirm the efficacy of interventional surgery, to characterise PMD natural history and to disseminate research findings. It is hoped that the clinical work presented in this MD thesis will inform and encourage further research into PMD and lead to a reduction in the incidence of malignancy and improved morbidity and mortality.

Magister Scientiae Dentium - MSc(Dent) / Oral epithelial dysplasia (OED) is a growth anomaly which occurs as a result of atypical, abnormal proliferation and a change in the architecture and cytological features of cells of epithelial origin, which ultimately results in the formation of a lesion with disturbed differentiation and maturation The purpose of this study was to describe the OED cases diagnosed at Tygerberg Oral health centre in a 7-year period between 2012 and 2019. The patients’ medical records from Tygerberg Oral Health Centre and National Health Laboratory Service (NHLS) were reviewed. All diagnosed cases of OED were identified and the data retrieved for further assessment and comparison. The individual medical records and follow up data were assessed. Seventy cases of OED were diagnosed in the period assessed. Of those 70 cases, the median age was 58 and the interquartile range was from 48 – 62. Thirty-six of the diagnosed patients were female and thirty-four were males. The majority of lesions diagnosed with OED were found on the tongue, floor of the mouth (FOM) and buccal mucosa. Majority of the lesions were found in non-smokers and non-alcohol consumers. These two categories both presented with mild cases of OED. From the results, it was derived that OED has no intra-oral location predilection. Moreover, OED is not directly associated with smoking.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2017-03-16T17:16:39Z No. of bitstreams: 2 Tese - Andréia de Souza Gonçalves - 2017.pdf: 2952330 bytes, checksum: 1e8c4975cba85c509fcbc41ceea34245 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-03-20T13:18:44Z (GMT) No. of bitstreams: 2 Tese - Andréia de Souza Gonçalves - 2017.pdf: 2952330 bytes, checksum: 1e8c4975cba85c509fcbc41ceea34245 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-03-20T13:18:44Z (GMT). No. of bitstreams: 2 Tese - Andréia de Souza Gonçalves - 2017.pdf: 2952330 bytes, checksum: 1e8c4975cba85c509fcbc41ceea34245 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Study Rationale: Oral potentially malignant disorders (OPMDs) consist of morphologically altered tissues, which present a greater risk of malignant transformation than normal tissue. The most frequently found OPMDs are leukoplakia (OL) and actinic cheilitis (AC). It is now known that mutated or genetically altered cells have developed immunomodulatory strategies which allow them to escape antitumor immune response. The immune escape mechanisms used by mutated cells include the expressions of HLA-G, HLA-E and PD-L1 molecules and the IL-10 and TGF-β cytokines. Objective: To evaluate tissue and salivary expressions of HLA-G, HLA-E and PD-L1 molecules and IL-10 and TGF-β1, -β2 e -β3 cytokines in OPMDs, and relate such immunomodulatory mediators with antitumor immune response and potential for malignant transformation of lesions. Methods: Samples of patients with OL (n= 80) were submitted to immunohistochemistry and ELISA techniques to evaluate tissue and salivary expressions of the HLA-G, HLA-E, PD-L1, IL-10, TGF-β1, TGF-β2 and TGF-β3. In addition, samples of patients with QA (n= 30) were submitted to immunohistochemistry technique to evaluation tissue expression of HLA-G, HLA-E, PD-L1 and IL-10. Control group (n= 20) consisted of saliva and tissue of healthy individuals. The immunostained tissue samples were measured using a semi-quantitative method in association with staining intensity. The expression of these molecules and cytokines were related with the malignant potential of the lesions (epithelial dysplasia grading, proliferation- Ki-67 and apoptosis index- mutated p53). Moreover, the association with the density of granzyme B (GB+) cells and regulatory T cells FOXP3+ was investigated. The statistical tests used were: Fisher’s exact or Pearson Chi-Squared, Mann-Whitney and Kruskal- Wallis tests. The significance level was set at P < 0.05. Results: Fifteen samples showed severe dysplasia, twenty moderate, thirty-two mild and thirteen non-dysplasia. Forty samples (50%) of OL presented combined high Ki-67/p53. Irrespective of the severity of epithelial dysplasia and proliferation/apoptosis index in OL, an overexpression of HLA-G, -E, PD-L1, IL-10, TGF-β2 and -β3 was found in OL when compared with control (P < 0.05). The number of GB+ and FOXP3+ cells in OL was similar to control. Salivary concentration of sHLA-G, IL-10 and TGF-β did not allow distinction between OL patients and healthy individuals (P > 0.05). As regards to AC, we showed that this lesion had an increase in expression of HLA-G, HLA-E, IL-10 and PD-L1 when compared to control; however this increase was statistically significant only for PD-L1 (P= 0.04). Conclusion: The OL showed a reduced cytotoxic immune response (low number of GB+ cells) associated with a high expression of immunomodulatory mediators; however, this expression was independent of epithelial dysplasia grading, proliferation and apoptosis index. Regarding AC we also showed an increase in expression of HLA-G, -E, IL-10 and PD-L1 when compared to the control. Thus, our findings suggest that this lesion present an immunosuppressive microenvironment which favors the escape of mutated keratinocytes in any stage dysplastic, proliferation or apoptosis which this disease finds itself. / Justificativa do estudo: As desordens potencialmente malignas (DPMs) de boca consistem em tecidos morfologicamente alterados, os quais apresentam maior risco de transformação maligna que o tecido normal. Dentre as DPMs de boca, as mais frequentes são a leucoplasia (LE) e a queilite actínica (QA). Atualmente, sabe-se que células mutadas ou geneticamente alteradas são capazes de desenvolver estratégias imunomodulatórias que lhes permitem a evasão à resposta imune antitumoral. Dentre esses mecanismos, pode-se citar a expressão das moléculas HLA-G, HLA-E e PD-L1 e das citocinas IL-10 e TGF-β. Objetivo: Avaliar a expressão tecidual e salivar das moléculas HLA-G, HLA-E e PD-L1 e das citocinas IL-10 e TGF-β1, -β2 e -β3 em DPMs de boca e relacionar tais mediadores imunomodulatórios com a resposta imune antitumoral e com o potencial de transformação maligna dessas lesões. Metodologia: Amostras de pacientes acometidos pela LE (n= 80) foram submetidas às técnicas da imunoistoquímica e ELISA para avaliação da expressão tecidual e salivar, respectivamente, das proteínas supracitadas. Adicionalmente, amostras de pacientes acometidos pela QA (n= 30) foram submetidas à técnica da imunoistoquímica para avaliação da expressão tecidual das proteínas HLA-G, HLA-E, PD-L1 e IL-10. O grupo controle (n= 20) consistiu de tecido e saliva de indivíduos saudáveis. As amostras teciduais imunomarcadas foram mensuradas por um método semi-quantitativo associado à intensidade de marcação. A expressão dos mediadores imunomodulatórios foi associada com o potencial de malignização das lesões (gradação de displasia epitelial, índice de proliferação celular- Ki-67 e apoptose- p53 mutado). Além disso, a associação com a densidade de células granzima B+ (GB+) e células T regulatórias FOXP3+ foi investigada. Os testes estatísticos utilizados foram: teste exato de Fisher ou qui-quadrado de Pearson, Mann-Whitney e Kruskal- Wallis. O nível de significância foi estabelecido em P < 0,05. Resultados: No que se refere a LE, o presente estudo demonstrou que 15 amostras apresentaram displasia severa, 20 moderada, 32 leve e 13 sem displasia. Quarenta amostras (50%) apresentram o combinado alto Ki-67/p53. Independente da severidade da displasia epitelial e do índice de proliferação celular/ apoptose, observou-se uma expressão aumentada de HLA-G, HLA-E, PD-L1, IL-10, TGF-β2 e -β3 quando comporado ao controle (P < 0.05). O número de células GB+ e FOXP3+ nas LE foi similar ao controle. Os níveis salivares de HLA-G solúvel (HLA-Gs), IL-10 e TGF-β não nos possibilitou uma diferenciação entre os pacientes com LE e indivíduos saudáveis (P > 0,05). Em relação aos resultados obtidos para QA, evidenciou-se que essa lesão teve um aumento na expressão de HLA-G, HLA-E, IL-10 e PD-L1 em comparação ao controle, todavia esse aumento só foi estatisticamente significativo para o PD-L1 (P= 0,04). Conclusão: As LEs apresentaram uma reduzida resposta imunológica citotóxica (baixo número de células GB+) associada a uma elevada expressão de mediadores imunomodulatórios; todavia essa expressão não possui relação com a gradação de displasia epitelial e índice de proliferação celular e apoptose. Em relação à QA, nós também evidenciamos uma maior expressão de HLA-G, HLA-E, IL-10 e PD-L1 se comparado ao controle. Desta forma, nossos achados sugerem que tais lesões apresentam um microambiente imunossupressor que favorece a evasão de queratinócitos mutados em qualquer estágio de alteração displásica, proliferação ou apoptose que essa doença se encontre.