Despite improvements in treatment, the prognosis for patients with advanced head and neck squamous cell carcinomas (HNSCC) has only improved to a minor degree. To raise the success rate and minimize morbidity further developments in diagnostics are highly desired. Radioimmunodiagnosis could offer a more specific and sensitive diagnostic method. Herein, we have evaluated different radioimmunoconjugates directed against CD44v6 and epidermal growth factor receptor (EGFR) for imaging of HNSCC. The studies were performed in a murine HNSCC xenograft model. Initially, the 111In-labeled anti CD44v6 chimeric monoclonal antibody U36 (cMAb U36) was evaluated. The novel radioimmunoconjugate showed high and accumulating tumor uptake. Since small molecules might be advantageous for imaging, due mainly to their shorter circulation half-life in the bloodstream, we then investigated antibody fragments F(ab’)2 and Fab’ derived from cMAb U36. The highest tumor-to-blood ratio was achieved with the dimeric antibody fragment F(ab’)2, compared with both the intact anti-body and monomeric Fab’. Furthermore, the possibility of improving EGFR-targeted imaging was explored by pre-blocking EGFR. The liver uptake of injected labeled human epidermal growth factor (hEGF) was significantly reduced when an excess of unlabeled hEGF was injected 30 minutes in advance. However, as hEGF stimulates cell proliferation it may be inadvisable to treat cancer patients with large amounts. Alternatively, pre-blocking with an anti-EGFR Affibody molecule (ZEGFR:955)2 demonstrated similar decrease in liver uptake as unlabeled hEGF. Finally, (ZEGFR:955)2 was compared with other Affibody molecules with higher affinity to EGFR, ZEGFR:1907 and (ZEGFR:1907)2, as pre-blocking agents. In addition, a novel hEGF radioimmunoconjugate, [67Ga]Ga-NOTA-Bn-NCS-hEGF was used for EGFR targeting. The dimeric (ZEGFR:1907)2 showed greatest reduction in non-tumor uptake, and highest tumor-to-organ ratio in EGFR expressing organs, when injected in advance of the radioimmunoconjugate. To summarize, the results presented here demonstrate how different radioimmunoconjugates as well as pre-blocking EGFR can improve the radioimmunodiagnosis of head and neck squamous cell carcinomas.

<p>Ultraviolet radiation (UVR) is a major risk factor for development of skin cancer. UVR-induced DNA damage and a dysfunctional p53 protein are important steps in the development of squamous cell carcinoman in human skin (SCC). The aim of the present investigation was to analyze incidence trends of SCC in Sweden, quantify the risk of second primary cancer after SCC and further analyze the effects of UVR and p53 protein in human skin <i>in vivo</i> and <i>in vitro</i>. The effect of photoprotection by sunscreens was also evaluated. </p><p>We found that the age-standardized incidence rate of SCC in Sweden increased substantially in both men and women during the period 1961-1995, especially in men and at chronically sun-exposed skin sites. Patients with SCC are also at increased risk of developing new primary cancers, especially in the skin, squamous cell epithelium, hematopoietic tissues and respiratory organs. In experimental studies <i>in vivo</i> and <i>in vitro</i> in human skin we observed that repair of UV-induced DNA damage appears to be more efficient in chronically sun-exposed skin despite a less uniform p53 response. Non-sun- exposed skin is more homogeneous with respect to the epidermal p53 response. Keratinocytes in skin exposed frequently to the sun may be prone to react more easily to cytotoxic stress. Two different modalities of photoprotection significantly reduced the amount of DNA damage and the number of p53-positive cells. In addition, we demonstrated that a well-defined system for <i>in vitro</i> culture of explanted skin provides an excellent alternative to <i>in vivo</i> experiments. </p><p>In conclusion, this study has increased our knowledge of SCC epidemiology in Sweden and of the effects of artificial and solar UVR and sunscreens on chronically sun-exposed and non-sun-exposed sites, respectively, of human skin.</p>

Ultraviolet radiation (UVR) is a major risk factor for development of skin cancer. UVR-induced DNA damage and a dysfunctional p53 protein are important steps in the development of squamous cell carcinoman in human skin (SCC). The aim of the present investigation was to analyze incidence trends of SCC in Sweden, quantify the risk of second primary cancer after SCC and further analyze the effects of UVR and p53 protein in human skin in vivo and in vitro. The effect of photoprotection by sunscreens was also evaluated. We found that the age-standardized incidence rate of SCC in Sweden increased substantially in both men and women during the period 1961-1995, especially in men and at chronically sun-exposed skin sites. Patients with SCC are also at increased risk of developing new primary cancers, especially in the skin, squamous cell epithelium, hematopoietic tissues and respiratory organs. In experimental studies in vivo and in vitro in human skin we observed that repair of UV-induced DNA damage appears to be more efficient in chronically sun-exposed skin despite a less uniform p53 response. Non-sun- exposed skin is more homogeneous with respect to the epidermal p53 response. Keratinocytes in skin exposed frequently to the sun may be prone to react more easily to cytotoxic stress. Two different modalities of photoprotection significantly reduced the amount of DNA damage and the number of p53-positive cells. In addition, we demonstrated that a well-defined system for in vitro culture of explanted skin provides an excellent alternative to in vivo experiments. In conclusion, this study has increased our knowledge of SCC epidemiology in Sweden and of the effects of artificial and solar UVR and sunscreens on chronically sun-exposed and non-sun-exposed sites, respectively, of human skin.

Made available in DSpace on 2014-08-20T14:30:10Z (GMT). No. of bitstreams: 1 Dissertacao_alessandro_menna_alves.pdf: 813449 bytes, checksum: 43f43131b0d8e54633d172ea87ffa722 (MD5) Previous issue date: 2013-03-18 / This dissertation was divided into two distinct works, which can be summarized as follows: Article 1: The oral squamous cell carcinoma (OSCC) is the most prevalent malignance in mouth, being an important public health problem. The aim of this study was to evaluate the clinical and epidemiological profile of the OSCC cases registered in a center of clinical and histopathological diagnosis, located in Southern Brazil. Eight hundred and six individuals with OSCC and its variants were included in this study, over 1959-2012 period. The variables recorded from the files were: age, gender, skin color, tumor location, size and evolution time of the lesions (referred by the patients), as well as, the presence of pain lymph nodes, habits of tobacco and alcohol, and also the profession. OSSC was more frequent in males (76.6%), with the majority of cases distributed between 51 and 70 years old (53.9%). The most prevalent sites were lower lip vermilion [23.3% (20.4; 26.4)], followed by lateral border/ventral surface of the tongue [20.2% (17.5; 23.2)], gingiva/alveolar ridge [18.1% (15.5; 21.0)], and floor of the mouth [14.9% (12.5; 17.5)]. A strong association between outdoor occupation and OSCC in lower lip vermilion was found. The OSCC lesions located in tongue, gingiva/alveolar ridge and floor of the mouth were commonly more painful, bigger than 2 cm, and frequently presenting lymph nodes involvement. Most of the results confirm the data from literature. Mouth self-examination should be recommended and campaigns of prevention and early detection of OSCC should be periodically performed in order to increase people s feelings of personal risk. Article 2: Inside the tumor microenvironment (TM) the neoplastic cells are in dynamic crosstalk with the vascular and lymphatic endothelial cells in order to allow the tumor to growth and metastasize. Hypothesizing that there is a crosstalk between lymphatic endothelial cells and tumor cells from squamous cell carcinoma (SCC) that plays an important role in metastasis, we aimed to identify potential interactions between lymphatic endothelial cells and tumor cells lines from SCCs, through some in vitro assays. Primary adult human dermal lymphatic microvascular endothelial cells (HMVECs) and the human head and neck SCC cell lines like A431, UM-SCC-1, UM-SCC-22A and UM-SCC-22B were cultured in their specific media. UM-SCC cells lines were treated with rhIL-6, being VEGF-C expression checked by Elisa. Baseline IL-6 was evaluated in HMEVCs using the same assay. Also the IL-6 receptor (IL-6R) was analyzed by Western blot in UM-SCC cells. Conditioned media from HMVECs were prepared with different treatments and incubated with SCC A431 cells, in order to verify the MMPs enzymatic activities by gelatin zymography. Our results demonstrated that there are interactions between tumor cells and LECs, since the LECs-CM were able to enhance MMP-2 gelatinolytic activity. Moreover, we showed that LECs secrete IL-6, and different SCC lines have receptors for this cytokine. Therefore, our results indicate some potential interactions between LECs and TCs, being necessary other studies to elucidate the involved signaling pathways / Esta dissertação foi dividida em dois trabalhos distintos, os quais podem ser resumidos da seguinte maneira: Artigo 1: O carcinoma espinocelular oral (CEO) é o tumor maligno mais prevalente na cavidade oral, sendo um importante problema de saúde pública. O objetivo deste estudo foi avaliar o perfil clínico e epidemiológico dos casos registrados de CEO em um centro de diagnóstico clínico e histopatológico, localizado no Sul do Brasil. Oitocentos e seis indivíduos com CEO e suas variantes histológicas foram incluídos neste estudo, num período entre 1959 e 2012. As variáveis anotadas dos arquivos foram: idade, sexo, cor da pele, sítio, tamanho, tempo de evolução (relatado pelo paciente), assim como a presença de dor, linfonodos palpáveis, hábitos de tabagismo e etilismo, e a profissão. CEO foi mais prevalente em homens (76,6%), com a maioria dos casos distribuídos entre os 51 e 70 anos de idade (53,9%). Os sítios mais prevalentes foram vermelhão do lábio inferior [23,3% (20,4; 26,4)], seguido por borda lateral/ventre de língua [20,2% (17,5; 23,2)], gengiva/rebordo alveolar [18,1% (15,5; 21,0)], e assoalho bucal [14,9% (12,5; 17,5)]. Foi encontrada uma forte associação entre ocupações ao ar livre e CEO de vermelhão de lábio inferior. As lesões localizadas na língua, gengiva/rebordo alveolar e assoalho bucal foram comumente mais dolorosas, maiores que 2 cm, a frequentemente apresentavam envolvimento de linfonodos. A maioria dos nossos resultados confirmam os dados da literatura. O autoexame bucal deveria ser recomendado e campanhas de prevenção e detecção precoce do CEO deveriam ser realizadas periodicamente na tentativa de aumentar o sentimento pessoal em relação ao CEO. Artigo 2: Dentro do microambiente tumoral (MT), as células neoplásicas estão numa constante crosstalk com células endoteliais linfáticas (CELs) e sanguíneas a fim de permitir o crescimento tumoral e metástase. Supondo que haja um crosstalk entre as CELs e as células do carcinoma espinocelular (CE) que exerce um importante papel na metástase, nosso objetivo foi identificar potenciais interações entre as CELs e linhagens celulares de CE, através de alguns ensaios in vitro. Células endoteliais linfáticas primárias adultas humanas da microvasculatura dérmica (HMVECs) e as linhagens de CE A431, UM-SCC-1, UM-SCC-22A e UM-SCC-22B foram cultivadas nos seus meios específicos. UM-SCC foram tratadas com rhIL-6, sendo a expressão de VEGF-C verificada por Elisa. Produção natural de IL-6 pelas HMVECs foi avaliada da mesma maneira. A presença de receptor de IL-6 (IL-6R) foi analisada por Western Blot nas linhagens UM-SCC. Meios condicionados(MC) das HMVECs foram preparados com diferentes tratamentos e incubados com a linhagem A431, a fim de verificar a atividade genatinolítica das MMPs por zimografia. Nossos resultados demonstraram que há interações entre as células tumorais e as CELs, uma vez que MC-CELS foram capazes de aumentar a atividade genatinolítica da MMP-2. Além disso, nós mostramos que as CELs secretam IL-6, e diferentes linhagens de CE possuem receptores para esta citocina. Sendo assim, nossos resultados indicam potenciais interações entre as LECs e as células tumorais, sendo necessário outros estudos para elucidar as vias de sinalização envolvidas