Thesis (doctoral)--Katholieke Universiteit te Nijmegen.

Submitted by INGRID DA SILVA SANTOS null (iingridsantos@hotmail.com) on 2017-06-26T03:09:39Z No. of bitstreams: 1 Dissertação de Mestrado_Repositório UNESP_INGRID DA SILVA SANTOS.pdf: 1875360 bytes, checksum: b760f2a2958eb68922d78501172c5f4e (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-06-28T16:45:33Z (GMT) No. of bitstreams: 1 santos_is_me_araca_par.pdf: 1437951 bytes, checksum: 9bb3ef4b8f15e4259996185982c7950e (MD5) / Made available in DSpace on 2017-06-28T16:45:33Z (GMT). No. of bitstreams: 1 santos_is_me_araca_par.pdf: 1437951 bytes, checksum: 9bb3ef4b8f15e4259996185982c7950e (MD5) Previous issue date: 2017-06-02 / Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM) / Evidências sugerem que o papilomavírus humano (HPV) está associado com um subgrupo de carcinomas de células escamosas da cabeça e pescoço (HNSCC). No entanto, a prevalência do HPV varia substancialmente dependendo do local anatômico e da região geográfica estudada. Aqui, nosso objetivo foi investigar a prevalência do HPV em amostras de tecido fresco de pacientes brasileiros com carcinoma de células escamosas (CEC) de boca e orofaringe combinando dois métodos confiáveis para a detecção do HPV. Foram recrutadas trinta e seis amostras de tecido fresco provenientes de CEC de boca (n= 27) e orofaringe (n= 9) para análises. As características sociodemográficas, estilo de vida e clinicopatológicas foram coletadas através dos prontuários. O DNA do HPV foi detectado por dois métodos: reação em cadeia da polimerase (PCR) em tempo real através de ensaio qualitativo de presença ou ausência do HPV-16, e testado para 37 genótipos usando Linear Array. A amplificação do gene β -globina funcionou como controle interno positivo para a análise do DNA em todas as amostras. O DNA do HPV não foi detectado em nenhum dos casos de amostras de tecido de pacientes com CEC em ambos os métodos utilizados. A ausência do HPV observada em nosso estudo pode sugerir que este não é um fator de risco prevalente nos CECs de boca e orofaringe nesta região geográfica. Os fatores de risco clássicos para o desenvolvimento desses tumores parecem ser ainda a principal causa nessa população brasileira. Investigações detalhadas do estilo de vida com maior amostragem precisam ser melhor exploradas para compreensão da baixa prevalência encontrada. / Evidence suggests that human papillomavirus (HPV) is associated with a subgroup of squamous cell carcinomas of the head and neck (HNSCC). However, the prevalence of HPV varies substantially depending on the anatomical site and geographic region studied. Here, our goal was to investigate the prevalence of HPV in fresh tissue samples from Brazilian patients with squamous cell carcinoma (SCC) of the oral cavity and oropharynx by combining two reliable methods for the detection of the HPV DNA. We recruited thirty-six fresh tissue samples from SCC of the oral cavity (n= 27) and oropharynx (n= 9) for analysis. The sociodemographic, lifestyle and clinicopathological characteristics were obtained from individual medical records. HPV DNA was detected by two methods: real-time polymerase chain reaction (PCR) through the qualitative assay of presence or absence for HPV-16, and tested for 37 genotypes by the Roche Linear Array. Amplification of the β-globin gene functioned as a positive internal control for DNA analysis in all samples. HPV DNA was detected in none of the tissue samples from patients with SCC in both methods. The absence of HPV observed in our study may suggest that this is not a prevalent risk factor in SCC of the oral cavity and oropharynx in this geographical region. The classic risk factors for the development of these tumors seem to be still the main cause in this Brazilian population. Detailed investigations of lifestyle with larger sample needs to be better explored to understand the low prevalence found. / FAPEAM: 120/2015

IMPORTANCE: Advanced stage head and neck cancers are often deemed unresectable due to the aggressive nature of the cancer. In evaluating survival patterns of patients with stage IVb tumors, it is valuable to determine whether patients who undergo oncological surgery have favorable outcomes in order to deem surgery as a viable treatment option and demonstrate that these patients can survive with adequate treatment. OBJECTIVE: To determine whether patients with stage IVb cancers who undergo oncological surgery have favorable survival outcomes. MATERIALS AND METHODS: Of 320 cases reviewed of patients treated for head and neck cancer at Boston Medical Center between June 2009 and October 2014, 18 patients with stage IVb tumors were identified. Information regarding date of initial diagnosis, date and type of treatment, and date of death were extrapolated from medical records. Mean survival rates were calculated to compare survival outcomes of those who received and those who did not receive surgical intervention. RESULTS: The mean survival rate for patients who underwent surgical intervention was found to be 29.5 months while those who did not receive surgical intervention had a mean survival of 20.83 months. CONCLUSION: Cancers of the head and neck are associated with poor prognoses and are often deemed unresectable. Patients should be offered definitive treatment despite recommended palliative treatment, as, with adequate treatment, favorable survival outcomes are attainable.

Head and neck cancer (HNC) accounts for 3-5% of all malignancies in the United States and is the sixth most common cancer worldwide. Over the past two decades, radiation therapy (RT) has become a frequent therapeutic strategy, however one of its side effects, dysphagia has had a huge impact on patients’ quality of life. The value of determining the true prevalence of dysphagia is remarkable, which is what prompted us to carry out a study to determine the prevalence, trends, and risk factors for dysphagia following completion of RT over one year in patients diagnosed with HNC at Boston Medical Center over a 7-year period. A retrospective cohort study was conducted that involved a chart review of the medical records of all patients who completed RT for HNC cancer from January 1, 2003 to December 31, 2009 at Boston University Medical Center. 113 eligible patients were who had comprehensive treatment and follow up data at 3, 6, 9 or 12 months post RT were analyzed. Outcome variables of interest included feeding tube status, diet status, subjective swallow status, and percent weight loss from end of RT. 113 patients were identified for this study, of which 31% (n=35) were female and 69% (n=78) were male. Average age was 58.6 years old (35 to 88). The most common cancer sites were oropharynx and nasopharynx (38.9%) as well as hypopharynx and larynx (31%). 71.7% of the cohort had chemotherapy (CT) in addition to RT, and about half the patients had some degree of surgery. Altogether, the most “clinically meaningful” indicator of dysphagia (diet level of moderate/severe diet restriction) showed that the prevalence or probability of dysphagia to be 49% at 3 months, 56% at 6 months, 45% at 9 months, and 31% at 12 months. Our results suggest that about half the patients who undergo RT may be expected to develop a significant swallowing dysfunction in the first year following RT. This is extremely useful data for a health care provider to present to a patient after diagnosis of HNC and should complement counseling provided to them at the time of creating a treatment plan. Interestingly most of the patients who developed moderate/severe dysphagia did so within the first 6 months of completion of RT. Only oral cavity as cancer site was associated with moderate/severe dysphagia in our cohort of patients.

Os microRNAs (miRNAs, miRs) são pequenos RNAs não codificadores presentes em diferentes organismos. Esses RNAs regulam a tradução de genes alvos por meio de ligação seqüência-específica a RNAs mensageiros (mRNAs). Dependendo do grau de complementaridade, podem inibir a tradução e/ou induzir a degradação desses mRNAs. No presente estudo, foi investigado por PCR em tempo real o padrão de expressão de quatro microRNAs (miR-21, -205, -342 e let-7a ) em quatro linhagens celulares derivadas de tumores da cavidade oral e da faringe (FaDu, Hep-2, SCC9 e UM-SCC-38), em queratinócitos orais normais e em amostras de tumor e margens cirúrgicas pareadas de 34 pacientes com carcinomas epidermóides de cabeça e pescoço (CECP). Foi também investigada a correlação da expressão dos MiRs de interesse com as características clinicopatológicas de pacientes com CECP. Nas linhagens celulares, os níveis dos miRs foram similares ou mais baixos que os de queratinócitos normais, ou os miRs não se expressaram. Somente o miR-342 mostrou níveis elevados na linhagem FaDu. Em células Hep-2 tratadas com estradiol, a expressão de miR-let-7a mostrou-se reduzida. Em tumores primários, níveis baixos de miR-let-7a foram observados em carcinomas de soalho de boca e laringe. A expressão de miR-21, -205 e -342 mostrou grande variabilidade entre as amostras e foi reduzida em um dos sítios anatômicos. Não foi observada correlação entre a expressão dos miRs e as características clinicopatológicas dos pacientes com CECP. A análise de três genes alvo candidatos (LYZ, MGLL e SPRR3) mostrou, em carcinomas de soalho de boca e laringe, associação positiva entre a expressão de miR-let-7a e de seu alvo predito MGLL, uma lipase que pode favorecer o fenótipo maligno aumentando os níveis de ácidos graxos livres e sinais lipídicos oncogênicos. O significado dessa associação não pode ser deduzida dos experimentos realizados pelo presente trabalho. / MicroRNAs (miRNAs, miRs) are small, non-coding RNAs present in different organisms. They regulate the translation of target genes through sequence specific binding to mRNA. Depending on the degree of sequence complimentary, they can inhibit translation and/or degradation of target mRNAs In the present study, we used real time PCR to investigate the expression pattern of four microRNAs (miR-21, -205, -342 e let-7a ) in four cell lines derived from tumors of oral cavity and pharinx (FaDu, Hep-2, SCC9 e UM-SCC-38), in normal oral keratinocytes and in matched tumor / surgical margin samples from 34 patients with head and neck squamous cell carcinomas (HNSCC). We also aimed to correlate the miR expression with the clinicopathological features in HNSCC. In cell lines, the miR levels were similar or lower than those in normal keratinocytes, or even absent. Only miR-342 showed high levels in FaDu cell line. In Hep-2 cells treated with estradiol, miR-let-7a expression was reduced. In primary tumors, low miR-let-7a levels were observed in floor of the mouth and larynx carcinomas. The expression of miR-21, -205 and -342 showed high variability between samples and was reduced in one anatomical site. No correlation was observed between miR expression and clinopathological features of head and neck cancer patients. The analysis of three potential target genes (LYZ, MGLL e SPRR3) showed, in floor of the mouth and larynx carcinomas, a positive correlation between the expression of miR-let-7a and its predicted target gene MGLL, a lipase that may support the malignant phenotype by increasing levels of free fatty acids and oncogenic lipid signals. The meaning of such association was not clear from our data.

Alterações no padrão normal de metilação do DNA têm sido caracterizadas como um importante mecanismo na gênese de neoplasias. Esta modificação do DNA é denominada de epigenética uma vez que altera o padrão de expressão das células sem alterar a seqüência do DNA. No câncer, as alterações epigenéticas observadas consistem na hipermetilação das ilhas CpG nos promotores dos genes acompanhada de uma hipometilação global dos dinucleotídeos CpG dispersos pelo genoma. Este evento mostra geralmente ser câncer-específico, ou seja, alguns genes que são metilados em um tipo de câncer, não o são na maioria dos outros tipos. O objetivo deste projeto foi identificar, através da construção de bibliotecas subtrativas de RaSH, genes silenciados por metilação nas linhagens de câncer de cabeça e pescoço FaDu, UM-SCC-14A, UM-SCC-17A e UM-SCC-38 e que possuem expressão induzida após o tratamento com o agente demetilante 5-aza-2-deoxicitidina. Uma vez que a metilação leva a diminuição gradual da expressão gênica, o método RT-PCR semi-quantitativo foi utilizado para validação da expressão diferencial dos genes candidatos PLAU, CD82, RBBP4, AOF2, TMSB10, HSPA5 e LAMC2 nas linhagens não tratadas e tratadas com o agente demetilante 5-aza-2-deoxicitidina. Para todos os genes candidatos foi observado aumento na expressão gênica após o tratamento em pelo menos uma das quatro linhagens. Na linhagem UM-SCC-14 A, os genes CD82, RBBP4, AOF2, HSPA5 e LAMC2 mostraram aumento na expressão após o tratamento com o agente demetilante, sendo que o gene LAMC2 também mostrou esse aumento de expressão na linhagem UM-SCC-17A. Na linhagem UM-SCC-38A todos os genes mostraram aumento de expressão após o tratamento. Embora novos estudos sobre a metilação da região promotora dos genes selecionados sejam necessários, aumentam as evidências de que os genes selecionados sejam regulados pelo mecanismo de metilação e que estejam metilados nas linhagens estudadas. / Abnormalities on the normal pattern of DNA methylation have been caracterized as an important mechanism on carcinogenesis. This modification is called epigenetic and can be defined as a heritable change in gene expression that is not accompanied by changes in DNA sequence. The epigenetic alterations observed on cancer include hypermethylation of selected CpG island gene promoters and simultaneous global hypomethylation. The aim of this project was to identify, by rapid subtraction hybridization, genes silenced by methylation on head and neck cancer lineages with alterations on gene expression after the treatment with 5-aza-2-deoxycytidine. The cancer lineages we used for our experiments were: FaDu, UM-SCC-14A, UM-SCC-17A e UM-SCC-387A and seven genes (PLAU, CD82, RBBP4, AOF2, TMSB10, HSPA5 and LAMC2) were analysed by semiquantitative RT-PCR and for all of them an increaseament of gene expression was observed. For the UM-SCC-14A lineage, the genes CD82, RBBP4, AOF2, HSPA5 and LAMC2 were upregulated after the treatment with demethylating agent as well as LAMC2 was uperegulated on UM-SCC-17A. For the UM-SCC-38A lineage all the genes showed increased expression after the treatmente with -aza-2-deoxycytidine. Our work is another evidence that some genes may be regulated by methylation during carcinogenesis.

Abstract Traditional clinicopathological factors are not accurate enough to predict the behavior of head and neck squamous cell carcinoma (HNSCC). The most powerful indicator of prognosis is the stage of the disease. New prognostic markers have, however, been searched for in order to better identify patient groups in need of different treatments or follow-up. Gelatinases (MMP-2, -9) are endopeptidases associated with tumor invasion and angiogenesis, and their tissue inhibitors (TIMP-1, -2) are also linked to cancer cell invasion and metastasis formation. In some cancer types they are even prognostic and relate with a more aggressive clinical course of the disease. In the present work the expression and the clinical significance of tumor tissue and circulating immunoreactive proteins for MMP-2, -9, TIMP-1 and -2 were assessed in HNSCC. The study group included 74 patients with HNSCC and 44 healthy controls. The expression of immunoreactive proteins was examined in paraffin-embedded tumor sections by immunohistochemical staining using specific antibodies, and the pretreatment serum levels of those proteins were quantitatively measured by ELISA assay. Immunohistochemical overexpression of MMP-9 in tumor was for the first time found to predict the prognosis for shortened survival in HNSCC, the cause-specific survival rates being 45% and 92% and relapse-free survival being 42% and 79% in MMP-9 positive or negative cases, respectively. Additionally, tissue TIMP-1, MMP-2 and TIMP-2 positivity were all also linked with poorer survival of patients with HNSCC. However, these differences remained less distinct than with MMP-9. The expression of gelatinases and their inhibitors in tumor tissue was also an indicator for later lymph node or hematogenic relapses in HNSCC patients. Circulating MMP-9 and TIMP-1 levels were significantly higher in HNSCC patients than in healthy controls. Further, the cause-specific and relapse-free survival rates were lower among HNSCC patients with high MMP-9 and TIMP-1 serum levels compared to patients with low levels of circulating MMP-9 and TIMP-1. A significant correlation was shown between circulating MMP-9 and MMP-9 immunohistochemical staining in the corresponding tumors. No correlation was found between tissue or circulating levels of gelatinases or their inhibitors and the traditional clinical or histopathological factors, except for the association between tissue and circulating TIMP-1 and the size of the primary tumor. Taken together, these results suggest that tissue expression of gelatinases and their inhibitors as well as pretreatment circulating MMP-9 and TIMP-1 levels could be prognostic in estimation of the clinical course of HNSCC. The results indicate further studies are needed with larger patient materials.

The goal of this thesis was to evaluate the cost-effectiveness of implantable Doppler technology (IDT) used to monitor free tissue transfer (FTT) procedures in the treatment of cancer of the upper aerodigestive tract (UADT). First, a systematic review of the literature on the effectiveness of traditional and IDT monitoring techniques was performed. Second, a utility survey using a time trade-off technique was created and administered. The results from this survey were used to establish utility values for health states common in patients undergoing FTT procedures. Third, a cost study using the microcosting data available through the Ottawa Hospital was performed. Finally, a decision analytic model was created and an economic evaluation from the payer perspective was completed. A probabilistic sensitivity analysis (PSA) and a value of information analysis (VOI) were performed. The thesis found that the currently available evidence supports IDT as a cost-effective intervention. Further research should be directed towards determining the effectiveness of both traditional and IDT monitoring.

Receptor Tyrosine Kinase (RTK) inhibitors, such as erlotinib/tarceva, have been introduced in the past decade as a promising therapeutic option in Head and Neck Squamous Cell Carcinoma (HNSCC), however, they lack significant efficacy as single agents. As a result, RTK inhibitors require a combination based therapeutic approach with other treatment modalities. To uncover such a combination of agents, we performed a high throughput Prestwick library screen that included 1200 compounds approved by the FDA on HNSCC cell lines and found that monensin, a coccidial antibiotic, synergistically enhanced the cytotoxicity of erlotinib. RT-PCR revealed that monensin induced the expression of Activation of Transcription Factor (ATF) 3 and its downstream target C/EBP homologous protein (CHOP) which are key regulators of apoptosis. Furthermore, RNA-Seq analysis suggests that monensin augments erlotinib cytotoxicity by disturbing lipid and sterol biosynthesis. Therefore, identifying the mechanism of action exerted by monensin may open alternative avenues of cancer treatment.

Cancer cells are believed to exist in a condition of metabolic oxidative stress compared to normal cells because of inherent mitochondrial dysfunction. Cancer cells up regulate antioxidant defense mechanisms to combat the toxic effect of reactive oxygen species (ROS). Many anticancer agents block ROS detoxification mechanisms and utilize oxidative stress to cause cytotoxicity to cancer cells. However, ROS also up-regulate many pro-survival signaling pathways that may mediate resistance to chemotherapy. I hypothesize that ROS induces both cytotoxicity and pro-survival mechanisms in cells treated with chemotherapeutic agents such as the EGFR inhibitor erlotinib. This thesis explores how oxidative stress may induce both pro-survival and pro-death mechanisms in HNSCC cells and how this can be exploited to increase the cytotoxicity of erlotinib. The combined use of buthionine-[S,R]-sulfoximine, an inhibitor of glutathione and auranofin, an inhibitor of thioredoxin metabolism enhanced human head and neck cancer cell killing by a mechanism involving oxidative stress both in vitro and in vivo and sensitized cells to erlotinib in vitro. However, in other studies erlotinib as a single agent induced oxidative stress and this was mediated by NADPH oxidase 4 (NOX4). NOX4 mediated oxidative stress activated a process called autophagy which protected cancer cells from cytotoxic effect of erlotinib and inhibition of autophagy sensitized cells to erlotinib in vitro. These studies show that oxidative stress may have a dual role in cancer chemotherapy. ROS generated from various drug treatments can cause oxidative damage of cells culminating in cell death. However, it may also activate autophagy protecting cells against the stress and leading to decreased efficacy of the treatment. Hence inhibiting autophagy and hydroperoxide metabolism can be effective treatment modalities to enhance the cytotoxicity of erlotinib and achieve maximum therapeutic efficacy.