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Objetivo: Avaliar por meio da Tomografia Computadorizada singleslice, alterações nas estruturas do espaço mastigador, por disseminação loco-regional das neoplasias malignas originárias da loja tonsilar, trígono retromolar, seio maxilar e nasofaringe; correlacionar a presença de trismo com os achados tomográficos e dimensões do tumor. Material e métodos: foram selecionados prontuários de pacientes portadores de tumores malignos envolvendo as 4 regiões a serem estudadas, junto aos arquivos do Hospital Heliópolis. Foram excluídos os pacientes submetidos a terapêutica antineoplásica prévia ou com doenças inflamatórias ou infeciosas locais. Os prontuários foram revisados por um único examinador, onde foram coletadas informações relativas a idade, gênero, localização do tumor primário, tempo de evolução da doença, estadiamento do tumor e presença ou ausência de trismo. Após a administração do meio de contraste iodado, o protocolo de aquisição de imagens tomográficas foi de cortes axiais de 3mm de espessura, aquisições coronais foram feitas quando necessário. A análise das imagens foi feita por dois examinadores médicos, residentes do 3° de Radiologia médica, em momentos distintos, sem o conhecimento prévio das informações clínicas. Na avaliação do EM, os achados foram transcritos para uma planilha específica, considerando a presença ou ausência dos seguintes itens: simetria com o EM contralateral, obliteração do plano gorduroso, do trígono retromolar e do espaço faríngeo, edema e/ou atrofia dos músculos pterigoideos medial e lateral, destruição do ramo da mandíbula. Resultados: O trismo esteve presente em 10 pacientes estudados. Na associação entre dimensão do tumor e trismo, observamos que 90% dos pacientes apresentam tamanho T4, enquanto essa porcentagem é de 43% nos pacientes sem trismo. Analisando-se em termos de razão de chances, verificou-se que pacientes com tumores T4 apresentam uma chance de trismo de 11,6 vezes maior que as demais dimensões (T0 a T3). As neoplasias da loja tonsilar e trígono retromolar perfizeram 95% das neoplasias avaliadas. Foram encontrados apenas 3 casos de neoplasia da nasofaringe, e em nenhum deles observamos a presença de trismo, e não foi encontrado nenhum caso de neoplasia de seio maxilar. Relacionando os achados tomográficos com o grupo de pacientes com trismo, observamos que 60% dos pacientes com trismo apresentaram edema e/ou atrofia dos músculos pterigoideos na TC, enquanto que essa porcentagem foi de 21,8% nos pacientes sem trismo. Em relação às dimensões do tumor e sua relação com os achados das imagens observamos associação apenas entre tumores T4 e detecção de edema e/ou atrofia dos músculos pterigoideos, neste item pacientes com edema e/ou atrofia dos músculos pterigoideos apresentam 5,4 vezes ais chance de apresentarem trismo. Conclusões: Observamos na TC as seguintes alterações no EM: obliteração do plano gorduroso em 69,2 % dos pacientes, assimetria em 27,7%, edema/atrofia em 26,2 % dos pacientes e destruição óssea em 20%. Os graus de concordância inter examinadores foram variáveis. Encontramos associação apenas entre pacientes com trismo e edema/atrofia dos músculos pterigoideos. Neste mesmo item foi observado associação com tumores de dimensão T4. Não encontramos associação do trismo com os demais itens avaliados na TC. / Objective: Evaluate by computed tomography (CT) singleslice changes in the structures of the masticator space, for loco-regional dissemination of malignancies originating in the tonsil, retromolar area, maxillary sinus and nasopharynx to correlate the CT findings with trismus and the tumor dimensions. Material and Methods: The medical records of patients with malignant tumors involving the four regions to be studied were selected from the archives of the Heliopolis Hospital. Patients submited previously to antineoplastic therapy or with local inflammatory or infectious diseases were excluded. The medical records were reviewed by a single examiner, where information was collected on age, gender, tumor location, disease evolution, tumor stage, and the presence or absence of trismus. After administration of iodinated contrast media, the protocol the image acquisition was axial slices 3mm, coronal acquisitions were made when necessary. The image analysis of the MS by two medical examiner residents in their 3rd year of Medical Radiology, at distinct moments , without prior knowledge of the clinical information. In the evaluation of the MS, the findings were transcribed to a specific worksheet, considering the presence or absence of the following items: symmetry with the contralateral MS, obliteration of the fat plane of the retromolar trigone and the pharyngeal space, edema, and/or the atrophy of the medial and lateral pterygoid muscles, and destruction of the mandibular ramus. Results: According to the adopted criteria for trismus in this study, the presence of trismus was observed in 10 patients. The association between tumor size and trismus shows that 90% of patients present size T4, while this percentage is 43% in patients without trismus. Analyzing in terms of odds ratios, the study showed that patients with T4 sized tumors had a chance of developing trismus 11.6 times greater than patients with T0 to T3 sized tumors. The neoplasms of tonsillar crypts and retromolar trigone aggregated 95% of the neoplasms evaluated. Only 5% of the cases accounted for neoplasms of nasopharynx, in which none of them presented the existence of trismus. No cases of neoplasm of maxillary sinus was found. Correlating the CT findings with the group of patients with trismus, the observation was made that 60% of patients with trismus showed edema and/or pterygoid muscle atrophy on CT, whereas this percentage was 21.8% in patients without trismus. Regarding the dimensions of the tumor and its relationship with the image findings, the detection of edema and/or atrophy of the pterygoid muscles was observed only in association with T4 sized tumors. In this instance, patients with edema and/or atrophy of the pterygoid muscles present a 5,4 times greater chance of having trismus. Conclusions: Observed in the following changes in TC IN: obliteration of fat plane in 69.2% of patients, 27.7% asymmetry, edema / atrophy in 26.2% of patients and 20% in bone destruction. The degree of inter examiners were variable. Association was found only among patients with trismus and edema / atrophy of the pterygoid muscles. In this same item was observed association with tumor size T4. We found no association of trismus with other items valued at TC.

Objetivo: Avaliar por meio da Tomografia Computadorizada singleslice, alterações nas estruturas do espaço mastigador, por disseminação loco-regional das neoplasias malignas originárias da loja tonsilar, trígono retromolar, seio maxilar e nasofaringe; correlacionar a presença de trismo com os achados tomográficos e dimensões do tumor. Material e métodos: foram selecionados prontuários de pacientes portadores de tumores malignos envolvendo as 4 regiões a serem estudadas, junto aos arquivos do Hospital Heliópolis. Foram excluídos os pacientes submetidos a terapêutica antineoplásica prévia ou com doenças inflamatórias ou infeciosas locais. Os prontuários foram revisados por um único examinador, onde foram coletadas informações relativas a idade, gênero, localização do tumor primário, tempo de evolução da doença, estadiamento do tumor e presença ou ausência de trismo. Após a administração do meio de contraste iodado, o protocolo de aquisição de imagens tomográficas foi de cortes axiais de 3mm de espessura, aquisições coronais foram feitas quando necessário. A análise das imagens foi feita por dois examinadores médicos, residentes do 3° de Radiologia médica, em momentos distintos, sem o conhecimento prévio das informações clínicas. Na avaliação do EM, os achados foram transcritos para uma planilha específica, considerando a presença ou ausência dos seguintes itens: simetria com o EM contralateral, obliteração do plano gorduroso, do trígono retromolar e do espaço faríngeo, edema e/ou atrofia dos músculos pterigoideos medial e lateral, destruição do ramo da mandíbula. Resultados: O trismo esteve presente em 10 pacientes estudados. Na associação entre dimensão do tumor e trismo, observamos que 90% dos pacientes apresentam tamanho T4, enquanto essa porcentagem é de 43% nos pacientes sem trismo. Analisando-se em termos de razão de chances, verificou-se que pacientes com tumores T4 apresentam uma chance de trismo de 11,6 vezes maior que as demais dimensões (T0 a T3). As neoplasias da loja tonsilar e trígono retromolar perfizeram 95% das neoplasias avaliadas. Foram encontrados apenas 3 casos de neoplasia da nasofaringe, e em nenhum deles observamos a presença de trismo, e não foi encontrado nenhum caso de neoplasia de seio maxilar. Relacionando os achados tomográficos com o grupo de pacientes com trismo, observamos que 60% dos pacientes com trismo apresentaram edema e/ou atrofia dos músculos pterigoideos na TC, enquanto que essa porcentagem foi de 21,8% nos pacientes sem trismo. Em relação às dimensões do tumor e sua relação com os achados das imagens observamos associação apenas entre tumores T4 e detecção de edema e/ou atrofia dos músculos pterigoideos, neste item pacientes com edema e/ou atrofia dos músculos pterigoideos apresentam 5,4 vezes ais chance de apresentarem trismo. Conclusões: Observamos na TC as seguintes alterações no EM: obliteração do plano gorduroso em 69,2 % dos pacientes, assimetria em 27,7%, edema/atrofia em 26,2 % dos pacientes e destruição óssea em 20%. Os graus de concordância inter examinadores foram variáveis. Encontramos associação apenas entre pacientes com trismo e edema/atrofia dos músculos pterigoideos. Neste mesmo item foi observado associação com tumores de dimensão T4. Não encontramos associação do trismo com os demais itens avaliados na TC. / Objective: Evaluate by computed tomography (CT) singleslice changes in the structures of the masticator space, for loco-regional dissemination of malignancies originating in the tonsil, retromolar area, maxillary sinus and nasopharynx to correlate the CT findings with trismus and the tumor dimensions. Material and Methods: The medical records of patients with malignant tumors involving the four regions to be studied were selected from the archives of the Heliopolis Hospital. Patients submited previously to antineoplastic therapy or with local inflammatory or infectious diseases were excluded. The medical records were reviewed by a single examiner, where information was collected on age, gender, tumor location, disease evolution, tumor stage, and the presence or absence of trismus. After administration of iodinated contrast media, the protocol the image acquisition was axial slices 3mm, coronal acquisitions were made when necessary. The image analysis of the MS by two medical examiner residents in their 3rd year of Medical Radiology, at distinct moments , without prior knowledge of the clinical information. In the evaluation of the MS, the findings were transcribed to a specific worksheet, considering the presence or absence of the following items: symmetry with the contralateral MS, obliteration of the fat plane of the retromolar trigone and the pharyngeal space, edema, and/or the atrophy of the medial and lateral pterygoid muscles, and destruction of the mandibular ramus. Results: According to the adopted criteria for trismus in this study, the presence of trismus was observed in 10 patients. The association between tumor size and trismus shows that 90% of patients present size T4, while this percentage is 43% in patients without trismus. Analyzing in terms of odds ratios, the study showed that patients with T4 sized tumors had a chance of developing trismus 11.6 times greater than patients with T0 to T3 sized tumors. The neoplasms of tonsillar crypts and retromolar trigone aggregated 95% of the neoplasms evaluated. Only 5% of the cases accounted for neoplasms of nasopharynx, in which none of them presented the existence of trismus. No cases of neoplasm of maxillary sinus was found. Correlating the CT findings with the group of patients with trismus, the observation was made that 60% of patients with trismus showed edema and/or pterygoid muscle atrophy on CT, whereas this percentage was 21.8% in patients without trismus. Regarding the dimensions of the tumor and its relationship with the image findings, the detection of edema and/or atrophy of the pterygoid muscles was observed only in association with T4 sized tumors. In this instance, patients with edema and/or atrophy of the pterygoid muscles present a 5,4 times greater chance of having trismus. Conclusions: Observed in the following changes in TC IN: obliteration of fat plane in 69.2% of patients, 27.7% asymmetry, edema / atrophy in 26.2% of patients and 20% in bone destruction. The degree of inter examiners were variable. Association was found only among patients with trismus and edema / atrophy of the pterygoid muscles. In this same item was observed association with tumor size T4. We found no association of trismus with other items valued at TC.

Breast Cancer susceptibility gene 1 (BRCA1) & Breast Cancer Breast cancer is one of the most common malignancies affecting women worldwide. About 5-10% of all cases are estimated to be familial. Mutations in the BRCA1 (Breast Cancer susceptibility gene 1) gene account for about 15-20% of inherited breast cancer cases and 60-80% of families predisposed to both breast and ovarian cancer. BRCA1 mutations also result in susceptibility to early-onset breast and ovarian cancer. The human BRCA1 gene encodes a multi-domain 1,863 amino acid nuclear protein that is expressed in a wide variety of adult human tissues. The N-terminal end of BRCA1 contains a RING-finger domain. Exon 11 of BRCA1 contains two nuclear localization signals towards its N-terminal for targeting BRCA1 to the nucleus. The carboxyl terminus contains two BRCT (BRCA1 C-terminal) domains and a transcriptional activation domain. This study was carried out to functionally characterize BRCA1 and to find out the percentage in which BRCA1 gene is mutated in Indian familial breast and/or ovarian cancer families. The work has been divided into three sections: 1. Identification & characterization of a BRCA1 Associated Protein 2 (BAP2). 2. Germ-line BRCA1 mutation Analysis in Indian Breast and/or Ovarian Cancer Families. 3. Characterization of a novel missense mutation (E116K) in BRCA1. BRCA1 is known to interact with large number of proteins and is involved in various cellular functions like tumorigenesis, transcription, DNA damage repair, cell-cycle control, ubiquitinylation, genetic stability, cell growth and apoptosis. The interacting partners of BRCA1 have given a lot of clue about the functions of this complex protein. In the first project, we used the yeast two-hybrid system to identify novel interacting proteins of BRCA1. We used the 1-500 amino acid region of BRCA1 as bait in library screen and picked up a novel clone (clone 89) showing interaction with BRCA1. Clone 89 contains approximately 2.3 Kb long cDNA sequence. Using the nucleotide blast search, we obtained a full-length cDNA of approximately 5.4 Kb (KIAA0657) that is located on chromosome 2, 2q36.1 region. We have named this new protein BRCA1 Associated Protein 2 (BAP2). Translation of this coding sequence gave a protein that has homology to Titin protein. This protein, which has 1,236 amino acids, contains 9 Immunoglobulin like domains. The homologues of this protein exists in many other organisms but the function is not known. We have confirmed the interaction between BRCA1 and c89 using in vitro GST pull-down assay. We have studied the influence of BAP2 on various functions of BRCA1 like transcription, colony suppression and cell cycle. In the transcription assays, BAP2 activated p21 promoter activity perhaps by using endogenous BRCA1 as simultaneous ectopic expression of truncated BRCA1 (containing aa 1-500) abolished this activity. Further, BAP2 also increased the ability of BRCA1 to activate p21 promoter suggesting that BAP2 may act as a co-activator of BRCA1 functions. Surprisingly, we observed that BAP2 inhibited p53-mediated transcription both in the absence and presence of BRCA1. BAP2 failed to inhibit colony growth by itself as well as in combination with BRCA1. In the cell-cycle study, we found that BAP2 did not have any significant effect on cell cycle profile by itself. However, it drastically augmented the G2/M arrest mediated by BRCA1. Thus we conclude that we have identified a novel interacting protein of BRCA1 that regulates certain functions of BRCA1. Detection of mutations is of central importance in the study of genetic and malignant diseases. Mutation detection helps us in understanding the protein structure, function and expression. More than that, it is also important for pre-symptomatic/antenatal diagnosis, confirmation of the genetic cause of the disease and the mode of inheritance of a disease in a particular family, the prediction of clinical phenotype and the potentiation of diagnostic analysis in the case of families with incomplete pedigrees or with new mutations. Therefore, the importance of direct mutation analysis cannot be understated. The second project deals with screening of mutations in BRCA1 gene in 50 familial breast and/or ovarian cancer families using the technique of Conformation Sensitive Gel Electrophoresis (CSGE). CSGE can be used to detect mismatches in DNA heteroduplexes that contain one strand of wild type and one strand of mutated DNA. In a collaborative study with Kidwai Memorial Hospital for Oncology, Bangalore, we screened 50 families suffering from breast and/or ovarian cancer. We detected 13 mutations in this study out of which 3 are novel and 10 have already been reported earlier (Breast Information Core). All the mutations obtained in our study result in truncation of the BRCA1 protein either because of non-sense mutation or frame-shift mutation. Interestingly, 8 of the mutations detected are 185delAG mutations – the most commonly occurring mutation in Ashkenazi Jewish population. From this study, we conclude that BRCA1 is mutated in 26% of familial breast and/or ovarian cancer cases in India. Genetic testing in individuals with family history of breast, ovarian or both has become very common. It is difficult to interpret the result of genetic screen if a DNA change in the gene does not result in truncation of the protein. Rare missense changes of unknown functional and pathogenic significance are called unclassified variants. It is important to study the functional implications of these unclassified variants in order to determine the risk associated with the presence of such variations. The third project deals with characterization of one such missense variation. In an earlier mutation analysis study for BRCA1 gene in breast cancer samples, we found a novel missense variation resulting in Glu116Lys (E116K) change. In order to determine if this variant is a disease associated missense mutation or a benign sequence alteration; we introduced this variation into full length BRCA1 cDNA and studied its effect on the known functions of BRCA1, namely, transcription, colony suppression and cell cycle. We found that E116K is defective for activating transcription. However, it continued to inhibit growth in colony formation assay and arrest cells in G2/M phase of cell cycle. We conclude that E116K mutation results in loss of transactivation function of BRCA1 but has no effect on colony formation and cell cycle regulation; thus it can be categorized as a novel missense mutation.

Background: Cancer in the head and neck region is an unusual form of cancer. The treatment for this cancer can cause permanent damage to vital functions. Nurses' have expressed frustration about feeling insufficient to accommodate the patients' needs. Patients' with head and neck cancer are reported to have low quality of life. Aim Describe patients' experiences of undergoing treatment of cancer in the head and/or neck region. Method: A qualitative systematic literature review with descriptive approach of qualitative articles. Result: Undergoing treatment for cancer in the head and neck region created challenges for the patients'. Many patients' have had experienced that the time between the diagnose and treatment went fast. The treatment caused side effects that led to constraints in daily life and many created a new identity because they did not recognize themselves in the same way as before the diagnose. Patients became dependent to the medical care and made them omitted to others. They had to handle a new life situation, and to manage that, the patients created strategies. Conclusion: The treatment provided side effects that affected the everyday lives of the patients. The side effects impacted on patients both physically, psychologically, socially and existentially. More knowledge is needed in order to improve the health care for patients with head and neck cancer.

No / Methionine dependence is unique to cancer cells and defined as the inability to grow in a methionine-deprived environment even if supplemented with the metabolic precursor homocysteine. Cobalamin-dependent methionine synthase (MS) catalyses the formation of methionine and tetrahydrofolate from homocysteine and methyltetrahydrofolate, thus linking the methionine and folate pathways. The apparent altered methionine metabolism in methionine-dependent cancer cells suggests a role for MS, although results to date are conflicting. We have analysed key metabolites of the MS-associated transmethylation, transsulphuration and folate pathways of the methionine-dependent MAC15A tumour model as a function of tumour progression over a 10-day period. MS activity increased 2-fold from day I to day 10. Cysteine, homocysteine, S-adenosylmethionine and S-adenosylhomocysteine levels in tumour cytosolic fractions decreased as a function of tumour progression. Plasma cysteine levels also decreased, whilst the distribution of folates in erythrocytes was altered, with a maximum increase in methyltetrahydrofolate observed by day 5. The increasing MS activity and decreasing cysteine levels suggest an increasing methionine requirement by the tumour, whilst the induction of enzyme activity indicates that MS is not defective in the methionine-dependent MAC15A tumour. The decrease in tumour S-adenosylmethionine and S-adenosylhomocysteine levels suggests that methionine is required for some function other than cellular methylation, e.g., incorporation into protein. Overall, the results support a theory of methionine conservation in response to tumour growth, where the methionine-dependent MAC15A tumour has a higher than normal methionine requirement.

Due to the constantly increasing epidemiological burden of our population on oncological diseases, nationwide preventive programs for selected types of malignant tumors have been introduced within the Czech health system. The aim of this thesis is to analyze and evaluate these screenings, such as mammographic screening, cervical screening and screening of the colon and rectum. The subject of the analysis is the mortality rates for breast cancer (C50), cervix (C53) and colon and rectum cancer (C18-21) between 1994 and 2015. The main focus is on question of whether the development of the mortality rate for selected neoplasms depends on the degree of coverage rate by a preventive program.

Insulin-like growth factor binding proteins (IGFBPs) modulate the bioavailability of IGFs in circulation. IGFBPs 1-6 bind IGFs with high affinity and can either potentiate or inhibit IGF signaling in a context dependent manner. IGFBP2 is a 36 kDa protein and the second most abundant IGFBP in serum. Numerous studies in the recent past have implied a pro-tumorigenic role of IGFBP2. Elevated expression of IGFBP2 has been observed in multiple malignancies, including glioblastoma multiforme (GBM), ovarian, pancreatic, gastric, prostate, colon, breast, thyroid cancer and leukemia. In addition, increased expression of IGFBP2 in both tissues and serum of patients has been correlated with poor prognosis in prostate, glioblastoma and colon cancers. Pro-tumorigenic actions of IGFBP2 have been supported by in vitro studies, where IGFBP2 increases the tumorigenic potential of adrenocortical tumor cells, epidermoid carcinoma cells, glioma cells and ovarian cancer cells. Further, using xenograft animal models, the role of IGFBP2 in the progression of glioma has been established. In breast cancer, IGFBP2 was found to be over expressed in ductal carcinoma in situ and invasive breast cancer samples. IGFBP2 over expression has been shown to confer drug resistance and an increased expression has been reported to correlate with lymph node metastasis in T1 breast carcinomas. These reports implicate IGFBP2 in breast cancer biology. However, its role in breast cancer progression is not well defined. With this background, the following objectives were set for the current study: Functional characterization of IGFBP2 with respect to its possible role in breast cancer progression. Elucidation of the molecular mechanisms of IGFBP2 actions. Towards this, immunohistochemistry was performed on 132 invasive ductal carcinoma (IDC) grade III tumors using IGFBP2 specific antibody. It was observed that IGFBP2 expression was significantly higher in tumors in comparison to normal tissues that showed no detectable staining for IGFBP2. It was also observed that expression of IGFBP2 significantly correlated with the expression of ER. To understand the functional significance of IGFBP2 over expression in breast cancer, IGFBP2 was characterized with respect to proliferation, survival and tumor forming ability (in vitro and in vivo) in BT474 breast cancer cells. The knockdown of IGFBP2 expression resulted in suppression of colony formation (nearly 70%) in these breast cancer cells, which could be partially reversed upon exogenous addition of IGFBP2 protein. Proliferation assays using stable clones with knockdown of IGFBP2 in BT474 cells showed a significant decrease in proliferation as compared to vector transfected cells in the presence of serum. Culturing of IGFBP2 knockdown breast cancer cells in serum free medium resulted in their growth arrest in G0/G1 phase of cell cycle as compared to control cells, which progressed through the cell cycle. Prolonged culturing of IGFBP2 knockdown cells in serum free condition (up to 72 h) resulted in the increase of cells in sub G1 phase of the cell cycle. Prolonged depletion of growth factors (serum free conditions) could result in apoptosis of these G1 arrested IGFBP2 knockdown cells. When serum starved IGFBP2 knockdown cells were treated with IGFBP2 protein, the cells arrested in G0/G1 phase were able to progress through the cell cycle and concomitant decrease in sub G1 fraction was observed. Knockdown of IGFBP2 resulted in significantly decreased number and visibly smaller colonies in anchorage independent conditions in vitro. Consistent with this observation, in vivo tumor xenograft formation with IGFBP2 knockdown cells also showed significant reduction in tumor weight as compared to vector generated tumors. These results imply that IGFBP2 has potent growth promoting effects on breast cancer and acts as a mitogen/survival factor for breast cancer cells. To elucidate the molecular mechanisms underlying the pro-tumorigenic effects of IGFBP2, the transcriptome profile following IGFBP2 perturbation in breast cancer cells was determined. IGFBP2 knockdown resulted in significant changes in the expression of genes associated with cellular proliferation and tumorigenicity. The down regulated genes were found to be associated with several events, notably cell cycle, p53 and Wnt signaling, as revealed by Gene Set Enrichment Analysis (GSEA). To further validate these results in breast cancer tissues, whole genome expression analysis was performed in 19 breast tumor samples which were categorized as IGFBP2 positive or negative based on immunohistochemical staining pattern. In comparison to IGFBP2 negative tumors, IGFBP2 positive tumors showed increased expression of genes belonging to MAPK, focal adhesion and Wnt signaling pathway. In order to identify the genes commonly regulated by IGFBP2 in cell lines and tumors, the gene expression profiles of IGFBP2 positive versus IGFBP2 negative tumors and IGFBP2 knockdown breast cancer cells were compared. 347 genes were found to be common among IGFBP2 regulated genes in tumors and cell line. The most significant networks representing the web of interactions among these genes were found to be associated with cellular growth and proliferation, cellular movement and nucleic acid metabolism, indicating an association of IGFBP2 expression phenotype to the distinct changes in expression of genes associated with the regulation of cellular growth and migration. Silencing of IGFBP2 in BT474 cells resulted in a reduced IGF signaling as evidenced by the reduced phosphorylation of IGF1R and concomitantly that of ERK. This effect could be reversed upon addition of the IGFBP2 protein, implying that IGFBP2 potentiates IGF signaling in breast cancer cells. Besides IGF ligand and their receptors, regulation of proliferation associated genes like CENPF, TOP2A, CCND1 and FOXM1 by IGFBP2 was observed, thus providing a molecular basis for the pro-proliferative effects of IGFBP2 on breast cancer cells. Addition of IGFBP2 to immortal breast cells resulted in reduced IGF1R signaling and reduced pERK and pAKT signaling. Additionally, the genes involved in cellular proliferation were down regulated upon IGFBP2 treatment in immortal cells. IGFBP2 knockdown clones had reduced expression of FOXM1, a key regulator of cell cycle for G1/S and G2/M transition, and M phase progression. The regulation of CENPF and CCND1 genes was established following over expression of FOXM1 in IGFBP2 knockdown cells. One of the important and novel finding of this study is the regulation of Wnt signaling pathway genes such as CCND1, MMP7, FGF18, MYCBP, FN1 and survivin by IGFBP2. In support of this, β-catenin protein was found to be regulated by IGFBP2 in breast cancer and GBM cells, as evidenced by knockdown and over expression studies. Furthermore, regulation of β-catenin by IGFBP2 was found to involve integrin-FAK and IGF1R signaling. Another important finding of this study is the correlation of IGFBP2 over expression with elevated β-catenin levels in breast tumors. When expression of both IGFBP2 and β-catenin was correlated with the lymph node status of breast cancers, a significant association of IGFBP2 and β-catenin staining with increased lymph node metastasis was observed in comparison with tumors that did not show staining for either protein. Altogether, in this study employing genomic, cellular and molecular approaches, a pro- tumorigenic role for IGFBP2 in breast cancer has been established. Furthermore, this study provides novel insights into the molecular mechanisms employed by IGFBP2 involving IGF1R, FAK and Wnt signaling pathways during breast cancer progression.

Breast cancer is a major global health problem and the most common form of cancer among women. Major advances in the technologies of imaging provide improved detection and sensitivity with fewer unnecessary biopsies. Commonly used imaging modalities include mammography, ultrasonography, magnetic resonance imaging (MRI), scintimammography, single photon emission computed tomography (SPECT) and positron emission tomography (PET). The current study is focused on breast MRI imaging, especially one of the most promising recent techniques, i.e. the Diffusion Weighted Imaging breast MRI (DWI). DWI is an unenhanced MRI technique, based on volume sequences on various b values (the b value identifies the measurement's sensitivity to diffusion and determines the strength and duration of the diffusion gradients) measuring the mobility of water molecules (Brownian motion) in vivo (in tissues) and provides different and potentially complementary information to Dynamic Contrast-Enhanced Magnetic Resonance Imaging (DCE-MRI) technique. As DWI based on the diffusive properties of water molecules, reflects their random motion resulting from thermal agitation. Water diffusion on breast can be quantified by measuring the mean diffusivity, which is the average of Apparent Diffusion Coefficient (ADC). The ADC can be calculated by making measurements at a low b factor, b1, and a higher b factor, b2. DWI allows the mapping of the diffusion process of molecules by the ADC map. ADC maps are calculated by collecting images with at least 2 different values, b1 and b2, of the b factor. The ADC map is a parametric image whose color scale or gray scale represents the ADC values of the voxels and is usually generated by proprietary or in house software. DWI apart from the 3D anatomical information, provides a noninvasive investigation of tissue vascularity, a novel contrast mechanism in MRI and has a high sensitivity in the detection of changes in the local biologic environment due to a pathologic process. Therefore, in addition to contrast enhancement-based characterization (DCE-MRI), measurement of the motion of water molecules in DWI provides an additional feature for lesion characterization that may further increase the specificity of MRI for classifying breast lesions. The diagnostic task that the current study deals with, accounts for the diagnosis of mass-like lesions in Diffusion Weighed Magnetic Resonance Imaging, based on low ADC values compared to high once in case of benign versus normal tissue. The hypothesis is that diffusivity of water molecules is restricted in environments of high cellularity, intracellular and extracellular edema, high viscosity, and fibrosis, such as malignant tumors, because these conditions become barriers to the movement of water molecules. Therefore, most of breast cancers show low ADC values compared with benign and normal tissue. Many studies have revealed the usefulness of ADC values in the differential diagnosis of breast lesions; however, the clinical effect remains limited because of the substantial overlap between benign and malignant lesions, which presents challenges for implementing a useful diagnostic ADC threshold. The majority of studies, similar to the current study, determined optimal cutoff levels of the ADC value between malignant and benign lesions by using ROC analysis, and ranged from 0.90 to 1.76 × 10-3 mm2/s while the sensitivity and specificity ranged from 63% to 100% and 46% to 97%, respectively. In addition, the methods for measuring ADC differ among reported studies, with the most representative method being the mean value of ADC (mean ± standard deviation) over a Region Of Interest representative of the breast lesion. The purpose of this study was to investigate the ability of histogram characteristics of Apparent Diffusion Coefficient (Apparent Diffusion Coefficient-ADC) to differentiate malignant from benign breast lesions in breast DWI. To this end the ADC maps of representative lesion ROIs were subjected to first order statistics analysis by calculating five first order textural features: Mean value, Standard Deviation, Kurtosis, Skewness and Entropy. This approach is intended to offer a more complete assessment of tumor texture and heterogeneity. The dataset analyzed is comprised of 92 histologically verified breast lesions, originating from 69 women with mammographically and/or ultrasonographically detected or palpable findings. Histology revealed 53 malignant lesions originating from 45 women and 39 benign lesions originating from 26 women. All of the breast MR examinations were performed with a 3T MR scanner, for b= 0, 900 s/mm2. Diagnostic performances of these parameters were compared by receiver operating characteristic (ROC) curve analysis. The mean of ADC of benign lesions [(1.470 ± 0.342) × 10-3 mm2/s] was found to be significantly higher than that of malignant tumours, [(0.965 ± 0.268) × 10-3 mm2/s, (p<0.00001)]. The standard deviation of ADC of benign lesions [(0.184 ± 0.999) × 10-3 mm2/s] was not significantly different from that of malignant tumours, [(0.192 ± 0.151) × 10-3 mm2/s, (p=0.6581)]. The skewness of ADC of benign [-0.303 ± 0.584] was significantly different than that of malignant tumours, 0.210 ± 0.725. (p = 0.0008)]. The kurtosis of ADC of benign [3.003 ± 1.065] was not significantly different from that of malignant tumours, [3.337 ± 1.334. (p=0.0987)]. The entropy of ADC of benign [4.794 ± 0.665] was significantly lower than that of malignant tumours, [5.569 ± 0.649, (p<0.00001)] The corresponding area under the empirical receiver operating characteristic curve was: 0.862 ± 0.042 (95% confidence interval: 0.754, 0.925) for mean of ADC, 0.705 ± 0.054 (95% confidence interval: 0.589, 0.800) for skeweness of ADC, 0.800 ± 0.046 (95% confidence interval: 0.691, 0.874) for entropy of ADC, resulting a good diagnostic performance of DWI for these parameters. On the other hand, an AUC of 0.527 ± 0.063 (95% confidence interval: 0.393, 0.640) and 0.601 ± 0.061 (95% confidence interval: 0.470, 0.707) for Standard deviation and kurtosis respectively, suggests a degree of overlap in ADC values between benign and malignant tumors. In an effort to identify optimal threshold values for differentiating benign versus malignant lesions these were selected to correspond to the points of highest accuracy of the ROC curves. In our study, we obtained two threshold values of mean ADC, both with an accuracy of 83.15%: 1.21 x 10-3 mm2/s with a sensitivity of 86.27% and specificity of 78.95%; and 1.32 x 10-3 mm2/s with a sensitivity of 92.16% and specificity of 71.05%. The threshold value of skeweness was -0.06 with an accuracy of 68.54%, a sensitivity of 66.03% and specificity of 66.67%. Finally, we found two threshold values of entropy, both with an accuracy of 76.40%: 5.17 with a sensitivity of 75.47% and specificity of 71.80%; and 5.21 with a sensitivity of 73.59% and specificity of 74.36%. In conclusion, results of the current study suggest the contribution of texture analysis methods in Diffusion-weighted MRI breast imaging for the quantification of tissue heterogeneity, providing important information for breast cancer diagnosis. Histogram analysis of ADC values in breast cancer has potential for differentiating benign and malignant tumors, providing information about the entire tumor. The mean, skewness and entropy of ADC are valuable parameters that are correlated with pathologic characterization of breast tumors. These 3 ADC parameters significantly elevated the quantitative diagnostic performance of breast DWI and would be effective parameters in distinguishing between malignant and benign breast lesions. Finally, future efforts will also focus on investigating the correlation of extracted texture features with histopathological findings, in order to verify the potential of the proposed texture analysis of ADC map in providing non-invasive prognostic factors of breast cancer. / Ο καρκίνος του μαστού είναι ένα σημαντικό παγκόσμιο πρόβλημα υγείας και η πιο διαδεδομένη μορφή καρκίνου στον γυναικείο πληθυσμό. Η ολοένα και πιο έγκαιρη διάγνωση του καρκίνου του μαστού έχει οδηγήσει σε σημαντική βελτίωση του ρυθμό θεραπείας της νόσου. Σημαντικές πρόοδοι στην τεχνολογία της απεικόνισης παρέχουν τη βελτιωμένη ανίχνευση και ευαισθησία του καρκίνου και οδηγούν σε όλο ένα και λιγότερες περιττές βιοψίες. Οι πιο συνηθισμένες μέθοδοι απεικόνισης, που χρησιμοποιούνται, περιλαμβάνουν την Μαστογραφία, την Υπερηχογραφία, την Μαγνητική Τομογραφία (MRI), την σπινθηρομαστογραφία, την Τομογραφία Εκπομπής Φωτονίων (SPECT) και την Τομογραφία Εκπομπής Ποζιτρονίων (PET). Η παρούσα μελέτη επικεντρώνεται στην τεχνολογία της Απεικόνισης Μαγνητικού Συντονισμού ειδικά σε μία πρόσφατη και ελπιδοφόρα τεχνική απεικόνισης του καρκίνου του μαστού, που ονομάζεται Απεικόνιση Σταθμισμένης Διάχυσης στη Τομογραφία Πυρηνικού Μαγνητικού Συντονισμού (Diffusion Weighted Imaging breast MRI (DWI)). Η DWI είναι μια MRI ακολουθία χωρίς χρήση σκιαγραφικής ουσίας, η οποία βασίζεται σε αλληλουχίες για διάφορες τιμές του παράγοντα διάχυσης b (η τιμή b προσδιορίζει την διαχυτότητα και καθορίζει την ένταση και τη διάρκεια των βαθμωτών πεδίων διάχυσης). Η DWI ποσοστικοποιεί την κινητικότητα των μορίων του νερού (Brownian κίνηση) in vivo (σε ιστούς) και παρέχει διαφορετικές και ενδεχομένως συμπληρωματικές πληροφορίες στην μαστογραφία μαγνητικής τομογραφίας με χρήση σκιαγραφικού (Dynamic Contrast-Enhanced Magnetic Resonance Imaging: DCE-MRI). Η DWI με βάση τις ιδιότητες διάχυσης των μορίων του νερού, αντανακλά την τυχαία κίνησής τους λόγω της θερμικής τους ενέργειας. Η διάχυση του νερού στον μαστό μπορεί να ποσοτικοποιηθεί με τη μέτρηση της μέσης διαχυτότητας, η οποία αναφέρεται ως Φαινόμενος Συντελεστής Διάχυσης (Apparent Diffusion Coefficient-ΑDC). Ο ADC υπολογίζεται ύστερα από μετρήσεις για δυο b τιμές, μια χαμηλή b1 και μια υψηλότερη b2 τιμή. Η DWI επιτρέπει την χαρτογράφηση της διάχυσης των μορίων του νερού μέσω του ADC χάρτη. Ο ADC χάρτης είναι μια παραμετρική εικόνα της οποίας η κλίμακα χρωμάτων ή κλίμακα των τόνων του γκρι, αντιπροσωπεύει τις ADC τιμές των voxels και συνήθως παράγεται από λογισμικό. Οι παραμετρικοί ADC χάρτες απεικόνισης DWI αναπαριστούν τη μικροδομή των ιστών για διάφορους συνδυασμούς τιμών της παραμέτρου b. Η DWI εκτός από 3D ανατομική πληροφορία, παρέχει μια μη επεμβατική διερεύνηση της αγγειοβρίθειας του ιστού, έναν νέο μηχανισμό αντίθεσης στην MRI, και χαρακτηρίζεται από υψηλή ευαισθησία στην ανίχνευση ενδεχόμενων αλλαγών στο τοπικό βιολογικό περιβάλλον, οι οποίες οφείλονται σε παθολογία. Ως εκ τούτου, εκτός από τον χαρακτηρισμό αλλοιώσεων βάση σκιαγραφικής ενίσχυσης (DCE - MRI), η ποσοτικοποίηση της κίνησης των μορίων του νερού στην DWI παρέχει επιπλέον στοιχεία για τον χαρακτηρισμό της αλλοίωσης, κάτι το οποίο μπορεί να αυξήσει περαιτέρω την ειδικότητα της MRI για την ταξινόμηση των αλλοιώσεων του μαστού. Το διαγνωστικό πρόβλημα το οποίο αντιμετώπισε/εστίασε η παρούσα διπλωματική εργασία, αφορά στο χαρακτηρισμό/διάγνωση χωροκατακτητικών αλλοιώσεων (mass-like) του μαστού στην Απεικονιση Μαγνητικου Συντονισμου Σταθμισμενης Διαχυσης (DWI) και την ποσοτικη αναλυση του Φαινομενου Συντελεστη Διαχυσης (ADC) για διαγνωση καρκινου του μαστου. Η ικανότητα διάχυσης των μορίων του νερού περιορίζεται σε περιβάλλον υψηλής κυτταροβρίθιας, ενδοκυττάριων και εξωκυττάριων οιδημάτων, υψηλού ιξώδους και ίνωσης, όπως συμβαίνει στους κακοήθεις όγκους, διότι οι παράγοντες αυτοί εμποδίζουν την κυκλοφορία των μορίων του νερού. Αποτέλεσμα αυτού είναι οι περισσότεροι καρκίνοι του μαστού να παρουσιάζουν χαμηλές ADC τιμές σε σύγκριση με τους καλοήθεις όγκους ή τον φυσιολογικό ιστό. Πολλές μελέτες έχουν δείξει τη χρησιμότητα των ADC τιμών στη διαφορική διάγνωση των αλλοιώσεων του μαστού. Εν τούτοις, το κλινικό αποτέλεσμα παραμένει περιορισμένο λόγω της σημαντικής επικάλυψης καλοήθων και κακοήθων αλλοιώσεων, γεγονός που αποτελεί πρόκληση για την εφαρμογή ενός χρήσιμου διαγνωστικού ορίου της μέσης ADC. Στη πλειοψηφία των μελετών, όπως και στη παρούσα μελέτη, τα βέλτιστα επίπεδα αποκοπής της ADC μεταξύ κακοήθων και καλοήθων αλλοιώσεων προσδιορίστηκαν με τη χρήση ROC ανάλυσης. Στις μέχρι τώρα μελέτες τα διαγνωστικά όρια της μέσης ADC κυμαίνονται από 0.90 έως 1.76 × 10-3 mm2 / s, με ευαισθησία και ειδικότητα να κυμαίνονται από 63% έως 100% και 46% έως 97%, αντίστοιχα. Γεγονός αποτελεί, επίσης, η διαφορετική μέθοδος υπολογισμού της ADC που ακολουθεί η κάθε μελέτη, με πιο συχνή μέθοδο, ο υπολογισμός της μέσης τιμής της ADC (μέση τιμή ± τυπική απόκλιση) σε μια περιοχή ενδιαφέροντος (ROI) μιας αλλοίωσης του μαστού. Σκοπός της παρούσας μεταπτυχιακής διπλωματικής εργασίας ήταν να διερευνηθεί η ικανότητα των χαρακτηριστικών ιστογράμματος του Φαινόμενου Συντελεστή Διάχυσης (Apparent Diffusion Coefficient-ADC) να διαφοροποιούν κακοήθεις από καλοήθεις αλλοιώσεις του μαστού στην Απεικόνιση Μαγνητικού Συντονισμού Σταθμισμένης Διάχυσης (Diffusion Weighted MRI-DWI). Για το σκοπό αυτό, δημιουργήθηκε ο ADC παραμετρικός χάρτης ο οποίος αποτέλεσε τη βάση για την εφαρμογή μεθόδου ανάλυσης υφής εικόνας, και τον υπολογισμό πέντε χαρακτηριστικών υφής πρώτης τάξης: την μέση τιμή, την τυπική απόκλιση, την κύρτωση, την λοξότητα και την εντροπία. Η προσέγγιση αυτή θεωρήθηκε ότι θα προσφέρει μια πιο ολοκληρωμένη αξιολόγηση της υφής του όγκου και της ετερογένειας. Η προσέγγιση εφαρμόσθηκε σε κλινικό δείγμα 92 ιστολογικά αποδεδειγμένων αλλοιώσεων του μαστού, οι οποίες προέρχονται από 69 γυναίκες οι οποίες είχαν νωρίτερα ανιχνευθεί μέσω μαστογραφίας ή/και υπερηχογραφίας ή από ψηλαφητά ευρήματα. Η ιστολογική εξέταση αποκάλυψε 53 κακοήθεις αλλοιώσεις που προέρχονταν από 45 γυναίκες και 39 καλοήθεις αλλοιώσεις από 26 γυναίκες. Όλες οι εξετάσεις μαγνητικής τομογραφίας του μαστού έγιναν με σύστημα MRI 3T και για b=0 και 900 s/mm2. Η διαγνωστική απόδοση/επίδοση των παραμέτρων αυτών συγκρίθηκε με την ανάλυση λειτουργικού χαρακτηριστικού δέκτη (ROC analysis). Τα αποτελέσματα υποδεικνύουν τον σημαντικό ρόλο της ανάλυσης ADC ιστογράμματος χρησιμοποιώντας τα 5 παραπάνω χαρακτηριστικά υφής για την ταυτοποίηση των αλλοιώσεων του μαστού. Οι μετρήσεις της μέσης τιμής, της λοξότητας και της εντροπία της ADC των καλοήθων και κακοήθων αλλοιώσεων του μαστού είχαν στατιστικώς σημαντική διαφορά. Ειδικότερα, η μέση ADC τιμή των καλοήθων όγκων [(1.470 ± 0.342) × 10-3 mm2/s] ήταν σημαντικά υψηλότερη από εκείνη των κακοήθων, [(0.965 ± 0.268) × 10-3 mm2/s, (ρ < 0.00001)]. Η λοξότητα της ADC των καλοήθων όγκων [-0.303 ± 0.584], διέφερε σημαντικά από εκείνη των κακοήθων, [0.210 ± 0.725. (ρ= 0.0008)]. Και η εντροπία της ADC των καλοήθων όγκων [4.794 ± 0.665], ήταν σημαντικά χαμηλότερη από εκείνη των κακοήθων, [5.569 ± 0.649, (ρ < 0.00001)]. Ωστόσο, η τυπική απόκλιση και η κύρτωση της ADC των καλοήθων και κακοήθων αλλοιώσεων του μαστού δεν είχαν στατιστικώς σημαντική διαφορά. Συγκεκριμένα, η τυπική απόκλιση της ADC των καλοήθων όγκων ήταν [(0.184 ± 0.999) × 10-3 mm2/s] ενώ των κακοήθων ήταν [(0.192 ± 0.151) × 10-3 mm2/s, (ρ = 0.6581)] και η κύρτωση της ADC των καλοήθων όγκων ήταν [3.003 ± 1.065] ενώ των κακοήθων ήταν [3.337 ± 1.334, (ρ = 0.0987)]. Η περιοχή κάτω από την ROC καμπύλη (AUC) για τη μέση ADC τιμή ήταν 0.862 ± 0.042 (95% διάστημα εμπιστοσύνης: 0.754, 0.925), για την λοξότητα της ADC ήταν 0.705 ± 0.054 (95% διάστημα εμπιστοσύνης: 0.589, 0.800) και για η εντροπία της ADC ήταν 0.800 ± 0.046 (95% διάστημα εμπιστοσύνης: 0.691, 0.874), και είχαν ως αποτέλεσμα μια καλή διαγνωστική απόδοση/ επίδοση της DWI για τις παραμέτρους αυτές. Από την άλλη πλευρά, η AUC με 0.527 ± 0.063 (95% διάστημα εμπιστοσύνης: 0.393, 0.640) και με 0.601 ± 0.061 (95% διάστημα εμπιστοσύνης: 0.470, 0.707) για την τυπική απόκλιση και την κύρτωση, αντίστοιχα, υποδηλώνει ένα βαθμό επικάλυψης στις ADC τιμές μεταξύ καλοήθων και κακοήθων όγκων. Τα βέλτιστα κατώφλια αποκοπής για διαφοροποίηση καλοήθων έναντι κακοήθων αλλοιώσεων καθορίστηκαν με τον εντοπισμό των σημείων όπου η ακρίβεια ήταν μέγιστη στις καμπύλες ROC. Από τη συγκεκριμένη μελέτη, προέκυψαν δύο τιμές κατωφλίου αποκοπής της μέσης ADC, με την ίδια ακρίβεια 83.15%. Το πρώτο κατώφλι με τιμή 1.21 x 10-3 mm2/s χαρακτηρίζεται με ευαισθησία 86.27% και ειδικότητα 78.95%. Και το δεύτερο κατώφλι με τιμή 1.32 x 10-3 mm2/s χαρακτηρίζεται με ευαισθησία 92.16% και ειδικότητα 71.05%. Το κατώφλι για την λοξότητα της ADC ήταν στα -0.06 με ακρίβεια 68.54%, ευαισθησία 66.03% και ειδικότητα 66.67%. Τέλος, προέκυψαν δύο τιμές κατωφλίου αποκοπής της εντροπίας της ADC με την ίδια ακρίβεια ακρίβεια 76.40%. Το πρώτο κατώφλι με τιμή 5.17 χαρακτηρίζεται με ευαισθησία 75.47% και ειδικότητα 71.80%. Και το δεύτερο κατώφλι με τιμή 5.21 χαρακτηρίζεται με ευαισθησία 73.59% και ειδικότητα 74.36%. Συμπερασματικά, τα αποτελέσματα της παρούσας μελέτης δείχνουν τη συνεισφορά των μεθόδων ανάλυσης υφής εικόνας στη Απεικονιση Μαγνητικου Συντονισμου Σταθμισμενης Διαχυσης (DWI) του μαστού για την ποσοτικοποίηση της ετερογένειας του ιστού, παρέχοντας σημαντικές πληροφορίες για τη διάγνωση του καρκίνου του μαστού. Η ανάλυση ιστογράμματος των ADC τιμών στον καρκίνο του μαστού έχει τη δυνατότητα διαφοροποίησης καλοήθων και κακοήθων όγκων, παρέχοντας πληροφορίες για το σύνολο του όγκου. Η μέση τιμή, η λοξότητα και η εντροπία του ADC είναι πολύτιμες παράμετροι που συσχετίζονται με παθολογικό χαρακτηρισμό των όγκων του μαστού. Αυτές οι 3 ADC παράμετροι αύξησαν σημαντικά την ποσοτική διαγνωστική απόδοση της DWI του μαστού και πιθανότατα να είναι αποτελεσματικές παράμετροι όσον αφορά τη διάκριση μεταξύ καλοήθων και κακοήθων αλλοιώσεων του μαστού Μελλοντικές προσπάθειες πρόκειται να εστιάσουν στη διερεύνηση της συσχέτισης των εξαχθέντων χαρακτηριστικών υφής με ιστοπαθολογικούς δείκτες, με σκοπό την περαιτέρω επιβεβαίωση των προτεινόμενων προσεγγίσεων και ενδεχομένως την χρήση συγκεκριμένων χαρακτηριστικών υφής ως μη επεμβατικών προγνωστικών δεικτών καρκίνου του μαστού.