A adequação das opções terapêuticas para o adenocarcinoma de reto é dependente do estadiamento da lesão primária, feito por ressonância magnética (RM) dirigida. A RM é capaz de detectar fatores de mau prognóstico associados a alto risco de recidiva pélvica, selecionando pacientes que se beneficiam de tratamento neoadjuvante pré-operatório e diferenciando-os daqueles que podem ser operados após o diagnóstico, sem risco aumentado de recidiva local. No contexto de escassez de recursos e baixa disponibilidade da RM, a tomografia computadorizada (TC) poderia auxiliar nesta seleção de pacientes com fatores de mau prognóstico, agindo como um método de triagem para detecção de lesões avançadas que teriam indicação de tratamento neoadjuvante. O objetivo do presente estudo é avaliar o desempenho da TC no estadiamento local da neoplasia de reto para seleção de pacientes com fatores de mau prognóstico associados a alto risco para recidiva local. Cento e oitenta pacientes com diagnóstico histopatológico de adenocarcinoma de reto e sem tratamento prévio foram retrospectivamente analisados por dois radiologistas cegos a outros dados clínicos. As imagens de tomografia foram submetidas a reconstrução multiplanar, e a lesão primária foi analisada no eixo axial do reto, semelhante ao que é utilizado para análise por RM. Os critérios avaliados por TC foram: estadiamento T3c-d ou T4, estadiamento N2, acometimento da fáscia mesorretal, presença de invasão vascular extramural ou linfonodos pélvicos laterais acometidos. Os achados da TC foram comparados aos obtidos por RM. Os resultados mostraram que a TC foi capaz de detectar 108 de 128 pacientes com critérios de mau prognóstico, com sensibilidade de 84%, especificidade de 79%, valor preditivo positivo de 91% e valor preditivo negativo de 67%. O desempenho da TC foi superior quando avaliou tumores a mais de 5 cm da borda anal, mostrando sensibilidade de 89%, especificidade de 86%, valor preditivo positivo de 93% e valor preditivo negativo de 80%. Caso fosse utilizada para seleção dos critérios de mau prognóstico nesta população, a TC teria reduzido o número necessário de exames de RM em 52% / In order to tailor the most appropriate treatment option for rectal cancer, accurate staging is necessary. Patients with high risk of local recurrence may benefit from preoperative neoadjuvant treatment. In contrast, in patients with no risk of local recurrence, upfront surgery avoids radiation-related toxicity with good oncologic prognosis. The optimal staging strategy includes magnetic resonance imaging (MRI), which is the most accurate method of detecting lesions with high risk of local recurrence. When MRI is not available to all patients, computed tomography (CT) could possibly detect high risk features, avoiding delays without an adverse effect on primary treatment decisions. The purpose of this study is to evaluate CT staging accuracy to detect high risk of local recurrence in patients with rectal cancer. One hundred and eighty patients with biopsy-proven adenocarcinoma and no previous treatment were retrospectively studied. CT and MR images were reviewed by two blinded and independent radiologists. CT multiplanar reformatting allowed true axial images as seen by MRI. High-risk for local recurrence features were as follows: T3c-d or T4 status; N2 status; extramural venous invasion, mesorectal fascia involvement, and lateral pelvic lymph nodes. MRI was considered the reference standard. The results showed CT sensitivity of 84%, specificity of 79%, positive predictive value of 91% and negative predictive value of 67% for detection of any high risk of local recurrence feature. When tumors lying 5 cm above the anal verge were considered, CT sensitivity was 89%, specificity was 86%, positive predictive value was 93% and negative predictive value was 80%. CT staging would have reduced the need for 52% of MRI scans

Colorectal cancer (CRC) remains a significant burden in contemporary society due to an aging population, unhealthy dietary choices and an increasingly sedentary lifestyle. While the underlying defects for many hereditary forms of CRC have been determined, many genetic and epigenetic changes promoting common sporadic CRCs have yet to be identified. The Mutated in Colorectal Cancer (MCC) gene, identified in 1991, was initially thought to be responsible for the hereditary form of CRC, familial adenomatous polyposis, before the discovery of the susceptibility gene Adenomatous Polyposis Coli (APC), which then became the focus of intense research. Recent data, however, suggests that MCC may also be important in the development of CRC. I have investigated the mechanism of MCC gene silencing, the putative structure, and multiple functions of MCC. MCC was frequently silenced by promoter hypermethylation in CRC cell lines and primary tumours. MCC methylation showed strong molecular and clinicopathological associations with hallmarks of the serrated neoplasia pathway. Furthermore, MCC methylation was more frequent in serrated precursor lesions compared with adenomas, thus occurring early during carcinogenesis. MCC is highly conserved in complex multicellular organisms. Re-introduction of MCC in CRC cell lines resulted in partial G1 to S phase, and G2/M phase cell cycle blocks, potentially by upregulating cell cycle inhibitor gene transcription and interfering with the process of mitotic checkpoints and division, respectively. Changes in MCC levels also modulated NF?B pathway signalling, the pathway required for maintaining cell viability and proliferation in colonic epithelial cells. In particular, MCC overexpression suppressed both TNF? and LPS-induced NF?B activation, decreasing both the magnitude and rate of cellular responses. Overexpression also resulted in downregulation of proteins involved in canonical NF?B pathway signalling, while increasing the transcription of non-canonical NF?B genes. Therefore, MCC may direct activation of this pathway to a specific subset of NF?B-regulated genes. These data provide a molecular basis for the role of MCC as a tumour suppressor gene in CRC. MCC may have multiple functions, regulating cell cycle progression and modulating NF?B pathway signalling, either through direct involvement in pathway signalling cascades, or by providing a scaffold on which signalling events can occur.

ABSTRACT The morphological premalignant changes in prostate epithelium such as high grade prostatic intraepithelial neoplasia (HGPIN) precede invasive prostate cancer (PC) by several decades. The overall aim of this project was to identify patterns of gene expression in HGPIN and early PC which increase our understanding of the early biology of PC and identify genes and pathways that correlate with an aggressive phenotype. A comprehensive tissue cohort of premalignant prostate lesions was collected in a tissue microarray (TMA) platform that was utilised for high-throughput validation of target genes. Using this unique resource, the expression of the tumour suppressor gene PTEN was assessed using immunohistochemistry in an initial candidate gene approach based on mouse models implicating PTEN in carcinogenesis. No significant difference in expression of PTEN was detected in premalignant and benign epithelium. A transcript profiling approach was undertaken by integrating laser capture microdissection, linear RNA amplification and oligonucleotide microarrays to perform a screen of matched patient samples of normal, HGPIN and PC cells. The expression patterns of two genes encoding secreted proteins, neuropeptide Y (NPY) and macrophage inhibitory cytokine (MIC-1) were validated using immunohistochemistry on TMAs representing the progression model of early PC. Increased expression of these proteins in PC was confirmed to occur early in the disease process and altered expression of NPY and MIC-1 was associated with worse clinical outcome. Further analysis of global gene expression patterns using a structured network knowledge base identified a notable aberration in the expression of extracellular matrix and extracellular matrix associated proteins in HGPIN and provided novel evidence for the role of this class of molecules in the development of PC. In summary, contrary to current dogma based on work in animal models, altered PTEN expression is unlikely to represent an important event in the development of malignancy in the human prostate. In contrast, the expression patterns and prognostic value of NPY and MIC-1 in HGPIN support their further evaluation as biomarkers for the development and progression of PC. The aberrant expression of genes and networks of genes detected in HGPIN will assist in further identification of biological pathways which may be targeted in therapeutic strategies against the development and progression of PC.

Understanding signalling pathways that control pancreatic endocrine tumour (PET) development and proliferation may reveal novel targets for therapeutic intervention. The pathogenesis for sporadic and hereditary PETs, apart from mutations of the MEN1 and VHL tumour suppressor genes, is still elusive. The protein product of the MEN1 gene, menin, regulates many genes. The aim of this thesis was to identify genes involved in pancreatic endocrine tumourigenesis, with special reference to Notch signalling. Messenger RNA and protein expression of NOTCH1, HES1, HEY1, ASCL1, NEUROG3, NEUROD1, DLK1, POU3F4, PDX1, RPL10, DKK1 and TPH1 were studied in human PETs, sporadic and MEN 1, as well as in tumours from heterozygous Men1 mice. For comparison, normal and MEN1 non-tumourous human and mouse pancreatic specimens were used. Nuclear expression of HES1 was consistently absent in PETs. In mouse tumours this coincided with loss of menin expression, and there was a correlation between Men1 expression and several Notch signalling factors. A new phenotype consisting of numerous menin-expressing endocrine cell clusters, smaller than islets, was found in Men1 mice. Expression of NEUROG3 and NEUROD1 was predominantly localised to the cytoplasm in PETs and islets from MEN 1 patients and Men1 mice, whereas expression was solely nuclear in wt mice. Differences in expression levels of Pou3f4, Rpl10 and Dlk1 between islets of Men1 and wt mice were observed. In addition, combined RNA interference and microarray expression analysis in the pancreatic endocrine cell line BON1 identified 158 target genes of ASCL1. For two of these, DKK1 (a negative regulator of the WNT/β-catenin signalling pathway) and TPH1, immunohistochemistry was performed on PETs. In concordance with the microarray finding, DKK1 expression showed an inverse relation to ASCL1 expression. Altered subcellular localisation of HES1, NEUROD1 and NEUROG3 and down-regulation of DKK1 may contribute to tumourigenesis.

Pancreatic endocrine tumors and gastrointestinal stromal tumors are rare. Evidence regarding prognostic factors, and in the former also treatment, is scarce. We evaluated the survival and prognostic factors in a consecutive series of 324 patients with pancreatic endocrine tumors treated at a single institution. Radical surgery, WHO classification, TNM stage, age and Ki67 ≥2% emerged as independent prognostic factors. Having a non-functioning tumor was not an independent prognostic marker, and neither was hereditary tumor disease. We present the first evaluation of the newly proposed TNM staging system for these patients. A separate analysis of well-differentiated neuroendocrine carcinomas is reported, suggesting tumor size ≥5cm and Ki67 ≥2% as negative prognostic markers in this group. The first 36 patients with advanced neuroendocrine tumors treated with temozolomide at our clinic were evaluated. The median time to progression was seven months. Fourteen percent showed partial regression and 53% stabilization of disease. Side effects were generally mild. Investigation of O6-methylguanine DNA methyltransferase revealed a low expression in a subset of tumors. Four out of five patients responding to treatment had tumors with low expression. Concomitant expression of the orexigen ghrelin and its receptor in pancreatic endocrine tumors is demonstrated. No significant difference in mean plasma ghrelin between patients and controls were found, but elevated plasma ghrelin was seen in five patients. We provide the first report of expression of ghrelin and its receptor in gastrointestinal stromal tumors. Concomitant expression was frequent, indicating the presence of an autocrine loop. The tumors also expressed the neuroendocrine marker synaptic vesicle protein 2. Together, these findings are suggestive of neuroendocrine features.

This thesis develops and evaluates three approaches for quantitative molecularly-targeted optical imaging of neoplasia. The first approach focuses on widefield imaging of biomarkers near the tissue surface for early detection applications; this approach is demonstrated in freshly resected oral tissue. Most oral cancers are not detected until the disease has spread, but topical application of targeted imaging agents allows rapid visualization of biomarker expression, giving real-time, objective information. Epidermal growth factor receptor (EGFR) expression was quantified in patient samples using fluorescent epidermal growth factor. Dysplasia (n=4) and cancer (n=13) had an average 2.3-fold and 3.8-fold increase in signal compared to normal tissue. EGFR expression was assessed along with metabolic activity using a fluorescent glucose analog, 2-NBDG, in 9 patient samples. A classification algorithm using quantitative image features resulted in an area under the curve (AUC) of 0.83, though the main advantage of this technique may be to understand spatial heterogeneity of biomarker expression and how this correlates with disease. The next approach focuses on high-resolution optical imaging through a needle to detect metastases in lymphoid tissue for clinical staging applications; this approach is demonstrated in resected lymph nodes from breast cancer patients. These patients often require removal of nodes, but an optical imaging strategy using topical application of imaging agents in vivo may classify nodes as normal or metastatic, thus reducing unnecessary removal of normal nodes and improving metastasis detection. Proflavine, a nuclear dye, was topically applied to 43 nodes. A classification algorithm developed from quantitative image features distinguished normal lymphoid tissue from metastases with an AUC of 0.84. Because optical imaging is depth limited, the final approach combines high-resolution optical imaging with magnetic resonance imaging (MRI) for multimodal evaluation of deep tissue. An imaging agent functional in both optical and MRI was developed by co-loading fluorescent EGFR antibodies and gadolinium-based contrast agents in silicon discs. These discs accumulate in tumors, resulting in localized delivery of imaging agents. The research presented here can be applied to understanding tumor biology and biomarker heterogeneity, with the future clinical goal of improving identification of disease and determination of appropriate therapy for cancer patients.

Einleitung: Neuroendokrine Tumoren (NET) des gastroenteropankreatischen Systems entstammen den Zellen des diffusen neuroendokrinen Systems und sind seltene Tumoren mit einer steigenden Inzidenz. Die Beurteilung der Dignität von NET, allein mit Hilfe histologischer Kriterien, ist problematisch. Die WHO-Klassifikation von 2000 unterscheidet nicht nur histomorphologisch in gut und wenig differenziert, beurteilt werden auch Tumorgröße, Angioinvasion, Invasion der Musculuaris propria, hormonales Tumorsyndrom, Proliferationsindex (Ki67) sowie vorhandene Metastasen. Die neue WHO-Klassifikation von 2010 nutzt ein standardisiertes Grading-System, in dem nach Proliferationsindex in G1 (Ki67≤2%) niedriggradig maligne, G2 (3-20%) intermediärgradig maligne und G3 (>20%) hochgradig maligne, neuroendokrine Neoplasien (NEN) eingeteilt wird. Das Tumormikroenvironment gastroenteropankreatischer NET, im Hinblick auf die Tumor-Klassifikationen von 2000 und 2010, ist bisher nicht untersucht. Methoden: In meiner Arbeit wurden Paraffinblöcke von 55 Patienten immunhistochemisch untersucht: 32 Dünndarm-, 13 kolorektale und 10 Pankreas-NET. Als Tumormarker wurden Chromogranin, CD56, und S100 beurteilt. Marker für das Tumorstroma waren α-SMA und Desmin, Endothelzellmarker waren CD34 und vWF, und Immunzellmarker waren CD3 für T-Lymphozyten, CD20 für B-Lymphozyten und CD68 für Makrophagen. Kim1p galt als Marker für fibrohistiozytäre Zellen, der CC-Chemokinrezeptor-2 (CCR2) wurde exemplarisch für die Zytokinrezeptoren gefärbt. Die Auswertung erfolgte computergestützt. Die Immunzellen wurden quantitativ ausgewertet. Ergebnisse: In meiner Arbeit konnte ich zeigen, dass NET mit zunehmender Malignität weniger Stromazellen aufweisen. Auch die o.g. Tumormarker nahmen, wenn vorhanden, ab. Fibrohistiozytäre Zellen, die dendritische Zellen beinhalten, stellten die häufigsten Immunzellen dar, gefolgt von CD3+ T-Zellen. Im Vergleich der Organlokalisationen waren CD68+ Makrophagen in kolorektalen NET signifikant häufiger als in Dünndarm-NET. Nach der WHO-Klassifikation (2000) waren in den NET der WHO-Gruppe 2 und 3 signifikant mehr Kim1p+ Zellen als in der WHO-Gruppe 1 nachweisbar. CD3+ T-Zellen waren signifikant häufiger in NET der WHO-Gruppe 3 im Vergleich zur WHO-Gruppe 2. CD68+ Makrophagen waren in der WHO-Gruppe 3 im Vergleich zu den WHO-Gruppen 1 und 2 signifikant häufiger vorhanden. Die dargestellten Unterschiede zeigten sich auch für die neue Klassifikation (2010), allerdings mit vermindertem Signifikanzniveau. In NET der WHO-Gruppe 3 traten die Immunzellen in engen Kontakt zu den Tumorzellen, bei geringerem Tumorstroma. Zusammenfassung: Es bestehen deutliche Unterschiede in der Zusammensetzung des Tumormikroenvironments der NET, abhängig von der Malignität. Meine Ergebnisse weisen auf einen möglichen Zusammenhang zwischen der Anzahl an Immunzellen und der Differenzierung hin. Ob dies relevant für eine maligne Transformation ist, bleibt zu klären.

Aukšto laipsnio prostatos intraepitelinė neoplazija (ALPIN, angl. – HGPIN – high grade prostatic intraepithelial neoplasia ) priskiriama prie priešvėžinių būklių ( arba prostatos vėžio prekursorių) ir susijusi su padidėjusia kartu esamo vėžio diagnozavimo rizika arba vėlesne jos progresija iki vėžio. Manoma, kad pacientai, kuriems nustatyta ALPIN prostatos biopsinėje medžiagoje, yra tinkami kandidatai chemoprevencijai. Šio darbo tikslas buvo nustatyti 5-alfa reduktazės inhibitoriaus dutasterido reikšmę prostatos vėžio prevencijai esant didelei šios ligos rizikai. Darbo uždaviniai : 1. Nustatyti prostatos vėžio dažnį esant didelei šios ligos rizikai (biopsinėje medžiagoje nustatyta aukšto laipsnio prostatos intraepitelinė neoplazija). 2. Įvertinti pakartotinių biopsijų reikšmę prostatos vėžio diagnozavimo dažniui. 3. Įvertinti tiriamojo vaisto reikšmę prostatos vėžio prevencijai (ar sumažina prostatos vėžio diagozavimo dažnį) esant didelei šios ligos rizikai. 4. Palyginti diagnozuoto prostatos vėžio diferenciacijos skirtumus tiriamosiose grupėse. / A high grade prostatic intraepithelial neoplasia (HPIN) is traditionally ascribed to pre-cancerous conditions or prostate cancer (PC) precursors, and associated with an increased risk of concomitant cancer or its later progression to malignant disease. After evaluation of the data indicating HPIN as a pre–cancerous condition, it is thought that the patients having HPIN in the prostate biopsy material are suitable candidates for chemoprevention.The aim of the study was to find out the significance of dutasteride, a 5–alpha reductase inhibitor, for the prevention of development of prostate cancer in case of a high risk for the disease. Objectives of the study: 1. To determine the rate of prostate cancer in case of a high risk for the disease (when a high grade prostatic intraepithelial neoplasia is found in biopsy material). 2. To find out the significance of repeat biopsies for the detection rate of prostate cancer. 3. To find out the significance of the investigatory medication for the prevention of development of prostate cancer in case of a high risk for the disease. 4. To compare the differences in the differentiation of diagnosed prostate cancer between the study groups.

Uncovering the chromosomal abnormalities associated with human malignancy can provide significant insights into the molecular basis of tumorigenesis, as well as identifying potential targets for therapy. The present study set out to examine the genetic characteristics of t(2;7)(p11-12;q21-22) translocations arising in conjunction with chronic B-cell neoplasia. Using long-range PCR, a t(2;7) was initially mapped in an individual presenting with the preclinical entity CD5- monoclonal B-cell lymphocytosis. This revealed a breakpoint at 2p11.2 localized to the recombination signal of the immunoglobulin kappa (IGK) variable gene IGKV3-15, and a breakpoint at 7q21.2 located 520 bp upstream of cyclin dependent kinase 6 (CDK6). The same approach was subsequently employed to elucidate near-identical t(2;7) breakpoints in 4 additional cases presenting with chronic lymphocytic leukemia or indolent non-Hodgkin lymphomas. The remarkable consistency of these translocations implicates the dysregulation of CDK6 via translocation to IGK as a recurrent pathomechanism during the emergence of B-cell lymphoproliferative disorders.

Uncovering the chromosomal abnormalities associated with human malignancy can provide significant insights into the molecular basis of tumorigenesis, as well as identifying potential targets for therapy. The present study set out to examine the genetic characteristics of t(2;7)(p11-12;q21-22) translocations arising in conjunction with chronic B-cell neoplasia. Using long-range PCR, a t(2;7) was initially mapped in an individual presenting with the preclinical entity CD5- monoclonal B-cell lymphocytosis. This revealed a breakpoint at 2p11.2 localized to the recombination signal of the immunoglobulin kappa (IGK) variable gene IGKV3-15, and a breakpoint at 7q21.2 located 520 bp upstream of cyclin dependent kinase 6 (CDK6). The same approach was subsequently employed to elucidate near-identical t(2;7) breakpoints in 4 additional cases presenting with chronic lymphocytic leukemia or indolent non-Hodgkin lymphomas. The remarkable consistency of these translocations implicates the dysregulation of CDK6 via translocation to IGK as a recurrent pathomechanism during the emergence of B-cell lymphoproliferative disorders.