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The liver fluke Opisthorchis felineus is a member of the triad of epidemiologically relevant species of the trematode family Opisthorchiidae, and the causative agent of opisthorchiasis felinea over an extensive range that spans regions of Eurasia. The International Agency for Research on Cancer classifies the infection with the liver flukes Opisthorchis viverrini and Clonorchis sinensis as group 1 agents and a major risk factor for cholangiocarcinoma. However, the carcinogenic potential of the infection with O. felineus is less clear. Here, we present findings that support the inclusion of O. felineus in the Group 1 list of biological carcinogens. Two discrete lines of evidence support the notion that infection with this liver fluke is carcinogenic. First, novel oxysterol-like metabolites detected by liquid chromatography-mass spectroscopy in the egg and adult developmental stages of O. felineus, and in bile, sera, and urine of liver fluke-infected hamsters exhibited marked similarity to oxysterol-like molecules known from O. viverrini. Numerous oxysterols and related DNA-adducts detected in the liver fluke eggs and in bile from infected hamsters suggested that infection-associated oxysterols induced chromosomal lesions in host cells. Second, histological analysis of liver sections from hamsters infected with O. felineus confirmed portal area enlargement, inflammation with severe periductal fibrosis and changes in the epithelium of the biliary tract characterized as biliary intraepithelial neoplasia, BilIN. The consonance of these biochemical and histopathological changes revealed that O. felineus infection in this rodent model induced precancerous lesions conducive to malignancy.

Bladder carcinogenesis and tumour progression is accompanied by profound alterations in protein glycosylation on the cell surface, which may be explored for improving disease management. In a search for prognosis biomarkers and novel therapeutic targets we have screened, using immunohistochemistry, a series of bladder tumours with differing clinicopathology for short-chain O-glycans commonly found in glycoproteins of human solid tumours. These included the Tn and T antigens and their sialylated counterparts sialyl-Tn(STn) and sialyl-T(ST), which are generally associated with poor prognosis. We have also explored the nature of T antigen sialylation, namely the sialyl-3-T(S3T) and sialyl-6-T(S6T) sialoforms, based on combinations of enzymatic treatments. We observed a predominance of sialoglycans over neutral glycoforms (Tn and T antigens) in bladder tumours. In particular, the STn antigen was associated with high-grade disease and muscle invasion, in accordance with our previous observations. The S3T and S6T antigens were detected for the first time in bladder tumours, but not in healthy urothelia, highlighting their cancer-specific nature. These glycans were also overexpressed in advanced lesions, especially in cases showing muscle invasion. Glycoproteomic analyses of advanced bladder tumours based on enzymatic treatments, Vicia villosa lectin-affinity chromatography enrichment and nanoLC-ESI-MS/MS analysis resulted in the identification of several key cancer-associated glycoproteins (MUC16, CD44, integrins) carrying altered glycosylation. Of particular interest were MUC16 STn+ -glycoforms, characteristic of ovarian cancers, which were found in a subset of advanced-stage bladder tumours facing the worst prognosis. In summary, significant alterations in the O-glycome and O-glycoproteome of bladder tumours hold promise for the development of novel noninvasive diagnostic tools and targeted therapeutics. Furthermore, abnormal MUC16 glycoforms hold potential as surrogate biomarkers of poor prognosis and unique molecular signatures for designing highly specific targeted therapeutics.

Estrogen-like metabolites have been identified in S. haematobium, the helminth parasite that causes urogenital schistosomiasis (UGS) and in patients´ blood and urine during UGS. Estrogen receptor (ER) activation is enriched in the luminal molecular subtype bladder cancer (BlaCa). To date, the significance of ER to these diseases remains elusive. We evaluated ERα and ERβ expression in UGS-related BlaCa (n = 27), UGS-related non-malignant lesions (n = 35), and noninfected BlaCa (n = 80). We investigated the potential of ERα to recognize S. haematobium-derived metabolites by docking and molecular dynamics simulations and studied ERα modulation in vitro using 3 BlaCa cell lines, T24, 5637 and HT1376. ERα was expressed in tumor and stromal cells in approximately 20% noninfected cases and in 30% of UGS-related BlaCa, predominantly in the epithelial cells. Overall, ERα expression was associated with features of tumor aggressiveness such as high proliferation and p53 positive expression. ERα expression correlated with presence of schistosome eggs. ERβ was widely expressed in both cohorts but weaker in UGS-related cases. molecular dynamics simulations of the 4 most abundant S. haematobium-derived metabolites revealed that smaller metabolites have comparable affinity for the ERα active state than 17β-estradiol, while the larger metabolites present higher affinity. Our in vitro findings suggested that ERα activation promotes proliferation in ERα expressing BlaCa cells and that this can be reverted with anti-estrogenic therapy. In summary, we report differential ER expression between UGS-related BlaCa and noninfected BlaCa and provide evidence supporting a role of active ERα during UGS and UGS-induced carcinogenesis. Highlights: Estrogen receptor (ERs) are expressed in both urogenital schistosomiasis and bladder cancer; ERα was associated with aggressive tumor features and presence of schistosome eggs; In silico analysis shows an agonistic putative action of S. haematobium-derived metabolites for ERα; ERα induced proliferation in bladder cancer cells can be reverted by anti-estrogens; Evidence suggests that activation of ERα could play a role in urogenital schistosomiasis and urogenital schistosomiasis-induced carcinogenesis. PJB acknowledges support from award R01CA164719 from the National Cancer Institute (NCI), National Institutes of Health (NIH), USA. info:eu-repo/semantics/publishedVersion

Purpose The aim of this study was to investigate the breast cancer (BC) molecular subtypes according to its surrogate immunohistochemistry (IHC) markers. We conducted a preliminary study, to correlate the clinical pathological profiles and molecular subtypes of breast cancer in Luanda, Angola. Methods From January 2011 to 30 December 2014, 140 consecutive cases of microscopically confirmed invasive breast carcinoma were classified regarding histology and IHC (ER, PR, HER2, and Ki-67). Surrogate molecular subtypes were classified according to ESMO recommendations. Results All patients were female; the median age was 47 years (24-84 years). Invasive carcinoma NST was the most common type (91.4%) and grade 2 was prevalent (70.7%). Most tumours were locally advanced (stage III - 65% and stage IV - 3.6%). In 140 studied cases, 74 (52.8%) malignancies were hormone receptor positive; 25.7% were luminal A like, 19.3% luminal B and HER2 negative like, 7.9% luminal B and HER2-positive like, 15.7% HER2 positive, and 31.4% were triple negative. Conclusion Women's BC in Luanda-Angola is diagnosed at a young age and at an advanced stage. The two predominant molecular subtypes are HR positive and triple negative. The percentage of HER2-positive BC cases was high. Determining the molecular subtype using surrogate IHC markers has important treatment and prognostic implications for Angolan women with BC. There is an urgent need to study a prospective BC series in order to confirm the present results.

The relapsing and progressive nature of bladder tumors, and the heterogeneity in the response to cisplatin-containing regimens, are the major concerns in the care of urothelial bladder carcinoma (UBC) patients. The metabolic adaptations that alter the tumor microenvironment and thus contribute to chemoresistance have been poorly explored in UBC setting. We found significant associations between the immunoexpressions of the microenvironment-related molecules CD147, monocarboxylate transporters (MCTs) 1 and 4, CD44 and CAIX in tumor tissue sections from 114 UBC patients. The presence of MCT1 and/or MCT4 expressions was significantly associated with unfavorable clinicopathological parameters. The incidence of CD147 positive staining significantly increased with advancing stage, grade and type of lesion, and occurrence of lymphovascular invasion. Similar associations were observed when considering the concurrent expression of CD147 and MCT1. This expression profile lowered significantly the 5-year disease-free and overall survival rates. Moreover, when selecting patients who received platinum-based chemotherapy, the prognosis was significantly worse for those with MCT1 and CD147 positive tumors. CD147 specific silencing by small interfering RNAs (siRNAs) in UBC cells was accompanied by a decrease in MCT1 and MCT4 expressions and, importantly, an increase in chemosensitivity to cisplatin. Our results provide novel insights for the involvement of CD147 and MCTs in bladder cancer progression and resistance to cisplatin-based chemotherapy. We consider that the possible cooperative role of CD147 and MCT1 in determining cisplatin resistance should be further explored as a potential theranostics biomarker.

Objective Bacillus Calmette-Guerin (BCG) immunotherapy is the gold standard treatment for superficial bladder tumors with intermediate/high risk of recurrence or progression. However, approximately 30% of patients fail to respond to the treatment. Effective BCG therapy needs precise activation of the type 1 helper cells immune pathway. Tumor-associated macrophages (TAMs) often assume an immunoregulatory M2 phenotype and may directly interfere with the BCG-induced antitumor immune response. Thus, we aim to clarify the influence of TAMs, in particular of the M2 phenotype in stroma and tumor areas, in BCG treatment outcome. Patients and methods The study included 99 patients with bladder cancer treated with BCG. Tumors resected before treatment were evaluated using immunohistochemistry for CD68 and CD163 antigens, which identify a lineage macrophage marker and a M2-polarized specific cell surface receptor, respectively. CD68+ and CD163+ macrophages were evaluated within the stroma and tumor areas, and high density of infiltrating cells spots were selected for counting. Hypoxia, an event known to modulate macrophage phenotype, was also assessed through hypoxia induced factor (HIF)-1α expression. Results Patients in whom BCG failed had high stroma-predominant CD163+ macrophage counts (high stroma but low tumor CD163+ macrophages counts) when compared with the ones with a successful treatment (71% vs. 47%, P = 0.017). Furthermore, patients presenting this phenotype showed decreased recurrence-free survival (log rank, P = 0.008) and a clear 2-fold increased risk of BCG treatment failure was observed in univariate analysis (hazard ratio = 2.343; 95% CI: 1.197–4.587; P = 0.013). Even when adjusted for potential confounders, such as age and therapeutic scheme, multivariate analysis revealed 2.6-fold increased risk of recurrence (hazard ratio = 2.627; 95% CI: 1.340–5.150; P = 0.005). High stroma-predominant CD163+ macrophage counts were also associated with low expression of HIF-1α in tumor areas, whereas high counts of CD163+ in the tumor presented high expression of HIF-1α in tumor nests. Conclusions TAMs evaluation using CD163 is a good indicator of BCG treatment failure. Moreover, elevated infiltration of CD163+ macrophages, predominantly in stroma areas but not in the tumor, may be a useful indicator of BCG treatment outcome, possibly owing to its immunosuppressive phenotype.

Background: High risk of recurrence/progression bladder tumours is treated with Bacillus Calmette-Gue´ rin (BCG) immunotherapy after complete resection of the tumour. Approximately 75% of these tumours express the uncommon carbohydrate antigen sialyl-Tn (Tn), a surrogate biomarker of tumour aggressiveness. Such changes in the glycosylation of cell-surface proteins influence tumour microenvironment and immune responses that may modulate treatment outcome and the course of disease. The aim of this work is to determine the efficiency of BCG immunotherapy against tumours expressing sTn and sTn-related antigen sialyl-6-T (s6T). Methods: In a retrospective design, 94 tumours from patients treated with BCG were screened for sTn and s6T expression. In vitro studies were conducted to determine the interaction of BCG with high-grade bladder cancer cell line overexpressing sTn. Results: From the 94 cases evaluated, 36 had recurrence after BCG treatment (38.3%). Treatment outcome was influenced by age over 65 years (HR¼2.668; (1.344–5.254); P¼0.005), maintenance schedule (HR¼0.480; (0.246–0.936); P¼0.031) and multifocallity (HR¼2.065; (1.033–4.126); P¼0.040). sTn or s6T expression was associated with BCG response (P¼0.024; Po0.0001) and with increased recurrence-free survival (P¼0.001). Multivariate analyses showed that sTn and/or s6T were independent predictive markers of recurrence after BCG immunotherapy (HR¼0.296; (0.148–0.594); P¼0.001). In vitro studies demonstrated higher adhesion and internalisation of the bacillus to cells expressing sTn, promoting cell death. Conclusion: s6T is described for the first time in bladder tumours. Our data strongly suggest that BCG immunotherapy is efficient against sTn- and s6T-positive tumours. Furthermore, sTn and s6T expression are independent predictive markers of BCG treatment response and may be useful in the identification of patients who could benefit more from this immunotherapy.

Objective To investigate whether carcinoma in situ (CIS) lesion could be considered a surrogate marker of urothelium genetic instability and field carcinogenesis or not, we evaluated DNA content, p53 overexpression, and proliferative index (Ki-67 expression) in primary tumor, in tumor-adjacent mucosa, and distant urothelial mucosa with and without presence of CIS. Patients and methods A retrospective study in radical cystectomy specimens from 49 patients was carried out. All the lesions present in each cystectomy specimen were studied, including the tumor area and the adjacent mucosa (AM). Whenever possible, the distant mucosa (DM) was also studied. When CIS was detected, this lesion and the surrounding normal mucosa were also studied. The 49 tumor areas included high grade papillary urothelial carcinoma (HGP) in 19 cases (38.8%) and invasive urothelial cell carcinomas in 30 cases (61.2%). The nuclear DNA content of cancer cells was evaluated using image cytometry allowing the determination of the DNA ploidy and 5cER parameters. The p53 and Ki-67 immunoexpression was evaluated by immunohistochemistry. Results CIS lesions were observed in the AM and DM of both tumor groups: 15.8% and 15.4% in AM and DM, for each one of them, in HGP group and 26.7% and 22.2% in AM and DM, for each one of them, in invasive tumors group. In CIS lesion aneuploid DNA content, p53 overexpression and high proliferative labeling index were observed. The so-called normal mucosa (AM and DM) with and without focus of CIS lesions were compared for genetic instability and molecular alterations profile. Statistical differences were observed between the normal mucosa with and without CIS: the so-called normal mucosa areas with focus of CIS revealed significantly higher frequencies of DNA content alterations, p53 overexpression, and higher proliferative index. These differences were significantly different in the invasive UCC group, but this profile it is also present in HPG group. Conclusion This study points out that CIS is a marker of genetic instability of the urothelium mucosa. The CIS surrounding morphologically normal urothelium showed a high frequency of abnormal DNA content, with high percentage of clear aneuploid cells (high 5cER), p53 mutated protein expression, and a proliferative status underlying a field carcinogenesis. These alterations in normal mucosa were not found when CIS was not present.

Follicular thyroid carcinoma is being diagnosed less and less frequently despite the increasing incidence of well-differentiated thyroid carcinomas everywhere. This review will discuss the reasons underlying such an observation focusing on the evolution of the morphological and immunohistochemical diagnostic criteria of follicular thyroid tumors. It will address the differential diagnosis between follicular carcinoma and three tumor types--follicular adenoma, follicular variant of papillary carcinoma and poorly differentiated carcinoma--as well as the problems raised by the newly described categories of follicular tumors: follicular tumor of uncertain malignant potential, well-differentiated tumor of uncertain malignant potential and well-differentiated carcinoma, not otherwise specified. Finally, the prognostic and therapeutic significance of some promising molecular biomarkers will be discussed within the frame of the aforementioned histopathological classification.

Monocarboxylate transporters (MCTs) are vital for intracellular pH homeostasis by extruding lactate from highly glycolytic cells. These molecules are key players of the metabolic reprogramming of cancer cells, and evidence indicates a potential contribution in urothelial bladder cancer (UBC) aggressiveness and chemoresistance. However, the specific role of MCTs in the metabolic compartmentalization within bladder tumors, namely their preponderance on the tumor stroma, remains to be elucidated. Thus, we evaluated the immunoexpression of MCTs in the different compartments of UBC tissue samples (n = 111), assessing the correlations among them and with the clinical and prognostic parameters. A significant decrease in positivity for MCT1 and MCT4 occurred from normoxic toward hypoxic regions. Significant associations were found between the expression of MCT4 in hypoxic tumor cells and in the tumor stroma. MCT1 staining in normoxic tumor areas, and MCT4 staining in hypoxic regions, in the tumor stroma and in the blood vessels were significantly associated with UBC aggressiveness. MCT4 concomitant positivity in hypoxic tumor cells and in the tumor stroma, as well as positivity in each of these regions concomitant with MCT1 positivity in normoxic tumor cells, was significantly associated with an unfavourable clinicopathological profile, and predicted lower overall survival rates among patients receiving platinum-based chemotherapy. Our results point to the existence of a multi-compartment metabolic model in UBC, providing evidence of a metabolic coupling between catabolic stromal and cancer cells' compartments, and the anabolic cancer cells. It is urgent to further explore the involvement of this metabolic coupling in UBC progression and chemoresistance., This study was supported by the Life and Health Sciences Research Institute (ICVS) from the School of Health Sciences of the University of Minho. JA received a postdoctoral fellowship from ICVS (reference ICVS-SSRL: ON.2 SR&TD Integrated Program, NORTE-07-0124-FEDER-000017)., info:eu-repo/semantics/publishedVersion

The aim of the present study was to assess if the presence of survivin mRNA in exfoliated cells present in urine samples can be a reliable marker of the presence of bladder tumour and recurrence. Urine samples from 30 patients with superficial urothelial cell carcinomas (UCC) were collected prior to transurethral resection (TUR) of the tumour and in the first routine follow-up, three months after TUR. Detection of survivin mRNA was performed by reverse transcription-polymerase chain reaction (RT-PCR). No correlation was observed between survivin detection and the clinico-pathological variables analysed, nevertheless, when patients were grouped into low-grade (G1) and high-grade (G2+G3) tumours, statistically significant differences were found between both groups (p=0.04). When we analysed the results of survivin detection and urinary cytology together, we observed that informative cases rose from 27.8% to 44.4%. Also, Kaplan-Meier curves for patients with negative cytology in the first follow-up, categorised according to survivin detection, revealed that survivin mRNA positive cases recurred earlier than negative ones. From our results we can conclude that detection of survivin expression can be a reliable tumour marker, but more studies are needed to clarify the potential of survivin to predict recurrences. These results showed that survivin detection in combination with conventional urinary cytology can be a useful tool to increase the sensitivity in detecting the presence of a recurrence after TUR.

Resumo: Introdução: Os tumores epiteliais tímicos (TET), a maioria timomas, são neoplasias desenvolvidas a partir das células epiteliais do timo e constituem cerca de 30% das massas do mediastino anterior em adultos. Os timomas são constituídos por células sem características citológicas de malignidade, sendo o comportamento maligno determinado pela invasão da cápsula e estru-turas adjacentes. Estes tumores apresentam um amplo espectro de características clínicas e morfológicas, e as pequenas séries de doentes conhecidas tornam difícil o estabelecimento de um tratamento standard.Material e métodos: Efectuouse um estudo retrospectivo dos doentes admitidos com diagnóstico de ti moma no Instituto Português de Oncologia - Centro do Porto (IPO-Porto), de 1983 a 2004. Foram anali-sadas as suas características clínicas, classificação histológica segundo a OMS, o estadiamento de Masaoka, e a sua relação com as modalidades de tratamento. Procedeu-se à revisão dos registos clínicos destes doentes e revisão do material histológico para a classificação segundo critérios da OMS de 1999.Resultados: No IPO-Porto, entre 1983 e 2004, fo-ram tratados 28 doentes com TET. Destes, 21 eram timomas invasivos, sendo estes o objecto deste estudo. Dos dados demográficos salienta-se que eram 11 homens, 10 mulheres, com uma idade mediana de 55 anos (24-79 anos). A classificação histológica da OMS foi a seguinte: 2 doentes (9,5%) Tipo A, 6 (28,6%) tipo AB, 4 (19%) tipo B1, 2 (9,5%) tipo B2, 7 (33,4%) tipo B3. O estadiamento segundo Masaoka foi 9 doentes (42,8%) com estádio II, 6 (28,6%) com estádio III e 6 (28,6%) com estádio IVa. A maioria dos doentes apresentava sintomas locais à apresen-tação, com apenas 1 doente com diagnóstico de aplasia eritrocitária e 5 com Mastenia gravis (MG).Os 6 doentes submetidos apenas a ressecção cirúrgica completa não tiveram evidência de recorrência da doença (2 tipo A-II, 2 tipo AB-II, 1 tipo B1-II, 1 tipo B2-IVa), com follow-up variando entre 8 e 144 meses. 10 doentes com ressecção completa receberam tratamento adjuvante, 6 radioterapia (4 doentes B3-II, 2 doentes B3-III), 2 quimioterapia (AB-IVa) e 2 radioterapia e quimioterapia (B1-IVa, B2-III). Apenas os 2 doentes que efectuaram quimioterapia adjuvante reci-divaram, aos 168 e 46 meses, e morreram aos 168 e 49 meses. Os restantes doentes que efectuaram tratamento adjuvante encontram-se sem evidência de doença. Dos 5 doentes com ressecção incompleta seguido de tratamento complementar (2 doentes AB-III, 2 B1-IVa, 1 B3-III), 3 morreram aos 11 meses (B3-III), aos 12 meses (B1-IVa) e aos 241 meses (AB-III), este últi-mo por MG.Conclusões: Apesar de se tratar de uma pequena série, os factores preditivos de mau prognóstico foram a ressecção incompleta, estádio avançado e o subtipo histológico B3. à necessário investigar o papel do tratamento adjuvante e neoadjuvante no grupo de doentes com doença avançada e subtipo histológico B3.Rev Port Pneumol 2007; XIII (4): 553-585 Abstract: Introduction: Epithelial thymic tumours (ETT), which comprise the majority of thymomas, are neoplasias developed from the epithelial cells of the thymus and constitute around 30% of anterior mediastinal masses in adults. Thymomas consist of cells with no cytological characteristics of malignity; malignant behaviour is determined by invasion of the capsule and adjacent structures. These tumours present a broad spectrum of clinical and morphological characteristics and the small series of known patients makes establishing a standard treatment difficult.Material and methods: A retrospective study was made into thymoma diagnosed patients admitted to the Portuguese Oncology Institute in Porto (IPO-Porto) from 1983 to 2004. Clinical characteristics were analysed and a histological classification made in accordance with World Health Organization criteria, Masaoka staging, and their relation to treatment methods. A review of the clinical records of these patients was then made, as well as a review of histological material for classification in line with 1999 WHO criteria.Results: Twenty-eight ETT patients were treated at the IPO-Porto between 1983 and 2004. Of these, 21 had invasive thymomas and these are the subject of this study. Eleven subjects were male and 10 female, with a median age of 55 years (24-79 years). The WHO histological classification was as follows: 2 patients (9.5%) type A, 6 (28.6%) type AB, 4 (19%) type B1, 2 (9.5%) type B2, 7 (33.4%) type B3. Masaoka staging was 9 patients (42.8%) with stage II, 6 (28.6%) with stage III and 6 (28.6%) with stage IVa. The majority of patients had local symptoms, with only one subject diagnosed with erythrocyte aplasia and five with Myasthenia Gravis (MG).The 6 patients who were given complete surgical resection only showed no evidence of disease recurrence (2 type A-II, 2 type AB-II, 1 type B1-II, 1 type B2-IVa), with follow-up from 8-144 months. Ten patients with complete resection received adjuvant treatment; 6 radiotherapy (4 B3-II patients, 2 B3-III patients), 2 chemotherapy (AB-IVa) and 2 chemo and radiotherapy (B1-IVa, B2-III). Only the 2 patients who under-went adjuvant chemotherapy relapsed, at 168 and 46 months, dying at 168 and 49 months, respectively. The remaining patients who were given adjuvant treatment did not present signs of disease. Of the 5 subjects having incomplete resection followed by complementary treatment (2 AB-III patients, 2 B1-IVa patients, 1 B3-III patient), 3 died, at 11 months (B3-III), 12 months (B1-IVa) and 241 months (AB-III), the latter with MG.Conclusions: Predictive factors of bad prognosis here were incomplete resection, advanced staging and B3 histological subtype, the smallness of this series not-withstanding. It is necessary to investigate the role of adjuvant and neoadjuvant treatment in a group of subjects with advanced disease of the B3 histological subtype.Rev Port Pneumol 2007; XIII (4): 553-585 Palavras-chave: Tumores epiteliais tímicos, timomas, Miastenia gravis, quimioterapia, radioterapia, Key-words: Epithelial thymic tumours, thymomas, Myasthenia gravis, chemotherapy, radiotherapy

Introduction: Epithelial thymic tumours (ETT), which comprise the majority of thymomas, are neoplasias developed from the epithelial cells of the thymus and constitute around 30% of anterior mediastinal masses in adults. Thymomas consist of cells with no cytological characteristics of malignity; malignant behaviour is determined by invasion of the capsule and adjacent structures. These tumours present a broad spectrum of clinical and morphological characteristics and the small series of known patients makes establishing a standard treatment difficult. Material and methods: A retrospective study was made into thymoma diagnosed patients admitted to the Portuguese Oncology Institute in Porto (IPOPorto) from 1983 to 2004. Clinical characteristics were analysed and a histological classification made in accordance with World Health Organization criteria, Masaoka staging, and their relation to treatment methods. A review of the clinical records of these patients was then made, as well as a review of histological material for classification in line with 1999 WHO criteria. Results: Twenty-eight ETT patients were treated at the IPO-Porto between 1983 and 2004. Of these, 21 had invasive thymomas and these are the subject of this study. Eleven subjects were male and 10 female, with a median age of 55 years (24-79 years). The WHO histological classification was as follows: 2 patients (9.5%) type A, 6 (28.6%) type AB, 4 (19%) type B1, 2 (9.5%) type B2, 7 (33.4%) type B3. Masaoka staging was 9 patients (42.8%) with stage II, 6 (28.6%) with stage III and 6 (28.6%) with stage IVa. The majority of patients had local symptoms, with only one subject diagnosed with erythrocyte aplasia and five with Myasthenia Gravis (MG).The 6 patients who were given complete surgical resection only showed no evidence of disease recurrence (2 type A-II, 2 type AB-II, 1 type B1-II, 1 type B2- IVa), with follow-up from 8-144 months. Ten patients with complete resection received adjuvant treatment; 6 radiotherapy (4 B3-II patients, 2 B3-III patients), 2 chemotherapy (AB-IVa) and 2 chemo and radiotherapy (B1-IVa, B2-III). Only the 2 patients who underwent adjuvant chemotherapy relapsed, at 168 and 46 months, dying at 168 and 49 months, respectively. The remaining patients who were given adjuvant treatment did not present signs of disease. Of the 5 subjects having incomplete resection followed by complementary treatment (2 AB-III patients, 2 B1-IVa patients, 1 B3-III patient), 3 died, at 11 months (B3-III), 12 months (B1-IVa) and 241 months (AB-III), the latter with MG. Conclusions: Predictive factors of bad prognosis here were incomplete resection, advanced staging and B3 histological subtype, the smallness of this series notwithstanding. It is necessary to investigate the role of adjuvant and neoadjuvant treatment in a group of subjects with advanced disease of the B3 histological subtype. Resumo: Introdução: Os tumores epiteliais tímicos (TET), a maioria timomas, são neoplasias desenvolvidas a partir das células epiteliais do timo e constituem cerca de 30% das massas do mediastino anterior em adultos. Os timomas são constituídos por células sem características citológicas de malignidade, sendo o comportamento maligno determinado pela invasão da cápsula e estruturas adjacentes. Estes tumores apresentam um amplo espectro de características clínicas e morfológicas, e as pequenas séries de doentes conhecidas tornam difícil o estabelecimento de um tratamento standard. Material e métodos: Efectuou-se um estudo retrospectivo dos doentes admitidos com diagnóstico de timoma no Instituto Português de Oncologia - Centro do Porto (IPO-Porto), de 1983 a 2004. Foram analisadas as suas características clínicas, classificação histológica segundo a OMS, o estadiamento de Masaoka, e a sua relação com as modalidades de tratamento. Procedeuse à revisão dos registos clínicos destes doentes e revisão do material histológico para a classificação segundo critérios da OMS de 1999. Resultados: No IPO-Porto, entre 1983 e 2004, foram tratados 28 doentes com TET. Destes, 21 eram timomas invasivos, sendo estes o objecto deste estudo. Dos dados demográficos salienta-se que eram 11 homens, 10 mulheres, com uma idade mediana de 55 anos (24-79 anos). A classificação histológica da OMS foi a seguinte: 2 doentes (9,5%) Tipo A, 6 (28,6%) tipo AB, 4 (19%) tipo B1, 2 (9,5%) tipo B2, 7 (33,4%) tipo B3. O estadiamento segundo Masaoka foi 9 doentes (42,8%) com estádio II, 6 (28,6%) com estádio III e 6 (28,6%) com estádio IVa. A maioria dos doentes apresentava sintomas locais à apresentação, com apenas 1 doente com diagnóstico de aplasia eritrocitária e 5 com Mastenia gravis (MG).Os 6 doentes submetidos apenas a ressecção cirúrgica completa não tiveram evidência de recorrência da doença (2 tipo A-II, 2 tipo AB-II, 1 tipo B1-II, 1 tipo B2-IVa), com follow-up variando entre 8 e 144 meses. 10 doentes com ressecção completa receberam tratamento adjuvante, 6 radioterapia (4 doentes B3-II, 2 doentes B3-III), 2 quimioterapia (AB-IVa) e 2 radioterapia e quimioterapia (B1-IVa, B2-III). Apenas os 2 doentes que efectuaram quimioterapia adjuvante recidivaram, aos 168 e 46 meses, e morreram aos 168 e 49 meses. Os restantes doentes que efectuaram tratamento adjuvante encontram-se sem evidência de doença. Dos 5 doentes com ressecção incompleta seguido de tratamento complementar (2 doentes AB-III, 2 B1- IVa, 1 B3-III), 3 morreram aos 11 meses (B3-III), aos 12 meses (B1-IVa) e aos 241 meses (AB-III), este último por MG. Conclusões: Apesar de se tratar de uma pequena série, os factores preditivos de mau prognóstico foram a ressecção incompleta, estádio avançado e o subtipo histológico B3. à necessário investigar o papel do tratamento adjuvante e neoadjuvante no grupo de doentes com doença avançada e subtipo histológico B3. Key-words: Epithelial thymic tumours, thymomas, Myasthenia gravis, chemotherapy, radiotherapy, Palavras-chave: Tumores epiteliais tímicos, timomas, Miastenia gravis, quimioterapia, radioterapia

Invasive bladder tumours express the cell-surface Sialyl-Tn (STn) antigen, which stems from a premature stop in protein O-glycosylation. The STn antigen favours invasion, immune escape, and possibly chemotherapy resistance, making it attractive for target therapeutics. However, the events leading to such deregulation in protein glycosylation are mostly unknown. Since hypoxia is a salient feature of advanced stage tumours, we searched into how it influences bladder cancer cells glycophenotype, with emphasis on STn expression. Therefore, three bladder cancer cell lines with distinct genetic and molecular backgrounds (T24, 5637 and HT1376) were submitted to hypoxia. To disclose HIF-1α-mediated events, experiments were also conducted in the presence of Deferoxamine Mesilate (Dfx), an inhibitor of HIF-1α proteasomal degradation. In both conditions all cell lines overexpressed HIF-1α and its transcriptionally-regulated protein CA-IX. This was accompanied by increased lactate biosynthesis, denoting a shift toward anaerobic metabolism. Concomitantly, T24 and 5637 cells acquired a more motile phenotype, consistent with their more mesenchymal characteristics. Moreover, hypoxia promoted STn antigen overexpression in all cell lines and enhanced the migration and invasion of those presenting more mesenchymal characteristics, in an HIF-1α-dependent manner. These effects were reversed by reoxygenation, demonstrating that oxygen affects O-glycan extension. Glycoproteomics studies highlighted that STn was mainly present in integrins and cadherins, suggesting a possible role for this glycan in adhesion, cell motility and invasion. The association between HIF-1α and STn overexpressions and tumour invasion was further confirmed in bladder cancer patient samples. In conclusion, STn overexpression may, in part, result from a HIF-1α mediated cell-survival strategy to adapt to the hypoxic challenge, favouring cell invasion. In addition, targeting STn-expressing glycoproteins may offer potential to treat tumour hypoxic niches harbouring more malignant cells.

Oxyphil or Hurthle cell tumours of the thyroid are characterised by their consistent excessive number of mitochondria. A recently discovered gene, GRIM-19 has been found to fulfil two roles within the cell: as a member of the interferon-beta and retinoic acid-induced pathway of cell death, and as part of the mitochondrial Complex I assembly. In addition, a gene predisposing to thyroid tumours with cell oxyphilia (TCO) has been mapped to chromosome 19p13.2 in one family. A cluster of genes involved in mitochondrial metabolism occurs in this region; one of these is GRIM-19. We have searched for GRIM-19 mutations in a series of 52 thyroid tumours. Somatic missense mutations in GRIM-19 were detected in three of 20 sporadic Hurthle cell carcinomas. A germline mutation was detected in a Hurthle cell papillary carcinoma arising in a thyroid with multiple Hurthle cell nodules. No mutations were detected in any of the 20 non-Hurthle cell carcinomas tested, nor in any of 96 blood donor samples. In one of the sporadic Hurthle cell papillary carcinomas positive for GRIM-19 mutation, we have also detected a ret/PTC-1 rearrangement. No GRIM-19 mutations were detected in any of the six cases of known familial Hurthle cell tumour tested, so that our results do not support the identification of GRIM-19 as the TCO gene. The GRIM-19 mutations we have detected are the first nuclear gene mutations specific to Hurthle cell tumours to be reported to date; we propose that such mutations can be involved in the genesis of sporadic or familial Hurthle cell tumours through the dual function of GRIM-19 in mitochondrial metabolism and cell death.

Background Of patients with superficial bladder cancer, a group are still at risk of disease recurrence, progression and death from their cancer after curative treatment. Angiogenesis is a crucial pathogenic mechanism for this type of urothelial cell carcinoma (UCC), and is a potential therapeutic target. However, the selection of the appropriate patients remains a dilemma. Patients and methods Vascular endothelial growth factor (VEGF) expression and the presence of angiogenesis and occurrence of CD31, CD34, endoglin and factor VIII immunoexpression, were evaluated in 66 superficial papillary UCCs of the bladder and were correlated with classical histopathological factors and disease outcome. Results VEGF immunoreactivity was observed in 100% of cases, and more intensely in the luminal surface. The presence of microvessel clusters independently of a fibrovascular core was observed in 22.7% of cases. Of these, the T1/G2 subgroup had an independent and significantly lower recurrence-free survival (P = 0.0002). Conclusions These results indicate that the presence of angiogenesis in tumour urothelium is a potential prognostic factor in superficial UCC, particularly in T1/G2 tumours, and may be used to select patients for anti-angiogenic treatments.

Clear cell sarcoma of tendons and aponeuroses (CCS) is an uncommon malignancy characterized by a t(12;22)(q13;q12) causing a fusion of the EWS and ATF1 genes. We describe the cytogenetic and molecular genetic analyses of two lung metastases from a CCS patient. Both lesions presented the defining t(12;22) and a type 1 EWS/ATF1 chimeric transcript. The additional cytogenetic changes present in the two lesions allowed us to obtain some insight into the pathogenetic basis of disease progression. Four related clones were identified in the right lung metastasis, permitting a partial reconstitution of the stepwise clonal evolution, whereas the left lung metastasis presented yet another subclone. The comparison of the two karyotypes enabled us to pinpoint which changes occurred in the primary tumor and which emerged independently after the two metastases had been established. We conclude that both clonal divergence and convergence may be operative during tumor progression of CCS.

Approximately 80% of bladder tumors are urothelial superficial papillary carcinomas (USPC). Despite a generally good prognosis, these tumors have a strong propensity to recur and about 1/3 of them compared to disease progression. Histological assessment of these superficial tumors is not sufficiently discriminator in predicting prognosis; therefore, we decided to evaluate the prognostic significance of p53 and Ki-67 immunoexpression in low-grade (GI-II) USPC in order to predict the potential outcome of these tumors. P53 and Ki-67 immunoexpression were studied in function of recurrence-free and progression-free survival in 159 primary superficial bladder tumors. A prognostic risk model based on grade, stage and multifocality was also evaluated. P53 accumulation was significantly related to tumor progression (p=0.006). High Ki-67 index (≥18%) and multifocality were significantly related to recurrence (both p=0.0001) and progression-free survival (both p=0.0001) and were independent prognostic factors in the multivariate analysis. The prognostic risk model based on grade, stage and multifocality was not an efficient discriminator of outcome. Adding the Ki-67 index into the risk model, single pTa/T1-GI Ki-67 positive tumors, usually classified as low risk, were reclassified as of intermediate risk. After this reclassification, the risk group model identified a subgroup of pTa/T1-G1 with a high risk of recurrence and progression. Ki-67 index is a reliable prognostic marker in urothelial superficial bladder carcinoma and, when included into a risk profile classification of the low-grade USPC, the accuracy of the prognostic discrimination is enhanced. © 2003 Wiley-Liss, Inc.

Urothelial bladder carcinoma (UBC), the most frequent type (90%) of bladder cancer and the second most common malignancy of the urogenital region, is a relatively well understood type of cancer, with numerous studies concerning pathogenetic pathways, natural history and bladder tumor biology being reported. Despite this, it continues to remain a challenge in the oncology field, mostly due to its relapsing and progressive nature, and to the heterogeneity in the response to cisplatin-containing regimens. Although the formulae based on clinical staging and histopathological parameters are classically used as diagnostic and prognostic tools, they have proven insufficient to characterize the individual biological features and clinical behaviour of the tumours. Understanding the pathobiology of the disease can add important information to these classical criteria, and contribute to accurately predict outcome and individualize therapy for UBC patients. In this line of investigation, we found that tumour angiogenesis and lymphangiogenesis, the process of invasion and metastasis and the energy metabolism reprogramming/tumour microenvironment encompass several potential biomarkers that seem to infl bladder cancer aggressiveness and chemoresistance. We particularly highlight the roles of lymphovascular invasion, and of RKIP, CD147 and MCT1 immunoexpressions, as relevant prognostic and/or predictive biomarkers, and as promising areas of therapeutic intervention, eliciting for the development of additional studies that can validate and further explore these biomarkers., O carcinoma urotelial da bexiga (CUB), o tipo de cancro mais frequente deste órgão (90%) e o segundo mais comum da região genitourinária, está relativamente bem caracterizado, fundamentado por inúmeros estudos sobre vias patogenéticas, histogénese e biologia tumoral. No entanto, permanece como um desafio na oncologia, principalmente devido à sua elevada taxa de recidiva e progressão, e à heterogeneidade na resposta a tratamentos de quimioterapia contendo cisplatina. As fórmulas baseadas no estadiamento tumoral e em parâmetros histopatológicos, embora geralmente utilizadas como ferramentas de diagnóstico e prognóstico, são insuficientes para caracterizar as propriedades biológicas e o comportamento clínico dos tumores. A compreensão detalhada da patobiologia da doença poderá adicionar informações importantes aos critérios clássicos, e contribuir para uma correta previsão individual do prognóstico e da terapêutica a utilizar nos doentes com CUB. Nesta linha de investigação, o nosso grupo sugere que na angiogénese e na linfangiogénese tumoral, no processo de invasão e metastização e na reprogramação do metabolismo energético/microambiente tumoral estão implicados potenciais biomarcadores que parecem influenciar a agressividade tumoral e a resistência à quimioterapia no CUB. Salienta-se o papel da invasão linfovascular, e da imunoexpressão das moléculas RKIP, CD147 e MCT1, como biomarcadores de prognóstico e/ou preditivos de resposta à terapêutica, e como áreas promissoras de intervenção terapêutica. É urgente desenvolver estudos adicionais que continuem a explorar e, eventualmente, validar as potencialidades destes biomarcadores.

Muscle invasive bladder cancer (MIBC, stage >= T2) is generally associated with poor prognosis, constituting the second most common cause of death among genitourinary tumours. Due to high molecular heterogeneity significant variations in the natural history and disease outcome have been observed. This has also delayed the introduction of personalized therapeutics, making advanced stage bladder cancer almost an orphan disease in terms of treatment. Altered protein glycosylation translated by the expression of the sialyl-Tn antigen (STn) and its precursor Tn as well as the activation of the PI3K/Akt/mTOR pathway are cancer-associated events that may hold potential for patient stratification and guided therapy. Therefore, a retrospective design, 96 bladder tumours of different stages (Ta, T1-T4) was screened for STn and phosphorylated forms of Akt (pAkt), mTOR (pmTOR), S6 (pS6) and PTEN, related with the activation of the PI3K/Akt/mTOR pathway. In our series the expression of Tn was residual and was not linked to stage or outcome, while STn was statically higher in MIBC when compared to non-muscle invasive tumours (p = 0.001) and associated decreased cancer-specific survival (log rank p = 0.024). Conversely, PI3K/Akt/mTOR pathway intermediates showed an equal distribution between non-muscle invasive bladder cancer (NMIBC) and MIBC and did not associate with cancer-specif survival (CSS) in any of these groups. However, the overexpression of pAKT, pmTOR and/or pS6 allowed discriminating STn-positive advanced stage bladder tumours facing worst CSS (p = 0.027). Furthermore, multivariate Cox regression analysis revealed that overexpression of PI3K/Akt/mTOR pathway proteins in STn+ MIBC was independently associated with approximately 6-fold risk of death by cancer (p = 0.039). Mice bearing advanced stage chemically-induced bladder tumours mimicking the histological and molecular nature of human tumours were then administrated with mTOR-pathway inhibitor sirolimus (rapamycin). This decreased the number of invasive lesions and, concomitantly, the expression of STn and also pS6, the downstream effector of the PI3K/Akt/mTOR pathway. In conclusion, STn was found to be marker of poor prognosis in bladder cancer and, in combination with PI3K/Akt/mTOR pathway evaluation, holds potential to improve the stratification of stage disease. Animal experiments suggest that mTOR pathway inhibition could be a potential therapeutic approach for this specific subtype of MIBC.

The prognostic significance of p53 accumulation and MDM2 overexpression in superficial and papillary urothelial cell carcinoma (UCC) of the bladder has not yet been established. Therefore, MDM2 and p53 protein were investigated focusing on tumour grade and muscularis mucosae invasion in order to identify their prognostic significance. Thirty-five archival UCC were studied by immunohistochemistry using monoclonal antibodies DO7 against p53, and IB10 against MDM2. MDM2 overexpression was observed in 51.4% of the cases. The recurrence rate for patients with MDM2 overexpression was 7.09 fold higher than for those without MDM2 overexpression. p53 accumulation was not related to prognosis. There was no significant difference between low and high grade tumours in regard to MDM2 overexpression and p53 accumulation. pTa cases presented the higher frequency of MDM2 positive cases (66.7%) and a lower frequency of p53 accumulation (33.3%). MDM2 overexpression does not seem to be related to muscular mucosae invasion. Thus, MDM2 status may well be a prognostic marker in superficial urothelial bladder carcinomas.

e16013 Background: Epidemiological and molecular findings suggest that estrogen may play a role in bladder carcinogenesis and in urogenital schistosomiasis (UGS). Methods: A comprehensive analysis of expression of estrogen receptors (ER) in urothelial carcinomas (UC), n = 80 from non-infected patients and UGS-related bladder carcinoma (BlaCa) n = 27 was undertaken. I n vitro, we evaluated the effect of estradiol (E2) and anti-estrogens on the survival of human BlaCa cells. In addition, molecular dynamics simulations are ongoing to assess the ability of estradiol-like metabolites produced by the Schistosoma haematobiumto bind ERs. Results: Tumor cell expression of ERα was found in 17.5% of the non-infected cases, and was associated with proliferative tumors (p = 0.004, ES = 0.326) and lower overall survival. Expression of ERα was also found in normal urothelium adjacent to the tumor and in tumor stromal cells, particularly in T2 and late stage tumors (p = 0.034, ES = 0.498and p = 0.037, ES = 0.357, respectively). In UGS-related BlaCa, 22% of tumor cells were ERα positive. Presence of parasite eggs was associated with higher ERα expression in cancer cells and adjacent urothelium (p = 0.038, ES = 0.399 and p = 0.020, ES = 0.600, respectively). ERβ was found in 91% of non-infected UC and 78% of the UGS-related tumors. ERβ was equally expressed across different tumor stages and grade but more intense in tumor cells than in urothelium of normal appearance and in non-infected than in UGS-related cases. In the latter group, staining intensity was increased in women and in samples with parasite eggs. Using 3 human BlaCa cell lines, HT1374, T24 and 5637, we observed that 5637 cells express ERα and can be stimulated by E2 and inhibited by the anti-estrogens (4OH-tamoxifen and ICI 182 780). T24 cells expressed ERα at very low levels and did not respond to E2, but were inhibited by anti-estrogens; HT1376 do not express ERα. ERβ mRNA was not detected in any of the cell lines. Conclusions: Our results document an association between expression of ERα and aggressive features. The potential of expression of ERs as a prognostic tool and as therapeutic target should be further investigated especially for tumors with aggressive profile and during UGS-related carcinogenesis.

Made available in DSpace on 2015-11-03T18:06:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-10-01. Added 1 bitstream(s) on 2015-11-04T10:13:22Z : No. of bitstreams: 1 WOS000342725400009.pdf: 652138 bytes, checksum: b4c7b188d4ab260cf65e7c3243e162c0 (MD5) Urothelial bladder carcinoma (UBC) is heterogeneous in its pathology and clinical behaviour. Evaluation of prognostic and predictive biomarkers is necessary, in order to produce personalised treatment options. The present study used immunohistochemistry to evaluate UBC sections containing tumour and non-tumour areas from 76 patients, for the detection of p-mTOR, CD31 and D2-40 (blood and lymphatic vessels identification, respectively). Of the non-tumour and tumour sections, 36 and 20% were scored positive for p-mTOR expression, respectively. Immunoexpression was observed in umbrella cells from non-tumour urothelium, in all cell layers from non-muscle-invasive (NMI) tumours (including expression in superficial cells), and in spots of cells from muscle-invasive (MI) tumours. Positive expression decreased from non-tumour to tumour urothelium, and from pTl/pTis to pT3/pT4 tumours; however, the few pT3/pT4 positive cases had worse survival rates, with 5-year disease-free survival being significantly lower. Angiogenesis occurrence was impaired in pT3/pT4 tumours that did not express p-mTOR. In conclusion, p-mTOR expression in non-tumour umbrella cells is likely a reflection of their metabolic plasticity, and extension to the inner layers of the urothelium in NMI tumours is consistent with an enhanced malignant potential. The expression in cell spots in a few MI tumours and absence of expression in the remaining tumours is intriguing and requires further research. Additional studies regarding the up- and downstream effectors of the mTOR pathway should be conducted. Univ Minho, Sch Healh Sci ECS, Life &Hlth Sci Res Inst ICVS, P-4710057 Braga, Portugal ICVS 3Bs, PT Govt Associate Lab, P-4806909 Guimaraes, Portugal Sao Paulo State Univ, Fac Med, LIM 14, BR-01246000 Sao Paulo, Brazil Barretos Canc Hosp, Mol Oncol Res Ctr, BR-14784400 Sao Paulo, Brazil IPO, Dept Urol, P-4200072 Oporto, Portugal IPO, Expt Pathol &Therapeut Res Ctr, P-4200072 Oporto, Portugal IPO, Dept Surg Oncol, P-4200072 Oporto, Portugal Univ Fernando Pessoa, Fac Hlth Sci, P-4200150 Oporto, Portugal Sao Paulo State Univ, Fac Med, LIM 14, BR-01246000 Sao Paulo, Brazil

More than 70% of muscle invasive bladder cancers (MIBC) express the cell-surface antigen sialyl-Tn (sTn) that promotes motility and invasive potential of tumor cells. Effective drug testing models to optimize therapy against these tumors are warranted.Fragments of sTn-positive MIBC were subcutaneously engrafted into nude mice and expanded until the third passage. Histology and immunoexpression of tumor markers (p53, p63, Ki-67, CK20, sTn) were studied in order to evaluate tumor phenotype maintenance.Tumor take rate was low in the first passage (1/9) but increased and became consistent, therefore suitable for drug testing, in the third passage (13/13). Histology and immunoexpression patterns were similar between primary tumors and xenografts. However, p53 and ki-67 levels increased with passages suggesting a selection of more proliferative clones. STn expression, even though decreased, was preserved in xenografts.We describe the first patient-derived sTn-positive xenograft model to be used for drug testing and identification of prognostic biomarkers.

Bacillus Calmette-Guérin (BCG) immunotherapy is the gold standard treatment for superficial bladder tumors with intermediate/high risk of recurrence or progression. However, approximately 30% of patients fail to respond to the treatment. Effective BCG therapy needs precise activation of the type 1 helper cells immune pathway. Tumor-associated macrophages (TAMs) often assume an immunoregulatory M2 phenotype and may directly interfere with the BCG-induced antitumor immune response. Thus, we aim to clarify the influence of TAMs, in particular of the M2 phenotype in stroma and tumor areas, in BCG treatment outcome.The study included 99 patients with bladder cancer treated with BCG. Tumors resected before treatment were evaluated using immunohistochemistry for CD68 and CD163 antigens, which identify a lineage macrophage marker and a M2-polarized specific cell surface receptor, respectively. CD68(+) and CD163(+) macrophages were evaluated within the stroma and tumor areas, and high density of infiltrating cells spots were selected for counting. Hypoxia, an event known to modulate macrophage phenotype, was also assessed through hypoxia induced factor (HIF)-1α expression.Patients in whom BCG failed had high stroma-predominant CD163(+) macrophage counts (high stroma but low tumor CD163(+) macrophages counts) when compared with the ones with a successful treatment (71% vs. 47%, P = 0.017). Furthermore, patients presenting this phenotype showed decreased recurrence-free survival (log rank, P = 0.008) and a clear 2-fold increased risk of BCG treatment failure was observed in univariate analysis (hazard ratio = 2.343; 95% CI: 1.197-4.587; P = 0.013). Even when adjusted for potential confounders, such as age and therapeutic scheme, multivariate analysis revealed 2.6-fold increased risk of recurrence (hazard ratio = 2.627; 95% CI: 1.340-5.150; P = 0.005). High stroma-predominant CD163(+) macrophage counts were also associated with low expression of HIF-1α in tumor areas, whereas high counts of CD163(+) in the tumor presented high expression of HIF-1α in tumor nests.TAMs evaluation using CD163 is a good indicator of BCG treatment failure. Moreover, elevated infiltration of CD163(+) macrophages, predominantly in stroma areas but not in the tumor, may be a useful indicator of BCG treatment outcome, possibly owing to its immunosuppressive phenotype.

This work was supported by Portuguese Foundation for Science and Technology (FCT) Postdoctoral grant SFRH/BPD/66288/2009 (Jose Alexandre Ferreira), PhD grant SFRH/BD/43399/2008 (Luis Lima), SFRH/BD/81860/2011 (Mariana Silva), SFRH/BD/45120/2008 (Paulo F. Severino) and by LPPC/Pfizer 2011 (Mylene Carrascal). This work was also financially supported by FCT (PTDC-SAU-ONC/112511/2009). FCT is co-financed by European Social Fund (ESF) under Human Potential Operation Programme (POPH) from National Strategic Reference Framework (NSRF). Little is known on the expression of the tumour-associated carbohydrate antigen sialyl-Tn (STn), in bladder cancer. We report here that 75% of the high-grade bladder tumours, presenting elevated proliferation rates and high risk of recurrence/progression expressed STn. However, it was mainly found in non-proliferative areas of the tumour, namely in cells invading the basal and muscle layers. STn was also found in tumour-adjacent mucosa, which suggests its dependence on a field effect of the tumour. Furthermore, it was not expressed by the normal urothelium, demonstrating the cancer-specific nature of this antigen. STn expression correlated with that of sialyltransferase ST6GalNAc. I, its major biosynthetic enzyme. The stable expression of ST6GalNAc. I in the bladder cancer cell line MCR induced STn expression and a concomitant increase of cell motility and invasive capability. Altogether, these results indicate for the first time a link between STn expression and malignancy in bladder cancer. Hence, therapies targeting STn may constitute new treatment approaches for these tumours. publishersversion published

Urothelial bladder cancer (UBC) is a heterogeneous type of disease. It is urgent to screen biomarkers of tumour aggressiveness in order to clarify the clinical behaviour and to personalize therapy in UBC patients. Raf kinase inhibitory protein (RKIP) is a metastasis suppressor, and its downregulation is associated with metastatic events in an increasing number of solid tumours. We evaluated the clinical and prognostic significance of RKIP expression in patients with high risk of progression UBC. Using immunohistochemistry, we determined RKIP expression levels in a series of 81 patients with high-grade pT1/pTis or muscle-invasive UBC. Staining of CD31 and D2-40 was used to assess blood and lymphatic vessels, in order to distinguish between blood and lymphatic vessel invasion (LVI). We found that 90 % of pT1/pTis tumours, 94 % of non-muscle invasive papillary tumours and 76 % of the cases without LVI occurrence expressed RKIP in > 10 % of cells. In this group, we observed a subgroup of tumours (42 %) in which the tumour centre was significantly more intensely stained than the invasion front. This heterogeneous pattern was observed in 63 % of the cases with LVI. Low RKIP expression was associated with poorer 5-year disease-free and overall survival rates, and remained as an independent prognostic factor for disease-free survival. Loss of RKIP expression may be an important prognostic factor for patients with high risk of progression bladder cancer.

Serviço de Oncologia Médica. Instituto Português de Oncologia Francisco Gentil. Porto. Portugal. A 27-year-old man, smoker, presented with three months history of fever. A left pulmonary mass inseparable from the heart was identified and serum alpha-fetoprotein was 4160 ng/ml. The morphologic aspects and immunohistochemistry of the biopsy specimen, in conjunction with the clinical findings were compatible with a diagnosis of pulmonary blastoma with germ cell differentiation. The tumour was considered unresectable. The patient was submitted to two cycles of primary chemotherapy with bleomycin, etoposide and cisplatin. Despite a reduction in serum alpha-fetoprotein, the tumor did not regress. Second line chemotherapy (with paclitaxel, ifosfamide and cisplatin) was instituted, but progressive disease was identified after 2 cycles. Six months after the diagnosis cerebral metastases were found and the patient died. This case illustrates a rare situation of difficult diagnosis and treatment.

Background Urothelial bladder carcinoma (UBC) is a chemo-sensitive tumour, but the response to treatment is heterogeneous. CD147 has been associated with chemotherapy resistance. We aimed to define tumours with an aggressive phenotype by the combined analysis of clinicopathological and biological parameters. Methods 77 patients with T1G3 or muscle-invasive UBC treated by radical cystectomy were studied. Immunohistochemistry was performed to detect CD147, heparanase, CD31 (blood vessels identification) and D2-40 (lymphatic vessels identification) expressions. The immunohistochemical reactions were correlated with the clinicopathological and the outcome parameters. 5-year disease-free survival (DFS) and overall survival (OS) rates were estimated using the Kaplan–Meier method. Multivariate analysis was performed by Cox proportional hazards analysis. Results The 5-year DFS and OS rates were significantly influenced by the classical clinicopathological parameters, and by the occurrence of lymphovascular invasion. CD147 and heparanase immunoexpression did not affect patients' outcome. However, patients with pT3/pT4 tumours had a median OS time of 14.7 months (95% CI 7.1–22.3, p = 0.003), which was reduced to 9.2 months (95% CI 1.5–17.0, p = 0.008) if the tumours were CD147 positive. We developed a model of tumour aggressiveness using parameters as stage, grade, lymphovascular invasion and CD147 immunoexpression, which separated a low aggressiveness from a high aggressiveness group, remaining as an independent prognostic factor of DFS (HR 3.746; 95% CI 1.244–11.285; p = 0.019) and OS (HR 3.247; 95% CI 1.015–10.388, p = 0.047). Conclusion CD147 overexpression, included in a model of UBC aggressiveness, may help surgeons to identify patients who could benefit from a personalized therapeutic regimen. Additional validation is needed.

Introduction: This study evaluates the influence of clinicopathological characteristics, bacillus Calmette-Guérin (BCG) therapeutic schedule [maintenance (mBCG) or induction (iBCG)], and TNF-α and IL-4 polymorphisms on the outcome of non-muscle-invasive bladder cancer patients treated with BCG. Material and Methods: DNA was extracted from 125 bladder cancer patients treated with BCG; TNF-308G/A and IL4-590C/T polymorphisms were genotyped. Results: The TNF-308A allele carriers had an increased risk of developing multiple tumors (OR: 2.80, p = 0.031). However, IL4-590 T carriers also had an increased risk of developing multiple and carcinoma in situ tumors (OR: 2.52, p = 0.033). For these polymorphisms, no association was found with BCG treatment outcome. When treated with iBCG, patients with multiple tumors had shorter recurrence-free survival (RFS) compared with those with a single tumor (p = 0.004); nevertheless, patients with multifocal tumors have improved RFS when treated with mBCG. Conclusions: Overall, the results suggest that multiple tumors and/or carcinoma in situ development are associated with the IL4-590C/T and TNF-308G/A polymorphisms, and emphasize the effectiveness of the mBCG schedule.

The present study investigated the similarities between rodent and human urothelial carcinogenesis models using DNA content, p53 and Ki-67 immunoexpression as surrogate markers of bladder carcinogenesis. Following N-butyl-N-(4-hydroxybutyl)-nitrosamine exposure, 49 human cystectomy specimens of bladder cancer and 53 rat bladder specimens were studied. All of the tumours and adjacent mucosa present in each specimen were evaluated. High similarities were observed between the rodent urothelium carcinogenesis process and the corresponding process in humans, in regards to the histopathological features and biological alteration profile: DNA aneuploidy, p53 overexpression and high proliferative index measured by Ki-67 immunoexpression. Despite these similarities, a higher frequency of alterations was observed in earlier stages in the rat chemical-induced carcinogenesis, namely in 5c aneuploid cells, p53 overexpression and higher Ki-67 labelling index. These results confirm that this experimental animal model is a suitable and reproducible model of bladder carcinogenesis, particularly in regards to high-risk non-invasive and invasive urothelial carcinomas. These features mandate its use in the identification of new molecular targets and evaluation of tumour response to new cytotoxic drugs or drug combinations in bladder cancer therapeutic intervention.

Bladder cancer is the second most common malignancy of the urogenital region. The majority of bladder cancer deaths occur as a consequence of metastatic disease. Blood vessel density (BVD), a surrogate marker for angiogenesis, has been shown to be predictive of progression and poor prognosis, as well as lymphatic vessel density (LVD). The aim of this study was to evaluate, in human urothelial bladder cancer (UBC), the clinical and prognostic significance of angiogenesis, lymphangiogenesis and lymphovascular invasion, assessed with the use of specific immunohistochemical markers., This work was supported by Minsaude Grant 215/2001

We performed a phase II trial to test whether a cyclooxygenase (COX-2) inhibitor, celecoxib, added to standard first-line combination chemotherapy (CT) and as maintenance therapy would improve outcomes in extensive-stage (ES) small-cell lung cancer (SCLC). This was a multicenter trial in CT-naive patients with ES-SCLC. They received standard cisplatin and etoposide (EP) up to 6 cycles and celecoxib 400 mg PO bid continuously until disease progression. Primary end points were response rate (RR), time to progression (TTP), and toxicity. Secondary were overall survival (OS) and quality of life. Of 74 expected patients, only 24 were enrolled and the study stopped earlier because of the published safety concerns about celecoxib. The patients, all male, were between 38 and 74 years. A total of 130 cycles of CT were administered. Toxicity associated with celecoxib was minimal. The RR was 56.5%. Median TTP and OS were 8.6 and 11.3 months, respectively. These data suggest that celecoxib may safely be combined with EP for treatment of ES-SCLC. This combination showed a promising activity and, despite the safety concerns regarding celecoxib, it would be interesting to further evaluate this regimen.

Approximately 80% of bladder tumors are urothelial superficial papillary carcinomas (USPC). Despite a generally good prognosis, these tumors have a strong propensity to recur and about 1/3 of them compared to disease progression. Histological assessment of these superficial tumors is not sufficiently discriminator in predicting prognosis; therefore, we decided to evaluate the prognostic significance of p53 and Ki-67 immunoexpression in low-grade (GI-II) USPC in order to predict the potential outcome of these tumors. P53 and Ki-67 immunoexpression were studied in function of recurrence-free and progression-free survival in 159 primary superficial bladder tumors. A prognostic risk model based on grade, stage and multifocality was also evaluated. P53 accumulation was significantly related to tumor progression (p=0.006). High Ki-67 index (/=18%) and multifocality were significantly related to recurrence (both p=0.0001) and progression-free survival (both p=0.0001) and were independent prognostic factors in the multivariate analysis. The prognostic risk model based on grade, stage and multifocality was not an efficient discriminator of outcome. Adding the Ki-67 index into the risk model, single pTa/T1-GI Ki-67 positive tumors, usually classified as low risk, were reclassified as of intermediate risk. After this reclassification, the risk group model identified a subgroup of pTa/T1-G1 with a high risk of recurrence and progression. Ki-67 index is a reliable prognostic marker in urothelial superficial bladder carcinoma and, when included into a risk profile classification of the low-grade USPC, the accuracy of the prognostic discrimination is enhanced.

Aims: Cell-cycle regulatory proteins are important indicators in determining progression trough the cell-cycle and progression to invasive cancer in patients presenting with superficial bladder cancer. We performed an immunohistochemical study in order to evaluate the prognostic value of the expression of p16, p27, pRb, p53 and Ki-67 in superficial grade I and II papillary urothelial cell carcinoma of the bladder. Methods: p16, p27, p53, pRb and Ki-67 immunoexpression was studied in 14 pTa, 35 pT1a and 7 pT1b bladder tumours at presentation and at recurrence of their tumours. The recurrence-free survival and the progression-free survival were analysed according to these regulatory cell-cycle proteins expression. Results: For survival in univariate analysis a high Ki-67 labelling index was a poor prognostic factor for recurrence-free and progression-free survival ( P =0.0014 and P =0.012, respectively). Ki-67 labelling index was also an independent recurrence-free survival prognostic factor ( P =0.0005). The p16, p27, p53 and pRb immunoreactivity was not significantly associated with recurrence or progression rate in this group of bladder carcinomas. Conclusions: These data suggest that the Ki-67 labelling index can be a reliable marker in predicting recurrence and/or progression in superficial low-grade bladder carcinomas and may be relevant in planning adjuvant therapy.

► Unusual survival of IVB endometrial carcinoma with peritoneal keratin granuloma(PKG). ► PKG can be secondary to endometrioid adenocarcinoma with squamous differentiation. ► Prognosis meaning of lesions is difficult to assess because few cases are published.

Urothelial bladder carcinoma (UBC) is an intricate malignancy with a variable natural history and clinical behavior. Despite developments in diagnosis/prognosis refinement and treatment modalities, the recurrence rate is high, and progression from non-muscle to muscle invasive UBC commonly leads to metastasis. Moreover, patients with muscle-invasive or extra-vesical disease often fail the standard chemotherapy treatment, and overall survival rates are poor. Thus, UBC remains a challenge in the oncology field, representing an ideal candidate for research on biomarkers that could identify patients at increased risk of recurrence, progression, and chemo-refractoriness. However, progress toward personalized medicine has been hampered by the unique genetic complexity of UBC. Recent genome-wide expression and sequencing studies have brought new insights into its molecular features, pathogenesis and clinical diversity, revealing a landscape where classical pathology is intersected by the novel and heterogeneous molecular groups. Hence, it seems plausible to postulate that only an integrated signature of prognostic/predictive biomarkers inherent in different cancer hallmarks will reach clinical validation. In this review, we have summarized ours and others' research into novel putative biomarkers of progression and chemoresistance that encompass several hallmarks of cancer: tumor neovascularization, invasion and metastasis, and energy metabolism reprogramming of the tumor microenvironment.