Infection with Opisthorchis felineus induces intraepithelial neoplasia of the biliary tract in a rodent model.

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.

CD147 and MCT1-potential partners in bladder cancer aggressiveness and cisplatin resistance.

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.

Prognostic Value of Tumor Budding for Early Breast Cancer.

BACKGROUND: Tumor budding (TB) is a dynamic process associated with the epithelial-mesenchymal transition and a well-established prognostic biomarker for colorectal cancer. As part of the tumor microenvironment, tumor buds demonstrate increased cell motility and invasiveness. Current evidence demonstrates that high levels of TB correlate with disease progression and worst outcomes across different solid tumors. Our work aims to demonstrate the clinical applicability of TB analysis and its utility as a prognostic factor for patients with early breast cancer (EBC). METHODS: Retrospective, single-center, observational study, enrolling patients with EBC diagnosed in a Portuguese hospital between 2014 and 2015. TB classification was performed according to the International Tumor Budding Conference 2016 guidelines. RESULTS: A statistically significant relation was found between higher TB score and aggressive clinicopathological features (angiolymphatic/perineural invasion-p < 0.001; tumor size-p = 0.012; nuclear grading-p < 0.001; and Ki-67 index-p = 0.011), higher number of relapses (p < 0.001), and short disease-free survival (DFS) (p < 0.001). CONCLUSION: We demonstrate that high TB correlates with shorter DFS and aggressive clinicopathological features used in daily practice to decide on the benefit of chemotherapy for EBC. TB represents a needed prognostic biomarker for EBC, comprising a new factor to be considered in the adjuvant decision-making process by identifying patients at a high risk of relapse and with higher benefit on treatment intensification. Clinical trials incorporating TB are needed to validate its prognostic impact.

EGFR exon mutation distribution and outcome in non-small-cell lung cancer: a Portuguese retrospective study.

Epidermal growth factor receptor (EGFR) mutations play a predictive role in advanced stages of non-small-cell lung cancer (NSCLC) patients. We conducted this study in order to assess EGFR status in a Portuguese population and its role in NSCLC patients' outcomes. Patients were submitted to EGFR assessment by high-resolution melting and/or direct sequencing. Kaplan-Meier curve was used to assess overall survival and progression-free survival (PFS). Two hundred forty eight out of 322 participants were assessed for EGFR status. Forty-two patients (16.9 %) presented EGFR-mutated status: one patient (2.4 %) presented exon 18; 21 patients (50 %), exon 19; one patient (2.4 %), exon 20; and 18 patients (45.2 %), exon 21 mutations, p < 0.001. PFS was not assessed (n.a.) for patient with exon 18 mutation, and for the other patients with mutations, it was 7 months (3.96-10.03) (exon 19), <1 month (exon 20), and 7 months (0-14.2) (exon 21) (p = 0.027). Overall survival (OS) was 11 months (exon 18), 11 months (1-18) (exon 19), 1 month (exon 20), and 7.5 months (2-70) (exon 21) (p = n.a). This study suggests that the EGFR mutation is herein observed in a higher proportion than expected for a Caucasian population, and OS is a little less than that published in the literature.

Follicular thyroid carcinoma.

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.

IL-4 and TNF-α polymorphisms are associated with risk of multiple superficial tumors or carcinoma in situ development.

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.

Non-Secretory Multiple Myeloma with Patchy Marrow Involvement and Aberrant Cytokeratin Expression.

Non-secretory multiple myeloma is a rare form of the disease that presents a diagnostic challenge. A 69-year-old woman presented to the emergency department with a pathological fracture of the right clavicle, along with a history of asthenia and middle back pain in the preceding three months. Workup revealed multiple focal lytic bone lesions in the clavicles, ribs, skull and thoracic- lumbar-sacral spine, without evidence of anemia, hypercalcemia or renal failure, with no abnormal immunofixation in the serum or urine and with normal serum free light chain ratios. The Iliac crest bone marrow aspiration and biopsy revealed a scarcely involved marrow, However, biopsy of one of the focal bone lesions revealed a hypercellular bone marrow with phenotypically abnormal plasmocytes, along with an intriguing, albeit aberrant, cytokeratin expression. Non-secretory multiple myeloma is in itself a rare diagnosis. However, the combination of a patchy marrow involvement and aberrant cytokeratin expression makes this a noteworthy presentation.

O mieloma múltiplo não-secretor é uma forma rara da doença e um desafio diagnóstico. Uma mulher de 69 anos recorreu ao serviço de urgência com uma fratura patológica da clavícula direita e uma história de astenia e dorsalgia com três meses de evolução. A investigação revelou múltiplas lesões ósseas focais osteolíticas nas clavículas, costelas, crânio e coluna dorso-lombo-sagrada, sem evidência de anemia, hipercalcemia ou insuficiência renal, sem imunofixação anormal no soro ou na urina e com rácios de cadeias leves livres normais. O aspirado e biópsia de medula óssea da crista ilíaca revelou um escasso envolvimento por plasmócitos. No entanto, a biópsia de uma das lesões focais revelou uma medula hipercelular com plasmócitos fenotipicamente anormais e com uma aberrante expressão de citoqueratinas. O mieloma múltiplo não-secretor é por si só um diagnóstico raro. Contudo, a combinação de envolvimento irregular da medula e a expressão aberrante de citoqueratinas são merecedoras de atenção.

Hysteroscopic Findings Related with the Assessment and Treatment of Uterine Florid Cystic Endosalpingiosis: A Case Report and Review of All the Published Cases.

INTRODUCTION: Endosalpingiosis is a rare benign condition characterized by the presence of tubal epithelium outside the Fallopian tube. The clinical presentation of endosalpingiosis is nonspecific, and the diagnosis is typically incidental in women undergoing surgery for pelvic pain, infertility, urinary symptoms, or a pelvic mass. It can only be confirmed with histopathological examination. CASE REPORT: We report the first case of uterine florid cystic endosalpingiosis, with unusual hysteroscopic findings. We reviewed all the published cases of uterine florid cystic endosalpingiosis and their clinical presentation including hysteroscopic characteristics. It is a rare benign condition, with only 32 cases described in the literature. This is the first hysteroscopic description of this condition to be made. DISCUSSION: The patient first underwent a hysteroscopy and a leiomyoma resection when she was 51 years old. At 55, she went through another hysteroscopy, and a polypoid lesion was excised. A third hysteroscopy, one year later, revealed a new polypoid lesion in a similar location. After the initial incisions, this polypoid lesion disappeared. By decreasing the intrauterine pressure, it became visible again, corresponding histologically to an endometrial polyp with tubal metaplasia. At last, she underwent a laparoscopic hysterectomy with a final histopathological diagnosis of uterine florid cystic endosalpingiosis. CONCLUSION: Florid cystic endosalpingiosis is a rare condition that may be associated with several bizarre hysteroscopic findings.

Introdução: A endossalpingiose é uma condição benigna rara caracterizada pela presença de epitélio tubário fora da trompa de Falópio. A apresentação clínica da endossalpingiose é inespecífica e o diagnóstico é geralmente incidental em mulheres submetidas a cirurgia por dor pélvica, infertilidade, sintomas urinários ou massa pélvica. O diagnostico só pode ser confirmado com exame histopatológico. Caso Clínico: Relatamos o primeiro caso de endosalpingiose cística florida uterina, no qual se encontrou achados histeroscópicos incomuns. Foram revistos todos os casos publicados de endosalpingiose cística florida uterina e suas apresentações clínicas, incluindo características histeroscópicas. Trata-se de uma condição benigna rara, com apenas 32 casos descritos na literatura. Apresentamos a primeira descrição histeroscópica desta patologia. Discussão: A doente foi submetida a uma primeira histeroscopia e resseção de leiomioma aos 51 anos de idade. Com 55, realizou uma nova histeroscopia e removeu uma lesão polipóide. Uma terceira histeroscopia, um ano depois, revelou uma nova lesão polipóide num local semelhante. Após as incisões iniciais, essa lesão polipóide desapareceu. Ao diminuir a pressão intrauterina, tornou-se visível novamente, correspondendo histologicamente a um pólipo endometrial com metaplasia tubária. A doente foi submetida a uma histerectomia laparoscópica com diagnóstico histopatológico final de endosalpingiose cística florida uterina. Foram revistos todos os casos publicados de endosalpingiose cística florida uterina e suas apresentações clínicas, incluindo características histeroscópicas. Conclusão: A endosalpingiose cística florida uterina é uma condição rara que pode estar associada a achados histeroscópicos bizarros.

Estrogen receptors in urogenital schistosomiasis and bladder cancer: Estrogen receptor alpha-mediated cell proliferation.

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 ERß 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. ERß 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 17ß-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.

The aggressiveness of urothelial carcinoma depends to a large extent on lymphovascular invasion--the prognostic contribution of related molecular markers.

AIMS: 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. METHODS AND RESULTS: Immunohistochemistry for CD31 (a blood vessel endothelial cell marker), D2-40 (a lymphatic vessel endothelial cell marker), vascular endothelial growth factor (VEGF)-C and VEGF-receptor 3 antibodies was performed in 83 patients with urothelial carcinoma who underwent radical cystectomy. The classic histopathological characteristics, associated with lymphovascular invasion and loco-regional dissemination, had a negative influence on 5-year overall survival (OS) rates. BVD and LVD were correlated with advanced and poorly differentiated UBC with lymphovascular invasion. Blood vessel invasion (BVI) by malignant emboli assessed by CD31 staining, and lymphatic vessel invasion (LVI) by isolated malignant cells assessed by D2-40 staining significantly affected OS. VEGF-C overexpression was correlated with both BVI and LVI by single malignant cells assessed by CD31 and D2-40, respectively. BVI by malignant emboli assessed by CD31 staining remained as an independent prognostic factor. CONCLUSIONS: Patients with UBC with embolic BVI assessed by CD31 and LVI by isolated malignant cells assessed by D2-40 have a worse prognosis and may benefit from adjuvant therapies.

Phase II study of celecoxib with cisplatin plus etoposide in extensive-stage small cell lung cancer.

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.

Malignant thymomas--the experience of the Portuguese Oncological Institute, Porto, and literature review.

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 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.

Breast cancer in Angola, molecular subtypes: a first glance.

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.

Targeted O-glycoproteomics explored increased sialylation and identified MUC16 as a poor prognosis biomarker in advanced-stage bladder tumours.

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.

Metabolic coupling in urothelial bladder cancer compartments and its correlation to tumor aggressiveness.

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.

Hypoxia enhances the malignant nature of bladder cancer cells and concomitantly antagonizes protein O-glycosylation extension.

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.

Abnormal Protein Glycosylation and Activated PI3K/Akt/mTOR Pathway: Role in Bladder Cancer Prognosis and Targeted Therapeutics.

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.

Karyotypic divergence and convergence in two synchronous lung metastases of a clear cell sarcoma of tendons and aponeuroses with t(12;22)(q13;q12) and type 1 EWS/ATF1.

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.