Sofia S Pereira

Ki-67 index enhances the prognostic accuracy of the urothelial superficial bladder carcinoma risk group classification.

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.

Overexpression of tumour-associated carbohydrate antigen sialyl-Tn in advanced bladder tumours

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.

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.

Adipocyte secreted factors enhance aggressiveness of prostate carcinoma cells.

Obesity has been associated with increased incidence and risk of mortality of prostate cancer. One of the proposed mechanisms underlying this risk association is the change in adipokines expression that could promote the development and progression of the prostate tumor cells. The main goal of this study was to evaluate the effect of preadipocyte and adipocyte secretome in the proliferation, migration and invasion of androgen independent prostate carcinoma cells (RM1) and to assess cell proliferation in the presence of the adiposity signals leptin and insulin. RM1 cells were co-cultured in with preadipocytes, adipocytes or cultured in their respective conditioned medium. Cell proliferation was assessed by flow cytometry and XTT viability test. Cell migration was evaluated using a wound healing injury assay of RM1 cells cultured with conditioned media. Cellular invasion of RM1 cells co-cultured with adipocytes and preadipocytes was assessed using matrigel membranes. Preadipocyte conditioned medium was associated with a small increase in RM1 proliferation, while adipocytes conditioned media significantly increased RM1 cell proliferation (p<0.01). Adipocytes also significantly increased the RM1 cells proliferation in co-culture (p <0.01). Cell migration was higher in RM1 cells cultured with preadipocyte and adipocyte conditioned medium. RM1 cell invasion was significantly increased after co-culture with preadipocytes and adipocytes (p <0.05). Insulin also increased significantly the cell proliferation in contrast to leptin, which showed no effect. In conclusion, prostate carcinoma cells seem to be influenced by factors secreted by adipocytes that are able to increase their ability to proliferate, migrate and invade.

Detailed characterization of incretin cell distribution along the human small intestine

BACKGROUND Incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1), are physiologic stimulants of insulin release that have been implicated in diabetes remission after bariatric surgery. The detailed distribution of incretin cells along the human small gut, so far unknown, is of utmost importance for the understanding of the metabolic changes observed after bariatric surgery because diabetes remission rate varies according to the type of anatomic rearrangement. OBJECTIVE To characterize the distribution of incretin producing cells along the human jejunum-ileum. SETTING Academic public institution. METHODS Small intestines (n = 30) from autopsies were sampled every 20 cm along their entire length and tissue microarrays were constructed. The percentage of immunohistochemistry-stained cell areas for GLP-1, GIP, and chromogranin A at each segment length was quantified using a computer-aided analysis tool. RESULTS The percentage of stained area for GLP-1 immunoreactive cells was found to be significantly higher from 200 cm from Treitz ligament onward compared with the first 80 cm of the small intestine, whereas GIP immunoreactive cells were predominant expressed in the first 80 cm. In contrast, chromogranin A expression was constant along the entire jejunum-ileum. CONCLUSION The uneven distribution of GLP-1-expressing cells, with a higher density from 200 cm of the jejunum-ileum, could contribute to explain the improvement of glycemic profile of diabetic patients observed after anatomic rearrangement of the intestinal tract, in particular when subjected to gastric bypass with longer biliopancreatic limbs.

The emerging role of the molecular marker p27 in the differential diagnosis of adrenocortical tumors

Malignant adrenocortical tumors (ACTs) are rare and highly aggressive; conversely, benign tumors are common and frequently found incidentally (the so-called incidentalomas). Currently, the use of molecular markers in the diagnosis of ACTs is still controversial. The aim of this study was to analyze the molecular profile of different ACTs with the purpose of identifying markers useful for differentiating between these tumors. The ACTs that were studied (n=31) included nonfunctioning adenomas (ACAn)/incidentalomas (n=13), functioning adenomas with Cushings syndrome (ACAc) (n=7), and carcinomas (n=11); normal adrenal glands (n=12) were used as controls. For each sample, the percentage area stained for the markers StAR, IGF2, IGF1R, p53, MDM2, p21, p27, cyclin D1, Ki-67, β-catenin, and E-cadherin was quantified using a morphometric computerized tool. IGF2, p27, cyclin D1, and Ki-67 were the markers for which the percentage of stained area was significantly higher in carcinoma samples than in adenoma samples. Ki-67 and p27 were the markers that exhibited the highest discriminative power for differential diagnosis between carcinomas and all type of adenomas, while IGF2 and StAR were only found to be useful for differentiating between carcinomas and ACAn and between carcinomas and ACAc respectively. The usefulness of Ki-67 has been recognized before in the differential diagnosis of malignant tumors. The additional use of p27 as an elective marker to distinguish benign ACTs from malignant ACTs should be considered.

Chromosome instability and progression in urothelial cell carcinoma of the bladder.

Superficial urothelial cell carcinoma (UCC) is a heterogeneous group of neoplasia with an unpredictable clinical course. Numerical alterations of chromosomes 7, 9 and 17 in superficial and invasive UCCs were analysed to evaluate the importance of chromosome instability in the progression of these tumours. Our sample consisted of 75 patients (47 with superficial and 28 with invasive bladder tumours). In situ hybridization using centromeric probes for chromosomes 7, 9 and 17 was done for the chromosome analysis in paraffin-embedded tissues. From the results obtained it can be concluded that losses of genetic material seem to be important early events in the carcinogenesis of the urothelium, but during progression of UCCs there seems to be a selection of those cells with gains of genetic material. This chromosome instability may be due to the acquisition of mechanisms involved in aneuploidization, namely p53 function disorders.

GLP1 and glucagon co-secreting pancreatic neuroendocrine tumor presenting as hypoglycemia after gastric bypass.

Summary Post-prandial hypoglycemia is frequently found after bariatric surgery. Although rare, pancreatic neuroendocrine tumors (pNET), which occasionally are mixed hormone secreting, can lead to atypical clinical manifestations, including reactive hypoglycemia. Two years after gastric bypass surgery for the treatment of severe obesity, a 54-year-old female with previous type 2 diabetes, developed post-prandial sweating, fainting and hypoglycemic episodes, which eventually led to the finding by ultrasound of a 1.8-cm solid mass in the pancreatic head. The 72-h fast test and the plasma chromogranin A levels were normal but octreotide scintigraphy showed a single focus of abnormal radiotracer uptake at the site of the nodule. There were no other clinical signs of hormone secreting pNET and gastrointestinal hormone measurements were not performed. The patient underwent surgical enucleation with complete remission of the hypoglycemic episodes. Histopathology revealed a well-differentiated neuroendocrine carcinoma with low-grade malignancy with positive chromogranin A and glucagon immunostaining. An extract of the resected tumor contained a high concentration of glucagon (26.707 pmol/g tissue), in addition to traces of GLP1 (471 pmol/g), insulin (139 pmol/g) and somatostatin (23 pmol/g). This is the first report of a GLP1 and glucagon co-secreting pNET presenting as hypoglycemia after gastric bypass surgery. Although pNET are rare, they should be considered in the differential diagnosis of the clinical approach to the post-bariatric surgery hypoglycemia patient. Learning points pNETs can be multihormonal-secreting, leading to atypical clinical manifestations. Reactive hypoglycemic episodes are frequent after gastric bypass. pNETs should be considered in the differential diagnosis of hypoglycemia after bariatric surgery.

Lymph Node Metastases in Papillary and Medullary Thyroid Carcinoma Are Independent of Intratumoral Lymphatic Vessel Density

Background: Blood and lymph vessel invasion are well-recognized markers of tumor aggressiveness, as these are the routes that lead to metastases. Thyroid tumors, depending on the histological variant, tend to have distinctive biological behaviors and use different vascular routes to metastasize, yet the mechanisms underlying the metastatic process are still poorly understood. Objectives: The aim of this study was to assess how the lymph vessel density (LVD) in different histological types of thyroid tumors, and in their surrounding tissue, correlate with the presence of lymph node metastases (LNM) and tumor pathological features. Methods: Lymph vessels of papillary thyroid carcinomas (PTC), of the classical (CVPTC, n = 50) and follicular variants (FVPTC, n = 18), and medullary thyroid carcinomas (MTC, n = 34) were immunohistochemically stained against antigen D2-40. The stained area was quantified using a computerized morphometric analysis tool and correlated with the tumor pathological characteristics. Results: LVD within all analyzed thyroid tumor subtypes was significantly lower than in the surrounding thyroid tissues (p < 0.001). Despite intratumoral LVD being significantly higher in CVPTC than in FVPTC, and peritumoral LVD being significantly higher in MTC than in PTC (p < 0.05), no correlations were found between LVD (either intratumoral or peritumoral) and the presence of lymph node metastasis. Conclusions: As no LVD differences were found amongst thyroid tumors with or without LNM, dissemination is more likely to depend on the tumor ability to invade the abundant lymph vessel network of the surrounding thyroid tissue than on the ability of the tumor to promote de novo lymphangiogenesis.

Telomerase and N-Cadherin Differential Importance in Adrenocortical Cancers and Adenomas†

Adrenocortical carcinomas (ACC) are most frequently highly aggressive tumors. We assessed the telomerase reverse transcriptase (TERT) and N‐cadherin role in the biology of ACC and their potential utility as molecular biomarkers, in different types of tumoral adrenocortical tissue. A total of 48 adrenal cortex samples (39 tumoral and 9 normal adrenal glands) were studied. TERT promoter mutations were searched by PCR and Sanger sequencing in two hotspots positions (−124 and −146). Also, telomerase and N‐cadherin expression were evaluated by immunohistochemistry. TERT promoter mutations were not detected in any of the samples either malignant or benign. Telomerase nuclear expression was present in 26.6% of ACC and in 45.5% of non‐functioning adenomas. It was absent in benign Cushings lesions and in normal adrenal glands. Contrarily, N‐cadherin was always expressed in the cellular membranes of benign adenomas or normal adrenals but no expression was detected in the majority of ACC. Nuclear telomerase and membrane N‐cadherin expression were positively correlated in ACCs. We conclude that in ACC, the loss of N‐cadherin is a frequent phenomenon while the existence of TERT promoter mutations is not and nuclear telomerase expression is present in only a minority of cases. Since the loss of N‐cadherin expression was identified in both high and low proliferative ACC, this marker should be considered important for diagnostic application. Our study also suggests the existence of a TERT non‐canonical function in cell adhesion. J. Cell. Biochem. 118: 2064–2071, 2017.