Expresión de PD-L1 en cáncer renal, características pronósticas y utilidad en la práctica clínica habitual / Expression of PD-L1 in renal cancer, prognostic features and clinical utility of its routine staining

Introducción La expresión de PD-L1 en el carcinoma de células renales (CCR) se asocia a tasas de sobrevida y características clínico-patológicas pronósticas peores. Sin embargo, estos parecen responder mejor ante nuevos agentes terapéuticos. Conocer el comportamiento del CCR según la presencia de PD-L1 puede tener implicancias en la consejería de los pacientes y el abordaje terapéutico. Objetivo Identificar la presencia de PD-L1 en las células tumorales renales y analizar su asociación con los factores pronósticos de los pacientes, la sobrevida global (SG) y la sobrevida cáncer-específica (SCE). Metodología Análisis retrospectivo a partir de muestras de tejido de CCR obtenidas entre 2018 y 2021. Estudio inmunohistoquímico con anticuerpo monoclonal de ratón anti PD-L1, clon 22C3. Se definió PD-L1 «positivo» como una puntuación de proporción tumoral ≥ 1%. Comparación de factores pronósticos según la presencia o ausencia de PD-L1, y análisis univariante para la SG y la SCE. Resultados Un 14% (n=11) de la muestra era PD-L1(+). La edad media era de 59 años. No hubo diferencias estadísticamente significativas entre el estatus de PD-L1 y el estadio TNM, el grado nuclear y el tipo histológico. Los pacientes PD-L1(+) tuvieron peor SG con un HR de 5,27 (IC: 1,1-23,7; p=0,03) y la SCE mostró una tendencia desfavorable para PD-L1(+) con un HR de 4,79 (IC: 0,79-28,95; p=0,08). Conclusión La prevalencia de PD-L1 en el CCR es considerable. En este estudio, PD-L1(+) se asoció con una SG y SCE desfavorables, lo que justifica incorporar su uso rutinario en el CCR (AU)

Introduction The expression of PD-L1 in renal cell carcinoma (RCC) is associated with worse survival and prognostic clinical-pathological features. However, they seem to respond better to new therapeutic agents. Knowing the behavior of RCC according to the presence of PD-L1 may have implications for medical counseling and therapeutic approaches. Objective To identify the presence of PD-L1 in renal tumor cells and analyze its association with patientś prognostic factors, overall survival (OS) and cancer-specific survival (CSS). Methodology Retrospective analysis of RCC tissue samples, obtained between 2018 and 2021. Immunohistochemistry analysis with mouse monoclonal Anti PD-L1, clone 22C3. Definition of PD-L1 “positive” as a Tumor Proportion Score ≥ 1%. Comparison of prognostic factors according to the presence or absence of PD-L1, and univariate analysis for OS and CSS. Results 14% (n=11) of the sample were PD-L1(+). Average age was 59 years. There were no statistically significant differences between PD-L1 status and TNM stages, nuclear grade and histology. PD-L1(+) had worse OS with a HR of 5.27 (CI: 1.1-23.7; p=0.03) and CSS showed a unfavorable tendency for PD-L1(+) with a HR of 4.79 (CI: 0.79-28.95; p=0.08). Conclusion The prevalence of PD-L1 in RCC is considerable. In this study PD-L1(+) was associated with unfavorable OS and CSS. It seems reasonable to incorporate its routine use in RCC (AU)

Expression of PD-L1 in renal cancer, prognostic features and clinical utility of its routine staining.

INTRODUCTION: The expression of PD-L1 in renal cell carcinoma (RCC) is associated with worse survival and prognostic clinical-pathological features. However, they seem to respond better to new therapeutic agents. Knowing the behavior of RCC according to the presence of PD-L1 has implications for medical counseling and therapeutic approaches. OBJECTIVE: To identify the presence of PD-L1 in renal tumor cells and analyze its association with patients' prognostic factors, overall survival (OS) and cancer-specific survival (CSS). METHODOLOGY: Retrospective analysis of RCC tissue samples, obtained between 2018 and 2021. Immunohistochemistry analysis with mouse monoclonal Anti PD-L1, clone 22C3. Definition of PD-L1 "positive" as a Tumor Proportion Score ≥1%. Comparison of prognostic factors according to the presence or absence of PD-L1, and univariate analysis for OS and CSS. RESULTS: 14% (n = 11) of the sample were PD-L1(+). Average age was 59 years. There were no statistically significant differences between PD-L1 status and TNM stages, nuclear grade and histology. PD-L1(+) had worse OS with a HR of 5.27 (CI: 1.1-23.7; P = .03) and CSS showed a unfavorable tendency for PD-L1(+) with a HR of 4.79 (CI: 0.79-28.95; P = .08). CONCLUSION: The prevalence of PD-L1 in RCC is considerable. In this study PD-L1(+) was associated with unfavorable OS and CSS. It seems reasonable to incorporate its routine use in RCC.

Proinflammatory environment and role of TNF-α in endometrial function of obese women having polycystic ovarian syndrome.

BACKGROUND/OBJECTIVES: A high percentage of women having polycystic ovarian syndrome (PCOS) exhibit hyperinsulinemia and obesity. Transforming necrosis factor-α (TNF-α) is an adipokine that increases in obesity and negatively affects insulin action in several tissues, including the endometrium. In fact, it has been reported that insulin signaling is altered in the endometrium of PCOS women, affecting its reproductive function. The aim of this study was to determine the proinflammatory environment and TNF-α signaling in endometrium from obese women with PCOS, and also to evaluate the effect of TNF-α on endometrial cell energy homeostasis. METHODS: Serum and endometrial tissues were obtained from four study groups: normal-weight, normal-weight-PCOS, obese and obese-PCOS (hyperandrogenemia/hyperinsulinemia) (n=7 per group). Serum TNF-α level was assayed by enzyme-linked immunosorbent assay (ELISA); endometrial TNF-α level and its receptors (TNFR1/TNFR2) as well as nuclear factor (NF)-κB content were determined by immunohistochemistry. Finally, we evaluated TNF-α effect on glucose uptake in cultured human endometrial stromal cells (T-HESC) treated or not with testosterone/insulin resembling partially the PCOS condition. RESULTS: TNF-α plasma levels were similar between groups, whereas cytokine levels and macrophage number increased in endometrium from obese-PCOS women (P<0.001). Both receptor types were higher in obese vs normal-weight women, particularly TNFR2 content in the obese-PCOS group (P<0.001). Furthermore, an increased NF-κB nuclear content in endometrium from obese-PCOS was observed (P<0.001). Finally, TNF-α treatment of T-HESC cultures exhibited a decrease of glucose uptake (P<0.05), although similar to cells treated with testosterone or testosterone/insulin/TNF-α. CONCLUSIONS: These results suggest that the PCOS condition induces an inflammatory state exacerbated when obesity is present, where a higher TNF-α signaling is observed, all of which could affect glucose uptake in the tissue and may cause fertility failures in these women.

Levels of Regulatory Proteins Associated With Cell Proliferation in Endometria From Untreated Patients Having Polycystic Ovarian Syndrome With and Without Endometrial Hyperplasia.

Polycystic ovarian syndrome (PCOS) has been associated with endometrial hyperplasia and cancer. The aim of this study was to establish whether the expression of proliferation regulatory proteins in the endometria of patients having PCOS, with or without hyperplasia, differs from control women. Control endometria (CE), patients having PCOS without and with endometrial hyperplasia (PCOSE and HPCOSE, respectively), and that of women with endometrial hyperplasia (HE) were used. The phosphorylated estrogen receptor form (pERα), similar to mother against decapentaplegic (SMAD) 2, SMAD3, and SMAD4, vascular epithelial growth factor (VEGF), and phosphorylated SMAD (pSMAD) 2 and pSMAD3 were detected by immunohistochemistry or Western blot. The results show higher levels of pERα in HE versus CE (P < .05), while higher VEGF levels were found in PCOSE and HE (P < .05) compared to CE; SMAD2 diminished in HE (P < .05) versus CE. Consequently, the higher levels of VEGF and pERα in PCOSE could represent early changes in the progression of PCOSE toward hyperplasia and cancer, whereas changes observed in SMAD proteins support the differential origin of the pathologies of HPCOSE and HE.

[Computer aided dosage management of phenprocoumon anticoagulation therapy. Clinical validation]. / Rechnergestützte Dosierung in der Antikoagulationstherapie mit Phenprocoumon. Validierung für den klinischen Einsatz.

UNLABELLED: A recently developed multiparameter computer-aided expert system (TheMa) for guiding anticoagulation with phenprocoumon (PPC) was validated by a prospective investigation in 22 patients. The PPC-INR-response curve resulting from physician guided dosage was compared to INR values calculated by "twin calculation" from TheMa recommended dosage. Additionally, TheMa was used to predict the optimal time to perform surgery or invasive procedures after interruption of anticogulation therapy. RESULTS: Comparison of physician and TheMa guided anticoagulation showed almost identical accuracy by three quantitative measures: Polygon integration method (area around INR target) 616.17 vs. 607.86, INR hits in the target range 166 vs. 161, and TTR (time in therapeutic range) 63.91 vs. 62.40 %. After discontinuation of anticoagulation therapy, calculating the INR phase-out curve with TheMa INR prognosis of 1.8 was possible with a standard deviation of 0.50 ± 0.59 days. CONCLUSION: Guiding anticoagulation with TheMa was as accurate as Physician guided therapy. After interruption of anticoagulant therapy, TheMa may be used for calculating the optimal time performing operations or initiating bridging therapy.

Decreased phosphorylation of Y¹4caveolin-1 in endometrial tissue of polycystic ovary syndrome patients may be related with an insulin resistant state in this tissue.

Endometrial tissue of patients with polycystic ovary syndrome (PCOS) shows an impaired expression of insulin signaling molecules. Tyrosine phosphorylation of the insulin receptor (IR) by insulin promotes glucose uptake by activating the PI3K/Akt pathway. IR stability and function depend on the presence of the protein caveolin-1. Activation of IR increases phosphorylation of Y¹4caveolin-1. Since the endometrium of PCOS patients is proposed to be insulin resistant, we evaluated the phosphorylation of IR and caveolin-1 in endometria of patients with insulin resistance (PCOSE-IR) compared to controls (CE). To explore the mechanism associated with this condition, cultured endometrial cells (T-HESC) were exposed to high glucose (25 mM, 24 h), an experimental condition that leads to insulin resistance in other cell types. Endometrial protein levels of phospho-Y97²IR, phospho-Y¹4caveolin-1 and caveolin-1 were determined by Western blotting. In cultured cells, protein levels of caveolin-1, IR, and Akt were evaluated by Western blotting. After acute insulin stimulation, phospho-S47³Akt, phospho-Y¹4caveolin-1, and 2-deoxyglucose (2-DOG) uptake were determined. PCOSE-IR samples showed high protein levels of caveolin-1, but reduced phospho-Y¹4caveolin-1 compared to CE. No differences were observed for phospho-Y97²IR between both groups. Cells pretreated with glucose showed a reduction in protein levels of IR and caveolin-1 and were unable to increase 2-DOG uptake, phospho-S47³Akt and phospho-Y¹4caveolin-1 after insulin stimulation. In conclusion, in PCOSE-IR the impaired phosphorylation of IR downstream molecules such as phospho-Y¹4caveolin-1 suggests a diminished insulin sensitivity in endometria, condition that could be supported in vitro by the ability of T-HESCs to become insulin resistant when they are exposed to high glucose.

Nerve growth factor induces the expression of chaperone protein calreticulin in human epithelial ovarian cells.

Epithelial ovarian cancer is highly angiogenic and high expression of Nerve Growth Factor (NGF), a proangiogenic protein. Calreticulin is a multifunctional protein with anti-angiogenic properties and its translocation to the tumor cell membrane promotes recognition and engulfment by dendritic cells. The aim of this work was to evaluate calreticulin expression in human normal ovaries, benign and borderline tumors, and epithelial ovarian cancer samples and to evaluate whether NGF regulates calreticulin expression in human ovarian surface epithelium and in epithelial ovarian cancer cell lines. Calreticulin mRNA and protein levels were analyzed using RT-PCR, Western blot and immunohistochemistry in 67 human ovarian samples obtained from our Institution. Calreticulin expression induced by NGF stimulation in cell lines was evaluated using RT-PCR, Western blot and immunocytochemistry. We found a significant increase of calreticulin mRNA levels in epithelial ovarian cancer samples as compared to normal ovaries, benign tumors, and borderline tumors. Calreticulin protein levels, evaluated by Western blot, were also increased in epithelial ovarian cancer with respect to benign and borderline tumors. When HOSE and A2780 cell lines were stimulated with Nerve Growth Factor, we found an increase in calreticulin protein levels compared to controls. This effect was reverted by GW441756, a TRKA specific inhibitor. These results suggest that NGF regulates calreticulin protein levels in epithelial ovarian cells through TRKA receptor activation.

The identification of two subgroups of obese women with differing endometrial proliferation levels: potential consequences in the development of endometrial cancer.

Enhanced endometrial proliferation correlates obesity to type-I (estrogen-dependent) endometrial cancer (EC). Our aim was to distinguish obese women (without EC) with differing endometrial proliferation. Endometrial and blood samples were obtained from normal-weight and obese women without EC. Type-I EC samples were obtained from obese patients. On measuring endometrial proliferation (Ki67 and phosphorylated histone H3 (p-H3)), two groups of obese women without EC were identified: obese(High Proliferating) (O(HP)) and obese(Low Proliferating) (O(LP)). Increased Ki67 (88.5%, P<0.001), p-H3 (62.6%, P<0.01), 17ß-estradiol/progesterone ratio (46.3%, P<0.01) and endometrial estrogen receptor alpha (ERα) (82.2%, P<0.001) were observed in O(HP) compared with O(LP) patients. ECs possessed similar ERα and enhanced proliferation as O(HP), suggesting that O(HP) women are at higher risk of type-I EC. O(LP) women were indistinguishable from normal-weight women regarding these determinants of endometrial proliferation, ERα and 17ß-estradiol/progesterone ratio. Our data may further define the obesity phenotype in regards to type-I EC risk and may help identify obese women more susceptible to develop type-I EC, allowing early intervention and a potential reduction in mortality.

Computer-aided dosage in oral anticoagulation therapy using phenprocoumon. Problems and approaches.

Oral anticoagulation using vitamin K antagonists has been established for over 50 years. Although it is highly effective in preventing thromboembolic incidents, its therapeutic control still remains problematic. Therefore, a computer-aided approach is recommended for deriving dosages. Up to now, the dosage is often based on the visual inspection of previous INR measurements, average weekly doses, and the INR target range. Statistical variations of measurement results and time-delayed effects of dosages, however, frequently result in the misinterpretation of data and suggest pseudo-trends. Treating physicians are not only responsible for determining the patient-specific maintenance dose, but must also respond to deviating INR values, overdosage or underdosage, initiate the oral anticoagulation therapy, and control the INR level in case of a new target range (bridging). Instructive examples are provided to illustrate the described difficulties. A computer-aided expert system is currently developed to ensure the therapeutic safety under the specified conditions. We present preliminary results from a study designed to validate mathematical models underlying such expert systems.

Levels of Rabs and WAVE family proteins associated with translocation of GLUT4 to the cell surface in endometria from hyperinsulinemic PCOS women.

BACKGROUND: Polycystic ovary syndrome (PCOS) is an endocrine-metabolic disorder highly associated with insulin resistance and compensatory hyperinsulinemia. It is known that the insulin signaling pathway is impaired in endometria from PCOS hyperinsulinemic women, but no information is available about molecules associated with cell surface GLUT4 translocation. We therefore evaluated the protein levels of AS160 target molecules, Rab8A and Rab10, and the WAVE family proteins involved in the cortical-actin remodeling, Neural Wiskott-Aldrich Syndrome Protein (N-WASP) and WASP, in endometria from hyperinsulinemic PCOS women and controls. METHODS: Protein levels were assessed by western blot, immunohistochemistry and immunofluorescence in proliferative (PE = 7) and secretory (SE = 7) phase endometria from control women and in endometria from hyperinsulinemic PCOS women (PCOS h-INS = 7). RESULTS: Similar levels were detected for Rab10 in the three studied groups; however, Rab8A levels decreased in SE (P < 0.05) while higher levels were obtained in PCOSE h-INS compared with PE (P < 0.05). In the normal menstrual cycle, Neural Wiskott-Aldrich syndrome protein (N-WASP) and WASP levels were increased in SE versus PE (P < 0.05), but in PCOSE h-INS, the levels were diminished compared with PE (P < 0.05). CONCLUSIONS: SE is characterized by protein expression changes associated with glucose uptake. In endometria from PCOS women with hyperinsulinemia, reduced levels of WAVE family proteins could compromise the cell surface GLUT4 exposure and the consequent glucose uptake in this tissue.

The conversion of dehydroepiandrosterone into androst-5-ene-3beta,17beta-diol (androstenediol) is increased in endometria from untreated women with polycystic ovarian syndrome.

The changes in endometrial homeostasis found in women with polycystic ovarian syndrome (PCOS) could be associated with alterations in the intracrine metabolism of steroid hormones. The uptake of dehydroepiandrosterone-sulphate (DHEA-S), precursor of the intracrine pathway, is achieved by transporters, such as organic anion transporter polypeptides (OATPs), and molecules with oestrogenic activity, such as androst-5-ene-3beta,17beta-diol (androstenediol), can be generated. We aimed to determine androstenediol generation and the expression of OATPs in human endometria throughout the menstrual cycle and in endometria from PCOS women. Endometrial samples were obtained from control women in the proliferative phase (control endometria (CEp), n=7), secretory phase (CEs, n=7), and from PCOS patients (PCOSEp, n=7). The mRNA levels of OATP-B, OATP-D and OATP-E were measured by reverse transcriptase polymerase chain reaction (RT-PCR) and protein levels of OATP-E by immunofluorescence; 3beta-hydroxysteroid dehydrogenase (HSD) by immunohistochemistry/Western blot; the metabolism of DHEA to androstenediol was evaluated by thin layer chromatography-high-performance liquid chromatography (TLC-HPLC). Lower levels of OATP-E transcript were obtained in PCOSEp (p<0.05) compared with CEp, while OATP-E protein levels (p<0.05) and DHEA conversion to androstenediol (p<0.01) were higher in PCOSEp. Lower 3beta-(hydroxysteroid dehydrogenase) HSD protein levels were found in PCOSEp (p<0.05) (Western blot, immunohistochemistry). These results reveal a higher capacity of the endometria from PCOS women to metabolise DHEA to androstenediol, which, coupled with the high oestrogen sensitivity previously found in these endometria, may account for the increase in cell proliferation in PCOSEp already reported.

The effect of overweight and obesity on proliferation and activation of AKT and ERK in human endometria.

OBJECTIVE: To examine whether overweight and obesity could lead to increased endometrial proliferation and activation of AKT and ERK1,2 in cycling premenopausal women. METHODS: Endometrial and blood samples were obtained from women with normal endometrial histology, and allocated into three groups-normal-weight, overweight and obese-according to the subject's body mass index (BMI). Samples from obese patients with type-I endometrial cancer (EC) were included as a control. Cell proliferation was measured by immunohistochemical detection of Ki67 and phosphorylated histone H3 (p-H3). AKT and ERK1,2 activation was assessed by Western blot. Circulating steroids, leptin and insulin were measured by immunoassays. RESULTS: In endometrial samples with normal histology, epithelial cell proliferation was higher in the overweight and obese groups versus the normal-weight set (P<0.05). Proliferation indexes were positively correlated with the subject's BMI and serum levels of estrogen, leptin and insulin (P<0.05). Increased phosphorylated AKT (pAKT) (1.6-fold) and ERK1,2 (pERK1,2) (8.7-fold) were observed in endometria from obese with respect to normal-weight subjects (P<0.05). Similarly, increased phosphorylation of AKT (0.7-fold) and ERK1,2 (2.3-fold) was detected in endometria from overweight as compared with the normal-weight group (P<0.05). In women with EC, we found a significant increase in endometrial proliferation, and in pAKT and pERK1,2 expression levels when compared to patients with normal endometrial histology. CONCLUSION: These results show correlation between obesity (and overweight) and increased endometrial cell proliferation, and the activation of AKT and ERK1,2. These features could be related with the higher risk to develop type-I EC in overweight and obese women.

Control of Postharvest Gray Mold of Table Grapes in the San Joaquin Valley of California by Fungicides Applied During the Growing Season.

Fungicides applied before harvest were evaluated to control postharvest gray mold of table grapes, caused by Botrytis cinerea. The concentrations of thiophanate methyl (THM), iprodione (IPR), cyprodinil (CYP), pyraclostrobin + boscalid (PS+BO), pyrimethanil (PYR), or fenhexamid (FEN) that inhibited the growth of four isolates sensitive to these fungicides by 50% (EC50) were 12.4, 2.5, 0.61, 0.29/0.57, 0.26, or 0.17 mg liter-1, respectively. THM, IPR, CYP, PS+BO, PYR, or FEN were applied to detached 'Thompson Seedless' berries at the equivalent of the maximum approved rates of 600, 500, 270, 59/116, 370, or 290 mg liter-1, respectively, except PS+BO, which were used at 54.2% of their current registered maximum rates. The berries were inoculated with B. cinerea 48 or 24 h before treatment or 24 or 48 h after treatment. Gray mold 2 weeks after treatment and storage at 15°C was lowest after FEN application, followed by PYR, CYP, IPR, PS+BO, and THM. In commercial vineyards, one application of FEN, PYR, CYP, or PS+BO, all at their current maximum approved rates, 2 weeks before harvest reduced postharvest gray mold by approximately 50%. When fungicides were applied repeatedly after berry set either in mixtures or alternated with fungicides of different mode of action classes, postharvest gray mold was reduced by about 50% using a commercial air-blast sprayer and by 70 to 87% using a hand-held sprayer that was directed into the clusters. The fungicide sensitivity of isolates collected in numerous vineyards indicated those with reduced sensitivity to all of the tested fungicides, except FEN, were common. The efficacy of preharvest fungicide regimes was not sufficient to replace postharvest sulfur dioxide fumigation.

Relación entre el diagnóstico clínico preoperatorio y el histopatológico posoperatorio, en pacientes con cáncer prostático localizado / Relationship between clinical preoperative diagnosis and postoperative histology in patients with localized prostate cancer

Introducción: Las variables relevantes preoperatorias con que cuenta el urólogo para una toma de decisión frente a un cáncer prostático localizado son: la edad, el tacto rectal, el antígeno prostático específico (APE) e informe histológico de la biopsia por punción con el Gleason. Además se pueden incluir otras variables como el volumen prostático, número de muestras de biopsias positivas, porcentaje de la muestra comprometida, etc. Nosotros quisimos evaluar el grado de concordancia entre el diagnóstico clínico-patológico preoperatorio con el hallazgo histológico, posoperatorio en pacientes prostatectomizados, debido a la implicancia pronóstica y en la toma de decisión que pudiese tener. Material y Método: Se estudiaron retrospectivamente 119 prostatectomías radicales entre marzo de 2004 y junio de 2009. Se consideraron: edad, tacto, antígeno prostático específico (APE) y score de Gleason. Se excluyeron pacientes con tratamiento antiandrogénico u hormonal neoadjuvante. Resultados: En el preoperatorio la mediana de edad fue de 66 años (61-68), de APE 7,35 ng/ml (5,38-11,8) y de Gleason fue de 6 (5-7). El 87,4 por ciento de los pacientes tenía un APE >4,0 ng/ml. El 54 por ciento (n= 64) tenía un estadio clínico T1c y el 46 por ciento (n= 55) un estadio T2. En el posoperatorio 23,5 por ciento (n= 28) tuvo un estadio pT2 y el 74 por ciento (n= 88) un estadio pT3. En pacientes con estadio pT2 el APE preoperatorio fue de 5,9 ng/ml (4,4-9,4), en el estadio pT3 fue de 7,9 ng/ml (5,7-12,8). El score de Gleason en pT2 fue de 5 (5-6), en el pT3 fue de 6 (5-7). No encontramos diferencia de edad en los estadios pT2 (67 años) y pT3 (68 años). Conclusiones: En el estudio histopatológico posoperatorio de pacientes con estadio clínico T1c y T2, se confirmó un estadio pT2 sólo en 23,5 por ciento, el 74 por ciento tenían un estadio pT3 (a, b). En el cáncer prostático localizado, el tacto rectal no fue útil en su correlación con el estadio histológico...

Introduction: Relevant preoperative variables in patients with localized prostate cancer are: age, digital rectal examination (DRE), prostatic specific antigen (PSA) level and Gleason score in the transrectal biopsy. Other variables include prostate volume, number of positive biopsy samples, percentage of involvement in the biopsy, etc. We evaluated the agreement between the preoperative clinico pathologic diagnosis and the postoperative histology report in patients submitted to prostatectomy. Material and method: This is a retrospective review of 119 radical prostatectomies performed between March 2004 and June 2009. We recorded age, DRE, PSA level, and Gleason score. Patients receiving anti-androgenic treatment or neoadjuvant hormonal treatment were excluded. Results: Preoperative findings: median age was 66 years (61-68), median PSA level was 7.35 ng/ml(5.38-11.8) and median Gleason score was 6 (5-7). PSA level >4 ng/ml was found in 87.4 percent of the patients. Clinical stage T1c was found in 54 percent (n=64) of the cases whereas 46 percent (n=55) were stage T2. Postoperative findings: stage pT2 was found in 23.5 percent (n=28) of the patients whereas 74 percent (n =88)were pT3 stage. In pT2 patients, preoperative PSA was 5,9 ng/ml (4.4-9.4). In pT3 patients, PSA was7.9 ng/ml (5.7-12.8). Gleason score in pT2 was 5 (5-6); in pT3 patients, Gleason score was 6 (5-7). No age difference was found between pT2 stage (67 years) and pT3 stage (68 years).Conclusions: Postoperative histology in patients with T1c and T2 stages confirmed a pT2 stage only in 23.5 percent of the cases; 74 percent of the cases were pT3 (a,b) stage. In localized prostate cancer, DRE was not useful for the correlation with pathologic staging, especially for stage pT3 cases. Preoperative Gleason score was relatively useful; we found understaging 36.2 percent of the cases and overstaging 21.8 percent of the patients. These variables should be considered in the initial evaluation of...

Involvement of Akt, Ras and cell cycle regulators in the potential development of endometrial hyperplasia in women with polycystic ovarian syndrome.

OBJECTIVE: To examine whether the abundance, localization, and/or activity of cell cycle regulators CDK2, Cyclin E, p27, and survival proteins AKT and Ras in PCOS-associated endometria (with and without hyperplasia) differ from non-PCOS endometria. METHODS: The expression of CDK2, Cyclin E, p27, AKT and Ras was measured by immunohistochemistry and/or Western blot in 9 normal endometria (NE), 12 endometria from PCOS patients without endometrial hyperplasia (PCOSE), 7 endometria from PCOS women with endometrial hyperplasia (HPCOSE), and 9 endometria from patients with endometrial hyperplasia (HE). The activity of CDK2 was assessed by an in vitro kinase assay. RESULTS: CDK2, Cyclin E and p27 proteins were expressed mainly in the endometrial epithelial cells of the studied groups. No change in the activity of CDK2 was observed in total extracts obtained from the tissue samples. However, the nuclear expression of CDK2 in epithelial cells was slightly elevated in PCOSE and significantly increased in HPCOSE when compared to NE. Higher expression of p27 was detected in the cytoplasm of epithelial cells of PCOSE and HPCOSE when compared to NE. Also, we found an increment in Ser473-AKT phosphorylation and an over-expression of the Ras oncogene in endometria of patients with PCOS. CONCLUSION: The PCOS condition is associated with increased Ser473-AKT phosphorylation, elevated expression of Ras, increased cytoplasmic abundance of p27, and increased nuclear abundance of CDK2 in the endometrial epithelial cells. These biological events could potentially provide a chance for endometrial cells from PCOS patients to exit the controlled cell cycle and become hyperplastic at a later stage.

In situ estrogen metabolism in proliferative endometria from untreated women with polycystic ovarian syndrome with and without endometrial hyperplasia.

The aim of the present investigation was to study whether the endocrinological status of women bearing polycystic ovarian syndrome (PCOS) affects the endometrial in situ steroid metabolism. For this purpose, we evaluated the mRNA levels (RT-PCR), and the activity of steroid metabolic enzymes: P450 aromatase, steroid sulfatase (STS), estrogen sulfotransferase (EST) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) in 23 samples of normal endometria (CE), 18 PCOS endometria without treatment (PCOSE), 10 specimens from PCOS women with endometrial hyperplasia (HPCOSE), and 7 endometria from patients with endometrial hyperplasia not associated to PCOS (EH). The data showed lower levels of STS mRNA for PCOSE and HPCOSE (p<0.05, p<0.01, respectively) and of EST for HPCOSE and EH compared to control (p<0.05). However, higher levels for EST mRNA were obtained in PCOSE (p<0.05) versus CE. The mRNA and protein levels for P450 aromatase were undetectable in all analyzed endometria. The relationship between the activities of STS and EST was lower in PCOSE and HPCOSE (p<0.05) versus CE. The ratio between the mRNA from 17beta-HSD type 1/type 2 was higher in PCOSE (p<0.05), whereas, a diminution in the 17beta-HSD type 2 activity was observed in PCOSE (p<0.05). These results indicate that the activity of enzymes related to the steroid metabolism in analyzed PCOSE differ from those found in the CE. Consequently, PCOSE may present an in situ deregulation of the steroid metabolism.

Deregulation of tissue homeostasis in endometria from patients with polycystic ovarian syndrome with and without endometrial hyperplasia.

OBJECTIVE: To study the proteins involved in endometrial homeostasis in PCOS women. METHODS: Protein expression of Ki67, Bcl-2, Bax, Pro-Caspase-3 and Caspase-3 by immunohistochemistry and/or Western blot, and DNA fragmentation using in situ 3'-end labeling of apoptotic cells, was measured in 9 samples of normal endometrium (NE), 12 PCOS endometria without treatment (PCOSE), 7 endometria from PCOS women with endometrial hyperplasia (HPCOSE) and 9 endometria from patients with endometrial hyperplasia (HE). RESULTS: Cell proliferation was higher in epithelium from PCOSE (P<0.05), HPCOSE and HE vs NE. A higher Bcl-2/Bax relative ratio in PCOSE and HPCOSE was observed, in absence of active Caspase-3 and scarce DNA fragmentation in the four groups of endometria studied. CONCLUSION: As the apoptosis was scarce in all of the groups studied, endometrial homeostasis deregulation in PCOS could be a result of increased proliferation. Therefore, the onset of endometrial hyperplasia in PCOS endometrium could be linked to inadequate cell proliferation, and concomitantly to inadequate cell survival.

Expression of molecules associated with tissue homeostasis in secretory endometria from untreated women with polycystic ovary syndrome.

BACKGROUND: The hormonal alterations observed in women with polycystic ovary syndrome (PCOS) may promote implantation failure as well as disruption of their endometrial homeostasis. To evaluate cell survival of mid-secretory endometrium from untreated women with PCOS, we measured the expression of apoptosis and proliferation-related proteins. METHODS: A case-control study of 11 patients with PCOS and 11 fertile women in the Hospital Research Unit was performed. Endometrial samples were obtained from PCOS women (PCOSE) and fertile healthy women (CE) during the mid-secretory phase of the menstrual cycle. Protein expressions for Akt, p-AktSer473 and p-AktThr308, Bad, p-BadSer136, Bcl-2, Bax and pro-caspase-3/caspase-3, were assessed by western blot, and Ki67 and p-histone-3 (p-H3) by immunohistochemistry. RESULTS: In CE and PCOSE, a predominance of p-AktThr308 over p-AktSer473 is observed; p-BadSer136 expression is higher in PCOSE than in CE (P < 0.05). Also, Bcl-2 protein is overexpressed in PCOSE (P < 0.05), with no changes in Bax expression among the two groups, resulting in a significantly higher Bcl-2/Bax ratio in PCOSE than in CE (P < 0.05). No changes in the expression of caspase-3 are obtained between both groups of endometria. Furthermore, cell proliferation detected by the expression of Ki67 and p-H3 proteins is higher in the epithelia than the stroma of PCOSE versus CE (P < 0.05). CONCLUSION: The abnormal tissue homeostasis exhibited by the secretory endometrium from PCOS patients with spontaneous ovulation may interfere with their endometrial receptivity.

Androgen and estrogen receptors and co-regulators levels in endometria from patients with polycystic ovarian syndrome with and without endometrial hyperplasia.

OBJECTIVE: To study if the endocrinological status of PCOS women affects the endometrial sensitivity to steroids by evaluating the expression of androgen receptor (AR), estrogen receptor alpha (ERalpha), estrogen receptor beta (ERbeta), co-activators AIB1 and ARA70, and co-repressor NCoR. METHODS: Gene and/or protein expression of steroid receptors and co-regulators was measured in 17 samples of normal endometrium (NE), 23 PCOS endometrium without treatment (PCOSE), 11 endometria from PCOS women and with endometrial hyperplasia (HPCOSE), and 10 endometria from patients with endometrial hyperplasia (HE), using RT-PCR and/or immunohistochemistry and Western blot. RESULTS: Gene and protein expression of AR was relatively elevated in PCOSE and HPCOSE compared with NE. A significant increase in ERalpha protein expression was observed in PCOSE, preferentially in the nucleus of endometrial cells, whereas ERbeta gene and protein expression increased gradually from PCOSE to HPCOSE and HE, mainly in the epithelial compartment. Importantly, we found a gradual increase in the ERbeta/ERalpha gene and protein expression ratio in endometria from the four groups of women. AIB1 showed increased nuclear protein expression in PCOSE compared to NE, in the presence of a high expression of ARA70 in all groups. High expression of ARA70 together with a normal expression level of AIB1 was observed in HPCOSE. The cytoplasmic immunostaining of NCoR was similar between the four groups of patients. CONCLUSION: The PCOS endometrium exhibits a higher sensitivity to steroid action. We can inferred that these alterations could deregulate the transcription of genes involved in the cell cycle, which may lead to the development of endometrial hyperplasia in PCOS women.