Chorionic Gonadotropin β-Subunit and Core Fragment in Bladder Cancer: mRNA and Protein Expression in Urine, Serum and Tissue

Objectives: Many transitional cell carcinomas (TCC) of the bladder express the β-subunit (CGβ) of chorionic gonadotropin (CG), and elevated serum levels occur especially in advanced disease. We have compared the diagnostic utility of various methods for detecting CG and CGβ expression at the protein and mRNA level.Methods: We used RT-PCR to detect CGβ mRNA in urinary cells and highly sensitive immunoassays to determine CG and CGβ in serum and the core fragment of CGβ (CGβcf) in urine from patients under follow-up for bladder cancer. Tissue expression was studied by immunohistochemistry.Results: CGβ mRNA was detected in urinary cells in 50% (n=84) of the cancer cases and in none of the healthy controls (n=15). Positive staining for CGβ in tissue samples was observed not only in 30% (n=96) of the TCC cases, but also in 5 of 20 histologically benign samples from TCC patients, and in 10 of 21 samples from benign bladder diseases. Serum and urinary concentrations of CGβ were elevated in 29% (n=66) and 8% (n=72), respectively, while serum CG was elevated in 18% of the TCC patients. Urinary CGβcf concentrations were higher in invasive (T1–T4) than superficial (T in situ and Ta) tumors (p=0.037), in cases positive for CGβ mRNA (p=0.03) and cases with suspicious or malignant urinary cytology (p=0.002). The ratio of urinary to serum concentration of CGβ showed the strongest correlation with tumor stage (p<0.00001), grade (p<0.00001), and staining for CGβ (p=0.019).Conclusions: Although CGβ expression may occur in benign bladder epithelium, CGβ mRNA in urinary cells is a potential marker of bladder cancer. Urinary and serum CGβ have low sensitivity in early disease, but the urine/serum ratio appears to indicate local release of CGβ into urine. Further studies are needed to evaluate the clinical usefulness of different forms of CGβ expression.     

Bilateral testicular germ-cell tumors – A single centre long-term experience

Objectives: The incidence of bilateral testiculartumors (BTT) had increased over the preceding decade. The aim of thepresent study is to analyse a group of patients with BTT and tohigh-light the need for long-term follow-up of patients treated in asingle centre. Material and methods: 27 (2.8%) out of960 patients with germ-cell testicular tumors (GCTT), treated between4/1977 and 8/2001, developed bilateral disease. All of themunderwent radical orchiectomy (in one patient was done delayedorchiectomy after primary chemotherapy due to advanced disease).Additional treatment was planned according to the histologic type andclinical stage of the disease, and previous treatment as well. Thesurvival data were reviewed. Results: 24 out of 27 patients(88.9%) developed the 2nd tumor metachronously (median interval66 months, range, 4–197 months) and three (11.1%) had synchronousBTT. Only 7 patients (25.9%) had identical histological types onboth sides (6 of them with pure seminomas, one with embryonalcarcinoma). Two of three synchronously developed BTT had differenthistologic types on both sides. GCTT of one histologic type wereobserved in respect of the first tumor: 11 seminomas, three embryonalcarcinomas, in respect of the 2nd tumor: 10 seminomas, three embryonalcarcinomas, in respect of the 2nd tumor: 10 seminomas, three embryonalcarcinomas and one mature teratoma. GCTT of more than one histologictype were observed in respect of the first and the 2nd tumors: 6 mixedGCTT with seminoma component and 7 without seminoma component. Majorityof BTT was presented in clinical stage I (in respect of the first tumorin 70.4%, in respect of the 2nd tumor in 62.9%). Themedian duration of the follow-up after the diagnosis of the first GCTTwas 149 months (range, 13–288 months) and after the diagnosis ofthe contralateral GCTT was 68 months (range, 1–167 months).Twenty-five patients (92.6%) were alive with NED at their lastfollow-up visit. Two patients died by mean of 22.5 months (range,21–24 months) after the 2nd orchiectomy. Conclusions: Allpatients with unilateral GCTT have an increased risk of developing acontralateral testicular tumor, even decades after diagnosis. Managementshould be individualised for each patient.     

超声鉴别诊断不同类型的睾丸恶性肿瘤

目的探讨超声鉴别诊断不同类型睾丸恶性肿瘤的价值。方法回顾性分析超声和术后病理证实为睾丸恶性肿瘤患者的资料。结果90例睾丸恶性肿瘤患者中,精原细胞瘤（50例）多表现为患侧睾丸增大,内见不均匀低回声;非精原细胞性生殖细胞肿瘤（胚胎癌5例、卵黄囊瘤2例、畸胎瘤2例、混合型生殖细胞肿瘤8例）多有程度不同液化及粗大钙化;非生殖细胞肿瘤（共9例）呈形态规则、边界清晰的低回声;而淋巴瘤10例、横纹肌肉瘤2例、类癌2例未见明显特征性表现。结论不同类型睾丸恶性肿瘤的超声表现各有特点。结合患者年龄、血清肿瘤学检查对临床判断睾丸恶性肿瘤类型具有一定意义。     

Reduced prostate branching morphogenesis in stromal fibroblast,but not in epithelial,estrogen receptor α knockout mice

Early studies suggested that estrogen receptor alpha (ERα) is involved in estrogen-mediated imprinting effects in prostate development. We recently reported a more complete ERα knockout (KO) mouse model via mating β-actin Cre transgenic mice with floxed ERα mice. These ACTB-ERαKO male mice showed defects in prostatic branching morphogenesis, which demonstrates that ERα is necessary to maintain proliferative events in the prostate. However, within which prostate cell type ERα exerts those important functions remains to be elucidated. To address this, we have bred floxed ERα mice with either fibroblast-specific protein (FSP)-Cre or probasin-Cre transgenic mice to generate a mouse model that has deleted ERα gene in either stromal fibroblast (FSP-ERαKO) or epithelial (pes-ERαKO) prostate cells. We found that circulating testosterone and fertility were not altered in FSP-ERαKO and pes-ERαKO male mice. Prostates of FSP-ERαKO mice have less branching morphogenesis compared to that of wild-type littermates. Further analyses indicated that loss of stromal ERα leads to increased stromal apoptosis, reduced expression of insulin-like growth factor-1 (IGF-1) and FGF10, and increased expression of BMP4. Collectively, we have established the first in vivo prostate stromal and epithelial selective ERαKO mouse models and the results from these mice indicated that stromal fibroblast ERα plays important roles in prostatic branching morphogenesis via a paracrine fashion. Selective deletion of the ERα gene in mouse prostate epithelial cells by probasin-Cre does not affect the regular prostate development and homeostasis.     

Growth, Differentiation and Senescence of Normal Human Urothelium in an Organ-Like Culture

Objective: To examine the kinetics of growth, differentiation and senescence of normal human urothelium in an organoid-like culture model.Materials and Methods: Micro-dissected normal human urothelium explants were grown on porous membranes pretreated with various matrix components. Between 5 and 30 days of culture, cell proliferation was assessed by BrdU incorporation. Differentiation was evaluated on the basis of cytokeratin (Ck) and uroplakin (UP) expression. Epidermal growth factor family mRNA expression was monitored during explant outgrowth. Senescence was assessed by measuring endogenous β-galactosidase activity and p16^INK4a mRNA expression.Results: Collagen IV was the most efficient matrix component for urothelial cell expansion. BrdU incorporation by urothelial cells was 5% between 15 and 30 days, corresponding to steady-state urothelium in vivo. Heparin-binding EGF (HB-EGF), Amphiregulin (AR) and Transforming Growth Factor α (TGFα) expression correlated with increased cell proliferation. UPII expression was stable throughout culture. P16^INK4a mRNA expression and β-galactosidase activity increased on day 25, giving signs of senescence.Conclusions: This model retains many characteristics of the urothelium in vivo. It can be used for pharmacological studies between 15 to 25 days and to study mechanisms such as wound healing, proliferation and senescence.     

Antidiabetic Actions of an Estrogen Receptor β Selective Agonist

The estrogen receptor β (ERβ) is emerging as an important player in the physiology of the endocrine pancreas. We evaluated the role and antidiabetic actions of the ERβ selective agonist WAY200070 as an insulinotropic molecule. We demonstrate that WAY200070 enhances glucose-stimulated insulin secretion both in mouse and human islets. In vivo experiments showed that a single administration of WAY200070 leads to an increase in plasma insulin levels with a concomitant improved response to a glucose load. Two-week treatment administration increased glucose-induced insulin release and pancreatic β-cell mass and improved glucose and insulin sensitivity. In addition, streptozotocin-nicotinamide–induced diabetic mice treated with WAY200070 exhibited a significant improvement in plasma insulin levels and glucose tolerance as well as a regeneration of pancreatic β-cell mass. Studies performed in db/db mice demonstrated that this compound restored first-phase insulin secretion and enhanced pancreatic β-cell mass. We conclude that ERβ agonists should be considered as new targets for the treatment of diabetes.Diabetes has become one of the most challenging health problems on a global scale, with an estimated 285 million people affected by this disease in 2010 (1,2). The most common form of diabetes is type 2 (T2D), which results from the interaction of a subject’s genetic background with the environment. Both insulin resistance and pancreatic β-cell dysfunction contribute importantly to the pathogenesis of this disease; however, T2D develops only when insulin secretion cannot meet the insulin demand (3–5). Therefore, the most effective therapy for T2D should control not only β-cell failure, but also the loss of β-cell mass. Today, although there is an extensive range of oral antidiabetic drugs that differ in their modes of action, none seem to be completely effective (6–9).Estrogen receptors are emerging as important molecules involved in modulating pancreatic β-cell function. 17β-estradiol (E2) modulates insulin content in an estrogen receptor α (ERα)-dependent manner (10). In addition, the activation of the estrogen receptor β (ERβ) triggers the closure of ATP-sensitive K⁺ (K_ATP) channels, enhancing glucose-induced [Ca²⁺] oscillations and insulin release cooperatively with glucose (11). Selective ERβ agonists, such as diarylpropionitrile (DPN), elicit this rapid phenomenon (1–7 min). The K_ATP channel–dependent pathway in the pancreatic β-cell is the major trigger for glucose-stimulated insulin secretion (GSIS). Accordingly, the fact that ERβ selective ligands can activate this mechanism raises the possibility that these compounds may behave as rapid insulinotropic agents and, thus, lead to new antidiabetic drugs.Since the discovery of ERβ in the mid-1990s, intense research efforts continue to focus on the biology of this receptor and on developing and evaluating the use of ERβ-specific agonists in animal models of human disease. Remarkably, some of the ERβ agonists are already under evaluation in clinical studies (12–14). At present, ERβ is a promising novel drug target for the treatment of cancer and multiple sclerosis because of distinct functional characteristics of this estrogen receptor subtype.Here, we evaluate the action of a selective ERβ agonist (WAY200070) on glucose homeostasis in different animal models of diabetes. We analyze the capacity of this compound to normalize fasting glucose levels, to enhance endogenous insulin secretion, and to regulate β-cell mass. We hypothesize that the use of selective ERβ agonists offers great hope in the treatment of T2D.     

Elevated plasma thymosin-α1 levels in lung cancer patients

Objective: Prothymosin-α, the precursor of thymosin-α1, may play a role in cell proliferation, and the plasma level of thymosin-α1 may reflect the degree of proliferation of the tumor cells. Methods: Recently, a new sandwich immunoradiometric assay for thymosin-α1 was developed using monoclonal and polyclonal antibodies. In this investigation, we used this assay to measure plasma and tissue level of thymosin-α1 in 131 lung cancer patients. Results: We found that the mean plasma thymosin-α1 levels in lung cancer patients were higher than in normal individuals (P<0.001). However, half of the patients showed normal levels. Thymosin-α1 levels correlated neither with the stage nor pathological subtype of the lung cancer, and did not decrease significantly in the 4 weeks after the resection of the tumor. Thymosin-α1 levels of lung cancer patients with another cancer were higher than those without evidence of other cancers (P=0.03). Survival of patients with normal levels of plasma thymosin-α1 was significantly better than that with higher levels (P=0.04). Conclusions: The plasma level of thymosin-α1 may be used as a marker for the prognosis of lung cancer patients. Further investigations are warranted to determine its role in the lung cancer.     

The equilibrium of XIAP and Smac/DIABLO expression is gradually deranged during the development and progression of testicular germ cell tumours

Overexpression of the inhibitor of apoptosis protein (IAP) XIAP (BIRC4) and downregulation of its antagonist Smac/DIABLO (DIABLO) are associated with the onset and progression of various malignancies. In this study, real-time RT-PCR was used to quantify the mRNA expression of XIAP and Smac/DIABLO in normal testicular tissue (n = 19), testicular carcinoma in situ (CIS; n = 4), testicular seminomas (n = 64) and non-seminomatous germ cell tumours (NSGCT; n = 35). XIAP and Smac/DIABLO were commonly expressed in normal and malignant testicular tissue with no apparent differences in XIAP mRNA levels among the histologic subgroups. Smac/DIABLO levels, on the other hand, gradually decreased from normal testicular tissue to CIS and seminomas and finally to NSGCT (p < 0.001). An inverse trend was observed when calculating the XIAP-to-Smac/DIABLO ratio (p < 0.001). This ratio differed when comparing normal testicular tissue with CIS (p = 0.014), seminomas (p < 0.001) and NSGCT (p < 0.001) and when comparing seminomas with NSGCT (p = 0.002), whereas no such difference was observed between CIS and seminomas (p = 0.302). TGCT patients dichotomized by the overall median XIAP-to-Smac/DIABLO ratio were more likely to present with a high ratio in clinical stage (CS) III than in CS I or II (p = 0.034). These data indicate that the balance of mRNA expression between XIAP and Smac/DIABLO is altered in favour of antiapoptotic XIAP during the development and progression of TGCT. Thus the expression of proapoptotic Smac/DIABLO is lowest in NSGCT and stage III tumours.     

Retroperitoneal lymph node dissection for testicular seminomas: population-based practice and survival outcomes

Purpose

While retroperitoneal lymph node dissection (RPLND) is traditionally reserved for nonseminomatous germ cell tumors, recent efforts to reduce long-term toxicities of radiation and chemotherapy have turned attention to its application for testicular seminomas. Currently, RPLND is reserved for the post-chemotherapy for stage II testicular seminomas; we aimed to describe current utilization of RPNLD for testicular seminomas by stage and implications for survival.

Methods

A national sample of men diagnosed with stage IA/IB/IS/IIA/IIB/IIC testicular seminoma (1988–2013) was evaluated from SEER Program registries. Stage-specific utilization of RPLND was determined. Cox proportional hazards models, adjusted for age, race, and radiotherapy, evaluated overall (OS) and cancer-specific survival (CSS) for the RPLND cohort. Adjusted models assessed predictors of RPLND.

Results

A total of 17,681 men (mean age 38.1 years) with testicular seminoma were included with low utilization of RPLND for stage I disease (1.3% overall) and higher rates for stage II disease (10.6% overall). There were no appreciable trends over time. Patients receiving RPLND did not appear to have worse OS or CSS on adjusted stage-by-stage analysis. Higher stage disease (IIA-IIC) was associated with greater need for RPLND while radiotherapy was associated with decreased use [OR 0.40 (0.32–0.51), p < 0.001].

Conclusions

Utilization of RPLND for testicular seminomas in the post-chemotherapy setting has remained stable over a 25-year period. Patients undergoing RPLND are a higher risk cohort but stage-by-stage survival outcomes appeared comparable to men not undergoing RPLND. Upcoming trials implementing RPLND as a first-line modality for testicular seminoma or isolated retroperitoneal relapse will help better quantify relative recurrence and survival.

     

Androgen and estrogen receptor expression in the spinal segments of the genitofemoral nerve during testicular descent

Aim

During testicular descent (TD), the genitofemoral nerve (GFN) is masculinized by androgen. This study aimed to test whether androgen receptor (AR), estrogen receptorα (ERA), or estrogen receptor β (ERB) are expressed during TD in the GFN spinal segments and dorsal root ganglia (DRG) in normal and flutamide-treated rats.

Methods

Time-mated Sprague-Dawley dams were injected with flutamide (75 mg/kg, subcutaneously (S/C) in sunflower oil) on embryonic (E) days 16 to 19. Embryonic and postnatal (P) male L1-2 spinal cord segments were collected (E16, E17, E19, P0, P2, and P4) in control and flutamide-treated groups (n = 5-10). Samples were fixed in 4% paraformaldehyde. Five-micrometer-thick sections were prepared immunohistochemically for AR, ERA, and ERB.

Results

During TD, ERB was expressed in L1-2 DRG. Surprisingly, AR was not expressed in prenatal DRG, only after P2. There was no ERA expression. Flutamide had no effect on AR, ERB, or ERA expression in the L1-2 DRG during TD.

Conclusion

During the E window of androgen sensitivity, the GFN is not directly masculinized, with little AR expression and no change with flutamide over this period. Estrogen receptor β is expressed in the DRG during TD. However, its relevance is yet to be determined.     

Association of estrogen receptor α gene microsatellite polymorphism with annual changes in bone mineral density in Korean women with hormone replacement therapy

Variation in drug response to hormone replacement therapy (HRT) may reflect genetic heterogeneity in the estrogen-related genes, possibly including estrogen receptor alpha (ERα) gene. However, only a few association studies of the drug response to HRT have been reported, focusing mainly on the intronic polymorphisms of the ERα gene. We therefore examined 284 postmenopausal women (mean age, 52.2 ± 5.0 years) for the microsatellite thymine–adenine (TA) repeat polymorphism in the promoter of the ERα gene and its relationship to drug response by measuring changes in bone mineral density (BMD) after 1 year of HRT. In our study population, the most common number of TA repeats was 14, with a range of values between 11 and 27. At baseline, the number of TA repeats was neither associated with measured lumbar spine or femoral neck BMD nor with bone markers. When we categorized the subjects by the TA repeat numbers into an L group (n = 142), with a low mean number of repeats (TA < 16), and an H group (n = 142), with a high mean number of repeats (TA ≥ 16), no significant genotypic differences were noted in spinal or femoral neck BMD or in bone markers. However, the drug response on lumbar spine BMD after 1 year of HRT correlated with the mean number of TA repeats (r = −0.131, P = 0.035) after adjustment for confounding factors such as body mass index and years since menopause. This correlation was also seen with the number of TA repeats on the shorter allele (r = −0.159, P = 0.012), which was defined as the allele with the lower number of TA repeats. However, this genotypic association was not found in the femoral neck BMD (r = 0.053, P = 0.396). When we defined the nonresponder group as women who had lost BMD even with HRT, 15.9% of the subjects were included, and this group was significantly younger and had higher initial BMD than the responder group. After further adjustment for age and initial BMD, the number of TA repeats on the shorter allele remained significantly associated with drug responsiveness (P = 0.005). These data indicate significant effects of the ERα TA repeat polymorphism on the estrogen responsiveness of lumbar spine BMD after 1 year of HRT in Korean women.     

Evaluation of the Clinical Benefit of Permixon and Tamsulosin in Severe BPH Patients—PERMAL Study Subset Analysis

Objective: To compare the efficacy of the lipido-sterolic extract of Serenoa repens, Permixon, to that of the α–blocker, tamsulosin, in the treatment of severe low urinary tract symptoms (LUTS) of benign prostatic hyperplasia (BPH).Methods: In a 12-month, double-blind, randomized study that showed equivalent efficacy of Permixon 320 mg/day and tamsulosin 0.4 mg/day (“PERMAL study”), 685 BPH patients with IPSS ≥10 had been analyzed for efficacy. Of these, the 124 patients with severe LUTS (IPSS >19) at randomization were retained for this subset analysis. After a 4-week run-in period, 59 and 65 patients had been randomized to tamsulosin and Permixon groups, respectively. Both treatment groups were compared regarding the evolution from baseline of total IPSS and its irritative and obstructive subscores, LUTS-related QoL, prostate volume, Q_max and MSF-4 (sexual activity questionnaire) at different time points over 1 year. An analysis of variance of changes from baseline to end point was performed for all the parameters. The over-time evolutions of total, irritative and obstructive IPSS were further compared using a variance analysis for repeated measurements.Results: At 12 months, total IPSS decreased by 7.8 with Permixon and 5.8 with tamsulosin (p=0.051); the irritative symptoms improved significantly more (p=0.049) with Permixon (−2.9 versus −1.9 with tamsulosin). The superiority of Permixon in reducing irritative symptoms appeared as soon as month 3 and was maintained up to month 12 (p=0.03).Conclusion: Permixon 320 mg/day was shown to be slightly superior to tamsulosin 0.4 mg/day in reducing LUTS in severe BPH patients after 3 months and up to 12 months of treatment.     

Facteurs pronostiques du myélome utilisables en pratique courante : suivi sur dix ans de 148 malades âgés de plus de 55 ans

Objective. – The objective of this study was to identify prognostic factors in a uniform population of older patients with myeloma.Methods. – Thirty-one study centers in France included 148 patients who were older than 55 years at diagnosis and were followed up until death or for at least ten years. The following tests were available for all patients: blood cell counts; serum and urinary protein electrophoresis; and serum levels of creatinine, calcium, β2-microglobulin (β2-m), lactic dehydrogenase (LDH), and C-reactive protein (CRP).Results. – Mean age was 71.9 years, median survival was 34 months, and mean survival was 47 months. In the univariate analysis, factors significantly associated with higher mortality were male gender (odds ratio [OR], 1–2.12), age older than 70 years (OR, 1.10–2.28), serum albumin lower than 30 g/l (OR, 1.16–3.28), serum creatinine greater than 100 μmol/l (OR, 1.34–2.81), β2-m greater than 6 mg/l (OR, 1.78–4), CRP greater than 6 mg/l (OR, 1.44–3.06), hemoglobin lower than 10 g/dl (OR, 1.8–2.23). In the multivariate analysis, only two factors significantly predicted a higher risk of death: β2-m greater than 6 mg/l (OR =2.439 [1.59–3.76]) and CRP greater than 6 mg/l (OR =1.76 [1.18–2.63]). β2-m level was greater than 6 mg/l in 41 (27.7%) patients and CRP was greater than 6 mg/l in 61 (43.6%) patients. Other potential prognostic factors such as chromosome 13 deletion were not investigated because they were not available for all study patients.Conclusions. – The strength of this study is the ten-year follow-up in a uniform patient cohort. β2-m and CRP independently predicted the risk of death.