A novel grading approach predicts worse outcomes in stage pT1 non-muscle-invasive bladder cancer.

OBJECTIVE: To develop a prognostically relevant scoring system for stage pT1 non-muscle-invasive bladder cancer (NMIBC) incorporating tumour budding, growth pattern and invasion pattern because the World Health Organisation grading system shows limited prognostic value in such patients. PATIENTS AND METHODS: The tissue specimens and clinical data of 113 patients with stage pT1 NMIBC who underwent transurethral resection of bladder tumour were retrospectively investigated. Tumour budding, and growth and invasion patterns were evaluated and categorised into two grade groups (GGs). GGs and other clinical and histopathological variables were investigated regarding recurrence-free survival (RFS), progression-free survival (PFS), cancer-specific survival (CSS) and overall survival (OS) using univariable and multivariable Cox regression analyses. RESULTS: The integration of two tumour budding groups, two growth patterns, and two invasion patterns yielded an unfavourable GG (n = 28; 24.7%) that had a high impact on oncological outcomes. The unfavourable GG was identified as an independent RFS and OS predictor (P = 0.004 and P = 0.046, respectively) and linked to worse PFS (P = 0.001) and CSS (P = 0.001), irrespective of the European Association of Urology risk group. The unfavourable GG was associated with higher rates of BCG-unresponsive tumours (P = 0.006). Study limitations include the retrospective, single-centre design, diverse therapies and small cohort. CONCLUSIONS: We present a morphology-based grading system for stage pT1 NMIBC that correlates with disease aggressiveness and oncological patient outcomes. It therefore identifies a highest risk group of stage pT1 NMIBC patients, who should be followed up more intensively or receive immediate radical cystectomy. The grading incorporates objective variables assessable on haematoxylin and eosin slides and immunohistochemistry, enabling an easy-to-use low-cost approach that is applicable in daily routine. Further studies are needed to validate and confirm these results.

Innervation pattern of the unclosed detrusor muscle in classic bladder exstrophy: a study of patients with urothelial overexpression of nerve growth factor.

PURPOSE: An overexpression of nerve growth factor (NGF) in the urothelium is discussed to lead to neuronal hyperinnervation of the bladder detrusor. The aim was to assess the sensory and sympathetic innervation of the detrusor in unclosed exstrophic bladders patients with known overexpression of NGF in the urothelium. METHODS: Full-thickness bladder biopsies were prospectively obtained from 34 infants at delayed primary bladder closure between 01/2015 and 04/2020. The bladder biopsies were immunohistochemically stained with antibodies against S100, calcitonin gene-related peptide (anti-CGRP), Neurofilament 200 (anti-NF200), and tyrosine-hydroxylase (anti-TH). Specimens from 6 children with congenital vesicoureterorenal reflux (VUR) served as controls. RESULTS: There was no statistically significant difference in nerve fiber density in any of the immunohistochemical assessments (anti-S100 [p = 0.210], anti-CGRP [p = 0.897], anti-NF200 [p = 0.897]), and anti-TH [p = 0.956]) between patients with BE and patients with VUR. However, we observed a trend toward lower nerve fiber densities in exstrophic detrusor. CONCLUSION: Overall our results showed an unharmed innervation pattern in this cohort but a lower density of nerve fibers in the detrusor compared to controls. Further studies in patients after successful primary closure are needed to clarify the potential impact of the urothelial overexpression of NGF modulating the innervation pattern in exstrophic bladders.

Prognostic impact of variant histologies in urothelial bladder cancer treated with radical cystectomy.

OBJECTIVES: To evaluate variant histologies (VHs) for disease-specific survival (DSS) in patients with invasive urothelial bladder cancer (BCa) undergoing radical cystectomy (RC). MATERIALS AND METHODS: We analysed a multi-institutional cohort of 1082 patients treated with upfront RC for cT1-4aN0M0 urothelial BCa at eight centres. Univariable and multivariable Cox' regression analyses were used to assess the effect of different VHs on DSS in overall cohort and three stage-based analyses. The stages were defined as 'organ-confined' (≤pT2N0), 'locally advanced' (pT3-4N0) and 'node-positive' (pTanyN1-3). RESULTS: Overall, 784 patients (72.5%) had pure urothelial carcinoma (UC), while the remaining 298 (27.5%) harboured a VH. Squamous differentiation was the most common VH, observed in 166 patients (15.3%), followed by micropapillary (40 patients [3.7%]), sarcomatoid (29 patients [2.7%]), glandular (18 patients [1.7%]), lymphoepithelioma-like (14 patients [1.3%]), small-cell (13 patients [1.2%]), clear-cell (eight patients [0.7%]), nested (seven patients [0.6%]) and plasmacytoid VH (three patients [0.3%]). The median follow-up was 2.3 years. Overall, 534 (49.4%) disease-related deaths occurred. In uni- and multivariable analyses, plasmacytoid and small-cell VHs were associated with worse DSS in the overall cohort (both P = 0.04). In univariable analyses, sarcomatoid VH was significantly associated with worse DSS, while lymphoepithelioma-like VH had favourable DSS compared to pure UC. Clear-cell (P = 0.015) and small-cell (P = 0.011) VH were associated with worse DSS in the organ-confined and node-positive cohorts, respectively. CONCLUSIONS: More than 25% of patients harboured a VH at time of RC. Compared to pure UC, clear-cell, plasmacytoid, small-cell and sarcomatoid VHs were associated with worse DSS, while lymphoepithelioma-like VH was characterized by a DSS benefit. Accurate pathological diagnosis of VHs may ensure tailored counselling to identify patients who require more intensive management.

Evaluation of Gas 6 as a Prognostic Marker in Papillary Renal Cell Carcinoma.

INTRODUCTION: Growth arrest-specific protein 6 (Gas 6) is a ligand that plays a role in proliferation and migration of cells. For several tumor entities, high levels of Gas 6 are associated with poorer survival. We examined the prognostic role of Gas 6 in renal cell carcinoma (RCC), especially in papillary RCC (pRCC), which is still unclear. METHODS: The patients' sample collection is a joint collaboration of the PANZAR consortium. Patients' medical history and tumor specimens were collected from n = 240 and n = 128 patients with type 1 and 2 pRCC, respectively. Expression of Gas 6 was determined by immunohistochemistry. RESULTS: In total, Gas 6 staining was evaluable in 180 of 240 type 1 and 110 of 128 type 2 pRCC cases. Kaplan-Meier analysis disclosed no significant difference in 5-year overall survival for all pRCC nor either subtype. Also, Gas+ and Gas- groups did not significantly differ in any tumor or patient characteristics. CONCLUSION: Gas 6 was not found to be an independent prognostic marker in pRCC. Future studies are warranted to determine if Gas 6 plays a role as prognostic marker or therapeutic target in pRCC.

Geriatric Patients and Symptomatic Urinary Tract Infections: Analysis of Bacterial Range and Resistance Rates at a 3rd Level of Care Hospital in Germany.

BACKGROUND: Increasing life expectancy and demographic change result in a rising number of geriatric patients, but knowledge about geriatric treatment options and adapted diagnostic pathways is low. Simultaneously, urinary tract infections (UTIs) are common in older people and often difficult to treat. METHODS: Patients with a UTI at a urological outpatient department between January 01, 2016, and December 31, 2017, were retrospectively identified in the data system. One thousand one hundred fifteen patients with "N30" and "N39" diagnosis were included in this study, 965 of which were under the age of 75 (group 1) and 150 of which 75 years or older (group 2). RESULTS: Of 150 patients in group 2, 41.6% had nitrite-positive urine (vs. 20.9% in group 1, p < 0.05 in χ2 testing). Patients in group 2 often had indwelling devices (30.3 vs. 6.0%, p < 0.05) and presented a sterile urine culture in only 20.7% (vs. 40.1% in group 1). Regarding calculated antibiotic therapy, there were significant differences concerning prescription of ciprofloxacin (34.9 vs. 25%, p < 0.05) and fosfomycin (12.7 vs. 40.7%, p < 0.05). CONCLUSION: UTI in geriatric patients should be treated differently than in younger patients because antibiotic resistance is high. The group with indwelling devices is complex. Individual strategies for geriatric patients should be considered.

Impact of Male Patient Information on Quality of Urine Examination (PIQUE Study).

BACKGROUND: Urine examination has relevance for treatment, and reliability of positive urine culture (UC) is of importance. The technique of urine sampling (US), storage, and transportation is important. The objective of this study was to investigate if detailed patient information for the technique of US and hygiene reduces rates of contaminated UC in screened male patients, as this group was not investigated yet. METHODS: All patients independently of complaints were enrolled prospectively and consecutively in an outpatient setting in 2 groups - the first group did not receive detailed information and the second group did. We examined 372 consecutive patients in 2017, 190 not receiving (median age 69 years) and 182 receiving information (median age 70 years), with comparable numbers of patients and age. The result of UC and age was imposed. RESULTS: In all,74.2% of preclarification UC showed a contamination (n = 95) and 75.5% after clarification (n = 83), without significant differences (p = 0.827). This study is limited by the fact that adherence could not be checked. CONCLUSIONS: Similar to studies with females, no difference occurred in rates of contaminated UC, so detailed information regarding the US technique does not decrease rates of contaminated UC and vice versa does not increase the quality of midstream-sampled UC in male patients.

The Prognostic Impact of PD-L2 in Papillary Renal-Cell Carcinoma.

INTRODUCTION: Programmed death-1 ligand (PD-L1) has been often studied in different types of renal-cell carcinoma (RCC). For example, in clear-cell renal carcinoma it is well established that programmed death-1 receptor and PD-L1 are important prognostic markers. In contrast, the role of programmed death-2 ligand (PD-L2) as prognostic marker remains unclear. The aim of this study was to evaluate if PD-L2 expression could play a role as a prognostic marker for papillary RCC (pRCC). METHODS: The patients' sample collection was a joint collaboration of the PANZAR consortium. Patients' medical history and tumor specimens were collected from n = 240 and n = 128 patients with type 1 and 2 pRCC, respectively. Expression of PD-L2 was determined by immunohistochemistry. In total, PD-L2 staining was evaluable in 185 of 240 type 1 and 99 of 128 type 2 pRCC cases. RESULTS: PD-L2 staining was positive in 67 (36.2%) of type 1 and in 31 (31.3%) of type 2 pRCC specimens. The prevalence of PD-L2+ cells was significantly higher in high-grade type 1 tumors (p = 0.019) and in type 2 patients with metastasis (p = 0.002). Kaplan-Meier analysis disclosed significant differences in 5-year overall survival (OS) for patients with PD-L2- compared to PD-L2+ in pRCC type 1 of 88.4% compared to 73.6% (p = 0.039) and type 2 of 78.8% compared to 39.1% % (p < 0.001). However, multivariate analysis did not identify the presence of PD-L2+ cells neither in type 1 nor type 2 pRCC as an independent predictor of poor OS. DISCUSSION/CONCLUSION: PD-L2 expression did not qualify as an independent prognostic marker in pRCC. Future studies will have to determine whether anti-PD-L2-targeted treatment may play a role in pRCC and expression can potentially serve as a predictive marker for these therapeutic approaches.

High Stroma T-Cell Infiltration is Associated with Better Survival in Stage pT1 Bladder Cancer.

Stage pT1 bladder cancer (BC) shows highly diverse outcomes. Predictive markers are required to stratify patients for personalized treatment. The present study aimed to validate immune response quantification as a prognostic marker. Patients with pT1 BC (n = 167) treated by transurethral resection of the bladder (TURB) were enrolled. Formaldehyde-fixed paraffin-embedded material was stained for CD3 and CD8. Corresponding T cells were counted in three regions with the highest immune response. Numbers of tertiary lymphoid structures (TLS) and lymphocyte aggregates (LA) were quantified. High CD3+ stroma T-cell infiltration was associated with improved survival (p = 0.045), especially in the G3 subgroup (p = 0.01). Cluster with higher immune response showed less recurrence (p = 0.034) and favorable overall survival (OS) (p = 0.019). In contrast, higher CD3+ and CD8+ tumor T-cell infiltration seemed to have a negative impact on prognosis. TLS and LA were more frequently observed in G3 tumors, indicating an increased anti-tumoral immune response. We proved the role of immune cell infiltration and showed that higher infiltration numbers of CD3+ (not CD8+) lymphocytes in the stroma are associated with favorable outcome. Immune cell quantification could be used as a marker to help stratify patients' risk and therefore, to optimize patients' management and follow-up examination as well as possible therapies.

Copper in Wood Preservatives Delayed Wood Decomposition and Shifted Soil Fungal but Not Bacterial Community Composition.

Copper-based fungicides are routinely used for wood and plant protection, which can lead to an enrichment of copper-tolerant microbial communities in soil. To investigate the effect of such wood preservatives on the soil fungal and bacterial community compositions, five different vineyard and fruit-growing soil environments were evaluated using incubation studies over time. Pine sapwood specimens were impregnated with either water or different biocide treatment solutions containing a mixture of copper, triazoles, and quaternary ammonium compounds (CuTriQAC), a mixture of triazoles and quaternary ammonium compounds (TriQAC), or copper alone (Cu). Specimens were incubated in soil from each sample site for 8, 16, 24, and 32 weeks. The effects of preservative treatment on the modulus of elasticity (MOE) of the wood specimens and on the soil fungal as well as bacterial community composition at the soil-wood interface were assessed by quantitative PCR and amplicon sequencing of the fungal internal transcribed spacer (ITS) region and bacterial 16S rRNA gene. Specimens impregnated with CuTriQAC and Cu showed decreased MOE and reduced fungal and bacterial copy numbers over time compared to those impregnated with water and TriQAC. Fungal but not bacterial community composition was significantly affected by wood preservative treatment. The relative abundance of members of the family Trichocomaceae compared to other genera increased in the presence of the Cu and CuTriQAC treatments at three sites, suggesting these to be Cu-tolerant fungi. In conclusion, the copper-containing treatments resulted in marginally increased MOE, lowered microbial gene copy numbers compared to those in the TriQAC and water treatments, and thus enhanced wood protection against soil microbial wood degradation.IMPORTANCE Copper-containing rather than TRIQAC formulations are efficient wood preservatives irrespective of the origin and composition of the soil microbial communities. However, some fungi appear to be naturally insensitive to copper and should be the focus of future wood preservative formulations to facilitate the life span of wooden construction in contact with soil while also minimizing the overall environmental impact.

FOXM1 overexpression is associated with adverse outcome and predicts response to intravesical instillation therapy in stage pT1 non-muscle-invasive bladder cancer.

OBJECTIVE: To investigate the role of forkhead box protein M1 (FOXM1) mRNA expression and its prognostic value in stage pT1 non-muscle-invasive bladder cancer (NMIBC). PATIENTS AND METHODS: Clinical data and formalin-fixed paraffin-embedded tissues from transurethral resection of the bladder from patients with stage pT1 NMIBC, treated with an organ-preserving approach, were analysed retrospectively. Total RNA was isolated using commercial RNA extraction kits, and mRNA expression of FOXM1, MKI67, KRT20 and KRT5 was measured by single-step quantitative RT-PCR using RNA-specific TaqMan Assays. Statistical analysis was performed using Spearman's Rho, Wilcoxon or Kruskal-Wallis tests, Kaplan-Meier estimates of recurrence-free (RFS), progression-free (PFS) and cancer-specific survival (CSS) and Cox regression analysis. RESULTS: Data from 296 patients (79.4% men, median age 72 years) were available for the final evaluation. Spearman correlation analysis showed that mRNA expression of FOXM1 was significantly correlated with MKI67 (ρ: 0.6530, P < 0.001) and with the luminal subtype, reflected by the positive correlation with KRT20 (ρ: 0.2113, P < 0.001). Furthermore, there was also a strong correlation of FOXM1 expression with adverse clinical and pathological variables, such as concomitant carcinoma in situ (P = 0.05), multifocal tumours (P = 0.005) and World Health Organization 1973 grade 3 disease (P < 0.001). Kaplan-Meier analysis showed overexpression of FOMX1 to be associated with worse PFS (P = 0.028) and worse CSS (P = 0.015). FOXM1 overexpression was also shown to be a predictive risk factor for CSS (hazard ratio 1.61 [1.13-2.34], L-R chi-squared: 7.19, P = 0.007). FOXM1 overexpression identified a subgroup of patients within the luminal subtype with worse RFS (P = 0.017), PFS (P < 0.001) and CSS (P = 0.015). Patients with low FOXM1 expression had better outcomes, irrespective of instillation therapy, whereas patients with high FOXM1 expression benefitted from intravesical chemotherapy with mitomycin C. CONCLUSION: High FOXM1 expression was associated with adverse clinical and pathological features and worse outcomes, and predicted response to intravesical instillation therapy in patients with stage pT1 NMIBC.

Strong Expression of Cancertestis Antigens CTAG1B and MAGEA3 Is Correlated with Unfavourable Histopathological Features and MAGEA3 Is Associated with Worse Progression-Free Survival in Urothelial Bladder Cancer.

BACKGROUND: Cancer/testis antigens (CTA) are expressed in urothelial bladder cancer (UBC). Their therapeutical and prognostic relevance remains unclear. We studied the correlation of MAGEA3 and CTAG1B with histopathological factors in UBC and their prognostic value. METHODS: Retrospective analysis of 93 patients who underwent treatment for UBC was conducted. Besides clinical and histopathological parameters, the expression of MAGEA3 and CTAG1B was assessed by immunohistochemistry. RESULTS: Median follow-up was 75 months. Fifteen per cent of patients showed strong positive reaction to MAGEA3 staining. These tumours were statistically and significantly more often correlated with unfavourable World Health Organization (WHO) grading (G1: 0%, G2: 10.3%, G3: 23.4%, p = 0.048; low grade 0%, high grade 18.4%, p = 0.046 respectively). Correlation of CTAG1B with WHO grading was impressive with strong expression in no G1, 31.1% of G2 and 51.1% of G3 tumours (low grade 0%, high grade 43.4%, p = 0.001, respectively). Concomitant carcinoma in situ (Cis) was associated with strong CTAG1B expression (54.2% in concomitant Cis vs. 29% without concomitant Cis, p = 0.026). Kaplan-Meier analysis revealed statistically and significantly worse 5 years progression-free survival (PFS) associated with a strong expression of MAGEA3 (59 vs. 84%, p = 0.032). CONCLUSIONS: Strong CTA expression was correlated with unfavourable histopathological features. A strong expression of MAGEA3 was statistically and significantly associated with worse PFS across all stages of UBC.

Impact of E-Cadherin and ß-Catenin as Prognostic Factor in Renal Cell Carcinoma with Tumor Thrombus of the Vena Cava.

PURPOSE: Renal cell carcinoma (RCC) forming tumor thrombus (TT) of vena cava (VC) is characterized by poor prognosis. Nevertheless, the outcome of patients after radical surgery varies. To date only limited data concerning prognostic biomarkers in this RCC subgroup are available. METHODS: Out of 159 patients with pT3b/c RCC, 95 patients without synchronous distant metastases at time of diagnosis were included in the study cohort. After immunohistochemical (IHC) evaluation of E-cadherin and ß-Catenin expression, association with clinical, histopathological and survival was assessed by univariate analysis, multivariate analysis, and Kaplan-Meier-analysis. Cancer-specific survival (CSS) rates and overall survival (OS) rates were estimated using Kaplan-Meier analysis and compared using Log rank test. RESULTS: We found a significant correlation between E-cadherin overexpression and initial lymph node metastasis (ρ = 0.300, p = 0.003), positive surgical margins (ρ = 0.210, p = 0.043), and the development of distant metastases (ρ = 0.258, p = 0.012). Furthermore, we observed a significant correlation of ß-Catenin overexpression with higher tumor stage pT3c (ρ = 0.230, p = 0.028) and initial lymph node metastases (ρ = 0.236, p = 0.025). Survival analysis revealed a statistically significant association of both E-cadherin and ß-Catenin overexpression with worse CSS (p < 0.001 and p = 0.007, respectively) and OS (p < 0.001 and p = 0.041, respectively). Multivariate analysis revealed initial lymph node metastasis as the only predictive factor for worse OS (HR 4.54, 95% CI 2.30-8.93; p < 0.001). E-Cadherin and ß-Catenin expression failed to be significant in multivariable analysis for OS and CSS. CONCLUSIONS: In a large series of RCC with TT of VC high IHC expression of E-cadherin and ß-Catenin was associated with initial lymph node metastasis and with both worse OS and worse CSS. This might help to identify patients at risk for recurrence who might benefit from adjuvant therapy or stricter follow-up.

High PDL1 mRNA expression predicts better survival of stage pT1 non-muscle-invasive bladder cancer (NMIBC) patients.

INTRODUCTION AND OBJECTIVES: Checkpoint inhibition has emerged as new therapeutic option in muscle-invasive bladder cancer. The objective of the present study was to evaluate the prognostic role of PD1 and PDL1 expression in non-muscle-invasive bladder cancer (NMIBC) and establish an objective measuring method using RNA quantification. MATERIALS AND METHODS: We retrospectively analyzed clinical data and formalin-fixed paraffin-embedded tissues (FFPE) of patients with stage pT1 NMIBC who underwent transurethral resection of the bladder. mRNA expression of PD1, PDL1 and CD3 was measured by single step RT-qPCR and correlated to clinicopathological parameters, recurrence-free survival (RFS), progression-free survival (PFS) and carcinoma-specific survival (CSS). RESULTS: We have analyzed 334 patients with NMIBC at stage pT1 for mRNA analysis. Data from 296 patients (79% male, median age: 72 years) could be used. Spearman correlation revealed significant associations between mRNA expressions of PD1/PDL1 (ρ: 0.6024, p < 0.0001), CD3/PDL1 (ρ: 0.5728, p < 0.0001) and CD3/PD1 (ρ: 0.7005, p < 0.0001). Kaplan-Meier analysis revealed that high PDL1 mRNA expression (≥ 33.83) is a favorable prognostic factor with regard to better RFS (p = 0.0018), PFS (p = 0.021) and CSS (p = 0.012). Multivariate Cox-regression analysis proved PDL1 expression to be an independent prognosticator for RFS [HR 0.48 (0.31-0.72), p = 0.0005], PFS [HR 0.45 (0.24-0.80), p = 0.0059] and CSS [HR 0.31 (0.13-0.67), p = 0.0021]. CONCLUSION: High mRNA expression of PDL1 predicts improved RFS, PFS and CSS of pT1 NMIBC. Following prospective validation, this objective measurement of PD-L1 might help stratify patients with NMIBC for immunotherapy and identify patients who might benefit from early cystectomy.

Differentiation-associated urothelial cytochrome P450 oxidoreductase predicates the xenobiotic-metabolizing activity of "luminal" muscle-invasive bladder cancers.

Extra-hepatic metabolism of xenobiotics by epithelial tissues has evolved as a self-defence mechanism but has potential to contribute to the local activation of carcinogens. Bladder epithelium (urothelium) is bathed in excreted urinary toxicants and pro-carcinogens. This study reveals how differentiation affects cytochrome P450 (CYP) activity and the role of NADPH:P450 oxidoreductase (POR). CYP1A1 and CYP1B1 transcripts were inducible in normal human urothelial (NHU) cells maintained in both undifferentiated and functional barrier-forming differentiated states in vitro. However, ethoxyresorufin O-deethylation (EROD) activity, the generation of reactive BaP metabolites and BaP-DNA adducts, were predominantly detected in differentiated NHU cell cultures. This gain-of-function was attributable to the expression of POR, an essential electron donor for all CYPs, which was significantly upregulated as part of urothelial differentiation. Immunohistology of muscle-invasive bladder cancer (MIBC) revealed significant overall suppression of POR expression. Stratification of MIBC biopsies into "luminal" and "basal" groups, based on GATA3 and cytokeratin 5/6 labeling, showed POR over-expression by a subgroup of the differentiated luminal tumors. In bladder cancer cell lines, CYP1-activity was undetectable/low in basal PORlo T24 and SCaBER cells and higher in the luminal POR over-expressing RT4 and RT112 cells than in differentiated NHU cells, indicating that CYP-function is related to differentiation status in bladder cancers. This study establishes POR as a predictive biomarker of metabolic potential. This has implications in bladder carcinogenesis for the hepatic versus local activation of carcinogens and as a functional predictor of the potential for MIBC to respond to prodrug therapies.

The World Health Organization 1973 classification system for grade is an important prognosticator in T1 non-muscle-invasive bladder cancer.

OBJECTIVES: To compare the prognostic value of the World Health Organization (WHO) 1973 and 2004 classification systems for grade in T1 bladder cancer (T1-BC), as both are currently recommended in international guidelines. PATIENTS AND METHODS: Three uro-pathologists re-revised slides of 601 primary (first diagnosis) T1-BCs, initially managed conservatively (bacille Calmette-Guérin) in four hospitals. Grade was defined according to WHO1973 (Grade 1-3) and WHO2004 (low-grade [LG] and high-grade [HG]). This resulted in a lack of Grade 1 tumours, 188 (31%) Grade 2, and 413 (69%) Grade 3 tumours. There were 47 LG (8%) vs 554 (92%) HG tumours. We determined the prognostic value for progression-free survival (PFS) and cancer-specific survival (CSS) in Cox-regression models and corrected for age, sex, multiplicity, size and concomitant carcinoma in situ. RESULTS: At a median follow-up of 5.9 years, 148 patients showed progression and 94 died from BC. The WHO1973 Grade 3 was negatively associated with PFS (hazard ratio [HR] 2.1) and CSS (HR 3.4), whilst WHO2004 grade was not prognostic. On multivariable analysis, WHO1973 grade was the only prognostic factor for progression (HR 2.0). Grade 3 tumours (HR 3.0), older age (HR 1.03) and tumour size >3 cm (HR 1.8) were all independently associated with worse CSS. CONCLUSION: The WHO1973 classification system for grade has strong prognostic value in T1-BC, compared to the WHO2004 system. Our present results suggest that WHO1973 grade cannot be replaced by the WHO2004 classification in non-muscle-invasive BC guidelines.

Serum metabolites and risk of myocardial infarction and ischemic stroke: a targeted metabolomic approach in two German prospective cohorts.

Metabolomic approaches in prospective cohorts may offer a unique snapshot into early metabolic perturbations that are associated with a higher risk of cardiovascular diseases (CVD) in healthy people. We investigated the association of 105 serum metabolites, including acylcarnitines, amino acids, phospholipids and hexose, with risk of myocardial infarction (MI) and ischemic stroke in the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam (27,548 adults) and Heidelberg (25,540 adults) cohorts. Using case-cohort designs, we measured metabolites among individuals who were free of CVD and diabetes at blood draw but developed MI (n = 204 and n = 228) or stroke (n = 147 and n = 121) during follow-up (mean, 7.8 and 7.3 years) and among randomly drawn subcohorts (n = 2214 and n = 770). We used Cox regression analysis and combined results using meta-analysis. Independent of classical CVD risk factors, ten metabolites were associated with risk of MI in both cohorts, including sphingomyelins, diacyl-phosphatidylcholines and acyl-alkyl-phosphatidylcholines with pooled relative risks in the range of 1.21-1.40 per one standard deviation increase in metabolite concentrations. The metabolites showed positive correlations with total- and LDL-cholesterol (r ranged from 0.13 to 0.57). When additionally adjusting for total-, LDL- and HDL-cholesterol, triglycerides and C-reactive protein, acyl-alkyl-phosphatidylcholine C36:3 and diacyl-phosphatidylcholines C38:3 and C40:4 remained associated with risk of MI. When added to classical CVD risk models these metabolites further improved CVD prediction (c-statistics increased from 0.8365 to 0.8384 in EPIC-Potsdam and from 0.8344 to 0.8378 in EPIC-Heidelberg). None of the metabolites was consistently associated with stroke risk. Alterations in sphingomyelin and phosphatidylcholine metabolism, and particularly metabolites of the arachidonic acid pathway are independently associated with risk of MI in healthy adults.

Expression, localisation and potential significance of aquaporins in benign and malignant human prostate tissue.

BACKGROUND: To study the expression pattern, localisation and potential clinical significance of aquaporin water channels (AQP) both in prostate cancer (PC) cell lines and in benign and malignant human prostate tissue. METHODS: The AQP transcript and protein expression of HPrEC, LNCaP, DU-145 and PC3 cell lines was investigated using reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence (IF) microscopy labelling. Immunohistochemistry (IHC) was performed to assess AQP protein expression in surgical specimens of benign prostatic hyperplasia as well as in PC. Tissue mRNA expression of AQPs was quantified by single-step reverse transcriptase quantitative polymerase chain reaction (qPCR). Relative gene expression was determined using the 40-ΔCT method and correlated to clinicopathological parameters. RESULTS: Transcripts of AQP 1, 3, 4, 7, 8, 10 and 11 were expressed in all four cell lines, while AQP 9 transcripts were not detected in malignant cell lines. IF microscopy confirmed AQP 3, 4, 5, 7 and 9 protein expression. IHC revealed highly heterogeneous AQP 3 protein expression in PC specimens, with a marked decrease in expression in tumours of increasing malignancy. Loss of AQP 9 was shown in PC specimens. mRNA expression of AQP3 was found to be negatively correlated to PSA levels (ρ = - 0.354; p = 0.013), D'Amico risk stratification (ρ = - 0.336; p = 0.012), ISUP grade (ρ = - 0.321; p = 0.017) and Gleason score (ρ = - 0.342; p = 0.011). CONCLUSIONS: This is the first study to systematically characterize human prostate cell lines, benign prostatic hyperplasia and PC in relation to all 13 members of the AQP family. Our results indicate the differential expression of several AQPs in benign and malignant prostate tissue. A significant correlation was observed between AQP 3 expression and tumour grade, with progressive loss in more malignant tumours. Taken together, AQPs may play a role in the progression of PC and AQP expression patterns may serve as a prognostic marker.

Infiltrative lamina propria invasion pattern as an independent predictor for cancer-specific and overall survival of instillation treatment-naïve stage T1 high-grade urothelial bladder cancer.

OBJECTIVES: To investigate established prognostic factors and relatively new histopathological tumor characteristics including metric substage and lamina propria invasion patterns in a large series of T1 high-grade non-muscle-invasive bladder cancer. METHODS: Between 1989 and 2012, 322 patients with initial stage T1 high-grade bladder cancer underwent transurethral resection, followed by re-transurethral resection and a conservative approach with follow-up regime alone or instillation treatment. Transurethral resection specimens were reassessed by two experienced urological pathologists for tumor grade according to the World Health Organization 1973 classification, metric T1 substage, lamina propria invasion pattern and associated carcinoma in situ. The median follow-up period was 42 months (interquartile range 25-72 months). In addition to Kaplan-Meier analyses, uni- and multivariable Cox regression analyses were used to compare progression-free survival, cancer-specific survival and overall survival for the studied parameters comparing two subcohorts. RESULTS: While in patients after instillation treatment no examined feature was shown as an independent predictor for prognosis, there were predictive histopathological features in multivariable Cox regression analyses in instillation treatment-naïve patients: associated carcinoma in situ (hazard ratio 2.278, 95% confidence interval 1.119-4.634, P = 0.023) and World Health Organization 1973 grade 3 (hazard ratio 2.950, 95% confidence interval 1.021-8.536, P = 0.046) for worse progression-free survival, infiltrative lamina propria tumor pattern for worse cancer-specific survival (hazard ratio 2.369, 95% confidence interval 1.034-5.429, P = 0.042) and overall survival (hazard ratio 1.049, 95% confidence interval 1.024-1.075, P = 0.001). CONCLUSIONS: The results of the present T1 high-grade bladder cancer series suggest that lamina propria invasion pattern is a promising parameter to predict the prognosis of T1 high-grade bladder cancer in an instillation treatment-naïve subcohort. Prospective multicenter evaluations are warranted. The need for instillation treatment in T1 high-grade bladder cancer is clearly demanded.

mRNA-Expression of KRT5 and KRT20 Defines Distinct Prognostic Subgroups of Muscle-Invasive Urothelial Bladder Cancer Correlating with Histological Variants.

Recently, muscle-invasive bladder cancer (MIBC) has been subclassified by gene expression profiling, with a substantial impact on therapy response and patient outcome. We tested whether these complex molecular subtypes of MIBC can be determined by mRNA detection of keratin 5 (KRT5) and keratin 20 (KRT20). Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) was applied to quantify gene expression of KRT5 and KRT20 using TaqMan®-based assays in 122 curatively treated MIBC patients (median age 68.0 years). Furthermore, in silico analysis of the MD Anderson Cancer Center (MDACC) cohort (GSE48277 + GSE47993) was performed. High expression of KRT5 and low expression of KRT20 were associated with significantly improved recurrence-free survival (RFS) and disease-specific survival disease specific survival (DSS: 5-year DSS for KRT5 high: 58%; 5-year DSS for KRT20 high: 29%). KRT5 and KRT20 were associated with rates of lymphovascular invasion and lymphonodal metastasis. The combination of KRT5 and KRT20 allowed identification of patients with a very poor prognosis (KRT20⁺/KRT5-, 5-year DSS 0%, p < 0.0001). In silico analysis of the independent MDACC cohorts revealed congruent results (5-year DSS for KRT20 low vs. high: 84% vs. 40%, p = 0.042). High KRT20-expressing tumors as well as KRT20⁺/KRT- tumors were significantly enriched with aggressive urothelial carcinoma variants (micropapillary, plasmacytoid, nested).

Biochemical Foundations of Health and Energy Conservation in Hibernating Free-ranging Subadult Brown Bear Ursus arctos.

Brown bears (Ursus arctos) hibernate for 5-7 months without eating, drinking, urinating, and defecating at a metabolic rate of only 25% of the summer activity rate. Nonetheless, they emerge healthy and alert in spring. We quantified the biochemical adaptations for hibernation by comparing the proteome, metabolome, and hematological features of blood from hibernating and active free-ranging subadult brown bears with a focus on conservation of health and energy. We found that total plasma protein concentration increased during hibernation, even though the concentrations of most individual plasma proteins decreased, as did the white blood cell types. Strikingly, antimicrobial defense proteins increased in concentration. Central functions in hibernation involving the coagulation response and protease inhibition, as well as lipid transport and metabolism, were upheld by increased levels of very few key or broad specificity proteins. The changes in coagulation factor levels matched the changes in activity measurements. A dramatic 45-fold increase in sex hormone-binding globulin levels during hibernation draws, for the first time, attention to its significant but unknown role in maintaining hibernation physiology. We propose that energy for the costly protein synthesis is reduced by three mechanisms as follows: (i) dehydration, which increases protein concentration without de novo synthesis; (ii) reduced protein degradation rates due to a 6 °C reduction in body temperature and decreased protease activity; and (iii) a marked redistribution of energy resources only increasing de novo synthesis of a few key proteins. The comprehensive global data identified novel biochemical strategies for bear adaptations to the extreme condition of hibernation and have implications for our understanding of physiology in general.