PD-L1 expression in tumor and inflammatory cells is associated with favorable tumor features and favorable prognosis in muscle-invasive urothelial carcinoma of the bladder not treated by immune checkpoint inhibitors.

BACKGROUND: A high level of PD-L1 expression is the most relevant predictive parameter for response to immune checkpoint inhibitor (CPI) therapy in urinary bladder cancer. Existing data on the relationship between PD-L1 expression and the natural course of disease are controversial and sparse. METHODS: To expand our understanding of the relationship between PD-L1 expression and parameters of cancer aggressiveness, PD-L1 was analyzed on tissue microarrays containing 2710 urothelial bladder carcinomas including 512 patients with follow-up data who underwent radical cystectomy and follow-up therapies in the pre-immune checkpoint inhibitor therapy era. RESULTS: Tumor cell positivity in ≥10% of cells were seen in 513 (20%) and an immune cell positivity occurred in 872 (34%) of 2566 interpretable cancers. PD-L1 positivity in tumor cells increased from pTaG2 low grade (0.9% positive) to pTaG3 high grade (4.1%; p = 0.0255) and was even higher in muscle-invasive (pT2-4) carcinomas (29.3%; p < 0.0001). However, within pT2-4 carcinomas, PD-L1 positivity was linked to low pT stage (p = 0.0028), pN0 (p < 0.0001), L0 status (p = 0.0005), and a better prognosis within 512 patients with cystectomy who never received CPIs (p = 0.0073 for tumor cells and p = 0.0086 for inflammatory cells). PD-L1 staining in inflammatory cells was significantly linked to PD-L1 staining in tumor cells (p < 0.0001) and both were linked to a positive p53 immunostaining (p < 0.0001). CONCLUSION: It cannot be fully excluded that the strong statistical link between PD-L1 status and favorable histological tumor features as well as better prognosis could influence the outcome of studies evaluating CPIs in muscle-invasive urothelial carcinoma.

1H-NMR metabolomics reveals the Glabrescione B exacerbation of glycolytic metabolism beside the cell growth inhibitory effect in glioma.

BACKGROUND: Glioma is the most common and primary brain tumors in adults. Despite the available multimodal therapies, glioma patients appear to have a poor prognosis. The Hedgehog (Hh) signaling is involved in tumorigenesis and emerged as a promising target for brain tumors. Glabrescione B (GlaB) has been recently identified as the first direct inhibitor of Gli1, the downstream effector of the pathway. METHODS: We established the overexpression of Gli1 in murine glioma cells (GL261) and GlaB effect on cell viability. We used 1H-nuclear magnetic resonance (NMR) metabolomic approach to obtain informative metabolic snapshots of GL261 cells acquired at different time points during GlaB treatment. The activation of AMP activated protein Kinase (AMPK) induced by GlaB was established by western blot. After the orthotopic GL261 cells injection in the right striatum of C57BL6 mice and the intranasal (IN) GlaB/mPEG5kDa-Cholane treatment, the tumor growth was evaluated. The High Performance Liquid Chromatography (HPLC) combined with Mass Spectrometry (MS) was used to quantify GlaB in brain extracts of treated mice. RESULTS: We found that GlaB affected the growth of murine glioma cells both in vitro and in vivo animal model. Using an untargeted 1H-NMR metabolomic approach, we found that GlaB stimulated the glycolytic metabolism in glioma, increasing lactate production. The high glycolytic rate could in part support the cytotoxic effects of GlaB, since the simultaneous blockade of lactate efflux with α-cyano-4-hydroxycinnamic acid (ACCA) affected glioma cell growth. According to the metabolomic data, we found that GlaB increased the phosphorylation of AMPK, a cellular energy sensor involved in the anabolic-to-catabolic transition. CONCLUSIONS: Our results indicate that GlaB inhibits glioma cell growth and exacerbates Warburg effect, increasing lactate production. In addition, the simultaneous blockade of Gli1 and lactate efflux amplifies the anti-tumor effect in vivo, providing new potential therapeutic strategy for this brain tumor.

Microvascular and lymphovascular tumour invasion are associated with poor prognosis and metastatic spread in renal cell carcinoma: a validation study in clinical practice.

OBJECTIVE: To validate microvascular (MVI) and lymphovascular (LVI) invasion as prognostic factors in patients with renal cell carcinoma (RCC). PATIENTS AND METHODS: Data of patients with RCC who underwent radical or nephron-sparing surgery were prospectively collected from three academic centres. The occurrence of MVI and LVI was determined with standard staining protocols by experienced pathologists at the time of diagnosis. The association of MVI and LVI with clinicopathological data, metastatic spread, and cancer-specific survival (CSS) were evaluated with Fisher's exact tests, binary logistic regression analyses, and univariable and multivariable Cox proportional hazard regression models. RESULTS: MVI was present in 201 of 747 patients (26.9%) and was associated with advanced Tumour-Node-Metastasis (TNM) stages, high Fuhrman grades, and sarcomatoid features (all P < 0.001). MVI was associated with a higher rate of metastatic spread. LVI was present in 32 of 573 patients (5.5%) and was associated with advanced TNM stages, high Fuhrman grade, and sarcomatoid features (all P < 0.001). Two-thirds of LVI-positive patients died (P < 0.001). Both LVI and MVI were significantly associated with CSS in all patients, clear cell RCC (ccRCC), and localised RCC in univariable analysis (all P < 0.001). On multivariable analysis, presence of MVI was identified as an independent prognostic factor (hazard ratio 2.09; P = 0.001). Moreover, MVI [odds ratio (OR) 2.7; P = 0.001] and not macrovascular invasion (P = 0.895) was an independent predictor of sychronuous metastatic spread. LVI was the strongest factor associated with sychronous metastatic spread (OR 4.73, 95% confidence interval 1.84-12.14; P = 0.001) in all patients and in the subgroup of patients with ccRCC (P = 0.001). CONCLUSIONS: The present study validated LVI and MVI as prognostic factors for poor outcome in RCC. These findings endorse an evaluation of both variables in the clinical routine setting to facilitate survival prognostication in follow-up protocols and for assignment to adjuvant treatment trials.

Single-Cell Analysis of the Dps Response to Oxidative Stress.

UNLABELLED: Microorganisms have developed an elaborate spectrum of mechanisms to respond and adapt to environmental stress conditions. Among these is the expression of dps, coding for the DNA-binding protein from starved cells. Dps becomes the dominant nucleoid-organizing protein in stationary-phase Escherichia coli cells and is required for robust survival under stress conditions, including carbon or nitrogen starvation, oxidative stress, metal exposure, and irradiation. To study the complex regulation of Dps in E. coli, we utilized time-lapse fluorescence microscopy imaging to examine the kinetics, input encoding, and variability of the Dps response in single cells. In the presence of an oxidative stressor, we observed a single pulse of activation of Dps production. Increased concentrations of H2O2 led to increased intensity and duration of the pulse. While lower concentrations of H2O2 robustly activated the Dps response with little effect on the growth rate, higher concentrations of H2O2 resulted in dramatically lower and highly varied growth rates. A comparison of cells exposed to the same concentration of H2O2 revealed that increased levels of Dps expression did not confer a growth advantage, indicating that recovery from stress may rely primarily upon variation in the amount of damage caused to individual cells. IMPORTANCE: We show for the first time the response of the DNA-binding protein from starved cells (Dps) to oxidative stress in single cells of E. coli Through time-lapse fluorescence microscopy, a single pulse of Dps production is observed in cells exposed to H2O2, with a duration and intensity of induction proportional to the concentration of the applied stress. More intense Dps expression did not provide a growth benefit to the bacteria, suggesting that healing from oxidative stress may largely depend upon the amount of damage in each individual cell.

Association of human telomerase reverse transcriptase gene polymorphisms, serum levels, and telomere length with renal cell carcinoma risk and pathology.

Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of the human telomerase and plays a key role in telomere restitution and gene regulation. Evidence suggests that hTERT is linked with the risk and progression of several malignancies, but there are no comprehensive data in renal cell carcinoma (RCC). In this case-control study, we assessed seven polymorphic hTERT gene variants (MNS16A, rs2736100, rs2736098, rs7726159, rs2853677, rs13172201, and rs10069690), hTERT serum levels, and the telomere length of 663 individuals, including 243 with clear cell RCC and 420 age- and gender-matched healthy controls. The SL and SS genotypes of MNS16A were associated with a decreased risk for RCC on the multivariable logistic regression analysis (SL-OR 0.72, SS-OR 0.37, P < 0.001). The GG genotype of rs2736098 was associated with a decreased risk for RCC compared with AA (OR 0.18, P < 0.001). Both telomere length and hTERT serum levels increased with every G allele in rs2736098 (P = 0.008). Pretherapeutic hTERT serum levels were higher in patients with advanced tumor stages (P = 0.037) and distant metastases (P = 0.006). Rs2736100, rs7726159, rs2853677, rs13172201, and rs10069690 were not linked with RCC risk, and none of the polymorphisms was associated with RCC pathology. In conclusion, the polymorphic number of tandem repeats in hTERT (MNS16A) and rs2736098 may be linked with the risk for RCC. Rs2736098 may have an important role in telomere length restitution and serum hTERT levels may represent a novel biomarker for RCC. © 2015 Wiley Periodicals, Inc.

Dynamic Prognostication Using Conditional Recurrence and Progression Estimates for Patients with Nonmuscle Invasive Bladder Cancer.

PURPOSE: Conditional estimates provide a dynamic prediction of outcomes but to our knowledge there are no data on nonmuscle invasive bladder cancer. We assessed changes in conditional recurrence and progression rates after transurethral resection of the bladder and explored the prognostic impact of established factors and risk groups with time. MATERIALS AND METHODS: We retrospectively analyzed data on 1,292 consecutive patients with newly diagnosed Ta/T1 bladder cancer who underwent transurethral resection of the bladder. Study end points were time to first recurrence and time to progression. RESULTS: The 2-year recurrence rate at baseline was 36%, which improved as a function of the time that patients were free of disease recurrence. After 6, 12, 24, 36 and 48 months the 2-year conditional recurrence rate improved to 31% (14% improvement vs baseline), 22% (39% improvement), 16% (56% improvement), 13% (64% improvement) and 11% (69% improvement), respectively. Comparably, conditional progression rates improved with increasing followup, although relative differences were less distinct. The prognostic impact of established factors and nonmuscle invasive bladder cancer risk groups progressively decreased with time and finally disappeared. However, bacillus Calmette-Guérin had a protective effect on progression even after 3 years. We provide tables with dynamic prognostic information at all analyzed time points. CONCLUSIONS: In patients with primary Ta/T1 bladder cancer recurrence and progression rates improve with time. The prognostic impact of established factors and risk groups decreases and finally disappears. The effect of bacillus Calmette-Guérin on progression is long-lasting. Conditional outcome estimates may improve patient counseling and individualize surveillance planning.

Prognostic role of ERCC1 protein expression in upper tract urothelial carcinoma following radical nephroureterectomy with curative intent.

BACKGROUND: Excision repair cross-complementing 1 (ERCC1) has been associated with outcomes of urothelial carcinoma of the bladder, but was not yet studied in upper tract urothelial carcinoma (UTUC). The aim of this study was to assess the prognostic role of ERCC1 expression in a large international cohort of UTUC patients. METHODS: Immunohistochemical ERCC1 expression was evaluated in 716 UTUC patients who underwent radical nephroureterectomy with curative intent. ERCC1 was considered positive when the H-score was >1.0. Associations with overall survival and cancer-specific survival were assessed using univariable and multivariable Cox models. RESULTS: ERCC1 was expressed in 303 tumors (42.3 %) and linked with the presence of tumor necrosis (16.2 vs. 10.4 %, p = 0.023), but not with any other clinical or pathological variable. ERCC1 status did not predict cancer-specific survival and overall survival on both univariable (p = 0.70 and 0.32, respectively) and multivariable analyses (p = 0.48 and 0.33, respectively). CONCLUSIONS: ERCC1 is expressed in a significant proportion of UTUC and is linked with tumor necrosis, but its expression appears not to be associated with prognosis following radical nephroureterectomy.

Dynamic Behavior of Clobazam on High-Performance Liquid Chromatography Chiral Stationary Phases.

Clobazam, a 1,5-benzodiazepin-2,4-dione, is a chiral molecule because its ground state conformation features a nonplanar seven-membered ring lacking reflection symmetry elements. The two conformational enantiomers of clobazam interconvert at room temperature by a simple ring-flipping process. Variable temperature HPLC on the Pirkle type (R)-N-(3,5-dinitronenzoyl)phenylglycine and (R,R)-Whelk-O1 chiral stationary phases (CSPs) allowed us to separate for the first time the conformational enantiomers of clobazam and to observe peak coalescence-decoalescence phenomena due to concomitant separation and interconversion processes occurring on the same time scale. Clobazam showed temperature dependent dynamic high-performance liquid chromatography (HPLC) profiles with interconversion plateaus on the two CSPs indicative of on-column enantiomer interconversion. (enantiomerization) in the column temperature range between Tcol = 10°C and Tcol = 30°C, whereas on-column interconversion was absent at temperature close to or lower than Tcol = 5°C. Computer simulation of exchange-deformed HPLC profiles using a program based on the stochastic model yielded the apparent rate constants for the on-column enantiomerization and the corresponding free energy activation barriers. At Tcol = 20°C the averaged enantiomerization barriers, ΔG(‡), for clobazam were found in the range 21.08-21.53 kcal mol(-1) on the two CSPs. The experimental dynamic chromatograms and the corresponding interconversion barriers reported in this article are consistent with the literature data measured by DNMR at higher temperatures and in different solvents.

Preoperative serum cholesterol is an independent prognostic factor for patients with renal cell carcinoma (RCC).

OBJECTIVE: To assess the prognostic role of preoperative serum cholesterol in patients with renal cell carcinoma (RCC), as increasing evidence suggests that alterations in the lipid profile are associated with the development, progression and prognosis of various cancers. PATIENTS AND METHODS: We analysed 867 patients, who underwent radical or partial nephrectomy for RCC between 2002 and 2012. Preoperative total cholesterol levels were determined in serum using colorimetric analysis (CHOD-PAP method). The association with cancer-specific survival (CSS) was assessed with Cox models. Discrimination was quantified with the C-index. The median follow-up was 52 months. RESULTS: The median (interquartile range) serum cholesterol was 195 (166-232) mg/dL. Decreasing serum cholesterol was associated with more advanced T, N and M stages (P < 0.001), higher grades (P = 0.001) and presence of tumour necrosis (P = 0.002). Continuously coded cholesterol was associated with CSS in both univariable (hazard ratio [HR] 0.87, P < 0.001) and multivariable analyses (HR 0.93, P = 0.001). The discrimination of a multivariable base model increased significantly from 88.3% to 89.2% following inclusion of cholesterol (P = 0.006). In patients with clinically localised disease (T1-3N0/+M0), cholesterol remained associated with CSS in multivariable analysis (HR 0.90, P = 0.002) and increased the discrimination from 74.6% to 76.9% (P = 0.002). CONCLUSIONS: Preoperative serum cholesterol is an independent prognostic factor for patients with RCC, with lower levels being associated with worse survival. Its use increases the discrimination of established prognostic factors. As cholesterol is a broadly available routine marker, its use may provide a meaningful adjunct in clinical practice. The biological rationale underlying this association remains to be clarified.

The role of adjuvant chemotherapy for lymph node-positive upper tract urothelial carcinoma following radical nephroureterectomy: a retrospective study.

OBJECTIVE: To evaluate the effect of adjuvant chemotherapy (AC) on mortality after radical nephroureterectomy (RNU) for upper tract urothelial carcinoma (UTUC) with positive lymph nodes (LNs) and to identify patient subgroups that are most likely to benefit from AC. PATIENTS AND METHODS: We retrospectively analysed data of 263 patients with LN-positive UTUC, who underwent full surgical resection. In all, 107 patients (41%) received three to six cycles of AC, while 156 (59.3%) were treated with RNU alone. UTUC-related mortality was evaluated using competing-risks regression models. RESULTS: In all patients (T(all) N+), administration of AC had no significant impact on UTUC-related mortality on univariable (P = 0.49) and multivariable (P = 0.11) analysis. Further stratified analyses showed that only N+ patients with pT3-4 disease benefited from AC. In this subgroup, AC reduced UTUC-related mortality by 34% (P = 0.019). The absolute difference in mortality was 10% after the first year and increased to 23% after 5 years. On multivariable analysis, administration of AC was associated with significantly reduced UTUC-related mortality (subhazard ratio 0.67, P = 0.022). Limitations of this study are the retrospective non-randomised design, selection bias, absence of a central pathological review and different AC protocols. CONCLUSIONS: AC seems to reduce mortality in patients with pT3-4 LN-positive UTUC after RNU. This subgroup of LN-positive patients could serve as target population for an AC prospective randomised trial.

Adjuvant cisplatin-based combined chemotherapy for lymph node (LN)-positive urothelial carcinoma of the bladder (UCB) after radical cystectomy (RC): a retrospective international study of >1500 patients.

OBJECTIVE: To compare outcomes of patients with lymph node (LN)-positive urothelial carcinoma of the bladder (UCB) treated with or without cisplatin-based combined adjuvant chemotherapy (AC) after radical cystectomy (RC). PATIENTS AND METHODS: We retrospectively analysed 1523 patients with LN-positive UCB, who underwent RC with bilateral pelvic LN dissection. All patients had no evidence of disease after RC. AC was administered within 3 months. Competing-risks models were applied to compare UCB-related mortality. RESULTS: Of the 1523 patients, 874 (57.4%) received AC. The cumulative 1-, 2- and 5-year UCB-related mortality rates for all patients were 16%, 36% and 56%, respectively. Administration of AC was associated with an 18% relative reduction in the risk of UCB-related death (subhazard ratio 0.82, P = 0.005). The absolute reduction in mortality was 3.5% at 5 years. The positive effect of AC was detectable in patients aged ≤70 years, in women, in pT3-4 disease, and in those with a higher LN density and lymphovascular invasion. This study is limited by its retrospective and non-randomised design, selection bias, the absence of central pathological review and lack in standardisation of LN dissection and cisplatin-based protocols. CONCLUSION: AC seems to reduce UCB-related mortality in patients with LN-positive UCB after RC. Younger patients, women and those with high-risk features such as pT3-4 disease, a higher LN density and lymphovascular invasion appear to benefit most. Appropriately powered prospective randomised trials are necessary to confirm these findings.

Comparison of the prognostic value of pretreatment measurements of systemic inflammatory response in patients undergoing curative resection of clear cell renal cell carcinoma.

PURPOSE: Pretreatment measurements of systemic inflammatory response, including the Glasgow prognostic score (GPS), the neutrophil-to-lymphocyte ratio (NLR), the monocyte-to-lymphocyte ratio (MLR), the platelet-to-lymphocyte ratio (PLR) and the prognostic nutritional index (PNI) have been recognized as prognostic factors in clear cell renal cell carcinoma (CCRCC), but there is at present no study that compared these markers. METHODS: We evaluated the pretreatment GPS, NLR, MLR, PLR and PNI in 430 patients, who underwent surgery for clinically localized CCRCC (pT1-3N0M0). Associations with disease-free survival were assessed with Cox models. Discrimination was measured with the C-index, and a decision curve analysis was used to evaluate the clinical net benefit. RESULTS: On multivariable analyses, all measures of systemic inflammatory response were significant prognostic factors. The increase in discrimination compared with the stage, size, grade and necrosis (SSIGN) score alone was 5.8 % for the GPS, 1.1-1.4 % for the NLR, 2.9-3.4 % for the MLR, 2.0-3.3 % for the PLR and 1.4-3.0 % for the PNI. On the simultaneous multivariable analysis of all candidate measures, the final multivariable model contained the SSIGN score (HR 1.40, P < 0.001), the GPS (HR 2.32, P < 0.001) and the MLR (HR 5.78, P = 0.003) as significant variables. Adding both the GPS and the MLR increased the discrimination of the SSIGN score by 6.2 % and improved the clinical net benefit. CONCLUSIONS: In patients with clinically localized CCRCC, the GPS and the MLR appear to be the most relevant prognostic measures of systemic inflammatory response. They may be used as an adjunct for patient counseling, tailoring management and clinical trial design.

Aurora A Kinase as a diagnostic urinary marker for urothelial bladder cancer.

OBJECTIVE: To evaluate urinary Aurora A Kinase (AURKA) mRNA expression as a diagnostic biomarker for urothelial bladder cancer (UBC). METHODS: One hundred and eighty-eight urine samples from patients with UBC (n = 122) and controls with hematuria (n = 66) were investigated. AURKA expression was quantified using real-time PCR and compared with voided urinary cytology. Associations with stage and grade were assessed. The area under curve was used to quantify the predictive accuracy (PA). RESULTS: The sensitivity and the specificity of AURKA for UBC were 83.6 and 65.2 %, respectively (PA = 74.4 %). Among those with detectable AURKA, the quantity of expression was similar in cases and controls. Compared with Ta, tumors staged T1 and T2 showed a 9.31-fold and 4.78-fold increased AURKA expression (p = 0.034), respectively. Further, high-grade tumors showed 5.33-fold higher expression levels than low-grade tumors (p = 0.031). AURKA and urinary cytology showed similar overall PA for UBC detection (74.4 vs. 72.1 %, p = 0.588). For low-grade tumors, AURKA was more accurate (72.5 vs. 59.0 %, p = 0.004), while cytology was more accurate for high-grade lesions (76.8 vs. 89.1 %, p = 0.011). CONCLUSIONS: In patients with hematuria, AURKA is associated with the presence and grade of UBC, suggesting a role as diagnostic and prognostic biomarker. As AURKA is more accurate in low-grade tumors but less accurate in high-grade tumors than urinary cytology, both could be complementary in detecting UBC.

Impact of ABO blood type on outcomes in patients with primary nonmuscle invasive bladder cancer.

PURPOSE: ABO blood type is an established prognostic factor for several malignancies but its role in bladder urothelial carcinoma is largely unknown. We determined whether ABO blood type is associated with the outcome of transurethral resection of nonmuscle invasive bladder urothelial carcinoma. MATERIALS AND METHODS: We retrospectively studied ABO blood types in 931 patients with primary nonmuscle invasive bladder urothelial carcinoma treated with transurethral bladder resection with or without intravesical instillation therapy. Disease recurrence and progression were analyzed with univariable and multivariable competing risks regression models. Median followup was 67 months. Discrimination was evaluated by the concordance index. RESULTS: The ABO blood type was O, A, B and AB in 414 (44.5%), 360 (38.7%), 103 (11.1%) and 54 patients (5.8%), respectively. ABO blood type was significantly associated with outcome on univariable and multivariable analysis. Overall, patients with blood type O had worse recurrence and progression rates than those with A (p = 0.015 and 0.031) or B (p = 0.004 and 0.075, respectively). The concordance index of multivariable base models increased after including ABO blood type. CONCLUSIONS: In patients with nonmuscle invasive bladder urothelial carcinoma the ABO blood type may predict the outcome. Those with blood type O showed the highest recurrence and progression rates. Including ABO blood type in multivariable models increases the accuracy of standard prognostic factors. Since the ABO blood type is available for most patients, it may represent an ideal adjunctive marker to predict recurrence and progression. The biological explanation and prognostic value of this finding must be further elucidated.

Evaluation of ABO blood group as a prognostic marker in renal cell carcinoma (RCC).

OBJECTIVE: To evaluate ABO blood group as a prognostic marker in patients with renal cell carcinoma (RCC). PATIENTS AND METHODS: This retrospective study included 556 consecutive patients who underwent surgery for RCC at a single institution. The associations of ABO blood group with clinical and pathological variables were assessed using Kruskal-Wallis and chi-squared tests. The impact on overall survival (OS) and RCC-specific survival (RCC-SS) was analysed using univariable and multivariable Cox proportional hazards regression models. RESULTS: Blood group O was associated with the absence of lymph node metastases (P = 0.034) and the presence of bilateral RCC (P = 0.017). No associations with age, gender, body mass index, Charlson comorbidity index, T stage, M stage, grade and histological subtype were observed. In univariable and multivariable survival analysis, ABO blood group was not associated with OS and RCC-SS. CONCLUSIONS: In the present study, ABO blood group was not linked with RCC prognosis. Blood group O may be associated with the absence of lymph node metastases and the presence of bilateral RCC. External validation in larger cohorts is necessary.

Loss of Upk1a and Upk1b expression is linked to stage progression in urothelial carcinoma of the bladder.

BACKGROUND: Uroplakin-1a (Upk1a) and uroplakin-1b (Upk1b) have recently been identified as diagnostic markers for the distinction of urothelial carcinomas from other solid tumor entities. Both proteins play an important role in the stabilization and strengthening of epithelial cells that line the bladder. METHODS: To evaluate the prognostic role of uroplakin expression in urothelial carcinomas, more than 2700 urothelial neoplasms were analyzed in a tissue microarray format by immunohistochemistry. To further assess the diagnostic role of uroplakin immunohistochemistry, results were compared with preexisting GATA3 data. RESULT: The fraction of Upk1a/Upk1b positive cases decreased slightly from pTaG2 low-grade (88% positive for Upk1a/87% positive for Upk1b) and pTaG2 high-grade (92%/89%) to pTaG3 (83%/88%; p > 0.05) and was lower in muscle-invasive (pT2-4) carcinomas (42%/64%; p < 0.0001/p < 0.0001 for pTa vs. pT2-4). Within pT2-4 carcinomas, high expression of Upk1a and Upk1b was linked to nodal metastasis and lymphatic vessel infiltration (p < 0.05) but unrelated to patient outcome. There were significant associations between Upk1a, Upk1b and GATA3 immunostaining (p < 0.0001 each), but 11% of GATA3 negative cancers were Upk1a/b positive and 8% of Upk1a/b negative cancers were GATA3 positive. Absence of GATA3/Upk1a/b staining was significantly linked to poor patient survival in the subgroup of 126 pT4 carcinomas (p = 0.0004) but not in pT2 and pT3 cancers. CONCLUSIONS: In summary, the results of our study demonstrate that Upk1a and/or Upk1b immunohistochemistry can complement GATA3 for the distinction of urothelial carcinomas. Furthermore, a progressive loss of Upk1a/b expression during stage progression and a prognostic role of the combination GATA3/Upk1a/Upk1b in pT4 carcinomas is evident.

CEA (CEACAM5) expression is common in muscle-invasive urothelial carcinoma of the bladder but unrelated to the disease course.

Objectives: Carcinoembryonic antigen (CEA) is a cell surface glycoprotein that represents a promising therapeutic target. Serum measurement of shedded CEA can be utilized for monitoring of cancer patients. Material and Methods: To evaluate the potential clinical significance of CEA expression in urothelial bladder neoplasms, CEA was analysed by immunohistochemistry in more than 2500 urothelial bladder carcinomas in a tissue microarray format. Results: CEA staining was largely absent in normal urothelial cells but was observed in 30.4% of urothelial bladder carcinomas including 406 (16.7%) with weak, 140 (5.8%) with moderate, and 192 (7.9%) with strong staining. CEA positivity occurred in 10.9% of 411 pTaG2 low-grade, 32.0% of 178 pTaG2 high-grade, and 43.0% of 93 pTaG3 tumours (p < 0.0001). In 1335 pT2-4 carcinomas, CEA positivity (34.1%) was lower than in pTaG3 tumours. Within pT2-4 carcinomas, CEA staining was unrelated to pT, pN, grade, L-status, V-status, overall survival, recurrence free survival, and cancer specific survival (p > 0.25). Conclusion: CEA increases markedly with grade progression in pTa tumours, and expression occurs in a significant fraction of pT2-4 urothelial bladder carcinomas. The high rate of CEA positivity in pT2-4 carcinomas offers the opportunity of using CEA serum measurement for monitoring the clinical course of these cancers. Moreover, CEA positive urothelial carcinomas are candidates for a treatment by targeted anti-CEA drugs.

The protease activated receptor 1 gene variation IVSn -14 A>T is associated with distant metastasis and cancer specific survival in renal cell carcinoma.

PURPOSE: PAR-1 mediates angiogenesis and impacts the process of tumor growth and disease progression. We evaluated the associations of the gene variations PAR-1 IVSn -14 A>T (rs168753), -506 Ins/Del (rs11267092) and -1426 C>T (rs32934) with renal cell carcinoma pathology and cancer specific survival. MATERIALS AND METHODS: DNA was extracted from the peripheral blood leukocytes of 236 consecutive patients with renal cell carcinoma. Genotyping was done using restriction fragment length polymorphism analysis of polymerase chain reaction amplicons and sequencing. RESULTS: The IVSn -14 AA genotype was associated with a 3.13-fold increased risk of distant metastases (p = 0.015). In addition, cancer specific survival of patients with IVSn -14 AA was significantly worse than in those with AT/TT (HR 2.98, p = 0.019). The 1 and 4-year cancer specific survival rate for AA vs AT/TT was 89% vs 99% and 82% vs 92%, respectively. After adjusting for the stage, size, grade and necrosis (SSIGN) score on multivariable analysis, IVSn -14 AA was identified as an independent adverse prognostic factor (HR 2.72, p = 0.044). The variations -506 Ins/Del and -1426 C>T were not significantly associated with pathological factors or cancer specific survival. CONCLUSIONS: Results suggest that the AA genotype of the PAR-1 variation IVSn -14 A>T is associated with an increased risk of metastasis and poorer prognosis of renal cell carcinoma. Therefore, assessing the individual risk based on genotypes may be a helpful adjunct to identify subgroups at high risk for a poor clinical outcome.

The CASP8 -652 6N insertion/deletion promoter polymorphism is associated with renal cell carcinoma risk and metastasis.

PURPOSE: Caspase-8 is a key regulator of apoptosis. Its cancer cell antigen induced cell death activity is strongly impacted by the insertion/deletion promoter polymorphism CASP8 -652 6N ins/del (rs3834129). We studied the association of this polymorphism with renal cell carcinoma risk and pathology. MATERIALS AND METHODS: In this hospital based case-control study 500 Austrian patients were genotyped, including 250 with renal cell carcinoma, and 250 age and gender matched healthy controls. Polymerase chain reaction amplified genomic DNA was evaluated by restriction fragment length polymorphism analysis and automatic sequencing. We assessed associations with renal cell carcinoma risk and pathological factors, and performed a meta-analysis of the literature. RESULTS: The CASP8 -652 6N ins/del polymorphism was significantly linked to renal cell carcinoma (chi-square for trend = 9.50, p = 0.002). Compared with ins/ins, del/del was associated with a 57% decreased risk of the disease (OR 0.43, 95% CI 0.26-0.73, p = 0.002). Furthermore, del/del was associated with a lower risk of distant metastases (p <0.05) but not with T stage, N stage or grade. On meta-analysis the CASP8 -652 6N ins/del polymorphism was associated with renal cell carcinoma risk (p <0.001). CONCLUSIONS: The del/del genotype of the CASP8 -652 6N ins/del promoter polymorphism decreases the overall risk of renal cell carcinoma. It may be associated with a decreased risk of metastasis. Larger studies are warranted to validate our findings.

Prognostic impact of preoperative neutrophil-to-lymphocyte ratio in localized nonclear cell renal cell carcinoma.

PURPOSE: The preoperative neutrophil-to-lymphocyte ratio was proposed as a prognostic factor for localized clear cell renal cell carcinoma. We evaluated its role in nonclear cell renal cell carcinoma. MATERIALS AND METHODS: We queried 2 prospective kidney cancer databases. Patients who underwent full resection of localized (T1-3 N0/+ M0) nonclear cell renal cell carcinoma by radical or partial nephrectomy were included in analysis. Associations of the continuously coded neutrophil-to-lymphocyte ratio with disease-free survival were assessed with univariable and multivariable Cox regression models. Prognostic accuracy was evaluated with the Harrell concordance index. RESULTS: Our final cohort included 281 patients. The 5-year disease-free survival rate was 88.1%. The neutrophil-to-lymphocyte ratio was significantly associated with disease-free survival. With each 1.0 increase in the ratio the risk of recurrence increased by 15% (HR 1.15, p=0.028). On multivariable analysis TNM group (HR 2.84, p=0.025), Fuhrman grade (HR 3.40, p<0.001) and the neutrophil-to-lymphocyte ratio (HR 1.17, p=0.022) were independently associated with disease-free survival. Adding the neutrophil-to-lymphocyte ratio improved the accuracy of a base model to predict disease-free survival from 78.8% to 80.8%. CONCLUSIONS: The neutrophil-to-lymphocyte ratio is an independent prognostic factor for disease-free survival after surgery with curative intent for localized nonclear cell renal cell carcinoma. It significantly increases the accuracy of established prognostic factors. The neutrophil-to-lymphocyte ratio may provide a meaningful adjunct for patient counseling and clinical trial design.