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1.  Hexaminolevulinate-guided fluorescence cystoscopy reduces recurrence in patients with non-muscle invasive bladder cancer 
The Journal of urology  2010;184(5):1907-1913.
To assess the impact that improved detection of non-muscle invasive bladder cancer with hexaminolevulinate (HAL) fluorescence cystoscopy may have on early recurrence rates.
Materials and methods
This prospective, randomized study enrolled 814 patients suspected of having bladder cancer at increased risk for recurrence. All patients underwent white light cystoscopy and mapping of lesions, followed by transurethral resection of the bladder (TURB) where indicated. Patients in the fluorescence group also received intravesical hexaminolevulinate solution at least one hour before cystoscopy to induce fluorescence of cancerous lesions, and underwent additional inspection with blue light before and after TURB. Adjuvant intravesical therapy was based on risk. Follow up cystoscopies at 3, 6 and 9 months were conducted with white light.
Detection was carried out as a within-patient comparison in the fluorescence group. In this group, 286 patients were found to have at least one Ta or T1 tumor (ITT). In 47 (16%), at least one of the tumors was seen only with fluorescence (p=0.001). During the 9 month follow-up period (ITT), there were tumor recurrences in 128/271 patients (47%) in the fluorescence group and 157/280 patients (56%) in the white light group (p=0.026). The relative reduction in recurrence rate was 16%.
HAL fluorescence cystoscopy significantly improves detection of Ta and T1 lesions and significantly reduces the rate of tumor recurrence at 9 months.
PMCID: PMC4327891  PMID: 20850152
bladder cancer; Hexvix; HAL; fluorescence cystoscopy; recurrence
2.  Robot-assisted radical cystectomy and intracorporeal neobladder formation: on the way to a standardized procedure 
Robot-assisted radical cystectomy (RARC) with intracorporeal diversion has been shown to be feasible in a few centers of excellence worldwide, with promising functional and oncologic outcomes. However, it remains unknown whether the complexity of the procedure allows its duplication in other non-pioneer centers. We attempt to address this issue by presenting our cumulative experience with RARC and intracorporeal neobladder formation.
We retrospectively identified 62 RARCs in 50 men and 12 women (mean age 63.6 years) in two tertiary centers. Intracorporeal Studer neobladders were created, duplicating the steps of standard open surgery. Perioperative and postoperative variables and complications were analyzed using standardized tools. Functional and oncological results were assessed.
The mean operative time was 476.9 min (range, 310 to 690) and blood loss was 385 ml (200 to 800). The mean hospital stay was 16.7 (12 to 62) days with no open conversion. Perioperative complications were grade II in 15, grade III in 11, and grade IV in 5 patients. The mean nodal yield was 22.9 (8 to 46). Positive margins were found in in 6.4%. The 90- and 180-day mortality rates were 0% and 3.3%. The average follow-up was 37.3 months (3 to 52). Continence was achieved in 88% of patients. The cancer-specific survival rate and overall survival rate were 84% and 71%, respectively.
A RARC with intracorporeal neobladder creation is safe and reproducible in ‘non-pioneer’ tertiary centers with robotic expertise with acceptable operative time and complications. Further standardization of RARC with intracorporeal diversion is a prerequisite for its widespread use.
PMCID: PMC4326337  PMID: 25560783
intracorporeal diversion; laparoscopy; neobladder; radical cystectomy; robot-assisted
3.  Stepwise Application of Urine Markers to Detect Tumor Recurrence in Patients Undergoing Surveillance for Non-Muscle-Invasive Bladder Cancer 
Disease Markers  2014;2014:973406.
Background. The optimal use of urine markers in the surveillance of non-muscle-invasive bladder cancer (NMIBC) remains unclear. Aim of the present study was to investigate the combined and stepwise use of the four most broadly available urine markers to detect tumor recurrence in patients undergoing surveillance of NMIBC. Patients and Methods. 483 patients with history of NMIBC were included. Cytology, UroVysion, fluorescence in situ hybridization (FISH), immunocytology (uCyt+), and NMP22 ELISA were performed before surveillance cystoscopy. Characteristics of single tests and combinations were assessed by contingency analysis. Results. 128 (26.5%) patients had evidence of tumor recurrence. Sensitivities and negative predictive values (NPVs) of the single tests ranged between 66.4–74.3 and 82.3–88.2%. Two-marker combinations showed sensitivities and NPVs of 80.5–89.8 and 89.5–91.2%. A stepwise application of the two-test combinations with highest accuracy (cytology and FISH; cytology and uCyt+; uCyt+ and FISH) showed NPVs for high-risk recurrences (G3/Cis/pT1) of 98.8, 98.8, and 99.1%, respectively. Conclusions. Combinations of cytology, FISH, immunocytology, and NMP22 show remarkable detection rates for recurrent NMIBC. Stepwise two-test combinations of cytology, FISH, and immunocytology have a low probability of missing a high-risk tumor. The high sensitivities may justify the use of these combinations in prospective studies assessing the use of urine markers to individualize intervals between cystoscopies during follow-up.
PMCID: PMC4284969  PMID: 25587206
4.  Chromosomal alterations in exfoliated urothelial cells from bladder cancer cases and healthy men: a prospective screening study 
BMC Cancer  2014;14(1):854.
Chromosomal instability in exfoliated urothelial cells has been associated with the development of bladder cancer. Here, we analyzed the accumulation of copy number variations (CNVs) using fluorescence in situ hybridization in cancer cases and explored factors associated with the detection of CNVs in tumor-free men.
The prospective UroScreen study was designed to investigate the performance of UroVysion™ and other tumor tests for the early detection of bladder cancer in chemical workers from 2003–2010. We analyzed a database compiling CNVs of chromosomes 3, 7, and 17 and at 9p21 that were detected in 191,434 exfoliated urothelial cells from 1,595 men. We assessed the accumulation of CNVs in 1,400 cells isolated from serial samples that were collected from 18 cancer cases up to the time of diagnosis. A generalized estimating equation model was applied to evaluate the influence of age, smoking, and urine status on CNVs in cells from tumor-free men.
Tetrasomy of chromosomes 3, 7 and 17, and DNA loss at 9p21 were the most frequently observed forms of CNV. In bladder cancer cases, we observed an accumulation of CNVs that started approximately three years before diagnosis. During the year prior to diagnosis, cells from men with high-grade bladder cancer accumulated more CNVs than those obtained from cases with low-grade cancer (CNV < 2: 7.5% vs. 1.1%, CNV > 2: 16-17% vs. 9-11%). About 1% of cells from tumor-free men showed polysomy of chromosomes 3, 7, or 17 or DNA loss at 9p21. Men aged ≥50 years had 1.3-fold more cells with CNVs than younger men; however, we observed no further age-related accumulation of CNVs in tumor-free men. Significantly more cells with CNVs were detected in samples with low creatinine concentrations.
We found an accumulation of CNVs during the development of bladder cancer starting three years before diagnosis, with more altered cells identified in high-grade tumors. Also, a small fraction of cells with CNVs were exfoliated into urine of tumor-free men, mainly exhibiting tetraploidy or DNA loss at 9p21. Whether these cells are preferentially cleared from the urothelium or are artifacts needs further exploration.
PMCID: PMC4247705  PMID: 25412927
Aneuploidy; Bladder cancer; Chromosomal instability; Copy number variation; DNA gain; DNA loss; Fluorescence in situ hybridization; Tetrasomy
6.  Accurate Risk Assessment of Patients with Asymptomatic Hematuria for the Presence of Bladder Cancer 
World journal of urology  2012;30(6):847-852.
Bladder cancer is frequently diagnosed during a workup for hematuria. However, most patients with microscopic hematuria and many with gross hematuria are not appropriately referred to urologists. We hypothesized that in patients presenting with asymptomatic hematuria, the risk of having bladder cancer can be predicted with high accuracy. Towards this end, we analyzed risk factors in patients with asymptomatic hematuria and developed a nomogram for the prediction of bladder cancer presence.
Data from 1,182 consecutive subjects without a history of bladder cancer undergoing initial evaluation for asymptomatic hematuria were collected at three centers. Clinical risk factors including age, gender, smoking status, and degree of hematuria were recorded. All subjects underwent standard workup including voided cytology, upper tract imaging, and cystourethroscopy. Factors associated with the presence of bladder cancer were evaluated by univariable and multivariable logistic regression analyses. The multivariable analysis was used to construct a nomogram. Internal validation was performed using 200 bootstrap samples.
Of the 1,182 subjects who presented with asymptomatic hematuria, 245 (20.7%) had bladder cancer. Increasing age (OR=1.03, p<0.0001), smoking history (OR=3.72, p<0.0001), gross hematuria (OR=1.71, p=0.002), and positive cytology (OR=14.71, p<0.0001) were independent predictors of bladder cancer presence. The multivariable model achieved 83.1% accuracy for predicting the presence of bladder cancer.
Bladder cancer presence can be predicted with high accuracy in patients who present with asymptomatic hematuria. We developed a nomogram to help optimize referral patterns (i.e., timing and prioritization) of patients with asymptomatic hematuria.
PMCID: PMC4004026  PMID: 23124847
urinary bladder neoplasms; hematuria; nomograms; early detection of cancer; carcinoma
7.  Galectin-1 and Galectin-3 mRNA expression in renal cell carcinoma 
Galectins are known to regulate cell differentiation and growth as well as cell adhesion and apoptosis. Galectins have been discussed as possible prognosticators for survival in renal cell cancer (RCC) and other urological tumors. They might also play an emerging role as possible new marker-proteins for RCC. In this study, we analyzed the expression of galectin-1 and galectin-3 mRNA in order to further investigate their clinical significance in RCC.
Tissue samples were obtained from 106 patients undergoing surgery for RCC. The expression of galectin-1 and galectin-3 mRNA in normal kidney and corresponding cancer tissue was analyzed using quantitative real time PCR. Differences in expression levels of paired tissue samples were assessed using paired two-sample tests. Associations of relative mRNA expression levels in tumor tissues with clinical findings were analyzed using univariate logistic regression.
The expression of galectin-1 (p < 0.001) and -3 (p < 0.001) mRNA were significantly higher in RCC when compared to the adjacent normal kidney tissue. For clear cell RCC, an association of male gender with higher galectin-1 and galectin-3 mRNA expression (p = 0.054, p = 0.034) was detected. For all RCCs, galectin-1 mRNA expression failed to show a significant association with advanced disease as well as a higher rate of lymph node metastases (p = 0.058, p = 0.059).
The mRNA expression of galectin-1 and galectin-3 is significantly increased in RCC cancer tissue. The higher mRNA expression in tumor tissue of male patients raises the question of a functional connection between galectins and the higher prevalence of RCC in men. Associations with advanced disease might lead to new ways of identifying patients at higher risk of recurrent disease and might even facilitate early metastasectomy with curative intent.
PMCID: PMC4026056  PMID: 24708743
Galectin; Renal cell carcinoma; Biomarker; Prognosis
8.  Neurofilament Heavy polypeptide CpG island methylation associates with prognosis of renal cell carcinoma and prediction of antivascular endothelial growth factor therapy response 
Cancer Medicine  2014;3(2):300-309.
Neurofilament Heavy polypeptid (NEFH) belongs to the group of type IV intermediate filament proteins. DNA methylation of the NEFH promoter and loss of expression have previously been shown to activate the AKT/β-catenin pathway in tumor cells. When identifying hypermethylation of the NEFH CpG island (CGI) in renal cell cancer (RCC) we asked whether methylation could provide clinical or prognostic information for RCC and/or predict therapy response in patients with metastatic RCC (mRCC) undergoing antiangiogenic therapy. Relative methylation of the NEFH CGI was analyzed in 132 RCC samples and 83 paired normal tissues using quantitative methylation-specific PCR. Results were statistically compared with tumor histology, clinicopathological parameters, progression-free survival (PFS) as well as with overall survival (OS) in a subset of 18 mRCC patients following antiangiogenic therapy regimens. The NEFH CGI methylation demonstrated a tumor-specific increase (P < 0.001), association with advanced disease (P < 0.001), and distant metastasis (P = 0.005). Higher relative methylation was also significantly associated with a poor PFS (HR = 8.6, P < 0.001) independent from the covariates age, gender, diameter of tumors, state of advanced disease, and local and distant metastasis. Median OS following targeted therapy was 29.8 months for patients with low methylation versus 9.8 months for the group with high methylation (P = 0.028). We identified NEFH methylation as a candidate epigenetic marker for prognosis of RCC patients as well as prediction of anti-vascular endothelial growth factor-based therapy response.
PMCID: PMC3987080  PMID: 24464810
Methylation; risk assessment; translational research; urological oncology
9.  GATA5 CpG island hypermethylation is an independent predictor for poor clinical outcome in renal cell carcinoma 
Oncology Reports  2014;31(4):1523-1530.
Transcriptional inactivation and CpG island (CGI) methylation of GATA transcription factor family members GATA3 and GATA5 have been reported for a few types of human cancer. Whether high-density CGI methylation of GATA3 or GATA5 is associated with the clinical course of patients with renal cell cancer (RCC) has not been clarified. Quantitative methylation-specific PCR assays were carried out to analyze 25 tumor cell lines including 6 RCC lines and 119 RCC and 87 adjacent normal tissues for the presence of densely methylated sequences. Methylation values were statistically compared with clinicopathological and recurrence-free survival (RFS) data for patients. Comparison of GATA3 and GATA5 methylation in different tumor cell lines revealed a marker-specific methylation characteristic with high and frequent signals for both methylation marks in RCC lines. GATA3 and GATA5 CGI relative methylation levels were found to be strongly associated with the state of metastasis (P=0.003 and P<0.001, respectively) and advanced disease (P=0.024 and P<0.001, respectively). Moreover, an independent decrease in RFS in Cox proportional hazard analysis was found for tumors exhibiting high GATA5 methylation (P<0.001, hazard ratio, 19.3; 95% confidence interval, 4.58–81.6). Epigenetic alterations in GATA family members may be associated with aggressive tumor phenotypes in RCC, and in the case of GATA5, may serve as a new independent molecular marker for aggressiveness and disease progression.
PMCID: PMC3975988  PMID: 24549248
GATA3; GATA5; renal cell cancer; DNA hypermethylation; survival; prognosis
10.  Decreased GATA5 mRNA expression associates with CpG island methylation and shortened recurrence-free survival in clear cell renal cell carcinoma 
BMC Cancer  2014;14:101.
GATA-5, a zinc-finger transcription factor and member of the GATA family proteins 1–6, is known to be involved in cellular differentiation. We recently found that tumor-specific hypermethylation of the GATA5 CpG island (CGI) occurs in renal cell carcinoma (RCC) and is associated with an adverse clinical outcome. In this study, we investigated whether epigenetic GATA5 alterations may result in changes in GATA5 mRNA expression levels and correlate with the observed prognostic impact of epigenetic changes in GATA5 in RCC.
Quantitative real-time reverse-transcribed polymerase chain reaction was applied to measure relative GATA5 mRNA expression levels in 135 kidney tissue samples, including 77 clear cell RCC (ccRCC) tissues and 58 paired adjacent normal renal tissue samples. Relative GATA5 expression levels were determined using the ΔΔCt method and detection of three endogenous control genes then compared to previously measured values of relative methylation.
The mean relative GATA5 mRNA expression level exhibited an approximately 31-fold reduction in tumor specimens compared with corresponding normal tissues (p < 0.001, paired t-test). Decreased GATA5 mRNA expression was inversely correlated with increased GATA5 CGI methylation (p < 0.001) and was associated with shortened recurrence-free survival in ccRCC patients (p = 0.023, hazard ratio = 0.25).
GATA5 mRNA expression is decreased in ccRCC, likely due to gene silencing by methylation of the GATA5 CGI. Moreover, reduced GATA5 mRNA levels were associated with a poor clinical outcome, indicating a possible role of GATA5 for the development of aggressive ccRCC phenotypes.
PMCID: PMC3930894  PMID: 24533449
GATA5; Renal cell carcinoma; mRNA; Prognosis; DNA methylation
11.  Prospective evaluation of fluorescence-guided cystoscopy to detect bladder cancer in a high-risk population: results from the UroScreen-Study 
SpringerPlus  2014;3:24.
To prospectively evaluate the role of fluorescence-guided cystoscopy in a high-risk bladder cancer population undergoing screening based on a multi-marker panel of urine-tests (UroScreen-study).
Patients and methods
UroScreen was conducted as a validation study for tumor markers within the frame of a health surveillance program of workers with occupational exposure to aromatic amines. Voluntary annual screens were done in 1,609 men. Cytology, quantitative NMP22® assay, and UroVysion (FISH) were applied to 7091 urine samples. Subjects with at least one positive urine-based tumor marker and/or persisting microscopic hematuria were offered fluorescence-guided (PDD) instead of white light cystoscopy. In case of suspicious findings histopathological evaluation by transurethral biopsy was performed. Data were statistically summarized and compared to tumors found by the standard algorithm of the screening study.
Twenty-two subjects with a mean age of 58 years (39–72) underwent PDD cystoscopy. Of those 3 had positive NMP22 tests, 14 positive FISH tests and 9 suspicious cytologies. Two had persisting microscopic hematuria only. PDD cystoscopy revealed enhanced unifocal fluorescence in 14. All had subsequent transurethral biopsy or resection. In total, 1 urothelial carcinoma (pTaG1, low grade) was diagnosed. In the other participants urothelial cancer of the bladder was ruled out. Chronic cystitis was revealed in 8 of 14 biopsies. No higher detection rate was found using PDD than with the standard algorithm of the UroScreen study in which 17 tumors were detected by white light cystoscopy.
The use of PDD does not lead to a higher cancer detection rate in a high-risk screening population. Larger sample sizes may be needed to ultimately asses the value of PDD for bladder cancer screening.
PMCID: PMC3905106  PMID: 24478941
Urothelial cancer of the bladder; Urine based tumor marker; Bladder cancer screening; NMP22; UroVysion; UroScreen; Cytology; Photodynamic diagnostics; Cystoscopy
13.  Intracorporeal ileal ureter replacement using laparoscopy and robotics 
Ileal ureter is a suitable treatment option for patients with long ureteric strictures. Minimally invasive techniques have been shown to be as safe as open techniques but superior in terms of post–operative recovery. We report our experience using minimally invasive techniques for total intracorporeal ureteral replacement.
Material and methods
A chart review revealed five patients who underwent intracorporeal ileal ureter using minimally invasive techniques in the preceding 5 years. 4 patients underwent conventional laparoscopic surgery and 1 patient underwent robotic–assisted surgery. Patient's characteristics, perioperative data and functional outcomes as well as a detailed description of surgical technique are reported. In all 5 of these patients, the ileal ureter was performed completely intracorporeally.
The median age of our patients is 61 (range 42–73). The median operative time was 250 minutes (range 150–320) and median blood loss was 100 ml (range 50–200). The median hospital stay was 8 days (range 6–10) and there were no major perioperative complications reported. At median follow up of 22 months (range 4–38), there were no recurrences of strictures or any other complications.
We have demonstrated the safety and feasibility of minimally invasive intracorporeal ileal ureter. Numbers are still small but its application is likely to grow further.
PMCID: PMC4310887  PMID: 25667767
ileal ureter; ileal interposition; minimally invasive; laparoscopy; intracorporeal; ureteric strictures; robotics
14.  Stem cell therapy for voiding and erectile dysfunction 
Nature reviews. Urology  2012;10.1038/nrurol.2012.111.
Voiding dysfunction comprises a variety of disorders, including stress urinary incontinence and overactive bladder, and affects millions of men and women worldwide. Erectile dysfunction (ED) also decreases quality of life for millions of men, as well as for their partners. Advanced age and diabetes are common comorbidities that can exacerbate and negatively impact upon the development of these disorders. Therapies that target the pathophysiology of these conditions to halt progression are not currently available. However, stem cell therapy could fill this therapeutic void. Stem cells can reduce inflammation, prevent fibrosis, promote angiogenesis, recruit endogenous progenitor cells, and differentiate to replace damaged cells. Adult multipotent stem cell therapy, in particular, has shown promise in case reports and preclinical animal studies. Stem cells have also enabled advances in urological tissue engineering by facilitating ex vivo construction of bladder wall and urethral tissue (using a patient's own cells) prior to transplantation. More recent studies have focused on bioactive factor secretion and homing of stem cells. In the future, clinicians are likely to utilize allogeneic stem cell sources, intravenous systemic delivery, and ex vivo cell enhancement to treat voiding dysfunction and ED.
PMCID: PMC3769422  PMID: 22710667
stem cell therapy; urology; tissue engineering; voiding; bladder dysfunction; urinary incontinence; erectile dysfunction; MSC; urothelium
15.  A biomarker based detection and characterization of carcinomas exploiting two fundamental biophysical mechanisms in mammalian cells 
BMC Cancer  2013;13:569.
Biomarkers allowing the characterization of malignancy and therapy response of oral squamous cell carcinomas (OSCC) or other types of carcinomas are still outstanding. The biochemical suicide molecule endonuclease DNaseX (DNaseI-like 1) has been used to identify the Apo10 protein epitope that marks tumor cells with abnormal apoptosis and proliferation. The transketolase-like protein 1 (TKTL1) represents the enzymatic basis for an anaerobic glucose metabolism even in the presence of oxygen (aerobic glycolysis/Warburg effect), which is concomitant with a more malignant phenotype due to invasive growth/metastasis and resistance to radical and apoptosis inducing therapies.
Expression of Apo10 and TKTL1 was analysed retrospectively in OSCC specimen (n = 161) by immunohistochemistry. Both markers represent independent markers for poor survival. Furthermore Apo10 and TKTL1 have been used prospectively for epitope detection in monocytes (EDIM)-blood test in patients with OSCC (n = 50), breast cancer (n = 48), prostate cancer (n = 115), and blood donors/controls (n = 74).
Positive Apo10 and TKTL1 expression were associated with recurrence of the tumor. Multivariate analysis demonstrated Apo10 and TKTL1 expression as an independent prognostic factor for reduced tumor-specific survival. Apo10+/TKTL1+ subgroup showed the worst disease-free survival rate in OSCC.
EDIM-Apo10 and EDIM-TKTL1 blood tests allowed a sensitive and specific detection of patients with OSCC, breast cancer and prostate cancer before surgery and in after care. A combined score of Apo10+/TKTL1+ led to a sensitivity of 95.8% and a specificity of 97.3% for the detection of carcinomas independent of the tumor entity.
The combined detection of two independent fundamental biophysical processes by the two biomarkers Apo10 and TKTL1 allows a sensitive and specific detection of neoplasia in a noninvasive and cost-effective way. Further prospective trials are warranted to validate this new concept for the diagnosis of neoplasia and tumor recurrence.
PMCID: PMC4235042  PMID: 24304513
Biomarker; DNaseX; Apo10; TKTL1; EDIM (epitope detection in monocytes); EDIM-blood test; Early detection and diagnosis
17.  A Double-Blind, Randomized Phase II Study to Evaluate the Safety and Efficacy of Acetyl-L-Carnitine in the Prevention of Sagopilone-Induced Peripheral Neuropathy 
The Oncologist  2013;18(11):1190-1191.
Peripheral neuropathy (PN) is a recognized side effect of microtubule-targeting agents and the most clinically relevant toxicity observed with the epothilone sagopilone (SAG). Studies suggest that acetyl-L-carnitine (ALC) may prevent chemotherapy-induced PN. We conducted a prospective, placebo (PBO)-controlled, double-blind, randomized trial to investigate the safety and efficacy of ALC for the prevention of SAG-induced PN.
Patients with ovarian cancer (OC) or castration-resistant prostate cancer (CRPC) and no evidence of neuropathy received SAG (16 mg/m2 intravenously over 3 hours every 3 weeks) with ALC (1,000 mg every 3 days) or placebo (PBO). The primary endpoint was incidence of PN within six or fewer cycles in both treatment groups.
Overall, 150 patients enrolled (98 OC patients, 52 CRPC patients), with 75 per treatment arm. No significant difference in overall PN incidence was observed between treatment arms. The incidence of grade ≥3 PN was significantly lower in the ALC arm in OC patients. Median duration of neuropathy was similar between treatment arms. The best overall response (according to the modified Response Evaluation Criteria in Solid Tumors), response according to tumor markers, time-to-event variables, and discontinuations because of adverse events (AEs) were comparable between treatment arms.
Administration of ALC with SAG did not result in a significant difference in overall PN incidence compared with a PBO. OC patients in the SAG/ALC arm had a significantly lower incidence of grade 3 or 4 PN compared with OC patients in the SAG/PBO arm.
PMCID: PMC3825303  PMID: 24105751
18.  Clonal expansion of renal cell carcinoma-infiltrating T lymphocytes 
Oncoimmunology  2013;2(9):e26014.
T lymphocytes can mediate the destruction of cancer cells by virtue of their ability to recognize tumor-derived antigenic peptides that are presented on the cell surface in complex with HLA molecules and expand. Thus, the presence of clonally expanded T cells within neoplastic lesions is an indication of ongoing HLA-restricted T cell-mediated immune responses. Multiple tumors, including renal cell carcinomas (RCCs), are often infiltrated by significant amounts of T cells, the so-called tumor-infiltrating lymphocytes (TILs). In the present study, we analyzed RCC lesions (n = 13) for the presence of expanded T-cell clonotypes using T-cell receptor clonotype mapping. Surprisingly, we found that RCCs comprise relatively low numbers of distinct expanded T-cell clonotypes as compared with melanoma lesions. The numbers of different T-cell clonotypes detected among RCC-infiltrating lymphocytes were in the range of 1–17 (median = 5), and in several patients, the number of clonotypes expanded within tumor lesions resembled that observed among autologous peripheral blood mononuclear cells. Moreover, several of these clonotypes were identical in TILs and PBMCs. Flow cytometry data demonstrated that the general differentiation status of CD8+ TILs differed from that of circulating CD8+ T cells. Furthermore, PD-1 and LAG-3 were expressed by a significantly higher percentage of CD8+ RCC-infiltrating lymphocytes as compared with PBMCs obtained from RCC patients or healthy individuals. Thus, CD8+ TILs display a differentiated phenotype and express activation markers as well as surface molecules associated with the inhibition of T-cell functions. However, TILs are characterized by a low amount of expanded T-cell clonotypes.
PMCID: PMC3820815  PMID: 24228230
T-cell phenotypes; T-cell receptor clonotype mapping; clonally expanded T cells; renal cell carcinoma; tumor-infiltrating lymphocytes
19.  Circulating Fibronectin Controls Tumor Growth12 
Neoplasia (New York, N.Y.)  2013;15(8):925-938.
Fibronectin is ubiquitously expressed in the extracellular matrix, and experimental evidence has shown that it modulates blood vessel formation. The relative contribution of local and circulating fibronectin to blood vessel formation in vivo remains unknown despite evidence for unexpected roles of circulating fibronectin in various diseases. Using transgenic mouse models, we established that circulating fibronectin facilitates the growth of bone metastases by enhancing blood vessel formation and maturation. This effect is more relevant than that of fibronectin produced by endothelial cells and pericytes, which only exert a small additive effect on vessel maturation. Circulating fibronectin enhances its local production in tumors through a positive feedback loop and increases the amount of vascular endothelial growth factor (VEGF) retained in the matrix. Both fibronectin and VEGF then cooperate to stimulate blood vessel formation. Fibronectin content in the tumor correlates with the number of blood vessels and tumor growth in the mouse models. Consistent with these results, examination of three separate arrays from patients with breast and prostate cancers revealed that a high staining intensity for fibronectin in tumors is associated with increased mortality. These results establish that circulating fibronectin modulates blood vessel formation and tumor growth by modifying the amount of and the response to VEGF. Furthermore, determination of the fibronectin content can serve as a prognostic biomarker for breast and prostate cancers and possibly other cancers.
PMCID: PMC3730044  PMID: 23908593
20.  Impact of an Altered Wnt1/β-Catenin Expression on Clinicopathology and Prognosis in Clear Cell Renal Cell Carcinoma 
In renal cell carcinoma (RCC), single members of the Wnt/β-catenin signaling cascade were recently identified to contribute to cancer progression. However, the role of Wnt1, one of the key ligands in β-catenin regulation, is currently unknown in RCC. Therefore, alterations of the Wnt1/β-catenin axis in clear cell RCC (ccRCC) were examined with regard to clinicopathology, overall survival (OS) and cancer specific survival (CSS). Corresponding ccRCCs and benign renal tissue were analyzed in 278 patients for Wnt1 and β-catenin expression by immunohistochemistry in tissue microarrays. Expression scores, including intensity and percentage of stained cells, were compared between normal kidney and ccRCCs. Data was categorized according to mean expression scores and correlated to tumor and patients’ characteristics. Survival was analyzed according to the Kaplan-Meier and log-rank test. Univariable and multivariable Cox proportional hazard regression models were used to explore the independent prognostic value of Wnt1 and β-catenin. In ccRCCs, high Wnt1 was associated with increased tumor diameter, stage and vascular invasion (p ≤ 0.02). High membranous β-catenin was associated with advanced stage, vascular invasion and tumor necrosis (p ≤ 0.01). Higher diameter, stage, node involvement, grade, vascular invasion and sarcomatoid differentiation (p ≤ 0.01) were found in patients with high cytoplasmic β-catenin. Patients with a high cytoplasmic β-catenin had a significantly reduced OS (hazard ratio (HR) 1.75) and CSS (HR 2.26), which was not independently associated with OS and CSS after adjustment in the multivariable model. Increased ccRCC aggressiveness was reflected by an altered Wnt1/β-catenin signaling. Cytoplasmic β-catenin was identified as the most promising candidate associated with unfavorable clinicopathology and impaired survival. Nevertheless, the shift of membranous β-catenin to the cytoplasm with a subsequently increased nuclear expression, as shown for other malignancies, could not be demonstrated to be present in ccRCC.
PMCID: PMC3709711  PMID: 23708097
renal cell cancer; Wnt1; β-catenin; carcinogenesis; prognosis; targeted therapy
21.  A Rare Case of Synchronous Renal Cell Carcinoma of the Bladder Presenting With Gross Hematuria 
Rare Tumors  2013;5(2):72-74.
A 57-year old man was referred to the Urology Department due to gross hematuria; abdominal ultrasound revealed an unspecific solid tumor of the left bladder wall. Ultrasound, transurethral resection of the bladder mass with subsequent histological analysis, thoracic and abdominal computed tomography-scan and brain magnetic resonance imaging were performed. He was diagnosed with a bladder metastasis of clear cell renal cell carcinoma (RCC) with concomitant bone, pulmonary, and cerebral metastatic disease of a primary RCC of the right kidney. Management: Transurethral resection of the bladder mass, cerebral and bone radiotherapy, removal of the primary tumor, targeted systemic therapy with mTOR followed by tyrosine kinase inhibition.
PMCID: PMC3719114  PMID: 23888219
renal cell cancer; bladder metastasis; transurethral resection; hematuria
22.  Reduced mrna expression level of corticotropin-releasing hormone-binding protein is associated with aggressive human kidney cancer 
BMC Cancer  2013;13:199.
Significance of Urocortin (Ucn or UcnI), Ucn2, Ucn3 and their receptors, Corticotropin Releasing Factor Receptor 1 and 2 (CRFR1 and CRFR2), and the binding protein, Corticotropin-Releasing Hormone-Binding Protein (CRHBP) in oncology is growing rapidly. The objective of our study was to assess the expression of the CRHBP mRNA and protein in renal cancer.
Tumoral tissues of 78 patients with clear cell renal cell cancer and their corresponding normal tissues were analyzed using quantitative mRNA expression analysis for detection of mRNA expression level. Protein expression and tissue localization of CRHBP protein in renal specimens was evaluated using western blotting, immunohistochemistry and double immunofluorescence, respectively.
We found an approx. 33 fold decrease of average CRHBP mRNA level in tumoral tissues compared to paired normal tissues (p<0.001). Diminished CRHBP mRNA expression was positively correlated with advanced, metastasized and higher stage of disease (p<0.001, p=0.026, p=0.028 respectively). CRHBP protein was detected in glomeruli and proximal tubules of normal kidney while none or weak immunopositivity was found in cc-RCC (p<0.001).
The expression analysis of CRHBP shows that cc-RCC is characterized by a significant loss of CRHBP mRNA expression that furthermore is associated with a more aggressive state of tumors. Depletion of CRHBP proteins also indicate that the protein as part of the UCN system may be involved in renal carcinogenesis.
PMCID: PMC3653809  PMID: 23607589
23.  Immunocytology Is Strong Predictor of Bladder Cancer Presence in Patients With Painless Hematuria: A Multicentre Study 
European urology  2011;61(1):185-192.
Although the performance of immunocytology has been established in the surveillance of patients with urothelial carcinoma of the bladder (UCB), its value in the initial detection of UCB in patients with painless hematuria remains unclear.
To determine whether immunocytology improves our ability to predict the likelihood of UCB in patients with painless hematuria. Further, to test the clinical benefit of immunocytology in this setting using decision curve analysis.
Design, setting, and participants
The subjects were 1182 consecutive patients without a history of UCB presenting with painless hematuria and were enrolled at three centres.
All patients underwent upper-tract imaging, cystourethroscopy, voided urine cytology, and immunocytology analysis. Bladder tumors were biopsied and histologically confirmed as UCB.
Multivariable regression models were developed. Area under the curve was measured and compared using the DeLong test. A nomogram was constructed from the full multivariable model. Decision curve analysis was performed to evaluate the clinical benefit associated with use of the multivariable models including immunocytology.
Results and limitations
Immunocytology had the largest contribution to a multivariable model for the prediction of UCB (odds ratio: 18.3; p < 0.0001), which achieved a 90.8% predictive accuracy. Decision curve analysis revealed that models incorporating immunocytology achieved the highest net benefit at all threshold probabilities.
Immunocytology is a strong predictor of the presence of UCB in patients who present with painless hematuria. Incorporation of immunocytology into predictive models improves diagnostic accuracy by a statistically and clinically significant margin. The use of immunocytology in the diagnostic workup of patients with hematuria appears promising and should be further evaluated.
PMCID: PMC3628750  PMID: 21924544
Cystoscopy; Decision curve analysis; Early detection of cancer; Hematuria; Immunocytology; Nomograms; Urinary bladder neoplasms
24.  Targeted therapy in renal cell carcinoma: moving from molecular agents to specific immunotherapy 
World Journal of Urology  2013;32(1):31-38.
Non-specific immunotherapy has been for a long time a standard treatment option for patients with metastatic renal cell carcinoma but was redeemed by specific targeted molecular therapies, namely the VEGF and mTOR inhibitors. After moving treatment for mRCC to specific molecular agents with a well-defined mode of action, immunotherapy still needs this further development to increase its accuracy. Nowadays, an evolution from a rather non-specific cytokine treatment to sophisticated targeted approaches in specific immunotherapy led to a re-launch of immunotherapy in clinical studies. Recent steps in the development of immunotherapy strategies are discussed in this review with a special focus on peptide vaccination which aims at a tumor targeting by specific T lymphocytes. In addition, different combinatory strategies with immunomodulating agents like cyclophosphamide or sunitinib are outlined, and the effects of immune checkpoint modulators as anti-CTLA-4 or PD-1 antibodies are discussed.
PMCID: PMC3901931  PMID: 23404195
Renal cell carcinoma; Tyrosine kinase inhibitor; Immune therapy; Vaccination; IMA901
25.  Insulin Receptor Isoforms A and B as well as Insulin Receptor Substrates-1 and -2 Are Differentially Expressed in Prostate Cancer 
PLoS ONE  2012;7(12):e50953.
In different cancers types, insulin receptor isoform composition or insulin receptor substrate (IRS) isoforms are different to healthy tissue. This may be a molecular link to increased cancer risk in diabetes and obesity. Since this is yet unclear for prostate cancer, we investigated IR isoform composition and IRS balance in prostate cancer compared to benign and tumor adjacent benign prostate tissue and brought this into relation to cell proliferation.
We studied 23 benign prostate samples from radical cystectomy or benign prostatic hyperplasia surgery, 30 samples from benign tissue directly adjacent to prostate cancer foci and 35 cancer samples from different patients. RNA expression levels for insulin receptor isoforms A and B, IRS-1, IRS-2, and IGF-1 receptor were assessed by quantitative real-time RT-PCR. In addition, RNA- and protein expression of the cell cycle regulator p27Kip1 was quantified by real-time RT-PCR and immunohistochemistry.
Insulin receptor isoform A to B ratio was significantly higher in cancer as well as in tumor adjacent benign prostate tissue compared to purely benign prostates (p<0.05). IRS-1 to IRS-2 ratios were lower in malignant than in benign prostatic tissue (p<0.05). These altered ratios both in cancer and adjacent tissue were significantly associated with reduced p27Kip1 content (p<0.02). Interestingly, IGF-1 receptor levels were significantly lower in patients with type 2 diabetes (p = 0.0019).
We found significant differences in the insulin signaling cascade between benign prostate tissue and prostate cancer. Histological benign tissue adjacent to cancer showed expression patterns similar to the malignancies. Our findings suggest a role of the insulin signaling pathway in prostate cancer and surrounding tissue and can hence be relevant for both novel diagnostic and therapeutic approaches in this malignancy.
PMCID: PMC3519512  PMID: 23251408

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