Noninvasive diagnosis of urothelial cancer in urine using DNA hypermethylation signatures-Gender matters.

Urothelial cancer (UCa) is the most predominant cancer of the urinary tract and noninvasive diagnosis using hypermethylation signatures in urinary cells is promising. Here, we assess gender differences in a newly identified set of methylation biomarkers. UCa-associated hypermethylated sites were identified in urine of a male screening cohort (n = 24) applying Infinium-450K-methylation arrays and verified in two separate mixed-gender study groups (n = 617 in total) using mass spectrometry as an independent technique. Additionally, tissue samples (n = 56) of mixed-gender UCa and urological controls (UCt) were analyzed. The hypermethylation signature of UCa in urine was specific and sensitive across all stages and grades of UCa and independent on hematuria. Individual CpG sensitivities reached up to 81.3% at 95% specificity. Albeit similar methylation differences in tissue of both genders, differences were less pronounced in urine from women, most likely due to the frequent presence of squamous epithelial cells and leukocytes. Increased repression of methylation levels was observed at leukocyte counts ≥500/µl urine which was apparent in 30% of female and 7% of male UCa cases, further confirming the significance of the relative amounts of cancerous and noncancerous cells in urine. Our study shows that gender difference is a most relevant issue when evaluating the performance of urinary biomarkers in cancer diagnostics. In case of UCa, the clinical benefits of methylation signatures to male patients may outweigh those in females due to the general composition of women's urine. Accordingly, these markers offer a diagnostic option specifically in males to decrease the number of invasive cystoscopies.

Soluble chemokine (C-X-C motif) ligand 16 (CXCL16) in urine as a novel biomarker candidate to identify high grade and muscle invasive urothelial carcinomas.

Information on biomarkers of urothelial carcinomas (UC) for clinical decision-making is limited. Here, we newly identified and verified CXCL16 as a promising novel biomarker in urine for high grade and muscle invasive UC in a cross-sectional cohort of 308 UC patients, 126 urological hospital controls, and 50 population controls using antibody arrays and ELISA. Median CXCL16 levels in urine was significantly higher in UC patients (273.2 pg/mg creatinine) compared to hospital (148.1 pg/mg) and population controls (85.1 pg/mg) with a particular preference for high grade (460.8 pg/mg), muscle invasive (535.7 pg/mg) and primary UC (327.8 pg/mg) (all p<0.0001). Group differences were confirmed after adjusting or stratifying for potential clinical and individual characteristics, such as leukocyte counts, haematuria, age, gender, and smoking status. In contrast, CXCL16 showed less discriminating power in low grade (244.3 pg/mg), non-muscle invasive (≤pT1, 251.2 pg/mg) and recurrent UC (203.9 pg/mg). In agreement with its function in immune defence, expression of CXCL16 in tissue samples of primary UC patients (n=53) showed only a weak or no immunoreactivity compared to urological hospital controls (n=32). Expression of CXCR6, the G-protein-coupled receptor of CXCL16, remained unchanged. Our findings suggest that evading the immune defence by shedding cell-surface CXCL16 and its increased elimination in urine is a molecular feature of high grade and muscle invasive UC. Therefore, urinary CXCL16 may serve as a useful, simple and non-invasive tool to identify high-risk UC with increased risk of progression at the molecular level.

Automated quantification of FISH signals in urinary cells enables the assessment of chromosomal aberration patterns characteristic for bladder cancer.

Targeting the centromeres of chromosomes 3, 7, 17 (CEP3, 7, 17) and the 9p21-locus (LSI9p21) for diagnosing bladder cancer (BC) is time- and cost-intensive and requires a manual investigation of the sample by a well-trained investigator thus overall limiting its use in clinical diagnostics and large-scaled epidemiological studies. Here we introduce a new computer-assisted FISH spot analysis tool enabling an automated, objective and quantitative assessment of FISH patterns in the urinary sediment. Utilizing a controllable microscope workstation, the microscope software Scan^R was programmed to allow automatic batch-scanning of up to 32 samples and identifying quadruple FISH signals in DAPI-scanned nuclei of urinary sediments. The assay allowed a time- and cost-efficient, automated and objective assessment of CEP3, 7 and 17 FISH signals and facilitated the quantification of nuclei harboring specific FISH patterns in all cells of the urinary sediment. To explore the diagnostic capability of the developed tool, we analyzed the abundance of 51 different FISH patterns in a pilot set of urine specimens from 14 patients with BC and 21 population controls (PC). Herein, the results of the fully automated approach yielded a high degree of conformity when compared to those obtained by an expert-guided re-evaluation of archived scans. The best cancer-identifying pattern was characterized by a concurrent gain of CEP3, 7 and 17. Overall, our automated analysis refines current FISH protocols and encourages its use to establish reliable diagnostic cutoffs in future large-scale studies with well-characterized specimens-collectives.

The impact of real-time elastography guiding a systematic prostate biopsy to improve cancer detection rate: a prospective study of 353 patients.

PURPOSE: We evaluated whether real-time elastography guided biopsy improves prostate cancer detection compared to conventional systematic gray scale ultrasound guidance. MATERIALS AND METHODS: A total of 353 consecutive patients suspicious for prostate cancer were prospectively randomized for real-time elastography (178) or gray scale ultrasound (175). Each patient enrolled in the study underwent a 10-core prostate biopsy. Six lateral prostate sectors (base, mid, apex) were scanned for cancer suspicious areas, defined as stiffer blue lesions using real-time elastography and hypoechoic lesions using gray scale ultrasound. Suspicious areas were sampled by a single targeted biopsy and considered representative of a defined prostate sector. If real-time elastography or gray scale ultrasound did not visualize a suspicious area in a sector, the biopsy core was taken systematically. Imaging findings were correlated with histopathological reports. Real-time elastography and gray scale ultrasound cases were compared in terms of cancer detection rate and imaging guidance accuracy. RESULTS: Characteristics of patients undergoing real-time elastography and gray scale ultrasound, including age, prostate specific antigen, prostate volume and digital rectal examination, were not significantly different (p>0.05). Prostate cancer was detected in 160 of 353 patients (45.3%). The prostate cancer detection rate was significantly higher in patients who underwent biopsy with the real-time elastography guided approach compared to the gray scale ultrasound guided biopsy at 51.1% (91 of 178) vs 39.4% (69 of 175) (p=0.027). Overall sensitivity and specificity to detect prostate cancer was 60.8% and 68.4% for real-time elastography vs 15% and 92.3% for gray scale ultrasound, respectively. CONCLUSIONS: Sensitivity to visualize and detect prostate cancer improved using real-time elastography in addition to gray scale ultrasound during prostate biopsy. Overall sensitivity did not reach levels to omit a systematic biopsy approach.

Comparison of real-time elastography with grey-scale ultrasonography for detection of organ-confined prostate cancer and extra capsular extension: a prospective analysis using whole mount sections after radical prostatectomy.

OBJECTIVE: •To evaluate whether transrectal real-time elastography (RTE) improves the detection of intraprostatic prostate cancer (PCa) lesions and extracapsular extension (ECE) compared with conventional grey-scale ultrasonography (GSU). PATIENTS AND METHODS: •In total, 229 patients with biopsy-proven PCa were prospectively screened for cancer-suspicious areas and ECE using GSU and RTE. •The largest tumour focus detected by RTE was defined as the index lesion. •The prostate gland was stratified into six sectors on GSU and RTE, which were compared with histopathological whole mount sections after radical prostatectomy. RESULTS: •Histopathologically, PCa was confirmed in 894 out of 1374 (61.8%) evaluated sectors and ECE was identified in 47 (21%) patients. •Of these 894 sectors, RTE correctly detected 594 (66.4%) and GSU 215 (24.0%) cancer suspicious lesions. •Sensitivity was 51% and specificity 72% using RTE compared to 18% and 90% for GSU. •RTE identified the largest side specific tumour focus in 68% of patients. •ECE was identified with a sensitivity of 38% and specificity of 96% using RTE compared to 15% and 97% using GSU. CONCLUSIONS: •Compared with GSU, RTE provides a statistically significant improvement in detection of PCa lesions and ECE. •RTE enhances GSU, although improvement is still needed to achieve a clinically meaningful sensitivity.

Effect of keratinocyte growth factor and activin on cell growth in the human prostatic cancer cell line LNCaP.

Keratinocyte growth factor (KGF) has paracrine properties in the human prostate which stimulate epithelial cell growth. Activins have profound effects on cell growth and function in the human prostate, and are expressed in LNCaP, DU 145 and PC3 cells. LNCaP cells were characterized by immuncytochemistry, an immunoassay and polymerase chain reaction. A 3[H]thymidine assay was used with 0.01-10 nM dihydrotestosterone, 10 micro M flutamide, 1-100 ng/ml KGF and 3 nM activin. LNCaP cells expressed Ki67, PSA, cytokeratins (8, 18, 19, 14, 15) androgenreceptor but no KGF protein. LNCaP cells showed telomerase activity. Furthermore, ARmRNA (365 bp), but no KGF or KGFRmRNA were expressed. KGF ELISA detected no intracellular or secreted KGF. DHT (1, 10 and 100 nM) and KGF (10 and 100 ng/ml) significantly stimulated LNCaP cell proliferation. However, flutamide and 3 nM activin A significantly decreased cell proliferation in the presence and absence of KGF. The results of our experiments support the hypothesis that cell growth and proliferative characteristics of LNCaP cells are modulated by KGF and activin A.

A prospective randomized study of combined visual laser ablation and transurethral resection of the prostate versus transurethral prostatectomy alone.

INTRODUCTION: Visual laser ablation of the prostate (VLAP) has a clinical failure rate of up to 18% which is 3 times higher than transurethral resection of the prostate (TURP) alone. Prolonged spontaneous passage of necrotic debris is the major shortcoming of this method. Therefore combined visual laser-assisted and transurethral prostatectomy was compared to TURP alone. MATERIALS AND METHODS: 105 patients were evaluated in a prospective randomized study comparing TURP alone and VLAP combined with TURP. The patients were evaluated 1, 3 and 12 months after surgery. First VLAP was performed using a neodymium:yttrium-aluminum-garnet laser with the prolase fiber followed by standard resection of the necrotic and remaining prostatic tissue. Treatment efficacy was assessed by the American Urological Association (AUA) symptom score, measurements of peak urinary flow, residual urine volume, intraoperative bleeding, and by the occurrence of intra- and postoperative complications. RESULTS: The use of VLAP and consecutive TURP improved the AUA symptom score, urinary flow and residual volume and was comparable in all patients treated. Intraoperative bleeding was significantly reduced from 522 +/- 45 ml by TURP to 214 +/- 33 ml by VLAP+TURP (p < 0.05). There was a significant improvement in the postoperative values of the parameters observed in both groups. CONCLUSIONS: It appears that the combined method for treatment of benign prostatic hyperplasia reduced the specific intraoperative morbidity of TURP while achieving the same clinical effect as TURP alone.