Telomerase activity as a potential marker in preneoplastic bladder lesions

OBJECTIVE: To assess telomerase activity (involved in cell immortalization and detectable in most malignant tumours but not in normal somatic tissues) as a marker in cancer diagnosis. PATIENTS AND METHODS: Tissue telomerase activity was assayed by two different techniques, the telomeric repeat amplification protocol-polymerase chain reaction (TRAP-PCR) and a telomerase PCR-enzyme linked immunosorbent assay. Malignant and inflammatory bladder lesions and their adjacent normal tissues were assessed for telomerase activity in a group of 18 patients, 14 of whom had urothelial carcinoma and four a nonspecific inflammatory lesion of the bladder. RESULTS: Eleven of the 14 tumour samples analysed were telomerase-positive and two of the three telomerase-negative tumour samples had a detectable 'telomerase inhibitor'. In the apparently normal tissues next to bladder tumours, four of the 14 specimens were telomerase-positive. Interestingly, these lesions were always next to high-grade muscle-invasive bladder tumours (pT2G3). Two of the four nonspecific inflammatory lesions (one of cystitis glandularis and one of severe dysplasia), known to be preneoplastic lesions, were also telomerase-positive. CONCLUSION: These results strongly suggest that the reactivation of telomerase may be an early event in bladder carcinogenesis, preceding morphological changes related to malignant transformation. Telomerase activity may therefore be useful both as an indicator of malignant potential in preneoplastic lesions, e.g. cystitis glandularis and severe dysplasia, and as a prognostic marker of bladder tumour relapse or progression.     

The effect of tumor grade heterogeneity on recurrence in non-muscle invasive bladder cancer

ObjectiveTo investigate the outcomes of mixed-grade non-muscle invasive bladder cancer (NMIBC) based on the degree of high-grade predominance.MethodsWe identified patients in our institutional database who had a transurethral resection of bladder tumor(s) for NMIBC. Tumors with mixed-grade features on pathology report were reanalyzed, assigned the percentage high-grade component, and stratified into ≤ 5% high-grade and > 5% high-grade groups. All others were classified as low-grade or high-grade NMIBC. Differences in recurrence-free survival were assessed by log-rank test. A multivariable Cox regression model was used to evaluate the impact of tumor grade on recurrence, controlling for tumor stage, size, multifocality, and intravesical therapy.ResultsTwo hundred and twenty patients were followed for a median of 2 years; 127 (58%) had low-grade NMIBC, 66 (30%) had high-grade NMIBC, and 27 (12%) had mixed-grade NMIBC. Of the mixed-grade patients, 14 had a ≤ 5% high-grade component, and 13 had a > 5% high-grade component. Recurrence rates across all groups ranged from 42% to 79%. There was no significant difference in intravesical recurrence-free survival among the grade categories as assessed by log-rank test. On multivariable Cox regression analysis, grade category was not significantly associated with likelihood of recurrence.ConclusionsThe prognosis of mixed-grade histology in NMIBC has not previously been well defined. Although grade category was not found to be an independent significant predictor of recurrence, the recurrence rate for mixed-grade tumors was quite high overall. Further studies are required to better understand appropriate risk stratification and treatment of mixed-grade NMIBC.     

Transurethral Resection of Non–muscle-invasive Bladder Cancer

Transurethral resection of the bladder (TURB) is the initial and critical step in the management of bladder tumours. The aim of the procedure is to establish the histologic diagnosis, determine the tumour stage and grade, and achieve complete removal of papillary non–muscle-invasive tumours. Although TURB is a frequently performed procedure, its results are limited by the high recurrence rate and by the risk of tumour understaging. The major prerequisite for optimal outcomes is a systematically and meticulously performed procedure by a well-trained urologist. Smaller tumours can be resected en bloc; tumours >1 cm should be resected separately in fractions. Deep resection, including the detrusor muscle, is essential for correct staging. The biopsy should be taken from all areas suggestive of carcinoma in situ (CIS), and biopsies from normal-looking mucosa are recommended only in patients with positive cytology or non-papillary tumours. TURB should be performed with modern equipment, including new telescopes and video systems. Moreover, urologists should be aware of promising innovations, including new imaging techniques, and their possible benefits.Re-TUR can improve recurrence-free survival (RFS) and tumour staging. It is recommended in any patient with a T1 or high-grade tumour at initial resection and when the pathologist has reported that the specimen contained no muscle. It should also be considered in cases where the urologist is not sure that the initial resection was complete, especially in extensive and multiple tumours.     

Exome Sequencing of Prostate Cancer Supports the Hypothesis of Independent Tumour Origins

Background

Prostate cancer (PCa) is a clinically and pathologically heterogeneous disease. The rapid development of sequencing technology has the potential to deliver new biomarkers with emphasis on aggressive disease and to revolutionise personalised cancer treatment. However, a prostate harbouring cancer commonly contains multiple separate tumour foci, with the potential to aggravate tumour sampling. The level of intraprostatic tumour heterogeneity remains to be determined.

Objective

To determine the level of intraprostatic tumour heterogeneity through genome-wide, high-resolution profiling of multiple tumour samples from the same individual.

Design, settings, and participants

Multiple tumour samples were obtained from four individuals following radical prostatectomy. One individual (SWE-1) contained >70% cancer cells in all tumour samples, whereas the other three (SWE-2 to SWE-4) required the use of laser capture microdissection for tumour cell enrichment. Subsequently, DNA was extracted from all tissue samples, and exome sequencing was performed. All tumour foci of SWE-1 were also profiled using a high-resolution array for the identification of copy number alterations (CNA).

Outcome measurements and statistical analysis

Shared somatic high-frequency single nucleotide variants (SNV) and CNAs were used to infer the level of intraprostatic tumour heterogeneity.

Results and limitations

No high-frequency mutations, common for the three tumour samples of SWE-1, were identified. Ten randomly chosen positions were validated with Sanger sequencing in all foci, which verified the exome data. The high level of intraprostatic heterogeneity was consistent in all individuals. In total, three out of four individuals harboured tumours without an apparent common somatic denominator. Although we cannot exclude the presence of common structural rearrangements, a high-density array was used for the detection of deletions and amplifications in SWE-1, which agreed with the exome data.

Conclusions

We present evidence for the presence of somatically independent tumours within the same prostate. This finding will have implications for personalised cancer treatment and biomarker discovery.     

Large-scale real-time reverse transcription-PCR approach of angiogenic pathways in human transitional cell carcinoma of the bladder: identification of VEGFA as a major independent prognostic marker

Background

Actors of the angiogenesis pathways are targets for the new promising targeted therapies already used in several malignancies. In bladder cancer, antiangiogenic molecules could also add to already existing treatment options.

Objective

To evaluate the involvement of angiogenesis pathways in bladder carcinogenesis and identify new molecular markers having a clinical implication.

Design, setting, and participants

Expression levels of 40 genes involved in angiogenesis were assessed by quantitative real time RT-PCR in 157 urothelial tumour bladder samples obtained from patients who underwent transurethral bladder resection or radical cystectomy between 2001 and 2005. Pathologic tumour staging showed: 73 non-muscle-invasive bladder tumours (30 low-grade pTa, 14 high-grade pTa, and 29 high-grade pT1), and 84 muscle-invasive tumours (≥ pT2), all of high grade. RT-PCR results were associated with a survival analysis.

Results and limitations

VEGFA, MET, CXCR4, and IL8 were significantly overexpressed in tumour samples as compared to normal bladder tissue. VEGFA overexpressions were found in 89% of non-muscle-invasive and 66% of muscle-invasive tumour samples. In univariate analysis, for invasive tumours, VEGFA overexpression was associated with a poorer outcome in both overall and disease-free survival (p = 0.011 and 0.026 respectively) at a 13-mo median follow-up. Multivariate analysis retained T stage, N status, and VEGFA overexpression as independent prognostic factors in both overall and disease-free survival (p = 0.02 and p = 0.04, respectively, for VEGFA).

Conclusions

This study shows that, in bladder cancer, VEGFA status could be used as a prognostic factor at the individual level. VEGFA overexpression could guide a rationalized use of the costly antiangiogenic therapies which could therefore become part of the treatment options in bladder cancer.     

Human papillomavirus associated with bladder carcinoma? Analysis by polymerase chain reaction

BACKGROUND: The aim of the present study was to assess the possible etiologic role of human papillomaviruses (HPV) in bladder tumors. METHODS: Forty-two fresh biopsy specimens from different grades and stages of bladder tumor cases and 10 normal bladder mucosa biopsies were studied. Specimens were analyzed by polymerase chain reaction (PCR) with HPV-specific general primer set for the detection of viral DNA. Polymerase chain reaction-positive samples were also tested with HPV 16- and 18-specific primers by the same method. RESULTS: We found two samples (4.8%) containing HPV DNA among the TaG1 bladder tumors. All other specimens, including the control group, were found to be negative by PCR. Neither of the two HPV-positive patients had immune deficiency and/or genital wars. Human papillomavirus 16 was detected by type-specific primers in one sample, but the other HPV-positive sample could not be typed. CONCLUSIONS: The low prevalence of HPV in this and many previous studies does not support an etiologic role of HPV in bladder carcinogenesis. We detected the virus in two early stage tumors, but none was detected in the high-grade samples. However, to clarify the positivity of HPV in these occasional cases, future studies must be designed by using in situ PCR techniques, including samples from tumors and normal bladder mucosa from the same patient.     

Centrosome hyperamplification and chromosomal instability in bladder cancer

OBJECTIVE: Chromosomal instability (CIN) is a common feature of malignant tumors. Centrosome hyperamplification (CH) occurs frequently in human cancers, and may be a contributing factor in CIN. In this study, we investigated the relationship between CH and CIN in bladder cancer. METHODS: Clinical samples obtained by transurethral resection from 22 patients with bladder cancer were examined (histological grade G1, 5 cases; G2, 6 cases; G3, 11 cases). CH was evaluated by immunohistochemistry using anti-pericentrin antibody. CIN was evaluated by fluorescence in situ hybridization (FISH). FISH probes for pericentromeric regions of chromosomes 3, 7, and 17 were hybridized to touch preparations of nuclei from frozen tissues. We also analyzed the centrosome replication cycle of bladder cancer by laser scanning cytometry (LSC). RESULTS: Of the 22 cases examined, 18 (81.8%) had centrosome hyperamplification: CH 0, 4 cases (18.1%); CH I, 5 cases (22.7%); CH II, 5 cases (22.7%); CH III, 8 cases (36.4%). The grade of CH was directly proportional to the histological grade (p=0.03, chi(2) test). LSC analysis showed that the centrosome replication cycle was well regulated in pathologically low-grade bladder cancer, which did not have chromosomal instability. In contrast, we found marked variability of centrosomes in pathologically high-grade bladder cancer, which had chromosomal instability. CH and CIN were both detected in pathologically high-grade tumors. The grade of CH was directly proportional to the CIN grade (p=0.0079, chi(2) test). CONCLUSION: The results of the present study suggest that CH may be involved in CIN in bladder cancer.     

Improved application of impulse cytophotometry for the diagnosis of urinary bladder carcinoma

Summary Using pulse cytophotometry, almost quantitative separation of the leucocyte fraction from DNA histograms was possible by means of an anticoincidence discrimination device. This modified technique was employed for biparametric DNA/protein measurements of voided urine samples, bladder washings, and tumour tissues. The results show a high degree of correlation between these samples so that, for tumour diagnosis from DNA histograms, voided urine specimens can be used rather than bladder washings. The criteria for the bladder tumour diagnosis are derived from DNA measurements of 32 controls and 35 tumour patients. The diagnostic sensitivity of this method is 0.91 and the specifity 0.75.Supported by grant No. 01 ZO 029-ZA/NT/MT 299 of the DEVLR (ministry of research and technology)     

Micropapillary carcinoma of the urinary bladder: a case report and review of the literature

Micropapillary carcinoma is an uncommon variant of urothelial carcinoma with high metastatic potential. The presence of micropapillary carcinoma component in bladder biopsies should alert urologists to its aggressive behaviour. We report the case of a 70-year-old man who presented with macroscopic hematuria lasting 2 weeks. Magnetic resonance imaging revealed a bladder tumour in the dome area extended to perivascular adipose. The transurethral biopsy showed a high-grade micropapillary carcinoma with muscle invasion. Radical cystectomy with lymph node dissection was then performed. The pathological examination revealed a high-grade purely micropapillary carcinoma invading the perivesical adipose. No tumour recurrence or metastasis were reported at the 6-month follow-up.     

LBH与肝纤维化进展及肝内免疫细胞分布的关联研究

背景与目的 研究发现,肝内免疫调节与肝纤维化发生发展密切相关。然而,在肝纤维化中与免疫细胞相关联的关键基因目前仍不清楚。因此,本研究探讨肝纤维化中的关键基因与疾病进展以及肝内免疫细胞分布的关联。方法 基于公共数据库中的不同肝纤维化程度的肝组织（GSE162694和GSE49541）,对转录组测序RNA-seq测序数据进行差异表达比较,以正常肝样本作为对照;通过相对表达丰度排秩（REO）算法结果获得相关逆转稳定基因对;分析与肝纤维化相关的关键基因。构建CCl₄诱导的肝纤维化小鼠模型,Masson染色鉴定病理组织学改变,qRT-PCR和Western blot方法检测关键基因的mRNA与蛋白表达;xCell工具分析关键基因与免疫细胞、肝纤维化进展的关系。结果 分析测序数据获得两个肝纤维化相关的交叠的逆转基因对（THBS2>RHDE及LBH>LRRC19）;其中THBS2、LBH均在肝纤维化组织中差异上调,并且LBH表达与肝纤维化分级、组织学评分和炎症评分明显相关（均P<0.05）。与对照组比较,模型组小鼠肝脏中LBH mRNA及蛋白水平均明显上调（均P<0.05）。基于LBH表达中位值,将肝纤维化芯片GSE162694、GSE49541中的样本分为LBH高表达组与LBH低表达组,xCell分析显示,CD4⁺记忆T细胞、中央记忆CD8⁺T细胞、树突状细胞、aDC、cDC等免疫细胞富集水平及免疫细胞评分在LBH高表达组中均明显上调（均P<0.05）。结论 转录辅助因子LBH可能参与调节肝内免疫细胞分布并促进肝纤维化的进展。     

Urothelkarzinom der Harnblase

Background

Transurethral resection of transitional cell carcinoma of the bladder provides a definitive surgical treatment and supplies tissue for histological evaluation. Superficial low-grade carcinomas with a small risk of progression are treated properly with fulguration alone. To justify fulguration as a definitive treatment of papillary bladder tumours, one must be able to safely distinguish low-grade, noninvasive tumours from those that are high grade and potentially invasive.

Material and methods

A total of 160 patients with a transitional cell carcinoma at cystoscopy underwent transurethral resection of the tumour. The macroscopic appearance of the tumour, the aspect with bimanual palpation and the perioperative urine cytology were compared with the histological report.

Results

In our study we were able to safely distinguish low-grade tumours from high-grade tumours. All noninvasive tumours could be identified visually as such.

Conclusion

Urologists skilled in the evaluation of urine cytology can distinguish low-grade noninvasive tumours of the bladder from high-grade and potentially invasive tumours by means of appearance at cystoscopy and perioperative urine cytology.     

Her2 amplification is significantly more frequent in lymph node metastases from urothelial bladder cancer than in the primary tumours

Background

Her2, an alias for the protein of v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian), might be an attractive therapeutic target in metastasising bladder cancer. Genotype and phenotype of primary tumours and their metastases may differ.

Objectives

Determine Her2 status in both tumour components to better assess the potential of anti-Her2 therapies.

Design, setting, and participants

Histologic examination revealed lymph node metastases in 150 patients with urothelial bladder cancer clinically staged as N0M0. A tissue microarray was constructed with four tumour samples per patient: two from the primary tumour and two from nodal metastases. Her2 status was determined at the gene level by fluorescence in situ hybridisation (FISH) and at the protein level by immunohistochemistry (IHC).

Interventions

All patients underwent cystectomy and standardised extended lymphadenectomy.

Measurements

Overall survival was assessed according to HER2 gene status and protein expression in primary bladder cancers and lymph node metastases.

Results and limitations

Her2 amplification was significantly more frequent in lymph node metastases (15.3%) than in matched primary bladder cancers (8.7%; p = 0.003). Her2 amplification in primary tumours was highly preserved in the corresponding metastases as indicated by only one amplified primary tumour without amplification of the metastasis. There was a high concordance in HER2 FISH results between both samples from the primary tumour (κ = 0.853) and from the metastases (κ = 0.930). IHC results were less concordant (κ = 0.539 and 0.830). FISH and IHC results were poorly correlated in primary tumours (κ = 0.566) and metastases (κ = 0.673). While Her2 amplification in the primary tumour significantly predicted poor outcome (p = 0.044), IHC-based survival prediction was unsuccessful.

Conclusions

Her2 amplification in metastasising bladder cancer is relatively frequent, is homogeneous in each tumour component, and predicts early death. This suggests a high potential for anti-Her2 therapies. For patient selection, FISH might be more accurate than IHC.     

Differential endostatin binding to bladder, prostate and kidney tumour vessels

OBJECTIVES: To define the anti-angiogenic mechanism and causes of the heterogeneous influence of endostatin, one of a group of matrix-derived inhibitors of tumour angiogenesis of increasing significance in tumour treatment, on various tissue types. MATERIALS AND METHODS: Variations in the binding behaviour of endostatin with vessels were assessed in different tumours (bladder, prostate and kidney) and compared with benign tissue vessels. Biotinylated endostatin was used and detected using extravidin CY3 and extravidin-gold immunolabelling. RESULTS: There were significant differences in the number of vessels showing endostatin binding among benign and malignant bladder, prostate and kidney tissues. While there was distinct endostatin binding on a mean (sd) of 94.2 (3.0)% bladder and 73.8 (19.5)% prostate tumour vessels, there was less binding, at 11.32 (3.9)%, on kidney tumour vessels. There was less binding to vessels of benign bladder, prostate and kidney tissue, at 2.0 (1.5), 1.7 (1.7) and 1.5 (1.7)%, respectively. At the ultrastructural level, different binding sites were detected both inside and outside the endothelial cells. CONCLUSION: Endostatin binds more to all tumour tissues than to benign tissue, but the degree of binding in malignant kidney tissue was significantly less than that in malignant prostate and bladder tissues. These divergent vascular endostatin-binding patterns could be responsible for a tumour type-dependent anti-angiogenic effect attributable to endostatin. Such selective behaviour would have therapeutic consequences for future anti-angiogenic therapy, in which different kinds of tumours could be further classified into those responding to endostatin or not.     

p130Cas,E‐cadherin and β‐catenin in human transitional cell carcinoma of the bladder: Expression and clinicopathological significance

Objectives: To investigate the effects of junction protein, p130 Crk‐associated substance (p130Cas), and adhesion molecules, E‐cadherin and β‐catenin, on the biological behavior of transitional cell carcinoma of the bladder. Methods: In 72 paraffin embedded specimens of transitional cell carcinoma of the bladder and 20 normal controls, the expression of p130Cas, E‐cadherin and β‐catenin was examined by quantum dot‐based immunofluorescence histochemistry (QD‐IHC) and conventional immunohistochemistry (IHC). Results: QD‐IHC was consistent with IHC in detecting the expression of the three molecules (P > 0.05 for all comparisons). The positive expression rate of p130Cas in bladder cancer tissues increased more significantly than that in normal bladder tissues (P < 0.001). Similarly, the aberrant expression rates of E‐cadherin and β‐catenin in bladder cancer tissues were significantly higher than those in normal bladder tissues (P < 0.001 for both comparisons). The expression of each molecule was correlated with tumor pathological grade and clinical stage (P < 0.05 for all comparisons), but not with tumor number and size (P > 0.05 for all comparisons). Furthermore, negative correlations were found between the expression intensities of p130Cas and E‐cadherin or β‐catenin in transitional cell carcinoma of the bladder (P < 0.05 for both comparisons). Conclusions: p130Cas, E‐cadherin and β‐catenin might represent useful predictors of malignant degree of transitional cell carcinoma of the bladder.