Immunohistochemical and molecular characterizations in urothelial carcinoma of bladder in patients less than 45 years.

Bladder tumours in early-onset patients are rare and seem to exhibit unique clinicopathological features. Only few studies have investigated somatic alterations in this specific age of onset group and evidence is accumulating of a distinct molecular behaviour of early-onset bladder tumours. We collected the largest cohort of early-onset tumours of patients 45 years old or younger and aimed to test genomic alterations typically found in bladder cancer. Tumours of 118 early-onset patients were compared with a consecutive group of 113 cases. Immunohistochemistry of TP53, CK20 and Ki-67 was carried out. Molecular analysis was conducted to test for loss of heterozygosity of chromosome 9 and 17, as well as TP53 and FGFR3 mutations. Fisher´s exact and chi-squared test were appropriately used. No differences in grade/stage characteristics were observed. Overexpressed TP53 was differentially distributed between the two groups. TP53 nuclear accumulation was significantly more frequent in early-onset papillomas, PUNLMPs and pTa low-grade tumours compared to the consecutive cohort (p=0.005). Moreover, chromosome 9 deletions (29.5% vs. 44.6%) and FGFR3 mutations (34.5% vs. 63.7%) were less often detected in early-onset patients (p=0.05 and p<0.0001). By comparing the largest cohort of early-onset bladder cancer patients with an unselected group, we demonstrated that the typical molecular features are not independent of age at diagnosis. Our study supports the hypothesis of a distinct biological behaviour in early-onset tumours.

TERT Core Promotor Mutations in Early-Onset Bladder Cancer.

Activating mutations in the core promoter of the TERT gene have been described in many different tumor entities. In vitro models showed a two- to fourfold increase in transcriptional activity of the TERT promoter through creation of a consensus binding motif for Ets/TCF transcription factors caused by these mutations. TERT core promoter mutations are the most common mutations in bladder cancer with a frequency between 55.6% and 82.8% described so far, and are independent of stage and grade. Since limited data on molecular alterations of early-onset bladder tumors exists, we assessed the frequency of TERT core promoter mutations in early-onset bladder cancer. Two cohorts of bladder tumors (early-onset patient group; n=144 (age of onset of disease ≤45 years); unselected, consecutive group; n=125) were examined for TERT core promoter mutations. After microdissection and extraction of DNA the corresponding hotspot regions in the TERT core promoter were examined by Sanger-sequencing or a SNaPshot approach. A significantly lower frequency of TERT core promoter mutations was found in tumors from the early-onset cohort compared to the consecutive cohort (57.6% vs. 84.8%, p<0.001). Among the early-onset cohort cases younger than the cohort's median age of 39 years at disease onset showed a significantly reduced number of TERT promoter mutations (31/67, 46,3%) than cases aged between 39 and 45 years (52/77, 67.5%; p=0.012). This association was not found in the consecutive cases. Mutation status was independent of tumor stage and grade. We conclude that in tumors from early-onset bladder cancer patients TERT core promoter mutations are not as frequent as in bladder tumors from consecutive cases, but seem to play an important role there as well. In patients below 39 years of age TERT core promoter mutations are a more infrequent event, suggesting different mechanisms of tumorigenesis in these young patients.

Low frequency of HNPCC-associated microsatellite instability and aberrant MMR protein expression in early-onset bladder cancer.

OBJECTIVES: Recently, it was shown that patients with Lynch syndrome due to an MSH2 mutation are at increased risk for the development of bladder cancer. To further this discussion, we screened the largest investigated cohort of patients with early-onset bladder cancer for microsatellite instability (MSI) and mismatch repair (MMR) deficiency to determine a possible role of Lynch syndrome in young patients with bladder cancer. METHODS: A total of 109 cases of bladder tumors from young patients (aged <45 years) were examined for MSI (Bethesda consensus panel). Expression of MMR proteins (hMLH1, hMSH2, and hMSH6) was evaluated by immunohistochemistry using a tissue microarray. Results were compared with a series of unselected consecutive bladder tumors (n = 95). RESULTS: Regarding the frequency of MSI high (1% vs 0%) or abnormal expression of MMR proteins (2% vs 6.5%), no significant difference between the early-onset and unselected patient group was found. CONCLUSIONS: In young patients with bladder tumors, MSI and defects in MMR protein expression were not more frequent than in a series of consecutive bladder tumors. Most bladder tumors in young patients are not to be attributed to Lynch syndrome.

Frequency of activating mutations in FGFR2 exon 7 in bladder tumors from patients with early-onset and regular-onset disease.

The FGF/FGFR-system plays an important role in embryogenesis, tissue homeostasis and carcinogenesis. Mutational activation of FGFR2 resulting in aberrant FGFR2 signaling activation is known from both hereditary germ line alterations and somatic mutations in various malignancies (e.g. breast, gastric or ovarian cancer). FGFR2 mutations are mainly located within the hinge between Ig-like domains (exon 7), around the 3rd Ig-like domains and within the kinase domain. For bladder cancer only sparse data on FGFR2 mutations are available. Most interestingly a case of early-onset papillary carcinoma of the bladder showing a FGFR2 p.Pro253Arg mutation in exon 7 in a patient with Apert Syndrome was reported recently. To further evaluate the importance of FGFR2 exon 7 alterations in bladder cancer a cohort of 254 bladder tumors (cohort 1: unselected cases: n=139; cohort 2: early-onset bladder cancer cases (age at time of diagnosis≤45 years): n=115) was analyzed. Sections from formalin-fixed, paraffin-embedded bladder tumors were used for DNA isolation. After precise microdissection exon 7 of the FGFR2 gene was analyzed by direct Sanger sequencing. All cases could be analyzed successfully. Mutations in exon 7 of FGFR2 could not be detected in any of the cases. All tumors showed wild type sequence. Our data demonstrate that the recently reported association between early-onset papillary carcinoma of the bladder with germ line FGFR2 p.Pro253Arg mutation could not be found in our cohorts of sporadic bladder tumors. These results indicate that FGFR2 gene mutations might only play a minor role in bladder carcinogenesis.

Does photodynamic transurethral resection of bladder tumour improve the outcome of initial T1 high-grade bladder cancer? A long-term follow-up of a randomized study.

OBJECTIVE: To evaluate, in a long-term follow-up of T1 high-grade bladder cancer treated in a prospective, randomized trial, whether fluorescence diagnosis (FD) increases recurrence-free survival (RFS) or reduces progression to muscle-invasive stages. PATIENTS AND METHODS: In all, 191 patients with suspected superficial bladder cancer were treated with transurethral resection under white light (WL) or with FD; 46 presented with initial T1 high-grade BC (WL, 25; FD, 21). There were no differences in multifocality of tumours, concomitant carcinoma in situ or tumour size in either group. RESULTS: Patients were followed for a median of 7.3 (WL) and 7.5 (FD) years to evaluate RFS. In the WL group there were 11, and in the FD group three, recurrent tumours of the same stage and grade. The RFS at 4 and 8 years was 69% and 52% in the WL, and 91% and 80% in FD group, respectively. With FD, the RFS was significantly longer according to Kaplan-Meier analysis (P = 0.025). In the WL group, three (12%), and in the FD group four (19%) patients progressed to muscle-invasive stages (>or= T2). CONCLUSION: In initial T1 high-grade bladder cancer, FD is significantly better than conventional WL transurethral resection for RFS. However, the progression rate to muscle-invasive disease was not reduced by FD. Thus the clinical course (progression) of T1 high-grade bladder cancer remains unaffected by FD.

Clinically relevant reduction in risk of recurrence of superficial bladder cancer using 5-aminolevulinic acid-induced fluorescence diagnosis: 8-year results of prospective randomized study.

OBJECTIVES: Several studies have shown that 5-aminolevulinic acid (5-ALA)-induced fluorescence cystoscopy improves the detection of superficial bladder cancer. The results have suggested a reduced rate of recurrent tumors with the use of 5-ALA fluorescence before bladder tumor resection. We performed a prospective, randomized trial to investigate whether the long-term tumor recurrence and residual tumor rates can be decreased using 5-ALA fluorescence diagnosis (FD). METHODS: A total of 301 patients with suspected superficial bladder carcinoma were randomized to transurethral resection (TUR) using conventional white light (WL) or FD. TUR was repeated to evaluate the residual tumor rate. In addition, patients were followed up for a median of 83 (WL) and 86 (FD) months to evaluate recurrence-free survival (RFS). RESULTS: Of the 301 patients, 191 were available for the efficacy analysis. The residual tumor rate was 25.2% in the WL arm versus 4.5% in the FD arm (P <0.0001). The RFS rate after 2, 4, 6, and 8 years was 73%, 64%, 54%, and 45% in the WL group and 88%, 84%, 79%, and 71% in the FD group, respectively, revealing a statistically significant difference in favor of fluorescent TUR (P = 0.0003). CONCLUSIONS: 5-ALA-induced FD is significantly superior statistically to conventional WL TUR with respect to the residual tumor rate and RFS. This advantage of decreased bladder tumor recurrence risk was maintained with high statistical significance for at least 8 years. The differences in RFS imply that FD offers a clinically relevant procedure to reduce the incidence of tumor recurrence.

Photodynamic diagnostics and noninvasive bladder cancer: is it cost-effective in long-term application? A Germany-based cost analysis.

OBJECTIVES: Noninvasive urothelial carcinoma of the bladder (UCB) causes an enormous economic burden to public health systems due to its life-long character and frequent recurrences. While white light (WL) cystoscopy is considered to be the gold standard for transurethral resection of the bladder, photodynamic diagnostic (PDD) has been shown to improve final outcome. Escalating healthcare costs warrant increased effectiveness in treating noninvasive UCB. No data based on assessment of costs have been published to date. METHODS: A series of 301 patients with noninvasive UCB were randomized prospectively to standard WL or PDD transurethral resections of the bladder. Intravesical adjuvant therapy was administered as reflected in the appropriate guidelines. Expenditures of subsequent procedures and PDD-associated costs were assessed. RESULTS: Median follow-up was 7.1 yr. Disease recurrence was found in 42% and 18% of WL and PDD patients, respectively (p=0.0003). In the WL group 2.0 and in the PDD group 0.8 transurethral resections of the bladder were noted per patient. In the WL group 1.0 and in the PDD group 0.3 recurring UCB occurred per patient, resulting in costs of 1750 euro per WL patient versus 420 euro per PDD patient in the follow-up period, respectively. Because a single expenditure of 135 euro was assessed for PDD, overall costs were significantly lower (by 1195 euro) in PDD patients. As the median follow-up was 7.1 yr, costs saved by PDD per patient per year were 168 euro. CONCLUSION: Our data suggest that PDD significantly cut costs related to recurring UCB. Further studies are needed from an economic point of view.

Gene expression of 5-, 12-, and 15-lipoxygenases and leukotriene receptors along the rat nephron.

The arachidonate signaling pathways comprise prostanoids formed by cyclooxygenases, EETs, and HETEs formed by cytochrome P-450 (CYP) enzymes and HETEs and leukotrienes generated by lipoxygenases. Whereas the intrarenal localization of cyclooxygenases and of some CYP enzymes along the nephron has already been determined, the localization of lipoxygenases and leukotriene-forming enzymes together with leukotriene receptors in the kidney is less clear. This study therefore aimed to determine the expression of 5-, 12-, and 15-lipoxygenases as well as the leukotriene receptors along the rat nephron. The kidneys were dissected into cortex and outer and inner medulla, and the microdissected nephron segments were collected after a collagenase digestion. mRNA abundance was determined by RT-PCR and real-time PCR. 15-LOX mRNA showed a characteristic expression pattern along the distal nephron. 12-LOX mRNA was only found in the glomerulus. Similarly, 5-LOX mRNAs together with 5-LOX-activating protein mRNAs were expressed in the glomerulus and also in the vasa recta. The leukotriene A4 hydrolase was found in all nephron segments, whereas leukotriene C4 synthase mRNA could not be found in any nephron segment. The leukotriene receptor B4 and the cysteinyl leukotriene receptor type 1 were selectively expressed in the glomerulus, whereas cysteinyl receptor type 2 was not found in any nephron segment. Our data suggest that the glomerulus is a major source and target for 5- and 12-HETE and for leukotrienes. The collecting duct system, on the other hand, appears to be a major source of 15-HETE.

A metachronous, atypical, multifocal renal oncocytoma with a concomitant renal cell carcinoma of the contralateral side and bilateral multifocal oncocytomas: two case reports and review of literature.

We present one case of a metachronous, atypical, multifocal renal oncocytoma with a concomitant chromophobe renal cell carcinoma (RCC) of the contralateral side and one case of bilateral and multifocal oncocytomas. Oncocytomas are benign renal tumours that rarely appear bilateral or multifocal or with coexisting RCC. A common pathogenic denominator of oncoytomas and RCC is being discussed. The first case was a 63 years old patient presenting with a history of nephrectomy for a pT1 G1 pN0 R0 papillary RCC 4 years prior to presentation, showed two tumours of a singular kidney. Upon nephron-sparing surgery one typical and one atypical oncocytoma with an invasion of the perinephric fat were found. Comparative genomic hybridisation was performed. Both tumours revealed genetic alterations with loss of genetic material on chromosome 1p. The second case was a 62 years old patient presenting with multifocal and bilateral renal tumours of undeclared dignity upon imaging. During open exploration all tumours could be removed by nephron-sparing surgery and were identified as oncocytomas. Again comparative genomic hybridisation was performed. All 4 tumours revealed genetic alterations with loss of genetic material on chromosome 1p, one of the tumours an additional loss of chromosome 10.

Deletions of chromosomes 9 and 8p in histologically normal urothelium of patients with bladder cancer.

OBJECTIVES: Multifocality and frequent recurrence of urothelial carcinoma of the urinary bladder indicate a disease of the entire "urothelial field", but little is known about chromosomal alterations in histologically normal urothelium. Histopathologically normal urothelium from patients with and without concurrent urothelial carcinoma was analyzed for loss of heterozygosity (LOH) on chromosomes 8p, 9 and 17p, regions that are known to play an important role in urothelial carcinogenesis. MATERIALS AND METHODS: Histologically inconspicuous urothelial mucosa samples (n = 160) from cystectomy specimens of patients with urothelial carcinoma (n = 15, max. diagnosis: CIS, pT1-4; all tumors grade 3) were studied. Control samples (n = 50) were obtained from patients with benign prostatic hyperplasia (n = 30) and from cystectomies performed for invasive non-urothelial carcinoma (n = 20). Tissue samples were laser-microdissected and DNA was isolated using standard protocols. LOH analyses were performed using 16 polymorphic markers on chromosomes 8p, 9p/q and 17p. RESULTS: All investigated samples were informative for at least one microsatellite marker on each chromosome/chromosomal arm. Deletions were found on chromosome 9 and/or 8p in 15/160 (9.4%) of normal urothelial samples from urothelial cancer patients. These alterations were only found in 5/15 patients with urothelial carcinoma. There were no deletions on chromosome 17p. The marker D9S1113 on chromosome 9q33-34 was most frequently affected (11/15 samples, 73%). No chromosomal deletions were found in any of the 50 urothelial control samples. CONCLUSION: Specific genetic alterations frequently associated with bladder cancer are detectable in histologically normal urothelium of patients with bladder cancer, supporting the field effect hypothesis in urothelial carcinogenesis. However, intramucosal spread of tumor cells that escape light microscopic detection remains a possibility, since normal urothelium and concurrent carcinomas showed matching deletions. Chromosomal deletions in normal bladder mucosa are not a common finding and argue against a frequent genomic alteration of the entire urothelial field in bladder carcinogenesis.

Fluorescence in situ hybridization detects frequent chromosome 9 deletions and aneuploidy in histologically normal urothelium of bladder cancer patients.

Investigation of early urothelial lesions is essential to gain insight into the development of bladder cancer. In order to evaluate if deletions of chromosome arms 9p and 9q are preceding histomorphological changes in the urothelium, histologically normal urothelial samples of patients with previous and/or simultaneous (pre)malignant urothelial lesions were investigated. Dual colour fluorescence in situ hybridization (FISH) was performed on 96 histologically inconspicuous urothelial biopsies from 41 patients. Centromeric probes for chromosomes 9 and 17 were combined with gene locus specific probes for 9p21 (p16/CDKI2), 9q22 (FACC) and 17p (p53). Deletions of chromosome 9 (defined as presence of only one copy of centromere 9 or fewer gene-specific than centromeric signals in at least 40% of evaluated cells) were found in 21% of the samples (18/87). Deletions of chromosome 9p were present in 16% of cases (14/89), whereas deletions of chromosome 9q were encountered in 10% of specimens (9/93). A hemizygous deletion of p53 was found only once. Polysomy in more than 10% of cells was encountered in 16% of cases for both chromosome 9 and chromosome 17. Non-diploidy (defined as polysomy of both chromosomes) was found in 6% of samples (5/80). In summary, chromosome 9 deletions are frequently found in histologically normal urothelium of patients with bladder cancer, although less frequently than in hyperplasias and dysplasias. These findings support the hypothesis of multi-focal development of bladder cancer from histologically inconspicuous but already genetically altered urothelium. FISH analysis of chromosome 9 regions could provide a useful tool to detect potentially premalignant lesions in the follow-up care of patients with bladder cancer.

No evidence for mutation of B-RAF in urothelial carcinomas of the bladder and upper urinary tract.

Mutational activation of the MAP kinase pathway is frequently found in various cancers. Recently, activating mutations in the B-RAF gene, an important activator of this pathway, have been described in several tumor types including melanoma, colorectal and papillary thyroid cancer. The most frequent mutation in exon 15 (V599E), as well as several other mutations within exons 11 and 15 result in constitutive activation of the oncoprotein. In addition, a significant association between mismatch-repair (MMR) deficiency and the V599E mutation in colorectal tumors has been found. The aim of our study was to investigate B-RAF mutations in 121 urothelial carcinomas of the urinary bladder (ranging from pTaG1 to pT4aG3) and 27 tumors from the upper urinary tract (UUT), including 16 tumors of the renal pelvis and 11 tumors of the ureter). Twelve of 27 UUT tumors were MMR-deficient and showed microsatellite instability. The V599E mutation was screened for by allele-specific PCR (PASA) and exons 11 and 15 of B-RAF including intron-exon-boundaries were sequenced. Overall, 116/121 bladder tumors and 23/27 tumors of the UUT were successfully investigated by PASA. None of the tumors showed the V599E mutation. Sequence analysis of exons 11 and 15 was successful in 46 urothelial tumors (bladder, n=31; UUT, n=15). No mutations within the coding region of exons 11 and 15 and the intron-exon junctions were found. The most frequent alterations in the B-RAF gene do not seem to be involved in urothelial carcinogenesis, and there is no correlation between MMR-deficiency and B-RAF mutations in urothelial tumors.

Histologic-genetic mapping by allele-specific PCR reveals intraurothelial spread of p53 mutant tumor clones.

Common and clinically important features of urothelial carcinomas are multifocality and a high rate of recurrence. Molecular studies demonstrated that multifocal tumors are frequently composed of one tumor clone spreading throughout the urothelial tract. A combination of histologic and genetic mapping of cystectomy specimens from bladder cancer patients is a valuable tool to study bladder carcinogenesis and tumor cell spread by correlating urothelial morphologic features and defined genetic alterations. In the present study, the primary tumors of 14 cystectomy specimens were investigated for p53 protein overexpression by immunohistochemistry and p53 gene mutation by genomic sequencing. Seven tumors showed a strong nuclear staining for the p53 protein. In six of seven tumors, a p53 gene mutation was detected. Allele-specific PCR of defined p53 mutations was established in five of six cases with a p53 mutation. Subsequent screening of the entire urothelial lining of each cystectomy specimen by allele-specific PCR revealed p53-mutant cell clones in urothelial patches with carcinoma in situ and dysplasia, but also frequently in histomorphologically normal urothelium adjacent to the tumor. The pattern of tumor cell spread indicated a continuous intraurothelial growth of the p53-mutant clone. P53 immunohistochemistry visually confirmed the presence of mutant cells in most of these samples. We conclude that allele-specific PCR is a highly sensitive and reliable method for tracking specific p53 mutant clones in the urothelium. Moreover, the detection of p53-mutant cells in histologically normal or preneoplastic urothelial areas in four patients with invasive bladder cancer indicates an extensive intraurothelial tumor cell spread. The excellent correlation of immunohistochemically positive urothelial patches with the presence of a specific mutation highlights the biologic significance of p53-positive cells in the urothelium of tumor patients.

Clinically relevant improvement of recurrence-free survival with 5-aminolevulinic acid induced fluorescence diagnosis in patients with superficial bladder tumors.

PURPOSES: Fluorescence diagnosis induced by 5-aminolevulinic acid enables more thorough transurethral resection of superficial bladder carcinoma compared with conventional white light. We performed a prospective, single institution, randomized trial to investigate whether the residual tumor rate and long-term tumor recurrence can be decreased by fluorescence diagnosis. MATERIALS AND METHODS: A total of 301 patients underwent transurethral resection of bladder tumors with white light or fluorescence diagnosis. Transurethral resection was repeated 5 to 6 weeks later to evaluate the residual tumor rate. To determine recurrence-free survival patient followup was performed every 3 months by white light cystoscopy and urine cytology. Recurrence-free survival was analyzed via Kaplan-Meier methods and multivariable Cox regression analysis. RESULTS: A total of 191 patients with superficial bladder carcinoma were available for efficacy analysis. The residual tumor rate was 25.2% in the white light arm versus 4.5% in the fluorescence diagnosis arm (p <0.0001). Median followup in the white light arm in 103 cases was 21.2 months (range 4 to 40) compared with 20.5 (range 3 to 40) in the 88 in the fluorescence diagnosis arm. Recurrence-free survival in the fluorescence diagnosis group was 89.6% after 12 and 24 months compared with 73.8% and 65.9%, respectively, in the white light group (p = 0.004). This superiority proved to be independent of risk group. The adjusted hazard ratio of fluorescence diagnosis versus white light transurethral resection was 0.33 (95% confidence interval 0.16 to 0.67). CONCLUSIONS: Fluorescence diagnosis is significantly superior to conventional white light transurethral resection with respect to the residual tumor rate and recurrence-free survival. The differences in recurrence-free survival imply that fluorescence diagnosis is a clinically relevant procedure for decreasing the number of tumor recurrences.