Superficial Papillary Tumors Research Articles

Simple and Efficient Stratification of Invasive Bladder Cancer Patients

Bladder cancer (BC) is the 4th most common cancer in men and the 11th most common in women (Kamat et al., 2016). The majority of bladder carcinomas (~ 90%) are non-invasive papillary tumors that are treated by transurethral resection followed by intravesical BCG installations or chemotherapy (Kiselyov et al., 2016). The superficial papillary tumors rarely progress to invasive carcinomas but they have a high propensity for recurrence. This necessitates a lifetime of invasive surveillance by repeated cystoscopy.

Abstract 4809: Validation of molecular subtypes of urothelial carcinoma of bladder in multi-institutional and public cohorts

Abstract BACKGROUND: Recently we have proposed a molecular sub-classification of conventional urothelial carcinoma which could be divided into two major categories designated as basal and luminal similar to the ones defined for breast cancer. In addition, luminal cancers contain a group of cases characterized by upregulation of p53 regulatory genes and is referred to as p53-like. The purpose of this project is to verify whether such subtypes can be identified in several large independent cohorts of bladder cancers. DESIGN: Gene expression profiles and molecular subtyping were performed by using previously described analytical algorithms on the following bladder cancer patient cohorts: (1) The Cancer Genome Atlas (TCGA) cohort containing tumor tissues from muscle invasive high grade urothelial carcinomas of 129 patients; (2) the MD Anderson Cancer Center (MDA) cohort of fresh frozen tumor tissues which included 65 superficial papillary carcinomas and 77 invasive carcinomas; (3) the Swedish cohort containing fresh frozen tumor samples of 116 superficial papillary tumors and 192 invasive tumors. RESULTS: The three distinct molecular subtypes defined as luminal, p53-like and basal were identified in all three cohorts. The distribution of molecular subtypes in individual cohorts was as follows: the TCGA cohort contained 50 (39%) luminal, 30 (23%) p53-like and 49 (38%) basal tumors; the MDA cohort contained 54 (38%) luminal, 61 (43%) p53-like and 27 (19%) basal; the Swedish cohort contained 102 (33%) luminal, 129 (42%) p-53 like and 77 (25%) basal tumors. Interestingly, in the MDA and Swedish cohorts virtually all superficial tumors, of either low or high grade, were of luminal type. CONCLUSION: The conventional bladder cancer can be separated into three distinct molecular subtypes with luminal phenotype defining the vast majority of superficial bladder cancers while muscle invasive tumors can be separated into luminal and basal forms. A subset of luminal cancers is characterized by upregulation of p53 regulatory genes. The identification of subtypes may help to stratify the patients for targeted therapies. Citation Format: Vipulkumar Dadhania, Charles G. Guo, Tadeusz Majewski, Li Zhang, Jolanta Bondaruk, Shizhen Zhang, Woonyoung Choi, David McConkey, Colin Dinney, Keith Baggerly, Bogdan A. Czerniak. Validation of molecular subtypes of urothelial carcinoma of bladder in multi-institutional and public cohorts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4809. doi:10.1158/1538-7445.AM2015-4809

Waterjet hydrodissection: first experiences and short-term outcomes of a novel approach to bladder tumor resection

TURB is the standard approach to bladder tumors but suffers from several disadvantages. Waterjet hydrodissection is a new technology for removing superficial tumors in the GI tract promising to preserve the histological structures of biopsy specimens with favorable long-term results as recent studies have shown. The aim of this study was to show the feasibility and applicability of waterjet hydrodissection for removing papillary superficial bladder tumors. In five patients diagnosed with superficial papillary bladder tumor, transurethral submucosal dissection was conducted using the T-type I-Jet HybridKnife (Erbe, Tuebingen). The resection edges were labeled by means of electrical coagulation with the HybridKnife. Subsequently, a submucosal fluid cushion specific to the tissue layer was formed by the waterjet implementation function of the HybridKnife, thereby elevating the tumorous tissue. The tumor was endoscopically extracted with a retrieval bag. Biopsy specimens of the tumor edges and base were subsequently collected. All tumors could be resected en bloc, and the lamina propria was intact in all specimens, allowing the pathologist to distinguish between superficial and invasive tumors. Pathological analysis confirmed R0 resection in all samples. These initial results prove the feasibility of waterjet hydrodissection for removing bladder tumors. In contrast to conventional TURB, this new technique allows the pathologist to assess the entire lamina propria and the resection edges due to the en-bloc resection and to determine invasiveness as well as R0 versus R1 resection. These first results are promising, long-term oncological follow-up, and prospective randomized surveys investigating the recurrence rate have to be evaluated.

Molecular alterations associated with bladder cancer initiation and progression

Bladder cancer is the fifth most commonly diagnosed non-cutaneous solid malignancy, and the second most commonly diagnosed genitourinary malignancy amongst people living in the United States, where it is estimated that more than 61,000 new cases of bladder cancer will be diagnosed in the year 2008. Approximately 90% of malignant tumors arising in the urinary bladder are of epithelial origin, the majority being transitional cell carcinomas. Early stage bladder tumors have been classified into two groups with distinct behavior and unique molecular profiles: low grade tumors (always papillary and usually superficial), and high-grade tumors (either papillary or non-papillary, and often invasive). Clinically, superficial bladder tumors (stages Ta and Tis) account for 75% to 85% of neoplasms, while the remaining 15% to 25% are invasive (T1, T2-T4) or metastatic lesions at the time of initial presentation. Studies from the author's group and others have revealed that distinct genotypic and phenotypic patterns are associated with early versus late stages of bladder cancer. Most importantly, early superficial diseases appear to segregate into two main pathways. Superficial papillary bladder tumors are characterized by gain-of-function mutations, mainly affecting classical oncogenes such as RAS and FGFR3. Deletions of chromosome 9, mainly allelic losses on the long arm (9q) are also frequent events in these tumors. Such genetic alterations are observed in most if not all superficial papillary non-invasive tumors (Ta), but only in a small subset of invasive bladder neoplasms. Flat carcinoma in situ (Tis) and invasive tumors are characterized by loss-of-function mutations, affecting the prototype tumor suppressor genes, including p53, RB and PTEN. These alterations are absent or very rare in the Ta tumors analyzed, but have been frequently identified in invasive bladder carcinomas. Based on these data, a novel model for bladder tumor progression has been proposed in which two separate genetic pathways characterize the evolution of superficial bladder neoplasms. Numerous individual molecular markers have been identified in the tissue specimens that correlate to some extent with tumor stage, and possibly with prognosis in bladder cancer. However, these molecular prognosticators do not play a role in the clinical routine management of patients with bladder tumors, mainly due to lack of large prospective validation studies. Thus, the need for development of specific tissue and serum tumor markers for prognostic stratification remains. The advent of high-throughput microarrays technologies allows comprehensive discovery of targets relevant in bladder cancer progression, which could be translated into new approaches for drug and biomarker development. Further investigation is warranted to define novel biomarkers specific for bladder cancer patients based on the molecular alterations of tumor progression, and multiplexed strategies for clinical management.

Holmium Laser Treatment for Low Grade, Low Stage, Noninvasive Bladder Cancer With Local Anesthesia and Early Instillation of Mitomycin C

We evaluated the results of laser photocoagulation of recurrent low stage noninvasive bladder cancer. The study included 36 patients with a recurrent superficial papillary tumor within 1 year of endoscopic resection. Patients underwent laser photocoagulation of the recurrence under local anesthesia and sedation. They received early instillation of 40 mg mitomycin C and were discharged home without a catheter a few hours after the operation. Patients completed a visual analog scale to quantify the perceived level of pain, including 1--no pain to 10--maximum pain. Patients were reviewed after 3, 6 and 12 months to evaluate tumor recurrence. The mean and median visual analog scale score was 3 points (range 1 to 10). No patient had urinary infection or a catheter at hospital discharge. The incidence of recurrence at 12 months was 25%, mainly in the first 15 cases. Laser photocoagulation with local anesthesia and sedation is easy to perform and well tolerated. There were no complications and the recurrence rate was similar to that of transurethral resection, as calculated using the recurrence calculator of the 2006 guidelines on TaT1 (nonmuscle invasive) bladder cancer from the European Association of Urology.

The histone deacetylase inhibitor belinostat (PXD101) suppresses bladder cancer cell growth in vitro and in vivo

BackgroundTreatment options for patients with recurrent superficial bladder cancer are limited, necessitating aggressive exploration of new treatment strategies that effectively prevent recurrence and progression to invasive disease. We assessed the effects of belinostat (previously PXD101), a novel histone deacetylase inhibitor, on a panel of human bladder cancer cell lines representing superficial and invasive disease, and on a transgenic mouse model of superficial bladder cancer.MethodsGrowth inhibition and cell cycle distribution effect of belinostat on 5637, T24, J82, and RT4 urothelial lines were assessed. Ha-ras transgenic mice with established superficial bladder cancer were randomized to receive either belinostat or vehicle alone, and assessed for bladder weight, hematuria, gene expression profiling, and immunohistochemistry (IHC).ResultsBelinostat had a significant linear dose-dependent growth inhibition on all cell lines (IC50 range of 1.0–10.0 μM). The 5637 cell line, which was derived from a superficial papillary tumor, was the most sensitive to treatment. Belinostat (100 mg/kg, intraperitoneal, 5 days each week for 3 weeks) treated mice had less bladder weight (p < 0.05), and no hematuria compared with 6/10 control mice that developed at least one episode. IHC of bladder tumors showed less cell proliferation and a higher expression of p21WAF1 in the belinostat-treated mice. Gene expression profile analysis revealed 56 genes significantly different in the treated group; these included the upregulation of p21WAF1, induction of core histone deacetylase (HDAC), and cell communication genes.ConclusionOur data demonstrate that belinostat inhibits bladder cancer and supports the clinical evaluation of belinostat for the treatment of patients with superficial bladder cancer.

Traitement conservateur des tumeurs des voies excrétrices supérieures

The histological appearance and the clinical behaviour of upper urinary tract urothelial tumours are almost identical to those of the bladder. Superficial papillary tumours rarely progress and turn to invasive disease despite a high frequency of recurrence. Technical developments in the endourology field have allowed full endoscopic access to upper tract tumours. Endoscopic resection or ablation of the tumour can be undertaken safely and effectively through ureteroscopy or percutaneous nephroscopy with low risk of extra-renal tumour seeding. For superficial (Ta, T1), low grade (I, II) tumours, a conservative approach can be selected without compromising survival and prognosis. For muscle invasive > T2 or high grade (III) tumours, nephroureterectomy remains the treatment of choice. Intracavitary BCG used after percutaneous resection reduces the risk of recurrence of upper tract urothelial tumours regardless of the grade. Finally, the world literature and our personal experience have shown that the tumour grade and stage are the two independent factors that affect survival of patients with upper urinary tract tumours.

Molecular subtypes of bladder cancer: Jekyll and Hyde or chalk and cheese?

Cancer of the bladder shows divergent clinical behaviour following diagnosis and it has been proposed that two major groups of tumours exist that develop via different molecular pathways. Low-grade, non-invasive papillary tumours recur frequently, but patients with these tumours do not often suffer progression of disease to muscle invasion. In contrast, tumours that are invading muscle at diagnosis are aggressive and associated with significant mortality. Molecular studies have identified distinct genetic, epigenetic and expression changes in these groups. However, it is not yet clear whether there is direct progression of low-grade superficial tumours to become invasive (a Jeckell and Hyde scenario) or whether in those patients who apparently progress from one form of the disease to the other, different tumour clones are involved and that the two tumour groups are mutually exclusive ('chalk and cheese'). If the latter is true, then attempts to identify molecular markers to predict progression of low-grade superficial bladder tumours may be fruitless. Similarly, it is not clear whether other subgroups of tumours exist that arise via different molecular pathways. There is now a large amount of molecular information about bladder cancer that facilitates examination of these possibilities. Some recent studies provide evidence for the existence of at least one further group of tumours, high-grade superficial papillary tumours, which may develop via a distinct molecular pathway. Patients with such tumours do show increased risk of disease progression and for these there may exist a real progression continuum from non-invasive to invasive. If this is the case, definition of the molecular signature of this pathway and improved understanding of the biological consequences of the events involved will be pivotal in disease management.

Stage-dependent increase of orosomucoid and zinc-alpha2-glycoprotein in urinary bladder cancer

Identification and characterization of biomarkers in body fluids such as serum or urine serve as a basis for early detection of diseases, particularly of cancer. Performing 2-DE with subsequent MS analyses, conventional immunoblotting and immunohistochemistry we identified two proteins, orosomucoid (ORM) and human zinc-alpha(2)-glycoprotein (ZAG), which were increased in the urine samples of patients with bladder cancer in comparison to the urine samples of healthy volunteers. The highest amount of both proteins was found in invasive bladder cancer stages such as pT2-3. Immunohistochemical studies showed ORM in inflammatory cells but also in endothelial cells of blood vessels within or adjacent to the tumor area and in part of the tumor cells. ZAG was prominent in tumor cells at the tumor invasion front. Additionally, ZAG was localized at the luminal surface of normal urothelium, which switches to the basal side when a superficial papillary tumor was observed. These results show that we have been able to identify two new proteins that may be related to the development of superficial bladder cancer and to its switch to an invasive phenotype.

IMPROVED DETECTION AND TREATMENT OF BLADDER CANCER USING HEXAMINOLEVULINATE IMAGING: A PROSPECTIVE, PHASE III MULTICENTER STUDY

We determined if improved tumor detection using hexaminolevulinate (HAL) fluorescence cystoscopy could lead to improved treatment in patients with bladder cancer. A total of 146 patients with known or suspected bladder cancer were assessed in this open, comparative, within patient, controlled phase III study. Patients received intravesical HAL for 1 hour and were assessed with standard white light cystoscopy and blue light fluorescence cystoscopy. All lesions were mapped onto a bladder chart and biopsies were taken from suspicious areas for assessment by an independent pathologist. An independent urologist blinded to the detection method used recommended treatment plans based on biopsy results and medical history according to European Association of Urology bladder cancer guidelines. Any differences in recommended treatment plans arising from the 2 cystoscopy methods were recorded. HAL imaging improved overall tumor detection. Of all tumors 96% were detected with HAL imaging compared with 77% using standard cystoscopy. This difference was particularly noticeable for dysplasia (93% vs 48%), carcinoma in situ (95% vs 68%) and superficial papillary tumors (96% vs 85%). As a result of improved detection, additional postoperative procedures were recommended in 15 patients (10%) and more extensive treatment was done intraoperatively in a further 10. Overall 17% of patients received more appropriate treatment at the time of the study following blue light fluorescence cystoscopy, that is 22% or 1 of 5 if patients without tumors were excluded. HAL imaging is more effective than standard white light cystoscopy for detecting bladder tumors and lesions. This leads to improved treatment in a significant number of patients (p <0.0001).

Sensitive Allele-Specific PCR Assay Able to Detect FGFR3 Mutations in Tumors and Urine from Patients with Urothelial Cell Carcinoma of the Bladder

Activating somatic point mutations in exons 7, 10, and 15 of the FGFR3 gene are frequently observed in urothelial cell carcinoma (UCC) (1)(2). These mutations have been found primarily in superficial papillary pTa tumors and were absent in carcinoma in situ (3). Given the high frequency of FGFR3 mutations and the possible implication of this receptor in the development of UCC, it was important to develop a simple, fast, and reliable method to identify these mutations in greater detail as a potential tool for the diagnosis and follow-up of UCC patients. Ten different FGFR3 mutations have been described in UCC, but 4 of them (R248C, S249C, G372C, and Y375C) account for >95% of cases (2). These mutations therefore represent an excellent target for assays, such as allele-specific PCR (AS-PCR), that depend on the specific detection of point mutations. The general principle underlying the AS-PCR technique is to design a mutation-specific primer that produces the preferential amplification of a specific mutant allele (4). We compared the results obtained with this assay with those obtained by direct sequencing in a series of 95 DNA samples extracted from UCC tumors. We also analyzed matching tumors and voided urine from 20 patients. Our results demonstrate the sensitivity, specificity, and reliability of this technique for detecting FGFR3 mutations in both tumors and urine from patients with UCC. Ninety-five primary tumor samples were collected from patients admitted to the Department of Urology of Hospitals Henri Mondor (Creteil) and CHRU (Besancon) and who had received no previous treatment. Patients gave written informed consent, and we collected matched tumors and blood samples. Tumors from each patient were snap-frozen and stored at −80 °C. DNA from tumor samples was extracted as described previously (5). DNA was extracted from the blood with use of the QIAamp system …

Differential Expression of Cell Cycle Regulators in Phenotypic Variants of Transgenically Induced Bladder Tumors: Implications for Tumor Behavior

Proteins controlling cell growth, differentiation, apoptosis, and oncogenic stress are often deregulated in tumor cells. However, whether such deregulations affect tumor behavior remains poorly understood in many tumor types. We recently showed that the urothelium-specific expression of activated H-ras and SV40 T antigen in transgenic mice produced two distinctive types of tumors strongly resembling the human superficial papillary tumors and carcinoma in situ of the bladder, respectively. Here we assessed the expression of a key set of cell cycle regulators in these mouse tumors and in a new transgenic line expressing a cyclin D1 oncogene in the urothelium. We found that urothelia of the wild-type and cyclin D1 transgenic mice exhibited a profile of cell cycle regulators found in quiescent (G(0)) cells, indicating that urothelium overexpressing the cyclin D1 (an 8-fold increase) is reminiscent of normal urothelium and remains slow-cycling. Low-grade superficial papillary tumors induced by activated H-ras had no detectable Rb family proteins (Rb, p107, and p130) and late cell cycle cyclins and kinases (cyclin A, E, and CDK1), but had increased level of p16, p53, and MDM2. These data suggest that the inactivation of the Rb pathway plays an important role in H-ras-induced superficial papillary tumors and that oncogenic H-ras can induce a compensatory activation of alternative tumor suppressor pathways. In contrast, carcinoma in situ of the bladder induced by SV40 T antigen had increased expression of cell cycle regulators mainly active in post-G(1) phases. The fact that phenotypically different bladder tumors exhibit different patterns of cell cycle regulators may explain why these tumors have different propensity to progress to invasive tumors. Our results indicate that the transgenic mouse models can be used not only for studying tumorigenesis but also for evaluating therapeutic strategies that target specific cell cycle regulators.

Absence ofFGFR3mutations in urinary bladder tumours of rats and mice treated withN-butyl-N-(-4-hydroxybutyl)nitrosamine

Frequent activating mutations of FGFR3 (fibroblast growth factor receptor 3) are found in human urothelial cell carcinomas, particularly in superficial papillary tumours (in 74%-84% of pTaG1-G2), but not in carcinomas in situ (CIS) and at a low rate in invasive tumours (in 16%-21% of pT1-4). In mice and rats, BBN (N-butyl-N-(4-hydroxybutyl)nitrosamine) specifically induces bladder tumours. In rats, superficial papillary tumours are mostly observed. In mice, tumour progression follows the CIS pathway: CIS are first observed, followed by tumours that invade surrounding muscle. Therefore, we looked for FGFR3 mutations in these two animal models of bladder cancer. Only the FGFR3b isoform is expressed in human urothelium and derived tumours. We identified the FGFR3b isoform in rats for the first time and showed that this is the main isoform expressed in the bladder urothelium and derived carcinomas in mice and rats, as in humans. SSCP and sequence analysis of FGFR3b showed sequence changes (polymorphisms or silent mutations) in four BBN-induced rat and mouse bladder tumours. The absence of activating mutations of FGFR3 in the mouse model was in agreement with the fact that mouse BBN-induced bladder tumour progression mimics the CIS pathway. The absence of FGFR3 mutations in the rat bladder tumours suggests that, at least at the genetic level, rat superficial papillary tumours differ from their human counterparts.

Motility-related protein 1 (MRP-1/CD9) expression in urothelial bladder carcinoma and its relation to tumor recurrence and progression.

CD9 has been implicated in cell adhesion, motility, and proliferation, and numerous studies have demonstrated its prognostic value in different solid tumors. The objective of this study was to determine the relation of CD9 expression to tumor grade and tumor stage of urothelial carcinoma of the bladder and to define the value of CD9 in predicting the behavior of superficial papillary tumors (SPTs) (pathologic Ta [pTa] and pT1). Three hundred twenty patients (118 patients with pTa tumors, 111 patients with pT1 tumors, and 91 patients with pT2 tumors) were examined for CD9 expression using immunohistochemistry applied on formalin fixed, paraffin embedded tissue. Patients were stratified into 3 categories, depending on CD9 expression: positive (> 50% positive cells), reduced (5-50% positive cells), or negative (< 5% positive cells). Loss of CD9 expression was found to be associated significantly with high-grade and high-stage urothelial tumors (P < 0.0001). A reduced/negative (altered) CD9 expression was associated with SPT progression, but not with recurrence (P < 0.001). Patients who had pTa or pT1 tumors with altered CD9 expression had a relative risk of 5.59 (P = 0.005; 95% confidence interval [95% CI], 1.69-18.48) for progression compared with patients who had tumors with positive CD9 expression. Kaplan-Meier curves showed that a lack of CD9 expression was associated significantly with progression free survival (P < 0.001; log-rank test), but not with recurrence. In patients with SPTs, multivariate Cox proportional hazards regression analysis revealed that negative CD9 expression was an independent prognostic marker for the prediction of tumor progression (P = 0.007; 95% CI, 0.11-0.70). In patients with urothelial bladder carcinoma, CD9 expression was associated significantly with tumor stage and grade, and a loss of CD9 expression was an independent prognostic factor for predicting progression in patients with SPTs. Thus, CD9 immunoexpression is a potential new predictor of tumor behavior in patients with SPTs of the urinary bladder.

Role of Ha-ras activation in superficial papillary pathway of urothelial tumor formation.

Urothelial tumors develop along two distinctive phenotypic pathways (superficial papillary non-invasive tumors versus flat carcinoma in situ lesions), with markedly different biological behavior and prognosis. Although multiple genetic alterations have been identified in human bladder cancer, their cause-effect relationship with the two pathways has not been firmly established. Using a urothelium-specific promoter of the uroplakin II gene, we showed earlier in transgenic mice that the urothelial expression of SV40T antigen, which inactivates p53 and pRb, induced carcinoma in situ and invasive and metastatic bladder cancer. In striking contrast, we demonstrate here that the urothelial expression of an activated Ha-ras in transgenic mice caused urothelial hyperplasia and superficial papillary non-invasive bladder tumors. These results provide strong, direct experimental evidence that the two phenotypical pathways of bladder tumorigenesis are caused by distinctive genetic defects. Our results indicate that Ha-ras activation can induce urothelial proliferation in vivo; and that urothelial hyperplasia is a precursor of low-grade, superficial papillary bladder tumors. Our transgenic models provide unique opportunities to study the detailed molecular events underlying different types of bladder neoplasms, and can serve as useful preclinical models for evaluating the in vivo efficacy of preventive and therapeutic agents that act on various signaling pathways in bladder cancer.

Clonality and Genetic Divergence in Multifocal Low-Grade Superficial Urothelial Carcinoma as Determined by Chromosome 9 and p53 Deletion Analysis

Multifocality and recurrence are clinically important features of urothelial carcinomas of the urinary bladder. Recent molecular genetic studies have suggested that multifocal urothelial carcinomas are monoclonally derived from an identical transformed progenitor cell. However, most of these studies investigated advanced and poorly differentiated tumors. The study presented focuses on early papillary tumors, including 52 superficial well-differentiated multifocal and recurrent bladder carcinomas from 10 patients. Microdissection separating urothelium from stromal cells was considered essential to obtain pure tumor cell populations. Genetic analysis was carried out by applying two different methods. Dual color fluorescence in situ hybridization (FISH) with centromeric probes for chromosomes 9 and 17 and gene-specific probes for chromosome loci 9q22, 9p21, and 17p13 was carried out in parallel to loss of heterozygosity (LOH) analyses applying 5 microsatellite markers on these chromosomes. Overall, deletions on chromosome 9p were found in 47 tumors (90%), at chromosome 9q in 36 tumors (69%) and at chromosome 17p in 3 tumors (6%). There was a very high correlation of the results between FISH and LOH analysis. Ten early superficial papillary tumors showed deletion of chromosome 9p without deletion of 9q, suggesting 9p deletions as a very early event in the development of papillary urothelial carcinoma. Although in four patients, all investigated tumors showed identical genetic alterations and one patient showed no genetic alterations at the loci investigated, in five patients, two or more clones with different deletions were found. In four of these patients, the results are compatible with clonal divergence and selection of different cell subpopulations derived from a common progenitor cell. However, in one patient different alleles in two markers at chromosome 9 were deleted, favoring an independent evolution of two recurring tumor cell clones. In summary, we could show that there is considerable genetic heterogeneity in early multifocal and recurring urothelial carcinoma and demonstrated the occurrence of two independent clones in at least one patient as an indicator of possible initial oligoclonality of bladder cancer.

Frequent Genetic Alterations in Simple Urothelial Hyperplasias of the Bladder in Patients with Papillary Urothelial Carcinoma

In order to understand the origin of bladder cancer, very early urothelial lesions must be investigated in addition to more advanced tumors. Tissue from 31 biopsies of 12 patients with urothelial hyperplasias and simultaneous or consecutive superficial papillary tumors were used to microdissect urothelium from 15- microm sections of biopsies. The biopsies were obtained with the recently developed highly sensitive diagnostic method of 5-aminolevulinic acid-induced fluorescence endoscopy (AFE). Besides flat and papillary urothelial neoplasms, the method of photodynamic diagnostics also detects simple urothelial hyperplasias as fluorescent positive lesions. In addition, 12 fluorescence-positive biopsies showing histologically normal urothelium were investigated. Fluorescence in situ hybridization was done using a dual color staining technique of biotinylated centromeric probes of chromosomes 9 and 17 and digoxigenin-labeled gene-specific P1 probes for chromosomes 9q22 (FACC), 9p21(p16/CDKI2), and 17p13(p53). Ten of 14 hyperplasias (70%) showed deletions of chromosome 9. In 7 out of 8 patients with genetic alterations in the hyperplasias the genetic change was also present in the papillary tumor. Six out of 12 samples of microdissected normal urothelium also showed genetic alterations on chromosome 9. Microdissection of urothelial lesions, obtained during AFE, has led to the first unequivocal documentation of genetic changes in urothelial lesions diagnosed as normal in histopathology. Thus, this technical approach is important to provide insight into the earliest molecular alterations in bladder carcinogenesis.

Four tumor suppressor loci on chromosome 9q in bladder cancer: evidence for two novel candidate regions at 9q22.3 and 9q31.

The most common genetic alteration identified in transitional cell carcinoma (TCC) of the bladder is loss of heterozygosity (LOH) on chromosome 9. However, localization of tumor suppressor genes on 9q has been hampered by the low frequency of subchromosomal deletions. We have analysed 139 primary, initial low stage TCC of the bladder using a panel of 28 microsatellite markers spanning chromosome 9 at an average distance of 5 Mb, following a primer-extension preamplification (PEP) technique. Sixty-seven (48%) tumors showed LOH at one or more loci and partial deletions were detected in 62 (45%) tumors; apparent monosomy 9 was detected in only five (4%) tumors. Deletions were more frequent on 9q (44%) than on 9p (23%), the latter being mostly associated with 9q deletion, suggesting that alteration of genes on 9q may be an early event associated with superficial papillary tumors. Combined data from the cases with partial 9q deletions displayed four candidate regions for tumor suppressor loci, based on the frequency of deletion observed and tumors with unique deletions at these sites. In two tumors, the unique partial deletion comprised D9S12 at 9q22.3, a region encompassing loci for the Gorlin syndrome and multiple self-healing squamous epithelioma gene. In two other tumors, the single LOH was identified at the D9S172 locus at 9q31-32 where the dysautonia and Fukuyama-type congenital muscular dystrophy genes have been located. One tumor showed unique LOH at the GSN locus at 9q33, a region frequently deleted in other sporadic tumors while the fourth region of deletion was observed at 9q34 between ASS and ABL-1, in two tumors. This region is frequently deleted in tumors and encompasses the locus for the hereditary hemorrhagic telangiectasia gene. These findings suggest four target regions on 9q within which suppressor genes for TCC may reside.

Interferon alpha for the treatment of superficial bladder cancer.

The main problems that the patient with superficial transitional cell carcinoma (TCC) faces are the lifelong risk of tumor recurrence, and the risk of tumor progression and death secondary to cancer. Historically, cystectomy was the primary and only availible treatment option for patients who had recurrent bladder cancer or carcinoma in situ (CIS). However, the development of topical (intravesical) therapies for transitional cell carcinoma of the bladder has revolutionized the management of both superficial papillary tumors, and of flat carcinoma in situ. Due to the discovery of intravesical agents that have biologic activity against superficial TCC of the bladder (such as Bacillus Calmette-Guerrin (BCG) and Mitomycin), the mortality rate from bladder cancer has decreased despite a concurrent increase in the incidence of this malignancy.1

Valrubicin.

Valrubicin (AD-32) is an N-trifluoroacetyl, 14-valerate derivative of the anthracycline doxorubicin. It has antineoplastic activity which probably results from interference with nucleic acid metabolism by the drug. Valrubicin entered individual cells more rapidly than doxorubicin in vitro. When valrubicin was administered intravesically to patients with bladder cancer, cytotoxic concentrations of the drug penetrated the superficial muscle layer of the bladder. Complete response rates were 18 and 29% in patients with carcinoma in situ of the bladder which was refractory to intravesical BCG in 2 non-comparative trials of prophylactic intravesical valrubicin. In patients with recurrent superficial papillary tumours, the complete response rate was 46%. Adverse events were generally transient in patients who received intravesical valrubicin. Bladder irritation occurred in 88% of patients. Systemic absorption of intravesically administered valrubicin was minimal. Accordingly, systemic adverse events generally occurred in < or =5% of patients. Valrubicin was less toxic to chick embryos and haematopoietic stem cells in vitro and produced a lower incidence of cardiotoxicity in rabbits, compared with doxorubicin.