Predictors of oncological outcomes in T1G3 patients treated with BCG who undergo radical cystectomy.

PURPOSE: To evaluate the oncological impact of postponing radical cystectomy (RC) to allow further conservative therapies prior to progression in a large multicentre retrospective cohort of T1-HG/G3 patients initially treated with BCG. METHODS: According to the time of RC, the population was divided into 3 groups: patients who did not progress to muscle-invasive disease, patients who progressed before radical cystectomy and patients who experienced progression at the time of radical cystectomy. Clinical and pathological outcomes were compared across the three groups. RESULTS: Of 2451 patients, 509 (20.8%) underwent RC. Patients with tumors > 3 cm or with CIS had earlier cystectomies (HR = 1.79, p = 0.001 and HR = 1.53, p = 0.02, respectively). Patients with tumors > 3 cm, multiple tumors or CIS had earlier T3/T4 or N + cystectomies. In patients who progressed, the timing of cystectomy did not affect the risk of T3/T4 or N + disease at RC. Patients with T3/T4 or N + disease at RC had a shorter disease-specific survival (HR = 4.38, p < 0.001), as did patients with CIS at cystectomy (HR = 2.39, p < 0.001). Patients who progressed prior to cystectomy had a shorter disease-specific survival than patients for whom progression was only detected at cystectomy (HR = 0.58, p = 0.024) CONCLUSIONS: Patients treated with RC before experiencing progression to muscle-invasive disease harbor better oncological and survival outcomes compared to those who progressed before RC and to those upstaged at surgery. Tumor size and concomitant CIS at diagnosis are the main predictors of surgical treatment while tumor size, CIS and tumor multiplicity are associated with extravesical disease at surgery.

Recurrence, progression and cancer-specific mortality according to stage at re-TUR in T1G3 bladder cancer patients treated with BCG: not as bad as previously thought.

PURPOSE: The goals of transurethral resection of a bladder tumor (TUR) are to completely resect the lesions and to make a correct diagnosis to adequately stage and treat the patient. Persistent disease after TUR is not uncommon and is why re-TUR is recommended in T1G3 patients. When there is T1 tumor in the re-TUR specimen, very high risks of progression (82%) have been reported. We analyze the risks of recurrence, progression to muscle-invasive disease and cancer-specific mortality (CSM) according to tumor stage at re-TUR in T1G3 patients treated with BCG. METHODS: In our retrospective cohort of 2451 T1G3 patients, 934 patients (38.1%) underwent re-TUR. 667 patients had residual disease (71.4%): Ta in 378 (40.5%), T1 in 289 (30.9%) patients. Times to recurrence, progression and CSM in the three groups were estimated using cumulative incidence functions and compared using the Cox regression model. RESULTS: During a median follow-up of 5.2 years, 512 patients recurred. The recurrence rate was significantly higher in patients with a T1 at re-TUR (P < 0.001). Progression rates differed according to the pathology at re-TUR, 25.3% in T1, 14.6% in Ta and 14.2% in case of no residual tumor (P < 0.001). Similar trends were seen in both patients with and without muscle in the original TUR specimen. CONCLUSIONS: Patients with T1G3 tumors and no residual disease or Ta at re-TUR have better recurrence, progression and CSM rates than previously reported, with a CSM rate of 13.1 and a 25.3% progression rate in re-TUR T1 disease.

The prognostic value and therapeutic target role of stathmin-1 in urinary bladder cancer.

BACKGROUND: The oncoprotein-18/stathmin 1 (STMN1), involved in cell progression and migration, is associated with clinical outcome in breast cancer. Here we aim to investigate its clinical significance in urinary bladder cancer and its possibilities as a therapeutic target. METHODS: Immunohistochemical analyses of STMN1 protein expression were performed in three patient cohorts: cohort I (n=115 Ta, n=115 T1, n=112 T2-4 stages), cohort II, based on randomised controlled trials (n=239 T1-T4), and cohort III of primary tumour/matched metastasis (n=90 T1-T4). The effects of STMN1 on cell proliferation and migration were evaluated in the urinary bladder cancer cell line, T24, by inhibiting STMN1-cellular expression using siRNA. RESULTS: In cohort I, high STMN1 expression correlated to shorter disease-specific survival hazard ratio (HR)=2.04 (95% confidence interval (CI) 1.13-3.68; P=0.02), elevated p53- (P<0.001) and Ki67-protein levels (P<0.001). The survival result was validated in cohort II: HR=1.76 (95% CI 1.04-2.99; P=0.03). In the metastatic bladder cancer material, 70% of the patients were STMN1-positive in both the primary tumour and matched metastases. In vitro, the growth and migration of the T24 cells were significantly reduced (P<0.01, P<0.0001, respectively), when transfecting the cells with STMN1-siRNA. CONCLUSIONS: STMN1 protein expression has prognostic significance but is primarily a potential treatment target in urinary bladder cancer.

Membranous expression of podocalyxin-like protein is an independent factor of poor prognosis in urothelial bladder cancer.

BACKGROUND: Membranous expression of the anti-adhesive glycoprotein podocalyxin-like (PODXL) has previously been found to correlate with poor prognosis in several major cancer forms. Here we examined the prognostic impact of PODXL expression in urothelial bladder cancer. METHODS: Immunohistochemical PODXL expression was examined in tissue microarrays with tumours from two independent cohorts of patients with urothelial bladder cancer: n=100 (Cohort I) and n=343 (Cohort II). The impact of PODXL expression on disease-specific survival (DSS; Cohort II), 5-year overall survival (OS; both cohorts) and 2-year progression-free survival (PFS; Cohort II) was assessed. RESULTS: Membranous PODXL expression was significantly associated with more advanced tumour (T) stage and high-grade tumours in both cohorts, and a significantly reduced 5-year OS (unadjusted HR=2.25 in Cohort I and 3.10 in Cohort II, adjusted HR=2.05 in Cohort I and 2.18 in Cohort II) and DSS (unadjusted HR=4.36, adjusted HR=2.70). In patients with Ta and T1 tumours, membranous PODXL expression was an independent predictor of a reduced 2-year PFS (unadjusted HR=6.19, adjusted HR=4.60) and DSS (unadjusted HR=8.34, adjusted HR=7.16). CONCLUSION: Membranous PODXL expression is an independent risk factor for progressive disease and death in patients with urothelial bladder cancer.

Analysis of molecular intra-patient variation and delineation of a prognostic 12-gene signature in non-muscle invasive bladder cancer; technology transfer from microarrays to PCR.

BACKGROUND: Multiple clinical risk factors and genetic profiles have been demonstrated to predict progression of non-muscle invasive bladder cancer; however, no easily clinical applicable gene signature has been developed to predict disease progression independent of disease stage and grade. METHODS: We measured the intra-patient variation of an 88-gene progression signature using 39 metachronous tumours from 17 patients. For delineation of the optimal quantitative reverse transcriptase PCR panel of markers, we used 115 tumour samples from patients in Denmark, Sweden, UK and Spain. RESULTS: Analysis of intra-patient variation of the molecular markers showed 71% similar classification results. A final panel of 12 genes was selected, showing significant correlation with outcome. In multivariate Cox regression analysis, we found that the 12-gene signature was an independent prognostic factor (hazard ratio=7.4 (95% confidence interval: 3.4-15.9), P<0.001) when adjusting for stage, grade and treatment. Independent validation of the 12-gene panel and the determined cut-off values is needed and ongoing. CONCLUSION: Intra-patient marker variation in metachronous tumours is present. Therefore, to increase test sensitivity, it may be necessary to test several metachronous tumours from a patient's disease course. A PCR-based 12-gene signature significantly predicts disease progression in patients with non-muscle invasive bladder cancer.

Risk factors for residual disease at re-TUR in a large cohort of T1G3 patients.

INTRODUCTION AND OBJECTIVES: The goals of transurethral resection of a bladder tumor (TUR) are to completely resect the lesions and to make a correct diagnosis in order to adequately stage the patient. It is well known that the presence of detrusor muscle in the specimen is a prerequisite to minimize the risk of under staging. Persistent disease after resection of bladder tumors is not uncommon and is the reason why the European Guidelines recommended a re-TUR for all T1 tumors. It was recently published that when there is muscle in the specimen, re-TUR does not influence progression or cancer specific survival. We present here the patient and tumor factors that may influence the presence of residual disease at re-TUR. MATERIAL AND METHODS: In our retrospective cohort of 2451 primary T1G3 patients initially treated with BCG, pathology results for 934 patients (38.1%) who underwent re-TUR are available. 74% had multifocal tumors, 20% of tumors were more than 3 cm in diameter and 26% had concomitant CIS. In this subgroup of patients who underwent re-TUR, there was no residual disease in 267 patients (29%) and residual disease in 667 patients (71%): Ta in 378 (40%) and T1 in 289 (31%) patients. Age, gender, tumor status (primary/recurrent), previous intravesical therapy, tumor size, tumor multi-focality, presence of concomitant CIS, and muscle in the specimen were analyzed in order to evaluate risk factors of residual disease at re-TUR, both in univariate analyses and multivariate logistic regressions. RESULTS: The following were not risk factors for residual disease: age, gender, tumor status and previous intravesical chemotherapy. The following were univariate risk factors for presence of residual disease: no muscle in TUR, multiple tumors, tumors > 3 cm, and presence of concomitant CIS. Due to the correlation between tumor multi-focality and tumor size, the multivariate model retained either the number of tumors or the tumor diameter (but not both), p < 0.001. The presence of muscle in the specimen was no longer significant, while the presence of CIS only remained significant in the model with tumor size, p < 0.001. CONCLUSIONS: The most significant factors for a higher risk of residual disease at re-TUR in T1G3 patients are multifocal tumors and tumors more than 3 cm. Patients with concomitant CIS and those without muscle in the specimen also have a higher risk of residual disease.

Molecular genetics of bladder cancer: an update.

Urinary bladder cancer is a heterogeneous disease with tumors ranging from papillary non-invasive to solid muscle infiltrating high grade tumors. There are mainly three problems after initial management: recurrence, progression to higher stage and metastases. The respective risk is well known for each of the stages of the disease but not sufficiently for individual optimal risk assessments. The clinical need is initially to establish the correct risk irrespective of later treatment that is to find prognostic factors. Secondarily it is important to develop predictive factors for each specific therapy. With the advent of array-based molecular profiling it is possible to obtain a more complete picture of the cancer biology and thus hope to improve the prediction of risk. Today the microarray approach is implemented at DNA, RNA and protein level. Reported chromosomal alterations in low-grade papillary tumors are few and the most common are 9q and 9p deletions. Activation of the MAPK pathway through mutations of FGFR3, RAS or PI3K seems to be crucial in the genesis of these low malignant tumors. Muscle infiltrating bladder tumors typically have more genetic aberrations than non-muscle invasive cancers. Key genes are related to the p53 and RB pathways. Gene-expression signatures correlated to stage, CIS, progression and recurrence have been proposed but require further validation.

Astatinated trastuzumab, a putative agent for radionuclide immunotherapy of ErbB2-expressing tumours.

The anti-ErbB2 antibody trastuzumab is used for the treatment of patients with advanced breast cancer, resulting in a response rate of 40-60%. Coupling with a cytotoxic nuclide, e.g. alpha-emitting 211At, may further increase tumour response. The tumour-targeting properties of trastuzumab, astatinated using N-succinimidyl-para-(tri-n-methylstannyl)-benzoate, were evaluated and compared with those of radioiodinated trastuzumab in this study. We found that astatinated trastuzumab retains high specificity towards ErbB2. While the immunoreactive fraction of radioiodinated trastuzumab was higher than that of astatinated trastuzumab (76+/-9% versus 54+/-28%), both radioconjugates showed high affinity (KD 0.75+/-0.16 nM versus 1.8+/-0.3 nM). A growth inhibition study indicated a dose-dependent cell deactivation, in which approximately 74 cell-associated astatine decays per cell gave a survival fraction of 4.5+/-0.8x10(-4). Results of a comparative animal study on normal mice gave no indication that astatination would have any adverse effects on the biodistribution of the antibody. In conclusion, the results of the study suggest that astatinated trastuzumab is a promising candidate for treating ErbB2-expressing tumours.

p53 deletion as a genetic marker in urothelial tumor by fluorescence in situ hybridization.

In order to investigate the significance of p53 deletion, 42 specimens of transitional cell carcinoma were analyzed by interphase cytogenetics with a fluorescence in situ hybridization technique and compared with clinicopathological and cytochemical parameters. In total, 27 (64%) and 16 (38%) specimens demonstrated p53 deletion and overexpression, respectively. The p53 deletion was significantly correlated with grade (P < 0.01), stage (P < 0.05), S-phase fraction (P < 0.05), and DNA ploidy (P < 0.01), while p53 overexpression correlated only with grade (P < 0.05). The close correlation of p53 deletion with clinicopathological parameters suggests p53 deletion to be of clinical importance to indicate the malignant potential of human urothelial tumors.

Optimization of the MB49 mouse bladder cancer model for adenoviral gene therapy.

Bladder cancer is regarded as a promising candidate for innovative therapies in the field of immune and gene therapy. In this paper, we present the subcutaneous, metastatic and a novel orthotopic model of murine MB49 bladder cancer in C57BL/6 mice. We further show the potential of using adenoviral vectors together with different transduction enhancers to augment in vivo gene delivery. Finally, we present candidate genes for tumour detection, therapy or targeting. The MB49 tumour grew rapidly in mice. The subcutaneous model allowed for tumour detection within a week and the possibility to monitor growth rate on a day-by-day basis. Injection of MB49 cells intravenously into the tail vein gave rise to lung metastases within 16 days, while instillation of tumour cells into pretreated bladders led to a survival time of 20-40 days. Adenoviral vectors can be used as a vehicle for gene transfer to the bladder. By far, the most potent transduction enhancer was Clorpactin, also known as oxychlorosene. Last, we show that MB49 cells express tumour-associated antigens like bladder cancer-4, prostate stem cell antigen and six-transmembrane epithelial antigen of the prostate. Given the possibility for efficient genetic modification of the bladder and the presence of known tumour antigens, the MB49 models can be used in innovative ways to explore immunogene therapy.

Increasing survival of patients with urinary bladder cancer. A nationwide study in Sweden 1960-1986.

Survival rates were analysed in 29,055 patients with urinary bladder cancer diagnosed in Sweden from 1960 to 1986 and followed up until 1987. The 2-, 5- and 10-year relative survival rates were 79, 70 and 64% for men and 75, 68 and 63% for women, respectively. Patients with a history of bladder cancer for at least 15 years ran a negligible risk of dying from their disease. Prognosis was consistently better in younger than in older patients; below 50 years of age the 5-year relative survival rate was 90%, as compared with 60% in patients aged 70-79 years. Patients diagnosed between 1960 and 1964 had a 60% 5-year relative survival, as compared to 71% in those diagnosed between 1980 and 1984. Multivariate analyses further confirmed that age but not sex is an important prognostic factor in bladder cancer and, further, that a substantial improvement in survival rates took place during the 1960-1986 period. Compared with 1960-1964 the risk of dying of bladder cancer within 5 years in patients diagnosed between 1980 and 1984 was 51% lower in men [relative risk (RR) = 0.49; 95% confidence interval (C.I.) 0.42-0.57] and 44% lower in women (RR = 0.56; 95% C.I. 0.45-0.70).

Expression of proliferative cell nuclear antigen (PCNA) in urinary bladder carcinoma. Evaluation of antigen retrieval methods.

Proliferative cell nuclear antigen (PCNA) expression was analyzed in formalin-fixed and paraffin-embedded specimens from patients with urinary bladder cancer using three different anti-PCNA monoclonal antibodies. In 20 recent cases a positive correlation was found between the extent and intensity of PCNA staining and grade of malignancy. In 95 specimens, three to six years old, extensive positive staining was detected in 15 and 23% of grade 2B and 3-4 tumors, respectively. No equivalent staining was found in the grade 1 and 2A tumors. In material more than six years old, a remarkably weak staining was observed regardless of grade. Similarly, in a test of archival material of tonsils a very weak immuno-reactivity was found as compared with fresh material. However, antigen retrieval by microwave heating of the tissue sections was possible in the majority of all cases, and the difference in extent and intensity of the staining between low and high grade tumors remained.

Flow cytometric analysis of DNA content of deparaffinized nuclei in urinary bladder carcinomas. Comparison of different isolation methods and relation to histological grade and stage.

A consecutive series of 230 patients with transitional cell carcinoma of the urinary bladder was analyzed with respect to the DNA content of the primary tumor, as determined by flow cytometry on material obtained from paraffin blocks. A new method for this analysis was elaborated, based on proteolytic digestion with protease (type VII, Sigma) and density separation of the nuclei by Percoll centrifugation. The method provided DNA flow histograms with less baseline debris and a smaller coefficient of variation in the peaks than published techniques. In eight of 70 cases tested (11%) the method revealed aneuploid peaks that were not distinguished with the original technique and in 197/230 case (86%) interpretable DNA histograms were obtained. The distribution of diploid and aneuploid tumors in different grades and T categories was assessed. None of the grade 1 tumors was aneuploid; 11% of grade 2A, 63% of grade 2B and 83% of grade 3-4, respectively, were aneuploid. The corresponding figures for categories Ta, T1, T2, T3 and T4 were 16, 55, 72, 65 and 100%, respectively.

Filling defect in ureter after ileal conduit surgery.

Two cases with a filling defect in the distal part of the ureter after operation with a nippled ureteroileal anastomosis are presented. The filling defects were due to an inversion of the nipple.

Paraffin section storage and immunohistochemistry. Effects of time, temperature, fixation, and retrieval protocol with emphasis on p53 protein and MIB1 antigen.

It has been observed that immunoreactivity in paraffin sections decreased during storage. In this study, stored paraffin sections from both biopsy material and cultured cells were assessed for changes in immunoreactivity, using color-based image analysis to quantitate extent and intensity of the stainings. For seven of the 11 antibodies studied, storage at 20 degrees C for 16 weeks reduced the extent of immunostaining compared with that of freshly cut sections. Furthermore, increased storage temperatures resulted in a progressive loss of immunoreactivity. After 2 weeks of storage, at both 4 degrees C and 20 degrees C, p53 protein- and MIB1-antigen expression was significantly reduced regarding extent and intensity. The extent of the immunoreactivity reduced more for p53 protein than for MIB1 antigen, but the intensity did not. Boric acid was used for antigen retrieval on sections stored for 12 weeks at 20 degrees C. For both p53 protein and MIB1 antigen, this resulted in an extent and intensity of immunostaining equal to or higher than (MIB1) that obtained in freshly cut sections, using citrate buffer. Staining of cultured cells confirmed the results from biopsy material on the influence of storage temperature. Fixation time only marginally influenced the storage-related decrease in immunoreactivity. In conclusion, storage of paraffin sections leads to a varying degree of decreased immunoreactivity for several antibodies. The degree is at least partly dependent on storage time and temperature but not fixation time. However, this may be compensated for by optimizing the antigen retrieval protocol.

A grading system for the immunostaining of A, B and H blood group isoantigens in bladder carcinoma.

A test system for the evaluation of ABH blood group isoantigens in bladder carcinoma was developed. A highly standardized staining technique and a set of criteria for the judgement of staining extent and intensity was used. These variables are given numerical values which are multiplied to give a score (0-12) representing a semiquantitative estimate of the ABH-antigen expression. A consecutive series of 230 patients with bladder carcinoma was analyzed, using the test. The correlation between staining scores on the one hand and the tumor grade and stage on the other was assessed. Thirty-three percent of the tumors, irrespective of grade or stage, were completely negative, whereas 15% were positive to the same extent and intensity as normal mucosa. The remaining tumors showed intermediate patterns with respect to both extent and intensity of the expression with a progressive loss of detectable antigens with increasing grade and stage. The material was categorized with respect to the staining patterns for future analysis of the influence on prognostic parameters.

Estramustine-binding protein (EMBP) content in four different cell lines and its correlation to estramustine induced metaphase arrest.

It is known that estramustine (EM) accumulates in cells at the G2/M-phase and causes metaphase arrest of various cell types. The inhibitory effect is mediated by interaction with microtubule-associated proteins (MAPs) and/or tubulin. Estramustine-binding protein (EMBP) is a secretory protein which has been found in a number of different tumor cells and has been shown to faciliate the uptake of EM into cells. In this study the efficacy of EM in arresting cells at metaphase was studied, using four different human cell lines; the prostatic cancer cell line DU 145, the breast cancer cell line MDA 231, the colon cancer cell line Colon 320, and the urinary bladder cancer cell line RT4. The cells were incubated with EM at a concentration of 10 micrograms/ml for 24 hours. The data reveal an increase in metaphase arrests in the DU 145 and in Colon 320 cell lines. Both of these cell lines were found to contain high amounts of EMBP using a dot-blot assay. The other two cell lines, MDA 231 and RT4 had undetectable intracellular amounts of the protein and exhibited a low increase in metaphase arrests. The cell lines were analysed regarding S-phase fraction with flow-cytometry (FCM) to exclude the growth rate of the cells as a limiting factor. The results from the FCM confirmed the cytogenic analysis, that is a higher percentage of cells were in the G2/M phase in both the DU 145 and Colon 320 cell line compared to MDA 231 and RT4. EM causes mitotic arrest in those cell lines that contain detectable amounts of EMBP.

Chromosome 8 numerical aberration and C-MYC copy number gain in bladder cancer are linked to stage and grade.

Chromosome 8 aberration and c-myc amplification have been suggested as playing important roles in the development of different human cancers. Using fluorescence in situ hybridization (FISH), chromosome 8 polysomy and c-myc amplification can be detected in cells from bladder cancer. We investigated the correlation of chromosome 8 polysomy, c-myc gene alteration and p53 deletion with histopathological parameters. Twenty-four tumors obtained from patients with bladder cancer were analyzed by interphase cytogenetics using FISH with chromosome 8 and 17 centromere probes together with an YAC clone covering the c-myc locus and three cosmid DNA probes covering the p53 locus. Chromosome 8 polysomy was found in 12 tumors. The average copy number of chromosome 8 centromere signals were significantly higher in high grade and stage, cancers. Also the c-myc copy gain and p53 deletion were significantly correlated with grade as well as stage (p<0.05, in both cases). Both polysomy 8 and c-myc copy gain were significantly correlated with p53 deletions (p<0.01) and DNA ploidy (p<0.001). On the contrary there was no significant correlation between c-myc protein over-expression and c-myc gene amplification. These results may indicate that alteration of chromosomal regions on 8q and 17p, including c-myc and p53 genes, may be linked to progression of bladder cancer.

Comparison of comparative genomic hybridization, fluorescence in situ hybridization and flow cytometry in urinary bladder cancer.

Comparative genomic hybridization (CGH) was applied to screen the genetic events in six invasive urinary bladder cancers. These cases were also studied by flow cytometry (FCM) and fluorescence in situ hybridization (FISH). Four samples showed partial gain on chromosome 8, with the common region involved was on 8q23-qter. Full or partial deletion on chromosome 2 and 17p in addition to gain on 20q was found in two cases. Interestingly one diploid tumor with low mitotic index, stage and grade showed more genetic aberrations (8 gains and 7 losses) by CGH than other aneuploid tumors with high mitotic index, stage and grade. The numerical chromosomal aberration detected by FISH for chromosomes 7, 8, 9, 10, 11 and 17 were 50% in T1 cases and 100% in T2-T4 cases. FISH was performed on chromosome 8q and 17p to compare and validate the sensitivity of CGH. The agreement was 100% for 8q24 locus and 50% for p53 locus. This indicates that different molecular genetic techniques showed relatively different aspect of genomic aberrations.

Salvage cystectomy for bladder and gynaecologic cancer after irradiation failure.

From November 1981 through 1983, salvage cystectomy was performed on five men with recurrent bladder carcinoma after definitive irradiation; three of the men were 75 years or older. There were no deaths, but the complication rate was fairly high, though acceptable. Salvage cystectomy was further performed, together with radical extirpation of reproductive organs, in two women with recurrent gynaecologic malignancy and a history of local irradiation. The authors conclude that salvage cystectomy is a fairly safe procedure, even in elderly patients, relieving discomfort and probably also prolonging survival.