Background Cystoscopy is the gold standard for bladder cancer detection, but is costly, invasive and has imperfect diagnostic accuracy. We aimed to identify novel and accurate DNA methylation biomarkers for non-invasive detection of bladder cancer in urine, with the potential to reduce the number of cystoscopies among hematuria patients. Results Biomarker candidates (n = 32) were identified from methylome sequencing of urological cancer cell lines (n = 16) and subjected to targeted methylation analysis in tissue samples (n = 60). The most promising biomarkers (n = 8) were combined into a panel named BladMetrix. The performance of BladMetrix in urine was assessed in a discovery series (n = 112), consisting of bladder cancer patients, patients with other urological cancers and healthy individuals, resulting in 95.7% sensitivity and 94.7% specificity. BladMetrix was furthermore evaluated in an independent prospective and blinded series of urine from patients with gross hematuria (n = 273), achieving 92.1% sensitivity, 93.3% specificity and a negative predictive value of 98.1%, with the potential to reduce the number of cystoscopies by 56.4%. Conclusions We here present BladMetrix, a novel DNA methylation urine test for non-invasive detection of bladder cancer, with high accuracy across tumor grades and stages, and the ability to spare a significant number of cystoscopies among patients with gross hematuria.

Retrospective studies have provided proof of principle that bladder cancer can be detected by testing for the presence of tumor DNA in urine. We have conducted a prospective blinded study to determine whether a urine-based DNA test can replace flexible cystoscopy in the initial assessment of gross hematuria. A total of 475 consecutive patients underwent standard urological examination including flexible cystoscopy and computed tomography urography, and provided urine samples immediately before (n=461) and after (n=444) cystoscopy. Urine cells were collected using a filtration device and tested for eight DNA mutation and methylation biomarkers. Clinical evaluation identified 99 (20.8%) patients with urothelial bladder tumors. With this result as a reference and based on the analysis of all urine samples, the DNA test had a sensitivity of 97.0%, a specificity of 76.9%, a positive predictive value of 52.5%, and a negative predictive value of 99.0%. In three patients with a positive urine-DNA test without clinical evidence of cancer, a tumor was detected at repeat cystoscopy within 16 mo. Our results suggest that urine-DNA testing can be used to identify a large subgroup of patients with gross hematuria in whom cystoscopy is not required. PATIENT SUMMARY:We tested the possibility of using a urine-based DNA test to check for bladder cancer in patients with visible blood in the urine. Our results show that the test efficiently detects bladder cancer and therefore may be used to greatly reduce the number of patients who would need to undergo cystoscopy.

PURPOSE:Due to the high recurrence risk of nonmuscle invasive urothelial carcinoma it is crucial to distinguish patients at high risk from those with indolent disease. In this study we used a machine learning algorithm to identify the genes in patients with nonmuscle invasive urothelial carcinoma at initial presentation that were most predictive of recurrence. We used the genes in a molecular signature to predict recurrence risk within 5 years after transurethral resection of bladder tumor. MATERIALS AND METHODS:Whole genome profiling was performed on 112 frozen nonmuscle invasive urothelial carcinoma specimens obtained at first presentation on Human WG-6 BeadChips (Illumina®). A genetic programming algorithm was applied to evolve classifier mathematical models for outcome prediction. Cross-validation based resampling and gene use frequencies were used to identify the most prognostic genes, which were combined into rules used in a voting algorithm to predict the sample target class. Key genes were validated by quantitative polymerase chain reaction. RESULTS:The classifier set included 21 genes that predicted recurrence. Quantitative polymerase chain reaction was done for these genes in a subset of 100 patients. A 5-gene combined rule incorporating a voting algorithm yielded 77% sensitivity and 85% specificity to predict recurrence in the training set, and 69% and 62%, respectively, in the test set. A singular 3-gene rule was constructed that predicted recurrence with 80% sensitivity and 90% specificity in the training set, and 71% and 67%, respectively, in the test set. CONCLUSIONS:Using primary nonmuscle invasive urothelial carcinoma from initial occurrences genetic programming identified transcripts in reproducible fashion, which were predictive of recurrence. These findings could potentially impact nonmuscle invasive urothelial carcinoma management.

Molecular analysis of cells from urine provides a convenient approach to non-invasive detection of bladder cancer. The practical use of urinary cell-based tests is often hampered by difficulties in handling and analyzing large sample volumes, the need for rapid sample processing to avoid degradation of cellular content, and low sensitivity due to a high background of normal cells. We present a filtration device, designed for home or point-of-care use, which enables collection, storage and shipment of urinary cells. A special feature of this device is a removable cartridge housing a membrane filter, which after filtration of urine can be transferred to a storage unit containing an appropriate preserving solution. In spiking experiments, the use of this device provided efficient recovery of bladder cancer cells with elimination of >99% of excess smaller-sized cells. The performance of the device was further evaluated by DNA-based analysis of urinary cells collected from 57 patients subjected to transurethral resection following flexible cystoscopy indicating the presence of a tumor. All samples were tested for FGFR3 mutations and seven DNA methylation markers (BCL2, CCNA1, EOMES, HOXA9, POU4F2, SALL3 and VIM). In the group of patients where a transitional cell tumor was confirmed at histopathological evaluation, urine DNA was positive for one or more markers in 29 out of 31 cases (94%), including 19 with FGFR3 mutation (61%). In the group of patients with benign histopathology, urine DNA was positive for methylation markers in 13 out of 26 cases (50%). Only one patient in this group was positive for a FGFR3 mutation. This patient had a stage Ta tumor resected 6 months later. The ability to easily collect, store and ship diagnostic cells from urine using the presented device may facilitate non-invasive testing for bladder cancer.

Bladder cancer is diagnosed by cystoscopy, a costly and invasive procedure that is associated with patient discomfort. Analysis of tumor-specific markers in DNA from sediments of voided urine has the potential for non-invasive detection of bladder cancer; however, the sensitivity is limited by low fractions and small numbers of tumor cells exfoliated into the urine from low-grade tumors. The purpose of this study was to improve the sensitivity for non-invasive detection of bladder cancer by size-based capture and enrichment of tumor cells in urine. In a split-sample set-up, urine from a consecutive series of patients with primary or recurrent bladder tumors (N = 189) was processed by microfiltration using a membrane filter with a defined pore-size, and sedimentation by centrifugation, respectively. DNA from the samples was analyzed for seven bladder tumor-associated methylation markers using MethyLight and pyrosequencing assays. The fraction of tumor-derived DNA was higher in the filter samples than in the corresponding sediments for all markers (p<0.000001). Across all tumor stages, the number of cases positive for one or more markers was 87% in filter samples compared to 80% in the corresponding sediments. The largest increase in sensitivity was achieved in low-grade Ta tumors, with 82 out of 98 cases positive in the filter samples (84%) versus 74 out of 98 in the sediments (75%). Our results show that pre-analytic processing of voided urine by size-based filtration can increase the sensitivity for DNA-based detection of bladder cancer.

You have accessJournal of UrologyBladder Cancer: Basic Research II1 Apr 2012887 USING ARTIFICIAL INTELLIGENCE AND MACHINE-LEARNING ALGORITHMS WITH GENE EXPRESSION PROFILING TO PREDICT SUPERFICIAL BLADDER CANCER RECURRENCE AT INITIAL PRESENTATION Anirban P. Mitra, Georg Bartsch, Sheetal A. Mitra, Arpit A. Almal, Kenneth E. Steven, David W. Fry, Peter F. Lenehan, Richard J. Cote, and William P. Worzel Anirban P. MitraAnirban P. Mitra Los Angeles, CA More articles by this author , Georg BartschGeorg Bartsch Los Angeles, CA More articles by this author , Sheetal A. MitraSheetal A. Mitra Los Angeles, CA More articles by this author , Arpit A. AlmalArpit A. Almal Ann Arbor, MI More articles by this author , Kenneth E. StevenKenneth E. Steven Copenhagen, Denmark More articles by this author , David W. FryDavid W. Fry Ann Arbor, MI More articles by this author , Peter F. LenehanPeter F. Lenehan Ann Arbor, MI More articles by this author , Richard J. CoteRichard J. Cote Miami, FL More articles by this author , and William P. WorzelWilliam P. Worzel Ann Arbor, MI More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.982AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES 50%-70% of patients with superficial (Ta/T1, N0M0) bladder cancer (SBC) recur within 5 years of initial presentation. Tumor grade and multifocality are routinely used indicators of recurrence. SBCs' propensity to recur persistently necessitates intense follow up and invasive treatment. It is therefore crucial to objectively determine SBCs' recurrence potential to identify patients at greatest risk and minimize invasive follow up schedules for those harboring relatively indolent disease. This study used a machine-learning algorithm to identify SBC genes at initial presentation that were most predictive of recurrence, and used them in a molecular signature to predict recurrence risk within 5 years after TURBT. METHODS Whole genome profiling was performed on 112 frozen SBCs obtained at first presentation by TURBT on Illumina Human WG-6 BeadChips. A genetic programming (GP) algorithm was used to evolve classifier mathematical models for outcome prediction. Cross-validation-based resampling and gene usage frequencies were used to identify the most prognostic genes, which were combined into rules used in a voting algorithm to predict a sample's target class. Key genes were validated by quantitative PCR. RESULTS 88 (79%) patients recurred within 5 years of initial presentation. A GP algorithm was used to select a minimal set of markers grouped as a classifier for predicting recurrence, and cross-validation estimated its robustness by analyzing its ability to generalize to unseen samples. The classifier set included 21 genes that could predict recurrence. Quantitative PCR was done on a subset of 100 patients for these genes. With amplicon sizes limited to 100 bases and Ct values >35 not being considered, a 4-fold cross-validation (n=83) resulted in a 5-gene combined rule that incorporated a voting algorithm to yield 77% sensitivity and 85% specificity in predicting recurrence in the training set. The respective values in the test set were 69% and 62%. A singular 3-gene rule was also constructed that predicted recurrence with 80% sensitivity and 90% specificity in the training set. The respective values in the test set were 71% and 67%. CONCLUSIONS Using primary SBCs from initial occurrences, GP identified transcripts in a reproducible fashion that were predictive of recurrence. These findings could potentially impact SBC management, including surveillance frequency, administration of adjuvant therapy, and selection of candidates for an expectant approach. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e361 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Anirban P. Mitra Los Angeles, CA More articles by this author Georg Bartsch Los Angeles, CA More articles by this author Sheetal A. Mitra Los Angeles, CA More articles by this author Arpit A. Almal Ann Arbor, MI More articles by this author Kenneth E. Steven Copenhagen, Denmark More articles by this author David W. Fry Ann Arbor, MI More articles by this author Peter F. Lenehan Ann Arbor, MI More articles by this author Richard J. Cote Miami, FL More articles by this author William P. Worzel Ann Arbor, MI More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

You have accessJournal of UrologyBladder Cancer: Superficial1 Apr 20111653 AN ANGIOGENESIS-MARKER PANEL PREDICTS NONINVASIVE PAPILLARY BLADDER CANCER RECURRENCE AT INITIAL PRESENTATION Claudia Bartsch, Anirban Mitra, Georg Bartsch, Eila Skinner, Donald Skinner, Kenneth Steven, and Richard Cote Claudia BartschClaudia Bartsch Los Angeles, CA More articles by this author , Anirban MitraAnirban Mitra Los Angeles, CA More articles by this author , Georg BartschGeorg Bartsch Los Angeles, CA More articles by this author , Eila SkinnerEila Skinner Los Angeles, CA More articles by this author , Donald SkinnerDonald Skinner Los Angeles, CA More articles by this author , Kenneth StevenKenneth Steven Copenhagen, Denmark More articles by this author , and Richard CoteRichard Cote Miami, FL More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1763AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Tumor grade is currently the best predictor of noninvasive papillary (Ta) bladder cancer outcome at first presentation. This study investigated the role of angiogenic markers in predicting Ta bladder cancer outcome, under the hypothesis that angiogenesis plays a role in Ta tumor recurrence and progression. METHODS Whole genome analysis was performed on frozen cold cup biopsies from 138 patients with Ta G2/3 bladder tumors at initial presentation on the Illumina Sentrix platform. 33 (24%), 76 (55%) and 29 (21%) patients did not recur, recurred and progressed to a higher T-stage during follow up. Adequate follow up ensured that patients likely to recur/progress did so by the end of the reporting period. 803 candidate angiogenesis-related genes were identified by GeneCards (v2.42). Mann-Whitney-U test examined associations of individual genes with outcome. Hierarchical clustering and log rank analysis was used to examine the discriminatory potential of the final gene panel for recurrence and progression. RESULTS Median follow up of patients who never recurred (n=33) and never progressed (n=109) was 7.7 and 5.9 years, respectively. To identify genes predictive of recurrence, profiles of patients who never recurred (n=33) were compared to those who recurred and/or progressed (n=105). Mann-Whitney-U test identified 270 genes that could individually predict recurrence in this cohort (all p≤0.05). To identify genes predictive of progression, profiles of patients who never progressed (n=109) were compared to those who progressed (n=29). 50 genes that were individually predictive for progression were identified (all p≤0.05). 18 genes were found to be common between these two panels and were selected for the final analysis. Hierarchical clustering using the 18-gene panel identified six patient groups with varying risks of recurrence and progression. Log rank analysis showed that the 18-gene panel could significantly discriminate between patients with varying risk for recurrence (p=0.037), although its ability to do the same for progression fell short of statistical significance (p=0.16). CONCLUSIONS Profiling angiogenic molecules is a promising approach for predicting Ta bladder tumor outcome at initial diagnosis. The 18-gene panel was able to categorize patients into several risk strata based on recurrence risk. Examining the interplay of angiogenic markers with other pathways may be essential to identify panels for progression. Such panels can help differentiate between patients who need aggressive versus expectant management. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e664 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Claudia Bartsch Los Angeles, CA More articles by this author Anirban Mitra Los Angeles, CA More articles by this author Georg Bartsch Los Angeles, CA More articles by this author Eila Skinner Los Angeles, CA More articles by this author Donald Skinner Los Angeles, CA More articles by this author Kenneth Steven Copenhagen, Denmark More articles by this author Richard Cote Miami, FL More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Purpose: The development of ureteral strictures is a complicated problem for patients who have had urinary reconstruction at the time of radical cystectomy. Management options range from conservative approaches with minimally invasive options such as balloon dilation and stent placement to the more invasive options with open or laparoscopic surgical repair. The purpose of this study was to review the outcomes and treatment of patients who developed ureteral stricture following Indiana pouch reconstruction at the time of robotic-assisted laparoscopic radical cystectomy.

The bladder cancer genome harbors numerous oncogenic mutations and aberrantly methylated gene promoters. The aim of our study was to generate a profile of these alterations and investigate their use as biomarkers in urine sediments for noninvasive detection of bladder cancer. We systematically screened FGFR3, PIK3CA, TP53, HRAS, NRAS and KRAS for mutations and quantitatively assessed the methylation status of APC, ARF, DBC1, INK4A, RARB, RASSF1A, SFRP1, SFRP2, SFRP4, SFRP5 and WIF1 in a prospective series of tumor biopsies (N = 105) and urine samples (N = 113) from 118 bladder tumor patients. We also analyzed urine samples from 33 patients with noncancerous urinary lesions. A total of 95 oncogenic mutations and 189 hypermethylation events were detected in the 105 tumor biopsies. The total panel of markers provided a sensitivity of 93%, whereas mutation and methylation markers alone provided sensitivities of 72% and 70%, respectively. In urine samples, the sensitivity was 70% for all markers, 50% for mutation markers and 52% for methylation markers. FGFR3 mutations occurred more frequently in tumors with no methylation events than in tumors with one or more methylation events (78% vs. 33%; p < 0.0001). FGFR3 mutation in combination with three methylation markers (APC, RASSF1A and SFRP2) provided a sensitivity of 90% in tumors and 62% in urine with 100% specificity. These results suggest an inverse correlation between FGFR3 mutations and hypermethylation events, which may be used to improve noninvasive, DNA-based detection of bladder cancer.

Background: Currently, tumor grade is the best predictor of outcome at first presentation of noninvasive papillary (Ta) bladder cancer. However, reliable predictors of Ta tumor recurrence and progression for individual patients, which could optimize treatment and follow-up schedules based on specific tumor biology, are yet to be identified.Objective: To identify genes predictive for recurrence and progression in Ta bladder cancer at first presentation using a quantitative, pathway-specific approach.Design, setting, and participants: Retrospective study of patients with Ta G2/3 bladder tumors at initial presentation with three distinct clinical outcomes: absence of recurrence (n = 16), recurrence without progression (n = 16), and progression to carcinoma in situ or invasive disease (n = 16).Measurements: Expressions of 24 genes that feature in relevant pathways that are deregulated in bladder cancer were quantified by real-time polymerase chain reaction on tumor biopsies from the patients at initial presentation.Results and limitations: CCND3 (p = 0.003) and HRAS (p = 0.01) were predictive for recurrence by univariate analysis. In a multivariable model based on CCND3 expression, sensitivity and specificity for recurrence were 97% and 63%, respectively. HRAS (p < 0.001), E2F1 (p = 0.017), BIRC5/Survivin (p = 0.038), and VEGFR2 (p = 0.047) were predictive for progression by univariate analysis. Multivariable analysis based on HRAS, VEGFR2, and VEGF identified progression with 81% sensitivity and 94% specificity. Since this is a small retrospective study using medium-throughput profiling, larger confirmatory studies are needed.Conclusions: Gene expression profiling across relevant cancer pathways appears to be a promising approach for Ta bladder tumor outcome prediction at initial diagnosis. These results could help differentiate between patients who need aggressive versus expectant management. (C) 2009 European Association of Urology. Published by Elsevier B. V. All rights reserved.

Ligation of two oligonucleotide probes hybridized adjacently to a DNA template has been widely used for detection of genome alterations. The multiplex ligation-dependent probe amplification (MLPA) technique allows simultaneous screening of multiple target sequences in a single reaction by using pairs of probes that carry tails for binding of common amplification primers. Resolution of the various targets is achieved by electrophoresis on the basis of predefined differences in amplicon length. In the conventional MLPA approach, one of the two target probes is generated by cloning in a single-stranded bacteriophage vector to introduce a sequence of defined length between the primer binding site and the specific target sequence. Here we demonstrate that differences in amplicon length can be achieved by using multiple short synthetic probes for each target sequence. When joined by a DNA ligase, these probes will form a single amplifiable template whose length is defined by the number and lengths of the individual probes. We have used this principle to establish a methylation-specific MLPA (MS-MLPA) assay that simultaneously determines the methylation status of five promoter CpG islands, and we have used this assay to analyze DNA from tumor tissue and corresponding urine samples from patients with bladder cancer. Our data show that the use of multiple short synthetic probes provides a simple means for custom-designed MS-MLPA analysis.

This Practice Point commentary discusses the paper by Dhar and colleagues, which compared outcomes between two cohorts of patients with muscle-invasive bladder cancer who received either 'limited' pelvic lymph node dissection (LND) or 'extended' pelvic LND at clinics in the US or Switzerland. The patients who received extended LND demonstrated improved overall and recurrence-free survival. Notably, however, the 'extended' and 'limited' dissections utilized in this study do not correspond to the commonly used anatomical templates defined by these terms. Nonetheless, despite this and other methodological limitations, this study does add to the growing evidence supporting the use of more-extensive pelvic LND in this setting. One prospective, randomized trial aiming to address this issue is currently accruing patients in Germany. While results are awaited, extended LND should be regarded as an essential component of radical cystectomy and applied to all patients undergoing radical surgery for bladder cancer.

PURPOSE:We assessed the clinical outcome in patients with invasive bladder cancer and lymph node metastasis above the bifurcation of the common iliac vessels treated with radical cystectomy including extended pelvic lymph node dissection without adjunct therapy. MATERIALS AND METHODS:Between 1993 and June 2005 a total of 336 consecutive patients underwent radical cystectomy and extended pelvic lymphadenectomy without preoperative or postoperative chemotherapy by 1 surgeon. A total of 263 patients (78.3%) had orthotopic bladder reconstruction. The pelvic lymph node dissection began at the distal aorta including the common and external iliac lymph nodes, and the periaortic, presacral and obturator fossa nodes. The lymphatic tissue removed above and below the bifurcation of the common iliac vessels was submitted separately for histopathological analysis. Data were prospectively entered into a database that forms the basis of this cohort study. RESULTS:The 5-year overall and recurrence-free survival rates in the entire study population of 336 patients were 68% and 69%, respectively. Overall 64 patients (19%) had lymph node metastases of whom 22 (34.4%) had lymph node involvement above the bifurcation of the common iliac vessels outside the template of the standard lymph node dissection. The median number of retrieved lymph nodes was 27 (range 7 to 78) and in those with lymph node metastases 27 (range 11 to 49) included 8 (range 0 to 17) above the bifurcation and 18 (range 8 to 41) below the bifurcation of the common iliac vessels in the true pelvis. Lymph node involvement proved a significant adverse prognostic factor with a 5-year probability of survival of 39% vs 76%. The overall 5-year survival rates was similar in patients with lymph node involvement above the bifurcation of the common iliac vessels (37%) compared to the entire population with lymph node metastasis (41%) and to those with lymphatic metastases in the true pelvis below the bifurcation of the common iliac vessels (42%). The survival rate was significantly higher in patients with 5 or less involved lymph nodes (50% vs 13%, p <0.002) and in those with a lymph node density (number of lymph nodes involved/total number of lymph nodes removed) less than 20% (25% vs 47%, p <0.05), but it did not relate to the total number of retrieved lymph nodes. CONCLUSIONS:Overall 34% of our patients with lymph node metastases had nodal involvement in the common iliac, periaortic and presacral regions after radical cystectomy for bladder cancer. Survival was similar in this group of patients with lymphatic metastasis outside the boundaries of the standard pelvic lymph node dissection template compared to the entire population with lymph node metastasis. This finding underscores the contention that extended dissection not only provides the most accurate staging but also offers the patient the best chance of survival. Following radical cystectomy patients can be stratified into risk groups according to tumor stage, lymph node involvement, number of metastatic nodes and lymph node density. Our results support the idea that the benchmark for radical cystectomy should include extensive pelvic lymph node dissection with anatomical boundaries including the common iliac and presacral nodes.

Somatic instability at microsatellite repeats was detected in 6 of 200 transitional cell carcinomas of the bladder. Instabilities were apparent as changes in (GT). repeat lengths on human chromosome 9 for four tumors and as alterations in a (CAG). repeat in the androgen receptor gene on the X chromosome for three tumors. Single locus alterations were detected in three tumors, while three other tumors revealed changes in two or more loci. In one tumor we found microsatellite instability in all five loci analyzed on chromosome 9. The alterations detected were either minor 2-base pair changes or larger (>2 base pairs) alterations in repeat length. All six tumors were low stage (Ta-T1), suggesting that these alterations can occur early in bladder tumorigenesis. I n t r o d u c t i o n Microsatel l i te markers play an impor tant role in the analysis of L O H 4 in cancer. Microsatel l i tes are t andem iterations o f s imple di-, tri-, or te t ranucleot ide repeats, and their usefulness can be attr ibuted to abundancy (1), hypervariabi l i ty (2), fairly even genom ic distr ibution (3), and ease of detect ion by the PCR. Microsatel l i tes have been reported to be unstable in some inheri ted diseases and in some types of cancer. This instability consists of expans ion or contract ion of D N A within repeat e lements (4). Expansion of a t r inucleot ide repeat is responsible for fragile X syndrome (5), spinobulbar muscu la r a t rophy (6), myo ton ic dys t rophy (7), Hunt ington ' s disease (8), and spinocerebel lar ataxia type 1 (9). Dinucleot ide repeat alterations have recently been l inked to predisposi t ion to colorectal cancer (10, 11), in which changes in the microsatel l i te repeats were shown to be variable, ranging f rom 2-base pair changes to larger alterations (11), and attr ibuted to RERs (10). Moreover , genet ic instability is present in some inheri ted syndromes (such as xeroderma p igmen tosum, ataxia-telangiectasia, and B l o o m ' s syndrome) in which there is an associated predisposi t ion to cancer (12). We have recently used microsateUites located on h u m a n ch romosomes 9 and 17p to de te rmine the sequence of molecular defects ocurr ing in a series o f 200 T C C of the bladder s (13, 14). Recent ev idence suggests that L OH of c h r o m o s o m e 9 is an early event in the generat ion of papil lary T C C but not in ca rc inoma in situ of the Received 8/30/93; accepted 10/18/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by USPFIS Grant R35 CA49758 from the National Cancer Institute, and a Collaborative Research agreement with Oncor, Inc., Gaithersburg, MD. 2 Recipient of a fellowship from The Government of Navarra, Spain. 3 To whom requests for reprints should be addressed, at the Kenneth Norris Jr. Comprehensive Cancer Center, University of Southern California, 1441 East Lake Ave. Los Angeles, CA 90033-0800. 4 The abbreviations used are: LOH, loss of heterozygosity; PCR, polymerase chain reaction; TCC, transitional cell carcinoma; RER, replication error. 5 C. H. Spruck III, P. E Ohneseit, M. Gonzalez-Zulueta, D. Esrig, N. Miyao, Y. C. Tsai, S. P. Lerner, A. S. Yang, R. Cote, L. Dubeau, P. W. Nichols, G. G. Hermann, T. Horn, K. Steven, D. G. Skinner, and P. A. Jones. Two molecular pathways to transitional cell carcinoma of the bladder, submitted for publication. bladder. 5 Allel ic losses of c h r o m o s o m e 17p and p53 muta t ions were c o m m o n l y seen in carc inoma in situ and in invasive tumors (15). 5 The purpose o f the current report is to show that microsatel l i te changes were apparent in some of these tumors , inc luding four tumors wi th d inucleot ide repeat al terations in c h r o m o s o m e 9 and three tumors wi th changes in a t r inucleot ide repeat in the androgen receptor gene located on the X ch romosome . The cases we report were very interest ing since one of them showed genet ic alterations at all loci examined in chrom o s o m e 9 and all were low stage tumors , sugges t ing that the ge nomic instabili ty g iv ing rise to these microsatel l i te changes migh t occur as an early event in b ladder tumorigenesis . Mater ia l s a n d M e t h o d s The current results were obtained from the analysis of 200 transitional cell carcinomas of the bladder, 154 of which were previously reported cases (13). 5 TCC specimens were obtained from hospitals in Los Angeles County, CA (n = 90), from the Herlev Hospital in Copenhagen, Denmark (n = 64), and from the Johns Hopkins tumor bank, Baltimore (n = 46). Of these, 112 were freshfrozen and 88 were paraffin-embedded tissues. Tumors were graded according to the criteria of Bergkvist et al. (16) and staged according to the tumor-nodesmetastasis staging system (17). High molecular weight DNA was prepared from fresh-frozen tumor specimens and matching blood samples by proteinase K digestion and phenol/chloroform extraction as described (18). DNA from archival paraffin-embedded specimens was isolated by microdissecting tumor and normal tissues from hematoxylin and eosin-stained frozen sections as described (19). Tumor DNA was examined for genetic alterations at seven separate microsatellites, five localized in chromosome 9 (D9S59, D9S63, D9S64, D9S146, D9S156), one in chromosome 17p (D17S513), and one in the X chromosome (androgen receptor gene locus). Loci D9S59, D9S63, and D9S64 were analyzed for 156 tumors; loci D9S146 and D9S156 were analyzed for 49 tumors; locus D17S513 was analyzed for 90 tumors; the androgen receptor gene locus was analyzed for 25 tumors. The dinucleotide repeat polymorphism, (GT),, at loci on chromosomes 9 and 17p was analyzed by PCR amplification followed by electrophoresis on denaturing 8% polyacrylamide gels as described (20, 21). The sequences of primers used are: locus D9S59, 5'-TTA CAC TAT ACC AAG ACT CC-3' and 5'-AAG GGA ATT CAT CCC CTG CT-3'; locus D9S63, 5'-TFA TAA TGC CGG TCAACC Tr-3' and 5'-CCG GAA G'rT ACT CTA GTC TA-3'; locus D9S64, 5'-GAA GGG CTC T I T ATI" AAC TGA T-3' and 5'-AAC CTG GGC GAC ACA GCAA-3'; locus D9S146, 5'-TGC AAT CAA ATT CCC AGC-3' and 5'-GAG GTG ACA TCT GGA AqT-3'; locus D9S156, 5 '-ATC ACT T I T AAC TGA GGA GG-3' and 5'-AGA TGG TGG TGA ATA GAG GG-3'; locus D17S513, 5'-TTC ACT TGT GGG CTG CTG TC-3' and 5'-TAA GAA AGG CTC CCA CAA GCA-3'. The trinncleotide repeat polymorphism, (CAG),, in the androgen receptor gene (22) was analyzed by PCR, performed in a final volume of 25 txl, containing 50 ng of genomic DNA, 1/xM concentrations of each oligonucleotide primer (5'-GTG CGC GAA GTG ATC CAG AA-3' and 5'-TCT GGG ACG CAA CCT CTC TC-3'), 200/xM of the nonradioactive deoxynucleotides, 2/xCi of [a-32P]dCTP, 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 1.5 mM MgC12, 0.01% gelatin, and 1 unit of Taq DNA polymerase (Boehringer Mannhein Biochemicals, Indianapolis, IN). Twentyfour cycles of 94~ for 1 min, 60~ for 1 min, and 72~ for 1.5 rain were performed with the initial denaturation step and final elongation step length-

Transitional cell bladder tumors (TCT) is prone to recurrence (60-80%) after tumor resection. Up to 25% of these patients will progress, so it is important to find reliable predictive markers. We analyzed for loss of heterozygosity (LOH) with respect to 13 microsatellites located on 10 different chromosomal arms. This analysis was performed on the urine sediment and tumor tissue from 59 patients with bladder TCT and on the urine and normal-looking mucosa from 25 patients with a history of bladder TCT but no evidence of disease at the time of the study inclusion. The median follow-up period was 23.1 months (range, 2-48 months) for the 59 patients with bladder TCT and 25 months (range, 4-57 months) for the 25 patients without evidence of ongoing active disease. Correlation between LOH and eventual recurrence, progression, and mortality was investigated. In patients with noninvasive TCT, correlation between 11p tumor tissue LOH and recurrence was found. Similarly, 8p LOH in both urine sediment and tumor tissue correlated with progression. Finally, in the group of patients with a history of bladder TCT, normal tissue 8p and/or 11p LOH correlated with recurrence.

BACKGROUND:To investigate whether the recently reported evidence of differences in the overall loss of heterozygosity (LOH) frequency between urine and tumour tissue in patients with transitional cell tumours (TCT) of the urinary bladder involved specific chromosomal sites, and their impact in diagnosis.MATERIALS AND METHODS:Blood, tissue and urine specimens were obtained from 55 patients and 25 controls. Sixteen microsatellites were PCR-amplified and blindly analyzed for LOH through a laser-based capillary electrophoresis system.RESULTS:Significant frequence differences between tumour tissue and urine sediment LOH were found in 9q and 11p in non-invasive disease and 14q in invasive disease. There was no significant difference for all the other chromosomal arms analyzed.CONCLUSION:The contribution in the urine sediment of cells belonging to tumours of the same histological classification differs according to the specific genetic alterations these cells carry. Furthermore, the location regarding these differences could indicate regions involved in tumour exfoliation or apoptosis.