American Journal ofPathology, Vol. 151, No. 3, September 1997 Copynight © American Societyfor Investigative Pathology Chromosome 8p Deletions Are Associated with Invasive Tumor Growth in Urinary Bladder Cancer Urs Wagner,* Lukas Bubendorf,* a third locus telomeric to 8p23.1.912 Previous studies Thomas C. Gasser,t Holger Moch,* using molecular analysis and comparative genomic hy- Jean-Philippe Gorog,* Jan Richter,* bridization (CGH) have found 8p deletions in more than Michael J. Mihatsch,* Frederick M. Waldman, 25% of bladder tumors.57' 13 Chromosome 8 mapping by and Guido Sauter* Takle et al13 revealed common areas of deletion span- ning from 8p11.2 to 8p12 and from 8p21.1-8pter. In From the Institute ofPathology* and Urologic Clinics,t University another study, we found a strong association between ofBasel, Basel, Switzerland, and the Division ofMolecular chromosome 8q gains and histological phenotype, sug- Cytometry,* Department ofLaboratory Medicine, University of gesting that a gain of chromosome 8q material might also California, San Francisco, San Francisco, California contribute to bladder cancer progression.14 To better understand the role of chromosome 8 alter- ations in bladder cancer 87 tumor specimens were ex- Alterations of chromosome 8, including deletions of amined by dual labeling fluorescence in situ hybridization (FISH). aims were to and 8p, occur frequently in many tumors. In this study, Specific explore prevalence fluorescence in situ hybridization was used to study extent of chromosome 8p losses in bladder cancer and to the relationship between 8p deletions, 8q gains, and determine the relationship of chromosome 8p losses with tumor phenotype in bladder cancer. Celis from 87 tumors 8q gains and histopathologic phenotype. were examined by dual-labeling fluorescence in situ hybridization with a centromere 8 probe (pJM12) and P1 probes for 8p22, 8p12, 8q12, and 8q24. Both 8p22 Materials and Methods deletions and 8q24 gains were strongly associated Tumor Material with tumor phenotype. There was a marked differ- ence in 8p22 deletions between noninvasive (pTa) Seventy-two formalin-fixed, paraffin-embedded bladder tumors (3/33) and minimally invasive (pTl) tumors tumors were randomly selected from the archives of the (8/19; P = 0.005) whereas there was no significant Institute of Pathology, University of Basel. Nuclei of for- difference between pTl and muscle-invasive (pT2-4) malin-fixed tissue blocks were dissociated and dropped tumors (19/35; P = 0.3926). Six tumors with 8p22 onto slides as previously described.15 Additionally, im- deletion were examined at 8p12. Three of these tu- print preparations were made immediately after surgery mors showed no 8p12 deletion, narrowing down the from 15 unfixed bladder tumor samples and stored at site of a putative tumor suppressor gene distal to -20°C. All hematoxylin and eosin slides of each case 8pl2. In one other case, there was a marked increase were reviewed by a single pathologist (G. Sauter). Tumor in 8p12 copy number (>40 per cell; amplification), stage and grade were defined according to UICC and suggesting the presence of an oncogene involved in World Health Organization classifications.16,17 Particu- bladder cancer at 8p12. The marked difference in larly stringent criteria were applied for the diagnosis of 8p22 deletions between noninvasive (pTa) and mini- stage pTl, which is defined by a tumor invasion of the mally invasive (pTl) tumors is consistent with a role suburothelial stroma but not of the muscular bladder wall. of a putative tumor suppressor gene on 8p for devel- An unequivocal stromal invasion was required to prevent opment of invasive tumor phenotype. (AmJ Pathol overstaging of pTa tumors. To minimize the risk of under- 1997, 151:753-759) staging muscle-invasive tumors (pT2-T4), the presence of tumor-free fragments of the muscular bladder wall in Recent cytogenetic and molecular studies have shown that deletions involving the short arm of chromosome 8 Supported by Krebsforschung Schweiz, Schweizerische Krebsliga (SKL are frequent in numerous tumors including prostate,1 137-7-1995), Krebsliga Beider Basel, and Thurgauer Krebsliga (G. Sau- lung,2 colon,3,4 and bladder cancer.5 It has been sug- ter, T. C. Gasser, and M. J. Mihatsch) and National Institutes of Health gested that 8p may be the site of at least one tumor grant CA47537 (F. M. Waldman). suppressor gene.5'6 Mapping studies have proposed Accepted for publication June 18, 1997. several different loci as sites of putative tumor suppressor Address reprint requests to Dr. G. Sauter, Institut fOr Pathologie, Uni- genes, 9 including an area between 8p21.3 and versitatskliniken, Kantonsspital Basel, Schonbeinstrasse 40, CH-4003 8p22,231011 another region centromeric to 8p21,4 and Basel, Switzerland. 753 754 Wagner et al AJP September 1997, Vol. 151, No. 3 the primary biopsy, and the absence of muscle invasion were counted for 100 nuclei. To avoid misinterpretation in a subsequent transurethral resection performed within due to insufficient hybridization efficiency, cells were 8 weeks after the first biopsy was required. Muscle inva- scored only when at least one bright P1 and one bright sion in a follow-up biopsy after less than 8 weeks was centromere signal were present. Two P1 signals were considered to indicate a sampling error at the initial bi- counted as one signal when they were situated very close opsy. Because of the limitations of transurethral biopsies to each other (O0.5 p.m) to avoid misinterpretation due to in accurately determining the depth of invasion of higher- sister chromatids of cells in S or G2M phase. The defini- stage bladder cancer, all tumors showing muscle inva- tions used for deletions and gains of loci were based on sion were categorized into one group (pT2-T4). Only the results obtained in normal urothelial cells from 10 tumors for which histological staging was unequivocal patients without a bladder cancer history (mean + 3 SD). were included in the study. DNA Probes for FISH Statistics A chromosome 8 centromere probe (pJM12) was used x2 tests were used to compare the frequency of genomic in combination with P1 probes mapping to 8p22 alterations between tumors of different grades and (RMC08POO5, LPL, Flpter 0.140), 8p12 (RMC08PO03, stages and to evaluate the relationship between 8p de- D8S549, Flpter 0.307), 8q12 (RMC08PO02, D8S285, letions and 8q gains. Flpter 0.420), and 8q24 (RMC08POO1, c-myc, Flpter 0.845) to determine gains and deletions of chromosome 8 sequences. All probes were kindly provided by Dr. Joe Gray, Resource for Molecular Cytogenetics, University of Results California, San Francisco. The centromere 8 probe was labeled with biotin-14-dATP and P1 probes were labeled Pathology with digoxigenin-1 1-dUTP by nick translation using stan- Eighty-seven primary bladder tumors (72 paraffin-disso- dard protocols. ciated and 15 fresh touch preparations) were success- fully analyzed by FISH (17 additional dissociated cases Fluorescence in Situ Hybridization had insufficient hybridization intensity and were exclud- ed). Thirty-three tumors were confined to the bladder In all hybridizations, a chromosome 8 centromere probe mucosa (pTa), nineteen showed invasion of the lamina (pJM12) was simultaneously used with a locus-specific propria (pTl), and thirty-five were muscle invasive (pT2- P1 probe to define relative gains and losses. FISH was as T4). Fourteen tumors were classified as grade 1, thirty- previously described.18 Briefly, cells on slides were de- nine were grade 2, and thirty-four were grade 3. natured in 70% formamide/2X SSC (1X SSC is 0.15 mol/L NaCI, 0.015 mol/L sodium Citrate), pH 7, at 75°C for 2.5 minutes. After dehydration in graded ethanol, samples were treated with proteinase K (Sigma Chemical Co., St. Normal Controls Louis, MO; 2.0 ,ug/ml for formalin-fixed cells and 0.2 Ten tissue samples (three unfixed bladder washes and ,ug/ml for unfixed specimens) in PBS (pH 7.0) for 7 min- dissociated nuclei from seven formalin-fixed biopsies) utes at 37°C, followed by ethanol dehydration. The hy- containing normal urothelial cells from patients without a bridization mixture was denatured for 5 minutes at 750C. bladder cancer history were used as normal controls. All A 10-,ul aliquot of hybridization mixture consisted of 10 ng normal controls showed an equal number of centromere of P1 probe, 30 ng of centromere 8 probe, and 10 ng of and P1 signals in .85% of cells. The fraction of cells with unlabeled, sonicated (200- to 500-bp) human placental fewer P1 signals than centromere signals ranged from 0 DNA (Sigma) in 50% formamide, 10% dextran sulfate, to 14% (7.5 ± 5.2, mean ± SD). These cells were attrib- and 2X SSC (pH 7). Re-annealing of hybridization mixture uted to a decreased hybridization efficiency of the small was allowed for 30 minutes at 37°C. Hybridization was P1 probes as compared with the much larger centromere overnight at 37°C. Lymphocyte metaphase spreads were probes. The fraction of cells with more P1 signals than used to assure probe specificity. The slides were washed centromere signals (considered to represent cells in S or in 50% formamide/2X SSC, pH 7, at 450 C. Immunohis- G2 phase with both sister chromatids visible) was gen- tochemical probe detection using Texas Red avidin (Vec- erally lower than the fraction of cells with deletion and tor Laboratories, Burlingame, CA) and FITC-conjugated ranged from 0 to 10% (4.5 ± 3.1, mean ± SD). Our cutoff sheep anti-digoxigenin (Vector) was as described.18 Nu- levels for definition of deletions and gains on a tumor clei were counterstained with 0.07 ptg/ml 4.5-diamino-2- basis were based on these results (mean number of phenyl-Indole (DAPI) in antifade solution. aberrant cells found in normal tissues + 3 SD). More than 25% of cells with fewer P1 signals than centromere 8 Scoring of FISH Signals signals were required to define a tumor as deleted for a specific locus.