(CANCER RESEARCH 51, 3807-3813. July 15, 1991] Centromeric Copy Number of Chromosome 7 Is Strongly Correlated with Tumor Grade and Labeling Index in Human Bladder Cancer1 Frederic M. Waldman,2 Peter R. Carroll, Russell Kerschmann, Michael B. Cohen,3 Frederick G. Field, and Brian H. Mayall Departments of Laboratory Medicine [F. M. W., F. G. F., B. H. M.], Pathology [R. K., M. B. C.¡,and Urology ¡P.R. C.J, University of California San Francisco, San Francisco, California 94143-0808 BrdUrd4 into tumor DNA5 or by immunohistochemical staining ABSTRACT of cell cycle-specific antigens such as Ki67 (21) or PCNA (22) The relationship between interphase cytogenetics and tumor grade, expressed only in proliferating cells. The present study was stage, and proliferative activity was investigated in 27 transitional cell undertaken in order to study the relationships in bladder cancer carcinomas of the urinary bladder. Using fluorescence in situ hybridiza between genotypic and phenotypic abnormalities. Tumor grade, tion with chromosome-specific DNA probes, the copy number of pericen- tumor stage, and tumor labeling index, as measured by BrdUrd tromeric sequences on chromosomes 7, 9, and 11 was detected within incorporation or PCNA labeling, were used to characterize interphase nuclei in touch preparations from tumor biopsies. Monosomy tumor phenotype, while tumor genotype was defined by cyto- of chromosome 9 was detected in 9 of 22 cases (41%), while tetrasomy genetic abnormalities, as detected by FISH with DNA probes for chromosomes 7 and 11 was detected in 10 of 26 (38%) and 6 of 23 specific for chromosomes 7, 9, and 11. (26%) cases, respectively. Copy number of chromosome 7 was the most highly correlated with increasing tumor grade (r = 0.616, P < 0.001, MATERIALS AND METHODS Spearman rank correlation) or increasing pathological stage (r = 0.356, Clinical Material. All tumor samples were obtained fresh from the P < 0.002). Copy number for chromosome 9 did not correlate with either Medical Center and affiliated hospitals. University of California, San grade or stage (P > 0.05). Tumor labeling index (LI) was determined Francisco. All patient protocols were reviewed by the Institutional after in vitro 5-bromodeoxyuridine incorporation, while proliferating cell Review Board. Tumors were analyzed for pathological stage (Ta, pap nuclear antigen LI was determined immunohistochemically. Increasing illary noninvasive; Tl, invading into lamina propria; T2, superficial LI by either method correlated with increasing copy number for all three invasion of muscularis; T3, deep invasion of muscularis; T4, metastatic) chromosomes tested (r2 = 0.473, P < 0.002 for 7; r2 = 0.384, P < 0.01 and pathological grade (23), without knowledge of interphase cytoge for 11; and r1 = 0.316, P < 0.05 for 9). Since high tumor grade, stage, netics or labeling index results. Fresh unfixed tumor samples were collected during cystoscopy and and LI are all indicative of more aggressive tumor behavior and worse stored in Hanks' balanced salt solution prior to sample processing. prognosis, these findings suggest that polysomy, especially for chromo Adequate biopsy material was always first collected for histopatholog- some 7, may be highly predictive for bladder tumor aggressiveness. ical diagnosis. Touch preparations were generated by gently touching the urothelial surface of the biopsy to a dry microscope slide. This brief contact allowed an adequate number of single tumor cells to adhere to INTRODUCTION the slide surface. Five to 10 such touch preparations were made from each biopsy specimen. Slides were then air dried at room temperature Numerous specific genetic aberrations have been implicated overnight and stored under nitrogen at -20°C. Whenever possible, 5- in bladder cancer. Cytogenetic studies have defined numerical mm x 5-mm x 1-mm slices were also cut from cystectomy specimens, and structural changes involving chromosomes 1, 3, 5, 7,9, 11, and touch preparations were made as described. and 17 (1-8). Flow cytometric analysis of DNA content has After touch preparations were made, tumor slices were incubated with BrdUrd (100 ¿iMBrdUrd, 10 nM fluorodeoxyuridine, hyperbaric shown a correlation between DNA ploidy, S-phase fraction, oxygen in RPMI) for 2 h at 37°C,to label cells undergoing DNA and tumor grade (9-14). Analyses of allelic loss rates for sites synthesis. Tissue was then fixed in 70% ethanol, embedded in paraffin, on 9q, 11p, and 17p have shown a high fraction of tumors with and sectioned for immunocytochemical staining using monoclonal an such losses (15). In particular, it has been shown (16) that losses tibodies to BrdUrd (IU4, 1:10,000; Caltag) and cyclin/PCNA (19A2, of 9q occur independently of tumor grade, whereas 17p allelic 1:200; Boehringer Mannheim). Standard indirect immunoperoxidase loss is more specifically associated with higher grade tumors. staining was used (Vectastain Elite ABC kit; Vector Laboratories). The proportion of tumor cells staining positively for BrdUrd and PCNA While the prognostic significance of these genetic and cytoge- was determined from 2000 cell counts, by selecting 4-6 high power netic changes has yet to be demonstrated in prospective studies, (x40) fields showing the greatest concentration of stained nuclei. there is increasing evidence that the specific genes involved may Controls used for in situ hybridization were peripheral blood lym modulate tumor proliferation (17-20). phocytes from healthy human volunteers. Lymphocytes in metaphase Tumor labeling index or S-phase fraction has been correlated were obtained after short term culture by standard procedures. Control slides were stored under nitrogen at -20°Cuntil use. with prognosis in bladder tumors (10,12). S-phase fraction can be measured directly by incorporation of [3H]thymidine or Flow Cytometry. When adequate material was available, single-cell suspensions were generated by finely mincing tissue, and cells were prepared according to the method of Vindelov et al. (24). Alternatively, Received 12/7/90; accepted 5/7/91. The costs of publication of this article were defrayed in part by the payment a vigorous bladder wash specimen taken at the time of biopsy was used of page charges. This article must therefore be hereby marked advertisement in for flow analysis. The FACScan flow cytometer (Becton-Dickinson, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This work was supported by NIH Grant CA47537, as part of the Bladder 4The abbreviations used are: BrdUrd, bromodeoxyuridine; PCNA, proliferat Marker Network of the National Cancer Institute. ing cell nuclear antigen: FISH, fluorescence in situ hybridization; SSC, standard 2To whom requests for reprints should be addressed, at Laboratory for Cell saline citrate: LI, labeling index; ADB, antibody-diluting buffer; FITC, fluorescein Analysis, MCB 232, Dept. of Laboratory Medicine, University of California San isothiocyanate. Francisco, San Francisco, CA 94143-0808. 5Waldman, F. M., Chew, K., Ljung, B. M., Goodson, W., Horn, J., Duarte, 3 Present Address: Department of Pathology, University of Iowa, Iowa City, I... Smith, H., and Mayall, B. A comparison between bromodeoxyuridine and IA. [3H]thymidine labeling in human breast tumors. Modern Pathol., in press, 1991. 3807 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1991 American Association for Cancer Research. BLADDER INTERPHASE CYTOGENETICS San Jose, CA) was used for analysis, with doublet discrimination gating age, sex, labeling indices, and interphase cytogenetics for the and CellFit (Becton-Dickinson) software. A clearly defined second peak three chromosomes tested. The worst pattern present for nu comprising more than 20% of total cells was necessary to define clear grade and pathological stage was recorded for each case. aneuploidy. The mean age was 68 years, and 90% of the tumors were from Chromosome-specific Probes. The probes used were specific for per- men. Four of 27 patients were treated with various intravesical icentromeric sequences on chromosome 7 (p7«tet;H. Willard, Stanford chemotherapeutic reagents prior to biopsy. University), chromosome 9 (pHUR98; B. Moyzis, Los Alamos Na tional Laboratory), and chromosome 11 (pLCl l A; H. Willard). Probes Interphase Cytogenetics. The copy number distributions of were labeled by nick translation using either biotin-11-dUTP (Bethesda chromosomes 7, 9, and 11 in control interphase lymphocytes Research Laboratories) or digoxigenin-11-dUTP (Boehringer Mann are shown in Fig. 1. The sensitivity of the reaction was apparent heim). Approximately 5-35% of thymidine residues were substituted from the high proportion of nuclei having two signals. Because during the reaction. only 3.3, 4.2, and 5.0% of control nuclei had one target copy In Situ Hybridization. The hybridization was carried out as previously described (25), with modifications (26). Slides were first fixed for 5 min each in three changes of freshly prepared Carnoy's solution (3/1 meth- Table 1 Summary of patient data and interphase cytogenetics for 27 bladder anol/acetic acid). Hybridizations containing 5-10 ng labeled probe, 2.5 tumors /¿gcarrierDNA, 55% formamide, and 10% dextran sulfate, in 2x SSC, were incubated overnight at 37°C.Slides were washed 3 times for 10 num min each in hybridization wash buffer (2x SSC, 50% formamide for ber17111222222224444543345544449131112311144323213421411I2111222222443344435342 LISex" LI(%)0.73.61.24.86.35.430.915.73.746.922.714.215.328.6DNAindex*111112.OB.OB.OT.OB.OT.OT2.0