Centromeric Copy Number of Chromosome 7 Is Strongly Correlated with Tumor Grade and Labeling Index in Human Bladder Cancer1

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(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.Â¡,and Urology Â¡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 -20Â°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 Â¿iMBrdUrd, 10 nM fluorodeoxyuridine, hyperbaric shown a correlation between DNA ploidy, S-phase fraction, oxygen in RPMI) for 2 h at 37Â°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 -20Â°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

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 (p7Â«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 /Â¿gcarrierDNA, 55% formamide, and 10% dextran sulfate, in 2x SSC, were incubated overnight at 37Â°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 chromosome 7, 55% formamide for chromosomes 9 and 11) at 45Â°C (%)FMMMMMMMMFMMMMMMMMMMMMMMMMM1.33.60.71.51.25.14.414.910.14.815.013.713.317.32013.6PCNA and then 2 times in 2x SSC, first at 45Â°Candthen at room temperature. Case123456789101112131415161718192021222324252627GradeIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIStageTaTa"TaTaTaTaTaTaTlTa"Ta"TaTaTaTlTlTlTlT3TlTlTlTlT2T3T3T3"Age(yr)537885455659778158707660647170677986808764556363636767BrdUrd To minimize nonspecific staining, slides were then incubated for 5 min in ADB (5% Carnation milk powder, 4x SSC, pH 7, 0.02% sodium azide, 0.1% Triton X-100). Slides on which cells had been hybridized to biotinylated probes were incubated for 25 min at room temperature in FITC-avidin (Vector Laboratories) diluted 1:200 in ADB. Slides on which cells had been hybridized to digoxigenated probes were incubated for 30 min in FITC-conjugated antidigoxigenin antibody (Boehringer Mannheim) in ADB. Slides were then rinsed 10 min each in 4x SSC, 4x SSC/0.1% Triton X-100, and 4x SSC. Slides were then rinsed in distilled water, air dried, and counterstained with either 0.25 Mg/ml propidium iodide (Sigma) or 0.2 Mg/ml 4',6-diamidino-2-phenylindole hydrochloride (Molecular Probes) in anti-fade solution (27). If neces T1.8 sary, biotinylated probe signals were amplified by preblocking in ADB for 5 min, incubating 30 min in 1:100 goat antiavidin/ADB. washing in 4x SSC, blocking again in ADB, and incubating 25 min in FITC- avidin diluted 1:200 in ADB. Scoring of Interphase Nuclei. A touch preparation from each biopsy was stained with Giemsa and analyzed to determine the proportion of T17.321.920.138.948.126.521.91.8 malignant cells present. Those cases with <50% tumor cells in the touch preparations (by conservative cytological estimate) were censored from the study. Additionally, six cases showed no tumor in hematoxy- TCopy lin- and eosin-stained sections of the biopsy and were excluded. When " M, male; F. female. the majority of cells were tumor cells by cytological criteria, benign * B, bladder wash; T. disaggregated tissue. ' Centramene copy number of major or dominant population. fibroblasts and inflammatory cells were readily recognized and were d Previously treated with intravesical chemotherapy. excluded from further cytogenetic analysis. After hybridization, slides were scored for the number of hybridiza tion signals in each nucleus, using an epifluorescence microscope equipped with a 63x na 1.3 oil immersion objective. If possible, 200- 100 H 500 nuclei were scored for each hybridization. Nuclei in which the nuclear boundary was broken or torn were considered damaged and were censored from analysis. Similarly, squashed, smeared, clumped, or overlapped nuclei were ignored. Each hybridization was accompanied by a control hybridization using normal lymphocytes. If the control 0) displayed increased monosomy (>10%), parallel tumor preparations 73 60 3 displaying weak signals were repeated, because the likelihood of missing signals during the counting procedure was high. 40- The copy number category (monosomic, disomic, trisomie, tetrasomic, pentasomic, and above) reflects the major (>50%) or dominant (20-50%) population present. If no aneusomic (i.e., other than two m 20 copies/nucleus) subpopulation was present at >50% of the total, then the largest aneusomic subpopulation having >20% of total cells was used. Thus, a tumor was considered disomic if no other subpopulations had >20% of total cells. 01234 RESULTS Centromeric Copy Number Fig. 1. Centromeric copy numbers within control interphase lymphocyte nuclei Clinical. Twenty-seven cases of transitional cell carcinoma of for chromosomes 7 (B, n = 7), 9 (Ã¼,n = 3), and 11 (D, n â€”4). Two hundred the bladder were analyzed. Table 1 shows tumor grade, stage, nuclei were scored for each hybridization. 3808

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1991 American Association for Cancer Research. BLADDER INTERPHASE CYTOGENETICS for chromosomes 7, 9, and 11, respectively, it was possible to to chromosome 9 probe, showing one copy in each cell. Cyto- define, with high confidence, true tumor monosomy when the plasmic shadows could be seen staining nonspecifically with proportion was above 20%. Conversely, the specificity of hy propidium iodide. Nonspecific hybridization to nuclear DNA bridization reactions was demonstrated in metaphase chromo was also apparent as much smaller and fainter fluorescein somes, in which only two specific targets were present. signals. Monosomy for chromosome 11 in case 5 is shown in Photomicrographs of four different hybridizations are shown Fig. 2D. A minor population of nuclei having two signals was in Fig. 2. Case 20 (Fig. 2A) was easily characterized, showing also present. In this photomicrograph, cytoplasmic fluorescence three small well defined signals in each nucleus with the probe appears green. for chromosome 7. Case 25 (Fig. 2B) also is the result of A summary of the interphase FISH results for two repre hybridization with probe specific for chromosome 7. Most sentative tumors is shown in Fig. 3. In case 11, two patterns of nuclei showed four signals, although in this case the signals copy number distribution were seen. Chromosome 9 was pres were larger than in case 20 and slightly spread. Two smaller ent predominantly as one copy/nucleus. Chromosomes 7 and nuclei having only two signals were also present. Some of the 11 were detected predominantly as two copies/nucleus. This signals were split into couplets, which were scored as single tumor was scored as monosomic for 9 and disomic for 7 and signals. Fig. 2C shows nuclei from case 11 after hybridization 11. Note that there were small populations of nuclei which had

Fig. 2. Photomicrographs of four different tumors after fluorescence in situ hybridization. Probes were biotinylated and then stained with FITC-avidin (green). Nuclei were counterstained with propidium iodide (red) or 4'.6-diamidino-2-phenylindole hydrochloride (blue). Green cytoplasmic fluorescence is the result of autofluorescence and nonspecific staining. Multiple exposures were used to combine colors. Original magnification, x63. /. case 20, showing trisomy 7. B, case 25, showing tetrasomy 7. C, case 11, showing monosomy 9. D. case 5, showing monosomy 11. 3809

Case 11 tumors had a negative imbalance for chromosome 7. 100 n Chromosome 9 was probed in 22 cases. Nine of the 22 cases (41%) were monosomic for chromosome 9. Five of these mon- 80 osomic cases (cases 9-11, 18, and 22) and one disomic case Â« (case 24) had a negative imbalance and two cases (cases 3 and u 60 8) had a positive imbalance for chromosome 9. Chromosome 11 was probed in 23 tumors. Eight cases (35%) 40 o were disomic and six (26%) tetrasomic. There was a positive â€¢s imbalance in one disomic (case 2) and one tetrasomic (case 19) 20 case and a negative imbalance in one disomic (case 27) and one trisomie (case 21) case. O 1 2 3 Table 2 Incidence of centromeric copy number in 27 bladder tumors Centromeric Copy Number Incidence (%) 100 Case 27 n I copy 2 copies 3 copies 4 copies 5 copies 80 Chromosome7Chromosome 9Chromosome 11262223124117311835g2317381826115 60

40- g 20- I o 20- E 0123456 Centromeric Copy Number 10 - Fig. 3. Actual distributions of Centromeric copy number for two bladder tumors. Chromosomes probed were 7 (â€¢).9(0), and 11 (D). l one copy of chromosomes 7 and 11, most likely due either to overlap causing apparent fusion of two signals into one or to Centromeric Copy Number weaker signals which were missed during scoring. These subpopulations, always <10%, were also present in control prepa rations. The small populations (~5%) of nuclei having three copies of chromosome 7 was less likely due to artifact, however. 30 -, These trisomy 7 nuclei most likely represented true heteroge neity of copy number distribution, because nuclei with such increased copy number are not present in control hybridizations 20 - in the absence of larger populations with four or more signals. Case 27 in Fig. 3 showed a more heterogeneous distribution of centromeric copy numbers. Large subpopulations (30-50%) 10 - of nuclei having either two or four copies of chromosomes 7 and 9 were present. Chromosome 11 was present predominantly at two copies/nucleus. This tumor was scored as tetrasomic for 7 and 9 and disomic for 11. 2345 Table 1 shows the clinical, proliferative, and cytogenetic Centromeric Copy Number information for all of the cases analyzed, ordered with increas ing pathological grade. Although sufficient tissue for flow cytometric analysis was available for only nine cases, a good correlation was seen between DNA index and total centromeric 30 -i copy numbers. In case 9, there was a small (10% of total cells) peak seen by flow cytometry with a DNA index of 1.8. Inter estingly, there was a similar minor population of nuclei ana 20 - lyzed by FISH with four copies of chromosome 7 (20%) and 11 (16%) and <4% tetrasomy for chromosome 9 (data not shown). The copy number distributions, for the 27 tumors, for each of the three chromosomes tested are summarized as percentages in Table 2. Chromosome 7 was probed in 26 cases. Ten of these l (38%) showed tetrasomy for chromosome 7, while eight cases (31 %) showed disomy. Note from Table 1 that five cases (cases CenÃ®romeric Copy Number 4, 5, 14, 16, and 24) showed a positive imbalance of chromo Fig. 4. Relationship between tumor grade and centromeric copy number for some 7 (i.e., 9 and 11 both had fewer copies than did 7), but no chromosomes 7, 9, and 11. B, grade 1; O. grade II; D, grade III. 3810

so n as strongly as grade (Table 1 and Fig. 5). Stage Ta tumors were more likely to be monosomic or disomic, and stage T2-T3 tumors were more likely to be polysomic. The correlation I 20 between stage and chromosome 7 copy number (Spearman rank correlation, r2 = 0.356, P < 0.002) was stronger than that between stage and chromosome 11 copy number (r2 = 0.284, P 10- < 0.01); there was no significant correlation between chromo some 9 copy number and tumor stage (r2 = 0.025, P > 0.25). BrdUrd Labeling Index. Tumors were labeled in vitro with BrdUrd, to detect the fraction of cells in the S (synthesis)-phase 12345 of the cell cycle (the tumor labeling index). The LI showed a Centromeric Copy Number strong correlation with pathological grade (Spearman rank correlation, r2 = 0.814, P < 0.001) and stage (r2 = 0.537, P < 0.001). BrdUrd LI was compared to the chromosome copy number, as determined by FISH. Fig. 6 shows the strong 30 -Â¡ correlation which existed for chromosomes 7 (Spearman rank correlation, r2 = 0.473, P < 0.002), 9 (r2 = 0.316, P < 0.05),

o 20-10

40 - -LUI'â€¢-1

30H

1iPm 20 - 12345 Centromeric Copy Number m 10 -

12345 30 -, Centromeric Copy Number

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12345 Centromeric Copy Number T> m 10 - Fig. 5. Relationship between tumor stage and Centromeric copy number for chromosomes 7, 9. and 11. B. stage Ta; m, TI; D, T2; B, T3.

Centromeric Copy Number The copy numbers for chromosomes 7, 9, and 11 were generally correlated with each other. The correlation between chromosomes 7 and 11 was the strongest (r2 = 0.619, P = 0.0001, simple regression), followed by 7 and 9 (r2 = 0.209, P = 0.037) and 9 and 11 (r2 = 0.127, P = 0.123). Tumor Grade and Interphase Cytogenetics. Tumor grade was strongly associated with chromosome copy number for chro C 30- mosomes 7 and 11 (Spearman rank correlation, r2 = 0.616, P < 0.001 and r2 = 0.524, P < 0.001, respectively) and was not 20 - quite significant for chromosome 9 (r2 = 0.140, P < 0.10). As shown in Fig. 4, all grade I tumors were either monosomic or disomic for chromosome 7, while all grade III tumors were polysomic. Grade II tumors were more evenly distributed. These relationships were also apparent for chromosome 11 but 012345 less strongly so for chromosome 9. Centromeric Copy Number Tumor Stage and Interphase Cytogenetics. Tumor stage was Fig. 6. Relationship between BrdUrd labeling index and Centromeric copy also correlated with chromosome copy number, although not number for chromosomes 7, 9, and 11. 3811

DISCUSSION 50 - (Z) â€” 40 - Numerical cytogenetic aberrations in 27 carcinomas of the urinary bladder have been characterized in interphase cells, -j 30- using fluorescence in situ hybridization with DNA probes spe cific for pericentromeric regions on chromosomes 7, 9, and 11. Z 20 - These tumors showed a high incidence of chromosomal aneu- O o. somy; only one of the 27 tumors analyzed was disomic for all 10 - three chromosomes tested. There was a high correlation seen between the BrdUrd and PCNA labeling indices and tumor 1 2 grade, tumor stage, and chromosomal pericentromeric copy Centromeric Copy Number number. This correlation was strongest for chromosome 7 copy number and weakest for chromosome 9 copy number. Chromosomal copy number, as defined by FISH with peri centromeric probes, is only an estimate of the copy number of sequences carried on that chromosome. It should be emphasized so - that each nuclear signal which was counted represented the hybridization target only. Other regions on these chromosomes â€” Â«O coding for other gene sequences may have been present at â€” 30 - greater or fewer copy number. It should be noted that centro meric copy number determined by FISH does not increase with 20 - DNA replication. G2 cells show only two hybridization spots U CL for each centromeric signal, because sister chromÃ¢tids have not 10- yet separated. This is evident from the virtual absence of nuclei with four signals in preparations of proliferating lymphocytes. 1 2 3 Analyses of primary bladder cancers using banded cytogenet- Centromeric Copy Number ics have shown numerous nonrandom aberrations, with increas ing numbers of numerical and structural aberrations present in more advanced disease. Both monosomy 9 and trisomy 7 have been proposed as primary (initiating) changes in bladder cancer, since they are sometimes the sole cytogenetic lesion detected so - (4, 8). Structural alterations of chromosome 11 have also been noted, particularly deletions on lip (1, 29). Although flow â€” 40-1 cytometric analysis of DNA content of tumor cells was only -j 30- possible in a fraction of cases in this study, the strong correla tion between FISH and flow analysis seen in other studies (30, 20 - 31) was substantiated. Flow cytometric analysis does not dis U a. criminate between chromosomes, however. Aneusomies, as de tectable by FISH, which affect just one or a few chromosomes, are frequently missed in tumors with a diploid DNA index.6 The correlation between chromosomal copy number and Centromeric Copy Number tumor proliferation or grade may be direct and specific or Fig. 7. Relationship between PCNA labeling index and centromeric copy indirect and nonspecific. Results reported here may specify a number for chromosomes 7, 9, and 11. direct connection between chromosome 7 copy number and tumor behavior. There may be genes on chromosome 7 (epider mal growth factor receptor, for example) which, when present and 11 (r = 0.384, P < 0.01). For chromosome 7, all mono- in extra copy number, lead to increased tumor proliferation. Alternatively, the strong correlation between chromosome 7 somic and disomic tumors had a BrdUrd LI less than 5.1%, copy number and tumor proliferation may be a general phenom while only one tumor with greater copy number had a labeling enon, in which genetic instability (and chromosomal aneusomy) index of <10% (this was a stage Ta tumor and had a LI of leads to any of a number of specific gene mutations or deletions 4.8%). A similar relationship was present for chromosome 11 on other chromosomes, and this, in turn, may cause increased but was not present for chromosome 9. tumor proliferation. The tighter correlation between chromo PCNA Labeling Index. PCNA is an auxiliary protein to DNA some 7 copy number and tumor proliferation, as compared to polymerase 6 (28). Like BrdUrd, it can be detected immunohis- chromosomes 11 and 9, might be due to an increased likelihood tochemically and is a reflection of the fraction of cycling tumor for random loss of 9 or 11, rather than an amplification of cells. The PCNA LI showed a good correlation with patholog ical grade (Spearman rank correlation, r2 = 0.444, P < 0.005) specific gene(s) on chromosome 7. and stage (r2 = 0.444, P < 0.005). PCNA Lis were compared A high incidence of alMie loss in chromosomes 9, 11, and 17 (and low levels of loss in the other chromosomes tested) has to chromosome copy number as determined by FISH. Fig. 7 been reported in bladder cancer (15, 16, 33). The pattern of shows the distribution of PCNA LI with chromosome copy number for chromosomes 7 (Spearman rank correlation, r2 = allelic loss, when detected, is usually consistent with >90% of 0.519, P < 0.001 ), 9 (r2 = 0.040, P > 0.50), and 11 (r2 = 0.569, tumor cells having that loss (32), strongly suggesting that a P < 0.002). ' Submitted for publication. 3812

O., and Ous, S. DNA flow cytometry in human bladder carcinoma. Pathol. genetic change associated with allelic loss will lead to an in Res. Pract., 181: 291-295, 1986. creased proliferative advantage for that tumor clone. Thus, 10. Helpap, B., Schwabe, H. W., and Adolphs. H. D. Proliferative pattern of finding a high S-phase fraction in tumors carrying specific urothelial bladder cancer and urothelial atypias. J. Cancer Res. Clin. Oncol., 109: 46-54, 1985. genetic aberrations is not surprising. 11. Jakobsen, A., Mommsen. S., and Olsen, S. Characterization of ploidy level A strong correlation was seen in this study between tumor in bladder tumors and selected site specimens by flow cytometry. Cytometry, grade and both BrdUrd and PCNA labeling indices. This as 4: 170-173, 1983. 12. Kirkhus, B., Clausen, O. P., Fjordvang, H., Helander, K., Iversen. O. H., sociation was also seen in a larger series of bladder tumors. The Reitan, J. B., and Vaage, S. Characterization of bladder tumours by multi- correlation between LI and grade is consistent with tumor grade parameter flow cytometry with special reference to grade II tumours. Apmis, being a descriptor of nuclear activity. Proliferating cells gener 96: 783-792, 1988. 13. Smeets, A. W., Pauwels, R. P., Beck, J. L., Geraedts, J. P., Debruyne, F. M., ally have larger, less condensed nuclei and more mitoses than Laarakkers. L., Feitz, W. F., Vooijs, G. P., and Ramaekers, F. C. Tissue- nonproliferating cells, characteristics used to define higher tu specific markers in flow cytometry of urological cancers. III. Comparing chromosomal and flow cytometric DNA analysis of bladder tumors. Int. J. mor grade. Pathological stage is less clearly a marker of prolif Cancer, _?9:304-310, 1987. eration than of tumor invasiveness and is less strongly associ 14. Wijkstrom, H.. Granberg-Ohman, I., and Tribukait, B. Chromosomal and ated with either labeling index or chromosome copy number in DNA patterns in transitional cell bladder carcinoma. Cancer (Phila.), 5.?: 1718-1723, 1984. our study. 15. Tsai, Y. C., Nichols, P. W., Hiti, A. L., Williams, Z., Skinner, D. G., and In summary, the results of this study demonstrate a strong Jones, P. A. Allelic losses of chromosomes 9, 11, and 17 in human bladder correlation in human bladder cancer between genotypic abnor cancer. Cancer Res., 50: 44-47, 1990. 16. Olumni, A. F., Tsai, Y. C, Nichols, P. W., Skinner, D. G., Cain, D. R., malities and tumor phenotype. Changes in centromeric copy Bender, L. I., and Jones, P. A. Allelic loss of chromosome 17p distinguishes number reflect genotypic modification. An increase in copy high grade from low grade transitional cell carcinomas of the bladder. Cancer Res., 50:7081-7083, 1990. number, particularly of chromosome 7, is associated with a less 17. Cooper, J. A., and Whyte, P. RB and the cell cycle: entrance or exit? Cell, favorable phenotype, including high nuclear grade, high path 5*: 1009-1110, 1989. ological stage, and high BrdUrd and PCNA labeling indices. 18. Fearon, E. R.. Cho, K. R., Nigro. J. M., Kern, S. E., Simons, J. W., Ruppert. J. M., Hamilton, S. R., Preisinger, A. C., Thomas, G., Kinzler, K. W., et al. Conversely, a disomic or monosomic genotype, particularly for Identification of a chromosome 18q gene that is altered in colorectal cancers. chromosome 7, is associated with a more favorable phenotype. Science (Washington DC). 247:49-56, 1990. The causal mechanism for this relationship is unclear, although 19. Liotta, L. A., and Steeg, P. S. Clues to the function of Nm23 and Awd proteins in development, signal transduction, and tumor metastasis provided it may possibly involve expression or suppression of specific by studies of Dictyostelium discoideum. J. Nati. Cancer Inst., 82:1170-1172, genes on chromosome 7. Prospective follow-up of these patients 1990. 20. Michalovitz, D., Halevy, O., and Oren, M. Conditional inhibition of trans will establish the relationship between the observed genotype formation and of cell proliferation by a temperature-sensitive mutant of p53. and phenotype and actual clinical outcome. Cell, 62:671-680, 1990. 21. Matsumura, K., Tsuji, T., Shinozaki, F., Sasaki, K., and Takahashi, M. Note Added in Proof Immunohistochemical determination of growth fraction in human tumors. Pathol. Res. Pract., 184:609-613, 1989. I [opinan el al. (Hopman, A. H. N., Moesker, O., Smeets, W. G. B., Pauwels, 22. Celis, J. E., and Celis, A. Cell cycle dependent variations in the distribution R. P. E., Vooijs, G. P., and Ramaekers, C. S. Numerical chromosome 1, 7, 9, of the nuclear protein cyclin proliferating cell nuclear antigen in cultured and 11 aberrations in bladder cancer detected by in situ hybridization. Cancer cells: subdivision of S phase. Proc. Nati. Acad. Sci. 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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1991 American Association for Cancer Research. Centromeric Copy Number of Chromosome 7 Is Strongly Correlated with Tumor Grade and Labeling Index in Human Bladder Cancer

Frederic M. Waldman, Peter R. Carroll, Russell Kerschmann, et al.

Cancer Res 1991;51:3807-3813.

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