[CANCER RESEARCH 51, 644-651, January 15. 1991] Numerical Chromosome 1, 7, 9, and 11 Aberrations in Bladder Cancer Detected by in Situ Hybridization1 Anton H. N. Hopman,2 Olof Moesker, A. Wim G. B. Smeets, Ruud P. E. Pauwels, G. Peter Vooijs, and Frans C. S. Ramaekers Department of Pathology, L'niversity //ospitai Nijmegen, fieert Grooteplein Zulu 24, 6525 (iA, .\ijmegen ¡A.H. N. H., O. .\t., C. P. ('./.' Stickling Ziekenkuisapotkeek en Klinisch Laboratorium l'enray [A. H'. G. B. S.J; Department of Urology, Hospital I enlo-1 'enray [R. P. K. P.], and Department of Molecular Cell Biology, L'nirersity ofLimhurg, Maastricht ¡A.H. N. H., F. C. S. R.], The Netherlands. ABSTRACT studies we demonstrated that this approach enables a routine screening of large tumor cell populations in, for example, Forty transitional cell carcinomas of the human urinary bladder (TCCs) TCCs4 (5, 10). Furthermore, ISH enables the detection of minor were examined for numerical aberrations of chromosomes 1, 7, 9, and 11 by in situ hybridization using chromosome-specific probes. Our inter- cell populations or imbalance in chromosome copy number phase cytogenetic study of 24 low-grade, noninvasive TCCs, which were within one tumor. near-diploid by flow cytometry, showed a numerical aberration for at By means of conventional karyotyping nonrandom chromo least I of these chromosomes in 14 of these cases. Most strikingly, a some aberrations involving chromosomes 1, 7, 9, and 11 have monosomy for chromosome 9 was found in 9 of 24 low-grade TCCs. A been detected in bladder cancer. Table 1 summarizes the chro trisomy for chromosomes 1, 7, and 11 was detected in 5, 2, and 1 case(s), respectively. In 1 case a monosomy for chromosome 1 was detected by mosomal aberrations in TCCs with a modal chromosome num in situ hybridization. Monosomy for chromosome 9 was the only detected ber of 2 n described in the literature thus far (17-25). Of 52 numerical change in 5 low-grade TCC cases. Examination of 16 invasive described cases, 32 tumors showed affected chromosomes 1, 7, TCCs showed extra copies for chromosomes 1 and 7 in 7 flow cytometr- 9, or 11. Monosomy for chromosome 9 was observed in 15 ically diploid cases with numerical chromosome aberrations; also, loss of cases, in which this aberration was the only karyotypic change chromosome 9 was detected. In 5 invasive and 2 noninvasive aneuploid/ observed in about 40% of these tumors. In the noninvasive tetraploid TCCs a profound imbalance between the different chromo cancers, monosomy 9 was detected in 6 of 12 cases. With somes was found. In 5 of these cases an evident underrepresentation of chromosome 9 in comparison to chromosomes 1, 7, and 11 was detected. progression of the disease numerical and structural abnormali This underrepresentation of chromosome 9 in diploid, as well as aneu ties of chromosomes 1, 7, and 11 have been suggested to occur ploid, TCCs, and in some cases the constant ratio between this chromo in higher numbers of tumors. Furthermore, allelic loss on some and the other chromosomes, may be explained by a process of chromosomes 9, 11, and 17 has been found recently in high- tetraploidization. Therefore, loss of chromosome 9 may be one of the stage, high-grade TCCs by screening of loci on these chromo primary genetic events in TCC oncogenesis, with secondary events, such somes by restriction fragment length polymorphism analysis as tetraploidization, correlated to tumor progression. Our results show (26,27). that in situ hybridization can be routinely used to study important For the underlying study we used probes that recognize highly- cytogenetic changes which occur during the development of a malignant disease. repetitive sequences in the centromeric regions of chromosomes 1,7,9, and 11 (28-31 ). The number of ISH signals, which was found to be constant during the entire cell cycle, in the inter- INTRODUCTION phase nucleus and in the condensed chromosomes indicates the The cellular DNA content of certain malignancies is regarded chromosome copy number independent from the cell cycle stage as a prognostic parameter. Therefore, the FCM analysis became (5, 6, 10). Although the DNA probes used in these experiments a rapid and objective screening method for the DNA content detect highly repetitive genomic sequences and their target of malignant tumor cells (1-3). However, no small variations covers only a limited part of the chromosome, their applicability in DNA content can be detected. Karyotyping of solid tumors is best demonstrated by comparing cytogenetic and flow cyto- is a more precise approach and detects numerical and/or struc metric data. tural chromosomal defects (4). However, when tumor cells are Screening of 40 noninvasive and invasive TCCs by in situ cultured to obtain more and better metaphases rather than hybridization with these chromosome probes is of particular using direct analysis, a potential danger of loss of genetic interest in view of the following questions: (a) Can ISH screen material and selection of certain fast growing subpopulations is ing of TCCs be applied to the detection of numerical chromo present. some aberrations described by karyotyping in literature? (b) In situ hybridization using specific probes and nonisotopic Can the recent findings of loss of chromosome 9q (26) in high- detection procedures allows the detection of numerical (5-12) stage, high-grade tumors be confirmed by the use of a chro and structural chromosome aberrations in nonmitotic cells (13- 16). This method of interphase cytogenetics has already been mosome 9 repetitive centromeric probe? and (c) Is tetraploidi applied to several types of malignancies (7-11). In earlier zation a crucial step in genetic progression of solid tumors, in which random as well as nonrandom loss of chromosomes Received 7/18/90; accepted 10/26/90. could lead to selection and growth of cells to aneuploid or The costs of publication of this article were defrayed in part by the payment heteroploid cells (32-36)? 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. 1This work was supported by the Netherlands Cancer Foundation. NUKC 'The abbreviations used are: TCC. transitional cell carcinoma of the human 88-6. urinary bladder; ISH, in situ hybridization; FCM, flow cytometry; DI. DNA 2To whom requests for reprints should be addressed, at Department of index; PBS, phosphate-buffered saline; SSC, standard sodium citrate; FITC, Molecular Cell Biology. University of Limburg. Faculty of Medicine, P.O Box fluorescein isothiocyanate; RFLP, restriction fragment length polymorphism; 616. 6200 M D Maastricht. The Netherlands. TRITC. rhodanin isothiocyanate. 644 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1991 American Association for Cancer Research. INTERPHASE CVTOGENETICS IN BLADDER CANCER Table 1 Summary of results described in the literature with respect to chromosome I, 7, 9, and 11 aberrations in handed near-diploid transitional cell carcinomas of the urinary bladder involving chromosome chromosomes I, Author(s)Gibas« tumors"65416587luCase25723456121135679234568367132\23GradeGno. orrange45474542-4544-4546464645424746464745-48464646-504746454746444245/4639-424941-454745Aberrations1-9. 7, 9. and 1 ft al.(18)Atkin 2Gjc;2G,G,G,G2G,G,-G2G3G2NGNGNGNGG2G2G2G,G2G2Ci;G3<;2G,G,Ci,G2G2G2G,StageT.T,T.T2T,T,T,T2T,T3T.NGNGNGNGT./T,T./T.T./T,Tu/T,T.T.T.T.T,T,T,T.T.T.T.T,Modal-11-7. -9-9lp-llp-llq+. (19)Gibasand Baker -9i(lq), llp-.-9t(X;l), llqder(l)+7+7-9, (20)Sandbergétal. (21)Bergerétal. (22)Sandberget al. ¡<9q)-9+7Examples, (23)Baba -9Deletion +7. et al.(24)Smeets llpDuplication7q, 9q, IqDeletion 11p.duplication Iq+79q--99q++ (25)Vanniet al. IP-+ lp-, -9.llp+-7, -9,(t7:9)-9/del(9)(ql2)-9-I,del(l)(q21), et al. (26)Ail -9-9+79p-, llp- ' Number of patients examined with a near-diploid chromosome number. *G. grade: T, tumor: NG. not given. MATERIALS AND METHODS 10% dextran sulfate-1 Mg/M'salmon sperm DNA as carrier DNA. Under these stringent conditions, hybridization to minor binding sites was avoided. Hybridization mixture, 5 /il, was added to the slides under a Samples. Forthy fresh TCC specimens (clinical and ISH data sum coverslip (18 x 18 mm) and denaturated on the bottom of a metal box marized in Tables 2 and 4) were obtained immediately after transure- in a water bath at 70°Cfor 3 min. Hybridization was performed thral resection. PCM and direct karyotyping was performed as de overnight at 37°C.Posthybridization washings were done twice in 60% scribed previously (24, 37). The DNA content is expressed as the DI. formamide-2x SSC at 42°Cfor 5 min and twice in PBS containing The TCCs were qualified FCM (near)-diploid (Dl = 0.9-1.1), FCM 0.05% Tween-20 at room temperature. Single-target ISH reactions aneuploid (DI = 1.2-1.7, >2.2), and FCM tetraploid (DI = 1.8-2.2). were performed using biotinylated probes, immunocytochemically proc Tumor Cell Processing for in Situ Hybridization. Preparation of slides essed using FITC-conjugated avidin, and. if necessary, immunologically from the cell suspensions, as well as steps necessary for removal of amplified using biotin-labeled goat anti-avidin followed by a second cytoplasm to improve DNA probe and antibody penetration, was per layer of FITC-conjugated avidin. Double-target ISH using biotin-la formed as previously described (10, 38). Briefly, 5 ¿/Iofa TCC cell beled and digoxigenin-labeled probes were hybridized simultaneously suspension was dropped onto poly-/-lysine-coated glass slides, air dried, and immunochemically processed using monoclonal anti-digoxin and heated at 80°Cfor 60 min. Thereafter digestion with pepsin from (Sigma) in PBS-4% normal rabbit serum, followed by incubation of porcine stomach mucosa (2500-3500 units/mg protein; Sigma Chem FITC-conjugated avidin and TRITC-conjugated rabbit anti-mouse an ical Co., St.