[CANCER RESEARCH 59, 5148 –5153, October 15, 1999] Pancolonic Chromosomal Instability Precedes Dysplasia and Cancer in Ulcerative Colitis 1 Peter S. Rabinovitch, 2 Shawna Dziadon, Teresa A. Brentnall, Mary J. Emond, David A. Crispin, Rodger C. Haggitt, and Mary P. Bronner Departments of Pathology [P. S. R., D. A. C., R. C. H., M. P. B.], Medicine [S. D., R. C. H., T. A. B., M. P. B.], and Biostatistics [M. J. E.], University of Washington, Seattle, Washington 98195 ABSTRACT Patients with long-standing ulcerative colitis (UC) are at increased risk for colon cancer. These cancers are thought to arise from preexisting dysplasia in a field of abnormal cells that often exhibits aneuploidy and p53 abnormalities. Using dual color fluorescence in situ hybridization with centromere probes and locus-specific arm probes for chromosomes 8, 11, 17, and 18, we demonstrate that chromosomal instability (CIN) is present throughout the colon of UC patients with high-grade dysplasia or cancer. In rectal biopsies that were negative for dysplasia, abnormalities in chro- mosomal arms, especially losses, were most common, whereas centromere gains were most common in dysplasia and cancer. The frequency and type of abnormalities varied between the chromosomes examined; chromo- some 8 was the least affected, and 17p loss was found to be an early and frequent event. Chromosomal arm instability showed 100% sensitivity and specificity for distinguishing control biopsies from histologically neg- ative rectal biopsies from these UC patients, raising the possibility that a screen for CIN might detect the subset of UC patients who are at greatest risk for development of dysplasia and cancer. These results suggest that dysplasia and cancer in UC arise from a process of CIN that affects the entire colon; this may provide the mutator phenotype that predisposes to loss of tumor suppressor genes and evolution of cancer. INTRODUCTION Extensive UC 3 of more than 8 years in duration confers an in- creased risk for the development of colorectal cancer (1, 2). The current standard of practice for managing this risk requires lifelong annual colonoscopic surveillance of all such patients to detect dys- plasia or early curable cancer (3). We undertook the present study to develop a better understanding of the earliest steps of UC carcino- genesis, in the hope that this knowledge would help identify the subset of patients at highest risk for progression to cancer. It has been suggested that progression toward cancer in UC pro- ceeds in a stepwise fashion of histological changes from negative for dysplasia 3 indefinite for dysplasia 3 dysplasia 3 cancer (4). Regions of cancer or HGD in UC are often surrounded by mucosa with indefinite or low-grade histology. We and others have shown that these fields of histologically abnormal mucosa are often accompanied by even larger fields of flow cytometrically detectable aneuploidy (5) and/or regions with loss of heterozygosity and mutation of p53 (6). Recently, using comparative genomic hybridization (CGH), we found that approximately 40% of flow cytometrically diploid biopsies near sites of dysplasia or cancer contained clonal cell populations with subtle chromosomal abnormalities. 4 These and other similar findings suggested the possibility that CIN is an early step in neoplastic progression in UC. To pursue this question, we used dual-color FISH with paired green fluorescent centromere probes and red fluorescent arm probes on four different chromosomes. FISH can identify CIN in small subpopulations of interphase cells (7), allowing the detection of infrequent, possibly random changes before they lead to clonal ex- pansion. These experiments demonstrate that CIN is present through- out the colon of patients with dysplasia or cancer in UC. MATERIALS AND METHODS Patients. The average age of the 10 non-UC control patients (5 males and 5 females) was 54 years, and the average age of the 17 UC patients (10 males and 7 females) was 48 years. Resections were performed in controls for diverticular disease (4), endometriosis, hernia, rectal prolapse, appendicitis, complications of decubitus ulcer, and tubular adenoma. One biopsy was examined by FISH from each of the non-UC control colons. The known duration of disease in UC patients was 17  12 years (mean  SD; range, 2–50 years). Seven of the UC patients had the highest histological grade of cancer, whereas 10 UC patients had dysplasia. Colonoscopy or colectomy biopsies were selected from the 17 UC patients to provide a full spectrum of histolog- ical grades for FISH analysis. These consisted of four biopsies with cancer, four with HGD (all from patients in whom this was the highest grade lesion), five with LGD, eight that were IND, seven that were negative for dysplasia (Neg) but near HGD or cancer, and eight rectal biopsies that were negative for dysplasia (NegR). The diagnosis of IND as a histological category between negative and LGD was made according to the consensus criteria (4), with the exception that the indefinite group was not subdivided into three categories. The average distance from the site of highest grade (cancer or HGD) was 10 cm for LGD biopsies, 16 cm for IND biopsies, 10 cm for Neg biopsies, and 49 cm for NegR biopsies. Aneuploidy was examined by flow cytometry as described previously (5) and defined as a distinct nondiploid peak in excess of 5% of cells (8); it was present in three of four cancer biopsies, one of four HGD biopsies, and two of seven Neg biopsies surrounding dysplasia/cancer. All biopsies were frozen at -70°C in MEM with 10% DMSO until use. Cell Preparation. Epithelial cells were isolated from biopsies by the following method: the nonmucosal side of biopsy specimens was affixed to the end of a 2.5-mm-diameter stick with cyanoacrylate glue and soaked for 5 min in Hank’s buffer with 20 mM DTT and 5 mM EDTA at 37°C. Samples were transferred to 2 ml of shaking solution (Hank’s buffer with 20 mM DTT, 5 mM CaCl 2 ,5mM MgCl 2 , and 10% DMSO at 4°C) and vortexed for 5 s, and then the stick with residual stroma was removed. Staining with anticytokeratin antibody showed that the cells in suspension at the end of this procedure were 90% epithelial. FISH. Cells were washed in 5 mM CaCl 2 with 0.1% NP40, spun (10 min at 1000  g), washed again in 1 ml of 5 mM CaCl 2 with 200 l chilled 3:1 methanol:acetic acid, and spun. Ten l of nuclear suspension were dropped on plain glass slides and fixed with 3:1 methanol:acetic acid, followed by 1% paraformaldehyde. Replicate slides from each biopsy were hybridized to pairs of FITC-labeled centromere and TRITC-labeled locus-specific probes for chromosomes 8, 11, 17, and 18: (a) D8Z2 (centromere), 8q21.3, D18Z1 (centromere), and 18q21.2 (Oncor, Inc., Gaithersburg, MD); and (b) p53 Received 3/16/99; accepted 8/19/99. 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 grants from the Crohn’s and Colitis Foundation of America and NIH Grants R01CA68124, P01CA74184, and R29CA77607. 2 To whom requests for reprints should be addressed, at Department of Pathology, Box 357707, University of Washington, Seattle, WA 98195. Fax: (206) 616-8271; E-mail: petersr@u.washington.edu. 3 The abbreviations used are: UC, ulcerative colitis; FISH, fluorescence in situ hy- bridization; HGD, high-grade dysplasia; CIN, chromosomal instability; LGD, low-grade dysplasia; IND, indefinite for dysplasia; Neg, negative for dysplasia; ROC, receiver operator curve; NegR, rectal biopsies negative for dysplasia; CGH, comparative genomic hybridization. 4 Robert F. Willenbucher, Peter Rabinovitch, Daniela E. Aust, Teresa Brentnall, Suzanne J. Zelman, Roger Haggitt, and Frederic M. Waldman. Ulcerative colitis-related dysplasia arises in a genetically abnormal nondysplastic epithelium, submitted for publi- cation. 5148 Research. on October 3, 2021. © 1999 American Association for Cancer cancerres.aacrjournals.org Downloaded from