Chromosomal alterations in ulcerative colitis-related neoplastic progression

GASTROENTEROLOGY 1997;113:791–801

Chromosomal Alterations in Ulcerative Colitis–Related Neoplastic Progression ROBERT F. WILLENBUCHER,* SUZANNE J. ZELMAN,‡ LINDA D. FERRELL,§ DAN H. MOORE II,\ and FREDERIC M. WALDMAN‡ ‡

Cancer Center and Department of Laboratory Medicine, Departments of *Medicine and §Pathology, University of California, San Francisco, and \Geraldine Brush Cancer Research Institute, California Pacific Medical Center, San Francisco, California

Background & Aims: It is unclear whether genomic derangement precedes the histological development of dysplasia in ulcerative colitis (UC)-related neoplastic progression. The primary aim of this study was to determine if chromosomal alterations occur early in the progression pathway of UC-related neoplasia. Methods: Fluorescence in situ hybridization (FISH) was performed on nuclei dissociated from sites of cancer, dysplasia, and UC-involved nondysplastic epithelium in five UC-related cancer colectomy specimens using a panel of pericentromeric probes. Comparative genomic hybridization (CGH) was used to detect clonal chromosomal losses and gains in DNA extracted from these sites. Results: FISH analysis revealed significant and often dramatic alterations in chromosome copy number compared with controls in all biopsy specimens of cancer, dysplasia, and nondysplastic UC-involved epithelium. Clonal chromosomal losses and gains were detected by CGH in all but one analyzed site of dysplasia and cancer and in two of the five nondysplastic sites. FISH and CGH frequently detected the relative loss of chromosome 18. Conclusions: Chromosomal alterations may occur early in UC-related neoplastic progression and seem to precede the histological development of dysplasia. Relative loss of 18q may be important in the progression of UC-related neoplasia. The detection of chromosomal alterations as an intermediate end point may prove useful in identifying patients at high risk for the development of colorectal cancer.

P

atients with ulcerative colitis (UC) are at high risk for the development of colorectal cancer.1 Cancer in patients with UC seems to develop through a multistep process similar to that described for sporadic colon cancer.2 Like sporadic colon cancer, alterations in both protooncogenes and tumor suppressor genes have been described in UC-related colon cancer.3 – 7 However, little is known of the earliest genetic changes that occur during development of UC-related neoplasia. Cancer surveillance in patients with long-term UC is problematic because their cancers typically do not arise / 5e20$$0015

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from endoscopically identifiable polyps, as is the case in sporadic colon cancer, but most often arise from flat, dysplastic epithelium that is not grossly distinguishable from nondysplastic epithelium. Currently practiced cancer surveillance of patients with long-term UC involves periodic random colonic mucosal biopsies to detect the presence of dysplasia,8 because there is a high incidence of coexistent invasive cancer in colons removed for dysplasia.9 Failure to detect cancer before surgery is related to the large sampling error inherent in random endoscopic biopsies. It has been estimated that 33–56 biopsy specimens are required to be 90%–95% confident of detecting dysplasia.10 In addition, it is difficult to diagnose histological dysplasia in the setting of acute and chronic inflammation because many of the morphological features of dysplasia can also be seen in response to inflammation.11 The identification of an earlier, more widespread marker that identifies patients at high risk for the development of colorectal cancer would facilitate cancer surveillance in UC. Ideally, this marker would not be influenced by coexistent inflammation. The aim of this study was to determine if chromosomal alterations occur early during the development of UCrelated neoplasia. This is based on the model that chronic inflammation, through the production of carcinogenic inflammatory mediators, produces a field effect in colonic epithelium that results in genomic instability and chromosomal aberrations. To accomplish this aim, we considered two approaches to investigate chromosomal alterations in UC-related neoplasia. In the first approach, biopsy specimens of nondysplastic epithelium, dysplasia, and cancer from colectomy specimens containing UCAbbreviations used in this paper: APC, adenomatous polyposis coli gene; bp, base pair; CGH, comparative genomic hybridization; DAPI, 4*,6-diamidino-2-phenylindole hydrochloride; DCC, deleted in colon cancer gene; dUTP, deoxyuridine triphosphate; FISH, fluorescence in situ hybridization; FITC, fluorescein isothiocyanate; kb, kilobase pair; PCR, polymerase chain reaction; SSC, standard saline citrate. q 1997 by the American Gastroenterological Association 0016-5085/97/$3.00

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related cancer would be analyzed. It is generally believed that colitis-related neoplasia progresses histologically from nondysplasia to dysplasia to cancer. By performing molecular cytogenetic analysis on these three tissue types from each colectomy specimen, we would be able to determine if chromosomal alterations are present at different histological stages of progression. The second approach would be to perform a longitudinal study to define carefully the time course of the development of chromosomal alterations in relation to dysplasia and cancer development. We chose the first approach as our initial approach to define chromosomal alterations in UC-related neoplasia. We hypothesize that chromosomal abnormalities are present in UC-related dysplasia and cancer. In addition, we hypothesize that histologically nondysplastic epithelium from individuals in whom UC-related colorectal cancer has developed is genetically different from normal colonic epithelium. If these hypotheses are true, chromosomal changes may represent an important intermediate end point in UC-related neoplasia. We applied techniques of molecular cytogenetics to samples from archival colectomy specimens that had UCrelated cancer to determine the extent of chromosomal abnormalities in colonic epithelium. Fluorescence in situ hybridization (FISH) was used to detect abnormal chromosome copy number (aneusomies) using a panel of five chromosome-specific pericentromeric probes. These probes allow the detection of monosomy (one chromosome copy) or polysomy (ú2 chromosome copies) for specific chromosomes in individual nuclei. Comparative genomic hybridization (CGH) was used to obtain an overview of clonal chromosomal gains and losses. In CGH, total genomic DNA from the colonic biopsy and normal human genomic DNA (detected in different colors) are simultaneously hybridized to a normal metaphase spread.12 The ratio of the colors along the normal chromosomes provides a quantitative map of the relative copy number of DNA sequences in the colon biopsy specimen. Thus, the entire genome can be surveyed in a single step without the need to select which genetic loci to test.

Materials and Methods Fluorescence In Situ Hybridization Tissue selection. Five colectomy cases from patients with UC-related colon cancer were identified from the University of California, San Francisco Moffitt–Long Hospital Department of Pathology archives. One tissue block each of cancer, dysplasia, and nondysplastic UC-involved epithelium was selected from each case. Blocks of normal surgical margins from five colon tissue samples removed for diverticular disease

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served as normal controls. In addition, one block each from five colectomy cases from patients with short-term UC (õ8 years’ duration) served as an additional set of controls. Patients with UC for õ8 years are at low risk for neoplasia. The histological diagnosis of dysplasia was made using standard criteria.11 In addition, the degree of inflammation in UC biopsy specimens was subdivided in quiescent, mild, moderate, and severe categories. This study was approved by the University of California, San Francisco Committee on Human Research. Cell preparation. Two to four 50-mm-thick sections (depending on the size of the region of interest) were cut from paraffin blocks, preceded and followed by thin (5-mm-thick) sections for H&E staining. These H&E sections were reviewed to assure that the thick sections contained the region of interest. If necessary, the thick sections were trimmed to exclude unwanted tissue, including muscle and submucosa. Nuclei were dissociated from thick sections by overnight xylene treatment at 557C to remove the paraffin, followed by 100%, 95%, and 80% ethanols at room temperature and 50% ethanol at 47C for 2.5 days, distilled deionized water for 1 hour, and 0.5% pepsin treatment at 377C for 1 hour. The action of the pepsin was stopped by adding an equal volume of fetal bovine serum. The cells were washed once in phosphatebuffered saline (PBS), and nuclei in suspension were cytospun onto uncharged glass slides. Slides were air dried at room temperature and then stored in nitrogen gas at 0207C. Before FISH, the slides were fixed (3 1 5 minutes) in methanol/acetic acid (3:1) and air dried. Hybridization. FISH was performed using probes for repeated chromosome-specific DNA sequences found at or near the centromere of chromosomes 1, 7, 8, 17, and 18. Probes for chromosomes 17 and 18 were chosen because of their frequent loss in colon cancer. Probes for chromosomes 1, 7, and 8 were chosen because of their frequent gain in colon cancer.13 Probe specificity was confirmed by hybridization to normal lymphocyte metaphase spreads. Probes were labeled with biotin or digoxigenin by nick translation using standard protocols with commercially available kits (Life Technologies, Gaithersburg, MD). FISH was performed as previously described.14 Briefly, slide preparations were denatured in 70% formamide/21 standard saline citrate (SSC) (11 SSC is 0.15 mol/L NaCl plus 0.015 mol/L sodium citrate) at 807C for 10 minutes, then treated with 20–50 mg/mL proteinase K (Sigma Chemical Co., St. Louis, MO) in PBS for 7.5 minutes at 377C. Twenty nanograms each of two probes labeled with either biotin or digoxigenin in hybridization mix (55% formamide/11 SSC/10% dextran sulfate) was denatured at 757C for 5 minutes and applied to warmed slides. Hybridization was overnight at 377C. Slides were then washed three times for 10 minutes each at 457C in 50% formamide/21 SSC, once in 21 SSC, and once in 21 SSC at room temperature. Hybridized probes were detected immunochemically. All staining reactions were performed at room temperature for 30– 45 minutes. Before each immunocytochemical staining, slides

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were preblocked with antibody dilution buffer, 41 SSC/1% bovine serum albumin, for five minutes. Probes were detected with either fluorescein isothiocyanate (FITC)-conjugated sheep antidigoxigenin (4 mg/mL) (Boehringer Mannheim, Indianapolis, IN) or Texas red avidin (5 mg/mL) (Vector Laboratories, Burlingame, CA) or FITC-avidin (5 mg/mL) (Vector) and washed in 41 SSC and 41 SSC/0.1% Triton X-100 (Fisher Scientific, Santa Clara, CA). Amplification of the signal was accomplished by further incubation in either biotinylated goat antiavidin immunoglobulin (Ig)G (5 mg/mL) (Vector) or rabbit anti-sheep FITC antibody (1:50 dilution) (Sigma), washing as above, and a final incubation in Texas red avidin or FITC avidin. Nuclei were then counterstained with 4*,6-diamidino2-phenylindole hydrochloride (DAPI) at 0.1 mg/mL in antifade solution. Scoring of FISH signals. Nuclei were scored for the number of hybridization signals per nucleus using an epifluorescence microscope equipped with a 631 NA:1.3 oil immersion objective. Approximately 200 nuclei were scored for each hybridization. Nuclei that were damaged (torn or broken) or overlapping were excluded from analysis. In addition, small, round nuclei (one half or less of the diameter of the epithelial nuclei) were presumed to be lymphocytes and were excluded from analysis. Statistical analysis. Chromosome copy numbers by FISH were classified into three categories: monosomy (a single copy), disomy (two copies), and polysomy (more than two copies) for each of the five chromosomes. For each of the UC cases (cancer, dysplasia, nondysplasia, and short-term UC) we formed a statistic equal to the sum of the squared differences between the observed copy number counts and those expected based on estimates of copy number frequency from normal colonic epithelium. This statistic (which we call a deviancy statistic) provides a convenient way of summarizing information from multiple copy number counts and multiple chromosomes. When test material is no different from the pool of normals, this statistic is expected to have a x2 distribution and to vary around the number 10, which is equal to the number of probes times the number of abnormal categories (monosomy and polysomy). The value of the statistic will increase as the test material deviates in copy number count from normals.

Comparative Genomic Hybridization Tissue dissection and DNA extraction. CGH analysis of DNA extracted from thin paraffin sections was performed using modifications of our previous protocols.15 Regions of nondysplastic epithelium, dysplastic epithelium, and tumor from each of the 5 UC cases as well as from normal colon samples were analyzed by CGH. For each site selected, two adjacent 5-mm-thick sections were cut from the paraffin block. One section was stained with H&E, and the second section was stained with methyl green (0.1%). All sections were photographed. Using the adjacent H&E section for orientation, the study pathologist identified on the photographs of the methyl green–stained sections the epithelium of interest for CGH

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analysis. The selected areas were recovered by first carefully scraping away surrounding tissue, and then the desired regions were removed under a droplet of DNA extraction buffer (10 mmol/L TRIS-HCl [pH 8.0], 1.5 mmol/L MgCl2 , 50 mmol/ L KCl, 0.5% Tween-20 [Fisher Scientific], and 0.4 mg/mL proteinase K [Sigma]) and transferred into 15 mL (30 mL for larger areas) of DNA extraction buffer. Mineral oil (20 mL) was placed over the sample, which was then incubated at 557C overnight. Fresh proteinase K (0.3 mL of 20-mg/mL stock) was added daily for 2 more days and was finally inactivated at 957C for 15 minutes. The underlying buffer was then stored at 47C. Polymerase chain reaction amplification. Amplification of the microdissected DNA was based on the degenerate oligonucleotide primed polymerase chain reaction (PCR) protocol of Guan et al.16 Briefly, a 1–2-mL aliquot of microdissected DNA was added to 5 mL of 11 PCR buffer and pretreated with 0.1 mL or 1 U TOPOisomerase I (Promega, Madison WI). The TOPO pretreatment was followed by five cycles of sequenase treatment (1 minute at 947C; 2 minutes at 307C, and 2 minutes at 377C). Preamplification was followed by one cycle at 957C for 10 minutes, and 45 mL of 11 PCR buffer with 2.0 U of Taq DNA Polymerase (Boehringer) was then added. This was followed by 35 cycles at 947C for 1 minute, 567C for 1 minute, and 727C for 3 minutes, with a final extension at 727C for 5 minutes. Each PCR run included samples of normal genomic DNA, MPE600 (a breast cancer cell line with known CGH aberrations), and a blank to check for contamination. Size ranged from 200 base pairs (bp) to 2 kilobase pairs (kb). Microdissected DNA yielded up to 1 mg of PCR product, averaging 400 bp in size (range, 200 bp–2 kb). Fifty nanograms of reference and MPE600 cell line DNA resulted in approximately 2–3 mg of amplified DNA. Probe labeling. Nick translation of the PCR product was more reliable than labeling during the PCR reaction. For normal reference DNA, PCR amplified DNA was labeled with Texas red–5-deoxyuridine triphosphate (dUTP) or with fluorescein-12-dUTP (Dupont NEN, Boston, MA). Forty microliters of amplified DNA from paraffin sections was used per 50 mL nick translation reaction and was labeled with fluorescein or digoxigenin. The optimum size for CGH was 500–2000 bp. Nick translated PCR products were close to the original PCR product size. Probes ú2 kb tended to yield more granular hybridizations. Hybridization. Test DNA was labeled in one color and a sex-matched normal reference DNA (unrelated to the test patient) was labeled in the other color. The hybridization was as previously described.12,17 Samples were hybridized onto normal male metaphases. Each sample was hybridized in duplicate with different fluorochromes. Image acquisition and analysis. Successful hybridizations were judged by the intensity of the tumor and normal signals, by the granularity vs. smoothness of the signals, by the homogeneity of the signal over the entire metaphase, and by the banding intensity of the DAPI signals used for chromosome identification. At least five metaphase spreads were cho-

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Table 1. UC-Related Colorectal Cancer Cases Case

Age/sex ( yr )

Colitis duration ( yr )

Tumor location; stage

Dysplasia location/grade

Nondysplasia location

1 2

36/M 37/M

20 17

Rectum; T1N1M0 Transverse colon; T3N2M0

Transverse colon Cecum

3 4

60/M 26/M

11 9

Transverse colon; T2N0M0 Rectum; T2N0M0

5

60/F

23

Rectum; T3N0M0

Rectum/high grade Transverse colon/high grade with focal low grade Transverse colon/low grade Rectum/low grade with focal high grade Hepatic flexure/low grade with focal high grade

sen for image acquisition based on these criteria and also on the optimal spreading of the metaphase chromosomes, so that there was little overlap. Acquisition was performed using our QUIPS analysis system (Piper et al. Cytometry 1995; 19[1]).12,17 Profile analysis. High-level amplifications were defined as a peak of the ratio intensity ú2.0, involving less than a whole chromosome arm. Low level gain or loss was defined as chromosome regions that have a ratio ú 1.25 or õ0.8. Our use of inverse CGH, using a second hybridization with reversed color labels, allowed greater confidence in making these interpretations. The inverse pair was examined together to allow better discrimination of significant changes. All changes must have been seen in both the forward and inverse hybridizations, and the ratio value must have been beyond the thresholds in one of the hybridizations. Interpretation of changes at 1pter, 19, and 22 required careful examination of all chromosome profiles, because these loci were likely to show more variability in their ratios. Definition of changes at these loci required the cutpoint to be exceeded in both hybridizations.

Results Five UC-related cancer colectomy cases were characterized by both FISH and CGH. The clinical data and biopsy locations are summarized in Table 1. The dysplastic biopsy specimens in the 5 UC-related cancer cases were all mildly inflamed. Of the nondysplastic UC-involved biopsy specimens from these cases, one was quiescent (case 5), two were mildly inflamed (cases 1 and 2), and two were moderately inflamed (cases 3 and 4). In the five short-term UC cases, the analyzed biopsy specimens were quiescent in 2 cases, moderately inflamed in 2 cases, and severely inflamed in the remaining case.

Cecum Transverse colon Right colon

8, 17, and 18 using chromosome-specific pericentromeric probes. Normal nuclei are expected to have two signals for each probe used, but in practice the copy number is altered in a small fraction of nuclei. Nuclei having one signal (monosomies) and more than two signals (polysomies) were infrequent in these normal specimens. Monosomy in normal tissue may be caused by õ100% hybridization efficiency or overlapping signals, and polysomy may be related to splitting of fluorescent signals or inadvertent scoring of overlapping nuclei. True aneusomy, caused by mutagenic loss or gain of individual chromosomes, is extremely rare in normal samples. UC-related cancer cases. Chromosomal copy number distributions were determined for biopsy specimens from cancer, dysplastic, and nondysplastic UC-involved epithelium from each of the 5 UC-related cancer colectomy cases. Distributions of chromosomal copy numbers for all cases are shown in Figure 2. A photomicrograph of nuclei from the dysplasia of 1 case showing abnormal chromosomal copy numbers is shown in Figure 3C.

Chromosomal Copy Number Distribution Determined by FISH Normal colon. Figure 1 summarizes the normal range of chromosome copy numbers detected by FISH in nuclei dissociated from normal colonic epithelium from five colon specimens resected for diverticular disease. FISH analysis was performed for chromosomes 1, 7,

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Figure 1. FISH chromosome distributions in normal (non-UC) colonic epithelium. Copy number frequencies per nucleus for chromosomes 1, 7, 8, 17, and 18 detected by FISH. Each histogram represents counts from 200 nuclei. Copy number distributions for chromosomes 1 (j), 7 ( ), 8 ( ), 17 ( ), and 18 ( ) represent average values from biopsy specimens from five different normal colon specimens.

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Figure 2. FISH chromosome distributions in UC-related neoplasia (cases 1–5). Histograms represent distributions of individual chromosome copy numbers in nondysplastic (h), dysplastic ( ), and carcinomatous (j) epithelium from each of the 5 UC-related cases.

Both monosomies and polysomies were common in UC-related cancer and dysplastic biopsy specimens. Abnormalities that were highly significant (P õ 0.001), compared with the copy number distribution in normal colonic epithelium, were present for at least two chromosomes in all sites of dysplasia and cancer. In all but case 3, chromosomal copy number in dysplastic epithelium was abnormal in all five chromosomes (P õ 0.001). The specific aneusomy identified in the dysplasia was almost always present in the cancer from the same case. Polysomies were identified considerably more frequently than monosomies. In case 3, monosomy for chromosome 18 was observed in all three tissue types (P õ 0.001). A less marked monosomy was also identified for chromosome 8 in the cancer of case 1 (P õ 0.05). Marked alterations in chromosomal copy number were also observed in nondysplastic epithelium from UC-related cancer colectomy cases (Figure 2; cases 1–3). In nondysplastic UC-involved epithelium, polysomies were / 5e20$$0015

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detected for all chromosomes in case 2 and for chromosomes 7, 8, and 18 in case 1 (P õ 0.001). Monosomy for chromosome 18 was identified in the nondysplastic site of case 3 (P õ 0.001). An even more prominent monosomy was observed for chromosome 18 in the dysplasia and cancer in the same case (case 3). There were also several nondysplastic sites that had less marked (0.05 ú P ú 0.001) polysomies (case 1, chromosome 17; case 3, chromosome 8; case 4, chromosome 1; case 5, chromosome 18) and monosomies (case 4, chromosomes 7 and 18). In all 5 cases, the nondysplastic site was located a considerable distance from the dysplasia and cancer (Table 1). Figure 4 is a summary plot of the total x2 test used for comparing cases with normals; it summarizes aneusomies (monosomies and polysomies) over all chromosomes for each biopsy specimen. Although the normal colonic nuclei fell within a normal range of values for a x2 distribution, it is readily apparent from this plot that the nondysWBS-Gastro

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Figure 3. FISH and CGH analysis of dysplastic epithelium. (A and B ) H&E sections of dysplastic epithelium from case 2. (C ) FISH. Dissociated nuclei from case 2 dysplasia section (see A ) were hybridized to pericentromeric probes for chromosomes 1 (red) and 18 (green), showing ¢3 copies of each in most cells. (D ) CGH. DNA was microdissected from dysplastic epithelium in a case 2 archival section (see A ), amplified by DOP-PCR, and nick translated with digoxigenin-dUTP, before hybridization to a normal lymphocyte metaphase spread in the presence of FITCdUTP–labeled normal reference DNA and digital image acquisition. Note chromosome loss of 5q and 18q (relatively green) and gain of 5p and 8q23-24 (red). (A, original magnification 251; B, 801; C, 631.)

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Figure 4. Statistical analysis of FISH copy number frequencies. Total deviancy was calculated to represent the deviation of each biopsy from normal (non-UC controls). The deviancy statistic is described in Materials and Methods. In this plot, this statistic is expected to vary around the value of 10 when the material comes from normals. The cut point representing a significance of P õ 0.001 (based on the x2 distribution) is represented by the horizontal dashed line. The total deviancy statistic values for the 5 non-UC normals ( ) and the individual biopsy specimens for cases 1 ( ), 2 (%), 3 (L), 4 ( ), and 5 ( ) are shown.

plastic, dysplastic, and tumor nuclei were well beyond the normal range of values. For all UC-related biopsy specimens, the level of significance for the x2 statistics was P õ 0.001. In all instances in which significant aneusomies were detected in the nondysplastic epithelium (cases 1, 2, and 3), that same aneusomy was also present in the dysplasia from the same case. For most chromosomes evaluated, if an aberrant copy number distribution was present in the nondysplasia and/or dysplasia, a similar pattern of distribution was also observed in the tumor. However, this did not appear to be the case for chromosome 18. In cases 1, 2, and 5, there was a shift downward in copy number distribution of chromosome 18 in the cancer relative to the dysplasia. This suggests there was a relative loss of chromosome 18 in the cancer. In case 3, there was already significant monosomy for chromosome 18 in the nondysplasia and dysplasia that did not change in the cancer. This apparent relative loss of chromosome 18 in these 4 cases (cases 1, 2, 3, and 5) was not explained by a generalized loss of all chromosomes associated with the transition from dysplasia to cancer. Only case 1 seemed to show a decline in copy number for most chromosomes in the cancer compared with the dysplasia. In the remainder of the cases, there did not appear to be an / 5e20$$0015

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Figure 5. FISH chromosome distributions in low-risk (disease duration õ 8 years) UC-involved colonic epithelium. Copy number frequencies per nucleus for chromosomes 1, 7, 8, 17, and 18 detected by FISH. Each histogram represents counts from 200 nuclei. Copy number distributions for chromosomes 1 (j), 7 ( ), 8 ( ), 17 ( ), and 18 ( ) represent average values from biopsy specimen from five different UC low-risk specimens.

overall pattern of decreasing copy number with histologic progression from dysplasia to cancer. Short-term UC cases. Chromosome copy number distribution in low-risk UC-involved epithelium from five colectomy specimens from individuals with short-term UC (õ8 years) was not significantly different from the normal control for each of the five chromosomes (Figure 5; P ú 0.20). By summary x2 analysis, nuclei dissociated from epithelium from patients with shortterm UC were overlapping with the normal controls. Chromosomal Losses and Gains as Determined by CGH Relative chromosomal losses and gains were identified by CGH analysis of microdissected epithelium from the same sites in the UC-related colectomies that were analyzed by FISH. Chromosomal abnormalities detected by CGH are summarized in Table 2. CGH-detectable chromosomal losses and gains were common in dysplasia and cancer with changes detectable in four of five sites of dysplasia and in all assessable sites of cancer. Two

Table 2. CGH Analysis of UC-Related Neoplasia Case

Nondysplasia

1 2

No changes 5q0, 18q0, 19p/

011, 18q12-qter0 5p/, 5q0, 8q2324.1/, 18q0

3 4

/19 No changes

5

No changes

018, /X 5q0, 8q21.1-qter/, 12q13-qter0, 017 No changes

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Dysplasia

Carcinoma Inadequate 5q0, 8p12pter0, 13q/, 18q0, /20 018, /X Inadequate 18q0

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sites of cancer yielded an inadequate amount of DNA, resulting in failure to PCR amplify or a small PCR product that was inadequate for CGH. CGH detected abnormalities in the nondysplastic sites in 2 of the 5 cases, including 5q and 18q losses and 19p gain in case 2, and chromosome 19 gain in case 3. CGH of epithelium from two low-risk colitis biopsy specimens and one normal (diverticulitis specimen) biopsy specimen did not show any chromosomal aberrations. Comparison of CGH and FISH Analyses FISH analysis with pericentromeric probes suggested that there was a loss of chromosome 18 in the tumor relative to the dysplasia in 4 of the 5 cases (cases 1, 2, 3, and 5). Relative loss of chromosome 18 or 18q was also observed in the tumor of each of these cases that was assessable by CGH, i.e., loss chromosome 18 in case 3 and loss of 18q in cases 2 and 5. FISH detected significant monosomy for chromosome 18 in the nondysplastic and dysplastic epithelium of case 3. CGH also detected a relative loss of chromosome 18 in the dysplasia of case 3 but not in the nondysplastic biopsy specimen. However, the chromosome 18 monosomy in this nondysplastic biopsy specimen demonstrated by FISH, although highly significant as compared with normal colonic biopsy specimens, was observed in only approximately 25% of the nuclei (and so would not be detectable by CGH) compared with a frequency in the dysplasia and tumor of approximately 60% of the nuclei.

Discussion These studies show that chromosomal abnormalities occur early during UC-related neoplasia. Marked genomic derangement seems to be a prominent feature of preinvasive neoplasia: FISH-detectable aneusomies were present in the dysplasias from all 5 UC-related cancer cases. Chromosomal copy number in nondysplastic epithelium from UC-related cancer cases deviated markedly from normal colonic epithelium in all 5 cases. A possible explanation for this is that UC-related neoplasia begins with a nondysplastic but chromosomally aberrant epithelium that subsequently gives rise to dysplasia. Support for this model is derived from the observation that when a specific, marked aneusomy was detected in the nondysplastic epithelium, it was also present in the dysplasia from the same case. This suggests a clonal relationship between the genetically aberrant nondysplastic epithelium and the dysplasia. Aneusomies do not appear to be a nonspecific consequence of mucosal inflammation, because none of the UC-involved epithelium from pa/ 5e20$$0015

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tients with short-term UC had FISH-detectable aneusomies. CGH identified relative chromosome losses and gains in both UC-related cancers and dysplasias. CGH also detected chromosomal abnormalities in nondysplastic epithelium from colectomy specimens containing UC-related colorectal cancer. These CGH findings not only confirm the FISH findings of chromosomal abnormalities in nondysplastic epithelium from patients with UC-related cancer but show that these genetic alterations in nondysplastic epithelium are clonal. CGH detects clonal changes and requires that at least 60% of nuclei contain the same genetic alteration.18 Perhaps most interesting is the frequent loss of all or just the long arm of chromosome 18. FISH demonstrated a relative loss of chromosome 18 in the cancer compared with the dysplasia in 4 of 5 cases. CGH analysis correlated well with the FISH findings. CGH showed relative loss of chromosome 18 or 18q in three of three assessable cancers, three of five dysplastic sites, and one of the five nondysplastic sites. These data suggest that relative allelic loss of 18q may be an important event during the progression of UC-related neoplasia. 18q is the location of the deleted in colon cancer (DCC) tumor suppresser gene19,20 and the more recently described candidate tumor suppresser genes, DPC421,22 and JV18-1,23 both Mad-related genes located just centromeric to DCC on 18q. The role of DCC has not been reported in UCrelated neoplasia, although the loss of 18q was reported in three of five UC-related cancers.24 Whereas allelic loss of 18q appears to be a late event in invasive sporadic colon cancer,19 the relative 18q loss detected by CGH in dysplastic and nondysplastic epithelium suggests that 18q allelic loss may be a relatively early event in UCrelated neoplasia. That CGH did not always detect aberrations in specific chromosomes that were markedly abnormal by FISH is not unexpected. As stated previously, CGH detects only clonal changes. In addition, CGH detects chromosomal losses and gains relative to the overall genome of interest but does not provide information about absolute copy number or ploidy. For example, a loss of a single specific chromosome in an otherwise diploid genome will appear identical by CGH to the loss of two copies of the same chromosome in an otherwise tetraploid genome. By contrast, FISH allows nucleus-by-nucleus analysis of absolute copy number for any given chromosome-specific probe. FISH, although limited to analysis with selected probes, also allows the assessment of copy number heterogeneity within a given population of cells. A potential limitation of FISH is in defining monosomy. When copy WBS-Gastro

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number distribution in a test sample is compared with control, nuclei with single signals may reflect impaired hybridization efficiency that is inherent to the test sample rather than true monosomy. This potential limitation can be overcome by the use of internal controls. For example, in case 3, significant monosomy populations were detected by FISH for chromosome 18 compared with normal colonic epithelium. However, significant monosomy populations were not present for the other four probes, making it unlikely that impaired hybridization efficiency accounted for this monosomy. Use of normal lymphocytes contained within the sample was also considered as an internal control for hybridization, but was discarded because of the observation that such lymphocytes required more aggressive proteinase treatment to achieve equivalent hybridization results. In addition, the loss of chromosome 18 in case 3 was also confirmed by CGH. CGH also detected relative chromosomal losses at loci other than chromosome 18 known to contain tumor suppresser genes previously implicated in colorectal cancer. For example, 5q was lost in all tissue sites from case 2 and in the dysplasia from case 4. 5q is the location of the adenomatous polyposis coli (APC) gene. Point mutation and allelic loss of the APC gene have been reported in UC-related dysplasia and cancer,7,25 although a recent study suggests that APC mutations occur much less commonly in UC-related neoplasia (6%) than in sporadic colorectal neoplasia (74%).26 However, this study failed to note whether accepted criteria were used to define the UC-related dysplasia that was analyzed. CGH also detected the loss of chromosome 17 in the dysplasia from case 4. 17p is the location of p53. Point mutations and/ or allelic loss of the p53 gene have been reported in the majority of UC-related cancers that have been investigated.3,6,27 In addition, allelic loss of p53 can be identified approximately 50% of the time in UC-related dysplasias.3 However, CGH may be less sensitive than other approaches for identifying deletions if they are especially small. CGH is also capable of detecting amplifications of oncogenes. In the dysplasia from case 2, the 8q2324.1 gain is in the location of the c-myc oncogene. The c-myc oncogene has been implicated in the development of sporadic and UC-related colorectal neoplasia.28,29 The present data suggesting that significant genetic alterations precede the histological development of UCrelated dysplasia are supported by studies using other analytic approaches. Abnormal DNA content (aneuploidy) by flow cytometry may occasionally be found in histologically nondysplastic UC-involved epithelium from colectomy specimens containing colorectal can/ 5e20$$0015

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cer.10,30,31 In addition, there have been two reports of aneuploidy preceding the development of dysplasia in patients monitored in prospective colonoscopic surveillance programs.10,31 Specific gene mutations have also been reported in nondysplastic UC-involved epithelium from patients with UC-related colorectal cancer. In one report, a specific p53 mutation at codon 248 (exon 7) was mapped in colectomy specimens from 2 patients with long-term UC.32 This specific mutation was found to be present in the cancer/dysplasia as well as in histologically nondysplastic/indefinite epithelium, suggesting that specific clonal genetic alterations may be present in nondysplastic epithelium before the development of dysplasia and cancer. However, these results were not confirmed by an independent assay. K-ras mutations have also been found in nondysplastic UC-involved epithelium,33 as well as in epithelium that was indefinite for dysplasia.7 Molecular cytogenetic analysis of UC-related neoplasia showed widespread and often dramatic chromosomal aberrations. Detection of chromosomal losses and gains may represent a useful intermediate marker of neoplastic progression. Assuming that UC follows the process of clonal expansion as described by Nowell,34 a genetically aberrant but predysplastic clone should be more widespread and therefore more easily detectable than histological dysplasia. The data in this report support the concept that predysplastic genetic changes are more widespread than abnormal histology in that chromosomal aberrations were detected in nondysplastic epithelium that was located a considerable distance from histologically defined dysplasia and cancer. Further studies are required to prove that chromosomal alterations are more widespread than histologic dysplasia. Extensive molecular cytogenetic analysis of histologically mapped UC colectomy specimens to further investigate the distribution of chromosomal alterations in colons containing UC-related neoplasia should address this question. Chromosomal markers may have the potential of being detected earlier, more easily, and subject to less interpretive difficulties than histological dysplasia. This may have important implications for cancer surveillance. Future studies will be directed at defining the time course of the development of chromosomal alterations in relation to dysplasia and cancer development. In such studies, chromosomal analysis will be performed at multiple time points before the development of histological dysplasia or cancer. Biopsy specimens from patients matched for UC extent and duration who do not progress to dysplasia or cancer will serve as controls so that the sensitivity and specificity of chromosomal alterations for the prediction WBS-Gastro

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of dysplasia and cancer development can be determined. If chromosomal markers are detectable at a time point that significantly predates the development of dysplasia/ cancer, this will allow an increase in the interval between surveillance examinations. Ultimately, the detection of specific chromosomal losses and gains may identify UC patients at high risk for the development of cancer, and these chromosomal aberrations may prove to be an important intermediate end point for chemoprevention studies. Insights gained in these studies will likely be generalizable to other gastrointestinal cancers that arise from chronically inflamed epithelium such as cancer complicating Barrett’s esophagus and chronic gastritis.

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30. Burmer GC, Rabinovitch PS, Haggitt RC, Crispin DA, Brentnall TA, Kolli VR, Stevens AC, Rubin CE. Neoplastic progression in ulcerative colitis: histology, DNA content, and loss of a p53 allele. Gastroenterology 1992;103:1602–1610. 31. Lofberg R, Brostrom O, Karlen P, Ost A, Tribukait B. DNA aneuploidy in ulcerative colitis: reproducibility, topographic distribution, and relation to dysplasia. Gastroenterology 1992;102: 1149–1154. 32. Brentnall TA, Crispin DA, Rabinovitch PS, Haggitt RC, Rubin CE, Stevens AC, Burmer GC. Mutations in the p53 gene: an early marker of neoplastic progression in ulcerative colitis. Gastroenterology 1994;107:369–378. 33. Chaubert P, Benhattar J, Saraga E, Costa J. K-ras mutations and p53 alterations in neoplastic and nonneoplastic lesions associ-

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ated with longstanding ulcerative colitis. Am J Pathol 1994;144: 767–775. 34. Nowell PC. The clonal evolution of tumor cell populations. Science 1976;194:23–28. Received November 5, 1996. Accepted April 22, 1997. Address requests for reprints to: Robert F. Willenbucher, M.D., Center for Inflammatory Bowel Disease, University of California, San Francisco, 2215 Post Street, Suite 1, San Francisco, California 94115. Fax: (415) 502-2249. Supported in part by the Department of Medicine Development Fund at the University of California, San Francisco–Mount Zion Medical Center, the Theodora Betz Foundation, and grants CA47537, and CA44768 from the National Cancer Institute.

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