Sialyl-Tn Antigen as a Marker of Colon Cancer Risk in Ulcerative Colitis: Relation to Dysplasia and DNA Aneuploidy

GASTROENTEROLOGY 1998;115:1395 1404 Sialyl-Tn Antigen as a Marker of Colon Cancer Risk in Ulcerative Colitis: Relation to Dysplasia and DNA Aneuploidy PER KARLÉN,* ERIC YOUNG, OLLE BROSTRÖM,* ROBERT LÖFBERG, BERNHARD TRIBUKAIT, ÅKE ÖST, CAROL BODIAN, # and STEVEN ITZKOWITZ *Division of Gastroenterology, Department of Medicine, Söder Hospital, Karolinska Institute, Stockholm, Sweden; Gastrointestinal Research Laboratory, Department of Medicine, Mount Sinai School of Medicine, New York, New York; Department of Gastroenterology, Huddinge University Hospital, Karolinska Institute, Stockholm, Sweden; Department of Radiobiology, Karolinska Institute, Stockholm, Sweden; Department of Pathology and Cytology, Medilab, Täby, Stockholm, and Karolinska Institute, Stockholm, Sweden; and # Department of Biomathematical Sciences, Mount Sinai School of Medicine, New York, New York Background & Aims: Expression of the mucin-associated carbohydrate antigen sialyl-tn (STn) and DNA aneuploidy has each been shown to correlate with malignant transformation in patients with sporadic colon cancer and in those with ulcerative colitis (UC). This study aimed to determine how STn expression topographically and temporally relates to aneuploidy and neoplasia in patients with long-standing UC. Methods: Twenty-six patients enrolled in a cancer surveillance program were studied, and 1691 mucosal from repeated colonoscopies and colectomies were assessed in a standardized, prospective fashion for the presence of dysplasia, aneuploidy, and STn antigen. Results: STn was expressed in 47% of from 6 patients who underwent colectomy for dysplasia and 7% of from 6 well-matched patients who underwent surgery for medical intractability. Seven other patients who never developed dysplasia or aneuploidy expressed STn in 6% of biopsy. STn expression was independent of aneuploidy in colons both with and without dysplasia. Of 5 patients with aneuploidy but without dysplasia, 4 expressed STn earlier than aneuploidy. Conclusions: In UC, STn antigen and DNA aneuploidy are independent markers of neoplastic transformation. Determination of STn expression may complement dysplasia and aneuploidy for identification of risk for colonic neoplasia in UC. There is consensus about the increased risk of colorectal cancer in patients with long-standing, extensive ulcerative colitis (UC). 1,2 The recognition of mucosal dysplasia as an early marker of colorectal cancer in patients with UC 3 provided an alternative to prophylactic colectomy for this group of patients. The principal method of colorectal cancer surveillance in patients with UC is repeated colonoscopy with examination of multiple biopsy for histological evidence of dysplasia and/or carcinoma to select high-risk patients for colectomy. However, both intraobserver and interobserver variation may effect the interpretation of dysplasia, and histological assessment can be even more difficult in the presence of active mucosal inflammation. Moreover, dysplasia is often patchy and may not be reliably sampled, and cancers can also arise without any apparent preexisting dysplasia. 4 These problems have prompted a need for molecular markers that might complement dysplasia in prediction of cancer risk. One such molecular marker is abnormal DNA content, or aneuploidy. Aneuploidy, measured by flow cytometry (FCM) on biopsy material, has been demonstrated in prospective and retrospective studies to correlate with dysplasia in long-standing UC, where it is often more widespread throughout the colon and can precede the first detection of dysplasia by several years. 5 10 The mucin-associated sialyl-tn (STn) antigen, as detected by immunohistochemistry with monoclonal antibody (MAb) TKH2, is another potentially useful marker in this respect and has recently been shown to precede dysplasia in UC. 11 STn is rarely expressed in normal colonic mucosa or hyperplastic polyps but is expressed in 87% 90% of colon cancers. 12,13 Adenomatous polyps show STn reactivity increasingly with size, degree of dysplasia, and villous components. 14 Furthermore, STn antigen expression has been associated with a poor prognosis in patients with sporadic colonic cancer independent of tumor stage, DNA ploidy status, or histological type. 13 In UC patients, STn antigen expression is increased in dysplastic and cancerous colonic mucosa, 15 and the finding of STn in nondysplastic mucosa may be a marker of cancer risk, not only in those areas of the colon where carcinoma is present, but also in nondysplastic mucosa remote from the cancer. 16 Abbreviations used in this paper: FCM, flow cytometry; MAb, monoclonal antibody; STn, sialyl-tn. 1998 by the American Gastroenterological Association 0016-5085/98/$3.00

1396 KARLÉN ET AL. GASTROENTEROLOGY Vol. 115, No. 6 Little is known of the relationship between STn and aneuploidy in terms of distribution in the colon and timing in relation to each other and to dysplasia. Simultaneous expression of both markers in a given biopsy specimen would suggest that they reflect the same molecular pathway of carcinogenesis. However, if they are expressed independently, they may provide complementary information. The present study was conducted to address this issue. STn expression was investigated in collected prospectively within a colonoscopic cancer surveillance program in which have been routinely analyzed for DNA aneuploidy and dysplasia in a standardized fashion since 1982. Patients and Methods Patients All patients included in the study were enrolled in a colonoscopic cancer surveillance program at the Gastroenterology Unit at Söder Hospital in Stockholm with a definite diagnosis of UC, disease duration exceeding 8 years, and extensive disease. 5,17 FCM analysis for aneuploidy was initiated in 1982 as a regular part of this surveillance program. Criteria used for definite diagnosis of UC included a history of diarrhea and/or rectal bleeding for 6 weeks or more, typical radiological or endoscopical findings, and characteristic microscopic changes on biopsy. 5,17 Disease duration was calculated from the date of diagnosis of UC, taken from the medical record; this was considered a more exact measure than the date of onset of symptoms. The extent of disease was evaluated mainly on the basis of reviewed radiological findings; changes on barium enema reaching at least proximal to the hepatic flexure were considered indicative of total or extensive colitis. If the radiological findings were inconclusive or not available, the extent was classified as total only if endoscopy showed marked alteration in vascular pattern and/or dehaustration and/or inflammatory polyps in the ascending colon or cecum. Study Design The patients were included in two separate but related studies. Study A represents a case-control study in which 6 patients who underwent colectomy for dysplasia (cases) were matched with 6 others who underwent colectomy for medical intractability (controls). Controls were selected for having never manifested definite dysplasia or aneuploidy during the course of surveillance. Control patients were matched with cases mainly on the basis of disease duration, but also as closely as possible according to number of surveillance colonoscopies, number of years under surveillance, and total number of colonoscopic biopsies (Table 1). For each of these 12 patients, the colectomy were analyzed along with all available previous surveillance colonoscopy biopsy. Study B included 14 patients in the surveillance colonoscopy program for long-standing UC who have not undergone a colectomy. The patients were divided into three groups depending on their pattern of dysplasia or aneuploidy over the years: Group I included patients (n 7) who had never demonstrated dysplasia or aneuploidy despite repetitive sampling over many years. These patients may therefore be considered as additional controls except that they did not undergo colectomy. Group II included patients (n 2) who were found to have dysplasia at some time during surveillance, but no aneuploidy. Group III included patients (n 5) who had aneuploidy at some time during the course of surveillance but never had any evidence of definite dysplasia. Altogether, 1691 colorectal mucosal from 26 patients were analyzed. This consisted of 725 in study A (323 from cases; 402 from controls) and 966 in study B. Colonoscopy In the surveillance program, colonoscopy was performed every second year between 8 and 20 years of disease Table 1. Matching of Cases and Controls Sex Age at diagnosis (yr) UC duration (yr) surveillance examinations Years of surveillance STn Case 1 F 11 20 5 5 60 43 (72%) Control 1 F 10 12 4 5 50 2 (4%) Case 2 M 22 25 3 4 36 14 (39%) Control 2 F 8 31 5 13 56 5 (9%) Case 3 M 24 29 5 5 60 18 (30%) Control 3 F 13 35 11 12 119 2 (2%) Case 4 M 10 43 9 9 99 27 (27%) Control 4 M 18 42 10 12 107 9 (8%) Case 5 M 17 16 2 2 28 15 (54%) Control 5 M 40 14 2 2 30 6 (20%) Case 6 F 17 21 3 2 40 23 (58%) Control 6 M 28 13 3 4 40 2 (5%) All cases, mean (range) 16.8 (10 24) 25.7 (16 43) 4.5 (2 9) 4.5 (2 9) 323 a 47% b All controls, mean (range) 19.5 (8 40) 24.5 (12 42) 5.8 (2 11) 8.0 (2 13) 402 a 7% b a Total number of. b Median percent STn-positive.

December 1998 SIALYL Tn AS A PRECANCEROUS MARKER IN UC 1397 duration and annually thereafter. For each patient in study A, results of 2 11 colonoscopies preceding the colectomy were analyzed, along with the colectomy specimen itself. In study B, results of 3 9 consecutive colonoscopies were reviewed in group I, results of 4 5 consecutive colonoscopies were reviewed in group II, and results of 6 11 consecutive colonoscopies performed at 1 2-year intervals were studied in group III. Biopsies At colonoscopy, 2 3 biopsy were obtained from flat mucosa at 10 predetermined locations (1, cecum; 2, ascending colon; 3, hepatic flexure; 4, proximal transverse colon; 5, distal transverse colon; 6, splenic flexure; 7, proximal descending colon; 8, distal descending colon; 9, sigmoid colon; and 10, rectum) at approximately 10-cm intervals in the colon. 5,17 Fluoroscopy was used when necessary to confirm the position of the tip of the colonoscope. At each location, 2 biopsy were taken for histological assessment, and 1 was taken immediately adjacent (within 0 2 mm) and placed in saline for FCM analysis of DNA content. From 1982 to 1985, FCM biopsy were pooled into three fractions (corresponding to locations 1 3, 4 6, and 7 10, respectively). 5 From the colectomy, corresponding pathology samples were obtained. Specimens were fixed in formalin, embedded in paraffin, cut in sections, and stained with H&E for histological assessment. One biopsy specimen from each location was sampled for FCM DNA analysis according to methods described previously. 5 Histological Interpretation Histological evaluation was performed by one pathologist (A.O.) in a blinded fashion throughout the study. Dysplasia was classified as indefinite probably positive and definite low-grade or definite high-grade, in line with the classification proposed by the Inflammatory Bowel Disease-Dysplasia Morphology Study Group. 18 Findings of dysplasia were confirmed by another experienced pathologist. In the present study, the term indefinite is used to refer to that were considered indefinite but probably positive for dysplasia. FCM DNA Analyses Biopsy were processed and analyzed for DNA content as previously described. 7,19,20 The DNA content of normal human lymphocytes was used as a diploid reference value with a coefficient of variation (CV) of less than 3%. The ploidy level of the analyzed cells was defined as the DNA value of the G1/G0 cells in relation to the ploidy level of the control cells. If the ploidy level deviated less than 10% from that of standard lymphocytes, it was regarded as diploid (2.0c). Hence, distinct peaks with c values exceeding 2.2c were considered aneuploid. The number of cells analyzed from each preparation varied from a few thousand to 50,000, with an average of approximately 25,000. The results were then presented as DNA histograms. Immunohistochemical Staining and Interpretation of Slides All were archival, formalin-fixed, paraffinembedded tissues that were prepared as 5-µm sections. Slides were stained using MAb TKH2 (mouse immunoglobulin G1; kindly provided by Dr. Sen-itiroh Hakomori, The Biomembrane Institute, Seattle, WA), which specifically recognizes STn antigen. 21 MAb TKH2 reacts with STn antigen that is not O-acetylated. 22 24 Biotinylated rabbit anti-mouse immunoglobulin (Ig) G IgA IgM and streptavidin peroxidase conjugate were obtained from Zymed Laboratories (South San Francisco, CA). A standard immunohistochemical staining method using streptavidin-peroxidase was used, and all steps were conducted at room temperature as described previously. 11 A negative control slide was created for each staining procedure by substitution of isotype-matched normal mouse IgG1 for MAb TKH2. This always resulted in negative staining. The reactivity of MAb TKH2 with endothelial cells of the colonic mucosa provided a useful internal positive control. Immunoperoxidase-stained slides were interpreted for STn expression in a blinded fashion by at least two examiners (S.I. and either P.K. or E.Y.) without knowledge of patient source, histopathology, or DNA ploidy status. For each specimen, the number of crypts showing any immunoreactive cells was expressed as a percentage of the total number of crypts. In keeping with our previous work, 11 scoring was as follows:, 5% crypts positive;, 6% 35% positive;, 36% 65% positive; and, 66% 100% positive. Previous experience with scoring of colonic mucosa for STn expression showed minimal if any interobserver variation in interpretation of slides as positive or negative for STn and a variation of less than 15% between grades of positivity (unpublished observations). Although each specimen was scored using this scoring system, we have found that intensity of STn expression (i.e.,,, ) is less important than negativity ( 5% crypts positive) or positivity ( 5% crypts positive) for STn. Therefore, this cutoff value is used in the data presented. Statistical comparisons were performed using the Mantel Haenszel test or Cochran Armitage exact trend test where appropriate. Results Study A Matching of cases and controls. Control patients were matched with cases primarily on the basis of disease duration, but as shown in Table 1, the cases and controls were also well matched with respect to number of surveillance colonoscopies and years of surveillance, and fairly well matched according to age at time of diagnosis of UC. If anything, the controls had more surveillance examinations, more years under surveillance, and more total biopsy examined than the cases

1398 KARLÉN ET AL. GASTROENTEROLOGY Vol. 115, No. 6 Table 2. Number of STn-Positive Colonoscopies At least 1 segment STn At least 2 segments STn At least 3 segments STn Pair Case Control Case Control Case Control 1 5/5 (100) a 0/4 5/5 (100) 0/4 5/5 (100) 0/4 2 3/3 (100) 2/5 (40) 3/3 (100) 1/5 (20) 2/3 (67) 1/5 (20) 3 5/5 (100) 2/11 (18) 4/5 (80) 0/11 2/5 (40) 0/11 4 7/9 (78) 4/10 (40) 7/9 (78) 2/10 (20) 5/9 (56) 1/10 (10) 5 2/2 (100) 0/2 2/2 (100) 0/2 2/2 (100) 0/2 6 3/3 (100) 2/3 (67) 3/3 (100) 0/3 3/3 (100) 0/3 Colonoscopies 25/27 (93) b 10/35 (29) 24/27 (89) b 3/35 (9) 19/27 (70) b 2/35 (6) Patients 6/6 (100) 4/6 (75) 6/6 (100) 2/6 (33) 6/6 (100) 2/6 (33) a Number of STn-positive colonoscopies/total number of colonoscopies (%); colectomy data excluded. b P 0.00001 case vs. control; Mantel Haenszel test. to avoid the potential bias of studying cases more intensively than controls. Overall percentage of STn-positive. Overall, the median percent of STn-positive was 47% of cases, compared with 7% of controls (Table 1). For each pair, STn was expressed much more frequently in the case than in the control. Cases were also significantly more likely than controls to have colonoscopy results with at least one STn-positive segment (Table 2). In fact, all of the cases, and 93% of their colonoscopies, expressed STn in at least 1 segment, and 70% of their colonoscopies showed STn expression in at least 3 simultaneous segments. In contrast, among controls, 29% of colonoscopies manifested STn in at least 1 segment, and only 6% of their colonoscopies had 3 or more segments manifesting STn expression. STn expression in relation to dysplasia. Comparison of STn expression and different degrees of dysplasia could be performed only in the cases because by definition none of the controls manifested definite dysplasia. For each of the cases, the frequency of STn expression increased with progressive degrees of dysplasia (Table 3). For the cases overall, the median percentage of STn positivity in biopsy that showed negative, indefinite, low-grade, or high-grade dysplasia/cancer results was 33%, 64%, 74%, and 84%, respectively, a statistically significant trend (P 0.003). Selection of only those that were negative for dysplasia permitted comparison between cases and controls. As shown in Table 4, STn expression was significantly more frequent in nondysplastic mucosa from cases than in those from controls (P 0.0001). Aneuploidy in relation to dysplasia. Aneuploid cell populations were present in 151 of 323 (47%) from cases but by definition in none of the control. The median percentage of aneuploidy in the cases did not show a progressive trend according to the degrees of dysplasia, being found in 45%, 40%, 70%, Table 3. STn Expression and Aneuploidy Relative to Dysplasia Degree of dysplasia STn Aneuploid Case 1 Negative 43 30 (70%) 28 (65%) Indefinite 4 3 (75%) 1 (25%) Low-grade 13 10 (77%) 7 (54%) High-grade 0 Total 60 43 (72%) 36 (60%) Case 2 Negative 8 2 (25%) 4 (50%) Indefinite 16 7 (44%) 8 (50%) Low-grade 9 3 (33%) 5 (56%) High-grade 3 2 (67%) 3 (100%) Total 36 14 (39%) 20 (57%) Case 3 Negative 48 11 (23%) 5 (10%) Indefinite 4 3 (75%) 0 Low-grade 7 3 (43%) 2 (29%) High-grade 1 1 (100%) 0 Total 60 18 (30%) 7 (12%) Case 4 Negative 88 24 (27%) 43 (49%) Indefinite 10 2 (20%) 3 (30%) Low-grade 1 1 (100%) 1 (100%) High-grade 0 Total 99 27 (27%) 47 (47%) Case 5 Negative 16 6 (38%) 6 (38%) Indefinite 3 2 (67%) 2 (67%) Low-grade 6 5 (83%) 5 (83%) High-grade/CA 3 2 (67%) 2 (67%) Total 28 15 (54%) 18 (64%) Case 6 Negative 17 8 (47%) 7 (41%) Indefinite 15 9 (60%) 10 (67%) Low-grade 7 5 (71%) 6 (86%) High-grade/CA 1 1 (100%) 0 Total 40 23 (58%) 23 (58%) All cases (median % positive) Negative 220 33% 45% Indefinite 52 64% 40% Low-grade 43 74% 70% High-grade/CA 8 84% 34% Total 323 140 (43%) a 151 (47%) b a P 0.003, b P 0.13; Cochran Armitage Trend exact test, stratified on cases.

December 1998 SIALYL Tn AS A PRECANCEROUS MARKER IN UC 1399 Table 4. STn Expression in Specimens Negative for Dysplasia Case Control P 1 30/43 (70%) 2/50 (4%) 0.0001 2 2/8 (25%) 5/56 (9%) 0.21 3 11/48 (23%) 2/119 (2%) 0.0001 4 24/88 (27%) 9/107 (8%) 0.001 5 6/16 (38%) 6/30 (20%) 0.29 6 8/17 (47%) 2/40 (5%) 0.001 Overall 0.00001 and 34% of biopsy with negative, indefinite, low-grade, and high-grade dysplasia/cancer results, respectively (Table 3). STn in relation to aneuploidy. Although the 43% frequency of overall STn positivity is very similar to the 47% frequency of aneuploidy (Table 3), analysis of individual indicated that the two markers did not regularly overlap but were expressed independently (Table 5). For example, 52% of aneuploid from the cases were positive for STn, indicating that a biopsy specimen with aneuploidy is just as likely to be positive as negative for STn. Likewise, among the 172 diploid from the cases, 35% were positive for STn. By definition, all from controls were diploid. Temporal and topographical relationship between STn, aneuploidy, and dysplasia. Figure 1 illustrates the temporal and topographical relationship between STn, aneuploidy, and dysplasia in the 6 casecontrol pairs. In case 1, STn was expressed extensively throughout the colon 1 year before the first detection of dysplasia (Figure 1A). The rather extensive STn expression persisted over the years, and colectomy eventually was performed because low-grade dysplasia was detected repeatedly throughout the colon. In contrast, control 1 never showed STn expression in any of the surveillance colonoscopy biopsy and showed only weak expression in 2 areas of the colectomy. Like STn, aneuploidy was expressed rather extensively in case 1 from the time of the initial colonoscopy through subsequent surveillance colonoscopies and finally in the colectomy. Table 5. STn Expression Relative to DNA Aneuploidy Aneuploid Diploid Case Total STn Total STn 1 36 27 (75%) 24 16 (67%) 2 20 9 (45%) 16 5 (31%) 3 7 4 (57%) 53 14 (26%) 4 47 15 (32%) 52 12 (23%) 5 18 10 (56%) 10 5 (50%) 6 23 13 (57%) 17 9 (53%) Total 151 78 (52%) 172 61 (35%) Case 2 already had dysplasia at the time of the initial colonoscopy, and in the colectomy specimen 4 years later there was extensive dysplasia, particularly in the proximal colon (Figure 1B). STn was present at the time of the initial colonoscopy and persisted until colectomy. Aneuploidy was also present at the initial colonoscopy and was found persistently thereafter. Control 2 had weak STn expression in a few segments 1 year before colectomy but essentially no STn expression in the colectomy specimen. Case 3 manifested low-grade dysplasia diffusely in the distal colon at the time of colectomy; dysplasia had been detected in this region 2 years earlier (Figure 1C). However, in biopsy from the colonoscopy performed 2 years before the first detection of dysplasia, STn expression was noted in distal locations, and expression persisted until the time of colectomy. Aneuploidy was also seen at the time of the initial colonoscopy, preceding the first detection of dysplasia by 2 years. This finding was not reproduced in 2 of the 4 subsequent surveillance colonoscopies. The corresponding control had weak STn expression in only 1 biopsy specimen at 2 separate colonoscopies during more than a decade of surveillance. Case 4 showed indefinite dysplasia in 1985, similar findings plus low-grade dysplasia in 1987, and then no dysplasia until 1992, at which time several areas of indefinite dysplasia were again detected (Figure 1D). Aneuploidy was detected at all surveillance endoscopies and strongly influenced the decision to perform colectomy. Aneuploidy also was seen extensively in the colectomy specimen, but no dysplastic changes were found. STn was expressed in the biopsy from the index colonoscopy and, except for two intervening examinations, persisted in biopsy from all other colonoscopies. Over time, the frequency of STn expression increased. In keeping with the absence of dysplasia in the colectomy specimen, there was no STn expression in the final colectomy. Control 4 had 1 colonoscopy result showing STn in 3 segments; otherwise there was little or no STn expression over more than a decade of surveillance. Case 5 underwent colectomy because of high-grade dysplasia and low-grade dysplasia primarily in the proximal colon and was found to have proximal colon cancer and multiple areas of dysplasia throughout the colon in the final surgical specimen (Figure 1E). STn was expressed in multiple segments of the colon in surveillance biopsy as well as the colectomy specimen, particularly in areas of dysplasia. Like STn expression, DNA aneuploidy was widespread and tended to occur in areas of dysplasia, but overall there was essentially no correlation between aneuploidy and STn expression.

1400 KARLÉN ET AL. GASTROENTEROLOGY Vol. 115, No. 6 Figure 1. (A F ) Comparison of STn, aneuploidy, and dysplasia in 6 case-control pairs in study A. Numbers at the top represent colonic segments from the cecum (1) to rectum (10) as described in Patients and Methods. Each segment was analyzed for STn expression, aneuploidy, and dysplasia. Unmarked segments indicate nondysplastic, diploid, STn-negative mucosa. Shaded boxes indicate STn-positive (defined as 5% crypts expressing STn antigen). Aneuploidy is indicated by An. Dysplasia, when present, is indicated below each entry. Slashes indicate tissue not available for STn analysis. LGD, low-grade dysplasia; HGD, high-grade dysplasia. Control 5 had no STn expression except in severely ulcerated distal segments of the colectomy specimen. Finally, case 6 had numerous biopsy with indefinite or low-grade dysplasia and even a suspicion of cecal carcinoma, prompting colectomy (Figure 1F). At the time of colectomy, only indefinite dysplasia was confirmed. STn expression was noted in numerous colonic segments simultaneous with dysplasia and persisted in the colectomy specimen in the areas considered indefinite for dysplasia. Aneuploidy was again widespread in the surveillance biopsy and colectomy without much relationship to STn expression. Control 6 showed minimal STn expression in only 2 biopsy. Study B Group I patients represent 7 additional controls in that they never had findings of definite dysplasia or aneuploidy. In 3 of these patients, no STn antigen expression was found, and another 3 expressed STn very rarely (3% 7%; Table 6). However, 1 patient (patient

December 1998 SIALYL Tn AS A PRECANCEROUS MARKER IN UC 1401 Table 6. Clinicopathologic Features of Patients in Study B Patient Sex Age of diagnosis (yr) UC duration (yr) surveillance examinations Years of surveillance STn aneuploid Group I I-1 M 21 40 9 12 89 0 (0%) NA I-2 M 18 26 8 11 80 0 (0%) NA I-3 F 20 19 3 4 26 0 (0%) NA I-4 F 24 19 3 10 30 2 (7%) NA I-5 F 8 28 7 12 68 2 (3%) NA I-6 F 21 27 8 11 65 3 (5%) NA I-7 F 23 32 9 12 85 20 (24%) NA Total 27/443 (6%) Group II II-1 F 23 18 5 7 49 22 (45%) NA II-2 F 27 14 4 3 40 1 (3%) NA Total 23/89 (26%) Group III III-1 F 25 34 11 12 109 26 (24%) 4/110 (4%) III-2 M 24 26 6 10 59 4 (7%) 7/60 (12%) III-3 M 18 30 10 11 99 10 (10%) 3/100 (3%) III-4 M 17 21 6 6 60 1 (2%) 9/60 (15%) III-5 F 12 31 11 12 107 6 (6%) 3/107 (3%) Total 47/434 (11%) 26/437 (6%) NA, not applicable. I-7) had STn expression in 24% of from 9 surveillance examinations performed over 12 years. Group II patients had transient dysplasia but no aneuploidy. Patient II-1 expressed STn rather extensively (45% of ; Table 6) in the first 3 surveillance colonoscopies and for 4 years before the first detection of dysplasia but then had 2 clean colonoscopies in the following 3 years. Patient II-2 was more like the controls; only 1 of 40 (3%) expressed STn. Group III patients had a history of aneuploidy at some time in their course but no definite dysplasia. Aneuploidy was found in 3% 15% of all biopsy in these patients, being noted in only 1 or 2 surveillance examinations over many years in any given patient (Table 6). The expression of STn was also rare. Four patients had STn frequencies in the control range (2%, 6%, 7%, 10%), whereas 1 had higher STn prevalence (24%). Only 1 patient (patient III-1) had STn expression that seemed to persist over time in particular regions of the colon (Figure 2). Comparison of STn and aneuploidy in group III patients indicates that except for patient III-4, all the other 4 patients had expression of STn before aneuploidy, often several years before. Moreover, the expression of STn was independent of aneuploidy; in fact, most of the aneuploid biopsy were negative for STn. Discussion The present study provides validation of the relationship between STn expression and cancer risk in UC and defines the relationship between STn and aneuploidy. All patients included in this study had a definite diagnosis and established disease extent. All were followed up prospectively since 1982 in a standardized fashion within the framework of a cancer surveillance program with synchronous biopsies for both histopathology and FCM DNA analyses. This made it possible to compare the expression of STn in matching locations using archival tissues. By analysis of STn antigen expression both throughout the colon topographically and temporally, a prospective clinical approach was simulated. In study A, the matching of cases and controls made it possible to compare results between individuals who had developed dysplasia leading to colectomy and individuals without definite dysplasia who underwent colectomy because of medical intractability. STn was frequently expressed in dysplastic as well as nondysplastic biopsy from UC patients who developed dysplasia leading to colectomy but was rarely expressed in the colorectal mucosa of those who did not develop dysplasia. The observation that STn was expressed in a median of 47% of all surveillance biopsy from cases but in only 7% of biopsy from well-matched controls who never developed dysplasia (despite comparable disease duration and intensity of surveillance) is remarkably similar to the results of our previous retrospective study. 11 In that study of patients with long-standing UC followed up at The Mount Sinai Hospital, we reported that among 11 case-control pairs, STn was expressed in 44% of surveillance biopsy from

1402 KARLÉN ET AL. GASTROENTEROLOGY Vol. 115, No. 6 cases and 11% from controls. 11 We also found that in contrast to controls, cases were more likely to have had colonoscopies with 2 or more segments expressing STn and to have repeated STn expression in 2 or more consecutive colonoscopies. 11 In addition, the appearance of STn preceded the initial detection of dysplasia by as much as 7 years. In the present study, a similar topographical and temporal relationship between STn and dysplasia was found. Our previous study did not determine the DNA ploidy status of, so conclusions could not be drawn regarding the relationship between STn and aneuploidy. In the present study A, we found essentially no correlation between expression of STn and the ploidy status of individual, despite similar frequencies of STn expression and aneuploidy in each category of dysplasia. For example, in the cases, half of all aneuploid were positive for STn, whereas the other half were negative for STn (Table 5). Likewise, in the cases, diploid expressed STn antigen at almost the same frequency as aneuploid. However, STn expression was considerably more common in diploid from cases (35%) than in diploid from controls (7%). This further supports the notion that STn is a marker of cancer risk in UC patients regardless of DNA ploidy status. An advantage of study A is that all patients underwent colectomy, allowing definite diagnosis of the presence and distribution of dysplasia. However, it can be argued that these patients represent a select group because of their need for colectomy (whether for neoplasia or for refractory disease). Therefore, in study B, we analyzed STn expression in patients who had not undergone colectomy. Of the 7 patients in group I, 6 expressed STn rarely if at all, thereby strongly resembling the control group of study A. One patient (patient I-7) was different from the others and expressed STn in 24% of during the course of 9 surveillance examinations over 12 years. The pattern of expression indicates that most of this STn expression occurred in the middle of the surveillance period, and STn expression diminished thereafter. These data emphasize the importance of follow-up of STn expression over time rather than on 1 or 2 colonoscopies. 11 It is possible that patient I-7 will still develop neoplasia in the future, which would explain the frequent STn expression. However, this patient has not had evidence of dysplasia or aneuploidy in colonoscopies from 1995 and 1997. Group II patients had transient dysplasia but no aneuploidy. As such, it is difficult to classify them as cases or controls. Indeed, 1 patient (patient II-1) expressed STn rather extensively, and another (patient II-2) was more like the controls, with only 1 of 40 (3%) expressing STn. Although in study A we observed that STn could precede the first detection of dysplasia (at least in cases 1, 3, and 4), STn and aneuploidy coexisted at the time of initial colonoscopy in all cases, so we could not determine which marker occurred earlier. Therefore, we studied group III patients who, like controls, never manifested definite dysplasia, but in whom aneuploidy was detected at some time. In 4 of the 5 group III patients, STn was expressed before aneuploidy, often several years earlier. The finding of aneuploidy was rather infrequent in group III patients, perhaps because they never manifested dysplasia and indeed represent a low-risk group. Aneuploidy was noted in only 1 or 2 surveillance examinations over many years in any given patient (Figure 2), with the frequency of aneuploidy ranging from 3% to 15% of all biopsy in these patients (Table 6). Likewise, the expression of STn was also rather rare in these patients. Despite the rather infrequent expression of either marker, these observations confirm the finding in study A that the expression of STn appears to be independent of aneuploidy. In fact, most of the aneuploid biopsy in group III patients were negative for STn. In our previous study of UC patients, we reported that STn expression was increased in biopsy manifesting active inflammation. 11 However, this increase was limited to patients who subsequently developed dysplasia or cancer. Biopsy with corresponding degrees of inflammation from patients who did not develop dysplasia rarely manifested STn expression. Thus, inflammatory changes seemed to enhance the sensitivity of STn antigen expression as an indicator of cancer risk. In the present study, we did not directly analyze the effect of inflammation on STn expression, but we believe the same observation applies. If anything, the controls should have had more active inflammation than the cases because they underwent colectomy because of medical intractability, yet most of them (5 of 6) demonstrated rare STn expression. Control 5 was the only exception. This patient had a fair amount of STn expression, albeit only in the colectomy specimen. This occurred in severely ulcerated mucosa with fissuring. In Crohn s colitis, STn expression is quite common, particularly in actively inflamed mucosa. 25 This difference between UC and Crohn s colitis may relate to differences in local cytokine production, which might regulate the expression of STn antigen. In the future, it would be worthwhile to formally test the effect of active inflammation on STn expression in UC mucosa in a controlled study. There are several reasons to consider using STn

December 1998 SIALYL Tn AS A PRECANCEROUS MARKER IN UC 1403 detected and therefore to be complementary to dysplasia as a marker of risk. Third, the immunohistochemical analysis of STn expression is technically very straightforward and reproducible and has the additional advantage of using the same tissue block that is already being processed for standard histological (H&E) interpretation without the need for fresh tissue. Fourth, there is little or no interobserver variation in interpreting a biopsy specimen as either positive or negative for STn. Fifth, unlike dysplasia, which can be difficult to interpret in the setting of active inflammation, STn interpretation appears to be less affected by inflammatory changes except perhaps in the presence of severe ulceration, although this issue requires further study. Finally, analysis is inexpensive using standard immunohistochemical reagents without the need for specialized FCM equipment. However, our studies of STn expression used formalin-fixed, paraffinembedded tissues and MAb TKH2, which recognizes STn antigen that is not O-acetylated. 21 23 Different results could occur if different fixatives or other anti-stn antibodies are used. In conclusion, STn antigen and aneuploidy are both markers of increased colon cancer risk in patients with long-standing UC. Each correlates with progressive degrees of dysplasia, and each is expressed more widely throughout the colon and chronologically earlier than dysplasia. However, the present study using prospectively acquired establishes that these two markers are independent of each other and that STn often precedes aneuploidy. Determination of STn may consequently complement dysplasia and aneuploidy in helping to identify patients at subsequent risk for neoplastic transformation of the colorectal mucosa. The potential utility of STn antigen in the clinical treatment of patients with long-standing, extensive UC warrants further investigation in prospective studies. Figure 2. Expression of STn antigen in patients in study B, group III, who had aneuploidy at some time in the past but no dysplasia. See legend to Figure 1 for definition of symbols. antigen to help determine cancer risk in patients with long-standing UC. First, expression of STn antigen correlates with the dysplasia-carcinoma sequence. Second, because STn expression is more widespread and occurs earlier than dysplasia, it is likely to be more readily References 1. Gyde SN, Prior P, Allan RN, Stevens A, Jewell DP, Truelove SC, Löfberg R, Broström O, Hellers G. Colorectal cancer in ulcerative colitis: a cohort study of primary referrals from three centers. Gut 1988;29:206 217. 2. Ekbom A, Helmick C, Zack M, Adami HO. Ulcerative colitis and colorectal cancer. A population based study. N Engl J Med 1990;323:1228 1233. 3. Morson BC, Pang LSC. Rectal biopsy and precancer in ulcerative colitis. Gut 1967;8:423 434. 4. Löfberg R, Lindquist K, Veress B, Tribukait B. Highly malignant carcinoma in chronic ulcerative colitis without preceding dysplasia or DNA-aneuploidy: report of a case. Dis Colon Rectum 1992;35:82 86. 5. Löfberg R, Broström O, Karlén P, Tribukait B, Öst Å. Colonoscopic surveillance in long-standing total ulcerative colitis a 15-year follow-up study. Gastroenterology 1990;99:1021 1031. 6. Melville DM, Jass JR, Shepherd NA, Northover JMA, Cepellaro D, Richman PI, Lennard-Jones JE, Ritchie JK, Andersen SN. Dysplasia and deoxyribonucleic acid aneuploidy in the assessment of

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