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Keywords:

  • Crohn's disease;
  • colorectal cancer;
  • dysplasia;
  • DNA aneuploidy;
  • Sweden

Abstract

  1. Top of page
  2. Abstract
  3. SUBJECTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Background:

There is uncertainty about how patients with Crohn's colitis should be monitored for colorectal cancer (CRC). By analogy to ulcerative colitis, regular colonoscopy with biopsies for dysplasia has been used. We describe the occurrence of dysplasia and DNA aneuploidy in a cohort of patients with Crohn's colitis.

Methods:

In all, 245 patients with extensive colitis (225 with a firm diagnosis of Crohn's disease, and 20 diagnosed as indeterminate colitis) at Stockholm Söder Hospital and Karolinska University Hospital, Huddinge were included. They were followed with regular colonoscopies with biopsies both for dysplasia and DNA aneuploidy. The cumulative occurrence of DNA aneuploidy and dysplasia was estimated using Kaplan–Meier curves. Time sequences and interactions between DNA aneuploidy, dysplasia, and CRC were studied using Cox regression analysis, adjusted for age, sex, and age at diagnosis.

Results:

During a median follow-up time of 9.2 person-years, DNA aneuploidy was found in 53 patients (22%), with 10 patients having multifocal aneuploidy and high S-phase values. Dysplasia was found in 42 patients (17%), 10 having multifocal dysplasia. Relative risk (RR) of dysplasia given DNA aneuploidy was 5.3 (95% confidence interval [CI] 2.3–12). RR of CRC given dysplasia was 10 (95% CI 2–50), and RR of CRC given aneuploidy was 1.5 (95% CI 0.3–9.3).

Conclusions:

Dysplasia and DNA aneuploidy including S-phase analysis may complement stratification of patients with Crohn's colitis with respect to risk of CRC and thus the need for surveillance. (Inflamm Bowel Dis 2010)

Long-standing inflammatory bowel disease (IBD) is associated with an increased risk of colorectal cancer (CRC).1, 2 This was first shown for ulcerative colitis (UC). Long-term experience of surveillance programs in handling this increased risk is now at hand; most centers employ some form of surveillance program for patients with long-standing (>8–10 years) and extensive UC. The value of this surveillance in terms of decreased mortality is debated, particularly the importance of histopathological low-grade dysplasia as a precancerous indicator.3, 4 There is still more controversy about the value of surveillance in Crohn's colitis. Only one study on colonoscopic surveillance in Crohn's disease (CD) has been performed, and in this study the authors found a cumulative incidence of dysplasia similar to that in UC.5

Apart from histopathological dysplasia, other precancerous markers have been used in surveillance.6 DNA aneuploidy (a numeric chromosomal imbalance) has been used together with histopathology in UC.7–9 Studies using DNA aneuploidy as an additional precancerous marker in Crohn's colitis are few, small, and have so far led to inconclusive results.10–12

At Stockholm Söder Hospital and Karolinska University Hospital, Huddinge patients with long-standing (>8 years duration) extensive Crohn's colitis have been followed with colonoscopic monitoring in the same way as those with UC. In this study, we describe the long-term results from this monitoring of patients with longstanding and extensive Crohn's colitis. In particular, we assessed the incidence of DNA aneuploidy, histopathological dysplasia, and colorectal cancer, and any possible interrelations between them.

SUBJECTS AND METHODS

  1. Top of page
  2. Abstract
  3. SUBJECTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Design and Setting

We performed a retrospective cohort study on two Swedish population-based cohorts with Crohn's colitis and indeterminate colitis at two metropolitan hospitals in Stockholm, Sweden, with follow-up evaluation for vital status, DNA aneuploidy, dysplasia, and cancer occurrence, through local registers where data have been prospectively recorded and through manual searches of patients' files and pathology reports.

Study Population

The patients comprised a clinically followed-up group of 245 patients with colitis (at least 1/3 of the colon involved) attending the gastroenterology departments at the Stockholm Söder Hospital and the Karolinska University Hospital, Huddinge, Stockholm. The patients were followed from 1982 when we started with both histopathological and DNA flow cytometry analyses.

The diagnosis of CD was made according to Lennard-Jones' criteria.13 All patients' files were scrutinized and patients, where the clinical picture, endoscopic appearance14 (taking possible medication effects into account), laboratory values and histopathological examinations15, 16 all pointed to a diagnosis of CD, were included. In this heterogeneous group there were also, not surprisingly, a number of patients where a definite diagnosis of CD could not be made (n = 20 of 245). In these 20 patients a diagnosis of UC could not be made either, and they were therefore classified as indeterminate colitis (IC) and were analyzed and described separately. Table 1 summarizes the patient characteristics.

Table 1. Patient Characteristics
Type of IBDCDIC
  1. A total of 225 consecutive patients with colonic Crohn's disease (CD), and 20 patients with indeterminate colitis (IC) from Stockholm, Sweden diagnosed from 1959 and onwards, monitored from 1982 with colonoscopies for histopathology and DNA flow cytometry up through 31 December 2007.

Sex (men/women)118/1079/11
Median age at diagnosis, y (range)25 (4–72)23 (5–59)
Calendar period of IBD diagnosis, n (%)  
Before 19606 (2.7)2 (10)
1960–196922 (9.8)2 (10)
1970–197946 (20.4)7 (35)
1980–198990 (40)5 (25)
1990–200461 (27)4 (20)
Extent of disease (colonic/ileocolonic)181/4419/1
Status at end of follow-up (alive/dead/lost)207/10/819/0/1
Primary sclerosing cholangitis (yes/no)10/2153/17
Colorectal surgery, n (%)66 (29%)11 (55%)
Total colectomy (n)3111
Partial colectomy (n)130
Ileocecal resection (n)220
Median duration of follow-up, y (range)5.4 (0–26)11 (0.7–24)

Follow-up

All patients were identified and followed in a local register over patients with extensive colitis monitored with regular colonoscopies with biopsies taken for both histopathology and DNA flow cytometry. In this register we could retrieve information on DNA aneuploidy, dysplasia, and CRC. The number and type of any colorectal surgery was also collected and verified by scrutinizing each patient's file.

Endoscopy

Colonoscopies were made using Olympus equipment. Preparation before examination included oral lavage with 3–4 L of polyethylene glycol-electrolyte lavage solution (Laxabon, Bio-Phausia, Sweden) or two doses of 45 mL of sodium phosphate (Phosphoral, Ferring, Sweden). No or only limited sedation (diazepam 5–10 mg intravenous [i.v.] and/or pethidine 25–50 mg i.v., or since 1985 midazolam and/or alfentanil) was used.

Biopsy Specimens

Biopsies were sampled from the large bowel at 10 predetermined locations (1 = cecum, 2 = ascending colon, 3 = hepatic flexure, 4 = proximal and 5 = distal transverse colon, 6 = splenic flexure, 7 = proximal and 8 = distal descending colon, 9 = sigmoid colon, and 10 = rectum). Two biopsies were taken from each location for histopathology and one biopsy immediately adjacent (0–2 mm apart) was analyzed by flow cytometry. The biopsies were taken randomly without use of narrow banding imaging (NBI) or chromendoscopy. Additional biopsy specimens were taken if macroscopic polypoid or raised lesions other than endoscopically typical inflammatory polyps were found.

Histopathological Grading

Biopsy specimens for histopathology were fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin. All specimens were assessed for histopathological dysplasia without knowledge of the results of the DNA analyses. Dysplasia was classified according to Riddell et al17 and graded as 1) negative, 2) indefinite, probably negative, 3) indefinite unknown, 4) indefinite, probably positive, 5) definite low-grade dysplasia (LGD), 6) high-grade dysplasia (HGD), and 7) cancer. If dysplasia was found in flat mucosa adjacent to a raised lesion it was classified as a dysplasia associated lesion or mass (DALM). Findings of sporadic adenomas were excluded from the analysis. Only biopsies without active inflammation (defined as no more than infiltration of lymphocytes and plasma cells in the lamina propria)18 were evaluated to avoid misinterpretation of reactive, inflammatory changes. We actively avoided taking biopsies for dysplasia in visibly inflamed areas. In cases with very active inflammation where this would have been difficult, and where the inflammatory changes would have made the pathological examination difficult to interpret, the patients were treated with high-dose cortisone and the examination was repeated in 3 months' time. No biopsies with LGD or worse were discarded because of inflammation. Dysplasia findings were classified as unifocal if occurring only once in one fraction, unifocal reproducible if occurring in one fraction but repeatedly in the same fraction over several examinations, and multifocal if occurring in several (at least four fractions). The most advanced neoplasia was included in the analysis. Patients with LGD repeated at two different occasions, confirmed by an independent pathologist, were recommended for colectomy. All patients with HGD were referred to surgery.

DNA Flow Cytometry

The fresh biopsies were fixed in buffered formalin and analyzed as previously described.19 Any DNA aneuploidy findings were classified as unifocal if occurring only once in one fraction, unifocal reproducible if occurring in one fraction but repeatedly in the same fraction over several examinations, and multifocal if occurring in several (at least four fractions). We also determined the S-phase (synthesis phase in the cell cycle used as a measure of proliferative activity) in all patients with DNA aneuploidy findings. The S-phase was graded as high if it was over 8% and as low if under 8%. This grading was based on the findings from a previous work20 where all biopsies from a healthy population had S-phase values below 8%.

Statistics

The cumulative incidences of dysplasia (LGD or HGD) and DNA aneuploidy, respectively, were estimated using Kaplan–Meier curves counting time from first colonoscopy until the most recent colonoscopy, death, or lost to follow-up. The cumulative incidence of CRC was estimated using Kaplan–Meier curves counting time from first colonoscopy until first of: death, lost to follow-up, or December 31 2007.

To assess the relative risk of dysplasia following DNA aneuploidy, and of CRC following DNA aneuploidy or dysplasia, we used Cox regression adjusted for age, sex, duration of disease, type of (CD or IC), and extent of IBD. In these models, time was counted identically to that in the above-mentioned Kaplan–Meier curves. We also estimated the positive predictive value of dysplasia for subsequent CRC and the positive predictive value of a finding of DNA aneuploidy for the subsequent development of dysplasia or CRC.

Ethical Considerations

The work was approved by the Ethics Committee at Karolinska Institutet.

RESULTS

  1. Top of page
  2. Abstract
  3. SUBJECTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Endoscopic Follow-up

Median attained age at first colonoscopy was 46 years (range 20–78), median IBD duration at first colonoscopy was 14.1 years (range 0.5–55), median number of colonoscopies per patient was 4.0 (range 1–33), median time between first and last colonoscopy was 6.1 years (range 0–25.6), median total follow-up time for CRC counting from first colonoscopy was 9.2 person-years.

Overall Occurrence and Cumulative Incidence of DNA Aneuploidy

In total, DNA aneuploidy was observed in 53 patients (22%). Of these, 26 patients (11%) had only unifocal, 17 (7%) had unifocal reproducible, and 10 (4%) had multifocal DNA aneuploidy (Table 2). The cumulative incidence of DNA aneuploidy is shown in Figure 1. The S-phase was high in patients with multifocal DNA aneuploidy: 10 of 17 patients with a high S-phase had multifocal DNA aneuploidy and no patients with multifocal DNA aneuploidy had low S-phase values.

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Figure 1. Cumulative incidence of DNA aneuploidy in a Swedish population based cohort of 245 patients with Crohn's colitis (n = 225) and indeterminate colitis (n = 20).

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Table 2. DNA Aneuploidy and Dysplasia Findings
 All IBD n Total 245CD n Total 225IC n Total 20
  • Swedish cohort of 225 patients with Crohn's disease (CD) and 20 patients with indeterminate colitis (IC).

  • *

    Aneuploidy findings were classified as unifocal if occurring only once in one fraction (of 9-10 possible), unifocal reproducible if occurring in one fraction but repeatedly in the same fraction over several examinations, and extensive if occurring in several (at least four) fractions. Dysplasia was low grade (LGD) or worse in accordance with the criteria for dysplasia by Riddell et al. Otherwise classification as for aneuploidy.

Aneuploidy*n (%)   
 No aneuploidy192 (78.4)186 (82.3)6 (30)
 Unifocal26 (10.6)24 (10.7)2 (10)
 Unifocal reproducible17 (6.9)10 (4.4)7 (35)
 Extensive10 (4.1)5 (2.2)5 (25)
Dysplasia*n (%)   
 No dysplasia203 (82.9)200 (88.9)3 (15)
 Unifocal30 (12.2)15 (6.7)15 (75)
 Unifocal reproducible2 (0.82)2 (0.82)0
 Extensive10 (4.1)8 (3.6)2 (10)

No patients had concurrent DNA aneuploidy and dysplasia, concurrent DNA aneuploidy and CRC, nor dysplasia preceding DNA aneuploidy. All instances of first DNA aneuploidy preceded the occurrence of dysplasia or CRC.

Overall Occurrence and Cumulative Incidence of Dysplasia

In total, 42 patients (17%) developed dysplasia. Thirty patients (12%) had only unifocal dysplasia, two (0.85%) had unifocal reproducible dysplasia, and 10 (4%) had multifocal dysplasia (Table 2). All cases of dysplasia occurred in flat mucosa and there were no documented DALMs. No patients fulfilling the criteria for LGD or HGD remained under surveillance. The two patients with HGD had their HGD confirmed at operation. The patients who underwent surgery for repeated, independently confirmed LGD, all had a final diagnosis of LGD; there were no instances of HGD or CRC in this group when the colectomy specimens were analyzed. Figure 2 shows the cumulative incidence of dysplasia.

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Figure 2. Cumulative incidence of dysplasia in a Swedish population based cohort of 245 patients with Crohn's colitis (n = 225) and indeterminate colitis (n = 20).

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Occurrence of DNA Aneuploidy and Subsequent Dysplasia and CRC

Of the 203 patients without dysplasia, 26 (13%) had DNA aneuploidy, the majority (n = 19) only unifocally, and there were no patients with multifocal aneuploidy. In patients with LGD, 21 (70%) had DNA aneuploidy, and the two patients with HGD both had multifocal DNA aneuploidy. In the 10 patients with CRC, four had preceding DNA aneuploidy and six had no aneuploidy (Table 3). Of the 17 patients with DNA aneuploidy and high S-phase values, 12 developed neoplasia (CRC, HGD, or LGD). The median time from first colonoscopy to first DNA aneuploidy in CD was 2 years (6.8 years to dysplasia, and 9.8 years to CRC). In IC the corresponding median times were: 0.3 years, 10.8 years, and 12.1 years, respectively.

Table 3. Cross-tabulation of Dysplasia and DNA Aneuploidy
 Dysplasia, n   
Aneuploidy, n (%)NormalLGDHGDCRC
  1. Occurrence for all IBD and by diagnosis (CD or IC) in a Swedish cohort of 225 patients with Crohn's colitis and 20 patients with indeterminate colitis.

No aneuploidy177 (92.2)9 (4.7)06 (3.1)
Sporadic19 (73.1)6 (23.1)01 (3.8)
Sporadic reproducible7 (41.2)9 (52.9)01 (5.9)
Extensive06 (60)2 (20)2 (20)
CDDysplasia, n   
Aneuploidy, nNormalLGDHGDCRC
No aneuploidy174705
Sporadic19401
Sporadic reproducible7300
Extensive0122
ICDysplasia, n   
Aneuploidy, nNormalLGDHGDCRC
No aneuploidy3201
Sporadic0200
Sporadic reproducible0601
Extensive0500

Of the 42 patients who developed neoplasia, five were diagnosed with CRC during our follow-up. The relative risk of CRC associated with dysplasia (LGD or HGD) was 10 (95% confidence interval [CI] 2–50). Of the 53 patients who developed DNA aneuploidy, 23 were diagnosed with dysplasia (LGD or HGD) during follow-up (RR = 5.3 95% CI 2.3–12), and four with CRC during follow-up (RR = 1.5 95% CI 0.3–9.3). When the relative risks were adjusted for age, sex, extent (colonic or ileocolonic), type of IBD (CD or IC), duration of disease, number of colonoscopies, these relative risks remained largely unchanged. The positive predictive value (PPV) of dysplasia for subsequent CRC was 14% (negative predictive value [NPV] was 98%). The PPV of DNA aneuploidy for subsequent dysplasia was 47% (NPV was 95%), and PPV of DNA aneuploidy for CRC was 14% (NPV was 97%).

DISCUSSION

  1. Top of page
  2. Abstract
  3. SUBJECTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

In this study we found that in a cohort of patients with Crohn's and indeterminate colitis the overall occurrence of dysplasia was of similar magnitude as in previous studies of CD as well as of UC.5, 21, 22 The majority of this dysplasia was, however, unifocal, and only a small fraction (4%) had multifocal dysplasia. Besides confirming the occurrence of dysplasia, our study adds important information on the occurrence of DNA aneuploidy in these patients, and the interrelationships between aneuploidy, dysplasia, and CRC. In this regard, our main findings was that: 1) the crude occurrence of DNA aneuploidy was in the same range as in previous studies on UC7, 9; 2) DNA aneuploidy was mostly found in patients with subsequent either low grade or high grade dysplasia; and 3) the finding that DNA aneuploidy with high S-phase values correlated to a subsequent finding of dysplasia.

There are three previous studies on CD-colitis where the occurrence of both DNA aneuploidy and dysplasia has been studied.10–12 The article by McKinley et al10 was a case report of one patient with CD colitis who had concomitant HGD and DNA aneuploidy. Lofberg et al12 studied 24 patients with extensive CD colitis and found no dysplasia but two cases of DNA aneuploidy. Porschen et al11 studied both CD colitis and UC, and found no aneuploidy among 51 patients with CD colitis.

There is one previous study on CD-colitis and surveillance5 in which the frequency of dysplasia paralleled that reported for UC (no other precancerous markers were used). In that study the cumulative incidence of HGD/CRC was 7% after ≈10 years of follow-up, which is in close agreement with our finding of a cumulative CRC incidence 7% after 10 years of follow-up. Further, in a case–control study on possible exposures and CRC risk in Crohn's colitis23 the authors found that previous colonoscopy for an indication of screening or surveillance was associated with a lower risk of CRC.

Based on their findings the authors made the recommendation that patients with extensive CD-colitis should be followed with periodic surveillance colonoscopies. It is tempting to include CD-colitis in similar surveillance programs as for UC. However, the benefit in terms of decreased mortality from such programs is even less certain for CD-colitis, where the absolute and relative risks for colitis associated cancer are uniformly lower than for UC.

A better approach would be to stratify which patients with CD-colitis should be included in such surveillance. Patients with extensive CD-colitis (at least 1/3 of colon), a positive family history for CRC, concomitant PSC, extensive, reproducible DNA aneuploidy with high S-phase values, or any other marker for neoplasia should probably be followed with yearly colonoscopies.

Our study has a several limitations. The lack of a control group, a weakness in common with the earlier studies on CD-colitis and precancerous markers, precluded a direct comparison with the normal population regarding prevalence of dysplasia and DNA aneuploidy. In an earlier study,20 however, we have shown that an unselected normal population without colitis does not have DNA aneuploidy or dysplasia. A further weakness was the incomplete (and hence not included) data on factors such as smoking status, 5-ASA-treatment, and family history of CRC. The anticipated difficulty in definitely separating colonic CD from IC or UC is also a limitation, although particular care was taken to minimize any misclassification of UC as CD. The 25 cases where a firm diagnosis of CD could not be made were retained as IC, since we believed the important factor might be long-standing colitis rather than the diagnosis per se. As always in studies involving histopathology there is the well-described problem of subjectivity in dysplasia grading.24, 25 All our analyses were made by experienced pathologists with a special interest in gastrointestinal pathology. To minimize any ambiguities, only LGD or worse were retained as dysplasia and all cases of indefinite probably dysplastic mucosa were excluded. All cases of dysplasia leading to colectomy were reviewed independently by a second gastrointestinal pathologist.

All patients were surveyed in a similar way, regardless of previous presence of DNA aneuploidy or not, according to the protocol of our local surveillance program. Since we follow this program for every patient undergoing surveillance, there is little risk of bias in real time. The presence of only DNA aneuploidy (without dysplasia) will according to this program not lead to a closer surveillance compared to patients with no DNA aneuploidy. There was no difference in the mean number of colonoscopies between patients with DNA aneuploidy and those without aneuploidy.

A particular difficulty with CD colitis is the occurrence of strictures and complicated anal disease with fistulas. However, in our patient group there were no cases of strictures, fistulas, or by-passed intestinal segments among the neoplasia cases.

Common to all surveillance there is always a risk of sampling error, since only a very small fraction of the entire mucosa can be covered with biopsies. This problem is particularly true for CD colitis, with its often patchy inflammation and possibility of strictures. Brush cytology could be a way to reduce sampling error because of strictures.

Our study also has several strengths: it is based on the largest cohort of patients with extensive CD colitis where information both on histopathological dysplasia as well as DNA aneuploidy were available. All patients were included according to strict diagnostic criteria to minimize the risk for misclassification of patients with UC as Crohn's colitis.

The addition of DNA aneuploidy as a second biological marker for neoplasia adds strength as the histopathological inflammation-dysplasia-carcinoma sequence is not as firmly established for CD as for UC. We increased the precision further by also studying the S-phase fraction and not only the occurrence of DNA aneuploidy per se. It seems that the crucial factor for neoplasia development is the increased S-phase and not just the mere presence of DNA aneuploidy. This seems also to be the case for UC, where high S-phase values correlated well to extensive LGD and HGD. We therefore suggest that in following CD colitis patients in surveillance programs an additional marker for neoplasia such as DNA aneuploidy with S-phase analysis, be used.

In conclusion, the results of our study suggest that although both dysplasia and DNA aneuploidy could be used in the surveillance of CD colitis, the value of merely measuring their crude occurrence may be moderate. By contrast, when additional aspects such as S-phase and extent are considered, the clinical value of both DNA aneuploidy and dysplasia increases. Thus, together with age, duration of disease, CRC heredity, and a recent change in symptoms, S-phase and extent of aneuploidy and dysplasia can help stratify risk and thereby find patients with CD colitis to follow more closely.

REFERENCES

  1. Top of page
  2. Abstract
  3. SUBJECTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES