Colonoscopic surveillance in long-standing total ulcerative colitis—A 15-year follow-up study

GASTROEh’TEROLOGY

1880;89:1021-1031

Colonoscopic Surveillance in Long-Standing Total Ulcerative ColitisA Is-Year Follow-up Study ROBERT LbFBERG, OLLE BROSTRijM, PER KARLl?N, BERNHARD TRIBUKAIT, and AKE 6ST Medical Department, Huddinge Hospital, Medical Department II, South Hospital, and Departments of Medical Radiobiology and Pathology, Karolinska Institute and Hospital, Stockholm, Sweden

In a 15-year surveillance program composed of 72 patients with total ulcerative colitis, 12 patients developed definite dysplasia. At endoscopy, low-grade dysplasia was detected in seven patients, high-grade in four, and a carcinoma (Dukes’ stage A at operation) in one. One of the patients with high-grade dysplasia and macroscopical lesions at colonoscopy had a carcinoma (Dukes’ A) detected at operation. A sequential development of dysplasia was found in seven patients. The cumulative risk of developing at least low-grade dysplasia was 14% after 25 years of disease duration. Using flow cytometric analyses, abnormal, aneuploid DNA content was detected in biopsies of 12 of 59 patients (20.3%); this correlated significantly with low-grade and high-grade dysplasia. Aneuploidy preceded dysplasia in two patients and was also detected in two dysplasia-free patients. The long-term use of colonoscopic surveillance in ulcerative colitis is a reliable way to select patients, in whom dysplasia is developing, for prophylactic surgery. Additionally, flow cytometric DNA analyses may help in the selection. The risk of missing a carcinoma until it becomes incurable appears to be low.

he increased risk of colorectal carcinoma in ulcerative colitis [UC] was previously considered high enough to warrant prophylactic colectomy in high-risk patients, i.e., those with total involvement of the disease and disease duration exceeding 3-15 years [l-3). The recognition of precancerous lesions (dysplasia] in the colorectal mucosa, which were associated with carcinoma in UC (4), initiated a prospective follow-up program at St. Mark’s Hospital in 1966 (5). In this study, patients in whom dysplasia, which was detected by rectal biopsies, was developing were

T

selected for cancer-prophylactic surgery. The evolution of flexible fiberendoscopes facilitated total colonoscopy (6), enabling removal of multiple biopsies from the entire colon. Several endoscopic surveillance programs of various length have been presented (7-15), giving further support to the concept of a dysplasia-carcinoma sequence in UC. Surveillance has now become common. However, dysplasia is difficult to interpret, especially when inflammation is present (16). Also, there is a considerable interobserver variation (17). A classification of dysplasia was presented in 1983 by Riddell et al. (18) to overcome some of these problems, but there is still a demand for other methods for selecting those UC patients who are prone to carcinoma development. Abnormal DNA pattern (aneuploidy) is closely related to malignancy and has been detected in biopsies of approximately 10% of patients with UC with long duration in prospective trials (19,ZO)using flow cytometric (FCM] analyses. In a retrospective study including 22 patients with UC and colorectal carcinoma, 17 displayed DNA aneuploidy (21). It has been suggested that detecting aneuploidy may be of additional value in assessing dysplasia in UC follow-up (19-21). The primary aims of the current prospective study were to evaluate the long-term reliability and efficacy of a colonoscopic surveillance program in longstanding total UC and to estimate the cumulative risk of dysplasia development. Furthermore, the use of FCM DNA analyses during the last 6 years of the study period was evaluated.

Abbreviations used in this paper: DALM, dysplasia-associated lesion or mass; FCM, flow cytometry. 0 1880by the American Gastroenterological Association 0016-5085/80/$3.00

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Materials and Methods Patients Patients eligible for this study included those patients attending the Gastroenterology Unit at South Hospital in Stockholm who had a definite diagnosis of UC, a disease duration exceeding 8 years, and a total extent of the disease. Criteria used for definite diagnosis included a typical history and endoscopic and radiological findings (22). Disease duration was calculated from the date of diagnosis of UC; for the patients in this study, it differed from the date of onset of symptoms by an average of 9 months. The date of diagnosis was used because it was considered to be more accurate than the date of onset of symptoms. Extent of disease was determined radiologically, and changes on barium enema reaching at least beyond the hepatic flexure were classified as total colitis (5,6). Consequently, if the radiological changes were inconclusive or if radiology had not been performed, the disease was classified as total colitis only if endoscopy showed changes such as inflammatory polyps, marked alteration in vascular pattern, and dehaustration in the cecum and ascending colon. Most of the patients resided in the hospital’s primary catchment area (230,000 inhabitants), but seven patients were referred from other neighboring areas. Patients were advised to participate in the surveillance, but prophylactic colectomy was offered as an alternative. The surveillance program was approved by the local ethical committee.

Study Period The surveillance program was initiated in November 1973, and the outcome until December 1988 has been evaluated. The entry time was defined as date of the first colonoscopy and the endpoints were dates of either death, colectomy, or last colonoscopy.

Endoscopy Colonoscopy was performed every second year between 8-20 years of disease duration and thereafter annually. If dysplasia or aneuploidy was detected before 20 years of disease duration, colonoscopy was repeated within 1 year. During the first 9 years of the study, rigid sigmoidoscopy was performed annually if colonoscopy was done the same year, otherwise it was done twice yearly. During the last 6 years of the study, rigid sigmoidoscopy was not regularly performed. Patients reaching the age of 70 years during the program and patients who underwent colectomy with ileorectal anastomosis were additionally surveyed by rigid or flexible sigmoidoscopy only.

Biopsies At colonoscopy, two to three biopsies were obtained from flat mucosa at 10 predetermined locations in the colon (13,19]. Fluoroscopy was used whenever needed to establish the position of the tip of the colonoscope. If macroscopical nodular, polypoid, or elevated lesions were detected, additional biopsies were sampled. Biopsy specimens were fixed

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in formalin, embedded in paraffin, cut in sections and stained with H&E for histological assessment. From April 1982, one additional biopsy from each location was sampled for FCM analysis. The FCM biopsies were pooled into three fractions, each comprising one third of the colon (191. Since 1985, each biopsy for the FCM analysis was analyzed separately if aneuploidy had been detected at a previous colonoscopy to obtain a direct comparison between histological and FCM findings.

Biopsy

interpretation

The histological evaluation of the biopsy specimens was done by the same pathologist (A.&] throughout the study in a blinded manner. Dysplasia was recorded only when the mucosa showed none or only slight inflammation to avoid misinterpretation of reactive, inflammatory changes. To ensure that patients were having as little inflammatory activity as possible, some patients received a low dose of prednisolone during 3 weeks before the examination. Until 1983, dysplasia was classified as slight, moderate, or severe. In accordance with the nomenclature adopted in 1983 (181, the grading has been reclassified as indefinite, probably positive (probably dysplastic), and definite, lowgrade or high-grade dysplasia. The indefinite changes, corresponding to slight dysplasia, will be referred to as “indefinite changes, probably dysplastic” in this report. Low-grade and high-grade dysplasia correspond to moderate and severe dysplasia, respectively. Findings of dysplasia leading to surgical intervention were assessed by at least one other senior pathologist with special interest in inflammatory bowel disease for consensus of the grading.

Flow Cytometric

DNA Analyses

The fresh biopsy specimens for DNA analysis were placed in cold saline, pressed through a nylon grid with 40-50 mesh counts/cm, and the resulting suspension was fixed in ice-cold 96% ethanol. The fixed cells were thereafter treated with an isotonic Tris ethylenediaminetetraacetic acid (EDTA) buffer (Tris 0.1 mol/L, NaCl 0.07 mol/L, and EDTA 0.005 mol/L with pH 7.5) containing ribonuclease (RNase) to eliminate the RNA. After pepsin treatment (0.5% pepsin solution with pH 2.0) for 10 minutes in a 37°C water bath, suspensions of single cell nuclei were obtained. This was followed by staining of the nuclei with 2.5 x lo- ’mol/L ethidium bromide. The staining was performed in Tris EDTA buffer with a molarity of 395 mOsm to reduce the risk of nonspecific binding of ethidium bromide. The DNA content of the cell nuclei was analyzed using a rapid flow cytofluorometer ICP 11 (Phywe, Federal Republic of Germany] equipped with a xenon lamp. The excitation and emission wavelengths were 455-490 nm and 590-630 nm, respectively. The output was sorted with a 256 multichannel analyzer. 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 G,/G,

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cells in relation to the ploidy level of the control cells. If the ploidy level deviated less than 10% from the standard lymphocytes, it was regarded as diploid (2.0~). Hence, distinct peaks with c values exceeding 2.2~were considered as aneuploid. The number of cells analyzed from each preparation varied from a few thousand to 50,000 with an average of around 25,000. The result was presented as a DNA histogram according to a method previously described in further detail (19.23). Indications

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findings of colonic Crohn’s disease were subsequently found, and they were eliminated from the program. None had dysplasia or DNA aneuploidy. Seventy-two patients with UC, 37 men and 35 women, remained for evaluation. Sixty-five patients had total colitis, diagnosed radiologically, and seven total colitis, as judged by colonoscopy only. Their median age at diagnosis was 21.0 years (range 6-62 years). At the endpoint of the study the median duration of disease was 20.0 years [range 8-44 years).

for Surgery

Patients in whom carcinoma or high-grade dysplasia or at least low-grade dysplasia in combination with a macroscopic dysplasia-associated lesion or mass (DALM) (24) was developing were selected for surgery. Patients not responding to medical treatment of a severe acute attack or chronic continuous activity were also referred for surgery.

Follow-up During the study period, 18 patients discontinued surveillance (Figure 1). Two patients died; one from a pulmonary carcinoma and the other because of a pulmonary embolus. [Both had inactive UC.) Six patients discontinued colonoscopic surveillance because of old age, and in one patient medical complications (renal failure with hemodialysis) made further surveillance impossible. Five patients moved to other areas but continued colonoscopy surveillance at other centers and did not develop definite dysplasia. Four patients were lost to further surveillance during the study period. One refused further colonoscopies on religious grounds but continued with sigmoidoscopy controls; another patient in whom mammary carcinoma developed missed her colonoscopy, but she reentered the program after the study period had ended. Two patients, one woman and one man who was an alcoholic, were completely lost to followUP* Of the remaining 54 patients, 12 underwent surgery and 42 continued in the surveillance program.

Statistical Analyses Fisher’s exact test was used for statistical analysis of the findings of DNA aneuploidy. The cumulative risk of development of at least low-grade dysplasia was calculated using the life table method (25). Differences in age at diagnosis between patients without dysplasia and patients with at least low-grade dysplasia were assessed for statistical significance using the life table method and Cox’variation analysis. Student’s t test was used in the comparison between differences in duration of disease. Results One patient chose prophylactic colectomy as an alternative to surveillance; altogether, 79 patients entered the program. In seven patients, histological

non-uc

r.mtea

Operated

n i

12

indefinite changes 2

Hlgh

ag.

ns3 indefinite changes 2

Indefinite changes 2

Figure 1. Outcome for 72 patients with UC entering the colonoscopic surveillance program. The 5ndings at colonoscopy of indefinite changes (probably dysplastic) and definite dysplasia are indicated under each subgroup. IRA, ileorectal anastomosis.

Compllcstlng direas. n=l k5ov.d to other hospital nr5 I

Dyrplasla nr9 Low grade 4 High grade 4 (1 with carcinomaat op.) caKjllWT0.1

-

Dysplasia Il.13 Indefinite changes 7 Ademma 3 Lowgrade t Continued

rurveillanco

n34

(2 colectomy+ IR4) ETigmiiscopy (1 c&cbmy + IfM) sigmidoscoW (1 segmental res.sctian)mlonosmpy

1024

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Colonoscopies A total number of 291 colonoscopies were performed, 273 of which were complete (94%). The mean number of colonoscopies per patient was 4.0 (range l-10). Forty-nine patients (68%) had at least three colonoscopies. There were no complications during colonoscopy. The length of the surveillance period ranged from l-14 years with a mean of 5.5 years. The average number of patients entering the surveillance program each year was four to five.

Dysplasia In 30 patients (41.7%), dysplastic lesions were detected at least once (Table 1). Three of these patients had isolated tubular or tubulovillous adenomas of the pedunculated type; all were radically excised using diathermal polypectomy. Dysplasia in flat mucosa was not detected in the colon in any of the three patients, and they remained in the surveillance program. Indefinite changes, probably dysplastic, were found in 15 patients. However, in 10 of these patients, the changes were not found at subsequent colonoscopies. In four patients whose surveillance was discontinued, changes classified as indefinite, probably dysplastic were found at the last colonoscopy or at time of surgery. Only one of seven patients with indefinite changes, probably dysplastic, who continued in the program had this finding at the most recent colonoscopy. Seven patients had low-grade dysplasia and two of them had also a DALM. Three of these patients had indefinite changes, probably dysplastic, preceding definite dysplasia of low grade. However, in three of these seven patients low-grade dysplasia was detected only once in one single biopsy; the finding was not made at subsequent colonoscopies or at time of surgery. One of these seven patients had preceding indefinite changes, probably dysplastic, associated with a small macroscopical mass, which was completely removed by snare biopsy. No further macroscopical lesions were found at later colonoscopies. High-grade dysplasia was found in four patients, two of whom also had DALM. All four patients displayed a sequential development from indefinite, Table 1. Findings

of Dysplastic

Lesions

at Endoscopy

n %

1" 1.4

High-grade 4b 5.6

probably dysplastic changes, and/or low-grade dysplasia to high-grade dysplasia. A polypoid carcinoma in the sigmoid colon was found in one other patient in whom preceding dysplasia had not been detected at colonoscopy 1 year earlier. No concomitant dysplasia in the colon adjacent or distant to the tumor was detected when the carcinoma was found. The findings of at least lowgrade dysplasia and/or DNA aneuploidy in individual patients are shown in Figure 2, which shows the development of dysplasia from one examination to the other and also how it relates to the duration of the disease. Reactive changes in the epithelium, which were interpreted as caused by inflammation, were found in seven patients at eight colonoscopies. DNA Aneuploidy Since 1982,59 patients under surveillance were also studied with FCM DNA analyses. A total of 616 analyses were carried out. In six analyses from two patients, the cell material had deteriorated and no interpretable histogram could be obtained. Subsequent FCM analyses in these two patients were adequate. In twelve patients, (20.3%) DNA aneuploidy was detected at least once (Figure 2). Two patients (no. 5 and 131 had aneuploidy without dysplasia at repeated examinations, and in two patients (no. 4 and 15) aneuploidy preceded the development of indefinite changes, probably dysplastic, or low-grade dysplasia. One patient [no. 5) had a persistent finding of aneuploidy in the sigmoid colon for 6 years, without developing dysplasia or carcinoma. No patient had persistent findings of definite dysplasia preceding DNA aneuploidy during the 6-year follow-up with FCM analyses. Five of six patients with low-grade dysplasia, who were observed since 1982, and both patients with high-grade dysplasia had aneuploidy. There was a statistically significant correlation between aneuploidy and definite dysplasia (P < 0.01). The proportion of aneuploidy in different groups of dysplasia findings is shown in Table 2. The findings of aneuploidy were widespread in some patients and persisted in the same part(s) of the colon and rectum in

in 72 Patients

With Total Colitis

Indefinite changes

Definite dysplasia Cancer

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Low-grade 7c 9.7

Probably dysplastic 15 20.8

“Dukes’ A at surgery. bT~o patients with DALM; one of these had a carcinoma Dukes’ A at surgery. Three patients with DALM.

Adenoma 3 4.2

No dysplasia 42 58.3

Total 72 100

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Op. local

sr

r-

, ,

I

I,

8

,I

resectIon

COLITIS

(Dukes’

1025

A)

Op. C I IRA LEGEND:

? ?No dysplasia ? *?Slgmoldoscopy IndefInIte changer, probably dyaplwtlc ??

.

8

op.

PC

I

H

Low grade dysplasia

High

I

grade

dysplasia

? ?DALY ??Carcinoma ? ?Severe Inflammation op.

PCIiAA*PP

+

9

SWI

of FCM DNA-analyses

DNA-aneuploid No FCY

Op.

.

Op.

PC / IAA + PP (Dukes’ A)

* lly+

0

I

PC / IAA + PP

I

C 1 IRA PC

=

A

DNA-dlploid

DNA-analyses

= Colectomy

and

performed

ileorecl~l

mmstomosla

Proclocolectomy

PC / IAA + PP = Proctocoleclomy + palvlc

and

lleoanal

?? nastomosls

pouch

Op.PC,;A+PP

Figure 2. Individual results in 17 patients with long-standing, total UC in whom at least low-grade dysplasia and/or DNA aneuploidy was developing during colonoscopb surveillance. The most severe finding at each colonoscopy is indicated.

eight of nine patients with repeated findings of DNA aneuploidy. In these nine patients, the ploidy level of the aneuploid peaks was consistent in seven patients from one examination to another. Additional peaks with lower or higher ploidy levels were detected in four patients indicating the existence of different clones of aneuploid cells. In three of four patients with Table 2. Findings of Dysplasia and DNA Aneuploidy in 59 Patients With Total Colitis Followed With Colonoscopic Biopsies From 1982 to 1988 Aneuploidy

No dysplasia Adenoma Indefinite changes, probably dysplastic Low-grade High-grade Total

n

%

n

%

37 3

63.6 5.2

2 0

5.4 0

11

19.0

6 2 59

8.6 3.4

3 5 2

27.3 83.0 100 20.3

100

12

DNA aneuploidy as well as dysplasia selected for prophylactic surgery (patients 12, 15, and 16), FCM analyses were performed in tissue samples from lo-30 different locations in the colectomy specimens. There was a good correlation between the preoperative colonoscopy grading of dysplasia and of DNA aneuploidy findings and those made in the colectomy specimen, although dysplasia as well as aneuploidy were found multifocally and with a patchy distribution throughout the colon and rectum. In eight patients with indefinite changes, probably dysplastic, that were not found again at repeat colonoscopy, no aneuploidy was detected. Nor was aneuploidy found in the patient with a macroscopical lesion which was removed by polypectomy (patient 7 in Figure 2). No patients with reactive, inflammatory epithelial changes had DNA aneuploidy.

P < 0.01

NOTE. When the findings of aneuploidy among patients without dysplasia were compared with aneuploidy in the group with definite dysplasia [low-grade or high-grade), a statistically significant difference was found.

Dysplasia and DNA Aneuploidy Age and Duration of Disease

in Relation to

Patients in whom indefinite changes developed had a median age at diagnosis of UC of 21.0 years (range, lo-62 years) and a median duration of UC

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since diagnosis of 25.0years (range, 11-36years). The median age at diagnosis was 17.0 years (range, 6-28 years] in those patients who developed at least lowgrade dysplasia after a median of 26.0 years (range, 13-42 years) duration. Their median age at time of development of at least low-grade dysplasia was 41.5 years (28-58 years]. Patients with DNA aneuploidy had a median age at diagnosis of 20.5 years (lo-32 years) and a median disease duration at the first detection of aneuploidy of 24.0 years (range, 13-38 years). The median age at the first detection of aneuploidy was 44.0 years (27-64 years]. In patients with neither indefinite changes, definite dysplasia, nor DNA aneuploidy, the median age at diagnosis was 22.0 years (range, 7-55 years). Compared with the group with at least low-grade dysplasia, the difference was not statistically significant (P = 0.69). The median disease duration at the time of follow-up was 15.5 years (range, 8-44 years), not significant (NS) compared with the group with at least low-grade dysplasia. Cumulative Risk of Developing

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Surgery During the surveillance program, a total of 12 patients underwent surgery. Three patients underwent colectomy because of failure of medical therapy (Figure 1). Indefinite changes, probably dysplastic, were found in one location in the colectomy specimen in one of the patients who also had such changes in uninflamed mucosa at colonoscopy 1 year before the operation. Nine patients were operated on because of the outcome of the surveillance program (Table 3). As can be seen from the table, all four patients who underwent surgery after the DNA analyses were included in the program displayed aneuploidy. The patient who developed a carcinoma in the sigmoid during surveillance (no. 2) had only a local resection performed because of the surgeon’s preference. The carcinoma was staged as Dukes’ A, and the patient, followed by annual colonoscopy since the operation, has subsequently developed low-grade dysplasia as well as aneuploidy in the distal part of the colon. Only one of the nine patients had a finding of dysplasia at the first colonoscopy severe enough to warrant surgery (no. 16). The other eight patients developed a higher degree of dysplasia during surveillance. Five of these had no dysplasia at all at their first colonoscopy (patients 1,2,6,15, and 17 in Figure 2). In two patients, the dysplasia, which led to surgery,

Dysplasia

The cumulative risk of developing dysplasia of at least low-grade in relation to the disease duration was calculated using the life-table method. The cumulative risk was 1.6% after 13 years, 10.0% after 17 years, 14.2% after 25 years, and 32.8% after 30 years (Figure 3). % 40

30

.-J

20

I ‘O-1

I

I

,__A I

_-

,_-I

4

m-r_. _ I

.-

20 66

53

I

I

lo smph *ire

1

45

42

3-o 25

23

13

17

16

t 40

Duratlon years

12

Figure 2. The cumulative risk of developing at least low-grade dysplasia among 72 patients with total UC followed by colonoscopy (solid line) The sample size is shown under each step on the curve. The cumulative risk of developing colorectal carcinoma in 466 patients with total UC as found in a joint study from Birmingham, Oxford, and Stockholm (26)is shown as comparison (dotted line).

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Table 3. Nine Patients With Long-Standing During Colonoscopic Surveillance Age at Patient no.

Duration (yrl

operation (yr)

Dysplasia at first colonoscopy

Ulcerative Dysplasia leading to surgery

1

42

49

0

2

32

56

0

6

30

50

0

a

26

43

10

25

42

Indefinite changes, probably dysplastic Low-grade

12 15

17 17

46 30

Low-grade 0

16

15

32

Low-grade + High-grade DALM DALM

17

13

42

0

Colitis Operated

DNAFCM

High-grade + DALM Polypoid carcinoma

ND

Low-grade + DALM Low-grade + DALM

ND

High-grade

ND

High-grade Low-grade

+ +

Low-grade

+

ND

ND

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orrBecause of Findings

Dysplasia at operation High-grade + DALM Carcinoma Dukes’ A in sigmoid colon

Su@cal procedure

of Dysplasia or Carcinoma

Continued surveillance

C/IRA

Sigmoidoscopy

Segmental resection

Colonoscopy annually

Low-grade + DALM Low-grade + DALM

C/IRA

Sigmoidoscopy

C/IRA

Sigmoidoscopy

(Severe inflammation) High-grade Low-grade

PC/IS

None

PC/IAA + PP None PC/IAA + PP None

+

Carcinoma Dukes’ A

PC/WA + PP None

+

Indefinite changes, probably dysplastic

PC/IAA + PP None

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Remarks No further dysplasia Low-grade dysplasia + aneuploidy at follow-up Alive 8 yr after operation No further dysplasia No further dysplasia

Operated on because of persistent low-grade dysplasia and aneuploidy Carcinoma in DALM. Alive 4 yr after operation Operated on because of widespread inflammatory polyps, lowgrade dysplasia + aneuploidy

NOTE. Patient numbers correspond to those in Figure 2. Duration, years of ulcerative colitis since diagnosis. DNA-FCM, + aneuploidy present. ND, not done; C/IRA, colectomy and ileorectal anastomosis; PC/IS, proctocolectomy and ileostomy; PC/IAA + PP. proctocolectomy with ileoanal anastomosis and pelvic pouch.

was detected in the rectum (patients 10 and 171, whereas the other seven patients had significant dysplasia predominantly in the proximal third of the colon. All patients selected for surgery because of findings of dysplasia had their preoperative biopsies reassessed by another pathologist, and a consensus of the grading reported here was made in all nine cases. At the time of operation, the median age of the nine patients with dysplasia leading to surgery was 43 years [range, 30-58 years], and the median duration of UC was 25 years (range, 13-42 years].

Discussion Study

Design

In this study, a substantial number of patients with longstanding total UC were observed prospectively with colonoscopy at regular intervals for a long period of time. This enabled monitoring of eventual

development of dysplasia in individual patients. Only the study from St. Mark’s Hospital (11) has been of comparable length, albeit covering more than twice as many patients with total UC, but it was not a dedicated colonoscopic surveillance. Few other surveillance programs have been of prospective design (7-9,13-15) and they are difficult to compare because selection of patients and length of follow-up differ. The interpretation and terminology of dysplasia also vary in these studies, and sometimes insufficient data makes it difficult to perform a direct comparison. By assessing the biopsies in a blinded manner by the same pathologist and by attempting to avoid the influence of inflammation, it was sought to optimize the conditions for a cor.rect interpretation of dysplasia in this study. Only a.few patients had inflammatory activity at time of colonoscopy. Biopsies were sampled from 10 different predetermined locations to enable comparison of results from one colonoscopy to another. The interval between colonoscopies was de-

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creased from 2 years to 1 year after a disease duration of 20 years. This schedule seemed to be sufficient. A duration-differentiated colonoscopy schedule has also been proposed by Lashner et al. (26) based on hazard rates of detection of high-grade dysplasia and carcinoma. Rigid sigmoidoscopy as a complement to colonoscopy was used in our previous study (13) but because the yield of dysplasia findings was low (more than low-grade dysplasia detected only once in more than 300 examinations], it was not regularly included for the last 6 years of this study.

Patient Selection Only UC patients with a disease duration exceeding 8 years and with total extent of the disease were included in this study. The criteria for radiological determination of disease extent have been the same as used by others (9,111. Barium enema, often of the single contrast type, has been the primary method in establishing the extent of the disease in the majority of patients with long duration of disease. Most reports of the increased risk of colorectal carcinoma in total colitis are based on retrospective studies in which the radiological extent has been used for patient selection (2,3,27). We previously found dysplasia in “substantial colitis” (defined as radiological signs of UC extending beyond the splenic flexure but not engaging the hepatic flexure) to be rare (13); this was confirmed also by other studies (91415). The risk of colorectal carcinoma may be increased in this group of patients too but probably to a lesser degree (27,281. Many new patients will have their diagnosis of total colitis based on endoscopic or histological grounds only. However, there are no generally accepted criteria for histological or endoscopic total colitis, making comparison of surveillance programs difficult. Whether or not patients with endoscopic or histological total colitis have the same risk of carcinoma development as the classical high-risk group with radiological total colitis remains to be settled. Only a small number of patients diagnosed as having endoscopic and/or histological total colitis has entered surveillance programs (7,14,15), and, therefore, long-term results of dysplasia frequency among such patients are not yet available. Patients with Crohn’s disease that is confined to the colon and rectum represent another problem when selecting patients for surveillance. We encountered seven patients in this category, all showing histologically characteristic features of Crohn’s colitis after two or more colonoscopies. The frequency of dysplasia in patients with Crohn’s colitis is not known but the occurrence in occasional patients has been reported in prospective studies (14). None of our patients with Crohn’s disease had dysplasia or DNA aneuploidy.

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Dysplasia Findings A sequential development of dysplasia was found in seven patients, one of which had a carcinoma at time of operation. This development supports the concept of a dysplasia-carcinoma sequence in UC. Only one out of nine patients subsequently operated on because of dysplasia had a finding severe enough to warrant surgery at time of the first colonoscopy. This indicates that our group of patients entered the surveillance program at a comparably early stage of their disease and was not highly selected. In a large study by Rosenstock et al. (12) a substantial yield of highgrade dysplasia and macroscopical lesions already at the first colonoscopy probably reflect a selection bias in the referral pattern of that patient series. The curve of the cumulative risk of developing at least low-grade dysplasia closely resembles the cumulative risk of developing colorectal carcinoma reported in a joint study (27)from Birmingham, Oxford, and Stockholm composed of 486 patients with total UC (Figure 3). The latter study calculated disease duration from onset of symptoms, but because the difference between the date of symptom onset and date of definite diagnosis was less than a year in the present study, the two curves are considered to be comparable. The dysplasia curve rises a few years before the cancer curve, which could be regarded as an indirect support of definite dysplasia as a marker of impending carcinoma. This was also noted by Lashner et al. (26) although they found a much higher cumulative risk of developing dysplasia. They argued that eventually, after 40 years of disease duration, the cumulative risk of at least low-grade dysplasia would be in the order of 85%. This may be an overestimation of the risk, because still only a limited number of patients will get cancer (27). The total frequency of definite dysplasia in this study was similar to that reported by others (ll,14-15). In most patients with indefinite changes, probably dysplastic, the findings were not detected at subsequent examinations. The appearance of these changes is thus of limited predictive value even though a few patients later developed definite dysplasia. Low-grade dysplasia found in four patients was not detected again at later examinations (patients 2, 7, and 14 in Figure 2) or at time of surgery (patient 17).Similar observations have been made in other studies (7,12,14), and the St. Mark’s group (11) even reported that in 5 out of 17 patients with findings of high-grade dysplasia at one endoscopy, the findings could not be redetected again at surgery or at subsequent colonoscopies. These observations may indicate that dysplasia is reversible, but they could also be caused by difficulties in dysplasia interpretation or, in the case of colonoscopy, sampling error. The latter could depend on the patchy distribution of dysplasia throughout the colon (13,16,29).

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Dysplasia may thus be missed from one examination to another, as only a minute fraction of the colorectal mucosa is sampled during colonoscopy (12). Two of the patients operated on in this study because of dysplasia (patients 12 and 15) had persistent low-grade dysplasia in different parts of the colon during surveillance, and both had a patchy distribution of dysplasia when their colectomy specimens were assessed. These two patients also displayed widespread DNA aneuploidy. It may be wise to consider even low-grade dysplasia as an additional indication for colectomy when dysplasia is found at repeated colonoscopies and in different parts of the bowel. Widespread inflammatory polyps, as illustrated by case no. 17, pose a special problem in surveillance because it is impossible to sample biopsies adequately from all macroscopical lesions. Our patient had dysplasia as well as aneuploidy in flat mucosa; colectomy was recommended, which probably is a good alternative for these patients, because a safe follow-up cannot be anticipated. In early retrospective studies, it has been suggested that biopsies from the rectum would be sufficient to detect the majority of cases developing dysplasia (30,311. In only two of the nine patients, operated on because of dysplasia, significant dysplasia developed in the rectum. Our results, therefore, reemphasize the necessity of total colonoscopy in a successful surveillance program (7,912). We achieved 94% total colonoscopies, a figure which may be further improved because many of the incomplete examinations were carried out at the beginning of the study. Pedunculated adenomas have been reported by Rosenstock et al. (12), and, as in this series, after polypectomy was performed no further dysplasia was detected in the colon. It is not clear if this type of adenomas should be regarded differently in UC compared with normal subjects (16). If no dysplasia in flat mucosa is detected and the patient is elderly, it may be appropriate to consider pedunculated adenomas as age-related and continue with colonoscopic surveillance after polypectomy. Patients in whom at least low-grade dysplasia develops had a somewhat lower age at diagnosis of UC than the rest of the patients. Although the number of patients developing definite dysplasia was small, this is an interesting finding because some previous studies have indicated that early onset of UC may be an additional risk factor for carcinoma development. Long duration of UC is, however, interrelated to an early age at onset: this was also noted in the patients with definite dysplasia as well as in patients who developed DNA aneuploidy. Patients without dysplasia or DNA aneuploidy had, not unexpectedly, a much shorter median duration of disease. No case with

definite dysplasia was detected before 13 years of disease duration in this study, supporting the initiation of colonoscopic surveillance after 8-10 years of duration. CoJorectaJ Carcinoma The main aim of surveillance in longstanding UC is to select patients for prophylactic surgery before carcinoma occurs. In this study, one of the patients with carcinoma did not have preceding or concomitant dysplasia at colonoscopy at time of diagnosis. Only a segmental resection was performed, but subsequent colonoscopic follow-up showed development of low-grade dysplasia as well as DNA aneuploidy. The frequency of carcinomas in UC not associated with findings of adjacent or distant dysplasia varies between 0%-20% in retrospective assessments of colectomy specimens (29,32). Again, the problem with patchy and focal distribution of dysplasia may be valid in these rather unusual cases. Single cases with “idiopathic” colorectal cancers may also occur, especially in elderly patients. Another carcinoma was found at time of surgery in a patient who had high-grade dysplasia and DALM detected at colonoscopy. Altogether, we found five patients with DALM, in whom four DALM had developed during the course of the surveillance. The clinical significance of DALM and carcinoma has previously been recognized in two retrospective reports (12,241.

In previous prospective surveillance programs, only two carcinomas have actually been found at endoscopy (ll,l3), and a small number (all Dukes’ A or B) has been detected after colectomy because of significant dysplasia, i.e., low-grade with DALM or highgrade (7,9,14). The exception is the St. Mark’s hospital study (11) in which there were as many as 13 carcinomas: eight of them were staged as Dukes’ A. Nevertheless, the majority of carcinomas occurring within the frame of prospective colonoscopic programs have been in early, and thus operable and potentially curable stages. Both patients who developed carcinoma in the present study are alive and well 8 and 4 years after surgery, respectively. DNA Aneuploidy The present study additionally aimed to investigate if the detection of DNA aneuploidy could provide further help in the selection of patients for prophylactic colectomy. We found a correlation between DNA aneuploidy and definite dysplasia. This is in accordance with the retrospective study by Melville et al. (211,in which also almost 80% of UC carcinomas were DNA aneuploid. In a previous study (19) we found DNA aneuploidy detected by FCM analyses from

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fresh biopsies obtained at colonoscopy to be persistent and reproducible. The results were similar in this study, although two patients, in whom aneuploidy had been detected in single locations, had negative findings at time of the latest colonoscopy (Figure 2). This most likely reflects sampling problems, but may also indicate that DNA aneuploidy as well as dysplasia could be reversible in some instances. Hypothetically, chromosomal changes detected as an abnormal increase in the cellular DNA content are one of the first cellular abnormalities in malignant transformation of the mucosa in longstanding UC, preceding morphological changes manifested as dysplasia. In this 6-year prospective follow-up, we found two patients with DNA aneuploidy preceding dysplasia and two other patients with DNA aneuploidy at repeated colonoscopies without signs of dysplasia. This indicates that DNA aneuploidy may sometimes be an earlier warning of malignancy than dysplasia: however, further prospective follow-up is needed before conclusions can be made in this respect. Interestingly, during the 6-year follow-up with FCM analyses no patient developed persistent, definite dysplasia that preceded findings of DNA-aneuploidy. DNA aneuploidy was often detected multifocally in the colon and rectum, with some variation between colonoscopies, and different ploidy levels of the aneuploid peaks could be seen in the same patient. This indicates that the cytogenetic changes are extensive and that the entire colonic mucosa may be at increased risk of transformation into the malignant phase. The patchy pattern of aneuploidy (as well as of dysplasia) may simply reflect a difference in the “schedule” of malignant transformation from one part of the colon to another. Because FCM DNA analyses can only show gross chromosomal abnormalities, diploid or near diploid malignant transformations are not detected by this method.

Clinical Implications The risk of developing colorectal carcinoma in total UC must be considered large enough (27) to warrant some kind of action, even though the risk apparently is lower than previously suggested t3). To ignore the risk would not be acceptable. Early prophylactic proctocolectomy is the only lOO%proof concept used to avoid subsequent carcinoma development. Proctocolectomy is also a curative measure for UC, but it involves morbidity and mortality. Colectomy, total mucosal proctectomy, and construction of .a pelvic pouch with an ileoanal anastomosis have become attractive alternative to conventional ileostomy but long-term results are not yet available. This type of operation is mainly applicable to younger

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patients (less than 55-60 years of age) with normal or slim constitution; however, it also carries some complications. Subtotal colectomy with ileorectal anastomosis is another, simpler alternative with less complications, but still requires follow-up surveillance with sigmoidoscopy since the cancer risk of the rectum remains (33). Overall operative mortality in elective surgery is small but not negligible. Patient compliance is another problem with the surgical approach. In a previous study (13). we found that some patients did not attend any regular checkups because of fear of colectomy. It must be assumed that some patients will always keep away from controls if they learn that they will face an operation after a certain period of time even if they are symptom free. Good patient compliance is therefore also essential for a surveillance program. In this respect, the current program was successful because only two patients were completely lost to follow-up. This could be result of the fact that a small group of interested gastroenterologists attended to the patients both at the outpatient visits and at the colonoscopies. A surveillance program based on colonoscopies at regular intervals offers the patients an alternative to compulsory prophylactic colectomy after a disease duration of a decade. The nine patients selected for surgery on the basis of dysplasia had their colons left for a median of 25 years. The additional 15 years for these patients with their colon in situ, may have important implications on their lives in terms of social function, marriage, and childbearing. For younger patients, initial surveillance and later colectomy with ileoanal anastomosis and pelvic pouch may be an alternative. The two last surgically treated patients (no. 15 and 17 in Table 3) illustrate this to some degree. Both patients had findings of low-grade dysplasia and DNA aneuploidy, but rather than continuing surveillance for another year or two to find out if the finding of dysplasia would be aggravated, they were advised to have a colectomy. A controlled study to evaluate the efficacy of colonoscopic surveillance in UC will probably never be performed for practical and ethical reasons. However, a report by Jones et al. (34) is very interesting in this respect. Although it was not a controlled study, in a group of UC patients who did not attend regular colonoscopies, significantly more colorectal carcinomas were found to develop compared with a surveillance group. Considering its limitations, this study further supports the benefit of surveillance. Conclusion In our experience, colonoscopic surveillance with multiple biopsies is a reliable solution to the long-term management of the increased risk for colo-

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rectal carcinoma in longstanding, total UC. With biopsy sampling from 10 locations in the colon and rectum at fixed time intervals, the diagnosis of definite dysplasia is usually reproducible and safe, and highrisk patients in whom at least multifocal low-grade dysplasia develops can be selected for cancerprophylactic surgery. The risk of missing a noncurable carcinoma appears to be low. The addition of FCM analysis for detecting DNA aneuploidy may increase the accuracy of patient selection at an earlier stage, although further prospective follow-up is needed before the role of this method can be fully determined. References I. Dennis C, Karlsson KE. Cancer risk in ulcerative

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Received May 5.1989. Accepted April 19,199O. Address requests for reprints to Robert Lofberg, M.D., Unit of Gastroenterology, Medical Department, Huddinge University Hospital, S-141 86 Huddinge, Sweden. This study was supported by grants from the Cancer Society in Stockholm, the Swedish National Cancer Society, the Julin foundation, and the Swedish Society for Medical Research.