Vol. 6. 1021-1027, December 1997 Cancer Epidemiologj�, Biomarkers & Prevention /021

Calcium and Colorectal Epithelial Cell Proliferation in

Ulcerative l

Roberd M. Bostick,2 Mark Boldt, Mohamed Darif,James R. Wood, Paula Oven,, and John D. Potter

Department of Public Health Sciences-Epidemiology, The Bowman Gray

School of Medicine, Wake Forest University, Winston-Salem, North Carolina

27157 IR. M. B.]; Digestive Healthcare, PA, Minneapolis, Minnesota 55404

EM. B., J. R. W.]; Division of Biostatistics, School of Public Health, University

of Minnesota, Minneapolis, Minnesota 55455 [M. DI; Abbott-Northwestem

Hospital, Minneapolis, Minnesota [P. 0.]; and Fred Hutchinson Cancer

Research Center, Seattle, Washington 98104 [J. D. P.]

Abstract

In persons at higher risk for colon cancer (e.g., those

with sporadic adenoma or ulcerative colitis), compared tothose at lower risk, colonic epithelial cell proliferation

kinetics are altered. We have shown previously thatcalcium supplementation appears to normalize thedistribution of proliferating cells without affecting theproliferation rate in the colorectal mucosa of sporadicadenoma patients. In a pilot randomized, double-blind,placebo-controlled, clinical trial conducted concurrentlywith our previously published sporadic adenoma trial, wetested whether calcium supplementation can alsomodulate cell proliferation kinetics in patients withulcerative colitis. Ulcerative colitis patients (n = 31) wererandomized to placebo or 2.0 g of supplemental calcium

daily. Colorectal epithelial cell proliferation was

determined by immunohistochemical detection ofproliferating cell nuclear antigen labeling of cells in “non-prep” rectal biopsies taken at randomization and after 2months treatment. All biopsies were scored by onereviewer. Differences in mean follow-up minus baselinelabeling index (LI; the proportion of colon cryptepithelial cells that were labeled) and in the 0h

(proportion of labeled cells that were in the upper 40%

of the crypts) were compared with analysis of covariance.Pill-taking adherence was 97%. Biopsy-scoring reliabilitywas high (r 0.89). The pooled baseline LI and 0h were6.3% and 5.6%, respectively. The LI in the calciumgroup decreased by 0.3% (proportionately, 3%) morethan in the placebo group (P 0.91). Similarly, the 0h inthe calcium group decreased by 0.5% (proportionately,10%) more than in the placebo group (P = 0.85). This

Received 5/8/97; revised 8/27/97; accepted 8/28/97.

The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement in

accordance with 18 U.S.C. Section 1734 solely to indicate this fact.I This study was supported in part by USPHS Biomedical Research Support Grant

507 RR 055448 (Division of Research Resources) and by PHS Grant POICA-

50305 (National Cancer Institute), NIH, Department of Health and Human

Services.

2 To whom requests for reprints should be addressed, at Department of PublicHealth Sciences-Epidemiology, The Bowman Gray School of Medicine, Wake

Forest University, Medical Center Boulevard, Winston-Salem, NC 27157.

pilot study does not suggest that 2.0 g of calcium ascalcium carbonate daily can substantially normalizeeither the rate or distribution of proliferating cells over a2-month period in the colon crypts of patients withulcerative colitis; a more definitive answer to the questionof whether calcium may be effective would require astudy with a larger sample size and/or other study designmodifications.

Introduction

Coborectal cancer is the second most common cause of cancerdeaths in the United States (1). Persons with ulcerative colitis,

especially those with pancobitis, are at increased risk of devel-

oping colon cancer (2). Dietary calcium supplementation hasbeen consistently found to reduce colon cancer occurrence in

experiments in animals (3-9), and the epidemiobogical litera-ture, although inconsistent, on balance provides additional sup-

port for the hypothesis that higher calcium consumption mayreduce colon cancer incidence in humans (10).

It has been hypothesized that calcium may reduce the risk

of colon cancer by normalizing cobonic crypt cell proliferationkinetics (1 1). Several studies (12-26) have reported that, com-

pared to patients at low risk for colon cancer, patients withcolon cancer (12-21) and patients in every category known to

be at higher risk for colon cancer [those with a history ofsporadic adenoma (12-15, 17-21), familial polyposis (16, 22),

or ulcerative colitis (12, 23, 24); those with a family history ofcolon cancer (16, 17, 25); and the elderly (13, 26)], on average,

exhibit in their normal-appearing mucosa both an increasedcobonic epithebiab cell proliferation rate and an extension of the

colon crypt proliferative zone from the lower (basal) 60% of thecrypt to include the upper (buminal) 40% of the crypt. In

patients with previous colon cancer or sporadic adenomas,these changes also predict adenoma recurrence (27, 28). Inbarge bowel tumors in humans, an upward shift in the prolif-

erative zone is found in colon cancers and adenomas but not in

hyperplastic pobyps (29). As reviewed elsewhere (30-32), pro-biferative changes in histologically normal-appearing mucosa

have been shown to be a consequence of both cancer-initiatingand cancer-promoting agents: proliferative changes both pre-

cede and accompany cobonic neoplasms in rodents given chem-icab carcinogens, and a high fat diet produces proliferativechanges in both rodents and humans. Evidence from animalexperiments and preliminary evidence from human studies

strongly suggest that these two proliferation abnormalities, i.e.,

hyperproliferation and upward shift of the proliferation zone,

are reversible biomarkers or precursors for colon neoplasia(30-33). In humans, the two proliferation abnormalities appearto be independent variables (19, 34), and rectal biopsy findings

on both measures reflect those throughout the colon (21 , 35).Calcium administration was found to ameliorate the pro-

liferative changes in rodents (36-38). Several (39-43), but notall (32, 44), small preliminary clinical trials suggested similar

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1022 Calcium Trial in Ulcerative Colitis Patients

effects in humans. More recently, there have been three larger

controlled trials in humans with sporadic adenoma (45-47). Afull-scale (n = 193) trial in sporadic adenoma patients con-ducted by our group concurrently with the present study found

a large, statistically significant, normalizing effect on the pro-liferative zone (45); however, a second full-scale trial (47) didnot, and the third trial (46) did not address this measurement.

Possible mechanisms of action of calcium in normalizing cob-

rectal epithebial cell proliferation and reducing the risk of coloncancer include the binding of calcium with bile acids (thoughtto be promoters of colonic neoplastic change) to form inert

soaps (1 1) and the direct induction by calcium of terminal

differentiation of the cobonic epithebial cells (30-33).There have been no reported randomized clinical trials to

assess the efficacy of higher calcium consumption in normal-izing cell proliferation kinetics in humans with ulcerative co-

litis. To address this need, we conducted a randomized, double-

blind, placebo-controlled clinical trial (n = 31) to determinewhether calcium supplementation will reduce the coborectal

epithelial cell proliferation rate and normalize the distribution

of proliferating cells within colorectal crypts (i.e., shift the zoneof proliferation from one that includes the entire crypt to one

that is confined to the lower 60%, or normal proliferative zone,

of the crypt) in patients with ulcerative colitis.

Materials and Methods

This study was approved by the Committee on Use of HumanSubjects in Research of the University of Minnesota. Writteninformed consent was obtained from each study participant.

Participant Population. Participants were recruited from the

patient population attending a private practice gastroenterobogygroup that performs approximately 60% of all cobonoscopies in

the Minneapolis-St. Paul area. To be eligible for the study,subjects must have been 2 1-74 years of age, in general goodhealth, and capable of informed consent. They must have had a

history of pathology-confirmed ulcerative colitis with diseaseinvolvement that included the rectum; had no contraindications

to calcium supplementation or rectal biopsy procedures; and

had no medical conditions, habits, or medication usage thatwould otherwise interfere with the study as described below.

Specific exclusions included: a history of ever havingtoxic megacolon or other life-threatening complication of ub-cerative colitis, hospitalization for treatment of ulcerative co-litis within the previous 2 years, Crohn’s disease, calcium

supplement use, vegetarian diet, major diet change within theprevious 6 months, supplemental daily intake of vitamin D

greater than 400 IU or vitamin A greater than 10,000 IU,regular antacid use, bile acid-binding resin use, long-term tet-

racycline or indomethacin use, inability to refrain from aspirinuse for 10 days, history of bleeding disorder or current use of

anticoagulant medication, history of endocarditis or artificialheart valve, lithium therapy, current use of thiazide diuretics inamounts greater than the equivalent of 50 mg of hydrochbo-

rothiazide daily, immunosuppression, childbearing potential,renal insufficiency, kidney stones within the previous 20 years,hyper- or hypoparathyroidism, hypo- or hyperthyroidism not

under control, abnormal serum calcium or creatinine levels ateligibility visit, familial polyposis or Gardner syndrome, intes-

tinal malabsorption syndromes, active liver or pancreatic dis-ease, gastrectomy, bowel resection, enema or laxative depend-ence, active peptic ulcer disease, active malignancy other thannonmelanoma skin cancer, cardiovascular or pulmonary disease

that moderately or severely limited activity, narcotic or alcohol

dependence, nondeliberate weight loss of � 10% in the previous

3 months, and less than 80% compliance to a medicationregimen in a 4-week placebo run-in trial.

Clinical Trial Protocol. All age-eligible practice patients whohad been diagnosed with ulcerative colitis were identified as

potential study participants. All patients passing initial chartscreening for eligibility were sent an introductory letter, fob-bowed by a brief telephone interview. Potential participantswere then scheduled for an eligibility visit, at which time theywere interviewed, completed questionnaires, and provided a

blood sample. Their diet was assessed with a semiquantitativefood frequency questionnaire (48). Medical and pathology

records were reviewed. Those eligible entered a 4-week pla-cebo run-in trial. Only participants without significant per-

ceived side effects and who had taken at least 80% of theirtablets were eligible for randomization. Compliance for therun-in was assessed by questionnaire, interview, and pill count.Eligible participants then received their baseline biopsy and, ifstill willing to continue, were randomized (stratified by sex to

ensure balance by sex between treatment groups) to one of two

groups. All involved in the trial, including study personnel,endoscopists, study participants, and laboratory personnel were

blinded to treatment category.Participants (n = 31) were randomly assigned to the two

treatment groups: a placebo-control group (n = 14) and a 2.0 g(n 17) elemental calcium supplementation group. The pla-cebo was free of calcium, magnesium, vitamin D, or chelatingagents. As with our calcium and colorectal epithelial cell pro-liferation in sporadic adenoma patients trial (45), the calcium

tablets were calcium carbonate tablets (OsCal; at the time of thestudy, from Marion Merrill Dow, Inc., Kansas City, MO; nowfrom SmithKiine Beecham, Pittsburg, PA) taken twice dailywith meals (i.e., 5.0 g of calcium carbonate daily). Placebo and

calcium tablets were identical in size, appearance, and taste.The treatment period was 2 months. At the single fob-

bow-up visit 2 months after randomization (baseline), pill-

taking adherence was assessed by questionnaire, interview, andpill count. Participants were instructed to remain on their usualdiet during the study. Factors hypothesized to be related to

colon cell proliferation were assessed at baseline, and severalwere reassessed at the follow-up (final) visit.

Rectal biopsies were taken from study participants at ran-dom assignment to their treatment groups (baseline) and againat 2 months after onset of treatment (follow-up). The rectalbiopsy procedure was performed without the participant first

taking a laxative, enema, or other bowel-cleansing preparation.Three biopsies that appeared grossly adequate were taken fromnormal-appearing mucosa of the rectum 10 cm above the level

of the anus.

Laboratory Protocol. The laboratory protocol is described inmore detail elsewhere (45). Briefly, the biopsies were stretchedout flat, lumen side up, on bibulous paper; fixed in 70%ethanol; processed in a tissue processor; embedded in a paraffin

block; cut with a microtome so that crypts were longitudinallysectioned from base to lumen (section bevels were 3 p�m thickand taken 50 p.m apart); and mounted on microscope slides. Tolabel proliferating cells, slides were then subjected to immu-

nohistochemical analysis for PCNA3 as summarized: slideswere deparaffinized; rinsed; received a blocking agent, 5%

normal horse serum (diluted in PBS; Vector Laboratories,

Burbingame, CA), followed by PC-b clone PCNA antibody

3 The abbreviations used are: PCNA, proliferating cell nuclear antigen; LI,

labeling index; BrdUrd, 5-bromodeoxyuridine.

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CancerEpidemiology, Biomarkers & Prevention 1023

(diluted 1:200 with PBS; Oncogene Science, Cambridge, MA);

rinsed; received the secondary antibody, rabbit antimouse IgG

(diluted 1 :200 with PBS; DAKO Corp., Carpinteria, CA);rinsed; received the endogenous peroxide blocker, 0.3% H2O2;

rinsed; received the tertiary agent, avidin-biotin complex (Vec-tor Standard Kit at half strength); rinsed; developed by immer-

sion with the chromogen 3,3’-diaminobenzidine (Sigma Chem-ical Co., St. Louis, MO), in a 1 mg/mb solution with 0.003%

H2O2; rinsed; counterstained with Harris’s hematoxybin; dehy-drated; and coversbipped.

The BrdUrd uptake immunohistochemical method of iden-

tifying S-phase cells and measuring cell proliferation, as de-scribed elsewhere (45), was also used; however, the labeling

failure rate using this method in the ulcerative colitis patients inthe present study was 100% (compared to 15% in our concur-

rently studied sporadic adenoma patients; Ref. 45).

Protocol for Scoring Colon Crypt Sections on Slide. Thebiopsy scoring protocol is described in more detail elsewhere

(45). Briefly, colon crypts longitudinally sectioned from base to

lumen were analyzed. The total number of cells and number oflabeled cells for each crypt scored were counted, and the cell

count position of each PCNA-babeled cell within a crypt was

recorded. A scorabbe crypt was defined as an intact crypt

extending from the muscubaris to the lumen. Only whole crypts

were counted. Countable cells were defined as crypt cells in linein a single column of nuclei that extended from base to lumen.

An unlabeled cell was defined as a cell with a blue nucleus. Aweakly labeled cell (a cell in late G, or early G2) was defined

as a cell with a nucleus that was light brown in color (1-2 �

intensity). A strongly labeled cell (a cell in S-phase) was

defined as a cell with a nucleus that was dark brown in color(3-4k intensity). These cells tended to stand out prominently

on the slides. The slide reader began with the first PCNA-

stained slide on the participant. The slide was oriented in a

standardized fashion, and the section levels on the slides wereviewed in sequence using light microscopy. Scoring was begun

at the first complete crypt found. The reader moved through asection level from left to right until a scorable crypt waslocated. Crypts were always counted under X400 magnifica-

tion. During counting, a number was entered into a computer

data entry program that permitted analysis of the cell countposition and other characteristics of each cell. Each cell was

identified as an unlabeled cell, a weakly labeled cell, or astrongly labeled cell. The crypt base center cell was also iden-

tified. The goal was to score a minimum of 10 crypts.The number of labeled cells in the crypt was determined

by direct count of all labeled cells in the crypt. The position or

height of a labeled cell was assigned by cell count number from

the center cell of the crypt base.One slide reader scored slides throughout the study. Slides

from a 10% sample of biopsies scored by the reader wereresubmitted in a blinded manner to be reread to determine

intrarater reliability.

Statistical Analysis. Treatment groups were assessed for com-

parability of characteristics at baseline and at final follow-up by

x2 test for categorical variables and by analysis of covariancefor continuous variables; sex was included as a covariate.

Reader reliability was analyzed using intraclass correlation

coefficients.The overall cell proliferation rate, the LI, was calculated

for each biopsy by dividing the total number of labeled cellscounted (LC) by the total number of cells counted (TC) from allscored crypts from the biopsy and multiplying by 100% (100%x LCTTC; Ref. 18). Crypts were subdivided horizontally by cell

count height into five equal-sized compartments (18). A meas-

ure of the distribution of proliferating cells in the crypt, the

proportion of proliferating cells that were in the upper (luminal)

40% of the crypt (#{248}h)’ was calculated on each biopsy bydividing the number of labeled cells counted in the upper 40%of the crypt (LC4_5) by the total number oflabebed cells counted

(LC) and multiplying by 100% (#{248}h= 100% X LC4_5/LC; Ref.16). Each of these measures was calculated separately using

both the 3-4k labeled cells (strongly labeled cells) as thelabeled cells and the l-4� labeled cells (all labeled cells) as the

labeled cells.Mean coborectal epithelial cell proliferation parameters

were calculated for each treatment group at baseline and at the

2-month follow-up. Changes in proliferation parameters over

time were computed within each treatment group as person-

specific follow-up minus baseline differences, and then meanchanges were calculated and compared using analysis of co-variance (with sex as a covariate). Analyses were performed on

both the raw data and on the data transformed to improvenormality as described previously (45). However, because theresults of the analyses did not differ substantially, for simplic-ity, only the analyses on the raw data are presented herein.

In addition, the baseline cell proliferation data from both

treatment groups were combined and compared with those fromthe sporadic adenoma patients accrued during the same time

period in our concurrently conducted calcium and colorectalepithelial cell proliferation trial (45). Mean age- and sex-adjusted cell proliferation values were computed and comparedusing analysis of covariance.

Results

All patients were white, none had a history of having anadenoma, and none had a history of a first-degree relative withcolon cancer. Treatment groups did not differ significantly on

any other characteristics measured at baseline (for examples,see Table 1) or at the end of the study.

Biopsies that were scorable were obtained on 28 partici-

pants at baseline and 24 at 2 months; scorable biopsies wereobtained at both baseline and 2 months on 21 participants. A

total of 85% of biopsies had one or more scorable crypts andadequate labeling, and 84% had eight or more scorable crypts

and adequate labeling. Treatment groups did not differ signif-icantly at baseline on the mean number of crypts scored perparticipant, on crypt characteristics, or on cell proliferation

characteristics (Table 2). As shown in Table 3, levels of cellproliferation in the ulcerative colitis patients were higher thanthose found in the adenoma patients in our concurrent trial in

adenoma patients. The S-phase and all-labeled cell measure-ment alternatives for each proliferative parameter were highly

correlated, and analyses using either closely mirrored the other.

Consequently, for succinctness, only the more traditionallyreported S-phase measures are presented hereafter.

Adherence to visit attendance was 100%. The mean pro-

portion of pills taken in each group was 97%, and over 92% ofall participants in each group took �80% of their pills. Therewere no treatment or biopsy complications. Intrareader reliabil-

ity (intraclass correlation coefficient) for biopsy slide scoring

was 0.89.Analyses of clinical trial biopsy results by treatment group

are summarized in Table 2. There were no substantial or sta-tistically significant differences in follow-up minus baselinedifferences between the two treatment groups. For the LI, theabsolute follow-up minus baseline differences for the placeboand calcium groups were - 1.4% and - 1.7%, respectively,

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1024 Calcium Trial in Ulcerative Colitis Patients

Table 1 Selected baseline characteristics of participants (n 31)

Treatment groupCharacteristic

Placebo (n = 14) Calcium” (n = 17)Pb

Demographics

Mean (SD) age (yr) 44 (9.5) 48 (8.3) 0.21

Men (%) 71 47 0.18

College graduate (%) 64 47 0.46

Ulcerative colitis history

Mean (SD) duration (yr) 15 (5.6) 19 (9.3) 0.06

Dysplasia (%) 7 12 0.84

On ulcerative colitis medications (%) 71 65 0.36

Symptoms stable (%) 64 47 0.27

Mean (SD) No. bowel movements/wk. 1 1 (6.5) 12 (5.9) 0.40

Degree of symptoms in the pa.st month (%)

None 64 47 0.27

Mild 29 41 0.41

Moderate 7 1 2 0.62

Degree of symptoms past 24 hrs. (%)

None 71 82 0.75

Mild 29 18 0.75

Moderate 0 0

Diet

Mean (SD) total energy intake (kcal/day) 2154 (546) 21 10 (793) 0.98

Mean (SD) dietary calcium intake (mg/day) 949 (402) 731 (382) 0.13

Mean (SD) dietary vitamin D intake (lU/day) 304 (256) 288 (214) 0.70

Mean (SD) total fat intake (% kcal) 30 (2.7) 28 (5.6) 0.33

Mean (SD) dietary fiber intake (g/day) 19 (7.2) 21 (10.6) 0.66

“ Calcium, 2.0 g of elemental calcium per day delivered as two 1 .25-g calcium carbonate tablets taken twice daily.

I, By analysis of covariance for continuous variables (adjusted for sex) and by extended � for categorical variables.

Table 2 Summary of clinical trial rectal biopsy results: placebo versus calcium

Placebo Calcium Pb

B F � � BL�

ase ifl�S -

Biopsy measurement Baseline (n = 14), Hi - BL” (n = 9), Baseline (n = 14), FU - BL” (n = 12),

Mean (SD) Mean (SD) Mean (SD) Mean (SD)

No. ofcrypts 9.5(1.0) 0.1 (1.1) 9.3(2.1) -1.3(4.6) 0.71 0.09

Total No. of cells/crypt 121.5 (13.1) 2.3 (24.5) 131.3 (20.3) -6.6 (35.7) 0.1 1 0.83

No. of labeled cells/cryp( 7.5 (5. 1 ) - 1 .4 (6.9) 8.9 (4.4) - 2. 1 (4.9) 0.33 0.80

LI” (%)

Overall 6.0 (3.7) - 1.4 (4.6) 6.6(2.6) - 1.7 (3.3) 0.47 0.91

Compartmental�

I 9.7 (7.9) -2.2 (10.6) 1 1.4 (4.5) -2.7 (6.0) 0.34 0.40

2 9.8 (6.5) -3.0 (8.4) 10.9 (5.6) -4.2 (6.8) 0.41 0.59

3 4.5 (3.6) -0.8 (6.0) 4.1 (4.1) -0.6 (5.3) 0.99 0.67

4 1.6 (2.2) -0.5 (2.6) 1.5 (1.6) 0.0 (1.7) 0.66 0.70

5 0.3 (0.6) -0.1 (0.5) 0.5 (0.9) -0.3 (0.9) 0.62 0.46

0h� 5.6 (6.4) -0.7 (6.3) 5.6 (5.3) - I .2 (4.3) 0.63 0.85

Height of highest labeled cell

As cell No. 32.4(8.6) 0.9(12.8) 36.4(13.5) -4.6(13.2) 0.37 0.33

As % of crypt height 52.5 (10.4) -0.3 (1 1.8) 53.3 (13.5) -5.9 (14.0) 0.89 0.26

“ Two-month follow-up minus baseline differences.h From analysis of covariance, with sex as a covariate for differences in baseline values and in follow-up minus baseline differences: placebo versus calcium.

C Labeled using PCNA immunohistochemistry; only S-phase cells were scored as labeled.

d Proportion of crypt cells that are labeled; only S-phase cells were scored a.s labeled.

� Crypt divided into fifths based on height of crypt by cell count, with compartment 1 being the lowest or basal compartment and compartment 5 being the highest or luminal

compartment.

I Proportion of labeled cells that are in the upper 40% of the crypt; only S-phase cells were scored as labeled.

corresponding to proportional differences of -23% and -26%, group was reduced, proportionately, 10% more than that in the

respectively; thus, the LI in the calcium group was reduced, placebo group (P = 0.85).proportionately, 3% more than that in the placebo group (P

0.91). For the 0h’ the absolute follow-up minus baseline dif- �ferences for the placebo and calcium groups were -0.7% and D1SCU5S1on

- 1.2%, respectively, corresponding to proportional differences This pilot study provides no substantial evidence that 2.0 g ofof - 12% and -22%, respectively; thus, the 0h in the calcium calcium as calcium carbonate daily can normalize either the rate

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Cancer Epidemiology, Biomarkers & Prevention 1025

Table 3 Comparison of ag e-, sex-adjusted colorectal epith dial cell p roliferation measurements in ulcerative colitis versus sporadic aden oma patients

Biopsy measurementSporadic adenoma (n =

M� (SE)

1 19) Ulcerative colitis (n = 28)

Mean (SE)

Proportional

difference” (%)

Pb

S-phase LI’ (%)

All-labeled cell LI” (%)

S-phase #{248}he(%)

All-labeled cell #{248}h1(’�’)

4.3 (0.3)

16.1 (0.8)

5.6 (0.6)

10.1 (0.8)

6.7 (0.6)

31.1 (2.8)

7.8 (1.5)

12.0 (1.9)

56

93

39

19

<0.0001

<0.0001

0.21

<0.0001

“ 100% x � - � cotiu,’Uacienoma) similarly for 0h�

b From analysis of covariance; adjusted for age and sex.

C Proportion of crypt cells that are labeled; only S-phase cells were scored as labeled.

d Proportion of crypt cells that are labeled; all labeled cells were scored as labeled.

e Proportion of labeled cells that are in upper 40% of the crypt; only S-phase cells were scored as labeled.

I Proportion of labeled cells that are in the upper 40% of the crypt; all labeled cells were scored as labeled.

or distribution of proliferating cells in the colon crypts of

patients with ulcerative colitis over a 2-month treatment period.However, it must be emphasized that the sample size was small

and the statistical power limited; thus, the findings may be due

to chance. Other possible reasons for our findings include that

(a) calcium may indeed have no substantial effect on colon cellproliferation in ulcerative colitis patients, because the colon

carcinogenic processes in ulcerative colitis patients differ fromthose in sporadic adenoma patients in ways that involve or do

not involve calcium, and (b) in contrast to our findings foradenoma patients, the calcium dose or duration may have been

insufficient for efficacy. Given the suggestion of a 3% propor-tionalby greater drop in the LI and the 10% proportionately

greater drop in the 0h’ as well as the caveats about sample sizeand chance and calcium dose and duration, the possibility that

calcium may beneficially contribute to a mubtifactorial ap-proach to normalizing coborectal epithelial cell proliferationand reducing the risk of colon cancer in ulcerative colitis

patients cannot be excluded.If the true possible treatment effects were the sizes esti-

mated by this pilot study, a sample size of 718 per treatment

group would have been required to detect the difference in theLI at P � 0.05, and 368 per group would have been required todetect the difference in the 0h#{149} To detect perhaps more mean-ingful proportional differences of 20% in the LI and #{216}�,,the

sample sizes would have needed to be 40 and 89 per group,respectively.

Other findings from this study include: (a) both the LI and

0h in ulcerative colitis patients are, on average, higher thanthose in sporadic adenoma patients; (b) investigating coborectal

epithelial cell proliferation in ulcerative colitis patients in gen-erab, and in clinical trials and using PCNA in particular, isfeasible; (c) the BrdUrd method for labeling proliferating colon

crypt cells in ulcerative colitis is ineffective when used in the

usual manner (the cause of this is unknown); and (d) theinadequate biopsy rate in the present study was 15% (compared

to 5% in our sporadic adenoma patients; Ref. 45), a factor thatshould be considered in deciding on sample sizes for colorectal

epithelial cell proliferation studies in ulcerative colitis patients.The present study has several strengths and limitations.

The most obvious limitation is, as pointed out above, the smallsample size and the consequent increased robe for chance in thelikelihood of detecting (or not detecting) a treatment effect. Thesmall sample size also precluded subgroup analyses. Another

limitation, common to nearby all coborectal epithebial cell pro-liferation trials, is that the only site sampled was the rectum;

thus, treatment effects higher in the colon cannot be ruled out.This is potentially important, because the physiology of the

colon and rectum and the epidemiology of colon and rectalcancer differ (2). The strengths of the study are that it is, to our

knowledge, the first clinical trial of calcium and coborectal

epithelial cell proliferation in ulcerative colitis patients; thehigh bevel of protocol adherence; the strict quality control and

consequent high level of biopsy-scoring reliability; and, per-haps most importantly, the randomized, controlled design.

There have been no previous reports of trials of calciumand coborectab epithelial cell proliferation; however, as re-viewed extensively elsewhere (49), there have been 14 previous

such trials in patients without inflammatory bowel disease whowere at increased risk for colon cancer (32, 39-47, 50-53).The results of these studies are inconsistent with one another:

the majority of the uncontrolled trials found mostly large de-

creases in the LI and 0h’ the small controlled trials yieldedmixed results, and the full-scale controlled trials found small(not statistically significant) proportional decreases in the LI of

about 3%. The full-scale trial in sporadic adenoma patientsconducted by our group concurrently with the present study

found a statistically significant proportional decrease of 100%

in the 0h (45), but a second full-scale trial (47), a trial withmore methodological problems (49), did not.

There have been no previous reports of comparisons be-

tween coborectal epithelial cell proliferation in ulcerative colitis

patients and sporadic adenoma patients, but three small studies(12, 23, 24) compared ulcerative colitis patients with normalpatients. All three studies used tritiated thymidine uptake and

microautoradiography as the S-phase cell labeling method, re-ported the LI but not the 0h’ and did not report biopsy-scoringreliability. One study compared eight normal subjects to four

patients with ulcerative colitis and found a 173% proportion-

ately higher LI in the ulcerative colitis patients (9.5% versus

25.9%; SE and P not given; Ref. 24). A second study comparedfour normal subjects with nine ulcerative colitis patients and

found a 3% proportionately lower LI in the ulcerative colitis

patients (1 1.1% versus 11.4%; SE and P not given; Ref. 23).The third study compared eight normal subjects with seven

ulcerative colitis patients and found a statistically significant168% proportionately higher LI in the ulcerative colitis patients

(9.5% versus 25.5%; SE 1 . 1 and 1 .8, respectively; Ref. 12).Taken altogether, the results of the present and past studiessuggest that, on average, ulcerative colitis patients may have

higher levels of colorectal epithelial cell proliferation than do

normal or sporadic adenoma patients.In summary, the sample size in this preliminary study does

not allow a definitive answer to the question of whether calcium

can reduce coborectab epithelial cell proliferation in ulcerativecolitis patients; therefore, although the results suggest that

calcium may have a modest effect, conservatively, they cannot

be considered to support the hypothesis. This study suggeststhat ulcerative colitis patients have higher levels of coborectab

epitheliab cell proliferation than do sporadic adenoma patients;

on July 16, 2018. © 1997 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from

1026 Calcium Trial in Ulcerative Colitis Patients

confirms the feasibility of colorectal epithelial cell proliferationtrials in ulcerative colitis patients; and points out the futility of

using BrdUrd labeling in ulcerative colitis patients, the feasi-

bility of using PCNA labeling in these patients, the need toconsider a biopsy inadequacy rate of approximately 15%, andthe importance of randomization and control groups in chemo-

prevention trials of colorectal epithelial cell proliferation or

other cancer-related end points.

Acknowledgments

We thank Colleen Forster for help with developmental work to adapt the PCNA

labeling technique for large-scale studies; Patricia Winkels for advice and work

on biopsy procurement and initial handling methods: Bryan Randall for devel-

opment of the computer software necessary to score biopsy slides processed by

the PCNA technique: and the physicians of Digestive Healthcare, PA for advice

and work on biopsy procurement methods and implementing epidemiological

studies at the university-private community interface that measure colorectal

epithelial cell proliferation.

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1997;6:1021-1027. Cancer Epidemiol Biomarkers Prev   R M Bostick, M Boldt, M Darif, et al.   colitis.Calcium and colorectal epithelial cell proliferation in ulcerative

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