Human Studies of Calcium Supplementation and Colorectal ... · ofcalcium consumed is absorbed (3),...

Vol. 6. 971-980. November 1997 Cancer Epidemiology, Biomarkers & Prevention 97/

Review

Human Studies of Calcium Supplementation and Colorectal Epithelial

Cell Proliferation

Roberd M. Bostick’

Department of Public Health Sciences, Epidemiology Section, The Bowman

Gray School of Medicine, Wake Forest University. Winston-Salem, North

Carolina 27157

Abstract

The kinetics of coborectal epithelial cell proliferation(CECP) have been found to be altered in patients atincreased risk for colon cancer. Altered CECP kineticsinclude an increase within the colon crypts of the overallproportion of proliferating cells (labeling index; LI) andthe proportion of proliferating cells that are in the upper40% of the crypts (4h). Use of CECP as a biomarker tomeasure effects of calcium interventions on the colon hasbeen reported in five small uncontrolled clinical trials,nine small randomized placebo-controlled trials, andthree full-scale randomized placebo-controlled trials. Allfive uncontrolled trials indicated substantial andsignificant decreases in proliferation. Of the nine small

controlled trials, three found statistically significantdecreases in the LI, and the remainder were inconclusivebecause of insufficient sample size. Of the three full-scaletrials, one found a decrease in, or normalization of, the

4�h but no effect on the LI; a second found an increase inthe 4�h but no effect on the LI; and the third, which didnot measure the 4�h� also found no effect on the LI.Differences between the two full-scale trials thatmeasured the 4�, were that the negative trial wasmulticentered, used multiple types of bowel-cleansingpreparations, had no baseline measurements of CECP,and had low intrareader reliability for CECP scoring.The positive trial was single centered, used no bowel-

cleansing preparations, measured CECP prior to, and atthree precise intervals after, randomization, and had highintrareader reliability for CECP scoring. The currentliterature indicates that in humans, it is unlikely thatcalcium supplementation can substantially lower CECPrates, but it may normalize the distribution ofproliferating cells within colon crypts.

Introduction

Colon cancer is the second leading cause of cancer deaths inWestern countries and one of the leading causes of cancerdeaths in the world as a whole (1 ). World-wide, average cab-

Received 3/4/97; revised 8/ l/97; accepted 8/5/97.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement in

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I To whom requests for reprints should be addressed, at Department of Public

Health Sciences. Epidemiology Section. The Bowman Gray School of Medicine.Wake Forest University. Medical Center Boulevard, Winston-Salem, NC 27157.

cium intake in modern times has been far lower than estimatedfor that in Paleobithic times when Honio sapiens evolved (wild

plant foods were a high source of calcium, thus providing

1500-2000 mg daily compared to the current American aver-age intake of740 mg; Ref. 2). Considering that only about 30%

ofcalcium consumed is absorbed (3), it is plausible that humans

evolved in an environment in which there was a generally highlevel of calcium in the colon. It is reasonable to speculate that

our modern dietary habits with low calcium intake and lowcalcium bevels in our enteral environments are having modernadverse consequences for cobonic diseases. Indeed. it is biobog-

icably plausible that a higher intake of calcium may preventcolon cancer. If calcium intake is in the range estimated for that

during the Pabeobithic period. then, unlike with the average

amounts consumed currently. there is enough calcium in thecolon left over after absorptive needs and binding with phos-

phate to bathe the colon mucosa with free calcium and to bindwith bile acids (4). These events may be beneficial because (a)

in cell culture, calcium slows cell proliferation and speeds up

cell differentiation, both thought to reduce the risk of cancer (5,

6), and (b) calcium binds bile acids, which have been shown todamage DNA (7) and, in animals, to have toxic effects on colon

epithelial cells, resulting in compensatory cobonic epithelial cellproliferation (8) and promotion of tumorigenesis (9. 10).

Thus, it is biologically plausible that raising calcium in-takes to 1500-2000 mg daily may reduce the risk of coloncancer in humans and that it may reduce risk by mechanisms

involving normalization of rates of cell proliferation and dif-ferentiation in the colon epithelium. Indeed, calcium has been

shown consistently to substantially normalize colorectal epithe-hal cell proliferation (I 1-13) and to reduce colon tumorigenesis

in rodents ( 14-20). However, studies in rodents may or may not

be of direct relevance to humans. In humans. ecological epi-demiobogicab studies show a correlation between per capita

calcium consumption and rates of colon cancer mortality ( 1):

however, these studies are subject to the ecological fallacy. Asreviewed elsewhere ( 1 , 2 1 ), the analytic observational epide-miobogicab literature on the association of calcium and colon

cancer is somewhat inconsistent, but inverse associations havemore frequently been found. However, several factors make it

difficult to draw strong conclusions from these studies. In

general. observational studies are hampered by the relativehomogeneity of diets within populations, the multitude of die-

tary variables, the problem of unmeasured nutrient-nutrientinteractions, and the back of precision in current dietary meas-

ures used in barge population studies. Although the majority ofthe published studies reported associations suggesting de-

creased risk of colon cancer with relatively high intakes ofcalcium, reporting and publication biases cannot be ruled out

(i.e., investigators finding no association may not have submit-ted their findings for publication or they may have been unable

to have them accepted for publicaton because ofjournal editors’back of interest in negative results). Residual confounding in the

reviewed studies also cannot be ruled out. Although the epide-

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972 Review: Calcium and Colon Cell Proliferation

miologicab appearance of colon cancer is different from that of

rectal cancer, many of these studies combined the two forreported analyses, possibly attenuating observed associations.

Furthermore, few of the studies reported taking into accountsupplement intake, a factor that is especially important giventhe homogeneity of diets within most populations. Most of thestudies were reported before the emergence of strong theoret-ical and experimental support for calcium as a potential pro-tective factor against colon cancer and did not focus specificallyon calcium intake; consequently, they did not fully address all

confounding and interaction issues related to current hypothe-

ses regarding this dietary constituent.Thus, although there is animal and epidemiobogical sup-

port for the hypothesis that a higher calcium consumption maycontribute to at least a modest reduction in the risk of colon

cancer, clinical trials will be needed to ultimately fully addressthe hypothesis. However, at this time, the evidence is not strong

enough to support such an expensive undertaking. This is acommon predicament for many hypotheses of cancer etiology

and primary prevention. A recent trend in thought to understandcarcinogenesis is to view it as a mubtistep process occurringover a relatively long period of time (22). Such a view implies

that there may be opportunity for measuring early steps in thecarcinogenic process (e.g., the well-established late “interme-

diate end point” for colon cancer, adenomatous pobyps) andusing their measurements as “intermediate end points” in in-tervention trials. Intermediate end points selected for thesetrials generally should have the properties of having been

validated as true intermediate end points, being more common

than the cancer, being likely to be reversible with the interven-tion, and having been shown to be easily and reliably measured.When an intermediate end point can be treated successfully orreversed in randomized controlled trials, then further support is

provided for whether or not a trial using the same interventionbut with the cancer itself as the end point is warranted. Cancerprimary prevention research using intermediate end points hasbeen most extensively put to use in colon cancer investigations.These investigations have included studies to validate whethervarious measures of coborectal epithelial cell proliferation ki-

netics are valid and reliably measured intermediate end pointsfor coborectal cancer and whether or not they can be modulated

in clinical trials, most of which have used calcium as the tested

intervention.Herein, after a brief description of the basic measurements

of colorectab epithelial cell proliferation kinetics and a briefreview of the evidence of whether colorectab epithelial cellproliferation kinetics are valid intermediate end points for cob-rectal cancer, a full review of human studies of calcium sup-

plementation interventions on colorectal epithelial cell prolif-eration is provided with the aim of assessing whether this lineof study has been explored adequately, and if so, whether itsresults provide the support required forjustifying a calcium andcolon cancer intervention trial.

Basic Definitions of Colorectab Epithelial CellProliferation Kinetics

There are two basic measurements of colorectab epithelial cellproliferation kinetics, one to indicate the rate of proliferation of

colon crypt epithelial cells and the second to indicate thedistribution of proliferating cells within the colon crypts. Themost commonly used methods to indicate these aspects ofcoborectal epithelial cell proliferation kinetics in humans usemeans of labeling proliferating cells and counting the labeled

and unlabeled cells in several colon crypts either in histological

cross-sections or in wet mounts of microdissected whole crypts.From these procedures, the resulting indicator of rate is termedthe LI,2 defined as the proportion of cells in the crypts that arelabeled (23). The most validated measurement of distribution isone that indicates a deviance from the proliferative zone of thecrypt being confined to the lower (basal) 60% of the crypt to

one that extends into the upper (luminal) 40% of the crypt. Thismeasurement has been termed the 4� and is defined as theproportion of labeled cells in the crypt that are in the upper 40%

of the crypt (23).

The most commonly used laboratory methods of meas-

uring colorectal epithelial cell proliferation have been the[3H]dThd method (23), the BrdUrd method (24), the PCNAmethod (25), and the CCPR method (26). In the [3H]dThd and

BrdUrd methods, biopsies are obtained and then incubated in

cell culture media for a specified length of time (e.g., 1 h), sothat cells in S-phase will incorporate the primary labeling agent

(i.e., the [3H]dThd or the BrdUrd). Following histological sec-tioning of the biopsies, the cells containing the labeling agent

(the proliferating cells) are identified as labeled by microauto-radiography in the [3H]dThd method and by immunohisto-chemical methods for the BrdUrd method. In the PCNAmethod, no incubation and incorporation of a labeling agent arerequired; rather, immunohistochemical methods are used to

identify an endogenous nuclear protein associated with the S

phase of the cell cycle. In the CCPR method, whole crypts aremicrodissected out, incubated in media to which vincristine isadded to arrest cells undergoing mitosis in metaphase, and then

stained with Feubgen’s stain to identify the cells arrested inmitosis. Most validation work in humans has been done with

the [3H]dThd method, some has been done with the BrdUrdmethod, a little with the PCNA method, and virtually none withthe CCPR method. The [3H]dThd and BrdUrd methods suffer

from variability of uptake of the labeling agent (27), and the

CCPR method suffers from not providing information on dis-tribution of proliferating cells and from not being validated inhumans. The PCNA method has been criticized for being moredifficult to count, because the method yields cells that are

labeled to varying intensity; however, (a) methods in which thelabeled cells are distinguished as darkly labeled (corresponding

to the S-phase cells) or lightly labeled (peri-S-phase cells) havefound that the dark cell count correlates very highly with, and

parallels, the all-labeled (dark plus bight) cell count; and (b) the

cell proliferation scoring reliability reported for this method has

actually been higher than that for any other method (25).

Coborectal Epitheliab Cell Proliferation as an IntermediateEnd Point for the Study of Coborectal Cancer

Several studies (28-44) have reported that, compared to pa-tients at low risk for colon cancer, patients with colon cancer (4,28-37) and patients in every category known to be at higher

risk for colon cancer [those with a history of sporadic adenoma(28-3 1 , 33-37), familial polyposis (32, 38), or ulcerative colitis

(28, 39, 40); those with a family history ofcobon cancer (32, 33,41); and the elderly (39, 42)], on average, exhibit in their

normal-appearing mucosa both an increased cobonic epithebial

cell proliferation rate and an extension of the colon crypt

proliferative zone from the lower (basal) 60% of the crypt to

2 The abbreviations used are: LI, labeling index; I3HldThd, tritiated thymidine;

BrdUrd, 5-bromodeoxyuridine; PCNA. proliferating cell nuclear antigen; CCPR.colon crypt production rate; BL - FU, baseline minus follow-up; bowel prep.

bowel cleansing preparation.



Cancer Epidemiology, Biomarkers & Prevention 973

Table I Summ ary of uncont rolled clinical trials of calci um supplem entation and colorectal epithelial cell proliferation

Reference, yearCalcium

Patients ndose (g)

Trial design

Biopsy

time points

(months)

Bowel

prep

Laboratory

method”

Readingreliability

(r)

LIproportional

change (%)

4�,

proportional

change (%)

Comments

Lipkin and Newmark Family history 10 1.25 Uncontrolled 0, 2 Tap water l3HldThd -4O�’ -19

(50), 1985 of sporadic

adenoma

enema

Buset et al. (51), Mixed 9 1.5 Uncontrolled 0, 1.5 Tap water [3HldThd -561986 enema

Lipkin et al. (52), Mixed 28 0.6-0.8 Uncontrolled 0. 3-4 Tap water l3HldThd -28 - I I1989 enema

Rozen et al. (53), Mixed 35 1.25, 1.5 Uncontrolled 0, 1, 1.5, None [3HldThd -36” Used two forms of

1989 2-3 calcium

Biopsies also I .5-2

months

posttreatment

O’Sullivan et al. Sporadic 30 1.0, 2.0 Randomized. 0, 1 ? BrdUrd 1.0 g: 47b 1.0 g Pilot; no real control

(54, 1993 adenoma parallel

group

2.0 g: -40” LI4’: -50

LI,”:

2.0 g

LI(: -44”

LI5”: -32”

group

,, [3H]dThd, tritiated thymidine uptake method of identifying S-phase cells; BrdUrd, BrdUrd uptake method of identifying S-phase cells.

S Statistically significant at P � 0.05.� LI4, LI of the fourth quintile of the crypt (the first quintile includes the base of the crypt).

,/ LI5, LI of the upper or luminal quintile of the crypt.

include the upper (luminal) 40% of the crypt. As an example ofthe magnitude of these differences, in sporadic adenoma, pa-

tients as compared to normal controls (across the eight studieshaving at least 10 persons per group), the mean LI was 43%higher (median, 24%), and the mean 4h was 582% higher(median, 94%). In patients with previous colon cancer or spo-radic adenomas, these changes also predict adenoma recurrence(43, 44). In large bowel tumors in humans, an upward shift in

the proliferative zone is found in colon cancers and adenomasbut not in hyperplastic polyps (45). As reviewed elsewhere (5,

6, 46), proliferative changes in normal-appearing mucosa havebeen shown to be a consequence of both cancer-initiating and

cancer-promoting agents; proliferative changes both precedeand accompany cobonic neoplasms in rodents given chemical

carcinogens, and a high-fat diet produces proliferative changesin both rodents and humans. Animal experimental evidence and

preliminary evidence in humans strongly suggest that these two

proliferation abnormalities (hyperproliferation and upward shiftof the proliferation zone) are reversible biomarkers or precur-

sors for colon neoplasia (5, 6, 46, 47). In humans, the twoproliferation abnormalities appear to be independent variables(35, 48), and rectal biopsy findings on both measures reflectthose throughout the colon (37, 49).

Human Trials of Calcium and Coborectab Epithebial CellProliferation

The results of the human trials of calcium and colorectalepithelial cell proliferation are summarized in Tables 1-3. Inviewing these tables, there are several points that should be

kept in mind: (a) the trials in Table 1 are all uncontrolled(50-54), and thus, although they represent seminal works,they should not be weighed as heavily as the controlledtrials; (b) the distinction between the smaller trials (46,55-62; Table 2) and the larger trials (25, 63, 64; Table 3) is

somewhat arbitrary, and it should be kept in mind that whatconstitutes a study of adequate size is related to the magni-tude of the size and clinical importance of the estimatedtreatment effect and the variability in the end point meas-

urement(s); (c) there are differences among the various trials

in the types of patients (some even have mixed-diagnosis

populations). Conceivably, different types of patients may be

affected differently by calcium supplementation; (d) thedifferent studies all used doses of calcium in approximately

the same range, most used calcium carbonate as the calcium

supplement form. and only one (53) deviated from optimal

clinical trial form by using a mixture of different forms anddoses of calcium supplements (i.e., there was not a random-

ization and comparison of the different forms and doses); (e)

the different trials took biopsies for cell proliferation meas-urements at different time points from one another. In the

tables, in the columns summarizing the results, for the stud-ies having more than one follow-up biopsy visit, only the

results involving the final follow-up visit are presented.

Results of studies with more than one follow-up indicate thatany response to calcium may vary by length of treatment,

and results do not stabilize in the first 2 months (25, 63).

Only one study (64), unfortunately one of the larger studies,

deviated from optimal clinical trial form by not having a

baseline (pretreatment) biopsy. A few studies (52, 53, 64),including, unfortunately, the same larger trial that did not

obtain a baseline biopsy (64), deviated from optimal clinicaltrial form by not standardizing the length of time from onsetof treatment to obtaining the follow-up biopsy of greatest

interest; (f) the studies differed on use of bowel preps. At

least some bowel preps have been shown to affect cellproliferation (65). The studies that have suggested that some

bowel preps do not affect measurements (65) have been

small and may not be conclusive. Some studies deviated

from optimal clinical trial form by using different bowelpreps on different patients in the study (60, 63, 64) and even

by using different bowel preps at baseline and follow-up on

the same patients (60, 63). At this point in our knowledge, it

may be safest to assume that the ideal bowel prep forpurposes of measuring cell proliferation is no prep untilproven otherwise; (g) the different studies used different

laboratory methods for assessing cell proliferation. Pros and




of smaller controlled clinical trials of calcium supplementation and colorectal epithelial cell proliferation

.Reference, year Patients

Calcium

n dose

(g)

. .Tnal design

Biopsy. .

time points

(months)

LaboratoryBowel prep

method

Reading. . .

reliability

(r)

LI

proportional

change(%)

proportional

change(%)

Comments

Gregoire et (11. Postoperative 30 1 .2 Randomized. 0, 1 None L3HldThd +36 LI4”: - 16 Pilot

(55, 1989 colon cancer double blind. Ll3�: +50

placebo

controlled

Stem it al. (56). Postoperative 31 1.2 Randomized, 0, 3, 6. 9 None l3HldThd -21 LI4”: -50 Pilot

199)) FAt” double blind, LI5’: +50

placebo

controlled

Barsoum ci al. Spordic 14 1 .25 Randomized, 0. 2 ? CCPR - l8� Pilot; CCPR not

(57, 1992 adenoma double blind,

placebo

controlled,

validated

cross-over

Wargovich et al. Sporadic 20 2 Randomized, 0, I PEG p.ot [3HbdThd 0.88 �‘ Pilot; did not adhere

(58). 1992 adenoma single blind,

placebo

controlled,

cross-over

to intention-to-treat

principles

Thomas et al. Postoperative 28 0.6 Randomized, 0, 6 None CCPR -42W Pilot; did not adhere

(59), 1993 FAt” double blind,placebo

controlled

to intention-to-treat

principles

Bostick ci al. Sporadic 21 1.2 Randomized, 0, 2 None I3HldThd 0.66 +23 LI4”: +66 Pilot

(46. 1993 adenoma double blind, LI5’: 0

placebo

controlled

Cats el al. (60), FH of HNPCC” 30 1 .5 Randomized, 0, 3 PEG pot and/or BrdU Rectum: - 1 1 Rectum: Pilot

1995 double blind,

placebo

controlled

PEG enema Sigmoid: -32

Descending

colon: +8

- I l2�

Sigmoid:

-ll3�

Descending

colon: +8

Weisgerber et al. Sporadic 30 2 Randomized, -3. 9 7 BrdU and +6 LI4”: +2 Pilot;

(61 ), 1996 adenoma double blind,

placeco

PCNA LI5’: -40 Biopsies

from sigmoid at

colonoscopy; 5

patients BrdUrd,

25 PCNA

Bostick et a!. Ulcerative 31 2 Randomized, 0, 2 None PCNA 0.91 -3 - 10 Pilot; PCNA/BrdUrd’

(62). 1996 colitis double blind,

placebo

controlled

“ I’HbdThd. i’HldThd uptake method of identifying S-phase cells; CCPR, crypt cell production rate method of identifying mitotic cells; BrdUrd and PCNA. methods ofidentifying S-phase cells.

1’ LI4. LI of the fourth quintile of the crypt (the first quintile includes the base of the crypt).

‘ LI,. LI of the upper or luminal quintile of the crypt.

,, Postop FAP. has familial adenomatous polyposis and has received colectomy for it.� Statistically significant at P � 0.05.

1PEG, polyethylene glycol.a � , decrease in LI”; numerical value not provided.

‘, FH of HNPCC, family history of hereditary nonpolyposis colon cancer.‘ PCNA/BrdUrd, S-phase cells identified by the PCNA methods, but only after biopsies underwent incubation process, as if they were to be processed for the BrdUrd

method.

cons of the different laboratory methods are discussed aboveat the end of the previous section; (h) very few studies (25,

46, 58, 62, 64) adhered to optimal clinical trial form bysubmitting blinded biopsy slides to assess biopsy scoringreliability. Of those that did, the scoring reliability was goodexcept for, unfortunately, the same larger trial (64) that did

not obtain baseline biopsies; (i) because different trials useddifferent methods of measuring cell proliferation, and sometrials that used the same or similar methods obtained mark-edly different levels of cell proliferation, to standardize the

magnitude of the estimated treatment effect, the results of

the studies pertaining to LI and 4h are summarized as pro-portional change. For the uncontrolled trials, this was de-fined as follows:

BL - FU

lOO%X BL

For the controlled trials, the definition was the placebo group

proportional change in values minus the calcium group propor-tionab change in values. It should be pointed out that some trials(56-60, 63) did not properly analyze their results as in this




Table 3 Summary oflarger controfled clinicaltrials ofcalcium supplementation and colorectal epithelial cell proliferation

Reference. year Patients nCalcium

dose (g)Trial design

Biopsy

time

points

(months )

Bowel prepL4boratory

method”

Readingreliability

(r)

LI

proportional

change

(%)

�,,

proportional

change

(%)

Comments

Armitage et al.

(63), 1995

Sporadic

adenoma

79 1.5 Randomized,

double

blind,

placebo

controlled

0, 3. 12 None or PEG

p.o.”

CCPR - 10 Full scale

Baron et (21. Sporadic 333 1.2 Randomized, 6-9 PEG poP and/ PCNA 0.40 - 30 +90’ Full scale:(64). 1995 adenoma double

blind.

placebo

controlled

or saline or

tap water

enema

multicentered,

no baseline

biopsy

Bostick ci al. Sporadic 193 1.0. 2.0 Randomized, 0, 1, 2, 6 None PCNA LI”: 0.91 -3 -100’ Full scale:

(25). 1995 adenoma double

blind,

placebo

controlled,

parallel

group

4,,’: 0.77 (1 g: -77

2g: -127’)

PCNAIBrdUrd”

“ CCRP. crypt cell production rate method of identifying mitotic cells: PCNA. proliferating cell nuclear antigen method of identifying S-phase cells.h PEG, polyethylene glycol.‘. Statistically significant at P � 0.05.d PCNA/BrdUrd, S-phase cells identified by the PCNA method but only after biopsies underwent incubation process as if there were to be processed for the BrdUrd uptake

method of identifying S-phase cells.

latter difference of differences [i.e., (BL - FU in the placebogroup) - (BL - Hi in the calcium group)], thus defeating the

point in having a control group. Thus, for these trials, it was

necessary to make the calculations from their published data.

Also, for some trials (50-52, 60), it was necessary to calculatethe LI and/or 4�h from data presented in the paper before making

the calculation for the proportional change. This was not alwayspossible for the 4�. In these cases, if possible, the compart-mental LIs for the fourth and fifth (luminal) compartments of

the crypts are presented as an approximation of the 4s�; and (j)in the “Comments” columns of the Tables, some of the com-ments point out potential concerns about the study summarized

on the intersecting row. Some of these concerns were pointed

out above. Additional concerns pointed out are that, to attainstatistical significance, two trials (58, 59) violated the clinical

trial “intention-to-treat” analysis principle; one trial was mul-

ticentered (64), a dubious design at this point for studies using

such a difficult measurement as cell proliferation; and one trial

(25) measured PCNA after incubating biopsies for BrdUrd, thusmaking the results intermediate in quality between those ofPCNA and BrdUrd studies.

Of the five uncontrolled trials (50-54), all suggested sub-

stantial (28-56%) decreases in the LI. Of the three that meas-

ured 4h in some form (50, 52, 54), all suggested substantial(1 1-44%) decreases in the 4h� One of the uncontrolled trials

(53) suggested that when the calcium intervention was stopped,the LI returned to baseline levels. The data of one uncontrolled

trial (54) suggested that there was no difference in response to1.0 g of calcium compared to 2.0 g of calcium (this interpre-

tation of the data, which is contrary to that of the authors’, was

a result of my own calculations that were based on the “differ-

ence of differences” described above). The major problem withthese seminal studies, of course, is that there was no control

group. Other problems with these small, short studies includedno evidence of blinding of treatment assignment to anyone

involved in the studies, and no reports of biopsy scoring reli-ability. The use in some studies of mixed patient groups, bowel

preps, and laboratory methods involving incubation steps were

potential problems.

Of the nine small controlled trials (46, 55-62), six sug-gested [three statistically significant (57-59)] decreases (3-

21%) in the LI, and three (46, 55, 61) suggested (none statis-

ticably significant) increases (6-36%). Of the two that

measured it (60, 62), both suggested decreases in the 4h (10 and1 12%, of which the larger decrease was statistically signifi-

cant). Four (46, 55, 56, 61) of the nine studies reported com-partmental LIs for the upper two quintile compartments sepa-rateby; of these, all yielded ambiguous results. The two studies

that used the CCPR laboratory method did not attempt to studythe distribution of proliferating cells in the colon crypts (57). In

the only trial that has investigated the effects of calcium inportions of the colon other than the rectum (60), small non-

statistically significant decreases in the LI were suggested to

have occurred in the rectum and sigmoid but not in the de-

scending colon, and large statistically significant decreases in

the 4h were found in the rectum and sigmoid but not in thedescending colon. The major problem with these smaller stud-ies, from the standpoint of statistical inference. of course, istheir small size and lack of statistical power. Despite this

problem, as noted, statistically significant reductions in the LIwere found in three (57-59), and in the 4h in one (60); none of

those that suggested increases in either measure of cell prolif-eration had statistically significant results (46, 55, 61). Otherproblems included, in three studies (55, 56, 59), patient popu-

lations composed of persons with colectomies, hardly a situa-

tion that physiologically replicates the average person at risk forcolon cancer; in two studies (57, 59), the CCPR laboratorymethod, an unvalidated method for human studies, was used; in

one study (58), single rather than double blinding was used, andstatistical significance was achieved in only one arm of the

cross-over and then only when the intention-to-treat principle ofanalysis of controlled trials was violated; in one other study(59), the intention-to-treat principle of analysis of controlledtrials was violated also; in one study (60). different bowel preps



0 2 4 6 8

Month

S-Phase

c)

V

Month

Fig. 1. Changes relative to baseline [relative change is presented on a scale like that

of the comparable relative risk or odds ratio (right verticalaxis) and as its equivalent

translation into proportional relative change on a pereentage scale (left vertical axis)l

in rectal mucosa crypt S-phase LI (fraction of crypt cells that are labeled as prolif-

crating) and S-phase 4’,, (fraction ofcrypt cells that are labeled as proliferating that are

in the upper 40% of the crypt) over the course of the Calcium and Colorectal

Epithelial Cell Proliferation clinical trial (n = 193; 25), by treatment group. (There

were no statistically significant differences in LI among treatment groups. Relative to

placebo, on average, the S-phase 4i�, was reduced by one-half(relative effect, 0.50 or

a proportional relative change of 100%) in the combined calcium groups (P = 0.05).

0. placebo; 0 . 1.0 g calcium; 0, 2.0 g calcium.


were used for different patients and for baseline and follow-up

biopsy visits in some patients; in one study (61), biopsies were

performed at cobonoscopy, but the colon prep was not specified;and only three studies (46, 58, 62) reported on biopsy scoringreliability. The use, in some studies, of bowel preps and labo-ratory methods involving incubation steps were potential prob-lems.

Of the three larger controlled trials (25, 63, 64), nonefound statistically significant reductions in the LI, althoughsmall reductions of 3-10% were suggested in all. One trial usedthe CCPR laboratory method and did not address the 4� (63).The other two trials diverged widely in their findings for the 4�h

One found a large. statistically significant reduction (25), and

the other a barge, statistically significant increase (64). Therewere also substantial differences in the designs and reported

quality assurance in these two trials. Differences between thetwo full-scale trials that measured the 4h were that the trialfinding an increase in the 4,, (64) was multicentered, used

multiple types of bowel preps, had bow intrareader reliabilityfor biopsy scoring, had no baseline measurements of cell pro-

biferation, and measured the 4,, once only (from biopsies ob-tamed during a cobonoscopy that occurred anywhere from 6 to9 months after randomization). The laboratory method for

measuring cell proliferation was the PCNA method. The trialfinding a decrease in the 4,, (25) was single centered; used nobowel preps; measured cell proliferation prior to, and at threeprecise intervals after, randomization; and had high intrareader

reliability for biopsy scoring. The laboratory method for meas-

uring cell proliferation was the PCNA method; however, the

immunohistochemical method for identifying PCNA was usedonly after the biopsies were incubated, as for the BrdUrdmethod. Thus, neither trial was ideal; however, the trial show-

ing an increase in the 4,, (64) had substantially more method-obogical problems, some of which potentially substantially aS-fected the results (i.e., the fasting involved with thecobonoscopy bowel preps, the multiple types of bowel preps,the poor intrareader reliability for biopsy scoring, and the lackof baseline end point measurements). Furthermore, the resultsof the smaller controlled trials were more in accord with the

larger trial showing the decrease in 4,,.The data from the larger trial showing a decrease in 4h

(25) are potentially instructive and are summarized in Table 3and in Fig. 1 . As stated earlier, some of the trials were uncon-

trolled, and some of the controlled trials did not take the controlgroup into account in their analyses. The purpose of the control

group is to account for any occurrence of the regression-to-the-mean phenomenon or any extraneous influences that may occur

and that presumably affect the treatment and control groups

equally, such as temporal influences, laboratory drift, or theHawthorne effect (i.e. , participants under the intense scrutinycharacteristic of the clinical trial setting unconsciously changetheir behavior and thus affect their end point measurements).Thus, changes that occur baseline to follow-up in the controlgroup must be subtracted from baseline to follow-up changes in

the treatment group (i.e., calculate a “difference of differences”to find the true treatment effect in the treatment group). The

data from the full-scale trial (see Fig. 1 ) showed that the LI of

the patients in the placebo group dropped just as much as thatof those in the calcium groups. (For an example of how errorsare propagated when differences of numbers, each with errors,are compared, see the tables in Ref. 25.) Without a randomlyassigned control group and using a proper difference of differ-ences analysis, the apparent drops in the LIs in the calciumgroups during the first 2 months of the trial could have beenfalsely interpreted as treatment effects. These results indicate

S-Phase Labeling Index

that the findings of uncontrolled trials of calcium and theoverall cell proliferation rate were likely the result of regressionto the mean and/or the Hawthorne effect. Alternatively, based

on the results of one uncontrolled trial, the authors of that trial




speculated that patients with relatively high proliferation ratesmay be calcium responsive and that those with relatively low

rates may be unresponsive (52). However, in the full-scale trialshown in Fig. 1 , relative changes (calcium versus placebo) in

the LI were similar in both those with high and low prolifera-tion at baseline (data not shown), thus indicating that the

findings of the uncontrolled trial were likely the result ofregression to the mean. In another uncontrolled trial, there was

a drop in the LI 2 months into the study, which returned tobaseline 2 months after cessation of treatment (53). The authorstook this as further evidence of a calcium treatment effect.However, as noted in Fig. 1, the LI of patients in the placebogroup of the full-scale controlled trial dropped as much as those

in the treatment groups during the first 2 months and, as didthose in the treatment groups, rose back to baseline by the next

time point (6 months), thus indicating that the findings of theuncontrolled trial were likely the result of the Hawthorne effect,which was wearing off after the initial months of the study. Not

only do control groups that are properly taken into account inthe analyses prevent falsely seeing treatment effects, but theymay also help prevent missing treatment effects. For example,

as noted in Fig. 1, without a control group, the treatment effecton the 4�h would have been underestimated because the time-related influences producing an increase in the 4h from 2 to 6

months in all treatment groups (seen most prominently in theplacebo group but also to some extent in the two calciumgroups) would not have been accounted for. A final point is that

from a trial without a baseline measurement, it would not beknown whether the treatment and control groups started out at,

on average, the same end point values; thus, differences at

follow-up may merely be a perpetuation of differences at base-

line rather than a treatment effect.

Conclusions

Taking the results of all of the published studies of calciumsupplementation and coborectab epithelial cell proliferation ab-together and considering all their strengths and weaknesses, thedata do not support the hypothesis that calcium supplementa-

tion reduces the LI, or rate of proliferation of the colon epithe-hum, in humans. If calcium does affect the LI in humans, itwould appear that the effect is small (say, on average, in adults

consuming an average intake of calcium, 5%). The data are notconclusive but do suggest that calcium supplementation mayreduce the 4sh (i.e., normalize the distribution of proliferating

cells in colon crypts) and may do so substantially (say, on

average, in adults consuming an average intake of calcium,100%). Ifcalcium affects the 4� and not the LI, then this is stillconsistent with the hypothesis that a higher consumption ofcalcium may reduce the risk of colorectal cancer. Previous

studies (35, 48) have shown that the LI and 4,,, are statisticallyindependent variables, and other preliminary controlled trials

testing other agents hypothesized to normalize colorectal pro-liferative kinetics and to reduce risk of colon cancer have found

reductions in the 4�h without reductions in the LI (66, 67).If calcium normalizes the distribution of proliferating cells

without affecting the overall proliferation rate in humans, themechanistic hypotheses for how calcium affects cell prolifera-

tion in viva and may reduce colon cancer needs to be reexam-med. The first and most cited hypothesis for how calcium mightreduce colorectal cancer risk has been that calcium binds in-traluminal bile acids, thus preventing their toxic effects with

their resultant promotion of compensatory hyperproliferation(4). However, the data provide little evidence for this explan-

atory hypothesis. First, the evidence does not support an effect

on the overall cell proliferation rate. Second, the hypothesiswould predict that, for a person consuming a level of fat and

calcium in the Western-style diet range, the 1 .0-g calcium dose(resulting in a total calcium intake of 1 .5-2.0 g daily) would

have provided as great as an effect as the 2.0-g dose in thelarger trial that found a reduction in 4h#{149} The data, however,were consistent with a greater effect provided by the higherdose. Furthermore, recent reports of studies that have examined

the effects of calcium supplementation on stool bile acids inhumans are inconsistent with one another (68 -7 1 ). These linesof evidence do not rule out a beneficial effect of calcium via

bile acid binding but do suggest that, if this is involved, themechanism is more complex than previously thought.

On the basis of in vitro data showing that calcium directlyaffects the cell cycle, modulating cell proliferation and inducingterminal differentiation, it has also been hypothesized that cal-

cium may exert a similar influence in vivo in the cells of thecolon mucosa (5, 46, 72). The data from the larger trial that

found a decrease in the 4h (25) are consistent with this hypoth-esis. Even when proliferation rates are fast, if differentiation

occurs rapidly as cells migrate up the crypt, they are more likelyto have completed proliferation bower in the crypt. A conse-quence of this may be that, because the DNA of a cell under-going replication is more vulnerable to damage by variousagents and because cells proliferating lower in the crypt may be

less likely to be exposed to injurious intraluminal agents, such

cells may be less likely to be involved in colorectal carcino-

genesis.The mechanisms by which calcium affects cell cycle are

not clear; however, several lines of research indicate that cal-

cium may exert such effects by interacting with cyclic AMP(73), calmodulin (6, 74), tyrosmne kinase (75), and ornithine

decarboxylase (5, 75). In addition, calcium may influence othermechanisms, e.g., cell adhesion mechanisms involving E-cad-hem, a calcium-dependent cell adhesion molecule that inter-acts in complex fashion with the adenomatous polyposis cobi

gene product (76, 77).Although studies in humans have found that cell prolifer-

ation kinetics found on rectal biopsies reflect those foundthroughout the colon (37, 49) and studies in rodents have found

that calcium affects cell proliferation kinetics throughout thecolon (1 1-13), there are insufficient data to assess whether

calcium affects cell proliferation throughout the colon in hu-mans. This remains an important question, especially given that

the epidemiology of rectal cancer appears different in severalrespects from that of colon cancer ( 1).

Another point is that coborectal epithelial cell proliferationkinetics remain unproven but logical and well-supported inter-mediate end points for colon cancer. The existing cell prolif-eration studies in humans therefore cannot prove that, because

calcium may normalize one of the cell proliferation end pointsin the rectum, calcium can reduce the risk of colon cancer; theydo, however, provide justification for additional study of thecalcium-colon cancer association. More work needs to be done

to determine whether calcium supplementation can normalizecolorectab epithelial cell proliferation kinetics in humans. A

clinical trial to test the efficacy of calcium on reducing the

incidence of colon cancer is not justified at this time.

Recommendations

Although it is not reviewed herein, there needs to be an ac-

knowledgment that measuring coborectal epithebiab cell prolif-eration is difficult and susceptible to numerous sources ofvariability due to error (27). More work is needed on quanti-




tating coborectal epithebial cell proliferation. More work isneeded to investigate the effects of bowel preps on coborectalepithelial cell proliferation.

Although it is not reviewed herein, more work is needed to

establish whether coborectab epithebiab cell proliferation is a trueintermediate end point for colon cancer. More work also needs

to be done on the number of crypts that should be counted perperson in clinical trials. One study (46) indicated that approx-imateby eight crypts should be scored in sporadic adenomapatients assessed using [3HJdThd; however, the number may

differ by cell proliferation measurement methodology, the lab-

oratory peforming the measurements, the patient populationunder study, and the sample size.

Improvements are needed in intermediate end point trials

in general and in coborectab epithebiab cell proliferation endpoint trials in particular.

(a) It is acknowledged that uncontrolled seminal studiesare often done on small budgets, are often focused on method-ology, and are legitimate for establishing whether additional,more definitive study is justified. However, such studies shouldprovide little weight in weighing the evidence of whether agiven intervention produces a treatment effect. Studies that

should be given the most weight should be randomized and

controlled, and where possible, double-blinded.(b) End points such as colorectal epithelial cell prolifera-

lion, should be measured at baseline and follow-up.(c) All participants of clinical trials should be treated

exactly alike, and the conditions at end point ascertainmentshould be exactly alike at baseline and follow-up for all par-

ticipants. For example, for coborectal epithelial cell prolifera-lion trials. because bowel preps may affect coborectal epithebiabcell proliferation, either no or only one proven type of bowelprep should be used for all patients at all biopsy visits.

(d) Quality assurance should be a priority in all trials,regardless of their size. A high level of end point (e.g.. cob-rectal epithelial cell proliferation) measurement reliability

should be attained before a trial begins, and intra- (and whereappropriate, inter-) rater reliability should be monitored

throughout the trial by submission of blinded repeat or splitsamples and presented in the final manuscript reporting the

results of the trial. Measures of adherence to treatment andclinical trial protocol should also be monitored and reported.

(e) Extreme caution should be used in attempting to use inmulticentered studies an end point that is so difficult to measure

and that is so sensitive to extraneous influences as is thecoborectab epithelial cell proliferation end point.

(11Proper controlled trial analysis methods should be used.Proper methods for controlled trials with baseline and fob-low-up end point measurements do not include control group

follow-up versus treatment group follow-up comparisons, ortreatment group baseline versus follow-up measurements.

Rather, in principle, the proper method should estimate thetreatment effect by taking the control group BL - FU minus the

treatment group BL - FU and then test this result for statisticalsignificance by comparing the control group BL - FU versus

the treatment group BL - FU. Of course, there are variationsof this. For example. to account for the need to include datafrom trials with multiple follow-up time points in repeated

measures mixed models in which the treatment effects areexpressed as odds ratios or relative effects, the following cab-culation is employed: (treatment group follow-up/treatmentgroup baseline):(control group follow-up/control group base-line). In addition, for the inferential statistical tests used, the

end point measures may need to be transformed to meet theassumptions of normality (25, 64, 78). For example, for cob-

rectal epithelial cell proliferation, the data usually need to betransformed by the natural logarithm before the data are enteredinto the tests of statistical significance (25, 78). Few of thereported calcium and coborectal epithelial cell proliferationstudies even mentioned whether the data were tested to seewhether they met the assumptions for the statistical tests used.

(g) The relationships among sample size, size of the treat-ment effect, and amount of variability in the end point meas-

urements should be kept in mind, both for planning samplesizes for studies and in interpreting the results of studies. A

common problem noted in this review of the human studies ofcalcium and colorectal epithelial cell proliferation was authors

authoritatively, unequivocally stating conclusions of no effectbecause the results were not statistically significant, when in

fact the estimated treatment effect was large, but the statisticalpower quite low due to the small sample size and/or the bargevariability of the LI or 4� (55, 56, 60). Such studies should beregarded as pilot studies that actually suggest that there may be

a treatment effect and that a full-scale trial is justified todefinitively address the tested hypothesis. Current evidence isthat full-scale clinical trials using colorectal epithelial cell pro-liferation as the end point need to be larger than originallyhoped for but still much smaller than trials of cancer or colonpolyp occurrence. For example, it has been estimated that for a

two-group controlled trial using current PCNA methods to havesufficient statistical power to detect a proportional reduction in

LI of 20% at P � 0.05 (recall that across eight validation

studies, the LI was proportionately higher in sporadic adenomapatients than in normal controls by a mean and median, respec-lively, of 43% and 24%), the sample size needs to be about 100

patients per group (79).Another calcium and coborectal epithelial cell proliferation

epitheliab cell proliferation clinical trial is needed. Such a trialshould only be carried out after sufficient improvement in

measurement and validation of coborectal epithelial cell prolif-eration as a true intermediate end point for colorectal cancer.

The trial should be full scale, i.e., with sufficient sample size/statistical power to answer the question, and should use the

suggestions outlined above. It would also be desirable for thetrial to address calcium effects proximal to the rectum as well

as in the rectum and to address mechanisms of action of

calcium on cell proliferation.

Summary

In summary, there are biologically plausible mechanisms ofaction for protective effects of calcium against colon cancer,

and it is plausible that these mechanisms involve normalizationof coborectal epitheliab cell proliferation kinetics. Currently,

animal experimental data are strongly supportive. Epidemio-logical data are inconsistent but, overall, are weakly supportive

as well. A review of the 17 human trials (5 small and uncon-trolled, 9 small and controlled, and 3 larger and controlled) ofcalcium and colorectal epithelial cell proliferation suggests that

calcium supplementation, without affecting the proliferationrate, may normalize the distribution of proliferating cells in the

rectal mucosa. This supports the hypothesis that higher calciumconsumption may reduce the risk of colon cancer. It alsosupports the hypothesis that the possible chemoprotective ac-lion of calcium may not be simply binding bile acids andthereby reducing compensatory hyperproliferation, but it isconsistent with the hypothesis that calcium exerts its possiblechemoprotective effect by directly affecting cell cycle andincreasing rates of cell differentiation. The mechanism(s) bywhich calcium may affect coborectal epithelial cell proliferation




in humans remain(s) to be resolved. Not only do the currently

available data indicate that a causal relationship between cal-cium intake and colorectal cancer incidence, although increas-

ingly supported, cannot be considered established, but theyeven indicate that a calcium and colon cancer prevention trial isnot yet justified. Much work needs to be done to improve themethodology of measuring colorectal epithelial cell prolifera-tion and to validate whether it is a true intermediate end point

for colorectal cancer. The quality of the conduct and analysis ofcoborectal epithelial cell proliferation trials needs to be im-

proved. When these issues are addressed adequately, another

full-scale calcium and colorectal epithelial cell proliferationclinical trial that addresses mechanisms of action and effects inmultiple sites of the colon will be needed.

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