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A)!. 2, 7 1 .- 78, January/Ie’bruary 1993 Cancer Epidemiology, Biomarkers & Prevention 71

3 The abbreviations used are: dThd, deoxythymidine; BCH, basal cell

hyperplasia.

A Randomized Double-Blind Intervention Study on the Effectof Calcium Supplementation on Esophageal Precancerous

Lesions in a High-Risk Population in China1

Li-Dong Wang, Song-Liang Qiu, Guan-Rui Yang, MartinLipkin, Harold L. Newmark, and Chung S. Yang2

Henan Institute of Medical Sciences, Henan Medical University,

Zhengzhou, China [L-D. W.. S-L. Q.. C-R. Y.[;Laboratory for Cancer Research, College of Pharmacy, RutgersUniversity. Piscataway, New lersey 08855-0789 [L-D. W., H. L. N.,C. S. Y.[; and Castroenterology and Nutrition Science, Memorial Sloan-

Kettering Cancer Center, New York, New York 10021 [M. L., H. L. N.]

AbstractTo determine whether dietary calcium supplementationaffects esophageal precancerous lesions, 200 subjectswith esophageal lesions in a high-risk area foresophageal cancer in China (Huixian, Henan) wererandomly divided into 2 groups (100 subjects/group).Subjects in one group received an oral supplementationof calcium carbonate tablets (1200 mg of calciumdaily), and subjects in the other group received placebopills for 1 1 months. At the entry and the end of thetrial, esophagoscopy was performed, and 2 or 3 biopsyspecimens were taken from the middle and lowerthirds of the esophagus and from macroscopic lesions,if any, of each subject for histopathology and cellproliferation analysis with deoxythymidine labeling. Incomparison to normal epithelium, increasedproliferative compartment sizt was observed inepithelia with hyperplasia or dysplasia. After theintervention, the percentage of individuals with“normal epithelium,” “basal cell hyperplasia,” “basalcell hyperplasia II,” and “basal cell hyperplasia III anddysplasia” were 44, 31, 13, and 11% in the calciumgroup and 35, 39, 17, and 6% in the placebo group,respectively. The labeling index was 0.046 in thecalcium group and 0.044 in the placebo group. Afterthe intervention, the labeling index in basal cell layers1 to 5, the major zone of cell proliferation, fell 38% inthe calcium group and 44% in the placebo group frombefore the intervention. Therefore, in this study,calcium supplementation was not shown to havebeneficial effects in alleviating precancerous lesionsand abnormal cell proliferation patterns.

Introduction

Carcinoma of the esophagus remains a leading cause ofcancer-related deaths in China, and its incidence rate has

not changed appreciably over the last decade (1, 2).Because of its poor prognosis and the difficulty of early

diagnosis, the importance of exploring practical meansfor the prevention of esophageal cancer is thus apparent(3). Lesions considered as precursors of esophageal can-cen have been described and are shown to be morefrequent and more severe in high-risk than in low-riskpopulations. In persons predisposed to esophageal can-cer, an early indicator of abnormality is increased prolif-eration of esophageal epithelial cells, morphologicallymanifested in basal cell hyperplasia, chronic esophagitis,dysplasia, and carcinoma in situ (4-6). Studies with triti-ated dThd3 labeling of esophageal biopsy specimenshave shown significant differences in the frequency anddistribution of labeled S-phase cells between populationsat high risk and low risk for esophageal cancer (7, 8).

Another feature associated with increased risk foresophageal cancer is a general background status ofnutritional deficiencies which can be due to chronicalcohol abuse, poor dietary habits, or poverty. A poormonotonous diet, mainly composed of corn with a lowintake of animal protein, fruits, and vegetables was foundin high esophageal cancer risk regions in China (1). Stud-ies in such high-risk populations in China revealed a wideprevalence of deficiencies of vitamin A, riboflavin, ascor-bate, zinc, and calcium (9-13). The lack ofcertain micro-nutrients may enhance carcinogenesis (9, 14, 15). Low

dietary calcium intake (300-550 mg daily) was prevalentin the high esophageal cancer incidence regions (ii, 16).In particular, the low calcium content of the diet iscompounded by the intake of large amounts of calciumchelators, such as phytic acid, which accounts for about1% of the dry weight of corn grains (9).

In culture, both rat and human esophageal epithelialcells tend to proliferate in low-calcium (0.1-0.3 mM)medium and to differentiate in high-calcium (1-1.5 mM)medium (17, 18). Markers of calcium-induced terminaldifferentiation include enhanced production of cornifiedenvelopes, cholesterol sulfate, and high-molecular-weight keratins (17, 18). Similar results on the control of

cellular proliferation and differentiation have also beenreported for mouse and human skin keratinocytes andbronchial, gastric, pancreatic, and colonic epithelial cells(19-22). Increasing calcium level in the medium evenresulted in the regression of urinary bladder epithelialdysplasia and hyperplasia to normal in organ culture (23).In animals, oral calcium supplementation was shown todecrease colonic and mammary epithelial cell hyperpro-

Received 6/1 1/92.

‘ Supported in part by NIH Grant CA37037.2 To whom requests for reprints should be addressed at Rutgers Univer-sity, Piscataway. NI 08855-0789.

on June 9, 2021. © 1993 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from

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7.? Calcium Supplementation and Esophageal Lesions

liferation induced by carcinogens (24, 25) and gastricepithelial cell hyperplasia and dysplasia induced by hy-pentonic sodium chloride (26) and calcium deficiency(27).

In patients at increased risk for familial colon cancer,supplementation with calcium has been shown in somestudies to reduce colonic epithelial cell hyperprolifera-tion (28-30). Supplementation with calcium has beenshown to suppress ornithine decarboxylase activity inelderly patients with adenomatous polyps (3 1 ). Althoughseveral mechanisms have been proposed (32-34), themolecular basis for these observations is not known.

As part of a general effort to find effective preventiveagents for esophageal cancer, we carried out a study todetermine whether calcium supplementation could re-duce the prevalence or severity of esophageal lesions inHuixian, a high-risk area of this cancer in northern China.A large-scale intervention study with multiple micronu-tnients (not including calcium) was ongoing in Linxian(35), a county north of Huixian. We report herein thedesign and results of the randomized double-blind pla-cebo-controlled trial with calcium in Huixian.

Materials and MethodsSubjects and Experimental Design

In October 1989 esophageal endoscopic and histopath-ological examinations were performed on 602 subjects

(26 to 65 years old) in Huixian county, Henan province,China. Huixian is a high-risk area for esophageal cancer,with standardized mortality rates of 120.9/100,000among men and 68.0/100,000 among women (4). Of thesubjects examined, 200 subjects identified as havingprecancerous lesions by histopathology were randomlyallocated to two groups, 100 in the calcium interventiongroup and 100 in the placebo group. The code numbersfor the subjects were kept locked at Rutgers Universityand were opened only after all the data collection wascompleted. All the participants, field supervisors, andparticipating investigators were blind to the treatmentassignment. During the study, all the subjects were askedto keep on their regular diet, and each subject receiveda supplementation of 1200 rng of calcium as 6 calciumcarbonate tables/day, 2 after each meal, or of placebotablets for 1 1 months (December 1989-November 1990).The calcium carbonate chewable tablets and placebotabletsi were identical in size and appearance. They weregenerously donated I)y NorclitI Thayer, Inc. (St. Louis,MO). The body weight and height and blood pressurewere measured for some subjects at the beginning andend of the intervention.

Pill Delivery and Compliance Assessment

Each pill bottle contained a 2-week supply of tablets.Two bottles were distributed to each individual every 4

_ .

weeks. Compliance was assessed by pill counting andinterviews. In the regular pill counting, the bottles usedfor the previous 4 weeks were retrieved at the time ofthe new bottle delivery, and the unused pills werecounted and recorded. In the unannounced pill counting,the compliance was checked 2 or 3 weeks after the pilldelivery. At that time, 25-30% of the subjects werevisited at their homes by one of the investigators or tabletdeliverers; the expected and actual pill numbers wererecorderl. In addition, the subjects were interviewedevery 3 to 4 weeks at their homes on in the village’sadministrative office. Any side effects due to the takingof the’ pills or irregularities in pill use were recorded.

Endoscopy, Biopsies, and Physical Examinations

Endos.opic examinations were performed at the entryand the end of the intervention. Two biopsy sampleswere taken at standard positions, one each from themiddle third (30-32 cm from incisor teeth) and the lowerthird (2-3 cm aEIR�)Ve the junction of the esophagus andcardia) of the esophagus. Additional biopsies were takenwhen there were macroscopic lesions. The biopsy Spec-inietis were irnniediately incul)ate(1 with tritiated dThd(5 .zCi/t131) itt a 95% basal nie’diuni with 10% fetal calfserum for 1 h in a shaking bath at 37CC. The biopsieswere theti lixed in 10% buffered forn3alin, embedded in

par�iffin, and sectioned at S pm. Each slide containedse’(tiot’ts fronl the 2 or 3 biopsies obtained from the same



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Fig. 2. Microphotograph of biopsies of esophageal mucosa with BCH. Based on the morphological changes and the expansion of the proliferative

compartments of esophageal epithelial cells, BCH was graded into BCH I (A, X20), BCH II (B, X10), and BCH III (C. X20). H&E.

Cancer Epidemiology, Biomarkers & Prevention 7 1

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subject. Five coded slides were prepared for each sub-ject. One set of slides was stained with hernatoxylin andeosin for histopathological observation. For the dThdincorporation study, another set of slides was dipped inNTB-2 emulsion (Eastman Kodak Co., Rochester, NY), air

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Fig. .3. Mk ropholograph of biopsy of esophageal mucosa with dysplasia.HAE, x20.

Fig. 4. Microphotograph of biopsy of esophageal mucosa with BCH andimcreased dThd-Iabeled cells (arrow). H&E, x20.

74 Calcium Supplementation and Esophageal Lesions

Criteria for Histological Diagnosis of EsophagealLesions

In histopathological analysis, the esophageal epitheliawere graded according to the cellular morphologicalchanges and tissue architecture. The criteria were asfollows.

Normal. The nonkeratinized stratified squamous epithe-hum consisted of three zones, i.e. , basal, intermediate,and superficial (Fig. 1 ). The basal cells were characterizedby a round nucleus, fine chrornatin, without mitosis, andwere usually composed of 1 -3 cell layers. The interme-diate zone was composed of 5-10 layers of larger poly-hedral spinous cells with abundant pink cytoplasm,round nucleus, and sparse chromatin. The superficialcells were flatten and had a nucleus of dense chromatin.The papillae usually I)enetrate no more than the lowerhalf of the epithelium. The inflammatory cells were rareboth in the larnina propnia and epitheliurn.

Basal Cell Hyperplasia. Both the cell volume and celllayer number increased in basal cell hyperplasia (Fig. 2).The cell nuclei were often enlarged, with prominentnucleoli. Mitosis figures were not uncommon. However,the cell polarity and normal maturation were still main-tamed. Based on the expansion of the proliferative corn-partments of esophageal epithelial cells, the BCH wasgraded into: BCH I (3-5 layers of proliferating basal cell;Fig. 2A); BCH II (more than 5 layers but less than one-half of the whole epitheliurn of proliferating basal cell;Fig. 2B); and BCH Ill (hyperproliferative basal cells ac-count for more than one-half of the whole epithelialthickness; Fig. 2C).

Dysplasia. The dysplasic cells usually involved part of theepithelium and were characterized by hyperchromaticnuclei with increased and variable size, by loss of cellpolarity and maturation, and by active mitosis (Fig. 3).The basal membrane was intact.

Evaluation of Histopathological Changes

The subjects were diagnosed based on the most severelesions and classified into four grades, i.e., normal, BCHI, BCH II, and BCH Ill and dysplasia. Based on thehistopathological diagnosis before and after intervention,the effects of the trial were evaluated as improved,

unchanged, and deteriorated. “Improved” refers to sub-jects with epithelial lesions improved by two grades afterintervention; e.g., “BCH Ill and dysplasia” returned to“normal” or “BCH I,” or “BCH II” returned to “normal.”“Deteriorated” means the lesions became more severeby at least one grade; e.g., “BCH I” developed into “BC HII.’, With a few samples, a reasonable diagnosis could notbe made, because of an inadequate amount or inappro-pniate handling of the biopsy samples, and these aredesignated as “uncertain.”

Assessment of the Cell Proliferation

For assessing the cell proliferation, a set of slides werecoded and analyzed, first under low power to select atrandom well-defined flat negions of the esophageal epi-thelium (Fig. 4) and then under oil immersion to countthe epithelial cells. For the recording of dThd labelingversus cell position in the epitheliurn, the basal epithelialcells between the two good orientation papillae weresegregated into cell columns oriented perpendicularly tothe basal cell layer. In each cell column, cells werecounted proceeding from the bottom of the basal layertoward the esophageal lumen. For each subject, a totalof 200 consecutive cell columns were recorded, skippingover the papillae. The detailed criteria for recordingdThd-labeled cells were the same as those previouslydescribed (7, 8, 36).

Serum Laboratory Tests

Blood samples (5 ml) were collected from 100 subjectsbefore the intervention and 84 subjects after the inter-vention. The samples were analyzed for blood calcium,cholesterol, triglycerides, low-density lipoprotein choles-terol, and high-density lipoprotein cholesterol.

Statistics

The data were expressed as the mean ± SE unless oth-erwise stated. Statistics of labeling index values werecomputed by averaging those values with equal weightsover the members of each group, by computing thecorresponding SD and SE, and subjected to unpairedtests (36). The x2 test was used for histopathologicalvalues (P < 0.05 was considered significant).



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Cancer Epidemiology, Biomarkers & Prevention 7.5

Table 1 Age and sex distribution of subjects in the calcium and theplacebo gnoups

Calcium group Placebo groupAge

(years) No. of � No. of 0/subjects #{176} subjects

Male30-39 14 33 17 34

40-49 18 42 16 32

50-59 9 21 12 24

60+ 2 5 5 10

Total 43 100 50 100

Female30-39 13 33 12 36

40-49 17 44 15 45

50-59 5 13 2 6

60+ 4 10 4 12

Total 39 100 33 100

Table 2 Histopathological results before and after the intervention trial

Calcium group Placebo group

. Before . BeforeLesions . After trial . After trial

trial trial

n (%) n (%) n (%) n (%)

MaleNormal 0 (0) 13 (30) 0 (0) 15 (30)

BCH I� 8 (19) 16 (37) 16 (32) 19 (38)BCH II 28 (65) 8 (19) 23 (46) 10 (20)

BCH III and dys. 7 (16) 5 (12) 1 1 (22) 3 (6)Uncertain 0 (0) 1 (2) 0 (0) 3 (6)

Total 43 (100) 43 (100) 50 (100) 50 (100)

FemaleNormal 0 (0) 23 (59) 0 (0) 14 (42)

BCH I 18 (46) 9 (23) 11 (33) 13 (39)BCH II 16 (41) 3 (8) 16 (49) 4 (12)

BCH III and dys. 5 (13) 4 (10) 6 (18) 2 (6)Uncertain 0 (0) 0 (0) 0 (0) 0 (0)

Total 39 (100) 39 (100) 33 (100) 33 (100)

a BCH I, basal cell hypenplasia I; BCH II, basal cell hyperplasia grade II;

BCH III and dys. basal cell hyperplasia III and dysplasia.

Results

General Observations. After the intervention period, 165of the 200 subjects participated in the final endoscopicbiopsy examination. Reasons for nonparticipation weretemporary absence from Huixian, illness, or refusal.There were two deaths, one from hepatic cirrhosis andanother from an accident. Of the 35 subjects who didnot take the final endoscopic examination, 18 were fromthe calcium group and 1 7 from the placebo group, andthese two groups had similar distributions of subjects interms of sex and severity of esophageal lesions. Of the165 subjects who participated in the final endoscopicexamination, 82 subjects were in the calcium group and83 subjects in the placebo group. The two groups weresimilar with respect to sex and age distribution (Table 1)and body mass index. Compliance for both groups wasgood; more than 90% of the subjects took at least 85%of the allocated pills in each 4-week period. No side

Table 3 Evaluation of histopathological results of subjects in the

calcium and the placebo groups after the intervention

tmpno- Un- Deteni- Uncer- T � I

ve& changed orated tam 0 a

n (%) n (%) n (%) n (%) n (%)

Calcium groupMale 13 (30) 24 (56) 5 (12) 1 (2) 43 (100)

Female 12 (31) 24 (62) 3 (8) 0 (0) 39 (100)

Total 25 (30) 48 (59) 8 (10) 1 (1) 82 (100)

Placebo groupMale 10 (20) 32 (64) 5 (10) 3 (6) 50 (100)Female 8 (24) 24 (73) 1 (3) 0 (0) 33 (100)

Total 18 (21) 56 (67) 6 (7) 3 (4) 83 (100)

a The definitionsof”irnproved,” “unchanged,” “deteriorated,” and ‘un-

certain” are described in Matenials and Methods.”

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Esophageal Cell Laye� Nw�h”

Fig. 5. Labeling index profiles of esophageal epithelium by cell layers in

the calcium group and the placebo group before and after calciumintervention trial. Points, mean; bars, SE. �-O, calcium group before trial)n = 77); #{149}-- - -#{149},placebo group before trial (n = 70); 0-0, calciumgroup after trial (n = 77); 0- - - -0, placebo group after trial )n = 70).

effect due to the supplementation of the calcium tabletswas observed.

The histological slides were read blind independ-

ently by two pathologists. The two pathologists reexam-med together the slides (about 10% of the total slides),which initially received different diagnosis from themand reached a consensus on the diagnosis. After obtain-ing the final diagnosis based on histopathology and ed-iting the results on cell proliferation patterns, the codefor the treatment assignment was broken.

Analysis Based on Histopathology. After the interventiontrial, the most clear-cut change was that many of thesubjects previously diagnosed with esophageal lesionswere diagnosed as “normal” (Table 2). The proportionsof subjects diagnosed as “normal” were 36 of 82 or 44%(30% in males and 59% in females) in the calcium group;and 29 of 88 or 35% (30% in males and 42% in females)in the placebo group. This was accompanied by a de-crease in the number of subjects classified as BCH II,observable in males and females in both the calcium andthe placebo groups. After the intervention, the numberof subjects diagnosed as BCH I and BCH III and dysplasiawere changed somewhat due to the aforementioned



Esophageal Cell Laye’ Numbe’

Esophageal Cell Lays’ Numbs’


TaI)le -3 Cell prolif eralion in e’sophage al epithelium in individuals befur e and after calcium intervention

Calcium group Placebo group

Before trial After trial Before trial After trial

No. of

Suhle’ Is studied 77 77 70 70Ce’II columns assayed 15,400 15,400 14,000 14,000

Epilhe’IiaI o’Ils counte’d 154,000 154,000 140,000 140,000I ,il)(’I(’d ( e’IIs foui�l 1 1 .507 7, 1 1 9� 1 1 040 6,141’

Labe’Ie’d ((‘lIs/eo)Iunln )me’an ± SE) 74.7 ± 2.4 46.24 ± 4.2#{176}’ 78.9 ± 3.4 43.9 ± 2.3�

% cells labeled’ for cell layer

1 17.1 ± 1.0 13.2± 1.0” 18.9± 1.1 11.32 36.0 ± 0.9 23.8 ± 1.3” 36.0 ± 1.0 24.6 ± 1.2k3 15.1 ± 1.0 5.6 ± 1.1#{176}’ 16.2 ± 1.2 5.7 ± 0.9”4 4.5±0.7 2.1 ±0.9” 4.5±0.8 1.4±0.5”S 1.3±0.3 1.1 ±0.6 1.8±0.5 0.6±0.2”

6 0.4 ± 0.1 0.4 ± 0.3 0.9 ± 0.3 0.2 ± 0.1’7 0.2 ± 0.1 0.1 ± 0 0.4 ± 0.2 0.1 ± 08 0.1 ± 0 0.0 ± 0 0.2 ± 0.1 0.0 ± 0

a Significantly different froni the’ pretrial results, with P < 0.001 by the x2 test.h Significantly different from the’ pretrial results, with P < 0.001 by unpaired t test.

‘ Percentage’ of C ells labeled (labeling index X 100).

‘I Significantly different from the’ pretrial results, with P < 0.01 by unpaired t test.� Significantly (lifferent from the? pretrial results, with P < 0.05 by unpaired t test.

fig. 6. Histopathology and labeling indices from the subjects before andafter intervention. The subjects from the calcium and the placebo groupswe?re analyzed together. Points, mean; bars, SE. A, before trial. �-&

BCH I In = 46); #{149}-#{149},BCH II )n = 78); 0-0, BCH III and dysplasia (n= 23). B, after trial. A-A, normal )n = 59); � BCH I (n = 53);.- -., BCH II In = 23); 0-0, BCH III and dysplasia )n = 12).

redistribution of subjects into different categories; but nosignificant difference between the calcium group and theplacebo group was observed (Table 2). Evaluation ofhistopathological changes showed that after the trial theproportion of “improved” cases in the calcium group was

somewhat higher (30%) than that in the placebo group(21%). However, the difference was not statistically sig-nificant (P > 0.05; x2 test). The figures for “unchanged”or “deteriorated” were similar in both groups (Table 3).Concerning the effect of intervention on more severelesions, 6 of 12 subjects with “BCH Ill and dysplasia”returned to normal in the calcium group (50%), in com-panison to 4 of 1 7 (23%) in the placebo group (data notshown). However, these numbers were too small toindicate a statistical significance.

The dThd Labeling Index. The labeling indices in differentcell layers are presented in Fig. 5. The proliferative corn-partments of the esophageal epitheliurn were chiefly

located in basal cell layers 1 to 5. The dThd cell labelingpatterns in the calcium group (n = 77) and the placebogroup (n = 70) were similar before intervention. After theintervention, fewer cells were labeled, but a differencebetween the calcium and the placebo groups was notobserved. The labeling indices of cell layers 1 -5, totallabeled cells found in the columns assayed, and labeledcells in the cell layers of 1 -8 were significantly lower

after the intervention than before the intervention inboth the calcium group and the placebo group (Table 4).The labeling index fell 38% in the calcium group and44% in the placebo group in basal cell layers 1 to 5.

Fig. 6 shows the labeling index profiles in tissueswith different pathology, i.e., normal, hyperplasia, and

dysplasia before (Fig. 6A) and after (Fig. 6B) calciumsupplementation. There was a gradient of increased ex-pansion in the proliferating cells, progressing from normalto BCH I, BCH II, and BCH III and dysplasia. The prolif-erating patterns observed in the calcium group and theplacebo group were similar, and therefore data from bothgroups were shown together in Fig. 6.



Cancer Epidemiology, Biomarkers & Prevention 77

Blood Calcium, Lipid Analysis, and Blood Pressure.Serum calcium, lipids, and blood pressure were meas-uned before and after calcium supplementation. Theserum calcium was 8.7 ± 0.7 mg/100 ml (mean ± SD;n = 46) in the calcium group and 9.0 ± 0.7 mg/100 ml(n = 40) in the placebo group before intervention; afterintervention, the serum calcium was 10.4 ± 1.0 mg/100ml (n = 46) and 10.2 ± 1.1 mg/100 ml (n = 40). Althoughthe calcium levels in the calcium-supplemented groupwere slightly increased (P < 0.05) after the intervention,we do not know whether this was a real increase or dueto variations in laboratory analyses, because the levels inthe placebo group also appeared to increase (P > 0.05)after the intervention. The diastolic and systolic bloodpressures in seated position were 86 ± 13 and 131 ± 18mrnHg (mean ± SD; n = 39), respectively, in the placebogroup. Similar values were found in the calcium group,

and the calcium supplementation did not change theblood pressure. Serum lipids were also not affected by

the supplementation.

Discussion

After the supplementation period, there were substantialdecreases in cell proliferation in the placebo group (44%)and in the calcium group (38%). However, no apparentdifference between the calcium and the placebo groupswas observed in this study. Although it appeared thatmore subjects in the calcium group showed improve-ment (30%) than did the placebo group (2i%) based onhistopathology (Table 3), the results are not statistically

significant.The most intriguing observation of this study is that

a large number of subjects diagnosed with lesions re-verted back to normal after the intervention period inboth the calcium group (44%) and the placebo group(35%) (Table 2). The placebo tablets contained starch

and small amounts of flavoring agents4 which are notbelieved to have any significant effect on esophageallesions. Hyperplastic and dysplastic lesions of the esoph-agus are known to be unstable; they may revert back tonormal, remain the same, or become more severe (1, 4).One possibility is that there was indeed a high rate ofreversion. Another possibility is that the observed differ-ence is due to the biopsy sampling procedure. Becausethe hyperplastic and dysplastic lesion sites cannot beobserved when taking the biopsies, there was a possibil-ity for taking normal tissues instead of tissues with lesions.If normal tissues were taken instead of tissues with lesionsin the pretrial examination, these subjects were screenedout (they were not included in the study); whereas in theexamination after the intervention period, the subjectswere recorded as “normal” and thus produced a falsenegative result. Assuming a false negative rate of 40%,we would expect 40% of the subjects to be diagnosed“normal” even when the intervention agent was ineffec-tive. This appears to be what we have obtained. Thisobservation demonstrates the importance of having aplacebo control group in intervention studies.

There are no reliable data on the reversion rates ofesophageal hyperplasia and dysplasia. Assuming a never-sion rate of 35%, our current study would have a 73%power to determine a 20% difference between the cal-cium group and the placebo group. Based on the resultsfrom histopathological and cell proliferation analyses, we

conclude that the 1 1 months of intervention with calciumdid not produce the expected beneficial effects on theprecancerous lesions of the esophagus. The results, how-ever, cannot exclude the possibility that low calciumdietary intake is a contributing factor to esophageal can-cer. In order to further assess this factor, interventionstudies with larger number of subjects and improvedsampling techniques for a longer period of time than thepresent study are needed. The interaction of calciumwith other nutritional factors, especially vitamin D,should also be an important concern for such studies.

AcknowledgmentsThe authors would like to acknowledge Norcliff Thayer, Inc., for their

generous contribution of calcium carbonate pills for this intervention trial.The authors would like to thank Qi Zhong Zhao, Kiu lin. Jian Guo Zhang,

and Ji Yang (Meng Zhuang Xiang, Huixian) and Cuo Rong Li, Cuo BaoLiu, and Guo Ying Li (Zheng Tun Village, Huixian) for their help in

organizing this trial; Drs. Shan Liang Feng and Bao Mi Huang (Zheng TunVillage, Huixian) for delivering the tablets; Dr. Ling Wang and Shu Li

Wang (Henan Medical University) for blood calcium and lipid analyses;Drs. Hua Qin Cuo, Yu Shui, Li Qun Zhao, lu Zhen Zhang, and Shan Li)Henan Institute of Medical Sciences, Zhengzhou) for field work; and Dr.

Wen Cheng You (Beijing Institute for Cancer Research, Beijing) and Dr.Sandra Mohr (UMDNJ-Robert Wood Johnson Medical School) for helpfuldiscussions.

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1993;2:71-78. Cancer Epidemiol Biomarkers Prev L D Wang, S L Qiu, G R Yang, et al. in a high-risk population in China.calcium supplementation on esophageal precancerous lesions A randomized double-blind intervention study on the effect of

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