2 The abbreviations used are: PGI or PGA, pepsinogen I or pepsinogen
A; PGII or PGC, pepsinogen II or pepsinogen C; Al, absorbance index.
Antibodies to Helicobacter pylon and Pepsinogen Levels inChildren from Costa Rica: Comparison of Two Areas with
Different Risks for Stomach Cancer
R. Sierra, N. Mu#{241}oz,1A. S. Pe#{241}a,I. Biemond,W. van Duijn, C. B. H. W. Lamers, S. Teuchmann,S. Hernandez, and P. Correa
Instituto de Investigaciones en Salud, Universidad de Costa Rica, San
lose, Costa Rica [R. S., S. H.]; Unit of Field and Intervention Studies,
International Agency for Research on Cancer, Lyon, France [N. M.,S. T.]; Department of Gastroenterology, University Hospital, Leiden, theNetherlands [A. S. P., I. B., W. v. D., C. B. H. W. L.]; Louisiana State
University Medical Center, New Orleans, Louisiana 70112-1393 [P. C.]
Abstract
In children and adolescents from two areas of CostaRica with contrasting gastric cancer risks, two factorssuspected to be linked to the natural history of thedisease were tested: serum antibodies to Hellcobacterpylon and serum pepsinogen levels. One hundred fifty-five subjeds from the high-risk area of Turrubareswere compared to 127 from the low-risk area ofHojancha. No significant differences were found in theprevalence of lgG or IgA antibodies to Helicobacterpylon between the two regions. The prevalence of lgGwas 65.8% in the high-risk area and 72.4 in the low-risk area, and that of IgA was 43% in both areas. Thelevels of pepsinogen, especially pepsinogen C, weresignificantly elevated in subjeds with H. pylonantibodies in their serum. The mean levels ofpepsinogen C in those negative, positive, and strongpositive for H. pylon antibodies were 8.7, 14.3, and21.1 ng/ml. These findings suggest that H. pylon-associated gastritis, predominantly of antrallocalization, is very prevalent in Costa Rican childrenand adolescents. Such gastritis might be associated witha high prevalence of intestinal metaplasia and a highgastric cancer risk in the inland, but not the coastalrural populations. H. pyloni may therefore be aninsufficient cause whose role in gastric carcinogenesisis contingent upon the presence of other fadors.
Introduction
Stomach cancer is the most frequently occurring malig-nant tumor in Costa Rica (1), and the mortality rate fromthis tumor is the highest for any type of cancer in theworld at the present time (2). Large intracountry differ-ences in the incidence rates of gastric cancer are reportedin this small country of2.5 million inhabitants and 51,000
km2. The regions with the highest rates of gastric cancer(84.2/100,000 in males) and of precancerous lesions aresituated in the highland area of the center of the country,and those with the lowest rates (25.4/100,000) are in thelowland and coastal areas (1, 3).
Helicobacter pylon infection has been linked tochronic gastnitis, and recent studies have shown that it isalso associated with an increased risk of gastric carcinoma(4, 5). Furthermore, it has been postulated that H. pyloninfection during childhood might be one of the maindeterminants of stomach cancer risk later in life (6).Serum pepsinogen levels reflect the degree of gastricatrophy and therefore have been used as markers ofprecancerous lesions of the stomach (7, 8). Pepsinogenis present in the serum in two immunochemically distinctforms: PGI,2 produced by the oxyntic cells of the bodyof the stomach decrease with increasing degrees ofchronic atrophic gastnitis, and PGII, which originates fromthe gland cells throughout the stomach and from theBrunner’s glands in the duodenum, shows less variationwith gastric atrophy (9).
In order to explore the reasons for contrasting gastriccancer risks in Costa Rica and to gather more informationon the role of H. pylon infection in the early stages ofgastric carcinogenesis, we have examined the prevalenceof IgG and IgA serum antibodies for H. pylon and theserum levels of pepsinogens in a group of children andyoung adults from a rural area with a high incidence ofgastric cancer (Turrubares) and from a rural area at lowrisk for gastric cancer (Hojancha).
Materials and Methods
Two areas were selected for the study: one county inTurrubares with an age-adjusted incidence rate of stom-ach cancer of 49.2/100,000 and another in Hojanchawith an incidence rate of 20.3/100,000 for the period of1984 to 1988. These two counties were selected be-cause, despite having different incidence rates of stom-ach cancer, they were similar in many respects: bothwere rural with a tropical climate (minimum temperature,26#{176}C;maximum temperature, 33#{176}C)and have mestizopopulations of low socioeconomic status, although theproportion of household heads who cultivated their ownland was greater in the low-risk area (37.5%) than in thehigh-risk area (0%). A dietary survey conducted in thetwo study areas revealed similar dietary habits, although
Received 2/26/92.‘ To whom requests for reprints should be addressed, at International
Agency for Research on Cancer, 1 50, cours Albert Thomas, F-69372 Lyoncedex 08, France.
Mean absorbance reading (n = 2) of referen..’e seruni
4 5?) Helicobacter pylon, Pepsinogens, and Gastric Cancer
- niean al)sorbance of blank reading
Table 1 Prevalence o f IgG and IgA-il. py Ion antibodies by age an d study area in Costa Rica
.Study areas
Age group(years)
No. ofsubjects
Anti-IgG Anti-IgA
% positive
(Al > 0.32)
Mein
‘
positive
(Al > 0.35)
‘Mein
‘
High-risk 7-1011-13
14-1617-20All ages
6148
2719
155
60.766.7
70.473.7
65.8
0.460.51
0.540.53
0.50
39.347.9
40.747.4
43.2
0.400.37
0.380.460.39
P (test forlinear trend)
0.22 0.60
Low-risk 7-1011-13
14-16
17-20
Unknown
All ages
365822
10
1
1 27
57.177.680.9
77.8
72.4
0.410.570.65
0.63
(1. 54
30.643.154.5
70.0
4 1. 1
0.290.420.460.47
O.4()
I’ (test orlinear trend)
0.05 ().() 1
the intake of certain nutrients was lower in the high-risk
area. Details of this survey will be published elsewhere’.Two schools that were’ easily accessible’ by car atici
served the less isolated regions of the county were se-lected in each study area. All children attending theselected schools were invited to participate, and over90% of them agreed to participate and were included in
the’ study.Blood samples were’ collected from children and
young adults from the two study areas: 1 53 from thehigh-risk area and 1 23 from the low-risk area. Children7 1 5 years of age were invited to participate at theSc hools, and those for whom consent was obtained fromthe parents (most of those invited) were included in thestudy. Subjects 15-20 years of age were invited to par-ti(’ipate through local leaders and local health workers.A 5- 10-mI blood sample was collected from each partic-iI)lnt. On the day of collection serum samples wereseparated and stored at -20#{176}C. After a few weeks, thefrozen samples were transferred to the Netherlands andanalyzed for I I. pylon antibodies. The analyses for pep-sinogens were done 2 years after collection and storage(it -20#{176}C in the same laboratory.
Anti-H. pyloniAntibodies. IgG and IgA specific antibodiesagainst II. pylon were measured by a modified enzyme-linked immunosorbent assay technique using conjugate’slabeled with immunoperoxidase specific for human IgA1n(I lgG as described by Pe#{241}aet a!, (10). For standardi-
zation of the measurement of these antibodies, test con-ditions were chosen such that the absorbance of thestandard reference serum was 0.5 ± 0.1 for IgA and 1.0± 0.1 for IgG. These values were used to correct the
absorbance given by the sera under study. The resultswere expressed as the Al:
Al =
Mean absonl)ance reading (n = 2) of patient’s serum
- mean absorbance of blank reading
Correlation analysis of the imniunoblotting and en-zyme-linked immunosorl)ent dSScly results showed that�it (1 serum dilution of 1:200 �uid under the conditions
defined above, a seruni with an Al > 0. 32 for IgG anti-I I. pylon W�S positive ‘tE)Ov(.’ the 95% level. A (‘orrelatR)nanalysis between lgG �m(l IgA showed that an Al > 0.353for IgA-anti-fI. pylon W�S positive (ibov(’ the 95% level,
Under these (Oriditiotis the sensitivities (in(l specific-ities of the serologi(’al assay were 89% itid 3�O/,� for lgGand 78% and 88% for IgA when (oriip�ire(l with I I. pylondetected by culture or Warthin-Starry stain (10).
Pepsinogen Levels. Pepsinogen A 1iticl C (PGI and PGII)were measured in duplicate’ by specific sensitive radio-inimunoassays ( 1 1 ). Purified hutitati Pepsinogetis A (111(1
C were used JS standar(l �)reparations. Serum sampleswere diluted 10-fold for Pepsinogen A (PGA) and 5-foldfor Pepsinogen (‘ (PGC) and were incul)at(’d with labeledpepsinogen atid atitibody in a final volunie of 1 ml, for 4(Jays, at 4#{176}C.Free’ pepsinogen was sul)sequetltly sepa-rated from antibody-bound pepsinoge’n by a double-antibody solid-phase’ technique, and tl�’ pellets werecounted in a gamnia scintillation (‘ounter. All sampleswe’re measured in the same assays. Intraassay variationvaried from 3 to 10% for PGA and from 13 to 15% forPGC. The detection limit for the PGA radioimrnunoassaywas 0. 12 .zI/Iiter �1ri(1 for the PGC radioimmunoassay 1 .8
gal/liter. Dilution curves of a seniini saniple containinghigh concentrations of the �)(‘psinogens were parallel tothe standard curve. Aliquots of a suhsariiple of 20 serawere also tested at Dr. Sanlloff’s laboratory rising a radio-immunoassay as dc’scribecl elsewhere (7).
Differences irt the �ieat� values of I I. pylon antibod-ies and pepsinogen levels were coniparecl using the I testafter logarithmic transformation and the Mann-WhitneyV test. Correlation between pepsinogen levels and H.pylon antibodies was tested using the Spearnian’s rank
test.
ResultsIn the high-risk area a total of 65.8% of the subjectstested were positive for lgC� I I. pylcini antibodies (Al >
Table 2 Mean values o f serum PG A and PGC levels (ng/ml) by ag e and study area
Study areasAge
(years)No. of
.sublects Means
PGA
(±SD) Means
PGC
(±SD)
PGA/PGC
Means (±SD)
High-risk 7-1011-1314-1617-20
All ages
61482719
155
35.539.8
39.738.4
37.9
(±11.3)(±13.8)
(±13.6)(±11.8)(±12.6)
13.516.113.412.714.2
(±7.2)(±13.1)
(±6.6)(±7.7)(±9.3)
3.33.23.34.13.4
(±2.5)(±1.5)(±1.3)(±3.0)(±2.1)
Low-risk 7-1011-1314-1617-20
UnknownAll ages
36582210
1
127
34.841.433.847.0
38.6
(±13.6)(±15.0)(±12.9)(±25.8)
(±15.5)
11.015.214.418.1
14.1
(±7.2)(±8.6)
(±11.2)(±12.7)
(±9.2)
4.94.33.64.0
4.3
(±4.9)(±5.8)(±2.2)(±2.6)
(±4.8)
0.32), and 43.2% were positive for IgA antibodies (Al >
0.35). The corresponding figures for the low-risk areawere 72.4% and 43.3%. Table 1 shows the prevalenceof IgG and IgA antibodies to H. pylon by age in the twostudy areas. An increase in the prevalence of H. pylonantibodies with increasing age was observed, but the testfor linear trend for the percentage of positives in the fourage groups was significant only in the low-risk area (P =
0.05 for lgG and 0.01 for gA). The distribution of IgGantibodies in both high- and low-risk areas was clearlybimodal. No significant differences in the prevalence ofIgG and IgA antibodies to H. pylon was found betweenmales and females or between the high- and the low-riskareas for gastric cancer.
Table 2 summarizes the results of the serum pepsi-nogen levels. No differences were observed in the me-dian or log-transformed mean levels of PGA, PGC, andin the PGA:PGC ratio between the two study areas. Inboth areas the distributions of PGA and PGC wereskewed to the right, and no difference between the high-and low-risk area was observed in any of the age groupswith the exception of 7-10-year-old children, in whomPGC levels were significantly higher in the high-risk areathan in the low-risk area (P = 0.022).
The mean levels of both PGA and PGC were signif-icantly higher for males than for females U test for differ-ence between means: P < 0.001 for PGA and P = 0.002for PGC). No correlation between PGA and PGC levelsand age was observed among H. pylon-positive or H.pylon-negative children.
The relationship between the levels of lgG H. pylonantibodies and pepsinogen levels is summarized in Table3. Higher levels of pepsinogens are observed in thosepositive or strongly positive for H. pylon antibodies, andthis tendency was more marked for PGC. A positive
correlation was found between the levels of both van-ables. The mean levels of PGC in those negative, positive,and strongly positive for H. pylon antibodies were 8.7,14.3, and 21.1 ng/ml, respectively.
The values for PGA obtained in Dr. Samloff’s Iabo-ratory were on average twice as high as those obtainedin the Netherlands, but those for PGC were only 10%higher. The correlation coefficient between the measure-ments in the two laboratories was 0.76 for PGA (P <
0.001 ), and for PGC it was 0.96 (P < 0.001).Based on the results of the 20 sera tested in the two
laboratories a correction index was applied to the rest ofthe sera, and the following results were obtained: theproportion of children with low levels of PGA (below 20ng/ml) decreased from 3.6% to 0.7% and those with alow PGA:PGC ratio (<1 .0) decreased from 2.5% to 0.4%.
Concerning the relationship between the levels ofH. pylon antibodies and pepsinogen levels, the propor-tion (percentage) of those negative, positive, and stronglypositive for H. pylon among those with high levels ofPGA changed from 8.0, 18.3, and 26.5% to 64.8, 73.8,and 85%, and these proportions among those with highlevels of PGC changed from 3.4, 18.3, and 42.7% to 5.7,32.5, and 57.4%.
Our pepsinogen Categories were chosen based oncutoff values used in previous studies to identify subjectslikely to be affected by moderate or severe chronicatrophic gastnitis (7). Since the use of these cutoff pointsin our study population might be questionable, we haveplotted the serum values of PGA and PGC against the H.pylon IgG titers of those considered positive, as shownin Fig. 1 . A positive correlation with H. pylon antibodieswas observed for both PGA and PGC, but it was clearerfor PGC (for PGA, r = 0.16, P = 0.025; for PGC, n = 0.36,P< 0.001).
Table 3 IgG H. pylon antibodies and pepsinogen levels
452 Helicobacter pylon, Pepsinogens, and Gastric Cancer
0.0
C
‘C0
1:
Serum pepstnog.n A (log)
Fig. 1. Correlation between serum pepsinogen levels and IgG H. pylonantibody levels.
Discussion
Our results show an extremely high prevalence (over60.0%) of lgG and IgA H. pylon antibodies in childrenand young adults in Costa Rica. We measured both IgGand IgA antibodies because although IgG correlates well
with infection and is more sensitive than IgA, the latterhas been found to be of particular value in the follow-upof patients (12). This finding is in sharp contrast withreports from developed countries at low risk for gastriccancer where H. pylon antibodies are found only excep-tionally in people under 10 years of age and the preva-lence in the age group between 1 0 and 20 years is usuallyless than 20% (13, 14). However, in Costa Rica the highprevalence of H. pylon antibodies was found both inhigh- and low-risk areas for precancerous lesions and forstomach cancer. Since H. pyloni infection has shownintrafamilial clustering (15) we also compared the meanfamily size in both study areas and found no difference;
it was 6.1 in the high-risk area and 6.3 in the low-riskarea. Our results should be regarded with caution since
they are based on a comparison of two areas only. In arecent ecological study conducted in serum samplesfrom 46 counties in China, a significant but weak come-lation was found (n = 0.40; P = 0.02) between theprevalence of H. pylon antibodies and mortality rates forgastric cancer (16).
Recent reports on the prevalence of H. pylon infec-
tion in different populations have pointed out remarkabledifferences between developing and developed coun-tries. The former display a higher prevalence (over 60%)and an earlier age of onset of infection than the devel-oped or affluent countries (13, 14, 17-21). This patternhas led to the hypothesis that an early age of H. pyloninfection is an etiological factor in gastric carcinogenesisand that a lower mate of such infection in children mayaccount for the decline in gastric cancer rates reportedin most developed countries. However, this hypothesis
does not account for some of the international differ-ences in gastric cancer risk. Thus, some developing coun-tries in which the H. pylon prevalence in children andadults is high display high rates of gastric cancer, i.e.,Peru (22), Colombia, China, and Costa Rica (23), butothers, such as Algeria, Ivory Coast, and Vietnam (22),appear to have low rates of cancer.
In addition, the association between H. pylon infec-tion, gastnitis, and gastric cancer is not straightforward:diffuse antral gastritis related to duodenal ulcer is prob-ably caused by H. pylon, but it is not associated with anincreased risk of gastric cancer. In the case of multifocalatrophic gastnitis, a precursor lesion of gastric cancer,other factors in addition to H. pylon are suspected toplay an important etiological role, i.e., excessively saltedfoods and low intake of certain micronutnients such as fi-carotene and vitamin C (17). In this type of gastritis, H.
pylon infection could be another source of chronic irri-tation of the gastric mucosa, inducing excessive cellularproliferation (17, 18) and in this way enhancing carcino-genesis. The role of H. pylon would then be similar to
that of salt (NaCI), which is not a carcinogen in itself butmarkedly enhances the carcinogenesis of genotoxic
agents (24). In Costa Rica, the critical genotoxic damageto the gastric cells might be induced by N-nitmosocom-
pounds. This possibility is supported by a parallel study
carried out in the children who are the subject of thepresent report. After proline ingestion, the excretion ofnitrosoproline in the urine was higher in the childrenfrom the high-risk area than in children from the low-riskarea, indicating a higher potential of in vivo formation of
Our results in Costa Rican children confirm thefindings of high levels of PGA in H. pylon-positive chil-dren with histologically confirmed gastritis as reportedby Oderda et al. (26-27). Although we did not interviewor examine the children for gastrointestinal symptoms, itis presumed that most of them were asymptomatic orhad minor symptoms that did not prevent them fromschool attendance. At the moment the present manu-script was submitted, no publication existed showing astrong correlation of PGC and H. pylon antibody levels.However, subsequently similar findings have been me-ported before and after treatment of H. pylon infection(28) and in Dutch healthy blood transfusion donors (29).
Errors in the measurement of pepsinogen levels areunlikely. PGC levels showed an excellent correlationbetween the U.S. and Dutch laboratories, but the con-relation of PGA was less satisfactory.
It has been shown that H. pylon stimulates pepsi-nogen secretion in vitro independently of toxic damageto cells and probably through mediation of a peptidesmaller than M, 12,000 (30). Thus, although endoscopyand gastric biopsies were not performed in the presentstudy, the serum pepsinogen levels and their correlationwith the H. pylon antibody titers suggest that a highproportion of these children actually had gastritis. Similarfindings have been reported in children with nonspecificabdominal pain (26, 27). The much higher levels of PGCobserved in the present study among those found to bepositive or strongly positive for H. pylon antibodies thanamong the negative subjects suggest that theme is in-creased liberation of PGC as a result of antral gastnitis.
In general, gastric cancer risk is higher in populationswith low levels of blood pepsinogens, especially PGA(31). On the other hand, our and other studies (27)suggest that H. pylon elevates blood levels of PG, espe-cially PGC. This apparent incongruence may be ex-plained by the fact that low PG levels reflect mostlyatrophic and intestinalized mucosa, which is not cob-nized by H. pylon. Anti-H. pylon antibodies are veryprevalent in populations at high gastric cancer risk, eventhough morphological detection of H. pylon in them islow (32). In patients with extensive metaplasia, H. pyloninfection in the nonatrophic mucosa is insufficient toraise PG blood levels.
Our results indicate that Costa Rican children andadolescents have a high prevalence of H. py!oni-associ-ated antral gastnitis. Such gastnitis might be associatedwith a high prevalence of intestinal metaplasia and a highgastric cancer risk in the highlands but not in the coastalmural populations. H. pylon infection could therefore beregarded as an insufficient cause of intestinal metaplasiaand of gastric cancer, whose effect is dependent on thesimultaneous presence of other factors, such as certainvitamin deficiencies and exposure to genotoxiccompounds.
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