RADIATION RESEARCH 180, 60–69 (2013)0033-7587/13 $15.00
Ó2013 by Radiation Research Society.
All rights of reproduction in any form reserved.
DOI: 10.1667/RR3183.1

Effects of IL-10 Haplotype and Atomic Bomb Radiation Exposure
on Gastric Cancer Risk
Tomonori Hayashi,a,1 Reiko Ito,a John Cologne,b Mayumi Maki,a Yukari Morishita,a Hiroko Nagamura,aKeiko Sasaki,a Ikue Hayashi,c Kazue Imai,a Kengo Yoshida,a Junko Kajimura,a Seishi Kyoizumi,aYoichiro Kusunoki,a Waka Ohishi,d Saeko Fujiwara,d Masazumi Akahoshie and Kei NakachiaaDepartment of Radiobiology/Molecular Epidemiology, Radiation Effects Research Foundation, Hiroshima, Japan; b Department of Statistics,
Radiation Effects Research Foundation, Hiroshima, Japan; c Central Research Laboratory, Hiroshima University Faculty of Dentistry, Hiroshima,
Japan; d Clinical Studies, Radiation Effects Research Foundation, Hiroshima, Japan; and e Department of Clinical Studies, Radiation Effects
Research Foundation, Nagasaki, Japancancer of the diffuse type, and that IL-10 haplotypes may
explain individual differences in the radiation-related risk ofgastric cancer. Ó 2013 by Radiation Research Society

Hayashi, T., Ito, R., Cologne, J., Maki, M., Morishita, M.,
Nagamura, H., Sasaki, K., Hayashi, I., Imai, K., Yoshida, K.,
Kajimura, J., Kyoizumi, S., Kusunoki, Y., Ohishi, W.,
Fujiwara, S., Akahoshi, M. and Nakachi, K. Effects of IL-10
Haplotype and Atomic Bomb Radiation Exposure on Gastric
Cancer Risk. Radiat. Res. 180, 60–69 (2013).

INTRODUCTION

Even now, more than 60 years after the atomic bombings,
rates of certain cancers in atomic bomb survivors are
significantly elevated in a dose-dependent manner (1–4).
Widely accepted mechanisms of radiation carcinogenesis
include direct damage to cellular oncogenes and tumor
suppressor genes, and other late effects by which mutationsof these genes occur in later years (e.g., bystander effects;
genomic instability), eventually leading to malignant
transformation of directly-exposed, surrounding and offspring cells. No definite conclusion has been reachedconcerning whether the immune system is involved in thesemechanisms. In the study of late effects of atomic bombradiation, it is important to elucidate gene-radiationinteraction—the impact of past radiation exposure may
work differently on cancer risks of individuals who havedifferent genetic backgrounds, particularly related toimmune and inflammatory responses.
Some reports suggest that functional polymorphisms ingenes regulating the immune and inflammatory response
may contribute to susceptibility to, and clinical outcome
with, gastric cancer (GC) (5–7). Interleukin-10 (IL-10) is an
important immunoregulatory cytokine mainly produced by
activated T cells, monocytes, B cells and thymocytes. It has
important anti-inflammatory and immunosuppressive activities, including the ability to downregulate T helper 1 (Th1)
cytokine and macrophage costimulatory molecule expression (8). IL-10 has been shown to inhibit various immune
functions, such as antigen presentation, cytokine production, macrophage activation and antigen-specific T-cellproliferation (9, 10). By interfering with antigen-presenting
cells, IL-10 reduces antigen-specific T-cell proliferation. It
has been postulated that IL-10 plays a key role in the

Gastric cancer (GC) is one of the cancers that reveal
increased risk of mortality and incidence in atomic bomb
survivors. The incidence of gastric cancer in the Life Span
Study cohort of the Radiation Effects Research Foundation(RERF) increased with radiation dose (gender-averagedexcess relative risk per Gy ¼ 0.28) and remains high morethan 65 years after exposure. To assess a possible role of geneenvironment interaction, we examined the dose response forgastric cancer incidence based on immunosuppressionrelated IL-10 genotype, in a cohort study with 200 cancer
cases (93 intestinal, 96 diffuse and 11 other types) among
4,690 atomic bomb survivors participating in an immunological substudy. Using a single haplotype block composed offour haplotype-tagging SNPs (comprising the major haplotype allele IL-10-ATTA and the minor haplotype allele IL-10GGCG, which are categorized by IL-10 polymorphisms at
819A.G and 592T.G, +1177T.C and +1589A.G),
multiplicative and additive models for joint effects ofradiation and this IL-10 haplotyping were examined. The
IL-10 minor haplotype allele(s) was a risk factor for intestinal
type gastric cancer but not for diffuse type gastric cancer.
Radiation was not associated with intestinal type gastriccancer. In diffuse type gastric cancer, the haplotype-specificexcess relative risk (ERR) for radiation was statisticallysignificant only in the major homozygote category of IL-10(ERR ¼ 0.46/Gy, P ¼ 0.037), whereas estimated ERR forradiation with the minor IL-10 homozygotes was close to 0
and nonsignificant. Thus, the minor IL-10 haplotype might
act to reduce the radiation related risk of diffuse-type gastriccancer. The results suggest that this IL-10 haplotyping might
be involved in development of radiation-associated gastric
1 Address for correspondence: Department of Radiobiology/Molecular Epidemiology, Radiation Effects Research Foundation, 5-2
Hijiyama Park, Minami-ku, Hiroshima 732-0815, Japan. e-mail:tomo@rerf.or.jp.

60

IL-10 AND RADIATION EFFECTS ON GASTRIC CANCER RISK

oncogenic and metastatic ability of neoplasms (11, 12).
However, a large body of evidence in different animal
tumor models shows that IL-10 can favor immune-mediatedcancer rejection (13–17). The IL-10 gene comprises fiveexons and is located on chromosome 1q31-32 (18). The
promoter region contains at least 40 polymorphic sites
according to dbSNP (http://www.ncbi.nlm.nih.gov/sites/snp). Three polymorphic promoter variants of IL-10, locatedat positions 1082 (A.G), 819 (T.C) and 592 (A.C),
are associated with IL-10 production (19, 20) and gastriccancer risk (21, 22). It has also been reported that IL-10
819 TT genotype is protective against gastric cancer
relative to the CC and TC genotypes in Asians (23) and that
the IL-10 1082 G allele is associated with an increased
risk of cardiac gastric cancer in Asians (24).
Several studies have demonstrated a possible involvementof IL-10 in the pathogenesis of gastric cancer (22, 25–28). A
nested case–control study previously carried out within the
Adult Health Study (AHS) cohort of atomic bomb survivors
at the Radiation Effects Research Foundation, using storedsera and blood cells (29, 30), reported that radiation
increases the risk of noncardia gastric cancer of diffuse
type but not intestinal type (31). The aim of the present
study was to examine the relationship between risk ofintestinal- or diffuse-type gastric cancer and radiation dose
based on inflammation-related IL-10 gene polymorphisms
among atomic bomb survivors. The effects of IL-10
genotype and radiation exposure on plasma IL-10 levels
were also examined.
MATERIALS AND METHODS
Study Population
The Radiation Effects Research Foundation (RERF) conducted acohort study within the AHS of approximately 20,000 atomic bomb
survivors. The AHS is a clinical research program based on biennial
health examinations established in July, 1957 as the clinical subcohortof the Life Span Study (LSS) cohort of atomic bomb survivors in
Hiroshima and Nagasaki, Japan. AHS subjects were selected from the
LSS cohort stratified according to distance from the hypocenter at thetime of bombing and presence or absence of acute symptoms. Subjects
for a broad immunology study including the genome study wereselected from the AHS subjects because blood samples are notavailable from other members of the LSS. The clinical data collectedduring these examinations facilitate long-term follow-up studies of
disease incidence and changes in physiological and biochemical
endpoints, which benefit participants and contribute to healthmanagement of the atomic bomb survivors. Between 1981–2002,
we obtained blood samples from 7,131 AHS participants who visitedthe clinic for examinations as part of the broader immunology study.
After excluding subjects who had a history of first primary cancer at
the time of blood collection, whose radiation dose could not be
estimated, who were exposed in utero (organ doses not estimable),
who were aged 80 or older at the time of blood collection or who
refused to provide informed consent (84 subjects), 4,690 subjects
remained for analysis [3,175 subjects provided informed consent(1,515 subjects who were deceased after blood collection wereapproved for this study by the RERF Ethical Committee)]: we call thisthe ‘‘cancer and immuno-genome (IMG) cohort’’. As a part of the LSS
study, incident cancer cases were detected by the Hiroshima Tumor

61

and Tissue Registries and the Nagasaki Cancer Registries, which also
provided data on histological classification. Histological classificationof gastric cancer was based on the Japanese Research Society for
gastric cancer classification until 1986, and subsequently on the WHO
coding system (ICD-O, ICD-O-2 and ICD-O-3), which was converted
into Lauren’s classification as reported elsewhere (32, 33). Baseline
for follow-up was defined as the date of the first blood sample for the
IMG study (collection began in 1981). The end of follow-up was
December 31, 2001, the latest date of complete cancer ascertainment
as of the time the data analysis was initiated. Follow-up of individual
subjects ended on the date of first primary cancer onset, date of death,or the end of cohort follow-up, whichever occurred first. During thefollow-up period (maximum 21 years), 200 gastric cancer cases (93intestinal, 96 diffuse and 11 other types) were identified.
Baseline characteristics of cases and cohort subjects are shown in
Table 1. We analyzed linkage disequilibrium (LD) with 300 controls,
performed association analysis between IL-10 haplotypes and plasma
IL-10 levels in 644 noncancer cohort members, and conducted risk
estimation for radiation and IL-10 haplotyping using 200 cases amidst
the 4,690 IMG cohort subjects.
Ethical Consideration
This study was approved by the Ethical Committee (Human
Investigation Committee) and by the Ethics Committee for Genome
Research at the RERF.
Measurement of Plasma IL-10 Levels
We measured plasma IL-10 levels in the IMG cohort members
using a highly sensitive enzyme-linked immunosorbent assay kit(Quantikine HS, R&D systems, Minneapolis, MN). The minimum
detectable dose of IL-10 was 0.5 pg/mL. Six hundred forty-four
subjects who did not have a cancer history were randomly selectedfrom the IMG cohort members to exclude an effect of cancer history
on the relationship between IL-10 haplotype and plasma IL-10 levels.
Identification and Genotyping of SNPs
The Celera Genomic database including Asian populations (34, 35)
was used to screen haplotype-tagging (ht) SNPs in the IL-10 generegion, along with the detection of novel SNPs over the region using
the NCBI database. We selected the 19 htSNPs with allele frequency.5% among Japanese. After examining allele frequency in the study
population, we found that ten of the 19 htSNPs showed variant allele
frequencies .5% in our study population. We therefore selected theseten htSNPs: IL-10 92952C.T (IL10-1, rs1400986), 64871T.C(IL10-2, rs2073186), 32510A.G (IL10-3, rs4347211),
23055T.C (IL10-4, rs11583394), 14378T.C (IL10-5,
rs880790), 1082T.C (IL10-6, rs1800896), 819G.A (IL10-7,
rs1800871), 592T.G (IL10-8, rs1800872), þ1177T.C (IL10-9,
rs1518111), and þ1589A.G (IL10-10, rs1554286). To reduce timeand labor, 300 noncancer cohort members from the nonexposed groupin the IMG cohort were analyzed to identify IL-10 haplotypes for these10 htSNPs. Primers and probes for these htSNPs were designed using
Primer Express software, version 2.1 (Applied Biosystems, Foster
City, CA). The TaqMan-Allelic Discrimination method was used forthe detection of SNPs. All of the assays were conducted in 384-well
PCR plates. The principle of TaqMan Real-Time PCR assay systemusing fluorogenic probes and the 5 0 nuclease is described by Livak(36). Amplification reactions (5 ll) were carried out in duplicate with
10 ng of template DNA, 13 TaqMan Universal Master Mix buffer(Applied Biosystems), 300 nM of each primer and 200 nM of eachfluorogenic probe. Thermal cycling was initiated with 2 min
incubation at 508C, followed by a first denaturation step of 10 min
at 958C, and then by 40 cycles of 15 s at 958C and of 1 min at 608C.
After PCR was completed, the plates were brought to room
temperature and read in an ABI PRISM 7900 Sequence Detection

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HAYASHI ET AL.

TABLE 1
Characteristics of the Study Subjects within the RERF Immuno-Genome Cohort and Others
TotalaAge at the time of bombingsb
Age at entryc
Gendera
Men
Women
Citya

Cases

CohortSubjects other than IMG cohort

200 (100)
21 (1–43)
59 (38–80)

4,690 (100)18 (0–43)
56 (37–80)

15,476 (100)33 (0–79)

111 (55.5)
89 (44.5)

1,642 (35.0)
3,048 (65.0)

6,328 (40.9)
9,148 (59.1)

Men

Women

Men

Women

Men

Women

77 (69.4)
34 (30.6)

65 (73.0)
24 (27.0)

1,013 (61.7)
629 (38.3)

2,064 (67.7)
984 (32.3)

4,362 (68.9)
1,966 (31.1)

6,731 (73.6)
2,417 (26.4)

Men

Women

Men

Women

Men

Women

53 (47.7)
27 (24.3)
31 (27.9)

28 (31.5)
32 (36.0)
29 (32.6)710 (43.2)
423 (25.8)
509 (31.0)

1,246 (40.9)
943 (30.9)
859 (28.2)

3,736 (59.0)
1,250 (19.8)
1,342 (21.2)

5,328 (58.2)
2,121 (23.2)
1,699 (18.6)

Smoking statusa

Men

Women

Men

Women

Nonsmoking
Quit smoking
Smoking
Unknown
Histological typee
Intestinal type
Diffuse type
Other types
IL-10 haplotype
Major homozygote
Heterozygote
Minor homozygote
Othersa f

41 (36.9)9 (8.1)46 (41.4)
15 (13.5)

64 (71.9)
3 (3.4)8 (9.0)14 (15.7)

728 (44.3)
233 (14.2)
636 (38.7)
45 (2.7)

2,672 (87.7)
110 (3.6)
190 (6.2)
76 (2.5)

Hiroshima
Nagasaki
Radiation dosea,5 mGy5–728d728

93 (46.5)
96 (48.0)
11 (5.5)
71 (35.5)
93 (46.5)
33 (16.5)
3 (1.5)2,048 (43.8)
1,988 (42.5)
492 (10.5)
145 (3.1)aNumber (%).
Median (range).
cMedian (5–95% percentiles).
d
728 mGy: median dose in exposed cohort members.
e
Gastric cancer cases are classified by intestinal, diffuse and other types (the intestinal type included tubular and papillary carcinomas, thediffuse type included adenocarcinomas, mucinous adenocarcinomas, and signet-ring adenocarcinomas, and the other types were five cases withcancer in adenoma, one with squamous or adenosquamous carcinoma and five not otherwise specified cases). These histological types are counted
individually.
fSeventeen of 4,690 subcohort subjects could not be determined one or two genotypes in the IL-10 haplotype.
b

System (Applied Biosystems). Results were analyzed using the Allelic
Discrimination software (SNPAlyze, Dynacom, Yokohama, Japan).
Haplotype and Radiation Risk Analysis
Rate ratios or excess rate ratios for gastric cancer incidence were
estimated using general risk models extending the standard proportional hazards time-to-event analysis for cohort follow-up data, withattained age as the underlying time axis and left truncation on age at
the time of blood collection (37, 38). Analyses were performed using
the Cox regression module (Peanuts) of Epicure (version 1.81,
Hirosoft Inc., Seattle, WA). All statistical models were stratified on
gender and included adjustment for year of birth, city (Hiroshima or
Nagasaki) and smoking intensity (cigarettes per day). City was
included in the background in all analyses to adjust for any potential
confounders, since IL-10 haplotype-specific risk for radiation may
possibly differ by city due to potential involvement of other gene
polymorphisms that are mechanistically associated with IL-10
functions and differ by city. Information on smoking was obtainedby interview at the time of blood collection. IL-10 haplotypes were
determined as described below. Atomic bomb radiation dose inweighted Gray (wGy) was estimated using the DS02 dosimetry system(39), based on weighted skin dose computed as the gamma dose plus
10 times the neutron dose. A radiation dose ,0.005 Gy was called
nonexposed when performing analyses based on dose group.
Regression analysis of gastric cancer incidence was based on
histological subtype – intestinal or diffuse. Cases in this study wereprimary gastric cancers, which included 17 synchronous multiple
cancer cases that experienced primary cancer(s) of other organs within
one year before or after gastric cancer diagnosis. Censoring was
imposed at the time of other primary cancer diagnosis, death or the endof cancer follow-up (December 31, 2001). An excess relative risk
(ERR: excess rate ratio) model was used for radiation risk estimation
with adjustment for gender using stratification and adjustment forsmoking intensity and year of birth using a log-linear model.
For IL-10 haplotypes, indicator variables for the three categories(major homozygote, heterozygote and minor homozygote) were used
to estimate either the rate ratio (RR, in the case of multiplicativemodels using the standard log-linear Cox model for IL-10 haplotypes)
or ERR (in the case of additive models for the joint effect of
haplotyping and radiation), with the major homozygotes treated as thereference category. In this study, we considered only heterozygotes
involving the most frequent (major) and second-most frequent (minor)
alleles among the numerous heterozygotes that appeared in the process

IL-10 AND RADIATION EFFECTS ON GASTRIC CANCER RISK

63

FIG. 1. Identification of haplotype block. Panel A: 10 SNPs examined in the 95 kb region spanning the IL-10, IL-19 and IL-20 loci (arrows withnumbers from 1 to 10). Panel B: LD analysis of the IL-10 htSNPs. The r2 values between the ten htSNPs are shown, indicating strong LD among
IL10-7, 8, 9 and 10.
of LD analysis. Four genomic models for IL-10 haplotype effects wereconsidered: arbitrary (no structure among the three IL-10 haplotypes;
the ‘‘categorical’’ genomic model); ‘‘linear’’ (a codominant model
where the effect in heterozygotes is assumed to be one-half of theeffect in minor homozygotes); ‘‘dominant’’ (the presence of one or
two minor haplotype alleles confers the same risk); and ‘‘recessive’’(risk occurs only in minor homozygotes). Statistical interaction (effectestimate modification) between radiation and IL-10 haplotypes wastested on both multiplicative and additive scales (40). The multiplicative joint risk model is r(g,d) ¼ edg[1 þ bd] and the additive joint
risk model is r(g,d) ¼ 1 þ /g þ cd, with g being the haplotype (a twoor three-level factor) and d the radiation dose (a continuous variable).
Both models specify a rate ratio r(g,d) that multiplies the backgroundincidence (incidence among nonexposed persons).

RESULTS

Identification of Haplotype Blocks

The ten htSNPs (IL-10-1 to 10) are located on one generegion spanning about 95 kb in length (Fig. 1A). When we
examined all combinations between the ten htSNPs for LD,
four of ten htSNPs formed one haplotype block with r2
values greater than 0.9 (Fig. 1B) and generated two major
haplotype alleles, wild allele IL-10 –ATTA and variant one
IL-10 -GGCG (IL-10-7, 8, 9 and 10). These two
haplotype alleles constituted 98.1% of all haplotype alleles
available in this block, 65.9% for wild IL-10 -ATTA and
32.2% for variant IL-10 -GGCG. Posterior probabilities forthese two haplotype alleles exceeded 0.99, so these
haplotypes were treated as known.
IL-10 Haplotypes and Plasma IL-10 Levels

As these htSNPs are located in untranslated regions
including the promoter regions, we examined the relationship between IL-10 haplotypes and plasma IL-10 levels in
664 cancer-free IMG cohort members. Mean plasma IL-10
protein levels were 2.79 pg/ml (SD 1.85, CV 0.68) among
persons with the major homozygote IL-10 haplotype, 3.02
pg/ml (SD 2.36, CV 0.78) among heterozygotes and 3.69
pg/ml (SD 2.97, CV 0.81) among minor homozygotes.
Using log transformed IL-10 protein levels, regression
analysis revealed no difference in IL-10 protein levels
between major homozygotes and heterozygotes (P ¼ 0.39),
but minor homozygotes had significantly higher levels (P ¼
0.030). Using a linear model for IL-10 haplotype, there wasa significant trend in number of minor haplotyope alleles (P¼ 0.034). Radiation dose was related to log plasma IL-10
protein levels with a relative change in actual protein levels(not log transformed) of e0.1278 ¼ 1.14 (14% increase) at 1 Gy
(P , 0.001). These results suggest that plasma IL-10 levels
varied not only by genetic factors but also by radiation
exposure, and might therefore be closely associated withgastric cancer risk.

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HAYASHI ET AL.

TABLE 2
Relative Risk of Intestinal Type Gastric Cancer for Radiation and IL-10 Haplotypes using a Multiplicative Model
Model

Haplotype RR (95% CI, P value)(log-linear model, adjusted for radiation)

P value is for heterogeneity test withcategorical haplotype or for comparison Major homozygote
with categorical haplotype(AA)
Numbers of cases

Heterozygote
(Aa)

Minor
homozygote
(aa)

41

20

31

Radiation ERR(95% CI, P value)Interaction(adjusted for haplotypes) P value

Categorical haplotype (P ¼ 0.071)(aa) or (Aa) vs. AA[ref]AICb ¼ 910.022

1 (referent)

1.38
2.22
(0.83 – 2.33, 0.21) (1.10 – 4.25, 0.027)

0.05(0.19 – 0.23, .0.5)

NAa

Linear (P . 0.5)aa(1) . Aa(½) . AA[ref]AIC ¼ 908.106

1 (referent)

exp flog(RR[aa])3 ½g ¼ 1.47

0.05
(0.19 – 0.23, .0.5).0.5

Dominant (P ¼ 0.17)(aa,Aa) vs. AA[ref]AIC ¼ 909.950

1 (referent)

0.05(0.19 – 0.23, .0.5)

0.32

0.06
(0.19 – 0.21, .0.5)

0.30

Recessive (P ¼ 0.20)
aa vs. (Aa,AA)[ref]AIC ¼ 909.646

2.16(1.11 – 4.17, 0.024)

1.55(0.96 – 2.53, 0.071)
1 (referent)

1.87(0.98 – 3.30, 0.057)aNA, could not be estimated.
Akaike’s information criteria.

b

The incidence rate ratio of all gastric cancer combined for
women compared with men was 0.41 [95% confidence
interval (CI) 0.28, 0.58; P , 0.001]. Incidence increased
with smoking intensity (RR ¼ 1.24 among smokers of 10
cigarettes/day compared with nonsmokers; 95% CI 1.07,
1.43; P , 0.001) and year of birth (rate increased with more
recent years of birth; P , 0.001). With those adjustments,
crude RRs for IL-10 haplotypes were: 1.26 (ATTA/GGCG
heterozygote; 95% CI 0.91, 1.75; P ¼ 0.17) and 1.59 (minor
homozygote; 95% CI 0.98, 2.50; P ¼ 0.062), withheterogeneity test P ¼ 0.13. The linear genomic model
was statistically significant: risk for the minor homozygote
was 1.60 (95% CI 1.03, 2.48; P ¼ 0.038), with the risk for
the ATTA/GGCG heterozygote being one-half of the risk forminor homozygote on the log scale (i.e., RR ¼
expf[ln(1.60)]/2g ¼ 1.26). The other two types of oneparameter genomic models (dominant and recessive) did not
produce significant effects (P ¼ 0.066 with the dominantmodel, P ¼ 0.13 with the recessive model).
There was no evidence of interaction between gender andIL-10 haplotypes (P ¼ 0.23). Crude (adjusted only forgender, smoking and birth year, but not for IL-10
haplotypes) ERR of all gastric cancers for radiation was
0.11 (95% CI 0.05, 0.34; P ¼ 0.22) and after adjustment forIL-10 haplotypes using the categorical genomic model the
ERR for radiation was 0.12 (95% CI 0.05, 0.36; P ¼ 0.19).
The main effect of IL-10 haplotypes after adjustment forradiation was essentially the same as the crude risk with eachgenomic model. The best fitting genomic model, and only
statistically significant parameter, was with the linear
genomic model. However, the apparent effects of radiation
and IL-10 haplotypes with all gastric cancers combinedreflect large differences in their effects according to subtypes(see below), hence results for interaction between these two
factors with all gastric cancers combined are not reported.
Results for Intestinal Type Gastric Cancer

Crude proportions of cohort members with intestinal typegastric cancer increased with number of variant (GGCG)
haplotype allele: 1.5% among those with the major (ATTA)
IL-10 homozygote, 2.0% with the ATTA/GGCG heterozygote, and 3.9% with the minor homozygote. Incidence wasabout twice as high in Hiroshima as in Nagasaki (RR for
Nagasaki 0.41; 95% CI 0.22, 0.71; P ¼ 0.001) and wassignificantly related to smoking intensity (RR for smoking
10 cigarettes/day 1.38; 95% CI 1.12, 1.66; P ¼ 0.004).
The cohort distribution of haplotypes (major homozygote,
ATTA/GGCG heterozygote, minor homozygote) was similarin the two cities: (0.45, 0.44, 0.11) in Hiroshima and (0.46,
0.43, 0.11) in Nagasaki. Given no apparent association
between city and IL-10 haplotypes, the city difference
would not be expected to confound the IL-10 risk, andindeed there was no noticeable difference in IL-10 risk
parameters depending on whether or not city was includedin the background model. There was no evidence of aneffect of radiation: after adjustment of background incidence for gender, birth year, city and smoking (not adjustingfor IL-10 haplotypes), radiation ERR/Gy was 0.06 (95%CI 0.19, 0.21; P . 0.5).
Tables 2 and 3 show the jointly estimated risks forradiation and IL-10 haplotypes with intestinal type gastriccancer. Main effects for radiation and haplotypes weresimilar with either the multiplicative or the additive model,
so ERRs for radiation and haplotypes in the additive model65

IL-10 AND RADIATION EFFECTS ON GASTRIC CANCER RISKTABLE 3
Excess Relative Risk of intestinal Type Gastric Cancer for Radiation by IL-10 Haplotypes using an Additive Model
Radiation ERR with interaction (95% CI, P value)(radiation ERR specific to haplotypes)

Modelb
P value is for heterogeneity test withcategorical haplotype or comparison
with categorical haplotype (crude risk)

Major homozygote(AA)

Heterozygote(Aa)

Minor homozygote(aa)

Numbers of cases

31

41

20

Categorical haplotype (P ¼ 0.071)(aa) or (Aa) vs. AA[ref]AICb ¼ 910.022

0.13(NA – 0.20, 0.30)

0.25(0.21, 1.00, .0.5)0.58(NAa – NA, 0.25)

0.23

Codominant (P . 0.5)aa(1) . Aa(½).AA[ref]AIC ¼ 908.337

0.11(NA – 0.28, 0.42)—

0.14(NA – NA, NA).0.5

Dominant (P ¼ 0.17)(aa,Aa) vs. AA[ref]AIC ¼ 909.950
Recessive (P ¼ 0.20)
aa vs. (Aa,AA)[ref]AIC ¼ 909.646

0.13(0.21 – 0.20, 0.30)

0.09(0.33 – 0.72, .0.5)

0.02(NA – NA, NA)0.53(NA– NA, NA)Interaction
P value

0.39

0.29

a

NA, could not be estimated.
Akaike’s information criteria.

b

are not shown. With intestinal type gastric cancer, there wasevidence of a significant effect of IL-10 haplotypes after
adjustment for radiation (Table 2). As with all gastriccancers combined, the fit of the categorical genomic model
was similar to that of the linear genomic model, but the RRs
were larger than with all gastric cancers combined. The
linear genomic model had the lowest value of Akaike
information criterion (AIC), and the categorical model
produced risk estimates for the minor homozygote and the
ATTA/GGCG heterozygote that were consistent with thosefrom the linear model (compare expfln[2.16]/2g ¼ 1.47
from the linear model with 1.55 for major heterozygotes).
Although the intestinal subtype evidenced no overall
effect of radiation, it is possible that radiation effects might
exist if there were biologically significant interaction
between radiation and IL-10 haplotypes. It was not possible
to estimate or test interaction between radiation andparticular IL-10 haplotypes with the categorical genomic
model, but none of the estimable radiation ERRs withinteraction in the multiplicative model were very far from 0(not shown) and none of the interaction tests for the oneparameter genomic models produced significant results(Table 2). With the additive model, again there were no
statistically significant interactions (Table 3) and there was
no evidence of trend in radiation ERRs with frequency ofminor haplotype allele (radiation ERR was higher with the
ATTA/GGCG heterozygote relative to the major homozygote but lower with the minor homozygote; Table 3).
Results for Diffuse Type Gastric Cancer

Crude proportions of cohort members with diffuse typegastric cancer evidenced no association with IL-10

haplotypes: 1.9% with the major (ATTA) homozygote,
2.3% with the ATTA/GGCG heterozygote and 2.0% withthe minor (GGCG) homozygote. Incidence of diffuse type
did not depend on city (P ¼ 0.49) and there was no impact
on rate ratios for IL-10 haplotypes depending on whether or
not city was included in the background model. Smoking
was not significantly related to diffuse type gastric cancer:
RR for smoking 10 cigarettes/day compared with nonsmokers was 1.15 (95% CI 0.91, 1.40; P ¼ 0.23). However,smoking was left in the analyses to avoid potential
confounding. The crude ERR for radiation (after adjustment
for gender, birth year, city and smoking but not for IL-10
haplotypes) was statistically significant (ERR/Gy ¼ 0.33;
95% CI 0.03, 0.83; P ¼ 0.027).
Tables 4 and 5 show the jointly estimated risks forradiation and haplotype for diffuse type gastric cancer. With
diffuse type, there was no evidence of an effect of IL-10
haplotypes after adjustment for radiation (Table 4). There
was no significant evidence of interaction between radiation
and IL-10 on either the multiplicative (Table 4) or additive(Table 5) scale. However, the haplotype-specific ERR forradiation was statistically significant only in the major
homozygote category of IL-10 (Table 5), being higher thanthe overall ERR for radiation, whereas estimated ERR forradiation with the minor IL-10 homozygote was close to 0
and nonsignificant. Thus, the minor IL-10 haplotype might
act to reduce radiation-related risk of diffuse type gastriccancer.
DISCUSSION

We investigated the association between gene polymorphisms in the anti-inflammatory cytokine IL-10 gene region

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TABLE 4
Relative Risk of Diffuse Type Gastric Cancer for Radiation and IL-10 Haplotypes using a Multiplicative Model
Haplotype RR (95% CI, P value)(log-linear model, adjusted for radiation)Model

P value is for heterogeneity test with
arbitrary haplotype or for comparison Major homozygote
with arbitrary haplotype(AA)
Numbers of cases

Heterozygote(Aa)

Minor homozygote(aa)

Radiation ERR
Inter(95% CI, P value)
action(adjusted for haplotypes) P value

38

46

10

Categorical haplotype (P . 0.5)(aa) or (Aa) vs. AA[ref]AICa ¼ 1,039.889

1 (referent)

1.15(0.73 – 1.82) .0.5

0.87
(0.35 – 1.84, .0.5)

0.33(0.03 – 0.83, 0.027).0.5

Codominant (P ¼ 0.46)
aa(1) . Aa(½) . AA[ref]AIC ¼ 1,038.444

1 (referent)

expflog(RR[aa])3 ½g ¼ 1.01

1.02
(0.52 – 1.94, .0.5)

0.32(0.03 – 0.82, 0.029)

.0.5

0.33
(0.03 – 0.82, 0.028)

.0.5

0.32
(0.03 – 0.82, 0.029).0.5

Dominant (P . 0.5)
(aa,Aa) vs. AA[ref]AIC ¼ 1,038.338
Recessive (P . 0.5)
aa vs. (Aa,AA)[ref]AIC ¼ 1,038.153

1 (referent)

1 (referent)

1.09(0.71 – 1.71, .0.5)
0.81(0.34 – 1.64, .0.5)

aAkaike’s information criteria.

and gastric cancer risk using a cohort study setting. IL-10
haplotypes were significantly related to the risk of intestinal
type gastric cancer, but not to the diffuse type. On the other
hand, radiation was significantly associated with anincreased risk of diffuse type gastric cancer. Regarding
statistical interaction between radiation and IL-10, there wasa suggestion of a trend towards lower radiation risk of the
diffuse type with an increased number of variant IL-10
haplotype allele, and only the IL-10 major homozygote
evidenced a statistically significant risk elevation byradiation when the radiation ERR was separately estimatedby IL-10 haplotype category. In addition, plasma IL-10
levels among noncancer cohort members were associatedwith IL-10 haplotypes and increased with radiation dose,demonstrating the functional significance of this IL-10
haplotyping and implying a joint effect of genetic factors
and radiation in IL-10 expression.
Although the linear model was the best-fitting genomic
model, we cannot conclude based on statistical criteria that
it is the correct model. Furthermore, four genomic models
were tested, leading to the possibility of spurious findings
due to multiple testing. A Bonferroni correction would
result in reducing the significance level from 0.05–0.0125,
but due to a high degree of correlation among the genomic
models the Bonferroni correction is inappropriate and amultiple-testing correction taking the correlation into
account would not result in much change of significancelevel. We are therefore confident that the effect of IL-10 for
intestinal type gastric cancer based on the linear genomic
model (P ¼ 0.038) is unlikely to be a spurious result.
Observational studies lack power for detecting statisticalinteractions (40), and biological interactions—usually
defined as departure from additive effects (41)—cantherefore go undetected. The advantages of our study are
long-term follow-up, detailed dosimetry reconstruction, anda well-defined radiation-exposed population. A limitation is
the small number of subjects, particularly cases of gastriccancer, due to the size of the original cohort and exclusion
criteria. Thus, lack of statistical significance does notnecessarily imply lack of meaningful biological interaction.
With intestinal type gastric cancer, not only were theinteraction tests not statistically significant, the haplotypespecific radiation ERR estimates did not suggest any
consistent pattern, being higher among the major heterozygotes and lower among the minor homozygotes, comparedwith the major homozygote. Nevertheless, were such apattern biologically significant, it could explain the absenceof overall radiation effect for intestinal type gastric cancer
because the haplotype-specific ERRs would tend to cancel
in the overall (no interaction) radiation ERR estimate. With
diffuse type gastric cancer, there was some evidence of lackof additivity for joint effects of IL-10 and radiation, withradiation risk being high and statistically significant among
the major homozygote but low and nonsignificant among
the minor homozygote. The lack of statistically significant
interaction test in the additive model could be due to lack of
power. Indeed, there was no statistically significant
interaction on either the additive or multiplicative scale,but one or the other must require a statistical interaction
term depending on which is the more appropriate model
representing the joint biological effects (42).
To increase statistical power we included synchronous
gastric cancer cases diagnosed within one year of other,first-primary cancer diagnoses. Although some of thesecases could have been diagnosed through greater surveillance resulting from prior cancer, we think that is unlikely in67

IL-10 AND RADIATION EFFECTS ON GASTRIC CANCER RISKTABLE 5
Excess Relative Risk of Diffuse Type Gastric Cancer for Radiation by IL-10 Haplotypes using an Additive Model
Radiation ERR with interaction (95% CI, P value)(radiation ERR specific to haplotypes)

Model bP value is for heterogeneity test with
arbitrary haplotype or comparison
with arbitrary haplotype (crude risk)

Major homozygote(AA)

Heterozygote(Aa)

Minor homozygote(aa)

Numbers of cases

38

46

10

Categorical haplotype (P . 0.5)(aa) or (Aa) vs. AA[ref]AICb ¼ 1,039.889

0.46(0.02 – 1.43, 0.037)

0.29(0.25 – 1.14, 0.33)

0.12(NAa – NA, .0.5).0.5

Codominant (P ¼ 0.46)
aa(1) . Aa(½).AA[ref]AIC ¼ 1,038.444

0.45(0.02 – 1.33, 0.034)—

0.08(NA – 1.08, .0.5).0.5

Dominant (P . 0.5)
(aa,Aa) vs. AA[ref]AIC ¼ 1,038.338

0.46(0.02 – 1.43, 0.037)

Recessive (P . 0.5)
aa vs. (Aa,AA)[ref]AIC ¼ 1,038.153

0.25(0.23 – 0.97, 0.33)

0.34(0.03 – 0.88, 0.027)

0.10(NA – NA, .0.5)

Interaction
P value

.0.5

.0.5

a

NA, could not be estimated.
Akaike’s information criteria.

bthe case of gastric cancer. It is also unlikely that such cases
are due to treatment for prior cancer because of theinduction and latency periods for cancer growth anddetection. However, we repeated the analyses of Tables
2–5 using as cases only first primary gastric cancers, withmultiple cancer diagnoses (other cancer diagnosed at thesame time as gastric cancer) or nonfirst-primary gastriccancers (gastric cancer diagnosed within one year of priorfirst-primary cancer) treated as censored at the time of firstcancer diagnosis (the same as was done with all other cancer
diagnoses in the analysis). There was no significant impact
on results, except for slightly lower precision as would be
expected with fewer cases. For example, with diffuse typegastric cancer, numbers of first-primary cases were 36, 44
and 10 (versus 38, 46 and 10 in Tables 4 and 5). The
radiation ERR among major homozygotes was the same
(0.45) but with slightly wider confidence interval (0.02–
1.36 vs. 0.02–1.33) and slightly higher P value (0.038 vs.
0.034).
It is difficult to directly compare our results with those ofprevious studies in the atomic bomb survivors (31) because
the nested case-control study that examined subtypes ofgastric cancer involves a separately selected sample of caseswith different follow-up period and the Life Span Study
incidence analyses do not include subtype. Subtype will beconsidered in future LSS analyses but results are not yet
available.
The Gram-negative bacterium Helicobacter pylori (H.
pylori) is a well established etiologic factor, which works toelicit a chronic inflammatory response in the gastric mucosa
and closely relates to the development of intestinal typegastric cancer (43). Differing inflammatory responses

among hosts may help to explain different outcomes for
persons infected with H. pylori, and therefore, considerationof H. pylori infectious status as well as gene polymorphisms
involved in the host response to this infection might help
further estimation of the radiation risks for H. pyloriinfected and noninfected populations. This will be considered in future studies.
Regulatory mechanisms of immune suppression andinflammatory cytokine production inhibition of IL-10 are
not fully understood. However, immune suppression of IL10 may play an important role in the development of gastriccancer. From these results, we assessed the possibility ofmeasurement of surrogate biomarkers, such as plasma IL-10
levels, for real-time estimation of gastric cancer risk as amodel for prevention of gastric cancer. This idea came to us
because the findings suggested that plasma IL-10 level isaffected by both genetic and environmental factors, such asradiation exposure, and is closely associated with gastriccancer risk. We hope that further details of the relationshipbetween gastric cancer risk and biomarkers clarified by
future studies will enable us to better estimate gastric cancer
risk, contributing to innovative preventive measures inatomic bomb survivors as well as other populations exposed
to high levels of radiation in general, on the basis of both
genetic and radiation factors.
ACKNOWLEDGMENTS
The Radiation Effects Research Foundation (RERF), Hiroshima andNagasaki, Japan is a private, nonprofit foundation funded by the Japanese
Ministry of Health, Labour and Welfare (MHLW) and the U.S.
Department of Energy (DOE), the latter in part through DOE Award
DE-HS0000031 to the National Academy of Sciences. This publication

68

HAYASHI ET AL.

was supported by RERF Research Protocols RPs 1–93, 4-04, 5-04, and 3–
09 and was supported in part by the Grant-in-Aid for Scientific Research(B) from the Ministry of Education, Culture, Sports, Science, andTechnology of Japan (24390162), and the Grant-in-Aid for Cancer
Research from the Ministry of Health and Welfare of Japan (22090501).
The views of the authors do not necessarily reflect those of the two
governments.
Received: September 4, 2012; accepted: March 5, 2013; published online:
June 17, 2013

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