Hormonal Markers and
Hepatitis B Virus-Related
Hepatocellular Carcinoma
Risk: a Nested Case–Control
Study Among Men
Ming-Whei Yu, Yu-Ching Yang,
Shi-Yi Yang, Shu-Wen Cheng,
Yun-Fan Liaw, Shi-Ming Lin,
Chien-Jen Chen
Background: The incidence ofhepatitis
B virus (HBV)-related hepatocellular
carcinoma (HCC) is higher in men than
in women. We examined whether
en-dogenous sex hormone levels or
hor-mone-related factors might affect the
risk ofHCC in men. Methods: Baseline
blood samples were collected from 4841
male Taiwanese HBV carriers without
diagnosed HCC from 1988 through
1992. Plasma testosterone and estradiol
levels and genetic polymorphisms in
the hormone-related factors
cyto-chrome P450c17
␣ (CYP17, A1 versus
A2 alleles), steroid 5
␣-reductase type II
(SRD5A2, valine [V] versus leucine [L]
alleles), and androgen receptor (AR,
number ofCAG repeats) were assayed
among 119 case patients who were
diag-nosed with HCC during 12 years
offol-low-up and 238 control subjects. All
sta-tistical tests were two-sided. Results: The
risk ofHCC increased with increasing
concentrations oftestosterone (odds ratio
[OR]
highest versus lowest tertile= 2.97; 95%
confidence interval [CI] = 1.54 to 5.70;
P
trend<.001) and with increasing number
ofthe V allele ofthe SRD5A2 V89L
poly-morphism (OR
VV versus LL genotype= 2.47;
95% CI = 1.21 to 5.03; P
trend= .011).
Fewer AR gene CAG repeats (<23
re-peats) were associated with a 1.64-fold
(95% CI = 1.00 to 2.68) increased risk
ofHCC. Although the CYP17 genotype
alone did not increase the risk ofHCC,
there was evidence ofa gene–gene
in-teraction, because the CYP17 A1 allele
statistically significantly increased the
risk ofHCC in the presence offewer
AR gene CAG repeats (OR = 2.51; 95%
CI = 1.06 to 5.94). We found a similar
interaction between the SRD5A2 VV
genotype and fewer AR gene CAG
re-peats (OR = 5.58; 95% CI = 1.86 to
16.71). Body mass index (BMI)
modi-fied the association of HCC with
testos-terone and SRD5A2 genotype; in men
with low BMI, multivariate-adjusted
ORs for the highest tertile of
testoster-one versus the lowest and the SRD5A2
VV genotype versus the LL genotype
were 7.63 (95% CI = 2.13 to 27.27) and
8.64 (95% CI = 2.75 to 27.14),
respec-tively. No clear associations were found
between estradiol or
testosterone-to-estradiol ratio and HCC. Conclusions:
Pathways involving androgen signaling
may affect the risk of HBV-related
HCC among men. [J Natl Cancer Inst
2001;93:1644–51]
Chronic infection with hepatitis B
vi-rus (HBV) or hepatitis C vivi-rus (HCV) has
a major role in the development of
hepa-tocellular carcinoma (HCC). HCC is more
prevalent in men than in women
through-out the world (1). In Taiwan, where HBV
infection is hyperendemic, the incidence
of HCC for men is approximatelythree
times that for women, despite similar
rates of chronic HBV infection (2). The
predominance of males with HCC has
also long been observed in various animal
models, including transgenic mice
ex-pressing HBV or HCV proteins (3–11).
Castration of male mice decreased the
in-cidence of chemicallyinduced HCC
com-pared with that of intact males, whereas
chronic testosterone administration to
fe-male or castrated fe-male animals increased
the risk of spontaneous or
chemicallyin-duced HCC (3–8). In addition, one animal
model (7) found that
ovariectomyin-creased the incidence of HCC because of
partial hepatectomyin
chemicallyin-duced hepatocarcinogenesis. However,
the long-term treatment effects of various
synthetic forms of estrogens on
hepato-carcinogenesis in animals remain
contro-versial (3,12–17). Although these
find-ings suggest that the pathogenesis of HCC
maybe influenced bythe hormonal
envi-ronment of the host, data about the role of
endogenous sex hormones in human
he-patocarcinogenesis are limited (18–22).
In addition to testosterone, proteins
in-volved in androgen transport and
metabo-lism also mayplaya critical role in the
development of HCC. We have begun to
explore the genes of three such proteins:
the androgen receptor (AR), the steroid
5
␣-reductase type II (SRD5A2), and the
cytochrome P450c17
␣ (CYP17). The AR
gene is responsible for androgen
trans-port; the SRD5A2 gene encodes the
en-zyme responsible for conversion of
tes-tosterone to the more active androgen (in
terms of AR affinity);
dihydrotestoster-one, and the CYP17 gene encodes an
en-zyme that catalyzes critical steps in
ste-roid genesis (23). The AR gene is located
on the X chromosome. Its exon 1 contains
a polymorphic CAG microsatellite that
codes for variable-length polyglutamine
in the AR protein (23). Our initial study
(21) has suggested that higher
testoster-one levels or fewer number of AR-CAG
repeats mayincrease HBV-related HCC
risk in men. There are several SRD5A2
polymorphisms, with the most common
being SRD5A2 V89L (valine [V] at
codon 89 to leucine [L]) and the least
common being SRD5A2 A49T (alanine
[A] at codon 49 to threonine [T]) (23–27).
Furthermore, the SRD5A2 is expressed in
the liver of adults (28). The CYP17
poly-morphism was detected byrestriction
di-gest with MspA1I. This didi-gestion
distin-guishes two alleles designated as A1 and
A2. The SRD5A2 V89L polymorphism
and the CYP17 polymorphism have been
associated with circulating sex hormone
levels (23–25,29,30).
In this nested case–control study, we
dissected the role of endogenous estradiol
in the etiologyof HBV-related HCC
among men who test positive for the HBV
surface antigen (HBsAg carriers) and
ex-tended our original study (21) on the
evaluation of the androgen hypothesis
concerning hepatocarcinogenesis to the
investigation of the possible association
between the SRD5A2 and CYP17
poly-morphisms and HCC development. We
also assessed whether there might be
in-teractions between hormone-related
fac-tors, including sex hormone levels and
genes involved in androgen biosynthesis
(CYP17), activation (SRD5A2), and
transport (AR), in determining HCC risk.
S
UBJECTS ANDM
ETHODSStudy Population
The cohort consisted of 4841 men, aged 30 years or older, who had tested positive for the HBsAg
Affiliations of authors: M.-W. Yu, Y.-C. Yang,
S.-Y. Yang, S.-W. Cheng, C.-J. Chen, Graduate In-stitute of Epidemiology, College of Public Health, National Taiwan University, Taipei; Y.-F. Liaw, S.-M. Lin, Liver Research Unit, Chang-Gung Memorial Hospital, Chang-Gung University, Taipei.
Correspondence to: Ming-Whei Yu, Ph.D.,
Graduate Institute of Epidemiology, College of Pub-lic Health, National Taiwan University, No. 1 Jen-Ai Rd., Section 1, Rm. 1550, Taipei 100, Taiwan.
See “Notes” following “References.”
(HBsAg carriers) and who attended a specific clinic for asymptomatic HBsAg carriers at the Liver Unit of Chang-Gung Memorial Hospital or the Govern-ment Employee Central Clinics (Taipei, Taiwan) for regular health examinations, from 1988 through 1992 (21). Written informed consent was obtained from all studyparticipants. The investigation was approved bythe research ethics committee at the College of Public Health, National Taiwan Univer-sity, Taipei, and by the appropriate institutional re-view board.
After an initial baseline examination, including ultrasonographymeasurements and conventional liver function tests, an in-person interview was con-ducted bytrained research assistants with the use of a structured questionnaire to obtain information on demographic and anthropometric characteristics, lifetime habits of alcohol and tobacco use, as well as personal and familyhistories of major chronic dis-eases. A blood specimen also was collected at the end of the interview.
Participants were monitored for incident HCC through various channels, including periodic ultra-sonographymeasurements and conventional liver function tests every6–12 months, a personal tele-phone interview, abstraction of medical records, and a data linkage to the national death certification and cancer registry systems. After 12 years of follow-up examinations, approximately70% of the HBsAg carriers who were still alive continued to return for their examinations. Subjects who did not participate in the follow-up examinations were traced and con-tacted bytelephone and bydata from the computer files of national cancer registryand death certifica-tion systems. In Taiwan, if a patient is diagnosed with or treated for cancer in a hospital with 50 or more beds, then the hospital has a legal obligation to report cancer patients to the national cancer registry. Case ascertainment bythe registrythrough the hos-pital system is estimated to be 85% complete. Com-puter files of the death certification system are rou-tinelymatched against profiles of members of the cohort. Thus, data on the vital status of all cohort members and the causes of all deaths are complete. When a case patient with HCC was identified, per-mission was sought from the hospital where the sub-ject was diagnosed with cancer to obtain medical charts and pathologyreports. Each case patient was diagnosed on the basis of either a histologic finding or elevated serum␣-fetoprotein level (艌400 ng/mL) combined with at least one positive image on angi-ography, sonangi-ography, and/or computed tomography. ByAugust 31, 2000, we confirmed 146 incident case patients with HCC.
For each case patient, two control subjects were selected at random from the cohort of HBsAg car-riers who were alive and remained unaffected with HCC throughout the follow-up period. The control subjects were matched to case patients bydate of blood collection (within 3 months) and year of birth (within 5 years, except for one elderly case patient, who was matched within 10 years). These control subjects have been included in a previous nested case–control studyon the role of the AR gene CAG repeats and elevated testosterone levels in the devel-opment of HCC (21). Five case patients who were included in the previous study (21) were not in-cluded in the present studybecause of insufficient DNA samples. Fourteen case patients identified since the end of the follow-up period in the previous
study (21) were added to the present study. Finally, a total of 119 case patients and 238 control subjects were included in the present study.
Laboratory Analyses
A blood sample was collected from each member of the cohort and processed to isolate white blood cells, serum, and plasma. All components were fro-zen at –70 °C until analysis.
The status of serum HBsAg was determined bya radioimmunoassay(Abbott Laboratories, Chicago, IL). DNA was extracted from frozen white blood cells according to a standardized protocol basically as described previously (31,32) but with some modi-fications. The CAG trinucleotide repeat found in exon 1 of the AR gene was amplified, and the num-ber of repeats was determined as described previ-ously (33). The CYP17 genotype was determined by use of the polymerase chain reaction (PCR)– restriction fragment length polymorphism (RFLP) method of Feigelson et al. (29), in which restriction digest by MspA1I (New England Biolabs, Inc., Bev-erly, MA) identifies the presence of the A2 allele. The SDR5A2 V89L polymorphism was determined byuse of PCR–RFLP, in which 100 ng of genomic DNA was amplified in the presence of 5 pmol of each of the primers 5 ⬘-TCGGGCCACCTGGGAC-GCTAC-3⬘ (SRD5A2–960) and 5⬘-GTTCCTCA-CAGCGCCCCACGC-3⬘ (SRD5A2R), 1× Taq buffer (i.e., 50 mmol/L potassium chloride, 1.5 mmol/L magnesium chloride, and 10 mmol/L Tris– HCl [pH 9.0]), 5 mmol/L of each deoxynucleoside triphosphate, and 0.5 U of Taq DNA polymerase (Amersham Pharmacia Biotech, Piscataway, NJ), in a total volume of 40L. The first primer contained a mismatched base (underlined) that leads to the loss of a RsaI restriction site in the amplified products. The amplification reaction consisted of an initial de-naturation step at 94 °C for 4 minutes, followed by 35 cycles of 40 seconds at 94 °C, 25 seconds at 65 °C, 30 seconds at 72 °C, and a final extension at 72 °C for 10 minutes. The PCR products then were digested overnight with 2 U of RsaI (Roche Molecu-lar Biochemicals, Mannheim, Germany) at 37 °C. The digested fragments were separated on a 2.5% agarose gel and visualized after staining with ethid-ium bromide. The VV, VL, and LL genotypes resulted in 137 and 40; 177, 137 and 40; and 177-base-pair fragments, respectively. We con-firmed PCR–RFLP genotypes by BigDye terminator cycle sequencing of the polymorphic region (Ap-plied Biosystems, Foster City, CA).
Testosterone was measured from the plasma bya competitive immunoassaythat uses direct chemilu-minescent technology(Chiron Diagnostics Corpora-tion, East Walpole, MA), and estradiol was mea-sured byuse of radioimmunoassaykits obtained from Diagnostic Systems Laboratories, Inc., Web-ster, TX (Catalog #DSL 4800). All assays were car-ried out and interpreted byindividuals blinded to the case–control status of the sample.
Statistical Methods
Odds ratios (ORs) and 95% confidence intervals (CIs) from unconditional logistic regression models were used to evaluate relative risks. Multivariate-adjusted ORs of HCC associated with various hor-mone-related factors were computed after adjust-ment for matching variables (e.g., age at recruitadjust-ment and the time that blood was drawn) and potential
confounders. Cigarette smoking and alcohol con-sumption are risk factors for HCC that might be associated with chronic liver disease (34), and chronic liver disease might influence the phenotype of the SRD5A2 and CYP17 gene byaltering the enzymatic activity. In the multivariate analyses on genotype and HCC, cigarette smoking, alcohol consumption, and chronic liver disease were thus also included in the logistic regression models as covariates.
The numbers of AR-CAG repeats were originally categorized into four groups (>24, 23–24, 21–22, and艋20 repeats) as described in our previous study
(21). To gain statistical power and to avoid sparse
cells occurring in stratified analyses on the interac-tions of the AR gene and other hormonal markers, a cut point of fewer than 23 repeats was finally chosen, after combining categories with similar risks. Although the estradiol levels are reported in picograms per milliliters, the ratio of testosterone to estradiol was calculated as the testosterone level in nanograms per milliliters divided bythe estradiol level in nanograms per milliliters. Tertile cut points were used for continuous hormone-related variables because of the distribution among the control group. Tertile cut points were chosen to avoid sparse cells so that both their independent effects and their in-teractive effects with other factors could be deter-mined. Categoric trends were tested in logistic re-gression byuse of an ordered categoric variable. Stratum-specific analyses were compared to evalu-ate the potential modification effect for each hor-mone-related factor. Statistical significance of the modification effect of each hormone-related factor on the HCC risk associated with other hormonal factors was determined bycomparing the fit of the logistic model that included the main effects and all potential confounders with a fullyparameterized model containing all possible two-factor inter-action terms for the variables of interest. All P val-ues were from two-tailed tests. All analyses were conducted using SAS release 6.12 (SAS Institute, Inc., Cary, NC).
R
ESULTSFrom the cohort of 4841 male HBsAg
carriers, 119 who developed HCC during
the follow-up period were included in this
study. On average, 4.8 years (range, 0.25–
11.25 years) elapsed between blood
col-lection and diagnosis. The mean age at
recruitment was 50.6 ± 9.3 years for case
patients and 50.3 ± 9.0 years for control
subjects (P
⳱ .746). For case patients, the
mean age at diagnosis of cancer was
55.5 ± 9.0 years (range, 36–78 years).
To estimate the risk associated with
each genetic polymorphism or hormone
level, we determined the OR. The crude
ORs associated with HCC for the VL and
VV genotypes of the SRD5A2 V89L
polymorphism were 1.34 (95% CI
⳱ 0.81
to 2.21) and 1.61 (95% CI
⳱ 0.85 to
3.05), respectively, compared with the LL
genotype as the reference group.
Com-pared with HBV carriers in the lowest
ter-tile of testosterone levels, the crude ORs
for those in the middle and highest tertile
were 1.28 (95% CI
⳱ 0.70 to 2.36) and
2.36 (95% CI
⳱ 1.35 to 4.12). The crude
ORs were 1.43 (95% CI
⳱ 0.92 to 2.22)
when comparing shorter (<23 repeats)
with longer CAG-repeat lengths (data not
shown). After adjustment for matching
factors (i.e., age at recruitment and the
time that blood was drawn), other HCC
risk factors (i.e., cigarette smoking,
alco-hol consumption, and historyof chronic
liver disease) (35–37), and selected
socio-demographic characteristics, the VV
geno-type of the SRD5A2 V89L polymorphism
was statisticallysignificantlyassociated
with an increased risk of HCC (OR
⳱
2.17; 95% CI
⳱ 1.10 to 4.29) compared
with the LL genotype. Inclusion of these
covariates did not substantiallyalter the
ORs for testosterone. Case patients were
more likelythan control subjects to have
AR-CAG repeats of fewer than 23
re-peats, yet this association was not
statis-ticallysignificant (P
⳱ .099) (model 1,
Table 1).
For the investigation of the impact of
the adjustment for the potential
confound-ing effects bybodymass index (BMI) and
other hormone-related factors on the OR
of HCC for each hormone-related factor,
BMI and other hormone-related factors
were further added as covariates in the
logistic regression model that analyzed
the main effect of each hormone-related
factor (model 2, Table 1). We found that
the association became even stronger for
HCC and testosterone or the SRD5A2
V89L polymorphism. An increasing trend
in HCC risk was observed with increasing
concentrations of testosterone (P
trend<.001) and with an increasing number of
Table 1. Association of selected hormone-related factors with risk of HCC among male HBsAg carriers in Taiwan*
Hormone-related factors Case patients (n⳱ 119) Control subjects (n⳱ 238) Model 1† Model 2‡ Adjusted OR 95% CI Adjusted OR 95% CI CYP17 polymorphism
A2/A2 43 (36.1%) 90 (37.8%) 1.00 Referent 1.00 Referent
A1/A2 56 (47.1%) 111 (46.6%) 1.07 0.64 to 1.77 1.15 0.67 to 1.96 A1/A1 20 (16.8%) 37 (15.6%) 1.18 0.60 to 2.34 1.23 0.59 to 2.56 SRD5A2 V89L polymorphism LL 35 (29.4%) 88 (37.0%) 1.00 Referent 1.00§ Referent VL 59 (49.6%) 111 (46.6%) 1.45 0.85 to 2.47 1.60 0.92 to 2.80 VV 25 (21.0%) 39 (16.4%) 2.17 1.10 to 4.29 2.47 1.21 to 5.03 Testosterone, ng/mL 0.87–4.73 25 (21.0%) 78 (32.8%) 1.00 Referent 1.00㛳 Referent 4.74–6.47 32 (26.9%) 78 (32.8%) 1.23 0.65 to 2.33 1.41 0.73 to 2.75 6.48–13.99 62 (52.1%) 82 (34.4%) 2.20 1.21 to 3.98 2.97 1.54 to 5.70 Estradiol, pg/mL 3.7–13.7 32 (27.3%) 74 (31.4%) 1.00 Referent 1.00 Referent 13.8–17.3 38 (32.5%) 77 (32.6%) 1.19 0.64 to 2.20 1.31 0.68 to 2.50 17.4–53.8 47 (40.2%) 85 (36.0%) 1.21 0.67 to 2.19 1.16 0.63 to 2.17 Missing 2 2 T/E2 ratio¶ 75.7–288.8 35 (29.9%) 78 (33.1%) 1.00 Referent 1.00 Referent 288.9–436.4 40 (34.2%) 77 (32.6%) 1.02 0.57 to 1.82 1.08 0.59 to 1.97 436.5–2539.7 42 (35.9%) 81 (34.3%) 1.03 0.57 to 1.88 1.16 0.61 to 2.17 Missing 2 2
No. of AR gene CAG repeats
25–35 33 (27.7%) 73 (30.7%) 1.00 Referent
23–24 26 (21.8%) 66 (27.7%) 0.80 0.42 to 1.51
21–22 43 (36.1%) 74 (31.1%) 1.29 0.72 to 2.32
14–20 17 (14.3%) 25 (10.5%) 1.44 0.66 to 3.14
Presence of AR alleles with <23 CAG repeats
No 59 (49.6%) 139 (58.4%) 1.00 Referent 1.00 Referent Yes 60 (50.4%) 99 (41.6%) 1.48 0.93 to 2.35 1.64 1.00 to 2.68 BMI, kg/m2 16.7–22.0 34 (28.6%) 79 (33.2%) 1.00 Referent 1.00 Referent 22.1–24.5 39 (32.8%) 78 (32.8%) 1.20 0.67 to 2.15 1.52 0.81 to 2.87 24.6–32.0 46 (38.7%) 81 (34.0%) 1.42 0.80 to 2.53 1.98 1.05 to 3.74
*HCC⳱ hepatocellular carcinoma; HBsAg ⳱ hepatitis B surface antigen; OR ⳱ odds ratio; CI ⳱ confidence interval; T/E2 ratio ⳱ testosterone-to-estradiol ratio; AR⳱ androgen receptor; BMI ⳱ bodymass index; SRD5A2 ⳱ steroid 5␣-reductase type II; CYP17 ⳱ cytochrome P450c17␣.
†Adjusted for age at recruitment (continuous variable), the time of blood draw (continuous variable), ethnicity(Fukien Taiwanese, Hakka Taiwanese, and Mainland Chinese), years of education (continuous variable), cigarette smoking, alcohol consumption, and history of chronic liver disease.
‡For the T/E2 ratio, ORs have been adjusted for covariates in model 1, BMI, and other hormone-related factors listed in the table, except for testosterone and estadiol. For other hormone-related factors, ORs have been adjusted for the same covariates that we treated for the hormone ratio, but testosterone and estradiol were included as covariates instead of the ratio. All variables were included in the logistic regression models as categorized in the table (the number of AR-CAG repeats was included as a dichotomous variable), except for BMI, which was included as a continuous variable for adjusting for its effect. Two case patients and two control subjects were excluded from analysis because of missing data on plasma estradiol.
§Ptrend⳱ .011.
㛳Ptrend<.001.
V alleles of the SRD5A2 polymorphism
(P
trend⳱ .011). After adjustment for
BMI and other hormone-related factors,
male HBsAg carriers with fewer than 23
AR-CAG repeats were found to have a
statisticallysignificantlyincreased risk of
HCC (OR
⳱ 1.64; 95% CI ⳱ 1.00 to
2.68) compared with those with at least
23 AR-CAG repeats (P
⳱ .048). Also,
the positive association between tertile
distribution of BMI and the risk of HCC
became statisticallysignificant (highest
versus lowest tertile; OR
⳱ 1.98; 95% CI
⳱ 1.05 to 3.74) after adjustment for other
hormone-related factors.
We found no statisticallysignificant
association between the risk of HCC and
estradiol, testosterone-to-estradiol ratio,
or the CYP17 genotype (Table 1).
More-over, when estradiol values were
catego-rized as quartiles in an unconditional
lo-gistic regression model adjusted for the
matching factors (i.e., age at recruitment
and the time that blood was drawn),
ciga-rette and alcohol use, historyof chronic
liver disease, years of education,
ethnic-ity, BMI, and other hormone-related
fac-tors, there was still little evidence for an
effect of estradiol (multivariate-adjusted
ORs byquartile
⳱ 1.00 [referent], 1.01
[95% CI
⳱ 0.49 to 2.08], 0.79 [95% CI
⳱ 0.37 to 1.68], and 1.39 [95% CI ⳱
0.69 to 2.79]). A similar finding was
ob-served for the testosterone-to-estradiol
ra-tio (multivariate-adjusted ORs byquartile
⳱ 1.00 [referent], 1.03 [95% CI ⳱ 0.51
to 2.07], 1.13 [95% CI
⳱ 0.56 to 2.27],
and 1.17 [95% CI
⳱ 0.57 to 2.41]).
We also examined the interactions
be-tween anytwo hormone-related factors.
Al-though the strength of the interaction
be-tween the SRD5A2 genotype and AR-CAG
repeats approached statistical significance
(P
interaction⳱ .084), the association
be-tween the SRD5A2 genotype and HCC
appeared to be stronger for male HBsAg
carriers with fewer than 23 AR-CAG
re-peats (Table 2). Among HBsAg carriers
with fewer than 23 AR-CAG repeats, the
multivariate-adjusted OR of HCC for
those with the SRD5A2 VV genotype was
5.58 (95% CI
⳱ 1.86 to 16.71) compared
with those with the SRD5A2 LL
geno-type. Conversely, there was no increased
risk of HCC associated with the SRD5A2
VV genotype among HBsAg carriers with
23 or more AR-CAG repeats. Similarly,
among HBsAg carriers with fewer than
23 AR-CAG repeats, the
multivariate-adjusted OR was 2.51 (95% CI
⳱ 1.06 to
5.94) for those with the CYP17 A1 allele
(A1/A1 and A1/A2 genotypes) compared
with those without the A1 allele (A2/A2
genotype). There was no evidence,
how-ever, that the presence of the A1 allele
increased risk among those with longer
repeats. The interaction between the
CYP17 genotype and AR-CAG repeats
was statisticallysignificant (P
interaction⳱
.04) (Table 2). Testosterone, estradiol, or
the testosterone-to-estradiol ratio did not
appear to modifythe effect of anyof the
other hormonal factors. No notable
inter-action was also observed between CYP17
and the SRD5A2 genotype (data not
shown).
Because high BMI has been associated
with hormone-related cancers (38,39),
stratified analyses were also performed
according to BMI (Table 3). This analysis
revealed that the SRD5A2 V89L
geno-type (VV versus LL genogeno-type; OR
⳱
8.64; 95% CI
⳱ 2.75 to 27.14) and
tes-tosterone levels (highest versus lowest
tertile; OR
⳱ 7.63; 95% CI ⳱ 2.13 to
27.27) were strong predictors for HCC
risk among HBsAg carriers with a BMI
less than or equal to the median value
among control subjects. However, among
those with a BMI above the median value,
we identified onlya weak positive
asso-ciation for testosterone (highest versus
lowest tertile; OR
⳱ 2.28; 95% CI ⳱
0.92 to 5.64). Furthermore, there was no
association between the SRD5A2
geno-type and the risk of HCC in HBsAg
car-riers with a BMI above the median level.
BMI also appeared to modifythe effect of
CYP17 genotype, but we failed to detect a
statisticallysignificant interaction
be-tween BMI and CYP17 genotype in the
risk of HCC. None of the other
hormone-related factors considered showed any
meaningful interaction with BMI (Table 3).
We also analyzed the association
be-tween BMI and HCC within strata of the
SRD5A2 genotype. In this analysis, BMI
was treated as a continuous variable.
Table 2. Association of cytochrome P450c17␣ (CYP17) and steroid 5␣-reductase type II (SRD5A2) genotypes with the risk of HCC by
the number of AR gene CAG repeats among male HBsAg carriers*
No. of AR-CAG repeats
艌23 repeats <23 repeats Case patients (n⳱ 59) Control subjects (n⳱ 139) Adjusted OR†,‡ 95% CI Case patients (n⳱ 60) Control subjects (n⳱ 99) Adjusted OR†,§ 95% CI SRD5A2 V89L polymorphism LL 18 48 1.00 Referent 17 40 1.00 Referent VL 33 65 1.72 0.78 to 3.82 26 46 1.46 0.60 to 3.53 VV 8 26 1.08 0.36 to 3.24 17 13 5.58 1.86 to 16.71
Test for interaction: P⳱ .084 CYP17 polymorphism
A2/A2 26 48 1.00 Referent 17 42 1.00 Referent
A1/A2 22 66 0.51 0.23 to 1.11 34 45 2.51 1.03 to 6.09
A1/A1 11 25 0.65 0.24 to 1.76 9 12 2.50 0.71 to 8.78
Test for interaction: P⳱ .04
*HCC⳱ hepatocellular carcinoma; AR ⳱ androgen receptor; HBsAg ⳱ hepatitis B surface antigen; OR ⳱ odds ratio; CI ⳱ confidence interval; BMI ⳱ body mass index.
†Adjusted for age at recruitment (continuous variable), the time of blood draw (continuous variable), ethnicity(Fukien Taiwanese, Hakka Taiwanese, and Mainland Chinese), years of education (continuous variable), cigarette smoking, alcohol consumption, history of chronic liver disease, BMI, and other hormone-related variable listed in Table 1, except for the ratio of testosterone to estradiol. All variables were included in logistic regression models as categorized in Table 1, except for BMI, which was included as a continuous variable.
‡One control subject was excluded from analysis because of missing data on plasma estradiol.
Among HBsAg carriers, there was a
posi-tive association in LL homozygotes
(mul-tivariate-adjusted OR
⳱ 1.37; 95% CI ⳱
1.09 to 1.71) but a negative association
among VV homozygotes
(multivariate-adjusted OR
⳱ 0.66; 95% CI ⳱ 0.45 to
0.95). We found no association among the
heterozygotes (multivariate-adjusted OR
⳱ 1.13; 95% CI ⳱ 0.98 to 1.31) (data not
shown).
D
ISCUSSIONNumerous reports (35,40) have
sug-gested that the therapeutic use of
andro-genic steroids or oral contraceptives may
cause benign hepatic adenoma and may
increase the risk of HCC. In this study,
the positive association between
testoster-one and HCC risk is consistent with our
earlier Taiwanese case–control study,
which was nested within another cohort
study (20), and with a recent Japanese
co-hort studyof male patients with liver
cir-rhosis predominantlyof HCV origin (22).
However, our results are somewhat in
contrast with two previous cohort studies
(18,19). One study (18), from an area of
China with a high-incidence of HCC,
re-ported that male HBsAg carriers with the
highest tertile of circulating testosterone
levels had a statisticallynonsignificant
50% increase in the risk of HCC
com-pared with those in the lowest tertile.
However, that study (18) included only50
HBsAg-positive case patients. The other
study (19) reported no association
be-tween testosterone levels and HCC risk in
a cohort of 101 male patients with
cirrho-sis, mainlycaused byalcohol abuse.
Be-cause alcoholic cirrhosis is
frequentlyas-sociated with hypogonadism (41), the
association between testosterone levels
and HCC risk observed in such patients
with cirrhosis might not be expected to be
the same as that observed in HBsAg
car-riers included in our study.
The positive association between
tes-tosterone levels and the incidence of HCC
that we observed raises the possibilitythat
genes involved in the regulation of
testos-terone mayalso playa role in the etiology
of HCC. One such gene is SRD5A2. Two
missense mutations have been identified
in the SRD5A2 gene (23–27). The
valine-Table 3. Association of selected hormone-related factors with the risk of HCC byBMI among male HBsAg carriers*
Hormone-related factors BMI 艋23.2† >23.2 Case patients (n⳱ 59) Control subjects (n⳱ 119) Adjsuted OR‡,§ 95% CI Case patients (n⳱ 60) Control subjects (n⳱ 119) Adjusted OR‡,§ 95% CI Testosterone, ng/mL 0.87–4.73 4 31 1.00 Referent 21 47 1.00 Referent 4.74–6.47 16 39 2.76 0.74 to 10.31 16 39 0.97 0.40 to 2.35 6.48–13.99 39 49 7.63 2.13 to 27.27 23 33 2.28 0.92 to 5.64
Test for interaction: P⳱ .023 SRD5A2 V89L polymorphism
LL 13 50 1.00 Referent 22 38 1.00 Referent
VL 30 55 3.22 1.30 to 8.01 29 56 0.86 0.39 to 1.93
VV 16 14 8.64 2.75 to 27.14 9 25 0.74 0.24 to 2.28
Test for interaction: P⳱ .003 CYP17 polymorphism
A2/A2 27 43 1.00 Referent 16 47 1.00 Referent
A1/A2 25 55 0.73 0.33 to 1.64 31 56 2.44 1.02 to 5.83
A1/A1 7 21 0.38 0.11 to 1.31 13 16 3.75 1.26 to 11.17
Test for interaction: P⳱ .302 No. of AR gene CAG repeats
艌23 29 66 1.00 Referent 30 73 1.00 Referent
<23 30 53 1.40 0.64 to 3.07 30 46 2.00 0.96 to 4.16
Test for interaction: P⳱ .708 Estradiol, pg/ml
3.7–13.7 17 46 1.00 Referent 15 28 1.00 Referent
13.8–17.3 20 38 1.92 0.74 to 4.98 18 39 1.35 0.49 to 3.77
17.4–53.8 21 34 1.68 0.65 to 4.38 26 51 0.76 0.30 to 1.97
Test for interaction: P⳱ .136 T/E2 ratio㛳
75.7–288.8 10 31 1.00 Referent 25 47 1.00 Referent
288.9–436.4 24 35 1.63 0.61 to 4.37 16 42 0.99 0.41 to 2.38
436.5–2539.7 24 52 1.55 0.57 to 4.17 18 29 1.38 0.55 to 3.45
Missing 1 1 1 1
Test for interaction: P⳱ .067
*HCC⳱ hepatocellular carcinoma; BMI ⳱ bodymass index; HBsAg ⳱ hepatitis B surface antigen; OR ⳱ odds ratio; CI ⳱ confidence interval; AR ⳱ androgen receptor; SRD5A2⳱ steroid 5␣-reductase type II; CYP17 ⳱ cytochrome P450c17␣.
†Cut point was chosen on the basis of median value among control subjects.
‡For the testosterone-to-estradiol ratio, ORs have been adjusted for age at recruitment (continuous variable), the time of blood draw (continuous variable), ethnicity (Fukien Taiwanese, Hakka Taiwanese, and Mainland Chinese), years of education (continuous variable), cigarette smoking, alcohol consumption, historyof chronic liver disease, BMI (continuous variable), and other hormone-related variables listed in Table 1, except for testosterone and estradiol. For other hormone-related factors, ORs have been adjusted for the same covariates that we treated for the hormone ratio, but testosterone and estradiol were included as covariates instead of the ratio. All variables were included in logistic regression models as categorized in the table.
§One case patient and one control subject were excluded from analysis because of missing data on plasma estradiol levels. 㛳T/E2 ratio ⳱ testosterone in nanograms per milliliters divided byestradiol in nanograms per milliliters.
to-leucine substitution at codon 89
ap-pears to be much more common in Asians
than in whites or African-Americans (24,
26). Studies (24,25) have shown a
statis-ticallysignificant association between the
SRD5A2 V89L polymorphism and in
vivo enzymatic activity, with VV
homo-zygotes having substantially higher levels
of activitythan LL homozygotes and
het-erozygotes having intermediate levels of
activity. We also found that, among male
HBsAg carriers, the V allele was
statisti-callysignificantlyassociated with an
in-creased risk of HCC, with an allele
dos-age effect. However, although the SRD5A2
V89L polymorphism appears to have an
association with the risk of HCC, it does
not appear to be associated with prostate
cancer risk, which is thought to be
andro-gen dependent (26). A second missense
mutation in the SRD5A2 gene has been
identified at codon 49 and results in a
sub-stitution of alanine with threonine. This
substitution increased enzymatic activity
in vitro and has been positivelyassociated
with advanced-stage prostate cancer (23,
27). However, the SRD5A2 A49T allele
is rare in various populations, such as
African-Americans and Latinos (23,27).
A sample size larger than that of the
current studymaybe required to have
sufficient power to examine the
associa-tion of this polymorphism with the risk
of HCC.
A second hormone-related gene is the
CYP17 gene, which encodes the CYP17
enzyme that functions at key steps in the
synthesis of both androgens and estrogens
(42). There are conflicting results
regard-ing whether the CYP17 polymorphism,
detected by MspA1I digestion, is a risk
factor for prostate cancer and female
breast cancer (26,30,43,44). Also, whereas
the CYP17 A2 allele was associated with
higher serum estrogen levels in women
(29,30), the A1 allele was associated with
higher serum androgen metabolite levels
in men (25). In our study, although the
CYP17 genotype alone did not influence
the risk of HCC, inheritance of at least
one CYP17 A1 allele was
specificallyas-sociated with an increased risk of HCC
among male HBsAg carriers who had
fewer AR-CAG repeats or higher BMI.
A third hormone-related gene is the
AR, which is responsible for the transport
of androgens. ARs have been
demon-strated in HCC and nontumorous liver
tis-sue, but AR expression is greater in HCC
(45). In normal male mice, the growth
rate of chemicallyinduced preneoplastic
hepatic foci is 20-fold faster than the rate
in mice with testicular feminization,
which lacks functional ARs (8). This
ob-servation suggests that the development
of HCC maybe AR dependent. In
addi-tion, the number of AR-CAG repeats is
inverselyassociated with transcriptional
activitybythe AR in vitro (46), with an
increase in transcriptional
activityassoci-ated with ARs containing fewer CAG
re-peats. Fewer AR-CAG repeats also have
been associated with an excess risk of
prostate cancer (23,33,47). Moreover, a
moderate expansion of the CAG-repeat
sequence has been suggested to playa
role in male infertility (48). In this study
and in our previous case–control
studyin-volving almost all of the case patients in
the present studyand a series of hospital
male patients with HBV-related HCC
(21), we also observed that HBsAg
carri-ers with fewer AR-CAG repeats had an
increased risk of HCC, suggesting that
in-creased AR-mediated transcriptional
ac-tivitymaycontribute to the development
of HBV-related HCC.
We did not find convincing evidence
for a modification effect of AR-CAG
re-peats on the association between
testos-terone levels and HCC risk. Nevertheless,
the effects of SRD5A2 and CYP17
geno-types on the risk of HCC appeared to
de-pend on the AR genotype. For both the
SRD5A2 V89L and the CYP17
polymor-phisms, the at-risk genotypes statistically
significantlyincreased disease risk among
male HBsAg carriers with fewer than 23
CAG repeats but posed no increased risk
among those with longer repeats.
Regard-less of the mechanisms underlying the
modifying effect of the AR-CAG repeats
on HCC risk associated with the SRD5A2
and CYP17 polymorphisms, it appears
that the development of HCC among male
HBsAg carriers is mediated bya
combi-nation of genes involved in the
metabo-lism and transport of androgens. Thus,
HBV-related hepatocarcinogenesis is
closelyassociated with the pathways
in-volving androgen signaling.
The development of HCC mayalso be
associated with obesity. Obesity has been
associated with steatohepatitis and
non-insulin-dependent diabetes mellitus,
which maylead to HCC development
(35,49). Patients with
non-insulin-dependent diabetes mellitus often
mani-fest insulin resistance, which, in addition
to increased adiposityas measured by
BMI, has been proposed to be a risk factor
for certain hormone-related cancers (38,
39,50). After adjusting for other
hormone-related factors and potentiallyrelevant
co-variates, we found that BMI was
posi-tivelyassociated with the risk of HCC.
However, we also found that the BMI
as-sociation differed according to the
cat-egoryof the SRD5A2 genotype. Of
par-ticular interest is the striking difference in
the estimated risks of HCC associated
with testosterone levels and the SRD5A2
genotype between male HBsAg carriers
with different BMI values (Table 3). This
difference raises the intriguing possibility
that men with different magnitudes of
obesitymayhave divergent responses to
the promoting effect of androgens on
he-patocarcinogenesis.
Our data, in conjunction with past
ani-mal studies (3–11), provide strong
evi-dence for a close relationship between
HBV-related HCC risk among men and
higher levels of androgen signaling,
re-flected byhigher testosterone levels,
increased metabolic activation of
testos-terone, and/or increased AR-mediated
transcriptional activity. The
predomi-nance of HCC among males maybe
at-tributed to the higher androgenic activity
and/or the lower estrogenic activityin
men than in women. Of note, similar to
two earlier cohort studies (19,22), we did
not find a relationship between estradiol
levels and the risk of HCC. In addition,
the results of animal studies have been
inconsistent regarding the role of
estro-gens in the development of HCC (3,7,12–
17). The major source of estrogens in men
is from the conversion of androgens to
estrogens in peripheral tissues, such as
adipose tissues. Unlike the small cohort
studyof Japanese patients with cirrhosis,
which reported a strong positive
associa-tion between the testosterone-to-estradiol
ratio and HCC risk (22), we did not find a
statisticallysignificant association in any
of our analyses. Instead, plasma
testoster-one levels appeared to be a better
predic-tor than the hormone ratio for HCC risk.
This is presumablybecause absolute
rather than relative hormone amounts
have a more profound effect on HCC
de-velopment. If so, pharmacologic
ap-proaches to decrease androgen action may
warrant investigation as a
strategyspecifi-callytargeted at male HBsAg carriers for
the treatment or prevention of HCC.
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OTESSupported bygrant NSC 89–2314-B-002–367 from the National Science Council and bygrant DOH89-TD-1130 (Frontier Medical Genomic Pro-gram) from the Department of Health, Executive Yuan, Taiwan.
Manuscript received February26, 2001; revised July31, 2001; accepted August 14, 2001.
