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Human papillomavirus status in southern Chinese women

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RESEARCH FUND FOR THE CONTROL OF INFECTIOUS DISEASES
Human papillomavirus status in southern
Chinese women
SS Liu, KKY Chan, RCY Leung, AMW Yip, LSK Lau, XY Liao, LL Jiang, MHM Luk, SST Lo, DYT Fong,
ANY Cheung, ZQ Lin, HYS Ngan *
Hong Kong Med J 2014;20(Suppl 6):S35-8
RFCID project number: 05050052
Key Messages
1. The
overall
and
type-specific
human
papillomavirus (HPV) prevalence differed
between Hong Kong and Guangzhou healthy
women. The prevalence of HPV was significantly
higher in Guangzhou than Hong Kong women.
Younger women had significantly higher risk of
HPV infection.
2. HPV16 remained the most common type detected
in both regions; the frequency increased with
increasing disease severity. The prevalence of
HPV58 and HPV52 was relatively high in women
with normal cervix and precancerous lesions.
SS Liu, 2 KKY Chan, 1 RCY Leung, 1 AMW Yip, 1 LSK Lau, 2 XY Liao,
LL Jiang, 3 MHM Luk, 4 SST Lo, 5 DYT Fong, 2 ANY Cheung, 3 ZQ Lin,
1
HYS Ngan
1
2
Department of Obstetrics and Gynaecology, Faculty of Medicine, The
University of Hong Kong, Hong Kong
2
Department of Pathology, Faculty of Medicine, The University of Hong
Kong, Hong Kong
3
Department of Obstetrics and Gynaecology, The Second Affiliated
Hospital of Sun Yat-Sen University, Guangzhou, China
4
Family Planning Association of Hong Kong, Hong Kong
5
School of Nursing, Faculty of Medicine, The University of Hong Kong,
Hong Kong
1
* Principal applicant and corresponding author: hysngan@hkucc.hku.hk
Introduction
Human papillomavirus (HPV) is a sexually
transmitted pathogen that plays an important role
in the pathogenesis of precancerous cervical lesions
and cervical cancer.1 The prevalence of HPV types
in Hong Kong differs from that in Sichuan, China.
Cross-border travel and marriage between people in
Hong Kong and Guangzhou have increased greatly,
as has the potential for disease transmission.
Integration of HPV correlates with poor
response to treatment and poor disease-free survival
in cervical cancer.2 Integration usually causes
deletion and/or disruption of the E2 gene of the HPV.
In this study, we aimed to determine (1) the spectrum
and prevalence of HPV in healthy women in Hong
Kong and Guangzhou, and (2) whether integration
of the high-risk HPV16 and HPV58 genomes in the
host is associated with progressive severity of the
precancerous cervical lesion and cervical cancer.
Methods
compared between Hong Kong and Guangzhou. The
association between co-factors and the risk of HPV
infection was analysed. The integrations of HPV16
and HPV58 viral DNA in host genomes were assessed
and the clinical significance was determined.
Cytology samples of 1280 and 1273 healthy
women in Hong Kong and Guangzhou were
collected, respectively. The exfoliated cervical cells
were collected by ThinPrep (Hologic, Bedford, MA,
USA) in Hong Kong or liquid-based cervical cytology
test in Guangzhou. In addition, 438 and 204 samples
of cervical precancerous lesions and cervical cancers
were retrospectively selected in the two regions,
respectively.
A Hybribio DNA extraction kit (Hybribio,
Hong Kong) and QIAamp DNA FFPE Tissue Kit
(Qiagen, Hilden, Germany) were used for DNA
extraction from the cytological remnants and the
paraffin-embedded tissues, respectively. Genotyping
of HPV was performed using the GenoArray HPV
genotyping test (Hybribio), which is a polymerase
chain reaction (PCR)-based assay that is capable of
amplifying 21 HPV genotypes, including 13 highrisk types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59,
and 68) and nine other-risk types.
The HPV viral DNA integration was determined
by quantitative real-time fluorescent PCR with five
sets of E2 and one set of E6 primers for HPV16 and
HPV58. The proportion of integrated value was
calculated by the ratio of E2 value (episomal) to E6
value (episomal and integrated).
This cross-sectional study was approved by the
local institutional review board and conducted from
October 2006 to September 2008, in collaboration
with the Department of Obstetrics and Gynaecology
and Department of Pathology of The University of
Hong Kong, the Family Planning Association of
Hong Kong, and the Department of Obstetrics and
Gynaecology of the Second Affiliated Hospital, Sun
Yet-Sen University, Guangzhou, Guangdong, China.
The prevalence of HPV and type-specific
infections in healthy women and women with Results
precancerous lesions and cervical cancer were Of the 1280 and 1273 cytology samples from Hong
Hong Kong Med J ⎥ Volume 20 Number 6 (Supplement 6) ⎥ December 2014 ⎥ www.hkmj.org
35
# Liu et al #
TABLE 1. Comparison of the prevalence of human papillomavirus (HPV) infections in Hong Kong and Guangzhou women
HPV prevalence
Hong Kong (% positive)
Normal
Low-grade
High-grade
(agecervical
cervical
adjusted) intraepithelial intraepithelial
neoplasia
neoplasia
Guangzhou (% positive)
Cervical
cancer
Normal
Low-grade
High-grade
(agecervical
cervical
adjusted) intraepithelial intraepithelial
neoplasia
neoplasia
Overall prevalence
5.6‡
22.4‡
30.8
40.2
10.1‡
28.9
39.2
Multiple infection
1.8
1.9
0.9
1.0
1.8
0
1.8
2.0
33.7†
8.3
2.8
2.4
15.3†
0
6.1
5.0
4.0‡
Multiple infection among HPVpositive
High-risk HPV
31.0†
28.2
71.9
79.2
91.7
100
75.5
83.3
81.8
HPV16
23.6*
16.7
27.8
56.1*
11.0*
16.7
36.4
77.5*
HPV18
14.6*
12.5
0*
22.0
4.9*
0
15.2*
17.5
High-risk types among HPVpositive
40.2
7.6‡
6.0‡
Cervical
cancer
5.0†
23.7
39.2
100
Type-specific (% HPV-positive)
HPV31
0‡
0
13.9*
4.9
16.6‡
33.3
0*
0
HPV52
19.1
20.8
13.9
7.3
23.9
33.3
9.1
0
HPV58
20.2*
29.2†
27.8
9.8
10.4*
0†
12.1
Total (5 types)
77.5
79.2
83.4
66.8
83.3
72.8
100
5.0
100
* P=0.01 to <0.05, Chi-squared test
† P=0.001 to <0.01, Chi-squared test
‡ P<0.001, Chi-squared test
36
frequencies of HPV58 and 52 were higher in women
with normal cervix and precancerous lesion in both
regions.
Age was a risk factor only in Hong Kong
women younger than 29 years, compared with other
age-groups (P=0.009). Previous Pap smear test
was another risk factor for women in Hong Kong
(P=0.037). Women with a previous abnormal Pap
smear test also had significantly higher HPV infection
(P=0.021). In Guangzhou, women with previous
abnormal cytological findings also had increased risk
6.0
HPV prevalence (%)
Kong and Guangzhou women, respectively, 1245
(97.3%) and 1209 (95.0%) had normal cytology.
The age-adjusted HPV prevalence was significantly
lower in Hong Kong than Guangzhou women (5.6%
[89/1245] vs 10.1% [163/1209], Table 1), particularly
in the three younger age-groups (Fig). The prevalence
of HPV infection varied between age-groups in the
two regions.
In Hong Kong, the HPV prevalences in women
with low-grade cervical intraepithelial neoplasia
(LG CIN), high-grade CIN (HG CIN), and cervical
cancer were 22.4%, 30.8%, and 40.2%, respectively.
In Guangzhou, the corresponding prevalences
were 6.0%, 28.9%, and 39.2%, respectively. The HPV
prevalence in women with LG CIN was significantly
higher in Hong Kong than Guangzhou (P<0.001).
The rate for age-adjusted multiple HPV infection
was 1.8% in women with normal cervix for both
regions, accounting for 33.7% and 15.3% of HPVpositive cases in Hong Kong and Guangzhou
cohorts, respectively (P=0.001).
In Hong Kong, HPV16 was most commonly
identified in women with normal cytology, HG
CIN, and cervical cancer, whereas HPV58 was most
commonly identified in women with LG CIN. In
Guangzhou, HPV16 was most commonly identified
in women with HG CIN and cervical cancer, whereas
HPV52 and HPV31 were most commonly identified
in women with normal cytology and LG CIN. HPV16,
52 and 58 were the most common high-risk types
detected in women with normal cytology in both
regions. The frequency of HPV16 in women with
normal cervix and cervical cancer was significantly
higher in Hong Kong than in Guangzhou (P=0.015
and P=0.036, respectively). The frequency of HPV16
increased with an increase of disease severity. The
Hong Kong
Guangzhou
5.0
4.0
† ‡
† ‡
3.0
2.0
† ‡
1.0
0.0
*
20-29
30-39
40-49
50-59
Age (years)
≥60
FIG. Age-adjusted human papillomavirus (HPV) infection
prevalences in five age-groups in Hong Kong and Guangzhou
women.
* In Hong Kong, the prevalence of HPV infection is
significantly lower in the age-group of 40-49 years than that
of 20-29 years and ≥60 years (P<0.05)
† The prevalence of HPV infection is significantly higher in
Guangzhou than Hong Kong women in the age-groups of
20-29 years (P<0.001), 30-39 years (P<0.001), and 40-49
years (P=0.009)
‡ In Guangzhou, the prevalence of HPV infection is
significantly higher in the age-groups of 20-29 years, 30-39
years, and 40-49 years than in the other groups (P<0.05)
Hong Kong Med J ⎥ Volume 20 Number 6 (Supplement 6) ⎥ December 2014 ⎥ www.hkmj.org
# Human papillomavirus status in southern Chinese women #
of HPV infection (P=0.044). Two sexual behaviour
variables (lifetime number of sexual partners and
age at first sexual intercourse) were associated with
the risk of HPV infection. In Guangzhou, the risk of
HPV infection was significantly higher in women
with ≥3 than 1-2 lifetime sexual partners (P<0.001).
In Hong Kong, women having first sexual intercourse
before the age of 20 years had higher risk than those
having first sexual intercourse after the age of 21
years (P=0.043). After multivariable analysis, only
the lifetime number of sexual partners remained a
significant factor in Guangzhou women (Table 2).
No association was noted with variables of gravidity,
use of condom or oral contraceptive, history of
sexually transmitted diseases, and smoking.
The complete episomal form was found in
only 3/110 (2.7%) HPV16- and 4/48 (8.3%) HPV58positive samples. HPV16 integration and HPV58
integration were detected in 107 and 44 samples,
respectively; 30 (27.3%) HPV16-positive samples
and only one (2.1%) HPV58-positive sample showed
complete integration. No significant difference in
the integration patterns of HPV16 or HPV58 with
regard to the various grades of cervical lesions was
noted. The distribution and frequency of HPV16 or
HPV58 DNA integration were similar between the
Hong Kong and Guangzhou regions.
Discussion
In Guangzhou, the age-adjusted HPV infection rate
was 10.1% in women with normal cytology, which is
TABLE 2. Multiple logistic regression for risk factors of human papillomavirus (HPV) infection in Hong Kong and Guangzhou women
Variable
Hong Kong
No.
HPVpositive (%)
20-29
248
10.9
30-39
243
4.9
40-49
249
3.2
50-59
250
≥60
255
OR (95% CI)
Guangzhou
No.
HPVpositive (%)
233
15.0
0.53 (0.24-1.15)
237
14.3
1.48 (0.79-2.77)
0.37 (0.14-1.00)
236
12.7
1.19 (0.61-2.33)
7.2
0.77 (0.29-2.01)
253
13.8
1.55 (0.77-3.10)
9.4
1.32 (0.50-3.48)
250
11.6
1.21 (0.59-2.49)
377
11.4
832
14.4
Age (years)
P value
OR (95% CI)
0.025
1
Previous Pap smear test
0.626
1
0.038
Yes
1040
6.4
No
205
10.7
1
0.49 (0.25-0.96)
Lifetime number of sexual partner
0.087
1
0.71 (0.48-1.05)
0.973
1
868
6.8
2
185
7.0
0.92 (0.45-1.88)
1
≥3
192
8.9
0.98 (0.46-2.09)
Age at first sexual intercourse (years)
0.015
1039
12.4
123
13.8
1.21 (0.67-2.18)
1
46
34.8
3.20 (1.45-7.04)
176
14.2
0.236
0.701
≤20
430
17.8
21-25
510
5.3
0.55 (0.30-1.00)
643
14.3
1.31 (0.76-2.26)
26-35
292
6.2
0.62 (0.30-1.28)
385
11.7
1.11 (0.58-2.10)
13
7.7
1.14 (0.13-10.26)
5
20.0
1.64 (0.17-16.20)
1170
13.2
17
29.4
1031
13.2
177
14.6
≥36
1
Sexually transmitted disease history
Never
Ever
7.2
16
6.3
1
0.94 (0.12-7.52)
Oral contraceptive use
0.368
1
1.74 (0.52-5.76)
0.197
Never
551
6.5
Ever
683
7.6
1
1.38 (0.85-2.26)
Condom use
0.588
1
1.14 (0.71-1.84)
0.152
Never or rare
672
8.3
Regular
549
5.6
0
354
6.5
1-2
455
8.4
3-4
334
7.2
≥5
100
4.0
1
0.888
877
13.7
318
12.9
86
20.9
1.87 (0.92-3.80)
548
12.4
0.55 (2.80-1.08)
1.25 (0.52-3.00)
455
13.6
0.52 (0.24-1.09)
0.55 (0.45-2.04)
120
12.5
0.52 (0.21-1.28)
1173
13.3
0.69 (0.42-1.15)
Gravidity
1
0.97 (0.64-1.47)
0.065
1
Smoking
Never
1
0.95
1214
0.348
1
0.808
1105
6.7
1
P value
0.958
1
Former
72
8.3
1.09 (0.43-2.77)
17
17.6
1.22 (0.32-4.65)
Current
68
11.8
1.34 (0.56-3.21)
19
21.1
1.01 (0.30-3.49)
Hong Kong Med J ⎥ Volume 20 Number 6 (Supplement 6) ⎥ December 2014 ⎥ www.hkmj.org
37
# Liu et al #
similar to other regions of China.3 In Hong Kong, the
rate was significantly lower at 5.6%. In Guangzhou,
younger women (age <39 years) had a significantly
higher infection rate than older women. In Hong
Kong, the highest infection rate was in women in
the age-groups of 20-29 years and >60 years. The
difference in HPV prevalence between the two
regions could be due to differences in the living
environments and life-styles.
In Hong Kong, the crude HPV infection rate in
healthy women was 7.2%, which was lower than that
in our previous study.3 The difference could be due
to the different HPV detection methods used. The
Hybribio HPV genotyping test detects only 21 HPV
genotypes, whereas the DNA sequencing method
detects more genotypes. In CIN and cancer samples,
the HPV prevalence was also significantly lower in
our study than in the previous study. These CIN and
cancer samples were paraffin-embedded tissues, and
the DNA extracted was frequently fragmented. As
the primers in the Hybribio HPV genotyping test
amplify 450 bp long products, a number of HPVpositive samples and genotypes might have been
missed.
HPV16 remained the most common high-risk
type detected in both Hong Kong and Guangzhou,
and HPV18 was the second most common type in
cervical cancer. The frequency of HPV16 increased
with increased disease severity. Consistent with
our previous finding,3 high prevalence of HPV58
and HPV52 was noted, especially in the normal and
precancerous groups.
Having previous cervical cancer screening
was an independent risk factor associated with
HPV infection in Hong Kong women, but not in
Guangzhou women. This might be due to the small
sample size. Only one third of women in Guangzhou
had a previous Pap smear test, compared with >80%
of women in Hong Kong. This might have contributed
to the higher incidence of HPV infection in healthy
women in Guangzhou. Cervical cancer screening
should be promoted to prevent cervical disease.
In Guangzhou, women having ≥3 lifetime
sexual partners had increased risk of HPV infection.
In Hong Kong, sexual behaviour factors had no such
association. A population-based study in Shanxi,
China also found no such association.4
Integration of HPV16 into the host DNA has
been proposed as a potential marker of cervical
neoplastic progression.2 E2 disruption may occur in
any part of the E2 genome, which spans about 1000
bp. To optimise the test sensitivity, we designed five
sets of primers to flank the entire E2 sequence for
HPV16 or HPV58 genes. Most of the HPV16-positive
samples harboured mixed forms of viral integration
products. The mixed integrated form of HPV16 was
the most prevalent physical state in normal cytology,
and the complete integrated form occurred in two
thirds of LG CIN cases. Similar findings have also
been reported.5
In this study, the prevalent physical state of
38
HPV58 integration was the mixed integrated form.
HPV58 commenced early during the course of lesion
progression, but this might not be essential for the
development of HPV58-related cervical cancer.
Significant differences in HPV16 and HPV58
integration patterns were not observed between
Hong Kong and Guangzhou women, although
the prevalence for both types was significantly
higher in Hong Kong women. Clinical association
with abnormal cytology was not observed with
integration. This may have been due to the limited
number of mixed swap samples and archived paraffin
samples analysed. The significant portion of normal
cytology samples harbouring the mixed integration
form suggested that HPV16 or HPV58 integration
occurred early in the cervical transformation
process leading to deregulated expression of the E6/7
oncoproteins resulting in major genomic instability
in HPV-infected cells. Therefore, detection of viral
DNA integration may have prognostic value in the
prediction of cervical lesion progression, especially
in women without detectable cervical abnormality.
Acknowledgements
This study was supported by the Research Fund
for the Control of Infectious Diseases, Food and
Health Bureau, Hong Kong SAR Government
(#05050052). We thank all doctors, research and
technical staff, and participants who contributed to
this project. Special thanks to Dr HJ Zhang in the
Department of Pathology, The University of Hong
Kong for assessment of the haematoxylin and eosin
slide, Ms Elaine Szeto in the Cytology Laboratory,
Department of Pathology, Queen Mary Hospital
for coordination of the cytology tests, and Hybribio
Ltd for technical support in HPV genotyping. We
also thank the Family Planning Association of Hong
Kong and Kwong Wah Hospital Well Women Clinic
for participant recruitment.
References
1. Schiffman MH, Bauer HM, Hoover RN, et al. Epidemiologic
evidence showing that human papillomavirus infection
causes most cervical intraepithelial neoplasia. J Natl
Cancer Inst 1993;85:958-64.
2. Jeon S, Allen-Hoffmann BL, Lambert PF. Integration of
human papillomavirus type 16 into the human genome
correlates with a selective growth advantage of cells. J Virol
1995;69:2989-97.
3. Liu SS, Tsang PC, Chan KY, et al. Distribution of six
oncogenic types of human papillomavirus and type 16
integration analysis in Chinese women with cervical
precancerous lesions and carcinomas. Tumour Biol
2008;29:105-13.
4. Dai M, Bao YP, Li N, et al. Human papillomavirus
infection in Shanxi Province, People's Republic of China: a
population-based study. Br J Cancer 2006;95:96-101.
5. Kulmala SM, Syrjanen SM, Gyllensten UB, et al. Early
integration of high copy HPV16 detectable in women with
normal and low grade cervical cytology and histology. J
Clin Pathol 2006;59:513-7.
Hong Kong Med J ⎥ Volume 20 Number 6 (Supplement 6) ⎥ December 2014 ⎥ www.hkmj.org
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