University of Groningen. New insights in methodology of screening for cervical cancer Wang, Rong

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1 University of Groningen New insights in methodology of screening for cervical cancer Wang, Rong IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2015 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Wang, R. (2015). New insights in methodology of screening for cervical cancer: Nieuwe inzichten in de methodologie van baarmoederhalskanker screening [Groningen]: University of Groningen Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:

2 Nationwide prevalence of HPV infection in China Chapter 2 Nationwide prevalence of human papillomavirus infection in 37 cities in China Rong Wang 1,2 *, Xiao-lei Guo 3 *, G. Bea. A. Wisman 2, Ed Schuuring 4, Wen-feng Wang 3, Zheng-yu Zeng 3, Hong Zhu 5, Shangwei Wu 3,1 1 Division of Clinical Microbiology, School of Laboratory Medicine, Tianjin Medical University, China 2 Department of Gynecologic Oncology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 3 Department of Microbiology, Kingmed Center For Clinical Laboratory, China 4 Department of Pathology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 5 Department of Epidemiology & Biostatistics, Tianjin Medical University, China *These authors contributed equally to this work Corresponding author Submitted for publication 37

3 Chapter 2 Abstract Background: Type-specific high-risk HPV (hrhpv) infection is related to cervical carcinogenesis. The prevalence of hrhpv infection varies geographically, which may reflect the epidemiological characteristics of cervical cancer in different populations. To lay the basis for HPV-based screening and vaccination programs in China, we investigated the latest HPV prevalence and genotypic distributions in different female age groups and geographical regions in China. Methods: In 2012, a total of 120,772 liquid-based cytological samples from women enrolled for population- or employee-based cervical screening in 37 Chinese cities were obtained by the Laboratory of Molecular Infectious Diseases of Guangzhou KingMed. Among those samples, 111,131 were tested by Hybrid Capture II and the other 9,641 were genotyped by TellgenplexTM HPV DNA Assay. Results: The total positive rate of hrhpv was 21.07%, ranging from 18.42% to 31.94% depending on regions. Age-specific prevalence showed a two peak pattern, with the youngest age group (15-19 yrs) presenting the highest hrhpv infection rate (30.55%) followed by the second peak for the old age group of yrs. Overall, the most prevalent genotypes were HPV16 (4.82%) and 52 (4.52%), followed by HPV58 (2.74%). Two genotypes HPV6 (4.01%) and 11 (2.29%) were predominant in the low risk HPV (lrhpv) type while mixed genotypes HPV16+52 and HPV52+58 were most common in women with multiple infections. Conclusions: This study shows that HPV infection in China has increased to the level of an HPV-heavy-burden country zone, with the prevalence rates varying significantly depending on ages and regions. The data from this study represents the most recent update on the nationwide prevalence of HPV infection in China, which can serve as valuable reference to guide nationwide cervical cancer screening and HPV vaccination programs. Keywords: Human papillomavirus, hrhpv prevalence, HPV genotyping, cervical cancer, China 38

4 Nationwide prevalence of HPV infection in China Background HPV infection may cause a variety of genital diseases, and type-specific persistence infection of high-risk HPV (hrhpv) is significantly relevant with the occurrence of cervical carcinogenesis 1. Cervical cancer is the third most common cancer in women worldwide 2. Effective implementation of cervical screening programs in developed countries has resulted in a steady decline in the incidence of cervical cancer 3. However, in China, the most populated country, cervical cancer remains the second leading cause of cancer deaths among the females aged from 15 to 44-year old 4. It is estimated that 75,434 women are diagnosed with cervical cancer yearly (11.3/100,000) and 33,914 (45.0%) of those die of the cancer 5,6. To date, more than 200 HPV genotypes have been identified, and approximately 40 HPV genotypes have been detected in the female genital tract. HPV16 and 18 are well known as oncogenic genotypes, in addition, HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 69 and 82 are also closely associated with cervical cancer, therefore, all of those are termed as high-risk HPV. On the other hand, as low-risk genotypes, HPV6, 11, 42, 43 and 44 are the causative agents for benign or low-grade changes in the cervical cells, for instance, genital warts 7,8. The HPV-based screening program is supposed to be an additional approach for early diagnosis of cervical cancer in order to complement the less sensitivity and non-objectiveness of cytology-based screening method 3. The high negative predictive value of hrhpv testing is applicable for the indication of low-risk population, in which the screening interval can be safely extended 3. Based on the results of clinical trials, several European countries will implement hrhpv testing as the primary screening modality 9. In addition to HPV screening, HPV vaccination has been certified as an effective strategy against HPV infection and has been implemented in most western countries recently. Although Cervarix (HPV16/18) and Gardasil (HPV6/11/16/18) protect against infection with HPV16 and 18, these vaccines provide no effect on some of the hrhpv types found in at least 25% of cervical cancers 10. Furthermore, the role of non-vaccine HPV types in the development of lesions is still unknown, it is possible that non-vaccine HPV types may replace the 39

5 Chapter 2 vaccine types as the causative agents for cervical precancerous and cancer in vaccinated cohorts without sufficient broad cross-protection 10. The prevalence of HPV infection and type-specific distribution vary greatly by geographical areas 11, as well as in the different regions within a country 12. In addition, several other risk factors may influence the prevalence of HPV, such as genetic variation, sexual behavior (age at the first sexual intercourse and the individual with multiple sex partners), biological predisposition of the immature cervix and immunodeficiency 13. Hence, surveillance in the general population is needed to assess the clinical benefit from screening and vaccination strategies. To obtain large scale information on epidemiological feature of HPV in China is crucial for the prevention strategies worldwide because of the diversities of geography and age characteristics. Although a pilot project regarding the hrhpv infection rate and cervical cancer screening were conducted in few Chinese areas in , and several similar investigations in larger scale and covering more regions were respectively achieved in 2003, 2008, 2009 and , the data are still far from a nationwide standard 9 and the information should be updated with time in order to provide reference for effective screening and vaccination as well. We hereby perform a national wide cross-sectional investigation in a large scale for the following purposes: 1) to reveal the HPV prevalence in the regions not yet investigated; 2) to follow up the potential changes of HPV infection in the regions previously studied; 3) to clarify the genotypic distribution of HPV in different regions and age-grouped populations; thereby enriching the informative resources of HPV related with cervical cancer for further study, screening and vaccination. Methods Ethics Statement The study was approved by the Ethics Committee of Tianjin medical University in accordance with the Ethical principles for Biomedical Research Involving Human Subjects (Ministry of health of the people's republic of china) and Declaration of Helsinki for Human Research of 1974 (last modified in 2000). Samples were 40

6 Nationwide prevalence of HPV infection in China originally obtained from clinical settings for laboratory diagnosis. After diagnostic testing, the excess samples were anonymized and kept for this study. An informed consent was obtained from each of the women. For the individuals under 18 years, the consent was signed by the parents. Study population Kingmed Diagnostics is the largest reference laboratory in China and provides diagnostic testing services for over 13,000 hospitals in 18 provinces and 4 municipalities in the national wide. From January to December of 2012, in the total of 120,772 samples was obtained for population- or employee-based screening from 37 cities, belonging to 18 regions (Guangdong province was regarded as one region since 20 cities in this investigation are located in that province). Figure 1 shows the map of all the geographical sites in China and the 18 regions were further grouped into 4 macro-geographical regions (East, West, South, and North). 41

7 Chapter 2 The women enrolled from population- or employee-based screening programs had an age range from 15 to 60 year-old, were sexually active and no history of cervical treatment before the screening. The exclusion criteria consisted of current pregnancy, <3 months post-partum, HIV-seropositivity and a history of either hysterectomy or treatment for cervical cancer. Out of all the samples, 111,131 collected from 15 regions (Hainan, Chongqing, Jinan, Jilin, Shenyang, Tianjin, Shanghai, Nanning, Guangdong, Guiyang, Fuzhou, Hangzhou, Chengdu, Changsha and Jiangxi) (Fig. 1) were detected by use of Hybrid Capture II (HCII) and 105,069 (94.5%) could be grouped by the ages. The other 9,641 samples from 10 regions (Shanghai, Guiyang, Xi an, Guangdong, Nanning, Changsha, Anhui, Kunming, Shenyang and Jilin) (Fig. 1) were genotyped using Tellgenplex HPV DNA Test and 9,194 (95.4%) had age information. Specimen collection According to the protocols of practice, the cervico-vaginal cells at the transformation zone of the uterine cervix were collected by a gynecologist or a trained gynecologist assistant with a standard cytobrush (with spatula), then suspended into a standard transport medium (STM) and stored at All specimens coded without knowledge of the subjects. Subsequently, all the samples were shipped to laboratory of Kingmed Diagnostics for HPV tests within 24hrs. Hybrid Capture II (HCII) Liquid-based samples were processed by following the instruction included in the Digene sample conversion kit and the hrhpv DNA panel of 13 pooled types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) was examined with the HCII HPV DNA test (Digene Corporation, Gaithersburg, Maryland). The results were determined as a ratio of mean relative light unit (RLU) for the sample to the mean RLU values for the assay of a positive calibrator (PC). A RLU-to-PC ratio >1 (~1.08 pg DNA/ml) was defined as a positive result. 42

8 Nationwide prevalence of HPV infection in China Tellgenplex TM HPV DNA Test HPV genotyping was performed using the Tellgenplex HPV DNA Test (Tellgen Life Science Co. Ltd. Shanghai, China). The assay is able to detect and genotype 26 HPV genotypes including 19 hrhpv genotypes (HPV16, 18, 26,31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 82, and 83) and 7 lrhpv genotypes (HPV6, 11, 40, 42, 44, 61 and 73). The Tellgenplex HPV kit was applied to examine the presence or absence of the most common 26 HPV genotypes simultaneously in a single test by a multiplex PCR combined with Luminex technology 20,21. The 3 steps of DNA extraction, PCR amplification and hybridization, were included in the procedure and the template of 10~20 pg/ml HPV DNA was needed for each assay. Statistical analysis Region-specific prevalence of HPV: The HPV infection rate in each region was calculated by dividing the number of HPV-positive samples by the total number of samples successfully tested for HPV. A binomial 95% confidence interval (95% CI) was estimated for each calculation of the HPV prevalence. Chi-square ( ²) tests were used to compare the differences among all the regions and each two regions. Age-specific prevalence: The HPV infection rate was estimated within 5 age groups (15-, 20-, 30-, 40- and 50-60). A binomial 95% confidence interval (95% CI) was estimated, P value for age trend of HPV infection was analyzed by using the linearby-linear association test. The difference between each two age group was compared by Chi-square ( ²) test. Multiple comparisons were performed using the Bonferoni step-down procedure to minimize the in ated risk of type 1 error 22 and P<0.05 as statistically significant. HPV-type-specific prevalence: The frequency of each hrhpv and lrhpv genotype was presented in hrhpv positive samples and lrhpv positive samples respectively. All statistical analyses were conducted using SPSS20.0 software (SPSS20, lnc., Chicago, IL). 43

9 Chapter 2 Results The total prevalence of hrhpv infection The total hrhpv infection rate was 21.07% (95%Cl 20.83%~21.31%) and the range was between 18.42% and 31.94% upon different regions. The prevalence of hrhpv infection differed significant among the various regions (P<0.001). The regions with highest hrhpv prevalence were Hainan (31.94%) and Chongqing (27.29%), followed by Jinan, Shenyang, Jilin and Tianjin. Relatively, Jiangxi, Changsha, Hangzhou, Chengdu, Fuzhou, Guangdong and Guiyang could be classified into the low burden regions (Table 1). Table 1 Region-specific prevalence of hrhpv infection by HCII Regions Positive samples Total samples Infection Rate(%) 95%CI of infection Rate(%) Hainan Chongqing Jinan Shenyang Jilin Tianjin Shanghai Nanning Guiyang Guangdong Fuzhou Chengdu Hangzhou Jiangxi Total HrHPV infection rate was different among all the regions using Chi-square test ( P<0.001). Subsequently, multiple comparisons were further performed using the Bonferoni step-down procedure : marginal difference between two most heavy-burden cities of Hainan and Chongqing (P=0.057), no difference among the second group cities including Jinan, Shenyang, Jilin and Tianjin (P=0.892), as well as the less infection rate group with Jiangxi, Changsha, Hangzhou, Chengdu, Fuzhou, Guangdong and Guiyang (P=0.758). 44

10 Nationwide prevalence of HPV infection in China As depicted in Figure 2, hrhpv infection was likely relevant with age. The group of 15-year old showed the highest prevalence (30.55%), followed by the group of (23.30%). The total infection rate of hrhpv was associated with age (P<0.001, Table S1). Next to the highest peak, the prevalence declined from 22.17% in the 20-year group to 19.71% in the 30-year group. Subsequently, the infection rate significantly increased again in 50-60year group over the 40-year group (P<0.001, Table S1). Among the four macro-regions, only the south showed the similar trend with the overall age-specific hrhpv prevalence, whereas the other three macro-geographic regions did not show significant differences in all five age groups (Figure 2, Table S1). The distribution of variant HPV genotypes Among the well-recognized 26 HPV genotypes, all of the genotypes but HPV73 were examined using Tellgenplex technique. The total infection rate of each genotype in all of the samples and the distributions of each genotype in the HPV positive portion were respectively evaluated. In descending order, the infection rate and distribution are shown in Table 2: the most common hrhpv types were HPV16 (18.02%) and HPV52 (16.9%) (P=0.288), followed by HPV58 (10.23%), then the third group, including HPV59, 56, 39, 18 and 68 (P=0.373) shared the similar proportion. 45

11 Chapter 2 Relatively, HPV6 (45.80%), HPV11 (26.15%) and HPV61 (14.20%) were common in lrhpv. Table 2 The prevalence of each HPV genotype by Tellgenplex HPV DNA Test HrHPV Genotypes Positive samples Infection Rate (in 9641 total samples)% HPV Proportion (in 2580 hrhpv positive samples)% HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV LrHPV Genotypes Positive samples Infection Rate (in 9641 total samples)% Proportion (in 844 lrhpv positive samples) HPV HPV HPV HPV HPV HPV Significant difference among the proportion of all the genotype using Chi-square ( ²) test (P<0.0001). Multiple comparisons were further performed using the Bonferoni step-down procedure, the proportion of the most common genotype HPV16 and 52 (P=0.288), as well as the proportion of HPV59, 56, 39,18 and 68 (P=0.583) were no significant difference. 46

12 Nationwide prevalence of HPV infection in China For the region-specific distribution of hrhpv, the top three genotypes were analyzed in each of the regions. HPV16, 58 and 52 were dominant in six regions, i.e., Guiyang, Xi an, Guangdong, Nanning, Changsha and Shenyang although the orders of the three genotypes may vary in different regions (Table S2a). However, different top three patterns were observed: such as HPV16, 18 and 83 in Shanghai, HPV16, 33 and 82 in Anhui, HPV16, 56 and 59 in Kunming, as well as HPV16, 52 and 58 in Jilin (Table S2a). For lrhpv, HPV11 was the most common genotype in Shanghai and Kunming, while HPV6 was the most frequent genotype in all the other regions (Table S2b). The distribution of top three HPV genotypes was also determined on the age basis. For hrhpv, HPV16, 52 and 58 were dominant among all of the age groups except the group of 15-year old, in which HPV52, 16 and 59 were the major genotypes (Table S3). As to lrhpv, HPV6 was the leading genotype in all age groups, and the second commonly detected genotype was HPV11 in the younger age groups (i.e., the groups of 15-, 20-, 30-), while in the older groups (40-, 50-60) HPV61 was the most prevalent lrhpv type (Table S3). Infection with multiple HPVs was detected in a total of 486 specimens (5.04%), among which 434 (16.82%) and 52 (6.16%) were infected with hrhpvs and lrhpvs respectively. In the multiple hrhpv infections, the frequencies of 6, 5, 4, 3 and 2 genotypes were 0.23%, 1.84%, 4.61%, 17.74% and 75.58%, respectively, and three genotypes showed higher positive rates, i.e., HPV16 (35.02%), 52 (32.26%) and 58 (21.20%) (Fig3.). The top two double-agents were detected in the decline order of HPV16+52 (26 cases) and HPV52+58 (14 cases) (Table S3.). On the regional basis, Shanghai had the highest incidence of multiple infections (19.51%), followed by Jilin (12.34%) and Nanning (6.25%) (Fig 4).Multiple infections were examined in 52 multiple lrhpv infections. The proportion of 4, 3 and 2 genotypes were 1.92%, 3.85%, and 94.23%, respectively. Nanning showed the highest incidence of multiple infections (3.13%), followed by Jilin (2.92%) and Xi an (1.40%) (Fig4). Similar to the overall age trend, within 472 cases with age information, after the first peak in the group of 15- years old, another high peak was observed in the group of 50-60years old both for hrhpv and lrhpv (P<0.001) ( Fig S1.) 47

13 Chapter 2 FIG3.The prevalence of each genotype hrhpv in multiple infections by Tellgenplex HPV DNA Test Discussion This is one of the few nation-wide investigations on high and low risk HPV in a large scale of Chinese screening population. Because of the regional difference, the population composition and the sampling period around the world, the prevalence varies study by study, the heavily burdened HPV regions are Sub-saharan Africa (24.0%), Eastern Europe (21.4%), Latin America (16.1%), and Southeastern Asia (14%) 23. In this surveillance, the overall hrhpv positive rate was 21.07% (95%Cl 48

14 Nationwide prevalence of HPV infection in China 20.83%~21.31%), which has increased into the levels of HPV-heavy-burden countries and higher than the global average level. According to the population-based screening results previously reported, the overall prevalence of hrhpv varies from 9.9%~27.5%, respectively 15. The highest infection rate is in Shanxi, a cervical cancer heavy burden region in China. And the lowest one is in Beijing, the capital of China with prosperous economic and better healthcare system. In addition to the Top and down, others are around 15%~20%, and a pool analysis including 17 populations from 9 regions shows the positive rates of hrhpv were 17.7% 24. Together, the overall hrhpv positive rate in this surveillance is increasing slightly. Compared with the region-based data, the results obtained in the present study are higher than those previously reported for Shanghai 25,26, Shenyang 25, Guangdong 27,28 and Hangzhou 29 (Table S5). In addition, some newly studied regions in this surveillance showed high hrhpv incidences, for instance, Hainan (31.94%) and Chongqing (27.29%), and several cities in north, including Jinan, Jilin and Tianjin. The data revealed that the hrhpv infection is becoming serious in both the increase of infection rate in the same regions and some of those reaching to the level of heavy burden regions as well. Of course, the great improvement in screening strategy and laboratory methods could also partially contribute to the increased prevalence. As to the age-relevant hrhpv prevalence, a meta-analysis conducted by Bruni et al 23 showed a bimodal age distribution, with the peak of HPV infection occurring within a younger age group (just after sexual debut). Globally, a lower prevalence plateau in the middle age group, followed by a gradual reduction of incidence in developing countries or a second peak in developed countries 23. The trend of hrhpv infection showing high in younger and low in middle age groups reflects the natural history of HPV infection. Young females are sensitive to HPV soon after beginning of sexual activity due to immature immune protection, nevertheless most of HPV infection are usually temporary; thus in 70% and 91% of women infected with HPV, the virus may be cleared within one or two years respectively 30. The slight increase of the HPV infection rate in older females might reflect the viral persistence or the 49

15 Chapter 2 reactivation of latent HPV assumingly because of the physiological and immunological disorders resulted from hormone fluctuations during the menopausal transition 31. In this investigation, the general age distribution showed the first peak of hrhpv in the age group of 15 years (30.55%), then gradually decreasing in the middle age, which is consistent with the data from Bruni et al 23. However, hrhpv infection rate was significantly increased in the age group of years compared to the women in 40s, a similar trend seen in most developed countries. The prosperous economy in most of the metropolis may have influence on the culture and sexual behavior of Chinese people. Nonetheless, the exact mechanisms for the increase of HPV prevalence remain unclear at this moment. Information concerning the distribution of HPV genotypes is important not only for vaccine development but also for HPV-based screening design, particularly for selecting the testing spectrum of HPV genotypes 32 and multiple HPV infection detection. It is a prerequisite for the genotyping assays in cervical cancer screening programs. However, HCII, the only Food and Drug Administration (FDA) approved test cannot provide any genotype-specific information 33. Therefore, Tellgenplex HPV DNA Test, a reported genotyping method 21, was performed in this surveillance. In the in-house clinical validation according to CAP(College of American Pathologists) standards, the correlation between Tellgenplex HPV DNA Test and HCII was shown acceptable (Coincident rate 90.5%, Kappa=0.88). Consistent with the results yielded from some of Chinese population-specific investigations, HPV16, 52 and 58 are the dominant hrhpv types 34. Compared with the data by Wu et al 25, a population-based investigation from 5 regions (Beijing, Shanghai, Xinjiang, Henan and Shangxi) in China, the infection rates of HPV16 (4.82%), 52 (4.52%) and 58 (2.74%) were all higher than the HPV16 (2.9%), 52 (1.7%) and HPV58 (1.5%) obtained in this study. Whereas, as to the proportion rate of these three genotypes, besides HPV52 (16.9% &11.9%: P=0.006), which is higher in the present study and there are no significant differences in the proportion of HPV16 (20.2%&18.02%: P=0.245) and 58 (10.8 %&10.23%: P=0.686). The proportion of HPV16 in this surveillance was even lower than that in the normal cytology samples reported by Guan et al and Bruni et al (20.4% and 22.5%, 50

16 Nationwide prevalence of HPV infection in China respectively) 23,35. Relatively, HPV52 and 58 accounted for 27.13%, which is dramatically higher than the global level of 14.37% 35. Interestingly, although both HPV52 and 58 were all common in Asian population, the significance of the two genotypes is still unknown. Zhao et al 36 reported that HPV52 infections are more common among healthy individuals, whereas HPV58 is reported to be related to cervical cancer. Some studies conducted in the South and West regions in China, only including CIN or cancer samples, have demonstrated that HPV58 is more prevalent than HPV52 18,27,37,38. HPV18, next to HPV16, is known to be most important for cervical carcinogenesis 39. However, in our study it was at the seventh position, and the infection rate (1.48%) is in line with the study of Wu et al. (P=0.871) 25. In addition to carcinogenesis, many benign cutaneous warts, mucosal lesions and low-grade cervical intraepithelial lesions generate a considerable health burden associated with lrhpv infection 40. Specifically, HPV types 6 and 11 cause 90% of genital warts, over 95% of recurrent respiratory papillomatosis cases, and approximately 10% of early cervical lesions 41. The infection will become more serious in immune-compromised individuals 40. In our surveillance, the incidence of HPV6 (4.01%) infection was inconsistent with the results of Bruni et al 23, showing that HPV6 is most frequent in Americans (2.9%) and is less frequent in Asian individuals (0.2%), followed by HPV11 and 61. Characterization of the prevalence of multiple HPV infections might be important for the effect on cervical carcinogenesis. Herrero et al 10 reported that women infected with HPV16 alone were at similar or higher risk for cervical cancer than those infected with both HPV16 and another HPV type. Lee et al 42 reported an association between infection with multiple HPV types and the increased risk of cervical cancer. In a recent study, Schmitt et al. confirmed that co-infection will increase the infection duration. Furthermore, patients with multiple high viral loads showed a 4- to 6-fold increased risk of cervical precancerous cytological lesions than patients with single high viral loads 43. In the present study, 434 hrhpv-positive samples (4.5%) were multiple-infections. The incidence is in the same range ( 3.5%-5.3%) as reported in China 34, but higher than the global average (3.2%) 23 and the proportion (14.19%) is lower than both domestic (25.8%) 25 and international (20%) 23. The most common 51

17 Chapter 2 combinations of two types were HPV16+52 (26 cases) and HPV (14 cases), and the genotypes 52 and 58 were more likely involved in co-infections. From the region-specific surveillance, some geographical features were observed, for instance in Shanghai, a metropolis with internalized population, showing a situation close to the world in the HPV prevalence, the genotypic distribution and multiple infections. This study has confirmed the high incidence of HPV in overall China and strongly argues the necessity for developing the national population-based screening programs. However, the appropriate management of this HPV screening program for the large number of women with HPV-positive specimens and no cytological evidence of cervical pre-cancer or cancer remains a major concern 44. HPV genotyping could be an option to stratify the HPV positive women. Simultaneously, a stainable HPV detection and closely follow-up for the hrhpv carried women should be implemented, thus more cost-effective techniques, for instance, Cervista TM HR HPV test, COBAS HPV test and some other genotyping platforms might be the alternatives for molecular HPV detection 45. Conclusions In conclusion, China has large population and a variety of territories; meanwhile, economic conditions, cultural habits and population migrations have affected on Chinese daily life and health situation dramatically, for instance, the cancers related with sexual transmitted diseases. Therefore, attention should be paid to the prevalence of HPV infection on the timely and regional basis because it has been commonly recognized that the HPV infection plays a crucial role in the occurrence of cervical cancer and the increase incidence. This surveillance results have indicated that a national plan for cervical screening program is urgently needed, not only because the increase in infection rate in some previously reported regions, but also the high infection rate in most of newly investigated regions. Shortly, the most significant discoveries by this investigation are following: (1). the prevalence of hrhpv infection has reached a level that could not be ignored, and the prevalence increase is ongoing with time obviously; (2). the prevalence of hrhpv infection showed population variations on age and region as well as reflected economic, cultural and lifestyle relevance; (3). the HPV16, 52 and 58 constituted the three 52

18 Nationwide prevalence of HPV infection in China dominant genotypes consistently in different Chinese populations, a characteristic pattern significantly different from the epidemiological features in most of industrial countries, which defined the principles for cervical screening and vaccination in China should be proposed distinguishably. List of abbreviations HPV: Human papillomavirus; hrhpv: high-risk HPV; lrhpv: low risk HPV; HCII: Hybrid Capture II; CAP: College of American Pathologists Competing interests Shangwei Wu is Medical Director of Kingmed Diagnostics. E. Schuuring is a member of the scientific advisory board of Roche, Hologic and QCMD, received travel reimbursements from Roche, Abbott, Hologic Inc. and QCMD. Authors contributions RW, XLG WFW and ZZY carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. GBAW, ES and HZ participated in the design of the study and performed the statistical analysis. SWW and RW conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. Acknowledgements Rong Wang is appointed to a collaborative project between University of Groningen in the Netherlands and Tianjin medical University of China. This study was supported by the grant from natural science foundation of Tianjin (12JCYBJC33700). References 1. Zur Hausen H: Papillomaviruses in human cancers. Mol Carcinog 1:147-50, Jemal A, Bray F, Center MM, et al: Global cancer statistics. CA Cancer J Clin 61:69-90, Priebe AM: 2012 cervical cancer screening guidelines and the future role of HPV testing. Clin Obstet Gynecol 56:44-50,

19 Chapter 2 4. Human Papillomavirus and Related Cancers. WHO/ICO HPV information centre summary report 5. Ferlay J, Shin HR, Bray F, et al: Estimates of worldwide burden of cancer in 2008: GLOBOCAN Int J Cancer 127: , Internation Agency for Research on Cancer(IRAC). IARC handbooks of cancer prevention: cervix cancer screening,chapter 2. Lyon:IARC Press 7. de Villiers EM, Fauquet C, Broker TR, et al: Classification of papillomaviruses. Virology 324:17-27, Munoz N, Bosch FX, de Sanjose S, et al: Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 348:518-27, Castle PE, de Sanjose S, Qiao YL, et al: Introduction of human papillomavirus DNA screening in the world: 15 years of experience. Vaccine 30 Suppl 5:F117-22, Herrero R: Human papillomavirus (HPV) vaccines: limited cross-protection against additional HPV types. J Infect Dis 199:919-22, de Sanjose S, Diaz M, Castellsague X, et al: Worldwide prevalence and genotype distribution of cervical human papillomavirus DNA in women with normal cytology: a meta-analysis. Lancet Infect Dis 7:453-9, Krul EJ, Van De Vijver MJ, Schuuring E, et al: Human papillomavirus in malignant cervical lesions in Surinam, a high-risk country, compared to the Netherlands, a low-risk country. Int J Gynecol Cancer 9: , Vinodhini K, Shanmughapriya S, Das BC, et al: Prevalence and risk factors of HPV infection among women from various provinces of the world. Arch Gynecol Obstet 285:771-7, Belinson J, Qiao Y, Pretorius R, et al: Prevalence of cervical cancer and feasibility of screening in rural China: a pilot study for the Shanxi Province Cervical Cancer Screening Study. Int J Gynecol Cancer 9: , Li J, Huang R, Schmidt JE, et al: Epidemiological features of Human Papillomavirus (HPV) infection among women living in Mainland China. Asian Pac J Cancer Prev 14: , Zhao R, Zhang WY, Zhang SW, et al: [Study on subtype of human papillomavirus infection among aged reproductive women in Beijing from 2006 to 2008]. Zhonghua Fu Chan Ke Za Zhi 46:184-7, Wu D, Cai L, Huang M, et al: Prevalence of genital human papillomavirus infection and genotypes among women from Fujian province, PR China. Eur J Obstet Gynecol Reprod Biol 151:86-90, Li J, Mei J, Wang X, et al: Human papillomavirus type-specific prevalence in women with cervical intraepithelial neoplasm in Western China. J Clin Microbiol 50: ,

20 Nationwide prevalence of HPV infection in China 19. Han J, Swan DC, Smith SJ, et al: Simultaneous amplification and identification of 25 human papillomavirus types with Templex technology. J Clin Microbiol 44: , Wei W, Shi Q, Guo F, et al: The distribution of human papillomavirus in tissues from patients with head and neck squamous cell carcinoma. Oncol Rep 28:1750-6, Hu Y, Qian HZ, Sun J, et al: Anal human papillomavirus infection among HIV-infected and uninfected men who have sex with men in Beijing, China. J Acquir Immune Defic Syndr 64:103-14, Ludbrook J: Multiple comparison procedures updated. Clin Exp Pharmacol Physiol 25:1032-7, Bruni L, Diaz M, Castellsague X, et al: Cervical human papillomavirus prevalence in 5 continents: meta-analysis of 1 million women with normal cytological findings. J Infect Dis 202: , Zhao FH, Lewkowitz AK, Hu SY, et al: Prevalence of human papillomavirus and cervical intraepithelial neoplasia in China: a pooled analysis of 17 population-based studies. Int J Cancer 131: , Wu EQ, Liu B, Cui JF, et al: Prevalence of type-specific human papillomavirus and pap results in Chinese women: a multi-center, population-based cross-sectional study. Cancer Causes Control 24: , Zhang R, Shi TY, Ren Y, et al: Risk factors for human papillomavirus infection in Shanghai suburbs: A population-based study with 10,000 women. J Clin Virol, Chen Q, Xie LX, Qing ZR, et al: Epidemiologic characterization of human papillomavirus infection in rural Chaozhou, eastern Guangdong Province of China. PLoS One 7:e32149, Wu RF, Dai M, Qiao YL, et al: Human papillomavirus infection in women in Shenzhen City, People's Republic of China, a population typical of recent Chinese urbanisation. Int J Cancer 121: , Ye J, Cheng X, Chen X, et al: Prevalence and risk profile of cervical Human papillomavirus infection in Zhejiang Province, southeast China: a population-based study. Virol J 7:66, Baseman JG, Koutsky LA: The epidemiology of human papillomavirus infections. J Clin Virol 32 Suppl 1:S16-24, Hildesheim A, Wang SS: Host and viral genetics and risk of cervical cancer: a review. Virus Res 89:229-40, Smith JS, Lindsay L, Hoots B, et al: Human papillomavirus type distribution in invasive cervical cancer and high-grade cervical lesions: a meta-analysis update. Int J Cancer 121:621-32, Cho EJ, Do JH, Kim YS, et al: Evaluation of a liquid bead array system for high-risk human papillomavirus detection and genotyping in comparison with Hybrid Capture II, DNA chip and sequencing methods. J Med Microbiol 60:162-71,

21 Chapter chung-yung chen c-hw: Human papillomavirus type distribution in southern china and taiwan,. human papillomavirus and related diseases-from bench to bedside-research aspects, Guan P, Howell-Jones R, Li N, et al: Human papillomavirus types in 115,789 HPV-positive women: a meta-analysis from cervical infection to cancer. Int J Cancer 131: , Zhao FH, Tiggelaar SM, Hu SY, et al: A multi-center survey of age of sexual debut and sexual behavior in Chinese women: suggestions for optimal age of human papillomavirus vaccination in China. Cancer Epidemiol 36:384-90, Wang S, Wei H, Wang N, et al: The prevalence and role of human papillomavirus genotypes in primary cervical screening in the northeast of China. BMC Cancer 12:160, Liu SS, Chan KY, Leung RC, et al: Prevalence and risk factors of Human Papillomavirus (HPV) infection in southern Chinese women - a population-based study. PLoS One 6:e19244, zur Hausen H: Papillomaviruses in the causation of human cancers - a brief historical account. Virology 384:260-5, Leto M, Santos Junior GF, Porro AM, et al: Human papillomavirus infection: etiopathogenesis, molecular biology and clinical manifestations. An Bras Dermatol 86:306-17, Preaud E, Largeron N: Economic burden of non-cervical cancers attributable to human papillomavirus: a European scoping review. J Med Econ, Lee SA, Kang D, Seo SS, et al: Multiple HPV infection in cervical cancer screened by HPVDNAChip. Cancer Lett 198:187-92, Schmitt M, Depuydt C, Benoy I, et al: Multiple HPV infections with high viral loads are associated with cervical lesions but do not differentiate grades of cervical abnormalities. J Clin Microbiol, Gage JC, Ajenifuja KO, Wentzensen NA, et al: The age-specific prevalence of human papillomavirus and risk of cytologic abnormalities in rural Nigeria: implications for screen-andtreat strategies. Int J Cancer 130:2111-7, Munson E, Du Chateau BK, Bellerose B, et al: Clinical laboratory evaluation of Invader(R) chemistry and hybrid capture for detection of high-risk human papillomavirus in liquid-based cytology specimens. Diagn Microbiol Infect Dis 71:230-5,

22 Nationwide prevalence of HPV infection in China Table S1. Age-specific HPV infection in each region by HCII regions 15-yrs 20-yrs 30-yrs 40-yrs 50-60yrs Shanghai 0.00% 22.81% 20.33% 27.27% 0.00% Jiangxi 25.00% 21.79% 16.60% 18.15% 23.66% Hangzhou 19.44% 18.83% 19.88% 19.82% 21.45% East 18.75% 20.62% 18.71% 19.25% 22.18% Chengdu 11.63% 22.55% 19.69% 18.32% 15.71% Guiyang 29.52% 17.88% 21.86% 20.86% 17.09% Chongqing 30.77% 29.02% 24.43% 26.35% 34.29% West 25.29% 20.98% 21.27% 20.59% 19.01% Guangdong 33.11% 21.76% 18.15% 18.85% 23.26% Nanning 21.15% 22.25% 22.22% 22.32% 23.48% Hainan 36.36% 22.13% 29.49% 37.30% 34.85% Changsha 50.00% 23.15% 13.60% 15.46% 25.00% Fuzhou 19.44% 20.91% 20.08% 20.13% 13.95% South 31.67% 21.81% 18.72% 19.51% 23.19% Tianjin 28.30% 26.78% 23.19% 25.10% 23.67% Jilin 37.50% 25.30% 24.21% 24.28% 30.16% Jinan 18.64% 25.34% 26.36% 26.18% 24.40% Shenyang 71.43% 17.11% 25.42% 24.19% 26.32% North 25.51% 25.58% 25.58% 25.73% 24.81% Total 30.55% 22.17% 19.71% 20.55% 23.30% In Total. P value for age trend of HPV infection was analyzed by using the linear-by-linear association test (P<0.001). Furthermore, Infection rate is difference in each age group in addition to the margin difference in the age group of 20-yrs and 50-60yrs (P=0.051). In the Four marco-geographic regions, in addition to the South(p<0.001), the other three including the East, West and North were all no significant.(p=0.401, 0.595, 0.974) in each age group. 57

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25 Chapter 2 Table S2b. Region-specific lr HPV subtype prevalence by Tellgenplex HPV DNA Test Total positive HPV6 HPV11 HPV40 HPV42 HPV44 HPV61 Shanghai Positive rate 29.27% 9.76% 14.63% 0.00% 0.00% 0.00% 4.88% % 33.33% 50.00% 0.00% 0.00% 0.00% 16.67% East % 9.76% 14.63% 0.00% 0.00% 0.00% 4.88% % 33.33% 50.00% 0.00% 0.00% 0.00% 16.67% Guiyang Infection rate 6.55% 2.43% 1.87% 0.47% 0.19% 0.56% 1.03% proportion % 37.14% 28.57% 7.14% 2.86% 8.57% 15.71% Xi'an Infection rate 16.53% 8.96% 4.20% 0.56% 0.28% 1.68% 0.84% proportion % 54.24% 25.42% 3.39% 1.69% 10.17% 5.08% West % 4.07% 2.45% 0.98% 0.84% 0.49% 0.21% % 44.96% 27.13% 10.85% 9.30% 5.43% 2.33% Guangdong Infection rate 4.96% 1.99% 1.20% 0.29% 0.11% 0.42% 0.95% proportion % 40.18% 24.11% 5.80% 2.23% 8.48% 19.20% Nanning Infection rate 10.94% 7.81% 3.13% 0.00% 0.00% 0.00% 0.00% proportion % 71.43% 28.57% 0.00% 0.00% 0.00% 0.00% Changsha Infection rate 7.01% 3.43% 1.65% 0.36% 0.14% 0.50% 0.93% proportion % 48.98% 23.47% 5.10% 2.04% 7.14% 13.27% Anhui Infection rate 7.14% 2.38% 4.76% 0.00% 0.00% 0.00% 0.00% proportion % 33.33% 66.67% 0.00% 0.00% 0.00% 0.00% Kunming Infection rate 8.39% 1.29% 2.58% 0.00% 0.65% 0.00% 3.87% proportion % 15.38% 30.77% 0.00% 7.69% 0.00% 46.15% South % 2.36% 1.38% 0.29% 0.13% 0.42% 1.00% % 42.32% 24.64% 5.22% 2.32% 7.54% 17.97% Shenyang Infection rate 26.79% 13.58% 7.53% 1.77% 0.84% 0.47% 2.60% proportion % 77.66% 43.09% 10.11% 4.79% 2.66% 14.89% Jilin Infection rate 7.58% 3.57% 1.52% 0.22% 0.43% 0.32% 1.52% proportion % 47.14% 20.00% 2.86% 5.71% 4.29% 20.00% North % 8.95% 4.75% 1.05% 0.65% 0.40% 2.10% % 50.00% 26.54% 5.87% 3.63% 2.23% 11.73% 60

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28 Nationwide prevalence of HPV infection in China Table S4. The common combinations of double infections by Tellgenplex HPV DNA Test Type1 Type2 Positive cases HPV16 HPV52 26 HPV 52 HPV HPV16 HPV59 13 HPV 52 HPV HPV16 HPV58 10 HPV 16 HPV HPV 52 HPV 68 9 HPV16 HPV56 8 HPV16 HPV39 7 HPV16 HPV68 7 HPV16 HPV51 7 HPV 16 HPV 66 6 HPV 52 HPV 59 6 HPV 52 HPV 18 6 HPV 58 HPV 56 6 HPV16 HPV18 5 HPV 16 HPV 82 5 HPV 52 HPV 56 5 HPV 58 HPV 18 5 HPV 58 HPV 33 5 HPV 58 HPV 66 5 HPV 59 HPV 56 5 HPV 39 HPV 68 5 HPV 39 HPV 53 5 HPV 68 HPV 31 5 FIG S1. Age-specific multiple infection by Tellgenplex HPV DNA Test 63

29 Chapter 2 Table S5. HPV Prevalence in Women from population-based Screening Studies Author /Pub year (Belinson et al.2001) (Zhao et al., 2001) (Shen et al., 2003) (Zou et al., 2011) (Li et al., 2007) (Li et al., 2006) (Dai et al., 2006) (Wu et al., 2007) (Zhao et al., 2012b) (Li et al., 2010) (Ye et al.,2010 (Hu et al., 2011) (Zhang et al.,2013) Study year Location Number(N) Age Range (yrs) 1999 Xiangyuan 1, HC Xiushui, Jiangxi 2, HC Labassays Xiangyuan and Yangcheng,shanxi 8, HC Xiangyuan and 9, HC Yangcheng,shanxi 2004 Yangcheng HC Shenyang GP5+/6+ mediated PCR Yangcheng GP5+/6+ mediated PCR Shenzhen 1, GP5+/6+ mediated PCR overall HPV prevalence (%) Shanxi, Beijing(18.8), Xinjiang(8.2), Henan(13.03), Shanghai(18.66) 13, HC Beijing 6, HC2 9.9 zhejiang 5, PCR (MY09/11 primer) Jiangsu HC Shanghai suburb 10, PCR (MY09/11 primer) 12.6 Compared with the region-based data, the results obtained in the present study are higher than those previously reported for Shanghai (this cohort & previously: 22.61% & 18.66% in urban and 12.6% in suburbs) 25,26, Shenyang (25.32% & 16.8%) 25, Guangdong (20.02% & 16.6% in urban and 9.03% in rural) 27,28 and Hangzhou (19.85% & 13.3%)