Genotype Distribution of Human Papillomavirus in Women with Abnormal Cervical Cytology in an Esophageal Carcinoma High Incidence Area of China

Cervical cancer is the third most commonly diagnosed cancer and the fourth leading cause of cancer death in females worldwide, which accounting for 9% of the total new cancer cases and 8% of the total cancer deaths among females with more than 85% of these cases and deaths occur in developing countries (Jemal et al., 2011). The incidence of cervical cancer was estimated about 8.7/100, 000 in females and accounts about 6.3% of all female cancer in China (Kim et al., 2009; Guo et al., 2012; Wang et al., 2012), with obviously geographic variation (Li et al., 2011; Shi et al., 2012). Approximately 9.09% cytological positive rate of cervical cancer and its precancerous lesions had been found in Guangdong, southern China (Zheng et al., 2009). Interestingly, the Shantou coastal area of eastern Guangdong has also a high incidence of esophageal cancer (EC) (Su et al., 2007). Histological type for both cancers is squamous cell carcinoma (SCC) in Shantou, and


Introduction
Cervical cancer is the third most commonly diagnosed cancer and the fourth leading cause of cancer death in females worldwide, which accounting for 9% of the total new cancer cases and 8% of the total cancer deaths among females with more than 85% of these cases and deaths occur in developing countries (Jemal et al., 2011).The incidence of cervical cancer was estimated about 8.7/100, 000 in females and accounts about 6.3% of all female cancer in China (Kim et al., 2009;Guo et al., 2012;Wang et al., 2012), with obviously geographic variation (Li et al., 2011;Shi et al., 2012).Approximately 9.09% cytological positive rate of cervical cancer and its precancerous lesions had been found in Guangdong, southern China (Zheng et al., 2009).Interestingly, the Shantou coastal area of eastern Guangdong has also a high incidence of esophageal cancer (EC) (Su et al., 2007).Histological type for both cancers is squamous cell carcinoma (SCC) in Shantou, and

Genotype Distribution of Human Papillomavirus in Women with Abnormal Cervical Cytology in an Esophageal Carcinoma High Incidence Area of China
Rui-Qin Mai 1 , Bo Huang 2 , Ling Shen 3 , Guo-Hong Zhang 2, 4 , Liang-Li Hong 5 *, Ying-Mu Cai 1 * esophageal SCC (ESCC) and cervical SCC (CSCC) have similar multi-stage histopathological progression pattern: from normal mucosa to dysplasia, carcinoma in situ, and invasive carcinoma.Moreover, the predilection site of both ESCC and CSCC is the junction of the squamous and columnar epithelium (Liu et al., 2014).Those similarities supported that common environment risk factors might have etiologic effect on both ESCC and CSCC.
It is well-established that human papillomavirus (HPV) infection is the central cause in the development of cervical cancer.Furthermore, the HPV as oncogenes or co-factors in the carcinogenesis of EC has been reported in the last 30 years (Petrick et al., 2014), especially combinative study of HPV infection rate on EC and cervical cancer tumor samples in geographic areas with a high incidence of EC (Zhang et al., 2010;Liu et al., 2014).According to epidemiologic classification, at least 15 oncogenic HPV types were found in cervical precancerous lesions and carcinoma, of which HPV16 and HPV18 were the most prevalent high-risk types worldwide, whereas HPV-58 has been found in a relatively higher proportion in eastern Asia (Chan et al., 2012).HPV prevalence of cervical smear in women shows different by geographic region varied from 6.7% to 29.6% found by population-based studies with different type-specific in China and 9.03% was found in Chaozhou population, which is closed to Shantou, with the most common type of HPV-52 and -58 (Lin et al., 2008).The incidence rate was 65.5% and 69.1% in tissues of ESCC cancerous and precancerous have been reported in Shantou (Shen et al., 2002).However, to our knowledge, no reliable epidemiologic data described HPV type-specific distributions in cervical precancerous lesions in Shantou have been reported.Current vaccines protect against 2 HPV types, HPV 16 and 18, which are associated with 70% of cervical cancers and 50% of highgrade cervical lesions.Perhaps, considering of geographic variation and type-specific HPV prevalence in women with cervical cytologic abnormalities could offer more benefit from cervical cancer (Clifford et al., 2005;Gage et al., 2013).
The aim of this present study was to address the description of HPV prevalence and type-specific distribution in women with cervical cytological abnormalities and to identify the most frequent multiple HPV types associated with different cervical lesions in Shantou population, which has high incidence of EC by a hospital-based study.

Study design and population
Shantou locates on the southeast coast of Guangdong province and is adjacent to Chaozhou, Jieyang city and southwestern Fujian province and Taiwan.Shantou City has a total area of 2, 064 square kilometer, with a population of about 5, 294, 400.The study group was consisted two kinds of consecutive participants who proceeded to routine screening or the Outpatient Gynaecological Clinic of the First Affiliated Hospital, Shantou University Medical College between September 2009 and August 2013.Eligible participants were identified from the database.Women were eligible if they had no previous diagnosis or treatment for cervical or vaginal disease, had not undergone hysterectomy and were not pregnant.All participants included in the study gave their written informed consent after discussing with Gyneacologists that there would be no implications to their health.The studied population was divided into 6 age groups according to the biological alterations in women's reproductive system (18-25 years, 26-35 years, 36-45 years, 45-55 year, 56-65 years and >65 years).Procedures for this study were approved by Research Ethics Boards at Shantou University Medical College.

Cervical specimen collection and HPV DNA extraction
Cervical exfoliated cell specimens were obtained by a gynecologist as part of routine investigative procedures at the hospital.Two separate cervical exfoliated cell specimens were collected independently for liquid-based cytological diagnosis and HPV DNA genotyping assays.
For HPV DNA extraction, cells stored at a specimen transport medium (Hybribio Biotechnology Limited Corp., Chaozhou, China).High-quality DNA was yielded from lysis of cells by isolation of DNA, precipitation, and purification according manufacturer's instruction (Hybribio Biotechnology Limited Corp., Chaozhou, China).

Cytological diagnosis criteria
Cervical slides were prepared using a liquid-based cytologymethod.Cytological classifications of disease grade were made in conformity to the Bethesda 2001 criteria (TBS2001) (Solomon et al., 2002).The evaluation system included: negative (0), atypical squamous cells of undetermined significance (ASC-US), low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL), squamous cell carcinoma (SCC) and atypical glandular cells (AGC).When a diagnosis of ASC-US was confirmed, the case was subclassified as ASC-US or ASC-H.The slides were evaluated for cervical cytology by three academic cytopathologists of the First Affiliated Hospital of Shantou University Medical College.

HPV genotyping
HPV DNA was amplified in a KP-TC48 (Chaozhou Hybribio Biotechnology, China) and genotyping for HPV was performed by flow-through hybridisation and gene chip by HybriMax (Chaozhou Hybribio Limited Corporation, Chaozhou, China).The gene chip contained type-specific oligonucleotides immobilised on a nylon membrane.The final results were detected by colourimetric change on the chip under direct visualisation.The chip allowed for the detection of the following 21 HPV genotypes: 15 highrisk (HR) types , 6 low-risk (LR) types ).

Statistical analysis
Categorical variables were summarized by absolute frequencies and percentages, and continuous variables by means and Standard Error (SE).The Chi-square test and Fisher's exact test were used to compare proportions; the t-test was used to compare continuous variables.The data were analyzed by using SPSS 16 software.

HPV prevalence in the study population
During the study period, total of 22, 989 consecutive women were subjected to gynecologic examination with cervical HPV-DNA testing.The final sample consisted of 22, 617 women with complete cytological and HPV-DNA testing results.Mean age of the study group was 36.41 ± 9.93 years.All of 21 different HPV types were identified in this study population.Overall, Table 1 showed the prevalence of any HPV including both low-risk and high-risk types in the study population was 19.76% (4468/22617), while high-risk HPV was positive in 16.97% (3837/22617) of all women in the study group.The 3 most prevalent HPV types were HPV-52 (4.27%,  2).

HPV prevalence according to age
The 22617 women were classified by their age into 6 groups, and the age-specific prevalence of HR-HPV types were shown in Figure 1.A U-shaped age-specific prevalence curve was observed in HR-HPV positive women, specifically, prevalence decreased from 23.49% at age 18-25 years to a peak of 14.75% at age 26-35 years and 14.94% at age 36-45 years, then prevalence increased progressively from 16.91% at 46-55 years to 24.69 and 28.89% at ages 56-65 and > 65 years, respectively.

HPV prevalence according to cervical cytology abnormalities
Cervical cytological abnormalities were observed in 2.22% of women, of which 0.06% ASC-H, 1.55% ASC-US, 0.27% HSIL, 0.34% LSIL and 0.01% AGC.The mean age was 37.6years.As far as cytology was concerned, the overall HPV prevalence in women with cervical cytological abnormalities was 52.49%, and by lesion type was 71.73% (ASC-H), 42.0% (ASCU-S), 81.79% (HSIL) and 73.68% (ILSL).Furthermore, the proportion of samples containing at least one of the 15 HR-HPV was greater in SIL (76.64%) than in ASC (40.38%).The prevalence of single and multiple HR-HPV according to histological diagnosis are shown in Table 3.To our knowledge, the current study is the first study of the prevalence of HPV in women with cervical cytologic abnormalities, a large sample size in Guangdong with participation of more than 500 patients.

Discussion
The HPV type-specific in women with cervical cytologic abnormalities remains uncertain in Shantou, which is also an ESCC high-incidence area.To our knowledge, this is the first study to describe prevalence of HPV infection and its subtypes and their association with cervical cytological abnormalities by analyzing the whole spectrum of all 15 known HR-HPV types in a large scale population and in a representative set of women with cervical abnormalities in Shantou.
In the absence of information on type-specific HPV infection found in cervical cytological abnormalities in Shantou population, our data from representative set of 503 women with cervical abnormalities showed the 3 dominant HR-HPV types were HPV-58 (14.12%, 71/503), -16 (13.72%, 69/503) and 52 (12.72%, 64/503).HPV-58 had been found as a much higher proportion of cervical cancers in East Asia than other types (Chan et al., 2011).Similarly, the 3 most common types have been found in Korea population (Kim et al., 2013).HPV 58 and 52 were among the five most common types in invasive cervical carcinoma in eastern and southeastern Asia (Bao et al., 2008).A high prevalence of HPV-58 among CSCC has been reported in 33% in Shanxi and 28% in Shanghai China (Chan, 2012;Chan et al., 2013), as the second types with prevalence 21.8% in Western China, respectively (Li et al., 2012).Previous studies suggested that testing for individual HPV genotypes can improve risk stratification in women with minor cytologic abnormalities (Gage et al., 2013), and knowledge of the prevalence and typespecific HPV, either as single or multiple infections, in the cervical cytological abnormalities will be important for an appropriate intervention in cervical cancer prevention, and to understand the potential impact of HPV vaccines in the population.Interesting, the prevalence of genital HR-HPV infection in male sexual partners of HPVpositive women in Chaozhou was lower (5.32%)than that expected, and the concordance of HR-HPV between couples was extremely low (2.63%).These findings suggested that infected men consitute an important viral reservoir, contributing to transmission of HR-HPV to women and maintenance of infection, but HR-HPV infection may be less likely to persist in men than in women (Huang et al., 2013).Therefore, our data provide further information about the type prevalence in women with cervical cytological abnormalities in Shantou and might be used for the screening individuals with the highrisk type 58 in the men.Moreover, it will be important to monitor possible changes in HR-HPV type distributions in the vaccinated and HPV-screened population to better understand cross-protection and to identify putative new HR-HPV types once HPV16 and HPV18 are eliminated.Heterogeneity in HPV type-specific distribution in Shantou should be taken into account for the secondgeneration vaccines, speciallyHPV-58, and 52 may offer higher benefit for women in Shantou and neighborhood.Although the significance of HPV-58 in cervical cytological abnormalities is poorly understood, HPV-58 and 18 was associated with lack of treatment response (Munagala et al., 2009).
Combined the HPV type and cytologic abnormalities, HPV-16 was the predominant type in ASC-H (42.86%) and HSIL (36.07%),HSIL is preferentially caused by HPV16 and less often caused by other genotypes (Kovacic et al., 2006).Our results show some interesting differences and comprehensive spectrum with cervical cytological abnormalities data in Shantou, comparing Chaozhou.The most frequent types we detected in ASC-US were HPV-52 (10.29%, 36/350), followed by HPV-58 (10%, 35/350) and -16 (8.29%, 29/350), which differ from HPV-52 as the most common types detected in only ASC in Chaozhou.HPV-58 was the most common type in women with LSIL (19/76), followed by HPV-16 (12/76) and -33 (11/76), while HPV-16 had been found as the most common type in LSIL in Chaozhou.In ASC-US, HSIL and ISIL lesions, HR-HPV 33 had been found as the fourth predominant HR-HPV type.In some level, those data suggested the infective context is special in the women with cervical cytological abnormalities in Shantou.
From prevalence, 16.70% (84/503) of women with cervical cytological abnormalities contained two or more HR-HPV types.Multiple HR-HPV positivity was significantly more common in SIL (31.39%, 43/137) than in ASC (11.26%, 41/364), and the HSIL (34.43%) had highest multiple infections, comparing ASC and ILSL.In our population, HPV 58 was the most prevalent type, either as a single infection or combined with other genotypes.Multiple infections were most prevalent of HPV-58/33 co-infection, followed by HPV-58/16 (14.29%; 12/84) and HPV58/52 (11.90%; 10/84).Furthermore, HPV-58/33 co-infection was most combination for ASC-US (20.00%, 8/40) and ISIL (18.18%, 4/22), while HPV-58/16 co-infection was common for HSIL (28.57%).Multiple HPV infections correlated most prominently with lack of treatment compared with single type infection (Munagala et al., 2009).And several studies indicated that the cumulative burden of HPV multiple infections maybe a marker of differential immune response.Our data provided some evidence that risk in the context of HPV-58 may be particularly modulated by coinfections with other types in women with cervical cytological abnormalities.Co-infection with multiple HPV types had been observed more frequently among younger women and among those with cytologic abnormalities (Trottier et al., 2006).However, there was no significant difference on age between single (37.40±7.99year) and multiple (36.98±9.41years) HR-HPV infection women (P = 0.714).
Before we consider the implications of these results, we need to assess the potential limitations of this investigation.The main limitation of our study is that our study was a retrospective, hospital-based observational analysis, and study participants included two groups of population, gynecologic clinic visitors and health medical examination women.Therefore population profile may not represent those of the general population since the low participation might introduce a self-selection bias, Another limitation is that we chose HPV types that have been associated with cancer; we were not able to measure all HPV subtypes.Consequently, we cannot rule out the possibility that unmeasured HPV types were confounding the associations reported.More studies will be necessary to examine these associations in study population.
In conclusion, in this large hospital-based study, we confirmed the HPV-52, -16 and -58 in women with normal cytology in Shantou; in addition, we comprehensively described HPV-58, -52 and -16 distributions in women with cytologic abnormalities, and particularly co-infection in women with ASC, HSIL, ISIL and multiple HR-HPV positive.

Figure 1 .
Figure 1.Age-Specific Prevalence of Human Papillomavirus in Women with Normal and Abnormal Cervical Cytology

Figure 2 .
Figure 2. Human Papillomavirus Type-specific Distribution and Cervical Cytology Abnormalities