Human papillomavirus genotype distribution in cervical intraepithelial neoplasia 2/3 and invasive cervical cancer among Hakka women in southern China

Background: Methods: Results of genotypes from women with histological diagnosis of CIN2/3 and ICC were collected from January, 2017 to December, 2018. HPV genotypes were analyzed by flow cytometry method. Association of HPV infection and lesions severity was estimated using prevalence ratio (PR). Results: Overall, 1,408 Hakka women with histological diagnosis of CIN2/3 and ICC were enrolled in this study. HPV infection prevalence was 92.92% in CIN2, 95.77% in CIN3 and 95.88% in ICC. Most frequent genotypes for CIN2 were HPV52 (31.42%), HPV16 (22.12%) and HPV58 (22.12%); for CIN3 were HPV16 (41.90%), HPV52 (20.77%) and HPV58 (18.31%); and for ICC were HPV16 (49.67%), HPV18 (11.25%) and HPV52 (9.80%). PR of HPV16 and HPV33 were significantly higher in CIN3 compared with CIN2 (PR = 2.372, 95%CI = 1.598-3.524; PR = 2.577, 95%CI = 1.250-5.310; respectively). HPV16 and HPV18 prevalence were significantly increasing in SCC compared with CIN3 (PR = 2.517, 95%CI = 1.095-5.786; PR = 2.473, 95%CI = 1.840-3.324; respectively). Most HPV infections were found in women aged 40 – 49 years in CIN2/3 and women aged 50 - 59 years in ICC. Conclusions: This is the first study of genotypes and age specific distribution of HPV infection among Hakka women with CIN2/3 and ICC in southern China. Our results provide available information for HPV vaccine development in China.


Background
Cervical cancer is known as the second most common cancers in women around the world, and most cervical cancer occur in developing countries [1]. Incidence of cervical cancer in China is contributed to be about 14% of the world, and therefore it becomes an crucial public health problem in China [2,3].
Molecular epidemiological evidence suggests that high risk human papillomavirus (HPV) genotypes are the principal cause of cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (ICC) [4,5]. More than 200 HPV genotypes have been recognized [6], and nearly 40 are associated with genital tract infections [7]. At least 15 genotypes (HPV16, 18, 31, 33, 35, 39, 45, 51, 56, 58, 59, 68, 73 and 82) are considered as high risk HPV genotypes [7]. A meta-analysis analyzed HPV genotypes results 3 among 115,789 women around the world, the most prevalent genotypes are HPV16 and HPV18 [8]. In a large study contained 51,345 cases in China showed that HPV52, HPV16 and HPV58 were top three most prevalent genotypes, while HPV18 was only the sixth most prevalent genotype [9]. China is a large country with unbalanced social and economic conditions from region to region, and hence HPV infections are varying [10]. Guangdong, as the most populous and economically developed province in China, the prevalence of high risk HPV infection is as high as 21.07% [11].
Meizhou is located in the northeastern of Guangdong Province and most people in this area were Hakka. The physical characteristics of Hakka in Meizhou are differ from the populations of northern and southern China [12]. HPV infection of Hakka people in Meizhou was observed up to 19.81% in our previous study [13]. There are no data available for Hakka area of southern China in relation to the HPV type-specific distribution in women with CIN2/3 and ICC.
HPV vaccine is an important measure to prevent persistent infection and cervical disease [14]. Three virus-like particle vaccines including HPV16/18 (Cervarix),HPV6/11/16/18 (Gardasil) and HPV6/11/16/18/31/33/45/52/58 (Gardasil) were designed based on studies from Western countries [15]. Whether the application of these vaccines in China can achieve the expected preventive effect has not been proven. Therefore, the knowledge of the prevalence of HPV genotypes in CIN2/3 and ICC patients can provide useful information for development of HPV vaccines for Chinese women. The objective of recent study is to estimate HPV type-specific distribution among Hakka women with CIN2/3 and ICC in southern China.  11,40,42,43,44,55,61,81,83). HPV DNA was amplified with 5'-biotiny-lated consensus PCR primers. PCR products were hybridized to beads with type-specific probes, then incubated with phycoerythrin (PE)-conjugated streptavidin (SA-PE) and final results were analyzed on a Luminex 200 system (Luminex Corporation, Texas) [16]. Quality controls were strictly performed throughout the experiments by applying positive and negative controls for PCR reaction.

Data analysis
Total accounts and proportion of women in different HPV infections were presented. A log binomial model was performed to estimate the prevalence rate (PR) of high risk HPV with 95% confidence intervals (CI) between different histological grades. The PR was adjusted with the age of patient at the time of histological diagnosis. Statistical analyses were performed using SPSS (Statistical Package for the Social Sciences, Chicago, IL). P< 0.05 was considered statistically significant.

Results
Participants and overall HPV infection prevalence and ICC with a high detection rate over 90% (Table 1), and single high risk HPV infection among these histological grades were the most common infection, ranged from 61.50% -74.65%. However, the multiple high risk HPV infection were detected in 29.20% of CIN2 samples, 18.66% in CIN3 samples and 8.46% in ICC samples, presented decreasing prevalence with severity of lesions. The participants only infected with low risk HPV genotypes were rarely detected in all histological grades. HPV genotype distribution 6 Table 2 and Fig.2 performed accounts and prevalence of high and low risk HPV genotypes and ten most prevalent HPV genotypes in CIN2/3. Type-specific distribution of high risk HPV infection was significantly different between CIN2 and CIN3. HPV52 (31.42%), HPV16 (22.12%) and HPV58 (22.12%) were most commonly detected in CIN2, however, HPV16 (41.90%), HPV52 (20.77%) and HPV58 (18.31%) were predominately detected in CIN3.

HPV prevalence ratio
To assess the risk of CIN progression ascribe to specific high risk HPV, the prevalence ratio (PR) of specific genotype was calculated by contrasting the frequency with CIN2 and CIN3. As presented in Table 2, HPV16 and HPV33 prevalence were positively correlated with progression from CIN2 to CIN3 As demonstrated in Table 3, high risk genotype prevalence between CIN3 and SCC was estimated.

HPV infection in different ages
The age-specific distribution of HPV infection among Hakka women in southern China was shown in   HPV16, HPV18, HPV31, HPV35, HPV52 and HPV58 presented a high risk of progression to CIN3. Similar to this study, we observed PR of HPV16 and HPV33 were significantly higher in CIN3 compared with CIN2, indicating a higher likelihood of progression from CIN2 to CIN3 than with other genotypes. Also, PR of HPV16 and HPV18 in SCC were significantly higher than that of other high risk genotypes in CIN3, supporting the important role of sustained HPV16 infection in increasing carcinogenicity. Reversely, low PR of HPV51, HPV52 and HPV53 were found in CIN3 compared with CIN2, suggesting a lower potential for progression to CIN3 than with HPV16 and HPV33 infection. Consistent with previous report [17], in our study PR of HPV33, HPV52 and HPV58 in SCC were significantly lower than in CIN3, which indicates progression to SCC is less likely than with HPV16 and HPV18 infection.
HPV DNA test as a primary method should be offer for women age 25 to 65 years to screen cervical cancer was recommended by the US Food and Drug Administration (FDA). Current study showed top age group of highest prevalence in CIN2/CIN3 was women with age of 40 -49 and the highest prevalence in ICC/SCC/ADC was observed in women aged 50 -59 years. These findings indicated that hormones level and immune function of women in these ages may be lower, so they can hardly resist HPV infection. However, our study presented 4.62% cases were negative for HPV DNA result before histological diagnosis of CIN2/3 and ICC. It remains the limitation of HPV DNA testing as a major for cervical cancer screen.
Currently, no study is available on the type-specific distribution of HPV in precancerous lesions / cervical cancerous among Hakka women in southern China. The present study found HPV16, HPV18, HPV52 and HPV58 were the main genotypes in CIN2/3 and ICC and women aged 40 -49 suffered severely HPV infection. It would be instructive to estimate the potential protection of current HPV 9 vaccines and future modified vaccines achieve in this area.

Conclusion
Our study presented prevalence and genotyping distribution of HPV infection among Hakka women in southern China, our findings will provide important information for the development and application of HPV vaccines and cervical screening in Chinese Hakka women in southern China.

Availability of data and materials
The datasets generated during the current study are not publicly available yet, due to privacy concerns and ongoing additional research. Data can be made available for peer review on reasonable request through contacting the corresponding author.

Competing interests
The authors declare that they have no competing interests.     Figure 1 Flow chart of study participants Type-specific prevalence of 10 most prevalent HPV genotypes in cervical intraepithelial neoplasia (CIN2) and cervical intraepithelial neoplasia (CIN3) among Hakka women in Southern China, prevalence (%) with single infections and overall type specific were shown at the bar.  Type-specific prevalence of 10 most prevalent HPV genotypes in squamous cell carcinoma (SCC) and adenocarcinoma (ADC) among Hakka women in Southern China, prevalence (%) with single infections and overall type specific were shown at the bar. adenocarcinoma.