Yaqi Pan

E3 ubiquitin ligase E6AP-mediated TSC2 turnover in the presence and absence of HPV16 E6

We previously found that HPV16 E6 causes the degradation of the tumor suppressor protein TSC2, resulting in the phosphorylation of S6 kinase and S6 even in the absence of insulin. In the present study, we investigated the role of E6‐associated protein (E6AP) in HPV16 E6‐induced TSC2 degradation. Our results demonstrated that TSC2 was targeted for degradation in the presence or absence of HPV16 E6. Over‐expression of E6AP enhanced the degradation of TSC2 by HPV16 E6, while expression of a dominant negative E6AP (C833A) inhibited the E6‐induced degradation. Additionally, by using shRNAs to block E6AP expression in HPV16 positive and negative cells, we found a significantly prolonged TSC2 half‐life. An in vivo ubiquitination assay was done to reveal that E6AP promoted the ubiquitination of TSC2 independent of HPV16 E6. We further found that TSC2 bound E6AP in the presence as well as in the absence of HPV16 E6. The binding regions on E6AP and TSC2 have been identified as amino acid (aa) 260–316, aa 428–500 and aa 1–175, aa 1251–1807, respectively. Taken together, degradation of TSC2 is mediated by E6AP ubiquitin ligase.

The Anyang Esophageal Cancer Cohort Study: Study Design, Implementation of Fieldwork, and Use of Computer-Aided Survey System

Background Human papillomavirus (HPV) has been observed repeatedly in esophageal squamous cell carcinoma (ESCC) tissues. However, the causal relationship between HPV infection and the onset of ESCC remains unknown. A large cohort study focusing on this topic is being carried out in rural Anyang, China. Methodology/Principal Findings The Anyang Esophageal Cancer Cohort Study (AECCS) is a population-based prospective endoscopic cohort study designed to investigate the association of HPV infection and ESCC. This paper provides information regarding the design and implementation of this study. In particular we describe the recruitment strategies and quality control procedures which have been put into place, and the custom designed computer-aided survey system (CASS) used for this project. This system integrates barcode technology and unique identification numbers, and has been developed to facilitate real-time data management throughout the workflow using a wireless local area network. A total of 8,112 (75.3%) of invited subjects participated in the baseline endoscopic examination; of those invited two years later to take part in the first cycle of follow-up, 91.9% have complied. Conclusions/Significance The AECCS study has high potential for evaluating the causal relationship between HPV infection and the occurrence of ESCC. The experience in setting up the AECCS may be beneficial for others planning to initiate similar epidemiological studies in developing countries.

Oral Human Papillomavirus Infection and Its Risk Factors among 5,410 Healthy Adults in China, 2009–2011

Background: Despite the established link between oral human papillomavirus (HPV) infection and a subset of oropharyngeal squamous cell carcinoma (OSCC), little is known about the epidemiology of oral HPV infection among healthy adults in China. Methods: Oral swab specimens and questionnaires were collected from 5,410 individuals (ages 25–65 years). HPV DNA in oral exfoliated cells was tested by general primer-mediated (SPF1/GP6+) PCR and sequencing. Univariate and multivariate analyses were performed to assess the associations between exposure factors and oral infection. Results: Alpha mucosal HPV types were detected in 0.67% [95% confidence interval (CI), 0.47%–0.93%] of 5,351 β-globin–positive specimens, and cutaneous HPV in 5.46% (95% CI, 4.86%–6.10%). HPV 16 and 3 were the most prevalent types of α mucosal (0.43%; 95% CI, 0.27%–0.64%) and cutaneous HPV (4.17%; 95% CI, 3.65%–4.74%), respectively. The prevalence of α mucosal HPV decreased with increasing age (25–65 years) from 0.93% to 0.36% (Ptrend = 0.033), and was associated with self-reported history of oral disease [adjusted odds ratio (OR), 4.78; 95% CI, 1.07–21.41]. In 1,614 heterosexual couples, cutaneous HPV in one partner was found to increase the other partners risk of cutaneous HPV infection (adjusted OR, 2.33; 95% CI, 1.22–4.48). Conclusions: Oral HPV infection, particularly with α mucosal types, is rare among healthy adults in China. A younger age and a history of oral disease imply higher risk of α mucosal HPV infection. Impact: This study addresses the paucity of epidemiological data on oral HPV infection among healthy population in China. Cancer Epidemiol Biomarkers Prev; 23(10); 2101–10. ©2014 AACR.

Whole Genome Sequencing and Evolutionary Analysis of Human Papillomavirus Type 16 in Central China

Human papillomavirus type 16 plays a critical role in the neoplastic transformation of cervical cancers. Molecular variants of HPV16 existing in different ethnic groups have shown substantial phenotypic differences in pathogenicity, immunogenicity and tumorigenicity. In this study, we sequenced the entire HPV16 genome of 76 isolates originated from Anyang, central China. Phylogenetic analysis of these sequences identified two major variants of HPV16 in the Anyang area, namely the European prototype (E(p)) and the European Asian type (E(As)). These two variants show a high degree of divergence between groups, and the E(p) comprised higher genetic diversity than the E(As). Analysis with two measurements of genetic diversity indicated that viral population size was relatively stable in this area in the past. Codon based likelihood models revealed strong statistical support for adaptive evolution acting on the E6 gene. Bayesian analysis identified several important amino acid positions that may be driving adaptive selection in the HPV 16 population, including R10G, D25E, L83V, and E113D in the E6 gene. We hypothesize that the positive selection at these codons might be a contributing factor responsible for the phenotypic differences in carcinogenesis and immunogenicity among cervical cancers in China based on the potential roles of these molecular variants reported in other studies.

T cell receptor β-chain repertoire analysis reveals intratumour heterogeneity of tumour-infiltrating lymphocytes in oesophageal squamous cell carcinoma.

Oesophageal squamous cell carcinoma (ESCC) has a generally poor prognosis, due to the lack of effective treatment methods. Immunotherapeutic approaches based on tumour‐infiltrating lymphocytes (TILs) have demonstrated that durable responses are produced in some patients with solid tumours, which suggests the potential feasibility of clinical application of immunotherapy for ESCC. However, many of the basic characteristics of TILs in ESCC are poorly understood, including clonality, specificity and spatial heterogeneity of the response of TILs, which depends on the interaction between antigens and T cell receptors (TCRs). We used ultra‐deep sequencing of rearranged genes in TCR β‐chain (TCRβ) to profile the basic characteristics of T cells in tumour tissues (four to six regions from each tumour) as well as matched adjacent normal tissue and peripheral blood from seven patients diagnosed with primary ESCC. We found that T cell clones within ESCCs were quite different from those of the peripheral blood and even the adjacent normal tissues in general. Although there was a relatively higher degree of overlap of intratumoural TCRβ repertoires than those between the tumour and other tissues, intratumoural TCRβ repertoires were spatially heterogeneous. Due to the restricted sampling, high‐throughput TCRβ sequencing could characterize the diversity and composition of a limited (compartment‐dependent) fraction of the respective T cell clones in any individual ESCC, expanding our understanding of immune behaviour and immune response and shedding more light on ESCC immunotherapy. Copyright

Genome-wide analysis of Epstein-Barr virus (EBV) isolated from EBV-associated gastric carcinoma (EBVaGC)

Epstein-Barr virus (EBV) is linked to the development of a variety of malignancies, including EBV-associated gastric carcinoma (EBVaGC). In this study, EBVaGC was detected in 15 (7.3%) of 206 GC cases. To identify the EBV genomic variation, EBV genomic sequences isolated from 9 EBVaGC biopsy specimens were successfully retrieved, designated EBVaGC1 to EBVaGC9. By comparative analysis of these strains with another 6 completely sequenced EBV strains, EBV-wild type, B95–8, AG876, GD1, GD2, and HKNPC1, it was demonstrated that EBVaGC1 to 9 were most closely related to the GD1 strain. Phylogenetic analysis of the GC biopsy specimen-derived EBV (GC-EBV) genomes was subsequently performed to assess their genomic diversity and it exhibited the greatest divergence from the type 2 strain, AG876. Compared with the reference EBV strain GD1, they harbored 961 variations in total, including 919 substitutions, 23 insertions, and 19 deletions. Single nucleotide polymorphism (SNP) density varied substantially across all known open reading frames and was highest in latency-associated genes. Moreover, we identified 2 interstrain recombinants at the EBNA1 locus, which provided a further mechanism for the generation of diversity. Some T-cell epitope sequences in EBNA1 and LMP2A genes showed extensive variation across strains, which implied their importance in the development of vaccines and T-cell therapy. In conclusion, we reported the first genome-wide view of sequence variation of EBV isolated from primary EBVaGC biopsy specimens, which might serve as an effective method for further understanding the genomic variations contribute to EBVaGC carcinogenesis and treatment.

Seroprevalence of Merkel cell polyomavirus in the general rural population of Anyang, China.

Background Despite the probably causal link between Merkel cell polyomavirus (MCPyV) infection and Merkel cell carcinoma (MCC), a rare but aggressive skin malignancy, little is known about the seroepidemiology of MCPyV among healthy adults in China. Methods Serum antibodies against MCPyV were evaluated by multiplex serology in a population-based study of 5548 adults (including 1587 heterosexual couples) aged 25–65 years who were enrolled from rural Anyang, China in 2007–2009. Univariate and multivariate logistic regression analyses were performed to assess the risk factors for the seropositivity of MCPyV. Results The seroprevalence for MCPyV was 61.0%. MCPyV seropositivity was significantly higher in males than in females (64.5% vs. 57.7%, P<0.001), and for both genders, showed a trend of increase with age (Male: P trend<0.001; Female: P trend<0.001). Furthermore, among antibody positives, antibody levels of MCPyV increased with advancing age (P trend = 0.017). MCPyV seropositivity of one spouse was significantly associated with that of the other partner (Adjusted OR = 1.32, 95% CI: 1.07–1.62). However, there was no association between sexual behaviors and the seropositivity of MCPyV. Conclusions High seroprevalence of MCPyV was observed in healthy Chinese individuals. Serological evidence suggests that nonsexual horizontal spread of MCPyV can occur among family members, and further research in this regard is needed.

Anti-HPV-E7 seropositivity and risk of esophageal squamous cell carcinoma in a high-risk population in China

Results of previous serologic studies on the association of human papillomavirus (HPV) with esophageal squamous cell carcinoma (ESCC) have been inconsistent. From 2007 to 2010, the authors collected blood samples and relevant demographic data from 1435 patients with ESCC and 2071 age- and sex-matched normal controls from Anyang, China. HPV-16, 18 and 57 E7 antibodies were evaluated with the glutathione-S-transferase capture ELISA. The proportions of subjects who were positive for antibodies against these three HPV antigens in the case group were all significantly higher than those in the control group. In multivariate analysis, the presence of HPV-16 E7 antibody was associated with an increased risk of ESCC [odds ratio (OR) = 3.6, 95% confidence interval (CI): 2.5-5.0], whereas the presence of HPV-18 (OR = 1.1, 95% CI: 0.7-1.7) and HPV-57 (OR = 1.3, 95% CI: 0.9-1.9) antibodies were not significant after adjustment for HPV-16. In multiple cutoff value analysis, the lowest OR for HPV-16 was obtained with the standard cut point mean + 3 SD. This study provides serological evidence in support of HPV-16 infection playing a role in the occurrence of ESCC in a high-incidence area of China.

Improved detection of human papillomavirus harbored in healthy skin with FAP6085/64 primers

FAP59/64, FAP6085/6319, and CUT primer sets were designed for detecting cutaneous HPV and have been used in many clinical and epidemiology studies. The FAP6085/64 primer set was first evaluated in this study and the FAP6085/64 combination was found to be much more sensitive than all three original primer sets by using HPV plasmids as a template. To confirm further the effectiveness of the FAP6085/64 primer set in human DNA templates, 90 palmar exfoliated cell DNA samples were used to detect the cutaneous HPV by both the FAP59/64 and FAP6085/64 primer sets. The overall proportion of HPV detection in those skin samples was 77.8% (70/90) using FAP6085/64, as compared to 55.6% (50/90) using FAP59/64. The FAP6085/64 primer set was also applied in a population based study. The proportion of HPV detection was 73.96% (2076/2807) in skin samples collected from healthy individuals, and a total of 336 different PV types were found. Sixty (17.9%) of them were fully characterized HPV types, 127 (37.8%) were putative HPV types which had been described previously, 149 (44.3%) were novel putative HPV types, and two animal PVs were also detected. These results suggest that the FAP6085/64 primer set was sensitive and effective for detection of cutaneous HPV in healthy skin samples.

Prevalence, Incidence, Clearance, and Associated Factors of Genital Human Papillomavirus Infection among Men: A Population-Based Cohort Study in Rural China

Background: The natural history of human papillomavirus (HPV) infection in men on a population base has rarely been studied in general, particularly among Chinese men. Methods: A total of 1,286 men ages 25 to 65 years from rural China were enrolled during 2009–2010 and their genital HPV infection status was assessed biannually for up to seven visits using PCR and sequencing methods. Prevalence analysis was performed among men with at least one valid HPV result (N = 1,279) and men with at least two consecutive HPV results (N = 1,059) were included in incidence and clearance analyses (median follow-up time, 31.8 months; interquartile range, 15.4–37.9 months). Results: The prevalence and incidence of any HPV type, oncogenic, and nononcogenic HPV were 17.8%, 6.4%, 12.4%, and 14.6, 4.9, 10.8 per 1,000 person months, respectively. The median duration of infection with any HPV type, oncogenic, and nononcogenic HPV was 11.5, 6.8, and 11.5 months, respectively. The number of lifetime sexual partners was consistently associated with increased risk of prevalent and incident infection of HPV. Men ages 25 to 50 years had a higher incidence and longer duration of HPV infection than older men (51–65 years). Conclusions and Impact: This epidemiologic investigation provides basic information of genital HPV infection among the Chinese male population; these data are crucial for the consideration of primary strategies against HPV-related carcinoma in the Chinese male and female population. Cancer Epidemiol Biomarkers Prev; 23(12); 2857–65. ©2014 AACR.