The natural history of cervical HPV infection: unresolved issues.

The identification of high-risk human papillomavirus (HPV) types as a necessary cause of cervical cancer offers the prospect of effective primary prevention and the possibility of improving the efficiency of cervical screening programmes. However, for these opportunities to be realized, a more complete understanding of the natural history of HPV infection, and its relationship to the development of epithelial abnormalities of the cervix, is required. We discuss areas of uncertainty, and their possible effect on disease prevention strategies.

The Major Constituent of Green Tea, Epigallocatechin-3-Gallate (EGCG), Inhibits the Growth of HPV18-Infected Keratinocytes by Stimulating Proteasomal Turnover of the E6 and E7 Oncoproteins.

Epigallocatechin-3-gallate (EGCG), the primary bioactive polyphenol in green tea, has been shown to inhibit the growth of human papilloma virus (HPV)-transformed keratinocytes. Here, we set out to examine the consequences of EGCG treatment on the growth of HPV18-immortalised foreskin keratinocytes (HFK-HPV18) and an authentic HPV18-positive vulvar intraepithelial neoplasia (VIN) clone, focusing on its ability to influence cell proliferation and differentiation and to impact on viral oncogene expression and virus replication. EGCG treatment was associated with degradation of the E6 and E7 oncoproteins and an upregulation of their associated tumour suppressor genes; consequently, keratinocyte proliferation was inhibited in both monolayer and organotypic raft culture. While EGCG exerted a profound effect on cell proliferation, it had little impact on keratinocyte differentiation. Expression of the late viral protein E4 was suppressed in the presence of EGCG, suggesting that EGCG was able to block productive viral replication in differentiating keratinocytes. Although EGCG did not alter the levels of E6 and E7 mRNA, it enhanced the turnover of the E6 and E7 proteins. The addition of MG132, a proteasome inhibitor, to EGCG-treated keratinocytes led to the accumulation of the E6/E7 proteins, showing that EGCG acts as an anti-viral, targeting the E6 and E7 proteins for proteasome-mediated degradation.

The ATM tumour suppressor gene is down-regulated in EBV-associated nasopharyngeal carcinoma.

A micro-array analysis using biopsies from patients with EBV-positive undifferentiated nasopharyngeal carcinoma (NPC) and from cancer-free controls revealed down-regulation of tumour suppressor genes (TSG) not previously associated with this disease; one such gene was the ataxia telangiectasia mutated (ATM) gene. Q-PCR confirmed down-regulation of ATM mRNA and ATM protein expression in tumour cells was weak or absent in almost all cases. In NPC cell lines, however, ATM was down-regulated only in the EBV-positive line, C666.1, and in none of five EBV-negative lines. In vitro infection of EBV-negative NPC cell lines with a recombinant EBV was followed by the down-regulation of ATM mRNA and protein, and only EBV-positive cells showed a defective DNA damage response following gamma-irradiation. Our data suggest that loss of ATM function could be an important step in the pathogenesis of NPC, and may have implications for the treatment of this disease.

Expression of the Epstein-Barr virus-encoded Epstein-Barr virus nuclear antigen 1 in Hodgkin's lymphoma cells mediates Up-regulation of CCL20 and the migration of regulatory T cells.

In approximately 50% of patients with Hodgkin's lymphoma (HL), the Epstein-Barr virus (EBV), an oncogenic herpesvirus, is present in tumor cells. After microarray profiling of both HL tumors and cell lines, we found that EBV infection increased the expression of the chemokine CCL20 in both primary Hodgkin and Reed-Sternberg cells and Hodgkin and Reed-Sternberg cell-derived cell lines. Additionally, this up-regulation could be mediated by the EBV nuclear antigen 1 protein. The higher levels of CCL20 in the supernatants of EBV-infected HL cell lines increased the migration of CD4(+) lymphocytes that expressed FOXP3, a marker of regulatory T cells (Tregs), which are specialized CD4(+) T cells that inhibit effector CD4(+) and CD8(+) T cells. In HL, an increased number of Tregs is associated with the loss of EBV-specific immunity. Our results identify a mechanism by which EBV can recruit Tregs to the microenvironment of HL by inducing the expression of CCL20 and, by doing so, prevent immune responses against the virus-infected tumor population. Further investigation of how EBV recruits and modifies Tregs will contribute not only to our understanding of the pathogenesis of virus-associated tumors but also to the development of therapeutic strategies designed to manipulate Treg activity.

Sphingosine-1-phosphate signalling drives an angiogenic transcriptional programme in diffuse large B cell lymphoma.

Although the over-expression of angiogenic factors is reported in diffuse large B-cell lymphoma (DLBCL), the poor response to anti-VEGF drugs observed in clinical trials suggests that angiogenesis in these tumours might be driven by VEGF-independent pathways. We show that sphingosine kinase-1 (SPHK1), which generates the potent bioactive sphingolipid sphingosine-1-phosphate (S1P), is over-expressed in DLBCL. A meta-analysis of over 2000 cases revealed that genes correlated with SPHK1 mRNA expression in DLBCL were significantly enriched for tumour angiogenesis meta-signature genes; an effect evident in both major cell of origin (COO) and stromal subtypes. Moreover, we found that S1P induces angiogenic signalling and a gene expression programme that is present within the tumour vasculature of SPHK1-expressing DLBCL. Importantly, S1PR1 functional antagonists, including Siponimod, and the S1P neutralising antibody, Sphingomab, inhibited S1P signalling in DLBCL cells in vitro. Furthermore, Siponimod, also reduced angiogenesis and tumour growth in an S1P-producing mouse model of angiogenic DLBCL. Our data define a potential role for S1P signalling in driving an angiogenic gene expression programme in the tumour vasculature of DLBCL and suggest novel opportunities to target S1P-mediated angiogenesis in patients with DLBCL.

Epstein-Barr virus (EBV) and its associated human cancers--genetics, epigenetics, pathobiology and novel therapeutics.

Epstein-Barr virus (EBV) is a B-lymphotropic virus that is associated with a range of human malignancies. Although for many of these tumors the association has long been established, unraveling the precise role of EBV in disease pathogenesis has been more difficult. This review summarizes current knowledge concerning the association between EBV and human cancers, and illustrates how a deeper insight into viral latent gene expression, regulation and functions in different cell environments is already helping towards a better understanding of both the natural history of infection in normal individuals and how EBV contributes to malignant transformation. Finally, therapeutic strategies targeting EBV in tumors are discussed.

Role of sexual behavior in the acquisition of asymptomatic Epstein-Barr virus infection: a longitudinal study.

BACKGROUND: The natural history of Epstein-Barr virus (EBV) infection is poorly defined. We report the prevalence and subsequent incidence of EBV infection in a cohort of sexually active young women and explore the social and sexual determinants of incident infections. METHODS: The study population was drawn from a cohort of young women, who were recruited for a longitudinal study of risk factors for early cervical neoplasia. A case-control analysis, nested within the cohort of 45 women for whom the first EBV sample tested was EBV-negative and who had further follow-up, was undertaken. EBV serostatus was determined in serum with a synthetic peptide-based enzyme-linked immunosorbent assay; EBV DNA was measured in cervical smears with the use of quantitative polymerase chain reaction. RESULTS: Of 1023 women 15-19 years of age included in this analysis, 978 (95.6%) tested positive for antibodies to EBV in their first serum sample. Of 45 women who tested negative, 22 subsequently acquired an asymptomatic EBV infection; the median time to seroconversion was 25 months (range, 1-60 months), and the median age at seroconversion was 18 years (range, 16-21 years). The risk of seroconversion increased with increasing number of sexual partners [compared with 1 partner, odds ratio (OR) was 1.28 for 2 partners and 2.23 for 3 or more; chiTREND 5.02; df 1; P < 0.05] and was greatest when a new sexual partner had been acquired in the 2 years before seroconversion (OR 4.78; chi 4.62; df 1; P < 0.05). EBV DNA was detected in 9 of 14 women who seroconverted and who also provided cervical samples. CONCLUSIONS: In susceptible young women, the acquisition of EBV infection is associated with their sexual behavior.

Cigarette smoking is an independent risk factor for cervical intraepithelial neoplasia in young women: a longitudinal study.

Repeated measurements of smoking, cervical human papillomavirus (HPV) status and sexual behaviour were used to measure the risk of high-grade cervical intraepithelial neoplasia (CIN) in relation to changes in smoking and cervical HPV status, and to explore the impact of smoking on the acquisition and duration of incident cervical HPV infection. Included in this longitudinal analysis are 1485 women aged 15-19 years: 1075 were HPV-negative and cytologically normal at recruitment; 410 were HPV-positive, cytologically abnormal or both, at this time. Women re-attended every 6 months, when samples were taken for cytological and virological examination. Current smoking intensity was associated with an increased risk of high-grade CIN, after controlling for cervical HPV status (compared to non-smokers, hazards ratio (HR) for 10 or more cigarettes per day=2.21, 95% confidence interval (CI) 1.19-4.12, p-trend=0.008). In women who were HPV-negative and cytologically normal at recruitment, current smoking was not significantly associated with the risk of acquiring a cervical HPV infection, after controlling for life-time number of partners and age of oldest partner (HR=1.13, 95% CI 0.90-1.41); nor did it prolong the length of time during which HPV could be detected (HR=1.03, 95% CI 0.78-1.34). Current smoking intensity is an independent risk factor for high-grade CIN in young women, after controlling for cervical HPV infection.

Is human papillomavirus viral load a clinically useful predictive marker? A longitudinal study.

BACKGROUND: It has been suggested that in women who test positive for high-risk human papillomavirus (HPV) types, viral load can distinguish women who are at increased risk of cervical neoplasia from those who are not. METHODS: Quantitative PCR (qPCR) was used to measure HPV copy number in serial samples taken from 60 and 58 young women previously found to have incident cervical HPV16 or HPV18 infections, respectively, using GP5+/GP6+ primers; women provided at least three samples for qPCR testing, at least one of which was positive. RESULTS: A 10-fold increase in HPV16 or HPV18 copy number was associated with a modestly increased risk of acquiring a cytologic abnormality [HPV16: hazards ratio, 1.76 (95% confidence interval, 1.38-2.25); HPV18: hazards ratio, 1.59 (95% confidence interval, 1.25-2.03)]. However, in most women, copy number increased during follow-up, before decreasing again. In women with a HPV16 infection, the median copy number per 1,000 cells was 7.7 in their first qPCR HPV-positive sample, 1,237 in the sample yielding the maximum copy number, and 7.8 in their last qPCR HPV-positive sample; corresponding copy numbers for women with HPV18 infection were 2.3, 87, and 2.4. Maximum HPV16 and HPV18 copy number did not differ significantly between women who acquired an incident cervical cytologic abnormality and those who did not. CONCLUSION: Whereas large relative increases in copy number are associated with an increased risk of abnormality, a single measurement of viral load made at an indeterminate point during the natural history of HPV infection does not reliably predict the risk of acquiring cervical neoplasia. Therefore, a single measure of HPV viral load cannot be considered a clinically useful biomarker.

Disruption of the E2 gene is a common and early event in the natural history of cervical human papillomavirus infection: a longitudinal cohort study.

Integration of high-risk human papillomavirus (HPV) types into the host-cell genome disrupts the HPV regulatory E2 protein, resulting in a loss of negative feedback control of viral oncogene expression; this disruption has been considered a critical event in the pathogenesis of cervical neoplasia, and a potential biomarker of progressive disease. However, using serial samples taken from a cohort of young women who were recruited soon after they first had sexual intercourse, we show that disruption of the E2 gene is a common and early event in the natural history of incident cervical HPV infections. The E2 gene was significantly more likely to be disrupted in women who tested positive for HPV18 in their baseline sample than in those who tested positive for HPV16 [26% versus 58%; relative risk, 2.26; 95% confidence interval (CI), 1.38-3.71; chi(2), 9.23; 1 degree of freedom (df); P = 0.002]. Among women with an intact E2 gene in their baseline sample, the median time to first detection of E2 disruption was also shorter for those who tested positive for HPV18 than HPV16 (5.7 versus 10.9 months; hazards ratio, 1.93; 95% CI, 0.84-4.44; chi(2), 2.49; 1 df; P = 0.11). This tendency for HPV18 to integrate early, coupled with the substantial reduction in viral load in HPV18-positive samples in which E2 is disrupted, may explain why HPV18-associated disease is often reported to be characterized by minor cytologic changes, which underestimate the severity of the underlying histologic abnormality.

Measurement of the humoral immune response following an incident human papillomavirus type 16 or 18 infection in young women by a pseudovirion-based neutralizing antibody assay.

We have evaluated a neutralizing antibody assay which uses human papillomavirus (HPV) type 16 (HPV-16) and HPV-18 pseudovirions carrying a secretory alkaline phosphatase reporter gene and which can potentially measure functionally relevant HPV type-specific neutralizing antibodies. The reproducibility of the assay was excellent; for HPV-16, the intra- and interassay kappa values were 0.95 and 0.90, respectively; and for HPV-18, the corresponding values were 0.90 and 0.90. This assay was used to describe the kinetics of the neutralizing antibody response in a cohort of 42 young women who were recruited soon after first intercourse and who first tested positive for HPV-16 DNA or HPV-18 DNA, or both, during follow-up. Most women seroconverted following the first detection of type-specific HPV DNA and remained seropositive until the end of follow-up. Our findings are broadly consistent with those of two other cohort studies which have measured the serological response following an incident infection by using the technically simpler virus-like-particle-based enzyme-linked immunosorbent assay.

Proximity of first intercourse to menarche and the risk of human papillomavirus infection: a longitudinal study.

Cross-sectional studies have suggested that compared with women who delay the start of their sexual career, those who first have intercourse soon after menarche are more susceptible to cervical human papillomavirus (HPV) infection and thus have a greater risk of cervical neoplasia. We describe, using longitudinal observations, how the risk of infection with HPV varies with the interval between menarche and first intercourse in 474 women aged 15-19 recruited within 12 months of first intercourse and before the acquisition of a second sexual partner. One hundred forty-five women became HPV-positive; the cumulative risk of HPV infection 3 years after first intercourse was 45.0% (95% CI = 37.9-51.2). In univariate analyses, the hazards ratio (HR) of HPV infection increased significantly with age at first intercourse (HR = 1.212 per year; 95% CI = 1.050-1.398), partner age (HR = 1.084 per year; 95% CI = 1.045-1.125) and when women reported a sexually experienced partner (HR = 2.794; 95% CI = 1.804-4.326); the interval between menarche and first intercourse was a significant predictor of infection, with an increase in the HR of 12.9% for every year of increase in this interval (95% CI = 2.1%-24.9%). In a multivariate analysis, compared with women who first had intercourse within 3 years of menarche, those who postponed first intercourse beyond this time had a greater risk of infection (HR = 1.581; 95% CI = 1.113-2.245) after controlling for age and sexual experience of partner.

High incidence of cervical human papillomavirus infection in women during their first sexual relationship.

The prevalence of cervical human papillomavirus increases with increasing numbers of sexual partners, leaving the impression that this infection is acquired only as a result of high risk sexual behaviour. Using longitudinal data from 242 women who had only had one sexual partner, we found that the risk of acquiring cervical human papillomavirus infection was 46% (95% CI 28-64) at three years after first intercourse and that the median time from first intercourse to first detection of human papillomavirus was only three months.

Human papillomavirus type 18 and rapidly progressing cervical intraepithelial neoplasia.

BACKGROUND: Human papillomavirus type 18 (HPV-18) is the second most frequent of the HPV types detected when squamous-cell cancer is diagnosed and the type most strongly associated with adenocarcinoma of the cervix. However, in cross-sectional studies, HPV-18 is rarely detected at the time of diagnosis of high-grade cervical intraepithelial neoplasia (CIN). We used a longitudinal study design to describe the occurrence of cytological abnormality after incident HPV-18 and HPV-16 infections. METHODS: The analysis was based on 1075 women aged 15-19 years, who had normal cytology and were negative for HPV at recruitment from a single family-planning clinic, and who had further follow-up. The women reattended every 6 months, and samples were taken for cytological and virological examination. FINDINGS: The relative risk of a cytological diagnosis of borderline nuclear abnormality after exposure to HPV-18 was 2.06 (95% CI 1.24-3.43) and that after exposure to HPV-16 was 1.99 (1.32-3.01). The relative risks of mild dyskaryosis were 3.11 (1.86-5.18) and 4.76 (3.15-7.18), and the relative risks of moderate or severe dyskaryosis were 0.80 (0.24-2.65) and 2.85 (1.36-5.97). Time to acquisition of cytological abnormality was unrelated to the infecting type (p=0.88). INTERPRETATION: Our findings do not support the long-held view that the reason why HPV-18 infection is under-represented at the time of diagnosis of high-grade CIN is because HPV-18-associated disease rapidly progresses through the preinvasive stages of neoplasia. We suggest that the cytological changes detected after HPV-18 infection might understate the severity of underlying disease. This feature could compromise the effectiveness of screening programmes in reducing the frequency of HPV-18-associated cancers.