Background
Infection with one or more high-risk human papillomavirus [HR-HPV] types increases the risk of the occurrence and progression of cervical intraepithelial neoplastic [CIN] lesions and invasive cervical cancer [
1]. Whilst the majority of infections are transient and resolve of their own accord, a recent meta-analysis gave a summary estimate for HR-HPV persistence [HR-HPV positive at 2 or more consecutive time-points] of about 40%, for both nontype-specific and type-specific HR-HPV [
2].Women with persistent infection with HR-HPV have a greater risk of developing cervical cancer; the risk is greater for those with type-specific persistence [
3]. A number of factors are thought to influence the likelihood of HR-HPV persistence, including immunocompetence, use of oral contraceptives, smoking, parity, genotype and diet, although results are not wholly consistent [
2,
4‐
6].
There is evidence that folate status influences the natural history of HPV infection. A prospective follow-up study that monitored 345 women over a 24-month period showed that women with low folate status were at a higher risk of acquiring a HR-HPV infection and of repeat infection with HR-HPV, than women with higher folate status [
7]. Furthermore, in our previous cross sectional study of 308 women with different cervical cytology, women with HR-HPV infection had a lower red blood cell folate concentration than women free of infection [P <0.05] [
8]. In the same study it was shown that women diagnosed with CIN grades 1, 2, or 3, or cancer had a significantly lower red cell folate status than those with normal cervical histology, independent of HPV status [P < 0.05] [
8].
The usefulness of tumour suppressor gene hypermethylation as a prognostic biomarker is under intense investigation in many different cancers, including cervical cancer and its precursor lesions. Several groups, including our own, have reported that high-grade cervical cell abnormality or invasive cervical cancer is associated with an increased likelihood of promoter methylation of selected tumour suppressor genes compared with normal cells [
8‐
10]. Folate, as a methyl donor, is considered to be an important determinant of normal DNA methylation, although direct evidence that folate status influences gene specific methylation in a predictable and consistent manner in humans is lacking.
The approach to cervical cancer prevention up until now has involved primary screening for cytological abnormalities followed by direct referral to colposcopy for women with persistent borderline changes, mild, moderate and severe dyskaryosis. HPV triage is now being implemented across England, which involves HPV testing in the event of cytology reported as borderline or mild dyskaryosis. Women who are HPV positive are referred to colposcopy and HPV negative women are referred back to routine recall. At colposcopy women with < CIN1 are not treated and referred back to routine recall whilst women with CIN1 have a repeat cytology and HPV test at 6 months [
11]. Predictive markers of HPV persistence may be clinically useful and inform patient management.
We conducted a case–control study, nested in a randomised trial [ARTISTIC;
A Randomised
Trial
In
Screening
To
Improve
Cytology] conducted within the routine NHS Cervical Screening Programme in Greater Manchester [
12], to assess the predictive power of cervical cell folate status and tumour suppressor gene methylation in determining HR-HPV clearance.
Discussion
Persistent infection with high-risk HPV increases the risk of cervical cancer. In this study, cervical cell folate concentration, tumour suppressor gene methylation, and particular HR-HPV types, were all shown to be associated with an increased likelihood of persistent HR-HPV infection, defined as infection with any HR-HPV type at follow-up. HPV persistence has been defined and estimated in a number of ways; a recent meta-analysis providing data on more than 100,000 women worldwide, found that 73% of studies defined persistence as HPV positivity at a minimum of two time points [
2].
Consistent with the literature, HPV-16 was the most prevalent infection [
22]. HPV-16 infection and multiple HR-HPV infections were found to be significant independent determinants of persistent HR-HPV infection, which is consistent with findings from a study of type-specific HPV persistence in Finnish women [
23].
Cervical cell folate concentration was higher in women with normal cytology than women with borderline or mild cytology, which is consistent with previous findings in which folate was measured in red blood cells [
8] or serum [
24]. Multivariate modelling, taking account of HR-HPV-type, infection with multiple HR-HPV types, and a woman’s age, showed higher cervical cell folate concentration to be associated with an increased likelihood of HR-HPV persistence. When the analysis was conducted in each of the three cytology groups separately, this effect was only evident in women who had mild cervical cell abnormalities.
Few other studies on folate status in cervical tissue have been published. The concentrations of folate in cervical cells collected by liquid-based cytology in this study were comparable to those measured in cervical biopsies by Fowler et al., who reported concentrations of 2.75 to 4.39 ng/mg protein [
25]. In their small cross-sectional study, Fowler et al. reported a higher mean concentration of cervical cell folate in women who tested HR-HPV positive [4.05 ng/mg] than in women who tested HR-HPV negative [3.13 ng/mg], but the difference did not reach statistical significance. In contrast, Flatley et al. [
8] found a lower red blood cell folate concentration in women carrying an HR-HPV infection compared with those free of infection and Piyathilake et al. [
7] showed that a higher circulating folate concentration was associated with a greater likelihood of clearing an HR-HPV infection. Unlike Piyathilake et al. (7) we were able to examine a possible role of folate in HPV persistence taking cytology into account as well as HR-HPV type and infection with multiple HPV types. This is important because the complex literature around folate and cancer suggests very strongly that the presence of cell abnormality influences the association. Generally, studies have suggested that a better folate status might protect against certain cancers but that where an underlying neoplasm exists, supplemental folate might accelerate carcinogenesis [
26,
27].
Finding from this study differ from the Piyathilake study (7) in which folate was measured in the blood. It is not clear whether the concentration of folate in the blood is strongly correlated with that in cervical tissue, particularly in non-normal tissue. Some cancer cells are known to upregulate folate receptors [
28], which facilitates folate uptake and could fuel enhanced cell proliferation. Although cervical cancer cells (HeLa cells) do express a high density of the folic acid receptor [
29], it is not known whether this represents an upregulation from the normal cell. An immunohistochemical study of folate receptor expression in cervical tissue showed no difference between normal cells and low-grade abnormalities and a reduced expression in higher-grade abnormalities and cancer [
30]. In our study, the higher concentration of folate in the cases would be expected to increase host cell proliferation and this would facilitate viral replication. The downstream effect on persistence is not clear; enhanced viral replication might lead to greater re-infection of adjacent sites, but it might also lead to adaptive immune responses. A recent elegant study of HPV integration in human keratinocytes showed that folate deficiency impaired the cells’ ability to make HPV-16 virion particles, and this was associated with enhanced integration into the host DNA [
31]. In our study, cells with higher folate concentration may have been able to produce more virus particles than cells with lower folate concentration, and therefore might have been more permissive of re-infection.
The temporal relationship might be between a lower folate concentration and low-grade cell abnormalities, as observed in the ‘baseline’ measurements is not known. Neither can we be certain what the temporal relationship between a persistent HPV infection and cell folate concentration might be. Although we have discussed how a higher folate concentration might increase the likelihood of HR-HPV persistence, given the understanding that HPV infection can lead to changes in DNA methylation and thereby alter expression of genes [
32], we cannot rule out the possibility that persistent HPV infection and cell changes have a synergistic influence on the expression of the folate receptor and folate uptake into cells.
In the multivariate analysis, the association between age and risk of HPV persistence was different, depending on the underlying cytology. In the total sample, age was not associated with risk of HPV persistence. We did not have access to demographic data on alcohol and tobacco use for women whose samples were used in this study; it is possible that there was a difference between younger and older women which could have confounded the association between age and HPV persistence. Cigarette smoking and alcohol consumption are both thought to influence HPV infection and the risk of cervical cancer and both show an age association. Studies have examined HR-HPV persistence by age but the meta-analysis of HR-HPV persistence by Rositch et al. [
2] reports no consistent trend.
Promoter methylation of the tumour suppressor gene, death-associated protein kinase,
DAPK, was also associated with an increased likelihood of HR-HPV persistence.
DAPK has a known role as a promoter of programmed cell death and
DAPK promoter methylation has been reported for several cancers including cervical cancer [
33,
34]. Promoter methylation of this gene is associated with gene silencing [
35]. A link with HR-HPV persistence has not been reported previously but there is a plausible mechanism for a causal relationship. Viruses have evolved different strategies to avoid the host immune response to infection. The inhibition of apoptosis is important to viral pathogenesis. Should
DAPK be silenced through promoter methylation it no longer promotes cell death via the normal apoptotic pathway of an HR-HPV-infected cell and the host cell may survive and differentiate. Under such circumstances the HR-HPV virus would have longer to replicate, increasing copy number and the likelihood of infecting other cells. A mechanistic link between
CDH1 methylation and HR-HPV persistence is less easy to explain. Promoter methylation of this gene can lead to gene silencing [
9].
CDH1 is a member of the cadherin family, loss of expression would be expected to reduce cell-cell contact, leading to an increase in cell motility and invasion, hallmarks of metastasis. Whilst cadherin expression is important to bacterial adherence and internalisation, this group of proteins has not been implicated in cellular uptake of viruses, although cellular uptake by endocytosis is common to both bacteria and viruses [
36].
The temporal relationship between gene methylation, cervical cell folate concentration and infection with HR-HPV in baseline samples is not clear. HR-HPV infection can induce change in gene methylation [
37]. This would require recruitment of the host cell methylation apparatus and utilisation of intracellular folate, as methyl donor. Folate status of the cell may influence DNA methylation through effects on DNA methyltransferases [DNMTs]. DNMT downregulation in response to folate depletion has been reported for human colon cancer cells in vitro [
38] and unpublished data from our laboratory show that methyl donor depletion of cervical cancer cells
in vitro leads to downregulation of DNA methyltransferases. By inference, higher cellular folate might increase DNMT expression, and facilitate
DAPK methylation. This provides a putative link between higher cell folate status and
DAPK methylation in HR-HPV infection. The low frequency of
DAPK methylation in women with normal cytology is compatible with our previous findings [
8] and other studies showing that
DAPK methylation occurs on the pathway of HR-HPV-induced cell transformation [
10] may explain the lack of association with HR-HPV persistence in women with normal cytology.
It would have been preferable to have had access to sufficient cervical cell material to allow the measurement of tissue folate concentration and gene methylation on the same samples, and to avoid sample pooling for folate measurements. This must be considered a limitation of the study but is unlikely to be resolved without access to biopsy material. It would also have been useful to have been able to include information about smoking and alcohol use into the multivariate analyses, as these are factors are thought to influence the process of viral infection and clearance.
Conclusions
Persistent infection with HR-HPV causes cervical cancer, and therefore factors which influence the natural history of HR-HPV infection may be important modulators of cervical cancer risk, but the mechanisms that favour HPV persistence are not understood. We have shown that a higher concentration of folate in cervical cells, and promoter methylation of the tumour suppressor gene DAPK, in women with cervical cell dyskaryosis, are associated with increased risk of HR-HPV persistence.
We hypothesize that HR-HPV infection induces DAPK methylation in dyskaryotic cells, supported by a high intracellular folate, and that DAPK methylation leads to dysregulation of apoptosis and promotes HR-HPV persistence. There is a need for in vitro studies to examine these hypotheses and so shed further light on mechanisms of viral persistence. An understanding of such mechanisms may have predictive value and inform patient management.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JEF, HK and HJP conceived and designed the study; JEF conducted all the molecular and biochemical analyses; JR carried out the statistical analysis of data; AS was responsible for sample and data retrieval; JEF and HJP drafted the manuscript, all authors contributed to the manuscript and read and approved the final version.