Background
Persistent infection with oncogenic genotypes of genital Human Papillomavirus (HPV) is associated with the development of cervical cancer, a significant cause of morbidity and mortality of women worldwide [
1]. Precancerous cervical disease is classified by cytological (low [LSIL] or high grade [HSIL] squamous intraepithelial lesions) and histological stages (cervical intraepithelial neoplasia [CIN] grades 1 to 3). There are about a dozen HPV types associated with the development of cervical cancer [
2], differing in their relative contributions to the prevalence of cervical disease [
3]. Testing for the presence of oncogenic HPV DNA offers improved sensitivity, though lower specificity, than cytology alone [
4] while the next generation of molecular tests, including those with limited genotyping capability, may improve upon this [
5]. Other potential molecular markers of cervical disease include type-specific viral load (VL), integration of HPV DNA into the host genome and methylation of the HPV genome. An improved understanding of the role of these potential molecular markers in cervical disease development may shed some light on HPV pathogenesis and may be helpful to guide future cervical cancer screening or treatment algorithms.
HPV DNA VL, usually estimated as the amount of HPV genome copies per cell, has been variably associated with cervical disease. Some studies were able to use HPV16 VL to differentiate between high grade (HSIL, CIN2+) and low grade (LSIL, CIN1) disease [
6‐
8], between cervical cancer and lower grades of disease [
9‐
11], or between any grade of cervical disease and normal samples [
12]. Other studies could not find any association between HPV16 VL and cervical disease [
13,
14]. There are few studies examining any potential link between VL and cervical disease for other HPV types with some finding a positive association between some stages of disease for HPV18 [
8,
9], HPV31 [
8], HPV33 [
8], and HPV52 [
9] while others have not [
9,
14‐
17].
HPV16 integration status has been able to distinguish between HSIL and LSIL samples [
6], between cervical cancer samples and those of a lower grade of disease [
9,
10], or in some studies a strong positive correlation with increasing disease severity has been found [
18]. In other studies no relationship between cervical disease grade and HPV16 integration status was apparent [
11‐
13,
19]. For HPV18 there are fewer studies overall with some finding an association with disease [
9,
18] and some not [
16]. One study [
18], found a strong positive correlation with increasing disease severity for HPV31, HPV33 and HPV45 while not for HPV52 [
9,
15] and HPV58 [
9,
17] in others.
Methylation of CpG sites within L1 [
20‐
22], the upstream regulatory region (URR) [
20,
21,
23] and/or other regions of the HPV16 genome [
24‐
26] have often, but not always [
27,
28], been associated with cervical disease. Data on the degree of CpG site methylation for genotypes HPV18 [
25,
29,
30], HPV31 [
29] and HPV45 [
29] are limited but appear to show a similar trend, suggesting that HPV methylation may be useful as a potential marker for cervical disease [
31].
Some studies have examined both VL and integration status for HPV16 [
6,
7,
9‐
13] but for other types including HPV18 [
9,
16], HPV52 [
9,
15] and HPV58 [
9,
17] the sources are limited. The VL of samples containing fully integrated HPV tends to be lower than that found in samples containing purely episomal or mixed forms [
6,
7,
10,
15], although this does not always appear to be the case [
16,
17]. Fewer studies have examined methylation status in relation to other parameters and then only for HPV16 infection [
22,
32].
Mixed infections are common throughout the course of cervical disease [
3]. Few of these studies have explicitly used, or separately analyzed, samples harboring a single HPV type wherein the association between the HPV type under evaluation and cervical disease can be made with some confidence. Within these limited number of studies, the VL of samples harboring single infections has been associated with disease severity in some [
7] but not in other studies [
13,
14,
17]. For integration-based studies that explicitly mentioned the use of single infection samples, one study found an association between HPV16 integration status and disease [
7] while another did not [
13]. The only study explicitly to examine methylation levels in single infections (HPV18 and HPV31) demonstrated that some CpG sites exhibited higher methylation levels in CIN3 cases harboring single infections than in CIN3 cases with multiple infections with these types [
29].
In this study, we evaluate the DNA viral load, integration and CpG methylation status of women singly infected with HPV16, HPV18, HPV31 or HPV45 in order better to understand the potential role for these molecular markers in cervical disease.
Conclusions
The purpose of this study was to evaluate any potential relationship between HPV type-specific viral load, integration and methylation status and current cervical disease stage with regard to a single infecting HPV genotype, wherein the association with disease can be assumed with some confidence.
There are potential shortcomings to this study. First, samples containing a single infecting genotype were identified using a generic PCR and genotyping test [
33]. While this is the most common approach used and the test used arguably one of the more robust [
39], such a determination is not without its problems including the potential for masking within mixed infections [
40]. The selection of single infection samples is, we believe, a significant improvement on the use of unselected samples, wherein the potential impact of such masking is likely to be far higher. Nevertheless, the possibility that a minority of samples in this study contained low levels of one or more other HPV types cannot be ruled out. Second, although these samples constitute a highly selected panel and a minority sample type collected during routine screening, they do nevertheless permit the evaluation of the potential for these markers to be used, with a limited number of other confounding factors. Finally, although cytology samples are the primary sample type collected during cervical screening and are readily amenable to such testing, the use of cytology samples alone to improve the definition of cervical disease stage is problematic given the often discontinuous relationship between cytology and histology stage designations [
1].
In summary, even under these optimized conditions, the sensitivities and specificities of many of these potential molecular markers were lower than the median sensitivity (98%) and specificity (86%) obtained in a recent evaluation of commercial HPV DNA tests for identifying CIN2+ cases during cytological screening [
5]. There were a few individual measurements that have the potential for use in triage, characterized by a similar or higher specificity than required for initial screening. HPV VL appears to have an overall specificity of
ca. 80% (range 76 - 91%) to differentiate normal cytology from abnormal cytology. Conversely, HPV methylation appears to be able to differentiate HG cytology from normal and LG cytology with a specificity of 77 - 100%. Both of these measures, therefore, have potential for use in screening and/or triage, but the utility of HPV genome methylation status may be improved if the disease-associated genotype-specific CpG site methylation patterns can be appropriately exploited.
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Competing interests
The authors declare no conflicts of interest.
Authors’ contributions
LM conceived and designed the experiments, performed the experiments, analyzed the data and contributed to drafting the manuscript. AG conceived and designed the experiments, analyzed the data and contributed to drafting the manuscript. SB conceived and designed the experiments, analyzed the data and contributed to drafting the manuscript. JVP contributed to drafting the manuscript. All authors read and approved the final manuscript.