Abstract
BackgroundPersistent infection with oncogenic Human Papillomavirus (HPV) is associated with the development of cervical cancer with each genotype differing in their relative contribution to the prevalence of cervical disease. HPV DNA testing offers improved sensitivity over cytology testing alone but is accompanied by a generally low specificity. 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. The aim of this study was to evaluate the relationship between HPV type-specific viral load, integration and methylation status and cervical disease stage in samples harboring HPV16, HPV18, HPV31 or HPV45.MethodsSamples singly infected with HPV16 (n = 226), HPV18 (n = 32), HPV31 (n = 75) or HPV45 (n = 29) were selected from a cohort of 4,719 women attending cervical screening in England. Viral load and integration status were determined by real-time PCR while 3’L1-URR methylation status was determined by pyrosequencing or sequencing of multiple clones derived from each sample.ResultsViral load could differentiate between normal and abnormal cytology with a sensitivity of 75% and a specificity of 80% (odds ratio [OR] 12.4, 95% CI 6.2–26.1; p < 0.001) with some variation between genotypes. Viral integration was poorly associated with cervical disease. Few samples had fully integrated genomes and these could be found throughout the course of disease. Overall, integration status could distinguish between normal and abnormal cytology with a sensitivity of 72% and a specificity of 50% (OR 2.6, 95% CI 1.0–6.8; p = 0.054). Methylation levels were able to differentiate normal and low grade cytology from high grade cytology with a sensitivity of 64% and a specificity of 82% (OR 8.2, 95% CI 3.8–18.0; p < 0.001). However, methylation varied widely between genotypes with HPV18 and HPV45 exhibiting a broader degree and higher magnitude of methylated CpG sites than HPV16 and HPV31.ConclusionsThis study lends support for HPV viral load and CpG methylation status, but not integration status, to be considered as potential biomarkers of cervical disease.
Highlights
Persistent infection with oncogenic Human Papillomavirus (HPV) is associated with the development of cervical cancer with each genotype differing in their relative contribution to the prevalence of cervical disease
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
HPV16 viral load (VL) was able to distinguish between cases and controls with a sensitivity of 70% and a specificity of 91% (AUC 0.859) at a VL threshold of 1.58 c/c
Summary
Persistent infection with oncogenic Human Papillomavirus (HPV) is associated with the development of cervical cancer with each genotype differing in their relative contribution to the prevalence of cervical disease. HPV DNA testing offers improved sensitivity over cytology testing alone but is accompanied by a generally low specificity. Testing for the presence of oncogenic HPV DNA offers improved sensitivity, though lower specificity, than cytology alone [4] while the 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
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