Risk stratification of cervical disease using detection of human papillomavirus (HPV) E4 protein and cellular MCM protein in clinical liquid based cytology samples

Andrew Stevenson,Kim Kavanagh,Jiafeng Pan,Lynne Stevenson,Heather Griffin,John Doorbar,Evelyn Scott,Miriam Deeny,Kate Cuschieri,Sheila V Graham

doi:10.1016/j.jcv.2018.08.011

Abstract

BackgroundWhile human papillomavirus (HPV) DNA testing offers high sensitivity for the detection of significant cervical disease, its specificity is suboptimal given the high prevalence of transient HPV infections (CIN1 or less). Biomarkers to identify those suffering from low grade disease from those with high grade disease could save healthcare costs and reduce patient anxiety. ObjectiveThe objective of the present work was to develop and test an immunohistochemistry (IHC)-based dual viral and cellular biomarker strategy which was applicable to liquid based cytology (LBC) samples. Study designWe developed a novel IHC assay for detection of HPV E4 and cellular minichromosome maintenance (MCM) proteins in routinely taken cervical LBC samples using cytospin-prepared slides. The assay was applied to a prospective cohort of Scottish women referred to a colposcopy clinic due to preceding cytological abnormalities. The performance of the biomarkers for detection of clinically insignificant (CIN1 or less) versus significant disease was determined. ResultsA total of 81 women were recruited representing 64 cases of <=CIN1 and 28 of CIN2 + . Biomarker performance relative to histopathology outcomes showed high levels of MCM detection was significantly associated with CIN2+ (p = 0.03) while E4 was detected more frequently in <=CIN1 (p = 0.06). ConclusionsCombined detection of a host proliferation marker and a marker of viral gene expression could allow triage of cases of clinically insignificant disease prior to colposcopy. However, there was overlap between distributions of MCM levels in CIN2+ and <=CIN1 suggesting that additional biomarkers would be required for improved specificity. Combined with cytospin-prepared slides this approach could provide a means of risk stratification of disease in low resource settings.

Highlights

While human papillomavirus (HPV) DNA testing offers high sensitivity for the detection of significant cervical disease, its specificity is suboptimal given the high prevalence of transient HPV infections (CIN1 or less)
The aim of the study was to determine the performance of E4, a biomarker of transient HPV infection (≤CIN1), in combination with MCM2, a biomarker of significant cervical disease in a cohort of patients who had an abnormal Pap smear test result, were called to attend a colposcopy clinic, and tested positive for HPV
To test specificity of the antibodies for cellular minichromosome maintenance (MCM) and viral E4 proteins, HeLa cells, and HeLa cells transfected with an expression plasmid for HPV16 E4 were suspended in PreservCyt, stained with E4 and MCM antibodies and visualised by immunofluorescence microscopy

Summary

Introduction

While human papillomavirus (HPV) DNA testing offers high sensitivity for the detection of significant cervical disease, its specificity is suboptimal given the high prevalence of transient HPV infections (CIN1 or less). Biomarkers to identify those suffering from low grade disease from those with high grade disease could save healthcare costs and reduce patient anxiety. The high prevalence of clinically insignificant HPV infections, in young women, means the HPV DNA tests in Abbreviations: HPV, human papillomavirus; CIN, cervical intraepithelial neoplasia; IHC, immunohistochemistry; LBC, liquid based cytology; MCM, minichromosome maintenance. In the mid layers of the epithelium, increased synthesis of HPV E1 and E2 proteins allows viral genome replication and HPV E4 protein, the most abundant viral protein, is synthesised and facilitates viral replication and virion egress

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