Abstract
High-risk human papillomavirus (HPV) infections are the cause of nearly all cases of cervical cancer. Although the detection of HPV DNA has proved useful in cervical diagnosis, it does not necessarily predict disease presence or severity, and cannot conclusively identify the causative type when multiple HPVs are present. Such limitations may be addressed using complementary approaches such as cytology, laser capture microscopy, and/or the use of infection biomarkers. One such infection biomarker is the HPV E4 protein, which is expressed at high level in cells that are supporting (or have supported) viral genome amplification. Its distribution in lesions has suggested a role in disease staging. Here we have examined whether type-specific E4 antibodies may also allow the identification and/or confirmation of causal HPV-type. To do this, type-specific polyclonal and monoclonal antibodies against three E4 proteins (HPV-16, -18, and -58) were generated and validated by ELISA and western blotting, and by immunohistochemistry (IHC) staining of epithelial rafts containing these individual HPV types. Type-specific detection of HPV and its associated disease was subsequently examined using formalin-fixed paraffin-embedded cervical intra-epithelial neoplasias (CIN, (n = 247)) and normal controls (n = 28). All koilocytotic CIN1 lesions showed type-specific E4 expression of their respective HPV types. Differences were noted amongst E4 expression patterns in CIN3. HPV-18 E4 was not detected in any of the 6 HPV-18 DNA-positive CIN3 lesions examined, whereas in HPV-16 and -58 CIN3, 28/37 (76%) and 5/9 (55.6%) expressed E4 respectively, usually in regions of epithelial differentiation. Our results demonstrate that type-specific E4 antibodies can be used to help establish causality, as may be required when multiple HPV types are detected. The unique characteristics of the E4 biomarker suggest a role in diagnosis and patient management particularly when used in combination.
Highlights
Human papillomavirus (HPV) DNA is found in most cases of cervical cancer (.99.7%) and high-grade pre-cancers [1], and has been used to assign causality to a HPV type in a lesion
To a large extent, such studies have moved from the analysis of HPV DNA alone, to the analysis of markers of active viral infection, such as viral transcripts, viral proteins [2], and/or cellular gene products that can be used as surrogate markers of viral E6/E7 gene activity, such as p16 [3] and/or minichromosome maintenance protein (MCM) [4]
We show that HPV type-specific anti-E4 antibodies (MoAb16E435–42, R18E453–60 and R58E423–30) can be generated using a short-peptide approach, and that such reagents can be applied to formalin fixed paraffin-embedded clinical tissue sections to identify sites of active infection by specific HPV types
Summary
Human papillomavirus (HPV) DNA is found in most cases of cervical cancer (.99.7%) and high-grade pre-cancers [1], and has been used to assign causality to a HPV type in a lesion. To a large extent, such studies have moved from the analysis of HPV DNA alone, to the analysis of markers of active viral infection, such as viral transcripts, viral proteins [2], and/or cellular gene products that can be used as surrogate markers of viral E6/E7 gene activity, such as p16 [3] and/or minichromosome maintenance protein (MCM) [4]. These approaches have considerable potential, they generally have limited ability to distinguish HPV type, and/or are difficult to use on standard formalin fixed paraffin-embedded (FFPE) tissue where RNA degradation may have occurred. They have not yet been widely applied to the problem of assigning causality or confirming causality when multiple HPV types are found
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