The prototype kit SILgrade-E4-1 is an immunohistochemistry (IHC) assay for the study of the human papillomavirus (HPV) E4 protein on formalin-fixed, paraffin-embedded tissue sections prepared from cervical and anal biopsies. E4 is intended to be used in conjunction with H&E and p16INK4a stained slides prepared from the same cervical or anal tissue specimen as an aid to study productive viral status.
Summary and explanation
Cervical and anal squamous intraepithelial lesions (SIL) are very heterogeneous groups, consisting of HPV productive lesions that will spontaneously regress and transforming lesions with a higher chance of progression to cancer. The morphology of potentially regressing viral productive lesions are hard to distinguish from transforming lesions that will progress to cancer if not treated and the histological threshold used routinely for treatment of high-grade squamous intraepithelial lesions (HSIL)/cervical intraepithelial neoplasia (CIN) grade 2 or anal intraepithelial neoplasia (AIN) grade 2 is not universally reproducible (Darragh et al, 2012). HPVs are small viruses containing a double-stranded, circular DNA genome of approximately 7,900 base pairs. The viral genome is divided into three regions: the long control region, which regulates viral gene expression and replication; the late region, which encodes the two viral structural proteins and six or more early (E) region open reading frames, which encode proteins required for viral gene expression, replication and survival (Doorbar, 2013). The early protein E4 is a marker for initiation of the productive phase of the HPV life cycle; and E7 protein action results in expression of p16INK4a and this is used to support a histological diagnosis of the presence of a significant transforming HPV infection recognised as -IN 2/3 or HSIL (Darragh et al, 2012).
The SILgrade-E4-1 kit is a prototype kit that allows investigation of productive HPV infection and life cycle completion. E4 in conjunction with p16INK4a offers a reproducible molecular classification of cervical and anal HSIL of potential value for natural history studies and supporting histological diagnosis (Leeman et al, 2018; van Baars et al, 2015; Zummeren et al, 2018).
Principles of the procedure
The anti-E4 (XR-E4-1) is a PAN-genotypic monoclonal antibody reactive against the HPV E4 protein from at least HPV 6, 11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 53, 56, 58, 59, 66, 67, 70 and 74; and skin HPV genotypes 27 and 57 (van Baars et al, 2015; and unpublished data). Anti-E4 (XR-E4-1) binds to E4 protein in paraffin-embedded tissue sections and exhibits a cytoplasmic staining pattern.
The prototype kit is available for research use only studies.
REF: K 162 SILgrade-E4-1 kit, version 1 50 tests
REF: K 162-C SILgrade-E4-1 kit, controls
Figure: HPV productive and transforming infections by E4 and p16INK4a staining. AIN1 lesion with extensive E4 staining and patchy p16INK4a in the lower one third of the epithelium. AIN2 lesion with HPV18, showing extensive E4 staining of the superficial layers and p16INK4a staining in the lower two third of the epithelium. AIN3 lesion with HPV35, showing focal E4 staining at the edge of the AIN3 lesion where it collides with low-grade AIN and full-thickness p16INK4a positivity (figure adapted from (Leeman et al, 2018) )
Darragh TM, Colgan TJ, Cox JT, Heller DS, Henry MR, Luff RD, McCalmont T, Nayar R, Palefsky JM, Stoler MH, Wilkinson EJ, Zaino RJ, Wilbur DC, Members of LPWG (2012) The Lower Anogenital Squamous Terminology Standardization Project for HPV-Associated Lesions: background and consensus recommendations from the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology. J Low Genit Tract Dis 16(3): 205-42
Doorbar J (2013) The E4 protein; structure, function and patterns of expression. Virology 445(1-2): 80-98
Leeman A, Pirog EC, Doorbar J, van de Sandt MM, van Kemenade FJ, Jenkins D, Quint WGV (2018) Presence or Absence of Significant HPVE4 Expression in High-grade Anal Intraepithelial Neoplasia With p16/Ki-67 Positivity Indicates Distinct Patterns of Neoplasia: A Study Combining Immunohistochemistry and Laser Capture Microdissection PCR. Am J Surg Pathol 42(4): 463-471
van Baars R, Griffin H, Wu Z, Soneji YJ, van de Sandt M, Arora R, van der Marel J, Ter Harmsel B, Jach R, Okon K, Huras H, Jenkins D, Quint W, Doorbar J (2015) Investigating Diagnostic Problems of CIN1 and CIN2 Associated With High-risk HPV by Combining the Novel Molecular Biomarker PanHPVE4 With P16INK4a. Am J Surg Pathol 39(11): 1518-1528
Zummeren MV, Kremer WW, Leeman A, Bleeker MCG, Jenkins D, Sandt MV, Doorbar J, Heideman DAM, Steenbergen RDM, Snijders PJF, Kenter GG, Quint WGV, Berkhof J, Meijer C (2018) HPV E4 expression and DNA hypermethylation of CADM1, MAL, and miR124-2 genes in cervical cancer and precursor lesions. Mod Pathol