Grading immunohistochemical markers p16 ^INK4a and HPV E4 identifies productive and transforming lesions caused by low‐ and high‐risk HPV within high‐grade anal squamous intraepithelial lesions

Abstract

Summary Objectives Because current guidelines recognise high‐grade anal squamous intraepithelial lesions (HSILs) and low‐grade SILs (LSILs), and recommend treatment of all HSILs although not all progress to cancer, this study aims to distinguish transforming and productive HSILs by grading immunohistochemical (IHC) biomarkers p16INK4a (p16) and E4 in low‐risk human papillomavirus (lrHPV) and high‐risk (hr)HPV‐associated SILs as a potential basis for more selective treatment. Methods Immunostaining for p16 and HPV E4 was performed and graded in 183 biopsies from 108 HIV‐positive men who have sex with men. The causative HPV genotype of the worst lesion was identified using the HPV SPF10‐PCR‐DEIA‐LiPA25 version 1 system, with laser capture microdissection for multiple infections. The worst lesions were scored for p16 (0–4) to identify activity of the hrHPV E7 gene, and panHPV E4 (0–2) to mark HPV production and life cycle completion. Results There were 37 normal biopsies, 60 LSILs and 86 HSILs, with 85% of LSILs caused by lrHPV and 93% of HSILs by hrHPV. No normal biopsy showed E4, but 43% of LSILs and 37% of HSILs were E4 positive. No differences in E4 positivity rates were found between lrHPV and hrHPV lesions. Most of the lesions caused by lrHPV (90%) showed very extensive patchy p16 staining; p16 grade in HSILs was variable, with frequency of productive HPV infection dropping with increasing p16 grade. Conclusions Combined p16/E4 IHC identifies productive and nonproductive HSILs associated with hrHPV within the group of HSILs defined by the Lower Anogenital Squamous Terminology recommendations. This opens the possibility of investigating selective treatment of advanced transforming HSILs caused by hrHPV, and a ‘wait and see’ policy for productive HSILs. What's already known about this topic? For preventing anal cancer in high‐risk populations, all patients with high‐grade squamous intraepithelial lesions (HSILs) are treated, even though this group of lesions is heterogeneous, the histology is variable and regression is frequent. What does this study add? By adding human papillomavirus (HPV) E4 immunohistochemistry to p16 INK4a (p16), and grading expression of both markers, different biomarker expression patterns that reflect the heterogeneity of HSILs can be identified. Moreover, p16/E4 staining can separate high‐risk HPV‐associated HSILs into productive and more advanced transforming lesions, providing a potential basis for selective treatment.

Characterisation of anal intraepithelial neoplasia and anal cancer in HIV‐positive men by immunohistochemical markers p16, Ki‐67, HPV‐E4 and DNA methylation markers

“…Our findings and previous studies support that E4 expression, as a marker for viral production, can help to further characterise AIN lesions. 19,20 The presence of a methylation positive, E4 negative biomarker pattern, mainly seen in anal cancer, the majority (73.3%) of AIN3 and a proportion (26.7%) of AIN2 lesions is supportive of a high shortterm progression risk to cancer of AIN lesions with this biomarker pattern. Conversely, the presence of an E4 positive, methylation negative biomarker pattern in a high proportion of AIN1 lesions (65.4%) and almost one-third of AIN2 lesions (31.1%) and the near, respectively, complete absence of this biomarker pattern in AIN3 and SCC support our assumption that these lesions have a low risk of progression to cancer.…”

mRNA Detection in Anal Cytology: A Feasible Approach for Anal Cancer Screening in Men Who Have Sex with Men Living With HIV

“…Another limitation is that the presence and effect of low-risk HPV types could not be evaluated with the genotyping test used in our study. However, although it has been suggested that low-risk HPV types may have a role in the appearance of anal lesions, the risk of low-risk HPV infection in the development and progression of anal premalignancies is clearly lower than the risk due to high-risk HPV infection [44,45].…”

Cervical cancer risk profiling: molecular biomarkers predicting the outcome of hrHPV infection

“…Similarly, Griffin et al compared the expression of E4 to p16 INK4A , another host marker, and the hypermethylation of some hostcell genes, which increases with CIN severity. They claim that E4 expression is correlated with low hypermethylation and the absence of p16 INK4A , which is not observed in cervical carcinoma, as described in additional studies [7,96,97].…”