Expression of p16 and HPV E4 on biopsy samples and methylation of FAM19A4 and miR124‐2 on cervical cytology samples in the classification of cervical squamous intraepithelial lesions

Abstract

The decision to treat a cervical squamous intraepithelial lesion (SIL) by loop electrosurgical excision procedure (LEEP) relies heavily on a colposcopy‐directed biopsy showing high‐grade (H)SIL. Diagnosis is often supported by p16, an immunohistochemical (IHC) biomarker of high‐risk (hr)HPV E7 gene activity. Additional potential markers include methylation of tumor suppressor genes FAM19A4/miR124‐2 in cervical cytology for advanced transforming HSIL and the IHC marker HPV E4 for productive, potentially regressing lesions. In 318 women referred for colposcopy, we investigated the relationship between staining patterns of p16 and E4 IHC in the worst biopsy, and the relation of these to FAM19A4/miR124‐2 methylation status in cytology. E4‐positive staining decreased with increasing SIL/CIN grade from 41% in LSIL to 3% in HSIL/CIN3. E4 positivity increased with grade of p16 when p16 expression was limited to the lower two third of the epithelium (r = 0.378), but fell with expression over. Loss of E4 expression in the worst lesion was associated with the methylation of FAM19A4/miR124‐2. We also examined whether these biomarkers can predict the histological outcome of the LEEP biopsy in a subgroup of 119 who underwent LEEP treatment. About 85% of women with ≥lower two third p16 staining/E4‐negative HSIL biopsies and 65% with limited p16 staining/E4‐positive HSIL biopsies had ≥HSIL in the LEEP specimen (P = .025). p16 expression in a biopsy is related both to viral production and transformation, while decreased E4 expression relates to methylation, indicating advanced HSIL. p16 expression in ≥2/3 of the epithelium and absent E4 indicate likely HSIL on a subsequent LEEP specimen.

Classification of high‐grade cervical intraepithelial neoplasia by p16ink4a, Ki‐67, HPV E4 and FAM19A4/miR124‐2 methylation status demonstrates considerable heterogeneity with potential consequences for management

“…36 However, Leeman et al found 3% positive E4 staining in CIN3. 29 We showed that in a small number of CIN3 lesions (9.8%; 23/235) and IS 5-6 lesions (12.0%; 38/316), E4 positivity was found, which is in agreement with our earlier findings. 20 Moreover, we now extend these findings by showing that methylation was present in 60.9% (14/23) of E4-positive CIN3 and in 65.8% (25/38) of E4-positive IS 5-6 lesions.…”

“…Based on the results of this study, we stratified all CIN2/3 lesions according to the immunoscore and methylation status into biomarker profiles (Table 4). E4 expression was not used in these biomarker 20,29,36 and the presence of E4 in a minority of methylation-positive CIN2/3, makes the potential clinical significance of absence or presence of E4 expression difficult to interpret in terms of regression or progression. Methylation status was included since methylation levels increase with increasing disease severity and are extremely high in cervical carcinomas (Figure 1).…”

“…Membranous and/or cytoplasmic E4 staining was scored as either negative (Score 0), focally positive (ie, limited staining of some cells restricted to the superficial layer of the epithelium, Score 1) or extensively positive (ie, widespread positive staining in the superficial layers of the epithelium extending to half of the epithelial width, Score 2). 29 Examples of the scoring of E4 staining are shown in Supplementary Figure 2.…”

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].…”

Association of high-risk human papillomavirus infection duration and cervical lesions with vaginal microbiota composition

“…HPVs are non-enveloped small DNA viruses that widely exist in nature and encompass a large family of viruses, both benign and highly carcinogenic (4). HPVs primarily replicate on the surface of squamous epithelia, such as at the junction of squamous epithelial cells and the columnar epithelium of the cervix; this viral replication can interfere with the normal replication of squamous cells, leading to abnormal proliferation of the squamous epithelia (5). Generally, as a non-lytic infection, this process does not result in a strong immune response or inflammation by activating Toll-like receptors and natural killer cells (6).…”