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.

Annemiek Leeman,Wim G V Quint,John Doorbar,Marta Del Pino,Chris J L M Meijer,Folkert J Van Kemenade,Jaume Ordi,David Jenkins,Aureli Torné

doi:10.1002/cam4.2855

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.

Highlights

In cervical cancer (CC) prevention, women with an abnormal screening result are subjected to colposcopy to detect of highgrade squamous intraepithelial lesions/cervical intraepithelial neoplasia grades 2 and 3 (HSIL/CIN2-3), or CC
Methylation positivity for markers FAM19A4 and miR124-2 increased with the severity of the lesion: 23.2% of women with no CIN biopsies were positive for one or more markers compared with 66.1% of women with HSIL/CIN3 biopsies
There is an inverse relation between productive infection as shown by HPV E4 positivity in the worst lesion present on biopsy, and methylation of markers FAM19A4 and miR124-2 on cytology which are associated with transforming HPV infection

Summary

Introduction

In cervical cancer (CC) prevention, women with an abnormal screening result are subjected to colposcopy to detect of highgrade squamous intraepithelial lesions/cervical intraepithelial neoplasia grades 2 and 3 (HSIL/CIN2-3), or CC. Hematoxylin and eosin stain (H&E) diagnosis is subjective and substantial interobserver and intra-observer variability has been reported.[1] Previous reports have shown that HSIL/CIN3 is more reproducible, but important variation exists in the diagnosis of HSIL/CIN2. This diagnosis includes both productive lesions that might regress and advanced transforming lesions that require treatment and can result in overtreatment.[2,3,4]. Grading patterns of expression of biomarkers such as E4 and p16 might play an important role in predicting progression of a lesion, deciding treatment and might reduce the overtreatment of productive lesions that can regress

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