Reliable identification of women with CIN3+ using hrHPV genotyping and methylation markers in a cytology‐screened referral population

Abstract

Cervical screening aims to identify women with high-grade squamous intraepithelial lesion/cervical intraepithelial neoplasia 2-3 (HSIL/CIN2-3) or invasive cervical cancer (ICC). Identification of women with severe premalignant lesions or ICC (CIN3+) could ensure their rapid treatment and prevent overtreatment. We investigated high-risk human papillomavirus (hrHPV) detection with genotyping and methylation of FAM19A4/miR124-2 for detection of CIN3+ in 538 women attending colposcopy for abnormal cytology. All women had an additional cytology with hrHPV testing (GP5+/6+-PCR-EIA+), genotyping (HPV16/18, HPV16/18/31/45), and methylation analysis (FAM19A4/miR124-2) and at least one biopsy. CIN3+ detection was studied overall and in women <30 (n = 171) and ≥30 years (n = 367). Positivity for both rather than just one methylation markers increased in CIN3, and all ICC was positive for both. Overall sensitivity and specificity for CIN3+ were, respectively, 90.3% (95%CI 81.3-95.2) and 31.8% (95%CI 27.7-36.1) for hrHPV, 77.8% (95%CI 66.9-85.8) and 69.3% (95%CI 65.0-73.3) for methylation biomarkers and 93.1% (95%CI 84.8-97.0) and 49.4% (95%CI 44.8-53.9) for combined HPV16/18 and/or methylation positivity. For CIN3, hrHPV was found in 90.9% (95%CI 81.6-95.8), methylation positivity in 75.8% (95%CI 64.2-84.5) and HPV16/18 and/or methylation positivity in 92.4% (95%CI 83.5-96.7). In women aged ≥30, the sensitivity of combined HPV16/18 and methylation was increased (98.2%, 95%CI 90.6-99.7) with a specificity of 46.3% (95%CI 40.8-51.9). Combination of HPV16/18 and methylation analysis was very sensitive and offered improved specificity for CIN3+, opening the possibility of rapid treatment for these women and follow-up for women with potentially regressive, less advanced, HSIL/CIN2 lesions.

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

“…A total of 538 women had a smear which was eligible for methylation testing and interpretation of the result . Of these women, 482 remained after exclusion of biopsies which had a worst diagnosis on the newly sectioned slide which was more than 1 grade aside from the consensus diagnosis on the locally produced histology slide.…”

“…Residual material after HPV testing and cytology was concentrated and sent to DDL Diagnostic Laboratory for methylation testing . DNA was isolated using the MagNA Pure 96.…”

“…Residual material after HPV testing and cytology was concentrated and sent to DDL Diagnostic Laboratory for methylation testing. 14 DNA was isolated using the MagNA Pure 96. The level of amplifiable human DNA was assessed using a qPCR of the in-house reference gene RNaseP with the phocine herpesvirus as an internal control for the absence of PCR-inhibition.…”

Novel biomarkers with promising benefits for diagnosis of cervical neoplasia: a systematic review

“…DNA methylation assays on the other hand recorded sensitivity, specificity, PPV, NPV, and AUC ranges of 59.7-92.9%, 67-98%, 15-95.4%, 65.5-98.3%, and 0.81-0.86, respectively, in detection of CIN2+ [23,[38][39][40][41][42][43][44], with a low sensitivity of 59.7% reported by Schmitz et al [23], compared to 92.9% sensitivity by Dong et al [39]; and extreme PPV of 15% reported by van Leeuwen et al [40], compared to 95.4% PPV by Kong et al, [38]. DNA methylation is a major epigenetic mechanism that involves the transfer of a methyl group to the C5 carbon residues (5mC) of cytosines that is mediated by a family of DNA methyltransferases, and plays an important role in various biological processes including the regulation of gene expression, genomic imprinting, cell differentiation, development, and inflammation [70].…”

CADM1, MAL, and miR124 Promoter Methylation as Biomarkers of Transforming Cervical Intrapithelial Lesions

“…Hypermethylation of CpG islands in the promoter regions of tumor suppressor genes leads to silencing of the gene and is an essential step in the development of many cancers, including CC [8]. Hypermethylation of promoter regions in certain genes has also been suggested as a molecular alteration appearing in transforming HSIL/CIN2-3 lesions at high risk of progression to CC [9,10,11,12,13,14]. Recent studies have shown that the combination of methylation-mediated silencing of CADM1 (cell adhesion molecule 1) and MAL (T-lymphocyte maturation-associated protein) promoter genes in cervical scrapes seems to be related to the duration of hrHPV infection and the severity of the SIL/CIN lesion [10,15].…”