HPV16 L1 and L2 DNA methylation predicts high‐grade cervical intraepithelial neoplasia in women with mildly abnormal cervical cytology

Abstract

DNA methylation changes in human papillomavirus type 16 (HPV16) DNA are common and might be important for identifying women at increased risk of cervical cancer. Using recently published data from Costa Rica we developed a classification score to differentiate women with cervical intraepithelial neoplasia grade 2 or 3 (CIN2/3) from those with no evident high-grade lesions. Here, we aim to investigate the performance of the score using data from the UK. Exfoliated cervical cells at baseline and 6-months follow-up were analyzed in 84 women selected from a randomized clinical trial of women undergoing surveillance for low-grade cytology. Selection of women for the methylation study was based on detectable HPV16 in the baseline sample. Purified DNA was bisulfite converted, amplified and pyrosequenced at selected CpG sites in the viral genome (URR, E6, L1 and L2), with blinding of laboratory personnel to the clinical data. The primary measure was a predefined score combining the mean methylation in L1 and any methylation in L2. At the second follow-up visit, 73/84 (87%) women were HPV16 positive and of these 25 had a histopathological diagnosis of CIN2/3. The score was significantly associated with CIN2/3 (area under curve = 0.74, p = 0.002). For a cutoff with 92% sensitivity, colposcopy could have been avoided in 40% (95% CI 27–54%) of HPV16 positive women without CIN2/3; positive predictive value was 44% (32–58%) and negative predictive value was 90% (71–97%). We conclude that quantitative DNA methylation assays could help to improve triage among HPV16 positive women.

Methylation of viral and host genes and severity of cervical lesions associated with human papillomavirus type 16

“…HPV16 L1 assays were done as previously described. 16 For EPB41L3 and LMX1A, the primers were designed using PyroMark Assay Design software version 2.0.1.15 (Qiagen). 21 The methods for amplification and pyrosequencing and the associated quality control procedures have been described in detail elsewhere.…”

“…21 The methods for amplification and pyrosequencing and the associated quality control procedures have been described in detail elsewhere. 16,18,21 …”

“…8,9,15,16 In cervical cancer specimens, a large number of host genes have been shown to be hypermethylated. 6,7,9,11,12 Changes in methylation of many single genes has high sensitivity for detecting cervical cancer but diagnostic performance decreases substantially to detect CIN2/3.…”

“…13 We report herein our analysis of methylation of two HPV16 L1 sites and host CpG sites in genes EPB41L3 and LMX1 that have in recent studies shown promising ability to discriminate among pre-invasive cervical lesion stages. 6,16,[18][19][20][21] We investigated the predictive biomarker ability of these methylation sites across the spectrum of HPV16 positive cervical lesions, from negative for intraepithelial lesions or malignancy (NILM) to cervical cancer.…”

HPV33 DNA methylation measurement improves cervical pre-cancer risk estimation of an HPV16, HPV18, HPV31 and \textit{EPB41L3} methylation classifier

“…These epigenetic differences can be accurately measured by molecular methods, and are linked with the development of a variety of cancers [10]. We and other research groups have observed that measurement of differential methylation of certain HPV CpG sites is useful for stratifying HPV type-specific risk [8,[11][12][13]19]. Additionally, methylation of CpGs in the promoters or introns of human genes has shown some value [5,6,9,14,20].…”