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. 2024 Dec:18:200288.
doi: 10.1016/j.tvr.2024.200288. Epub 2024 Jul 1.

DNA methylation at individual CpG-sites of EPB41L3, HTERT and FAM19A4 are useful for detection of cervical high-grade squamous intraepithelial lesions (HSIL) or worse: Analysis of individual CpG-sites outperforms averaging

Monica Molano  1 , Dorothy A Machalek  2 , Samuel Phillips  3 , Grace Tan  4 , Suzanne M Garland  5 , David Hawkes  4 , Prisha Balgovind  3 , Reza Haqshenas  5 , Steve G Badman  6 , John Bolnga  7 , Josephine Gabuzzi  7 , Zure Kombati  8 , Gloria M Munnull  9 , Julia Ml Brotherton  4 , Marion Saville  4 , John M Kaldor  6 , Pamela J Toliman  6 , Andrew J Vallely  6 , Gerald L Murray  5
Affiliations

DNA methylation at individual CpG-sites of EPB41L3, HTERT and FAM19A4 are useful for detection of cervical high-grade squamous intraepithelial lesions (HSIL) or worse: Analysis of individual CpG-sites outperforms averaging

Monica Molano et al. Tumour Virus Res. 2024 Dec.

Abstract

Global methylation analysis of gene promoters is promising for detection of high-grade squamous intraepithelial lesions or worse (HSIL+) in high-risk human papillomavirus (hrHPV)-positive women. However, diagnostic performance of methylation data at individual CpG-sites is limited. We explored methylation for predicting HSIL+ in self- and clinician-collected samples from Papua New Guinea. Methylation of EPB41L3 (1-6 CpG-sites), hTERT (1-10 CpG-sites) and FAM19A4 (1-5 CpG-sites) was assessed through pyrosequencing from 44 HPV+ samples (4 cancers, 19 HSIL, 4 low-grade squamous intraepithelial lesions (LSIL), 17 normal). New primers were designed for FAM19A4 directed to the first exon region not explored previously. In clinician-collected samples, methylation at CpG-sites 4 and 5 of EPB41L3 were the best HSIL predictors (AUC >0.83) and CpG-site 4 for cancer (0.925). Combination of EPB41L3 sites 2/4 plus FAM19A4 site 1 were the best HSIL+ markers [100% sensitivity, 63.2% specificity]. Methylation at CpG-site 5 of FAM19A4 was the best HSIL predictor (0.67) in self-collected samples, and CpG-sites 1 and 3 of FAM19A4 for cancer (0.77). Combined, FAM19A4 site 1 plus HPV 16/18 detection yielded sensitivity of 82.6% and specificity of 61.9%. In conclusion, methylation at individual CpG-sites of EPB41L3 and FAM19A4 outperformed global analysis and improved HSIL+ detection, warranting further investigation.

Keywords: Cervical cancer; DNA methylation; Diagnostic test; Epigenetics; Human papillomavirus; Molecular diagnostics.

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Conflict of interest statement

Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. AJV, JG, JB, GMM, PJT, SGB, JMK have received subsidized test kits for research from Cepheid. MS, JMLB, GT, DH have received donated test kits for research from Roche, Abbott, Seegene, Cepheid, Aus Diagnostics and Becton Dickinson. AJV and MS jointly lead the Elimination of Cervical Cancer in theWesternPacific (ECCWP) program with philanthropic funding support from the Minderoo Foundation and the Frazer Family Foundation; and equipment, tests and consumables donated by Cepheid for HPV-based cervical screening in Papua New Guinea and Vanuatu. SMG is a member of the Global Advisory Board HPV Merck, and has led investigator-initiated grants from Merck on HPV in young women. All other authors report no potential conflicts.

Figures

Fig. 1
Fig. 1
Percentage of DNA methylation at individual CpG-sites of EPB41L3 gene according to cytology diagnosis in hrHPV+ women. Clinician-collected cervical samples (Cervical) are presented in the top panel, and self-collected vaginal samples (Vaginal) in the bottom panel. Comparisons were assessed by the nonparametric Wilcoxon test, with whiskers corresponding to the first and third quartiles (the 25th and 75th percentiles). Significance values p ≤ 0.05.
Fig. 2
Fig. 2
Percentage of DNA methylation at individual CpG-sites of hTERT gene according to cytology diagnosis in hrHPV+ women. Clinician-collected cervical samples (Cervical) are presented in the top panel and self-collected vaginal samples (Vaginal) in the lower panel. Comparisons were assessed by the nonparametric Wilcoxon test, with whiskers corresponding to the first and third quartiles (the 25th and 75th percentiles). Significance values p ≤ 0.05.
Fig. 3
Fig. 3
Percentage of DNA methylation at individual CpG-sites of FAM19A4 gene according to cytological diagnosis in hrHPV+ women. Clinician-collected cervical samples (Cervical) are presented in the top panel, and self-collected vaginal (Vaginal) samples in the bottom panel. Comparisons were assessed by the nonparametric Wilcoxon test, with whiskers corresponding to the first and third quartiles (the 25th and 75th percentiles). Significance values p ≤ 0.05.
Fig. 4
Fig. 4
Area under the curve values of FAM19A4 (CpG-sites 1–5), EPB41L3 (CpG-sites 1–6) and hTERT (CpG-sites 1–10) methylation for distinguishing HSIL, HSIL+ and SCC from normal/LSIL, stratified by sample type Orange bars and green bars represent clinician-collected cervical and self-collected vaginal samples respectively. HSIL, in the top panel, HSIL+ in the middle panel and SCC in the bottom panel. The p values in the figure represent p < 0.05 = *, p < 0.01 = **, p < 0.005 = ***, p < 0.001 = ****. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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References

    1. Gravitt P.E., Silver M.I., Hussey H.M., Arrossi S., Huchko M., Jeronimo J., et al. Achieving equity in cervical cancer screening in low- and middle-income countries (LMICs): Strengthening health systems using a systems thinking approach. Prev. Med. 2021;144 - PubMed
    1. Cuschieri K., Ronco G., Lorincz A., Smith L., Ogilvie G., Mirabello L., et al. Eurogin roadmap 2017: triage strategies for the management of HPV-positive women in cervical screening programs. Int. J. Cancer. 2018;143(4):735–745. - PubMed
    1. Polman N.J., de Haan Y., Veldhuijzen N.J., Heideman D.A.M., de Vet H.C.W., Meijer C.J.L.M., et al. Experience with HPV self-sampling and clinician-based sampling in women attending routine cervical screening in The Netherlands. Prev. Med. 2019;125:5–11. - PubMed
    1. Maver P.J., Poljak M. Primary HPV-based cervical cancer screening in Europe: implementation status, challenges, and future plans. Clin. Microbiol. Infect. 2020;26:579–583. - PubMed
    1. Machalek D.A., Roberts J.M., Garland S.M., Thurloe J., Richards A., Chambers I., et al. Routine cervical screening by primary HPV testing: early findings in the renewed National Cervical Screening Program. Med. J. Aust. 2019;211:113–119. - PubMed

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