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Discovery of new methylation markers to improve screening for cervical intraepithelial neoplasia grade 2/3

BACKGROUND: Assessment of DNA promoter methylation markers in cervical scrapings for the detection of cervical intraepithelial neoplasia (CIN) and cervical cancer is feasible, but finding methylation markers with both high sensitivity as well as high specificity remains a challenge. In this study, w...

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Autores principales: Boers, A., Wang, R., van Leeuwen, R. W., Klip, H. G., de Bock, G. H., Hollema, H., van Criekinge, W., de Meyer, T., Denil, S., van der Zee, A. G J., Schuuring, E., Wisman, G. B. A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4784352/
https://www.ncbi.nlm.nih.gov/pubmed/26962367
http://dx.doi.org/10.1186/s13148-016-0196-3
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author Boers, A.
Wang, R.
van Leeuwen, R. W.
Klip, H. G.
de Bock, G. H.
Hollema, H.
van Criekinge, W.
de Meyer, T.
Denil, S.
van der Zee, A. G J.
Schuuring, E.
Wisman, G. B. A.
author_facet Boers, A.
Wang, R.
van Leeuwen, R. W.
Klip, H. G.
de Bock, G. H.
Hollema, H.
van Criekinge, W.
de Meyer, T.
Denil, S.
van der Zee, A. G J.
Schuuring, E.
Wisman, G. B. A.
author_sort Boers, A.
collection PubMed
description BACKGROUND: Assessment of DNA promoter methylation markers in cervical scrapings for the detection of cervical intraepithelial neoplasia (CIN) and cervical cancer is feasible, but finding methylation markers with both high sensitivity as well as high specificity remains a challenge. In this study, we aimed to identify new methylation markers for the detection of high-grade CIN (CIN2/3 or worse, CIN2+) by using innovative genome-wide methylation analysis (MethylCap-seq). We focused on diagnostic performance of methylation markers with high sensitivity and high specificity considering any methylation level as positive. RESULTS: MethylCap-seq of normal cervices and CIN2/3 revealed 176 differentially methylated regions (DMRs) comprising 164 genes. After verification and validation of the 15 best discriminating genes with methylation-specific PCR (MSP), 9 genes showed significant differential methylation in an independent cohort of normal cervices versus CIN2/3 lesions (p < 0.05). For further diagnostic evaluation, these 9 markers were tested with quantitative MSP (QMSP) in cervical scrapings from 2 cohorts: (1) cervical carcinoma versus healthy controls and (2) patients referred from population-based screening with an abnormal Pap smear in whom also HPV status was determined. Methylation levels of 8/9 genes were significantly higher in carcinoma compared to normal scrapings. For all 8 genes, methylation levels increased with the severity of the underlying histological lesion in scrapings from patients referred with an abnormal Pap smear. In addition, the diagnostic performance was investigated, using these 8 new genes and 4 genes (previously identified by our group: C13ORF18, JAM3, EPB41L3, and TERT). In a triage setting (after a positive Pap smear), sensitivity for CIN2+ of the best combination of genes (C13ORF18/JAM3/ANKRD18CP) (74 %) was comparable to hrHPV testing (79 %), while specificity was significantly higher (76 % versus 42 %, p ≤ 0.05). In addition, in hrHPV-positive scrapings, sensitivity and specificity for CIN2+ of this best-performing combination was comparable to the population referred with abnormal Pap smear. CONCLUSIONS: We identified new CIN2/3-specific methylation markers using genome-wide DNA methylation analysis. The diagnostic performance of our new methylation panel shows higher specificity, which should result in prevention of unnecessary colposcopies for women referred with abnormal cytology. In addition, these newly found markers might be applied as a triage test in hrHPV-positive women from population-based screening. The next step before implementation in primary screening programs will be validation in population-based cohorts. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13148-016-0196-3) contains supplementary material, which is available to authorized users.
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spelling pubmed-47843522016-03-10 Discovery of new methylation markers to improve screening for cervical intraepithelial neoplasia grade 2/3 Boers, A. Wang, R. van Leeuwen, R. W. Klip, H. G. de Bock, G. H. Hollema, H. van Criekinge, W. de Meyer, T. Denil, S. van der Zee, A. G J. Schuuring, E. Wisman, G. B. A. Clin Epigenetics Research BACKGROUND: Assessment of DNA promoter methylation markers in cervical scrapings for the detection of cervical intraepithelial neoplasia (CIN) and cervical cancer is feasible, but finding methylation markers with both high sensitivity as well as high specificity remains a challenge. In this study, we aimed to identify new methylation markers for the detection of high-grade CIN (CIN2/3 or worse, CIN2+) by using innovative genome-wide methylation analysis (MethylCap-seq). We focused on diagnostic performance of methylation markers with high sensitivity and high specificity considering any methylation level as positive. RESULTS: MethylCap-seq of normal cervices and CIN2/3 revealed 176 differentially methylated regions (DMRs) comprising 164 genes. After verification and validation of the 15 best discriminating genes with methylation-specific PCR (MSP), 9 genes showed significant differential methylation in an independent cohort of normal cervices versus CIN2/3 lesions (p < 0.05). For further diagnostic evaluation, these 9 markers were tested with quantitative MSP (QMSP) in cervical scrapings from 2 cohorts: (1) cervical carcinoma versus healthy controls and (2) patients referred from population-based screening with an abnormal Pap smear in whom also HPV status was determined. Methylation levels of 8/9 genes were significantly higher in carcinoma compared to normal scrapings. For all 8 genes, methylation levels increased with the severity of the underlying histological lesion in scrapings from patients referred with an abnormal Pap smear. In addition, the diagnostic performance was investigated, using these 8 new genes and 4 genes (previously identified by our group: C13ORF18, JAM3, EPB41L3, and TERT). In a triage setting (after a positive Pap smear), sensitivity for CIN2+ of the best combination of genes (C13ORF18/JAM3/ANKRD18CP) (74 %) was comparable to hrHPV testing (79 %), while specificity was significantly higher (76 % versus 42 %, p ≤ 0.05). In addition, in hrHPV-positive scrapings, sensitivity and specificity for CIN2+ of this best-performing combination was comparable to the population referred with abnormal Pap smear. CONCLUSIONS: We identified new CIN2/3-specific methylation markers using genome-wide DNA methylation analysis. The diagnostic performance of our new methylation panel shows higher specificity, which should result in prevention of unnecessary colposcopies for women referred with abnormal cytology. In addition, these newly found markers might be applied as a triage test in hrHPV-positive women from population-based screening. The next step before implementation in primary screening programs will be validation in population-based cohorts. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13148-016-0196-3) contains supplementary material, which is available to authorized users. BioMed Central 2016-03-09 /pmc/articles/PMC4784352/ /pubmed/26962367 http://dx.doi.org/10.1186/s13148-016-0196-3 Text en © Boers et al. 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Boers, A.
Wang, R.
van Leeuwen, R. W.
Klip, H. G.
de Bock, G. H.
Hollema, H.
van Criekinge, W.
de Meyer, T.
Denil, S.
van der Zee, A. G J.
Schuuring, E.
Wisman, G. B. A.
Discovery of new methylation markers to improve screening for cervical intraepithelial neoplasia grade 2/3
title Discovery of new methylation markers to improve screening for cervical intraepithelial neoplasia grade 2/3
title_full Discovery of new methylation markers to improve screening for cervical intraepithelial neoplasia grade 2/3
title_fullStr Discovery of new methylation markers to improve screening for cervical intraepithelial neoplasia grade 2/3
title_full_unstemmed Discovery of new methylation markers to improve screening for cervical intraepithelial neoplasia grade 2/3
title_short Discovery of new methylation markers to improve screening for cervical intraepithelial neoplasia grade 2/3
title_sort discovery of new methylation markers to improve screening for cervical intraepithelial neoplasia grade 2/3
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4784352/
https://www.ncbi.nlm.nih.gov/pubmed/26962367
http://dx.doi.org/10.1186/s13148-016-0196-3
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