Methylation analysis for the identification of cervical lesions to improve cervical cancer screening in a Chinese population
Li, Na DOI:
10.33612/diss.134442856
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date: 2020
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Li, N. (2020). Methylation analysis for the identification of cervical lesions to improve cervical cancer screening in a Chinese population. University of Groningen. https://doi.org/10.33612/diss.134442856
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.
Chapter 1
General introduction
1. Epidemiology of cervical cancer
Cervical cancer is the fourth leading cause of cancer death in women globally, with an estimated 570,000 new cases and 311,000 deaths in 2018 worldwide 1. Nearly 90% of cervical cancer deaths occur in developing countries 2. The latest statistics show ~700 new cervical cancer patients and ~250 deaths per year in the Netherlands, while the number of new cases in China is ~106,500 and ~48,000 women die from the disease every year 1.
Cervical cancer develops from a premalignant phase defined as cervical intraepithelial neoplasia (CIN), including CIN1, CIN2 and CIN3. However, not all CIN lesions will progress into invasive cancer 3. Most CIN1 lesion will regress (60%), however, 30% persist, 10% proceed to CIN3 and 1% to cervical cancer 4. CIN2 lesions progress to CIN3 in 20% and to cancer in 5%, 40% regress and 40% will persist 5, while ~50% of CIN3 women will develop cancer if these lesions are not treated 6, 7. Progression of CIN towards invasive cervical cancer generally takes 10-15 years. Taking advantage of this long gradually developmental process, cervical cancer could be effectively prevented by early diagnosis and treatment of CIN lesions through cervical screening 8. The most widely used cervical screening approaches are based on cytological examinations of cervical cells obtained by cervical scraping. Routine cytological classifications include the Pap and the Bethesda systems (TBS), which are well correlated with the histopathology of the various stages of (pre)malignant cervical lesions (Figure 1 and Table 1). The cytological classification is used to select women who need referral for colposcopy. In 1977 infection with human papillomavirus (HPV) was reported to be associated with cervical cancer 9 and now has been identified as the main cause of cervical cancer. In many (richer) countries cytology and HPV testing are nowadays both regularly combined in cervical cancer screening to improve correct referrals of women with disease.
Figure 1. Morphological alterations in cervical carcinogenesis with the histological CIN classification and cytology Pap classification (adapted from Umar et al. Nat. Rev. Cancer 2012).
Table 1. Overview of the cytomorphological and histological nomenclature of cervical cancer and its premalignant lesions.
Histology Cytology
Dysplasia CIN Bethesda Papanicolaou
Normal Normal Within normal limits Pap 1
Benign atypia Inflammatory
atypia Benign celluar changes Pap 1
Atypical cells Squamous atypia ASCUS Pap 2
Mild Dysplasia CIN1 Low-grade SIL (LSIL) Pap 3A1
Moderate Dysplasia CIN2
High-grade SIL (HSIL)
Pap 3A2 Severe Dysplasia
CIN3 Pap 3B
Carcinoma in situ Pap 4
(Microinvasive) cancer (Microinvasive)
cancer (Microinvasive) cancer Pap 5
ASCUS: Atypical Squamous Cells of Undetermined Significance. CIN: cervical intraepithelial neoplasia; PAP: Papanicolaou; SIL: Squamous Intraepithelial Lesion. (adapted from Nijhuis ER et al. Cell Oncol. 2006).
2. Human papillomavirus
The association of HPV with cervical cancer was first discovered by Zur Hausen in 1977 9. Papillomavirus has a circular double-stranded DNA structure. Currently, over 200 types of HPV have been fully sequenced 10, 11. Individual HPV types are subdivided into high-risk HPV (hrHPV) and low-risk HPV (lrHPV) groups according to their association with various malignant and benign lesions. According to the classification of the International Agency for Research on Cancer, HPV6 and HPV11 belong to lrHPV group which are correlated with benign genital
warts 10. Twelve HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59) are classified as hrHPV due to their carcinogenic capacity in cervical cancer. HPV68 is recognized as probably oncogenic. Another 12 HPV types (26, 30, 34, 53, 66, 67, 69, 70, 73, 82, 85 and 97) are considered as possibly carcinogenic 12. However, some studies showed that HPV66 and 68 are significantly more common in cancer cases with very similar biomarker patterns as those fully recognized carcinogenic hrHPV types 13, 14. HPV16 and 18 are present in around 70% of all cervical cancers 15.Viral proteins E6 and E7 are main HPV oncoproteins. E6 stimulates the degradation of tumor suppressor p53, via the formation of a trimeric complex comprising E6, p53 and the cellular ubiquitination enzyme E6-AP, which results in resistance to apoptosis and an increase in chromosomal instability. E7 competes with EF2 for binding the retinoblastoma tumor suppressor pRB, which inactivates its negative regulator of cell cycle and leads to cell cycle-promotion 16, 17. The majority of hrHPV infections will be cleared within 1-2 years because of the temporary natural history of hrHPV infection, especially due to the host’s immune response 18. Only persistent hrHPV infection may lead to neoplastic transformation. As mentioned above, only a subset of CIN will progress to cancer which also appears to be dependent on the hrHPV type present. This is likely a reflection of the heterogeneity of CIN lesions associated with different oncogenic potential of different hrHPV types combined with various viral persistent infection and immune system evasion 19-21.
3. Cervical cancer screening program
Cytology has been used as a traditional program in many countries for cervical cancer screening 22. Cytology is considerable successful with high specificity. The moderate sensitivity (~60%) however for the detection of CIN2+ due to the subjective nature of the cytological interpretation, still leads to a large number of missed diagnoses of cervical cancer 23-25. Primary hrHPV testing shows advantages of objectivity and high-throughput productivity accompanied with more detection of CIN2+ (90%) and CIN3+ (95%) lesions 26, 27. However, transient hrHPV infection, particularly in young women, results in a rather low specificity, leading to unnecessary referrals of many women for colposcopy without having a disease 26, 28, 29. Therefore, triage testing is mandatory to decrease the number of unnecessary referrals. Recent data revealed that colposcopy
referral rate after primary hrHPV testing with cytology as triage test is significantly higher than observed in the primary cytology-based program (from 5% to 9%) 30.
In China, the presence of cervical cancer screening programs is largely dependent on the area where women live. In the urban areas opportunistic screening is frequently observed 31. Liquid-based cytology and TBS reporting terminology have been widely adopted in China 32. However, the majority of women living in rural areas still can not get access to cervical screening due to the lack of specialist cytology training programs 33. The more objective hrHPV tests might be a very useful tool in China to overcome these challenges. With the increasing application of hrHPV testing in China, huge numbers of hrHPV-positive women are referred for colposcopy. Due to the inevitable large proportion of unnecessary referrals an efficient triage test is compulsory. In addition, the overall hrHPV-positive rate in China is very high (around 20%) with even much higher infection rates (31%) in younger women (15-19 years old). This high frequency of (transient) hrHPV infections poses a major challenge for application of hrHPV screening in China 31, 34.
Until the end of 2016, primary cytology-based screening was used in the Dutch cervical cancer screening program. Women were invited for screening every 5 years from ages 30 to 60. Women with abnormal cytology of high-grade squamous intraepithelial lesion (HSIL) and worse were referred to a gynecologist. For those with abnormal cytology of atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous intraepithelial lesion (LSIL), a repeated cytology or combined hrHPV test at 6 months was performed, of which women with cytology worse than LSIL and/or positive hrHPV result were referred to the gynecologist. Women with normal cytology and positive hrHPV or with abnormal cytology of ASCUS/LSIL and negative hrHPV were offered a repeated cytology triage test at 18 months, then colposcopy was recommended for women with any abnormal cytology result.
In 2017, the Netherlands changed to a primary hrHPV screening program. In this program cytology is used as a triage test to select hrHPV-positive women with high risk of disease for referral to a gynecologist 30. HrHPV-positive women without cytological abnormalities are invited to have another triage cytology test at 6 months and women with abnormal cytology ≥ASCUS are referred for colposcopy. In general, women between the ages of 30 and 60 are invited to participate approximately every 5 or 10 years. In this current population-based
screening program, women who do not visit their general practitioner for cervical scraping upon invitation (considered as non-responders) are offered a self-sampling kit that is directly sent to a central laboratory for hrHPV testing 30. In contrast to general practitioner (GP)-collected cervical scrapings, self-sampled material is only suitable for DNA testing. Women with a hrHPV-positive self-sampling test need to visit GP to collect cervical scraping for triage cytological examination.
4. Triage test of hrHPV-positive women
A variety of triage options of hrHPV-positive women have been reported, including cytology, p16/Ki67 dual-staining, HPV genotyping, RNA-based biomarkers, HPV E6 protein and host or viral DNA methylation analysis. Currently, cytology and specific HPV genotyping are the most applied triage approaches after a positive hrHPV test 35, whereas p16/Ki67 dual-staining and DNA methylation are still in research setting. The evaluation of E6 protein and E6/E7 mRNA has only been reported in some clinical cohorts with hrHPV infection 23, 36 and is therefore not further described below.
4.1 Cytology
Cytology is the most commonly used triage strategy after hrHPV screening and those women with abnormal cytology (≥ASCUS) are referred for colposcopy 37. When using cytology as a primary screening, the cyto-pathologists have to identify and recognize abnormal cytology present in less than 3% of all scrapings when analyzing women in the population-based screening program resulting in a sensitivity of ~60% with a very high specificity of ~98% 27. When using cytology as a triage test in hrHPV-positive samples, the cyto-pathologists are likely to deliver a biased cytological assessment (being aware of the hrHPV status), thereby resulting in sensitivities ranging between 62-94% and specificities between 50-77% 23, 38-43. In the Netherlands, evaluation of the first 2 years of population-based screening revealed many referrals per screening round with only ~50% of women having a CIN2+ lesion 30, 44. So, cytology is not the ideal triage test and optimizing triage to decrease unnecessary referrals is urgently needed.
4.2 HPV16/18 genotyping
Specific genotyping refers generally to tests for HPV types 16 and 18, which together are responsible for 70% of cervical cancers 45. HPV16/18 genotyping has been implemented as a triage test for hrHPV-positive individuals in the guideline of the American Society for Colposcopy and Cervical Pathology and is also recommended in China 35. The performance of HPV16/18 genotyping in triaging hrHPV-positive women has been evaluated in multiple studies, in which sensitivity and specificity for CIN2+ detection is ~60% and ~80%, respectively 23, 46-48. However, HPV genotype distribution varies by region geography. In Asia, HPV types 52 and 58 are found to play dominant roles in cervical cancer as well as HPV16 and 18 34, 49. In addition, in many countries national prophylactic vaccination programs against HPV16/18 have been started and in the coming years it is expected to result in a significant decrease in cervical cancer and in cancers with other than HPV16/18 genotypes 50. Thus, it is still necessary to seek for other triage strategies.
4.3 P16/Ki67 dual staining
P16/Ki-67 dual-staining (CINtec Plus Cytology test (Roche)), the immunocytochemical detection of co-expression of tumor-suppressor p16 and cell proliferation marker Ki-67, has been proposed as another useful method for triaging hrHPV-positive women 51-53. Abnormal co-expression of p16/Ki67 within the same cell implies cell cycle dysregulation assumed to be caused by integration of HPV infections 54. Currently, studies of p16/Ki-67 triaging hrHPV-positive women show a weighted mean sensitivity of 85% (range 83-98%) to detect CIN2+ with a weighted mean specificity of 60% (range 39-77%) 52, 55-58. However, similar with cytology, p16/Ki-67 dual-staining testing depends on subjective microscopic assessment by qualified professionals. Furthermore, self-sampling material is not suitable for p16/Ki-67 dual-staining 59.
5. DNA methylation
DNA methylation is a heritable epigenetic modification by which a methyl group is added to the C5 carbon residues of cytosines (5mC) mediated by DNA methyltransferases (Figure 2A). DNA
methylation occurs almost exclusively at cytosines located next to a guanidine nucleotide in the CpG site (Figure 2B) 60, 61. Methylation can change the epigenetic structure of a DNA segment without changing the nucleotide sequence structure itself and is involved in the regulation of binding of DNA binding complexes. DNA hypermethylation of regulatory sequences in gene promoter sequences is typically associated with gene transcription silencing (Figure 2C). This phenomenon is often seen in cancer cells for the inactivation of tumor suppressor genes 62-64.
Figure 2. (A) Schematic representation of DNA methylation, which converts cytosine to 5′methyl-cytosine catalyzed by DNMT.
(B) DNA methylation typically occurs at cytosines that are followed by a guanine (i.e., CpG motifs) 65.
(C) DNA promoter methylation is associated with gene inactivation 66.
NOTES: SAM = S-adenosylmethionine; SAH = S-adenosylhomocysteine; DNMT = DNA methyltransferase.
Bisulfite treatment of genomic DNA converts unmethylated cytosines to uracil, but leaves methylated cytosines unaffected. After bisulfite conversion, Methylation-specific PCR (MSP) assays can be designed to distinguish methylated DNA target sequences from unmethylated counterpart sequences 67. Accurate quantification can be achieved through real-time measurement of the methylated amplicons with an additional fluorescence-labeled specific probe referred to as quantitative MSP (QMSP) 68. QMSP is a highly sensitive, specific and reproducible technique which makes it suitable for implementation in cervical cancer screening 69-71. In addition to (Q)MSP various other methods to detect methylated DNA have been reported such as combined bisulfite restriction analysis (COBRA), bisulfite pyrosequencing, methylation
microarray profiling and bisulfite next-generation sequencing 72. In this thesis, we will focus on methylation markers detected with (Q)MSP only.
Besides the functional implication of DNA methylation, these DNA methylation patterns can also be used as a diagnostic target. Classic examples of DNA methylation markers in different diagnostic clinical settings are methylation detection of the GSTP1 and MGMT gene. Hypermethylation of the GSTP1 promoter was demonstrated as a powerful diagnostic tool for prostate cancer 73, 74. Hypermethylation of the MGMT promoter predicts a better response to treatment with chemotherapy using alkylating agents, and is currently used in treatment decision making in glioblastomas 75, 76.
Studies on DNA methylation in cervical cancer have focused on the detection of methylation of the host genes and hrHPV DNA. A recent review described that only methylation of HPV16 L1 and L2 regions is consistently associated with increasing disease severity in different studies with pooled sensitivity and specificity of 77% and 64% to detect CIN2+ lesions. However, information for other HPV types is lacking 77.
In the last few decades, numerous studies have been reported on the discovery of methylation markers of the host genome associated with CIN2, CIN3 and/or invasive cervical cancer, resulting in the identification of several methylation markers with a pooled sensitivity of 69% and 71% to detect respectively CIN2+ and CIN3+ at a set specificity of 70%, reported in a recent meta-analysis 78. Using (Q)MSP, most of these methylation markers with a potential relevance as a triage test in cervical cancer screening programs, were identified and reported by the Taiwanese group (e.g. PAX and ZNF582 ) 79-92, the Amsterdam group (VUMC) (e.g. CADM1, MAL, Hsa-miR124-2, and FAM19A4) 93-95 and our group at University Medical Centre Groningen (UMCG). The UMCG group identified and validated several host DNA methylation markers with high sensitivity and specificity to detect CIN2+ lesions 70, 96-102. To determine the clinical relevance of these methylation markers, both the discovery and validation were performed using mainly cytological abnormal scrapings selected from Dutch cohorts. Six specific methylation markers (ANKRD18CP, C13ORF18, EPB41L3, JAM3, ZSCAN1 and SOX1) have been identified with sensitivities between 43-72% and specificities between 78-94% for CIN2+ detection 99-104. Sensitivities could be improved further with acceptable loss of specificity through different combinations of several methylation markers. Three methylation
panels (C13ORF18/EBP41L3/JAM3, C13ORF18/ANKRD18CP/JAM3 and ZSCAN1/SOX1) revealed a similar sensitivity of ~75% for the detection of CIN2+ compared to hrHPV testing, but with a better specificity (72-83%), leading to less unnecessary colposcopy referrals. Furthermore, in hrHPV-positive scrapings similar sensitivity and specificity to detect CIN2+ were found with these same marker combinations 98, 99, suggesting that these same markers might be relevant also as a triage test in women primarily screened with hrHPV tests. However, we are still awaiting the results of analysis of such cohorts in the near future. Only very few studies using (Q)MSP have been performed to validate UMCG markers in cohorts in China 68, 105. Some markers were evaluated, with methods other than (Q)MSP (eg. bisulfite pyrosequencing) and therefore direct comparison with studies using Dutch cohorts remains difficult 106.
6. Outline of this thesis
Gene promoter methylation is an early event in cervical carcinogenesis. Therefore, detection of methylation markers may be an excellent screening test for detection of (pre)malignant cervical neoplasia. At UMCG, for six methylation markers (ANKRD18CP, C13ORF18, EPB41L3, JAM3, ZSCAN1 and SOX1), it has been observed that their methylation status increases with gradual increasing severity of the cervical lesions. Using three methylation panels (C13ORF18/EBP41L3/JAM3, C13ORF18/ANKRD18CP/JAM3 and ZSCAN1/SOX1) a high combined sensitivity and specificity for the detection of CIN2+ lesions was revealed. The main goal of this thesis is to evaluate whether these Dutch methylation markers are also associated with severity of disease as well as predictive for CIN2+/CIN3+ (using sensitivity/specificity) in cervical scrapings collected globally from women from different geographic areas and races. For this purpose, a cohort of Chinese women was selected of whom cervical scrapings were prospectively collected and stored in the Department of Pathology at the Tianjin Central Hospital of Gynecology Obstetrics. These women visited the hospital because of complaints (70%), for opportunistic screening (27%) or for unknown reasons (3%). Scrapings were examined routinely using cytology and hrHPV testing. Women with hrHPV16/18 or abnormal cytology were referred for colposcopy. A cohort of 246 Chinese women with all cytology, hrHPV typing, histology and clinical information was used in this thesis to investigate the performance of methylation markers compared to a similar Dutch cohort.
In chapter 2, we validated the methylation panels previously evaluated in Dutch cohorts (C13ORF18/EBP41L3/JAM3, C13ORF18/ANKRD18CP/JAM3 and ZSCAN1/SOX1) in this Chinese cohort. The same QMSP assays developed at UMCG were used at the lab in Tianjin (China). Many studies have been performed to identify ideal methylation markers to detect (pre)malignant cervical neoplasia 99-104. As more and more countries are changing to primary hrHPV screening for cervical cancer, an optimal triage test is needed to mainly refer those women with disease to the gynecologist. As most available methylation markers were identified and validated on cohorts based on primary cytological screening, in chapter 3 we performed a systematic review on methylation markers to predict clinical outcome restricted to hrHPV-positive women. In this review, we performed a comprehensive analysis of published studies that report on methylation markers in hrHPV-positive cervical samples by (Q)MSP and data on sensitivity and specificity to detect CIN2+ or CIN3+ lesions.
Based on this systematic review, two other promising methylation markers (PAX1 and ZNF582) were identified to play potential roles for early cervical cancer detection. To understand the diagnostic role of ZNF582, we performed another meta-analysis of published studies, however irrespective of hrHPV status and including only Chinese women (chapter 4).
In addition to ZNF582, also PAX1 methylation was mainly studied in cervical scrapings in Asian populations 79-88. To further evaluate the diagnostic performance of PAX1 and ZNF582 methylation using QMSP, we investigated and directly compared the methylation status in Dutch and Chinese populations (chapter 5). Chapter 6 contains the summary and chapter 7 the general discussion and future perspectives of this thesis.
Reference
1. Arbyn, M., E. Weiderpass, L. Bruni, et al., Estimates of incidence and mortality of cervical cancer in 2018: a
worldwide analysis. The Lancet Global Health, 2020. 8(2):e191-e203.
2. Shrestha, A.D., D. Neupane, P. Vedsted, et al., Cervical Cancer Prevalence, Incidence and Mortality in Low
and Middle Income Countries: A Systematic Review. Asian Pac J Cancer Prev, 2018. 19(2):319-24.
3. Kudela, E., V. Holubekova, A. Farkasova, et al., Determination of malignant potential of cervical
intraepithelial neoplasia. Tumour Biol, 2016. 37(2):1521-5.
4. Moscicki, A.-B., S. Shiboski, N.K. Hills, et al., Regression of low-grade squamous intra-epithelial lesions in
young women. The Lancet, 2004. 364(9446):1678-83.
5. Ostor, A.G., Natural history of cervical intraepithelial neoplasia: a critical review. Int J Gynecol Pathol, 1993. 12(2):186-92.
6. Agency for Healthcare Research and Quality. Evidence synthesis No. 158: screening for cervical cancer with
high-risk human papillomavirus testing: a systematic evidence review for the US Preventive Services Task Force.
7. Peto, J., C. Gilham, O. Fletcher, et al., The cervical cancer epidemic that screening has prevented in the UK. The Lancet, 2004. 364(9430):249-56.
8. Fang, C., S.Y. Wang, Y.L. Liou, et al., The promising role of PAX1 (aliases: HUP48, OFC2) gene methylation
in cancer screening. Mol Genet Genomic Med, 2019. 7(3):e506.
9. zur Hausen, H., Human papillomaviruses and their possible role in squamous cell carcinomas. Curr Top Microbiol Immunol, 1977. 78:1-30.
10. Groves, I.J.,N. Coleman, Pathogenesis of human papillomavirus-associated mucosal disease. J Pathol, 2015. 235(4):527-38.
11. Egawa, N., K. Egawa, H. Griffin, et al., Human Papillomaviruses; Epithelial Tropisms, and the Development
of Neoplasia. Viruses, 2015. 7(7):3863-90.
12. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Biological agents. A review of
human carcinogens. IARC Monogr Eval Carcinog Risks Hum, 2012. 100(B):1-441.
13. Halec, G., L. Alemany, B. Lloveras, et al., Pathogenic role of the eight probably/possibly carcinogenic HPV
types 26, 53, 66, 67, 68, 70, 73 and 82 in cervical cancer. J Pathol, 2014. 234(4):441-51.
14. Bzhalava, D., P. Guan, S. Franceschi, et al., A systematic review of the prevalence of mucosal and
cutaneous human papillomavirus types. Virology, 2013. 445(1-2):224-31.
15. Tyson, N., HPV Update. J Pediatr Adolesc Gynecol, 2017. 30(2):262-4.
16. zur Hausen, H., Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer, 2002. 2(5):342-50.
17. Georgescu, S.R., C.I. Mitran, M.I. Mitran, et al., New Insights in the Pathogenesis of HPV Infection and the
Associated Carcinogenic Processes: The Role of Chronic Inflammation and Oxidative Stress. J Immunol Res,
2018. 2018:5315816.
18. Rodriguez, A.C., M. Schiffman, R. Herrero, et al., Rapid clearance of human papillomavirus and
implications for clinical focus on persistent infections. J Natl Cancer Inst, 2008. 100(7):513-7.
19. Doorbar, J., Host control of human papillomavirus infection and disease. Best Pract Res Clin Obstet Gynaecol, 2018. 47:27-41.
20. Herfs, M.,C.P. Crum, Laboratory management of cervical intraepithelial neoplasia: proposing a new
21. Verlaat, W., R.W. Van Leeuwen, P.W. Novianti, et al., Host-cell DNA methylation patterns during high-risk
HPV-induced carcinogenesis reveal a heterogeneous nature of cervical pre-cancer. Epigenetics, 2018.
13(7):769-78.
22. Cubie, H.A.,C. Campbell, Cervical cancer screening - The challenges of complete pathways of care in
low-income countries: Focus on Malawi. Womens Health (Lond), 2020. 16:1745506520914804.
23. Luttmer, R., L. De Strooper, J. Berkhof, et al., Comparing the performance of FAM19A4 methylation
analysis, cytology and HPV16/18 genotyping for the detection of cervical (pre) cancer in high‐risk HPV‐ positive women of a gynecologic outpatient population (COMETH study). International Journal of Cancer,
2016. 138(4):992-1002.
24. Mustafa, R.A., N. Santesso, R. Khatib, et al., Systematic reviews and meta-analyses of the accuracy of HPV
tests, visual inspection with acetic acid, cytology, and colposcopy. Int J Gynaecol Obstet, 2016. 132(3):259-65.
25. Kitchener, H.C., P.E. Castle, J.T. Cox, Chapter 7: Achievements and limitations of cervical cytology screening. Vaccine, 2006. 24 Suppl 3:S3/63-70.
26. Arbyn, M., G. Ronco, A. Anttila, et al., Evidence regarding human papillomavirus testing in secondary
prevention of cervical cancer. Vaccine, 2012. 30 Suppl 5:F88-99.
27. Nanda, K., D.C. McCrory, E.R. Myers, et al., Accuracy of the Papanicolaou test in screening for and
follow-up of cervical cytologic abnormalities: a systematic review. Ann Intern Med, 2000. 132(10):810-9.
28. Cuzick, J., C. Clavel, K.U. Petry, et al., Overview of the European and North American studies on HPV
testing in primary cervical cancer screening. Int J Cancer, 2006. 119(5):1095-101.
29. Whitlock, E.P., K.K. Vesco, M. Eder, et al., Liquid-based cytology and human papillomavirus testing to
screen for cervical cancer: a systematic review for the U.S. Preventive Services Task Force. Ann Intern Med,
2011. 155(10):687-97, w214-5.
30. Aitken, C.A., H.M.E. van Agt, A.G. Siebers, et al., Introduction of primary screening using high-risk HPV DNA
detection in the Dutch cervical cancer screening programme: a population-based cohort study. BMC Med,
2019. 17(1):228.
31. Tao, X., R.M. Austin, H. Zhang, et al., Histopathologic follow-up and HPV test results with HSIL
Papanicolaou test results in China's largest academic women's hospital. Cancer Cytopathol, 2017.
125(12):947-53.
32. Tao, X., R.M. Austin, H. Zhang, et al., Pap Test Reporting Rates for Conventional Smear and Liquid-Based
Cervical Cytology from the Largest Academic Women's Hospital in China: Analysis of 1,248,785 Pap Test Reports. Acta Cytol, 2015. 59(6):445-51.
33. Zheng, B., R.M. Austin, X. Liang, et al., Conventional Pap smear cervical cancer screening in 11 rural
counties in Hainan Province, China: analysis of Bethesda system reporting rates for 218,195 women (predominantly ages 35-64 years) screened in China's National Cervical Cancer Screening Program in Rural Areas (NCCSPRA). J Am Soc Cytopathol, 2017. 6(3):120-5.
34. Wang, R., X.L. Guo, G.B. Wisman, et al., Nationwide prevalence of human papillomavirus infection and
viral genotype distribution in 37 cities in China. BMC Infect Dis, 2015. 15:257.
35. Cuschieri, K., G. Ronco, A. Lorincz, et al., Eurogin roadmap 2017: Triage strategies for the management of
HPV-positive women in cervical screening programs. Int J Cancer, 2018. 143(4):735-45.
36. Zhao, F.H., J. Jeronimo, Y.L. Qiao, et al., An evaluation of novel, lower-cost molecular screening tests for
human papillomavirus in rural China. Cancer Prev Res (Phila), 2013. 6(9):938-48.
37. Ebisch, R.M., A.G. Siebers, R.P. Bosgraaf, et al., Triage of high-risk HPV positive women in cervical cancer
38. Bosgraaf, R.P., V.M. Verhoef, L.F. Massuger, et al., Comparative performance of novel self‐sampling
methods in detecting high‐risk human papillomavirus in 30,130 women not attending cervical screening.
International Journal of Cancer, 2015. 136(3):646-55.
39. Wentzensen, N., B. Fetterman, P.E. Castle, et al., p16/Ki-67 dual stain cytology for detection of cervical
precancer in HPV-positive women. Journal of the National Cancer Institute, 2015. 107(12):djv257.
40. Bergeron, C., P. Giorgi-Rossi, F. Cas, et al., Informed cytology for triaging HPV-positive women: substudy
nested in the NTCC randomized controlled trial. Journal of the National Cancer Institute, 2015.
107(2):dju423.
41. Verhoef, V.M., R.P. Bosgraaf, F.J. van Kemenade, et al., Triage by methylation-marker testing versus
cytology in women who test HPV-positive on self-collected cervicovaginal specimens (PROHTECT-3): a randomised controlled non-inferiority trial. Lancet Oncology, 2014. 15(3):315-22.
42. Richardson, L.A., M. El‐Zein, A.V. Ramanakumar, et al., HPV DNA testing with cytology triage in cervical
cancer screening: Influence of revealing HPV infection status. Cancer Cytopathology, 2015. 123(12):745-54.
43. Wright, T.C., Jr., M.H. Stoler, S. Aslam, et al., Knowledge of Patients' Human Papillomavirus Status at the
Time of Cytologic Review Significantly Affects the Performance of Cervical Cytology in the ATHENA Study.
Am J Clin Pathol, 2016. 146(3):391-8.
44. Monitor Cervical Cancer Population Screening 2018.; Available from:
https://www.rivm.nl/documenten/monitor-bevolkingsonderzoek-baarmoederhalskanker-2018.
45. Stoler, M.H., E. Baker, S. Boyle, et al., Approaches to triage optimization in HPV primary screening:
Extended genotyping and p16/Ki-67 dual-stained cytology-Retrospective insights from ATHENA. Int J
Cancer, 2020. 146(9):2599-607.
46. Rijkaart, D.C., J. Berkhof, F.J. van Kemenade, et al., Evaluation of 14 triage strategies for HPV DNA-positive
women in population-based cervical screening. Int J Cancer, 2012. 130(3):602-10.
47. Castle, P.E., M.H. Stoler, T.C. Wright, et al., Performance of carcinogenic human papillomavirus (HPV)
testing and HPV16 or HPV18 genotyping for cervical cancer screening of women aged 25 years and older: a subanalysis of the ATHENA study. The Lancet Oncology, 2011. 12(9):880-90.
48. Ebisch, R.M., G.M. de Kuyper-de Ridder, R.P. Bosgraaf, et al., The clinical value of HPV genotyping in triage
of women with high-risk-HPV-positive self-samples. Int J Cancer, 2016. 139(3):691-9.
49. Luo, G., X. Sun, M. Li, et al., Cervical human papillomavirus among women in Guangdong, China
2008-2017: Implication for screening and vaccination. J Med Virol, 2019. 91(10):1856-65.
50. Harper, D.M.,L.R. DeMars, HPV vaccines - A review of the first decade. Gynecol Oncol, 2017. 146(1):196-204.
51. Petry, K.U., D. Schmidt, S. Scherbring, et al., Triaging Pap cytology negative, HPV positive cervical cancer
screening results with p16/Ki-67 Dual-stained cytology. Gynecol Oncol, 2011. 121(3):505-9.
52. Wentzensen, N., B. Fetterman, P.E. Castle, et al., p16/Ki-67 Dual Stain Cytology for Detection of Cervical
Precancer in HPV-Positive Women. J Natl Cancer Inst, 2015. 107(12):djv257.
53. Clarke, M.A., L.C. Cheung, P.E. Castle, et al., Five-Year Risk of Cervical Precancer Following p16/Ki-67
Dual-Stain Triage of HPV-Positive Women. JAMA Oncol, 2019. 5(2):181-6.
54. Wentzensen, N.,M. von Knebel Doeberitz, Biomarkers in cervical cancer screening. Dis Markers, 2007. 23(4):315-30.
55. Luttmer, R., M.G. Dijkstra, P.J. Snijders, et al., p16/Ki-67 dual-stained cytology for detecting cervical
56. Gustinucci, D., P. Giorgi Rossi, E. Cesarini, et al., Use of Cytology, E6/E7 mRNA, and p16INK4a-Ki-67 to
Define the Management of Human Papillomavirus (HPV)-Positive Women in Cervical Cancer Screening. Am
J Clin Pathol, 2016. 145(1):35-45.
57. Stanczuk, G.A., G.J. Baxter, H. Currie, et al., Defining Optimal Triage Strategies for hrHPV Screen-Positive
Women-An Evaluation of HPV 16/18 Genotyping, Cytology, and p16/Ki-67 Cytoimmunochemistry. Cancer
Epidemiol Biomarkers Prev, 2017. 26(11):1629-35.
58. Arean-Cuns, C., M. Mercado-Gutierrez, I. Paniello-Alastruey, et al., Dual staining for p16/Ki67 is a more
specific test than cytology for triage of HPV-positive women. Virchows Arch, 2018. 473(5):599-606.
59. Satake, H., N. Inaba, K. Kanno, et al., Comparison Study of Self-Sampled and Physician-Sampled Specimens
for High-Risk Human Papillomavirus Test and Cytology. Acta Cytol, 2020:1-9.
60. Bird, A., DNA methylation patterns and epigenetic memory. Genes Dev, 2002. 16(1):6-21.
61. Greenberg, M.V.C.,D. Bourc'his, The diverse roles of DNA methylation in mammalian development and
disease. Nat Rev Mol Cell Biol, 2019. 20(10):590-607.
62. Hernandez-Juarez, J., O. Vargas-Sierra, L.A. Herrera, et al., Sodium-coupled monocarboxylate transporter is
a target of epigenetic repression in cervical cancer. Int J Oncol, 2019. 54(5):1613-24.
63. Jones, P.A., Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat Rev Genet, 2012. 13(7):484-92.
64. Li, J., H. Jin, X. Wang, Epigenetic biomarkers: potential applications in gastrointestinal cancers. ISRN Gastroenterol, 2014. 2014:464015.
65. Schematic-representation-of-DNA-methylation-which-converts-cytosine-to-5methyl-cytosine. Available
from: https://www.researchgate.net/figure/Schematic-representation-of-DNA-methylation-which-converts-cytosine-to-5methyl-cytosine_ fig1_ 259209496, 16-06-2020.
66. DNA promoter methylation and gene inactivation Available from: https://www.ncc.go.jp/en/ri/division/
epigenomics/project/230/20170913152903.html,16-06-2020.
67. Herman, J.G., J.R. Graff, S. Myöhänen, et al., Methylation-specific PCR: a novel PCR assay for methylation
status of CpG islands. Proc Natl Acad Sci U S A, 1996. 93(18):9821-6.
68. Zhang, L., J. Yu, W. Huang, et al., A Sensitive and Simplified Classifier of Cervical Lesions Based on a
Methylation-Specific PCR Assay: A Chinese Cohort Study. Cancer Manag Res, 2020. 12:2567-76.
69. Reesink-Peters, N., G.B. Wisman, C. Jeronimo, et al., Detecting cervical cancer by quantitative promoter
hypermethylation assay on cervical scrapings: a feasibility study. Mol Cancer Res, 2004. 2(5):289-95.
70. Wisman, G.B., E.R. Nijhuis, M.O. Hoque, et al., Assessment of gene promoter hypermethylation for
detection of cervical neoplasia. Int J Cancer, 2006. 119(8):1908-14.
71. Wentzensen, N., M.E. Sherman, M. Schiffman, et al., Utility of methylation markers in cervical cancer early
detection: appraisal of the state-of-the-science. Gynecol Oncol, 2009. 112(2):293-9.
72. Clausen, M., Tumor methylation markers and clinical outcome of primary oral squamous cell carcinomas, 2020, University of Groningen. p. 21.
73. Van Neste, L., J.G. Herman, G. Otto, et al., The epigenetic promise for prostate cancer diagnosis. Prostate, 2012. 72(11):1248-61.
74. Zhou, X., D. Jiao, M. Dou, et al., Association of glutathione-S-transferase p1 gene promoter methylation
and the incidence of prostate cancer: a systematic review and meta-analysis. J Cancer Res Clin Oncol, 2019.
145(8):1939-48.
75. Hegi, M.E., A.C. Diserens, T. Gorlia, et al., MGMT gene silencing and benefit from temozolomide in
76. Butler, M., L. Pongor, Y.T. Su, et al., MGMT Status as a Clinical Biomarker in Glioblastoma. Trends Cancer, 2020. 6(5):380-91.
77. Bowden, S.J., I. Kalliala, A.A. Veroniki, et al., The use of human papillomavirus DNA methylation in cervical
intraepithelial neoplasia: A systematic review and meta-analysis. EBioMedicine, 2019. 50:246-59.
78. Kelly, H., Y. Benavente, M.A. Pavon, et al., Performance of DNA methylation assays for detection of
high-grade cervical intraepithelial neoplasia (CIN2+): a systematic review and meta-analysis. Br J Cancer, 2019.
121(11):954-65.
79. Lai, H.C., Y.W. Lin, T.H. Huang, et al., Identification of novel DNA methylation markers in cervical cancer. Int J Cancer, 2008. 123(1):161-7.
80. Liou, Y.L., T.L. Zhang, T. Yan, et al., Combined clinical and genetic testing algorithm for cervical cancer
diagnosis. Clin Epigenetics, 2016. 8:66.
81. Huang, T.H., H.C. Lai, H.W. Liu, et al., Quantitative analysis of methylation status of the PAX1 gene for
detection of cervical cancer. Int J Gynecol Cancer, 2010. 20(4):513-9.
82. Lin, C.J., H.C. Lai, K.H. Wang, et al., Testing for methylated PCDH10 or WT1 is superior to the HPV test in
detecting severe neoplasms (CIN3 or greater) in the triage of ASC-US smear results. Am J Obstet Gynecol,
2011. 204(1):21.e1-7.
83. Chang, C.C., R.L. Huang, Y.P. Liao, et al., Concordance analysis of methylation biomarkers detection in
self-collected and physician-self-collected samples in cervical neoplasm. BMC Cancer, 2015. 15:418.
84. Liou, Y.L., Y. Zhang, Y. Liu, et al., Comparison of HPV genotyping and methylated ZNF582 as triage for
women with equivocal liquid-based cytology results. Clin Epigenetics, 2015. 7:50.
85. Kan, Y.Y., Y.L. Liou, H.J. Wang, et al., PAX1 methylation as a potential biomarker for cervical cancer
screening. Int J Gynecol Cancer, 2014. 24(5):928-34.
86. Lai, H.C., Y.C. Ou, T.C. Chen, et al., PAX1/SOX1 DNA methylation and cervical neoplasia detection: a
Taiwanese Gynecologic Oncology Group (TGOG) study. Cancer Med, 2014. 3(4):1062-74.
87. Lai, H.C., Y.W. Lin, R.L. Huang, et al., Quantitative DNA methylation analysis detects cervical intraepithelial
neoplasms type 3 and worse. Cancer, 2010. 116(18):4266-74.
88. Lim, E.H., S.L. Ng, J.L. Li, et al., Cervical dysplasia: assessing methylation status (Methylight) of CCNA1,
DAPK1, HS3ST2, PAX1 and TFPI2 to improve diagnostic accuracy. Gynecol Oncol, 2010. 119(2):225-31.
89. Chang, C.C., Y.C. Ou, K.L. Wang, et al., Triage of Atypical Glandular Cell by SOX1 and POU4F3 Methylation:
A Taiwanese Gynecologic Oncology Group (TGOG) Study. PLoS One, 2015. 10(6):e0128705.
90. Lin, H., T.C. Chen, T.C. Chang, et al., Methylated ZNF582 gene as a marker for triage of women with Pap
smear reporting low-grade squamous intraepithelial lesions - a Taiwanese Gynecologic Oncology Group (TGOG) study. Gynecol Oncol, 2014. 135(1):64-8.
91. Chang, C.C., R.L. Huang, H.C. Wang, et al., High methylation rate of LMX1A, NKX6-1, PAX1, PTPRR, SOX1,
and ZNF582 genes in cervical adenocarcinoma. Int J Gynecol Cancer, 2014. 24(2):201-9.
92. Huang, R.L., C.C. Chang, P.H. Su, et al., Methylomic analysis identifies frequent DNA methylation of zinc
finger protein 582 (ZNF582) in cervical neoplasms. PLoS One, 2012. 7(7):e41060.
93. Wilting, S.M., R.A. van Boerdonk, F.E. Henken, et al., Methylation-mediated silencing and tumour
suppressive function of hsa-miR-124 in cervical cancer. Molecular cancer, 2010. 9(1):167.
94. Overmeer, R.M., J.A. Louwers, C.J. Meijer, et al., Combined CADM1 and MAL promoter methylation
analysis to detect (pre‐) malignant cervical lesions in high‐risk HPV‐positive women. International Journal
95. Strooper, L.M.A.D., V.M.J. Verhoef, J. Berkhof, et al., Validation of the FAM19A4/mir124-2 DNA
methylation test for both lavage- and brush-based self-samples to detect cervical (pre)cancer in HPV-positive women. Gynecologic oncology, 2016. 141(2):341-7.
96. Eijsink, J., N. Yang, A. Lendvai, et al., Detection of cervical neoplasia by DNA methylation analysis in
cervico-vaginal lavages, a feasibility study. Gynecologic Oncology, 2011. 120(2):280-3.
97. Yang, N., J.J. Eijsink, Á. Lendvai, et al., Methylation markers for CCNA1 and C13ORF18 are strongly
associated with high-grade cervical intraepithelial neoplasia and cervical cancer in cervical scrapings.
Cancer Epidemiology and Prevention Biomarkers, 2009. 18(11):3000-7.
98. van Leeuwen, R.W., A. Oštrbenk, M. Poljak, et al., DNA methylation markers as a triage test for
identification of cervical lesions in a high risk human papillomavirus positive screening cohort.
International Journal of Cancer, 2019. 144(4):746-54.
99. Boers, A., R. Wang, R.W. van Leeuwen, et al., Discovery of new methylation markers to improve screening
for cervical intraepithelial neoplasia grade 2/3. Clin Epigenetics, 2016. 8:29.
100. Wang, R., R.W. van Leeuwen, A. Boers, et al., Genome-wide methylome analysis using MethylCap-seq
uncovers 4 hypermethylated markers with high sensitivity for both adeno- and squamous-cell cervical carcinoma. Oncotarget, 2016. 7(49):80735-50.
101. Yang, N., E.R. Nijhuis, H.H. Volders, et al., Gene Promoter Methylation Patterns throughout the Process of
Cervical Carcinogenesis. Cellular Oncology the Official Journal of the International Society for Cellular
Oncology, 2010. 32(1-2):131-43.
102. Eijsink, J.J., A. Lendvai, V. Deregowski, et al., A four-gene methylation marker panel as triage test in
high-risk human papillomavirus positive patients. Int J Cancer, 2012. 130(8):1861-9.
103. Ongenaert, M., G.B. Wisman, H.H. Volders, et al., Discovery of DNA methylation markers in cervical cancer
using relaxation ranking. BMC Med Genomics, 2008. 1:57.
104. Hoque, M.O., M.S. Kim, K.L. Ostrow, et al., Genome-wide promoter analysis uncovers portions of the
cancer methylome. Cancer Res, 2008. 68(8):2661-70.
105. Yin, A., Q. Zhang, X. Kong, et al., JAM3 methylation status as a biomarker for diagnosis of preneoplastic
and neoplastic lesions of the cervix. Oncotarget, 2015. 6(42):44373.
106. Yuan, L., Y. Hu, Z. Zhou, et al., Quantitative methylation analysis to detect cervical (Pre)-cancerous lesions
in high-risk HPV-positive women. International Journal of Clinical & Experimental Medicine, 2017.
