Methylation analysis for the identification of cervical lesions to improve cervical cancer screening in a Chinese population
Li, Na
DOI:
10.33612/diss.134442856
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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
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Chapter 7
General discussion
Early detection and subsequent treatment of precancerous lesions can effectively prevent development of cervical cancer that is primarily induced by persistent infection with high-risk human papillomavirus (hrHPV). Population-based screening programs for cervical cancer in developed countries have a significant role in reducing the incidence and mortality of cervical cancer 1. Cytology is by far the most widely used test in cervical cancer screening programs. However, the cytological screening tests demand an expensive national infrastructure with well-defined protocols for population-based cytological sample collection, and high-quality laboratories with well-trained cytologists, who have a high level of expertise to allow adequate assessment 2. Even when all requirements for adequate cytology based screening are fulfilled, a
substantial number of cervical cancer cases are still missed because of the false negative findings of cytology 3, 4. Given the improved sensitivity by hrHPV testing, several developed countries,
including the Netherlands, have switched from cytology-based to hrHPV-based primary screening for early cervical cancer detection in their national population-based programs 5.
Although hrHPV testing allows more CIN2+ lesions to be diagnosed, hrHPV screening is less specific, resulting in a significant increase of unnecessary colposcopy referrals because of transient hrHPV infections, which resolve spontaneously in most cases, especially in a young population 5, 6. Triage testing in hrHPV-positive individuals is therefore warranted to reduce
over-diagnosis and treatment. The three most studied triage strategies in hrHPV-positive women are cytology, HPV16/18 partial genotyping and p16/Ki67 dual-staining 7. Cytology and p16/Ki67 dual-staining are morphological diagnosis methods with requirement of professional experts and high quality specimen preparation. HPV16/18 genotyping is a reproducible molecular analysis, but is not useful in cases infected with other HPV subtypes. Thus, it is compulsory to explore more specific and reliable molecular biomarkers to better distinguish true cervical (pre)cancer lesions from transient hrHPV infections.
DNA methylation analysis: quantitative methylation-specific PCR (QMSP)
DNA methylation of specific host cell genes has also been evaluated as triage method to further improve the early detection of cervical neoplastic lesions. Various methods have been reported for DNA methylation analysis, such as quantitative methylation-specific PCR (QMSP), bisulfite pyrosequencing, combined bisulfite restriction analysis (COBRA), methylation microarray profiling and bisulfite next-generation sequencing 8. QMSP is the most commonly used technique for methylation analysis of specific genes 9. QMSP serves as a specific and sensitive method with advantages such as accurate quantification analysis, high-throughput testing and the requirement of a minimal amount of input DNA, which makes QMSP suitable for implementation in cervical cancer screening 10, 11. The benefit of bisulfite pyrosequencing is that
this method can provide an absolute level of methylation by determining the ratio of methylated and unmethylated cytosine residues separately 8, 12. Disadvantages of bisulfite pyrosequencing
however are that this method 1) is less reliable to quantify the methylated CpG’s from dysplastic cells in an environment of many normal cells, such as cervical specimens, 2) is only possible with sufficient CpG-free flanking sequences available and 3) has relatively high costs 8. Other techniques, mainly used for methylation identification, are not suitable for extensive methylation validation in clinical practice. Detection of methylation status as a method for cervical cancer screening needs to be applicable by a wide-range of laboratories. QMSP is the preferred technique because of its relative ease to analyze large number of DNA samples, its accurate quantitation power and low costs. Therefore, in this thesis, only studies performing methylation analysis using QMSP were included.
Diagnostic performance of methylation analysis in cervical scrapings from Europe and Asia
Methylation markers may have fluctuations in diagnostic performance related to differences in geographic areas and race. As shown in chapters 2 and 5, the diagnostic power to detect CIN2+ lesions in cervical scrapings was highly comparable for several single methylation markers (such as ANKRD18CP, C13ORF18, EPB41L3, PAX1 and ZNF582) and methylation panels (e.g. C13ORF18/EBP41L3/JAM3 and C13ORF18/ANKRD18CP/JAM3) in both cytological
abnormal and hrHPV-positive women in both Chinese and Dutch cohorts. For the ZSCAN1/SOX1 panel, the diagnostic performance was similar in the hrHPV-positive samples in both cohorts.
Recently, these three methylation marker panels were evaluated by us in a Slovenian hrHPV-positive cohort, derived from a prospective population-based screening program. This study revealed that all three marker panels showed comparable diagnostic performance to detect CIN2+ lesions, as observed in the Dutch cohort 13. There are no other studies using these methylation panels in cervical cancer in the Chinese population yet to confirm our results in chapter 2. However, some studies reported on our single markers (C13ORF18, EPB41L3, JAM3 and SOX1) using Chinese samples. All these studies were performed in hospital-based cohorts from China with most studies using QMSP, one other pyrosequencing and one methylation-sensitive high-resolution melting method 14-20. Diverse discordant results (i.e. regarding
sensitivity and specificity) of each single marker were shown among these published studies and our present study (Chapter 2). The inconsistent performance of methylation analysis as a diagnostic test might be related to the different methylation test methods and cut-off values used by different research groups in these studies, thus making comparison difficult 14-20. In contrast, for the two methylation markers (PAX1 and ZNF582) which were identified and validated only in studies using an Asian population, the diagnostic performance was very similar between these 12 studies mostly from the same research group 17, 21-31 and also to our Dutch study (Chapter 5).
Based on our study (Chapter 2), there is no obvious explanation for the different diagnostic performances of ZSCAN1/SOX1 in cytological abnormal scrapings between European and Chinese women. One possible explanation might be that especially the methylation status of ZSCAN1/SOX1 is affected by differences in hrHPV genotyping, because HPV52 and HPV58 are also dominant genotypes in cervical cancer in the Chinese population 32, 33 while rarely seen in European women 34. In our study, the Cobas 4800 hrHPV test was used for determination of HPV status, which only includes HPV16 and HPV18 versus other types. Therefore, we cannot confirm whether ZSCAN1/SOX1 methylation was more related to specific other HPV types. More studies are needed to investigate the association between hrHPV genotyping and methylation status of CIN2+-specific markers, but our data showed that methylation markers and
panels are both suitable as a triage test in women with cytological abnormal scrapings or hrHPV-positive samples, independent of geographic areas and race.
What are currently the best methylation markers for triage of hrHPV-positive women?
In order to find methylation markers with the highest sensitivity and specificity to detect CIN2+/CIN3+ for triage of hrHPV-positive women using QMSP, we performed a systematic literature search and our analysis revealed 13 genes (AJAP1 35, CADM1 36, 37, CDH6 38,
EPB41L3 38-41, FAM19A4 3, 37, 42, 43, GHSR 44, JAM3 15, 38-41, MAL 36, 37, 45, POU4F3 35, 46, SST 44,
TERT 38, 41, 44, ZIC1 44 and ZNF582 47) that showed at least in one study a sensitivity ≥70% and specificity of ≥60% (Chapter 3). Of these 13 genes, six genes (CADM1, EPB41L3, FAM19A4, JAM3, MAL and POU4F3) were most promising as they showed a sensitivity of 53%-87% and specificity of 60%-95%. In addition, methylation marker panels of JAM3/ANKRD18CP, C13ORF18/ANKRD18CP/JAM3, C13ORF18/EPB41L3/JAM3, ASTN1/DLX1/ITGA4/RXFP3/ SOX17/ZNF671 and different combinations of Hsa-miR124-2, FAM19A4, CADM1 and MAL with improved diagnostic performance were identified as putative triage possibilities to detect CIN2+ in hrHPV-positive women.
Three other reviews on (host and viral) gene methylation analysis in early detection of cervical cancer were recently published 9, 48, 49. Kelly et al 48 did not describe the best methylation markers reported in numerous studies separately, but reported on a pooled analysis of all included methylation markers and marker panels in cervical cancer irrespective of the used method. They reported a similar set of host gene markers with the addition PAX1 and SOX1, and some viral methylation markers (HPV16 L1/L2) with a pooled sensitivity of 69% for CIN2+ and 71% for CIN3+, based on a threshold to achieve a specificity of 70% in women with a hrHPV-positive or abnormal cytology result. In this latter review only methylation markers were included if evaluated in 4 or more studies or evaluated as part of a large population-based screening study. In addition inclusion was not restricted to studies using QMSP only and not classified as only hrHPV-positive or abnormal cytological samples, thus making comparison with our systematic review difficult. Another review from Kremer et al 9 mainly focused on the evaluation of reported methylation markers with good performance for the detection of CIN3+
lesions. Most studies included in this review were using QMSP. As might be expected, the main marker panels to detect CIN3+ lesions with the best performance are similar as those we described in our systematic review in chapter 3, thereby confirming our findings. In addition, Kremer et al found that methylation of PAX1, also described by Kelly and reported separately by us (Chapter 5), was mostly analyzed without prior hrHPV testing for the detection of CIN3+. And finally, a review on HPV DNA methylation from Bowden et al 49 described only methylation of HPV16 L1 and L2 regions, which was 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 the other hrHPV types were lacking. Moreover, as also described in paragraph “DNA methylation analysis: quantitative methylation-specific PCR (QMSP)”, the application of viral methylation determination remains difficult as it can only be analyzed by bisulfite pyrosequencing and it will only determine methylation of specific hrHPV types.
In our review we focused specifically on host methylation markers analyzed by QMSP on scrapings from women with CIN2+ lesions (with exceptions of collecting data for only CIN3+ when CIN2+ data could not be extracted). Methylation analysis by QMSP is currently considered to be most suitable for being implemented in cervical screening. Moreover, we also focused on methylation markers used to triage hrHPV-positive scrapings, which is more in line with the current clinical needs, as more and more countries are adopting hrHPV testing as the primary methodology for their cervical cancer screening program. Despite these restrictions, our review presents all data of methylation markers and panels separately to analyze their consistency and/or variation. As illustrated in our review, highly consistent results for most markers are difficult to obtain. The large variation between different studies and the few independent studies done with the same markers, point to an urgent need to reliably confirm the relevance of selected promising methylation markers in studies with well-defined methodology. Standardization of methylation testing and analysis in well-defined populations in that respect is essential. Therefore, despite a large number of already available studies there still is a need to select the most effective and reproducible methylation markers and to evaluate them in large randomized controlled population-based studies.
Our current review and this thesis support further validation of the markers ANKRD18CP, CADM1, C13ORF18, EPB41L3, FAM19A4, JAM3, MAL, Hsa-miR124-2, PAX1, POU4F3, SOX1, ZNF582 and ZSCAN1, and a combination of ASTN1, DLX1, ITGA4, RXFP3, SOX17 and ZNF671 using QMSP to find the best panel with the highest sensitivity and combined highest specificity to detect CIN2+ lesions in hrHPV-positive women.
Future perspectives
Application prospects of methylation analysis in China
In China, there currently is no well-organized cervical cancer screening system due to insufficient public health infrastructure 50. In 2009, a National Cervical Cancer Screening
Program for women aged 35–64 years in Rural Areas (NACCSPRA) was initiated by the Chinese government. Women residing in rural areas have access to the free cervical cancer screening services 51. However, only a small fraction (4%) of eligible women could be served by
the program, which is about 10 million women every year. So most of the women should initiate themselves to go to the hospital for cervical screening, which has to be paid by themselves 52, 53.
The Ministry of Health of China has launched a guideline for screening. This guideline recommends region-driven protocols involving cytology plus hrHPV test in more developed areas. In low-resource settings as an alternative method visual inspection with acetic acid (VIA) is applied 54. VIA does not require a cytopathology laboratory and therefore can be performed
with little infrastructure and low costs.
It is difficult to introduce cytology-based primary screening in China due to the large population, its complicated technology and highly different (regional) levels of economic development. Simultaneously VIA is unsuitable for a mass-screening program because of its limited utility, related to a low sensitivity (~60%) to detect CIN2+ lesions, and a high risk for overtreatment 55, 56. The application of hrHPV testing is gradually increasing in China due to its higher sensitivity.
More recently HPV16/18 genotyping is recommended as a triage test after a hrHPV-positive test in order to reduce unnecessary colposcopy referrals. However, HPV52 and HPV58 are found to play dominant roles in cervical cancer in addition to HPV16 and HPV18 in the Chinese population 32, 33. In this thesis, our CIN2+-specific methylation panels showed much higher
specificity for CIN2+ than hrHPV testing in cytological abnormal scrapings and noninferiority to HPV16/18 partial genotyping to triage hrHPV-positive samples for CIN2+ detection in the Chinese cohort. Therefore, DNA methylation markers might serve as an alternative triage test compared to HPV16/18 genotyping or cytology in cervical cancer screening in China. Currently no methylation markers are available that could be directly implemented for clinical use or replace the existing screening methods in China. But based on the results in this thesis, it would
early detection of cervical (pre)cancers in China. However, large prospective randomized population-based studies are still needed to investigate the best practice resulting in (1) identifying all women with disease; (2) a perfect triage test to decrease the number of referrals especially those without disease, and (3) a cost-effective testing procedure with requiring minimum medical resources. To perform such national-scaled studies, strong support from the Chinese government is needed and there should be a hospital in each major region (e.g. Easter/Southern/Western/Northern part of China) responsible for the implementation of the project.
Application prospects of methylation analysis in the Netherlands
In the Netherlands, hrHPV primary screening with a reflex cytology test was implemented since January 2017 5. Recent data based on the first two years of hrHPV primary screening program
showed that cyto-pathologists with prior knowledge of the HPV status deliver a higher frequency of cytological abnormal (≥ASCUS) results in hrHPV-positive samples; approximately 50% of women referred for colposcopy per screening round had a CIN2+ lesion 5, 57. Methylation marker panels analyzed in this thesis showed a significant higher specificity accompanied with a lower sensitivity to triage hrHPV-positive women compared to the published data of cytology with cyto-pathologists being aware of hrHPV status 3, 58-63. As shown in this thesis the specificity of our methylation panels is also significantly higher than HPV16/18 genotyping (88-94% vs 52% for CIN2+) with a similar sensitivity. These results suggest that these methylation markers might be relevant as a triage test in women primarily screened with hrHPV tests. It has been reported that combining cytology (≥ASCUS) with HPV16/18 genotyping after hrHPV testing gives promising results with a high negative predictive values (NPV), but is generally accompanied with a higher colposcopy referral rate 64, 65. Even more interesting is that unnecessary referrals
might be decreased through combining either cytology or HPV genotyping with methylation analysis after primary hrHPV screening in clinician-collected samples 42, 66-68. Considering economic benefits, it would be expected that the decreased number of unnecessary referrals could offset the high cost of combined testing ultimately leading in higher cost-effectiveness. Before implementation of methylation testing, the promising methylation panels described in this thesis should be validated on scrapings collected by general practitioner (GP) and primarily
tested for the presence of hrHPV, from women participating in the current population-based screening program. The performance of our methylation panels so far have been validated in scrapings from the “old” cytology-based screening program, and therefore hrHPV-positive women with normal cytology (account for ~70% of cases) 5 were not available and have not been tested to determine the sensitivity and specificity for CIN2+ lesions. Therefore, as a next step, further validation of our marker panels needs to be performed in samples prospectively collected from the current hrHPV-based population screening program and this is currently ongoing.
Methylation analysis in self-sampling materials
HrHPV testing on self-samples may be one strategy to improve screening attendance for women who hesitate to participate in routine screening 69-71. In the current population-based screening program, around 8% of participating women used self-sampling in the first year of hrHPV primary screening in the Netherlands 5. The self-sampling device is not suitable for cytological examination 69, 70, therefore, hrHPV-positive women will be invited to visit their GP for cervical scraping, and this GP-taken scraping material will be used for the triage by cytological examination and decision-making regarding referral for colposcopy. Of the hrHPV positive women who used self-sampling, 79% will visit their GP after 15 months with an increase to 89% after 27 months 57, showing a big delay before decisions can be made for referral to the gynecologist. Moreover 11% will not see their GP at all. Screening might be significantly improved when we would be able to apply molecular marker analysis on the same self-sampled DNA as was used for hrHPV testing, thus avoiding the need for women to visit GP to obtain a sample for cytological examination. Potential molecular triage tests that could be performed on the self-sampled material are HPV16/18 genotyping or methylation analysis. Several studies showed that HPV16/18 genotyping is applicable in hrHPV-positive self-samples with acceptable clinical performance 67, 72, 73. However, there are still limitations due to infections with other hrHPV subtypes. Methylation analysis has been shown to be feasible on self-sampled material 41,
61, 74. A previous study from our group showed a very high concordance of methylation
(C13ORF18, EPB41L3 and JAM3) between matched self- and physician-taken scrapings 41. In
will be used to quantify the amount of methylation of each methylation marker in self-samples, and sensitivity and specificity will be determined to identify the best combination for CIN2+ detection in samples from hrHPV-positive self-sample participants of the population-based screening program. In parallel, this same validation will also be performed in GP-collected samples to determine whether the selected methylation marker panels can reach similar diagnostic performance in self-sampled materials compared to physician-taken samples.
Methylation analysis in the era of HPV vaccination
Primary prevention of cervical cancer by prophylactic HPV vaccination decreases the prevalence of CIN lesions and cervical cancer 79-83. However, the effect of vaccination is not completely
clear yet, as not every girl before becoming sexual active gets the HPV vaccine (participation rates in the countries that implemented a national vaccination program vary) and hrHPV vaccination provides no therapeutic benefit against hrHPV infections that are already present 82.
Therapeutic HPV vaccines have not yet clinically demonstrated consistent success 84.
Infrastructure and financial barriers still exist for the implementation of national vaccination programs, especially in the low- and middle-income countries, which carry the greatest burden of cervical cancer 85. Until additional evidence emerges on the longer-term effects of
HPV vaccination at a population level, vaccinated women should be screened in the same way as unvaccinated women 86. Furthermore, an overall decrease in hrHPV and CIN prevalence caused
by vaccination will reduce the positive predictive value (PPV) of cervical screening performance. It has been reported that the PPV of cytology for CIN2+ detection decreased significantly for vaccinated compared to unvaccinated women 87. Thus, a triage test with high
specificity is therefore becoming even more important. As the efficacy of viral DNA methylation may also be affected by a decreased hrHPV prevalence, studies to find the optimal screening models using host DNA methylation markers as triage tests in HPV vaccinated populations are anticipated in the near future.
Future directions for methylation analysis in cervical cancer screening
Many groups including ours already did multiple genome-wide studies to identify and validate methylation markers, e.g. Taiwanese group (e.g. PAX1 and ZNF582 ) 17, 21, 23-30, 75, the Amsterdam group (VUMC) (e.g. CADM1, MAL, Hsa-miR124-2, and FAM19A4) 36, 76, 77 and our group in the University Medical Centre Groningen (UMCG) (e.g.ANKRD18CP, C13ORF18, EPB41l3, JAM3, ZSCAN1 and SOX1) 32, 38, 40, 78. All these studies indicate that there is currently no single methylation marker suitable for cervical cancer screening. A high specificity could be achieved by the tested methylation markers. However, the sensitivity is still not adequate for the detection of CIN2+ lesions 48. To improve current sensitivity, studies on different combinations of methylation markers appears to be the preferred way. However, it might also be taken into consideration for future studies, that sensitivity could be improved, when only detection of more severe disease (e.g. CIN3+ lesions) is strived for. The post-hoc analysis of POBASCAM screening trial in the Netherlands reported that after 14 years follow-up of hrHPV-positive women, the cervical cancer risk was lower among FAM19A4/Hsa-miR124-2 methylation-negative (1.7%) than cytology-methylation-negative women (2.4%), and the cumulative CIN3+ risks were comparable between women with a negative methylation result and women with a negative cytology result (16.3% and 15.6%, respectively) 68, 88. One may hypothesize, that e.g.
hrHPV-positive/methylation negative CIN2 and 3 lesions are less likely to progress and do not need immediate treatment and therefore could be labeled as “test negative”. However, to implement such a policy long-term follow up is needed of hrHPV-positive/methylation negative CIN2+ lesions, that are left untreated. Such studies are difficult and therefore unlikely to be performed on a large scale.
Regardless of the current low sensitivity, the relevance of selected promising methylation markers all together need to be analyzed on a large series of samples collected during regular population-based screening. Furthermore, as demonstrated in this thesis, in different populations different marker panels may have to be used to reach the best combined sensitivity and specificity. So, in order to find the most optimal marker panel, we need to validate the identified markers simultaneously in different geographic areas and races. With QMSP, high-throughput analysis can be performed to find a methylation panel with combined high sensitivity and
studies described in this thesis the same QMSP assays as developed at UMCG were implemented in the Tianjin hospital to analyze a Chinese cohort, illustrating the ease of performing such methylation-based screening programs.
It is expected that objective and high-throughput molecular diagnostic methods will eventually replace morphological analysis as a suitable triage method in cervical cancer screening. A five-phase framework is recommended for biomarker development. Phase 1: Preclinical exploratory studies. Phase 2: Clinical assay in non-invasive samples in selected subjects with known outcome. Phase 3: Retrospective longitudinal repository studies, for example from biobank-based case-control studies. Phase 4: Prospective screening studies. Phase 5: Prospective intervention studies 89. Until now, only one randomized prospective study was performed with
only two methylation markers (MAL/Hsa-miR124-2) evaluated 61. The Chinese methylation
analysis in this thesis is classified as phase 3, while the current ongoing study in the UMCG analyzing samples from the Dutch population-based screening is classified as a prospective screening study (phase 4). After completion of this study, according to the recommended strategy, it is suggested to perform a prospective intervention study. However, as our project will provide data on the number of women with a CIN2+ lesion detected by our methylation test in a cohort normally attending the national population-based screening program, a randomized controlled study (phase 5) may not be indicated anymore.
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