Methylation testing for the detection of recurrent cervical intraepithelial neoplasia

Women treated for CIN2/3 remain at increased risk of recurrent CIN and cervical cancer, and therefore posttreatment surveillance is recommended. This post hoc analysis evaluates the potential of methylation markers ASCL1/LHX8 and FAM19A4/miR124‐2 for posttreatment detection of recurrent CIN2/3. Cervical scrapes taken at 6 and 12 months posttreatment of 364 women treated for CIN2/3 were tested for methylation of ASCL1/LHX8 and FAM19A4/miR124‐2 using quantitative multiplex methylation‐specific PCR. Performance of the methylation tests were calculated and compared with the performance of HPV and/or cytology. Methylation levels of recurrent CIN were compared between women with a persistent HPV infection, and women with an incident HPV infection or without HPV infection. Recurrent CIN2/3 was detected in 42 women (11.5%), including 28 women with CIN2 and 14 with CIN3. ASCL1/LHX8 tested positive in 13/14 (92.9%) of recurrent CIN3 and 13/27 (48.1%) of recurrent CIN2. FAM19A4/miR124‐2 tested positive in 14/14 (100%) of recurrent CIN3 and 10/27 (37.0%) of recurrent CIN2. Combined HPV and/or methylation testing showed similar positivity rates as HPV and/or cytology. The CIN2/3 risk at 12 months posttreatment was 30.8% after a positive ASCL1/LHX8 result at 6 months posttreatment. Methylation levels of CIN2/3 in women with a persistent HPV infection were significantly higher compared with women with an incident or no HPV infection. In conclusion, posttreatment monitoring by methylation analysis of ASCL1/LHX8 and FAM19A4/miR124‐2 showed a good performance for the detection of recurrent CIN. DNA methylation testing can help to identify women with recurrent CIN that require re‐treatment.


What's new?
Methylation markers are promising tools for posttreatment detection of recurrent cervical intraepithelial neoplasia grade 2 or 3 (CIN2/3).Whether such markers can consistently detect recurrent lesions however, requires further evaluation.Here, the authors investigated the performance of ASCL1/LHX8 and FAM19A4/miR124-2 methylation for CIN2/3 lesion detection and compared the results to detection via HPV testing and cytology.Analyses show that ASCL1/LHX8 and FAM19A4/miR124-2 methylation can reliably detect recurrent CIN2/3 lesions in women with persistent HPV infection following treatment for high-grade CIN.The findings indicate that DNA methylation can effectively distinguish lesions requiring re-treatment from lesions requiring only conservative management.

| INTRODUCTION
2][3][4][5] Therefore, strategies to monitor these women after treatment are implemented in many countries.[8] Recurrent CIN represent a heterogeneous disease category including both residual lesions (resulting from incomplete excision) and newly developed lesions.Residual and newly developed CIN look morphologically similar and, at present, all recurrent lesions are treated similarly to prevent progression to cervical cancer.Yet, the shortterm risk of progression to cervical cancer of residual and newly developed lesions might be different.Newly developed lesions probably have a lower short-term risk of progression to cancer compared with residual lesions, and therefore these lesions may not need immediate treatment.In addition, CIN lesions can show spontaneous regression in 40% to 50% of CIN2 and 30% of CIN3. 2,9,10[13] DNA methylation of human genes has shown to be particularly sensitive for the detection of cervical cancer and advanced CIN2/3, that is, lesions caused by a long-lasting persistent high-risk HPV infection with a presumed increased short-term risk of progression to cervical cancer. 14,15Absence of host-cell DNA methylation, on the other hand, is associated with regression of CIN2/3. 16DNA methylation could therefore be an interesting strategy to detect recurrent CIN after treatment, in particular for the detection of residual lesions in need of re-treatment.The use of DNA methylation markers in posttreatment surveillance was evaluated for methylation of CADM1/MAL in the SIMONATH trial, a multicentre prospective clinical cohort study among women treated for CIN2/3. 179][20] In the current study, the performance of methylation markers ASCL1/LHX8 and FAM19A4/miR124-2 is evaluated and compared with the performance of HPV testing and/or cytology for the detection of recurrent CIN2/3 after treatment.

| Study population
This study is a post hoc analysis of the SIMONATH study (Trial registration ID: NTR1964).A detailed description of the SIMONATH study has been published before. 17This multicentre prospective clinical cohort study was conducted within six outpatient clinics between April 2010 and June 2012 in the Netherlands.Women treated with LLETZ because of CIN2/3 were included.Cervical scrapes were collected prior to treatment, 6 months posttreatment and 12 months posttreatment.These cervical scrapes were tested for HPV, cytology and CADM1/MAL methylation.HPV testing was performed using the GP5+/6+ PCR with an enzyme-immuno assay read out followed by reverse line blot analysis for HPV genotyping. 21Cytology was classified according to the CISOE-A classification and translated into the Bethesda system for analysis. 22At 6 months posttreatment, women with a cytology result of Atypical Squamous Cells of Undetermined Significance or worse (≥ASC-US), a positive HPV test and/or a positive CADM1/MAL methylation result were referred for colposcopy.At 12 months posttreatment, all women were referred for colposcopy with mandatory biopsies independent of test results.Histology results were classified as: no dysplasia, CIN1, CIN2, CIN3, AIS or cervical cancer.Women with recurrent disease, that is, CIN2 or worse, were treated according to national guidelines with LLETZ or conisation. 232 | Methylation testing of ASCL1/LHX8 and FAM19A4/miR124-2 For this post hoc analysis, DNA methylation testing of ASCL1/LHX8 and FAM19A4/miR124-2 was performed on left-over material of the cervical scrapes taken at 6 and 12 months posttreatment.Bisulphite converted DNA was subjected to methylation analysis using a quantitative multiplex methylation-specific PCR as described previously for marker panels ASCL1/LHX8 18,24 and FAM19A4/miR124-2. 25A prototype version of the QIAsure Methylation Test (Qiagen, Hilden, Germany) was used for methylation analysis of host-cell genes FAM19A4 and miR124-2. 26The bi-marker methylation panel ASCL1/ LHX8 was obtained by dichotomizing a weighted linear combination of ddCt ratio scores using an earlier defined threshold based on the Youden J-index. 18The FAM19A4/miR124-2 test was labeled positive if the ddCt ratio of any of the two markers exceeded a pre-set threshold as per the QIAsure Methylation Test protocol.Methylation testing was performed blinded for HPV, cytology and histology outcomes.

| Sample size calculation
The primary objective of the SIMONATH study was to determine whether testing for molecular markers, that is, HPV, CADM1/MAL methylation and combinations thereof, yielded a higher sensitivity and specificity for the detection of CIN2/3 or cancer after treatment in comparison with cytology.
In order to calculate a sample size with a power of 80% and alpha of 0.05, the two-sided McNemar test for power calculation has been used and resulted in a total of 34 CIN2/3 lesions to be developed posttreatment.Assuming that 10% of treated women will present with residual/recurrent disease at least 340 women needed to be included.
To correct for possible drop out, at least 360 women were included.

| Statistical analysis
The endpoint for recurrent CIN was defined as histologically confirmed CIN2 or worse (CIN2+) at 6 or 12 months posttreatment.Adjusted endpoints were used if no histology result was available at 6 or 12 months posttreatment.An HPV-negative cervical scrape with normal cytology was categorized as "adjusted endpoint ≤CIN1."An HPV-positive cervical scrape with normal cytology or a cervical scrape with abnormal cytology result were considered as 'no endpoint' and excluded from analysis.
Positivity rates with Wilson 95% confidence intervals (95% CI) were determined for HPV, cytology, ASCL1/LHX8 methylation, FAM19A4/miR124-2 methylation and combinations thereof using the test results of the cervical scrape taken at diagnosis of recurrent CIN or for women without recurrent CIN, the cervical scrape taken at

| Study population and test positivity
The study population consisted of 364 women of whom 42 (11.5%)developed recurrent CIN during 12 months of follow-up: 28 CIN2 and 14 CIN3 (Figure 1).One woman with no recurrent CIN2/3 at 6 months posttreatment and one woman with a recurrent CIN2 at 12 months posttreatment were excluded from the analysis, because no cervical scrape was collected.Another three women were excluded from the analysis because of a missing endpoint.Positivity rates in ≤CIN1, recurrent CIN2 and recurrent CIN3 of HPV, cytology, ASCL1/LHX8 methylation, FAM19A4/miR124-2 methylation and combinations thereof are shown in Table 1  which was lower compared with the other single strategies (P < .001).
The combination strategies showed similar detection of CIN2 and CIN3, but positivity was also higher in ≤CIN1.Comparison of the performance between different test strategies can be found in Table S1.

| Methylation levels of women with recurrent CIN lesions stratified for HPV infection
Results of the methylation tests in women with recurrent CIN2/3 stratified for HPV genotype infection (ie, no HPV infection, incident HPV genotype infection or persistent HPV genotype infection) and histology are shown in Figure 2 and Table 3. Methylation levels in women with a persistent HPV infection were significantly higher compared with women with an incident HPV infection or no HPV infection (Figure 2).All women with a recurrent CIN3 (14/14) had a persistent HPV infection, and 92.9% (13/14) of them tested positive for ASCL1/LHX8 methylation.Of women with a recurrent CIN2, 57.7% (15/26) had a persistent HPV infection, and 73.3% (11/15)   tested positive for ASCL1/LHX8 methylation.Recurrent CIN2 in women with an incident HPV infection or no HPV infection were barely positive for ASCL1/LHX8 methylation (0% (0/5) and 16.7% (1/6), respectively).ASCL1/LHX8 methylation positivity was higher in women with recurrent CIN2/3 and a persistent HPV infection compared with women with recurrent CIN2/3 and an incident HPV infection or no HPV infection (P < .001).Methylation levels of FAM19A4 and miR124-2 showed comparable results, although women with CIN2 and a persistent HPV genotype infection showed a somewhat lower positivity rate compared with ASCL1/LHX8 (Figure 2 and Table 3).

| DISCUSSION
6][7][8] In this study, we evaluated the performance of different test strategies including methylation markers ASCL1/LHX8 and FAM19A4/miR124-2 to detect recurrent disease in a cohort of 364 women who were treated for CIN2/3.Both methylation marker F I G U R E 2 Methylation levels of (A) ASCL1, (B) LHX8, (C) FAM19A4, and (D) miR124-2 in recurrent CIN2/3 lesions in women with no HPV infection, an incident HPV infection or a persistent HPV infection in the corresponding cervical scrape.HPV status of one CIN2 at 6 months posttreatment was unknown and was therefore not included in this figure.CIN, cervical intraepithelial neoplasia.panels showed excellent positivity in cervical scrapes for the detection of recurrent CIN3 (>92.9%), while positivity in ≤CIN1 were the lowest (<8.9%) of the evaluated strategies.The CADM1/MAL methylation test, originally applied in this cohort, tested positive on the cervical scrapres in 64% of the recurrent CIN3 and 17 to 22% in ≤CIN1, thus the discriminatory power of the novel methylation markers was considerably higher. 17Methylation positivity in recurrent CIN2 was somewhat lower compared with the other test strategies, which is most likely explained by the heterogeneity within this group.Notably, DNA methylation analysis has also shown to be particularly sensitive for the detection of cervical cancer and advanced CIN2/3. 14,15This is further supported by our finding that most of the recurrent CIN2 and CIN3 in women with a persistent HPV infection were methylation positive.In contrast, recurrent lesions in women with an incident HPV infection or no HPV infection were mostly methylation negative.This illustrates that DNA methylation mainly identifies persistent CIN lesions, and that methylation-negative CIN lesions are probably the newly developed lesions.These results are in line with earlier research in which we demonstrated that methylation-negative CIN2/3 have a higher chance of regression compared with methylation-positive CIN2/3 when left untreated. 16A detection rate of 37.0 to 48.1% for CIN2 in the current study by the DNA methylation markers was therefore expected.
Since surgical treatment of CIN is associated with an increased risk of preterm birth and other obstetric complications, especially after two or more surgical excisions, it is important to limit the number of excisions. 11A benefit of DNA methylation is that it differentiates between lesions with a high risk of progression to cancer and a low risk of progression to cancer.A strategy for women with recurrent CIN could be to re-treat in case of a positive methylation test, while a watchful waiting approach could be applied in women with a negative methylation test.Another advantage of DNA methylation is that it can be performed on self-collected samples, which would further simply posttreatment monitoring.
Interestingly, women with a positive ASCL1/LHX8 or FAM19A4/ miR124-2 methylation result at 6 months posttreatment had a CIN2/3 risk at 12 months posttreatment of 30.8% and 21.1%, respectively, while the CIN2/3 risk after a negative methylation test was <4%.This high risk at 12 months posttreatment after a positive test result indicates that these women need close monitoring with colposcopy and biopsies for histological evaluation.On the other hand, we showed that recurrent lesions with a negative methylation result were associated with incident HPV infections or no HPV infection and therefore this risk also includes CIN2/3 with a low risk of progression to cancer.
The currently most used posttreatment surveillance method to detect recurrent CIN is HPV and/or cytology co-testing.In this study, we demonstrated that the combination strategies HPV and/or FAM19A4/miR124-2 and HPV and/or ASCL1/LHX8 showed similarly high detection rates for CIN2 and CIN3 compared with HPV and/or cytology.A disadvantage of any strategy including HPV testing is that it is less specific, because an HPV infection can still be present after removal of the lesion, and it does not distinguish between newly developed lesions and residual lesions.An advantage of combination strategies in general is the increased detection rate, however, at the cost of a higher positivity in women without recurrent CIN2/3.[29][30][31][32] In most countries, posttreatment surveillance takes 2 years.A limitation of this study is the short follow-up period of 12 months.More recurrent CIN lesions may have been identified with a longer followup period.Although our study cohort is large with 364 women treated for CIN2/3, recurrent CIN lesions were detected in 42 women resulting in wide 95% CI's because of limited numbers in the subgroup analysis.Furthermore, a few samples could not be included in this post hoc analysis, because no left-over material was available for additional testing.Also, some of the recurrent CIN2/3 lesions in women that were classified as caused by a persistent HPV infection might have been misclassified, because they could have been caused by a re-infection with the same HPV genotype.
To conclude, ASCL1/LHX8 and FAM19A4/miR124-2 methylation on cervical scrapes showed a good performance with a similar positivity in CIN3 and a lower positivity in ≤CIN1 compared with HPV testing and cytology.We showed that CIN2/3 lesions with a persistent HPV infection were mostly methylation positive, supporting the concept that DNA methylation especially detects advanced CIN2/3 lesions.Thereby, methylation testing can help to discriminate women in need of re-treatment from those who can be managed conservatively to prevent overtreatment.

AUTHOR CONTRIBUTIONS
the last study visit at 6 or 12 months posttreatment.Outcomes of the different test strategies were compared using McNemar tests.Cytology was labeled positive at the threshold of ASC-US.The combination strategies were labeled positive in case one or two of the tests were positive.Missing samples or samples with invalid results were excluded from analysis.To evaluate the CIN2/3 risk at 12 months posttreatment according to the 6 months posttreatment test results, the positive predictive value (PPV) and 1-negative predictive value (1-NPV) were calculated.HPV genotype persistence was defined as the detection of an identical HPV genotype at 6 or 12 months posttreatment compared with the HPV genotype prior to treatment.An incident HPV infection was defined as the detection of a different HPV genotype at 6 or 12 months posttreatment compared with the HPV genotype prior to treatment.Methylation levels and positivity rates of recurrent CIN lesions were compared between women with a persistent HPV genotype infection and women with an incident HPV infection or without HPV infection using pairwise Wilcoxon-Mann-Whitney U tests or Fisher's exact test.Statistical analyses were performed in SPSS Statistics (version 26, IBM Corp, Armonk, NY), Stata (version 17.0, StataCorp, College Station, TX) and RStudio (version 4.2.1) using the binom (version 1.1-1.1)package.

1
Clinical performance of HPV, cytology, ASCL1/LHX8, FAM19A4/miR124-2 and combinations of these tests for the detection of recurrent CIN2/3.Absolute CIN2/3 risk at 12 months posttreatment based on the 6-months test results a One woman had an invalid cytology result and two women had an invalid HPV result at 12 months posttreatment.Left-over material for DNA methylation testing was missing of three women and one woman had an invalid FAM19A4/miR124-2 DNA methylation result.bIncludingadjustedendpoints.Abbreviations: CI, confidence interval; CIN, cervical intraepithelial neoplasia; HPV, human papillomavirus.T A B L E 2 aWomen with recurrent CIN at 6 months posttreatment were excluded.bIncluding adjusted endpoints.Abbreviations: CIN, cervical intraepithelial neoplasia; HPV, human papillomavirus; NPV, negative predictive value; PPV, positive predictive value.