Discussion
This is the first study to assess the predictive potential of the S5 methylation classifier in a larger screening setting of patients with abnormal results between HPV and cytology in an Asian country. S5 classifier, a multigene methylation test, which has shown good performance in the United Kingdom30,32 , Canada33, and Mexico31, Colombia34 and recently in a China colposcopy referral population conducted by our team13. In this study, we enrolled the screening population as study setting, and we evaluated the predictive potential of S5 methylation for progression of specific untreated squamous intraepithelial lesion.
Here, we validated the effectiveness of S5 classifier at the cutoff 2.85 in the present screening population setting. We validated a DNA methylation classifier of HSIL+ histology, among women with abnormal results between HPV and cytology from a China screening population. Our study included 938 baseline samples (including HPV, cytology and S5 methylation) from women who were referred colposcopy at first screening or had 2 years of active follow-up and culminating in colposcopy clinics directed biopsy diagnoses if positive for either HPV or cytology tests. To address potential bias in methylation levels by age at diagnosis or time of follow-up, our controls were age and time to diagnosis matched and randomly chosen among all baseline ≥HSIL+ women. We conducted an independent analysis in the RF arm and IC arm in Table 2, although the sensitivity and specificity of S5 is different, the result is still in line with the overall conclusion (which means that the sensitivity and specificity of S5 in both cohorts are better than the performance of HPV16/18 and cytology). The genotyping and methylation assays, as well as the verification of the histological diagnoses, were conducted independently and blindly after the end of the study. To date, there are few reports comparing the performance of cytology and HPV16/18 genotyping with DNA methylation assays to detect HSIL+ in women with abnormal results between HPV and cytology. The study compared the performance of S5 for HSIL+ detection to repeatedly cytology testing and HPV genotyping in women with abnormal results between HPV and cytology in developing countries setting, objectively.
The AUC obtained in this study was 0.80 (95% CI 0.74-0.85). At a specificity set at 79.9% that corresponds to using S5 at a cut-off value of 2.85, the sensitivity of S5 for HSIL+ were 76.1%, which were significantly higher than the sensitivities of HPV16/18 (P = 0.039) or cytology (P < 0.001). In addition, S5 methylation classifier also outperformed the comparator triage testes (HPV and cytology) and their combination regarding the specificity. Considering that we are comparing different triage tests among women with above the average risk for HSIL+ (cytology≥(TCT)HSIL or/and hrHPV+), S5 acts as a good triage alternative since this test decreased the false positive rate by near 35% (Table 4) and exhibited higher sensitivity than HPV 16/18, cytology, or combination of these two tests for both HSIL+ endpoints. This characteristic of S5 is especially valuable for remote areas of developing countries, where higher sensitivity is crucial to identify at-risk women in fewer screening visits and decreasing the use of resources to follow-up women with low risk of disease.
In our study, cytology had a specificity of 54.4%, but had the test with by far the highest false negative rate. We recognized that the sensitivity of our cytology was lower than the performance of cytology in several studies in developed countries, but several studies have demonstrated that the sensitivity in developing countries may be as low as 30%. Although the selection bias about population occurs, it is worth noting that despite many efforts to improve cytology quality and sensitivity, difficulties in quality controlling and delays in diagnosis still prevail. We used the predefined cutoff at UK population (S5 cutoff = 0.8) that corresponded to previous studies with S531,32, while the S5 at cutoff 0.8 (95.5%) vs the sensitivity of S5 at cutoff 2.85 (76.1%), the specificity(19.4%, 95% CI 17.8-21.7) at 0.8 cutoff is much lower than 79.9% at 2.85 cutoff. Future work is planned to assess the performance of S5 as a triage to determine appropriate cutoffs within a screening population. Also, further work is needed to determine the performance of the S5 methylation assay in vaccinated populations.
Also, we analyzed the distribution of S5 panels scoring and host-cell gene named EPB41L3 scoring among a screening population (Figure 2 and Figure 3 ), the results were in line with the distribution among the colposcopy referral population. Additionally, the changes in the level of methylation of S5 panels and host-cell gene named EPB41L3 compared if methylation of EPB41L3 was different among different cytology results and different HPV status, the result showed very little difference (P = 0.31) (Figure 4 ). This result indicates that it is difficult to conduct a screening test only by measuring the methylation levels EPB41L3, which was in line with some other previous studies29,32.
What’s more, we reveal a new utility of the S5 DNA methylation classifier measurement, specifically as a classifier not only for an enlarged screening population, but also for assessing risk of progression specific untreated squamous intraepithelial lesions. A promising prognostic test for SIL could greatly improve the current screening and even alter further treatment algorithms. A generally acknowledged difficulty of current strategies lies in the subjectivity and inter-observer variability in not only cytological but also histological diagnoses. These limitations result in misclassification of lesions, unnecessary colposcopy referrals, multiple follow-up visits, and either delayed treatment or over-treatment, exacerbating the potential harm to the patient meanwhile excessive burdening to medical practice. An improved predictive test could optimize the current management so that cases with progressive potential could be treated sooner and cases with regressive potential perhaps be indicated untreated for longer periods to allow more regressions. It should be considered that the natural history of long-term HPV persistence with respect to eventual true progression of abnormal screening results (abnormal screening results between HPV and cytology) to ≥HSIL+ beyond 2 years remains unclear. According to the current Chinese clinical algorithm, persistence of abnormal screening results (abnormal screening results between HPV and cytology) for 2 years was an indication for colposcopy referrals or even further treatment; however, we do not know what proportion of these colposcopy referrals were necessary to prevent cervical cancer.
In this study, of all the women (abnormal results between HPV and cytology; meanwhile HPV16/18- and < (TCT)HSIL result of cytology) progressed to ≥HSIL+ within 2 years, most of these cases were positive for the S5 methylation classifier (S5 methylation classifier ≥ 2.85) at the first visiting. All the cases with HPV16/18 positive or cytology ≥ (TCT)HSIL+ were referred to immediate colposcopy according to the current Chinese algorithms, thus it is not clear the proportion of progressed women with HPV16/18 positive or cytology ≥ (TCT)HSIL+, and it is plausible that further prospective study is needed to demonstrate the issue. Even so, these results fully indicate that S5 methylation classifier with great potential to predict the progression of women with abnormal results between HPV and cytology.
According to the call for “action to eliminate cervical cancer” issued by the World Health Organization in 20199, increasing effective screening of women with hrHPV testing is necessary. However, it is difficult to implement the HPV DNA testing frequently for populations living in remote areas where there few opportunities to screen women at proper intervals and for follow-up after screening4,35. What’s more, most HPV infections are transient so immediate ablative treatments can lead to unnecessary gynecological harms for women with low risk of disease36. For this reason, primary screening with HPV testing and/or cytology requires other triage tests to identify women at high-risk of high-grade disease among those who are with abnormal results between HPV and cytology. Considering recalling women for a second test after screening is challenging or impossible in developing countries; therefore, risk triage should ideally occur at the screening visit in these settings. Hence, these settings have unique needs for triage strategies.
Considering these results, it is possible to envisage a screening test that simultaneously the current tests (HPV genotyping and cytology) and measurement of S5 methylation. As depicted in Figure 8 , such potential screening strategy would provide the benefit of immediate and more accurate results for triaging women as below: (I) negative for all biomarkers, who would go back to routine screening; (II) referred to colposcopy (according to the current algorithm: HPV16/18 positive or cytology ≥ (TCT)HSIL+ or repeated abnormal result among HPV and cytology) and methylation positive, would be given priority for colposcopy inspection than who referred colposcopy and methylation negative, and (III) repeated follow up (according to the current algorithm) and methylation positive, would be given priority for follow-up at proper intervals than who were repeated follow up and methylation negative, in developing countries especially some remote areas.
The strength of this study is the validation of the S5 classifier in a routine screening study in the China with blinding of all results to the lab technicians, and the use of prespecified cutoffs for the methylation classifiers which minimized the risks for bias and overfitting. In practice, hrHPV-positive women could have the methylation tests performed on the original samples in a reflex manner, triaging women at risk to colposcopy and thereby reducing anxiety and overtreatment in the low-risk women. Possible concerns over missing some of the LSIL and HSIL might be addressed by referring women negative or low risk by the DNA methylation classifier to repeat HPV testing and cytology in proper follow-up duration.
A weakness is that our study was restricted to the length of follow-up short, which may result in some bias. While potential statistical bias, it is under the circumstance of the current algorithm in China based on the guideline: all the women (with HPV16/18 positive and/or cytology results ≥(TCT)HSIL+) were advised to refer colposcopy. Thus, it is difficult to arranged women for immediate colposcopy or follow up randomly, also difficult for RF cohort undergo colposcopy before the following-up. Considering that in this study we focused the screening population (with abnormal screening results) at the first visiting, IR cohort and RF cohort were both included. Since the RF cohort including women with (“HPV positive except HPV16/18” and “abnormal cytology results except ≥(TCT)HSIL”), who were followed-up every 6 months for 2 years follow-up period, and need 2 years following up period to determine whether to be enrolled in, the current study (including IC cohort and RF cohort) lasted for a long time to complete the data collection. However, a larger population is needed in the further study. In addition, our results might not be directly generalized to other histological diagnosis (diagnosis using cervical intraepithelial neoplasia in some studies) properly. Despite the differences in study designs and in the proportion of the HSIL+ cases be included, our results are remarkably similar to other studies30,32,33. The S5 methylation test accurately identified women with higher risk of cervical high-grade disease and cancer among those who with abnormal result between HPV and cytology. Even further, our study demonstrated that S5 outperforms both cytology and HPV16/18 in detecting HSIL+ in Chinese women. Currently, S5 DNA methylation test is labor intensive and costly. Based on the practice of our study13, the cost of an S5 test is not higher than that of a routine HPV screening and reflex genotyping, and it could be balanced against the expected 30 to 50% reduction in colposcopy referral costs. The recent developments of affordable and scalable next-generation sequencing assays strengthen our proposal that the S5 DNA methylation classifier test may be an acceptable strategy for the triage of women with abnormal result between HPV and cytology in developing countries. Strategies combing screening and immediate clinical management are urgently needed for developing countries settings where infrastructure for follow-up after screening is limited. S5 might be a potential classifier in developing countries for reducing costs and encouraging doctors to focus on the women at real risk of cervical cancer. Our results warrant further clinical validation of S5 in larger prospective population-based screening trials.