Selection of methylation sub-group participants and study protocol
Cases were women identified after the end of multi-center study as women who had abnormal screening results at baseline (first visiting) and with a colposcopy-directed biopsy diagnosis of (His)HSIL or worse (including (His)HSIL, carcinoma in situ, and cervical cancer (CC)) at any time during the 2-year follow-up. Controls were randomly selected regardless of arm allocation from women who were abnormal screening results at baseline, had a biopsy with a diagnosis of less than (His)LSIL during the follow-up confirming that they were at low risk of cervical cancer and with enough remainder of archived baseline samples in specimen transport medium for further testing. Controls were individually matched to cases by age. As shown in the flowchart in Figure 1 , the 471 cases and 471 matched controls were identified for the study. Briefly, we enrolled samples (with initially an HPV positive or abnormal cytology result) derived from IC cohort and RF cohort, and then grouped as “cases group (with histology ≥HSIL+)” and age-matched “control group (with histology ≤LSIL)”.
In this study, according to “The Lower Anogenital Squamous Terminology Standardization Project for HPV-associated Lesions” (LAST), we categorized histopathological results as “No-Lesion”, “LSIL”, “HSIL” and “CC”. And we categorized all the cytology results as “NILM”, “(TCT)LSIL”, “(TCT)HSIL” and “CC”, “ASCUS” was grouped in “(TCT)LSIL”, and “ASC-H” was grouped in “(TCT)HSIL”. Based on the histopathological diagnoses as “golden standard”, we compared the diagnostic performance of S5 methylation with other existing classifier among women with abnormal results with HPV or cytology.
All the cervical samples were obtained from the first visiting in multi-center clinics. Samples for cytology were collected using Cervi-Brush® bush (Rovers® Medical Devices, North Brabant, Netherlands) and stored in a Sure-Path vial for further cytology analysis, and samples for HPV and methylation detection was also collected and then stored at 2°C to 8°C. The vials used for testing were stored 2°C to 8°C upon arrival the central cytology laboratory of Gy&Ob hospital of Fudan University in Shanghai, China.