Study design and baseline screening procedure
Between June to August in 2018, a total of 2,000 women aged 25-64 from Tuoli County, Xinjiang, China, were recruited to this prospective cervical cancer screening cohort. Details on the study design and baseline information have been previously published[10]. In brief, women who were not pregnant, had not been treated for cervical intraepithelial neoplasia (CIN) in the last 5 years and consented to participate in the study were eligible for inclusion. Exclusion criteria were pregnancy, previous total hysterectomy and inability to comply with the study protocol. The study was approved by the Ethical Committee of The Affiliated Cancer Hospital of Xinjiang Medical University, China (Approval number: K-201802).
Informed consent was obtained by local health care personnel after explaining the study procedure. All women were invited to answer a questionnaire regarding demographic, gynecologic and obstetric history, then underwent a pelvic examination followed by collection of cervical samples by a gynecologist in following order: a cervical swab (dacron) sample, a second cervical cytology sample collected with sample collection brush and placed in Sample Preservation Solution (Shenzhen Senying Biotechnology Co. Ltd, China); a third sample collected using the careHPV collection device and placed in specimen transport medium (Qiagen). All laboratory tests were performed totally blinded to the other screening results.
careHPV test
careHPV is a nucleic acid hybridization assay with signal amplification using microplate chemiluminescence for the detection of 14 high- risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) in bulk. careHPV test was proceeded at the HPV laboratory of Tuoli Maternal and Child Health Hospital according to the standard protocol. Samples were considered hrHPV positive if relative light units (RLU/CO) were ≥1.0.
GenPlex® HPV test
GenPlex® HPV test (Human Papillomavirus Genotyping Kit (Microfluidic Chip), BOHUI, China), is a multiplex Polymerase Chain Reaction (PCR) test. Type-specific primers target the L1 region of the HPV genome, whereas identification of amplicons is performed by reverse DNA hybridization using DNA chip technology. The test detects separately 24 HPV genotypes, including HPV6, 11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 81, 82 and 83. In the present study, GenPlex® HPV test was performed according to manufacturer’s instructions using 0.5mL from the third cervical specimen remnant after careHPV testing.
Cytology
The cytology specimen in the Sample Preservation Solution was used to prepare a slide for liquid-based cytology (LBC) using the Papanicolaou staining method. Results were interpreted according to the Bethesda 2001 classification system by experienced cytology technicians blinded to other screening results[11]. Atypical squamous cells of undetermined significance or worse (ASC-US+) were considered abnormal. All cytology slides were double read, namely they were examined by a cytologist from the Affiliated Cancer Hospital of Xinjiang Medical University and re-reviewed by a senior expert from Cancer Hospital/Chinese Academy of Medical Sciences (CHCAMS).
Methylation
The swab samples were stored at -80℃ until tested for methylation. careME methylation test (careLYFE, China) was used, which is based on methylation-specific real-time PCR technique and targets the host cell gene EPB41L3 and viral HPV16L1/HPV18L2 genes. Firstly, the swab sample was vortexed for 2 min in 600uL lysis buffer, then used for DNA extraction by the Magnetic DNA Puri Kit (careLYFE, China) according to manufacturer`s instructions. The 40uL eluted and purified DNA was used for the bisulfite conversion reactions where unmethylated cytosine was converted to uracil, then the converted DNA was used for desulphonation and clean-up with the Magnetic DNA methylation kit (careLYFE, China). careME methylation assay was based on 2 tubes of methylation-specific multiplex real-time PCR. A pair of methylation-specific EPB41L3 primers/probe covering targeted CpG positions were used for EPB41L3 CpG detection. Another pair of methylation-nonspecific ACTB primers/probe were used as internal control for total bisulfite conversion to normalize the methylation level of EPB41L3 precisely. For the 2-plex EPB41L3 PCR reaction, different fluorescent signals labeled in different probes were used for different gene testing. Similarly, a 4-plex HPV16L1&HPV18L2 methylation assay was established containing 4 pairs of primers/probe, one specific for HPV16L1 methylation and one for the internal control of HPV16, another one specific for HPV18L2 methylation and one for the internal control of HPV18. Briefly, for 2-plex EPB41L3 PCR, 10uL of PCR master mix, 5uL of converted DNA, 1uL of primer (0.4 umol/L of each primer), 1uL of probe (0.2 umol/L of each probe), 0.2uL HotStar Taq DNA polymerase(1U) were used; for 4-plex HPV16L1&HPV18L2 PCR, 10uL of PCR master mix, 5uL of converted DNA, 1uL of primer (0.4 umol/L of each primer), 1uL of probe (0.2 umol/L of each probe), 0.25uL HotStar Taq DNA polymerase(1.25U) were used; both reactions were adjusted with water to give a final 25uL reaction volume and run at thermal cycling conditions initiated at 94℃ for 10min, followed by 45 cycles: 20s at 94℃, 45s at 62℃, then final 10s at 12℃ for hold. Signals were collected in the stage of 45s at 62℃. From DNA extraction to methylation-specific PCR, high-methylation positive control (PC), non-methylation negative control (NC) and non-template blank control (BC) were tested in each run in parallel. According to the result of EPB41L3 and HPV16L1/HPV18L2 PCR assays, a Risk value (R value) for each sample was calculated by the Ct value of each gene and then normalized by each internal control gene. A cutoff Risk value of ≥1.8 derived in a previous cutoff study by logistic regression was regarded as positive methylation results, indicating a high risk of high-grade cervical lesions and cervical cancer, which means further referral for colposcopy or other follow-up tests were required. When R value < 1.8 indicates, the result was regarded as methylation negative, indicating a low risk of high-grade cervical disease and no need for referral to colposcopy.
Outcome verification
Women positive for one or more of the 14 hrHPV types by either HPV test and/or with a cytology result of ASCUS+ were referred to colposcopy examination. Colposcopy was performed according to accepted diagnostic standards[12]. Biopsies were taken if clinically indicated. Cases with negative colposcopic impression where no biopsies were taken were considered negative for disease. Biopsy confirmed CIN2+ were used as clinical outcome endpoint. All histology slides were reviewed by an experienced pathologist from Affiliated Cancer Hospital of Xinjiang Medical University and re-confirmed by the pathologist from CHCAMS.
Follow-up procedure
All women with either hrHPV positive or cytology ASC-US+ results, except of those histologically confirmed CIN2+ at baseline were called back for annual follow up in 2019 and 2020. HrHPV and cytology tests were used in follow-up visits and any positive results were referred to colposcopy examination with biopsy if necessary. All laboratory tests and clinical diagnosis were conducted as described in previous text.
Statistical Analysis
Pearson chi-square test was used to assess differences in methylation positivity rates by population characteristics. Chi-square test for trend was used to assess whether methylation test positivity increased by severity of the cervical lesions and respective odds ratios (ORs) with 95% confidence intervals (CIs) were calculated. Twelve-month (12M) and 24-month (24M) risk of hrHPV persistence was evaluated by risk ratio (RR) with respective 95% CIs. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) with respective 95%CIs for detecting CIN2+ were calculated for different triage strategies. A difference in sensitivity and specificity between given triage strategies and the reference triage was considered significant if the 95% CIs around the relative sensitivity and specificity did not include 1. We also calculated the referral rates (based on % triage test positivity) and the number needed to refer to colposcopy to find one case of CIN2+ (NNR) to evaluate the triage efficiency. All statistical tests were two-sided using a 0.05 significance level. Stata (version 12.0, StataCorp) was used for statistical analyses.