In Denmark, cervical cancer screening is a nationwide program inviting women aged 23–64 years for liquid-based cervical cytology sampling at their GP (cervical sample) . At present, women aged 23–59 years are screened with cytology, whereas women aged 60–64 years undergo hrHPV-based screening . Women aged 30–59 years who are diagnosed with atypical squamous cells of undetermined significance (ASC-US) undergo reflex hrHPV triage testing, and ASC-US/hrHPV positive women are referred for colposcopy, whereas ASC-US/hrHPV negative women are referred back to the routine screening program . This study was conducted in the Central Denmark Region (CDR), where all cervical cytologies are routinely handled and analyzed by the Department of Pathology, Randers Regional Hospital. In the CDR, the COBAS® 4800 (Roche Diagnostics, Switzerland) test is the routine test platform for hrHPV DNA analysis.
Women eligible for this study were aged 30–59 years and diagnosed with ASC-US based on a cervical sample between June 2015 and December 2016. Exclusion criteria were pregnancy and having given birth within the last 3 months. The recruitment procedure has been described in detail in a previous publication assessing the hrHPV concordance between home-based vaginal self-sampling and cervical sampling when using COBAS for analysis . In brief, eligible women were consecutively mailed a consent form and an information letter about the study explaining that they had to contact the investigator for oral information regarding the study and return a signed informed consent if they wanted to participate.
Sample collection, processing and storage
As per routine, the women had a cervical sample collected with a cervical cytobrush (Cervex-Brush® Combi, Rovers Medical Devices, B. V, Oss, Netherlands) at their GP . The Cervex-Brush device enables simultaneous collection of cells from the ectocervix, the endocervix and the transformation-zone in a single sample . After collecting cervical epithelial cells, the cervical cytobrush was rinsed in 10 mL SurePath medium (BD Diagnostics, Burlington, NC) and mailed to the Department of Pathology, Randers Regional Hospital, for routine processing and testing as previously described . The COBAS hrHPV testing was performed as per routine using the sample cell pellet from 1 mL SurePath medium. For this study, 100 μl of the residual purified DNA material was subsequently stored at -80 °C prior to CLART testing.
After undergoing cervical sampling at the GP and written informed consent was obtained, the women were mailed a self-sampling package to their home. The package included a dry brush device (Evalyn® Brush, Rovers Medical Devices, B. V, Oss, Netherlands) for vaginal self-sampling , a transportation tube with preservative media (Genelock, ASSAY ASSURE, Sierra Molecular, CA) for urine sampling, written and picture-based instructions showing the order of the self-sampling, pre-addressed return envelope, and a questionnaire. The women were further asked to firstly collect the vaginal sample and secondly collect a first-void urine sample (the first of 10–12 mL of urine voided) in a plastic cup during their first urination in the morning. The urine was transferred to the provided transportation tube by the participant. The women were urged to collect both samples and return the samples and the accompanying questionnaire by ordinary mail on the same day as the samples were taken and before an eventual colposcopy examination .
Upon arrival in the laboratory, the urine sample was stored overnight at 4 °C and then vortexed for 5 min. A 10–12 mL volume of urine was centrifuged at 3000 x RPM for 20 min at room temperature. After centrifugation, the cell pellet was re-suspended in 1 mL 25% ethanol-buffered (TRIS) and stored at -80 °C until further hrHPV testing. The dry brush head was transferred into 10 mL SurePath medium (BD Diagnostics, Burlington, NC) also stored overnight at 4 °C and then vortexed for 5 min. A 6.4 mL volume of the self-sample material was centrifuged at 3000 x RPM for 20 min at room temperature . With the supernatant removed, the cell pellet was placed in 1 mL 25% ethanol-buffered (TRIS) and stored at -80 °C until further hrHPV testing . A volume of 6.4 mL was chosen to correct for the material volume used for cytology examination performed on the cervical sample . The median time interval between collecting the self-samples at home and the samples being processed and stored at -80 °C at the laboratory was 3 days (range: 2 to 8 days).
HrHPV DNA testing
Before the day of COBAS hrHPV DNA testing, the cell pellet material from the urine and vaginal self-samples were thawed overnight at 4 °C (after storage for 4 days to 18 months, median: 10 months). Subsequently, the self-samples (1 mL volume) were vortexed for 15 s before being transferred to empty test tubes for DNA purification and hrHPV testing . DNA was purified using the COBAS × 480, and amplification and detection of hrHPV DNA were undertaken using the COBAS z480 analyzer . From each self-sample, 100 μl of the residual purified DNA material were stored at -80 °C, until further CLART hrHPV testing. The COBAS® assay is a fully automated real-time PCR-based method, separately detecting HPV16, HPV18, and 12 other hrHPV types (HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) including the beta-globin gene as an extraction and amplification control .
Before the day of the CLART hrHPV DNA testing (HPV4S, GENOMICA, Madrid, Spain), the residual purified DNA material from the self-samples and the cervical samples gained using COBAS × 480, were thawed overnight at 4 °C (after storage for 2 to 25 months, median: 12 months). Five μl of the purified DNA material from the samples were used for the PCR amplification. The PCR amplification was performed using the CLART HPV4S amplification kit (GENOMICA) . Detection was performed on the CLART microarray. The genotyping results were analyzed and automatically performed on the Clinical Array Reader (GENOMICA) . CLART HPV4S detects 14 hrHPV genotypes individually (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) and two low-risk HPV genotypes (6 and 11). Amplification of a spiked CFTR plasmid served as an internal control of the PCR process, while the internal control for human CFTR gene validates specimen quality in the sample .
For both assays, samples with an invalid test result (i.e. COBAS: no betaglobin gene detected, CLART: no human CFTR amplification detected, or no spiked CFTR plasmid amplification detected) were retested once on diluted samples, and the second result was considered definitive. To avoid between-run discrepancy, the self-samples belonging to the same woman were processed in the same run for the COBAS analysis; and similarly for the CLART analysis the self-samples and the cervical sample belonging to the same woman were processed in the same run. Every run included four water samples to measure contamination . The laboratory personnel performed the hrHPV testing without knowledge of the hrHPV status of the cervical samples .
HrHPV positivity and histological results
Any hrHPV positivity was defined as samples being positive for HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, or 68, whereas positivity for specific hrHPV genotypes were grouped into “HPV16/18” and “hrHPV other” including HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. If the sample was positive for low-risk HPV types only (HPV6 and 11), it was classified as hrHPV negative. As per routine, histological results were only available for women with an hrHPV positive cervical sample . The results were classified following the CIN classification and grouped into normal tissue, CIN (not specified), CIN1, and CIN2+ (including CIN2, CIN3/AIS, and carcinoma). The most severe histological result was used if more were available. Test results of the cervical samples and the histological samples were obtained from the nationwide Danish Pathology Data Bank , while test results of the self-samples were retrieved from the Department of Pathology.
Acceptability and preferences
Together with the self-sampling package, the women received a questionnaire (see supplementary material) addressing among others the acceptance of urine collection, the clarity of the user instructions, and the confidence in correct execution of the urine collection. For analysis, the five response categories were grouped into three: “Agree” (totally agree and agree), “Disagree” (disagree or totally disagree), and “Do not know”. Women were also requested to rank if they preferred urine collection, vaginal self-sampling, or cervical sampling at the GP as their future screening examination. For analysis, women who refrained to rank their preferences or ranked two or more sampling methods as their preferred method were coded as missing. Additionally, the women were asked to report the date of collecting their self-sample and whether they had engaged in sexual intercourse between the cervical sampling at the GP and self-sampling .
For analysis, only women with valid hrHPV results from paired urine, vaginal, and cervical samples for both hrHPV assays were included. In the following, the term concordance refers to kappa values, while the term agreement corresponds to percentage agreement. Cohen’s kappa (κ) was used to measure concordance in hrHPV positivity (any hrHPV and specific genotypes) for urine vs vaginal self-samples, urine vs cervical samples by the COBAS and CLART assays. Concordance was defined as “poor” (κ ≤ 0.20), “fair” (0.21 ≤ κ ≤ 0.40), “moderate” (0.41 ≤ κ ≤ 0.60), “good” (0.61 ≤ κ ≤ 0.80), or “very good” (κ ≥ 0.81) . Concordance estimates were presented with 95% confidence intervals (CIs). Comparing the presence of HPV16/18 and hrHPV other types between the samples; concordance was determined by the presence of at least one identical genotype in both samples (i.e. HPV16/18 only, HPV16/18 and other types, and other types only); discordance was determined as no genotype similarities . McNemars test was performed to compare proportions of hrHPV positive results between the paired sample types. We also calculated the overall percentage of agreement between the paired samples including 95% CIs . Stratified by hrHPV assay, the sensitivity and specificity of hrHPV positivity (any hrHPV and specific genotypes) in urine samples was calculated with 95%CIs using the vaginal self-sample or the cervical sample as comparator test.
The acceptability of urine collection and preferences regarding the different sampling procedures were evaluated by descriptive statistics (proportions and 95% CIs). We tested whether women’s preferences differed between age groups (30–39, 40–49 and 50–59 years). The χ2-test was used to test for differences in categorical data. For continuous data, medians and interquartile ranges (IQR) were calculated; the Mann Whitney rank sum test was used to test for differences. P-values < 0.05 were considered statistically significant. The statistical analyses were performed using STATA, version 16 (STATA College). A sample size calculation has been reported elsewhere .
According to the EUs General Data Protection Regulation, the project was listed at the record of processing activities for research projects in the CDR (j.no.:1–10–72-69-15). The study was approved by the local Ethical Committee of the CDR (j.no.:1–16–02-209-15).