Sample selection
SurePath cervical cancer screening samples
For the specificity analysis (the “no disease” control population), 1,395 residual SurePath samples were collected from Danish women ≥30 years undergoing routine cervical cancer screening at Hvidovre Hospital, Denmark. Collection of the control panel was completed in October 2016. In total, 211 samples were excluded due to one of the following reasons 1) women with previous cytological diagnosis of ASCUS within the past 15 months; 2) a cytological diagnosis of more than ASCUS (>ASCUS) in the past 12 months; 3) previous cervical cancer or CIN in the previous three years; or 4) insufficient/incomplete histological follow-up in the Danish register or diagnosis of ≥CIN2 follow-up after baseline analysis, 5) laboratory processing and/or technical errors. The final control population incorporated 1184 samples (mean age 43.4, range 30–65).
For the sensitivity analysis (the “disease” case population) residual SurePath material from 411 consecutive, unselected samples were collected from Danish women undergoing screening between September and October 2012 at Hvidovre Hospital. The samples were derived from women with ≥ASCUS cytology. After collection, samples with insufficient material for testing were excluded and from the remainder women ≥30 years with confirmed ≥CIN2 histology were selected, yielding 57 samples in total. In June 2016, an additional 24 samples were selected from 70 consecutive ≥ASCUS samples using the same criteria. In total, the case population consisted of 81 samples from women ≥30 years of age with confirmed ≥CIN2 (mean age 40.3, range 30 to 73).
For assay reproducibility, 474 samples included in the control population were selected. In addition, 70 samples with ≥ASCUS cytology were collected from the routine cervical screening at Hvidovre hospital, to ensure compliance with the requirement for a 30 % HPV positive rate within the reproducibility element (4). Four samples were excluded due to technical invalidity in one of three runs for the reproducibility element. In total, DNA from 540 samples were included constituting 379 MGP-PCR negative and 161 MGP-PCR positive samples. An aliquot of extracted DNA from the 540 reproducibility samples was shipped to the HPV Research Group, University of Edinburgh, who performed the inter-laboratory agreement testing.
ThinPrep cervical cancer screening samples.
For the specificity analysis (the “no disease” control population), all women between 01-jan–2013 and 31-dec–2015 undergoing routine cervical screening in Stockholm county, Sweden was included, in total 290,793 samples, of these 117,365 had sample residuals stored in the Clinical Cytology Biobank, Karolinska University Laboratory, Stockholm, Sweden. Subsequently all women with both the current and previous cytology classified as normal (n = 92,695) were identified. From these, a random set of 1,169 samples with sufficient material was drawn (mean age 38.3, range 30–63).
For the sensitivity analysis (the “disease” case population), all women with ≥CIN2 diagnosed in routine cervical screening in Stockholm, Sweden, who had residual samples stored in the Clinical Cytology Biobank, Karolinska University Laboratory, Stockholm, Sweden, were identified from 01-jan–2013 to 31-dec–2015 (n = 4,274). From here 80 consecutive samples from women ≥23 years of age with confirmed ≥CIN2 (mean age 34, range 23–60) was selected. Of these, 21 samples were derived from women <30 and 59 samples derived from women ≥30 years of age.
For the reproducibility analysis, samples derived from women participating in primary HPV-based screening in Stockholm, stored in the Clinical Cytology Biobank, Karolinska University Laboratory, were identified starting from 1-sep–2014 (the start of primary HPV-screening above 30) to 30-nov–2014. The first 160 consecutive samples registered as HPV-positive and the first 360 consecutive samples registered as HPV-negative were included, for a total of 520 samples. An aliquot of extracted DNA from the 520 reproducibility samples was shipped to the HPV Research Group, University of Edinburgh, where inter-laboratory agreement testing was performed. In total 491 and 495 valid samples were included in the intra and inter-laboratory reproducibility element, respectively.
DNA extraction
The MagNA Pure 96 platform (Roche diagnostics, Rotkreutz, Switzerland) with MagNA Pure LC total nucleic acid isolation kit (Roche Diagnostics) was used for both the SurePath and ThinPrep samples. For SurePath; 1 ml of material was preprocessed with heat treatment for 1 hour at 56°C with proteinase K, followed by 1 hour at 90°C to reverse formaldehyde-induced cross-linking prior to extraction. Extracted DNA was stored refrigerated prior to CLART4S and MGP-PCR testing. For ThinPrep, an aliquot of 100 µL from all included ThinPrep samples was extracted, and the resulting DNA was stored in –20°C prior to CLART 4S and (MGP-PCR) Luminex testing.
GENOMICA CLART® HPV4S Assay
GENOMICA CLART® HPV4S is a PCR-based microarray assay that targets the HPV L1 region, and detects 16 individual genotypes: HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 and HPV6 and 11. The assay has two internal controls: one for PCR performance and one for sample sufficiency and assay performance. The internal control for PCR processing relies on amplification of a spiked CFTR plasmid, and is used to validate the individual PCR run, the internal control for human CFTR is used to validate sufficient human material in the sample. The assay is fully automated after PCR amplification using the autoclart®plus platform. In short, 5 µl aliquots of extracted DNA were used for the CLART HPV4S PCR amplification. Prior to visualization on low-density microarrays, the PCR products were denaturated at 95°C for 10 minutes. Visualization and reporting of genotyping results were done automatically on the Clinical Array Reader (CAR®) as part of the automated autoclart®plus workflow. All samples with an invalid result (no human CFTR amplification detected or no spiked CFTR plasmid amplification detected) were retested once, and the second result was considered definitive. The CLART4S assay run-protocol was independent of sample media. As part of the validation, a posteriori optimization of genotype specific cut off values was conducted against detection of ≥CIN2 and <CIN1, resulting in two LBC specific, optimized assay reading software versions. The final dataset was analyzed using the ThinPrep and SurePath specific assay reading software versions.
MGP-PCR and HPV typing using Luminex
All samples were HPV genotyped using MGP-PCR, primer targeting L1, and type-specific probes using Luminex detection technology, as previously described (34–36). Briefly, 5 µL aliquots of extracted DNA were used in the MGP-PCR in a total volume of 25 µL. Forty-two beads, 37 different HPV-types, three HPV variants, and two ‘universal’ HPV probes, were included in the Luminex assay. Samples with a grey-zone result were retested in duplicate and HPV type(s) that were reproducible were considered definitive. All MGP-PCR and Luminex testing was performed at the Karolinska Institute, Stockholm, Sweden. MGP-PCR and HPV typing using Luminex was performed with the same protocol for both SurePath and ThinPrep collected samples.
Cytology
SurePath procedure (Denmark)
Cytology was read following the Bethesda 2001 criteria. Hvidovre Hospital employs computer assisted screening using FocalPoint™ GS imaging system and SlideWizard™ (BD diagnostics, Burlington, NC), prior to cyto-screener review. HPV testing was performed after cytology evaluation; hence the cyto-screener was blinded to the HPV result upon evaluation, except for ASCUS cases which are reflex tested, routinely for HPV in accordance with current Danish National screening guidelines. All abnormal cytology findings were routinely adjudicated by a pathologist. According to National guidelines, women with LSIL were invited for repeat cytology testing after 6 months. Women with normal cytology were returned to routine screening after three years if aged 23–49 or five years if aged 50–59. All women included in the study were managed according to the routine guidelines for the Danish cervical cancer screening program.
ThinPrep procedure (Sweden)
Cytology was read following the Bethesda 2001 criteria. Manual screening review was performed by especially trained cyto-diagnosticians, with ambiguous cases resolved by specialist cytologist review. The Karolinska University Laboratory is the central cervical screening diagnostic laboratory for the Stockholm region, the capital region of Sweden. Within the cervical screening program, a randomized health services study was performed during 2012–2016 (37) for women aged 30–60. Half of the population was randomized to primary cytology, and half to primary HPV-based screening. In the cytology arm, ASCUS cases were routinely tested for HPV in accordance with guidelines. In the HPV arm, HPV-positive samples were tested with reflex cytology. In 2015, Sweden issued new guidelines for cervical screening recommending HPV-based screening for all women 30–64 years of age (38). The Stockholm-Gotland region has been biobanking residuals from screening samples gradually since 2011 and all cervical screening samples since 2013 at the Clinical Cytology Biobank, Karolinska University Laboratory, Karolinska University Hospital.
Histology
Danish procedures
In Denmark, women ≥30 years with ASCUS and a concurrent HPV-positive test result are referred to colposcopy with biopsies, as are women with high-grade squamous intraepithelial lesions (HSIL), atypical squamous cells—cannot exclude HSIL (ASC-H), atypical glandular cells (AGS) or cytological sign of carcinoma and women with continued ASCUS and LSIL cytological diagnosis. Danish screening guidelines requires biopsies from all aceto-white lesions or 4-counter clockwise random biopsies from all four quadrants in cases where no lesions are visible upon colposcopy. All histological data included in the study were retrieved from the Danish Pathology Data Register.
Swedish procedures
In Sweden, women are referred to colposcopy with biopsies according to similar guidelines as listed above for Denmark. For women with suspected high-grade disease, biopsies from lesions or random biopsies in the similar fashion should be performed. All histological data included in the study were retrieved from the Swedish National Cervical Screening Register.
Data analysis
For CLART4S HPV, a sample was considered positive if at least one of the 14 genotypes (16, 18, 31,33, 35, 39, 45, 51, 52, 56, 58, 59, 66, & 68) was detected. HPV6, and/or HPV11 present alone without any of the other 14 HPV genotypes were considered HPV screen negative. The same was true for MGP-PCR. The CLART4S assay automatically reports genotype findings detected in an “uncertainty” range, if the visualization outcome falls close to the manufacturer cut-off. Reflecting routine practice at our facility, these genotypes are considered positive only if part of a multiple infection.
Clinical specificity and sensitivity values for CLART4S were compared to those of MGP-PCR using the non-inferior score test, where non-inferiority is defined as a relative specificity for <CIN2 of ≥98% and a relative sensitivity for ≥CIN2 of ≥90%. For the intra-laboratory reproducibility and inter-laboratory agreement, a lower confidence bound of ≥87% was used as a threshold (4). The non-inferior score excel sheet was provided by VU University, Amsterdam, The Netherlands (4). In the Swedish study population, Fisher’s exact test of homogeneity was applied to test homogeneity of distribution of HPV-status in women of <30, and ≥30 years, respectively. For other statistical computations incl. 95% CI, the SPSS statistics 22 software was used.