Jesper Bonde

Participation and relative cost of attendance by direct‐mail compared to opt‐in invitation strategy for HPV self‐sampling targeting cervical screening non‐attenders: A large‐scale, randomized, pragmatic study

AbstractBroad accessibility to cervical cancer screening and high participation rate is essential to reduce cervical cancer incidence. HPV self‐sampling is an alternative to clinician collected cervical samples to increase accessibility and screening coverage. To inform on deployment strategies of HPV self‐sampling, this large‐scale, randomized, pragmatic study compared two invitation modalities; direct‐mail and opt‐in. The study included screening non‐attenders from the Capital Region of Denmark randomly allocated (1:4) to a direct‐mail or opt‐in invitation for cervical screening by HPV self‐sampling. Primary endpoint was screening participation; secondary endpoints were HPV prevalence and histology outcome. Adherence to follow‐up and cost were also evaluated. After exclusion of hysterectomized/non‐accessible women, 49,393 women were invited: 9639 by direct‐mail, and 39,754 by the opt‐in offer. A direct‐mail invitation for HPV self‐sampling yielded a significant higher participation than an opt‐in invitation. HPV self‐sample participation for direct‐mail was 25.2% (n = 2426), opt‐in participation was 20.2% (n = 8047), adjusted OR = 1.27, 95% CI 1.20–1.34. Participation increased with age (p < .0001) for both strategies and decreased with screening history of non‐attendance (p < .0001). Interaction between invitation strategy and age/screening history was found; more women below 50 years of age participated by direct‐mail compared to opt‐in (p < .0001) and higher participation by direct‐mail group was found in women with a short history of non‐attendance (p < .0001). Participation of long‐term unscreened women was similar between arms. The relative cost was ≈14 HPV self‐sample kits distributed per additional participant by direct‐mail over opt‐in. HPV prevalence, adherence to follow‐up, and detection of high‐grade cervical intraepithelial neoplasia was similar between invitation strategies.

Evaluation of targeted next‐generation sequencing for detection of HPV genotypes and sublineages in cervical liquid‐based cytology SurePath samples from the Danish screening program

Abstract The carcinogenicity of HPV genotypes is well established. However, HPV genotypes have sublineages with individual risk profiles, and these are much less described with respect to carcinogenicity. Research to characterize HPV sublineages by next‐generation sequencing (NGS) on screening‐derived liquid‐based cytology (LBC) samples is limited because of the technical and quality assurance challenging nature of sublineage analysis. This study aimed to evaluate the feasibility of detecting HPV sublineages from 14 HPV genotypes in SurePath LBC samples from Danish cervical cancer screening. We included 41 HPV plasmids (the Global HPV LabNet DNA Genotyping Proficiency Panel 2023) to quality assure the NGS approach and 120 SurePath LBC samples from the screening program for proof of concept. Our results of the HPV plasmids showed the correct sublineage for all included HPV genotypes except for HPV68b, where the coverage was inadequate for sublineage analysis. The NGS analysis enabled HPV sublineage analysis in 99.1% (112/113) of HPV‐positive SurePath LBC samples. Sublineages belonging to the A lineage were most frequent for HPV16, 18, 31, 33, 35, 39, 51, 52, 58, 59, and 68, while B‐type sublineages showed the highest frequency in HPV45, 56, and 66. The most diverse sublineage data was obtained for HPV31 with sublineages from the A, B, and C lineages. In conclusion, our method enables the identification of HPV sublineages in SurePath LBC screening samples. This information can be used in future studies to determine the usefulness of HPV sublineage analysis in screening settings for risk stratification and clinical management of HPV‐positive women.

Large‐Scale Study Comparing Analytical and Diagnostic Quality of Three HPV Self‐Sampling Devices for At‐Home Cervical Cancer Screening

ABSTRACT Elimination of cervical cancer requires broad access to and participation in cervical screening. Self‐sampling has emerged as a robust technology that simplifies access; however, few self‐sampling devices are independently validated regarding sample quality. This study compares the quality of three self‐sampling devices: Evalyn (Rovers), currently used in Denmark, the FLOQSwab (Copan) and the modified FLOQSwab: SensiGrip. Women residing in the Capital Region of Denmark, accepting screening by self‐sampling, were offered a kit containing the following combinations of devices: (1) Evalyn, FLOQSwab, (2) Evalyn, SensiGrip or (3) FLOQSwab, SensiGrip. Returned kits were analyzed using the validated BD Onclarity HPV assay on the COR instrument (BD). A total of 1677 women participated. Sample quality was similar across devices, also when stratifying into age groups. Two hundred thirty‐two women (13.9%) tested positive for one or more oncogenic HPV types. Pairwise concordance analysis for each group showed an overall agreement between 93.5% and 95%. Analytical and diagnostic performance of samples collected by the three self‐sampling devices resulted in similar quality and HPV detection. Hence the sample quality is not a determinant in the choice of sampling device and focus on other factors such as cost, women's preference or size and weight can take precedence.

Methylation markers FAM19A4 and miR124‐2 as triage strategy for primary human papillomavirus screen positive women: A large European multicenter study

AbstractIn human papillomavirus (HPV) cervical cancer screening, cytology is used as triage to counter the low specificity of HPV testing. VALID‐SCREEN is a EU‐multicenter, retrospective study conducted to evaluate the clinical performance of the FAM19A4/miR124‐2 methylation‐based molecular triage test as a substitute or addition to cytology as reflex testing of HPV screen positive women. FAM19A4/miR124‐2 methylation test (QIAsure Methylation Test) was evaluated in 2384 HPV‐positive cervical screening samples, from women 29‐76 years of age, derived from four EU countries. Specimens were collected in ThinPrep or SurePath media, HPV‐status, concurrent cytology, and histology diagnosis were provided by the parent institutes. The control population consisted of women with no evidence of disease within 2 years of follow‐up. A total of 899 histologies were retrieved; 527 showed no disease, 124 CIN2 (5.2%), 228 CIN3 (9.6%) and 20 cervical cancers (0.8%); 19 of 20 screen‐detected cervical cancers were found methylation‐positive (sensitivity 95%). Overall specificity of FAM19A4/miR124‐2 methylation test was 78.3% (n = 2013; 95%CI: 76‐80). The negative predictive value of hrHPV positive, methylation‐negative outcomes were 99.9% for cervical cancer (N = 1694; 95%CI: 99.6‐99.99), 96.9% for ≥CIN3 (95%CI: 96‐98), and 93.0% for ≥CIN2 (95%CI: 92‐94). Overall sensitivity for CIN3 using FAM19A4/miR124‐2 methylation test was 77% (n = 228; 95%CI: 71‐82). CIN3 sensitivity was uniform between centers independent of sample collection medias, DNA extraction methods and HPV screening tests. Being objectively reported compared to the subjectivity of cytology, equally performing across settings and screening methods, the FAM19A4/miR124‐2 methylation constitute an alternative/supplement to cytology as triage method to be investigated in real‐life pilot implementation.

Clinical validation of the Cobas 4800 HPV assay using cervical samples in SurePath medium under the VALGENT4 framework

The VALidation of HPV Genotyping Tests (VALGENT) framework is an international cooperation designed for comparison and clinical validation of HPV assays with genotyping capabilities. Here we addressed the accuracy of the Roche cobas 4800 HPV test using SurePath samples from the Danish cervical cancer screening program under the VALGENT framework. The VALGENT4 panel comprises 998 consecutive SurePath cervical samples from routine screening and 297 SurePath samples enriched for disease (100 ASC-US, 100 LSIL, 97 HSIL). The cobas HPV test is a real-time PCR assay which detects HPV16 and 18 individually and 12 other high-risk (hr) HPV genotypes in one bulk. The clinical performance of the cobas test was assessed relative to that of the comparator assay GP5+/6 + PCR Enzyme ImmunoAssay (GP-EIA) by a non-inferiority test. The relative sensitivity for ≥ CIN2 was 1.00 (95% CI: 0.97-1.04) and relative specificity for the control group was 1.02 (95% CI: 1.01-1.04). The cobas test was found non-inferior to that of GP-EIA for both sensitivity and specificity (p-0.0006 and p < 0.0001, respectively). The type specific performance of the cobas test was evaluated using the GP5+/6 + PCR with Luminex genotyping (GP-LMNX) as comparator. The cobas test showed excellent to good concordance (Kappa: 0.70 to 0.90) with GP-LMNX for all three genotype groups in the overall VALGENT population but good to moderate concordance in the Screening population (kappa from 0.56 to 0.80). The cobas HPV test demonstrated non-inferiority to the comparator assay on cervical SurePath screening samples using the VALGENT4 panel.

FAM19A4/miR124‐2 methylation in invasive cervical cancer: A retrospective cross‐sectional worldwide study

Widespread adoption of primary human papillomavirus (HPV)‐based screening has encouraged the search for a triage test which retains high sensitivity for the detection of cervical cancer and precancer, but increases specificity to avoid overtreatment. Methylation analysis of FAM19A4 and miR124‐2 genes has shown promise for the triage of high‐risk (hr) HPV‐positive women. In our study, we assessed the consistency of FAM19A4/miR124‐2 methylation analysis in the detection of cervical cancer in a series of 519 invasive cervical carcinomas (n = 314 cervical scrapes, n = 205 tissue specimens) from over 25 countries, using a quantitative methylation‐specific PCR (qMSP)‐based assay (QIAsure Methylation Test®). Positivity rates stratified per histotype, FIGO stage, hrHPV status, hrHPV genotype, sample type and geographical region were calculated. In total, 510 of the 519 cervical carcinomas (98.3%; 95% CI: 96.7–99.2) tested FAM19A4/miR124‐2 methylation‐positive. Test positivity was consistent across the different subgroups based on cervical cancer histotype, FIGO stage, hrHPV status, hrHPV genotype, sample type and geographical region. In conclusion, FAM19A4/miR124‐2 methylation analysis detects nearly all cervical carcinomas, including rare histotypes and hrHPV‐negative carcinomas. These results indicate that a negative FAM19A4/miR124‐2 methylation assay result is likely to rule out the presence of cervical cancer.

Classification of high‐grade cervical intraepithelial neoplasia by p16ink4a, Ki‐67, HPV E4 and FAM19A4/miR124‐2 methylation status demonstrates considerable heterogeneity with potential consequences for management

AbstractHigh‐grade cervical intraepithelial neoplasia (CIN2 and CIN3) represents a heterogeneous disease with varying cancer progression risks. Biomarkers indicative for a productive human papillomavirus (HPV) infection (HPV E4) and a transforming HPV infection (p16ink4a, Ki‐67 and host‐cell DNA methylation) could provide guidance for clinical management in women with high‐grade CIN. This study evaluates the cumulative score of immunohistochemical expression of p16ink4a (Scores 0‐3) and Ki‐67 (Scores 0‐3), referred to as the “immunoscore” (IS), in 262 CIN2 and 235 CIN3 lesions derived from five European cohorts in relation to immunohistochemical HPV E4 expression and FAM19A4/miR124‐2 methylation in the corresponding cervical scrape. The immunoscore classification resulted in 30 lesions within IS group 0‐2 (6.0%), 151 lesions within IS group 3‐4 (30.4%) and 316 lesions within IS group 5‐6 (63.6%). E4 expression decreased significantly from CIN2 to CIN3 (P &lt; .001) and with increasing immunoscore group (Ptrend &lt; .001). Methylation positivity increased significantly from CIN2 to CIN3 (P &lt; .001) and with increasing immunoscore group (Ptrend &lt; .001). E4 expression was present in 9.8% of CIN3 (23/235) and in 12.0% of IS group 5‐6 (38/316). Notably, in a minority (43/497, 8.7%) of high‐grade lesions, characteristics of both transforming HPV infection (DNA hypermethylation) and productive HPV infection (E4 expression) were found simultaneously. Next, we stratified all high‐grade CIN lesions, based on the presumed cancer progression risk of the biomarkers used, into biomarker profiles. These biomarker profiles, including immunoscore and methylation status, could help the clinician in the decision for immediate treatment or a “wait and see” policy to reduce overtreatment of high‐grade CIN lesions.

Lack of consensus in calculation of interval cancer rates for cervical cancer screening

AbstractIntroductionIn 2018, nondisclosure of results of retrospective audits of cytology in interval cancers precipitated a crisis in the Irish national cervical screening programme. In response, an Expert Reference Group was convened which recommended a collaborative approach to the development of a new key performance indicator, the interval cancer rate. The Expert Reference Group also recommended that the Irish programme should collaborate with international colleagues to reach consensus on (i) the definition of an interval cervical cancer, (ii) the methodology to calculate the interval cancer rate, and (iii) benchmarking with other international programs. This study was undertaken to determine if a consensus regarding the definition of an interval cervical cancer and the calculation of an interval cancer rate exists.Material and MethodsA web‐based questionnaire was sent to 18 population‐based cervical screening programs. Inclusion criteria involved (1) a national or regional population‐based cervical screening prograe; (2) a country or region with a population ≥population of Ireland; (3) programs located in Europe, Australia, or Canada; (4) programs that had responded to a previously published international survey on the disclosure of retrospective cytology reviews in cervical cancer cases.ResultsThe response rate was nine out of 18. Of nine respondents, six had an agreed definition of interval cervical cancer, and four of these calculated an interval cancer rate. Three programs neither calculated interval cancer rates nor had any guidelines related to this. Of the six with an agreed definition, all respondents defined the numerator as invasive cancers in the screening age group, with four including microinvasive disease. Respondents included cancers diagnosed 3–5 years after the last screening test had been taken. Three respondents also included cancers diagnosed in women up to 3.5 years after they exited the screening program. Countries use different denominators, including (i) per women years, (ii) per number of screens, and (iii) per total cancers in screened population.ConclusionsThere is variation in the parameters used in interval cancer rate calculation. To allow benchmarking of cervical screening program performance, there is a need for consensus on a standardized method of interval cancer definition and interval cancer rate calculation.