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. 2023 Jul;129(1):104-111.
doi: 10.1038/s41416-023-02277-z. Epub 2023 Apr 26.

Evaluation of DNA methylation biomarkers ASCL1 and LHX8 on HPV-positive self-collected samples from primary HPV-based screening

Lisanne Verhoef  1   2 , Maaike C G Bleeker  1   2 , Nicole Polman  1   2 , Renske D M Steenbergen  1   2 , Renée M F Ebisch  3 , Willem J G Melchers  4 , Ruud L M Bekkers  5   6 , Anco C Molijn  7 , Wim G Quint  7 , Folkert van Kemenade  8 , Chris J L M Meijer  1   2 , Johannes Berkhof  9 , Daniëlle A M Heideman  10   11
Affiliations

Evaluation of DNA methylation biomarkers ASCL1 and LHX8 on HPV-positive self-collected samples from primary HPV-based screening

Lisanne Verhoef et al. Br J Cancer. 2023 Jul.

Abstract

Background: Host-cell DNA methylation analysis can be used to triage women with high-risk human papillomavirus (HPV)-positive self-collected cervicovaginal samples, but current data are restricted to under-/never-screened women and referral populations. This study evaluated triage performance in women who were offered primary HPV self-sampling for cervical cancer screening.

Methods: Self-collected samples from 593 HPV-positive women who participated in a primary HPV self-sampling trial (IMPROVE study; NTR5078), were tested for the DNA methylation markers ASCL1 and LHX8 using quantitative multiplex methylation-specific PCR (qMSP). The diagnostic performance for CIN3 and cervical cancer (CIN3 + ) was evaluated and compared with that of paired HPV-positive clinician-collected cervical samples.

Results: Significantly higher methylation levels were found in HPV-positive self-collected samples of women with CIN3 + than control women with no evidence of disease (P values <0.0001). The marker panel ASCL1/LHX8 yielded a sensitivity for CIN3 + detection of 73.3% (63/86; 95% CI 63.9-82.6%), with a corresponding specificity of 61.1% (310/507; 95% CI 56.9-65.4%). The relative sensitivity for detecting CIN3+ was 0.95 (95% CI 0.82-1.10) for self-collection versus clinician-collection, and the relative specificity was 0.82 (95% CI 0.75-0.90).

Conclusions: The ASCL1/LHX8 methylation marker panel constitutes a feasible direct triage method for the detection of CIN3 + in HPV-positive women participating in routine screening by self-sampling.

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Conflict of interest statement

DAMH, RDMS and CJLMM are minority shareholders of Self-screen B.V., a spin-off company of Amsterdam UMC, location VUmc; Self-screen B.V. develops, manufactures and licences high-risk HPV and methylation marker assays for cervical cancer screening and holds patents on these tests; JB had financial support from the European Commission (RISCC, grant number 847845); CJLMM is part-time CEO of Self-screen B.V., and has a very small number of shares of MDXHealth and previously QIAGEN, has received speakers fees from GSK, QIAGEN and SPMSD/Merck, and served occasionally on the scientific advisory boards of these companies; LV, MCGB, NP, RMFE, WJGM, RLMB, ACM, WGQ and FJvK declare no competing interests.

Figures

Fig. 1
Fig. 1. Venn diagram showing the relation between the sample series used in this study.
Data analysis comprised methylation results of self-collected samples from 593 HPV-positive women (white ellipse; study population), methylation results of paired clinician-collected samples from 485 (out of 593) women (light-grey ellipse; subset A) and methylation results of corresponding formalin-fixed paraffin-embedded (FFPE) tissue samples of 116 (out of 485) women (dark grey ellipse; subset B). HPV human papillomavirus, n number of, NILM negative for intraepithelial lesion of malignancy, CIN cervical intraepithelial neoplasia.
Fig. 2
Fig. 2. Stacked histograms showing the relative frequency of methylation levels of ASCL1 and LHX8 per disease category.
Methylation levels were categorised in sextiles. CIN cervical intraepithelial neoplasia, 2x NILM two consecutive normal cytology results.
Fig. 3
Fig. 3. ASCL1/LHX8 marker panel outcome according to HPV genotype stratified for disease category.
Multiple HPV infections were counted as separate attributions. HPV human papillomavirus, CIN cervical intraepithelial neoplasia, N total number of samples, CI confidence interval. *Other alpha-7/9 types: HPV31, 33, 35, 39, 45, 52, 58, 59 and 68. **Non-alpha-7/9 types: HPV51, 56 and 66.
Fig. 4
Fig. 4. Conditional scatterplots displaying the methylation levels for ASCL1 and LHX8 for paired self-collected samples (S), clinician-collected cervical samples (C) and cervical tissue specimens (F) stratified for histology.
Differences in methylation levels of (A) ASCL1 and (B) LHX8 between paired sample types were assessed using the Mann–Whitney U test: self-collected samples compared to clinician-collected cervical samples, both P values <0.0001; self-collected samples compared to cervical tissue specimens, P value = 0.134 and 0.002, respectively; and clinician-collected cervical samples compared cervical tissue specimens, P value = 0.130 and 0.179, respectively. Cq quantification cycle, CIN cervical intraepithelial neoplasia, sqrt square root.
See this image and copyright information in PMC

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