Validation of ASCL1 and LHX8 Methylation Analysis as Primary Cervical Cancer Screening Strategy in South African Women with Human Immunodeficiency Virus

Abstract

Background: Compared with women who are human immunodeficiency virus (HIV) negative, women with human immunodeficiency virus (WWH) have a higher human papillomavirus (HPV) prevalence and increased cervical cancer risk, emphasizing the need for effective cervical cancer screening in this population. The present study aimed to validate methylation markers ASCL1 and LHX8 for primary screening in a South African cohort of WWH.

Methods: In this post hoc analysis within the DIAgnosis in Vaccine And Cervical Cancer Screen (DiaVACCS) study, a South African observational multicenter cohort study, cervical scrape samples from 411 HIV-positive women were analyzed for hypermethylation of ASCL1 and LHX8 genes, HPV DNA, and cytology. Sensitivities, specificities, and positive and negative predictive values of primary methylation-based, HPV-based and cytology-based screening were calculated for the detection of cervical intraepithelial neoplasia of grade 3 or higher.

Results: Single markers ASCL1 and LHX8 resulted in a good performance for the detection of cervical intraepithelial neoplasia of grade 3 or higher, with sensitivities of 85.9% (95% confidence interval [CI], 78.2%-93.6%) and 89.7% (83.0%-96.5%), respectively, and specificities of 72.9% (67.3%-78.5%) and 75.0% (69.5%-80.5%). Combining markers ASCL1 and LHX8 resulted in a lower sensitivity compared with HPV testing (84.6% vs 93.6%, respectively; ratio, 0.90 [95% CI, .82-.99]) and a higher specificity (86.7% vs 78.3%; ratio 1.11 [1.02-1.20]) and reduced the referral rate from 46.8% to 33.4%. ASCL1/LHX8 methylation had a significantly higher sensitivity than cytology (threshold, high-grade intraepithelial squamous lesion or worse), (84.6% vs 74.0%, respectively; ratio, 1.16 [95% CI, 1.01-1.32]) and similar specificity (86.7% vs 91.0%; ratio, 0.95 [.90-1.003]).

Conclusions: Our results validate the accuracy of ASCL1/LHX8 methylation analysis for primary screening in WWH, which offers a full-molecular alternative to cytology- or HPV-based screening, without the need for additional triage testing.

Keywords: HIV; cervical cancer; cervical cancer screening; cervical intraepithelial neoplasia; host cell DNA methylation analysis.

Figure 1.

Methylation levels of ASCL1 and LHX8 genes increase with severity of disease. Methylation levels are represented by the log₁₀-transformed ΔΔ cycle threshold (Ct) ratios (y-axis) in different histology groups. Abbreviations: Ca, cervical carcinoma; CIN, cervical intraepithelial neoplasia; CIN1, CIN2, and CIN3, CIN grade 1, 2, and 3; NS, not significant. **P < .01; ***P < .001.

Figure 2.

Proportion of hypermethylated ASCL1 and LHX8 genes testing positive in relation to severity of underlying cervical disease. The proportions of samples testing positive for 0, 1, or 2 markers within the different histology subgroups (x-axis) are represented on the y-axis. Abbreviations: Ca, cervical carcinoma; No CIN, CIN1, CIN2, and CIN3, cervical intraepithelial neoplasia, grade 0, 1, 2, and 3.

Figure 3.

Forest plots showing the relative sensitivity and specificity for the detection of cervical intraepithelial neoplasia (CIN) of grade 3 or higher (CIN3+) of different primary screening tests compared with human papillomavirus (HPV) (A) and cytology (threshold, high-grade squamous intraepithelial lesion or worse [≥HSIL]) (B). Abbreviations: CI, confidence interval; ≤CIN1, CIN grade 1 or below.