Belinda Nedjai

A longitudinal pilot study in pre-menopausal women links cervicovaginal microbiome to CIN3 progression and recovery

Abstract Increasing evidence suggests vaginal dysbiosis is associated with persistent high-risk human papillomavirus (hrHPV) infection and cervical intraepithelial neoplasia (CIN) development. In this pilot longitudinal study, we investigate the potential of vaginal microbiome biomarkers to predict CIN3 development in hrHPV-positive (hrHPV+) women of reproductive age and assess loop electrosurgical excision procedure (LEEP) outcomes. Fifty-nine non-menopausal women 20–53 years old, with normal cytology, were selected from the ARTISTIC trial and followed up twice over six years. Vaginal microbiome was analysed by 16S rRNA sequencing. HrHPV+ women with CIN3 showed a significant overrepresentation of Sneathia amnii, Megasphaera genomosp., Peptostreptococcus anaerobius and Achromobacter spanius (p < 0.05). Successfully LEEP-treated hrHPV-negative women exhibited increased Lactobacillus species, especially Lactobacillus gasseri. Additionally, Lactobacillus helveticus, suntoryeus and vaginalis showed a potential protective role against CIN3 development. These unique microbial biomarkers associated with CIN3 development and recovery following LEEP treatment bring new insights into the vaginal microbiome’s role on disease progression.

Long‐term prediction by DNA methylation of high‐grade cervical intraepithelial neoplasia: Results of the ARTISTIC cohort

AbstractMethylation markers have shown potential for triaging high‐risk HPV‐positive (hrHPV+) women to identify those at increased risk of invasive cervical cancer (ICC). Our aim was to assess the performance of the S5 DNA methylation classifier for predicting incident high‐grade cervical intraepithelial neoplasia (CIN) and ICC among hrHPV+ women in the ARTISTIC screening trial cohort. The S5 classifier, comprising target regions of tumour suppressor gene EPB41L3 and L1 and L2 regions of HPV16, HPV18, HPV31, and HPV33, was assayed by pyrosequencing in archived hrHPV+ liquid‐based samples from 343 women with high‐grade disease (139 CIN2, 186 CIN3, and 18 ICC) compared to 800 hrHPV+ controls. S5 DNA methylation correlated directly with increasing severity of disease and inversely with lead time to diagnosis. S5 could discriminate between hrHPV+ women who developed CIN3 or ICC and hrHPV+ controls (p <.0001) using samples taken on average 5 years before diagnosis. This relationship was independent of cytology at baseline. The S5 test showed much higher sensitivity than HPV16/18 genotyping for identifying prevalent CIN3 (93% vs. 61%, p = .01) but lower specificity (50% vs. 66%, p <.0001). The S5 classifier identified most women at high risk of developing precancer and missed very few prevalent advanced lesions thus appearing to be an objective test for triage of hrHPV+ women. The combination of methylation of host and HPV genes enables S5 to combine the predictive power of methylation with HPV genotyping to identify hrHPV‐positive women who are at highest risk of developing CIN3 and ICC in the future.

Clinical performance of methylation as a biomarker for cervical carcinoma in situ and cancer diagnosis: A worldwide study

AbstractThe shift towards primary human papillomavirus (HPV)‐based screening has necessitated the search for a secondary triage test that provides sufficient sensitivity to detect high‐grade cervical intraepithelial neoplasia (CIN) and cancer, but also brings an improved specificity to avoid unnecessary clinical work and colposcopy referrals. We evaluated the performance of the previously described DNA‐methylation test (S5) in detecting CIN3 and cancers from diverse geographic settings in high‐, medium‐ and low‐income countries, using the cut‐off of 0.80 and exploratory cut‐offs of 2.62 and 3.70. Assays were performed using exfoliated cervical specimens (n = 808) and formalin‐fixed biopsies (n = 166) from women diagnosed with cytology‐negative results (n = 220), CIN3 (n = 204) and cancer stages I (n = 245), II (n = 249), III (n = 28) and IV (n = 22). Methylation increased proportionally with disease severity (Cuzick test for trend, P < .0001). S5 accurately separated women with negative‐histology from CIN3 or cancer (P < .0001). At the 0.80 cut‐off, 543/544 cancers were correctly identified as S5 positive (99.81%). At cut‐off 3.70, S5 showed a sensitivity of 95.77% with improved specificity. The S5 odds ratios of women negative for cervical disease vs CIN3+ were significantly higher than for HPV16/18 genotyping at all cut‐offs (all P < .0001). At S5 cut‐off 0.80, 96.15% of consistently high‐risk human papillomavirus (hrHPV)‐negative cancers (tested with multiple hrHPV‐genotyping assay) were positive by S5. These cancers may have been missed in current primary hrHPV‐screening programmes. The S5 test can accurately detect CIN3 and malignancy irrespective of geographic context and setting. The test can be used as a screening and triage tool. Adjustment of the S5 cut‐off can be performed considering the relative importance given to sensitivity vs specificity.

Consistency of the S5 DNA methylation classifier in formalin‐fixed biopsies versus corresponding exfoliated cells for the detection of pre‐cancerous cervical lesions

AbstractMethylation biomarkers are promising tools for diagnosis and disease prevention. The S5 classifier is aimed at the prevention of cervical cancer by the early detection of cervical intraepithelial neoplasia (CIN). S5 is based on pyrosequencing a promoter region of EPB41L3 and five late regions of HPV types 16, 18, 31, and 33 following bisulfite conversion of DNA. Good biomarkers should perform well in a variety of sample types such as exfoliated cells, fresh frozen or formalin‐fixed paraffin‐embedded (FFPE) materials. Here, we tested the performance of S5 on 315 FFPE biopsies with paired exfoliated cervical samples using four different conversion kits (Epitect Bisulfite, Epitect Fast Bisulfite, EZ DNA Methylation, and EZ DNA Methylation‐Lightning). The S5 values from FFPE biopsies for all kits were significantly correlated with those obtained from their paired exfoliated cells. For the EZ DNA Methylation kit, we observed an average increased methylation of 4.4% in FFPE. This was due to incomplete conversion of DNA (73% for FFPE vs. 95% for cells). The other kits had a DNA conversion rate in FFPE similar to the cells (95%–97%). S5 performed well at discriminating <CIN2 lesions from CIN2+ lesions on the FFPE with all kits given optimized adjustments to the cut‐off. The area under the curve (AUC) for S5 on FFPE was not significantly different from the paired cells (0.74–0.79 vs. 0.81). The best sensitivity and specificity were obtained for EZ DNA Methylation after the adjustment of the cut‐off to reflect its lower conversion rate. Consistent methylation results can be obtained from FFPE material regardless of the conversion kit used. The S5 classifier performed as well on FFPE material as on exfoliated cells with adjusted cut‐off allowing easier clinical implementation.

A Randomized Comparison of Different Vaginal Self-sampling Devices and Urine for Human Papillomavirus Testing—Predictors 5.1

Abstract Background: Human papillomavirus (HPV)-based screening is rapidly replacing cytology as the cervical screening modality of choice. In addition to being more sensitive than cytology, it can be done on self-collected vaginal or urine samples. This study will compare the high-risk HPV positivity rates and sensitivity of self-collected vaginal samples using four different collection devices and a urine sample. Methods: A total of 620 women referred for colposcopy were invited to provide an initial stream urine sample collected with the Colli-Pee device and take two vaginal self-samples, using either a dry flocked swab (DF) and a wet dacron swab (WD), or a HerSwab (HS) and Qvintip (QT) device. HPV testing was performed by the BD Onclarity HPV Assay. Results: A total of 600 vaginal sample pairs were suitable for analysis, and 505 were accompanied by a urine sample. Similar positivity rates and sensitivities for CIN2+ and CIN3+ were seen for DF, WD, and urine, but lower values were seen for QT and HS. No clear user preferences were seen between devices, but women found urine easiest to collect, and were more confident they had taken the sample correctly. The lowest confidence in collection was reported for HS. Conclusions: Urine, a DF swab, and WD swab all performed well and were well received by the women, whereas the Qvintip and HerSwab devices were less satisfactory. Impact: This is the first study to compare five self-sampling methods in the same women taken at the same time. It supports wider use of urine or vaginal self-sampling for cervical screening.

DNA methylation testing with S5 for triage of high‐risk HPV positive women

AbstractMethylation of host and viral genes is promising for triage of women with high‐risk human papillomavirus infections (hrHPV). Using a population‐based sample of hrHPV positive women with cervical biopsies within 12 months after cervical screening, the clinical value of the S5 methylation classifier (S5), HPV genotyping and cytology were compared as potential triage tests, for outcomes of cervical intraepithelial neoplasia (CIN) grade 3 or greater (CIN3+), CIN2+ and CIN2, and the area under the curve (AUC) calculated. S5 scores increased with histopathology severity (Ptrend < .001). For CIN3+, the AUC was 0.780 suggesting S5 provides good discrimination between <CIN3 and CIN3+. AUCs were significant for all pairwise comparisons of <CIN2, CIN2 and CIN3+ (P < .001). The positive predictive value (PPV) of HPV16/18 genotyping for women with any abnormal cytology was greater than S5 (25.36% vs 20.87%, P = .005) for CIN3+, while sensitivity was substantially greater for S5 (83.33% vs 59.28%, P < .001). Restricting to women with abnormal cytology, but excluding those with high‐grade cytology, both S5 and HPV16/18 provided CIN3+ PPVs high enough to recommend colposcopy. Triage with S5 also appeared useful for hrHPV positive women negative for HPV16/18 (CIN3+ PPV: 7.33%, sensitivity: 57.52%). S5 provided increased sensitivity for CIN3+ compared to HPV16/18 genotyping for hrHPV positive women, overall and when restricted to women with abnormal cytology, suggesting S5 may improve colposcopy referral. S5 also has the ability to distinguish between <CIN2, CIN2 and CIN3+, a finding of importance for managing CIN2, given the complexity and uncertainty associated with this diagnosis.