Characterization of cervical biopsies of women with HIV and HPV co-infection using p16ink4a, ki-67 and HPV E4 immunohistochemistry and DNA methylation.

This study aims to characterize cervical intraepithelial neoplasia (CIN) in women living with HIV using biomarkers. Immunohistochemical (IHC) staining for human papillomavirus (HPV) E4 protein indicates CIN with productive HPV infection, whereas Ki-67 and p16ink4a indicate CIN with transforming characteristics, which may be further characterized using DNA hypermethylation, indicative for advanced transforming CIN. Cervical biopsies (n = 175) from 102 HPV positive women living with HIV were independently reviewed by three expert pathologists. The consensus CIN grade was used as reference standard. IHC staining patterns were scored for Ki-67 (0-3), p16ink4a (0-3), and E4 (0-2) and correlated to methylation levels of four cellular genes in corresponding cervical scrapes. Reference standards and immunoscores were obtained from 165 biopsies:15 no dysplasia, 91 CIN1, 31 CIN2, and 28 CIN3. Ki-67 and p16ink4a scores increased with increasing CIN grade, while E4 positivity was highest in CIN1 and CIN2 lesions. E4 positive CIN1 lesions had higher Ki-67 and p16ink4a scores and higher methylation levels compared with E4 negative CIN1 lesions. E4 positive biopsies with low cumulative Ki-67/p16 ink4a immunoscores (0-3) had significantly higher methylation levels compared with E4 negative biopsies. No significant differences in Ki-67 and p16ink4a scores and methylation levels were observed between E4 negative and positive CIN2 or CIN3 lesions. The presence of high methylation levels in scrapes of CIN lesions with IHC characteristics of both productive (E4 positive) and transforming infections (increased Ki-67/p16ink4a expression) in women living with HIV might indicate a rapid aggressive course of HPV infections towards cancer in these women.

HPV16-Related Cervical Cancers and Precancers Have Increased Levels of Host Cell DNA Methylation in Women Living with HIV.

Data on human papillomavirus (HPV) type-specific cervical cancer risk in women living with human immunodeficiency virus (WLHIV) are needed to understand HPV⁻HIV interaction and to inform prevention programs for this population. We assessed high-risk HPV type-specific prevalence in cervical samples from 463 WLHIV from South Africa with different underlying, histologically confirmed stages of cervical disease. Secondly, we investigated DNA hypermethylation of host cell genes ASCL1, LHX8, and ST6GALNAC5, as markers of advanced cervical disease, in relation to type-specific HPV infection. Overall, HPV prevalence was 56% and positivity increased with severity of cervical disease: from 28.0% in cervical intraepithelial neoplasia (CIN) grade 1 or less (≤CIN1) to 100% in invasive cervical cancer (ICC). HPV16 was the most prevalent type, accounting for 9.9% of HPV-positive ≤CIN1, 14.3% of CIN2, 31.7% of CIN3, and 45.5% of ICC. HPV16 was significantly more associated with ICC and CIN3 than with ≤CIN1 (adjusted for age, ORMH 7.36 (95% CI 2.33⁻23.21) and 4.37 (95% CI 1.81⁻10.58), respectively), as opposed to non-16 high-risk HPV types. Methylation levels of ASCL1, LHX8, and ST6GALNAC5 in cervical scrapes of women with CIN3 or worse (CIN3+) associated with HPV16 were significantly higher compared with methylation levels in cervical scrapes of women with CIN3+ associated with non-16 high-risk HPV types (p-values 0.017, 0.019, and 0.026, respectively). When CIN3 and ICC were analysed separately, the same trend was observed, but the differences were not significant. Our results confirm the key role that HPV16 plays in uterine cervix carcinogenesis, and suggest that the evaluation of host cell gene methylation levels may monitor the progression of cervical neoplasms also in WLHIV.

Posttreatment monitoring by ASCL1/LHX8 methylation analysis in women with HIV treated for cervical intraepithelial neoplasia grade 2/3.

OBJECTIVE: Women with HIV (WWH) have an increased risk to develop recurrent cervical intraepithelial neoplasia grade 2/3 (rCIN2/3) after treatment compared with HIV-negative women. Therefore, appropriate posttreatment monitoring of WWH is important. This study evaluates the performance of ASCL1 and LHX8 methylation analysis as posttreatment monitoring test in WWH treated for CIN2/3, as alternative to cytology or human papillomavirus (HPV) as follow-up test. DESIGN: Prospective observational cohort study. METHODS: WWH treated for CIN2/3 by large loop excision of the transformation zone (LLETZ) (n  = 61) were invited for follow-up study visits at 1, 2.5 and 4 years after baseline. Baseline and follow-up cervical scrapes were tested for cytology, HPV and DNA methylation of ASCL1 and LHX8 genes. The performance of these strategies for the detection of rCIN2/3 was evaluated in the first follow-up cervical scrape. RESULTS: Thirteen (21.3%) rCIN2/3 lesions were detected within 4 years of follow-up. In women without rCIN2/3 in follow-up, methylation levels of ASCL1 and LHX8 decreased significantly after LLETZ treatment (P  = 0.02 and 0.007, respectively). In women with rCIN2/3, methylation levels remained high after LLETZ treatment. The 4-year rCIN2/3 risk was 4.9% (95% CI: 0.6-16.5) for ASCL1/LHX8-negative women, 8.1% (95% CI: 1.7-21.9) for HPV-negative women and 7.7% (95% CI: 2.1-18.5) for cytology-negative women. CONCLUSION: A negative ASCL1/LHX8 methylation test in follow-up is associated with a low rCIN2/3 risk and could serve as an objective test of cure and well tolerated alternative for HPV and/or cytology screening in the posttreatment monitoring of WWH.

The use of molecular markers for cervical screening of women living with HIV in South Africa.

OBJECTIVE: To determine the performance of molecular screening strategies for detection of cervical intraepithelial neoplasia grade 3 or worse (CIN3+) in comparison with cytology screening in women living with HIV. DESIGN: Post-hoc analysis using data from a South African study cohort. METHODS: Cytology and human papillomavirus (HPV)-based strategies were evaluated, including single test and FAM19A4/miR124-2 methylation triage strategies. Participants underwent cytology screening and a colposcopy-directed biopsy. Valid results on cytology, HPV status, 16/18 genotyping and histology were available for 318 women. Detection of HPV and FAM19A4/miR124-2 hypermethylation was performed on DNA from cervical scrapes. Histological diagnosis of CIN3+ was used as outcome. RESULTS: Cytology provided highest specificity (91.6%), but lowest sensitivity (59.3%), whereas a single HPV test provided highest sensitivity (83.1%), but lowest specificity (66.4%). Combining cytology with methylation did not improve the performance compared with cytology alone: a slight increase in sensitivity was seen, at the cost of a decrease in specificity. Triage of high-risk HPV positive women with methylation increased specificity (76.1%) compared with a single HPV or cytology test, while maintaining acceptable sensitivity (72.9%). Similar performance was observed for HPV16/18 with methylation triage (sensitivity 79.7%, specificity 74.8%). The number of women needed to refer to detect one CIN3+ ranged from 1.5 (cytology) to 2.6 (single HPV test). CONCLUSION: Molecular screening strategies using HPV, with or without HPV16/18 genotyping, and FAM19A4/miR124-2 methylation have higher sensitivity with an acceptable loss in specificity compared with current cytology screening and are efficient for the detection of CIN3+ in South African women living with HIV.

Detection of hypermethylated genes as markers for cervical screening in women living with HIV.

INTRODUCTION: To evaluate the performance of hypermethylation analysis of ASCL1, LHX8 and ST6GALNAC5 in physician-taken cervical scrapes for detection of cervical cancer and cervical intraepithelial neoplasia (CIN) grade 3 in women living with HIV (WLHIV) in South Africa. METHODS: Samples from a prospective observational cohort study were used for these analyses. Two cohorts were included: a cohort of WLHIV who were invited for cervical screening (n = 321) and a gynaecologic outpatient cohort of women referred for evaluation of abnormal cytology or biopsy proven cervical cancer (n = 108, 60% HIV seropositive). Cervical scrapes collected from all subjects were analysed for hypermethylation of ASCL1, LHX8 and ST6GALNAC5 by multiplex quantitative methylation specific PCR (qMSP). Histology endpoints were available for all study subjects. RESULTS: Hypermethylation levels of ASCL1, LHX8 and ST6GALNAC5 increased with severity of cervical disease. The performance for detection of CIN3 or worse (CIN3+ ) as assessed by the area under the receiver operating characteristic (ROC) curves (AUC) was good for ASCL1 and LHX8 (AUC 0.79 and 0.81 respectively), and moderate for ST6GALNAC5 (AUC 0.71). At a threshold corresponding to 75% specificity, CIN3+ sensitivity was 72.1% for ASCL1 and 73.8% for LHX8 and all samples from women with cervical cancer scored positive for these two markers. CONCLUSIONS: Hypermethylation analysis of ASCL1 or LHX8 in cervical scrape material of WLHIV detects all cervical carcinomas with an acceptable sensitivity and good specificity for CIN3+ , warranting further exploration of these methylation markers as a stand-alone test for cervical screening in low-resource settings.

Three-tiered score for Ki-67 and p16ink4a improves accuracy and reproducibility of grading CIN lesions.

AIMS: To investigate the accuracy and reproducibility of a scoring system for cervical intraepithelial neoplasia (CIN1-3) based on immunohistochemical (IHC) biomarkers Ki-67 and p16ink4a. METHODS: 115 cervical tissue specimens were reviewed by three expert gynaecopathologists and graded according to three strategies: (1) CIN grade based on H&E staining only; (2) immunoscore based on the cumulative score of Ki-67 and p16ink4a only (0-6); and (3) CIN grade based on H&E supported by non-objectified IHC 2 weeks after scoring 1 and 2. The majority consensus diagnosis of the CIN grade based on H&E supported by IHC was used as the Reference Standard. The proportion of test positives (accuracy) and the absolute agreements across pathologists (reproducibility) of the three grading strategies within each Reference Standard category were calculated. RESULTS: We found that immunoscoring with positivity definition 6 yielded the highest proportion of test positives for Reference Standard CIN3 (95.5%), in combination with the lowest proportion of test positives in samples with CIN1 (1.8%). The proportion of test positives for CIN3 was significantly lower for sole H&E staining (81.8%) or combined H&E and IHC grading (84.8%) with positivity definition ≥CIN3. Immunoscore 6 also yielded high absolute agreements for CIN3 and CIN1, but the absolute agreement was low for CIN2. CONCLUSIONS: The higher accuracy and reproducibility of the immunoscore opens the possibility of a more standardised and reproducible definition of CIN grade than conventional pathology practice, allowing a more accurate comparison of CIN-based management strategies and evaluation of new biomarkers to improve the understanding of progression of precancer from human papillomavirus infection to cancer.

Selection of women at risk for cervical cancer in an HIV-infected South African population.

OBJECTIVE: Cervical cancer is the leading cause of cancer-related death in women in South Africa. This study evaluates DNA methylation levels in cervical (pre)cancer and aims to assess the value of high-risk human papillomavirus (hrHPV) testing and methylation analysis, alone or in combination, on physician-taken cervical scrapes to detect cervical cancer, and cervical intraepithelial neoplasia grade 3 (CIN3) in an HIV-infected South African population. DESIGN: Prospective observational multicentre cohort study. METHODS: Women from a cohort of women living with HIV (n = 355) and a referral cohort (n = 109, 60% HIV seropositive) were included. Cervical scrapes were collected for hrHPV testing and methylation analysis of cell adhesion molecule 1, T-lymphocyte maturation-associated protein, and microRNA124-2 genes. Histologic endpoints were available for all participants. Performance for detection of CIN3 or worse (CIN3+) was determined in the cohort of women living with HIV and different testing strategies were compared. RESULTS: HrHPV and methylation positivity rates increased with severity of cervical disease in the two study cohorts, each reaching 100% in samples of women with carcinoma. HrHPV testing showed a sensitivity for CIN3+ of 83.6%, at a specificity of 67.7%. Methylation analysis showed a comparable CIN3+ sensitivity of 85.2%, but a significantly lower specificity of 49.6%. HrHPV testing with reflex methylation analysis showed a CIN3+ sensitivity of 73.8%, at a specificity of 81.5%. CONCLUSION: In this HIV-infected South African population, stratifying hrHPV-positive women with reflex methylation analysis detects all cervical carcinomas and yields an acceptable sensitivity and specificity for CIN3+.

Safety of extending screening intervals beyond five years in cervical screening programmes with testing for high risk human papillomavirus: 14 year follow-up of population based randomised cohort in the Netherlands.

OBJECTIVES:  To provide an early risk assessment of extending screening intervals beyond five years for a human papillomavirus (HPV) based cervical screening programme in the Netherlands. DESIGN:  14 year follow-up of a population based randomised cohort from the POBASCAM randomised trial. SETTING:  Organised cervical screening in the Netherlands, based on a programme of three screening rounds (each round done every five years). PARTICIPANTS:  43 339 women aged 29-61 years with a negative HPV and/or negative cytology test participating in the POBASCAM trial. INTERVENTIONS:  Women randomly assigned to HPV and cytology co-testing (intervention) or cytology testing only (control), and managed accordingly. MAIN OUTCOME MEASURES:  Cumulative incidence of cervical cancer and cervical intraepithelial neoplasia (CIN) grade 3 or worse (CIN3+). Associations with age were expressed as incidence rate ratios. In HPV positive women, reductions in CIN3+ incidence after negative cytology, HPV type 16/18 genotyping, and/or repeat cytology were estimated. RESULTS:  The cumulative incidence of cervical cancer (0.09%) and CIN3+ (0.56%) among HPV negative women in the intervention group after three rounds of screening were similar to the cumulative among women with negative cytology in the control group after two rounds (0.09% and 0.69%, respectively). Cervical cancer and CIN3+ risk ratios were 0.97 (95% confidence interval 0.41 to 2.31, P=0.95) and 0.82 (0.62 to 1.09, P=0.17), respectively. CIN3+ incidence was 72.2% (95% confidence interval 61.6% to 79.9%, P<0.001) lower among HPV negative women aged at least 40 years than among younger women. No significant association between cervical cancer incidence and age could be demonstrated. CIN3+ incidence among HPV positive women with negative cytology, HPV 16/18 genotyping, and/or repeat cytology was 10.4 (95% confidence interval 5.9 to 18.4) times higher than among HPV negative women. CONCLUSIONS:  Long term incidences of cervical cancer and CIN3+ were low among HPV negative women in this study cohort, and supports an extension of the cervical screening interval beyond five years for women aged 40 years and older. HPV positive women with subsequent negative cytology, HPV16/18 genotyping, and/or repeat cytology have at least a fivefold higher risk of CIN3+ than HPV negative women, indicating that HPV based programmes with long intervals (>five years) should be implemented with risk stratification.Trial registration POBASCAM trial number ISRCTN20781131.

CADM1, MAL and miR124-2 methylation analysis in cervical scrapes to detect cervical and endometrial cancer.

AIMS: Gene promoter hypermethylation is recognised as an essential early step in carcinogenesis, indicating important application areas for DNA methylation analysis in early cancer detection. The current study was set out to assess the performance of CADM1, MAL and miR124-2 methylation analysis in cervical scrapes for detection of cervical and endometrial cancer. METHODS: A series of cervical scrapes of women with cervical (n=79) or endometrial (n=21) cancer, cervical intraepithelial neoplasia grade 3 (CIN3) (n=16) or CIN2 (n=32), and women without evidence of CIN2 or worse (n=120) were assessed for methylation of CADM1, MAL and miR124-2. Methylation analysis was done by the PreCursor-M assay, a multiplex quantitative methylation-specific PCR. RESULTS: All samples of women with cervical cancer (79/79, 100%), independent of the histotype, and 76% (16/21; 95% CI 58.0% to 94.4%) of women with endometrial cancer scored positive for DNA methylation for at least one of the three genes. In women without cancer, methylation frequencies increased significantly with severity of disease from 19.2% (23/120; 95% CI 12.1% to 26.2%) in women without CIN2 or worse to 37.5% (12/32; 95% CI 20.7% to 54.3%) and 68.8% (11/16; 95% CI 46.0% to 91.5%) in women with CIN2 and CIN3, respectively. Overall methylation positivity and the number of methylated genes increased proportionally to the lesion severity. CONCLUSIONS: DNA methylation analysis of CADM1, MAL and miR124-2 in cervical scrapes consistently detects cervical cancer and the majority of CIN3 lesions, and has the capacity to broaden its use on cervical scrapes through the detection of a substantial subset of endometrial carcinomas.