CADM1 and MAL promoter methylation levels in hrHPV‐positive cervical scrapes increase proportional to degree and duration of underlying cervical disease

Combined detection of cell adhesion molecule 1 (CADM1) and T‐lymphocyte maturation‐associated protein (MAL) promoter methylation in cervical scrapes is a promising triage strategy for high‐risk human papillomavirus (hrHPV)‐positive women. Here, CADM1 and MAL DNA methylation levels were analysed in cervical scrapes of hrHPV‐positive women with no underlying high‐grade disease, high‐grade cervical intraepithelial neoplasia (CIN) and cervical cancer. CADM1 and MAL methylation levels in scrapes were first related to CIN‐grade of the corresponding biopsy and second to CIN‐grade stratified by the presence of ‘normal’ or ‘abnormal’ cytology as present in the accompanying scrape preceding the cervical biopsy. The scrapes included 167 women with ≤CIN1, 54 with CIN2/3 and 44 with carcinoma. In a separate series of hrHPV‐positive scrapes of women with CIN2/3 (n = 48), methylation levels were related to duration of preceding hrHPV infection (PHI; <5 and ≥5 years). Methylation levels were determined by quantitative methylation‐specific PCR and normal cytology scrapes of hrHPV‐positive women with histologically ≤CIN1 served as reference. CADM1 and MAL methylation levels increased proportional to severity of the underlying lesion, showing an increase of 5.3‐ and 6.2‐fold in CIN2/3, respectively, and 143.5‐ and 454.9‐fold in carcinomas, respectively, compared to the reference. Methylation levels were also elevated in CIN2/3 with a longer duration of PHI (i.e. 11.5‐ and 13.6‐fold, respectively). Moreover, per histological category, methylation levels were higher in accompanying scrapes with abnormal cytology than in scrapes with normal cytology. Concluding, CADM1 and MAL promoter methylation levels in hrHPV‐positive cervical scrapes are related to the degree and duration of underlying cervical disease and markedly increased in cervical cancer.     

Combined CADM1 and MAL promoter methylation analysis to detect (pre-)malignant cervical lesions in high-risk HPV-positive women

Given the lower specificity for high-grade cervical lesions of high-risk human papillomavirus (hrHPV) testing compared to cytology, additional triage testing for hrHPV test-positive women is needed to detect high-grade cervical lesions. Here, we tested whether combined methylation analysis for cell adhesion molecule 1 (CADM1) and T-lymphocyte maturation associated protein (MAL), both functionally involved in cervical carcinogenesis, could serve as such a triage marker. Four quantitative methylation-specific PCRs (qMSP), two for CADM1 (regions M12 and M18) and MAL (regions M1 and M2) each, were applied to 261 cervical tissue specimens ranging from no neoplasia to carcinoma. When qMSPs were combined and positivity for at least one of the qMSPs in the combination was taken into account, the highest positivity rates for cervical intraepithelial neoplasia grade 3 (CIN3) lesions (97%) and squamous cell- and adeno-carcinomas (99%) were obtained by combining a single CADM1 marker with a single MAL marker. Subsequent qMSP analysis of 70 GP5+/6+-PCR hrHPV-positive scrapings revealed that a two-marker panel consisting of CADM1-M18 and MAL-M1 was most discriminative, detecting 90% of women with CIN3 (n = 30), whereas it showed a positive result in only 13.5% of women without cervical disease (n = 40). Finally, we applied hrHPV GP5+/6+-PCR testing followed by CADM1-M18/MAL-M1 methylation analysis to a cohort of 79 women visiting the outpatient colposcopy clinic. hrHPV testing revealed a sensitivity of 97% and a specificity of 33% for CIN3+. Additional CADM1-M18/MAL-M1 methylation analysis on the hrHPV-positive women increased the specificity to 78% with a sensitivity of 70%. In conclusion, the methylation marker panel CADM1-M18 and MAL-M1 may serve as an alternative molecular triage tool for hrHPV-positive women.     

Comparing the performance of FAM19A4 methylation analysis,cytology and HPV16/18 genotyping for the detection of cervical (pre)cancer in high‐risk HPV‐positive women of a gynecologic outpatient population (COMETH study)

Recently, DNA methylation analysis of FAM19A4 in cervical scrapes has been shown to adequately detect high‐grade cervical intraepithelial neoplasia and cervical cancer (≥CIN3) in high‐risk HPV (hrHPV)‐positive women. Here, we compared the clinical performance of FAM19A4 methylation analysis to cytology and HPV16/18 genotyping, separately and in combination, for ≥CIN3 detection in hrHPV‐positive women participating in a prospective observational multi‐center cohort study. The study population comprised hrHPV‐positive women aged 18–66 years, visiting a gynecological outpatient clinic. From these women, cervical scrapes and colposcopy‐directed biopsies (for histological confirmation) were obtained. Cervical scrapes were analyzed for FAM19A4 gene promoter methylation, cytology and HPV16/18 genotyping. Methylation analysis was performed by quantitative methylation‐specific PCR (qMSP). Sensitivities and specificities for ≥CIN3 were compared between tests. Stratified analyses were performed for variables that potentially influence marker performance. Of all 508 hrHPV‐positive women, the sensitivities for ≥CIN3 of cytology, FAM19A4 methylation analysis, and cytology combined with HPV16/18 genotyping were 85.6, 75.6 and 92.2%, respectively, with corresponding specificities of 49.8, 71.1 and 29.4%, respectively. Both sensitivity and specificity of FAM19A4 methylation analysis were associated with age (p ≤ 0.001 each). In women ≥30 years (n = 287), ≥CIN3 sensitivity of FAM19A4 methylation analysis was 88.3% (95%CI: 80.2–96.5) which was noninferior to that of cytology [85.5% (95%CI: 76.0–94.0)], at a significantly higher specificity [62.1% (95%CI: 55.8–68.4) compared to 47.6% (95%CI: 41.1–54.1)]. In conclusion, among hrHPV‐positive women from an outpatient population aged ≥30 years, methylation analysis of FAM19A4 is an attractive marker for the identification of women with ≥CIN3.     

Comparison of HPV DNA testing in cervical exfoliated cells and tissue biopsies among HIV‐positive women in Kenya

HIV‐positive women are infected with human papillomavirus (HPV) (especially with multiple types), and develop cervical intraepithelial neoplasia (CIN) and cervical cancer more frequently than HIV‐negative women. We compared HPV DNA prevalence obtained using a GP5+/6+ PCR assay in cervical exfoliated cells to that in biopsies among 468 HIV‐positive women from Nairobi, Kenya. HPV prevalence was higher in cells than biopsies and the difference was greatest in 94 women with a combination normal cytology/normal biopsy (prevalence ratio, PR = 3.7; 95% confidence interval, CI: 2.4–5.7). PR diminished with the increase in lesion severity (PR in 58 women with high‐grade squamous intraepithelial lesions (HSIL)/CIN2–3 = 1.1; 95% CI: 1.0–1.2). When HPV‐positive, cells contained 2.0‐ to 4.6‐fold more multiple infections than biopsies. Complete or partial agreement between cells and biopsies in the detection of individual HPV types was found in 91% of double HPV‐positive pairs. The attribution of CIN2/3 to HPV16 and/or 18 would decrease from 37.6%, when the presence of these types in either cells or biopsies was counted, to 20.2% when it was based on the presence of HPV16 and/or 18 (and no other types) in biopsies. In conclusion, testing HPV on biopsies instead of cells results in decreased detection but not elimination of multiple infections in HIV‐positive women. The proportion of CIN2/3 attributable to HPV16 and/or 18 among HIV‐positive women, which already appeared to be lower than that in HIV‐negative, would then further decrease. The meaning of HPV detection in cells and random biopsy from HIV‐positive women with no cervical abnormalities remains unclear.     

Experience with high-risk human papillomavirus testing on vaginal brush-based self-samples of non-attendees of the cervical screening program

We evaluated the effect of offering brush-based vaginal self-sampling for high-risk human papillomavirus (hrHPV) testing to non-attendees of the cervical screening program on response rate, compliance to follow-up and cervical intraepithelial neoplasia grade 2 or 3 (CIN2+/CIN3+) yield. In addition, concordance of hrHPV test results between physician-taken cervical scrapes and vaginal self-samples was determined. A total of 26,409 nonattending women were randomly assigned to receive a vaginal brush device for hrHPV testing by Hybrid Capture-2 method (i.e., self-sampling group, n = 26,145) or a reinvitation for regular cytology-based screening (i.e., recall control group, n = 264). hrHPV-positive self-sampling responders were invited for a physician-taken scrape for cytology and blinded hrHPV testing. If cytology was abnormal, women were referred for colposcopy. Response rate in the self-sampling group was significantly increased compared to the recall control group (30.8% versus 6.5%; p < 0.001). The concordance rate between hrHPV detection in self-samples and corresponding physician-taken cervical scrape samples was 68.8%. Amongst women with CIN3+ and CIN2+, the concordance rates in hrHPV positivity between both samples were 95.5% and 93.8%, respectively. Adherence at baseline to cytology triage of hrHPV-positive self-sampling women (89.1%) and colposcopy referral of those with abnormal cytology (95.8%) was high. The CIN2+/CIN3+/carcinoma yields were 1.5%, 1.0% and 0.1%, respectively, in self-sampling responders. In conclusion, offering hrHPV testing on self-sampled vaginal material with a brush device to non-attendees significantly increases the attendance to the regular screening program, yields hrHPV test results that are in very good concordance with those of physician-taken scrapes in women with CIN2+/CIN3+, and is effective in detecting CIN2+/CIN3+.     

Impact of HPV testing in opportunistic cervical screening: Support for primary HPV screening in the United States

Human papillomavirus (HPV) testing for cervical screening increases diagnosis of precancer and reduces the incidence of cervical cancer more than cytology alone. However, real-world evidence from diverse practice settings is lacking for the United States (U.S.) to support clinician adoption of primary HPV screening. Using a population-based registry, which captures all cervical cytology (with or without HPV testing) and all cervical biopsies, we conducted a real-world evidence study of screening in women aged 30 to 64 years across the entire state of New Mexico. Negative cytology was used to distinguish cotests from reflex HPV tests. A total of 264 198 cervical screening tests (with exclusions based on clinical history) were recorded as the first screening test between 2014 and 2017. Diagnoses of cervical intraepithelial neoplasia grades 2 or 3 or greater (CIN2+, CIN3+) from 2014 to 2019 were the main outcomes. Of cytology-negative screens, 165 595 (67.1%) were cotests and 4.8% of these led to biopsy within 2 years vs 3.2% in the cytology-only group. Among cytology-negative, HPV tested women, 347 of 398 (87.2%) CIN2+ cases were diagnosed in HPV-positive women, as were 147 of 164 (89.6%) CIN3+ cases. Only 29/921 (3.2%) CIN3+ and 67/1964 (3.4%) CIN2+ cases were diagnosed in HPV-negative, cytology-positive women with biopsies. Under U.S. opportunistic screening, across a diversity of health care delivery practices, and in a population suffering multiple disparities, we show adding HPV testing to cytology substantially increased the yield of CIN2+ and CIN3+. CIN3+ was rarely diagnosed in HPV-negative women with abnormal cytology, supporting U.S. primary HPV-only screening.     

Methylation testing for the detection of recurrent cervical intraepithelial neoplasia

Women treated for CIN2/3 remain at increased risk of recurrent CIN and cervical cancer, and therefore posttreatment surveillance is recommended. This post hoc analysis evaluates the potential of methylation markers ASCL1/LHX8 and FAM19A4/miR124-2 for posttreatment detection of recurrent CIN2/3. Cervical scrapes taken at 6 and 12 months posttreatment of 364 women treated for CIN2/3 were tested for methylation of ASCL1/LHX8 and FAM19A4/miR124-2 using quantitative multiplex methylation-specific PCR. Performance of the methylation tests were calculated and compared with the performance of HPV and/or cytology. Methylation levels of recurrent CIN were compared between women with a persistent HPV infection, and women with an incident HPV infection or without HPV infection. Recurrent CIN2/3 was detected in 42 women (11.5%), including 28 women with CIN2 and 14 with CIN3. ASCL1/LHX8 tested positive in 13/14 (92.9%) of recurrent CIN3 and 13/27 (48.1%) of recurrent CIN2. FAM19A4/miR124-2 tested positive in 14/14 (100%) of recurrent CIN3 and 10/27 (37.0%) of recurrent CIN2. Combined HPV and/or methylation testing showed similar positivity rates as HPV and/or cytology. The CIN2/3 risk at 12 months posttreatment was 30.8% after a positive ASCL1/LHX8 result at 6 months posttreatment. Methylation levels of CIN2/3 in women with a persistent HPV infection were significantly higher compared with women with an incident or no HPV infection. In conclusion, posttreatment monitoring by methylation analysis of ASCL1/LHX8 and FAM19A4/miR124-2 showed a good performance for the detection of recurrent CIN. DNA methylation testing can help to identify women with recurrent CIN that require re-treatment.     

HPV prevalence, viral load and physical state of HPV-16 in cervical smears of patients with different grades of CIN

Human papillomavirus (HPV) infection is the most important event in malignant transformation of human cervical epithelium. We analysed in cervical smears, HPV genotypes with a focus on single/multiple infections, then characteristics of HPV-16 infections (presence of other genotypes, viral load and physical state) according to the grade of histological lesions. The purpose of this study was to know if these parameters could allow to differentiate histological diagnoses. DNA was extracted from 363 cervical samples corresponding to 24 cases without lesion, 96 CIN1, 92 CIN2, 144 CIN3 and 7 cancers. Our results show that HPV-16 was predominant and its prevalence increased with the severity of lesions (CIN1: 27.1%; CIN3: 65.3%). In addition, we showed that the frequency of single infections, as compared with multiple infections, increased with the severity of the lesion (CIN1: 25.0%; CIN3: 54.8%). Among HPV-16 positive samples (n = 170), we found that viral load, determined on cervical samples by real-time PCR, did not vary significantly according to the different CIN grades. Concerning HPV-16 integration, the mixed and integrated HPV-16 forms, already present in women with normal histology, increased to the benefit of pure episomal forms with the severity of lesions (normal cervix: 28.6%; CIN3: 73.8%). Thus, our data raise the question of the viral load as a valuable clinical parameter to discriminate between lesion grades. Moreover, we emphasize integration as an early event in cervical carcinogenesis, increasing with the severity of lesions. Finally, this study underlines the importance of single versus multiple infections linked to the severity of CIN.     

子宫颈癌SOX9基因甲基化早期诊断价值探讨

目的检测宫颈癌和宫颈上皮内瘤变（cervical intraepithelial neoplasia,CIN）脱落细胞中SOX9基因甲基化水平,为子宫颈癌的早期诊断提供新的分子标志。方法收集2010-03-01-2011-06-30就诊于首都医科大学附属北京妇产医院肿瘤科患者的宫颈脱落细胞156例,采用双探针实时定量聚合酶链式反应（polymerase chain reaction,PCR）技术,定量检测正常宫颈组（48例）、CINⅠ组（30例）、CINⅡ-Ⅲ组（30例）和宫颈癌组（48例）的宫颈脱落细胞中SOX9基因甲基化水平,分析SOX9基因甲基化与各级别宫颈病变的关系。结果正常宫颈组SOX9基因甲基化水平（甲基化评分）17.93±4.33,CINⅠ组为10.35±4.06,CINⅡ-Ⅲ组为33.17±15.19,宫颈癌组为48.04±24.03。SOX9基因甲基化水平随宫颈病变级别的增加而升高,且在CINⅡ-Ⅲ组和宫颈癌组中显著升高。CINⅠ组和正常宫颈组脱落细胞甲基化水平差异无统计学意义,P=0.062;SOX9基因甲基化水平在其余任意两组间差异均有统计学意义,P〈0.001。SOX9基因甲基化评分可有效区分宫颈癌/CINⅡ-Ⅲ和CINⅠ/正常宫颈,受试者工作特征曲线下面积（area under the receiver operating characteristic curve,AUC）为0.961（95%CI：0.933-0.990）,P〈0.001。在最佳临界值,SOX9基因甲基化评分检测宫颈癌/CINⅡ-Ⅲ的敏感度为92.3%,特异度为89.7%。结论甲基化SOX9基因能够有效诊断宫颈癌/CINⅡ-Ⅲ,可以作为子宫颈癌早期诊断的分子标志。     

Grading the severity of cervical neoplasia based on combined histopathology,cytopathology, and HPV genotype distribution among 1,700 women referred to colposcopy in Oklahoma

Diagnosis and treatment of cervical cancer precursors rely on colposcopic biopsy, which is sometimes hampered by incorrect biopsy placement and the unclear prognostic value of poorly reproducible diagnoses such as cervical intraepithelial neoplasia (CIN) Grade 1 and 2. Searching for discrete disease categories that incorporate the value of cytology and that reflect the causal role of particular HPV types, we analyzed histology, cytology and HPV genotype distributions in the Study to Understand Cervical Cancer Endpoints and Early Determinants (SUCCEED). This cross‐sectional study comprises ～1,700 women referred to colposcopy or treatment for the spectrum of cervical disease, including 439 women with <CIN1, 429 CIN1, 322 CIN2, 297 CIN3 and 107 with cancer. Using hierarchical clustering of histology‐cytology groups based on HPV genotype distributions, we could plainly distinguish in this referral population 5 increasingly severe diagnostic groups of HPV‐positive women: (i) HPV‐positive women with <CIN2 histology and normal cytology, (ii) HPV positive women with <CIN2 histology and ASC or LSIL cytology; (iii) CIN2, including histologic CIN2 and HSIL cytology with any histology <CIN2; (iv) CIN3; and (v) invasive cervical cancer. The grouping of women with HSIL cytology, but without histological abnormalities to women with CIN2 suggests that in these cases the worst lesion was missed during colposcopy‐biopsy. We are now using these sharpened diagnostic categories to search for novel biomarkers predicting the risk of progression and invasion. © 2008 Wiley‐Liss, Inc.     

Methylation of viral and host genes and severity of cervical lesions associated with human papillomavirus type 16

Methylation of human papillomavirus (HPV) and host genes may predict cervical cancer risk. We examined the methylation status of selected sites in HPV16 and human genes in DNA extracted from exfoliated cervical cell samples of 244 women harboring HPV16‐positive cancer or cervical intraepithelial neoplasia (CIN) or negative for intraepithelial lesions or malignancy (NILM). We quantified the methylation of CpG sites in the HPV16 L1 gene (CpG 6367 and 6389) and in the human genes EPB41L3 (CpG 438, 427, 425) and LMX1 (CpG 260, 262, 266, 274) following bisulfite treatment and pyrosequencing. Receiver operating characteristic (ROC) curves were used to analyze the diagnostic utility of methylation level for the different sites and for a joint predictor score. Methylation in all sites significantly increased with lesion severity (p < 0.0001). Area under the curve (AUC) was highest among the CIN2/3 vs. cancer ranging from 0.786 to 0.853 among the different sites. Site‐specific methylation levels strongly discriminated CIN2/3 from NILM/CIN1 and cancer from CIN2/3 (range of odds ratios [OR]: 3.69–12.76, range of lower 95% confidence bounds: 1.03–4.01). When methylation levels were mutually adjusted for each other EPB41L3 was the only independent predictor of CIN2/3 vs. NILM/CIN1 contrasts (OR = 9.94, 95%CI: 2.46–40.27). High methylation levels of viral and host genes are common among precancerous and cancer lesions and can serve as independent risk biomarkers. Methylation of host genes LMX1 and EPB41L3 and of the viral HPV16 L1 sites has the potential to distinguish among precancerous lesions and to distinguish the latter from invasive disease.     

Methylation marker analysis of self‐sampled cervico‐vaginal lavage specimens to triage high‐risk HPV‐positive women for colposcopy

Methylation markers were studied for their suitability to triage human papillomavirus (HPV)‐positive women by testing self‐collected cervico‐vaginal lavage specimens. For this purpose, we analyzed 355 hrHPV‐positive self‐collected specimens with three methylation markers, that is, CADM1‐m18, MAL‐m1 and miR‐124‐2 by quantitative methylation‐specific PCR. The areas under the receiver‐operating characteristic (ROC) curve for end‐point cervical intraepithelial neoplasia grade 3 or worse (CIN3+) were 0.637 for CADM1‐m18, 0.767 for MAL‐m1 and 0.762 for miR‐124‐2. This indicates that CADM1‐m18 is not suitable as single marker. By varying the thresholds of both markers in the bi‐marker panels CADM1‐m18/MAL‐m1, CADM1‐m18/miR‐124‐2 and MAL‐m1/miR‐124‐2 upper and lower ROC curves were obtained, depicting the maximum and minimum CIN3+ sensitivity, respectively, at given specificity. For all these bi‐marker combinations, the upper curves were similar. However, for the MAL‐m1/miR‐124‐2 panel, the distance between upper and lower ROC curves was closest and this panel displayed the highest assay thresholds, indicating that this combination was most robust. At clinical specificities of 50 and 70%, the MAL‐m1/miR‐124‐2 sensitivity for detection of CIN3+ ranged from 77.0 to 87.8% and from 64.9 to 71.6%, respectively. At 70% specificity thresholds no carcinomas were missed. By comparison, the CIN3+ sensitivity of HPV16/18 genotyping on the self‐sampled lavage specimens was 58.1% (95%CI: 46.6–68.8) at a specificity of 87.7% (95%CI: 83.2–91.2). In conclusion, methylation analysis is a promising triage tool that in combination with HPV‐DNA testing offers feasible, full molecular screening on self‐collected cervico‐vaginal lavage specimens.     

The role of genotype-specific human papillomavirus detection in diagnosing residual cervical intraepithelial neoplasia

We assessed prospectively whether residual cervical intraepithelial neoplasia (CIN) after treatment for high-grade CIN can be predicted by genotype-specific high-risk HPV (HR-HPV) detection in follow-up cervical scrapes. A broad spectrum, highly sensitive SPF(10)-LiPA-PCR HPV detection technique was used on cervical scrapes before large loop excision of the transformation zone (LLETZ), on the LLETZ biopsy and on follow-up scrapes of 90 patients treated for high-grade CIN. HR-HPV was detected in the biopsies of 93% (n = 84) of the patients and in the follow-up scrapes of 48% (n = 43) of the patients. In 12 patients, genotype-specific HR-HPV persistence was detected in both follow-up scrapes. In 10 patients, residual CIN was detected. In 5 of these patients (including all patients with residual CIN 3), the follow-up scrapes showed genotype-specific HR-HPV persistence. In 2 patients, a different HR-HPV was detected, and 3 patients had HR-HPV-negative follow-up scrapes. Conventional cytologic follow-up was abnormal in 13 patients including all 10 patients with residual CIN. The negative predictive value (NPV) of HR-HPV detection on follow-up scrapes was high (94%). Repeat detection of genotype-specific HR-HPV showed a lower sensitivity and NPV than repeat detection of any HR-HPV, but its specificity was higher. Repeat conventional cytologic follow-up showed the highest sensitivity and NPV. In conclusion, the presence of HR-HPV in cervical scrapes after LLETZ for high-grade CIN is a risk factor for the presence of residual CIN. HR-HPV genotype-specific persistence is specifically present in patients with residual CIN 3. However, HR-HPV detection cannot predict or exclude the presence of residual CIN in the individual patient and additional procedures remain necessary.     

Effectiveness, safety and acceptability of 'see and treat' with cryotherapy by nurses in a cervical screening study in India

We evaluated a 'see and treat' procedure involving screening, colposcopy, biopsy and cryotherapy by trained nurses in one-visit in field clinics in a cervical screening study in South India for its acceptability, safety and effectiveness in curing cervical intraepithelial neoplasia (CIN). Women positive on visual inspection with acetic acid (VIA) were advised colposcopy, directed biopsies and cryotherapy if they had colposcopic impression of CIN in one visit by nurses in field clinics supervised by a doctor. Side effects and complications were assessed and cure rates were evaluated with VIA, colposcopy and biopsy if colposcopic abnormalities were suspected. Cure was defined as no clinical or histological evidence of CIN at > or =6 months from treatment. Of the 2513 women offered 'see and treat' procedure, 1879 (74.8%) accepted. Of the 1397 women with histologically proved CIN treated with cryotherapy, 1026 reported for follow-up evaluation. Cure rates were 81.4% (752 out of 924) for women with CIN 1; 71.4% (55 out of 77) for CIN 2 and 68.0% (17 out of 25) for CIN 3. Minor side effects and complications were documented in less than 3% of women. 'See and treat' with cryotherapy by nurses under medical supervision is acceptable, safe and effective for cervical cancer prevention in low-resource settings.     

A higher degree of methylation of the HPV 16 E6 gene is associated with a lower likelihood of being diagnosed with cervical intraepithelial neoplasia

BACKGROUND:

Although HPV 16 is the most common HPV genotype associated with cancerous lesions of the cervix, only a fraction of HPV 16 infected women are diagnosed with precancerous lesions of the cervix. Therefore, molecular changes in HPV 16, rather than infections per se, may serve as better screening or diagnostic biomarkers. The purpose of the study was to evaluate whether methylation status of specific regions of the HPV E6 gene promoter and enhancer is independently associated with the likelihood of being diagnosed with higher grades of cervical intraepithelial neoplasia (CIN 2+).

METHODS:

The study included 75 HPV 16‐positive women diagnosed with CIN 2+ or ≤CIN 1. Pyrosequencing technology was applied to quantify methylation at 6 cytosine guanine dinucleotide (CpG) sites of the HPV 16 E6 promoter and enhancer. CIN 2+ (yes/no) was the dependent variable in logistic regression models that specified the degree of methylation of the CpG sites of the HPV 16 E6 gene as the primary independent predictors. All models were adjusted for demographic, lifestyle, known risk factors for cervical cancer, and circulating concentrations of “cancer‐protective” micronutrients.

RESULTS:

The odds of being diagnosed with CIN 2+ were 79% lower when the degree of methylation of the HPV 16 enhancer and promoter sites was ≥9.5% (OR = 0.21; 95% CI, 0.06‐0.79; P = .02).

CONCLUSIONS:

Results suggested that CpG methylation is independently involved in the biology of HPV 16 as well as in the development of higher grades of CIN. Cancer 2011. © 2010 American Cancer Society.     

HPV16 L1 and L2 DNA methylation predicts high‐grade cervical intraepithelial neoplasia in women with mildly abnormal cervical cytology

DNA methylation changes in human papillomavirus type 16 (HPV16) DNA are common and might be important for identifying women at increased risk of cervical cancer. Using recently published data from Costa Rica we developed a classification score to differentiate women with cervical intraepithelial neoplasia grade 2 or 3 (CIN2/3) from those with no evident high‐grade lesions. Here, we aim to investigate the performance of the score using data from the UK. Exfoliated cervical cells at baseline and 6‐months follow‐up were analyzed in 84 women selected from a randomized clinical trial of women undergoing surveillance for low‐grade cytology. Selection of women for the methylation study was based on detectable HPV16 in the baseline sample. Purified DNA was bisulfite converted, amplified and pyrosequenced at selected CpG sites in the viral genome (URR, E6, L1 and L2), with blinding of laboratory personnel to the clinical data. The primary measure was a predefined score combining the mean methylation in L1 and any methylation in L2. At the second follow‐up visit, 73/84 (87%) women were HPV16 positive and of these 25 had a histopathological diagnosis of CIN2/3. The score was significantly associated with CIN2/3 (area under curve = 0.74, p = 0.002). For a cutoff with 92% sensitivity, colposcopy could have been avoided in 40% (95% CI 27–54%) of HPV16 positive women without CIN2/3; positive predictive value was 44% (32–58%) and negative predictive value was 90% (71–97%). We conclude that quantitative DNA methylation assays could help to improve triage among HPV16 positive women.     

妊娠期生理特点与宫颈癌的关系及围妊娠期宫颈癌筛查的意义

目的探讨妊娠期生理特点与宫颈癌的关系及加强宫颈疾病史妇女围妊娠期宫颈癌筛查的意义。方法选取2018年1月至2020年1月间陕西省西安医学院第二附属医院妇产科门诊建立档案并接受正规产前检查的22141例孕妇作为研究对象,根据是否处于妊娠期进行分组,妊娠期3328例,非妊娠期18813例。按照自愿原则,受检者接受液基细胞学检查(TCT),共计3328例。TCT异常者及发现宫颈病变的孕妇,在产后6周进行TCT复查。对选择同期接受宫颈癌筛查项目的非孕期妇女开展TCT检查结果分析。结果TCT检查结果显示,妊娠期和非妊娠期妇女细胞学阳性比组间比较,差异无统计学意义(P>0.05)。非妊娠组患者高度鳞状上皮内病变(HSIL)高于妊娠组,差异有统计学意义(P<0.05)。两组患者的不典型鳞状细胞(ASC-US)、低度上皮内病变(LSIL)、不典型鳞状细胞(ASC-H)、鳞状细胞癌(SCC)和不典型腺细胞(AGC)比较,差异无统计学意义(P>0.05)。两组患者宫颈组织学结果中,宫颈上皮内瘤样病变(CIN)及宫颈癌发生率、活检率和阳性率比较,差异无统计学意义(P>0.05)。随访134例细胞学异常的妊娠期妇女,细胞学转阴率为68.7%(92/134),阴道镜检查结果显示,炎症或人乳头瘤状病毒(HPV)感染6例,CINⅠ5例、CINⅡ3例,CINⅢ2例,没有浸润癌出现。结论多数妊娠合并宫颈病变患者在产后可自行缓解或无进展,预后效果良好。     

Genome-wide DNA methylation profiling identifies two novel genes in cervical neoplasia

DNA methylation analysis may improve risk stratification in cervical screening. We used a pan-epigenomic approach to identify new methylation markers along the continuum of cervical intraepithelial neoplasia (CIN) to cervical cancer. Physician-collected samples (54 normal, 50 CIN1, 40 CIN2 and 42 CIN3) were randomly selected from women at a single-center colposcopy clinic. Extracted DNA was subjected to Illumina Infinium EPIC array analysis, and methylation was assessed blinded to histopathological and clinical data. CpG sites whose state of methylation correlated with lesion grade were assessed (Spearman correlation), and a weighted methylation score was calculated comparing normal to CIN3. Validation of the top selected genes was performed in an independent cohort (100 normal, 50 CIN1, 50 CIN2, 50 CIN3 and 8 cervical cancers) of new patients, referred for colposcopic examination at three hospitals, using targeted DNA methylation Illumina amplicon sequencing. The relationship between a combined weighted marker score and progression from normal through precancerous lesions and cervical cancer was compared using one-way ANOVA. Our analyses revealed 7,715 CpGs whose methylation level correlated with progression (from normal to CIN1, CIN2 and CIN3), with a significant trend of increased methylation with lesion grade. We shortlisted a bigenic (hyaluronan synthase 1, HAS1 and ATPase phospholipid transporting 10A, ATP10A corresponding to cg03419058 and cg13944175 sites) marker set; r = 0.55, p < 0.0001. Validation of the four most discriminating genes (CA10, DPP10, FMN2 and HAS1) showed a significant correlation between methylation levels and disease progression (p-value < 2.2 × 10⁻¹⁶, adjusted R² = 0.952). Translational research of the identified genes to future clinical applications is warranted.     

Efficacy of thermal ablation for treatment of biopsy-confirmed high-grade cervical precancer among women living with HIV in Kenya

The World Health Organization recommends thermal ablation (TA) as an alternative to cryotherapy within “screen-and-treat” cervical cancer programs in low- and middle-income countries (LMICs), including among women living with HIV (WLWH). Data on TA efficacy among WLWH are limited, however. We conducted a clinical trial to evaluate efficacy of TA for treatment of biopsy-confirmed cervical intraepithelial neoplasia grades 2 and 3 (CIN2/3) among WLWH in Kenya. Nonpregnant HPV-positive WLWH age 25 to 65 years underwent colposcopy-directed biopsy, and same-day treatment with TA, if eligible. Women with biopsy-confirmed CIN2/3 at baseline had colposcopy-directed biopsies at 12 months to determine cure. A total of 376 participants underwent TA during the study period. At baseline, 238 (63.3%) had normal histology, 39 (10.4%) had CIN1, 15 (4.0%) had CIN2, 55 (14.6%) had CIN3, 7 (1.9%) had microinvasive cancer and 22 (5.6%) had indeterminate results. Twelve-month follow-up pathology results are available for 59 of 70 (84.3%) participants with CIN2/3 at baseline. Of these, 39 (66.1%, 95% CI 0.54-0.99) had successful treatment, defined as biopsy-confirmed CIN1 or normal findings, while 20 (33.9%, 95% CI 0.22-0.46) had treatment failure, defined as persistent biopsy-confirmed CIN2 or worse. Treatment failure was 23.1% (95% CI 0.17-0.46) and 39.9% (95% CI 0.23-0.51) among women with CIN2 and CIN3 at baseline, respectively. HIV-positive women with CIN2/3 have high rates of treatment failure at 1-year following thermal ablation. This highlights a significant limitation in the current WHO cervical cancer secondary-prevention strategy and calls for strategies to optimize cervical precancer treatment in this population.     

Prevalence of human papillomavirus and cervical intraepithelial neoplasia in China: A pooled analysis of 17 population-based studies

High-risk (HR) human papillomavirus (HPV) prevalence has been shown to correlate well with cervical cancer incidence rates. Our study aimed to estimate the prevalence of HR-HPV and cervical intraepithelial neoplasia (CIN) in China and indirectly informs on the cervical cancer burden in the country. A total of 30,207 women from 17 population-based studies throughout China were included. All women received HPV DNA testing (HC2, Qiagen, Gaithersburg, MD), visual inspection with acetic acid and liquid-based cytology. Women positive for any test received colposcopy-directed or four-quadrant biopsies. A total of 29,579 women had HR-HPV testing results, of whom 28,761 had biopsy confirmed (9,019, 31.4%) or assumed (19,742, 68.6%) final diagnosis. Overall crude HR-HPV prevalence was 17.7%. HR-HPV prevalence was similar in rural and urban areas but showed dips in different age groups: at age 25–29 (11.3%) in rural and at age 35–39 (11.3%) in urban women. In rural and urban women, age-standardized CIN2 prevalence was 1.5% [95% confidence interval (CI): 1.4–1.6%] and 0.7% (95% CI: 0.7–0.8%) and CIN3+ prevalence was 1.2% (95% CI: 1.2–1.3%) and 0.6% (95% CI: 0.5–0.7%), respectively. Prevalence of CIN3+ as a percentage of either all women or HR-HPV-positive women steadily increased with age, peaking in 45- to 49-year-old women. High prevalence of HR-HPV and CIN3+ was detected in both rural and urban China. The steady rise of CIN3+ up to the age group of 45–49 is attributable to lack of lesion removal through screening. Our findings document the inadequacy of current screening in China while indirectly raising the possibility that the cervical cancer burden in China is underreported.