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.     

DNA methylation markers as a triage test for identification of cervical lesions in a high risk human papillomavirus positive screening cohort

Objective triage strategies are required to prevent unnecessary referrals for colposcopy in population-based screening programs using primary high-risk human papillomavirus (hrHPV) testing. We have identified several DNA methylation markers with high sensitivity and specificity for detection of high-grade cervical intraepithelial neoplasia or worse (CIN2+) in women referred for colposcopy. Our study assessed diagnostic potential of these methylation markers in a hrHPV-positive screening cohort. All six markers (JAM3, EPB41L3, C13orf18, ANKRD18CP, ZSCAN1 and SOX1) showed similar association across histology in the hrHPV-positive cohort when compared to the Dutch cohort (each p > 0.15). Sensitivity for CIN2+ was higher using methylation panel C13orf18/EPB41L3/JAM3 compared to the other 2 panels (80% vs. 60% (ANKRD18CP/C13orf18/JAM3) and 63% (SOX1/ZSCAN1), p = 0.01). For CIN3+ all three methylation panels showed comparable sensitivity ranging from 68% (13/19) to 95% (18/19). Specificity of SOX1/ZSCAN1 panel (84%, 167/200) was considerably higher compared to ANKRD18CP/C13orf18/JAM3 (68%, 136/200, p = 2 × 10⁻⁵) and C13orf18/EPB41L3/JAM3 (66%, 132/200, p = 2 × 10⁻⁷). High negative predictive value (NPV) (91–95% and 96–99%) was observed for CIN2+ and CIN3+, for all three methylation panels, while positive predictive value (PPV) varied from 25 to 40% for CIN2+ and 15–27% for CIN3+. Interestingly, 118/235 samples were negative for all six markers (including 106 controls (89.8%), 6 CIN1 (5.1%), 5 CIN2 (4.2%) and 1 CIN3 (0.8%)). Methylation results from both independent cohorts were comparable as well as high sensitivity for detection of cervical cancer and its high-grade precursors in hrHPV-positive population. Our study therefore validates these methylation marker panels as triage test either in hrHPV-based or abnormal cytology-based screening programs.     

A four-gene methylation marker panel as triage test in high-risk human papillomavirus positive patients

Cervical neoplasia‐specific biomarkers, e.g. DNA methylation markers, with high sensitivity and specificity are urgently needed to improve current population‐based screening on (pre)malignant cervical neoplasia. We aimed to identify new cervical neoplasia‐specific DNA methylation markers and to design and validate a methylation marker panel for triage of high‐risk human papillomavirus (hr‐HPV) positive patients. First, high‐throughput quantitative methylation‐specific PCRs (QMSP) on a novel OpenArray? platform, representing 424 primers of 213 cancer specific methylated genes, were performed on frozen tissue samples from 84 cervical cancer patients and 106 normal cervices. Second, the top 20 discriminating methylation markers were validated by LightCycler® MSP on frozen tissue from 27 cervical cancer patients and 20 normal cervices and ROCs and test characteristics were assessed. Three new methylation markers were identified (JAM3, EPB41L3 and TERT), which were subsequently combined with C13ORF18 in our four‐gene methylation panel. In a third step, our methylation panel detected in cervical scrapings 94% (70/74) of cervical cancers, while in a fourth step 82% (32/39) cervical intraepithelial neoplasia grade 3 or higher (CIN3+) and 65% (44/68) CIN2+ were detected, with 21% positive cases for ≤CIN1 (16/75). Finally, hypothetical scenario analysis showed that primary hr‐HPV testing combined with our four‐gene methylation panel as a triage test resulted in a higher identification of CIN3 and cervical cancers and a higher percentage of correct referrals compared to hr‐HPV testing in combination with conventional cytology. In conclusion, our four‐gene methylation panel might provide an alternative triage test after primary hr‐HPV testing.     

Evaluation of a validated methylation triage signature for human papillomavirus positive women in the HPV FOCAL cervical cancer screening trial

Human papillomavirus (HPV)-based cervical cancer screening requires triage of HPV positive women to identify those at risk of cervical intraepithelial neoplasia grade 2 (CIN2) or worse. We conducted a blinded case–control study within the HPV FOCAL randomized cervical cancer screening trial of women aged 25–65 to examine whether baseline methylation testing using the S5 classifier provided triage performance similar to an algorithm relying on cytology and HPV genotyping. Groups were randomly selected from women with known HPV/cytology results and pathology outcomes. Group 1: 104 HPV positive (HPV+), abnormal cytology (54 CIN2/3; 50 <CIN2); Group 2: 103 HPV+, normal cytology with HPV persistence at 12 mo. (53 CIN2/3; 50 <CIN2); Group 3: 50 HPV+, normal cytology with HPV clearance at 12 mo. (assumed <CIN2), total n=257. For the combined groups, S5 risk score CIN2/3 relative sensitivity, specificity and positive predictive value (PPV) were compared with other triage approaches. Methylation showed a highly significant increasing trend with disease severity. For CIN3, S5 relative sensitivity and specificity were: 93.2% (95%CI: 81.4–98.0) and 41.8% (35.2–48.8), compared to 86.4% (75.0–95.7) and 49.8% (43.1–56.6) respectively for combined abnormal cytology/HPV16/18 positivity (differences not statistically significant at 5% level); adjusted PPVs were 18.2% (16.2–20.4) and 19.3% (16.6–22.2) respectively. S5 was also positive in baseline specimens from eight cancers detected during or after trial participation. The S5 methylation score had high sensitivity and PPV for CIN3, compatible with US and European thresholds for colposcopy referral. Methylation signatures can identify most HPV positive women at increased risk of cervical cancer from their baseline screening specimens.     

Combined use of cytology,p16 immunostaining and genotyping for triage of women positive for high-risk human papillomavirus at primary screening

Human papillomavirus (HPV) testing is very sensitive for primary cervical screening but has low specificity. Triage tests that improve specificity but maintain high sensitivity are needed. Women enrolled in the experimental arm of Phase 2 of the New Technologies for Cervical Cancer randomized controlled cervical screening trial were tested for high-risk HPV (hrHPV) and referred to colposcopy if positive. hrHPV-positive women also had HPV genotyping (by polymerase chain reaction with GP5+/GP6+ primers and reverse line blotting), immunostaining for p16 overexpression and cytology. We computed sensitivity, specificity and positive predictive value (PPV) for different combinations of tests and determined potential hierarchical ordering of triage tests. A number of 1,091 HPV-positive women had valid tests for cytology, p16 and genotyping. Ninety-two of them had cervical intraepithelial neoplasia grade 2+ (CIN2+) histology and 40 of them had CIN grade 3+ (CIN3+) histology. The PPV for CIN2+ was >10% in hrHPV-positive women with positive high-grade squamous intraepithelial lesion (61.3%), positive low-grade squamous intraepithelial lesion (LSIL+) (18.3%) and positive atypical squamous cells of undetermined significance (14.8%) cytology, p16 positive (16.7%) and, hierarchically, for infections by HPV33, 16, 35, 59, 31 and 52 (in decreasing order). Referral of women positive for either p16 or LSIL+ cytology had 97.8% sensitivity for CIN2+ and women negative for both of these had a 3-year CIN3+ risk of 0.2%. Similar results were seen for women being either p16 or HPV16/33 positive. hrHPV-positive women who were negative for p16 and cytology (LSIL threshold) had a very low CIN3+ rate in the following 3 years. Recalling them after that interval and referring those positive for either test to immediate colposcopy seem to be an efficient triage strategy. The same applies to p16 and HPV16.     

DNA methylation analysis in self-sampled brush material as a triage test in hrHPV-positive women

Background:

Primary high-risk human papillomavirus (hrHPV) testing in cervical cancer screening shows relatively low specificity, which makes triage testing necessary. In this study, DNA methylation analysis was compared with cytology for triage testing in hrHPV-positive women. Moreover, feasibility of DNA methylation analysis directly on brush-based self-sampled specimens was assessed.

Methods:

Non-responding women from population-based screening were invited to self-collect a cervico-vaginal specimen for hrHPV testing; hrHPV-positive women were referred to a physician for triage liquid-based cytology. DNA methylation analysis was performed on 128 hrHPV-positive physician-collected triage samples and 50 matched brush self-samples with QMSP for C13ORF18, EPB41L3, JAM3 and TERT.

Results:

In physician-taken triage material, DNA methylation analysis of JAM3 showed the highest combined specificity (88%) and sensitivity (82%) for detection of CIN3+, whereas cytology showed a specificity of 48% and a sensitivity of 91%. Out of 39 women with abnormal cytology and normal histology (false-positive by cytology), 87% were negative for JAM3 and 90% for C13ORF18 methylation. Agreement between DNA methylation analysis performed directly on the matched self-sampled material and physician-taken samples was 88% for JAM3 (κ=0.75, P<0.001) and 90% for C13ORF18 (κ=0.77; P<0.001).

Conclusions:

DNA methylation analysis as a triage test in hrHPV-positive women is an attractive alternative to cytology. Furthermore, DNA methylation is feasible directly on brush-based self-samplers and showed good correlation with matched physician-taken samples. Direct molecular triage on self-collected specimens could optimise the screening program, especially for non-responders, as this would eliminate the need for an additional physician-taken scraping for triage testing.     

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。     

The clinical performance of APTIMA human papillomavirus and Hybrid Capture 2 assays in the triage of lesser abnormal cervical cytologies

Objective

This study was performed to evaluate the clinical performance of APTIMA human papillomavirus (AHPV) assay and Hybrid Capture 2 (HC2) assay in screening for cervical disease, especially in women with atypical squamous cell of undetermined significance (ASC-US) and low-grade squamous intraepithelial lesion (LSIL).

Methods

A total of 411 women diagnosed with ASC-US or LSIL were referred and further triaged by HC2 test. Prior to colposcopy, liquid-based cytology specimens were collected for the AHPV assay. Sensitivity and specificity were established based on the histological findings of cervical intraepithelial neoplasia (CIN).

Results

In all 411 subjects, the positive detection rate of AHPV assay was 70.8% (95% confidence interval [CI], 66.4 to 75.2), which was significantly lower than the positive detection rate of 94.9% obtained using HC2 test (95% CI, 92.3 to 96.8). Only one CIN 3-positive case was detected among the 120 AHPV-negative women, which was then confirmed by Pap smear test to be LSIL. The sensitivities of AHPV and HC2 for CIN 3 were similar (94.1% and 100%, respectively). However, AHPV showed a significantly higher specificity than HC2 test (30.2% and 5.3%, respectively; p<0.001).

Conclusion

AHPV assay is effective in identifying CIN 3-positive cases because of its high specificity and lower false-negative rate. The use of AHPV for the triage of ASC-US and LSIL might help to reduce the referral rate of colposcopy during cervical cancer screening.     

Hierarchical clustering of human papilloma virus genotype patterns in the ASCUS-LSIL triage study

Anogenital cancers are associated with ～13 carcinogenic human papilloma virus (HPV) types in a broader group that cause cervical intraepithelial neoplasia (CIN). Multiple concurrent cervical HPV infections are common, which complicates the attribution of HPV types to different grades of CIN. Here we report the analysis of HPV genotype patterns in the atypical squamous cells of undetermined significance-low-grade squamous intraepithelial lesion triage study with the use of unsupervised hierarchical clustering. Women who underwent colposcopy at baseline (n = 2,780) were grouped into 20 disease categories based on histology and cytology. Disease groups and HPV genotypes were clustered with the use of complete linkage. Risk of 2-year cumulative CIN3+, viral load, colposcopic impression, and age were compared between disease groups and major clusters. Hierarchical clustering yielded four major disease clusters: cluster 1 included all CIN3 histology with abnormal cytology; cluster 2 included CIN3 histology with normal cytology and combinations with either CIN2 or high-grade squamous intraepithelial lesion cytology; cluster 3 included older women with normal or low-grade histology/cytology and low viral load; and cluster 4 included younger women with low-grade histology/cytology, multiple infections, and the highest viral load. Three major groups of HPV genotypes were identified: group 1 included only HPV16; group 2 included nine carcinogenic types, plus noncarcinogenic HPV53 and HPV66; and group 3 included noncarcinogenic types, plus carcinogenic HPV33 and HPV45. Clustering results suggested that colposcopy missed a prevalent precancer in many women with no biopsy/normal histology and high-grade squamous intraepithelial lesion. This result was confirmed by an elevated 2-year risk of CIN3+ in these groups. Our novel approach to study multiple genotype infections in cervical disease with the use of unsupervised hierarchical clustering can address complex genotype distributions on a population level.     

Validation of a DNA methylation HPV triage classifier in a screening sample

High‐risk human papillomavirus (hrHPV) DNA tests have excellent sensitivity for detection of cervical intraepithelial neoplasia 2 or higher (CIN2+). A drawback of hrHPV screening, however, is modest specificity. Therefore, hrHPV‐positive women might need triage to reduce adverse events and costs associated with unnecessary colposcopy. We compared the performance of HPV16/18 genotyping with a predefined DNA methylation triage test (S5) based on target regions of the human gene EPB41L3, and viral late gene regions of HPV16, HPV18, HPV31 and HPV33. Assays were run using exfoliated cervical specimens from 710 women attending routine screening, of whom 38 were diagnosed with CIN2+ within a year after triage to colposcopy based on cytology and 341 were hrHPV positive. Sensitivity and specificity of the investigated triage methods were compared by McNemar's test. At the predefined cutoff, S5 showed better sensitivity than HPV16/18 genotyping (74% vs 54%, P = 0.04) in identifying CIN2+ in hrHPV‐positive women, and similar specificity (65% vs 71%, P = 0.07). When the S5 cutoff was altered to allow equal sensitivity to that of genotyping, a significantly higher specificity of 91% was reached (P < 0.0001). Thus, a DNA methylation test for the triage of hrHPV‐positive women on original screening specimens might be a valid approach with better performance than genotyping.     

Identification of progressive cervical epithelial cell abnormalities using DNA image cytometry

BACKGROUND: The objectives of the current study were to compare the capabilities of conventional cervical cytology and of DNA image cytometry (DNA-ICM) in the prediction of progressive or regressive behavior in atypical squamous cells (ASC), low-grade squamous intraepithelial lesions (LSIL), and atypical glandular cells (AGC). METHODS: One hundred ninety-six women with Papanicolaou (Pap) smears that yielded diagnoses of ASC, LSIL, or AGC were included in a prospective cohort study. Slides were classified according to the Bethesda system. DNA-ICM was performed according to the consensus reports of the European Society of Analytical Cellular Pathology. RESULTS: Reference standard verification was available in 108 patients. The rate of DNA aneuploidy in Pap smears increased significantly from cervical intraepithelial neoplasia 1 (CIN1) (54%) and CIN2 (64.3%) to CIN3 or greater (CIN3+) (83.3%) in subsequent biopsies (P < 0.05). Using ASC, LSIL, and AGC as input cytologic diagnoses and >/= CIN2 as the output histologic diagnosis, the positive predictive values (PPVs) for conventional cytology and DNA-ICM were 35.2% and 65.9%, respectively (P < 0.001). The negative predictive value (NPV) of DNA-ICM was 85.0%. When >/= CIN3 was used as the output histologic diagnosis, conventional cytology had a PPV of 22.2%. The PPV and NPV of DNA-ICM were 43.9% and 93.3%, respectively. CONCLUSIONS: The results of the current study confirmed the prognostic validity of DNA image cytometry for differentiation between progressive and regressive lesions in patients with ASC, LSIL, and AGC diagnoses.     

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.     

Evaluation of cytology and visual triage of human papillomavirus‐positive women in cervical cancer prevention in India

Although virtually all cervical cancers and most cervical intraepithelial neoplasia (CIN) are caused by persistent human papillomavirus (HPV) infection, only a small proportion of HPV‐positive women have or will develop CIN. Triaging HPV‐positive women has been suggested to reduce the false‐positive rate and proportion of women referred for CIN confirmation and/or treatment. In two cross‐sectional studies and one randomized trial in India, we evaluated the impact of using cytology or visual inspection with acetic acid (VIA) to triage HPV‐positive women on the proportion of women who would be referred for CIN confirmation and on the detection rates of high‐grade CIN. We present the numbers of HPV test‐positive women found and the CIN detected among them. We further assess the proportions that would be referred for CIN confirmation with colposcopy/biopsy and CIN that would be detected if cytology triage or VIA triage were used. Using cytology triage at atypical squamous cells of undetermined significance threshold or VIA triage reduced referrals for colposcopy by about 62% and 59%, respectively (p‐value = 0.012), but missed around 16% and 18%, respectively, of the high‐grade CIN (p‐value = 0.539) indicating similar performance of both triaging approaches. The choice of a triage test in different low‐ and middle‐income countries (LMIC) would depend on the availability and affordability in the particular setting. Cytology triage may be considered in settings where adequate infrastructure exists, whereas VIA triage may be suitable in settings with limited or no cytology infrastructure.     

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.     

宫颈病变组织CK17蛋白表达临床意义的研究

目的:检测宫颈上皮内瘤变及宫颈癌组织中CK17的表达,并探讨其临床意义.方法:应用免疫组织化学SP 法,检测15例正常宫颈组织、15例宫颈上皮内瘤变(C1N)Ⅰ、17例CINⅡ、21例CINⅢ、36例宫颈鳞癌和8例宫颈腺癌组织中CK17的表达情况.结果:CK17在正常宫颈组织、CIN和宫颈鳞癌中的阳性表达率分别为0、56.6％和83.3％,3组相比差异有统计学意义,x2=30.177,P＜0.05.CIN Ⅰ、CINⅡ和CINⅢ中,CK17的阳性表达率分别为20.0％、64.7％和76.2％,CINⅡ组阳性率高于CINⅠ组,差异有统计学意义,P=0.016;CINⅢ组阳性率高于CINⅠ组,差异有统计学意义,P=0.002;CIN Ⅱ组和CINⅢ组阳性率差异无统计学意义,P＞0.05.随着宫颈病变程度的加重,CK17的阳性表达率逐渐增高,其阳性表达同宫颈病变程度呈正相关,r=0.958,P＜0.05.结论:CK17可作为高级别CIN和浸润性鳞状细胞癌诊断及预测病变进展趋势的标志.     

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.     

Assessing the risk of cervical neoplasia in the post-HPV vaccination era

This review is based on the recent EUROGIN scientific session: “Assessing risk of cervical cancer in the post-vaccination era,” which addressed the demands of cervical intraepithelial neoplasia (CIN)/squamous intraepithelial lesion (SIL) triage now that the prevalence of vaccine-targeted oncogenic high-risk (hr) human papillomaviruses (HPVs) is decreasing. Change in the prevalence distribution of oncogenic HPV types that follows national HPV vaccination programs is setting the stage for loss of positive predictive value of conventional but possibly also new triage modalities. Understanding the contribution of the latter, most notably hypermethylation of cellular and viral genes in a new setting where most oncogenic HPV types are no longer present, requires studies on their performance in vaccinated women with CIN/SIL that are associated with nonvaccine HPV types. Lessons learned from this research may highlight the potential of cervical cells for risk prediction of all women's cancers.     

Promoter methylation of DAPK1, FHIT,MGMT, and CDKN2A genes in cervical carcinoma

Background and objectives

Aberrant DNA methylation contributes to the malignant phenotype in virtually all types of human cancer. This study explored the relationship between promoter methylation and inactivation of the DAPK1, FHIT, MGMT, and CDKN2A genes in cervical cancer.

Methods

The promoter methylation of DAPK1, FHIT, MGMT, and CDKN2A was investigated by using a methylation-specific polymerase chain reaction in 53 specimens of cervical cancer (42 squamous cell carcinoma, 11 adenocarcinoma), 22 specimens of intraepithelial neoplasia tissues, and 24 control normal cervical tissue specimens. The correlation of promoter methylation with the clinicopathological features of cervical cancer was analyzed. The expressions of DAPK1, FHIT, MGMT, and CDKN2A were detected by measuring relative mRNA levels.

Results

The promoter methylation of DAPK1, FHIT, MGMT, and CDKN2A in cervical cancer vs. intraepithelial neoplasia vs. normal cervical tissue was 75.5 vs. 31.8 vs. 4.2 % (p < 0.0001), 66.0 vs. 59.1 vs. 25.0 % (p = 0.0033), 34.0 vs. 27.3 vs. 20.8 % (p = 0.76), and 17.0 vs. 31.8 vs. 8.3 % (p = 0.11), respectively. The methylation of the promoter region significantly decreased the expression of only DAPK1 (p = 0.03). The methylation rate of the DAPK1 gene promoter was significantly higher in cervical cancer tissues than in cervical intraepithelial neoplasia and normal cervical tissues.

Conclusion

Promoter methylation may therefore lead to the inactivation of the DAPK1 gene, and may be related to the progression of cervical oncogenesis.     

Effectiveness of two strategies to follow-up ASC-US and LSIL screening results in The Netherlands using repeat cytology with or without additional hrHPV testing: a retrospective cohort study

Purpose

The purpose of the study was to assess the effectiveness of repeat cytology with and without additional high-risk human papilloma virus (hrHPV) testing after atypical squamous cells of undetermined significance/low-grade squamous intraepithelial lesion (ASC-US/LSIL) screening results.

Methods

In the Netherlands, ASC-US/LSIL is triaged by repeat cytology at 6 months or repeat cytology at 6 months with additional hrHPV testing. ASC-US/LSIL results from 13,734 screenees in 2008 were extracted from “Dutch Pathology Registry” including cytology, histology, and/or HPV follow-up results. Proportions of compliance, repeat cytology, referral, and detected cervical intraepithelial neoplasia (CIN) were assessed.

Results

With additional hrHPV testing, 46.8 % was send back to regular screening at 6 months, 28.6 % needed second repeat cytology, and 24.6 % was referred for colposcopy. Without additional hrHPV testing, this was 0.0, 76.1, and 23.9 %, respectively. With additional hrHPV testing, significantly higher proportions of persisting ASC-US/LSIL; compliance with repeat/referral advices; and histological detection of CIN0 (no CIN or cancer), CIN1, and CIN2 were found but equal proportions CIN3+.

Conclusions

Additional hrHPV testing shortens follow-up without altering CIN3+ detection. Detection of CIN0, CIN1, and CIN2 was higher, presumably by hrHPV-driven biased cytology and detection bias. Restricting additional hrHPV testing to older women, reading cytology without knowledge of hrHPV status, and addition of more specific triage tests could further improve the effectiveness of additional hrHPV testing.     

Performance of Siriraj Liquid-Based Cytology: a Single Center Report Concerning over 100,000 Samples

Background: To evaluate the performance of Siriraj liquid-based cytology (LBC) for cervical neoplasiascreening after increasing use of this technology. Materials and Methods: Cytological reports of 103,057Siriraj-LBC specimens obtained in 2007-2009 were compared with those of 23,676 specimens obtained in 2006.Results: Comparing with the year 2006, the 2007-2009 patients were slightly older (43.4±12.yr vs 42.7±12.2yr, p <0.001), and their specimens had much lower proportion of unsatisfactory slides (OR=0.06, 95%CI0.04-0.09) with comparable detection rates (3.96% vs 3.70%, p=0.052) but different proportions of variouscytological abnormalities (p<0.001). The 2007-2009 Siriraj-LBC had a negative predictive value (NPV) forcervical intraepithelial neoplasia 2+ (CIN2+) of 97.6% and an overall positive predictive value (PPV) of 43.9%.The PPV for CIN2+ varied with types of abnormal cytology, from 13.7% to 93.8% in atypical squamous cellsof undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LSIL), atypical squamouscells cannot exclude HSIL (ASC-H), high-grade squamous intraepithelial lesion (HSIL), atypical glandularcells (AGC), to squamous cell carcinoma (SCC), respectively. The PPVs for CIN2+ in ASCUS and LSIL werecomparable, but the PPV for CIN1 was higher for LSIL than for ASCUS (41.63% vs 16.32%). Conclusions:Siriraj-LBC has demonstrated a stable detection rate and NPV for CIN2+ of >95% since the first year of use.The comparable PPVs for CIN2+ of ASCUS and LSIL suggests that these two conditions may undergo similarmanagement; other cytological abnormalities need immediate evaluation.