FAM19A4/miR124-2 methylation in invasive cervical cancer: A retrospective cross-sectional worldwide study

Widespread adoption of primary human papillomavirus (HPV)-based screening has encouraged the search for a triage test which retains high sensitivity for the detection of cervical cancer and precancer, but increases specificity to avoid overtreatment. Methylation analysis of FAM19A4 and miR124-2 genes has shown promise for the triage of high-risk (hr) HPV-positive women. In our study, we assessed the consistency of FAM19A4/miR124-2 methylation analysis in the detection of cervical cancer in a series of 519 invasive cervical carcinomas (n = 314 cervical scrapes, n = 205 tissue specimens) from over 25 countries, using a quantitative methylation-specific PCR (qMSP)-based assay (QIAsure Methylation Test®). Positivity rates stratified per histotype, FIGO stage, hrHPV status, hrHPV genotype, sample type and geographical region were calculated. In total, 510 of the 519 cervical carcinomas (98.3%; 95% CI: 96.7–99.2) tested FAM19A4/miR124-2 methylation-positive. Test positivity was consistent across the different subgroups based on cervical cancer histotype, FIGO stage, hrHPV status, hrHPV genotype, sample type and geographical region. In conclusion, FAM19A4/miR124-2 methylation analysis detects nearly all cervical carcinomas, including rare histotypes and hrHPV-negative carcinomas. These results indicate that a negative FAM19A4/miR124-2 methylation assay result is likely to rule out the presence of cervical cancer.     

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.     

Evaluation of partial genotyping with HPV16/18 for triage of HPV positive,cytology negative women in the COMPACT study

ObjectiveWhile cytology-based screening programs have significantly reduced mortality and morbidity from cervical cancer, the global consensus is that primary human papillomavirus (HPV) testing increases detection of high-grade cervical intraepithelial neoplasia (CIN) and invasive cancer. However, the optimal triage strategy for HPV+ women to avoid over-referral to colposcopy may be setting specific. We compared absolute and relative risk (RR) of >CIN2/3 within 12 months of a negative cytologic result in women HPV16/18+ compared to those with a 12-other high-risk HPV (hrHPV) genotype to identify women at greatest risk of high-grade disease and permit less aggressive management of women with other hrHPV infections.MethodsParticipants were 14,160 women aged 25–69 years with negative cytology participating in the COMparison of HPV genotyping And Cytology Triage (COMPACT) study. Women who were HPV16/18+ were referred to colposcopy. Those with a 12-other hrHPV type underwent repeat cytology after 6 months and those with >abnormal squamous cells of undetermined significance went to colposcopy.ResultsAbsolute risk of >CIN2 in HPV16/18+ women was 19.5% (95% CI=12.4%–29.4%). In women 25–29 years and HPV16+ it was 40.0% (95% CI=11.8%–76.9%). Absolute risk of >CIN3 in women HPV16/18+ was 11.0% (95% CI=5.9%–19.6%). For women 30–39 years and HPV16+ it was 23.1% (95% CI=5.0%–53.8%). Overall risk of >CIN2, >CIN3 in women with a 12-other hrHPV HPV type was 5.6% (95% CI=3.1%–10.0%) and 3.4% (95% CI=1.6%–7.2%) respectively. RR of >CIN2, >CIN3 in HPV16/18+ vs. 12-other hrHPV was 3.5 (95% CI=1.7–7.3) and 3.3 (95% CI=1.2–8.8), respectively.ConclusionPrimary HPV screening with HPV16/18 partial genotyping is a promising strategy to identify women at current/future risk of >CIN2 in Japan without over-referral to colposcopy.Trial RegistrationUMIN Clinical Trials Registry Identifier: UMIN000013203     

Cervical cancer risk in HPV‐positive women after a negative FAM19A4/mir124‐2 methylation test: A post hoc analysis in the POBASCAM trial with 14 year follow‐up

DNA methylation analysis of cervical scrapes using FAM19A4 and mir124‐2 genes has shown a good clinical performance in detecting cervical cancer and advanced CIN lesions in need of treatment in HPV‐positive women. To date, longitudinal data on the cancer risk of methylation test‐negative women are lacking. In our study, we assessed the longitudinal outcome of FAM19A4/mir124‐2 methylation analysis in an HPV‐positive screening cohort with 14 years of follow‐up. Archived HPV‐positive cervical scrapes of 1,040 women (age 29–61 years), who were enrolled in the POBASCAM screening trial (ISRCTN20781131) were tested for FAM19A4/mir124‐2 methylation. By linkage with the nationwide network and registry of histo‐ and cytopathology in the Netherlands (PALGA), 35 cervical cancers were identified during 14 years of follow‐up comprising three screens (baseline, and after 5 and 10 years). The baseline scrape of 36.1% (n = 375) women tested positive for FAM19A4/mir124‐2 methylation, including 24 women with cervical cancer in follow‐up, and 30.6% (n = 318) had abnormal cytology (threshold borderline dyskaryosis or ASCUS), including 14 women with cervical cancer in follow‐up. Within screening round capability of FAM19A4/mir124‐2 methylation to detect cervical cancer was 100% (11/11, 95% CI: 71.5–100). Kaplan–Meier estimate of 14‐year cumulative cervical cancer incidence was 1.7% (95% CI: 0.66–3.0) among baseline methylation‐negative and 2.4% (95% CI: 1.4–3.6) among baseline cytology‐negative women (risk difference: 0.71% [95% CI: 0.16–1.4]). In conclusion, a negative FAM19A4/mir124‐2 methylation test provides a low cervical cancer risk in HPV‐positive women of 30 years and older. FAM19A4/mir124‐2 methylation testing merits consideration as an objective triage test in HPV‐based cervical screening programs.     

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.     

Prevalence and determinants of HPV infection among Colombian women with normal cytology

Human papillomavirus is the principal risk factor associated with cervical cancer, the most common malignancy among women in Colombia. We conducted a survey, aiming to report type specific prevalence and determinants of human papillomavirus infection in women with normal cytology. A total of 1859 women from Bogota, Colombia were interviewed and tested for human papillomavirus using a general primer GP5+/GP6+ mediated PCR-EIA. The overall HPV DNA prevalence was 14.8%; 9% of the women were infected by high risk types, 3.1% by low risk types, 2.3% by both high risk/low risk types and 0.4% by uncharacterized types (human papillomavirus X). Thirty-two different human papillomavirus types were detected, being human papillomavirus 16, 58, 56, 81(CP8304) and 18 the most common types. The human papillomavirus prevalence was 26.1% among women younger than 20 years, 2.3% in women aged 45-54 years, and 13.2% in women aged 55 years or more. For low risk types the highest peak of prevalence was observed in women aged 55 years or more. Compared to women aged 35-44 years, women aged less than 20 years had a 10-fold increased risk of having multiple infections. Besides age, there was a positive association between the risk of human papillomavirus infection and number of regular sexual partners and oral contraceptive use. In women aged below 25 years, high educational level and having had casual sexual partners predicted infection risk. In conclusion, there was a broad diversity of human papillomavirus infections with high risk types being the most common types detected. In this population multiplicity of sexual partners and, among young women, high educational level and casual sexual partners seem to determine risk.     

宫颈癌组织miR-221的表达及其与HPV感染的关联

目的探讨miR-221在宫颈癌组织中的表达情况及其与HPV感染的关系。方法经HC2法检测宫颈癌患者高危型HPV病毒,分别收集30例高危型HPV（HR-HPV）阳性及5例阴性的宫颈癌标本。同时选取30例同期因良性疾病住院患者的正常宫颈组织为对照,通过RT-PCR方法检测宫颈组织中miR-221的表达情况,初步探讨miR-221与宫颈癌的发生发展及HPV感染的关系。并通过转染的方法分别将miR-221和anti-miR-221转入HPVl6阴性的宫颈癌细胞株C33a中和HPVl6阳性的宫颈癌细胞株Caski,观察其对C33a和Caski转移和侵袭的影响。结果miR-221在宫颈癌组织中的阳性表达率显著高于正常宫颈组织（P〈0．01）,miR-221的表达水平与患者有无淋巴结转移、临床病理分级、临床分期密切相关（P〈0．01）;miR-221在HR—HPV阳性癌组织中的阳性表达率显著高于HR-HPV阴性癌组织（P〈0．01）;转染miR-221可促进Caski和C33a细胞转移和侵袭,而转入anti-miR-221可降低Caski和C33a细胞转移和侵袭能力,转染后两组细胞增殖率和凋亡率变化情况比较差异具有统计学意义（P〈0．05）。结论宫颈癌组织中miR-221表达升高与宫颈癌的发生发展及HPV感染密切相关。     

Validity of combined cytology and human papillomavirus (HPV) genotyping with adjuvant DNA-cytometry in routine cervical screening: results from 31031 women from the Bonn-region in West Germany

Our aim was to improve the accuracy of routine cervical screening by a risk-adapted multimodal protocol with special focus on possible reduction and prognostic assessment of false positive results. A cohort of 31031 women from the Bonn-region in West Germany, median age 36 years, were screened by cytology (conventional or liquid-based), followed by PCR-based HVP detection with genotyping and adjuvant DNA image cytometry, if indicated, in a sequential manner. The true prevalence of high-grade cervical intraepithelial neoplasia and carcinoma (>/=CIN2) was 0.32% in the population as projected from cervical biopsies of 123 women (0.4%), of whom 100 showed >/=CIN2. Sensitivity of the cytology screening program at PapIIID/HSIL threshold for detecting histologically confirmed >/=CIN2 cases was 81%, with specificity, positive predictive value (PPV) and negative predictive value (NPV) of 99, 20.9 and 99.9%, respectively. Of 38 women receiving the complete screening protocol, all the 31 >/=CIN2 cases were correctly detected by cytology alone, 30 by positive high-risk HPV genotype and 30 by aneuploid DNA profile. The combination of the three methods resulted in an up to 6.9% increase in PPV for >/=CIN2 at practically unchanged detection rate with the additional benefit of being able to predict the probable outcome of CIN1 lesions detected as false positives with any single test. Multimodal cervical screening might permit identification of those women with low-grade squamous intraepithelial lesions likely to progress at an earlier and curable stage of disease and lengthen the screening interval in those with transient minor lesions caused by productive HPV infection.     

A pilot study of HPV DNA and cytology testing in 50,159 women in the routine Mexican Social Security Program

Introduction  

We present a large feasibility evaluation of high-risk HPV (HR-HPV) DNA testing and cervical cytology as a primary screening strategy for cervical cancer precursor lesions in Mexican women, as part of a routine cancer control program (CCP).     

Reliable identification of women with CIN3+ using hrHPV genotyping and methylation markers in a cytology-screened referral population

Cervical screening aims to identify women with high-grade squamous intraepithelial lesion/cervical intraepithelial neoplasia 2-3 (HSIL/CIN2-3) or invasive cervical cancer (ICC). Identification of women with severe premalignant lesions or ICC (CIN3+) could ensure their rapid treatment and prevent overtreatment. We investigated high-risk human papillomavirus (hrHPV) detection with genotyping and methylation of FAM19A4/miR124-2 for detection of CIN3+ in 538 women attending colposcopy for abnormal cytology. All women had an additional cytology with hrHPV testing (GP5+/6+-PCR-EIA+), genotyping (HPV16/18, HPV16/18/31/45), and methylation analysis (FAM19A4/miR124-2) and at least one biopsy. CIN3+ detection was studied overall and in women <30 (n = 171) and ≥30 years (n = 367). Positivity for both rather than just one methylation markers increased in CIN3, and all ICC was positive for both. Overall sensitivity and specificity for CIN3+ were, respectively, 90.3% (95%CI 81.3–95.2) and 31.8% (95%CI 27.7–36.1) for hrHPV, 77.8% (95%CI 66.9–85.8) and 69.3% (95%CI 65.0–73.3) for methylation biomarkers and 93.1% (95%CI 84.8–97.0) and 49.4% (95%CI 44.8–53.9) for combined HPV16/18 and/or methylation positivity. For CIN3, hrHPV was found in 90.9% (95%CI 81.6–95.8), methylation positivity in 75.8% (95%CI 64.2–84.5) and HPV16/18 and/or methylation positivity in 92.4% (95%CI 83.5–96.7). In women aged ≥30, the sensitivity of combined HPV16/18 and methylation was increased (98.2%, 95%CI 90.6–99.7) with a specificity of 46.3% (95%CI 40.8–51.9). Combination of HPV16/18 and methylation analysis was very sensitive and offered improved specificity for CIN3+, opening the possibility of rapid treatment for these women and follow-up for women with potentially regressive, less advanced, HSIL/CIN2 lesions.     

TCT及HC2-HPV—DNA检测对绝经后妇女宫颈癌筛查的应用价值评估

目的：评价TCT、HC2-HPV—DNA单一及联合检测在绝经后妇女宫颈癌筛查中的应用价值。方法：收集北京市海淀妇幼保健院155例TCT检测提示异常的绝经后妇女,进一步行HC2-HPV—DNA检测和病理学活检。以病理诊断结果为金标准,计算TCT、HC2-HPV—DNA及两者联合检测筛查宫颈CINl／湿疣及以上病变的敏感度、特异度、假阳性率、假阴性率等指标,评价其诊断价值。结果：HC2-HPA—DNA对绝经后妇女CINl／湿疣以上病变的筛查敏感性高于TCT检测（分别为84．0％及76．0％）,但其特异度明显低于后者（分别为52．4％及74．3％）,两种检测均存在较高的假阳性率（25．7％及47．6％）,两者联合检测可明显降低假阳性率（19．0％）,并且明显提高诊断的特异度（81．0％）及诊断符合率（78．1％）,两种检测方法单一或联合检测的阴性预测值无差异显著性。结论：TCT、HC2-HPA—DNA联合检测可降低筛查假阳性率,提高特异度及诊断符合率,在绝经后妇女宫颈癌筛查中具有较高的应用价值。     

Alteration of MMP-2 and -14 expression by imatinib in HPV-positive and -negative squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is an aggressive epithelial malignancy. It is known to be the most common neoplasm appearing in the upper aerodigestive tract. The poor 5?year survival rate has remained unchanged in the last decades even though improved techniques in surgery, radiation and chemotherapy have been established. In contrast to the overall decreasing incidence of head and neck cancer in the US, the incidence of HPV-associated oropharyngeal cancer is increasing, indicating the importance of viral etiology. Furthermore, growth and invasion of HNSCC are strongly influenced by the extracellular matrix (ECM). Matrix metalloproteinases (MMP) have been shown to play key roles in the remodeling of the ECM. Imatinib (STI 571) was originally designed to inhibit the BCR-ABL tyrosine kinase in chronic myeloid leukaemia. But it also has an inhibitory impact, e.g., on the protein-tyrosine-kinase (PTK) receptor c-kit and on its PTK activity in HNSCC. In this study, we incubated the HNSCC cell lines HNSCC 11A and 14C and the p16-positive SCC line CERV196 with increasing concentrations of imatinib or carboplatin. After an incubation time of up to 10 days, we evaluated MMP-2 and -14 expression by ELISA techniques and immunohistochemistry. MMP-2 and -14 expression was demonstrated in all incubated tumor cell lines. Especially incubation with imatinib resulted in a significant decrease in MMP expression in incubated cell lines. Our results indicate that the expression of MMP-2 and -14 is suppressed in the presence of imatinib. Thus, imatinib may exert in part its inhibitory effect on malignant cell growth via the blockage of the signal transduction of PTK receptors. Further studies are warranted, especially keeping in mind the moderate toxicity of imatinib.     

Multi-site study of HPV type-specific prevalence in women with cervical cancer, intraepithelial neoplasia and normal cytology, in England

Background:

Knowledge of the prevalence of type-specific human papillomavirus (HPV) infections is necessary to predict the expected, and to monitor the actual, impact of HPV immunisation and to design effective screening strategies for vaccinated populations.

Methods:

Residual specimens of cervical cytology (N=4719), CIN3/CGIN and cervical cancer biopsies (N=1515) were obtained from sites throughout England, anonymised and tested for HPV DNA using the Linear Array typing system (Roche).

Results:

The prevalence of HPV 16 and/or 18 (with or without another high-risk (HR) type) was 76% in squamous cell carcinomas, 82% in adeno/adenosquamous carcinomas and 63% and 91% in CIN3 and CGIN, respectively. Of all HR HPV-infected women undergoing cytology, non-vaccine HPV types only were found in over 60% of those with mild dyskaryosis or below, and in <20% of those with cancer. In women of all ages undergoing screening, HR HPV prevalence was 16% and HPV 16 and/or 18 prevalence was 5%.

Conclusion:

Pre-immunisation, high-grade cervical disease in England was predominantly associated with HPV 16 and/or 18, which promises a high impact from HPV immunisation in due course. Second-generation vaccines and screening strategies need to consider the best ways to detect and prevent disease due to the remaining HR HPV types.     

Preferential risk of HPV16 for squamous cell carcinoma and of HPV18 for adenocarcinoma of the cervix compared to women with normal cytology in The Netherlands

We present the type-distribution of high-risk human papillomavirus (HPV) types in women with normal cytology (n=1467), adenocarcinoma in situ (ACIS) (n=61), adenocarcinoma (n=70), and squamous cell carcinoma (SCC) (n=83). Cervical adenocarcinoma and ACIS were significantly more frequently associated with HPV18 (OR(MH) 15.0; 95% CI 8.6-26.1 and 21.8; 95% CI 11.9-39.8, respectively) than normal cytology. Human papillomavirus16 was only associated with adenocarcinoma and ACIS after exclusion of HPV18-positive cases (OR(MH) 6.6; 95% CI 2.8-16.0 and 9.4; 95% CI 2.8-31.2, respectively). For SCC, HPV16 prevalence was elevated (OR(MH) 7.0; 95% CI 3.9-12.4) compared to cases with normal cytology, and HPV18 prevalence was only increased after exclusion of HPV16-positive cases (OR(MH) 4.3; 95% CI 1.6-11.6). These results suggest that HPV18 is mainly a risk factor for the development of adenocarcinoma whereas HPV16 is associated with both SCC and adenocarcinoma.