2020 list of human papillomavirus assays suitable for primary cervical cancer screening

BackgroundOnly clinically validated HPV assays can be accepted in cervical cancer screening.ObjectivesTo update the list of high-risk HPV assays that fulfil the 2009 international validation criteria (Meijer-2009).Data SourcesPubMed/Medline, Embase, Scopus, references from selected studies; published in January 2014 to August 2020.Study eligibility criteriaHPV test validation studies and primary screening studies, involving testing with an index HPV test and a comparator HPV test with reporting of disease outcome (occurrence of histologically confirmed cervical precancer; CIN2+).ParticipantsWomen participating in cervical cancer screening.InterventionsTesting with an index and a comparator HPV test of clinician-collected cervical specimens and assessment of disease outcome (<CIN2, CIN2+). Comparator HPV assays were HC2, GP5+/6+ PCR-EIA, recommended in validation guidelines, or tests with consistent previous validations.MethodsAssessment of relative clinical accuracy (including non-inferiority statistics index vs comparator assay) and test reproducibility in individual studies; random effects meta-analyses of the relative clinical sensitivity and specificity of index vs comparator tests.ResultsSeven hrHPV DNA tests consistently fulfilled all validation criteria in multiple studies using predefined test positivity cut-offs (Abbott RealTime High Risk HPV, Anyplex II HPV HR Detection, BD Onclarity HPV Assay, Cobas 4800 HPV Test, HPV-Risk Assay, PapilloCheck HPV-Screening Test and Xpert HPV). Another assay (Alinity m HR HPV Assay) was fully validated in one validation study. The newer Cobas 6800 HPV Test, was validated in two studies against Cobas 4800. Other tests partially fulfilled the international validation criteria (Cervista HPV HR Test, EUROArray HPV, Hybribio's 14 High-Risk HPV, LMNX Genotyping Kit GP HPV, MALDI-TOF, RIATOL qPCR and a number of other in-house developed assays) since the non-inferior accuracy was reached after a posteriori cut-off optimization, inconsistent accuracy findings in different studies, and/or insufficient reproducibility assessment. The APTIMA HPV Assay targeting E6/E7 mRNA of hrHPV was fully validated in one formal validation study and showed slightly lower pooled sensitivity but higher specificity than the standard comparator tests in seven screening studies. However, the current international validation criteria relate to DNA assays. The additional requirement for longitudinal performance data required for non-DNA based HPV assays was not assessed in this review.ConclusionsEleven hrHPV DNA assays fulfil all requirements for use in cervical cancer screening using clinician-collected specimens.     

Association of human papillomavirus type 16 and its genetic variants with cervical lesion in Korea

Persistent human papillomavirus type 16 (HPV16) is the major risk factor for cervical cancer. HPV16 intratypic variants differ in their geographical distribution and oncogenic potential. This study aimed to analyze the distribution of HPV16 variants and their association with cervical lesion histopathology in Korean women. In total, 133 HPV16‐positive cervical samples from women admitted to Seoul National University Boramae Hospital were analyzed by sequencing E6, E7, and L1 genes and the long control region (LCR), and the variant distribution according to cervical lesion grade was determined. Isolates were grouped into a phylogenetic lineage, and A1‐3, A4, C, and D sublineages were detected in 54.1, 37.8, 0.7, and 7.4% of samples, respectively. The most commonly observed LCR variations were 7521G>A (91.5%), 7730A>C (59.6%), and 7842G>A (59.6%). Furthermore, A4 or D sublineage‐positive women had a higher risk for cervical cancer than women who were positive for A1–3. Among HPV phylogenetic clusters, A1–3 was the predominant sublineage, and within A1–3, the 350G polymorphism was highly frequent. These results differed from those of previous studies in Korea and other Asian countries. The findings suggest that cervical neoplasia incidence in HPV16‐infected patients could be affected by the distribution of HPV16 variants in the population.     

The human papillomavirus (HPV) vaccine,HPV related diseases and cervical cancer in the post-reproductive years

Prophylactic HPV vaccines have demonstrated high efficacy against a range of HPV related diseases. They have been widely adopted as population health interventions in many jurisdictions and their routine use has been endorsed by the WHO. The development of these vaccines comes after an increased understanding of the natural history and epidemiology of HPV infection and disease in both males and females. Persistent HPV infection with oncogenic types induces malignant transformation in a range of epithelia including the cervix, anogenital regions, the penis and a number of head and neck sites. In relation to HPV disease prevention in the post-reproductive years, most infections occur soon after commencement of sexual activity but new infections do occur throughout the age spectrum. This reduces the likely impact of prophylactic vaccines in this population. The major impact on HPV related disease in this age group will come from advances in screening and early detection of HPV and neoplastic precursors. The most appropriate prevention for any individual man or women in this age group will be an individualised combination of vaccination, screening and early detection depending on the individual's own circumstances.     

Human papillomavirus (HPV) E6/E7 mRNA as a triage test after detection of HPV 16 and HPV 18 DNA

High‐risk human papillomavirus (HPV) DNA detection provides high sensitivity but low specificity for moderate‐grade cervical intraepithelial neoplasia or worse histological identification. A prospective study evaluated mRNA testing efficacy for predicting this histological diagnosis in case of HPV 16 and/or 18 DNA detection. A total of 165 endocervical samples harboring HPV 16 and/or 18 DNA were tested with NucliSENS‐EasyQ® HPV E6/E7‐mRNA‐assay (Biomerieux, Marcy l´Etoile, France). Women with cytological alterations were referred to colposcopy (n = 111). Moderate‐grade cervical intraepithelial neoplasia or worse was diagnosed in 25.8% of women presenting atypical squamous cells of undetermined significance or low‐grade squamous intraepithelial lesions and in 89.8% of women with high‐grade squamous intraepithelial lesions. mRNA sensitivity was 81.3% and 84.1%, respectively. Specificity was 52.2%, and 80.0%, respectively. Negative predictive value (NPV) was 88.9% in undetermined or low‐grade squamous lesions. Positive predictive value (PPV) was 97.4% in high‐grade squamous lesions. mRNA reduced colposcopies by 44.3% in undetermined or low‐grade squamous lesions. Direct treatment of mRNA‐positive cases reduced 77.5% of colposcopies in high‐grade squamous lesions. Women without cytological alterations were followed for 18 months (n = 35), and moderate‐grade cervical intraepithelial neoplasia or worse was diagnosed in 34.3%; mRNA sensitivity and specificity were 83.3% and 86.9%, respectively. PPV and NPV were 76.9% and 90.9%, respectively for predicting moderate‐grade cervical intraepithelial neoplasia or worse in 18 months. mRNA reduced the number of visits for follow‐up in 62.2%. In conclusion, NucliSENS‐EasyQ® HPV E6/E7‐mRNA‐assay (Biomerieux) can serve as a triage test in case of HPV 16 and/or 18 DNA detection. J. Med. Virol. 85: 1063–1068, 2013. © 2013 Wiley Periodicals, Inc.     

Prevalence of human papillomavirus types in women screened by cytology in Germany

Incidence and mortality rates of cervical cancer are higher in Germany than in other Western European countries. Type-specific human papillomavirus (HPV) distribution was investigated for the first time in Germany in an epidemiological study including 8,101 women. Women above the age of 30 years, self-referring for cervical cancer screening, were enrolled in two study centers in Hannover (Northern Germany) and Tübingen (Southern Germany). Participants were screened by the Pap smear and the hybrid capture 2 (HC2) test using the high-risk probe. All samples that were positive by the HC2 test were genotyped using the prototype PGMY09/11 PCR line blot assay. Most women in the study population had a negative Pap smear (96.7%). Prevalence of high-risk type HPV detected by HC2 was 6.4% and prevalence of carcinogenic types detected by PGMY09/11 was 4.3%. Of the PGMY09/11 PCR-positive women, 70.2% had a single infection, 28.1% had multiple infections and 1.7% remained uncharacterized. 32 different HPV types were detected using PGMY09/11 PCR. HPV 16, 31, 52, 51, 18, and 45 were the most common carcinogenic types in the study population. Among women with histologically confirmed high-grade lesions HPV 16, 45, 58, 18, 31, 33, and 52 were the predominant types. These results provide valuable information for the management of HPV infections in Germany, both in terms of future strategies of screening and vaccination.     

FAM19A4/miR124-2 methylation analysis as a triage test for HPV-positive women: cross-sectional and longitudinal data from a Dutch screening cohort

ObjectivesThe aim was to evaluate the cross-sectional and long-term triage performance of FAM19A4/miR124-2 methylation analysis in human papillomavirus (HPV)-based cervical screening.MethodsWe conducted a post hoc analysis within a Dutch population-based HPV-positive study cohort of women aged 30–60 years (n = 979). Cross-sectional cervical intraepithelial neoplasia (CIN) 3+ sensitivity, specificity, positive predictive value and negative predictive value as well as cumulative CIN3+ or cervical cancer risks after 9 and 14 years were compared for three baseline triage strategies: (1) cytology, (2) FAM19A4/miR124-2 methylation analysis and (3) combined FAM19A4/miR124-2 methylation with cytology.ResultsCIN3+ sensitivity of FAM19A4/miR124-2 methylation analysis was similar to that of cytology (71.3% vs 76.0%, ratio 0.94, 95% CI 0.84 to 1.05), at a lower specificity (78.3% vs 87.0%, ratio 0.90, 95% CI 0.86 to 0.94). Combining FAM19A4/miR124-2 methylation analysis with cytology resulted in a CIN3+ sensitivity of 84.6% (95% CI 78.3 to 90.8) at a specificity of 69.6% (95% CI 66.5 to 72.7). Similar 9- and 14-year CIN3+ risks for baseline cytology-negative women and baseline FAM19A4/miR124-2 methylation-negative women were observed, with risk differences of –0.42% (95% CI –2.1 to 1.4) and –0.07% (95% CI –1.9 to 1.9), respectively. The 14-year cumulative cervical cancer incidence was significantly lower for methylation-negative women compared to cytology-negative women (risk difference 0.98%, 95% CI 0.26 to 2.0).DiscussionFAM19A4/miR124-2 methylation analysis has a good triage performance on baseline screening samples, with a cross-sectional CIN3+ sensitivity and long-term triage-negative CIN3+ risk equalling cytology triage. Therefore, FAM19A4/miR124-2 methylation analysis appears to be a good and objective alternative to cytology in triage scenarios in HPV-based cervical screening.     

Concordant testing results between various human papillomavirus assays in primary cervical cancer screening: systematic review

ObjectivesHuman papillomavirus (HPV) assays are increasingly used for primary cervical screening and HPV-vaccination-effect monitoring. We undertook a systematic literature review to determine the concordance in positive test results (i.e. detection of HPV infections) between Hybrid Capture 2 (HC2) and other assays.MethodsWe searched PubMed, Embase and Scopus for studies of primary screening with HC2 and one or more assays, with cross-tabulated testing results for the assays. Two authors applied inclusion criteria and three authors extracted data from included studies. For each inter-assay comparison, we calculated the concordance by comparing the number of concordant samples with the number of samples that tested positive on at least one assay.ResultsSixteen studies fulfilled inclusion criteria, comparing nine assays to HC2, and including 392 to 9451 patients each. The calculated concordance varied between 48% and 69% for HC2 and APTIMA, Cobas, Abbott RealTime, Cervista, GP5+/6+, CLART, BD HPV test, Amplicor and Linear Array, i.e. 31%–52% of all positive tests on any pair of compared assays were discordant. Although modest variation in the degree of concordance with HC2 was suggested for particular assays, the numbers of studies per assay were generally low. No pronounced systematic patterns were observed by study (e.g. liquid medium) or population characteristics.ConclusionsThe ten commercially available assays do not detect the same HPV infections. Even in the most favourable case, the two assays provided discordant test results in 31% of all detected infections.     

宫颈癌HPV16,18 DNA定量分析

目的为了研究HPV16/18病毒载量与宫颈癌危险性的关系.方法 FQPCR用于HPVDNA的检测,卡方检验用于阳性率的分析,t检验用于病毒DNA量的分析.结果宫颈癌妇女高危型阳性率显著高于湿疣组妇女;病毒载量也显著高于后者.结论宫颈癌与高危型HPV感染相关,随病毒载量增加致癌危险性增大.     

宫颈癌组织中人乳头瘤病毒16型L1基因的克隆及序列分析

目的人乳头瘤病毒１６型（ＨＰＶ１６）晚期基因区（Ｌ１）编码病毒的主要衣壳蛋白,且ＨＰＶ１６感染与宫颈癌发生、发展关系密切,故对中国妇女感染的ＨＰＶ１６基因进行克隆及序列分析有重要实际意义。方法采用聚合酶链反应技术（ＰＣＲ）扩增了３例中国妇女宫颈癌组织中ＨＰＶ１６的Ｌ１基因片段,并对其进行了克隆及全序列分析。结果发现３个ＨＰＶ１６Ｌ１基因核苷酸序列有４处均与最初报道的ＨＰＶ１６Ｌ１ＤＮＡ序列不同,并由此引起所编码的氨基酸亦发生变化。结论中国妇女宫颈癌组织中ＨＰＶ１６Ｌ１核苷酸有一定程度变异。     

Prognostic value of human papillomavirus types 16 and 18 DNA physical status in cervical intraepithelial neoplasia

The aim of this work was to assess the value of the physical status of human papillomavirus (HPV) DNA as a disease marker for cervical cancer development in a set of 248 DNA samples previously genotyped as HPV 16 or 18, by calculating the E2/E6 ratio through real-time PCR. There was a significant difference in integration status according to disease grade for both genotypes (p <0.001). Furthermore, especially for HPV 18, determining the DNA physical status could be a useful biomarker in predicting cervical cancer risk development, with a lower E2/E6 ratio clinically associated with the development of a precancerous lesion.     

Detection and typing of human papillomavirus DNA in the uterine cervix of japanese women by nonradioactive dot blot and southern blot hybridization

HPV infection was examined with Cytobrush in exfoliated cervical cells sampled from 347 women. HPV DNA analysis was conducted in two steps. The presence of HPV DNA was demonstrated by dot blotting and typing of HPV DNA was made by Southern blotting using biotinylated probes. Of 167 cases with cervical intra-epithelial neoplasia (CIN) and cervical carcinoma, HPV DNA was detected and typed in 25 cases (15.0%). HPV 16, 18, and 33 were found mainly, and no HPV 6 or 11 was detected. The frequency of HPV DNA was 7.1% of CIN I, 28.6% of CIN II, 54.5% of CIN III, and 50.0% of invasive carcinoma. HPV occurred in patients newly diagnosed as CIN at 25.8% and in those of diagnosed as CIN in the past and followed up, 7.6%. Of 180 healthy women, the screening test of F08830 was positive for HPV DNA in four women (2.2%). The present system proved to be useful for facilitating large-scale clinical research.     

Detection of human papillomavirus in sanitary napkins: a new paradigm in cervical cancer screening

Human papillomavirus was successfully detected by polymerase chain reaction (PCR) in menstrual blood or vaginal discharge collected in sanitary napkins in 100% of 17 women having koilocytosis, cervical intraepithelial neoplasia, or squamous carcinoma. We advocate this form of cervical cancer screening because of its high sensitivity and acceptance by patients.     

HPV16/18和HPV16E6/E7DNA在宫颈癌组织中的表达

目的检测HPV16/18和HPV16E6/E7 DNA在宫颈癌组织中的表达,探讨其在宫颈癌发病中的作用.方法应用PCR和琼脂糖凝胶电泳方法检测46例宫颈癌组织中HPV16/18和HPV16E6/E7DNA.结果 46例宫颈癌中56.5%(26/46)扩增HPV16/18 DNA,其中宫颈鳞癌25例,宫颈腺癌1例.正常对照组20例HPV16/18DNA均为阴性,与宫颈癌组相比差异有显著性(P<0.01).HPV16/18 DNA阳性拷贝对数值为4.32±2.45.HPV16E6,E7DNA分别有53.8%(14/26)、46.2%(12/26)扩增.结论 HPV16/18和HPV16E6/E7 DNA与宫颈癌的发生密切相关,是宫颈癌恶性转化的关键之一,预示着宫颈癌有较强的增殖能力和转移能力.     

Molecular detection and genotyping of human papillomavirus in cervical carcinoma biopsies in an area of high incidence of cancer from Moroccan women

Cervical cancer is a leading cause of cancer‐related deaths in developing countries, and the human papillomavirus (HPV) is linked etiologically to cervical cancer. Eighty nine cervical carcinoma biopsies collected from women visiting the Oncologic Center in Casablanca (Centre Hospitalier Universitaire Ibn Rochd, Morocco) for cervical cancer symptoms, were screened for HPV DNA by polymerase chain reaction amplification with subsequent typing by hybridization with specific oligonucleotides for HPV types 16, 18, 31, 33, 45, and 59. Using very high stringency hybridization the HPV types could be easily distinguished. After preliminary clinical sorting, 92% (82/89) of the samples were found to be HPV‐positive. Among the samples infected by a single HPV, type 16 was the most frequent 36.6% (30/82) of the positive samples, followed by HPV 18; 19.5% (16/82). Double or even multiple infections by the different HPV types were also detected (35.5% of the positive samples); dual infections were the more frequent, with the following combinations of HPVs: HPV16/HPV18 (21% of the positives samples) and HPV16/HPV45 (8.5%). J. Med. Virol. 81:678–684, 2009 © 2009 Wiley‐Liss, Inc.     

Molecular variants of human papillomavirus type 16 and 18 and risk for cervical neoplasia in Portugal

Persistent high-risk human papillomavirus (HPV) infection is considered as the central cause of invasive cervical cancer. Specific HPV 16 and 18 sequence variations were associated with an increased risk for progression. The purpose of this study was to analyze intratypic variations of HPV 16 and 18 within the E6 gene, MY09/11 and LCR regions, and to evaluate the risk of these variants for cervical neoplasia among Portuguese women. Cervical samples from 187 HPV 16-positive and 41 HPV 18-positive women with normal epithelium, cervical intraepithelial neoplasia, or invasive cervical cancer were amplified by type-specific PCR, followed by sequence and phylogenetic analysis. Sixteen new HPV 16 and 18 patterns are described in this paper. European HPV 16 variants were the most frequent (74.3%), particularly Ep-T350 (44.4%), followed by African (16.1%), and Asian-American (9.6%). Non-European HPV 16 variants were more frequent in pre-invasive lesions than in normal tissue and low-grade lesions. However, when analyzed separately, only African variants were associated significantly with an increased risk for cervical cancer. For HPV 18, the AsAi variant showed a trend, which was not statistically significant to an enhanced oncogenicity. European variants seemed to be significantly associated with a lower risk for cervical cancer development. The distribution of HPV 16 and 18 variants was not related to age or race among women living in the same geographical region. Knowledge of variants will be important for risk determination as well as for designing primers or probes for HPV detection methods, and for appropriate cervical cancer prevention strategies.     

子宫颈癌、子宫颈上皮内病变组织中SIRT1、HPV 16/18E6的表达及其意义

目的检测子宫颈癌及癌前病变中SIRT1的表达情况,探讨其与临床病理特征的关系。同时检测早期癌蛋白中HPV16/18E6的表达,分析两者的相关性。方法应用免疫组化En Vision法检测30例子宫颈炎、100例子宫颈上皮内病变(高级别、低级别各50例)、30例子宫颈鳞状细胞癌组织中SIRT1和HPV 16/18E6的表达。结果子宫颈癌组织中SIRT1阳性率为93.33%(28/30),高于子宫颈炎组织(13.33%,4/30),差异有统计学意义(P0.05)。子宫颈高级别上皮内病变SIRT1阳性率(88%,44/50)高于低级别上皮内病变(14%,7/50),差异有统计学意义(P0.05)。但高级别上皮内病变与子宫颈癌SIRT1阳性率差异无统计学意义(P0.05);低级别上皮内病变与子宫颈炎中SIRT1阳性率差异无统计学意义(P0.05)。在子宫颈癌中,SIRT1的阳性率与临床分期和组织学分级有关,晚期、分化差的子宫颈癌阳性率高于早期、分化好的子宫颈癌,差异有统计学意义(P0.05)。在子宫颈癌中SIRT1的阳性率(93.33%,28/30)高于HPV 16/18E6(30%,9/30),在子宫颈高级别上皮内病变中SIRT1的阳性率(88%,44/50)高于HPV 16/18E6(28%,14/50),但在低级别上皮内病变中SIRT1的阳性率(14%,7/50)低于HPV 16/18E6(36%,18/50),两者差异有统计学意义(P0.05)。结论 SIRT1的阳性率随着病变程度增加而升高,提示SIRT1与细胞恶性转变有关,其过表达可能促进上皮向高级别内病变乃至子宫颈癌进展。     

Immunohistochemical detection of p53 in cervical epithelial lesions with or without infection of human papillomavirus types 16 and 18

Using formalin-fixed and paraffin-embedded cervical tissues, we examined infection with human papillomavirus (HPV) types 16 and 18 by Southern blot analysis following polymerase chain reaction (PCR), and the accumulation of p53 protein by immunohistochemistry in 30 cases of normal or metaplastic cervix, 17 cases of cervical intraepithelial neoplasia grade I (CIN I), 20 cases of CIN II, 37 cases of CIN III and 23 cases of invasive squamous cell carcinoma (ISCC). In addition, we examined the ratio of HPV-infected cells by in situ hybridization (ISH) and the alteration of p53 gene using PCR followed by single-strand conformation polymorphism (PCR-SSCP) in 2 cases of CIN III and 12 cases of ISCC, in which overexpression of p53 was immunohistochemically detected. HPV DNA was detected in 5 cases (16.7%) of normal or metaplastic cervix, 5 cases (29.4%) of CIN I, 9 cases (45.0%) of CIN II, 26 cases (70.3%) of CIN III and 15 cases (65.2%) of ISCC. Positivity for HPV in the groups of CIN III and ISCC was significantly higher than in the normal or metaplastic cervix (P<0.05). The accumulation of p53 was not detected in the normal or metaplastic cervix, CIN I and CIN II. High-level p53 accumulation was identified in basal and suprabasal atypical cells in 27.0% (10/37) of CIN III and in carcinoma cells in 43.5% (10/23) of ISCC cases, and low-level accumulation was identified in atypical cells of 35.1% (13/37) of CIN III and in carcinoma cells in 30.4% (7/23) of ISCC cases. The accumulation of p53 was found to coexist with infection by HPV in 17 (46.0%) of 37 CIN III cases and 12 (52.2%) of 23 ISCC cases, and high-level p53 accumulation was more frequently detected in HPV-positive ISCC cases. Either HPV infection or accumulation of p53 was found in 16.7% (5/30) of the cases of normal or metaplastic cervix, 29.4% (5/17) of CIN I, 45.0% (9/20) of CIN II, 86.5% (32/37) of CIN III and 87.0% (20/23) of ISCC cases. These results suggest that the inactivation of p53 function by HPV infection or alteration of p53 protein itself precedes the development of tumours with a fully malignant and invasive phenotype and plays an important role in tumorigenesis in the uterine cervix. ISH study provided no correlation between the degree of immunohistochemical positivity for p53 and the ratio of HPV-positive cells in the same lesions. PCR-SSCP detected the alteration of p53 gene in at least 4 cases of ISCC, 2 of which were accompanied by HPV infection.