High‐risk human papillomavirus DNA load in a population‐based cervical screening cohort in relation to the detection of high‐grade cervical intraepithelial neoplasia and cervical cancer

In a population‐based cervical screening cohort, we determined the value of type‐specific viral load assessment for the detection of high‐grade cervical intraepithelial neoplasia and cervical cancer (≥CIN2). Viral load was determined by type‐specific real‐time PCR in women with single HPV16,‐18,‐31 and ‐33 infections, as determined by GP5+/6+‐PCR. Study endpoints were the detection of cumulative ≥CIN2 or ≥CIN3 within 18 months of follow‐up. High viral loads of HPV16,‐31, and ‐33 were predictive for ≥CIN2 (relative risk of 1.6 (95% CI: 1.3–1.9), 1.7 (95% CI: 1.1–2.7) and 1.9 (95% CI: 1.1–3.1) per 10‐fold change in viral load, respectively). For HPV18, the relative risk was of similar magnitude (1.5, 95% CI: 0.7–3.1), though not significant (p = 0.3). Subsequently, we determined the sensitivities of viral load for ≥CIN2 and ≥CIN3 in HPV DNA‐positive women using viral load thresholds previously defined in a cross‐sectional study. These thresholds were based on the 25th, 33rd and 50th percentiles of type‐specific HPV16,‐18,‐31 or ‐33 viral load values found in women with normal cytology. For all types, combined sensitivities for ≥CIN2 were 93.5%, 88.8% and 77.7% for the 25th, 33rd and 50th percentile thresholds, respectively. Response‐operator‐characteristics (ROC) curve analysis showed that viral load testing on HPV DNA‐positive women in addition to or instead of cytology may result in an increased sensitivity for ≥CIN2, but at the cost of a marked decrease in specificity in relation to cytology. Similar results were obtained when using ≥CIN3 as endpoint. In conclusion, in a cervical screening setting viral load assessment of HPV16, 18, 31 and 33 has no additive value to stratify high‐risk HPV GP5+/6+‐PCR‐positive women for risk of ≥CIN2 or ≥CIN3. © 2008 Wiley‐Liss, Inc.     

Prevalence of types 16 and 33 is increased in high-risk human papillomavirus positive women with cervical intraepithelial neoplasia grade 2 or worse

High-risk human papillomavirus (hrHPV) types are causally related to cervical cancer and its high-grade precursor lesions. The risk posed by the different hrHPV types for the development of cervical intraepithelial neoplasia grade 2 or worse (> or =CIN2) needs to be established. Here, we present the hrHPV type-distribution in relation to cytology and histology for women participating in a cervical screening program. From 44,102 women who participated in a population-based cervical screening program in the Netherlands, 2,154 hrHPV GP5+/6+ PCR positive women were recruited to determine the distribution of 14 hrHPV types by reverse line blotting of GP5+/6+ PCR products. For each HPV type, associations with cytology and histologically confirmed > or =CIN2 were measured by odds ratios. HPV types 16 and 33 were more prevalent in women, amongst those containing a single hrHPV type, with moderate dyskaryosis or worse (>BMD) than in women with normal cytology, but only in case of underlying > or =CIN2 (OR 4.10, 95%CI 2.98-5.64 and OR 2.68, 95%CI 1.39-5.15, respectively). Similar results were obtained for women with double infections (OR 3.29, 95% CI 1.61-6.75 and OR 4.37, 95% CI 1.17-16.34). Coexisting types did not influence the prevalence of > or =CIN2 in HPV 16 or 33 positive women. The increased prevalence of type 16 and 33 in hrHPV positive women with > or =CIN2, compared to women with normal cytology, suggests that infection with these types confers an increased risk for development of > or =CIN2. Distinguishing these types may therefore have implications for future cervical screening strategies.     

Comparison of hybrid capture 2 with in situ hybridization for the detection of high-risk human papillomavirus in liquid-based cervical samples

BACKGROUND: The performance of two commercially available detection systems for high-risk HPV (hrHPV), Hybrid Capture 2 (HC2) and in situ hybridization (ISH), were compared on cervical scrapings. METHODS: Using general primer (GP)-mediated GP5+/6+-polymerase chain reaction (PCR)-enzyme immunoassay and reverse line blot genotyping, 76 liquid-based cervical samples were identified with > or = 1 of the 12 hrHPV types present in the probes of the HC2 and ISH assays. The positivity rate of the assays and the HC2 viral load were determined and related to cytologic findings (n = 76 samples) and histologic findings (n = 43 samples). RESULTS: Overall, HC2 scored significantly more samples positive compared with ISH (P < 0.01). Seventy-four of 76 samples (97%) were positive according to HC2. Forty-six of 76 samples (61%) were positive according to ISH, including 80% and 70% of samples that were classified cytologically as moderate dysplasia and severe dysplasia, respectively. All women with underlying cervical intraepithelial neoplasia (CIN) lesions and 67% of women without CIN had positive HC2 samples. ISH scored 33%, 66%, 88%, and 73% of samples positive of women with no CIN, Grade 1 CIN (CIN 1), CIN 2, and CIN 3, respectively. The HC2 viral load was significantly higher in women who had a cytologic diagnosis of dysplasia (P < 0.01) and in women who had an underlying diagnosis of CIN (P < 0.01) compared with women who had neither. In addition, the viral load was significantly higher in ISH positive samples compared with ISH negative samples (P < 0.01). CONCLUSIONS: An increased HC2 viral load was associated with an increased chance of underlying high-grade CIN disease in women who tested hrHPV GP5+/6+-PCR positive. Moreover, although positive ISH results were associated with an increased overall viral load in the sample, the analytic sensitivity of ISH was too low to detect all women with prevalent high-grade CIN.     

Long‐term risk of cervical intraepithelial neoplasia grade 3 or worse according to high‐risk human papillomavirus genotype and semi‐quantitative viral load among 33,288 women with normal cervical cytology

In this prospective cohort study, we estimated the long‐term risk of cervical intraepithelial neoplasia grade 3 or cancer (CIN3+) by high‐risk human papillomavirus (hrHPV) genotype and semi‐quantitative viral load at baseline among 33,288 women aged 14–90 years with normal baseline cytology. During 2002–2005, residual liquid‐based cervical cytology samples were collected from women screened for cervical cancer in Copenhagen, Denmark. Samples were HPV‐tested with Hybrid Capture 2 (HC2) and genotyped with INNO‐LiPA. Semi‐quantitative viral load was measured by HC2 relative light units in women with single hrHPV infections. The cohort was followed in a nationwide pathology register for up to 11.5 years. In women aged ≥30 years at baseline, the 8‐year absolute risk for CIN3+ following baseline detection of HPV16 was 21.8% (95% confidence interval [CI]: 18.0–25.6%). The corresponding risks for HPV18, HPV31, HPV33, and other hrHPV types, respectively, were 12.8% (95% CI: 7.6–18.0%), 11.3% (95% CI: 7.7–14.9%), 12.9% (95% CI: 7.0–18.8%) and 3.9% (95% CI: 2.7–5.2%). Similar absolute risk estimates were observed in women aged <30 years. Higher HPV16‐viral load was associated with increased risk of CIN3+ (hazard ratio = 1.34, 95% CI: 1.10–1.64, per 10‐fold increase in viral load). A similar trend, although statistically nonsignificant, was found for viral load of HPV18. The 8‐year absolute risk of CIN3+ in women with HPV16‐viral load ≥100.0 pg/ml was 30.2% (95% CI: 21.9–38.6%). Our results support that hrHPV genotyping during cervical cancer screening may help identify women at highest risk of CIN3+.     

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.     

High-risk HPV type-specific clearance rates in cervical screening

We assessed clearance rates of 14 high-risk human papillomavirus (hrHPV) types in hrHPV-positive women with normal cytology and borderline/mild dyskaryosis (BMD) in a population-based cervical screening cohort of 44,102 women. The 6-month hrHPV type-specific clearance rates, that is, clearance of the same type as detected at baseline, in women with normal and BMD smears were 43% (95% confidence interval (CI) 39-47) and 29% (95% CI 24-34), respectively. Corresponding 18-month clearance rates were markedly higher, namely 65% (95% CI 60-69) and 41% (95% CI 36-47), respectively. The lowest clearance rates in women with normal cytology were observed for HPV16, HPV18, HPV31, and HPV33. Significantly reduced 18-month clearance rates at a significance level of 1% were observed for HPV16 (49%, 95% CI 41-59) and HPV31 (50%, 95% CI 39-63) in women with normal cytology, and for HPV16 (19%, 95% CI 12-29) in women with BMD. Among women who did not clear hrHPV, women with HPV16 persistence displayed an increased detection rate of >or=CIN3 (normal P<0.0001; BMD, P=0.005). The type-specific differences in clearance rates indicate the potential value of hrHPV genotyping in screening programs. Our data support close surveillance (i.e. referral directly, or within 6 months) of women with HPV16 and are inconclusive for surveillance of women with HPV18, HPV31, and HPV33. For the other hrHPV-positive women, it seems advisable to adopt a conservative management with a long waiting period, as hrHPV clearance is markedly higher after 18 months than after 6 months and the risk for >or=CIN3 is low.     

Human papillomavirus type-specific 18-month risk of high-grade cervical intraepithelial neoplasia in women with a normal or borderline/mildly dyskaryotic smear.

INTRODUCTION: High-risk human papillomavirus (hrHPV) DNA testing is an increasingly used instrument in cervical cancer prevention along cervical cytology. The inclusion of hrHPV testing in cervical screening requires efficient management as many hrHPV infections are transient. We investigated the potential value of hrHPV genotyping in normal and borderline/mildly dyskaryotic (BMD) smears. MATERIALS AND METHODS: From a screening population of 44,102 women in the Netherlands, we included hrHPV-positive women with a normal or BMD smear. We assessed the type-specific 18-month risk of high-grade cervical intraepithelial neoplasia (CIN). RESULTS: In hrHPV-positive women, 18-month risk of CIN grade 3 or invasive cancer (> or =CIN3) was 6% [95% confidence interval (95% CI), 4-9] after normal cytology and 20% (95% CI, 16-25) after BMD. If positive for HPV16, > or =CIN3 risks were 14% (95% CI, 9-21) and 37% (95% CI, 28-48), respectively. In the subset of hrHPV-positive women without HPV16, HPV18 was associated with an increased risk of high-grade CIN after normal cytology and HPV31 and HPV33 were associated with an increased risk, particularly after BMD. HPV16 and HPV18 were also associated with an increased risk of high-grade CIN in women with an hrHPV-positive normal baseline smear and a repeat normal smear at 6 months. DISCUSSION: HrHPV-positive women without type 16, 18, 31, or 33 had a relatively low risk of high-grade CIN. Among women with baseline normal cytology and among women with a baseline and repeat normal smear, HPV16/18-positive women showed an increased risk of high-grade CIN. This warrants more aggressive management of HPV16/18-positive women compared with other hrHPV-positive women.     

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.     

Physical state and viral load as predictive biomarkersfor persistence and progression of HPV16-positive cervical lesions: results from a population based long-term prospective cohort study

Persistent infection with a high risk (hr) human papillomavirus (HPV) has been established as the main cause of cervical cancer and high-grade cervical intraepithelial neoplasia (CIN3). Because most infections are transient, testing for hrHPV lacks specificity and has a low positive predictive value. It has been suggested that additional parameters like viral load and physical status of the viral genome could improve the effectiveness of HPV-based screening. We investigated the association between HPV16 viral load and physical state with viral persistence or risk of incident CIN3 or worse in a population-based prospective cohort study comprising 8656 women (20-29 years). All participants had two gynecological examinations two years apart and were followed through the nationwide Danish Pathology Data Bank (median follow-up: 12.9 yrs). Seventynine cervical swabs from women with a persistent HPV16 infection were available for analysis. For comparison we selected a random age-matched sample of transiently HPV16 infected women (N=91). Persistently infected women with incident CIN3 or cancer (CIN3+; N=31) were compared to women with normal cytology during follow up (non-progressors; N=39). Quantitative real-time PCR for HPV16E6, E2 and IFNb1 was done to determine the HPV16 viral load and the E2/E6 ratio was used as a surrogate marker for integration. Women with normal cytology who became persistently HPV16 infected had a significantly lower HPV16 load at baseline than women who cleared the infection (median 4.72 copies/cell versus median 20.0 copies/cell, respectively; p=0.0003). There was no difference in viral load at enrollment between women who progressed to CIN3+ and women who stayed cytologically normal (p=0.85). At the second examination viral load tended to be higher in women who progressed, but the difference was not statistically significant (p=0.39). The E2/E6 ratio was shown to be lower in the persistently infected group (p<0.0001) already at the first examination, but no difference between non-progressors and CIN3+ cases was observed at any of the two examinations (p=0.61 and 0.86). Lower viral load and integration of the viral genome are predictive for the persistence of HPV16 DNA, but not for the progression of a persistent HPV16 infection to CIN3+ in women with normal cytology.     

Guidelines for human papillomavirus DNA test requirements for primary cervical cancer screening in women 30 years and older

Given the strong etiologic link between high-risk HPV infection and cervical cancer high-risk HPV testing is now being considered as an alternative for cytology-based cervical cancer screening. Many test systems have been developed that can detect the broad spectrum of hrHPV types in one assay. However, for screening purposes the detection of high-risk HPV is not inherently useful unless it is informative for the presence of high-grade cervical intraepithelial neoplasia (CIN 2/3) or cancer. Candidate high-risk HPV tests to be used for screening should reach an optimal balance between clinical sensitivity and specificity for detection of high-grade CIN and cervical cancer to minimize redundant or excessive follow-up procedures for high-risk HPV positive women without cervical lesions. Data from various large screening studies have shown that high-risk HPV testing by hybrid capture 2 and GP5+/6+-PCR yields considerably better results in the detection of CIN 2/3 than cytology. The data from these studies can be used to guide the translation of high-risk HPV testing into clinical practice by setting standards of test performance and characteristics. On the basis of these data we have developed guidelines for high-risk HPV test requirements for primary cervical screening and validation guidelines for candidate HPV assays.     

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.     

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.     

POBASCAM, a population-based randomized controlled trial for implementation of high-risk HPV testing in cervical screening: design, methods and baseline data of 44,102 women

Cytological cervical screening is rather inefficient because of relatively high proportions of false negative and false positive smears. To evaluate the efficiency of high-risk human papillomavirus (hrHPV) testing, by GP5+/6+ PCR-enzyme immunoassay (EIA), in conjunction with cytology (Intervention Group) to that of the classical cytology (Control Group), we initiated the Population Based Screening Study Amsterdam (POBASCAM). POBASCAM is a population-based randomized controlled trial for implementation of hrHPV testing in cervical screening. The outcome measure is the proportion of histologically confirmed > or =CIN3 lesions in each study arm up to and including the next screening round after 5 years. We present the design, methods and baseline data of POBASCAM. When, in the next 5 years, the follow-up will be completed, the data obtained will be used in model studies, including a cost-effectiveness study, to advise the Dutch Ministry of Public Health in deciding whether cervical screening should be based on combined hrHPV and cytology testing instead of cytology alone. Between January 1999 and September 2002, 44,102 women (mean age = 42.8 years; range = 29-61) that participated in the regular Dutch screening program were included in our study. In the Intervention Group the distribution of cytology and hrHPV by cytology class was as follows: normal cytology 96.6% (3.6% hrHPV positive); borderline and mild dyskaryosis (BMD) 2.5% (34.6% hrHPV positive); and moderate dyskaryosis or worse (>BMD) 0.8% (88.3% hrHPV positive), i.e., 0.4% moderate dyskaryosis (82.9% hrHPV positive), 0.3% severe dyskaryosis (92.5% hrHPV positive), 0.1% carcinoma in situ (95.2% hrHPV positive), <0.1% suspected for invasive cancer (hrHPV positive 100.0%). In the Control Group 96.5% of the women had normal cytology, 2.4% BMD and 0.8% >BMD, i.e., 0.4% moderate dyskaryosis, 0.3% severe dyskaryosis, 0.1% carcinoma in situ, <0.1% suspected for invasive cancer. The presence of hrHPV was age-dependent, decreasing from 12.0% at 29-33 years to 2.4% at 59-61 years. Among women with a positive hrHPV test, the prevalence of BMD was age-dependent ranging from 20.2% at 29-33 years to 7.8% at 54-58 years. In contrast, the risk of >BMD of 13.7% among women with a positive hrHPV test was not age-dependent. Our study indicates that large-scale hrHPV testing by GP5+/6+ PCR-EIA in the setting of population-based cervical screening is practically feasible, is accepted by both participating women and general practitioners and yields highly reproducible results.     

Human papillomavirus 16 load in normal and abnormal cervical scrapes: an indicator of CIN II/III and viral clearance

The relation between human papillomavirus type 16 (HPV 16) viral load in cervical scrapes and development of high-grade cervical intraepithelial neoplasia (CIN II or III) was studied in a nested case-control study of women with normal cytology (group A) and in a cohort of women with abnormal cytology (group B). HPV 16 DNA load was determined using a quantitative real-time PCR assay. In group A, case women (women with CIN II/III, n = 12) had a significantly higher viral load than control women (women with CIN < or = I, n = 47). This resulted in an increased relative risk of women with the 50% highest viral load for development of CIN II/III (OR 7.7; CI 1.6-33). In group B, women with CIN II/III (n = 38) had a significantly higher viral load than women with CIN < or = I (n = 25). Women with the 50% highest viral load had an increased relative risk of CIN II/III (OR 3.2; CI 1.1-9.3) and a decreased chance of both viral clearance and cytologic regression. Our data suggest that in women with normal cytology an increased HPV 16 load confers an increased risk of developing a CIN lesion. A sustained high viral load is subsequently informative for progression to a high-grade CIN lesion.     

高危型 HPV 载量与分型检测对宫颈高级别病变预测价值的前瞻性队列研究*

目的:评价高危型人乳头瘤病毒（highriskhumanpapillomavirus,hrHPV ）载量和分型检测在中国农村地区妇女人群中预测宫颈鳞状上皮高级别病变发生的价值。方法:2012年5 月至2015年5 月以农村妇女人群为基础选取江西省兴国县、靖安县和玉山县2 257 例,年龄35～64岁纳入本前瞻性队列研究。同时采用HC- 2（hybridcapture-2）和导流杂交技术（HybriMax ）两种方法分别检测hrHPV 载量和亚型,两种方法中任一亚型阳性者行阴道镜及活检检查,并将HC- 2 检测阳性结果中病毒载量< 10.0 RLU/CO认定为低病毒载量,病毒载量≥ 10.0 RLU/CO为高病毒载量。对hrHPV 结果阴性或病理诊断为CIN 1 的2 211 例妇女行24个月无干预随访。根据随访分别评价hrHPV 载量和HybriMax 分型两种检测方法预测宫颈鳞状上皮高级别病变（CINgrade2 orworse,CIN 2 +）的效果。结果:纳入基线、随访数据完整的女性共1 636 例。2 年内采用HC- 2 检测的132 例基线高病毒载量妇女中CIN 2 +的发生率为3.03%（4/ 132）,其相对危险度（RR）值为42.24（95%CI 为4.76～375.2）;采用HybriMax 分型检测的159 例基线分型HPV 16或18型阳性妇女中CIN 2 + 的发生率为2.51%（4/ 159）,RR值为33.06（95%CI 为3.72～293.9）。 对2 年内HC- 2 检测中高病毒载量例数和HybriMax 分型检测中HPV 16/ 18型别阳性例数进行比较,CIN 2 + 的发病率差异无统计学意义（P > 0.05）。 结论:HPV高载量和HPV 16/ 18型别阳性妇女人群进展为CIN 2 + 的风险均较高。在不具有持续监测hrHPV 条件的农村地区,HC- 2 检测的病毒载量≥ 10.0 RLU/CO阈值设定,与HybriMax 分型检测HPV 16/ 18型别均对hrHPV 初筛有分流作用,并对CIN 2+ 发生有预测作用。      

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.     

The clinical value of HPV genotyping in triage of women with high‐risk‐HPV‐positive self‐samples

Cytology alone, or combined with HPV16/18 genotyping, might be an acceptable method for triage in hrHPV‐cervical cancer screening. Previously studied HPV‐genotype based triage algorithms are based on cytology performed without knowledge of hrHPV status. The aim of this study was to explore the value of hrHPV genotyping combined with cytology as triage tool for hrHPV‐positive women. 520 hrHPV‐positive women were included from a randomised controlled self‐sampling trial on screening non‐attendees (PROHTECT‐3B). Eighteen baseline triage strategies were evaluated for cytology and hrHPV genotyping (Roche Cobas 4800) on physician‐sampled triage material. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), referral rate, and number of referrals needed to diagnose (NRND) were calculated for CIN2+ and CIN3+. A triage strategy was considered acceptable if the NPV for CIN3+ was ≥98%, combined with maintenance or improvement of sensitivity and an increase in specificity in reference to the comparator, being cytology with a threshold of atypical cells of undetermined significance (ASC‐US). Three triage strategies met the criteria: HPV16+ and/or ≥LSIL; HPV16+ and/or ≥HSIL; (HPV16+ and/or HPV18+) and/or ≥HSIL. Combining HPV16+ and/or ≥HSIL yielded the highest specificity (74.9%, 95% CI 70.5–78.9), with a sensitivity (94.4%, 95% CI 89.0–97.7) similar to the comparator (93.5%, 95% CI 87.7–97.1), and a decrease in referral rate from 52.2% to 39.5%. In case of prior knowledge of hrHPV presence, triage by cytology testing can be improved by adjusting its threshold, and combining it with HPV16/18 genotyping. These strategies improve the referral rate and specificity for detecting CIN3+ lesions, while maintaining adequate sensitivity.     

Approaches to triage optimization in HPV primary screening: Extended genotyping and p16/Ki-67 dual-stained cytology—Retrospective insights from ATHENA

The objective of our study was to assess the performance of different triage strategies for high-risk human papillomavirus (hrHPV)-positive results utilizing either extended genotyping or a p16/Ki-67 dual-stained cytology (DS) approach, with or without partial genotyping. A subset of women with hrHPV infections participating in the Addressing the Need for Advanced HPV Diagnostics (ATHENA) study were analyzed to determine the number of cervical intraepithelial neoplasia grade 3 or worse (≥CIN3) cases detected, and the absolute risk for ≥CIN3 of each genotype. A clinical utility table was constructed to compare the impact of different triage strategies. In all, 2,339 women with single-genotype hrHPV infections were identified. Among these were 171 ≥CIN3 cases. The U.S. Food and Drug Administration (FDA)-approved algorithm (HPV16/18 positive, or 12-other hrHPV positive and Pap positive, i.e., ≥ atypical squamous cells of undetermined significance) for primary HPV screening detected 132/171 (77.2%) ≥CIN3 cases and required 964 colposcopies (colposcopies per ≥CIN3 ratio: 7.3). An approach that uses DS instead of cytology in the FDA-approved algorithm detected 147/171 (86.0%) ≥CIN3 cases, requiring 1,012 colposcopies (ratio: 6.9). Utilizing DS for triage of all hrHPV-positive women identified 126/171 (73.7%) ≥CIN3 cases, requiring 640 colposcopies (ratio: 5.1). A strategy that detected HPV16/18/31/33/35+ captured 130/171 (76.0%) ≥CIN3 cases, requiring 1,025 colposcopies (ratio: 7.9). Inclusion of additional genotypes resulted in greater disease detection at the expense of higher colposcopy ratios. Substituting cytology with a DS triage approach improved disease detection and the colposcopy detection rate. Further reduction of colposcopy rates can be achieved by using DS without partial genotyping. Extended genotyping strategies can identify a comparable number of cases but requires an increased number of colposcopies.     

hrHPV DNA载量分型和E6蛋白对宫颈癌前病变进展的预测作用

  目的  探讨预测高危型人乳头瘤病毒（high risk human papillomavirus,hrHPV）阳性和宫颈上皮内瘤样病变1级（cervical intraepithelial neoplasia grade 1,CIN1）妇女进展的生物学标志物。  方法  2010年10月至2012年8月在山西省、河南省和江西省招募7 543名妇女,采用hrHPV DNA检测和E6蛋白检测（Onco E6）等方法进行宫颈癌筛查,任一结果阳性者转诊阴道镜并取活检,于1年后随访。纳入基线hrHPV阳性或病理诊断CIN1者。  结果  共纳入794例满足条件妇女,1年后88例妇女病理级别发生进展。基线hrHPV DNA中高载量者进展的风险是低载量者的2.9倍（95%CI为1.8~4.8）,hrHPV16/18/45、E6蛋白阳性者进展的风险分别是阴性者的2.4倍（95%CI为1.5~3.9）、2.9倍（95%CI为1.5~5.9）,其进展至宫颈上皮内瘤样病变2级及以上（cervical intraepithelial neoplasia grade 2 or worse,CIN2+）的绝对风险分别为4.9%（95%CI为3.2~7.4）、9.0%（95%CI为5.5~14.3）、18.8%（95%CI为10.2~31.9）。hrHPV16/18/45中高载量且E6蛋白（Onco E6）阳性妇女进展的绝对风险高达32.4%（11/34）。  结论  hrHPV DNA中高载量、hrHPV16/18/45分型以及E6蛋白（Onco E6）可作为妇女hrHPV阳性和CIN1进展的生物学标志物,特别是hrHPV中高载量且E6蛋白（Onco E6）阳性妇女,临床应给予密切随访。