Triaging HPV‐positive women with normal cytology by p16/Ki‐67 dual‐stained cytology testing: Baseline and longitudinal data

Primary human papillomavirus (HPV)‐based screening results in a 2–5% lower specificity for cervical intraepithelial neoplasia Grade 2 or worse (CIN2+) compared to Pap cytology. To identify HPV‐positive women with CIN2+, we retrospectively evaluated the cross‐sectional and longitudinal performance of p16/Ki‐67 dual‐stained cytology in HPV‐positive women with normal cytology participating in population‐based cervical screening. Conventional Pap cytology specimens of 847 of these women derived from the VUSA‐Screen study were dual‐stained for p16/Ki‐67. Cross‐sectional clinical performance in detecting CIN3 or worse (CIN3+), and CIN2+ was compared to that of baseline HPV genotyping. Moreover, 5‐year cumulative incidence risks (CIR) for CIN3+ (CIN2+) were determined. The sensitivity of p16/Ki‐67 dual‐stained cytology for CIN3+ (CIN2+) was 73.3% (68.8%) with a specificity of 70.0% (72.8%). HPV16/18 genotyping showed a sensitivity for CIN3+ (CIN2+) of 46.7% (43.8%), with a specificity of 78.3% (79.4%). The 5‐year CIR for CIN3+ in HPV‐positive women with normal cytology was 6.9%. Testing these women with p16/Ki‐67 dual‐stained cytology resulted in a significantly lower CIN3+ 5‐year CIR of 3.3% (p = 0.017) in case of a negative test result. A negative HPV16/18 genotyping test result also led to a lower 5‐year CIN3+ CIR of 3.6%. p16/Ki‐67 dual‐stained cytology detects more than 70% of underlying CIN3+ lesions in HPV‐positive women with normal cytology at baseline and is therefore suitable for triaging these women to colposcopy. Furthermore, the CIN3+ 5‐year CIR of 3.3% after a negative dual‐stain result is significantly lower compared to the 5‐year CIR of 6.9% in women without p16/Ki‐67 dual‐stained cytology triage.     

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+.     

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.     

Post‐treatment CIN: Randomised clinical trial using hrHPV testing for prediction of residual/recurrent disease

We investigated in a randomised clinical trial whether addition of hrHPV testing (high‐risk human papillomavirus) to cytological follow‐up after treatment for high‐grade CIN (cervical intraepithelial neoplasia 2/3) can lead to a better selection of women at risk for residual/recurrent CIN. We included 210 women with high‐grade CIN undergoing treatment in outpatient clinics in The Netherlands. Follow‐up was based on cytology alone and cytology combined with hrHPV detection. Our primary outcome measurement was improving specificity for residual/recurrent CIN after treatment. Secondary, we compared health‐care costs and impact of individual hrHPV type on the risk of residual/recurrent CIN. Follow‐up by abnormal cytology alone (6, 12 and 24 months after treatment according to the Dutch protocol) showed a lower specificity for detection of residual/recurrent CIN than follow‐up by abnormal cytology and presence of hrHPV (80 vs. 91%, relative risk 0.87 (95% CI 0.77–0.99)). Both methods showed no significant difference in sensitivity ((86 vs. 100%) RR 0.86 (95% CI 0.63–1.16)). Comparing different post hoc modifications in the strategy of combined testing showed similar test characteristics when low‐risk women (normal cytology and hrHPV negative at 6 months) omitted the 12 months visit (specificity 91%, p = 1.00 z = 0.00). Prediction of residual/recurrent CIN by typing of hrHPV could not be confirmed. Total health‐care costs using cytology and hrHPV testing during follow‐up decreased when low‐risk women omit the 12 months visit. Follow‐up after treatment for high‐grade CIN can be improved by combining cytology with hrHPV testing. We advise combined cytology and hrHPV testing at 6, 12 and 24 months after treatment. Low‐risk women may omit the 12 months visit, resulting in cost reduction. © 2008 Wiley‐Liss, Inc.     

Human papillomavirus testing versus cytology in primary cervical cancer screening: End‐of‐study and extended follow‐up results from the Canadian cervical cancer screening trial

The Canadian Cervical Cancer Screening Trial was a randomized controlled trial comparing the performance of human papillomavirus (HPV) testing and Papanicolaou cytology to detect cervical intraepithelial neoplasia of grades 2 or worse (CIN2+) among women aged 30–69 years attending routine cervical cancer screening in Montreal and St. John's, Canada (n = 10,154). We examined screening and prognostic values of enrollment cytologic and HPV testing results. Extended follow‐up data were available for St. John's participants (n = 5,754; 501,682.6 person‐months). HPV testing detected more CIN2+ than cytology during protocol‐defined (82.9 vs. 44.4%) and extended (54.2 vs. 19.3%) follow‐up periods, respectively. Three‐year risks ranged from 0.87% (95% CI: 0.37–2.05) for HPV‐/Pap‐ women to 35.77% (95% CI: 25.88–48.04) for HPV+/Pap+ women. Genotype‐specific risks ranged from 0.90% (95% CI: 0.40–2.01) to 43.84% (95% CI: 32.42–57.24) among HPV? and HPV16+ women, respectively, exceeding those associated with Pap+ or HPV+ results taken individually or jointly. Ten‐year risks ranged from 1.15% (95% CI: 0.60–2.19) for HPV?/Pap? women to 26.05% (95% CI: 15.34–42.13) for HPV+/Pap+ women and genotype‐specific risks ranged from 1.13% (95% CI: 0.59–2.14) to 32.78% (95% CI: 21.15–48.51) among women testing HPV? and HPV16+, respectively. Abnormal cytology stratified risks most meaningfully for HPV+ women. Primary HPV testing every 3 years provided a similar or greater level of reassurance against disease risks as currently recommended screening strategies. HPV‐based cervical screening may allow for greater disease detection than cytology‐based screening and permit safe extensions of screening intervals; genotype‐specific testing could provide further improvement in the positive predictive value of such screening.     

p16/ki-67 dual-stain cytology in the triage of ASCUS and LSIL papanicolaou cytology: results from the European equivocal or mildly abnormal Papanicolaou cytology study

BACKGROUND:

The objective of this study was to analyze the diagnostic performance of a newly established immunocytochemical dual‐stain protocol, which simultaneously detects p16^INK4a and Ki‐67 expression in cervical cytology samples, for identifying high‐grade cervical intraepithelial neoplasia (CIN2+) in women with Papanicolaou (Pap) cytology results categorized as atypical squamous cells of undetermined significance (ASCUS) or low‐grade squamous intraepithelial lesions (LSIL).

METHODS:

Residual liquid‐based cytology material from 776 retrospectively collected ASCUS/LSIL cases that were available from a recent study evaluating p16 cytology and HPV testing were subjected to p16/Ki‐67 dual staining. The presence of 1 or more double‐immunoreactive cell(s) was regarded as a positive test outcome, irrespective of morphology. Test results were correlated to histology follow‐up.

RESULTS:

Sensitivity of p16/Ki‐67 dual‐stain cytology for biopsy‐confirmed CIN2+ was 92.2% (ASCUS) and 94.2% (LSIL), while specificity rates were 80.6% (ASCUS) and 68.0% (LSIL), respectively. Similar sensitivity/specificity profiles were found for both age groups of women aged <30 years versus women aged ≥30 years. Dual‐stain cytology showed comparable sensitivity, but significantly higher specificity, when compared with human papillomavirus (HPV) testing.

CONCLUSIONS:

The results of this study show that p16/Ki‐67 dual‐stain cytology provided a high sensitivity for the detection of underlying CIN2+ in women with ASCUS or LSIL Pap cytology results, comparable to the rates previously reported for HPV testing and p16 single‐stain cytology. However, the specificity of this morphology‐independent interpretation of p16/Ki‐67 dual‐stain cytology testing was further improved compared with the earlier p16 single‐stain cytology approach, which required morphology interpretation, and it is significantly higher when compared with HPV testing. Cancer (Cancer Cytopathol) 2011;. © 2011 American Cancer Society.     

Interlaboratory variation in the performance of liquid‐based cytology: Insights from the ATHENA trial

Although it is recognized that cervical cytology is highly subjective, and that there is considerable interlaboratory variation in how slides are evaluated, little is known as to how this impacts the performance of cytology. In the ATHENA trial, liquid‐based cytology specimens from 46,887 eligible women ≥21 years of age were evaluated at four large regional US laboratories, providing a unique opportunity to evaluate the impact of interlaboratory variations on the performance of cervical cytology. All women with abnormal cytology (atypical squamous cells of undetermined significance or higher) were referred to colposcopy, as were all high‐risk human papillomavirus (hrHPV)–positive women ≥25 years of age and a random subset of those ≥25 years of age who were negative by both hrHPV testing and cytology. Sociodemographics, risk factors for cervical disease, and prevalence of cervical intraepithelial neoplasia (CIN) were similar across the laboratories. There were considerable differences among the laboratories both in overall cytological abnormal rates, ranging from 3.8 to 9.9%, and in sensitivity of cytology to detect CIN grade 2 or worse (CIN2+), from 42.0 to 73.0%. In contrast, the hrHPV positivity rate varied only from 10.9 to 13.4%, and the sensitivity of hrHPV testing from 88.2 to 90.1%. These observations suggest that hrHPV testing without cytology should be considered as the initial method for cervical cancer screening.     

Comparative performance of novel self‐sampling methods in detecting high‐risk human papillomavirus in 30,130 women not attending cervical screening

We determined whether the participation rate for a brush‐based cervicovaginal self‐sampling device is noninferior to the participation rate for a lavage‐based one for testing for hrHPV (high‐risk human papillomavirus). Additionally, positivity rates for hrHPV, the detection rates for cervical intraepithelial neoplasia grades 2 and 3 or worse (CIN2+/3+), and user comfort were compared. A total of 35,477 non‐responders of the regular cervical screening program aged 33–63 years were invited to participate. Eligible women (n = 30,130) were randomly assigned to receive either a brush‐based or a lavage‐based device, and a questionnaire for reporting user convenience. Self‐sampling responders testing hrHPV‐positive were invited for a physician‐taken sample for cytology; triage‐positive women were referred for colposcopy. A total of 5,218 women participated in the brush‐based sampling group (34.6%) and 4809 women in the lavage‐based group (31.9%), i.e. an absolute difference of 2.7% (95%CI 1.8–4.2). The hrHPV‐positivity rates in the two groups were identical (8.3%, relative risk (RR) 0.99, 95%CI 0.87–1.13). The detection of CIN2+ and CIN3+ in the brush group (2.0% for CIN2+; 1.3% for CIN3+) was similar to that in the lavage group (1.9% for CIN2+; 1.0% for CIN3+) with a cumulative RR of 1.01, 95%CI 0.83–1.24 for CIN2+ and 1.25, 95%CI 0.92–1.70 for CIN3+. The two self‐sampling devices performed similarly in user comfort. In conclusion, offering a brush‐based device to non‐responders is noninferior to offering a lavage‐based device in terms of participation. The two self‐sampling methods are equally effective in detecting hrHPV, CIN2+/CIN3+ and are both well accepted.     

Human papillomavirus testing on self-sampled cervicovaginal brushes: an effective alternative to protect nonresponders in cervical screening programs

Women not attending cervical screening programs are at increased risk of cervical cancer. We investigated in these nonresponders to what extent offering self-sampling devices for cervicovaginal brushes for high-risk human papillomavirus (hrHPV) testing would induce participation and, if so, what the yield of precursor (i.e. CIN2 or worse) lesions following self-sampling would be. In addition, we assessed screening history of participants and costs per detected high-grade CIN2 or worse ("CIN2+") lesion in comparison to the regular program in the Netherlands. Nonresponders received a device for hrHPV testing (self-sampling group, n=2,546) or an extra recall for conventional cytology (control group, n=284). The percentage of self-sampling responders were compared with responders in the recall group. hrHPV positive self-sampling responders were invited for cytology and colposcopy. CIN2+ yield and costs per detected CIN2+ were evaluated. Active response was higher in the self-sampling than in the control group (34.2 vs. 17.6%; p<0.001). hrHPV positive self-sampling responders were less likely to have a prior screening history than screening participants (p<0.001), indicating that they are regular nonresponders. hrHPV prevalence was similar (8.0 vs. 6.8%; p=0.11), but CIN2+ yield was higher in self-sampling responders compared to screening participants (1.67 vs. 0.97%; OR=2.93, 95% CI 1.48-5.80; p=0.0013). Costs per CIN2+ lesion detected via self-sampling were in the same range as those calculated for conventional cytological screening (euro 8,836 vs. euro 7,599). Offering self-sampling for hrHPV testing in nonresponders is an attractive adjunct to effectively increase population coverage of screening without the adverse effect of markedly increased costs per detected CIN2+ lesion.     

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.     

Prognostic value of p16‐INK4A protein in women with negative or CIN1 histology result: A follow‐up study

P16‐INK4A overexpression has been proposed as a prognostic marker to manage the follow up of women with positive cytology and/or HPV test but without high‐grade cervical intraepithelial neoplasia (CIN2+). This study measures the relative risk (RR) of CIN2+ of p16 positive versus negative in these women. All the women referred to colposcopy from October 2008 to September 2010 with negative or CIN1 colposcopy‐guided biopsy were included in the study; women surgically treated or having a CIN2–3 were excluded. All baseline biopsies were dyed with hematoxylin and eosin and p16. Women were followed up according to screening protocols, with cytology or colposcopy at 6 or 12 months. CIN2/3 RRs and 95% confidence intervals (95%CI) were computed. Of 442 eligible women, 369 (83.5%) had at least one follow‐up episode. At baseline, 113 (30.6%) were CIN1, 248 (67.2%) negative, and 8 (2.2%) inadequate histology; 293 (79.4%) were p16‐negative, 64 (17.3%) p16 positive and 12 (3.2%) not valid. During follow up, we found ten CIN2 and three CIN3; of these, six were p16 positive (sensitivity 46%, 95% CI 19–75). The absolute risk among p16 positives was 9.4/100 compared to 1.7/100 of the p16 negatives (RR 5.5; 95% CI 1.7–17.4). The risk was also higher for CIN1 than for histologically negative women (RR 4.4; 95% CI 1.3–14.3). The RR for p16 in CIN1 did not change (RR 5.2; 95% CI 0.6–47.5). P16 overexpression is a good candidate for modulating follow‐up intensity after a negative colposcopy but is limited by its low prospective sensitivity.     

High‐risk HPV testing in the management of atypical glandular cells: A systematic review and meta‐analysis

Whereas the utility of high‐risk HPV (hrHPV) testing is widely accepted in triage of women with atypical squamous lesions, its role in managing atypical glandular cells (AGC) is not fully elucidated. A systematic review and meta‐analysis were performed to evaluate the accuracy of hrHPV testing in the management of women with AGC to detect underlying high‐grade intraepithelial neoplasia or worse, and adenocarcinoma in situ or worse (AIS+). Additionally, the diagnosis of extra‐cervical cancer was considered as an outcome in this review. A bibliographic database search (PubMed, EMBASE, CENTRAL) identified twelve eligible studies. The occurrence of cervical intraepithelial neoplasia grade two or worse including AIS+ (CIN2+/AIS+), was 19.8% among women with AGC, and 55.7% among women with AGC and concurrent squamous lesions (atypical squamous cells of undetermined significance or worse, ASC‐US+). The pooled sensitivity and specificity of hrHPV‐testing with Hybrid Capture 2 (HC2) to detect CIN2+/AIS+ in women with AGC was 90.0% (95% CI = 85.1–93.4%) and 75.1% (95% CI = 64.8–83.2%), respectively. Women who were hrHPV‐negative, demonstrated an increased risk for extra‐cervical malignancy (endometrium, fallopian tube, ovary). In women of 50y and older, a hrHPV‐negative result was linked with a 18.0% chance of extra‐cervical malignancy, while the chance of cervical pre‐cancer and cancer was 0.4 and 0.0%, respectively. In conclusion, given the high risk of underlying CIN2+/AIS+, women with AGC should be referred directly to colposcopy. However, hrHPV test results in combination with the age, appears to improve the diagnostic process by distinguishing the risk for cervical versus non‐cervical lesions.     

Human papilloma virus genotyping for the cross‐sectional and longitudinal probability of developing cervical intraepithelial neoplasia grade 2 or more

Human papilloma virus (HPV) testing is more sensitive but less specific than cytology. We evaluated stand‐alone genotyping as a possible triage method. During a multicentre randomised controlled trial comparing HPV testing to conventional cytology, HPV‐positive women were referred to colposcopy and followed up if no high‐grade lesion was detected. HPV‐positive samples were genotyped by GP5+/GP6+ primed polymerase chain reaction followed by reverse line blot. Genotypes were hierarchically ordered by positive predictive value (PPV) for CIN grade 2 or more (CIN2+), and grouped by cluster analysis into three groups (A, B and C in decreasing order). Receiver operating characteristic curves were computed. Among 2,255 HPV‐positive women with genotyping, 239 CIN2+ (including 113 CIN3+) were detected at baseline or during a 3‐year follow‐up. HPV33 had the highest PPV with CIN2+ and CIN3+ as the endpoint and when considering lesions detected at baseline or also during follow‐up. HPV16 and HPV35 were the second and third, respectively. Cross‐sectional sensitivity for CIN2+ at baseline was 67.3% (95% CI 59.7–74.2), 91.8% (95% CI 86.6–95.5) and 94.7% (95% CI 90.2–97.6), respectively, when considering as “positive” any of the HPV types in group A (33, 16 and 35), A or B (31, 52, 18, 59 and 58) and A or B or C (39, 51, 56, 45 and 68). The corresponding cross‐sectional PPVs for CIN2+ were 15.8% 95% (CI 13.2–18.7), 12.0% (95% CI 10.3–13.9) and 9.6% (95% CI 8.2–11.1), respectively. HPV33, 16 and 35 confer a high probability of CIN2+ but this rapidly decreases when adding other genotypes.     

Human papillomavirus mRNA and DNA testing in women with atypical squamous cells of undetermined significance: A prospective cohort study

In this prospective cohort study, we compared the performance of human papillomavirus (HPV) mRNA and DNA testing of women with atypical squamous cells of undetermined significance (ASC‐US) during cervical cancer screening. Using a nationwide Danish pathology register, we identified women aged 30–65 years with ASC‐US during 2005–2011 who were tested for HPV16/18/31/33/45 mRNA using PreTect HPV‐Proofer (n = 3,226) or for high‐risk HPV (hrHPV) DNA using Hybrid Capture 2 (HC2) (n = 9,405) or Linear Array HPV‐Genotyping test (LA) (n = 1,533). Women with ≥1 subsequent examination in the register (n = 13,729) were followed for up to 9.5 years for high‐grade cervical intraepithelial neoplasia (CIN) or cancer. After 3 years' follow‐up, mRNA testing had higher specificity for CIN3 or worse (CIN3+) than HC2 testing (88.1% [95% confidence interval (CI): 86.8–89.6%] versus 59.3% [95% CI: 58.1–60.4%]) and higher positive predictive value (PPV) (38.2% [95% CI: 33.8%–43.1%] versus 19.5% [95% CI: 17.8–20.9%]). However, the sensitivity of mRNA testing was lower than that of HC2 testing (66.7% [95% CI: 59.3–74.5%] versus 97.0% [95% CI: 95.5–98.4%]), and women testing mRNA negative had higher 3‐year risk for CIN3+ than those testing HC2 negative (3.2% [95% CI: 2.2–4.2%] versus 0.5% [95% CI: 0.3–0.7%]). Patterns were similar after 18 months and 5 years'; follow‐up; for CIN2+ and cancer as outcomes; across all age groups; and when comparing mRNA testing to hrHPV DNA testing using LA. In conclusion, the HPV16/18/31/33/45 mRNA test is not optimal for ASC‐US triage due to its low sensitivity and the substantial risk for precancer following a negative test.     

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.     

Assessing the time dependence of prognostic values of cytology and human papillomavirus testing in cervical cancer screening

Accurate assessment of risks for developing cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) after a given set of screening test results is instrumental to reaching valid conclusions and informing cervical cancer screening recommendations. Using data from the Canadian Cervical Cancer Screening Trial (CCCaST), we assessed prognostic values of enrollment screening test results to predict CIN2+ among women attending routine cervical screening using multivariable Cox proportional hazards (PH) regression and its flexible extension during each of two follow-up periods (protocol-defined and extended). Nonproportional (time-dependent (TD)) and/or nonlinear effects were modeled, as appropriate. Women with abnormal cytology had hazard ratios (HRs) for CIN2+ detection of 17.61 (95% CI: 11.25–27.57) and 10.46 (95% CI: 5.41–20.24) relative to women with normal cytology during the protocol-defined and extended follow-up periods, respectively. High-risk human papillomavirus (HR-HPV) positivity was an even stronger predictor of CIN2+ risk, with significant TD effects during both follow-up periods (p <0.001 for both TD effects). Risks among women co-testing HR-HPV+ with and without abnormal cytology (relative to women co-testing negative) were highest immediately after baseline, and decreased significantly thereafter (p <0.001 for both TD effects). HRs for HPV16+ and HPV18+ women (relative to those testing HR-HPV-) did not vary significantly over time (HR = 182.96; 95% CI: 95.16–351.77 and HR = 111.81; 95% CI: 44.60–280.31, respectively). Due to TD effects, conventional Cox model estimates considerably underestimated adjusted HRs associated with positive HR-HPV testing results early on in the follow-up periods.     

The APTIMA HPV assay versus the hybrid capture 2 test in triage of women with ASC‐US or LSIL cervical cytology: A meta‐analysis of the diagnostic accuracy

Testing for DNA of 13 high‐risk HPV types with the Hybrid Capture 2 (HC2) test has consistently been shown to perform better in triage of women with cervical cytology results showing atypical squamous cells of undetermined significance (ASC‐US) but often not in triage of low‐grade squamous intraepithelial lesions (LSIL) detected in cervical cancer screening. In a meta‐analysis, we compared the accuracy of the APTIMA HPV test, which identifies RNA of 14 high‐risk HPV types, to HC2 for the triage of women with ASC‐US or LSIL. Literature search‐targeted studies where the accuracy of APTIMA HPV and HC2 for detection of underlying CIN2/3+ was assessed concomitantly including verification of all cases of ASC‐US and LSIL. HSROC (Hierarchical Summary ROC) curve regression was used to compute the pooled absolute and relative sensitivity and specificity. Eight studies, comprising 1,839 ASC‐US and 1,887 LSIL cases, were retrieved. The pooled sensitivity and specificity of APTIMA to triage ASC‐US to detect underlying CIN3 or worse was 96.2% (95% CI = 91.7–98.3%) and 54.9% (95% CI = 43.5–65.9%), respectively. APTIMA and HC2 showed similar pooled sensitivity; however, the specificity of the former was significantly higher (ratio: 1.19; 95% CI = 1.08–1.31 for CIN2+). The pooled sensitivity and specificity of APTIMA to triage LSIL were 96.7% (95% CI = 91.4–98.9%) and 38.7% (95% CI = 30.5–47.6%) for CIN3+. APTIMA was as sensitive as HC2 but more specific (ratio: 1.35; 95% CI = 1.11–1.66). Results were similar for detection of CIN2 or worse. In both triage of ASC‐US and LSIL, APTIMA is as sensitive but more specific than HC2 for detecting cervical precancer.     

Specimen self‐collection and HPV DNA screening in a pilot study of 100,242 women

Since cervical cancer remains common in Mexico despite an established cytology screening program, the Ministry of Health recently introduced pilot front‐line HPV testing into the Mexican cervical cancer screening program (CCSP). Here, we present the key field performance metrics of this population‐based study. High‐risk HPV DNA (hrHPV) testing was conducted on self‐collected vaginal specimens from 100,242 women aged 25–75 years residing in Morelos State. All hrHPV positive women and a random sample of 3.2% (n = 2,864) of hrHPV negative participants were referred for colposcopic examination. The main disease endpoint of interest was cervical intraepithelial neoplasia grade 2 or higher (CIN2+). We calculated relative risk, positive predictive value and negative predictive value adjusted for screening test verification bias. The overall prevalence of hrHPV was 10.8% (95%CI 10.6–11.0). Women positive for hrHPV had a relative risk of 15.7 for histologically detectable CIN2+. The adjusted positive predictive value of the hrHPV test was 2.4% (95%CI 2.1–2.7); whereas the adjusted negative predictive value was 99.8% (95%CI 99.8–99.9). These findings suggest that large‐scale vaginal hrHPV testing in a middle‐income country can identify women at greater risk of advanced cervical abnormalities in a programmatically meaningful way but care is warranted to ensure that disease not detectable at colposcopy is kept to a minimum. PASS shows areas that need improvement and sets the stage for wider use of hrHPV screening of self‐collected vaginal specimens in Mexico.     

Diagnostic value of p16INK4A,Ki‐67, and human papillomavirus L1 capsid protein immunochemical staining on cell blocks from residual liquid‐based gynecologic cytology specimens

BACKGROUND:

This study was conducted to evaluate the reliability and role of cell block preparations in the diagnosis of neoplastic and preneoplastic lesions of the cervix and to improve the value of cell block preparations in diagnosing and predicting the prognosis of cervical lesions through immunostaining of p16INK4A (p16), Ki‐67, and human papillomavirus (HPV) L1 capsid protein (HPV L1).

METHODS:

In total, 138 specimens were diagnosed on liquid‐based cytology (LBC) and cell block preparations, and 63 specimens were subjected subsequently to tissue follow‐up and immunostaining for p16, Ki‐67, and HPV L1 on cell block sections.

RESULTS:

In 42 specimens that were diagnosed as low‐grade squamous intraepithelial lesion, high‐grade squamous intraepithelial lesion (HSIL), and squamous cell carcinoma (SCC) on cell blocks, 38 specimens (90.5%) were confirmed by histopathologic reports, and there was slightly better than 81.6% agreement between LBC and tissue follow‐up. Immunointensity and cells that were positive for p16 were enhanced according to increased pathologic grade and differed statistically between cervical intraepithelial neoplasia 1 (CIN‐1) and CIN‐2/CIN‐3 as well as SCC. The positive rates of HPV L1 decreased gradually according to the severity of cervical neoplasia, and HPV L1/p16 expression patterns were related to the severity of cervical lesions.

CONCLUSIONS:

The cell block preparation technique was complementary to LBC, and the authors concluded that the application of LBC combined with cell block preparations may improve the diagnostic accuracy of cytology. Immunostaining for p16 and Ki‐67 on cell block preparations can help to improve the diagnostic accuracy of HSIL and SCC. A combined expression pattern of p16 and HPV L1 may serve as a valuable index for predicting prognosis and follow‐up of cervical dysplastic lesions. Cancer (Cancer Cytopathol) 2010. © 2010 American Cancer Society.     

Long‐term HPV type‐specific risks for ASCUS and LSIL: A 14‐year follow‐up of a randomized primary HPV screening trial

Human papillomavirus (HPV) infections result in a significant burden of low‐grade cervical lesions. Between 1997 and 2000, our randomized trial of primary HPV screening enrolled 12,527 women participating in population‐based screening. Women between 32 and 38 years of age (median: 34, interquartile range: 33–37) were randomized to HPV and cytology double testing (intervention arm, n = 6,257 enrolled, n = 5,888 followed‐up) or to cytology, with samples frozen for future HPV testing (control arm, n = 6,270 enrolled, n = 5,795 followed‐up). We estimated the HPV type‐specific, long‐term absolute risks (AR), and population attributable proportions (PAR) for cytological diagnoses of atypical squamous cells of undetermined significance (ASCUS) or low‐grade squamous intraepithelial lesion (LSIL) and for histopathologically diagnosed cervical intraepithelial neoplasia grade 1 (CIN1). The women were followed using comprehensive, nationwide register‐based follow‐up. During a mean follow‐up time of 11.07 years, 886 ASCUS and LSIL lesions were detected, 448 in the intervention arm and 438 in the control arm. Poisson regression estimated the incidence rate ratios (IRRs) of low‐grade lesions by HPV type. The IRRs were strongly dependent on follow‐up time. The IRRs for ASCUS/LSIL associated with high‐risk HPV positivity were 18.6 (95% CI: 14.9–23.4) during the first screening round, 4.1 (95% CI: 2.8–6.2) during the second, 2.6 (95% CI: 1.7–4.1) during the third, and 1.1 (95% CI: 0.7–1.8) for >9 years of follow‐up, with similar declines seen for the individual types. Type 16 contributed consistently to the greatest proportion of ASCUS, LSIL, and CIN1 risk in the population (first screening round PAR: ASCUS: 15.5% (95% CI: 9.7–21.9), LSIL: 14.7% (95% CI: 8.0–20.9), and CIN1: 13.4% (95% CI: 3.2–22.5)), followed by type 31 [8.4% (95% CI: 4.2–12.5) for ASCUS to 17.3% (95% CI: 6.8–26.6) for CIN1]. In summary, most ASCUS/LSIL lesions associated with HPV infection are caused by new HPV infections and most lesions are found during the first screening round.