Robert G. Pretorius

Improved sensitivity of vaginal self-collection and high-risk human papillomavirus testing

Self‐collected vaginal specimens tested for high‐risk human papillomavirus (HR‐HPV) have been shown to be less sensitive for the detection of cervical intraepithelial neoplasia or cancer (≥CIN 3) than physician‐collected endocervical specimens. To increase the sensitivity of self‐collected specimens, we studied a self‐sampling device designed to obtain a larger specimen from the upper vagina (POI/NIH self‐sampler) and a more sensitive polymerase chain reaction (PCR)‐based HR‐HPV assay. Women (10,000) were screened with cervical cytology and HR‐HPV testing of vaginal self‐collected and endocervical physician‐collected specimens. Women were randomly assigned to use either a novel self‐collection device (POI/NIH self‐sampler) or conical‐shaped brush (Qiagen). The self‐collected and clinician‐collected specimens were assayed by Cervista (Hologic) and the research only PCR‐based matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF). Women with any abnormal screening test underwent colposcopy and biopsy. Women (8,556), mean age of 38.9, had complete data; 1.6% had ≥ CIN 3. For either HR‐HPV assay, the sensitivity was similar for the two self‐collection devices. Tested with Cervista, the sensitivity for ≥CIN 3 of self‐collected specimens was 70.9% and for endocervical specimens was 95.0% (p = 0.0001). Tested with MALDI‐TOF, the sensitivity for ≥CIN 3 of self‐collected specimens was 94.3% and for endocervical specimens was also 94.3% (p = 1.0). A self‐collected sample using a PCR‐based assay with the capability of very high throughput has similar sensitivity as a direct endocervical specimen obtained by a physician. Large population‐based screening “events” in low‐resource settings could be achieved by promoting self‐collection and centralized high‐throughput, low‐cost testing by PCR‐based MALDI‐TOF.

Inappropriate gold standard bias in cervical cancer screening studies

As acetic acid‐aided visual inspection (VIA) and colposcopic‐directed biopsy miss small ≥cervical intraepithelial neoplasia (CIN) 2, inflation of sensitivity of VIA may occur when colposcopic‐directed biopsy is the gold standard for ≥CIN 2. To determine whether such inflation occurs, we reviewed 375 women with ≥CIN 2 from the Shanxi Province Cervical Cancer Screening Study II. These women had positive self or physician‐collected tests for high‐risk human papillomavirus or abnormal cervical cytology and had VIA followed by colposcopy with directed biopsy and endocervical curettage (ECC). If a cervical quadrant had no lesion, a random biopsy at the squamocolumnar junction within that quadrant was obtained. Sensitivity of colposcopic‐directed biopsy was higher for ≥CIN 2 involving 3–4 cervical quadrants (81.3%) than for ≥CIN 2 involving 0–2 quadrants (49.0%, p < 0.001). Sensitivities of VIA, cytology of ≥ASC‐US, ≥LSIL, and ≥HSIL were higher for ≥CIN 2 involving 3–4 quadrants than for ≥CIN 2 involving 0–2 quadrants. When a colposcopic‐directed biopsy gold standard was compared with that of a 5‐biopsy standard (which included ≥CIN 2 from colposcopic‐directed biopsy, random biopsy, or ECC), the sensitivity for ≥CIN 2 of VIA was inflated by 20.0% (65.9% vs. 45.9%, p < 0.001). Sensitivities of other screening tests were not affected. Similar inflation of sensitivity of VIA was found with an endpoint of ≥CIN 3 (70.4% vs. 52.0%, p = 0.0013). Inflation of sensitivity of VIA depended upon agreement between colposcopic‐directed biopsy and the screening tests as measured by kappa. Studies of VIA that used colposcopic‐directed biopsy as the gold standard require reevaluation.

Prevalence of type‐specific human papillomavirus in endocervical, upper and lower vaginal, perineal and vaginal self‐collected specimens: Implications for vaginal self‐collection

To determine why a vaginal self‐collection tested for high‐risk human papillomavirus (HR‐HPV) by Hybrid Capture 2® (hc2) has lower sensitivity and specificity for cervical intraepithelial neoplasia Grade 2 or worse (≥CIN 2), we collected 5 specimens (endocervix, upper and lower vagina, perineum, vaginal self‐collection) from 2,625 women. Endocervical and self‐collected specimens had HR‐HPV tests by hc2. All 5 anogenital specimens were tested for 37 HPV genotypes [Linear Array®, (LA)] from 397 women hc2 positive in endocervical or self‐collected specimens and for a randomly selected 71 of 2,228 women hc2 negative on both specimens. Three hundred nintey‐five women who screened positive by hc2 or had abnormal cytology underwent colposcopic evaluation. Of 47 women with ≥CIN 2, hc2 was positive in 97.9% (46/47) of endocervical and 80.9% (38/47), p = 0.008 of self‐collected specimens. Seven of 9 women with ≥CIN 2 and negative self‐collected hc2 tests were positive for HR‐HPV by LA. Of 2,578 women without ≥CIN 2, hc2 was positive in 9.8% (253/2,578) of endocervical and 11.4% (294/2,578), p = 0.001 of self‐collected specimens. Of the 41 more women without ≥CIN 2 that tested hc2 positive on the self‐collected but negative on endocervical specimen, LA tested positive for HR‐HPV in 24, negative for HPV in 11 and negative for HR‐HPV but positive for low‐risk HPV in 6. Lower sensitivity of self‐collected specimens is secondary to lower levels of vaginal HR‐HPV. The principal cause of the lower specificity of self‐collected specimens is HR‐HPV present solely in the vagina, which is not associated with ≥CIN 2.

A Population-Based Clinical Trial Comparing Endocervical High-Risk HPV Testing Using Hybrid Capture 2 and Cervista From the SHENCCAST II Study

Our objective was to directly compare the accuracy of the high-risk human papillomavirus (HPV) assays, Hybrid Capture 2 (hc2; Qiagen, Gaithersburg, MD) and Cervista (Hologic, Bedford, MA), in diagnosing cervical intraepithelial neoplasia (CIN) 3 or worse (cancer). A population-based, cross-sectional study (The Shenzhen Cervical Cancer Screening Trial II) was conducted in Guangdong Province in China. Three high-risk HPV assays, self and direct cervical sampling and cytology, were studied. Abnormal results on any of 6 study tests (33%) resulted in referral to colposcopy. At colposcopy, every patient had at least 5 cervical biopsy specimens obtained. For 8,556 women between the ages of 25 and 59 years (mean, 38.9 years), the rate for CIN 3 or worse was 1.6% (141/8,556). The sensitivity (confidence interval) values for CIN 3 or worse were 97.9% (94.0%-99.6%) and 95.1% (90.0%-98.0%) for hc2 and Cervista, respectively (P > .05). The specificity (confidence interval) values were 87.8% (87.1%-88.5%) and 90.3% (89.6%-90.9%), respectively (P < .05). Differences in accuracy in diagnosing CIN 3 or worse with the hc2 and Cervista tests are minor and result from the decisions made in selecting the cut points.

Inflation of sensitivity of cervical cancer screening tests secondary to correlated error in colposcopy.

Objective To determine whether the sensitivity of screening tests that miss small cervical intraepithelial neoplasia (CIN) 2 or worse (e.g., acetic acid-aided visual inspection) is inflated when the criterion standard (colposcopic-directed biopsy) misses the same small CIN 2 or worse. Materials and Methods One thousand nine hundred twenty-eight women were screened using acetic acid-aided visual inspection, self-tests, and direct tests for high-risk human papillomavirus, and using liquid-based cytologic screening. All women underwent colposcopy with biopsy. If a cervical quadrant had no lesion, a random biopsy at the squamocolumnar junction within that quadrant was obtained. All patients underwent endocervical curettage (ECC). Differences in sensitivity for CIN 2 or worse of screening tests were compared with criterion standards of colposcopically directed biopsy and colposcopically directed biopsy, random biopsy, plus ECC. Results Sixty-two of 83 women with CIN 2 or worse were diagnosed by colposcopically directed biopsy, 19 by random biopsy, and 2 solely by ECC. Fifty-six of the 83 women had CIN 2 or worse involving 0 to 2 quadrants (2 with 0 quadrants were diagnosed solely by positive ECC) and 27 of 83 had CIN 2 or worse results involving 3 to 4 quadrants. Colposcopically directed biopsy detected 35 of 56 women (62.5%) with CIN 2 or worse involving 0 to 2 cervical quadrants and 27 of 27 women (100%) with CIN 2 or worse involving 3 to 4 quadrants (p < .005). Acetic acid-aided visual inspection and cytologic analysis of high-grade squamous intraepithelial lesion or cancer detected 35 of 56 (62.5%) and 36 of 56 (64.3%) lesions involving 0 to 2 quadrants and 27 of 27 (100%) (p < .005) and 24 of 27 (88.9%; p < .05) lesions involving 3 to 4 quadrants. The sensitivity of direct human papillomavirus tests (97.6%) and cytologic analysis of atypical squamous cells of undetermined significance or worse (94.0%) for CIN 2 or worse were high and unaffected by lesion size. When the criterion standard was changed from colposcopically directed biopsy, random biopsy, plus ECC to colposcopically directed biopsy, the sensitivity of acetic acid-aided visual inspection and cytologic analysis of high-grade squamous intraepithelial lesion or worse for CIN 2 or worse increased from 75.9% to 85.5% (p > .1) and from 71.1% to 79.0% (p > .25). Sensitivities of other screening tests were unaffected by changing the criterion standard. Conclusions Criterion standards that miss the same CIN 2 or worse as the screening tests likely cause inflation of the sensitivity of those tests.

Human Papillomavirus Testing for Cervical Cancer Screening: Results From a 6-Year Prospective Study in Rural China

Long-term follow-up evaluations of cervical screening approaches are limited in low-resource areas. This prospective study assessed the risk of future cervical intraepithelial neoplasia grade 2 or worse (CIN2+) associated with baseline human papillomavirus (HPV) and cytologic status. In rural China, 1,997 women were screened with 6 screening tests, including colposcopic evaluations, and underwent biopsies in 1999. In December 2005, 1,612 women with cervical intraepithelial neoplasia grade 1 or less at baseline were rescreened by visual inspection, liquid-based cytology, and HPV-DNA testing. All women underwent colposcopy at follow-up, with biopsies taken from women with visually apparent lesions or cytologic abnormalities. Twenty women developed incident CIN2+. The crude relative risk of CIN2+ for baseline HPV-positive women was 52 (95% confidence interval: 12.1, 222.5). The crude relative risk of CIN2+ was 167 (95% confidence interval: 21.9, 1,265) for baseline and follow-up repeatedly HPV-positive women compared with repeatedly HPV-negative women. Among 1,374 baseline HPV-negative women, 2 and no incident CIN2+ cases were detected in baseline cytologically normal and abnormal subgroups, respectively. Among 238 baseline HPV-positive women, 6 of 18 incident cases of CIN2+ developed in the cytologically normal group. This study demonstrates that a single oncogenic HPV-DNA test is more effective than cytology in predicting future CIN2+ status.

Risk factors for HPV infection and cervical cancer among unscreened women in a high-risk rural area of China

We report a prevalence rate of 23.6% human papillomavirus (HPV) infection with oncogenic subtypes and 2.4% cervical intraepithelial neoplasia (CIN) III and cervical cancer (CC) in rural middle‐aged women in 2 counties with the highest CC mortality in Shanxi Province, China. We examined the association of risk factors to HPV infection and to CIN III and CC in 8,798 unscreened women aged 35–50 years. Multivariate odds ratios (OR) and 95% confidence intervals (CI) for each endpoint were obtained for risk factors after adjustment for covariates. The OR of oncogenic HPV were: 1.41 (95% CI = 1.25–1.60) and 1.42 (95% CI = 1.24–1.61) for the participant and her husband having multiple sexual partners, respectively; 1.67 (95% CI = 1.37–2.04), 1.15 (95% CI = 1.04–1.26), and 0.82 (95% CI = 0.72–0.94) for ever (vs. never) diagnosed with tuberculosis, cervical inflammation and vaginal trichomoniasis, respectively; while bathing in a public (v. private) facility had an OR of 1.23 (95% CI =1.11–1.35). Seasonal fluctuations in HPV infection, but not CC, appeared in Xiangyuan County, with OR of 1.23 (95% CI = 1.14–1.33) and 1.51 (95% CI = 1.35–1.67) in Spring and Winter compared to Summer, respectively. The OR of CIN III and CC in the HPV positives were: 2.03 (95% CI = 1.63–2.53) for ages ≥45 years (vs. <40); and 4.01 (95% CI = 1.46–11.0) for ≥3 (vs. no) home births. Public health interventions and control strategies for improving the reproductive health of women in these rural populations need to be developed to reduce risk of HPV and subsequent CC.

A thin-layer, liquid-based pap test for mass screening in an area of China with a high incidence of cervical carcinoma. A cross-sectional, comparative study.

OBJECTIVE To confirm the accuracy of the ThinPrep Pap Test (Cytyc Corp., Boxborough, Massachusetts, U.S.A.) on the basis of histologic diagnosis by biopsy and the detection of human papillomavirus (HPV) DNA in mass screening. STUDY DESIGN A total of 1,997 women residing in Xiangyuan County, Shanxi Province, P.R.C., an area with a high incidence of cervical carcinoma, were enrolled in this study. We collected exfoliative cervical samples from all subjects into a liquid buffer (Preserv-Cyt [Cytyc]) and utilized for both cytologic screening using the ThinPrep Pap Test and HPV DNA testing. Subsequent colposcopic biopsies were taken on all subjects. All the tests were performed in an independent and blinded fashion. We compared the ThinPrep Pap test with colposcopic biopsy and HPV test. RESULTS High grade squamous intraepithelial lesions (HSIL) (CIN 2/3) were identified in 74 (3.7%) of 1,993 women adequately assessed, and there were 12 cases of squamous cell carcinoma (SCC). The false negative rate of ThinPrep cytology was 3.2% for biopsy-confirmed CIN 3 and 9.3% for CIN 2. Twenty-seven (87%) of the 31 women with biopsy-confirmed CIN 3 and 12 (100%) of 12 with biopsy-confirmed SCC had a diagnosis of either HSIL or greater abnormalities on ThinPrep cytology. In addition, the HPV DNA detection rates offered a good correlation between cytology and biopsy. CONCLUSION The ThinPrep Pap performed extremely well in this primary screening trial. We found a good correlation between ThinPrep cytology and colposcopic biopsy on detection of HSIL and SCC; cervical specimens collected in ThinPrep liquid buffer serve as a direct test for HPV as well.

Utility of Random Cervical Biopsy and Endocervical Curettage in a Low-Risk Population

Objective The study aimed to determine the increase in the yield of cervical intraepithelial neoplasia 3 (CIN 3) or cancer (CIN 3+) from random cervical biopsy in quadrants without visible lesions and endocervical curettage (ECC) in a low-prevalence setting. Materials and Methods Random biopsy and ECC (unless pregnant) have been obtained in the colposcopy clinic of the Southern California Permanente Medical Group (SCPMG)-Fontana since 2004. We reviewed the colposcopy experience of SCPMG-Fontana for January 1, 2007, to December 31, 2009, to determine the method of diagnosis of CIN 3+. Results Between January 1, 2007, and December 31, 2009, 4677 women with median age 32 years had 4932 colposcopies in the SCPMG-Fontana colposcopy clinics. Cervical intraepithelial neoplasia 3+ was diagnosed in 295 women. Cervical biopsy detected 64.4% of CIN 3+; ECC diagnosed 5.1%; loop electrocautery excision procedure (LEEP) or cervical conization for cervical biopsy and/or ECC of CIN 2 diagnosed 27.8%; LEEP for the cytology of high-grade squamous intraepithelial lesion with cervical biopsy result of negative or CIN 1 diagnosed 1.4%; and LEEP, cervical conization, or biopsy in follow-up of CIN 2 diagnosed 1.4%. Sixty-one of the 295 cases of CIN 3+ (20.7%) were diagnosed after evaluation of random cervical biopsy and/or ECC of CIN 2+. Conclusions Random biopsy in cervical quadrants without visible lesions and ECC increased the yield of CIN 3+ in this low-risk colposcopy setting. Endocervical curettage can be omitted in women younger than 25 years.

False negative colposcopy is associated with thinner cervical intraepithelial neoplasia 2 and 3

OBJECTIVE To assess whether thinner lesion epithelium or lower nuclear density contribute to false negative colposcopy for dysplasia, we determined epithelial thickness and nuclear density and correlated this with the accuracy of colposcopic impression for cervical quadrants with biopsies of normal, CIN 1, CIN 2 and CIN 3. METHODS The thickness and nuclear density of squamous epithelium of 261 selected cervical biopsies (CIN 2/CIN 3, N=144; Normal/CIN 1, N=117) from the Shanxi Province Cervical Cancer Screening Study (where a colposcopic impression and biopsy were obtained in each cervical quadrant) were measured. Average epithelial thickness was defined as the thinnest area plus the thickest area divided by two. Average nuclear density was defined as the number of nuclei in a 2,500 microm(2) grid at the junction of the superficial and intermediate zones plus that at the junction of the intermediate and parabasal zones divided by two. Differences in means were measured by Wilcoxon Rank-Sum Test. Trends among means were measured by a generalized linear mixed model. RESULTS Mean average epithelial thickness for 33 biopsies of CIN 2/CIN 3 from cervical quadrants with colposcopic impression of normal (184 microm) was less than that of 111 biopsies of CIN 2/CIN 3 from quadrants with colposcopic impressions of low, high, or cancer (321 microm, p<.001). CIN 2/CIN 3 had higher mean average nuclear density (p<.001) and was thinner than normal/CIN 1 (p<.001). CONCLUSION The inability of expert colposcopists to visualize some CIN 2/CIN 3 is associated with thinner epithelium.