Clinical Experience with the Cervista HPV HR Assay

Transcription

1 The Journal of Molecular Diagnostics, Vol. 13, No. 2, March 2011 Copyright 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. DOI: /j.jmoldx Clinical Experience with the Cervista HPV HR Assay Correlation of Cytology and HPV Status from 56,501 Specimens Kenneth E. Youens,* Gregory A. Hosler, Paula J. Washington, E. Patrick Jenevein, and Kathleen M. Murphy From the Department of Pathology,* Duke University Medical Center, Durham, North Carolina; and ProPath, Dallas, Texas Testing for high-risk (HR) human papillomavirus (HPV) is a key component of current recommendations for cervical cancer screening. Herein is described our clinical experience using Cervista HPV HR, a testing platform recently approved by the US Food and Drug Administration for clinical use. Using data from a high-volume commercial laboratory, a retrospective analysis of cytologic and Cervista HPV HR test results from 56,501 samples was performed, and an indirect comparison was made with previous experience with 53,008 samples tested using the Hybrid Capture 2 platform. Of samples analyzed using Cervista HPV HR, 1.5% were of insufficient volume for testing and 1.1% yielded an insufficient signal from the internal control to be reported. In samples with a cytological interpretation of atypical squamous cells of undetermined significance, 48.5% (95% confidence interval [CI], 47.5 to 49.5) tested positive using Cervista HPV HR, compared with 59.4% (95% CI, 58.3 to 60.5) of samples using Hybrid Capture 2. Of samples from women aged 30 years or older with a negative cytological interpretation, 5.8% (95% CI, 5.6 to 6.1) tested positive using Cervista HPV HR, compared with 5.5% (95% CI, 5.3 to 5.7) of samples using Hybrid Capture 2. When stratified by five-year age groups between 30 and 65 years, positivity rates for high-risk human papillomavirus were similar in the Cervista HPV HR and Hybrid Capture 2 populations, and were consistent with expectations established by the literature. (J Mol Diagn 2011, 13: ; DOI: /j.jmoldx ) 160 The relationship of cervical cancer and cervical intraepithelial neoplasia grades 2 and 3 to persistent infection with human papillomavirus (HPV) is well established. Since the identification of HPV as the primary etiologic agent of cervical cancer, 1 14 sexually transmitted oncogenic variants have been identified. These variants, referred to as the high-risk subtypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68), are responsible for most cervical cancers worldwide. 2 4 The remarkable success of Papanicolaou test based cervical cancer screening programs demonstrates that cervical cancer is largely preventable. However, despite its many advantages and historical success, morphologic examination of cervical cytologic material alone has limitations as a screening test. The addition of high-risk HPV (HR-HPV) DNA testing to screening algorithms has enabled better identification of clinically significant precancerous cervical lesions and more accurate evaluation of the risk of developing cervical cancer. 5,6 HR-HPV DNA testing is currently recommended by the American Society for Colposcopy and Cervical Pathology screening guidelines in two principal clinical settings. One setting in which HR-HPV DNA testing is beneficial is when cytologic findings are ambiguous or equivocal. HR- HPV DNA testing for triage of patients with atypical squamous cells of undetermined significance (ASC-US) cytological interpretations decreases colposcopic referrals by roughly half without sacrificing screening sensitivity. 7 A second setting in which HR-HPV testing is useful is for primary screening ( co-testing, along with cytology) in patients aged 30 years or older. Women in this group with both normal cytological interpretations (ie, negative for intraepithelial lesion or malignancy [NILM]) and negative HR-HPV test results are at extremely low risk of developing cervical cancer over the next several years. 8 Accordingly, the current (2006) American Society for Colposcopy and Cervical Pathology screening guidelines and the 2010 American Cancer Society screening guidelines recommend that the repeat screening interval should be increased to three years in this double-negative population. 6,9 Until 2009, clinical testing for HR-HPV was limited to a single US Food and Drug Administration (FDA) ap- Accepted for publication November 10, Supplemental material for this article can be found at amjpathol.org or at doi: /j.jmoldx Address reprint requests to Kenneth Youens, M.D., Duke University Medical Center, Department of Pathology, DUMC 3712, Durham, NC kenneth.youens@duke.edu.

2 Cervista HPV HR Clinical Experience 161 proved method, the Hybrid Capture 2 (hc2; Digene Corp./QIAGEN, Gaithersburg, MD) assay. This platform remains the most widely used clinical method for HR-HPV DNA testing. A second testing platform, the Cervista HPV HR Assay (Hologic, Inc., Bedford, MA) was FDA-approved for clinical use in March Both tests involve hybridization of nucleic acid probes that are complimentary to the viral genome, followed by a signal amplification method for detection. Given its recent approval for clinical use, few data about Cervista clinical test performance are available Cervista HPV HR package insert. Herein we present our experience using the Cervista HPV HR platform in a commercial laboratory, along with an indirect comparison with previous experience using the hc2 assay. In addition, estimates of the prevalence of HR-HPV infection in women with ASC-US and NILM cytologic interpretations in the present patient population are provided. Materials and Methods The present study was a retrospective evaluation of cervical screening data from specimens subjected to both cytologic examination and HR-HPV DNA testing at a physician-owned commercial laboratory (ProPath, Dallas, TX). Identified were 56,501 samples analyzed between August 2009 and April 2010 using the Cervista platform and 53,008 samples analyzed during 2008 using the hc2 platform. The Western Institutional Review board determined this study to be exempt under 45 CFR.101(b) (4). Test samples were received in SurePath (Becton, Dickinson and Co., Franklin Lakes, NJ) liquid-based cytology medium or in ThinPrep vials containing PreservCyt (Hologic, Inc.) medium, and were collected by clinical providers referring specimens for cytologic analysis and HPV testing. For all samples, HR-HPV DNA testing and cytologic examination were performed using material from the same specimen container. Papanicolaou tests for all patients were evaluated using manual cytotechnologist review or the ThinPrep Imaging System (Hologic, Inc.) or the FocalPoint Slide Profiler (Becton, Dickinson and Co.), followed by manual cytotechnologist review. Papanicolaou tests suspicious for reactive, reparative, or dysplastic changes were reviewed by board-certified anatomic pathologists (G.A.H., P.J.W., and E.P.J.). Cytologic findings were classified according to the 2001 Bethesda System terminology for reporting results of cervical cytology. 12 All HR-HPV DNA testing was performed according to the manufacturers instructions. On the Cervista platform, sequence-specific probe and Invader (Third Wave Technologies, Inc., Madison, WI) oligonucleotides cycle rapidly on and off the 14 HR-HPV target DNA sequences, creating substrate for the proprietary Cleavase enzyme (Hologic, Inc.). The action of the enzyme results in production of cleaved 5= oligonucleotide flaps. These flaps bind to a universal hairpin fluorescence resonance energy transfer oligonucleotide, creating a second substrate for the Cleavase enzyme. Cleavage of the fluorescence resonance energy transfer oligonucleotide results in production of a fluorescent signal. Oligonucleotides specific for the human histone 2 gene are present in each mixture and act as an internal control (Cervista HPV-HR package insert.). The hc2 platform uses a signal amplification method in which target HPV DNA from any of the 13 high-risk HPV types tested (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) is hybridized with HPV-specific RNA probes. Some cross-reactivity of the probes with other HPV types (including type 66) also occurs. 13 The resulting DNA-RNA hybrids are captured on the surface of a microplate well coated with antibodies specific for DNA- RNA hybrids. In the detection step, multiple signaling antibodies conjugated to alkaline phosphatase bind to each hybrid. Cleavage of a chemiluminescent substrate by alkaline phosphatase results in light emission, measured using a luminometer (Digene Hybrid Capture 2 High-Risk HPV DNA Test package insert. Gaithersburg, MD: Digene Corp., 2004, 1 24). Before commencing the present study, 176 liquidbased cytologic specimens were analyzed, 79 in Sure- Path medium and 97 in PreservCyt medium, using both the Cervista and hc2 assays. Four specimens yielded a low internal control signal using the Cervista assay, and tested negative for HR-HPV using the hc2 assay. Of the remaining 172 specimens, 147 (85.5%) demonstrated concordant results (62 positive, 85 negative). One specimen tested HR-HPV positive using the Cervista assay and negative using the hc2 assay, and 24 specimens tested negative using Cervista and positive using hc2. Discordant samples were submitted to SeqWright, Inc. (Houston, TX) for testing using PCR and sequencing. All of the discordant specimens tested negative for HR-HPV by PCR and sequencing. As part of the Cervista validation process, it was determined that a FAM dye fold over zero (FOZ) ratio threshold for positivity for SurePath specimens of 2.0 was optimal. This cutoff was used for all subsequent analyses. The manufacturer-recommended FAM FOZ ratio was used as the positivity threshold for PreservCyt specimens. Performance characteristics of the Cervista assay that were evaluated included number and percentage of specimens classified as having a low internal control, number and percentage of specimens with insufficient volume for testing, and number and percentage of total reportable results. Rates of HR-HPV positivity were calculated. Results were stratified according to patient age and were compared by specimen preservative type and cytologic interpretation. When appropriate, results from the Cervista population were compared with data from the hc2 population, and the Fisher s exact test was used to determine statistical significance. No corrections or adjustments for patient demographic characteristics were applied. Confidence intervals were calculated at 95% using the binomial method. Statistical computations were performed using commercially available software (Microsoft Excel 2003 for Windows; Microsoft Corp., Redmond, WA) and the R software package (R Foundation for Statistical Computing, Vienna, Austria).

3 162 Youens et al Table 1. Patient Demographics and Specimen Preservative Type Variable Cervista (n 56,501) hc2 (n 53,008) Age, years Mean (SD) 40.9 (12.9) 40.6 (12.0) Median (range) 40 (12 97) 39 (11 92) Preservative, % SurePath ThinPrep Results Specimens were referred for analysis primarily from outpatient private practices including gynecology and family practice clinics, from multiple states in all regions of the United States. Aggregated demographic characteristics of the women undergoing testing and information about the specimens according to preservative type used are given in Table 1. The age distribution of patients undergoing testing in the Cervista and hc2 populations was similar (see Supplemental Table S1 at org). A summary of nonreportable results is given in Table 2. Overall, 97.5% of samples from the Cervista population [55,065 of 56,501; 95% confidence interval (CI), 97.4 to 97.6] could be reported, compared with 96.3% of samples from the hc2 population (51,071 of 53,008; 95% CI, 96.1 to 96.5). For the Cervista population, 1.5% of samples (848 of 56,501; 95% CI, 1.4 to 1.6) were of insufficient volume to perform the HPV HR test, compared with 3.6% of samples (1908 of 53,008; 95% CI, 3.4 to 3.8) for the hc2 population. For the Cervista population, 1.1% of tested specimens (622 of 56,501) provided an insufficient signal from the internal control; the hc2 platform lacks an internal control. Overall, the rate of HR-HPV positivity in reportable samples from the entire Cervista population was 15.9% (8778 of 55,065; 95% CI, 15.6 to 16.2). In all reportable samples from the hc2 population, 16.9% (8635 of 51,071; 95% CI, 16.6 to 17.2) were positive for HR-HPV (Figure 1A). For specimens classified as unsatisfactory for cytologic evaluation, 11.7% (24 of 205; 95% CI, 8.0 to 16.8) were of insufficient volume to perform the HR-HPV test in the Cervista population, compared with 15.6% (32 of 205; 95% CI, 11.3 to 21.2) in the hc2 population. In the Cervista population, 31.7% (65 of 205) of these cytologically unsatisfactory samples provided an insufficient signal from the internal control; hc2 lacks an internal control. Figure 1. Percent HR-HPV positive by cytologic interpretation. A: All samples. B: ASC-US. C: Women aged 30 years or older, NILM. All calculations are for reportable samples only. Whiskers indicate 95% confidence intervals. The HR-HPV positivity rate for cytologically unsatisfactory specimens that yielded a reportable HR-HPV result was 8.6% (10 of 116; 95% CI, 4.7 to 15.1) in the Cervista population and 9.8% (17 of 173; 95% CI, 6.2 to 15.1) in the hc2 population. For specimens with an ASC-US cytologic interpretation, the HR-HPV positivity rate in the Cervista population was 48.5% (4438 of 9151; 95% CI, 47.5 to 49.5), compared with 59.4% (4495 of 7568; 95% CI, 58.3 to 60.5) in the hc2 population (Table 3 and Figure 1B). Relative to the total number of reportable results in each test population, the percentage of specimens with both an ASC-US cytologic interpretation and a positive HR-HPV test result was 8.1% (4483 of 55,065; 95% CI, 7.9 to 8.3) in the Cervista population and 8.8% (4495 of 51,071; 95% CI, 8.6 to 9.0) in the hc2 population (Table 3). For specimens from patients younger than 30 years with a NILM cytologic interpretation, the HR-HPV positivity rates were 18.2% (847 of 4648; 95% CI, 17.1 to 19.3) and 21.9% (769 of 3507; 95% CI, 20.6 to 23.3), respectively, in the Cervista and hc2 populations. For specimens from patients aged 30 years or older with a NILM cytologic interpretation, the HR-HPV positivity rates were 5.8% (2289 of 39,619; 95% CI, 5.6 to 6.1) and 5.5% (2117 of 38,448; 95% CI, 5.3 to 5.7), respectively, in the Cervista and hc2 populations (Table 4 and Figure 1C). Relative to the total number of reportable results in each test population, the percentage of specimens from women aged 30 years or older with both a NILM cytologic interpretation and a positive HR-HPV test result was 4.16% Table 2. Summary of Nonreportable Results Variable Cervista (n 56,501)* hc2 (n 53,008)* P value Low internal control 1.1 ( ) NA NA Quantity not sufficient 1.5 ( ) 3.6 ( ) Borderline positive NA 0.1 NA Total reportable results 97.5 ( ) 96.3 ( ) NA, not applicable. *Data are given as % (95% CI).

4 Cervista HPV HR Clinical Experience 163 Table 3. HR-HPV Results for All Samples with ASC-US Cytologic Interpretations Variable Cervista (n 9151) hc2 (n 7568) Age, years Mean (SD) 32.5 (12.4) 31.4 (11.9) Median (range) 29 (13 86) 28 (12 89) HR-HPV positive, No. (%; 95% CI)* 4438 (48.5; ) 4495 (59.4; ) Reportable results with both ASC-US cytologic interpretation and positive HR-HPV test, % (95% CI)* 8.1 ( ) 8.8 ( ) *P (2289 of 55,065; 95% CI, 4.00 to 4.33) and 4.15% (2117 of 51,071; 95% CI, 3.98 to 4.33), respectively, in the Cervista and hc2 populations. Age-specific rates of HR- HPV positivity in the Cervista and hc2 populations in samples from women aged 30 years or older with a NILM cytologic interpretation are shown in Figure 2. For specimens from patients aged 30 years or older with a NILM cytologic interpretation, the HR-HPV positivity rates were compared according to specimen preservative type. In the Cervista population, the HR-HPV positivity rates were 4.7% (666 of 14,133; 95% CI, 4.4 to 5.1) and 6.4% (1624 of 25,486; 95% CI, 6.1 to 6.7), respectively, for SurePath and PreservCyt. In the hc2 population, the HR-HPV positivity rates were 6.3% (631 of 9957; 95% CI, 5.8 to 6.8) and 5.2% (1486 of 28,491; 95% CI, 6.1 to 6.7), respectively, for SurePath and PreservCyt (Table 5). Discussion The present study describes our overall experience with the Cervista HPV HR testing platform in a high-volume commercial laboratory with more than 50,000 specimens over nine months. The comparison of performance of the Cervista and hc2 platforms provides some insight into the overall comparability of the two tests in a high-volume clinical laboratory setting. Given the indirect nature of the comparison, the conclusions derived about the relative performance of the two platforms should be interpreted with caution. For example, the usefulness of comparing HR- HPV positivity rates in the Cervista and hc2 populations depends on the assumption that the prevalence of HR- HPV infection was the same in each patient group compared. Although it is likely that the characteristics of the populations tested were similar, some differences are to be expected. The populations studied were not random samplings, but represent an analysis of specimens from women for whom clinicians believed that HR-HPV testing was needed for clinical management. This likely introduces bias, in particular because testing may have been requested more frequently in patients with other risk factors for sexually transmitted diseases or cervical cancer. In most of the groups analyzed (including samples from patients with ASC-US and unsatisfactory cytologic interpretations, as well as patients younger than 30 years with NILM cytologic interpretations), the HR-HPV positivity rate was higher in the hc2-tested population compared with the Cervista population. One potential explanation for this finding would be if the hc2 assay had greater sensitivity compared with Cervista, possibly because of the larger starting volume used for hc2. Another possible explanation would be if the hc2 assay were less specific than the Cervista assay. Cross-reactivity of the hc2 assay with multiple low-risk HPVs is well documented and could account for an increased rate of false-positive results. 13 Several studies that directly compared hc2 with an Invader-based HR-HPV assay (pre-fda-approval, analyte-specific reagent-based versions) demonstrated a higher false-positive rate with hc2 compared with Invader. 11,14 16 In all tested groups, an inverse relationship was observed between HR-HPV positivity rate and increasing age, a pattern consistent with findings in numerous studies of HPV prevalence in developed countries Samples with ASC-US cytological interpretations demonstrated a higher HR-HPV positivity rate in the hc2 population (59.4%) than in the Cervista population (48.5%). Previously reported rates of HR-HPV positivity in samples with ASC-US cytological interpretations vary Table 4. HR-HPV Results for All Samples, Women Aged 30 Years or Older with NILM Cytologic Interpretations Variable Cervista (n 39,619) hc2 (n 38,448) Age, years Mean (SD) 44.8 (10.7) 43.8 (9.9) Median (range) 43 (30 88) 42 (30 90) HR-HPV positive, No. (%; 95% CI)* 2289 (5.8; ) 2117 (5.5; ) Reportable results with both NILM cytologic interpretation and positive HR-HPV test, % (95% CI) 4.16 ( ) 4.15 ( ) *P Women aged 30 years or older. P

5 164 Youens et al 15% 11% 8% 4% 0% Present Study (Cervista) Present Study (hc2) Datta et al., 2008 Castle et al., Thrall et al., 2010* Figure 2. HR-HPV positivity rates, stratified by age, for women aged 30 years or older with NILM cytologic interpretations, United States studies. Whiskers indicate 95% confidence intervals. *Reported in intervals of 10 years (ie, 30 39, 40 49, 50). widely, primarily because of differing HR-HPV prevalence in tested populations; however, in the present study, results in both populations were comparable to the benchmark of 50.7% established by the ASCUS/LSIL Triage Study for Cervical Cancer. 7,21 The different HR-HPV positivity rates observed in samples with ASC-US cytological interpretations from the Cervista and hc2 populations may be due, in part, to small differences in the patient populations tested. The hc2-tested population was slightly younger (median age, 28 years) than the Cervista-tested population (median age, 29 years). Although this age difference is small, it could have significantly affected HR-HPV prevalence in these populations. Numerous studies have reported higher HR-HPV prevalence in women younger than 30 years compared with women aged 30 years or older In the present test populations, the HR-HPV positivity rate in women with NILM cytologic interpretations was threefold to fourfold higher in women younger than 30 years compared with women aged 30 years or older. In the present study, the median ages of the two ASC-US populations were near this age threshold of 30 years, which may have contributed to the higher prevalence of HR- HPV observed in the younger hc2-tested population. Another contributing factor could have been differences in cytologic diagnostic evaluation in the two test populations. Despite standardized morphologic criteria, the ASC-US category exhibits substantial variability in practice. 26 To reduce bias introduced by differences in ASC-US cytologic interpretation, the percentage of reportable cases from each test population that had both an ASC-US cytological interpretation and a positive HR- HPV test result was calculated. This calculation should have reduced any bias that may have occurred if there were a consistently lower or higher threshold for ASC-US interpretation during the test intervals. The difference in results between the Cervista and hc2 populations was much smaller with application of this correction. In women aged 30 years or older with NILM cytologic interpretations the HR-HPV positivity rate in the Cervista population (5.8%) was not significantly different from that in the hc2 population (5.5%). Multiple studies have investigated the HR-HPV positivity rate in women aged 30 years or older with NILM cytologic interpretations (Table 6), most of which used the hc2 assay because the Cervista assay has only recently been approved by the FDA. Reported results range from approximately 4% to greater than 16%. Even when comparing only studies that used hc2, the results are quite variable, indicating that the variability likely reflects differences in the populations analyzed. Data from the present study provide additional evidence that the different HR-HPV positivity rates reported in various studies are likely due to differences in the tested populations rather than to differences in test method. First, the result in women aged 30 years or older with NILM cytologic interpretations using Cervista (5.8%) was significantly lower than the rate reported in the Cervista package insert (18.5%), which demonstrates the variability in HR-HPV positivity rates between studies that used the same test method but tested different patient populations. Second, although not identical, the two populations tested were likely demographically similar. The comparable HR-HPV positivity rates observed between the Cervista (5.8%) and hc2 (5.5%) populations provide some evidence that in similar populations, the Cervista and hc2 tests perform similarly. Third, when stratified by age, the HR-HPV positivity rates in the Cervista population are comparable to those observed in multiple previous studies using hc2 (Figure 2). Although HPV testing is most useful because of its improved sensitivity compared with cytology, an excessively sensitive or inadequately specific HR-HPV test could substantially decrease the utility of the test. As noted in a recent commentary, an excessively sensitive test could result in an unnecessary increase in surveillance testing, colposcopic referrals, and excisional treatments of the cervix. 27 Considerable data exist about the sensitivity, specificity, and clinical performance of the hc2 assay. In comparison, because of its recent implementation into clinical practice, we do not know the real-world performance of the Cervista HR. 27 The present study reports real-world experience with the Cervista HPV HR assay Table 5. HR-HPV Results According to Preservative Type, Women Aged 30 Years or Older with NILM Cytologic Interpretations Variable SurePath Preservative PreservCyt Cervista group No. of samples (%) 14,133 (35.7) 25,486 (64.3) Patient age, mean, y Positive for HR-HPV No. (%; 95% CI)* ( ) ( ) hc2 group No. of samples (%) 9957 (25.9) 28,491 (74.1) Patient age, mean, y Positive for HR-HPV No. (%; 95% CI) 631 (6.3; ) 1486 (5.2; ) *P P

6 Cervista HPV HR Clinical Experience 165 Table 6. HR-HPV Positivity Rates in Women Aged 30 Years or Older with NILM Cytologic Interpretation, Previous US Studies Source/year HR-HPV positivity rate, % No. of samples HPV test Papanicolaou test Present study ,619 Cervista Liquid-based Present study ,448 hc2 Liquid-based Cervista HPV-HR package insert Cervista Liquid-based (Bedford, MA: Hologic, Inc.) Thrall et al 22 / hc2 Liquid-based Castle et al 17 / ,594 hc2 Conventional Datta et al 18 / hc2 Liquid-based/conventional Baseman et al 29 / hc2 Liquid-based Evans et al 23 / PCR Liquid-based and provides some reassurance that its performance in the cohort aged 30 years or older with NILM cytologic interpretations is comparable to that of the hc2 assay. Additional studies, preferably direct comparisons of Cervista and hc2 in the same patient population, are needed to better understand the relative performance of the two platforms. Initial results from these types of direct comparison are only now becoming available. For example, data from a direct head-to-head comparison of hc2 and Cervista were presented at the International Papillomavirus Congress in July Using both testing platforms, Wu et al 28 tested 4965 cervical samples and demonstrated a 12.2% positivity rate using Cervista and a 14.6% rate using hc2.at present, given the scarcity of this kind of head-to-head comparison data and lack of a clear criterion standard for clinical HR-HPV test performance, a conservative approach is warranted during internal laboratory validations of the Cervista assay. In the present study, samples submitted in PreservCyt medium had a higher HR-HPV positivity rate in the Cervista population, whereas samples submitted in SurePath medium were more likely to test positive for HR-HPV in the hc2 population. It is unclear whether this was because of differences in the populations tested or to true differences in assay performance based on the specimen collection system and/or preservative type. The SurePath system is widely used to collect specimens for both cytology and HR-HPV DNA testing; however, clinical studies that validate the SurePath system for HR-HPV testing are few and are limited to the hc2 platform At present, only the ThinPrep system, which uses Preserv- Cyt media, has FDA approval for this application. Therefore, internal laboratory validation of SurePath specimens is required for each assay. The differences in HR-HPV positivity observed in the present study may in part reflect our laboratory-validated assay positivity cutoff values. Further investigation is needed to determine whether the SurePath and ThinPrep collection systems are equivalent when used for HR-HPV testing using the Cervista platform. Two potentially important advantages of the Cervista platform compared with the hc2 platform are the reduced sample volume required and the presence of an internal control sample. The Cervista platform requires 2.0 ml of residual PreservCyt or SurePath preservative solution, compared with 4.0 ml required by the hc2 platform. It was observed that in the Cervista population, approximately half as many specimens were classified as of insufficient volume. The contribution of this factor to the total percentage of reportable results was partially offset by those Cervista samples that could not be reported because of an insufficient control signal; however, compared with the hc2 population, the Cervista population retained a slightly higher overall percentage of reportable results. In addition to decreasing the number of samples deemed insufficient, the lower required sample volume meant that more material was available for additional tests such as molecular testing for other infectious diseases. Approximately 1% of samples tested in the Cervista population generated a low internal control signal, which indicates that insufficient nucleic acid was present for testing. This could occur under various circumstances, the most likely being that the specimens were inadequately cellular. It is reasonable to assume that a like percentage of specimens in the hc2 population were similarly inadequate but were reported as negative because of lack of an internal control sample. This assumption is supported by previous studies that reported occasional false-negative results for hc2 when comparing hc2 and Invader-based assays. 11,14,15 In addition, results from nearly one-third of the cytologically unsatisfactory samples in the Cervista population could not be reported because of low internal control signal; however, all samples of this type from the hc2 population that had sufficient volume for testing were reported as being definitively positive or definitively negative for HR-HPV. The clinical usefulness of HR-HPV testing largely relies on its high sensitivity and strong negative predictive value; thus, false-negative results are of particular concern because they fail to identify women who require further testing. The addition of the internal control sample to the Cervista assay likely reduces a significant number of these false-negative results. The Cervista assay is performed using three different reagent mixtures, each containing oligonucleotide probes for a phylogenetically related subset of the 14 tested HR- HPV subtypes. The test reaction for each reagent mixture is performed separately, resulting in a relatively large number of total reactions and raising questions about the scalability of the platform in a high-volume practice. However, the assay is amenable to automation, and the test software interprets the three reactions as a single test. In addition, there are several points in the Cervista procedure at which testing can be set aside and resumed later. This makes the

7 166 Youens et al workflow flexible, and enables it to be modified according to scheduling and staffing needs. In the present study, the sample size was large and from a broad geographic distribution across the United States. The results should be generalizable to US laboratories that test primarily outpatient samples, but may not apply to all populations. In keeping with previously published data, it was observed that the rate of HR-HPV positivity depends on patient age and that various other population characteristics and risk factors likely influence test results. Several of the practical benefits of the Cervista platform were confirmed including the smaller sample size required and the presence of an internal control. In summary, comparisons between Cervista and hc2 provide some reassurance that no dramatic differences in HR-HPV positivity rates are to be expected between these two platforms. References 1. Schwarz E, Freese UK, Gissmann L, Mayer W, Roggenbuck B, Stremlau A, zur Hausen H: Structure and transcription of human papillomavirus sequences in cervical carcinoma cells. Nature 1985, 314: Cogliano V, Baan R, Straif K, Grosse Y, Secretan B, El Ghissassi F: Carcinogenicity of human papillomaviruses. Lancet Oncol 2005, 6: Meijer CJ, Snijders PJ, Castle PE: Clinical utility of HPV genotyping. Gynecol Oncol 2006, 103: Muñoz N, Bosch FX, de Sanjosé S, Herrero R, Castellsagué X, Shah KV, Snijders PJ, Meijer CJ: Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003, 348: Apgar BS, Kittendorf AL, Bettcher CM, Wong J, Kaufman AJ: Update on ASCCP consensus guidelines for abnormal cervical screening tests and cervical histology. Am Fam Phys 2009, 80: Smith R, Cokkinides V, Brooks D, Saslow D, Brawley O: Cancer screening in the United States, 2010: a review of current American Cancer Society guidelines and issues in cancer screening. CA Cancer J Clin 2010, 60: ASCUS-LSIL Triage Study Group: Results of a randomized trial on the management of cytology interpretations of atypical squamous cells of undetermined significance. Am J Obstet Gynecol 2003, 188: Sherman ME, Lorincz AT, Scott DR, Wacholder S, Castle PE, Glass AG, Mielzynska-Lohnas I, Rush BB, Schiffman M: Baseline cytology, human papillomavirus testing, and risk for cervical neoplasia: a 10- year cohort analysis. J Natl Cancer Inst 2003, 95: Wright TC, Massad LS, Dunton CJ, Spitzer M, Wilkinson EJ, Solomon D: 2006 Consensus guidelines for the management of women with abnormal cervical screening tests. J Low Genit Tract Dis 2007, 11: Einstein MH, Martens MG, Garcia FA, Ferris DG, Mitchell AL, Day SP, Olson MC: Clinical validation of the Cervista HPV HR and 16/18 genotyping tests for use in women with ASC-US cytology. Gynecol Oncol 2010, 17: Ginocchio CC, Barth D, Zhang F: Comparison of the Third Wave Invader human papillomavirus (HPV) assay and the Digene HPV Hybrid Capture 2 assay for detection of high-risk HPV DNA. J Clin Microbiol 2008, 46: Solomon D, Davey D, Kurman R, Moriarty A, O Connor D, Prey M, Raab S, Sherman M, Wilbur D, Wright T, Young N: The 2001 Bethesda System: terminology for reporting results of cervical cytology. JAMA 2002, 287: Poljak M, Marin IJ, Seme K, Vince A: Hybrid Capture II HPV test detects at least 15 human papillomavirus genotypes not included in its current high-risk probe cocktail. J Clin Virol 2002, 25(suppl):3:S89 S Johnson LR, Starkey CR, Palmer J, Taylor J, Stout S, Holt S, Hendren R, Bock B, Waibel E, Tyree G, Miller GC: A comparison of two methods to determine the presence of high-risk HPV cervical infections. Am J Clin Pathol 2008, 130: Schutzbank TE, Jarvis C, Kahmann N, Lopez K, Weimer M, Yount A: Detection of high-risk papillomavirus DNA with commercial Invadertechnology based analyte-specific reagents following automated extraction of DNA from cervical brushings in ThinPrep media. J Clin Microbiol 2007, 45: Wong AK, Chan RC, Nichols WS, Bose S: Human papillomavirus (HPV) in atypical squamous cervical cytology: the Invader HPV test as a new screening assay. J Clin Microbiol 2008, 46: Castle PE, Fetterman B, Poitras N, Lorey T, Shaber R, Kinney W: Five-year experience of human papillomavirus DNA and Papanicolaou test cotesting. Obstet Gynecol 2009, 13: Datta SD, Koutsky LA, Ratelle S, Unger ER, Shlay J, McClain T, Weaver B, Kerndt P, Zenilman J, Hagensee M, Suhr CJ, Weinstock H: Human papillomavirus infection and cervical cytology in women screened for cervical cancer in the United States, Ann Intern Med 2008, 148: Franceschi S, Herrero R, Clifford GM, Snijders PJ, Arslan A, Anh PT, Bosch FX, Ferreccio C, Hieu NT, Lazcano-Ponce E, Matos E, Molano M, Qiao YL, Rajkumar R, Ronco G, de SanjoséE S, Shin HR, Sukvirach S, Thomas JO, Meijer CJ, Muñoz N: Variations in the age-specific curves of human papillomavirus prevalence in women worldwide. Int J Cancer 2006, 119: Dunne EF, Unger ER, Sternberg M, McQuillan G, Swan DC, Patel SS, Markowitz LE: Prevalence of HPV infection among females in the United States. JAMA 2007, 297: Allen GL, Klobocista MM, Sugarman S, Gravel K, Feldman D, Schnatz PF: Prevalence of high-risk human papillomavirus in an inner-city population with atypical squamous cells of undetermined significance. J Low Genit Tract Dis 2009, 13: Thrall MJ, Russell DK, Facik MS, Yao JL, Warner JN, Bonfiglio TA, Giampoli EJ: High-risk HPV testing in women 30 years or older with negative Papanicolaou tests: initial clinical experience with 18-month follow-up. Am J Clin Pathol 2010, 33: Evans MF, Adamson CS, Papillo JL, St John TL, Leiman G, Cooper K: Distribution of human papillomavirus types in ThinPrep Papanicolaou tests classified according to the Bethesda 2001 terminology and correlations with patient age and biopsy outcomes. Cancer 2006, 106: Jacobs MV, Walboomers JM, Snijders PJ, Voorhorst FJ, Verheijen RH, Fransen-Daalmeijer N, Meijer CJ: Distribution of 37 mucosotropic HPV types in women with cytologically normal cervical smears: the age-related patterns for high-risk and low-risk types. Int J Cancer 2000, 87: Sargent A, Bailey A, Almonte M, Turner A, Thomson C, Peto J, Desai M, Mather J, Moss S, Roberts C, Kitchener HC: Prevalence of typespecific HPV infection by age and grade of cervical cytology: data from the ARTISTIC trial. Br J Cancer 2008, 98: Stoler MH, Schiffman M: Interobserver reproducibility of cervical cytologic and histologic interpretations: realistic estimates from the ASCUS-LSIL Triage Study. JAMA 2001, 85: Kinney W, Stoler MH, Castle PE: Patient safety and the next generation of HPV DNA tests. Am J Clin Pathol 2010, 134: Wu R, Du H, Belinson S, Yang B, Qu X, Wang C, Wu R, Wang G, Yang G, Belinson JL: P-191: The Shenzhen Cervical Cancer Screening Trial II (SHENCCAST II). Presented at the 26th International Papillomavirus Conference; July 3 8, 2010; Montreal, QC, Canada 29. Baseman JG, Kulasingam SL, Harris TG, Hughes JP, Kivat NB, Mao C, Koutsky LA: Evaluation of primary cervical cancer screening with an oncogenic human papillomavirus DNA test and cervical cytologic findings among women who attended family planning clinics in the United States. Am J Obstet Gynecol 2008, 199:26.e1 26.e8 30. Knoepp SM, Kuebler DL, Wilbur DC: Resolution of equivocal results with the Hybrid Capture II High-Risk HPV DNA Test: a cytologic/ histologic review of 191 cases. Diagn Mol Pathol 2007, 16: Ko V, Tambouret RH, Kuebler DL, Black-Schaffer WS, Wilbur DC: Human papillomavirus testing using Hybrid Capture II with SurePath collection: initial evaluation and longitudinal data provide clinical validation for this method. Cancer, 2006, 108: Siddiqi A, Spataro M, McIntire H, Akhtar I, Baliga M, Flowers R, Lin E, Guo M: Hybrid ccapture 2 human papillomavirus DNA testing for women with atypical squamous cells of undetermined significance: Papanicolaou results in SurePath and ThinPrep specimens. Cancer Cytopathol 2009, 117: