18 genotyping tests for use in women with ASC-US cytology

Gynecologic Oncology 118 (2010) 116–122

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Gynecologic Oncology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y g y n o

Clinical validation of the Cervista® HPV HR and 16/18 genotyping tests for use in women with ASC-US cytology Mark H. Einstein a,⁎,1, Mark G. Martens b,1, Francisco A.R. Garcia c, Daron G. Ferris d, Amy L. Mitchell c, Stephen P. Day e, Marilyn C. Olson e a

Montefiore Medical Center and Albert Einstein College of Medicine, Bronx, NY, USA Jersey Shore University Medical Center, Neptune, NJ, USA The University of Arizona, Tucson, AZ, USA d Gynecologic Cancer Prevention Center, Medical College of Georgia; Augusta, GA, USA e Hologic, Inc, Madison, WI, USA b c

a r t i c l e

i n f o

Article history: Received 10 February 2010 Available online 21 May 2010 Keywords: High-risk HPV Genotyping Colposcopy Triage ASC-US

a b s t r a c t Objective. High-risk (HR) human papillomavirus (HPV) testing is important in cervical cancer screening for triage to colposcopy. This study evaluated the clinical performance of the Cervista® HPV HR and 16/18 genotyping tests for detection of HPV in cervical cytology specimens. Methods. The tests were prospectively evaluated in a multicenter clinical study. DNA was extracted from approximately 4000 residual liquid-based cytology specimens collected during routine liquid-based Papanicolaou tests at standard of care visits and was assessed for the presence of HR HPV and/or HPV types 16 and 18. All women with cytology results of atypical squamous cells of undetermined significance (ASC-US) or greater underwent colposcopic examination and biopsies were collected. Test results were compared with local colposcopy and histology results from a central pathology review panel. Results. There were 1347 subjects with complete data sets of cytology, HR HPV, colposcopy, and histology included in the analysis of the HPV HR test. Sensitivity of the HPV HR test for detection of cervical intraepithelial neoplasia (CIN) 2+ among women with ASC-US cytology was 92.8% (95% confidence interval [CI]: 84.1–96.9) and the negative predictive value (NPV) was 99.1% (95% CI: 98.1–99.6). Sensitivity for detection of ≥ CIN 3 in women with ASC-US was 100% (95% CI: 85.1–100) and the NPV was 100% (95% CI: 99.4–100). The specificity of the test for detection of ≥ CIN 2 and ≥ CIN 3 was 44.2% (95% CI: 41.5–46.9) and 43% (95% CI: 40.3–45.7), respectively. The HPV 16/18 genotyping test also performed as expected in women with ASC-US cytology who were positive for HR HPV. Conclusion. The Cervista® HPV HR test can be clinically used for detecting HR HPV types in conjunction with cervical cytology for use in triage of women with ASC-US cytology during routine cervical cancer screening. © 2010 Elsevier Inc. All rights reserved.

Introduction The role of oncogenic human papillomavirus (HPV) as the causative agent of cervical cancer has been well documented [1–3]. There are 14 oncogenic, high-risk (HR) HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) that have been recognized as the cause of nearly all cervical cancers [2,3]. HPV 16 and 18 are responsible for over 65% of all cervical cancers in the United States [4,5]. As a result of the multiple natural history studies that have proven the causality of HPV with cervical cancer, patient management guidelines have been published by various professional societies and public health entities in the United

⁎ Corresponding author. Albert Einstein College of Medicine and Albert Einstein Cancer Center, Montefiore Medical Center, 1695 Eastchester Rd, Suite 601, Bronx, NY 10461, USA. Fax: +1 718 405 8087. E-mail address: meinstei@montefiore.org (M.H. Einstein). 1 Drs. Einstein and Martens contributed equally to this manuscript. 0090-8258/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2010.04.013

States to guide cervical cancer screening practices [6–8]. Most recommend testing for the presence of HR types for HPV triage of atypical squamous cells of undetermined significance (ASC-US) cytology. As an alternative screening strategy to cervical cytology, HR HPV testing is recommended as a co-test in combination with cytology in women 30 years of age and older to guide referral to colposcopy, if needed. In general, patient management decisions, with particular regard to triage to colposcopy, reflect patients' overall cytology history in addition to the presence or absence of HR HPV types [6,8,9]. In the clinical setting, most HPV testing in the United States before 2009 has been performed using the Hybrid Capture II test (hc2: QIAGEN, Inc; Valencia, CA), which was the only test approved by the US Food and Drug Administration (FDA) [10]. The strengths and weaknesses of this test have been well documented in peer-reviewed literature [11–13]. We present results from a prospective, multicenter clinical study establishing the clinical performance characteristics of 2 new HPV tests that use novel molecular methods, the Cervista® HPV HR and Cervista®

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HPV 16/18 tests (Hologic, Inc; Marlborough, MA). The former is a qualitative test for the detection of DNA from 14 HR HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68), while the latter is a genotyping test for the detection of HPV 16 and 18. Both tests use the chemistry (HologicTM, Inc), a signal amplification method for detection of specific nucleic acid sequences [14]. The subject population was comprised of women who were diagnosed with ASC-US, including both HPV-positive and -negative subjects. The study goals were to establish the clinical performance characteristics (ie, sensitivity, specificity, and negative predictive value [NPV]) of the Cervista® HPV HR and 16/18 genotyping tests for the detection of CIN 2 and CIN 3 in women with ASC-US, as well as estimate the risk for progression to cervical disease over a 3-year follow-up period in both HPV-positive and -negative subjects 30 years or older with normal Papanicolaou (Pap) results at the time of enrollment.

Materials and methods A prospective, multicenter clinical study was conducted. The study evaluated the performance of a novel HPV HR pooled-probe DNA-based test (Cervista® HPV HR test) for triage of subjects with ASC-US cytology and to determine the need for referral to colposcopy. In addition, the study evaluated the performance of a HPV 16/18 DNA-based genotyping test (Cervista® HPV 16/18 test) for screening patients with ASC-US cytology and those with concomitant HR HPV results. The study was conducted in the United States, with participation from 89 sites across 22 states. ThinPrep® (Hologic, Inc) liquid cytology specimens were used for all HPV testing. These were “standard of care” studies with respect to patient management, with the addition of performing colposcopy on all subjects who had ASC-US cytology, regardless of HPV testing. Cytology results were classified according to the 2001 Bethesda System Classification [9]. Each site obtained approval by local or central institutional review board with written informed consent obtained from each subject prior to enrollment. The study population was comprised of 2 arms. The first included women age 18 or older with ASC-US cytology results during routine cervical cancer screening who were invited to participate in the study prior to knowledge about their HPV status. This arm of the study was conducted between July 2006 through the end of December 2007, prior to implementation of guidelines that recommend limiting ASC-US HPV testing to women 21 years or older [15]. The second arm consisted of women ≥ 30 years and negative for intraepithelial neoplasia or malignancy (NILM). The data for the NILM arm of this study are not included in this publication as subjects are still in the follow-up phase. All enrolled subjects in the ASC-US arm underwent colposcopic examination as pre-specified in the protocol to determine the clinical performance of the HPV testing. To avoid clinical bias, investigators and subjects remained blinded to the subject's Cervista® HPV status. Women were excluded from the study if they were pregnant at the time of enrollment. If a subject became pregnant before the study-associated Pap sample was collected, they were discontinued and their data were not used in the final analysis. Exclusion criteria also included women who received chemotherapy or radiation therapy to the pelvis prior to enrollment or at any time during the trial. Cervical specimens were collected with a Rovers® Cervex-Brush® (Oss, the Netherlands), Wallach Papette® (Orange, CT), or other FDAapproved broom-type device or endocervical brush/spatula combination, and stored in PreservCyt® medium (the specimen collection medium for the ThinPrep® Pap test). ThinPrep® samples were sent to local or reference laboratories for cytology analysis and in some cases for clinically indicated HPV testing (using the hc2 test) as part of the routine standards of care specific to the site. Residual cervical samples were then returned to the clinical trial center where the sample was initially collected. These residual samples from enrolled subjects were then deidentified and shipped to a central sample repository (Fisher BioSer-

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vices; Rockville, MD) for random redistribution to 1 of 4 molecular testing centers for study-related blinded testing. The molecular testing centers extracted DNA from the residual liquid cytology samples that contained at least 2 mL of sample volume, using the Genfind™ DNA Extraction Kit (Hologic, Inc), to perform the HPV HR and 16/18 genotyping tests [16–18]. DNA was extracted twice, from 2 mL aliquots of the original sample, and placed into 2 separate cryovials labeled B and C. Cryovial B was used for analysis of HR HPV and 16/18 genotyping. Cryovial C was retained for long-term storage except when required as backup material. The technology for the HPV HR and 16/18 genotyping tests has been previously described [14]. The test uses the Invader® chemistry, a signal amplification method for detection of specific nucleic acid sequences. This method is based on enzymatic cleavage by the Cleavase® enzyme (Hologic,™ Inc), a structure specific 5′ nuclease. In brief, the sequence specific probe and Invader® oligonucleotides bind to the DNA target sequence to create a substrate structure for the enzyme. Probes cycle rapidly on and off the target sequence so that multiple cleaved 5′ flaps are generated per target sequence. The cleaved flaps then bind to a universal hairpin fluorescence resonance energy transfer (FRET) oligonucleotide, creating another substrate structure for the enzyme. The enzyme cleaves the FRET oligonucleotides and produces fluorescence signal as the cleaved flaps cycle on and off. The reagents for the HPV HR test are provided as 3 oligonucleotide mixtures, which altogether detect the 14 types of HPV grouped according to their HPV phylogenetic relatedness. Probes directed against multiple gene regions of all 14 HPV types tested are included to account for potential loss of portions of the HPV genome integration into human cellular chromosomes. The reagents for the 16/ 18 genotyping test are provided as 2 oligonucleotide mixtures, containing probes specific for multiple genomic regions of either HPV 16 or 18. For the HPV HR test, a positive HPV result indicates that at least 1 of the 14 HR types is present in the DNA sample, whereas a positive result for the 16/18 genotyping test indicates that HPV 16, HPV 18, or HPV 16 and 18 have been detected in the sample. Oligonucleotides targeting the human histone 2 gene (H2be, HIST2H2BE) are present in each of the oligonucleotide mixtures to serve as an internal control. Biopsy samples were collected from women with ASC-US or worse cytology at the discretion of the colposcopist and based on colposcopic findings. Histological analysis of biopsy specimens was conducted locally at clinical centers or reference pathology laboratories, then reported to the subject's physician per local clinical standards of care. Subsequently, the original slides that were used by the local pathology laboratory were retrieved, de-identified by the clinical trial site, and sent to the central pathology review panel. In few instances (6%), due to local pathology policies, these biopsies were re-cuts of the original biopsy block. Pathology results provided by the central review panel were reported as a consensus report from 2 expert pathologists blinded to each other's interpretation. If the pathologists disagreed, a third pathologist provided an independent review. If that pathologist disagreed with both initial reviews, a final review was performed by all 3 pathologists to achieve a consensus diagnosis. The clinical performance of the HPV HR test was assessed by calculating sensitivity, specificity, NPV, and positive predictive value (PPV) for detecting colposcopically-directed, centrally-confirmed histology results in all subjects (using ≥ CIN 2 histology results as positive for disease) with 2-sided 95% confidence intervals (CI). Clinical performance data for the 16/18 genotyping test were calculated in conjunction with HPV HR test results. All ASC-US subjects with both HPV HR test and colposcopy or histology results were considered evaluable and included in the analysis of the HR HPV test. For the 16/18 genotyping test, all ASC-US subjects with an HPV HR test, 16/18 genotyping test, and colposcopy or histology results were considered evaluable and included in the analysis. Negative results were defined by colposcopy or as “no CIN or CIN 1” by central review panel histology. The ASC-US enrollment target was calculated based on an anticipated prevalence rate of approximately 7% to 8% for CIN 2 or greater. The actual prevalence of

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Table 1 Subject demographics. N Age (years) at consent Mean ± SD Median Min. Max. Race Asian Black or African American Native American or Alaska Native Native Hawaiian or Pacific Islander White or Caucasian Other Ethnicity Hispanic or Latino Non-hispanic or Latino

1514 33.7 ± 11.8 31.0 18.0 79.0 33 (2.2) 282 (18.6) 6 (0.4) 4 (0.3) 1172 (77.4) 17 (1.1) 132 (8.7) 1382 (91.3)

HR HPV prevalence by age

HR HPV

HPV 16

HPV 18

HPV 16 and 18

Overall 18 to b21 21 to b30 30 to b39 ≥39

57.1% (769/1347) 77.8% (105/135) 71.7% (352/491) 55.3% (167/302) 34.6% (145/419)

16.8% (229/1363) 31% (40/129) 23.7% (117/493) 11.9% (37/312) 8.2% (35/429)

4.2% (57/1363) 7.8% (10/129) 6.3% (31/493) 2.6% (8/312) 1.9% (8/429)

0.9% (12/1363) 1.6% (2/129) 1.6% (8/493) 0.6% (2/312) 0% (0/429)

SD = standard deviation; HR = high-risk; HPV = human papillomavirus.

CIN 2 and CIN 3 within the ASC-US sample population was monitored without knowledge of any HR HPV results; if the observed prevalence differed from the estimated 7% to 8%, the enrollment goal for ASC-US subjects was to be adjusted as part of a continuous enrollment to reach the pre-specified event goal. All computations were performed using SAS® software version 9.1 (Cary, NC).

Results The participant flow is presented in Fig. 1. Pap results from subjects who were included in the study yielded 1514 ASC-US subjects. Complete data sets of cytology, HR HPV, colposcopy, and histology (when biopsy was warranted based on colposcopic findings) were available for 1347 of

Fig. 1. Study design of the Cervista® HPV HR and 16/18 genotyping tests.

M.H. Einstein et al. / Gynecologic Oncology 118 (2010) 116–122

those subjects. For the 16/18 genotyping analysis, complete data sets of cytology, HR HPV, 16/18 genotyping, colposcopy, and histology (when biopsy was taken by clinical judgment based on colposcopic findings) were available for 1312 of the original ASC-US subject population. Subjects in this trial were a broad representative sampling of the US population with regards to race and ethnicity (Table 1). The prevalence of HR HPV, HPV 16, HPV 18, and HPV 16/18 among the study population, stratified by subject age, is shown in Table 1. As expected, the highest prevalence of any HPV tested occurred in younger women and declined as subject age increased. Of the HR HPV subjects, 69 had histology-defined ≥CIN 2 of which 22 had ≥CIN 3 (Table 2A). This represented a disease prevalence rate of 5.2% for ≥CIN 2 among enrolled ASC-US subjects. Moreover, 63.8% (44/69) of these subjects were positive for HPV 16 or 18 (Table 2B). The overall and age-stratified clinical performance of the HPV HR and 16/18 genotyping tests were compared with colposcopically-directed, centrally-confirmed histology results among subjects with ASC-US cytology. For the HPV HR test, the clinical sensitivity and NPV calculated for ≥CIN 2 was 92.8% (95% CI: 84.1–96.9) and 99.1% (95% CI: 98.1–99.6), respectively (Table 3). The specificity of the test for detection of ≥CIN 2 was 44.2% (95% CI: 41.5– 46.9). For the 16/18 genotyping test, the observed sensitivity and NPV calculated for ≥CIN 2 was 68.8% (95% CI: 56.6–78.8) and 96.0% (95% CI: 93.9–97.4), respectively, which are in agreement with estimated prevalence of these types in cervical disease (Table 3) [4,5,19]. For ≥CIN 3, the clinical sensitivity and NPV of the HPV HR test was 100% (95% CI: 85.1–100) and 100% (95% CI: 99.4–100), respectively. The specificity of the test for detection of ≥CIN 3 was 43% (95% CI: 40.3–45.7). For the 16/18 genotyping test, sensitivity and NPV were 77.3% (95% CI: 56.6–89.9) and 99.0% (95% CI: 97.7–99.6), respectively (Table 3). As previously observed for hc2 [20], sensitivity appeared to decrease with increasing age for the HPV HR and 16/18 genotyping tests (Table 4). Discussion This study demonstrated the Cervista® HPV HR test accurately detects the presence of clinically-relevant DNA from 14 HR HPV types in women with ASC-US cytology. The clinical sensitivity for the detection of ≥CIN 2 and ≥CIN 3 was 93% and 100%, respectively, and NPV of ≥CIN 2 and ≥CIN 3 was 99.1% and 100%, respectively. In addition, the clinical performance for the Cervista® HPV 16/18 test in women infected with HR HPV demonstrates the test performed as expected. The observed sensitivities

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Table 3 Clinical performance summary of the Cervista® HPV HR and Cervista® HPV 16/18 genotyping tests compared with colposcopy/central histology results (≥ CIN 2 and ≥CIN 3) among women with ASC-US cytology. Central histology result

Cervista® HPV HR test

Cervista® HPV 16/18 testa

≥ CIN 2 Sensitivity (95% CI) Specificity (95% CI) NPV (95% CI) PPV (95% CI)

92.8% (84.1–96.9) 44.2% (41.5–46.9) 99.1% (98.1–99.6) 8.3% (7.6–8.9)

68.8% 69.3% 96.0% 17.1%

≥ CIN 3 Sensitivity (95% CI) Specificity (95% CI) NPV (95% CI) PPV (95% CI)

100% (85.1–100) 43% (40.3–45.7) 100% (99.4–100) 2.9% (2.4–3.0)

77.3% (56.6–89.9) 67.3% (63.9–70.6) 99.0% (97.7–99.6) 6.6% (4.2–10.3)

(56.6–78.8) (65.7–72.6) (93.9–97.4) (13.0–22.2)

HPV = human papillomavirus; HR = high-risk; CIN = cervical intraepithelial neoplasia; ASC-US = atypical squamous cells of undetermined significance; CI = confidence interval; PPV = positive predictive value; NPV = negative predictive value. a Performance values for the 16/18 genotyping test were calculated for subjects who had positive HPV HR results.

of the 16/18 genotyping test for ≥CIN 2 (68.8%) and ≥CIN 3 (77.3%) are in agreement with estimated prevalence of these types in cervical disease [4,5,19]. Professional societies have established evidence-based guidelines for HPV testing to help guide clinical practice and management of women with cervical disease since the initiation of molecular testing for HPV. The ASC-US/LSIL Triage Study (ALTS) for cervical cancer [21], the largest trial conducted supporting the use of clinical testing of HR HPV (with hc2), provided a baseline of performance . What remains important is the clinical performance of any HR HPV DNA test must show correlation with the presence and absence of clinically-relevant cervical disease (≥CIN 2) before use in clinical practice [22]. When used in combination with cytology, HR HPV testing improves the sensitivity of a single Pap test [9,23]. The Cervista® HPV HR test has 2 important characteristics; it requires a 2 mL sample volume for test performance that reduces the potential of yielding a “quantity not sufficient” result. Additionally, it includes an internal control to assess sample adequacy. The latter is an improvement over currently available testing methodologies [24,25]. This should enhance reliability by reducing or eliminating the number of specimens

Table 2 Cervista® HPV HR (A) and Cervista® HPV 16/18 genotyping (B) results compared with colposcopy/central histology results among women with ASC-US cytology. A. Cervista® HPV HR HR HPV positive HR HPV negative HR HPV indeterminate Total

Negative colposcopy, no biopsy

Central histology No CIN

CIN 1

CIN 2

≥CIN 3

164 214 4 382

389 314 11 714

152 30 0 182

42 5 0 47

22 0 0 22

B.

Total

769 563 15 1347

Central histology

HPV HR result

HPV 16/18 genotyping result

Negative colposcopy, no biopsy

No CIN

CIN 1

CIN 2

CIN 3

Total

HR HPV positive

HPV HPV HPV HPV HPV HPV

39 11 1 109 3 210 373

83 22 3 273 3 304 688

40 9 5 98 1 29 182

25 0 2 15 0 5 47

14 1 2 5 0 0 22

201 43 13 500 7 548 1312

HR HPV negative Total

16 18 16 16 16 16

positive positive and 18 positive and 18 negative and/or 18 positive and 18 negative

Among those with HR HPV determinate results and disease status data, percent of indeterminate HPV genotyping results in the clinical study of women with ASC-US cytology was 0% (0/1312) with 95% CI: 0% to 0.3%. HPV = human papillomavirus; HR = high-risk; ASC-US = atypical squamous cells of undetermined significance; CIN = cervical intraepithelial neoplasia; CI = confidence interval.

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Table 4 Age-stratified clinical performance summary of the (A) Cervista® HPV HR and (B) Cervista® HPV 16/18 genotyping tests compared with colposcopy/central histology results (≥ CIN 2) among women with ASC-US cytology. Age 4A 18 to b 21 years HR HPV result Positive Negative Total 21 to b 30 years HR HPV result Positive Negative Total 30 to b 39 years HR HPV result Positive Negative Total ≥ 39 years HR HPV result Positive Negative Total

bCIN 2

96 28 124

321 136 457

157 126 283

131 268 399

≥ CIN 2

Sensitivity (95% CI)

Specificity (95% CI)

100% (70.1–100.0)

22.6% (16.1–30.7)

100% (89.0–100.0)

29.8% (25.8–34.1)

76.9% (49.7–91.8)

44.5% (38.8–50.3)

87.5% (64.0–96.5)

67.2% (62.4–71.6)

9 0 9

31 0 31

10 3 13

14 2 16

Note: Among women with ASC-US cytology, there were 1.1% (15 out of 1347) HPV HR indeterminate results with 95% CI: 0.7% to 1.8%. HPV = human papillomavirus; HR = high-risk; ASC-US = atypical squamous cells of undetermined significance; CIN = cervical intraepithelial neoplasia; CI = confidence interval. 4B 18 to b 21 years HR HPV positive HPV 16 and/or 18 (+) HPV 16 and 18 (−) HR HPV negative Total 21 to b 30 years HR HPV positive HPV 16 and/or 18 (+) HPV 16 and 18 (−) HR HPV negative Total 30 to b 39 years HR HPV positive HPV 16 and/or 18 (+) HPV 16 and 18 (−) HR HPV negative Total ≥ 39 years HR HPV positive HPV 16 and/or 18 (+) HPV 16 and 18 (−) HR HPV negative Total

40 55 27 122

116 198 135 449

31 124 124 279

26 103 264 393

77.8% (45.3–93.7)

57.9% (47.8–67.3)

67.7% (50.1–81.4)

63.1% (57.6–68.2)

80.0% (34.9–94.3)

80.0% (73.0–85.5)

57.1% (32.6–78.6)

79.8% (72.1–85.9)

7 2 0 9

21 10 0 31

8 2 3 13

8 6 2 16

Note: Among women with Cervista® HPV HR determinate results and disease status data, percent of indeterminate Cervista® HPV 16/18 results in women with ASC-US cytology was 1.5% (20 out of 1332) with 95% CI: 1.0% to 2.3%. CIN =cervical intraepithelial neoplasia; CI = confidence interval; HR = high-risk; HPV = human papillomavirus.

that are hypocellular or contain insufficient DNA to provide valid test results, reducing the potential for false-negative HPV results. Internal controls are a requirement for most other molecular diagnostic tests currently being performed in a Clinical Laboratory Improvement Amendments (CLIA) and College of American Pathologists (CAP)-certified setting. A reduced sample volume requirement also minimizes the percentage of HPV samples with insufficient volume remaining after cytological examination, something that can occur N4% of the time with other HPV testing methods [21]. Future studies may help determine the clinical importance of these characteristics. Studies have demonstrated cross-reactivity with additional HPV types using other molecular HPV testing methodologies [11–13]. While this may not appear to be a disadvantage with respect to HR HPV types, cross-reactivity with low-risk, nononcogenic types may have clinically-significant consequences if such false-positive results lead to unneeded or frequent invasive diagnostic and therapeutic

procedures. The HPV HR test does not exhibit any cross-reactivity to common low-risk HPV types 6, 11, 42, 43, 44, and 53 [14]. Invariably, many clinicians will compare this current study with ALTS (Table 5). For the ALTS data, disease assessment was derived from the immediate colposcopy arm, while clinical performance data was derived from the HPV triage arm [21]. This analysis illustrates the lower disease prevalence in the present trial (5.2%) versus that of ALTS (11%). Several factors may have contributed to this difference. Principally, the observed difference in disease prevalence rate could be attributed to more wide-scale HPV screening that may increase detection of disease. Less likely, the mean age of ASC-US subjects in this study and ALTS was different; 33 years of age versus 29 years of age, respectively. Because the age of the subject population is older than ALTS, the referral rates were lower, since prevalence of HPV decreases with age. The present study also included HPV-negative ASC-US subjects who were subject to colposcopy. This study also had 89 subject recruitment sites located in

M.H. Einstein et al. / Gynecologic Oncology 118 (2010) 116–122 Table 5 Comparison of the Cervista® HPV HR Clinical Trial (ASC-US cytology study arm) and ALTS. Criterion

ALTS

Cervista® trial

No. of enrollment sites/states Mean age of subjects Subjects with colposcopy completed Subjects with no lesion; no biopsy performed (%) Subjects with no pathologic lesion on biopsy (%) Subjects with ≥ CIN 1 (%) Subjects with ≥ CIN 2 (%) Detection rate for ≥CIN 2 Detection rate for ≥CIN 3 Negative predictive value for ≥CIN 2 Negative predictive value for ≥CIN 3 Referral rate to colposcopy PCR concordance

4/4 29 1149a 25%a 49%a 15%a 11%a 96%c 96%c 98.9%c 99.5%c 56%c 82.7%

89/22 33 1347b 28% 53% 14% 5% 93% 100% 99.1% 100.0% 57%d 86.1%

HPV = human papillomavirus; HR = high-risk; ASC-US = atypical squamous cells of undetermined significance; ALTS = ASC-US/LSIL Triage Study for Cervical Cancer; CIN = cervical intraepithelial neoplasia; PCR = polymerase chain reaction. a Immediate colposcopy arm of ALTS. b Number of subjects with known disease status and Cervista® HPV HR results. c Combined HPV triage and immediate coloposcopy arms of ALTS. d Referral rate for women 30 years of age and older was 43%.

22 states, providing a diverse geographic estimate of disease prevalence throughout the United States with a mix of private practice as well as hospital or clinic-based subjects who were enrolled. In the context of an ASC-US cytological diagnosis, the Cervista® HPV HR test provides a sensitive triage method for the detection of ≥CIN 2. In this subject population, approximately half of the women would be referred for colposcopy based on a positive HR HPV result while those with a negative result can be assured that little risk of cervical disease is present based on NPVs of 99.1% and 100% for ≥CIN 2 and CIN 3, respectively. Consistent with the biology of infection and similar to results from other studies, many of the subjects in the ASCUS population tested positive for HPV without the presence of disease. Hence the low PPV and specificity values that were calculated for the HPV HR test in this population. These values are similar to those reported for other HPV tests such as hc2 in an ASC-US population [21]. Despite these strengths of the study, there are limitations, such as lower disease prevalence of the subject population and validation of the HPV HR and 16/18 genotyping tests only for use with cervical cytology specimens collected with specific devices and reagents. Also, PPVs decrease when testing populations with low disease prevalence or individuals with no risk of infection. Subsequent to the development of the Cervista® HPV HR test, the International Agency for Research on Cancer determined that there is limited evidence to conclude HPV 66 is carcinogenic [26]. Although the prevalence of HPV 66 in women is low [4,5], this reclassification may have a marginal effect on the false-positivity rate of the test. The identification of women with ASC-US cytology in conjunction with a HR HPV infection is a useful aid for clinicians when determining who should be screened at increased intervals or undergo colposcopy [3,20,27,28]. Determining which HR HPV-positive women are infected with HPV 16 or 18 may also be a beneficial management tool for clinicians. Current patient management guidelines recommend HR HPV testing in women with ASC-US cytology who are 21 years of age and older as well as routine cytology and HR HPV testing for primary screening of women 30 years of age and older in conjunction with cytology [15]. Guidelines also state that HPV 16/18 genotyping would be clinically useful in conjunction with cytology in HR HPV-positive women who are ≥ 30 years of age [29]. This clinical trial substantiates the use of the Cervista® HPV HR test as a clinically-validated test for detecting HR HPV types in conjunction with cervical cytology for routine cervical cancer screening patients who have ASC-US.

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Conflict of interest statement Dr. Einstein has participated in scientific advisory boards for Hologic and Qiagen but does not receive an honorarium. Dr. Martens is the chair for DSMB for Contraceptive Devices, Hologic, Inc. Dr. Garcia has participated in an advisory board for Hologic, Inc. but does not receive an honorarium. Dr. Ferris has received research support from Third Wave Technologies (now part of Hologic, Inc.). Drs. Olson and Day are employees of Hologic, Inc. Dr. Mitchell has nothing to disclose.

Acknowledgments The Principal Investigators for the clinical trial were: Matthew L. Anderson, Baylor College of Medicine, Houston, TX; Eugene Andruczyk, Philadelphia Clinical Research, Philadelphia, PA; Keith Aqua, Visions Clinical Research, Boynton Beach, FL; David A. Baker, Stony Brook University, Stony Brook, NY; Kurt Barnhart, University of Pennsylvania Medical Center, Philadelphia, PA; Deborah A. Bartholomew, The Ohio State University, Columbus, OH; Jerome Belinson, Cleveland Clinic, Cleveland, OH; Debra L. Birenbaum, Dartmouth-Hitchcock Medical Center, Lebanon, NH; Stephen Blank, Mount Vernon Clinical Research, Sandy Springs, GA; Rory K. Brening, San Diego State University, San Diego, CA; LaRoyce F. Chambers, Aurora Health Care, Milwaukee, WI; Arthur Donovan, Bluegrass Clinical Research, Inc., Louisville, KY; Mark H. Einstein, Montefiore Medical Center, Bronx, NY; Robert Feldman, Miami Research Associates, Miami, FL; Daron G. Ferris, Medical College of Georgia, Augusta, GA; Donald Galen, Compass Clinical Research, San Ramon, CA; Cynthia Goldberg, Visions Clinical Research Tucson, Tucson, AZ; Elizabeth Graul, Salt Lake Research, LLC, Salt Lake City, UT; Linda Grover, Southeastern Integrated Medical, PL d/b/a Florida Medical Research, Gainesville, FL; Mark Alan Jacobs, TMC Life Research, Inc., Houston, TX; Andrew M. Kaunitz, University of Florida, Jacksonville, FL; Ilana Kirsch, Memorial Medical Group Clinical Research, South Bend, IN; Robin Kroll, Women’s Clinical Research Center, Seattle, WA; Leroy J. Leeds, The Women’s Hospital of Texas Clinical Research Center, Houston, TX; Cherie LeFevre, Saint Louis University, St. Louis, MO; Bruce Levine, Phoenix OB-GYN Associates, Moorestown, NJ; Tyrone Malloy, Soapstone Center for Clinical Research, Decatur, GA; John McKenna, North Spokane Women’s Clinic Research, Spokane, WA; Jeffrey A. Michelson, Eastern Carolina Women’s Center, New Bern, NC; Amy Mitchell, University of Arizona, Tucson, Arizona; Harold Moore, SC Clinical Research Center, Columbia, SC; Rovena R. Reagan, Women’s Health Care, San Diego, CA; Christine Riley, Welborn Clinic, Evansville, IN; Gloria E. Sarto, University of Wisconsin, Madison, WI; Gerrit Schipper, Capitol Women’s Care Frederick Ob-Gyn, Frederick, MD; Alan Schwartz, Helping Hands Clinical Research, Santa Ana, CA; Robert Schwartz, University of Miami, Miami, FL; Melvin Seid, Lyndhurst Gynecologic Associated, Winston-Salem, NC; Marilyn D. Short, Johns Hopkins Community Physicians, Baltimore, MD; Scott Spear, Planned Parenthood of Arkansas and Eastern Oklahoma, Tulsa, OK; Michael Thomas, Greater Cincinnati OB/GYN, Cincinnati, OH; Daniel A. Tomlinson, Advanced Clinical Research, Medford, OR; Suzanne Trupin, Women’s Health Practice, Champaign, IL; Arthur S. Waldbaum, Downtown Women’s Healthcare, Denver, CO; Frank White, Granite Run Ob/Gyn, Media, PA; Daryl Wieland, Jacobi Medical Center, Bronx, NY. Grateful acknowledgement is made to Jorge Garces, PhD, for his oversight of this clinical trial and contribution to the drafting of this manuscript. We would also like to thank Ping-Yu Liu for statistical analysis, Dayna Geralts (Hologic,™ Inc.) as the clinical trial project manager, Joe King (Hologic,™ Inc.) for data analysis, and Health Decisions Inc., (Chapel Hill, NC) for data management and analysis. Editorial support was sponsored by Hologic,™ Inc. and provided by David E. Kaminsky, PhD, at AlphaBioCom.

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