Materials and Methods
Study Design, Conduct, and Participants
The CLEAR study consisted of two parts: the ASC-US (Atypical Squamous Cells of Unknown Significance) Study and the Adjunct Study described here Figure 1. Women 30 years and older undergoing routine Pap testing who had a NILM cytology result were eligible to participate in the Adjunct Study and were recruited from 19 US family planning and obstetric/gynecologic clinics (private and academic), family practice medical groups, and clinical research centers encompassing a wide geographic area representative of the US population. Informed consent was obtained prior to enrollment of participants. The study protocol was approved by institutional review boards at the participating centers, and the study was conducted in accordance with applicable regulatory requirements and good clinical practices.
Clinical evaluation of Aptima mRNA study participant disposition. aReasons for withdrawal: did not meet eligibility criteria (70); Pap volume insufficient for AHPV testing (117); specimen expired or unsuitable for testing (190); specimen lost (58); noncompliant site (320); other reasons (26). bReasons for withdrawal: collection site did not participate in follow-up (243); subject terminated participation (37); participant had hysterectomy (22); participant not eligible (17); participant treated prior to CIN2+ diagnosis (8); other reasons (4). AHPV, Aptima HPV (Hologic, San Diego, CA); ASC-US, atypical squamous cells of unknown significance; CIN2+, cervical intraepithelial neoplasia grade 2 or higher; HC2, Hybrid Capture 2 (Qiagen, Gaithersburg, MD); HPV, human papillomavirus; NILM, negative for intraepithelial lesions or malignancy; Pap, Papanicolaou test.
Women were excluded from the study if they were pregnant, were vaccinated against HPV, had a history of cervical disease (cancer or precancerous) or an abnormal Pap test result in the previous 12 months, or had a history of illness that could interfere with the study or create an unacceptable risk to the participant. Demographic information and relevant medical information (cervical cancer history, prior HPV diagnosis, and any abnormal cytology history) were collected from each participant. The study employed a baseline evaluation and a 3-year follow-up period with annual cytology visits for longitudinal disease ascertainment. Participants completed and exited the study once they had a CIN2+ diagnosis.
Cytology (Referral Pap)
At the baseline evaluation and each annual visit thereafter, a cervical specimen was collected with a broom-like device (Papette; Wallach Surgical Devices, Orange, CT) or an endocervical brush and spatula (Cytobrush Plus GT and Pap Perfect Plastic Spatula; Medscand, Trumbull, CT) and placed into a ThinPrep Pap Test (Hologic) vial containing PreservCyt Solution ("referral Pap" specimen). Pap specimens were processed locally using the ThinPrep 2000 System (Hologic) and evaluated for cytologic abnormalities. Cytology results were classified using the 2001 Bethesda System for reporting cervical cytology.
Baseline PreservCyt specimens (1-mL aliquot) were tested with the AHPV (Hologic) on both the automated Tigris DTS System and Panther System. Results from the two systems were similar; Panther System results are presented here. AHPV is a target amplification assay that uses transcription-mediated amplification to detect the E6/E7 oncogene mRNA of 14 HR-HPV genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). Three clinical laboratories each tested approximately one-third of all samples with AHPV. Most of the PreservCyt specimens were also tested at one laboratory for HR-HPV DNA using the Hybrid Capture 2 assay (HC2; Qiagen, Gaithersburg, MD), an FDA-approved test that detects HR-HPV DNA of 13 of the 14 HR-HPV types detected by the AHPV and is known to cross-react with the 14th type. Testing and results interpretation of both HR-HPV tests were done according to the manufacturer's instructions.[38,39] Technicians performing HC2 and AHPV assays were masked to the other HPV test results and the participants' clinical status and colposcopic/histology results.
At baseline, women who tested positive in either the AHPV or the HC2 assay (HPV-positive women) and, to adjust for verification bias, approximately 6% of women who tested negative in both HPV assays (HPV-negative women) were randomly selected and referred to colposcopy. Most colposcopy visits (>60%) were completed within 16 weeks from the baseline visit (median, 14 weeks; interquartile range, 8 weeks).
Colposcopists were masked to HPV results and collected cervical punch biopsy specimens from each visible lesion ("directed" biopsy) and an endocervical curettage (ECC) biopsy specimen. The biopsy specimens were processed according to the normal site procedures to produce H&E-stained slides. After local pathologist review, slides were reviewed by two central panel pathologists (T.C.W. and M.H.S.) and classified using the three-tiered CIN terminology. Slides with discordant central panel diagnoses were reviewed by a third central pathologist to reach a consensus diagnosis (two out of three agreement). If agreement was not achieved, the three central panel pathologists reviewed the slides in conference to reach consensus. A participant's cervical disease status represents the highest grade consensus histology result from the colposcopy biopsy specimen. Review pathologists were masked to all other pathologists' diagnoses, the participants' clinical status, enrollment status (ASC-US Study or Adjunct Study), and HPV test results.
During follow-up, women with ASC-US or more severe cytology results were referred to colposcopy. Colposcopists collected the same types of biopsy specimens, which were processed and submitted to the same central pathology review as done at baseline to obtain the consensus histology result for that visit. Women with ASC-US or more severe cytology results who did not have a colposcopy were considered to have indeterminate disease status. Participants with NILM cytology at a follow-up visit were not referred to colposcopy and were considered to have a normal cervix.
The final disease status after 3-year follow-up was determined for each participant who completed the study. To complete the study, a woman must have either (1) a consensus result of CIN2 or worse or (2) at least one cytology visit during the first or second year of follow-up and one cytology visit during the third year of follow-up, including colposcopies for those with ASC-US or more severe cytology. Final disease status for women meeting the second criterion was based on their final consensus histology result or they were considered to have a normal cervix if they had NILM cytology at the last visit. Women with CIN2 or worse did not have further follow-up in the study.
Participants with an ASC-US or more severe cytology during follow-up who did not have a colposcopy or who attended the colposcopy visit but biopsy specimens were not collected, were lost, or the slides were inadequate to determine disease status were classified as indeterminate for cervical disease status.
Test performance was evaluated with participants having a consensus histology result of CIN2+ (CIN2, CIN3, carcinoma in situ, or invasive cancer) classified as positive for cervical disease. A diagnosis of CIN1 or normal disease status classified participants as negative for cervical disease. In addition, test performance was evaluated using a more definitive disease end point where a consensus histology result of CIN3+ (CIN3, carcinoma in situ, or invasive cancer) classified participants as positive for cervical disease, and CIN2, CIN1, or normal classified participants as negative for cervical disease.
For the baseline risk analysis, a disproportionately smaller subset (3.4%) of HPV-negative vs HPV-positive women had disease status determined from the baseline colposcopy visit, resulting in verification bias. To adjust for this bias, a multiple imputation method was used to impute missing disease status based on the observed consensus histology results and AHPV and HC2 assay results from women who had a baseline colposcopy. Verification bias-adjusted risk estimates and 95% confidence intervals (CIs) were generated using these imputed results.
The follow-up risk analysis included disease identified at baseline and during follow-up. Because all women did not have a colposcopy at baseline, individual by-year risk estimates may reflect disease that was either present but not detected at baseline or incident or progressive disease. Cumulative risks with 95% CIs were generated using the life table method, with participants not completing the study censored after the follow-up year last attended.
Sensitivity and specificity estimates with 95% CIs were generated including women who completed the study. The McNemar exact test of discordant matched pairs was performed to compare the assays, including only participants with results for both assays. All statistical tests and 95% CIs were two-tailed and performed at the 5% significance level, using SAS version 9.1 or higher (SAS Institute, Cary, NC).
Am J Clin Pathol. 2015;144(3):473-483. © 2015 American Society for Clinical Pathology