BD-ProExC as Adjunct Molecular Marker for Improved Detection of CIN2⁺ after HPV Primary Screening

Subject enrollment

From August 2005 to February 2007, 3,126 women with a median age of 42.7 years (range 18.0–84.3) undergoing routine screening agreed to participate in this prospective, colposcopy controlled study. We evaluated samples collected in 9 gynecological practices in Flanders (Belgium). Study specific subject identification codes were assigned and transmitted in such a manner that subject confidentiality was preserved. All women gave written informed consent and this study was approved by the local ethical committee (Ziekenhuis Oost Limburg, ZOL, Genk, Belgium).

Inclusion and exclusion criteria

Exclusion criteria included pregnancy and history of cervical disease (previous history of CIN2⁺); 221 women were excluded.

Study design and assessment of study endpoints

Power-based sample size calculations were performed for the relative ratios of PPVs. Using the Inequality Tests for Two Proportions based on the assumption of a reference proportion of 0.15, a test strategy sample of at least 80 cases and a reference strategy sample of at least 160 cases were determined sufficient to detect an effect corresponding to a ratio of at least 2.1 between Cytology (reference) and the test strategy, with α = 0.05, power = 80%, and the z-test based relative risk comparison to 1.

The study algorithm is summarized in Figure 1. After the exclusion criteria were applied 2,905 women underwent liquid-based cytology, HPV DNA testing, BD ProExC immunocytochemistry (ICC), and colposcopy verification. All colposcopies at baseline were performed according to a standardized protocol by 9 different gynecologists. First a 5% acetic acid solution was applied to assist in identifying undifferentiated epithelia or inflammation as well as true CIN. After the application of the acetic acid, a judgment of the transformation zone was made. After visual inspection a cervical smear was taken with the Cervex-Brush. Biopsies were only taken from all acetowhite lesions after smear taking. Those with positive colposcopic examinations received immediate cervical biopsy with subsequent histologic analysis and were given treatment as required. All other subjects were followed up based on their HPV and cytology results and either received a second colposcopy and biopsy or were monitored based on recommended Belgian follow-up guidelines. (21)

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Figure 1.

Study flow diagram.

Liquid-based cytology samples from all the eligible women were prepared for routine cervical screening. The cytology leftover was used for hrHPV detection by real-time PCR analysis, and an extra slide was prepared for BD ProExC ICC staining. All the results from cytology, ICC, biopsies, treatment/follow-up, and HPV status were entered into a database. The unique subject ID number was used to link the different cytological, histological, and virological data. For all samples included in the study the database was searched to identify cases with histology-proven CIN2⁺ within a 24-month follow-up period.

Cervical sample processing and classification

Cervical cells were collected using the Cervex-Brush (Rovers). After collection, the brush head of the sampling device was deposited directly into the vial containing the ethanol-based BD SurePath Preservative Fluid (BD SurePath; BD Diagnostics—TriPath). The vials were then transported to the laboratory for testing. All samples were prepared and analyzed in the laboratory of Clinical Pathology (RIATOL). A density sedimentation method (BD PrepMate; BD Diagnostics–Tripath) was used to enrich the cell samples by excluding blood and the majority of inflammatory cells, as well as necrotic debris, mucus, and other contaminants. Thin-layer slide preparations were then made with the fully robotic BD PrepStain (BD Diagnostics–Tripath) according to manufacturer instructions. Thin-layer cell dispersions with discrete staining for cytological analysis were generated with the BD PrepStain slide processor.

The cytological results were classified according to the Bethesda system 2001 (22): negative for intraepithelial lesion or malignancy (NILM), ASC-US, atypical squamous cells of undetermined significance, cannot exclude high-grade squamous intraepithelial lesions (ASC-H), atypical glandular cells of undetermined significance (AGC, 3 cases which were included in the ASC-US group), low-grade squamous intraepithelial lesions (LSIL), and high-grade squamous intraepithelial lesions (HSIL).

BD FocalPoint guided screening

All slides were scanned by the BD FocalPoint Guided Screener Imaging System according to manufacturer instructions. As a positive control, a known sample, previously diagnosed as HSIL (CIN3 biopsy confirmed) and positive for HPV 16, was loaded in between the study samples each day. Cytology screening was performed without knowledge of the HPV status.

Isolation of DNA from cervical cells for HPV DNA testing

DNA isolation from the cellular pellet remaining after processing of the BD SurePath cytology specimen was performed as previously described (22–25). The DNA extracts were stored at −20°C until PCR was performed.

Real-time type specific PCR analysis of HPV DNA

Each sample was initially subjected to real-time PCR amplification for the detection of β-globin to confirm that the DNA quality was suitable for PCR analysis. All samples were subsequently tested for the following hrHPV types using type specific-PCR (as previously described; refs.22 and 23): 16 E7, 18 E7, 31 E6, 33 L1, 33 E6, 35 E6, 39 E7, 45 E7, 51 E7, 52 L1, 52 E7, 56 E7, 58 L1, 58 E7, 59 E7, and 68 E7. Low-risk HPV types 6 E6, 53 E6, 66 E6, and 67 L1 were also tested.

BD ProExC immunocytochemistry

An additional thin-layer slide was prepared from the residual BD SurePath vial using the BD PrepStain instrument. Slides were treated with a pretreatment buffer for antigen retrieval (BD SureDetect; BD Diagnostics–TriPath). Slides were processed on an SMS 3600 automated stainer using the BD ProExC immunohistochemistry kit according to manufacturer instructions (BD Diagnostics–TriPath). Briefly, the liquid-based cytology slide was incubated with the BD ProExC antibody reagent, this was followed by the use of a detection reagent that included a 3,3′-diaminobenzidine tetrahydrochloride (DAB)-based chromagen (BD SureDetect detection reagent). The DAB-based chromogen imparts a brown nuclear stain to cells that over express MCM-2 and TOP2A. The specimen was then counterstained with hematoxylin–a bluing agent (BD SureDetect counterstains), and the slide was coverslipped for viewing under a light microscope. Moderate-to-intense brown nuclear staining observed in atypical epithelial cells was considered a positive result. Enlarged naked nuclei that stained positive were not considered positive. In some slides, bacilli and abundant mucus gave rise to background staining, whereas inflammatory cells did not stain. In some cases, staining of glandular cells and tubal metaplasia was seen and, for the most part, these showed a light cytoplasmic staining with occasional staining of the nuclei. They could be clearly identified as cases of tubal metaplasia or glandular cells by morphology and were not considered positive. All slides were screened by experienced cytotechnologists. The staining of the 2,905 slides was performed in 97 batches, with negative and positive controls included in each batch.

Statistical analysis

The main analyses were calculated on intention-to-treat populations. Although CIN2⁺ was used as the primary endpoint, we repeated the calculations with CIN3⁺ as the endpoint. All histologically verified cases of CIN2⁺ detected within 24 months after trial enrollment were included in this cross-sectional analysis. Women who did not have a CIN2⁺ lesion recorded in the database were classified as not having a high-grade CIN lesion. In ASC-US triage only cytology with result ASC-US are tested (not LSIL and HSIL). Women who had a cytological diagnosis of ASC-US or worse (i.e., ASC-US, ASC-H, AGC, LSIL, or HSIL) were classified as having abnormal cytology (ASC-US⁺).

We evaluated the sensitivity, specificity, PPV, and negative predictive value (NPV) of HPV DNA testing and cytology. Separate analyses were performed using histologically confirmed CIN2⁺ and histologically confirmed CIN3 or worse (i.e., CIN3⁺) as endpoints. We compared the efficacy–defined as the sensitivity (proportion of CIN2⁺ or CIN3⁺ detected)–PPV, and number of screening tests required of 8 different screening strategies involving HPV DNA testing and ProEx C immunostaining with that of primary screening with cytology alone:

• hrHPV DNA test alone;

• ProEx C immunostaining alone;

• Double testing, both hrHPV testing and cytology screening performed independently, with only LSIL, ASC-H, and HSIL irrespective of HPV status and ASC-US/hrHPV⁺ were considered for colposcopy;

• Cytology with ASC-US triage by hrHPV;

• Cytology with ASC-US triage by HPV 16/18;

• HPV DNA test targeting 16/18 only;

• Cytology with ASC-US triage by BD ProExC; and

• hrHPV DNA test with triage by BD ProExC.

The PPV describes the proportion of test-positive women who actually have the disease being screened for. We used the total number of screening tests and the number of tests needed to detect one case of CIN2⁺ and CIN3⁺ as indicators of the cost-effectiveness of a screening strategy. The following screening strategies were considered: (i) a single HPV DNA test; (ii) an HPV DNA test in combination with cytology (without knowledge of HPV results); and (iii) screening for specific hrHPV types 16 and 18, which in other cohort studies were found to confer a particularly high risk for the development of CIN3⁺ (26, 27). All proportions were calculated with exact 95 binominal CIs. We compared the efficacy of different screening strategies by calculating risk ratios with 95 CI.

Overall numbers of CIN2⁺/CIN3⁺ were calculated for primary screening and the subsequent triage. The number of referrals to colposcopy and the number of CIN2⁺/CIN3⁺ lesions per referral were also determined. Statistical analysis was performed using the MedCalc programme (MedCalc Software; ref. 28).