Objective
The objective of the study was to investigate whether knowledge of human papillomavirus (HPV) deoxyribonucleic acid test results increases sensitivity of guided cytology screening for the detection of cervical intraepithelial neoplasia (CIN)-2 or higher-grade cervical lesions.
Study Design
This was a prospective colposcopy-controlled study of 2905 BD SurePath samples to identify cases with CIN2+ within a 24 month follow-up period. Sensitivity and specificity to detect CIN2+ was evaluated, comparing guided cytology screening with and without prior knowledge of HPV status.
Results
Prior knowledge of HPV status resulted in significantly higher detection rate of CIN2+ compared with screening blinded to HPV status ( P = .005) with limited loss of specificity ( P = .026). Gain in sensitivity is higher in older women (43.8%, P = .008) vs in younger women (10.2%, P = .317), whereas loss of specificity is more pronounced in younger women ( P < .001) vs older women ( P = .729).
Conclusion
Guided cytological screening performed with prior knowledge of HPV status results in an improved detection of CIN2 or higher-grade lesions.
The recognition of the strong causal relationship between persistent infection with high-risk (HR) human papillomavirus (HPV) (HR-HPV) types, and cervical cancer and its precursors, has resulted in the development assays that detect viral nucleic acids as an alternative for or as an adjunct to cervical cytology. One can distinguish assays that detect all HR-HPV types as a group and genotyping tests that distinguish individual HPV types. Liquid-based cytology is now gradually replacing conventional cytological testing, because of practical advantages (quicker interpretation, easy ancillary molecular testing, and possibility of computerized guided screening), in spite of the lack of evidence that it increases the detection of cervical intraepithelial neoplasia (CIN)-2 or higher-grade cervical abnormalities.
Cytological screening combined HR-HPV testing and HPV-based screening followed by cytology triage have been evaluated as more sensitive than cytology screening alone. Compared with cytology alone, this screening strategy improves detection of precancerous growths but with a certain increase in the number of false-positive tests. Recent randomized trials have confirmed that HPV-based screening, in women older than 30-35 years followed by cytology triage results in detection of more CIN2 or worse lesions compared with cytology screening. Moreover, longitudinal results of these trials have demonstrated that women with a negative HPV test have a lower risk of CIN3 and even invasive cancer.
This study aimed to evaluate the influence of knowing the different HR-HPV genotypes present in cervical specimens before performing guided cytological screening.
Materials and Methods
Study population
In this prospective, colposcopy-controlled study, we enrolled 3126 voluntary participants from August 2005 until February 2007 ( Figure 1 ) . Samples were collected during opportunistic routine health checks by 11 selected gynecologists in Flanders (Belgium). All women gave written informed consent.
Exclusion criteria included pregnancy and history of cervical disease (previous history of CIN2+); 221 women were excluded. At enrollment, participants underwent a colposcopy after smear taking. Colposcopy was performed in the framework of this study to obtain a gold standard. Study-specific patient identification codes were assigned and transmitted in such a manner that patient confidentiality was preserved and linked with follow-up and histology results. This study was approved by the local ethical committee (Ziekenhuis Oost Limburg, Genk, Belgium).
Cervical sample processing
Slide preparation
Cervical cells were collected using the Cervex-Brush (Rovers, Oss, The Netherlands).
After collection, brush heads were transferred directly into alcohol-based preservative (SurePath; Tripath Imaging Inc, Burlington, NC), and the vials were transported to the Laboratory for Clinical Pathology (labo RIATOL, Antwerp, Belgium). Thin-layer slide preparations were made with the fully robotic AutoCyte PREP System (AutoCyte; Tripath Imaging) and were prepared as described elsewhere.
High-risk HPV testing
All specimens from the screening visit were tested for HPV deoxyribonucleic acid (DNA) by polymerase chain reaction (PCR) amplification.DNA isolation from liquid-based cytology was performed as previously described. Briefly, HPV DNA was extracted from cervical cells using standard proteinase K-based digestion according to the manufacturers’ protocol. Washed cell pellets were incubated with proteinase K solution (100 μg/mL) for 3 hours at 55°C. First, each sample was subjected to quantitative PCR (qPCR) amplification for the detection of β-globin to confirm that the DNA quality was suitable for PCR analysis.
All samples were tested for the presence of 14 different HR-HPV genotypes using TaqMan-based real-time qPCR, targeting type-specific sequences of viral genes: 16 E7, 18 E7, 31 E6, 33 E6, 35 E6, 39 E7, 45 E7, 51 E6, 52 E7, 56 E7, 58 E6, 59 E7, 66 E6, and 68 E7. Presence of low-risk HPV types, for instance, HPV6, HPV53, and HPV67, were considered negative for HPV infection.
Slide classification
BD FocalPoint reading
All liquid-based cytology samples were first scanned with the BD-FocalPoint system followed by guided assisted screening with BD-SlideWizard, according to the manufacturer’s instructions (BD Diagnostics-TriPath, Burlington, NC). The FocalPoint system classifies 25% of all slides as no further review (NFR) in which the probability of intraepithelial lesions is extremely low. The other 75% are categorized in quintile 1-5, in which quintile 1 has the highest probability of abnormality, based on slide scores. In this study, also, the NFR slides were screened cytologically.
BD SurePath liquid-based cytology using guided screening with a BD-SlideWizard was used to compare screening with and without prior knowledge of HR-HPV status for each selected sample.
Bethesda classification
The cytological results were classified according to the Bethesda system 2001, using the classes negative for intraepithelial lesions or malignancy, atypical squamous cells of undetermined significance (ASC-US), atypical squamous cells of undetermined significance that cannot exclude high-grade squamous intraepithelial lesions (ASC-H), low-grade squamous intraepithelial lesions (LSIL), and high-grade squamous intraepithelial lesions (HSIL). Slides were read by 2 independent cytologists in arbitrary order so that for any sample, it was equally likely that the cytology with or without prior knowledge would take the first reading. High-grade cervical disease was considered to comprise histological grade CIN2 or higher (CIN2, CIN3, adenocarcinoma in situ, invasive squamous cell carcinoma).
Cytology with/without prior knowledge
Cytology was performed twice on every sample, with and without knowledge of HPV DNA test results. Reading was done in arbitrary order, and it was ensured that every slide was read by different cytologists, arbitrarily selected from a pool of cytologists. In both cytological readings, demographic and clinical information was provided. Cytological interpretation with knowledge of HPV status included information on the type and the type-specific viral load. All slides were reviewed by cytopathologists, blinded from HPV DNA tests results but aware of the screening results of the cytologists.
Follow-up and assessment of study endpoints
All women underwent colposcopy at enrollment and participants with cervical abnormalities were referred for biopsy ( Figure 1 ). Women with a normal colposcopy result (or a negative histology result if a biopsy was taken) were considered as free of CIN. Women with a precancerous lesion or cervical cancer were referred for further management. The number of detected CIN 2 or higher grade cases in a 24 month follow-up period was used as study endpoint.
Data management
All results from cytology, histology, treatment/follow-up, and HPV status were entered in a database. Each patient was allocated a unique patient identification (ID) number. This patient ID number was used to link the different cytological, histological, and virological data.
Statistical analysis
The statistical package R version 2.10.1 was used for data analysis. HR-HPV positivity was defined as the presence of 1 or more of the following 14 HPV types: HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, and HPV68. Estimates of sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) for detection of CIN 2+ were obtained for both screening strategies: cytology with prior knowledge of HPV types and cytology without prior knowledge. Confidence intervals (CIs) for proportions were calculated by Wilson’s method. Differences in proportions were tested using McNemar statistics for paired comparison. The 95% CIs were computed for proportions, differences, and ratios of proportions. All statistical tests were 2 sided, and P ≤ .05 was considered statistically significant.
Women were divided by age into 2 categories; younger (<30 years old) and older (≥30 years old). The difference in sensitivity to detect CIN2+ in the 2 age groups and corresponding specificities and predictive values were assessed. A receiver operating characteristics (ROC) plot was used to evaluate/visualize the true positive rate vs the false-negative rate.
Histologically verified CIN 2+ cases that were detected within 24 months after enrollment at routine screening were included. Women who had a cytological diagnosis of ASC-US or worse were classified as having abnormal cytology. Separate analyses were performed to compare the sensitivity in the detection of CIN 2 or worse.
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