The Wolfsburg HPV Epidemiological Study (WOLVES) is a population-based cohort study on the prevalence and incidence of HPV infections and associated diseases in women of two predefined birth cohorts (born in 1984/4 or 1988/9) recruited between 19 October 2009 and 31 December 2010. The study was designed to measure the role of HPV infection in the pre-vaccination era and changes over a 5-year observation period. The residents’ registration office provided a list of all women born either 1983/84 or 1988/89 with a first residency in Wolfsburg city and 2850 women were invited by letter to attend cervical cancer screening. The invitation included information on the intention of the study and the voluntary participation. To be recruited, women had to attend one of 18 gynecologists in private practices in the city of Wolfsburg for routine Pap smear screening. Participants had to give written consent and complete a short standardized questionnaire in the private practice. Then they underwent a pelvic examination with visualization of the uterine cervix. Pap smears were taken using spatula and endocervical brush. A second sample was then obtained with a Qiagen Cervical Sampler (Medscan, Uppsala, Sweden), and suspended in 1 ml of specimen transport medium (STM/Qiagen Inc., Hilden, Germany) for HPV DNA testing.
The questionnaire included questions on education, birth country, marital status, pregnancies, parity, contraception, smoking, number of sexual partners, age at sexual debut, history of abnormal Pap smears, STI and GW. Furthermore, the referring gynecologist collected information on HPV vaccination status by checking the certificate of vaccination.
Patients were transferred for colposcopy if they suffered from GW and/or had abnormal Pap smears conspicuous of high-grade lesions or had Pap smears classified as borderline/low-grade and tested positive for high-risk (HR)-HPV.
Colposcopists classified the type of transformation zones according to the Barcelona nomenclature of the International Federation for Cervical Pathology and Colposcopy (IFCPC) [7
]. In cases of type 1 or type 2 transformation zone with visible squamous columnar junction (SCJ), colposcopy was regarded as satisfactory. Any visible lesion underwent histological assessments with punch biopsies. No random punch biopsies were taken if colposcopy findings were normal. In cases of type 3 transformation zones, colposcopy with not fully visible SCJ was regarded as unsatisfactory and endocervical curettage (ECC) was obligatory. Type 3 transformation zones with visible lesions underwent punch biopsies and ECC.
A variety of skin and mucosal lesions may be classified as GW. Lesions were included in the category “genital warts” only if they were classified on colposcopy as:
1. “typical condylomata acuminata” (TCA) for lesions that showed the typical acuminate morphology (Figure )
A: Typical condyloma acuminatum; B: Flat condyloma; C: Seborrheic wart like lesion.
2. “flat condylomata” (FC) for genital papillomas with a more hyperkeratotic and papillomatous surface and flat condylomata with a smooth surface and non-pigmented papules (Figure )
3. “seborrheic wart-like lesions” (SWL) of the dry skin of the external anogenital area if intraepithelial neoplasia was excluded on histology (Figure ).
Naevi, vulvar intraepithelial neoplasia (VIN), hirsutoidpapillomas and molluscacontagiosa were excluded.
Colposcopists and pathologists were blinded to HPV genotyping results at the time of classification of GW.
All primary HPV testing was undertaken using the HC2 assay (HC2/Qiagen Inc., Hilden Germany). All samples were analyzed for the presence of at least 13 HR-HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68) following the manufacturer’s instructions. Therefore, a positive HR-HPV result in this study refers to a patient positive for one or more of the 13 HR-HPV types included in the probe mix. Infection with LR-HPV types was also investigated by HC2. The LR probe mix included HPV types 6, 11, 42, 43 and 44. Samples were considered positive if they attained or exceeded the FDA-approved threshold of 1.0 pg HPV DNA/ml, which corresponds to 1.0 relative light unit (RLU).
All samples that tested positive for LR-HPV and/or HR-HPV with HC2 and 10% (every tenth) of all HC2-negative samples underwent HPV genotyping. HPV genotyping was performed as described previously using SPF-10-PCR, followed by Reverse Line Probe Assay LiPA Extra (SPF-10-PCR) [8
]. Briefly, total DNA was isolated from the cervical samples with the use of a MagNAPure device (Roche, Indianapolis, IN) and analyzed with the use of the INNO-LiPA Extra HPV prototype assay (Innogenetics, Inc, Gent, Belgium) according to the manufacturer’s instructions. According to good laboratory practice guidelines, all PCR manipulations were performed in a laboratory that was separate from the other laboratory rooms. All PCR reactions were undertaken with 10 μl input DNA in a final volume of 50 μl using provided reagents from Innogenetics, 10 min 37°C, 9 min 94°C, and 40 cycles of 30 sec of denaturation at 94°C, followed by 45 sec of 52°C annealing temperature and 45 sec of extension at 72°C run on a MJ Thermocycler PCT 200. The PCR product was then denatured and a 10 μl aliquot hybridized to one strip at 49°C for 60 min, followed by multiple washing steps. The INNO-LiPA Extra test allowed identification of 13 established high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68), five known or putative high-risk types (26, 53, 66, 73 and 82) [9
], seven LR-HPV types (6, 11, 40, 43, 44, 54 and 70), additional non-differentiated HPV types, and types with undefined risk (74 and 69/71) [11
]. The strips were analyzed on a flatbed scanner with the use of LiRAS prototype software (Innogenetics, Inc), which displays the patterns and relative intensity of positive bands as arbitrary grey-tone values between 0.1 and 1.0 and allows direct data transfer to Excel spreadsheets. All HC2-LR positive samples, which were negative in the LiPA Extra, were retested with the CP4/5 PCR. The positive PCR products were purified with the Qiagen Gel Extraction Test QIAquick and then directly sequenced with the CP4 primer [13
]. Sequencing allowed the identification of HPV subtypes that were not detectable by LiPA Extra.
Any lesion suspicious of high-grade neoplasia had to undergo histological assessment and patients with GW were tested for histological confirmation of the diagnosis with biopsies. Paraffin embedded tissue blocks of GW and/or cervical intraepithelial neoplasia (CIN), vulvar intraepithelial neoplasia (VIN), and vaginal intraepithelial neoplasia (VAIN) underwent HPV-genotyping with SPF-10 and LiPA. Data for the prevalence of HR-HPV, cofactors associated with the risk of HR-HPV infection, and the association between specific HR-HPV subtypes and abnormal Pap smear test results will be published separately.
The study was designed to provide a one-time cross sectional study of the 1983/84 cohort, whereas the 1988/89 cohort will be followed by annual examination for 5 years. In the year 2014/15, women born 1993/94 will be invited for a one-time examination. Overall WOLVES will allow investigation of individual changes in HPV infection during follow-up and a comparison of prevalence of HPV infections in different age cohorts over time.
WOLVES was approved by the ethics committee of the physicians’ association of Lower Saxony in Hannover, Germany (Bo/07/2009).