Between 2013 and 2016, women 60-65 years of age, with normal cytology in the southern region of Sweden (Skåne) (n=5925) were tested for the presence of HR-HPV.
When the Exit test was introduced in 2013 the labaratory had access only to the MGP-PCR Luminex HPV DNA assay and it was therefore used as the primary HPV assay throughout the study.
Cervical HPV DNA positivity was found in 286 (4.8%) individuals with a mean age of 61.9 years (SD +/- 1.7).
Exclusion criteria from further follow-up were history of cervical neoplasia and/or treatment of cervical disease such as the loop electrical excision procedure (LEEP), hysterectomy or trachelectomy and ongoing oncological treatment at the time the double test was performed. A total of 271 HR-HPV DNA positive women with normal cervical cytology were eligible for inclusion (Figure 1).
The double test consisted of a LBC sample (Thinprep, Hologic, Inc.) that was analyzed for HR-HPV DNA by using the MGP-PCR-Luminex assay 6,7. In women testing positive for HR-HPV DNA, a concomitant HPV E6/E7 mRNA assay (APTIMA, Hologic, Inc.) was performed. Women with normal cytology and a positive HR-HPV DNA result were scheduled for a new follow-up examination after 12 months including a new LBC specimen and a HPV DNA / mRNA co-testing procedure. All women diagnosed with cervical pathology and/or a positive HR-HPV DNA and/or mRNA result, were planned for a further clinical evaluation with colposcopic assessment. The same accounts for women with a positive HR-HPV DNA and/or mRNA outcome on two subsequent controls. The next routine co-testing procedure was scheduled after twelve months including even those women who underwent a clinical examination.
At all further follow-up controls which were performed at intervals of 12 to 18 months, the same selection criteria were applied to determine which women were in need of a further clinical investigation. During our surveillance period at least three consecutive follow-ups could be documented.
Women presenting with normal cytology and negative HR-HPV DNA results left the routine screening service.
Classification of LBC and Histology Results
LBC results were defined as normal, atypical squamous cells of undetermined significance (ASCUS), atypical glandular cells (AGCs), low-grade squamous intraepithelial lesions (LSILs), high-grade intraepithelial lesions (HSILs) according to the Bethesda classification 8. Histopathological results were defined as LSIL and HSIL lesions using a two tired classification system 9. Histologically confirmed HSIL or worse was used as primary study endpoint. Low-grade lesions based on cytological or histological findings are presented separately. Recurrent cytological abnormalities of the same severity level were considered as one incident case.
In women with HSIL lesions on LBC and corresponding colposcopic findings, a LEEP was performed for therapeutic management. Also patients with cytological ASCUS or LSIL but a colposcopic picture suggestive of an underlying precancer lesion were scheduled for a LEEP procedure.
In case of an inaccessible transformation zone located within the cervical channel, cervical biopsy or conisation specimen were obtained for diagnostic reasons.
HR-HPV testing
The MGP-PCR Luminex HPV DNA assay detects several HPV types simultaneously 6,7.
From each LBC vial (Thinprep) 2ml was centrifuged at 3500 x g for five minutes and then liquid was removed so that 500 uL remained. From each sample, DNA was purified by total NA-kit (200 uL input and 100 uL output) using Magna Pure LC (Roche) and then HPV DNA was amplified by PCR with modified GP5+/6+ (MGP) primers 7.
After amplification, the Luminex-based HPV genotyping allows the identification of the following HR-HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and the probable high-risk type 68 (A and B) as well as the possibly high-risk types 26, 53, 66, 67, 69, 73 and 82 as described by IARC classification from year 2012 10. In the present study, probable and possible HR-HPV types were classified as HR-HPV types.
The HPV E6/E7 mRNA (APTIMA) assay detects qualitatively E6/E7 mRNA from 14 HR-HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68.
From each LBC vial (Thinprep) 1 ml was automatically transferred to 2.9 ml APTIMA transfer solution (ATS, Hologic, Inc.) by a Tomcat instrument (Hologic, Inc.). Thereafter an aliquot of 400 ul was further processed for the HPV E6/E7 mRNA (APTIMA) assay by the Panther system (Hologic, Inc.).
We calculated the proportion of HPV E6/E7 mRNA positivity for each of these 14 HR-HPV types as well as for HPV67 (APTIMA is known to cross-react with HPV67, Kit insert, APTIMA HPV Assay, nr 503744), as determined by the MGP-PCR Luminex HPV DNA assay.
The presence of the same HR-HPV genotype at inclusion and at follow-up was defined as a persistent infection. At follow-up, women with benign cytology who tested negative for HR-HPV DNA but were positive for low-risk (LR) HPV DNA according to the Luminex assay, the HR-HPV status was considered as negative or as cleared infection.
Endpoints
The endpoint was the development of histologically confirmed HSIL or worse over a follow-up period of four years.
Statistical evaluation
Statistical comparisons were based on two-sided chi-square tests. All comparisons were two-sided, and a 5% level of significance was applied. The strength of association between the HPV mRNA results and the development of cervical abnormalities was measured using relative risk and the corresponding 95% confidence intervals.
Cumulative incident rates (CIR) during the follow- up period were calculated according to Kaplan-Meier survival analysis and presented as percentages with the corresponding 95% Confidence Interval (CI).
The statistical analyses were performed using SPSS version 19.0 or higher (IBM Corp., Amonk, NY, USA) and Omnistat (SBU, Trelleborg, Sweden).