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Human papillomavirus (HPV) infections may result in benign hyperplasia, caused by low-risk HPV types, or (pre)malignant lesions caused by high-risk HPV types. The molecular basis of this difference in malignant potential is not completely understood. Here, we performed gene profiling of different HPV infected vulvar tissues (condylomata acuminata (n = 5), usual type vulvar intraepithelial neoplasia (uVIN) (n = 9)) and control samples (n = 14) using Affymetrix Human U133A plus 2 GeneChips. Data were analyzed using OmniViz (R), Partek (R) and Ingenuity (R) Software. Results were validated by real-time RT-PCR and immunostaining. Although similarities were observed between gene expression profiles of low- and high-risk HPV infected tissues (e.g., absence of estrogen receptor in condylomata and uVIN), high-risk HPV infected tissues showed more proliferation and displayed more DNA damage than tissues infected with low-risk HPV. These observations were confirmed by differential regulation of cell cycle checkpoints and by increased expression of DNA damage-biomarkers p53 and ?H2AX. Furthermore, FANCA, FANCD2, BRCA1 and RAD51, key players in the DNA damage response, were significantly upregulated (p < 0.05). In addition, we compared our results with publicly available gene expression profiles of various other HPV-induced cancers (vulva, cervix and head-and-neck). This showed p16INK4a was the most significant marker to detect a high-risk HPV infection, but no other markers could be found. In conclusion, this study provides insight into the molecular basis of low- and high-risk HPV infections and indicates two main pathways (cell cycle and DNA damage response) that are much stronger affected by high-risk HPV as compared to low-risk HPV.

Imiquimod has been shown to be an effective treatment for usual type vulvar intraepithelial neoplasia (uVIN). Since local inflammation and burning are common side effects, patients often use nonsteroidal anti-inflammatory drugs (NSAIDs). Our study investigated whether NSAID-use, which has been documented to inhibit the cell-mediated immune response, interferes with the outcome of imiquimod treatment. Monocyte-derived dendritic cells (moDCs) and Langerhans cells (moLCs) were cultured in the presence of NSAIDs. The expression of relevant surface markers (CD80, CD86, CD40, HLA-DR, CCR6 and CCR7), stimulatory function, and cytokine production were evaluated. Furthermore, we analyzed in uVIN patients whether frequent NSAID-use had an effect on the clinical response and on immunocompetent cell counts before and after imiquimod treatment. Although an effect was observed on the expression of moDC and moLC maturation markers, NSAIDs did not affect the ability of moDCs and moLCs to stimulate allogeneic T-cell proliferation, or the production of cytokines in an allogeneic T-cell stimulation assay. In agreement with this, in uVIN patients treated with imiquimod, no interference of frequent NSAID-use with clinical outcome was observed. However, we did notice that high CD1a(+) and CD207(+) cell counts in frequent NSAID-users before treatment seemed to predict a favourable response to imiquimod treatment. Our data indicate that NSAID-use does not seem to interfere with moDC and moLC function and does not interfere with immunomodulatory properties of imiquimod in uVIN patients. Therefore, NSAIDs can safely be used to reduce imiquimod side effects in uVIN patients during treatment.

We studied the health and economic effects of human papillomavirus (HPV) DNA testing in cervical screening using a simulation model. The key data source was a Dutch longitudinal screening trial. We compared cytological testing with repeat cytology (for borderline/mildly abnormal smears) to HPV testing with cytology triage (for HPV-positive smears), combination testing (combined HPV and cytology) and cytological testing with HPV triage (for borderline/mildly abnormal smears). We varied the screening interval from 5 to 10 years. The main outcome measures were the number of cervical cancer cases, the number of quality-adjusted life years (QALYs), and the incremental cost-effectiveness ratio (ICER). The base-case estimates were accompanied with ranges across 118 calibrated parameter settings (calibration criteria: cervical intraepithelial neoplasia 2/3, cancer and mortality rates). In comparison to 5-yearly cytology, 5-yearly HPV testing with cytology triage gave a reduction in the number of cancer cases of 23% (range, 9-27%). The reduction was 26% (range, 10-29%) for combination testing and 3% (range, -1 to 8%) for cytology with HPV triage. For strategies with primary HPV testing, the model also estimated a reduction in cancer cases when the screening interval was extended to 7.5 years. Five-yearly cytology with HPV triage and 5 to 7.5-yearly HPV testing with cytology triage were cost effective for the base-case settings and the majority of calibrated parameter settings (ICER below Dutch willingness-to-pay threshold of -20,000/QALY). Our model indicates that HPV testing with cytology triage is likely to be cost effective. An extension of the screening interval may be considered to control costs.

The conventional direct referral to colposcopy of persistent borderline or mildly dyskaryotic (BMD) smears in cervical cancer screening leads to considerable unnecessary referrals and associated anxiety and costs. This may be improved by including testing for oncogenic human papillomavirus (HPV) in the triage. We assessed costs and side effects (referrals, treatments and time in follow-up) for 3 possible HPV triage strategies (immediate HPV testing, a 6-month delay in HPV testing, a 2-stage combination of both) and compared them with the conventional strategy. The assessments are based on recent Dutch data from various national databases and trials. We estimated that the referral rate could be reduced by 49, 58 and 58% with immediate, delayed and 2-stage HPV testing, respectively. As a consequence, the average length of follow-up, as well as average costs, also decrease. Therefore, we advocate including HPV testing before referring to colposcopy. Among the 3 HPV strategies, analysis of additional aspects favors implementation of immediate HPV testing.

Vulvar intraepithelial neoplasia (VIN) is a premalignant disorder caused by human papillomaviruses. Basic knowledge about the molecular pathogenesis of VIN is sparse. Therefore, we have analyzed the gene expression profile of 9 VIN samples in comparison to 10 control samples by using genome wide Affymetrix Human U133A plus2 GeneChips. Results were validated by quantitative real-time RT-PCR analysis and immunostaining of a few representative genes (TACSTD1, CCNE2, AR and ESR1). Significance analysis of microarrays (SAM) showed that 1,497 genes were differentially expressed in VIN compared to controls. By analyzing the biological processes affected by the observed differences, we found that VIN appears to be a highly proliferative disease; many cyclins (CCNA, CCNB and CCNE) and almost all prereplication complex proteins are upregulated. Thereby, VIN does not seem to depend for its proliferation on paracrine or endocrine signals. Many receptors (for example ESR1 and AR) and ligands are downregulated. Furthermore, although VIN is not an invasive disease, the inhibition of expression of a marked number of cell-cell adhesion molecules seems to indicate development towards invasion. Upon reviewing apoptosis and angiogenesis, it was observed that these processes have not become significantly disregulated in VIN. In conclusion: although VIN is still a premalignant disease, it already displays several hallmarks of cancer.

The relation between human papillomavirus type 16 (HPV 16) viral load in cervical scrapes and development of high-grade cervical intraepithelial neoplasia (CIN II or III) was studied in a nested case-control study of women with normal cytology (group A) and in a cohort of women with abnormal cytology (group B). HPV 16 DNA load was determined using a quantitative real-time PCR assay. In group A, case women (women with CIN II/III, n = 12) had a significantly higher viral load than control women (women with CIN ≤ I, n = 47). This resulted in an increased relative risk of women with the 50% highest viral load for development of CIN II/III (OR 7.7; CI 1.6–33). In group B, women with CIN II/III (n = 38) had a significantly higher viral load than women with CIN ≤ I (n = 25). Women with the 50% highest viral load had an increased relative risk of CIN II/III (OR 3.2; CI 1.1–9.3) and a decreased chance of both viral clearance and cytologic regression. Our data suggest that in women with normal cytology an increased HPV 16 load confers an increased risk of developing a CIN lesion. A sustained high viral load is subsequently informative for progression to a high-grade CIN lesion. © 2002 Wiley-Liss, Inc.