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Several countries offer HPV self-sampling for screening non-attendees. It is assumed that screening attendees also prefer self-sampling to clinician-based sampling, however, little research has been conducted with respect to this. Women participating in the IMPROVE-study were randomised (1:1) to self- or clinician-collected HPV testing, and HPV-positive women were retested using the other collection method. Three different questionnaires were sent out among a subset of participating women: Q1) HPV-positive women from both study groups were asked about their experiences with self-sampling and clinician-based sampling (n = 497); Q2) HPV-negative women from the self-sampling group were asked about their experiences with self-sampling (n = 2366); and Q3) HPV-negative women in the clinician-collection group were asked about their experiences with clinician-based sampling (n = 2092). Response rates ranged from 71.6 to 79.4%. Women reported significantly lower levels of shame, nervousness, discomfort and pain during self-sampling compared to clinician-based sampling. However, trust in correct sampling was significantly higher during clinician-based sampling. The majority of women in group Q1 preferred self-sampling (76.5%) to clinician-based sampling (11.9%) in future screening, while 11.6% of women reported to have no preference for either method. To conclude, women from a regular screening population have a positive attitude towards self-sampling but express some concerns with respect to accuracy. The majority prefers self-sampling to clinician-based sampling in future screening. Based on these results, a screening approach where women can choose for either self-sampling or clinician-based sampling seems highly justifiable. [ABSTRACT FROM AUTHOR]

Abstract Objective HPV16/18 genotyping and detection of hypermethylation of human cell genes involved in cervical oncogenesis have shown promising results in triage of high-risk HPV (hrHPV)-screen positive women on cervical smears. These tests can be performed on self-samples, which contain cervical and vaginal cells. We studied whether a self-sample represents the hrHPV type causing the worst cervical lesion and whether any differences in hypermethylation of FAM19A4/miR124-2 exist between CIN lesions caused by different hrHPV types. These results have important implications for reflex triage of self-samples. Methods Correlation between genotype found on self-sample using GP5+/6+-PCR-EIA-LMNX and causative hrHPV genotype in the worst lesion on histology was studied using laser capture microdissection (LCM)-SPF10-PCR (N = 152). Hypermethylation of FAM19A4/miR124-2 in the self-sample was tested in a quantitative methylation specific PCR and compared between lesions caused by HPV16/18 and other hrHPV genotypes. Results Causative hrHPV genotype of the worst lesion (CIN1, CIN2, CIN3, invasive cervical cancer) was detected on self-sample in 93.4%. HPV16 was the most frequently found genotype on self-sampling (39.2%, 73/186) and causative genotype in CIN3+ (51.4%, 38/74, all detected on self-sample). There were no differences in the percentages of positive FAM19A4/miR124-2 methylation assays between lesions caused by HPV16/18 (73.8% in CIN3+) or other hrHPV genotypes (66.7% in CIN3+) (p = 0.538). Conclusions Our results show that hrHPV genotypes found on self-sample were a good representation of hrHPV in the worst CIN lesion and that methylation testing on self-sample for detection of CIN3+ was not significantly different between lesions caused by HPV16/18 and other hrHPV genotypes. Highlights • Genotyping of self-samples finds the causative genotype of the worst underlying lesion in almost all HPV screen-positives. • Hypermethylation of FAM19A4/miR124-2 shows no difference between lesions caused by HPV16/18 or other hrHPV genotypes. • Our results support HPV16/18 genotyping and hypermethylation of FAM19A4/miR124-2 for triage of HPV self-screen-positives. [ABSTRACT FROM AUTHOR]

Objectives DNA methylation analysis of cancer-related genes is a promising tool for HPV-positive women to identify those with cervical (pre)cancer (CIN3+) in need of treatment. However, clinical performance of methylation markers can be influenced by the sample type utilized. We describe a multiplex quantitative methylation-specific PCR that targets FAM19A4 and mir124-2 loci, to detect CIN3+ using both HPV-positive lavage- and brush self-samples. Methods We determined methylation thresholds for clinical classification using HPV-positive training sets comprising lavage self-samples of 182 women (including 40 with CIN3+) and brush self-samples of 224 women (including 61 with CIN3+). Subsequently, independent HPV-positive validation sets of 389 lavage self-samples (including 78 with CIN3+), and 254 brush self-samples (including 72 with CIN3+) were tested using the preset thresholds. Furthermore, the clinical performance of combined methylation analysis and HPV16/18 genotyping was determined. Results Training set analysis revealed similar FAM19A4 and mir124-2 thresholds for both self-sample types to yield highest CIN3+ sensitivity at 70% specificity. Validation set analysis resulted in a CIN3+ sensitivity of 70.5% (95%CI: 60.4–80.6) at a specificity of 67.8% (95%CI: 62.7–73.0) for lavage self-samples, and a CIN3+ sensitivity of 69.4% (95%CI: 58.8–80.1) at a 76.4% (95%CI: 70.2–82.6) specificity for brush self-samples. In combination with HPV16/18 genotyping, CIN3+ sensitivity and specificity were 88.5% (95%CI: 81.4–95.6) and 46.0% (95%CI: 40.4–51.5) for lavage self-samples, and 84.7% (95%CI: 76.4–93.0) and 54.9% (95%CI: 47.7–62.2) for brush self-samples. Conclusions FAM19A4 / mir124-2 methylation analysis performs equally well in HPV-positive lavage- and brush self-samples to identify women with CIN3+. In combination with HPV16/18 genotyping, significantly higher CIN3+ sensitivities are obtained, at decreased specificity. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: High attendance rates in cervical screening are essential for effective cancer prevention. Offering HPV self-sampling to non-responders increases participation rates. The objectives of this study were to determine why non-responders do not attend regular screening, and why they do or do not participate when offered a self-sampling device. Methods: A questionnaire study was conducted in the Netherlands from October 2011 to December 2012. A total of 35,477 non-responders were invited to participate in an HPV self-sampling study; 5347 women did opt out. Finally, 30,130 women received a questionnaire and self-sampling device. Results: The analysis was based on 9484 returned questionnaires (31.5%) with a self-sample specimen, and 682 (2.3%) without. Among women who returned both, the main reason for non-attendance to cervical screening was that they forgot to schedule an appointment (3068; 32.3%). The most important reason to use the self-sampling device was the opportunity to take a sample in their own time-setting (4763; 50.2%). A total of 30.9% of the women who did not use the self-sampling device preferred after all to have a cervical smear taken instead. Conclusions: Organisational barriers are the main reason for non-attendance in regular cervical screening. Important reasons for non-responders to the regular screening to use a self-sampling device are convenience and self-control. [Copyright &y& Elsevier]

Abstract: Objective: To review and characterise by clinical evaluation, immunohistochemistry and HPV typing a group of adenocarcinomas initially diagnosed with primary localisation in the cervix. Furthermore, to assess the prevalence and prognostic significance of HPV genotypes in a large series of HPV positive cervical adenocarcinomas (AC). Methods: One hundred and seventy-one cases of adenocarcinomas (AC) with a primary localisation in the cervix and diagnosed between 1989 and 2008 in the region of Rotterdam, the Netherlands were retrieved. Slides and blocks were reviewed and immunohistochemically stained for CEA and vimentin. HPV testing for high-risk HPV (hrHPV) by PCR (GP5+/6+) and genotyping by reversed line blot were performed. Results: In 113 of 171 patients HPV evaluation was possible. 101 were HPV-positive (89%) and 11 were HPV-negative (11%). The 5-year disease free survival was 80% in the HPV-positive group versus 74% in the HPV-negative group (ns). The distribution of HPV types was type 18 in 55 patients (54%), type 16 in 37 (37%), type 45 in 7 (7%), types 53 and 39 were found in 2 respective patients. 5-year overall-survival in patients with HPV-18 was not significantly worse than in patients with HPV-16 (81 versus 87%). Patients with HPV-45 had a worse 5-year survival, 57%. Conclusions: AC is hrHPV related in most cases (89%) and HPV-18 is the most frequent type (54%). With the exception of HPV-45, HPV-positivity or type in endocervical AC has no significant influence on survival. [Copyright &y& Elsevier]

Abstract: Objective: To evaluate the sensitivity of sentinel node (SN) ultrastaging and to define parameters that may reduce the overall false-negative rate in women with early-stage cervical cancer. Methods: We analyzed data from a large retrospective multicenter cohort group with FIGO stages IA–IIB cervical cancer in whom at least one SN was identified and systematic pelvic lymphadenectomy was uniformly performed. All who were SN negative by initial evaluation were subjected to ultrastaging. Results: In all, 645 patients were evaluable. SN were detected bilaterally in 72% of cases and unilaterally in 28%. Patients with optimal bilateral SN detection were significantly more likely to have any metastasis detected (33.3% vs. 19.2%; P <0.001) as well as micrometastasis detected in their SN (39.6% vs. 11.4%). SN ultrastaging resulted in a low overall false-negative rate of 2.8% (whole group) and an even lower false-negative rate of 1.3% for patients with optimal bilateral mapping. Patients with false-negative SN after ultrastaging had a higher prevalence of LVSI and more frequent unilateral SN detection. Sensitivity of SN ultrastaging was 91% (95% CI: 86%–95%) for the whole group and 97% (95% CI: 91%–99%) in the subgroup with bilateral SN detection. Conclusion: These data confirm previous observations that optimal bilateral SN detection substantially decreases the false negative rate of SN ultrastaging and increases detection of micrometastasis. In patients with bilateral SN detection, the sensitivity of SN ultrastaging is not reduced in more advanced stages of the disease. SN mapping and ultrastaging should become standard practice in the surgical management of early-stage cervical cancer. [Copyright &y& Elsevier]

Abstract: Objective: Currently, women treated for high-grade cervical intraepithelial neoplasia (CIN 2/3) are followed-up by cytology to monitor them for residual and recurrent (post-treatment) disease. This systematic review and meta-analysis determine the test performance of testing for high-risk types of the human papillomavirus (hrHPV), cytology and co-testing (combined hrHPV testing and cytology) in predicting high-grade post-treatment disease (CIN2+). Methods: Studies that compared at least two of three post-treatment surveillance methods, and were published between January 2003 and May 2011, were identified through a bibliographic database search (PubMed, Embase.com and Wiley/Cochrane Library). Identification of relevant studies was conducted independently by two reviewers with a multi-step process. The reference standard used to diagnose post-treatment disease was histologically confirmed CIN2+. Sensitivity, specificity, diagnostic odds ratios and relative sensitivity and specificity were calculated for each study. Pooled estimates were calculated using a random effects model if heterogeneity among studies was significant, otherwise by using a fixed effects model. Estimates were reported with 95% confidence intervals (95%CI). Results: Out of 2410 potentially relevant citations, 8 publications, incorporating 1513 treated women, were included. Pooled sensitivities were 0.79 (95%CI 0.72–0.85) for cytology, 0.92 (0.87–0.96) for hrHPV testing, and 0.95 (0.91–0.98) for co-testing. HrHPV testing was more sensitive than cytology to predict post-treatment CIN2+ (relative sensitivity 1.15; 95%CI 1.06–1.25). Pooled specificities were 0.81 (95%CI 0.74–0.86) for cytology, 0.76 (0.67–0.84) for hrHPV testing and 0.67 (0.60–0.74) for co-testing. HrHPV testing and cytology had a similar specificity (relative specificity 0.95, 95%CI 0.88–1.02). Conclusions: This review indicates that the hrHPV test should be included in post-treatment testing 6months after treatment, because hrHPV testing has a higher sensitivity than cytology in detecting high-grade post-treatment disease and has a similar specificity. [Copyright &y& Elsevier]

Abstract: Cervical carcinogenesis is driven by deregulated E6/E7 expression in dividing cells. A potential deregulating mechanism is methylation of E2 binding sites in the viral long control region, thereby prohibiting HPVE2-mediated transcription regulation. Here the frequency of HPV16E2BS methylation in cervical lesions (SCC, n=29; CIN3, n=17) and scrapes (controls, n=17; CIN3, n=21) was investigated. Three E2BSs were amplified using methylation independent PCR followed by specific detection of methylated CpGs using the Luminex® xMAP™ system. The frequency of E2BS1, E2BS3 and E2BS4 methylation was significantly higher in SCC compared to CIN3, i.e. 93% vs. 21% (p<0.01), 90% vs. 47% (p<0.01) and 69% vs. 5% (p<0.01), respectively and ranged from 6 to 15% in controls. In scrapings of women with CIN3 methylation ranged from 24 to 33%. In conclusion, we showed that the MIP–Luminex system is a highly sensitive method for methylation analysis. HPV16 E2BSs methylation appeared highly frequent in SCC, with particularly E2BS3 methylation occurring proportional to severity of cervical disease. [Copyright &y& Elsevier]

Abstract: Objective: Over 90% of all cervical adenocarcinoma are caused by a transforming infection with a high-risk type human papillomavirus (hrHPV). Previous studies demonstrated that the association between hrHPV positivity and cervical clear-cell adenocarcinoma (CCAC) varies between 0% and 100%. As approximately 60% of all CCAC are associated with intra-uterine diethylstilbestrol (DES) exposure, we determined in a cohort of both DES-exposed and DES-unexposed women the prevalence of hrHPV infections, and the potential etiological role of hrHPV by additional analysis of p16INK4a and p53 expression. Methods: Representative slides of 28 women diagnosed with CCAC were tested for hrHPV by two PCR methods (the clinically validated GP5+/6+ PCR and the very sensitive SPF10PCR/LiPA25). Fifteen women were DES-exposed, 10 unexposed and of 3 women DES-exposure was unknown. Twenty-one cases with sufficient material were immuno-histochemically stained for p16INK4a and p53. Results: Seven tumors, of which four DES-exposed and two unexposed tested positive for hrHPV with GP5+/6+ PCR. Thirteen tumors, of which five DES-exposed and seven unexposed, tested positive with SPF10PCR/LiPA25. In one women with unknown exposure, a CCAC tested positive in both assays. Only three cases, none in DES-exposed women, and all positive with both hrHPV assays, revealed diffuse p16INK4a immuno-staining and weak p53 staining as well, supporting indisputable hrHPV involvement. Conclusions: Although the prevalence of hrHPV was high, only two DES-unrelated CCAC (25%) and one tumor in a woman with unknown exposure could be attributed to hrHPV. [Copyright &y& Elsevier]

Abstract: Immunotherapy aiming at the combined activation of tumor associated antigen (TAA) specific cytotoxic T lymphocytes (CTL) and Natural Killer (NK) cells may be crucial to eradicate both MHC-I positive and negative tumors. Vaccination with mature dendritic cells (DC) transfected with mRNA encoding for TAA and the pro-inflammatory cytokines interleukin (IL)-12 and IL-18 may increase NK cell and TAA specific CTL activity. We demonstrate here that IL-12 over-expressing human DC induces increased NK cell activation and effector function and confirm the increase in TAA specific CTL by TAA/IL-12 double transfected DC. The effects of IL-18 transfection were limited to phenotypic activation and down-regulation of tissue homing receptors and did not add to the effect of IL-12 on NK cell effector function. In conclusion, co-transfection of TAA and IL-12 mRNA into mature DCs offers a vaccine for the induction of an anti-tumor immune response mediated by CTL and NK effector cells. [Copyright &y& Elsevier]

Abstract: Expression of native transgenic T cell receptors in recipient human T cells is often insufficient to achieve highly reactive T cell bulks. Here we show that codon modification of an HPV16E7-specific T cell receptor (TCR), together with omission of mRNA instability motifs and (cryptic) splice sites, leads to a dramatic increase in the expression levels of the transgenic TCRs in human CD8+ T cells. The codon-modified TCRs have been tested in three different configurations in the retroviral vector LZRS: (1) TCRα-IRES-GFP in combination with TCRβ-IRES-NGFR, (2) TCRα-IRES-TCRβ, and (3) TCRα-2A-TCRβ. T cells carrying the codon-modified TCRs are functionally active against target cells loaded with relevant peptide, model tumor cells expressing the specific epitope as well as cervical carcinoma cells. The significant improvements we report here in the functional expression of specific human TCRs will hopefully expedite clinical application of TCR transfer-based immunotherapy. [Copyright &y& Elsevier]

Abstract: Objectives: Plasmacytoid dendritic cells (PDC) play an important role in the innate immune response to viral infections through the secretion of high levels of IFNα. We investigated whether PDC play a role in Human Papillomavirus (HPV) associated cervical carcinoma. Methods: Frozen sections of 18 cervical carcinomas were analyzed for the presence of myeloid and plasmacytoid DC. To study whether the HPV virus can activate PDC, expression of putative VLP receptors (CD49f and CD16) was analyzed on PDC in peripheral blood mononuclear cells of healthy donors. Furthermore, CD83 induction and IFNα production by purified blood-derived PDC was measured after incubation with HPV 16 virus like particles (VLP). Results: PDC were detected in 83% of the CxCa cases, primarily in the stroma. PDC express one of the putative VLP receptors (CD49f). IFNα production but no CD83 expression was induced in PDC upon incubation with VLP. Conclusion: Our data suggest that PDC, which are at hand locally in the cervix, play a role in the natural immune response against HPV and identify PDC as possible targets for VLP-based vaccines. [Copyright &y& Elsevier]

Abstract: Human papilloma virus (HPV) type 16 infections of the genital tract are associated with the development of cervical cancer (CxCa) in women. HPV16-derived oncoproteins E6 and E7 are expressed constitutively in these lesions and might therefore be attractive candidates for T-cell-mediated adoptive immunotherapy. However, the low precursor frequency of HPV16E7-specific T cells in patients and healthy donors hampers routine isolation of these cells for adoptive transfer. To overcome this problem, we have isolated T cell receptor (TCR) genes from four different HPV16E7-specific healthy donor and patient-derived human cytotoxic T lymphocyte (CTL) clones. We examined whether genetic engineering of peripheral blood-derived CD8+ T cells in order to express HPV16E711–20-specific TCRs is feasible for adoptive transfer purposes. Reporter cells (Jurkat/MA) carrying a transgenic TCR were shown to bind relevant but not irrelevant tetramers. Moreover, these TCR-transgenic Jurkat/MA cells showed reactivity towards relevant target cells, indicating proper functional activity of the TCRs isolated from already available T cell clones. We next introduced an HPV16E711–20-specific TCR into blood-derived, CD8+ recipient T cells. Transgenic CTL clones stained positive for tetramers presenting the relevant HPV16E711–20 epitope and biological activity of the TCR in transduced CTL was confirmed by lytic activity and by interferon (IFN)-γ secretion upon antigen-specific stimulation. Importantly, we show recognition of the endogenously processed and HLA-A2 presented HPV16E711–20 CTL epitope by A9-TCR-transgenic T cells. Collectively, our data indicate that HPV16E7 TCR gene transfer is feasible as an alternative strategy to generate human HPV16E7-specific T cells for the treatment of patients suffering from cervical cancer and other HPV16-induced malignancies. [Copyright &y& Elsevier]

: ObjectiveWe evaluated high-risk human papillomavirus (HPV) testing by Hybrid Capture II (HC II) in addition to cytology to predict recurrent/residual cervical intraepithelial neoplasia (CIN) 2/3 and cervical cancer in women treated for CIN 3.: MethodsFollow-up study of 108 women with histologically confirmed CIN 3.: ResultsPretreatment, in 96% (104/108) of the smears high-risk HPV DNA was present. Posttreatment, 71% (77/108) of the women had normal cytology and negative HC II test and none developed recurrent/residual disease during a median follow-up of 28.8 months with a range of 2.4–64.8 months. One of the 12% (13/108) of women with normal cytology and positive HC II test was diagnosed with cervical adenocarcinoma. One of the 7% (8/108) of women with abnormal cytology (borderline dyskaryosis or worse) and negative HC II test was diagnosed with CIN 2. Three of the 9% (10/108) of women with abnormal cytology and a positive HC II test were diagnosed with CIN 2/3. These results show an increased risk for recurrent/residual CIN 2/3 and cervical carcinoma when at least one posttreatment test is positive. The highest relative risk (72.9, 95% CI 25–210) was present in women with both tests positive.: ConclusionsHPV testing with Hybrid Capture II in conjunction with cytology can be used as a tool to select women with an increased risk for recurrent/residual CIN 2/3 and cervical cancer. The standard policy in The Netherlands is cytology at 6, 12, and 24 months posttreatment. However, for women with both normal cytology and negative HC II test at 6 months the chance to develop recurrent/residual CIN 2/3 and cervical carcinoma is so low that retesting at 12 months can be omitted. [Copyright &y& Elsevier]

Abstract: Objective. : Addition of high-risk human papillomavirus (hrHPV) testing to post-treatment monitoring policies of women treated for high-grade cervical intraepithelial neoplasia (CIN) may improve the effectiveness of detecting recurrent/residual disease. Recent studies have shown that HPV type 16 confers an increased risk of high-grade CIN and cervical cancer. This study aimed to find out whether the post-treatment CIN3 rate is increased in HPV16-positive women treated for CIN3. Methods. : We included 229 hrHPV-positive women treated for CIN3. HPV typing was performed by GP5+/6+-PCR followed by reverse line blotting on a cervical scrape taken before treatment. HPV typing data were related to the occurrence of post-treatment CIN3 within a median follow-up time of 20.1 months (range 3–85.4 months) following treatment. Results. : Twenty nine of the 151 (19%) HPV16-positive women versus 6 of the 78 (8%) women with other hrHPV types had recurrent/residual CIN3. Post-treatment CIN3 rate was significantly increased in women with HPV16 compared to those harboring other hrHPV types (p =0.03). None of the other hrHPV types were associated with higher post-treatment CIN3 rates. Conclusion. : Women treated for HPV16 containing CIN3 should be monitored more intensively because of their increased risk of post-treatment CIN3. Thus, the HPV genotype should be considered in post-treatment monitoring policies. [Copyright &y& Elsevier]