With a lifetime risk of high-risk human papillomavirus (hrHPV) infection around 80% and less than 1% of those infections leading to cervical cancer, the development of a cervical carcinoma is a rare complication of hrHPV infection. How this happens is discussed in detail in Section 1 of the book and in Chapter 19, Clinical trials of human papillomavirus vaccines. Most infections are cleared by the host immune system within 2 years and do not always produce a definite dysplastic lesion. Several determinants have been found to influence the progression of oncogenic HPV infection, such as smoking, number of sexual partners, contraceptive use, and an effective immune response. Cervical cancer develops through a series of stages and takes years. The large majority of HPV lesions, however, are productive lesions which produce and shed viral particles without signs of cellular transformation. The morphology of HPV-induced lesions is such that it is not possible to reliably distinguish which lesions are transforming to cancer from those with a high chance of spontaneous regression. The transforming process is driven by viral oncogenes E6 and E7 overexpression, but these genes are also relevant to productive infection, leading to formation and release of new virus particles. Which hrHPV infections lead to productive infections which will never progress and which ones lead to progressive transforming lesions and warrant treatment is not clear. The site of infection is the basis of one hypothesis that has been formulated. Squamo-columnar junction cells, which are localized ectocervical squamous epithelium and endocervical glandular epithelium, are thought to be highly susceptible to transforming HPV infection while productive infections are thought to arise from squamous epithelium from the ectocervix.

AbstractThe decision to treat a cervical squamous intraepithelial lesion (SIL) by loop electrosurgical excision procedure (LEEP) relies heavily on a colposcopy‐directed biopsy showing high‐grade (H)SIL. Diagnosis is often supported by p16, an immunohistochemical (IHC) biomarker of high‐risk (hr)HPV E7 gene activity. Additional potential markers include methylation of tumor suppressor genes FAM19A4/miR124‐2 in cervical cytology for advanced transforming HSIL and the IHC marker HPV E4 for productive, potentially regressing lesions. In 318 women referred for colposcopy, we investigated the relationship between staining patterns of p16 and E4 IHC in the worst biopsy, and the relation of these to FAM19A4/miR124‐2 methylation status in cytology. E4‐positive staining decreased with increasing SIL/CIN grade from 41% in LSIL to 3% in HSIL/CIN3. E4 positivity increased with grade of p16 when p16 expression was limited to the lower two third of the epithelium (r = 0.378), but fell with expression over. Loss of E4 expression in the worst lesion was associated with the methylation of FAM19A4/miR124‐2. We also examined whether these biomarkers can predict the histological outcome of the LEEP biopsy in a subgroup of 119 who underwent LEEP treatment. About 85% of women with ≥lower two third p16 staining/E4‐negative HSIL biopsies and 65% with limited p16 staining/E4‐positive HSIL biopsies had ≥HSIL in the LEEP specimen (P = .025). p16 expression in a biopsy is related both to viral production and transformation, while decreased E4 expression relates to methylation, indicating advanced HSIL. p16 expression in ≥2/3 of the epithelium and absent E4 indicate likely HSIL on a subsequent LEEP specimen.

Objectives Because current guidelines recognise high-grade anal squamous intraepithelial lesions (HSILs) and low-grade SILs (LSILs), and recommend treatment of all HSILs although not all progress to cancer, this study aims to distinguish transforming and productive HSILs by grading immunohistochemical (IHC) biomarkers p16(INK)(4a) (p16) and E4 in low-risk human papillomavirus (lrHPV) and high-risk (hr)HPV-associated SILs as a potential basis for more selective treatment. Methods Immunostaining for p16 and HPV E4 was performed and graded in 183 biopsies from 108 HIV-positive men who have sex with men. The causative HPV genotype of the worst lesion was identified using the HPV SPF10-PCR-DEIA-LiPA25 version 1 system, with laser capture microdissection for multiple infections. The worst lesions were scored for p16 (0-4) to identify activity of the hrHPV E7 gene, and panHPV E4 (0-2) to mark HPV production and life cycle completion. Results There were 37 normal biopsies, 60 LSILs and 86 HSILs, with 85% of LSILs caused by lrHPV and 93% of HSILs by hrHPV. No normal biopsy showed E4, but 43% of LSILs and 37% of HSILs were E4 positive. No differences in E4 positivity rates were found between lrHPV and hrHPV lesions. Most of the lesions caused by lrHPV (90%) showed very extensive patchy p16 staining; p16 grade in HSILs was variable, with frequency of productive HPV infection dropping with increasing p16 grade. Conclusions Combined p16/E4 IHC identifies productive and nonproductive HSILs associated with hrHPV within the group of HSILs defined by the Lower Anogenital Squamous Terminology recommendations. This opens the possibility of investigating selective treatment of advanced transforming HSILs caused by hrHPV, and a 'wait and see' policy for productive HSILs. What's already known about this topic? For preventing anal cancer in high-risk populations, all patients with high-grade squamous intraepithelial lesions (HSILs) are treated, even though this group of lesions is heterogeneous, the histology is variable and regression is frequent. What does this study add? By adding human papillomavirus (HPV) E4 immunohistochemistry to p16 (INK4a) (p16), and grading expression of both markers, different biomarker expression patterns that reflect the heterogeneity of HSILs can be identified. Moreover, p16/E4 staining can separate high-risk HPV-associated HSILs into productive and more advanced transforming lesions, providing a potential basis for selective treatment.

The aim of this study is to determine whether immunohistochemistry for Stathmin-1 enhances diagnostic accuracy of anal dysplasia. The study included 40 biopsies with diagnosis of benign anal transitional zone (n=10), low-grade anal intraepithelial neoplasia (AIN) (AIN1, n=10), and high-grade AIN (AIN2, n=10, AIN3, n=10). The cases were selected to represent classic features. Immunohistochemistry for Stathmin-1, p16, and Ki-67 was performed and assessed for distribution within epithelial thickness. Stathmin-1 was expressed only in the basal layer of benign anal epithelium. Similar pattern of distribution was seen in all low-grade AIN cases (100%). In total, 40% of AIN2 showed Stathmin-1 staining pattern similar to AIN1. The other 60% of cases showed staining extending into the middle third of the epithelial thickness. Of AIN3 cases, 20% showed staining confined to the lower third epithelium, 20% showed staining extending to the middle third, and 60% showed staining extending into the upper third epithelium. The pattern of stain distribution suggested that staining extending above the lower one-third of the epithelial thickness discriminates between low-grade and high-grade AIN. With this cutoff, the sensitivity for the diagnosis of high-grade AIN was 70%, specificity was 100%, positive predictive value equaled 100%, and negative predictive value equaled 77%. P16INK4a showed 100% sensitivity for AIN2 and AIN3, whereas Ki-67 had 100% sensitivity for any AIN grade. In conclusion, Stathmin-1 has excellent specificity for the diagnosis of high-grade AIN; however, Stathmin-1 alone may not be sufficiently sensitive. Use in conjunction with other sensitive markers, such as p16 or Ki-67 may be considered.

Aims To investigate the accuracy and reproducibility of a scoring system for cervical intraepithelial neoplasia (CIN1–3) based on immunohistochemical (IHC) biomarkers Ki-67 and p16 ink4a . Methods 115 cervical tissue specimens were reviewed by three expert gynaecopathologists and graded according to three strategies: (1) CIN grade based on H&E staining only; (2) immunoscore based on the cumulative score of Ki-67 and p16 ink4a only (0–6); and (3) CIN grade based on H&E supported by non-objectified IHC 2 weeks after scoring 1 and 2. The majority consensus diagnosis of the CIN grade based on H&E supported by IHC was used as the Reference Standard . The proportion of test positives ( accuracy ) and the absolute agreements across pathologists ( reproducibility ) of the three grading strategies within each Reference Standard category were calculated. Results We found that immunoscoring with positivity definition 6 yielded the highest proportion of test positives for Reference Standard CIN3 (95.5%), in combination with the lowest proportion of test positives in samples with CIN1 (1.8%). The proportion of test positives for CIN3 was significantly lower for sole H&E staining (81.8%) or combined H&E and IHC grading (84.8%) with positivity definition ≥CIN3. Immunoscore 6 also yielded high absolute agreements for CIN3 and CIN1 , but the absolute agreement was low for CIN2 . Conclusions The higher accuracy and reproducibility of the immunoscore opens the possibility of a more standardised and reproducible definition of CIN grade than conventional pathology practice, allowing a more accurate comparison of CIN-based management strategies and evaluation of new biomarkers to improve the understanding of progression of precancer from human papillomavirus infection to cancer.

Background: Immunohistochemical (IHC) biomarkers enable standardised, reproducible grading of anal intraepithelial neoplasia (AIN) based on progression of HPV infection from transient productive infection to complete neoplastic transformation. We investigated the expression of IHC biomarker HPV E4, a marker of productive HPV infection, in different grades of AIN caused by low-risk (lr) and high-risk (hr) HPV infection. Methods: A reference grading of AIN was established using expert, consensus, subjective HE diagnoses supported by p16 expression for 183 anal biopsies: normal squamous epithelium 37, AIN1 67, AIN2 43, AIN3 36. Causative HPV genotype of the worst lesion was identified using laser capture microdissection and SPF10-PCR. Results: Most AIN1 lesions were caused by lrHPV (72%) whereas most AIN2 (84%) and AIN3 (92%) was caused by hrHPV. None of the normal biopsies showed E4 positivity and 49% (33/67) of AIN1, 56% (24/43) of AIN2 and 3% (1/36) of AIN3 showed E4 positivity. While the large majority of E4 positive AIN1 (26/33) and AIN2 (16/24) lesions showed extensive positivity, the E4 positive AIN3 lesion showed only focal, superficial E4 positivity. HPV E4 positivity was seen in lesions caused by lr and hr HPV. Conclusions: Our results show that when using HPV E4 in addition to p16, productive infections among both low-grade and high-grade AIN can be identified. Studies that further explore progression and regression in transforming (E4 negative) and productive (E4 positive) high-grade AIN, are necessary.

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. (C) 2018 Published by Elsevier Inc.

Cervical screening aims to identify women with high-grade squamous intraepithelial lesion/cervical intraepithelial neoplasia 2-3 (HSIL/CIN2-3) or invasive cervical cancer (ICC). Identification of women with severe premalignant lesions or ICC (CIN3+) could ensure their rapid treatment and prevent overtreatment. We investigated high-risk human papillomavirus (hrHPV) detection with genotyping and methylation of FAM19A4/miR124-2 for detection of CIN3+ in 538 women attending colposcopy for abnormal cytology. All women had an additional cytology with hrHPV testing (GP5+/6+-PCR-EIA+), genotyping (HPV16/18, HPV16/18/31/45), and methylation analysis (FAM19A4/miR124-2) and at least one biopsy. CIN3+ detection was studied overall and in women <30 (n = 171) and ≥30 years (n = 367). Positivity for both rather than just one methylation markers increased in CIN3, and all ICC was positive for both. Overall sensitivity and specificity for CIN3+ were, respectively, 90.3% (95%CI 81.3-95.2) and 31.8% (95%CI 27.7-36.1) for hrHPV, 77.8% (95%CI 66.9-85.8) and 69.3% (95%CI 65.0-73.3) for methylation biomarkers and 93.1% (95%CI 84.8-97.0) and 49.4% (95%CI 44.8-53.9) for combined HPV16/18 and/or methylation positivity. For CIN3, hrHPV was found in 90.9% (95%CI 81.6-95.8), methylation positivity in 75.8% (95%CI 64.2-84.5) and HPV16/18 and/or methylation positivity in 92.4% (95%CI 83.5-96.7). In women aged ≥30, the sensitivity of combined HPV16/18 and methylation was increased (98.2%, 95%CI 90.6-99.7) with a specificity of 46.3% (95%CI 40.8-51.9). Combination of HPV16/18 and methylation analysis was very sensitive and offered improved specificity for CIN3+, opening the possibility of rapid treatment for these women and follow-up for women with potentially regressive, less advanced, HSIL/CIN2 lesions.

BACKGROUNDOur objective was to identify virological and serological predictors of anal high-grade squamous intraepithelial lesions (HSIL) in human immunodeficiency virus (HIV)-positive men who have sex with men (MSM).METHODSHIV-positive MSM were recruited from a longitudinal study during which anal self-swabs and serum were collected at up to 5 bi-annual visits. Swabs were human papillomavirus (HPV) genotyped, and the type-specific HPV viral load in the anal swabs was determined. Serum antibodies to the E6, E7, E1, E2, and L1 proteins of 7 high-risk HPV (hrHPV) types and HPV6 and 11 were analyzed. The participants who had a high-resolution anoscopy after the last study visit were included in the current analysis. Anal HSIL was diagnosed by histopathological examinations of anal biopsies. The causative HPV type of anal HSIL was determined in whole tissue sections (WTS) and by laser capture micro-dissection if more than one HPV-type was found in WTS. Multivariable logistic regression was used to study whether persistent anal HPV infections, HPV viral loads, and seropositivity for HPV were predictors of anal HSIL, either in general or caused by the concordant HPV type.RESULTSOf 193 HIV-positive MSM, 50 (26%) were diagnosed with anal HSIL. HrHPV persistence in anal swabs was common, varying by hrHPV type between 3-21%. Anal HPV persistence was the only determinant independently associated with anal HSIL, both in general and by concordant, causative HPV type.CONCLUSIONSPersistent HPV infections were strongly associated with anal HSIL, in general as well as for the concordant HPV type.

Progression of anal intraepithelial neoplasia (AIN) involves transition from productive to transforming human papillomavirus (HPV) infection. Grading aims to distinguish productive low-grade AIN from high-grade anal intraepithelial neoplasia (HGAIN) with risk of cancer. We describe immunohistochemical patterns in AIN adding a novel marker for initiation of the productive phase of the HPV life cycle (panHPVE4) to those for cell cycle activity (Ki-67) and transforming activity of HPVE7 gene (p16). We studied 67 anal biopsies for suspected anal neoplasia (17 normal, 15 AIN1, 20 AIN2, 15 AIN3) from 54 men who have sex with men at New York Presbyterian Hospital, USA. Two pathologists generated consensus AIN and immunogrades. Whole tissue and laser capture microdissection samples from multiple HPV-infected biopsies were tested for HPV with SPF10-PCR-DEIA-LiPA25, version 1. (Para)basal Ki-67 expression distinguished normal from AIN (≥lower-third Ki-67) with sensitivity 0.92 and specificity 1.0. Ki-67 did not distinguish grades of AIN. Null/patchy p16 versus diffuse ≥lower-third patterns discriminated HGAIN (sensitivity, 1.0; specificity, 0.84). There was marked heterogeneity in E4 expression within HGAIN. Most AIN2 (14/20) was E4+ versus 0/15 AIN3 (sensitivity, 0.70; specificity 1.0). HPV was detected in 63 (94%) biopsies, with 49 (77.8%) high-risk HPV+. HPV16 was the most frequent (13%). Multiple HPV genotypes were found in 15 (24%) biopsies and laser capture microdissection -polymerase chain reaction confirmed specific HPV types in E4 +/− AIN. Although Ki-67 discriminated AIN and p16 HGAIN, E4+/p16+ staining shows that most AIN2 is different from transformed AIN3 in showing both entry into productive HPV infection and transforming activity.

In this study, we evaluate the expression of human papillomavirus E4 protein (marker for the onset of a productive infection) and hypermethylation of host-cell CADM1, MAL, and miR124-2 genes (marker for an advanced, transforming infection) in cervical intraepithelial neoplasia (CIN) and cancer. A total of 115 cervical lesions were categorized by 3 pathologists into no dysplasia, CIN1, CIN2, CIN3, or cancer by classical histomorphological grading criteria, and by an immunoscore (cumulative value: 0–6) grading system based on Ki-67 (score: 0–3) and p16ink4a (score: 0–3) expression. Lesions were immunostained for E4 protein and analyzed for hypermethylation of CADM1, MAL, or miR124-2 genes. Expression of E4 and hypermethylation levels were related to CIN grade based on both classical and immunoscore grading. Hypermethylation increased with severity of the lesion as defined by both classical histomorphological grading and immunoscore criteria, and was always present in carcinomas (22/22). Extensive E4 expression decreased with increasing CIN grade and immunoscore, being most frequent in classically graded CIN1 or in lesions with cumulative immunoscore 1–3 and absent in carcinomas. High-grade lesions (CIN2/3 or immunoscore: 4–6) showed less E4 expression, which was inversely related to an increasing hypermethylation. Extensive E4 expression, as observed in a small proportion of high-grade lesions (6/49 and 8/43, respectively), was mostly associated with a negative methylation marker status (5/6 and 7/8, respectively). Our results illustrate the gradual transition of productive CIN (reflected by extensive E4 expression), to advanced transforming CIN (reflected by extensive hypermethylation) and cancer. Expression patterns of E4 and hypermethylation status of host-cell genes, may be used to identify cervical lesions at risk for cervical cancer, providing a better guidance for clinicians on treatment decisions.

Objective To compare the sensitivity of high‐risk human papillomavirus (hr HPV ) and genotype detection in self‐collected urine samples in the morning (U1), and later on (U2), brush‐based self‐samples ( SS ), and clinician‐taken smears ( CTS ) for detecting cervical intraepithelial neoplasia grade 2+ ( CIN 2+) in a colposcopic referral population. Design Cross‐sectional single‐centre study. Setting A colposcopy clinic in Spain. Population A cohort of 113 women referred for colposcopy after an abnormal Pap smear. Methods Women undergoing colposcopy with biopsy for abnormal Pap smears were sent a device (Colli‐Pee™, Novosanis, Wijnegem, Belgium) to collect U1 on the morning of colposcopy. U2, CTS , and SS (Evalyn brush™, Rovers Medical Devices B.V., Oss, the Netherlands) were also analysed. All samples were tested for HPV DNA using the analytically sensitive SPF 10‐ DEIA ‐Li PA 25 assay and the clinically validated GP 5+/6+‐ EIA ‐ LMNX . Main outcome measures Histologically confirmed CIN 2+ and hr HPV positivity for 14 high‐risk HPV types. Results Samples from 91 patients were analysed. All CIN 3 cases ( n = 6) tested positive for hr HPV in CTS , SS , U1, and U2 with both HPV assays. Sensitivity for CIN 2+ with the SPF 10 system was 100, 100, 95, and 100%, respectively. With the GP 5+/6+ assay, sensitivity was 95% in all sample types. The sensitivities and specificities for both tests on each of the sample types did not significantly differ. There was 10–14% discordance on hr HPV genotype. Conclusions CIN 2+ detection using HPV testing of U1 shows a sensitivity similar to that of CTS or brush‐based SS , and is convenient. There was substantial to almost excellent agreement between all samples on genotype with both hr HPV assays. There was no advantage in testing U1 compared with U2 samples. Tweetable abstract Similar CIN 2+ sensitivity for HPV testing in first‐void urine, physician‐taken smear and brush‐based self‐sample.

Background: Cervical cancer incidence and mortality rates in Sub-Saharan Africa (SSA) remain high due to several factors including low levels of uptake of cervical cancer screening. Self-collection of cervicovaginal samples for HPV DNA testing may be an effective modality that can increase uptake of cervical cancer screening in SSA and hard to reach populations in developed countries. We investigated whether self-collection of cervicovaginal samples for HPV DNA tests would be associated with increased uptake of screening compared with clinic based collection of samples. Furthermore, we compared the quality of samples collected by both approaches for use in HPV genotyping.Methods: We conducted a community based randomized trial in a semi-urban district of Abuja, Nigeria with 400 women, aged 30 to 65 years randomized to either hospital-collection or self-collection of cervicovaginal samples. We compared cervical cancer screening uptake among the 2 groups and evaluated the concentration of human DNA in the samples by measuring RNase P gene levels using qPCR. High-risk HPV DNA detection and typing was done using the GP5+/6+ Luminex system.Results: Most participants in the self-collection arm (93%, 185/200) submitted their samples while only 56% (113/200) of those invited to the hospital for sample collection attended and were screened during the study period (p value < 0.001). Human genomic DNA was detected in all but five (1.7%) participants, all of whom were in the self-collection arm. The prevalence of high-risk HPV in the study population was 10% with types 35, 52 and 18 being the commonest.Conclusions: Our study shows that self-sampling significantly increased uptake of HPV DNA based test for cervical cancer screening in this population and the samples collected were adequate for HPV detection and genotyping. Cervical cancer screening programs that incorporate self-sampling and HPV DNA tests are feasible and may significantly improve uptake of cervical cancer screening in SSA.

Objectives: To compare the SPF10 system to the GP5+/6+ system for high risk HPV (hrHPV) screening on cervicovaginal samples in Nigeria. Methods: In Karu, a suburban district of Abuja, Nigeria, 400 women were randomized for a self- or hospital-collection after flyer recruitment. A volume of 250 μL was used for DNA isolation with the MagNA Pure and 750 μL was used for nucliSENS easyMAG isolation. MagNA Pure isolates were tested with the SPF10-PCR-DEIA-LiPA25, version 1 system, a test often used in epidemiological studies. GP5+/6+-PCR-EIA, a clinically validated cervical cancer screening method, followed by Luminex genotyping was performed on the nucliSENS easyMAG isolate. qPCR was done (RNaseP) to assess the level of human DNA. Results: Samples of 298 women (74.5% response), with a mean age of 41.1 (SD 7.8, Range 30–62) years were included. The SPF10 showed 23.8% hrHPV+ versus 10.4% with the GP5+/6+. The SPF10 found 9 (3%) samples positive for HPV genotype 16 or 18 versus 4 (1%) by GP5+/6+. Comparing HPV genotypes identified by both systems, 22/29 (75.9%) had concordant genotypes, with the SPF10 system finding additional (low risk) genotypes. Conclusions: Cervical cancer screening by hrHPV testing seems feasible in Nigeria. Corresponding with previous studies (Hesselink et al, 2008), the level of hrHPV+ found by SPF10 was higher compared to GP5+/6+. HrHPV genotyping with both systems showed good agreement. To assess the clinical relevance of any hrHPV+ result, a follow-up study collecting a colposcopy directed biopsy and a repeat hrHPV test is being performed. Results are expected by September 2015.

Objectives: To assess the feasibility of the Precursor-M methylation test as a triage marker on self-collected and hospital-collected samples amongst a hrHPV+ population in a resource limited setting. Methods: A total of 400 women were invited to participate through flyer recruitment in Abuja, Nigeria. After randomization, 200 were asked to self-collect and deposit cervical samples at designated collection points and 200 were invited to the hospital for a hospital-collected sample (HCS). A dry flocked swab was used in both groups. DNA was isolated with the nucliSENS easyMAG, tested with the GP5+/6+-PCR-EIA. A qPCR (RNaseP) to assess the level of DNA was performed. EIA+ samples with a Cq<28 (human genomic DNA concentration >1 ng/μL) were tested with the Precursor-M methylation kit. Cut-offs for positivity were applied as described by the test manufacturer. Results: Samples from all 298 responding women (74.5% response) were included. The mean age of the women was 41.1 (SD 7.8, range 30–62). A total of 29 samples were found hrHPV+ by GP5+/6+; 28 (96.6%) contained enough DNA for Precursor-M testing (11 HCS, 17 self-samplers). 28.6% (8/28), including 3 HCS and 5 self-samplers, were methylation positive. Conclusions: The percentage of 28.6% methylation positive samples corresponds with previous findings (De Strooper et al, 2014). In order to assess the feasibility of methylation testing as a triage marker in the detection of high grade CIN lesions in an all-molecular screening setting, a follow-up study collecting a colposcopy directed biopsy will be performed. Results are expected by September 2015.