Abstract Book 2 APSC

27th INTERNATIONAL PAPILLOMAVIRUS CONFERENCE AND CLINICAL WORKSHOP
September 17-22, 2011

Berlin Germany
APPLIED/CLINICAL SCIENCE
ABSTRACT BOOK
www.hpv2011.org
Content
Session 11: Clinical immunology Sunday Sept 18, 10:30 - 12:00 Oral presentation abstracts Poster abstracts Session 12: Clinical aspects of HPV testing Monday Sept 19, 8:30 - 10:00 Oral presentation abstracts Poster abstracts Session 13: HPV related benign diseases Monday Sept 19, 10:30 - 12:00 Oral presentation abstracts Poster abstracts Session 14: Clinical use of biomarkers Monday Sept 19, 15:45 - 17:15 Oral presentation abstracts Poster abstracts Session 15: Screening and patient management Tuesday Sept 20, 8:30 - 10:00 Oral presentation abstracts Poster abstracts Session 16: Clinical aspects of non-cervical infections Tuesday Sept 20, 10:30 - 12:00 Oral presentation abstracts Poster abstracts Session 17: Cervical screening Wednesday Sept 21, 8:30 - 10:00 Oral presentation abstracts Poster abstracts Session 18: Prophylactic vaccination: clinical studies Wednesday Sept 21, 10:30 - 12:00 Oral presentation abstracts Poster abstracts Session 19: Standard and experimental treatment Wednesday Sept 21, 15:45 - 17:15 Oral presentation abstracts Poster abstracts Session 20: Oral poster presentations 201-236 Tuesday Sept 20, 15:45 - 17:15 Oral poster presentation abstracts 3 5 10 25 27 32 39 41 46 49 51 56 75 77 82 91 93 98 117 119 124 149 151 156 159 161 166 177 179
Poster session
Poster session Applied/clinical science Tuesday Sept 20 Odd poster numbers: 15:45-16:30* Even poster numbers: 16:30-17:15*
*Please be at your poster for scientific discussion
Session 11: Clinical immunology

Oral presentation abstracts
Chair: Margaret Stanley
Poster abstracts
Clinical immunology | 11 O-11.00

INTRODUCTION BY CHAIRPERSON
Margaret Stanley
FOR YOUR NOTES:
O-11.01
HPV16/18 SEROPOSITIVITY AND SUBSEQUENT INFECTION RISK: COMPARISON OF SEROLOGICAL ASSAYS
S W Lin, National Cancer Institute, Rockville, UNITED STATES, A Ghosh, National Cancer Institute, Rockville, United States, C Porras, Proyecto Epidemiologico Guanacaste, Fundacion INCIENSA, Guanacaste, Costa Rica, M H Shiffman, National Cancer Institute, Rockville, United States, S Wacholder, National Cancer Institute, Rockville, United States, L A Pinto, National Cancer Institute, Rockville, United States, M T Esser, MedImmune, Gaithersburg, United States, S Poncelet, GlaxoSmithKline Biologicals, Rixensart, Belgium, R Herrero, Proyecto Epidemiologico Guanacaste, Fundacion INCIENSA, Guanacaste, Costa Rica, M Safaeian, National Cancer Institute, Rockville, United States, M Safaeian, National Cancer Institute, Rockville, United States Background: Several serological assays have been developed to detect antibodies elicited against infections with oncogenic human papillomavirus (HPV) types 16 and 18. However, seropositivity from natural infection among these assays is not equivalent, and associations with subsequent risk of HPV infection are not clear. Objectives: We compared four serological assays: a virus-like particle (VLP)-based polyclonal enzyme-linked immunoassay (ELISA); a VLP-based competitive luminex immunoassay (cLIA); a secreted alkaline phosphatase protein neutralization assay (SEAP-NA); and an epitope-specific inhibition ELISA. We assessed the association of assay seropositivity (at manufacturers cutoff and tertile categories) and risk of subsequent HPV infection. Methods: Replicate enrollment serum aliquots from 500 unvaccinated women (enrollment cervical HPV16/18 DNA-negative) in the control arm of an ongoing Costa Rican HPV vaccine trial were measured for HPV16 or HPV18 seropositivity. New cervical HPV16 or HPV18 DNA infections over four years were assessed. Odds ratios (OR) and 95% confidence intervals (CI) were calculated to determine the association between enrollment antibody levels and risk of subsequent infection. Results: Seropositivity (at the manufacturers cutoff ) from the cLIA was significantly associated with protection from subsequent HPV16 infection (OR=0.49, CI=0.27-0.88, compared with seronegatives). The highest tertile antibody levels from the SEAP-NA (OR=0.20, CI=0.06-0.63, compared with seronegatives) as well as the VLP-based ELISA (OR=0.53, CI=0.28-0.90, compared with seronegatives) were also significantly associated with protection from subsequent infection. The VLP-based ELISA was the most sensitive assay (25%), and the epitope-specific ELISA was the most specific assay (100%) for protection from subsequent HPV16 infection. Similar analyses are ongoing for the HPV18 assays. Conclusions: Seropositivity was associated with protection from subsequent infection but was dependent on data categorization. Understanding the utility of serological assays that measure HPV-specific antibodies will contribute to comprehending the mechanisms of immune protection in HPV natural infection.
Declaration of interest S Poncelet is an employee at GSK Biologicals. MT Esser is an employee at MedImmune.
11 | Clinical immunology O-11.02

HPV ACQUISITION AND CLEARANCE ASSOCIATED WITH ENROLLMENT SEROSTATUS IN MEN
B LU, H. Lee Moffitt Cancer Center and Research Institute, Tampa, United States, R P Viscidi, Johns Hopkins University, Baltimore, United States, A G Nyitray, H. Lee Moffitt Cancer Center and Research Institute, Tampa, United States, L L Villa, Ludwig Institute for Cancer Research, So Paulo, Brazil, E Lazcano - Ponce, Instituto Nacional de Salud Pblica, Cuernavaca, Mexico, M Abrahamsen, H. Lee Moffitt Cancer Center and Research Institute, Tampa, United States, D C Smith, H. Lee Moffitt Cancer Center and Research Institute, Tampa, United States, M Papenfuss, H. Lee Moffitt Cancer Center and Research Institute, Tampa, United States, A R Giuliano, H. Lee Moffitt Cancer Center and Research Institute, Tampa, United States Background: The mechanisms through which natural immunity confers protection against subsequent HPV infection remain poorly understood in men. Objectives: We determined whether enrollment serum antibody status affects the likelihood of acquiring or clearing incident HPV infection among healthy adult men enrolled in a multi-national longitudinal study. Methods: 3377 baseline HPV-negative men were followed every 6 months for up to 4 years. At each study visit, exfoliated skin cells were collected from the penis and scrotum and tested for presence of HPV using PGMY09/11 and the Linear Array Genotyping assays. 10 ml venous blood was collected at enrollment for serum antibody testing using VLP-based ELISA assays. The likelihood of type-specific HPV acquisition and clearance by baseline serostatus was estimated from Cox regression models and presented as Hazard Ratios (HR) and 95% confidence intervals (95% CI). HPV acquisition and clearance was also examined according to the time from initial antibody detection to HPV acquisition to determine if protection is time dependent. Results: The median duration of follow-up was 19.1 months (range: 4.4-56.7), with a median study interval of 6.0 months. Detection of type-specific seropositivity at enrollment was not associated with acquisition of the corresponding HPV infection during the study period for HPV 6, 11, 16 or 18. Similarly, no significant association was observed between HPV 6, 11 or 16 seropositivity and clearance of incident infections with the same HPV type. However, men who were HPV 18 seropositive at enrollment were significantly more likely to clear incident HPV 18 infection (HR, 2.44, 95% CI: 1.16-5.15). No time-dependent effect of seropositivity on HPV acquisition or clearance was observed. Conclusion: Our data suggest that more efficient HPV clearance may be a protective mechanism of natural immunity and the protection may be genotype dependent.
Declaration of interest BL -- None declard. RPV-- None declared. AGN-- Research support from Merck & Co. LLV-- Consultant and Speakers Board of Merck & Co. EL -- None declared. MA -- None declared. DCM-- None declared. MP -- None declared. ARG-- Consultant and Speakers bureau of Merck & Co.
O-11.03
SEROLOGY IN RRP SIMILAR TO PATIENTS WITH GENITAL HPV INFECTION
F Buchinsky, Allegheny General Hospital, Pittsburgh, UNITED STATES C Derkay, Eastern Virginia Medical School, Norfolk, United States D Radley, Merck & Co, North Wales, United States J McClay, University of Texas Southwestern Medical School- , Dallas, United States C Myer, University of Cincinnati College of Medicine, Cincinnati, United States R Bastian, Bastian Voice Institute, Downers Grove, United States K Lollar, University of Missouri Hospital & Clinics, Columbia, United States D Marino, Merck & Co, North Wales, United States D Guris, Merck & Co, North Wales, United States Background: Recurrent respiratory papillomatosis (RRP) is a rare, but devastating, illness caused by HPV 6 and 11. With the advent of a quadrivalent HPV (qHPV) vaccine (HPV 6/11/16/18), it is useful to know if RRP patients develop a serologic response to the viruses. Objective: To assess HPV 6 and 11 antibody levels measured by competitive Luminex Immunoassay (cLIA) among children (age < 11) and adolescents / adults (age > 11) with RRP. Methods: Four institutions (and two support groups) in the USA recruited a cross-sectional convenience sample of RRP patients. Serum samples were tested with cLIA according to protocols used in qHPV vaccine trials. A subject was declared double negative if geometric mean titres (GMT) for anti-HPV 6 and anti-HPV 11 antibody were <20 milli Merck units (mMu) and < 16 mMu, respectively. Subjects provided demographic, vaccination and clinical data. HPV typing was not available. Results: Seventeen of 36 (47%) adolescents and adults with active RRP versus 13/16 (81%) children were double negative (p=0.03). The double negative rate for subjects who had surgery >18 months ago was 6/10 (60%). Amongst all seropositives, the titres were similar to that seen amongst PCR positive subjects in the qHPV vaccine trials. There appeared to be no relationship between titres and time since last surgery. Eight subjects reported receiving qHPV vaccine before enrolling (number of doses unknown); they had GMT of 521 mMu for HPV 6 and 235 mMu for HPV 11; one person had anti-HPV 11 titer below detectable levels. Conclusions: Many adults with RRP have no measurable serologic response to HPV 6 or 11 at a rate similar to subjects with genital infection (59% in qHPV vaccine trials). Children with RRP have a higher rate of seronegativity. RRP patients appeared to mount HPV 6 and 11 serologic response to the qHPV vaccine.
Declaration of interest Farrel Buchinsky - Extramural research funding by Merck & Co.

HIGH PREVALENCE OF INTERMEDIATE-RISK HPV IN WOMEN INFECTED WITH HIV
T Sasagawa, Kanazawa Medical University, Kahoku-gun, JAPAN M Rahman, Kanazawa Medical University, Kahoku-gun, JAPAN R Yamada, Ishikawa Prefectural Hospital, Kanazawa, JAPAN A Kingoro, Kenya Medical Research Institute, Nairobi, KENYA H Ichimura, Kanazawa University, Kanazawa, JAPAN S Makinoda, Kanazawa Medical University, Kahoku-gun, JAPAN The aim of this study was to investigate an association between certain human papillomavirus (HPV) types and human immunodeficiency virus (HIV) infections. Sexually active females (n = 487; 1961 years old) were enrolled in the study. Subjects underwent Pap testing and evaluations of HIV and HPV infection status on uterine cervical cell samples. HPV genotyping was performed using a Kurabo GeneSQUARE DNA microarray test. Overall, 23 HPV genotypes were detected, and the most prevalent HPV genotype was HPV-52, followed by HPV-39, -54, -45, -56, -53, -31, -42, -16, -68, and -51. HPV-30, -53, -54, -61, and -66, which are associated with abnormal cytology, are categorized as intermediate-risk in this study. Detection of both high- and intermediate-risk HPV types was significantly associated with cervical abnormality and HIV infection. Multivariate analysis revealed that some highrisk HPV types (HPV-31, -45, -51, -56, and -59) and most intermediate-risk HPV types were associated with HIV infection, while the high-risk types (HPV-16, -18, -33, -35, -39, -52, -58, and -68) were not. The oncogenic effect of the most malignant HPV types (e.g., HPV-16 and -18) appear to be lower, while that of intermediate-risk types are greater, in areas with a high prevalence of HIV infection.
Declaration of interest None declared
O-11.05
HPV TRANSMISSION IN SOUTH AFRICAN HETEROSEXUALLY ACTIVE COUPLES
Z Mbulawa, University of Cape Town, Cape Town, SOUTH AFRICA D J Marais, University of Cape Town, Cape Town, SOUTH AFRICA L F Johnson, University of Cape Town, Cape Town, SOUTH AFRICA D Coetzee, University of Cape Town, Cape Town, SOUTH AFRICA A L Williamson, University of Cape Town, Cape Town, SOUTH AFRICA This study investigated factors influencing genital HPV transmission. Methods: Participants were black, heterosexually active couples aged 19 to 65 years that were either both HIV-seropositive or HIV-seronegative or HIV-discordant. There were 486 couples at the baseline visit, 285 at the 6-month visit, 209 at the 12-month visit, 152 at the18-month visit and 65 at the 24-month visit. Cervical and penile HPV types were determined by Roche Linear Array HPV genotyping assay. HPV transmission was defined as the acquisition of an HPV type that was detected in their partner at the previous visit. Results: The female to male HPV transmission rate (transmission events per 1000 person-months) was 28.0, while the male to female HPV transmission rate was 11.7. Male to female transmission of LR-HPV types occurred at a higher rate (15.8) than male to female transmission of HR-HPV types (6.0). In contrast, female to male transmission of HR-HPV types and LR-types was similarly frequent (29.2 and 26.4 respectively). HIV-positive women were found to be at higher risk of HPV acquisition from their male partners compared to HIVnegative women (RR: 2.31, 95% CI: 1.08-4.92, P=0.03). HIV-positive men with a CD4 count <350/mL had a higher risk of HPV acquisition from their female partners compared to HIV-positive men with CD4 counts 350/mL (RR: 3.17, 95% CI: 1.05-9.55, P=0.04). In women, the risk of HPV infection from their male partners was significantly associated with young age at sexual debut (P=0.03). Conclusion: HIV infection and a low CD4 count increases the rate of HPV acquisition from sexual partners, indicating a high risk of HPV associated cancers in both HIV-positive women and men.
11 | Clinical immunology O-11.06

ASSOCIATION OF INTERLEUKIN-10 PROMOTER POLYMORPHISMS WITH CIN AND CERVICAL CANCER
K Torres Poveda, Chronic Infection Diseases and Cancer Division, INSP, Cuernavaca, MEXICO A Burguete Garca, Chronic Infection Diseases and Cancer Division, INSP, Cuernavaca, MEXICO G Martnez Nava, Chronic Infection Diseases and Cancer Division, INSP, Cuernavaca, MEXICO M Bahena Romn, Chronic Infection Diseases and Cancer Division, INSP, Cuernavaca, MEXICO E Ortz Flores, Chronic Infection Diseases and Cancer Division, INSP, Cuernavaca, MEXICO A Ramrez Gonzlez, Chronic Infection Diseases and Cancer Division, INSP, Cuernavaca, MEXICO K Delgado Romero, Health Services of State of Morelos, CAPASAM, Cuernavaca, MEXICO D Cant, Cancer Biomedical Research Unit, INCan, Mexico City, MEXICO A Garca Carranc, Unit of Biomedical Research in Cancer, INCan, Mexico , MEXICO V Madrid Marina, Chronic Infection Diseases and Cancer Division, INSP, Cuernavaca, MEXICO Background. An immunosuppressive state had been identified in women with HPV persistence, characterized by high levels of IL-10; this may allow the cervical intraepithelial neoplasia (CIN) and cervical cancer (CC) development. Objectives. a) To analyze the association of polymorphisms in IL-10 promoter gene with the risk of developing CIN and CC. b) Assess the systemic IL-10 mRNA expression level (IREL) and in cervix and the IL-10 protein level in serum in the study population. Methods. Peripheral blood samples from patients with CIN (n=204), with CC (n=80) and non-lesions controls (NLC, n = 166) were used to detect polymorphisms at -592, -819, -1082 and -1352 sites by allelic discrimination. The IREL were measured with RT-PCR. Cervical swab in NLC and biopsy in CIN and CC were used for HPV typing and to evaluate IREL. Genotypic and allelic frequencies of the SNPs were evaluated in each group; we analyzed the association of these polymorphisms with the risk of CIN and CC by logistic regression. Results. Significant differences were not found between genotype frequencies at locus (-819, -1082, -1352). CIN individuals and carriers of heterozygous -592 C/A polymorphism were twice greater odds of having CIN (OR 2.02 (95% CI, 1.26-3.25), p<0.003, as compared with NCL. Similarly, CC individuals and carriers of this SNP had an OR of 1.70 (95% CI, 1.06-2.71), p<0.02. The IREL and the level in serum was significantly higher in CIN and CC compared with NCL (p<0.00), however none of the polymorphisms was statistically associated with the IREL. Conclusions. The levels of mRNA and IL-10 protein were progressively higher with increasing the degree of malignancy, so that IL-10 is relevant to viral persistence and progression of the disease. The -592 C/A polymorphism of the IL-10 promoter appear to be associated with the risk of CIN and CC in Mexican women.
O-11.07
IMMUNE CORRELATES OF SUCCESS AND FAILURE OF THERAPEUTIC CANCER VACCINATION.
M Welters, Leiden University Medical Center, Leiden, NETHERLANDS G Kenter, Leiden University Medical Center, Leiden, Netherlands C Melief, Leiden University Medical Center, Leiden, Netherlands S van der Burg, Leiden University Medical Center, Leiden, Netherlands Persistent infection with oncogenic HPV16 results in anogenital lesions and their subsequent progression to carcinoma. Development of HPV16-induced diseases is associated with immune failure at three different levels. Patients either fail to induce HPV16-specific responses or mount weak T-cell responses. When present HPV-specific effector T cells may not infiltrate the lesions and third the immune response can be suppressed by HPV16-specific CD4+ regulatory T-cells. To overcome this immune deficit we have developed an immunotherapeutic vaccine based on synthetic long overlapping HPV16 E6/E7 peptides (HPV16-SLP). Vaccination of patients with HPV16+ high-grade vulvar intraepithelial neoplasia (VIN3) resulted in objective clinical response rate of 79% and complete and durable (>24 months) regression (CR) in 47% of the patients. Clinical outcome was related to lesion size at study entry. Patients with smaller lesions displayed much stronger vaccine-prompted HPV16-specific effector T-cell responses (already ex-vivo detectable) with higher IFN (P=0.0003) and IL-5 (P<0.0001) levels than patients with large lesions. Characteristically, this response was accompanied by a distinct peak in cytokine levels after the first vaccination. Additionally, the CR patient group harboured earlier and significant higher levels of HPV16 E6/E7-specific IgG levels showing a well-induced Th2 response upon vaccination compared to non-CR patients (P<0.05). In contrast, patients with larger lesions displayed weaker effector and stronger HPV16-specific regulatory immune responses, as witnessed by significantly lower IFN/IL-10 ratio and higher frequency of HPV16-specific CD4+CD25+Foxp3+ T cells (P=0.005) upon vaccination. In conclusion, HPV16-SLP vaccine-induced immunity in patients, who were or were not successfully treated, revealed that therapeutic vaccination can be truly effective but also that HPV-specific CD4+CD25+Foxp3+ T cells expanded by vaccination, mainly in patients with larger lesions, can blunt vaccine-induced T-cell reactivity and clinical responses. Therefore, progressive disease may deregulate specific immunity to such an extent that for those patients the current immunotherapy strategy needs further adjustment.
Declaration of interest This study has been conducted by the Leiden University Medical Center (LUMC), which holds a patent on the use of synthetic long peptides as vaccine (US 7.202.034). C.J.M. Melief and S.H. van der Burg are named as inventors on this patent. The LUMC does not share the financial benefit from this patent with its employees. C.J.M. Melief has been employed part-time (75%) since January 20, 2008, by ISA Pharmaceuticals, which exploits this long-peptide vaccine patent, and has been granted options on ISA Pharmaceuticals stock.

CD8+ AND FOXP3+ T-CELLS IN HPV+/- TONSILLAR CANCER AND SURVIVAL
T Ramqvist, Karolinska Institutet, Stockholm, SWEDEN A Nsman, Karolinska Institutet, Stockholm, SWEDEN M Romanitan, Karolinska Institutet, Stockholm, SWEDEN E Munck - Wikland, Karolinska Institutet, Stockholm, SWEDEN T Dalianis, Karolinska Institutet, Stockholm, SWEDEN Background: HPV is a major causative factor for tonsillar squamous cell carcinomas (TSCC) and patients with HPV positive (HPV+) TSCC have an improved 5-year survival after treatment as compared to patients with HPV negative (HPV-) TSCC. There is therfore a need to find biomarkers that can be used in combination with HPV status to predict response to therapy. The presence of tumour infiltrating cytotoxic T-cells (CD8+) and regulatory T-cells (FoxP3+) cells were investigated as possible biomarkers for treatment response in TSCC. Objectives: To investigate if the number of tumour infiltrating CD8+ T-cells and FoxP3+ regulatory T-cells in TSCC can be correlated to the presence of high risk HPV and/or disease specific outcome after treatment. Methods: Formalin fixed paraffin embedded TSCC from 83 patients, formerly analyzed for the presence of HPV DNA, were divided into four groups depending on HPV status and disease specific outcome. Tumours were stained by immunohistochemistry and evaluated for the number of tumour infiltrating cytotoxic (CD8+) and regulatory (FoxP3+) T-cells. Results: For both HPV+ and HPV- tumours, response to treatment was positively correlated to the number of CD8+ cells. In addition, the number of CD8+ and FoxP3+ cells were increased in HPV+ tumours regardless of response to treatment. However, for FoxP3+ cells no differences with regard to disease specific outcome was found between the two HPV+ groups. Conclusions: The positive correlation between number of infiltrating CD8+ cells and disease specific outcome indicates that CD8+ cells may contribute to a positive response to treatment in TSCC patients regardless of HPV status and could thus potentially serve as a biomarker for response to treatment. The increased number of infiltrating regulatory T-cells in HPV+ tumours indicates that these may be exposed to a more active immunosupression.
11 | Clinical immunology P-11.09

WOMEN CYTOKINES PROFILE WITH LSIL, HSIL AND INVASIVE SCC
J Grisi Candeias, Botucatu Biosciences Institute, So Paulo State University, Botucatu, BRAZIL L Doddi Marcolino, Department of Pathology, Botucatu Medical School, So Paulo State University, Botucatu, BRAZIL M Msch, Faculty of Earth and Life Sciences, Vrije Universiteit, Amsterdam, HOLAND D Ferreira Silva, Department of Pathology, Botucatu Medical School, So Paulo State University, Botucatu, BRAZIL J Polettini, Department of Pathology, Botucatu Medical School, So Paulo State University, Botucatu, BRAZIL M Marques, Department of Pathology, Botucatu Medical School, So Paulo State University, Botucatu, BRAZIL P Traiman, Department of Gynecology and Obstetrics, Botucatu Medical School, So Paulo State University, Botucatu, BRAZIL L Mauad, Preventative Gynaecological Ambulatory at the Amaral Carvalho Hospital, Jau, BRAZIL M Guimaraes da Silva, Department of Pathology, Botucatu Medical School, So Paulo State University, Brazil, Botucatu, BRAZIL Immune response has an important role in the natural history of HPV infection in uterine cervix and is involved in viral persistence and progression of lesions. The Th1 pattern contributes to develop cellular immunity against HPV infection and neoplasia, and is related to clearance of infection. The Th2 pattern is associated with persistence of viral infection and the progression of lesions. The concentration of IL-2, IL-4, IL-6, IL-10, IL-12, IFN- and TNF- was evaluated in cervical secretion and serum of women with LSIL, HSIL and invasive squamous cell carcinoma (SCC) our study included 109 women with histological diagnosis of LSIL (n = 16), HSIL (n = 40), SCC (n = 13) and 40 women with suspected of HPV-induced disease, but no pathological changes on cervix biopsy (control group) attending in the Colposcopy Clinic of the Botucatu Medicine School (UNESP) and in the Preventive Gynecology, Hospital Amaral Carvalho, Jau, SP. During speculum examination cervical secretion was collected with cytobrush to determine the cytokines profile. Peripheral blood of women enrolled in the study was collected and cytokine patterns in serum and cervical secretion were assessed by ELISA. HPV DNA was amplified by MY09/11 and GP5+/ GP6+ PCR and genotyping was done by PCR specific primers and confirmed by RFLP. HPV DNA was found in 90% of samples from the control group, 93.7% in LSIL, 100% in HSIL and 84.6% in the EC. The levels of IL-4, IL-6 and IL-10 in cervical secretion were significantly increased in patients with EC when compared with control groups, LSIL and HSIL. In the serum, the level of IL-6 was significantly increased in patients with HSIL. Our results corroborate the finding that the increase of Th2 cytokines in serum and cervical secretion of women with HSIL and SCC is related to the progression of intraepithelial lesions
P-11.10
CHARACTERISTICS OF HPV INFECTION IN HIV-POSITIVE EUROPEAN WOMEN
I Heard, HPV National Reference Centre, Pasteur Institute, Groupe Hospitalier Piti-Salptrire, Universit Pierre et Marie Curie, PARIS , FRANCE H Cubie, Scottish HPV Reference Laboratory, Royal Infirmary of Edinburgh, EDINBURGH, UNITED KINGDOM D MESHER, Cancer Research UK Centre for Epidemiology, Mathematics & Statistics, Wolfson Institute of Preventive Medicine, Queen Mary University of London, Barts & The London School of Medicine and Dentistry, LONDON, UNITED KINGDOM P SASIENI, Cancer Research UK Centre for Epidemiology, Mathematics & Statistics, Wolfson Institute of Preventive Medicine, Queen Mary University of London, Barts & The London School of Medicine and Dentistry, LONDON, UNITED KINGDOM Background: The objectives of the study were to investigate high risk human papillomavirus (HRHPV) infection in HIV-1 infected women in Europe in the era of combination antiretroviral therapy availability; to describe HPV type distribution in HRHPV positive women according to age, CD4 count and link to cytological findings. and to demonstrate the association of HRHPV positivity with persistence/loss to the development of high-grade cervical disease in HIV-infected women. Methods: Regular follow-up of a cohort of European women infected with HIV was undertaken. Residual cervical brush samples in PreservCyt medium were used for HPV screening using Hybrid Capture II and genotyping of positives using HPV Line Blot Assay. Results were analysed in relation to cytological and colposcopic /histological findings. Results: HRHPV prevalence at baseline was estimated in 923 women. Prevalence of any HR-HPV type was 49.5%, with 10% for HPV 16 ( the most common type) and 4.4% for HPV 18. Prevalence was similar whether women were below or above 35 years of age. Prevalence increased with level of immunosuppression. HR-HPV genotypes were detected in 34.5% of cases of normal cytology, 78.2% of ASCUS/LSIL and 89.6% of HSIL/cancer. The prevalence of HPV16 in HSIL was 38.1% with the three most common types thereafter having prevalence rates of 19.5% (HPV58), 18.3% (HPV53) and 16.0% (HPV52) ; prevalence of HPV16/18 infection was 49.5%. Rate of multiple infection was 26.7%. Conclusion: A wide diversity of HPV types was evident and multiple infections were common. HPV genotyping in HIV women contributes to knowledge and understanding of pathogenesis but may offer little in terms of clinical management. The lower prevalence of HPV16 and 18 in HIV-positive women with disease may decrease the benefit of current HPV vaccines in this population.
10
Clinical immunology | 11 P-11.11

HPV 16 AND 18 NEUTRALIZING ANTIBODIES IN HIV-SEROPOSITIVE MSM
R Sharma, University of California San Francisco, San Francisco, United States J T Efird, university of California, San Francisco, United States A Chein, University of California, San Francisco, United States E A Holly, University of California, San Francisco, United States J M Berry, University of California, San Francisco, United States N Jay, University of California, San Francisco, United States T M Darragh, University of California, San Francisco, United States J M Palefsky, University of California, San Francisco, United States Background: HIV-seropositive men who have sex with men (MSM) are at high risk of high-grade anal intraepithelial neoplasia (AIN) and anal cancer. Little is known about neutralizing antibodies (NA) to HPV16 or 18 in this population. To determine risk factors for seropositivity, we performed HPV NA testing and correlated the data with clinical parameters, HPV DNA, and AIN status. Methods: Pseudovirus-based NA assay was used to screen for type-specific HPV16 and HPV18 antibodies. Baseline analyses were conducted in serum from a prospective AIN cohort study beginning in 1998. 296 HIV-seropositive MSM were tested with serology, anal cytology and biopsy for detection of AIN, and anal HPV DNA using PCR with quantitation. Results: Overall, 132 (45%) were HPV16-seropositive, 119 (40%) were HPV16 DNA-positive and 175 (59%) were HPV16 DNA- or sero-positive. For HPV18, these were 141 (48%), 79 (27%) and 167 (56%), respectively. HPV16 antibodies correlated with HPV16 DNA detection but not HPV18 DNA, and vice versa. Higher HPV16 and 18 DNA levels correlated with higher NA titer of HPV16 (ptrend<0.008) and HPV18 (ptrend<0.003), respectively. In multivariate analysis for HPV16 seropositivity, significant risk factors included higher HPV16 DNA level (ptrend<0.0001) and number of receptive anal partners in previous year (ptrend<0.02). Years of HIV positivity, CD4+ level, HIV viral load and AIN status were not significant. For HPV18, these included older age (ptrend<0.019), HPV18 DNA level (ptrend<0.0001) and number of receptive anal partners in previous year (ptrend<0.001). Conclusion: In this cohort of HIV-seropositive MSM followed in the post-antiretroviral treatment era, a high proportion showed evidence of prior exposure to HPV16 or 18. Seropositivity was correlated with type-specific DNA positivity and level, and with recent sexual exposures. Anal HPV DNA testing alone under-estimates the true degree of prior exposure.
P-11.12
PERSISTENT HPV18 INFECTION LASTED LONGER THAN PERSISTENT HPV16 INFECTION
M Nakagawa, University of Arkansas for Medical Sciences, Little Rock, AR, UNITED STATES A Moscicki, University of California at San Francisco, San Francisco, CA, United States S Farhat, University of California at San Francisco, San Francisco, CA, United States S Gupta, University of Arkansas for Medical Sciences, Little Rock, AR, United States X Wang, China Medical University, Shenyang, China H Coleman, University of Arkansas for Medical Sciences, Little Rock, AR, United States Background: A large majority of HPV infection is cleared within a few years. Objectives: CD8 T-cell responses were examined in subjects with incident or prevalent (> 4 months) HPV16 or HPV18 infection. Methods: Two groups were chosen from a cohort of women being followed every 4 months with cervical cytology and HPV-DNA testing: (1) women with an incident HPV16 (n=12) or HPV18 (n=5) infection (undetectable HPV-DNA results for two consecutive visits followed by a positive), and (2) women with a prevalent HPV16 (n=11) or HPV18 (n=4) infection (those with at least two consecutive visits positive for either HPV). The HPV-DNA testing results one year later were then used to determine whether the infection was cleared. ELISPOT assay was performed at enrollment into the current study, and one year later. Twenty additional subjects who never had HPV16 and HPV18 detected were enrolled as negative controls, and ELISPOT assay was performed once for HPV 16 (n=10) or HPV18 (n=10). Results: For the subjects with prevalent infection, the mean duration of persistence was 513139 days for the HPV16 group and 916208 days for the HPV18 group (p=0.1). All of the 11 women with prevalent HPV16 infection showed clearance one year later. In comparison, only 1 (25%) of 4 women with pervalent HPV18 infection demonstrated clearance one year later (p=.009). None of the negative control subjects had any positive ELISPOT results. These was a significant difference between the ELISPOT results of the negative control group and the ELISPOT results at one year after the incident HPV16 or HPV18 infection [6 (35%) of 17 women had positive ELISPOT results (p=.005)]. Conclusions: Of those women who were found to have prevalent infection at enrollment, persistence one year later was more common for HPV18 compared to HPV16.
11

CLINICOPATHOLOGIC BEHAVIOR OF CERVICAL CARCINOMA IN RENAL TRANSPLANT RECIPIENTS
S T Park, The Catholic University of Korea, Seoul, KOREA, REPUBLIC OF E Y Ki, The Catholic University , Seoul, KOREA, REPUBLIC OF S J Lee, The Catholic University , Seoul, KOREA, REPUBLIC OF J S Park, The Catholic University , Seoul, OREA, REPUBLIC OF S Y Hur, The Catholic University , Seoul, REA, REPUBLIC OF Background: Renal allograft recipients are reported to have a higher incidence of malignancy than the general population. This single hospital based study examined the incidence and clinicopathologic behavior of uterine cervical carcinoma in renal transplant recipients. Methods: Among 453 women receiving renal transplantation from January 1990 to December 2008, 5 cervical carcinoma patients were detected. The medical records of the 5 patients were retrospectively reviewed, and the clinicopathologic data were collected and analyzed. Results: The incidence of cervical carcinoma in renal transplant recipients was 58.1 out of 100,000 per year, which is 3.5 times higher than general Korean population. The mean interval between the time of renal transplantation and the time to be diagnosis with cervical carcinoma was 80.7 months. After a median follow-up of 96.2 months, there was no recurrence or death of disease. In 4 patients who were positive in human papillomavirus in situ hybridization (HPV ISH), high or probably high risk HPV DNA was detected in all. And punctate staining of HPV ISH was detected in 3 out of 4 patients. Conclusion: Higher incidence of cervical carcinoma is expected in renal transplant recipients, therefore the appropriate surveillance is needed to ensure early detection and treatment of cervical carcinoma.
P-11.14
SYSTEMIC AND LOCAL HPV16-SPECIFIC T-CELLS IN HEAD AND NECK CANCER
M Heusinkveld, leiden university medical center, Leiden, NETHERLANDS R Goedemans, leiden university medical center, Leiden, NETHERLANDS R J P Briet, leiden university medical center, Leiden, NETHERLANDS A J Gelderblom, leiden university medical center, Leiden, NETHERLANDS J W R Nortier, leiden university medical center, Leiden, NETHERLANDS V T H B M Smit, leiden university medical center, Leiden, NETHERLANDS A P M Langeveld, leiden university medical center, Leiden, NETHERLANDS J C Jansen, leiden university medical center, Leiden, NETHERLANDS S H van der Burg, leiden university medical center, Leiden, NETHERLANDS Squamous cell carcinomas of the head and neck (HNSCC) can be caused by an infection with human papilloma virus type 16 (HPV16). Notably, patients with an HPV positive tumor have better disease free survival and display evidence of a strong local immune infiltration. Potentially, the expression of the viral HPV16 E6 and E7 oncoproteins in HNSCC may evoke an adaptive T-cell response and contributes to these characteristics. Therefore, we performed an exploratory study on the presence, type and function of systemic and local (tumor and/or metastatic lymph node) HPV16-specific T cells in an unselected group of 50 patients with HNSCC by several immune assays. Tumor tissue was analyzed for the presence of HPV16 DNA. In 11 tumors HPV16 was present and except for one, all these tumors were located in the oropharynx. Circulating HPV16-specific T cells were found in 16/47 tested patients. T cells were isolated from tumor cultures and/or lymph nodes of 19 patients. HPV16-specific T cells were readily detected in 5 of 7 patients with HPV16+ tumors that could be tested, but in none of the 12 HPV-negative tumors. In depth analysis of the HPV16-specific T-cell response by the isolation of T-cell clones from tumor-infiltrating lymphocytes revealed that this response comprised a broad repertoire of CD4+ T-helper type 1 and 2 cells, CD4+ regulatory T cells and CD8+ T cells reactive to HPV16. The clear local presence of HPV16-specific T-cell immunity in HPV16-induced HNSCC indicates that HPV16-specific T-cells may contribute to the local antitumor response supporting the development of immunotherapeutic approaches aiming at reinforcing HPV-specific immunity in HNSCC.
Declaration of interest none declared
12

HIV PROTEASE INHIBITORS EFFECT TITERS AND INFECTIVITY OF HPV16.
M Israr, Penn State College of Medicine, Hershey, UNITED STATES C Meyers, Penn State College of Medicine, Hershey, USA Human papillomavirus type 16 (HPV16) is a major risk factor for the development of oral and cervical cancers. HPV replication is intimately connected to the differentiation program of its host tissue squamous epithelium. The molecular biology of HPV infections of the oral cavity is poorly understood. HIV positive patients taking antiretroviral drugs have shown a significant increase in the development of HPV positive oral lesions. However, the effects of these drugs on the life cycle of HPVs have not been widely studied. The present study showed that HIV protease inhibitor Lopinavir/Ritonavir (Kaletra) treatments increased infectivity of HPV16 virions in gingival as well as in tonsil tissues. However, Amprenavir treatments decreased infectivity of HPV 16 virions in both tissues in a concentration dependent manner. Further studies are in progress to compare the effects of both drugs on titers, infectivity and morphogenesis of HPV16 virions among oral and cervical tissues.
P-11.16
DIFFERENCES IN HPV CROSS-REACTIVITY IN NATURALLY INFECTED AND VACCINATED INDIVIDUALS.
R Schepp, Public Health Institute The Netherlands (RIVM), Bilthoven, NETHERLANDS M Scherpenisse, Public health Institute The Netherlands (RIVM), Bilthoven, Netherlands C Meijer, VU Medical Centre, Amsterdam, Netherlands G Berbers, Public Health Institute The Netherlands (RIVM), Bilthoven, Netherlands F van der Klis, Public health Institute The Netherlands (RIVM), Bilthoven, Netherlands Objective: The aim of this study was to investigate cross-reactivity of antibodies against phylogenetically related papillomaviruses induced by infection with HPV, or by immunisation with the bivalent HPV16/18 vaccine (Cervarix, GSK). Methods: A fluorescent bead-based multiplex assay (Luminex technology) was used for the detection of VLP antibodies for seven HR-HPV serotypes (16, 18, 31, 33, 45, 52 and 58) . From a large serosurveillance study 200 sera from naturally infected individuals were selected which were seropositive for 2 HPV serotypes. From a vaccine study 50 sera from vaccinated healthy individuals were selected. All sera were analyzed with and without preabsorption with intact VLP-16 and/or VLP-18. Results: In the large serosurveillance study we found that 10% of the naturally infected individuals was seropositive for 2 HPV serotypes. Pre-absorbing these sera with VLP-16 and/or 18 resulted in a 80% homologous inhibition and 30% to 65% reduction in antibody concentration and seropositivity for the other 5 HPV serotypes. After vaccination seroconversion against the 5 non-vaccine types was common (40% to 88%), although antibody levels just exceeded the cut-off value. In contrast to naturally induced cross-reactive antibodies, vaccine induced crossreactive antibodies could be inhibited almost completely (>90%) by pre-absorbtion with VLP 16 and/or18. In addition, less cross-reactivity between clades 7 and 9 was observed for vaccine induced antibodies. Conclusions: Cross-reactivity in serum of naturally infected has been observed within clades but also between clades. Discrimination between type-specific responses after infection with HPV and responses due to crossreactivity with related HPV types is therefore difficult. Although cross reactivity in naturally infected is relatively rare, it can lead to an overestimation of seropositives. Vaccine induced antibodies appear to be more clade specific.
13

IMMUNE RESPONSE GENES AND PATHWAYS INFLUENCE HPV 18/18-LIKE INFECTION OUTCOMES
S L Sudenga, University of Alabama at Birmingham, Birmingham, UNITED STATES J Kravchenko, Duke University, Durham, UNITED STATES I Akushevich, Duke University, Durham, UNITED STATES L K Vaughn, University of Alabama at Birmingham, Birmingham, UNITED STATES C M Wilson, University of Alabama at Birmingham, Birmingham, UNITED STATES J Tang, University of Alabama at Birmingham, Birmingham, UNITED STATES S Shrestha, University of Alabama at Birmingham, Birmingham, UNITED STATES Background HPV infection is one of the most common STIs, worldwide. Most individuals infected with HPV clear the infection naturally; however, in 15-20% individuals the virus persists, and is associated with development of 5% of all human cancer, mostly cervical cancer. Objectives To assess genetic variants and networks related to HPV 18 and 18-like clearance/persistence in HIV+ African-American adolescent females. Methods Cervical lavage samples collected every 6 months were used in PCR assays to identify 30 HPV types in adolescents in the Reaching for Excellence in Adolescent Care and Health (REACH) cohort; 106 HIV-positive African American females were found to have high-risk HPV-18 and 18-like (18, 39, 45, 59, 68, 70, 26, 69, and 51) infections over a median of 3 years of follow-up. We examined the influence of 631 SNPs in the coding, 5 and 3 UTR within 94 immune response genes with a) a logistic-type stochastic model, b) Cox-proportional hazard model provided with maximum likelihood estimates for the effects of specific SNPs on clearance probabilities while controlling for CD4 count level and other HPV co-infections. HPV clearance was defined as absence of type-specific infection for two consequent visits after initial diagnosis. Ingenuity Pathway Analysis (IPA) was used to identify an interaction network based on genes with significantly associated SNPs in both models. Results There were 31 SNPs in 15 genes putatively associated (p-value =0.00008-0.05) with clearance of HPV 18 and 18-like in both models, after adjusting for relevant covariates. IPA suggested a distinct network of interaction between genes, including an enrichment for genes involved in cytokine mediated communication of inflammation and immune activation in innate and adaptive immunity. Conclusions Clearance or persistence of HPV 18 and 18-like virus in adolescents appears to be dictated by a network of genes that regulate inflammation and immune activation.
P-11.18
DISTINCT CERVICAL IMMUNE MARKER PATTERNS IN OLDER HPV POSITIVE WOMEN
M Marks, Johns Hopkins Bloomberg School of Public Health, Baltimore, UNITED STATES A Burke, Johns Hopkins University, Baltimore, UNITED STATES K Chang, Johns Hopkins Bloomberg School of Public Health, Baltimore, UNITED STATES M Silver, Johns Hopkins Bloomberg School of Public Health, Baltimore, UNITED STATES L Howard, Johns Hopkins Bloomberg School of Public Health, Baltimore, UNITED STATES R Viscidi, Johns Hopkins University, Baltimore, UNITED STATES P Gravitt, Johns Hopkins Bloomberg School of Public Health, Baltimore, UNITED STATES Background: Women over 45 years of age with persistent HPV infections have been shown to have reduced in vitro PBMC lymphoproliferative responses and increased serum concentrations of pro-inflammatory immune markers. Objective: We sought to determine whether similar immunologic profiles were observed at the site of cervical HPV infections in older women. Methods: Cervical secretion specimens were collected from 34 high risk HPV (HR-HPV) positive and 44 HR-HPV negative women (mean age (SD): 45 (6.9)) enrolled in an ongoing prospective cohort study to assess the natural history of human papillomavirus across the menopausal transition. Twenty-seven immune markers were measured from these samples using multiplexed bead-based immunoassays. Raw values were normalized to total protein content and expressed as ug (marker) /mg (total protein). Median immune marker concentrations, as well as inter-marker correlations were compared by HPV DNA status using Wilcoxon rank sum test and Spearman rank correlation with correction for multiple comparisons, respectively. Results: HR-HPV positive women had higher median concentration of IL-5 (0.11 vs. 0.08; p=0.03), IL-9 (2.7 vs. 2.1; p=0.04), IL-13 (2.1 vs. 0.9; p=0.01), IL-17 (2.9 vs. 1.1; p=0.003), EOTAXIN (4.1 vs. 1.1; p=0.04), GM-CSF (4.3 vs. 3.3; p=0.01), and MIP-1a (3.5 vs. 1.9; p=0.005) compared to HR-HPV negative women. A shift in the correlation of T-cell and inflammatory cytokines (IFN-g, IL-5, IL-9, IL-10, IL-12, IL-13, IL-15, and TNF-a) from IL-2 to EOTAXIN was observed between HR-HPV negative and positive women. Conclusions: Higher local concentrations of anti-inflammatory cytokines among HR-HPV positive older women are consistent with previous studies. The strong correlation between these cytokines and EOTAXIN, rather than IL-2, in HR-HPV positive women should be further evaluated to determine the relationship of HR-HPV and host immunity.
Declaration of interest None Declared
14

KIR IN RELATION TO HPV-MEDIATED CERVICAL DISEASE IN KOREAN WOMEN
C W Lee, The Catholic Unversity of Korea/Yeouido St. Mary's Hospital, Seoul, KOREA, REPUBLIC OF M J Song, The Catholic Unversity of Korea/Daejeon St. Mary's Hospital, Daejeon, KOREA, REPUBLIC OF J H Kim, The Catholic Unversity of Korea/Uijeongbu St. Mary's Hospital, Uijeongbu, KOREA, REPUBLIC OF S J Lee, The Catholic Unversity of Korea/St. Vincent's Hospital, Suwon, KOREA, REPUBLIC OF S T Park, The Catholic Unversity of Korea/Seoul St. Mary's Hospital, Seoul, KOREA, REPUBLIC OF C J Kim, The Catholic Unversity of Korea/Seoul St. Mary's Hospital, Seoul, KOREA, REPUBLIC OF S Y Hur, The Catholic Unversity of Korea/Seoul St. Mary's Hospital, Seoul, KOREA, REPUBLIC OF T G Kim, The Catholic Unversity of Korea/Seoul St. Mary's Hospital, Seoul, KOREA, REPUBLIC OF J S Park, The Catholic Unversity of Korea/Seoul St. Mary's Hospital, Seoul, KOREA, REPUBLIC OF Objective: HPV infection is the major risk factor to develop cervical cancer. However, host and viral genetic factors play a role in this process. NK cells play a pivotal role to protect from viral infections. KIRs are a kind of receptors which are expressed on NK cells and it bind to HLA class I molecules. In this study, we intended to investigate the influence of the KIR genes and HLA-C alleles on genetic susceptibility in HPV mediated cervical diseases in Korean women. Methods: A total of 148 Korean women with HPV related cervical disease who presented to the department of obstetrics and gynecology at Seoul St. Mary's Hospital from July 2009 through July 2010 were selected for this study. These patients were compared to 159 healthy controls selected during the same study period without HPV infection. KIR genotypes were determined using PCR-SSP method and HLA-C genotyping was performed by ARMS-PCR method. Results: The frequencies of KIR3DL1 (a) and KIR2DS4 (b) significantly decreased with low risk (LR)-HPV group compared to the control group (a, b: OR=0.14, p-value < 0.05) and that of KIR2DS5 (c) meaningfully increased with LRHPV group compared to the control group (c: OR=3.27, p-value < 0.05). The expression of KIR2DS1 and KIR2DS5 was more frequent in LR-HPV group compared to high risk (HR)-HPV group. The frequency of HLA-C*01 allele decreased with HPV related cervical disease group (OR=0.53, p<0.05), compared to the control group and that of HLA-C*0303 allele significantly increased in HPV related cervical disease group compared to the control group (OR=2.04, p<0.05). In combination of KIR and HLA-C, the frequency of 2DS1-C2 group decreased in HR-HPV group (OR=0.37, p<0.05) compared to the control group. Conclusion: a few KIR genes, HLA-C alleles and KIR/HLA combination appeared to have significant correlation with disease protection or progression following HPV infection.
P-11.20
ORAL HPV PREVALENCE AND PERSISTENCE ARE ELEVATED AMONG HIV-POSITIVE ADULTS
D C Beachler, Johns Hopkins School of Public Health, Baltimore, United States, K M Weber, Hektoen Institute of Medicine, Chicago, United States, R D Cranston, University of Pittsburgh, Pittsburgh, United States, H D Strickler, Albert Einstein College of Medicine, Bronx, United States, D J Wiley, University of California-Los Angeles, Los Angeles, United States, R D Burk, Albert Einstein College of Medicine, Bronx, United States, H Minkoff, Maimonides Medical Center, Brooklyn, United States, J B Margolick, Johns Hopkins Bloomberg School of Public Health, Baltimore, United States, M L Gillison, Ohio State University, Columbus , United States, G D' Souza, Johns Hopkins Bloomberg School of Public Health, Baltimore, United States Background: Initial studies suggest oral HPV prevalence is higher among HIV-positive than HIV-negative individuals. Objectives: To evaluate the natural history and risk factors for oral HPV among HIV-positive and -negative individuals Methods: 490 HIV-positive and 305 HIV-negative men and women were recruited from the Multicenter AIDS Cohort Study and the Womens Interagency HIV Study. Oral rinse samples were collected semiannually between 20092010 using a Scope mouthwash rinse and gargle, and tested for 37 types of HPV DNA using PGMY09/11 consensus primers and reverse lineblot hybridization. Oral HPV persistence was defined as type-specific HPV detection at two or more visits, and was evaluated by person and infection. Risk factors for prevalent oral HPV infection were explored using generalized estimating equations. Results: Oral HPV infection was common (38%), including HPV16 infection in 5.5% of those studied. The most significant risk factor for prevalent oral HPV infection was the number of recent oral sex or rimming partners among HIV-negative (p-trend<0.001) but not HIV-positive (p-trend=0.64) individuals, for whom low CD4 T-cell count was the most significant risk factor (p-trend<0.001). While 57% of participants had no oral HPV infection at baseline or follow-up, 24% had persistent infection. Among those without persistent infection, 10% cleared all infections and 8% had at least one newly-detected (incident) infection. Oral HPV persistence was more common among HIV-positive than HIV-negative individuals (30% vs. 16% p<0.001) and in those with CD4 T cell count<200/ul (50%, p=0.007). The majority of the 432 baseline oral HPV infections were persistent six (60%) and twelve (54%) months later. Conclusion: Oral HPV prevalence and persistence were elevated among HIV-positive individuals, particularly those with reduced CD4 T-cell count, suggesting that immunosuppression may alter the natural history of oral HPV Infection. This suggests that HIV-positive individuals may be at increased risk of HPV-associated oral cancer.
15

TYPE-SPECIFIC HUMAN PAPILLOMAVIRUS (HPV) L1 ANTIBODY SEROREVERSION IN HOMOSEXUAL MEN
F Jin, University of New South Wales, Darlinghurst NSW, AUSTRALIA, I M Poynten, University of New South Wales, Darlinghurst NSW, Australia, D J Templeton, University of New South Wales, Darlinghurst NSW, Australia, G P Prestage, University of New South Wales, Darlinghurst NSW, Australia, B Donovan, University of New South Wales, Darlinghurst NSW, Australia, C Fairley, Melbourne Seuxal Health Centre, Melbourne, Australia, S Garland, Royal Women's Hospital, Melbourne, Australia, M Pawlita, German Cancer Research Center (DKFZ), Heidelberg, Germany, A E Grulich, University of New South Wales, Darlinghurst NSW, Australia, T Waterboer, German Cancer Research Center (DKFZ), Heidelberg, Germany Background: Type-specific antibodies to HPV L1 are measured as a marker of lifetime HPV exposure. Multiplex type specific HPV antibody assays allow the simultaneously analysis of the serological response to a broad range of HPV antigens. However, the durability of HPV antibody responses has not been well documented. Objectives: To report the incidence of HPV seroreversion in a cohort of HIV-negative homosexual men in Sydney, Australia. Methods: Participants were 1,427 men from the Health in Men (HIM) cohort study recruited from community-based settings from 2001 to 2004 and followed to June 2007. Multiplex type-specific HPVL1 serological testing (Luminex) was performed on stored sera collected at each annual visit. HPVL1 seroreversion was defined by a status change from seropositive to seronegative and a two-fold decrease in antibody reactivity compared with the previous year. Results: Among the 10 HPV types (6, 11, 16, 18, 31, 33, 35, 45, 52, and 58) examined, the incidence of HPVL1 seroreversion ranged from 3.11 per 100 person-years (PY) for HPV31 to 6.66 per 100 PY for HPV58. None of those who seroconverted during the study seroreverted during a median of 3.9 years follow up. Younger age was associated with higher seroreversion incidence for HPV 6, 11, 45 and 58. More recent onset of homosexual activity was associated with higher seroreversion rates for HPV 6, 45, 52 and 58, as was more recent onset of anal intercourse for HPV 6, 11, 33, 45, 52 and 58. Conclusion: HPV seroreversion is not uncommon in HIV-negative homosexual men, but the absence of seroreversions in recent seroconverters suggests that antibody responses are reasonably durable. Younger men and those who have become recently homosexually active had higher rates of seroreversion for low-risk and less common high-risk HPV types, but not for HPV16 and HPV18.
P-11.22
CORRELATION OF TOTAL IGG ASSAY WITH PSEUDOVIRION BASED NEUTRALIZATION ASSAY
D Brown, Indiana University, Indianapolis, UNITED STATES INTRODUCTION: A concordance study was conducted to compare serologic response to the qHPV L1 virus-likeparticle vaccine in three different assays: the 4-HPV type competitive Luminex Immunoassay (cLIA), the 9-HPV type Total IgG Luminex assay (Total IgG), and the Pseudovirion Based Neutralization Assay (PBNA). OBJECTIVE: The goal of this study was to determine the correlation of qHPV-induced antibody responses in the cLIA/total IgG/PBNA. A secondary goal of the study was to broaden the number and type of serological assays available for anti-HPV detection. METHODS: A total of 648 samples were selected for evaluating anti-HPV-16 response in the cLIA, IgG-LIA, and HPV-16 PBNA, and a total of 623 samples were selected for evaluating anti-HPV-18 response. All quantitative comparisons were performed on the log-transformed data and were limited to the subset of sera having a quantifiable result in the assays being compared. The functional relationship between pairs of assay methods (cLIA, IgG, and PBNA) was estimated separately for each HPV type, and sampling interval using the linear statistical relationship model of Tan and Iglewicz. The Pearson correlation coefficient was also calculated for each protocol and sampling interval sub-grouping. RESULTS: For both HPV-16 and HPV-18, for all samples tested, the cLIA and IgG assays were strongly associated, and the PBNA was strongly associated with both the cLIA and IgG assays. For all samples combined, the correlation coefficients computed on the log transformed data were high across all assays and two HPV types. The estimates of slope from the fitted linear statistical relationship model were all near unity, providing further evidence of strong agreement between assays. CONCLUSION: The cLIA, total IgG assay and PBNA are highly correlated and reflect measurement of neutralizing antibody against HPV types 16 and 18 L1 VLPs.
Declaration of interest Presenter will provide
16

CORRELATION OF CYTOKINE EXPRESSION IN THE ANUS AND CERVIX
A Moscicki, University of California, San Francisco, San Francisco, UNITED STATES Y Ma, University of California, San Francisco, San Francisco, UNITED STATES S Nozzari, University of California, San Francisco, San Francisco, UNITED STATES D Vo, University of California, San Francisco, San Francisco, UNITED STATES M Scott, University of California, San Francisco, San Francisco, UNITED STATES Objective: Little is known about immune responses to HPV in the anus. Some speculate that there are similarities in type of mucosal immune responses including cross-talk. We examined cytokine expression in the anus and cervix in the same women in relation to their HPV status. Methods: Samples obtained from the anus (swab) and the cervix (lavage) from 58 women participating in a longitudinal study of HPV were analyzed for HPV (Roche Linear Array) and ILs 1, 1, 6, 8, 10, 12, IFN2, and IFN (Luminex). Pearson Correlations () were performed between anal and cervical samples for cytokine expression (log transformed) depending on HPV status. Results: Among visits (n=38) where women shared HPV type at both sites, IL-10 (=0.44; p=.01) and IL-12 (=.32; p=.05) were found to be correlated. Similar findings were found if both sites were HPV+ with different types. Among women (n=9) who cleared HPV at the both sites, IFN ( =.9; p<.001), IL-10 (=.6; p=.1) and IL1- (=.6; p=.1) were correlated. Among women (n=19) who had HPV persistence at both sites, IFN2 (=.6; p=.01), IL-10 (=.5; p=0.03) and IL-12 (=.5; p=.04) were correlated. There were no correlations between the anus and cervical cytokines for women (n=10) who were HPV negative at both sites or had discordant clearance status (n=12). Conclusion: There appears to be similar immune responses at both the anus and cervix associated with HPV clearance and persistence. The lack of correlation between the anus and cervix in women who were negative or discordant for HPV status suggests that these immune compartments work independently.
P-11.24
NEUTRALIZING ANTIBODY AND AVIDITY RESPONSES INDUCED BY CERVARIX
T Kemp, SAIC-Frederick, Inc, Frederick, UNITED STATES, M Safaeian, DCEG, Rockville, United States, A Hildesheim, DCEG, Rockville, United States, Y Pan, SAIC-Frederick, Inc, Frederick, UNITED STATES, K Penrose, SAIC-Frederick, Inc, Frederick, UNITED STATES, C Porras, Proyecto Epidemiolgico , San Jose, Costa Rica, J Schiller, Laboratory of Cellular Oncology, CCR, NCI, NIH, Bethesda, UNITED STATES, D Lowy, Laboratory of Cellular Oncology, CCR, NCI, NIH, Bethesda, UNITED STATES, R Herrero, Proyecto Epidemiolgico , San Jose, Costa Rica, L Pinto, SAIC-Frederick, Inc, Frederick, UNITED STATES Background: Cervarix protects against targeted and some phylogenetically-related HPV types. We previously demonstrated that Cervarix elicits antibody responses against related types for which efficacy was demonstrated (HPV-31 and 45) but not for types for which no efficacy was observed (HPV52/58). Objectives: 1) Assess kinetics of antibody responses to vaccine targeted and related types up to 36 months; 2) Compare antibody titers and avidity developed after vaccination between HPV infected women and unexposed women. Methods: Pre- and post-vaccination (through 36-months) sera from HPV16 DNA+ (n=18), HPV31 DNA+ (n=13), HPV58 DNA+ (n=7), HPV DNA negative (n=12), and virgin (HPV-, n=15) women at enrollment, who participated in the Costa Rica Vaccine Trial, were tested for antibody responses to HPV16/18/31/45/58 using a pseudovirion-based neutralization assay, and anti-HPV16 antibody avidity using HPV16 L1 VLP-based ELISA. Results: HPV-31 and 45 neutralizing antibody titers increased significantly after the 3rd dose, and persisted up to month 36. Individuals infected with HPV16 or HPV31 at enrollment developed a significantly higher median antibody response to the corresponding HPV type after one dose, but there was not a difference between median titers after three doses compared to HPV- group. Median HPV16 antibody avidity and titer increased up to 12 months, but avidity did not correlate well with antibody titers at most time points examined. Median avidity levels were higher in HPV16infected women than in HPV- group after one dose of Cervarix (HPV16+ 1.3, and HPV- 0.8; p=0.04), but was lower after three doses of Cervarix (HPV16+ 1.8, and HPV- 2.4; p=0.006). Conclusions: Cross-neutralizing antibody responses, while significantly weaker than neutralizing antibody responses against targeted types, persist for several years. Neutralizing antibody titers correlated weakly with antibody avidity, raising the need for a better understanding of the role of avidity in long-term protection against infection.
Declaration of interest Troy J. Kemp, No conflict; Allan Hildesheim, No conflict; Mahboobeh Safaeian, No conflict; Kerri Penrose, No conflict; Yuanji Pan, No conflict; Carolina Porras, No conflict; John T. Schiller, listed as inventor on US governmentowned patents covering the papillomavirus virus-like-particlebased vaccine technology. These patents have been licensed coexclusively to Merck and GlaxoSmithKline; Douglas R. Lowy, listed as inventor on US governmentowned patents covering the papillomavirus virus-like-particlebased vaccine technology. These patents have been licensed coexclusively to Merck and GlaxoSmithKline; Rolando Herrero, No conflict; Ligia A. Pinto, No conflict.
17

HPV DETECTION AND CERVICAL PATHOLOGY IN HIV POSITIVE WOMEN
F Carozzi, Institute of Cancer Prevention ISPO, Florence, ITALY F Vichi, ASF 10, Florence , italy E Burroni, ISPO, Florence, italy C Sani, ISPO, Florence, Italy Background: Several studies have consistently identified that HIV-infected women, especially those with low CD4 T-cell counts, are at increased risk for infection with HPV the etiologic agent of cervical cancer and its precursor lesions. Objectives: Aim of the study is to understand the relationship of HAART therapy on the incidence of high-grade CIN and on the regression of existing CIN in women with HIV. Methods: From June 2006 up to December 2010, 147 HIV-positive women, aged 21- 76 years, receiving HAART, were enrolled in a gynecological prospective study. All subject underwent gynecologic examination including a HPV cervical sample collection, Pap smear, a colposcopic examination and, if necessary, a cervical biopsy. HPV test was carried out using typing with reverse dot blot. Details of antiretroviral therapy, cytologigal assessment, and clinical examination were related to HPV types. Results and Conclusions: The median age of the 147 women included in the study was 41, median CD4=476, median viral load 50 in 74.15%(109/147) of cases. Abnormal cytology was detected in 24 subjects (16.32%): HSIL in 2/147 (1.36%), LSIL in 12/147 (8.16%) and ASCUS in 10/147 (6.80%). One-hundred-forty subjects underwent colposcopy and a positive histological result was observed in 25 (17.86%): CIN 1 in 17.14%(24/140) and invasive cancer in 0.71%(1/140). HPV HR and/or LR was positive in 48.30 %(71/147) of subjects with multiple HPV types in 39.44%(28/71). Oncogenic HPV types were found in 74.65 %(53/71): HPV 16 18.31% (13/71), HPV 18 4.43% (3/71), HPV 31 2.82% (2/71), HPV 33 8.45% (6/71), HPV 45 12.68% (9/71).Few data are available on HAART and its role in HPV-associated cervical disease in HIV positive women. Immune restoration may decrease the severity and recurrence of HPV infection and potentially impact cervical disease although infection occur more often in immunodepressed HIV-positive patients.
Declaration of interest Conflicts of interest: F Carozzi participated occasionally in advisory board of GSK, Sanofi-Pasteur MSD, Abbott, Geneprobe None declared from the other authors
P-11.26
ANOGENITAL HPV-TYPE DISTRIBUTION AND PERSISTENCE IN HIV-POSITIVE MEN
A Potthoff, Ruhr-University Bochum/ St. Josef Hospital, Bochum, GERMANY A Kreuter, Ruhr-University Bochum, Bochum, Germany S Silling, University of Cologne, Koeln, Germany N H Brockmeyer, Ruhr-University Bochum, Bochum, Germany H Pfister, University of Cologne, Koeln, Germany U Wieland, University of Cologne, Koeln, Germany Background: Anogenital HPV-infections and associated benign and (pre)malignant lesions are frequent in HIVpositive men who have sex with men (HIV+MSM). Objectives: Determination of HPV-type distribution and persistence in anal and penile swabs of HIV+MSM. Methods: Consecutive anal and penile swabs of 397 HIV+MSM were collected within 12-18 months between 09/2008 and 03/2011. HPV-DNA detection and typing were performed by PCR and hybridization with 18 high-risk (HR)- and 18 low-risk (LR) type-specific probes using a bead-based multiplex genotyping assay. Results: At baseline, 354 (89.2%) of anal swabs were HPV-DNA-positive, 42 (10.6%) were HPV-negative and 1 (0.3%) was not evaluable. The respective numbers for penile swabs were 157 (41.3%), 174 (45.8%), and 49 (12.9%). Of the HPV-positive swabs (HPV+swabs), 311 (87.9%)/235 (66.7%) anal and 106 (67.5%) /72 (45.9%) penile swabs were HR- and LR-HPV-positive, respectively. Infections with >1 HPV-type were found in 75.7% of anal and 35.7% of penile HPV+swabs. Vaccine-included HPV-types 16, 18, 6, 11 were found in 48.0%, 18.6%, 15.0%, 14.7% of anal and in 24.8%, 8.9%, 1.3%, 3.8% of penile HPV+swabs. 67.0% of anal and 36.3% of penile HPV+swabs contained >=1 vaccine-included HPV-type. The most frequent non-vaccine included HR-types were HPV35 (12.1%), HPV58 (11.9%), HPV73 (11.9%), HPV52 (10.2%) for anal and HPV59 (8.9%), HPV52 (8.3%), HPV73 (7.6%), HPV35 (7.0%) for penile HPV+swabs. The most frequent non-vaccine included LR-types were HPV42 (11.9%/7.0%) and HPV61 (12.7%/6.4%) in anal/penile HPV+swabs. Persistent type-specific infections (detection of the same type >= 12 months) were found in 44.7% (anal) and 18% (penile) of HPV16-positive patients. The respective numbers for HPV18 were 39.4% (anal) and 7.1% (penile), 30.2% (anal) for HPV6, 30.8% (anal) and 16.7% (penile) for HPV11, 20.9% (anal) for HPV35, 54.8% (anal) for HPV58. Conclusions Persistent penile and anal infections with multiple HR- and LR-HPV-types are frequent in HIV+MSM.
Declaration of interest Conflict of interest: The study was supported by Sanofi Pasteur MSD.
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DIFFERENTIAL ANTIBODY RESPONSES TO HPV16 IN CERVICAL AND OROPHARYNGEAL CANCERS
K S Anderson, Dana Farber Cancer Institute, Boston, MA, UNITED STATES G Panicker, Centers for Disease Control and Prevention, Atlanta, GA, United States J Wong, Dana Farber Cancer Institute, Boston, MA, United States R Haddad, Dana Farber Cancer Institute, Boston, MA, United States M Posner, Mt Sinai School of Medicine, New York City, NY, United States E R Unger, Centers for Disease Control and Prevention, Atlanta, GA, United States Background: HPV type 16 is associated with the majority of CIN II/III, invasive cervical cancers, and oropharyngeal cancers. HPV DNA testing alone has low specificity for the detection of high risk cervical disease. Antibodies (Abs) to HPV16-derived proteins are potential biomarkers of HPV-related malignancies. Methods: Using a custom bead array ELISA, Abs to the HPV16 proteome were measured in sera from patients with no cervical disease (CIN 0, n=33), high-grade cervical dysplasia (CIN II/III; n=52), invasive cervical carcinoma (ICC, n=13), and oropharyngeal cancer (OPC; n=15). The median fluorescent intensity (MFI) ratios of IgG specific for each HPV-GST antigen to control p21-GST antigen were determined. All cervical cases and controls were typed for HPV DNA by Roche linear array. Results: In comparison to CIN 0, CIN II/III sera had an increased MFI ratio of Abs to HPV16 E2, E6, and E7 (p<0.05). There were no detectable differences in Abs to E1, E4, and E5 for CIN II/III. There was a trend to the detection of L1 and L2 specific Abs in CIN II/III (p=0.1). In comparison, sera from 4/13 patients with ICC had detectable Abs E4, E6, and/or E7. OPC sera had significantly higher MFI ratios of Abs to E1, E2, E6, and E7 antigens than ICC patients (p<0.05). Conclusions: Serum IgG Abs to HPV16 E2, E6, and E7 proteins are detected in high grade cervical dysplasia. The response broadens in ICC; in contrast to ICC, OPC sera contain strong E1 and E2 Abs, suggesting differences in biology reflected in the antibody responses between these two HPV-associated malignancies.
P-11.28
SOLUBLE HLA-G IN WOMEN WITH CERVICAL INTRAEPITHELIAL NEOPLASIAS AND CONTROLS
M G BICALHO, FEDERAL UNIVERSITY OF PARAN, CURITIBA-PARAN, BRAZIL R SLOWIK, FEDERAL UNIVERSITY OF PARAN, CURITIBA-PARAN, BRAZIL F S NARDI, FEDERAL UNIVERSITY OF PARAN, CURITIBA-PARAN, BRAZIL S J SILVA, FEDERAL UNIVERSITY OF PARAN, CURITIBA-PARAN, BRAZIL N S CARVALHO, FEDERAL UNIVERSITY OF PARAN, CURITIBA-PARAN, BRAZIL Human leukocyte antigen (HLA)-G molecule acts as modulator of innate and adaptive immune responses. Human papillomavirus (HPV) infection is associated with cervical intraepithelial lesions (CIN) of high (CIN 2 and CIN 3) and low (CIN 1) grades, and with cervical cancer. HLA-G expression, either its membrane or soluble (sHLA-G) forms, is frequently detected in a wide variety of human cancers and viral infections, and its levels negatively correlate with poor clinical outcome. This study aimed to investigate correlations between the serum levels of sHLA-G and the development and progression of CIN lesions. We performed the quantification of sHLA-G by ELISA (sHLA-G ELISA, BioVendor, Czech Republic) in the serum of 242 women. Out of 117 women (48,35%) were under the detection limit. The concentration of sHLA-G ranged from 0,6773-355,14 U/ml in the serum of the remaining women (n=125 ; 51,65%) as followed: CIN 2 (n=42), CIN 3 (n= 40) and Controls (n= 43). The statistical analyses revealed (Kruskal-Wallis Test ) significant differences among the sHLA-G level in the three groups (p=0.0037). The two-by-two comparisons carried out between the three groups showed the following results: CIN 2 vs CIN 3 (p=0.9385), CIN 2 vs Controls (p=0.0032) and CIN 3 vs Controls (p=0.0047). No differences were observed when comparisons were performed concerning sHLA-G serum levels between CIN 2 and CIN 3 patients (p=0.9385). Nevertheless, the serum levels of sHLA-G detected in CIN 2 and CIN 3 patients were significantly higher when compared to control women. Such findings are very suggestive that serum sHLA-G levels seem not to be a good prognostic marker of evolution of cervical lesions, as observed to other kinds of pathologies, although patients with lesions CIN 2 and CIN 3 express increased serum sHLA-G levels than controls (p=0.0032 and p= 0.0047, respectively).
Declaration of interest " None declared"
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THERAPEUTIC HPV16-SLP VACCINATION IN RECURRENT CERVICAL CANCER
M I E van Poelgeest, Leiden University Medical Center, Leiden, NETHERLANDS, S H van der Burg, Leiden University Medical Center, Leiden, Netherlands, M J P Welters, Leiden University Medical Center, Leiden, Netherlands, D M A Berends - van der Meer, Leiden University Medical Center, Leiden, Netherlands, M J G Lowik, Leiden University Medical Center, Leiden, Netherlands, E M G van Esch, Leiden University Medical Center, Leiden, Netherlands, L F M Stynenbosch, Leiden University Medical Center, Leiden, Netherlands, G G Kenter, Acadenic Medical Center, Amsterdam, Netherlands, C J M Melief, Leiden University Medical Center, Leiden, Netherlands Background: The median survival of patients with recurrent cervical cancer is 10-12 months. About 50-60% of cervical cancer is caused by HPV type 16. An HPV16 synthetic long peptide (HPV16-SLP) vaccine showed high immunogenicity in patients at different stages of disease and clinical benefit in patients with HPV16-induced vulvar lesions. Objectives: To test the clinical and immunological efficacy of the HPV16-SLP vaccine in patients with clinical and radiological evidence of HPV16-positive recurrent cervical cancer without curative treatment options. Methods: Patients were maximally 4 times injected subcutaneously with the HPV16-SLP vaccine at 300 g per peptide emulsified in Montanide ISA51, at 3 week intervals. Imaging (CT- or MRI scan) was performed 6-8 weeks after the last vaccination. Blood samples for immunological analysis were taken at study entry, and after 2 and 4 vaccinations. Immunological assays included lymphocyte proliferation and cytokine production as well as IFNgELISPOT. Results: Twenty patients were accrued and vaccinated. The median overall survival in this group was 14.7 months after diagnosis of recurrence and 29,6 months after primary diagnosis. Vaccine-induced proliferative HPV16specific T-cell responses were detected in 9 out of 16 patients tested after vaccination, while HPV16-specific IFNg-producing T-cells were detected in 14 of 16 patients by IFNg-ELISPOT. Patients were grouped according the median overall survival and those with a longer survival (i.e. >11 months) displayed a stronger HPV16-specific T-cell response than the patients with a shorter survival (i.e. 11 months) but also to recall antigens. Conclusions: HPV16-SLP vaccination is able to induce HPV16-specific T-cell immunity in patients with advanced recurrent cervical cancer. The strength of the recall and vaccine-induced T-cell responses was higher in patients with longer survival (>11 months) compared to patients with a survival of 11 months.
Declaration of interest Prof. Dr. Melief is a paid member of the steering committee of ISA Pharmaceuticals.
P-11.30
PLASMACYTOID DENDRITIC CELLS, T-REGS AFFECT HPV PERSISTENCE IN HIV+/HIV- WOMEN
H Strickler, Albert Einstein College of Medicine, Bronx, New York, UNITED STATES S Desai, Rush University, Chicago, IL, United States J Palefsky, University of California, San Francisco, San Fran, CA, United States X Xie, Albert Einstein College of Medicine, Bronx, New York, United States H Minkoff, Maimonides Medical Center, Brooklyn, New York, United States L Massad, Washington University School of Medicine, St Louis, MO, United States K Anastos, Albert Einstein College of Medicine, Bronx, New York, United States R Burk, Albert Einstein College of Medicine, Bronx, New York, United States Background: Beyond a low total CD4+ count, the immune cell deficits that drive the association of HIV/AIDS with HPV and cervical neoplasia are not well understood. Alpha-interferon producing plasmacytoid dendritic cells (pDCs) are of special interest, since they bridge the innate (as effector cells) and adaptive immune systems (as antigen presenting cells). Further, low pDC levels predict AIDS risk independent of CD4+. Regulatory T-cells (Tregs) down-regulate immune responses and may be elevated in HIV+ patients. A prior report correlated high Treg levels with HPV16 persistence. Methods: We examined 25 HIV+ women with CD4+ >350/L and a current high-risk (hr)-HPV infection that either persisted 1.5 years (N=14) or resolved within 6 months (N=11). Eight HIV- women (3 persisters, 5 regressers) were also studied. Subjects had semiannual follow-up. Cryopreserved PBMCs from the first visit when oncogenic HPV was detected and PBMCs from one year later were assessed using flow cytometry to determine the percent (%) Tregs and pDCs. Results: Among HIV+ women, the Treg% was higher in hr-HPV persisters (median=4.3%) than regressers (median=3.0%), a difference that was significant in logistic regression models even after adjustment for age and HAART use (P=0.05). The pDC% was, as hypothesized, lower in hr-HPV persisters (0.04%) than regressers (0.06%), though this difference did not reach significance (P=0.12). Similar findings were obtained in the few HIV- women studied, including for Treg% (4.7% verus 2.7%;P=0.23), and pDC% (0.03% versus 0.19%;P=0.07). Given these similar findings in HIV+ and HIV- women we combined all data. In models adjusted for age and HIV-serostatus, hr-HPV persistence was associated with high Treg (P=0.01) and low pDC (P=0.03) levels. Conclusions: These data suggest that high T-reg and low pDC levels may be associated with hr-HPV persistence in both HIV+ and HIV- women. More comprehensive, larger studies are warranted.
Declaration of interest None
20

CYTOKINE PROFILE IN WOMEN WITH AND WITHOUT CERVICAL CANCER
A Bedoya, University of Antioquia/Group Infection and Cancer/School of Microbiology, Medellin, COLOMBIA D Tate, Stanley S. Scott Cancer Center, LSUHSC, New Orleans, United States V Flrez, University of Antioquia/Group Infection and Cancer, Medellin, Colombia C Crdoba, University of Antioquia/Department of ginecology Hospital San Vicente de Pal, Medellin, Colombia M Borrero, University of Antioquia/Department of ginecology Hospital San Vicente de Pal/Clnica Las Amricas, Medelln, Colombia J Zabaleta, Stanley S. Scott Cancer Center, LSUHSC, New Orleans, United States R Herrero, International Agency for Research on Cancer, Lyon, France A H Zea, Stanley S. Scott Cancer Center, LSUHSC, New Orleans, United States G I Snchez, University of Antioquia/Infection and Cancer, Medellin, Colombia Background: Cellular immune response is essential to control HPV infection and cancer. Cytokines play an important role for activation, maintenance and regulation of the immune response. It has been postulated that immune response may be impaired in cervical cancer. Objective: To compare the systemic cytokine levels in women with and without cervical cancer and determine the cytokine gene expression in cervical cancer tissues. Methods: Samples from women with (n=86) and without (n=79)invasive cervical cancer from Medellin-Colombia were included. Levels of GM-CSF, IFN-, IL-1Beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, MCP-1, tumor necrosis factor- (TNF-), VEGF and TGF-Beta were measured in sera using Milliplex Human cytokine/chemokine from Millipore. RNA extracted from 42 biopsies of women with cervical cancer was used to determine gene expression by qRT-PCR of IL12, IL2, IFN-, IL-1Beta, IL-6, IL-8, TNF-, IL-10, VEGF and TGF-Beta. Data are presented as median and the Mann-Whitney test was used to assess significance between groups. Results: Analysis of systemic cytokines profile revealed a statistically significant decrease in GM-CSF, IFN-, IL-2, IL-4, IL-5, IL-7, IL-8, IL-12p70, IL-13, MCP-1, tumor necrosis factor- (TNF-) and VEGF in sera from women with cervical cancer compared to controls (MannWhitney test, p<0.0001). An increased in IL-10 levels in women with cervical cancer (median 0.0 pg/mL) compared to controls (median 0.17(pg/mL), (Mann-Whitney test, p<0.0001.) was observed. There were no differences in levels of IL-1Beta and IL-6. Tissue gene expression of IL-12, IL-2, IFN-, IL-1Beta, IL-6, IL-8, TNF-, IL-10 and TGF-Beta was low in cervical cancer tissues. Conclusions: A shift towards a systemic Th2/Tc2 cytokine profile due to low levels of IFN-, IL-2 and IL-12 and high levels of IL-10 was observed in invasive cervical cancer. The profile of expression of cytokines in tumor tissue did not explain the Th2 cytokine profile observed in sera.
P-11.32
HIGH INCIDENT DETECTION OF HPV SUBTYPES IN HIV WOMEN
M D Zeier, Stellenbosch University, Tygerberg Hospital, Cape Town, SOUTH AFRICA J B Nachega, Stellenbosch University, Tygerberg Hospital, Cape Town, SOUTH AFRICA M H Botha, Stellenbosch University, Tygerberg Hospital, Cape Town, SOUTH AFRICA D Mason, Stellenbosch University, Tygerberg Hospital, Cape Town, SOUTH AFRICA M Van Schalkwyk, Stellenbosch University, Tygerberg Hospital, Cape Town, SOUTH AFRICA S Engelbrecht, Stellenbosch University, Tygerberg Hospital, Cape Town, SOUTH AFRICA Background: Limited data are available on the impact of ART on HPV subtype detection. Objectives: We aim to determine HPV type specific differences over the first 6 months of a study that examines the effect of ART on women with cervical dysplasia and HPV/HIV co-infection. Methods: In a prospective cohort study enrolling in South Africa, 208 cervical samples from the 93 study participants that reached 6 month follow-up were tested with the Roche HPV Linear Array to differentiate 37 subtypes. Of the participants, 38 commenced ART. Decision to initiate was based on HIV markers and not cervical pathology. We compared incident and persistent detection of HPV between the ART groups. Results: At least one high-risk type HPV was detected in all 93 participants. Most (88/93) had incident detection at 6 months for at least one HPV type. The most common of the incident HPV types were 84 (42%), 16 (29%) and 62 (29%) of ART participants and 84 (33%), 51 (29%) and 35 (25%) in the non-ART group. The incident detection of HPV type 16 at 6 months was marginally higher in the ART than the non-ART group (29 vs. 13% of women) (p=0.05). Of the women that had incident detection of HPV 16, 3/18 reported abstinence from sexual activity for preceding 12 months. Persisting most at 6 months were HPV 16 (21/34 women), and least was 68 (4/22 women). Conclusion: The incident detection was high at 6 months, and for HPV 16 it seemed higher in the ART group in spite of good test sensitivity for this subtype. Incident detection could be due to new infection, reactivation or another reason for increased viral shedding. It may present a limiting factor in a study examining early impact of ART on HPV infection.
21

WHAT IS THE ROLE OF MICA GENE IN CIN PROGRESSION?
P P FRANA, Federal University of Parana, Curitiba, BRAZIL R SLOWIK, Federal University of Parana, Curitiba, BRAZIL M B S XAVIER, Federal University of Parana, Curitiba, BRAZIL G Y REZENDE, Federal University of Parana, Curitiba, BRAZIL C. MAESTRI, Erasto Gaertner Hospital, Curitiba, BRAZIL N S CARVALHO, Clinical Hospital of the Federal University of Parana, Curitiba, BRAZIL M G BICALHO, Federal University of Parana, Curitiba, BRAZIL Background: Persistent infection with high-risk-HPV(hrHPV) is necessary for the development of cervical cancer (CC) and its precursors. Although CC evolves from cervical intraepithelial neoplasia(CIN), not all CIN progress to CC, suggesting that other risk factors are essential on the evolution or regression of CIN. Investigations of the immunogenetic factors involved in the disease might help to understand the biology of CC development. The MHC Class I Chain-related Gene A (MICA) is a non classical HLA-I gene. MICA is expressed by keratinocytes and epithelial cells and interacts with gamma-delta-T and NK cells. It is therefore possible that MICA might influence the pathogenesis of CIN and CC. Objectives: The purpose of this study was to investigate the association between MICA polymorphism and CIN2/ CIN 3, in a preliminary case-control study from Curitiba-Brazil. Methods: Women, aged 15-45years, with CIN II (n=40) and CIN III (n=40) from Erasto Gaertner Hospital were investigated. Genotyping was conducted using SSP-PCR and SSOP-Luminex technology. The results were compared with those from 81 healthy women using Chi-square-test. Epidemiological questionnaire and a written informed consent term were obtained from all participants. Hybrid capture for hrHPV was performed. Results: A significant association of CIN II and CIN II with MICA polymorphisms was observed. MICA*008:01/04 was associated with an increased risk of developing CIN 2 (OR=3.36; 95% CI: 1.33-8.25, p=0,0126) and CIN 3 (OR=2.53; 95% CI: 1.10-5.83; p=0,04). MICA*020/055 emerged as a protective factor against CIN 2 (OR=0.16; 95% CI: 0.04-0.71; p=0,01) and CIN 3 (OR=0.15; 95% CI: 0.04 0.51; p=0,0012). Conclusion: Our study provides evidence for genetic susceptibility to CIN due to allelic variation in MICA gene. MICA*008:01/04 seems to be important on the progression of CIN and MICA*020/055 provokes a protective effect to prevent CIN/CC.
Declaration of interest None declared.
P-11.34
CHARACTERIZATION OF HLA-C ALLELES IN CERVICAL INTRAEPITHELIAL LESIONS
N S Carvalho, Federal University of Parana, Curitiba, BRAZIL M B S Xavier, Federal University of Parana, Curitiba, BRAZIL G Y T Rezende, Federal University of Parana, Curitiba, BRAZIL P P Frana, Federal University of Parana, Curitiba, BRAZIL M G Bicalho, Federal University of Parana, Curitiba, BRAZIL Background:The squamous cervical cancer (SCC) is the second most common cancer among women worldwide, especially in developing-country, like Brazil. The role of HPVs in the etiology of SCC precursor lesions and invasive carcinoma development has been well known, associated on other risk factors. The genetic susceptibility to HPV infection seems to be important in determining individual risk for the development of virus-induced cancer. The cellular immune response is an important mechanism for the elimination of HPV infections already established. Variation in human leucocytes antigens (HLA) may influence the risk of SCC by altering the efficiency of the T cell or NK cell immune response to HPV antigens. The combination of HLA-C allotypes and KIR receptors has been associated with diseases as viral infections and cancer. Objectives:This preliminary case-control study aimed to compare the HLA-C genetic profile, in high-definition HLA-C typing, of women in different cervical intraepithelial neoplasia (CIN) degrees: 37 CIN II and 37 CIN III, and 80 women with normal cervix cytology, to explore the relationship between HLA-C with the SCC development in a south Brazilian population. Methods:We studied women between 15-45 years old. The case group was diagnosed CIN II and CIN III after conization surgery. After HLA-C genotyping, statistical analyses were made using direct counting and Contingence table analyses. Results were considered statistically significant if p 0,05. Results: We found statistically significant association between HLA-C*04 and CIN. A protective effect of this allelic group was observed for CIN III (OR=0,2883; 95% IC:0,1369-0,6073;p=0,0015) and CIN II (OR=0,4349; 95% IC:0,21770,8685; p=0,027). Conclusions:The preliminary results show that individual allelic frequency may be not considered statistically significant, but when classifying HLA-C groups, we found significant differences in HLA-C*04 on a stratified sample, showing that, among south Brazilian, the outcome of HPV-infected CINs is associated with HLA-C polymorphism.
22

ANTIBODIES AGAINST HPV16 AND POLYOMAVIRUSES IN ADOLESCENTS AND YOUNG ADULTS.
E Liais, University of TOURS, TOURS, France A Carpentier, University of TOURS, TOURS, France M J Fleury, University of TOURS, TOURS, France J Nicol, University of TOURS, TOURS, France E Mazzoni, University of Ferrara, Ferrara, Italy M Tognon, University of Ferrara, Ferrara, Italy A Touz, University of TOURS, TOURS, France P Coursaget, University of TOURS, TOURS, France Little is known concerning the human papillomavirus type 16 infection in adolescents and young adults. In this study, we investigated the HPV16 seroprevalence in 2-30 years old subjects from Italy. As a comparison, antibodies against two newly discovered human polyomaviruses were investigated: Merkel cell polyomavirus associated with Merkel cell carcinoma, and HPyV9, a polyomavirus closely related to the simian lymphotropic polyomavirus (LPV). Detection of antibodies was investigated using virus-like particles produced in insect cells infected with the respective recombinant baculoviruses encoding VP1 protein. The results obtained confirmed that anti-HPV antibodies are detected only after sexual activity debut, and that MCV antibodies are detected very early in life with a kinetic of apparition similar to that observed for BK polyomavirus.
23
Session 12: Clinical aspects of HPV testing

Chair: Joel Palefsky
Poster abstracts
Clinical aspects of HPV testing | 12 O-12.00

Joel Palefsky
FOR YOUR NOTES:
O-12.01
HPV RNA TESTING IN A CLINICAL SETTING IN THE U.K.
K Cuschieri, Royal Infirmary of Edinburgh, Edinburgh, UNITED KINGDOM H Kitchener, University of Manchester, Manchester, UNITED KINGDOM A Horne, University of Edinburgh, Edinburgh, UNITED KINGDOM C Busby - Earle, University of Edinburgh, Edinburgh, UNITED KINGDOM A Hardie, Royal Infirmary of Edinburgh, Edinburgh, UNITED KINGDOM J Rowan, Royal Infirmary of Edinburgh, Edinburgh, UNITED KINGDOM A Bailey, Manchester Royal Infirmary, Manchester, UNITED KINGDOM C Graham, University of Edinburgh, Edinburgh, UNITED KINGDOM E Crosbie, University of Manchester, Manchester, UNITED KINGDOM H Cubie, Royal Infirmary of Edinburgh, Edinburgh, UNITED KINGDOM Background: Detection of E6/E7 mRNA may be more specific for the detection of clinically significant HPV infection compared to HPV DNA testing. Objectives To evaluate clinical sensitivity and specificity of the APTIMA HPV RNA based assay (AHPV, Gen-Probe Incorporated) in comparison to the Hybrid Capture 2 DNA based assay (HC2, Qiagen Ltd) in a clinical setting. Methods: Women attending two NHS colposcopy clinics (for all referral indications) in two large teaching hospitals were invited to participate. Cervical samples in PreservCyt were tested via the two HPV assays described. Biopsies were taken where clinically indicated. Relative sensitivity and specificity of each assay for CIN2+ and CIN3+ was calculated in the all indication population and a subgroup referred for low-grade disease. The effect of age (> and < 30) on test performance was measured. Results: Of 1329 cervical samples, 365 were associated with CIN2+. Sensitivity for CIN2+ detection was identical for both assays, 94% (95% CI 91.0, 96.2) whereas specificity was significantly higher for AHPV:, 49.5% (95% CI 46.3, 52.7) vs. 45.4% (95% CI 42.3, 48.6) for AHPV and HC2, respectively (p<0.001, McNemars test based on paired comparison of AHPV and HC2 in women with <CIN2). Results were similar for CIN3+ detection. Subgroup analysis revealed similar CIN2+ detection rates in women referred with low grade cytological abnormalities with sensitivity 89.6% (95% CI 80.6, 95.4) vs. 90.9% (95% CI 82.2, 96.3) and specificity 42.9% (95% CI 37.9, 48.0) vs. 42.4% (95% CI 37.9, 47.5) for AHPV and HC2, respectively. Specificity of both assays was significantly higher in women >30 in both the all indication and low-grade referral population. Conclusions: The two tests exhibit similar sensitivity with the AHPV showing higher specificity in the all referral population. Longitudinal data on a post treatment population will be presented.
Declaration of interest Genprobe funded the work described in the abstract however the analysis was performed independently. The presenting author has also attended advisory board meetings with GenProbe.
27
12 | Clinical aspects of HPV testing O-12.02

HPV MRNA TEST IN PRIMARY SCREENING OF WOMEN 20-34 YEARS
S Srbye, University Hospital of North Norway, Troms, NORWAY S Fismen, University Hospital of North Norway, Troms, NORWAY T Gutteberg, University Hospital of North Norway, Troms, NORWAY E Mortensen, University Hospital of North Norway, Troms, NORWAY F Skjeldestad, University of Troms, Troms, NORWAY Background. Primary cervical screening using HPV test relative cytology has been advocated because of higher sensitivity for detection of CIN2+. However, HPV DNA testing is not cost-effective in women 20-34 years due to a high positivity rate of HPV infection. Objective was to assess the performance of HPV mRNA test in primary screening. Methods. In 2003-2004 18 852 women were tested with HPV mRNA (PreTect HPV-Proofer, NorChip AS) in primary and secondary screening. Women with a history of abnormal PAP-smear, with biopsy with CIN2+ before screening or until 3 months after, were excluded. Eligible were 12 958 women 20-69 years in a situation resembling primary screening. Follow-up through December 31st 2009 were done through national surveillance of CIN2+ in three registries administered by the Norwegian Cancer Registry (CIN treatment registry, CIN biopsy registry, Cancer registry). All analyses were done by survival analysis in SPSS (version 17.0). Results. 5.2% were HPV mRNA positive at screening. The cumulative rate of CIN2+ was 1.8% through 81 months of follow-up. For women 20-34 years (n=5 085) 9.4% were HPV mRNA positive at baseline and the cumulative rate of CIN2+ was 2.9%. For women 35-69 years (n=7 873) 2.5% were HPV mRNA positive at baseline and the cumulative rate of CIN2+ was 1.1%. Cumulative rates by baseline status for HPV mRNA positive and HPV mRNA negative in women 20-34 years were 20.7% and 1.0%, respective 20.1% and 0.6% in women aged 35-69 years. Except for HPV18, the cumulative incidence rates for CIN2+ were relative constant for HPV-16 and HPV-31,-33,-35 by age. Conclusion. The HPV mRNA test may be used in primary screening for both women 20-34 and 35-69 years. Due to differences in test properties and understanding of oncogenesis of cervical cancer, studies comparing head-tohead DNA and mRNA tests in primary screening are warranted.
O-12.03
TRIAGE OF WOMEN WITH ABNORMAL CYTOLOGY USING SEVERAL HPV TESTS
D Mesher, Queen Mary University of London, London, UNITED KINGDOM A Szarewski, Queen Mary University of London, London, UNITED KINGDOM L Cadman, Queen Mary University of London, London, UNITED KINGDOM J Austin, Queen Mary University of London, London, UNITED KINGDOM L Ho, Queen Mary University of London, London, UNITED KINGDOM G Terry, Queen Mary University of London, London, UNITED KINGDOM S Liddle, The Doctors Laboratory, London, UNITED KINGDOM C Bergeron, Laboratoire Pasteur-Cerba, Cergy Pontoise, FRANCE D Lyons, St Marys Hospital, London, UNITED KINGDOM J Cuzick, Queen Mary University of London, London, UNITED KINGDOM Background: It is well established that low grade cytology has poor positive predictive value and triage tests are needed to improve specificity (including HPV DNA, HPV RNA, HPV typing and p16 tests). Objectives: In women referred with abnormal cytology, we aim to compare sensitivity, specificity and positive predictive value (PPV) for high-grade cervical disease (CIN2+ and CIN3+) for different triage strategies using several different HPV tests to form a consensus result. Methods: A total of 2,052 were eligible for analysis from two studies which compared a number of biomarkers (953 from Predictors 1 and 1099 from Predictors 2). HPV tests from Predictors 1 included Hybrid Capture 2 (Qiagen), APTIMA (GenProbe), RealTime PCR (Abbott) and Linear Array (Roche); and in Predictors 2 using Hybrid Capture 2 (Qiagen), APTIMA (GenProbe), RealTime PCR (Abbott), Cobas (Roche) and BD HPV (BD). HPV was taken as positive if three or more of these HPV tests were positive and negative only if none of these tests gave a positive result. PreTect HPV-Proofer (Norchip) and p16INK4a (mtm laboratories) were also performed. Sensitivity, specificity and PPV were based on the worst histology at baseline. Results: 1,272 (62%) of women had mild dyskaryosis cytology at referral. A total of 243 (19.1%) of these women had CIN2+ and 106 (8.3%) CIN3+. Restricting analysis to only women with a mild dyskaryosis smear at referral, for the detection of CIN2+: HPV testing had a sensitivity of 98.7%, specificity of 17.4%, and PPV of 24.0%. p16INK4a had a sensitivity of 77.4%, specificity of 47.8%, and PPV of 27.6%. PreTect HPV Proofer had a sensitivity of 67.8%, specificity of 72.5%, and PPV of 36.7%. Testing for high-risk type 16 only had a sensitivity of 54.1%, specificity of 82.5%, and PPV of 43.0%. Results will be presented separately for those with a borderline referral smear.
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COMPARISON OF APTIMA AND HC2 IN ROUTINE SCREENING IN GERMANY
T Iftner, University Hospital, Dept. Med. Virology and Epidemiol. of Viral Diseases, Tbingen, GERMANY, S Becker, University Hospital, Dept. Gynecology and Obstetrics, Tbingen, GERMANY, K Neis, Frauenrzte am Staden, Saarbrcken, GERMANY, B Holz, University Hospital, Dept. Med. Virology and Epidemiol. of Viral Diseases, Tbingen, GERMANY, A Castanon, Queen Mary University of London, London, UNITED KINGDOM, A Staebler, University Hospital, Dept. Pathology and Neuropathology, Tbingen, GERMANY, C Hann von Weyhern, University Hospital, Dept. Pathology and Neuropathology, Tbingen, GERMANY, A Clad, University Hospital, Dept. Gynecology, Obstetrics and med. Microbiology, Freiburg, GERMANY, M Henes, University Hospital, Dept. Gynecology and Obstetrics, Tbingen, GERMANY, P Sasieni, Centre for Cancer Prevention, Queen Mary University of London, London, UK Background: E6/E7 mRNA testing of high-risk HPV might improve the specificity for cervical precancer compared to HPV DNA by reducing the false positive rate. Objectives: As the APTIMA HPV Assay (AHPV) has been compared to HPV DNA tests mostly in triage settings we compared both tests with liquid-based cytology (LBC) in women aged 30 attending routine cervical screening. Methods: Women aged 3065 were screened at office based gynecologists in three different areas: Tbingen, Saarbrcken and Freiburg. All specimens were collected with a cytobrush and tested centrally by LBC in Saarbrcken, and by AHPV and HC2 in Tuebingen. Women were referred to colposcopy if they had an abnormal cytology result and/or tested positive on either HPV assay. So far 60% of the histology has been reviewed. Sensitivity, specificity and positive predictive values were calculated based on all available test results. Results: Preliminary screening results are available for 7649 women. There were 68 with CIN2+ detected on histology of which 31 were CIN3+. AHPV and HC2 were highly sensitive for CIN2+: 92.6% (95% CI 83.7-97.6) and 95.6% (87.6-99.1), respectively; and for CIN3+: 93.5% (78.6-99.2) and 100% (88.8-100), respectively. Out of the two CIN3 missed by AHPV one was 0.2mm in size and the other had HPV62. LBC had a sensitivity of 37% (25.4-49.3) for CIN2+ and 48.4% (30.2-66.9) for CIN3+. Specificity of AHPV for CIN2+ was higher than that of HC2: 96.1% (95.7-96.6) versus 95.2% (94.7-95.6), with a corresponding reduction in the false positivity rate of 20%. The positive predictive value for CIN2+ amongst those who have attended colposcopy were 20%, 18.3% and 23.0%, for AHPV, HC2 and LBC respectively. Conclusions: The AHPV assay is both specific and sensitive for the detection of high-grade lesions and can be used as an option for routine cervical screening for women 30 years old.
Declaration of interest Conflicts of interest: S. Becker: no conflict. P. Sasieni: Participated in a Gen-Probe Scientific Advisory Board meeting. K. Neis: Travel support from Hologic, support for lectures from Gen-Probe, Hologic, MTM Laboratories, Sanofi Pasteur MSD. A. Castanon: no conflict. A. Staebler: no conflict. C. Hann von Weyhern: no conflict. A. Clad: no conflict. M. Henes: no conflict. T. Iftner: institutional research grants from Gen-probe, GSK, Hologic, Roche, Sanofi Pasteur MSD.
O-12.05
META-ANALYSIS: APTIMA VERSUS HYBRID-CAPTURE-2 TO TRIAGE ASCUS OR LSIL CYTOLOGY
M Arbyn, Scientific Institute of Public Health, Brussels, BELGIUM, K Cuschieri, Scottish HPV Reference Laboratory, Royal Infirmary of Edinburgh, Edinburgh, SCOTLAND, UK, J Cuzick, Cancer Research UK, Centre for Epidemiology, Mathematics and Statistics, Wolfson Institute of Preventive Medicine, London, UK, A Szarewski, Cancer Research UK, London, UK, S Ratnam, Public Health Laboratory, St. John's, Canada, J Dockter, Gen-Probe Incorporated, San Diego, USA, M Reuschenbach, Department of Applied Tumor Biology, Institute of Pathology, University of Heidelberg, Heidelberg, Germany, J L Belinson, Technology Evaluation Center, Blue Cross Blue Shield Association, Chicago, USA, J Monsonego, Institute of the Cervix, Paris, France Background and objectives: Systematic reviews and randomised trials have consistently shown superior accuracy of the Hybrid Capture-2 (HC2) assay in triage of women with ASCUS (atypical squamous cells of undetermined significance) cytology to detect underlying high-grade cervical intraepithelial neoplasia or worse (CIN2/3+) compared to repeat cytology. However, LSIL triage with HC2 showed no enhanced sensitivity and was substantially less specific than a repeat Pap test. In the current meta-analysis, we compared the accuracy of the APTIMA test, which identifies RNA of 14 high-risk HPV types, with that of HC2 for the triage of women with ASCUS and LSIL. Methods: Literature searching targeted studies where the accuracy of APTIMA and HC2 for detection of underlying CIN2/3+ was assessed concomitantly including verification of all cases of ASCUS and LSIL. Authors were contacted to provide data separately for ASCUS or LSIL. HSROC curve regression was used to compute the pooled absolute and relative sensitivity and specificity. Results: Seven studies (comprising 900 ASC-US and 2383 LSIL cases) were retrieved. The pooled sensitivity and specificity of APTIMA to triage ASCUS was 97.9% (95% CI [CI]: 86.8-99.7%) and 53.0% (CI: 37.7-67.8%), respectively, for CIN2+ and 97.6% (CI: 93.0-100%) and 53.3% (CI: 37.8-68.8), respectively, for CIN3+. APTIMA and HC2 showed similar pooled sensitivity and specificity for CIN2+ and CIN3+. The pooled sensitivity and specificity of APTIMA to triage LSIL were 92.1% (CI: 87.5-96.8%) and 43.1% (CI: 34.5-51.7%), respectively, for CIN2+ and 98.9% (51.9-99.9%) and 42.1% (CI: 35.2-49.4%), respectively, for CIN3+. APTIMA was as sensitive as HC2 but more specific (ratio of 1.44; CI: 1.19-1.75) for CIN2+ and CIN3+ (ratio: 1.35; CI: 1.11-1.66). Conclusions: The accuracy of APTIMA and HC2 for detecting CIN2+ or CIN3+ in women with ASCUS was similar. However, in triage of LSIL, APTIMA is as sensitive but significantly more specific than HC2.
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12 | Clinical aspects of HPV testing O-12.06

A COMPARISON OF TESTS FOR HIGH GRADE CIN: PREDICTORS 3
A Szarewski, Queen Mary University of London, London, UNITED KINGDOM, L Cadman, Queen Mary University of London, London, UNITED KINGDOM, D Mesher, Queen Mary University of London, London, UNITED KINGDOM, J Austin, Queen Mary University of London, London, UNITED KINGDOM, L Ho, Queen Mary University of London, London, UNITED KINGDOM, G Terry, Queen Mary University of London, London, UNITED KINGDOM, S Liddle, The Doctors Laboratory, London, UNITED KINGDOM, W P Soutter, Hammersmith Hospital, London, UNITED KINGDOM, D Lyons, St Marys Hospital, London, UNITED KINGDOM, J Cuzick, Queen Mary University of London, London, UNITED KINGDOM Background: Studies have shown that HPV testing is substantially more sensitive than cytology for the detection of high grade cervical lesions, but lacks specificity. Earlier phases of the Predictors study compared several tests in women referred for colposcopy. We compared specificity, sensitivity and positive predictive value (PPV) of several tests for the detection of high-grade disease (CIN2+ and CIN3+) in a screening setting. Methods: 6000 women aged 25 to 66 attended for cervical cytology. Six DNA or mRNA tests were performed using residual material from ThinPrep vials: Hybrid Capture 2 (Qiagen), PreTect HPV-Proofer (Norchip), APTIMA HPV assay (Gen-Probe), Real-time PCR (Abbott), BD HPV (BD) and Cobas (Roche). Sensitivity, specificity and PPV were based on the worst histology at baseline. Colposcopic referral was based solely on cytology results so women with a negative cytology result but HPV positive were considered negative on histology. Results: Median age of women was 37 years. 318 women (5.3%) had an abnormal cytology result (borderline or worse). Overall, 40 women had high grade disease (CIN2+), of whom 19 had a CIN3+ result. For the detection of CIN2+, APTIMA had a sensitivity of 97.5%, specificity of 90.2%, and PPV of 6.3%. Abbott Real-time PCR had a sensitivity of 95.0%, specificity of 87.2%, and PPV of 4.7%. Hybrid Capture 2 had a sensitivity of 97.5%, specificity of 85.4%, and PPV of 4.3%. Cobas had a sensitivity of 97.5%, specificity of 84.5%, and PPV of 4.1%. BD HPV had a sensitivity of 97.5%, specificity of 84.3%, and PPV of 4.0%. Using McNemars test, APTIMA was significantly more specific than all other tests. There was also a significant difference in specificity between Abbott and Hybrid Capture 2. The least specific tests were Cobas and BD. PreTect HPV-Proofer testing is ongoing and results will be included in the final presentation.
O-12.07
THE CLINICAL PERFORMANCE OF PAPILLOCHECK HPV DNA DETECTION ASSAY
A Bailey, Central Manchester University Hospitals NHS Foundation Trust, Manchester , UNITED KINGDOM E J Crosbie, Central Manchester University Hospitals NHS Trust, Manchester, UK A Sargent, Central Manchester University Hospitals NHS Trust, Manchester, UK C Gilham, Institute of Cancer Research, London, UK J Peto, Institute of Cancer Research, London, UK H Kitchener, Central Manchester University Hospitals NHS Trust, Manchester, UK Objective: To compare the clinical performance of Papillocheck (Greiner Bio-one), a PCR-based DNA microarray system with Hybrid Capture-2 (HC2) (QIAGEN) for the detection of CIN2+. Methods: 1) Archived (Round 1 ARTISTIC) and 2) prospectively collected (Round 3 ARTISTIC trial) cervical liquidbased cytology (LBC) samples were tested by both HC2 and PapilloCheck. HPV positive samples by either assay were genotyped using Roche Prototype Line Blot (RLB)/Linear Array (LA) assays. Results: From ARTISTIC Round 1, 3792 LBC specimens were tested (2223 HC2 positive/normal cytology, 1099 borderline/mild dyskaryosis, 420 moderate/severe dyskaryosis). CIN2+ was found in 46/2223 (2%) women with HC2 positive/normal cytology samples; seven of these 46 (15%) were HPV negative by Papillocheck. Among the 2223 HC2 positive/cytology negative samples, 1007 (45%) were PapilloCheck negative and 667 (66%) were negative by RLB/LA. In women with minor cytological abnormalities, HC2 and PapilloCheck detected underlying CIN2+ with similar accuracy (sensitivity 89% vs. 88%; specificity 53% vs. 52%; and negative predictive value 95% vs. 94% for HC2 vs. PapilloCheck respectively). Overall, there were 660 underlying CIN2+ lesions in women with abnormal cytology; HC2 failed to detect 4 of these (0.6%) while PapilloCheck missed 21 (3.2%). From ARTISTIC Round 3, 5132 LBC specimens were tested (4849 normal cytology, 249 borderline/mild dyskaryosis, 34 moderate/ severe dyskaryosis). The overall HPV positive rate was 13% by HC2 and 12% by PapilloCheck, including 9% of normal cytology samples (both tests). There were only 13 underlying CIN2+ lesions; all of these were HPV positive by both HC2 and PapilloCheck. Conclusions: In the archived normal cytology samples, using PapilloCheck instead of HC2 would have resulted in 1007 fewer colposcopy referrals and just 7 missed CIN2+ lesions. In the archived abnormal cytology samples, HC2 and PapilloCheck performed similarly in terms of CIN2+ detection, picking up 656 (99.4%) and 639 (96.8%) of 660 lesions respectively.
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GENOTYPE ATTRIBUTION IN HIGH-GRADE CIN SHOWS ETIOLOGICAL DOMINANCE OF HPV16
J van der Marel, DDL Diagnostic Laboratory, Voorburg, NETHERLANDS, W G V Quint, DDL Diagnostic Laboratory, Voorburg, NETHERLANDS, M Schiffman, National Cancer Institute, NIH, DHHS, Rockville, USA, M M van de Sandt, DDL Diagnostic Laboratory, Voorburg, NETHERLANDS, R Zuna, University of Oklahoma Health Sciences Center, Oklahoma City, USA, J Walker, University of Oklahoma Health Sciences Center, Oklahoma City, USA, S T Dunn, University of Oklahoma Health Sciences Center, Oklahoma City, USA, M A Gold, University of Oklahoma Health Sciences Center, Oklahoma City, USA, N Wentzensen, National Cancer Institute, NIH, DHHS, Rockville, USA Background: Women with HGCIN frequently present with multiple concomitant lesions of similar or lower grade, and up to 50% of women with HGCIN have multiple concurrent HPV genotypes. The topographic and clonal relationship between multiple HPV genotypes and heterogeneous CIN lesions in the same woman is not well understood. Objectives: To perform molecular mapping of cervical disease in women with HGCIN and elucidate HPV genotype attribution in individual lesions. Methods: We selected 13 subjects who were referred to colposcopy for abnormal cytology results at the University of Oklahoma. A cervical smear and 4 biopsies were taken from different areas on the cervix. Laser Capture Microdissection (LCM) was performed on individual biopsies. Whole tissue biopsy samples and LCM samples were analyzed using SPF10PCR/LiPA25(version1). HPV genotypes detected by LCM-PCR were compared to types found in cervical smears and whole tissue sections with single or multiple HPV types. Results: Eleven of 13 women had at least two biopsies with HGCIN, and in 4 women, HGCIN was detected at all four biopsy sites. Eight of the 13 cervical smears (62%) showed multiple genotype infections. When analyzing whole tissue sections of individual biopsies, only 9 of 52 biopsies (17%) had multiple infections and after performing LCM, only 6 of 132 areas (5%) had multiple genotype infections. Eight of 13 women had the same carcinogenic type in all CIN lesions detected in multiple biopsies. HPV16 was identified as the causal type of HGCIN in all women with HPV16 in cytology, while a large proportion of HPV genotypes found in the smear was not detected on the tissue level. Conclusions: Tissue-based genotyping and LCM-PCR analysis can attribute an individual HPV type to each area of CIN. Our data show that HPV16 is even more etiologically dominant than previously thought based on various genotype attribution models.
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12 | Clinical aspects of HPV testing P-12.09

EVALUATION OF PAPTYPE HPV ASSAY IN WOMEN WITH ABNORMAL PAP
S N Tabrizi, The Royal Women's Hospital, Parkville, AUSTRALIA M P Stevens, The Royal Women's Hospital, Parkville, AUSTRALIA M A Devitt, Mobius Medical Pty Ltd, Sydney, AUSTRALIA Z Khan, Genera Biosystems, Melbourne, AUSTRALIA C Chow, Genera Biosystems, Melbourne, AUSTRALIA S M Garland, The Royal Women's Hospital, Parkville, AUSTRALIA Background: The role of HPV DNA testing is being considered in the follow-up of patients recently treated for precancerous lesions in order to identify women with residual disease and or recurrence. The aim of this study was to evaluate performance of a new detection and genotyping assay in comparison to commercially available Hybrid Capture 2, Roche Amplicor and Linear Array. Objective: To evaluate the performance of the novel PapType HPV test for the detection and identification of highrisk (HR) HPV genotypes by comparison with Hybrid Capture 2 (HC2), Amplicor (Amp) and Linear Array (LA) HPV tests in a high risk population. Methods: Eight hundred and ninety-four endocervical brush specimens collected in PreservCyt (liquid-based) from women undergoing management for a high-grade Pap abnormality were evaluated. DNA extracts from each clinical specimen were tested by PapType HPV test and results compared with HC2, Amp and LA HPV test results, in addition to histological diagnosis of a contemporaneously collected biopsy. Results: The sensitivity in detection of underlying high-grade histological diagnosis by PapType was 90.3% (95% CI, 88.4-92.3%), for HC2 79.8% (95% CI, 77.2-82.5%), whilst for Amp and LA HPV 92.4% (90.6% - 94.1%) and 91.6% (95% CI, 89.8% - 93.5%) respectively. Detection of any of the 13 HR genotypes (for 884 specimens) by PapType showed a concordance of 84.5% ( = 0.638) when compared to HC2, 94.6% ( = 0.858) and 95.9% ( = 0.895) for both Amp, and LA. Conclusions: The performance of PapType was equivalent to the Amp and LA HPV tests for HR HPV detection. PapType identified more underlying histologically-confirmed high-grade lesions than the HC2 HPV test, with the added advantage of identifying specific HPV genotype(s) present.
Declaration of interest MA Devitt is the medical director of Mobius Medical Pty Ltd who is retained as a Consultant Medical Director by Genera Biosystems Limited. SN Tabrizi and SM Garland are both on the technical advisory board of Genera. ZA Khan and C Chow are employees of Genera Biosystems.
P-12.10
SELF-COLLECTED HR-HPV SPECIMENS ARE AS SENSITIVE AS PHYSICIAN COLLECTED SAMPLES
J Belinson, Preventive Oncology International / Cleveland Clinic, Cleveland Heights, UNITED STATES, H Du, Peking University Shenzhen Hospital, Shenzhen, P.R. China, B Yang, Cleveland Clinic, Cleveland, United States, R Wu, Preventive Oncology International, Cleveland Heights, United States, X Qu, Preventive Oncology International, Shenzhen, P.R. China, R Pretorius, Kaiser Permanente, Fontana, United States, X Yi, BGI Shenzhen, Shenzhen, P.R. China, P Castle, American Society of Clinical Pathology, Washington, United States Background: Our prior work defined the need for the collection of more cells selectively from the cervix and upper vagina in order to increase the clinical sensitivity of self-collected specimens for precancerous lesions. We also had noted that by using a PCR-based assay self-collection was highly sensitive for precancerous lesions. Therefore, we studied a self-sampling device designed to obtain a larger specimen from the upper vagina and a more sensitive PCR-based HR-HPV assay. Materials and Methods: 10,000 women were screened with cervical cytology and HR-HPV testing of vaginal self-collected and endocervical physician-collected specimens. Women were randomly assigned to use either a novel self-collection device (POI/NIH self-sampler) or conical-shaped brush (Qiagen). Self-collected and clinician-collected specimens were assayed by Cervista (Hologic) and a research-only PCR-based MALDI-TOF. Women with any abnormal screening test underwent colposcopy and biopsy. Results: 8,556 women, mean age of 38.9 years, had complete data; 1.6% had CIN 3. For either HR-HPV assay, the sensitivity was similar for the two self-collection devices. Tested with Cervista, the sensitivity for CIN 3 of self-collected specimens was 70.9% and for endocervical specimens was 95.0%. (p= .0001). Tested with MALDI-TOF the sensitivity for CIN 3 of self-collected specimens was 94.3% and for endocervical specimens was also 94.3%. (p=1.0). Conclusion: Clearly the critical element in the performance of self-collection is the assay. PCR-based assays (Linear Array in our prior studies and MALDI-TOF in the current study) appearsto have an analytical sensitivity that is optimally balanced for self-collected samples. In this trial, combining a self-collected sample with MALDI-TOF with high-throughput capacity achieved similar sensitivity as a direct endocervical specimen obtained by a physician. Large population based screening events in low resource settings could be achieved by promoting self-collection and centralized high-throughput, low-cost per case testing.
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Clinical aspects of HPV testing | 12 P-12.11

A CLINICAL TRIAL OF THE CERVISTA 16/18 HPV ASSAY
J Belinson, Preventive Oncology International / Cleveland Clinic, Cleveland Heights, UNITED STATES R Wu, Peking University Shenzhen Hospital, Shenzhen, P.R. China B Yang, Cleveland Clinic, Cleveland, United States S Belinson, Preventive Oncology International, Cleveland Heights, United States X Qu, Preventive Oncology International, Shenzhen, P.R. China R Pretorius, Kaiser Permanente, Fontana, United States Background: It has been suggested and recommended that using a specific 16/18 assay as a secondary screen would be an excellent method of increasing the specificity of primary HR-HPV screening. Methods: From the SHENCCAST II trial women were eligible if they were 25-59, not pregnant, no cervical cancer screening for 3 years, no hysterectomy, and no pelvic radiation. 9179 women with primary HR-HPV testing and who returned for colposcopy and biopsy if indicated are analyzed. Results: Sensitivity and Specificity for CIN 3+ (%) (C.I.): PRIMARY SCREENING HYBRID CAPTURE II 97.9 (94.0 -99.6), 87.8 (87.1- 88.5); CERVISTA HR-ASSAY 95.1 (90.0 - 98.0), 90.3 (89.6 - 90.9); CYTOLOGY ASCUS (I2 imager assisted) 88.7(82.2, 93.4), 89.2 (88.6, 89.9); CERVISTA 16/18 ASSAY 52.4(44.0-60.8), 98.3(98.0-98.6). PRIMARY/SECONDARY SCREENING - CERVISTA HR ASSAY (n=968/9179) / CERVISTA 16/18 (n=191) 51.7% (43.3, 60.1),98.7% (98.5, 98.9); CERVISTA HR ASSAY and NLM CYTOLOGY (n=458) / CERVISTA 16/18 (n=191) 61.5% (31.6, 86.1), 89.6% (86.5, 92.3); CERVISTA HR ASSAY and ASCUS-US CYTOLOGY (n=217) / CERVISTA 16/18 (n=39) 52.6% (28.9,75.6), 85.4% (79.6, 90.1); ASCUS-US (n=649/9179)/ CERVISTA 16/18(n=39)6.9% (3.4, 12.3), 99.7% (99.5, 99.8) Conclusion: Whichever primary test is used you significantly improve specificity by secondary screening with the Cervista 16/18 assay. The fact that world-wide 70% of the cervical cancers are either 16 or 18 positive makes this a relevant strategy to consider even with the loss of sensitivity for CIN3.
Declaration of interest Investigator initiated study funded by a Grant from Hologic Inc.
P-12.12
HPV-MRNA DETECTION IN PATIENTS WITH HPV INFECTIONS
M Rongioletti, San Giovanni Calibita Hospital, Rome, ITALY, F Papa, San Giovanni Calibita Hospital, Rome, ITALY, M B Majolini, San Giovanni Calibita Hospital, Rome, ITALY, C Vaccarella, San Giovanni Calibita Hospital, Rome, ITALY, M C Giustiniani, San Giovanni Calibita Hospital, Rome, ITALY, U Rasulo, San Giovanni Calibita Hospital, Rome, ITALY, B D'Andrea, San Giovanni Calibita Hospital, Rome, ITALY, P Pasqualetti, Fatebenefratelli Association For the Research, Rome, ITALY, R Mari, San Giovanni Calibita Hospital, Rome, ITALY, G M Liumbruno, San Giovanni Calibita Hospital, Rome, ITALY Background. HPV is associated with almost all cervical cancerous lesions. To date, the HPV-DNA testing is recommended, but data from previous studies suggest that high-risk HPV mRNA testing may distinguish clinically relevant from irrelevant high-risk HPV infections. Objectives. This retrospective observational study was carried out for the comparative evaluation of the clinical utility of the HPV-Proofer assay results by evaluating the possible correlation between this test, the DNA HPV-test and pap smear results. Methods. 362 samples were tested by: conventional pap smear; HPV-DNA test and typing (Innogenetics N.V. Belgium); E6/E7-mRNA expression from the carcinogenic HPV types 16, 18, 31, 33, 45 (PreTect HPV-Proofer assay, NorChip, Italy). Data obtained were correlated through K-statistic value with the aim of identifying possible significant associations. Results. Pap-smear: negative (NEG) = 248 (68.5%); atypical cells of undetermined significance (ASCUS) = 50 (12.4%); low-grade squamous intraepithelial lesion (LSIL) = 36 (9.9%); high-grade squamous intraepithelial lesion (HSIL) = 28 (7.7%). The HPV-DNA test was positive in 192/362 (53.04%) samples; the HPV-mRNA test was positive in 80/362 (22.10%) samples. In addition, the HPV-DNA test was positive in 110/248 (44.4%) of NEG, 37/50 (74.0%) of ASCUS, 22/36 (61.1%) of LSIL and 23/28 (82.1%) of HSIL; the E6/E7-mRNA test was positive in 25/248 (10.1%) of NEG, 17/50 (34.0%) of ASCUS, 15/36 (41.7%) of LSIL and 23/28 (82.1%) of HSIL. We followed a subgroup of 39 patients for two years : at baseline 16/39 were mRNA negatives and LSIL to the cytology test and became cytologically negatives, whereas 4/39 were mRNA positives and ASCUS/LSIL and became HSIL. Colposcopy and biopsy confirmed these data. Conclusions. The detection of the rate of activity of the oncogenes E6 and E7 of high-risk HPV might be used as a second level tool to distinguish patients with transient or persistent HPV infection.
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HUMAN PAPILLOMAVIRUS GENOTYPES AMONG WOMEN ATTENDING THE UKUROVA UNIVERSITY HOSPITAL
F Yarkn Yldrm, University of ukurova, Adana, TURKEY D Yldrm, University of ukurova, Adana, TURKEY A B Gzel, University of ukurova, Adana, TURKEY B eflek, University of ukurova, Adana, TURKEY D Gmrdl, University of ukurova, Adana, TURKEY M A Vardar, University of ukurova, Adana, TURKEY Background: Human papillomavirus (HPV) is the primer etiologic agent of the cervical cancer. Objectives: To detect the prevalence of HPV genotypes among women admitted to Gynecology Clinic of Hospital of Faculty of Medicine, ukurova University, Adana, Turkey. Methods: A total of 450 cervical smear samples were collected from women aged 30 years and above. HPV genotyping was performed by the f-HPV typing Kit (Molgentix, Barcelona, Spain) which is multiplex flourescent PCR test. Results: The prevalence of HPV was found to be 16.2% (73) in the study group. Among HPV DNA positive women, 62 (13.8%) had single high risk HPV infection and 11 (2.4%) had multiple HPV infection. The most common HPV genotype was HPV 39 (26.4%), followed by HPV 51 (17.2%), HPV 16 (16.1%), HPV 52 (10.3%), HPV 18 5.7%), HPV 35 (5.7%), HPV 68 (4.6%), HPV 56 (3.4%), HPV 58 (3.4%), HPV 31 (2.3%), HPV 45 (2.3%), HPV33 (1.2%) and HPV 59 (1.2%). Conclusions: The detection of distribution of HPV genotypes among women is important to evaluate the effectiveness of HPV vaccines and will contribute to the development of HPV vaccine strategies.
P-12.14
HPV ONCOGENE MRNA-ANALYSIS FOR DIAGNOSING ANAL DYSPLASIA IN HIV-INFECTED MEN
S Silling, University of Cologne, Kln, GERMANY A Kreuter, Ruhr-University Bochum, Bochum, Germany N H Brockmeyer, Ruhr-University Bochum, Bochum, Germany J Swoboda, Institute of Cytology, Bonn, Germany H Pfister, University of Cologne, Kln, Germany U Wieland, University of Cologne, Kln, Germany Background: Anal HPV-infection and anal dysplasia are very frequent in HIV-positive men who have sex with men (HIV+MSM), and progression of low-grade (LSIL) to high-grade lesions (HSIL) occurs faster than in HIV-negative persons. Detection of high-risk (HR)-HPV-E6/E7 oncogene-mRNAs has been reported to have higher specificity and positive predictive value (PPV) for the detection of high-grade cervical intraepithelial lesions compared to HR-HPV-DNA testing. Objectives: We have evaluated the APTIMA HPV Assay (Gen-Probe Incorporated) for diagnosing anal dysplasia in HIV+MSM. Methods: So far, 239 intra--anal swabs from HIV+MSM participating in a screening program including anal cytology, histology, and high-resolution anoscopy were analyzed. HR-HPV-DNA detection and typing were performed by PCR and hybridization with 18 HR type-specific probes using a bead-based multiplex genotyping assay. E6/7mRNA detection of 14 HR-HPV-types was performed using the APTIMA HPV assay. Results: 236/239 swabs had valid results in both test-formats (105 normal, 12 ASCUS, 89 LSIL/AIN1, 30 HSIL/AIN2-3). For the detection of ASCUS+-lesions (ASCUS+LSIL+HSIL) sensitivity, specificity, negative predictive value (NPV) and PPV were 87.0%, 26.7%, 62.2%, and 59.7% for HR-DNA-testing, compared to 81.7%, 40.0%, 63.6% and 62.9% for E6/ E7-mRNA-testing. The respective values for the detection of LSIL+ (LSIL+HSIL) were 86.6%, 24.8%, 64.4%, 53.9% for HR-HPV-DNA-testing and 82.4%, 38.5%, 68.2%, 57.6% for E6/E7-mRNA-testing. For the detection of HSIL, the values were 100%, 21.8%, 100%, 15.7% for HR-HPV-DNA-testing and 93.3%, 31.1%, 97.0%, 16.5% for E6/E7-mRNA-testing, respectively. A shift of the cut-off of the Aptima assay (from 1 to 5 S/CO) did not affect sensitivity for the detection of HSIL and slightly improved specificity (48.6%/46.2%/36.4%) and PPV (66.0%/60.4%/17.6%) for the detection of ASCUS+/LSIL+/HSIL, respectively. Conclusions: HPV oncogene mRNA analysis has an increased specificity, similar sensitivity and NPV, and a slightly increased PPV for the detection of anal dysplasia in HIV+MSM compared to HR-HPV-DNA-testing.
Declaration of interest APTIMA HPV mRNA test kits were provided by Gen-Probe GmbH, Wiesbaden, Germany
34

RELIABILITY AND REPRODUCIBILITY OF THE HIGHLY SENSITIVE SPF10/LIPA-HPV DETECTION METHOD
A King, RIVM, Bilthoven, NETHERLANDS E van Logchem, RIVM, Bilthoven, Netherlands H Vriend, RIVM, Bilthoven, Netherlands M Fetlkamp, RIVM, Bilthoven, Netherlands H Boot, RIVM, Bilthoven, Netherlands Introduction: To monitor the prevalence of specific HPV types in the general population of adolescents and young adults in the Netherlands, several HPV prevalence surveys were initiated in 2009. In one of these studies, young STD-clinic visitors (n=1800, age 16-24) were asked to collect anogenital-samples at 12 different STD clinics in the Netherlands. HPV detection and -genotyping was performed in 7 different medical laboratories. Here we evaluated the reliability and reproducibility of the SPF10/LiPA type-specific HPV testing method performed in the different laboratories. Methods: A collection of anogenital self-swabs from 1800 STD-clinic visitors (female & male; age = 16 - 24) from 12 STD-clinics was tested for the presence of HPV. HPV-DNA in swabs was determined in 7 different laboratories including the RIVM using a uniform PCR-based protocol (SPF10/LiPA, version 1, DDL) with high analytical sensitivity. A standardized protocol for spiking (PhHV-1), extraction (MagnaPure 32, Roche), amplification (SPF10), detection (DEIA) and genotyping (LiPA) was used in all laboratories. Technicians were trained to perform the assay uniformly. To test for accuracy among the different laboratories 5% of both the HPV-positive and -negative DNA extracts were retested at the RIVM. Furthermore, the RIVM laboratory participated in the 2010 WHO HPV Labnet Profiency panel. Results: The reproducibility of HPV detection in STD samples was high, 100% of positive HPV-DEIA STD-samples were positive on DEIA-retesting and 82.6% of the DEIA negatives were negative on DEIA-retesting. Of the 146 HPVtypes detected in 61 samples, 126 were found on retesting (86%). Non-matching results were often (3/7) associated with new DNA isolation. Partly matching results were almost exclusively found in samples with multiple infections (avr=3.8 HPV types). Conclusions: SPF10/LiPA detection of HPV in anogenital self-swabs is a reliable and reproducible assay to determine the prevalence of high risk HPV types in a multicentre surveillance study.
P-12.16
HPV TEST QUALITY CONTROL IN A SCREENING SETTING IN ITALY
F Carozzi, Institute of Cancer Prevention ISPO, Florence, ITALY C Sani, ISPO, Florence, italy E Burroni, ISPO, Florence, italy C D Q quality assurance group, italy Objective Aim of this study is to evaluate the reproducibility of HPV test in a quality control program, involving several different laboratories. Methods The setting involved several laboratories with different previous experiences processing a high number of samples. Therefore, it approached the conditions of a field application of HPV testing in mass screening for cervical cancer. Two different panel of samples (five 100 l aliquots of clinical blinded samples collected in STM Solution and three 2ml aliquots of clinical blinded PreservCyt samples each time) were sent from Florence laboratory to the other participating centres. The blinded samples were used as external control samples and processed by each laboratory in duplicate in two different sessions in the same way as the screening samples. Results and Conclusions In these two mailing, 30 test sets were sent out, and valid answers were received from 29 laboratories by the due date. Results were sent to Florence laboratory and data were classified as positive and negative or indeterminate for HPV. Twenty-seven participants (90%) used the Hybrid Capture Digene method and two (10%) Cobas 4800 System Roche. High consensus (> 90%) was achieved with twelve samples across all methods. In contrast four samples, that essentially fell into the high titer negative or low titer positive range, showed much less uniformity. This Proficiency Test for human papilloma virus (HPV) determination. was designed to monitor the accuracy, reproducibility and detection steps in each laboratory. Preliminary data indicate high accuracy. In conclusion, our study stresses the importance of using standardized protocols and proficiency panels for the comparability of results among laboratories involved in screening programme.
Declaration of interest F Carozzi participated occasionally in advisory board of GSK, Sanofi-Pasteur MSD, Abbott, Geneprobe. None declared by other authors
35

COMPARISON OF COBAS AND HC2-HPV-TESTING IN A GERMAN ROUTINE LABORATORY
H Ikenberg, Cytomol, Laboratory for Cytology and Molecular Biology, Frankfurt, Germany T Iftner, Section for Experimental Virology, Institute for Virology, University Tbingen, Tbingen, Germany C Brsch, Cytomol, Laboratory for Cytology and Molecular Biology, Frankfurt, Germany B Pittel, Cytomol, Laboratory for Cytology and Molecular Biology, Frankfurt, Germany A Xhaja, Cytomol, Laboratory for Cytology and Molecular Biology, Frankfurt, Germany F Britz, 3Roche Diagnostics, Mannheim, Germany Background: Up to now the Digene HC2 test (Qiagen, Hilden, Germany) is regarded the gold standard for human papillomavirus (HPV) DNA testing. Meanwhile several new test systems for HPV are available, among them the cobas HPV assay (Roche Diagnostics, Mannheim, Germany [Roche]) which allows simultaneous genotyping for HPV 16 and 18. Objective: To compare the performance of the cobas HPV with the HC2 assay in cervical samples collected with the PreservCyt collection medium (Hologic, Frankfurt, Germany). Methods: 1781 anonymized routine specimens pretested with the HC2 test were available for analysis with cobas HPV. Cases with discrepancies between the two tests were retested with the Linear Array HPV genotyping test (LA) (Roche). Partially, histologic diagnoses were available. Results: In 1566 (87.9%) of the cases HPV results were concordant. Of the 215 cases (12.1%) with discrepancies LA results are available in 214 cases. 94 cases were LA-negative: 13 of 105 cobas-pos/HC2-neg and 81 of 99 cobas-neg/ HC2-pos cases. 110 cases were LA-positive: 92 of 105 cobas-pos/HC2-neg and 18 of 99 cobas-neg/HC2-pos cases. 325 cases with histologically confirmed CIN2+ were included. In 261 out of 293 cases (89.1%) where a HC2 result was available this was positive, while 298 out of 318 of the cases (93.7%) tested with cobas HPV were positive. The rate of HPV positivity in cytologically normal cases and in HSIL was slightly higher with cobas HPV, while it was in the same range in ASC-US and in LSIL cases. With increasing severity of the cytologic findings the rate of HPV 16 and 18 positivity increased proportionally. Conclusion: In routine specimens from a German commercial laboratory the cobas HPV test showed similar performance compared to the HC2 test. Preliminary data point to a potentially higher sensitivity and specificity with cobas HPV while adding information by HPV 16 and 18 genotyping.
Declaration of interest Potential conflicts of interest: occasional adviser to and/or speakers honoraria/travel grants from Abbott, B&D, Genprobe, GSK, Hologic, MTM, Qiagen, Roche, SPMSD
P-12.18
HPV16/18/45 GENOTYPING BY THE DIGENE HPV GENOTYPING PS TEST.
L Ho, Queen Mary University of London, London, UNITED KINGDOM, G Terry, Queen Mary University of London, London, UNITED KINGDOM, W Chen, QIAGEN, Gaithersburg, USA, A Shrestha, QIAGEN, Gaithersburg, USA, A Szarewski, Queen Mary University of London, London, UNITED KINGDOM, L Cadman, Queen Mary University of London, London, UNITED KINGDOM, J Austin, Queen Mary University of London, London, UNITED KINGDOM, H a Thai, QIAGEN, Gaithersburg, USA G Roeder, QIAGEN, Gaithersburg, USA, J Cuzick, Queen Mary University of London, London, UNITED KINGDOM Background. The digene HC2 High-Risk HPV DNA Test (HC2) which detects 13 HR HPV genotypes has high sensitivity but relatively low specificity for high grade intraepithelial neoplasia (CIN2+). Objectives. We evaluated triage of the HC2 positive PreservCyt specimens for the 3 most oncogenic genotypes, HPV16, HPV18 or HPV45, for improved specificity using the digene HPV Genotyping PS test (based on hc2 technology, RUO). Methods. 140 specimens collected from women following an abnormal smear were tested for HR HPV DNA using HC2. Positive specimens were further genotyped by the PS test. DNA was extracted from unconverted PreservCyt HC2 positive specimens and genotyped by qPCR. Results. 123 of 140 specimens were positive by HC2. Of these 36, 7 and 2 were positive for HPV16, HPV18, HPV45 respectively and 42 for any of these genotypes (cut-off=5000 copies/genotype/2ml PreservCyt). There was 100% agreement between PS and qPCR for the detection of HPV16 and HPV45. Three specimens had discrepant HPV18 results. Two were missed by PS but positive by qPCR. One had no CIN on biopsy and one was not biopsied (assumed normal). HPV viral load for these 2 specimens was 2.5x105 and 1.4x104 copies per 2ml PreservCyt respectively. One specimen positive by PS but negative by qPCR had CIN1. In this cohort, the positive predictive value (PPV) for CIN2+ by HR HPV was 30.1%. Triage based on HPV16/18/45 resulted in PPVs for HPV16 of 55.6% (p=0.0001;OR=5.0 8;95%CI=1.98-13.03) and for 18/45 combined of 33.3% (p=0.34) compared to 19.8% for women positive for HR HPV other than these genotypes. Conclusion. The digene PS test demonstrated good concordance with qPCR. Detection of HPV16 is a useful adjunct for predicting CIN2+ in HC2 positive specimens. The role of HPV18/45 requires further exploration, but may be more relevant for detecting occult lesions in the endocervical canal.
Declaration of interest Jack Cuzick is on the Speakers Bureau at Qiagen.
36

TAMPONS AND SELF-VAGINAL SWABS ARE VIABLE SCREENING ALTERNATIVES FOR HPV
N Nelson, Louisiana State University Health Sciences Center, New Orleans, UNITED STATES K Sutton, Louisiana State University Health Sciences Center, New Orleans, UNITED STATES W Nichols, Louisiana State University Health Sciences Center, New Orleans, UNITED STATES A Clark, Louisiana State University Health Sciences Center, New Orleans, UNITED STATES H Oddo, Louisiana State University Health Sciences Center, New Orleans, UNITED STATES N Love, Louisiana State University Health Sciences Center, New Orleans, UNITED STATES M Hagensee, Louisiana State University Health Sciences Center, New Orleans, UNITED STATES Background: Human Papillomavirus (HPV) is the most common sexually transmitted disease and also causes cervical cancer. Studies have shown that a single HPV DNA test is more sensitive to predict cervical pathology than a single cervical pap smear. If validated, self-collected genital samples could be effective for large scale cervical cancer prevention. Objectives: Three different less-invasive sampling techniques were compared to determine if they were concordant with the HPV results obtained from the cervical sample obtained by a health-care provider. Methods: A self-collected urine, tampon, and vaginal swab sample were obtained from HIV+ women and compared to a provider obtained cervical swab. These women were enrolled in a natural history HPV study. The presence of human DNA was determined from isolated cellular DNA by PCR for the beta-globin gene, HPV was detected using the PGMY primers targeting the L1 gene and genotype determined by reverse line blot. Results: All 4 samples were obtained for 55 visits representing 34 women. The cervical swabs, self-vaginal swabs, and tampons had higher amplification rates as compared to the urine specimens. In a paired analysis utilizing the HPV status of the provider-obtained cervical swab as the gold standard, the self-swab, tampon, and urine samples were 85%, 68%, and 58% concordant, respectively. Similar concordance rates were found when restricting the analysis to only high-risk HPV types. Conclusions: HPV DNA can be detected by all of the sampling methods. The tampons and self-vaginal swabs are more concordant with the cervical samples than the urine samples. Further studies are ongoing to validate these findings in larger cohorts of HIV+ and HIV-negative women.
P-12.20
DIFFERENT PERSISTENT HRHPV GENOTYPES IN CERVICO-VAGINAL PRE-NEOPLASTIC LESIONS: A CASE-REPORT
J van der Marel, DDL Diagnostic Laboratory, Voorburg, NETHERLANDS W G V Quint, DDL Diagnostic Laboratory, Voorburg, NETHERLANDS J. Lindeman, DDL Diagnostic Laboratory, Voorburg, NETHERLANDS F M Smedts, Reinier de Graaf Groep, Delft, NETHERLANDS T J M Helmerhorst, Erasmus MC, Rotterdam, NETHERLANDS Background: Up to 50% of women with CIN, irrespective of grade, are reported to be infected with multiple HPV types. Evidence suggests that multiple HPV infections and persistent high-risk HPV infection highlights individuals at greater risk for disease progression. A drawback of studies investigating multiple HPV infections is that they give an overall picture of the HPV types present, and no information of HPV infection at a cellular level. Utilization of laser capture microdissection (LCM), as done in this study, enables examination of HPV in subpopulations of cells under direct microscopic visualization. Objectives: To designate the responsible HPV genotype for recurrent and persistent CIN and VAIN in a patient with a history of cervico-vaginal disease. Methods: In a 41 year old woman, treated and surveyed yearly for high-grade cervico-vaginal lesions for 9 years, archival cytology samples and biopsies were taken for HPV analysis using the HPV SPF10/LiPA25 (version 1) system. Microscopically selected regions of CIN, normal tissue and VAIN were isolated by LCM and analyzed by the same PCR system. Results: Eight cervical smears performed between 2003 and 2011 showed multiple HPV genotype infection. HPV33,44,51,55 and 82 were persistently present in several smears, while type 31 and 52 showed recurrence. Whole tissue analyses on biopsy material also showed multiple HPV infections, and LCM-PCR assigned HPV33 to a CIN2 and VAIN2 lesion in 2003. HPV67 was found in a CIN2 lesion in 2005. Regions from borderline CIN/VAIN lesions and normal tissue were negative for HPV. Conclusions: This case-report describes a patient in whom multiple persistent HPV types were found in cytology. Assignment toward a specific HPV type per lesion was done using LCM-PCR and we found a switch from HPV33 to 67 on lesional level, while HPV33 continued to persist in cytology and whole tissue sections.
37

QUANTIFICATION OF CERVICAL HPV DNA AND RNA OF CIN1/CIN2 PATIENTS
R Schubbert, Eurofins Medigenomix GmbH, Ebersberg, GERMANY S Hoffmeyer, Eurofins Medigenomix GmbH, Ebersberg, GERMANY S Rittler, Eurofins Medigenomix GmbH, Ebersberg, GERMANY S Zorbas - Seifried, Eurofins Medigenomix GmbH, Ebersberg, GERMANY E Amtmann, DKFZ, Heidelberg, GERMANY M Schulz, Lumavita AG, Basel, SWITZERLAND Background: No therapeutic alternatives to surgery of advanced cervical HPV lesions are available. A validated quantitative PCR detection method for HPV DNA and RNA must be developed in order to monitor the early therapeutic antiviral activity of intra-vaginal drug treatment. The phosphatidylcholine specific phospholipase C (PC-PLC) inhibitor LMV-601 acts on HPV transcription and has just entered clinical studies with topical application. Objective: Develop and validate a quantitative Real Time PCR method for the detection of HPV-16 and HPV-18 E7 DNA and RNA in clinical studies with topical treatment of cervical HPV infections. Methods: Primer-probe systems specific for HPV-16 and HPV-18 E7 DNA and RNA were established. Assay set-up was performed by using the HPV-16 (CaSki) and HPV-18 (HeLa) cell lines. Amongst other GAPDH and ApoB were used as human reference genes. An internal control plasmid was designed containing specific HPV-16 and HPV-18 sequences, as well as defined sequence regions of reference genes. Cervical swabs were received from CIN1 and CIN2 patients diagnosed positive for HPV-16 and/or HPV-18. Results: A quantitative RT PCR method was set up with comparable sensitivity and limit of detections for HPV-16 and HPV-18. Viral DNA and RNA content of known HPV cell lines were confirmed. No influence of the foreseen drug formulation on assay performance could be detected. Data will be presented from the assay validation by studying cervical swabs from patients positive for HPV-16 and/or HPV-18. Conclusion: A validated method has been established for quantitative analysis of HPV-16 and HPV-18 RNA expression and viral DNA load. This method will be applied for monitoring the antiviral efficacy of topical treatment of cervical HPV infections in clinical studies. A respective study is ongoing with the PC-PLC inhibitor LMV-601, which inhibits effectively HPV transcription.
38
Session 13: HPV related benign diseases

Chair: Reinhard Kirnbauer
Poster abstracts
HPV related benign diseases | 13 O-13.00

Reinhard Kirnbauer
FOR YOUR NOTES:
O-13.01
HUMAN PAPILLOMAVIRUS IN ORAL ATROPHIC LICHEN PLANUS LESIONS
J Rautava, University of Turku, Turku, FINLAND R Mattila, University of Turku, Turku, FINLAND S Syrjnen, University of Turku, Turku, FINLAND BACKGROUND: Oral lichen planus (OLP) is a potentially premalignant disorder in which human papillomavirus (HPV) DNA detection has been significantly higher (OR=5.12; 95% CI:2.40-10.93) than in normal oral mucosa. OBJECTIVES: The present study assessed the HPV genotype distribution in atrophic OLPs and its association with DNA content and repair, proliferation activity, apoptosis, epithelial adhesion and lymphocyte cell infiltration. METHODS: A series of 69 patients (74.2% women) with OLP were studied with a mean FU-time of 62.4 months. OLPs fulfilled the WHO-criteria and histological diagnosis was re-evaluated from formalin-fixed paraffin-embedded samples. DNA was extracted with high salt method and HPV-genotyping was made with Luminex-based assay detecting 24 HPV genotypes. Earlier, a large panel of immunohistochemical markers and static cytometry were performed from these samples, data now compared with HPV data. RESULTS: HPV DNA was found in 15.9% of OLPs, including the following genotypes: HPV6, 11, 16, 31 and 33 and one multiple-type infection. Two of the five patients who developed cancer had low-risk HPV6/11 infection and three were HPV-negative. There was a statistically significant correlation between HPV DNA in OLP and DNA content and ploidy markers determined with static cytometry: in the HPV-positive samples, the proliferation index was higher (p=0.016), the nuclei were larger (p=0.033), cells were less in resting state G1/G0 (p=0.021) and more often in the S-phase (p=0.036) than the epithelial cells in HPV-negative lesions. HPV positivity was also related to Topoisomerase-II- (p=0.051), Caspase-3 (p=0.049) and CD20 (p=0.014) expression. CONCLUSIONS: HPV DNA was found in atrophic OLPs. Static cytometry is a sensitive method to identify HPVassociated changes in cellular DNA content and proliferation. Of all 15 markers, only topoisomerase-II (proliferation and DNA repair), Caspase-3 (apoptosis) and CD20 (B-lymphocytes) were related to HPV. None of the HR-HPVs but LR-HPVs were associated with the lesions developed to cancer.
Declaration of interest No conflict of interest.
41
13 | HPV related benign diseases O-13.02

LARYNGEAL PAPILLOMATOSIS: IMMUNOLOGICAL AND CLINICAL RESPONSES TO HPV AND VACCINATION
T Fromm, Charit Berlin, Berlin, GERMANY K Stlzel, Charit Berlin, Berlin, Germany M Gross, Charit Berlin, Berlin, Germany L Schdlich, Charit Berlin, Berlin, Germany A M Kaufmann, Charit Berlin, Berlin, Germany A Albers, Charit Berlin, Berlin, Germany Objective: Recurrent respiratory papillomatosis (RRP) is a sometimes life threatening disease of the larynx that is associated in >90% with low-risk HPV6 and 11. Juvenile and adult forms are known and standard treatment is surgical ablation to open the airway. However, this treatment rarely cures the disease. We vaccinated 5 patients with Gardasil and observed the clinical and immunological effects. Patients and Methods: Fife patients between age 5 and 64 with persistent and recurrent RRP were vaccinated with Gardasil after surgical removal of RRP. Treatment intervals and extent of surgery were recorded. Peripheral blood was analysed for immune responses to HPV 6, 11, 16, and 18 L1 antigens. Serologic responses were investigated by VLP capture ELISA and ex vivo memory CD4 T cell quantitation of antigen specific cytokine (IFN-gamma, IL-2) and CD154 cellular responses was done by flow cytometry. Results: Patients had reduced regrowth of papillomas and treatment intervals increased. All vaccinees developed or boosted antibody responses to L1 and developed significant CD4 T cell responses that increased during the course of vaccination. Conclusion: Vaccination of RRP patients with Gardasil showed in the observed cases no adverse effects and was immunogenic as shown by the induction of high specific antibody titres and T cell frequencies. Post-vaccination, the frequency of operations was clearly reduced in some patients, however it remains unclear if this effect will last. Although designed as a prophylactic vaccine there may be a beneficial effect also in established RRP in combination with surgery.
O-13.03
JUVENILE RESPIRATORY PAPILLOMATOSIS RISK FACTOR FOR SEVERITY
C Rodier, Department of Social and Preventative Medecine, University of Montreal. /Merck Canada. , Montreal, Canada A Lapointe, Department of Otorhinolaryngology, St-Justine Hospital, University of Montreal. , Montreal, Canada D Dal Soglio, Departement of Pathology, St-Justine Hospital, University of Montreal. , Montreal, Canada M H Mayrand, Departement of Obstetric and Gynecology, St-Luc Hospital, University of Montreal./ Department of Social and Preventative Medecine, University of Montreal., Montreal, Canada F Coutlee, Department of Microbiology and Infectious Diseases, Notre-Dame Hospital, University of Montreal., Montreal, Canada M Roger, Department of Microbiology and Infectious Diseases, Notre-Dame Hospital, University of Montreal, Montreal, Canada H Trottier, Department of Social and Preventative Medecine, University of Montreal. , Montreal, Canada Background: Juvenile-Onset Recurrent Respiratory Papillomatosis (RRP) is mainly caused by HPV genotypes 6 or 11 and often acquired vertically from an infected mother. It is characterised by recurring warts most commonly in the larynx. The severe form of the disease has a devastating impact on the health and the quality of life of the child and its family because of the consequences of multiple surgical procedures and the constant risk of airway obstruction. Objectives: To study the determinants of severe RRP. Method: We conducted a retrospective case series of the cases of RRP diagnosed between January 1995 and December 2008 in a pediatric tertiary care hospital. We analyzed demographic variables abstracted from medical charts. Viral factors (HPV genotyping by consensus L1 PCR, type-specific HPVviral load by real-time PCR, molecular HPV variant analysis by PCR-sequencing) were assessed on paraffin-embedded biopsies. We contacted the patients to get a saliva sample and evaluated the DQB1 and DRB1 HLA class II alleles. We had accessed to the delivery chart and a questionnaire was filled by the mother. Multivariate logistic regression was performed to evaluate the impact of each of independent variables on the disease severity measured with the standardized Derkay scoring system. Results: Over the study period, 26 cases of laryngeals papillomas and 5 cases of oropharynx papillomas were diagnosed. Median number of surgeries was 5 (range 1-37). Among these cases, 61.3% were classified as severe. Preliminary results from the sociodemographic variable analysis showed that the history of genital warts of the mother was significantly associated with disease severity OR 22.00 (p=0.023) but not the age of the mother OR 0.838 (p=0.154). Conclusions: RRP is a rare but very morbid disease. Further understanding of the risk factor associated with the severity may have important implications for prevention.
Declaration of interest Presenting author is working for Merck Canada.
42

HPV EPIDEMIOLOGY IN GENITAL WARTS IN COLOMBIAN MEN & WOMEN
G Hernandez - Suarez, Research & Innovation Fundation, Bogota, COLOMBIA M Pieros, Research & Innovation Fundation, Bogota, COLOMBIA L Orjuela, Research & Innovation Fundation, Bogota, COLOMBIA Background: Epidemiological studies on benign lesions related to HPV infection are scarce in the Latin American context.Objectives: To Describe the HPV genotype distribution in genital warts (GW) diagnosed in patients attending an STD consult in Bogota,Colombia. Methods: A cross sectional study in a mayor private outpatient clinic in Bogot, Colombia, was carried out to invite patients between 18 and 45 years attending due to genital lesions suspicious of GW. All patients underwent confirmation biopsy and GP5/GP6 HPV testing of GW frozen tissue. Detailed clinical and anatomic features of GWs were also collected. Results: From 352 enrolled patients, 97% (138 male and 204 female) got a satisfactory sample for pathology diagnosis and HPV testing. GW was confirmed by pathology in 81% of cases.f, 8% were diagnosed as Papillomatous hyperplasia, 4% and 6% showed chronic inflammation or no histological changes. Overall, B-globine positive rate was 84%. From 261 confirmed GW, 88% were HPV positive, being HPV6 (67%), HPV11(23%) and HPV16(4%) the most frequent gentotypes detected. Single infections were identified in 70% of cases and when it dealt with multiple infections, HPV 6/HPV11 and HPV6/HPV16, were the most frequent combinations. No difference of genotype distribution was observed across sex, age, or number of sexual partners. Women consulting for GW were younger than men (mean age 25.3 vs 29, p<0.01).Conclusions: This study indicates, as previous studies did, that HPV 6 and HPV11 are responsible of vast majority of Genital Warts. HPV16 found as single infection may indicate causative association with GW. At least 1/5 of suspected GW corresponded to diagnosis other than GW. This information is valuable as baseline data for future estimation of HPV burden in benign disease in men and women and confirms the potential benefit to be obtained by prophylactic vaccination against HPV 6 and 11 types
Declaration of interest G. Hernndez, MSD, Grant support for the present study M. Pieros, MSD, has been speaker and member of an advisory board for MSD-Gardasil
O-13.05
A LARGE SPECTRUM OF HPV-TYPES IN GENITAL WARTS
H Johansson, Lund University, Malm, SWEDEN E Sturegrd, Skne University Hospital, Malm, SWEDEN A Johnsson, Skne University Hospital, Malm, SWEDEN E Gustafsson, Skne University Hospital , Malm, SWEDEN J Dillner, Skne University Hospital, Malm, SWEDEN O Dillner, Skne University Hospital, Malm, SWEDEN Background. It has generally been reported that about 90% of genital warts (GWs) are caused by HPV6 or 11, both of which are included in the quadrivalent HPV vaccine. Objectives. In this study we analyzed the prevalence of HPV-types in clinically diagnosed GWs prior to induction of Swedish vaccination programs in order to set a baseline for future monitoring. Methods. Swab samples from 703 clinically diagnosed GWs were collected between 2006 and 2009 from patients (mean age 28.5 years) attending a STI-clinic in Malm, Sweden. Of the material, 621 samples (376 males and 245 females) were adequate for HPV-DNA analysis. The swab samples were analyzed for HPV-types using a PCR-based Luminex method, targeting 33+ genital HPV-types. HPV-DNA negative samples were subjected to extended analysis, by rolling circle amplification and FAP-PCR, in order to possibly identify additional HPV types not detectable by the Luminex method. Results. Genital HPV-types were found in 94% of the samples. Thirty-four different genital HPV-types were detected, of which HPV6 (62%), HPV16 (13%), HPV11 (10%), HPV42 (7%), HPV66 (6%), HPV51 (5%), HPV18 (5%), HPV31 (5%), HPV59 (4%), and HPV56 (3%) were most common. After extended analysis, HPV-DNA was detected in 36% (14/39) of the previously HPV-negative samples. These findings contained HPV-types and FA-isolates that belong to the Beta-, Gamma-, or Nupapillomavirus genus, including the novel HPV-types 112 and 153 of the Gammapapillomavirus genus. The isolation of HPV153 is described in this report. Conclusions. We found that 71% of the GWs were infected by HPV6 or 11, which is lower than expected. We also show that GWs are infected with a large spectrum of genital HPV-types and some cutaneous HPV types, but at least one of four HPV-types covered by the quadrivalent vaccine was detected in 79% of GWs.
Declaration of interest Annika Johnsson have participated in a HPV-vaccination trial (Merck). Joakim Dillner have recieved grants from Merck and SP MSD.
43
13 | HPV related benign diseases O-13.06

PERSISTENCE OF HPV-6 GENOMIC VARIANTS IN RECURRENT ANOGENITAL WARTS
K Fujs Komlo, Faculty of Medicine, University of Ljubljana, Ljubljana, SLOVENIA B J Kocjan, Faculty of Medicine, University of Ljubljana, Ljubljana, SLOVENIA K Seme, Faculty of Medicine, University of Ljubljana, Ljubljana, SLOVENIA M Poljak, Faculty of Medicine, University of Ljubljana, Ljubljana, SLOVENIA Background: Anogenital warts (AGW) are the most common benign neoplastic lesions in anogenital region, and are etiologically associated with infection with HPV-6 and HPV-11 in more than 90% of cases. The traditional treatment for AGW is surgical excision of warts; however the recurrence of AGW is very frequent. At present, it is not clear whether the recurrence of AGW is a result of reinfection with novel HPV variants or is it caused by persistent HPV infection. Objectives: To investigate whether the recurrence of HPV-6-linked AGW is a consequence of persistence of a single viral genomic variant or of reinfection with novel genomic variant. Methods: Fourteen HPV-6 isolates from freshfrozen tissue specimens (5 initial and 9 recurrent samples), obtained from five Slovenian patients with recurrent AGW over a two-year period, were included in the study. The number of recurrent AGW specimens (isolates) per patient ranged from 1 to 4 and the follow-up period ranged from 2 to 16 months. HPV-6 genomic variants were determined by sequencing of HPV-6 L1, E5a, E5b, and LCR genomic regions. Results: Altogether, three different HPV-6 L1-E5a-E5b-LCR genomic variants were identified among five patients with recurrent AGW, all of which corresponded to non-prototypic HPV-6a (one variant) and HPV-6vc genetic lineage (two variants). Within each of the five patients included in the study, HPV-6 L1, E5a, E5b, and LCR sequences obtained from the recurrent samples matched completely corresponding sequences obtained from the initial AGW sample. Conclusions: The presence of the same and unique HPV-6 genomic variant in initial and all recurrent AGW samples within individual patient with recurrent AGW indicates that the recurrence of AGW is a consequence of persistence of a single viral genomic variant and not of reinfection with a novel genomic variant.
O-13.07
GENOMIC DIVERSITY OF HPV GENOTYPES HPV40, HPV42, HPV43 AND HPV44
P Maver, University of Ljubljana, Faculty of Medicine, Ljubljana, SLOVENIA B Kocjan, University of Ljubljana, Faculty of Medicine, Ljubljana, SLOVENIA K Seme, University of Ljubljana, Faculty of Medicine, Ljubljana, SLOVENIA M Poljak, University of Ljubljana, Faculty of Medicine, Ljubljana, SLOVENIA BACKGROUND: Low-risk HPV genotypes HPV40, HPV42, HPV43 and HPV44 are involved in the etiology of genital warts and low-grade squamous intraepithelial lesions of cervix, vulva, vagina, penis and anus. Since they are usually not associated with malignant transformation, the knowledge about their genomic variation is extremely limited. OBJECTIVES: To perform first molecular analysis within 3 different genomic regions of HPV40, HPV42, HPV43 and HPV44, and to present the first comprehensive data on their genomic diversity. METHODS: Altogether 108 isolates (14 HPV40, 49 HPV42, 10 HPV43 and 35 HPV44) were sequenced across approximately one third of the viral genome. The identification and characterization of variants was based on the analysis of L1, LCR and E6 genomic regions. The coding regions were analyzed to determine the prevalence and distribution of amino acid changes that could have an impact on biological properties and structure of viral proteins. RESULTS: After combining nucleotide data in all 3 genomic regions for each of 108 isolates, a total of 9 HPV40, 30 HPV42, 3 HPV43 and 19 HPV44 genomic variants were identified. Several new, potentially important nucleotide substitutions, as well as amino acid changes were identified. One HPV40 LCR variant contained a 17 bp long deletion, two HPV43 variants contained 21 bp and 29 bp inserts and one 19 bp deletion, and one HPV42 E6 variant contained one 93 bp deletion which resulted in shortening of E6 protein. No other deletions or inserts identified were longer than 2 bp. CONCLUSIONS: The present study provides first comprehensive data on genomic diversity of low-risk HPV genotypes other than HPV6 and HPV11, which is potentially important for future molecular assay development, functional, epidemiological and phylogenetic studies.
44

INTRA-ANAL CONDYLOMA FREQUENTLY CONTAIN HIGH-GRADE DYSPLASIA
O Richel, Academic Medical Center, University of Amsterdam, Amsterdam, NETHERLANDS A Kreuter, Ruhr-University Bochum, Bochum, GERMANY N D L Hallensleben, Academic Medical Center, University of Amsterdam, Amsterdam, NETHERLANDS C M van Noesel, Academic Medical Center, University of Amsterdam, Amsterdam, NETHERLANDS J M Prins, Academic Medical Center, University of Amsterdam, Amsterdam, NETHERLANDS H J C de Vries, Academic Medical Center, University of Amsterdam, Amsterdam, NETHERLANDS BACKGROUND High resolution anoscopy (HRA) is increasingly advocated to screen HIV+ MSM for anal cancer and its precursor, anal intraepithelial neoplasia (AIN). However, guidelines to identify AIN are limited and often derived from studies on cervical intraepithelial neoplasia. Therefore, systematic comparison is needed between macroscopic characteristics and histopathology of such lesions. OBJECTIVE In this study we retrospectively compared photos of suspect anal lesions with their histopathological outcome. METHODS HRA was performed in 163 HIV+ MSM by a single investigator. After acetic acid application, suspect lesions were photographed and biopsies taken. All biopsies were examined by the same pathologist. Next, two HRAexperienced dermatologists from two different centres and blinded for histopathological outcome, retrospectively studied the photos. Firstly, condylomatous (papulous) lesions were identified. Secondly, non-condylomatous (flat) lesions were judged on leukoplakia, punctation, mosaicism and vessel abnormalities. RESULTS In the 163 patients 299 biopsies were taken. AIN was found in 116 patients (71%), high grade (HG) AIN in 62 (38%). On biopsy level, 167 (56%) of 299 samples showed AIN and 66 (22%) HGAIN. According to both experts 90 of 299 lesions (30%) were condyloma-like. Pathological analysis showed HGAIN in 16 (17.8 %; 95% CI 9.7-25.9). Flat leukoplakia was seen in 140, of which 38 (27.1%; 95% CI 19.6-34.6) showed HGAIN. Of lesions with punctuation (n=135) 43 (31.9%; 91% CI 23.9-39.9) showed HGAIN. A combination of flat leukoplakia and punctation showed HG AIN in 34.4% (95% CI 24.7-44.1). Mosaicism was only seen in 3 lesions without HGAIN. Twenty-one lesions had atypical vessels on HRA, of which the vast majority (15) showed normal mucosa and only 1 HGAIN. CONCLUSION This study demonstrated that HGAIN is present in a high proportion of intra-anal condyloma and flat lesions with leukoplakia and/or punctation. Therefore, lesions with these characteristics need additional histopathological examination, to not overlook HGAIN.
45
13 | HPV related benign diseases P-13.09

DMEP-CRYOTHERAPY IN A VETERINARY SETTING CASE STUDY REPORT
A Manns, NA, Bethlehem, PA, UNITED STATES L Damico, NA, Bethlehem, PA, United States Background - Papilloma virus (HPV)-related warts are common in animals. In dogs, contagious warts appear quite often and lesion location tends to be age-dependent. In older dogs, warts can occur anywhere on the body, are typically benign, and injury to the affected tissue can result in bleeding which can cause additional infections. Common treatment options are surgical excision or cryotherapy. Objectives - A veterinary practice for domestic animals conducted a usability study of Dimethylether-PropaneIsobutene (DMEP)-based Cryosurgical System Histofreezer to treat HPV-related skin lesions in dogs. The focus was to assess the practicability and treatment efficacy. Methods & Results - After confirmation of the diagnosis of HPV-infection, the dog owner consented to the treatment method. Prior to treatment, the Practice photographed the animal, first as a general photograph and then the specific skin lesions identified for treatment. Over a period of four months, four dogs with eight different skin lesions each less than 4mm in size and on different body locations (i.e. ear, paw, cheek, eyelid, foot and neck) received treatments according to the Cryosurgery Kit Directions for Use. With the exception of one lesion on one dog, all other patients were successfully treated. Conclusions Seven of eight different HPV-related skin lesions of four dogs were successfully treated. The success rate of treating small canine warts is dependent upon the lesion location and the temperature sensitivity of the skin. Due to animals pain-response, it is easier to treat warts on the torso and tail. The use of cryotherapy avoids surgical disadvantages such as bleeding, need for anesthesia, which can be a risky, costly procedure, risk of post-infection and cost of post-surgical care. The product performance, efficacy, safety, ease-ofuse and portability are the main benefits of utilizing this medical device in domestic veterinary practices.
Declaration of interest Presenting author declaration of interest Andreas Manns, Ph.D. Affiliated OraSure Technologies, Inc.; grant research support
P-13.10
UNUSUAL WART CASE REPORT CRYOSURGERY AS CUT- & STITCH-ALTERNATIVE
M Almasry, NA, Stockelsdorf (Lbeck), GERMANY A Manns, NA, Lbeck, GERMANY Background Surgeons operate to repair injuries, correct deformities and improve patient health. Through examination, they determine whether surgery is necessary, evaluate the risk involved and select the appropriate procedure, normally cutting & stitching. The patient scenario sometimes dictates the use of Cryosurgery (Cryotherapy) as alternative to routine surgical treatment. Objective A 68-year-old diabetic male presented with an exceptionally large wart centrally located on his forehead. This location is challenging for classical surgical ablation due to insufficient skin tissue for proper closure. Diabetic patients with wounds are at increased risk of infection due to impaired healing. Additionally, the patient was on a Marcuma (oral anticoagulant) regiment and hemorrhage was a substantial hazard. To mitigate these risks, the practice chose a portable Dimethylether-Propane-Isobutene (DMEP)-based Cryosurgical System as the treatment method. Method & Result After confirmation of the HPV-wart diagnosis, the 1cm base-diameter wart was sectored for cryo-treatment two times per week for 4 weeks. During the last treatment session, the physician tied a thin sewing thread around the treated wart-tissue creating a slight constriction that caused the treated wart tissue to release immediately. Thereafter the affected skin area was treated two more times that week. No bleeding occurred and they left the treatment area uncovered to heal. Nineteen months after treatment the patient is relapse free. Conclusions A diabetic, Marcumar-treated patient was treated with cryosurgery. Several health-critical aspects dictated cryosurgery to be the best treatment option, and the overall and cosmetic results were very satisfactory. The use of cryotherapy in this specific case avoided the need for plastic-surgery, and the surgical risk such as bleeding, post-surgical care and potentially unsightly cosmetic appearance. The product efficacy, safety, ease-ofuse and portability are the main benefits of utilizing this medical device as alternative to other procedures in a domestic medical practice.
Declaration of interest None declared Mohamed Almasry, Ph.D. Surgery Physician & Paediatric Surgery no conflict of interest
46
HPV related benign diseases | 13 P-13.11

PREVALENCE OF GENITAL WARTS IN FEMALE OUTPATIENTS: HOSPITAL-BASED STUDY
A KUCUKUNAL, SISLI ETFAL TRAINING AND RESEARCH HOSPITAL, ISTANBUL, TURKEY A KUCUKUNAL, SISLI ETFAL TRAINING AND RESEARCH HOSPITAL, ISTANBUL, TURKEY I ALTUNAY, SISLI ETFAL TRAINING AND RESEARCH HOSPITAL, ISTANBUL, TURKEY Background: Cervical cancer is a potentially fatal outcome of genital HPV infection. Genital HPV infections are mostly presented by genital warts (GW). While prevalence of GW is increasing worldwide, the importance of GW is often underestimated. It is important to detect prevalence figures regarding both preventive public health and therapeutic strategies. Turkey is a developing country in which no study of GW has yet been carried out in dermatology. Objectives: This study was aimed to demonstrate the prevalence of GW in females outpatients and to assess the link between sociodemographic factors, clinical complaints and the presence of GW. Methods: 1000 consecutive women included into the study. Age, marital status and educational levels were registered in a form. Dermatologic examination including vulvar examination were done in every patient. Clinical findings and signs were all listed and patients were grouped according to the existence of their initial complaints about vulvar area . Results: Two hundred and twelve patients (21,2%) of the study group had vulvar skin diseases. Of those with vulvar diseases, 34,9% presented with a vulvar complaint, although 65,1% didnt have any subjective complaint. Twenty one patients (%9,9) had visible signs of genital warts , 5 of those didn t have any complaint. The frequency of genital warts in the patients with vulvar complaints was statistically significant than those without any vulvar complaints (p<0,05). The number of women graduated from high school and university were also statistically significantly higher in the group suffering from genital warts than the other group. Conclusion: In fact, it is very likely the figures obtained in this study to be bigger. We need studies with far more patients to determine preventive and therapeutic strategies for HPV and GW in our society.
P-13.12
SADDLE SORE AND OTHER FEMALE COMPLAINTS
M Lwik, Leiden University Medical Centre, Leiden, NETHERLANDS D Berends, Leiden University Medical Centre, Leiden, Netherlands M van Poelgeest, Leiden University Medical Centre, Leiden, Netherlands What its like to suffer from cancer is something that we all can envisage, but having to live for many years with the threat of developing cancer has just as much impact on someones life. This presentation investigates the impact of a HPVrelated abnormality on the body, mental state and social environment of patients. Years of experience with these patients during clinical trials has provided valuable insight into this patient group. Patients diagnosed with HPV-related abnormalities suffer the rest of their lives from physical discomfort, isolation, loneliness and lack of understanding from the people around them. The objective of this presentation is to give the women behind the immuno data a face.
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13 | HPV related benign diseases P-13.13

IMPACT OF GENITAL WARTS ON SELF-ESTEEM OF MEN AND WOMEN
G Hernandez - Suarez, Research & Innovation Fundation, Bogota, COLOMBIA M Pieros, Research & Innovation Fundation, Bogota, COLOMBIA L Orjuela, Research & Innovation Fundation, Bogota, COLOMBIA Background: Most studies on HPV awareness and knowledge are focused on women and malignant lesions; information including men and benign lesions is scarce. Objectives: To assess differences in knowledge of HPV and the impact on self-esteem in men and women with genital warts. Methods: A cross-sectional study in a mayor private out-patient clinic in Bogot, Colombia, included 138 male and 204 female patients between 18 and 45 years with clinical diagnosis of genital warts (GW). All patients underwent biopsy for pathological diagnosis confirmation and HPV DNA testing. Participants responded a questionnaire assessing HPV knowledge, risk factors, and consequences of GW on self-esteem and sexual performance. Patients without pathological confirmation of GW were excluded from the analysis. Differences in proportions were assessed with a chi2 test. Results: 261 patients (106 men and 155 women) had GW pathologic confirmation and were included for this analysis. To the question Have GW affected your self-esteem? 90% of women and 62% of men responded affirmatively (p<0.01). 51% had heard about HPV before consultation (no gender differences), the majority in massive media (51,7%). In women, impact on self-esteem showed no differences according to age, education or knowledge of HPV. However, women with GW in the introitus reported a lower impact on self-esteem than women without GW in the introitus (82% vs. 95%; p< 0.01). In contrast, among men the impact of GW on self-esteem was greater among those older than 25 years, and those with knowledge on HPV, but there was no difference according to the location of GW. Conclusions: GW impact on self-esteem is greater in women than men, probably reflecting the higher health consciousness of women. As reported previously in other studies, there is a large gap in HPV knowledge even in patients suffering an HPV related disease.
Declaration of interest G. Hernndez, MSD, Grant support for the present study M. Pieros, MSD, has been speaker and member of an advisory board for MSD-Gardasil
48
Session 14: Clinical use of biomarkers

Chair: Barbara Moscicki
Poster abstracts
Clinical use of biomarkers | 14 O-14.00

Barbara Moscicki
FOR YOUR NOTES:
O-14.01
PROGNOSTIC SIGNIFICANCE OF HPV VACCINE PROTEIN L1 DETECTION WITH CYTOACTIV
R Hilfrich, Cytoimmun diagnostics, Pirmasens, Germany, G Mehlhorn, University Hospital for Women, Erlangen, Germany, M Beckmann, University Hospital for Women, Erlangen, Germany, L Bubendorf, Institute for Pathology, University Hospital, Basel, Switzerland, E Obermann, Institut for Pathology Univesity Hospital, Basel, Switzerland, G Negri, Institute for Pathology, Bolzano, Italy, H Sander, Cytology Lab, Einbeck, Germany, A Weiss, Center of Pathology and Cytology, Cologne, Germany, M Ltge, Cytology Lab, Salzgitter Bad, Germany, H Griesser, Center for Pathology and Cytodiagnostics, Cologne, Germany Background: Cervical cancer screening programs for the detection of precancerous cervical neoplasia are highly effective and caused a decline in the incidence of invasive cervical carcinomas. Consequently, dysplastic epithelial lesions are being diagnosed frequently. We and others have shown that HPV-L1 capsidprotein detection results with Cytoactiv predict spontaneous remission or progression to histologically confirmed CIN3 for such precancerous lesions. Objective: The aims of this prospective, randomized study are 1.)to validate the prognostic relevance of HPV-L1 capsid protein detection for early dysplastic lesions. 2.) to evaluate the impact of different preparation techniques (conventional Pap smear versus FDA approved LBC) on the sensitivity of L1 detection and its prognostic significance. Material and Methods: Beginning in 2007, HIV negative, non-pregnant, non HPV-L1 vaccinated women reported as LSIL (internationally) or (Germany) as group IIID with subclassification into mild (LSIL) or moderate (HSIL) dysplasia were included in this study. Follow up will be until June 2011. Results and Conclusion: 53-85% of the LSIL and 23-50% of the HSIL cases were positive for L1 capsidprotein immune staining. After 53 month follow up records of 642 cases are completed. Only 16% of 349 L1 positive, but 63,4% of the 293 L1 negative precancerous lesions showed a progression to histologically confirmed CIN3. A spontaneous remission was observed in 53,3% of the L1 positive, but only 4,8% of the L1 negative cases. A stable disease was reported in 30% of the L1 positive and L1 negative cases. L1 positive mild and moderate dysplasias, reflecting productive HPV infection, had low malignant potential, justifying a watch and wait strategy to prevent overtreatment especially in women of child-bearing age. L1 negative early dysplastic lesions, as non-productive infections or precancerous lesions, have a considerable malignant potential and close follow up with colposcopy and histological evaluation is advised.
Declaration of interest R. Hilfrich is chief scientific officer of cytoimmun dignostics GmbH, Germany Other authors none declared
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14 | Clinical use of biomarkers O-14.02

MANAGEMENT OF PAP-NEGATIVE-HPV-POSITIVE WOMEN WITH P16/KI-67-DUAL-STAINED CYTOLOGY: PALMS-TRIAL-RESULTS
H Ikenberg, Cytomol, Laboratory for Cytology and Molecular Biology, Frankfurt, Germany, M Sideri, European Institute of Oncology, Unit of Preventive Gynaecology, Milan, Italy, C Bergeron, Laboratoire Cerba , Cergy Pontoise, France, M von Knebel Doeberitz, Applied Tumor Biology, University of Heidelberg, Heidelberg, Germany, H Griesser, Center for Pathology and Cytodiagnostics, Cologne, Germany, J Bogers, Labo Lokeren - Campus Riatol, Antwerp, Belgium, H Neumann, Institute for Pathology, Schttorf/Leer, Germany, D Schmidt, Institute of Pathology, Mannheim, Germany, R Ridder for the PALMS Study Group, 9mtm laboratories, Heidelberg, Germany Background: Adding HPV-testing to cytology based cervical cancer screening (co-testing) in women aged 30 has the potential to increase sensitivity for the detection of high-grade cervical intraepithelial neoplasia (CIN2+). However, there is a need for an efficient clinical management algorithm for women with discrepant, i.e. Papnegative/HPV-positive test results. Objectives: We analyzed the performance of a novel immuno-cytochemical dual biomarker approach that detects co-localization of p16- and Ki-67-expression within the same cervical epithelial cell as an indicator of cell-cycle deregulation, in triaging all Pap-negative/HPV-positive screening results from the PALMS-study, a prospective screening trial comprising more than 27,000 women. Methods: Women prospectively enrolled to the PALMS-trial were tested using Pap cytology (LBC or conventional Pap smears), HPV (hc2, Qiagen) testing, and p16/Ki-67 Dual-stained cytology (CINtec PLUS, mtm laboratories) from samples collected at the enrolment visit. Women aged 30 with any positive test result were referred to colposcopy. Adjudicated tissue diagnosis on cervical biopsies was used as reference standard. Results: Out of a total of 19,205 women aged 30 enrolled to the PALMS-trial, 1,023 women (5.3%) were tested Pap-negative, HPV-positive. p16/Ki-67 Dualstained cytology testing at baseline provided positive test results in 225/1,023 (22.0%) women. Within this Dualstain positive group, 29 out of a total of 36 (80.6%) biopsy-confirmed CIN2+ cases were found. Specificity of Dualstained cytology at the CIN2+ threshold was 79.1%. The negative predictive value (NPV) of a negative Dual-stained cytology result was 98.7%. Conclusions: These results from the prospective PALMS-trial confirm the high sensitivity and specificity of Dual-stained cytology testing recently reported for triaging Pap-negative/HPV-positive screening results. The detection of co-localization of p16/Ki-67 expression in cervical cells identifies women that may benefit most from immediate colposcopy, whereas negative Dual-stain test results may exclude pre-cancerous cervical disease within this cohort with a high NPV.
Declaration of interest Potential conflicts of interest: occasional adviser to and/or speakers honoraria/travel grants from Abbott, B&D, Genprobe, GSK, Hologic, MTM, Qiagen, Roche, SPMSD
O-14.03
HPV/P16INK4A STATUS AND LOCAL IMMUNE RESPONSE IN ANAL CANCER PATIENTS
S Stiegler, University of Heidelberg, Heidelberg, GERMANY, M Reuschenbach, University of Heidelberg, Heidelberg, GERMANY, S Maiof Heidelberg, Mannheim Medical Centre, University of Heidelberg, Mannheim, GERMANY, M Ottstadt, Mannheim Medical Centre, University of Heidelberg, Mannheim, GERMANY, M Trunk, Mannheim Medical Centre, University of Heidelberg, Mannheim, GERMANY, N Wentzensen, University of Heidelberg, Heidelberg, GERMANY, M von Knebel Doeberitz, University of Heidelberg, Heidelberg, GERMANY, F Lohr, Mannheim Medical Centre, University of Heidelberg, Mannheim, GERMANY, F Wenz, Mannheim Medical Centre, University of Heidelberg, Mannheim, GERMANY Background: The carcinogenesis of anal cancers may be associated with a transforming HPV infection or may be induced by genetic and other cellular alterations unrelated to an HPV infection. Objectives: We evaluated the outcome in anal cancer patients associated with the HPV-DNA and p16INK4a expression status of the tumor. As the density of tumor infiltrating and surrounding immune cells has been shown to have prognostic significance in other cancers, we also analyzed the density of different T cell subsets and their relation to the HPV/p16INK4a status and the course of the disease. Methods: A total of 48 anal cancer patients who received combined radio-chemotherapy were included in the study. Tumor tissue was analyzed for HPV-DNA by GP5+6+ PCR, p16INK4a expression by immunohistochemistry and T cell infiltration by immunohistochemistry with antibodies for global T cells (CD3/CD8), activated T cells (CD45RO), regulatory T cells (Foxp3) and granzyme B expressing cytotoxic T cells. Results: 25 out of 48 (52.1%) patients had HPV and p16INK4a positive tumors suggestive of HPV-associated transformation. In 6 tumors HPV was detectable without p16INK4a expression, while in 6 tumors p16INK4a was positive but HPV-DNA was not detected. Infiltration with immune cells was highest in HPV/p16INK4a-positive tumors and lowest in HPV-negative/p16INK4a-positive tumors. Overall 5-year survival of the patients was 81.0% and 5-year disease specific survival was 67.7%. A better outcome could be observed in patients with HPV-positive/ p16INK4a-positive tumors (5-y survival 96.0%; p=0.04) and in patients with high density of immune cells (5-y survival 88.0%, p=0.05). Conclusions: Our findings of higher levels of immune infiltration in HPV/p16 positive tumors point to a pronounced immunogenicity of this phenotype which may affect the clinical course of the disease. The study is currently continued to increase the number of investigated patients.
Declaration of interest mtm Laboratories donated antibodies for the study. Magnus von Knebel Doeberitz is shareholder and member of the supervisory board of mtm Laboratories.
52

ELEVATED METHYLATION OF HPV16 DNA IS ASSOCIATED WITH CIN2+ DEVELOPMENT
L Mirabello, M Schiffman, A Ghosh, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, UNITED STATES, A C Rodriguez, Proyecto Epidemiolgico Guanacaste, Fundacin INCIENSA, San Jos, Costa Rica, N Wentzensen, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, UNITED STATES, R Herrero, Proyecto Epidemiolgico Guanacaste, Fundacin INCIENSA, San Jos, Costa Rica, S Wacholder, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, UNITED STATES, N Vasiljevic, Queen Mary, University of London, Centre for Cancer Prevention, Wolfson Institute of Preventive Medicine, London, UK, R D Burk, Departments of Pediatrics; Epidemiology and Population Health; and, Obstetrics, Gynecology and Women's Health, at Albert Einstein College of Medicine, Bronx, UNITED STATES, A Lorincz, Queen Mary, University of London, Centre for Cancer Prevention, Wolfson Institute of Preventive Medicine, London, UK Background: HPV16 DNA methylation has been suggested as a marker for cervix intraepithelial neoplasia, although rigorous epidemiological studies have not been performed. Objectives: To determine the association of HPV16 DNA methylation with prevalent CIN2+, HPV16 persistence and the development of persistent and/or CIN2+ in serially collected specimens from women in the Guanacaste, Costa Rica cohort. Methods: DNA was extracted from cervical cells collected from 196 women: 92 with HPV16 DNA clearance (controls), 37 with HPV16 DNA persistence (without CIN2+), 22 with CIN2, 31 with CIN3, and 14 with cervical cancer. Methylation was quantified after bisulfite treatment using pyrosequencing assays at 66 CpGs throughout the genome. The Kruskal-Wallis test was used to determine significant differences among groups. Results: In the cross-sectional study, methylation levels were significantly associated with disease outcome at 89.4% of CpG sites, after correction for multiple tests. All sites in E2-E4, E6, L2 and L1, and select sites in E7 and URR, had significantly higher methylation levels in CIN2+, particularly in the L1 and L2 ORFs, compared with the controls. Prospectively, 1 CpG site in URR and E2-E4, 3/14 in L2, and 6/19 in L1 had significantly higher methylation in prediagnostic CIN2+ specimens (median time of 5 years prior to diagnosis) vs. controls. In logistic regression models, increased methylation (>2nd tertile) was associated with an increased risk of CIN2+ compared with controls at all of these CpG sites (odds ratios ranged from 2 to 20.8). The odds ratio estimate of the joint effects of methylation at the 3 strongest CpG sites in L1, L2, and E6 as predictors of CIN2+ under a multiplicative model was OR=83.9 (95% CI 9.5-740). Conclusions: Methylation of specific CpG sites appears to be a robust biomarker associated with and indicating progression to cervix precancer and cancer.
O-14.05
CYTOLOGY, HPV TYPING, AND E6/E7 MRNA PERFORMANCE FOR HGAIN DETECTION
N Phanuphak, The Thai Red Cross AIDS Research Centre, Bangkok, THAILAND, S Kerr, HIV Netherlands Australia Thailand Research Collaboration, Bangkok, THAILAND, N Teeratakulpisarn, The Thai Red Cross AIDS Research Centre, Bangkok, THAILAND, A Sukjitpaiboonphol, The Thai Red Cross AIDS Research Centre, Bangkok, THAILAND, T Pankam, The Thai Red Cross AIDS Research Centre, Bangkok, THAILAND, J Barisri, The Thai Red Cross AIDS Research Centre, Bangkok, THAILAND, S Triratanachat, Chulalongkorn University, Bangkok, THAILAND, S Keelawat, Chulalongkorn University, Bangkok, THAILAND, J Palefsky, University of California at San Francisco, California, USA, J Ananworanich, South East Asia Research Collaboration with Hawaii, Bangkok, THAILAND Background: MSM are at risk of having high-grade anal intraepithelial neoplasia (HGAIN). Data on performance of different tests to screen HGAIN are limited. Objectives: To evaluate performance characteristics of cytology, HPV typing, and E6/E7 mRNA for detecting HGAIN. Methods: 246 MSM (123 HIV-positives and 123 HIV-negatives) attending the Thai Red Cross AIDS Research Centre in Bangkok had anal swabs collected in LiquiPrep for cytology, HPV typing (Roche Linear Array), and E6/E7 mRNA flow cytometry (Oncotect). All underwent high-resolution anoscopy (HRA)-directed biopsy of abnormal areas (histology). Cytology, HPV typing, E6/E7 mRNA were compared with histology (gold standard) for HGAIN diagnosis. Results: Complete testing (histology, cytology, HPV typing and E6/E7 mRNA) were available for 218 MSM at baseline, 76 at month 6 and 64 at month 12. At baseline, HGAIN was diagnosed on biopsy in 33 participants (13.5%); 18.9% in HIV-positives and 8.1% in HIV-negatives (p=0.01). An additional 9 and 5 HGAIN cases were detected at months 6 and 12, respectively. To detect overall HGAIN, the sensitivity, specificity, negative and positive predictive values (NPV and PPV) for abnormal cytology (ASC-US and above) were 35, 81, 87 and 26%; for high-risk HPV detection were 65, 48, 88 and 19%; for positive E6/E7 mRNA were 68, 45, 88 and 19%; for combined high-risk HPV and E6/E7 mRNA were 91, 23, 9 and 18%, respectively. High-risk HPV was the only test whose performance was affected by HIV status, with lower specificity among HIV-positives (40% vs. 60%, p=0.0005). Conclusion: Performance of HRA and training clinicians in HRA is time-consuming and expensive for developing countries. Optimization of screening tests that maximize the sensitivity and specificity of detecting HGAIN are therefore critical to minimizing HRA need. A combination of high-risk HPV and E6/E7 mRNA detection provided the best sensitivity and NPV for HGAIN.
53
14 | Clinical use of biomarkers O-14.06

PROSPECTIVELY IDENTIFIED EPIGENETIC SIGNATURES THAT PREDICT CLINICAL OUTCOMES IN CIN
M H Einstein, Albert Einstein College of Medicine, Bronx, NY, UNITED STATES T J Belbin, Albert Einstein College of Medicine, Bronx, NY, UNITED STATES M A Abadi, Albert Einstein College of Medicine, Bronx, NY, UNITED STATES L M Nathan, Albert Einstein College of Medicine, Bronx, NY , UNITED STATES G L Goldberg, Albert Einstein College of Medicine, Bronx, NY, UNITED STATES H D Strickler, Albert Einstein College of Medicine, Bronx, NY, UNITED STATES R D Burk, Albert Einstein College of Medicine, Bronx, NY , UNITED STATES N F Schlecht, Albert Einstein College of Medicine, Bronx, NY`, UNITED STATES Objective: Most CIN I will regress spontaneously. DNA hypermethylation of tumor suppressor genes has been described for CIN but its relation with CIN regression has not been previously studied. Materials: Women with newly detected oncogenic HPV-positive CIN I were followed every 6 months with 1) cytology, 2) colposcopy directed biopsies of acetowhite lesions and adjacent cervix, and 3) biopsies of histologically confirmed normal cervix. CpG methylation analyses of over 27,578 CpG loci covering over 14,000 genes were performed using the Infinium array HumanMethylation27 BeadChip (Illumina, CA). Candidate genes were confirmed by bisulfite sequencing. HPV typing was performed by MY09/MY11 PCR. Results: Sufficient DNA for methylation analysis was available from 15/16 subjects, of which 7/15 (47%) regressed, 7/15 (47%) continued to have CIN I, and 1/15 (1%) progressed to CIN3 over 18 months of follow-up. Ten adjacent confirmed normal cervical biopsies from 9 subjects were also analyzed. There was little to no difference in methylation levels between each lesion and normal adjacent biopsies within the same subject (mean beta=0.0020.026). When comparing the initial lesion biopsy with follow-up biopsies at a later time point in the same subject, one geneMGMT, had a significant change in methylation predictive of persistent disease. Comparing baseline biopsies from subjects who persisted versus regressed to normal, 94 genes were identified, of which 3 (CCND2, MTMR4 and TEX2) were found to differ in methylation levels, including lower levels than observed in normal biopsies. Conclusions: In this prospective study of epigenetic profiles in women with persistent versus regressed CIN I, methylation of MGMT may be correlated with early CIN persistence. Recruitment and analysis to confirm and identify additional gene methylation associated with cervical disease progression is ongoing.
O-14.07
METHYLATION ANALYSIS ON SELF-SAMPLES AS TRIAGE TOOL FOR HPV-POSITIVE WOMEN
B Hesselink, VU University medical center, Amsterdam, NETHERLANDS, D Heideman, VU University medical center, Amsterdam, NETHERLANDS, S Wilting, VU University medical center, Amsterdam, NETHERLANDS, R Steenbergen, VU University medical center, Amsterdam, NETHERLANDS, M Gok, VU University medical center, Amsterdam, NETHERLANDS, F van Kemenade, VU University medical center, Amsterdam, NETHERLANDS, V Coup, VU University medical center, Amsterdam, NETHERLANDS, H Berkhof, VU University medical center, Amsterdam, NETHERLANDS, P Snijders, VU University medical center, Amsterdam, NETHERLANDS, C Meijer, VU University medical center, Amsterdam, NETHERLANDS Background: Recently, we studied quantitative methylation-specific PCR (qMSP) assays assessing the methylation status of two human genes for their potential value as triage marker for hrHPV-positive women. A combination of two qMSPs yielded a methylation marker panel that, when applied to cervical scrapes, was equally discriminatory for CIN3+ as cytology or cytology/HPV16/18 genotyping combination in hrHPV-positive women (Hesselink et al.,ClinCancerRes 2011). Objective: Analyzing whether promoter methylation analysis can also serve as triage tool for hrHPV positive women when applied to cervico-vaginal lavage samples self-collected with a Delphi-screener (Delphi-bioscience). Methods: hrHPV-positive self-sampled lavage specimens of 355 screening non-attendees who participated in the PROHTECT trial (Gok et al., BMJ, 2011) were tested by qMSP for three promoter regions. The series included 74 specimens of women who were subsequently diagnosed with CIN3+. Cross-validated ROC curves for panels consisting of one to three qMSP markers (total 7 combinations) were constructed. The most discriminating panel for CIN3+ was considered the panel with the highest cross-validated partial area-under-the-curve (partial AUC). Results: After cross-validation a two marker panel displayed the highest partial AUC (0.740) for detection of CIN3+. Depending on the qMSP threshold settings the CIN3+ sensitivities ranged from 97.3% (95%CI:91.799.3) to 63.5% (95%CI:53.9-73.6), with corresponding specificities ranging from 20.6% (95%CI:18.8-22.6) to 80.1% (95%CI:75.9-83.7). A trade-off point that appeared optimal on the ROC curve was achieved with thresholds yielding a CIN3+ sensitivity of 85.1% (63/74; 95%CI:77.0-93.2) at a specificity of 53.7% (151/281; 95%CI:48.1-59.4) amongst these hrHPV positive women. Conclusions: The two marker methylation panel is an attractive triage tool for selfsampled cervico-vaginal lavage specimens of hrHPV positive women. Its application could obviate the need for an extra visit to the general practitioner for a cervical scrape when a woman has a hrHPV positive self-sample. This will save costs and reduce loss to follow-up.
Declaration of interest PS and DH provided occasional consultation to Roche. CM, PS, RS, and DH are shareholders of self-screen, a recent spin-off company of VUmc medical center. All other authors declare that they have no conflict of interest. The sources of funding did not have any influence on the design and the analysis of the results.
54

HPV METHYLATION DISTINGUISHES CIN3 FROM INFECTION: A GENERAL PHENOMENON
M Schiffman, National Cancer Institute, Rockville, UNITED STATES N Wentzensen, National Cancer Institute, Rockville, UNITED STATES C Sun, Albert Einstein College of Medicine, Bronx, UNITED STATES L Mirabello, National Cancer Institute, Rockville, UNITED STATES A Ghosh, National Cancer Institute, Rockville, UNITED STATES S Wacholder, National Cancer Institute, Rockville, UNITED STATES W Kinney, Kaiser Permanente Northern California, Oakland, UNITED STATES P E Castle, American Society of Clinical Pathology, Washington, UNITED STATES R D Burk, Albert Einstein College of Medicine, Bronx, UNITED STATES Background: A critical step in cervical carcinogenesis is the transition from common, acute HPV infection to uncommon, persistent infection and precancer (cervical intraepithelial neoplasia grade 3 or CIN3). Using casecontrol studies nested within our Guanacaste cohort, we recently observed that methylation of the HPV16 viral genome, particularly of L1 and L2, is more common in CIN3 than in infections that cleared. There are >100 CpG sites in HPV genomes. Objectives: To examine, in the larger Kaiser Permanente Northern California cohort, viral methylation for other HPV types to see whether the association with CIN3 is a general phenomenon. In case-control comparisons, we have analyzed HPV18 (38 CIN3, 67 cleared), HPV31 (38 CIN3, 58 cleared), and HPV45 (12 CIN3, 22 cleared). We will add HPV33, HPV35, HPV52 and HPV58 by the meeting. Methods: DNA isolated from cytologic brushings was bisulfite-converted, and PCR amplified using type-and sitespecific primers. To determine methylation status, CpG sites were pyrosequenced yielding values from 0 to 100%. The difference at each site between CIN3 cases and controls (clearing HPV infection) was analyzed by the Wilcoxon rank-sum test adjusted for multiple comparisons. Results: For HPV18, 22/24 L1 CpG sites assayed were significantly elevated in CIN3 (mean percentage of methylation in significant cases 25-59%). For HPV31, 2/21 L1 CpG sites analyzed were significantly elevated in CIN3 (20% and 39% methylation for the 2 cases). For HPV45, 8/13 L1 CpG sites analyzed were significantly elevated in CIN3 (levels from 25%-83%). We will present combinations of genes and sites that best discriminated CIN3 from infection. Conclusion: Especially for L1, high methylation levels indicate increased risk for underlying CIN3 while low/no methylation indicates that the infection is likely to clear. A clinically useful risk bioassay might be conceivable.
55
14 | Clinical use of biomarkers P-14.09

HPV 16&18 VIRAL LOAD AND RISK FACTORS FOR CERVICAL CANCER
R URSU, UNIVERSITY OF MEDICINE AND PHARMACY, IASI, ROMANIA M ONOFRIESCU, UNIVERSITY OF MEDICINE AND PHARMACY, IASI, ROMANIA D NEMESCU, UNIVERSITY OF MEDICINE AND PHARMACY, IASI, ROMANIA R NEMESCU, UNIVERSITY OF MEDICINE AND PHARMACY, IASI, ROMANIA L S IANCU, UNIVERSITY OF MEDICINE AND PHARMAC , IASI, ROMANIA Background: one of the posible virus related biomarkers used to early detect the evolution to cervical cancer is the viral load of HPV 16 and 18. The HPV viral load and risk factors such smoking and oral contraceptive seems to influence the evolution to cervical cancer. Objectives: purpose of the study was to assess the clinical utility of viral load determination for HPV 16 and 18 in relation with the severity of cervical dysplasia and in association with classical risk factors Methods: viral load was detected with Path-HPV16/18 Real-time PCR detection kit for Human Papillomavirus and 2xPrecision TM Mastermix (PrimerDesign). PCR amplification was performed in MX3005P Stratagene platform. Statistical analysis had used SPSS 16.0 program. Results: from 267 genotyped samples, we identified 26 (9.7 %) positive for HPV 16 and 9 (3.4 %) positive for HPV 18. The Ct / Qs values were 19.68 - 33.94. The mean of viral load of HPV 16 was 684515,68 copies (limits 3,45 - 7.177 x 106 copies). Ct values for HPV 18 were between 25.44 46.93. The viral load of HPV 18 was lower than the viral load of HPV 16 (limits 1.138 x 103 - 7.119 x 104). The highest viral load of HPV 16 was detected in LGSIL and in HGSIL cases (> 106 copies) and in 30 40 years group. From all HPV 16 positive women, 15.4% was smoker, 34.6% had used oral contraceptive and 38.5% had multiple other HPV type infections. From HPV 18 positive women, 50% was smoker. Conclusions: viral load of high risk HPV types seems to be one sensitive and objective biological marker in detecting women at risk for developing cervical cancer. No significant association was found for the risk factors investigated in the study group.
P-14.10
E2 AND E6 GENE DELETIONS IN HPV16 CERVICAL INFECTIONS
R Lpez - Revilla, IPICYT, San Luis Potos, MEXICO S Hernndez - Arteaga, IPICYT, San Luis Potos, MEXICO A Garca - Carranc, INCAN, Mexico City, MEXICO G A Anguiano - Vega, IPICYT, San Luis Potos, MEXICO J Jurez - lvarez, IPICYT, San Luis Potos, MEXICO BACKGROUND. Human papillomavirus type 16 (HPV16), whose circular DNA replicates as an episome, is the most common cause of cervical cancer (CC). Viral genome integration, apparently essential for neoplastic transformation, is usually preceded by disruption of the E2-HPV16 gene. Initiation of cervical intraepithelial neoplastic (CIN) lesions requires E6 oncogene expression and may progress to CC passing through low degree CIN1 and high degree CIN23 precancerous lesions. Some researches propose that in CIN1 the episomal form of the viral genome predominates (similar number of E2 and E6 genes and E2/E6 ratios ~ 1), whereas in CC the integrated form predominates (loss of E2 and E2/E6 ratios close to 0). OBJECTIVES. To assess E2 and E6 loads and E2/E6 ratios in HPV16-infected women with precancerous lesions and cancer. METHODS. E2 and E6 loads (copies per ng of DNA) and E2/E6 ratios were determined in cervical scrapes of 35 women with diverse lesions (16 CIN1, 7 CIN2, 3 CIN3, 9 CC) using nested real time PCR calibrated with constructs containing E2 and E6 sequences. RESULTS. Ranges of E2 and E6 loads and E2/ E6 ratios per lesion group: CIN1 16-18,076, 55-816,656, 0.006-153.5; CIN2 30-58,535, 119-538, 0.006-15.5; CIN3 21808,000, 444-5,200,000, 0.736-25.0; CC: 15-1,152,000, 18-17,200,000, 0.020-182.0. Significant differences of E6 loads were found only in CIN1 vs. CC (P = 0.028) and CIN1 vs CIN2 (P = 0.045). Unexpected features of E2/E6 ratios were that their range (0.006-625.5) exceeded the maximum expected value of 1, 80% of the scrapes had E2/E6 ratios higher than 1, and log (E2/E6) values had a gaussian distribution. CONCLUSIONS. E2 and E6 loads and E2/E6 ratios do not correlate with the degree of neoplastic lesions caused by HPV16, where deletions of the E2 and E6 genes occur spontaneously with unknown effects on viral replication and neoplastic progression.
56
Clinical use of biomarkers | 14 P-14.11

HPV L1 PROTEIN IMMUNOCYTOCHEMISTRY AND E2/E6 RATIO IN CERVICAL CYTOLOGY
C W Lee, Yeouido St. Mary's Hospital, the Catholic University of Korea, Seoul, KOREA, REPUBLIC OF J S Park, Seoul St. Mary's Hospital, the Catholic University of Korea, Seoul, KOREA, REPUBLIC OF S J Lee, St. Vincent Hospital, the Catholic University of Korea, Suwon, Gyeonggi-do , KOREA, REPUBLIC OF Background: Human papillomavirus (HPV) L1 negative in immunostaining was reported to be related with the progression of cervical cytology and E2/E6 ratio of HPV showed different values according to the cervical grades. Objectives: The aim of this study was to evaluate the immunocytochemistry of L1 capsid protein and E2/E6 ratio in various cytology grades infected with HPV 16. Methods: A total of 124 cervical cytology specimens were collected at Seoul St. Marys Hospital, the Catholic University of Korea, from January 2006 to December 2010. Only cervical specimens with HPV 16 single infection were entered in the study. Cytological specimens were evaluated by immunostaining of HPV L1 capsid protein and E2/E6 ratio of HPV 16 with real time-polymerase chain reaction (RT-PCR). The ANOVA test was used to compare mean ratio of E2/E6 of HPV 16 in cytological group. The linear by linear association test was used to evaluate the correlation between HPV L1 capsid protein and HPV physical status. Results: A total of 124 cases were finally eligible for the study, consisting of 45 cases of normal cervical cytology, 16 cases of ASCUS, 29 cases of LSIL, 23 cases of HSIL, and 11 cases of cervical cancer. The HPV L1 positivity were found in 11.1%, 37.5%, 51.7%, 30.4%, 0.0% of normal, ASCUS, LSIL, HSIL, cancer groups, respectively. All of cancer cytology showed HPV L1 capsid protein negative for immunocytochemistry. The mean of E2/E6 ratio in cancer cytology was 0.23 and significantly lower than those of the other groups (p < 0.05) Conclusions: All of cancer cytology showed negative for the immunocytochemistry of HPV L1 capsid protein. Also, cancer cytology expressed significantly low ratio of E2/E6. Hence, low ratio of E2/E6 can be suggested to be closely associated with tendency of mixed and integrated form of HPV infection in cancer cytology.
P-14.12
CORRELATION BETWEEN HPV L1 AND BEHAVIOR OF LSIL IN KOREAN.
E Y Ki, The Catholic University of Korea./Seoul St. Mary's Hospital, Seoul, KOREA, REPUBLIC OF S T Park, The Catholic University of Korea./Seoul St. Mary's Hospital, Seoul, KOREA, REPUBLIC OF S Y Hur, The Catholic University of Korea./Seoul St. Mary's Hospital, Seoul, KOREA, REPUBLIC OF S J Lee, The Catholic University of Korea./St. Vincent's Hospital, Suwon, OREA, REPUBLIC OF J S Park, The Catholic University of Korea./Seoul St. Mary's Hospital, Seoul, KOREA, REPUBLIC OF Aim: The aim of this study was to evaluate the behavior of low-grade squamous intraepithelial lesion (LSIL) in Korean women infected with human papillomavirus (HPV) in relation to the immunocytochemical detection of the HPV L1 capsid protein. Material and Methods: From January 2006 to December 2007, a total of 353 immunocytochemistry tests were performed on specimens from HPV-infected patients with LSIL. Due to exclusions, the study population was reduced to 318. Subjects were monitored at 4-6 month intervals. The regression, persistence, and progression of the cytologic abnormalities of the 318 cases were compared with the results of HPV L1 capsid protein immunocytochemical detection. Results: Of the 137 patients negative for the HPV L1 capsid protein, 38 (27.7%) showed progression to high-grade lesions, 50 (36.5%) showed persistence, and 49 (35.8%) showed regression to normal cytological features. In contrast, of the remaining 181 patients positive for the HPV L1 capsid protein, 15 (8.3%) showed progression to high-grade lesions, 74 (40.9%) showed persistence, and 92 (50.8%) showed regression. The results of immunocytochemical testing for the HPV L1 capsid protein show a linear association with the progression or regression behavior of lowgrade cervical cytology in patients infected with HPV (linear by linear association test, P < 0.05). Conclusion: Immunocytochemical detection of HPV L1 was significantly related with the biological patterns of LSIL in Korean women. Hence, immunocytochemistry for the detection of HPV L1 is beneficial in providing further information for LSIL.
57

PERFORMANCE OF PROEX C IN DETECTING CERVICAL SQUAMOUS INTRAEPITHELIAL LESIONS
R Alaghehbandan, Memorial University of Newfoundland, St. John's, CANADA D Fontaine, University of Calgary, Calgary, CANADA A Lear, Dr H Bliss Murphy Cancer Centre, St. John's, CANADA S Ratnam, Public Health Laboratory, Department of Health; Memorial University of Newfoundland, St. John's, CANADA The clinical usefulness of the ProEx C test (BD) for the detection of cervical intraepithelial neoplasia (CIN) was determined in a multicentre study carried out in Canada, supplemented with HPV genotypic analysis. The study population comprised of women representing five of the ten Canadian provinces referred to colposcopy for further assessment of cervical cancer risk and follow-up, and those routinely screened. Cervical specimens were collected in PreservCyt and cytology was performed using the ThinPrep method (Hologic), and were processed for HPV genotyping using the Linear Array method (Roche Molecular System). HPV types 16, 18, 31, 33, 35, 45, 51, 52, 56, 57, 58, 59, 68 were deemed as high risk (HR)-HPV. Histology confirmed CIN served as the disease endpoint to assess the test performance. Both descriptive and inferential statistical analyses were performed using the Statistical Package for the Social Sciences. The study analysis was based on a total of 1625 women (mean age 30.8 years) with 427 (26.3%) having CIN 2+. Overall, ProEx C was positive in 39% (634/1625). ProEx C detecting CIN was best found at CIN2+ with a concordance rate of 73.5% (95% CI: 72.4-74.6). The overall positive rate of HR-HPV was 69.8% (1135/1625), with a concordance of 54.0% (95% CI: 52.8-55.3) for detecting CIN2+. The sensitivities of ProEx C and HR-HPV in detecting CIN2+ lesions were 74.2% and 95.3% while the specificities were 73.3% and 39.3%, respectively. A significant correlation between ProExC and HR-HPV in terms of detecting CIN2+ lesions was found (p = 0.044). In conclusion, ProEx C was found to be more specific but less sensitive than HR-HPV marker in detecting CIN2+ lesions. ProEx C may have the potential to serve as a useful adjunct test for the detection of CIN2+ lesions in cervical cancer screening.
P-14.14
MRNA FOR TRIAGE OF HPV 16/18 DNA POSITIVE WOMEN
S Perez, Complejo Hospitalario Universitario de Vigo (CHUVI), VIGO, SPAIN, M J Lamas, Complejo Hospitalario Universitario de Vigo (CHUVI), VIGO, SPAIN, M T Saran, Complejo Hospitalario Universitario de Vigo (CHUVI), VIGO, SPAIN, A Cid, Complejo Hospitalario de Ourense, OURENSE, SPAIN, M J Alvarez, Complejo Hospitalario Universitario de Vigo (CHUVI), VIGO, SPAIN, A Iarrea, Complejo Hospitalario Universitario de Vigo (CHUVI), VIGO, SPAIN, J Rubio, Complejo Hospitalario Universitario de Vigo (CHUVI), VIGO, SPAIN, M C Reboredo, Complejo Hospitalario Universitario de Vigo (CHUVI), VIGO, SPAIN, I Lpez- Miragaya, Complejo Hospitalario Universitario de Vigo (CHUVI), VIGO, SPAIN, S Meln, Hospital Universitario Central de Asturias, OVIEDO, SPAIN BACKGROUND: Testing for HPV messenger RNA (mRNA) is more specific than testing for HPV DNA. OBJECTIVES: To evaluate the mRNA detection for triage of HPV 16/18 DNA positive women. METHODS: This study investigated 94 women from a routine based screening population with a baseline cytological(C)/ histological (H) result from no-alteration to carcinoma and HPV 16/18 DNA detection. DNA was detected by MY09/MY11/GP5+/6+ consensus primers nested PCR followed by sequencing (HPV 16, n=78. HPV 18, n=13. Coinfection HPV 16 and 18, n=3). Baseline mRNA was detected by Nuclisens EasyQ E6/E7 mRNA HPV assay (bioMerieux) after RNA extraction (RNeasy Plus Mini Kit, QIAgen). Patients (n=51) without baseline histologyconfirmed CIN2 or worse (CIN2+), were followed up for progression to CIN2+ (PRO) and HPV DNA persistence (PER). Association between variables was determined with Fisher's exact test. p<0.05 was considered statistically significant. RESULTS: BASELINE: According to baseline C (n=73) and/or H (n=48) mRNA positivity was: no-alteration 25% (7/28), ASCUS 60% (9/15), LSIL/CIN1 68%(13/19), ASC-H/HSIL/CIN2/CIN3/carcinoma 90% (37/41). Nine C/H discordances were double counted. All ASC-H and HSIL -except 1- were CIN2+. mRNA detection was more frequent in women with baseline CIN2+ (90%) than in women with other baseline C/H (47%) (p<0.001). FOLLOW-UP: For detecting PRO, sensitivity of mRNA testing was 75%, specificity 69%, positive predictive value (PPV) 43% and negative predictive value (NPV) 90%. mRNA detection in PRO (9/12) was more frequent than in regression/persistence (no PRO) (12/39) (p<0.05). Average time (months) until CIN2+ in PRO: 115 (5-18). Average follow-up time (months) in no PRO: 163 (12-24). For detecting PER, sensitivity of mRNA testing was 47%, specificity 68%, PPV 71% and NPV 43%. CONCLUSIONS: (1) Detection of E6/E7 mRNA increases with the severity of the cervical lesion. (2) mRNA testing may be a biomarker for progression to CIN2+ in case of detection of HPV 16/18 DNA.
58

EPSTEIN-BARR VIRUS AS A POTENTIAL BIOMARKER FOR CERVICAL DYSPLASIA
W Nichols, Louisiana State University Health Science Center, New Orleans, UNITED STATES K Sutton, Louisiana State University Health Science Center, New Orleans, United States N Nelson, Louisiana State University Health Science Center, New Orleans, United States A Clark, Louisiana State University Health Science Center, New Orleans, United States H Oddo, Louisiana State University Health Science Center, New Orleans, United States N Love, Louisiana State University Health Science Center, New Orleans, United States M Hagensee, Louisiana State University Health Science Center, New Orleans, United States Background: Despite the high prevalence rate of HPV infections, only a small fraction of infected women develop cervical cancer. This suggests that HPV is required for tumorgenesis, but is not sufficient alone and requires a cofactor for progression to carcinoma. A potential co-factor is oncogenic EBV. Preliminary studies demonstrated co-detection of EBV with high-risk HPV in cervical fluids did increase the risk of cervical dysplasia in HIV+ women. Objectives: To determine if EBV can be detected in Pap smear fluid and if its detection could improve the current cervical cancer screening process. Methods: Residual Pap smear fluid from HIV+ women were processed for DNA extraction using a Qiagen kit. HPV was detected and genotyped using the reverse line blot assay while EBV was detected by BamH1 targeted PCR. The presence of EBV was correlated with HPV results and concurrent Pap smear findings. Results: A cross-sectional study of discarded residual Pap smear fluids (n=134) detected EBV in 48% of the samples, HPV in 12%, and co-shedding in 6%. Dysplasia was found in 75% of both co-shedding and HPV-only shedding women. A follow-up comparative study (n=56) showed an increase in dysplasia for co-shedders (75% vs. 54%) utilizing cervical swab specimens, but the residual fluid did not demonstrate this. In both studies, the rates of HPV DNA detection were markedly lower in the Pap smear fluid (12%) versus the cervical swabs (40%) raising a concern for inadequate cells remaining in the residual fluid. Conclusions: EBV can be readily detected in residual Pap smear fluid from HIV+ individuals. Women with an abnormal Pap smear have high rates of detectable EBV in residual fluid. Additional studies are ongoing to improve detection of HPV and EBV co-shedding, which may lead to an improved biomarker for abnormal Pap smears.
P-14.16
CYCLINS - IMMUNOCYTOCHEMICAL MARKERS FOR CERVICAL LESIONS
R Stanculescu, University of Medicine Carol Davila Bucharest - St Pantelimon Hospital, Bucharest, ROMANIA V Bausic, University of Medicine Carol Davila Bucharest - St Pantelimon Hospital, Bucharest, ROMANIA M Russu, University of Medicine Carol Davila Bucharest - I. Cantacuzino Hospital, Bucharest, ROMANIA C Tihoanu, St. Pantelimon Hospital, Bucharest, ROMANIA A Cucu, St. Pantelimon Hospital, Bucharest, ROMANIA T Vladescu, St. Pantelimon Hospital, Bucharest, ROMANIA Background: Cervical HPV infections cause changes in cell cycle control whose activity is regulated by cyclins. Objective: Immunocytochemical highlighting of B, D, E cyclins, p53, p63, p27, Keratin Pan AB1, p16ink4a, Ki67 and p27 in liquid based cytology envisaging cervical intraepithelial and invasive lesions. Methods: Improved methods of immunocytochemical identification of cyclins allowed us to obtain 60 samples that were interpreted by three pathologists trained in cytology. p16ink4a identification was performed with monoclonal antibodies produced by MDM-US Laboratory; the antibodies for other cyclins were provided by Thermo Fischer Scientific Lab Vision Named Product. Results: Immunocytochemical positive test results showed following percentages: cyclin B1 - 15 cases (26.31%), cyclin D1 - 18 cases (31.57%), cyclin E - 10 cases (17.54%), Keratin Pan AB1 - 30 cases (52.63%), Ki67 - 20 cases (35.08%), p53 -16 cases (28.07%), p63 - 20 cases (35.08%), p27 - 12 cases (21.05%), p16ink4a - 28 cases (46%). The study of simultaneously positive cyclins for the same type of lesion analysis revealed the presence of all investigated cyclins in all L-SIL lesions. H-SIL lesions are positive for the same cyclins except p27. The search for location of cyclins in dysplastic and malignant cells disclosed the constant presence of B1, D1, E, Ki67, p16ink4a, p53, p63 and p27 in dysplastic cells. Keratin Pan AB1 is present in cytoplasm of dysplastic and malignant cells. p16ink4a proved to be the only marker present in both nucleus and cytoplasm. Ki67 is a nuclear antibody only. Ki67 was present in L-SIL lesions in no more than 5% and in H-SIL lesions in no more than 10%. Conclusions: Intensity of expression of cyclins is higher in H-SIL and squamous cervical cancer, exception being p27. From all investigated markers, p16ink4a and Ki67 can be considered the most reliable diagnostic markers for cervical lesions aggravation potential.
59

DETECTION OF EARLY VIRAL TRANSCRIPTS IN PERSISTENTLY HPV INFECTED WOMEN
N Silvestre, Strasbourg, FRANCE D Schmitt, Strasbourg, FRANCE A Brochot, INSERM UMR-S 903, CHU de Reims, Reims, FRANCE C Orange, Strasbourg, FRANCE G Inchauspe, Lyon, FRANCE P Ancian, Strasbourg, FRANCE C Clavel, INSERM UMR-S 903, CHU de Reims, Reims, FRANCE A Fournillier, Lyon, FRANCE V Dalstein, INSERM UMR-S 903, CHU de Reims, Reims, FRANCE Persistent infections with high-risk HPV (HR-HPV) are associated with an increased risk for the development of cervical cancer. Identification of markers of persistent infection very early in the infection could be useful for specific treatment. In order to identify such markers, we analyzed the expression of the early antigens E1, E2 and E6/E7 at the transcriptional level in persistently infected women with normal cervical cytology. Among the subjects enrolled in the Reims HPV Cohort Study (France) with a positive Hybrid Capture 2 test, we selected women with HPV16 or HPV18 persistent infection showing normal cytology and stored prospectively material for subsequent RNA isolation. RNA extracts were analyzed with the Nuclisens Easy-Q HPV kit (bioMrieux) for the detection of E6/E7 mRNA and RT-qPCR assays were developed to detect E1 and E2 mRNA of HPV16 and HPV18. Thirty-three women were eligible for the study, 31 of which with HPV16 and 2 with HPV18 persistent infection. The median age was 37.3 years (23.4-75.7) and the median time of persistence was 10.6 months (5-24.2). Four samples were invalidated in the RT-qPCR assays and 2 of them were also invalidated in the Nuclisens Easy-Q assay. E1 mRNA was detected in cervical cells of 16/29 (55 %) women while E2 mRNA was detected in 13/29 (45 %) women. On the other hand E6/E7 mRNA was detected in 20/31 (64.5%) women. It should be noticed that all samples positive for E1 and/or E2 were also E6/E7 positive. In conclusion, E1 and E2 viral transcripts, but also E6/E7 transcripts could be detected in this study in more than the half of persistently infected women with normal cytology, which could have an impact on the choice of antigens for the development of therapeutic vaccine
Declaration of interest This work was supported by the ADNA program, coordinated by Institut Mrieux and supported by research and innovation aid from the French public agency, OSEO
P-14.18
HPV 16 VARIANTS IN YOUNG WOMEN WITH CERVICAL CANCER
Y Jayasinghe, Royal Women's Hospital, Melbourne, AUSTRALIA, V Sasongko, University of Melbourne, Melbourne, Australia, E Moore, Royal Women's Hospital , Melbourne, Australia, T Leong, Austin Hospital , Melbourne, Australia, M Stevens, Royal Women's Hospital , Melbourne, Australia, S Tabrizi, Royal Women's Hospital , Melbourne, Australia, S Grover, Royal Women's Hospital , Melbourne, Australia, S Garland, Royal Women's Hospital , Melbourne, Australia, for the EOCC study Group, Australia Background: Sequence variations in viral oncogenes may contribute to early-onset cervical cancer (CC) in young women. Objectives: To compare HPV variants (in E6, E2, L1 genes) and possible amino acid (aa) changes in HPV16-positive CC biopsies in women 25 years of age, compared to those found in biopsies from women >25 years of age. Methods: A hospital based case-control study across three states in Australia. Subjects were diagnosed with CC between 1983-2007. HPV genotyping of archival sectioned paraffin blocks was performed with INNO-LiPA using SPF 10 primers. HPV 16-positive samples were analyzed for variants in L1 (nucleotide [nt] 6638-6806), E2 (nt 3112 to 3222) and E6 (nt 100-218 and 273-375) by type-specific PCR and Sanger sequencing. Results: HPV16 positive biopsies from 18 cases and 52 controls underwent sequence analysis. All subjects originally diagnosed with microinvasive disease (14/70, 20%) had residual CIS/CIN3 or ACIS in the remainder of the block, while the remainder had residual cancer in the block (56/70, 80%). Genomic variability in L1 was 1.1%, E2 was 4.5%, and E6 5.9% with no significant difference between cases and controls (p=0.5). 15 SNPs were identified in cases and 54 in controls, resulting in 13 aa changes in cases and 54 in controls. Variant lineages were similar between cases and controls, with the majority being European (94% & 86% respectively), then Asian American [AA](5.6% & 11.8% respectively ) and Asian (none in cases and 2.0% in controls)[ p=0.8]. The E-350T prototype was seen in 69% of cases and 56% of controls (p=0.4). A higher proportion of AA variants were seen in AC compared to SCC (p=0.05, OR 5.0, 95%CI 0.6-41.6). Conclusions: In our sample HPV16 variants did not account for increased carcinogenesis in young women. European variants were the most predominant in Australian cervical cancers
Declaration of interest Dr Yasmin Jayasinghe: is a coinvestigator on Glaxo Smith Kline Vaccine trials (HPV-015 and extension - 066) and (HPV008 extension - 052) Professor Suzanne Garland: has received funding from Merck & Co.,Inc. (Whitehouse Station, NJ) through her institution to conduct clinical trials of the HPV vaccine. Professor Garland has received advisory board fees and grant support from Commonwealth Serum Laboratories (Victoria, Australia) and GlaxoSmith- Kline (Research Triangle Park, NC), and lecture and consultancy fees from Merck &Co., Inc. She has previously owned stock in Commonwealth Serum Laboratories and has received grant support through her institution from GlaxoSmithKline to conduct clinical trials for human papillomavirus (HPV) and cervical cancer vaccines.
60

IMMUNOHISTOCHEMICAL VALIDATION OF SCCA1/2 EXPRESSION IN CERVICAL CARCINOMA
K Shroyer, Stony Brook University Medical Center, Stony Brook, UNITED STATES E Chen, Stony Brook University Medical Center, Stony Brook, UNITED STATES S Mehmood, Stony Brook University Medical Center, Stony Brook, UNITED STATES S Burke, Stony Brook University Medical Center, Stony Brook, UNITED STATES J Chu, Stony Brook University Medical Center, Stony Brook, UNITED STATES L Fernandez - Escobar, Stony Brook University Medical Center, Stony Brook, UNITED STATES D Pal, Stony Brook University Medical Center, Stony Brook, UNITED STATES Background: Squamous Cell Carcinoma Antigen 1/2 (SCCA1/2), a member of the serpin family of endogenous serine protease inhibitors, has been explored as a serologic marker of cervical squamous cell carcinoma (CSCC) but has not been well characterized at the tissue level. Objective: To determine if the expression of SCCA1/2 is related to CSCC tumor grade and stage. Methods: Archival formalin-fixed paraffin-embedded CSCC tissue blocks were selected from the Department of Pathology at Stony Brook University Medical Center. By coupling liquid chromatography with tandem mass spectrometry, we resolved over 100,000 tandem mass spectra from laser captured FFPE tissues and identified over 3,000 proteins, including a distinct signature of SCCA1/2 as a potential marker of biologically aggressive cervical squamous cell carcinoma (SCC). Tissue microarrays were constructed from triplicate cores, representative of 16 low grade and 9 high grade CSCCs. Immunohistochemical staining of CSCC tissue microarrays was performed for SCCA, p16ink4a, and Ki-67 by indirect immunoperoxidase methods. Results: Strong nuclear and cytoplasmic SCCA1/2 staining was detected in 14/25 (56%) and weak to absent staining was detected in 11/25 (44%) CSCCs. SCCA1/2 expression was inversely correlated with tumor grade and also showed a trend for decreased expression in cases with nodal metastases: staining was strongly positive in 2/9 (22%) of high grade versus 13/16 (81.3%) of low grade tumors and in 2/5 (40%) of N1/N2 tumors versus 12/19 (63.2%) of N0/NX CSCCs. p16ink4a was strongly positive in 23/25 (92%) and a high Ki-67 labeling index was detected in 7/25 (28%) of CSCCs but neither marker correlated to the SCCA1/2 status. Conclusions: SCCA1/2 expression is highly correlated with early tumor development and may also be indicative of the lymph node status but is unrelated to p16ink4a and Ki-67. SCCA1/2 should be further investigated as an independent prognostic marker for CSCC.
P-14.20
STUDY OF HPV16 VIRAL LOAD IN SQUAMOUS CERVICAL CANCER
K Sundstrm, Karolinska Institutet, Stockholm, SWEDEN N Ylitalo, Karolinska Institutet, Stockholm, SWEDEN A Ploner, Karolinska Institutet, Stockholm, SWEDEN L Arnheim Dahlstrm, Karolinska Institutet, Stockholm, SWEDEN J Palmgren, Karolinska Institutet, Stockholm, SWEDEN J Dillner, Karolinska Institutet, Stockholm, SWEDEN H O Adami, Karolinska Institutet, Stockholm, SWEDEN P Sparn, Karolinska Institutet, Stockholm, SWEDEN BACKGROUND: A strong association has been shown between high viral load (VL) of HPV16 and increased risk for cervical cancer in situ (CIN3). However, significantly fewer data are available for the significance of VL in invasive squamous cell carcinoma (SCC). OBJECTIVES: To contrast VL levels in CIN3 and SCC by using a strict epidemiological study design, and state-of-theart laboratory assays. METHODS: In two nested case-control series among women participating to cervical screening, with a first cytologically normal smear, we collected 6471 cervical smears from 717 women with CIN3, 549 with SCC, and individually matched controls. All smears were tested for HPV with Luminex, and the VL of all HPV16 positive smears was quantified using RT-PCR. The median follow-up until diagnosis was 5 to 8 years. RESULTS: Average age at entry was 33 years (range 15-85) for the CIS/SCC group. The average number of smears was 3 (range 1-25) for both cases and controls. Around 46% of cases were positive for HPV16, but only 5% of controls. The average VL at entry was ~45 copies/ul for women with CIN3 and ~35 copies/ul for women with SCC. VLs among the two control groups were consistently lower. In a case-case analysis, VL levels were higher in CIN3 than in SCC; culminating at time of diagnosis, when the VL in CIN3 was on average double that in SCC. VL levels also increased more rapidly before diagnosis in CIN3 than SCC. CONCLUSIONS: In this comprehensive study, we demonstrate that HPV16 VL levels in SCC are raised compared to controls, but not as high as those in CIN3. One may speculate that a high VL primarily drives the progression to CIN3, and then is overtaken by other factors such as viral integration (lowering virion production), which could explain the paradoxically lower loads in invasive cancer.
61

HPV SECONDARY MARKER HETEROGENEITY IN CERVICAL CARCINOMAS
M F Evans, University of Vermont, Burlington, United States V Rajendran, University of Vermont, Burlington, United States C S C Adamson, University of Vermont, Burlington, United States Z Peng, University of Vermont, Burlington, United States K Cooper, University of Vermont, Burlington, United States S Nandekar, Sri Aurobindu Institute of Medical Sciences, Indore, India K Munjal, Sri Aurobindu Institute of Medical Sciences, Indore, India Background: HPV is detectable by PCR in nearly all invasive cervical carcinomas (ICCs) supporting the primary role of HPV in ICC etiology; however, there have been few large studies of secondary markers of HPV status. Objectives: HPV16 positive ICCs were examined for viral DNA load, chromogenic in situ hybridization (CISH) signal patterns, and p16INK4a protein staining. Design: DNA extracts from whole tissue sections of ICC from Madhya Pradesh, India, were tested for HPV16 L1 and NADPH DNA by quantitative real-time PCR. Biotinyl-tyramide-based HPV DNA CISH and p16INK4a immunohistochemistry (IHC) were also performed. Results: Data were collected from 192 patients: ages 25.0-90.0; mean 48.7; SD 12.5; histopathology: 174 squamous cell carcinoma, 12 adenocarcinoma, 6 other. Viral loads ranged from 0.006 to 1780.0 HPV16 copies per cell equivalent (mean 69.0; SD: 214.7). CISH positive status (67.9% specimens) was associated with viral load (P<0.0001) as was p16INK4a diffuse staining (78.6% ICC, P=0.046). There was also a significant relationship between CISH and p16INK4a staining (P=0.0002). One CISH positive sample showed only diffuse (episomal HPV) signals; all others showed punctate (integrated HPV) staining, with 12 showing punctate and diffuse patterns. A wide variation was observed with respect to CISH signal intensity, number of punctate signals per cell, and percentage of tumor cells showing staining. Conclusion: These data demonstrate that there is considerable HPV secondary marker heterogeneity among ICCs. Specimens showing a low viral load, negative CISH and negative p16INK4a staining, may indicate ICCs positive for passenger (rather than driver) HPV. Further studies are warranted to assess the relationship of viral load, CISH and p16INK4a staining to other tumor markers and to evaluate their possible diagnostic value for the management of patients with cervical carcinoma.
P-14.22
CERVICAL LESION GRADE BIOMARKER ASSAY BY RNA IN SITU HYBRIDIZATION
K Cooper, University of Vermont, Burlington, United States Z Peng, University of Vermont, Burlington, United States X J Ma, Advanced Cell Diagnostics, Hayward, United States X Wu, Advanced Cell Diagnostics, Hayward, United States Y Luo, Advanced Cell Diagnostics, Hayward, United States M F Evans, University of Vermont, Burlington, United States Background: Cervical carcinogenesis proceeds subsequent to an HPV infection and via increasingly dysplastic epithelia. Much remains to be understood about the biology underlying this process. Additionally, there remains a need for markers to aid the diagnosis of cervical intraepithelial neoplasia (CIN) grade and the potential for progression to invasive cervical carcinoma (ICC). Objectives: This study investigated a novel highly sensitive chromogenic in situ hybridization (CISH) assay called RNAscope for high-risk (HR) HPV E6/E7 and p16 gene expression in cervical specimens in comparison with HPV DNA CISH and p16INK4a immunohistochemistry (IHC) to assess the potential of the method for HPV research and diagnostics. Methods: Formalin-fixed, paraffin-embedded specimens: 2 normal, 13 CIN1, 37 CIN2/3 (preselected on the basis of HPV16 positive cytology) and 5 ICC, from 52 patients were examined for HPV E6/E7 (18 HR types) and p16 RNA expression. Biotinyl-tyramide-based HPV DNA CISH and p16INK4a IHC (E6H4 clone) were also performed. Results: Staining patterns for E6/E7 and p16 RNA and p16INK4a protein correlated strongly with severity of CIN grade (all p<0.001), with 1/3 thickness staining of the lesion epithelium in CIN1, and 2/3 to 3/3 in CIN2/3. Exceptions included one CIN1 and one CIN2/3 that showed E6/E7 and p16 RNA staining but which were p16INK4a IHC negative. HPV DNA CISH signal patterns (diffuse and/or punctate) matched CIN grade in the 69.5% that stained positive; 98.0% CIN stained for E6/E7 RNA (p=0.0002). All four assays showed data concordance with normal and ICC tissues. Conclusions: The RNAscope E6/E7 assay demonstrated superior sensitivity to HPV DNA CISH. The data support CISH RNA expression analysis in the investigation of cervical lesion biology. The findings also support the potential of E6/E7 and/or p16 RNA expression analysis in the diagnosis of CIN grade.
Declaration of interest K Cooper, Z Peng, MF Evans - none declared X-J Ma, Y Luo and X Wu are employees and stockholders of Advanced Cell Diagnostics
62

HPV DETECTION, P16/PR EXPRESSION AND HISTOLOGICAL DIAGNOSIS OF CERVICAL ADENOCARCINOMA
D JENKINS, Nottingham University, tynemouth, UNITED KINGDOM, X Zhang, CICAMS, Beijing, China, A Molijn, DDL, Voorburg, Netherlands, J E Schmidt, GSKbiologicals, Wavre, Belgium, Q Li, CICAMS, Beijing, China, E Pirog, Cornell-Weill Medical College, New York, USA, W Chen, CICAMS, Beijing, China, Y L Qiao, CICAMS, Bejing, China, Chinese HPV typing group, CICAMS, Beijing, China Background: Cervical adenocarcinoma (CADC) has multiple histological subtypes. The diagnosis of these and the relation to Human Papilomavirus (HPV) remains a challenge. Objectives: To investigate whether correlating the results from HPV DNA testing and immunohistochemical staining improves the diagnostic accuracy of CADC. Methods: Paraffin-embedded tissue blocks diagnosed locally as CADC are being obtained from study sites covering all regions of China. Cases are being subjected to central histological review (CICAMS), and external expert consensus review supported by immunohistochemical staining using p16 and progesterone receptor (PR). HPV DNA is detected using SPF10 PCR-DEIA-LiPA25 version 1 (Labo Biomedical Products, The Netherlands, based on SPF10 licensed Innogenetics technology). Additional HPV testing will be performed on HPV DNA negative cases. Results: Altogether 1019 CADC cases have been collected to date. 196 cases (average age 45 years) have been processed at CICAMS using sandwich cutting, histological review and HPV DNA testing, of which 115 have been examined and validated by expert pathology review. 57/115(49.6%) were diagnosed as true cervical adenocarcinoma (ADC-CX), 8/115(7.0%) as endometrioid adenocarcinoma (ADC-ENDO), and 50 cases of other histological subtypes. HPV prevalence in CADC varied by subtype: 75.4%(43/57) in ADC-CX, 60%(3/5) in Clear-cell adenocarcinoma, 50%(4/8) in ADC-ENDO, 33.3%(1/3) in minimal deviation ADC, 80.5(33/41) in unspecified ADC, 0.0%(0/1) in Serous ADC. Diffuse p16 immunohistochemical staining is correlated to HPV infection (2=45.1, P<0.001) and is seen in 68.4%(39/57) in ADC-CX, 60.0%(3/5) in ADC-CC, 50%(4/8) in ADC-ENDO, 0%(0/3) in ADC-MIN, 80.5%(33/41) in ADCNOS, and 0.0%(0/1) in ADC-SER. PR staining was frequently positive only in ADC-ENDO(75% 6/8). Conclusion: Correlating results from HPV DNA testing and immunohistochemical staining appears to improve accuracy of histological diagnosis of CADC and predict the presence of oncogenic HPV.
Declaration of interest This work was sponsored by GlaxoSmithKline Biologicals through a grant to Chinese Academy of Medical Sciences. The presenting author is a consultant to GlaxoSmithKline Biologicals and JE Schmidt is an employee of GSK biologicals. Anco Molijn is an employee of DDL and Edyta Pirog is a consultant to DDL.
P-14.24
CERVICAL NITRIC OXIDE IN CHLAMYDIA TRACHOMATIS AND HUMAN PAPILLOMAVIRUS INFECTION
P Rahkola, Helsinki University Central Hospital, Helsinki, FINLAND M Visnen - Tommisk, Helsinki University Central Hospital, Helsinki, FINLAND E Hiltunen - Back, Helsinki University Central Hospital, Helsinki, FINLAND E Auvinen, Helsinki University Central Hospital, Helsinki, FINLAND O Ylikorkala, Helsinki University central Hospital, Helsinki, FINLAND T S Mikkola, Helsinki University Central Hospital, Helsinki, FINLAND Background: Venereal Chlamydia trachomatis escapes cervical immunologic defense and favors the pathogenesis of high-risk human papillomavirus (hrHPV). Nitric oxide is produced by uterine cervical cells and is stimulated by hrHPV. Objectives: To compare cervical nitric oxide release in women with and without Chlamydia trachoma-tis and hrHPV infection. Methods: Thirty-nine women were studied regarding infection with Chlamydia trachomatis and hrHPV by using specific RNA- and DNA-based tests. Levels of cervical fluid nitric oxide metabolite (NOx) were assessed by the Griess reaction. Results: Twenty-one (54%) women showed the presence of Chlamydia trachomatis. Fourteen (67%) Chlamydia trachomatis-infected women and three (17%) Chlamydia trachomatis noninfected women had concomitant hrHPV infection. The level of cervical fluid NOx in women with Chlamydia tra-chomatis (median 37.5 mol/L, 95% CI: 26.150.9) was higher (p = 0.02) than in Chlamydia trachoma-tis noninfected women (median 19.7 mol/L, 95% CI: 5.630.0). The presence of hrHPV did not associ-ate with any difference in NOx levels between Chlamydia trachomatis infected or noninfected women. Conclusions: Chlamydia trachomatis was associated with increased release of nitric oxide metabolite in the uterine cervix. This stimulus was stronger than that of hrHPV, because no additional rise in NOx in was seen in women with concomitant Chlamydia trachomatis and hrHPV infection.
63

ANALYSIS OF BIOMARKER PROFILES TO PREDICT HPV16 INFECTION OUTCOME
A Brochot, CHU Reims, Hpital Maison Blanche, REIMS , FRANCE V Dalstein, CHU Reims, Hpital Maison Blanche, REIMS , FRANCE E Cailliez, CHU Reims, Hpital Maison Blanche, REIMS , FRANCE M Lorenzato, CHU Reims, Hpital Maison Blanche, REIMS , FRANCE S Caudroy, CHU Reims, Hpital Maison Blanche, REIMS , FRANCE P Birembaut, CHU Reims, Hpital Maison Blanche, REIMS , FRANCE C Clavel, CHU Reims, Hpital Maison Blanche, REIMS , FRANCE Background: In the perspective of a future cervical screening based on primary HPV testing, the management of HPV positive women, and mostly HPV16 positive women, will be a challenge. There will be a need for risk stratification to distinguish between the vast majority of transient HPV infections and the few persistent infections that need intensified follow-up. Objectives: The aim of this study is to analyze putative biomarkers, alone or in combination, to evaluate their use to predict the outcome of HPV16 infection as transient or as persistent and progressive infection. Methods: Patients from the Reims HPV Cohort Study, with an HPV16 infection and normal cytology at baseline and archival DNA/RNA available, are selected according to the follow-up data: (i) patients with subsequent negative HPV16 and normal cytology smears, referred to as transient/benign HPV16 infection group; (ii) patients who presented a progressive disease leading to CIN2/3 lesion with persistent HPV16 infection, referred to as persistent/ progressive HPV16 infection group. The following biomarkers are being assessed on the baseline sample: coinfections with other HPV genotypes (InnoLipa test), ploidy, full-length E6/E7 mRNA detection (EasyQ), E2 gene integrity (5 overlapping PCR and 1 full-length E2 gene PCR), L1 methylation status (pyrosequencing). Results: The selection of patients is ongoing, as well as the analysis of the biomarkers baseline status. To date, we identified 49 and 10 patients in the benign and progressive HPV16 groups, respectively. Cumulative probabilities of CIN2/3 development will be estimated using the Kaplan-Meier method for each selected biomarker, alone or in combination. Conclusions: The definition of such a biomarker profile to predict cervical precancerous lesion would be useful for the management of HPV16 infections associated with a normal cytology. The potential impact would also be important in the near future for the follow-up of vaccinated women who could be HPV16 positive.
P-14.26
DETECTION OF HPV16 E6T350G VARIANT AND PROGRESSION OF CERVICAL INJURY.
R Carballo, Hospital Universitario Central de Asturias, Oviedo, SPAIN, M E Alvarez - Argelles, Hospital Universitario Central de Asturias, Oviedo, SPAIN, M Torralba, Hospital Universitario Central de Asturias, Oviedo, SPAIN, J A Boga, Hospital Universitario Central de Asturias, Oviedo, SPAIN, S Meln, Hospital Universitario Central de Asturias, Oviedo, SPAIN, S Perez, Hospital Meixoeiro, Vigo, SPAIN, M de Oa, Hospital Universitario Central de Asturias, Oviedo, SPAIN INTRODUCTION: The HPV16 and HPV16 E6 variants, specially European-T350G(L83V)E6 variant, are risk factors for viral progression to high-grade lesions. OBJECTIVES:1.-To determine the incidence of T350G variant in our environment. 2.-To associate the presence of T350G with cytological/histology characteristics of patients and with the age. 3.-To study the T350G variant as a risk factor of progression. MATERIALS AND METHODS: A total of 150 women (36.212,2;17-76) and 19 men (35.913.8;24-73) who attended to a STI or gynecology consultations were analyzed. Among the women, 5 had warts, 62 had different grades of cytological alterations and 83 were subjected to a screening. T350G variant detection test was performed by a RT-PCR Taqman allelic discrimination assay using two primers (forward/reverse) and two TaqMan MGB probes labeled with VIC and FAM to detect 350T and 350G, respectively. RESULTS: T350G variant was detected in 66(46.7%) women [30(43.5%) with cytological alterations, 24(40.7%) subjected to a screening and 12(54.5%) from the STI consultation]. T350G variant was also found in 13 men (68.4%). The lesions progressed in 6(24%) women with T350 and in 8 (32%) women with T350G (16663.8;52-221 vs. 297222.8;81-772 days)(OR=1.33). Among women with cytological alterations, the mean age of those with T350G and with T350 was 35.9310.47;20-54 vs 31.258.33;18-49, respectively (p=0.041). Among women with normal cytology, the mean age of those with T350G and with T350 was 34.310.7;17-66 vs 42.64.5;19-76, respectively (p=0.0067). CONCLUSIONS: 1. T350G is highly prevalent and is distributed in the same way in the population studied. 2. After a following for 6-10 months, T350G was not associated with progression of cervical lesions, but a higher number of patients would verify such relationship. 3. Women with cytological alterations and with T350G were most older than those with T350 and women with normal cytological and with T350G were most younger than those with T350.
64

CIN 3, P16INK4 AND POST-CONIZATION EVOLUTION
F CARDOSO, SANTA CASA DE SO PAULO, SO PAULO, BRAZIL M EDUARDO, SANTA CASA, SO PAULO, BRAZIL A CAMPANER, SANTA CASA, SO PAULO, BRAZIL M A SILVA, SANTA CASA, SO PAULO, BRAZIL A COELHO, SANTA CASA, SO PAULO, BRAZIL F CARDOSO, SANTA CASA, SO PAULO, BRAZIL Background: Several markers have been investigated for their ability to identify women at risk of recurrence after cervical conization. One such marker is p16NK4a. The lack of relevant studies regarding this protein and postconization evolution after CIN 3 formed the rationale behind conducting the present investigation. Objective: to assess the prognostic value of p16INK4a as a marker of post-conization relapse in patients treated for cervical intraepithelial neoplasia grade 3 (NIC 3). Methods: A retrospective study of 76 women with NIC 3 diagnoses, treated at the Hospital da Santa Casa de Misericrdia de So Paulo (Brazil) between January 2003 and September 2004, was performed. The study samples were obtained from cervical conization procedures, where paraffin blocks containing the most representative areas of intraepithelial lesions were selected. Immunohistochemical techniques were used on individual paraffin blocks for each case to detect p16INK4a protein expression. The p16INK4a cell counts were performed in ten different high-amplification fields (400x) by light microscopy and total cell count expressed as number of cells per mm2. Patients involved in this study were followed up at the colposcopy outpatient unit for at least 48 months after cervical conization. The correlation of p16INK4a values with post-conization evolution in the patients (disease relapse or disease free) was determined. Results: A significantly higher count of cells expressing p16INK4a was found in those patients with disease relapse during follow up (p< 0.001). The variables age, number of gestations and births correlated positively with number of cells expressing p16INK4a cells (p<0.001; p= 0.001; 0.009, respectively). No correlation was found for the variables menopause, hormonal contraception or smoking (p = 0.369, 0.425 and 0.853, respectively). Conclusion: p16INK4a can be considered a biomarker of cervical intraepithelial neoplasia grade 2 and 3 cases presenting high risk of relapse or evolution to invasive carcinoma.
P-14.28
BCL-2, SQUAMOUS CELL CARCINOMAS AND ADENOCARCINOMAS OF THE UTERINE CERVIX
F CARDOSO, SANTA CASA, SO PAULO, BRAZIL A CAMPANER, SANTA CASA, SO PAULO, BRAZIL M A SILVA, SANTA CASA, SO PAULO, BRAZIL A COELHO, SANTA CASA, SO PAULO, BRAZIL M EDUARDO, SANTA CASA, SO PAULO, BRAZIL Background: There is a great deal of debate in the literature with regard to the prognosis for cervical adenocarcinomas in relation to squamous cell carcinomas cases. Objective: To compare the imunoexpression of the Bcl-2 protein in squamous carcinomas and adenocarcinomas of the uterine cervix. Methods: The present study involved the assessment of surgical specimens from 74 women with cervical carcinomas FIGO stage IB1 (54 squamous cell carcinomas and 20 adenocarcinomas). The study samples were obtained from selected paraffin blocks containing specimens from patients submitted to surgical procedures. The respective medical charts of patients were reviewed and epidemiologic, clinical and disease-related data were collected. Cervical specimens were assessed by the immunohistochemistry technique using the Bcl-2 protein as a marker. The reactions were considered positive when the cells became stained in brown color. Bcl-2 positive cells were counted in 10 fields under a high magnification (400x) using light microscopy, in the slides area containing squamous carcinoma and adenocarcinoma of the cervix. The total cell count was expressed as the number of positive Bcl-2 cells per mm2. Results: No significant difference in the number of cells marked by the Bcl-2 protein was found for the variables age, tumor diameter, angiolymphatic invasion or number of lymph nodes affected. Comparison of the number of cells marked by the Bcl-2 protein in the two histological groups revealed a statistically significant difference, with squamous tumors presenting a greater number of marked cells. Conclusion: Squamous cervical tumors present a greater number of positive Bcl-2 cells per mm2, suggesting that that the rate of cell death in squamous cell carcinomas of the cervix is lower than in adenocarcinomas.
65

P53, SQUAMOUS CELL CARCINOMAS AND ADENOCARCINOMAS OF THE UTERINE CERVIX
F CARDOSO, SANTA CASA , SO PAULO, BRAZIL A CAMPANER, SANTA CASA, SO PAULO, BRAZIL M A SILVA, SANTA CASA, SO PAULO, BRAZIL M EDUARDO, SANTA CASA, SO PAULO, BRAZIL A COELHO, SANTA CASA, SO PAULO, BRAZIL Background: There is a great deal of debate in the literature with regard to the prognosis for cervical adenocarcinomas in relation to squamous cell carcinomas cases. Objective: To compare the imunoexpression of the p53 protein in squamous carcinomas and adenocarcinomas of the uterine cervix. Methods: The present study involved the assessment of surgical specimens from 74 women with carcinomas of the uterine cervix, stage IB1 (54 squamous cell carcinomas and 20 adenocarcinomas). The study samples were obtained from selected paraffin blocks containing specimens from patients submitted to surgical procedures. The respective medical charts of patients were reviewed and epidemiologic, clinical and disease-related data were collected. Cervical specimens were assessed by the immunohistochemistry technique using the p53 protein as a marker. The reactions were considered positive when the cells became stained in brown color. p53 positive cells were counted in 10 fields under a high magnification (400x) using light microscopy, in the slides area containing squamous carcinoma and adenocarcinoma of the cervix. The total cell count was expressed as the number of positive p53 cells per mm2. Results: No significant difference in the number of cells marked by the p53 protein was found for the variables age, tumor diameter, angiolymphatic invasion or number of lymph nodes affected. Comparison of the number of cells marked by the p53 protein in the two histological groups revealed no statistically significant difference. Conclusion: There was no significant correlation between p53-immunostaining and the histological type of cervical carcinoma.
P-14.30
BIOMARKERS OF IRON STORAGE AND TYPE SPECIFIC HUMAN PAPILLOMAVIRUS CLEARANCE
E Siegel, Moffitt Cancer Center, Tampa, UNITED STATES, N Patel, James A. Haley Veterans' Hospital, Tampa, UNITED STATES, L Galan, Ludwig Institute for Cancer Research, Sao Paulo, BRAZIL, B Lu, Moffitt Cancer Center, Tampa, UNITED STATES, J H Lee, Moffitt Cancer Center, Tampa, UNITED STATES, A Nyitray, Moffitt Cancer Center, Tampa, UNITED STATES, X Huang, NYU Cancer Institute and School of Medicine, New York, UNITED STATES, L Villa, Ludwig Institute for Cancer Research, Sao Paulo, BRAZIL, E Franco, McGill University, Montreal, CANADA, A Giuliano, Moffitt Cancer Center, Tampa, UNITED STATES Background: Several studies have documented that elevated iron promotes cancer cell proliferation and causes oxidative DNA damage through its interaction with oxygen and hydrogen peroxide. Increased reactive oxygen species may adversely affect the natural history of HPV infections by increasing the oxidant:antioxidant balance and directly influence transcriptional activity. Thus, elevated iron levels are hypothesized to be an HPV-cofactor that is associated with longer duration of infection and HPV-associated carcinogenesis. To date, the association between iron status and early events in cervical carcinogenesis, such as HPV clearance, has not been investigated. Objectives: The purpose of this study was to determine if iron status is associated with clearance of incident typespecific HPV infections. Methods: Ferritin and soluble transferrin receptor (sTfR) were measured in baseline serum samples from 413 HPVpositive women enrolled in the Ludwig-McGill Cohort Study. HPV clearance rates (any type, oncogenic HPV, nononcogenic HPV, and HPV 16) over 36 months were estimated from Cox-proportional hazard models with a robust sandwich estimator covariance matrix to account for within-subject correlations due to inclusion of multiple infection events from one participant. Results: Among this population of pre-menopausal women, the median ferritin level was 27.6 ug/L (Range 1.0391.5 ug/L). Women with ferritin levels above the median were less likely to clear an incident oncogenic HPV infection [Adjusted Hazard Ratio (AHR) = 0.71; 95% CI 0.52-0.96]. Using physiological cut-points, women with overloaded iron stores (120 ug/L) were less likely to clear incident oncogenic HPV infections compared to those with low-levels of iron (<20ug/L) [AHR=0.34; 95% CI 0.14-0.85]. Conclusion: We found that women with increased ferritin levels are less likely to clear incident HPV infections compared to women with the low-levels of ferritin. Rising iron stores may increase risk for cervical dysplasia or cancer.
Declaration of interest E. Franco: Consultant to Qiagen, Roche, and Cytyc All other authors: None Declared
66

ASSOCIATION BETWEEN DELTANP73 EXPRESSION AND HPV INFECTION IN CERVICAL CANCER
H Ngan, The University of hong Kong, Hong Kong, CHINA S Liu, The University of hong Kong, Hong Kong, CHINA R Leung, The University of hong Kong, Hong Kong, CHINA A Cheung, The University of hong Kong, Hong Kong, CHINA Background: Cervical cancer is a common genital tract cancer in women. Persistent infection of high-risk Human papillomavirus (HPV) is recognized as a necessary cause of cervical cancer. The high-risk HPV encodes two oncoproteins E6 and E7, which disrupt the tumour suppressive functions of p53 and pRb. p73 is a member of p53 tumor suppressor family and shares both structural homology and functional similarities with p53. It exists as two major forms: transactivation active TAp73 isoform and dominant negative DeltaNp73 isoform, with opposing pro- and anti-apoptotic functions, respectively. Our previous study has demonstrated the differential expressions of p73 and its prognostic significance in cervical cancer. As HPV is the important pathogen for the development of cervical cancer, the relationship between p73 and HPV warrants further study. Objective: To assess the association between the expression of DNp73 and HPV16/18 infections in cervical cancer. Methods: Totally 118 cervical cancers and 113 normal cervical tissues were recruited and paraffin sections were prepared for immunohistochemical staining of DNp73. Genomic DNAs were extracted from cervical cancer tissues for detection of HPV infection. Results: The expression of DNp73 was significantly higher in cervical cancer cells than that in normal cervical epithelial cells (p<0.001). HPV16 and HPV18 infections were detected in 48.3% and 21.2% of cancer patients, respectively. Cancers with HPV infection tended to have higher DNp73 expression. Most of HPV16 positive cancers showed significantly higher DNp73 expression than HPV16 negative cancers (p=0.035). Conclusions: Oncogenic DNp73 was over-expressed in cervical cancer, especially in HPV16 infected cells. The underlying mechanism is under investigation.
P-14.32
A DOSE-RESPONSE RELATIONSHIP BETWEEN HPV VIRAL LOADS AND CERVICAL NEOPLASIA
H C Chen, Academia Sinica, Taipei, TAIWAN, B H Lee, King Car Food Industrial Co. Ltd., I-Lan, TAIWAN, M H Pan, Academia Sinica, Taipei, TAIWAN, C Y Lin, Taipei Medical University, Taipei, TAIWAN, Y C Chou, National Taiwan University, Taipei, TAIWAN, S L You, Academia Sinica, Taipei, TAIWAN, C Y Hsieh, National Taiwan University Hospital, Taipei, TAIWAN, C J Chen, Academia Sinica, Taipei, TAIWAN BACKGROUND Human papillomavirus (HPV) is the major cause of cervical neoplasia. The role of HPV quantity has rarely been studied. OBJECTIVES To assess the associations between viral loads of major HPV types and cervical neoplasia prevalence in Taiwan. METHOD There were 11,923 women from seven townships participated in this Community-Based Cervical neoplasia Screening Project (CBCSP), which provided two health examinations in 1991-1993 and 1993-1995. Participants signed their informed consent for Pap smear examination, cervical cells collection, and cancer status follow-up through computerized data linkage with national cancer registry profile. Deep-frozen cervical cells were used for HPV DNA amplification by polymerase chain reaction and genotyping by HPV Blot to identify 39 types of HPV. Viral loads of HPV16, 18, 31, 33, 52 and 58 were measured by types-specific real-time PCR and expressed as copies/50ng DNA. RESULTS There were 10,602 women with available Pap smear and cervical cells at enrollment. Participants positive for HPV16 (n=210), HPV18 (n=170), HPV31 (n=72), HPV33 (n=123), HPV52 (n=254) and HPV58 (n=138) were included in this analysis. Consistently, the mean viral loads of these carcinogenic HPVs increased from cytologically normal women to women with cytological ASCUS/LSIL and HSIL+. High viral load (10,000 vs. <10,000 copies/50 ng DNA) of HPV16, HPV18, HPV31, HPV33, HPV52 and HPV58 was associated with an increased age-adjusted prevalence odds ratio (95% confidence interval) of cytological HSIL+ of 33.39 (11.80-94.47), 3.46 (0.90-13.32), 1.64 (0.48-5.63), 24.76 (5.90-103.17), 3.93 (1.66-9.27), and 11.38(4.90-30.87), respectively. For each log (viral load) increase, the ageadjusted prevalence odds ratio (95% confidence interval) of cytological HSIL+ for HPV16, HPV18, HPV31, HPV33, HPV52 and HPV58 was 2.61 (1.88-3.63), 1.42 (0.94-2.15), 1.98 (1.16-3.37), 2.98 (1.78-5.00), 1.88 (1.35-2.61) and 2.31 (1.59-3.35). CONCLUSION Our findings suggest quantitative biomarker (viral load) of HPV is associated with an increased prevalence of cytological HSIL+ among HPV-infected women.
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CO-INFECTIONS OF EBV AND HPV: A COFACTOR OF CERVICAL CANCER
T Ekalaksananan, Khon Kaen University, Khon Kaen, THAILAND S Aromseree, Khon Kaen University, Khon Kaen, THAILAND N Sunthamala, Khon Kaen University, Khon Kaen, THAILAND P Swangphon, Khon Kaen University, Khon Kaen, THAILAND C Pientong, Khon Kaen University, Khon Kaen, THAILAND A Chaiwongkot, Khon Kaen University, Khon Kaen, THAILAND B Kongyingyoes, Khon Kaen University, Khon Kaen, THAILAND N Patarapadungkit, Khon Kaen University, Khon Kaen, THAILAND B Chumworathayee, Khon Kaen University, Khon Kaen, THAILAND Background: Cervical cancer is caused by human papillomavirus (HPV). Approximately 80% of genital HPV infection will regress within 2 years in women under 35 years old and only small percentage of HPV infected women will develop cervical cancer indicates that there are other cofactors which influence the disease development. Some studies found that Epstein-Barr virus (EBV) might have a role in cervical carcinogenesis but there is no strongly supportive experimental evidence. In contrast, someone could not identify EBV in the cervical cancer samples. Objectives: (i) To evaluate the prevalence of EBV and HPV in cervical biopsies with no squamous intraepithelial lesion (noSIL), low grade SIL (LSIL), high grade SIL (HSIL) and cervical cancer (CA). (ii) To determine the association between the EBV and HPV co-infections and cervical cancer development. Methods: DNA was extracted from cervical biopsies and qualified by beta-globin gene amplification. The presence of EBV and HPV DNA were detected by PCR using specific primers to EBV DNA polymerase and HPV L1 genes, respectively. HPV genotypes were determined by reverse line blot hybridization assay. Data were analyzed by SPSS 13.0. Results: The presence of EBV DNA in noSIL, LSIL, HSIL and CA groups were 37.50% (12/32), 14.29% (5/35), 60.0% (27/45) and 38.71% (12/31), respectively while the HPV DNA were detected in 43.75% (14/32), 42.86% (15/35), 82.22% (37/45) and 93.55% (29/31), respectively. All of 143 cases, co-infections of HPV and EBV were detected in 39 cases and more frequently found in HSIL/CA group than in noSIL/LSIL (p < 0.05, OR = 5.081). We also found that most of these cases (18/39) were co-infected with HPV16. Conclusions: The data indicate that there is the association between co-infections of EBV and HPV and the progression of cervical cancer. Thus, EBV might be a possible cofactor of cervical cancer development.
P-14.34
BIOMARKERS IN CERVICAL SCREENING: ANALYSIS PRC1, CDC20, CCNB2, AND CDKN3
A Espinosa, Genomic Medicine Unit,Hospital General de Mxico, Mxico, MEXICO A Alfaro, Genomic Medicine Unit, HGM, Mxico I Palma, Escuela Superior de Medicina IPN, Mxico C Serralde, Genomic Medicine Unit, HGM, Mxico I Medina, Genomic Medicine Unit, HGM, Mxico E Juarez, Genomic Medicine Unit, HGM, Mxico E Marquez, IIMAS,UNAM, Mxico E Roman - Basaure, Oncology Service,HGM, Mxico S Muoz - Cortez, Gynecology Sevice, HGM, Mxico J Berumen, Genomic Medicine Unit, HGM Facultad de Medicina UNAM, Mxico Background: sensitive and specific molecular markers are needed to identify the cervical intraepithelial neoplasia (CIN). The detection of cellular proteins that are up-regulated by HPV-infected cells might play an important role in future cervical cancer screening strategies. Objectives: the aim of this study is to investigate the sensitivity and specificity of four genes, yet uncovered: PRC1, CDC20, CCNB2 and CDKN3, which correlate with progression to neoplasia in cervical cancer. Methods: a total of 146 biopsies were analyzed from different grades, normal (n=25), CIN-I (n=35), CIN2/3 (n=21) and invasive tumors (n=65). In this work, four potential markers were screened with quantitative reverse transcription PCR (QRT-PCR) and immunohistochemistry (IHC). Results and Conclusions: Based on a receiver operating characteristic (ROC) curve analysis, genes that showed the higher sensitivity and specificity and clearly separated control from tumors samples. PRC1 showed greater sensitivity and specificity, 100% and 97% respectively, associated exclusively with invasion, CDC20 with high grade lesion (CIN-2/3), finally, CCNB2 and CDKN3, either to low or high grade neoplasia, including invasion. Additionally, selected genes were confirmed by IH in another independent group of samples. This is the first study to simultaneously compare normal cervical tissue, cervical intraepithelial neoplasia, and invasive cervical cancer using a panel of four markers for screening, which allows the detection and differentiation of neoplasic lesions of cervix. Thus, by targeting specific biomarkers, aids in the screening, diagnosis and in the assessment of the prognosis of the disease.
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FOUR SNPS IN RELATION TO CERVICAL DYSPLASIA AND CANCER DEVELOPMENT
H von Keyserling, Charit, Berlin, Berlin, GERMANY, T Bergmann, MPI Mol.Gen., Berlin, GERMANY, M Schuetz, Charit, Berlin, Berlin, GERMANY, U Schiller, Charit, Berlin, Berlin, GERMANY, J Stanke, Charit, Berlin, Berlin, GERMANY, C Hoffmann, Charit, Berlin, Berlin, GERMANY, A Schneider, Charit, Berlin, Berlin, GERMANY, H Lehrach, MPI Mol.Gen., Berlin, GERMANY, A Dahl, MPI Mol.Gen., Berlin, GERMANY, AM Kaufmann, Charit, Berlin, Berlin, GERMANY Here, we describe a retrospective screening study on archived material for HPV type and single nucleotide polymorphisms (SNP) in genetic markers TP53, MTHFR, CYP1A1, and CYP2E1. Methods: EHR of 749 patients were reviewed. A highly specific ligation-detection reaction procedure in combination with a sensitive TaqMan PCR approach was developed that allowed simultaneous allelic discrimination of SNPs in a multiplexed approach. The method was successfully applied to low input DNA derived from HPV diagnostic procedures and was applicable in a high-throughput protocol. For association studies, controls were healthy or had only mild dysplasia. Cases were defined to have high grade dysplastic lesions or invasive cervical cancer. Results: A strong association with cervical cancer and high grade dysplasia was found for the rare homozygous CC genotype (rs4646903) in CYP1A1 (OR = 8.862, CI(95%) = 1.11970.212, P = 0.034). Significantly elevated odds ratios were also found in MTHFR SNP genotype (rs1801133) (OR = 1.457, CI(95%) = 1.056-2.01, P = 0.022). No significant association was found in TP53 (rs1042522) and CYP2E1 (rs3813867). Additionally, we found smokers at higher risk (OR = 2.688 CI(95%) = 1.718-4.205, P <0.001) and identified pregnancies as a significant risk factor (OR 1.54, CI(95%) 1.25-2.108, P = 0.012). Conclusion: The particularity of our study is i) the use of archived material from bio banks, ii) the integration of the corresponding clinical data by semantic integration, a method to identify and evaluate the clinical status by information from electronic health records (EHR), and iii) the development of a multiplexed genotyping assay for the analysis of SNPs in TP53, MTHFR, CYP1A1, and CYP2E1. We present a protocol enabling large retrospective screening studies on archived material derived from residual DNA extracts (e.g. HPV typing) that is high-throughput compatible. Additionally, this study supports association of cervical cancer with certain genetic and environmental factors.
Declaration of interest Conflict of interest statement The authors declare that there are no conflicts of interest.
P-14.36
BIOMARKERS AND HR-HPV RESULTS IN HIGH PRECISION SCREENING DIAGNOSIS
C Jeney, Genoid Ltd., Budapest, Hungary J Mzes, Genoid Kft., Budapest, Hungary N Varga, Genoid Kft., Budapest, Hungary M Benczik, Genoid Kft., Budapest, Hungary R Koiss, Szent Istvn Hospital, Budapest, Hungary C Martin, University of Dublin, Dublin, Ireland H Keegan, University of Dublin, Dublin, Ireland J O' Leary, University of Dublin, Dublin, Ireland Background. We have developed a novel, mRNA based expression biomarker set for improved cervical cancer screening. The method is based on liquid based cytology specimen and offers application in the framework of the current screening protocols. Objectives. The effective application of the diagnostic criteria of cervical cytology is operator dependent and varies as a function of experience and training. Our method is an objective surrogate marker for cytological lesions offering possibilities to improve the overall screening performance and reducing colposcopic referral rate. Methods. The performance accessed on a colposcopy referral population (n=700, prevalence of CIN1+: 0.55) and a screening population (n=249, prevalence of HR-HPV: 0.15). Results. The combined test has a sensitivity for CIN1+ of 0.75 (95%CI 0.72-0.81) and high specificity (0.96 95%CI 0.94-0.99). For CIN2+ the corresponding values are 0.85 95%CI 0.81-0.89 and 0.97 95%CI 0.94-0.99. In this study the HPV detection sensitivity (0.87 95%CI 0.84-0.89) was lower than expected. Conclusion. The marker combination has high sensitivities and specificities. Alone or with a combination of the existing screening methods our results justify the assessment of performance in large screening population based study.
Declaration of interest C Jeney, J Mzes and N Varga are employees of Genoid Ltd.
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EXPRESSION OF HPV-RELATED BIOMARKERS ASSOCIATED WITH THE GRADE OF CIN
S M Stasinou, Department of Obstetrics & Gynaecology, University Hospital of Ioannina, Ioannina, GREECE G Valasoulis, Department of Obstetrics & Gynaecology, University Hospital of Ioannina, Ioannina, Greece M Kyrgiou, Department of Gynaecologic Oncology - Obstetrics & Gynaecology, Queen Charlotte's & Chelsea Hammersmith Hospital, London, United Kingdom I Tsoumpou, Department of Obstetrics and Gynecology, University Hospitals of South Manchester, Manchester, United Kingdom G Koliopoulos, Department of Obstetrics & Gynaecology, University Hospital of Ioannina, Ioannina, Greece M Nasioutziki, Second Department of Obstetrics and Gynecology, Aristotle University of Thessaloniki, Hippokration Hospital, Thessaloniki, Greece A Loufopoulos, Second Department of Obstetrics and Gynecology, Aristotle University of Thessaloniki, Hippokration Hospital, Thessaloniki, Greece P Karakitsos, Department of Cytopathology, Attikon Hospital, University of Athens, Athens, Greece E Paraskevaidis, Department of Obstetrics & Gynaecology, University Hospital of Ioannina, Ioannina, Greece Objective: To assess the alterations of HPV-related biomarkers in relation with the grade of cervical intraepithelial lesion Materials & Methods: Design: Retrospective observational study. Inclusion criteria: Women referred to colposcopy with abnormal smear and available histological diagnosis (Punch Biopsies/LLETZ). Intervention: Prior to the biopsy an LBC sample was obtained and was tested for HPV typing, mRNA E6&E7 (NASBA technique), E6&E7 mRNA by flow cytometry and p16INK4a. Outcomes: Assessment of HPV-related biomarkers positivity or negativity rates correlated with the grade of the cervical intraepithelial lesion (chi square test for trend). Results: A total of 216 women were recruited, Twenty three were found to have negative histology (10.6%), 79/216 (36.6%) HPV and CIN1, 50/216 (23.1%) CIN2, 48/216 (22.2%) CIN3. In 16 cases the histology reported micro-invasive or invasive disease (7.4%). All the biomarkers have revealed an increased linear positivity rate with the progression of the grade of the intraepithelial lesion (chi-square for trend p<0,05). Amongst 23 women with negative histology, 6 were found to be positive for hr HPV, 3 for NASBA, 5 for mRNA E6&E7 estimated by flow cytometry and 2 had positive p16INK4a expression. Conclusions: The linear alteration between HPV-related biomarkers and the grade of cervical intraepithelial lesion suggest that these biomarkers may prove to be useful in the prediction of CIN grade. These biomarkers may be incorporated in a prediction Scoring System that could allowed tailored management and interventions.
P-14.38
OESTROGEN RECEPTOR EXPRESSION IN HPV-ASSOCIATED GENITAL NEOPLASIA
J C Sterling, University of Cambridge, Cambridge, UNITED KINGDOM M F Griffin, University of Cambridge, Cambridge, United Kingdom D A Winder, University of Cambridge, Cambridge, United Kingdom S L R Ball, University of Cambridge, Cambridge, United Kingdom P Goon, University of Cambridge, Cambridge, United Kingdom R A F Crawford, Addenbrooke's Hospital, Cambridge, United Kingdom J C Sterling, University of Cambridge, Cambridge, United Kingdom Background: No human model of cervical cancer currently exists, yet animal models have shown the involvement and the potential therapeutic target of oestrogen receptors. Oestrogen plays a significant role in other gynaecological cancers and the interplay between both the alpha (ER-1) and beta (ER-2) oestrogen receptor is of great interest. Objectives: The aim was to define alpha- and beta-oestrogen receptor (ER-1 and ER-2) expression in cervical HPVassociated disease Methods: Human genital samples were obtained. HPVs present in the tissue were identified by Linear Array HPV genotyping test and HPV 16 viral load was determined by quantitative PCR. Expression of both estrogen receptors was quantitatively assessed by qRT-PCR. Results: 54 adequate samples were obtained. The histological distribution in the samples was CIN1: 16% (n=8), CIN2: 30% (n=15), CIN3: 32% (n=16), squamous cell carcinoma (SCC):18% (n=9), and normal tissue: 4% (n=2). 49 (98%) samples contained HPV 16, whilst only 3 (6%) contained HPV 18. 72% of samples were infected with multiple types (36/50). 40 and 38 samples had detectable levels of alpha and beta oestrogen receptor mRNA respectively. ER-1 mRNA level was not significantly associated with histological status of CIN but was reduced in cervical cancer, however there was a trend seen with ER-2 expression increasing with grade of CIN. ER-2 expression in CIN of all grades was significantly greater than that in SCC samples. Neither ER-1 or -2 mRNA levels correlated with HPV 16 viral load. Conclusions: Alpha and beta oestrogen receptor expression is detectable in cervical HPV-associated intraepithelial disease but ER-1 is reduced and ER-2 possibly lost upon development of squamous cell carcinoma.
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PRESENCE AND PERSISTENCE OF E6/E7HPV58 VARIANTS IN WOMEN FROM COLOMBIA
M Molano, Instituto Nacional de Cancerologia, Bogota, COLOMBIA O Buitrago, Instituto Nacional de Cancerologia, Bogota, COLOMBIA N Morales, Instituto Nacional de Cancerologia, Bogota, COLOMBIA C Martin, Instituto Nacional de Cancerologia, Bogota, COLOMBIA A Huertas, Instituto Nacional de Cancerologia, Bogota, COLOMBIA T Martinez, Instituto Nacional de Cancerologia, Bogota, COLOMBIA P Moreno, Instituto Nacional de Cancerologia, Bogota, COLOMBIA Introduction: Certain variants of HPV58 have shown an enhanced oncogenicity for HGSIL and cervical cancer. However, there are not studies about its role in the persistence of the infection. Objectives: We report results of presence and persistence of E6/E7HPV58 variants in 71 women with incident HPV58 infections and normal cytology that belong to the Bogot cohort that were followed during seven years. In total 639 samples were analyzed. Methodology: HPV detection and typing was done using the GP5+/GP6+ PCR-RLB assay. The E6 and E7 regions of HPV 58 positive samples were amplified using E6 F1-E6 R1 and E7P1-E7P2 primers that amplify a fragment of 521 and 335bp respectively. E6/E7/HPV 58 variants were detected using a RLB assay and automated direct sequencing. Results: 117/639 samples (18.39%) were HPV58 positive. In 85.5% of the samples was possible identify E6/E7HPV58 variants. 88% of the samples showed the T307/A694/G744/A761 variant, 9% showed the T307/G744 variant and 3% showed the T187/T307/A367/G744/G793/T798/A801/T840/C852 variant. Globally 20% of the infections were still present at 2 years of follow up and 2% at five years. During follow up, women that had first sexual intercourse at >15 years of age were associated with a lower persistence rate than those at <15 years of age (RR=0.29; 95% CI 0.09-0.92). On the other hand, parous women were associated with a higher persistence rate than nulliparous women (RR=3.43, 95% CI 1.23-9.60). Conclusion: After five years of follow up only 2% of the HPV58 infections persist. There is not clear association of variants presence with persistence of the infection.
P-14.40
HLA-CIN ASSOCIATION ON A SCIENTIFIC STUDY: A POINT OF VIEW
M B S XAVIER, FEDERAL UNIVERSITY OF PARANA, CURITIBA, BRAZIL P P FRANA, FEDERAL UNIVERSITY OF PARANA, CURITIBA, BRAZIL G Y T REZENDE, FEDERAL UNIVERSITY OF PARANA, CURITIBA, BRAZIL N.S CARVALHO, FEDERAL UNIVERSITY OF PARANA, CURITIBA, BRAZIL C A MAESTRI, ERASTO GAERTNER CANCER HOSPITAL, CURITIBA, BRAZIL R SLOWIK, FEDERAL UNIVERSITY OF PARANA, CURITIBA, BRAZIL M G BICALHO, FEDERAL UNIVERSITY OF PARANA, CURITIBA, BRAZIL Background: Multiple determinants are involved in the progression of human papillomavirus (HPV)-infected cervical lesion to invasive cancer. Only a minority of women infected with oncogenic HPV types develop cervical intraepithelial neoplasia (CIN) or cervical cancer (CC), indicating that other factors such as an inadequate immune function are necessary for the development of progressive CIN lesions and CC. Human leukocyte antigens (HLAs) are important in the presentation of foreign antigens to the immune system and HLA-C genotypes have been associated with viral infections and cancer. Objectives: Our objective was to investigate the relations between HLA-C polymorphism and non-stratified-CIN in a sub-population of south Brazilian women before conization results. Methods: DNA from peripheral blood samples was examined by PCR and the HLA-C genotyping was performed using SSOP method on a group of 67 women presenting CIN, before conization results, treated in the Erasto Gaertner Hospital, Curitiba, Brazil and a control group of 154 women recruited from a cervical cancer prevention campaign. Statistical analysis was carried out using Chi-square test. Results: There were no differences in genotypic or allelic frequencies between CIN patients and controls. Before conization, the diagnostic by biopsy were: 42 CINII, 13 CINIII and 12 others lesions (cervicitis and CINI). After conization procedure, we observed that 40 women presented CIN II, 16 CIN III, 4 CC and 7 other lesions (cervicitis and CINI). No additional risk association was observed when the sample was stratified by cervical disease. Conclusion: The present study did not find association between HLA-C polymorphism and non-stratified CIN group from the results of biopsies. The present data indicate that immune response is different in specific degrees of CIN. Sample stratification by cervical disease well defined and large enough sample in each subgroup is necessary to evaluate the real impact of HLA-C in CIN and CC natural history.
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EXPRESSION OF SOD2 CORRELATES WITH REGIONAL METASTASIS IN PENILE CARCINOMA
L Termini, Ludwig Institute for Cancer Research, So Paulo, BRAZIL M A Andreoli, Ludwig Institute for Cancer Research, So Paulo, BRAZIL M C Costa, Ludwig Institute for Cancer Research, So Paulo, BRAZIL K B Ribeiro, HPV Institute - Santa Casa de Misericrdia, So Paulo, BRAZIL L Kagohara, Hospital AC Camargo, So Paulo, BRAZIL S Nonogaki, Hospital AC Camargo, So Paulo, BRAZIL G C Guimares, Hospital AC Camargo, So Paulo, BRAZIL A Longatto-Filho, So Paulo University School of Medicine, So Paulo, BRAZIL F A Soares, Hospital AC Camargo, So Paulo, BRAZIL L L Villa, Ludwig Institute for Cancer Research, So Paulo, BRAZIL Background: Penile carcinoma is a rare neoplasm and the highest incidences occur in developing countries. HPV infection has been identified as a possible etiologic agent of a significant proportion of cases. These tumors spread locally to regional lymph nodes and are treated surgically with high morbidity rates ranging from 30% to 90%. Therefore, one important goal is to identify molecular markers that can predict the presence of lymph node metastasis. Among other molecules, we have analyzed the expression of superoxide dismutase 2 (SOD2) protein levels in primary penile cancers. This protein belongs to a family of enzymes involved in the conversion of superoxide radicals in molecular oxygen. Importantly, numerous in vivo studies have shown that the superoxide dismutases can be highly expressed in aggressive human solid tumors. Objective: Identify molecular markers in penile carcinoma and study their association with HPV presence. Methods: Immunoreactivity of SOD2 and other molecular markers previously shown to be associated with cervical lesions progression was determined. HPV status and clinicopathological variables, including patient age, tumor stage, histological grade, tumor thickness, lymphatic and venous embolization, infiltration in corpora cavernosa, corpus spongiosum and urethra were analyzed. Results: Samples were grouped depending on the percentage of stained cells as weak or moderate (<50% of stained cells) and intense (>50% of stained cells). We observed that intense staining for SOD2 in primary penile carcinomas significantly correlates with the existence of lymph nodal metastasis (p=0.03). In this subset analysis, SOD2 positivity was not associated with HPV presence. Conclusions: High expression of SOD2 is correlated with regional metastasis in penile carcinoma.
P-14.42
C677T MTHFR POLYMORPHISM AS A PREDISPOSITION BIOMARKER TO CERVIX CANCER
M Bicho, Lisbon, Lisbon, PORTUGAL, M Carvalho, Lisbon, Lisbon, Portugal, I Alho, Lisbon, J Ferreira, Lisbon, A Silva, Lisbon, R Medeiros, Porto, M Bicho, Lisbon, C Marinho, Lisbon Introduction:The methylenetetrahydrofolate reductase(MTHFR) is a key enzyme in the folate cycle and is envolved in the S-Adenosylmethionine (SAM) sythesis,a methyl donor essential for the DNA methylation,particularly of oncogenes.This enzyme is inhibited by oxidative stress.The T allele of MTHFR C677T polymorphism is associated with increased thermolability and decreased affinity for FAD(derived from riboflavin),causing lower activity and inhibiting the formation of 5-methyltetrahydrofolate,required for SAM synthesis.However,MTHFR is also crucial for the synthesis of dTMP from dUMP,which accumulates an is incorporated instead of dTMP,leading to DNA instabillity. Material and Methods:The C677T polymorphism was evaluated in a population of 370 women(238 healthy and a case group constituted from 132 patients with cervix cancer and its precursor lesions.The MTHFR genotype was determined by PCR-RFLP(with Hinf I restriction enzyme) using DNA extracted from peripheral blood.Analysis for linear trend in proportions was done by Xsquare test with a confidence interval of 95% and a significance level of p<0.05. Results:The distribution of MTHFR genotypes in these two Portuguese populations subgroups (controls vs cases were CC=118(49.6%),CT=109(45.8%),TT=11(4.6%) and CC=57(43.2%),CT=60(45.5%),TT=15(11.4%) with an OR=2.823;95%CI(1.219-6.538);p=0.04.The linear trend of odds in the presence of T allele number has Xsquare=3.837 and p=0.05.When we take on account the interaction of polymorphism with steroid sex hormones the linear trend of odds for TT genotype has a Xsquare=8.003,p=0.005. Discussion and conclusions:These data sugested that the presence of MTHFR T allele may represent a higher risk for cancer,and aggravate that of sex steroid hormones,leading to decreased methylation(hypomethylation) of important regulatory genes,namely those with a relevant role on oncogenesis processes.
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A META-ANALYSIS OF CHROMOSOMAL INSTABILITY IN LOWER GENITAL TRACT LESIONS
L Thomas, Department of Applied Tumor Biology, Institute of Pathology, University of Heidelberg, Heidelberg, GERMANY S Vinokurova, Department of Applied Tumor Biology, Institute of Pathology, University of Heidelberg, Heidelberg, GERMANY M von Knebel Doeberitz, Department of Applied Tumor Biology, Institute of Pathology, University of Heidelberg, Heidelberg, GERMANY M Reuschenbach, Department of Applied Tumor Biology, Institute of Pathology, University of Heidelberg, H, M Reuschenbach, Department of Applied Tumor Biology, Institute of Pathology, University of Heidelberg, Heidelberg, GERMANY Background: Carcinomas of the lower genital tract may be associated with transforming HPV infections or may be induced by other cellular alterations. Objectives: We wanted to identify the most common chromosomal aberrations which are characteristic for HPV/ non HPV-induced lesions of different grades and localizations. Methods: A systematic literature search was performed in Medline for studies that analyzed chromosomal alterations by comparative genomic hybridization (CGH), in cancers or corresponding premalignant lesions of the anogenital tract (cervix, anus, vagina, penis, vulvar). All results, except for cervical carcinoma, were separated into HPV-positive and HPV-negative samples. Results: In total 26 studies were identified (cervix 15 studies, 482 samples; anal 3 studies, 60 samples; vagina 1 study, 16 samples, penile 1 study, 26 samples; vulva 6 studies, 84 samples).The most common alterations in cervical cancer were gains at 3q and 5p (in 53.4%, 174/326 and 51.8%, 169/326 respectively) and 11q losses (41.7%, 136/326). 3q gains and 11q losses were also observed in some high grade CIN (36/120) and 17/120, respectively). Data for other localizations are very scarce, but indicate similar pattern when compared to cervical cancer. Compared to HPVpositive carcinomas losses at 5q appeared more frequent in HPV-negative cancers. Conclusions: The results point to an increase of chromosomal aberrations during the progression from precancerous lesions to cervical squamous cell carcinoma with 3q gains and 11q losses predominating. These alterations were also observed in the limited number of analyzed vulvar, anal, vaginal and penile lesions. Compared to non-HPV induced carcinomas some differences were seen for 5q losses, however the number of so far analyzed HPV-negative tumors is low.
P-14.45
EVALUATION OF P16 PROTEIN IN HIV/HPV CERVICAL TMA SPECIMENS
N S Oliveira, LIPMED, Rio de Janeiro, BRAZIL S M Amaro- Filho, LIPMED-OC - Fiocruz, Rio de Janeiro, Brazil J Golub, Johns Hopkins Univ, Baltimore, USA W R Menezs, LIPMED, Rio de Janeiro, Brazil C Pirmez, LIPMED -IOC - Fiocruz, Rio de Janeiro, Brazil C A Viana, IFF - Fiocruz, RJ, Brazil F Russomano, IFF-Fiocruz, RJ, Brazil G Nuovo, OSU, Columbus, USA B Grinsztejn, IPEC - Fiocruz, Rio de Janeiro, Brazil A F Nicol, LIPMED - IOC - Fiocruz, RJ, Brazil Background: Although p16INK4a immunostaining has been correlated with the severity of cytological and histological abnormalities in cervical lesions, the reproducibility is limited due, in part, to insufficiently standardized interpretation of the immunostaining. We examined the potential utility of p16INK4a as a biomarker for detecting CIN and invasive cervical cancer in tissue microarray (TMA) specimens. Methods: Immunohistochemical analysis of p16INK4a was performed in 326 cervical TMA specimens of normal control, low grade CIN, high grade CIN and invasive tumors. 38 specimens were from HIV positive women. HPV DNA was determined by in situ hybridization. Evaluation of p16INK4a expression was done by ImagePro Software. Statistical analysis was carried out by SAS 9.1. Results: Marked over-expression of p16 protein was found in the progression from Low to high-grade CIN and invasive tumors (p<0.0001). All controls were negative and low-grade CIN showed focal and weak immunohistochemical staining of p16. A cutoff of 75% immunopositivity for p16INK4a resulted in high sensitivity (94.6%), however a low specificity was found. Positive predictive value was 66.6% to diagnose high-grade CIN and invasive tumor. HIV/HPV co-infected cervices showed lower p16INK4a expression in low grade CIN (p =0.003) compared to only HPV infected cervices however no difference was found among high grade CIN (p=0.55). Conclusions: Immunohistochemical p16INK4a is over-expresseduring progression from low to high-grade CIN and invasive tumors, though change in expression due to HIV co-infection needs to be assessed further.
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Session 15: Screening and patient management

Chair: Tom Wright
Poster abstracts
Screening and patient management | 15 O-15.00

Tom Wright
FOR YOUR NOTES:
O-15.01
UTILITY OF RANDOM BIOPSY AND ENDOCERVICAL CURETTAGE IN LOW-RISK POPULATION
R Pretorius, S.C.P.M.G.-Fontana, Fontana, CA, UNITED STATES J L Belinson, Preventive Oncology International, Cleveland, OH, UNITED STATES P Peterson, S.C.P.M.G.-Fontana, Fontana, CA, UNITED STATES F Azizi, S.C.P.M.G.-Fontana, Fontana, CA, UNITED STATES S E Belinson, Preventive Oncology International, Cleveland, OH, UNITED STATES Background: Random biopsy in cervical quadrants without visible lesions and endocervical curettage (ECC) diagnosed 36.5% of the cervical intraepithelial neoplasia 3 or cancer (CIN 3+) at colposcopy in high-prevalence areas in China. Objective: Determine whether random biopsy and ECC are as useful in low-prevalence areas. Methods: We reviewed the S.C.P.M.G.-Fontana colposcopy experience for 1/1/07 to 12/31/09 where up to four random biopsies and ECC (unless pregnant) were obtained to determine the method of diagnosis of CIN 3+. Results: Between 1/1/07 and 12/31/09, 4,677 women with median age 32 years had 5,126 colposcopies in the S.C.P.M.G.-Fontana colposcopy clinics. Indications for colposcopy were cytology of high-grade squamous intraepithelial lesion (HSIL) or cancer (5.2%), low-grade squamous intraepithelial lesion (LSIL) (32.4%), atypical squamous cells of uncertain significance (ASCUS) with positive high-risk human papillomavirus (HR-HPV) (32.2%), ASCUS with negative HR-HPV times two (10.9%), negative cytology with positive HR-HPV for two years or associated with prior history of HR-HPV or CIN (11.3%), follow-up of CIN 2 or CIN 3 (5.2%), and other (2.3%). CIN 3+ was diagnosed in 279 women. Cervical biopsy detected 68.5% of CIN 3+, ECC diagnosed 4.3%, loop electrocautery excision procedure (LEEP) for ECC of CIN 2 diagnosed 5.0%, LEEP for cervical biopsy of CIN 2 diagnosed 21.1%, and LEEP for cytology of HSIL with cervical biopsy of negative or CIN 1 diagnosed 1%. Of the 191 women diagnosed with CIN 3+ by a cervical biopsy, 23.6% had negative colposcopic impressions in all four cervical quadrants. None of the 32 women under age 25 were diagnosed with CIN 3+ solely by ECC showing CIN 2+. Conclusions: Random biopsy in cervical quadrants without visible lesions increased the yield of CIN 3+ at colposcopy. ECC can be omitted in women under age 25 years.
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15 | Screening and patient management O-15.02

THE INCREMENTAL BENEFIT OF TAKING MULTIPLE BIOPSIES FOR DETECTING HGCIN
N Wentzensen, National Cancer Institute, Rockville, UNITED STATES, J Walker, University of Oklahoma, Oklahoma City, UNITED STATES, R Zuna, University of Oklahoma, Oklahoma City, UNITED STATES, K Smith, University of Oklahoma, Oklahoma City, UNITED STATES, C Mathews, University of Oklahoma, Oklahoma City, UNITED STATES, K Moxley, University of Oklahoma, Oklahoma City, UNITED STATES, M Tenney, University of Oklahoma, Oklahoma City, UNITED STATES, R Zhang, University of Oklahoma, Oklahoma City, UNITED STATES, M Gold, Vanderbilt University, Nashville, UNITED STATES, M Schiffman, National Cancer Institute, Rockville, UNITED STATES Background: A conventional colposcopic examination detects only 50-60% of prevalent precancers. Objectives: To systematically evaluate the benefit of taking multiple biopsies and to study cervical precancer on the lesion level. Methods: Previously-untreated women referred to the University of Oklahoma colposcopy clinic for abnormal screening results were enrolled. Before colposcopy, a specimen was taken for liquid-based cytology and HPV genotyping. During colposcopy, a digital image of the cervix was taken for annotating lesions and biopsy sites. Up to four biopsies were taken from acetowhite lesions; if less than four targeted biopsies were obtained, a random biopsy was added. All biopsies were ranked by visual impression and evaluated individually in histology. Results: Over 630 women have been enrolled in the study so far; 568 had histological diagnoses available. As worst diagnoses, 61 women (10.7%) had CIN3, 169 (29.8%) had CIN2, 208 (36.6%) had CIN1, and 130 (22.9%) had no dysplasia. 116/383 women (30.3%) with a low grade or benign colposcopic impression had CIN2+ in one of the biopsies. Conversely, 71/185 (38.4%) with a high grade colposcopic impression had <CIN2 in their worst biopsy result. In 61.7% of women with CIN2+, the worst lesion was detected in the first biopsy, in 26.1% it was found at the second biopsy and in 12.2% it was detected in the third or fourth biopsies. Similarly, 68.9% of CIN3 were detected with the first biopsy, 21.3% with the second biopsy, and 9.8% with the third and fourth biopsies. Only one CIN3 was detected with a random biopsy (1.6%). Conclusion: Colposcopic impression is unreliable to guide biopsy placement for confirmation of cervical precancer. Our data suggest that current colposcopy-biopsy protocols miss about 30% of prevalent CIN3. Performing 2-3 biopsies targeting acetowhite lesions increases the yield of CIN2+ substantially.
O-15.03
INCIDENCE OF CIN-2+ IN CYTOLOGICALLY NORMAL, ONCOGENIC HPV-NEGATIVE HIV-INFECTED WOMEN
H D Strickler, Albert Einstein College of Medicine, Bronx, UNITED STATES, R D Burk, Albert Einstein College of Medicine, Bronx, United States, X Xie, Albert Einstein College of Medicine, Bronx, United States, L S Massad, Washington University School of Medicine, St. Louis, United States, H Minkoff, Maimonides Medical Center, Brooklyn, United States, G D' Souza, Johns Hopkins University Bloomberg School of Public Health, Baltimore, United States, A M Levine, City of Hope National Medical Center, Duarte, United States, P E Castle, American Society of Clinical Pathology Institute, Washington, United States, M J Keller, Albert Einstein College of Medicine, Bronx, United States, J M Palefsky, University of California, San Francisco, San Francisco, United States Background: U.S. cervical cancer screening guidelines state that HIV-uninfected women 30 years and older who have both a negative Pap and oncogenic human papillomavirus (HPV) test can be re-screened in three years. Whether similar guidelines would be safe in HIV-infected women is unknown. Objectives: To determine the risk of high-grade cervical neoplasia defined cytologically (high-grade squamous intraepithelial lesions or more severe [HSIL+]) or histologically (cervical intraepithelial neoplasia 2 or more severe [CIN-2+]) in HIV-infected women who had a normal Pap test and were oncogenic HPV-negative at enrollment. Methods: Subjects included 420 HIV-infected and 279 HIV-uninfected women with normal cytology at enrollment in a longitudinal cohort. Semi-annual visits included Pap testing and, if indicated, cervical biopsy for histology. Cervicovaginal lavages from enrollment were tested for HPV DNA using a well-established PCR assay. Outcome: The cumulative incidence of high-grade cervical neoplasia during four years of follow-up. Results: Only two cases of HSIL+ were observed; an HIV-uninfected woman and an HIV-infected woman with a CD4 count > 500/mL. The four-year cumulative incidence of CIN-2+ was 3% (95% CI, 0%-8%) in HIV-infected women with CD4 counts less than or equal to 350/mL, 3% (95% CI, 0%-7%) in those with CD4 counts between 350 and 500/ mL, 5% (95% CI, 0%-10%) in those with CD4 counts greater than or equal to 500/mL, and 5% (95% CI, 0%-9%) in HIV-uninfected women. Only 2 cases were CIN-3, none were cancer. Risk of CIN-2+ was higher in oncogenic HPVpositive (hazard ratio, 6.57; 95% CI, 1.9-23;P=0.004) than oncogenic HPV-negative cytologically normal women. Conclusion: The four-year cumulative incidence of HSIL+ and CIN-2+ was low in HIV-infected and HIV-uninfected women who were cytologically normal and oncogenic HPV-negative at enrollment. Thus, a three-year screening interval would have had similar safety in HIV-infected and HIV-uninfected women in this cohort.
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IMPACT OF STRATEGIES FOR HPV-POSITIVE, CYTOLOGY-NEGATIVE WOMEN ON COLPOSCOPY UTILIZATION
E Burger, University of Oslo, Oslo, NORWAY J Ortendahl, Harvard School of Public Health, Boston, USA S Sy, Harvard School of Public Health, Boston, USA I Kristiansen, University of Oslo, Oslo, NORWAY J Kim, Harvard School of Public Health, Boston, USA Background: New cervical cancer screening strategies that involve cytology at younger ages, followed by a switch to primary HPV testing with reflex cytology are actively being considered by the Norwegian government. However, there is concern regarding the potential increased demand on colposcopy resources due to the enhanced sensitivity and reduced specificity of HPV testing. Objectives: We evaluated the impact of alternative follow-up strategies for HPV-positive, cytology-negative (HPV+/ Cyt-) women on colposcopy utilization within the context of Norway. Methods: We used likelihood-based methods to calibrate a first-order Monte Carlo simulation model to reflect the natural history of HPV-induced cervical cancer in Norway. The current screening strategy involving cytology only was compared to alternative strategies that switch to primary HPV-based screening every 5 years for older women. For HPV+/Cyt- women, we varied the follow-up interval (6-12 months), and the number of additional persistent HPV+/Cyt- results required before being referred to colposcopy (1-3). Outcomes included the number of colposcopies and the reduction in lifetime cancer risk compared to the current screening strategy. Results: A strategy requiring 3 additional persistent HPV+/Cyt- results, 12 months apart prior to colposcopy referral, could reduce cancer risk by 7.6% but increase the number of colposcopies by 9%, compared to current screening. The most intensive follow-up strategy, requiring 1 persistent result, 6 months later, reduces cancer by 12% but would more than double the number of colposcopies. However, as vaccination uptake increases, colposcopies can be reduced by as much as 15%, compared to a fully vaccinated population adhering to current screening guidelines. Conclusions: Strategies involving primary HPV-based screening may increase colposcopy utilization while further reducing cancer risk; however, colposcopy resources may be more than offset by vaccination. More intensive strategies require explicit tradeoffs when faced with capacity constraints.
O-15.05
NEGATIVE COLPOSCOPY AFTER HPV TRIAGE: IS ROUTINE RECALL SAFE?
H Kitchener, The University of Manchester, Manchester, UNITED KINGDOM R S Kelly, The Institute of Cancer Research, Sutton, UNITED KINGDOM P Walker, The Royal Free Hospital, London, UNITED KINGDOM S M Moss, The Institute of Cancer Research, Sutton, UNITED KINGDOM Background: The use of HPV testing to triage women with low grade cytological abnormalities is being increasingly implemented. The management of HPV+ve women who have negative adequate colposcopy/biopsy at referral, requires clarification in order to streamline the patient pathway and avoid unnecessary extended follow up. We wished to determine whether such women could be returned to routine recall, which is three years in the English Cervical Screening Programme for women less than 50 years. Methods: We followed up HPV+ve women who had been found to have negative colposcopy with or without a biopsy following triage referral at NHS Pilot Sites [1] between 2001-2002. Information on additional management and follow up cytology was obtained. Survival analysis was used to examine whether grade of cytology impacted on the detection of subsequent disease. Results: Out of 1063 eligible women who had negative colposcopy negative biopsy following referral with HPV+ve low grade cytology (borderline or mild dyskaryosis/ASCUS or LSIL), 965 had documented follow-up. The median time from colposcopy to final results was 27 months. The cumulative rate of CIN2+ incidence at 3 years was 4.4% (95%CI 4.0%-7.0%). The risk of CIN3 was 2.4% and CIN2 2%. The initial grade of cytology did not have a significant impact. Conclusion: Negative colposcopy has valuable clinical utility in this setting. Although higher than the rate of CIN2+ detection amongst routinely screened women in England (1.2%), the risk of acquiring CIN2+, and particularly CIN3 was considered sufficiently low to consider return to recall to be a safe policy. [1] Moss et al (2006) BMJ; 332: 83-85.
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15 | Screening and patient management O-15.06

HYBRID CAPTURE 2 USE WHEN SCREENING FOR ANAL DYSPLASIA
S Goldstone, Mount Sinai School of Medicine, New York, UNITED STATES B Lowe, Qiagen, Gaithersburg, United States T Rothmann, Qiagen, Gaithersburg, United States I Nazarenko, Qiagen, Gaithersburg, United States Background. HPV-related anal cancer is increasing in men and women, but especially in men who have sex with men. Similarities in anatomy and etiology within HPV-related anal and cervical disease may warrant similar diagnostic screening. The hc2 HPV DNA Test (HC2) is a FDA-approved, proven aid in detecting HPV infections of the cervix and as an aid in diagnosing, with Pap, cervical disease. The applications presented here are for research use only and not for use in diagnostic procedures Methods. This prospective study (n=298 male and female, 44% HIV positive) of patients undergoing anal screening included cytology, histology and HC2 results for two specimens co-collected: a swab into liquid-based cytology medium (PreservCyt), reflex, and a randomized swab or brush into Specimen Transport Medium (STM). Highresolution anoscopy was performed on all patients, with biopsy of lesions suspicious for AIN2+. Results. Cytology results were benign 42%, ASCUS 30%, LSIL 18%, HSIL 1%, ASC-H 1.7% and non-diagnostic 7%. AIN2+ was detected in 36% of patients. Cytology percent sensitivities/specificities were 77/52 for all patients, 75/44 for HIV+ and 81/56 for HIV-. For STM specimens, HC2 sensitivities/specificities were 91/40 for all, 96/31 for HIV+ and 83/45 for HIV-. For reflex HC2 (n=120), the percent sensitivities/specificities were 85/56; in comparison with STM, the p=0.3250 for sensitivity and p=0.0076 for specificity. In this study, 20% of patients with benign cytology and 36% with ASCUS cytology had AIN2+ lesions, but only 9 AIN2+ patients were HC2 negative. The increase in sensitivity for HC2 over cytology was statistically significant (p=0.0025). There were no significant differences in test sensitivities or specificities between newly referred and previously diagnosed patients. Conclusions. Feasibility of HC2 to detect HPV in anal specimens is indicated, and further research may indicate the use of HC2 for anal cancer screening.
Declaration of interest Qiagen sponsored this research with a grant to Dr. Stephen Goldstone I Nazarenko, B Love and T Rothmann are employees of Qiagen
O-15.07
SIX-YEAR PROSPECTIVE STUDY ON CERVICAL OUTCOMES ASSOCIATED WITH HPV LOAD
S WANG, Cancer Institute/Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, CHINA J Li, Cancer Institute/Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, CHINA F Zhao, Cancer Institute/Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, CHINA J Shi, Cancer Epidemiology Research Unit, Cancer Council New South Wales, Sydney, Australia W Zhang, Cancer Institute/Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, CHINA X Zhang, Cancer Institute/Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, CHINA Q Pan, Cancer Institute/Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, CHINA J BELINSON, Department of Gynecology and Obstetrics,Cleveland Clinic Foundation, Cleveland, America Y Qiao, Cancer Institute/Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, CHINA Background: Data on cervical outcomes of women with variegated HPV viral load and pathological results are limit. Objects: To provide data for the long-term natural regression and progression of cervical lesions associated with different HPV viral loads and histological statuses. Methods: 1,997 women were screened in rural China with 6 screening tests and underwent random biopsies in 1999. In 2005, 1,601 women with CIN1- or CIN2+ at baseline but refused for therapy were rescreened by visual inspection, cytology, and HPV testing. All women underwent colposcopy with biopsies taken if any visual lesion was found. HPV was detected by the HC2 system in both baseline and follow-up study, and viral load was measured by the ratio of relative light units to standard positive control (RLU/PC). RLU/PC was categorized into four groups: negative [0, 1.0), low viral load [1.0, 10.0), moderate viral load [10.0, 100.0] and high viral load>=100.0. Results: 9.8% of women with a positive HPV result at baseline developed CIN2+ lesions in 6 years, showing an increasing trend of 3.66%, 10.61% and 15.63% for HPV viral load categories of low, moderate and high subgroup, respectively. If combining two studies together: 0.17% of women with repeat negative HPV result developed CIN2+, and 2.75% women with moderate/high load at baseline and negative/low load result at follow-up developed CIN2+, while 33.33% women with repeat moderate/high load result at baseline and follow-up developed CIN2+. Specifically, for women with CIN2+, 100%(9/9) of women who had a positive HPV result at baseline but negative result at follow-up regressed to normal, while 38.46%(5/13) of women with repeat moderate/high HPV load both at baseline and follow-up regressed to CIN1, and 46.15%(6/13) of women remain as CIN2+. Conclusions: Risk of CIN2+ increases with HPV viral load, and significantly increased by repeat results of moderate/high HPV viral load.
Declaration of interest HC2 kits are donated by Digene Corporation.
80

ACCEPTABILITY OF MAIL BASED HOME-HPV TEST AMONG US WOMEN.
R Ghebre, University of Minnesota, Minneapolis, UNITED STATES S Kulasingam, University of Minnesota, Minneapolis, United States R Nguyen, University of Minnesota, Minneapolis, United States T Church, University of Minnesota, Minneapolis, United State Background: Cervical cancer prevention guidelines include recommendations for clinician-obtained human papillomavirus (HPV) tests. Studies have shown that self-collected HPV tests are as sensitive as cytology tests for detection of cervical intraepithelial neoplasia grades 2-3 (CIN 2-3), the cervical cancer precursor lesion. However, few studies have evaluated self-collected HPV testing outside of a clinic setting among women in the United States (US). Objective: To determine the acceptability and feasibility of a mail based home-HPV test among women who have performed self-collection at home instead of a clinic setting. Methods: Women aged 25-70 years scheduling routine gynecologic clinic visits were invited to participate. Consenting women collected a vaginal HPV sample at home and mailed it in along with an acceptability survey prior to the clinic visit. During the clinic visit two HPV tests (vaginal and cervix) and a liquid based Pap test (LBC) were collected by a clinician. HPV identification and typing was performed using PCR. Results: From October, 2009 to May, 2010, 254 consecutive eligible women were enrolled. A total of 222 women (87.4%) completed the home-HPV test and 211 (83.1%) completed the acceptability survey. Women reported a high level of confidence with collecting the home-HPV test (90.6% very confident/confident). Sixty four percent (134 women) reported that the sample collection took less than 3 minutes; this did not vary by age (p<0.5). Although all home-HPV samples were adequate for laboratory analysis, 24% of women expressed concern about sample adequacy. There was a significant difference for how women preferred results: women less < 35 years of age preferred phone call or electronic mail compared to women > 35 years, who preferred traditional letter mail. Conclusion: Among this population of women, home self-collected HPV tests showed high feasibility and acceptability and can potentially be utilized for cervical cancer screening.
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15 | Screening and patient management P-15.09

HUMAN PAPILLOMAVIRUS PREVALENCE IN WOMEN TREATED FOR HIGH-GRADE (CIN)
J Jones, Cardiff University, Cardiff, UNITED KINGDOM A Saleem, Cardiff University, Cardiff, UNITED KINGDOM N Rai, Royal Victoria Infirmary, Newcastle, UNITED KINGDOM T Shylasree, University Hospital Llandough, Llandough, Cardiff, UNITED KINGDOM S Ashman, Cardiff University, Cardiff, UNITED KINGDOM K Gregory, Cervical Screening Wales, Cardiff, UNITED KINGDOM N Powell, Cardiff University, Cardiff, UNITED KINGDOM A Tristram, Cardiff University, Cardiff, UNITED KINGDOM A Fiander, Cardiff University, Cardiff, UNITED KINGDOM S Hibbitts, Cardiff University, Cardiff, UNITED KINGDOM Objectives: To evaluate an HPV genotyping platform as a test of cure in management of women treated for high grade CIN using Large Loop Excision of the Transformation Zone (LLETZ). Methods: The PapilloCheck assay was evaluated in a test of cure setting to determine its clinical utility as a predictor of disease recurrence. Ninety-eight women (age range 19-52 years) treated for CIN2+ by LLETZ were evaluated and samples taken for HPV testing before and 6 months after treatment. Cytology and histology were available from recruitment and up to 24 months post treatment. Results: Recurrent disease was evident in 4% of patients with 2 cases of low-grade and 2 cases of high-grade disease. In women with no disease recurrence, 40% cases (95% CI 30.42-51.05%) were High Risk (HR) HPV negative post LLETZ. The 2 cases with high-grade disease had a persistent HPV16 infection detected. Genotyping before and after treatment revealed 83% (95% CI 75.74-88.78%) of total viral infections were cleared whilst 17% (95% CI 11.2224.26) of viral infections persisted. Combining viral persistence with abnormal cytology at 6 months predicted CIN2+ with 100% sensitivity, 95% specificity, 100% NPV and 28.6% PPV. Conclusion: Genotyping before and after treatment will identify persistent infections that have a high risk of recurrence. Acquisition of new HPV infections in this patient group also requires monitoring and HPV genotyping will enable consideration of the type-specific risk for high-grade disease recurrence and help refine patient management.
P-15.10
PREFERENCES FOR MANAGING LOW-GRADE ABNORMAL CERVICAL SCREENING TESTS: SYSTEMATIC REVIEW
M Rebolj, University of Copenhagen, Copenhagen, DENMARK M Frederiksen, University of Copenhagen, Copenhagen, DENMARK E Lynge, University of Copenhagen, Copenhagen, DENMARK Background. The choice between the available alternatives for follow-up of low-grade abnormal cervical screening abnormalities, active follow-up and observation, is equivocal. Because the frequency of these tests may double, the problem will become more prominent when Human Papillomavirus (HPV) testing will replace cytology in primary screening. Objectives. To determine womens preferences for follow-up of low-grade cervical screening abnormalities. Methods. Using MeSH terms, PubMed was systematically searched for articles published until December 2010. The reference lists of retrieved studies were checked for additional studies. Studies were included if women were asked to state a preference between active follow-up and observation for managing low-grade abnormalities on screening cytology or HPV tests. Information on study designs, participants and outcomes was retrieved using a pre-specified form. Studies were sorted by their designs. Results. 13 studies could be included in the review. In all five studies that surveyed women with low-grade abnormal tests before their own management had started, two thirds preferred active follow-up, predominantly as immediate colposcopy, to observation, predominantly as repeated Pap smears. In all, but two, studies testing other situations, women more often expressed a preference for active follow-up than for observation. Women though appeared somewhat more willing to accept observation if reassured of low risk of cervical cancer. Conclusions. Even for low-grade abnormal cervical tests, women tend to prefer active management strategies. It may be a challenge to meet their expectations of optimal follow-up when HPV testing will be used in primary screening.
Declaration of interest Elsebeth Lynge and Matejka Rebolj are currently undertaking a comparative study of new-generation HPV tests, involving collaboration with Roche Diagnostics A/S, Genomica S.A.U., Qiagen Gaithersburg Ltd., and GenProbe Inc. Concerning the present paper, there has been no collaboration with, or support from any of the companies. Elsebeth Lynge has served as unpaid scientific advisor to GenProbe and Norchip.
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Screening and patient management | 15 P-15.11

ACCEPTABILITY OF SELF-COLLECTION FOR HIV TESTING USING FTA CARTRIDGE
Y Guan, Johns Hopkins University School of Medicine, Baltimore, MD, UNITED STATES S Wang, B Li, C Feng, B Ci, X Li, W Zhang, Cancer Institute/Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, CHINA P Gravitt, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, UNITED STATES P Castle, American Society for Clinical Pathology Institute, Washington DC, UNITED STATES Y L Qiao, Cancer Institute/Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, CHINA Objectives: To assess the relative acceptability, preference, and accuracy of self-collected HPV samples versus clinician-collected samples using FTA cartridge. Background: The Whatman indicating FTA cartridge has been shown to be an equally sensitive and more convenient transport option for HPV specimen than liquid-based transport media. This new transport method along with self-collection holds great promise for HPV screening. However, few data exist assessing the acceptability of self-collection using FTA cartridge. Method: 174 women who were participating in the national cervical cancer screening program in Xiangyuan County, China, were enrolled in our study and underwent HPV self-collection, clinician-collection, colposcopy exam, and questionnaire session. The questionnaire assessed the patients preference and acceptability of collection method including trust, comfort, convenience, ability to collect specimen, and perceived effect of screening. HPV DNA testing was done using Roche HPV Linear Array Test. Results: 99% of participants thought FTA cartridge was easy to use. The acceptability scores for self-collection and clinician-collection were not significantly different on scales measuring comfort and convenience (p>0.05). The scores were significantly lower for self-collection on scales measuring trust, ability to collect specimen, and perceived effects of testing (p < 0.01). 74% of participants preferred clinician-collection, and of these participants, 86% chose it because they thought the results were more accurate. HPV Linear Array test showed 93% overall agreement (k=0.84) between self and clinician collection in detecting HPV DNA eluted from the FTA cartridge. Conclusion: Our results demonstrate that FTA cartridge is an acceptable method for transport, and that even though self- and clinician-collection are equally comfortable and convenient; our participants still preferred clinician-collection because of a lack of trust in the results of self-collection. This indicates that selfcollection using FTA cartridge is an effective potential method for screening; however, more patient education about the validity of self-collection is needed.
P-15.12
THE UTILITY OF P16INK4A/KI-67 CYTOLOGY IN SAMPLES FROM KENYAN WOMEN
C Ngugi, Jomo Kenyatta University of Agriculture and Technology, Nairobi, KENYA, M Reuschenbach, Department of Applied Tumor Biology, Institute of Pathology, University of Heidelberg, and Clinical Cooperation Unit, German Cancer Research Cancer (DKFZ), Heidelberg, Germany, D Schmidt, Institute of Pathology, Mannheim, Mannheim, Germany, R K Wanyoro, Thika District Hospital, Thika, Kenya, H I Boga, Faculty of Science, Jomo Kenyatta University Of Agriculture and Technology, Nairobi, Kenya, P Wanzala, Centre for Public Health Research- Kenya Institute of Medical Research, Nairobi, Kenya, A W Muigai, Faculty of Science, Jomo Kenyatta University Of Agriculture and Technology, Nairobi, Kenya, J N Mbithi, Department of Medical Laboratory Sciences, Kenyatta University, Nairobi, Kenya, M von Knebel Doeberitz, Department of Applied Tumor Biology, Institute of Pathology, University of Heidelberg, and Clinical Cooperation Unit, German Cancer Research Cancer (DKFZ), Heidelberg, Germany Objectives: The prevalence of high risk human papillomavirus (HR-HPV) infection and related diseases in Kenya is high. Cervical cancer screening rates are low due to limited infrastructure. This warrants for evaluation of markers that specifically detect high grade cervical lesions which need treatment, in order to avoid repeated testing. We evaluated the performance of p16INK4a/Ki-67 dual-stain cytology in a screening population in Kenya and compared it to Hybrid Capture 2 HR-HPV DNA test. Methods: A total of 498 PreservCyt samples were collected from women without known history of cervical dysplasia, cancer or HPV infection in Thika district, Kenya. ThinPrep Pap cytology (Hologic), HC2 (Qiagen) and p16INK4a/Ki-67 dual-stain cytology (CINtec PLUS, mtm Laboratories) was performed. Conclusions: Valid results for all tests were obtained in 485 samples. HR-HPV DNA by HC2 was detected in 104/485 (21.4%) of the samples. Most women had normal cytology 431 (88.9%), 14 (2.9%) LSIL, 1 (0.2%) AGUS, 28 (5.8%) ASCUS and 11 (2.3%) had HSIL. p16INK4a/Ki-67 cytology was positive in 39/485 (8.0%) of the samples. HC2 was positive in 9/11 (81.8%) of HSIL and p16INK4a/Ki-67 positive in 8/11 (72.7%) of HSIL samples. In samples diagnosed as normal in Pap cytology, the rate of positive HC2 results was 77/431 (17.9%) higher than the rate of positive p16INK4a/Ki-67 samples (15/431, 3.5%). No histology is available for most of the women as the situation at the study site precluded systematic colposcopy and biopsy follow-up. Thus diagnostic sensitivity and specificity of p16INK4a/Ki-67 dual-stain cytology in Kenya remains to be adequately evaluated. However, our study is the first demonstrating general technical applicability of p16INK4a/Ki-67 cytology in a developing country screening population. Our finding points to a lower fraction of false positive test results using p16INK4a/Ki-67 cytology compared to HC2, which would be important for a point-of care test in limited resource settings.
83

HPV GENOTYPE AND COLPOSCOPY PERFORMANCE IN YOUNG WOMEN
M Cruickshank, University of Aberdeen, Aberdeen, UNITED KINGDOM C Gillespie, University of Aberdeen, Aberdeen, UK S Cotton, University of Aberdeen, Aberdeen, UK C Busby - Earle, Simpson Centre for Reproductive Health, Royal Infirmary of Edinburgh , Edinburgh, UK C Moore, Specialist Virology Centre, Edinburgh, Edinburgh, UK H Cubie, 3Scottish HPV Reference Laboratory, Edinburgh, Edinburgh, UK K Cuschieri, 3Scottish HPV Reference Laboratory, Edinburgh, Edinburgh, UK Background: In 2008, vaccination against the two most common oncogenic HPV types, 16 and 18, was introduced across the UK as a further step towards cervical cancer eradication. However, CIN could be easier to miss in immunised women if high risk HPV infection is responsible for the colposcopic features widely considered by colposcopists to represent high grade CIN. Aims: To determine if there is an association between HPV genotype and colposcopic features identified by colposcopists in young women with abnormal cytology. Methods: This study is recruiting women aged 20-25 years in Aberdeen and Edinburgh referred with abnormal cervical cytology in the national cervical screening programme. Colposcopic findings and a Reid's Colposcopic Index are recorded and a cervical sample is obtained for HPV DNA and mRNA genotyping at this examination. The colposcopist has the results of the referral cytology but the HPV status is not known. Results: The mean age of the first 105 women recuited was 23 years. Only one had received HPV vaccination, 20% of women had an infection with one HPV type, 31% with two types, 17% with three types, and 19% with four types, and the remaining 10% had five or more types. Only 4 of the 105 women had no HPV infection detected. One third of women had CIN2/3 on biopsy. The proportion with CIN2/3 was higher in those with HPV16 infection (50%) compared to those with no HPV16 infection (21%). The PPV of colposcopic impression for high grade CIN was 85% in women with HPV16 and/or18 but only 50% with other HPV types. This study is ongoing with 200 women recruited. Further data and associated analysis will be presented.
P-15.14
TOLERABILITY OF SCREENING PROCEDURES FOR ANAL DYSPLASIA
S Goldstone, Mount Sinai School of Medicine, New York, UNITED STATES T Davis, Sackler School of Medicine, Tel Aviv, Israel G Chen, Qiagen, Gaithersburg, Maryland Background: Anal cancer rates are rising with high-grade dysplasia the probable precursor. Screening procedures can be uncomfortable and may lead to decreased patient compliance. Objectives: to determine the tolerability of screening procedures for anal dysplasia. Methods: 293 patients enrolled for a 2 visit study. On visit one (V1), cytology was collected with a swab and patients were randomized to HPV collection by brush or swab followed by digital exam and standard anoscopy. Patients completed a survey regarding procedure tolerability. For visit two (V2), patients had repeat HPV sampling and high resolution anoscopy (HRA) with biopsy if indicated. Patients reported the presence and duration of pain, bleeding, or other symptoms after V1 and levels of discomfort for V2 procedures. Results: V1 anoscopy caused the most discomfort (mean 1.9). V2 biopsy caused the least discomfort (mean 1.04). Mean difference between swab (1.56) and brush (1.87) discomfort at V1 was significant (p=0.03), but no significant difference was found at V2. Mean difference between V2 brush (1.63) and V1 brush (1.86) discomfort was significant (p=0.02), but there was no significant difference between V1 and 2 swab. Mean difference between V1 anoscopy (1.90) and V2 HRA (1.53) was insignificant. Eight patients reported discomfort from 1 procedure would prevent them from having a procedure again yet all returned for their second visit. Less than 10% of patients reported any pain or bleeding post V1 without difference in duration between brush or swab. HRA with biopsy was significantly more painful than without biopsy but number of biopsies did not increase perceived pain. Conclusions: Screening procedures for anal dysplasia are well tolerated, with minimal to no difference in tolerability, post procedure bleeding or pain between brush or swab.
Declaration of interest Quiagen supported this research and G. Chen is an employee of Qiagen.
84

HPV IN THE ANAL CANAL DETECTED WITH HYBRID CAPTURE 2.
S Goldstone, Mount Sinai Medical Center, New York, NY, UNITED STATES B Lowe, Qiagen Inc, Gaithersburg, MD, UNITED STATES T Rothmann, Qiagen Inc, Gaithersburg, MD, UNITED STATES I Nazarenko, Qiagen Inc, Gaithersburg, MD, UNITED STATES Background. HPV-related anal cancer is increasing in men and women, but especially in men who have sex with men. Similarities in anatomy and etiology within HPV-related anal and cervical disease may warrant similar diagnostic screening. The hc2 HPV DNA Test (HC2) is a FDA-approved, proven aid in detecting HPV infections of the cervix and as an aid in diagnosing, with Pap, cervical disease. The applications presented here are for research use only and not for use in diagnostic procedures Methods. This prospective study (n=298 male and female, 44% HIV positive) of patients undergoing anal screening included cytology, histology and HC2 results for two specimens co-collected: a swab into liquid-based cytology medium (PreservCyt), reflex, and a randomized swab or brush into Specimen Transport Medium (STM). Highresolution anoscopy was performed on all patients, with biopsy of lesions suspicious for AIN2+. Results. Percent cytologies were 42, 30, 54, 3 and 7 for benign, ASCUS, LSIL, HSIL and non-diagnostic, respectively. The histological end-point, AIN2+ lesions, was detected in 36% of patients. Cytology percent sensitivities/ specificities were 77/52 for all patients, 75/44 for HIV+ and 81/56 for HIV-. For STM specimens, HC2 sensitivities/ specificities were 91/40 for all, 96/31 for HIV+ and 83/45 for HIV-. For reflex HC2 (n=120), the percent sensitivities/ specificities were 85/56; in comparison with STM, the p=0.3250 for sensitivity and p=0.0076 for specificity. In this study, 20% of patients with benign cytology and 36% with ASCUS cytology had AIN2+ lesions, but only 5% of these AIN2+ specimens were HC2 negative. The increase in sensitivity for HC2 over cytology was statistically significant (p=0.0025). There were no significant differences in test sensitivities/specificities between newly referred and previously diagnosed patients. Conclusions. Feasibility of HC2 to detect HPV in anal specimens is indicated, and further research may indicate the use of HC2 for anal cancer screening.
Declaration of interest Research funded by Qiagen; presenting author is from Mount Sinai Medical Center; all other authors are from Qiagen.
P-15.16
COLPOSCOPY RESOLUTION OF CIN1 LESIONS AFTER HPV CLEARANCE
G Hernandez - Suarez, National Cancer Institute of Colombia, Bogota, COLOMBIA M Gonzalez, National Cancer Institute of Colombia, Bogota, COLOMBIA O Gamboa, National Cancer Institute of Colombia, Bogota, COLOMBIA Background: Despite the large acceptance of HPV test by gynecologists for follow up of cervical lesions, the is no published data referring the time of CIN1 resolution after HPV clearance. Objectives: To estimate the time to CIN1 resolution after HPV clearance and identify related risk factors Methods: Cohort study. 2000 omen were followed on average 10 years to asses the natural history of HPV infection and related lesions in Bogota Colombia. Women with incident CIN1 lesions and PCR-HPV valid result which did not progress during follow up were included into the analysis. Time to resolution of CIN1 Cox regression after HPV clearance was done using Cox regression. Results: 69 women were included into the analisis. Mean time to CIN1 resolution after HPV clearance was 19.5 months. Having new sexual partners in the last 6 months (HR: 0.43 95%CI 0.33-1.03) and Alpha 9 HPV species (vs low risk HPV HR:0.36 95%CI 0.15-0.88) were factors that showed association with longer time of CIN1 resolution. Conclusion: Colposcopy minor changes related to CIN1 lesions are still present after HPV clearance. Colposcopy follow up should be done at least18 months after initial CIN1, to avoid misinterpretation of results. Before the 18 months frame period colposcopy changes observed will correspond to the same original diagnosis.
Declaration of interest G. Hernndez, MSD, Grant support O. Gamboa. Non Decleared M. Gonzalez. Non Decleared
85

SMALLER CIN 3 OR CANCER IS LESS EFFICIENTLY DIAGNOSED
R Pretorius, S.C.P.M.G.- Fontana, Fontana, CA, UNITED STATES J L Belinson, Preventive Oncology International, Cleveland, OH, UNITED STATES M Arbyn, Unit of Cancer Epidemiology, Scientific Institute of Public Health, Brussels, BELGIUM F Azizi, S.C.P.M.G.-Fontana, Fontana, CA, UNITED STATES P Peterson, S.C.P.M.G.-Fontana, Fontana, CA, UNITED STATES S E Belinson, Preventive Oncology International, Cleveland, OH, UNITED STATES Background and Objectives: We determined the relationship between the number of cervical quadrants involved with cervical intraepithelial neoplasia 3 or cancer (CIN 3+) and the efficiency of testing as measured by the area under the receiver operating characteristic curve (AUC). Methods: The Shanxi Province Cervical Cancer Screening Studies (SPOCCS) I and II which were cross sectional trials of multiple techniques to detect CIN in which biopsies were obtained in all four cervical quadrants were reviewed. Proportions and AUCs were compared with Chi-Square. Results: Within SPOCCS I and II, 244 cases of CIN 3+ were diagnosed. The proportion of CIN 3+ involving 3 or 4 cervical quadrants was greater for cytology of high grade squamous intraepithelial lesion or cancer (HSIL) [21.7% (38/175)] than for other cytologic results [4.4% (3/69), p=.001]. The proportion of CIN 3+ with cytology of HSIL in SPOCCS I (90.5%, 38/42) was greater than for SPOCCS II [67.8% (137/202), p=.003]. CIN 3+ was larger in SPOCCS I, involving 3 or 4 cervical quadrants in 33.3% (14/42) of cases compared with 13.4% (27/202), p=.002 in SPOCCS II. The AUC for diagnosis of CIN 3+ with cervical cytology and HR-HPV testing for SPOCCS I (0.9808) was greater than for SPOCCS II (0.9512, p<.05). Within SPOCCS II, the AUC for CIN 3+ involving 4 cervical quadrants (0.9821) exceeded that of CIN 3+ in 3 quadrants (0.9716, p<.001) which exceed that for 2 quadrants (0.9494, p<.001) which exceeded that for 1 quadrant (0.930, p<.001). Conclusion: Smaller CIN 3+ is less efficiently diagnosed with cervical cytology and HR-HPV testing. Smaller CIN 3+ is also associated with a lower proportion of cytology of HSIL.
P-15.18
COLPOSCOPIC, CYTOLOGIC, AND HISTOPATHOLOGIC CORRELATIONS OF HYPERKERATOSIS IN REPRODUCTIVE WOMEN
N Chogovadze, Research Institute of Clinical Medicine, Tbilisi, GEORGIA M Jugheli, Research Institute of Clinical Medicine, Tbilisi, GEORGIA M Gachechiladze, N.Kipshidze Central University Clinic, Tbilisi, GEORGIA G Burkadze, N.Kipshidze Central University Clinic, Tbilisi, GEORGIA Background: Leukoplakia or hyperkeratosis considered as benign structural change of cervical squamous epithelium, but sometimes dysplastic epithelium may exhibit abnormal keratinization on the surface as well. Objectoves: We aimed to investigate the correlation of colposcopically identified hyperkeratosis with cytopathological and histopathologycal data, and its possible relationship with dysplastic lesions of uterine cervix. Methods: We studied 123 cases of colposcopically identified hyperkeratosis. Patients were distributed into two groups: I group 42 patients with thin leukoplakia and II group 81 patients with thick leukoplakia. Cytological reports were available in all cases. Directed biopsy with subsequent histopathological examination of H&E stained sections was performed in patients with cytologically identified epithelial cell abnormalities (ECAs), including atypical squamous cells of undetermined significance (ASCUS), Low-grade squamous intraepithelial lesion (LSIL) and high-grade squamous intraepithelial lesion (HSIL). Results: I group: 28 (67%) cases were negative for intraepithelial lesion or malignancy (NILM), ECAs were detected in 14 (33%) patients: ASCUS - in 7(16,6%), LSIL in 4 (9,5%) and HSIL in 3 (7,1%) cases; From II group 68 (84%) cases were NILM and ECAs were detected in 13 (16%) patients: ASCUS in 9 (11,1%), LSIL in 4 (4,9%) and HSIL in 0 (0%) patients. The results of histopathological examination of 14 patients with ECA from I group showed cervical intraepithelial neoplasia (CIN) in 10 (71%) patients (CIN1 - 4(28,5%), CIN2 4(28,5%), CIN3 3(21,4%)). Dysplasias were detected in all 13(100%) patients (CIN1 -3(3,7%), CIN2 6(7,4%), CIN3-4(4,9%)) with ECA from II group. Conclusions: 1) cervical dysplastic lesions are identified 2 times less frequently in conventional Pap smears from patients with thick leukoplakia, than in patients with thin leukoplakia and 2) histopathologically identified cervical dysplasias are 1,5 times more common in patients with thick leukoplakia. This difference should be considered by pathologists and gynecologists.
Declaration of interest I don't have any conflict of interest.
86

DETERMINANTS OF HPV-MRNA POSITIVITY IN SELF- AND PHYSICIAN-COLLECTED SPECIMENS, KENYA
N Mugo, University of Nairobi/ Kenyatta National Hospital, Nairobi, KENYA, J Ting, University of North Carolina, Chapel Hill, USA, J Kwatampora, University of Nairobi/University of Manitoba, Nairobi/Manitoba, KENYA/USA, C Hill, Gen-Probe Incorporated, San Diego, USA, A Schroeder, Gen-Probe Incorporated, San Diego, USA, M Chitwa, University of Nairobi/ University of Manitoba, Nairobi/Manitoba, KENYA/USA, S Patel, University of North Carolina, Chapel Hill, USA, H Gakure, University of Nairobi/University of Manitoba, Nairobi/Manitoba, KENYA/USA, J Kimani, University of Nairobi/University of Manitoba, Nairobi/Manitoba, KENYA/USA, J Smith, University of North Carolina, Chapel Hill, USA Background: Little is known of the prevalence high-risk(hr) HPV-mRNA, as well as factors which potentially affect hrHPV-mRNA positivity in self- and physician-collected samples in Kenya. Objectives: To determine the prevalence and determinants of hrHPV-mRNA infection in self- and physician-collected samples from women with normal cervical cytology. Methods: A cross-sectional analysis was conducted of 235 female sex workers(FSW) with normal cervical cytology in Nairobi. Participants conducted self-collection in privacy using a brush, according to standardized instructions. A physician conducted a pelvic exam to obtain cervical cell samples. Samples were tested for hrHPVmRNA using APTIMA HPV Assay (Gen-Probe Incorporated). Cervical cytology was conducted using physician-collected samples. Blood samples were obtained for HIV testing. Odds ratios were adjusted for age and HIV-serostatus. Results: Among 235 women with normal cervical cytology, hrHPV-mRNA prevalence was 22.1% and 24.3% in self- and physiciancollection, respectively. In both self- and physician-collection, hrHPV-mRNA was more common among women reporting older age of first intercourse (self-collection OR=1.4,95%CI:0.7-2.7; physician-collection OR=1.5,95%CI:0.8-2.8 for 16 years vs. <16 years), although estimates were imprecise. HrHPV-mRNA infection appeared to be significantly lower in women reporting 3 compared with <3 sexual clients per day in physician-collection (OR=0.4,95%CI:0.2-0.7), although a similar association in self-collection did not reach statistical significance (OR=0.8;95%CI:0.4-1.5). Women reporting higher number of sexual clients per week (10 vs. <10) also appeared to have somewhat lower likelihood of hrHPV-mRNA infection in physician-collection (OR=0.6,95%CI:0.3-1.0), an occurrence not observed in self-collection (OR=1.1,95%CI:0.6-2.2). A reported history of condom use and smoking did not appear to significantly protect against HPV in either self- or physician-collection. Conclusion: Among women with normal cervical cytology, there appeared to be no statistically significant differences in characteristics of women with hrHPV-mRNA in self- or physician-collected samples. HrHPV-mRNA infection did appear lower, however, in physician-collected samples among FSW reporting a higher number of sexual clients, results which deserve further investigation.
Declaration of interest HPV-mRNA testing was provided and conducted by Gen-Probe Incorporated, San-Diego
P-15.20
REDUCING ANXIETY BEFORE COLPOSCOPY: FOCUS-GROUP INVESTIGATION OF WOMENS EXPERIENCES/NEEDS
R P Bosgraaf, Radboud University Nijmegen Medical Centre, Nijmegen, NETHERLANDS P Servaes, Radboud University Nijmegen Medical Centre, Nijmegen, NETHERLANDS P H Struik - van der Zanden, Radboud University Nijmegen Medical Centre, Nijmegen, NETHERLANDS R L M Bekkers, Radboud University Nijmegen Medical Centre, Nijmegen, NETHERLANDS Background: Women experience high levels of anxiety and have negative emotional responses when they attend for colposcopy. High levels of anxiety before and during colposcopy can have adverse consequences, including pain and discomfort during the procedure and subsequently may result in defaulting follow-up. It is shown that anxiety levels are not decreased following individually targeted information given before colposcopy. Objectives: This qualitative study therefore investigates womens experiences towards a colposcopy appointment and womens solutions for reducing anxiety levels. Methods: Three focus groups were conducted with women who had recently attended a colposcopy appointment. The semistructured, researcher-facilitated discussions covered factors that cause anxiety in women and how to influence these anxiety levels. Audiotapes of the group discussions were transcribed and analyzed qualitatively. Results: Most of the women indicated high levels of anxiety and discomfort before their colposcopy appointment. Participants emphasized their reasons for anxiety: they do not exactly know what to expect (despite information leaflets), anxiety for the examination itself, shame, long waiting times in the outpatients clinic. Participants prefer provision of adequately tailored visual information prior to their attendance at the clinic especially about the examination and about the environment (e.g. the hospital, the waiting room, the colposcopy room, the colposcopist). Conclusions: The provision of appropriate information appears an important aspect of reducing anxiety before colposcopy. Our findings suggest that there is scope for improvement in content of information provision. Visual information in the form of an explanatory video, with more specific information prior to attendance may reduce fear and stress in women with an abnormal cervical smear result referred to the colposcopy clinic.
87

MANAGEMENT OF WOMEN WITH HIGH-GRADE CYTOLOGY:A COMPARISON OF TRIAGE OPTIONS
M Kyrgiou, Department of Gynaecologic Oncology - Obstetrics & Gynaecology, Queen Charlotte's and Chelsea Hammersmith Hospital, London, UNITED KINGDOM, G Valasoulis, Department of Obstetrics & Gynaecology, University Hospital of Ioannina, Ioannina, Greece, S Gritzeli, Department of Obstetrics & Gynaecology, University Hospital of Ioannina, Ioannina, Greece, S M Founta, Department of Obstetrics & Gynaecology, University Hospital of Ioannina, Ioannina, Greece, P Karakitsos, Department of Cytopathology, Attikon Hospital, University of Athens, Athens, Greece, M Nasioutziki, Second Department of Obstetrics and Gynecology, Aristotle University of Thessaloniki, Hippokration Hospital, Thessaloniki, Greece, C Founta, Department of Obstetrics & Gynaecology, University Hospital of Ioannina, Ioannina, Greece, A Loufopoulos, Second Department of Obstetrics and Gynecology, Aristotle University of Thessaloniki, Hippokration Hospital, Thessaloniki, Greece, G Koliopoulos, Department of Obstetrics & Gynaecology, University Hospital of Ioannina, Ioannina, Greece, E Paraskevaidis, Department of Obstetrics & Gynaecology, University Hospital of Ioannina, Ioannina, Greece Background: A proportion of women referred for high-grade cytology will be proven to have only low grade histology. This study aims to investigate possible combinations of tests that could safely identify these women. Methods: Design: Diagnostic study. Inclusion criteria: Women referred with high grade cytology. Intervention: An LBC specimen obtained prior to colposcopic evaluation was tested for HPV typing, E6 & E7 mRNA (NASBA), E6 & E7 mRNA by flow cytometry, p16INK4a and microspectroscopy. All women underwent LLETZ (gold standard). Outcomes: The sensitivity, specificity, PPV, NPV, positive and negative likelihood ratio (LR) were calculated for each parameter alone or in combination for CIN2+ histology. Results: A total of 336 women have been recruited. The colposcopic assessment appeared to have the best sensitivity [94 %(95%CI:89-100)], NPV [69%(95%CI:32-100)] and negative LR [0.17(95%CI:0.03-0.51)]. NASBA had the best specificity [82%(95%CI:48-100)], PPV [96%(95%CI:84-100)] and positive LR [3.8(95%CI:0.91-12.17)]. The combination of colposcopy with high-risk HPV had the best sensitivity [91%(95%CI:75-99)] and negative LR 0.29(95%CI:0.06-0.59)]; flow cytometry with p16 had the best specificity [98%(95%CI:89-100)], negative PV 81%(95%CI:63-97) and positive LR [22.1(95% CI:3-126.77)], while NASBA with p16 the best PPV 99%(95%CI:89-100). Conclusions: Some of the combinations might have significant accuracy for the prediction of high-grade histology. This could allow conservative management for women at low risk and avoidance of unnecessary intervention and/or treatment. All the above markers should be evaluated in a cost analysis and could be integrated in highgrade triage prediction scoring system, allowing tailored selection for treatment. The above findings need to be confirmed in larger cohorts.
P-15.22
MANAGEMENT OF WOMEN WITH LOW-GRADE CYTOLOGY:A COMPARISON OF TRIAGE OPTIONS
M Kyrgiou, Department of Gynaecologic Oncology - Obstetrics & Gynaecology, Queen Charlotte's and Chelsea Hammersmith Hospital, London, UNITED KINGDOM, G Valasoulis, Department of Obstetrics & Gynaecology, University Hospital of Ioannina, Ioannina, Greece, I Tsoumpou, Department of Obstetrics and Gynecology, University Hospitals of South Manchester, Manchester, United Kingdom, S M Stasinou, Department of Obstetrics & Gynaecology, University Hospital of Ioannina, Ioannina, Greece, M Nasioutziki, Second Department of Obstetrics and Gynecology, Aristotle University of Thessaloniki, Hippokration Hospital, Thessaloniki, Greece, C Founta, Department of Obstetrics & Gynaecology, University Hospital of Ioannina, Ioannina, Greece, V Malamou - Mitsi, Department of Cytopathology, University Hospital of Ioannina, Ioannina, Greece, P Karakitsos, Department of Cytopathology, Attikon Hospital, University of Athens, Athens, Greece, G Koliopoulos, Department of Obstetrics & Gynaecology, University Hospital of Ioannina, Ioannina, Greece, E Paraskevaidis, Department of Obstetrics & Gynaecology, University Hospital of Ioannina, Ioannina, Greece Background: A proportion of women referred for low-grade cytology will be proven to have a high-grade histology. However, the majority will have lesions of low malignant potential and this study investigates possible methods that could identify these women. Methods: Design: Diagnostic study. Inclusion criteria: Women referred with ASCUS or LSIL cytology; histological diagnosis was available for all women (punch biopsies or LLETZ). Interventions: An LBC specimen obtained prior to colposcopic evaluation was tested for HPV typing, E6 & E7 mRNA (NASBA), E6 & E7 mRNA by flow cytometry, p16INK4a and microspectroscopy. Outcomes: The sensitivity, specificity, PPV, NPV, positive and negative likelihood ratio (LR) were calculated for each parameter alone or in combination for CIN2+ histology. Results: A total of 589 women were included. High-risk HPV testing showed the highest sensitivity [96%(95%CI:8697)], moderate specificity [69%(95%CI:54-82)] and positive LR [2.92(95%CI:1.97-4.7)]. HPV16-specific typing showed the best specificity [94%(95%CI:82-97)], PPV (89%) and positive LR [11.6(95%CI:2.93-47.75)]. Amongst the various combinations, p16 with high-risk HPV testing showed a specificity of 98% and a sensitivity of 58%. Conclusions: High-risk HPV testing has the highest sensitivity, whilst HPV 16-specific genotype achieves the best specificity for the detection of high-grade lesions. These test and some of the above combinations could discriminate women at high-risk that need referral to colposcopy +/-treatment from those at low risk that do not require further unnecessary intervention. All the above markers should be evaluated in a cost analysis and could be integrated in low-grade triage prediction scoring system, allowing tailored management of women.
88

PREVALENCE OF HPV INFECTION IN PREGNANT LITHUANIAN WOMEN
Z Gudleviciene, Institute of Oncology, Vilnius University, Vilnius, LITHUANIA G Domza, Vilnius university, Faculty of Medicine, Clinic Obstetrics and Gynecology, Vilnius, LITHUANIA J Didziapetriene, Institute of Oncology, Vilnius University, Vilnius, LITHUANIA Background. HPV infection is one of the most prevalent infections in the world. It is well known risk factors for cervical and other mucosal cancers development. Many studies conducted to date to evaluate the HPV prevalence in many populations with mean prevalent Objective of the study: to detect the prevalence of HPV in I and III trimester of pregnant women in Lithuania. Methods. 213 pregnant women were invited to participate in the study and tested for HPV infection in period of 2008 2010 years. HPV test was done two times in the beginning of pregnancy (I trimester) and the in the end (III trimester). HPV testing and typing was performed using Polymerase chain reaction. The questionnaire regarding other risk factors was filled. Results. In the first trimester 17.8% (95% PI=13.2523.57) of pregnant women (38 from 213) were infected by HPV; 52.6% of them were infected by high risk group HPV. In the end of pregnancy (III trimester) 10.3% (95% PI=6.7617.16) of pregnant women (15 from 146) were infected by HPV, high risk types identified for 66.7% of them. 33.3% (5 from 15) of women have been infected by HPV in III trimester de novo. Analysis of other risk factors shows that younger women (<29 years) and women with previous gynecological diseases (inflammation) had the bigger risk to be infected by HPV during the pregnancy (OR=0.291, 95% PI=0.1860.456, p=0.063 and OR=0.383, 95% PI=0.2220.659, p=0.013 respectively). Conclusions. Biggest prevalence of HPV was stated in the beginning of pregnancy (17.8 vs 10.3%, p< 0.05); younger women (<29 years) and women with previous gynecological diseases (inflammation) had the bigger risk to be infected by HPV during the pregnancy.
P-15.24
RISK ASSESSMENT AFTER ABNORMAL SCREENING: WHAT ROLE FOR COLPOSCOPIC GRADING?
S Igidbashian, European Institute of Oncology, Milan, ITALY S Boveri, European Institute of Oncology, Milan, Italy N Spolti, European Institute of Oncology, Milan, Italy C Casadio, European Institute of Oncology, Milan, Italy D Radice, European Institute of Oncology, Milan, Italy M T Sandri, European Institute of Oncology, Milan, Italy M Sideri, European Institute of Oncology, Milan, Italy BACKGROUND: Data from studies investigating highly sensitive molecular tests have challenged the ability of colposcopy to detect cervical pre-cancers. Colposcopy is a technique to identify the area to place a biopsy, and so colposcopy result equals the biopsy pathology report. It has been suggested that the entire cervical cancer screening process can be viewed as a risk stratification system where the result of different exams, cytology, molecular analysis etc, acts as risk stratifier which trigger intervention (closer follow up or treatment). In this way the patient is managed by level of risk and not on the basis of the histologic diagnosis of a colposcopic directed biopsy. OBJECTIVE: We tested the hypothesis that colposcopic impression alone can act as risk stratifier in patients with abnormal screening. METHODS: 657 patients submitted to LEEP or laserconization were analyzed. The pathology results of the excised specimen served as gold standard. Results of cervical cytology, HR-HPV test, HPV genotyping and colposcopy at the time of treatment were recorded. Cervical cytology was classified according to the 2001 Bethesda system; HR-HPV test as positive or negative; HPV genotyping as HPV16 positive or HR-HPV non-16 positive; colposcopy as negative, grade (G) 1, G2 or suspicion of invasive cancer. RESULTS: When G2+ colposcopies were associated with a positive cervical cytology at an ASC-US threshold, the histology of the excised specimen was CIN2+ in 87.7% of the cases and when it was associated with a positive HRHPV test in 90.1% of the cases. Conversely the finding of a G1 colposcopy in the presence of an HSIL cytology or in the presence of a positivity to HPV16 predicted CIN2+ respectively in 73.3% and 77% of the cases. CONCLUSIONS: The results suggest that colposcopic grading without histology can be used as risk stratifier in a management algorithm based on risk stratification.
Declaration of interest M. Sideri is advisor with no personal salary of Sanofi Pasteur, GSK, Qiagen, Innogenetics, and MTM. The salary is given to the European Institute of Oncology.
89
Session 16: Clinical aspects of non-cervical infections

Chair: Anna Giuliano
Poster abstracts
Clinical aspects of non-cervical infections | 16 O-16.00

Anna Giuliano
FOR YOUR NOTES:
O-16.01
PERSISTENT BETAPAPILLOMAVIRUS INFECTIONS IN ORGAN TRANSPLANT RECIPIENTS
M de Koning, DDL Diagnostic Laboratory, Voorburg, NETHERLANDS, S Weissenborn, Institute for Virology, Cologne, Germany, S Euvrard, Hpital Edouard Herriot, Lyon, France, M Feltkamp, Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands, C Harwood, Centre for Cutaneous Research, Blizard Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom, L Naldi, Department of Dermatology and GISED Study Center, Ospedali Riuniti, Bergamo, Italy, I Nindl, DKFZ Charit, Viral Skin Carcinogenesis, Division Viral Transformation Mechanisms, German Cancer Research Center (DKFZ), Heidelberg, Germany, H Pfister, Institute for Virology, Cologne, Germany, W Quint, DDL Diagnostic Laboratory, Voorburg, the Netherlands, J Bouwes Bavinck, Department of Dermatology, Leiden University Medical Center, Leiden, the Netherlands Background and objectives: Betapapillomaviruses (betaPV) have been implicated as a co-factor in non-melanoma skin carcinogenesis, and organ transplant recipients (OTR) have a much higher risk of developing skin cancer than healthy persons. In this study the cumulative number of betaPV types (persistently) infecting OTR in the first two years after transplantation was determined. Methods: One-hundred and one OTR from The Netherlands, 96 from France, 100 from Italy, 24 from the UK and 88 from Germany were included in this analysis. Ten eyebrow hairs were plucked from each person at approximately 3-monthly intervals. The presence of 25 beta-PV types was determined by PCR and reverse hybridization. Data from three more study populations will be analyzed and presented. Results: At baseline, prior to transplantation, 91% of the OTR were betaPV positive and all 25 betaPV types were found with a maximum of 14 betaPV types in one eyebrow hair sample. The cumulative number of betaPV types increased during follow-up but no significant type-specific increase was observed except for HPV23 and HPV47 (p=0.03). When persistence was defined as having the same type present at least 12 months apart, 258/296 (87%) OTR had a total of 1147 persistent infections with an average of 3.9 persisting types. Conclusions: The high cumulative number of betaPV types and the relatively high percentage of persistently infected OTR provide further evidence for a possible role of betaPV as a co-factor in skin carcinogenesis. Matched case-control or longer lasting follow-up studies are needed to understand which host factors are critical in HPV infection and tumour development in OTR.
93
16 | Clinical aspects of non-cervical infections O-16.02

NON-MELANOMA SKIN CANCER AND CUTANEOUS HPV PSEUDOVIRION ANTIBODIES: PROSPECTIVE STUDY
K Andersson, Skne University Hospital, Malm, SWEDEN H Faust, Lund University, Malm, SWEDEN T Luostarinen, Finnish Cancer Registry, Helsinki, FINLAND J Dillner, Karolinska Institute, Stockholm, SWEDEN Background: Cutaneous HPV is commonly found in NMSC, but whether cutaneous HPV infection is associated with NMSC risk is not known. Serology using native pseudovirions appears to have an improved correlation to HPV infection. Objectives: To determine whether antibodies to HPV pseudovirions associate with the risk for future squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) over a follow-up of up to 30 years. Methods: Serum samples from healthy subjects who during follow-up later developed SCC (633 subjects) or BCC (1990 subjects) and an equal number of matched subjects who did not develop NMSC were tested with a heparinpseudovirion Luminex method to detect IgG antibodies against 16 different HPV types (3,5,6,11,15,16,18,31,32,33, 38,45,52,58,68 and 76) and 2 polyomaviurses (MCV and JCV). Results: Weak associations to BCC risk were found for baseline seropositivity for HPV5 (OR=1.1 95%CI 1.0-1.3), HPV15 (OR=1.2 95%CI 1.0-1.4), HPV38 (OR=1.2 95%CI 1.0-1.4) and MCV (OR=1.1 95%CI 1.0-1.3). Analysis of subjects with multiple serial samples taken before diagnosis found that seroconversion for HPV 31 and HPV5 during follow-up was strongly associated to SCC (OR=10.0 95%CI 1.3-78 and OR=3.2 95%CI 1.3-7.6 respectively). Seroconversions to HPV38 and 76 were weakly associated with SCC risk (OR=2.0 95%CI 1.0-4.0) and OR=1.9 95%CI 1.0-3.8 respectively). Conclusion: Baseline seropositivity for certain cutaneous HPV types showed a weak association to BCC risk, whereas seroconversion (acquisition of new seropositivity in serial samples taken before diagnosis) to in particular HPV31 and 5 associated with SCC risk.
Declaration of interest K Andersson - None declared H Faust - None declared T Luostarinen - None declared J Dillner - Recipient of research grants from Merck and SPMSD
O-16.03
EPIGENOMIC MARKERS OF RESPONSE AND HPV GENOTYPING IN ANAL CANCERS
E Siegel, Moffitt Cancer Center, Tampa, FL, UNITED STATES, B Riggs, Moffitt Cancer Center, Tampa, FL, UNITED STATES, W Grady, FHCRC, Seattle, WA, UNITED STATES, S Eschrich, Moffitt Cancer Center, Tampa, FL, UNITED STATES, A Kaz, FHCRC, Seattle, WA, UNITED STATES, A Giuliano, Moffitt Cancer Center, Tampa, FL, UNITED STATES, D Coppola, Moffitt Cancer Center, Tampa, FL, UNITED STATES, D Shibata, Moffitt Cancer Center, Tampa, FL, UNITED STATES Background: The incidence of anal cancer, an HPV-associated malignancy, is on the rise and a significant burden among HIV populations. Chemoradiation treatment for anal cancer is associated with substantial short and longterm morbidity. Biomarkers that identify patients who may or may not respond to standard treatment would be valuable to allow for alternative treatment strategies, including dosage reductions or avoidance of ineffective treatment. Evidence suggests that HPV status and DNA methylation patterns may represent useful predictive biomarkers of treatment response. Objectives: In this pilot study, we sought to identify differential methylation patterns between responders and nonresponders and characterize HPV genotypes in anal cancer patients treated by 5FU, Mitomycin C and radiation. Methods: DNA was extracted from formalin-fixed paraffin embedded tumors from 24 non-immunosuppressed patients from the RTOG trial 98-11 (10 non-responders with 3-year locoregional recurrence and 14 responders). Methylation status was analyzed by the Illumina HumanMethylation27 Array (27,578 CpG loci) and HPV status determined by SPF10-LiPA HPV genotyping assay. Differential methylation was analyzed using Mann-Whitney and t-tests. Results: Non-responders demonstrated differential methylation in 162 genes compared to responders, including Thromobospondin-1 (p=7x10-6; an inhibitor of angiogenesis), MSX-1 (p=9x10-5; an effectors of apoptosis), FOXL2 (p=3x10-5; associated with 5-FU sensitivity) and EPHB1 (p=1x10-4; underexpressed in more aggressive GI cancers). Using pathway enrichment analysis, 37 biologic pathways were identified with differential methylation, including apoptosis, DNA damage repair and cell adhesion/cytoskeletal remodeling pathways. Preliminary data suggest that all anal cancers are HPV-positive and the number of HPV infections and type distribution varies. Conclusions: We have demonstrated biologically relevant alterations in individual methylated targets and cellular pathways that discriminate between anal cancer treatment responders and non-responders. Larger scale studies to further evaluate and validate the impact of epigenetic signatures and HPV status on anal cancer prognosis are warranted.
94

RISK FACTORS FOR ANAL INTRAEPITHELIAL NEOPLASIA IN HIV+ MSM
O Richel, Academic Medical Center, University of Amsterdam, Amsterdam, NETHERLANDS H J C de Vries, Academic Medical Center, University of Amsterdam, Amsterdam, NETHERLANDS M G W Dijkgraaf, Academic Medical Center, University of Amsterdam, Amsterdam, NETHERLANDS C.M. van Noesel, Academic Medical Center, University of Amsterdam, Amsterdam, NETHERLANDS J M Prins, Academic Medical Center, University of Amsterdam, Amsterdam, NETHERLANDS BACKGROUND Anal Intraepithelial Neoplasia (AIN) is present in the majority of HIV+ men who have sex with men (MSM) and routine screening and treatment of AIN is subject of discussion. It would be helpful to identify risk factors for having AIN. OBJECTIVE To analyse a wide range of potential risk factors for AIN in HIV+ MSM. METHODS We screened 311 HIV+ MSM for AIN by high resolution anoscopy and histopathological examination of suspect lesions. Medical history, HIV parameters and questionnaires on sexual practices and illicit substance use were collected. Risk factors were identified by uni- and multivariate logistic regression. RESULTS AIN was found in 175/311 MSM (56.3%) and HG AIN in 92 (29.6%). Univariate analysis showed that antiretroviral therapy (ART)-use (odds ratio [OR] =0.45 p=0.04), number of years using ART (OR=0.92 per year, p=0.001) and a history of hepatitis A (OR=0.52, p=0.02) had a negative association with AIN. Factors associated with an increased risk were a history of condyloma (OR=1.91, p=0.006) and use of GHB (-Hydroxybutyric acid) (OR=2.1, p=0.009). Amphetamine use was marginally significant (OR=1.8, p=0.079). Nadir and baseline CD4, number of lifetime passive anal sex partners, years of sexual activity, anal fisting and anal substance use were not significantly associated with AIN. In the multivariate analysis three parameters remained significant: number of years using ART (OR=0.93 per year, p=0.009), a history of hepatitis A (OR=0.47, p=0.017) and GHB use (OR=1.97, p=0.023). The only parameter significantly associated with HG AIN was enema use prior to sex (life time > 50 enemas OR 1.79, p=0.03). CONCLUSION Anal enemas and GHB use independently increase the risk on (HG) AIN, ART duration and a history of hepatitis A are negatively correlated with AIN. However, given the high prevalence of AIN in HIV+ MSM, these associations are not helpful to guide a screening programme.
O-16.05
HPV GENOTYPE ATTRIBUTION IN ANAL INTRAEPITHELIAL NEOPLASIA AMONG HIV-INFECTED MSM
V Sahasrabuddhe, National Cancer Institute, Rockville, UNITED STATES, P Castle, American Society for Clinical Pathology, Washington, UNITED STATES, S Follansbee, Kaiser Permanente Northern California, San Francisco, UNITED STATES, S Borgonovo, Kaiser Permanente Northern California, San Francisco, UNITED STATES, B LaMere, Kaiser Permanente Northern California, San Francisco, UNITED STATES, D Tokugawa, Kaiser Permanente Northern California, San Francisco, UNITED STATES, T Darragh, University of California, San Francisco, San Francisco, UNITED STATES, M Sadorra, Roche Molecular Systems, Pleasanton, UNITED STATES, S Tange, Roche Molecular Systems, Pleasanton, UNITED STATES, N Wentzensen, National Cancer Institute, Rockville, UNITED STATES Background: Evaluating causal attribution of HPV genotypes to high-grade anal intraepithelial neoplasia (AIN) is important to establish a type spectrum for HPV-based anal cancer screening and to estimate the potential impact of HPV vaccination. Objectives: To characterize HPV genotypes and evaluate type-specific attribution to AIN. Methods: 363 HIV-infected, men who have sex with men (MSM) underwent anal cytology and high-resolution anoscopy/biopsy at an anal cancer-screening clinic at Kaiser Permanente Northern California. A composite histology/cytology endpoint was used to classify disease stages as no AIN, AIN1 [AIN1 histology or ASC-US/LSIL cytology], AIN2 [AIN2 histology or HSIL-AIN2/ASC-H cytology] and AIN3 [AIN3 histology or HSIL-AIN3 cytology]. HPV was measured using the Roche Linear Array (LA) and cobas 4800 assays. Results: Using LA assay, the prevalence of any of 37 HPV genotypes and any of 13 carcinogenic HPV genotypes were 94.4% and 75.4%, respectively. Multiple concurrent HPV genotypes [80.7%, median 4(IQR:2-6) types/participant], and multiple carcinogenic HPV types [47.4%, median 1(IQR: 1-3)] were very common. 118(32.5%) MSM had no AIN, 129(35.5%) had AIN1, 59(16.3%) had AIN2, and 57(15.7%) had AIN3. Prevalence of HPV16 ranged from 9% in MSM with no AIN to 62.5% in MSM with AIN3. Restricted to single HPV infections, prevalence of HPV16 ranged from 3.6% in MSM with no AIN to 40.0% in MSM with AIN3. The next most common carcinogenic HPV types detected singly in AIN3 were 31, 33, 35, and 52 (13.3% each) and HPV51 (6.7%). We observed excellent agreement between cobas 4800 and LA assays for detection of HPV16 [kappa:0.93 (95%CI 0.89-0.97)], HPV18 [kappa:0.86 (95%CI 0.76-0.94)], and any of 11 other carcinogenic HPV types plus HPV66 [kappa:0.76 (95%CI 0.68-0.83)]. Conclusion: Similar to cervix, multiple HPV genotype infections complicate attribution of HPV genotypes to AIN disease stages. HPV16 is strongly associated with AIN2/3 in HIV+ MSM.
Declaration of interest M. Sadorra and S. Tang are employed by Roche Molecular Systems, manufacturer of the Linear Array and cobas 4800 HPV assays used in this study.
95
16 | Clinical aspects of non-cervical infections O-16.06

IMMUNOHISTOCHEMICAL IDENTIFICATION OF OROPHARYNGEAL SQUAMOUS CELL CARCINOMAS WITH ACTIVE HPV16
D Holzinger, Experimental Head and Neck Oncology, Department of Otolaryngology, Head and Neck Surgery, University Hospital, Heidelberg, GERMANY, C Flechtenmacher, Institute of Pathology, University Hospital, Heidelberg, GERMANY, M Schmitt, Division of Genome Modifications and Carcinogenesis, Infection and Cancer Program, German Cancer Research Center (DKFZ), Heidelberg, GERMANY, J He, Experimental Head and Neck Oncology, Department of Otolaryngology, Head and Neck Surgery, University Hospital, Heidelberg, GERMANY, M Pawlita, Division of Genome Modifications and Carcinogenesis, Infection and Cancer Program, German Cancer Research Center (DKFZ), Heidelberg, GERMANY, F X Bosch, Experimental Head and Neck Oncology, Department of Otolaryngology, Head and Neck Surgery, University Hospital, Heidelberg, GERMANY Background: HPV16 DNA-positive oropharyngeal squamous cell carcinomas (OPSCC) are heterogeneous regarding viral oncogene expression and clinical behavior. HPV is actively involved only in tumors with viral oncogene RNA expression. Objectives: To determine whether the classical analysis of the cellular proteins p16INK4a, pRb, Cyclin D1 and p53 can provide surrogate marker combinations that identify OPSCC with an active HPV16 involvement using tissue microarraybased immunohistochemistry (IHC). Methods: Expression of the marker proteins was analyzed on tissue microarrays created from 188 OPSCC with known HPV DNA and RNA status. Associations of the markers and marker combinations with cervical carcinoma-like (CxCa-like) viral RNA patterns (RNA+ group) were evaluated and sensitivity, specificity, negative and positive predictive values were calculated. Cox proportional hazard models were used to estimate the association between the markers and marker combinations with survival. Results: The cellular protein expression profiles in the RNA+ group were significantly different from HPV-negative tumors (HPV- group) and from HPV16 DNA-positive tumors without CxCa-like viral RNA patterns (RNA- group). RNA+ OPSCC were characterized by high p16INK4a, reduced pRb, low Cyclin D1 and low p53 expression levels. Among all possible IHC marker combinations, the strongest associations with RNA+ tumors (OR 59.1, 95%CI 15.3-228.8, p<.0001) and with a longer survival (HR 0.34, 95%CI 0.1-0.8, p=0.02), and best predictive values (79% sensitivity, 94% specificity, 79% PPV, 94% NPV) were found for the combination of high p16INK4a and reduced pRb expression levels. Conclusion: The clinical heterogeneity among HPV16 DNA-positive OPSCC is also reflected by the expression patterns of cellular surrogate markers. RNA- tumors are similar as HPV- tumors. The marker combination p16INK4a-high/pRb-reduced is best suited to predict and to identify RNA+ OPSCC with biologically active HPV16 which represent a distinct OPSCC entity with better prognosis.
O-16.07
INCREASED RADIATION SENSITIVITY IN HPV-POSITIVE HEAD AND NECK CANCERS
R Kimple, Univ of Wisconsin, USA, Madison, UNITED STATES, R Yang, Univ of Wisconsin, Madison, USA, G Blitzer, Univ of Wisconsin, Madison, USA, M Smith, Univ of Wisconsin, Madison, USA, E Armstrong, Univ of Wisconsin, Madison, USA, P Harari, Univ of Wisconsin, Madison, USA, P Lambert, Univ of Wisconsin, Madison, USA Background/objectives: HPV is associated with a growing proportion of head and neck squamous cell carcinomas (HNSCCs), which show improved clinical outcome compared with non-HPV associated HNSCC. Utilizing primary tumor and cell line xenograft models as well as existing cell lines and novel cell strains, we are investigating combinations of therapy for HPV-positive and HPV-negative head and neck cancers and attempting to understand the mechanisms underlying improved outcomes in HPV-positive HNSCC. Methods: Patient biopsies were implanted directly into NOD-SCID gamma mice for initial passage. Athymic nude mice were used for both primary and cell line xenograft studies to assess single and combination therapies of radiation, cisplatin, and/or cetuximab. Southern blot was used to confirm HPV status in HNSCC cell lines and directfrom-patient primary tumor xenografts. Clonogenic survival and proliferation assays were used to assess radiation and chemotherapy sensitivity, respectively. Results: Thirteen primary tumor xenografts have been established and passaged from P0 to P1. Xenografts retain characteristics of the primary tumor through initial passage. Using in vitro assays, HPV-positive HNSCC cell lines are more sensitive than HPV-negative HNSCC cell lines to single-fraction radiation exposure, but have similar sensitivity to the chemotherapeutic agents cisplatin, docetaxel, cetuximab, and erlotinib. HPV-positive cell line xenografts show a significantly improved response to chemoradiation with concurrent cetuximab compared to either radiation alone or radiation with concurrent cisplatin. Primary tumor xenografts are being expanded to validate their usefulness as co-clinical models to inform newly initiated phase 3 clinical trials exploring alternative chemo-radiotherapy regimens. Conclusions: Consistent with clinical reports, HPV-positive HNSCC cells exhibit increased sensitivity to radiation. Both primary tumor and cell line xenografts provide a useful system for testing existing and novel chemoradiation approaches. In addition to in vitro systems, these models provide a framework for understanding the molecular mechanisms for altered response profiles in HPV-positive HNSCC.
96

VALUE OF HPV IN NECK METASTASIS OF UNKNOWN PRIMARY TUMOURS.
J Straetmans, Maastricht University Medical Centre, Maastricht, NETHERLANDS, J Vent, University Hospital Cologne, Cologne, Germany, M Henfling, Maastricht University Medical Centre, Maastricht, Netherlands, A Haesevoets, Maastricht University Medical Centre, Maastricht, Netherlands, B Haild, University Hospital of Cologne, Cologne, Germany, C Huebbers, Universtiy Hospital of Cologne, Cologne, Germany, F Ramaekers, Maastricht University Medical Centre, Maastricht, Netherlands, E Speel, Maastricht University Medical Centre, Maastricht, Netherlands, P Klussmann, University of Giessen, Giessen, Germany, B Kremer, Maastricht University Medical Centre, Maastricht, Netherlands Background. There is no consensus on optimal diagnostic and therapeutic strategies for patients with neck metastasis of unknown primary tumours (UPT). UPTs are heterogeneous in their clinical and biological characteristics, and a pre-operative prognostic marker is desirable to optimize therapy and improve outcome. Human papillomavirus (HPV) is an etiologic factor in a subgroup of head and neck squamous cell carcinomas and a significant prognostic biomarker. Objective. We analysed the value of HPV concerning the origin and prognosis of UPTs. Methods. 47 patients (mean age 58.7 years; 40 men, 7 women) presenting with UPTs between 1994 and 2008 in Cologne were examined by standard diagnostic procedures including radiological imaging of head, neck, thorax and abdomen, positron emission tomography (PET), panendoscopy, nasopharyngeal curettage, bilateral tonsillectomy and blind probes of base of tongue. All patients were surgically treated with neck dissection and adjuvant chemoradiation. The mean follow-up time was 34 months. Formaldehyde-fixed, paraffin-embedded tissue specimens of the 47 metastases were examined retrospectively by means of p16INK4A immunohistochemistry, HPV-specific PCR and fluorescence in situ hybridization. Results were correlated with clinical follow-up data. Results. Oncogenic HPV was present in 12/47 (26%) metastases (10 with HPV 16), which showed also p16INK4A overexpression. In 42% of UPTs, the primary tumor was discovered during follow-up. A significant correlation between HPV positivity and later detection of the primary tumor in the oropharynx was found (p=0.038). Moreover, patients with a p16INK4A- (13/47) or HPV-positive tumor had a more favorable overall 5-year survival rate then p16INK4A- and HPV-negative tumors (69% vs 33%, p=0.05; 65% vs 37%, p=0.093; respectively). Conclusion. HPV is present in a quarter of neck metastases of UPTs and the presence of oncogenic HPV and p16INK4A expression can serve to locate the primary tumor in the oropharynx. In addition, both biomarkers are indicators of a favorable prognosis.
97
16 | Clinical aspects of non-cervical infections P-16.09

HPV DNA DETECTION IN OESOPHAGEAL LESIONS BY REAL TIME PCR.
B Dinc, T. Yuksek Ihtisas Hospital, Microbiology Laboratory, Ankara, TURKEY G Bozdayi, Gazi University, Faculty of Medicine, Department of Medical Microbiology, Ankara, TURKEY G Aydog, T. Yuksek Ihtisas Hospital, Pathology Laboratory, Ankara, TURKEY M Meral, Gazi University, Faculty of Medicine, Department of Medical Microbiology, Ankara, TURKEY S Kuran, T. Yuksek Ihtisas Hospital, Department of Gastroenterology, Ankara, TURKEY Background: Human papillomavirus (HPV) has been found repeatedly in oesophageal lesions. However, reported detection rates of HPV DNA in these lesions have varied markedly according to the differences in detection methods, sample types, and geographic regions of sample origin. Objectives: The aim of this study was to detect HPV DNA and HPV type 16 in oesophageal papillomas and squamouse cell carcinoma (SCC) of oesophagus. Methods: 76 tissue samples (50 oesophageal papillomas and 26 SCC) were included in the study. After deparaffinization by xylene, HPV DNA extraction was performed by phenol chloroform isoamylalchol. Seventy six tissue samples were amplified by using a glucose 6-phosphatase dehydrogenase (G6PDH) control kit (Eurogentec, Seraing, Belgium), and 65 G6PDH-positive samples were thought to have appropriate tissues. HPV DNA was analyzed in 65 (44 oesophageal papilloma and 21 SCC) cases according to G6PDH amplification. HPV DNA and HPV type 16 were detected by Real Time polymerase chain reaction (RT-PCR) using the L1 region. Real Time PCR nested amplifications of MY09/11 products were done by GP5+/GP6+ primers and Cyanine-5 labeled HPV DNA and HPV 16 DNA specific probe. Results: Of 65 DNA positive tissues 44 (67.7%) were papillomas and 21 (32.3%) were SCC of oesophagus. We found one (2.3%) HPV DNA positive and 12 (27.3%) HPV type 16 positive in 44 squamous papilloma samples. On the other hand of 21 SCC samples 11 (52.4 %) were found HPV type 16 end one (4.8 %) was mixed type. Conclusions: HPV is one of the most important DNA viruses in carcinogenesis and our results suggest that HPV infection is common in oesophageal papillomas and oesophageal carcinoma. Due to the well-known association of HPV with premalignant and malignant conditions, follow-up of patients with esophageal lesions accompanied by HPV should be implemented.
P-16.10
REAL TIME PCR OF HPV DNA IN COLORECTAL CANCER TISSUES.
G Bozdayi, Gazi University, School of Medicine, Ankara, TURKEY B Dinc, Ministry of Health, T. Yuksek Ihtisas Hospital, Microbiology Laboratory, Ankara, TURKEY G Aydog, Ministry of Health, T. Yuksek Ihtisas Hospital, Pathology Laboratory, Ankara, TURKEY M Akoglu, Ministry of Health, T. Yuksek Ihtisas Hospital, Pathology Laboratory, Ankara, TURKEY A Pnar, Hacettepe University, Faculty of Medicine, Department of Medical Microbiology, Ankara, TURKEY M Meral, Gazi University, School of Medicine, Ankara, TURKEY Backround: Recent publications have reported an association between colon cancer and human papillomaviruses (HPV), suggesting that HPV infection of the colonic mucosa may contribute to the development of colorectal cancer. The aim of this study was to reveal the prevalence of HPV DNA and HPV type 16 in colorectal cancer tissues. Methods: One hundred and twenty parafin embedded colorectal adenocarcinoma tissues were included in the study. After deparaffinization by xylene, HPV DNA extraction was performed by phenol chloroform isoamylalcohol. One hundred and twenty tissue samples were amplified by using a glucose 6-phosphatase dehydrogenase (G6PDH) control kit (Eurogentec, Seraing, Belgium), and G6PDH-positive samples were accepted as appropriate and adequate tissues. HPV DNA and HPV type16 were detected by realtime PCR targeting the L1 region. Real-time nested amplifications of MY09/11 products were done by GP5+/GP6+ primers and Cyanine-5 labeled HPV and HPV 16 DNA specific probe. Real time PCR product analysis was done by melting curve analysis on LightCycler Software version 3.5.3 (LC 2.0 Roche Diagnostics, Germany). Melting peaks of 78-82C showed the detection of HPV DNA in the sample. Probe melting peaks of positive samples has been analyzed in the same run and HPV16 positive samples gave peaks around 68C. Results: We found 111 (92.5) G6PDH positivity. Twenty (18%) of the 111 DNA positive tissues from colorectal adenocarcinoma tissues were HPV DNA (other than 16) positive, 22 (19.8 %) were HPV type 16, and five (4.5%) were mixed type (type 16+other than 16). Conclusion: Our results suggest that colorectal HPV infection is common in patients with colorectal cancer and as well as type 16, HPV types other than 16 also have a critical role in colorectal carcinogenesis.
Declaration of interest none declerated
98
Clinical aspects of non-cervical infections | 16 P-16.11

ROLE OF HUMAN PAPILLOMAVIRUS IN HEAD AND NECK CANCER
G Alp Avc, Department of Medical Microbiology, School of Medicine, Gazi University, , Ankara, Turkey G Bozdayi, Department of Medical Microbiology, School of Medicine, Gazi University, , Ankara, , Turkey Y. K. Kemaloglu, Department of Otorhinolaryngology, School of Medicine, Gazi University,, Ankara, , Turkey O. Erdem, Department of Pathology, School of Medicine, Gazi University, , Ankara,, Turkey S. Ozkan, Department of Public Health, School of Medicine, Gazi University, , Ankara, , Turkey M. Meral, Department of Clinical Microbiology, School of Medicine, Gazi University, , Ankara, , Turkey Y. A Beyazit, Department of Otorhinolaryngology, School of Medicine, Gazi University, , Ankara, , Turkey I. E Inal, Department of Otorhinolaryngology, School of Medicine, Gazi University, , Ankara,, Turkey Background: Human papillomavirus (HPV) infections are thought to be one of the causal factors in the development of head and neck squamous cell carcinomas (HNSCC) Approximately 25% of HNSCC worldwide are associated with high-risk HPVs. HPV-positive HNSCC's have a more favorable outcome and greater response to therapy. Objectives: To determinate the prevalence of HPV types in patients with head and neck cancers in our region by PCR was aimed in this study. Methods: Tissue samples of 45 patients with head and neck cancer were included in the study. After deparafinization by xylene, HPV DNA extraction was performed by phenol-chloroform-isoamylalcohol. HPV DNA was analyzed in 45 cases from larynx tissue. HPV DNA and HPV type 16 were detected by Real Time polymerase chain reaction (RTPCR) targeting the L1 region. Real Time PCR nested amplifications of MY09/11 products were done by GP5+/GP6+ primers and Cyanine-5 labeled HPV DNA and HPV 16 DNA specific probe. Results and Conclusion: HPV type 16; 46.6%, and non-typing HPV; 9.0% were found in patients with head and neck cancer. Also, HPV positivity was determined as 75% in glottic area and 43% in supraglottic area. According to results, T1-8.8%, T2-46.6%, T3-31.2% and T4-16.7% of HPV positivity detected in patients with head and neck cancer. Furthermore, HPV positivity was observed in 28% of 16 patients who have a lymph node metastasis. Thirty-nine of the patients were smokers and 56.4% of these were also positive for HPV. The average age of the population participated in the study were between 4624 and HPV-positive individuals were between 36-58 years. Analysis of HPV positivity could be used as a prognostic factor. Consequently screening the presence of HPV in larynx lesions is important and must be added in routine analysis of larynx lesions.
Declaration of interest I have no any sponsor
P-16.12
DETECTION AND PROGNOSTIC VALUE OF HPV IN HEAD NECK CANCER
Z Deng, University of the Ryukyus, Nishihara, Okinawa, JAPAN M Suzuki, University of the Ryukyus, Nishihara, Okinawa, JAPAN Background: HPV has been shown to be etiologically related to development of cervical cancers, but its roles in HNSCCs remain unestablished. Objectives: To determine human papillomavirus (HPV) load and physical status, especially HPV-16, and its prognostic value in different types of head and neck squamous cell carcinomas (HNSCCs) using fresh-frozen samples. Methods: HPV DNA and E6/E7 mRNA expression in 184 patients with HNSCC were examined by polymerase chain reaction (PCR) and reverse-transcription PCR (RT-PCR), respectively. Viral load and physical status of HPV-16 were determined by real-time PCR of the E6 and E2 genes. Survival outcomes, including recurrence-free survival and disease-specific survival were also examined. Results: HPV was detected in 54 HNSCC samples (29.3%), and HPV-16 was identified in 87.0% of positive specimens. The HPV genome was most frequently observed in the oropharyngeal cancers (50.0%). In the oropharynx, 69.6% of tonsillar carcinomas were HPV positive and the viral copy numbers were significantly higher than in non-tonsillar HNSCCs (P < 0.001; median 4.13 105/50ng DNA vs. 9.7 101/50ng DNA). Analysis of viral physical status in HPV16-positive samples demonstrated the presence of either integrated or mixed forms in 75.6%. RT-PCR detected E6/E7 mRNA transcription in 13 of 51 (25.5%) HPV DNA-positive cases. High HPV-16 DNA load was significantly correlated with E6/E7 mRNA expression of HPV-16 (P < 0.001). Of the 184 patients, 144 cases received curative surgery and/or concurrent chemoradiation treatment. HPV positivity significantly correlated with recurrence-free survival (P = 0.0152 for HNSCC and P = 0.0364 for oropharyngeal SCC). Conclusion: This study demonstrates a significant relationship between HPV and HNSCC, especially for cancers of the palatine tonsil. Integrated status is frequently found in HPV-associated HNSCC. The presence of HPV is a favourable prognostic factor for survival among patients with HNSCC, especially with oropharyneal SCC.
99

HPV SURROGATE MARKERS IN BENIGN, PREMALIGNANT, AND MALIGNANT VULVAR LESIONS
J Hariri, Sonderborg Hospital, Sonderborg, DENMARK Background: Currently recognised precursors of vulvar squamous cell carcinoma (SCC) are HPV-related VIN, NonHPV-related (simplex) VIN, Lichen Sclerosus (LS), and chronic vulvar granulomatous lesions. It is also believed that there are 2 types of vulvar SCC; one is HPV-related and seen in a younger age-group, and one in an older age-group and is probably LS-related. Objectives: The objective of this retrospective pilot-study was to test the ability the HPV surrogate protein markers (HPV-SM) to mirror these assumptions. Material and methods: The study included 20 cases of inflammatory / benign lesions, 20 cases of LS, 20 cases of VINIII, and 20 cases of SCC, all were analysed during 2008 and 2009. Newe sections were stained by HE, p16, p53, Ki67, and PreoEx-C were cut. The immunostaining was performed on BenchMark-XT / Ventana-Roche. A case was regarded positive for HPV-SM when at least 2 of p16, Ki67 and ProEx-C were positive. Results: 4/20 (20%), 13/20 (65%), 8/20 (40%), and 13/20 65% of the cases in the benign group, LS group, VINIII group, and vulvar SCC respectively were older than 60 years. All of the benign and LS cases were negative for both HPV-SM and p53. HPV-SM and p53 were positive in 20/20 and 14/20 of the VINII cases. All of the 6 p53 negative cases in this group were 60 years. 6 of the 7 60 years (86%) and 4 of the 13 > 60 years (31%) cases of SCC were positive in HPV-SM, while p53 was positive in 4 of 7 (57%), and 12 of 13 (92%) cases of SCC in the same order. Conclusion: The results support the assumption of 2 types of vulvar SCC; one is HPV related in a younger group and the other is mostly p53 positive. We found no correlation between LS and SCC.
P-16.14
CLINICAL VALUE OF HPV-125/150/151 AND IDENTIFICATION OF NOVEL HPV-150-RELATED GENOTYPE
A Kovanda, Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Ljubljana, SLOVENIA B Luzar, Institute of Pathology, Faculty of Medicine, University of Ljubljana, Ljubljana, SLOVENIA B Kocjan, Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Ljubljana, SLOVENIA B Pir, Department of Dermatovenereology, University Medical Centre Ljubljana, Ljubljana, SLOVENIA M Potonik, Department of Dermatovenereology, University Medical Centre Ljubljana, Ljubljana, SLOVENIA M Poljak, Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Ljubljana, SLOVENIA Three novel HPV genotypes HPV-125, HPV-150 and HPV-151 are phylogenetically related to cutaneous HPVs of Alphapapillomavirus species 2 (HPV-125), and Betapapillomavirus species 5 (HPV-150) and 2 (HPV-151). Because phylogenetic relationships among HPVs do not necessarily reflect their tissue tropism, the tissue predilection of the three novel genotypes was assessed by testing 601 clinical samples for HPV-125 and 540 clinical samples for HPV-150 and HPV-151 by using quantitative real-time PCR assays. The panel of samples was representative of various sites (mucosal and skin) and outcomes of HPV-infection, and included most important HPV-associated malignant neoplasms (cervical carcinoma, squamous cell (SCC) and basal cell carcinoma of the skin (BCC)), the most important HPV-associated benign neoplasms (genital warts, laryngeal papillomas, common warts) and additionally hair follicles of immuno-competent individuals, a known reservoir of commensalic HPV genotypes. The testing showed the absence of HPV-125, HPV-150 and HPV-151 in all mucosal samples, showing lack of mucosal tropism of all three novel HPV types. HPV-125 was found only in benign skin lesions (2/102 of common warts), while HPV-150 and HPV-151 were found in 4/101 and 4/101 of common warts; in 3/52 and 1/52 of SCC; and in 1/49 and 3/49 of BCC, respectively. All three genotypes were present in hair follicles of immuno-competent individuals, and HPV-150 was shown to persist for at least 12 months in a single individual. In a single SCC sample from an immuno-compromised individual, partial L1 sequence of a putative novel genotype (SI-HPV-Beta5; GenBank Acc. No. FR822732), related to HPV-150, was obtained. The results obtained indicate HPV-125, HPV-150 and HPV-151 to be rare genotypes with a cutaneous tropism. HPV125 can be etiologically linked with sporadic cases of common warts, while HPV-150 and HPV-151 can be etiologically linked with sporadic cases of common warts and SCC and BCC of the skin in immuno-competent individuals.
100

HIGH BETAHPV LOADS ASSOCIATED WITH AGE, IMMUNOSUPPRESSION AND SKIN CANCER
S Weissenborn, University of Cologne, Cologne, GERMANY R Neale, Queensland Institute of Medical Research, Brisbane, Australia D Abeni, Istituto Dermopatico dell'Immacolata IDI-IRCCS, Rome, Italy JN Bouwes Bavinck, Dermatology, LUMC, Leiden, The Netherlands M C W Feltkamp, Medical Microbiology, LUMC, Leiden, The Netherlands AC Green, Queensland Institute of Medical Research, Brisbane, Australia M N C De Koning, Medical Microbiology, LUMC, Leiden, The Netherlands U Wieland, University of Cologne, Cologne, GERMANY H Pfister, University of Cologne, Cologne, GERMANY Background: There is increasing evidence for an association between beta human papillomaviruses (betaHPV) and the development of cutaneous squamous cell carcinoma (SCC). The viral DNA load may be an important determinant of pathogenicity, but there are currently no epidemiological data relating to load in people with or without SCC and/or immunosuppression. Objectives: Determination of DNA loads of eight betaHPV types previously associated with risk of SCC. Methods: We collected eyebrow hairs from immunocompetent people (ICP) and organ transplant recipients (OTR) with and without SCC, determined load by quantitative PCR, and obtained demographic, phenotypic and sun exposure information. Results: Viral loads in ICP and OTR with and without SCC spanned seven orders of magnitude. The median loads for all types were below one viral DNA copy per 60 cells, and were highest for HPV5, 8 and 20. None of the populations had consistently higher viral loads for all 8 types. However, among individuals without SCC a higher proportion of OTR were in the top deciles of viral load distributions for six of the eight betaHPV-types examined. These findings were significant in a nested analysis of Italian OTR and ICP for six types. Increasing age and the time elapsed since transplantation in the OTR was associated with higher viral loads. Between Australian cases and controls significant differences in top loads were observed for HPV types 5, 23, 36 and 38. Data from the OTR case-control study will be shown. Conclusion: We observed a wide spectrum of betaHPV loads with OTR and cases significantly more likely to have the highest viral loads. Thus viral loads may be an important contributor to the higher risk of SCC in OTR and the general population.
P-16.16
HPV-31: A DOMINANT GENOTYPE IN BLACK SOUTH AFRICAN OROPHARYNGEAL CARCINOMAS
C Paquette, University of Vermont/Fletcher Allen Health Care, Burlington, UNITED STATES M Evans, University of Vermont, Burlington, UNITED STATES S Meer, University of the Witwatersrand, Johannesburg, SOUTH AFRICA V Rajendran, University of Vermont, Burlington, UNITED STATES C Adamson, University of Vermont, Burlington, UNITED STATES K Cooper, University of Vermont/Fletcher Allen Health Care, Burlington, UNITED STATES Background: HPV infection, predominantly HPV-16, has been implicated as an important etiologic agent in a subset of oropharyngeal squamous cell carcinomas (OSCC) worldwide. However, data are scarce regarding OSCC in black South African patients; three prior studies suggested no significant role for HPV. Objectives: To assess OSCC from black South African patients for HPV and determine genotype. In parallel, to investigate p16INK4a and p53 markers in the context of three proposed pathways for OSCC: Class I (tobacco/ alcohol; p53 positive, p16 and HPV negative); Class II (tobacco/alcohol/HPV; HPV and p53 positive, p16 negative); and Class III (HPV; HPV and p16 positive, p53 variable). Methods: Formalin-fixed, paraffin-embedded tissues from 55 OSCC collected between 2005 and 2010 at the University of the Witwatersrand were analyzed for HPV DNA via PCR (positives genotyped by sequencing), chromogenic in situ hybridization (CISH), and immunohistochemistry for p16INK4a and p53. Twelve cases were excluded (pediatric, insufficient tissue, and duplicate tumors). Results: In the analysis of 43 OSCCs from 41 patients (mean age 58, 88% male), 35 (85%) samples were HPV positive by PCR: 24 (69%) HPV-31, 5 (14%) HPV-16, 4 (11%) HPV-18, and one each (6%) HPV-16/31 and HPV-18/31. CISH was negative in all cases. Three OSCCs (7%) met criteria for Class I, 11 (26%) for Class II, 20 (46%) for Class III, and nine (21%) were unclassifiable. Conclusion: In accordance with worldwide trends, HPV appears to play a role in the pathogenesis of OSCC in black South Africans. Contrary to data from research in Caucasians, HPV-31, and not HPV-16, was the predominant genotype identified. The absence of assays for HPV-31 in previous black South African studies may explain our present findings. Further studies, including HPV-31 viral load and alternative CISH methodologies, are being investigated to corroborate our data.
101

PERSISTENCE OF ANAL HPV IN HIV-INFECTED HOMOSEXUAL MEN (MSM)
I E Salit, University of Toronto, Toronto, CANADA S Blitz, University Health Network, Toronto, CANADA J Raboud, University of Toronto, Toronto, CANADA M Sano, University Health Network, Toronto, CANADA A Rebbapragada, Ontario Agency for Health Protection and Promotion, Toronto, CANADA J Tinmouth, Sunnybrook Health Sciences Centre, Toronto, CANADA Background: HPV persistence has been associated with neoplasia. Anal dysplasia, anal cancer and anal HPV occur at high rates in HIV+ MSM. It is uncertain if the high rates of anal HPV are due to persistence or frequent HPV infections. Objectives: To determine a) if anal infection with HPV remains persistent over years in HIV+ MSM and b) whether persistence of HR HPV/oncogenic types was associated with anal neoplasia. Methods: The subjects had high resolution anoscopy with directed biopsy and anal HPV genotyping by Linear Array. For 67 subjects, more than one anal canal sample (separated by at least 2 months) was available for HPV testing. We determined if there was HPV concordance in the multiple samples from each subject using the persistent presence of the following as the criteria: a) any HPV b) any oncogenic HPV and c) presence of certain HPV types (16, 18, 31 or 33). Results: The 67 subjects had 176 anal samples from 2003-2011; 53% of subjects had 2 samples and the remainder had 3-5 samples. The maximum sampling interval was 40 (15-47) months (median (IQR)). All subjects had at least one HPV type detected at one or more visits and 55/67 had repeated detection of any HPV. Oncogenic HPV was detected at one or more visits in 64/67 and 39/64 had repeated detection of at least 1 oncogenic HPV type. Persistent detection of certain high risk HPV types occurred as follows: HPV 16 (18/39), HPV 18 (7/15), HPV 31(9/21) and HPV 33(4/10). HPV 16 was present in 36 and persistent in 18 of the 55 subjects with high-grade dysplasia (AIN2+). All of those with persistent HPV16 had had AIN2+. Conclusion: In HIV+ MSM anal HPV infection may persist over years. Anal HPV 16 persistence may be associated with a risk of high-grade dysplasia.
P-16.18
HPV PREVALENCE IN DIFFERENT OROPHARYNX SUB-SITES AND CORRELATION TO PROGNOSIS
T Dalianis, Karolinska Institutet, Stockholm, SWEDEN A Nsman, Karolinska Institutet, Stockholm, SWEDEN L Marklund, Karolinska Institutet, Stockholm, SWEDEN L Hammarstedt, Karolinska Institutet, Stockholm, SWEDEN J Ferreira, Karolinska Institutet, Stockholm, SWEDEN T Ramqvist, Karolinska Institutet, Stockholm, SWEDEN E Munck - Wikland, Karolinska Institutet, Stockholm, SWEDEN T Dalianis, Karolinska Institutet, Stockholm, SWEDEN Background: Several studies report a high prevalence of human papilloma virus (HPV) in oropharyngeal squamous cell carcinoma (OSCC) and HPV is now regarded by the International Agency for Cancer Research (IARC) as a risk factor for OSCC. In addition, after similar treatment, patients with HPV-positive OSCC have a better 5-year survival compared to those with HPV-negative cancer. Less is known however to whether the prevalence of HPV varies according to OSCC sub-site and to whether its presence and influence as a positive predictive factor in response to treatment applies to all oropharynx sites. Objectives: In this study we examine the prevalence of HPV and the influence of HPV for clinical outcome per subsite within the oropharynx. Methods: In a retrospective design, 381 pre-treatment primary OSCC (tonsil n=228, base of tongue n=89 and other sites n=64) samples from patients diagnosed between 2000-2007 in the County of Stockholm were obtained. These samples are presently being analyzed for presence of HPV DNA by PCR and p16INK4a expression by immunohistochemistry and the findings will be correlated to patient clinical outcome after treatment. Results: HPV DNA was present in 259/381 (68%) of the oropharyngeal samples. The prevalence of HPV varied at different sites, with roughly 80%, 70% and around 20% respectively, of the samples from the tonsillar-, base of tongue-, and non-tonsil and non-base of tongue sites respectively, being HPV-positive. Evaluation of p16INK4a expression and correlation to prognosis per sub-site is still in progress. HPV is a positive predictive factor for clinical outcome in tonsillar and base of cancer, and is being evaluated for clinical outcome in non-tonsillar and base of tongue cancer. Conclusion: The prevalence of HPV varies in OSCC depending on site and OSCC should be separated into sub-sites when reporting HPV prevalence and the influence of HPV on clinical outcome.
102

IMMUNE AND GENETIC ANALYSES IN AIN, THE HIV POSITIVE COHORT.
P Fox, Chelsea & Westminster Hospital, London, UNITED KINGDOM P Goon, Cambridge University, Cambridge, UK P Holmes, Chelsea & Westminster Hospital, London, UK S Mandalia, Chelsea & Westminster Hospital, London, UK Background: A three year observational study of AIN diagnostic parameters. Objectives: better insight into the natural history of AIN. Method: Random selection; annual anal cytology and virology. Results: Provisional analysis on 106 patients, 90% MSM. Cytology:48% normal 33% mild dyskaryosis, 15% moderatesevere dsykaryosis. More smokers had HPV 6/11 in the anal canal ( 46% v 31% non smokers). No relationship between smoking status and HPV 16/18. 56% non smokers had negative cytology versus 34% smokers. 41% reporting recent anal sexual activity had HPV 16/18, versus 27% with no such activity for years. Patients with CD4 count >500 were less likely to have HPV 16 or 18. 37 patients had HPV 16 detected, half with normal cytology. Most of the 11 patient with HPV 18 had normal cytology. 44 patients lacked any of the major high risk HPV types of whom less than half had normal cytology. 8 patients with only HPV 6 or 11 had cytological abnormality. Conclusions: There is debate about the optimum follow up of patients with abnormal anal cytology, and whether HPV typing can be utilised to reduce the need for high resolution anoscopy ( HRA). While there is no question that individuals with moderate to severe dyskaryosis require HRA, it would be helpful to find ways of reducing the need for HRA in the large proportion of HIV positive patients with minor cytological abnormality. The selective HPV sampling of patients with cytological abnormality might represent a less expensive and less invasive way of providing reassurance. Whether quantitative HPV 16/18 analysis would be helpful as part of that secondary screening will be investigated in the ongoing follow up of this cohort.
P-16.20
IMPLICATION OF HPV IN INVASIVE ANAL CARCINOMA
S VALMARY - DEGANO, Besanon University Hospital - IFR133/EA3181 : carcinogense pithliale. UFR SMP. 25000, Besanon, FRANCE E JACQUIN, Besanon University Hospital - IFR133/EA3181 : carcinogense pithliale. UFR SMP. 25000, Besanon, FRANCE F MONNIEN, Besanon University Hospital - IFR133/EA3181 : carcinogense pithliale. UFR SMP. 25000, Besanon, FRANCE R HAMLAOUI, Besanon University Hospital - IFR133/EA3181 : carcinogense pithliale. UFR SMP. 25000, Besanon, FRANCE B KANTELIP, Besanon University Hospital - IFR133/EA3181 : carcinogense pithliale. UFR SMP. 25000, Besanon, FRANCE J F BOSSET, Besanon University Hospital - IFR133/EA3181 : carcinogense pithliale. UFR SMP. 25000, Besanon, FRANCE J L PRETET, Besanon University Hospital - IFR133/EA3181 : carcinogense pithliale. UFR SMP. 25000, Besanon, FRANCE C MOUGIN, Besanon University Hospital - IFR133/EA3181 : carcinogense pithliale. UFR SMP. 25000, Besanon, FRANCE Background: A French molecular epidemiological study (EDiTH V) showed HPV16 as the most frequent HPV type in anal cancers with a rate of 75%. Objectives: We attempt to describe more precisely HPV implication on 87 invasive anal cancers from patients treated by radiochemotherapy at the Hospital of Besanon between 1997 and 2007. Methods: Tumoral tissues taken before treatment were formalin-fixed and paraffin-embedded. Histological types were mostly squamous cell carcinoma. Genotyping was performed with the INNO-LiPA assay. HPV16 load and HPV16 DNA physical state were measured by real-time PCR. Expression of p16, p53, p21 and Ki-67 was evaluated by immunohistochemistry (IHC). Results: Now, 69 samples were tested by INNO-LiPA and 100% were HPV positive. HR-HPV were present in 98% of cases: the major type was HPV16 (93%) and only one sample harboured HPV18. One case was HPV6. Real time PCR performed on all samples showed a HPV16 prevalence of 90% (78/87 cases). Among 55 cases with HPV16 DNA copy number above the limit of quantification (LOQ), viral loads ranged from 2 copies to 15x103 copies/cell. Three samples presented only integrated HPV16 genomes, 12 displayed mostly episomes (E2/E6 ratio > 0,8), and 40 a mix of integrated and episomal HPV16 genomes. As for p16 IHC, 10/87 cases were negative: 8 were HPV16- or HPV16+ with a load below the LOQ and 2 were HPV16+. As for p53 IHC, 15/87 cases showed an overexpression: 12 were HPV16- or HPV16+ with a load below the LOQ and 3 were HPV16+. Other results are under analysis. Conclusion: We confirm the high prevalence of HPV16 and show a high viral load in anal carcinoma. This is consistent with the etiologic role of HPV16 in these tumours. Next, we plan to correlate these data with OS and PFS of patients to highlight theranostic factors in anal cancer.
103

HUMAN PAPILLOMA VIRUS DETECTION IN BARRETT'S OESOPHAGUS
S Rajendra, Bankstown-Lidcombe Hospital, Sydney, AUSTRALIA E T Snow, School of Human Life Sciences, University of Tasmania, Launceston, AUSTRALIA A Hodges, School of Human Life Sciences, University of Tasmania, Launceston, AUSTRALIA M Ball, School of Human Life Sciences, University of Tasmania, Launceston, AUSTRALIA I K Robertson, School of Human Life Sciences, University of Tasmania, Launceston, AUSTRALIA S Bart, School of Human Life Sciences, University of Tasmania, Launceston, AUSTRALIA BACKGROUND: Human papilloma virus (HPV) has been detected in some patients with squamous cell carcinoma of the oesophagus. We have previously hypothesized that HPV might also be found in patients with Barretts oesophagus and or oesophageal adenocarcinoma. OBJECTIVES: To determine whether HPV prevalence rates are more common in patients with Barretts oesophagus as compared to controls. METHODS: Tissue samples were obtained during elective gastroscopy from consecutive patients aged between 18 and 80 years. Cases were defined as those with Barretts oesophagus (metaplasia, n=62 or dysplasia, n=12), gastro-oesophageal reflux disease (GERD) (n=46), and patient controls (n=37). Exclusion criteria were: prior HPV vaccination, known HPV associated disease, concomitant peptic ulcer disease, secondary causes of GERD and any contraindication to performance of endoscopic biopsies. The biopsies were obtained from the squamo-columnar junction (SCJ) in all patients and from within Barretts lesions or, for control samples, from 2 cm above the SCJ. Tissue was tested for the presence of HPV DNA by polymerase-chain reaction (PCR), using MY09/MY11 and GP5+/ GP6+ primers. HeLa, SiHa and HaCaT cells were used as HPV16 positive controls, HPV18 positive controls and HPV negative controls, respectively. Beta-actin was the internal PCR control. RESULTS: HPV prevalence rate in the study patients was low (14 of 157). Significantly more short segment Barretts oesophagus (SSBE) patients had HPV at the SC junction (6/36) compared to long segment Barretts oesophagus (LSBE) patients (0/26) [p=0.035]. The HPV detection rate at the SCJ (13 of 157) was significantly higher than within the BE lesion or comparable oesophageal position(1 of 157, p=0.002). CONCLUSION: Although overall prevalence of HPV is low, the results indicate a significant difference in HPV prevalence rates between SSBE and LSBE patients at the SCJ. The SCJ appears the primary site for HPV detection.
P-16.22
EYEBROW HAIRS REPRESENT THE STATUS OF CUTANEOUS HPV INFECTIONS
I Schneider, Charit, Berlin, GERMANY M Lehmann, Charit, Berlin, GERMANY M Kizzie, Charit, Berlin, GERMANY E Stockfleth, Charit, Berlin, GERMANY I Nindl, Charit, Berlin, GERMANY Cutaneous human papillomavirus (HPV) infections seem to be associated with the onset of actinic keratosis (AK) and cutaneous squamous cell carcinoma (SCC). This study compares the prevalence of betaPV in eyebrow hairs to that in tissues of normal skin and AK lesions. Biopsies from AK lesions, normal skin, and plucked eyebrow hairs were collected from 75 patients diagnosed with AK. DNA from these specimens was tested for the presence of 25 beta and 3 gamma HPV types by a PCR based BGC-RLB method. The highest numbers of HPV infections were detected in eyebrow hairs (84%) compared to AK (47%) and normal skin (37%). The total number of HPV types was 228 in eyebrow hairs versus 92 in AK and 69 in normal skin. The highest prevalence was found with HPV20 and HPV37 independent of the specimens. In 35 patients HPV infections in eyebrow hairs as well as in AK were observed, which corresponds to 56% (35/63) overlapping infections. The number of overlapping infections in eyebrow hairs compared to normal skin was 44%. Thus, eyebrow hairs represent the betaPV infections and are a useful marker for the cutaneous HPV infection status in epidemiological studies.
104

RISK STRATIFICATION BASED ON P16-EXPRESSION AND OTHER FACTORS IN OPSCC
M A Broglie, Kantonsspital St. Gallen , St. Gallen, SWITZERLAND A Soltermann, University Hospital of Zurich, Zurich, SWITZERLAND M Pawlita, German Cancer Research Center, Heidelberg, GERMANY R Probst, University Hospital of Zurich, Zurich, SWITZERLAND S J Stoeckli, Kantonsspital St. Gallen , St. Gallen, SWITZERLAND Background: HR-HPV infection and its surrogate marker p16 are established independent risk factors for oropharyngeal squamous cell carcinoma (OPSCC) associated with better survival. Efforts are made towards treatment de-escalation to avoid late toxicities impairing patients outcome. Factors for reliable risk stratification are needed to guide treatment decisions. Objective: Risk stratification based on multivariate analysis identifying the factors most influential for survival Methods: Retrospective analysis in a single institutional, consecutive OPSCC patient cohort treated by primary intensity modulated chemoradiation (XRT) (n=140) or surgery with adjuvant XRT (n=60) between 2002 and 2007 at the Zurich University Hospital. Tissue micro array construction for p16 immunohistochemistry (2 cores/tumor) was possible in 181/200 (91%) patients. Results: Inclusion of 200 patients (79% male), median age 61 years (range 42-91yrs). Tumors of 93/181 (51%) patients were p16 positive. Comparable tumor stages within the p16 positive and -negative subgroup (84% UICC stage IV). Patients with p16-positive OSCC were significantly less often drinkers (p=0.0001) or smokers (p=0.0001). Overall (OS) (p=0.0001), disease-specific (DSS) (p=0.0001) and recurrence free survival (RFS) (p=0.002) was significantly higher in p16-positive tumors compared to p16-negative. Analyzing the impact of risk factors on survival, cox proportional hazard regression revealed significantly higher adjusted Hazard Ratios (HR) in OS, DSS and RFS for advanced N-stage (>N2a), alcohol and smoking and lower HR for p16-positivity. We modified the algorithm described by Ang et al. and defined three risk groups based on p16 overexpression, smoking (>10py) and/or drinking as well as N-stage. Survival differences within the three groups were highly significant even when stratified by therapeutic modality. Conclusion: p16 protein expression in OSCC is a significant and in clinical routine useful prognosticator for survival independent of treatment modality. Further factors like smoking, alcohol and N-stage have to be considered for patients risk stratification.
P-16.24
HIGH-THROUGHPUT SEQUENCING REVEALS DIVERSITY OF HUMAN PAPILLOMAVIRUSES IN CUTANEOUS LESIONS
J Ekstrm, Dept. of Laboratory Medicine, Division of Medical Microbiology, Lund University, Sknes Universitetssjukhus, Malm, SWEDEN D Bzhalava, Dept. of Laboratory Medicine, Karolinska Institute and Karolinska Hospital, Stockholm, SWEDEN D Svenback, Dept. of Experimental Medical Sciences, Lund University, Lund, SWEDEN O Forslund, Dept. of Laboratory Medicine, Division of Medical Microbiology, Lund University, Sknes Universitetssjukhus, Malm, SWEDEN J Dillner, Dept. of Laboratory Medicine, Division of Medical Microbiology, Lund University, Sknes Universitetssjukhus and Depts. of Laboratory Medicine, Karolinska Institute and Karolinska Hospital, Malm and Stockholm, SWEDEN Background: There are at least 120 completely characterized human papillomavirus (HPV) types and putative new types are continuously found. Both squamous cell carcinoma of the skin (SCC) and other skin lesions commonly contain multiple cutaneous HPV types. Objectives: The objective of this study was to achieve an improved resolution of the diversity of HPV types in lesions such as SCCs, actinic keratoses (AKs) and keratoacanthomas (KAs). Methods: Fresh frozen biopsies from 37 SCC lesions, 36 AK lesions and 92 KA lesions and swab samples from the top of the lesion from 86 SCCs and 92 AKs were amplified using the general HPV primers FAP and mixed to three pools followed by high throughput sequencing. Results: We obtained 2196 reads with homology to HPV. In the pool of SCC/AK biopsies 48 different HPV types were found. Eighty-three types were found in the pool of SCC/AK swab samples and 64 types in the KA biopsies, respectively. For 9 novel putative HPV types most of the amplimer sequence was obtained, whereas for an additional 35 novel putative HPV types only partial amplimer sequences were obtained. Most of the novel putative types belonged to the genus Gamma. Conclusion: In conclusion, high throughput sequencing was an effective means to identify both known and previously unknown HPV types in putatively HPV-associated lesions and has revealed an extended diversity of HPV types.
105

HPV DISTRIBUTION IN WARTS IS DIFFERENT IN HIV INFECTED PATIENTS
M de Koning, DDL Diagnostic Laboratory, Voorburg, NETHERLANDS, R Meys, Departments of Dermatology, Genitourinary Medicine and Immunology, Faculty of Medicine, Imperial College, London, United Kingdom, K Purdie, Centre for Cutaneous Research, Blizard Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom, M Nelson, Departments of Dermatology, Genitourinary Medicine and Immunology, Faculty of Medicine, Imperial College, London, United Kingdom, D Asboe, Departments of Dermatology, Genitourinary Medicine and Immunology, Faculty of Medicine, Imperial College, London, United Kingdom, D Hawkins, Departments of Dermatology, Genitourinary Medicine and Immunology, Faculty of Medicine, Imperial College, London, United Kingdom, F Gotch, Departments of Dermatology, Genitourinary Medicine and Immunology, Faculty of Medicine, Imperial College, London, United Kingdom, C Harwood, Centre for Cutaneous Research, Blizard Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom, C Bunker, Departments of Dermatology, Genitourinary Medicine and Immunology, Faculty of Medicine, Imperial College, London, United Kingdom, W Quint, DDL Diagnostic Laboratory, Voorburg, Netherlands Background and objectives: Little is known about the HPV type distribution in warts from HIV infected patients. The goal of the study was to compare the HPV prevalence in cutaneous and external genital warts in HIV infected patients (64 and 21 warts, respectively) with healthy individuals (45 and 28 warts, respectively). Methods: We have profiled them using newly developed Luminex technology (HSL-PCR/MPG) for cutaneous wart associated HPV types and using the SPF10-LiPA25, version 1 for the presence of genital HPV types. Warts were fresh frozen and clinically and/or histologically confirmed. Results: The prevalence of multiple infections was slightly higher in cutaneous and genital warts from HIV patients than in warts from healthy individuals but this was not significant (19% vs 7% in cutaneous warts and 14% vs 7% in genital warts, p > 0.05). In cutaneous warts from HIV patients HPV7 appeared to be more prevalent (22% vs 0%, p =0.002) while HPV2 was more prevalent in cutaneous warts from healthy individuals (29% vs 3%, p = 0.004). HPV6 appeared to be significantly more prevalent in genital warts from healthy individuals compared to the HIV patients (82% vs 33%, p = 0.002). Conclusions: The HPV genotype profile in both cutaneous and genital warts is different in HIV infected and healthy persons. However, contrary to what was expected multiple infections were not detected significantly more often in the HIV group.
P-16.26
HRHPV BUT NO BETAPV TYPES DETECTED IN PENIS IN-SITU CANCERS
M de Koning, DDL Diagnostic Laboratory, Voorburg, NETHERLANDS, R Meys, Dermatology and Histopathology, Faculty of Medicine, Imperial College, London, United Kingdom, S Krishna, Dermatology and Histopathology, Faculty of Medicine, Imperial College, London, United Kingdom, K Purdie, Centre for Cutaneous Research, Blizard Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom, N Francis, Dermatology and Histopathology, Faculty of Medicine, Imperial College, London, United Kingdom, T Ryder, Dermatology and Histopathology, Faculty of Medicine, Imperial College, London, United Kingdom, M Walker, Dermatology and Histopathology, Faculty of Medicine, Imperial College, London, United Kingdom, C Harwood, Centre for Cutaneous Research, Blizard Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom, C Bunker, Dermatology and Histopathology, Faculty of Medicine, Imperial College, London, United Kingdom, W Quint, DDL Diagnostic Laboratory, Voorburg, Netherlands Background and objectives: High risk (HR) genital human papillomavirus (HPV) infection is strongly associated with cervical and anal cancers but the association with penile cancers is dependent on histological type. Low risk and HR genital HPV DNA has been found in 70 to 90% of penis in-situ cancer samples. Betapapillomaviruses (betaPV), constituting a group of HPV that are associated with non-melanoma skin cancer have also been detected in penile lesions (36 to 100%). The purpose of this study was to re-evaluate the spectrum of HPV types present in penis insitu cancer. Methods: We investigated 21 penis biopsies from 21 patients with invasive cancer (n=5) or in-situ cancer (n=16). Host and viral DNA was extracted from formalin-fixed paraffin embedded archival tissue using laser capture microscopy to select cancerous/pre-cancerous zones. DNA derived from lesions was analysed with the SPF10LiPA25, version 1 for detection and genotyping of genital HPV types and with the PM-PCR RHA method for the detection and genotyping of betaPV types Results: HPV was present in 13/16 (81%) of the in-situ cancer samples. HPV16 was detected in 12/16 (75%) of them and HPV68 in 1/16 (6%). Three samples were negative. HPV18 was found in one of the five (20%) penis cancer samples. No betaPV types were present in these samples. Conclusions: The genital HPV infections that were detected in these lesions corroborates previous work. However the high prevalence of betaPV types could not be confirmed. This may be accounted for by our use of old archival tissue, the exclusion of surface contamination with microdissection and/or stringent laboratory standards.
106

LESIONAL HPV TYPES OF CUTANEOUS WARTS ARE IDENTIFIED BY SWABS
M de Koning, DDL Diagnostic Laboratory, Voorburg, NETHERLANDS L Khoe, Department of Dermatology, Leiden University Medical Centre, Leiden, Netherlands J Eekhof, Department of Public Health and Primary Care, Leiden University Medical Centre, Leiden, Netherlands M Kamp, DDL Diagnostic Laboratory, Voorburg, Netherlands J Gussekloo, Department of Public Health and Primary Care, Leiden University Medical Centre, Leiden, Netherlands J ter Schegget, DDL Diagnostic Laboratory, Voorburg, Netherlands J Bouwes Bavinck, Department of Dermatology, Leiden University Medical Centre, Leiden, Netherlands W Quint, DDL Diagnostic Laboratory, Voorburg, Netherlands Background: Large numbers of HPV types infect the human skin and members from the HPV genera alpha, gamma and mu are associated with cutaneous warts. Objectives: The aim of this study was to test if the HPV genotypes in swabs of the overlying skin are identical to the types present within these warts. Methods: To this purpose, 25 persons being treated for persistent cutaneous warts were enrolled. Swabs of the overlying skin of the wart were collected from each participant. Additionally, scabs of the wart and deeper portions of the warts were surgically removed. HPV genotyping was performed on all samples using the novel HSL-PCR/ MPG assay and the HPV genotyping results were compared. Results: From the 25 wart biopsies one was HPV negative. 15 were positive for HPV27, 3 for HPV57, 2 for HPV2, 2 for HPV1, 1 for HPV3 and 1wart biopsy was positive for both HPV41 and HPV65. Scabs and swabs of the warts both showed identical typing results as the biopsies in 24 of the 25 cases (sensitivity: 96%). Conclusions: There was an excellent agreement between HPV types in the swabs of the skin that overlies the warts and the biopsies of these warts validating the use of wart swabs for future studies of wart-associated HPV types. HPV27 was highly prevalent (70%) in the in adults of the investigated population of patients with persistent cutaneous warts.
P-16.28
SKIN WART HPV TYPES REMAIN DETECTABLE AFTER WARTS HAVE RESOLVED
M de Koning, DDL Diagnostic Laboratory, Voorburg, NETHERLANDS S Bruggink, Department of Public Health and Primary Care, Leiden University Medical Center, Leiden, Netherlands J Gussekloo, Department of Public Health and Primary Care, Leiden University Medical Center, Leiden, Netherlands P Egberts, Department of Public Health and Primary Care, Leiden University Medical Center, Leiden, Netherlands J ter Schegget, DDL Diagnostic Laboratory, Voorburg, Netherlands M Feltkamp, Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, Netherlands J Bouwes Bavinck, Department of Dermatology, Leiden University Medical Center, Leiden, Netherlands W Assendelft, Department of Public Health and Primary Care, Leiden University Medical Center, Leiden, Netherlands J Eekhof, Department of Public Health and Primary Care, Leiden University Medical Center, Leiden, Netherlands W Quint, DDL Diagnostic Laboratory, Voorburg, Netherlands Background and objectives: Little is known about the persistence of infections by HPV genotypes associated with cutaneous warts. The newly developed HSL-PCR/MPG assay for genotyping of all known wart-associated HPV types from the alpha- (HPV2, 3, 7, 10, 27, 28, 29, 40, 43, 57, 77, 91 and 94), gamma- (4, 65, 95, 48, 50, 60 and 88), mu- (HPV1 and 63) and nu-genus (HPV41) was used to determine HPV persistence in a cohort study. Methods: The study comprised 246 children and adults attending their family physician for treatment of one or more warts. Treatment consisted either of freezing by liquid nitrogen, application of salicylic acid or an expectant policy. At the start of the study swabs were taken from wart(s) and the forehead of the participants. The exact positions of the warts were annotated for each participant. The skin areas where the warts were located were tested again after 26 weeks. In some cases the warts were still present and in other cases the lesions had resolved. Analyses are presented for one wart of each participant. Results: In case of persisting warts 61% (71/117) of the type specific HPV infections persisted. Partial remission of warts at the second time point might explain that only 61% of HPV types persisted. However, even in case the wart had completely resolved, still 39 % (45/114) of the HPV types persisted. Interestingly, the prevalence of HPV27 was significantly higher in the patients with persisting warts as opposed to the patients with resolved warts. In contrast, the prevalence of HPV4 and HPV63 was significantly lower. Conclusions: Cutaneous wart associated HPV types remain detectable in a considerable percentage of the patients also when the original lesions have resolved.
107

THE RELATIONSHIP BETWEEN ANAL WARTS AND ANAL CANCER
M Nathan, Homerton University Hospital, London, UNITED KINGDOM K Klein, Homerton University Hospital, London, UNITED KINGDOM N Hickey, Homerton University Hospital, London, UNITED KINGDOM A Umaipalan, Homerton University Hospital, London, UNITED KINGDOM D Awosika, Homerton University Hospital, London, UNITED KINGDOM N Singh, Barts and the London NHS Trust, London, UNITED KINGDOM M Sheaff, Barts and the London NHS Trust, London, UNITED KINGDOM Background: Epidemiological data suggests anal warts (low grade disease) as a risk factor for anal cancer. In contrast, cervical or vulval warts are not recognised risk factors for cervical cancer. Some studies have shown coexistence of high-grade anal neoplasia (AIN 2/3)with anal warts. Objective: To characterise better the relationship between anal warts and anal cancer. Methods: 1) Retrospective analysis of those presenting with high-grade anal neoplasia (AIN 2/3) regarding their prior history of anal warts. 2) Prospective observation of those presenting with anal warts (low-grade neoplasia) for the development of AIN 2/3 after treatment of their low-grade disease. Results: 1) One hundred and fifty two patients presented with AIN 2/3 that was proven through histology, during their initial visit. Eighty one (53.3%) of these patients gave a history of anal warts (70) or external warts only (11), ranging from less than one year to 24 years (median 2 years). 2) Two hundred and nine patients presented with anal warts (low-grade disease). On prospective follow-up, 40 patients (19.1%) developed biopsy proven high-grade disease (AIN2/3), at a median 19 months (mean 26.2 months; range 4 - 85), after their initial presentation. Ten other cases showed high-grade anal cytology changes (range 12 72 months). In summary, 50 of 209 patients (23.9%) developed incident AIN 2/3. Conclusion: It appears that those who develop anal warts have an inherent risk of developing AIN 2/3 and therefore anal cancer. This may reflect their defect in mucosal immunity in dealing with HPV and/or other acquired immune suppressive causes. Further work is needed to elucidate the risk of anal cancer in those presenting with anal warts.
P-16.30
HPV AND LUNG CANCER: THE VIRUSES IN PULMONARY CARCINOMA STUDY
M Schabath, H. Lee Moffitt Cancer Center, Tampa, UNITED STATES D Cress, H. Lee Moffitt Cancer Center, Tampa, UNITED STATES E Haura, H. Lee Moffitt Cancer Center, Tampa, UNITED STATES K Jonathan, H. Lee Moffitt Cancer Center, Tampa, UNITED STATES D Smith, H. Lee Moffitt Cancer Center, Tampa, UNITED STATES G Bepler, The Barbara Ann Karmanos Cancer Institute, Detroit, UNITED STATES D Rollison, H. Lee Moffitt Cancer Center, Tampa, UNITED STATES A Giuliano, H. Lee Moffitt Cancer Center, Tampa, UNITED STATES Background: HPV is hypothesized to play a role in lung cancer. Globally, association studies have reported a wide variation in the prevalence (0% to 100%) of HPV in lung tumors. A recent systematic review reported that ~25% of lung cancers had detectable HPV DNA; HPV-16/18 were the most common types detected. Only two studies have been conducted in the US, both with relatively small number of cases and utilized less sensitive methods of HPV detection. Objectives: Assess the prevalence and HPV type distribution in fresh frozen lung tumor tissue and matched non-involved tissue. Methods: DNA isolated from tumor (n=106) and adjacent non-involved (n=11) tissues were assayed using broad-spectrum HPV typing (SPF10 LiPA assay). A subset of tumor specimens was sequenced for mutations in KRAS, EGFR, and p53. Results: Among the tumors, prevalence of any HPV was 43%; the prevalence of oncogenic and non-oncogenic HPV was 23% and 21%, respectively. The prevalence of HPV-6+ and HPV-16+ was 26% and 20% for multiple infections, respectively, and 18% and 6% for single infections, respectively. The prevalence of any HPV was 52% among ever smokers versus 35% among never smokers (p=0.08). The prevalence of any HPV was 19% among EGFR mutant adenocarcinoma versus 51% without the mutation (p=0.03). There was no difference in HPV status by age, gender, histology, and p53 and KRAS mutations. HPV-16+ was present in only 20% of non-involved tissue among the subset of HPV-16+ tumors that had adjacent non-involved tissue. Conclusions: These preliminary data provide compelling evidence that HPV is involved in lung cancer. Also, the molecular profile of HPV-related lung cancers may be distinct from non-HPV lung cancer. Validation and further analyses is currently underway, including additional samples and LCM of tumor blocks.
108

RARE HPV GENOTYPES IN VULVAR, VAGINAL, PENILE AND ANAL CARCINOMAS
D Geraets, DDL Diagnostic Laboratory, Voorburg, NETHERLANDS W Quint, DDL Diagnostic Laboratory, Voorburg, NETHERLANDS N Guimer, IDIBELL, Institut Catal dOncologia - Catalan Institute of Oncology, Barcelona, SPAIN M de Koning, DDL Diagnostic Laboratory, Voorburg, NETHERLANDS L J van Doorn, DDL Diagnostic Laboratory, Voorburg, NETHERLANDS S de Sanjose, IDIBELL, Institut Catal dOncologia - Catalan Institute of Oncology, Barcelona, SPAIN L Alemany, IDIBELL, Institut Catal dOncologia - Catalan Institute of Oncology, Barcelona, SPAIN X Bosch, IDIBELL, Institut Catal dOncologia - Catalan Institute of Oncology, Barcelona, SPAIN Background: High-risk HPV types are considered the causative agents of virtually all cases of cervical carcinoma. Their role in the development of other carcinoma, e.g. vulvar, vaginal, anal and penile (VVAP), remains to be determined. Archival specimens of VVAP cases have been globally collected to study presence of HPV genotypes by the SPF10 LiPA algorithm. This algorithm comprises SPF10 PCR-based DEIA (broad-spectrum detection for over HPV 54 types), and HPV genotyping by LiPA25 (version 1). The positivity rate and prevalence of HPV by this algorithm is described in more detail by others. Objectives: In this study, the aim was to characterize HPV in a small fraction of VVAP carcinoma cases (n=77) that were SPF10 DEIA-positive but negative by LiPA25. Methods: Sequence analysis was carried out on the SPF10 amplimers of HPV-positive cases that could not be typed by LiPA25. Results: HPV sequences were obtained in 52 of the 77 VVAP cases, of which the majority comprised rare HPV types. Several of these types (i.e., HPV26, 30, 67, 69, 82) have already been classified as possibly carcinogenic (belonging to the high-risk clade of species -5, 6, 7, 9, and 11). Genotypes from other species were found in the remaining cases as single infections (i.e., HPV6, 27, 32, 44, 61, 76, 83, 87, 89, 102, and 114). Conclusions: Rare and possibly carcinogenic HPV types, already characterized in cervical carcinomas in a related study, were also present as single infections in vulvar, vaginal, anal and penile carcinomas. Their causative role will be further investigated by HPV analysis on laser-capture microdissected lesional tissue.
Declaration of interest SdS, Qiagen & Sanofi & GSK, occasional travel support NG, GSK & MSD, occasional travel support LA, MSD, occasional travel support XB, MSD, Sanofi & GSK, consultancy and occasional travel support
P-16.32
HPV 38 IS TRANSCRIPTIONALLY ACTIVE IN A RARE CUTANEOUS DISEASE
F Paolini, Regina Elena Nacional Cancer Istitute, Rome, ITALY P Donati, San Gallicano Dermatologic Institute I.R.C.C.S, Rome, ITALY S Trincone, San Gallicano Dermatologic Institute I.R.C.C.S, Rome, ITALY C Cota, San Gallicano Dermatologic Institute I.R.C.C.S, Rome, ITALY A Venuti, Regina Elena Nacional Cancer Istitute, Rome, ITALY Background. Kerinokeratosis Papulosa is a sporadic or familiar keratotic appearing at birth or during the first year of life. An unusual case of kerinokeratosis in an adult man is reported in which the histological analysis led to suspect the presence of HPV infections as for other keratotic disorders. Mutations in the EVER1 and or EVER2 gene have been detected in multiple HPV skin infections and are linked to epidermodysplasia verruciformis (EV), a genodermatosis with multiple papules and macules in sun-exposed areas. Objectives. In this unusual pathology the presence of HPV and mutations in EVER1 and EVER2 have been analysed to define a possible link to this disease. Methods DNA and RNA were extracted from paraffin embedded sections of bioptical samples. Extracted DNA was utilized for the EVER analysis and for the HPV presence with primers amplifying a large number of HPV types. All the amplified products were subject to direct sequencing. RT-PCR was performed to ascertain the presence of viral transcripts. Results. HPV38 was found in one sample and other HPVs in another sample. Multiple infections are a characteristic of EV but no mutation in these two genes was found except a single nucleotide change, causing the N306I aminoacid substitution. Thus genetic analysis demonstrated a homozygous single nucleotide polymorphism in EVER2 gene that is linked to high susceptibility to HPV infection and squamous cell transformation. Data from RT and nested real time PCR revealed the presence of HPV 38 E7 transcripts indicating that only this type of HPV is transcriptionally active in the lesion and it may play a pathogenetic role. Conclusions. On the basis of this molecular diagnosis this patient could represent an unusual case of kerinokeratosis associated to the presence of HPV38 and of EVER2 gene polymorphism, further strengthening the beta papillomavirus link to host genetic background.
109

BIOLOGICAL ROLE OF HPV IN LARYNX CARCINOMA
G Halec, German Cancer Research Center (DKFZ), Heidelberg, GERMANY M Schmitt, German Cancer Research Centre (DKFZ), Heidelberg, GERMANY G Dyckhoff, Heidelberg University Hospital, Heidelberg, GERMANY F X Bosch, Heidelberg University Hospital, Heidelberg, GERMANY M Pawlita, German Cancer Research Center (DKFZ), Heidelberg, GERMANY Background: In head and neck carcinogenesis, causal relationship has been demonstrated between mucosal HRHPV and a subset of oropharyngeal squamous cell carcinoma (OP-SCC). For laryngeal (L-)SCC, HPV prevalences up to 25% were reported, but molecular evidence of HPV activity is largely lacking. We previously reported (HPV workshop Montreal) for a series of FFPE tumor tissues, a DNA prevalence of 34.6% but found active HPV involvement based on DNA, RNA and immunohistochemistry analysis of only 4.0%. Objective: To analyze HPV DNA and RNA in corresponding fresh frozen biopsies of 105 L-SCC patients. Methods: Fresh frozen biopsies (n=61), were genotyped for 51 mucosal HPV types. HPV DNA-positive tumors were analyzed for HPV E6*I transcripts by newly developed RTPCR assays extended to all 20 HR/pHR-HPV types. Results: Fourteen (23%) fresh frozen biopsies contained HPV sequences, of which 13 were HPV-16 single and one was HPV-16, -18 double infection. Three of those tumours contained HPV-16 and one HPV-18 E6*I, accounting for 7% RNA positivity. Combining FFPE and fresh frozen biopsy results, 94/105 (90%) patients had analyzable DNA (61/61, 100% in fresh frozen and 81/105, 77% in FFPE material). HPV positivity was 35.1% (33/94). Of 33 DNA positive cases, 29 had sufficient material for RNA and IHC analysis. Active HPV contribution including RNA positivity with down-regulated pRb and up-regulated p16INK4a as IHC surrogate markers of HPV involvement was found in 4/90 cases (4.4%). Three of those cases contained HPV-16 and one pHR-HPV-70. Conclusions: This study provides evidence for the etiological role of HPV-16 but also pHR-HPV-70 in a very small subset of German L-SCC. Better analyzable fresh frozen material confirmed previously observed, very low active contribution of HPV in laryngeal carcinogenesis. The presence of HPV DNA alone in 25/90 (27.7%) is not compatible with active viral contribution to the transformed phenotype in these tumors.
P-16.34
PREVALENCE OF BETAPAPILLOMAVIRUS IN EARLY ONSET SQUAMOUS CELL SKIN CANCER.
G St George, Westmead Institute for Cancer Research,University Of Sydney, Westmead, AUSTRALIA M N C de Koning, DDL Diagnostic Laboratory, Voorburg, The Netherlands R A Dalziell, Westmead Institute for Cancer Research,University Of Sydney, Westmead, AUSTRALIA W G V Quint, DDL Diagnostic Laboratory, Voorburg, The Netherlands G J Mann, Westmead Institute for Cancer Research,University Of Sydney, Westmead, AUSTRALIA M Feltkamp, Leiden University Medical Center, Leiden, The Netherlands Background and objectives: Cutaneous squamous cell carcinoma (cSCC) are frequently diagnosed and account for most of the 410 deaths that occur due to non-melanoma skin cancer in Australia each year. Evidence is mounting that HPV also contributes to cSCC. The objective of this study was to test this hypothesis further by determining the betaPV distribution in Australian cSCC cases diagnosed at a relatively early age, using both related and population controls. Methods: Cases (n=111), recruited from sequential records of a dermatopathology referral service in Sydney, had been diagnosed with cSCC of any stage <50 yr (<10% of cases in Australia are diagnosed <50yr). The cases nominated both sibling (n=53) and unrelated partner (n=66) controls. Eyebrow hair bulbs were collected for detection and genotyping of betaPV using the PM-PCR reverse hybridization assay (RHA) method. Results: Results from half the sample show an overall carriage frequency of 81% in cases, 65% in sibling controls and 77% in unrelated controls (p=0.018). HPV types 5, 15, 23, 24, 38 were the most commonly recurring, each present in >25% of subjects. Four or more types were present in 42% of cases, 10% of siblings and 34% of unrelated controls (p=0.336). Putatively high cancer risk types (5, 8, 15, 20, 24, 36, 38) were present in 67% of cases, 48% of sibling controls and 66% of unrelated controls; 23% of the cases and unrelated controls, but none of the siblings, carried three or more types from the high-risk group. The remaining samples are being analysed and will be presented at the conference. Conclusion: The data suggest that control siblings were less likely than case siblings to exhibit carriage of (multiple) betaPV types. However, there is no evidence at this stage for association of betaPV with early-onset cSCC, especially when cases were compared with unrelated controls.
110

PENISCOPIC EVALUATION OF MALE PARTNERS OF WOMEN WITH GENITAL WARTS
C LIVERANI, UNIVERSITY OF MILAN, MILANO, ITALY E MONTI, UNIVERSITY OF MILAN, MILANO, ITALY D PUGLIA, UNIVERSITY OF MILAN, MILANO, ITALY S MANGANO, UNIVERSITY OF MILAN, MILANO, ITALY Objectives: To evaluate the prevalence of penile lesions in male sexual partners of women diagnosed and treated for anogenital warts. Methods: Male partners of 219 women treated for anogenital warts were assessed by clinical examination and peniscopy before and after application of acetic acid (5%). HPV testing was not performed. Conclusions: After careful peniscopic examination 81 patients resulted free of disease. In 129 patients condylomata acuminata of the penis, pubis and/or the perineal area were detected. Treatment consisted of laser CO2 vaporization and/or application of imiquimod cream. Three patients had a white epithelium of the glans and the biopsy revealed a penile intraepithelial neoplasia grade 1 (PIN 1). Six patients were diagnosed with molluscum contagiosum, a member of the Poxvirus family not necessarily related to sexual contact. In 27 patients pearly penile papules were described. Peniscopy is helpful in revealing tiny lesions which may escape to the naked eye. Diagnosing and treating external genital warts in the whole anogenital tract in both men and women is essential in reducing the relapse of the disease. References (1) CDC Atlanta - MMWR 2010;59(No RR-12):69-78. (2) de Carvalho JJ, de Carvalho JZ, Rosa NT et al. Identification of males at increased risk for genital human papillomavirus (HPV) infection among patients referred for urological consultation. Scand J Infect Dis 2007;39(1112):1029-37. (3) Hippelinen MI, Hippelinen M, Saarikoski S, Syrjnen K. Clinical course and prognostic factors of human papillomavirus infections in men. Sex Transm Dis 1994;21(5):272-9. (4) Spuhler S, Pasche N, Sauthier P et al. Value of penis endoscopy in sexual partners of women with condylomatous or dysplastic lesions. J Gynecol Obstet Biol Reprod 1994;23(6):660-4. (5) Wolf R, Davidovici B. Treatment of genital warts: facts and controversies. Clin Dermatol 2010;28:546-8.
Declaration of interest NONE DECLARED
P-16.36
ORAL HPV PREVALENCE AMONG HIGH RISK YOUNG ADULTS
G DSouza, Johns Hopkins, Baltimore, UNITED STATES E Stammer, Johns Hopkins, Baltimore, UNITED STATES R Youngfellow, Baltimore County Health Department, Baltimore, UNITED STATES M Gillison, Ohio State University, Columbus, UNITED STATES Background: Oral HPV infection and risk factors have not been well evaluated among young adults. Objective: To evaluate risk factors for prevalent oral HPV infection among high-risk young adults. Methods: So far we have enrolled 156 men and 121 women ages 18-25, who had not received the HPV vaccine, from Baltimore County STD clinics. Each participant completed a risk factor survey including questions about lifetime and recent behavior. Exfoliated oral cells were collected using a 30-second Scope mouthwash rinse and gargle and tested for 37 types of HPV DNA using PGMY09/11 consensus primers and reverse line blot hybridization. Results: Prevalent oral HPV infection was common (13.8%), including infection with any oncogenic HPV type (10.7%) and HPV16 infection (1.7%). The most common oral HPV infections detected were HPV51, 59 and 84 (each >2% prevalence). Oral HPV prevalence was associated with the number of recent oral sex (p-trend=0.05) and vaginal sex (p-trend=0.005) partners, but not with the number of deep-kissing partners. Oral HPV prevalence was 9%, 13% and 30% in those with 0, 1 and 2 recent oral sex partners (p-trend=0.01), but was only 6% among 18 subjects with four or more recent oral sex partners. In multivariate analysis factors associated with increase oral HPV prevalence included number of sexual partners in the past three months (p-trend=0.04), having a primary sexual partner (OR=3.0 95%CI=1.2-7.5), ever having used marijuana (OR=4.6, 95%CI= 1.0-21) and never mouthwash use compared to daily use (OR=4.1 95%CI=0.95-18). Gender, tobacco, alcohol, cocaine use and number of deep kissing partners were not associated with HPV prevalence. Among a subset of participants with follow-up oral rinses, prevalent infections were repeatedly detected in 40% of three month and 33% of six month samples. Discussion: Oral HPV infection is common among young sexually active adults and was associated with recent sexual behavior
Declaration of interest This study was supported by funding from Merck Inc. to G. DSouza. G DSouza and M Gillison have received research funding from and consulted for Merck.
111

HPV TYPES IN HIV-INFECTED MSM: RELEVANCE FOR QUADRIVALENT VACCINE USE
J Tinmouth, Sunnybrook Health Sciences Centre, Toronto, CANADA S Blitz, University Health Network, Toronto, Canada J Raboud, University Health Network, Toronto, Canada M Sano, Toronto General Hospital, Toronto, Canada E Collins, Toronto General Hospital, Toronto, Canada A Rebbapragada, Ontario Agency for Health Protection and Promotion, Toronto, Canada I Salit, Toronto General Hospital, Toronto, Canada Background: Human papillomavirus (HPV) is found in most anal, penile and some oral cancers. HIV-infected men who have sex with men (MSM) are at increased risk for these cancers. Although developed to prevent cervical cancer, the quadrivalent HPV vaccines may be beneficial in other HPV-related cancers. Objectives: In a cohort of HIV-infected MSM in an anal cancer screening program, to determine: a) prevalence of HPV at multiple body sites and b) type-specific concordance of HPV across these sites. Methods: Using linear array, we tested for HPV genotypes in the anal canal, perianal region, penis/scrotum (PS) and mouth. Prevalence of HPV by type was determined for each body site. Type-specific concordance was calculated for site by site comparisons. Prevalence of concurrent infection with vaccine strains (6, 11, 16 and 18) within subjects was determined. Results: 94 HIV-infected MSM were studied. The prevalence of any HPV and high risk HPV, respectively, were: anal (85%, 63%), perianal (81%, 57%), PS (42%, 19%) and oral (23%, 5%). Type specific concordance was highest for the anal/perianal comparison (median kappa (IQR): 0.58 (0.44, 0.72)). Prevalence of the 4 vaccine strains at any site ranged from 10-31%. None of the subjects were concurrently infected with all 4 vaccine strains and 43% of subjects were infected with none of the strains. 36%, 17% and 4% of subjects had 1, 2 and 3 vaccine strains present, respectively. Conclusions: Within the same subjects, anal infection with HPV is common while oral infection is not. Type-specific concordance across body sites is minimal except for anal/perianal. Among HIV-infected MSM with a high burden of HPV, none were infected with all 4 vaccine strains, suggesting that the quadrivalent vaccine may be useful in this high risk population.
P-16.38
PREVALENCE OF HIGH-GRADE ANAL NEOPLASIA AFTER REFERRAL FROM ANAL SURGEONS
J Berry, University of California San Francisco, San Francisco, UNITED STATES D Neumark, University of California San Francisco, San Francisco, UNITED STATES N Jay, University of California San Francisco, San Francisco, UNITED STATES M Rubin, University of California San Francisco, San Francisco, UNITED STATES I Park, University of California San Francisco, San Francisco, UNITED STATES J Palefsky, University of California San Francisco, San Francisco, UNITED STATES Background: High-resolution anoscopy (HRA) consists of inspection of the anal mucosa under magnification after applying acetic acid to identify otherwise invisible high-grade anal neoplasia (HGAIN) and occasionally, anal carcinoma (SCCA). Routine use of HRA by anal surgeons is limited and not standard of care. Objectives: We compared the extent and presence of HGAIN/SCCA determined by HRA by experienced HRA providers with findings on standard anoscopy (no acetic acid or magnification) performed by experienced anal surgeons. Methods: Convenience series of 35 patients referred for HRA for management of AIN within 7 months of standard anoscopy. Results: 9 HIV-seropositive men-who-have-sex-with-men (MSM) and 26 HIV-seronegative patients (14 women, 3 heterosexual men, and 9 MSM). The median age was 53 years (range: 34-68). Biopsy-proven HGAIN was found in 27 of 35 patients, SCCA in 5, and no lesions in 3. Referral notes indicated that extent of HGAIN was underestimated, often found incidentally while treating hemorrhoids, tags, and condyloma. Of 6 patients referred with warts, all had HGAIN. Of 12 referred for management of documented HGAIN, SCCA was found in 3 and moderate or extensive HGAIN in 9. Of 8 patients referred post-excision of SCCA, 3 had no lesions, 2 had residual SCCA on HRA, 1 had limited HGAIN within 6 months of excision and 1 each had moderate or extensive concurrent HGAIN. Nine were referred for management of persistent HGAIN after attempted treatment; all had moderate or extensive lesions. Two of these 9 patients had normal surgical biopsies prior to referral. Conclusions: A high prevalence of HGAIN/SCCA was found on HRA following standard anoscopy by anal surgeons. Undiagnosed SCCA, normal biopsies in face of extensive HGAIN, and persistent lesions after non-HRA-guided treatment suggest that standard anoscopy is suboptimal for diagnosis of HGAIN/SCCA. HRA should be considered an integral part of management of HGAIN/SCCA.
112

ANXA1 GENE EXPRESSION IN PENILE CARCINOMA INFECTED WITH HPV
M Calmon, So Paulo State University, So Jos do Rio Preto, BRAZIL E Babeto, So Paulo State University, So Jos do Rio Preto, BRAZIL M Mota, So Paulo State University, So Jos do Rio Preto, BRAZIL N Candido, So Paulo State University, So Jos do Rio Preto, BRAZIL A Girol, So Paulo State University, So Jos do Rio Preto, BRAZIL S Oliani, So Paulo State University, So Jos do Rio Preto, BRAZIL J Bonilha, So Paulo State University, So Jos do Rio Preto, BRAZIL J vassallo, Hospital A. C. camargo, So paulo, Brazil P Rahal, So Paulo State University, So Jos do Rio Preto, BRAZIL Penile cancer (PC) is an invasive epithelium tumor, representing more than 10% of the malignancies in men in some developing countries, mainly in Brazil. Studies on the aetiopathogenesis of this cancer have focused on its association with the HPV. The aim of this study is evaluating the possible role of HPV in the development of PC and the use of the RaSH technique to analyze gene expression in normal tissues and penile tumors. HPV detection was done by PCR with generic primers GP5+/GP6+ and HPV typing was done by direct sequencing. RaSH methodology identified differentially expressed genes, generating subtractive cDNA libraries. The presence of HPV was analyzed in 58 samples of patients with PC. It has been observed high prevalence (85.12%) of HPV. Of the 16 samples sequenced, HPV-16 was found in 14 (87.5%) samples, HPV-11 in 1 (6.3%) and HPV-35 in 1 (6.3%). The genes PBEF1, ANX1, RPL6 and KIAA1033 were overexpressed in the tumors. On the other hand, the gene p16 was overexpressed in the normal tissue. Finally, the expression of the selected genes was confirmed by qPCR. Only ANX1 was validated with overexpression in 80% of all samples. It was also analyzed ANX1 protein expression by immunohistochemistry in 25 samples of penile cancer. It was observed overexpression of ANX1 protein 100% of PC samples compared to normal tissue. The results obtained are capable of revealing differences in patterns of gene expression between normal and tumor tissues. Moreover, it was observed high prevalence of HPV, suggesting an important role of this virus in penile carcinogenesis. Besides, overexpression of ANXA1 was observed in most of the penile cancer samples and such information will contribute to develop a possible marker for penis tumor diagnostic and prognostic, improving the development of directed therapies against this new putative marker.
P-16.40
ETIOLOGICAL ROLE OF HUMAN PAPILLOMAVIRUS IN BLADDER CARCINOMA AND PAPILLOMA
T Sasagawa, Kanazawa Medical University, Kahoku-gun, JAPAN, K Shigehara, Kanazawa University Graduate School of Medical Science, Kanazawa, JAPAN, S Kawaguchi, Senboku Fujii Hospital, Osaka, JAPAN, K Nakashima, Kanazawa University Graduate School of Medical Science, Kanazawa, JAPAN, M Shimamura, Ishikawa Prefectural Central Hospital, Kanazawa, JAPAN, T Nakashima, Ishikawa Prefectural Central Hospital, Kanazawa, JAPAN, Y Kobori, Dokkyo Medical School, Koshigaya Hospital, Koshigaya, JAPAN, E Koh, Kanazawa University Graduate School of Medical Science, Kanazawa, JAPAN, M Namiki, Kanazawa University Graduate School of Medical Science, Kanazawa, JAPAN Background: Information on the relationship between human papillomavirus (HPV) infection and the development of bladder tumor. Objectives: We elucidated an etiological role of HPV infection in bladder tumor. Methods: One hundred thirty patients with bladder tumor (117 carcinoma and 8 bladder papillomas) treated between 1997 and 2009 were enrolled in this study. The presence of HPV-DNA was tested on frozen carcinoma tissues or paraffin-embedded tissues obtained by transurethral resection using a PCR-based method. Localization of HPV was observed on archival tissue specimens by in situ hybridization (ISH) for high-risk HPV-DNA. P16-INK4a and HPV-L1 protein expression were evaluated by immunohistochemistry (IHC). Results: HPV-DNA was detected in 18 (15%) of 117 bladder carcinoma cases. All were single high-risk HPV type infections, and HPV16 was identified in 6 cases, HPV18 in 4 cases, and HPV33 in 3 cases. HPV prevalence of the bladder carcinomas was 38% (12/28) of grade 1, 8.5% (6/71) of grade 2, and none (0/18) of grade 3 bladder carcinomas, suggesting that grade 1 tumor was associated with HPV infection. On the other hand, HPV-DNA was detected in 7 (87.5%) of 8 bladder papilloma samples, and multiple types infection was shown in 2 cases. ISH analysis showed that high-risk HPV-DNA was localized in the nuclei of tumor cells of all HPV-positive cases including carcinoma or papilloma. P16-INK4a, which are surrogate markers for HPV-E7 expression, were expressed in 94% of HPV-positive carcinoma cases, and its expression level was higher than that of HPV-negative ones. P16-INK4a was also detected in 100% of HPV-positive papilloma cases. On the other hands, HPV-L1 protein expression suggesting reproductive HPV infection was not observed in any carcinoma, but was observed in the well-differentiated parts of the bladder papilloma tissue. Conclusions: High-risk HPV is likely to be a causative agent of some low-grade bladder carcinomas and bladder papilloma.
113

DO HPV-POSITIVE OPSCC PATIENTS HARBOUR HPV IN OTHER PHARYNGEAL SITES?
A Stokes, King's College London Dental Institute, London, UNITED KINGDOM K Masuno, King's College London Dental Institute, London, UNITED KINGDOM R Henley - Smith, King's College London Dental Institute, London, UNITED KINGDOM S Thavaraj, King's College London Dental Institute, London, UNITED KINGDOM Background: It is now recognised that the increase in incidence of oropharyngeal squamous cell carcinoma (OPSCC) is attributed to oncogenic HPV. Although there are reports of multi-centric HPV-associated OPSCC, the relative risk of developing a second HPV-associated head and neck tumour is still unknown. There are no established genotypic or phenotypic criteria of HPV-associated premalignancy and it is not known whether the rest of the pharyngeal mucosa is at risk of developing a second primary HPV-associated malignancy. Determining whether other pharyngeal sites contain viral DNA in patients presenting with primary HPV-associated OPSCC may provide preliminary information as to whether tumours arise in wide fields of HPV-infected mucosa. Objectives: To determine whether patients with HPV-associated OPSCC harbour HPV DNA in other pharyngeal sites in addition to their primary tumour. Methods: Fourteen patients with primary OPSCC had pan-endoscopy and multiple biopsies at the time of initial diagnosis. Histological review was carried out on a total of forty-five biopsy specimens (fourteen carcinoma specimens and thirty-one synchronously biopsied non-tumour pharyngeal specimens). DNA was extracted from these specimens and was followed by PCR using consensus HPV primers (GP5+/GP6+) and -actin primers. HPV positive and negative cell line DNA served as positive and negative PCR controls respectively. In addition, all histologically normal pharyngeal biopsies were also subject to p16 immunohistochemistry. Results: All synchronously biopsied pharyngeal specimens showed no histological evidence of dysplasia. HPV DNA was not detected in any of biopsies. All biopsies were negative for p16 immunohistochemistry. Conclusions: These preliminary findings suggest that OPSCCs do not arise in wide areas of HPV infected mucosa. Further work is necessary to increase patient numbers to further confirm these findings and to investigate whether these tumours arise in areas of genetically altered mucosa.
P-16.42
HPV IN HEAD AND NECK CANCER: A BRAZILIAN CASE-CONTROL STUDY
M Oliveira - Silva, Oswaldo Cruz Institute, Rio de Janeiro, BRAZIL V Perrusi, National Institute of Cancer, Rio de Janeiro, BRAZIL R A Arcuri, National Institute of Cancer, Rio de Janeiro, BRAZIL R Hassan, National Institute of Cancer, Rio de Janeiro, BRAZIL L P Mota, National Institute of Cancer, Rio de Janeiro, BRAZIL M C M Santos, National Institute of Cancer, Rio de Janeiro, BRAZIL FL Dias, National Institute of Cancer, Rio de Janeiro, BRAZIL S E Vianna, National Institute of Cancer, Rio de Janeiro, BRAZIL C R Bonvicino, National Institute of Cancer, Rio de Janeiro, BRAZIL Background: Recent studies suggest an association between Human Papillomavirus (HPV) infection and several head and neck cancers, especially oropharyngeal and oral cavity invasive carcinomas. Objectives: The aim of this study is to assess HPV prevalence and HPV type in a case-control study in Brazil to evaluate associations between HPV infection and head and neck cancer. Methods: We performed a hospital-based study including 81 patients diagnosed with hypopharynx, oropharyngeal or oral cavity cancer and 188 individuals without history of cancer. The HPV molecular testing was carried out by nested-PCR using PGMY and GP05/06+ primers and the HPV typing using cloning followed by sequencing. Results: The overall HPV prevalence of 81 cases was 13.6%, among which 14.3% were detected in oral cavity, 20% in oropharyngeal and 11.1% in hypopharyngeal carcinomas. A similar general HPV prevalence (19.1%) was observed in 188 control individuals that showed higher prevalence than previously reported in Latin America and Central Europe. Partial results of control group showed a diversity of types as HPV6, HPV16, HPV18 and HPV70, similar to a recent Brazilian study of cervical samples in women with and without lesions. Conclusions: Our data indicated a high prevalence and diversity of HPV types in control group which will be further investigate in this study with multivariate regression models for casecontrol comparisons also analyzing sociodemographic data and specific sexual behaviors.
114

P16-EXPRESSION AS A PROGNOSTIC MARKER FOR HEAD AND NECK CANCER
C Salazar, Columbia University, Manhattan, USA, R Smith, Montefiore Medical Center, Bronx, USA, M Brandwein Gensler, University of Alabama at Birmingham, Birmingham, USA, M Haigentz, Montefiore Medical Center, Bronx, USA, M Garg, Montefiore Medical Center, Bronx, USA, C Keller, Montefiore Medical Center, Bronx, USA, B Schiff, Montefiore Medical Center, Bronx, USA, R Burk, Albert Einstein College of Medicine, Bronx, USA, M Prystowsky, Albert Einstein College of Medicine, Bronx, USA, N Schlecht, Albert Einstein College of Medicine, Bronx, USA Objective: P16-INK4 immuno-histochemistry (IHC) is increasingly being used by surgical pathologists to characterize head and neck squamous cell carcinoma (HNSCC). Recent evidence suggests that expression of p16 expression is correlated with human papillomavirus (HPV) infection in HNSCC, and may help predict cancer progression. To evaluate the prognostic role of p16 expression in HNSCC, we assessed the survival in HNSCC patients presenting with primary disease by p16 IHC and HPV PCR analysis. Methods: We prospectively collected 108 primary tumor specimens from histologically confirmed HNSCC patients prior to treatment at Montefiore Medical Center in the Bronx (NY). Formalin fixed tumor specimens were tested for p16-INK4 expression by IHC on tissue microarrays. HPV-DNA and RNA was detected by MY09/11-PCR and E6/E7 RT-PCR on matched frozen tissue from the same tumor in a research laboratory. Cox regression analyses were used to build multivariable survival models with covariate adjustment using an inverse probability weighting approach. Results: Using pre-defined criteria, positive p16-expression was detected in 17 (15.7%) HNSCC overall. Comparing by tumor subsite, 31.0% of oropharyngeal, 16.1% of laryngeal, and 7.7% of lip/oral cavity tumors were p16-positive (p=0.04). After adjustment for clinical and pathological indicators and confounding, overall survival was better among p16-positive cases as compared to p16-negative cases for oropharyngeal SCC (albeit not significantly; HR=0.28, 95%CI:0.05-1.45), whereas this was not observed for non-oropharyngeal sites (HR=0.83, 95%CI:0.22-3.11). Subgroup analyses showed moderate overlap between p16 and HPV16-RNA detection (kappa=0.47, 95%CI:0.270.67) and similar agreement with HPV16-DNA detection. HNSCC cases with p16+/RNA+ tumors exhibited a significantly better survival than those with p16-/RNA- tumors (adjusted HR=0.22, 95%CI:0.06-0.78), independent of subsite. Conclusions: P16-expression shows potential as a biomarker for predicting HNSCC survival, particularly for oropharyngeal SCC or HPV-associated HNSCC.However, further research is needed to investigate the clinical utility of p16 and HPV-RNA testing for clinical management of HNSCC.
Declaration of interest N. Schlecht, NIH grant CA115243, Principal Investigator; C. Salazar, NIH T32 training grant DE007255, Recipient
P-16.44
AN EVALUATION OF AN OROPHARYNGEAL PAP-TEST EQUIVALENT IN HIGH-RISK POPULATIONS
C Fakhry, Johns Hopkins, baltimore, UNITED STATES M Gillison, Ohio State University, columbus, United States D Clark, Johns HOpkins, Baltimore, United STates B Rosenthal, JOhns HOpkins, Baltimore, United States Background: Human papillomavirus (HPV) is responsible for the rising incidence of oropharyngeal squamous cell cancers (OSCC) in the United States (U.S.) and yet no screening strategies have been considered. Secondary prevention by means of HPV detection and cervical cytology have led to a decline of cervical cancer in the U.S. Objectives: Here, we explored an analogous strategy by evaluating associations between HPV16 infection, cytopathology and histopathology in two populations at elevated risk for OSCC. Methods: In the first, a crosssectional study population (PAP1), cytology specimens were collected by means of brush biopsy from patients presenting with oropharyngeal abnormalities. The second (PAP2), a nested case-control study, bilateral tonsillar cytology samples were collected at 12-month intervals from HIV-infected individuals. HPV16 was detected in samples by consensus primer PCR and/or type-specific PCR. The presence of cytopathology in HPV16-positive tonsil biopsies (cases) was compared to HPV16-negative tonsil biopsies (controls). Results: In PAP1, the presence of HPV16 was associated with abnormal cytology (OR 3.6, 95%CI 1.08-11.9) and OSCC (OR 6.1, 95%CI 1.6-22.7). In PAP2, among HIV-infected individuals without clinical abnormalities, the prevalence of tonsillar HPV16 was 4.5%. No dysplasia or malignancy was found. ASCUS was the only cytologic abnormality observed and there was no association with tonsillar HPV16 infection. Conclusion:vHPV16 is associated with cytopathology and OSCC among individuals with oropharyngeal lesions accessible to biopsy, but not among HPV16-positive individuals without clinically identifiable lesions. An oropharyngeal pap equivalent may not be feasible, likely due to a limitation of sampling the relevant epithelium in tonsillar crypts.
115
Session 17: Cervical screening

Chair: Jack Cuzick
Poster abstracts
Cervical screening | 17 O-17.00

Jack Cuzick
FOR YOUR NOTES:
O-17.01
HPV TESTING PROTECTS AGAINST CIN3+ IN SUBSEQUENT SCREENING ROUND
C Meijer, VU-University Medical Center, Amsterdam, NETHERLANDS D Rijkaart, VU-University Medical Center, Amsterdam, Netherlands J Berkhof, VU-University Med, Amsterdam, Netherlands F van Kemenade, VU-University Med, Amsterdam, Netherlands V Coupe, VU-University Med, Amsterdam, Netherlands L Rozendaal, VU-University Medical Center, Amsterdam, Netherlands G Kenter, VU-University Medical Center, Amsterdam, Netherlands N Bulkmans, VU-University Medical Center, Amsterdam, Netherlands D Heideman, VU-University Medical Center, Amsterdam, Netherlands P Snijders, VU-University Medical Center, Amsterdam, NETHERLANDS Introduction: HPV testing is now on the brink for implementation in cervical cancer screening. We report the efficacy of HPV DNA testing in cervical screening over two rounds (interval 5 years) in the POBASCAM trial. Methods: Between January 1999 and September 2002, women aged 30-60 years who participated in the regular cervical screening programme in The Netherlands were randomly assigned to control (conventional cytology) or intervention (HPV DNA/cytology co-testing) group. During the subsequent screening round after 5 years, HPV DNA/cytology co-testing was performed on both groups. Primary outcome measure was detection of CIN3+ during the baseline and subsequent round. Analysis was done by intention to treat. Findings: 40,105 women met eligibility criteria for our analyses at entry (19,999 in the intervention group and 20,106 in the control group). Over two screening rounds, detection rates of CIN2+ were similar in the intervention and control groups., indicating that HPV DNA screening does not lead to overdiagnosis of regressive lesions. This is evident for both women between 29-34 years and women of 34-59 years. However, when comparing the intervention group to the control group more CIN2+ lesions were observed at baseline (relative risk [RR] 1.25 95%CI 1.05-1.50; p=0.015), and fewer cervical cancer cases and CIN3+ at the subsequent round (RR 0.29, 95%CI 0.10-0.87; p=0.031 and RR 0.73, 95%CI 0.55-0.96; p=0.023, respectively). Conclusion: Replacement of cytology by HPV DNA testing in cervical screening results in earlier detection of relevant CIN2+ and consequently better protection against CIN3+ and cervical cancer.
Declaration of interest C.Meijer Qiagen, scientific advisory board. C.Meijer GSK, speakers bureau. P.Snijders Roche speakers buro. P. Snijders Gen-Probe speakers buro. D.Heideman: Roche Speakers buro
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17 | Cervical screening O-17.02

FIVE-YEAR CERVICAL CANCER RISK FOLLOWING HPV TRIAGE OF EQUIVOCAL CYTOLOGY
H Katki, US National Cancer Institute, Rockville, UNITED STATES, W Kinney, Kaiser Permanente Northern California, Berkeley, UNITED STATES, M Schiffman, US National Cancer Institute, Rockville, UNITED STATES, B Fetterman, Kaiser Permanente Northern California, Berkeley, UNITED STATES, T Lorey, Kaiser Permanente Northern California, Berkeley, UNITED STATES, N Poitras, Kaiser Permanente Northern California, Berkeley, UNITED STATES, L Cheung, Information Management Services, Inc., Silver Spring, UNITED STATES, F Demuth, Information Management Services, Inc., Silver Spring, UNITED STATES, S Wacholder, US National Cancer Institute, Rockville, UNITED STATES, P Castle, American Society for Clinical Pathology, Washington, DC, UNITED STATES Background: Clinical trials show that cervical intraepithelial neoplasia grade 3 or more severe (CIN3+) risk in women with ASC-US (Atypical Squamous Cells of Undetermined Significance) cytology who are HPV-positive have a risk that resembles the risk in women with LSIL (Low-grade Squamous Intraepithelial Lesion) cytology. Furthermore, CIN3+ risk in women with ASC-US cytology who are HPV-negative is closer to risk in women with normal cytology. However, data are lacking on actual cancer risks in routine clinical practice. Objectives/Methods: We estimated the cumulative 5-year risks of CIN3+ and cancer for HPV-positive and HPV negative women with ASC-US cytology for 331,818 women aged 30 and older who enrolled in co-testing at Kaiser Permanente Northern California in 2003-2005 and were followed through 2009. Results: 76% of the 8,517 enrollment ASC-US Pap tests were HPV-negative. Among HPV-negative women with ASC-US cytology, 5-year cumulative risks of CIN3+ and cancer were 0.54% (95%CI 0.19% to 1.6%) and 0.031% (95%CI 0.008% to 0.12%). By comparison, the 5-year cumulative CIN3+ and cancer risks in 319,177 women with normal cytology were 0.36% (95%CI 0.24% to 0.55%) and 0.037% (95%CI 0.016% to 0.087%). In contrast, 5-year cumulative risks were much higher in HPV-positive women with ASC-US cytology: 8.5% (95%CI 5.5% to 13%) for CIN3+ and 0.15% (95%CI 0.05% to 0.5%) for cancer. By comparison, the 2517 women with LSIL cytology had 5-year cumulative CIN3+ risk of 4.8% (95%CI 2.9% to 8.1%) and cancer risk of 0.30% (95%CI 0.04% to 2.0%). Conclusions: In routine clinical practice, women with HPV-negative ASC-US cytology and women with normal cytology had very similar CIN3+ and cancer risks. Similarly, women with HPV-positive ASC-US cytology and women with LSIL cytology had similar CIN3+ and cancer risks. Women who have similar risks should be managed similarly.
Declaration of interest Dr. Schiffman reports working with Qiagen, Inc. on independent evaluations of non-commercial uses of CareHPV (a low-cost HPV test for low-resource regions) for which he has received research reagents and technical aid from Qiagen for free. No other authors report any conflicts of interest.
O-17.03
P16/KI-67 DUAL-STAINING USING THINPREP CYTOLOGY-SUB-ANALYSES OF >9,000 PALMS PARTICIPANTS
C Bergeron, Laboratoire Cerba , Cergy Pontoise, FRANCE, F Alameda, Hospital del Mar, Barcelona, SPAIN, H Griesser, Center for Pathology and Cytopathology, Cologne, GERMANY, M Labadie, Laboratoire GRC, Limonest, FRANCE, V Maccalini, Unita Gestionale screening regionale ospedale, Atri, ITALY, M Sideri, European Institute of Oncology Unit of Preventive Gynaecology, Milan, ITALY, R Dachez, Institute Alfred Fournier, Paris, FRANCE, R Ridder, mtm Laboratories, Heidelberg, GERMANY Background: The PALMS trial (Primary ASC-US LSIL Marker Study) evaluated the diagnostic performance of the p16/Ki-67 Dual-staining in cervical cancer screening as well as in the triage of ASC-US or LSIL results. The outcomes were compared to Pap cytology and HPV testing in the screening setting, and to HPV testing in the triage of ASC-US or LSIL. For both Pap cytology and Dual-staining, liquid-based and conventional cytology methods were included in the PALMS trial. Objectives and Methods: We performed an analysis of the diagnostic performance of Dual-staining, Pap cytology and HPV testing (hc2, Qiagen) limited to the sub-cohort of 9,231 women enrolled to the PALMS trial who had ThinPrep (Hologic) liquid-based cytology (LBC) for both Pap and Dual-staining. Results: Test positivity rates for Dual-stained cytology, Pap, and HPV testing over all ages were 5.7%, 5.6%, and 11.2%, respectively. Sensitivity of Dual-stained cytology for CIN2+ (n=67 cases) was found at 95.6% [95%CI 87.1-98.6%], significantly higher than Pap cytology (80.2% [95%CI 68.5-88.3%]; p=0.0082). Specificity levels were identical for both methods (95.0% vs. 95.1%). In women aged 30, sensitivity (specificity) of Dual-stained cytology for CIN2+ was 91.2% (96.0%), compared to 77.9% (95.9%) for Pap testing and 96.1% (92.1%) for HPV testing. For the triage of abnormal Pap cytology results, dual-stained cytology showed identical (ASC-US triage: 100% for both tests) or similar (LSIL triage: 94.5 vs. 100%) sensitivity as HPV testing for the detection of underlying CIN2+, at significantly higher specificity rates. Conclusions: p16/Ki-67 Dual-stained cytology performed on ThinPrep LBC may achieve a sensitivity level as high as 95% for underlying CIN2+ in primary screening of women of all ages, combined with a 95% specificity. Furthermore, dual-stained cytology was shown to be a highly efficient tool for triaging ASC-US or LSIL when performed as a reflex test on ThinPrep LBC specimens.
Declaration of interest Laboratory services related to this clinical study were reimbursed to the authors institution as one of the study centers of the PALMS trial.
120

DURATION OF PROTECTION OF CERVICAL SCREENING FOR WOMEN AGED 65+
A Castanon, Queen Mary University of London, London, UNITED KINGDOM D Mesher, Queen Mary University of London, London, UNITED kINGDOM P Sasieni, Queen Mary University of London, London, UNITED kINGDOM Background: There is a lack of consensus regarding the appropriate upper age for cervical screening in previously well screened women and little direct evidence on which to base policy. In England screening is offer to age 65. Objectives: to establish the duration of protection offered by screening at ages 50-64 to women over age 65. Methods: We used data from the UK NHSSCP Audit of Cervical Screening which includes screening histories (since 1988) from 1463 cases over age 65.5 at diagnosis and under age 65 when the programme started in 1988 together with their 2882 matched (on age and area of residence) controls. Conditional logistic regression was performed to look at the effect of time since last adequate smear (under age 65) on the risk of subsequent cervical cancer. Results: The risk of developing cervical cancer at ages 65.5-79.9 is reduced by 76% (OR 0.24, 95% CI 0.19-0.31) within 8 years of an adequate screening test between the ages of 55-64. This risk reduction remains significant, but at a lower magnitude 8- 15 years after an adequate test (OR 0.42, 95% CI 0.34-0.51).Women with cervical cancer are more likely than cases (47% vs. 30%) not to have been screened before age 65 and less likely to exit the screening programme with a negative smear aged 60-64 (36% vs. 60%). Women exiting with a abnormal smear are 5.0 (95% CI 3.6-6.8) times more likely to develop cancer aged 65+ than those exiting with a normal smear. Conclusions: Offering screening up to age 64 halves the risk of developing cervical cancer between ages 65 and 80. Unresolved abnormal smears in older women carry a substantially increased risk of future cancer.
O-17.05
INTEROBSERVER VARIABILTY AND PREDICTIVE VALUE OF VIA BY AGE GROUP
O Ajenifuja, Obafemi Awolowo Universit Teaching Hospitaly, Ile-Ife, NIGERIA J Gage, NCI/NIH, Bestheda, USA A Adepiti, ANWLGA, Oke-Agbe, NIGERIA R Burk, Albert Einstein University, New York, USA N Wentzzensen, NCI, Bestheda, USA C Eklund, NCI, Bestheda, USA M Schiffman, NCI, Bestheda, USA BACKGROUND: Visual Inspection with Acetic Acid (VIA) was introduced for cervical cancer screening in developing countries due to inability of these countries to implement high quality cytologic services. OBJECTIVE: In this project we looked at the inter observer variability between seven health workers trained to perform VIA using IARC educational materials. METHODS: In a population-based project, we recruited women who had never had cervical cancer screening in a rural village (Irun) in South Western Nigeria. Each woman had cervical screening done by VIA. Liquid-based cytology was performed in the US; the residual fluid was tested for oncogenic HPV-DNA by PCR (Burk lab). Based on cytology and HPV results, women were classified as either 1) high risk (high grade cytology and oncogenic HPV positive), 2) increased risk (abnormal cytology or HPV+), or 3) low risk (normal cytology and HPV-). VIA results were classified as suspect cancer, positive, negative or squamo-columnar junction (SCJ) not visible. Results were stratified by health worker and participant age. RESULTS: Complete results were available for 1163 women. 18 women with a visible SCJ were considered high risk, of whom only 6 had a positive or suspect-cancer VIA result (33.3%). With the exception of SCJ visibility, VIA positivity (7.0% overall) and suspect cancer results (6.9% overall) were each similar across age groups (Chi-square p=.48 and .39, respectively). Positive predictive value was highest in women age 30-49 (7.3% with VIA+/suspect cancer were high risk). Across age groups providers had wide ranges of VIA results: 0-21% suspect cancer and 0.025% VIA+. Providers did not visualize the SCJ in 3-78% of women age 50+. CONCLUSIONS: In our project, VIA was not reproducible, nor was it sensitive compared to cytology and HPV testing. Histology results are pending.
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17 | Cervical screening O-17.06

IMPLEMENTATION OF SELF-SAMPLING WITH PAP SMEAR REFERRAL AMONG HIGH-RISK WOMEN
J S Smith, UNC, Chapel Hill, UNITED STATES, N T Brewer, UNC, Chapel Hill, UNITED STATES, A Rinas, UNC, Chapel Hill, UNITED STATES, M Kamradt, UNC, Chapel Hill, UNITED STATES, K Ricchetti, UNC, Chapel Hill, UNITED STATES, K Murphy, UNC, Chapel Hill, UNITED STATES, A Richman, East Carolina University, Greensville, UNITED STATES, B Li, Cleveland Clinic, Cleveland, UNITED STATES, L Barclay, ASHA, Research Triangle Park, UNITED STATES, J Belinson, Cleveland Clinic, Cleveland, UNITED STATES Background: The United States has about 4,000 cervical cancer deaths annually, of which an estimated 50% are among women who have not been screened regularly or at all. Objective: To test the feasibility of a mailed self-collection HPV test among women not recently screened for cervical cancer. Methods: Women were eligible to participate in the MybodyMytest study if they had not been screened in 4 years; 30 years of age, not pregnant, without a hysterectomy; North Carolina residents, and had incomes 250 percent of the poverty line. Recruitment flyers were distributed in local venues and referrals were made from 2-1-1 helpline callers seeking housing assistance. All women were asked to self-collect cervico-vaginal samples at home; return specimens by mail; receive HPV results by telephone; visit a local clinic for Pap screening; and respond to questionnaires on experiences of self-sampling and Pap screening. Self-samples were collected using the Rovers brush, placed in Scope mouthwash, and tested for high-risk HPV infection with Hybrid-Capture II. Results: Of 929 high-risk women who called the MybodyMytest toll-free number, 539 (58%) were eligible, and 333 successfully returned the kits (62% participation rate). The 282 women who completed post-testing questionnaires ranged in age from 30-65 years. Most were black (61%) and without private insurance(64%). Women reported high satisfaction with the use and instructions of self-collection by mail. Most preferred self-sampling at home(79%), fewer preferred it in a clinic(1%), and 20% had no preference. Of 324 HPV test results, 315(98%) were adequate, of which 15% HPV positive. Completion of HPV testing and Pap smear follow-up is ongoing and will be presented. Conclusions: Our results among hard-to-reach, underserved women indicate that mailed self-sampling is feasible and well-accepted. This study is the first we are aware of to study mailed HPV screening among high risk women in the United States.
Declaration of interest Dr. Jennifer S. Smith has received research grants and consultancies from Genprobe, Hologic and Qiagen over the past four years. Dr. Jerome Belinson has served as a speaker and received research funding from Gen-Probe, Hologic and Qiagen.
O-17.07
VIA BUT NOT HPV RESULTS ARE HETEROGENEOUS IN ROUTINE SCREENING
M Almonte, Centre for Cancer Prevention, London, UNITED KINGDOM C Ferreccio, Pontificia Universidad Catolica de Chile, Santiago, Chile S Luciani, PAHO, Washington DC, USA M Gonzales, Direccion Regional de Salud San Martin, Tarapoto, Peru J M Delgado, Direccion Regional de Salud San Martin, Lima, Peru C Santos, Instituto de Enfermedades Neoplasicas, Lima, Peru M Alvarez, Instituto de Enfermedades Neoplasicas, Lima, Peru J Cuzick, Centre for Cancer Prevention, London, United Kingdom P Sasieni, Centre for Cancer Prevention, London, United Kingdom Background: Conventional cytology (Pap) and visual inspection after the application of acetic acid (VIA) are currently used in primary screening in Peru. Studies suggest that the quality of VIA is highly variable. Objective: To evaluate the variation of positivity rates of Pap, VIA, liquid-based cytology (LBC) and HPV testing (HC2) over time and between screening centres within the TATI (Tamizaje y Tratamiento Inmediato de Lesiones Cervico-uterinas) project. Methods: Over 36 months, 31,341 women aged 25-49 (main intervention) were screened with VIA and Pap in Amazonian Peru. Additionally, 5435 women (sub-cohort) were also screened with LBC and HC2 over 11 months. Midwives collected three cervical samples for Pap, LBC and HC2 before performing VIA. Pap was done locally, LBC in Lima and HC2 in London. Pap and LBC were positive if classified as ASCUS or worse. The variations in positivity rates of screening tests were measured by the dispersion factor (D=Pearson chi-square divided by degrees-offreedom). Because Pap positivity rates were very low, we also evaluated the variation in inadequate rates of Pap and LBC. Results: In the sub-cohort, monthly positivity rates of VIA varied the most (D=27.6, p<0.001), followed by those of LBC (D=8.5, p<0.001), Pap (D=0.75, p=0.68) and HC2 (D=0.58, p=.83). Pap inadequate rates varied substantially more (D=11.8, p<0.001) than those of LBC (D =4.7, p<0.001). Similar results were found between 16 centres (D=31.4, 3.2, 1.4 and 1.2 for VIA, LBC, Pap and HC2). The monthly variation of VIA positivity rates was high in the main intervention (D=9.7, p<0.001, 36 months) but lower than in the sub-cohort. Conclusion: The lack of overdispersion for HC2 suggests that the variable results for VIA do not reflect true variation in underlying disease, but a lack of consistency in human judgement. The low variation of Pap positivity rates reflects major under-reporting of high-grade disease.
Declaration of interest Maribel Almonte has been sponsored by QIAGEN to attend the 27th International Papillomavirus Conference. Jack Cuzick is on the Speaker's Bureau for QIAGEN
122

HPV FOCAL: ROUND 1 RESULT OF CERVICAL CANCER SCREENING TRIAL.
G Ogilvie, BC Centre for Disease Control, Vancouver, CANADA, D van Niekirk, BC Cancer Agency, Vancouver, CANADA, M Krajden, BC Centre for Disease Control, Vancouver, CANADA, K Ceballos, BC Cancer Agency, Vancouver, CANADA, T G Ehlen, BC Cancer Agency, Vancouver, CANADA, R E Martin, University of British Columbia, Vancouver, CANADA, L Smith, BC Cancer Agency, Vancouver, CANADA, G C E Stuart, University of British Columbia, Vancouver, CANADA, E L Franco, McGill University, Montreal, Quebec, CANADA, A J Coldman, BC Cancer Agency, Vancouver, CANADA Objectives: The HPV Focal trial is the first North American trial comparing the efficacy of HR-HPV DNA with liquid based cytology (LBC) triage of HR-HPV -positive women compared to LBC followed by HR-HPV triage of ASCUS with CIN3 as the outcome. Results from Round 1 of screening and follow up are presented. Method: Women aged 25-65 who are assigned to 1 of 2 study arms- Control: LBC testing with reflex HR-HPV testing in ASCUS +; Negatives screen again at 2 and 4 years. Colposcopy referral at LSIL of ASCUS + HPV positive. Combined safety and Interventions (HPV:) HR-HPV testing with reflex cytology in HPV positive women. Colposcopy referral where HPV positive and ASCUS. Exit screen at either 2 to 4 years. Exit colposcopy referral at ASC-US threshold or HPV-positive. Outcomes measures: Confirmed CIN3 detected at exit screen in control and intervention arms. Results: To date, 15,589 women of 28,000 participants have completed Round 1 of screening. Overall referral to colposcopy was 33.01000 women screened in control and 52.0/1000 in the HPV arms. Overall CIN2+ and CIN3+ rates were 11.0/1000 and 4.1/1000 in the control and 14.7/1000 and 6.7/1000 in the HPV arms. In the control arm, ASCUS+/HPV-women who had repeat cytology in 12 months had no cases of CIN2+ detected (PPV=0%). In contrasts, in women in the HPV arms who were initially HPV+/Cytology negative and remained HPV positive 12 months later, the CIN2+ rate in follow-up was 236.4/1000 (PPV=23.6%). Findings: At round 1 screening, overall referral rates to colposcopy were higher in the HPV than in the control arm. Comparing the follow-up screening round, the PPV in HPV arm was substantially higher than women in cytology arm (0% vs 23.6%), indicating the greater risk associated with persistent hr-HPV infection for precancerous lesions.
Declaration of interest "None declared"
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17 | Cervical screening P-17.09

HPV-GENOTYPE DISTRIBUTION IN CYTOLOGICAL SCREENING, HISTOLOGY AND IMPACT OF VACCINATION.
M de Jonge, Institute of Pathology and Cytology Schttorf/Leer, Schttorf, GERMANY G Busecke, Medical office of General Medicine, Weener, GERMANY A Heinecke, Department of Medical Informatics and Biomathematics, University of Mnster, Mnster, GERMANY O Bettendorf, Institute of Pathology and Cytology Schttorf/Leer, Schttorf, GERMANY Background: HPV-genotyping became a powerful tool for the management of cervical dysplasia during the last years. About 40 HPV types can infect the anogenital tract, but not all of these types lead directly to cervical cancer since every type has its own oncogenic potentials. The distribution of HPV types varies between countries and little is known about HPV-distribution in our rural area of Northwest-Germany. Objective: We analyzed HPV-type-distribution and the distribution in the different cytological and histological lesions in our cohort. Furthermore we studied the possible impact of HPV-vaccination. Methods: 3381 cytological screened women with an HPV-genotyping were analyzed retrospectively. PapilloChecktest was used for molecular investigations. Results of HPV-genotyping and corresponding cytological diagnosis and histological outcomes underwent statistical analysis (SPSS 17.0, SAS9.3, Pearsons chi-square test). The minimum-impact-calculation and the maximum-impact-calculation were used for estimation the possible effect of HPV-vaccination on our cohort. Results: The HPV-type distribution showed marked differences among cervical lesions and age. HPV-51 was common in all cervical lesions, but rarely detected as single-type HPV infection in high-grade cervical intraepithelial neoplasia (CIN3). Only 58% of the CIN3 lesions showed an infection with HPV-16 and/or HPV-18 but these genotypes were predominant compared to CIN2 (p<0.0001). The impact of HPV vaccination showed a probable loss of: 13.1% of ASC-US; 10.0% of LSIL; 32.7% of ASC-H; 20.7% of HSIL; 17.2% of CIN1; 11.1% of CIN2 and 31.3% of CIN3 (MINIcalculation). Conclusion: HPV genotyping is a useful tool for triage especially for women with CIN2. However, in our cohort, primary cervical HPV screening only for HPV-16 and HPV-18 would have been insufficient to detect high-grade intraepithelial neoplasia. Vaccination would especially have prevented high-grade cervical lesions in our cohort but would have less impact on low grade lesions. Systematic screening remains important for preventing cervical cancer even after HPV-vaccination.
P-17.10
PREVALENCE OF HPV IN WOMEN WITH SEVERAL CERVICAL PATHOLOGIES
G Alp Avc, Department of Clinical Microbiology, School of Medicine, Gazi University, Ankara, TURKEY G Bozdayi, Department of Clinical Microbiology, School of Medicine, Gazi University, Ankara, Turkey C. Taskiran, Department of Obstetrics and Gynecology, School of Medicine, Gazi University, Ankara, Turkey M. A Onan, Department of Obstetrics and Gynecology, School of Medicine, Gazi University, Ankara, Turkey S. Ozkan, Department of Public Health, School of Medicine, Gazi University, Ankara, Turkey M. Meral, Gazi University, School of Medicine,Department of Medical Microbiology, Ankara, Turkey Human papillomavirus infection is one of the most common sexually transmitted diseases. The relation between HPV infection and cervical cancer is demonstrated. Objectives: To determinate the prevalence of HPV types in patients with several cervical pathology by PCR and DNA sequence analyse the phylogenetic character were aimed in this study. Methods: Seventy seven patients directed to colposcopy from January to October 2010 were included in the study. HPV-DNA extraction was performed by phenol-chloroform-isoamylalcohol. HPV-DNA and HPV type 16 were detected by Real Time polymerase chain reaction targeting the L1 region. Real Time PCR nested amplifications of MY09/11 products were done by GP5+/GP6+ primers and Cyanine-5 labeled HPV-DNA and HPV 16 specific probe. HPV types determined by Big Dye Terminator Cycle Sequencing (Applied Biosystem, ABD) kit, GP5+/GP6+ primers and ABI Prism 3130XL Genetic Analyzer (Applied Biosystems, ABD). Phylogenetic analysis of sequences was done by Kimuras two parameters method (K2P). Statistically analysis was performed by using Pearson chi-square and odss ratio tests. Results and Conclusion: The incidence of HPV 16; 52%, HPV 16+11; 4%, HPV 16+6; 1% and non-typing HPV 4% were found in cervical samples. HPV positivity was observed between the 34-56 years old patiens and 80% of the positivity was detected between the 31-40 ages. Also positivity decreased 52.2% in patients between 41-50 years and a second peak was determined between the age of 51-60 years with 83.3 % positivity. Among the different cases, 60.0% of 20 ASC-H cases, 63.8% of 36 ASC-US cases, 100% of 9 HSIL cases and 25.0% of 12 LSIL cases positive for HPV-DNA. To reveal the distribution of HPV-genotypes in women with cervical cancer and precancerous lesions in our hospital is important. Early diagnosis of HPV by using improved technological assays, play a key role to prevent the turn precancerous lesions into invasive cancers.
Declaration of interest I have no any sponsor
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Cervical screening | 17 P-17.11

HUMAN PAPILLOMAVIRUS INFECTIONS IN 624 CYTOLOGICALLY NORMAL SMEARS
N H Othman, Universiti Sains Malaysia, Kota Bharu, MALAYSIA N Othman, Institue of Medical Research, Kuala Lumpur, MALAYSIA Background: In countries with low resource, screening for cervical cancer is mainly by cytological examination. In routine cytology for cervical cancer screening, majority of diagnosis made falls under Normal [NILM] category in the Bethesda classification. Objective: This study was conducted to ascertain the presence of HPV-DNA and the distribution of HPV specific types among cases diagnosed as Normal by cytology. Methodology: Cervical scrapings from consecutive 624 samples which were diagnosed to have Normal cervical cytology [NILM] on ThinPrep Pap smears were subjected to HPV testing. We used polymerase chain reactionbased assays using universal primer capable of detecting more than 25 different HPV types. The positive cases were proceeded to specific genotyping by cycle-sequencing. Results: HPV DNA was detected in 3.2% of these samples. 70% were typed and revealed 6 different genotypes; 92.9% were HR-HPVs and the rest were LR-HPVs. HPV 16 was the predominant type, present in 50% followed by HPV 58 (21.4%). HPV 6, 18, 33 and 61 made up of 7.1% of these samples. Conclusion: This study revealed cytologically normal smears show presence of HPVs. Though the proportion is low, the majority is of the HR types. The findings affirms the need of HPV testing on all cases including those with normal cervical smears.
P-17.12
PREDICTORS OF CERVICAL CANCER SCREENING AMONG INUIT WOMEN IN NUNAVIK
P Brassard, McGill University Health Centre, Montreal, CANADA H Cerigo, McGill University, Montreal, CANADA F Coutle, Universit de Montral, Montreal, CANADA E L Franco, McGill University, Montreal, CANADA Background: The Canadian circumpolar Inuit population has a higher incidence of cervical cancer than the general population. The majority of cervical cancer cases occur among underscreened women; however no information is available in regards to predictors of screening use among Inuit women of Quebec. Objectives: The objectives of this study were to determine Pap smear utilization rates and to determine factors associated with time inappropriate use of cervical cancer screening among a cohort of Inuit women from Nunavik. Methods: We used baseline information collected from a cohort formed between January 2002-December 2007 to study the natural history of HPV among Inuit women in Nunavik. Medical and cervical cancer screening history was obtained from baseline questionnaire and medical chart reviews. Unconditional logistic regression was used to estimate the odds ratios and 95% confidence intervals for the association between having not had a Pap smear within the previous 3 years and each covariate among women aged 20-69. Results: A total of 446 women who had complete baseline information were included. The mean age of the study population was 35.5 years. 28.5% of women aged 20-69 had not had a Pap smear within the previous three years before study entry. Women in the 50-59 years old group showed rates of time inappropriate screening of 61% and reaches 83% in the 60-69 age group. Predictors of not having had a Pap smear within the previous 3 years were age (per 10 years) (AOR: 1.79, 95% CI:1.4-2.4) and use of birth control (AOR:0.51, 95% CI:0.3-0.9). Conclusions: Increasing age is associated with reduced uptake of cervical cancer screening, which is of concern as the prevalence of high risk HPV among women 50 years and older is high in this population. Strategies to reach this age group may include HPV-DNA self-sampling.
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RECRUITING VIA SOCIAL NETWORKING SITES FOR HUMAN PAPILLOMAVIRUS (HPV) RESEARCH
S M Garland, The Royal Women's Hospital, Dept. Of Microbiology and Infectious Diseases, University of Melbourne Department of Obstetrics & Gynaecology, Murdoch Children's Resarch Institute, Melbourne, AUSTRALIA, J D Wark, University of Melbourne, Department of Medicine, Royal Melbourne Hospital, Bone and Mineral Service, Royal Melbourne Hospital, Melbourne, Australia, S T Tabrizi, The Royal Women's Hospital, Dept. of Microbiology and Infectious Diseases, University of Melbourne, Dept. of Medicine, Murdoch Children's Research Institute, Melbourne, Australia, Y Jayasinghe, The Royal Women's Hospital, Department of Microbiology and Infectious Diseases, University of Melbourne, Department of Obstetrics & Gynaecology, Melbourne, Australia, E Moore, A Fletcher, The Royal Women's Hospital, Department of Microbiology and Infectious Diseases, Murdoch Children's Research Institute, Melbourne, Australia, B Gunasekaran, N Ahmed, The Royal Women's Hospital, Department of Microbiology and Infectious Diseases, Melbourne, Australia, Y Fenner, The Royal Women's Hospital, Department of Microbiology and Infectious Diseases, Murdoch Children's Research Institute, Melbourne, Australia Background: There is limited research exploring HPV knowledge among young Australian women post-implementation of a national HPV vaccination program. This study explored HPV knowledge among young women recruited using the social networking site (SNS), Facebook. SNSs are commonly used for communication by young people and provide a promising alternative to traditional recruitment strategies, which have limitations including decreasing participation rates and increasing costs. Method: Women aged 16-25 years from Victoria, Australia were eligible to participate. An advertisement placed on Facebook between May-October 2010 was visible to a random sample of eligible Facebook users. Participants were asked to complete a survey containing demographic questions, plus sensitive questions about sexual history, knowledge of cervical cancer, HPV, HPV vaccines, and chlamydia. Participants could complete the survey at the study site or online. Results: 551 women responded to the advertisement, of whom 426 were enrolled (125 were unreachable by telephone, SMS and email): 278 completed the survey (50% at the study site, 50% online), and 148 were lost-to-follow-up (35 withdrew, 113 were unreachable after enrolling). Respondents age and geographical distribution (urban, regional, rural) were similar to the target population. Women 18-25 were more likely to enroll and complete the survey than 16-17 year-olds (odds ratio=1.78, p<0.05). 98% of participants found the survey not at all or only slightly embarrassing. Overall, 63% had heard of HPV: of these, 73% knew HPV is sexually-acquired and 94% knew it causes cancer. 59% self-reported being vaccinated; 18% did not know their vaccination status. This recruitment method was cost-effective ($20/participant). Conclusions: Our study found high levels of HPV awareness; however, we identified gaps in knowledge that should be addressed through educational initiatives. Results demonstrated excellent potential for using SNSs to engage young women in sexual health research, including women from regional and rural communities.
P-17.14
DISTRIBUTION OF HUMAN PAPILLOMAVIRUS GENOTYPES IN CERVICAL SAMPLES IN FRANCE
I Heard, HPV National Reference Laboratory, Pasteur Institute, Groupe Hospitalier Piti-Salptrire, Universit Pierre et Marie Curie, PARIS , FRANCE L AROWAS, HPV National Reference Laboratory, Pasteur Institute, PARIS , FRANCE M C DEMAZOIN, HPV National Reference Laboratory, Pasteur Institute, PARIS , FRANCE R DACHEZ, Laboratoire Biomnis, PARIS , FRANCE N DUPORT, INVS, SAINT-MAURICE, FRANCE M FAVRE, HPV National Reference Laboratory, Genetics, Papillomavirus and Human Cancer unit, Pasteur Institute, PARIS, FRANCE Background. Data on prevalence of HPV type infection are necessary for assessing the impact of HPV vaccination programs. We report the distribution of 18 high-risk HPV (HR HPV) type prevalence in relation to cervical cytology obtained through organised cervical cancer screening (OCCS) program in France. Methods. Residual specimens of cervical cytology (n = 2 804) were obtained from women participating in OCCS program. Genotyping was carried out using the PapilloCheck assay (Greiner Bio-one) at the HPV National reference laboratory. Prevalence rates for each HR-HPV and for multiple infections were assessed according to cytology results. Results. The most common HR HPV types were HPV16, 53, 56 and 31 which accounted for 47 % of all HR-HPV detected. The prevalence of HRHPV infection was 14.8% in women with normal Pap smears, 49.5% in ASC-US, 86.3% in LSIL and 96.4% in ASC-H and HSIL. There was a significant decrease in the prevalence of HRHPV infection with age. On the whole, HPV16 prevalence remained higher than 10% in women aged below 40 years then decreased with aging. HPV16/18 were detected in 26% of LSIL and 60% of HSIL. Conclusion. This study provides baseline HPV genotyping data in the French population and its association with cervical lesions. The overall prevalence of HRHPV infection is similar to the one observed in other West European studies using assays with high analytical sensitivity. Distribution of HPV genotypes in vaccinated women is in progress. These data are relevant for estimating the putative impact of available vaccines on HPV infection and ecology.
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USE AND RELEVANCE OF ASC-US MANAGEMENT OPTIONS IN OLDER WOMEN
M Barthoulot, Hpital universitaire de Strasbourg, Illkirch, FRANCE M Fender, Association Eve, Illkirch, FRANCE Mal BARTHOULOT (1), Muriel FENDER (1) and Jean-Jacques BALDAUF (2) (1) EVE Association, 69 Route du Rhin, 67400 Illkirch GRAFFENSTADEN (2) University Hospital, 67091 Strasbourg Cedex. Use and relevance of ASC-US management options in older women. Objectives: For ASC-US smears, three options are recommended: close-control cytology, colposcopy or HPV testing. Within the framework of an organized cervix screening program, we studied the use and relevance of these options in two age groups 25 to 49 years and 50 to 65 years since anatomical and physiological conditions differ among them. Methods: 6148 ASC-US Pap smears occurred during 2007 among women aged 25 to 65. Previous abnormal smears (n = 1931) and not valid follow-ups (n = 855) were excluded. Follow-up was considered to be normal when virology or cytology were normal and had normal smears control until 2010, or when colposcopy was normal. Results: The three management options were used in similar proportions before and after 50 years. Prevalence of histological lesions did not differ significantly between the two age groups. Cytology showed the highest false negative rate (2.7% before age 50 and 2.3% after), and had consequently lowest sensitivity (80.2% before age 50 and 79.5% after). False positive rates were high for colposcopy (23.8% before age 50 and 22.9% after) which was associated with poor specificity (59.0% and 66.7%) but high sensitivity (96.8% and 94.6%). HPV testing had lowest false positive rate (5.5% and 5.1%) leading to the best specificity (94.1% and 94.8%) but also low false negative rates(0.8% and 1.3%). Conclusion: The different procedures for the follow-up of ASC-US remain valid after 50 years. Their reliability was not worse.
P-17.16
SELF-SAMPLE HPV-TESTS FOR NON-ATTENDEES OF CERVICAL CANCER SCREENING IN FINLAND
A Virtanen, Finnish Cancer Registry, Helsinki, FINLAND P Nieminen, Helsinki University Central Hospital, Helsinki, FINLAND T Luostarinen, Finnish Cancer Registry, Helsinki, FINLAND A Anttila, Finnish Cancer Registry, Helsinki, FINLAND Background: Attendance in screening is an important determinant of cervical cancer. Previous experience on hrHPV-DNA detection based testing on patient obtained samples (self-sampling tests) suggests a good effect among non-attendees of screening. Objectives: We assessed the increase in cervical cancer screening attendance and coverage (coverage of any Pap-smear within the 5-year screening interval) achieved by hrHPV self-sampling kits, reminder letters or the combination of these two. Methods: In the routine screening programme of a Finnish municipality in 2008-2009, non-attendees after the primary invitation were randomised to receive either a self-sampling kit (2,397 women) or a reminder letter (6,302 women). One fourth (1,315 women) of non-attendees after a reminder letter also received a self-sampling kit as a third intervention. Effects on screening coverage were assessed according to self-reported previous Pap smear history of the participants. Results: Participation rate by self-sampling, 31.5% (CI: 29.7, 33.4%), was significantly higher than with a reminder letter, 25.9% (CI: 24.8, 26.9%). The adjusted relative risk for participation by self-sampling was 1.21 (CI 1.13-1.3). Total attendance increased from 65 to 76% with self-sampling and to 74% with a reminder letter. Combining the interventions reached 40% of non-attendees and increased total attendance from 63% to 78%. Only app. 20% of the participants in all three intervention groups increased screening coverage (previous Pap-smear 5 years ago or never). Self-obtained samples were more often HPV-positive than provider obtained ones, 12-13% versus 7%. Conclusions: Self-sample HPV-tests are a feasible option in enhancing the attendance at organised screening, particularly as an addition to a reminder letter. Our results suggest that if self-sampling is used as a third intervention after two written invitations, the overall attendance in Finland could reach the desired 80-85%.
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PROGRESSION OF CERVICAL DYSPLASIA AND CYTOLOGIC FINDINGS ASSOCIATED WITH HPVTYPES
J Saito, Kansai Medical University, hirakashi Osaka, JAPAN H Kanzaki, Kansai Medical University, Hirakatashi Osaka, Japan Object: The aim of this study is to clarify the specific cytological changes of HPV infection and the progression of cervical dysplasia among various HPV types. Method: Four hundred seventy-seven women who visit to our-patient clinic for abnormal cytology had Pap test,pathological findings under colposcopy, and HPVDNA test. Among three hundred sixty-eight cases who were HPV positive and one hundred nine cases who were HPV negative, we compared cytologic findings ( koilocyte,parakeratotic cells, multinucleation, smudged nuclei, giant cell, and immmature metaplastic cells) to types of HPV. One hundred eighty-four women were selected in which follow-up survey for more than five years. Results: Frequencies of cytological findings of HPV infection were significantly higher in HPV-positive cases than HPV-negative cases. Regarding the association of HPV types with cytological findings, koilocyte were detected more often in patients with types other than HPV16. The high risk HPV(HPV16,18,33,52) was detected at the higher frequencies in the progression state of dyslasia. Conclusion: By characteristic cytological findings of HPV infection, we could suspect HPV infection in women with abnormal cytology. Interestingly koilocytes provided a negative finding for HPV16 infection. Cytologic findings of HPV infection other than koilocyte were associated with the progression of dysplasia. THese datas suggesteed that HPVDNA testing could be helpful in monitoring cervical dyslasia.
P-17.18
KNOWLEDGE ABOUT HPV AND CERVICAL CANCER: AN INTERNATIONAL STUDY
J Waller, University College London, London, UNITED KINGDOM L Marlow, University College London, London, UNITED KINGDOM K Mc Caffery, University of Sydney, Sydney, AUSTRALIA R Ostini, University of Queensland, Ipswich, AUSTRALIA G Zimet, Indiana University, Indianapolis, UNITED STATES OF AMERICA Background: As HPV testing and vaccination are introduced into cervical cancer control programmes, it is increasingly important for the public to be aware of the virus. Many studies have evaluated awareness of HPV in different samples but international comparisons have been difficult due to inconsistency of measurement. Objectives: To evaluate HPV knowledge in the UK, the US and Australia. Methods: Participants (n=2442) were recruited from a market research database. They completed an online survey and provided demographic information as well as answering questions about HPV, testing and vaccination. Quotas were used to ensure the sample included equal numbers of men and women from the three countries. Results: Overall awareness of HPV was highest in the US (76%), followed by Australia (57%) and the UK (51%). Womens awareness was significantly higher than mens (p>.05). Among those who were aware of HPV (n=1499) the mean knowledge score was 9.0/16 (95% CI: 8.8-9.2), with no differences between countries. 84% knew that HPV can cause cervical cancer, but only 9% knew that HPV infection usually does not need treatment. Awareness of HPV testing among those who had heard of the virus was high in the US at 63% but lower in the UK (44%) and Australia (41%). Higher numbers of people had heard of the HPV vaccine (US: 82%; Australia: 78%; UK: 75%). Among women aged 18-29 (n=261), 62% in Australia had received at least one dose of the vaccine, compared with 37% in the US and 16% in the UK. Conclusions: Awareness of HPV and its link with cervical cancer is rising but is still far from universal, particularly among men. Awareness was higher in the US than the UK or Australia. Even when people had heard of HPV, their knowledge was generally poor.
128

BARRIERS TO CERVICAL SCREENING AMONG SEXUALLY ABUSED WOMEN: EXPLORATORY STUDY
L Cadman, Queen Mary, University of London, London, UNITED KINGDOM J Waller, University College,, London, United Kingdon L Ashdown - Barr, Quenn Mary, University of London, London, United Kingdon a Szarewski, Queen Mary, University of London, London, United Kingdon Background: Childhood sexual abuse is known to be a risk factor for cervical cancer, probably because of early exposure to human papillomavirus (HPV). Women who have been abused may be less likely to attend for cervical screening, which is concerning given their high-risk status. This study aimed to explore screening participation and perceived barriers among women who have been sexually abused. Methods: A short, web-based survey was developed, asking for basic demographic information, cervical screening history and type of abuse experienced. Two open questions asked about barriers to cervical screening attendance, and suggested improvements to the service. A link to the survey was placed on the website of NAPAC, the National Association for People Abused in Childhood. Data were collected between June and October 2010. Results: 136 women aged 20-64 years completed the survey. 77% had taken part in cervical screening but only 54% had been screened within the last 5 years. The main barriers to screening that emerged were: 1) emotional responses, including stigma and anxiety; 2) impact of abuse, including feelings of vulnerability and violation; 3) characteristics of the test itself, including pain and intrusiveness; and 4) past screening experiences, including inability to complete the test due to distress or flashbacks, and a lack of sensitivity from health professionals. Women had a variety of suggestions for improving screening, including developing ways to make it easier for them to disclose their abuse, longer appointments to give time for discussion prior to the test, sensitivity and acknowledgement of their distress by the screener, and the use of self-testing, or the option to insert the speculum themselves. Conclusions: Further research is needed to see whether the findings hold true in a more representative sample, and to develop ways to improve screening access for this vulnerable group of women.
P-17.20
PRIMARY HUMAN PAPILLOMAVIRUS DNA SCREENING BY AGE: SYSTEMATIC REVIEW
M Rebolj, University of Copenhagen, Copenhagen, DENMARK S Njor, University of Copenhagen, Copenhagen, DENMARK E Lynge, University of Copenhagen, Copenhagen, DENMARK Background. Cervical screening with Human Papillomavirus (HPV) testing is less specific for high-grade cervical intraepithelial neoplasia (CIN3) than cytology. As HPV infections are more common below age 30, it has been suggested that the problem of low specificity could be resolved by restricting HPV DNA screening to women above age 30. Objectives. The aim of this systematic review was to determine the adverse effects of HPV DNA testing relative to cytology for women below and women above age 30 based on data from randomized controlled trials on primary cervical screening. Methods. Based on published trial data, we calculated the relative detection of CIN1 and CIN2, and the relative risks of false-positive tests (positive tests without subsequent CIN3) per age group and trial for HPV testing versus cytology. Results. In trials with a low cytology-abnormality rate (4%), the relative risks of false-positive tests among women aged 30 years ranged between 1.63 (95% CI: 1.37-1.94) and 3.81 (3.05-4.76); the relative baseline detection rates of CIN1 were 1.54 (1.13-2.09) and 1.87 (1.38-2.53) in the 2 trials with reported data; the relative baseline detection rates of CIN2 ranged between 1.04 (0.59-1.85) and 2.07 (1.32-3.24), whereas the relative cumulative detection rates of CIN2 ranged from 1.05 (0.69-1.59) to 1.77 (1.18-2.67). In trials with high cytology abnormality rates in women aged 30 years, the risks of false-positive tests, CIN1 and CIN2 were similar for HPV testing and cytology. The relative risks of adverse effects of HPV testing were for both types of cytology settings generally higher for women below age 30. Conclusions. Screening-related adverse effects were less common among women aged 30 than among younger women. However, in women above age 30 HPV testing still led to about twice as many false-positive tests than cytology, and increased the frequency of CIN1 and CIN2 diagnoses.
Declaration of interest Elsebeth Lynge and Matejka Rebolj are currently undertaking a comparative study of new-generation HPV tests, involving collaboration with Roche Diagnostics A/S, Genomica S.A.U., Qiagen Gaithersburg Ltd., and GenProbe Inc. Concerning the present paper, there has been no collaboration with, or support from any of the companies. Elsebeth Lynge has served as unpaid scientific advisor to GenProbe and Norchip.
129

TRIAGING ASC-US PAP SMEAR WITH COLPOSCOPY: DATA FROM PATER STUDY.
S Costa, S. Orsola-Malpighi Hospital University of Bologna, Italy, Bologna, ITALY, S Venturoli, S. Orsola-Malpighi Hospital, University of Bologna, Italy, Bologna, ITALY, A Marcellusi, University of Tor Vergata, Rome, Italy, Rome, ITALY, G Garganese, Catholic University of the Sacred Heart, Rome, Italy, Rome, ITALY, G Favato, Kingston University, London, UK, London, UK, F S Mennini, University of Tor Vergata, Rome, Italy, Rome, ITALY, D Barbieri, S. Orsola-Malpighi Hospital, University of Bologna, Italy, Bologna, ITALY, M Pesaresi, S. Orsola-Malpighi Hospital University of Bologna, Italy, Bologna, ITALY, A Falasca, S. Orsola-Malpighi Hospital, University of Bologna, Italy, Bologna, ITALY, M Zerbini, S. Orsola-Malpighi Hospital, University of Bologna, Italy, Bologna, ITALY Background. An immediate colposcopy is assumed to be the safest option when an ASC-US is diagnosed. However, this category is often associated with an underlying benign condition rather than a real cell abnormality generating relevant costs to National Health Services (NHS). This analysis was focused on colposcopy for the detection of cervical intraepithelial neoplasia (CIN) lesions from ASC-US pap smears. Methods. In this retrospective and observational study, all patients aged 23-65 who referred to the Department of Gynecology and Obstetrics in Bologna University Hospital for an ASC-US between January 2000 and December 2007 were enrolled. Deterministic analyses were stratified by age, histology, colposcopy, and DNA-HPV testing. Results. Among 1,047 patients enrolled (mean age 38.49.6 years), a punch biopsy and/or an endocervical curettage was performed in 765. In 357 patients (46.7%), the colposcopy was abnormal while 408 (53.3%) had a normal colposcopy and squamo-columnar junctions not entirely visible. Overall, 357 (34.1%) women had cervical lesions: 279 CIN1 (26.6%) and 78 CIN2+ (7.4%). Among 279 CIN1, 250 (89.6%) showed a positive colposcopy which resulted HPV-negative in 127 (51.0%) cases. The remaining 29 negative colposcopies were HPV-negative in 96.6% of cases. Interestingly, 20% of CIN1 resulted positive for low-risk HPV. Among 78 CIN2+ patients, 74 (94.9%) had a positive colposcopy, 70of whom (94.6%) were HPV-positive for either high-risk or high-risk+low-risk genotypes. Residual 4 negative colposcopies harboured high-risk HPV. Conclusions. These data suggest that an immediate colposcopy following ASC-US pap smears may increase the rate of CIN1 that are HPV-negative. The majority of them are HPV-negative probably due to a physiologic squamous cytological repair. This policy might lead to overtreatment and significant avoidable costs for NHS. A quadrivalentbased vaccination and screening should provide an improvement of CIN1, high-grade lesions and anogenital warts management. This should reduce healthcare costs and optimize diagnostic and treatment procedures.
P-17.22
SCREENING PATHWAY HISTORIES OF WOMEN WITH CERVICAL CARCINOMA
M Duggan, University of Calgary, Calgary, CANADA J Nation, University of Calgary, Calgary, CANADA Background: Women who develop cancer of the uterine service when cervical cancer screening is available represent screening failures. Objectives: The Calgary Health Region (CHR) provides all health care including opportunistic cervical cancer screening to a population of 1.3M. The screening histories of women with carcinoma and resident in the CHR between 1996 and 2001 were audited to characterize factors in the screening pathway contributing to the failures. Methods: The cohort consisted of 246 women. Information on their Pap tests and colposcopic/gynecologic exams was obtained from the files of Calgary Laboratory Services and their colposcopic/cancer centre treatment charts. Factors in the screening pathway contributing to screening failures were defined and frequencies calculated. Results: Screening failure factors were: 1) 16.7% not screened i.e., no Pap test screening, 2) 11.8% under screened i.e., no Pap test within 12 months, 3) 13.7% under sampled i.e., the Pap test result was negative, 4) 13.8% no referral for a colposcopy/gynecology exam and/or it was delayed more than 3 months, 5) 13.3% delayed referral for examination of an HSIL + Pap test of more than 3 months, and 6) 33.6% under diagnosis i.e., the colposcopy/ gynecology exam diagnosis was less than malignant. Under reported Pap tests and delayed Pap test reporting could not be fully investigated. Limited evidence suggested they too were factors. Conclusions: Factors other than recruitment to cytological screening need to be targeted for improvement if the regions cervical cancer prevention program is to be more effective.
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CHANGING SCREENING PROGRAM IN NORTH-WEST SPAIN, BACK TO THE PAST?
A Cid, Hospital of Ourense-Dept of Microbiology, Ourense, SPAIN, S Prez, Hospital Meixoeiro-Dept of Microbiology, Vigo, SPAIN, M Pato, Hospital of Ourense-Dept of Obstetrics and Gynecology, Ourense, SPAIN, B Couso, Hospital of Ourense-Dept of Obstetrics and Gynecology, Ourense, SPAIN, R Rodriguez, Hospital of Ourense-Dept of Obstetrics and Gynecology, Ourense, SPAIN, M A Gmez, Hospital of Ourense-Dept of Microbiology, Ourense, SPAIN, M C Blanco, Hospital of Ourense-Dept of Microbiology, Ourense, SPAIN, M Losada, Hospital of Ourense-Dept of Microbiology, Ourense, SPAIN, M Gestal, Hospital of Ourense-Dept of Obstetrics and Gynecology, Ourense, SPAIN, J L Doval, Hospital of Ourense-Dept of Obstetrics and Gynecology, Ourense, SPAIN BACKGROUND: Different strategies are followed for Cervical Cancer Screening (CCS). Until 2008 in Ourense (northwest of Spain) we followed the co-testing (Pap + HPV) as primary screening in women >35 years, but in 2010 the public health-care system changed it, and we came back to cytology-based programs,using only HPV test for triage of ASC-US, and follow-up of positive smears or post-treatment cases. OBJECTIVES: To compare the influence of two different CCS(2008 / 2010) in laboratory workload and in the genotypes detected . METHODS: Amplicor HPV Test(Roche) was initially performed in cervical scrapes collected in PreservCyt medium . The samples with positive result or due to clinical requirements were genotyped by Linear Array HPV Genotyping Test (Roche). RESULTS: Year 2008 versus 2010: 4238 samples, mean age 43.4 (16.5-83.5) vs. 1426 samples, mean age 38.3 (16.982.6); 655 Amplicor HPV Test positive (15.5%) vs. 559 (39.2%), 841 samples genotyped (19.8%) finding some HPV in 709 samples vs. 942 samples genotyped (66%) finding HPV in 694 . HPV-16: 27.3% vs. 24.8%, HPV-51: 14% vs. 15%, HPV-31: 13,7% vs. 12.2%, HPV-53: 11.1% vs. 11.2%, HPV-66: 8.6% vs. 9.6%, HPV-18: 7.2% vs. 7.6%, HPV-45: 5.5% vs. 5.3%, HPV-33: 5.2% vs. 5.2% , HPV-35: 5.2%vs. 3%. Statistical significance was found (p<0.001) in the mean age of women (5 years; IC 95% 4.371-5.685), in the number of samples positive for HR HPV , and in the samples that were genotyped. No differences were found in the prevalence of genotypes. CONCLUSIONS: In 2010 , the number of samples decreases but more % is genotyped . The cost has been reduced, but many patients are surely under-diagnosed due to the sub-optimal sensitivity of cytology . Economic problems should not be an excuse for this changes in CCS.
P-17.24
HPV SELF-SAMPLING AMONG WOMEN NON-ADHERENT TO PAPANICOLAOU IN CHILE
J LENIZ, PONTIFICIA UNIVERSIDAD CATLICA DE CHILE, Santiago, CHILE H POGGI, Departamento de Laboratorios Clnicos. PONTIFICIA UNIVERSIDAD CATLICA DE CHILE, Santiago, CHILE M I BARRIGA, PONTIFICIA UNIVERSIDAD CATLICA DE CHILE, Santiago, CHILE C IBAEZ, PONTIFICIA UNIVERSIDAD CATLICA DE CHILE, Santiago, CHILE S TERRAZAS, PONTIFICIA UNIVERSIDAD CATLICA DE CHILE, Santiago, CHILE K PUSCHEL, PONTIFICIA UNIVERSIDAD CATLICA DE CHILE, Santiago, CHILE C FERRECCIO, PONTIFICIA UNIVERSIDAD CATLICA DE CHILE, Santiago, CHILE Background: Cervical cancer (CxCa) screening program, Papanicolaou (PAP) test every 3 years for women aged 2564 years, is associated with moderate decrease in CxCa in Chile, with coverage around 60%. Objectives: Our aim was to evaluate the HPV vaginal self-sampling to reach women non-compliant with the national program. Methods: We systematically visited the households in the catchment area of the Puente Alto Health Center, in Santiago, to identify women aged 30-64 years lacking their Pap screening for > 3 years. Eligible women were offered to attend the health center for her Pap or to provide a vaginal self-sample. Accepting women signed an informed consent, answered a questionnaire and provided a vaginal self-sampling using the HPV Hybrid Capture 2 brush and transport medium (Qiagen, Dusseldorf ). HC2 was performed School of Medicine of Catholic University Clinical Laboratory. Women HPV positive were referred to colposcopy and biopsied if needed. Results: We identified and contacted 1,254 women non-adherent to the Pap; 86.5% of eligible women accepted and provided a vaginal self-sample. Among them 3.5% had never had a PAP, with an average of 6.35 years since last Pap. Women mean age was 46.2 years, 66% were married, 48.9% had more than 8 years of education. HC2 was positive in 124 (11.5%) women who were referred to colposcopy and 85.5% attended to the colpo clinic; 12 (1.1% of total screened) women presented CIN2 or worse lesions (5 CIN2, 6 CIN3 and 1 cancer) as confirmed by histology. Most women (91.6%) reported less discomfort with self-sampling than with PAP. Conclusions: We demonstrated that HPV self-vaginal sampling permits to identify and refer for diagnoses and treatment women who otherwise would go on unattended until their disease become symptomatic. It should be established as an adjunct to the conventional screening program to increase its coverage and impact.
131

ADVANCED CERVICAL LESIONS AFTER CYTOLOGICAL ATYPICAL CELLS AND LOW-GRADE LESIONS
Y Chen, National Taiwan University, Taipei City, TAIWAN S You, Academia Sinica, Taipei City, Taiwan S Koong, Department of Health, Taipei City, Taiwan C Chen, National Taiwan University, Taipei City , Taiwan C Chen, Academia Sinica, Taipei City, Taiwan Background & Objectives: The study aimed to examine the incidence of invasive cervical cancer(ICC), cervical carcinoma in situ(CIS), and cytologic high-grade squamous intraepithelial lesions(HSIL) or more severe cytologic lesions among women detected their first atypical squamous cell undetermined of significance(ASC), atypical glandular cells(AGC), and low-grade squamous epithelial lesions(LSIL), and to estimate the associations between the subsequent follow-up attending and risks of developing cervical cancer. Methods: Cervical cancer screening program in Taiwan has been implemented since 1995, all screening records are mandatory to report, and a centralized screening database has been established as well. The cytologic abnormalities, included 110,561 ASC cases, 8,339 AGC cases, and 45,671 LSIL cases, and cytologic HSIL or above lesions were identified from screening registry. Women received Pap smear or biopsy in one year after their first cytologic abnormalities were considered have attended subsequent follow-up. Cervical cancers were ascertained from cancer registry until Dec, 31st, 2007. Hazard ratios were estimated by Coxs proportional hazards model, and cumulative incidences were estimated by Nelson-Aalen method. Results: There was at least 14% reduction of CIS and 23% reduction of ICC if women attended screening two years before their first abnormalities, and at least 32% reduction of CIS and 58% reduction of ICC if they attended screening within two years. Follow-up attending for the first abnormality significantly reduced 13% to 53% of cytologic HSIL, 40% to 49% of CIS, and 52% to 71% of ICC. Compared to LSIL, ASC is less risky to develop cytologic HSIL, similar risk to develop CIS, and possesses more risk to develop ICC. Subsequent follow-up attendance for their first abnormality and previous negative smear detection were inversely associated with cervical cancer occurrence. Conclusions: AGC is the most clinical significant lesion compared with ASC and LSIL, and is suggestive to include in the positivity follow-up system.
P-17.26
SCREENING AND TRIAGE USING PRIMARY MRNA HPV AND VIA
J Belinson, Preventive Oncology International, Cleveland Heights, UNITED STATES, L Nieves Arriba, The Cleveland Clinic , Cleveland, UNITED STATES, C Enerson, Prueba para La Vida, Morelia, MEXICO, S Belinson, Preventive Oncology International, Cleveland Heights, UNITED STATES, C Booth, The Cleveland Clinic , Cleveland, UNITED STATES, J Brainard, The Cleveland Clinic , Cleveland, UNITED STATES, A Chiesa - Vottero, The Cleveland Clinic , Cleveland, UNITED STATES, J Belinson, Preventive Oncology International, Cleveland Heights, UNITED STATES, L Nieves Arriba, The Cleveland Clinic , Cleveland, UNITED STATES, C Enerson, Prueba para La Vida, Morelia, MEXICO, S Belinson, Preventive Oncology International, Cleveland Heights, UNITED STATES, C Booth, The Cleveland Clinic , Cleveland, UNITED STATES, J Brainard, The Cleveland Clinic , Cleveland, UNITED STATES, A Chiesa - Vottero, The Cleveland Clinic , Cleveland, UNITED STATES BACKGROUND: In rural Mexico it has been difficult to demonstrate success for cytology based cervical cancer screening. OBJECTIVE: MECCS II seeks to develop a highly sensitive and highly specific screening program able to be adapted in Mexico. 1) Determine sensitivity and specificity of primary HPV screening using a mRNA based assay (APTIMA). 2) Study role of VIA as a triage technology prior to cryotherapy. METHODS: The study was conducted in Patzcuaro and Zitacuaro, Mexico. Women ages 30-50, non-pregnant, varied histories of screening, no history of hysterectomy or pelvic irradiation participated. A direct endocervical sample was tested for cytology, HC II and APTIMA assay (AHPV).(Comapred by McNemar test) Subjects positive on any test were recalled for Triage VIA, biopsies, and immediate cryotherapy. RESULTS: 2096 patients have complete results. Mean age (SD) = 39.2. 7.7 % with ASCUS; 1.8% LGSIL; and 0.5 % HGSIL. 2.1% of patients had CIN 2 and 0.77% had CIN 3 (4 with invasive disease). The sensitivity of ThinPrep >ASCUS, HC-II and AHPV for >CIN3 were similar, 86.7%, 100% and 100% respectively. The specificity of ThinPrep >ASCUS, HC-II and AHPV for >CIN3 was 94.1%, 90.2%, and 92.7% respectively. Specificities of HC-II and AHPV differed significantly. The overall percent of agreement among HPV assays (HC II versus Aptima) is 97%. 470 women returned for VIA and 300 were treated with cryotherapy. Theoretical under-treatment: 14 women with (+) ECC, 3 women with 3 or 4 quadrant lesions. 6 month follow-up data will be reported. CONCLUSIONS: The specificity of the APTIMA assay is an advantage for primary screening. Follow-up evaluation is important to determine the true impact of potential under-treatment of the screening algorithm. Self-sampling applications will be explored.
Declaration of interest "None declared".
132

HPV TRIAGE FOR LOW GRADE CYTOLOGY: ENGLISH SENTINEL SITES STUDY.
H Kitchener, The University of Manchester, Manchester, UNITED KINGDOM R S Kelly, The Institute of Cancer Research, Sutton, UNITED KINGDOM J Patnick, NHS Cancer Screening Programmes, Sheffield, UNITED KINGDOM S M Moss, The Institute of Cancer Research, Sutton, UNITED KINGDOM Background: Following successful piloting of HPV triage in the English Cervical Screening Programme (CSP) it was decided to broaden the implementation into six Sentinel Sites screening 300,000 women. The purpose was to determine variation in outcomes and predict the likely impact on services of national roll out. Methods: Women aged between 25-64 with either borderline (ASCUS) or mild dyskaryosis (LSIL) were included. Relevant liquid cytology samples were HPV tested at one of two regional designated service laboratories. HPV testing was based on Hybrid Capture 2 set at a cut-off of 2RLU/Co [1]. Women who tested HPV+ve were referred for colpsocopy and those who were HPV-ve were discharged to routine recall. Results: A total of 10,051 women entered the protocol; 6507 (64.7%) had borderline (ASCUS) cytology and 3544 (35.3%) mild dyskaryosis (LSIL). 64.4% tested positive for HPV; 53.7% of those with borderline and 83.9% of mild dyskaryosis. HPV positive rates ranged from 35% to 73% between the six sites for borderline and 73%-92% for mild dyskaryosis. Of the 6470 HPV+ve women referred, attendance at colposcopy was over 90%. The positive predictive value for detecting CIN2+ amongst those referred for colposcopy was 16.3%, and the range between sites was 9.3% to 21.5%. The proportion of women with negative colpsocopy negative biopsy was 54.6%. Conclusion: Variation in HPV+ve rates between sites and hence colposcopy referral rates between sites is likely to reflect difference in cytology reporting. This also leads to significant variation in the PPV for CIN2+. The large proportion of women with negative colposcopy reinforces the need for an efficient care pathway. The relatively low PPV suggests scope for improving specificity. [1] Sargent et al (2010) J Clin Microbiol; 48 (2): 554-8.
P-17.28
HR-HPV-TYPE ASSOCIATED RISK FOR CIN2 IN THE WOLFSBURG SCREENING PROJECT
A Luyten, Dept. of Gynecology and Obstetrics, Klinikum Wolfsburg, Wolfsburg, GERMANY A Laksana, Dept. of Gynecology and Obstetrics, Klinikum Wolfsburg, Wolfsburg, GERMANY T Iftner, Dept. of Med. Virology and Epidemiol. of Viral Diseases, Tbingen, GERMANY K U Petry, Dept. of Gynecology and Obstetrics, Klinikum Wolfsburg, Wolfsburg, GERMANY Background: In 2006 a primary screening project was started in the region of Wolfsburg combining Pap smear and HPV co-testing to improve cervical cancer prevention, in Germany. The risk of high-grade lesions and carcinoma is known to correlate with specific HR-HPV types. Objectives: To identify HPV genotypes and estimate the associated risk for developing high grade dysplasia (CIN2) in patients with positive HC2-HR probe results and normal cytology. Methods: 8170 screening samples of the Wolfsburg project were randomised for HPV-Genotyping. For each highrisk HPV-type the prevalence and odds ratio for CIN2 and CIN3 was calculated. Results: 349 samples with HR- HPV-infection (4.3%) were identified, 292 with concomitant normal cytology und 57 with abnormal cytology (Pap IIw). HPV 16 was the most common HPV type with the highest risk for CIN2 and CIN3. HPV 31 and HPV 52 were next in prevalence. HPV 31 showed an elevated risk for CIN3 and HPV 52 for CIN2. In spite of their lower incidence HPV 33 und 45 yielded a higher risk for high-grade dysplasia. In patients with adenocarcinoma in situ only HPV 16 und HPV 18 were detected. In general, HPV 16, 18, 31 and 33 showed a higher rate of persistence during follow up HPV testing (53,5%; 85,7%; 65%; 75%) compared to other HR HPV types. Conclusions: HPV-genotyping demonstrated good performance in the triage of high-risk HPV infected patients with normal cytology. HPV 16, 18, 31, 33 and 45 infected women should be referred within a short time frame to colposcopy.
Declaration of interest T. Iftner: institutional research grants from Gen-probe, GSK, Hologic, Roche, Sanofi Pasteur MSD
133

ESTABLISHMENT OF A CERVICAL CYTOLOGYBIOBANKSWEDISH CERVICAL CANCER SCREENING
N Perskvist, Karolinska universitetssjukhuset, Huddinge , Stockholm, SWEDEN Establishment of a Liquid-Based Cervical Cytology Biobank in the Swedish Cervical Cancer Screening Program Nasrin Perskvist and Joakim Dillner, Institute for Laboratory Medicine and the BioBanking & Molecular Resource Infrastructure of Sweden, Karolinska Institute, Stockholm, Sweden Large parts of the Swedish organized cervical cancer screening program has switched to Liquid Based Cytology (LBC). LBC samples contain large amounts of cells that in the methanol-based fixative and promise high-quality of well-preserved cellular morphology, DNA, RNA, proteins and is therefore potentially useful for biomarker research. In addition, analysis of biobanked specimens for HPV, can provide information on the length of persistence of the HPV infection and thus on the cervical cancer risk. However, the large 20 ml LBC vials are too bulky to be stored routinely and the samples have hitherto been discarded. As part of a development project in the Swedish national biobanking program BBMRI.se, we have developed an automated system that transfers the cells to 270ul storage vials for automated storage at minus 20 degrees. A national system for storage of LBC samples will greatly facilitate the implementation of screening strategies based on screening for HPV persistence in Sweden.
P-17.30
HPV PREVALENCE AND ACCURACY OF HPV TESTING FOR CERVICAL SCREENING
G Ronco, CPO Piemonte, Torino, ITALY P Giorgi - Rossi, ASP Lazio, Roma, ITALY S Franceschi, IARC, Lyon, FRANCE BACKGROUND. Concern was raised on using testing for high-risk (HR) human papillomavirus (HPV) in cervical cancer screening in populations where HPV prevalence is high. OBJECTIVES: Directly evaluating the effect of HR HPV prevalence on sensitivity and PPV of HPV test-based screening. Methods. A meta-regression of the relationship between HR HPV prevalence and the specificity and positive predictive value (PPV) of HPV DNA testing for the presence of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) was performed. Only studies that used Hybrid Capture 2 (HC2) were included. Country income (lowmedium vs. high) was used as a proxy of previous screening. RESULTS. Twenty-six populations from 20 studies were included. For a 10% increase in HR HPV prevalence HC2 specificity decreased by 8.41% (95% confidence interval (CI): 8.02-8.81%) whereas PPV increased by 4.74% (95% CI: 2.45-7.03%). HR HPV prevalence explained 98% of the variability in HC2 specificity and 38% of the variability in PPV. Country income did not affect specificity but low-medium income was associated with higher PPV (3.81%; 95% CI: 1.53-6.10%) after adjustment for HR HPV prevalence. CONCLUSIONS. When HR HPV prevalence is high, the specificity of HPV testing for CIN2+ decreases, but PPV does not decrease and it is high in inadequately screened populations. The number of HPV-positive women needing further assessment or treatment per CIN2+ case detected will therefore decrease and screening efficiency will improve. This behavior is unusual compared to other screening tests and is explained by the fact that HR HPV causes CIN2+: an increase in HR HPV prevalence is inevitably accompanied by an increase in CIN2+.
134

IMPLEMENTATION OF CERVICAL CANCER SCREENING WITH HPV TESTING IN COLOMBIA
C Wiesner, Instituto Nacional de Cancerologa, Bogota, COLOMBIA Objective: To carry out situation analysis as the basis for implementation of cervical cancer screening based on DNA-HPV testing (Hybrid Capture II) in a demonstrative area of a low-middle income country (Colombia). Study Design A descriptive study was designed using the PRECEDE-PROCEED model to carry out situation analysis in six Colombian municipalities where 20,000 women will be screened for HPV with hybrid capture as primary test. A survey among 15 health insurance companies, 40 screening centres, 9 cytological laboratories and 12 colposcopy centres was developed. Epidemiological assessment included secondary data analysis. 10 focus groups were held in each area with 134 subjects (community leaders, health managers, health professionals) and 6 interviews (3: health authorities, 3: religious authorities. Results Community participants were aware of cervical cancer, but did not consider it as great a priority as water pollution, alcoholism and teen pregnancy; for them, political action is more effective than community organization. HPV testing is highly acceptable. Insurance companies administrative requisites create access barriers for colposcopy and biopsy; furthermore, they provide scant follow up on positive women, and insufficient treatment procedure records Health professionals generally believe that women do not sufficiently participate in cervical cancer screening, even though Pap smear coverage register is acceptable. Not all cytology labs have internal quality control or reports unsatisfactory Pap smears. According to focus groups, women under 30 with encompassing HPV/mild dysplasia/CIN tend to be over treated. Human resources availability is similar among municipalities. Non professional nurses play an important role in cervical cancer screening. Conclusion: For successful DNA-HPV test implementation process, there should be Colombian Health Ministry support, improved and sustained communication with community organizations, agreements from insurance companies to overcome administrative obstacles, greater awareness of natural history of HPV and Pap smear limitations among Colombian health professionals.
Declaration of interest I received QIAGEN suppport to attend the International Papillomavirus Conference
P-17.32
HPV-DNA AND PAP-SMEAR CO-TESTING IN 29,000 DANISH WOMEN 30+ YEARS
C Munk, Department of Viruses, Hormones and Cancer, Institute of Cancer Epidemiology, Danish Cancer Society, Copenhagen, DENMARK, K Frederiksen, Department of Statistics, Institute of Cancer Epidemiology, Danish Cancer Society, Copenhagen, DENMARK, J Junge, Department of Pathology, Hvidovre Hospital, Copenhagen, DENMARK, T Iftner, Department of Medical Virology, University Hospital of Tbingen, Tbingen , GERMANY, S K Kjaer, Department of Viruses, Hormones and Cancer, Institute of Cancer Epidemiology, Danish Cancer Society, Copenhagen, DENMARK Background and Objective: Testing for HPV DNA has been suggested as a tool in primary screening for cervical cancer. Concerns have included over-diagnosis due to high prevalence of HPV in younger age groups. Our aim was to evaluate HPV DNA testing in Danish women 30 years or older taking HPV genotype and previous Pap smear history into account. Methods: We used data from our population-based study of more than 40,000 women enrolled from 2002-2005. All women had liquid based cytology samples taken. After cytological examination, HPV DNA testing was performed on the left-over material using the HC2 test. Samples positive by HC2 was genotyped using the INNO LiPAv2. By linkage with the Pathology Data Bank, we obtained information about index sample and previous screening history. Results: In all, 28,830 women were 30 years or older and 3,063 women had normal cytology and were HPV-positive (10.6%). This ranged from 17.6% in the age group 30-34 years to 4.7% among women 65 years or older. Only 1.5% was HPV-negative and had cytological abnormalities of which 83.2% had ASCUS/LSIL. Previous screening history did not seem to affect these associations. Among 3,844 women with HPV-positive samples (HC2), 11.6% had ASCUS/LSIL and 8.7% had HSIL on the corresponding cytology. HPV16, 18 and 33 were significantly more prevalent in HSIL compared with normal cytology (e.g. HPV16 prevalence odds ratio: 1.97; 95% CI: 1.69-2.30). The same types were equally prevalent in ASCUS/LSIL compared with normal cytology (e.g. HPV16 POR: 1.04; 95% CI: 0.86-1.27). These associations were not modified by age. Conclusion: A substantial proportion of women would be considered at risk for disease with HPV DNA testing compared to cytology in this population. However, this applies to prevalent disease and the value on incident disease will have to be determined. Adding genotyping may improve performance.
Declaration of interest C Munk: support for travel and conference participation from Merck and Sanofi Pasteur MSD. K Frederiksen: has no conflict of interest. J Junge: member of the Advisory Board for Sanofi Pasteur MSD Denmark. T Iftner: institutional research grants from Gen-Probe, GSK, Hologic , Roche, and Sanofi Pasteur MSD. SK Kjaer: travel and research grants from Merck and Sanofi Pasteur MSD, and institutional research grants from Merck and Sanofi Pasteur MSD.
135

CANCER DETECTION BY PRIMARY HPV TESTING IN OLDER WOMEN
J Peto, London School of Hygiene and Tropical Medicine, London, UNITED KINGDOM C Gilham, London School of Hygiene and Tropical Medicine, London, UNITED KINGDOM M Almonte, Wolfson Institute for preventive Medicine, London, UNITED KINGDOM A Sargent, Central Manchester University NHS Foundation Trust, Manchester, UNITED KINGDOM A Bailey, Central Manchester University NHS Foundation Trust, Manchester, UNITED KINGDOM H Kitchener, University of Manchester, Manchester, UNITED KINGDOM Background: CIN3 detection rates in ARTISTIC and in other trials show that primary HPV testing is at least as sensitive as cytology, and suggest that the screening interval can be safely increased following a negative HPV test. However, in older women CIN3+ cases are rare and invasive cancers constitute a substantial proportion of them. Early detection of cancer may be more important than CIN3 detection when screening older women. Objective: The most appropriate HPV test for cancer detection needs to be defined, particularly in older women. Methods: Invasive cervical cancers and CIN3s in the ARTISTIC Trial (24,510 women, including 5591 aged 50 or over) were identified through national cancer registration during a mean follow-up 6.5 years. Results: 429 CIN3s and 17 cervical cancers were diagnosed, including 13 CIN3s and 4 cervical cancers aged 50 years or over. The proportion of CIN3+ cases that were invasive cancers in this screened cohort was 4% (6/164) aged 3039, 12% (7/59) aged 40-49 and 24% (4/17) aged 50-69. The corresponding proportions in England in 2008 were 7% aged 30-39, 13% aged 40-49 and 35% aged 50-69. Entry LBC samples for 5/17 (29%) cancers were HC2 negative, and 4 of these were also tested with the Roche Line Blot Assay. HPV16 was detected in the 2 younger cases (aged 28 and 36 at entry), but the 2 older cases (aged 45 and 53 at entry) were HR-HPV negative. Four cancers remained cytology negative, and were diagnosed solely through HPV testing. Conclusion: Our results suggest that a more sensitive PCR-based assay may be required to detect invasive cancer. The screening histories and histologies of cancers arising in this and other trials should be reviewed and the results pooled to obtain better evidence on this small but important subset of women.
P-17.34
ACCEPTABILITY OF CERVICAL CANCER SCREENING IN INDIGENOUS AMAZONIAN WOMEN
M Blas, Universidad Peruana Cayetano Heredia, Lima, PERU I Alva, Universidad Peruana Cayetano Heredia, Lima, PERU C Carcamo, Universidad Peruana Cayetano Heredia, Lima, PERU P Garcia, Universidad Peruana Cayetano Heredia, Lima, PERU S Montano, NAMRU-6, Lima, PERU J Zunt, University of Washington, Lima, PERU Background: Cervical cancer is the leading cause of cancer death among Peruvian women. The percentages and factors associated with seeking and receiving cervical cancer screening (PAP smear) among indigenous Amazonian women have not been studied. Objectives: To determine the percentage of women who had Pap smears and the factors associated with uptake within the Shipibo-Konibo ethnic group Methods: A cross-sectional study to assess sexual and reproductive health behaviors and history of Pap smear utilization in 1,253 women, 15 to 39 years of age, members of the Shipibo-Konibo ethnic group living in Lima and in the Peruvian Amazon city of Pucallpa. Results: Overall, 41.3% (517) women had ever received a Pap smear. Of these women, 26.7% (138) did not return for their results. In the multivariate analysis, the probability of receiving a Pap nearly doubled (OR: 1.87, 95% CI: 1.68-2.09) for each 5 year increase in age. Women who completed post-high school education were more likely to have a Pap smear (OR: 1.63, 95% CI: 1.07-2.47), as were women who had a previous pregnancy (OR: 8.72, 95% CI: 4.53-16.79) or history of genital warts (OR: 2.09, 95% CI: 1.08-4.05). Conclusion: Cervical cancer screening via Pap smear is uncommon in indigenous Amazonian women. Efforts to promote Pap testing and increase awareness of the importance of following-up on test results could decrease the rate of cervical cancer among Shipibo-Konibo women, especially among younger, less-educated women.
136

EVALUATION OF HUMAN PAPILLOMAVIRUS TESTING IN CERVICAL CANCER SCREENING
T Maehama, Tomishiro Central Hospital, Tomigusuku, JAPAN M Azama, Tomishiro Central Hospital, Tomigusuku, JAPAN H Uechi, Tomishiro Central Hospital, Tomigusuku, JAPAN S Karibe, Tomishiro Central Hospital, Tomigusuku, JAPAN H Hamakawa, Tomishiro Central Hospital, Tomigusuku, JAPAN M Toma, Tomishiro Central , Tomigusuku, JAPAN Objective: To evaluate the performance of human papillomavirus(HPV) assays plus pap cytology for cervical cancer screening in Japan. Methods: Between December in 2010 and April in 2007, 12172 women who had pap smears for cancer cervical cancer screening. HPV testing plus Pap cytology was performed on 1390 women April in 2008 to December in 2010. HPV testing was performed in women who desired it with informed concent. Pap smears were evaluated at Tomishiro central hospital and diagnosed using the Bethesda syatem. HPV testing was performed using the Hybrid capture II HPV DNA Assay and PCR method. HPV DNA typing was performed using direct sequencing of PCR products. Results: Pap smear abnormalities were observed in 0.76% (93/12172) April in 2007 to December 2010. HPV DNA was detected in 7.0%(97/1390). The prevalence rates of HPV in the group aged 20-29, 30-39, 40-49, 50-59, 60 years upwards were 20.0%,8.2%,4.6%,6.4%,8.1%,respectively. The women with HPV positive-but normal cytology were detected in 91 cases and 82 cases of them were able to be examined for coloposcopy and biopsy. 27 cases of them were diagnosed as cervical intraepithelial neoplasia (CIN1:14, CIN2:10,CIN3:3) finally. Conclusion: We found CIN cases in 32.9%(27/82) of HPV positive-cytology negative cases. Our study suggests that HPV testing could be an effective way to improve the performance of cervical cancer screening.
P-17.36
HPV NEGATIVE AT BASELINE: RISK OF SUBSEQUENT ABNORMAL SMEARS
K U Petry, Klinikum Wolfsburg, Wolfsburg, GERMANY G Bhmer, Wagner, Stibbe, Bad Mnder, GERMANY T Iftner, University of Tbingen, Tbingen, GERMANY Background/Objectives: A negative HPV test should exclude almost any risk for cervical cancer for 6 years and allow for increased screening intervals. In theory shorter intervals should increase the rate of false positive screening results without any improvement of cervical cancer prevention but this was not examined systematically. Methods: 3,389 women (30-65 yrs) who were tested negative for HR-HPV (HC2) and had normal Pap smears at baseline were followed for 5 years with annual Pap smears within the German cervical cancer prevention program and had at least one follow-up smear. At study end a random sample of 350 women were retested with HC2 and invited for colposcopy. Results: 1178 (27.81%) double negative participants attended for 4 or more annual Pap smear screening rounds during the 5-year follow-up in accordance to the German screening program. The risk of atypical Pap smear findings ranged from 2.2% to 3.03% per screening round. The accumulated risk of receiving at least one atypical Pap smear was 12.8% in women who attended for 5 subsequent annual screening visits. However, cases of CIN2+ were neither reported in the complete double negative group nor found among 96 randomly selected women undergoing colposcopy at study entry and another 296 participants at study end. Conclusion: Pap smear screening of HPV negative women is associated with a high rate of false positive cytology results. Screening intervals of less than 3 years should be discouraged in HPV negative women
Declaration of interest K.U.P. received speakers honorarium from Qiagen
137

CAN PRIMARY HPV SCREENING PREVENT CANCERS OF VULVA AND VAGINA?
A Luyten, Klinikum Wolfsburg, Wolfsburg, GERMANY K U Petry, Klinikum Wolfsburg, Wolfsburg, GERMANY Background/Objectives: Primary HPV screening improves the prevention of cervical cancer in women who are 30 yrs or older. As the majority of VIN and VaIN is associated with HR-HPV, primary HPV testing could additionally help to prevent cancers of vulva and vagina. Methods: Within the Wolfsburg HPV screening pilot project, all women transferred to colposcopy underwent a careful evaluation of cervix, vagina and vulva using acetic acid and if indicated Lugols solution. Any suspicious lesion underwent histological assessment. Results: 19,675 women were recruited between 2006 and 2011in the pilot project. During the 5 year follow-up 653 participants were transferred to colposcopy because of HPV persistency or abnormal Pap smears & positive HC2 results. 158 women were diagnosed with CIN3 or cervical cancer, while 10 patients were found to suffer from cancer of the vulva or high-grade VIN / VaIN. 5/10 external lesions were associated with CIN3+ while the remaining patients had no cervical disease or CIN1. Only 3/10 patients showed atypical Pap smears. Conclusions: 1.53% of all HPV+ women transferred to colposcopy were diagnosed with high-grade lesions of vagina, vulva or anus. Apart from an increased detection rate of CIN3+, HPV screening allowed for an improved diagnosis of VIN/VaIN and may prevent cancers of vagina and vulva.
P-17.38
INFLUENCE OF REVEALED HR-HPV STATUS ON PAP CYTOLOGY TRIAGE PERFORMANCE
L A Richardson, McGill University, Montreal, CANADA A V Ramanakumar, McGill University, Montreal, CANADA E L Franco, McGill University, Montreal, CANADA for the PEACHS Study Consortium Background: Evidence is mounting for primary Human Papillomavirus (HPV) DNA screening followed by Pap cytology triage. Since Pap interpretation is subjective, revealing HPV positivity could influence detection of cytological abnormalities. We simulated this triage algorithm within three separate datasets and assessed the influence of revealing HPV DNA status on cytotechnician smear readings. Objectives: To determine if diagnostic performance of Pap cytology is superior when HPV status is revealed versus concealed. Methods: We conducted a post-hoc analysis of previously collected cervical slides and clinical data from (A) the Canadian Cervical Cancer Screening Trial (CCCaST), (B) a screening study from the Democratic Republic of Congo, and (C) the Brazilian Investigation into Nutrition and Cervical Cancer Prevention (BRINCA). Cervical slides were obtained and re-read with knowledge of HPV status for all HPV-positive and a control sample of HPV-negative women. Where appropriate, verification bias was corrected for. Results: A total of 1767 smears were re-read. Among 915 re-reads for HPV-positive women, contrast between revealed and concealed Pap readings demonstrated upgrades from negative to positive for n=109 and n=124 at cut-offs ASC-US and LSIL, respectively. Re-reads led to increases in false-positives which reduced Pap re-read specificity. At disease endpoint CIN2+, specificity significantly declined at ASC-US cut-off for studies (A) 86.6% to 75.3% and (C) 42.5% to 15.5%, and at LSIL cut-off for study (C) 61.9% to 37.6%. Sensitivity remained nearly unchanged between readings, with an exception in study (C) where re-read performance was superior (91.3% vs. 71.9%, LSIL cut-off ). Overall findings were similar for disease endpoint CIN3+. Conclusions: We observed declines in Pap specificity when HPV-positivity was revealed, possibly due to heightened awareness of potential abnormalities. Utility of Pap cytology in triage context will require strict quality control adherence and performance may benefit from a higher threshold cut-off (e.g. LSIL).
Declaration of interest Consultant to Merck, GSK, Qiagen, Roche, Gen-Probe, Ikonisys and Cytyc. Grant support from Merck.
138

CERVICAL LESION RISK PREDICTION WITH HPV DNA TESTING AND CYTOLOGY
M Chevarie - Davis, McGill University, Montreal, CANADA A V Ramanakumar, McGill University, Montreal, CANADA S Ferreira, Ludwig Institute for Cancer Research, So Paulo, BRAZIL A Ferenczy, Jewish General Hospital, Montreal, CANADA L L Villa, Ludwig Institute for Cancer Research, So Paulo, BRAZIL E Franco, McGill University, Montreal, CANADA Background: Although many studies have compared human papillomavirus (HPV) DNA testing to cervical cytology for the detection of pre-invasive lesions of the cervix, most used cytology as an endpoint, thus being subject to outcome misclassification. Objectives: Our objective was to use the gold standard of histopathology to ascertain lesion outcomes in a repeated-measurement, longitudinal study of HPV and cytology testing. Methods: A cohort study was conducted in Brazil and enrolled 2462 women for interviews, cervical cytology, cervicography, and HPV testing. HPV DNA testing and cytology were performed every 4 months in the first year and then every 6 months. Detection of high-grade lesions on cytology or cervicography prompted referral to colposcopy. The specificity, sensitivity and predictive values of HPV testing and cytology, both cross-sectionally and as repeated longitudinal measurements, were calculated. Time-to-event analyses using Kaplan-Meier plots and Cox regression were performed comparing the long term predictions of different screening modalities. Results: Amongst the 344 women who underwent biopsies, the risk of cervical intra-epithelial neoplasia (CIN) grade 2 or worse was 11 times higher (95%CI: 5.7-23.7) when cytology results were of atypical squamous cells of undetermined significance (ASCUS) or low grade squamous intra-epithelial lesion (SIL) as compared to negative cytology, and 17 times higher (95%CI: 8.7-31.8) when cytology showed high grade SIL. When HPV DNA testing identified high risk HPV, the risk of CIN 2 or greater was 10 times higher (95%CI: 4.5-24.1) as compared to cases with absent or strictly low risk HPV. The combination of both screening tests demonstrated that the addition of HPV testing when cytology results were negative or ASCUS was beneficial, whereas this was of no added value if cytology detected SIL. Conclusions: These results emphasize the importance of considering both HPV DNA testing and cytology for developing an optimal primary screening model.
Declaration of interest M Chevarie-Davis: None declared. AV Ramanakumar: None declared. S Ferreira: None declared. A Ferenczy: None declared. LL Villa: None declared. E Franco: GSK, Consultant. Quiagen, Consultant. Roche, Consultant. Gen-Probe, Consultant. Ikonisys, Consultant. Cytyc, Consultant. Merck, Consultant and Grant support.
P-17.40
CERVICAL CANCER SCREENING AMONG HIV-INFECTED WOMEN
I HEARD, HPV National Reference Centre, Pasteur Institute, 25 rue du Dr Roux, 75015, Paris. INSERM U943, F-75013 PARIS, FRANCE. Groupe Hospitalier Piti-Salptrire, Inserm U943, Paris, F-75013 France, Universit Pierre et Marie Curie, Paris 06, UMR S943, Paris, France , Paris, FRANCE V POTARD, Inserm U943 Paris, F-75013 France, 3 UPMC Univ Paris 06, UMR S943, Paris, F-75013 France, PARIS , FRANCE H CUBIE, Scottish HPV Reference Laboratory, Royal Infirmary of Edinburgh, 51 little France Cres, Edinburgh EH16 4SA, Edimburgh, Scotland, UK D COSTAGLIOLA, AP-HP, Groupe Hospitalier Piti-Salptrire, Service de maladies infectieuses et tropicales, Inserm U943 Paris, F-75013 France, 3 UPMC Univ Paris 06, UMR S943, Paris, F-75013 France, PARIS, FRANCE Background: The cervical cancer incidence among HIV-infected women remains high since the introduction of highly active antiretroviral therapy (HAART) in 1996. Data are still missing to determine appropriate cervical cancer screening guidelines in HIV-infected females. Objectives: To determine the incidence for squamous intra-epithelial lesions (SILs) in HIV infected women with normal cytology by baseline CD4 cell counts and HPV test results. Method: The VIHGY Study is a French observational cohort study of HIV-infected women. At each visit, patients provided cervical swabs for cytologic examination and HPV detection. Hybrid Capture 2 (HC2) was used for HPV testing with HPV linear array for genotyping of screen positive samples. Results: Results from the first 635 enrolled women were analysed. The overall prevalence of abnormal cytologic findings at baseline was 18% with 12.5% of low-SILs and 5.5% of High (H)-SILs. The rate of infection with High Risk (HR)-HPV genotypes was 23% in normal Pap test and 100% in H-SILs. Among patients with normal cytology, incidence rate of CIN2+ in patients infected with HPV16 was 9,4 (2,3- 37,5), significantly higher than in HC2 negative patients (0,6 (0,2-1,9), p=0.003); incidence rate was higher, although not significantly, than in HC2 positive patients infected with other HPV types (1.7 (0.4-6.6), p=0.08). The association of CD4 cell level with incident CIN2+ was not significant. Conclusion: In our study, 1 in 5 women had cytologic abnormalities at baseline. The use of genotyping in HCII+ patients may not improve the selection of patients at higher risk of developing CIN2+. The best strategy to screen HIV-infected women remains challenging.
139

START-HPV: FRENCH HPV-BASED PRIMARY SCREENING PILOT PROGRAM FOR CERVICAL CANCER
V DALSTEIN, CHU de Reims (University Hospital), REIMS, FRANCE, E ROUSSELOT - MARCHE, SAC08 (Ardennes' Cancer Society), CHARLEVILLI-MEZIERES, FRANCE, B CHARLIER, SAC08 (Ardennes' Cancer Society), CHARLEVILLI-MEZIERES, FRANCE, J BOTOKEKY, SAC08 (Ardennes' Cancer Society) , CHARLEVILLI-MEZIERES, FRANCE, E MEREB, CHR de Charleville (Regional Hospital), CHARLEVILLI-MEZIERES, FRANCE, O GRAESSLIN, CHU de Reims (University Hospital), REIMS, FRANCE, N JOVENIN, ONCOCHA (Regional Cancerology Network), REIMS, FRANCE, P BIREMBAUT, CHU de Reims (University Hospital), REIMS, FRANCE, The START - HPV Study Group, , , FRANCE, C CLAVEL, CHU de Reims (University Hospital), REIMS, FRANCE Background: New cervical screening algorithms need to be tested in the real life before implementation as national screening programs. The INCa (French National Institute of Cancer) is supporting the first French primary screening pilot study using solely HPV testing for 31-65 year-old women and cytology for 25-30 year-old women, referred to as START-HPV (STudy of primary screening in the ARdennes department by Testing for HPV infection). Objectives: To determine in a general population: (i) the optimal management of women tested HPV positive; (ii) the compliance for an extended screening interval (3 to 5 yrs) between 2 negative HPV tests; (iii) the feasibility of a distinct screening strategy according to age. Methods: 50,000 women aged 25-65 years leaving in the Ardennes Department, who did not get a Pap smear since more than 3 years, will be invited to participate in the screening program by mail. Non-responders will receive a second and third invitation. At the third invitation, an auto-sampling device will be proposed for HPV testing (DelphiScreener). Women aged 25-30 yrs will be screened using cytological testing. Women aged 31-65 years will be screened using solely HPV testing (Hybrid Capture 2) using STM or DelphiScreener devices. Cytology positive women will be managed according to the French national guidelines. HPV+ women will be triaged by cytology and HPV genotyping will also be performed. Women with normal or negative tests will be recalled at 3 or 5 years, according to the primary screening test, cytology or HPV respectively. Conclusion: The detailed methodology of the study will be presented. The START-HPV pilot study will provide a considerable amount of data concerning the organization, the management of HPV+ women and the education and feedback of physicians and population. These data will serve for the guidance of further implementation of large-scale HPV-based screening programs.
P-17.42
ESTIMATION OF TEST PERFORMANCE WITH MISSING TRIAGE AND COLPOSCOPY RESULTS
P Sasieni, Centre for Cancer Prevention, London, UNITED KINGDOM S Pineda, Centre for Cancer Prevention, London, UNITED KINGDOM M Almonte, Centre for Cancer Prevention, London, UNITED KINGDOM Introduction: The inevitable implementation of HPV primary screening demands the use of a triage test before full evaluation or treatment. Results of the triage test could be missing because of insufficient sampling, nonattendance to a second visit or simply because they are not yet available. Ignoring missing results of triage tests and of disease status leads to incorrect estimation of sensitivity and specificity of HPV testing. Objective: To evaluate a new method to estimate test performance in the presence of missing triage results and disease status. Methods: We did 10,000 simulations of a trial of 5435 women screened with liquid-based cytology (LBC), HPV testing and VIA, followed by magnified triage of positive VIA (VIAM). VIA_M is the combination of VIAM in VIA positive women. VIAM was not done on some VIA positives and disease status was not available on all women with an abnormal screen. Our method allows all possible outcomes for the missing data and assigns probabilities to these hypothetical outcomes. We first imputed missing VIAM values using logistic regression based on LBC and HPV results and disease status (when not missing). Next we imputed missing disease by a logistic model based on screening results. Within each simulation, 100 bootstraps samples were used to estimate confidence intervals. Estimated sensitivities and specificities were compared to those obtained from simulations with no missing values. Results: In the absence of missing data the sensitivities were: 50.4% for VIA_M and 91.7% for HPV. Ignoring the missing data lead to estimated sensitivities of 62.9% (VIA_M) and 90.8% (HPV). Our weighted imputation approach yielded estimated sensitivities of 46.3% for VIA_M and 92.9% for HPV. The bootstrap confidence intervals performed well. Conclusions: Appropriate adjustment for missing triage results and incomplete verification of disease is essential and can be done using this weighted imputation approach.
140

HPV INFECTION IN ADOLESCENTS AND UNIVERSITY STUDENTS FROM PORTUGAL: SELF-SAMPLING
J Ribeiro, Virology Service, Portuguese Institute of Oncology of Porto, Porto, PORTUGAL J Silva, Molecular Oncology Group; Portuguese Institute of Oncology of Porto, Porto, PORTUGAL H Sousa, Virology Service, Portuguese Institute of Oncology of Porto, Porto, PORTUGAL T Osrio, Portuguese League Against Cncer, Ncleo Regional do Norte, Porto, PORTUGAL R Medeiros, Virology Service, Portuguese Institute of Oncology of Porto, Porto, PORTUGAL Background: Human papillomavirus (HPV) is one of the most common sexually transmitted infections worldwide; nevertheless, few studies have been performed regarding the characterization of HPV infection in adolescents and university students. Self-sampling has been referred as a useful tool for the molecular characterization of HPV prevalence. Objectives: The aim of this study was to evaluate self-sampling as a tool for the characterization of HPV infection in young non-symptomatic women. Material and Methods: This study intends to characterize HPV infection in exfoliated cervical cells obtained by selfsampling from 435 young women (aged from 14 to 30 years old) resident in the northern region of Portugal. HPV infection was detected by PCR using two sets of consensus primers (MY09/11 and GP5+/6+) and genotyping was performed by restriction fragment length polymorphism (RFLP). Result: The overall frequency of HPV infection was of 11.5% among all women and 16.6% in sexually active women. High-risk genotypes were among the most frequently found among all women and genotypes 31, 16, 53 and 61 were the most represented. Moreover, our study identified median age of 17 years old (OR=3.56; p=0.001), the number of lifetime sexual partners (OR=4.50; p<0.001), and years of sexual activity (OR=2.36; p=0.008) as risk factors for HPV infection acquisition. Conclusion: Our study revealed that HPV infection is common in young asymptomatic women with age over 17 years old, having 2-5 sexual partners and over 2 year of sexual activity. Results also showed that HPV detection might be performed efficiently in self-collected samples. The exact knowledge of the HPV profile in young women may be important to assess the vaccination strategies within the different populations.
P-17.44
KNOWLEDGE AND PRACTICE OF PAP SMEAR IN WOMEN FROM BOLIVIA
B Alejos Ferreras, National Center of Epidemiology, Institute of Health Carlos III, Madrid, SPAIN C Tern Caldern, San Francisco Xavier of Chuquisaca University. School of Medicine, Sucre, BOLIVIA C Gonzalez Blzquez, National Center of Epidemiology, Institute of Health Carlos III, Madrid, SPAIN D Gorena Urizar, Simon Bolivar Andean University, Health Area, Sucre, BOLIVIA J Lpez Gallardo, Research, Education and Services Center, CIES, La Paz, BOLIVIA M Ortiz Rivera, National Center of Microbiology, Institute of Health Carlos III, Madrid, SPAIN J Del Amo Valero, National Center of Epidemiology, Institute of Health Carlos III, Madrid, SPAIN Objective: To describe and analyze the knowledge and practice regarding PAP smear testing in women from Sucre, Bolivia, in 2010 Methods: Cross sectional study of randomly selected women aged 20-59 resident in Sucres districts. Women were invited to answer a questionnaire which inquired about socio-demographic variables and on knowledge and practice regarding PAP smear. We considered adequate knowledge if the women answered correctly the following questions; What is PAP smear for?, Where are PAP smears obtained from?, How often should you have your PAP smear done?. Adequate practice was assessed by asking Have you ever had a PAP smear done?. Descriptive analyses were performed and multiple logistic regression used to study associations with the outcomes. Results: Of 1563 women, inadequate knowledge was present in 59% (95%CI 56.6-61.6) and inadequate practice in 33% (95%CI 30.7-35.4). Factors associated with inadequate knowledge were educational level [OR 2.07 (95%IC 1.58-2.71) for low educational level compared to high] and a negative perception of the need for PAP smear [OR 2.94 (95%CI 1.88-4.59)]. Factors associated with inadequate practice were age [20-29 vs 30-59 OR 4.96 (95% CI 3.84-6.41)], inadequate knowledge of PAP smear [OR 1.37 (95%CI 1.06-1.77)], low educational level [OR 2.45 (95%CI 1.31-4.59) compared to high level], negative perception of the need of PAP smear [OR 2.81 (95%CI 1.82-4.34)], previous sexual transmitted infections [OR 0.39 (95%CI 0.25-0.62)], information of PAP smear gained through health service [OR=0.35 (95%CI 0.27-0.48)] compared to that obtained from radio/TV. Conclusion: The proportions of women with poor knowledge regarding PAP smear and who have never had a smear done are very high. Future interventions need to improve the knowledge on PAP smear and raise awareness in order to increase uptake of PAP smear, especially in young women of low educational level. These actions should be led by the healthcare service.
141

HPV DNA VERSUS MRNA TESTING IN TRIAGE AMONG NORWEGIAN WOMEN
J Lmo, Oslo University Hospital, Oslo, NORWAY K Lie, Oslo University Hospital, Oslo, NORWAY T Sauer, Oslo University Hospital, Oslo, NORWAY E Morland, Norchip AS, Oslo, NORWAY H Skomedal, Norchip AS, Oslo, NORWAY Background: In 2005 national guidelines for secondary HPV testing were implemented in Norway among women aged 25-69 with a diagnosis of inadequate, ASC-US or LSIL cytology. A repeat smear and HPV testing was recommended within 6 months. The HPV test used should be validated and CE-marked. We have investigated the impact of three commercially available HPV assays on detecting CIN2+ during 2005-2009. Methods: An observational, population based study of 21958 women with an index cytology of ASC-US, LSIL or inadequate. The women were tested with HC II, Amplicor or PreTect HPV-Proofer which detects full length mRNA transcripts from HPV 16,18,31,33 and 45. The follow-up data included HPV results, cytology and histology from five different sources at the Cancer Registry of Norway until December 2009. Women were excluded if concomitant cytology and HPV test were not present (N=617), if CIN2+ was diagnosed before the HPV test was taken (N=21) or if women were tested with more than one type of HPV test (N=268). In total 21052 women were included in the study. 1249 were tested by HC II, 5687 by PreTect HPV-Proofer and 5116 by Amplicor. The women were split into five diagnostic groups: 1) negative HPV test and normal cytology, 2) negative HPV test and inadequate/ASCUS/LSIL cytology, 3) positive HPV test and low-grade cytology, 4) high-grade cytology and 5) positive HPV test and normal cytology. Group 1-2 were referred back to screening after three years. Group 3 - 4 were immediately referred for colposcopy and biopsy and group 5 was referred for colposcopy after 12 months if HPV persisted. Results: Predictive values for the different HPV tests for detecting CIN2 versus CIN3+ will be presented for women aged 25-34 and 35-69. Cost-effectiveness of HPV testing in triage will be discussed.
Declaration of interest Norchip AS is the manufacturer of one of the HPV tests (PreTect HPV-Proofer) in this study.
P-17.46
HPV-MRNA TESTING OF SELF-COLLECTED SPECIMENS TO DETECT CERVICAL LESIONS, KENYA
J Ting, University of North Carolina, Chapel Hill, UNITED STATES, N Mugo, University of Nairobi/Kenyatta National Hospital, Nairobi, KENYA, J Kwatampora, University of Nairobi/University of Manitoba, Nairobi/Manitoba, KENYA/USA, C Hill, GenProbe Incorporated, San Diego, USA, A Schroeder, Gen-Probe Incorporated, San Diego, USA, M Chitwa, University of Nairobi/ University of Manitoba, Nairobi/Manitoba, KENYA/USA, S Patel, University of North Carolina, Chapel Hill, UNITED STATES, H Gakure, University of Nairobi/University of Manitoba, Nairobi/Manitoba, KENYA/USA, J Kimani, University of Nairobi/ University of Manitoba, Nairobi/Manitoba, KENYA/USA, J Smith, University of North Carolina, Chapel Hill, UNITED STATES Background: Data on the performance of self-collection for HPV testing informs its usefulness as a potentially rapid method of cervical cancer screening. Objectives: To determine the prevalence, and the sensitivity and specificity of high-risk(hr) HPV-mRNA in self- and in physician-collected samples for the detection of HSIL. Methods: A crosssectional analysis was conducted of 295 female sex workers in Nairobi. Participants conducted self-collection in privacy using a brush, according to standardized instructions. A physician conducted a pelvic exam to obtain cervical cell samples. Samples were tested for hrHPV-mRNA using APTIMA HPV Assay (Gen-Probe Incorporated). Cervical cytology was conducted using physician-collected samples. Women with cytological HSIL underwent biopsy. Blood samples were obtained for HIV testing. Results: Among 295 women (mean age 29.5 years), hrHPVmRNA prevalence was similar in self- and physician-collected samples (28.8% and 29.8%, respectively). Overall, 24.7% of participants were HIV-seropositive, 79.9% had normal cytology, 4.1% ASCUS/AGUS, 11.5% LSIL and 4.8% HSIL/SCC. HPV prevalence in CIN 2 was similar in self- and physician-collected samples (90%). HPV prevalence in normal cytology was lower (22.1% self-;24.3% physician-collection) than in ASCUS/AGUS (66.7%;50.0%), LSIL (41.2%;38.2%) or HSIL/SCC (78.6%;85.7%). Sensitivity of self- and physician collection for HSIL (n=14) were similar; hrHPV-mRNA testing detected 1 less HSIL by self-collection than physician-collection (11/14;12/14). Specificity estimates were also comparable (73.7%;73.0%). Self-collection had a similar specificity in age strata (<30, 30 years), yet sensitivity appeared somewhat lower in women <30 years (71.4%) than in women 30 years (85.7%) (p=0.5). In physician-collection, sensitivity and specificity estimates were similar in women of both age-groups. Among 73 HIV-seropositive women, sensitivity for detection of HSIL (n=10) appeared more sensitive (100% in selfand physician-collection), albeit less specific (61.9%;65.1%) than the overall cohort. Conclusion: In a low-resource setting, hrHPV-mRNA testing of self-collected samples appears valid and is potentially a reasonable choice given the alternative of VIA or other less effective options.
Declaration of interest Craig Hill and Astrid Schroeder are full time employees of Gen-Probe Incorporated, San Diego Jennifer S. Smith has received research grants and consultancies from Genprobe, Qiagen, and Hologic within the last three years.
142

HPV TESTING AS AN ALTERNATIVE SCREENING TOOL IN TAIWAN
C Ho, National Taiwan University, Taipei City, TAIWAN Y Chen, National Taiwan University, Taipei City, TAIWAN S You, Academia Sinica, Taipei City, TAIWAN S Hsiao, Bureau of Health Promotion, Taipei City, TAIWAN S Koong, Bureau of Health Promotion, Taipei City, Taiwan S Chiou, Bureau of Health Promotion, Taipei City , Taiwan C Chen, National Taiwan University, Taipei City, Taiwan C Chen, Academia Sinica, Taipei City, Taiwan Background: To increase screening participation in Taiwan, women who aged 36 and did not attend past 6 years national screening program would be invited to attend self-sampled HPV testing in 2010. Objectives: The study aimed to assess participation to HPV self-sampling or Pap smear screening, screening positivity, and follow-up attending among women did not attend screening for 6 years. Methods: Self-sampled specimens were tested for HPV by Hybrid Capture 2 (HC2), and the testing results were mandatory to report to a centralized screening registry in Taiwan. Women detected HC2 positive would be recommended to receive Pap smear, and the subsequent managements were according to cytologic results. Screening participation and screening results were all obtained from the centralized screening registry database. Results: A total of 176,004 target women attended screening, and 13.6% of them participated in HPV self-sampling program. Proportion of never-screenees attended HPV screening (53.0%) was more than those attended Pap smear (40.2%). Compared with participation of the target women in 2008-2009 (7.7%), HPV program contributed 0.8% more in 2010 (8.5%). More women whose age >=50 (66.1% vs. 50.2%) or schooling year <6 (40.7% vs. 29.7%) attended HPV screening. The HC2 positivity was 7.9% among women attended HPV screening, 47.6% of HC2 positives followed by Pap smear, and detected 18.0% ASCUS+ cytologic abnormalities, which was 4.9 fold found in women attended Pap smear only (3.7%). Conclusion: In the first year of nationwide program of HPV testing from self-sampled specimens targeted at women did not attend screening for at least 6 years may encourage elder and less educated women to participate in screening program. To increase follow-up proportion among women detected HC2 positive is the major goal in the near future.
P-17.48
WOMEN'S PREFERENCES LEADING TO NEW SELF SAMPLE DEVICES
R Bosgraaf, Radboud University Nijmegen Medical Centre, Nijmegen, NETHERLANDS N van der Veen, National Institute for Public Health and the Environment, Bilthoven, NETHERLANDS R L M Bekkers, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands Objective: Vaginal self-sampling forms the ideal sampling method for population based HPV detection within a national cervical cancer screening programme. The development of vaginal self-samples has been investigator driven, without taking womens opinions into consideration. This study therefore investigates womens preferences for vaginal self-sampling. Methods: Qualitative research methods were used consisting of structured interviews of 12 women regarding two different self-sampling devices; vaginal lavage with the Delphi screener, and the Viba brush with FTA cartridge. Of these women, 6 were regularly screened (responders), and 6 had refused screening thus far (non-responders). Conclusions: Both subgroups indicated that they would participate in screening if self-sampling was introduced. The ideal self-sampling device should be packed in a small box, sent by regular mail, easy to open, contain clear instructions (with cartoons) with reference to a website/telephone number for further information. The device itself should be thin (< 1 cm) with a soft round tip, indicates clearly when it is inserted far enough, with maximal one additional instruction to take the sample. After sampling the device should be put in a box/envelop, without further handling, in order to be sent by regular mail for further analysis. In conclusion, women are very motivated to partake in screening using self-sampling. The sample itself has to be woman friendly, with clear instructions and minimal handling. On the basis of this study, new devices have been developed and will be shown during the congress.
143

SELF-SAMPLING HPV TESTING PILOT PROGRAM IN 11 COUNTIES IN TAIWAN
Y Chen, National Taiwan University, Taipei City, TAIWAN S You, Academia Sinica, Taipei City, Taiwan C Ho, National Taiwan University, Taipei City, Taiwan L Shih, Bureau of Health Promotion, Taipei City, Taiwan S Koong, Bureau of Health Promotion, Taipei City, Taiwan C Chen, Academia Sinica, Taipei City, Taiwan C Chen, National Taiwan University, Taipei City, Taiwan Background: Taiwan national cervical cancer screening for 30 years old women has been implemented since 1995. Lower participation has hindered the efficacy improvement and hence self-sampled HPV testing pilot program had implemented during 2005-2008 in 11 counties. Objectives: The study aimed to investigate the screening participation, HPV prevalence and CIN2+ lesions detection among women received HPV testing, Pap smear, and both tests. Methods: Women who did not attend screening during 1995-2004 were targeted to the program. Their attendances of HPV testing, Pap smear or both were identified from centralized screening databases. HPV testing was carried out by Hybrid Capture 2. Follow-up by Pap smear or biopsy after an abnormal screening was obtained, and CIN2+ diagnosis was ascertained from cervical biopsy and cancer registry. Results: A total of 9,671 women participated in the program, included 1,438 women received both HPV test and Pap smear, and 8,233 women received HPV test, and there were 75,106 women who had never attend screening received Pap smear in 2005. More >50 years old (68.7%) and less uneducated (8.4%) women received HPV test than those received Pap smear only (53.6% and 30.0%, respectively). Hybrid Capture 2 positivity was 9.0% and 7.5% in women received both tests and HPV test only; while cytological ASCUS+ was 2.99% and 3.25% in women received both tests and received Pap smear only, respectively. The proportion of CIN2+ lesions detected within half year after screening was 1.4%, 0.47%, 0.65% among women received both tests, HPV test only, and Pap smear only; while proportion of these lesions detected after half year of screening was 0.14%, 0.28%, and 0.36%, respectively. Conclusions: HPV testing facilitated to detect more CIN2+ lesions and to decrease subsequent CIN2+ occurrence in women unwilling to attend Pap smear screening previously.
P-17.50
QUANTITATIVE RT-PCR ANALYSIS IN LIQUID BASED SPECIMENS IN CERVICAL SCREENING
J Ordi, Hospital Clinic. University of Barcelona, Barcelona, SPAIN, M del Pino, Hospital Clinic. University of Barcelona, Barcelona, SPAIN, I Alonso, Hospital Clinic. University of Barcelona, Barcelona, SPAIN, P Castillo, Hospital Clinic. University of Barcelona, Barcelona, SPAIN, A Torne, Hospital Clinic. University of Barcelona, Barcelona, SPAIN, L Marimon, Hospital Clinic. University of Barcelona, Barcelona, SPAIN, J Gaber, Cepheid , Bronma, SWEDEN, D H Persing, Cepheid, Sunnyvale, U.S.A., C Svanholm-Barrie, Cepheid, Bronma, SWEDEN Background: The detection of cellular proteins overexpressed by HPV-infected cells is a possible alternative for cervical cancer screening. p16 immunostaining correlates with the severity of cyto-histological abnormalities, but shows some interobserver variability. Objective: To evaluate quantitative reverse transcriptase PCR as an alternative tool to detect overexpression of different biomarker candidates for transforming HPV infections in liquid based cytology specimens. Material and Methods: Liquid PAP specimens (PreservCyt, Hologic) from 129 patients (48 with CIN2-3, 51 with CIN1 and 30 without lesion) were analyzed. 5 mL of suspension were centrifuged and the pellet was mixed with QIAzol lysis reagent (Qiagen). Total RNA was extracted using RNeasy kit (Qiagen). Reverse transcription was performed with 10 L total RNA in a 20 L reaction volume using random hexamers and the High Capacity cDNA RT-kit (Applied Biosystems). The expression of CDKN2A (p16), MKI67 (Ki67), BIRC5, MCM5, TOP2A and MMP9 was analyzed. PKG1 and GUSB were used as reference genes. Expression data were analyzed as Ct (Ct target Ct (mean PKG1 + GUSB)). A ROC analysis was performed . Results: Total RNA yield varied between 0.5g 64,3g, reflecting variability of cell numbers in the specimens, but 91.5% of the samples were considered as adequate and only 8.5% were excluded due to low expression of reference genes and/or low RNA yield. Delta Ct values expression below 1.7241 for CDKN2A and below 4.22 for TOP2A identified CIN2-3 with a sensitivity of 75.6% and 80.5% and a specificity of 74.1% and 75.3% respectively. The combination of CDKN2A and TOP2A had a sensitivity of 82.9% and a specificity of 84.4%. Conclusions: mRNA expression in liquid PAP specimens is feasible and could be used for cervical screening purposes. The combination of CDKN2A and TOP2A is a good marker to separate premalignant from normal and low risk specimens.
Declaration of interest J. Ordi, Grant PI09/1084 Fondo de Investigaciones Sanitarias A. Torn, Grant PI09/1524. Fondo de Investigaciones Sanitarias J. Gaber, Cepheid, Employee D.H. Persing, Cepheid, Employee C. Svanholm-Barrie, Cepheid, Employee
144

SCREENING HISTORY DOES NOT MODIFY CERVICAL CANCER RISK IN CHILE
S Terrazas, Pontificia Universidad Catlica de Chile, Santiago, CHILE C Ibanez, Pontificia Universidad Catlica de Chile, Santiago, CHILE F Gonzalez, ontificia Universidad Catlica de Chile, Santiago, CHILE M T Urrutia, Pontificia Universidad Catlica de Chile, Santiago, CHILE C Ferreccio, Pontificia Universidad Catlica de Chile, Santiago, CHILE J Branes, Pontificia Universidad Catlica de Chile, Santiago, CHILE Background: Cervical Cancer Screening Program in Chile is based on Papanicolaou every three years, for women aged 25-64. The decrease in cervical cancer (CC) mortality has been attributed to this program. Nevertheless, CC is the first cause of Years of Life Lost in women in Chile and has a huge socioeconomic differential. Our aim was to evaluate the impact of the program in preventing CC in Santiago. Objectives: Compare screening history and CC risk factors in women with CC vs. healthy women. Methods: Population-based incident case-control study. Histologically confirmed CC cases entered in 2009-2010 in two hospitals of Santiago were compared with healthy controls matched by age and county of residence. Accepting women answered a questionnaire, administered in their households by trained personnel. Results: 79 cases and 233 controls were enrolled. Only 50% of cases and controls had a history of regular Pap. Cases and controls had no difference in years between Paps (5.8 vs. 5.9) and number of Paps for the woman's age (2.5 vs. 4.1 in < 30 years; 7.7 vs. 6.7 in 30-50 years; 9.9 vs. 9.3 in > 50 years). Cases compared with controls had significant less education (8.2 vs. 9.4, p=0.02), younger age at first sexual intercourse (18.1 vs. 19.6, p=0.001), younger age at first pregnancy (20.1 vs. 21.9, p=0.004), higher number of sexual partners before age 20 (1.1 vs. 0.7, p=0.01), more risk of having occasional partners (OR 2; 95% IC=1.06-3.86) and began to smoke at younger age (17.3 vs. 19.4, p=0.07). Conclusions: Cases and controls did not differ in Pap screening history; Pap was not a protective factor in this population. These results suggest that CC screening strategies must be reviewed and more sensitive tests should be incorporated to the National Prevention Program, where adherence is less than 50%.
P-17.52
ASC-US/SIL RATIO AND HPV RESULTS USING THE IMAGER SYSTEM
B Lloveras, Hospital del Mar, Barcelona, SPAIN F Alameda, Hospital del Mar, Barcelona, SPAIN I Soler, Hospital del Mar, Barcelona, SPAIN M Muset, Hospital del Mar, Barcelona, SPAIN M Bosch, Hospital del Mar, Barcelona, SPAIN L Pijuan, Hospital del Mar, Barcelona, SPAIN B Bellosillo, Hospital del Mar, Barcelona, SPAIN S Serrano, Hospital del Mar, Barcelona, SPAIN Background: The introduction of the Imaging system (Hologic) in cervical cytology is expected to diminish false negative results due to screening errors. The ASC-US/SIL ratio and the rate of HR-HPV positivity by HC2 (Qiagen) in ASC-US are considered useful indices for quality control in cervical cytology. Objective: To analyze the impact of the Imaging system in the ASCUS/SIL ratio and in the percentage of HPV positive results among ASC-US diagnosis. Material and methods: Liquid cytology (ThinPrep) was performed in all cases and we compared the results obtained before (2006) and after (2009) the introduction of Imaging assisted screening. HPV detection using HC2 was performed in 84% and 86% of the ASC-US cases respectively. Results: In year 2006, 19080 cervical samples were diagnosed using liquid cytology and manual screening, while in 2009 the same cytotechnologists using the Imager system screened 16821 samples. In the first period 282 (1,47%) ASC-US were diagnosed and in the second there were 699 (4,1%) ASC-US. In the first period the ratio ASC-US/SIL was 0.56 and in the second period it was 0.68. The rate of HC2 positive results in ASC-US was 70.5% in 2006 and 64.2% in 2009. HSIL following an ASC-US was detected in 19 (6.7%) patients from the first period and in 29 (4.14%) patients from the second one. Conclusions: The number of ASC-US after the introduction of the Imaging in screening increased by x 2.7. However, neither the ASC-US/SIL ratio nor the percentage of HPV positive cases changed significantly. The number of HSIL diagnosed after an ASC-US increased by x1.5. Imaging assisted screening seems an efficient tool in the detection of cervical cancer precursors.
Declaration of interest The presenting author has received support for attending conferences from Qiagen and Roche.
145

THE CLUE CELLS AND CLEANING OF HIGH RISK HPV INFECTION
L Geng, Peking University the Third Hospital, Beijing, CHINA Y Guo, Peking University , Beijing, CHINA K You, Peking University , Beijing, CHINA J Qiao, Peking University , Beijing, CHINA Objective: to investigate the correlation of high risk HPV infection and the bacterial infection in the vagina. Methods: totally 609 cases with high risk HPVDNA positive and abnormal cervical cytology (ASC-US 350, LSIL 259) were chosen, and 396 cases with high risk HPVDNA negative and normal cervical cytology as control. The liquid-based cytological test was applied and high risk HPVDNA was detected by Hybrid Capture II. The clue cells positive means the percentage of clue cells were above 20% on cervical smear. After 6-12 months the cases with high risk HPVDNA positive were followed. Results: The clue cell positive slides were 11.2% and 4.8% in both HPV positive and control groups, and much more clue cell positive slides were detected in the HPV positive group than that in control group (P<0.001). In the 6-12 months follow-up period, 343/609 (56.3%) cases still were high risk HPVDNA positive which were defined as persistent HPV positive in this study, the other 266 cases became HPV negative. The clue cell positive slides were 9.6% and 4.9% in persistent HPV positive and HPV negative cases respectively, the percentage of the clue cell positive slide in persistent HPV positive cases was higher than that in HPV negative cases (P<0.05), and the percentage of clue cell positive slides was much lower in the follow-up period than that in the beginning of the study (P<0.05). Conclusions: the results suggested that the women with HPV infection may be more susceptible for genital infection, e.g. bacterial vaginosis, and showed a strong association between HPV infection and bacterial vaginosis.
P-17.54
THE STUDY OF 361 CASES OF CERVICAL CYTOLOGY AS ASC-H
L Geng, Peking University the Third Hospital, Beijing, China K You, Peking University the Third Hospital , Beijing, China Y Guo, Peking University the Third Hospital, Beijing, China J Qiao, Peking University the Third Hospital, Beijing, China Objective: To explore the risk of CIN II or greater in patients with cytology interpreted as ASC-H. Study Design: Patients with ASC-H accepted HPV test, colposcopy-directed biopsy, and endocervical curettage. Surepath (TriPath) was used for cytologic diagnosis. Hybrid capture II was applied to detect HPV DNA. Results: 440 were diagnosed as ASC-H accounting for 0.7% of total cervical cytologic reports between Jan 2009 and Dec 2010. 361 patients accepted HPV testing , colposcopy-directed biopsy, and endocervical curettage. 47.1% (170/361) patients were diagnosed CIN II or greater, including 49 cases of CIN II,67 cases of CIN III, 6 cases of cervical squamous carcinoma, 1 case of cervical adenocarcinoma. 63.2% (168/266) patients with HPV positive were diagnosed CIN II or greater and 2.1% (2/95) in patients with HPV negative. Divided the patients into 30 years or younger group(group I) and older group(group II). In group I , the HPVDNA positive rate was 78.2% (54/69), and 39.1% (27/69) patients were diagnosed CIN II or greater, there is no invasive cancer. In group II,the HPVDNA positive rate was 72.6% (212/292), and 32.2% (94/292) patients were diagnosed CIN II or greater, there were 7 cases of invasive cancer. Conclusions: 47.1% patients with cervical cytology interpreted as ASC-H were diagnosed CIN II or greater and 2.4% were invasive cervical cancer. The negative prospective value of HPVDNA in this study were 97.9%(93/95) Key words: Cervical cytological examination, ASC-H, Cervical intraepithelial neoplasia, High-risk HPV.
146

THE STUDY OF 148 CASES OF CERVICAL CYTOLOGY AS AGC
K You, Peking University the Third Hospital, Beijing, CHINA L Geng, Peking University the Third Hospital, Beijing, CHINA Y Guo, Peking University the Third Hospital, Beijing, CHINA J Qiao, Peking University the Third Hospital, Beijing, CHINA Objective: To explore the risk of CIN II or greater in patients with cytology interpreted as atypical gland cells (AGC) Study Design: Patients with AGC accepted high-risk human papillomavirus (HPV) testing , colposcopy-directed biopsy, and endocervical curettage. Patients with irregular vaginal bleeding were accepted fraction diagnostic cuttege. Surepath was used for cytologic diagnosis. HPV DNA hybrid capture II was applied to detect for 13 most high-risk types of HPV DNA. Patients with CIN history or conducted hysterectomia were excluded. Results: Among 62801 patients in our diagnosis center between Jan 2009 and Dec 2010, 251 cases were diagnosed as AGC accounting for 0.4% of total cervical cytologic reports. 148 patients accepted high-risk human papillomavirus (HPV) testing , colposcopy-directed biopsy, and endocervical curettage. The average age was 44.8 years old. The HrHPV positive rate was 35.1%(52/148). 13.5% (20/148) patients were diagnosed CIN II or greater, including 7 cases of CIN II,5 cases of CIN III, 1 cases of cervical squamous carcinoma, 4 case of cervical adenocarcinoma and 4cases endometrial carcinoma. 28.8% (15/52) patients with HPV positive were diagnosed CIN II or greater and 7.3% (7/96) in patients with HPV negative. Conclusions: 13.5% patients with cervical cytology interpreted as AGC were diagnosed CIN II or greater and 3.3% were invasive cervical cancer and 2.7% were endometrial carcinoma. The negative prospective value of HPVDNA in this study were 97.9%(93/95). Key words: Cervical cytological examination, AGC, Cervical intraepithelial neoplasia, High-risk HPV.
P-17.56
THE STUDY OF CERVICAL CYTOLOGY IN PATIENTS WITH ENDOMETRIAL CARCINOMA
K You, Peking University the Third Hospital, Beijing, CHINA L Geng, Peking University the Third Hospital, Beijing, CHINA Y Guo, Peking University the Third Hospital, Beijing, CHINA J Qiao, Peking University the Third Hospital, Beijing, CHINA Objectives: Retrospective analyze the cervical cytology result from the patients with endometrial carcinoma preoperation. Methods: All the patient who had received operation for carcinoma of endometrial and taken a cervical Liquidbased cytology test pre-operation were enrolled our study. Results: There were 108 patients enrolled our study . 1.The average age of the group was 59.2 years old. 2.90.7% (98/108) of the cases were diagnosed as stage I by operation, 6.5% (7/108) stage II and 2.8% (3/108) stage III, respectively. 75% (81/108) were endometrioid endometrial adenocarcinoma; 9.3% (10/108) were uterine papillary serous carcinoma. 3.There were 48 cases (43.6%) with negative cytology result as negative of introepithelia lesion and malignant diseases .In this group there were 8 cases reports found uterin endometrial cell in women over 40years old, 7 cases high reflected by estrogen and 5 cases reported as unsatisfied sample 4.There were 46 cases (41.8%) with abnormal cytology result. There were 27 cases reported as abnormal glandulor cell (19 cases as AGC, 8 cases as AGC suspected adenocarcinoma )and 19 abnormal squamous cell(16 cases ASC-US ,2 cases LSIL ,1 case HSIL ). 5. There were 44 patient had HPVDNA test pre-operation and the positive rate were 27.2% (12/44).5 case were diagnosed as CIN1 and 1 case CIN2 by colposcopy directed biopsy. 6.The average tumor size in patients with abnormal cytology were 3.3 cm and patient with negative cytology were 2.1cm.The difference between the two group was significant by statistic. Conclusions: The sensitivity of cervical cytology to detect endometrial carcinoma is 24.5%. The size of the tumor related with cytology positive rate.
147

THE LEVEL OF AWARENESS OF CERVICAL CANCER SCREENING IN NIGERIA
O ADETULE, UNIVERSITY COLLEGE TEACHING HOSPITAL, IBADN, NIGERIA Background: Cervical cancer prevention efforts worldwide have focused on screening women at risk of the disease using Papanicolaou (Pap) smears and treating precancerous lesions. However, most developing countries have been unable to implement comprehensive Pap smear screening-based programs. Women in these countries are often not aware of cervical cancer screening and its treatment options. Objectives:This paper focused on the level of awareness of Pap smear screening test and Loop Electrosurgical Excision Procedure (LEEP) among the women in Ibadan North Local Government of Oyo State. Methods:The research design adopted was purely descriptive using a simple random sampling which was carried out on a population of 100 respondents who were all market women. Results:The result showed that 55% of the women have heard about cervical cancer before the study while 45% of the respondents have never heard about the disease. In Addition, 65% of the respondents have never heard of cervical screening while 35% have heard about it before the study. 97% of the women have a positive attitude that screening will help in detection of cervical cancer while 3% disagreed. On the respondent awareness of LEEP , only 26% of the respondents strongly agreed that they have heard about LEEP while 15% were undecided and 59 disagreed that they have never heard about it, which means that majority of the respondents were not aware of LEEP. Considering whether the awareness of LEEP is low, 79% agreed that the awareness is very low and it will be good if the primary, secondary, and tertiary institutions can pass the information across to the community, while 18% are undecided and 3% disagreed. Conclusion:The awareness on screening and necessary follow-up care for cervical cancer should be considered an integral part of reproductive health by a range of international organizations for developing countries such as Nigeria.
148
Session 18: Prophylactic vaccination: clinical studies

Chair: John Schiller
Poster abstracts
Prophylactic vaccination: clinical studies | 18 O-18.00

John Schiller
FOR YOUR NOTES:
O-18.01
DOES MERCK CLIA ACCURATELY DETECT HPV-18 ANTIBODIES IN VACCINATED SUBJECTS?
M Krajden, BC Centre for Disease Control and Univ. of British Columbia, Vancouver, CANADA, S Dobson, Vaccine Evaluation Centre, Children's & Women's Hospital, Vancouver, CANADA, D Cook, BC Centre for Disease Control, Vancouver, CANADA, R Chow, BC Centre for Disease Control, Vancouver, CANADA, A Yu, BC Centre for Disease Control, Vancouver, CANADA, S McNeil, Centre for Vaccinology, Dalhousie University, Halifax, CANADA, D Money, Children's & Women's Hospital, Vancouver, CANADA, M Dionne, Institut national de sant publique du Qubec, Qubec, CANADA, G Ogilvie, BC Centre for Disease Control, Vancouver, CANADA, M Petric, University of British Columbia, Vancouver, CANADA Background: Among individuals who initially sero-convert after Gardasil vaccination, HPV antibody levels measured by the Merck Competitive Luminex Immunoassay (cLIA) may decline to undetectable within as little as 2 years, especially for anti-HPV 18, despite continued evidence of vaccine efficacy in preventing subsequent infection. Objective: We tested for antibody to HPV 18 using a pseudovirus neutralizing antibody (PsV NAb) assay in a subset of subjects enrolled in a 2- vs. 3-dose Gardasil trial who had documented vaccine-induced sero-conversion and in whom anti-HPV 18 subsequently became undetectable by cLIA. Methods: The trial enrolled 828 subjects, with sera collected at baseline and months 7, 18, 24, and 36. cLIA results were expressed in milli-Merck units and PsV NAb results as geometric mean titres using three titration endpoints: NT100 (100% neutralization), NT90 (90%) and NTpartial (>10% and <90%). 100 infectious units of the respective pseudovirus (HPV 16 or HPV 18) were used in each well. Results: By month 7, all baseline sero-negative subjects sero-converted to HPV 16 and 18 by both PsV NAb and cLIA assays. At month 24, 620/620 (100%) were sero-positive for HPV 16 and 589/620 (95%) for HPV 18 by cLIA. Of the 31 HPV 18 cLIA negative subjects, PsV NAb was detected using the following endpoints: 31/31 (100%) had NTpartial; 13/31 (42%) had NT90; and 6/31 (19%) had NT100. This demonstrates that all 31 subjects displayed some level of HPV 18 neutralizing antibody at month 24. Conclusions: Although previous studies have shown very good correlation between the PsV NAb and cLIA assays, some subjects display discordant results. The detection of PsV NAb in subjects testing cLIA sero-negative for HPV 18 may reflect limitations in cLIA antibody detection sensitivity. Therefore, the cLIA test may provide a suboptimal measure of immune protection, especially for HPV 18.
Declaration of interest None declared, however future research grant funding by Merck, USA has been awarded.
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18 | Prophylactic vaccination: clinical studies O-18.02

CORRELATION BETWEEN ELISA, CLIA AND SECRETED ALKALINE-PHOSPHATASE PROTEIN NEUTRALIZATION ASSAYS
M Safaeian, National Cancer Institute, Rockville, UNITED STATES, S w Lin, National Cancer Institue, Rockville , United States, P Gonzales, Proyecto Epidemiologico Guanacaste, Guanacaste, Costa Rica, A Ghosh, National Cancer Institue, Rockville , United States, A C Rodriguez, Proyecto Epidemiologico Guanacaste, Guanacaste, Costa Rica, S Wacholder, National Cancer Institue, Rockville , United States, T Kemp, National Cancer Institue, Frederick, United States, M Esser, MedImmune, Gaithersburg, United States, A Meuree, GSK, Rixensart, Belgium, A Hildesheim, National Cancer Institue, Rockville , United States Background: Several serological assays have been developed to measure humoral responses to human papillomavirus (HPV) 16 and 18 infection and vaccination. The assay used by Merck is based on a virus-like particle (VLP)-based competitive luminex immunoassay (cLIA); the assay used by GSK is a VLP-based polyclonal enzymelinked immunoassay (ELISA). These are qualitatively and quantitatively different assays. Objectives: To understand the relation between cLIA and ELISA and how each relates with the secreted alkaline phosphatase protein neutralization assay (SEAP-NA) which is considered to be the gold-standard for measuring neutralization potential. Methods: Identical enrollment serum from 500 unvaccinated women (enrollment cervical HPV16/18 DNA-negative) in the control arm of an ongoing Costa Rica HPV vaccine trial were measured for antibodies elicited against HPV16 infections using the above mentioned assays. Agreement between the two assays and SEAP-NA was estimated. Results: Seropositivity by ELISA was 25%, compared with 7% by cLIA and 13.5% by SEAP-NA. Comparing the raw values, based on the manufacturers cutoff (ELISA seropositivity of 8 EU/ml and cLIA seropositivity of 20 mMU/ml) 32% of the samples were negative by both tests. There were no ELISA-negatives that were cLIA-positive, however, 68% of the samples were ELISA-positive but cLIA-negative, resulting in overall agreement of 53% (positive agreement=49%). Increasing ELISA cutoff to 50 (from the manufacturers recommended 8; keeping cLIA cutoff at 20) overall agreement increased to 90% (positive-agreement=79%). Correlation between cLIA and SEAP-NA was 0.72, and between ELISA and SEAP-NA was 0.63. Conclusions: cLIA is a more specific assay than ELISA. Using a more stringent cutoff for positivity by the ELISA, the cLIA and ELISA correlate well with each other and with neutralizing potential. Understanding the relation of ELISA and cLIA can provide a perspective on comparing immunogenicity results from Merck and GSK trials.
Declaration of interest PonceletArine Meuree is an employee at GSK Biologicals. Mark Esser is an employee at MedImmune.
O-18.03
TWO DOSE VACCINE TRIAL OF Q-HPV: RESULTS AT 36 MONTHS
S Dobson, University of British Columbia, Vancouver, CANADA, M Dawar, Vancouver Coastal Health Authority, Vancouver, CANADA, D Money, Women's Health Research Institute, Vancouver, CANADA, J Bettinger, University of British Columbia, Vancouver, CANADA, M Krajden, BC Centre for Disease Control, Vancouver, CANADA, J Langley, Dalhousie University, Halifax, CANADA, S McNeil, Dalhousie University, Halifax, CANADA, T Kollmann, University of British Columbia, Vancouver, CANADA, G Ogilvie, BC Centre for Disease Control, Vancouver, CANADA, M Dionne, Institut national de sant publique du Qubec, Quebec City, CANADA Background: Antibody responses to HPV-6,11,16,18, after the second dose of a 0, 6 month schedule using quadrivalent HPV vaccine (Q-HPV) in 9-13 year old girls were non-inferior to a 3 dose schedule in young adult women up to 24 months after dose one. Presented are the 36 month follow up immunogenicity data. Methods: In this post licensure randomized controlled multi-centre trial, three vaccine groups and 2 dosing regimens were assessed: Group 1, 9-13 years old2 doses at 0, 6 months (n=194); Group 2, 9-13 years old3 doses at 0, 2 and 6 months (n=187). Group 3, 16-26 year olds3 doses at 0, 2 and 6 months (n=203). At Month 36 blood of half of the subjects was evaluated using the Merck Competitive Luminex ImmunoAssay for HPV-6, 11, 16 and 18 serum antibody. Analysis of Variance to test differences in the Geometric Mean Titres (GMTs) was performed. Non-inferiority of any treatment arm was declared if the lower bounds of the 95% confidence intervals of GMT ratios were greater than 0.5. Results: At Month 36, GMT ratios (95% CI) were: Group 1/Group 3 HPV-6:1.38 (0.99, 1.94), HPV-11:1.45 (1.04, 2.02), HPV-16:1.70 (1.15, 2.50), HPV-18:1.47 (0.88, 2.44) Group 1/Group 2 HPV-6:0.65 (0.46, 0.92), HPV-11:0.74 (0.53, 1.05), HPV-16:0.82 (0.55, 1.21),HPV-18:0.44 (0.26, 0.74) Group 2/Group 3 HPV-6:2.12 (1.52, 2.97), HPV-11:1.95 (1.40, 2.72), HPV-16:2.08 (1.41, 3.06),HPV-18:3.34 (2.00, 5.58) Conclusions: At 36 months, HPV-16, 18, 6, 11 antibody responses following a 2 dose regimen in girls remained noninferior, to a 3-dose regimen in young adult women. Compared to a 3 dose regimen, a 2 dose regimen in girls was non-inferior for HPV-genotypes 16, 11 but not for HPV- 6 nor 18, where the lower bounds of the 95% CI fell below 0.5.
152

HPV-16/18 VACCINE: SUSTAINED IMMUNOGENICITY AND EFFICACY UP TO 9.4 YEARS
P Naud, Federal University of Rio Grande do Sul, Porto Alegre, BRAZIL, C M Roteli - Martins, Hospital Leonor Mendes de BarrosSecretaria da Sade de So Paulo, So Paulo, BRAZIL, N De Carvalho, Federal University of Parana, Parana, BRAZIL, J Teixeira, State University of Campinas - UNICAMP, Campinas, BRAZIL, P Borba, Ceara Cancer Prevention Institute, Ceara State, BRAZIL, N Sanchez, GlaxoSmithKline Biologicals, Rio de Janeiro, BRAZIL, T Zahaf, GlaxoSmithKline Biologicals, Wavre, BELGIUM, B Geeraerts, GlaxoSmithKline Biologicals, Wavre, BELGIUM, D Descamps, GlaxoSmithKline Biologicals, Wavre, BELGIUM Background and objectives: We present the final analysis results of immunogenicity, efficacy and safety of the HPV16/18 AS04 adjuvanted vaccine (HPV-16/18 vaccine) up to 9.4 years. Methods: Healthy women (1525 years; N=1113), oncogenic HPV DNA-negative, HPV-16/18 seronegative, and with normal cytology at baseline, received 3 doses of HPV-16/18 vaccine or placebo in a double-blind, randomised, multicentre study (HPV-001, NCT00689741). A subset of 776 subjects participated in a 3-year extension study (HPV-007, NCT00120848). Subjects from the five Brazilian sites (N=437) participated in the follow-up for 3 additional years, up to 9.4 years after vaccination (HPV-023, NCT00518336). Anti-HPV-16/18 antibodies were measured annually by ELISA and pseudovirion-based neutralisation assay (PBNA). Cervical samples were tested for HPV DNA every 6 months, and cytopathological examinations (ThinPrep) were performed annually. Results: Up to 9.4 years post-vaccination, all women remained seropositive for HPV-16 and HPV18 antibodies measured by ELISA and PBNA (according-to-protocol [ATP] cohort for immunogenicity; N=304). AntiHPV-16/18 antibody titres peaked at Month 7, establishing a plateau from 1824 months at least 10-fold (ELISA) and 4-fold (PBNA) above natural infection levels. In the combined analysis (HPV-001/007/023), vaccine efficacy (95% CI) against HPV-16/18-associated virological endpoints was 95.6% (86.2, 99.1), 100% (84.1, 100) and 100% (61.4, 100) for incident infection, 6-month persistent infection and 12-month persistent infection, respectively (ATP efficacy cohort; N=399). Vaccine efficacy against HPV-16/18-associated cytohistological endpoints was 97.1% (82.5, 99.9), 95.0% (68.0, 99.9), 100% (45.2, 100) and 100% (-128.1, 100) against ASCUS+, LSIL+, CIN1+ and CIN2+, respectively (total vaccinated cohort; N=437). The safety profile was clinically acceptable for both vaccine and control groups. Conclusions: The bivalent HPV-16/18 AS04-adjuvanted vaccine offers high and sustained immunogenicity and efficacy against HPV16/18-associated endpoints, with a clinically acceptable safety profile up to 9.4 years after vaccination. This is the longest follow-up study reported for a licensed HPV vaccine.
Declaration of interest Institutions of Paulo Naud, Cecilia M Roteli-Martins and Newton De Carvalho received grants from GlaxoSmithKline Biologicals. Paulo Naud and Newton De Carvalho received consulting fee/honorarium from GlaxoSmithKline Biologicals. Paulo Naud, Newton De Carvalho and Julio Teixeira received support for travel to meetings from GlaxoSmithKline Biologicals. Cecilia M Roteli-Martins, Newton De Carvalho, Julio Teixeira, and Paola Borba received fees for participation in advisory boards and/or lectures from GlaxoSmithKline Biologicals. Nervo Sanchez, Toufik Zahaf, Brecht Geeraerts and Dominique Descamps are employees of GlaxoSmithKline Biologicals. Nervo Sanchez and Dominique Descamps hold stock options from GlaxoSmithKline Biologicals.
O-18.05
A LONG-TERM EXTENSION STUDY OF GARDASIL IN ADOLESCENTS
D Ferris, Georgia Health Sciences University, Augusta, UNITED STATES BACKGROUND: Quadrivalent HPV vaccine has previously been shown to be safe and immunogenic in adolescents though 30 months after vaccination. OBJECTIVE: We describe the first interim effectiveness data for a long-term immunogenicity, safety, and effectiveness study of GARDASIL among adolescents. METHODS: In the base study, 1781 sexually nave boys and girls were assigned (2:1) to GARDASIL or saline placebo at day 1, months 2 and 6. At the end of the base study (month 30), the placebo group received GARDASIL following the same regimen. Those vaccinated with GARDASIL in the base study are the early vaccination group (EVG). Those vaccinated with GARDASIL during months 30-36 are the catch-up vaccination group (CVG). As this extension study does not have a placebo arm, effectiveness was assessed by calculating the incidence of the primary endpoints (HPV6/11/16/18 persistent infection or related disease) and comparing these rates with those from previous phase 3 studies in men and women aged 16-26. The median follow-up time for effectiveness was 1.8 years in both the EVG and CVG. RESULTS: For each gender, anti-HPV 6/11/16/18 responses at 4 weeks post dose-3 of GARDASIL were comparable in the EVG and CVG. Rates of acquisition of new sexual partners and common STIs were similar to previous studies in men and women. In females and males, there were no cases of disease related to any of the four vaccine HPV types during 6 years of follow-up. One SAE was reported, a fatal road traffic accident. CONCLUSION: For any HPV vaccine, long-term follow-up of disease endpoints is required to establish the duration of protection. This study of GARDASIL provides the first long-term effectiveness data among males and females vaccinated during adolescence and underscores the importance of early vaccination.
153
18 | Prophylactic vaccination: clinical studies O-18.06

HPV-16/18 AS04-ADJUVANTED VACCINE EFFICACY IN 26-YEAR-OLD WOMEN AFTER 4-YEAR FOLLOW-UP
D Descamps, GlaxoSmithKline Biologicals, Wavre, Belgium, R Skinner, University of Sydney, Sydney, AUSTRALIA, A Szarewski, Wolfson Institute of Preventive Medicine, London, UK, B Romanowski, University of Alberta, Alberta, Canada, S Garland, University of Melbourne and the Royal Women's Hospital, Melbourne, Australia, E Lazcano, Hospital General Dr. Ernesto Meana, SanRoman-Jojutla, Morelos-Mexico, M P David, GlaxoSmithKline Biologicals, Wavre, Belgium, B Geeraerts, GlaxoSmithKline Biologicals, Wavre, Belgium, K Hardt, GlaxoSmithKline Biologicals, Wavre, Belgium, G Dubin, GlaxoSmithKline Biologicals, King of Prussia, PA, USA, D Descamps, GlaxoSmithKline Biologicals, Wavre, Belgium Background-objectives: The HPV-16/18 AS04-adjuvanted vaccine has been shown to be well-tolerated, immunogenic and highly efficacious in the prevention of HPV-16/18 infections and precancerous lesions in women 1525 years. We present four-year follow-up results from the Human PapillomaVirus: Vaccine Immunogenicity and Efficacy study in women 26 years or older (VIVIANE; NCT00294047), a double-blind, randomised, placebo-controlled, multinational study. Methods: 5752 women were randomised (1:1) to receive 3 doses of HPV-16/18 vaccine (n=2881) or Al(OH)3 control (n=2871) at Months 0, 1, 6. Efficacy analyses were performed in the according-to-protocol cohort (ATP; 3 doses per protocol; vaccine, n=2264; control, n=2241) and in the total vaccinated cohort (TVC; 1 dose), including 15% of subjects with history of HPV disease/infection. Safety was assessed in TVC. Results: Vaccine efficacy (VE) against HPV-16/18 cervical intraepithelial neoplasia grade 1 or greater (CIN1+) and/or 6-month persistent infection (PI) was 81.1% (97.7% CI: 52.1; 94.0) in ATP and 43.9% (23.9; 59.0) in TVC. In the same cohorts, VE against HPV-16/18 6-month PI was 82.9% (53.8; 95.1) and 47.0% (25.4; 62.7). VE against HPV-16/18 CIN1+ in ATP was 86.1% (-35.4; 99.9) in HPV-16/18 seronegative women and 91.1% (25.4; 99.9) irrespective of serostatus; VE was 37.8% (-3.2; 63.1) in TVC. VE against 6-month PI associated with any oncogenic HPV types was 23.8% (3.4; 40.0) in ATP and 16.0% (2.4; 27.7) in TVC. Corresponding VE for HPV-31/45 was 77.6% (45.4; 92.3) and 43.6% (16.7; 62.2). VE was shown against HPV-16/18 atypical squamous cells of undetermined significance or greater (ATP: 93.7% [71.5; 99.5]; TVC: 57.2% [32.9; 73.3]). Incidences of adverse events, serious adverse events and medically significant conditions were generally similar between groups. Conclusions: The HPV-16/18 AS04-adjuvanted vaccine showed high efficacy against HPV-16/18 CIN1+ and/or 6-month PI, evidence of cross-protection and a clinically acceptable safety profile, in women aged 26 years.
Declaration of interest S. R. Skinner1, A. Szarewski2, B. Romanowski3, S. Garland4, E. Lazcano5, M-P. David6, B. Geeraerts6, K. Hardt6, G. Dubin7 and D. Descamps6 1Vaccine Trials Group, Telethon Institute for Child Health Research, and Sydney University Discipline of Paediatrics and Child Health, Childrens Hospital at Westmead, Sydney, New South Wales, Australia. 2Centre for Cancer Prevention, Wolfson Institute of Preventive Medicine, London, UK. 3University of Alberta, Edmonton, AB, Canada. 4Microbiology and Infectious Diseases Department, Royal Women's Hospital and Department of Obstetrics and Gynecology, University of Melbourne, Melbourne, Victoria, Australia. 5National Institute of Public Health, Cuernavaca Morelos, Mexico. 6GlaxoSmithKline Biologicals, Wavre, Belgium. 7GlaxoSmithKline Biologicals, King of Prussia, PA, USA
O-18.07
HPV VACCINE EFFICACY AGAINST BOTH CERVICAL AND ANAL HPV16/18 INFECTION
H Katki, US National Cancer Institute, Rockville, UNITED STATES A Kreimer, US National Cancer Institute, Rockville, UNITED STATES P Gonzalez, Proyecto Epidemiolgico Guanacaste, Liberia, COSTA RICA C Porras, Proyecto Epidemiolgico Guanacaste, Liberia, COSTA RICA M Schiffman, US National Cancer Institute, Rockville, UNITED STATES A Rodriguez, Proyecto Epidemiolgico Guanacaste, Liberia, COSTA RICA D J Lowy Schiller, US National Cancer Institute, Rockville, UNITED STATES W Quint, DDL Diagnosic Laboratory, Voorburg, THE NETHERLANDS A Hildesheim, US National Cancer Institute, Rockville, UNITED STATES R Herrero, Proyecto Epidemiolgico Guanacaste, Liberia, COSTA RICA Background: It is unknown how HPV vaccine efficacy varies for HPV infections in the anus or in the cervix. Objectives: Cervarix vaccine efficacies (VE) against HPV16/18 positivity solely at the anus, solely at the cervix, or positive at both sites, and by anal sexual behavior. Methods: In the Costa Rica Vaccine Trial, a randomized double-blind trial designed to evaluate Cervarix VE against persistent cervical HPV16/18 positivity and precancerous lesions, we evaluated VE against anal HPV16/18 positivity at the final blinded study visit 4 years after vaccination in 3,901 participants aged 22-29 who underwent anal specimen collection (71% of eligible women). HPV DNA testing was performed using SPF10 PCR/DEIA/LiPA25 (Labo Medical Products, based on SPF10 technology licensed by Innogenetics). Results: Vaccine efficacy 4-years post-vaccination against solely anal HPV16/18 positivity was 26% (95%CI -13% to 51%; 38 vs. 51 events in the HPV and control arms, respectively), against solely cervical HPV16/18 positivity was 64% (95%CI 46% to 75%; 34 vs. 93), and against concordant HPV16/18 positivity at both sites was 90% (95%CI 76% to 96%; 6 vs. 58); p<0.02 comparing each pair of VEs. Women reporting engaging in anal sex had VE against solely anal HPV16/18 positivity of 47% (95%CI -22% to 72%; 11 vs. 19), solely cervical HPV16/18 positivity of 49% (95%CI -20% to 74%; 10 vs. 18), and against concordant HPV16/18 positivity at both sites of 96% (95%CI 65% to 99%; 1 vs. 21). Conclusions: Cervarix protected strongly against concordant HPV16/18 positivity at both sites, less strongly against solely cervical HPV16/18 positivity, and weakly, if at all, against solely anal HPV16/18 positivity. Women reporting anal sex had higher vaccine efficacy against solely anal HPV16/18 positivity than women overall. Vaccine efficacy against anal HPV depended strongly on cervical HPV positivity and anal sexual behavior.
154

PREVENTION OF HGAIN WITH QUADRIVALENT HPV VACCINATION OF MSM
S Goldstone, Mount Sinai School of Medicine, New York, UNITED STATES K Swedish, Mount Sinai School of Medicine, New York, United States S Factor, Mount Sinai School of Medicine, New York, United States Background: Men who have sex with men (MSM) have high prevalence of human papillomavirus (HPV) infection and HPV-related disease. Randomized control trials of quadrivalent HPV vaccine (qHPV) demonstrated significant efficacy in reducing high-grade anal intraepithelial neoplasia (HGAIN) and anal cancer among young MSM with few lifetime sexual partners and without HPV infection. Objectives: To evaluate qHPV among older MSM likely to have HPV infection and/or HPV-related disease. Methods: This non-concurrent cohort study evaluated HIV-negative MSM at one clinical site. Patients who received all three doses of qHPV composed the vaccinated cohort; patients who had not received any doses of qHPV composed the unvaccinated cohort. Relative risk and vaccine efficacy were calculated. Kaplan-Meier plots were used to determine time to development of HGAIN; Cox Proportional Hazards Ratio adjusted for possible confounders. Sub-group analyses were performed on patients with history of HGAIN and patients high-risk HPV positive prior to time zero. Results: Two hundred fifty two patients received full three-dose series of qHPV. More than half were high-risk HPV positive; over 40% had history of HGAIN. Relative risk of developing HGAIN was 0.53 (0.36, 0.81) for vaccinated compared to unvaccinated patients. Vaccine efficacy was 46.3% (19.1, 64.4). Log-rank approached significance (p=0.096). Among patients with history of HGAIN, relative risk was 0.30 (0.16, 0.57) for vaccinated patients. Vaccine efficacy was 69.8% (42.7, 84.1). Log-rank was significant (p=0.006); adjusted hazard ratio was 0.39 (0.18, 0.83). Among patients who were high-risk HPV positive prior to time zero, relative risk was 0.53 (0.35, 0.82) for vaccinated patients. Vaccine efficacy was 46.7% (18.4, 65.1). Log-rank was significant (p=0.045). Conclusion: Overall, vaccinated patients were half as likely to develop HGAIN. Vaccination with qHPV significantly decreases risk of HGAIN recurrence among those with history of HGAIN. QHPV demonstrated efficacy among patients with history of high-risk HPV.
Declaration of interest Dr. Goldstone (the Principal Investigator in this study) receives financial compensation as a consultant, lecturer, and for service on a scientific advisory board for Merck & Co. Merck & Co. manufactures Gardasil, the only licensed quadrivalent HPV vaccine.
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18 | Prophylactic vaccination: clinical studies P-18.09

HPV4 COMPLETION AND IMMUNE RESPONSE AMONG PERUVIAN FEMALE SEX WORKERS
B Brown, UCLA , Laguna Niguel, UNITED STATES N Halsey, Johns Hopkins School of Public Health, Baltimore, UNITED STATES M Blas, Universidad Peruana Cayetano Heredia, Lima, PERU C Carcamo, Universidad Peruana Cayetano Heredia, Lima, PERU Background: Studies of HPV vaccines were conducted in girls and young women. As a result, there are limited data on vaccine induced antibody responses in highly exposed persons such as female sex workers (FSWs). Objectives: To compare antibody response to HPV vaccination by baseline serostatus, and determine completion rates for HPV vaccine among FSWs. Methods: Two hundred FSWs in Lima, Peru received HPV4 vaccine in the standard (0,2,6 months) or a modified schedule (0,3,6 months). Adherence was measured as receiving all 3 doses within a 30 day window. Blood was collected at baseline and one month following final dose. Antibody concentrations following vaccination for baseline seropositive women were compared with seronegative women. Results: One hundred and eighty four (92%) participants completed 3 doses of vaccine, and 95% of those adhered to the schedule. There were no significant differences in the final geometric mean titers (GMTs) of antibody by study schedule (p>0.2). Baseline seropositive rates by HPV genotype were HPV6-58% HPV11-22.5%, HPV16-41.5%, and HPV18-13%. Four women (2%) were seropositive for all 4 genotypes, and 79% were seropositive for any HPV4 genotype. The final antibody concentrations following vaccination were significantly greater for women who were seropositive at baseline compared to seronegative for HPV 6 (GMT ratio=2.3, p<0.01), HPV 11 (GMT ratio=2.7, p<0.01), HPV 16 (GMT ratio=1.3, p=0.04), and HPV 18 (GMT ratio=2.4, p<0.01). Almost all (99%) women, regardless of baseline serostatus responded to each of the 4 vaccine types. Conclusions: High rates of completion for the HPV vaccine series are possible in FSWs, although additional recruitment and retention efforts may be necessary for this population. Vaccination early in sexual careers is needed to assure maximum protection. Nearly all women mounted an immune response to vaccine, but women primed by natural HPV infection responded better than baseline seronegative women.
P-18.10
HPV EPIDEMIOLOGY IN HIV-POSITIVE WOMEN RECEIVING THEIR FIRST HPV VACCINATION
J Kahn, Cincinnati Children's Hospital Medical Center, Cincinnati, UNITED STATES, R Burk, Albert Einstein College of Medicine, Bronx, UNITED STATES, K Squires, Jefferson Medical College, Philadelphia, UNITED STATES, B Kapogiannis, National Institutes of Health, Rockville, UNITED STATES, B Rudy, New York University, New York, UNITED STATES, J Xu, Westat, Rockville, UNITED STATES, R Gonin, Westat, Rockville, UNITED STATES, N Liu, Westat, Rockville, UNITED STATES, C Worrell, National Institutes of Health, Rockville, UNITED STATES, C Wilson, University of Alabama at Birmingham, Birmingham, UNITED STATES OBJECTIVES: To describe the epidemiology of and risk factors for HPV infection among HIV-infected young women at the time of first HPV vaccination. METHODS: Data were collected at the baseline visit of an HPV-6, -11, -16, -18 vaccine clinical trial in 16- to 23-yearold HIV-infected young women who acquired HIV through sexual behaviors or IV drug use. Participants completed a questionnaire, and cervicovaginal swabs were collected and tested for HPV DNA using an MY09/11 assay and dot blot method to detect 41 HPV types. Univariate and multivariable logistic regression were used to examine whether independent variables (demographic characteristics, CD4+ count, viral load, HPV knowledge, sexual behaviors, and diagnosis of a sexually transmitted infection) were associated with high-risk HPV infection (positive for >= 1 of 13 high-risk types). RESULTS: Of the 99 participants (mean age 21.4 years, 79.8% Black, 40.4% with HIV viral load < 400 copies/mL, 30.3% on antiretrovirals), 71.7% were positive for >= 1 HPV type, 52.5% for >= 1 high-risk type, 11.1% for HPV-16, and 5.1% for HPV-18; mean number of types was 3.2 (range 1-8). In multivariable analyses, three variables were associated with >= 1 high-risk type: non-Hispanic vs. Hispanic ethnicity (AOR 5.56, 95% CI 1.45-20.0), HIV viral load >= 400 vs. < 400 (AOR 2.99, 95% CI 1.16-7.69), and frequency of vaginal sex in past 90 days (for the category 1 vs. 0, AOR 8.83, 95% CI 1.66-47.0). CONCLUSIONS: In a cohort of HIV-positive young women, the prevalence of HPV-16 and -18 was higher than in the general population. However, most participants were HPV DNA negative for these types at the time of vaccination, suggesting that HPV vaccination of HIV-positive women could be effective. Higher HIV viral load was associated with high-risk HPV infection, suggesting that adherence to or treatment with antiretroviral medications may prevent HPV.
Declaration of interest Vaccine and HPV Mean Geometric Titers were provided through the Investigator-Initiated Studies Program of Merck & Co., Inc. For the remaining authors no other conflicts were declared.
156
Prophylactic vaccination: clinical studies | 18 P-18.11

ANTIBODY RESPONSES IN JUVENILE IDIOPATHIC ARTHRITIS PATIENTS VACCINATED WITH CERVARIX
M Scherpenisse, National Institute for Public Health and the Environment, Laboratory for Infectious Diseases and Screening/VU University Medical Centre, Department of Pathology , Bilthoven/Amsterdam, The Netherlands M Heijstek, Wilhelmina Children's Hospital, University Medical Centre Utrecht, Department of Paediatric Immunology, Utrecht, The Netherlands G Berbers, National Institute for Public Health and the Environment, Laboratory for Infectious Diseases and Screening, Bilthoven, The Netherlands N Wulffraat, Wilhelmina Children's Hospital, University Medical Centre Utrecht, Department of Paediatric Immunology, Utrecht, The Netherlands F van der Klis, National Institute for Public Health and the Environment, Laboratory for Infectious Diseases and Screening, Bilthoven, The Netherlands Introduction: In the Netherlands, the bivalent HPV vaccine (Cervarix) was included in the national immunization program in 2010 for girls 12 years of age accompanied by a catch-up campaign for girls up to 16 years. In healthy children this vaccine has proven to be effective and induces high antibody concentrations. However, no vaccine immunogenicity data were available from immunosuppressed children, like patients with juvenile idiopathic arthritis (JIA). Methods: JIA patients (12-18 years-of-age, n=49 at baseline) and healthy children of the control group (12-16 yearsof-age, n=44 at baseline) were vaccinated in a 2+1 schedule with Cervarix. On four time points, sera were collected and tested for high-risk (hr) HPV16, 18, 31, and 45 antibodies. Sera were analyzed using a fluorescent microspheresbased multiplex immuno-assay with virus-like particles for the four HPV serotypes coupled to the microspheres. Results: No significant differences in antibody concentrations for HPV16, 18, 31 and 45 were found pre- and postvaccination in JIA patients compared with the control group at all time points. After the third vaccination, antibody concentrations were highest for HPV16 (GMC >15000 LU/ml) and HPV18 (GMC >5500 LU/ml). A significant decline in antibody concentrations for all four HPV types was shown 6 months after the third vaccination in both groups. Following vaccination, cross-reactivity was observed for HPV31 and 45 but antibody concentrations were 100 and 10 times lower, respectively, compared with HPV16 and 18. Conclusion: This study provides information about the vaccine immunogenicity in JIA patients. Antibody responses, pre- and post-vaccination, against the HPV vaccine serotypes and the cross-reactive HPV serotypes were not significantly different among adolescent JIA patients and healthy children of the same age.
P-18.13
THE CLINICAL IMPACT OF CROSS PROTECTION TO NON-VACCINE HPV TYPES
D Brown, Indiana University, Indianapolis, UNITED STATES Background: Prophylactic HPV vaccines have demonstrated a high degree of protection against infection and disease caused by HPV types included as L1 virus like particles (VLPs) in the vaccines, and in addition, a limited degree of protection against infection and disease caused by HPV types not present in the vaccines. The clinical significance of this apparent 'cross protection' is poorly understood, and the duration of such protection has not been established. Objective: To characterize the clinical significance of cross protection against persistent infection and high grade lesions caused by non-vaccine HPV types. Results: Protection against persistent infection and high grade cervical intraepithelial neoplasia grade 2 or worse and due to HPV types 31, 33 or 45 has been described as a result of vaccination with HPV vaccines that contain HPV types 16 and 18 L1 VLPs. Such protection is attributed to cross reacting antibodies that are generated by HPV types 16 and 18 against epitopes of phylogenetically related HPV types. The affinity, durability and neutralization capacity of antibodies that are generated in response to similar, but not type-specific epitopes are relevant areas for exploration. In clinical trials, the role of co-infection of lesions from placebo recipients who are HPV PCR positive for both vaccine and non-vaccine HPV types makes causal attribution to non-vaccine types particularly difficult. Such data will be presented. Conclusions: Efficacy of HPV vaccines against non-vaccine HPV types requires new analytical methodology in order to understand the true clinical significance of cross protection.
Declaration of interest Presenter will provide information.
157
Session 19: Standard and experimental treatment

Chair: Walter Kinney
Poster abstracts
Standard and experimental treatment | 19 O-19.00

Walter Kinney
FOR YOUR NOTES:
O-19.01
PREMATURE DELIVERY AFTER CIN TREATMENT, A BELGIAN MULTICENTRIC RETROSPECTIVE STUDY
C Simoens, Scientific Institute of Public Health, Brussels, BELGIUM, P Simon, Hpital Erasme, Brussels, BELGIUM, F Goffin, Hpital de la Citadelle, Lige, BELGIUM, M Bossens, Hpital Brugmann, Brussels, BELGIUM, P Barlow, Hpital St Pierre, Brussels, BELGIUM, S Merodio, Centre Hspitalier du Bois de l'Abbaye, Lige, BELGIUM, M Van Hentenrijk, Hpital Erasme, Brussels, BELGIUM, C Gerday, Hpital de la Citadelle, Lige, BELGIUM, M Timmermans, Hpital de la Citadelle, Lige, BELGIUM, M Arbyn, Scientific Institute of Public Health, Brussels, BELGIUM Background and objectives: Recent meta-analytical work indicates that surgical excision of cervical intra-epithelial neoplasia (CIN) is associated with adverse obstetrical outcomes. Currently, large loop excision of the transformation zone (LLETZ) is the most frequent procedure used to treat CIN in industrialised countries. The aim of the study was to estimate the burden of adverse obstetrical outcomes after CIN treatment in women delivering in Belgium. Methods: Cases with a prior history of CIN treatment were enrolled before delivery and matched with 2 women without history of a CIN lesion-delivering immediately after the case. Demographic data, risk factors and obstetrical outcomes were recorded for all subjects as well as surgery features for CIN treatment. 540 pregnant women fulfilled the inclusion criteria. Poisson regression was used to obtain the relative risk (RR) for preterm delivery (PD<37w) and low birth weight (LBW<2500g) adjusted for confounding factors. Results: There were significantly more black and HIV-positive women in the treated versus the control group. The smoking status was not significantly different. 48.4% had a LLETZ treatment, 30.9% laser conisation, 10.7% cold knife conisation and 10.0% other treatments. 23.7% of women with prior excisional treatment delivered prematurely compared to 9.2% of the controls (RR=2.96; 95%CI:1.85-4.73). A LBW occurred in 14.8% of the treated and in 6.5% of the untreated women (RR=2.64; 95%CI:1.49-4.68). The attributible risk in women with CIN treatment was 66.2% (95%CI:45.9%-78.9%) and 62.1% (95%CI:32.9%-78.6%) for PD and LBW, respectively. Conclusions: The incidence of PD and LBW in CIN treated women was more than two times higher than in women without treatment. Currently it is estimated that about 500 PDs occur each year, in Belgium, due to the consequences of CIN treatment. This could be reduced to approximately 170 by a vaccine preventing all CINs, ablative treatment or less deep excision in young reproductive women.
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19 | Standard and experimental treatment O-19.02

HPV TEST OF CURE: EFFECTIVE PROTECTION FOR 5 YEARS
M Cruickshank, University of Aberdeen, Aberdeen, UNITED KINGDOM R Hadwin, Royal Free Hospital, London, UNITED KINGDOM L Nelson, University of Manchester, Manchester, UNITED KINGDOM P Walker, Royal Free Hospital, London, UNITED KINGDOM C Moore, Royal Infirmary of Edinburgh, Edinburgh, UNITED KINGDOM H Kitchener, University of Manchester, Manchester, UNITED KINGDOM Background: The value of HPV test of cure following treatment of CIN is well established, particularly in terms of negative predictive value. Our previous report(1) showed that women who are cytology negative and HPV negative can be safely returned to 3 year recall which is now being implemented in the English Cervical Screening Programme. In this analysis we report on extended follow-up. Methods: Women treated for CIN underwent test of cure at 6 and 12 months post treatment with HC2 and LBC and followed up annually with cytology only. If either test were positive women were referred for colposcopy. Annual follow-up cytology and histology results have been obtained out to seven years. Results: 917 women, mean age 31.5 years had been treated (365 CIN3; 326 CIN2; 217 CIN1 and 9 CGIN). 70% of excised specimens had clear margins. At six months non-negative cytology was found in 10.7% and 14.6% were HPV positive. To date we have follow-up data at 36, 60 and 84 months on 617 (67.3%), 445 (48.5%) and 159 (17.3%) respectively. Among women who were HPV-ve post treatment, the cumulative rates of ASCUS+ from 12 months was 10.3%, 15.7% and 15.7% at 36, 60 and 84 months respectively. The corresponding data for HPV+ve women was 46.8%, 50.9% and 53.5%. Women who were HPV negative at baseline had a cumulative CIN2+ rate of 2.6% at 60 months compared with 12.6% for HPV positive. Women who were HPV+ve/cytology+ve at 6 months had a RR of 7.8(95% CI 3.6-16.9) for CIN2+ at 60 months compared with HPV-ve/cytology-ve. Conclusion: Women treated for CIN who are cytology-ve/HPV-ve at 6 month follow-up could have recall safely extended to 5 years. 1. Kitchener et al, BJOG 2008; 115: 1001-7
O-19.03
APTIMA HPV TESTING OF FOLLOW UP PATIENTS TREATED FOR CIN
A Clad, UNIVERSITAETSKLINIKUM FREIBURG, Universitaets-Frauenklinik, Freiburg, GERMANY J Waibel, UNIVERSITAETSKLINIKUM FREIBURG, Universitaets-Frauenklinik, Freiburg, GERMANY J Weinschenk, UNIVERSITAETSKLINIKUM FREIBURG, Universitaets-Frauenklinik, Freiburg, GERMANY P Zeitlow, Boyce & Bynum Pathology Labs, Columbia, USA Background: Despite treatment, some women diagnosed with cervical intraepithelial neoplasia (CIN) will develop recurrent disease. Surveillance options are cytology and high-risk human papillomavirus testing, but the long-term performance of these follow-up testing methods in this high-risk population is unknown. Objective: To evaluate the performance of the APTIMA HPV Assay (AHPV, Gen-Probe Incorporated) and the Hybrid Capture 2 test (HC2, Qiagen) in women followed-up after treatment of CIN. Materials and Methods: Liquid-based Pap samples were obtained at one or more follow-up visits for cytology, AHPV and HC2 testing from 83 women treated for CIN. Colposcopy was performed and biopsies obtained from women with an abnormal colposcopy. Women with a normal colposcopy were considered disease negative (<CIN1). Results from AHPV and HC2 were compared to histology results. Results: Of 20 women with positive AHPV and HC2 results at follow-up visits, 8 women were cytology and/or histology positive. Four of the 12 women that were cytology and histology negative were histology positive at the next follow-up visit. The remaining 8 women will be invited for additional follow-up visits. Of 39 women with negative AHPV and HC2 results, 28 were disease negative and 11 had CIN1. Seven of the 11 women with a CIN1 returned for a second follow up visit with no visible lesions observed. Of 13 women with negative AHPV and positive HC2 results, 3 had CIN2+ (1 CIN3, 2 CIN2), 2 had CIN1 and 8 were disease negative at the last follow-up visit. Of 11 women with positive AHPV and negative HC2 results, 9 had CIN2+ (1 CxCa, 4 CIN3, 4 CIN2) and 2 had CIN1 at follow-up. Conclusion: While additional follow-up of these and other women will continue, this study demonstrates that the AHPV is a very sensitive and specific follow-up test after treatment of CIN.
Declaration of interest APTIMA HPV testing was performed at Gen-Probe
162

TREATMENT OF CERVICAL INTRAEPITHELIAL NEOPLASIA WITH TOPICAL IMIQUIMOD.
C Grimm, Medical University of Vienna, Vienna, AUSTRIA S Polterauer, Memorial Sloan-Kettering Cancer Center, New York, USA C Natter, Medical University of Vienna, Vienna, AUSTRIA J Rahhal, Medical University of Vienna, Vienna, AUSTRIA L Hefler, Medical University of Vienna, Vienna, AUSTRIA A Reinthaller, Medical University of Vienna, Vienna, AUSTRIA P Speiser, Medical University of Vienna, Vienna, AUSTRIA BACKGROUND: Alternatives to surgical therapy are needed for the treatment of high-grade cervical intraepithelial neoplasia (CIN). OBJECTIVE: We investigated the effectiveness of a topical treatment with imiquimod (IMQ), an immune-response modulator, for patients with CIN 2 and 3. METHODS: In this placebo-controlled trial 59 patients with CIN 2 and 3 were randomized to receive either a 16-week treatment with topical IMQ or placebo. The main outcome was effectiveness, defined as histologic regression (to CIN1 or less) four weeks after end of treatment; secondary outcomes were complete histologic remission, human papillomavirus (HPV) clearance and treatment tolerability. RESULTS: Of the patients 27 (46%) and 32 (54%) presented with CIN 2 and 3, respectively. Histologic regression was significantly increased in the IMQ group (75%) compared to the placebo group (41%) (P=0.03). Complete histologic remission was significantly increased in the IMQ group (46%) as compared with the placebo (15%) group (P=0.02). At baseline, all 59 patients (100%) were tested positive for HPV infection. HPV clearance rate was increased in the IMQ group (61%), as compared with the placebo (15%) group (P<0.001). The lesion progressed to microinvasive disease in 2 of 59 patients (3%) - both within the placebo group. Topical IMQ treatment was acceptably tolerated. Due to local and/or systemic side effects, eight patients (27%) within the IMQ group switched to 50% dose medication and one patient (3%) prematurely quit the topical application. CONCLUSION: Topical IMQ is an effective and acceptably tolerable treatment for high grade cervical intraepithelial neoplasia. (ITIC-trial: ClinicalTrials.gov Identifier: NCT00941252)
Declaration of interest Of note, this study will also be presented as a poster presentation at the ASCO meeting 2011 in June in Chicago, USA.
O-19.05
SIRNA THERAPIES FOR CERVICAL NEOPLASIA.
N McMillan, University of Queensland, Brisbane, AUSTRALIA S Wu, University of Queensland, Brisbane, AUSTRALIA N Khairuddin, University of Queensland, Brisbane, AUSTRALIA W Gu, University of Queensland, Brisbane, AUSTRALIA S Blake, University of Queensland, Brisbane, AUSTRALIA S Blake, University of Queensland, Brisbane, AUSTRALIA S Blake, University of Queensland, Brisbane, AUSTRALIA G Purdie, University of Queensland, Brisbane, AUSTRALIA E Payne, University of Queensland, Brisbane, AUSTRALIA M Burgess, University of Queensland, Brisbane, AUSTRALIA The use of siRNA for cancer treatment is only now starting to reach the clinic. However, this is currently limited to a few cancers of the hepatic system, as most nanoparticle delivery systems efficently distribute siRNA to the liver. Indeed, a major barrier for the use of RNAi has been the inability to deliver siRNA to the target tumours and sites beyond the liver. It has been shown by several groups that siRNA targeting HPV E6/E7 is effective at inducing apoptosis or senescence in vitro. CIN and VIN offers the additional challenge of delivery to the cervix/vaginal tract and thus must overcome several additional barriers including mucus, the hyperkerototic cell barrier, a normal microflora, liquid flow through and acidic conditions. To overcome this we have developed the PLAS system (PEGlipoplex in Alginate Scaffold) that is highly effective at delivering siRNA cells in the genital tract. We are able to achieve siRNA delivery to vaginal epithelial cells and specifically silence target genes. We will also show for the first time that the use of immunostimulatory, bifunctional siRNAs are more effective at treating cervical cancer tumours in vivo compared to those that silence alone and that dual targeting of VEGF and E7 gives improved tumour outcomes. Overall, our work demonstrates the utility of RNAi therapy in this setting and offers a clear path to clinical trials for the use of siRNA in cervical neoplasia.
Declaration of interest International DNA Technologies provided siRNAs for some of these experiments.
163
19 | Standard and experimental treatment O-19.06

EFFECT OF REBACIN IN TREATMENT OF HIGH RISK HPV INFECTION
C Zhang, SR Life Sciences Institute Inc, Clarksburg, UNITED STATES D Wu, International Peace MCH Hospital, China Welfare Institute, Shanghai, CHINA R N Li, International Peace MCH Hospital, China Welfare Institute, Shanghai, CHINA H R Zhou, Changning District MCH Hospital, Shanghai, CHINA G Y Wang, SR BioPharma Inc, Haikou, CHINA Background: High-risk human papillomaviruses (HPVs) are recognized cause for cervical cancer. We have previously demonstrated that REBACIN, a novel antiviral factor, can efficiently suppress both the expression of E6 and E7 genes of HPV16 and HPV18 viruses, and the growth of tumors induced by these two types of viruses in a mouse model. Objective: To evaluate the clinical effect of REBACIN in treatment of patients with high risk HPV infection. Methods: 300 cases (patients) positive in Hybrid Capture II (HC2) test but Pap smear or liquid-based cytology test (LCT) normal were subject to this present investigation. They were randomly and even-number (100/100/100) divided into three groups. These include an experimental group (100 cases, G1) treated with REBACIN via vaginal injection, a positive control group (100 cases, G2) administrated with interferon suppository, and a negative control group (100 cases, G3) that received no drug treatment. The efficacy against HPV infection was evaluated by analysis of the negative conversion rates using HC2 tests at the 3rd and 6th month after the drug treatment ceased. Results: The negative conversion rates of HPV in REBACIN-treated group were 62% and 67% at the 3rd and 6th month follow ups, respectively. In comparison, 24% and 25% negative conversion rates were observed for the interferon treatment group. The self-clearance rates in the third group of patients receiving no drug interferences are 18% and 19% at these two follow ups. Conclusively, these results have demonstrated that REBACIN is significantly effective in high risk HPV infection clearance with notable virus negative conversion rate.
O-19.07
TOPICAL CIDOFOVIR -- FOR HIGH-GRADE PERIANAL DYSPLASIA, HIV+ INDIVIDUALS
E Stier, Boston University Medical Center, West Newton, UNITED STATES, S Goldstone, Mt Sinai Hospital, New York, united States, M Einstein, Montefiore Medical Cener, Bronx, United States, N Jay, UCSF, San Francisco, united States, M Berry, UCSF, san Francisco, united States, T Wilkin, Weill-Cornell Medical College, New York, united States, j lee, University of Arkansas for Medical Sciences, Little Rock, united States, j lee, University of Arkansas for Medical Sciences, Little Rock, united States, L Panther, Beth Israel Deaconness Medical Center, Boston, united States, J Palefsky, ucSF, san Francisco, united States Objective: Treatments for perianal intraepithelial neoplasia (PAIN) 2-3 include surgical ablation and excision. These are associated with significant morbidity and recurrence rates, particularly in HIV-infected patients. Cidofovir, a cytidine nucleotide analogue, has broad-spectrum antiviral activity, including activity against genital warts. This multi-center prospective study evaluated the efficacy, safety and tolerability of topical cidofovir for treatment of PAIN2-3 in HIV-infected individuals. Methods: HIV-infected patients with biopsy-proven PAIN2-3 3 cm2 were eligible. Participants applied 1% topical cidofovir for 6 two-week cycles to lesion(s) for 5 consecutive days, followed by 9 days off-treatment. Lesion measurement and biopsy were performed 6 weeks after the last cycle. Results were scored as stable disease (SD), partial response (PR) (> 50% reduction), complete response (CR) or progressive disease (PD) based on size and histology. Logistic regression analysis was used to correlate clinical factors with responses. Results: 24 men and 9 women were enrolled. Mean age at baseline=44 years (range, 24-66), mean CD4+ count=412 cells/l (range, 2-1152) and pretreatment PAIN2-3 size=8.3cm2 (range, 3-21.3 ). HPV was identified in all evaluable perianal specimens; HPV16 was the most common type (67%). 26 evaluable participants completed treatment per protocol. CR:5; PR:11; SD:8; PD:2 (one superficially invasive cancer and one new PAIN2-3 lesion). The overall response (CR + PR) rate=49% (95% CI:31%-67%). CD4+ count and lesion size were not significantly related to overall response. 32/33 (97%) participants who received treatment had adverse events; most commonly perianal pruritis and ulceration (grades 1-2). The only serious adverse event possibly related to treatment was the one superficially invasive cancer. Conclusions: Topical cidofovir was well-tolerated and had activity in 49% of participants with PAIN2-3. Further evaluation of topical cidofovir for PAIN is warranted.
164

POOR SURVIVAL OF CERVICAL CANCER PATIENTS UNDERGOING RADIOTHERAPY IN KENYA.
O Maranga, Universities of Manchester & Nairobi, Nairobi, Kenya L Hampson, St Mary's Hospital, University of Manchester, Manchester, UK A W Oliver, St Mary's Hospital, University of Manchester, Manchester, UK X T He, St Mary's Hospital, University of Manchester, Manchester, UK H C Kitchener, St Mary's Hospital, University of Manchester, Manchester, UK P Gichangi, University of Nairobi , Nairobi, Kenya F Rana, Aga Khan University Hospital, Nairobi, Kenya A Opiyo, Kenyatta National Hospital, Nairobi, Kenya I N Hampson, St Mary's Hospital, University of Manchester, Manchester, UK Background: Globally there are 270,000 deaths from HPV related cervical cancer per annum, with over 85% of these occurring in low resource settings. Although it is the most common adult cancer in Kenya (18-23% of all cancer cases) the survival rate and factors influencing this are poorly defined. Objectives: To determine the tumour response and survival rate of cervical cancer patients attending for radiotherapy at Kenyatta National Hospital (KNH) and to analyse factors affecting such outcomes. Methods: Between 2008 and 2010, 355 patients with histologically confirmed invasive cervical cancer were recruited and followed up at KNH Departments of Radiotherapy and Gynaecology. Structured questionnaires were completed recording socio-demographics, HIV status, tumour response and survival following treatment with external beam radiation (EBRT), brachytherapy and adjuvant chemotherapy. Results: Of the 355 patients, 146 (42%) were lost to follow-up while 64 died during the two year period. 80.5% of patients presented with advanced stage IIB disease or above, with only 6.7% of patients receiving optimal combined EBRT, brachytherapy and adjuvant chemotherapy. The remainder could not afford the cost of complete treatment. As part of normal KNH patient management protocol, 189 women (53.2%) were given an HIV test prior to starting radiotherapy, with 52 (27.5%) testing positive of which 27 were already on HAART. Kaplan Meier survival curves projected two year survival at < 20%. Conclusion: Poverty, low educational level, lack of cancer awareness coupled with absence of regular screening programs, late patient presentation and sub-optimal treatments are major factors contributing to the alarmingly low survival rate of cervical cancer patients in Kenya. There is clearly a need to focus increased resources on both prevention and treatment for this neglected yet preventable HPV-related cancer in developing countries.
165
19 | Standard and experimental treatment P-19.09

HPV16-E6-E7 SILENCING BY SIRNA INDUCES DEATH OF CERVICAL CANCER CELLS
O PERALTA - ZARAGOZA, Instituto Nacional de Salud Pblica, Cuernavaca, Morelos, MEXICO J Salazar - Len, Instituto Nacional de Salud Pblica, Cuernavaca, Morelos, Mxico V Guadarrama - Prez, Instituto Nacional de Salud Pblica, Cuernavaca, Morelos, Mxico F De la O- Gmez, Universidad Autnoma de Guerrero, Chilpancingo, Guerrero, Mxico A Meneses - Acosta, Universidad Autnoma del Estado de Morelos, Cuernavaca, Morelos, Mxico H Merchant, Universidad Nacional Autnoma de Mxico, Mxico D. F., Mxico C Gmez - Cern, Instituto Nacional de Salud Pblica, Cuernavaca, Morelos, Mxico V H Bermdez - Morales, Instituto Nacional de Salud Pblica, Cuernavaca, Morelos, Mxico V Madrid - Marina, Instituto Nacional de Salud Pblica, Cuernavaca, Morelos, Mxico BACKGROUND: HPV infection is the main etiologic agent in cervical cancer. HPV16 covers 50% of the cases and E6 and E7 oncoproteins have properties of cell transformation and immortalization cells. Prophylactic and therapeutic strategies had been developed against cervical cancer with limited results to patients. The targeted inhibition of E6 and E7 oncogenes in tumor cells infected with HPV may provide a rational approach towards the development of novel anticancer therapies. OBJECTIVE: Using HPV16-transformed cells as a model system, we analyzed whether small interference RNAs (siRNAs) can silence HPV16 E6 and E7 oncogene expression and if can be employed in order to overcome the carcinogenesis process. METHODS: For this aim, we generated two siRNA expression plasmids (psiRNAE6 and psiRNAE7) in a DNA-vector, specifically directed against HPV16 E6 and E7 oncogenes. SiHa cells (HPV16) were transfected with these plasmids to evaluate the effect on E6 and E7 oncogenes silencing by real time and end point RT-PCR and western blot, flow citometry and electron microscope. RESULTS: Our results indicate a selective reduced expression of mRNA E6 and E7 oncogenes in HPV16 transformed cells. In addition, we observed inhibition of HPV16 E6 and E7 oncoproteins expression as well as functional effects in cell proliferation, an increase of p53 and hipophosphorylated pRb isoform protein expression, and autophagy and apoptosis features in SiHa cells by silencing of E6 and E7 oncogenes. CONCLUSIONS: Thus, these results suggest that HPV16 E6 and E7 oncogene expression can be repressed by siRNAs and this strategy may represent an alternative of treatment against the cervical cancer development.
P-19.10
INTRAVAGINALLY ADMINISTERED INTERFERON ALPHA-2B IN PATIENTS WITH CIN 1/2
G Cichon, Charite Universittsmedizin - Campus Benjamin Franklin, Berlin, GERMANY R Kurzeja, Charite Universittsmedizin - Campus Mitte, Berlin, GERMANY A Schneider, Charite Universittsmedizin, Berlin, GERMANY Background: Intravaginally administered Interferon alpha-2b in patients with CIN 1/2. Objectives: To determine the pharmacokinetic (PK) profile of Interferon alpha-2b Cream (2MIU/g, 5g) in women with a history of cytologically diagnosed Pap IIID, histologically confirmed CIN 1/2 and confirmed HPV+ status when applied every other day for 27 days. Secondary endpoints included efficacy and safety following treatment with 35 doses. Methods: A mono-centre, open label design with application of study drug over 10-14 weeks. Results: None of the patients showed systemic interferon levels above LLOQ (6.25 pg/ ml, by ELISA) at any time point following single dose administration or drug administration every other day for 27 doses. 71.4% (10/14) of the women resolved their CIN 1/2 lesions following treatment upon colposcopic directed biopsy. 5/13 patients improved from Pap IIID to Pap II. 2/14 experienced HPV clearance. In 5 women, interferon application was accompanied by mild local reactions, but in the majority treatment was tolerated without any side effects. Conclusions: Since no interferon levels above LLOQ could be detected in any of the serum samples, it can be concluded that no significant systemic exposure to interferon occurs with the investigated regimen and, therefore, no associated significant toxicological risks are to be expected. Efficacy results from all 14 patients were assessed. 10/14 patients with CIN 1 or 2 at baseline improved to normal or atypical histology. Pap and HPV response were less pronounced, presumably because the study did not employ a long enough post-treatment observation period to facilitate potent effects on the superficial cervix. Treatment was safe and well-tolerated with the most frequent AE being local mild or moderate application site reactions. The positive effects on CIN show a clear tendency for a substance related effect, worth studying in further prospective, randomized controlled trials.
Declaration of interest Prof. Schneider, Dr. Cichon and Dr. Kurzeja were Gynecological Assessors on Helix BioPharma's clinical study described herein. Prof. Schneider acts as a medical advisor to Helix BioPharma on its Interferon Alpha 2b product/program.
166
Standard and experimental treatment | 19 P-19.11

DERMAL DELIVERY OF INTERFERON ALPHA 2B USING BIPHASIXTM TECHNOLOGY
P Kumar, Helix BioPharma Corp., Saskatoon, CANADA D Michel, Helix BioPharma Corp., Saskatoon, CANADA R Batta, HelixBioPharma Corp., Saskatoon, Canada J Docherty, Helix BioPharma Corp., Saskatoon, Canada M Foldvari, University of Waterloo, Waterloo, Canada M King, HelixBioPharma Corp., Saskatoon, Canada Objectives: The aim of this study was to demonstrate localised dermal delivery of interferon alpha-2b(IFN2b) ecapsulated in a topical BiphasixTM cream. Interferon alpha 2b cream is being developed for the treatment of HPV infections (cervical cancer and genital warts). Methods: IFN2b solution (INTRONA) was administered intravenously (5 MIU), intradermally(10 individual injections: 0.5 MIU/50 L injection) or topically via IFN2b BiphasixTM cream: 1 g, 2 MIU/g). Skin and serum samples collected over time were analyzed using antiviral assay based on the protection of the cytopathic effect of viral insult on a cytokine-sensitive cell line. Half-life (t ) and elimination rates (K) were calculated for each treatment. Results: IFN2b was rapidly cleared from the serum after intravenous (t 0.75 hrs, K 0.9271 hr-1) and intradermal injections (t 1.28 hrs, K 0.5421 hr-1). IFN2b peaked in the serum 3 hr after intradermal application. Clearance from the skin after dermal injections was initially fast (t 0.62 hr, K 1.1179 hr-1), followed by a slower steady decrease after 6 hr. Application of biphasic IFN2b cream resulted in peak skin levels of IFN2b within 6-12 hr, followed by a slow steady decrease over time (63% decrease by 72 hr). IFN2b remained largely localised within the skin after topical application of biphasic IFN2b cream (2 MIU/g). No adverse observations were noted in guinea pigs after treatment with biphasic IFN2b cream, but dermal irritation was observed in 4 out of 100 animals that received multiple doses. Presence of encapsulated Interferon alpha2b was confirmed by confocal microscopy. Conclusions: Dermal application of IFN2b by biphasic TIFN2b cream resulted in sustained delivery of biologically active IFN2b localised within the skin of guinea pigs, with minimal levels of IFN2b being observed systemically. Thus BiphasixTM technology provides an efficient means of dermal delivery of macromolecules.
P-19.12
2LPAPI IMMUNE-MODULATING TREATMENT FOR HPV INFECTED PATIENTS IN LONG-TERM FOLLOWUP
S Mazzoli, Santa Maria Annunziata Hospital, STDs Centre, Firenze, ITALY T Cai, Santa Chiara Hospital, Dpt. of Urology, Trento, ITALY F Meacci, Santa Maria Annunziata Hospital, STDs Centre, Firenze, ITALY P Addonisio, Santa Maria Annunziata Hospital, STDs Centre, Firenze, ITALY P Dorfman, LaboLife , Gembloux, Belgium Background: HPV infections are not treatable at moment by antiviral compounds. Most infections are either latent or subclinical and the majority occurs as asymptomatic both in females and males. Certain HR-HPV types, HPV 16 and other HR-HPVs, are associated with increased rates of persistence: this occurs in more than 30% of our STDs Centre population studied, without any conventional therapies to offer. Micro-immunotherapy medications using high dilutions of cytokines and specific nucleotide sequences have been developed to treat targeted viral infections and are currently prescribed in medical practice as immune regulators: 2LPAPI (LaboLife) is dedicated for HPV infection and may represent a new therapeutic approach. Objectives: this exploratory study was to assess the impact of 2LPAPI on HPV infection eradication in HR-HPV infected asymptomatic patients attending our STDs Centre in a long-term microbiological follow-up population survey. Methods: patients of both genders, aged 18-55 years, diagnosed with HR-HPV infection, with no evidence of symptomatic HPV or ano-genital cancer, were followed for two years (2009-2010) by HPV-DNA and by E6-E7mRNA. HPV-positives were asked by their urology specialist if they wanted or not to be treated by 2LPAPI for 4 months. Globally 46 patients were included, 23 treated and 23 not treated. Results: 15/46 (32,6%) patients were lost at follow-up. Clearance of HR-HPV infections was 50% (8/16) in the treated group and 7% (1/15) in controls (p=0,01071) at the end of follow-up. Clearance of HPV-16 was more efficient in the treated group (60%, versus 0% in controls: p=0,022; 2=5,1;df 1). Conclusion: the relevant percentage of lost patients at follow-up reflects the scarce information and awareness of this infection within asymptomatic people in Italy. 2LPAPI was efficient in eradicating 50% of HR-HPV infections in treated patients, including 60% of HPV-16. These promising data emphasize the importance to redirect immune responses in viral infections.
167

PREGNANCY INCIDENCE AND OUTCOME AFTER TREATMENT OF CIN
P Nieminen, University of Helsinki, Helsinki, FINLAND I Kalliala, University of Helsinki, Helsinki, FINLAND T Dyba, Cancer Registry of the Finnish Cancer Registry, Helsinki, FINLAND A Anttila, Mass Screening Registry of the Finnish Cancer Registry, Helsinki, FINLAND T Hakulinen, Cancer Registry of the Finnish Cancer Registry, Helsinki, FINLAND M Halttunen, University of Helsinki, Helsinki, FINLAND P Nieminen, University of Helsinki, Helsinki, FINLAND Background: Several studies exist regarding pregnancy or delivery complications among CIN-patients. Studies about pregnancy incidence or outcome among these women are scarce and based on small samples. Objective: To study the effect of cervical intraepithelial neoplasia (CIN) treatment on pregnancy incidence and pregnancy outcome. Methods: Based on nationwide registers, 6 179 women treated for CIN between 1974 and 2001 and a randomly selected, age- and municipality-matched, reference population of 30 436 women were followed up for pregnancy outcomes until the end of 2004 or sterilization, emigration or death. Pregnancy incidence, that is live births, miscarriages, extra-uterine pregnancies, molar pregnancies, and terminations of pregnancies (TOPs) before and after CIN treatment, were estimated by calculating hazard ratios (HR) with stratified Cox- regression and Poisson regression. Results: After CIN treatment, both pregnancy incidence (HR 1.20, 95% confidence interval 1.15 to 1.26, p< 0.001) and incidence of live births (HR 1.12, CI 1.06 to 1.18, p<0.001) were higher among the treated women than among the reference population. Before treatment, only pregnancy incidence had been elevated among those treated (HR 1.06, CI 1.04 to 1.09, p<0.001). The incidence of extra-uterine pregnancies and of TOPs was significantly elevated among those treated both before and after CIN treatment. Conclusions: No clear evidence emerged of adverse effects due to the CIN treatment itself. The treated women had higher incidence of pregnancies and more children than did their reference population. TOPs and extra-uterine pregnancies had been more common among the treated already before CIN treatment. Reasons and frequencies of induced abortions (TOPs) vary between countries, and our results are valid for our own health care
Declaration of interest PN has been working as a consultant for GSK and Sanofi Pasteur-MSD. IK none declared. AA none declared. TD none declared. TH none declared. MH none declared.
P-19.14
PROSPECTIVE COMPARISON OF LOOP CONISATION UNDER COLPOSCOPIC-GUIDANCE VERSUS VITOM-GUIDANCE
G Vercellino, Charite-Universitaetsmedizin Berlin, Berlin, GERMANY E Erdemoglu, Zekai Tahir Burak Women's Education and Research Hospital, Ankara, TURKEY V Chiantera, Charit-Universitaetsmedizin Berlin, Berlin, GERMANY J Vasilieva, Charit-Universitaetsmedizin Berlin, Berlin, GERMANY I Drechsler, Charit-Universitaetsmedizin Berlin, Berlin, GERMANY S Frangini, Charit-Universitaetsmedizin Berlin, Berlin, GERMANY S Younes, Charit-Universitaetsmedizin Berlin, Berlin, GERMANY G Boehmer, MVZ wagnerstibbe, Bad Mnder, GERMANY Objective: Comparison of quality of conizations for cervical premalignant lesions under magnification using a colposcope versus a VITOM system with respect to intra and post-operative complications, positive resection margins, and cervical volume removed. Material and methods: Women referred to the Charit-Universitaetsmedizin CervixCentre, Berlin, Germany or to Colposcopy Clinic Wagner Stibbe, Bad Mnder, Germany, were included. Patients had cytologic and colposcopic or histologic confirmed high grade CIN. The VITOM system, consisting of the VITOM scope, xenon light source, HD camera system , AIDA HD documentation system, 1 monitor and a mechanical holder (Karl Storz, Tuttlingen, Germany), was used for video exocolposcopy. 100 patients underwent loop conization under colposcopic guidance (colposcopic group) and 100 patients under VITOM guidance (VITOM group). Results: Six patients in the VITOM group and no patient in the colposcopy group had a history of previous conisations. In the VITOM group there were 2 late and one early complications and the percentage of positive margins was 12%. The mean cervical volume removed was 1.2 (+/-0.8) cc per patient. In the colposcope group no complications were observed. Positive resection margin were present in 8% of treated women in this group and the mean cervical volume removed per patient was 1.3 (+/-0.5) cc. Conclusion: VITOM colposcopy is as safe and effective as standard colposcopic guided conization. The potential advantage include broader availability of endoscopic systems, involvement of trained gynecologic laparoscopic surgeons, video- documentation for quality control, all resulting in improvement of womens health.
168

CARRAGEENAN-GEL AGAINST TRANSMISSION OF CERVICAL HPV: THE CATCH TRIAL
J Tota, McGill University, Montreal, CANADA A Burchell, McGill University, Montreal, CANADA P Tellier, McGill University, Montreal, CANADA F Coutle, Universit de Montral , Montreal, CANADA E L Franco, McGill University, Montreal, CANADA Background and Objectives: NIH investigators have identified carrageenan (an anionic polymer that is naturally derived from red algae) as a potent HPV infection inhibitor. There has been interest in carrageenan as a vaginal microbicide targeting HIV and herpes viruses, but laboratory tests have found that it is much more effective against HPV. Other than vaccination, which confers type-specific protection and whose immunity benefits may wane, there is no other highly effective intervention against HPV infection. Our group at McGill University (Montreal, Canada) will begin a double-blind randomized controlled trial to evaluate the efficacy of carrageenan in (i) preventing new HPV infections and, (ii) accelerating clearance of existing infections. Methods: 465 sexually active female students living in Montreal will be randomized to receive either Divine 9 (treatment) or Divine 1 (placebo) vaginal gel. Participants will be asked to self-apply the gel they are assigned every other day for the first month and prior to each act of vaginal and anal intercourse during the entire study period (1 year). The primary outcomes are (i) presence of newly-detected vaginal HPV infections, and (ii) clearance of HPV infections observed at baseline. HPV DNA detection and genotyping of vaginal samples will be done at baseline/time 0, 14 days and 1, 3, 6, 9 and 12 months after enrolment using the Linear array assay with PGMY primers (Roche Diagnostic) to permit genotyping of 36 genital types. Participants will self-complete a computerized questionnaire at each clinic visit that measures known and potential HPV risk factors, as well as compliance with the intervention. Implications: If carrageenan is proven effective against HPV, this will have enormous public health implications. It will be a useful adjunct to HPV vaccination in developed countries, and immediately serve as a primary means of preventing HPV infection and cervical cancer in developing countries.
P-19.16
CERVICAL EXCISION PROCEDURES AND OBSTETRIC OUTCOME
C LIVERANI, UNIVERSITY OF MILAN, MILANO, ITALY E MONTI, UNIVERSITY OF MILAN, MILANO, ITALY D PUGLIA, UNIVERSITY OF MILAN, MILANO, ITALY F FANETTI, UNIVERSITY OF MILAN, MILANO, ITALY G LIBUTTI, UNIVERSITY OF MILAN, MILANO, ITALY S MANGANO, UNIVERSITY OF MILAN, MILANO, ITALY Objectives: The aim of this study was to evaluate the association between loop electrosurgical excision procedure (LEEP) and obstetric outcome in a large urban area population in northern Italy. Methods: Out of 61.730 women delivered at Mangiagalli Hospital (University of Milano, Italy) between 2000 and 2009, 608 had previously undergone LEEP for cervical intraepithelial neoplasia (CIN). Preterm singleton deliveries were 3.875 (6,3%) in the general population and 12 (2,4%) in women who underwent LEEP. Conclusions: Conservative cervical excisions for high-grade CIN do not seem to cause preterm birth when carried out under careful colposcopic guidance. Employing cervical conization techniques, a variable amount of healthy tissue is removed during the procedure; this is unnecessary for therapeutical purposes and may alter cervical function. In our clinical practice we usually adopt different surgical approaches depending on the extent of the lesion inside the cervical canal. Young women in child bearing age usually have smaller or larger HPV-related lesions who rarely extend into the cervical canal. Thus these cases are being treated with LEEP in a disk shaped manner. On the contrary in older women lesions often affect the endocervix and LEEP is performed in a rugby ball shape. The amount of tissue removed in these cases may be the same or even smaller than that of a disk shaped LEEP, but the physiology of the cervical canal is impaired. Since the vast majority of precancerous lesions of the uterine cervix occur in women less than 35 years of age, cervical excision procedures must take into account these important differences.
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STUDY OF SUPERPARAMAGNETIC IRON OXIDE NANOPARTICLES IN HELA CELL LINE.
P Rahal, So Paulo State University, So Jos do Rio Preto, BRAZIL M Calmon, So Paulo State University, So Jos do Rio Preto, Brazil N Candido, So Paulo State University, So Jos do Rio Preto, Brazil A Tofanello, So Paulo State University, So Jos do Rio Preto, Brazil S Taboga, So Paulo State University, So Jos do Rio Preto, Brazil G Nery, So Paulo State University, So Jos do Rio Preto, Brazil Cervical cancer is the second most common cancer in women worldwide, and the most common cause of death from cancer in women in the developing world. An association between human papillomavirus (HPV) infection and the development of cervical cancer was initially reported over 30 years ago and today there is overwhelming evidence that certain subtypes of HPV are the causative agents of these malignancies. The HPV 16 and 18 types are most commonly described in pre-neoplastic and cervical cancer cases. So, the aim of this study was to study the action of superparamagnetic iron oxide nanoparticles (SPIONs) in a cervical cancer cell line infected with HPV18. The nanoparticles were transfected in HeLa cell line in three different concentrations (10, 50 e 100g/mL) and after the transfection, Prussian blue staining, apoptosis assay and MTT assay were performed in the cell lines. Prussian blue stain of HeLa cells revealed abundant deep blue granules in the cytoplasm of cells treated with 100 g/mL and could also be found at lesser degree in cells exposed to MNPs at the concentration of 10 g/mL. In the apoptosis assay, the cells transfected with higher concentrations of SPIONs showed morphologic changes characteristic of apoptotic cells as nuclei fragmentation in apoptotic bodies. In the MTT assay, the cells with higher concentrations of SPIONs didnt proliferate as the cells with lower concentrations of SPIONs. So, probably, the SPIONs may interact with HPV oncoproteins and alter the growth behavior in this cervical carcinoma cell line.
P-19.18
RECRUITMENT TO A RANDOMISED TRIAL OF TOPICAL TREATMENT OF VIN3
A Tristram, Cardiff University, Cardiff, UNITED KINGDOM G Griffiths, Cardiff University, Cardiff, UNITED KINGDOM C Hurt, Cardiff University, Cardiff, UNITED KINGDOM T Madden, Cardiff University, Cardiff, UNITED KINGDOM S Ashman, Cardiff University, Cardiff, UNITED KINGDOM S Jones, Cardiff University, Cardiff, UNITED KINGDOM S Man, Cardiff University, Cardiff, UNITED KINGDOM N Powell, Cardiff University, Cardiff, UNITED KINGDOM A Fiander, Cardiff University, Cardiff, UNITED KINGDOM Background: Historically Vulval Intraepithelial Neoplasia (VIN) has been managed with surgery. However, this is often associated with significant physical and psychosocial morbidity, particularly in extensive disease and a high risk of recurrence. Several small studies have evaluated medical treatments and small, separate trials of two topical treatments (cidofovir and imiquimod) showed similar promising results. In order to assess these treatments in the same setting, a phase 2 randomised controlled trial was designed. Since VIN is an uncommon, a multicentre study was required in order to complete recruitment in a timely manner. Objectives: The aim of the trial is not to make a direct comparison between arms but to assess each arm for complete response rate by 30 weeks,feasibility of use and toxicity. A Flemings single stage design was assigned to each of the treatment arms and assuming a dropout rate of 15%, a total of 204 patients were required. The primary outcome measure is complete response. Secondary outcome measures are: toxicities, treatment compliance, HPV presence, type and integration status, and recurrence rate at two years. Methods: The trial was advertised widely through the British Gynaecological Cancer Society, the British Society for the Study of Vulval Disease and the British Society for Colposcopy and Cervical Pathology. The study is being run by the Wales Cancer Trials Unit. Results: Recruitment is ongoing. There are currently 24 open centres throughout the UK and 93 patients have been recruited. Conclusion: Alternatives to surgery need assesment. Randomised trials in uncommon conditions require multicentre studies that are challenging to set up, but can be achieved. The next challenge will be to set up an international phase 3 study.
170

GENITAL WARTS: CANADIANS PERCEPTION AND HEALTH RELATED BEHAVIORS
M Steben, Institut national de sant publique du Qubec, Montral, CANADA D Labelle, Graceway Canada Co., London, CANADA Background: A high level of anxiety is associated with a diagnosis of genital warts but health-related behaviours are less well known. Objective: To gauge the perceptions of Canadians toward genital warts and associated treatments. Methods: A survey supported by an unrestricted grant from Graceway Canada was conducted in February 2011 by Leger Marketing using their online panel (LegerWeb). It included 17 questions (2 multiple-choice, 15 4-point rating from strongly agree to strongly disagree]) relating to genital wart perception plus 9 demographic questions. Results: The survey was completed by 1,520 Canadian adults aged 18 to 75+ years, of which 52% were female. Fiftytwo percent of respondents stated that they would monitor an unrecognized spot on their genitals and only seek medical assistance if it did not go away. Only 43% said they would stop having sex until the spots were gone. While, only 10% of respondents stated they would not inform their partner, this was much higher among men (14%) than women (7%). Concerns of being judged by friends/family were high (44%), especially among younger (1834 y) Canadians (60%). Also, this younger demographic generally believed their self-esteem would dramatically decrease (69%), they would feel ashamed (72%), and they would be embarrassed to talk to their doctor (49%), if they contracted genital warts. Regarding prevention, 32% of respondents believed that monogamy would protect against genital warts, and 25% believed they are not at risk if they use a condom. Treatment preference was in favour of a cream rather than an invasive treatment (58%), particularly among younger (67%) and male respondents (63%). 60% would worry that genital warts could not be resolved and 44% that they would recur. Conclusion: Among Canadians, genital warts were associated with a fair degree of social stigma and potential negative impact on their psyche, especially for younger Canadians.
Declaration of interest I have received, in my own name, my companys name (Communications Action-Sant inc), or through HPV 2010 or Institut national de Sant publique du Qubec: Sponsorship, consultation fees, speaker honoraria and travel allocations for scientific presentation from Graceway Canada Co. Research grants, consultation fees, speaker honoraria and travel allocations for scientific presentation from Merck
P-19.20
LMV-601 INHIBITS HPV-16 INDUCED TUMOR GROWTH AND E7 EXPRESSION
M Schulz, Lumavita AG, Basel, SWITZERLAND H Pink, DKFZ, Heidelberg, GERMANY F Mayer, Lumavita AG, Basel, SWITZERLAND E Amtmann, DKFZ, Heidelberg, GERMANY Background: It had been shown previously that the phosphatidylcholine specific phospholipase C (PC-PLC) inhibitor LMV-601 inhibits HPV E7 expression in vitro, both in cells containing HPV genomes in extra-chromosomal and integrated state. Objectives: Demonstrate (1) the efficacy of LMV-601 in vivo by topical treatment of HPV-16 CaSki lesions (i.e. tumors) and (2) the efficacy of the clinical service form (cream) depending on drug concentration and treatment duration. Methods: CaSki cells were transplanted into the skin of nu/nu mice. After growth of lesions these were treated topically for up to 14 days with LMV-601 creams or vehicle. The effect of LMV-601 on HPV-16 E7 RNA expression was determined by Real Time PCR at 24 and 72 hrs. The effect of treatment on lesion size was followed over a treatment period of 14 days. Results: In the absence of significant inhibition of the GAPDH reference gene LMV-601 creams showed a rapid and dose-dependent inhibitory effect on HPV-16 E7 expression: 3.5% (1.0%) cream 6.8 (2) fold after 24 hrs and 119.4 (7.6) fold after 72 hrs. There was no significant effect of the vehicle cream on either GAPDH or HPV-16 E7 expression. After 14 days of treatment the ratio of the mean lesion size in the vehicle treated animals to those treated with the 3.5% cream was 0.52 (p=0.0034). Conclusion: The efficacy of LMV-601 against HPV-16 was proven in vivo with the clinical service form. Within the ranges investigated the efficacy correlated with dose and treatment duration. As in vitro anti-HPV-18 efficacy of LMV-601 had been proven previously a clinical program for topical treatment of HPV-16 and HPV-18 infection of the cervix has meanwhile been initiated.
171

EFFECT OF CONDOM USE AFTER CIN TREATMENT ON HPV POSITIVITY:ANRCT.
G Valasoulis, Department of Obstetrics & Gynaecology, University Hospital of Ioannina, Ioannina, Greece, M Kyrgiou, Department of Obstetrics and Gynaecology, Queen Charlotte's and Chelsea Hospital, Hammersmith Hospital, London, United Kingdom, S M Stasinou, University Hospital of Ioannina, Ioannina, Greece, M Arbyn, Unit of Cancer Epidemiology, Scientific Institute of Public Health, Brussels, Belgium, I Tsoumpou, Department of Obstetrics and Gynecology, University Hospitals of South Manchester, Manchester, United Kingdom, C Founta, Department of Obstetrics & Gynaecology, University Hospital of Ioannina, Ioannina, Greece, P Martin - Hirsch, Department of Obstetrics and Gynaecology, Central Lancashire Teaching Hospitals, Preston, United Kingdom, P Karakitsos, Department of Cytopathology, Attikon Hospital, University of Athens, Athens, Greece, G Koliopoulos, Department of Obstetrics & Gynaecology, University Hospital of Ioannina, Ioannina, Greece, E Paraskevaidis, Department of Obstetrics & Gynaecology, University Hospital of Ioannina, Ioannina, Greece Objective: to determine whether consistent condom use after treatment of CIN reduces HPV positivity postoperatively and possibly as a result, the risk of treatment failure. Materials & Methods: Design: Single-blinded randomised controlled trial. Inclusion criteria: Women planned to undergo LLETZ for CIN. Intervention: Women randomly allocated to Group A were given recommendation for condom use, whilst women in Group B received routine information. An LBC sample was tested for HPV typing, E6 & E7 mRNA (NASBA technique), E6 & E7 mRNA by flow cytometry, p16INK4a and microspectroscopy at 0 (pre-treatment), 6 and 12 months. A questionnaire to assess compliance was also completed. Outcomes: The relative risk (RR) and absolute RR (ARR) were calculated in an intention-to-treat analysis. The number needed to treat (NNT) and compliance were also assessed. Results: A total of 204 women were recruited, 98 in Group A and 104 in Group B. The positivity for all the tested markers at follow-up was significantly reduced in Group A (p<0.05). In particular, 32% (31/98) of women tested positive for HPV in Group A in comparison to 61% (65/104) in Group B [p<0.0001; RRR: -0.484; ARR: -0.297; NNT: 3). Consistent compliance with condom use was notably low (59%). Conclusions: Post-treatment condom use significantly reduces HPV positivity at 6 months and as a result possibly the risk of treatment failure. However, compliance, particularly after 6 months, is difficult. It remains uncertain whether HPV vaccination might be effective in women that test negative for HPV after treatment.
P-19.22
MULTIPLE ELECTROSURGICAL EXCISIONS TREATMENT FOR PRECANCEROUS CERVICAL LESION
A C Anton, Gr. T. Popa University of Medicine and Pharmacy, Iasi, ROMANIA C Cojocaru, Gr. Ghica Voda Hospital, Iasi, ROMANIA G Anton, Stefan S. Nicolau Institute of Virology, Bucharest, ROMANIA R Ursu, Gr. T. Popa University of Medicine and Pharmacy, Iasi, ROMANIA D Socolov, Gr. T. Popa University of Medicine and Pharmacy, Iasi, ROMANIA M Grigore, Gr. T. Popa University of Medicine and Pharmacy, Iasi, ROMANIA S Chelemen, Gr. Ghica Voda Hospital, Iasi, ROMANIA Objectives: The purpose of the analysis of the cases with cervical pre-cancerous lesions through LEEP and conizations, which requested repetition of conservatory treatment, is to establish: Diagnosis in one of the three categories: residual lesion, recurrent or re-infection. Facilitating risk factors and arguments for diagnosing the cases in the three categories, with the purpose to observe the evolutions. The role of the HPV typing and of the molecular markers. Method: Between the 1.01.2006 and 31.12.2010 in the II Gynecological Clinic Iasi, 518 LEETZ and conizations were realized, out of which 63 were re-interventions. The HPV typing was performed on 45 cases. Results: 23.7% of the cases were cervixes treated conservatory in other clinics, long before the analyzed period. 4.7% were diagnosed with invasive cervical carcinoma. 41% were re-interventions for residual lesions, which all had either the same histological diagnosis or an inferior one to the initial diagnosis; they were all practiced in the first post-surgery year. 25.3% are cases which did not respect the follow-up algorithm post-excision. The association of more viral types and the persistence of those HR types were found in 52% of the patients. 10% presented negative typing, discordant with histology. Conclusions: 1. The histopathology results on our cases with cervical re-interventions were diagnoses as residual lesions. HPV typing and molecular markers are useful investigations in the diagnosis and follow-up of the cases with repeated conservatory treatments for pre-cancerous cervical lesions.
172

HPV-DNA AND E6/E7 MRNA PROGNOSTIC FACTORS FOR EXPECTANT MANAGEMENT CIN2
L Zeferino, State University of Campinas-UNICAMP, Campinas, BRAZIL M Discacciati, State University of Campinas-UNICAMP, Campinas, BRAZIL I Silva, Federal University of Sao Paulo, Sao Paulo, BRAZIL L Villa, Ludwig Institute for Cancer Research, Sao Paulo, BRAZIL S Rabelo-Santos, Federal University of Goias, Goiana, BRAZIL L Reis, Salomo & Zoppi Laboratory, Sao Paulo, BRAZIL P Hayashi, Salomo & Zoppi Laboratory, Sao Paulo, BRAZIL M Costa, Ludwig Institute for Cancer Research, Sao Paulo, BRAZIL Background: CIN2 lesions are prevalent in young women and then more conservative management should be considered. Some CIN2 lesions may represent cytopathological effects of a productive HPV infection, but others may exhibit changes in viral gene expression similar to CIN3 lesions. Objectives: To evaluate the outcome for expectant management for women with biopsy-proven CIN2, and to verify whether DNA-HPV and E6/E7 mRNA at patient admission could be prognostic factors. Methods: This cohort study included 50 consecutive women with ASC-US/LSIL and biopsy-proven CIN2. These women were submitted to cervical cytology and colposcopy examinations every three months for 12 months. At admission, HPV-DNA genotyping was tested using reverse lineblot hybridization assay of 450-nt PCR amplicon for 18 high risk HPV (Roche Linear Array HPV Genotyping). E6/ E7 mRNA of HPV types 16,18,31,33 and 45 was tested using NucliSENS EasyQ HPV v1, (bioMrieux). Results: The CIN2 regression rates after 12-month follow-up were 69.4% for women with HPV species alpha-9, 91.7% for other HPV species or HPV negative, and this difference was statistically significant (p<0.05). Similar results were observed for HPV 16. The CIN2 regression was 68.3% for women with positive E6/E7 mRNA, 82.0% for negative E6/E7 mRNA, but this difference was not statistically significant. Negative E6/E7 mRNA expression for HPV 16 seemed predicted better the CIN2 regression at short-term (3-month follow-up) than long-term outcome (6 to 12-month followup), but these findings were not statistically significant. Conclusions: The observed high rate of CIN2 regression could be due to the inclusion of women with previous LSIL, that could select lesions representing cytopathological effect of a productive HPV infection; HPV species alpha-9 and HPV 16 infection are associated with higher CIN2 progression rate; E6/E7 mRNA was not associated to CIN2 outcome but the sample statistical power was low for conclusive analysis.
P-19.24
HPV IN LYMPH NODE AS A PROGNOSTIC MARKER OF RECURRENCE
J Gonzalez - Montoya, Instituto Nacional de Cancerologia, Mexico, MEXICO J J Zermeo Nava, Instituto Nacional de Cancerologa , Mexico, MEXICO S Bernal Silva, Universidad Autnoma de Nuevo Len, Monterrey, MEXICO M Guido Jimnez, Universidad Nacional Autnoma de Mxico , Mexico, MEXICO M D Prez Montiel, Instituto Nacional de Cancerologa , Mexico, MEXICO H A Barrera Saldaa, Universidad Autnoma de Nuevo Len, Monterrey, MEXICO A Garca Carranc, Instituto Nacional de Cancerologa, Mexico, MEXICO D F Cant de Len, Instituto Nacional de Cancerologa, Mexico, MEXICO In early surgically treatment stages of cervical cancer where ganglionic state is the main prognostic factor, there is however a percentage (10% - 15%) of women who show unexpected recurrence despite of being lymph node negative by histopatological diagnostic. Although some well-known factors (age, ganglionic state, tumor size, invasivity depth) explain a percentage of recurrence, many times there is homogeneity among them and not everyone present recurrence. In the present study, we sought to investigate the relation between recurrence and HPV presence in lymph node. Our main goal was to identify if HPV could be used as an early predictive marker for early stages of cervical cancer recurrence. The case-control study was constructed with surgically treated women who underwent radical hysterectomy and bilateral pelvic lymphadenectomy, the groups we formed by recurring and non-recurring women. Every patient included were lymph node negative for metastases and identical for clinical and histological factors. Samples were obtained by a retrospective study in our hospital tumor bank. HPV genotyping of formalin-fixed paraffin-embedded tissues were achieved by multiple genotyping with INNO-LiPA (Innogentics) technology. As expected, VPH-16 was the most prevalent in primary tumor, present in 71.4% of recurring and 80% of non-recurring patient. Co-infections in primary tumors were found in 23.8% of both groups. The presence HPV presence in lymph node was 42%, being more prevalent in recurring (52.3%) than in nonrecurring (33.3%) women. HPV-16 was also the most prevalent in lymph node, being higher in recurring (38%) than in non-recurring (28.5%) women. Multiple infections prevalence was null in the non-recurring group, while a 14.3% was found in recurring women. Disease-free period and survival was 16.45 and 50 months for lymph node HPV positive patients vs. 18.9 y 65.7 months for lymph node HPV negative patients
173

HPV FOLLOW-UP OF CIN 1 OR LESS AT ONE YEAR
M Gold, Vanderbilt University Medical Center, Nashville, TN, UNITED STATES V Mazzoni, Vanderbilt University Medical Center, Nashville, TN, UNITED STATES Objectives: To evaluate the Vanderbilt University Medical Center experience with HPV follow-up of women with a histologic diagnosis of CIN 1 or less preceded by ASC-US, ASC-H, or LSIL cytology by reporting demographic data, pathology, treatment, and clinical outcomes. Methods: Retrospective chart review. Inclusion criteria were patients with a histologic diagnosis of CIN 1 or less preceded by ASC-US, ASC-H, or LSIL cytology managed with repeat HPV testing in 12 months. Inclusion was limited to women who underwent initial colposcopy after January 1, 2007. Patients were identified through use of CPT and ICD-9 billing codes. Statistical analysis performed using SAS, version 9.2. Results: Univariate analysis using chi square or Fishers exact tests demonstrated HPV positivity rates at one year for smokers to be 79%, compared to 52% for non-smokers; 75% positivity for patients with biopsy pathology results of CIN I compared to 44% for those with biopsy results less than CIN I; and HPV positivity for patients 23 years old and younger to be 75% as compared to 51% for patients older than 23. Multivariate analysis using a logistic regression model confirmed association with HPV positivity at one year for smokers and those with biopsy pathology of CIN I. Conclusion: HPV testing alone at one year for patients who are smokers or have biopsy pathology of CIN I is not adequate or cost-effective surveillance.
P-19.26
LOPINAVIR OR ZINC-FINGER EJECTING COMPOUNDS AS TREATMENT FOR HPV-RELATED LESIONS?
I Zehbe, Thunder Bay Regional Research Institute, Thunder Bay, CANADA C Richard, Thunder Bay Regional Health Sciences Centre, Thunder Bay, CANADA K F Lee, Thunder Bay Regional Research Institute, Thunder Bay, CANADA M Campbell, Thunder Bay Regional Research Institute, Thunder Bay, CANADA L Hampson, University of Manchester, Manchester, UK I N Hampson, University of Manchester, Manchester, UK Background: Non-surgical, antiviral treatment options are desirable for HPV-related lesions within the genitourinary and upper digestive tract. Objectives: We compared the toxicity of three zinc finger-ejecting (ZFE) compounds (4,4-dithiodimorpholine, azodicarbonamide and diamide) to the HIV protease inhibitor lopinavir using HPV-positive SiHa, CaSki, HeLa, ME180 and HPV-negative C33A cervical carcinoma cell lines as well as primary human foreskin keratinocytes (PHFKs. Methods and Results: Colorimetric growth assays revealed selective toxicity when treated with lopinavir. All carcinoma cell lines, except CaSki, were sensitive to 20M lopinavir whereas primary PHFKs were highly resistant. In contrast, 4,4-dithiodimorpholine was uniformly toxic to all cells tested while azodicarbonamide and diamide showed no effect at all. Conclusion: Lopinavir may be an attractive candidate to treat pre-cancerous and cancerous HPV-positive lesions.
174

DOSE ESCALATION TRIAL EXAMINING SAFETY AND TOLERABILITY OF INTRAVAGINAL CURCUMIN
L Flowers, Emory University, Atlanta, UNITED STATES BACKGROUND: Curcumin is a major active component from turmeric, a spice used for its anti-inflammatory and anti-infectious properties that has been shown in oral doses to have anticancer activity and cause regression of premalignant lesions. OBJECTIVE: The objective of this study was to assess the safety, pharmacokinetics of intravaginal curcumin in HIVseronegative women with normal Pap tests. METHODS: A Phase 1 dose escalation trial was conducted in a group of HIV-seronegative women with Pap tests negative for cervical intraepithelial lesions. Subjects were given one of four doses of curcumin powder: 500 mg, 1000 mg, 1500 mg, 2000 mg packed in gelatin capsules intravaginally everyday for a 14 day treatment period. Four study visits were performed on all subjects (Screening, Day 0, Day 7, and Day 14). For each visit a pelvic exam, colposcopy exam, labs, and adverse events were performed and documented. Data for curcumin pharmacokinetics was collected on Day 7 study visit. Thirteen subjects completed the trial. RESULTS: Twenty-three adverse events occurred during the course of the trial. The adverse events fell in 5 categories: genital pruritus (23% of subjects), vaginal discharge (100%), vaginal dryness (15%), abnormal PT (23%), and hypokalemia (8%). Curcumin and curcumin conjugates were not detectable in any plasma sample. Curcumin and curcumin conjugates were detected in the day 7 urine samples but were below 100 ng/ml. All events were grade 1 and no dose limiting toxicities were experienced. Of these events, 56% were definitely related to the study drug and 17% were possibly or probably related to the study drug. All events were resolved by the end of the study in an average of 8.7 days. CONCLUSIONS: Intravaginal curcumin was shown to be safe, with a favorable pharmacokinetic profile and welltolerated by all patients.
Declaration of interest I do not have any financial interest in the product or research in this abstract
P-19.28
D1000 APLICATION IN CERVIX DECREASES THE VIRAL LOAD: 41 CASES
G VALERIO TELLO, HOSPITAL , Mexico, D.F., MEXICO E DRAGUSTINOVIS RUIZ, HOSPITAL , Mexico, D.F., MEXICO A GANEM RONDERO, FES CUAUTITLAN, Mexico, D.F., MEXICO G LARA FIGUEROA, HOSPITAL "DR. MANUEL GEA GONZALEZ" , Mexico, D.F., MEXICO Background: The cervical cancer (cc) is associated among 99.7% of the cases with high risk Human Papilloma Virus (hr-HPV) infection, is a important cause of mortality by cancer in women in developing countries. The persistent infection cause cervical intraepithelial neoplasy (CIN) which across time evolution to CC. 12% of CIN-1 progresses and 32% remains constantly, 27 % of CIN-2 progresses and 35% persists, 12% of CIN-3 progresses and 56% remains constantly. We have been proposed a new compound (D1000), for management this persistent infection which are the causes of CIN anda cancer. D-1000 have effects anti-viral and anti-carcinogenic properties. Objective: To evaluate the effect of D-1000 in patients who are only infected with HPV or have CIN. Material and methods: Doses of D1000, between 250-500mg. were applied in patients from 7 to 15 days of compound gel. The viral load was measure with Hybrid Capture-II at the beginning of the study and after of 15-21 days post-treatment and the cytology was performed with negative report. Result: The treatment report result with the D-1000 (thermo reversible gel) in 41 patients. This is the viral load before and after the treatment: 23.14/0.16; 241/0.18; 2306/0.72; 2306.26/0.27; 5.59/0.09; 377.51/0.30, 271.92/0.18; 264.52/0.11; 6.41/0.25; 23.14/0.13; 1.25/0.52; 4.32/0.09; 187.76/0.91, 1336.49/0.12; 1.32/0.35; 78.56/0.16; 2.81/0.74; 1.9/0.16; 31.6/0.16; 254.52/0.36; 12.02/0.17; 1588.14/0.22; 103.54/9.23; 2068.42/94.83; 16.71/0.78; 48.51/0.09; 237.37/0.27; 1.29/0.22; 3.88/0.43; 1788.02/0.21; 310.12/0.16; 209.22/2.26; 798.23/0.11; 11.43/2.17; 242.49/0.17; 2.64/0.23; 700.41/2.18; 526.24/0.30; 546.83/0.21; 43.54/0.22; 783.47/0.18 URL. Conclusion: The decreased viral load observed in this cases lead our spectatives to use D-1000 as a therapeutic for persistent infections caused by the HPV-HR. We can interfere in the natural history of this disease that without treatment will increase the risk for develop CIN or cervical cancer.
175

THERAPEUTIC APPROACHES TO RECURRENT HPV GENITAL WARTS: A CASE REPORT
E Dobao, Santa Casa da Misericordia do Rio de Janeiro, Rio de Janeiro, BRAZIL A F Nicol, LiPMED - IOC - Fiocruz, Rio de Janeiro, Brazil N S Oliveira, LiPMED - IOC - Fiocruz, Rio de Janeiro, Brazil W R Menezes, LiPMED - IOC - Fiocruz, Rio de Janeiro, Brazil C Pires, Santa Casa - RJ, Rio de Janeiro, Brazil B Kawa Kac, Santa Casa - RJ, Rio de Janeiro, Brazil S Amaro - Filho, Santa Casa - RJ, Rio de Janeiro, Brazil S M B Cavalcanti, UFF - RJ, Rio de Janeiro, Brazil J A C Nery, Santa Casa - RJ, Rio de Janeiro, Brazil Background: Coinfection with HIV and HPV increases the risk for genital intraepithelial neoplasia, and this increase reflects the severity of immunosuppression. Objective: To report the clinical course of one HIV patient with extensive penial condylomatosis in relation to treatment modalities. Report: A male, 24 years old, HIV-seropositive, under HAART, CD4+ cell counts between 240 to 296 cells/mm, came to our clinic with the diagnostic of condyloma acuminatum and showed a verrucous lesion, 1.5 cm in diameter, in glans near the frenulum along with several small lesions less than 3mm. He was treated at this time with surgical resection and eletrocauterization of the base of the larger lesion, and only eletrocauterization of the smallest. After 60 days recurrent lesions appeared with the same size and appearance and also there were a new lesion 2cm in diameter near the frenulum. A new eletrocauterization of larger lesions was done with prescription of Imiquimod in the minors lesions. After 10 weeks there was a strong local reaction to the drug with pruritus, rash, eczema, erythema and great pain without changing the appearance of all lesions and recurrence of the greater. After healing this reaction we did a new eletrocauterization in one big lesion and 90% trichloroacetic acid on the other. After 30 days the patient returned without local change. Then we opted for a mass decrease with 90% acid trichloacetic acid and getting the local use in the next day of Podophyllotoxin to prevent recurrence. After 7 days all lesions were disappearing and in 20 days the patient returned completely healed without any lesion or injury in the penis. Conclusions: The present report described a low cost, fast and effective treatment to recurrent HPV lesions in immunocompromised patient.
Declaration of interest no conflict to declare
176
Session 20: Oral poster presentations 201-236

Oral poster presentation abstracts
Oral poster presentations 201-236 | 20 OP-201

METRONOMIC CYCLOPHOSPHAMIDE ENHANCES ANTITUMOR EFFECT OF THERAPEUTIC HPV DNA VACCINE
S Peng, Johns Hopkins University, Baltimore, UNITED STATES S Lyford - Pike, Johns Hopkins Univ, Baltimore, UNITED STATES C Hung, Johns Hopkins Univ, Baltimore, UNITED STATES T C Wu, Johns Hopkins Univ, Baltimore, UNITED STATES S Pai, Johns Hopkins Univ, Baltimore, UNITED STATES Human papillomavirus (HPV) is etiologically associated with 20 to 25% of head and neck squamous cell carcinomas (HNSCC), with 60-70% of those tumors localized to the oropharynx. HPV-16 DNA vaccines have been developed that enhance specific CD8+ T cell responses against the HPV viral oncoproteins E6 and E7. We report high frequencies of inhibitory CD4+CD25+Foxp3+ T cells in the tumor microenvironment of patients with HPV-HNSCC. These regulatory T cells (Treg) may inhibit the potency of vaccine-induced anti-tumor effects. Low dose cyclophosphamide (CTX) has been shown to have immune-potentiating activity by depleting the Treg population. In the current study, we evaluated the role as well as the optimal dosing regimen of CTX in combination with a therapeutic HPV DNA vaccine. TC-1 tumor-bearing mice received CTX administration in combination with weekly pNGVL4a-CRT/HPV16 E7(detox) vaccination by electroporation. Tumor volumes were monitored twice a week and survival was recorded. HPV-16 E7-specific CD8+ T cell responses were analyzed with E7aa49-57 peptide loaded H-2Db tetramer or IFN- intracellular staining and flow cytometry. Tregs were analyzed by CD4, Foxp3 and CD25 staining. We compared single low dose (50 mg/kg) administered one day prior to vaccination to metronomic daily dosing of CTX (10 mg/kg). We found that combination of therapeutic HPV DNA vaccine with either CTX administration protocol generated significantly enhanced antitumor effect and improved survival of HPV-tumor-bearing mice as compared to individual modality therapy alone. However, combination of therapeutic HPV DNA vaccine with daily low dose CTX generated enhanced antitumor effects than combination with single low dose prior to vaccination. Depletion of Tregs by CTX and preferential accumulation of E7-specific CD8+ T cells in the tumor contributed to this enhanced antitumor effect. This data provides support for the combination of daily low dose CTX and therapeutic HPV DNA vaccines to treat HPV-positive HNSCC patients.
OP-202
CIN IS ASSOCIATED WITH CHANGES IN GENITAL MUCOSAL IMMUNE ENVIRONMENT
M Mhatre, Albert Einstein College of Medicine, Bronx, NY, UNITED STATES T Mc Andrew, Albert Einstein College of Medicine, Bronx, NY, UNITED STATES C Carpenter, Albert Einstein College of Medicine, Bronx, NY , UNITED STATES R D Burk, Albert Einstein College of Medicine, Bronx, NY , UNITED STATES B C Herold, Albert Einstein College of Medicine, Bronx, NY, UNITED STATES M H Einstein, Albert Einstein College of Medicine, Bronx, NY, UNITED STATES Background: Papillomaviruses may modulate the local host immunity to favor its survival, and recent data suggests that HPV is associated with an increased risk of acquiring other STIs, including HIV. The biological mechanisms, however, are not well-understood. We hypothesize that women with CIN have an altered genital mucosal immune environment, which may facilitate HIV acquisition and HPV persistence. Methods: We conducted a pilot study comparing soluble immune mediators and endogenous antimicrobial activity in genital tract secretions collected by CVL in a cohort of HIV- women with CIN-1 (n=12), CIN-3 (n=37), or PAP- controls matched for age (n=57). CVLs were clarified and supernatants evaluated for concentrations of cytokines, chemokines, antimicrobial peptides and total protein and endogenous anti-E. coli activity, a potential biomarker of intact mucosal host defense. CIN with HPV types 16,18,31,45,and 52 were chosen after HPV typing on cell pellets using MY09/MY11 PCR. Results: Women with CIN-1 and CIN-3 displayed significantly higher levels of pro-inflammatory cytokines and chemokines including IL-1, IL-1, and IL-8 (p<0.01). Women with CIN-3 also had higher levels of RANTES (p<0.01). Conversely, women with CIN-1 and CIN-3 displayed significantly lower levels of anti-inflammatory mediators including IL-1 receptor antagonist and secretory leukocyte inhibitor (SLPI) (p<0.01) and lower levels of protective immune mediators such as human beta defensins 2 and 3 (p< 0.02). Women with CIN-3 had the lowest endogenous anti-E. coli activity (p<0.02). Conclusion: Investigation of mucosal immune mediators in the genital tract indicates that oncogenic-HPV infected CIN is associated with an increase in inflammatory and loss in protective immune mediators. These changes are consistent with the mucosal environment described in women who seroconvert to HIV and suggests that HPVassociated dysplasia disruption of local immunity may contribute to an increased risk of HIV acquisition and other STIs.
179
20 | Oral poster presentations 201-236 OP-203

HUMAN PAPILLOMAVIRUS16 E2 PROTEIN REGULATES TUMOR NECROTIC FACTOR-ALPHA GENE EXPRESSION
N Sunthamala, Khon Kaen University, Khon Kaen, THAILAND, C Pientong, Khon Kaen University, Khon Kaen, THAILAND, B Kongyingyoes, Khon Kaen University, Khon Kaen, THAILAND, A Chaiwongkot, Khon Kaen University, Khon Kaen, THAILAND, P Kleebkaow, Khon Kaen University, Khon Kaen, THAILAND, N Patarapadungkit, Khon Kaen University, Khon Kaen, THAILAND, B Chumworathayee, Khon Kaen University, Khon Kaen, THAILAND, T Ekalaksananan, Khon Kaen University, Khon Kaen, THAILAND Background: Cervical cancer is a major cause of mortality in women worldwide and relies on high risk human papillomavirus (HR-HPV) infection. Tumor necrotic factor (TNF) -alpha, a pro-inflammatory cytokine, the expression of which is induced by pathogen infection and stress conditions, plays an important role in innate immunity and triggers various immuno-modulator gene expression. This cytokine had been decreased in HPV infected patients. However, the associated factor which regulates TNF-alpha gene expression in HPV associated cancer remains elusive. Objective: The aim of this study is to investigate the effect of viral HPV16 E2 protein on the TNF-alpha gene expression. Methods: Seventy-four fresh cervical tissue specimens with various histopathological diagnosis (18 No-SIL, 49 LSIL, and 7 HSIL-CA) were determined for HPV16 E2 and TNF-alpha gene expression by real-time PCR. In vitro study was performed to elucidate the effect of HPV16 E2 to TNF-alpha gene expression by transfection of 4 HPV16 E2 expression vectors containing HPV16 E2-wild type and mutants to SiHa and C33A cell lines. Results: The level of HPV16 E2 expression in each histopathological grade were significantly different (p<0.05). TNF-alpha expression was suppressed in 45.45% in No-SIL cases with HPV16 E2 positive. This suppression displayed significant difference in each group (p<0.05). The result of transfection determined by real-time PCR using HPV16 E2 primers showed no significant difference between cell types and vector types (p>0.05). TNF-alpha expression was strongly suppressed by both HPV16 E2-wild type and mutants (p<0.05). Conclusion: The result suggested that TNF-alpha expression in early stage of infection might be affected by HPV16 E2 protein and might alter the immune environment, particularly immune against HPV infection leading to persistent infection and cancer progression. The regulatory functions of HPV16 E2 protein to TNF-alpha expression should be further determined.
OP-204
EVALUATION OF HPV-SPECIFIC CROSS-REACTIVE CD4+ MEMORY T CELL RESPONSES
M van den Hende, Leiden University Medical Center, Leiden, NETHERLANDS M J P Welters, Leiden University Medical Center, Leiden, NETHERLANDS C J M Melief, Leiden University Medical Center, Leiden, NETHERLANDS G G Kenter, Leiden University Medical Center, Leiden, NETHERLANDS S H van der Burg, Leiden University Medical Center, Leiden, NETHERLANDS R Offringa, Leiden University Medical Center, Leiden, NETHERLANDS Background and Objective: Cellular immune responses in relation to progression and regression of HPV related diseases have been studied intensively. HPV16-specific T cell responses are frequently detected in healthy subjects, but largely absent in patients with HPV16-positive anogenital neoplasia. HPV16, 31, 33, 35, 52 and 58 belong to clade A9 and share up to 70% nucleotide homology, suggesting immunological cross-reactivity may exist. Crossprotective immunity to related HPV types is described for humoral immune responses. No data is available on cross-reactivity in HPV-specific cellular immunity. Insight in this matter is important for correct interpretation of analysis of HPV specific T-cell responses in relation to health and disease and to inform us whether the impact of therapeutic HPV-specific vaccines would only be type specific or target multiple HPV types. Methods: Memory T cell responses against the E6 oncoprotein of the HPV types of clade A9 were evaluated by IFNELISPOT assays in peripheral blood mononuclear cell cultures of 44 healthy donors and 10 HPV16-positive patients who participated in our HPV16E6/E7 immunotherapeutic vaccination trial. Potential cross-reactivity of HPV-specific cellular immune responses was studied in detail in HPV16 E6-specific T cell clones (N=14) from healthy donors (ICS, proliferation assay, ELISA). Results: Overlapping peptide arrays showed one-half of the responding healthy subjects, as well as vaccinated patients displaying reactivity against corresponding E6 peptides from 2 or more related HPV types. This suggested immunological cross-reactivity between the E6 antigens. Further dissection of T cell responses by means of HPV16enriched and clonal T cell cultures using whole protein antigens, revealed that CD4+ T-cells capable of efficiently reacting against E6 antigen from multiple HPV types are rare and only occur when epitope sequences are highly conserved. Conclusion: Natural and vaccine-induced HPV16E6-specific CD4+ T-cell responses are unlikely to mediate efficient cross-protection against other clade A9 members.
180

QUADRIVALENT HPV VACCINATION INDUCES NEUTRALIZING ANTIBODIES IN ORAL MUCOSAL FLUIDS
A Handisurya, National Institutes of Health, NCI , Bethesda, USA V Gruber, Medical University of Vienna, DIAID, Vienna, Austria T Senger, German Cancer Research Center (DKFZ), Heidelberg, Germany C Schellenbacher, Medical University of Vienna, DIAID, Vienna, Austria R Kirnbauer, Medical University of Vienna, DIAID, Vienna, Austria Background: Infection with HPV16 and to a lesser extent HPV18 is associated with the development of a subset of head and neck squamous cell carcinomas (HNSCC), in particular oro-pharyngeal cancer. HPV vaccination induces neutralizing antibodies in sera and cervical secretions that prevent anogenital HPV infection and disease. Objectives: To determine whether quadrivalent (q) HPV (Gardasil) vaccination induces neutralizing antibodies in oral mucosal fluids (OMT). Methods: OMT and corresponding serum samples were collected from healthy female volunteers (n=20; age: 19-25 yrs) prior (day 0) and after 3x qHPV vaccinations (month 7), and from non-vaccinated controls (n=14; age: 22-26 yrs). Eligible participants reported fewer than five lifetime sexual partners and no history of previous anogenital HPV infection. Sera and OMT were analyzed for HPV16- and HPV18-specific antibodies by VLP-ELISA and pseudovirion (PsV)-based neutralization assays (PBNA). Results: All vaccinees seroconverted to HPV16 and HPV18 with titers up to 64,000 as determined by VLP-ELISA; non-vaccinated controls lacked HPV-specific antibodies. In OMT samples HPV16- and HPV18-specific antibodies were detected in 60% and 25% of the vaccinated participants, respectively. To determine the proportion of neutralizing antibodies OMT samples were diluted 1:4 to 1:16 and subjected to PBNA. To account for non-specific assay inhibition by oral fluids, post qHPV results were compared to those obtained from samples collected prior to vaccination from the same individual. OMT samples from 55% vaccinated participants neutralized HPV16 PsV, and 30% neutralized HPV18. OMT samples from non-vaccinated controls were not neutralizing, with the exception of one control (7%) becoming positive against HPV18 after the study period of 7 months. Conclusions: Following qHPV vaccination neutralizing antibodies against HPV16 and HPV18 are detected in oral fluids in a proportion of vaccinated individuals. This suggests that HPV vaccination may also confer protection against oral infection with high-risk HPV types involved in the development of HNSCC.
Declaration of interest none
OP-206
HEXAMINOLEVULINATE PHOTODYNAMIC THERAPY OF PATIENTS WITH CIN 1
P Soergel, Hannover Medical School, Hannover, GERMANY C Dannecker, University Clinic Grohadern, Munich, GERMANY K U Petry, Hospital Wolfsburg, Wolfsburg, GERMANY P Collinet, University Clinic Lille, Lille, FRANCE I Runnebaum, University Clinic Jena, Jena, GERMANY P Hillemanns, Hannover Medical School, Hannover, GERMANY Background: Cervical HPV-induced lesions affect seven million women in Europe and the US each year. Current treatment of precancerous lesions involves invasive surgical procedures to remove the precancerous lesions, which can result in the unnecessary removal of normal cervical tissue. Besides discomfort and recovery time, this can affect the patients ability to carry a child full-term, impact fertility and increase the risk of post-surgical infections. For a large number of patients with persistent HPV infections and low-grade cellular abnormalities, there are no currently available non-surgical treatment alternatives, and frequent follow up visits to avoid further disease progression causes patient anxiety and adds to the burden for the health care system. Objectives: To assess the effectiveness of photodynamic therapy (PDT) with hexaminolevulinate (HAL) in patients with CIN 1. Methods: 70 patients with CIN 1 received HAL 5% suppositories or placebo or follow-up only in a double-blinded 4 : 1 : 1 randomisation. PDT was carried out after five hours with a light dose of 50J/cm2 for both HAL and placebo patients. If cytology after one month was ASCUS or greater, a re-PDT was carried out. Six months after last PDT, final follow-up was evaluated. Results: At six months, CIN 1 lesions were completely cleared in 57% of women in the treatment arm as compared to 25% in the control group (p<0.05). In the active treatment arm of the study, the HPV response rate correlated with the lesion response in 86% of the women. Conclusions: PDT of low-grade CIN has the potential to offer patients and gynaecologists an easy, safe and highly selective treatment, thus preserving normal surrounding tissue.
Declaration of interest P. Soergel received travel grants from companies involved in photodynamic therapy. P. Hillemanns received travel and research grants from companies involved in photodynamic therapy.
181

GENE EXPRESSION CHANGES AFTER CIDOFOVIR TREATMENT OF PERIANAL INTRAEPITHELIAL NEOPLASIA
M M Da Costa, University of California, San Francisco, San Francisco, UNITED STATES E A Stier, Boston Medical Center, Boston, UNITED STATES J M Palefsky, University of California, San Francisco, San Francisco, UNITED STATES For the AMC 046 team OBJECTIVE: Cidofovir has been used to treat genital warts with some success but the mechanism of action of cidofovir against HPV-infected tissue is unknown. We characterized changes in gene expression after treating perianal intraepithelial neoplasia (PAIN) with cidofovir. METHODS: Participants enrolled in the AIDS Malignancy Consortium 046 Study, which used topical cidofovir to treat PAIN 2-3 in HIV-infected patients, had pre-treatment biopsies clinically consistent with PAIN 2-3. One % cidofovir ointment was administered topically for 6 cycles with each cycle consisting of 5 consecutive days of treatment followed by 9 days without treatment (3 months). Six months after baseline, the area of treatment was re-biopsied. The biopsy specimens were flash-frozen. RNA was extracted and then hybridized to the Affymetrix HG U133A 2.0+ chip. Histopathologic assessment of the frozen specimens was done to confirm diagnosis. RESULTS: Analysis included matched biopsy pairs from 3 participants with PAIN 2-3 at baseline who had reverted to PAIN 1 or normal after cidofovir treatment. Compared with the pre-treatment biopsies, 184 genes were up-regulated and 363 genes were down-regulated by a factor of 2 or more. There was a significant increase in expression (p<.05 unadjusted for multiple testing) of 6 of 18 genes involved in the complement activation Wikipathway including MASP1, C3 and C6. Eight of 35 genes were up-regulated (p<.05 unadjusted for multiple testing) in the inflammatory response Wikipathway, including IL2, IL4, interferon-gamma. CONCLUSION: Immune response genes showed the largest increase in expression after treatment with cidofovir in the patients who responded to treatment. These data suggest that the effect of cidofovir on PAIN 2-3 may be mediated in part through modulation of immune response.
OP-208
NOVEL HPV MICROBICIDES: THEIR POTENCY IN CELLS AND IN MICE
H Huang, University of Wisconsin-Madison, Madison, United States D Pyeon, University of Wisconsin-Madison, Madison, United States P Ahlquist, University of Wisconsin-Madison, Madison, United States P Lambert, University of Wisconsin-Madison, Madison, United States The future incidence of cervical cancer is forecast to decline because of the remarkably effective prophylactic vaccines against human papillomaviruses. However, lack of access to these expensive vaccines in the developing countries where cervical cancer is most frequent, and the restricted genotypes these vaccines protect against, will limit their impact. Clearly, there is still a need for identifying other modalities for preventing HPV infections. Ready access to effective, inexpensive microbicides represents one potentially valuable approach to the prevention of genital HPV infections. We developed a well-validated high throughput screening (HTS) assay for identifying compounds that inhibit HPV infection and applied this assay to identify lead compounds that are to be applied topically and act by inhibiting viral binding or uptake. We have screened approximately 43,000 small molecules that are available at the University of Wisconsin Small Molecule Screening Facility (UW-SMSF). The top 20 compounds were chosen for further analyses based upon the pharmacological property, scaffold diversity, strength of the inhibitory activity and lack of nonspecific cytotoxicity. Of these compounds, compounds 13 and 14 had the most acceptable properties of submicromolar IC50 and little to no cytotoxicity. Optimal microbicidal activities were elicited by exposure of cells to the compound 13 and 14 during the initial 12 hours following infection. Twenty-nine 13-like and twelve 14-like analogs were identified in silico and tested for their microbicidal activities corresponded to the altered structures comparing to compounds 13 and 14. The efficacy of compounds 13 and 14 in vivo was assessed using a mouse model for HPV infection.
182

MRNA TESTING IN THE FOLLOW-UP AFTER CONISATION
A Trop, Akershus University Hospital, LRENSKOG, NORWAY C M Jonassen, Akershus University Hospital, LRENSKOG, NORWAY K D Sjborg, Oestfold Hospital Trust, Fredrikstad, NORWAY M Nygrd, Cancer Registry Norway, Oslo, NORWAY F A Dahl, Akershus University Hospital, LRENSKOG, NORWAY G C Alfsen, Akershus University Hospital, LRENSKOG, NORWAY A K Lie, Norwegian Radium Hospital, Oslo, NORWAY Background. A combination of cytology and HR HPV DNA testing has shown a 100% sensitivity to detect residual CIN2+ after conisation. To our knowledge, no published studies have analysed HR HPV mRNA and DNA testing during follow-up after conisation. Objective. To evaluate testing for high-risk human papillomavirus (HR HPV) E6/E7 mRNA transcripts 6 months after conisation for CIN2+ to determine the risk of residual CIN2+. Methods. We prospectively followed 344 women treated for CIN2+ by conisation. HR HPV mRNA testing (PreTect HPV-Proofer, NorChip), HR HPV DNA testing (AMPLICOR HPV Test, Roche Diagnostics) and cytology was performed at 6 and 12 months after conisation. Biopsies were taken within 18 months of conisation if indicated by abnormal cytology, abnormal colposcopy, or positive HPV test. The LINEAR ARRAY HPV Genotyping Test (Roche Diagnostics) was used to genotype the cases with histologically confirmed residual disease diagnosed within 18 months after conisation. Results. 6.4% (22/344) of study women had detected residual CIN2+. Among women with detected residual CIN2+, 54.5% (12/22) had positive resection margins, 63.6% (14/22) had abnormal cytology, and 95.5% (21/22) had a positive HR HPV DNA test at 6 months. Sensitivity of HR HPV mRNA testing was 45.5% (95% confidence interval: 26.8-65.5%) at 6 months to predict detected residual CIN2+. Eight of 12 women who were HR HPV mRNA-negative at 6 months were HR HPV DNA-positive for one of the HPV types included in the mRNA test. Conclusion. Detection of E6/E7 mRNA transcripts by PreTect HPV Proofer does not seem suitable for short-term follow-up to detect residual CIN2+ after conisation.
OP-210
HIGH LOAD OF HR-HPV: PREDICTOR FOR PRESENCE OF CERVICAL LESIONS
M Schmitt, German Cancer Research Center (DKFZ), Heidelberg, GERMANY C E Depuydt, RIATOL, Sonic Healthcare Benelux, Antwerp, Belgium I H Benoy, RIATOL, Sonic Healthcare Benelux, Antwerp, Belgium J Antoine, Scientific Institute of Public Health, Brussels, Belgium M Pawlita, German Cancer Research Center (DKFZ), Heidelberg, GERMANY M Arbyn, Scientific Institute of Public Health, Brussels, GERMANY Background: Infections with high-risk human papillomaviruses can cause malignant transformation of the human cervical epithelium. HPV DNA tests generally are very sensitive to detect cervical neoplastic lesions but also identify transient HPV infections. As a consequence, the specificity and positive predictive value is low. Viral load assessment could improve clinical specificity. Objective: In this study, we present the clinical accuracy of two quantitative HPV assays to identify CIN2+. Methods: We analyzed viral load of all high-risk and possibly high-risk HPV types over 7 orders of magnitude (on a log10 scale) in 999 consecutive cervical smears from women participating in cervical cancer screening in Belgium enriched with ASC-US (n=100), LSIL (n=100) and HSIL (n=97) using a type-specific multiplex real-time qPCRs and the BSGP5+/6+-PCR/Multiplex HPV Genotyping (MPG) assay. Results: Both assays showed a strong correlation with respect to type-specific viral load. Using an empirically determined viral load cutoff for 14 high-risk HPV types, the sensitivity for prevalent CIN2+ was reduced slightly (qPCR, from 98.4 to 93.8%; BSGP5+/6+-PCR/MPG, from 98.4 to 95.3%) compared to the minimal threshold. The specificity for absent disease (corresponding to double negative cytology at subsequent screening episodes) increased substantially (qPCR, from 89.6 to 96.2%; BSGP5+/6+-PCR/MPG, from 80.5 to 96.1%). There was no significant difference in mean viral load between LSIL and HSIL. Conclusions: Type-specific HPV-DNA assays show flexibility in defining thresholds and targeting HPV types, which could optimize clinical accuracy for cervical cancer precursors.
Declaration of interest Part of this work was supported by Qiagen.
183

EVALUATION OF HPV MOLECULAR TESTS IN SCREENING FOR CERVICAL CANCER
J Levi, University of Sao Paulo, Sao Paulo, BRAZIL A Longatto - Filho, Fundao Pio XII, Barretos, Brazil J Eluf, University of So Paulo, So Paulo, Brazil C Rodrigues, University of So Paulo, So Paulo, Brazil C Oliveira, University of So Paulo, So Paulo, Brazil A Cruvinel, Fundao Pio XII, Barretos, Brazil M Tacla, University of So Paulo, So Paulo, Brazil J Fregnani, Fundao Pio XII, Barretos, Brazil A Saber, University of So Paulo, So Paulo, Brazil L Villa, University of So Paulo, So Paulo, Brazil Background: Incorporation of HPV tests into cervical cancer screening programs may be advantageous over conventional cytology especially in developing nations, where the largest incidence of cervical cancer is observed. In Brazil, incidence and mortality rates due to this cancer continue to be high despite the existence of screening programs which in general have unsatisfactory population coverage and poor quality control. Objectives: We conducted a comparative evaluation of commercially available molecular HPV tests and cervical cytology in Brazilian women attended at two large cervical cancer screening clinics. Methods: Two groups of women were recruited at the Hospital das Clnicas of the School of Medicine of the largest public university in So Paulo and Hospital do Cncer de Barretos, Brazil. Group A consisted of 2098 patients under routine screening. Group B was composed of 511 women at-risk for developing cervical neoplasia with a previous abnormal Pap test. Cervical samples were collected using SurePath liquid cytology, and split into aliquots which were submitted to the following HPV tests: APTIMA (Gen-Probe), Cobas 4800 (Roche) and Hybrid Capture High-risk probes (Qiagen). Moreover, all positive samples were genotyped by the Papillocheck kit (Greiner Bio-One). Results: Prevalence of HSIL was 1% on Group A and 6.7% on Group B. HPV tests results are as follows: 8% and 22.7% of the groups A and B respectively were positive for High-risk Hybrid Capture assay. Correspondingly, the Cobas 4800 resulted in 6.6% and 31.4% positive samples while APTIMA revealed 4.3 % and 12.5%. Rate of invalid tests was very low for all three methods. Conclusions: Molecular screening is feasible and represents an attractive tool for decreasing CC incidence in Brazil. Evaluation of the sensitivity and specificity of each method is underway.
Declaration of interest Villa LL received honoraria and research support from Qiagen and Roche Mol Diagnostics. Levi JE received honoraria (speaker fee) from Qiagen and Roche Mol Diagnostics.
OP-212
HUMAN PAPILLOMAVIRUS IN ANAL SPECIMENS DETECTED USING HYBRID CAPTURE 2.
S Rus, Qiagen Inc, Gaithersburg, MD, UNITED STATES B Lowe, Qiagen Inc, Gaithersburg, MD, UNITED STATES G Chen, Qiagen Inc, Gaithersburg, MD, UNITED STATES H Salim, Qiagen Inc, Gaithersburg, MD, UNITED STATES T Rothmann, Qiagen Inc, Gaithersburg, MD, UNITED STATES S E Goldstone, Qiagen Inc, Gaithersburg, MD, UNITED STATES I Nazarenko, Qiagen Inc, Gaithersburg, MD, UNITED STATES Background. HPV-related anal cancer is increasing in men and women, but especially in men who have sex with men. Similarities in anatomy and etiology within HPV-related anal and cervical disease may warrant similar diagnostic screening. The hc2 HPV DNA Test (HC2) is FDA-approved and proven in detecting HPV infections of the cervix and as an aid in diagnosing, with Pap, cervical disease. Presented applications are for research only, not for use in diagnostic procedures. Methods. This prospective study (n=292) determined that HC2 signal and HC2 agreement with a histological endpoint may be useful to diagnose anal specimens collected under various conditions. Results. HC2 sensitivity was 91% for co-collected specimens in a Sample Transport Medium and 85% for specimens collected in a liquid-based cytology (LBC) medium processed by a Gyn method (not significant difference, p=0.1264). HC2 sensitivity was significantly lower (62%, p=0.0135) for LBC processed by a Non-Gyn method, and HC2 signal and specimen cellularity were reduced. The HC2 agreement was similar for specimens collected by brush or swab, but median HC2 signal was 6X higher with the brush. Agreement was similar for first or second order collections for brush or swab, but second-collection, swab (217 mean RLU/CO) specimens had significantly lower HC2 signal (p=0.0108) than first collection (356 mean RLU/CO), and the swab cellularity was lower. Some specimens had maximum stool quality (10%, subjective). Swab specimens (not brush) with maximum stool had decreased agreement and HC2 signal, which may warrant the use of the brush or a cleaning swab. Presence of maximum stool decreased the HC2 signal in model experiments. Consensus Gp-PCR confirmed that the 13 HPV probe types in HC2 were optimal for performance. Conclusions. The feasibility of HC2 to detect HPV in anal specimens is indicated, further research may indicate the use of HC2 for anal cancer screening.
Declaration of interest This study was funded by Qiagen, all authors are employees of Qiagen, except S.G. of Mount Sinai Medical Center, N.Y., N.Y.
184

METHYLATION AT HPV18 E2 BINDING SITES INCREASES DURING CERVICAL CARCINOGENESIS
T W Leung, The University of Hong Kong , Hong Kong , China S S Liu, The University of Hong Kong , Hong Kong , China A N Y Cheung, The University of Hong Kong , Hong Kong , China H Y S Ngan, The University of Hong Kong , Hong Kong , China Background: There are four E2 binding sites (E2BSs) at the LCR region of HPV18 genome. Binding of E2 protein to different E2BSs may exert either transcriptional activation/repression on E6 and E7 oncoprotein, depending on their distance from the TATA box of the promoter. Methylation status at the E2BSs may affect the relative binding of E2 protein to them and may change during cervical carcinogenesis. Objectives. To investigate and compare the methylation status of E2BSs 1, 2 and 4 in HPV18-positive cervical cancer, high-grade CIN, low-grade CIN/ASCUS and normal cervical epithelium. Methods. DNA was extracted from paraffin sections of 24 cases of HPV18-positive cervical cancer, as well as from cytology swabs / paraffin sections of 11 cases of HPV18-positive high-grade CIN, 12 cases of low-grade CIN/ ASCUS and 7 cases of normal cervical epithelium. Methylation percentage of individual CpGs at E2BS 1, 2 and 4 were assessed by pyrosequencing. The average percentage of CpG methylation at each E2BS was calculated and compared among different histological groups. Results. HPV18 E2BS 1&2 remains more methylated than E2BS 4 in all histological groups. Both HPV18 E2BS 1&2 and E2BS 4 methylation increases significantly from ASCUS/ low-grade CIN to high-grade CIN (2.7% to 6.9% , p=0.019 and 1.2% to 6.7%, p=0.001 respectively) and then further increases form high-grade CIN to cervical cancer (6.9% to 55.4%, p<0.001 and 6.7% to 23.4%, p=0.005 respectively). Conclusions. Differential methylation at different E2BSs was observed in HPV18-postive cervical cancer, cervical premalignant lesions and normal cervical epithelium. Increasing methylation at HPV18 E2BSs is a potentially useful adjunctive molecular marker for predicting progression from ASCUS / low-grade CIN to high-grade CIN and from high-grade CIN to cervical cancer.
OP-214
HPV52 GENOME METHYLATION IN ASYMPTOMATIC INFECTION AND CERVICAL NEOPLASIA
I Murakami, School of Medicine, Keio University, Tokyo, JAPAN T Fujii, School of Medicine, Keio University, Tokyo, JAPAN A Ohno, School of Medicine, Keio University, Tokyo, JAPAN M Saito, School of Medicine, Keio University, Tokyo, JAPAN T Iwata, School of Medicine, Keio University, Tokyo, JAPAN D Aoki, School of Medicine, Keio University, Tokyo, JAPAN Background and Objectives; In previous studies examining HPV16 and HPV18 genome methylation, the authors concluded that the methylation status of the L1 gene might be a biomarker for neoplastic progression (Badal et al., 2003; Turan et al., 2006; Kalantari et al., 2010). HPV52 is a major cause of cervical intraepithelial neoplasia (CIN) and invasive squamous cell carcinoma (SCC) in Asia, especially in Japan. This study investigated whether the methylation status of HPV52 was correlated with asymptomatic infection and cervical neoplasia. Methods; Clinical samples positive for HPV52 from women with cervical cervicitis (n=8), CIN1 (n=9), CIN2 (n=15), CIN3 (n=22), and SCC (n=1) were used to investigate the methylation status of the HPV52 L1 gene, the long control region (LCR), and the E6 gene. The HPV genome isolated from the clinical samples was examined using bisulfite modification, PCR amplification, and sequencing. Results; The methylation status was determined for the HPV52 L1 gene, the LCR, and the E6 gene CpGs in all the samples using bisulfite sequencing. The CpGs were unmethylated or methylated in only one sample in the HPV52 LCR and E6 gene. However, the CpGs were hypermethylated in the HPV52 L1 gene. Moreover, the methylation frequencies of the HPV52 L1 gene were related to the severity of cervical neoplasia, with average rates of 15%, 34%, and 52% for cervical cervicitis and CIN1, CIN2, and CIN3 and SCC, respectively. Conclusions; These results indicate that increased methylation at the CpG sites in the HPV52 L1 gene are correlated with the severity of cervical neoplasia. The HPV52 genome methylation status may constitute a potential biomarker for HPV52-associated neoplastic progression.
185

BIOMARKER OVERLAY OF PATHOLOGY FOR DISEASE GRADING AND HPV ASSIGNMENT
J Doorbar, MRC National Institute for Medical Research, London, UNITED KINGDOM H Griffin, MRC National Institute for Medical Research, London, UNITED KINGDOM Y Soneji, MRC National Institute for Medical Research, London, UNITED KINGDOM A Khan, Whittington Hospital, London, UNITED KINGDOM R Jach, Jagiellonian University Medical College, Krakow, POLAND G Dyduch, Jagiellonian University Medical College, Krakow, POLAND K Oko, Jagiellonian University Medical College, Krakow, POLAND Z Wu, MRC National Institute for Medical Research, London, UNITED KINGDOM T Freeman - Wang, Whittington Hospital, London, UNITED KINGDOM A Singer, Whittington Hospital, London, UNITED KINGDOM High-risk HPV infections are graded on the basis of pathology. CIN2+ lesions are routinely treated, while lower grade lesions are not. Despite its importance in patient management, pathology grading is highly subjective and has proved difficult to standardise. Classification of CIN2, and the discrimination between viral and non-viral CIN1 can be particularly problematic. To improve accuracy and reduce subjectivity, molecular markers can be used to distinguish low-grade disease from transforming infections. Here we have used a six-colour staining-approach to correlate five biomarkers of HPV infection with pathology as determined by H&E staining on the same tissue section. This overlay technique allows correlation of molecular pathology based on viral gene expression with conventional pathology based on cell morphology. It appears that a combination of just two classes of marker, which discriminate between early and late life-cycle events, can allow establishment of disease severity and HPV causality. The first marker-class (MCM, p16 and Ki-67) is already in widespread use, and help identify cells driven into cycle by E7. The extent to which they persist above the basal layer correlates with CIN grade. The second marker-class includes E4, which becomes massively expressed as E7 levels decline during the switch to the late stages of the virus life cycle. Its presence in CIN1 and CIN2 can conclusively identify HPV causality and can be used to confirm HPV type. Thus, the presence of E7-surrogate markers at the epithelial surface reveals a need for treatment irrespective of the infecting HPV type. E4 at the epithelial surface indicates lower-grade disease, HPV causality and possibly also HPV type. The mapping of lesion-surface expression patterns is now allowing development of an in situ staining approach for the non-invasive identification of lesion grade and position prior to colposcopy.
OP-216
HPV CAPSID PROTEINS (L1 AND L2) IN SQUAMOUS INTRAEPITHELIAL LESIONS
A Yemelyanova, Johns Hopkins University, Baltimore, UNITED STATES P E Gravitt, Johns Hopkins University, Baltimore, UNITED STATES B M Ronnett, Johns Hopkins University, Baltimore, UNITED STATES A Ogurtsova, Johns Hopkins University, Baltimore, UNITED STATES R B S Roden, Johns Hopkins University, Baltimore, UNITED STATES Background: While cervical cancer screening relies on cervical cytology and high-risk HPV detection, histologic diagnosis, and specifically lesion grade, is the main parameter that drives clinical management of the screenpositive women. Morphologically diagnosed squamous intraepithelial lesions (SIL) regress spontaneously in more than half of the cases. Lack of major capsid protein L1 expression has been shown to be a feature of progressive lesions. Expression of the minor L2 protein in clinical lesions has not been extensively evaluated. Objectives: The goal of this study is to evaluate immunohistochemical expression of L1 and L2 in SILs in correlation with lesion grade. Methods: 101 SILs were selected based on HPV 16 or 18 detection by RT-PCR. These included 54 low-grade SILs (LSIL/CIN1) and 47 high-grade SILs(23 HSIL/CIN2 and 24 HSIL/CIN3). The presence and grade of SIL was confirmed by thorough histologic review and immunohistochemistry for p16. Immunohistochemical expression (nuclear staining) of L1 and L2 was assessed with MAB 837 for L1 and RG-1 for L2. Results: Co-expression of L1 and L2 was detected in 18/101 (18%) cases (all grades included), including 17/54 (32%) LSIL/CIN1 and 1/23 (4%) HSIL/CIN2. L1 and L2 were co-expressed in the same area of the lesion in these cases. Neither L1 nor L2 was expressed in 24 HSIL/CIN3 cases. In addition, 7 CIN1 lesions had discordant expression of L1 and L2 (L1 alone in 2 and L2 alone in 5). Conclusions: HPV capsid proteins are expressed almost exclusively in LSIL/CIN1. Additional studies are necessary to establish the utility of L1 and L2 expression as predictors of lesion behavior.
186

THE PREDICTIVE VALUE OF THE HPV MLPA-ASSAY IN CERVICAL CYTOLOGY
R Litjens, Maastricht University Medical Centre, Maastricht, NETHERLANDS M Ummelen, Maastricht University Medical Centre, Maastricht, NETHERLANDS W Theelen, Maastricht University Medical Centre, Maastricht, NETHERLANDS J Ossel, PathoFinder BV, Maastricht, NETHERLANDS M Reijans, PathoFinder BV, Maastricht, NETHERLANDS G Simons, PathoFinder BV, Maastricht, NETHERLANDS E Speel, Maastricht University Medical Centre, Maastricht, NETHERLANDS F Ramaekers, Maastricht University Medical Centre, Maastricht, NETHERLANDS R Kruitwagen, Maastricht University Medical Centre, Maastricht, NETHERLANDS A Hopman, Maastricht University Medical Centre, Maastricht, NETHERLANDS Background: The current screening programs (cytology and/or high risk HPV typing) for uterine cervical cancer have a low positive predictive value (PPV) for the presence of CIN 2 or higher. Therefore new parameters are needed. Objectives: The HPV multiplex ligation dependent probe amplification (MLPA) assay detects HPV type 16/ 18/31/33/45, viral load, viral integration and gain of telomerase associated genes in a single reaction. We determined the impact of viral load and viral integration of HPV16 and 18 on the PPV of the assay in cervical cytology. Methods: Cytological specimens (n=170) were analyzed with the MLPA assay. The GP5+/6+ assay and qPCR were used as a control for high risk HPV typing. The results were correlated with the matching histology. Results: A total of 170 samples were analyzed. Matching histological follow-up was available in 127 samples. With increasing severity of both the cytology and the matching histology, the MLPA showed an increasing percentage of high risk classification. The HPV MLPA assay was positive (high viral load and/or viral integration) in 39 cases, 31 of these had a CIN 2 lesion or higher (positive predictive value of 79%). In comparison cytology and/or high risk HPV typing had a positive predictive value of 55%. The negative predictive value of the HPV MLPA-assay was 69%. Conclusion: The HPV MLPA-assay determines HPV type 16/18, viral load and viral integration in cytological specimens. A high positive predictive value for the detection of CIN 2 or higher lesions was found. The addition of viral load and viral integration strongly improves the positive predictive value in comparison to the conventional methods (cytology and/or HPV typing).
Declaration of interest J. Ossel, PathoFinder BV, employee M. Reijans, PathoFinder BV, employee G. Simons, PathoFinder BV, CEO
OP-218
HUMAN PAPILLOMAVIRUS AND P16INK4A IN THE HEAD AND NECK REGION
M Reuschenbach, University of Heidelberg, Heidelberg, GERMANY, P Elena, University of Heidelberg, Heidelberg, GERMANY, C Toth, University of Heidelberg, Heidelberg, GERMANY, F Mueller, University of Giessen, Giessen, GERMANY, S Wagner, University of Giessen, Giessen, GERMANY, G Dyckhoff, University of Heidelberg, Heidelberg, GERMANY, K Freier, University of Heidelberg, Heidelberg, GERMANY, S Vinokurova, University of Heidelberg, Heidelberg, GERMANY, J P Klussmann, University of Giessen, Heidelberg, GERMANY, M von Knebel Doeberitz, University of Heidelberg, Heidelberg, GERMANY Background: High risk human papillomavirus (HR-HPV) has been acknowledged as a causal factor in a proportion of head and neck squamous cell carcinomas (HNSCC). Diffuse overexpression of p16INK4a in HR-HPV-positive HNSCC is commonly observed, but little is known on HR-HPV prevalence in conjunction with p16INK4a expression in premalignant and non-malignant epithelia of the head and neck region. Objectives: In order to investigate whether there is evidence for transforming HPV-infections already at noninvasive stages of dysplastic squamous epithelia in the head and neck region, we determined the prevalence of HR-HPV infection in normal, premalignant and malignant tissue of the head and neck region and correlated it to p16INK4a/Ki-67 co-expression. In transformed and thus proliferating cells of the uterine cervix p16INK4a is overexpressed, while physiological p16INK4a expression is restricted to cell cycle arrested cells. Accordingly, cervical HR-HPV transformed cells can be identified by simultaneous detection of p16INK4a and the proliferation marker Ki-67 in the same cell. Methods: Normal (n=50), premalignant (n=34) and malignant (HNSCC; n=148) head and neck tissue was evaluated using p16INK4a/Ki-67 immunohistochemistry. 14 HR-HPV genotypes were detected. Results: 8% (4/50) of normal, 41.2% (14/34) of premalignant head and neck samples and 14.2% (21/148) of HNSCC were HPV-positive. In normal tissue no p16INK4a/Ki-67 co-expressing cells were detected. p16INK4a/Ki-67-positive cells were identified in 28.6% of HPV-positive and 20.0% of HPV-negative premalignant samples and in 60.0% of HPV-positive and 3.1% of HPV-negative HNSCC. Conclusions: Co-expression of p16INK4a/Ki-67 in the same cell is a hallmark of neoplastic conversion as it is found in premalignant head and neck tissue and in HNSCC but not in normal epithelia. Our findings indicate association of p16INK4a/Ki-67 expression with HR-HPV in HNSCC. A small percentage of samples, particularly among premalignant tissues however show p16INK4a/Ki-67 co-expression without being HPV-positive, necessitating further analysis on the relevance of this finding.
Declaration of interest mtm Laboratories donated antibodies for the study. Magnus von Knebel Doeberitz is shareholder and member of the supervisory bord of mtm Laboratories.
187

HPV DETECTION AND GENOTYPING IN SELF-SAMPLES STORED ON FTA CARTRIDGE
R van Baars, VU Medical Centre, Amsterdam, NETHERLANDS D G Geraets, DDL Diagnostic Laboratory, Voorburg, NETHERLANDS I Alonso, Hospital Clinic, Barcelona, SPAIN J Ordi, Hospital Clinic, Barcelona, SPAIN A Torne, Hospital Clinic, Barcelona, SPAIN W J Melchers, Radboud University Nijmegen Medical Center, Nijmegen, NETHERLANDS W G V Quint, DDL Diagnostic Laboratory, Voorburg, NETHERLANDS Background: Cervical cancer incidence could be decreased by offering cervicovaginal self-sampling to nonresponders in screening programmes. Storage of vaginal self-samples on the Indicator FTA-elute cartridge (FTA) provides easy transportation and simple DNA elution for HPV testing. Objectives: To study HPV detection and to quantify DNA amounts in self-collected and physician-obtained cervical smears. Methods: Specimens were collected from women (n=189) referred for colposcopic evaluation because of an abnormal smear or for hrHPV detected in a first screening. Prior to colposcopy, women self-collected a cervicovaginal smear using a Vibabrush that was subsequently applied on FTA. Then a conventional cervical smear was obtained by a physician and suspended in ThinPrep medium. After DNA elution (FTA) or extraction (ThinPrep), hrHPV detection was performed using the SPF10-DEIA-LiPA25 and the clinically validated GP5+/6+-PCR-EIA. Quantification of the amount of DNA using a human reference gene was performed with qPCR. Clinical data were included to investigate clinically relevant differences in hrHPV detection. Results: The hrHPV positivity by both SPF10-DEIA-LiPA25 and GP5+/6+-EIA was significantly higher in Thinprep (resp. 78% and 63%) compared to FTA (68% and 51%)(p=0.007 and p<0.0001). The DNA quantity was also significantly higher (p<0.0001) for ThinPrep (median number of cells in original specimen 21040000; mean Ct=24.66) compared to FTA (median number of cells in original specimen 176556; mean Ct=28.53). In women with <CIN2 (n=141) and CIN2 (n=48), the hrHPV detection rate by GP5+/6+-EIA was significantly increased in Thinprep (resp. 51% and 98%) compared to FTA (40% and 85%) (p=0.002 and p=0.031). Conclusions: A self-obtained sample with a Vibabrush on FTA does not seem to be effective for HPV detection compared to physician taken smears. This seems to be in contrast to previous published reports and might be due to the way the cervicovaginal smears was obtained by the patient. Alternative self-collection methods need to be investigated.
Declaration of interest Non declared
OP-220
ADVERSE EFFECTS OF ANAL CANCER SCREENING STRATEGIES IN HOMOSEXUAL MEN
A Grulich, University of New South Wales, Sydney, Australia I M Poynten, University of New South Wales, Sydney, Australia F Jin, University of New South Wales, Sydney, Australia R Hillman, University of Sydney, Sydney, Australia D Templeton, University of New south Wales, Sydney, Australia C Fairley, Melbourne Sexual Health Centre, Melbourne, Australia S Garland, Royal Women's Hospital, Melbourne, Australia K Mc Caffery, University of Sydney, Sydney, Australia Background: Anal cytology, high resolution anoscopy (HRA), and digital anal examination (DAE) have been proposed as screening tools for anal cancer. Objectives: To determine the psychological morbidity and other adverse effects associated with DAE, anal Pap tests and HRA in a community-based cohort of homosexual Australian men. Methods: The Study for the Prevention of Anal Cancer (SPANC) is a prospective cohort study, aiming to recruit over 500 homosexual men in Sydney, Australia. All participants undergo DAE, anal Pap test, HRA and behavioural questionnaires at baseline and follow-up. Psychological and adverse affects questionnaires are completed at 2 weeks and 3 months post-HRA. Participants will be followed up every 6 months for 36 months. Results: By May 2011, 77 men were enrolled (39.0% HIV positive). No serious adverse events have occurred. Moderate pain was more commonly associated with the HRA (32.8%), than the anal swab (15.5%) or the DAE (5.2%). After HRA, 56.9% reported a little or some bleeding while 5 (8.6%) men reported a lot of bleeding. Bleeding lasted a few days in 52.6% of men and a few weeks in 2 men (5.3%). There was no change in the proportion of participants reporting psychological distress over time. Increased psychological distress was not significantly associated with a diagnosis of high grade intra-epithelial neoplasia (HGAIN) (p=0.270) but men diagnosed with HGAIN were significantly more likely to worry about developing anal cancer compared to men who were not diagnosed with HGAIN (p = 0.022). Conclusions: Preliminary study results demonstrate that moderate pain and bleeding were common after HRA. Thus far, psychological distress was not significantly related to a diagnosis of HGAIN, however men with HGAIN had elevated anal cancer worry. The moderate adverse effects and psychological impact associated with HRA may affect its utility and acceptability as a screening tool.
188

HIGH ACCEPTABILITY OF A MODIFIED LAVAGE DEVICE FOR SELF-SAMPLING
J A M Coebergh van den Braak, Mxima Medical Centre, Veldhoven, Netherlands C J M Verhoeven, Mxima Medical Centre, Veldhoven, Netherlands A T Hesselink, VU University Medical Centre, Amsterdam, Netherlands D A M Heideman, VU University Medical Centre, Amsterdam, Netherlands B W J Mol, Amsterdam Medical Centre, Amsterdam, Netherlands Introduction: Offering self sampling for HPV testing will increase the coverage of cervical screening programmes. High-risk HPV detection in self-collected cervico-vaginal samples using a lavage device (Delphi Screener, Pantarhei Screener) has shown to be reliable. Based on interviews and users feedback the Delphi Screener was modified to further optimize user comfort while keeping the same sampling quality. Objective: To evaluate the acceptability of the modified Delphi Screener. Primary outcomes were acceptance and user-friendliness of the screener, secondary outcomes were evaluation of user instructions and yield of cervicovaginal cell material. Methods: Fifty women between 31 and 65 years eligible for cervical examination were included in a prospective cohort study. Participants received the Delphi Screener kit, and were asked to perform self-sampling at home. They completed a questionnaire on acceptability using a Visual Analogue Scale from 0 to 10, and compared the comfort to their last physician-taken cervical smear. The self-collected specimen was posted to the laboratory. The cell yield for each sample was estimated by visually scoring sizes of pellets that appeared after centrifugation. The study was approved by the Medical Ethical Review Board. Results: Mean scores were 9.3 for ease of use, 9.3 for comfort, 9.2 for ease to follow user instruction, and 9.3 for preference for self-sampling. The comfort of their last physician-taken cervical smear was scored as 5.2. The yield of cells was comparably high as obtained at previous studies with the first generation lavage device. Conclusions: Women reported high acceptance of the modified Delphi Screener with high rates for ease of use and comfort. The modified Delphi Screener showed equivalence of cell yield with the first generation device, suggesting that in combination with HPV testing it is an attractive tool to increase the efficiency of the cervical screening programme.
OP-222
HPV TESTING VS. CYTOLOGY FOR CERVICAL CANCER SCREENING IN CONGO
S Mahmud, University of Manitoba, Winnipeg, CANADA, G Sangwa - Lugoma, S H Nasr, J Liaras, P K Kayembe, R R Tozin, P Drouin, A Lorincz, A Ferenczy, E L Franco Objectives: We compared the screening performance of conventional Pap cytology and two human papillomavirus (HPV) DNA assays, the original Hybrid Capture 2 (HC2; Qiagen Corporation) and an expanded version that tests for 4 additional HPV types (HC2+4; Qiagen), in the detection of cervical neoplasia among unscreened women in a primary care setting in a suburb of Kinshasa, Democratic Republic of Congo. Methods: All women aged 30 years residing in the area were invited to participate, and 1,528 (response rate=92%) were evaluated by Pap cytology (smears read in a reference laboratory in Lyon, France), both HPV assays (performed in Qiagens laboratories, Maryland, USA) and colposcopy. Cervical biopsies were obtained from all women with abnormal colposcopy and from 290 randomly chosen women with normal colposcopy (to correct for verification bias). The study was approved by the PATH Human Subjects Protection Committee and the IRB of the Kinshasa School of Public Health. Results: Using an RLU of 1 as the cutoff for positivity, 169 and 168 (11%) women tested positive using HC2 and HC2+4, respectively. HC2 and HC2+4 were in agreement in 98.6% of cases, corresponding to Kappa=0.94 (95% confidence interval: 0.91-0.96). Both assays were sensitive (~83%) and specific (~91%) for the detection of cervical intraepithelial neoplasia-2 or worse disease. Regardless of the cutoff point used to define positivity, Pap cytology was both less sensitive and more specific than HC2 or HC2+4. For instance, Pap cytology was 63% sensitive and 97% specific when a cutoff point of low-grade squamous intraepithelial lesions (LSIL) or worse was used to define test positivity. Conclusions: Among unscreened women, HC2 and HC2+4 had similar screening accuracy for cervical neoplasia, and both were more sensitive but less specific than Pap cytology. The high response rate may be an indication of the high level of acceptance of these screening methods.
Declaration of interest S Mahmud, GSK, Research grant A Lorincz, Qiagen, consultant E Franco, Merck, GSK, Qiagen, Roche, Gen-Probe, Ikonisys, Cytyc, Consultant E Franco, Merck, Grant support
189

NOVEL SCREENING OPPORTUNITIES: COMBINING P16/KI-67 DUAL-STAINED CYTOLOGY WITH HPV TESTING
R Ridder, mtm laboratories, Heidelberg, GERMANY, M Sideri, European Institute of Oncology, Unit of Preventive Gynaecology, Milan, ITALY, C Bergeron, Laboratoire Cerba, Cergy Pontoise, FRANCE, M von Knebel Doeberitz, Applied Tumor Biology, University of Heidelberg , Heidelberg, GERMANY, L Puig - Tintore, University of Barcelona, Barcelona, SPAIN Background: Pap cytology-based screening for cervical cancer has unsatisfactory sensitivity, but good specificity, whereas HPV testing is very sensitive, but has lower specificity, especially in the younger age groups. Ideally, a single test or a combination of two tests that combines high sensitivity with high specificity would be used for screening. The clinical performance of a novel biomarker approach, p16/Ki-67 Dual-stained cytology, has been recently assessed. Objectives: The objectives of this study was to evaluate the clinical performance characteristics and efficiency of p16/Ki-67 Dual-stained cytology in conjunction with HPV testing as compared to both tests alone or Pap cytologytesting for predicting underlying high-grade CIN in primary screening. Methods: Results from the PALMS trial, a prospective multi-national, multi-center screening trial evaluating Pap cytology, Dual-stained cytology and HPV-testing with cross-sectional disease ascertainment were used to estimate the performance of potential test combinations. Results: Combining Dual-stained cytology testing with HPV testing in women of all ages and referring only women positive for both tests to colposcopy revealed a sensitivity for CIN2+ of 86.8%, compared to 90.1% (Dual-stain), 96.4% (HPV), and 66.4% (Pap). Specificity for combined Dual-stained cytology/HPV testing was 97.1% and improved over any single testing approach, i.e. Dual-stain (95.3%), HPV (90.2%), or Pap (95.4%). Positive predictive value for the combined Dual-stain/HPV approach was increased to 25.3%, compared to 18.4% (Dual-stain), 10.2% (HPV), and 14.6% (Pap). There was a significant reduction of the number of colposcopies required for the combined Dualstain/HPV approach, even below those for single Pap cytology-testing. The combined approach works efficiently irrespective of age, i.e. also in women aged below 30/35. Conclusions: Dual-stained cytology provides high sensitivity and specificity in a single test. Combining it with HPV-testing allows for novel potential screening algorithms which may be even more efficient than any currently available screening technology.
Declaration of interest RR is an employee of mtm laboratories (study sponsor) and discloses a financial interest. M. Sideri, C. Bergeron, and L. Puig-Tintore have been occasional clinical advisors to mtm. L. Puig-Tintore is an occasional adviser to GSK, Sanofi PMSD, and Qiagen. M. von Knebel Doeberitz is a member of the supervisory board and has a financial interest in mtm.
OP-224
NEGATIVE PREDICTIVE VALUE OF PAP TESTING IN WOMEN WITH HIV
L Massad, Washington University, St Louis, MO, UNITED STATES, G D' Souza, F Tian, H Minkoff, M Cohen, L Sanchez Keeland, N Hessol, R Wright, C Colie, H Watts Background: Negative predictive value determines recommended screening interval, but this information is not know for Pap tests among women with HIV. Objective: To estimate the negative predictive value of Pap testing for women with human immunodeficiency virus. Methods: Women in the Womens Interagency HIV Study, the U.S. cohort study of HIV-seropositive and comparison seronegative women, have been followed with Pap smears at six month intervals since 1994. After excluding those with a history of abnormal Pap, cervical disease, or hysterectomy, women with negative initial Pap results were followed for 15-39 months for development of precancer, defined as a Pap read as HSIL, AGC favor neoplasia, AIS, or cancer, or a cervical biopsy read as CIN2+. Correlations between one or more consecutive negative Pap results and subsequent precancer were determined using Cox proportional hazards models, as were effects of other cancer risk factors. Results: Among 942 HIV seropositive women, 8 (1%) developed precancer within 15 months and 41 (4%) within 39 months of an initial negative Pap. After 3 initial consecutive negative Paps, precancer was rare, with no cases seen within 15 months and 11/539 (2%) within 39 months. No women developed precancer within 39 months after 10 initial negative Pap tests. Precancer within 15 months of follow-up was associated with any abnormal Pap at index visit (HR 6.25, 95% CI 4.2- 9.1), smoking (HR 1.8, 95% CI 1.15-2.8 vs nonsmokers), and lower CD4 count (HR 1.5, 95% CI 0.98, 2.37 for CD4 200-500, HR 2.0, 95% CI 1.24, 3.33 for CD4 <200, vs CD4 >500). Conclusion: Annual Pap testing appears safe for HIV infected women; for those with multiple serial negative tests, especially older women, 3-year of longer intervals may be appropriate.
190

HPV VERSUS PAPANICOLAOU PRIMARY SCREENING FOR CERVICAL CANCER: TRIAL RESULTS.
C Ferreccio, Escuela de Medicina/Pontificia Universidad Catlica de Chile, Santiago, Chile C Ibaez, Escuela de Medicina/Pontificia Universidad Catlica de Chile, Santiago, Chile S Terrazas, Escuela de Medicina/Pontificia Universidad Catlica de Chile, Santiago, Chile F Gonzalez, Escuela de Medicina/Pontificia Universidad Catlica de Chile, Santiago, Chile H Poggi, Escuela de Medicina/Pontificia Universidad Catlica de Chile, Santiago, Chile M Lagos, Escuela de Medicina/Pontificia Universidad Catlica de Chile, Santiago, Chile J Braes, Escuela de Medicina/Pontificia Universidad Catlica de Chile, Santiago, Chile M I Barriga, Hospital Stero del Rio/Servicio de Salud MetropolitanoSur Oriente, Santiago, Chile Background: Cervical cancer (CC) causes over 600 deaths per year in Chile with highly unequal distribution. As no significant reduction of this figure has been seen in the past 10 years, it is necessary to evaluate new screening options to improve the national prevention program and decrease inequalities.Objectives: To compare the accuracy of HPV-DNA test (Hybrid Capture 2 (HC2), Qiagen, Dsseldorf, Germany) and Conventional Pap test to detect prevalent precancerous lesions (CIN2 or more) in the primary care setting of the Chilean public health system. Methods: Women aged 25-64 years residents in the catchment area of 3 primary care centers of Santiago, Chile were invited through community outreach and direct communication at the health center to participate in the study. Accepting women were examined and tested with HC2 and Conventional Pap. Women with any positive test and a sample of double negative women (including women with abnormal visual inspection) were referred to colposcopy. Results: 8,407 women were enrolled; 8,266 were eligible (98.3%). These were aged 42.210.3 years, had 9.4 years of education and 6.1% reported no previous Pap. Abnormal Pap (ASCUS or worse) was 1.7% and HC2 positivity was 10.7%; 1.1% were positive in both tests. In all, 931 (11.3%) women with a positive screening were referred to colposcopy, of them 94.3% attended, 35.9% were biopsed. Additionally, 301 double negative women were referred to colposcopy, 78.1% attended. We identified 47 cases of CIN2, 40 of CIN3 and 9 cases of cervical cancer. Detection rate of CIN2 or worse was 100.0% for HC2 and 36.3% for Pap test (p<0.001). Detection bias corrected sensitivity was 92.4% for HC2 and 33.4% for Pap test. Conclusions: In this setting HC2 detected 3 times more CIN2 or worse than Pap, therefore is a more appropriate technique for primary screening in this population.
Declaration of interest The HPV kits were provided by Qiagen, no other
OP-226
RISK AND REFERRAL FOR HRHPV POSITIVITY IN HRHPV-CERVICAL SCREENING
F Van Kemenade, VUmc , Amsterdam, Netherlands, D C Rijkaart, VUmc, Amsterdam, Netherlands, J Berhof, VUmc, Amsterdam, netherlands, V M H Coupe, VUmc, Amsterdam, Netherlands, L Rozendaal, VUmc, Amsterdam, Netherlands, A T Hesselink, VUmc, Amsterdam, Netherlands, D A M Heideman, VUmc, Amsterdam, Netherlands, S Bulk, UUmc , Utrecht, Netherlands, P J F Snijders, VUmc, Amsterdam, Netherlands, C J L M Meijer, VUmc , Amsterdam, Netherlands Objective To measure risk profiles and referral rates for hrHPV DNA test positive women within programmed cervical cancer screening in the Netherlands. Intervention We recruited, after informed consent, a cohort of 25,871 women [30-60] invited for cytology screening in the Netherlands and added hrHPV testing to cytology. HrHPV test results were unknown to the cytotechnicians and women. A subcohort of women with normal cytology was randomized between HrHPV-positive and hrHPVnegative results and repeated after 1 and/or 2 years. Women with progressive cytology or persistent HrHPV positivity were referred for colposcopy. Post hoc risk profiling was performed on the results of all 1303 hrHPV women to identify a feasible triage and follow-up strategy Outcome measures Cumulative CIN grade 3 or worse (CIN3+) at 2 years, negative predictive value, and colposcopy referral rate. All estimates were adjusted for non-attendance at repeat testing. Results Two year cumulative CIN3+ risk for hrHPV-positive women with abnormal cytology was 42.2% (95%CI 36.448.2) in comparison to 5.22% (95%CI 3.72-7.91).for women with hrHPV-positive and normal cytology. An additional cytology test after 1 year, in hrHPV-positive women with normal cytology at baseline, reduced the residual CIN3+ risk to 0.7% (95%CI: 0.2-1.9) and yielded a colposcopy referral rate of 1 out of 3 hrHPV-positive women. Conclusions Primary hrHPV screening in women 30 years and older is feasible if hrHPV-positive women are triaged by cytology at baseline. In case of abnormal cytology, CIN3+ risk is high and direct referral for colposcopy and biopsy is warranted. In case of normal cytology, repeat cytology after 1 year for these hrHPV-positive women at baseline yields a high negative predictive value and a low colposcopy referral rate.
Declaration of interest CJLM is a member of the advisory board of Qiagen (formerly Digene) and received lecture fees from GSK. PJFS provided occasional consultation to Roche and Gen-Probe. Qiagen, Gen-Probe and Roche are companies involved with HPV diagnostics.
191

UNDER- AND NEVER-SCREENED WOMEN ARE EFFECTIVELY TARGETED BY OFFERING HRHPV-SELFSAMPLING
P Snijders, VU University medical center, Amsterdam, NETHERLANDS, M Gk, VU University medical center, Amsterdam, NETHERLANDS, D Heideman, VU University medical center, Amsterdam, NETHERLANDS, F van Kemenade, VU University medical center, Amsterdam, NETHERLANDS, A de Vries, VU University medical center, Amsterdam, NETHERLANDS, J Berkhof, VU University medical center, Amsterdam, NETHERLANDS, L Rozendaal, VU University medical center, Amsterdam, NETHERLANDS, L Overbeek, PALGA,, Utrecht, NETHERLANDS, M Babovi, Regional Screening Organisation, Amsterdam, NETHERLANDS, C Meijer, VU University medical center, Amsterdam, NETHERLANDS Background: Self-sampling for hrHPV testing (HPV-SS) is accepted by up to 30% of non-attendees to the regular cervical screening programme (Gok et al., 2010; 2011), but it is still unknown which subgroups of non-attendees are targeted when offering HPV-SS. Objective: To answer the following question: Which subgroup amongst non-attendees, in terms of age, ethnicity and screening history, is effectively targeted by HPV-SS and how do these risk factors relate to CIN2+/CIN3+ yield? Method: Pooled analysis of non-attendees invited for previous PROHTECT studies (n=52,985; Gok et al., 2010; 2011). Age and ethnic status were retrieved from the invitational screening database and screening history from the centralized PALGA database. A logistic regression model was fitted to analyze the risk factors independently. The significance of the effect of each risk factor to the overall model was evaluated with the Wald test. Results: Indigenous Dutch women responded better than immigrants (32.4% vs 21.8%; Wald(1:402.57, p<0.001). Amongst women 39 years, those who were screened 7 years ago revealed a higher response rate than under(i.e. last smear >7 years ago) or never-screened women (35.0% vs 23.7%; Wald(1: 420.94, p<0.001). Strikingly, neverscreened women responded better than under-screened women (24.8% vs 22.6%; Wald(1: 33.345, p<0.001). CIN2+/ CIN3+ rates were higher among participating indigenous Dutch women than immigrants (CIN2+: Wald(1): 13.021, p<0.01; CIN3+: Wald(1): 8.716, p<0.01). Amongst women 39 years, those who were under- or never-screened displayed a higher CIN2+/CIN3+ yield than women screened 7 years ago (CIN2+: Wald (1): 8.485, p<0.01; CIN3+: Wald(1): 7.390, p<0.01). Conclusions: Offering HPV-SS to non-attendees of the regular screening program increases the efficacy of the screening program by targeting a substantial portion of under-or never-screened women who have the highest risk of CIN2+.
Declaration of interest Occasional participation in advisory board meetings of Gen-probe and Roche.
OP-228
COBAS HPV TEST PERFORMANCE INCLUDING HPV16/18 IN CERVICAL CANCER SCREENING
M Stoler, University of Virginia Health System, Charlottesville, UNITED STATES P Castle, American Society for Clinical Pathology Institute, Washington DC, T Wright, Jr., Columbia University School of Medicine, New York, NY, A Sharma, Roche Molecular Diagnostics, Pleasanton, CA, T Wright, Roche Molecular Diagnostics, Pleasanton, CA, C Behrens, Roche Molecular Diagnostics, PLeasanton, CA, Background: There are no comprehensive U.S. trials that evaluate the relative utility of cytology compared to HPV testing with individual detection of HPV16 and HPV18 in cervical cancer screening. Objective:Our objective was to compare the screening performance of HPV testing and liquid-based cytology (LBC) and the various management strategies for HPV-positive women in a large, multi-centric U.S. study Methods: The study population included 41,955 women age 25 and older enrolled from 61 U.S. clinical centers. Cervical specimens were collected for LBC and HPV DNA testing by the cobas HPV Test, with individual HPV16 and HPV18 detection. Colposcopy and diagnostic biopsies were performed on all women who tested HPV or cytology positive and a random sample (25%) of women who tested negative by both tests. Disease status was determined by central pathology review of all biopsies and outcome measure was cervical intraepithelial neoplasia grade 3 or more severe (CIN3). Results: Overall, 104% of women tested positive for HPV and 64% had non-normal cytology (p < 0.0001). HPV testing (unadjusted crude estimate) was more sensitive for detection of CIN3 than LBC (920% vs 533%, p < 0.0001). Adjusting for verification bias, HPV testing remained more sensitive for CIN3 than LBC (751% vs 432%). The addition of LBC to HPV testing (co-testing) increased diagnostic yield for CIN3 by 5% while increasing the number of screen positives by 36%. Triage strategies incorporating HPV16 and/or HPV18 detection alone or in combination with >ASC-US cytology yielded equal or superior performance for identifying HPV-positive women with CIN3 compared to triage strategies based solely on non-normal cytology, the current model of colposcopy triage for HPV-primary screen positive women. Conclusions: HPV testing with individual detection of HPV16 and HPV18 may provide an alternative, more efficient strategy for cervical cancer screening than more traditional methods.
Declaration of interest M. Stoler, consultant for Hologic, QIAGEN, Gen-Probe and Roche Molecular Diagnostics the study sponsor. P Castle, no declarations. T Wright Jr, consultant and speaker for Merck, GlaxoSmithKline and Roche Molecular Diagnostics; consultant for Gen-Probe. A. Sharma, T.Wright and C. Behrens, are employed by Roche Molecular Diagnostics the study sponsor
192

INCIDENCE OF CIN3 BY AGE AND CYTOLOGY: ATHENA RESULTS
W Huh, University of Alabamha, Birmingham, UNITED STATES M Stoler, University of Virginia Health System, Charlottesville, VA, T Wright, Jr, Columbia University School of Medicine, New York, NY, A Sharma, Roche Molecular Diagnostics, PLeasanton, CA, T Wright, Roche Molecular Diagnostics, Pleasanton, CA, C Behrens, Roche Molecular Diagnostics, Pleasanton CA, Background: Adjunctive high risk (hr) HPV testing is currently not recommended for women below the age of 30, due to the higher prevalence of transient hrHPV infections not associated with disease in younger women. However, women below the age of 30 can present with >=CIN3 but are typically identified through follow-up of abnormal cytology Objective: To define the prevalence of >CIN3 disease in a screening population at baseline by age, and cytology, Methods: The ATHENA HPV study is a randomized trial designed to evaluate testing for pooled hrHPV as well as HPV16 and 18 genotyping using the cobas HPV Test, among women undergoing cervical cancer screening. 47,208 U.S. women age 21 or older were enrolled and cervical samples were collected into PreservCyt media for both cytology and hrHPV DNA testing. Colposcopy with biopsy was performed in all women with >ASCUS cytology results, hrHPV positive women >=25 years, and a subset of women >=25 years negative by both cytology and hrHPV testing. Disease status was determined by central pathology review of all biopsies. Results: Women 25-29 years of age with normal cytology were found to have the highest rate of >= CIN3- 1.0% (58/5,996). In comparison, women 30-39, 40-49, 50-59, and >60 years of age, were found to have similar or lower rates of > CIN3 (0.1-.6%). Conclusion: Women 25-29 years of age have abnormal cytology that is hr HPV positive and associated with CIN3 or greater, as followed by current guidelines. However, when compared to other age groups, similar rates of CIN3 or greater are found among women 25-29 years of age with NILM cytology , thus supporting the inclusion of women 25-29 in hr HPV primary screening programs.
Declaration of interest W. Huh is a consultant to Hologic, QIAGEN, and Roche Molecular Diagnostics -the stuidy sponsor. M. Stoler is a consultant for Hologic QIAGEN, and Roche Molecular Diagnostics T. Wright Jr. is a consultant and speaker for Merck, GlaxoSmithKline and Roche Molecular Diagnostics A. Sharma, T. Wright and C. Behrens are employed by Roche Molecular Diagnostics,the study sponsor
OP-230
SEXUAL BEHAVIOR AS RISK FACTOR FOR ORAL SQUAMOUS CELL CARCINOMA
K Dahlstrom, The University of Texas M.D. Anderson Cancer Center, Houston, TX, UNITED STATES A Richter, The University of Texas M.D. Anderson Cancer Center, Houston, TX, United States Q Wei, The University of Texas M.D. Anderson Cancer Center, Houston, TX, United States E M Sturgis, The University of Texas M.D. Anderson Cancer Center, Houston, TX, United States G Li, The University of Texas M.D. Anderson Cancer Center, Houston, TX, United States Background: Along with smoking and alcohol use, exposure to human papillomavirus type 16 (HPV16) has been established as an important risk factor for oral squamous cell carcinoma (OSCC). Objectives: To determine the magnitude of risk for OSCC associated with HPV16 exposure and smoking and alcohol use as well as to compare sexual behaviors between HPV16-positive and HPV16-negative OSCC patients. Methods: We performed multivariable analysis that included over 200 newly diagnosed OSCC patients and over 300 cancer-free controls. OSCC included tumors arising from the oral cavity and oropharynx. HPV16 exposure was measured by seropositivity for the L1 capsid protein and in situ hybridization for evidence of HPV DNA in tumor tissue. Results: HPV16 L1 seropositivity and history of smoking and alcohol use were independently associated with risk for OSCC. Furthermore, we found a greater than multiplicative interaction for both smoking and HPV16 seropositivity and alcohol use and HPV16 seropositivity. We also found that sexual behaviors, specifically oral-genital sex, were associated HPV16-positive OSCC. Ever having engaged in oral-genital sex was associated with HPV16-positive OSCC compared with never having engaged in oral-genital sex and a dose-response relationship for increasing numbers of oral-genital sex partners was observed. Conclusions: HPV16 exposure alone is strongly associated with risk for OSCC and, moreover, has a significant interaction effect with smoking and alcohol. Furthermore, having an HPV16-positive tumor is significantly associated with a history of having engaged in oral-genital sex and having had multiple oral-genital sex partners. These results suggest that risk for OSCC may be reduced through behavior modification and provide an opportunity for primary cancer prevention through smoking cessation programs, reducing alcohol intake, and sexual behavior modification. Finally, given the strong association of HPV16 with OSCC, these results indicate that the HPV vaccine may be useful in preventing a subset of OSCC.
193

MULTI-CENTRIC HPV-ASSOCIATED HEAD AND NECK SQUAMOUS CELL CARCINOMA.
S Thavaraj, King's College London, London, UNITED KINGDOM M Evans, Velindre Cancer Centre, Cardiff, UNITED KINGDOM N Powell, Cardiff University, Cardiff, UNITED KINGDOM M Robinson, Newcastle University, Newcastle, UNITED KINGDOM Background: It is now recognised that the recent rise in the incidence of oro-pharyngeal squamous cell carcinoma (SCC) is due to oncogenic HPV infection. It is well known that a proportion (around 10%) of patients with head and neck SCC develop second primary tumours as a consequence of field cancerisation. Currently there is little information about the development of multi-centric HPV-associated SCC in the head and neck region. It is also unknown whether persistent oncogenic HPV infection is associated with field cancerisation. Objective: To describe the clinico-pathological features of multi-centric HPV-associated SCCs of the head and neck region. Methods: Six patients with multi-centric HPV-associated head and neck SCC were included in this study. Individual primary tumours were characterised using p16 immunohistochemistry (IHC), high-risk HPV DNA in-situ hybridisation (ISH) and consensus PCR (GP5+/6+ primers) followed by HPV genotyping using PCR enzyme immuno assay. Results: There were six patients with a total of 14 primary tumours. Three patients had synchronous tumours and three developed metachronous tumours. One of the patients with metachronous disease developed synchronous second primary tumours. Five of the patients developed disease in the tonsil and/or tongue base and in one case the first primary tumour arose in the nasopharynx. All SCCs were strongly positive for p16 by IHC and the majority (13 of 14) were positive for high-risk HPV by ISH. HPV genotyping demonstrated that all the cases harboured a single high-risk HPV genotype; the majority of cases contained HPV16 (5 of 6) and one case HPV33. Conclusion: We report a series of six multi-centric HPV-associated head and neck SCCs which, to our knowledge, is the largest series to include genotype analysis. The demonstration of a single genotype in synchronous and metachronous carcinomas raises the possibility of virally-induced field cancerisation.
OP-232
DURATION OF HPV 6/11/16/18 INFECTION IN MALES
A Giuliano, Moffitt Cancer Center, Tampa, UNITED STATES J Palefsky, University of California San Francisco, San Francisco, USA S Goldstone, Mt. Sinai School of Medicine, New York, USA K L Liaw, Merck, Whitehouse Station, USA BACKGROUND: HPV causes a variety of cancers and genital warts in both men and women. Despite the large body of knowledge of HPV natural history accumulated in women, little is known about the natural history and the rate of clearance of HPV infection in men. OBJECTIVE: Examine the median duration of HPV 6/11/16/18 infection in a total of 2033 men between ages 15 and 27 in the placebo arm of a quadrivalent HPV vaccine clinical trial. METHODS: Three external genital swabs, (penile/scrotal/perineal/perianal) were collected from each man (including 1732 heterosexual men (HM) and 301 men who have sex with men [MSM]), and an additional intra-anal swab was collected from MSM for detection of HPV 6/11/16/18, approximately every 6 months during the study period (median follow-up time 35.3 months; mean 29.7 months). Clearance of type-specific incident infection were calculated by sexual orientation and sample collection site in men who were nave to the corresponding HPV type(s) at baseline RESULTS: Overall, the duration of prevalently detected HPV infections at enrollment had a longer median duration than infections that were newly acquired, regardless of the anatomic site of detection or sexual orientation. Median duration of incident HPV 16 infections was highest at the scrotum (8.3 months) and penile epithelium (8.9 months) and lowest at the perineal/perianal site (6.1 months) among HM. In contrast, among MSM duration of HPV 16 was highest at the anal canal 9.8 months) followed by the perineal/perianal site (7.7 months). CONCLUSIONS: Different HPV types appeared to behave differently, depending on the infection site. HPV 16 appeared to persist longer at the perianal sites in HM and anal sites in MSM. This is consistent with the predominant role of HPV 16 in HPV-related anal diseases.
194

HPV IN HEAD AND NECK CARCINOMA OF UNKNOWN PRIMARY SITE
S Tribius, University Medical Center Hamburg-Eppendorf, Hamburg, GERMANY, A S Ihloff, University Medical Center Hamburg-Eppendorf, Hamburg, GERMANY, T Grgh, Christian Albrechts University Kiel , Kiel, GERMANY, T Clauditz, University Medical Center Hamburg-Eppendorf, Hamburg, GERMANY, J Haag, Christian Albrechts University Kiel, Kiel, GERMANY, S Bastrop, Christian Albrechts University Kiel , Kiel, GERMANY, A Borcherding, University Medical Center Hamburg-Eppendorf, Hamburg, GERMANY, P Tennstedt, Martini Clinic, University Medical Center Hamburg-Eppendorf, Hamburg, GERMANY, H Henry, Christian Albrechts University Kiel , Kiel, GERMANY, M Hoffmann, Christian Albrechts University Kiel , Kiel, GERMANY Background: Cancer of unknown primary (CUP) presenting as neck metastasis is rare and often managed with multi-modality treatment. Standard RT involves all potential mucosal sites (nasopharynx, oropharynx, oral cavity, larynx/hypopharynx) and neck. Such RT can result in severe acute and long-term toxicity (xerostomia, dental decay, dysphagia and lymphedema). Studies have shown that infection with human papillomavirus is often linked to oropharyngeal cancer. This analysis was undertaken to investigate a possible association between CUP and HPV and to suggest where clinical examination should begin for patients (pts) with HPV and neck metastases and no apparent primary tumor. Methods: A population was defined at the University Medical Centers of Hamburg and Kiel consisting of pts with CUP during 20072010 for whom formalin-fixed paraffin-embedded tissue was available. Genotyping by HPV-DNA amplification was performed using polymerase chain reaction by applying HPV type-specific oligonucleotides; p16 expression was measured using immunohistochemistry. Results: In 63 pts, 33 (52%) had HPV16- or HPV33-DNA -positive samples; 36 (57%) had p16-positive samples. Most pts were positive (38%) or negative (29%) for both markers. The previously reported discordance between HPV and p16 status was observed: 12 pts (19%) were HPV-negative and p16 -positive, and 9 (14%) were HPV- positive and p16-negative. A statistically significant association was defined between tobacco smoking and p16 status: 96% of p16-negative pts were smokers versus 67% of p16 positive pts (p=0.003). Conclusion: This study, the largest to date investigating HPV status in pts with head and neck CUP, identified the presence of HPV-DNA and p16 overexpression in approximately 50% of pts. We postulate that identification of HPV positive nodal metastases may suggest the oropharynx as the potential source of the primary, allowing RT volume to be limited to these mucosal sites thereby limiting toxicity. Assessment of outcome by HPV/p16 status is ongoing.
OP-234
ASSOCIATIONS BETWEEN RACE, SEXUAL BEHAVIORS AND HPV SEROSTATUS
C Fakhry, Johns Hopkins , baltimore, UNITED STATES P Gravitt, Johns Hopkins, Baltimore, United states R Viscidi, Johns Hopkins, Baltimore, United States A Burke, Johns Hopkins, Baltimore, US K Chang, Johns Hopkins, Baltimore, US L Hackett, Johns Hopkins, Baltimore, US E Seay, Johns Hopkins, Baltimore, US Background: Human papillomavirus is responsible for a growing subset of oropharyngeal squamous cell cancers (OSCC). HPV-OSCCs are more prevalent among whites (than non-whites). Reasons that HPV-OSCC affects whites more than other racial groups are not understood. HPV-OSCC is also strongly associated with sexual behaviors. Therefore, we hypothesize that racial differences in sexual behavior may in part account for racial distribution of HPV-OSCC. Methods: HPV in Perimenopause is a prospective cohort study of women at Johns Hopkins. Data from baseline visit were used for cross-sectional analysis. Serologic HPV status was determined by ELISA. Race was considered a categorical variable (white, black and other). Descriptive statistics were used to summarize data and univariate logistic regression was performed to evaluate associations between race and behaviors of interest. Results: The majority of 827 subjects were white (n=620, 75.0%) and black (n=161, 19.5%) with a median age of 47 years (IQR 41, 52). Whites were more likely to report same-sex partner (p=0.07), ever oral sex (<0.001) and recent oral sex(p=0.03). There were no racial differences for recent vaginal sex, recent and lifetime anal sex. When considering number of partners, whites were less likely to have greater than 5 lifetime vaginal sex partners (p=0.003), however they were more likely to report a greater mean number of lifetime oral sex partners (2.7 vs. 1.7 vs. 1.9, p-trend<0.001). Serostatus was available for 85% of the study population. Black women were more likely to be HPV-seropositive (p=0.001). Among seropositive women, a greater proportion of whites had ever given oral sex (n=307, 96.5%, p<0.001) than non-whites, however these behavioral differences were not observed among seronegative women. Conclusion: Whites more commonly perform sexual behaviors that are associated with HPVOSCC and therefore may explain racial differences seen in HPV-OSCC.
195

ANTI-RETROVIRAL THERAPY INCREASES HPV-16 PERSISTENT ORAL INFECTION
M Hagensee, LSUHSC, New Orleans, LA , UNITED STATES A D' Angelo, LSUHSC, New Orleans, LA , UNITED STATES K Stuckey, LSUHSC, New Orleans, LA, UNITED STATES A Clark, LSUHSC, New Orleans, LA, UNITED STATES N Herrel, LSUHSC, New Orleans, LA , UNITED STATES Background: HPV-16 is linked to many oral squamous cell cancers, whereas HPV-32 is detected in 50% of oral warts in HIV+ individuals. Anti-retroviral therapy (ART) has been shown to be a risk factor for oral HPV infection. Objective: To examine if ART is a risk factor for persistent HPV 16 or 32 infection. Methods: HIV+ individuals on (n=43) or off (n=17) ART were followed at 3 month intervals for 18 months. A swab of the buccal mucosa, labia, gingival, tonsils, sublingual were obtained, gargle and saliva samples. HPV-16 or 32 were detected by type-specific PCR assays. Demographic variables, ART usage, CD4 counts, and HIV viral loads were collected. All oral sites were combined for analysis. Persistence was defined as being positive for the same virus type on at least 2 consecutive visits. Results: At the first visit, 26% of the HIV+ individuals were positive for either HPV-16 (15%) or 32 (17%). ART increased the risk of HPV-16 detection from 5.8% to 18.6%, HPV-32 detection from 11.6% to 18.6% or detection of either virus from 11.6% to 32.5% (p=.18). At any study visit, ART increased HPV-16 detection from 3.1% to 16.2% (p=.09) but HPV-32 rates did not change (22%). Persistent HPV-16 infection was seen in 40% and exclusively in those on ART. Persistent HPV-32 infection was seen in 60%. Discussion: The prevalence rates of HPV-16 and 32 in the oral cavity are modest but persistent infections are common. ART increases the detection rates and of HPV-16 and 32 and may be influencing persistence oral infection. Studies on larger cohorts of HIV+ individuals on and off ART are warranted to further explore these risk factors.
OP-236
RECURRENT RESPIRATORY PAPILLOMATOSIS AND HPV VACCINATION
E Hamsikova, Institute of Hematology and Blood Transfusion, Praha 2, CZECH REPUBLIC J Smahelova, Institute of Hematology and Blood Transfusion, Praha 2, CZECH REPUBLIC V Ludvikova, Institute of Hematology and Blood Transfusion, Praha 2, CZECH REPUBLIC J Stasikova, Institute of Hematology and Blood Transfusion, Praha 2, CZECH REPUBLIC J Dubova, Medical Healthcom, Praha 2, CZECH REPUBLIC J Vydrova, Medical Healthcom, Praha 2, CZECH REPUBLIC R Tachezy, Institute of Hematology and Blood Transfusion, Praha 2, CZECH REPUBLIC BACKGROUND: Recurrent respiratory papillomatosis (RRP) is a rare benign chronic disease of the larynx etiologically linked with the infection of low-risk human papillomavirus (HPV) types 6 and 11. RRP is characterized by voice changes, stridor, dyspnoea etc., tends to recur and spread throughout the aerodigestive tract. OBJECTIVES: Treatment involves combination of surgical and immunomodulatory therapy, but repeated surgery often results in permanent voice disorders, so the subsequent care is extremely important. As the immunotherapy presents unconvincing results, possible use of prophylactic tetravalent HPV vaccine that includes HPV 6 and 11 antigens has been studied. METHODS: Patients who signed the information consent form were included in the study (#20). Data on demographics, risk factors for RRP, and risks related to HPV exposure were collected by a questionnaire. The medical report was completed for each patient. All enrolled subjects underwent laryngostroboscopy (digital video recording) and tissue and/or smear for HPV DNA detection and typing and blood for antibody detection were taken. The presence of HPV DNA was detected by reverse line blot hybridization, HPV-specific antibodies were detected by ELISA using individual virus-like particles as antigens. RESULTS: HPV DNA detection was performed in 12 patients. Eleven were HPV 6 (91.7%) and one HPV 11 (8.3%) positive. Nine of these 12 HPV DNA positive patients were also positive for VLP 6 or VLP 6/11 (75%). Of 20 subjects 11 have already been vaccinated with tetravalent HPV vaccine. During clinical follow-up six (54.5%) were free of disease recurrence, two (18.2%) with no change in clinical status and in three (27.3%) recurrent lesions were observed. CONCLUSIONS: During the follow-up, 73% of cases vaccinated with tetravalent HPV vaccine were free of recurrent disease, suggesting that this procedure might improve the quality of life of RRP patients.
Declaration of interest R. Tachezy, GlaxoSmithKline, Member of the Czech advisory board for the prevention of cervical carcinoma
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27th IPV Conference and Clinical Workshop 17-22 Sept 2011 Berlin Germany
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27th INTERNATIONAL PAPILLOMAVIRUS CONFERENCE AND CLINICAL WORKSHOP

September 17-22, 2011

*Please be at your poster for scientific discussion

Session 11: Clinical immunology

Clinical immunology | 11 O-11.00

11 | Clinical immunology O-11.02

Clinical immunology | 11 O-11.04

11 | Clinical immunology O-11.06

Clinical immunology | 11 O-11.08

11 | Clinical immunology P-11.09

Clinical immunology | 11 P-11.11

11 | Clinical immunology P-11.13

Clinical immunology | 11 P-11.15

11 | Clinical immunology P-11.17

Clinical immunology | 11 P-11.19

11 | Clinical immunology P-11.21

Clinical immunology | 11 P-11.23

11 | Clinical immunology P-11.25

Clinical immunology | 11 P-11.27

11 | Clinical immunology P-11.29

Clinical immunology | 11 P-11.31

11 | Clinical immunology P-11.33

Clinical immunology | 11 P-11.35

Session 12: Clinical aspects of HPV testing

Clinical aspects of HPV testing | 12 O-12.00

12 | Clinical aspects of HPV testing O-12.02

Clinical aspects of HPV testing | 12 O-12.04

12 | Clinical aspects of HPV testing O-12.06

Clinical aspects of HPV testing | 12 O-12.08

12 | Clinical aspects of HPV testing P-12.09

Clinical aspects of HPV testing | 12 P-12.11

12 | Clinical aspects of HPV testing P-12.13

Clinical aspects of HPV testing | 12 P-12.15

12 | Clinical aspects of HPV testing P-12.17

Clinical aspects of HPV testing | 12 P-12.19

12 | Clinical aspects of HPV testing P-12.21

Session 13: HPV related benign diseases

HPV related benign diseases | 13 O-13.00

13 | HPV related benign diseases O-13.02

HPV related benign diseases | 13 O-13.04

13 | HPV related benign diseases O-13.06

HPV related benign diseases | 13 O-13.08

13 | HPV related benign diseases P-13.09

HPV related benign diseases | 13 P-13.11

13 | HPV related benign diseases P-13.13

Session 14: Clinical use of biomarkers

Clinical use of biomarkers | 14 O-14.00

14 | Clinical use of biomarkers O-14.02

Clinical use of biomarkers | 14 O-14.04

14 | Clinical use of biomarkers O-14.06

Clinical use of biomarkers | 14 O-14.08

14 | Clinical use of biomarkers P-14.09

Clinical use of biomarkers | 14 P-14.11

14 | Clinical use of biomarkers P-14.13

Clinical use of biomarkers | 14 P-14.15

14 | Clinical use of biomarkers P-14.17

Clinical use of biomarkers | 14 P-14.19

14 | Clinical use of biomarkers P-14.21

Clinical use of biomarkers | 14 P-14.23

14 | Clinical use of biomarkers P-14.25