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Context.— Detection of human papillomavirus (HPV) in formalin-fixed, paraffin-embedded (FFPE) tissues may identify the cause of lesions and has value for the development of new diagnostic assays and epidemiologic studies. Seegene Anyplex II assays are widely used for HPV screening, but their performance using FFPE samples has not been fully explored. Objective.— To validate Anyplex II HPV HR Detection (Anyplex II, Seegene) using FFPE samples. Design.— We used 248 stored DNA extracts from cervical cancer FFPE samples collected during 2005–2015 and that tested HPV positive using the RHA kit HPV SPF10-LiPA25, v1 (SPF10, Labo Biomedical Products) HPV genotyping assay, manufacturer-validated for FFPE samples. Results.— Of the selected 248 samples, 243 were used in our analysis. Consistent with SPF10 genotyping results, Anyplex II detected all 12 oncogenic types and had an overall HPV detection rate of 86.4% (210 of 243 samples). Anyplex II and SPF10 showed very high agreement for the detection of the 2 most important oncogenic genotypes: HPV 16 (219 of 226; 96.9%; 95% CI, 93.7–98.75) and HPV 18 (221 of 226; 97.8%; 95% CI, 94.9–99.3). Conclusions.— Overall results showed that both platforms produced comparable HPV genotyping results, indicating the suitability of Anyplex II for FFPE samples. The Anyplex II assay has the added convenience of being an efficient, single-well semiquantitative polymerase chain reaction assay. Further optimization of Anyplex II may enhance its performance using FFPE samples by improving the detection limit.

Background: It is unknown whether reactivation of human papillomavirus (HPV) after latency occurs in the anus. We measured incidence and predictors of incident anal HPV in sexually inactive gay and bisexual men (GBM) as a surrogate of HPV reactivation. Methods: The Study of the Prevention of Anal Cancer collected data on sexual behavior, anal cytology, HPV DNA, histology and HPV serology. HPV incidence during periods when zero sexual partners were reported in the last six months at both the current and previous annual visit (“no sexual activity”) was analyzed by Cox regression using the Wei-Lin-Weissfeld method to determine univariable predictors. Results: Of 617 men enrolled, 525 had results for ≥2 visits, of whom 58 (11%) had ≥ one period of “no sexual activity”. During sexually inactive periods, there were 29 incident high risk HPV infections in 20 men, which occurred more commonly in older men (Ptrend = 0.010), HIV-positive men (HR = 3.12; 95% CI, 0.91–16.65), longer duration of HIV (Ptrend = 0.028), history of AIDS defining illness (P = 0.010), lower current (P = 0.010) and nadir CD4 count (P = 0.014). For 18 of 29 infections with available results, 12 men remained type-specific HRHPV L1 seronegative. None were consistently seropositive. A new diagnosis of HSIL occurred in only two men, caused by an HPV type other than the incident type. Conclusions: Our findings suggest that in sexually inactive GBM, anal HRHPV incidence is relatively common, and is associated with increasing age and immune dysfunction, a pattern consistent with HPV reactivation. Impact: Reactivation of anal HPV may occur.

Background Gay and bisexual men (GBM) are at increased risk of human papillomavirus (HPV)–associated anal high-grade squamous intraepithelial lesions (HSILs). Understanding the fractions of HSILs attributable to HPV genotypes is important to inform potential impacts of screening and vaccination strategies. However, multiple infections are common, making attribution of causative types difficult. Algorithms developed for predicting HSIL-causative genotype fractions have never been compared with a reference standard in GBM. Method Samples were from the Study of the Prevention of Anal Cancer. Baseline HPV genotypes detected in anal swab samples (160 participants) were compared with HPV genotypes in anal HSILs (222 lesions) determined by laser capture microdissection (LCM). Five algorithms were compared: proportional, hierarchical, maximum, minimum, and maximum likelihood estimation. Results All algorithms predicted HPV-16 as the most common HSIL-causative genotype, and proportions differed from LCM detection (37.8%) by algorithm (with differences of −6.1%, +20.9%, −20.4%, +2.9%, and +2.2% respectively). Fractions predicted using the proportional method showed a strong positive correlation with LCM, overall (R = 0.73 and P = .002), and by human immunodeficiency virus (HIV) status (HIV positive, R = 0.74 and P = .001; HIV-negative, R = 0.68 and P = .005). Conclusions Algorithms produced a range of inaccurate estimates of HSIL attribution, with the proportional algorithm performing best. The high occurrence of multiple HPV infections means that these algorithms may be of limited use in GBM.

AIM: Validate the Roche, MagNAPure96 (MP96) nucleic acid extraction platform for Seegene Anyplex II HPV28 (Anyplex28) detection of Human Papillomavirus. METHODS AND RESULTS: Comparisons were made for Anyplex28 genotyping from 115 cervical samples extracted on the Hamilton, STARlet and the MP96. Two DNA concentrations were used for the MP96, one matched for sample input to the STARlet and another 5× concentration (laboratory standard). Agreement of HPV detection was 89·8% (κ = 0·798; P = 0·007), with HPV detected in 10 more samples for the MP96. There was a high concordance of detection for any oncogenic HPV genotype (κ = 0·77; P = 0·007) and for any low-risk HPV genotype (κ = 0·85; P = 0·008). DNA extracted at laboratory standard had a lower overall agreement 85·2% (κ = 0·708; P

Background Incidence of anal cancer is highest in gay and bisexual men (GBM). Better understanding of the natural history of anal high-risk human papillomavirus (hrHPV) infection is needed for anal cancer prevention. Methods The Study of the Prevention of Anal Cancer was a 3-year study of Australian GBM, aged 35 years or older. We examined incidence, clearance, and risk factors for 13 hrHPV types at baseline and 3 annual visits. Results In 525 men with ≥ 2 visits, 348 (66.3%) acquired ≥ 1 incident hrHPV infection. HPV16 incidence rates were similar, but non-16 hrHPV incidence was higher in HIV-positive (51.8/100 person years [PY]) than HIV-negative men (36.5/100 PY, P Conclusions Higher incidence of non-16 hrHPV types, coupled with lower clearance of non-16 hrHPV types in those with past impaired immune function, is consistent with the greater role of non-16 hrHPV in anal cancer in HIV-positive people. Australia New Zealand Clinical Trials Registry ANZCTR365383.

Background: Anal high-grade squamous intraepithelial lesion (HSIL) can be histomorphologically categorized into anal intraepithelial neoplasia (AIN) grade 2 (AIN2) and grade 3 (AIN3). Different risk factors for these two categories have been described. We investigated whether there were also differences in lesion-specific human papillomavirus (HPV) genotypes. Methods: The Study of the Prevention of Anal Cancer (SPANC) recruited 617 gay and bisexual men (GBM); 36% of participants were HIV positive. At baseline, 196 men (31.8%) had histologic HSIL lesions. Tissue was available for genotyping in 171, with a total of 239 HSIL lesions (183 AIN3 and 56 AIN2). Using laser capture microdissection, each lesion revealed a maximum of one genotype. Results: High-risk HPV (HR-HPV) genotypes were found in 220 (92.1%) HSIL lesions, with no significant difference between AIN3 (93.4%) and AIN2 (87.5%). AIN3 lesions had significantly more HPV16 (42.1%) than AIN2 lesions (12.5%; P < 0.001) and AIN2 lesions had significantly more non-16 HR-HPV types (75.0%) than AIN3 lesions (51.4%; P = 0.002). These associations were similar for HIV-negative men with HPV16 in 51.1% AIN3 and 18.2% AIN2 (P = 0.001) and non-16 HR-HPV in 40.0% AIN3 and 75.8% AIN2 (P < 0.001). For HIV-positive men, HPV16 remained more frequently detected in AIN3 (33.3% vs. 4.4% for AIN2; P = 0.004), but there was no difference between AIN3 and AIN2 for non-16 HR-HPV (62.4% vs. 73.9%; P = 0.300). Conclusions: As HPV16 has the strongest link with anal cancer, the subcategorization of HSIL may enable stratification of lesions for anal cancer risk and guide anal HSIL management. Impact: Stratification of anal cancer risk by histologic HSIL grade.

Background Gay and bisexual men (GBM) are disproportionately affected by anal cancer. Prevention is hindered by incomplete understanding of the natural history of its precursor, anal high-grade squamous intraepithelial lesions (HSIL). Methods The Study of the Prevention of Anal Cancer, conducted between 2010 and 2018, enrolled human immunodeficiency virus (HIV)-positive and HIV-negative GBM aged ≥35 years. Anal cytology and high-resolution anoscopy (HRA) were performed at baseline and 3 annual visits. A composite HSIL diagnosis (cytology ± histology cHSIL) was used. Cytological high-grade squamous intraepithelial lesions (cHSIL) incidence and clearance rates were calculated with 95% confidence intervals (CIs). Predictors were calculated using Cox regression with hazard ratios (HRs) and 95% CIs. Results Among 617 men, 220 (35.7%) were HIV-positive, median age 49 years. And 124 incident cHSIL cases occurred over 1097.3 person-years (PY) follow-up (11.3, 95% CI 9.5–13.5 per 100 PY). Significant bivariate predictors of higher incidence included age Conclusion These data strongly suggest that not all anal HSIL detected in screening requires treatment. Men with persistent HPV16 were less likely to clear HSIL and are more likely to benefit from effective HSIL treatments. Clinical Trials Registration Australia New Zealand Clinical Trials Registry (ANZCTR365383).

Anal cancer is preceded by high-risk human papillomavirus (HRHPV) infection, predominantly HPV16. No HPV assay is licenced for use in anal screening. We aimed to determine the sensitivity and specificity of four anal canal swab HPV assays to predict high-grade squamous epithelial lesions (HSIL).In a cohort of Australian HIV-positive and negative gay and bisexual men, we compared the sensitivity and specificity of detection of 13 anal HRHPV genotypes by Linear Array (LA), Cobas 4800, EuroArray, and Anyplex II HPV28 (+ and ++ cut offs), compared their ability to predict prevalent anal HSIL, and compared anal canal HRHPV detection with HRHPV isolated from HSIL using laser capture microdissection (LCM).A total of 475 participants had baseline results available for all four assays (166, 35.0% HIV positive), and 169 participants had a diagnosis of cytological and/or histological HSIL. The HPV16 and any HRHPV detection were highest with Anyplex II HPV28 (+) (156, 32.8% 95% CI 28.6-37.2 and 359, 75.6%, 95% CI 71.5-79.4, respectively). For detection of concurrent HSIL and HPV16, the assay sensitivity was similar, ranging from 49.1%, 95% CI 41.4-56.9 (Anyplex II HPV28 ++) to 55.0%, 95% CI 47.2-62.7 (Anyplex II HPV28 +). For concurrent HSIL and any HRHPV detection, EuroArray was more specific than Anyplex II HPV28 (+) (45.9% 95% CI 40.2-51.7 vs 36.7%, 95% CI 31.3-42.4, p = 0.021) and had comparable specificity with Anyplex II HPV28 (++) (45.9% vs 47.2%, 95% CI 41.5-53.0, p = 0.75). All assays had high sensitivities for predicting HPV16 detected on LCM (92.5-97.5%). Anyplex II HPV28 and EuroArray were significantly more sensitive than LA for lesions caused by non-HPV16 HRHPV types on LCM.Anyplex II HPV28 and EuroArray detected more non-16 HRHPV genotypes than LA. Increasing the Anyplex II HPV28 cutoff improved specificity without compromising sensitivity for detection of concurrent HSIL.

Background Few studies have analyzed the association between human telomerase reverse transcriptase (hTERT) protein expression (nuclear and cytoplasmic localization), hTERT methylation status, and human papillomavirus (HPV) genotype infection in cervical cancer. Patients and methods One hundred seventy-three patients with cervical cancer were analyzed. hTERT protein expression was detected by immunohistochemistry. hTERT DNA methylation analysis was performed using a PCR-RLB-hTERT assay, targeting two regions of the hTERT promoter. Type specific HPV infection was detected by using GP5+/GP6+PCR-RLB. Results hTERT protein expression was found in both cytoplasm and nucleus (78.0% of the samples showed a cytoplasmic localization and 79.8% had a nuclear localization). A statistically significant association was found between alpha 9 and 7 HPV species with a non-methylation pattern of the hTERT promoter and between these species and high expression of hTERT protein with nuclear localization. Conclusion hTERT protein is found in both the nucleus and cytoplasm of patients with cervical cancer and confirm the relationship between the non-methylated status of hTERT promoter and some HPV species as well as the relationship between these species and hTERT protein expression.

Human papillomavirus (HPV) is detected in up to 96% of anal squamous cell cancers, where screening programs needed. However, the best methodology is still undetermined. Host DNA methylation markers CADM1, MAL and miR124 have been identified in cervical disease, but not anal disease. Anal swabs varying by disease grade were assessed for DNA methylation of CADM1, MAL and miR124-2. Each marker was compared across disease grades, stratified by HPV and HIV status. Receiver operating characteristic curves identified the predictive value of significant gene candidates. CADM1 methylation was significantly higher in high-grade squamous intraepithelial lesions (HSIL) compared with low-grade (LSIL) (p = 0.005) or normal (p 0.001) samples with 67.2% correctly identified as HSIL. MAL methylation was significantly (p = 0.002) increased in HSIL compared with LSIL in HIV positive participants with 79.8% correctly indicated as HSIL. Gene miR124-2, showed no difference between disease grades. Biomarkers with established diagnostic value in cervical disease have limited utility in the prediction of anal disease, with CADM1 identified as a marker with screening potential in a gay and bisexual men (GBM) population and MAL in HIV positive GBM population. New markers specific to the anal mucosa are required to improve triage of high-risk individuals.

BACKGROUND: In Australia, high and widespread uptake of the quadrivalent human papillomavirus (HPV) vaccine has led to substantial population-level reductions in the prevalence of quadrivalent vaccine targeted HPV genotypes 6/11/16/18 in women aged ≤ 35 years. We assessed risk factors for HPV detection among 18-35 year old women, 9-12 years after vaccine program introduction. METHODS: Women attending health services between 2015 and 2018 provided a self-collected vaginal specimen for HPV genotyping (Roche Linear Array) and completed a questionnaire. HPV vaccination status was validated against the National Register. Adjusted odds ratios (aORs) and 95% confidence intervals (CI) were calculated for factors associated with HPV detection. RESULTS: Among 1564 women (median age 24 years; IQR 21-27 years), Register-confirmed ≥ 1-dose vaccine coverage was highest at 69.3% and 68.1% among women aged 18-21 and 22-24 years respectively, decreasing to 42.9% among those aged 30-35 years. Overall prevalence of quadrivalent vaccine-targeted HPV types was very low (2.0%; 95% CI: 1.4-2.8%) and influenced only by vaccination status (5.5% among unvaccinated compared with 0.7% among vaccinated women; aOR = 0.13 (95% CI: 0.05-0.30)). Prevalence of remaining HPV types, at 40.4% (95% CI: 38.0-42.9%), was influenced by established risk factors for HPV infection; younger age-group (p-trend

ObjectiveHigh-risk human papillomavirus (HRHPV) causes anal cancer, which disproportionately affects gay and bisexual men (GBM). We examined sexual behaviours associated with incident anal HRHPV in an observational cohort study of GBM in Sydney, Australia.MethodsGBM aged 35 years and above were enrolled in the Study of the Prevention of Anal Cancer. Detailed information on sexual practices in the last 6 months, including receptive anal intercourse (RAI) and non-intercourse receptive anal practices, was collected. Anal human papillomavirus (HPV) testing was performed at the baseline and three annual follow-up visits. Risk factors for incident HRHPV were determined by Cox regression using the Wei-Lin-Weissfeld method.ResultsBetween 2010 and 2015, 617 men were recruited and 525 who had valid HPV results at baseline and at least one follow-up visit were included in the analysis. The median age was 49 years (IQR 43–56) and 188 (35.8%) were HIV-positive. On univariable analysis, incident anal HRHPV was associated with being HIV-positive (pConclusionGBM living with HIV and those who reported RAI were at increased of incident anal HRHPV. Given the substantial risk of anal cancer and the difficulty in mitigating the risk of acquiring anal HRHPV, HPV vaccination should be considered among sexually active older GBM.Trial registration numberANZCTR365383.

Purpose Although the large impact of Human papilloma virus (HPV) in cervical cancer is established, its place as a therapeutic target is new and according to the growing literature, could be promising. In the present study, radiosensitivity's difference based on HPV-16 variants is assessed. Patients and methods Variants of Human papilloma virus were identified before the exclusive radiotherapy in patients with cervical cancer. Data were prospectively collected. Fifty-nine patients were screened. Results Among the 59 screened patients, 34 (57.6%) were identified to be HPV-16 (+), with 13 European and two non-European variants. Of the 34 patients, 15 experienced exclusive radiotherapy. Among them, eight had complete response (seven with European and one with non-European variants), four with European variant had partial response, three with European variant had tumour persistence and one with non-European variant progressed at 3 months. Conclusion No radiosensitivity difference was established, probably because of the limited population. Non-European variant aggressiveness might be suggested in accordance with the literature, as it was associated with the only tumour progression. Exclusive radiotherapy provides a unique and “pure” model of radioresistance in cervical cancer and could be the missing link between in vitro studies and state of the art chemoradiotherapy studies that probably feature too many parameters to identify radioresistance causes. The present study was a first step, with the future prospects of building a larger cohort study in order to better understand HPV-induced radioresistance and then to be able to propose new made-to-measure treatments.

Introduction Australia has recently implemented major changes in cervical cancer prevention policies including introduction of primary human papillomavirus (HPV) screening starting at age 25, and replacement of the quadrivalent HPV vaccine with the nonavalent vaccine in the national school-based program. We assessed the feasibility and utility of conducting HPV testing in residual clinical specimens submitted for routine Chlamydia trachomatis screening, as a means of tracking HPV vaccine program impact among young sexually active women. Methods De-identified residual specimens from women aged 16–24 years submitted for chlamydia testing were collected from three pathology laboratories in Victoria and New South Wales. Limited demographic information, and chlamydia test results were also collected. Patient identifiers were sent directly from the laboratories to the National HPV Vaccination Program Register, to obtain HPV vaccination histories. Samples underwent HPV genotyping using Seegene Anyplex II HPV 28 assay. Results Between April and July 2018, 362 residual samples were collected, the majority (60.2%) of which were cervical swabs. Demographic data and vaccination histories were received for 357 (98.6%) women (mean age 21.8, SD 2.0). Overall, 65.6% of women were fully vaccinated, 9.8% partially, and 24.7% unvaccinated. The majority (86.0%) resided in a major city, 35.9% were classified in the upper quintile of socioeconomic advantage and chlamydia positivity was 7.8%. The prevalence of quadrivalent vaccine-targeted types (HPV6/11/16/18) was 2.8% (1.5–5.1%) overall with no differences by vaccination status (p = 0.729). The prevalence of additional nonavalent vaccine-targeted types (HPV31/33/45/52/58) was 19.3% (15.6–23.8%). One or more oncogenic HPV types were detected in 46.8% (95% CI 41.6–52.0%) of women. Conclusions HPV testing of residual chlamydia specimens provides a simple, feasible method for monitoring circulating genotypes. Applied on a larger scale this method can be utilised to obtain a timely assessment of nonavalent vaccine impact among young women not yet eligible for cervical screening.

Background In Australia, high and widespread uptake of the quadrivalent human papillomavirus (HPV) vaccine has led to substantial population-level reductions in the prevalence of HPV16/18 in women aged ≤35 years. We assessed risk factors for oncogenic HPV detection among 18–35 year old women in 2015–2018. Methods Women attending health services across Australia provided a self-collected (vaginal) or clinician-collected (cervical) specimen for HPV genotyping (Roche Linear Array) and completed a questionnaire. HPV vaccination status was validated against the National HPV Vaccination Program Register. Odds ratios (ORs) and 95% confidence intervals (CI) were calculated for factors associated with detection of any oncogenic HPV (HPV16/18/31/33/35/39/45/51/52/56/58/59/66/68). Results Among 1,643 women, vaccine coverage (≥one dose) was 61.5% (69.1%, 58.7% and 41.1% among those 18–24, 25–29 and 30+ years, respectively). Oncogenic HPV prevalence was 25.4% (95% CI: 23.2–27.6%). In univariable analysis, risk factors for detection included younger age (p-trend Conclusion Oncogenic HPV was commonly detected among young Australian women; prevalence was influenced by risk factors related to sexual behaviour. In contrast, prevalence of quadrivalent vaccine-targeted types 16/18 was very low and influenced only by vaccination status. Vaccination has changed the epidemiology of HPV infection in Australia. Disclosure No significant relationships.

Purpose. Chlamydiatrachomatis is responsible for trachoma-associated blindness as well as the most common sexually transmitted bacterial infection worldwide, although the genovars for the former are typically A-C, whilst for the latter they are D-K and for the uncommon infection lymphogranuloma venereum they are L1-3. Nucleotide variations within the ompA gene facilitate the identification of C. trachomatis genovars. This study describes a colorimetric multiplex PCR/RLB typing assay (mPCR-RLB) directed to the VD2 region of the ompA gene for general C. trachomatis positivity and the identification of 14 individual C. trachomatis genovars.Methodology. The assay was validated by analysing 40 blinded samples that included reference strains of C. trachomatis genovars and other non-chlamydial micro-organisms that had been analysed previously using quantitative PCR (qPCR). Ninety clinical samples that had previously been found to be C. trachomatis-positive by qPCR were also evaluated using the mPCR-RLB assay.Results. The mPCR-RLB assay showed 100% agreement with the qPCR in the detection of C. trachomatis reference strains and no cross-reaction of non-chlamydial micro-organisms was observed. In the analysis of the chlamydial clinical samples, 97.8% were C. trachomatis-positive by mPCR/RLB assay and there was a 96.6% concordance with the qPCR at the group identification level and a 92.2% concordance at the genovar level.Conclusion. The mPCR-RLB assay is a rapid and sensitive methodology for the identification of C. trachomatis genovars associated with urogenital infections, trachoma or lymphogranuloma venereum diseases that can be implemented in clinical settings, helping to identify reinfections and treatment failures and establish the appropriate treatment course.

Human papillomavirus (HPV) is a nearly ubiquitous infectious organism. It is estimated that 80% of sexually active adults will be exposed to anogenital HPVs in their lifetime, and detection of multiple genotypes in an anogenital sample is common. Detection and genotyping of HPV is usually performed by DNA testing, and less frequently by mRNA testing. HPV genotype testing and characterization of DNA methylation patterns of HPV-related lesions can provide important biological, epidemiological, and potentially relevant clinical information in individuals and populations. The use of laser capture microdissection to isolate cells within a specific lesion allows for very precise molecular characterization and hence causal attribution. This chapter describes detailed protocols for the capture of lesion-specific tissue from formalin-fixed, paraffin-embedded (FFPE) biopsy tissue, and downstream DNA testing for lesion-specific HPV genotype and their methylation patterns.

Human papillomavirus (HPV) is a nearly ubiquitous infectious organism. It is estimated that 80% of sexually active adults will be exposed to anogenital HPVs in their lifetime, and detection of multiple genotypes in an anogenital sample is common. Detection and genotyping of HPV is usually performed by DNA testing, and less frequently by mRNA testing. HPV genotype testing and characterization of DNA methylation patterns of HPV-related lesions can provide important biological, epidemiological, and potentially relevant clinical information in individuals and populations. The use of laser capture microdissection to isolate cells within a specific lesion allows for very precise molecular characterization and hence causal attribution. This chapter describes detailed protocols for the capture of lesion-specific tissue from formalin-fixed, paraffin-embedded (FFPE) biopsy tissue, and downstream DNA testing for lesion-specific HPV genotype and their methylation patterns.

Epidemiological information on telomerase activity (TA) and development of cervical lesions is scarce. A nested case-control study was carried out within a cohort of Colombian women tested for Human Papillomavirus (HPV). Measurement of TA was done in cervical scrapes of 25 women who developed High Grade Squamous Intraepithelial Lesion (HGSIL) during the first 6 years of follow-up and was compared with that of 104 control women who maintained normal cytology during the entire follow-up. TA was measured by a telomerase repeat amplification protocol-ELISA. TA and HPV infections were significantly more frequent in cases than in controls. Likewise, 68% of the cases were positive for both TA and HPV compared with only 7.7% of the controls (P

Background Clinical parameters and proteins have recently been suggested as possible causes of radiotherapy (RT) resistance in cervical carcinoma (CC). The objective of the present study was to validate prognostic biomarkers of radiation resistance. Methods The present prospective study included patients undergoing RT with curative intent for histologically proven locally advanced squamous cell CC. Tissues and blood samples were systematically collected before RT initiation. Immuno-histochemistry was performed (IGF-IR α and β, GAPDH, HIF-1 alpha, Survivin, GLUT1, CAIX, hTERT and HKII). Response to radiation was assessed through tumour response 3 months after RT completion, through overall survival (OS) and through progression-free survival (PFS). Results One hundred forty nine patients with a mean age of 46 years were included, with FIGO IIB (n = 53) and FIGO IIIB (n = 96) CCs. 61 patients were treated with exclusive RT + brachytherapy and 88 underwent chemo-radiotherapy + brachytherapy. Our findings suggest an association between hemoglobin level (Hb) (>11 g/dL) and 3 months complete response (p = 0.02). Hb level

Incidence and mortality rates of anal cancer are increasing globally. More than 90% of anal squamous cell carcinomas (ASCC) are associated with human papillomavirus (HPV). Studies on HPV-related anogenital lesions have shown that patterns of methylation of viral and cellular DNA targets could potentially be developed as disease biomarkers. Lesion-specific DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissues from existing or prospective patient cohorts may constitute a valuable resource for methylation analysis. However, low concentrations of DNA make these samples technically challenging to analyse using existing methods. We therefore set out to develop a sensitive and reproducible nested PCR-pyrosequencing based method to accurately quantify methylation at 10 CpG sites within the E2BS1, E2BS2,3,4 and Sp1 binding sites in the viral upstream regulatory region of HPV16 genome. Methylation analyses using primary and nested PCR-pyrosequencing on 52 FFPE tissue [26 paired whole tissue sections (WTS) and laser capture microdissected (LCM) tissues] from patients with anal squamous intraepithelial lesions was performed. Using nested PCR, methylation results were obtained for the E2BS1, E2BS2,3,4 and Sp1 binding sites in 86.4% of the WTS and 81.8% of the LCM samples. Methylation patterns were strongly correlated within median values of matched pairs of WTS and LCM sections, but overall methylation was higher in LCM samples at different CpG sites. High grade lesions showed low methylation levels in the E2BS1 and E2BS2 regions, with increased methylation detected in the E2BS,3,4/Sp1 regions, showing the highest methylation at CpG site 37. The method developed is highly sensitive in samples with low amounts of DNA and demonstrated to be suitable for archival samples. Our data shows a possible role of specific methylation in the HPV16 URR for detection of HSIL.

The analysis of expression of the hTERT protein, the methylation status of the hTERT gene as the type of HPV infection, will contribute to deepen the role played by HPV infection in the expression and methylation of hTERT. The study included a group of one hundred fifty-five samples of women with squamous cell carcinomas of the uterine cervix in stage FIGO IIB-IIIB treated at the National Cancer Institute in Bogotá, Colombia. The expression of the hTERT protein was detected by immunohistochemistry both at the nuclear level and at the cytoplasmic level. In 79.7% of the cases, the protein was expressed at the nuclear level and 78% at the cytoplasmic level. HPV16, the most frequent viral type found, was detected by means of a GP5+/GP6+ mediated PCR- RLB in 84.4% and in 82.2% of the cases that expressed hTERT at the nuclear level and at the cytoplasmic level, respectively; with respect to HPV species, both at the nuclear level and at the cytoplasmic level, the species most frequently found was the alpha 9 species (HPVs 16, 31, 35, 52.58) followed by alpha 7 (HPVs 18, 45.59) and alpha 6 (HPVs 56, 66). In the multiple correspondence analyzes, a close relationship was observed between the cytoplasmic expression as nuclear of hTERT and the alpha species 9. A distant relationship was also observed between the expression of the protein and methylation, as well as between the non-methylation and the alpha 7 family of HPV; likewise, a distant relationship was observed between the non-methylation of hTERT and the alpha 9 species. The analyzes carried out show a possible relationship between the expression of the hTERT protein and the alpha 9 HPV species. Citation Format: Pablo Moreno-Acosta, Mónica Molano, Nicolas Morales, Alfredo Romero-Rojas, Oscar Gamboa, Jinneth Acosta, Raynner Alvarez, Nicolas Magné, Nubia Muñoz. hTERT protein expression, hTERT methylation and HPV infection in uterine cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4328.

International audience; High hypoxic, glycolytic and acidosis metabolisms characterize cervical cancer tumors and have been described to be involved in chemoradioresistance mechanisms. Based on these observations, the present study assessed four selected novel biomarkers on the prognosis of locally advanced cervical carcinoma. A total of 66 patients with stage IIB/IIIB cervical cancer were retrospectively included. The protein expression levels of glucose transporter 1 (GLUT1), carbonic anhy-drase 9 (CAIX) and hexokinase 1 (HKII) were investigated by immunohistochemistry on tumor biopsies, hemoglobin was measured and the disease outcome was monitored. A total of 53 patients (80.3%) presented a complete response. For these patients, the protein expression levels of GLUT1, CAIX and HKII were overexpressed. A significant difference was observed (P= 0.0127) for hemoglobin levels (≤11 g/dl) in responsive compared with non-responsive patients. The expression of GLUT1 is associated with a lower rate of both overall and disease-free survival, with a trend of decreased risk of 1.1x and 1.5x, respectively. Co-expression of GLUT1 and HKII is associated with a decreased trend risk of 1.6x for overall survival. Patients with hemoglobin levels ≤11 g/dl had a 4.3-fold risk (P=0.02) in decreasing both to the rate of overall and disease-free survival. The presence of anemic hypoxia (hemoglobin ≤11 g/dl) and the expression of GLUT1 and/or HKII influence treatment response and are associated with a lower overall and disease-free survival. The present results demonstrated that these biomarkers may be used as predictive markers and suggested that these metabolic pathways can be used as potential novel therapeutic targets.

Background There exists limited information on the role of hTERT methylation, and its association with type-specific HPV infections in cervical cancer. Materials and methods Eighty-seven frozen samples were analyzed for type-specific HPV infection using a GP5+/GP6+ PCR-RLB assay (RLB). hTERT DNA methylation analysis was performed using a newly developed PCR-RLB-hTERT. Results Ninety-three percent of samples were HPV-positive and fifteen different types were detected. hTERT methylation analysis of region 1 revealed no methylation in 78.8% of the samples and partial methylation in 21.2%. In region two, 68.2% showed no methylation and 31.8% showed a pattern of partial methylation. An association between the alpha 9 and alpha 7 species with a pattern of no methylation of hTERT in the region 1 was established (p=0.02 and p=0.03, respectively). Conclusion Differences in patterns of methylation of the hTERT core promoter [region 1 (nt -208 to -1) and region 2 (nt +1 to +104) relative to first ATG] are related to the HPV species present.

Introduction Anal squamous cell carcinoma is preceded by persistent infection with high-risk human papillomavirus (HPV) and the cancer precursor, high-grade squamous intraepithelial lesion (HSIL). Detection of specific HPV genotypes and HPV-related biomarkers may be an option for primary anal screening. However, more data on the natural history of HPV-related anal lesions are required. The outcomes from this study will enhance our understanding of the clinical and biological behaviour of HPV-related anal lesions and inform the development of future HPV genotype and/or biomarker screening tests. Methods and analysis HIV-negative and HIV-positive men who have sex with men, aged 35 years and over, recruited from community-based settings in Sydney, Australia, attend 6 clinic visits over 3 years. At the first 5 visits, participants undergo a digital anorectal examination, an anal swab for HPV genotyping and anal cytology, and high-resolution anoscopy with directed biopsy of any visible abnormalities that are suggestive of any abnormality suspicious of SIL. Tissue sections from participants diagnosed with histologically confirmed HSIL at the baseline clinic visit will undergo laser capture microdissection, HPV detection and genotyping, and quantitation of CpG methylation in baseline and follow-up biopsies. Histological and cytological findings in combination with HPV genotyping data will be used to identify persistent HSIL. HSIL will be stratified as non-persistent and persistent based on their status at 12 months. The performance of HPV genotype and methylation status in predicting disease persistence at 12 months will be assessed, along with associations with HIV status and other covariates such as age. Ethics and dissemination The St Vincent9s Hospital Ethics Committee granted ethics approval for the study. Written informed consent is obtained from all individuals before any study-specific procedures are performed. Findings from this study will be disseminated to participants and the community through study newsletters, and through peer-reviewed publications and international conferences.

Coinfection with multiple types of human papillomavirus (HPV) and its implications for the development of efficacious HPV vaccines is a subject of great interest. To describe the occurrence of concurrent infection with multiple HPV types and to determine whether genital HPV infection modifies the risk of acquiring a new HPV infection with another HPV type, 1610 subjects were monitored for an average of 4.1 years in Bogota, Colombia. Information on risk factors for HPV infection and cervical cells was collected for detection of HPV DNA of 36 types at study entry and at 6 consecutive 6-month follow-up visits. Clustering or the concurrent acquisition of multiple types occurred more often than would be expected by chance. Subjects with incident HPV-16 or -18 infection had 5-7 times higher odds of acquiring a subsequent HPV-58 infection than subjects not infected with HPV-16 or -18. This might affect the protection conferred by effective HPV vaccines.

Uterine cervical cancer (UCC) is one of the most prevalent malignant neoplasms in the world. UCC develops beyond the stage in situ and is frequently treated by a combination of intracavitary radiation therapy and external beam radiation therapy; 30-40% of patients with similar prognosis factors do not respond equally to a comparable standard treatment. Therefore, the study and identification of prognostic biomarkers, which indicate the probable course of the disease in an untreated individual, and predictive biomarkers, which allow identification of subpopulations of patients most likely to respond to a given therapy, would be extremely useful in the selection of patients for the development of innovative and effective therapies for locally advanced, metastatic, and refractory uterine cervical cancer. Within work that we have been developing, we reported that gene expression of IGF1R is a strong predictive marker for lack of response to radiotherapy (p=0.018, 95% CI (1.7-41.2)), and patients HPV16 (+) have 28.6 times higher risk of failure treatment; consequently, with the present study, we proposed to determine whether expression of IGF-IR, GAPDH, HIF-1 alpha, Survivin, GLUT1, CAIX, HKII, and clinic-pathologic parameters can be used as prognostic and predictive biomarkers and as possible molecular targets for individualized treatment. Patients and Methods: This prospective cohort study included 149 patients with squamous cell carcinomas of the uterine cervix in FIGO stages IIB (n= 53) and IIIB (n=96). Of the 149 patients, 61 were treated with radiotherapy and 88 with concurrent radio-chemotherapy. Expression of the proteins CAIX, GLUT-1, HIF1α, HKII, IGF-IRα, IGF-IRβ, and Survivin was determined by immunohistochemistry in biopsies taken before treatment. Results: An overexpression was found for GAPDH (100%), followed by Survivin (87%), IGF-IRα (76.5%), IGF-IRβ (74.5%), HIF1α (74.1%), and IGF-IRα and IGF-IRβ (73%); strong expression was observed with low frequency for GLUT-1 (31.1%), CAIX (16.2%), and HKII (10.6%). Hgb level was significantly correlated with treatment response (p=0.01). With a median follow-up of 2.1 years, OS was decreased for patients overexpressing IGF-1R β (p=0.04). The OS of the subgroup of patients with anemia (Hgb < 11 g/dL) and concomitantly overexpressing IGF-1R and GLUT-1 was significantly decreased compared to the opposite control group (p=0.015). Conclusions: The expression of GLUT-1, IGF-1R, and Hgb (≤ 11 g/dl) is associated with poor prognosis, and thus appear to be interesting biomarkers of radiation resistance. In order to contribute to appropriate therapeutic management as a personalized neoadjuvant treatment, a prospective phase II study should be designed in the near future to evaluate the effect of turmeric (curcumin) as an inhibitor of IGF-1Rβ and GLUT1. Note: This abstract was not presented at the conference. Citation Format: Pablo Moreno-Acosta, Schyrly Carrillo, Oscar Gamboa, Alfredo Romero-Rojas, Jinneth Acosta, Diana Mayorga, Mónica Molano, Martha Cotes, Nicolas Magne. Potential biomarkers for personalized oncology radiation in uterine cervical cancer [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B56.

The incidence of cervical cancer in Latin America and the Caribbean (LAC) is among the highest in the world. Because there are major demographic shifts happening in LAC countries (population growth, urbanization and ageing) cervical cancer incidence and mortality will likely continue to be a significant public health problem. Overall human papillomavirus (HPV) prevalence in the LAC general population has been found to be 2-fold higher than the average worldwide prevalence. The large HPV and cancer burden may be explained by the highly prevalent HPV variants of HPV types -16 and 18, which have an increased oncogenic potential. Given the major mode of transmission of genital HPV is sexual, certain, patterns of sexual behaviour (early age at first sexual intercourse, number of sexual partners and sexual behaviour of the partner) are associated with an increased risk of HPV genital acquisition. Although HPV infection is necessary for carcinogenesis, certain co-factors (high parity, long term use of oral contraceptives, smoking and co-infection with the human immunodeficiency virus (HIV)) help in the progression from infection to cancer. Many studies that have contributed to this evidence have been carried out in LAC and are reviewed and summarised in this article. Since HPV vaccines will likely take years to implement, and many more years to show impact on disease, cervical cancer screening programmes remain as the key intervention to control disease in LAC in the years to come. © 2008 Elsevier Ltd. All rights reserved.

Background: There is limited information of the role of hTERT methylation, hTERT expression and their association with type specific HPV infections in patients with invasive cervical cancer Objective: To analyze the possible association between methylation status of the hTERT promoter gene, hTERT expression and HPV infection in patients with invasive cervical cancer. Methodology: Eighty seven frozen samples of women with invasive cervical cancer were analyzed for type specific HPV infection using a GP5+/GP6+ mediated PCR- RLB. hTERT DNA methylation analysis was performed on bisulfite modified DNA using a new PCR-RLB-hTERT methylation assay targeting two regions flanking the hTERT [region 1 (nt -240 to -1) and region 2 (nt +1 to +120) relative to first ATG]. Expression of hTERT was detected by immunohistochemistry using an Anti-hTERT (348-358) rabbit pAb). Results: All samples with HPV types belonging to the α7 species (HPV 18, 45 and 59) showed no hTERT methylation in region 1 (core promoter) and an increase in% of partial methylation in region 2. Samples with HPV types belonging to the α9 specie (16, 52, 35, 31 and 58) showed similar% of methylation in region 1 and region 2 according viral type but the percentages of methylation were different between them (ranging from 0% for HPV58 samples to 66.7% for HPV31). Strong expression of hTERT was observed in 77% of the samples independent of the HPV genotype. Conclusion: hTERT methylation seems to be associated with specific genotype HPV infection; however expression of the hTERT protein seems to depend of additional molecular events and worth further investigation. Citation Format: Pablo Moreno-Acosta, Nicolas Morales, Marcela Burgos, Oscar Gamboa, Juan Carlos Mejia, Alfredo Romero-Rojas, Alba Lucia Combita, Monica Molano. HPV infections, hTERT methylation and hTERT expression in patients with invasive cervical cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C88.

Telomerase activation have been considered as a fundamental step in the immortalization process because majority nonmalignant somatic tissue shows undetectable or no telomerase activity. A few numbers of studies in cervical cancer have showed an increase in the telomerase activity with grade of lesion, and Human Papillomavirus (HPV) infection has been associated in some studies with telomerase activation. However until now there are not follow up studies that measure the telomerase activity along the time. We describe here results about telomerase activity and HPV infection during 7 years of follow up in women that belong to the Bogotá cohort. Samples were obtained every six months during follow up and we had in average four samples by woman. Telomerase activity was analyzed in 58 samples of 17 women that during follow up developed High Grade Squamous Intraepithelial Lesions (HGSIL) and 149 samples of 34 women that during follow up had normal cytology using a TeloTAGGG Telomerase PCR ELISA kit (Roche Applied Science). The HPV infection was previously tested using a GP5+/GP6+mediated PCR-EIA and typing using a Reverse Line Blot assay (RLB). Of the 17 women that developed HGSIL along follow up; 12 (70.5%) had HPV infection and telomerase activity at the same time of HGSIL diagnosis or in a previous visit, these women had infections with high risk types (HR) principally with those belong to the alpha 9 specie, 2 (17.7%) showed only transitory events of HPV infection and 2 (11.8%) had normal cytology without HPV infection or telomerase activity. Average time to develop HGSIL was 13.5 months. In normal women we observed diverse results. Of the 34 women; 21 (61.8%) had HPV transitory infections with high risk and/or low risk types sometime during follow up and only 12 (35.2%) of them were positive for telomerase activity sometime in follow up independently of HPV infection. Interestingly, 8 (23.5%) women had HPV-HR infection during follow up and they were never positive for telomerase activity and 5 (14.7%) women didn't have HPV infection and telomerase activity. Detection of HR-HPV infection and telomerase activity at the same time likes to predict the risk of HGSIL. Our results support the idea that telomerase activity and HR-HPV infection can be used as progression markers for the development of HGSIL. Citation Information: Cancer Res 2009;69(23 Suppl):C22.