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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.

OBJECTIVE: High-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. METHODS: GBM 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. RESULTS: Between 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 (p<0.001), having a higher number of recent RAI partners regardless of condom use (p<0.001 for both), preference for the receptive position during anal intercourse (p=0.014) and other non-intercourse receptive anal sexual practices, including rimming, fingering and receptive use of sex toys (p<0.05 for all). In multivariable analyses, being HIV-positive (HR 1.46, 95% CI 1.09 to 1.85, p=0.009) and reporting condom-protected RAI with a higher number of sexual partners (p<0.001) remained significantly associated with incident HRHPV. When stratified by recent RAI, non-intercourse receptive anal practices were not associated with incident HRHPV in men who reported no recent RAI. CONCLUSION: GBM 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

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: Anal symptoms may indicate serious pathology. Receptive anal intercourse (RAI) and sexually transmissible infections (STIs) may contribute to a higher prevalence of symptoms among gay and bisexual men (GBM). This study investigated associations with anal symptoms among GBM. Methods: The Study of the Prevention of Anal Cancer was a longitudinal study of anal human papillomavirus and related lesions in Sydney, Australia. GBM aged ≥35 years were recruited from community settings between September 2010 and August 2015. Information about anal symptoms (discharge, itch, pain defecating, lump, bleeding, 'sores', tearing, tenesmus), STIs and sexual behaviours was collected. High-resolution anoscopy (HRA) and STI testing were performed. Logistic regression analyses on baseline data were performed to assess associations with each symptom. Results: Among 616 participants (median age 49 years, 35.9% HIV positive), 35.3% reported at least one anal symptom within the past week and 65.3% were diagnosed with fistula, fissure, ulcer, warts, haemorrhoids and/or perianal dermatoses at HRA. Anal symptoms were not associated with anal chlamydia, gonorrhoea, warts or syphilis. Self-reported 'sores' were associated with previous anal herpes simplex virus (HSV; P < 0.001). 'Sores' (P < 0.001), itch (P = 0.019), discharge (P = 0.032) and lump (P = 0.028) were independently associated with ulceration. Among participants diagnosed with fissure, fistulae, haemorrhoids and perianal dermatoses, 61.9%, 100%, 62.0% and 63.9% respectively were asymptomatic. Only self-reported anal tear was independently associated with recent RAI. Conclusions: Previous anal HSV was the only STI associated with any symptom. Anal pathology was highly prevalent, but often asymptomatic. Anal symptoms do not appear to be useful markers of most anal pathology in GBM.

Transplant recipients are at high-risk of anal squamous cell cancer. We aimed to estimate the prevalence of high-risk human papillomavirus (HPV) and high-grade squamous intraepithelial lesion (HSIL) and assess characteristics associated with results METHODS: We recruited kidney transplant recipients in a single-center, 2015-2018. Participants completed a clinical questionnaire and received an anal-swab sent for HPV-DNA and cytological testing RESULTS: A total of 97 (74%) of 125 recipients approached consented to participate. Participants were median 47 (IQR 40-55) years, 60% male and median 4.5 (IQR .9-13) months-since-transplant. Of 86 assessable samples, at least one HPV genotype was detected in 15 (17%) participants; 1 (1%) HPV16, 8 (9%) other high-risk HPV. Of 76 assessable cytology samples, 9 (12%) showed evidence of abnormality; 1 (1%) HSIL, 1 (1%) atypical-squamous-cells, cannot exclude HSIL. Both HSIL recipients had high-risk HPV and biopsy confirmed HSIL. High-risk HPV was detected in six (9%) recipients with normal cytology. History of sexually transmitted infection, and abnormal cervical pap smear in women, was associated with high-risk HPV and HSIL CONCLUSIONS: High-risk HPV and HSIL testing may identify kidney transplant recipients at higher risk of anal cancer. Longitudinal studies are needed to describe the natural history of anal cancer in transplant recipients.

Kidney recipients have anal cancer rates 3 times higher than the general population in Australia and New Zealand. High-risk human papillomavirus (HPV) genotypes are implicated in the majority of anal cancers. Establishing the epidemiology of anal HPV infection and precursors of anal cancer in transplant recipient populations is 1 consideration in any potential screening program. The Transplant and Anal Neoplasia Study is a cross-sectional study of the prevalence of anal cytological abnormalities and HPV deoxyribonucleic acid in kidney transplant recipients, as well as evaluating the acceptability of an anal cancer screening intervention. The study aims to recruit 100 kidney transplant recipients, older than 18 years, in Australia. Transplant recipients attending for a protocol biopsy at 3 and 12 months and annually posttransplant are approached to participate. Participants undergo an anal swab, which is then analyzed using liquid-based cytological examination and tested for the detection of 37 anogenital HPV deoxyribonucleic acid genotypes. Participants also complete a demographic and behavioral questionnaire that covers sexual behavior, history of anal symptoms, and possible anal cancer risk factors. Associations will be tested using multiple regression analysis. Recruitment for the study began in 2015 and is ongoing. To date, 96 (77%) of 125 kidney transplant recipients approached have consented to the study. The mean age is 48 (median, 47 y; range, 20-76 y), 59% are male, and Northwest European (58%) represented the largest ethnic group. No participants self-identified as Aboriginal or Torres Strait Islander. High consent rates and positive qualitative results suggest that a larger screening program may be well received by kidney transplant recipients, with increased resources and some modification to the timing of approach. Further results of the study will inform the possible implementation of a larger screening trial for prevention of anal cancers in kidney an

OBJECTIVE: HPV causes ~90% of anal cancer and HPV16 is the type most commonly associated with anal cancer. Gay and bisexual men (GBM) are at greatly increased risk. We investigated patterns of vaccine-preventable anal HPV in older GBM. METHODS: The Study of the Prevention of Anal Cancer (SPANC) is an ongoing, prospective cohort study of HIV-positive and HIV-negative Australian GBM. Participants completed questionnaires and underwent an anal swab for HPV genotyping using Roche Linear Array. We analysed baseline data from SPANC by HPV type, mean number of types, stratified by age and HIV status. RESULTS: Anal HPV results from 606 (98.2%) of 617 participants (median age 49 years, 35.7% HIV-positive) showed 525 (86.7%) had ≥1 HPV type and 178 (29.4%) had HPV16. Over one third of participants (214, 35.3%) had no nonavalent vaccine-preventable types detected. Two (0.3%) participants had all quadrivalent types and none had all nonavalent vaccine types. HIV-positive participants (p<0.001) and younger participants (p=0.059) were more likely to have more vaccine-preventable HPV types detected. CONCLUSION: Anal HPV was highly prevalent in this largely community-based GBM cohort. Vaccine-preventable HPV16 was detected in approximately one third of participants. These findings suggest that the potential efficacy of HPV vaccination of older GBM should be explored.

Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved. In a natural history study of anal human papillomavirus (HPV) infection and HPV-related lesions among homosexual men in Sydney, Australia, we identified 15 examples of papillary immature metaplasia (PIM) in anal biopsy samples. PIM has previously been described in the cervix, but not in the anal canal. PIM is a form of exophytic low-grade squamous intraepithelial lesion (eLSIL) also known as condyloma. In contrast to the maturing keratinocytes and koilocytosis seen in conventional eLSIL, the slender papillary structures of PIM have a surface population of immature squamous cells. In our anal samples PIM was characterized by close proximity to conventional eLSIL, was negative for p16 INK4A (p16) expression, and revealed the presence of a single low-risk HPV genotype (either 6 or 11) in laser capture microdissected lesions. The clinical significance of recognizing PIM lies in preventing misdiagnosis as high-grade squamous intraepithelial lesion, (the presumed precursor to anal cancer), due to the morphologic immaturity of the cell population. In routine practice, awareness of anal canal PIM and p16 immunostaining will prevent this. Further study of the natural history of anal canal PIM is needed.

BACKGROUND: Studies regarding the performance of anal cytology in which both the screening test (cytology) and the diagnostic test (high-resolution anoscopy [HRA]) are performed in all members of a screening population are rare. The authors evaluated the performance of liquid-based anal cytology in a cohort of homosexual men in Sydney, New South Wales, Australia. METHODS: The Study of the Prevention of Anal Cancer (SPANC) is a 3-year prospective study of the natural history of anal human papillomavirus infection in homosexual men aged ≥35 years. At baseline, all participants underwent a liquid-based anal cytology test and HRA at the same clinical visit. Biopsies were obtained for histological assessment if lesions suspicious for human papillomavirus infection were visible during HRA. Using any cytological abnormality as the threshold, the sensitivity, specificity, and positive and negative predictive values were calculated against histologically diagnosed high-grade squamous intraepithelial lesions (HSILs). RESULTS: Among 617 men recruited, the median age was 49 years (range, 35-79 years) and 35.7% were positive for the human immunodeficiency virus. Overall, the sensitivity of cytology was 83.2%, the specificity was 52.6%, the positive predictive value was 45.8%, and the negative predictive value was 86.7%. Specificity improved with increasing age (P for trend =.041). Sensitivity was significantly higher in men with >1 anal octant of biopsy-confirmed HSIL (92.9% vs 77.7%; P =.010), and in those who had ≥10 metaplastic cells present on their cytology slides (87.5% vs 70.2%; P =.007). CONCLUSIONS: Anal cytology was found to have a higher specificity in older men while maintaining sensitivity. Sensitivity was higher among those with more extensive HSILs and men with metaplastic cells present on cytology. Cancer Cytopathol 2016;124:415–24. © 2016 American Cancer Society.

BACKGROUND: Anal intraepithelial neoplasia grade 2 (AIN2) and AIN grade 3 (AIN3) are commonly grouped together as high grade squamous intraepithelial lesions (HSIL). We assessed risk factors for HSIL-AIN2 and HSIL-AIN3 in a cohort of homosexual men. METHODS: At the baseline visit in the Study for the Prevention of Anal Cancer (SPANC), all men completed a questionnaire and underwent anal swabbing for cytology and HPV genotyping, followed by high resolution anoscopy. RESULTS: Composite-HSIL prevalence was 47% and 32% among 220 HIV-positive and 396 HIV-negative men, respectively. HSIL-AIN3 (37.7% versus 24.7%; p<0.001), but not HSIL-AIN2 (9.5% versus 7.6%; p=0.395) was more common in HIV-positive men. Recent receptive anal partners (p-trend=0.045), and increasing number of high-risk (HR)-HPV types (p-trend<0.001) were associated with HSIL-AIN2. Lifetime receptive partners (p-trend<0.001), HIV status (OR 1.74; 95% CI: 1.05-2.87) and HPV16 (OR 3.00; 95% CI: 1.56-5.75) were associated with HSIL-AIN3. HPV16 was the most common HR-HPV type detected in men with HSIL-AIN3, both HIV-negative (61.1%) and HIV-positive (54.9%). HPV16 was less commonly detected in men with HSIL-AIN2. CONCLUSIONS: Grouping HSIL-AIN2 and HSIL-AIN3 as HSIL may mask considerable heterogeneity in anal cancer risk. Given the strong link between HPV16 and anal cancer, men with HSIL-AIN3 and HPV16 are likely to be at greatest risk of cancer.

BACKGROUND: The ThinPrep Imaging System (TIS) is an accurate time-saving method of reading cervical ThinPrep slides in screening programs. As anal and cervical cytology are morphologically similar, TIS can potentially be used for anal cytology. We assessed the performance of TIS on anal ThinPrep slides from homosexual men in a natural history study of human papillomavirus-related anal abnormalities. METHODS: Four hundred nineteen anal cytology slides were processed by TIS and classified by a cytologist as either No further review (slide archived) or Manual review (slide requiring full manual screen). The results were compared with the original manual screening report for all slides and specifically for those screening episodes accompanied by a high-grade squamous intraepithelial lesion (HSIL) on concurrent biopsy. RESULTS: One hundred seventy six of 419 (42.0%) slides were classified as No further review, with a trend of decreasing proportions as the degree of severity of the cytological abnormality increased. Thirteen (27.7%) slides with an original unsatisfactory report were classified as No further review. Eighty two (92.1%) of those with biopsy HSIL and cytological abnormality were classified for Manual review, including all 45 (100%) with cytological HSIL. CONCLUSION: The cervical algorithm of TIS performed best on anal samples when HSIL was present both cytologically and histologically. The 27.7% unsatisfactory slides classified as No further review may indicate need for use of different criteria from cervical cytology. Because of the high prevalence of abnormalities, and hence the large proportion of slides needing manual review, the cytologist time-saving would compare unfavorably with use of TIS in cervical screening.

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.

Introduction: Anal squamous cell carcinoma is preceded by persistent infection with high-risk human papillomavirus (HPV) and the cancer precursor, highgrade 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 followup biopsies. Histological and cytological findings in combination with HPV genotyping data will be used to identify persistent HSIL. HSIL will be stratified as nonpersistent 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 Vincent's 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 parti

In a natural history study of anal human papillomavirus (HPV) infection and HPV-related lesions, we examined the reproducibility of histological high-grade squamous intraepithelial lesion (HSIL). Three expert anogenital pathologists share the reporting of histological specimens from the Study of the Prevention of Anal Cancer (SPANC), utilising Lower Anogenital Squamous Terminology (LAST) criteria. In total, 194 previously reported biopsies were randomly chosen within diagnostic strata [50 HSIL–anal intraepithelial neo-plasia (AIN) 3; 45 HSIL–AIN 2; 49 ‘flat’ low-grade squamous intraepithelial lesion (LSIL); 50 ‘exophytic’ LSIL; and 50 negative for squamous intraepithelial lesion] and reviewed by each of these three pathologists. Consensus was defined as agreement between at least two review diagnoses, using a binary classification of HSIL and non-HSIL, or if consensus was not obtained in this way, it was achieved through a multiheader microscope session by the three pathologists. We found very high agreement between original and consensus diagnoses (Kappa = 0.886) and between each pathologist's review and consensus (Kappas = 0.926, 0.917 and 0.905). Intra-observer agreement for the three pathologists was 0.705, 1.000 and 0.854. This high level of diagnostic reproducibility indicates that the findings of SPANC should be robust and provide reliable information about HPV-related anal canal disease.

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 Vincent's 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 pa

Anal cancer is one of the most common non-AIDS-defining malignancies in the era of combination antiretroviral therapy. Its precursor lesion, anal intraepithelial neoplasia (AIN), is highly prevalent in HIV-infected populations. More than 90% of anal squamous cell cancers are attributable to human papillomavirus (HPV). While the biology of HPV-related intraepithelial neoplasia is consistent across lower anogenital sites, the natural history of AIN is not well established and cannot be assumed to be identical to that of cervical intraepithelial neoplasia. Screening strategies to prevent anal cancer should be developed based on robust natural history data in HIV-infected and uninfected populations. Likewise, treatments need to be tested in randomized clinical trials, and reserved for those at significant risk of progression to cancer. This review covers the epidemiology, pathogenesis and immunology of HPV infection, AIN and anal cancer, and summarizes the current diagnosis, screening and treatment strategies in HIV-infected adults. © 2013 British HIV Association.

BACKGROUND: The incidence of human papillomavirus (HPV)-associated anal cancer is increasing in men who have sex with men (MSM). Screening for the presumed cancer precursor, high-grade anal squamous intraepithelial lesions (HSIL) in a manner analogous to cervical cancer screening has been proposed. Uncertainty remains regarding anal HPV natural history and the role of anal cytology and high-resolution anoscopy (HRA) as screening tests. Well-designed cohort studies are required to address these issues. METHODS/DESIGN: The SPANC study is a prospective study of the epidemiology of low-risk and high-risk anal HPV infection and related cytological and histological abnormalities in HIV-negative and HIV-positive homosexual men aged 35 years and over. The study aims to recruit 600 men from community-based settings in Sydney, Australia. There are six study visits over three years. At the first five visits men undergo a digital ano-rectal examination (DARE), an anal "Papanicolaou" (Pap) test for HPV detection, genotyping and anal cytology, followed by HRA and directed biopsy of any visible abnormalities. The men also complete a behavioural questionnaire before each visit. Questions include a detailed history of sexual behaviour, of anal symptoms, possible anal cancer risk factors and validated quality of life and psychosocial questions. Questionnaires are also completed 2 weeks and 3 months following the provision of test results and include questions on participant experience during the procedure and post-procedure symptoms, including pain and bleeding in addition to quality of life/ psychosocial outcomes. DISCUSSION: Recruitment for the study began in September 2010 and will conclude in mid-2015, with follow up continuing to 2018. Thus far, over 350 men have been recruited from a variety of community-based settings and are broadly representative of the target screening population. The SPANC study is one of only a small number of cohort studies globally to perform HPV, cytol

Genetic variation of 49 human papillomavirus (HPV) 6 and 22 HPV11 isolates from recurrent respiratory papillomatosis (RRP) (n = 17), genital warts (n = 43), anal cancer (n = 6) and cervical neoplasia cells (n = 5), was determined by sequencing the long control region (LCR) and the E6 and E7 genes. Comparative analysis of genetic variability was examined to determine whether different disease states resulting from HPV6 or HPV11 infection cluster into distinct variant groups. Sequence variation analysis of HPV6 revealed that isolates cluster into variants within previously described HPV6 lineages, with the majority (65%) clustering to HPV6 sublineage B1 across the three genomic regions examined. Overall 72 HPV6 and 25 HPV11 single nucleotide variations, insertions and deletions were observed within samples examined. In addition, missense alterations were observed in the E6/E7 genes for 6 HPV6 and 5 HPV11 variants. No nucleotide variations were identified in any isolates at the four E2 binding sites for HPV6 or HPV11, nor were any isolates found to be identical to the HPV6 lineage A or HPV11 sublineage A1 reference genomes. Overall, a high degree of sequence conservation was observed between isolates across each of the regions investigated for both HPV6 and HPV11. Genetic variants identified a slight association with HPV6 and anogenital lesions (p = 0.04). This study provides important information on the genetic diversity of circulating HPV 6 and HPV11 variants within the Australian population and supports the observation that the majority of HPV6 isolates cluster to the HPV6 sublineage B1 with anogenital lesions demonstrating an association with this sublineage (p = 0.02). Comparative analysis of Australian isolates for both HPV6 and HPV11 to those from other geographical regions based on the LCR revealed a high degree of sequence similarity throughout the world, confirming previous observations that there are no geographically specific variants for these HPV types.

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.