Emmanuel Masongsong

Smokers at Higher Risk for Undetected Antibody for Oncogenic Human Papillomavirus Type 16 Infection

Objective: To determine the association between tobacco smoking and serologic evidence of human papillomavirus type 16 (HPV16)–specific antibodies among HPV16 DNA–positive women. Design, Setting, and Participants: Baseline health history, physical examination, and laboratory data for 205 HPV16 DNA–positive women with no prior cytologic evidence of squamous intraepithelial lesions who were enrolled subsequently in a randomized clinical trial. Main Outcome Measure: HPV16-L1 antibody (anti-HPV16 antibody) detected from serum using RIA or ELISA. Results: Eighty-seven percent (179 of 205) of women tested positive for HPV16 DNA using cervicovaginal swabs or lavage specimens, and 26 women showed similar results using swab specimens of external genitalia alone. HPV16-infected women who reported increasingly greater levels of daily cigarette smoking were less likely to test positive for anti-HPV16 antibodies than nonsmoking women (P = 0.02). Smokers were twice as likely as nonsmokers to test negative for anti-HPV16 antibodies, even after controlling for the effects of other covariates in the analyses (adjusted odds ratio, 0.5; 95% confidence limits, 0.2-0.9). Although Papanicolaou test findings and smoking characteristics were poorly correlated (r2 = 0.01), women who showed atypical cells of unknown significance or squamous intraepithelial lesion were twice as likely to test anti-HPV16 antibody positive as women who showed normal Papanicolaou tests (adjusted odds ratio, 2.0; 95% confidence limits, 1.1-3.7). Conclusion: These data suggest that smoking may influence the long-term risk for cancer by perturbing early immune responses to the virus and may increase the likelihood of persistent infection. Patient education messages should alert women to this additional risk of smoking. A clinical trial of smoking cessation should be explored as a therapeutic intervention for primary HPV16 infection. (Cancer Epidemiol Biomarkers Prev 2006;15(5):915–20)

Anal Cancer Screening: Barriers and Facilitators Among Ethnically Diverse Gay, Bisexual, Transgender, and Other Men Who Have Sex With Men

ABSTRACT Knowledge and beliefs about anal cancer screening among gay and other men who have sex with men remains unclear, despite data that suggests significant risk for intra-anal HPV-related cancers. Nevertheless, community-based screening activities may be most effective when stakeholder perspectives are addressed. We conducted four focus groups among 16 male and 3 female health care advocates experienced in working with diverse gay and other men who have sex with men in Los Angeles. Barriers to anal cancer screening included lack of awareness, stigma, psychological and physical discomfort, the anus as hidden/private, primary concern with HIV, and mens lack of healthcare seeking. Facilitators were community screening sites, novel strategies such as home testing, health care system changes and targeted educational campaigns, which may increase anal cancer awareness and screening among ethnically diverse men who have sex with men.

Smoking enhances risk for new external genital warts in men

Repeat episodes of HPV-related external genital warts reflect recurring or new infections. No study before has been sufficiently powered to delineate how tobacco use, prior history of EGWs and HIV infection affect the risk for new EGWs. Behavioral, laboratory and examination data for 2,835 Multicenter AIDS Cohort Study participants examined at 21,519 semi-annual visits were evaluated. Fourteen percent (391/2835) of men reported or were diagnosed with EGWs at 3% (675/21,519) of study visits. Multivariate analyses showed smoking, prior episodes of EGWs, HIV infection and CD4+ T-lymphocyte count among the infected, each differentially influenced the risk for new EGWs.

Behavioral and sociodemographic risk factors for serological and DNA evidence of HPV6, 11, 16, 18 infections

INTRODUCTION Risk for HPV6/11/16/18 infections in young sexually active, behaviorally low-risk females is not well described and may inform public policy. METHODS To assess exposure risk for HPV/6/11/16/18 among 16-23 year old low-risk females, data for 2409 female clinical trial participants were evaluated. Baseline visit self-reported sexual, behavioral and demographic characteristics; and results from HPV genotyping and serology, and other clinical laboratory assays were analyzed. All subjects reported <5 lifetime male sexual partners and no prior abnormal cytology at baseline. RESULTS While 98% (2211/2255) were naïve to HPV16 or 18 and 99.6% (2246/2255) were naïve for 1-3 index HPVs, 27% (616/2255) showed antibody, DNA or both for ≥1 index HPV. While 18% (409/2255) tested HPV16- or -18-antibody- or -DNA-positive, only 2% (44/2255) tested positive for both types. Against this high background, other sexually transmitted infections (STIs) were uncommonly detected, suggesting low sexual risk-taking behavior. The adjusted analyses showed race, age, alcohol consumption, current Chlamydia trachomatis (chlamydia) and Trichamonas vaginalis (trichomoniasis), bacterial vaginosis (BV), number of lifetime male sex partners predicted positive index-HPV antibody test results. However, only the number of male sex partners predicted positivity for HPV6/11- and 16/18-DNA, and chlamydia infection predicted positivity for HPV6/11-DNA alone. CONCLUSIONS Taken together, type-specific HPV-DNA and -antibody evidence of HPV6/11/16/18 infections among behaviorally low-risk 16-23 year old females is high. Since almost all participants would have benefited by either currently available bivalent or quadrivalent vaccine strategies, delaying vaccination beyond menarche may be a missed opportunity to fully protect young females against HPV6/11/16/18 infections and related dysplasias. Early diagnosis and treatment of chlamydia and trichomonas may be important in HPV pathogenesis.

Comparison of 2 anal cytology protocols to predict high-grade anal intraepithelial neoplasia.

Objectives Nylon-flocked and Dacron swab anal cytology collection procedures were evaluated for detecting high-grade anal intraepithelial neoplasia. Materials and Methods Cross-sectional data for 42 HIV-infected and 16 uninfected men who have sex with men have been used. Sequentially collected anal cytology specimens, high-resolution anoscopy, and medical biopsy evaluated the sensitivity and specificity of cytology for predicting high-grade anal intraepithelial neoplasia. Men showing atypical squamous cells (ASC) or more severe findings by cytology were compared with those showing negative for intraepithelial lesions. Results The prevalence of high-grade anal intraepithelial neoplasia was 35% (21/58), and findings were approximately 1.5 times higher among HIV-infected compared with uninfected men. Unsatisfactory cytology was twice as common among Dacron compared with nylon-flocked swab protocol specimens (14% [8/58] vs 7% [4/58]). Sensitivity and specificity for the nylon-flocked protocol cytology showing ASC or more severe findings were 81% (58%–95%) and 73% (50%–89%), respectively. Dacron protocol specimens showed 52% (30%–74%) and 58% (34%–80%) sensitivity and specificity, respectively. Men showing ASC or more severe findings using the nylon-flocked protocol cytology showed 3-fold higher odds for high-grade anal intraepithelial neoplasia compared with men with negative results (p < .05), but no statistically significantly higher odds of high-grade anal intraepithelial neoplasia for men showing ASC or more severe findings compared with those with negative results for Dacron protocol cytology (p > .05). Conclusions The nylon-flocked protocol better detects high-grade anal intraepithelial neoplasia than does the Dacron protocol, yields more interpretable results, and classifies men with high-grade anal intraepithelial neoplasia as cytologically abnormal 2.5 times more often, even in this small clinical trial. Clinical trials registration number: NCT00955591.

The prevalence of examiner-diagnosed clitoral hood adhesions in a population of college-aged women.

Objective. To determine the prevalence of clitoral hood adhesions in college-aged women. Materials and Methods. A retrospective review of medical records was conducted for 589 college-aged women undergoing a routine womens annual examination at a student health clinic by a single examiner (E.W.) using a standardized procedure. The severity of adhesions was classified and recorded by the examiner based on the extent of visualization of the clitoral glands and sulcus. Recorded age, sexual activity, contraceptive use, pregnancy, and marital status were abstracted from records. Results. Two thirds (393/589) of women were unaffected by any clitoral hood abnormality; of the affected, 58% (113/196) showed 25% involvement. Only 12% (24/196) showed adhesions involving 75% to ≥90% of the region (&khgr;2 = 857.1, 4 df, p <.0001). The youngest females, born after 1981, were at highest risk for being affected (compared with women born 1976-1981, adjusted OR = 1.7 [1.1, 2.6]). Relationships with other demographic and behavioral characteristics were not statistically significant. Conclusions. Adhesions may be more common than previous published reports imply. Study participants were evaluated for routine health screening and presented with few or no symptoms. These data suggest that adhesions may be a normal variant in otherwise asymptomatic college-aged women and that no treatment is necessary.

Nylon-flocked swab collection method better predicts high-grade AIN than does dacron swab method

Author(s): Wiley, Dorothy; Bolan, Robert; Voskanian, Alen; Young, Stephen; Elashoff, David; Hsu, Hilary; Masongsong, Emmanuel; Barman, Provaboti; Detels, Roger

Abstract LB-177: HPV16 CpG and de novo-cytosine methylation is differentially associated with low-grade versus high-grade anal intraepithelial neoplasia in HIV-infected men

Objective: To determine if cytosine methylation in CpG and de novo sites is differentially associated with high- or low-grade anal intraepithelial neoplasias (HG-, LG-AIN), HPV16 DNA from182 clinical specimens. Background: Intra-anal cancer (IAC) is an emerging health crisis for gay, bisexual, transgender and other men who have sex with men (MSM) and rates have risen sharply among HIV-infected MSM despite introduction of HAART. High-risk HPVs are a causal risk factor for IAC and are especially common where MSM show HIV-coinfection. Further, cytosine methylation has been posited as an epigenetic transcription regulator that is poorly described in intra-anal dysplasias and cancers. Methods: HPV16 DNA was extracted from anal swab specimens and tested using bisulfite modification, PCR, cloning and sequencing of ∼10 clones/sample. Cervical cancer cell lines, CaSki and SiHa, and paraffin-embedded anal cancer specimens were similarly characterized as controls. Genomic sequences were evaluated using CLUSTAL and BiQ Sequence Alignment analysis software. Descriptive, tabular and multivariate analyses were performed using SAS (Version 9.2). Graphical representations were compiled using SIGMAPLOT (Version 9.0). Findings: Overall, methylation was relatively low in clinical AIN specimens. For 25,085 cytosines, the mean prevalence of methylation was 2.7% (SEM=0.4%) across 3′ L1 and LCR; the prevalence me-CpG, -CpA, -CpT, and -CpC was 3.2% (0.2%), 2.6% (0.1%), 2.5% (0.1%), and 2.7% (0.1%), respectively. However, some variation in HPV16 genomes was observed across specific functional gene sequences for HG- and LG-AIN specimens. Specifically, LG-AINs showed 1.30–2.62 times greater mean prevalence of methylation when compared to HG-AINs, across 24 of the 31 cytosines between nt7428–7564 ( p -values Conclusions: Data suggest the HPV16 5′LCR/enhancer region contains a large concentration of cytosine-containing transcription regulatory elements where binding interference by methylation might alter expression of the p97 promoter. Methylation in the HPV16 5′LCR/enhancer may decrease risk for HG-AIN. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-177. doi:10.1158/1538-7445.AM2011-LB-177

Cytosine methylation in the HPV16 3' L1/ 5'LCR region characterized from anal epithelia of HPV-HIV coinfected men

Anal specimens derived from 83 HIV-HPV16 co-infected men were analyzed for cytosine methylation using bisulfite modification, PCR amplification, cloning, and sequencing. Data were processed using the ClustalW alignment algorithm within BiQ Analyzer Software and input into SAS 9.2 to generate heat maps and histograms(Figure ​histograms(Figure1).1). Approximately 10 clones were characterized for each specimen across 81 CpA, CpT, CpC (denovo methylation) and 10 CpG sites within the 3’-L1 and 5’LCR region of HPV16 genomes. Analyses were confined to 64 denovo cytosine residues and 10 CpGs outside the primer annealing regions. Clinical outcome evaluation by a single examiner showed seven men with no anal intraepithelial neoplasias (AIN), 26 with AIN-1, and 50 with AIN-2. Data showed the average prevalence (standard deviation) of methylcytosines was 11% (0.3) across 7,553 bases/residues and did not vary across maintained or denovo cytosine groups, i.e., 6,723 denovo sites and 830 CpGs. Evaluation by a single examiner using cytology and histology showed 7 with no anal intraepithelial neoplasias (AIN), 26 showed AIN-1, and 50 showed AIN-2. Mean and median prevalence of denovo and CpG methylation were closely approximated across AIN groups. Maxima were observed in two regions: 7111 to 7119, and 7505 to 7514 that showed a methylcytocine prevalence of 24% (0.4), and 18% (0.4), respectively. Logistic regression suggests that with each year of age, risk for moderate-severe methylation ≥30% at each site decreases; however, among smokers risk for ≥30% methylation increases. Specifically, moderate cytosine-methylation decreased by 5% (OR=0.95, 95% CI, (0.92, 0.99) for each year of age, centered around the mean of the sample. Smokers were nearly twice as likely to show moderate cytosine methylation overall (OR=1.9, (1.1, 3.3) and these estimates did not vary across CpG-denovo sites. Although not statistically significant, moderate cytosine methylation was inversely associated with grade of AIN in these data (when compared to high-grade, low-grade and no AIN showed OR=1.5 (0.8, 2.7) and 2.0 (0.9, 4.5), respectively, p=0.2). Figure 1 Heatmap of Methylcytosines in the 3’L1 and 5’LCR Region of HPV16.

Abstract 2807: The relationship between age and efficiency of cervical cancer screening using population-based surveillance

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Data for in situ cervical carcinomas are not routinely collected by U.S. tumor registries. Some experts suggest that comparing preinvasive and invasive cervical cancers (ICC) rates may represent our best population-based appraisal of screening strategies. From 1991-1997, a total of 5,921 women diagnosed with in situ or ICC were reported to the Markey Cancer Registry in Lexington, Kentucky, a population-based registry. Maximum-likelihood Poisson regression analyses were used to estimate the incidence rate of in situ and ICC (per 100,000), summarized over age-and race-specific groups for each of seven years, 1991 to 1997. U.S. Census age and race data for 1990 were used to estimate denominators for two race groups: African American (AA) and all others (white). Incidence rates and rate ratios are reported. A total of 4,290 and 1,631 were diagnosed with in situ and ICC in the study period. While more than 80% of cases among women 65 years, only 42% (+4%) of tumors were diagnosed as in situ lesions. Specifically, among 20-39 year olds, AA were less likely than white women to be diagnosed with in situ carcinoma (p 55 were less likely to be diagnosed with in situ disease than ICC. While year of diagnosis was significant in the analyses, relationships between age and diagnostic stage persisted across each of 7 years examined. These data suggest that Pap test most efficiently identifies treatable, high-grade precancerous lesions among women younger than 50 years. HPV infection prevalence varies by age, but not race, while ICC disproportionately affects women of color, especially as they age. Several factors may explain these findings. Screening may be performed less often overall or more inconsistently among older women and among AA. Also, if screening in the population decreases with advancing age, symptomatic older women with higher likelihood of malignancy will be over-represented in the data. Directly tailoring screening programs to recruit and retain non- and under-screened women and women with medical record evidence of poorly triaged Pap test abnormalities may improve detection of cervical cancers at a preinvasive stage, where there is a very high likelihood of survival. We believe that these findings are consistent with those of others, and together they suggest our best public health strategy may be to actively recruit middle-age and older women into cervical cancer screening programs who have undergone Pap test screening fewer than three times in a ten year period. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2807.