Cañadas, María-Paz BSc*†; Darwich, Laila DVM, PHD*‡; Sirera, Guillermo MD, PHD§¶; Bofill, Margarita PHD*∥; Piñol, Marta MD**; Garcia-Cuyas, Francesc MD**; Llatjos, Mariona MD††; Corbasi, Patricia BSc¶; Clotet, Bonaventura MD, PHD*§¶; Videla, Sebastián MD, PHD¶; on behalf of the HIV-HPV Can Ruti Study Group
*Retrovirology Laboratory-IrsiCaixa Foundation, Barcelona, Spain; †General Lab, Barcelona, Spain; ‡Department of Sanitat i Anatomia Animal, University Autonomous of Barcelona (UAB), Barcelona, Spain; §HIV Clinical Unit, Department of Medicine, University Hospital Germans Trias i Pujol, Badalona (Barcelona), Spain; ¶Lluita Contra La SIDA Foundation, Barcelona, Spain; ∥Institució Catalana de Recerca i Estudis Avancats (ICREA), University Hospital Germans Trias i Pujol, Badalona (Barcelona), Spain; **Department of Surgery, University Hospital Germans Trias i Pujol, Badalona (Barcelona), Spain; and ††Department of Pathology, University Hospital Germans Trias i Pujol, Badalona (Barcelona), Spain
The authors thank Rafael Molina for statistical analyses, Dr. Marià Sust (external biostatistician) for his methodological advice, Dr. Belén LLoveras (ICO, Barcelona, Spain) for kindly providing SiHa cervical cells and to Mr. Navil Khamlichi for English correction. Special thanks also go to the male patients of our HIV Unit.
HIV-HPV Can Ruti Study Group: University Hospital Germans Trias i Pujol, Badalona (Barcelona). University Autonomous of Barcelona: HIV Clinical Unit and Internal Medicine Department: Dr. J. Coll, Dr. C. Rey-Joly, Dr. A. Bonjoch, Dr. M. Jabaloyas, Dr. T. Jou, Dr. J. Moltó, Dr. E. Negredo, Dr. J. Romeu, Dr. C. Tural; Department of Pathology: Eva Castellà; Nurses of Department of Proctology: Ms. Inés Fernández; Nurses of HIV Clinical Unit: Ms. Carmen Alcalde, Ms. Rosa Guerola, Ms. Anna Salas; Lluita contra la SIDA Foundation staff: Ms. Ingrid Martínez, Mr. Eduard Solaz; Department of Molecular Biology of General Lab.
Supported by the Spanish AIDS network “Red Temática Cooperativa de Investigación en SIDA” (RD06/0,006) and by a grant from the Lluita Contra La SIDA Foundation.
S.V. has received honoraria for collaborating with Laboratorios Dr Esteve for work unrelated to HPV/HIV. B.C. has received honoraria for speaking and participating in advisory boards from Abbott, Bristol-Myers Squibb, Boehringer-Ingelheim, Gilead Sciences, GlaxoSmithKline, Pfizer, Merck, Janssen-Tibotec. All others authors report no potential conflicts.
M.P.C. and L.D. have contributed equally to this study.
Correspondence: María-Paz Cañadas, BSc, IrsiCaixa Foundation, Hospital Universitari Germans Trias i Pujol, Badalona (Barcelona)-08916, Spain. E-mail: PCC@general-lab.com.
Received for publication June 5, 2009, and accepted October 21, 2009.
Highly oncogenic human papillomavirus 16 (HPV-16) is etiologically associated with approximately half of all cervical cancers.1 HPV-16 is also the most common HPV type detected in cervical high-grade squamous intraepithelial lesions, a precancerous lesion.2 Although most research has focused on women, an increase in HPV related malignancies has recently been reported in HIV-positive men.3
The integration of HPV-16 DNA into the host genome is considered an important event in the progression of premalignant cervical lesions to cervical cancer.4–6 During HPV-infection, products of viral E6 and E7 oncogenes are important in the process of transformation and immortalization of infected cells and, consequently, in the development of malignant lesions. These genes are controlled by the E2 protein, which represses the transcription of E6 and E7 genes. The preferential site of HPV integration has been identified as the E2 open reading frame, a region that shows more disruptions or deletions than other sites of the HPV genome.7,8 Therefore, the lack of control of E2 over E6 and E7 gene transcription, as a consequence of the integration process, is probably an important episode in the progression and development of carcinogenic lesions. However, little is known about this phenomenon in anal lesions of HIV-positive patients.
Our aim was to assess the prevalence of anal HPV-infection, anal HPV-16 infection, its physical status (HPV-16 integrated forms) in anal samples of HIV-1 infected men with no previous anal pathology background, and the predictive factors associated with them.
PATIENTS AND METHODS
This cross-sectional study included all HIV-positive men ≥18 years old, which were consecutively visited and interviewed in the outpatient HIV Clinical Unit between January 2005 and December 2006. These patients were the first patients enrolled in the CARH-MEN cohort (CAn Ruti HIV+ MEN). Patients included in this cohort are systematically screened for anal HPV infection and HPV-related anal pathology. Men with previous anal pathology were excluded. Written informed consent was obtained from all participants. The protocol and other materials were approved by independent ethics committee of the Hospital.
In this study we collected: age, sexual behavior (men who have sex with men (MSM), and heterosexual, receptive anal intercourse background, number of sexual partners), duration of HIV infection (years), CD4 cell counts (the closest value determined before the cytologic sample collection) and CD4 nadir (the lowest CD4 value of each patient's medical records), HIV viral load in plasma at baseline time-point (the closest value determined before the cytologic sample collection, test detection limit 50 copies/mm3), and duration (years) of highly active antiretroviral therapy (HAART) at the time of sampling.
Anal Cytologic Evaluation
Anal samples were obtained using a cytobrush (Eurogine SL, Sant Boi del Llobregat, Spain), which was introduced into anal canal and softly rotated during 30 to 45 seconds. The cytobrush was included and shaken into 20 mL of PreservCyt/ThinPrep Pap test solution (Cytyc Iberia SL, Barcelona, Spain) during 30 seconds. The cytobrush was removed and the PreservCyt/ThinPrep solution was stored until analysis at 4°C. Cytologic changes were classified according to the Bethesda System: normal (no cell changes), atypical squamous cells of uncertain significance (ASCUS) or low- or high-grade squamous intraepithelial lesions (LSIL or HSIL, respectively). Samples were checked by 2 cytopathologists.
HPV Detection and Genotyping
HPV detection and typing were performed in all samples, using the F-HPV Typing Kit (Molgentix, BCN, Spain) in accordance with the manufacturer's instructions. Briefly, extracted DNA was amplified using a multiplex F-HPV PCR with a set of 15 fluorescently labeled primers recognizing HPV Types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 within the E6 and E7 regions of the HPV genome. This assay can generate type-specific PCR products which can then be differentiated according to their size and color. A human short tandem repeat sequence included in the same multiplex was amplified as internal control to check for DNA integrity and absence of PCR inhibitors. The F-HPV amplification was performed in a final PCR volume of 25 μL containing 20 μL of ready-to-use reaction mixture and 5 μL of extracted DNA for 35 repeating cycles of 30 seconds at 95°C, 30 seconds at 64°C, and 30 seconds at 72°C. Products were analyzed by capillary electrophoresis on ABI 3,130 XL genetic analyzer and GeneMapper 4.0 Software (Applied Biosystems, CA).
Physical Status of HPV-16
Control Samples of HPV-DNA Integration.
SiHa and Caski cervical carcinoma cells were used as control DNA integration and episomal of HPV-16 respectively. Siha cell line has 1 to 2 copies of integrated HPV DNA in which the E2 gene is deleted and the E6 is retained.9
A multiplex real-time PCR method was carried out for simultaneous amplification of the E2 and E6 regions.10 The physical status defined as not integrated (episomal), totally integrated (integrated) or coexisting both forms (mixed) was identified examining differences in the threshold cycle (Ct) between E2 and E6. The same Ct was expected for episomal forms (same amount of E6 and E2 products), an increase up to one for E2 Ct for mixed forms, and a complete lack of amplification for E2 in integrated form.
Each PCR assay included HPV positive and negative controls. Particular care was taken to prevent carry-over contamination by separating pre- and post-PCR areas.
A descriptive analysis was performed for baseline population characteristics. Prevalence and its corresponding 95% confidence intervals (95% CI) were calculated for anal HPV infection (yes/no), anal HPV-16 infection (yes/no), HPV-16 integration (yes/no), and anal cytologic lesions (normal/abnormal). Predictive factors associated (odds ratios, OR and 95% CI) were analyzed using univariate and multivariate logistic regression models, or nominal regression analyses when cytology was categorized into normal, ASCUS or LSIL, and HSIL. The multivariate model included age, sexual behavior (MSM and number of sexual partners), CD4+ counts, plasma HIV load, time on HAART, and time since HIV diagnosis. A P value of ≤0.05 was considered statistically significant. All data were recorded in a database program (Microsoft Access for Windows XP, Redmont, CA). Data analysis was performed using the statistical software program SPSS version 15.0.
The study included 269 white HIV-1 infected men: 67% MSM and 33% heterosexual. The median age was 44 years (IQR: 38–49) and 12 years from HIV diagnosis (IQR: 6–18). The median CD4 cell count was 469 cell/mm3 (IQR: 298–643) at baseline, and of 222 cell/mm3 (IQR: 94–341) CD4 nadir count. Seventy-six percent of patients (205/269) were on HAART regimen. The median of years on HAART was 9 (IQR: 4–11). The median HIV viral load was 50 copies/mm3 (IQR: 50–420).
Prevalence of Anal HPV and HPV-16 Type Specific Infections
The overall HPV infection prevalence was 78% (209/269, 95% CI: 72%–83%), Single anal infection was detected in 21% of patients (56/269, 95% CI: 16%–26%), and multiple anal infections in 57% (153/269, 95% CI: 51%–63%).
Anal HPV-16 type specific prevalence was 29% (77/269, 95% CI: 23%–34%). Among these patients, 13% (10/77, 95% CI: 6%–23%) had a single anal HPV-16 infection, and 87% (67/77, 95% CI: 77%–94%) showed multiple HPV-infection. Twenty-four percent (16/67, 95% CI: 14%–36%) showed HPV-16 plus other HPV type, and 76% (51/67; 95% CI: 64%–86%) HPV-16 infection plus other 2 or more HPV types. Among patients with multiple infections including HPV-16, the most prevalent types found were HPV-33 (30%); HPV-39 (21%); HPV-59 (19%); HPV-51, HPV-52, HPV-58 (18%); and HPV-6 (14%) and HPV-11 (17%).
HPV-16 Integration and Anal Cytology
The overall HPV-16 integration was 9% (25/269, 95% CI: 6%–13%). Integration was detected in 32% (25/77) of samples positive for HPV-16 infection. Of the 25 samples, 23 were found as mixed (episomal and integrated) and the other 2 as completed integrated forms.
Overall, 77% (59/77, 95% CI: 66%–86%) of patients with anal HPV-16 infection had cytologic abnormalities. Pathologic cytologies were diagnosed as follows: 22% (17/77) ASCUS, 41.5% (32/77) LSIL, and 13% (10/77) HSIL. We found correlation between HPV-16 integration and severity of anal pathology. Thus, among 59 samples with cytologic abnormalities, 24 samples showed HPV-16 integration, 18% (3/17) in ASCUS, 44% (14/32) in LSIL, 70% (7/10) in HSIL. HPV-16 was found integrated in only 1 of 18 sample with normal cytology (Table 1).
Predictive Factors Associated With HPV and HPV-16 Infections, HPV-16 Integration and Anal Cytology
Anal HPV-infection was associated with MSM practices (OR = 4.4, 95% CI:1.3–15), receptive anal intercourse (OR = 3.4, 95% CI:1–11), detectable HIV load (OR = 3.1, 95% CI:1.3–7.3), low CD4 nadir counts (<200 cells/mm3) (OR = 2.1, 95% CI:1–4.8), and longer time of HIV infection (OR = 1.1, 95% CI:1.0–1.2) in the multivariate logistic regression model. MSM practice and receptive anal intercourse were highly correlated (r = 0.74, P < 0.001).
The only risk factor associated with anal HPV-16 integration was the time since HIV diagnosis (OR = 1.2, 95% CI: 1.0–1.3; P = 0.01) after adjusting for all covariables. Table 2 shows the risk factors associated with anal HPV-16 infection.
The risk factors associated with abnormal cytology results were: HPV infection (OR = 17.8, 95% CI: 6.8–46.6), HPV-16 infection (OR = 4.6, 95% CI: 2.5–8.4), and HPV-16 integrated forms (OR = 11.7, 95% CI: 1.5–93.5). Moreover, HPV-16 integration represented the most important risk factor (OR = 20, 95% CI: 1.6–226) for anal abnormalities in the multivariate analysis. We performed the same analyses classifying the cytology into 3 groups: normal cytology (as referent variable), low-grade (ASCUS and LSIL diagnosis), and high-grade (HSIL diagnosis) anal intraepithelial lesions. The integrated HPV-16 forms was related to higher prevalence of LSIL (OR = 11.3, 95% CI: 1.1–116) and HSIL (OR = 48, 95% CI: 2.4–960). Low nadir value of CD4 cells was together the HPV-16 integration, another risk factor for anal HSIL (Table 3).
This cross-sectional study provides significant data about the relationship between HPV-16 integration forms in anal cells and the presence of premalignant lesions in HIV-1 infected men with no previous history of anal pathology.
It is generally accepted that integration of high risk HPV into the host cell genome is an important factor in the process of cervical carcinogenesis. Differences in the integration profile seem to depend on the virus genotype.11 Although HPV-18, HPV-31, and HPV-33 are usually found in total integrated form, HPV-16 is found in episomal, total, and mixed forms. In this context, our study was focused on HPV-16 integration. The high overall HPV-16 integration prevalence observed in our study (9% among HIV infected men) was related to a high prevalence of HPV-16 anal infection and cytologic intraepithelial lesions. The high prevalence of HPV-16 anal infection observed was similar to results recent published in anal-related lesions among HIV-positive men.12
It is noteworthy that the HPV-16 integration was strongly associated with cytologic precancerous lesions. The prevalence of integration increased to 44% in LSIL and to 70% in HSIL. In a study with 16 anal biopsies samples categorized as HSIL and all of them positives for HPV-16 infection, showed 25% of integrated forms.13 Our results, suggest that HPV-16 allows identifying patients with a higher risk of developing anal lesions. In consequence, HPV-16 integration could be a good prognostic marker for anal precancerous lesions. Nonetheless, longitudinal studies are required to verify this first evidence.
The only risk factor associated with a higher prevalence of HPV-16 integration was the time since HIV diagnosis. This relation could be explained because HPV infections are more persistent in HIV patients, favoring the HPV-16 integration in anal cells and in consequence the development of anal pathology. Nevertheless, this result must be interpreted with caution due to the small sample size.
Recent studies have reported a high prevalence of anal HPV infection men. MSM practice or the number of sex partners are the most important risk factors associated to anal HPV-infection and to anal HPV-16 type specific infection.14–16 According to our findings, MSM, and the practice of receptive anal sex are a risk factors for anal HPV infection rather than the number of sex partners, despite of MSM had a larger number of partners (70% with more than 10 partners) compared to the heterosexual group (6%). Also, it has been reported that plasma HIV RNA levels and CD4+ T-cell count together had a strong interactive association with the prevalence and the incidence of HPV infections.17 In our study, detectable HIV RNA levels in plasma or CD4 nadir counts below 200 cells/mm3 were associated with a 3- and 2-fold higher prevalence of presenting anal HPV infection, respectively. On the other hand, when both variables were combined in a stratified analysis, no significant relations were observed for HPV infection or cytologic abnormalities. Having most patients with undetectable HIV load, it was not possible to categorize them by combined CD4+ and HIV RNA strata. Another risk factor associated to a major prevalence of anal HPV or HPV-16 specific infections was the time since HIV diagnosis (10% of increment per year of HIV infection), probably related to the fact of being immunocompromised for a longer period.
The effect of HAART on coinfections in HIV-infected patients is explained because HAART improves immunologic status by increasing CD4 cell counts and controls HIV replication, and these effects on HIV/AIDS illness could play a role in the copathologies. Nevertheless, the use of HAART does not seem to have effect on modification the natural history of HIV-HPV coinfection.18,19 Accordingly, in our study we did not observe any effect between time of HAART and prevalence of anal HPV infection.
A few limitations of this study should be acknowledged; the small sample size, the study design (cross-sectional), and to study integration only in anal cytologic samples, all of these factors together could underestimate or overestimate the obtained results.
In summary, this study taken as a whole provides information about the prevalence of HPV-16 infection and the role of its integration in anal cells of HIV infected men. Our results support the need for more closely monitoring of anal HPV infection and especially HPV-16 integration, since the presence of integrated HPV-16 was directly associated with a higher prevalence of anal precancerous lesions.
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