Role of Low-Risk HPV PCR Monoinfection in Screening for HSIL and Anal Cancer in Men Who Have Sex with Men Living with HIV

To determine the value of low-risk human papillomavirus (HPV) PCR to screen for “high-grade anal squamous intraepithelial lesion and anal cancer” (HSIL-plus), rate of patients with low-grade anal squamous intraepithelial lesion (LSIL) progressing to HSIL-plus, and progression-related factors. Prospective, longitudinal study of consecutive MSM-LHIV attended between May 2010 and December 2021 and followed for 43 months (IQR: 12–76). HIV-related variables were gathered at baseline, performing anal cytology for HPV detection/genotyping, thin-layer cytological study, and high-resolution anoscopy (HRA). Follow-up was annual when HRA was normal or LSIL, and post-treatment in cases of HSIL-plus, re-evaluating sexual behavior, viral-immunological status, and HPV infection of anal mucosa. The 493 participants had mean age of 36 years: CD4 nadir < 200 cells/uL in 23.1%, virological failure in 4.1%, and tetravalent HPV vaccine > 5 years earlier in 15%. HSIL-plus was ruled out in patients with monoinfection by low-risk HPV genotype and normal cytology (100% sensitivity, 91.9% specificity, PPV 2.9%, and NPV 100%). Progression from LISL to HSIL-plus occurred in 4.27% of patients within 12 months (IQR: 12–12): risk factors were acquisition of high-risk (HR: 4.15; 95% CI: 1.14–15.03) and low-risk (HR: 3.68 95% CI: 1.04–12.94) HPV genotypes, specifically genotype 6 (HR: 4.47, 95% CI: 1.34–14.91), and history of AIDS (HR: 5.81 95% CI: 1.78–18.92). Monoinfection by LR-HPV genotypes in patients with normal cytology is not associated with anal cancer or precursor lesions. Progression from LSIL to HSIL-plus, observed in <5% of patients, was related to acquisition of HR and LR HPV genotypes, especially 6, and a history of AIDS.


Introduction
Anogenital infection by human papillomavirus (HPV) is the most common sexually transmitted infection (STI) worldwide [1]. It has a predilection for skin and mucosal cells and is associated with a wide range of lesions, from warts to low-and high-grade squamous intraepithelial lesions (LSIL and HSIL) and vulvar, vaginal, penis, oropharyngeal, and anal carcinomas [2].
Immunosuppression is a risk factor for HPV acquisition and is produced by infection with the human immunodeficiency virus (HIV). The degree of immunodepression has been related to the incidence of anal dysplasia from chronic infection by HPV genotypes [3,4]. This infection commonly involves the anal mucosa, and anal cancer is one of the most frequent non-AIDS-defining diseases in people living with HIV (PLHIV), mainly in women and men who have sex with men (MSM) [5,6].
Research to date has established a relationship between high-risk (HR) genotypes and a greater likelihood of precursor lesions of anogenital or oropharyngeal cancer/carcinomas [7,8]. MSM living with HIV (MSM-LHIV) who have normal anal cytology and are not infected by an oncogenic HPV genotype have shown no increased risk of HSIL or anal cancer [9].
Inadequate data are available on the presence/absence of HSIL or anal cancer in patients with monoinfection by LR HPV genotype. The objectives of this study were to verify the sensitivity (S), specificity (Sp), and negative (NPV) and positive (PPV) predictive value of the presence of anal mucosal infection by LR HPV genotypes to screen for HSIL and/or anal cancer (HSIL-plus) in MSM-LHIV; to determine the percentage of these patients progressing from LSIL to HSIL-plus; and to analyze the factors related to this progression.

Diagnostic Value of Cytology and HPV PCR for Anal Dysplasia
LR-HPV PCR achieved 79.6% sensitivity (S), 27.9% specificity (Sp), 12.7% positive predictive value (PPV), and 91.6% negative predictive value (NPV) for the diagnosis of HSIL-plus, while cytology demonstrated 72.7% S, 43.7% Sp, 14.2% PPV, and 82.4% NPV for this diagnosis. HSIL-plus could be ruled out in all patients with anal mucosa monoinfection by LR-HPV genotypes and normal cytology (100% S, 91.9% Sp, 2.9% PPV, and 100% NPV) ( Table 5). Correlations between biopsy, cytology, and HR and LR HPV PCR results for HSILplus never exceeded 0.4, the minimum value for analyzing agreement between techniques (Table 6). Table 5. Sensitivity, specificity, PPV, and NPV of anal cytology and HPV PCR for diagnosis of HSIL-plus.
In the study of factors favoring progression from AIN1 to HSIL-plus in patients with AIDS, no statistically significant association was found in bivariate analyses, which considered age, age at start of sexual relations, number of sexual partners in previous 12 months, VPH vaccination, time since HIV diagnosis, CD4 count, CD8 count, viral load, and number of HR and LR HPV genotypes and of all genotypes.

Discussion
The participants in this study were followed for a median of 43 months. One-third were aged under 30 years, and three-fifths between 30 and 50 years; around one-half of them smoked, and one-fifth reported a history of >300 sexual partners throughout life. In regard to HIV infection, their viroimmunological status was excellent, with only 4.1% in virologic failure and a median CD4 count of 698.9 cells; 71.2% were infected with LR genotypes, 74% with HR genotypes, and 56% were coinfected with LR and HR genotypes. Less than half of the participants had normal anal mucosa, around one-tenth had HSIL at enrolment, and three (0.6%) had anal cancer, a similar profile to that of other European cohorts [10], and even of a North American study population that mainly differed in ethnic make-up, with a predominance of Afro-Americans [11] With respect to the value of LR-HPV PCR as the sole screening test for HSIL or anal cancer, it achieved S and NPV values that were higher than obtained with anal cytology and very similar to those observed with HR-HPV PCR. HSIL-plus could be ruled out in all participants with monoinfection by LR-HPV genotypes and normal cytology. This is the first report on the value of LR-HPV PCR in the diagnosis of these lesions in MSM-LHIV, showing that the combination of LR-HPV PCR and anal cytology ruled out HSIL-plus with 100% S and 100% NPV. These findings are in line with previously published results supporting the combination of HR-HPV PCR with anal cytology to screen for anal cancer and precursor lesions in HIV-positive MSM [12,13] LSIL progressed to HSIL-plus (1 ASCC and 22 AIN2/3) in less than 5% of participants during the follow-up period, largely within one year of follow-up, lower than the progression rates of 16.4% [14] and 36% [15] described in previous studies. However, in comparison to the present study, the mean age was higher and a larger percentage of patients had CD4 nadir < 200 cells/uL in the former study [14] and the cohort was more heterogeneous in the latter, including men and women with and without HIV infection [15], which may in part account for the differences in progression rates; in addition, more than half of participants were smokers or ex-smokers in both previous studies, whereas less than half of the present patients were smokers and only 9.1% were ex-smokers. The objective of the present study was to investigate the screening, early diagnosis, treatment, and prophylaxis of anal dysplasia, resulting in a highly treated cohort. Thus, 15% had been vaccinated against HPV, and those with HSIL or intra-anal or genital condylomas had been treated with imiquimod, achieving HR-HPV and LR-HPV clearance rates of 30.4% and 43.5%, respectively, which would have contributed to the lower percentage who progressed to high-grade lesions or cancer of the anal mucosa. Participants were not vaccinated in the other two studies [14,15], those receiving treatment for HSIL-plus were excluded from one of them [14], and neither study described the virus clearance rate. In the present investigation, progression from LSIL to HSIL-plus in anal mucosa was associated with the acquisition of HR-and LR-HPV genotypes (specifically HPV-6) and a history of AIDS, while no association was observed with coinfection by HR-and LR-HPV genotypes.
The degree of immunosuppression, defined by current CD4 count and CD4 nadir < 200 cells/uL, has been associated with the risk of lesion progression [16,17]. A recent HPV genotyping study of anal warts/condylomas in MSM-LHIV found that >20% of these lesions were HSIL and infected by LR and HR genotypes; the authors concluded that oncogenic and non-oncogenic HPV genotypes were involved in the genesis of HSILplus [18]. HR genotypes, in particular HPV-16, have frequently been related to anal lesion progression [19]. A meta-analysis published in 2018 described HPV-16 as the carcinogenic genotype par excellence in anal mucosa and responsible for both precursor lesions and anal cancer [20]. HPV-6 has historically been classified as an LR genotype and associated with the development of benign verrucose lesions or LSIL; however, an association was recently found between monoinfection by HPV-6 and progression to HSIL [21]. Furthermore, chronic monoinfection by LR genotypes 6, 11, 42, 44, or 70 has also been reported as the cause of a small number of anal cancers [22].
The results of this study are limited to a specific population of HIV-positive MSM and cannot be extrapolated to other types of subjects. Study strengths include the prospective and longitudinal design, large sample size, and systematic long-term follow-up. Notably, this is the first published report on monoinfection by LR-HPV genotypes in the anal canal of MSM-LHIV.
In conclusion, monoinfection due to LR-HPV genotypes is not associated with anal cancer or precursor lesions in patients with normal cytology. The progression rate from LSIL to HSIL-plus was <5% and was related to the acquisition of HR-and LR-HPV genotypes (specifically HPV-6) and a history of AIDS. These findings are directly relevant to the care of anal dysplasia in MSM-LHIV, because the presence of LR-HPV genotypes alone with normal cytology rules out precancerous lesions and anal cancer, allowing for a longer interval between anoscopy examinations. Furthermore, given that only a very small percentage of patients with low-grade AIN1 progress, and over a prolonged period, these can be monitored by HRA every year rather than every three or six months.

Materials and Methods
This prospective, longitudinal, single-center study included consecutive MSM-LHIV who participated between May 2010 and December 2021 in a program for the screening, diagnosis, treatment, and follow-up of anal mucosa dysplastic lesions. All participants were attended at Virgen de las Nieves University Hospital (HUVN) of Granada, after referral from Infectious Disease Units of the HUVN of Granada and Hospital Complex of Jaen (Jaen, Spain). This study was approved by the ethical committee of the coordinating center. All patients had given consent to the use of their data for research purposes, and all data were gathered in accordance with Spanish personal data protection legislation (Organic Law 3/5 December 2018).
The inclusion criterion was to be MSM-LHIV aged over 17 years. Exclusion criteria were being a heterosexual man or woman living with HIV and having a history of anal canal neoplasm in the previous five years.
At the baseline visit (V0), patients were informed about the conditions and objectives of this investigation, and data were gathered on their age, history of perianal-genital condylomas, number of sexual partners throughout life, number of sexual partners over past 12 months, age at onset of sexual relations, utilization of condoms and their percentage utilization, smoking (yes/no and packs/year), alcohol consumption (yes/no and standard drink units, SDUs), injection drug use (IDU), ex-IDU, nationality, schooling, months with HIV diagnosis, CDC-classified HIV stage, months under antiretroviral treatment (ART) and lines used, virological failure (RNA ≥ 50 copies/mL in ≥2 determinations during previous 6 months), CD4 nadir, CD4 and CD8 lymphocyte counts, viral load at HIV diagnosis, and the presence of other infections, including chronic hepatopathy by hepatitis B virus (HBV) or hepatitis C virus (HCV), syphilis, other sexual transmitted infections (STIs), perianalgenital warts, and latent, treated, or active tuberculous infection. Information was also collected on CD4 and CD8 cell counts, CD4/CD8 ratio, and viral load at V0.
At the same visit, cotton swabs impregnated with physiological saline were used to take two anal canal mucosal samples: one for HPV detection and genotyping by qualitative polymerase chain reaction (PCR) (Linear Array HPV Genotyping Test) with a "GeneAmp PCR System 9700" thermocycler (Applied Biosystems, Roche, Switzerland), and the other for thin-layer cytological study with a ThinPrep 2000 Processor (Hologic, Mississauga, Canada). Both samples were immersed in thin-layer liquid and sent to the hospital pathology laboratory for analysis. Genotypes [23].
After an interval of 4-12 weeks, participants underwent rectal inspection and digital rectal examination followed by high-resolution anoscopy (HRA) with a Carl Zeiss 150 fc © colposcope (Carl Zeiss, Oberkochen, Germany), introducing a transparent disposable anoscope through which 5 mL acetic acid was instilled and left for around 3 min, removing it for examination of the mucosa. Next, 5% Lugol's iodine was instilled for 1 min and the anoscopic examination was repeated. Samples were taken from quadrants of apparently normal mucosa and from areas with Lugol-negative aceto-white lesions. Biopsies were performed with endoscopic retrograde cholangiopancreatography (ERCP).
Patients with normal anoscopy or LSIL(AIN1) were followed up at one year with cytology, HPV PCR, and anoscopy studies. Patients with HSIL either underwent mucosectomy by electrical scalpel (offered from May 2010 onwards) in the Coloproctology Unit of the General Surgery Department or self-administered 5% imiquimod three times a week for 16 weeks (offered from 2014 onwards). Anoscopy was performed in these patients at the end of their treatment, scheduling a follow-up at one year if the outcome was normal/LSIL or retreatment if HSIL, and referring the patient to the hospital oncology department if anal cancer was detected. Information was gathered at the follow-up visit on the number of sexual partners in the previous 12 months, the emergence of STIs (conducting syphilis serol-ogy in all patients but ordering PCR and anal-urethral exudate culture solely in patients with symptoms or infected partners), genital/anal condylomas, ART experience (change, virological failure, adherence rate), CD4 and CD8 lymphocyte counts, CD4/CD8 ratio, and HIV viral load.
In the cytology study, the Bethesda classification [24] was used to categorize lesions as atypical squamous cells (ASC), atypical squamous cells-high (ASC-H), LSIL, or HSIL. The study variable "·abnormal cytology" includes ASCUS, LSIL, or HSIL. In the histology study, the proposal of the Lower Anogenital Squamous Terminology (LAST) Standardization Project for HPV served to classify lesions as LSIL (AIN1/condyloma), HSIL (AIN2, AIN3, C. in situ), or invasive carcinoma (ASCC) [25]. The study variable "HSIL plus" includes anal lesions ranging from HSIL to invasive cancer (high-grade SIL/cancer).

Statistical Analysis
In descriptive analysis, means, standard deviations, medians, and percentiles were calculated for quantitative variables and absolute and relative frequencies for qualitative variables. In bivariate analyses, the Student's t-test for independent samples was applied for quantitative variables when normally distributed according to the Kolmogorov-Smirnov test and the Mann-Whitney U test when non-normally distributed. Qualitative variables were analyzed with Pearson's chi-square test or, when application criteria were not met, Fisher's test. Multiple logistic regression analysis was then performed, entering variables that were significant in bivariate analyses or considered relevant in the literature. SPSS 21.0 (IBM SPSS, Armonk, NY, USA) was used for data analyses, and the level of significance was 0.05 in all tests.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The researchers confirm the accuracy of the data provided for the study, as well as their availability.