Prevalence of Human Papilloma Virus Infection in Bladder Cancer: A Systematic Review

The etiology of bladder cancer is known to be associated with behavioral and environmental factors. Moreover, several studies suggested a potential role of HPV infection in the pathogenesis with controversial results. A systematic review was conducted to assess the role of HPV. A total of 46 articles that reported the prevalence of HPV infection in squamous (SCC), urothelial (UC), and transitional cell carcinomas (TCC) were selected. A pooled prevalence of 19% was found, with a significant difference in SCC that was mainly driven by HPV-16. Moreover, infection prevalence in case-control studies showed a higher risk of bladder cancer in HPV-positive cases (OR: 7.84; p-value < 0.00001). The results may suggest an etiologic role of HPV in bladder cancer. HPV vaccine administration in both sexes could be key to prevent the infection caused by high-risk genotypes.


Introduction
Bladder cancer (BCa) is the 10th most prevalent cancer globally, with >572,000 and >212,000 incident cases and deaths [1]. The highest rates of BCa are registered in Southern and Western Europe and North America, with a higher incidence in men [2]. Several risk factors were found; most of them are related to personal behavior (i.e., diet and smoking), socioeconomic status (i.e., the accessibility to health services and delay in diagnosis), and environmental and occupational exposure to chemical substances or infectious diseases [3]. Moreover, epidemiological studies revealed a higher risk in men and a worse prognosis in black males [4]. BCa histological subtypes can vary: urothelial BCa, previously classified as transitional cell carcinoma (TCC) and the predominant histological type, accounts for~90% and is mainly related to chemical exposure, whereas squamous cell carcinoma (SCC, 5%) is associated with chronic inflammation and persistent infections (Schistosoma spp. in Africa) [5]. Adenocarcinoma (2%), sarcoma, and small cell carcinoma are less incident forms [6].
While the role played by human papillomavirus (HPV) in the development of cervical, anogenital, and oropharyngeal cancers was proven [7][8][9], the causative relationship between HPV and BCa still remains controversial, with a high variable prevalence attributed to the study design, the enrolled population, and the HPV detection methods [10]. Understanding the role of HPV in BCa could have relevant diagnostic, therapeutic, and preventive implications. The current systematic review is aimed to assess the prevalence of HPV infection in BCa, focusing on patients' clinical and epidemiological characteristics.

Search Strategy
A systematic literature review aimed at retrieving papers focused on the prevalence of HPV infection in BCa was carried out from its inception to 31st December 2021. The literature search was performed using PubMed and Scopus, selecting the key words "Human Papillomavirus", "HPV", or "Papillomavirus" and "bladder cancer" or "bladder carcinoma", combined in different strings. No restrictions related to age of patients, setting, or time of the study were chosen.
Lists of references of all selected articles were screened to find other eligible studies not included in the above-mentioned databases.

Study Selection and Inclusion Criteria
Case-control, cross-sectional, and cohort studies reporting HPV infection prevalence were selected.
The following inclusion criteria were considered: (1) Studies dealing with patients with primary BCa; (2) Studies describing molecular and non-molecular HPV detection methods on fresh or FFPE (Formalin Fixed Paraffin Embedded) bladder biopsies; (3) Studies focused on the following medical conditions: SCC, urothelial carcinoma (UC), and transitional cell carcinoma (TCC); Articles were excluded for the following reasons: (1) Review articles, abstracts, letters, commentaries, correspondences, case-reports, and case-series enrolling <10 subjects; (2) Use of languages other than English; (3) Secondary malignancies located in the bladder.
Article selection and data extraction were performed by two Authors and doublechecked (M.N. and D.B.), while discrepancies of opinions or disagreement were resolved by a third investigator (S.G.).

Data Extraction
Qualitative and quantitative variables were collected in an ad hoc electronic form. The following variables were collected: first author's last name; title of the article; year and country/countries of the study; period of the study; epidemiological study design; sample size; sex and age; type of samples; HPV detection methods; histological subtypes; tumor grading; HPV prevalence.
No ethical approval was needed given the anonymized and aggregated nature of the data.

Study Quality Assessment
Inter-rater agreement was~100% for the phases of study selection and data extraction, and the few inconsistencies were resolved by consensus and with the support of a third investigator (G.S.).
Guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) were followed to guide the process of the systematic review [11].
The Newcastle-Ottawa Scale [12] was used to assess the quality of the included casecontrol studies by evaluating selection, comparability, and exposure criteria, through four, two, and three items, respectively. The Joanna Briggs Institute Critical Appraisal tools (JBI), applied for analytical cross-sectional studies where the control group is missing, consists of eight items aimed at evaluating the risk of bias: high, moderate, or low risk of bias was assigned when positive answers were ≤49%, between 50% and 75%, or >75%, respectively [13] (Table S1a,b).

Statistical Analysis
Qualitative and quantitative variables were summarized with absolute and relative (percentage) frequencies and means/medians [standard deviation (SD), interquartile range (IQR)] respectively.
Forest plots were used to show pooled risk differences of the selected outcomes and interval (95% confidence interval, CI) estimates, as well as the weight of the sample size of the recruited studies. The I2 indicator (low, medium, and high heterogeneity expressed as <25%, ≥25%-<50%, ≥50%, respectively) showed the association between true variability and overall variation.
Fixed and random-effects models were chosen depending on the estimated betweenstudy heterogeneity. A two-tailed p-value less than 0.05 was deemed statistically significant. The statistical software Stata version 17 (StataCorp, College Station, TX, USA) and StatsDirect version 3.1.12 (StatsDirect Ltd., Willar, UK).

Study Selection
A total of 637 articles were identified through electronic database searches; 162 (26.2%) were excluded for being duplicates, and then, a total of 475 studies were screened by titles and abstracts. Fifty-six (11.8%) full texts were evaluated, and ten (17.9%) were excluded for the following reasons: tissue samples did not include bladder tissues (n = 5), full text was not available (n = 2), case-report (n = 1), review (n = 1), and dataset described in another study (n = 1). A total of 46 (46/56; 82.1%) manuscripts were included in the review (Table 1; Figure 1).

Quality Assessment
Twelve (34.3%) cross-sectional studies were deemed at moderate risk of bias, whereas 23 (65.7%) were classified as low-risk (Table 2). Nine (81.8%) cross-sectional studies were deemed to be medium-quality, whereas two (18.2%) were high-quality (Table 3). Table 2. JBI risk of bias assessment table. Eight items per study were evaluated and the risk of bias was calculated on the number of positive answers. y = yes, n = no, u = unclear. Moderate = where positive answers were between 50% and 75%; low = where positive answers were above 75%.

Ref. First Author
Were  "* " : Each study was awarded one star per item. The score is the sum of the awarded stars, ranged from 0 to 9 (high-quality, >7 stars; medium-quality, 4-6 stars; poor-quality, <4 stars).

Discussion
This systematic review was performed to evaluate the prevalence of HPV infection in BCa, keeping into consideration confounding demographic, histological, and diagnostic variables.
An overall HPV prevalence of 19% was found in 46 studies, in line with previous meta-analyses which reported a prevalence of 16.88% [10] and 14.3% [61]. Despite the growing number of reported studies, the role of HPV in cancers other than genital, anal, head, and neck cancers is still debated, due to the heterogeneity in the study design, population enrolled, and HPV detection methods [6]. In fact, several systematic reviews, which evaluated the prevalence of infection of DNA-based vs. non-DNA-based methods confirmed the higher specificity and sensitivity of molecular-based methods [62,63]. Moreover, the use of genotype primers designed for shorter DNA sequences reduced the risk of "false negative" results in comparison with broad-spectrum primers (i.e., GP5+/6+), especially for FFPE specimens often undergoing DNA damage [10]. Therefore, the implementation of a standardized procedure for HPV detection could better clarify the impact of HPV in BCa pathogenesis.
A statistically significant difference in the prevalence of infection was found in different histological subtypes, with higher estimates in SCC than those in TCC and UC. The high affinity of HPV to differentiating squamous epithelium, previously demonstrated in cervical, head and neck, and anogenital carcinomas [64], as well as the ability of the virus to evade and inactivate the immune response, could explain the mechanism of carcinogenesis in the bladder epithelium. Since SCC has poor prognosis and is associated with worse outcomes in different sites [65], the confirmation of the role of HPV in SCC could improve the epidemiological burden and the prognosis of potential cases.
The assessment of the role of HPV as causative agent in different histological subtypes could have been affected by the adoption of the new WHO BCa classification [66]. This re-cent recommendation changed the traditional nomenclature of "transitional cell carcinoma" in "urothelial carcinoma", causing potential misinterpretations of the results. On this basis, the identification of standard accurate procedures for the detection and diagnosis of HPV infection could be helpful to estimate the burden of cancers associated with HPV. However, the available molecular techniques show a high diagnostic accuracy.
Although several studies suggested a relationship between advanced stage (i.e., ≥T2) and HPV prevalence [27,43,44], the poor information collected in the selected studies did not prove this association.
Similarly to other HPV-related cancers, HPV-16 was the most prevalent genotype in BCa, supporting previous findings on the increased risk of BCa in cases of infection caused by high-risk HPV genotypes, mainly by HPV-16 [10]. The high detection rate of HPV-16, together with HPV-18 and the low-risk HPV-6, strongly supports the administration of HPV vaccines to prevent HPV-related cancers in both sexes [67].
Finally, our study showed a significant association between HPV and BCa (OR: 7.84), confirming the findings of recent meta-analyses on the risk of BCa [10,62,65]. However, these results are in contrast with a previous study published by Khatami et al. [61], who described a non-significant association between infection and cancer and highlighted the scientific need of larger case-control studies. Although the majority of case-control studies were classified as "medium-quality", controls showed a prevalence of infection <10%, regardless of HPV detection methods, suggesting the etiological role of HPV in BCa.
Previous systematic reviews investigated the role of HPV infection in BCa and described a moderate association. Our study selection and analysis showed an important role of HPV infection in SCC BCa. However, some limitations should be acknowledged. More stratified analyses related to demographic (i.e., geographical area, behavioral factors, occupational exposures) and clinical (i.e., stage, HPV detection methods, prognosis of patients) confounders should be performed. The cross-sectional assessment does not help prove the temporal relationship between an exposure and the development of cancer.

Conclusions
The present systematic review highlights a potential role of HPV in the development of bladder cancer, indirectly supporting the adoption of primary preventive strategies in both sexes, as recommended by international authorities. Further epidemiological studies are needed to confirm those findings and assess the role of diagnostic and preventive strategies for HPV-related bladder cancer.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/diagnostics12071759/s1, Figures S1-S3, Tables S1-S5.  Institutional Review Board Statement: Ethical review and approval were waived due to the design of the study.
Informed Consent Statement: Patient consent was waived due to the design of the study.

Data Availability Statement:
The data is available in case it is requested for motivated reasons.

Conflicts of Interest:
The authors declare no conflict of interest.