Human papillomavirus and bladder cancer: literature review and meta-analysis

Background The aim of this study was to evaluate data over the past 25 years to assess the prevalence of the virus in patients with bladder cancer and to evaluate the association between human papillomavirus (HPV) and bladder cancer risk. Main text Major databases were searched for published studies from October 1995 to May 2022. The present study evaluated the prevalence of HPV in patients with bladder cancer and the prevalence according to patients’ ethnicity. The incidence of HPV in bladder cancer patients varies widely, ranging from complete absence of the virus in tissues to 64.6%. The meta-analysis was performed using Meta-Essentials_1.5 software. Begg’s and Egger’s methods were used to assess publication bias. Cochran’s Q test was used to assess heterogeneity and the I 2 index was employed for calculating the variation in the pooled estimations. Conclusions A weak association of HPV infection with the risk of bladder cancer was found. The risk of bladder cancer with HPV infection has clear ethnic characteristics and is statistically significant in Arabs and Asians.


Introduction
Bladder cancer is a malignant neoplasm from the epithelial tissue of the bladder mucosa.According to literature it is included in the top 10 most common cancer disease in the world.There were 573,278 new cases of bladder cancer and 212,536 deaths in 2020, the incidence is growing every year.It should be noted that bladder cancer occurs in men much more often than in women [1].The following histological types exist: transitional cell carcinoma (or urothelial carcinoma), squamous cell carcinoma, and adenocarcinoma.Transitional cell carcinoma accounts for more than 90% of all bladder cancer cases; while, the latter two types are much less common [2].There are many factors for the development of bladder cancer, including effects of industrial carcinogens, lifestyle, or existing chronic medical conditions.However, researchers have focused on HPV infection as a key factor in the development of bladder cancer.To date, it has been demonstrated that the human papillomavirus is an etiological factor in cervical cancer, and it is also often found in tumors of the anogenital region [3].However, the role of HPV infection in urological tumors is still unclear.There is a need to systematize the available data from recent studies to establish the association of HPV infection with the risk of bladder cancer.

Epidemiology and etiology of bladder cancer
According to the Global Cancer Observatory, bladder cancer is the 10th most common cancer among all types and the 13th leading cause of cancer death worldwide [4].This pathology is typical for residents of developed countries.Despite the rapid development of science and new medical technologies, the incidence of bladder cancer continues to grow every year around the world.In 2020, according to the GCO, there were about 580,000 cases of bladder cancer, which is about 3% of all new diagnoses in the field of oncology.It is known that 18 thousand new cases of bladder cancer have been registered in Russia [4] (Fig. 1).
The highest incidence of bladder cancer is typical for the countries of Southern and Western Europe and North America.Greece has the highest incidence of this cancer type among men, but among women, the highest incidence is shown in Lebanon.However, the region with the highest incidence of bladder cancer in both sexes is Southern Europe.The regions with the lowest incidence are Central Africa, Central America and West Africa [5].
In 2018, 624,709 cases of malignant neoplasms were detected for the first time in Russia.Bladder cancer takes 13th place in the oncology disease (2.8%).It most often affects the male population.Tumors of the genitourinary system in men account for 25.1% of all malignant neoplasms, of which 4.7% are bladder tumors.The absolute number of diagnoses made for the first time in Russia for bladder cancer was 13,479 cases in 2018.The average age of patients suffering from this pathology is 67.6 years (for men 66.9 years, for women 69.7 years) [6].
Bladder cancer is a malignant neoplasm that forms from the elements of bladder mucous membrane epithelium.According to the 10th revision international classification of diseases, bladder cancer has the C67 code [7].The main histological types of bladder cancer include urothelial or transitional cell carcinoma, adenocarcinoma, and squamous cell carcinoma.Urothelial bladder cancer has a calm course and rare metastasis.According to the literature, transitional cell carcinoma of the bladder is diagnosed in 95% of cases [8].Chen and colleagues showed in 2017 that the incidence of the urothelial type was 86% (86 out of 100 samples) [9].
Many studies have shown that the etiology of bladder cancer is mainly associated with tobacco smoking, secondhand smoke, and various background diseases of the urinary system, such as urolithiasis.A 2018 metaanalysis found a statistically significant increase (22%) in bladder cancer risk with lifelong exposure to secondhand smoke in non-smokers compared to non-smokers not exposed to secondhand smoke [10].Smoking cigarettes significantly increases the risk of bladder cancer, especially when combined with infectious diseases such as Fig. 1 The number of new cases of cancer in the world in 2020 schistosomiasis [11].It has also been shown that smokers with a family history of bladder cancer have a four times higher risk than non-smokers [12].There is evidence that bladder adenocarcinoma often results from chronic inflammation or infectious of the bladder wall.Schistosoma haematobium infection provokes urogenital schistosomiasis and correlates with bladder cancer occurrence [13].
However, there is more and more studies focusing on the viral etiology of bladder cancer.In particular, scientists focus on the presence of HPV infection in patients with bladder cancer.
Human papillomaviruses are a large family of shoeless epitheliotropic viruses containing double-stranded circular DNA within the capsid.There are more than 150 different HPV genotypes.HPV is a widespread sexually transmitted infection.To date, a large number of studies have been published looking at the role of HPV in the development of other cancer types.For example, HPV is frequently found in anogenital tumors, head and neck tumors, and even in lung tumors [14][15][16][17].However, a clear association has only been shown for cervical cancer: HPV DNA has been shown to be detected in 99.7% of cervical cancer samples [18].
At this time, the exact mechanism of HPV-associated bladder tumor development has not been described.However, there are studies that suggest that HPV DNA can be transported by the bloodstream from the original site of infection to other organs and possibly contribute to tumor development, including bladder cancer [19].Also, a recent study showed a putative mechanism for the development of HPV-induced bladder cancer.Given that the urinary tract is anatomically close to the genital tract, it is hypothesized that HPV may spread upward through the urethra to the bladder epithelium, thereby causing the development and progression of bladder cancer.It is hypothesized that after exposure of bladder uroepithelial cells to the virus, DNA repair returns damaged cells to normal.In this case, cells that failed to repair become precancerous after persistent chronic infection.In the absence of tumor suppressors and under genomic changes, precancerous cells gradually turn into malignant cells [20,21].
The data available to date are insufficient to state that HPV is an etiologic factor in the development of bladder malignancies, but it is important to realize that HPV infection may be an important co-factor in the development of this malignancy.In this regard, it is particularly relevant to investigate the association of HPV with the risk of bladder cancer.
To date, few studies have been conducted to determine the association of HPV with bladder cancer.A 2011 metaanalysis reviewed 21 case-control studies that analyzed the prevalence of HPV in this type of cancer, using different methods of virus identification.The pooled odds ratio was 2.13 [22].Li and colleagues (2011) presented a paper that included 19 case-control studies, where samples of fresh and fixed tissue were used.The presence of HPV in the material was detected using PCR analysis, as well as in situ hybridization and Southern blotting.It has been shown that the HPV prevalence among patients with bladder cancer was 16.88% [23].
However, the presented results of HPV infection studies in bladder cancer are ambiguous; the data show both a high association of HPV infection with bladder cancer and a complete lack of association, which makes the study of the HPV role in bladder carcinogenesis relevant question.

Literature search strategy
A systematic literature search was performed using PRISMA guidelines.We searched PubMed, Web of Science, and Google Scholar for articles published between October 1995 and May 2020.The terms used to search for potentially relevant studies were: "cancer," "human papillomavirus," "bladder cancer," "association," "urologic cancer," and "bladder."

Research selection criteria
Mandatory criteria for inclusion in the meta-analysis were: 1. Study design: we included case-control studies published between October 1995 and May 2020.2. Participants: studies including patients with bladder cancer.3.Primary outcome: presence of human papillomavirus in bladder cancer patients.4. Secondary outcome: prevalence of human papillomavirus in bladder cancer patients.
We excluded a series of cases where urothelial smears and urine were used as material, as well as studies involving positive controls.No language restrictions were applied.

Statistical analysis
The meta-analysis was performed using Meta-Essen-tials_1.5 software.We used Begg's and Egger's methods to assess publication bias.The funnel plots shape is used to show symmetry and Egger's methods are applied to indicate significant publication bias for the analysis exploring association between risk of prostate cancer and HPV.The Egger test uses linear regression to assess the relationship between standardized effect estimates and standard error.For this test, a significant result is an indication that publication bias may influence the results.Egger's test reduces the standardized effect size by their precision; in the absence of publication bias, the regression crossover point is expected to be zero.Cochran's Q test was used to assess heterogeneity and the I 2 index was employed for calculating the variation in the pooled estimations.All p values are two-tailed, α ≤ 0.05 was considered statistically significant (p ≤ 0.05).

Results
This meta-analysis provides literature data for the last 25 years (1995-2020).The chronology of data is observed over the years.There were 56 studies selected, the total number included for the analysis of HPV infection was 4613 cases of bladder cancer and 636 control samples (Table 1).
The prevalence of HPV in bladder cancer was approximately 16.3%.The virus was detected in 5.95% of cases in the control group.The incidence of HPV in patients with bladder cancer varies widely from the complete absence of tissue virus to 64.6%.The most common HPV types are 16 and 18, with HPV types 31 and 33 in second place.
Next, we performed meta-analysis with 23 studies with the selection criteria for inclusion in the meta-analysis of the data to determine the risk of bladder cancer with HPV infection.
The studies of the "case-control" type were taken into account, samples of tumor (case) and normal tissue (control) were used as the material, and the main methods were real-time PCR and quantitative PCR.In particular, case-control studies using frozen tumor tissue or paraffin-embedded (FFPE) tissue (case) were included.Frozen normal tissue or FFPE samples obtained from the same patients or from healthy donors were used as controls.The main methods were real-time PCR and quantitative PCR.The PRISMA tool (Preferred Reporting Items for Systematic reviews and Meta-analysis) was used to improve the quality of data and the review result [24].A flowchart of study search and analysis for inclusion in meta-analysis is shown in Fig. 2.
It should be noted that studies that urothelial smears and urine were used as the test material, as well as studies involving a positive control, were excluded.A total of 1324 patients' bladder tumors and 599 normal bladder tissues were included in the meta-analysis.Meta-analysis was performed using the Meta-Essentials_1.5 program.The result of a meta-analysis of 23 studies is presented in Table 2 and Fig. 3.
Analysis of HPV prevalence according to the metaanalysis studies showed that HPV occurs in bladder cancer in 20.2% of cases, while in samples of the control group the virus was detected in 6.7% of cases.
In accordance with the literature data, the most common HPV genotypes were 16 and 18, in addition, 6, 31, 33 and 45 types are often found.A high incidence of human papillomavirus among patients with bladder cancer was shown in five studies [22,25,29,32,37].
At the same time, the total RR (RR 95CI) of developing bladder cancer with HPV infection was 1.21 (1.09-1.34),with p = 0.00253.The heterogeneity index of the sample I2 = 84.68%and the Cochrane Q-test was p = 4.5*10-20, with the required level p < 0.1, therefore the Random model was used.The funnel plot does not show significant asymmetry (Fig. 1) and according to Begg's test there was no significant publication bias in this metaanalysis (p = 0.054).Egger's test result was not significant (p = 0.265).
At the next stage, a subgroup analysis of the data was carried out depending on the ethnic origin of the patients.Table 3 shows the results of the subgroup analysis.
The highest risk of bladder cancer with HPV infection is observed in patients of Arab ethnicity OR (95CI OR) 1.49 (1.30-1.70).In Asians, the risk of developing bladder cancer with HPV infection was RR (RR 95CI) 1.15 (1.05-1.26)with p = 0.007.In patients of European ethnicity, the risk of developing bladder cancer in the presence of HPV infection RR (RR 95CI) 1.04 (0.99-1.09) at p = 0.129 does not reach statistically significant values, as well as in patients of Turkish ethnicity: OR (RR 95CI) 1.03 (0.81-1.33), at p = 0.438.Thus, the risk of developing bladder cancer with HPV infection has different ethnic characteristics and is statistically significant in Arabs and Asians.

Discussion
There is a large number of studies on the relationship of putative etiologic factors with bladder cancer, each of which plays a significant role in carcinogenesis.For example, a recent meta-analysis showed that smoking is associated with a high risk of recurrence and mortality in patients with bladder cancer [78].
However, the presence of papillomavirus infection is also of greater interest as a risk factor for bladder cancer.Human papillomavirus is an etiologic factor for cervical cancer, which in recent decades makes it relevant to study the association of this virus with the etiology and risk of other malignant pathologies, especially tumors of the genitourinary system.
To date, the global literature has presented studies examining the correlation between HPV infection and the risk of bladder malignancy.Nevertheless, unequivocal results remain elusive due to the relatively small number of homogeneous studies and small sample sizes.In addition, it remains unclear whether there are clear geographic differences in the risk of bladder malignancy with HPV infection.Thus, in the present study, we evaluated the overall impact of papillomavirus infection on the development of bladder cancer as well as the dependence of the bladder tumor risk of HPV infection on the ethnic aspect.
According to the presented meta-analysis, HPV was found in bladder cancer in 20.2% of cases, whereas in control group samples the virus was detected in 6.7% of cases.The findings are consistent with earlier metaanalyses [23,79] as well as recent studies [80,81].In an   L., the prevalence of HPV in US patients with bladder cancer was shown to be: HPV type 18 was 5.5%, HPV 16 was 13.2%, HPV 11 was 6.4%, and HPV 6 was 17. 4%.A high prevalence of the virus was noted in male patients (80% vs. 53%, p = 0.0056).In logistic regression adjusting for sex, smoking status, weight gain (OR 1.01, p < 0.0001) and HPV type 6 infection (OR 1.9, p < 0.0001) were associated with increased odds of developing bladder cancer [82].Review article by K.R Jorgensen published in 2020 reviewed 7 publications with 100 or more cases.A total of 877 patients were analyzed for the presence of HPV.The study material in all studies was frozen tumor tissue or tissue fixed with formalin and encapsulated in paraffin.The presence of HPV was determined by PCR, but in some studies PCR was combined with other tests to determine other HPV genotypes.As a result, the incidence of HPV in bladder tumors was found to vary widely, approximately 0-37.6%, and the most common HPV types, as in other localizations, were types 16, 18, 33, 6, and 31 [79].
In the present study, we also showed that the risk of bladder cancer in HPV infection has different ethnic characteristics.We found that the highest risk of bladder cancer with HPV infection was observed in Arab patients RR (RR 95CI) 1.49 (1.30-1.70)and Asian patients RR (RR 95CI) 1.15 (1.05-1.26)at p = 0.007.In Europeans and Turkish patients, the risk of developing bladder cancer with HPV infection was RR (RR 95CI) 1.04 (0.99-1.09) at p = 0.129 and RR (RR 95CI) 1.03 (0.81-1.33), at p = 0.438, respectively.The results for these patient groups do not reach statistically significant values.
Thus, in the presented work we analyzed both the general impact of papillomavirus infection on bladder cancer development and the risk of bladder tumor development in HPV infection for different ethnic groups of patients.
The analysis of literature data showed that HPV, though it cannot be recognized as an etiological factor in the development of bladder cancer, is definitely an important concomitant factor in the occurrence of this oncopathology.It is important to note that the obtained results confirm that the presence of HPV infection is important in order to create risk groups for the development of the studied oncopathology, as well as the need to monitor papillomavirus infection in patients with bladder pathology.

Conclusion
The data obtained are consistent with previously published studies.Despite the fact that this meta-analysis found a weak association of HPV infection with the risk of developing bladder cancer, it should be noted that the risk of developing bladder cancer with HPV infection has clear ethnic characteristics.Thus, the highest risk of bladder cancer with HPV infection is observed in Arab and Asian patients, which is not the same for European and Turkish patients.

Fig. 3
Fig. 3 Results of a meta-analysis of the relationship between the presence of HPV infection and the risk of developing bladder cancer

Table 1
Prevalence of HPV in bladder cancer

Table 2
Meta-analysis results for 23 studies

Table 3
Results of a subgroup meta-analysis by ethnicity of patientsMeta-analysis results obtained for patients of different ethnic groups are shown in bold