Urothelial cell carcinoma (UCC) is the most common type of bladder cancer (BC) (1, 2). Several factors are associated with an increased risk of BC development, including but not limited to schistosomiasis infection, smoking, and genetic predisposition (3). To date, it is estimated that up to 15% of the world’s cancer case burden is attributed to viral infections (4, 5). JC polyomavirus (JCPyV) and BK polyomavirus (BKPyV) are small, unenveloped, circular, and double-stranded DNA viruses, and both were the first human polyomaviruses isolated from patients in 1971 (6, 7). Recently, the urotheliotropic BKPyV has been increasingly well-studied and has been discussed as a possible oncogenic virus in the development of UCC in immunocompromised populations (8). JCPyV (HPyV2) is a neurotropic virus with a genome that shares about 75% homology with BKPyV (9). It has been identified as the etiological agent in the development of progressive multifocal leukoencephalopathy (PML), most commonly in immunocompromised individuals (9).

JCPyV (HPyV2) is acquired during early childhood, has shown about 80% seropositivity in humans, and has been described as a possible oncogenic virus in immunocompromised patients (10–12). In addition, JCPyV is known to remain latent or persistent in tubular epithelial cells of the kidney and urothelial tissue after primary infection (13). JCPyV large tumor antigen (LTAg) is located in the early region and has shown the capability to bind specifically with the p53 protein and retinoblastoma (pRB) protein, as known for some of the human polyomaviruses (HPyVs) (14–16). The presence of JCPyV-DNA was previously reported in many different tumor tissues, such as cervical, colorectal, gastric, lung, breast, brain, and urothelial cancers (17–23). The current number of members of HPyVs detected in various types of cancers has recently risen. Among them, Merkel cell polyomavirus (MCPyV) is the most recently identified human DNA tumor virus, which is clonally integrated into the majority of Merkel cell carcinoma (MCC) (24–26). MCPyV (HPyV5) has also been detected in the fresh frozen tissues of bladder cancer (27). Additionally, Husseiny and colleagues reported low levels of MCPyV viral load in urine specimens from immunosuppressed patients who were prospectively enrolled in kidney transplants (28). However, the potential role of HPyVs in UCC tumorigenesis is not fully elucidated.

Urine cytology using Papanicolaou staining is well accepted to reliably detect virally infected epithelial cells in the voided urine also known as “decoy cells”. These cells are characterized by enlarged nuclei and intranuclear inclusion cellular changes, as noticed in UCC neoplasia (29). The presence of decoy cells in urine has been used mostly as a marker to predict BKPyV and JCPyV reactivation (30, 31). In addition, immunohistochemical (IHC) immunolabeled with mouse anti-SV40 large T antigen monoclonal antibody, which is known to cross-react with both JCPyV and BKPyV LTAg, is used as an alternative marker to detect the reactivation of both respected viruses in urothelial cells in voided urine (10, 32, 33).

Recently, we studied the association between the BKPyV infection and UCC in patients with urine cytology positive for decoy cells (30). However, in our patient cohort, both primary and recurrent UCC tissues tested negative for BKPyV by PCR and IHC (30). Recent studies have investigated the role of JCPyV (HPyV2) in UCC tumorigenesis (34, 35). The aims of this study were to evaluate the presence of JCPyV, HPyV6, HPyV7, and MCPyV (HPyV5) in the UCC samples and in the voided urine of the patients diagnosed with UCC and with decoy cells in urine cytology.

We recently reported that we did not find an association between BKPyV detection in the urine sediments of patients with prior resected urothelial cell carcinomas. BKPyV reactivation was not restricted to immunosuppression and was possibly related to intravesical BCG or mitomycin treatment in immunocompetent patients (30). JCPyV is closely related to BKPyV, and the large- and small-tumor antigens of both viruses were found to be capable of transforming cells in vitro (44–47). In contrast to the relationship of BKPyV with UCC, limited information is available regarding the role of JCPyV in UCC tumorigenesis, especially in immunocompetent cases (34, 35).

We analyzed the presence of JCPyV on the protein level in combination with DNA-PCR in FFPE tissues and voided urine sediments obtained from UCC immunocompetent patients. Previously, we demonstrated that the detection of decoy cells was infrequent, present in only 0.2% of a large urine specimen cohort from our institution (30). The detection of decoy cells in urine cytology is used in a diagnostic setting for identifying possible polyomavirus infections (48). However, the decoy cells were identified in the urine cytology of all 14 UCC cases, which suggests polyomavirus reactivation (30). Indeed, SV40-LTAg expression was detectable in the urine sediments, which confirmed polyomavirus reactivation in all 14 UCC patients ( Figure 2B ). Although the SV40-LTAg monoclonal antibody is commonly used in diagnostic pathology, it is known to cross-react with the closely related human polyomaviruses BKPyV and JCPyV (49). We confirmed the presence of JCPyV-LTAg by PCR among the five available urine sediments obtained from five UCC patients. Also, we previously reported the detection of BKPyV-LTAg by PCR in all of the five urine sediments (30). Thus, it can be speculated that the tubular epithelial cells are coinfected with both JCPyV and BKPyV, and both may contribute to the presence of the decoy cells.

Here, we performed JCPyV-LTAg and JCPyV-NCCR PCR in UCC FFPE samples obtained from patients who were diagnosed with decoy cells in their urine. It is important to mention that the patients were diagnosed with UCC, and urine cytology was collected during the postoperative follow-up. The main finding in our data is that 21.4% of UCC FFPE tissues tested positive using JCPyV LTAg and NCCR PCR, as confirmed by sequencing. Furthermore, the JCPyV-NCCR positive tissues and urine specimens were identical to the archetype strain, as confirmed by the presence of the 64 and 23 bp sequences that are absent in the Mad strains, which have been associated with human brain tumors. However, there is a study that reported the detection of archetype strain sequences in two cases of oligodendroglioma (50). The detection of JCPyV-DNA was previously reported in many different tumors, such as cervical, colorectal, gastric, lung, breast, brain, and urothelial cancers (17–23). Infection with JCPyV has been suggested to be associated with bladder carcinoma, which still remains a controversial hypothesis (34, 35). Results from a study that screened for JCPyV among urothelial bladder cancer patients in the UK concluded that 0.9% of bladder tumors were positive for JCPyV (34). In contrast, Fioriti et al. suggested that JCPyV might play a role in bladder cancer and reported the detection of JCPyV-DNA in 19% of bladder urothelial carcinoma (35).

JCPyV is known to remain latent in the kidney after infection and reactivate in immunosuppressed patients (11). However, in our study, JCPyV-PCR positivity in urine sediments was seen in immunocompetent UCC patients; most of the patients were known to have received either mitomycin or intravesical BCG treatment. Thus, our results suggest that the reactivation of latent JCPyV could be related to the locally administered UCC intravesical treatment since the patients were diagnosed with UCC of the urinary bladder before presenting with decoy cells in the urine cytology.

MCPyV is an oncogenic virus discovered in 2008 and has been linked to the pathogenesis of the majority of MCC. MCC are rare, highly aggressive neuroendocrine nonmelanoma skin cancers (24–26). In contrast, there is yet no evidence established regarding the role of MCPyV in bladder carcinoma. One study reported the presence of MCPyV-DNA in voided urine specimens of transitional cell carcinomas of the bladder and revealed 2.7% positivity by PCR (51). Also, Loyo et al. reported the detection of MCPyV-DNA in 75% of bladder cancer tissues with a low viral load (< 0.001 copies per genome) (27). Another study revealed that MCPyV was detected in 30% of the urine specimens from prospectively enrolled immunosuppressed kidney transplant patients by using qPCR with a relatively low viral load (10 to 3.7 × 10² genome copies/mL) (28). In our study, MCPyV was not detectable in the urine sediments of UCC, while MCPyV-DNA was positive in two 14.3% UCC FFPE tissues, and specific nuclear MCPyV-LTAg expression was seen in the neoplastic cells of one UCC tissue. Indeed, it is very interesting that MCPyV immunoreactivity was found to be abundantly present in UCC tumor cells. However, a direct contributing role of MCPyV to UCC carcinogenesis seems unlikely, at least based on our findings unlikely (24–26). The observed pattern of MCPyV protein expression in UCC tissues possibly might imply an indirect role for MCPyV in the development of UCC by, e.g., inflammation as it is known for hepatotropic viruses (e.g., hepatitis B and C viruses) (52). However, further investigations are needed to identify any oncogenic mutations or clonal integration of MCPyV LTAg or sTAg into the genome of UCC.

Noteworthy, the mouse anti-SV40-LTAg monoclonal antibody (clone PAb416) is commonly used in clinical pathology settings and is well known to cross-react with both JCPyV and BKPyV LTAg (53). However, Toptan et al. reported that anti-SV40-LTAg (PAb416) is not restricted to JCPyV and BKPyV. Of note, PAb416 also detects LTAg proteins of KIV, WUV, HPyV6, HPyV7, TSV, HPyV10, and HPyV11 (54). HPyV6 and HPyV7 share approximately 68% genome sequence identity and reveal high seropositivity in healthy populations (55–57). Since anti-SV40-LTAg showed the ability to detect both HPyV6 and HPyV7, we have tested all 14 UCC tissues and the five urine sediments for the presence of HPyV6- and HPyV7-DNA. Our results showed that all UCC specimens were negative for both HPyV6 and HPyV7, and to our knowledge, this is the first study to test UCC specimens for HPyV6 and HPyV7.

Some limitations of this study need to be mentioned. First, the size of the study cohort is relatively small, which partially reflects the rarity of UCC and the number of UCC patients admitted to our hospital. Second, obtaining healthy control tissues is practically impossible. In addition, parallel screening for HPyV-DNA in blood specimens would have been an interesting add-on to our study; however, we were unable to carry out these experiments due to the unavailability of blood.

To the best of our knowledge, this is the first study to investigate the presence of several HPyVs in UCC samples using diverse molecular techniques and the possible contribution of HPyVs in immunocompetent UCC etiopathogenesis. In total, five UCC cases showed the presence of either JCPyV or MCPyV. Both HPyV6 and HPyV7 were not detected. Further studies are warranted to confirm the relevance of JCPyV or MCPyV as a risk factor for developing UCC and to identify any oncogenic mutations or clonal integration into the genome of urothelial cell carcinoma.

JCPyV-DNA is detected in the urine and urothelial cells, and MCPyV was detected in urothelial cell carcinoma. Since there is inadequate evidence of a role for JCPyV in carcinogenicity in UCC, these findings support the hypothesis that JCPyV infection could contribute to urothelial carcinoma tumorigenesis. Moving forward, it is important to define whether or not both JCPyV and MCPyV are involved in UCC tumorigenesis.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

The study was approved by the Medical Ethics Review Committee of the Maastricht University Medical Centre+, The Netherlands (2019-0977). All specimens were collected and studied in accordance with the protocol of the Dutch Code of Conduct for Observational Research with Personal Data (2004) and Tissue. The studies were conducted in accordance with the local legislation and institutional requirements. The human samples used in this study were acquired from a by- product of routine care or industry. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and institutional requirements.

All authors have contributed substantially to conceptualizing the research design planning and continued supervision of the work (FK, GM, AH, IS), aacquisition of data (FK, GM, SS, TM, RA, MM, JR), performed and processed the experimental data and analysis (FK GM SS RA, SA), analysis and interpretation of data (FK, GM, TM, MM, SA, AH, IS), drafting the manuscript (FK, GM, AH, IS), and revising it critically (JR, AH, IS). All authors contributed to the article and approved the submitted version.