A chimeric bladder tumor in a renal transplant recipient: the oncogenic convergence of JC virus and PIK3CA mutation

Organ transplant recipients face a markedly elevated risk of developing cancer, with posttransplant malignancies accounting for up to 20% of long-term complications [1]. The most common malignancies developing after solid organ transplantation and directly attributable to the transplant procedure include Kaposi's sarcoma, post-transplant lymphoproliferative disorders, and non-melanoma skin cancers [2,3]. Less frequently, yet with an incidence higher than in the general population, urothelial carcinomas are also diagnosed [4]. The primary driver of this increased oncologic burden is chronic immunosuppressive therapy, both due to the direct oncogenic effects of immunosuppressive drugs and, more importantly, the impaired immune surveillance they induce [5]. For certain cancers with clearly increased incidence, oncogenic viruses play a decisive role, with immunosuppression facilitating viral persistence and chronic infection in predisposed individuals [6,7]. Post-transplant malignancies are categorized as either de novo tumors or chimeric tumors [8]. The latter are further subdivided, based on timing, into "donor-transmitted" tumors (DTTs), occurring within the first two years post-transplant, and "donor-derived" tumors, arising thereafter (DDTs) [9]. DTTs are rare and typically reflect undetected malignancies present in the donor. Despite stringent screening protocols, a residual risk of DTTs persists [10]. DDTs, in contrast, appear later and originate from donor cells that migrate and engraft in the recipient. These cells are not malignant per se but behave as tumor-initiating cells (TICs), leading to the development of recipient malignancies [11].Here, we report a highly unusual case of urothelial carcinoma diagnosed two years after kidney transplantation from a deceased donor. This stands out both for its rarity-a donorderived bladder carcinoma in a recipient of two renal transplants-and for the coexisting JC polyomavirus infection, allowing investigation of its potential role in post-transplant oncogenesis [12].The patient is a 59-year-old Caucasian male with a history of chronic glomerulonephritis (not histologically confirmed) that progressed to end-stage renal disease, initially treated with peritoneal dialysis. After two years, he underwent a first kidney transplantation from a deceased male donor (karyotype XY), with implantation in the right iliac fossa and immediate graft function. Immunosuppressive induction therapy included basiliximab, tacrolimus, mycophenolate, and steroids; maintenance therapy consisted of tacrolimus, mycophenolate, and steroids. Renal function remained stable for seven years. Subsequently, progressive renal deterioration prompted a biopsy of the transplanted kidney, which revealed polyomavirusassociated nephropathy due to JC virus (JCV). This histopathological diagnosis, coupled with a concurrent plasma JC virus viremia of 39,650 copies/mL, established JCPyV-associated nephropathy as the primary cause of graft dysfunction, leading to chronic kidney failure. Immunosuppressive therapy was modified with tapering and eventual discontinuation of tacrolimus, and everolimus was introduced for its antiviral potential. Two years after JC virus diagnosis, further investigations were conducted as part of a staging work-up to assess potential systemic involvement, although no focal neurological symptoms were documented at that time. Lumbar puncture revealed JC virus in cerebrospinal fluid (20 copies/mL), and urinary viral load was 26,582,889,960 copies/mL. Four years later, renal failure progressed to end-stage disease, necessitating hemodialysis via a native arteriovenous fistula. The patient was re-listed for kidney transplantation and, after three years on the waitlist, received a kidney from a deceased female donor (karyotype XX). The second graft was implanted in the left iliac fossa with immediate functional recovery. Induction immunosuppression included thymoglobulin, basiliximab, tacrolimus, mycophenolate, and steroids; maintenance therapy consisted of tacrolimus, mycophenolate, and steroids. At that time, JC virus viremia was detected at low levels (467 copies/mL), and routine post-transplant brain MRI was unremarkable. Renal function remained satisfactory for approximately six months, after which a rapid deterioration was observed. A graft biopsy showed no histological evidence of JC virus infection or acute rejection. During this phase, a concomitant Escherichia coli urinary tract infection was diagnosed and treated with antibiotics, resulting in only partial and transient improvement of renal function. Five months later, a repeat graft biopsy revealed cytological features consistent with JC virusassociated cytopathic effect. As a consequence, tacrolimus was discontinued and everolimus was reintroduced in an attempt to modulate immunosuppression. Despite these interventions, renal function continued to decline, and hemodialysis was resumed one year later. Notably, JC virus infection persisted throughout follow-up, with a progressive increase in viral replication. Plasma viral load reached a peak of 2,957,144 copies/mL, while cerebrospinal fluid viral load rose to 2,427,850 copies/mL approximately one year after the second transplant, without the manifestation of overt clinical neurological deficits. Two years after the second transplant and one year after dialysis resumed, the patient presented with macrohematuria. Cytological analysis revealed malignant urothelial cells. Ultrasound showed a bladder mass on the left lateral wall. Transurethral Resection of Bladder Tumor (TURBT) revealed multiple exophytic lesions and tissue protruding from the left ureteral orifice. Histopathology confirmed high-grade invasive urothelial carcinoma with angioinvasion. (FIGURE 2) Ureteroscopy was not performed to avoid upper tract tumor seeding. Abdominal CT showed diffuse bladder wall thickening. Given the bladder tumor location, wall infiltration, and absent residual renal function, complete excision of the urothelial tract was indicated: bilateral native and graft nephroureterectomy and cystectomy were performed.(FIGURE 1) Post-operative recovery was uneventful, and the patient was discharged on day 5. Inspection of the surgical specimen revealed a 0.9 cm exophytic papillary lesion 6 cm from the urethral meatus, diagnosed as non-invasive urothelial carcinoma; a 1 cm depressed area 3.8 cm from the meatus with foci of urothelial carcinoma in situ, dysplasia, and chronic cystitis. The remaining bladder mucosa and all ureters showed no malignancy, except for signs of chronic inflammation.To investigate the potential presence of chimeric cells, we collected six fresh tissue samples from various sites and immediately stored them at -80°C. These included healthy bladder tissue, ureters from both native and transplanted kidneys (right and left), and a hyperemic area of the bladder mucosa. Genetic analysis was carried out using the Elucigene QST*Rplusv2 kit, a multiplex QF-PCR system designed to detect polymorphic short tandem repeats (STRs) across chromosomes 13, 18, 21, X, and Y. The assay employs fluorescently labeled primers, allowing the amplified products to be separated via capillary electrophoresis and quantified by fluorescence intensity using the ABI3500 Genetic Analyzer and GeneMapper software. To further explore cellular composition, small tissue fragments from the ureter of the left transplanted kidney and from the hyperemic bladder area were enzymatically dissociated and cultured in Chang medium. The culture from sample 3, initially composed of a heterogeneous cell population, progressively became dominated by cells displaying features suggestive of neoplastic transformation. These cells were successfully expanded over five passages and exhibited signs of immortalization in vitro (data not shown). Healthy bladder tissue, right and left native ureters, and hyperemic bladder area displayed an identical haplotype profile, indicating a shared genetic origin and confirming their derivation from the recipient's native tissues. In contrast, left transplanted ureter, suspected neoplastic lesion, XX karyotype and right transplanted ureter, XY karyotype showed distinct genetic profiles, both differing from each other. Notably, however, both samples retained allelic peaks shared with the recipient's profile, suggesting the coexistence of recipient-derived cells within these tissues. Primary cell cultures derived from sample 3 revealed a homogeneous population after several passages, consistent with a clonal expansion of cells of presumptive neoplastic origin, with XX karyotype. In order to obtain a comprehensive molecular profile, the tumour sample was subjected to NGS. Genomic analysis was performed with the Archer VariantPlex Solid Tumor Kit (ArcherDX, Boulder, CO), a targeted NGS assay optimised for the detection of single nucleotide variants (SNVs) and insertions/deletions (indels) across a panel of 67 cancerrelated genes. The preparation of sequencing libraries was undertaken in strict accordance with the protocol stipulated by the manufacturer. Subsequent processing was then conducted on an Illumina sequencing platform. NGS analysis of the left transplanted ureter revealed PIK3CA c.1571G>A variant, resulting in the missense substitution p.Arg524Lys. This variant, located within the helical domain of the protein, is an uncommon substitution currently categorized as a Variant of Uncertain Significance (VUS) according to publicly available databases. The same sequencing protocol was applied to hyperemic bladder area, but no pathogenic alterations were identified in any of the targeted genes. Given the patient's prior diagnosis of JC virus (JCV) infection, all tissue samples were also screened for viral DNA using real-time PCR with the JCV ELITe MGB Kit (ELITech Group, Puteaux, France). JCV DNA was detected in all six tissue samples, confirming widespread viral persistence. However, no JC virus DNA was identified in either the supernatant or the cellular pellet of the cultured cells, including those derived from ureter of the transplanted kidney.Organ transplant recipients live in a paradoxical balance: the same immunosuppressive therapies that protect graft viability also erode the body's innate defences against both infection and neoplastic transformation. Over time, this immunological compromise not only permits viral reactivation, but also disrupts tumor immune surveillance and accelerates carcinogenic processes, either directly or indirectly. Indeed, some immunosuppressive drugs exert pro-tumorigenic effects independently of their immunemodulating functions, adding another layer of complexity to post-transplant risk management [14]. While most post-transplant malignancies are de novo and arise years after transplantation, a small subset originates from donor tissues: donor-transmitted tumors (DTTs), in which malignant cells are already present at the time of transplantation, and donor-derived tumors (DDTs), which develop from donor cells initially healthy. Although a two-year cutoff has been proposed to distinguish between the two, evidence shows that over 30% of DTTs manifest beyond this threshold, emphasizing the need for lifelong oncologic vigilance in transplant recipients [15,16].Alternative explanations, including mixed donor-recipient chimerism, infiltration of recipient-derived stromal or immune cells, or chromosomal mosaicism, were carefully considered. However, these hypotheses are unlikely in light of the molecular findings. The concordant STR profiles obtained from tumor tissue and cultured cells, together with the clonal expansion of XX cells in vitro and the detection of the same PIK3CA mutation in both ureteral and bladder lesions, strongly support a donor-derived origin of the tumor-initiating cells rather than a secondary infiltration or mosaic event. Rather than acting as a direct oncogenic driver, JC virus may have contributed indirectly to tumor development by shaping a permissive inflammatory and immunosuppressed microenvironment. In this context, persistent JC virus infection could have favored the survival and expansion of donor-derived tumor-initiating cells, without necessarily inducing direct mutagenic events. Rather than acting as a direct cell-autonomous mutagenic driver, JC virus likely contributed indirectly to tumor development by shaping a permissive, chronically inflamed microenvironment. We hypothesize a "field effect" mechanism where persistent JC virus infection induced a severe localized cystitis, which was histopathologically validated by the presence of chronic inflammation and tissue injury in non-neoplastic bladder sectors. This inflammatory field effect, acting in synergy with long-term immunosuppression, provided a fertile biological niche that favored the survival, engraftment, and eventual clonal expansion of the donor-derived tumor-initiating cells, without necessarily inducing direct viralmediated mutagenic events.One of the rarest scenarios involves tumors that originate from donor cells but developing far from the transplanted organ, or tumor that appears in the grafted organ but coming from the recipient's own cells, without any evidence of metastasis [17].Only a small number of studies have explored the phenomenon of chimerism in transplant-associated tumours, and even fewer have conclusively demonstrated a donor-derived oncogenesis based on genomic profiling [20][21][22]. The present case constitutes a novel and instructive addition to this limited body of evidence.The patient, with a 10-year history of chronic JC virus infection, was diagnosed with urothelial carcinoma during routine post-transplant follow-up. Bladder biopsies tested positive for the JCV genome and revealed a chimeric genetic profile, retaining several allelic peaks from the recipient-suggesting the coexistence of both donor-and recipientderived cell populations. The sample also harbored a PIK3CA variant (PIK3CA p.Arg524Lys c.1571G>A). While this variant has been infrequently reported (PMID: 21103049; PMID: 28551381; PMID: 27899992; PMID: 30219970; PMID: 24341335) and is classified as a VUS according to publicly available databases, its presence warrants further investigation given the patient's clinical presentation. Indeed, the PI3K/AKT/mTOR pathway is a critical oncogenic signaling cascade, with its aberrant activation frequently observed in urothelial carcinoma. Indeed, mutations in PIK3CA, a key component of this pathway, are found in approximately 20-30% of urothelial tumors, establishing it as one of the most prevalent molecular alterations (PMID: 23714499). Interestingly, when cells from the same lesion were isolated and expanded in culture, they exhibited an XX karyotype, clearly indicating donor origin, and showed characteristics of immortalization, including sustained proliferative capacity across multiple in vitro passages. The same XX donor genotype and identical PIK3CA mutation were also detected in the left transplanted ureter, which appeared macroscopically normal and showed no histologic abnormalities. Cells derived from this site similarly displayed features of immortalization and are postulated to represent tumor-initiating cells (TICs). Their presence in the bladder is most likely explained by cell migration-a well-documented phenomenon in the urothelium, occurring in both tissue regeneration and tumorigenesis. Although the PIK3CA p.Arg524Lys variant is currently classified as a Variant of Uncertain Significance, its presence in donor-derived cells from both ureter and bladder lesions supports a shared clonal origin. This finding suggests that the mutation may represent an early event in the expansion of donor-derived tumor-initiating cells rather than a late passenger alteration. This combination of viral presence, cellular chimerism, and PIK3CA variant suggests a complex interplay between viral inflammation, immunosuppression, and donor-derived tumor-initiating cells, which may have contributed to neoplastic transformation in a permissive microenvironment. We propose a model in which donor-derived TICs, originally located in the ureter, migrated to an area of chronic bladder injury-possibly mediated by JC virus-induced cystitis-and engrafted within a permissive inflammatory niche shaped by immunosuppression. There, they acquired additional mutations or cellular adaptations that unleashed their tumorigenic potential. Notably, no neoplastic behavior was observed in the ureter itself, suggesting that the bladder microenvironment was uniquely conducive to malignant transformation. The genomic identity of the TICs in both sites-bladder and ureter-supports their clonal relationship, while the long latency from transplantation excludes a pre-existing malignancy in the donor. Histopathological examination of the graft confirmed the absence of neoplastic lesions, further validating this as a bona fide donor-derived tumor. This case is more than a rare oncologic curiosity-it provides a living model of early carcinogenesis. In a scenario where donor and recipient cells are genomically distinguishable, chimerism could enable the tracking of tumor initiation from its earliest steps, allowing the isolation of the first altered cells, those that silently pave the way for clinical cancer long before dysplasia becomes evident. Current sequencing efforts focus largely on late-stage tumors, where all cancer cells share the same set of "public mutations," masking the founding event of transformation. Here, we had the rare opportunity to detect that founding event in a context where the neoplastic population emerged from a known origin and timeline, offering an unparalleled view of tumor initiation in vivo. Furthermore, the use of tumor spheroid cultures proved invaluable in isolating and expanding the dysregulated cell population. This not only enabled molecular characterization but also created a renewable model system for functional studies-a bridge between diagnosis and mechanistic understanding. Lastly, our findings open new hypotheses about the oncogenic role of JC virus.Rather than acting as a direct cell-autonomous mutagenic driver, JC virus likely contributed indirectly to tumor development by shaping a permissive, chronically inflamed microenvironment. We hypothesize a "field effect" mechanism where persistent JC virus infection induced a severe localized cystitis, which was histopathologically validated by the presence of chronic inflammation and tissue injury in non-neoplastic bladder sectors. This inflammatory field effect, acting in synergy with long-term immunosuppression, provided a fertile biological niche that favored the survival, engraftment, and eventual clonal expansion of the donor-derived tumor-initiating cells, without necessarily inducing direct viralmediated mutagenic events.This case offers compelling evidence for the presence and clinical relevance of donorderived tumor-initiating cells (TICs) in the setting of solid organ transplantation. The discovery of chimeric, genetically immortalized urothelial cells carrying PIK3CA mutation-absent from the donor's medical history-illustrates how malignancy can arise de novo from engrafted donor cells when exposed to a permissive host environment. We propose that a combination of chronic inflammation, JC virus infection, and long-term immunosuppression may create a biologically fertile niche that supports the survival, expansion, and eventual transformation of these cells. In this context, molecular chimerism analysis-integrated with in vitro functional models and next-generation sequencingemerges as a powerful tool to investigate the earliest stages of tumor development in immunocompromised patients. Beyond its mechanistic implications, this case underscores the importance of extending oncologic surveillance beyond traditional post-transplant timelines, and suggests that genomic profiling of histologically normal tissues may offer novel predictive markers for cancer risk in transplant recipients. These insights not only deepen our understanding of post-transplant malignancies but also open new avenues for early detection, risk stratification, and potentially preventive strategies in vulnerable patient populations.