Cell-Free Mitochondrial DNA: An Upcoming Non-Invasive Tool for Diagnosis of BK Polyomavirus-Associated Nephropathy.

Mitochondria are essential organelles that possess their own DNA. Mitochondrial dysfunction has been revealed in many kidney diseases, including BK polyomavirus-associated nephropathy (BKPyVAN). In this study, we introduce an innovative approach for non-invasive monitoring of mitochondrial impairment through urinary donor-derived cell-free mitochondrial DNA (ddcfmtDNA), addressing the crucial challenge of BKPyVAN diagnosis. Urinary samples were collected at the time of biopsy from a total of 60 kidney transplant recipients, comprising 12 with stable function, 22 with T cell-mediated rejection, and 21 with biopsy-proven BKPyVAN. Our findings reveal that the ddcfmtDNA-to-ddcfDNA ratio exhibits superior capability in distinguishing BKPyVAN from other conditions, with a cutoff value of 4.96% (area under curve = 0.933; sensitivity: 71.4%; and specificity: 97.1%). Notably, an elevation of ddcfmtDNA levels is associated with mitochondrial damage, as visualized through electron microscopy. These results underscore the promise of non-invasive monitoring for detecting subtle mitochondrial damage and its potential utility in BKPyVAN diagnosis. Further investigations are required to advance this field of research.

A review of Merkel cell carcinoma.

ABSTRACT: Merkel cell carcinoma (MCC) is a rare and aggressive type of metastatic, nonmelanoma skin cancer derived from Merkel cells in the epidermis. MCC can be induced by sun exposure or via Merkel cell polyomavirus (MCV) gene expression. MCV is found in most patients with MCC and is associated with a lower recurrence rate of MCC. MCC has a wide range of clinical presentations that make diagnosis challenging. Histologic examination is performed using unique markers to differentiate it from other diagnoses. This article reviews the pathogenesis, clinical presentation, histopathology, differential diagnosis, and treatment of MCC.

Cultured Renal Proximal Tubular Epithelial Cells Resemble a Stressed/Damaged Kidney While Supporting BK Virus Infection.

BK virus (BKV; human polyomavirus 1) infections are asymptomatic in most individuals, and the virus persists throughout life without harm. However, BKV is a threat to transplant patients and those with immunosuppressive disorders. Under these circumstances, the virus can replicate robustly in proximal tubule epithelial cells (PT). Cultured renal proximal tubule epithelial cells (RPTE) are permissive to BKV and have been used extensively to characterize different aspects of BKV infection. Recently, lines of hTERT-immortalized RPTE have become available, and preliminary studies indicate they support BKV infection as well. Our results indicate that BKV infection leads to a similar response in primary and immortalized RPTE. In addition, we examined the patterns of global gene expression of primary and immortalized RPTE and compared them with uncultured PT freshly dissociated from human kidney. As expected, PT isolated from the healthy kidney express a number of differentiation-specific genes that are associated with kidney function. However, the expression of most of these genes is absent or repressed in cultured RPTE. Rather, cultured RPTE exhibit a gene expression profile indicative of a stressed or injured kidney. Inoculation of cultured RPTE with BKV results in the suppression of many genes associated with kidney stress. In summary, this study demonstrated similar global gene expression patterns and responses to BKV infection between primary and immortalized RPTE. Moreover, results from bulk transcriptome sequencing (RNA-seq) and SCT experiments revealed distinct transcriptomic signatures representing cell injury and stress in primary RPTE in contrast to the uncultured, freshly dissociated PT from human kidney. IMPORTANCE Cultured primary human cells provide powerful tools for the study of viral infectious cycles and host virus interactions. In the case of BKV-associated nephropathy, viral replication occurs primarily in the proximal tubule epithelia in the kidney. Consequently, cultured primary and immortalized renal proximal tubule epithelial cells (RPTE) are widely used to study BKV infection. In this work, using bulk and single-cell transcriptomics, we found that primary and immortalized RPTE responded similarly to BKV infection. However, both uninfected primary and immortalized RPTE have gene expression profiles that are markedly different from healthy proximal tubule epithelia isolated directly from human kidney without culture. Cultured RPTE are in a gene expression state indicative of an injured or stressed kidney. These results raise the possibility that BKV replicates preferentially in injured or stressed kidney epithelial cells during nephropathy.

Expression of Genes Associated With Epithelial-Mesenchymal Transition in Merkel Cell Polyomavirus-Negative Merkel Cell Carcinoma.

Two accepted possible pathways for Merkel cell carcinoma (MCC) pathogenesis include the clonal integration of the Merkel cell polyomavirus (MCPyV) into the neoplastic cells and by UV irradiation. We hypothesize that, in UV etiology, the expression of genes associated with epithelial-mesenchymal transition (EMT) would be higher in MCPyV-negative MCCs. We compared RNA expression in 16 MCPyV-negative with that in 14 MCPyV-positive MCCs in 30 patients using NanoString panel of 760 gene targets as an exploratory method. Subsequently, we confirmed the findings with a publicly available RNA sequencing data set. The NanoString method showed that 29 of 760 genes exhibited significant deregulation. Ten genes (CD44, COL6A3, COL11A1, CXCL8, INHBA, MMP1, NID2, SPP1, THBS1, and THY1) were part of the EMT pathway. The expression of CDH1/E-cadherin, a key EMT gene, and TWIST1, regulator gene of EMT, was higher in MCPyV-negative tumors. To further investigate the expression of EMT genes in MCPyV-negative MCCs, we analyzed publicly available RNA sequencing data of 111 primary MCCs. Differential expression and gene set enrichment analysis of 35 MCPyV-negative versus 76 MCPyV-positive MCCs demonstrated significantly higher expression of EMT-related genes and associated pathways such as Notch signaling, TGF-ß signaling, and Hedgehog signaling, and UV response pathway in MCPyV-negative MCCs. The significance of the EMT pathway in MCPyV-negative MCCs was confirmed independently by a coexpression module analysis. One of the modules (M3) was specifically activated in MCPyV-negative MCCs and showed significant enrichment for genes involved in EMT. A network analysis of module M3 revealed that CDH1/E-cadherin was among the most connected genes (hubs). E-cadherin and LEF1 immunostains demonstrated significantly more frequent expression in MCPvV-negative versus MCPyV-positive tumors (P < .0001). In summary, our study showed that the expression of EMT-associated genes is higher in MCPyV-negative MCC. Because EMT-related proteins can be targeted, the identification of EMT pathways in MCPyV-negative MCCs is of potential therapeutic relevance.

A review on the oncogenesis of Merkel cell carcinoma: Several subsets arise from different stages of differentiation of stem cell.

Merkel cell carcinoma (MCC), a rare primary cutaneous neuroendocrine neoplasm, is extremely aggressive and has a higher mortality rate than melanoma. Based on Merkel cell polyomavirus (MCPyV) status and morphology, MCCs are often divided into several distinct subsets: pure MCPyV-positive, pure MCPyV-negative, and combined MCC. MCPyV-positive MCC develops by the clonal integration of viral DNA, whereas MCPyV-negative MCC is induced by frequent ultraviolet (UV)-mediated mutations, that are characterized by a high mutational burden, UV signature mutations, and many mutations in TP53 and retinoblastoma suppressor gene (RB1). Combined MCC consists of an intimate mix of MCC and other cutaneous tumor populations, and is usually MCPyV-negative, with rare exceptions. Based on the existing subsets of MCC, it is speculated that there are at least 4 stages in the natural history of stem cell differentiation: primitive pluripotent stem cells, divergent differentiated stem cells, unidirectional stem cells, and Merkel cells (or epidermal/adnexal cells). In the first stage, MCPyV may integrate into the genome of primitive pluripotent stem cells, driving oncogenesis in pure MCPyV-positive MCC. If MCPyV integration does not occur, the stem cells enter the second stage and acquire the ability to undergo multidirectional neuroendocrine and epidermal (or adnexal) differentiation. At this stage, accumulated UV-mediated mutations may drive the development of combined MCC. In the third stage, the stem cells differentiate into unidirectional neuroendocrine stem cells, UV-mediated mutations can induce carcinogenesis in pure MCPyV-negative MCC. Therefore, it has been speculated that several subsets of MCCs arise from different stages of differentiation of common stem cells.

An Unusual Presentation of Metastatic BK Virus-Associated Urothelial Carcinoma Arising in the Allograft, Persisting After Transplant Nephrectomy.

We report a 32-year-old male 14 years post-living-related kidney transplant presenting with new-onset hematuria and BK viremia. He was found to have BK virus-associated urothelial carcinoma originating in the renal allograft with locally advanced disease and metastases to multiple sites. He also developed acute T-cell-mediated rejection in the setting of immunosuppression reduction for BK viremia prior to undergoing transplant nephrectomy. Eight months following transplant nephrectomy and immunosuppression cessation, distant metastases persisted with partial response to chemotherapy and immunotherapy. Here, we discuss this unique presentation and compare it with other BK virus-associated allograft carcinomas reported in the literature, in addition to discussing evidence for the role of BK virus in oncogenesis.

MCPyV-Negative Merkel Cell Carcinoma In Situ Associated With Immunosuppression.

ABSTRACT: Merkel cell carcinoma (MCC) is a rare and aggressive cutaneous neoplasm that is almost always intradermal. Immunosuppression increases the risk of MCC, which is believed to be due to increased susceptibility to Merkel cell polyomavirus (MCPyV). Intraepidermal MCC, or MCC in situ (MCCis), is extremely rare and usually associated with other cutaneous lesions. Here, we describe a case of MCPyV-negative MCCis arising in an immunocompromised patient. This case adds to only 9 previously reported cases of MCCis without a coexisting neoplasm and suggests that immunosuppression can lead to MCCis by mechanisms other than MCPyV. Although previously reported cases of MCCis demonstrated excellent prognosis, local recurrence and metastasis are still possible. Prognostication, treatment, and follow-up of MCCis should be similar to MCC.

BK DNAemia and native kidney polyomavirus nephropathy following lung transplantation.

BK virus DNAemia (BKPyV) and nephropathy are common after kidney transplant; however, there are limited data on BK infections in nonrenal solid organ transplant recipients. We examined the frequency, clinical and pathologic features, and kidney and lung outcomes of BKPyV and BK virus native kidney nephropathy (BKVN) in lung transplant recipients at our center. Among 878 recipients transplanted from 2003 to 2019, 56 (6%) developed BKPyV at a median of 30.1 months after transplant (range, 0.6-213) and 11 (1.3%) developed BKVN at a median of 46 months after transplant (range, 9-213). The incidence of end-stage kidney disease was significantly higher in patients with peak viral load ≥10 000 copies/mL (39% vs 8%, P < .001). All cases of BKVN were in patients with peak viral load of ≥10 000 copies/mL, and 55% of these patients developed end-stage kidney disease. Despite the reduction of immunosuppression to treat BKVN, only 1 patient developed acute rejection, and lung function was stable >1 year. BKPyV and nephropathy are more common after lung transplantation than previously reported. Routine screening for BKPyV should be considered in all lung transplant recipients.

Evaluation of the risk factors for BK virus-associated hemorrhagic cystitis in pediatric bone marrow transplantation patients: Does post-transplantation cyclophosphamide increase the frequency?

BACKGROUND: BKV-HC is one of the most significant complications of HSCT. This retrospective study aimed to determine the frequency of BKV-HC in pediatric patients undergoing HSCT, detect the associated risk factors for the development of BKV-HC, and explore the effects of post-transplantation Cy use. METHODS: Three hundred twenty-seven patients (girls: 121, boys: 206) were analyzed according to sex, conditioning regimen, transplantation type, donor relatedness, stem cell source, the presence and grade of aGVHD, CMV co-existence, and Cy use. RESULTS: Multivariate analysis confirmed the prognostic importance of age (OR: 4.865), TBI use, the presence of aGVHD (OR: 2.794), CMV coinfection (OR: 2.261), and Cy use (OR: 27.353). A statistically significant difference was found between the mean BKV-HC follow-up times compared with post-transplantation Cy intake (p < .001). The BKV-HC rate increased as the number of risk factors of the patient increased. CONCLUSION: BKV-HC is an essential complication of HSCT primarily associated with Cy use, the presence of aGVHD, and donor relatedness. The present study shows that the use of Cy in the post-transplantation period further increases BKV-HC risk in pediatric patients, regardless of dose.

Impact of BK Polyomavirus Plasma Viral Load in Kidney Transplant Outcomes.

BACKGROUND: BK polyomavirus infection (BKVi) is an important cause of kidney transplant (KT) loss, but there is scarce evidence on the impact of BK plasma viral load on graft function and long-term KT survival. METHODS: A retrospective cohort study including all KT recipients with BKVi (BK viremia identified in ≥3 consecutive samples by polymerase chain reaction) in our center from January 2010 to December 2020 was performed. A case-control study (1:2) was performed. We grouped the cases according to their highest peak viral load: low-level viremia (<10,000 copies/mL) and high-level viremia (≥10,000 copies/mL). To identify risk factors for BKVi, a logistic regression analysis was achieved, and a multivariable Cox regression was used to describe risk factors for graft loss. RESULTS: A total of 849 KTs were performed, and 67 presented BKVi (low-level viremia, n = 35 and high-level viremia, n = 26). In logistic regression analysis male sex (odds ratio [OR], 4.226; 95% CI, 1.660-10.758, P = .002), age (OR, 1.047; 95% CI, 1.008-1.088; P = .018), and retransplant (OR, 4.162; 95% CI, 1.018-17.015; P = .047) were predictors of BKVi. Acute rejection was more frequent in the BKVi group (18% vs 4.9%, P = .004), and graft survival was lower in patients with BKVi and high-level viremia (P = .027). In Cox regression analysis, BKVi (hazard ratio, 3.657; 95% CI, 1.146-11.670; P = .029) and specific BKV (BK polyomavirus) high-level viremia (hazard ratio, 1.988; 95% CI, 1.012-3.907; P = .046) were predictors of shorter graft survival. CONCLUSIONS: BKV high-level viremia was associated with BKV nephropathy and poorer graft survival. Additionally, acute rejection is more frequent after BKVi. It is necessary to develop strategies safe and effective for these patients.

S100A8/A9, an Upregulated Host Factor in BK Virus Infection after Kidney Transplantation, Is Associated with Allograft Function Impairment.

BK virus (BKV) is one of the most common pathogens in post-transplantation infections. For kidney transplantation, BKV infection results in the impairment of allograft function and thus increases the risk of allograft loss. However, clinical evaluation of the prognosis of BKV-associated allograft impairment is difficult. In the present study, differential plasma proteins were screened using proteomic methods from ten patients with a transition from BKV-negative to BKV activation. We identified 12 differentially expressed proteins, and S100A8 and S100A9 were the top two upregulated proteins. Data from a cross-sectional study with 66 BKV-negative and 66 BKV-positive recipients of renal transplantation indicated that plasma S100A8/A9 was upregulated in BKV-infected recipients. Plasma S100A8/A9 positively correlated with the 1 month creatinine increase (ρ = 0.499, P = 0.021) and negatively correlated with the 1 month estimated glomerular filtration rate change (ρ = -0.618, P = 0.003) in recipients with BK viremia. Using least absolute shrinkage and selection operator regression models, we found that S100A8/A9 was an independent risk factor for the decrease in allograft function after BKV infection. In conclusion, S100A8/A9 is a potential host biomarker for the clinical evaluation of BKV-associated allograft function impairment in kidney transplantation.

Bovine Polyomavirus-1 (Epsilonpolyomavirus bovis): An Emerging Fetal Pathogen of Cattle That Causes Renal Lesions Resembling Polyomavirus-Associated Nephropathy of Humans.

Bovine polyomavirus-1 (BoPyV-1, Epsilonpolyomavirus bovis) is widespread in cattle and has been detected in commercialized beef at supermarkets in the USA and Germany. BoPyV-1 has been questioned as a probable zoonotic agent with documented increase in seropositivity in people exposed to cattle. However, to date, BoPyV-1 has not been causally associated with pathology or disease in any animal species, including humans. Here we describe and illustrate pathological findings in an aborted bovine fetus naturally infected with BoPyV-1, providing evidence of its pathogenicity and probable abortigenic potential. Our results indicate that: (i) BoPyV-1 can cause severe kidney lesions in cattle, including tubulointerstitial nephritis with cytopathic changes and necrosis in tubular epithelial cells, tubular and interstitial inflammation, and interstitial fibroplasia; (ii) lesions are at least partly attributable to active viral replication in renal tubular epithelial cells, which have abundant intranuclear viral inclusions; (iii) BoPyV-1 large T (LT) antigen, resulting from early viral gene expression, can be detected in infected renal tubular epithelial cells using a monoclonal antibody raised against Simian Virus-40 polyomavirus LT antigen; and (iv) there is productive BoPyV-1 replication and virion assembly in the nuclei of renal tubular epithelial cells, as demonstrated by the ultrastructural observation of abundant arrays of viral particles with typical polyomavirus morphology. Altogether, these lesions resemble the "cytopathic-inflammatory pathology pattern" proposed in the pathogenesis of Human polyomavirus-1-associated nephropathy in immunocompromised people and kidney allograft recipients. Additionally, we sequenced the complete genome of the BoPyV-1 infecting the fetus, which represents the first whole genome of a BoPyV-1 from the Southern Hemisphere. Lastly, the BoPyV-1 strain infecting this fetus was isolated, causing a cytopathic effect in Madin-Darby bovine kidney cells. We conclude that BoPyV-1 is pathogenic to the bovine fetus under natural circumstances. Further insights into the epidemiology, biology, clinical relevance, and zoonotic potential of BoPyV-1 are needed.

Glypican-3 (GPC3) is associated with MCPyV-negative status and impaired outcome in Merkel cell carcinoma.

INTRODUCTION: Merkel cell carcinoma (MCC) is an aggressive skin cancer, related to the Merkel Cell Polyomavirus (MCPyV) in 80% of cases. Immune checkpoint inhibitors provide sustained benefit in about 50% of MCC patients with advanced disease. Glypican-3 (GPC3) is an oncofetal tumor antigen that is an attractive target for chimeric antigen receptor T cell therapy due to its highly restricted expression on normal tissue and high prevalence in several solid tumors. GPC3 is known to be expressed in MCC but its association with tumor characteristics or prognosis has not been reported. We investigated MCC GPC3 expression by immunohistochemistry (IHC) and its association with tumor characteristics, MCPyV status, and patient outcome. METHODS: The GC33 antibody clone was validated for GPC3 IHC staining of tumor specimens in comparison to an established GPC3 IHC antibody. An MCC tissue microarray was stained for GPC3 by IHC using GC33 antibody. Association of GPC3+ IHC with baseline characteristics, MCPyV status (qPCR) and outcome (death from MCC/recurrence) were assessed. RESULTS: Forty-two of 62 samples (67.7%) were GPC3+. GPC3 expression was more frequently observed in females (p = 0.048) and MCPyV-negative tumors (p = 0.021). By multivariate analysis, GPC3 expression was associated with increased death from disease (CSS) (hazard ratio [HR] 4.05, 95% CI 1.06-15.43), together with advanced age (HR 4.85, 95% CI 1.39-16.9) and male gender (HR 4.64, 95% CI 1.31-16.41). CONCLUSIONS: GPC3 expression is frequent in MCC tumors, especially MCPyV-negative cases, and is associated with increased risk of death. High prevalence of surface GPC3 makes it a putative drug target.

BK polyomavirus in Iranian patients with papillary thyroid cancer: Is it a major future challenge?

BK polyomavirus (BKPyV) is a well-known cause of nephropathy in renal transplant recipients. It has recently received much attention from researchers as a major predisposing factor for various cancers. This study aimed to investigate how BKPyV affected the advancement of papillary thyroid carcinoma (PTC). A total of 1057 samples were tested for BKPyV DNA and RNA, comprising 645 paraffin-embedded PTC biopsy samples (PEBS), 412 fresh biopsy samples (FBS), and 1057 adjacent noncancerous samples. The BKPyV DNA was found in 511 (48.3%) of the specimens, including 347 (84.2%) FBS and 164 (25.4%) PEBS. The mean BKPyV copy number was significantly lower in patients with PEBS (0.5 × 10-4 ± 0.1 × 10-4 copies/cell) than in FBS (1.3 × 10-1 ± 0.2 × 10-1 copies/cell) and non-PTC normal samples (0.3 × 10-5 ± 0.04 × 10-5 copies/cell). The PEBS had lower LT-Ag RNA expression than FBS, and no VP1 gene transcript expression was detected. In conclusion, although our findings indicated the presence of BKPyV in some Iranian PTC patients, more research is needed to corroborate these findings.

Impact of antimetabolite discontinuation following cytomegalovirus or BK polyoma virus infection in kidney transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) and BK polyoma virus (BKV) infection following kidney transplantation have been associated with allograft dysfunction and allograft loss. Reduction in immunosuppression is a mainstay of management yet has been associated with increased risk of rejection. According to international consensus guidelines, one approach to management of these viral infections is to discontinue the antimetabolite. Little is known surrounding long-term outcomes in these patients, and it remains unclear if consideration should be given to resuming the antimetabolite as variable re-escalation strategies have been reported. The objective was to describe episodes of rejection and identify risk factors for rejection following antimetabolite withdrawal after CMV or BKV DNAemia in kidney transplant recipients. METHODS: This single-center, retrospective review evaluated adult kidney transplant recipients with a serum CMV or BKV DNA PCR ≥500 copies/ml who underwent antimetabolite discontinuation. The primary outcome assessed was the incidence of biopsy-proven acute rejection (BPAR). RESULTS: One hundred fifty-nine patients were included. Overall, 14 patients (8.8%) experienced BPAR at a median of 1.6 years after antimetabolite discontinuation. Compared to CMV, discontinuation after BKV DNAemia was associated with a higher incidence of BPAR. Characteristics observed more frequently in patients with BPAR included younger age, female sex, higher initial viral load, and development of de novo donor-specific antibody (DSA). CONCLUSION: These findings suggest that antimetabolite discontinuation after CMV or BKV DNAemia in kidney transplant recipients is a reasonable and safe approach. Further prospective studies investigating optimal immunosuppression management following CMV or BKV DNAemia in kidney transplant recipients are warranted.

Clinicopathological features, MCPyV status and outcomes of Merkel cell carcinoma in solid-organ transplant recipients: a retrospective, multicentre cohort study.

BACKGROUND: The proportion of Merkel cell carcinomas (MCCs) in solid-organ transplant recipients (SOTR) harbouring Merkel cell polyomavirus (MCPyV) is unknown, as are factors affecting their outcomes. OBJECTIVE: To describe clinicopathological features of MCC in SOTR, investigate the tumoral MCPyV-status and identify factors associated with tumour outcomes. METHODS: Retrospective, international, cohort-study. MCPyV-status was investigated by immunohistochemistry and polymerase chain reaction. RESULTS: A total of 30 SOTR and 44 consecutive immunocompetent patients with MCC were enrolled. SOTR were younger at diagnosis (69 vs. 78 years, P < 0.001). Thirty-three percent of SOTR MCCs were MCPyV-positive vs. 91% of immunocompetent MCCs (P = 0.001). Solid-organ transplantation was associated with an increased cumulative incidence of progression (SHR: 3.35 [1.57-7.14], P = 0.002), MCC-specific mortality (SHR: 2.55 [1.07-6.06], P = 0.034) and overall mortality (HR: 3.26 [1.54-6.9], P = 0.002). MCPyV-positivity and switching to an mTOR inhibitor (mTORi) after MCC diagnosis were associated with an increased incidence of progression (SHR: 4.3 [1.5-13], P = 0.008 and SHR: 3.6 [1.1-12], P = 0.032 respectively) in SOTR. LIMITATIONS: Retrospective design and heterogeneity of SOTR cohort. CONCLUSIONS: MCPyV appears to play a less prominent role in the aetiopathogenesis of MCC in SOTR. SOTR have a worse prognosis than their immunocompetent counterparts and switching to an mTORi after the diagnosis of MCC does not improve progression.