Search Results (24)

Background: Ischemia–reperfusion injury (IRI) is a key contributor to graft dysfunction in kidney transplantation. Cell-free mitochondrial DNA (mtDNA) is increasingly recognized as a damage-associated molecular pattern (DAMP) and biomarker in IRI, but its prognostic role in living donor kidney transplantation (LDKT) remains unclear. Methods: This post hoc analysis of the VAPOR-1 study evaluated urinary mtDNA (UmtDNA) in 57 LDKT recipients. MtDNA levels (ND1, ND6, and D-loop) were measured at five early timepoints post-transplantation using qPCR. Associations between early UmtDNA and long-term graft function, defined by estimated glomerular filtration rate (eGFR) at 1, 12, and 24 months, were analyzed. Results: Higher UmtDNA levels in the first urine after reperfusion were significantly associated with improved eGFR at 12 months and a positive change in eGFR between month 1 and 24. These associations were not attributable to urine creatinine levels or mitochondrial copy number. Conclusions: In this LDKT cohort, elevated early UmtDNA may reflect a well-functioning graft capable of clearing systemic mtDNA rather than ongoing tubular injury. These findings suggest that the biological interpretation of mtDNA as a biomarker is context-dependent and call for careful reconsideration of its role in early transplant monitoring. Full article

Background: Growth arrest and DNA damage 45G (GADD45G) is a family of proteins involved in DNA damage response and cell growth arrest. In this study, we show evidence that urinary GADD45G protein is associated with the progression of IgA nephropathy. Methods: Patients diagnosed with IgA nephropathy without reversible acute kidney injury at study initiation and with at least one subsequent serum creatinine (SCr) measurement were included. A 50% or greater increase in SCr level was used as an endpoint for the deterioration of renal function. Enzyme-linked immunosorbent assay (ELISA) was performed using a Human GADD45G ELISA kit. Renal biopsy tissues were stained with a monoclonal mouse anti-GADD45G antibody. Results: Forty-five patients whose renal biopsy revealed IgA nephropathy were enrolled. Urinary GADD45G and urinary protein concentrations were 1.26 [0.69–2.20] μg/g creatinine and 0.65 [0.24–1.60] g/g creatinine, respectively. Urinary GADD45G showed significant positive correlations with SCr-slopes and urinary protein. The SCr-slope of the highest tertile group of urinary GADD45G (above 1.95 μg/g creatinine) was significantly higher than that of the lowest tertile group (below 0.90 μg/g). Univariate Cox regression analysis showed that urinary GADD45G was significantly associated with deterioration of renal function. A Kaplan–Meier test showed a significant difference in event-free survival for deterioration of renal function between the highest urinary GADD45G tertile group and other tertile groups. The area under the receiver operating characteristics (ROC) curve indicated urinary GADD45G had a good performance in predicting renal outcome (cut-off point 1.67 μg/g, positive predictive value 36.8%, negative predictive value 100%). Immunohistochemistry showed that GADD45G was expressed across all pathologic grades of IgA nephropathy and mainly detected in the cytoplasm of renal tubules, whereas no staining was noted in normal tissues. Conclusions: Urinary GADD45G excretion was significantly associated with kidney disease progression in patients with IgA nephropathy. Full article

Precision medicine is mainly based on reliable and noninvasive biomarkers. The aim of this review was to describe the newest biomarkers in the field of kidney transplantation and kidney rejection, one of the most common and severe complications. The standard tools used to identify acute rejection largely result in errors and have many drawbacks. In recent years, new and reliable biomarkers have been identified. These methods avoid risks, are noninvasive, and are able to detect rejection even in cases in which acute rejection is clinically asymptomatic and not otherwise identifiable, which is a frequent occurrence. In recent years, several biomarkers have been identified. Very recently, new relevant biomarkers with high positive predictive value and low negative predictive value have been identified. These are the donor-derived cell-free DNA found in the recipient, the gene expression profile of the donor found in the recipient, and the urinary cytokines that are modified in the graft tissue. The aim of this study was to identify the most recent findings in the literature on this topic and to describe the utility and possible limitations of such new biomarkers for kidney rejection. Full article

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. Full article

Background: The “Liquid Biopsy” has become a powerful tool for cancer research during the last decade. Circulating cell-free DNA (cfDNA) that originates from tumors has emerged as one of the most promising analytes. In contrast to plasma-derived cfDNA, only a few studies have investigated urinary cfDNA. One reason might be rapid degradation and hence inadequate concentrations for downstream analysis. In this study, we examined the stability of cfDNA in urine using different methods of preservation under various storage conditions. Methodology: To mimic patient samples, a pool of healthy male and female urine donors was spiked with a synthetic cfDNA reference standard (fragment size 170 bp) containing the T790M mutation in the EGFR gene. Spiked samples were preserved with three different buffers and with no buffer over four different storage periods (0 h; 4 h; 12 h; 24 h) at room temperature vs. 4 °C. The preservatives used were Urinary Analyte Stabilizer (UAS, Novosanis, Wijnegem, Belgium), Urine Conditioning Buffer (UCB, Zymo, Freiburg, Germany) and a self-prepared buffer called “AlloU”. CfDNA was extracted using the QIAamp MinElute ccfDNA Mini Kit (Qiagen, Hilden, Germany). CfDNA concentration was measured using the Qubit™ 4 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Droplet digital PCR (ddPCR) was used for detection and quantification of the T790M mutation. Results: Almost no spiked cfDNA was recoverable from samples with no preservation buffer and the T790M variant was not detectable in these samples. These findings indicate that cfDNA was degraded below the detection limit by urinary nucleases. Stabilizing buffers showed varying efficiency in preventing this degradation. The most effective stabilizing buffer under all storage conditions was the UAS, enabling adequate recovery of the T790M variant using ddPCR. Conclusion: From a technical point of view, stabilizing buffers and adequate storage conditions are a prerequisite for translation of urinary cfDNA diagnostics into clinical routine. Full article

Prostate cancer (PCa) is the most common cause of cancer death among African men. The presence of tumor-specific variations in cell-free DNA (cfDNA), such as mutations, microsatellite instability, and DNA methylation, has been explored as a source of biomarkers for cancer diagnosis. In this study, we investigated the diagnostic role of cfDNA among South African PCa patients. We performed whole exome sequencing (WES) of urinary cfDNA. We identified a novel panel of 31 significantly deregulated somatic mutated genes between PCa and benign prostatic hyperplasia (BPH). Additionally, we performed whole-genome sequencing (WGS) on matching PCa and normal prostate tissue in an independent PCa cohort from South Africa. Our results suggest that the mutations are of germline origin as they were also found in the normal prostate tissue. In conclusion, our study contributes to the knowledge of cfDNA as a biomarker for diagnosing PCa in the South African population. Full article

Methylation sequencing is a promising approach to infer the tissue of origin of cell-free DNA (cfDNA). In this study, a single- and a double-stranded library preparation approach were evaluated with respect to their technical biases when applied on cfDNA from plasma and urine. Additionally, tissue of origin (TOO) proportions were evaluated using two deconvolution methods. Sequencing cfDNA from urine using the double-stranded method resulted in a substantial within-read methylation bias and a lower global methylation (56.0% vs. 75.8%, p ≤ 0.0001) compared to plasma cfDNA, both of which were not observed with the single-stranded approach. Individual CpG site-based TOO deconvolution resulted in a significantly increased proportion of undetermined TOO with the double-stranded method (urine: 32.3% vs. 1.9%; plasma: 5.9% vs. 0.04%; p ≤ 0.0001), but no major differences in proportions of individual cell types. In contrast, fragment-level deconvolution led to multiple cell types, with significantly different TOO proportions between the two methods. This study thus outlines potential limitations of double-stranded library preparation for methylation analysis of cfDNA especially for urinary cfDNA. While the double-stranded method allows jagged end analysis in addition to TOO analysis, it leads to significant methylation bias in urinary cfDNA, which single-stranded methods can overcome. Full article

Objective: To assess the ability of cell-free urinary and plasma tumor DNA (cfDNA) to predict pathologic stage at radical cystectomy for patients with clinical muscle-invasive bladder cancer. Methods: A total of 25 patients with clinical muscle-invasive bladder cancer were enrolled before undergoing radical cystectomy. Blood and urine were collected before surgery. The 600-gene PredicineATLAS panel was used to sequence blood buffy-coat germline DNA, plasma cfDNA, and urine cfDNA samples. Low-pass whole genome sequencing was performed on plasma- and urine-derived cfDNA. CfDNA tumor fraction (TF), genome-wide copy number burden (CNB), and estimated tumor mutational burden (TMB) were measured in both plasma and urine samples and their correlation with pathologic T-stage was examined. Results: Three of 25 plasma samples had insufficient cfDNA. In 22 of 22 plasma samples and 24 of 25 urine samples, at least one nonsynonymous somatic variant was detected. Across the cohort, 44% of plasma variants were concordant with paired urine variants. The mean number of variants did not differ between noninvasive (< pT1/pN0) and invasive disease (≥ pT1 or N+) for both plasma (8 vs. 9.5 variants; P = 0.85) and urine (33.7 vs. 30 variants; P = 0.45). A strong correlation was observed between urine TF and urine CNB score within patients (rv = 0.92). Plasma TF (r = 0.38), urine TF (r = 0.21), and urine CNB score (r = 0.16) exhibited positive correlations with pT stage. Patients with carcinoma in situ (CIS) had higher mean urine TF and CNB scores (P = 0.07 and P = 0.05, respectively). Plasma TF and CNB score did not correlate with the presence of CIS. Conclusions: Combining plasma- and urine-based cfDNA analysis may help identify patients with residual disease at radical, although we were unable to predict pathologic T-stage based on these metrics.The presence of CIS may contribute to greater urinary CNB and TF levels. Considering CIS in the analysis may improve the ability to correlate tumor metrics with pathologic stage. Low-pass whole genome sequencing–derived urinary CNB correlates strongly with urinary TF and may provide a less resource-intensive method for future longitudinal disease monitoring. Full article

Extracellular DNA (ecDNA) is a promising candidate marker for the early diagnosis and monitoring of urinary tract infections (UTIs). The aim of our study is to describe the dynamics of ecDNA in the plasma and urine of patients with urosepsis as well as in a mouse model of UTI. Samples of blood and urine were collected from adult patients with UTIs and obstructive uropathy (n = 36) during the first 3 days at the hospital and during a follow-up. Bacterial burden and urinary ecDNA were evaluated in a mouse UTI model (n = 26) at baseline; 24, 48, and 72 h after UTI induction; and 7 days after UTI induction. The plasma ecDNA did not change during urosepsis, but the plasma DNase activity increased significantly at the follow-up. The urinary ecDNA decreased significantly during hospitalization and remained low until the follow-up (90% lower vs. admission). No change was seen in the urinary DNase activity. C-reactive protein (CRP) and procalcitonin are positively correlated with plasma and urinary ecDNA. A UTI caused sepsis in 23% of mice. The urinary ecDNA decreased by three-fold and remained low until day 7 post-infection. Urinary bacterial burden is correlated with urinary ecDNA. Urinary ecDNA is a potential non-invasive marker for monitoring the effects of treatment during urosepsis and is related to UTI progression in the experimental animal model. Full article

Liquid biopsy is a revolutionary tool that is gaining momentum in the field of cancer research. As a body fluid, urine can be used in non-invasive diagnostics for various types of cancer. We investigated the performance of UAS™ as a preservative for urinary analytes. Firstly, the need for urine preservation was investigated using urine samples from healthy volunteers. Secondly, the performance of UAS™ was assessed for cell-free DNA (cfDNA) and host cell integrity during storage at room temperature (RT) and after freeze-thaw cycling. Finally, UAS™ was used in a clinical setting on samples from breast and prostate cancer patients. In the absence of a preservative, urinary cfDNA was degraded, and bacterial overgrowth occurred at RT. In urine samples stored in UAS™, no microbial growth was seen, and cfDNA and cellular integrity were maintained for up to 14 days at RT. After freeze-thaw cycling, the preservation of host cell integrity and cfDNA showed significant improvements when using UAS™ compared to unpreserved urine samples. Additionally, UAS™ was found to be compatible with several commercially available isolation methods. Full article

Murine sepsis models are typically polymicrobial, and are associated with high mortality. We aimed to develop a high-throughput murine model that mimics a slow-paced, monomicrobial sepsis originating from the urinary tract. A total of 23 male C57Bl/6 mice underwent percutaneous insertion of a 4 mm catheter into the bladder using an ultrasound-guided method, previously developed by our group. The following day, Proteus mirabilis (PM) was introduced percutaneously in the bladder in three groups: g1—50 µL 1 × 10⁸ CFU/mL solution (n = 10); g2—50 µL 1 × 10⁷ CFU/mL solution (n = 10); and g3 (sham mice)—50 µL sterile saline (n = 3). On day 4, mice were sacrificed. The number of planktonic bacteria in urine, adherent to catheters, and adherent to/invaded into the bladder and spleen was assessed. Cell-free DNA, D-dimer, thrombin–antithrombin complex (TAT), and 32 pro-/anti-inflammatory cytokines/chemokines were quantified in the blood. All mice survived the 4 day postinterventional period. Mean weight loss was 11% in g1, 9% in g2, and 3% in the control mice. Mean urine CFU counts were highest in group 1. All catheters showed high catheter-adhered bacterial counts. Of the infected mice, 17/20 had CFU counts in the splenic tissue, indicating septicemia. Plasma levels of cell-free DNA, D-dimer, and the proinflammatory cytokines IFN-γ, IL-6, IP-10, MIG, and G-CSF were significantly elevated in infected mice versus controls. We present a reproducible, monomicrobial murine model of urosepsis that does not lead to rapid deterioration and death, and is useful for studying prolonged urosepsis. Full article

Donor-derived cell-free DNA (dd-cfDNA) is used as a biomarker for detection of antibody-mediated rejection (ABMR) and other forms of graft injury. Another potential indication is guidance of immunosuppressive therapy when no therapeutic drug monitoring is available. In such situations, detection of patients with overt or subclinical graft injury is important to personalize immunosuppression. We prospectively measured dd-cfDNA in 22 kidney transplant recipients (KTR) over a period of 6 months after conversion to belatacept for clinical indication and assessed routine clinical parameters. Patient and graft survival was 100% after 6 months, and eGFR remained stable (28.7 vs. 31.1 mL/min/1.73 m², p = 0.60). Out of 22 patients, 2 (9%) developed biopsy-proven rejection—one episode of low-grade TCMR IA and one episode of caABMR. While both episodes were detected by increase in creatinine, the caABMR episode led to increase in absolute dd-cfDNA (168 copies/mL) above the cut-off of 50 copies/mL, while the TCMR episode did show slightly increased relative dd-cfDNA (0.85%) despite normal absolute dd-cfDNA (22 copies/mL). Dd-cfDNA did not differ before and after conversion in a subgroup of 12 KTR with previous calcineurin inhibitor therapy and no rejection (12.5 vs. 25.3 copies/mL, p = 0.34). In this subgroup, 3/12 (25%) patients showed increase of absolute dd-cfDNA above the prespecified cut-off (50 copies/mL) despite improving eGFR. Increase in dd-cfDNA after conversion to belatacept is common and could point towards subclinical allograft injury. To detect subclinical TCMR changes without vascular lesions, additional biomarkers or urinary dd-cfDNA should complement plasma dd-cfDNA. Resolving CNI toxicity is unlikely to be detected by decreased dd-cfDNA levels. In summary, the sole determination of dd-cfDNA has limited utility in the guidance of patients after late conversion to belatacept. Further studies should focus on patients undergoing early conversion and include protocol biopsies at least for patients with increased dd-cfDNA. Full article

In breast cancer, the genetic profiling of circulating cell-free DNA (cfDNA) from blood plasma was shown to have good potential for clinical use. In contrast, only a few studies were performed investigating urinary cfDNA. In this pilot study, we analyzed plasma-derived and matching urinary cfDNA samples obtained from 15 presurgical triple-negative breast cancer patients. We used a targeted next-generation sequencing approach to identify and compare genetic alterations in both body fluids. The cfDNA concentration was higher in urine compared to plasma, but there was no significant correlation between matched samples. Bioinformatical analysis revealed a total of 3339 somatic breast-cancer-related variants (VAF ≥ 3%), whereof 1222 vs. 2117 variants were found in plasma-derived vs. urinary cfDNA, respectively. Further, 431 shared variants were found in both body fluids. Throughout the cohort, the recovery rate of plasma-derived mutations in matching urinary cfDNA was 47% and even 63% for pathogenic variants only. The most frequently occurring pathogenic and likely pathogenic mutated genes were NF1, CHEK2, KMT2C and PTEN in both body fluids. Notably, a pathogenic CHEK2 (T519M) variant was found in all 30 samples. Taken together, our results indicated that body fluids appear to be valuable sources bearing complementary information regarding the genetic tumor profile. Full article

Early detection of primary bladder cancer (BCa) is vital, because stage and grade have been generally accepted not only as categorical but also as prognostic factors in patients with BCa. The widely accepted screening methods for BCa, cystoscopy and urine cytology, have unsatisfactory diagnostic accuracy, with high rates of false negatives, especially for flat-type BCa with cystoscopy and for low-risk disease with urine cytology. Currently, liquid biopsy has attracted much attention as being compensatory for that limited diagnostic power. In this review, we survey the literature on liquid biopsy for the detection of BCa, focusing on circulating tumor cells (CTCs), urinary cell-free DNA (ucfDNA), and urinary microRNA (umiRNA). In diagnostic terms, CTCs and umiRNA are determined by quantitative analysis, and ucfDNA relies on finding genetic and epigenetic changes. The ideal biomarkers should be highly sensitive in detecting BCa. Currently, CTCs produce an unfavorable result; however, umiRNA and ucfDNA, especially when analyzed using a panel of genes, produce promising results. However, given the small cohort size in most studies, no conclusions can yet be drawn about liquid biopsy’s immediate application to clinical practice. Further large studies to validate the diagnostic value of liquid biopsy for clinical use are mandatory. Full article

Identification of mismatch repair (MMR)-deficient colorectal cancers (CRCs) is recommended for Lynch syndrome (LS) screening, and supports targeting of immune checkpoint inhibitors. Microsatellite instability (MSI) analysis is commonly used to test for MMR deficiency. Testing biopsies prior to tumour resection can inform surgical and therapeutic decisions, but can be limited by DNA quantity. MSI analysis of voided urine could also provide much needed surveillance for genitourinary tract cancers in LS. Here, we reconfigure an existing molecular inversion probe-based MSI and BRAF c.1799T > A assay to a multiplex PCR (mPCR) format, and demonstrate that it can sample >140 unique molecules per marker from <1 ng of DNA and classify CRCs with 96–100% sensitivity and specificity. We also show that it can detect increased MSI within individual and composite CRC biopsies from LS patients, and within preoperative urine cell free DNA (cfDNA) from two LS patients, one with an upper tract urothelial cancer, the other an undiagnosed endometrial cancer. Approximately 60–70% of the urine cfDNAs were tumour-derived. Our results suggest that mPCR sequence-based analysis of MSI and mutation hotspots in CRC biopsies could facilitate presurgery decision making, and could enable postal-based screening for urinary tract and endometrial tumours in LS patients. Full article