Urinary DNA Research Articles

Abstract A052: Circulating tumor DNA (ctDNA) in plasma and urine of renal cell carcinoma patients with localized disease

Abstract Introduction: Circulating tumor DNA (ctDNA) is increasingly valuable in cancer care. Early diagnosis of renal cell carcinoma (RCC) significantly improves prognosis. Clear cell RCC (ccRCC), the most prevalent and aggressive subtype, often leads to recurrence in over 20% of localized cases post-nephrectomy. Thus, risk-stratifying tools are crucial for tailored treatment strategies. In this study, we tracked tumor mutations in DNA from plasma (ctDNA) and urine (utDNA) in patients with localized ccRCC to explore their potential as risk-stratifying biomarkers for recurrence. We evaluated ctDNA/utDNA pre-surgery in all RCC patients and conducted longitudinal assessments post-surgery in ccRCC patients. Additionally, for one ccRCC patient with aggressive disease, we analyzed tumor mutations in the plasma of patient- derived xenograft (PDX) replicates. Methodology: RCC patients were recruited from A.C.Camargo Cancer Center. Urine and plasma samples were taken pre-surgery (BL-baseline), and for ccRCC patients, samples were collected at 6-8 weeks and 6, 18 and 30 months post- surgery (M1, M2, M3 and M4, respectively). Patients were monitored for recurrence for up to 6,3 years. We used a customized 28-gene RCC panel and a commercial 409-gene solid tumor panel to identify tumor somatic mutations using Illumina NextSeq 500 and Ion S5 platforms, respectively. Tumor specific mutations were tracked in DNA from patient plasma and urine, and PDX plasma, using the PATS (Personalized amplicon target sequencing) method on Ion S5 Plus platform. Results: Out of 79 RCC patients (58% ccRCC), 57 (72%) had tumor somatic mutations, including 39 (68%) in ccRCC. VHL and PBRM1 were the most frequently mutated genes. Baseline ctDNA/utDNA were analyzed in 51 RCC patients, with 18% (13% plasma, 12% urine) positivity in RCC and 19% (12% plasma, 12% urine) in ccRCC. During ccRCC monitoring, ctDNA/utDNA was positive only in M1 and M3, with 9% (9% plasma, 0% urine) and 18% (12% plasma, 6% urine), respectively. ctDNA-positive baseline plasma was associated to disease progression (p=0.0155) and tumor staging ≥pT3 (p=0.002); these patients also experienced reduced progression-free (p=0.004) and overall (p98%, resembling the patient’s tumor aggressiveness. Conclusions: ccRCC tumors release low levels of tumor DNA in urine and plasma. Pre-surgery plasma positivity for ctDNA appears to indicate a higher risk of recurrence in ccRCC patients. The ctDNA release of PDX tumors replicated that observed in one ccRCC patient with aggressive disease. Citation Format: Ana Carolina Kerekes Miguez, Isabella Tanus Job e Meira, Stephania Martins Bezerra, Adriano de Oliveira Beserra, Walter Henriques da Costa, Tiago Goss dos Santos, Stenio de Cassio Zequi, Giovana Tardin Torrezan, Dirce Maria Carraro. Circulating tumor DNA (ctDNA) in plasma and urine of renal cell carcinoma patients with localized disease [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A052.

Kidney hyperfiltration and mitochondrial changes are associated with eGFR decline in young people with type 1 diabetes.

To examine the relationship between kidney hyperfiltration during adolescence and subsequent changes in estimated glomerular filtration rate (eGFR) and urinary albumin creatinine ratio (UACR) in a young cohort of participants with type 1 diabetes. Additionally, to explore urinary mitochondrial DNA:nuclear DNA ratio (mtDNA:nDNA) as a marker of metabolic stress and its association with early changes in kidney function. Eighty adolescents were studied at baseline [mean (SD) age 14.2 (1.5) years; mean diabetes duration 6.7 (3.0) years] and followed up 9.2 (1.3) years later. Blood pressure, HbA1c, lipids, eGFR, UACR and heart rate variability were assessed at each visit. Urinary mtDNA:nDNA was measured by quantitative PCR (qPCR). Overall, 4.2% of participants had diabetic kidney disease (DKD) at follow-up. Hyperfiltration at baseline (>135 mL/min/1.73m2) was seen in 31% of adolescents and was associated with a decline in eGFR at follow-up when adjusted for sex, diabetes duration and HbA1c [hyperfiltration -1.46 (3.07) mL/min/1.73 m2/year vs non-hyperfiltration -0.51 (2.48) mL/min/1.73m2/year, P=0.02]. Participants with hyperfiltration also had higher odds of undergoing rapid eGFR decline (>3 mL/min/1.73m2/year) compared to those without hyperfiltration [OR 14.11, 95% CI (2.30-86.60), P=0.004]. Baseline urinary mtDNA:nDNA was significantly associated with both greater annual rate of eGFR decline and rapid eGFR decline in univariable but not multivariable modelling. Hyperfiltration during adolescence is significantly associated with greater reduction in eGFR and higher risk of rapid eGFR decline after ∼9 years, following transition into young adulthood in type 1 diabetes. Urinary mtDNA:nDNA measured during adolescence may be a novel predictor of early changes in kidney function.

A Novel Urine DNA Predictor for Noninvasive Early Diagnosis and Monitoring Minimal Residual Disease of Upper Tract Urothelial Carcinoma.

For early detection and postoperative monitoring of upper tract urothelial carcinoma (UTUC), the traditional detection method was limited to its invasiveness and insufficient sensitivity. We aim to use urine tumour DNA (utDNA) for detecting minimal residual disease (MRD), early diagnosis and perioperative monitoring in UTUC. We previously established a utDNA multidimensional bioinformatic valuation model, named utLIFE, using low-coverage whole-genome sequencing and targeted deep sequencing. This prospective cohort enrolled 93 patients diagnosed with UTUC without metastasis. We collected morning urine samples on the day of surgery and the discharge day after the operation for utLIFE testing. In addition, we also enrolled 80 healthy controls to further validate the specificity of the utLIFE model in the study. The utLIFE of preoperative samples could discriminate UTUC with high specificity (96.25%, 77/80), and high sensitivity (96.77%, 90/93) regardless of stage and grade. The sensitivity of utLIFE was significantly higher than urine cytology (p < 0.001) and fluorescence insitu hybridisation (FISH) (p < 0.001) (N = 19), especially in early-stage and low-grade UTUC. Postoperative utLIFE scores were significantly decreased compared with those of preoperative samples (79 vs. 36, p < 0.001), indicating its association with tumour burden. For special pathology types, utLIFE performed less well in sensitivity and perioperative alteration. In conclusion, we established a bioinformatic utDNA valuation model, utLIFE, which was validated to be a rapid and noninvasive approach with high sensitivity for early detection and MRD monitoring for UTUC.

Diagnostic Study Of Bladder Cancer Using Urine Methylation Biomarkers Of Iraqi Patients

Background: Bladder cancer (BC) is the 4th most frequently occurring malignancy and the 9th most common cause of death worldwide in men. The invasive and metastatic form of the cancer is the main cause of death or unfavorable prognosis for BC patients. The presence of a bladder tumor is often discovered after episodes of painless macroscopic hematuria. At initial diagnosis, the disease is non-muscle-invasive in approximately 75% of patients. In recent years, detecting aberrantly methylated DNA in urine has emerged as a promising and noninvasive approach to aid BC diagnosis. Objective: To detect bladder tumors in Iraqi patients. Methods: In this cross-sectional study, sixty cases were suspected of having bladder tumors according to the opinion of the physician and some signs and symptoms, the best sign was hematuria in urine (per diagnosis). An additional 30 healthy controls with similar age and sex were recruited as control groups, with an average age of 37_85 years. from Baghdad Medical City / Ghazi al–Hariri Teaching Hospital and AlKindi Specialized Hospital during the period from September / 2023 to April / 2024. Using molecular polymerase chain reaction (high-resolution melting-PCR). Results: Out of 60 bladder inflammations, the result of hypermethylation genes ZNF154, EOMES, and POU4F2, Sensitivity were (71.7, 85, and 75) %, and Specificity (60, 33.3, and 50) % respectively with high significant differences (p &lt; 0.01). In addition. Conclusions: Detecting DNA hypermethylation is considered a good diagnosis method for bladder tumors.

Analysis of urine cell-free DNA in bladder cancer diagnosis by emerging bioactive technologies and materials.

Urinary cell-free DNA (UcfDNA) is gaining recognition as an important biomarker for diagnosing bladder cancer. UcfDNA contains tumor derived DNA sequences, making it a viable candidate for non-invasive early detection, diagnosis, and surveillance of bladder cancer. The quantification and qualification of UcfDNA have demonstrated high sensitivity and specificity in the molecular characterization of bladder cancer. However, precise analysis of UcfDNA for clinical bladder cancer diagnosis remains challenging. This review summarizes the history of UcfDNA discovery, its biological properties, and the quantitative and qualitative evaluations of UcfDNA for its clinical significance and utility in bladder cancer patients, emphasizing the critical role of UcfDNA in bladder cancer diagnosis. Emerging bioactive technologies and materials currently offer promising tools for multiple UcfDNA analysis, aiming to achieve more precise and efficient capture of UcfDNA, thereby significantly enhancing diagnostic accuracy. This review also highlights breakthroughs in detection technologies and substrates with the potential to revolutionize bladder cancer diagnosis in clinic.

1978P Accurate detection of urothelial carcinoma by whole-genome methylation profiling of urinary cell-free DNA

1978P Accurate detection of urothelial carcinoma by whole-genome methylation profiling of urinary cell-free DNA

93P UriMee: A novel non-invasive test for diagnosis of urothelial carcinoma by detection of methylation markers in urinary sediment DNA

93P UriMee: A novel non-invasive test for diagnosis of urothelial carcinoma by detection of methylation markers in urinary sediment DNA

An assessment of gynecological manifestations in women with female genital schistosomiasis with reference to Schistosoma biomarkers, sexually transmitted infections and bacterial vaginosis

BackgroundAlthough a variety of different gynecological manifestations have been reported in women with female genital schistosomiasis (FGS), causality remains to be established. This study aimed to evaluate the gynecological manifestations in women with FGS in accordance with the status of Schistosoma biomarkers, sexually transmitted infections (STIs), and bacterial vaginosis (BV).MethodsThe study was conducted in an endemic Schistosoma haematobium (Sh) area in northern Madagascar in conjunction with a randomized controlled trial investigating the effects and safety of a praziquantel repeated-dosing regimen for women with FGS-associated cervical lesions. Urogenital complaints, pelvic exam abnormalities, and cervical lesion types were assessed in relation to cervicovaginal Schistosoma DNA, circulating anodic antigen (CAA) in serum, and urinary Sh egg count, in addition to STIs and BV.ResultsAmong the included 116 women with a median of 26 years (range 15 to 35), the distribution of Schistosoma DNA and CAA outcomes, specified as either positive (+) or negative (-), were as follows: +/+ (18.1%), +/- (0%), -/+ (58.6%), and -/- (23.3%). Of the three Schistosoma biomarkers, only Schistosoma DNA and the urogenital complaint of blood in the urine were significantly associated. None of the biomarkers were significantly associated with pelvic exam abnormalities or cervical lesions. Sixty women (52.6%) were diagnosed with STIs and/or BV. A positive status was not significantly associated with any of the gynecological manifestations, except BV and homogeneous yellow sandy patches.ConclusionIt remains uncertain whether biomarkers such as cervicovaginal Schistosoma DNA, serum CAA, and Schistosoma eggs in urine adequately cover the full spectrum of gynecological manifestations reported in women with FGS, including urogenital complaints, pelvic exam abnormalities, and cervical lesions. Moreover, it seems difficult to determine the origin of the different manifestations due to the common co-existence of STIs and/or BV as potential confounders.

An epigenome-wide study of selenium status and DNA methylation in the Strong Heart Study

BackgroundSelenium (Se) is an essential nutrient linked to adverse health endpoints at low and high levels. The mechanisms behind these relationships remain unclear and there is a need to further understand the epigenetic impacts of Se and their relationship to disease. We investigated the association between urinary Se levels and DNA methylation (DNAm) in the Strong Heart Study (SHS), a prospective study of cardiovascular disease (CVD) among American Indians adults. MethodsSelenium concentrations were measured in urine (collected in 1989–1991) using inductively coupled plasma mass spectrometry among 1,357 participants free of CVD and diabetes. DNAm in whole blood was measured cross-sectionally using the Illumina MethylationEPIC BeadChip (850 K) Array. We used epigenome-wide robust linear regressions and elastic net to identify differentially methylated cytosine-guanine dinucleotide (CpG) sites associated with urinary Se levels. ResultsThe mean (standard deviation) urinary Se concentration was 51.8 (25.1) μg/g creatinine. Across 788,368 CpG sites, five differentially methylated positions (DMP) (hypermethylated: cg00163554, cg18212762, cg11270656, and hypomethylated: cg25194720, cg00886293) were significantly associated with Se in linear regressions after accounting for multiple comparisons (false discovery rate p-value: 0.10). The top hypermethylated DMP (cg00163554) was annotated to the Disco Interacting Protein 2 Homolog C (DIP2C) gene, which relates to transcription factor binding. Elastic net models selected 425 hypo- and hyper-methylated DMPs associated with urinary Se, including three sites (cg00163554 [DIP2C], cg18212762 [MAP4K2], cg11270656 [GPIHBP1]) identified in linear regressions. ConclusionsUrinary Se was associated with minimal changes in DNAm in adults from American Indian communities across the Southwest and the Great Plains in the United States, suggesting that other mechanisms may be driving health impacts. Future analyses should explore other mechanistic biomarkers in human populations, determine these relationships prospectively, and investigate the potential role of differentially methylated sites with disease endpoints.

A Novel Poly(cytosine)-Based Electrochemical Biosensor for Sensitive and Selective Determination of Guanine in Biological Samples.

The novel poly(cytosine)-modified glassy carbon electrode-based electrochemical sensor was fabricated potentiodynamically for the detection of Guanine (G) in clinical and biological samples. The surface of the electrode was successfully activated by electropolymerization, and about a 7.5-fold current improvement due to modification was achieved. From the analysis of the dependence of peak current and peak potential on a scan rate, a higher R 2 for the peak current on the square root of scan rate (R 2 = 0.999) than the dependence of peak current on scan rate (R 2 = 0.982) indicated that the oxidation of G at poly(cytosine)/GCE was predominantly diffusion controlled. The oxidative peak response of the electrode revealed a high linear range of G concentration (0.1-200 μM) under optimized conditions. The detection limit and limit of quantification were 6.10 and 20.13 nM, respectively, associated with the %RSD of under 1%. The validation of the developed electrochemical sensor for the determination of G was investigated by analyzing human urine DNA and serum samples with spike recovery results in the range of 98.20-103.70% with the interferent recovery percentage in the range of 97.86-103.10% containing 50-300% of potential interferents. The newly designed sensor demonstrated the highest level of performance for the G detection in real samples.

From Detection to Cure - Emerging Roles for Urinary Tumor DNA (utDNA) in Bladder Cancer.

This review sought to define the emerging roles of urinary tumor DNA (utDNA) for diagnosis, monitoring, and treatment of bladder cancer. Building from early landmark studies the focus is on recent studies, highlighting how utDNA could aid personalized care. Recent research underscores the potential for utDNA to be the premiere biomarker in bladder cancer due to the constant interface between urine and tumor. Many studies find utDNA to be more informative than other biomarkers in bladder cancer, especially in early stages of disease. Points of emphasis include superior sensitivity over traditional urine cytology, broad genomic and epigenetic insights, and the potential for non-invasive, real-time analysis of tumor biology. utDNA shows promise for improving all phases of bladder cancer care, paving the way for personalized treatment strategies. Building from current research, future comprehensive clinical trials will validate utDNA's clinical utility, potentially revolutionizing bladder cancer management.

Tumor fraction and copy number burden from urinary cell-free tumor DNA (utDNA) to predict minimal residual disease prior to repeat-transurethral resection in high-risk non-muscle invasive bladder cancer (HR-NMIBC).

Tumor fraction and copy number burden from urinary cell-free tumor DNA (utDNA) to predict minimal residual disease prior to repeat-transurethral resection in high-risk non-muscle invasive bladder cancer (HR-NMIBC).

Non-invasive detection of urothelial carcinoma through ensembled methylation profiles and fragmentomic patterns in urinary cell-free DNA.

Non-invasive detection of urothelial carcinoma through ensembled methylation profiles and fragmentomic patterns in urinary cell-free DNA.

Predicting exacerbation of renal function by DNA methylation clock and DNA damage of urinary shedding cells: a pilot study

Recent reports have shown the feasibility of measuring biological age from DNA methylation levels in blood cells from specific regions identified by machine learning, collectively known as the epigenetic clock or DNA methylation clock. While extensive research has explored the association of the DNA methylation clock with cardiovascular diseases, cancer, and Alzheimer's disease, its relationship with kidney diseases remains largely unexplored. In particular, it is unclear whether the DNA methylation clock could serve as a predictor of worsening kidney function. In this pilot study involving 20 subjects, we investigated the association between the DNA methylation clock and subsequent deterioration of renal function. Additionally, we noninvasively evaluated DNA damage in urinary shedding cells using a previously reported method to examine the correlation with the DNA methylation clock and worsening kidney function. Our findings revealed that patients with an accelerated DNA methylation clock exhibited increased DNA damage in urinary shedding cells, along with a higher rate of eGFR decline. Moreover, in cases of advanced CKD (G4-5), the DNA damage in urinary shedding cells was significantly increased, highlighting the interplay between elevated DNA damage and eGFR decline. This study suggests the potential role of the DNA methylation clock and urinary DNA damage as predictive markers for the progression of chronic kidney disease.

Abstract IA006: Towards ctDNA-guided treatment of muscle-invasive bladder cancer

Abstract Cancer cells release cell-free DNA with tumor-specific molecular alterations into circulation, known as circulating tumor DNA (ctDNA). Numerous studies have underscored the biomarker potential of ctDNA in detecting minimal residual disease across various cancer types, including bladder cancer. The rapid clearance of ctDNA from circulation, typically under two hours, makes it an ideal candidate for real-time monitoring of tumor burden following surgical interventions and throughout oncological treatments. For ctDNA measurements to be effectively incorporated into clinical decision-making, it is crucial to conduct ctDNA-guided intervention trials that demonstrate tangible clinical benefits. Current trials focusing on muscle-invasive bladder cancer (MIBC) are supported by recent exploratory sub-analyses of completed clinical trials, which have shown that ctDNA can effectively guide treatment decisions in this context. Beyond guiding adjuvant treatments, ctDNA holds potential for directing bladder preservation strategies, potentially in combination with urinary tumor-derived DNA (utDNA) measurements. The need for treatment de-escalation, such as in neoadjuvant chemotherapy (NAC), is underscored by the observation that a significant proportion of patients undergo treatments that result in considerable adverse effects without achieving pathologic response. Traditional decision-making in cancer treatment, largely reliant on imaging-based criteria like the Response Evaluation Criteria in Solid Tumors (RECIST), often does not adequately reflect the ultimate clinical endpoint of overall survival. A ctDNA-based measure that considers ctDNA dynamics during treatment could serve as a superior tool or complement to current strategies. In conclusion, ctDNA has demonstrated significant promise as a biomarker in both retrospective and prospective studies for risk assessment in MIBC. The clinical value of ctDNA-guided treatment will soon be determined by ongoing clinical trials (TOMBOLA, IMvigor011, MODERN). In my presentation I will address the potential clinical impact of ctDNA analyses, the technological aspects involved, how urine analyses may supplement these findings, and future directions in the field. Citation Format: Lars Dyrskjøt. Towards ctDNA-guided treatment of muscle-invasive bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr IA006.

Abstract A001: Urine-based liquid biopsy test for the detection of residual bladder cancer in radical cystectomy patients

Abstract One commonly employed management strategy for muscle-invasive bladder cancer (MIBC) is neoadjuvant chemotherapy followed by radical cystectomy (RC) and urinary diversion. Approximately 35% of patients achieve a pathologic complete response (pCR) when evaluated from RC specimens. RC with urinary diversion is a life-altering operation with high complication rates and therefore determining who are complete responders would improve quality of life and could decrease costs and still achieve a high cure rate via RC avoidance. Unfortunately, current methods cannot accurately identify complete responders. Our lab has developed a urine-based liquid biopsy test in which DNA isolated from urine and germline is isolated and subjected to panel exome sequencing for enumeration of somatic variants. This occurs under the premise that tumor DNA identified (as mutant alleles) is a marker of residual disease while the absence of tumor DNA (meaning the absence of mutant alleles) can indicate a pCR. A 60 gene panel was designed to be used for exonic sequencing of urinary DNA (uDNA) to identify mutations indicative of bladder cancer or therapeutic targets. We have shown that most mutations in tumor tissue (Mt) are detectable as mutations in urine (Mu). We also showed that the presence or absence of residual Mu after completion of chemotherapy strongly associates with residual disease or pCR at the time of RC, respectively. Therefore, this test could be used after neoadjuvant therapy to better identify patients for RC avoidance. We optimized this test by studying pre-analytical factors affecting the yield, purity, and integrity of uDNA and the quality of sequencing data using prospectively collected samples from patients with MIBC. We are also in the process of testing commercial and novel preservatives to optimize DNA integrity, yield, and mutation detection accuracy to enable temporary ambient temperature storage without compromising mutation detection, permitting shipping/centralized testing. Lastly, we are comparing two library preparation approaches. Study of these preanalytical factors has increased the number of Mu that can be detected and were applied to a cohort of patients who underwent bladder sparing curative-intent treatment in a prospective clinical trial. Studies described herein will enable liquid biopsy tests to accurately identify patients with pCR after neoadjuvant therapy and might safely avoid radical cystectomy. Citation Format: Philip H. Abbosh, Henkel Valentine, Rhea Arya. Urine-based liquid biopsy test for the detection of residual bladder cancer in radical cystectomy patients [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A001.

Investigating the discriminatory potential of urinary DNA adductomics in smokeless tobacco-treated rats and head-neck cancer patients

The genotoxicity of smokeless tobacco (ST) remains an essential public health issue. We conducted a comprehensive study of rats exposed to ST and head and neck cancer patients to elucidate the DNA adduct profiles. ST exposure assessment used alkaloids and tobacco-specific nitrosamines in rat urine. High ST doses led to significantly elevated nicotine, cotinine, N-nitrosonornicotine (NNN), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) levels, indicating substantial exposure. DNA adductomics identified 17 significant DNA adducts using a neutral loss scan in liquid chromatography-mass spectrometry in rat urine samples upon ST exposure, highlighting the intricate landscape of DNA modifications associated with ST using univariate and multivariate statistical analyses. LC-ESI-QTOF-MS/MS analysis and accurate mass measurements of DNA adducts unveiled specific adducts upregulated in response to ST exposure, particularly those associated with methylation and NNK release. Intriguing correlations emerged between DNA adducts and alkaloids-TSNA in ST-exposed rat urine. Nicotine correlated positively with N2-paraldol-dG and O2/O4-[4-(3-Pyridyl)-4-oxobut-1-yl] thymidine in high-dose rats. NNK correlated with 6O-methylgaunine, while NNN correlated with 6-hydroxy-1-N2-propanodeoxyguanosine and 7-methylguanine. With this aim, we also applied analysis to human samples, where we observed specific DNA adducts for ST users, controls, and subjects with HNC. Two-way ANOVA showed that ST users and cancer patients had higher DNA adduct levels in human urine, which aids cancer risk assessment, early detection, and environmental exposure evaluation. This comprehensive study explores DNA adducts caused by ST exposure across rats and head and neck cancer patients, illuminating genotoxic mechanisms and health risks and providing valuable insights for future preventive and therapeutic strategies.

EarlyTect BCD, a Streamlined PENK Methylation Test in Urine DNA, Effectively Detects Bladder Cancer in Patients with Hematuria

The current noninvasive diagnostic approaches for detecting bladder cancer (BC) often exhibit limited clinical performance, especially for the initial diagnosis. This study aims to evaluate the validity of a streamlined urine-based PENK methylation test called EarlyTect BCD in detecting BC in patients with hematuria scheduled for cystoscopy in Korean and American populations. The test seamlessly integrates two steps, linear target enrichment and quantitative methylation-specific PCR within a single closed tube. The detection limitation of the test was approximately two genome copies of methylated PENK per milliliter of urine. In the retrospective training set (n=105), an optimal cutoff value was determined to distinguish BC from non-BC, resulting in a sensitivity of 87.3% and a specificity of 95.2%. In the prospective validation set (n=210, 122 Korean and 88 American patients), the overall sensitivity for detecting all stages of BC was 81.0%, with a specificity of 91.5% and an area under the curve value of 0.889. There was no significant difference between the two groups. The test achieved a sensitivity of 100% in detecting high-grade Ta and higher stages of BC. The negative predictive value of the test was 97.7%, and the positive predictive value was 51.5%. The findings of this study demonstrate that EarlyTect BCD is a highly effective noninvasive diagnostic tool for identifying BC among patients with hematuria.

Abstract LB105: Sensitive urothelial cancer detection via high volume urine DNA analysis

Abstract Background: The detection of cancer-associated DNA in urine samples has significant potential in urothelial cancer (UC) detection and surveillance. Every year millions of patients with urinary symptoms undergo cystoscopy, an examination of the bladder using a flexible scope. Only 10% of these invasive examinations result in a cancer diagnosis. Cystoscopy is also used in post-operative surveillance, with many UC patients requiring life-long cystoscopies. Methods: We developed a high-volume urine DNA collection kit and laboratory platform and prospectively analyzed 433 urine samples collected from diagnostic and post-operative timepoints from 167 UC patients and 95 negative controls from January 2021 to September 2023. We used a hybridization capture assay targeting the coding regions of 21 commonly mutated genes in urothelial tumors, as well as 8 frequently copy number altered loci. Samples were sequenced using Illumina NovaSeq instruments. Mutations, copy number alterations, and chromosomal rearrangements were computationally identified. Key Findings: In the diagnostic settings our assay achieved a sensitivity of 100% for pT1+ bladder cancer, 97% for pTa bladder cancer, and 91% for upper tract urothelial cancer (UTUC) (100% for pT1+ or high grade) in an all-comers UC cohort, at a specificity of 93%. Post-operative urine DNA surveillance informed by preoperative urine mutations detected 28/30 (93%) residual cancer cases found in cystoscopy or surgery, with two pTa low grade recurrences missed. Using a secondary, highly stringent urine tumor DNA detection threshold (100% specificity for UC), we show that urine DNA testing in the diagnostic setting may allow up to 68% of UC patients to be scheduled directly to operating room procedures, reducing hospital workload and expediting care. In addition, we demonstrate the feasibility of urine DNA for detecting underlying Lynch syndrome in UC patients by identifying three UTUC patients who carried germline defects in mismatch repair genes MSH2 or MSH6, indicative of Lynch syndrome. In two of these patients our assay also found a somatic second hit and high mutation load. Conclusions and Clinical Implications: These results show promise in urine DNA based cancer diagnostics even in patients with low grade tumors. The assay is designed to offer a non-invasive and cost-effective alternative for cystoscopies. Citation Format: Jussi Nikkola, Lauri Ryyppö, Juuso Vuorinen, Heini Kallio, Hanna Selin, Pyry Jämsä, Jonne Åkerla, Tarmo Pekkarinen, Antti Kaipia, Matti Nykter, Thea Veitonmäki, Matti Annala. Sensitive urothelial cancer detection via high volume urine DNA analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB105.

Urinary Mitochondrial DNA Induces an Inflammatory Response in Peripheral Blood Mononuclear Cells

Background: Mitochondria are semiautonomous cell organelles having its own nucleic acid. Mitochondrial DNA (Mt-DNA) remain in hypomethylated (CpG) state and impose an immunogenic response by binding to the toll-like receptor (TLR-9) through the NF-kB pathway. Innate immune cells recognize the hypomethylated pattern of mt-DNA and quickly trigger the innate immune response. The immunomodulatory effects of urinary mt-DNA derived from renal transplant recipients with COVID-19-associated acute kidney injury (AKI) have not been studied. Materials and Methods: Healthy donor peripheral blood mononuclear cell (PBMC) was cultured with the urinary Mt-DNA derived from the renal transplant recipients, who previously developed SARS-CoV-2 infection associated AKI. Cell activation was measured by the flow cytometry. In cell pellets, interleukin IL-6, IL-10, and Myd88, TLR-9 mRNA transcript expression was measured by the reverse transcription polymerase chain reaction. The IL-6 and IL-10 cytokine levels were measured by the enzyme-linked immunosorbent assay in culture supernatants. Results: The urinary mitochondrial DNA (umt-DNA) significantly induces the activation of &gt; 75% of PBMCs. The m-RNA transcript expression of the inflammatory gene in control versus umt-DNA treated PBMCs was for IL-6 (0.99 ± 0.05 vs. 2.18 ± 1.15 au; P = 0.004), MYD88 was (1.00 ± 0.05 vs. 1.55 ± 0.31; P &lt; 0.001), TLR-9 (1.00 ± 0.05 vs. 3.33 ± 1.37 au; P &lt; 0.001) was upregulated, and the IL-10 (1.00 ± 0.13 vs. −1.73 ± 0.58; P &lt; 0.001) level was downregulated. However, in PBMC culture supernatants, IL-6 level in control versus umt-DNA-treated groups were (37.50 ± 13.79 vs. 186.9 ± 15.50 pg/mL; P &lt; 0.001), which was significantly higher in umt-DNA-treated groups and the IL-10 (8.80 ± 2.16 vs. 7.60 ± 3.12 pg/mL; P = 0.32) level was similar between the control- and umt-DNA-treated groups. Conclusions: Urinary Mt-DNA significantly induces the inflammatory cytokine IL-6 secretion from the PBMCs through the Myd88-dependent pathway.