Urinary DNA Research Articles

The Challenge to Stabilize, Extract and Analyze Urinary Cell-Free DNA (ucfDNA) during Clinical Routine.

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. 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. 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. From a technical point of view, stabilizing buffers and adequate storage conditions are a prerequisite for translation of urinary cfDNA diagnostics into clinical routine.

Oxidative DNA damage estimated by urinary 8-Hydroxy-2’ –Deoxyguanosine (8-OHdG) and 8-Oxoguanine DNA Glycosylase (OGG1) in cigarette and non-cigarette smokers in South West Nigeria

BACKGROUND: The thousands of chemicals and compounds found in cigarette smoke, including many free radicals and oxidants, can cause oxidative damage to DNA either directly or by producing reactive oxygen species in cultured cells. This study was carried out to analyze the urinary 8-Oxoguanine DNA Glycosylase (OGG1) and 8-Hydroxy-2’ –Deoxyguanosine (8-OHdG) among cigarette and non-cigarette smokers in South-West, Nigeria. METHODS: Urine samples were collected from 250 cigarette smokers and 200 non-cigarette smokers who live in south-west, Nigeria. Questionnaire was administered first to both cigarette and non-cigarette smokers prior to specimen collection. About 10mls of urine samples were collected from each of the subjects and was used to carry out Oxoguanine Glycosylase and 8-Hydroxyl-2-deoxyguanosine using spectrophotometer at 450 nm wavelength. RESULTS: There was a significant (P < 0.05) increase in Urinary 8-OHdG (345.84±90.98 vs 223±68.48) and OGG1 (220.71±59.55 vs 97.20±56.14) of the cigarette smokers when compared with that of non-cigarette smokers. CONCLUSION: On the basis of this study, it has been observed and established that indulging in cigarette smoking can result in oxidative stress and DNA damage.

1621. "False-Positive" Cytomegalovirus (CMV) Screening Tests in Neonates: A Real Dilemma!

Abstract Background PCR testing of clinical specimens has been associated with false-positive test results. Detection of CMV DNA by PCR in saliva and urine has been used to identify neonates with congenital CMV infection, but the frequency of false-positive PCR results is not fully known. Our objective is to describe the frequency of "false-positive" saliva and urine CMV PCR tests when performed as screening for congenital CMV infection. Methods From 2018 to 2023, neonates were screened for CMV infection if they had abnormal clinical, laboratory, or radiographic findings, referred on the newborn hearing screen, and since 2019, on admission to the 9 neonatal intensive care units (NICU) of the Nationwide Children’s Hospital neonatal network, Columbus, OH. Pertinent demographic, clinical, audiologic, laboratory, and radiographic data were obtained and managed using REDCap. Infants who had an initial positive PCR screening test for CMV but subsequent negative PCR tests were identified. Results During the 6 year study period, 139 infants were diagnosed with congenital CMV infection. An additional 28 (17%) neonates tested positive for CMV DNA in saliva (n=26) or urine (n=2) but subsequent tests of urine specimens were negative for CMV. Most of the infants were tested as part of universal NICU screening (n=22), 5 had symptomatic disease, 3 referred on the newborn hearing screen, and 1 had thrombocytopenia. The infants were tested at a median (IQR) age of 3 (2-6) days. Their median (IQR) birth weight and gestational age were 2458 (1865-3360) grams and 37 (33-39) weeks, respectively. Four infants had their dried blood spot tested for CMV by PCR and were negative. 17 (77%) of 22 infants had known maternal milk exposure in the neonatal period. Conclusion Neonatal saliva and/or urine screening for congenital CMV infection was associated with a “false-positive” test result in 17% of neonates who tested positive for CMV DNA by PCR. These results suggest that confirmation of a diagnosis of congenital CMV infection by PCR requires testing of multiple specimens with one of them being urine. Disclosures Asuncion Mejias, MD, PhD, MsCS, Astra-Zeneca: Advisor/Consultant|Merck: Grant/Research Support|Pfizer: Advisor/Consultant|Sanofi-Pasteur: Advisor/Consultant Alexandra Katherine Medoro, MD, Merck: Grant/Research Support Amy Leber, PhD, Biomeriux: Grant/Research Support|BioRad: Advisor/Consultant|Cephied: Grant/Research Support|Diasorin: Grant/Research Support

Role of Polyomavirus BK and Adenovirus in the Development of Urinary Tract Complications in Pediatric Hematopoietic Stem Cell Transplantation

One of the major challenges in pediatric hematopoietic stem cell transplantation (HSCT) is the susceptibility to viral infections and reactivations that can lead to potentially life-threatening complications and increase the transplant-related mortality (TRM). Polyomavirus BK (BKPyV) and Adenovirus (AdV) represent the predominant causes of urinary tract complications, in particular hemorrhagic cystitis (HC), in HSCT recipients. Treatment options for BKPyV and AdV infections are limited and there is no accepted standard of care for the prevention and treatment of these HSCT complications. The aim of our retrospective study was to analyze the incidence of BKPyV and AdV infection in urinary and plasma samples of pediatric patients undergoing HSCT for both malignant and non-malignant disease at a single pediatric Hematology-Oncology unit. Endpoints were the cumulative incidence of infection, measured on blood and urine samples, in the first 100 days after HSCT, prevalence of asymptomatic and symptomatic infection, and correlation between BKPyV and AdV DNAemia and DNAuria and the development of HC. A total of 79 patients were analyzed. Thirty-seven (47%) patients were positive for BKPyV on urine samples and 20 (30%) on blood samples within the first 30 days after transplantation. As for AdV, 11 (9%) patients were positive on urine samples and 18 (15%) on blood samples. In our pediatric cohort, BKPyV DNAuria was a frequent event after HSCT, while BKPyV DNAemia was observed less frequently, and was accompanied by higher viruria values (p-value <0,001, with a median viral load in the urine of 3.8 x 10 9 copies/mL). Conversely, AdV positivity in urine is rare, while AdV DNAemia was more frequent, due to the modality of BKPyV and AdV distribution (urothelium is the main reservoir for BKPyV, while intestinal lymphocytes represents the main reservoir for AdV). Of the 37 patients with BKPyV DNA on urine samples, 11 (30%) developed a symptomatic infection with grade II (5 patients, 40%) and grade III (6 patients, 60%) HC. We did not observe any correlation between BKPyV DNA urinary levels and the development of HC, as higher viral loads have not been shown to be good predictors of occurrence or severity of HC. Cumulative incidence (CI) of BKPyV infection was higher in haploidentical HSCT (51%) and matched unrelated donors (MUD) (48%) than in HSCT from matched familiar donors (31%). Similarly, CI of AdV reactivation was higher in haploidentical HSCT (16%) than MUD (6%) and matched familiar donors (9%). Choice of donor, the type of immunosuppression used and the intensity of conditioning regimen represented predictors of both blood and urine DNA positivity for both viruses. In our cohort, positivity for BKPyV on blood and/or urine did not increase the risk of transplant-related mortality and did not affect the overall survival after HSCT. On the basis of our data, we now perform screening of BKPyV viruria only in symptomatic patients or in transplant recipients with specific risk factors (type of donor, severity of immunosuppression). Similarly, search for BKPyV DNAemia is reserved to subjects with a high viral load on urine. Likewise, search for AdV on urine samples is performed on symptomatic patients with high-load AdV DNAemia.

Performance evaluation of fully automated cobas® 6800 CMV PCR for the detection and quantification of cytomegalovirus DNA in neonatal urine and saliva, and adult urine, saliva, and vaginal secretion.

Laboratory testing for cytomegalovirus (CMV) in bodily fluids is essential to manage congenital and prenatal CMV infection. The rapid and fully automated cobas® CMV PCR is approved only for the testing of plasma in transplant patients. To evaluate the performance of the cobas® CMV to detect and quantify CMV DNA in neonatal and adult female urine, saliva, and vaginal secretion, the limit of detection (LoD), limit of quantification (LoQ), imprecision, linearity, PCR efficiency, bias, analytical specificity, cross-reactivity, and cross-contamination of the cobas® CMV for urine, saliva, and vaginal secretion was determined. The performance of the assay was evaluated prospectively with two laboratory-developed PCR assays using neonatal and adult urine, saliva swabs, and vaginal swabs. The LoD and LoQ were 31 and 100 IU/mL, respectively, for urine, and 81 and 100 IU/mL, respectively, for vaginal secretion. The LoD and LoQ for saliva were the same (200 IU/mL). The cobas® CMV was precise (coefficient of variation ≤10%), linear (R2 ≥ 0.995), and efficient (1.07 and 1.09) between 100 and 250,000 IU/mL for the sample types. The bias and analytical specificity was <±0.30 log10 IU/mL and 100%, respectively. Cross-reactivity with non-CMV pathogens was not detected. Cross-contamination rate was 0.28%. The diagnostic accuracy, sensitivity, and specificity of the cobas® CMV for neonatal urine and saliva were ≥95.0%, ≥93.3%, and ≥90.4%, respectively. The overall percent agreement for adult urine, saliva, and vaginal secretion was 86.6%, 94.5%, and 89.4%, respectively. Taken together, the cobas® CMV demonstrated acceptable analytical and diagnostic performance, and is suitable for routine diagnostic laboratory investigation of CMV infection in neonates and adults.

Urinary mitochondrial DNA may be useful in diagnosing early diabetic nephropathy.

The present study aimed to determine whether urinary mitochondrial (mt)DNA could be combined as a non-invasive biomarker with other clinical findings of kidney injury to help diagnose early diabetic nephropathy (DN). A total of 165 patients with type 2 diabetes mellitus (T2DM) were enrolled in the present study and the mtDNA levels in urine were measured using quantitative PCR. The diagnostic value of urinary mtDNA levels in patients with T2DM was compared using estimated glomerular filtration rate (eGFR) or albumin-to-creatinine ratio staging. Spearman correlation analysis was used to analyze the correlation between urinary mtDNA and other clinical findings. Correlation factors for early DN were assessed using univariate logistic regression analysis. Urinary leukocyte and glucose levels do not interfere with urinary mtDNA levels. In patients with T2DM, the level of urinary mtDNA increases in the early stages of kidney injury and further increases with the severity of kidney injury. Urinary mtDNA levels in patients with eGFR 60-90 ml/min/1.73 m2 were higher than that in patients with eGFR >90 ml/min/1.73 m2. The levels of urinary mt89DNA and mt349DNA were negatively correlated with the eGFR level (ρ=-0.437; P<0.001; ρ=-0.390; P<0.001) and positively correlated with the level of cystatin C (ρ=0.177; P=0.025; ρ=0.144; P=0.070). Urinary mtDNA is positively correlated with early DN occurrence [odds ratio (OR), 1.330; 95% confidence interval (CI), 1.175-1.507; P<0.001; OR, 1.328; 95% CI, 1.156-1.525; P<0.001]. In conclusion, urinary mtDNA combined with other clinical indicators of kidney injury may help the diagnosis of early DN.

GeneXpert Ultra in Urine Samples for Diagnosis of Extra-Pulmonary Tuberculosis.

Extra-pulmonary tuberculosis (EPTB) continues to be difficult to diagnose. Novel biomarkers in biological specimens offer promise. Detection of Mycobacterium tuberculosis (Mtb) DNA in urine could prove useful in diagnosis of EPTB, possibly due to disseminated disease or micro-abscesses reported in kidneys. The current study was designed to detect Mtb DNA in stored urine samples from patients with EPTB. Diagnosis of EPTB was reached using Microbiological Reference Standards (MRS) on samples from the disease site using WHO Recommended Diagnostics (WRD), [smear microscopy, liquid culture (MGIT-960)] and GX (molecular WRD, mWRD) and Comprehensive reference standards [CRS, clinical presentation, microbiological reference standards, radiology, histopathology]. GX-Ultra was performed on urine samples stored in -80oC deep freezer, retrospectively. Of 70 patients, 51 (72.9%) were classified as confirmed TB, 11 (15.7%) unconfirmed TB, and 8 (11.4%) unlikely TB. GX-Ultra in urine samples demonstrated sensitivity of 52.9% and specificity of 57.9% against MRS, and higher sensitivity of 56.5% and specificity of 100% against CRS. The sensitivity and specificity of GX-Ultra in urine was 53.6% and 75% for pus sample subset and 52.2% and 53.3% for fluid sample subset. Urine being non-invasive and easy to collect, detection of Mtb DNA using mWRD in urine samples is promising for diagnosis of EPTB.

High urinary oxidative DNA damage in wild chimpanzees ranging in proximity of agricultural fields in Sebitoli area, Uganda.

Agriculture expansion is a major cause of habitat loss and exposure to phytochemical pollution for non-human primates. In addition to endocrine disruption, exposure to pesticides may have other sublethal physiological consequences for animals, such as generation of oxidative damage to macromolecules. In this study, we analyzed the pesticides contained in the river water across the home range of wild chimpanzees (Pan troglodytes) in Sebitoli area located on the Northern part of Kibale National Park (Uganda). We tested whether levels of three urinary markers of oxidative damage vary among individuals in relation to their ranging patterns, as a proxy for pesticide exposure intensity. To better characterize the foraging habitat use, the trophic level, and the energetic status of study individuals, we also quantified urinary levels of carbon and nitrogen stable isotope signatures and of C-peptide. Among the 511 pesticides screened, 18 compounds including herbicides, insecticides, and fungicides were found in the water sampled in the Western part of the home range of chimpanzees. In this area, chimpanzees used to feed on maize crops. By contrast, in the Eastern part where crop feeding was never observed, we found only seven pesticides. According to their ranging patterns and thus crop feeding frequency, the 139 urine samples collected from 43 Sebitoli chimpanzees were categorized as belonging to low, medium, and high exposure level. Chimpanzees from the high exposure zone had higher oxidative DNA damage (8-OHdG) than chimpanzees from both the low and medium exposure groups, who had similar levels of oxidative DNA damage. In addition, individuals with higher C-peptide tended to have significantly higher oxidative DNA damage and lipid peroxides. The three exposure groups had similar levels of urinary 8-isoprostanes and of urinary lipid peroxides. These results were robust for any potential confounding effect of other variables because neither age category nor sex or isotope levels were significantly associated with markers of oxidative damage. Our study points to genotoxic effects as one potential sublethal consequence of ranging in proximity of agricultural fields owing to exposure to pesticides or other unidentified sources of stress. Given our phylogenetic proximity, this information is relevant for the conservation of this species which is endangered and also sentinel for human health.

2401P Detection of molecular recurrence in early-stage bladder cancer patients using a urinary tumor DNA assay after transurethral resection of bladder tumor (TURBT)

2401P Detection of molecular recurrence in early-stage bladder cancer patients using a urinary tumor DNA assay after transurethral resection of bladder tumor (TURBT)

2404P Concordance of genomic alterations by next-generation sequencing in tumor tissue versus urinary tumor DNA and circulating tumor DNA in bladder cancer

2404P Concordance of genomic alterations by next-generation sequencing in tumor tissue versus urinary tumor DNA and circulating tumor DNA in bladder cancer

Portable and visual quantification of urine cell-free DNA through smartphone-based colorimetric biosensor

The trace amount of cell-free DNA (cfDNA) in urine provides a promising diagnosis of urological disease, but its clinical applications are restricted by complicated operations for enrichment and detection. Herein, a portable and visual smartphone-based colorimetric platform is created for urine cfDNA quantification in quick, low-cost, and instrument-free manners. cfDNA in urine is enriched by the zinc ions-dipicolylamino complexes on glass slides and labeled by PicoGreen (PG) to form the DNA-PG complexes with photocatalysis for the generation of a visible output signal. The high sensitivity is related to the amplification of the optical signal of enriched cfDNA through the photocatalysis strategy, which yields greater color changes without interference from other ingredients. Notably, accurate quantitation of cfDNA is achieved with the aid of the smartphone App equipped with collecting and processing Color Grab. As a powerful alternative, such a robust colorimetric biosensor platform is employed for urine cfDNA quantification of clinical samples with excellent linearity compared with the two-stage commercial kits (R2 > 0.95), helping to distinguish the difference between healthy and urological disease. The comprehensive performance of our developed platform will be the first to enable visual quantification of urine cfDNA in terms of quick detection, low cost, and high portability.

Detection of Cancer-Associated Gene Mutations in Urinary Cell-Free DNA among Prostate Cancer Patients in South Africa.

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.

Reconsideration of Maternal Serological Testing for Predicting Congenital CMV Infection.

The value of the widely applied maternal cytomegalovirus (CMV) serological testing approach in predicting intrauterine transmission in highly seroprevalent regions remains unknown. A nested case-control study was conducted based on a maternal-child cohort study. Newborns with congenital CMV (cCMV) infection were included, and each of them was matched to 3 newborns without cCMV infection. Retrospective samples were tested for immunoglobulin G (IgG) avidity and immunoglobulin M (IgM) antibodies in maternal serum and CMV DNA in maternal blood and urine to analyze their associations with cCMV infection. Forty-eight newborns with cCMV infection and 144 matched newborns without infection were included in the study. Maternal IgM antibodies and IgG avidity during pregnancy were not statistically associated with intrauterine transmission. The presence of CMV DNAemia indicated a higher risk of cCMV infection, with odds ratio values of 5.7, 6.5, and 13.0 in early, middle, and late pregnancy, respectively. However, the difference in CMV shedding rates in transmitters and nontransmitters was not significant in urine. The value of current maternal CMV serological testing in regions with high seropositivity rates is very limited and should be reconsidered. The detection of DNAemia would be helpful in assessing the risk of intrauterine transmission.

Influence of PAHs and Genetic Polymorphisms on Urinary PAHs Metabolite and DNA Damage among Malaysian Children in Urban Traffic Area

Influence of PAHs and Genetic Polymorphisms on Urinary PAHs Metabolite and DNA Damage among Malaysian Children in Urban Traffic Area

Field Cancerization Is Associated with Tumor Development, T-cell Exhaustion, and Clinical Outcomes in Bladder Cancer

BackgroundField cancerization is characterized by areas of normal tissue affected by mutated clones. Bladder field cancerization may explain the development and recurrence of bladder cancer and may be associated with treatment outcomes. ObjectiveTo investigate the predictive and prognostic roles of field cancerization in patients with high-risk non–muscle-invasive bladder cancer (NMIBC) treated with bacillus Calmette-Guérin (BCG). Design, setting, and participantsWe conducted comprehensive genomic and proteomic analyses for 751 bladder biopsies and 234 urine samples from 136 patients with NMIBC. The samples were collected at multiple time points during the disease course. Field cancerization in normal-appearing bladder biopsies was measured using deep-targeted sequencing and error correction models. Outcome measurements and statistical analysisEndpoints included the rates of recurrence and progression. Cox regression and Wilcoxon rank-sum and Fisher’s exact tests were used. Results and limitationsA high level of field cancerization was associated with high tumor mutational burden (p = 0.007), high tumor neoantigen load (p = 0.029), and high tumor-associated CD8 T-cell exhaustion (p = 0.017). In addition, high field cancerization was associated with worse short-term outcomes (p = 0.029). Nonsynonymous mutations in bladder cancer–associated genes such as KDM6A, ARID1A, and TP53 were identified as early disease drivers already found in normal-appearing bladder biopsies. Urinary tumor DNA (utDNA) levels reflected the bladder tumor burden and originated from tumors and field cancerization. High levels of utDNA after BCG were associated with worse clinical outcomes (p = 0.027) and with disease progression (p = 0.003). High field cancerization resulted in high urinary levels of proteins associated with angiogenesis and proliferation. Limitations include variation in the number of biopsies and time points analyzed. ConclusionsField cancerization levels are associated with tumor development, immune responses, and clinical outcomes. utDNA measurements can be used to monitor disease status and treatment response. Patient summaryMolecular changes in the tissue lining the bladder result in tumor recurrence. Urinary measurements may be used to monitor bladder cancer status and treatment responses.

The Diagnostic, Prognostic, and Therapeutic Potential of Cell-Free DNA with a Special Focus on COVID-19 and Other Viral Infections.

Cell-free DNA (cfDNA) in human blood serum, urine, and other body fluids recently became a commonly used diagnostic marker associated with various pathologies. This is because cfDNA enables a much higher sensitivity than standard biochemical parameters. The presence of and/or increased level of cfDNA has been reported for various diseases, including viral infections, including COVID-19. Here, we review cfDNA in general, how it has been identified, where it can derive from, its molecular features, and mechanisms of release and clearance. General suitability of cfDNA for diagnostic questions, possible shortcomings and future directions are discussed, with a special focus on coronavirus infection.

Detection of equine herpesvirus-1 (EHV-1) in urine samples during outbreaks of equine herpesvirus myeloencephalopathy.

Real-time PCR is the diagnostic technique of choice for the diagnosis and control of equine herpesvirus-1 (EHV-1) in an outbreak setting. The presence of EHV-1 in nasal swabs (NS), whole blood, brain and spinal cord samples has been extensively described; however, there are no reports on the excretion of EHV-1 in urine, its DNA detection patterns, and the role of urine in viral spread during an outbreak. To determine the presence of EHV-1 DNA in urine during natural infection and to compare the DNA detection patterns of EHV-1 in urine, buffy coat (BC) and NS. Descriptive study of natural infection. Urine and whole blood/NS samples were collected at different time points during the hospitalisation of 21 horses involved in two EHV-1 myeloencephalopathy outbreaks in 2021 and 2023 in Spain. Quantitative real-time PCR was performed to compare the viral DNA load between BC-urine samples in 2021 and NS-urine samples in 2023. Sex, age, breed, presence of neurological signs, EHV-1 vaccination status and treatment data were recorded for all horses. A total of 18 hospitalised horses during the 2021 and 2023 outbreaks were positive for EHV-1, and viral DNA was detected in urine samples from a total of 11 horses in both outbreaks. Compared with BC samples, DNA presence was detected in urine samples for longer duration and with slightly higher concentration; however, compared with NS, detection of EHV-1 in urine was similar in duration with lower DNA concentrations. Limited sample size, different sampling times and protocols (BC vs. NS) in two natural infection outbreak settings. EHV-1 was detected in the urine from naturally infected horses. Urine should be considered as complimentary to blood and NS in diagnosis of EHV-1 infection.

Nuclear and Urinary Measurements Show the Efficacy of Sun-Protection Factor 50+ Sunscreen against DNA Photoproducts upon Real-Life Recreational Exposure

Sunscreens have been shown to protect against UVR-induced DNA damage in human skin under laboratory conditions. We presently extended these observations to real-life conditions in volunteers after their ordinary exposure habits during summer holidays. Volunteers were randomly assigned to a control group and an educated group supplied with a SPF ≥50 sunscreen and receiving instructions for use. A questionnaire was used to determine the extent of exposure. No difference in average solar UVR exposure was found between the two groups. DNA photoprotection was first assessed by, to our knowledge, a previously unreported noninvasive assay on the basis of the quantification of pyrimidine dimers released by DNA repair in urine. Damage was also quantified in the nuclear DNA extracted from the roof of suction blisters collected after recreational exposure. The urinary concentration of photoproducts was significantly higher in the control than in the educated group. The same trend was observed for the level of photoproducts in the DNA from suction blisters. The unambiguous observation of an efficient photoprotection against DNA damage afforded by sunscreen under real-life conditions provides strong support for the efficiency of the sunscreens. In addition, the results validate the use of urinary DNA photoproducts as a noninvasive assay applicable to photoprotection.

Combination of DNA methylation biomarkers with multiparametric magnetic resonance and ultrasound imaging fusion biopsy to detect the local spread of prostate cancer.

This study aimed to investigate the extent of field cancerization adjacent to index lesions in prostate cancer (PCa) by measuring DNA methylation of selected tumor suppressor genes in the perifocal tissue of PCa not visible on multiparametric magnetic resonanse imaging (mpMRI) for the safe zone of focal therapy identification. A total of 272 patients were enrolled in this study, 44 patients' tissue biosamples were included in the field cancerization research, and 272 urine samples were included in the urine-based test development. Targeted biopsies were performed using the mpMRI/ultrasoundimage fusion system. Quantitative analysis revealed significantly higher DNA methylation levels of RARB, RASSF1, GSTP1 &APC genes in the index lesion compared with perifocal tissue samples 10 mm away from it (p < 0.0001). Notably, the RARB, GSTP1 & APC and RARB, RASSF1, GSTP1 & APC biomarker combinations exhibited the highest sensitivity and specificity comparing the extent of DNA methylation in index lesions and noncancerous prostate tissues 20 mm away (both area under the curve [AUC] = 0.98; p < 0.0001). The analysis of the potential urinary biomarkers showed that the combination of all four DNA methylation biomarkers with prostate-specific antigen (PSA) or PSA density (PSAD) in the blood significantly improves the detection of clinically significant PCa (csPCa). The combination of the four-biomarker test with PSAD allowed the identification of csPCa with ≥90% sensitivity and specificity. Thus, this study suggests that for focal therapy by region target hemi-ablation, the safe distance from the index lesion is no less than 10 mm. Noninvasive urine DNA methylation tests in combination with PSAD could be used for further follow-up of the patients, but larger prospective studies with external validation are needed.

Urinary tumor DNA MRD analysis to identify responders to neoadjuvant immunotherapy in muscle-invasive bladder cancer.

Bladder preservation is a viable option for some muscle-invasive bladder cancer (MIBC) patients, but an effective noninvasive biomarker test to accurately identify promising candidates is lacking. Here we present the clinical application of a novel tissue-agnostic, urine-based minimal residual disease (MRD) assay in the neoadjuvant setting for personalized disease surveillance and actionable target identification to facilitate bladder-sparing treatment approaches. The urinary tumor DNA (utDNA) analysis was evaluated in an investigator-initiated phase I trial RJBLC-I2N003 in which 20 patients diagnosed with resectable MIBC were treated presurgically with the PD-1 inhibitor toripalimab followed by radical cystectomy (RC). We showed that neoadjuvant toripalimab therapy was feasible, safe, and induced a 40% rate (8/20) of pathological complete response. Longitudinal utDNA profiling outperformed radiographic assessment and conventional biomarkers to predict the pathologic outcome of immune checkpoint blockade. In addition to detecting 3 exceptional responders with molecular MRD-negative status, we identified 7 other individuals characterized for utDNA clearance and 4 harboring FGFR3 mutants, all of whom (60%, 12/20) could have postponed or avoided RC. These findings demonstrate the safety and efficacy of neoadjuvant toripalimab, and suggest the immense potential of noninvasive utDNA MRD testing to guide tailored decision-making with regard to bladder preservation and change the current treatment paradigm for MIBC patients.