Cancer Special Issue: Early detection and minimal residual disease.

Beryne Odeny discusses PLOS Medicine's Special Issue on early cancer detection and minimal residual disease.

Correlation of transrenal DNA with non-small-cell lung cancer in noninvasive disease monitoring.

Background: The study aims to use noninvasive transrenal DNA in advanced non-small-cell lung cancer (NSCLC) patients for treatment monitoring and prognosis. Methods: Urine specimens were collected longitudinally for 103 late-stage NSCLC patients. Detection of targetable mutations in transrenal DNA was achieved by digital droplet PCR. Patients' overall survival outcomes were correlated with levels of transrenal DNA. Results: Corresponding patients' matched tumor results demonstrated concordance rate of 95.6% with transrenal DNA. A significant decline in levels was observed after treatment initiation. We observed changes in transrenal DNA levels to be significantly associated with survival for patients (p < 0.0001). Conclusion: Our results demonstrated strong predictive values of transrenal DNA to better identify patients with poorer survival outcomes and may further complement disease management.

A urine-based DNA methylation assay to facilitate early detection and risk stratification of bladder cancer.

BACKGROUND: Current non-invasive tests have limited sensitivities and lack capabilities of pre-operative risk stratification for bladder cancer (BC) diagnosis. We aimed to develop and validate a urine-based DNA methylation assay as a clinically feasible test for improving BC detection and enabling pre-operative risk stratifications. METHODS: A urine-based DNA methylation assay was developed and validated by retrospective single-center studies in patients of suspected BC in Cohort 1 (n = 192) and Cohort 2 (n = 98), respectively. In addition, a prospective single-center study in hematuria patient group (Cohort 3, n = 174) was used as a second validation of the model. RESULTS: The assay with a dual-marker detection model showed 88.1% and 91.2% sensitivities, 89.7% and 85.7% specificities in validation Cohort 2 (patients of suspected BC) and Cohort 3 (patients of hematuria), respectively. Furthermore, this assay showed improved sensitivities over cytology and FISH on detecting low-grade tumor (66.7-77.8% vs. 0.0-22.2%, 0.0-22.2%), Ta tumor (83.3% vs. 22.2-41.2%, 44.4-52.9%) and non-muscle invasive BC (NMIBC) (80.0-89.7% vs. 51.5-52.0%, 59.4-72.0%) in both cohorts. The assay also had higher accuracies (88.9-95.8%) in diagnosing cases with concurrent genitourinary disorders as compared to cytology (55.6-70.8%) and FISH (72.2-77.8%). Meanwhile, the assay with a five-marker stratification model identified high-risk NMIBC and muscle invasive BC with 90.5% sensitivity and 86.8% specificity in Cohort 2. CONCLUSIONS: The urine-based DNA methylation assay represents a highly sensitive and specific approach for BC early-stage detection and risk stratification. It has a potential to be used as a routine test to improve diagnosis and prognosis of BC in clinic.

A systematic review on mutation markers for bladder cancer diagnosis in urine.

OBJECTIVES: To systematically summarise the available evidence on urinary bladder cancer (BC) mutation markers. Gene mutations are expected to provide novel biomarkers for urinary BC diagnosis. To date, evidence on urinary BC mutation markers has not proven sufficient to be adopted by clinical guidelines. In the present systematic review, diagnostic accuracy of urinary mutation analysis is separately assessed for primary BC diagnosis (BC detection) and for follow-up of BC patients (BC surveillance). METHODS: A literature search (PubMed, Embase.com and Wiley/Cochrane Library) and systematic review was performed up to 31 October 2019. As studies were too heterogeneous, no quantitative analysis could be performed. RESULTS: In total, 25 studies were summarised by qualitative analysis. For BC detection, diagnostic accuracy differed considerably for single mutation markers (sensitivity 1-85%, specificity 84-100%), and for marker panels (sensitivity 50-94%, specificity 43-97%). Similarly, for BC surveillance, diagnostic accuracy was highly variable for single mutation markers (sensitivity 0-85%, specificity 66-100%), and for marker panels (sensitivity 51-84%, specificity 66-96%). CONCLUSION: Urinary mutation analysis showed to be a promising diagnostic tool for non-invasive BC diagnosis. Nonetheless, we observed substantial differences in diagnostic accuracy of urinary BC mutation markers among publications. To translate the data summarised in the present review to future clinical practice, heterogeneity in research design, BC population, mutation analysis technique and urinary DNA should be considered. Eventual clinical implementation of urinary BC mutation markers can only be achieved by collecting more and stronger evidence. Combining different molecular assays might overcome current shortcomings of urinary mutation analysis.

Urine DNA methylation assay enables early detection and recurrence monitoring for bladder cancer.

BACKGROUNDCurrent methods for the detection and surveillance of bladder cancer (BCa) are often invasive and/or possess suboptimal sensitivity and specificity, especially in early-stage, minimal, and residual tumors.METHODSWe developed an efficient method, termed utMeMA, for the detection of urine tumor DNA methylation at multiple genomic regions by MassARRAY. We identified the BCa-specific methylation markers by combined analyses of cohorts from Sun Yat-sen Memorial Hospital (SYSMH), The Cancer Genome Atlas (TCGA), and the Gene Expression Omnibus (GEO) database. The BCa diagnostic model was built in a retrospective cohort (n = 313) and validated in a multicenter, prospective cohort (n = 175). The performance of this diagnostic assay was analyzed and compared with urine cytology and FISH.RESULTSWe first discovered 26 significant methylation markers of BCa in combined analyses. We built and validated a 2-marker-based diagnostic model that discriminated among patients with BCa with high accuracy (86.7%), sensitivity (90.0%), and specificity (83.1%). Furthermore, the utMeMA-based assay achieved a great improvement in sensitivity over urine cytology and FISH, especially in the detection of early-stage (stage Ta and low-grade tumor, 64.5% vs. 11.8%, 15.8%), minimal (81.0% vs. 14.8%, 37.9%), residual (93.3% vs. 27.3%, 64.3%), and recurrent (89.5% vs. 31.4%, 52.8%) tumors. The urine diagnostic score from this assay was better associated with tumor malignancy and burden.CONCLUSIONUrine tumor DNA methylation assessment for early diagnosis, minimal, residual tumor detection and surveillance in BCa is a rapid, high-throughput, noninvasive, and promising approach, which may reduce the burden of cystoscopy and blind second surgery.FUNDINGThis study was supported by the National Key Research and Development Program of China and the National Natural Science Foundation of China.

Current status of genetic urinary biomarkers for surveillance of non-muscle invasive bladder cancer: a systematic review.

BACKGROUND: Genetic biomarkers are a promising and growing field in the management of bladder cancer in all stages. The aim of this paper is to understand the role of genetic urinary biomarkers in the follow up of patients with non muscle invasive bladder cancer where there is increasing evidence that they can play a role in avoiding invasive techniques. METHODS: Following PRISMA criteria, we have performed a systematic review. The search yielded 164 unique articles, of which 21 articles were included involving a total of 7261 patients. Sixteen of the articles were DNA based biomarkers, analyzing different methylations, microsatellite aberrations and gene mutations. Five articles studied the role of RNA based biomarkers, based on measuring levels of different combinations of mRNA. QUADAS2 critical evaluation of each paper has been reported. RESULTS: There are not randomized control trials comparing any biomarker with the gold standard follow-up, and the level of evidence is 2B in almost all the studies. Negative predictive value varies between 55 and 98.5%, being superior in RNA based biomarkers. CONCLUSIONS: Although cystoscopy and cytology are the gold standard for non muscle invasive bladder cancer surveillance, genetic urinary biomarkers are a promising tool to avoid invasive explorations to the patients with a safe profile of similar sensitivity and negative predictive value. The accuracy that genetic biomarkers can offer should be taken into account to modify the paradigm of surveillance in non muscle invasive bladder cancer patients, especially in high-risk ones where many invasive explorations are recommended and biomarkers experiment better results.

Assessment of somatic mutations in urine and plasma of Wilms tumor patients.

Tumor DNA has been detected in body fluids of cancer patients. Somatic tumor mutations are being used as biomarkers in body fluids to monitor chemotherapy response as a minimally invasive tool. In this study, we evaluated the potential of tracking somatic mutations in free DNA of plasma and urine collected from Wilms tumor (WT) patients for monitoring treatment response. Wilms tumor is a pediatric renal tumor resulting from cell differentiation errors during nephrogenesis. Its mutational repertoire is not completely defined. Thus, for identifying somatic mutations from tumor tissue DNA, we screened matched tumor/leukocyte DNAs using either a panel containing 16 WT-associated genes or whole-exome sequencing (WES). The identified somatic tumor mutations were tracked in urine and plasma DNA collected before, during and after treatment. At least one somatic mutation was identified in five out of six WT tissue samples analyzed. Somatic mutations were detected in body fluids before treatment in all five patients (three patients in urine, three in plasma, and one in both body fluids). In all patients, a decrease of the variant allele fraction of somatic mutations was observed in body fluids during neoadjuvant chemotherapy. Interestingly, the persistence of somatic mutations in body fluids was in accordance with clinical parameters. For one patient who progressed to death, it persisted in high levels in serial body fluid samples during treatment. For three patients without disease progression, somatic mutations were not consistently detected in samples throughout monitoring. For one patient with bilateral disease, a somatic mutation was detected at low levels with no support of clinical manifestation. Our results demonstrated the potential of tracking somatic mutations in urine and plasma DNA as a minimally invasive tool for monitoring WT patients. Additional investigation is needed to check the clinical value of insistent somatic mutations in body fluids.

Treatment and relapse in breast cancer show significant correlations to noninvasive testing using urinary and plasma DNA.

Aim: Circulating tumor DNA is promising for routine monitoring of breast cancer. Noninvasive testing allows regular probing using plasma and urine samples. Methods: Peripheral blood and simultaneous urine collection from patients were quantified. Concordance between methods were made. Serial time-point measurements were correlated to disease outcome. Results: Index measurements demonstrate over 90% concordance with biopsy. Receiver operating characteristics curves showed over 0.95 for both plasma and urine results comparing with controls. Patients with lower risk of relapse experienced greater declines in detected DNA levels. Maximal declines were registered at 4.0- and 6.8-fold for plasma and urine results, respectively. Conclusion: Measuring and monitoring DNA levels complement existing testing regimes and provides better risk profiling of patients for possible relapse.

HOXA9, PCDH17, POU4F2, and ONECUT2 as a Urinary Biomarker Combination for the Detection of Bladder Cancer in Chinese Patients with Hematuria.

BACKGROUND: DNA methylation biomarkers for bladder cancer (BCa) have not been evaluated extensively in the Chinese population. OBJECTIVE: To develop and validate a urinary biomarker combination of methylation assays in a group of Chinese patients with hematuria. DESIGN, SETTING, AND PARTICIPANTS: A total of 192 urine samples were collected and evaluated from patients with microscopic or gross hematuria, including 97 BCa patients and 95 controls with benign diseases. A two-stage study was conducted: the first stage being assay construction and the second stage being assay validation. Eighty-one urine samples were analyzed for the hypermethylation of eight selected genes in stage 1 and then a four-gene panel was constructed. An additional 111 urine samples were analyzed using the four-gene panel (including HOXA9, PCDH17, POU4F2, and ONECUT2) for independent validation. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The positive predictive value (PPV) and negative predictive value (NPV) were calculated for the combination methylation assay. Uni- and multivariate binary logistic regression analyses (backward elimination, conditional) were performed to calculate the association between BCa and each predictor variable. RESULTS AND LIMITATIONS: The combination assay of HOXA9, PCDH17, POU4F2, and ONECUT2 was selected based on the results of multivariate logistic regression analysis in stage 1. Using a strategy of three-level risk stratification, the assay yielded a consistent PPV of 100%. With an estimated BCa prevalence of 10% in a general hematuria population, the assay would result in an overall NPV of 98%. This combined methylation biomarker would yield an overall area under the receiver operating characteristic curve of 0.871 (with a sensitivity of 90.5% and a specificity of 73.2%) if using the prediction model from multivariate regression analysis. In addition, over half of BCa cases would be predicted accurately and ∼60% of unnecessary cystoscopies could be spared. This study had several limitations. First, the sample size was relatively small. Second, it was performed in a case-control population rather than in a natural hematuria cohort. CONCLUSIONS: A combination methylation assay of HOXA9, PCDH17, POU4F2, and ONECUT2 resulted in high PPV and NPV in Chinese patients with hematuria. With accurate risk prediction, the urinary biomarker combination could spare a sizeable proportion of low-risk patients from extensive and invasive examination. PATIENT SUMMARY: In the present study, we looked at the predictive performance of a urinary biomarker combination of HOXA9, PCDH17, POU4F2, and ONECUT2. We found that this urinary biomarker combination may help discriminate bladder cancer from other benign diseases in patients with hematuria, resulting in a reduction of unnecessary invasive examination in patients at low risk.

Urinary DNA methylation biomarkers for prediction of prostate cancer upgrading and upstaging.

BACKGROUND: Significant numbers of prostate cancer (PCa) patients experience tumour upstaging and upgrading in surgical specimens that cause serious problems in timely and proper selection of the treatment strategy. This study was aimed at the evaluation of a set of established epigenetic biomarkers as a noninvasive tool for more accurate PCa categorization before radical prostatectomy (RP). METHODS: Quantitative methylation-specific PCR was applied for the methylation analysis of RARB, RASSF1, and GSTP1 in 514 preoperatively collected voided or catheterized urine samples from the single-centre cohort of 1056 treatment-naïve PCa patients who underwent RP. The rates of biopsy upgrading and upstaging were analysed in the whole cohort. RESULTS: Pathological examination of RP specimens revealed Gleason score upgrading in 27.2% and upstaging in 20.3% of the patients with a total misclassification rate of 39.0%. DNA methylation changes in at least one gene were detected in more than 80% of urine samples. Combination of the PSA test with the three-gene methylation analysis in urine was a significant predictor of pathological upstaging and upgrading (P < 0.050), however, with limited increase in overall accuracy. The PSA test or each gene alone was not informative enough. CONCLUSIONS: The urinary DNA methylation assay in combination with serum PSA may predict tumour stage or grade migration post-RP aiding in improved individual risk assessment and appropriate treatment selection. Clinical utility of these biomarkers should be proven in larger multi-centre studies.

Targeted deep sequencing of urothelial bladder cancers and associated urinary DNA: a 23-gene panel with utility for non-invasive diagnosis and risk stratification.

OBJECTIVES: To develop a focused panel of somatic mutations (SMs) present in the majority of urothelial bladder cancers (UBCs), to investigate the diagnostic and prognostic utility of this panel, and to compare the identification of SMs in urinary cell-pellet (cp)DNA and cell-free (cf)DNA as part of the development of a non-invasive clinical assay. PATIENTS AND METHODS: A panel of SMs was validated by targeted deep-sequencing of tumour DNA from 956 patients with UBC. In addition, amplicon and capture-based targeted sequencing measured mutant allele frequencies (MAFs) of SMs in 314 urine cpDNAs and 153 urine cfDNAs. The association of SMs with grade, stage, and clinical outcomes was investigated by univariate and multivariate Cox models. Concordance between SMs detected in tumour tissue and cpDNA and cfDNA was assessed. RESULTS: The panel comprised SMs in 23 genes: TERT (promoter), FGFR3, PIK3CA, TP53, ERCC2, RHOB, ERBB2, HRAS, RXRA, ELF3, CDKN1A, KRAS, KDM6A, AKT1, FBXW7, ERBB3, SF3B1, CTNNB1, BRAF, C3orf70, CREBBP, CDKN2A, and NRAS; 93.5-98.3% of UBCs of all grades and stages harboured ≥1 SM (mean: 2.5 SMs/tumour). RAS mutations were associated with better overall survival (P = 0.04). Mutations in RXRA, RHOB and TERT (promoter) were associated with shorter time to recurrence (P < 0.05). MAFs in urinary cfDNA and cpDNA were highly correlated; using a capture-based approach, >94% of tumour SMs were detected in both cpDNA and cfDNA. CONCLUSIONS: SMs are reliably detected in urinary cpDNA and cfDNA. The technical capability to identify very low MAFs is essential to reliably detect UBC, regardless of the use of cpDNA or cfDNA. This 23-gene panel shows promise for the non-invasive diagnosis and risk stratification of UBC.

Cell-free DNA diagnostics: current and emerging applications in oncology.

Liquid biopsy is a noninvasive dynamic approach for monitoring disease over time. It offers advantages including limited risks of blood sampling, opportunity for more frequent sampling, lower costs and theoretically non-biased sampling compared with tissue biopsy. There is a high degree of concordance between circulating tumor DNA mutations versus primary tumor mutations. Remote sampling of circulating tumor DNA can serve as viable option in clinical diagnostics. Here, we discuss the progress toward broad adoption of liquid biopsy as a diagnostic tool and discuss knowledge gaps that remain to be addressed.

Application of the Z-scan technique for the detection of CFCDNA (cell-free circulating DNA) and urine DNA (uDNA) in patients with bladder cancer.

BACKGROUND: Patients with BC have a higher amount of cell-free circulating DNA (CFCDNA) in the blood and urine than healthy people. We aimed to verify if the Z-Scan method could analyze the concentrations of uDNA (urinary) and pDNA (plasma) in relation to the time of collection during treatment for patients with bladder cancer. METHODS: Peripheral blood and urine samples were obtained from 30 patients with BC at the time of diagnosis, 45, 90 and 180 days after initiating treatment; 5 µL of k-DNA (k = u or p) was added in 250 µL a solution of 1:1000 Ethidium Bromide dye (EtBr) in water. Continum laser Nd:YVO4, wavelength λ = 532 nm was used. Samples of uDNA and pDNA in water were submitted to the laser with an incident power of 84.5 mW and an exposure time of 30 ms. RESULTS: There was a different concentration of pDNA and uDNA during the treatment of patients using both optical techniques. However, the reaction rate of pDNA and uDNA was similar with spectrophotometry, whereas the z-scan technique presented different values. CONCLUSION: Z-scan technique has potential for use in the differentiation of pDNA and uDNA concentrations, which are distinct in patients with BC and healthy people.

Whole exome sequencing analysis of urine trans-renal tumour DNA in metastatic colorectal cancer patients.

BACKGROUND: The analysis of circulating free tumour DNA (ctDNA) in blood, commonly referred as liquid biopsy, is being used to characterise patients with solid cancers. Tumour-specific genetic variants can also be present in DNA isolated from other body fluids, such as urine. Unlike blood, urine sampling is non-invasive, can be self-performed, and allows recurrent longitudinal monitoring. The features of tumour DNA that clears from the glomerular filtration barrier, named trans-renal tumour DNA (trtDNA), are largely unexplored. PATIENTS AND METHODS: Specimens were collected from 24 patients with KRAS or BRAF mutant metastatic colorectal cancer (mCRC). Driver mutations were assessed by droplet digital PCR (ddPCR) in ctDNA from plasma and trtDNA from urine. Whole exome sequencing (WES) was performed in DNA isolated from tissue, plasma and urine. RESULTS: Out of the 24 CRC cases, only four had sufficient DNA to allow WES analyses in urine and plasma. We found that tumour alterations primarily reside in low molecular weight fragments (less than 112 bp). In patients whose trtDNA was more than 2.69% of the urine derived DNA, cancer-specific molecular alterations, mutational signatures and copy number profiles identified in urine DNA are comparable with those detected in plasma ctDNA. CONCLUSIONS: With current technologies, WES analysis of trtDNA is feasible in a small fraction of mCRC patients. Tumour-related genetic information is mainly present in low molecular weight DNA fragments. Although the limited amounts of trtDNA poses analytical challenges, enrichment of low molecular weight DNAs and optimised computational tools can improve the detection of tumour-specific genetic information in urine.

Diagnostic value of combined IQGAP3/BMP4 and IQGAP3/FAM107A expression ratios in urinary cell-free DNA for discriminating bladder cancer from hematuria.

BACKGROUND: Urinary cell-free DNA (ucfDNA) has great potential as a "liquid biopsy" for use in diagnosis of urological cancers. In this study, we compared ucfDNA gene expression levels between patients with bladder cancer (BC) and those with hematuria, and determined whether they could be used as a noninvasive urine-based marker. METHODS: The study cohort of 355 patients included a screening group (40 BC and 41 hematuria controls) and a validation cohort (149 BC and 125 hematuria controls). Expression levels ratios of 1 up-regulated gene (IQGAP3) to those of 7 down-regulated genes were examined in ucfDNA in the screening group to identify ratios that differed significantly between BC and hematuria patients. IQGAP3/BMP4 and IQGAP3/FAM107A ratios were selected and combined to develop a discriminant score (DS) index, which was tested in the validation cohort. Receiver operating characteristic curves and areas under the curve were calculated to evaluate the performance of the DS index. RESULTS: IQGAP3/BMP4 and IQGAP3/FAM107A ratios in ucfDNA were both significantly higher in BC patients than in hematuria patients (both P < 0.001). The DS index had an area under the curve of 0.862, a sensitivity of 71.0%, a specificity of 88.6%, a positive predictive value of 90.3%, and a negative predictive value of 67.2%. CONCLUSIONS: Both IQGAP3/BMP4 and IQGAP3/FAM107A ratios in ucfDNA were significantly higher in patients with BC than in those with hematuria. The DS index exhibits good diagnostic performance as a noninvasive biomarker.

Evaluation of TERT promoter mutations in urinary cell-free DNA and sediment DNA for detection of bladder cancer.

BACKGROUND: Cell-free DNA (cfDNA) is proposed to be a valuable source of biomarkers in liquid biopsies for various diseases as it is supposed to partially originate from tumor cells. However, data about the diagnostic implications of cfDNA in urine for the detection of bladder cancer (BCa) is sparse. METHODS: We evaluated the usability of urinary cfDNA for diagnostic purposes compared to urine sediment DNA (sDNA) in 53 BCa patients and 36 control subjects by analyzing two abundant point-mutations (C228T/C250T) in the TERT promoter using Next-Generation Sequencing. RESULTS: Mutations were detected in 77% of the urinary sDNA compared to 63% of the cfDNA samples. Moreover, the TERT mutation allele frequencies (MAF) were highly correlated in cfDNA and sDNA. In comparison, the accuracy of the TERT assay was higher in sDNA (84%) compared to cfDNA or voided urine cytology (both 77%). Interestingly, MAFs from leukocyte-rich urines were higher in cfDNA than in sDNA, indicating a diagnostic advantage of cfDNA in such urines. CONCLUSIONS: Urine-based mutation detection has the ability to augment and surpass voided urine cytology as the current gold-standard for the non-invasive detection and surveillance of BCa. The analysis of cell-free DNA provides no general diagnostic advantage compared to urine sediment DNA.

Estrogen Metabolism in African-American Women with and without Breast Cancer: A Pilot Study.

Studies in Caucasian women have shown that the formation of estrogen-DNA adducts is greater in women at high risk for breast cancer or already diagnosed with the disease. To begin investigating whether the role of estrogens in the etiology of breast cancer is similar in African-American (AA) women, a saliva sample and a spot urine sample were collected from 19 AA women with breast cancer and 23 AA women not diagnosed with breast cancer. In the urine samples, 20 estrogen metabolites, conjugates, and DNA adducts were analyzed by using ultraperformance liquid chromatography/tandem mass spectrometry, and then the ratio of adducts to metabolites and conjugates was calculated for each sample. The ratio of depurinating estrogen-DNA adducts to estrogen metabolites and estrogen conjugates was significantly greater in cases compared to controls (92.4 ± 46.4 vs 38.5 ± 18.9, p < 0.0001). From the saliva samples, genomic DNA was purified and analyzed for genetic polymorphisms in the genes for two estrogen-metabolizing enzymes, catechol- O-methyltransferase (rs4680) and cytochrome P450 1B1 (rs1056836). There was no association between rs4680 and rs1056836 genotypes and adduct ratios or breast cancer status. This pilot study found higher DNA adduct ratios in women with breast cancer, which suggests that estrogen metabolism is out of balance, and the formation of estrogen-DNA adducts may exert a critical role in breast cancer initiation in AA women.

Detection and Surveillance of Bladder Cancer Using Urine Tumor DNA.

Current regimens for the detection and surveillance of bladder cancer are invasive and have suboptimal sensitivity. Here, we present a novel high-throughput sequencing (HTS) method for detection of urine tumor DNA (utDNA) called utDNA CAPP-Seq (uCAPP-Seq) and apply it to 67 healthy adults and 118 patients with early-stage bladder cancer who had urine collected either prior to treatment or during surveillance. Using this targeted sequencing approach, we detected a median of 6 mutations per patient with bladder cancer and observed surprisingly frequent mutations of the PLEKHS1 promoter (46%), suggesting these mutations represent a useful biomarker for detection of bladder cancer. We detected utDNA pretreatment in 93% of cases using a tumor mutation-informed approach and in 84% when blinded to tumor mutation status, with 96% to 100% specificity. In the surveillance setting, we detected utDNA in 91% of patients who ultimately recurred, with utDNA detection preceding clinical progression in 92% of cases. uCAPP-Seq outperformed a commonly used ancillary test (UroVysion, P = 0.02) and cytology and cystoscopy combined (P ≤ 0.006), detecting 100% of bladder cancer cases detected by cytology and 82% that cytology missed. Our results indicate that uCAPP-Seq is a promising approach for early detection and surveillance of bladder cancer. SIGNIFICANCE: This study shows that utDNA can be detected using HTS with high sensitivity and specificity in patients with early-stage bladder cancer and during post-treatment surveillance, significantly outperforming standard diagnostic modalities and facilitating noninvasive detection, genotyping, and monitoring.This article is highlighted in the In This Issue feature, p. 453.

Assessment of microsatellite instability for screening bladder cancer in high-risk population.

AIMS: This study aims to determine the diagnostic efficacy of microsatellite markers for screening bladder cancer in population at high risk. MATERIALS AND METHODS: A population of 200 people was screened for bladder cancer using a set of microsatellite markers. Urine samples were obtained from four different types of population groups - Group 1 (healthy population group), Group 2 (current smokers with a smoking history of more than 10 years), Group 3 (bladder cancer group), and Group 4 (bladder cancer group who were former smokers with a history of more than 10 years). Polymerase chain reaction (PCR) was performed to amplify microsatellite sequences at D9S63, D9S156, and D9S283. PCR products were separated on 1.8% agarose gel and were scanned using ultraviolet transilluminator. RESULTS: In Group 2 (high-risk population group, mainly current smokers with a history of more than 10 years), microsatellite alterations were found in 36 out of 50 people. We observed microsatellite alterations in 38 out of 50 people in Group 3 (bladder cancer group) and in 39 out of 50 people in Group 4 (bladder cancer group, mainly former smokers with a history of more than 10 years). The sensitivity of this test in Group 2, Group 3, and Group 4 was found to be 72%, 76% and 78%, respectively. The specificity of this test in each group was found to be 90%. CONCLUSION: Using these set of microsatellite markers, medium sensitivity and high specificity were reported for this test. The current findings suggest that a set of microsatellite markers (D9S63, D9S156, and D9S283) can be used to detect bladder cancer in high-risk population.