Urinary cfDNA Research Articles

Abstract 544: Urine as a non-invasive alternative to blood for germline and somatic mutation detection in hepatocellular carcinoma

Abstract BACKGROUND: Cell-free DNA (cfDNA) from blood has become a promising analyte for cancer diagnostics. However, because blood contains high concentrations of proteins and enzyme inhibitors, plasma cfDNA quality can be variable. Urinary cfDNA has been shown to contain mutations associated with non-genitourologic cancers including hepatocellular carcinoma (HCC). In this study, we evaluate urine as a noninvasive alternative to blood-based liquid biopsy for both germline and ctDNA genotyping in HCC. METHOD: Using qPCR, whole-genome sequencing (WGS), and targeted NGS, DNA isolated from blood or urine of patients with HCC was evaluated and analyzed for overall genome coverage, HCC hotspot coverage, and germline or somatic mutation concordance. Targeted NGS of plasma and urine cfDNA was also performed for detection of somatic variants. RESULTS: Similar to plasma cfDNA, urine cfDNA showed a major mononucleosomal species of 165-170 bp, with dinucleosomal species also discernable in most healthy individuals and patients with HCC. Overall genome coverage breadth was similar between urine and plasma cfDNA, with higher fraction of covered cancer-associated mutation hotspots in urine cfDNA. Quantitative PCR analyses of HCC-associated mutations (TP53, CTNNB1, and hTERT) in 102 patients with HCC, revealed 78% overall concordance between plasma and urine. Targeted NGS of HCC-associated gene regions in additional 15 patients showed an 97% overall position-level concordance between plasma and urine cfDNA. Furthermore, urine outperformed plasma in both the sensitivity and specificity of detection of CTNNB1 and hTERT mutations. CONCLUSION urine DNA can potentially be used as a completely noninvasive diagnostic biomarker for HCC. Citation Format: Amy Kim, Selena Lin, Yixiao Cui, Terence Gade, Fwu-Shan Shieh, Max Chao, John Shieh, Surbhi Jain, Jonathan Cheng, James Hamilton, Hie-Won L. Hann, Dmitry Goryunov, Ying-Hsiu Su. Urine as a non-invasive alternative to blood for germline and somatic mutation detection in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 544.

Abstract 2102: Development and validation of Predicine methylation assay towards early cancer detection

Abstract DNA methylation is one of the earliest signatures during cancer development. DNA methylation patterns from different cancer types can provide distinct epigenetic signatures. Several methods are available for sequencing DNA methylation throughout the genome, including whole genome bisulfite sequencing (WGBS) and antibody-dependent DNA immunoprecipitation (MeDIP). As a non-invasive approach, plasma cfDNA and urinary cfDNA (ucfDNA) have been widely explored for clinical applications. More recently, in virtue of its cancer-specific methylation signatures, cfDNA has been adopted for early cancer detection and further identification of cancer tissue of origin. Predicine has developed a liquid biopsy methylation assay that can robustly detect methylation using genomic and circulating cell-free DNA in body fluids. Predicine's methylation assay has great improvement over WGBS in reducing DNA damage, GC bias and increasing NGS reads mapping rate, and mapping quality score. In this study, we report the clinical utility of Predicine's methylation assay, which efficiently distinguishing healthy donors from patients of various types of cancer. Combined with its genomics NGS counterpart, Predicine's methylation platform is a powerful tool for early cancer detection and real-time monitoring of cancer progression. Citation Format: Binggang Xiang, Chao Dai, Ming Wang, Zhixin Zhao, Amy Xiaohong Wang, Kemin Zhou, Shidong Jia, Shujun Luo, Pan Du. Development and validation of Predicine methylation assay towards early cancer detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2102.

A novel urine cell-free DNA preservation solution and its application in kidney transplantation.

Urine cell-free DNA (cfDNA) is a new type of liquid biopsy biomarker used in tumours and allograft injury detection but is highly susceptible to degradation by the high nuclease activity of urine. This study presents a newly developed urine cfDNA preservation solution (AlloU), efficient for examining allograft injury in kidney transplant recipients (KTx). We established urine-preserve solution called AlloU based on the response-surface methodology, with two commercial collection reagents (Streck and K2 EDTA preservation solution) included for analysis. A total of 120 urine samples from KTx patients, including morning, nocturnal and random urine from specific storage time were subjected to investigation. The urine total cfDNA concentration was quantified by fluorometry, fragment distribution was analysed by qPCR, and donor-derived cfDNA (ddcfDNA) was detected by next-generation sequencing. Urine total cfDNA concentration and fragment size of samples preserved with AlloU and Streck did not change significantly within 5 days whereas the ddcfDNA also did not change significantly within 7 days. However, compared with EDTA, the total cfDNA concentration increased significantly on the third day. When compare with different urine types, it was found that samples preserved with AlloU showed no significant differences in total cfDNA concentration, fragment size, and ddcfDNA concentration, however, the SD for morning urine was significantly smaller in total cfDNA and ddcfDNA concentration. To the best of our knowledge, this is the first report to verify the dynamics of urine cfDNA in KTx, especially in the analysis impact of different urine types on cfDNA detection.

Toward urinary cell-free DNA-based treatment of urothelial carcinoma: a narrative review.

Liquid biopsy technique targeting urinary cell-free DNA (cfDNA) is getting a lot of attention to overcome limitations of the present treatment strategy for urothelial carcinoma, including urothelial bladder carcinoma (UBC) and upper tract urothelial carcinoma (UTUC). Analysis of tumor-derived DNA in urine focusing either on genomic or epigenomic alterations, holds great potential as a noninvasive method for the detection of urothelial carcinoma with high accuracy. It is also predictive of prognosis and response to drugs, and reveals the underlying characteristics of different stages of urothelial carcinoma. Although cfDNA methylation analyses based on a combination of several methylation profiles have demonstrated high sensitivity for UBC diagnosis, there have been few reports involving epigenomic studies of urinary cfDNA. In mutational analyses, frequent gene mutations (TERT promoter, TP53, FGFR3, PIK3CA, RAS, etc.) have been detected in urine supernatant by using remarkable technological innovations such as next-generation sequencing and droplet digital PCR. These methods allow highly sensitive detection of rare mutation alleles while minimizing artifacts. In this review, we summarize the current insights into the clinical applications of urinary cfDNA from patients with urothelial carcinoma. Although it is necessary to conduct prospective multi-institutional clinical trials, noninvasive urine biopsy is expected to play an important role in the realization of precision medicine in patients with urothelial carcinoma in the near future.

Diagnosing Pulmonary Tuberculosis by Using Sequence-Specific Purification of Urine Cell-Free DNA.

ABSTRACTTransrenal urine cell-free DNA (cfDNA) is a promising tuberculosis (TB) biomarker, but is challenging to detect because of the short length (<100 bp) and low concentration of TB-specific fragments. We aimed to improve the diagnostic sensitivity of TB urine cfDNA by increasing recovery of short fragments during sample preparation. We developed a highly sensitive sequence-specific purification method that uses hybridization probes immobilized on magnetic beads to capture short TB cfDNA (50 bp) with 91.8% average efficiency. Combined with short-target PCR, the assay limit of detection was ≤5 copies of cfDNA in 10 ml urine. In a clinical cohort study in South Africa, our urine cfDNA assay had 83.7% sensitivity (95% CI: 71.0 to 91.5%) and 100% specificity (95% CI: 86.2 to 100%) for diagnosis of active pulmonary TB when using sputum Xpert MTB/RIF as the reference standard. The detected cfDNA concentration was 0.14 to 2,804 copies/ml (median 14.6 copies/ml) and was inversely correlated with CD4 count and days to culture positivity. Sensitivity was nonsignificantly higher in HIV-positive (88.2%) compared to HIV-negative patients (73.3%), and was not dependent on CD4 count. Sensitivity remained high in sputum smear-negative (76.0%) and urine lipoarabinomannan (LAM)-negative (76.5%) patients. With improved sample preparation, urine cfDNA is a viable biomarker for TB diagnosis. Our assay has the highest reported accuracy of any TB urine cfDNA test to date and has the potential to enable rapid non-sputum-based TB diagnosis across key underserved patient populations.

Ultrasensitive hybridization capture: Reliable detection of

Urine cell-free DNA (cfDNA) is a valuable non-invasive biomarker with broad potential clinical applications, but there is no consensus on its optimal pre-analytical methodology, including the DNA extraction step. Due to its short length (majority of fragments <100 bp) and low concentration (ng/mL), urine cfDNA is not efficiently recovered by conventional silica-based extraction methods. To maximize sensitivity of urine cfDNA assays, we developed an ultrasensitive hybridization method that uses sequence-specific oligonucleotide capture probes immobilized on magnetic beads to improve extraction of short cfDNA from large-volume urine samples. Our hybridization method recovers near 100% (95% CI: 82.6–117.6%) of target-specific DNA from 10 mL urine, independent of fragment length (25–150 bp), and has a limit of detection of ≤5 copies of double-stranded DNA (0.5 copies/mL). Pairing hybridization with an ultrashort qPCR design, we can efficiently capture and amplify fragments as short as 25 bp. Our method enables amplification of cfDNA from 10 mL urine in a single qPCR well, tolerates variation in sample composition, and effectively removes non-target DNA. Our hybridization protocol improves upon both existing silica-based urine cfDNA extraction methods and previous hybridization-based sample preparation protocols. Two key innovations contribute to the strong performance of our method: a two-probe system enabling recovery of both strands of double-stranded DNA and dual biotinylated capture probes, which ensure consistent, high recovery by facilitating optimal probe density on the bead surface, improving thermostability of the probe-bead linkage, and eliminating interference by endogenous biotin. We originally designed the hybridization method for tuberculosis diagnosis from urine cfDNA, but expect that it will be versatile across urine cfDNA targets, and may be useful for other cfDNA sample types and applications beyond cfDNA. To make our hybridization method accessible to new users, we present a detailed protocol and straightforward guidelines for designing new capture probes.

Superior molecular pathology of urothelial bladder cancer using urinary cell-free DNA.

491 Background: Both urinary and blood cell-free DNA (cfDNA) have been implicated in noninvasive detection and surveillance of urothelial bladder cancer (UBC). However, a direct comparison of their diagnostic performance in the real-world setting is lacking. Methods: 59 eligible cases with pathologically confirmed disease and accompanying tissue/urine pairs were prospectively enrolled and consented to Institutional Review Board-approved protocols. Baseline peripheral blood mononuclear cell (PBMC) and plasma specimens were collected during clinic visit. The 180-gene Predicine liquid biopsy assay was applied for ultra-deep targeted sequencing and somatic alteration identification in tumor tissue-based DNA (tDNA), urinary cfDNA (ucfDNA) and blood cfDNA (bcfDNA). Results: The 59 studied subjects constituted a natural UBC cohort of non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC), including 48 (81.4%) NMIBC and 42 (71.2%) male patients. Diverse quantitative metrics such as VAF (variant allele frequency) and TMB (tumor mutational burden) were invariably concordant between tDNA and ucfDNA, but not bcfDNA. The mutational landscape captured by tDNA or ucfDNA highly resembled each other and mirrored previously described genomic panorama of UBC, whereas a significant proportion of bcfDNA aberrations stemmed from clonal hematopoiesis. Using tDNA-informed variants as the ground truth, ucfDNA assays achieved a specificity of 99.3%, a sensitivity of 86.7%, a positive predictive value (PPV) of 67.2%, a negative predictive value (NPV) of 99.8%, and a diagnostic accuracy of 99.1%, which were generally lower in the case of bcfDNA analysis. Conclusions: Urine-based molecular pathology provides valid and complete genetic information about neoplastic lesions, and represents a faithful surrogate for genotyping and monitoring UBC.

Jagged Ends of Urinary Cell-Free DNA: Characterization and Feasibility Assessment in Bladder Cancer Detection.

Double-stranded DNA in plasma is known to carry single-stranded ends, called jagged ends. Plasma DNA jagged ends are biomarkers for pathophysiologic states such as pregnancy and cancer. It remains unknown whether urinary cell-free DNA (cfDNA) molecules have jagged ends. Jagged ends of cfDNA were detected by incorporating unmethylated cytosines during a DNA end-repair process, followed by bisulfite sequencing. Incorporation of unmethylated cytosines during the repair of the jagged ends lowered the apparent methylation levels measured by bisulfite sequencing and were used to calculate a jagged end index. This approach is called jagged end analysis by sequencing. The jagged end index of urinary cfDNA was higher than that of plasma DNA. The jagged end index profile of plasma DNA displayed several strongly oscillating major peaks at intervals of approximately 165 bp (i.e., nucleosome size) and weakly oscillating minor peaks with periodicities of approximately 10 bp. In contrast, the urinary DNA jagged end index profile showed weakly oscillating major peaks but strongly oscillating minor peaks. The jagged end index was generally higher in nucleosomal linker DNA regions. Patients with bladder cancer (n = 46) had lower jagged end indexed of urinary DNA than participants without bladder cancer (n = 39). The area under the curve for differentiating between patients with and without bladder cancer was 0.83. Jagged ends represent a property of urinary cfDNA. The generation of jagged ends might be related to nucleosomal structures, with enrichment in linker DNA regions. Jagged ends of urinary DNA could potentially serve as a new biomarker for bladder cancer detection.

Enhanced Detection of Genitourinary Cancers Using Fragmentation and Copy Number Profiles Obtained from Urinary Cell-Free DNA.

Recent studies have reported that examining the fragmentation profiles (FP) of plasma cell-free DNA (cfDNA) further improves the clinical sensitivity of tumor detection. We hypothesized that considering the differences of the FP of urinary cfDNA would increase the clinical sensitivity of genitourinary (GU) cancer detection. 177 patients with GU cancer and 94 individuals without tumors were enrolled in the discovery cohort. An independent validation dataset comprising 30 patients without tumors and 66 patients with GU cancer was also collected. We constructed an ensemble classifier, GUIDER, to detect and localize GU cancers using fragmentation and copy number profiles obtained from shallow whole-genome sequencing of urinary cfDNA. Urinary cfDNA of patients with GU cancer had a higher proportion of long fragments (209-280 bp) and a lower proportion of short fragments (140-208 bp) compared to controls. The overall mean classification accuracy of the FP was 74.62%-85.39% for different algorithms, and integration of the FP and copy number alteration (CNA) features further enhanced the classification of samples from patients with GU cancer. The mean diagnostic accuracy was further improved by the ensemble classifier GUIDER, which integrated the FP and CNA profiles and resulted in a higher mean accuracy (87.52%) compared to the analysis performed without FP features (74.62%). GUIDER performed well in an independent validation dataset. The lengthening and shortening of urinary cfDNA within specific size ranges were identified in patients with GU cancer. Integration of the FP should further enhance the ability to use urinary cfDNA as a molecular diagnostic tool.

Diagnostic urinary cfDNA detected in human cystic echinococcosis

Cystic echinococcosis (CE) is a major neglected tropical zoonotic disease caused by the tissue-dwelling larval stage of the cestode parasite Echinococcus granulosus. For individuals suspected of CE, the diagnostic standard is imaging using ultrasonography, X rays, or computed tomography. These resource-demanding and expensive procedures are rarely available in endemic rural areas where CE is most prevalent. There is a critical need for a new approach to identify CE patients so that they can be managed early in the course of their infection. This study reports on the results of a diagnostic approach that identifies E. granulosus-derived cell-free DNA (cfDNA) in the urine of CE patients. Utilizing PCR to amplify a fragment of a major tandem repeat element found in E. granulosus nuclear DNA, urine samples from all seven imaging-confirmed CE patients who harbored active liver cysts were positive. In addition, the urine samples from 2/4 patients who presented with non-viable/calcified liver cysts were also PCR positive for the repeat fragment. To our knowledge, this is the first report of using parasite cfDNA from urine to diagnose CE. This approach provides an easy to implement and cost-effective method to survey for the prevalence of E. granulosus in humans populations.

Abstract 722: Urine for noninvasive liquid biopsy for germline and somatic mutations

Abstract Circulating tumor DNA (ctDNA) from blood and peripheral blood mononuclear cells (PBMCs) are used to determine comprehensive cancer genetics and germline genomics for precision medicine. To evaluate the use of urine samples as a noninvasive alternative to blood samples, we performed a comprehensive characterization of urine DNA as an alternative to PBMC DNA for germline genomics. Whole genome sequencing (WGS) was performed in PBMC and total urine DNA in six normal subjects to compare the quality and comprehensiveness of the genomic data. There was no significant difference between the sequencing data obtained from PBMC and urine DNA upon comparing the variant calling, percent reads passing Phred score Q30 (p&amp;gt;0.05, paired t-test), and coverage of human genome. Similar to plasma cfDNA, mononucleosomal-sized DNA was the most predominant species present in urine cfDNA. Samples from three hepatocellular carcinoma (HCC) patients were further analyzed for insert sizes by NGS, which demonstrated predominantly mononucleosomal-sized DNA fragments, with a series of peaks occurring with 10-bp periodicity, in both urine and plasma cfDNA. With shallow WGS, the genome coverage overlap was 53-61%, and by increasing the depth of coverage the overlap increased from 60% to 74%. Interestingly, 8-10% of the genome covered in plasma DNA was not detected in urine DNA, and 4-8% of the genome covered in urine DNA was not covered in plasma DNA. Next, we compared the detectability of three HCC-associated genetic mutations in the matched urine and plasma DNA obtained from 76 HCC patients including mutations at TP53 codon 249 (TP53 249T), CTNNB1 exon 3 regions 32-37 (CTNNB1 32-37), and hTERT promoter region position 124 (hTERT 124). We observed higher levels of the TP53 mutation, similar levels of CTNNB1 mutation, and lower levels of the TERT mutation in urine cfDNA compared to plasma ctDNA. This suggests a potential for urine as a potential noninvasive source for ctDNA analysis, and a combination of both urine and plasma ctDNA may increase the overall sensitivity of ctDNA detection. In conclusion, our data suggest that (1) total urine DNA can replace PBMC DNA for providing comprehensive genomic sequencing data and (2) urine can complement blood for liver cancer liquid biopsy, precision medicine, and potential applications in other cancers. Citation Format: James V. Vowles, Amy K. Kim, James P. Hamilton, Selena Y. Lin, Fwu-Shan Shieh, Harry Luu, Gabrielle Villafana, Chi-Tan Hu, Ying-Hsiu Su. Urine for noninvasive liquid biopsy for germline and somatic mutations [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 722.

Abstract 3098: Urine-based molecular biomarker signatures for bladder cancer

Abstract Introduction: By 2020 the estimated incidence of genitourinary (GU) cancers (prostate, bladder, and kidney) will be over 2 million worldwide and responsible for ∼800 000 deaths. Current diagnosis and monitoring methods of GU cancer patients are often invasive and/or lack sensitivity and specificity. The tremendous success in cell-free DNA (cfDNA) blood test provides a strong rationale for using body fluids such as blood and urine as minimally invasive “liquid biopsies” to identify DNA-based molecular biomarkers in urologic malignancies. Urinary cfDNA as a liquid biopsy holds greater potential for a more sensitive alternative to blood biopsies-based tests for clinical use in GU cancers. Urinary biomarker test offers advantages including the potential for more convenient testing, monitoring, and home use. Methods: Analytic validation was conducted using urine cfDNA samples containing different SNPs with different titrations. Urine samples were collected from two different groups, including treatment-naïve patients with pathology-confirmed bladder cancer (BLCA) and healthy donors. Both tumor and germline tissues were collected from the BLCA patients. Results: We report the development, validation, and clinical application of the urine-based next-generation sequencing for integrated genomic cfDNA profiling of bladder cancers, including genomic alterations, tumor mutation burden (TMB) and microsatellite instability (MSI). The analytic sensitivity goes down to 0.5% with 100% analytical specificity. High concordance for mutations between tumor tissue and urinary cfDNA suggests a higher sensitivity alternative for the non-invasive early detection of BLCA. In addition to the NGS-based test, we also developed a Bio-Rad digital PCR assay for orthogonal confirmation of the detected biomarkers such as FGFR3 hotspot mutations. Conclusions: Our work has demonstrated the promise of using urinary cfDNA for detecting genomic alterations at the early stage of BLCA for biomarker detections. Citation Format: Zhixin Zhao, Amy Chang, Binggang Xiang, Feng Xie, Xiaohong Wang, Kemin Zhou, Jianjun Yu, Pan Du, Shidong Jia. Urine-based molecular biomarker signatures for bladder cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3098.

P-283 Analysis of KRAS mutation allele in circulating cell-free DNA derived from urine in colorectal cancer patients

Liquid biopsy is widely introduced in various malignant diseases and it provides crucial clinical information on genetic profiles. Cell-free DNA (cfDNA) derived from peripheral blood has been studied mainly. However, other body fluids also have potential to provide molecular information. Urine could be a novel and completely noninvasive source to provide genetic information. The aim of this study was to evaluate the quantity of cfDNA derived from urine in colorectal cancer patients. The accuracy of KRAS mutation allele detection was also analyzed using droplet digital PCR (ddPCR). Urine, peripheral blood and tissue samples were collected from consecutively resected colorectal cancer patients. DNA was extracted from each sample and the quantity was measured. From each DNA sample, ddPCR was performed to detect common point mutations in KRAS oncogene. A total of 143 patients were enrolled. In all patients, plasma and urine cfDNA were successfully extracted and the quality and quantity were appropriate for genetic analysis. The concentration of urine cfDNA was significantly higher than that of plasma (2448 ng/ml vs. 267 ng/ml, p< 0.05). Neither plasma nor urine cfDNA concentration correlated with cancer staging. In tumor tissue DNA, KRAS mutation allele was detected in 95 patients (66.4%), KRAS mutation was detected in 21 patients from plasma and 13 patients from urine. The concordant rate of KRAS mutation allele detection between tumor tissue DNA and plasma cfDNA was 22.1%, and between tumor tissue DNA and urine cfDNA was 13%. In the early stage patients (stage I and II), the detection rate of KRAS mutation allele was similar in plasma and urine cfDNA (15.6% vs. 15.6%), however, in advanced-stage patients (stage III and IV), the detection rate in urine cfDNA was lower than that in plasma DNA (15.9% vs. 25.4%). Furthermore, urine cfDNA had a lower detection rate of KRASmutation than plasma cfDNA in stage I and IV patients (stage I; plasma cfDNA 14%, urine cfDNA 0%, stage IV; plasma cfDNA 52%, urine cfDNA 12%), however, the detection rates were equivalent in stage II and III patients (stage II; plasma cfDNA 16%, urine cfDNA 20%, stage III; plasma cfDNA 16%, urine cfDNA 16%). Our data indicated that urine cfDNA is a useful source in complementing genetic analysis in colorectal cancer patients. The sensitivity seems to be inferior especially in advanced stage, however, it will be considered useful for evaluation of minimal residual disease in stage II and III colorectal cancer patients.

Abstract PR14: Sub-nucleosomal fragmentation in urine cell-free DNA

Abstract Cell-free DNA (cfDNA) in plasma has been shown to be a promising analyte for noninvasive diagnostics. The collection of blood plasma requires venipuncture, and plasma volume obtainable at a single point is limited. In contrast, cfDNA in urine can be collected noninvasively, with minimal assistance and in larger volumes. However, so far there has been limited success in diagnostic development using urine cfDNA as urine cfDNA is highly fragmented, and whether the characteristics of these fragments reflect underlying genomic architecture is unknown. Here, we perform a comprehensive characterization of fragmentation patterns in urine cfDNA using high-depth whole-genome sequencing from 30 healthy volunteers. We show the distribution of fragment sizes and genome-wide distribution of urine cfDNA fragments are consistent with transient protection from complete degradation by stable intermediates of nucleosome disassembly. Genome-wide nucleosome occupancy and fragment sizes in urine cfDNA are informative of the cell of origin and renal epithelial cells are among the highest contributors in urine. Based on a reference nucleosome map for urine cfDNA positioning, we developed a computational method to measure the fraction of urine cfDNA fragments with aberrant ends at unexpected genomic loci. We observe a higher fraction of fragments with aberrant ends in pediatric and adult cancer patients, distinguishing cancer samples with an area under the curve of 0.89. Our results demonstrate genomic architecture is preserved to an unexpected degree in urine cfDNA and are proof of principle that genome-wide fragmentation analysis of urine cfDNA can enable cancer diagnostics. This abstract is also being presented as Poster B12. Citation Format: Havell Markus, Jun Zhao, Tania Contente-Cuomo, Elizabeth Raupach, Ahuva Odenheimer-Bergman, Sydney Connor, Bradon McDonald, Elizabeth Hutchins, Marissa McGilvrey, Michelina C. de la Maza, Kendall Van Keuren-Jensen Van Jensen, Patrick Pirrotte Pirrotte, Ajay Goel, Carlos Becerra, Daniel D. Von Hoff, Scott Celinski, Pooja Hingorani, Muhammed Murtaza. Sub-nucleosomal fragmentation in urine cell-free DNA [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr PR14.

Abstract B03: Urine cell-free DNA (cfDNA) concentration and stability test for future clinical use

Abstract Introduction: Urine was shown to be a liquid biopsy source with high sensitivity for cfDNA mutation detection in both early- and late-stage cancer patients, outperforming plasma cfDNA and tissue samples in some patients. In addition, obtaining a urine sample is completely noninvasive. Limitations of using urine samples in the clinical setting include their unknown nucleic acid stability and the large total volume of one deposit, and therefore the presence of highly diluted cfDNA. Urine volume concentration, cfDNA stability, cfDNA quantity, and detection of wild-type (wt) EGFR alleles were explored to determine the future suitability of urine cfDNA as a diagnostic or prognostic biosource. Methods: First-void urine samples, with a higher fraction of nucleic acids due to overnight concentration, were collected from 5 healthy donors and 5 cancer patients. Cell-free urine samples were concentrated using Amicon filter tubes to determine differences in cfDNA concentration. In addition, we tested the ability to detect wild-type EGFR exon 19 in cfDNA from concentrated and unconcentrated urine samples using a peptide nucleic acid (PNA) Taqman Assay. Hereafter, stability of urine cfDNA was explored using variable storage temperatures and times and addition of a urine cfDNA preservative directly after sample collection. cfDNA was extracted using the automated QIAsymphony system with the DSP virus/pathogen kit and quantified using the Qubit dsDNA HS Assay in all experiments. Results: Amicon filter-based concentration of urine samples from 15 mL to 1.2 mL yielded a two-fold increase in cfDNA concentration. Importantly, while wild-type EGFR could not be detected in first-void urine using a PNA Taqman Assay, detection was possible using concentrated urine samples. Therefore, in all following experiments, samples were concentrated unless specified otherwise. Stability tests indicated that storage temperature matters when samples are not processed within two hours, with nearly doubled cfDNA concentrations found in samples stored at four degrees for 26 hours, compared to room temperature (RT). Addition of a cfDNA preservative yielded a six-fold higher cfDNA concentration after 26 hours of storage at RT, compared to samples without a preservative added. For this reason, we tested the use of only cfDNA preservative without concentrating the sample and found a four-fold increase in cfDNA concentration when adding only preservative, versus a 15-fold increase when using both after sample storage at RT for 24 hours. Conclusion: Patient urine samples should be either concentrated and processed within two hours, or a urine cfDNA preservative should be added directly after sample collection to prevent cfDNA degradation. Depending on the downstream application, samples where preservative was added may be concentrated to enhance the cfDNA yield, for example, when downstream NGS analysis will be performed. Citation Format: Jillian W. P. Bracht, Niki Karachaliou, Jordi Berenguer, Manuel Fernandez-Bruno, Mónica Garzón, Ana Gimenez-Capitan, Clara Mayo-de-las-Casas, Miguel Angel Molina-Vila, Rafael Rosell. Urine cell-free DNA (cfDNA) concentration and stability test for future clinical use [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B03.

A novel cell-free single-molecule unique primer extension resequencing (cf-SUPER) technology for bladder cancer non-invasive detection in urine.

BackgroundThe clinical diagnostic method for bladder cancer is cystoscopy, an invasive, expensive and inconvenient clinical test. Using urinary cell-free DNA (cfDNA) to develop non-invasive test for bladder cancer was a promising liquid biopsy.MethodsTo improve the using rate of cfDNA template and decrease the PCR bias for liquid biopsy using urinary cfDNA, we developed a cell-free single-molecule unique primer extension resequencing (cf-SUPER) technology which was done for 29 matched urinary cfDNA and tumor DNA samples of bladder cancer patients to evaluate consistency of mutation profiles. Then, a 22 high mutational frequence genes was selected to form an uriprier panel, which was analyzed in 100 patients (47 bladder cancer cases and 53 controls) using cf-SUPER technology. This performance of the technology was evaluated using bioinformatic tools and clinical samples.ResultsThe study showed that cf-SUPER technology can accurately detect mutations with allele fractions even low as 0.01% and the DNA input as low as 1 ng. The consistency of mutation profiles and clinical pathological diagnose between 29 matched urinary cfDNA and tumor DNA samples was respectively 82.76% and 89.66% by using cf-SUPER technology. Using cf-SUPER technology, the sensitivity and specificity were 98%, 94% respectively for uriprier panel in non-invasive test.ConclusionsThe preliminary work shows that cf-SUPER technology will be a promising method for liquid biopsy. Focusing urinary cfDNA, the non-invasive diagnose and monitoring of bladder cancer can come true by using cf-SUPER technology.

Urinary cell-free DNA analysis for tumor mutation detection in patients with oligometastatic colorectal cancer.

e15545 Background: Cell-free DNA (cfDNA) analysis from blood is a useful liquid biopsy strategy. However, it requires phlebotomy and moderate volumes of plasma. Here, we explored the feasibility of urinary cfDNA (ucfDNA) analysis in patients with oligometastatic colorectal cancer (CRC). Methods: We applied error-corrected targeted next-generation sequencing (NGS) (AVENIO ctDNA surveillance assay) to 10 patients with oligometastatic CRC to the liver who underwent curative-intent surgery. 40 paired samples (tumor, plasma, urine supernatant, peripheral blood mononuclear cells (PBMCs)) were collected, from which DNA was extracted. Plasma and urine cfDNA samples (acquired preoperatively) were sequenced to a median de-duplicated depth of 8,065X with on-target rate of 71%. Similarly high depth targeted NGS was applied to tumor and PBMC samples. Variants were called from plasma cfDNA and tumoral DNA and queried in ucfDNA using methods derived from CAncer Personalized Profiling by deep Sequencing (CAPP-Seq) with integrated digital error suppression (iDES). Clonal hematopoiesis was accounted for by removing mutations also present in PBMCs. Results: Patients had a median of 2 tumors resected (range 1-7) with average sum of longest diameters of 8.7 cm (range 0.6-11). We used an average of 10 ml of blood and 37 ml of urine for cfDNA extraction. Circulating tumor DNA (ctDNA) was detected in all preoperative plasma samples (n = 10) at a mean mutant allele fraction (MAF) of 2.35% (range 0.09-11.78). Plasma ctDNA MAFs correlated significantly with tumor SLD (Pearson r = 0.81, P = 0.03). The average number of single nucleotide variants detected in plasma cfDNA, tumor and ucfDNA was 8 (range 1-42), 4 (range 1-7) and 2 (range 0-11), respectively. Indicative of concordance, 79% of the mutations called in tumor were detected in plasma. Tumor-derived DNA was detected in the preoperative urine of 6 patients (60%) with a MAF of 0.05% (range 0.03-0.12), ~35-fold less than in plasma (MAF of 1.78%, range 0.09-7.94) (P = 0.004). 38% of mutations identified in tumor and 26% from plasma were detected in ucfDNA for these 6 patients. In two patients, targetable mutations (BRAF p.Asp594Gly and PIK3CA p.Glu545Lys) present in tumor were also detected in ucfDNA, with the latter not detected in plasma. Conclusions: Our results indicate that ucfDNA analysis in patients with oligometastatic CRC is feasible; however, the mutational levels and detection sensitivity were much lower than from plasma. Yet, there may be situations where actionable mutations are missed in plasma but detected in urine.

Clinical Significance of Hotspot Mutation Analysis of Urinary Cell-Free DNA in Urothelial Bladder Cancer.

Recent studies showed the clinical utility of next-generation sequencing of urinary cell-free DNA (cfDNA) from patients with urothelial bladder cancer (UBC). In this study, we aimed to develop urinary cfDNA analysis by droplet digital PCR (ddPCR) as a high-throughput and rapid assay for UBC detection and prognosis. We analyzed urinary cfDNA of 202 samples from 2 cohorts. Test cohort was designed for investigating clinical utility of urinary cfDNA, and was composed of 74 samples from patients with UBC, and 52 samples of benign hematuria patients. Validation cohort was designed for validation and assessment of clinical utility comparing urinary cfDNA with UroVysion (Abbott, Illinois, USA), and was composed of 40 samples from patients with UBC, and 36 prospectively collected samples from patients under surveillance after surgery for urothelial carcinoma. We performed ddPCR analysis of hotspot gene mutations (TERT promoter and FGFR3). In the test cohort, the sensitivity of urinary cfDNA diagnosis was 68.9% (51/74) and the specificity was 100% in patients with UBC. The sensitivity increased to 85.9% when used in conjunction with urine cytology. In addition, patients with high TERT C228T allele frequency (≥14%) had significantly worse prognosis in bladder tumor recurrence than patients with low TERT C228T allele frequency or negative TERT C228T (p = 0.0322). In the validation cohort, the sensitivity of urinary cfDNA was 57.5% (23/40) and the specificity was 100% in UBC patients. The sensitivity of the combination of urine cytology with our hotspot analysis (77.5%) was higher than that of urine cytology with UroVysion (68.9%). In the post-surgical surveillance group, patients positive for the TERT C228T mutation had significantly worse prognosis for bladder tumor recurrence than mutation negative patients (p < 0.001). In conclusion, ddPCR analysis of urinary cfDNA is a simple and promising assay for the clinical setting, surpassing UroVysion for detection and prognosis determination in UBC.

Comparison of Four Commercial Kits for Isolation of Urinary Cell-Free DNA and Sample Storage Conditions.

Urinary cell-free DNA (cfDNA) is an attractive body fluid for liquid biopsy. In this study, we compared the efficiencies of four commercial kits for urinary cell-free DNA (cfDNA) isolation and of various sample storage conditions. Urinary cfDNA was isolated from 10 healthy individuals using four commercial kits: QIAamp Circulating Nucleic Acid Kit (QC; Qiagen), MagMAX™ Cell-Free DNA Isolation Kit (MM; Applied Biosystems), Urine Cell-Free Circulating DNA Purification Midi Kit (NU; Norgen Biotek), and Quick-DNA™ Urine Kit (ZQ; Zymo Research). To assess the isolation efficiency, an Agilent 2100 Bioanalyzer with High Sensitivity DNA chips was used, and cfDNA yield was defined as the amount of cfDNA obtained from 1 mL of urine. MM and QC provided the highest cfDNA yield in the 50–300 bp range, and MM and NU gave the highest cfDNA yield in the 50–100 bp range. In particular, the NU kit was efficient for isolation of more fragmented cfDNA in the range of 50–100 bp with the lowest cellular genomic DNA contamination. ZQ had the best cost-efficiency for isolating the same amount of urinary cfDNA. Samples stored at −70 °C with the addition of 10 mM EDTA resulted in the highest cfDNA yield 3 months after sample collection.

MP01-12 DETECTION OF UROTHELIAL CARCINOMA BASED ON COPY NUMBER PROFILES OF URINARY CFDNA USING SHALLOW WHOLE-GENOME SEQUENCING

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I (MP01)1 Apr 2020MP01-12 DETECTION OF UROTHELIAL CARCINOMA BASED ON COPY NUMBER PROFILES OF URINARY CFDNA USING SHALLOW WHOLE-GENOME SEQUENCING Yanqing Gong*, Guangzhe Ge, Ding Peng, Bao Guan, Yuanyuan Zhou, Weimin Ci, Xuesong Li, and Liqun Zhou Yanqing Gong*Yanqing Gong* More articles by this author , Guangzhe GeGuangzhe Ge More articles by this author , Ding PengDing Peng More articles by this author , Bao GuanBao Guan More articles by this author , Yuanyuan ZhouYuanyuan Zhou More articles by this author , Weimin CiWeimin Ci More articles by this author , Xuesong LiXuesong Li More articles by this author , and Liqun ZhouLiqun Zhou More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000815.012AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Current non-invasive assays for urothelial cancer (UC) lack sensitivity and specificity. Given the utility of plasma cell-free DNA (cfDNA) biomarkers, the development of urinary cfDNA biomarkers may improve the diagnostic sensitivity. METHODS: We assessed copy number aberrations (CNAs) by shallow genome-wide sequencing of urinary cfDNA in 95 cancer-free individuals and 65 UC, 58 kidney cancer and 45 prostate cancer patients. We used a support vector machine (SVM) to develop a diagnostic classifier based on CNA profiles to detect UC (UCdetector). The model was further validated in an independent cohort (52 cases). Genome sequencing data of tumor specimens from 90 upper tract urothelial cancers (UTUC) and CNA data for 410 urothelial carcinomas of bladder (UCB) from TCGA were used to validate the classifier. Genome sequencing data for urine sediments of 32 UC patients were compared with cfDNA. To monitor the treatment efficacy, we collected cfDNA from 7 post-treatment patients. RESULTS: We showed that urinary cfDNA could be a more sensitive alternative to urinary sediment. The UCdetector can detect UC at a median sensitivity of 86.5% and specificity of 94.7%. The UCdetector performed well in an independent validation dataset. Notably, the CNA features selected by UCdetector were specific markers for both UTUC and UCB. Moreover, the CNA changes in cfDNA were consistent with the treatment effects. Meanwhile, the same strategy could localize genitourinary cancers to tissue-of-origin in 70.1% of patients. CONCLUSIONS: Our findings underscore the potential utility of urinary cfDNA CNA profiles as a basis for non-invasive UC detection and surveillance. Source of Funding: This work was supported by CAS Strategic Priority Research Program (XDA16010102 to W.C.), the National Key R&D Program of China (2018YFC2000100 to W.C.), CAS (QYZDB-SSW-SMC039 to W.C.), the National Natural Science Foundation of China (81672541 to W.C.), and K.C.Wong Education Foundation to W.C. © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e6-e6 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yanqing Gong* More articles by this author Guangzhe Ge More articles by this author Ding Peng More articles by this author Bao Guan More articles by this author Yuanyuan Zhou More articles by this author Weimin Ci More articles by this author Xuesong Li More articles by this author Liqun Zhou More articles by this author Expand All Advertisement PDF downloadLoading ...