Urinary cfDNA Research Articles

P113 Developing an assay to detect and quantify donor-derived dna in the blood and urine of solid organ transplant patients

Aim Detection of circulating donor-derived cell-free DNA (cfDNA) as a non-invasive biomarker of graft injury shows tremendous potential for transplant monitoring. Increases in the amount of cfDNA in blood or urine indicate injury to the transplanted organ, including rejection. Also known as a liquid biopsy, this method is less invasive and more sensitive than an actual biopsy to detect early rejection. We proposed to develop a clinical test based on the detection and quantification of donor single-nucleotide polymorphisms (SNPs) using cfDNA extracted from plasma and urine of solid organ transplant patients. Methods Using genomic DNA, informative SNP markers from donor and recipient (ThermoFisher) will be identified using real-time PCR (Roche). cfDNA from transplant recipients will be extracted from plasma and urine (Qiagen), and informative SNPs will be quantified using digital-droplet PCR (ddPCR), (Bio-Rad). For development of the assay blood and urine samples were collected from 25 healthy individuals, genomic and cfDNA were extracted, and analytical parameters determined including limits of detection (LOD) and limits of quantification (LOQ) using ddPCR. Results cfDNA extraction was successfully performed from blood and urine. The average cfDNA concentration was 1.36 ± 0.54 ng/ul from plasma, and 1.15 ± 2.1 ng/ul from urine. Determined from genomic DNA, an average of 4.9 informative markers was present between any two of the 25 individuals, although one pairing had no informative markers present, indicating the need for a larger panel using more than 36 SNPs. LOD studies determined the false positivity rate to be less than 0.0025%. Preliminary LOQ testing indicates that 0.5–0.75% donor-derived DNA can be detected. Conclusions We have established a laboratory developed test to detect donor-derived cfDNA from multiple sample types such as blood and urine at a sensitivity that appears acceptable for clinical testing. In collaboration with the local solid organ transplant program, the clinical utility of this assay will be evaluated in kidney transplant recipients.

Abstract 1595: Detection of tumor-derived DNA in urine cell-free DNA of pre-operative renal cell carcinoma patients

Abstract The current clinical approaches lack reliable methods (i.e. imaging and serum biomarkers) to identify patients with renal cell carcinoma (RCC) resulting in late detection, where the 5-year survival rate is poor (<8%). Thus, there is an urgent need for better minimally-invasive screening and diagnostic blood/urine assays for earlier detection. Given the logistical ease of collecting urine, urinary cell-free DNA (cfDNA) represents an advantageous source to detect tumor biomarkers and permit for routine/repeat sampling. We previously demonstrated urine cfDNA contains tumor-derived DNA mutations in liver cancer, distal of the kidney filtration barrier. However to date, urinary cfDNA as a source of RCC biomarkers remains unexplored. We hypothesize due to the direct contact of urinary flow-through with the kidney organ that urinary cfDNA is enriched for RCC-derived DNA mutations. To test this, we evaluated the feasibility of detecting tumor-derived DNA mutations utilizing a hotspot 50-cancer gene panel droplet next generation sequencing (dNGS, Raindance Technologies) assay in a pilot cohort of 42 specimens obtained from 13 unique RCC patients and 3 controls (2 RCC cell-lines and 1 normal DNA). The specimens evaluated include matched tumor formalin-fix paraffin-embedded (FPPE) DNA (n=11), urine cfDNA (n=15), and peripheral blood lymphocytes (PBL) DNA (n=13), serving as a germline control. 150ng of genomic DNA from FFPE or PBLs and ≥5ng of urine cfDNA was used for the dNGS assay. An average of 5.5E10 reads were obtained of which 86% were on-target for all 42 specimens providing an average 3.9E3x coverage. Initial dNGS variant analysis utilizing a conservative 5% variant frequency (VF%) cut-off was performed in 11 unique RCC patients containing matched FFPE-urine cfDNA-PBL, of which five patients also contained post-operative urine. 100% of tumor FFPEs (n=11) contained detectable single nucleotide variants (SNVs) (1-10 SNVs) upon germline variant removal. 80% of matched urine cfDNA contained ≥1 tumor-concordant SNV. Additionally, 37.5% of patients contained previously reported somatic mutations (i.e. PIK3CA T1025T, RET L769L, TP53 H233Y, etc) that were tumor-urine concordant. Interestingly, three patients harbored known somatic mutations that were not detected in the matched tumor and three post-operative urine cfDNA specimens contained tumor-derived SNVs despite surgical resection. Ongoing follow-up will reveal if urine cfDNA is a suitable source for identifying tumor heterogeneity and/or residual disease in RCC patients. Furthermore, evaluation of matched plasma is ongoing. Overall, this proof-of-concept study demonstrates feasibility of utilizing urinary cfDNA as a noninvasive source to detect RCC-related biomarkers and provides a potential platform to study and discover novel RCC biomarkers for earlier detection of new/recurrent RCCs. Citation Format: Selena Lin, Jennifer A. Linehan, Ruby Kuang, Selvi Guharaj, Timothy G. Wilson, Dave SB Hoon. Detection of tumor-derived DNA in urine cell-free DNA of pre-operative renal cell carcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1595.

Abstract 1602: Urine cell-free DNA as early biomarker in immunotherapy response in NSCLC

Abstract Introduction: Immunotherapy shows a durable tumor response in approximately 20% of advanced NSCLC patients. Imaging by CT is used to evaluate tumor response. PD-L1 immunohistochemistry is generally used to predict survival advantages, however with low predictive value. Therefore, it is important that alternative predictive biomarkers are investigated. Minimal invasive techniques may be a solution. The role of circulating cell-free DNA (cfDNA) was found to be an early biomarker for tumor response in KRAS positive Non-Small Cell Lung Cancer (NSCLC) patients treated with immunotherapy (N=6). In this study we will determine the role of urine cell-free DNA (ucfDNA) in an extended cohort. The non-invasive method of urine collection makes it an excellent source for mutation detection and follow-up. Methods: In an ongoing study KRAS positive patients treated with immunotherapy, are being tested for cfDNA at baseline (before treatment, 1, 2, 4, 6 and thereafter every 12 weeks until disease progression) with a KRAS G12-13 or Q61H digital droplet PCR (ddPCR) screening assay (BioRad). In a pilot study, urine from these patients is collected in a tube with 5 ml of STRECK reagent at baseline and at 6 weeks. Results: In 16/27 patients with KRAS positive advanced NSCLC, the mutation was detected in plasma at baseline and subsequent evaluation time points. Urine cfDNA as a predictive marker seems promising and results will be reported and presented at the AACR meeting. Conclusion: Among patients with advanced NSCLC treated with immunotherapy plasma and urine cfDNA are collected and will be presented as non-invasive early biomarker for tumor response evaluation. Citation Format: Anthonie J. van der Wekken, Arja ter Elst, Anneke Miedema, Thijo J. Hiltermann, Harry J. Goen, Ed Schuuring. Urine cell-free DNA as early biomarker in immunotherapy response in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1602.

Abstract 581: Feasibility test of KRAS mutation in urine circulating tumor DNA in metastatic pancreatic ductal adenocarcinoma

Abstract Circulating tumor DNA (ctDNA) has been known to be released from tumor cells and evaluated as potential biomarkers for therapeutic responses. In our previous study, we selected KRAS mutation and evaluated applicability as a prognostic marker through the quantitative analysis of plasma ctDNA. Then we proved that KRAS mutation of ctDNA from plasma is significant biomarker for prediction of clinical outcome in pancreatic ductal adenocarcinoma (PDA). Here we investigated whether KRAS mutation in urine ctDNA had the prognostic impact as shown of plasma ctDNA. Of the 106 patients enrolled, 67 were males and median age was 66 years. Total of 51 PDA has been enrolled in this study and the median age was 63 years old and 30 males (58.8%). Urine was separated by established centrifugation method and ctDNA were extracted using QIAamp Circulating Nucleic Acid Kit (Qiagen, Germany) from 4 mL of urine. Extracted ctDNA was quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, USA). QX200 Droplet Digital PCR System (Biorad, USA) was applied to measure frequency of KRAS mutation by KRAS screening multiplex droplet digital PCR kit, which covers seven common mutation sites (Biorad, USA). Mutant concentration and fractional abundance were analyzed by QuantaSoft software (Biorad, USA). There was no significant difference between resectable (N=21), locally advanced (N=11) and metastatic (N=19) groups. When compared high versus low KRAS mutation concentration and KRAS fractional abundance group in each stage, high KRAS mutation groups showed short overall survival (p = 0.0321) in metastatic group. However, the correlation between plasma and urine was low (r=0.193) in metastatic group. This study represented the possibility of KRAS mutation concentration and fractional concentration of urine ctDNA as prognostic factors in metastatic PDA. Urine cfDNA role has not been well investigated in PDA. Urine is easy to obtain and is not well known for its mechanism. Therefore, we have conducted a pilot study and we will explore KRAS mutation status in urine ctDNA in further studies. (This study was supported by National Cancer Center, Korea, Grant no. 1510203.) Citation Format: Min Jeong Kwon, Min Kyeong Kim, Sang Myung Woo, Kyong-Ah Yoon, Yun Hee Kim, Boram Park, Jung Nam Joo, Sang Jae Park, Sun-Young Kong. Feasibility test of KRAS mutation in urine circulating tumor DNA in metastatic pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 581.

Urinary cell-free DNA is a versatile analyte for monitoring infections of the urinary tract

Urinary tract infections are one of the most common infections in humans. Here we tested the utility of urinary cell-free DNA (cfDNA) to comprehensively monitor host and pathogen dynamics in bacterial and viral urinary tract infections. We isolated cfDNA from 141 urine samples from a cohort of 82 kidney transplant recipients and performed next-generation sequencing. We found that urinary cfDNA is highly informative about bacterial and viral composition of the microbiome, antimicrobial susceptibility, bacterial growth dynamics, kidney allograft injury, and host response to infection. These different layers of information are accessible from a single assay and individually agree with corresponding clinical tests based on quantitative PCR, conventional bacterial culture, and urinalysis. In addition, cfDNA reveals the frequent occurrence of pathologies that remain undiagnosed with conventional diagnostic protocols. Our work identifies urinary cfDNA as a highly versatile analyte to monitor infections of the urinary tract.

Quantifying Cell Free DNA in Urine: Comparison Between Commercial kits, Impact of Gender and Inter-Individual Variation

DNA can enter the blood circulation from living cells by extracellular vesicles or at cell death, and pass into urine through the kidney barrier. Urine can be collected non-invasively, making it an interesting source of cell-free DNA (cfDNA) for research studies and ultimately for clinical diagnostics. However, there is currently a lack of data on the quantity and variability of cfDNA in urine. Here, we benchmark two commercial urine cfDNA isolation kits with respect to the quantity of DNA, the labor time, and cost. The results show distinctive differences between each kit. Furthermore, the cfDNA amount from the same probands varied strongly from day to day and may be higher in female samples than in male samples (p=0.003).

MP54-16 URINARY EXOSOMAL AND CELL-FREE DNA DETECTS SOMATIC MUTATION AND COPY NUMBER ALTERATION IN UROTHELIAL CARCINOMA OF BLADDER

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I1 Apr 2018MP54-16 URINARY EXOSOMAL AND CELL-FREE DNA DETECTS SOMATIC MUTATION AND COPY NUMBER ALTERATION IN UROTHELIAL CARCINOMA OF BLADDER Kwang Hyun Kim, Bong Suk Sim, and Dong Hyeon Lee Kwang Hyun KimKwang Hyun Kim More articles by this author , Bong Suk SimBong Suk Sim More articles by this author , and Dong Hyeon LeeDong Hyeon Lee More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.1707AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Urothelial bladder canrcinoma (UBC) is characterized by a large number of genetic alteration. Urinary DNA is promising resources for liquid biopsy in urological malignancies. In this study, we performed genomic profiling of UBC and matched urinary cell free DNA (cfDNA) and exosomal DNA (exoDNA). METHODS We included 9 patients who underwent surgery for UBC. Fresh frozen tumor sample and normal blood sample was used for genomic profiling of UBC. We also performed genomic profiling of matched urinary DNA to investigate whether genomic alteration in tumor samples are echoed in urinary DNA. Urinary exoDNA was extracted from urinary exosome which was isolated by ExoQuick and urinary cfDNA was extracted by commercial kit using magnetic bead. We performed 9 gene target sequencing for somatic mutation analysis and low depth whole genome sequencing (ldWGS) for copy number analysis. RESULTS We found 17 somatic mutations in 6 patients, and 17 included 6 nonsynonymous SNVs, 3 stopgain SNVs, 2 frameshift deletion and 6 synonymous SNVs. Of 17 somatic mutations, 12 were identified in cfDNA and exoDNA with the mean allele frequency of 54.5% and 65.6%, respectively. Mean depth of cfDNA and exoDNA was 1721X and 1627X, respectively (Figure 1). In copy number analysis, mean 20.4% of whole genome region was covered by >1X. Copy number plots of cfDNA and exoDNA showed similar pattern with those of tumor samples. When we compare the log2 ratio of 100k bin size in whole genome regions, Pearson correlation coefficients of tumor vs cfDNA (0.481) and tumor vs exoDNA (0.455) were higher than that of tumor vs normal (0.086) (Figure 2). CONCLUSIONS Both urinary cfDNA and exoDNA were representative of the entire human genome and allowed genomic profiling of UBC. Specifically, copy number analysis using ldWGS has potential to be used as tools developing biomarker with low cost and whole genome coverage. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e718 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Kwang Hyun Kim More articles by this author Bong Suk Sim More articles by this author Dong Hyeon Lee More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

1011 - ddPCR measurement of the TERT 228 G>A/T mutation in urinary cfDNA: A useful tool for non-invasive bladder cancer detection?

1011 - ddPCR measurement of the TERT 228 G>A/T mutation in urinary cfDNA: A useful tool for non-invasive bladder cancer detection?

1076 - Evaluation of hTert-mutations in urinary cfDNA for bladder cancer detection

1076 - Evaluation of hTert-mutations in urinary cfDNA for bladder cancer detection

Toward Personalised Liquid Biopsies for Urothelial Carcinoma: Characterisation of ddPCR and Urinary cfDNA for the Detection of the TERT 228 G>A/T Mutation.

Background:TERT promotor mutations are present in >75% of bladder tumours; these mutations are also detectable in urine. Previous studies have used urinary pellet DNA, and semi-quantitative methods unsuitable for detecting very low mutant allele frequencies.Objective:In this proof-of-principle study we use ddPCR to count the DNA molecules with wt and mutant TERT sequences in urinary cfDNA from patients whose bladder cancers harbour TERT mutations.Methods:Urinary cfDNA prepared from the urine from 104 bladder cancer patients was analysed. We determined the mutant allele frequency across stages and grades of disease, analysed concordance between cfDNA and tumour DNA, compared cfDNA with pellet DNA, and analysed the quantity and size distribution of cfDNA.Results:In 71 of 77 patients with a 228 G>A/T mutant tumour, the mutation was also detected in urinary cfDNA by ddPCR; all 6 “false negatives” were low grade pTa tumours. Overall concordance between tissue and cfDNA mutation status was 92%, and 100% was achieved for high grade disease. Median mutant allele frequencies in urinary cfDNA were 3.4, 13.4 and 32.1% in grade 1, 2 and 3 disease. The 228 G>A/T mutation was not detected in urinary cfDNA in 26 out of 27 mutation-negative patients (96% specificity).Conclusions:Concordance between tumour DNA and urinary cfDNA is high, and TERT 228 G>A/T ddPCR may prove useful for monitoring patients that harbour this mutation. Mutant allele frequencies in cfDNA are often high, but assays capable of detecting very low mutant allele frequencies will be required to achieve high sensitivity in low grade disease.

Urine cell-free DNA is a biomarker for nephroblastomatosis or Wilms tumor in PIK3CA-related overgrowth spectrum (PROS).

PurposeWe set out to facilitate the molecular diagnosis of patients with PIK3CA-related overgrowth spectrum (PROS), a heterogeneous somatic disorder characterized by variable presentations of segmental overgrowth, vascular malformations, skin lesions, and nephroblastomatosis, rare precursor lesions to Wilms tumor (WT). Molecular diagnosis of PROS is challenging due to its mosaic nature, often requiring invasive biopsies.MethodsDigital droplet polymerase chain reaction was used to analyze tissues including urine, saliva, buccal cells, and blood, from eight patients with PROS. Further analyses were performed on plasma and urine cell-free DNA (cfDNA).ResultsPIK3CA variants were detected in plasma cfDNA at levels up to 0.5% in 50% of tested samples. In addition, high levels of PIK3CA variants in urine cfDNA correlated with a history of nephroblastomatosis compared to patients without renal involvement (p<0.05).ConclusionDigital droplet PCR is a sensitive molecular tool that enables low-level variant detection of PIK3CA in various tissue types, providing an alternative diagnostic method. Furthermore, urine cfDNA is a candidate biomarker for nephroblastomatosis in PROS, which may be useful to refine screening guidelines for tumor risk in these patients.

Cell-free DNA: the role in pathophysiology and as a biomarker in kidney diseases.

Cell-free DNA (cfDNA) is present in various body fluids and originates mostly from blood cells. In specific conditions, circulating cfDNA might be derived from tumours, donor organs after transplantation or from the foetus during pregnancy. The analysis of cfDNA is mainly used for genetic analyses of the source tissue -tumour, foetus or for the early detection of graft rejection. It might serve also as a nonspecific biomarker of tissue damage in critical care medicine. In kidney diseases, cfDNA increases during haemodialysis and indicates cell damage. In patients with renal cell carcinoma, cfDNA in plasma and its integrity is studied for monitoring of tumour growth, the effects of chemotherapy and for prognosis. Urinary cfDNA is highly fragmented, but the technical hurdles can now be overcome and urinary cfDNA is being evaluated as a potential biomarker of renal injury and urinary tract tumours. Beyond its diagnostic application, cfDNA might also be involved in the pathogenesis of diseases affecting the kidneys as shown for systemic lupus, sepsis and some pregnancy-related pathologies. Recent data suggest that increased cfDNA is associated with acute kidney injury. In this review, we discuss the biological characteristics, sources of cfDNA, its potential use as a biomarker as well as its role in the pathogenesis of renal and urinary diseases.

Mutation-Enrichment Next-Generation Sequencing for Quantitative Detection of KRAS Mutations in Urine Cell-Free DNA from Patients with Advanced Cancers.

Purpose: Tumor-derived cell-free DNA (cfDNA) from urine of patients with cancer offers noninvasive biological material for detection of cancer-related molecular abnormalities such as mutations in Exon 2 of KRASExperimental Design: A quantitative, mutation-enrichment next-generation sequencing test for detecting KRASG12/G13 mutations in urine cfDNA was developed, and results were compared with clinical testing of archival tumor tissue and plasma cfDNA from patients with advanced cancer.Results: With 90 to 110 mL of urine, the KRASG12/G13 cfDNA test had an analytical sensitivity of 0.002% to 0.006% mutant copies in wild-type background. In 71 patients, the concordance between urine cfDNA and tumor was 73% (sensitivity, 63%; specificity, 96%) for all patients and 89% (sensitivity, 80%; specificity, 100%) for patients with urine samples of 90 to 110 mL. Patients had significantly fewer KRASG12/G13 copies in urine cfDNA during systemic therapy than at baseline or disease progression (P = 0.002). Compared with no changes or increases in urine cfDNA KRASG12/G13 copies during therapy, decreases in these measures were associated with longer median time to treatment failure (P = 0.03).Conclusions: A quantitative, mutation-enrichment next-generation sequencing test for detecting KRASG12/G13 mutations in urine cfDNA had good concordance with testing of archival tumor tissue. Changes in mutated urine cfDNA were associated with time to treatment failure. Clin Cancer Res; 23(14); 3657-66. ©2017 AACR.

A Study of Cell-free DNA Fragmentation Pattern and Its Application in DNA Sample Type Classification.

Plasma cell-free DNA (cfDNA) has certain fragmentation patterns, which can bring non-random base content curves of the sequencing data's beginning cycles. We studied the patterns and found that we could determine whether a sample is cfDNA or not by just looking into the first 10 cycles of its base content curves. We analysed 3189 FastQ files, including 1442 cfDNA, 1234 genomic DNA, 507 FFPE tumour DNA and 6 urinary cfDNA. By deep analysing these data, we find the patterns are stable enough to distinguish cfDNA from other kinds of DNA samples. Based on this finding, we build classification models to recognise cfDNA samples by their sequencing data. Pattern recognition models are then trained with different classification algorithms like k-nearest neighbours (KNN), random forest and support vector machine (SVM). The result of 1000 iteration .632+ bootstrapping shows that all these classifiers can give an average accuracy higher than 98%, indicating that the cfDNA patterns are unique and can make the dataset highly separable. The best result is obtained using random forest classifier with a 99.89% average accuracy (σ = 0.00068). A tool called CfdnaPattern (http://github.com/OpenGene/CfdnaPattern) has been developed to train the model and to predict whether a sample is cfDNA or not.

Abstract 726: Ultrasensitive quantification of promoter methylation in cell-free circulating DNA for early detection of lung cancer

Abstract Lung cancer is the leading cause of cancer-related deaths worldwide. Early detection of lung cancer using Low Dose Computed Tomography (LDCT) screening has been shown to decrease the mortality rate. However, most nodules found are deemed to be benign upon further invasive testing. Thus, complementary minimally-invasive tests are being sought that will help discriminate malignant from benign nodules. Molecular biomarkers are increasingly becoming part of routine clinical practice for the diagnosis, prognosis or prediction of treatment response, with improved disease management and survival outcome. Cell-free circulating DNA (cfDNA) in body fluids, including serum, plasma and urine, has recently emerged as a surrogate for tumor DNA. In addition to providing a minimally-invasive source of tumor DNA, cfDNA reflects molecular alterations and tumor heterogeneity. Epigenetic changes, including DNA methylation, occur early in carcinogenesis. Cancer cells are characterized both by global hypomethylation and hypermethylation of CpG islands in gene promoter regions. Analysis of tumor-specific DNA methylation in cfDNA is a promising strategy for applying epigenetic biomarkers to the detection of cancers at an early-stage. In a prior genome-wide analysis of DNA methylation, we identified a locus methylated de novo in fresh frozen tumor tissues resected from stage I lung cancer patients, that had high discriminatory power to distinguish tumor from non-tumor tissues in multiple patient cohorts. High promoter methylation was also associated with shorter cancer-specific survival. The present study aims at evaluating the diagnostic significance of promoter methylation in cfDNA, and the prognostic value in formalin-fixed paraffin-embedded (FFPE) tissues. We developed a methylation-specific droplet digital PCR (ddPCR) assay to quantify rare methylation events. DNA was subjected to bisulfite treatment to convert unmethylated cytosine residues to uracil. We designed specific primers and probe containing 7 CpGs to only amplify the methylated promoter. Experimental conditions were first optimized using fully methylated and unmethylated control DNAs, DNA extracted from lung cancer cell lines, germline cells and lung tissues (paired tumor and non-tumor). The ddPCR assay has a limit of detection of 30 haploid genomes equivalent of methylated promoter DNA, and a limit of quantification of a single methylated allele present at 0.2% (i.e., 1 methylated copy among 500 unmethylated copies). We demonstrated the assay linearity, reproducibility and specificity for the methylated locus. Differences in methylation levels between tumors and adjacent tissues were also observed. We have thus established a robust and ultrasensitive method for standardized determination of promoter methylation status in cfDNA. We are currently evaluating its potential value for noninvasive diagnosis and prognosis of lung cancer patients. Citation Format: Delphine Lissa. Ultrasensitive quantification of promoter methylation in cell-free circulating DNA for early detection of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 726. doi:10.1158/1538-7445.AM2017-726

Genomewide bisulfite sequencing reveals the origin and time-dependent fragmentation of urinary cfDNA

Urinary cell-free (cf) DNA holds great potential as a completely noninvasive form of liquid biopsy. Knowledge of the composition of cfDNA by tissue of origin is useful for guiding its clinical uses. We conducted a global survey of urinary cfDNA composition using genomewide bisulfite sequencing. While previous studies focused on detecting cfDNA from a single source at a time, genomewide tissue specific methylation signatures allow us to simultaneously deduce the proportional contribution from each contributing tissue. The proportional contributions derived from methylation deconvolution are highly correlated with those calculated using allograft-derived donor-specific genetic markers in the urine of hematopoetic stem cell and renal transplant recipients. We found a large variation of proportional contributions from different tissues. We then assessed if cfDNA undergoes time-dependent fragmentation in urine by conducting in vitro incubation experiments. In vitro incubation at 37°C showed that urinary cfDNA concentration decreased under first order kinetics with a half-life of 2.6 to 5.1h. This is reflected in parallel by a decrease in the proportion of long fragments and increase in amplitude of 10bp periodicity seen in the cfDNA size profile. This global survey of urinary cfDNA has deepened our understanding of the composition, degradation and variation of cfDNA in the urinary tract and has laid a foundation for the use of genomewide urinary cfDNA sequencing as a molecular diagnostics tool.

Mutation enrichment next-generation sequencing for quantitative detection of KRAS mutations in urine cell-free DNA from patients with advanced colorectal and other cancers.

602 Background: Molecular testing of cell-free (cf) DNA from urine is a completely non-invasive approach for detection of actionable mutations in cancer. Methods: A quantitative mutation enrichment next-generation sequencing (NGS) urine cell-free (cf) DNA KRASG12/G13mutation test was developed and results compared to clinical testing of archival tumor tissue and research testing of plasma cfDNA from patients with advanced colorectal (n=56, 79%) and other advanced cancers (n=15, 21%). Results: The analytical sensitivity of the KRASG12/G13 cfDNA test was 0.002%-0.006% mutant copies in wild-type background. In 71 patients, the agreement between urine cfDNA and tumor was 73% (sensitivity 63%; specificity 96%); the agreement increased to 89% for patients with recommended 90-110mL of urine. In 33 patients with available plasma samples, the agreement with tumor was 94% (sensitivity 92%; specificity 100%). In patients treated with systemic therapy there was lower number of KRASG12/G13 copies in urine and plasma cfDNA on therapy compared to baseline and progression ( P&lt;0.003). Decrease in urine and plasma cfDNA KRASG12/G13 copies on therapy compared to no change/increase was associated with longer median time to treatment failure ( P&lt;0.05). Conclusions: Mutation enrichment NGS detection of KRASG12/G13 mutations in urine cfDNA has good concordance with archival tumor tissue. Changes in urine cfDNA correspond with time to treatment failure.

Abstract 424: Urine cf-DNA SNPs as early biomarkers of prostate cancer

Abstract Background: The current strategies of prostate cancer (PC) detection, such as serum PSA, digital rectal examination (DRE) and prostate biopsy have been hampered by many limitations, including the degree of aggressiveness, invasiveness, non-specificity to PCa, over diagnosis and over treatment. Recent studies have proposed the use of cell-free circulating DNA (cf-DNA) as non-invasive biomarkers for cancer diagnosis, prediction of tumor burden and prognosis. We hypothesize that a selective subset of PC associated Single Nucleotide Polymorphisms (SNPs) in urine cf-DNA detects PCa with high specificity and sensitivity. Aim: To identify PC specific SNPs in urine cf-DNA from prostate cancer (PC) patients and further validate their clinical utilities as non-invasive biomarkers for early detection of PC in comparison to other genitourinary conditions, such as benign prostate hyperplasia (BPH) and prostatitis. Method and Results: A protocol was optimized for collection, processing and storage of urine samples and isolation of urine cf-DNA. Our discovery dataset was constructed by comparative Illumina HiSeq next generation sequencing (NGS) of urine cf-DNA from three different cohorts of patients presenting with PC (Gleason score 6), benign prostatic hyperplasia (BPH) or prostatitis. The data was analyzed by SNAPE-pooled and annotated using ANNOVAR; revealing 24 PC-specific non-synonymous SNPs. The selective detection of urine cf-DNAs in PC patients was verified by conventional PCR using primers flanking their genomic sites. Our validation analysis was performed in patients (n = 50/group) diagnosed with PC (Gleason score ≤ 3+4), Gleason score ≥ than 4 + 3, or patients deemed as “no cancer seen”. This was performed by Droplet Digital PCR (ddPCR) targeting wild-type versus variant (SNP) DNA in each patient sample. The results have revealed detection of two PC specific SNPs in urine cf-DNA (&amp;gt;60% for SNP1 and &amp;gt;75% SNP2 in 91 patients; patent pending). Conclusion: Our study brings forth the potential clinical utility for two novel urine cf-DNA SNPs as early biomarkers for prostate cancer. Citation Format: Amrita Datta, Hogyoung Kim, Allison Feibus, Melody Badoo, Adedoyin Johnson, Ahmed Moustafa, Jonathan Silberstein, Krishnarao Moparty, Raju Thomas, Asim B. Abdel-Mageed. Urine cf-DNA SNPs as early biomarkers of prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 424.

Abstract 1382: A complete workflow for high-throughput isolation and analysis of cell-free DNA from urine

Abstract Circulating cell-free DNA (cfDNA) shed from tumors has gained considerable attention as a source of nucleic acid for testing cancer biomarkers. Critical to finding and implementing a diagnostic biomarker for cfDNA is the consistent and efficient isolation of the nucleic acid from blood. Sample preparation technologies are now commercially available for cfDNA isolation from plasma and serum, making these sample types used for most applications. However, plasma and serum require blood drawn by trained phlebotomist and only limited amounts can be obtained; additionally, individuals with advanced disease usually require routine monitoring and may not be able to spare additional blood drawn for cfDNA testing. Recently, it has been appreciated that urine may also serve as a valuable source for cfDNA. DNA from tissues and organs of the genitourinary system may be shed directly into urine and cfDNA circulating in blood can filter through the glomeruli in the kidneys to end up in urine. Compared to plasma and serum, urine is much easier to obtain, does not require a needle stick, and it can be collected in larger volumes making longitudinal studies more accessible. Urine presents a number of new challenges for the preparation of cfDNA that need to be overcome before this sample source can truly be utilized. The objective of this project was to develop reagents and workflows optimized for analysis of cfDNA from urine. Through our studies, we found that the slightly shorter cfDNA in urine requires optimized chemistry to maximize yield, a larger volume of urine may be necessary to isolate sufficient cfDNA compared to plasma/serum and urine must be treated with stabilization agents to minimize further degradation of cfDNA after collection. Using the MagMAX™ cell-free DNA isolation kit, we have developed a magnetic bead-based sample preparation protocol specific for isolating cfDNA from urine. Workflows for preparing cfDNA from urine through manual processing or by automated high throughput sample processing on the KingFisher™ instruments were developed. A small cohort of healthy donors was used to demonstrate compatibility of the cfDNA with qPCR, dPCR and next generation sequencing platforms. The effectiveness of this fast and easy workflow will be further tested on cfDNA from urine samples from donors with and without metastatic disease. We will analyze cfDNA from paired urine and plasma to understand the applicability to different tumor types. Citation Format: Alex J. Rai, Robert A. Setterquist, Xingwang Fang, Hannah E. Saunders, Matthew Carter, Charmaine San Jose Hinahon, Sarah E. Larocca, Susan M. Magdaleno. A complete workflow for high-throughput isolation and analysis of cell-free DNA from urine. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1382.

Genomic complexity of urothelial bladder cancer revealed in urinary cfDNA.

Urothelial bladder cancers (UBCs) have heterogeneous clinical characteristics that are mirrored in their diverse genomic profiles. Genomic profiling of UBCs has the potential to benefit routine clinical practice by providing prognostic utility above and beyond conventional clinicopathological factors, and allowing for prediction and surveillance of treatment responses. Urinary DNAs representative of the tumour genome provide a promising resource as a liquid biopsy for non-invasive genomic profiling of UBCs. We compared the genomic profiles of urinary cellular DNA and cell-free DNA (cfDNA) from the urine with matched diagnostic formalin-fixed paraffin-embedded tumour DNAs for 23 well-characterised UBC patients. Our data show urinary DNAs to be highly representative of patient tumours, allowing for detection of recurrent clinically actionable genomic aberrations. Furthermore, a greater aberrant load (indicative of tumour genome) was observed in cfDNA over cellular DNA (P<0.001), resulting in a higher analytical sensitivity for detection of clinically actionable genomic aberrations (P<0.04) when using cfDNA. Thus, cfDNA extracted from the urine of UBC patients has a higher tumour genome burden and allows greater detection of key genomic biomarkers (90%) than cellular DNA from urine (61%) and provides a promising resource for robust whole-genome tumour profiling of UBC with potential to influence clinical decisions without invasive patient interventions.