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Detection of circulating tumor DNA (ctDNA) mutations, which are molecular biomarkers present in bodily fluids of cancer patients, can be applied for tumor diagnosis and prognosis monitoring. However, current profiling of ctDNA mutations relies primarily on polymerase chain reaction (PCR) and DNA sequencing and these techniques require preanalytical processing of blood samples, which are time-consuming, expensive, and tedious procedures that increase the risk of sample contamination. To overcome these limitations, here the engineering of a DNA/γPNA (gamma peptide nucleic acid) hybrid nanoreporter is disclosed for ctDNA biosensing via in situ profiling and recording of tumor-specific DNA mutations. The low tolerance of γPNA to single mismatch in base pairing with DNA allows highly selective recognition and recording of ctDNA mutations in peripheral blood. Owing to their remarkable biostability, the detached γPNA strands triggered by mutant ctDNA will be enriched in kidneys and cleared into urine for urinalysis. It is demonstrated that the nanoreporter has high specificity for ctDNA mutation in peripheral blood, and urinalysis of cleared γPNA can provide valuable information for tumor progression and prognosis evaluation. This work demonstrates the potential of the nanoreporter for urinary monitoring of tumor and patient prognosis through in situ biosensing of ctDNA mutations., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Background: Urinary cell-free deoxyribonucleic acid (DNA) (ucfDNA) holds promise as a biomarker; however, its potential remains largely unexplored. We examined the fragmentation pattern of ucfDNA and identified somatic mutations within urine samples from metastatic breast cancer (MBC) patients., Methods: Urine and blood specimens were collected before treatment from 45 MBC patients and posttreatment urine samples from 16 of the 45 patients at the China National Cancer Center. Somatic mutations and tumor mutational burden (TMB) in the urine and plasma of 10 patients were analyzed by next-generation sequencing (NGS). Fragmentation patterns of cfDNA were displayed using electropherograms. Differences in the extracted amount of cfDNA, length of cfDNA fragments, and TMB between urine and plasma were compared using a Wilcoxon test., Results: The fragmentation patterns of ucfDNA were categorized as follows: (1) profile A (n = 26) containing a short peak (100-200 bp) and a long peak (>1500 bp); (2) profile B (n = 8) containing only a long peak; and (3) profile C (n = 11) containing flat pattern. For profile A patients, the short-peaked ucfDNA circulating in the bloodstream was much shorter compared with plasma cfDNA (149 vs. 171 bp, Wilcoxon test, P = 0.023). The fragmentation patterns in lung metastasis patients exhibited a higher propensity toward profile C ( P = 0.002). After treatment, 87.5% of the patients exhibited consistent fragmentation patterns. The concordance rate for somatic mutations in the plasma and urine was 30%, and the median TMB of urine and plasma was not significantly different., Conclusions: This study established a fragmentation pattern for ucfDNA and detected somatic mutations in the urine of MBC patients. These results suggest the potential application of ucfDNA as a biomarker for MBC., (Copyright © 2024 Copyright: © 2024 Journal of Cancer Research and Therapeutics.)

Background: Hepatocellular carcinoma (HCC) occurs in a well-defined high-risk patient population, but better screening tests are needed to improve sensitivity and efficacy. Therefore, we investigated the use of urine circulating tumour DNA (ctDNA) as a screening test., Methods: Candidate markers in urine were selected from HCC and controls. We then enrolled 609 patients from five medical centres to test the selected urine panel. A two-stage model was developed to combine AFP and urine panel as a screening test., Results: Mutated TP53, and methylated RASSF1a, and GSTP1 were selected as the urine panel markers. Serum AFP outperformed the urine panel among all cases of HCC, but the urine panel identified 49% of HCC cases with low AFP < 20 ng/ml. Using the two-stage model, the combined AFP and urine panel identified 148 of the 186 HCC cases (79.6% sensitivity at 90% specificity), which was 30% more than the cases detected with serum AFP alone. It also increased early-stage HCC detection from 62% to 92% (BCLC stage 0), and 40% to 77% (BCLC stage A)., Conclusion: Urine ctDNA has promising diagnostic utility in patients in HCC, especially in those with low AFP and can be used as a potential non-invasive HCC screening test., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Circulating tumour DNA (ctDNA) is very useful for purposes of cancer genetics; however, it has some limitations. Recently, ctDNA in body fluids, such as urine, sputum, and pleural effusion, has been investigated. The aim of this study was to evaluate the quantity of ctDNA derived from urine (trans-renal ctDNA) and the accuracy of KRAS mutation detection in relation to disease stage in colorectal cancer., Methods: Urine, plasma, and tissue samples were collected from consecutively resected colorectal cancer patients. DNA was extracted from each sample and the quantity was determined. From each DNA sample, KRAS mutations were detected using droplet digital PCR., Results: 200 patients participated and KRAS mutations were detected in 84 patients (42.0%) from tumour tissue. The concentration of trans-renal ctDNA (trtDNA) was significantly lower than that of plasma; however, there was no significant difference between the sensitivity using ctDNA and that using trtDNA (29.8% VS 33.3%, p = 0.62). Concordance between these two tests was only 17.5%. Combination analysis (ctDNA + trtDNA) improved the sensitivity to 53.6%, and sensitivity was significantly higher than that of corresponding single assays (p = 0.003). In early cancer stages, trtDNA had greater sensitivity for detecting KRAS mutations than ctDNA (37.7% vs. 21.3%, p = 0.047). Conversely, it was less useful for advanced cancer stages (21.7% vs. 52.2%, p = 0.07). Notably, KRAS mutations were detected using ctDNA or trtDNA in 12 of 116 (10.3%) patients who had no KRAS mutations in their tissue samples., Conclusions: trtDNA and ctDNA have equal potential and combination analysis significantly improved the sensitivity., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2021 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

Purpose: Next-generation sequencing (NGS)-based profiling of both urinary tumor DNA (utDNA) and circulating tumor DNA (ctDNA) shows promise for noninvasive detection and surveillance of urothelial bladder cancer (UBC). However, the analytical performance of these assays remains undefined in the real-world setting. Here, we sought to evaluate the concordance between tumor DNA (tDNA) profiling and utDNA or ctDNA assays using a UBC patient cohort from the intended-use population., Materials and Methods: Fifty-nine cases with pathologically confirmed disease and matching tissue/urine pairs were prospectively enrolled. Baseline peripheral blood mononuclear cell and plasma specimens were collected during clinic visits. The PredicineCARE TM NGS assay was applied for ultra-deep targeted sequencing and somatic alteration identification in tDNA, utDNA and ctDNA., Results: Diverse quantitative metrics including cancer cell fraction, variant allele frequency and tumor mutation burden were invariably concordant between tDNA and utDNA, but not ctDNA. The mutational landscapes captured by tDNA or utDNA were highly similar, whereas a considerable proportion of ctDNA aberrations stemmed from clonal hematopoiesis. Using tDNA-informed somatic events as reference, utDNA assays achieved a specificity of 99.3%, a sensitivity of 86.7%, a positive predictive value of 67.2%, a negative predictive value of 99.8% and a diagnostic accuracy of 99.1%. Higher preoperative utDNA or tDNA abundance correlated with worse relapse-free survival. Actionable variants including FGFR3 alteration and ERBB2 amplification were identified in utDNA., Conclusions: Urine-based molecular pathology provides a valid and complete genetic profile of bladder cancer, and represents a faithful surrogate for genotyping and monitoring newly diagnosed UBC.

Monitoring of early-stage breast cancer is critical in promptly addressing disease relapse. Circulating cell-free DNA provides a minimally invasive and sensitive means to probing the disease. In a longitudinal analysis of 250 patients with early breast cancer, we compared the circulating cell-free DNA recovered from both plasma and urine specimens. For comparison, 50 healthy controls were also recruited. Specific mutations associated with the disease were profiled to determine the clinical sensitivity and specificity. Correlations of recovered concentrations of cell-free DNA with outcomes were examined to address early prognostication. PIK3CA mutation profiling in both plasma and urinary cell-free DNA showed an agreement of 97.2% compared with the results obtained for tumor tissues. The analysis of healthy controls revealed that cell-free DNA measurements were stable and consistent over time. Over the short 6-month period of monitoring, our analyses showed declines in recovered cell-free DNA; these findings may aid physicians in stratifying patients at higher risk for relapse. Similar results were observed in both plasma and urine specimens (hazard ratios: 2.16 and 2.48, respectively). Cell-free DNA presents a novel and sensitive method for the monitoring of early-stage breast cancer. In the present study, serial measurements of both plasma and urine specimens were useful in probing the disease., (© 2020 The Author(s).)

Objectives: Real-time monitoring of disease status would be beneficial for timely decision making in the treatment of urothelial cancer (UC), and may accelerate the evaluation of clinical trials. Use of cell free tumor DNA (cftDNA) as a biomarker in liquid biopsy is minimally invasive and its successful use has been reported in various cancer types, including UC. The objective of this study was to evaluate the use of digital droplet PCR (ddPCR)-based assays to monitor UC after treatment., Method and Materials: Blood, urine and matching formalin fixed, paraffin embedded diagnostic specimens were collected from 20 patients diagnosed with stage T1 (n = 2) and T2/T3 (n = 18) disease. SNaPshot assays, Sanger sequencing and whole exome sequencing were used to identify tumor-specific mutations, and somatic mutation status was confirmed using patient-matched DNAs extracted from buffy coats and peripheral blood mononucleocytes. The ddPCR assays of the tumor-specific mutations were used to detect the fractional abundance of cftDNA in plasma and urine., Results: SNaPshot and Sanger sequencing identified point mutations in 70% of the patients that were assayable by ddPCR. Cases of remission and relapse monitored by assays for PIK3CA E542K and TP53 Y163C mutations in plasma and urine concurred with clinical observations up to 48 months from the start of chemotherapy. A new ddPCR assay for the telomerase reverse transcriptase (TERT) promoter (-124) mutation was developed. The TERT assay was able to detect mutations in cases below the limit of detection by SNaPshot. Whole exome sequencing identified a novel mutation, CNTNAP4 G727*. A ddPCR assay designed to detect this mutation was able to distinguish mutant from wild-type alleles., Conclusions: The study demonstrated that ddPCR assays could be used to detect cftDNA in liquid biopsy monitoring of the post-therapy disease status in patients with UC. Overall, 70% of the patients in our study harbored mutations that were assayable by ddPCR., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Since the discovery of circulating tumor cells in 1869, technological advances in the study of biomarkers from liquid biopsy have made it possible to diagnose disease in a less invasive way. Although blood-based liquid biopsy has been used extensively for the detection of solid tumors and immune diseases, the potential of urine-based liquid biopsy has not been fully explored. Advancements in technologies for the harvesting and analysis of biomarkers are providing new opportunities for the characterization of other disease types. Liquid biopsy markers such as exfoliated bladder cancer cells, cell-free DNA (cfDNA), and exosomes have the potential to change the nature of disease management and care, as they allow a cost-effective and convenient mode of patient monitoring throughout treatment. In this review, we addressed the advancement of research in the field of disease detection for the key liquid biopsy markers such as cancer cells, cfDNA, and exosomes, with an emphasis on urine-based liquid biopsy. First, we highlighted key technologies that were widely available and used extensively for clinical urine sample analysis. Next, we presented recent technological developments in cell and genetic research, with implications for the detection of other types of diseases, besides cancer. We then concluded with some discussions on these areas, emphasizing the role of microfluidics and artificial intelligence in advancing point-of-care applications. We believe that the benefits of urine biopsy provide diagnostic development potential, which will pave opportunities for new ways to guide treatment selections and facilitate precision disease therapies., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Cell-free tumor-derived DNA (ctDNA) allows non-invasive monitoring of cancers, but its utility in renal cell cancer (RCC) has not been established., Methods: Here, a combination of untargeted and targeted sequencing methods, applied to two independent cohorts of patients (n = 91) with various renal tumor subtypes, were used to determine ctDNA content in plasma and urine., Results: Our data revealed lower plasma ctDNA levels in RCC relative to other cancers of similar size and stage, with untargeted detection in 27.5% of patients from both cohorts. A sensitive personalized approach, applied to plasma and urine from select patients (n = 22) improved detection to ~ 50%, including in patients with early-stage disease and even benign lesions. Detection in plasma, but not urine, was more frequent amongst patients with larger tumors and in those patients with venous tumor thrombus. With data from one extensively characterized patient, we observed that plasma and, for the first time, urine ctDNA may better represent tumor heterogeneity than a single tissue biopsy. Furthermore, in a subset of patients (n = 16), longitudinal sampling revealed that ctDNA can track disease course and may pre-empt radiological identification of minimal residual disease or disease progression on systemic therapy. Additional datasets will be required to validate these findings., Conclusions: These data highlight RCC as a ctDNA-low malignancy. The biological reasons for this are yet to be determined. Nonetheless, our findings indicate potential clinical utility in the management of patients with renal tumors, provided improvement in isolation and detection approaches.

Background: Cancer recurrence for patients with early breast cancer is significant. Patients will benefit from more non-invasive modes of monitoring and we aim to study the feasibility of urinary circulating tumor DNA (ctDNA) to monitor for residual disease (MRD)., Methods: In this longitudinal study, 300 early breast cancer patients were recruited prospectively. Measurements were taken prior to treatment and at different time points thereafter for a total of 8 measurements. Comparisons were made with healthy volunteers and patients without detectable mutations in urine specimens. Disease free relapse were correlated to both urinary DNA quantity and ctDNA concentration., Results: Baseline index measurements showed 38% of patients with detectable mutations. The concordance with biopsy tissues was 97.3%. Overall, breast cancer patients had higher urinary DNA compared with healthy volunteers. Over time, fluctuations in urinary DNA was negligible in healthy volunteers, indicating the stability of the marker. Among the patients with detectable mutations, we observed that higher urinary DNA quantity measurements at 6-month and patients with positive mutations were associated with greater risk of relapse. Hazard ratios for patients in this category was 1.65 (95% CI 1.26-2.16) and 1.98 (95% CI 1.48-2.63) respectively., Conclusion: Urinary DNA offers non-invasive probing and real-time monitoring of breast cancer relapse. Our results demonstrated clear clinical relevance in breast cancer and significant risk profiling of early breast cancer patients. This potentially aids to complement current cancer relapse monitoring and may help in early intervention.

Background: Tissue biopsy remains the conventional technique for tumor genotyping. The main limitations are it is invasive and provides only partial snapshot during disease progression. Liquid biopsy approaches via plasma and urine are possible alternatives, and the current study aims to provide comparative analyses for plasma and urine derived disease genotyping., Methods: Blood and urine specimens were collected from 150 individuals with metastatic colorectal cancer (mCRC). Patients had multiple metastases and advanced stages of cancer. Common genetic mutations including KRAS and BRAF genetic abnormalities were evaluated. Patients were also serially monitored and compared., Results: In all cases, plasma and urine cell free DNA were successfully recovered and were of good quality for genetic analysis. Median recovered DNA from both urine and plasma samples were higher in mCRC patients than healthy volunteers indicating disease associations. Among the identified mutations, matched tumor tissue profiles compared to results from plasma ctDNA was 92%. For urine cell free DNA, the concordance among the identified mutations was 91%. Both sample types were closely matched to reference standards of tissue biopsy and indicated good clinical utility. Serial measurements indicated trends within each patient group that was linked with disease outcome., Conclusions: In the current study, our data indicated that both plasma and urine cell free DNA can be utilized to address possible disease progression in colorectal cancer patients. More importantly, this also provide risk stratifications that correlated to disease outcome. This can potentially aid in early clinical intervention for patients with possibly worse outcomes.

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., Competing Interests: Competing interests: AB is a consultant for Biocartis and Guardant Health. AS-B is an advisory board member for Amgen, Bayer and Sanofi., (© Author (s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. Published by BMJ on behalf of the European Society for Medical Oncology.)

Liquid biopsy can quantify and qualify cell-free (cfDNA) and tumour-derived (ctDNA) DNA fragments in the bloodstream. CfDNA quantification and mutation analysis can be applied to diagnosis, follow-up and therapeutic management as novel oncologic biomarkers. However, some tumor-types release a low amount of DNA into the bloodstream, hampering diagnosis through standard liquid biopsy procedures. Several tumors, as such as brain, kidney, prostate, and thyroid cancer, are in direct contact with other body fluids and may be alternative sources for cfDNA and ctDNA. Non-blood sources of cfDNA/ctDNA useful as novel oncologic biomarkers include cerebrospinal fluids, urine, sputum, saliva, pleural effusion, stool and seminal fluid. Seminal plasma cfDNA, which can be analyzed with cost-effective procedures, may provide powerful information capable to revolutionize prostate cancer (PCa) patient diagnosis and management. In the near future, cfDNA analysis from non-blood biological liquids will become routine clinical practice for cancer patient diagnosis and management., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: The current diagnosis and monitoring of bladder cancer are heavily reliant on cystoscopy, an invasive and costly procedure. Previous efforts in urine-based detection of bladder cancer focused on targeted approaches that are predicated on the tumor expressing specific aberrations. We aimed to noninvasively detect bladder cancer by the genome-wide assessment of methylomic and copy number aberrations (CNAs). We also investigated the size of tumor cell-free (cf)DNA fragments., Methods: Shallow-depth paired-end genome-wide bisulfite sequencing of urinary cfDNA was done for 46 bladder cancer patients and 39 cancer-free controls with hematuria. We assessed ( a ) proportional contribution from different tissues by methylation deconvolution, ( b ) global hypomethylation, ( c ) CNA, and ( d ) cfDNA size profile., Results: Methylomic and copy number approaches were synergistically combined to detect bladder cancer with a sensitivity of 93.5% (84.2% for low-grade nonmuscle-invasive disease) and a specificity of 95.8%. The prevalence of methylomic and CNAs reflected disease stage and tumor size. Sampling over multiple time points could assess residual disease and changes in tumor load. Muscle-invasive bladder cancer was associated with a higher proportion of long cfDNA, as well as longer cfDNA fragments originating from genomic regions enriched for tumor DNA., Conclusions: Bladder cancer can be detected noninvasively in urinary cfDNA by methylomic and copy number analysis without previous knowledge or assumptions of specific aberrations. Such analysis could be used as a liquid biopsy to aid diagnosis and for potential longitudinal monitoring of tumor load. Further understanding of the differential size and fragmentation of cfDNA could improve the detection of bladder cancer., (© 2019 American Association for Clinical Chemistry.)

Background : Qualitative and quantitative analysis of circulating cell-free DNA (cfDNA) is a potential detection method for bladder cancer. Many studies have focused on the reliability of these results, but the conclusions have not been consistent. Methods : We performed a diagnostic meta-analysis to investigate the diagnostic significance of serum and urine cfDNAs with tumor tissues as the standard control. We searched the MEDLINE, EMABASE, and Cochrane Central Controlled Trials Register (CCTR) databases until January 2019. Results : A total of 11 studies involving early and/or advanced bladder cancer were finally included. The overall diagnostic accuracy was measured as follows: pooled sensitivity and specifcity were 0.69 (95%CI: 0.67, 0.71) and 0.72 (95%CI: 0.70, 0.74). Pooled positive likelihood ratio and negative likelihood ratio were 3.10 (95%CI: 2.35, 4.07) and 0.41 (95%CI: 0.34, 0.49). Combined diagnostic odds ratio was 8.26 (95%CI: 5.64, 12.11). A high diagnostic accuracy was demonstrated by the summary receiver operating characteristic curve, with area under the curve of 0.80 (95%CI: 0.77, 0.83). Conclusions : CfDNA assay has high diagnostic value for the detection of bladder cancer. Larger sample studies are needed to further confirm our conclusions and to make this approach more sensitive and specific.

Aims: The use of circulating cell-free DNA for detection of cancer genetics has been studied extensively. Liquid biopsy often refers to the use of blood as a minimally invasive source of body fluid for detecting circulating tumor DNA (ctDNA). However, urine collection, which is completely noninvasive, has been shown to also have great promise to serve as an alternate body fluid source for ctDNA. In this review article, we focus on the clinical utility of urine for genetic liquid biopsy of nonurological cancers., Conclusion: Although still in early stages as compared with blood-based liquid biopsy, recent studies have demonstrated the value of urine-based liquid biopsies for: nonurological cancer screening; early detection; monitoring for recurrence and metastasis; and therapeutic efficacy. Overall, the completely noninvasive and patient-friendly nature of the urine-based biopsy warrants further development and offers a promising alternative to blood-based biopsies.

The use of circulating tumor nucleic acids (ctNA) in patient liquid biopsies for targeted genetic analysis is rapidly increasing in clinical oncology. Still, the call for an integrated methodology, which is both rapid and sensitive for analyzing trace ctNA amount in liquid biopsies, has unfortunately not been fully realized. Herein, we performed complex liquid biopsy sample-to-targeted genetic analysis on a biochip with a 50 copies-detection limit within 30 min. Our biochip uniquely integrated the following: (1) electrical lysis and release of cellular targets with minimal processing; (2) nanofluidic manipulation to accelerate molecular kinetics of solid-phase isothermal amplification; and (3) single-step capture and amplification of multiple NA targets prior to nanozyme-mediated electrochemical detection. Using prostate cancer liquid biopsies, we successfully demonstrated multifunctionality for cancer risk prediction; correlation of serum and urine analyses; and cancer relapse monitoring.

Urothelial bladder carcinoma (UBC) is characterized by a large number of genetic alterations. DNA from urine is a promising source for liquid biopsy in urological malignancies. We aimed to assess the availability of cell-free DNA (cfDNA) and exosomal DNA (exoDNA) in urine as a source for liquid biopsy in UBC. We included 9 patients who underwent surgery for UBC and performed genomic profiling of tumor samples and matched urinary cfDNA and exoDNA. For mutation analysis, deep sequencing was performed for 9 gene targets and shallow whole genome sequencing (sWGS) was used for the detection of copy number variation (CNV). We analyzed whether genetic alteration in tumor samples was reflected in urinary cfDNA or exoDNA. To measure the similarity between copy number profiles of tumor tissue and urinary DNA, the Pearson's correlation coefficient was calculated. We found 17 somatic mutations in 6 patients. Of the 17 somatic mutations, 14 and 12 were identified by analysis of cfDNA and exoDNA with AFs of 56.2% and 65.6%, respectively. In CNV analysis using sWGS, although the mean depth was 0.6X, we found amplification of MDM2, ERBB2, CCND1 and CCNE1, and deletion of CDKN2A, PTEN and RB1, all known to be frequently altered in UBC. CNV plots of cfDNA and exoDNA showed a similar pattern with those from the tumor samples. Pearson's correlation coefficients of tumor vs. cfDNA (0.481) and tumor vs. exoDNA (0.412) were higher than that of tumor vs. normal (0.086). We successfully identified somatic mutations and CNV in UBC using urinary cfDNA and exoDNA. Urinary exoDNA could be another source for liquid biopsy. Also, CNV analysis using sWGS is an alternative strategy for liquid biopsy, providing data from the whole genome at a low cost.

Purpose: The need for less invasive procedures for lung cancer probing is critically needed to better understand the disease. The purpose of the current study aims to explore the use of circulating tumor DNA (ctDNA) derived from plasma and urine specimens., Methods: Matched peripheral blood and morning urine specimens were obtained from 160 late stage NSCLC patients. The amount of ctDNA was quantified for each of the patients. Activating and sensitizing EGFR mutations commonly found in NSCLC patients were profiled. Longitudinal analysis was performed to compared DNA variations during disease progression., Results: Measurement of EGFR mutations in NSCLC patients using plasma and urinal DNA demonstrated strong concordance to conventional tissue biopsy profiling. Baseline matched tumor samples yielded 82.8% and 84.0% for plasma and urinal DNA respectively. For these measurements, the positive predictive value was 100% for plasma and urinal DNA. In the longitudinal study, we observed strong links to disease severity and survival analysis showed a clear trend with patients having higher DNA concentrations to have worse outcome especially for urinal DNA. HR for patients stratified using plasma and urinal DNA were 1.23 and 2.55 respectively., Conclusion: Measurements of circulating DNA within body fluids presented potentially new tools for the disease management of NSCLC patients with EGFR mutations. We demonstrated both plasma and urinal DNA correlated well to tissue biopsies and were potentially prognostic to address patients' survival outcome.

Purpose: Non-invasive methods of molecular profiling for non-small cell lung cancer (NSCLC) are useful for monitoring disease progression. The aim of the current study was to ascertain if transrenal DNA is sensitive for clinical correlation and EGFR detection in NSCLC patients., Methods: 160 patients at various stages of the disease participated and samples were collected prospectively at 2-month intervals. A baseline sample was taken before treatment commencement. To ascertain the sensitivity of transrenal DNA, we compared its results with plasma DNA. ddPCR was used to profile the urine and blood samples for key EGFR mutations., Results: Using tumor tissues as references, our study showed good concordance in EGFR mutations with transrenal DNA before treatment. Results were highly matching in late-stage NSCLC patients, with stage III/IV patients yielding an agreement of more than 90%. The assay was also sensitive to detect early-stage patients after surgical procedures. Profiles were highly concordant with results derived from plasma DNA, demonstrating the specificity of transrenal DNA assays. Serial monitoring of these patients showed stable molecular signatures and correlated to different treatments. Survival analysis showed good prognostic utility for late-stage patients with high transrenal DNA variations and patients that acquired T790M mutation., Conclusion: The study demonstrated the feasibility of using transrenal DNA in mutation profiling for different stages of NSCLC patients. It highlights the importance of continual monitoring and has potential clinical utility in the clinical management of NSCLC.

Purpose: Noninvasive drug biomarkers for the early assessment of tumor response can enable adaptive therapeutic decision-making and proof-of-concept studies for investigational drugs. Circulating tumor DNA (ctDNA) is released into the circulation by tumor cell turnover and has been shown to be detectable in urine. Experimental Design: We tested the hypothesis that dynamic changes in EGFR activating (exon 19del and L858R) and resistance (T790M) mutation levels detected in urine could inform tumor response within days of therapy for advanced non-small cell lung cancer (NSCLC) patients receiving osimertinib, a second-line third-generation anti-EGFR tyrosine kinase inhibitor. Results: Eight of nine evaluable NSCLC patients had detectable T790M-mutant DNA fragments in pretreatment baseline samples. Daily monitoring of mutations in urine indicated a pattern of intermittent spikes throughout week 1, suggesting apoptosis with an overall decrease in fragment numbers from baselines to day 7 preceding radiographic response assessed at 6 to 12 weeks. Conclusions: These findings suggest drug-induced tumor apoptosis within days of initial dosing. Daily sampling of ctDNA may enable early assessment of patient response and proof-of-concept studies for drug development. The modeling of tumor lysis through the day-to-day kinetics of ctDNA released into the blood and then into the urine is demonstrated in this proof-of-concept study in lung cancer patients receiving anti-EGFR tyrosine kinase inhibitors. This strategy may determine the specific clonal populations of cells which undergo apoptosis within the first week of therapy. This has important implications for developing combinational strategies to address inter- and intralesional heterogeneity and characterizing residual disease after initial drug exposure. Clin Cancer Res; 23(16); 4716-23. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Background: Disease surveillance in patients with bladder cancer is important for early diagnosis of progression and metastasis and for optimised treatment., Objective: To develop urine and plasma assays for disease surveillance for patients with FGFR3 and PIK3CA tumour mutations., Design, Setting, and Participants: Droplet digital polymerase chain reaction (ddPCR) assays were developed and tumour DNA from two patient cohorts was screened for FGFR3 and PIK3CA hotspot mutations. One cohort included 363 patients with non-muscle-invasive bladder cancer (NMIBC). The other cohort included 468 patients with bladder cancer undergoing radical cystectomy (Cx). Urine supernatants (NMIBC n=216, Cx n=27) and plasma samples (NMIBC n=39, Cx n=27) from patients harbouring mutations were subsequently screened using ddPCR assays., Outcome Measurements and Statistical Analysis: Progression-free survival, recurrence-free survival, and overall survival were measured. Fisher's exact test, the Wilcoxon rank-sum test and Cox regression analysis were applied., Results and Limitations: In total, 36% of the NMIBC patients (129/363) and 11% of the Cx patients (44/403) harboured at least one FGFR3 or PIK3CA mutation. Screening of DNA from serial urine supernatants from the NMIBC cohort revealed that high levels of tumour DNA (tDNA) were associated with later disease progression in NMIBC (p=0.003). Furthermore, high levels of tDNA in plasma samples were associated with recurrence in the Cx cohort (p=0.016). A positive correlation between tDNA levels in urine and plasma was observed (correlation coefficient 0.6). The retrospective study design and low volumes of plasma available for analysis were limitations of the study., Conclusions: Increased levels of FGFR3 and PIK3CA mutated DNA in urine and plasma are indicative of later progression and metastasis in bladder cancer., Patient Summary: Urine and plasma from patients with bladder cancer may be monitored for diagnosis of progression and metastasis using mutation assays., (Copyright © 2016 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

In this review, we discuss the types of liquid biopsies currently available, and their recent and potential clinical applications, especially as they relate to the detection of molecular biomarkers of bladder cancer. The advances in research using high-throughput next-generation genomic techniques have identified a number of promising molecular biomarkers of bladder urothelial carcinoma. Liquid biopsy has recently received enormous attention as a potential tool for real-time monitoring of disease status in cancer patients, and genomic profiles are reported to closely match those of corresponding tumors. It is noninvasive and easily repeated, and allows monitoring during treatment and detection of different genomic alterations that are potentially accessible to targeted therapy or are associated with treatment resistance in bladder cancer patients. Moreover, liquid biopsy is a tool that allows rapid access to biomarker assessment in patients for whom solid biopsies are not available. Liquid biopsy will be widely applied to daily clinical practice and serve as an important tool for the screening, surveillance, prediction of outcomes, and selection of effective targeted therapies in bladder carcinoma.

We have examined in situ detection of single-nucleotide KRAS mutations in urine using a (Pb(Mg 1/3 Nb 2/3 )O 3 ) 0.65 (PbTiO 3 ) 0.35 (PMN-PT) piezoelectric plate sensor (PEPS) coated with a 17-nucleotide (nt) locked nucleic acid (LNA) probe DNA complementary to the KRAS mutation without DNA isolation and amplification. In situ mutant (MT) DNA in urine in a wild type (WT) background was carried out at a flow rate of 4 mL/min and at 63 °C with the PEPS vertically situated at the center of the flow. Both the temperature and the impingement flow force discriminated the wild type. Under these conditions PEPS was shown to specifically detect KRAS MT in situ within 30 min with an analytical sensitivity of 60 copies/mL in a clinically relevant background of WT with concentrations 1000-fold greater than that of MT without DNA isolation, amplification, or labeling. For validation, detection was performed in a mixture of blue MT fluorescent reporter microspheres (FRMs) (MT FRMs) that bound to only the captured MT, and orange WT FRMs that bound to only the captured WT. The captured blue MT FRMs still outnumbered the orange WT FRMs by a factor of 4-1 even though WT was 1000-fold of MT in urine, illustrating the specificity of the point mutation detection.