Liquid biopsy in the clinical management of tumor of urinary system: current status and future developments.

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Learn more: PMC Disclaimer | PMC Copyright Notice Cell Oncol (Dordr) . 2026 Apr 20;49(3):76. doi: 10.1007/s13402-026-01216-3 Search in PMC Search in PubMed View in NLM Catalog Add to search Liquid biopsy in the clinical management of tumor of urinary system: current status and future developments Runsen Xu Runsen Xu 1 Department of Urology, Shengjing Hospital of China Medical University, #36 Sanhao Street, Heping District, Shenyang, 110004 China Find articles by Runsen Xu 1, # , Yiming Zhao Yiming Zhao 1 Department of Urology, Shengjing Hospital of China Medical University, #36 Sanhao Street, Heping District, Shenyang, 110004 China Find articles by Yiming Zhao 1, # , Peng Su Peng Su 2 Medical Research Center, Shengjing Hospital of China Medical University, #7 Mulan Road, Xihu District, Shenyang, 110004 China Find articles by Peng Su 2 , Lin Li Lin Li 3 Department of Rehabilitation, Shengjing Hospital of China Medical University, #36 Sanhao Street, Heping District, Shenyang, 110004 China Find articles by Lin Li 3, ✉ , Hongyuan Liang Hongyuan Liang 4 Department of Radiology, Shengjing Hospital of China Medical University, #36 Sanhao Street, Heping District, Shenyang, 110004 China Find articles by Hongyuan Liang 4, ✉ , Dan Dong Dan Dong 5 College of Basic Medical Science, China Medical University, #77 Puhe Road, Shenbei New District, Shenyang, 110122 China Find articles by Dan Dong 5, ✉ , Kefeng Wang Kefeng Wang 1 Department of Urology, Shengjing Hospital of China Medical University, #36 Sanhao Street, Heping District, Shenyang, 110004 China Find articles by Kefeng Wang 1, ✉ Author information Article notes Copyright and License information 1 Department of Urology, Shengjing Hospital of China Medical University, #36 Sanhao Street, Heping District, Shenyang, 110004 China 2 Medical Research Center, Shengjing Hospital of China Medical University, #7 Mulan Road, Xihu District, Shenyang, 110004 China 3 Department of Rehabilitation, Shengjing Hospital of China Medical University, #36 Sanhao Street, Heping District, Shenyang, 110004 China 4 Department of Radiology, Shengjing Hospital of China Medical University, #36 Sanhao Street, Heping District, Shenyang, 110004 China 5 College of Basic Medical Science, China Medical University, #77 Puhe Road, Shenbei New District, Shenyang, 110122 China ✉ Corresponding author. # Contributed equally. Received 2025 Dec 11; Accepted 2026 Apr 12; Collection date 2026 Jun. © The Author(s) 2026 Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/ . PMC Copyright notice PMCID: PMC13096305  PMID: 42010034 Abstract Urinary system tumors are a major threat to human health. However, there is a lack of reliable non-invasive examinations for urinary system tumors. Liquid biopsy is an emerging method for detecting biomarkers in patients in recent years. Its components mainly include circulating tumor cells, circulating tumor DNAs, circulating-free DNAs, circulating-free RNAs, exosomes and tumor educated platelets. Liquid biopsy is considered to be of great significance for the diagnosis, prognosis, treatment monitoring and medication guidance of cancers. The non-invasive characteristics of liquid biopsy significantly improve patients’ compliance and make it possible to collect patient information in real time and continuously. In this review, we selected urinary system tumors as the clinical application subjects of liquid biopsy. We believe that liquid biopsy has great potential value and application prospects in the clinical treatment of urinary system tumors. Keywords: Liquid biopsy, Urinary system tumor, Biomarker, Clinical management Introduction Urinary system tumor is a major disease that endangers human health. According to the report in 2025, the three tumors with the highest incidence in the urinary system are prostate cancer (PCa), bladder cancer (BCa), and renal cell carcinoma (RCC) [ 1 ]. PCa was the first most common cancer and the second leading cause of cancer death in male. BCa was the seventh most common tumor, with 84,870 new cases. And there were 80,980 new cases of RCC [ 1 ]. Compared with 2024, the number of new cases and deaths of these three tumors have increased, and the clinical management situation is becoming more urgent [ 2 ]. To date, the clinical management of urological tumors is still based on the pathological biopsy of the tumor, but this approach has some limitations. A study by Gerlinger et al. [ 3 ] showed that traditional single tissue biopsy often produced bias due to the heterogeneity of tumors. The proposed solution to this bias, multi-region sequential detection, greatly avoids the heterogeneity [ 4 , 5 ]. However, this approach has drawbacks in clinical application due to financial and safety concerns for patients, as well as the possibility that multiple biopsies may reduce patient compliance [ 6 , 7 ]. Therefore, post-treatment evaluation and monitoring of recurrence and metastasis are still largely dependent on imaging examination. However, due to the limitation of resolution and shooting time, it can not reflect the whole situation of tumor [ 8 ]. The limitation of traditional methods has brought a new direction for the clinical treatment of tumor. There is an urgent need for non-invasive biopsy to complete the diagnosis, treatment and prognosis assessment of tumors. Liquid biopsy might be the solution to this dilemma. Liquid biopsy uses body fluids, mainly blood, urine, pleural fluid, ascites and cerebrospinal fluid, as biopsy specimens to minimize the harm to patients [ 9 ]. In addition, the biopsy signatures enter body fluids from the tumor by secreting or brushing out after being destructed, which greatly avoided the interference of intratumor heterogeneity [ 10 ]. In theory, the detection of circulating signatures can reflect all changes in the tumor microenvironment [ 7 , 11 ]. The purpose of this manuscript was to review the clinical application of liquid biopsy in urinary system tumors. We comprehensively summarized the application of liquid biopsy components as biomarkers in the screening, diagnosis, prognosis and monitoring of urinary system tumors. Studies we have summarized were included if they were about liquid biopsy and urinary system tumor which published in English in recent 10 years. Furthermore, during the reading of the papers, if there are references to earlier papers that have made crucial progress, they will also be included in our summary. Letters-to-the-editor, editorials, and commentaries were excluded. Classification and research progress of liquid biopsy At the beginning of the research field of liquid biopsy, circulating tumor cells (CTCs) were the only components had been found [ 11 ]. It was first discovered in 1869, and to date, CTCs were still a relatively well-studied and important component of liquid biopsies [ 12 ]. With the development of technology and in-depth research, more and more biopsy molecular signatures have been added to the components of liquid biopsy. Circular cell-free nucleic acids (cfNAs, including circulating tumor DNAs (ctDNAs), messenger RNAs (mRNAs), microRNAs (miRNAs) and circular RNAs (circRNAs)), extracellular vesicles (EVs, including exosomes and ectosomes), and tumor-educated platelets (TEPs) are becoming emerging components of liquid biopsy in subsequent studies [ 11 , 13 – 15 ]. They have different advantages in liquid biopsy and may play different roles in the clinical treatment of urinary system tumors (Fig. 1 ). These liquid biopsy molecules are closely related to each other and cannot be classified completely independently. We will try to describe their characteristics and advantages in the following introduction. Fig. 1. Open in a new tab Overview of biomarkers profiling using liquid biopsy. Body fluid specimens are collected through minimally invasive surgery. After processing, several different components can be obtained, include CTCs, cfNAs, exosomes and TEPs CTCs CTCs are cells that shed from primary or metastatic tumors and enter the bloodstream. They can be divided into two categories: singular CTCs and clustered CTCs. These CTCs stay in the blood for only 1 to 2.5 h before being attacked by the immune system or destroyed by mechanical forces [ 16 ]. CTCs exhibit remarkable phenotypic plasticity, undergoing epithelial–mesenchymal transition to acquire invasive and stem-like properties [ 17 ]. This transition enhances their ability to survive in the bloodstream, evade anoikis, and initiate metastatic outgrowth at distant sites [ 18 ]. Some clustered CTCs can exist in the blood circulation for a long time due to their resistance to anoikis [ 19 ]. These surviving CTCs may be associated with distant metastasis of tumors. Therefore, they can be used as a tool to detect tumor and monitor metastasis. CTCs are considered to be useful in the diagnosis, prognosis and therapeutic response of malignant tumors [ 20 ]. In the future, CTCs may also be used to test drug sensitivity and further guide treatment [ 18 , 21 ]. Compared with other liquid biopsy techniques, CTCs are intact cells that can better reflect changes in tumor morphology, metabolic status and genetic information [ 19 ]. Patients have extremely low CTCs in their blood, with only 5–50 cells per 7.5 ml of blood [ 22 ]. Therefore, the analysis of CTCs in tumor patients requires some technical means. The CTCs were first isolated and enriched for biopsy, followed by RT-PCR or immunocytochemical assay. Through the above process, the CTCs can be analyzed more reliably [ 23 ]. Recently, microfluidic techniques have also been used for the isolation, formation and characterization of CTCs [ 24 ]. Although there are still defects and uncertainties in the above approaches, with the deepening of research and the dynamic development of technology, the application of CTCs in liquid biopsy will be more and more stable and have greater clinical value in the future. CfNAs CfNAs are DNA or RNA molecules present in body fluids and are an important component of liquid biopsies. Early studies of DNAs in body fluids showed that they originated in dead cells and were released mainly through apoptosis or necrosis [ 25 ]. Recent literatures uncovered that they can be released into body fluids by living cells through active transport [ 26 , 27 ]. At present, the research objects of cfNAs mainly include ctDNAs, miRNAs and long non-coding RNAs (lncRNAs) [ 28 ]. CfNAs can be widely present in human body fluids. Some reports suggested that the detection of cfNAs by acupuncture in urine may be a good indicator of both urological and non-urological tumors [ 7 , 8 , 29 ]. The current research on cfNAs mainly focuses on ctDNAs. CtDNAs, derived from both tumor and normal cells, are a double-stranded DNA molecule with a length of 150–200 bp [ 30 ]. Next-generation sequencing analysis enables the application of ctDNAs in liquid biopsy [ 30 ]. Research on ctDNAs began about 30 years ago. A study published in 1989 by Stroun pointed out that a portion of free cell DNA was present in the plasma of tumor patients, but not detectable in non-tumor patients. This can rule out the possibility that this DNA was released due to cell necrosis, so these cfDNAs can reflect the presence of tumors [ 31 ]. This study laid the foundation for the concept of ctDNAs. Subsequent studies used the proportion of ctDNAs to total circulating DNAs as a tumor score to assess tumor prognosis [ 30 , 32 ]. At the same time, cohort studies of ctDNAs biopsy patients can help search for specific genes that may promote tumorigenesis [ 33 ]. The level of ctDNAs produced by a particular cancer changes over time and can be affected by the stage of the cancer and treatment [ 32 ]. Therefore, the long-term monitoring of ctDNAs may become an important means to evaluate tumor progression and treatment effect. As the research progressed, the researchers discovered that the release of ctDNA into the bloodstream is not merely a passive consequence of cell death. Emerging evidence suggests that active secretion mechanisms, including the formation of apoptotic bodies, phagocytosis of necrotic debris by macrophages, and even direct extrusion of DNA from viable tumor cells, contribute to the heterogeneity and fragmentation patterns of ctDNA [ 34 ]. These biological processes influence ctDNA integrity, size distribution, and methylation status, which in turn provide functional information about tumor dynamics and treatment response [ 32 , 34 ]. It is worth noting that the Methylation Profiling of ctDNA is becoming increasingly important in the application of liquid biopsy. Detection based on DNA methylation signals has multiple advantages. Changes in methylation in plasma and urine occur early in tumors, even earlier than gene mutations, making it suitable for early screening and detection in cases of low tumor burden [ 35 ]. The methylation pattern is tissue-specific and can be used to determine the source of metastatic foci and assist in locating the primary tumor [ 36 , 37 ]. Compared to gene mutations, methylation information is more consistent between different tumor samples and is suitable as a stable detection indicator [ 38 ]. In addition to methylation profiling, fragmentomics is also a currently effective and promising detection method that has the potential to be applied in clinical settings. Fragmentomics mainly focuses on detecting the differences between ctDNA and cfDNA in normal cell [ 39 , 40 ]. For instance, the fragments of ctDNA are relatively short in size, so they can be analyzed based on their lengths [ 41 ]. Due to the changes of tumor cells in the chromatin structure and nucleosome positions, the open state of the binding sites of transcription factors will also change [ 39 ]. These changes are reflected in the coverage of cfDNA and can be detected through nucleosome footprints [ 39 ]. The terminal sequences of the cfDNA fragments are fixed and reflect the specificity of the nucleases cutting sites [ 40 ]. The terminal motifs of ctDNA derived from tumors are different from those of normal cfDNA, so they can also be detected based on the terminal motifs [ 40 ]. By using these detection methods, fragmentomics can achieve higher detection sensitivity at lower abundance levels compared to traditional ctDNA sequencing [ 40 , 42 ]. In addition to ctDNAs, cfNAs used for liquid biopsies also include cell-free RNA molecules, commonly called cfRNAs, also known as extracellular RNAs. RNA is more unstable outside the cell than DNA, so cfRNAs are often tightly bound to proteins or lipoproteins [ 43 ]. Some of them are encapsulated in exosomes [ 44 , 45 ], have longer half-lives [ 46 ], and can even resist degradation caused by RNases [ 47 ]. The biogenesis of cfRNAs may be due to the retention of partial RNA encapsulated during cell necrosis or apoptosis or the active synthesis and release of living cells [ 48 ]. Thanks to the progress of sequencing technology, the determination of cfRNAs in body fluid samples is becoming more and more accurate, and its clinical application as a biopsy marker is gradually becoming possible [ 49 ]. Subsequent studies have gradually revealed the value of cfRNAs in early diagnosis and prognosis [ 11 , 50 ]. A study has shown that the frequency of detection of PD-L1 mRNA in cfRNAs of tumor patients is consistent with the immunohistochemical analysis of solid tumor tissues [ 51 ]. It provides a potentially more economical and less invasive way to guide tumor patients to drugs. Previous studies on cfNAs mainly focused on cfNAs in the circulatory system, while lymphatic-based biopsies have been almost blank. Research by Zawieja et al. 52 revealed that lymph can also be biopsied as a carrier of cfDNAs. There is evidence that cfNAs, which can be detected in plasma, are also present in lymph [ 52 ]. Other studies have also focused on lymphatic liquid biopsy and verified its sensitivity and specificity [ 53 ]. Perhaps lymphatic-based liquid biopsy will become an important research direction in the future. EVs EVs can be divided into three main categories: exosomes, ectosomes and oncosomes [ 13 , 54 ]. Currently, the main components that can be applied to liquid biopsy are mainly exosomes [ 13 ]. The diameter of exosome is mainly between 40 nm and 160 nm [ 55 ]. Exosomes can transport specific proteins, nucleic acids, and lipids to achieve specific biological functions [ 56 ]. Donor cells can secrete exosomes to convey information to recipient cells [ 57 ]. The biogenesis of exosomes involves a highly regulated process beginning with endosomal invagination to form multivesicular bodies, which subsequently fuse with the plasma membrane to release vesicles as exosomes [ 56 , 58 ]. Importantly, tumor cells exploit this machinery to selectively package oncogenic cargo—including mutant DNA fragments, microRNAs, and signaling proteins—into exosomes [ 58 ]. Thus, exosomes secreted by tumor cells are closely associated with tumor existence, metastasis, and immunosuppression [ 13 , 22 , 56 ]. Therefore, liquid biopsy with exosomes as components can effectively help the early diagnosis, prognosis, and medication guidance of tumors [ 59 ]. At present, the detection of exosomes mainly focuses on the analysis of DNAs, RNAs, proteins and other substances encapsulated in exosomes. However, compared with the direct detection of cfNAs in body fluids mentioned above, nucleic acids and other biomarkers in exosome are better preserved and have a larger amount in body fluids [ 60 ]. In addition, exosomes are more likely to be released by living tumor cells than other biomarkers, making them a more reliable indicator of living tumor cells rather than necrotic tumor cells [ 57 , 61 ]. Although the number of studies on exosome in the field of liquid biopsy is significantly less than that of CTCs and cfNAs, the potential clinical value of exosomes cannot be ignored. Beyond exosomes, however, emerging evidence highlights the potential clinical significance of large oncosomes (LO), a distinct and less explored subtype of EVs. LOs are a distinct subtype of EVs, typically ranging from 1 to 10 μm in diameter, that are from the plasma membrane blebbing of highly aggressive tumor cells [ 62 ]. Unlike exosomes, which are also produced by normal cells, LOs are predominantly originate from tumor cells and are generally absent in healthy tissues, conferring higher specificity for oncological applications [ 63 ]. Their larger size also facilitates detection through flow cytometry and imaging-based platforms, which may overcome certain technical limitations of nanoscale vesicles (such as exosomes) [ 63 , 64 ]. Given their biological specificity and diagnostic potential, large tumor bodies may become one of the most promising subtypes of EVs in precision oncology of urinary system tumors. TEPs Platelets are small discoid shaped non-nucleated cells in the human circulating system, originated from bone marrow megakaryocytes and are the second most abundant cells in the human body [ 65 ]. Their important role in blood clotting is well known. Earlier studies found that the RNA profiles in platelets from cancer patients differed from those of healthy people [ 66 , 67 ]. Subsequent studies have revealed and enriched the mechanisms that lead to this difference. Tumor cells can release cytokines, tumor-derived RNAs and other substances that are sometimes absorbed by platelets [ 68 , 69 ]. In addition, tumor cells can activate platelets by making direct contact with the adhesive G-protein-coupled receptor CD97 on platelets [ 70 ]. This process is called education, and these platelets that are altered by tumor cells are called TEPs [ 69 ]. TEPs are often closely associated with tumor metastasis. As mentioned above, some tumor cells enter the circulatory system and become CTCs. Most of these CTCs are attacked by the immune system and undergo necrosis or apoptosis [ 23 ]. TEPs attach to and wrap around the surface of CTCs, acting as a protective layer to help CTCs escape the immune system [ 71 ]. This process increases the possibility of metastatic tumors forming (Fig. 2 ). Therefore, the detection of TEPs can effectively help the discovery and prognosis of metastatic tumors in clinical practice. Fig. 2. Open in a new tab Normal platelets are educated by tumor cells to become TEPs. TEPs can coat the surface of naked CTCs, which are usually attacked by immune system, leading to necrosis or apoptosis. Coated CTCs are more likely to escape the immune system. This process makes the tumor more prone to metastasis Clinical application of liquid biopsy in PCa At present, the blood test for PCa mainly relies on prostate-specific antigen (PSA), which is widely used in diagnosis, prognosis and post-treatment monitoring of PCa. However, low specificity makes it necessary for many patients to undergo invasive and expensive prostate biopsy to identify benign and malignant tumors by pathology. Studies have shown that liquid biopsies, such as blood and urine, can be used to aid screening and long-term monitoring, which can effectively improve patient compliance and reduce the risk of PCa [ 6 , 72 ] (Table 1 ). Table 1. Summary of key clinical trials of liquid biopsy in patients with PCa Biomarkers Years Cohorts Main findings References cfRNA 2022 40 patients with CRPC, 40 healthy controls Revealed associations of cell subtype genes in patients’ blood with therapeutic regimens and clinical outcome Liquid biopsy-based targeted gene screening highlights tumor cell subtypes in patients with advanced Pca CTC 2023 98 mCRPC patients treated with enzalutamide Developed prognostic prediction models including CTC, AR-V7, and AR gain A correlative biomarker study and integrative prognostic model in chemotherapy-naïve mCRPC treated with enzalutamide CTC, cfRNA 2017 50 patients with mCRPC CTC and circulating miRNA biomarkers may have value as prognostic biomarkers Circulating microRNAs and treatment response in the Phase II SWOG S0925 study for patients with new mHSPC CTC 2021 1313 patients with mCRPC CTC count is an important prognostic indicator of PSA response and can distinguish patients with a lower survival rate Baseline circulating tumor cell count as a prognostic marker of PSA response and disease progression in mHSPC (SWOG S1216) CTC 2022 103 patients with mCRPC Patients with mCSPC who had higher CTC counts had a significantly higher number of altered genes and total alterations External validation of association of baseline circulating tumor cell counts with survival outcomes in men with mCSPC cfDNA 2015 97 mCRPC patients treated with abiraterone CtDNA is representative of tumor clones that are driving disease progression in CRPC Plasma AR and abiraterone-resistant PCa cfDNA 2016 65 mCRPC patients treated with enzalutamide CtDNA provide important insights into enzalutamide response and resistance Genomic alterations in cell-free DNA and enzalutamide resistance in CRPC ctDNA 2024 759 mCRPC patients treated with enzalutamide after abiraterone CtDNA tumor fraction provides a minimally invasive, complementary biomarker to PSA testing and may refine personalized treatment approaches Circulating tumor DNA assessment for treatment monitoring adds value to PSA in mCRPC ctDNA 2021 188 patients with mCRPC Plasma tumor fraction was prognostic for OS in univariable and stratified multivariable analyses Elucidating PCa behaviour during treatment via low-pass whole-genome sequencing of circulating tumor DNA Open in a new tab Liquid biopsy based on CTCs in PCa The clinical application of CTCs in PCa mainly focuses on prognosis and treatment monitoring. CTCs-based liquid biopsy biomarker studies have focused on the detection of androgen receptor (AR) splice variants (AR-Vs), which are formed by splicing of AR-mRNA [ 73 , 74 ]. The overexpression of AR-V7 often indicates drug resistance to anti-androgen therapy, which provides a basis for treatment monitoring [ 60 , 74 ]. A recent clinical trial developed a prognostic prediction model that included CTCs, AR-V7, and AR gain [ 75 ]. This also provides a further theoretical basis for the subsequent clinical prognostic application of CTCs and AR-Vs. In addition to AR-Vs, the count of CTCs is also considered to be correlated with tumor prognosis. Currently, CTCs count is approved by the Food and Drug Administration of the United States as a prognostic marker for both metastatic hormone sensitive PCa (mHSPC) and metastatic castration-sensitive PCa (mCRPC), which have been demonstrated in well-conducted prospective trials [ 76 ]. In a Phase II trial of mCRPC (SWOG 0925), CTCs count was correlated with miRNA-375 levels and further correlated with PSA response [ 77 ]. Preliminary data from another Phase III trial for mCRPC (SWOG 1216), published in 2021, confirmed that baseline CTCs count was associated with poor prognostic factors such as elevated PSA, presence of bone metastases, and disease severity [ 78 ]. A recent study confirmed this idea and on this basis proposed that TP53 gene alterations lead to a significant increase in CTCs count in PCa patients [ 79 ]. In turn, TP53 suggested a poor prognosis in mCRPC patients [ 80 ], which is also associated with an elevated CTCs count. These studies suggest that CTCs count may have value as a prognostic biomarker. Due to the low sensitivity of the detection of CTCs in non-metastatic tumors, the application of CTCs in the early stage of tumor progression is limited, but this cannot deny its clinical value in mCRPC patients [ 81 ]. Liquid biopsy based on cfDNAs in PCa Compared with CTCs, cfDNAs are more widely used in the clinical application of PCa, and play an important role in the diagnosis, screening, prognosis, and guidance of targeted drug therapy for advanced patients [ 81 , 82 ]. In terms of diagnosis, a study published a decade ago showed that cfDNAs level is elevated in the body fluids of PCa patients [ 83 ]. Other studies have investigated epigenetic changes as biomarkers for PCa diagnosis and screening [ 80 , 84 ]. In another study, PSA combined with cfDNAs and GADD45a methylation increased the diagnostic sensitivity and specificity to 87.5% and 94.1%, respectively [ 85 ]. A subsequent study revealed that the combined detection of GSTP1 promoter methylation and PSA in multiple body fluid samples could significantly improve the diagnostic accuracy [ 86 ]. A recent study, using a new algorithm based on fragmentomics, effectively distinguished clonal hematopoietic variants that were not derived from tumors. These variants would reduce the accuracy of liquid biopsy [ 42 ]. Furthermore, liquid biopsy based on ctDNA can assist us in classifying the risk of PCa at the early stage of diagnosis. Some studies have shown that genomic alterations in genes such as SPOP, CHD1, PTEN, NKX3-1, FOXA1, and APC can help distinguishing between primary PCa and advanced PCa [ 87 ]. In terms of prognosis, a high ctDNAs score indicates a poor prognosis in both mHSPC and mCRPC patients [ 88 ]. However, after the treatment of abiraterone acetate, enzalutamide and other commonly used drugs, ctDNAs level will be significantly reduced [ 88 ]. In addition, some patients whose cfDNAa sequencing detected AR amplification or two AR point mutations, L702H and T878A, had a non-significant PSA reduction after treatment, a poorer prognosis, and a lower overall survival (OS) rate [ 89 ]. In another study, data indicated that ctDNA fragments obtained through lpWGS could help determine prognostic information, suggesting that cfDNA testing can predict PCa prognosis [ 90 ]. A study conducted by Zang et al. [ 91 ] focused on minimal residual disease (MRD) in PCa. Before the surgery, 45% of the patients had detectable ctDNA in their bodies. After the surgery, more than 60% of the patients still had this substance in their bodies, and the average tumor proportion was less than 0.1%. The persistent ctDNA present in the body one month after the surgery seemed to be associated with the subsequent biochemical recurrence [ 91 ]. Another study showed that patients who could be detected ctDNA before treatment had much more severe clinical manifestations after treatment than those who could not be detected [ 92 ]. Its characteristics included early recurrence and metastasis of the disease, poor response to systemic treatment, and rapid deterioration of the condition, which usually leads to death within 2.5 years [ 92 ]. CfDNAs are also considered to be useful for long-term treatment monitoring and guidance of medication. Some studies have used cfDNAs to identify the presence of AR gain and some specific gene mutations, which are closely related to treatment resistance of PCa [ 88 , 89 ]. One of these studies pointed out that enzalutamide resistance may be related to AR amplification by detecting the AR axon 8 in plasma cfDNA [ 93 ]. A follow-up study of enzalutamide resistance was conducted in 65 mCRPC patients, and cfDNA was collected through continuous integrated genomic profiling [ 89 ]. Multiple mutations associated with drug resistance were identified, including AR amplification, RB1 deletion, AR-L702H mutations, AR-T878A mutations, PI3K pathway alterations, and CTNNB1 mutations [ 89 ]. Reversing these processes may allow for the early identification of patients who have developed resistance to enzalutamide and the choice of alternative drugs from the start. Another study on abiraterone found that patients with L702H and T878A mutations were more likely to develop abiraterone resistance [ 88 ]. A recent study revealed that ctDNA tumor fraction has prognostic significance and can be continuously updated through continuous monitoring, providing real-time individual treatment plan for patients [ 94 ]. In conclusion, cfDNAs have great clinical value in predicting PCa treatment. Liquid biopsy based on cfRNAs in PCa At present, the application of cfRNAs in liquid biopsy of PCa is mainly focused on PCa antigen 3 (PCA3). PCA3 is a lncRNA that is overexpressed in 95% of PCa [ 95 ]. PCA3 has high specificity and is not expressed in normal prostate tissue, but is only low expressed in benign hyperplastic prostate tissue [ 96 ]. Earlier studies have shown that at a cut-off point of 35, the specificity and sensitivity of PCa diagnosis are 72% and 58%, respectively, superior to conventional PSA testing [ 97 ]. The setting of the cut-off points of PCA3 is still highly controversial, therefore, more research investment is needed to clarify the clinical application of PCA3 cut-off point. Current studies are more inclined to believe that PCA3 should be combined with other biomarkers such as PSA to achieve joint diagnosis and improve sensitivity and specificity [ 97 – 99 ]. A recent study also found that overexpression of certain cell subtype genes via cfRNA sequencing may help in clinical treatment selection and prognosis prediction [ 100 ]. Liquid biopsy based on EVs in PCa EVs, particularly exosomes, have emerged as a promising platform for liquid biopsy in PCa. Unlike conventional urine based tests that require a digital rectal examination prior to sample collection, EVs based liquid biopsy analyze first catch urine samples without the need for prostate manipulation, significantly simplifying the sampling process and improving patient compliance [ 101 , 102 ]. In 2016, McKiernan et al. [ 101 ] conducted the first study on ExoDx Prostate IntelliScore (EPI) as the biomarker of PCa liquid biopsy. This exosome detection use urine as samples and it is based on measuring the expression of PCA3, ERG, and SPDEF [ 101 ]. Later research by McKiernan et al. [ 103 ] pointed out that the AUC of this detection method could reach 0.70. In 2023, Dudinec et al. [ 104 ] conducted a study on the combination of EPI with traditional PSA testing, and found that the combination of the two was helpful in determining the grade of PCa and might improve patient compliance. Nowadays, EPI testing has successfully been promoted from research to clinical practice and is expected to be widely applied in the future [ 105 ]. In addition, EVs can also be used for stratifying high-risk patients with PCa, and they are of great significance for the early detection of metastasis [ 106 ]. A study has shown that miR-4287 derived from EVs can predict the occurrence of metastasis at an early stage, with a specificity of 88.24% [ 107 ]. Clinical application of liquid biopsy in BCa BCa mainly relies on invasive cystoscopy or transurethral resection of bladder tumor (TURBT) for diagnosis and prognosis based on pathology. Because of the high recurrence rate of BCa, it is often inevitable that patients will have multiple surgeries [ 108 ]. As an expensive and invasive procedure, TURBT is not a convenient method for diagnose. Therefore, there is an urgent need for a non-invasive method to help patients with early diagnosis, prognosis and long-term monitoring of relapse after treatment. Liquid biopsy has great clinical potential and may play an important role in the clinical practice of patients with BCa (Table 2 ). Table 2. Summary of key clinical trials of liquid biopsy in patients with BCa Biomarkers Years Cohorts Main findings References CTC 2012 100 patients with BCa underwent cystectomy The presence of preoperative CTC is a powerful predictor of disease recurrence and cancer-specific mortality in patients with BCa underwent cystectomy Prognostic role and HER2 expression of circulating tumor cells in peripheral blood of patients prior to radical cystectomy: A prospective study CTC 2019 102 patients with high-grade T1 BCa CTCs are able to provide accurate risk stratification, which is essential to determine the best monitoring strategy for patients after diagnosis Circulating Tumor Cells Identify Patients with Super-High-Risk Non-Muscle-Invasive Bladder Cancer: Updated Outcome Analysis of a Prospective Single-Center Trial CTC 2022 315 patients with BCa CTCs have prognostic value for cancer-specific mortality and incidence of relapse in non-metastatic MIBC patients treated by radical cystectomy Circulating tumour cells to drive the use of neoadjuvant chemotherapy in patients with muscle-invasive bladder cancer ctDNA 2019 68 patients with BCa CtDNA serves as a prognostic biomarker; CtDNA dynamics during chemotherapy reflect response to treatment and patient outcome; CtDNA identifies disease recurrence better than radiographic imaging Early Detection of Metastatic Relapse and Monitoring of Therapeutic Efficacy by Ultra-Deep Sequencing of Plasma Cell-Free DNA in Patients With Urothelial Bladder Carcinoma mt-utDNA 2024 417 UC patients; 391 benign urologic diseases; 139 non-UC cancers Mt-utDNA test has high sensitivity for detecting early-stage and low-grade tumors Clinical effectiveness of a multitarget urine DNA test for urothelial carcinoma detection: a double-blinded, multicenter, prospective trial ctDNA 2019 68 patients with BCa CtDNA assessment for early risk stratification, therapy monitoring, and early relapse detection in BCa is feasible Early Detection of Metastatic Relapse and Monitoring of Therapeutic Efficacy by Ultra-Deep Sequencing of Plasma Cell-Free DNA in Patients With Urothelial Bladder Carcinoma ctDNA 2024 130 patients with BCa received pembrolizumab; 130 patients with BCa received chemotherapy Distinct patterns in early ctDNA changes with immunotherapy and chemotherapy and differences in their association with long-term outcomes Pembrolizumab for advanced urothelial carcinoma: exploratory ctDNA biomarker analyses of the KEYNOTE-361 phase 3 trial miRNA 2019 392 BCa patients; 100 non-cancer controls; 480 patieants with other types of cancers A combination of 7 miRNAs could discriminate BCa from non-cancer and other types of cancers Circulating miRNA panels for specific and early detection in bladder cancer miRNA 2015 250 patients with BCa; 240 healthy controls A combination of 6 miRNAs can be a novel non-invasive biomarker for BCa detection Serum microRNA expression signatures identified from genome-wide microRNA profiling serve as novel noninvasive biomarkers for diagnosis and recurrence EV, mRNA 2018 8 patients with UC; 4 healthy and disease controls Three urinary EV mRNAs were discovered to be elevated in BCa Bladder cancer detection by urinary extracellular vesicle mRNA analysis Open in a new tab Liquid biopsy based on CTCs in BCa CTCs are mainly detected in patients with metastatic tumors. Therefore, the potential early diagnostic value of CTCs for BCa is low [ 109 , 110 ]. At present, the application of CTCs in the clinical management of BCa is mainly to predict the prognosis, monitoring recurrence/progression and guide therapy. Rink et al. [ 111 ] reported that the detection of CTCs in patients undergoing cystectomy was associated with a higher risk of recurrence and a poorer prognosis. The sensitivity and specificity of CTCs for the stage and grade of BCa were 35% and 97%, respectively [ 112 ]. An early study found that 87.5% of non-muscle-invasive BCa (NMIBC) patients who tested positive for CTCs developed muscle-invasive BCa (MIBC), while those who did not test positive for CTCs showed no progression [ 113 ]. In another earlier study, it was confirmed for patients with high-risk NMIBC that the presence of CTCs is an independent predictor of disease progression to MIBC, with a predictive value of up to 75% [ 114 ]. A meta-analysis conducted by Andy et al. [ 115 ] in 2024 indicated that patients with positive CTCs had a significantly increased risk of recurrence, and the risk of progression from NMIBC to MIBC also significantly increased. A recent study has shown that CTCs can provide accurate risk stratification for NMIBC patients, which is critical for predicting the optimal monitoring strategy for patients after diagnosis [ 116 ]. In addition, they found that patients with at least one CTCs detected had poor metastasis-free survival, cancer-specific survival, and OS [ 116 ]. In other words, BCa patients with detectable CTCs have a poorer prognosis. In addition, since CTCs are strongly associated with hematogenous dissemination of tumors, and the aforementioned studies have shown that CTCs can be detected in patients with NMIBC, this undoubtedly indicates that hematogenous dissemination does not occur only in the advanced stage of the tumor, but also appears earlier in NMIBC patients [ 18 , 113 – 116 ]. Detection of CTCs can provide earlier treatment enhancement guidance for some patients. Rink et al. [ 111 ] conducted a study on 22 patients whose CTCs were positive. Among them, 64% (14/22) of the patients had the same HER2 status as their primary tumors. Among the 5 patients with lymph node metastasis, the HER2 status was completely consistent with the CTCs and the primary tumor. 14% (3/22) of the patients had strong positive HER2 expression in their CTCs, suggesting that these patients might benefit from anti-HER2 targeted therapy [ 111 ]. A recent study has shown that baseline detection and continuous assessment of PD-L1 through CTCs may be an effective approach, which can help select treatment options for PD-L1 inhibitor candidate patients after the failure of Bacillus Calmette-Guérin therap [ 117 ]. CTCs are also considered to be useful in predicting treatment response. Neoadjuvant chemotherapy (NAC) is commonly used clinically for metastatic tumors, but the OS benefit after treatment is only 5% to 6% [ 118 ]. However, patients with detectable CTCs by liquid biopsy had a higher response to NAC than those without CTCs [ 119 ]. This will undoubtedly provide a basis for CTCs to predict the therapeutic effect and guide further treatment. Liquid biopsy based on cfDNAs in BCa Both urine and blood can be used as samples for ctDNA testing. Apoptotic or necrotic urothelial cells and plasma DNA filtered by the kidneys are the main sources of ctDNA in urine [ 120 ]. Early studies on the diagnosis of BCa by cfDNAs often selected some markers that did not have clinical application value due to low sensitivity or specificity. For example, Kim et al. [ 121 ] studied Topoisomerase-II alpha, a cfDNA overexpressed in patients with BCa, as a biomarker with high sensitivity but low specificity. The research conducted by Roperch et al. [ 122 ] was based on a urine sample combined detection of FGFR3 and other ctDNA methylations (HS3ST2, SEPTIN9, and SLIT2), achieving a high diagnostic efficiency, the specificity and sensitivity were 97.6% and 84.8% respectively, and the AUC was 0.96. Another study on ctDNA using urine samples found that 36% of the patients carried at least one mutation in FGFR3 or PIK3CA [ 123 ]. Furthermore, the levels of FGFR3 or PIK3CA are significantly correlated with the size, grade and risk stratification of BCa [ 123 ]. Furthermore, patients with higher levels of FGFR3 or PIK3CA in NMIBC are more likely to experience disease progression [ 123 ]. A study published in 2022 focused on DNA methylation biomarkers and found that DMTRA2 was the best biomarker for BCa diagnosis with sensitivity and specificity of 82.9% and 92.5%, respectively [ 124 ]. More recently, another study on the screening and diagnosis of urothelial carcinoma (UC) used multi-target urine tumor DNA (mt-utDNA) as biomarkers, which is a series of cfDNA including FGFR3, TERT mutations, ONECUT2, and VIM [ 125 ]. Interestingly, in this study, the overall sensitivity and specificity of mt-utDNA for the diagnosis of UC were 91.37% and 95.09%, respectively, which increased with the change of UC stage and grade [ 125 ]. If more studies confirm this conclusion in the future, mt-utDNA is likely to be used in the clinical screening and diagnosis of BCa. In the research conducted by Ma et al. [ 41 ], the integration of fragmentomics and the low methylation levels in heterochromatin regions resulted in an AUC value of 0.93 for BCa detection. cfDNA can also be used for the prognosis of BCa. Studies have shown that cfDNA can be detected in patients who progress to MIBC several months before clinical progression [ 126 ]. Many studies have shown that the ability of ctDNA to detect recurrence and metastasis of BCa is much better than that of traditional radiographic imaging methods [ 127 , 128 ]. Another study claimed that among patients who underwent cystectomy, ctDNA was detected in 75% of them, and the recurrence-free survival rate of these patients was poor [ 128 ]. The study by Powles et al. [ 129 ] was based on the ctDNA detection using whole-genome sequencing. In the identification of recurrence after cystectomy, the detection sensitivity reached 91%, the specificity was 92%, and it was 131 days earlier than the imaging results [ 129 ]. A study conducted by Birkenkamp et al. [ 128 ] had similar results. Based on the monitoring of ctDNA, recurrence could be detected 101 days before it was diagnosed by imaging examinations. Another study found that the persistent presence of ctDNA after cystectomy can predict metastatic recurrence, with a sensitivity of 94% and a specificity of 98% [ 127 ]. Based on these previous studies, it can be concluded that longitudinal ctDNA monitoring can identify MRD and predict recurrence, detecting recurrence several weeks to several months earlier than imaging tests. This enables individualized supportive interventions and may help increase the detection rate of systemic metastasis [ 128 , 130 ]. In a recent study, the ctDNA of some patients who received pembrolizumab or chemotherapy decreased, and the radiological tumors also shrank [ 131 ]. This suggests that continuous detection of ctDNA may reflect the treatment response of BCa, which undoubtedly indicates that cfDNA also has great potential in clinical treatment monitoring. Liquid biopsy based on cfRNAs in BCa At present, the research on the application of cfRNAs in BCa mainly focuses on early diagnosis and screening, and a few studies are also related to prognosis, including miRNA and circRNA. The efficiency of a single miRNA in the diagnosis of BCa is not high. Therefore, recent studies often use multiple different miRNAs as panels for joint analysis. A total of 13 miRNAs were analyzed in two studies. The panels with the highest sensitivity and specificity for the diagnosis of BCa were 98% and 91%, respectively [ 132 , 133 ]. Some studies have linked miRNAs to tumor stage and grade. miR-210 was considered to be positively correlated with pathological stage [ 134 ], while miR-92 and miR-33 were considered to be negatively correlated with pathological stage [ 135 ]. CircRNAs mainly play a role in tumor progression by regulating multiple pathways [ 136 , 137 ]. For example, overexpression of circTRF25 can affect tumor progression by regulating the PI3K-AKT pathway [ 136 ]. Since various circRNAs usually play a role in the upstream of the pathways, they are considered closely related to the early diagnosis and prognosis of BCa [ 138 ]. However, more clinical trials are needed to verify this view. The types of BCa-related cfRNAs are numerous and the mechanisms are complex. Compared with other biomarkers, there is a lack of clinical trial support. Therefore, there are still great challenges in applying cfRNA as a biomarker in the clinical management of BCa patients. Liquid biopsy based on EVs in BCa A study published in 2018 showed that the levels of three mRNAs detected by EVs in urine were elevated, indicating that the three corresponding genes SLC2A1, GPRC5A and KRT17 were overexpressed in pT1 stage and advanced BCa [ 139 ]. This conclusion suggests that we may diagnose BCa by detecting the expression of these three genes in EVs. Although this study demonstrated the potential diagnostic value of EVs for BCa, more studies are required to support the clinical application. Ghoreifi et al. [ 140 ] conducted a study in 2024 that analyzed the preoperative blood samples of 28 patients with upper urinary tract UC and compared them with those of normal donors and postoperative samples. The results showed that the CTCs and LOs in the patients’ preoperative blood were significantly higher than those of the normal donors, while the LOs significantly decreased after surgery. Moreover, for patients with higher LOs, their prognosis was worse [ 140 ]. Another research conducted by Magri et al. [ 64 ] revealed that even in NMIBC patients with no CTCs detected, tumor-derived EVs ≥ 7 indicated a poorer prognosis; while in patients with CTCs ≥ 1, tumor-derived EVs ≥ 7 was significantly associated with worse time to progression and cancer-specific survival. This suggests that the count of tdEVs can further assist in risk stratification and has important prognostic value in early-stage cancers. The combined analysis of CTCs and tdEVs can significantly enhance the predictive ability for the risk of NMIBC patients [ 64 ]. Clinical application of liquid biopsy in RCC Due to the lack of powerful molecular diagnostic tools and the absence of relatively obvious specific symptoms in the early stage of the disease, RCC becomes a silent and unexpected tumor. Most patients were not adequately evaluated and screened within the first 6 months after the first appearance of symptoms, thereby reducing the number of patients diagnosed at an early stage [ 141 ]. Like BCa mentioned above, the early diagnosis of RCC depends on imaging, while the prognosis depend on post-operative solid tumor biopsy. In addition, as some patients with RCC develop drug resistance after long-term targeted drug therapy, non-invasive examinations, such as liquid biopsies, are also needed to assist in the long-term monitoring of the patients [ 142 ]. Fortunately, clinical trials in recent years have gradually revealed the clinical value of liquid biopsies of various components in diagnosis, prognosis and monitoring (Table 3 ). Table 3. Summary of key clinical trials of liquid biopsy in patients with RCC Biomarkers Years Cohorts Main findings References CTC 2021 195 patients with RCC The presence of three or more CTCs at baseline is associated with a significantly shorter progression free survival and OS in patients with mRCC Prognostic Role of Circulating Tumor Cells in Metastatic Renal Cell Carcinoma: A Large, Multicenter, Prospective Trial CTC 2006 55 patients with mRCC Disseminated CK+ cells play a role in the biology of tumor spread of RCC; Immunocytochemical detection of CK+ CTCs can be useful in assessing the prognosis of patients with M1 disease Frequency and Prognostic Relevance of Disseminated Tumor Cells in Bone Marrow of Patients with Metastatic Renal Cell Carcinoma CTC 2019 58 patients with nmRCC; 11 patients with mRCC The recurrence or metastasis of RCC was uncorrelated with initial CTCs counts but probably related with the variation trend of CTCs, especially mesenchymal CTCs and Beclin1 positive CTCs Dynamic changes of different phenotypic and genetic circulating tumor cells as a biomarker for evaluating the prognosis of RCC CTC 2021 20 RCC patients treated with ICI; 22 RCC patients treated with TKI CTC enumeration and expression of PD-L1 and HLA-I correlated with disease progression and treatment response Development and initial clinical testing of a multiplexed circulating tumor cell assay in patients with clear cell renal cell carcinoma cfDNA 2018 38 patients with RCC; 34 healthy controls CfMeDIP-seq of cfDNA is sensitive for mRCC detection Increased level and fragmentation of plasma circulating cell-free DNA are diagnostic and prognostic markers for renal cell carcinoma cfDNA 2021 82 patients with RCC; 20 healthy controls Plasma levels of cfDNA from GADPH and hTERT genes were correlated to tumor diagnosis and progression Clinical value of perioperative levels of DNA and mRNA in plasma of patients with renal cell carcinoma cfDNA; mRNA 2021 20 patients with RCC The levels of ctDNA could be an early predictor of treatment response in patients with mRCC who receive ICI therapy Potential of circulating tumor DNA as a predictor of therapeutic responses to immune checkpoint blockades in metastatic renal cell carcinoma miRNA 2018 52 patients with RCC; 15 patients with oncocytoma MiR-15a expression measured in urine may be used as diagnostic molecular biomarker for RCC. MicroRNA-15a expression measured in urine samples as a potential biomarker of renal cell carcinoma miRNA 2020 146 patients with RCC; 150 healthy controls The 3-miRNA panel in serum could serve as a non-invasive diagnostic biomarker of RCC A Three-microRNA Panel in Serum: Serving as a Potential Diagnostic Biomarker for Renal Cell Carcinoma miRNA 2020 50 patients with RCC; 30 patients with oncocytoma The diagnostic and prognostic potential of miR-30a-5p for ccRCC in urine samples is validated MicroRNA-30a-5p(me): a novel diagnostic and prognostic biomarker for clear cell renal cell carcinoma in tissue and urine samples miRNA 2018 86 patients with RCC; 55 patients with benign renal tumor; 28 healthy controls Serum miR-122-5p and miR-206 are novel non-invasive prognostic biomarkers for patients with ccRCC Serum miR-122-5p and miR-206 expression: non-invasive prognostic biomarkers for renal cell carcinoma Open in a new tab Liquid biopsy based on CTCs in RCC Studies have shown that CTCs detection is a potential method for the diagnosis and staging of RCC [ 143 ]. More and more studies revealed that CTCs were of great value in prognostic assessment in RCC. When CTCs counts of RCC patients reached 3, the OS and progression-free survival were significantly reduced compared with those of patients with CTCs less than 3 [ 144 ]. The other two studies both found that the primary tumors of CTC-positive patients were larger, and the tumor size was positively correlated with the CTCs count [ 145 , 146 ]. Song et al. [ 145 ] also noticed that CTCs count in postoperative patients decreased significantly compared with that before the operation. Another study published by Marchioni et al. [ 146 ] found that CTC-positive patients were more likely to metastasis or lymph node infiltration, which may provide a theoretical basis for clinical detection of CTCs to predict tumor metastasis. In addition, cytokeratin positive (CK+) CTCs are considered to be associated with poor prognosis of RCC [ 143 ]. The risk of death for patients with detectable CK+ CTCs is 2.3 times higher than that for patients without detectable CK+ CTCs [ 147 ]. However, another study based on metastatic RCC (mRCC) patients showed that CK+ CTCs played an important role in the metastasis of RCC and has a worse prognosis than patients with CK- CTCs [ 148 ]. This indicates that CK+ CTCs can be used for the prognosis assessment of mRCC patients. A recent study found that Beclin-1 positive CTCs in patients with mRCC were significantly higher than Beclin-1 negative CTCs, while this phenomenon was not observed in patients with non-mRCC (nmRCC) [ 149 ]. This trial may provide a basis for the clinical prognosis of tumor metastasis. CTCs can also be used to guide medication and monitor treatment. Studies have pointed out that the decreased expression of HLA-I in CTCs indicates a poor response to tyrosine kinase inhibitor (TKI) treatment [ 150 ]. A recent study indicates that PD-L1 and HLA-I have a good correlation with the responses of immune checkpoint inhibitor (ICI) treatment and TKI treatment, respectively [ 151 ]. In addition, their research indicates that, compared with traditional radiological imaging methods, the additional detection of CTCs may offer patients better personalized treatment options [ 151 ]. Liquid biopsy based on cfDNAs in RCC The application of cfDNAs in the diagnosis of RCC has been extensively studied. A study by Yamamoto et al. [ 152 ] showed that the level of plasma cfDNAs has diagnostic value, with a sensitivity and specificity of 63.0% and 78.1%, respectively. Although a variety of RCC related DNA mutations can be detected, the detection rates of corresponding cfDNAs in patients are very low and has no clinical value for the time being [ 153 ]. The detection of DNA methylation is a relatively promising research direction. In a study conducted in 2020, 21 candidate cfDNA variants were identified in patients with mRCC. cfDNAs were used to test 34 patients with mRCC, and the results were 100% positive [ 154 ]. This trial provided a potential diagnostic biomarker with a sensitivity of 100% and a specificity of 88%. RCC usually has no symptoms in its early stage, which leads to a large number of cases being discovered at an advanced stage. Fragmentomics has the potential to enhance the early detection rate of RCC and has already made certain progress [ 40 ]. A model proposed by Peng et al. [ 39 ] for the early detection of RCC, had an AUC value of 0.966 in the validation cohort and 0.952 in the external cohort. The sensitivity and specificity of the validation cohort were 90.5% and 93.8% respectively, while those of the external cohort were 76.7% and 92.9% respectively [ 39 ]. There are few studies on the application of cfDNAs in the prognosis and treatment of RCC. It has been reported that cfDNA levels of GAPDH and hTERT decreased significantly after surgical treatment, and the higher their concentrations in the blood, the higher the risk of RCC progression [ 155 ]. Another study by Kim et al. [ 156 ] indicated that ctDNA levels decreased in patients who responded well to ICI treatment, whereas ctDNA levels increased in those who still progressed after treatment. An earlier study showed that an increase in cfDNA levels was associated with a shortened RFS, indicating that cfDNA can be used for early detection of metastasis [ 157 ]. Further studies have shown that ctDNA has great potential in monitoring MRD in RCC [ 158 ]. It can sensitively detect residual tumor cells after treatment [ 158 ]. An increase in ctDNA levels can indicate that molecular-level recurrence has occurred before the appearance of imaging evidence, and can also reveal new drug-resistant mutations [ 40 , 158 ]. Liquid biopsy based on cfRNAs in RCC Studies on the application of cfRNA in RCC mainly focused on miRNAs. In addition to blood, urine can also be used as a miRNA detection specimen for the diagnosis of RCC. MiR-15a had a high differential diagnostic value in distinguishing RCC from benign tumors, with a sensitivity of 100% and a specificity of 98.1% [ 159 ]. The sample size of this trial is small, and further studies are needed to confirm its clinical value. Another study found that the combined detection of miR-122, miR-1271 and miR-15b had diagnostic value for RCC, with a sensitivity of 100% and a specificity of 86% [ 160 ]. However, the sample size of this study is also small, and further studies with large samples are needed for verification. Another study proposed by Huang et al. [ 161 ] focused on the application of multiple miRNAs as panels for detection. They first selected 30 different miRNAs and tried various combinations. Finally, it was determined that miR-224-5p, miR-34b-3p and miR-182-5p were the best panels for diagnosis [ 161 ]. The diagnostic area under the curves of these three-miRNA panel was 0.855, which has great clinical potential value [ 161 ]. In addition, the relationship between miRNA methylation levels and RCC has also attracted the attention of some scholars. Outeiro-Pinho et al. [ 162 ] demonstrated that the methylation level of miR-30a-5p could be used to detect RCC, with a sensitivity of 80.3% and a specificity of 66.3% in differentiating mRCC. Some studies have suggested that miRNAs can be applied in the prognosis assessment of RCC. It has been proposed that miR-122-5p and miR-206 are associated with the stage, grade and distant metastasis of RCC [ 163 ]. The level of miR-15a in the specimens was also related to tumor size, and the level of miR-15a decreased significantly after the operation [ 159 ]. Limitations for the applications of liquid biopsy The technology of liquid biopsy is still not fully developed today, and there are still many technical obstacles that need to be overcomed. The following text will list several of the current most significant technical challenges. Low levels of biomarkers The levels of most biomarkers are very low in samples such as blood or urine. In the previous two studies, the detection rates of CTC in patients with metastatic urothelial bladder carcinoma were all no more than 60%, while in patients with localized urothelial bladder carcinoma, CTC was not detected in all cases [ 110 , 164 ]. As for ctDNA, the proportion of certain ctDNA in total cfDNA varies greatly, and in some cases it can be lower than 0.1%. In the presence of a large number of different types of DNA copies, it is difficult to identify and track rare mutant DNA fragments [ 165 ]. Extraction and transportation difficulties Most of the biomarkers that can be used in liquid biopsy encounter technical difficulties in extraction or transportation. Because the ctDNA molecules are cleared from the blood very quickly, their half-life is usually less than one hour [ 14 ]. This means the concentration of ctDNA will decrease rapidly over time, which imposes strict requirements on the sample processing time [ 14 ]. Other studies on EVs have indicated that the most common storage temperature for EVs is -80 °C, but it is difficult to maintain such low temperature during handling or transportation [ 166 ]. And when EVs from different sample sources are stored under the same conditions, their stability may vary significantly [ 57 ]. Lack of standardized protocols Biomakers such as CTCs, ctDNA, ctRNA, and EVs may vary in sensitivity and specificity among different platforms depending on different collection, storage, and transportation conditions [ 12 , 32 , 166 , 167 ]. Therefore, an international standardized protocols is needed to reduce pre-analytical variables and conduct cross-platform validation and comparative studies. Conclusions For urinary system tumors, including PCa, BCa, and RCC, traditional clinical management approaches are often expensive and invasive, which will significantly reduce patients’ compliance and hinder the early diagnosis and treatment of the disease. Compared with existing clinical methods, liquid biopsy is a minimally invasive examination, which is more conducive to multiple collections and repeated detections, achieving early screening and long-term monitoring. Many components of liquid biopsy, such as CTCs, cfNAs, EVs etc., have been demonstrated by current studies to be applicable to the clinical management of urinary system tumors. Liquid biopsy can now help screen for and diagnose urinary system tumors at an early stage or differentiate unknown pathological types of masses. For patients who have been diagnosed, liquid biopsies can also provide prognostic judgments, and predict the risk of survival and death. For patients who have already undergone surgical treatment, liquid biopsies can provide inexpensive long-term monitoring, identify recurrence or metastasis in a timely manner, and guide changes in treatment. Up to now, liquid biopsy is still difficult to completely replace traditional detection methods and is more often used as an auxiliary method. Moreover, most of its research is limited to specific cancers, and many tumors with relatively low incidence have little relevant research. There are still many technical obstacles preventing the application of liquid biopsy. For instance, the low abundance of biomarkers makes them difficult to detect. Most biomarkers are also challenging to preserve and transport. The absence of standardized protocols also contribute to the limitations of liquid biopsy. Even if the biopsy results are proven to be effective, their clinical application is still very challenging. However, liquid biopsy still shows great potential and value, if the technical obstacles mentioned earlier can be overcome, it will be an important direction for the clinical management of urinary system tumors in the future. Good news is that more and more researchers and clinical workers have already noticed this field, and a lot of clinical research has already been done. There is also study that subtly reduce the high cost due to high sequencing coverage to make it more clinical applicable [ 168 ]. Furthermore, with the advancement of artificial intelligence, through machine learning, more meaningful and potential liquid biopsy biomarkers can be discovered. If liquid biopsy technique can be further perfected and applied to the clinical management of urinary system tumors, it may bring some benefit to the patients. Acknowledgements Not applicable. Abbreviations PCa Prostate cancer BCa Bladder cancer RCC Renal cell carcinoma CTCs Circulating tumor cells CfNA Cell-free nucleic acid CtDNA Circulating tumor DNA MRNA Messenger RNA MiRNA MicroRNA CircRNA Circular RNA EV Extracellular vesicle TEP Tumor-educated platelet LncRNAs Long non-coding RNAs LO Large oncosomes PSA Prostate-specific antigen AR Androgen receptor AR-V Androgen receptor splice variant MHSPC Metastatic hormone sensitive prostate cancer MCRPC Metastatic castration-sensitive prostate cancer OS Overall survival MRD Minimal residual disease PCA3 Prostate cancer antigen 3 EPI ExoDx Prostate IntelliScore TURBT Transurethral resection of bladder tumor NMIBC Non-muscle-invasive bladder cancer MIBC Muscle-invasive bladder cancer NAC Neoadjuvant chemotherapy UC Urothelial carcinoma Mt-utDNA Multi-target urine tumor DNA CK+ Cytokeratin positive MRCC Metastatic renal cell carcinoma NMRCC Non-metastatic renal cell carcinoma TKI Tyrosine kinase inhibitor ICI Immune checkpoint inhibitor Author contributions KW, DD and HL conceived the review; LL and PS reviewed the information. YZ arranged the format of the figures. RX, YZ, and XZ wrote the manuscript. KW, DD, HL, and LL critically reviewed the manuscript. All authors read and approved the final manuscript. Funding This work was supported by National Natural Science Foundation of China (Grant No. 82072835) to K Wang, 345 Talent Project of Shengjing Hospital of China Medical University (Grant No. M0366) to K Wang, Outstanding Scientific Fund of Shengjing Hospital (Grant No. 202205) to K Wang, Natural Science Foundation of Liaoning Province (Grant No. 2024-MSLH-562) to K Wang, and Department of Science and Technology of Liaoning Province (2022-BS-121) to D Dong. Data availability No datasets were generated or analysed during the current study. Declarations Ethical approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. 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Accessed February 28, 2023. https://www.frontiersin.org/articles/10.3389/fonc.2022.1014592 [ DOI ] [ PMC free article ] [ PubMed ] Associated Data This section collects any data citations, data availability statements, or supplementary materials included in this article. Data Availability Statement No datasets were generated or analysed during the current study. 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