PD-L1+ exosomes act as a useful biomarker in early cancer diagnosis, therapeutic monitoring, prognostic assessment, and immunotherapy for non-invasive liquid biopsy. There exists urgent clinical need for developing rapid, portable, and cost-effective immediate-response assays for PD-L1+ exosomes. Here, we proposed a smartphone-assisted colorimetric sensor using the triple-helix molecular switch (THMS) combined with Y-shaped catalytic hairpin assembly (Y-CHA) reaction. Namely, this strategy initiates the Y-CHA cycle and forms Y-DNA G-quadruplex/Hemin DNAzyme with K+ and Hemin after specific recognition of PD-L1+ exosomes by THMS. Then, PD-L1+ exosomes could be quantified via the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB)-mediated color change. The method demonstrates excellent specificity and sensitivity, with a limit of detection (LOD) of 5.45 × 103 particles/mL and an LOD of 8.56 × 103 particles/mL using smartphone analysis. With this strategy, we found that PD-L1+ exosome levels were significantly elevated in the peripheral blood of patients with colorectal cancer liver metastases (CRLM), indicating great potential for identifying CRLM patients. Additionally, this colorimetric sensor can be used to quantify PD-L1+ exosomes across wide range of cancers, which possess a great prospect in the point-of-care testing filed for cancer companion diagnosis.

High salt buffer may be used for the UV/VIS or radiometric based detection of trihybrid DNA but would contaminate the electrospray mass spectrometer. Colorimetric DNA hybridization assays with the substrate BCIP/NBT reacted with the alkaline phosphatase-streptavidin (APSA) enzyme conjugate that showed a linear range for HIV DNA from 1 pM to 100 pM DNA in presence of high salt concentrations. Ammonia bicarbonate (AMBIC) or ethanolamine resulted in strong DNA hybridization similar to NaCl but was compatible with specific and sensitive mass spectrometry. Linear and Gaussian analysis of HIV DNA with 10 % error was achieved across the pico Molar range from APSA amplification that converted the substrate AMP to adenosine for detection by monitoring the precursor ion at m/z 268 and plotting the fragment intensity at m/z 136 (m/z 268→ m/z 136) that was linear to 100 fM after log transformation. The novel observation that specific DNA hybridization in NaCl may be substituted with AMBIC permitted the direct analysis of a target DNA in femto molar to pico molar range by enzyme linked mass spectrometric assay (ELiMSA) using as little as 0.1 μL (100 nL) of sample reaction injected on column.

Liquid biopsy has emerged as a promising non-invasive, cost-effective and real-time approach for cancer diagnosis and monitoring, allowing for the detection of biomarkers in bodily fluids. Among these, microRNAs (miRNAs) are a valuable choice due to their stability and ability to reflect the tumor's heterogeneity. Concerning triple negative breast cancer (TNBC), an aggressive subtype, several studies have demonstrated consistent upregulation of miRNA-21. Its elevated levels, linked to poor prognosis, make it valuable for early detection, risk stratification, and targeted therapies. While traditional miRNA quantification methods are accurate, they often require complex procedures and skilled personnel, limiting their accessibility in low-resource environments. These challenges can be addressed by electrochemical point-of-care (POC) platforms, inspired by the glucose strip, offering a good alternative by reducing matrix effects, integrating cost-effective and eco-friendly substrates. In this work, miRNA-21 was selectively detected using a complementary DNA probe modified with methylene blue, as a redox mediator, immobilized onto an AuNPs-functionalized, paper-based screen-printed electrode. Significant experimental parameters and sensor's selectivity were carefully evaluated, allowing miRNA-21 detection in both standard solution and human serum with limit of detection (LOD) 1.2 nM and satisfactory repeatability of about 8%. The platform's performance improved tenfold with an external paper-based origami pre-concentration device, enabling pM-level miRNA detection and advancing its potential for real clinical practice applications. The platform is envisioned as a starting point for developing accessible, rapid, cost-effective POC testing, with significant implications for personalized medicine and early TNBC detection.

Ferroptosis is a type of iron‑dependent cell death characterized by excessive lipid peroxidation and may serve as a potential therapeutic target in cancer treatment. While the mechanisms governing ferroptosis continue to be explored and elucidated, an increasing body of research highlights the significant impact of epigenetic modifications on the sensitivity of cancer cells to ferroptosis. Epigenetic processes, such as DNA methylation, histone modifications and non‑coding RNAs, have been identified as key regulators that modulate the expression of ferroptosis‑related genes. These alterations can either enhance or inhibit the sensitivity of gastrointestinal cancer (GIC) cells to ferroptosis, thereby affecting the fate of GICs. Drugs that target epigenetic markers for advanced‑stage cancer have shown promising results in enhancing ferroptosis and inhibiting tumor growth. This review explores the intricate relationship between epigenetic regulation and ferroptosis in GICs. Additionally, the potential of leveraging epigenetic modifications to trigger ferroptosis in GICs is investigated. This review highlights the importance of further research to elucidate the specific mechanisms underlying epigenetic control of ferroptosis and to advance the development of novel therapeutic approaches.

Circulating tumor DNA (ctDNA) testing provides valuable prognostic and predictive information for guiding therapeutic choices and monitoring disease progression and drug resistance for urological tumors. Our review focuses on emerging opportunities for ctDNA analysis in urological tumors and the development of potential circulating biomarkers within a multidisciplinary framework to improve personalized treatment.

A nonsystematic literature review was conducted in the PubMed and MEDLINE databases. Prospective and retrospective peer-reviewed studies, review articles, and research abstracts on the use of ctDNA for urological tumors were included.

Several studies have demonstrated that ctDNA analysis is a promising tool that can help clinicians in the diagnosis and clinical management of urological tumors. In prostate and urothelial cancers, the ctDNA fraction increases proportionally from localized to metastatic disease, indicating a higher tumor burden and more aggressive behavior. Thus, ctDNA seems to be a useful tool for improving prognostic risk stratification and treatment selection. Data on the use of liquid biopsy in renal cell carcinoma are still limited, and assessment of prognostic and predictive biomarkers is a critical unmet need.

ctDNA analysis promises to revolutionize the management of urological tumors in different disease settings. Integration of ctDNA testing in routine clinical practice will require a multidisciplinary approach that involve patients, clinicians, and molecular biologists.

We reviewed how testing for tumor DNA in blood (circulating tumor DNA, ctDNA) is used in urological cancers. A great deal of evidence supports the usefulness of this noninvasive test. However, further research via a multidisciplinary approach is needed before ctDNA testing becomes part of routine patient care.

Metronomic therapy is one of the modalities used to treat OSCC. The present study is the first comprehensive analysis evaluating the potential of circulating endothelial cells (CECs) and their progenitors (CEPs) as prognostic biomarkers in OfSCC patients receiving metronomic therapy.

91 patients were included in the study group. ORR was 42.9 %, while the median OS and PFS were 6.67 months and 4.3 months, respectively. The counts of CECs and CEPs were enumerated by flow cytometry at baseline and follow-up (8, 30, 60, and 90 days after metronomic therapy).

We correlated the baseline and reduction of CECs after treatment with clinical response, PFS, and OS. Patients with high CEP levels at day 60 showed longer PFS, while patients with low CEC levels at day 30 had better PFS and OS. Change in CEC levels after 8 and 30 days was also correlated with PFS and OS, respectively. The change in CECs 8 days after metronomic therapy as a prognostic biomarker was further validated by multivariate Cox regression. A decline in CECs after 8 days had a favourable impact on OS, while none of the CEP measurements at any time point were statistically associated with OS or PFS.

The reduction of CECs after treatment might serve as a prognostic biomarker, whereas the baseline CEC counts as a predictive or prognostic factor in patients treated with metronomic therapy. The correlation between clinical response and survival further supports the biological relevance of these CECs and CEPs as prognostic biomarkers.

Current diagnostic tools are inaccurate and not specific to prostate cancer (PCa) diagnosis. Cancer-derived small extracellular vehicles (sEVs) play a key role in intercellular communication. In this study, we examined the diagnostic value of plasma sEV-derived carbonic anhydrase IX (CAIX) protein for PCa and clinically significant prostate cancer (csPCa) diagnosis and avoiding unnecessary biopsies.

Plasma samples (n = 230) were collected from the patients who underwent prostate biopsy with elevated prostate-specific antigen (PSA) levels. sEVs were isolated and characterized, and sEV protein CAIX was measured using an enzyme-linked immunosorbent assay. Independent predictors of csPCa (Gleason score ≥ 7) were identified, and a predictive model was established. A Nomogram for predicting csPCa was developed using data from the training cohort.

The expression of sEV protein CAIX was significantly higher in both PCa and csPCa compared to benign patients and nonsignificant PCa (nsPCa) (Gleason score < 7, p < 0.001). sEV protein CAIX performed well in distinguishing PCa from benign patients. The predictive model defined by sEV protein CAIX and PSA density (PSAD) demonstrated the highest discriminative ability for csPCa (AUC = 0.895), with diagnostic sensitivity and specificity of 82.5% and 85.8%, respectively. Furthermore, sEV protein CAIX is an effective predictor of 2-year biochemical recurrence (BCR) in PCa patients (p = 0.013), and its high expression is significantly associated with poorer BCR-free survival (p < 0.05).

Our findings demonstrate the excellent performance of sEV protein CAIX in PCa and csPCa diagnosis. The Nomogram-based csPCa predictive model incorporating sEV protein CAIX and PSAD exhibits strong predictive value. Additionally, assessing plasma sEV protein CAIX expression levels can further aid in evaluating patient prognosis and provide a basis for making effective treatment decisions.

Non-invasive diagnostic monitoring techniques have become essential for treating lung cancer (LC), which continues to be the primary cause of cancer-related death worldwide. The new diagnostic biomarkers called tumour-educated platelets (TEPs) show strong prospects for providing vital information about tumor biology, tumor spread pathways, and treatment reaction patterns. Despite lacking a nucleus, platelets exhibit an active RNA profile that develops through interactions with tumor-derived compounds and the tumor microenvironments (TME). This review explains platelet-tumour interaction regulatory mechanisms while focusing on platelet contributions toward cancer development, immune system avoidance, and blood clot formation. The detection and classification of LC show promise through the analysis of RNA molecules extracted from platelets that encompass mRNAs and non-coding RNAs. RNA sequencing technology based on TEP demonstrates excellent diagnostic power by correctly identifying LC patients alongside their oncogenic alterations of EGFR, KRAS, and ALK. Treatment predictions have proven successful using platelet RNA profiles, specifically in immunotherapy and targeted therapy. Integrating next-generation sequencing with machine learning and artificial intelligence enhances TEP-based diagnostic tools, improving detection accuracy. Standardizing platelet extraction methods and vesicle purification from tumor material needs better development for effective and affordable clinical use. Future investigations should combine TEPs with circulating tumor DNA and exosomal RNA markers to enhance both earliest-stage LC diagnosis and patient-specific therapeutic approaches. TEPs introduce a groundbreaking technique in oncology since they can transform non-invasive medical diagnostics and therapeutic monitoring for cancer.

Gastric cancer (GC) is a leading cause of cancer-related deaths worldwide, with late-stage diagnoses frequently leading to poor outcomes. This underscores the need for effective early-stage gastric cancer (ESGC) diagnostics.

We introduce ESGCmiRD, an innovative artificial intelligence-driven strategy that identifies a miRNA signature for ESGC detection by integrating robust expression patterns, ESGC relevance, and regulatory capabilities of microRNA (miRNA) based on multiple networks. Expression and biological roles of miRNAs in GC were validated and explored via bioinformatics analysis and in vitro studies. miRNA-target interaction was confirmed by dual-luciferase reporter assay. Molecular docking predicted miRNA-drug binding affinities, assessing the miRNA signature's therapeutic potential.

ESGCmiRD identified a blood miRNA signature (miR-320b, miR-222-3p, miR-181a-5p, miR-103a-3p, miR-107) for ESGC detection, demonstrated high diagnostic accuracy with AUC values of 0.986, 0.977, 0.815, and 0.811 in the test and three validation sets (GSE211692, TCGA-STAD, and our cohort), respectively. The five miRNAs were overexpressed in ESGC plasma and directly target PTEN, promoting GC cell proliferation, migration, and invasion. Molecular docking suggested Paclitaxel had the strongest potential interaction with these miRNAs.

This method identifies a robust miRNA signature for ESGC detection and sheds light on gastric carcinogenesis mechanisms, opening doors for potential therapeutic strategies.

Breast cancer (BC) remains a leading cause of morbidity and mortality among women worldwide, necessitating the development of innovative diagnostic and monitoring strategies. Liquid biopsy (LB), a minimally invasive approach that analyzes circulating tumor cells (CTCs), cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), extracellular vesicles (EVs), and other tumor-derived biomarkers in body fluids, has emerged as a transformative tool in BC management. This review comprehensively explores the role of LB in early detection, disease monitoring, treatment stratification, and resistance surveillance in BC. We discuss the latest advancements in LB technologies, including next-generation sequencing (NGS), digital PCR, and single-cell analysis, highlighting their sensitivity and specificity. Additionally, we examine the clinical utility of LB in guiding personalized therapy, particularly in the context of hormone receptor-positive, HER2positive, and triple-negative BC subtypes. Despite its promise, several challenges, including standardization, validation, and integration into clinical practice, remain to be addressed. By summarizing current evidence and future directions, this review underscores the potential of LB to revolutionize BC diagnosis and treatment, paving the way for a more precise and dynamic approach to disease management.

Liquid biopsies using circulating tumour DNA (ctDNA) have emerged as an alternative to conventional biopsies. They can be used to aid in diagnosing and selecting an agent for treatment and can possibly be used to monitor disease response to treatment. In this report, we present a patient who initially presented with lower abdominal pain. Imaging showed extensive retroperitoneal lymphadenopathy and lymph node biopsy demonstrated poorly differentiated carcinoma. Further workup did not reveal a primary lesion, but his genetic analysis revealed a BRAF V600E mutation and CD274 amplification which was used to guide treatment of the adenocarcinoma as a melanoma of unknown primary. He was initiated on ipilimumab and nivolumab and his ctDNA levels showed rapid improvement. After treatment was stopped due to adverse events, he was monitored via ctDNA, with an increase prompting repeat imaging that demonstrated enlargement of his lesions prompting a resumption of treatment.

Tumor-associated cells derived from a liquid biopsy are promising biomarkers for cancer detection, diagnosis, prognosis, and monitoring. However, their rarity, heterogeneity and plasticity make precise identification and biological characterization challenging for clinical utility. Enrichment-free approaches using whole slide imaging of all circulating cells offer a comprehensive and unbiased strategy for capturing the full spectrum of tumor-associated cell phenotypes. However, current analysis methods often depend on engineered features and manual expert review, making them sensitive to technical variations and subjective biases. These limitations highlight the need for a better feature representation to improve performance and reproducibility of applications in large-scale patient cohort analyses. In this study, we present a deep contrastive learning framework for learning features of all circulating cells, enabling robust identification and stratification of single cells in whole slide immunofluorescence microscopy images. We demonstrate performance of learned features in classification of diverse cell phenotypes in the liquid biopsy, achieving an accuracy of 92.64%. We further demonstrate that learned features improve performance in downstream applications such as outlier detection and clustering. Lastly, our feature representation enables automated identification and enumeration of distinct rare cell phenotypes, achieving average F1-score of 0.93 across cell lines mimicking circulating tumor cells and endothelial cells in contrived samples and average F1-score of 0.858 across CTC phenotypes in clinical samples. This workflow has significant implications for scalable analysis of tumor-associated cellular biomarkers in clinical prognosis and personalized treatment strategies.

Gynaecological cancers, including endometrial, ovarian, and cervical cancers as well as breast cancer, despite numerous studies, still constitute a challenge for modern oncology. For this reason, research aimed at the application of modern diagnostic methods that are useful in early detection, prognosis, and treatment monitoring deserves special attention, Great hopes are currently being placed on the use of liquid biopsy (LB), which examines various tumour components, including cell-free RNA (cfRNA), circulating tumour cells (CTCs), circulating tumour DNA (ctDNA), exosomes, and tumour-educated platelets (TEPs). LB has shown promise as a minimally invasive means of early diagnosis of cancers, detection of recurrence, prediction of therapy response, treatment monitoring, and drug selection. The integration of this test into clinical practice in modern oncology is challenging, but offers many benefits, including reducing the risks associated with invasive procedures, improving diagnostic and therapeutic efficacy, and improving the quality of life of oncology patients. The aim of this review is to present recent reports on the use of ctDNA in diagnosing, predicting the outcome of, and monitoring the treatment of gynaecological and breast cancers.

Circulating tumor DNA (ctDNA) analysis is transforming oncology, but challenges such as insufficient analytical sensitivity, difficult variant interpretation, suboptimal turnaround time, limited deployment flexibility, and high costs hinder its broader adoption and raise concerns about reimbursement sustainability across European health care systems.

To address these challenges, we created the OncNGS consortium, comprising academic, public, and private hospitals (buyers' group) and several supporting entities, to run a European precommercial procurement (PCP) initiative. The consortium defined ctDNA diagnostic testing requirements, conducted an open market consultation, and launched a call for tender. Suppliers were invited to develop an end-to-end, Conformité Européenne In Vitro Diagnostic (CE-IVD)-compliant solution integrating wet laboratory, dry laboratory, and reporting workflow in a single procedure, offering short turnaround time and reasonable cost.

The OncNGS consortium defined criteria for a versatile, modular, cost-effective solution, deployable centrally or on-site, and adaptable to advancements in precision oncology. Launched in July 2022, the tender attracted seven companies, with four selected for phase I-OncNGS solution(s) design. From these, three advanced to phase II-prototyping. Ultimately, two contractors were awarded contracts for phase III to assess the clinical performance of their prototypes.

By leveraging the PCP approach, OncNGS aims to deliver innovative, affordable solutions to standardize ctDNA testing and reporting across European Union countries, improving diagnostic and therapeutic strategies for oncology patients.

Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer-related deaths. The current standard of care for patients with early-stage CRC includes surgical resection and, in selected patients, adjuvant chemotherapy. Circulating tumor DNA (ctDNA) testing is an important component of liquid biopsy, and with the development of testing technology, its value for clinical application has attracted widespread attention. The aim of this study was to help researchers review what has been achieved and better understand the direction of future research through bibliometric analysis.

We used the Web of Science Core Collection database to search for ctDNA in CRC-related articles published between 2014 - 2023. Bibliometric analyses of major keywords, authors, countries, institutions, literature and journals in the field were performed using CiteSpace and VOSviewer.

The number of publications in the field has continued to increase over the last decade. The United States has the highest number of publications, and Italian research scholars have made outstanding contributions. Cancers is the journal with the highest number of publications.

This study systematically summarizes the research findings in the field of ctDNA in CRC from 2014 to 2023 and describes the research hotspots and trends worldwide that can guide future research.

Small-cell lung cancer (SCLC) remains one of the deadliest cancers worldwide. Patients' survival remains poor due to its rapid growth, high metastatic rate and limited possibilities of treatment. For many years, SCLC management has been based mostly on chemo and radiotherapy. However, new therapeutic approaches have been proposed in the past few years, including immunotherapy, which is currently implemented in clinical practice. Unfortunately, in many cases, response to therapy, especially chemotherapy, remains poor, or the patient becomes resistant to initially effective treatment. One of the crucial problems during SCLC patient care is a lack of appropriate predictive biomarkers for various therapeutic approaches. Another critical issue is the scarcity of collected tissue during biopsy, which may be insufficient or of too poor quality for analysis. A liquid biopsy might be the key to solving both of those problems as it is collected in a non-invasive way and enables the measurement of various biomarkers, including circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs). In this review, we discuss various approaches to potentially incorporating liquid biopsy into clinical application - as a companion to imaging during SCLC diagnostics, a new approach to molecular subtyping, and a material enabling predictive or prognostic biomarkers assessment. We also summarize ongoing clinical trials encompassing SCLC patients in which liquid biopsy is collected and examined.

Papillary thyroid carcinoma (PTC) is the most prevalent form of thyroid cancer with generally favorable outcomes. However, surgeons often face challenges regarding optimal surgical timing, extent of surgery, and identifying patients at risk for metastasis or progression to more aggressive subtypes. The ongoing debate over immediate surgery versus active surveillance emphasizes the need for reliable, minimally invasive diagnostic tools to inform surgical decision-making. This study aims to develop an epigenetic biomarker-based prediction system using fine-needle aspiration biopsy (FNAB) samples to assess PTC aggressiveness preoperatively. We conducted a comprehensive analysis of methylome data to identify approximately 7200 CpG islands with altered methylation levels in thyroid cancer tissues. These candidate regions were further examined in our cohort of 55 PTC patients to develop methylation-specific primers suitable for FNAB samples. Methylation patterns allowed us to stratify patients into two distinct prognostic groups, one of which exhibited a poorer survival rate. Our methylation-specific primers effectively classified FNAB samples into these groups, demonstrating their potential as a preoperative tool for assessing tumor aggressiveness. This stratification aids in informing surgical planning and personalizing treatment strategies. DNA methylation profiling of PTC identifies key epigenetic biomarkers associated with tumor aggressiveness. Utilizing these biomarkers in FNAB samples provides a minimally invasive method for preoperative risk assessment, assisting surgeons in tailoring surgical interventions and potentially improving patient outcomes.

As a non-invasive liquid biopsy technology, the detection of circulating tumor cells (CTCs) overcomes the limitations of traditional tissue biopsy methods, enabling continuous sample collection and long-term dynamic monitoring. However, current CTCs analysis methods typically rely on cell size to separate and identify tumor cells, which fails to effectively distinguish tumor cells from different sources. In addition, existing methods are often constrained by limited antibody species, typically detecting only 2-3 molecular phenotypes. This narrow detection scope does not fully capture the heterogeneity of CTCs at the single-cell level, thus limiting its utility in precision diagnosis and personalized treatment. To address these challenges, it is urgent to develop CTCs detection methods that can simultaneously integrate comprehensive target and cell morphology information.

Using breast cancer as a research model, we developed a computer-aided design-based hybridization chain reaction (CAD-HCR) by combining DNA encoding and antibody coupling technologies with orthogonal DNA self-assembly to achieve multiple detection and heterogeneity analysis of breast cancer mimic samples. This technology overcomes the limitation of antibody species in traditional CTCs detection and utilizes antibody-trigger strand coupling to convert target protein signals into DNA signals, thereby circumventing throughput limitation of existing detection methods. By utilizing the signal amplification effect of DNA self-assembly, this technology enhances sensitivity significantly, allowing for accurate single-cell level detection of CTCs.

This technology provides spatial positioning and cell morphological characteristics information for CTCs analysis of breast cancer, which is expected to provide a more accurate basis for diagnosis and treatment decision-making for in-depth understanding of tumor heterogeneity and clinical applications.

Liquid biopsy offers a noninvasive method to identify and monitor tumor-derived biomarkers, including circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), exosomes, microRNAs, and tumor-educated platelets, that provide real-time insights into the biological behavior of gynecological cancers. The detection of these markers has the potential to revolutionize cancer management by enabling earlier detection, providing novel data to personalize treatments, and predicting disease recurrence before clinical imaging and predicting disease recurrence before clinical imaging can confirm progression, thereby also guiding complex clinical decision-making. However, because this new "omics" layer introduces additional complexity, it must be fully understood, from its biological rationale to technical development and clinical integration, to prevent confusion or misapplication. That is why, focusing on 14 critical fields of inquiry, our goal is to map the current state of liquid biopsy from bench to bedside while highlighting practical considerations for clinical integration. Each topic integrates recent advances in assay sensitivity, biomarker variability, and data interpretation, underscoring how standardized protocols and robust analytical methods are pivotal for reliable results. We then translate these findings into disease-specific insights, examining how liquid biopsy could refine early detection, minimal residual disease assessment, and therapy guidance in endometrial, cervical, and ovarian cancers. Although several FDA-approved assays and promising commercial tests illustrate the field's rapid evolution, many translational hurdles remain, including the need for harmonized protocols, larger prospective clinical trials, and cost-effectiveness analyses. Crucially, our synthesis clarifies the pivotal role of interdisciplinary collaboration. Oncologists, laboratory scientists, and industry partners must align on standardized procedures and clinically relevant endpoints. Without such coordination, promising biomarkers may remain confined to research settings, limiting their practical benefit. Taken together, our review offers a translational view designed to contextualize liquid biopsy in gynecological oncology.

Nucleic acid probes, which are short sequences of nucleic acids designed to complement specific DNA or RNA targets, have broad applications in biosensing, genetic studies, and various other fields. In tumor diagnosis and treatment, nucleic acid probes offer a precise and accessible approach that is essential for improving patient care and quality of life. Despite substantial research on nucleic acid probes over the past three decades, few comprehensive reviews have retrospectively examined the field.

This study extracted 30 years of nucleic acid probe-related research articles from the Web of Science Core Collection database. We used CiteSpace, VOSviewer, and R tools to systematically analyze the field's current status and developmental trends, with an emphasis on applications in oncology.

Our findings indicate a continuous growth trend in nucleic acid probe research, with the United States and China, along with their leading institutions and authors, making the most significant contributions. In oncology specifically, nucleic acid probe research has focused primarily on signal amplification, liquid biopsy, and drug delivery. The emergence of novel biomarkers and assay techniques has been a pivotal factor driving advancements in this field.

Nucleic acid probes show strong potential for applications in tumor precise diagnosis and treatment. Continued innovation and closer interdisciplinary collaboration will be vital for further advancements, while large-scale clinical studies are needed to validate their clinical utility.

Circulating melanoma cells (CMCs) are responsible for the hematogenous spread of melanoma and, ultimately, metastasis. However, their study has been limited by the low abundance in patient blood and the heterogeneous expression of surface markers. The FDA-approved CellSearch platform enriches CD146-positive CMCs, whose number correlates with progression-free survival and overall survival. However, a single marker may not be sufficient to identify them all. The Parsortix system allows enrichment of CMCs based on their size and deformability, keeping them viable and suitable for downstream molecular analyses. In this study, we tested the strengths, weaknesses and potential convergences of both platforms to integrate the counting of CMCs with a protocol for their genetic analysis. Samples run on Parsortix were labeled with a customized melanoma antibody cocktail, which efficiently labeled and distinguished CMCs from endothelial cells/leukocytes. The capture rate of CellSearch and Parsortix was comparable for cell lines, but Parsortix had a higher capture rate in real-life samples. Moreover, double enrichment with both CellSearch and Parsortix succeeded in removing most of the leukocyte contamination, resulting in an almost pure CMC sample suitable for genetic analysis. In this regard, a proof-of-concept analysis of CMCs from a paradigmatic case of a metastatic uveal melanoma patient led to the identification of multiple genetic alterations. In particular, the GNAQ p.Q209L was identified as homozygous, while a deletion in BAP1 exon 9 was found hemizygous. Moreover, an isochromosome 8 and a homozygous deletion of the CDKN2A gene were detected. In conclusion, we have optimized an approach to successfully enrich and retrieve viable CMCs from metastatic melanoma patients. Moreover, this study provides proof-of-principle for the feasibility of a marker-agnostic CMC enrichment followed by CMC phenotypic identification and genetic analysis.Kindly check and confirm the processed contributed equally is correctly identify We confirm.

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults, characterized by rapid growth and resistance to chemotherapy. Conventional treatments remain largely ineffective, with patient survival averaging around 18 months after diagnosis. Current diagnostic methods rely on invasive tissue biopsies and imaging tests. While traditional biopsies involve extracting tissue samples, their routine use is often limited by surgical risks and the challenge of accessing sensitive brain regions. Liquid biopsy has emerged as a promising noninvasive alternative, analyzing circulating tumor components-such as DNA, RNA, extracellular vesicles, and microRNAs-found in body fluids. This approach enables initial diagnosis and continuous disease monitoring, offering a significant advantage over traditional biopsies, which are impractical for frequent repetition during treatment follow-up. This review highlights recent advances in liquid biopsy-derived biomarkers for the clinical management of GBM. The discussion includes the advantages, limitations, and potential of these biomarkers as tools for early diagnosis and disease monitoring. A narrative review of the literature published over the last decade (2014-2024) was conducted using major health-focused scientific databases. The analysis focuses on evaluating the clinical relevance and applicability of liquid biopsy in GBM, offering insights into its potential as a minimally invasive and effective tool for improving glioblastoma management.

Exosomes are nanoscale vesicles wrapped in lipid bilayers that are secreted by cells and carry a variety of proteins, lipids, nucleic acids, and metabolites. Exosomes are widely present in various bodily fluids and mediate intercellular communication. They participate in a variety of physiological and pathological processes, including immune regulation, angiogenesis, tumorigenesis, and metastasis, and have significant clinical diagnosis and treatment potential. Exosomes are source-rich, structurally stable, and reflect the states of their parental cells. Therefore, they are expected to serve as novel diagnostic markers for various diseases. In addition, stem-cell-derived exosomes show therapeutic potential and have the advantages of low immunogenicity, high safety and easy storage, and exhibit therapeutic potential for neurodegenerative disorder, cardiovascular disease, and cancer. Furthermore, exosomes are highly biocompatible, have natural homing properties, and are capable of easily penetrating biological barriers, making them excellent drug-delivery carriers. Isolation and enrichment of exosomes is a prerequisite for downstream analysis and application. High-purity, high-yield, and high-throughput exosome-isolation methods are expected to be used in clinical diagnosis and treatment applications. Based on the physicochemical properties of exosomes, including density, size, charge, and surface composition, exosome-isolation methods are mainly divided into density-based (e.g., differential ultracentrifugation, density-gradient ultracentrifugation), size-based (e.g., ultrafiltration, size-exclusion chromatography, field-flow fractionation), polymer-precipitation (e.g., polyethylene-glycol-based precipitation), and chemical affinity (e.g., antibody-based, aptamer-based, and surface-lipid-based lipid probes) methods. Currently, basic research into exosomes and their clinical applications face a number of challenges. Firstly, the complexity and heterogeneity of exosomes and the lack of standardized isolation methods has led to highly variable research results that hinder comparing and reproducing results between different laboratories and clinical settings. Current isolation methods are generally hindered by insufficient purity, low yield, low throughput, and difficulties separating specific subpopulations, which seriously restrict the development of the exosome field. Secondly, exosome-isolation methods that are easy to use in the clinic, have few technical requirements, and are highly efficient and inexpensive are lacking. Commonly used classical methods, such as ultracentrifugation, are time-consuming, labor-intensive, require large sample volumes, and are inappropriate for clinical settings. Methods such as immunoaffinity can be used to isolate exosomes from precious trace samples in clinical practice; however, high costs, low recoveries, and high operating requirements are shortcomings that restrict sample analysis in the clinic. In addition, robust large-scale methods for preparing exosomes are lacking. There is an urgent need to develop repeatable and scalable methods for preparing batches of high-quality exosomes owing to the rapid development of exosomes for the treatment of clinical diseases. Generally, exosome research progress is expected to greatly improve our understanding of the biological functions and components of exosomes, which will help transform the exosome research into effective diagnostic and therapeutic strategies and lead to new precision-medicine and personalized-treatment applications. This article summarizes the latest progress in exosome-isolation and -enrichment technologies and introduces the application of exosomes as disease diagnostic markers, therapeutic agents, and drug delivery carriers. Finally, the future developmental trends in exosome isolation and enrichment technologies for disease diagnosis and treatment are discussed.

The magnitude of combined analytical errors of urinary extracellular vesicle (uEV) preparation and measurement techniques (CVA) has not been thoroughly investigated to determine whether it exceeds biological variations. We utilized technical replicates of human urine to assess the repeatability of uEV concentration and size measurements by nanoparticle tracking analysis (NTA) following differential velocity centrifugation (DC), silicon carbide, or polyethylene glycol uEV isolation methods. The DC method attained the highest precision. Consequently, DC-derived uEV size, most abundant protein levels, and optical redox ratio (ORR) were further assessed by dynamic light scattering (DLS), immunoblotting or multi-photon (SLAM) microscopy. Procedural errors primarily affected uEV counting and uEV-associated protein quantification, while instrumental errors contributed most to the total variability of NTA- and DLS-mediated uEV sizing processes. The intra-individual variability (CVI) of uEV counts assessed by NTA was smaller than inter-individual variability (CVG), resulting in an estimated index of individuality IOI < 0.6, suggesting that personalized reference interval (RI) is more suitable for interpretation of changes in subject's test results. Population-based RI was more appropriate for ORR (IOI > 1.4). The analytical performance of DC-NTA and DC-SLAM techniques met optimal CVA < 0.5 × CVI criteria, indicating their suitability for further testing in clinical laboratory settings.

Colorectal cancer (CRC) is a significant global health issue, with early detection being critical to improving patient survival. Dysregulation of the glycolysis pathway plays a pivotal role in CRC progression, but specific gene-level mechanisms remain underexplored.

This study aimed to investigate the role of glycolysis-related genes in CRC development and identify potential diagnostic and prognostic biomarkers.

We utilized The Cancer Genome Atlas (TCGA) dataset to perform differential expression analysis of glycolysis-related genes in CRC. Protein-protein interaction (PPI) network analysis was conducted to identify central hub genes. The diagnostic potential of selected genes was evaluated using ROC curve analysis, while their expression levels were validated through RT-qPCR.

IER3 and AGRN were identified as significantly upregulated genes associated with reduced survival rates in CRC patients. PPI analysis revealed their roles as central hub genes within the glycolysis pathway. ROC curve analysis demonstrated their ability to distinguish CRC patients from healthy individuals. Validation through RT-qPCR confirmed their significant overexpression in CRC samples, highlighting their involvement in disease progression.

IER3 and AGRN are critical components of the glycolysis pathway, driving CRC development and progression while also showing potential as biomarkers for predicting outcomes, diagnosing CRC, and serving as treatment targets.

This review explores the current understanding and recent advancements in neuroblastoma, one of the most common extracranial solid pediatric cancers, accounting for ~ 15% of childhood cancer-related mortality. The hallmarks of NBL, including angiogenesis, metastasis, apoptosis resistance, cell cycle dysregulation, drug resistance, and responses to hypoxia and ROS, underscore its complex biology. The tumor microenvironment's significance in disease progression is acknowledged in this study, along with the pivotal role of cancer stem cells in sustaining tumor growth and heterogeneity. A number of molecular signatures are being studied in order to better understand the disease, with many of them serving as targets for the development of new therapeutics. This includes inhibitor therapies for NBL patients, which notably concentrate on ALK signaling, MDM2, PI3K/Akt/mTOR, Wnt, and RAS-MAPK pathways, along with regulators of epigenetic mechanisms. Additionally, this study offers an extensive understanding of the molecular therapies used, such as monoclonal antibodies and CAR-T therapy, focused on both preclinical and clinical studies. Radiation therapy's evolving role and the promise of stem cell transplantation-mediated interventions underscore the dynamic landscape of NBL treatment. This study has also emphasized the recent progress in the field of diagnosis, encompassing the adoption of artificial intelligence and liquid biopsy as a non-intrusive approach for early detection and ongoing monitoring of NBL. Furthermore, the integration of innovative treatment approaches such as CRISPR-Cas9, and cancer stem cell therapy has also been emphasized in this review.

Urine-based therapy, an ancient practice, has been utilized across numerous civilizations to address a wide range of ailments. Urine was considered a priceless resource in numerous traditional therapeutic applications due to its reported medicinal capabilities. While the utilization of urine treatment is contentious and lacks significant support from modern healthcare, the discovery of urine-derived stem cells (UDSCs) has introduced a promising avenue for cell-based therapy. UDSCs offer a noninvasive and easily repeatable collection method, making them a practical and viable option for therapeutic applications. Research has shown that UDSCs contribute to organ preservation by promoting revascularization and decreasing inflammatory reactions in many diseases and conditions. This review will outline the contemporary status of UDSCs research and explore their potential applications in both fundamental science and medical practice.

Metastatic colorectal cancer (mCRC) remains a major clinical challenge, despite therapeutic advancements. Mutations in KRAS and NRAS (RAS) oncogenes drive resistance to anti-EGFR drugs, necessitating RAS mutational analysis prior to treatment. While tissue biopsy remains the gold standard for molecular profiling, it has limitations such as invasiveness, intra-tumoral heterogeneity, and delayed results. Liquid biopsy (LB), on the other hand, offers a non-invasive alternative by analyzing circulating tumor DNA (ctDNA) and it provides a dynamic view of molecular changes over time. Indeed, ctDNA analysis has expanded the understanding of the mCRC's molecular landscape, revealing that RAS mutated (MT) subclones undergo both a positive and a negative selection during treatment. This negative selection has been described as the "NeoRAS WT phenomenon." The temporary disappearance of RAS mutations opens "RAS WT windows," thus making potential candidates for anti-EGFR therapies even patients initially diagnosed as RAS MT. This review examines numerous studies investigating the clinical significance of the "NeoRas WT phenomenon" as a distinct pathological entity. It also highlights the key limitations arising from the variability in study designs, detecting methods and ctDNA shedding rates. The results of ongoing prospective trials are necessary to determine whether NeoRAS WT stands as a reliable marker for guiding anti-EGFR treatment strategies in RAS-mutated patients.

The concept of liquid biopsy is based on the knowledge that blood or secretions from the body contain tumor cells, nucleic acids, cellular components, and tumor metabolites. Detection of circulating DNA (ctDNA) in liquid biopsy material shows the most promise due to the advances in DNA technologies that have made detection and sample screening possible. Clinical trials have begun to evaluate ctDNA monitoring for response to cancer treatment in clinical settings for non-small cell lung cancer (NSCLC), breast cancer, and colorectal cancer (CRC). However, most liquid biopsy tests introduced into the clinic have only been able to identify one or two features of tumor DNA, which limits the specificity of the test. In early 2025, a study led by a research team at Oxford University identified a new blood test, TriOx, developed using machine learning to detect multiple types of cancer at an early stage. These new blood tests may revolutionize oncology and make early cancer detection as routine as other diagnostic blood tests, such as blood glucose testing. This article aims to review ctDNA and liquid biopsy in the diagnosis, early detection, and monitoring of treatment response in cancer.

Simultaneous analysis of multiple biomarkers can typically improve the sensitivity and specificity of a disease diagnosis. Low-abundance serum proteins have recently emerged as a novel class of biomarkers for diseases. Due to the low concentration, the low-abundance protein analysis relies on single-molecule immunoassay, which has a very limited dynamic range. As a result, simultaneous analysis of low- and high-abundance protein markers requires multiple instruments, which demands larger sample volumes and is cost-/labor-consuming. To overcome these limitations, we developed a single-molecule imaging technique that can detect low- and high-abundance protein markers simultaneously in one chip. By employing a hybrid biomarker capture strategy that involves both glass surface and bead immobilization, our method greatly extended the detection range of the single-molecule assay. We used the method for pancreatic cancer diagnosis and analyzed three serum biomarkers of different abundances, including LIF, CA19-9, and CA125. Combined analysis of the three biomarkers yielded exceptional sensitivity and specificity (AUC = 0.996), which is better than using any of the markers alone, including CA19-9 that is used in clinical practice (AUC = 0.804). Overall, we demonstrated a simple and cost-effective method that greatly extended the dynamic range of single-molecule imaging while maintaining the sensitivity, which has great potential in various clinical applications.

Uveal melanoma is an aggressive ocular malignancy. Early molecular characterisation of primary tumours is crucial to identify those at risk of metastatic dissemination. Although tumour biopsies are being taken, liquid biopsies of ocular fluids may form a less invasive but relatively unexplored alternative. In this study, we aim to evaluate the DNA content of vitreous fluid from eyes with a uveal melanoma to obtain molecular tumour information.

DNA was isolated from 65 vitreous fluid samples from enucleated eyes with a uveal melanoma and studied using digital PCR. Primary and additional driver mutations (in GNAQ, GNA11, PLCB4, CYSLTR2, BAP1, SF3B1 and EIF1AX) were investigated using accustomed targeted and drop-off assays. The copy numbers of chromosome 3p and 8q were measured using multiplex and single-nucleotide polymorphism-based assays. Our findings were compared to the molecular profile of matched primary tumours and to the clinicopathological tumour characteristics.

Almost all (63/65) vitreous fluids had measurable levels of DNA, but melanoma-cell derived DNA (containing the primary driver mutation) was detected in 45/65 samples (median proportion 15.5%, range 0.03-94.4%) and was associated with a larger tumour prominence, but not with any of the molecular tumour subtypes. Among the vitreous fluids with melanoma-cell derived DNA, not all samples harboured (analysable) other mutations or had sufficient statistical power to measure copy numbers. Still, additional mutations in BAP1, SF3B1 and EIF1AX were detected in 15/17 samples and chromosome 3p and 8q copy numbers matched the primary tumour in 19/21 and 18/20 samples, respectively. Collectively, a clinically-relevant molecular classification of the primary tumour could be inferred from 29/65 vitreous fluids.

This proof-of-concept study shows that substantial amounts of DNA could be detected in vitreous fluids from uveal melanoma patients, including melanoma-cell derived DNA in 69% of the samples. Prognostically-relevant genetic alterations of the primary tumour could be identified in 45% of the patients. A follow-up study is needed to evaluate our approach in a prospective clinical context. Additionally, our work highlights improved possibilities to sensitively analyse scarce and heterogeneous tumour biopsies, with potential application in other malignancies.

The diagnosis of cholangiocarcinoma (CCA) remains challenging. Although new technologies have been developed and validated, their routine use in clinical practice is needed. Conventional cytology obtained during endoscopic retrograde cholangiopancreatography-guided brushings is the first-line technique for the diagnosis of CCA, but it has shown limited sensitivity when combined with endoscopic ultrasound-guided biopsy. Other diagnostic tools have been proposed for the diagnosis of CCA, with their respective advantages and limitations. Cholangioscopy with biopsy or cytology combined with FISH analysis, intraductal biliary ultrasound and confocal laser microscopy have made significant advances in the last decade. More recently, developments in the analytical "omics" sciences have allowed the mapping of the microbiota of patients with CCA, and liquid biopsy with proteomic and extracellular vesicle analysis has allowed the identification of new biomarkers that can be incorporated into the predictive diagnostics. Furthermore, in the preoperative setting, radiomics, radiogenomics and the integrated use of artificial intelligence may provide new useful foundations for integrated diagnosis and personalized therapy for hepatobiliary diseases. This review aims to evaluate the current diagnostic approaches and innovative translational research that can be integrated for the diagnosis of CCA.