Search Results (371)

Background: Hepatocellular carcinoma (HCC) is one of the most common and deadliest cancers worldwide. Alpha-fetoprotein (AFP), a widely used and accessible tumoral marker, has limited performance in the early detection of HCC among high-risk populations. This study aims to evaluate the potential added value of ccfDNA (circulating cell-free DNA) fragment size, alone or in a multiparameter panel, using accessible, feasible ccfDNA analysis. Methods: A prospective cohort of 125 patients with chronic liver disease was analyzed. Patients with incomplete clinical or laboratory data and patients without cirrhosis were excluded from the final analysis. Nonparametric tests, logistic regression and ROC curve analysis were performed. ccfDNA fragment size was measured using on-chip electrophoresis. Results: ccfDNA fragment size was significantly lower in the cirrhosis-HCC subgroup compared to the cirrhosis-only subgroup (p < 0.001). While AFP remains an independent predictor of HCC among cirrhosis patients, ccfDNA fragment size did not prove to be an independent predictor in this cohort. AUROC (area under the receiver operating characteristic curve) analysis revealed that a combined model of AFP, age, liver reserve, and ccfDNA fragment size did not perform better than the corresponding panel without ccfDNA. Moreover, after DeLong comparison, the difference between the two AUROCs proved statistically insignificant. Age and platelet count remain the strongest independent predictors in our exploratory cohort. Conclusions: Although ccfDNA fragment size proved to be lower in the HCC subgroup, its statistical significance fades when included into a multimarker panel. However, all panels should undergo further validation in a larger cohort, in order to better assess the individual contribution of each parameter and to discriminate between added diagnostic value and confounding effect of age and liver reserve parameters. Full article

Background/Objectives: Merkel cell carcinoma (MCC) is a rare but aggressive skin cancer with a 40% recurrence rate. However, reliable biomarkers for early recurrence detection or treatment guidance are lacking, especially for virus-negative tumors. Circulating tumor DNA (ctDNA), a fragment of tumor-derived cell-free DNA in blood, has emerged across multiple cancers as a minimally invasive precision biomarker to detect minimal residual disease (MRD); predict recurrence; and monitor treatment response. This review’s objective was to summarize recent advances in ctDNA as a tool for therapeutic decision-making in MCC, contextualized by findings in other malignancies. Methods: A comprehensive literature review was performed, focusing on studies published between 2016 and 2026 that evaluate ctDNA in MCC and other cancers. Key prospective trials, observational studies, and case reports were identified through PubMed and relevant conference proceedings. Data on ctDNA assay methods (tumor-informed vs. tumor-agnostic), clinical sensitivity, lead time for recurrence detection, and predictive value for therapy response were extracted and synthesized. Results: Across cancers such as colorectal, lung, and melanoma, ctDNA positivity after curative treatment predicts relapse months in advance of imaging and can guide adjuvant therapy decisions. In MCC, recent studies demonstrate that ctDNA levels correlate with MCC tumor burden and exhibit high sensitivity and specificity for clinically evident disease. Stage I-III MCC patients who were ctDNA-positive within four months of treatment had a 7.4-fold higher recurrence risk within the subsequent 12–18 months of follow-up. Serial ctDNA monitoring may enable earlier intervention in otherwise asymptomatic ctDNA-positive MCC cases, helping distinguish responders from non-responders. Conclusions: ctDNA is an emerging precision biomarker that offers significant prognostic and surveillance utility in MCC. It enables earlier detection of recurrence, potentially allowing treatment to begin before clinical disease manifests. It also helps stratify patients by risk and treatment response, informing personalized surveillance intensity and therapeutic choices. Integrating ctDNA monitoring into MCC management could improve outcomes by guiding timely interventions, although prospective trials are needed to confirm that ctDNA-guided decisions translate to improved patient survival. Formal cost-effectiveness analyses have not yet been conducted and represent an important area for future investigation. Full article

One of the major obstacles in early cancer detection in dogs is the limited sensitivity in detecting circulating tumor DNAs (ctDNAs) with low abundances. Standard next-generation sequencing (NGS) without error correction typically achieves detection limits around ~1% mutant allele frequency (MAF). We sought to improve the detection sensitivity using a sequential CRISPR-EspCas9 enrichment strategy in which iterative in vitro cleavage (IVC) was combined with PCR amplification to selectively deplete wild-type DNA and enrich rare tumor mutations. Applying the strategy to genomic DNA and cell-free DNA mimics from canine mammary gland tumor cell lines demonstrated that IVC enrichment enabled the detection of cancer-associated PIK3CA H1047R mutations that were undetectable by conventional Sanger sequencing. To evaluate detection sensitivity, we characterized enrichment using synthetic templates for PIK3CA H1047R and other cancer-related mutations, BRAF V596E, and KRAS G12C. We observed that three iterations of sequential IVC achieved ~160, ~15, and ~2.2-fold enrichment for PIK3CA H1047R, BRAF V596E, and KRAS G12C, respectively. Under the present synthetic-template conditions, the analytical LOD reached 0.001% MAF for PIK3CA and 0.01% MAF for BRAF, whereas KRAS showed only modest enrichment and remained practically limited under the current guide design. Together, the results show that the CRISPR-EspCas9 IVC strategy enables selective enrichment of low-frequency single-nucleotide mutant alleles. We anticipate that the finding could be utilized to develop a highly sensitive veterinary liquid biopsy application with further optimization and validation using canine plasma cfDNA. Full article

Background: Pancreatic ductal adenocarcinoma (PDAC) exhibits high post-resection relapse and early systemic dissemination rates. The level of circulating tumor cells (CTCs) correlates with early metastatic failure, motivating CTC interception strategies. Methods: In this hypothesis-driven review, we synthesized the contemporary evidence on PDAC staging and therapy, CTC detection (including portal versus peripheral sampling), and circulating tumor DNA (ctDNA)-based minimal residual disease (MRD), and evaluated the translational rationale for CTC-targeted adoptive immunotherapy focusing on CEACAM6 and CAR-T cells. Results: Prospective studies report higher portal versus peripheral CTC yields and stronger associations with relapse; tumor-informed ctDNA positivity in peri-operative and surveillance windows predicts shorter disease-free survival. CEACAM6 is overexpressed in PDAC and linked to invasion and metastasis, supporting antigen selection. However, target overexpression alone does not establish clinical suitability for adoptive cell transfer. Consequently, its therapeutic implementation must contend with assay heterogeneity, on-target/off-tumor risks, and the lack of interventional outcome data in PDAC, all of which remain key hurdles. Conclusions: CTC-targeting is biologically plausible and operationally measurable in PDAC. Consequently, a CEACAM6-directed CAR-T approach is proposed as a potential strategy for the interception of minimal residual disease (MRD). Randomized and biomarker-selected trials with composite MRD-clearance endpoints (CTC < LOQ and ctDNA-negative) may be justified to validate this interventional hypothesis. Full article

Relapse remains the leading cause of treatment failure following hematopoietic cell transplantation (HCT) for hematologic malignancies. Circulating tumor DNA (ctDNA) has emerged as a promising minimally invasive biomarker for measurable residual disease (MRD) assessment and early relapse detection; however, the prognostic significance of ctDNA in the post-transplant setting has not been comprehensively synthesized. We conducted a systematic review and meta-analysis in accordance with PRISMA guidelines and registered the protocol in PROSPERO (CRD420261392100). PubMed, Embase, Web of Science, EBSCO, Cochrane CENTRAL, and supplementary sources were searched through November 2025. Eligible studies evaluated tumor-specific ctDNA or tumor-informed/tumor-associated cfDNA in patients undergoing allogeneic or autologous HCT for hematologic malignancies. Random-effects meta-analyses were performed for relapse/progression, overall survival (OS), and relapse-free/progression-free survival (RFS/PFS). Studies evaluating total cfDNA quantity, methylation-based cfDNA profiling, cfRNA, or chimerism-only monitoring were synthesized narratively. Ten observational cohort studies comprising 883 patients met inclusion criteria. Across acute leukemias, lymphomas, multiple myeloma, and myelodysplastic syndromes, ctDNA/cfDNA positivity was consistently associated with adverse outcomes. The pooled hazard ratio (HR) for relapse or disease progression was 12.57 (95% CI: 4.59–34.46; p < 0.001), while pooled HRs were 7.45 (95% CI: 4.11–13.48; p < 0.001) for OS and 4.46 (95% CI: 2.22–8.97; p < 0.001) for RFS/PFS. Although statistical heterogeneity was low, interpretation was limited by the relatively small number of studies contributing to each pooled endpoint. Narrative evidence additionally suggested that broader circulating nucleic acid approaches may provide complementary information regarding graft-versus-host disease, infection, and other post-transplant complications. Tumor-specific ctDNA positivity is consistently associated with increased relapse risk and inferior survival outcomes following HCT. These findings support further investigation of ctDNA-based MRD monitoring as a promising non-invasive biomarker for post-transplant molecular surveillance and risk stratification. However, prospective multicenter validation studies, assay standardization, and ctDNA-guided interventional trials remain necessary before routine clinical implementation can be recommended. Full article

Thyroid cancer comprises biologically diverse entities ranging from largely indolent differentiated thyroid cancer (DTC) to aggressive poorly differentiated/anaplastic thyroid cancer and medullary thyroid cancer, generating a need for minimally invasive biomarkers that can be repeatedly sampled. This review summarizes recent advances in liquid biopsy for thyroid cancer, focusing on analytes and technologies spanning circulating tumor DNA (ctDNA)/cell-free DNA, circulating microRNAs (miRNAs), extracellular vesicles (EVs), and circulating tumor cells (CTCs). For ctDNA, we contrast qPCR/ddPCR and next-generation sequencing, tumor-informed versus tumor-agnostic strategies, the impact of low tumor fraction in DTC, clonal hematopoiesis confounding, and emerging methylation-based multi-cancer detection paradigms. For miRNAs, we highlight that bulk serum/plasma and EV-enriched compartments are not interchangeable and that regulated EV loading supports fraction-resolved biomarker development. We review recent translational EV-miRNA studies, including externally validated classifiers for metastatic disease and follicular-patterned/indeterminate nodules, and summarize the evolution of CTC research from enumeration to preoperative risk stratification and postoperative or radioiodine-related kinetics. We conclude with an indications-first framework that pairs analyte choice with clinical intent (preoperative diagnosis, initial risk stratification, response to treatment and minimal residual disease and identification of actionable alterations and resistance mechanisms) and prioritizes standardized workflows and prospective multicenter validation. Multi-analyte integration, epigenetic/fragmentomic cfDNA signals, and higher-resolution EV analytics are likely to accelerate clinical adoption, particularly in advanced thyroid cancer where circulating signal and therapeutic actionability are highest. Full article

Pancreatic ductal adenocarcinoma (PDAC) is the most complex and aggressive disease with the worst rates of unresectable or metastatic disease at diagnosis, resistance to systemic therapy, and case fatality rate (CFR) among leading cancers. In non-metastatic disease, neoadjuvant treatment with modern chemotherapeutic regimens followed by surgical resection and/or adjuvant mFOLFIRINOX has significantly improved oncological outcomes. However, recurrence rates remain alarmingly high, while immune checkpoint inhibitors (ICIs) or molecularly targeted therapy have not yet demonstrated clinical benefits. Comprehensive genomic profiling through NGS-based approved assays such as TruSight Oncology 500 (TSO500) could guide targeted therapy. Rapidly evolving mRNA cancer vaccines and circulating tumor DNA (ctDNA)-based prediction of minimal residual disease (MRD) and recurrence risk hold great promise towards the realization of rational combination therapy to improve recurrence-free survival (RFS) and overall survival (OS). More recently, single-cell multiomics (SC MO), spatial proteomics and transcriptomics (SPT), artificial intelligence (AI), and systems biology have revolutionized cancer research, enabling holistic tumor microenvironment (TME) analysis. In this comprehensive review, we describe the latest advances and unmet needs in the standard of care of PDAC. Moreover, we discuss the expectations of ongoing randomized clinical trials of adjuvant mRNA vaccine-based therapy and ctDNA MRD testing as prognostic biomarkers, towards personalized treatment to improve RFS and OS in a medium-term perspective. With a longer perspective, we explore how harnessing SC MO, SPT, AI, and systems biology can reveal the 3D spatial organization of interacting cancer, immune, and stromal cells. Multi-dimensional TME-, TSO500- and ctDNA-based framework of dynamic biomarkers are of paramount importance to achieve an optimal patient-specific perioperative multimodal treatment combining precision immunotherapy, targeted drugs, and modern chemotherapy, translated into future practice-changing clinical trials, that could eliminate MRD towards recurrence prevention. Full article

Pediatric brain and other central nervous system (CNS) tumors remain a leading cause of cancer-related death in children, while contemporary management increasingly depends on molecular classification, risk stratification, and longitudinal disease assessment. Yet tissue-based profiling has major limitations in pediatric neuro-oncology, particularly for deep-seated, eloquent, or surgically hazardous tumors and when repeat sampling is impractical. For primary CNS tumors, cerebrospinal fluid is generally more informative than plasma because it is anatomically closer to the tumor and more enriched for tumor-derived material. This narrative review summarizes current and emerging applications of cerebrospinal fluid in pediatric neuro-oncology, from conventional staging to molecular diagnosis, methylation-based classification, measurable residual disease detection, pharmacodynamic monitoring, and relapse surveillance. We discuss the biological rationale for cerebrospinal fluid analysis, major pre-analytical and technical determinants of assay performance, and the strengths and limitations of key analyte classes, including cytology, circulating tumor cells, cell-free DNA, RNA, extracellular vesicles, proteins, and metabolites. We also summarize how these approaches are being applied across major pediatric central nervous system tumor entities. Cerebrospinal fluid liquid biopsy is unlikely to replace tissue or imaging, but is increasingly positioned to complement both in precision pediatric neuro-oncology. Full article

Background/Objective: Blood-based assays for colorectal cancer (CRC) screening have emerged as promising non-invasive screening modalities, yet their real-world diagnostic performance across platforms remains unclear. Existing studies evaluating circulating tumor DNA (ctDNA) and cell-free DNA (cfDNA) based assays as non-invasive alternatives to stool-based tests vary substantially in methodology, population, and study design. In our systematic review and meta-analysis, we quantify the diagnostic yield of contemporary cfDNA/ctDNA for CRC detection through pooled sensitivity and specificity. Methods: A systematic search of diagnostic accuracy studies published through December 2025 was conducted by two researchers in PubMed and the Cochrane Library. Studies reporting sensitivity and specificity were included. Pooled proportions with the 95% confidence interval (CI) were calculated using a hierarchical bivariate random-effects model. Subgroup analyses were conducted stratified by cancer stage and advanced adenoma. (PROSPERO CRD420261290686). Results: Fifty-eight studies involving 63,309 subjects were included. The pooled sensitivity and specificity for colorectal cancer detection were 82.9% (95% CI: 79.9–85.5%) and 90.7% (95% CI: 89.2–92.0%), respectively. HSROC analysis demonstrated excellent overall diagnostic accuracy with an AUC of 0.937. Pooled sensitivity for advanced adenoma was suboptimal at 46% (CI: 32–59%). Detection sensitivity increased progressively by stage, from 70% (CI: 62–78%) in stage I CRC to 90% (CI: 87–92%) in stage IV CRC. Conclusions: These findings position next-generation cfDNA/ctDNA assays as promising, scalable, patient-friendly CRC screening tools with performance characteristics comparable to established non-invasive tests. Integration of cfDNA/ctDNA assays into population-level screening programs may substantially expand screening uptake by overcoming key barriers associated with stool-based modalities. Full article

In recent years, liquid biopsy has emerged as a promising non-invasive strategy for the identification of tumor-derived biomarkers. Among circulating analytes, cell-free DNA (cfDNA), including both nuclear and mitochondrial fractions, has been extensively investigated in a variety of biological fluids for its potential applications in cancer diagnosis, disease monitoring, and prognostic stratification. Owing to its higher copy number per cell compared with nuclear DNA, mitochondrial DNA (mtDNA) is typically present at higher concentrations in body fluids and is therefore potentially detectable. Circulating cell-free mitochondrial DNA (cfmtDNA) is closely associated with pro-inflammatory signaling pathways and cellular damage responses, including apoptosis, necrosis, and neutrophil extracellular trap formation (NETosis). This review provides a comprehensive overview of the reported alterations of cfmtDNA in the most prevalent gynecological malignancies, namely ovarian and endometrial cancers, which are characterized by a chronic inflammatory microenvironment. We further critically assess the current evidence supporting cfmtDNA as a potential non-invasive biomarker in these malignancies, highlighting current limitations and future research directions. Full article

Background/Objectives: Locoregional anal squamous cell carcinoma (ASCC) is usually cured with chemoradiotherapy; however, some patients relapse, require salvage abdominoperineal resection or develop metastatic disease. Approximately 90% of cases are driven by high-risk human papillomavirus, most commonly HPV16. Conventional surveillance using clinical examination and imaging may have limited sensitivity and specificity and is not individualized by recurrence risk. Circulating biomarkers (CBs), particularly circulating tumor DNA (ctDNA), have emerged as promising methods for real-time disease monitoring. We systematically reviewed available evidence evaluating CBs across treatment timepoints in localized ASCC. Methods: A PROSPERO-registered review (ID: 1133987) was conducted according to PRISMA guidelines. PubMed, EMBASE, Cochrane CENTRAL, Web of Science, and major conference proceedings (ASCO, ESMO, ASTRO) were searched on 30 November 2025. We included prospective or retrospective studies (≥10 patients) with stage I–III disease treated with curative-intent chemoradiotherapy that reported CBs, assay characteristics, and at least one clinical outcome. Studies with predominantly localized cohorts were included even if small proportions of metastatic patients were present, provided results relevant to curative-intent populations could be interpreted. Data were synthesized narratively by timepoint (baseline, mid-treatment, end of treatment, post-treatment/surveillance) and assay type given methodological heterogeneity. Results: Fifteen studies were included. CB assays comprised four categories: viral HPV ctDNA assays, tumor-informed ctDNA assays, non-specific total cell-free DNA (cfDNA) quantification, and circulating tumor cell (CTC)-based assays. Baseline detection rates varied by assay type. Viral HPV ctDNA assays demonstrated detection rates of 59–100%, while tumor-informed ctDNA assays showed rates of 79–89%. Across studies, higher CB detection rates and levels were generally associated with greater tumor burden, including more advanced T and N stage disease. Mid-treatment ctDNA clearance identified patients with excellent locoregional control and progression-free survival, whereas persistent ctDNA was associated with treatment failure. End-of-treatment and surveillance ctDNA positivity predicted recurrence within individual cohorts, with reported sensitivities of 80–90%, specificities of 95–99%, and molecular lead times preceding clinical or radiographic detection. In contrast, non-tumor-specific cfDNA dynamics showed more variable prognostic associations and were less consistently linked to tumor burden. Conclusions: Across heterogeneous assays, CB dynamics provide clinically meaningful prognostic information in localized ASCC, particularly when measured during treatment and early surveillance. Viral HPV and tumor-informed ctDNA may have the potential to guide follow-up intensity and inform future escalation or de-escalation strategies; however, prospective, standardized trials are needed to define actionable thresholds and test ctDNA-guided management. Full article

Small-cell lung cancer (SCLC) is an aggressive neuroendocrine carcinoma characterized by poor survival. Despite a high tumor mutation burden, biomarker discovery in SCLC remains challenging due to rapid tumor plasticity and limited tissue availability, highlighting the promise of liquid biopsy-based approaches. Epigenetic dysregulation of DNA 5-hydroxymethylcytosine (5hmC) has emerged as a cancer hallmark. However, its role in SCLC remains largely unexplored. Here, we characterized the cell-free DNA (cfDNA) 5hmC landscape in SCLC and evaluated its potential applications. We profiled the cell-free hydroxymethylomes of 107 pre-treatment SCLC patients and 53 matched controls using the 5hmC selective chemical labeling (5hmC-Seal) assay. SCLC displayed higher global 5hmC levels and distinct enrichment at neurodevelopmental and synaptic pathways, consistent with the neuroendocrine identity of SCLC. Concordance between plasma and matched circulating tumor cell patient-derived xenograft (CDX) demonstrated that cfDNA 5hmC reflects tumor epigenetic states and correlates with transcriptomic-derived molecular subtypes. Elevated SCLC-specific 5hmC levels and extensive stage (ES) disease were associated with inferior survival, with ES disease showing enrichment of pathways linked to cellular plasticity and neurodevelopment. Together, these findings indicate that cfDNA 5hmC profiling has potential as a biologically informative and clinically relevant biomarker in SCLC, with possible applications in tumor subtyping and risk stratification. Full article

Pancreatic cancer (PC) remains one of the most aggressive and lethal malignancies worldwide, largely due to late diagnosis, aggressive biology, limited therapeutic options and responsiveness. Conventional diagnostic and monitoring strategies, including imaging and serum biomarkers such as CA 19-9, provide limited sensitivity for early detection and suboptimal accuracy for the dynamic assessment of treatment response and disease evolution. These limitations highlight the urgent need for innovative, minimally invasive approaches capable of improving patient stratification and guiding personalized management. In this context, liquid biopsy has emerged as a promising, minimally invasive approach able to capture tumor-derived molecular information through the analysis of circulating cell-free nucleic acids, including circulating cell-free DNA (cfDNA) and circulating cell-free RNA (cfRNA). Released into the bloodstream by tumor cells, these analytes offer a real-time and comprehensive snapshot of tumor biology, capturing genetic, epigenetic, and transcriptional alterations through a simple blood draw. Liquid biopsy-based analyses hold significant potential for early detection, prognostic assessment, therapeutic decision-making, monitoring of minimal residual disease, and identification of resistance mechanisms. This review discusses the current state of research on circulating cell-free nucleic acids in PC, highlighting their biological basis, methodological approaches, clinical potential, and the challenges limiting their widespread implementation. By underscoring their translational relevance, we aim to outline how integrated liquid biopsy strategies, alongside the need for standardization and cross-study harmonization, may contribute to a more precise and dynamic approach to PC management. Full article

Background/Objectives: Liquid biopsy (LB) is transforming cancer care by enabling minimally invasive tumor profiling. While current research and clinical pathways mostly focus on blood LB, sputum represents a non-invasive, readily available respiratory specimen that may offer unique advantages for lung cancer (LC) care. Despite its potential, the maturity, breadth, and clinical applicability of sputum-based LB remain elusive. Methods: We conducted a scoping review to systematically map the existing literature on sputum LB in LC. Electronic databases were searched for studies evaluating sputum-derived biomarkers—cytologic, genomic, epigenetic, transcriptomic, proteomic, metabolomic, metagenomic, and extracellular vesicle–derived products—across the LC care continuum. Study designs, technologies, clinical contexts, and reported outcomes were extracted and synthesized qualitatively. Results: The literature demonstrated substantial heterogeneity in sputum collection, processing, and analytical platforms. Early work focused on cytometry and genetic alterations, while recent studies increasingly explore DNA methylomics, microRNAs, extracellular vesicle-derived products, and multi-omics approaches. The evidence suggests potential utility of sputum biomarkers for early detection and risk stratification, particularly in high-risk populations, with emerging data supporting roles in molecular subtyping, response monitoring, prognostication, and surveillance. However, few studies report prospective validation, direct comparison with blood-based LB, or impact on actual patient outcomes. Conclusions: Sputum LB is a promising yet underdeveloped modality in LC care. This scoping review highlights technological innovations alongside significant methodological heterogeneity and translational gaps. Future research should focus on standardization, prospective validation, impact on patient outcomes, and integration with blood- and other body fluid–based LB, as well as imaging biomarkers. This will enable incorporation of sputum-based LB into actual clinical pathways of LC care. Full article

Multiple myeloma (MM) is a malignancy of plasma cells that is characterized by a complex and spatially heterogeneous genomic landscape. Despite this complexity, clinical monitoring remains largely dependent on localized bone marrow (BM) assessments. This dependence creates a significant diagnostic gap, as the primary monitoring tools fail to account for the spatial and temporal heterogeneity that drives tumor relapse. Liquid biopsy can serve as an adjunctive approach in assessing the pan-clonal landscape in MM through the molecular profiling of circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs). In this review, we examine the clinical utility of liquid biopsy components in capturing mutational profiles, clonal evolution, treatment resistance mechanisms, and minimal residual disease (MRD), including early detection of relapse and extramedullary progression. We will further discuss current limitations, including variability in assay sensitivity, lack of standardization, and the need for prospective validation. Full article