Search Results (36)

Liquid biopsy, the analysis of tumor-derived material in blood, saliva, and other body fluids, is increasingly explored for the diagnosis, surveillance, and molecular characterization of head and neck squamous cell carcinoma (HNSCC). Its performance, however, is not uniform across the disease, and the reason is fundamentally molecular. human papillomavirus (HPV)-positive oropharyngeal cancers carry viral oncogenes that are absent from the host genome and therefore provide a near ideal, tumor specific circulating marker, whereas HPV-negative tumors are driven by a heterogeneous somatic landscape that offers no single universal target. In this narrative review, we adopt a molecular perspective. We first examine the biological origin of circulating tumor DNA and of the other analytes that liquid biopsy can interrogate including circulating tumor HPV DNA, viral transcripts, microRNAs, extracellular vesicles, and methylation signatures. We then consider how analytical platforms, from droplet digital PCR to next generation and ultrasensitive whole-genome sequencing, translate these molecules into measurements. Only afterward do we discuss the clinical questions, organized by clinical objective rather than by individual study: diagnosis and early detection, prognosis and risk stratification, treatment response monitoring, minimal residual disease and surveillance, and biomarker guided de-escalation in HPV-positive disease. Twelve registered clinical trials, involving approximately 1183 patients, are presented as illustrations of these questions. We close on the biological and technical gaps that still separate promising signals from clinical practice, and on the multi analyte and dynamic strategies most likely to bridge them. At present, liquid biopsy should be regarded as a complementary tool rather than as a replacement for established clinicopathological assessment. Full article

Clonal hematopoiesis of indeterminate potential (CHIP) is an age-associated premalignant state defined by somatic mutations in hematopoietic cells at a variant allele frequency (VAF) ≥2% in the absence of overt hematologic malignancy. Among CHIP-associated mutations, JAK2 V617F is of particular interest because it occupies a dual biological and clinical role: it is both the principal driver of BCR::ABL1-negative myeloproliferative neoplasms (MPNs) and a clonal hematopoiesis variant conferring approximately 12-fold cardiovascular risk in selected cohorts, exceeding that reported for common DTA CHIP variants. Quantitative assessment of JAK2 V617F allele burden is therefore clinically relevant across the full disease continuum—from subclinical clonal expansion to MPN diagnosis, prognostic stratification, and therapeutic monitoring—as VAF thresholds correlate with disease phenotype, thrombotic risk, molecular response, and fibrotic progression. Digital PCR platforms, including droplet digital PCR (ddPCR) and chip-based digital PCR, have emerged as highly sensitive and reproducible methods for absolute JAK2 V617F quantification without the need for standard curves, with reported limits of detection as low as 0.01%. In this review, we synthesize current evidence on the molecular biology of JAK2-driven clonal hematopoiesis, the clinical significance of allele burden quantification, and the analytical performance of digital PCR compared with quantitative PCR and next-generation sequencing. We interpret these findings through a systems biology lens that draws together JAK-STAT signaling networks and thrombo-inflammatory pathways including inflammasome-dependent IL-1 signaling, clonal architecture, and bone marrow microenvironmental remodeling. We also discuss published quantitative models in which JAK2 V617F allele burden is treated as a dynamic state variable, while emphasizing that the present review offers a conceptual synthesis rather than a new computational model. We provide a structured comparative synthesis of published digital PCR analytical performance data, a stage-adapted proposal for clinical monitoring, and schematic models to guide future implementation. Overall, the evidence supports digital PCR as a precision tool for monitoring JAK2 V617F clonal dynamics across the CHIP–MPN spectrum, and points to several priorities: assay standardization, harmonized reporting, external quality assessment, and prospective clinical validation. Full article

Background: Quantitative PCR measurement of BCR::ABL1 is essential for monitoring molecular response and detecting relapse in chronic myeloid leukemia (CML) patients. Given the availability of multiple commercial kits for cDNA synthesis and minimal residual disease assessment, analytical accuracy and reliability are critical. This study evaluated two commercial RT and qPCR kits for BCR::ABL1 quantification and fusion transcript variant identification. Methods: Total RNA was isolated from peripheral blood, bone marrow, and external quality control samples from the UK NEQAS for Leucocyte Immunophenotyping program. cDNA synthesis was performed using two kits: AffinityScript (ASK) and RT Kit (RTK). BCR::ABL1 transcript levels were determined using the ipsogen^® BCR-ABL1 Mbcr IS-MMR Kit (IPS) and the LightMix^® bcr-abl t(9;22) M/m/µ Kit (TMB). Results were compared with UK NEQAS LI reference data. Fusion transcript variants were analyzed using nested PCR and a commercial qPCR assay. Results: Substantial variability was observed between the TMB and IPS assays, with moderate, non-significant correlation and wide limits of agreement. Established discrepancies resulted in different classifications of molecular response, and IPS results showed better concordance with external quality assessment data. ABL1 quantification revealed significantly higher copy numbers with the RTK compared to the ASK (p < 0.0001), enabling more reliable assessment of deep molecular responses. Statistical analyses indicated systematic and proportional bias between the methods. For variant detection, nested PCR demonstrated higher specificity, while the commercial assay showed limited discriminatory capability. Conclusions: Significant methodological differences may affect clinical interpretation, underscoring the importance of validation and standardization in CML molecular monitoring. Full article

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of aggressive non-Hodgkin lymphoma and is characterized by pronounced molecular heterogeneity that is not always fully captured by standard histopathological assessment. Circulating tumor DNA (ctDNA) is increasingly regarded as a promising liquid-biopsy biomarker that enables non-invasive molecular tumor profiling, assessment of tumor burden, dynamic monitoring of treatment response, and detection of measurable/minimal residual disease (MRD). Modern analytical platforms, ranging from PCR-based assays to next-generation sequencing approaches, including CAPP-Seq and PhasED-Seq, have substantially expanded the possibilities of molecular monitoring in DLBCL. This review summarizes current data on the biological characteristics of ctDNA, contemporary methods for its analysis, concordance between ctDNA and tumor-tissue mutational profiles, and the clinical significance of baseline ctDNA levels, early molecular response, post-treatment MRD status, and molecular surveillance during remission. Special attention is given to ctDNA monitoring in patients receiving novel immunotherapies, including CAR-T cell therapy, bispecific antibodies, and antibody–drug conjugates. Emerging multi-omic approaches integrating genomic, epigenomic, and fragmentomic data are discussed as promising future directions. Key limitations of clinical implementation include insufficient standardization of preanalytical and analytical workflows, the confounding effect of clonal hematopoiesis of indeterminate potential, variability across technological platforms, and the lack of completed prospective randomized interventional studies demonstrating improved outcomes when therapy is modified according to ctDNA status. Overall, ctDNA is currently a highly informative prognostic biomarker in DLBCL; however, its full implementation as a predictive tool for treatment selection requires further harmonization, prospective validation, and confirmation in interventional clinical trials. Full article

Circulating tumor DNA (ctDNA) has emerged as a transformative tool in cancer diagnostics, enabling the non-invasive detection of tumor-derived DNA fragments released into the bloodstream through cellular lysis or active secretion. ctDNA measurement has demonstrated its clinical usefulness, including early cancer detection, identification of resistance mechanisms, and screening of asymptomatic individuals. In addition to prognosis, ctDNA analysis is increasingly used to guide adaptive treatment strategies by detecting minimal residual disease and tracking tumor evolution in real time. Recent advances in artificial intelligence are poised to further enhance the clinical impact of ctDNA, transforming it from a passive monitoring biomarker into a dynamic molecular sensor integrated into predictive clinical decision models. However, broad implementation of ctDNA-based assays in routine practice requires rigorous prospective validation, cross-platform standardization, and regulatory approval to unlock its full potential in precision oncology. 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

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

After many years of stagnation in the treatment of acute myeloid leukemia (AML), there is currently a rapid move towards personalized medicine. Improvements in molecular diagnostics, risk assessment tools, targeted therapies, overall patient fitness assessments, and quality-of-life assessments have significantly changed how patients are treated. Genetic and molecular analyses, risk and health assessments, and measurable residual disease (MRD) monitoring are now integral to the treatment plan for evaluating patient responses and recurrence. In this regard, lower-intensity treatments are provided to older or unfit individuals. On the other hand, younger patients are usually subjected to curative therapies such as intensive chemotherapy to induce remission. Depending on their fitness and disease risk, they can be considered for hematopoietic cell transplantation, which is done after close observation for MRD. In addition, newer therapeutic drugs and immunotherapy techniques are being applied for patient management. Tremendous strides have been made in improving the efficiency of treatment programs in the relatively new area of personalized AML therapy, with a focus on functionality. Full article

Multiple myeloma is a complex hematologic malignancy characterized by significant biological heterogeneity, a relapsing–remission clinical course, and a continuously evolving therapeutic landscape. Accurate and timely laboratory assessment is central to disease management, supporting diagnosis, risk stratification, evaluation of treatment response, and long-term monitoring. Despite major advances in therapy, a critical need remains for laboratory tools that can detect disease with greater sensitivity, capture spatial and clonal tumor heterogeneity, and reflect the true depth of treatment response beyond conventional serological and bone marrow-based criteria. Recent laboratory innovations have the potential to transform myeloma care by enabling earlier detection, more accurate prognostication, and personalized therapeutic strategies. This review focuses specifically on innovative laboratory technologies for the diagnosis of multiple myeloma and the evaluation of treatment response. Within this scope, we examine the current diagnostic approaches and the role of high-throughput technologies for measurable residual disease assessment. We explore the emerging role of liquid biopsy approaches, including circulating tumor cells, cell-free DNA/RNA, and mass spectrometry for ultrasensitive detection of monoclonal proteins. We further discuss novel molecular biomarkers and the integration of artificial intelligence and machine learning tools to enhance data interpretation. The innovations reviewed here represent a shift in the contribution of laboratory medicine to myeloma care, offering a more precise, less invasive, and biologically informative framework for targeted and adaptive clinical decisions. Full article

Circulating tumour DNA (ctDNA) assays are increasingly applied in haematological malignancies for non-invasive genotyping, quantitative response assessment, measurable residual disease (MRD) detection, and relapse surveillance, often complementing bone marrow-based testing and, in selected scenarios, potentially reducing its frequency. Yet, translating ctDNA results into comparable clinical decisions across laboratories, platforms, and time remains challenging because ctDNA measurements are influenced by the definition of the measurand (for example, variant allele fraction versus mutant molecules per mL), pre-analytical variables, end-to-end workflow losses, and lineage-specific confounders such as clonal haematopoiesis of indeterminate potential (CHIP), therapy-related clonal haematopoiesis, and compartmental disease (marrow, plasma, cerebrospinal fluid, extramedullary sites). This review proposes a harmonisation framework for haematological ctDNA based on three linked concepts—metrological traceability, which connects reported values to reference systems with stated uncertainty, commutability, which ensures that reference materials behave like patient specimens across diverse workflows and fit-for-purpose reference materials that support calibration, and quality control, external quality assessment, and cut-off setting for intended uses such as early molecular response in large B-cell lymphoma, molecular MRD in acute myeloid leukaemia, and deep response monitoring in multiple myeloma. This framework is accompanied by harmonised CHIP-aware reporting rules for settings without matched cellular DNA and practical change-control/bridging strategies to preserve clinical decision thresholds when platforms or bioinformatic pipelines evolve. Full article

Background/Objectives: Clonal evolution is mainly defined based on the appearance or expansion of clones harboring specific somatic mutations and/or cytogenetic abnormalities, whereas few studies have investigated immunophenotypic heterogeneity assessed by flow cytometry and its relationship with disease progression. In this study, flow cytometry immunophenotyping of acute myeloid leukemia (AML) was carried out to identify phenotypic subclones based on antigen expression and to investigate clonal sweep. Methods: A total of 24 patients diagnosed with AML followed at the Hematology and Transplant Center of Salerno were included. Bone marrow or peripheral blood specimens were subjected to flow cytometry immunophenotyping and leukemic cell characterization. Phenotypic profiles were also compared to molecular alterations detected by next-generation sequencing. Results: We found that flow cytometry-defined clonal heterogeneity was more complex than molecular heterogeneity at diagnosis and disease relapse. Flow cytometry enabled the identification of small phenotypic subclones that were not detected by molecular profiling and that, in several cases, expanded over time, consistent with a phenotypic clonal sweep. The presence of small clones was associated with shorter progression-free survival and overall survival. Conclusions: Flow cytometric clonal heterogeneity, especially the presence of small clones (defined by antigen expression from 2 to 30%), may serve as an additional prognostic factor in AML. Immunophenotyping integrated with molecular data may improve risk stratification, enhance measurable residual disease assessment, and contribute to a more personalized disease monitoring strategy. Full article

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) poses a serious threat to the swine industry in China. As a major pig-producing province, Shandong requires continuous epidemiological monitoring of PRRSV. To elucidate the molecular epidemiology of the virus, 1621 clinical samples were collected from suspected cases across different regions of Shandong Province between 2023 and 2025, primarily from Tai’an, Linyi, Jining, and Liaocheng. RT-qPCR detection showed that the positive rate for PRRSV-2 was 20.05% (325/1621). Genetic analysis based on ORF5 and NSP2 genes indicated that Sublineage L1C (NADC30-like) was the dominant strain for 38.38% of ORF5 gene and 72.73% of NSP2 sequencing results. This was followed by Sublineage L8E and L1A and L5A strains. Key virulence-related mutations were identified at residues R13 and R151 in the GP5 protein, which are associated with enhanced pathogenicity. Additionally, variations in neutralizing epitope and the number of N-glycosylation sites (ranging from 2 to 5 per strain) suggested potential immune evasion. Notably, 26.79% (15/56) of sequenced samples showed discordant ORF5 and NSP2 genotyping results, indicating widespread recombination among PRRSV strains in Shandong Province. These finding demonstrated that the genetic diversity, high recombination frequency, and key amino acid variations in circulating PRRSV strains collectively undermine vaccine effectiveness. This study highlights the need to optimize vaccination strategies, enhance biosecurity measures, and implement effective disease control and elimination programs to reduce the impact of PRRSV in Shandong Province. Full article

The FLT3-ITD mutation is a critical prognostic marker in acute myeloid leukemia (AML) and recent clinical trials demonstrate that FLT3-based measurable residual disease (MRD) is both prognostic and predictive, guiding therapeutic interventions in intensive and post-transplant settings. Conventional detection methods lack the sensitivity required for effective MRD monitoring. We developed a patient-specific droplet digital PCR (ddPCR) approach achieving analytical sensitivity of 10⁻⁵ (0.001%) for FLT3-ITD quantification. In our cohort, ddPCR enabled longitudinal monitoring of clonal dynamics, allowing the detection of re-emerging FLT3-ITD clones months before hematologic relapse and earlier than standard capillary electrophoresis. Notably, 25% of patients who relapsed as FLT3-ITD positive despite being classified as FLT3-negative at diagnosis harbored detectable microclones when retrospectively analyzed by ddPCR, suggesting that FLT3-ITD-positive relapse frequently originates from pre-existing subclones below conventional detection thresholds. These findings challenge current diagnostic classification and may influence risk stratification and treatment decisions, particularly regarding FLT3 inhibitor eligibility. While ddPCR is limited to tracking known dominant clones, it represents a practical, cost-effective solution for high-sensitivity MRD surveillance. In the era of targeted FLT3 therapies, integrating sensitive molecular monitoring into routine AML management may enable timely therapeutic adjustments and improve patient outcomes. Full article

Background: In acute myeloid leukemia (AML), the most sensitive measurable residual disease (MRD) methods are single-gene approaches, but these are applicable only in ~60% of AML cases. Methods: We applied multi-omics single-cell analysis on diagnostic and first remission samples to identify leukemia-specific molecular markers for subsequent MRD monitoring in six AML patients lacking AML-defining variants. Results: Five selection criteria were defined to identify suitable MRD markers. Markers of primordial leukemic clones were identified by combining data from single-cell sequencing and immunophenotyping. Specific markers suitable for use in MRD follow-up were identified in 6/6 patients, in some cases in myelodysplasia-related genes and clonal hematopoiesis-related genes usually not recommended for use in MRD determinations. Patient-specific ddPCR (limits of detection: 0.06–0.0011%) or EC-NGS assays correlated with therapeutic responses: 0/4 markers displayed molecular relapses in three non-relapsing patients, contrary to 4/4 markers of three relapsing patients. Of these, 3/4 and 1/4 markers detected molecular relapses earlier than or simultaneous with conventional methods, respectively (−115 to −338 days). Conclusions: Our results demonstrate that single-cell subclonal mapping at diagnosis and during first remission enables selection of reliable MRD targets for personalized disease surveillance in patients lacking conventional MRD markers. Full article

Background: Multiple myeloma (MM) is a hematologic malignancy characterized by clonal plasma cell expansion and diverse genomic rearrangements, including immunoglobulin heavy chain (IGH) translocations. Although RNA sequencing enables the comprehensive detection of IGH-associated fusions, routine molecular monitoring remains limited, particularly in non-secretory MM (NSMM), which lacks measurable serologic markers. Methods: Here, we contracted an integrated system combining RNA sequencing (RNA-seq) and reverse transcription quantitative polymerase chain reaction (RT-qPCR) to identify and validate fusion gene-based molecular markers for minimal residual disease (MRD) monitoring. Results: The global fusion landscape was delineated by the sequencing analysis of bone marrow samples from 22 newly diagnosed patients with MM. A total of 362 fusion events were identified, of which 190 non-immunoglobulin fusions were selected for detailed characterization. Recurrent breakpoints were concentrated on chromosomes 1 and 19, and five recurrent fusions, DDX5::EEF1A1, OAZ1::KLF2, OAZ1::KLF16, PFKFB3::LINC02649, and PLXNB2::SCO2, were detected across nine patients. Functional enrichment analyses indicated the significant involvement of these genes in RNA splicing regulation, transcriptional misregulation in cancer-related pathways, and focal adhesion processes. Twenty-three fusion transcripts were validated using RT-PCR and Sanger sequencing, demonstrating high specificity for MM. Longitudinal monitoring revealed that the quantitative assessment of fusion transcript levels enabled earlier relapse detection than flow cytometry, including in NSMM, where conventional MRD tools are ineffective. Conclusions: These findings suggest that individualized fusion transcripts serve as robust molecular markers for MRD surveillance. The proposed RNA-seq–RT-qPCR pipeline offers a clinically practical strategy to enhance precision diagnosis and personalized treatment in MM. Full article