Breast cancer is one of the most prevalent cancers and has emerged as a major global challenge. Circulating tumor DNA (ctDNA), a liquid biopsy method, overcomes the accessibility limitations of tissue-based testing and is widely used for monitoring minimal residual disease and molecular relapse, predicting prognosis, evaluating the response of neoadjuvant therapy, and optimizing treatment decisions in non-metastatic breast cancer. However, the application of ctDNA still faces many challenges. Here, we survey the clinical applications of ctDNA in non-metastatic breast cancer and discuss the significant biological and technical challenges of utilizing ctDNA. Importantly, we investigate potential avenues for addressing the challenges. In addition, emerging technologies, including fragmentomics detection, methylation sequencing, and long-read sequencing, have clinical potential and could be a future direction. Proper utilization of machine learning facilitates the identification of meaningful patterns from complex fragment and methylation profiles of ctDNA. There is still a lack of clinical trials focused on the subsets of ctDNA (e.g., circulating mitochondrial DNA), ctDNA-inferred drug-resistant clonal evolution, tumor heterogeneity, and ctDNA-guided clinical decision-making in non-metastatic breast cancer. Due to regional differences in the number of registered clinical trials, it is essential to enhance communication and foster global collaboration to advance the field.

Circulating tumor DNA (ctDNA) is emerging as a transformative biomarker in the management of non-small cell lung cancer (NSCLC). This review focuses on its role in detecting minimal residual disease (MRD), predicting treatment response, and guiding therapeutic decision-making in radiation oncology and immunotherapy. Key studies demonstrate ctDNA's prognostic value, particularly in identifying relapse risk and refining patient stratification for curative-intent and consolidative treatments. Future research is essential to standardize ctDNA assays, optimize integration into clinical workflows, and expand its clinical utility. This biomarker holds substantial promise by enabling non-invasive, real-time monitoring and improving outcomes for patients with NSCLC and beyond.

Aims/Background Effective molecular biomarkers for predicting prognosis and guiding treatment in patients with initially unresectable colorectal liver metastases (CRLMs) undergoing conversion therapy are currently lacking. This study investigated the predictive value of circulating tumor DNA (ctDNA) conversion therapy outcomes in initially unresectable CRLMs. Methods A retrospective analysis was conducted on 81 patients with CRLMs treated at the Sixth Affiliated Hospital, Sun Yat-sen University from January 2017 to April 2021. The relationships between baseline and treatment ctDNA levels and clinical responses were evaluated using group comparisons based on data type. The impact of ctDNA on survival outcomes was analyzed through Cox regression survival analysis. Results Analysis of 81 patients with ctDNA-positive at baseline showed that patients in the ctDNA low-level group had a significantly longer median progression-free survival (mPFS) (p = 0.039). Among 45 patients who underwent ctDNA testing during systemic therapy, the proportion of patients in the ctDNA-negative group receiving local ablative treatment (LAT) was significantly higher (70.0% vs 26.7%, p = 0.006). Furthermore, 50% of patients in the ctDNA-negative group achieved no evidence of disease (NED) status, compared to 6.7% in the ctDNA-positive group (p = 0.004). Both mPFS and median overall survival (mOS) were significantly longer in ctDNA-negative patients compared to ctDNA-positive patients (p < 0.05). Of the 61 patients who underwent LAT, 37 received ctDNA testing at the same time as imaging assessment for NED. The proportion of patients with ctDNA clearance who achieved NED status was markedly higher than that of patients with ctDNA non-clearance (78.6% vs 33.3%, p = 0.036). Patients with ctDNA clearance demonstrated significantly improved mOS compared to those with ctDNA non-clearance (not reached vs 30.1 months, p = 0.036). Conclusion Dynamic changes in ctDNA levels can predict both long-term survival and the effectiveness of conversion therapy in patients with initially unresectable CRLMs.

Circulating tumor DNA (ctDNA) has emerged as a dynamic biomarker in cancer, as evidenced by its increasing integration into clinical practice. Carrying tumor specific characteristics, ctDNA can be used to inform treatment selection, monitor response, and identify drug resistance. In this review, we provide a comprehensive, up-to-date summary of ctDNA in monitoring treatment response with a focus on lung, colorectal, and breast cancers, and discuss current challenges and future directions.

Cholangiocarcinoma (CCA) is a highly aggressive and heterogeneous malignancy arising from the epithelial cells of the biliary tract. The limitations of the current methods in the diagnosis of CCA highlight the urgent need for new, accurate tools for early cancer detection, better prognostication and patient monitoring. Liquid biopsy (LB) is a modern and non-invasive technique comprising a diverse group of methodologies aiming to detect tumour biomarkers from body fluids. These biomarkers include circulating tumour cells, cell-free DNA, circulating tumour DNA, RNA and extracellular vesicles. The aim of this review is to explore the current and potential future applications of LB in CCA management, with a focus on diagnosis, prognostication and monitoring. We examine both its significant potential and the inevitable limitations associated with this technology. We conclude that LB holds considerable promise, but further research is necessary to fully integrate it into precision oncology for CCA.

Cancer, characterized by the uncontrolled proliferation of cells, is one of the leading causes of death globally, with approximately one in five people developing the disease in their lifetime. While many driver genes were identified decades ago, and most cancers can be classified based on morphology and progression, there is still a significant gap in knowledge about genetic aberrations and nuclear DNA damage. The study of two critical groups of genes-tumor suppressors, which inhibit proliferation and promote apoptosis, and oncogenes, which regulate proliferation and survival-can help to understand the genomic causes behind tumorigenesis, leading to more personalized approaches to diagnosis and treatment. Aberration of tumor suppressors, which undergo two-hit and loss-of-function mutations, and oncogenes, activated forms of proto-oncogenes that experience one-hit and gain-of-function mutations, are responsible for the dysregulation of key signaling pathways that regulate cell division, such as p53, Rb, Ras/Raf/ERK/MAPK, PI3K/AKT, and Wnt/β-catenin. Modern breakthroughs in genomics research, like next-generation sequencing, have provided efficient strategies for mapping unique genomic changes that contribute to tumor heterogeneity. Novel therapeutic approaches have enabled personalized medicine, helping address genetic variability in tumor suppressors and oncogenes. This comprehensive review examines the molecular mechanisms behind tumor-suppressor genes and oncogenes, the key signaling pathways they regulate, epigenetic modifications, tumor heterogeneity, and the drug resistance mechanisms that drive carcinogenesis. Moreover, the review explores the clinical application of sequencing techniques, multiomics, diagnostic procedures, pharmacogenomics, and personalized treatment and prevention options, discussing future directions for emerging technologies.

Radiotherapy is an integral component in the treatment of many types of cancer, with approximately half of patients with cancer receiving radiotherapy. Systemic therapy applies pressure that can select for resistant tumor subpopulations, underscoring the importance of understanding how radiation impacts tumor evolution to improve treatment outcomes. We integrated temporal genomic profiling of 120 spatially distinct tumor regions from 20 patients with undifferentiated pleomorphic sarcomas (UPS), longitudinal circulating tumor DNA analysis, and evolutionary biology computational pipelines to study UPS evolution during tumorigenesis and in response to radiotherapy. Most unirradiated UPSs displayed initial linear evolution, followed by subsequent branching evolution with distinct mutational processes during early and late development. Metrics of genetic divergence between regions provided evidence of strong selection pressures during UPS development that further increased during radiotherapy. Subclone abundance changed after radiotherapy with subclone contraction tied to alterations in calcium signaling, and inhibiting calcium transporters radiosensitized sarcoma cells. Finally, circulating tumor DNA analysis accurately measured subclone abundance and enabled noninvasive monitoring of subclonal changes. These results demonstrate that radiation exerts selective pressures on UPSs and suggest that targeting radioresistant subclonal populations could improve outcomes after radiotherapy. Significance: Radiotherapy mediates tumor evolution by leading to the expansion of resistant subclonal cancer cell populations, indicating that developing approaches to target resistant subclones will be crucial to improve radiotherapy response.

Objectives: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer globally, with HPV-positive cases emerging as a distinct subtype with unique clinical and molecular characteristics. Current diagnostic methods, including tissue biopsy and imaging, face limitations in terms of invasiveness, static disease assessment, and difficulty in distinguishing recurrence from treatment-related changes. This review aimed to assess the potential of liquid biopsy as a minimally invasive tool for the diagnosis, treatment monitoring, and surveillance of HPV-associated HNSCC. Methods: This systematic review analyzed literature from PubMed/MEDLINE, Embase, and Web of Science, focusing on original research and reviews related to liquid biopsy applications in HPV-positive HNSCC. Included studies were evaluated based on the robustness of the study design, clinical relevance, and analytical performance of liquid biopsy technologies. Biomarker types, detection methods, and implementation strategies were assessed to identify advancements and challenges in this field. Results: Liquid biopsy technologies, including circulating HPV DNA, ctDNA, and extracellular vesicles, demonstrated high sensitivity (90-95%) and specificity (>98%) in detecting HPV-positive HNSCC. These methods enabled real-time monitoring of tumor dynamics, early detection of recurrence, and insights into treatment resistance. Longitudinal analysis revealed that biomarker clearance during treatment correlates strongly with patient outcomes. Conclusions: Liquid biopsy is a transformative diagnostic and monitoring tool for HPV-associated HNSCC, offering minimally invasive, real-time insights into tumor biology. While challenges remain in standardization and clinical implementation, ongoing research and technological innovations hold promise for integrating liquid biopsy into personalized cancer care, ultimately improving patient outcomes.

Lymphoma, a malignant tumor derived from lymphocytes and lymphoid tissues, presents with complex and heterogeneous clinical manifestations, requiring accurate patient classification for appropriate treatment. While invasive pathological examination of lymph nodes or lymphoid tissue remains the gold standard for lymphoma diagnosis, its utility is limited in cases of deep-seated tumors such as intraperitoneal and central nervous system lymphomas. In addition, biopsy procedures carry an inherent risk of complications. Computed tomography (CT) and positron emission tomography/computed tomography (PET/CT) imaging are essential for treatment assessment and monitoring, but lack the ability to detect early clonal evolution and minimal residual disease (MRD). Liquid biopsy-based analysis of circulating tumor DNA (ctDNA) offers a non-invasive alternative that allows for repeated sampling and overcomes the limitations of spatial heterogeneity and invasive biopsies. ctDNA provides genetic and epigenetic insights into lymphoma and serves as a dynamic, quantifiable biomarker for diagnosis, risk stratification, and treatment response. This review comprehensively summarizes common genetic variations in lymphoma and systematically evaluates ctDNA detection technologies, including PCR-based assays and next-generation sequencing (NGS). Applications of ctDNA detection in noninvasive genotyping, risk stratification, therapeutic response monitoring, and MRD detection are discussed across various lymphoma subtypes, including diffuse large B-cell lymphoma, Hodgkin lymphoma, follicular lymphoma, and T-cell lymphoma. By integrating recent research findings, the review highlights the role of ctDNA profiling in advancing precision medicine, enabling personalized therapeutic strategies, and improving clinical outcomes in lymphoma.

Breast cancer management has traditionally relied on tissue biopsies and imaging, which offer limited insights into the disease. However, the discovery of circulating tumor DNA (ctDNA) and minimal residual disease (MRD) detection has revolutionized our approach to breast cancer. ctDNA, which is fragmented tumor DNA found in the bloodstream, provides a minimally invasive way to understand the tumor's genomic landscape, revealing heterogeneity and critical mutations that biopsies may miss. MRD, which indicates cancer cells that remain after treatment, can now be detected using ctDNA and other advanced methods, improving our ability to predict disease recurrence. This allows for personalized adjuvant therapies based on individual MRD levels, avoiding unnecessary treatments for patients with low MRD. This review discusses how ctDNA and MRD represent a paradigm shift towards personalized, genomically guided cancer care, which has the potential to significantly improve patient outcomes in breast cancer.

Uterine carcinosarcomas (UCS) are high-grade biphasic neoplasms with generally poor outcomes. Based on The Cancer Genome Atlas molecular classification of endometrial carcinomas, the majority of UCS are classified as copy-number high/serous-like (p53-abnormal); however, a small subset represent other molecular subtypes, including those that harbor POLE mutations. We identified 11 POLE-mutated (POLEmut) UCS across 3 institutions and assessed the clinical, histopathologic, immunohistochemical, and molecular features of these tumors. POLEmut UCS occurred in adult women (median age, 64 years; range, 48-79 years) and usually presented as The International Federation of Gynecology and Obstetrics 2009 clinical stage IA (n = 4) or IB (n = 3). Almost all tumors were predominantly carcinomatous (n = 10), with most showing endometrioid morphology (n = 7), followed by ambiguous (n = 4) and serous (n = 3) histotypes. By immunohistochemistry, 7 tumors showed aberrant or subclonally aberrant expression of p53, 6 of which harbored pathogenic mutations in TP53 by sequencing. Other frequent mutations included PIK3CA (10/11), PTEN (8/11), RB1 (7/11), ARID1A (7/11), ATM (6/11), PIK3RA (5/11), and FBXW7 (4/11). Two tumors demonstrated loss of mismatch repair protein expression, and 1 had subclonal loss. Heterologous differentiation was uncommon, and only chondrosarcomatous type (n = 2) was observed. Mean and median follow-ups were 24.3 and 14.1 months, respectively (range, 1.4-61.1 months). Ten patients (91%) had no recurrences or death from disease, although 3 of these had follow-up periods <1 year. One patient, with the subclonal POLE variant, presented with stage IV disease and died 1.4 months after surgery. In conclusion, POLEmut UCS demonstrate unique morphologic and immunohistochemical features compared with their p53-abnormal counterparts and may have significant prognostic differences. Our study supports full molecular classification of UCS. We also raise awareness for potentially assessing POLE mutation allele frequency and clonality in consideration of classifying a tumor as POLEmut.

Currently, due to the invasive nature of colonoscopy and the associated pain, people avoid undergoing the procedure, making it difficult to detect the majority of potential early stage colorectal carcinoma/precancerous lesions or advanced adenoma. Advanced colorectal adenoma is the main precursor to the development of colorectal carcinoma. Therefore, improving advanced colorectal adenoma detection rate can significantly decrease the development and morbidity of colorectal carcinoma. Accordingly, a non-invasive method to screen high-risk people for colonoscopy in clinical practice is urgently needed.

With the development of medical technology, screening methods for colorectal carcinoma are emerging rapidly, and diverse non-invasive methods are being developed. Cell-free DNA (cfDNA), commonly referred to as liquid biopsy, has promising application prospects as a minimally invasive strategy for early screening of colorectal cancer. CfDNA has already been applied in the field of prenatal diagnosis, advanced carcinoma, and organ transplantation, and the application cfDNA in advanced colorectal adenoma is at the cutting-edge of current research. Thus, this review summarizes the progress in research on different biological characteristics of cfDNA and its utility in the screening of advanced colorectal adenoma, including sizes of cfDNA molecules, end signature of cfDNA (preferred ends, end motifs, jagged ends), nucleosomal footprints, cfDNA topology, cfDNA methylation, and cfDNA integrity.

We hope that this review will advance this promising research field.

Krukenberg tumors are rare cancers involving metastatic disease in the ovaries but classically originate from gastrointestinal malignancies, and often present diagnostic challenges due to their nonspecific symptoms and advanced stage at detection. Traditional imaging techniques like ultrasound, CT, and MRI are common methods of cancer monitoring but are limited in detecting micrometastatic disease and early-stage metastases. Circulating tumor DNA (ctDNA) testing, a noninvasive liquid biopsy method, offers a promising alternative to traditional screening methods, enabling earlier detection and precise molecular profiling of metastatic tumors. We present a case study involving a female patient who initially presented with stage IV colon cancer with oligometastatic disease to a single mesenteric lymph node. Despite neoadjuvant chemotherapy and resection of known disease, postresection ctDNA returned positive. Imaging after metastectomy failed to reveal any sites of ongoing disease, although did show a small, 2.4-cm hypodensity in the right ovary interpreted by radiology as likely an ovarian follicle. Given her ctDNA positivity, she was started on capecitabine. ctDNA levels improved, but her serum carcinoembryonic antigen (CEA) tumor marker continued to rise, and imaging subsequently revealed increased bilateral ovarian masses. She underwent bilateral salpingo-oophorectomy and total abdominal hysterectomy, with pathology confirming metastatic colon adenocarcinoma, and subsequent normalization of her CEA and ctDNA levels. Our findings underscore ctDNA's potential to complement imaging, particularly for high-risk patients, for disease monitoring and to refine therapeutic management when treating Krukenberg tumors.

Despite advances in treatments, multiple myeloma (MM) remains an incurable cancer where relapse is common. We developed a circulating tumor DNA (ctDNA) approach in order to characterize tumor genomics, monitor treatment response, and detect early relapse in MM. By sequencing 412 specimens from 64 patients with newly diagnosed or relapsed/refractory disease, we demonstrate the correlation between ctDNA and key clinical biomarkers, as well as patient outcomes. We further extend our approach to simultaneously track CAR-specific cell-free DNA (CAR-cfDNA) in patients undergoing anti-BCMA CAR T-cell (BCMA-CAR) therapy. We demonstrate that ctDNA levels following BCMA-CAR inversely correlate with relative time to progression (TTP), and that measurable residual disease (MRD) quantified by peripheral blood ctDNA (ctDNA-MRD) was concordant with clinical bone marrow MRD. Finally, we show that ctDNA-MRD can anticipate clinical relapse and identify the emergence of genomically-defined therapy-resistant clones. These findings suggest multiple clinical uses of ctDNA for MM in molecular characterization and disease surveillance.

Detection of low-abundance mutations for the early discovery of fungicide-resistant fungal pathogens is highly demanded, but remains challenging. Herein, we developed a dual-recognition strategy, termed PARPA, involving Pyrococcus furiosus Argonaute (pfAgo)-mediated elimination of wild-type fungal genes and CRISPR/Cas12a-based amplicon recognition. This assay can detect fungicide-resistant Puccinia striiformis at relative abundances as low as 0.05% and has potential for achieving early screening of fungicide-resistant fungal pathogens.

Local recurrence after radiotherapy is common in locally advanced head and neck cancer (HNC) patients. Re-irradiation can improve local disease control, but disease progression remains frequent. Hence, predictive biomarkers are needed to adapt treatment intensity to the patient's individual risk. We quantified circulating tumor DNA (ctDNA) in sequential plasma samples and correlated ctDNA levels with disease outcome. Ninety four longitudinal plasma samples from 16 locally advanced HNC patients and 57 healthy donors were collected at re-radiotherapy baseline, after 5 and 10 radiation fractions, at irradiation end, and at routine follow-up visits. Plasma DNA was subjected to low coverage whole genome sequencing for copy number variation (CNV) profiling to quantify ctDNA burden. CNV-based ctDNA burden was detected in 8/16 patients and 25/94 plasma samples. Ten additional ctDNA-positive samples were identified by tracking patient-specific CNVs found in earlier sequential plasma samples. ctDNA-positivity after 5 and 10 radiation fractions (both: log-rank, p = .050) as well as at the end of irradiation correlated with short progression-free survival (log-rank, p = .006). Moreover, a pronounced decrease of ctDNA toward re-radiotherapy termination was associated with worse treatment outcome (log-rank, p = .005). Dynamic ctDNA tracking in serial plasma beyond re-radiotherapy reflected treatment response and imminent disease progression. In five patients, molecular progression was detected prior to tumor progression based on clinical imaging. Our findings emphasize that quantifying ctDNA during re-radiotherapy may contribute to disease monitoring and personalization of adjuvant treatment, follow-up intervals, and dose prescription.

Achieving remission is the first step toward a cure in treating aggressive B-cell lymphomas. Radiographic imaging, such as fluorodeoxyglucose-positron emission tomography/computed tomography scans are the current standard to define remission at the end of therapy but lack specificity for lymphoma and cannot detect disease at the molecular level. Identifying measurable residual disease with ultrasensitive detection of circulating tumor DNA potentially offers the possibility of improving clinical outcomes.

Early studies of circulating tumor DNA in aggressive B-cell lymphomas showed a strong association with overall tumor burden, and baseline quantitative levels are associated with clinical outcomes after frontline chemotherapy. Next-generation sequencing methods that detect lymphoma-relevant genetic aberrations in circulating tumor DNA can also be used for noninvasive genotyping that strongly mirror tissue biopsies. Rapid changes in circulating tumor DNA dynamics after 1 or 2 cycles of frontline chemotherapy or within weeks of treatment with chimeric antigen receptor T-cell salvage therapy are also highly prognostic. Although serial monitoring of circulating tumor DNA can detect molecular relapse 3 to 6 months before clinical relapse, improved analytical thresholds are required to detect measurable residual disease at a singular point at the end of therapy. Modern advances in circulating tumor DNA methods now allow for the reliable detection of measurable residual disease, with an analytical detection threshold of 1 in 1 million cell-free DNA molecules.

The results of this review suggest that modern ultrasensitive methods of detecting circulating tumor DNA may improve the current definition of remission in aggressive B-cell lymphomas. Incorporating circulating tumor DNA at the end of therapy assessment identifies patients who do not require surveillance monitoring and introduces paradigms of treating measurable residual disease within clinical trials. Practical barriers, including standardization of collection, availability, turnaround times, and cost, remain hurdles preventing widespread implementation into clinical practice.

The analysis of circulating tumor DNA (ctDNA) in liquid biopsy specimens has an established role for the detection of predictive molecular alterations and acquired resistance mutations in several tumors. The low-invasiveness of this approach allows for repeated sampling and dynamic monitoring of disease evolution. Originating from the entire body tumor bulk, plasma-derived ctDNA reflects intra- and interlesional genetic heterogeneity. In the management of lymphoma patients, ctDNA quantification at various timepoints of the patient's clinical history is emerging as a complementary tool that may improve risk stratification, assessment of treatment response and early relapse detection during follow-up, most prominently in patients with diffuse large B-cell lymphoma or classic Hodgkin lymphoma. While liquid biopsies have not yet entered standard-of-care treatment protocols in these settings, several trials have provided evidence that at least a subset of lymphoma patients may benefit from the introduction of liquid biopsies into daily clinical care. In parallel, continuous technological developments have enabled highly sensitive ctDNA assessment methods, which span from locus-specific techniques identifying single hotspot mutations, to sequencing panels and genome-wide approaches that explore broader genetic and epigenetic alterations. Here, we provide an overview of current methods and ongoing technical developments for ctDNA evaluation. We also summarize the most important data from a selection of clinical studies that have explored the clinical use of ctDNA in several lymphoma entities.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by late detection and poor prognosis. Recent research highlights the pivotal role of epigenetic alterations in driving PDAC development and progression. These changes, in conjunction with genetic mutations, contribute to the intricate molecular landscape of the disease. Specific modifications in DNA methylation, histone marks, and non-coding RNAs are emerging as robust predictors of disease progression and patient survival, offering the potential for more precise prognostic tools compared to conventional clinical staging. Moreover, the detection of epigenetic alterations in blood and other non-invasive samples holds promise for earlier diagnosis and improved management of PDAC. This review comprehensively summarises current epigenetic research in PDAC and identifies persisting challenges. These include the complex nature of epigenetic profiles, tumour heterogeneity, limited access to early-stage samples, and the need for highly sensitive liquid biopsy technologies. Addressing these challenges requires the standardisation of methodologies, integration of multi-omics data, and leveraging advanced computational tools such as machine learning and artificial intelligence. While resource-intensive, these efforts are essential for unravelling the functional consequences of epigenetic changes and translating this knowledge into clinical applications. By overcoming these hurdles, epigenetic research has the potential to revolutionise the management of PDAC and improve patient outcomes.

Colorectal cancer (CRC) represents a heterogeneous group of diseases that imposes a considerable global and national health burden. Although most CRC patients are diagnosed at an early stage and undergo potentially curative treatment, a significant proportion experience recurrence. Currently, adjuvant chemotherapy decisions are primarily based on clinicopathological characteristics, which have well-recognized limitations in accurately identifying patients harboring minimal residual disease (MRD), often resulting in unnecessary chemotherapy exposure. Circulating tumor DNA (ctDNA) has emerged as a promising surrogate marker for MRD, offering a more precise approach to identifying patients at risk of recurrence after curative-intent surgery and refining adjuvant chemotherapy decisions. Growing evidence from multiple studies has demonstrated that ctDNA outperforms traditional clinicopathological factors as a marker for MRD. This review synthesizes key studies supporting the role of ctDNA in MRD detection for CRC patients and evaluates clinical trials investigating the application of ctDNA in guiding adjuvant therapy decisions. This emerging strategy holds the potential to transform the adjuvant treatment paradigm in colorectal cancer by optimizing therapeutic precision and minimizing unnecessary treatment.

Hemangiosarcoma is a highly malignant tumor commonly affecting canines, originating from endothelial cells that line blood vessels, underscoring the importance of early detection. This canine cancer is analogous to human angiosarcoma, and the development of liquid biopsies leveraging cell-free DNA (cfDNA) represents a promising step forward in early cancer diagnosis. In this study, we utilized Whole Genome Sequencing (WGS) to analyze fragment sizes and copy number alterations (CNAs) in cfDNA from 21 hemangiosarcoma-affected and 36 healthy dogs, aiming to enhance early cancer detection accuracy through machine learning models. Our findings reveal that similar to trends in human oncology, hemangiosarcoma samples exhibited shorter DNA fragment sizes compared to healthy controls, with a notable leftward shift in the primary peak. Interestingly, canine hemangiosarcoma DNA fragment sizes demonstrated eight distinct periodic patterns diverging from those typically observed in human angiosarcoma. Additionally, we identified seven novel genomic gains and nine losses in the hemangiosarcoma samples. Applying machine learning to the cfDNA fragment size distribution, we achieved an impressive average Area Under the Curve (AUC) of 0.93 in 10-fold cross-validation, underscoring the potential of this approach for precise early-stage cancer classification. This study confirms distinctive cfDNA fragment size and CNA patterns in hemangiosarcoma-affected vs. healthy dogs and demonstrates the promise of these biomarkers in canine cancer screening, early detection, and monitoring via liquid biopsies. These findings establish a foundation for broader research on cfDNA analysis in various canine cancers, integrating methodologies from human oncology to enhance early detection and diagnostic precision in veterinary medicine.

Esophageal cancer (EC) is one of the leading causes of cancer-related deaths globally. Surgery is the standard treatment for resectable EC after preoperative chemoradiotherapy or chemotherapy, followed by postoperative adjuvant chemotherapy in certain cases. Upper gastrointestinal endoscopy and computed tomography (CT) are predominantly performed to evaluate the efficacy of these treatments, but their sensitivity and accuracy for evaluating minimal residual disease remain unsatisfactory, thereby requiring the development of alternative methods. In recent years, interest has been increasing in using liquid biopsy to assess treatment responses. Liquid biopsy is a noninvasive technology for detecting cell components in the blood and other body fluids. It involves collecting a small sample of body fluid, which is then analyzed for the presence of components, including circulating tumor DNA (ctDNA), microRNA (miRNA), or circulating tumor cells (CTCs). Further, ctDNA and miRNA are analyzed with various techniques, including digital polymerase chain reaction (dPCR) and next-generation sequencing (NGS). CTCs are isolated by determining surface antigens using immunomagnetic techniques or by filtering the blood according to cell size and rigidity. Several studies indicate that investigating these materials helps predict EC prognosis and recurrence and possibly stratifies high-risk groups. Liquid biopsy may also apply to the selection of cases that have achieved a complete response through preoperative treatment to prevent surgery and preserve the esophagus, as well as identifying the suitability of postoperative chemotherapy and the timing of conversion surgery for unresectable EC. The potential of liquid biopsy to enhance treatment decisions will further advance EC treatment.

This review provides a comprehensive overview of the evolving role of minimal residual disease (MRD) for patients with Colon Cancer (CC). Currently, the standard of care for patients with non-metastatic CC is adjuvant chemotherapy (ACT) for all patients with stage III and high-risk stage II CC following surgical intervention. Despite a 5-20% improvement in long-term survival outcomes, this approach also results in a significant proportion of patients receiving ACT without any therapeutic benefit and being unnecessarily exposed to the risks of secondary side effects. This underscores an unmet clinical need for more precise stratification to distinguish patients who necessitate ACT from those who can be treated with surgery alone. By employing liquid biopsy, it is possible to discern MRD enabling the categorization of patients as MRD-positive or MRD-negative, potentially revolutionizing the management of ACT. This review aimed to examine the heterogeneity of methodologies currently available for MRD detection, encompassing the state-of-the-art technologies, their respective advantages, limitations, and the technological challenges and multi-omic approaches that can be utilized to enhance assay performance. Furthermore, a discussion was held regarding the clinical trials that employ an MRD assay focusing on the heterogeneity of the assays used. These differences in methodology, target selection, and performance risk producing inconsistent results that may not solely reflect biological/clinical differences but may be the consequence of the preferential use of particular products in studies conducted in different countries. Standardization and harmonization of MRD assays will be crucial to ensure the liquid revolution delivers reliable and clinically actionable outcomes for patients.

Metastatic castration-resistant prostate cancer (mCRPC) resistant to androgen receptor signaling inhibitors (ARSI) is often lethal. Liquid biopsy biomarkers for this deadly form of disease remain under investigation, and underpinning mechanisms remain ill-understood.

We applied targeted cell-free DNA (cfDNA) sequencing to 126 patients with mCRPC from three academic cancer centers and separately performed genome-wide cfDNA methylation sequencing on 43 plasma samples collected prior to the initiation of first-line ARSI treatment. To analyze the genome-wide sequencing data, we performed nucleosome positioning and differential methylated region analysis. We additionally analyzed single-cell and bulk RNA sequencing data from 14 and 80 patients with mCRPC, respectively, to develop and validate a stem-like signature, which we inferred from cfDNA.

Targeted cfDNA sequencing detected AR/enhancer alterations prior to first-line ARSIs that correlated with significantly worse progression-free survival (P = 0.01; HR = 2.12) and overall survival (P = 0.02; HR = 2.48). Plasma methylome analysis revealed that AR/enhancer lethal mCRPC patients have significantly higher promoter-level hypomethylation than AR/enhancer wild-type mCRPC patients (P < 0.0001). Moreover, gene ontology and CytoTRACE analysis of nucleosomally more accessible transcription factors in cfDNA revealed enrichment for stemness-associated transcription factors in patients with lethal mCRPC. The resulting stemness signature was then validated in a completely held-out cohort of 80 patients with mCRPC profiled by tumor RNA sequencing.

We analyzed a total of 220 patients with mCRPC, validated the importance of cell-free AR/enhancer alterations as a prognostic biomarker in lethal mCRPC, and showed that the underlying mechanism for lethality involves reprogramming developmental states toward increased stemness. See related commentary by Nawfal et al., p. 7.

Circulating tumor DNA (ctDNA) quantification surpasses cancer antigen 15 to 3 for metastatic breast cancer surveillance. Clinical translation, however, is limited because of uncertainties about the optimal method and clinically valid ctDNA decision thresholds. Plasma-SeqSensei Breast Cancer IVD kit (PSS) is a novel assay for ctDNA molecular counting, detecting ≥0.06% variant allele fractions in AKT1, ERBB2, ESR1, KRAS, PIK3CA, and TP53. PSS was validated against droplet digital PCR (ddPCR) in 201 samples from 16 subjects with PIK3CA/TP53-mutated cancers, longitudinally sampled for a median of 93 (range, 18 to 113) weeks, three to five weekly. PSS and ddPCR ctDNA levels correlate significantly (Spearman ρ, 0.923; 95% CI, 0.898-0.941) across 0% to 43% variant allele frequency (VAF) range. PSS predicts 12-week progression with high clinical accuracy (area under the curve, 0.848; 95% CI, 0.790-0.894). PSS validates a previously developed ddPCR classifier: <10 copies/mL (0.25% VAF); excludes >100 copies/mL (2.5% VAF); and confirms progression, with negative predictive value (95% CI) of 83% (76%-88%) and positive predictive value (95% CI) of 91% (81%-96%) (weighted κ, 0.856; 95% CI, 0.797-0.915). PSS thus confirms robust clinical thresholds (10 to 100 copies/mL, 0.25% to 2.5% VAF) for metastatic breast cancer surveillance, using absolute molecular counting.

General population cancer screening in the United Kingdom is limited to selected cancers. Blood-based multi-cancer early detection tests aim to detect potential cancer signals from multiple cancers in the blood. The use of a multi-cancer early detection test for population screening requires a high specificity and a reasonable sensitivity to detect early-stage disease so that the benefits of earlier diagnosis and treatment can be realised.

To undertake a systematic literature review of the clinical effectiveness evidence on blood-based multi-cancer early detection tests for screening.

Comprehensive searches of electronic databases (including MEDLINE and EMBASE) and trial registers were undertaken in September 2023 to identify published and unpublished studies of multi-cancer early detection tests. Test manufacturer websites and reference lists of included studies and pertinent reviews were checked for additional studies. The target population was individuals aged 50-79 years without clinical suspicion of cancer. Outcomes of interest included test accuracy, number and proportion of cancers detected (by site and stage), time to diagnostic resolution, mortality, potential harms, health-related quality of life, acceptability and satisfaction. The risk of bias was assessed using the quality assessment of diagnostic accuracy studies-2 checklist. Results were summarised using narrative synthesis. Stakeholders contributed to protocol development, report drafting and interpretation of review findings.

Over 8000 records were identified. Thirty-six studies met the inclusion criteria: 1 ongoing randomised controlled trial, 13 completed cohort studies, 17 completed case-control studies and 5 ongoing cohort or case-control studies. Individual tests claimed to detect from 3 to over 50 different types of cancer. Diagnostic accuracy of currently available multi-cancer early detection tests varied substantially: Galleri® (GRAIL, Menlo Park, CA, USA) sensitivity 20.8-66.3%, specificity 98.4-99.5% (three studies); CancerSEEK (Exact Sciences, Madison, WI, USA) sensitivity 27.1-62.3%, specificity 98.9- 99.1% (two studies); SPOT-MAS™ (Gene Solutions, Ho Chi Minh City, Vietnam) sensitivity 72.4-100%, specificity 97.0-99.9% (two studies); Trucheck™ (Datar Cancer Genetics, Bayreuth, Germany) sensitivity 90.0%, specificity 96.4% (one study); Cancer Differentiation Analysis (AnPac Bio, Shanghai, China) sensitivity 40.0%, specificity 97.6% (one study). AICS® (AminoIndex Cancer Screening; Ajinomoto, Tokyo, Japan) screens for individual cancers separately, so no overall test performance statistics are available. Where reported, sensitivity was lower for detecting earlier-stage cancers (stages I-II) compared with later-stage cancers (stages III-IV). Studies of seven other multi-cancer early detection tests at an unclear stage of development were also summarised.

Study selection was complex; it was often difficult to determine the stage of development of multi-cancer early detection tests. The evidence was limited; there were no completed randomised controlled trials and most included studies had a high overall risk of bias, primarily owing to limited follow-up of participants with negative test results. Only one study of Galleri recruited asymptomatic individuals aged over 50 in the United States of America; however, study results may not be representative of the United Kingdom's general screening population. No meaningful results were reported relating to patient-relevant outcomes, such as mortality, potential harms, health-related quality of life, acceptability or satisfaction.

All currently available multi-cancer early-detection tests reported high specificity (> 96%). Sensitivity was highly variable and influenced by study design, population, reference standard test used and length of follow-up.

Further research should report patient-relevant outcomes and consider patient and service impacts.

This study is registered as PROSPERO CRD42023467901.

This award was funded by the National Institute for Health and Care Research (NIHR) Health Technology Assessment programme (NIHR award ref: NIHR161758) and is published in full in Health Technology Assessment; Vol. 29, No. 2. See the NIHR Funding and Awards website for further award information.

Despite the advancements in early lung cancer detection attributed to the widespread use of low-dose computed tomography (LDCT), this technology has also led to an increasing number of pulmonary nodules (PNs) of indeterminate significance being identified. Therefore, this study was aimed to develop a model that leverages plasma methylation biomarkers and clinical characteristics to distinguish between malignant and benign PNs.

In a training cohort of 210 patients with PNs, we evaluated plasma circulating tumor DNA (ctDNA) for the presence of three lung cancer-specific methylation markers: SHOX2, SCT, and HOXA7. Subsequently, we constructed a combined model utilizing methylated SHOX2/SCT/HOXA7 (mSHOX2/SCT/HOXA7) ctDNA levels, the largest nodule size measured by LDCT, and age, employing the binary logistic regression algorithm. Furthermore, we compared the diagnostic performances of the combined model with the Mayo Clinic model and the single mSHOX2/SCT/HOXA7 model by analyzing the area under the receiver operating characteristic curve (AUC) for each.

The combined model demonstrated an impressive AUC of 0.87 and an accuracy of 0.75 in the training cohort, using pathologic diagnoses as the gold standard. This performance was significantly superior to that of the single mSHOX2/SCT/HOXA7 panel (AUC = 0.81, P < 0.0001) and the Mayo model (AUC = 0.65, P = 0.0005). Further validation in a cohort of 82 patients with PNs confirmed the diagnostic value of the combined model. Additionally, we observed that as the size of the nodule increased, the diagnostic accuracy of the combined model also improved.

A combined model incorporating the ctDNA-based methylation status of SHOX2/SCT/HOXA7 genes, the largest nodule size measured by LDCT, and age can serve as a supplementary approach to LDCT for lung cancer. This model enhances the precision in identifying high-risk individuals and optimizes the clinical management strategies for PNs detected by CT.

Lung cancer is the leading cause of cancer death world-wide. Along the entire timeline of lung cancer identification, diagnosis and treatment, clinicians and patients face challenges in clinical decision-making that could be aided by useful biomarkers. In this review, we discuss the development of biomarkers and qualities that are ideal in a biomarker candidate, types of biospecimens that can be utilized for biomarker development in lung cancer, and how biomarkers could be clinically useful at various points along lung cancer timeline. We then review biomarkers that have been validated and are clinically available to assist with the management of lung nodules and diagnosis of lung cancer, which includes blood-based biomarkers to assist with decision-making prior to an invasive diagnostic procedure, as well as specimens obtained during a bronchoscopy and applied in cases of an inconclusive biopsy result. Finally, we discuss challenges in biomarker application and recent publications relevant to future lung cancer biomarker development.

Liquid biopsy has become an opportunity in lymphoma diagnostics, since plasma circulating tumor DNA (ctDNA) is an easily accessible source of tumor DNA, which can be complementary to tissue biopsy. Through ctDNA, lymphomas can be molecularly characterized, tumor clonal evolution can be tracked, while monitoring minimal residual diseases during and after therapy. Here, we describe the methodology of cancer personalized profiling by deep sequencing (CAPP-seq) that we use for ctDNA qualification and quantification.

Due to current challenges in the early detection, less than 40% of individuals diagnosed with hepatocellular carcinoma (HCC) are viable candidates for surgical intervention. Therefore, validating and launching of a novel precise diagnostic approach is essential for early diagnosis. Based on developing evidence using circulating tumor cells and their derivatives, circulating miRNAs, and extracellular vesicles (EVs), liquid biopsy may offer a reliable platform for the HCC's early diagnosis. Each liquid biopsy analyte may provide significant areas for diagnosis, prognostic assessment, and treatment monitoring of HCC patients depending on its kind, sensitivity, and specificity. The current review addresses potential clinical applications, current research, and future developments for liquid biopsy in HCC management.

Liquid biopsy (LB) involves the analysis of circulating tumour-derived DNA (ctDNA), providing a minimally invasive method for gathering both quantitative and qualitative information. Genomic analysis of ctDNA through next-generation sequencing (NGS) enables comprehensive genetic profiling of tumours, including non-driver alterations that offer prognostic insights. LB can be applied in both early-stage disease settings, for the diagnosis and monitoring of minimal residual disease (MRD), and advanced disease settings, for monitoring treatment response and understanding the mechanisms behind disease progression and tumour heterogeneity. Currently, LB has limited use in clinical practice, primarily due to its significant costs, limited diagnostic yield, and uncertain prognostic role. The application of artificial intelligence (AI) in the medical field is a promising approach to processing extensive information and applying it to individual cases to enhance therapeutic decision-making and refine risk assessment.

The advent and success of new drugs for treating HER2-positive metastatic breast cancer has led to a constant improvement in disease and progression-free survival as well as overall survival. Despite these advantages, the overall survival and quality of life of patients with HER2-positive breast cancer brain metastases are significantly worse than the ones of patients with HER2-positive breast cancer metastases outside the brain. For this reason, prevention and treatment of brain metastasis remain a major clinical challenge and the keys to further improving the clinical and survival outcomes of HER2-positive breast cancer patients. This review discusses the etiopathogenesis of brain metastasis, the currently available treatments, and the future perspective on new treatment strategies and diagnostic tools.

Circulating tumor nucleic acids (CTNAs), which consist of cell-free DNA or RNA released from tumor cells, are utilized as potential biomarkers for diagnosing and managing tumor prognosis. There is a significant demand for developing a highly sensitive and reliable assay for CTNAs detection. In this study, we engineered a CRISPR/Cas12a corona nanomachine capable of detecting circulating tumor DNA and RNA in serum. This nanomachine consists of a protein shell incorporating Cas12a/crRNA ribonucleoprotein complexes and a scaffold AuNP core decorated with substrate ssDNA strands. The protective CRISPR corona shields the nucleic acid core from degradation by nuclease DNase/RNase, thereby enhancing the stability of the CRISPR/Cas12a corona nanomachine in biological fluids, even tolerating up to undiluted human serum and FBS. Upon encountering target CTNAs, the CRISPR/Cas12a is activated through the sequence-specific hybridization between crRNA and CTNAs. Subsequently, the activated CRISPR/Cas12a autonomously cleaves the collateral ssDNA substrates on AuNPs, releasing the fluorophore-labeled fragment and generating an increasing fluorescent signal. The CRISPR corona nanomachine was successfully employed to detect various CTNAs, including circulating tumor (ct)DNA/RNA (EGFR L858R) and microRNA-21, achieving a limit of detection of 0.14 pM for ctDNA and 1.0 pM for RNA. This CRISPR corona nanomachine enables simultaneous detection of both DNA and RNA in complex biological samples, offering a promising tool for early diagnosis.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although many people with CF (pwCF) are treated using CFTR modulators, some are non-responsive due to their genotype or other uncharacterized reasons. Autologous airway stem cell therapies, in which the CFTR cDNA has been replaced, may enable a durable therapy for all pwCF. Previously, CRISPR-Cas9 with two AAVs was used to sequentially insert two-halves of the CFTR cDNA and an enrichment cassette into the CFTR locus. However, the editing efficiency was <10% and required enrichment to restore CFTR function. Further improvement in gene insertion may enhance cell therapy production. To improve CFTR cDNA insertion in human airway basal stem cells (ABCs), we evaluated the use of the small molecules AZD7648 and ART558, which inhibit non-homologous end-joining (NHEJ) and micro-homology mediated end-joining (MMEJ). Adding AZD7648 alone improved gene insertion by 2- to 3-fold. Adding both ART558 and AZD7648 improved gene insertion but induced toxicity. ABCs edited in the presence of AZD7648 produced differentiated airway epithelial sheets with restored CFTR function after enrichment. Adding AZD7648 did not increase off-target editing. Further studies are necessary to validate if AZD7648 treatment enriches cells with oncogenic mutations.

Melanoma of the uveal tract or uveal melanoma (UM) originates from melanocytes of the eye and is the most common intraocular malignancy in adults. Despite considerable advances in diagnostic procedures and treatments, prognosis remains poor in those with advanced disease. Accordingly, although current treatments have an excellent local disease control rate, approximately 50% of patients develop metastatic relapse within 10 years. The high risk for metastatic disease with a variable and often long latency period is thought to be due to early spread of cancer cells disseminating into organs such as the liver, followed by a period of dormancy, before the eventual emergence of radiologically measurable disease. Early detection of disease relapse or metastasis is therefore crucial to allow timely treatment and ultimately improve patient outcome. Recently, advances in minimally-invasive liquid biopsy techniques and biomarkers such as circulating tumor DNA (ctDNA) have demonstrated potential to transform the field of cancer care by aiding diagnosis, prognosis and monitoring of various cancer types. UM is particularly suitable for ctDNA-based approaches due to the relatively well-characterized spectrum of genetic mutations, along with the inherent difficulties and risks associated with getting sufficient tumor samples via traditional biopsy methods. Key potential advantage of ctDNA are the detection of molecular residual disease (MRD) in patients post definitive treatment, and in the early identification of metastasis. This is particularly relevant contemporarily with the recent demonstration of tebentafusp improving survival in metastatic UM patients, and opens avenues for further research to investigate the potential utilization of tebentafusp combined with ctDNA-based strategies in adjuvant settings and early intervention for MRD. The present review illustrates the current understanding of ctDNA-based strategies in UM, discusses the potential clinical applications, explores the potential of utilizing ctDNA in UM MRD in the context of an ongoing clinical trial, and highlights the challenges that need to be overcome prior to routine clinical implementation.

Leptomeningeal disease (LMD) is associated with significant morbidity and mortality for metastatic non-small cell lung cancer (NSCLC). We describe our clinical experience in evaluating the use of cerebrospinal fluid (CSF)-derived circulating tumor cells (CTCs) for the diagnosis of LMD and the detection of genomic alterations in CSF cell-free DNA (cfDNA).

Patients with NSCLC who had CSF collection as part of routine clinical care for suspected LMD were included in the study. CSF was evaluated for CTCs and cfDNA using a commercial assay (CNSide; Biocept, San Diego, CA), and molecular profiling was performed. Molecular testing results from sequencing of tumor tissue and plasma circulating tumor DNA were collected. cMET and human epidermal growth factor receptor 2 (HER2) expression analysis was performed using fluorescence in situ hybridization (FISH).

Twenty-two patients were included (77% female; median age 60 years). Sixty-four percent had sensitizing EGFR mutations, and 32% had an atypical EGFR mutation. Thirteen of the 22 patients (59%) were diagnosed with LMD using the CSF CTC assay. Five of these 13 patients (38%) had negative CSF cytology for LMD, and two patients (15%) had normal magnetic resonance imaging brain imaging. Seven of the 13 patients (54%) had sufficient CTCs to perform molecular profiling. The concordance with tissue next-generation sequencing was 100%, and the driver mutation was identified in all seven patients with the CSF cfDNA assay. cMET expression and HER2 expression via FISH were noted in 11 patients (50%) and four patients (18%) respectively.

We detected higher sensitivity to diagnose LMD using CSF CTC-based assay; 38% of LMD cases identified using this assay were missed by standard CSF cytology. CSF molecular testing using CSF cfDNA demonstrated high concordance with tissue-based molecular testing.