Pancreatic cancer has become one of the most challenging malignant tumors. This type of cancer shows a high mortality paralleling morbidity and a rather low 5-year survival rate. Early diagnosis of the disease is the key to prevent and treat the pancreatic cancer. Cellular free DNA (cfDNA) methylation detection is a promising non-invasive method in early cancer screening, diagnosis, and prognostic monitoring. However, there are still shortcomings in developing fast, sensitive, low-cost, and quantitative methods for detecting target methylated DNA. Herein, new transcription activator-like effectors (TALEs)-mediated electrochemical sensor by rolling circle amplification (RCA)-assisted "silver-link" crossing electrode was developed for detecting DNA methylation of Ras association domain family 1 isoform A (RASSF1A) tumor suppressor genes (R-5mC). The TALEs-Ni magnetic beads could specifically recognize R-5mC and bind to target methylated DNA to trigger the RCA and catalytic hairpin assembly (CHA), thereby duplexes (H1-H2) were generated. By introducing H3 probe to cause CHA amplification and Exo III-assisted cyclic amplification strategy, large amounts of H2 probes were generated to trigger a new round of RCA reaction, long crossing DNA strands were formed between the gaps of electrodes. The "silver-link" crossing electrode was formed by metallization of "gene-link" and target was detected by recording this change in conductivity. The limit of detection (LOD) was 0.5 fM. The proposed electrochemical sensor showed good stability, high sensitivity and low detected background signal in the R-5mC detection. In addition, the R-5mC detection of cfDNA in plasma samples of cancer patients was satisfactory. This strategy is of great significance in the early screening, treatment and prognosis monitoring of pancreatic cancer.

Liquid biopsy encompasses a variety of molecular approaches to detect circulating tumor DNA (ctDNA) and has become a powerful tool in the diagnosis and treatment of solid tumors. Current applications include comprehensive genomic profiling for identifying targetable mutations and therapeutic resistance mechanisms, with emerging applications in minimal residual disease detection and treatment response monitoring. Increasingly, the potential for liquid biopsy in guiding treatment decisions is under active investigation through prospective clinical trials using ctDNA-adaptive interventions in patients with early-stage and metastatic cancers. Limitations arise on the basis of the sensitivity and feasibility of individual liquid biopsy assays; nonetheless, emerging technologies set the stage for improving these shortcomings. As the global oncology community continues to ascertain the clinical value of liquid biopsy across the continuum of patient care, this minimally invasive approach heralds a significant advancement in the promise of precision oncology.

Circulating tumour DNA (ctDNA)-based detection of molecular residual disease (MRD) presents a strategy to identify patients at high risk of relapse. In this article, we profile early breast cancer patients with an ultrasensitive, whole genome sequencing (WGS)-based, tumour-informed ctDNA platform.

We analysed 617 plasma samples (median 8, range 2-14) from 78 patients (23 triple-negative breast cancer, 35 human epidermal growth factor receptor 2-positive, 18 hormone receptor-positive, and 2 unknown). Samples were collected at diagnosis before therapy, cycle 2 of neoadjuvant chemotherapy, post-surgery after neoad'juvant therapy if administered, every 3 months during the first year, and every 6 months thereafter. Plasma DNA was analysed using the NeXT Personal MRD platform, a tumour-informed WGS approach to produce personalized ctDNA sequencing panels tracking a median of 1451 variants per patient. MRD detection was correlated with clinical outcomes.

ctDNA was detected at levels ranging from 2.19 parts per million (PPM) to 204 900 PPM (median 405 PPM), with 39% of all ctDNA detections in the ultra-low range <100 PPM. Of patients with samples at diagnosis, 98% (49/50) had ctDNA detected before treatment. At a median follow-up of 76 months (range 5-118 months), detection of ctDNA was associated with high risk of future relapse (P < 0.0001; log-rank test) and shortened overall survival (P < 0.0001) with a median lead time from ctDNA detection to clinical relapse of 15 months (range 0.9-61.5 months). MRD was identified in 100% (11/11) of patients who relapsed, with a median level of ctDNA at first MRD detection of 13.1 PPM. No ctDNA-undetected patients relapsed throughout follow-up (64/64). Comparison with exome-powered MRD detection assays showed improved sensitivity and lead time.

A whole genome-powered MRD assay detected breast cancer relapse with a long lead time over clinical relapse, and was strongly associated with relapse-free survival. Rates of ctDNA detection at diagnosis were higher than those reported with exome-based tumour-informed assays.

KRAS exon 2 mutations are highly prevalent in human malignancies, making them attractive targets for detection and monitoring in cell-free DNA (cfDNA) of cancer patients. Drop-off assays designed for digital polymerase chain reaction (ddPCR drop-off) span entire mutational hotspots and detect any mutated allele within the covered region, overcoming a major limitation of mutation-specific ddPCR assays. We therefore set out to develop a novel KRAS codon 12/13 ddPCR drop-off assay for the robust, highly sensitive and specific detection of KRAS exon 2 hotspot mutations in cfDNA.

We designed, optimized and extensively validated a KRAS codon 12/13 ddPCR drop-off assay. We compared assay performance to a commercially available KRAS multiplex assay. For clinical validation, we analyzed plasma samples collected from patients with KRAS-mutated gastrointestinal malignancies.

Limit of detection of the newly established ddPCR drop-off assay was 0.57 copies/µL, limit of blank was 0.13 copies/µ. The inter-assay precision (r2) was 0.9096. Our newly developed KRAS ddPCR drop-off assay accurately identified single nucleotide variants in 35/36 (97.2%) of circulating tumor DNA-positive samples from the patient validation cohort. Assay cross-validation showed that the newly established KRAS codon 12/13 ddPCR drop-off assay outperformed a commercially available KRAS multiplex ddPCR assay in terms of specificity. Moreover, the newly developed assay proved to be suitable for multiplexing with mutation-specific probes.

We developed and clinically validated a highly accurate ddPCR drop-off assay for KRAS exon 2 hot-spot detection in cfDNA with broad applicability for clinic and research.

Determining response to therapy for patients with pancreatic cancer can be challenging. We evaluated methods for assessing therapeutic response using cell-free DNA (cfDNA) in plasma from patients with metastatic pancreatic cancer in the CheckPAC trial (NCT02866383). Patients were evaluated before and after initiation of therapy using tumor-informed plasma whole-genome sequencing (WGMAF) and tumor-independent genome-wide cfDNA fragmentation profiles and repeat landscapes (ARTEMIS-DELFI). Using WGMAF, molecular responders had a median overall survival (OS) of 319 days compared to 126 days for nonresponders [hazard ratio (HR) = 0.29, 95% confidence interval (CI) = 0.11-0.79, P = 0.011]. For ARTEMIS-DELFI, patients with low scores after therapy initiation had longer median OS than patients with high scores (233 versus 172 days, HR = 0.12, 95% CI = 0.046-0.31, P < 0.0001). We validated ARTEMIS-DELFI in patients with pancreatic cancer in the PACTO trial (NCT02767557). These analyses suggest that noninvasive mutation and fragmentation-based cfDNA approaches can identify therapeutic response of individuals with pancreatic cancer.

Fragmentomics features of cell-free DNA represent promising non-invasive biomarkers for cancer diagnosis. A lack of systematic evaluation of biases in feature quantification hinders the adoption of such applications. We compare features derived from whole-genome sequencing of ten healthy donors using nine library kits and ten data-processing routes and validated in 1182 plasma samples from published studies. Our results clarify the variations from library preparation and feature quantification methods. We design the Trim Align Pipeline and cfDNAPro R package as unified interfaces for data pre-processing, feature extraction, and visualization to standardize multi-modal feature engineering and integration for machine learning.

Invasive central nervous system (CNS) cancers are an area where the development of breakthrough therapies is urgently needed. For instance, conditions such as glioblastoma multiforme (GBM) are associated with poor clinical prognosis, with the majority of trials offering no improvement to marginally enhanced survival. Unleashing the potential of targeting the immune system in CNS cancers has gained attention in recent years. Inhibition of immune checkpoints such as CTLA-4, PD-1/PD-L1, TIM-3, and LAG-3 has been attempted in recent trials. While potentially offering a notable edge over other immunotherapies, multi-organ adverse events have been found with the administration of immune checkpoint inhibitors (ICIs). The present review captures the state-of-the-art evidence on ICI treatments in different CNS cancers. Also, we discuss the value of combinational therapies involving ICIs as well as next-generation therapeutics such as bispecific antibodies targeting PD-1/LAG-3/TIM-3 and CRISPR-Cas9-edited PD-1-knock-out checkpoint-resistant CAR T-cells.

Diagnosing primary or secondary CNS lymphoma (CNSL) is a clinical challenge due to the limitations of standard biopsy and imaging procedures despite established guidelines. Therefore, accurate biomarkers and analytical methods that are convenient for practical routine use are needed to diagnose and manage these aggressive lymphomas effectively. We evaluated the utility of minimally invasive circulating tumor DNA (ctDNA) detection in a prospective real-world scenario, moving this approach closer to clinical practice.

A total of 164 plasma, cerebrospinal fluid (CSF), and tumor samples from 56 CNSL patients were collected to analyze tumor DNA by the diagnostic next-generation sequencing (NGS) panel LYNX, enabling simultaneous analysis of gene variants, chromosomal aberrations, and antigen receptor rearrangements in targeted regions.

The well-known genetic heterogeneity of CNSL was refined with integrative molecular data, showing the most frequent MYD88, PIM1, and KMT2D mutations and a broad spectrum of chromosomal aberrations, reflecting high genomic complexity. The multi-target approach achieved a substantially higher detection rate of CNS infiltration (90%) than tracking a single variant in gene MYD88 (46%). CSF clearly surpasses plasma if applying a routine (non-ultrasensitive) NGS approach and allows for more reliable evidence of CNS involvement than conventional flow cytometry (91% vs. 21%, p < 0.001). Parallel analysis of tumor DNA in both cell-free and cellular DNA from CSF makes the probability of primary or secondary CNS malignancy detection even higher.

Our prospective, tissue-agnostic approach highlights the feasibility of ctDNA sequencing by a commonplace and affordable method, offering higher sensitivity to detect CNS infiltration with lymphoma than standard cell-analyzing techniques. We accentuate the benefit of a multi-target NGS approach and adequate CSF sampling to obtain satisfactory diagnostic yield. Less invasive liquid biopsy testing by comprehensive NGS complements standard procedures in the diagnostics and management of CNSL patients, especially when encountering limitations.

Genomic analyses of cell-free DNA (cfDNA) in plasma are enabling noninvasive blood-based biomarker approaches to cancer detection and disease monitoring. Current approaches for identification of circulating tumour DNA typically use targeted tumour-specific mutations or methylation analyses. An emerging approach is based on the recognition of altered genome-wide cfDNA fragmentation in patients with cancer. Recent studies have revealed a multitude of characteristics that can affect the compendium of cfDNA fragments across the genome, collectively called the 'cfDNA fragmentome'. These changes result from genomic, epigenomic, transcriptomic and chromatin states of an individual and affect the size, position, coverage, mutation, structural and methylation characteristics of cfDNA. Identifying and monitoring these changes has the potential to improve early detection of cancer, especially using highly sensitive multi-feature machine learning approaches that would be amenable to broad use in populations at increased risk. This Review highlights the rapidly evolving field of genome-wide analyses of cfDNA characteristics, their comparison to existing cfDNA methods, and recent related innovations at the intersection of large-scale sequencing and artificial intelligence. As the breadth of clinical applications of cfDNA fragmentome methods have enormous public health implications for cancer screening and personalized approaches for clinical management of patients with cancer, we outline the challenges and opportunities ahead.

Differentiating sequencing errors from true variants is a central genomics challenge, calling for error suppression strategies that balance costs and sensitivity. For example, circulating cell-free DNA (ccfDNA) sequencing for cancer monitoring is limited by sparsity of circulating tumor DNA, abundance of genomic material in samples and preanalytical error rates. Whole-genome sequencing (WGS) can overcome the low abundance of ccfDNA by integrating signals across the mutation landscape, but higher costs limit its wide adoption. Here, we applied deep (~120×) lower-cost WGS (Ultima Genomics) for tumor-informed circulating tumor DNA detection within the part-per-million range. We further leveraged lower-cost sequencing by developing duplex error-corrected WGS of ccfDNA, achieving 7.7 × 10-7 error rates, allowing us to assess disease burden in individuals with melanoma and urothelial cancer without matched tumor sequencing. This error-corrected WGS approach will have broad applicability across genomics, allowing for accurate calling of low-abundance variants at efficient cost and enabling deeper mapping of somatic mosaicism as an emerging central aspect of aging and disease.

Circulating tumor DNA (ctDNA) has the potential to become a reliable biomarker for identifying minimal residual disease (MRD) and predicting recurrence in patients with non-small cell lung cancer (NSCLC) following curative treatment. However, there is a lack of studies that investigate the clinical validity of ctDNA using a tumor-agnostic approach, which can provide significant clinical benefits.

We analyzed samples from 45 NSCLC patients recruited in a prospective national multicenter study, all of whom had undergone curative treatment. A total of 38 pre-treatment plasma samples and 76 post-treatment plasma samples were examined using a commercially available cancer personalized profiling by deep sequencing (CAPP-seq) strategy, and a tumor-agnostic approach. Post-treatment samples were collected at two distinct landmark time points: Follow-up 1 (0.5-4.5 months post-treatment) and Follow-up 2 (4.5-7.5 months post-treatment).

Detectable ctDNA post-treatment was significantly associated with increased risk of tumor recurrence and shorter recurrence-free survival (RFS). Using only a single blood sample taken from Follow-up 2, we correctly identified MRD in 50% of the patients who later experienced recurrence. However, subgroup analysis further revealed that in patients treated with radiotherapy or chemoradiotherapy (CRT), ctDNA detection was significantly linked to shorter RFS in the MRD analysis from Follow-up 2, but not in the MRD analysis from Follow-up 1.

These findings suggest that post-treatment ctDNA, detected using a tumor-agnostic approach, is a reliable biomarker for predicting recurrence in NSCLC patients following curative treatment. However, the optimal timing for blood sampling to detect MRD appears to depend on the type of curative treatment received.

In stage 2-3 colon cancer (CC), postsurgery circulating tumor DNA (ctDNA) assessment is crucial for guiding adjuvant chemotherapy (ACT) decisions. While existing assays detect ctDNA and help identify high-risk persons with CC for recurrence, their limited sensitivity after surgery poses challenges in deciding on ACT. Additionally, a substantial portion of persons with CC fail to clear ctDNA after ACT, leading to recurrence. In this study, we performed whole-exome sequencing (WES) of ctDNA at different time points in participants with relapsed CC in two independent cohorts, alongside transcriptomic and proteomic analyses of metastases, to enhance comprehension of progression mechanisms. A plasma WES-based tumor-agnostic assay demonstrated higher sensitivity in detecting minimal residual disease (MRD) compared to current assays. Immune evasion appears to be the primary driver of progression in the localized CC setting, indicating the potential efficacy of immunotherapy for microsatellite stability in persons with CC. Organoid modeling further supports the promising potential of targeted therapy in eradicating MRD, surpassing conventional treatments.

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.

Precision medicine based on biomarkers, such as genetic abnormalities and PD-L1 expression, has been established for the treatment of nonsmall cell lung cancer. Recently, liquid biopsy has emerged as a valuable and minimally invasive alternative. This method analyzes blood and other bodily fluids to detect cancer-related genetic abnormalities and molecular residual disease (MRD). Liquid biopsy, which includes testing for circulating tumor cells, circulating tumor DNA (ctDNA), and microRNA (miRNA), offers several advantages over conventional methods. It is minimally invasive, can be performed repeatedly, and provides crucial information for early cancer diagnosis, genotyping, and treatment monitoring. Elevated ctDNA levels and miRNA markers show promise for early diagnosis. Liquid biopsy complements traditional tissue biopsy during genotyping, particularly when tumor samples are insufficient. Tests such as Cobas® EGFR Mutation Test v2 and Guardant360® CDx have been shown to be effective in detecting genetic mutations and guiding treatment decisions. Although the accuracy of liquid biopsy is still lower than that of tissue biopsy, its clinical utility continues to improve. For cancer prediction recurrence and treatment monitoring, ctDNA analysis can detect MRD earlier than conventional imaging, offering potential benefits for treatment adjustment and early relapse detection. The continuous development and validation of liquid biopsy methods are essential for improving personalized lung cancer treatment strategies.

Liquid biopsies, indicating the sampling of body fluids rather than solid-tissue biopsies, have the potential to revolutionize cancer care through personalized, noninvasive disease detection and monitoring. Circulating tumour DNA (ctDNA) and circulating tumour cells (CTCs) are promising blood-based biomarkers in bladder cancer. Results from several studies have shown the clinical potential of ctDNA and CTCs in bladder cancer for prognostication, treatment-response monitoring, and early detection of minimal residual disease and disease recurrence. Following successful clinical trial evaluation, assessment of ctDNA and CTCs holds the potential to transform the therapeutic pathway for patients with bladder cancer - potentially in combination with the analysis of urinary tumour DNA - through tailored treatment guidance and optimized disease surveillance.

Shallow genome-wide cell-free DNA sequencing holds great promise for noninvasive cancer monitoring by providing reliable copy number alteration (CNA) and fragmentomic profiles. Single-nucleotide variations (SNVs) are, however, much harder to identify with low sequencing depth due to sequencing errors. Here, we present Nanopore Rolling Circle Amplification (RCA)-enhanced Consensus Sequencing (NanoRCS), which leverages RCA and consensus calling based on genome-wide long-read nanopore sequencing to enable simultaneous multimodal tumor fraction (TF) estimation through SNVs, CNAs, and fragmentomics. The efficacy of NanoRCS is tested on 18 cancer patient samples and seven healthy controls, demonstrating its ability to reliably detect TFs as low as 0.24%. In vitro experiments confirm that SNV measurements are essential for detecting TFs below 3%. NanoRCS provides an opportunity for cost-effective and rapid sample processing, which aligns well with clinical needs, particularly in settings where quick and accurate cancer monitoring is essential for personalized treatment strategies.

There is much debate regarding optimal selection in patients with metastatic cancer who should undergo local treatment (surgery or radiation treatment) to the primary tumor and/or metastases. Additionally, the optimal treatment of newly diagnosed metastatic cancer is largely unclear. Current prognostication systems to best inform these clinical scenarios are limited, as all metastatic patients are grouped together as having Stage IV disease without further incorporation of patient and disease-specific covariates that significantly impact patient outcomes. Therefore, improving current prognostic scoring systems and incorporation of these covariates is essential to best individualize treatment for patients with metastatic cancer. In this narrative review article, we provide a detailed review of prognostication systems that can be used for both the site of metastasis and primary site to best tailor treatment in these patients. Additionally, we discuss the incorporation and ongoing developments in radiographic, genomic, and biostatistical techniques that can be used as prognostication tools.

Acute myeloid leukemia (AML) is a genetically heterogeneous disease with high rates of relapse after initial treatment. Identifying measurable residual disease (MRD) following initial therapy is essential to assess response, predict patient outcomes, and identify those in need of additional intervention. Currently, MRD analysis relies on invasive, serial bone marrow (BM) biopsies, which complicate sample availability and processing time and negatively impact patient experience. Additionally, finding a positive result can generate more questions than answers, causing anxiety for both the patient and the provider. Peripheral blood (PB) evaluation has shown promise in detecting MRD and is now recommended by the European Leukemia Net for AML for certain genetic abnormalities. PB-based sampling allows for more frequent testing intervals and better temporal resolution of malignant expansion while sparing patients additional invasive procedures. In this review, we will discuss the current state of PB testing for MRD evaluation with a focus on next-generation sequencing methodologies that are capable of MRD detection across AML subtypes.

Background: Circulating tumor DNA (ctDNA) may be released from neoplastic cells into biological fluids through apoptosis, necrosis, or active release. In patients with non-small-cell lung cancer (NSCLC), ctDNA analysis is being introduced in clinical practice only for advanced disease management. Nevertheless, an interesting and promising field of application is the analysis of ctDNA in the management of early stage non-small-cell lung cancer, both for evaluation before treatment, such as diagnosis and screening, and for prediction of histology or pathological features. Methods: A thorough review of the literature published between 2000 and 2024 was performed on PubMed, utilizing the advanced search feature to narrow down titles and abstracts containing the following keywords: ctDNA, early stage, and NSCLC. A total of 20 studies that met all inclusion criteria were chosen for this review. Results: In this review, we summarize the increasing evidence suggesting that ctDNA has potential clinical applications in the management of patients with early stage NSCLC. ctDNA levels in early stage cancers are very low, posing many technical challenges in improving the detection rate and sensitivity, especially in clinical practice, if it is to be implemented for early detection. Presently, the main limitation of ctDNA experimental and clinical studies, especially in early stage settings, is the lack of definitive standardization and consensus regarding methodology, the absence of systematically validated analyses, and the lack of adoption of sensitive approaches. Conclusions: Possible applications of this analyte open up new fields of diagnosis, treatment, and follow up, which are less invasive and more precise than other approaches currently in use, especially in early stage NSCLC patients.

In recent years, significant advancements have been achieved in the treatment of non-small cell lung cancer (NSCLC), leading to prolonged patient survival; however, a subset of NSCLC patients may experience recurrence or distant metastasis following initial successful treatment. This phenomenon may be attributed to the presence of minimal residual disease (MRD) that remains undetectable by conventional imaging or laboratory techniques post-treatment. The potential sources of tumor recurrence (MRD), are significantly associated with adverse patient prognosis; therefore, the monitoring of these lesions is critically important in the management of NSCLC. This review seeks to examine the current evidence regarding the application of MRD in NSCLC clinical practice, as well as the challenges encountered in its role as a biomarker.

We performed a narrative review by systematically searching the PubMed and Web of Science databases for pertinent literature published from 2005 to 2024, with the objective of identifying significant literature related to clinical research and detection techniques for MRD in NSCLC.

The detection of circulating tumor DNA (ctDNA) for MRD has emerged as a significant focus in high-sensitivity genetic testing for monitoring NSCLC. This method may facilitate the assessment of recurrence risk in NSCLC and inform clinical decision-making to identify high-risk patients who are likely to benefit from treatment, thereby providing a rationale for treatment escalation or de-escalation. Nevertheless, the clinical application of ctDNA MRD continues to encounter several challenges, among which improving detection sensitivity and selecting the best detection timing are urgent issues that need to be addressed.

ctDNA MRD testing offers robust evidence to assist clinicians in the early identification of NSCLC recurrence and in guiding clinical treatment. We recommend integrating ctDNA MRD with traditional biomarkers and imaging modalities for a comprehensive evaluation aiming at optimizing treatment strategies.

The introduction of bispecific antibodies (BsAbs) has led to significant improvements in survival for patients with relapsed and refractory B-cell lymphomas. Despite these advances, there remains a significant number of patients who experience disease progression after these novel therapies. Predicting which patients may respond to certain treatments and the durability of their responses remains challenging. Measurable residual disease (MRD) has become easier to detect and quantify through the use of genomic next-generation sequencing tools and has been studied as a possible biomarker to predict long-term outcomes and risk-stratify patients after BsAb therapy in several lymphoma subtypes. Here, we review recent data demonstrating that MRD negativity is associated with radiographic response and improved progression-free survival. Because of heterogeneity in assay choice, assessment timing, and technical parameters, further work is needed before MRD testing is ready to be incorporated into clinical practice in the context of BsAb treatment for B-cell lymphomas.

Aggressive variant and androgen receptor (AR)-independent castration resistant prostate cancers (CRPC) represent the most significant diagnostic and therapeutic challenges in prostate cancer. This study examined a case of simultaneous progression of both adenocarcinoma and squamous tumors from the same common origin. Using whole-genome and transcriptome sequencing from 17 samples collected over >6 years, we established the clonal relationship of all samples, defined shared complex structural variants, and demonstrated both divergent and convergent evolution at AR. Squamous CRPC-associated circulating tumor DNA was identified at clinical progression prior to biopsy detection of any squamous differentiation. Dynamic changes in the detection rate of histology-specific clones in circulation reflected histology-specific sensitivity to treatment. This dataset serves as an illustration of non-neuroendocrine transdifferentiation and highlights the importance of serial sampling at progression in CRPC for the detection of emergent non-adenocarcinoma histologies with implications for the treatment of lineage plasticity and transdifferentiation in metastatic CRPC.

Soft tissue sarcomas (STSs) constitute a group of tumors with heterogeneous alterations and different biological behavior. Genetic profiling techniques have immense potential to revolutionize sarcoma classification, detection, and treatment. Cell-free DNA (cfDNA) analysis offers a minimally invasive approach to profiling tumor alterations, including tracking specific mutations or targeted panels of cancer-related genes via DNA sequencing methods. Circulating tumor DNA (ctDNA) platforms have gained popularity as a noninvasive alternative to tissue biopsies, offering a less invasive approach to tumor profiling. Nonetheless, ctDNA profiling in concordance with standard solid tumor comprehensive genomic profiling (CGP) is poorly characterized for STSs. Ultra-low-pass whole-genome sequencing and whole exome sequencing of cfDNA have yet to be fully leveraged in patients with sarcomas. This comprehensive review provides an overview of the application of ctDNA in STSs.

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.

Outcomes under anti-PD-(L)1 therapy have been variable in advanced non-small cell lung cancer (NSCLC) without reliable predictive biomarkers so far. Targeted next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) has demonstrated potential clinical utility to support clinical decisions, but requires prior tumor genetic profiling for proper interpretation, and wide adoption remains limited due to high costs.

Tumor-agnostic low-coverage ctDNA whole genome sequencing (lcWGS) was used to longitudinally track genome-wide copy number variations (CNVs) and fragmentation features in advanced NSCLC patients (n = 118 samples from 49 patients) and healthy controls (n = 57). Tumor PD-L1 expression was available for comparison.

Fragmentation features and CNVs were complementary indicators, whose combination significantly increased ctDNA detection compared to single-marker assessments (+ 20.3% compared to CNV analysis alone). Baseline fragment length alterations, but not CNVs, were significantly associated with subsequent progression-free survival (PFS; hazard ratio [HR] = 4.10, p = 6.58e-05) and could improve PFS predictions based on tumor PD-L1 expression alone (HR = 2.70, p = 0.019). Residual CNVs or aberrant fragmentation of ctDNA under ongoing therapy could stratify patients according to the subsequent response duration (median 5.8 vs. 47.0 months, p = 1.13e-06). The integrative analysis of ctDNA fragment characteristics at baseline, tumor PD-L1 expression, and residual ctDNA under ongoing treatment constituted the strongest independent predictor of PFS (p = 6.25e-05) and overall survival (p = 1.3e-03) in multivariable analyses along with other clinicopathologic variables.

This study demonstrates the feasibility and potential clinical utility of lcWGS for the tumor-agnostic stratification and monitoring of advanced NSCLC under PD-(L)1 blockade based on CNV and fragmentomic profiling.

Circulating tumor DNA (ctDNA) analysis has emerged as a minimally invasive tool for detecting minimal residual disease (MRD) in colorectal cancer (CRC) patients. This enables dynamic risk stratification, earlier recurrence detection and optimized post-surgical treatment. Two primary methodologies have been developed for ctDNA-based MRD detection: tumor-informed strategies, which identify tumor-specific mutations through initial tissue sequencing to guide ctDNA monitoring, and tumor-agnostic approaches, which utilize predefined panels to detect common cancer-associated genomic or epigenomic alterations directly from plasma without prior tissue analysis. The debate over which is superior in terms of sensitivity, specificity, cost-effectiveness and clinical feasibility remains unsolved.

This review summarizes studies published up to November 2024, exploring the utility and performance of tumor-informed and tumor-agnostic approaches for ctDNA analysis in CRC. We evaluate the strengths and limitations of each methodology, focusing on sensitivity, specificity and clinical outcomes.

Both strategies demonstrate clinical utility in post-operative risk stratification and guiding adjuvant chemotherapy decisions in CRC patients. Tumor-informed approaches generally exhibit superior sensitivity and specificity for recurrence prediction, attributed to their personalized tumor profile designs. However, these methods are limited by the need for prior tissue sequencing and higher associated costs. In contrast, tumor-agnostic approaches offer broader applicability due to their reliance on plasma-only analysis, although with relatively lower sensitivity. Technological advancements, including fragmentomics and multi-omic integrations, are expanding the capabilities of ctDNA-based MRD detection, enhancing the performance of both approaches.

While tumor-informed strategies currently offer higher precision in MRD detection, tumor-agnostic approaches are gaining traction due to their convenience and improving performance metrics. The integration of novel technologies in ongoing clinical trials may redefine the optimal approach for MRD detection in CRC, paving the way for more personalized and adaptive patient management strategies.

Colorectal cancer (CRC) remains a significant health concern in the world. The existing standard of care guidelines for CRC surveillance fall short of effectively and timely detecting recurrence or metastasis.

In recent years, circulating tumor DNA (ctDNA) has emerged as a promising material for minimal residual disease (MRD) detection. In this article, we provide an exhaustive review of the methods utilized for MRD detection via ctDNA, present evidence supporting the potential of ctDNA MRD as a valuable biomarker in clinical applications, and engage in a discussion regarding ongoing ctDNA MRD-based clinical trials in CRC. Finally, we offer insights into future prospects of ctDNA-based MRD methodological advancements and clinical research.

It is foreseeable that more sensitive, flexible, and economical MRD detection methods will emerge with the deeper research on cell-free DNA (cfDNA) genomics, fragmentomics, methylomes, and nucleosome imprinting. At the same time, MRD-guided intervention studies will evolve for revolutionizing the treatment paradigm of CRC.

Circulating tumour DNA (ctDNA) can be released by cancer cells into biological fluids through apoptosis, necrosis or active release. In patients with non-small-cell lung cancer (NSCLC), ctDNA levels correlate with clinical and pathological factors, including histology, tumour size and proliferative status. Currently, ctDNA analysis is recommended for molecular profiling in patients with advanced-stage NSCLC. In this Review, we summarize the increasing evidence suggesting that ctDNA has potential clinical applications in the management of patients with early stage and locally advanced NSCLC. In those with early stage NSCLC, detection of ctDNA before and/or after surgery is associated with a greater risk of disease recurrence. Longitudinal monitoring after surgery can further increase the prognostic value of ctDNA testing and enables detection of disease recurrence earlier than the assessment of clinical or radiological progression. In patients with locally advanced NSCLC, the detection of ctDNA after chemoradiotherapy is also associated with a greater risk of disease progression. Owing to the limited number of patients enrolled and the different technologies used for ctDNA testing in most of the clinical studies performed thus far, their results are not sufficient to currently support the routine clinical use of ctDNA monitoring in patients with early stage or locally advanced NSCLC. Therefore, we discuss the need for interventional studies to provide evidence for implementing ctDNA testing in this setting.

Molecular residual disease detected by circulating tumor DNA (ctDNA) has been reported to be predictive of patients' outcomes in various types of cancers after curative intent treatment. Nevertheless, additional detailed information regarding the association of longitudinal ctDNA detection with long-term follow-up in lung cancer is needed. Here, we report on a cohort of patients with NSCLC who underwent definitive surgery and ctDNA analysis in the pre-operative, adjuvant, and surveillance settings.

Plasma samples were collected from 46 patients with clinical stage II-III NSCLC before surgery (n = 46), after surgery (n = 45), and every six months until two years thereafter (n = 78). A clinically validated, personalized, tumor-informed 16-plex polymerase chain reaction-next-generation sequencing assay was used for the detection and quantification of ctDNA in retrospectively analyzed plasma samples.

Circulating tumor DNA was detected in the first postoperative (within 51 days after surgery) plasma samples in 13% (6/45) of patients (landmark analysis). All of them had disease recurrence within a median of 9.1 months. These patients had shorter recurrence-free and overall survivals than those without detectable ctDNA at a landmark time point (p < 0.01) and in multivariate analyses (p < 0.03). Longitudinally (considering all postoperative follow-up time points), ctDNA was detected in 13 patients, all of whom experienced disease recurrence (positive predictive value = 100%). Three patients who had central nervous system-only metastases did not have detectable ctDNA.

The presence of ctDNA post-surgery or during surveillance identifies patients with NSCLC at high risk of recurrence. Serial testing is important to detect disease recurrence earlier (lead-time: 3.2 months).