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Through various pathways of cell death, degradation, and regulated extrusion, partial or complete genomes of various origins (e.g., host cells, fetal cells, and infiltrating viruses and microbes) are continuously shed into human body fluids in the form of segmented cell-free DNA (cfDNA) molecules. While the genetic complexity of total cfDNA is vast, the development of progressively efficient extraction, high-throughput sequencing, characterization via bioinformatics procedures, and detection have resulted in increasingly accurate partitioning and profiling of cfDNA subtypes. Not surprisingly, cfDNA analysis is emerging as a powerful clinical tool in many branches of medicine. In addition, the low invasiveness of longitudinal cfDNA sampling provides unprecedented access to study temporal genomic changes in a variety of contexts. However, the genetic diversity of cfDNA is also a great source of ambiguity and poses significant experimental and analytical challenges. For example, the cfDNA population in the bloodstream is heterogeneous and also fluctuates dynamically, differs between individuals, and exhibits numerous overlapping features despite often originating from different sources and processes. Therefore, a deeper understanding of the determining variables that impact the properties of cfDNA is crucial, however, thus far, is largely lacking. In this work we review recent and historical research on active vs. passive release mechanisms and estimate the significance and extent of their contribution to the composition of cfDNA.

Background It was recently demonstrated that the size of cell-free DNA (cfDNA) fragments that originates from tumor cells are shorter than cfDNA fragments that originates from non-malignant cells. We investigated whether cfDNA fragment size and cfDNA levels might have prognostic value in patients with advanced pancreatic cancer. Methods Blood samples were obtained from patients with advanced pancreatic cancer, before (n = 61) initiation of chemotherapy and after the first cycle of chemotherapy (n = 39). Samples were separated with density centrifugation and plasma DNA was isolated. Mode cfDNA fragment size and cfDNA levels were then determined using a 2100 Bioanalyzer. A cohort of partially age-matched healthy volunteers (n = 28) constituted the control group. Results Both a pre-treatment cfDNA fragment size of ≤ 167 bp (mode) and high pre-treatment cfDNA levels were associated with shorter progression-free survival (PFS) (p = 0.002 and p < 0.001, respectively) and overall survival (OS) (p = 0.001 and p = 0.001, respectively). Furthermore, multivariable Cox regression analyses demonstrated that pre-treatment cfDNA levels could independently predict prognosis for both PFS (HR = 3.049, p = 0.005) and OS (HR = 2.236, p = 0.028). Conclusion This study demonstrates that cfDNA fragment size and cfDNA levels can be used to predict disease outcome in patients with advanced pancreatic cancer. The described approach, using a rapid, economic and simple test to reveal prognostic information, has potential for future treatment stratification and monitoring.

Background Cell-free circulating DNA (cfDNA) is becoming a useful biopsy for noninvasive diagnosis of diseases. Microbial sequences in plasma cfDNA may provide important information to improve prognosis and treatment. We have developed a stringent method to identify microbial species via microbial cfDNA in the blood plasma of early-onset breast cancer (EOBC) patients and healthy females. Empirically, microbe-originated sequence reads were identified by mapping non-human PE reads in cfDNA libraries to microbial databases. Those mapped concordantly to unique microbial species were assembled into contigs, which were subsequently aligned to the same databases. Microbial species uniquely aligned were identified and compared across all individuals on MCRPM (Microbial CfDNA Reads Per Million quality PE reads) basis. Results The predominant microbial cfDNAs in all plasma samples examined are originated from bacteria and these bacteria were limited to only a few genera. Among those, Acinetobacter johnsonii XBB1 and low levels of Mycobacterium spp. were commonly found in all healthy females, but also present in an EOBC patient. Compared to those in healthy counterparts, bacterial species in EOBC patients are more diverse and more likely to present at high levels. Among these three EOBC patients tested, a patient who has record high titer (2,724 MCRPM) of Pseudomonas mendocina together with 8.82 MCRPM of Pannonibacter phragmitetus has passed away; another patient infected by multiple Sphingomonas species remains alive; while the third patient who has similar microbial species ( Acinetobacter johnsonii XBB1) commonly seen in normal controls is having a normal life. Conclusions Our preliminary data on the profiles of microbial cfDNA sequences suggested that it may have some prognostic value in cancer patients. Validation in larger number of patients is warranted.

Background Urine holds promise as a source for cell‐free DNA (cfDNA) analysis of cancer genetics due to its nature as a self‐collectable biospecimen available in large quantities. However, pre‐analytical variables such as preservation of cfDNA or efficiency of up‐scaling specimen volume need to be better explored to increase analysis sensitivity. Patients and Methods Initially effects of pH levels on cfDNA stability of urine preserved with EDTA were investigated in three healthy probands. Furthermore, the efficiency of urine volume up‐scaling was examined using a simple DNA extraction method and cfDNA in urine of 32 individuals. Quantification was performed by PCR detection of three relevant targets used for EGFR and KRAS gene mutational analysis. Results Only samples preserved with EDTA at alkaline pH levels showed cfDNA stability of up to 10 days at room temperature. Moreover, it was found that increasing the volume up to 100 mL was highly efficient. A similar amount of copies was detected in three different gene sites in all specimens indicating both a good availability and a non‐random distribution pattern across genes. Since the median cfDNA copy number was 1642 copies/mL, abundance of cfDNA in this type of liquid biopsy is low. Conclusion Predictable sensitivities with different urine volumes on the ground of detectable cfDNA in our study population revealed that up‐scaling (>5 mL) is mandatory if the mutation allele frequency is less than 1%. Only at alkaline pH levels EDTA is a reliable preservative for cfDNA in urine. Moreover, it was found that urine volume up‐scaling (>5 mL) is mandatory if the mutation allele frequency is less than 1%. A similar amount of small‐sized cfDNA targets were detected in three different gene sites indicating a non‐random distribution pattern across genes.

Liquid biopsies are increasingly used for cancer molecular profiling that enables a precision oncology approach. Circulating extracellular nucleic acids (cell-free DNA; cfDNA), circulating tumor DNA (ctDNA), and circulating tumor cells (CTCs) can be isolated from the blood and other body fluids. This review will focus on current technologies and clinical applications for liquid biopsies. ctDNA/cfDNA has been isolated and analyzed using many techniques, e.g., droplet digital polymerase chain reaction, beads, emulsion, amplification, and magnetics (BEAMing), tagged-amplicon deep sequencing (TAm-Seq), cancer personalized profiling by deep sequencing (CAPP-Seq), whole genome bisulfite sequencing (WGBS-Seq), whole exome sequencing (WES), and whole genome sequencing (WGS). CTCs have been isolated using biomarker-based cell capture, and positive or negative enrichment based on biophysical and other properties. ctDNA/cfDNA and CTCs are being exploited in a variety of clinical applications: differentiating unique immune checkpoint blockade response patterns using serial samples; predicting immune checkpoint blockade response based on baseline liquid biopsy characteristics; predicting response and resistance to targeted therapy and chemotherapy as well as immunotherapy, including CAR-T cells, based on serial sampling; assessing shed DNA from multiple metastatic sites; assessing potentially actionable alterations; analyzing prognosis and tumor burden, including after surgery; interrogating difficult-to biopsy tumors; and detecting cancer at early stages. The latter can be limited by the small amounts of tumor-derived components shed into the circulation; furthermore, cfDNA assessment in all cancers can be confounded by clonal hematopoeisis of indeterminate potential, especially in the elderly. CTCs can be technically more difficult to isolate that cfDNA, but permit functional assays, as well as evaluation of CTC-derived DNA, RNA and proteins, including single-cell analysis. Blood biopsies are less invasive than tissue biopsies and hence amenable to serial collection, which can provide critical molecular information in real time. In conclusion, liquid biopsy is a powerful tool, and remarkable advances in this technology have impacted multiple aspects of precision oncology, from early diagnosis to management of refractory metastatic disease. Future research may focus on fluids beyond blood, such as ascites, effusions, urine, and cerebrospinal fluid, as well as methylation patterns and elements such as exosomes.

[This corrects the article DOI: 10.3389/fvets.2026.1768078.].

Keywords: cfDNA, Fragmentomics, cfDNA-FEP, Liquid biopsy, Early cancer detection, Renal cancer, Colorectal cancer

The detection of circulating tumor DNA (ctDNA) in liquid biopsy samples as an oncological marker is being used in clinical trials at every step of clinical management. As ctDNA-based liquid biopsy kits are developed and used in clinics, companies work towards increased convenience, accuracy, and cost over solid biopsies and other oncological markers. The technology used to differentiate ctDNA and cell-free DNA (cfDNA) continues to improve with new tests and methodologies being able to detect down to mutant allele frequencies of 0.001% or 1/100,000 copies. Recognizing this development in technology, the FDA has recently given pre-market approval and breakthrough device designations to multiple companies. The purpose of this review is to look at the utility of measuring total cfDNA, techniques used to differentiate ctDNA from cfDNA, and the utility of different ctDNA-based liquid biopsy kits using relevant articles from PubMed, clinicaltrials.gov, FDA approvals, and company newsletters. Measuring total cfDNA could be a cost-effective, viable prognostic marker, but various factors do not favor it as a monitoring tool during chemotherapy. While there may be a place in the clinic for measuring total cfDNA in the future, the lack of standardization means that it is difficult to move forward with large-scale clinical validation studies currently. While the detection of ctDNA has promising standardized liquid biopsy kits from various companies with large clinical trials ongoing, their applications in screening and minimal residual disease can suffer from lower sensitivity. However, researchers are working towards solutions to these issues with innovations in technology, multi-omics, and sampling. With great promise, further research is needed before liquid biopsies can be recommended for everyday clinical management.

We investigated whether DNA methylation and cell‐free DNA (cfDNA) fragmentation patterns can improve circulating tumor DNA (ctDNA) detection in advanced pancreatic cancer. In a cohort of 33 patients, ctDNA detection was performed in a tumor‐agnostic fashion using DNA methylation, cfDNA fragment lengths, and 4‐mer 5′ end motifs. Machine learning models estimating ctDNA levels were built for each individual detection method and their combination. All models significantly differentiated ctDNA levels in patients from healthy individuals (P < 0.001). Using the highest estimated levels in healthy volunteers as cutoffs, ctDNA was detected in 79%, 67%, 67%, and 55% of patients using methylation, fragment length, end motifs, and the combined model, respectively. Univariable Cox regression showed that all ctDNA level estimates were associated with increased hazard ratios (HR, all P < 0.001) for progression‐free survival (PFS) and overall survival (OS). Multivariable Cox regression confirmed ctDNA levels as an independent predictor of PFS (HR = 1.9, P < 0.001) and OS (HR = 2.7, P < 0.001). Our findings suggest that machine learning models based on DNA methylation, cfDNA fragment lengths, and cfDNA end motifs can estimate ctDNA levels and predict clinical outcomes in advanced pancreatic cancer. We evaluated circulating tumor DNA (ctDNA) detection in advanced pancreatic cancer using DNA methylation, cell‐free DNA fragment lengths, and 5′ end motifs. Machine learning models were trained to estimate ctDNA levels from each feature and their combination. All models significantly distinguished patients from healthy individuals, and the ctDNA level estimations could be used to stratify patients according to survival.

Background DNA methylation biomarkers in circulating cell-free DNA (cfDNA) have great clinical potential for cancer management. Most methods for DNA methylation analysis require bisulfite conversion, causing DNA degradation and loss. This is particularly challenging for cfDNA, which is naturally fragmented and normally present in low amounts. The aim of the present study was to identify an optimal combination of cfDNA isolation and bisulfite conversion kits for downstream analysis of DNA methylation biomarkers in plasma. Results Of the five tested bisulfite conversion kits (EpiJET Bisulfite Conversion Kit, EpiTect Plus DNA Bisulfite Kit (EpiTect), EZ DNA Methylation-Direct Kit, Imprint DNA Modification Kit (Imprint) and Premium Bisulfite Kit), the highest and lowest DNA yield and recovery were achieved using the EpiTect kit and the Imprint kit, respectively, with more than double the amount of DNA for the EpiTect kit. Of the three tested cfDNA isolation kits (Maxwell RSC ccfDNA Plasma Kit, QIAamp Circulating Nucleic Acid Kit (CNA) and QIAamp MinElute ccfDNA Mini Kit), the CNA kit yielded around twice as much cfDNA compared to the two others kits, although with more high molecular weight DNA present. When comparing various combinations of cfDNA isolation kits and bisulfite conversion kits, the CNA kit and the EpiTect kit were identified as the best-performing combination, resulting in the highest yield of bisulfite converted cfDNA from normal plasma, as measured by droplet digital PCR (ddPCR). As a proof of principle, this kit combination was used to process plasma samples from 13 colorectal cancer patients for subsequent ddPCR methylation analysis of BCAT1 and IKZF1 . Methylation of BCAT1 and/or IKZF1 was identified in 6/10 (60%) stage IV patients and 1/3 (33%) stage III patients. Conclusions Based on a thorough evaluation of five bisulfite conversion kits and three cfDNA isolation kits, both individually and in combination, the CNA kit and the EpiTect kit were identified as the best-performing kit combination, with highest DNA yield and recovery across a range of DNA input amounts. The combination was successfully used for detection of clinically relevant DNA methylation biomarkers in plasma from cancer patients.