Body fluids are constantly replenished with a population of genetically diverse cell-free DNA (cfDNA) fragments, representing a vast reservoir of information reflecting real-time changes in the host and metagenome. As many body fluids can be collected non-invasively in a one-off and serial fashion, this reservoir can be tapped to develop assays for the diagnosis, prognosis, and monitoring of wide-ranging pathologies, such as solid tumors, fetal genetic abnormalities, rejected organ transplants, infections, and potentially many others. The translation of cfDNA research into useful clinical tests is gaining momentum, with recent progress being driven by rapidly evolving preanalytical and analytical procedures, integrated bioinformatics, and machine learning algorithms. Yet, despite these spectacular advances, cfDNA remains a very challenging analyte due to its immense heterogeneity and fluctuation in vivo. It is increasingly recognized that high-fidelity reconstruction of the information stored in cfDNA, and in turn the development of tests that are fit for clinical roll-out, requires a much deeper understanding of both the physico-chemical features of cfDNA and the biological, physiological, lifestyle, and environmental factors that modulate it. This is a daunting task, but with significant upsides. In this review we showed how expanded knowledge on cfDNA biology and faithful reverse-engineering of cfDNA samples promises to (i) augment the sensitivity and specificity of existing cfDNA assays; (ii) expand the repertoire of disease-specific cfDNA markers, thereby leading to the development of increasingly powerful assays; (iii) reshape personal molecular medicine; and (iv) have an unprecedented impact on genetics research.

Plasma cell-free DNA (cfDNA) originates from various tissues and cell types and can enable minimally invasive diagnosis, treatment and monitoring of cancer and other diseases. Proper extraction of cfDNA is critical to obtain optimal yields and purity. The goal of this study was to compare the performance of six commercial cfDNA kits to extract pure, high-quality cfDNA from human plasma samples and evaluate the quantity and size profiles of cfDNA extracts—among them, two spin-column based, three magnetic bead-based and two automatic magnetic bead-based methods. Significant differences were observed in the yield of DNA among the different extraction kits (up to 4.3 times), as measured by the Qubit Fluorometer and Bioanalyzer. All kits isolated mostly small fragments corresponding to mono-nucleosomal sizes. The highest yield and reproducibility were obtained by the manual QIAamp Circulating Nucleic Acid Kit and automated MagNA Pure Total NA Isolation Kit. The results highlight the importance of standardizing preanalytical conditions depending on the requirements of the downstream applications.

Given the crucial role of mitochondria as the main cellular energy provider and its contribution towards tumor growth, chemoresistance, and cancer cell plasticity, mitochondrial DNA (mtDNA) could serve as a relevant biomarker. Thus, the profiling of mtDNA mutations and copy number variations is receiving increasing attention for its possible role in the early diagnosis and monitoring therapies of human cancers. This applies particularly to highly aggressive pancreatic cancer, which is often diagnosed late and is associated with poor prognosis. As current diagnostic procedures are based on imaging, tissue histology, and protein biomarkers with rather low specificity, tumor-derived mtDNA mutations detected from whole blood represents a potential significant leap forward towards early cancer diagnosis. However, for future routine use in clinical settings it is essential that preanalytics related to the characterization of mtDNA in whole blood are thoroughly standardized, controlled, and subject to proper quality assurance, yet this is largely lacking. Therefore, in this study we carried out a comprehensive preanalytical workup comparing different mtDNA extraction methods and testing important preanalytical steps, such as the use of different blood collection tubes, different storage temperatures, length of storage time, and yields in plasma vs. whole blood. To identify analytical and preanalytical differences, all variables were tested in both healthy subjects and pancreatic carcinoma patients. Our results demonstrated a significant difference between cancer patients and healthy subjects for some preanalytical workflows, while other workflows failed to yield statistically significant differences. This underscores the importance of controlling and standardizing preanalytical procedures in the development of clinical assays based on the measurement of mtDNA.

Epigenetic dysregulation is a hallmark of many human malignancies, with DNA methylation being a primary mechanism influencing gene expression and maintaining genomic stability. Genome-wide hypomethylation, characteristic of many cancers, is partly attributed to the demethylation of repetitive elements, including LINE-1, a prevalent non-LTR retrotransposon. The methylation status of LINE-1 is closely associated with overall genomic methylation levels in tumors. cfDNA comprises extracellular DNA fragments found in bodily fluids such as plasma, serum, and urine, offering a dynamic snapshot of the genetic and epigenetic landscape of tumors. This real-time sampling provides a minimally invasive avenue for cancer diagnostics, prognostics, and monitoring. The methylation status of LINE-1 in cfDNA has emerged as a promising biomarker, with several studies highlighting its potential in diagnosing and predicting outcomes in cancer patients. Recent research also suggests that cfDNA-based LINE-1 methylation analysis could serve as a valuable tool in evaluating the efficacy of cancer therapies, including immunotherapy. The growing clinical significance of cfDNA calls for a closer examination of its components, particularly repetitive elements like LINE-1. Despite their importance, the role of LINE-1 elements in cfDNA has not been thoroughly gauged. We aim to address this gap by reviewing the current literature on LINE-1 cfDNA assays, focusing on their potential applications in diagnostics and disease monitoring.

Pancreatic cancer (PC) is a highly aggressive malignancy characterized by limited opportunities for early diagnosis and poor clinical outcomes, underscoring the need for minimally invasive biomarkers to improve detection and patient stratification. Given emerging evidence that mitochondrial DNA (mtDNA) alterations reflect cancer-related biological processes, this study investigated whether blood-derived mtDNA profiles could provide clinically relevant information in PC. In this exploratory study, whole-blood mtDNA from 33 PC patients and 10 healthy individuals were analyzed using next-generation sequencing to assess single-nucleotide variants (SNVs), allele frequencies, and mtDNA copy number. A total of 252 unique mtDNA SNVs were identified, including variants exclusive to PC patients, variants unique to controls, and variants shared between groups. While the overall SNV burden did not differ significantly between groups, PC patients showed distinct mutation distributions and allele frequency patterns, with cancer-exclusive variants occurring predominantly at low allele frequencies. Mutation hotspots were observed in the ND5, COI, and D-loop regions, implicating genes involved in oxidative phosphorylation and mtDNA maintenance. Although mtDNA copy number did not differ significantly between groups, greater variability was observed among PC patients and was associated with differences in survival outcomes. Overall, these findings indicate that blood-based mtDNA profiling captures biologically relevant variation associated with PC and supports further development of integrated mtDNA-based approaches for improved risk assessment and patient stratification.

Background: Systematic studies providing differentiated insight into the contribution of immunity directed against conserved and non-conserved epitopes of SARS-CoV-2 Spike on long-term protection are rare and insufficiently guide future pan-variant vaccine research. The present observational cohort study aimed to evaluate the correlation of neutralizing antibody levels and cellular immunity against the Spike protein with symptomatic Omicron breakthrough infection. Methods: Neutralizing antibody levels against multiple (sub)variants were analyzed 6 months following the second wild-type mRNA vaccination and 6 months after booster in 107 subjects using a multiplex surrogate virus neutralization assay. To assess cellular immunity, cytokine mRNA expression levels were determined after peptide pool stimulation in whole blood samples of a study subgroup. Results: Neutralizing antibody titers were found to serve as a reasonably reliable correlate of protection prior to booster immunization. However, the predictive power of neutralizing antibody titers was diminished after boosting. This loss appears to be due to a critical remodeling of the antibody repertoire—a process that was dose-dependent on pre-boost humoral immunity. Vaccination against Omicron infection was most effective when a balanced immune response to both conserved and non-conserved epitopes of the viral Spike protein was induced. While neutralizing antibodies against receptor-binding domain epitopes affected by mutations were specifically associated with protection from symptomatic variant infection, cellular immunity was most effective when targeting conserved Spike epitopes. Conclusions: Optimal long-term protection against Omicron infection requires balanced immunity to both conserved and non-conserved epitopes of the viral Spike protein. The limited availability of cross-neutralizing antibodies targeting non-conserved epitopes and their inherently lower efficacy renders them a limiting factor as humoral immunity wanes over time. Future pan-SARS-CoV-2 variant vaccines that primarily target conserved epitopes may therefore provide less effective long-term protection against symptomatic variant infection than vaccines targeting a broader epitope spectrum including both conserved and non-conserved epitopes.