In the era of personalized and precision therapies, where clinical trials are increasingly tailored to enhance therapeutic efficacy, disease detection and monitoring strategies should also evolve to embrace a more personalized approach, aiming to improve diagnostic specificity and sensitivity. Liquid biopsy has emerged as one of the most promising and innovative techniques in this context, offering a minimally invasive method for diagnosing and monitoring oncological diseases [16]. By analyzing circulating tumor cells, cfDNA, RNA, and other biomarkers in bodily fluids such as blood, liquid biopsy provides real-time insights into the molecular profile of tumors. This approach not only facilitates early detection and monitoring of disease progression but also enables the identification of actionable genetic alterations, paving the way for targeted therapies. Its dynamic adaptability aligns perfectly with the needs of precision medicine, addressing inter- and intra-tumor heterogeneity and overcoming the limitations of traditional tissue biopsies.

For hematological diseases, including leukemias, lymphomas, and myelomas, liquid biopsy is particularly relevant due to its ability to provide dynamic, non-invasive insights into disease status. While traditional techniques like bone marrow or lymph node biopsies are informative, they are limited by their invasive nature and challenges in continuous disease monitoring [17]. The measurement of cancer-specific cfDNA variants in the blood is currently considered the clinical standard for several hematological malignancies, but reaching a clinically adequate sensitivity and specificity might be problematic, and the concomitant presence of clonal hematopoiesis might be misleading [18, 19].

Despite the “circulating” nature of many hematological neoplasms, data on cfDNA in this setting remain sparse. First identified by Mandel and Metais in 1948, cfDNA refers to short fragments of double-stranded DNA found in the blood of healthy individuals [20]. These fragments are released from cells undergoing apoptosis or necrosis, as well as from tissues damaged by ischemia, trauma, infection, or inflammation [21]. In cancer, apoptotic or necrotic tumor cells are processed by phagocytes, which release small DNA fragments into circulation [22].

The analysis of cfDNA has become a promising non-invasive approach in cancer diagnosis. Its diagnostic and prognostic potential was demonstrated as early as 1977, when a study showed that patients with various neoplasms had elevated initial cfDNA levels that decreased by up to 90% after radiotherapy [4]. A recent study by Mattox et al. in solid cancer patients suggested that cfDNA could serve as an early detection method for preclinical neoplasms, with 83% of patients showing higher cfDNA levels compared to healthy controls [12].

In our study, we conducted a comprehensive analysis of cfDNA levels in a cohort of 98 patients with onco-hematological diseases and 80 healthy donors, evenly distributed by sex. Among the key findings, the association between cfDNA levels and sex is particularly intriguing. We confirmed significantly higher cfDNA concentrations in cancer patients compared to healthy individuals, consistent with previous reports. Notably, men exhibited significantly higher cfDNA levels than women, suggesting that sex-related differences in cfDNA are not solely disease-dependent but may also reflect inherent biological or physiological variations influencing cfDNA production, release, or clearance. This observation aligns with previous evidence, such as the study by Cherubini et al., which highlighted the regulatory role of estrogens in gene expression related to metabolic processes, pointing toward a potential hormonal basis for sex-specific differences in circulating DNA [23]. These results emphasize that the difference in cfDNA concentration between sexes is not solely linked to the presence of disease but may reflect an intrinsic biological distinction in the production, release, or turnover of cfDNA into the bloodstream. This difference may be attributable to biological and physiological variations between sexes.

This aspect remains underexplored in scientific literature and suggests that sex should be considered as an important variable in comparative analyses between patients and control groups. We further investigated the correlation between age and plasma cfDNA levels, hypothesizing that aging—due to associated biological processes like increased cell death and DNA damage accumulation—could lead to higher cfDNA concentrations. However, our statistical analysis revealed a non-significant correlation, suggesting that age is not likely to be a major determinant of cfDNA levels. This finding is consistent with some previous reports [24,25,26], but contrasts with other studies that have described an age-related increase in cfDNA among healthy individuals, as recently reviewed by Tessier et al. [27]. Several factors may account for this discrepancy. First, the relatively small sample size in our study may have limited the statistical power to detect subtle associations. More importantly, our analysis did not account for smoking status, medical comorbidities, medication use, and levels of physical activity. These factors have been shown to impact cfDNA concentrations even in otherwise healthy populations and may mask or mimic age-related effects. Furthermore, our study population included patients with hematological malignancies, where cfDNA levels are likely driven predominantly by disease-related mechanisms, such as tumor burden, inflammation, and cell turnover, potentially overriding baseline age-related differences. Taken together, these considerations highlight the need for future studies with larger, well-characterized cohorts that incorporate relevant clinical and lifestyle variables to fully elucidate the relationship between age and cfDNA dynamics in both healthy and diseased states.

Over the past two decades, cfDNA has been hypothesized to primarily originate from cell death processes, particularly apoptosis and necrosis [28]. Early studies using less sophisticated techniques like electrophoresis identified mononucleosome fragments (150–180 bp) typical of apoptotic DNA degradation [6]. Necrosis, on the other hand, produces larger cfDNA fragments (> 10,000 bp) due to chaotic cell membrane rupture [2]. However, recent studies suggest that cfDNA release results from a complex combination of mechanisms, including apoptosis, cellular senescence, and active processes such as NETosis [29]. This enriches the interpretive framework beyond the traditional view attributing cfDNA release predominantly to apoptosis.

A critical aspect in this context is the role of neutrophils and NETosis in cfDNA production. Neutrophil extracellular traps (NETs) are complex chromatin structures released by neutrophils in response to infection or inflammation and contribute to the cfDNA pool upon degradation. This phenomenon is particularly significant in oncology. Studies, such as one by Paunel-Görgülü et al. [30], indicate that cfDNA can promote NET formation, creating a vicious cycle where free DNA further activates neutrophils, amplifying inflammation and immune responses. In cancer patients, particularly those with metastatic colorectal cancer, a significant portion of cfDNA may originate from NETosis and activated neutrophils rather than solely apoptotic or necrotic tumor cells [31, 32].

Emerging evidence suggests a possible correlation between citrullinated histone H3 (H3Cit) levels and the concentration of cfDNA, suggesting a potential interplay in inflammatory and pathological processes [33]. NETs consist of nuclear DNA mixed with proteins and granular components, linking neutrophil activity to cfDNA presence. Elevated H3Cit is a hallmark of NET formation and is associated with tissue damage and systemic inflammation. These significant findings support the hypothesis that NETs might be a key source of cfDNA, highlighting the potential role of neutrophil activation in cfDNA release in pathological contexts. In our analysis, we investigated the origin of cfDNA in patient samples by measuring plasma levels of H3Cit, a known marker of NET formation in experimental models of thrombosis and sepsis [34]. Citrullination of histone H3, mediated by the PAD4 enzyme, leads to chromatin decondensation and NET formation [35]. Despite a smaller-than-anticipated availability of plasma samples, we found a direct correlation between H3Cit levels and cfDNA concentrations in onco-hematological patients. These results support the hypothesis that NETs represent a significant source of cfDNA, offering new perspectives on their pathological role and clinical impact in hematological malignancies.

Recent advances have highlighted the complexity of NET formation, distinguishing between suicidal and vital NETosis [36]. Suicidal NETosis, a lytic form of cell death induced by strong stimuli such as PMA or bacterial infections, depends on reactive oxygen species (ROS) generation and culminates in the rupture of the neutrophil membrane with the release of chromatin fibers decorated with granular enzymes. In contrast, vital NETosis allows neutrophils to extrude nuclear or mitochondrial DNA while maintaining cellular integrity and functional activity. It can occur rapidly in response to different stimuli, including platelets or bacterial components, without inducing cell death. In hematological malignancies, where inflammation, infection, and aberrant immune activation are common, both pathways may be active. Suicidal NETosis likely releases larger amounts of cfDNA due to cell lysis, whereas vital NETosis may result in lower but more sustained cfDNA release. Understanding this balance may offer insight into the dynamics of cfDNA release in patients with hematologic cancers and could have implications for biomarker development or therapeutic targeting.

Our study has several limitations that should be acknowledged. First, the relatively small sample sizes, particularly in subgroup analyses, may limit the statistical power and generalizability of our findings. We also recognize that we did not account for potential confounders such as smoking status, comorbidities, medication use, or physical activity, all of which have been shown to affect cfDNA concentrations and could impact data interpretation [37,38,39]. Another important limitation is the use of only one method for cfDNA quantification and one surrogate marker (H3Cit) for NET detection. While these approaches are widely used and validated, the inclusion of additional techniques—such as qPCR-based cfDNA quantification or immunoassays targeting NE-DNA and MPO-DNA complexes [40]—would have strengthened our conclusions.

In conclusion, these findings suggest that cfDNA levels and NET markers such as H3Cit deserve further investigation as potential biomarkers for cancer diagnosis and prognosis in hematological malignancies. The combined analysis of cfDNA and NET markers, which requires a minimal budget and avoids the need for expensive sequencing technologies, may provide valuable insights for monitoring patients during therapies and controlling tumor progression, enhancing our understanding of immune system interactions and homeostasis in cancer patients.

This study underscores the multifaceted role of cfDNA in hematologic malignancies, reflecting disease burden, inflammatory processes, and potential sex-based biological differences. While cfDNA holds significant promise as a biomarker, challenges such as pre-analytical variability, the lack of standardization, and the need for large-scale validation studies remain. Future investigations should focus on the integration of cfDNA analysis with other biomarkers, longitudinal studies to monitor cfDNA dynamics, and exploration of its utility in guiding therapeutic decisions. By addressing these gaps, cfDNA could evolve into a pivotal tool in precision medicine for hematologic malignancies.