Search Results (96)

Cell-free DNA (cfDNA) analysis-based liquid biopsy is a rapidly emerging diagnostic and prognostic tool in pancreatic ductal adenocarcinoma (PDAC). KRAS point mutations are the main biomarkers used for the detection of tumor cfDNA. However, there is another less studied yet frequent genetic alteration in this gene, namely copy number gain (CNG). The aim of this study was to evaluate the diagnostic and prognostic potential of KRAS CNG analysis in plasma and bile of patients with PDAC using ddPCR. This study included healthy volunteers (n = 69), patients with PDAC (n = 94), and other pancreatic neoplasms (OPN) (n = 17). The sensitivity and specificity of KRAS CNG compared to the control group were 16% and 100% (AUC-ROC—0.580), and compared to the OPN group, 16% and 94% (AUC-ROC—0.554), respectively. Addition of KRAS point mutations to the analysis increased the sensitivity to 65% (AUC-ROC—0.824 and 0.801, respectively). Bile exhibited an equal KRAS CNG detection rate compared to plasma (20% vs. 16%). KRAS CNG was not associated with clinical parameters, except prognosis. The probability of survival was worse in patients with KRAS CNG (HR—3.54; 95% CI: 1.55–8.12; p = 0.001). KRAS CNG in cfDNA might be a promising biomarker for both diagnostic and prognostic purposes in PDAC. Full article

Background: Theophylline, which is biologically important and found in tea, coffee, and cocoa beans, can be synthesized chemically or by direct extraction and concentration from natural sources. Theophylline derivatives have garnered attention in recent years for their potential therapeutic effects on Mycobacterium tuberculosis, antihistaminic, anti-inflammatory, and anticancer. Also, trifluoromethyl (CF₃) group has also been widely used in drug and agrochemical design. Methods: In this study, a series of new theophylline derivatives containing substituted trifluoromethyl and trifluoromethoxy groups were synthesized. The structures of these new compounds were confirmed by NMR, FT-IR, and elemental analyses. Additionally, the anticancer activities of the molecules were analyzed against VEGFR-2, CYP P450, and estrogen receptor by molecular docking method. Furthermore, in vitro biological effects of the compounds were comprehensively evaluated in cancer (A549 and HeLa) and normal (BEAS-2B) cells. Cell viability was assessed by MTT assay, and selectivity index (SI) values were calculated to determine tumor-specific toxicity. Results: N(7)-substituted theophyllines were prepared by the reaction of 1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione (theophylline) and trifluoromethyl substituted benzyl halide compounds. The synthesized N(7)-substituted theophyllines were obtained as white powder in high yield. The structure of synthesized compounds was confirmed by various spectroscopic techniques such as ¹H, ¹³C, ¹⁹F NMR, and FT-IR spectroscopy, and elemental analysis. The highest interaction was recorded as −5.69 kcal/mol for 3-CF₃ substituted against VEGFR-2 structure while the best binding affinity was determined for 4-OCF₃ substituted with −6.69 kcal/mol against Human Cytochrome P450 with in silico analysis. The in vitro anticancer activities of the molecules were also evaluated against A549 and HeLa cells, and displayed considerably higher cytotoxicity with 2-CF₃, 3-CF_3, and 4-CF₃ substituted molecules in Hela and A549 cell line. To elucidate the molecular mechanism, apoptosis-related gene expression changes were analyzed by RT-qPCR in A549 and HeLa cells treated with compound 2-CF₃. Significant upregulation of pro-apoptotic markers and downregulation of anti-apoptotic genes were observed. Consistently, ELISA-based quantification confirmed increased protein levels of Caspase-3, BAX, and Cytochrome C, and decreased BCL-2, validating the apoptotic mechanism at the protein level. Also, the antibacterial and antibiofilm activity details of the molecules were evaluated against DNA Gyrase, and SarA crystal structures by molecular docking method. The highest interaction was recorded as −5.56 kcal/mol for 2-CF₃ substituted with H-bonds with Asn46, Val71, Asp73, and Thr165 against DNA Gyrase crystal structure while 3-CF₃ substituted has the best binding affinity against SarA. The in vitro antimicrobial effects of the molecules were also evaluated. Conclusions: The synthesized molecules may provide insight into the development of potential therapeutic agents to the increasing antimicrobial resistance and biofilm-forming capacity of microorganisms. Additionally, compound 2-CF₃ substituted exhibited promising and selective anticancer activity through apoptosis induction, supported by gene and protein level evidence. Full article

Background/Objectives: Circulating cell-free DNA (cfDNA) consists of genomic DNA (cf-nDNA) and mitochondrial DNA (cf-mtDNA) fragments released from cells primarily through apoptosis and necrosis. In healthy individuals, the main source of cfDNA is apoptosis, whereas in cancer patients, necrosis predominates. Considering that in vitro cfDNA models are valuable research tools, this study presents an in vitro characterization of cf-mtDNA patterns released into the culture medium by four human cell lines: normal dermal fibroblasts (Hs27), induced pluripotent stem cells (iPSCs), melanoma cells (BMel), and prostate cancer cells (PC3). Furthermore, as fetal bovine serum (FBS)—a widely used supplement in cell culture media—has been shown to contain bovine cfDNA, species-specific primers were employed to eliminate potential artifacts arising from this contamination in in vitro experiments. Methods: Fragmentation analysis of cf-mtDNA was conducted by amplifying the human MT-CYB gene and the D-loop region in four cell lines using species-specific primers. Two indices, Q and λ, were employed to quantify fragmentation. Results: These indices reveal that cancer cells exhibit the highest degree of fragmentation compared to fibroblasts, whereas stem cells show the lowest degree of fragmentation. This study identified species-specific primers for the human and bovine MT-CYB gene, confirming the presence of bovine cf-mtDNA in cell culture media supplemented with FBS. Conclusions: in vitro cellular models are useful for studying the mechanisms of cfDNA release and fragmentation; designed primers provide a reliable tool for assessing contamination across different growth time points minimizing interference errors and non-specific amplifications. Full article

Background: The gold standard for diagnosing primary central nervous system lymphoma (PCNSL) is brain biopsy, an invasive procedure with significant risks. The role of cerebrospinal fluid (CSF) examination, limited to cytology and flow cytometry in current practice, is acknowledged as a less invasive diagnostic method. We aimed to summarize available data concerning the efficacy and actual use of current standard CSF diagnostics in the diagnosis of PCNSL. Methods: A systematic review and meta-analysis of 144 studies (n = 9493 patients) was conducted, assessing detection rates of cytology and flow cytometry and the proportion of diagnoses based on CSF analysis. The QUADAS-2 tool was used to evaluate study quality and bias. Results: Meta-analysis showed an 18% pooled detection rate for positive CSF results, with 17% for cytology and 20% for flow cytometry. Only 8% of diagnoses were made using CSF analysis. Most studies had a high risk of bias. Conclusions: Despite its established role in guidelines, CSF analysis remains underutilized for diagnosing PCNSL, with room to improve its clinical impact. Novel techniques, such as chemokines and circulating tumor DNA (cfDNA) analysis, hold promise to unlock the untapped potential of CSF diagnostics, offering significant advancements in non-invasive PCNSL diagnosis. Full article

Post-viral conditions, Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and Long COVID (LC), share > 95% of their symptoms, but the connection between disturbances in their underlying molecular biology is unclear. This study investigates DNA methylation patterns in peripheral blood mononuclear cells (PBMC) from patients with ME/CFS, LC, and healthy controls (HC). Reduced Representation Bisulphite Sequencing (RRBS) was applied to the DNA of age- and sex-matched cohorts: ME/CFS (n = 5), LC (n = 5), and HC (n = 5). The global DNA methylomes of the three cohorts were similar and spread equally across all chromosomes, except the sex chromosomes, but there were distinct minor changes in the exons of the disease cohorts towards more hypermethylation. A principal component analysis (PCA) analysing significant methylation changes (p < 0.05) separated the ME/CFS, LC, and HC cohorts into three distinct clusters. Analysis with a limit of >10% methylation difference and at p < 0.05 identified 214 Differentially Methylated Fragments (DMF) in ME/CFS, and 429 in LC compared to HC. Of these, 118 DMFs were common to both cohorts. Those in promoters and exons were mainly hypermethylated, with a minority hypomethylated. There were rarer examples with either no change in methylation in ME/CFS but a change in LC, or a methylation change in ME/CFS but in the opposite direction in LC. The differential methylation in a number of fragments was significantly greater in the LC cohort than in the ME/CFS cohort. Our data reveal a generally shared epigenetic makeup between ME/CFS and LC but with specific, distinct changes. Differences between the two cohorts likely reflect the stage of the disease from onset (LC 1 year vs. ME/CFS 12 years), but specific changes imposed by the SARS-CoV-2 virus in the case of the LC patients cannot be discounted. These findings provide a foundation for further studies with larger cohorts at the same disease stage and for functional analyses to establish clinical relevance. Full article

Background: Ovarian cancer remains the most lethal gynecological cancer, primarily due to its asymptomatic nature in early stages and consequent late diagnosis. Early detection improves survival, but current biomarkers lack sensitivity and specificity. Cell-free DNA (cfDNA) released from tumor cells captures tumor-associated epigenetic alterations and represents a promising source for minimally invasive biomarkers. Among these, aberrant DNA methylation occurs early in tumorigenesis and may reflect underlying disease biology. This study aimed to investigate genome-wide cfDNA methylation profiles in patients with ovarian cancer, benign ovarian conditions, and healthy controls to identify cancer-associated methylation patterns that may inform future biomarker development. Results: We performed genome-wide cfDNA methylation profiling using cell-free methylated DNA immunoprecipitation sequencing (cfMeDIP-seq) on plasma samples from 40 patients with high-grade serous ovarian carcinoma, 38 patients with benign ovarian conditions, and 38 healthy postmenopausal women. A total of 536 differentially methylated regions (DMRs) were identified between ovarian cancer and controls (n = 76), with 97% showing hypermethylation in ovarian cancer. DMRs were enriched in CpG islands and gene bodies and depleted in repetitive elements, consistent with known cancer-associated methylation patterns. Fifteen genes showed robust hypermethylation across analyses. These genes exhibited methylation across intronic, exonic, and upstream regulatory regions. Separate comparisons of ovarian cancer to each control group (benign and healthy) supported the reproducibility of these findings. Gene Ontology enrichment analysis revealed enrichment in gland development, embryonic morphogenesis, and endocrine regulation, suggesting biological relevance to ovarian tumorigenesis. Conclusions: This study identifies consistent cfDNA hypermethylation patterns in ovarian cancer, affecting genes involved in developmental regulation and hormone-related processes. Our findings underscore the potential of cfMeDIP-seq for detecting tumor-specific methylation signatures in plasma and highlight these 15 hypermethylated genes as biologically relevant targets for future studies on cfDNA methylation in ovarian cancer. Full article

Background: Cell-free DNA (cfDNA) released during tissue damage has attracted interest as a marker of patient outcomes. However, limited research has examined its predictive utility in thermally injured patients. Methods: This study measured cfDNA concentrations across days 1, 3, 7, 14, and 28 post-burn in a total cohort of 98 adult patients with total body surface area (TBSA) burns ≥ 15% and healthy controls (HC). CfDNA concentrations in survivors (n = 79) versus non-survivors (n = 16) were compared and area under the receiver operating curve (AUROC) models generated to evaluate cfDNA as a predictor of mortality. Results: Patient cfDNA levels were significantly elevated at all time points compared to HC. Positive correlations were identified between day 1 cfDNA concentrations (n = 95) and %TBSA (r = 0.413, p < 0.0005), rBAUX (r = 0.365, p = 0.0005) and SOFA (r = 0.391, p = 0.0002). On day one, cfDNA levels showed good discriminatory ability for distinguishing between survivors and non-survivors (AUROC 0.778), with an optimal cut-off value of 446.37 pg/mL exhibiting a sensitivity of 0.80 and specificity of 0.70. Predictive models built on rBAUX, SOFA, interleukin(IL)-6 and IL-10 generated AUROC values of 0.733, 0.743, 0.472, and 0.688 respectively. Conclusions: Major burns result in immediate and persistent cfDNA elevation, with concentrations on day one higher in non-survivors. Plasma cfDNA concentrations on day one post-burn showed good performance as a prognostic marker for mortality. CfDNA therefore represents a rapid objective measure that may be useful during acute burn assessments to aid mortality predictions. Full article

This study evaluated the post-thaw motility and in vitro fertility of ejaculated and epididymal semen from Pantaneiro bulls and characterized cell-free DNA (cfDNA) in fresh seminal plasma. Semen from five bulls was collected via electroejaculation or post-mortem epididymal extraction. Fresh semen parameters and cfDNA concentrations were assessed before cryopreservation. Post-thaw sperm kinetics were evaluated using CASA at 0 and 6 h of incubation, and in vitro embryo development was analyzed following IVF. Data were assessed using ANOVA and logistic regression. Ejaculate samples exhibited more morphological defects than epididymal samples (15.8% vs. 1.8%, p ≤ 0.05). Post-thaw, epididymal semen showed higher total (87.2% vs. 32.4%) and progressive (67.1% vs. 14.4%) motility at 0 h (p ≤ 0.05), and higher motility at 6 h (38.9% vs. 11.0%, p ≤ 0.05). In vitro fertility did not differ significantly between ejaculated (n = 525 oocytes) and epididymal (n = 500 oocytes) semen groups in terms of cleavage (49.6% vs. 44.2%) and blastocyst formation on D7 (26.1% vs. 22.2%, p > 0.05). cfDNA concentration in fresh semen ranged from 11.4 to 50.9 ng/µL. These findings indicate that epididymal sperm from Pantaneiro bulls retain high post-thaw motility and fertility. Additionally, cfDNA characterization in seminal plasma contributes to indigenous cattle preservation and advances in male fertility research. Full article

Background and objective: Multiple markers have been proposed, but there are no reliable pre-treatment markers that predict tumor response to total neoadjuvant therapy in patients with locally advanced rectal cancer. The objective of this study is to evaluate the usefulness of pre-treatment SEPTIN9 gene methylation ratio as a predictor of tumor response to total neoadjuvant therapy and its correlation with tumor size and tumor stage in patients with locally advanced rectal cancer. Methods: Patients with locally advanced rectal cancer (T3/4 and/or N+ histologically confirmed rectal cancer) undergoing total neoadjuvant therapy were included. Tumor size and tumor stage were determined by magnetic resonance. SEPTIN9 gene methylation in plasmatic cfDNA was analyzed by droplet digital PCR at the time of diagnosis. After completing total neoadjuvant therapy, tumor response was assessed by magnetic resonance and proctoscopy. The correlation between pre-treatment SEPTIN9 gene methylation ratio, tumor size, tumor stage and tumor response was analyzed. Results: 39 patients with locally advanced rectal cancer were included. Pre-treatment SEPTIN9 gene methylation ratio (p = 0.033) and tumor size (p = 0.026), but not tumor stage, significantly correlated with tumor response to total neoadjuvant therapy. Pre-treatment SEPTIN9 gene methylation ratio also correlated with N stage (p = 0.040) and tumor size (p = 0.001), but not with T stage (p = 0.846). Conclusions: Pre-treatment SEPTIN9 gene methylation ratio correlates with tumor size and N stage and can predict tumor response to total neoadjuvant therapy in patients with locally advanced rectal cancer. Full article

Background/Objectives: BRAF^V600E-mutant colorectal cancer (CRC) is associated with poor prognosis, and despite the introduction of BEACON therapy, significant treatment challenges remain. This study investigates the clinical utility of BRAF^V600E in cell-free DNA (cfDNA BRAF^V600E) as a biomarker for real-time treatment monitoring in metastatic cases and for evaluating minimal residual disease (MRD) after curative resection. Methods: This single-center, prospective observational study included 37 patients with BRAF^V600E-mutant CRC treated at Nippon Medical School Hospital between April 2017 and June 2024. Patients were divided into two cohorts: Cohort 1 (Stage IV cases): Evaluated cfDNA BRAF^V600E for treatment monitoring. Cohort 2 (Stage I–III curatively resected cases): Assessed cfDNA BRAF^V600E for recurrence risk prediction. Blood samples were collected before and during treatment and analyzed using droplet digital PCR (ddPCR) to measure cfDNA BRAF^V600E levels. Results: Cohort 1 (Stage IV, n = 14): Pre-treatment cfDNA BRAF^V600E was detected in 93% of cases. Patients with a decrease in cfDNA BRAF^V600E variant allele frequency (VAF) after chemotherapy had significantly longer overall survival (511 vs. 189 days, p = 0.03) than those without a decrease. Cohort 2 (curatively resected, n = 23): cfDNA BRAF^V600E was detected in 4/23 patients (17.4%) at 1 month post-surgery. cfDNA BRAF^V600E showed better recurrence prediction compared to CEA (100% vs. 18.8%, p = 0.004). Among the seven patients who experienced recurrence, those with postoperative cfDNA BRAF^V600E positivity had significantly shorter disease-free survival compared to cfDNA BRAF^V600E-negative patients (179 vs. 840 days, p = 0.04). Conclusions: These findings support cfDNA BRAF^V600E as a promising biomarker for monitoring treatment response and MRD detection in BRAF^V600E-mutant CRC, reinforcing its role in guiding personalized treatment strategies and postoperative surveillance. Full article

Pulmonary fibrosis, a chronic progressive lung disorder, can be the result of previous acute inflammation-associated lung injury and involves a wide variety of inflammatory cells, causing the deposition of extracellular matrix (ECM) components in the lungs. Such lung injury is often associated with excessive neutrophil function and the formation of DNA networks in the lungs, which are also some of the most important factors for fibrosis development. Acute lung injury with subsequent fibrosis was initiated in C57Bl/6 mice by a single intranasal (i.n.) administration of LPS. Starting from day 14, human recombinant DNase I in the form of Pulmozyme for topical administration was instilled i.n. twice a week at a dose of 50 U/mouse. Cell-free DNA (cfDNA), DNase activity, and cell content were analyzed in blood serum and bronchoalveolar lavage fluid (BALF). Inflammatory and fibrotic changes in lung tissue were evaluated by histological analysis. The gene expression profile in spleen-derived neutrophils was analyzed by RT-qPCR. We demonstrated that Pulmozyme significantly reduced connective tissue expansion in the lungs. However, despite the reliable antifibrotic effect, complete resolution of inflammation in the respiratory system of mice treated with Pulmozyme was not achieved, possibly due to enhanced granulocyte recruitment and changes in the nuclear/mitochondrial cfDNA balance in the BALF. Moreover, Pulmozyme introduction caused the enrichment of the spleen-derived neutrophil population by those with an unusual phenotype, combining pro-inflammatory and anti-inflammatory features, which can also maintain lung inflammation. Pulmozyme can be considered a promising drug for lung fibrosis management; however, the therapy may be accompanied by residual inflammation. Full article

Background: Cell-free DNA (cfDNA) is present in healthy individuals but is elevated in those undergoing physical exertion, trauma, sepsis, and certain cancers. Maintaining cfDNA concentrations is vital for immune homeostasis and preventing inflammatory responses. Understanding cfDNA release and clearance is essential for using cfDNA as a biomarker in clinical diagnostics. We focused on the fragment size of cfDNA and investigated cfDNA dynamics and half-life, particularly the 100–250 base pair fragments. Methods: Healthy, adult men (n = 5; age 40 ± 4.1 years) were subjected to a 30 min treadmill exercise. Blood samples were collected at 0, 5, 10, 15, 30, and 60 min post-exercise using PAXgene^® Blood ccfDNA tubes to stabilize and prevent nuclease-mediated cfDNA degradation and minimize genomic DNA contamination risk. The cfDNA concentration was measured using an electrophoresis-based technique (4150 TapeStation system) to quantify the concentration based on cfDNA fragment size. Results: The results showed a cfDNA half-life of 24.2 min, with a transient increase in 100–250 base pair cfDNA fragments post-exercise, likely due to nuclease activity. These levels rapidly reverted to the baseline within an hour. Conclusions: The rapid clearance of cfDNA underscores its potential as a biomarker for real-time disease monitoring and the evaluation of treatment efficacy. This study is expected to standardize cfDNA investigations, enhancing diagnosis and treatment monitoring across various disease conditions. Full article

Background: Intense exercise leads to neutrophil extracellular traps (NETs) formation, which triggers cell disintegration. NET, as well as other processes of apoptosis, necrosis, and spontaneous secretion, result in increased levels of cell-free DNA (cf-DNA) in the circulation. An increment of cf-DNA is also observed in autoimmune diseases, such as type 1 diabetes mellitus (T1DM). Repeated exhaustive exercises are an impulse for physiological adaptation; therefore, in this case–control study, we compared the exercise-induced increase in cf-DNA in men with T1DM and healthy controls to determine the development of the tolerance to exercise. Methods: Volunteers performed a treadmill run to exhaustion at a speed matching 70% of their personal VO2 max at three consecutive visits, separated by a 72 h resting period. Blood was collected before and after exercise for the determination of plasma cell-free nuclear and mitochondrial DNA (cf n-DNA, cf mt-DNA) by real-time PCR, blood cell count and metabolic markers. Results: Each bout of exhaustive exercise induced a great elevation of cf n-DNA levels. An increase in cf mt-DNA was observed after each run. However, the significance of the increase was noted only after the second bout in T1DM participants (p < 0.02). Changes in cf-DNA concentration were transient and returned to baseline values during 72 h of resting. The exercise-induced increment in circulating cf n-DNA and cf mt-DNA was not significantly different between the studied groups (p > 0.05). Conclusions: Cf-DNA appears to be a sensitive marker of inflammation, with a lower post-exercise increase in individuals with T1DM than in healthy men. Full article

Background: Patients with heart failure (HF) with improved ejection fraction (HFimpEF) demonstrate better clinical outcomes when compared with individuals without restoration of cardiac function. The identification of predictors for HFimpEF may play a crucial role in the individual management of HF with reduced ejection fraction (HFrEF). Cell-free nuclear (cf-nDNA) DNA is released from damaged cells and contributes to impaired cardiac structure and function and inflammation. The purpose of the study was to elucidate whether cf-nDNA is associated with HFimpEF. Methods: The study prescreened 1416 patients with HF using a local database. Between October 2021 and August 2022, we included 452 patients with chronic HFrEF after prescription of optimal guideline-based therapy and identified 177 HFimpEF individuals. Circulating biomarkers were measured at baseline and after 6 months. Detection of cf-nDNA was executed with real-time quantitative PCR (qPCR) using NADH dehydrogenase, ND2, and beta-2-microglobulin. Results: We found that HFimpEF was associated with a significant decrease in the levels of cf-nDNA when compared with the patients from persistent HFrEF cohort. The presence of ischemia-induced cardiomyopathy (odds ration [OR] = 0.75; p = 0.044), type 2 diabetes mellitus (OR = 0.77; p = 0.042), and digoxin administration (OR = 0.85; p = 0.042) were negative factors for HFimpEF, whereas NT-proBNP ≤ 1940 pmol/mL (OR = 1.42, p = 0.001), relative decrease in NT-proBNP levels (>35% vs. ≤35%) from baseline (OR = 1.52; p = 0.001), and cf-nDNA ≤ 7.5 μmol/L (OR = 1.56; p = 0.001) were positive predictors for HFimpEF. Conclusions: We established that the levels of cf-nDNA ≤ 7.5 μmol/L independently predicted HFimpEF and improved the discriminative ability of ischemia-induced cardiomyopathy, IV NYHA class, and single-measured NT-proBNP and led to a relative decrease in NT-proBNP levels ≤35% from baseline in individuals with HFrEF. Full article

We aimed to investigate the use of free glycosaminoglycan profiles (GAGomes) and cfDNA in plasma to differentiate between lung cancer and benign lung disease, in a cohort of 113 patients initially suspected of lung cancer. GAGomes were analyzed in all samples using the MIRAM^® Free Glycosaminoglycan Kit with ultra-high-performance liquid chromatography and electrospray ionization triple quadrupole mass spectrometry. In a subset of samples, cfDNA concentration and NGS-data was available. We detected two GAGome features, 0S chondroitin sulfate (CS), and 4S CS, with cancer-specific changes. Based on the observed GAGome changes, we devised a model to predict lung cancer. The model, named the GAGome score, could detect lung cancer with 41.2% sensitivity (95% CI: 9.2–54.2%) at 96.4% specificity (95% CI: 95.2–100.0%, n = 113). When we combined the GAGome score with a cfDNA-based model, the sensitivity increased from 42.6% (95% CI: 31.7–60.6%, cfDNA alone) to 70.5% (95% CI: 57.4–81.5%) at 95% specificity (95% CI: 75.1–100%, n = 74). Notably, the combined GAGome and cfDNA testing improved the sensitivity, compared to cfDNA alone, especially in ASCL stage I (55.6% vs 11.1%). Our findings show that plasma GAGome profiles can enhance cfDNA testing performance, highlighting the applicability of a multiomics approach in lung cancer diagnostics. Full article