Search Results (42)

Cell membrane glycosylations play a critical role in regulating cell signaling, adhesion, and immune recognition. Abnormal glycosylation is a hallmark of cancer and promotes tumor progression. Sulfatide (SM4), a glycosphingolipid synthesized by galactosylceramide sulfotransferase (CST), is frequently altered in cancers, including breast cancer. Our previous studies identified SM4 as an adhesive molecule that enhances metastasis through interactions with platelets and endothelium; however, its elevated levels increase apoptotic sensitivity and reduce tumorigenicity. Here, we elucidate the molecular mechanisms linking sulfatide metabolism to apoptosis and gene regulation. Using MDA-MB-231 and MDA-MB-468 breast cancer cells with altered CST/SM4 levels, RNA sequencing and functional analyses revealed that overproduction of the CST/SM4 significantly downregulated BOLA2, a gene in the CIAPIN1 pathway involved in apoptosis. RT-qPCR and Western blot confirmed an inverse relationship between CST/SM4 and BOLA2. Overexpressing BOLA2 provided resistance to doxorubicin-induced apoptosis, suggesting that SM4-mediated repression of BOLA2 increases apoptotic sensitivity. Luciferase assays showed reduced BOLA2 promoter activity in SM4-enriched cells. Transcription factor profiling and Electrophoretic Mobility Shift Assay demonstrated that SM4 suppresses STAT5 activation, which directly binds and regulates the BOLA2 promoter. SM4 also altered integrin profiles by upregulating β4/β5 and downregulating β1 subunits. Reintroducing β1 integrin restored STAT5 activation and BOLA2 expression, positioning β1 integrin upstream of STAT5. Collectively, these findings identify a novel sulfatide-dependent β1 integrin–STAT5–BOLA2 pathway controlling apoptosis in breast cancer cells. SM4 suppresses β1 integrin and STAT5-mediated BOLA2 transcription, promoting apoptosis, while β4/β5 upregulation may facilitate invasion. This pathway represents a potential therapeutic target in breast cancer. Full article

Fibrin monomer (FM) is a transient intermediate of blood coagulation that functions as both an active regulator of haemostasis and a sensitive biomarker for prothrombotic states. Clinically, FM is measured indirectly as its derivative, soluble fibrin monomer complexes (SFMC), which is also often referred to as FM throughout the clinical literature. FM participates in a complex regulatory network modulating thrombin generation and fibrinolysis, interacting with platelet receptors, including integrin αIIbβ3 and GPVI, and engaging GPIb-vWF interactions. This comprehensive review examines FM’s molecular mechanisms in haemostatic regulation and evaluates clinical evidence for FM as a biomarker. Particular focus is placed on FM’s utility for risk stratification across thrombotic conditions, including disseminated intravascular coagulation, venous thromboembolism, ischemic stroke, myocardial infarction, and COVID-19-associated coagulopathy. Current challenges, including assay standardization and universal cut-off values, are discussed. By synthesizing mechanistic insights with clinical data, this integrated perspective may accelerate the translation of FM biology into improved risk assessment tools and novel therapeutic strategies. Full article

Conventional whole-blood flow assays for quantifying thrombus formation are typically performed at room temperature and are technically demanding, which limits their translational applicability. We engineered a novel, disposable, mountable, and single-channel microfluidic chip (MC-2S), which is based on the Maastricht chamber (MC) and designed for automated evaluation of platelet function, coagulation and fibrinolysis under physiological conditions. The MC-2S chip allows customizable choices of thrombogenic surfaces, such as collagen and tissue factor. The chip was used in combination with an adapted, 1.3 kg brightfield/fluorescence microscope, operating at physiological temperature (37 °C), and with scripts for automated multicolor analysis of image features. The integrated system enables a robust, rapid, and high-content quantification of the kinetics of thrombus formation and dissolution. In platelet-sensitive mode, MC-2S demonstrated high sensitivity to antiplatelet therapy with aspirin or cangrelor. In coagulation-sensitive mode, it detected the anticoagulant effect of rivaroxaban plus its reversal by andexanet-α. In fibrinolysis-sensitive mode, it monitored tissue-type plasminogen activator-induced thrombus dissolution, inhibited by tranexamic acid. Collectively, the MC-2S platform was found to provide a versatile, physiologically relevant tool for functional hemostasis testing, with high potential for the acute and subacute evaluation of patient blood samples in the context of bleeding disorders, thrombosis risk, and drug monitoring. Full article

Introduction: For pregnant women with a history of fetal and neonatal alloimmune thrombocytopenia (FNAIT) or hemolytic disease of the fetus and newborn (HDFN), prenatal intervention in subsequent pregnancies may be necessary to prevent complications for the fetus. A non-invasive prenatal diagnostic procedure (NIPD) is recommended for fetal blood group genotyping. RT-PCR is used for fetal RHD determination as a reliable screening method with high sensitivity and specificity. For other antigens with variants involving single-base substitutions, droplet digital PCR (ddPCR) and next-generation sequencing (NGS) are recommended to reduce the risk of false-negative results. Only NGS offers the possibility of determining the cell-free fetal DNA (cffDNA) fraction in maternal plasma by sequencing additional gene fragments in parallel, but no standard exists for assay validation. Material and Methods: A custom-made primer panel was designed to target the common platelet and red cell antigens involved in fetal red cell and platelet incompatibilities, as well as additional anonymous single-nucleotide polymorphism (SNP) targets for use as an internal control. Amplicon-based NGS was carried out using semiconductor sequencing. For HPA-1a (HPA*1A, ITGB3) and K (KEL*01.01, KEL) assay validation, the limit of detection (LOD) and limit of quantification (LOQ) were estimated, as were false-positive antithetic alleles, linearity, and inter-assay variation, using cell-free DNA (cfDNA) extracted from the blood samples of healthy blood donors. An additional analysis was performed using 23 diagnostic samples from 21 pregnant women. Results: Regression analysis of dilution series using HPA-1a- and K-positive cell-free plasma samples in antigen-negative donor plasma showed that recovery is definitely feasible up to an HPA*1A and KEL*01.01 allele frequency of 1%. Base calls of false-positive antithetic alleles were detected with a maximum of 0.25% using 21 healthy blood donors. The LOD was estimated to be 0.2057% (mean + 3 SD) for HPA*1A with a LOQ of 0.6298% (mean + 10 SD). For KEL*01.01, the LOD was 0.1706% (mean + 3 SD) and the LOQ was 0.5314% (mean + 10 SD). The analysis of 15 of 21 cases with diagnostic samples from pregnant women with neonatal blood available for confirmatory testing resulted in 100% concordant results. The fetal fraction of these samples was calculated with a median of 11.03% (95% CI: 8.89, 13.20). Conclusions: NGS for non-invasive fetal blood group genotyping is an accurate and reliable method. In-house validation of the used assays can be performed using healthy donors to determine the LOD, LOQ and sensitivity. The threshold for paternally inherited fetal HPA*1A and KEL*01.01 alleles could be set at 1% (i.e., 2% fetal fraction) to obtain reliable test results. Internal controls for assessing the fetal fraction are essential to avoid false-negative test results. Full article

During pregnancy, the maternal hemostatic system undergoes significant changes to support placental angiogenesis, maintain fetal blood flow, and ensure safe delivery. This study investigates the dysregulation of hemostasis in placental insufficiency and explores potential markers for diagnosing and managing this gestational complication. Thromboelastography, coagulation and fibrinolysis functional assays, ELISA, and immunoblotting were employed to assess hemostasis dysregulation in placental dysfunction of two cohorts of pregnant women with placental dysfunction and healthy controls. Thromboelastographic analysis revealed no significant differences in clot lysis indices between the control and placental dysfunction groups, with values remaining within normal ranges, suggesting this method’s limitations for assessing fibrinolysis in pregnancy. The placental dysfunction group demonstrated moderately increased fibrinogen levels and platelet sensitivity to ADP, indicating hemostasis reactiveness. Significantly lower D-dimer levels, decreased plasminogen activator inhibitor activity (total PAI-1 + PAI-2), and increased plasminogen activator activity, driven primarily by uPA in the placental dysfunction group, indicated abnormal fibrinolysis. Immunoblotting confirmed elevated uPA/uPA-PAI complexes and reduced tPA/tPA-PAI complexes, indicating that shutdown of tPA-mediated fibrinolysis and induction of uPA-driven vessel-wall-associated proteolysis are linked to placental dysfunction. Placental dysfunction involves fibrinolytic system dysregulation, marked by decreased PAI and tPA, uPA overproduction, and hypofibrinolysis, contributing to thrombotic risks, impaired placental flow, and complications like fetal growth retardation. PAI/PA ratio and D-dimer levels have diagnostic potential for placental-dysfunction-associated complications. Full article

Liquid biopsy has emerged as a valuable tool for the detection and monitoring of colorectal cancer (CRC), providing minimally invasive insights into tumor biology through circulating biomarkers such as circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs). Additional biomarkers, including tumor-educated platelets (TEPs) and exosomal RNAs, offer further potential for early detection and prognostic role, although ongoing clinical validation is still needed. This review summarizes the current evidence on the diagnostic, prognostic, and predictive capabilities of liquid biopsy in both metastatic and non-metastatic CRC. In the non-metastatic setting, liquid biopsy is gaining traction in early detection through screening and in identifying minimal residual disease (MRD), potentially guiding adjuvant treatment and reducing overtreatment. In contrast, liquid biopsy is more established in metastatic CRC for monitoring treatment responses, clonal evolution, and mechanisms of resistance. The integration of ctDNA-guided treatment algorithms into clinical practice could optimize therapeutic strategies and minimize unnecessary interventions. Despite promising advances, challenges remain in assay standardization, early-stage sensitivity, and the integration of multi-omic data for comprehensive tumor profiling. Future efforts should focus on enhancing the sensitivity of liquid biopsy platforms, validating emerging biomarkers, and expanding multi-omic approaches to support more targeted and personalized treatment strategies across CRC stages. Full article

Background: Leucine rich α-2 glycoprotein (LRG) is a glycoprotein that is an acute-phase protein produced by neutrophils, macrophages, hepatocytes, and intestinal epithelial cells. This study aimed to determine the serum LRG (s-LRG) and urine LRG (u-LRG) expression levels in children with inflammatory bowel disease (IBD) and evaluated their correlation with clinical disease activity, other inflammatory markers, laboratory results, and endoscopic activity scoring. Methods: This prospective observational study was conducted at a tertiary centre and included children aged 2–18 years with IBD. Clinic activity scoring was used to assess clinical disease activity. Haemoglobin levels, platelet counts, albumin, C-reactive protein, and erythrocyte sedimentation rate were analysed in the blood sample. LRG levels were measured in both blood and urine samples. The endoscopic assessment was scored according to the simple endoscopic score and Mayo endoscopic score. Serum and urine LRG levels were measured using commercial enzyme-linked immunosorbent assay kits. Disease activation was defined based on clinical activity scoring, laboratory results, and endoscopic evaluation. The results were compared between the active IBD and remission groups. Results: Forty-two (50%) patients with active IBD and forty-two (50%) patients in remission were included in this study. The serum levels of LRG were elevated in the patients with active IBD compared with the levels in the patients with IBD in remission (p = 0.020). However, there was no difference in the u-LRG level between the two groups (p = 0.407). In patients with IBD, positive correlations were observed between s-LRG, platelet count, C-reactive protein (CRP), and the erythrocyte sedimentation rate. The serum LRG was negatively correlated with albumin and haemoglobin levels. Urine LRG was not correlated with s-LRG in any patients with IBD included or in patients with active IBD. The cutoff value for s- LRG (77.03 μg/mL) had a sensitivity and specificity of 40.4% (95% CI 25.6–56.7%) and 88.1% (95% CI 74.3–96.0%), respectively. It was found that s-LRG was a more significant parameter than CRP in predicting disease activation. Conclusions: This prospective study demonstrated that the s-LRG level is a useful biomarker for predicting disease activation in children with IBD and appears to be a more significant parameter than the CRP level. However, the u-LRG level is not effective in predicting disease activation in children with IBD. Full article

Colorectal cancer (CRC) is the third most common cancer and a leading cause of cancer-related mortality worldwide, with prognosis significantly deteriorating at advanced stages. While current diagnostic methods, such as colonoscopy and tissue biopsy, are widely employed in clinical practice, they are invasive, expensive, and limited in assessing tumor heterogeneity and monitoring disease processes, including therapy response. Therefore, early and accurate detection, coupled with minimal invasion and cost-effective strategies, are critical for improving patient outcomes. Liquid biopsy has emerged as a promising, minimally invasive alternative, enabling the detection of tumor-derived components. This approach is increasingly utilized in clinical settings. The current key liquid biopsy modalities in CRC include circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), and RNA-based biomarkers such as long non-coding RNAs (lncRNAs), microRNAs(miRNAs), and circular RNAs (circRNAs), and tumor-educated platelets (TEPs). These methods provide valuable insights into genetic and epigenetic tumor alterations, and serve as indicators for early detection, treatment monitoring, and recurrence prediction. However, challenges such as assay standardization and variability in sensitivity persist. This review delves into the clinical applications of liquid biopsy in CRC management, highlighting the transformative roles of ctDNA, CTCs, and non-coding RNAs, TEPs in early detection, prognostic assessment, and personalized therapy. In addition, it addresses current limitations and explores potential advancements to facilitate their integration into routine clinical practice. Full article

Objectives: This study sought to determine the platelet function and viscoelastic blood properties in the pre- and postoperative period using three different point-of-care (POC) devices (Multiplate^®, Siemens PFA-200^® and ROTEM^®). We aimed to investigate the association between preoperative POC test results and bleeding outcomes. Postoperative changes in blood hemostatic properties were also evaluated, as well as the agreement between two platelet function analyzers and rotational thromboelastometry parameters. Methods: The study was conducted in a prospective observational fashion. Patients undergoing elective coronary artery bypass graft surgery (CABG) were enrolled. Hemostatic blood properties were assessed using three different POC devices; two platelet function analyzers were used: (1) Impedance aggregometry (Multiplate^®) with the arachidonic acid (ASPI) test and adenosine diphosphate (ADP) test. (2) The Siemens INNOVANCE^® PFA-200 System with the following assays: the PFA Collagen/EPI test, PFA Collagen/ADP test, and the INNOVANCE^® PFA P2Y test. Viscoelastic blood properties were assessed using ROTEM^® delta (TEM Innovations GmbH, Munich, Germany). POC tests were performed simultaneously at two different time points: (1) before surgery and (2) on postoperative day 4, respectively. The primary outcome was defined as amounts of perioperative bleeding and transfusion requirements, classified according to the universal definition for perioperative bleeding (UDPB) score. Results: The study recruited a total number of 63 patients undergoing elective isolated coronary artery bypass graft surgery (CABG). Based on the packed red blood cell (PRBC) transfusion requirements, patients with excessive bleeding were not just only frequently transfused (87.5% vs. 48.9%, p = 0.007) but were also transfused with higher amounts of PRBCs (1338.75 mL ± SD 1416.49 vs. 289.36 mL ± 373.07, p < 0.001). The FIBTEM A30 results significantly correlated with excessive bleeding (Correlation Coefficient Rho = −0.280, p = 0.028). Regression analysis revealed FIBTEM A 30 as a strongest predictor of 24 h chest tube output (CTO) (R Square 0.108, p = 0.009). The receiver operating characteristics curve (ROC) analysis showed that a preoperative FIBTEM A30 < 10.86 mm predicted excessive bleeding with 94% sensitivity and 50% specificity (ROC AUC 68.4%). The multiplate ASPI test results were significantly higher (35.24 AUC ± SD 22.24 vs. 19.43 AUC ± SD 10.74) and the proportion of Aspirin responders was significantly lower (42.4% vs. 76.7%, p = 0.006) in patients considered to have insignificant bleeding. On postoperative day 4, we found platelet hyperreactivity in the ASPItest coupled with a ROTEM-documented shift towards hypercoagulability. Conclusions: Modern hemostatic management and perioperative antiplatelet therapy (APT) administration/discontinuation management should be guided by thromboelastometry and platelet function testing. Prospective interventional trials are necessary to validate such an approach in multicentric studies. Full article

Engineered light-sensitive molecules offer a sophisticated toolkit for the manipulation of biological systems with both spatial and temporal precision. Notably, artificial “caged” compounds can activate specific receptors solely in response to light exposure. However, the uncaging process can lead to the formation of potentially harmful byproducts. For example, the photochemical release of adrenaline (epinephrine) is accompanied by the formation of adrenochrome, which has neuro- and cardiotoxic effects. To investigate this effect in detail, we synthesized and compared two “caged” epinephrine analogs. The first was a classical compound featuring an ortho-nitrobenzyl protecting group attached to the amino group of epinephrine. The second analog retained the ortho-nitrobenzyl group but included an additional carbamate linker. The photolysis of both compounds was conducted under identical conditions, and the resulting products were analyzed using UV–Vis spectroscopy, chromatography, and NMR techniques. Surprisingly, while the classical compound led to the formation of adrenochrome, the carbamate-type caged epinephrine did not produce this byproduct, resulting in the clean release of the active substance. Subsequently, we assessed the novel compound in an in vitro platelet activation assay. The results demonstrated that the uncaging of epinephrine significantly enhances platelet activation, making it a valuable tool for advanced signaling studies. Full article

Background: Aldehyde dehydrogenase class 1 (ALDH1) is an enzyme that is ubiquitously distributed in adult tissues and may serve as a prognostic marker in various cancer types. In blood, 99% of ALDH1 is found in erythrocytes; although, it was also demonstrated that leukocytes and platelets exhibit ALDH activity. No ALDH activity was detected in plasma, even when employing the highly sensitive fluorometric method with 7-methoxy-1-naphthaldehyde as a substrate. However, some reports have been released describing stable and measurable ALDH1 activity in the serum of healthy subjects using 6-methoxy-2-naphthaldehyde as a substrate and a Shimadzu RF—5301 spectrofluorometer. Methods: Our study aimed to verify whether ALDH1 activity can be measured in plasma or serum (n = 80) using 6-methoxy-2-naphthaldehyde as a substrate and a highly sensitive Hitachi F7000 spectrofluorometer, which offers a higher signal-to-noise ratio compared to the Shimadzu RF-5301. Additionally, HPLC with fluorometric detection was used to validate the results (n = 25) and analyze the influence of hemolysis (n = 5) and liver cell damage (n = 15) on ALDH1 activity in serum. Results: Measurable ALDH activity in serum/plasma was very rarely detected using a spectrofluorometer (2 cases out of 80). However, background drift in assays without coenzyme addition was observed, and it may be easily mistaken for ALDH or oxidase activity. Therefore, the spectrofluorometer drift observed in blank assays and modified by a matrix, e.g., enhanced in protein-rich samples, should be considered in ALDH1 activity assays. Conclusions: The spectrofluorometric method has limited applicability for determining ALDH activity in plasma and serum. HPLC can measure ALDH1 activity in plasma or serum; however, factors like hemolysis and elevated liver enzymes significantly affect activity and must be considered in diagnostic interpretations. To enhance research quality on ALDH1 as a biomarker for diseases, including cancers, we recommend using control samples, reference materials, and purifying commercially available aldehyde substrates to improve method sensitivity. Full article

Background: Patients with a HeartMate 3 (HM3) left ventricular assist device (LVAD) typically receive anticoagulation and antiplatelet therapy. The HM3 has shown a marked reduction in hemocompatibility-related adverse events (HRAEs) like stroke, bleeding, and pump thrombosis. This study evaluated whether aspirin (ASA) response influences HRAE incidence and if ASA sensitivity changes over time in HM3 recipients. Methods: This single-center, cross-sectional study included 32 HM3 patients (age: 59.0 ± 10.0 years, 15.6% female). ASA sensitivity was assessed twice using the VerifyNow assay, with ASA resistance defined by ASA reactivity units (ARU) > 550. The primary endpoint was HRAE incidence in ASA responders vs. non-responders over two consecutive follow-ups; the secondary endpoint was temporal changes in ASA resistance. Results: At the first follow-up, 13 (40.6%) patients were ASA-resistant, and 8 (28.6%) were resistant at the second follow-up, without significant change (p = 0.22). ASA non-responders and responders had similar ASA doses and baseline characteristics. No significant difference in HRAE incidence was found between ASA non-responders and responders (0.0% vs. 15.8%, p = 0.14), and no additional HRAEs occurred during follow-up. Conclusions: ASA resistance varied considerably among HM3 patients without significant temporal changes, and the demonstrated excellent hemocompatibility supports recent evidence that ASA may have a limited role in the antithrombotic regimen for HM3 recipients. Full article

Objectives: We aimed to assess the relationship among circulating extracellular vesicles (EVs), hypoxia-related proteins, and the conventional risk factors of life-threatening coronary artery disease (CAD) to find more precise novel biomarkers. Methods: Patients were categorized based on coronary CT angiography. Patients with a Segment Involvement Score > 5 were identified as CAD patients. Individuals with a Segment Involvement Score < 5 were considered control subjects. The characterization of EVs and analysis of the plasma concentration of growth differentiation factor-15 were performed using multicolor or bead-based flow cytometry. The plasma protein levels of glycogen phosphorylase, muscle form, clusterin, and carboxypeptidase N subunit 1 were determined using an enzyme-linked immunosorbent assay. Multiple logistic regression was used to determine the association of the biomarkers with the CAD outcome after accounting for established risk factors. The analysis was built in three steps: first, we included the basic clinical and laboratory variables (Model 1), then we integrated the plasma protein values (Model 2), and finally, we complemented it with the circulating EV pattern (Model 3). To assess the discrimination value of the models, an area under (AUC) the receiver operating curve was calculated and compared across the three models. Results: The area under the curve (AUC) values were 0.68, 0.77, and 0.84 in Models 1, 2, and 3, respectively. The variables with the greatest impact on the AUC values were hemoglobin (0.2 (0.16–0.26)) in Model 1, carboxypeptidase N subunit 1 (0.12 (0.09–0.14)) in Model 2, and circulating CD41+/CD61+ EVs (0.31 (0.15–0.5)) in Model 3. A correlation analysis showed a significant impact of circulating CD41+/CD61+ platelet-derived EVs (p = 0.03, r = −0.4176) in Model 3. Conclusions: Based on our results, the circulating EV profile can be used as a supportive biomarker, along with the conventional laboratory markers of CAD, and it enables a more sensitive, non-invasive diagnostic analysis of CAD. Full article

Background/Objectives: Chronic skin wounds (CSWs) are a worldwide healthcare problem with relevant impacts on both patients and healthcare systems. In this context, innovative treatments are needed to improve tissue repair and patient recovery and quality of life. Cord blood platelet lysate (CB-PL) holds great promise in CSW treatment thanks to its high growth factors and signal molecule content. In this work, thermo-sensitive hydrogels based on an amphiphilic poly(ether urethane) (PEU) were developed as CB-PL carriers for CSW treatment. Methods: A Poloxamer 407^®-based PEU was solubilized in aqueous medium (10 and 15% w/v) and added with CB-PL at a final concentration of 20% v/v. Hydrogels were characterized for their gelation potential, rheological properties, and swelling/dissolution behavior in a watery environment. CB-PL release was also tested, and the bioactivity of released CB-PL was evaluated through cell viability, proliferation, and migration assays. Results: PEU aqueous solutions with concentrations in the range 10–15% w/v exhibited quick (within a few minutes) sol-to-gel transition at around 30–37 °C and rheological properties modulated by the PEU concentration. Moreover, CB-PL loading within the gels did not affect the overall gel properties. Stability in aqueous media was dependent on the PEU concentration, and payload release was completed between 7 and 14 days depending on the polymer content. The CB-PL-loaded hydrogels also showed biocompatibility and released CB-PL induced keratinocyte migration and proliferation, with scratch wound recovery similar to the positive control (i.e., CB-PL alone). Conclusions: The developed hydrogels represent promising tools for CSW treatment, with tunable gelation properties and residence time and the ability to encapsulate and deliver active biomolecules with sustained and controlled kinetics. Full article

Neuroendocrine neoplasms (NENs) present a diagnostic challenge due to their heterogeneous nature and non-specific clinical manifestations. This study aimed to explore novel biomarkers for NENs. Serum chromogranin A (CgA) levels and a panel of 48 inflammatory cytokines were analyzed in a cohort of 84 NEN patients and 40 healthy controls using enzyme-linked immunosorbent assay (ELISA) and multiplex ELISA. Significant alterations in cytokine levels were observed in the NEN patients compared to the controls, including elevated levels of pro-inflammatory cytokines, such as interleukin (IL)-6, IL-8, and tumor necrosis factor alpha (TNF-α), and reduced levels of angiogenic factors like platelet-derived growth factor-BB (PDGF-BB) and tumor necrosis factor beta (TNF-β). Notably, cytokines such as growth-regulated alpha protein (GRO-α) and TNF-β demonstrated strong potential as diagnostic markers, with receiver operating characteristic (ROC) curve analyses showing high sensitivity and specificity. Additionally, a positive correlation was found between CgA levels and several inflammatory cytokines, suggesting their synergistic role in tumor progression. These findings highlight the limited reliability of CgA alone as a diagnostic marker and underscore the importance of a multi-marker approach in diagnosing and monitoring NENs. Further research on a larger cohort is necessary to validate these biomarkers and their potential clinical applications. Full article