Background: Laboratory diagnosis is essential for identifying inherited bleeding disorders and monitoring therapy. We report results of a national survey by the Italian Association of Hemophilia Centers (AICE) that assesses test and reagent availability relative to guidelines and identifies improvement areas in reference laboratories (RLs) affiliated with hemophilia centers. Methods: Forty-three RLs participating in the ECAT External Quality Assessment completed an online questionnaire of 51 items across 11 sections. Results: Of surveyed RLs, 46.5% are located within hemophilia centers, and 53.5% are external, most within the same hospital. Most employ dedicated hemostasis personnel and use analytical platforms from Werfen, Siemens, or Stago. Nearly all perform one-stage assays for FVIII and FIX activity; 86% also use chromogenic substrate assays (CSA). Availability of CSA for FIX and capacity for CSA-based inhibitor titration are limited. Significant heterogeneity exists in reagent selection and application, notably for activated partial thromboplastin time and FVIII/FIX assays, and in von Willebrand disease (VWD) diagnostic workflows. Turnaround times are generally longer, and reagent use is more variable in external RLs. Conclusions: Practices across several RLs do not fully align with national and international recommendations for the diagnosis and monitoring of hemophilia and VWD. Regional procurement policies influence test availability. Findings support AICE’s initiative to establish a working group to improve laboratory diagnostic standards and highlight the need for targeted training and collaboration among clinical, laboratory, and healthcare management stakeholders. These data provide a baseline for harmonizing practices, guiding procurement decisions, and prioritizing education, research, and quality improvement initiatives nationally.

Introduction. A well-known connection exists between cancer and a prothrombotic state, where hemostatic biomarkers can signal disease progression and survival beyond just thrombosis. For metastatic breast cancer patients, identifying clinical and biochemical factors affecting short-term survival is vital for tailored treatment and better prognostic predictions. Aim. To evaluate the cumulative incidence of 1-year mortality and identify clinical characteristics and hemostatic biomarkers that independently predict mortality risk in patients with newly diagnosed metastatic breast cancer (MBC). Methods. This observational multicenter study enrolled patients with newly diagnosed MBC before chemotherapy as part of the HYPERCAN project. At baseline, clinical characteristics like ECOG status and molecular subtype were recorded, and hemostatic biomarkers (D-dimer, fibrinogen, FVIII, F1+2, thrombin generation) were measured. Patients were followed for 1 year to assess mortality. Survival analysis used Cox models; univariable analysis identified potential predictors, and multivariable forward selection determined independent risk factors. Results. A cohort of 189 patients was prospectively monitored. The cumulative one-year mortality was 12%. Univariable analysis identified elevated leukocyte counts, lower progesterone receptor expression, ECOG status ≥ 2, and the triple-negative (TN) molecular subtype as clinical predictors of mortality. Conversely, targeted therapy, tamoxifen, and aromatase inhibitors were protective. Regarding hemostatic biomarkers, high baseline levels of D-dimer, fibrinogen, and FVIII were independent predictors of death. In the multivariable model, the strongest independent predictors were the TN subtype, elevated leukocytes, and FVIII levels. Additionally, VTE occurring within the first six months was associated with a 3-fold increase in the risk of one-year mortality. Conclusions. One-year mortality is significantly influenced by biological subtype, inflammatory markers, and baseline coagulation profiles. The TN subtype and elevated FVIII emerged as particularly potent independent predictors of poor prognosis. Furthermore, the strong correlation between early VTE and a threefold increase in mortality risk underscores the critical importance of thromboprophylaxis strategies. These findings suggest that baseline hemostatic profiling may offer valuable prognostic insights beyond traditional clinical parameters.

Background. Anticoagulant management of cancer-associated thrombosis (CAT) is complex due to high risk of both recurrent venous thromboembolism (VTE) and bleeding. The START2 Registry ONCO-VTE prospectively collects real-world data of patients with active malignancy to monitor the evolution of clinical practice, particularly the shift toward DOACs. Aims. To describe the clinical characteristics of patients with CAT and evaluate variations in prescribing patterns and clinical outcomes between two enrollment periods: Jan. 2022–April 2024 (Group 1) and April 2024–Dec. 2025 (Group 2). Methods. We analyzed 333 patients (database as of 18/12/2025). Group 1 included 270 patients (enrolled through 14/04/2024), and Group 2 included 63 patients (enrolled from 15/04/2024 via REDCap). Primary outcomes were treatment choice, VTE recurrence, major bleeding, and mortality. Results. The cohort had a median age of 69 years, with 29% ≥75 years. Median follow-up was 12 months (14 months for Group 1 vs. 6 months for Group 2). Most patients (84%) had solid tumors, predominantly lung (18%) and colorectal (13%). Deep vein thrombosis (DVT) was the most frequent event (52%), followed by pulmonary embolism (PE) (22%) and both (12%). Of these events, 65% were symptomatic, and 35% were incidental. Central venous catheter (CVC)-related thrombosis accounted for 14%. Comorbidities included hypertension (47%), diabetes (15%), cardiovascular disease (11%), and chronic respiratory diseases (10%). A progressive shift toward DOACs was observed: in Group 1, DOACs were prescribed to 59% of patients, increasing to 64% in Group 2. Conversely, parenteral anticoagulants (LMWH/Fondaparinux) decreased from 39.8% to 34%. Overall, VTE recurrence occurred in 7.5%, major bleeding in 6.6%, and all-cause mortality was 15%. To ensure comparability, a subgroup analysis was performed on patients with at least 6 months of follow-up (n=227). In this cohort, major bleeding was significantly lower in Group 2 than in Group 1 (7.1% vs. 11.2%), whereas VTE recurrence rates were comparable (7.1% vs. 8.1%). Conclusions. The START2 ONCO-VTE registry analysis demonstrates a shift toward DOACs as the predominant strategy. This transition is associated with a significant reduction in major bleeding without compromising antithrombotic efficacy. These findings confirm that current management ensures high safety and effectiveness even in a complex and fragile oncological scenario.

Thrombosis remains a major cause of morbidity and mortality in patients with cancer. Existing risk models fail to reliably predict venous thromboembolism (VTE), underscoring the need for more accurate predictive models. In this study, we conducted a high-throughput proteomic analysis of 1105 plasma proteins in peripheral blood samples from patients with newly diagnosed lung or gastric cancer who were prospectively monitored for VTE development. Using a Bayesian probabilistic machine learning approach, we developed a predictive model incorporating 11 protein biomarkers and five clinical parameters (age, sex, history of VTE, body mass index, and hemoglobin), which outperformed the standardly used Khorana prediction score [c statistic 0.84 (0.79 to 0.90) as compared with 0.36 (0.27 to 0.45)]. Orthogonal validation in an external placebo cohort from a phase 3 trial confirmed the model's predictive power. Further investigation into the mechanistic role of CD200 receptor 1 (CD200R1), an immune checkpoint receptor known to limit leukocyte inflammatory response that contributed strongly to the model, showed that reduced concentrations in plasma correlated with higher D-dimer concentrations and thrombosis risk. CD200R1-deficient mice were characterized by features of a prothrombotic state, with elevated thrombin-antithrombin complexes, increased interleukin-17A (IL-17A), and endothelial inflammation. Administration of anti-IL-17A antibodies to CD200R1-deficient mice normalized thrombin-antithrombin complexes in vivo, and a meta-analysis of human COVID-19 studies showed reduced pulmonary thromboembolism in those on anti-IL-17A antibodies. These findings highlight the utility of plasma proteomics to improve prediction of thrombosis in patients with cancer and to identify unanticipated mechanistic insights and therapeutic targets in thrombo-inflammatory disease.

Background: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is characterized by thrombosis in unusual sites triggered by anti-PF4 antibodies. To the best of our knowledge, no studies with very long follow-up on anti-PF4 antibodies persistence have been reported. Methods: We carried out a multicenter study in 16 VITT patients studied at T0 (acute episode), T1 and T2 (after 6 and 29 months) assessing the persistence of anti-PF4/heparin antibodies and of neutrophil activation. Results: At T0 80%, at T1 75%, and at T2 11% of VITT patients were positive for anti-PF4/heparin antibodies by ELISA, while 75% at T0, 56% at T1, and 0% at T2 had a positive platelet activation assay. Plasmatic MMP-9 and MMP-9/NGAL were strikingly elevated at diagnosis, but they normalized at T1. No clinical relapses were observed. Conclusions: Although anti-PF4 antibodies may persist for a long-time following an acute VITT episode, they seem to be clinically irrelevant.

Introduction and aims: Immune Thrombotic Thrombocytopenic Purpura (iTTP) is a rare, life-threatening thrombotic microangiopathy caused by severe ADAMTS13 deficiency due to autoantibodies against ADAMTS13. Current treatment with therapeutic plasma exchange (TPE), immunosuppression, and the nanobody caplacizumab has dramatically reduced mortality to <10%, although the clinical severity of each acute episode remains unpredictable. While ADAMTS13 deficiency is central to pathogenesis, growing evidence implicates systemic inflammation and NETosis in disease progression. In the frame of a large longitudinal observational study involving iTTP patients, we aim to investigate the role of biomarkers of NETosis (citrullinated H3 histone, H3Cit) and inflammation (tumor necrosis factor-alpha, TNF-α, and interleukin-6, IL-6). Methods: Six patients (67% female; median age: 57 years, range 20–79) diagnosed with acute iTTP at the Hospitals of Bergamo and Brescia, Italy, between Feb 2024 and Apr 2025 were evaluated. Plasma samples were collected at baseline (D0), and on days 6 (D6), 9 (D9), and 15 (D15) following diagnosis, during treatment. ADAMTS13 activity, anti-ADAMTS13 antibodies, and ADAMTS13 inhibitory activity were assessed by ELISA (Technozym). Levels of H3Cit were quantified by ELISA (Cayman Chemical), and TNF-α and IL-6 levels by multiplex assay (Millipore Luminex). The analyses were performed at the Hemostasis and Thrombosis Center of Bergamo Hospital. Concurrent laboratory assessments comprised schistocyte detection, complete blood counts, and general biochemistry, including hemolysis parameters (reticulocytes, lactate dehydrogenase (LDH), bilirubin, haptoglobin). Clinical data and administered treatments were documented. Statistical analyses were conducted using SPSS v21.0 (IBM, Armonk, NY, USA) and R v4.2.2 (R Foundation, Vienna, Austria). Results: At onset, five patients presented neurological symptoms, ranging from mild headache to aphasia and acute ischemic cerebellar lesions. HIV infection, myelodysplastic syndrome (MDS), and autoimmune disorders were the most significant comorbidities. As first-line treatment, all patients received daily TPE, immunosuppression (including rituximab), and caplacizumab. Hematological remission (i.e., platelet count >150×10⁹/L and LDH<250 mg/dL) was achieved in a median time of 3 days, except for the patient with MDS who reached a stable platelet count >20 ×10⁹/L by day 9. Platelets' rise correlated with LDH reduction (p=0.001). Median time to ADAMTS13 >20% was 8 days; from D0 to D15, ADAMTS13 activity increased (1–43%; p=0.03), antibody titers declined (50 to 5 U/mL), as well as inhibitory activity (73 to 33%; p=0.045). During treatment, a significant decrease in H3Cit (p=0.0018) and TNF-α (p=0.015) plasma levels was observed over the different time points, while no significant changes were seen in the levels of IL-6. Specifically, H3Cit levels markedly decreased from D0 [5.90 ng/mL (IQR: 4.0–13.2)] to D6 [1.32 (0.9–2.9), p=0.017], D9 [0.55 (0.3–2.1), p=0.009], and D15 [0.69 (0.4–2.1), p=0.016]. TNF-α also significantly (p=0.035) declined from baseline to D9 [14.83 pg/mL (9.9–32.0) vs 4.90 (3.2–11.8) pg/mL]. Furthermore, a significant positive correlation was found between TNF-α levels and anti-ADAMTS13 antibodies (r=0.783; p<0.001). A subgroup analysis revealed that patients who received caplacizumab within 2 days of diagnosis (n=3) had significantly lower post-treatment TNF-α levels [4.6 (3.3–5.3) vs 7.2 (5.2–25.4), p=0.009] and showed a trend toward lower IL-6 levels [2.4 (1.2–6.1) vs 7.3 (2.9–16.5)] compared to those who initiated treatment later (n=3). Conclusions: Our data show significant reductions in H3Cit and TNF-α during treatment, paralleling hematologic and biochemical recovery. This suggests that such biomarkers may serve as indicators of NET-driven inflammation and immune activation. The positive correlation between TNF-α and anti-ADAMTS13 antibodies further supports the link between inflammation and the autoimmune process in iTTP. Additionally, our preliminary data suggest that early initiation of caplacizumab may be associated with reduced inflammatory markers. Despite the small sample size, these results highlight the potential for using NETosis and inflammation markers to monitor disease activity and optimize therapeutic strategies in iTTP.

(1) Background: The hemostatic system and tumor biology display a tight and reciprocal interaction where clotting products enhance tumor growth and dissemination, and the tumor, in turn, triggers a hypercoagulable and inflammatory state. Evaluating circulating biomarkers related to thrombo-inflammatory may provide a promising tool for predicting tumor outcomes, especially in non-small cell lung cancer (NSCLC) characterized by unfavorable outcomes. (2) Aim: In a prospective cohort of NSCLC patients, we evaluated whether thromboinflammatory biomarkers could predict early disease progression (DP) during the first 6 months of first-line anticancer treatment. (3) Methods: 719 newly diagnosed advanced-stage NSCLC patients were included. Complete blood cell count, high-sensitivity C-reactive protein (hs-CRP), FVIII, fibrinogen, D-dimer, thrombin-antithrombin (TAT) complexes, and prothrombin fragment1+2(F1+2) were tested in blood samples collected before starting chemotherapy. DP was gathered during follow-up. (4) Results: The 6-month cumulative incidence rate for DP was 49%. Univariable Cox regression analysis identified metastatic status, BMI, hemoglobin, leukocytes, hs-CRP, FVIII, fibrinogen, TAT, and D-dimer as significant predictors of DP. In a multivariable analysis that included all previously significant variables, only hs-CRP and D-Dimer levels remained strongly associated with DP. The two variables were used to establish a risk stratification model that significantly identified patients at high risk of DP at 6 months (HR 2.9; 95% CI, 2.3-3.7), which can be applied to 3, 9, and 12 months. (5) Conclusions: Our model easily and precisely estimates early DP during chemotherapy. If externally validated, this model can significantly enhance the allocation of medical resources in managing advanced NSCLC, ensuring that patients receive the most effective care possible.

Background: Patients with non-small cell lung cancer (NSCLC) are at high risk of venous thromboembolism (VTE), especially during chemotherapy. Even though the contact system is implicated in the pathogenesis of thrombosis, limited data are available on the role of contact system activation in NSCLC-associated VTE. Objectives: In a prospective cohort of patients with NSCLC starting chemotherapy, contact system activation and thrombin generation biomarkers were assessed in relation to 6-month VTE occurrence and mortality. Methods: Prechemotherapy plasma samples of 719 newly diagnosed patients with NSCLC were tested for in vivo biomarkers of contact system activation (ie, kallikrein [pKa]:antithrombin [AT; PKa:AT], activated factor [F]XI:AT [FXIa:AT], FXIa:C1-esterase inhibitor C1Inh [FXIa:C1Inh], activated FIX:AT [FIXa:AT]), and thrombin generation (ie, prothrombin fragment 1+2 [F1+2] and thrombin-antithrombin complex [TAT]). Clinical data, VTE, and mortality were recorded prospectively. Results: The 6-month VTE and mortality cumulative incidences were 11% and 27%, respectively. Basal levels of FXIa:AT complexes, F1+2, and TAT were higher in patients who developed VTE than those in VTE-free patients. Differently, PKa:AT, FIXa:AT, and TAT were lower in survivors than those in nonsurvivors. The multivariable analysis identified FXIa:AT (subdistribution hazard ratio, 1.17; 95% CI, 1.00-1.37) and TAT (subdistribution hazard ratio, 1.28; 95% CI, 1.10-1.50) as VTE-independent risk factors during chemotherapy. A score based on these biomarkers was generated, which was able to discriminate patients at significantly higher rates of VTE and mortality. Conclusion: Elevated in vivo contact pathway activation and thrombin generation were observed in patients with NSCLC who developed VTE. Furthermore, a score based on both FXIa:AT and TAT levels was developed to identify those patients at higher risk of VTE and mortality.

(1) Background: The presence of metastatic disease significantly increases the risk of venous thromboembolism (VTE) in breast cancer, particularly during chemotherapy. Although not categorized as a highly thrombogenic malignancy, the elevated global prevalence of this cancer places a substantial number of patients at risk of thrombosis, which cannot yet be accurately predicted by validated risk assessment models (RAMs), highlighting the need for a dedicated model. (2) Aim: This study aims to develop a RAM for VTE in newly diagnosed metastatic breast cancer patients enrolled in a prospective, observational, and multicenter study. (3) Methods: A cohort of 189 patients beginning antitumor therapy were enrolled and prospectively monitored for VTE and mortality. Blood samples collected at enrollment were tested for D-dimer, fibrinogen, FVIII, prothrombin fragment 1 + 2 (F1 + 2), and thrombin generation (TG). Competing risk analyses were performed to identify significant predictors. (4) Results: Within one year, the cumulative incidences of VTE and mortality were 7.0% and 12%, respectively. Univariable analysis identified high Ki-67, D-dimer, FVIII, fibrinogen, and TG levels, along with low hemoglobin levels, as independent predictors of VTE. Only Ki-67, fibrinogen, FVIII, and hemoglobin were retained as significant predictors in multivariable analysis. These variables were further examined by multiple linear regression, which revealed Ki-67 and fibrinogen as the most significant parameters. A continuous RAM was then developed based on Ki-67 and fibrinogen (c-statistics 0.78), categorizing patients into low-risk and high-risk groups for VTE (2% vs. 13%; SHR 3.6, p = 0.018). This stratification could not be achieved using currently validated models for VTE risk. (5) Conclusions: We developed an accurate RAM for VTE that enables the identification of metastatic breast cancer patients at high risk for VTE, which supports clinicians in personalized thromboprophylaxis strategies if externally validated.

Venous thromboembolism (VTE) is a frequent complication in patients with multiple myeloma (MM) that leads to increased morbidity, mortality, treatment interruptions, and reduced quality of life. Αn Expert Panel Consensus Guideline on behalf of the European Myeloma Network provides evidence-based recommendations for VTE prevention and treatment in patients with MM. Key recommendations include the following: Patient education. Informing patients about VTE risk and signs/symptoms of deep vein thrombosis and pulmonary embolism. Specialized care. Establishing a dedicated diagnostic and therapeutic pathway for the prevention and treatment of VTE that occurs in an outpatient setting. Risk assessment. Regular evaluation of VTE risk using validated scores. Primary thromboprophylaxis. Initiating pharmacological thromboprophylaxis with low-molecular-weight heparin (LMWH) or specific direct factor Xa inhibitors (anti-Xa agents) in high-risk patients. Prompt diagnosis and treatment. Early diagnosis of VTE and immediate initiation of antithrombotic therapy with LMWH or anti-Xa agents. Secondary prophylaxis. Personalized long-term antithrombotic therapy based on individual profiles of risk of VTE recurrence or bleeding. By implementing these guidelines, healthcare providers can improve the prevention and treatment of VTE in patients with MM, leading to better clinical outcomes and quality of life.

Background: In hospitalized patients, the SARS-CoV2 infection induces a thromboinflammatory state characterized by endothelial cell dysfunction, platelet activation, and hypercoagulability, leading to an increased risk of thrombosis. In ACS patients with concomitant SARS-CoV2 infection, a higher in-hospital mortality rate is reported; however, no information on the impact of this infection on the thromboinflammatory profile of patients presenting with ACS is currently available. Similarly, no role of hemostatic biomarkers in predicting in-hospital or long-term mortality risk has been established in this population. Aim: In a prospective cohort of ACS patients, we aimed to 1) characterize the contribution of concomitant SARS-CoV2 infection to the thromboinflammatory status by measuring a panel of hemostatic and inflammatory biomarkers; and 2) assess the role of these biomarkers in predicting in-hospital and/or two-year mortality. Methods: Consecutive patients admitted to interventional cardiology for ACS (i.e., ST-elevation myocardial infarction [STEMI], non-STEMI [NSTEMI], or unstable angina [UA]) during the first wave of the pandemic in Bergamo, Italy, from March to May 2020, were enrolled and followed-up for 2 years. Coronary angiography and percutaneous coronary intervention (PCI) data were collected. At admission, patients underwent complete blood cell count and an extensive coagulation profiling, including biomarkers of endothelial-cell activation (von Willebrand factor [vWF] antigen and activity, and ADAMTS13); hypercoagulation (prothrombin fragment 1+2 [F1+2], and D-dimer); and fibrinolytic proteins (tissue plasminogen activator [t-PA], and inhibitor [PAI-1]). In addition, the inflammatory biomarkers fibrinogen, factor VIII (FVIII), neutrophil extracellular traps (NETs), and C reactive protein (CRP) were tested. The occurrence of in-hospital and two-year mortality was prospectively recorded. Results: A total of 99 (76M/23F, median age 67 years) ACS patients (56 STEMI, 34 NSTEMI, 9 UA) were analyzed. Based on nasal swabs, 24 positive and 75 negative SARS-CoV-2 patients were identified. At admission, all patients showed significantly (p<0.05) higher levels of thromboinflammatory biomarkers compared to normal range values. Furthermore, ACS SARS-CoV-2-positive patients displayed significantly (p< 0.05) higher levels of fibrinogen, FVIII, D-dimer, vWF, t-PA, PAI-1, CRP, and NETs, together with significantly (p<0.05) lower levels of lymphocytes, hemoglobin, platelets, and ADAMTS-13 as compared to negative patients. The STEMI SARS-CoV2-positive group exhibited the highest values of the biomarkers. When performed, PCI ended more frequently with partial coronary reperfusion (TIMI grade flow <3) in SARS-CoV2-positive compared to negative patients (p= 0.004). The cumulative incidence of in-hospital mortality was 10%. The univariable analysis identified SARS-CoV-2 positivity as the strongest independent risk factor for mortality (HR 5.9 [95%CI 1.2-31.1]). Regarding the other clinical and laboratory parameters, the multivariable analysis identified dyspnea at presentation, vWF antigen, and leukocytes as independent risk factors for in-hospital mortality. These variables were used to develop a continuous score. Based on this score, the cumulative incidence of mortality was 0 % in the low-risk group, 19% in the intermediate-risk group (95% CI 6.1- 48.2%), and 71% in the high-risk group (95% CI 28.2-113.2%). After a 2-year observation, the mortality rate was 8%. Age was the sole independent risk factor for a 2-year mortality, with a HR of 1.2 (95% CI 1.04-1.3), regardless of the initial SARS-CoV2 status. Conclusions: For the first time, our study demonstrates that SARS-CoV2 infection worsens the thromboinflammatory state of ACS patients. This harmful effect might explain the observed heightened mortality rate among ACS patients concomitantly infected with SARS-CoV-2, which in our study was six times higher than in non-infected ACS patients. Additionally, the proposed scoring system, based on thromboinflammatory biomarkers, has proven effective in identifying ACS patients with elevated risks of in-hospital mortality. The potential role of this scoring system in identifying high-risk patients may possibly extend beyond our study to other highly inflammatory settings.

Introduction: Breast cancer is the most prevalent malignancy among women worldwide and is the cancer type in which the thrombogenic effect of anti-cancer drugs was first recognized (Levine et al, NEJM 1988). The occurrence of VTE in patients with metastatic breast cancer upon commencing anticancer treatment significantly affects disease burden, quality of life, and healthcare expenditure. Consequently, it is essential to accurately discern patients with a significant risk of developing thrombosis, thereby warranting medical prophylaxis. However, the stratification of outpatients on chemotherapy for metastatic breast cancer initiating an anticancer treatment by validated risk assessment models (RAMs) remains an unmet clinical need. In a prospective, observational cohort study of newly diagnosed metastatic breast cancer patients beginning chemotherapy, we aim to evaluate the incidence of VTE and the role of hypercoagulation biomarkers in predicting VTE and mortality within a 12-month follow-up. Methods: Newly diagnosed, metastatic breast cancer patients were enrolled and prospectively followed for VTE and mortality (HYPERCAN study, ClinicalTrials.gov ID#NCT02622815). Blood samples were collected at enrolment, before starting chemotherapy, and tested for biomarkers of blood clotting activation (i.e. D-dimer, fibrinogen, prothrombin-fragment 1+2 [F1+2]). Univariable and multivariable analyses were performed using the Cox proportional hazard model to identify statistically significant prognostic factors (SPSS Statistics, version 21.0). Fine and Gray competing risk analysis was also performed (Stata Corp, version 16). Results: A prospective cohort of 189 metastatic breast cancer patients with a median age of 59 years (range 32-91 years) was analyzed. Ductal carcinoma was diagnosed in 80% of patients. The most represented molecular subtypes were Luminal B HER2-neg (32.3%) > Luminal B HER2-pos (24.3%) > Luminal A (13.2%) > triple negative (TN, 9.5%) > HER2-pos (9%). Within 1 year of enrollment, 13 patients experienced VTE in a median time of 173 days (IQR: 52-258), resulting in a VTE cumulative incidence of 7.0% (95% CI 3.7-11.5). During the same follow-up period, the cumulative incidence of mortality was 12% (95% CI 7.4-17), within a median time to death of 212 days (IQR 106-306). Patients who experienced VTE had significantly higher baseline levels of D-dimer and fibrinogen than those who remained VTE-free during the same follow-up period (p<0.02). No significant differences were found in F1+2 levels between the two groups. In the Fine and Gray competing univariable analysis, D-dimer [HR 1.02 (95% CI 1.00-1.04), p=0.008] and Fibrinogen (HR 1.06 (95% CI 1.03-1.10), p<0.001), considered as continuous variables, emerged as significant predictors of VTE. Following correction for clinical variables (age, BMI, cardiovascular risk factors, and tumor histological characteristics), both biomarkers retained their independent associations with VTE occurrence. To establish specific cut-off values for each biomarker, D-dimer and fibrinogen levels were categorized into quartiles based on their distribution within the cohort. The results showed that D-dimer values greater than 533 ng/mL (2nd quartile) [HR 3.8 (95% CI 1.7 - 8.7), p=0.032] or fibrinogen values exceeding 428 mg/dL (3rd quartile) [HR 2.6 (95% CI 1.03-6.4), p=0.042] were significantly associated with an increased risk of VTE. Subsequently, the patients were categorized into two risk groups based on whether their biomarker values were above or below the identified thresholds. Specifically, analysis using Kaplan-Meier curves based on the D-dimer threshold revealed a VTE incidence of 3.3% versus 11% (p=0.029) in the low- and high-risk groups, respectively. Similarly, stratification by the fibrinogen threshold demonstrated a VTE incidence of 3.0% versus 18% (p<0.001) in the low- and high-risk groups. In this cohort, the occurrence of VTE did not affect the mortality rate, which was 64% in patients with VTE versus 51% in patients with no VTE (p=ns). Conclusion: Our data indicated that pre-chemotherapy D-dimer and fibrinogen levels are effective in identifying metastatic breast cancer patients at higher risk of VTE. These findings highlight the potential of these biomarkers in enhancing risk stratification and guiding personalized treatment strategies for breast cancer patients.

Topic: 31. Transfusion medicine Background: cTTP is an ultra-rare thrombotic microangiopathy, with two presentation peaks: in childhood and adulthood, in the latter typically presenting in pregnancy and puerperium. Although there are limited data on genotype-phenotype correlation in cTTP, some mutations are more likely associated with the pregnancy onset. Aims: Aim of this study was to evaluate the genotype-phenotype correlation in our cohort of pregnancy-related cTTP patients. Methods: Three pregnant women with cTTP were enrolled into the study. ADAMTS-13 activity (chromogenic assay), inhibitors (mixing studies), and anti-ADAMTS-13 antibodies (ELISA) were measured in plasma. The genetic analysis was performed by Sanger method. Results: Patient (P) 1 was a 30 y.o. Italian healthy blood donor. After delivery of her first child in a peripheral Hospital, she became severely anaemic and thrombocytopenic. She received RBC transfusion and steroids, meanwhile, blood samples sent to our Centre revealed ADAMTS13 of 5% and no auto-antibodies. Platelet count recovered after delivery. Genetic analysis revealed homozygosity for the c.3178C>T variant (p.R1060W). The familiar study showed a moderate reduction in the levels of ADAMTS13 activity in the mother (47%) and father (43%), both carrying the same variant in heterozygosity. Prophylactic plasma infusions were provided during her second pregnancy, that was carried through the 38th week without complications pre- and post-partum. P2 was a 21 y.o. Pakistani woman, who developed TTP during her first pregnancy (15th week). ADAMTS13 activity was 0% and no anti-ADAMTS13 antibodies were detected. Admitted to our hospital, from the 15th to 20th weeks of gestation, she received 9 PEX plus steroids. For persistence of severely reduced ADAMTS13 activity, molecular analysis was performed, revealing homozygosity for two rare missense mutations: c.2351G>A in exon 19 (R784S) and c.2746C>T (R916C) in exon 22 of ADAMTS13 gene. After the diagnosis of cTTP, she received treatment with plasma infusion. The pregnancy was continued throughout the 40th week, when she delivered a healthy baby. Plasma was also infused from day 1 to day 4 postpartum. Two years later, pregnant for the second time, she received prophylactic plasma infusions from the 13th week through the 38th, and delivered a healthy baby without problems. P3 was a 28 y.o. Italian woman with a previous diagnosis of HELLP syndrome in her first pregnancy. She was diagnosed with TTP in the 3rd trimester of her second pregnancy. PEX treatment was started, however, she underwent a caesarean section at 32 weeks due to clinical worsening, with a successful outcome. She had persistent low ADAMTS13 activity levels (10%) with no auto-antibodies, and at molecular analysis, compound heterozygosity for two novel ADAMTS13 mutations was found: a frameshift mutation in exon 6 (Gly149ArgfsX54) and a missense in exon 11 (Arg421Cys). Summary/Conclusion: In our series of pregnancy-onset cTTP, the genotype-phenotype correlation was confirmed in two out of three cases. Indeed, the first patient carried the p.R1060W, which is commonly associated with first presentation in pregnancy. The second patient was homozygous for the complex allele c.2351G>A; c.2746C>T (of which the variant c.2746C>T is probably pathogenic, while the c.2351G>A benign), described only in an adult-onset cTTP case. Finally, two new variants in compound heterozygosity, involving exons 6 and 11, were found in the third patient, classifiable as prespacer mutations, which are usually described in childhood-onset cases. The molecular study allowed to diagnose congenital cases, improving the management and outcome of subsequent pregnancies. Keywords: Pregnancy, Thrombotic thrombocytopenic purpura (TTP), Mutation analysis, ADAMTS13

Patients with chronic Myeloproliferative Neoplasms (MPN) including polycythemia vera (PV) and essential thrombocythemia (ET) exhibit unique clinical features, such as a tendency toward thrombosis and hemorrhage, and risk of disease progression to secondary bone marrow fibrosis and/or acute leukemia. Although an increase in blood cell lineage counts (quantitative features) contribute to these morbid sequelae, the significant qualitative abnormalities of myeloid cells that contribute to vascular risk are not well understood. Here, we address this critical knowledge gap via a comprehensive and untargeted profiling of the platelet proteome in a large (n= 140) cohort of patients (from two independent sites) with an established diagnosis of PV and ET (and complement prior work on the MPN platelet transcriptome from a third site). We discover distinct MPN platelet protein expression and confirm key molecular impairments associated with proteostasis and thrombosis mechanisms of potential relevance to MPN pathology. Specifically, we validate expression of high-priority candidate markers from the platelet transcriptome at the platelet proteome (e.g., calreticulin (CALR), Fc gamma receptor (FcγRIIA) and galectin-1 (LGALS1) pointing to their likely significance in the proinflammatory, prothrombotic and profibrotic phenotypes in patients with MPN. Together, our proteo-transcriptomic study identifies the peripherally-derived platelet molecular profile as a potential window into MPN pathophysiology and demonstrates the value of integrative multi-omic approaches in gaining a better understanding of the complex molecular dynamics of disease. Highlights MPN patient platelet proteome identifies key pathobiological mediators of thrombosis and proteostasis. The MPN platelet proteomic profile validates our prior findings from the platelet transcriptome.

Introduction: Given the great variability of the individual response to SARS-CoV2 infection, it is important to understand whether and how biological and genetic factors might predispose to the infection and to the degree of severity, including the development of systemic coagulopathy and thrombosis. In this study, we evaluated the frequency of prothrombotic allelic variants (AVs) in coagulation genes in a population of COVID-19 patients, to verify whether these polymorphisms, alone or in combination, are associated to an increased susceptibility to develop COVID-19 and/or a greater severity. Methods: A cohort of 358 patients (257M/101F; median age: 55 years) from the Bergamo area with two different degrees of COVID-19 severity, i.e., severe disease (hospitalized, n=212) or mild disease (non-hospitalized, n=146), was enrolled from April 2020 to June 2021 and followed-up for disease outcome and thrombosis. A cohort of 377 healthy subjects (177M/200F; median age: 49 years) from the same area without COVID-19 acted as a control group. DNA was analyzed for a panel of prothrombotic/inflammatory single nucleotide polymorphisms (SNPs) including FII rs1799963, FV rs6025, FV rs118203907, FXIIIA1 rs5985, FGB rs1800790, MTHFR rs1801131 and MTHFR rs1801133 as well as two SNPs located in the coding region of angiotensin-converting enzyme (ACE2) gene, namely ACE2 rs140312271 and ACE2 rs41303171. Statistical analysis was performed using SPSS statistical software. Results: Among the AVs analyzed, the rs6025 polymorphism of FV gene (FV Leiden) was more frequent in COVID-19 patients than in the control group. By a logistic regression corrected for age and gender, FV Leiden was associated with a 3-fold risk of developing COVID-19 (OR: 3.2 [95% CI 1.02-10]). In contrast, the rs41303171 polymorphism of the ACE2 gene was more frequent in the control group and was a protective factor for COVID-19 (OR: 0.12 [95% CI 0.02-0.99]) even after correction for age and gender. No significant differences appeared in the frequency of the other SNPs alone or in combination. Within the COVID-19 patient cohort, the rs1801131 SNP of MTHFR gene was associated with less severe COVID-19 disease. In the hospitalized severe cohort, 17 (8%) patients developed an overt venous thromboembolic event, and 7 COVID-19-related deaths (4%) were recorded. No significant associations were found between any of the AVs analyzed and the outcomes of thrombosis and death. However, the analyses of circulating biomarkers of clotting (i.e. D-dimer, FVIII, fibrinogen, prothrombin fragment 1+2 [F1+2], and FXIII), fibrinolysis (i.e. tissue plasminogen activator [tPA], and plasminogen activator inhibitor-1 [PAI-1]) and endothelial cell activation (i.e. thrombomodulin [TM], and von Willebrand Factor [vWF]), and the neutrophil-to-lymphocyte ratio (NLR) as an inflammatory biomarker revealed thathaving fibrinogen (OR: 4.9 [95% CI 1.04-23.1]), vWF:Ag (OR: 11.8 [95% CI 2.1-66.9]) and NLR (OR: 26.1 [95% CI 2.9-237] levels above the 75th percentile was significantly associated with death. Of interest, in the same hospitalized patient group, but not in the control group, the presence of FXIII SNPs was significantly associated with high levels of F1+2. Conclusions: Our study shows that the FV Leiden polymorphism predisposes to COVID-19 infection, while the rs41303171 ACE2 polymorphism has a protective role. In addition, the rs1801131 SNP of gene MTHFR correlates with a milder COVID-19 disease. In patients with severe COVID-19, the inflammatory marker NLR, and the circulating hemostatic proteins vWF and fibrinogen are associated with mortality.