Thrombotic biomarkers for risk prediction of malignant disease recurrence in patients with early stage breast cancer.

In cancer patients, hypercoagulability is a common finding. It has been associated with an increased risk of venous thromboembolism, but also to tumor proliferation and progression. In this prospective study of a large cohort of breast cancer patients, we aimed to evaluate whether pre-chemotherapy abnormalities in hemostatic biomarkers levels: (i) are associated with breast cancer-specific clinico-pathological features; and (ii) can predict for disease recurrence. D-dimer, fibrinogen, prothrombin fragment 1+2, and FVIIa/antithrombin levels were measured in 701 early-stage resected breast cancer patients candidate to adjuvant chemotherapy and prospectively enrolled in the HYPERCAN study. Significant prognostic parameters for disease recurrence were identified by Cox regression multivariate analysis and used for generating a risk assessment model. Pre-chemotherapy D-dimer, fibrinogen, and pro-thrombin fragment 1+2 levels were significantly associated with tumor size and lymph node metastasis. After 3.4 years of follow up, 71 patients experienced a recurrence. Cox multivariate analysis identified prothrombin fragment 1+2, tumor size, and Luminal B HER2-negative or triple negative molecular subtypes as independent risk factors for disease recurrence. Based on these variables, we generated a risk assessment model that significantly differentiated patients at low- and high-risk of recurrence (cumulative incidence: 6.2 vs 20.7%; Hazard Ratio=3.5; P<0.001). Our prospective clinical and laboratory data from the HYPERCAN study were crucial for generating a scoring model for assessing risk of disease recurrence in resected breast cancer patients, candidate to systemic chemotherapy. This finding stimulates future investigations addressing the role of plasma prothrombin fragment 1+2 in the management of breast cancer patients to provide the rationale for new therapeutic strategies. (The HYPERCAN study is registered at clinicaltrials.gov identifier 02622815).

Effect of the combinations between pea proteins and soluble fibres on cholesterolaemia and cholesterol metabolism in rats.

Many functional foods and dietary supplements have been reported to be beneficial for the management of dyslipidaemia, one of the major risk factors for CVD. Soluble fibres and legume proteins are known to be a safe and practical approach for cholesterol reduction. The present study aimed at investigating the hypocholesterolaemic effect of the combinations of these bioactive vegetable ingredients and their possible effects on the expression of genes regulating cholesterol homeostasis. A total of six groups of twelve rats each were fed, for 28 d, Nath's hypercholesterolaemic diets, differing in protein and fibre sources, being, respectively, casein and cellulose (control), pea proteins and cellulose (pea), casein and oat fibres (oat), casein and apple pectin (pectin), pea proteins and oat fibres (pea+oat) and pea proteins and apple pectin (pea+pectin). Administration of each vegetable-containing diet was associated with lower total cholesterol concentrations compared with the control. The combinations (pea+oat and pea+pectin) were more efficacious than fibres alone in modulating cholesterolaemia ( - 53 and - 54%, respectively, at 28 d; P< 0·005). In rats fed the diets containing oat fibres or apple pectin, alone or in combination with pea proteins, a lower hepatic cholesterol content (P< 0·005) and higher hepatic mRNA concentrations of CYP7A1 and NTCP were found when compared with the control rats (P< 0·05). In summary, the dietary combinations of pea proteins and oat fibres or apple pectin are extremely effective in lowering plasma cholesterol concentrations in rats and affect cellular cholesterol homeostasis by up-regulating genes involved in hepatic cholesterol turnover.

Characterization of the thrombin generation potential of leukemic and solid tumor cells by calibrated automated thrombography.

BACKGROUND: Thrombin, the final enzyme of blood coagulation, is a multifunctional serine protease also involved in the progression of cancer. Tumor cells may activate blood coagulation proteases through the expression of procoagulant activities. However, specific information about the thrombin generation potential of malignant tissues is lacking. In this study we applied a single global coagulation test, the calibrated automated thrombogram assay, to characterize the specific procoagulant phenotypes of different tumor cells. DESIGN AND METHODS: Malignant hematologic cells (i.e. NB4, HEL, and K562) or solid tumor cells (i.e. MCF-7 breast cancer and H69 small cell lung cells) were selected for the study. The calibrated automated thrombo-gram assay was performed in normal plasma and in plasma samples selectively deficient in factor VII, XII, IX or X, in the absence or presence of a specific anti-tissue factor antibody. Furthermore, cell tissue factor levels were characterized by measuring antigen, activity and mRNA expression. RESULTS: In normal plasma, NB4 induced the highest thrombin generation, followed by MCF-7, H69, HEL, and K562 cells. The anti-tissue factor antibody, as well as deficiencies of factors VII, IX and XII affected the thrombin generation potential of malignant cells to different degrees, allowing differentiation of the two different pathways of blood clotting activation - by tissue factor or contact activation. The thrombin generation capacity of NB4 and MCF-7 cells was tissue factor-dependent, as it was highly sensitive to inhibition by anti-tissue factor antibody and factor VII deficiency, while the thrombin generation capacity of H69, HEL and K562 was contact activation-dependent, as no thrombin was generated by these cells in factor XII-deficient plasma. CONCLUSIONS: This study demonstrates that the calibrated automated thrombogram assay is capable of quantifying, characterizing, and comparing the thrombin generation capacity of different tumor cells. This provides a useful tool for understanding the key factors determining the global pro-coagulant profile of tumors, which is important for addressing specific targeted therapy for the prevention of thrombosis and for cancer.

Reduced biliary sterol output with no change in total faecal excretion in mice expressing a human apolipoprotein A-I variant.

BACKGROUND/AIMS: Apolipoprotein (apo)A-I(M) (ilano), is a molecular variant of apoA-I(wild-type), associated with dramatically low HDL-cholesterol levels, but no increased risk for cardiovascular disease. In view of the present uncertainties on the role of apoA-I in liver cholesterol removal by way of bile acids and neutral sterols, and of the greater capacity of apoA-I(M) (ilano) to remove arterial cholesterol, biliary sterol metabolism was evaluated in transgenic mice expressing apoA-I(M) (ilano). METHODS: ApoA-I(M) (ilano) mice were fed a high-cholesterol/high-fat diet, and compared with human apoA-I(wild-type) mice. Plasma lipid levels, hepatic bile flow and composition, hepatic and intestinal cholesterol and bile acid content, and faecal sterol content were measured. Moreover, the expression of hepatic ABCA1, SR-B1 and that of hepatic and intestinal genes involved in bile acid metabolism were evaluated. RESULTS: The dietary treatment led to a strong elevation in HDL-cholesterol levels in A-I(M) (ilano) mice, associated with an increased expression of hepatic ABCA1. ApoA-I(M) (ilano) mice showed lower cholesterol output from the liver compared with apoA-I(wild-type) mice, in the absence of liver sterol accumulation. Faecal excretion of neutral sterols and bile acids was similar in the two mouse lines. CONCLUSIONS: In spite of a different response to the dietary challenge, with an increased ABCA1 expression and a lower hepatic cholesterol output in apoA-I(M) (ilano) mice, the net sterol excretion is comparable in the two transgenic lines.

LMWH bemiparin and ULMWH RO-14 reduce the endothelial angiogenic features elicited by leukemia, lung cancer, or breast cancer cells.

Low-molecular-weight heparins (LMWH) are anticoagulant drugs that also possess antitumor properties. We evaluated whether "second generation" LMWH bemiparin and the Ultra-Low-MWH (ULMWH) RO-14 are able to inhibit in vitro the angiogenic response of microvascular endothelium stimulated by tumor-cell-conditioned media (TCM) from human leukemia, lung cancer, and breast cancer cells. Bemiparin and RO-14 dose dependently inhibited the increase of capillary-like tube formation (Matrigel-based assay) and endothelial migration (wound-healing assay) induced by TCM. Both drugs also inhibited angiogenic response elicited by purified VEGF and FGF-2. These findings support a possible role of these molecules as adjuvant drugs in cancer treatment.

Platelet-induced thrombin generation by the calibrated automated thrombogram assay is increased in patients with essential thrombocythemia and polycythemia vera.

The platelet contribution to the thrombophilic state of patients with myeloproliferative neoplasms (MPNs), i.e., essential thrombocythemia (ET) and polycythemia vera (PV), remains uncertain. In this study we aimed to characterize the thrombin generation (TG) potential expressed by platelets from these subjects, compare it to normal platelets, and identify what factors might be responsible for platelet TG. In a group of 140 MPN patients (80 ET and 60 PV) and 72 healthy subjects, we measured the global procoagulant potential of platelet-rich plasma (PRP) utilizing the TG assay by the calibrated automated thrombogram (CAT). To characterize the procoagulant contribution of platelets in PRP, the TG of both isolated platelets and platelet-poor plasma was measured, and the platelet surface expression of TF was determined. Finally, the activation status of platelets was assessed by the levels of P-selectin expressed on platelet surface. MPN patients had significantly increased PRP and isolated platelet TG potential compared to controls. This was associated to the occurrence of platelet activation. Patients carriers of the JAK2V617F mutation showed the highest values of TG and platelet surface TF and P-selectin. Platelet TG potential was significantly lower in hydroxyurea(HU) compared to non-HU-treated patients and was lowest in HU-treated JAK2V617F carriers. In subjects not receiving HU, platelet TG significantly increased by JAK2V617F allele burden increment (P < 0.05).This study demonstrates a platelet-dependent form of hypercoagulability in MPN patients, particularly in those carriers of the JAK2V617F mutation. The cytoreductive therapy with HU significantly affects this prothrombotic phenotype.

Depletion of human histone H1 variants uncovers specific roles in gene expression and cell growth.

At least six histone H1 variants exist in somatic mammalian cells that bind to the linker DNA and stabilize the nucleosome particle contributing to higher order chromatin compaction. In addition, H1 seems to be actively involved in the regulation of gene expression. However, it is not well known whether the different variants have distinct roles or if they regulate specific promoters. We have explored this by inducible shRNA-mediated knock-down of each of the H1 variants in a human breast cancer cell line. Rapid inhibition of each H1 variant was not compensated for by changes of expression of other variants. Microarray experiments have shown a different subset of genes to be altered in each H1 knock-down. Interestingly, H1.2 depletion caused specific effects such as a cell cycle G1-phase arrest, the repressed expression of a number of cell cycle genes, and decreased global nucleosome spacing. On its side, H1.4 depletion caused cell death in T47D cells, providing the first evidence of the essential role of an H1 variant for survival in a human cell type. Thus, specific phenotypes are observed in breast cancer cells depleted of individual histone H1 variants, supporting the theory that distinct roles exist for the linker histone variants.

A Systematic Review and a Meta-Analysis Comparing Prophylactic and Therapeutic Low Molecular Weight Heparins for Mortality Reduction in 32,688 COVID-19 Patients.

Background: Antithrombotic treatment, including low molecular weight heparin (LMWH) or unfractionated heparin (UFH), has been proposed as a potential therapy for coronavirus disease 2019 (COVID-19) to lower diffuse intravascular clotting activation. However, it is unclear whether prophylactic or therapeutic doses have similar efficacy in reducing mortality. Methods: We performed a systematic review (PROSPERO registration CRD42020179955) and meta-analysis including observational cohort studies and randomized controlled trials (RCT) evaluating the effectiveness of heparins (either LMWH, UFH, or fondaparinux) in COVID-19 patients. Heparin treatment was compared to no anticoagulation. A subgroup analysis on prophylactic or therapeutic doses compared to no anticoagulation was performed. Prophylactic dose was also compared to full dose anticoagulation. Primary endpoint was all-cause mortality. Secondary endpoints were major bleeding and length of hospital stay (LOS). Results: 33 studies (31 observational, 2 RCT) were included for a total overall population of 32,688 patients. Of these, 21,723 (66.5%) were on heparins. 31 studies reported data on all-cause mortality, showing that both prophylactic and full dose reduced mortality (pooled Hazard Ratio [HR] 0.63, 95% confidence interval [CI] 0.57-0.69 and HR 0.56, 95% CI 0.47-0.66, respectively). However, the full dose was associated with a higher risk of major bleeding (Odds Ratio [OR] 2.01, 95% CI 1.14-3.53) compared to prophylactic dose. Finally, LOS was evaluated in 3 studies; no difference was observed between patients with and without heparins (0.98, -3.87, 5.83 days). Conclusion: Heparin at both full and prophylactic dose is effective in reducing mortality in hospitalized COVID-19 patients, compared to no treatment. However, full dose was associated with an increased risk of bleeding. Systematic Review Registration: https://clinicaltrials.gov/, identifier CRD42020179955.

Thrombin generation predicts early recurrence in breast cancer patients.

BACKGROUND: Cancer patients present with a hypercoagulable state often associated with poor disease prognosis. OBJECTIVES: This study aims to evaluate whether thrombin generation (TG), a global coagulation test, may be a useful tool to improve the identification of patients at high risk of early disease recurrence (ie, E-DR within 2 years) after breast cancer surgery. PATIENTS/METHODS: A cohort of 522 newly diagnosed patients with surgically resected high-risk breast cancer were enrolled in the ongoing prospective HYPERCAN study. TG potential was measured in plasma samples collected before starting systemic chemotherapy. Significant predictive hemostatic and clinic-pathological parameters were identified in the derivation cohort by Cox regression analysis. A risk prognostic score for E-DR was generated in the derivation and tested in the validation cohort. RESULTS: After a median observation period of 3.4 years, DR occurred in 51 patients, 28 of whom were E-DR. E-DR subjects presented with the highest TG values as compared to both late-DR (from 2 to 5 years) and no relapse subjects (P < .01). Multivariate analysis in the derivation cohort identified TG, mastectomy, triple negative and Luminal B HER2-neg molecular subtypes as significant independent predictors for E-DR, which were utilized to generate a risk assessment score. In the derivation and validation cohorts, E-DR rates were 2.3% and 0% in the low-risk, 10.1% and 6.3% in the intermediate-risk, and 18.2% and 16.7%, in the high-risk categories, respectively. CONCLUSIONS: Inclusion of TG in a risk-assessment model for E-DR significantly helps the identification of operated breast cancer patients at high risk of very early relapse.

Hypolipidaemic and anti-atherosclerotic effects of lupin proteins in a rabbit model.

The biological activities of a protein isolate from lupin (Lupinus albus) were studied in a rabbit model of atherosclerosis. Focal plaque development was induced at both common carotid arteries by perivascular injury. After surgery, animals were fed three different diets for 90 d, all with 1% cholesterol, 15% SFA and 20% protein; the protein source was casein (CAS), lupin proteins (LUP) or 50% CAS+50% LUP (CAS+LUP). Lower cholesterolaemia was detected in the LUP v. the CAS group at 60 and 90 d of treatment (-40·3 and -33·5%, respectively; P<0·05). Cryosection analyses of the carotids indicated a significant reduction in focal lesion progression in the LUP v. the CAS group (-37·4%; P<0·05). In summary, in a rabbit model of atherosclerosis, a protein isolate from L. albus reduced cholesterolaemia and exerted a remarkable protective activity against atherosclerosis progression.

Comparative assessment of low-molecular-weight heparins in cancer from the perspective of patient outcomes and survival.

Patients with cancer are at high risk of developing venous thromboembolism (VTE), including deep venous thrombosis and pulmonary embolism. Compared to non-cancer patients, VTE in cancer is more frequently associated with clinical consequences, including recurrent VTE, bleeding, and an increase in the risk of death. Low-molecular-weight heparins (LMWHs) are commonly recommended for the prevention and treatment of VTE in cancer patients because of their favorable risk-to-benefit profile. Indeed, compared with vitamin K antagonists, LMWHs are characterized by a reduced need for coagulation monitoring, few major bleeding episodes, and once-daily dosing, which make these drugs more suitable in the cancer setting. Guidelines have been published recently with the aim to improve the clinical outcomes in cancer patients at risk of VTE and its complications. Coagulation activation in cancer may have a role not only in thrombosis but also in tumor growth and dissemination. Hence, inhibition of fibrin formation has been considered a possible tool against the progression of malignant disease. Clinical studies show that anticoagulant drugs may have a beneficial effect on survival in cancer patients, with a major role for LMWHs. Recently a number of prospective randomized clinical trials to test LMWHs to improve cancer survival as a primary endpoint in cancer patients have been conducted. Although the results are controversial, the interest in this research area remains high.

Hypolipidemic effect of dietary pea proteins: Impact on genes regulating hepatic lipid metabolism.

Controversial data on the lipid-lowering effect of dietary pea proteins have been provided and the mechanisms behind this effect are not completely understood. The aim of the study was to evaluate a possible hypolipidemic activity of a pea protein isolate and to determine whether pea proteins could affect the hepatic lipid metabolism through regulation of genes involved in cholesterol and fatty acid homeostasis. Rats were fed Nath's hypercholesterolemic diets for 28 days, the protein sources being casein or a pea protein isolate from Pisum sativum. After 14 and 28 days of dietary treatment, rats fed pea proteins had markedly lower plasma cholesterol and triglyceride levels than rats fed casein (p<0.05). Pea protein-fed rats displayed higher hepatic mRNA levels of LDL receptor versus those fed casein (p<0.05). Hepatic mRNA concentration of genes involved in fatty acids synthesis, such as fatty acid synthase and stearoyl-CoA desaturase, was lower in pea protein-fed rats than in rats fed casein (p<0.05). In conclusion, the present study demonstrates a marked cholesterol and triglyceride-lowering activity of pea proteins in rats. Moreover, pea proteins appear to affect cellular lipid homeostasis by upregulating genes involved in hepatic cholesterol uptake and by downregulating fatty acid synthesis genes.