Alfonso Vignoli

Coagulation and cancer: biological and clinical aspects.

Summary.  Malignancy affects the hemostatic system and the hemostatic system affects malignancy. In cancer patients there are a number of coagulation abnormalities which provide the background for an increased tendency of these patients to both thrombosis and hemorrhage. The causes of this coagulation impairment rely on general risk factors which are common to other categories of patients, and other factors which are specific to cancer, such as tumor type and disease stage. In addition, data from basic research indicate that the hemostatic components and the cancer biology are interconnected in multiple ways. Notably, while cancer cells are able to activate the coagulation system, the hemostatic factors play a role in tumor progression. This opens the way to the development of bifunctional therapeutic approaches that are both capable of attacking the malignant process and resolving the coagulation impairment. On the other hand, the management of thrombosis and hemorrhages in cancer patients can be different. To approach these problems, some guidelines have been released by prominent international scientific societies. Also actively investigated is the issue of identifying new biomarkers to classify the subjects at a higher risk, thus improving the prevention of thrombohemorrhagic events in these patients. Finally, novel prophylactic and therapeutic approaches are currently under development. This review provides an overview of the hemostatic complications in cancer, together with new insights into the interaction between hemostasis and cancer biology. We also review the assessment of the risk of thrombohemorrhagic events in cancer patients, and the prophylaxis and treatment of such manifestations.

Nitric oxide derivatives and soluble plasma selectins in patients with myeloproliferative neoplasms.

Essential thrombocythaemia (ET) and polycythaemia vera (PV) are characterised by a high incidence of thrombotic complications due to high-shear stress of the vessel wall, blood hyperviscosity and hypoxaemia, all factors responsible for chronic endothelial dysfunction and platelet and leukocyte activation. We evaluated the activation status of vascular cells in 18 consecutive ET and 14 PV patients by measuring the plasma levels of the nitric oxide derivatives (NOX) (i.e. nitrites and nitrates) and of soluble selectins of platelet (P-selectin), endothelial cell (P-selectin and E-selectin) and leukocyte (L-selectin) origin. The effect of hydroxyurea (HU) therapy on these parameters was also investigated. NOX were significantly (p<0.01) increased in ET patients treated with HU (11.5 +/- 2.6 nM) compared to non-HU treated ET (1.41 +/- 0.3 nM) and to controls (4.78 +/- 2.49 nM). Multivariate analysis confirmed HU therapy as an independent predictor of higher NOX levels in ET. In addition, NOX significantly correlated with haematocrit. Plasma P-selectin was significantly elevated in ET (350 +/- 40 ng/10(6) platelets) and PV (482 +/- 53 ng/10(6) platelets) patients compared to controls (120 +/- 8 ng/10(6) platelets). In PV, also E-selectin (23.8 +/- 4.2 ng/ml) was significantly increased compared to controls (11.2 +/- 1.1 ng/ml; p<0.01). P-selectin was significantly correlated to platelet (R=0.33; p=0.01) and leukocyte count (R=0.6; p=0.000), while E-selectin (R=0.34; p=.014) and sL-selectin (R=0.3; p=0.03) were correlated with leukocyte count only. In the multivariate analysis, NOX predicted increased levels of E-selectin in ET, but not in PV patients. Our data demonstrate that ET and PV are characterised by an altered pattern of soluble selectins and NOX. HU-mediated increase of NOX levels could represent an additional antithrombotic mechanism of this drug.

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.

Tissue Factor Expression on Platelet Surface during Preparation and Storage of Platelet Concentrates

Background: Tissue factor (TF), the main activator of blood coagulation, is expressed on platelet surface and, together with procoagulant phospholipids, contributes to the global coagulation potential of these blood components. The present study evaluated, for the first time, the expression of TF on platelet surface during preparation and storage of platelet concentrates (PC) for transfusional use. Methods: Platelet TF was measured by flow cytometry in healthy donor whole blood (WB) and in pooled buffy-coat-derived PC on the day of preparation and up to 4 days of storage in parallel with classical markers of platelet activation, i.e., fibrinogen, P-selectin, and glycoprotein GPIIb. Data were analyzed according to donor age and blood ABO group. Results: TF was detected on whole blood platelets and was found highest in O donors. Compared to whole blood, platelet surface TF was higher upon PC preparation and further increased during storage. The rise in TF levels positively correlated with the elevations of the other platelet markers. Conclusions: Our findings show that platelet surface TF is maintained in PC obtained by the pooled buffy coat method. Further studies are warranted to investigate a possible correlation between TF levels and the hemostatic response of the platelet transfusion recipient.

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.

Hypercoagulation screening as an innovative tool for risk assessment, early diagnosis and prognosis in cancer: the HYPERCAN study

The HYPERCAN is a prospective observational Italian multicentre study started in 2012, structured in two main projects (i.e. Projects A and B) that involve both healthy subjects and cancer patients. The HYPERCAN study aims to assess whether the occurrence of a hypercoagulable state may be predictive of cancer diagnosis in healthy individuals, or may be predictive of disease recurrence, clinical progression and thrombosis in cancer patients. Project A involves two different large cohorts of subjects: The first cohort (Project A-1) consists of 10,000 healthy volunteer blood donors to be enrolled and prospectively follow-up for cancer occurrence, while the second cohort (Project A-2) consists of 25,000 people already enrolled in the framework of the general population-based Moli-Sani study. Project B involves 4,000 adult patients with a confirmed diagnosis of four different cancer types (both limited/resected or metastatic diseases), i.e. non-small cell-lung, gastric, colorectal, and breast cancer, to be enrolled and followed up for 5years or death. Blood samples from all enrolled subjects are collected at baseline and then at different time intervals according to specific time schedules set up for either normal subjects, or patients with limited cancers, or patients with metastatic cancers. Samples will be analysed for a panel of hemostatic proteins, clotting activation biomarkers, thrombin generation, procoagulant microparticles, and thrombophilic polymorphisms. As of November 2015, 6,189 healthy blood donors have been enrolled in project A-1 and 2,532 cancer patients in project B. Clinical follow-up and biological assays are ongoing. The HYPERCAN study wants to explore in different subset of individuals, affected and non-affected by malignant disease, the relationship between coagulation and cancer. The prospective design and the involvement of a large number of individuals will definitively clarify whether alterations in circulating thrombotic markers may be predictive of cancer diagnosis in an otherwise healthy subject and/or may be prognostic of cancer outcome, or of disease progression/relapse in cancer-affected individuals. Finally, the proposed screening with relatively simple and non-high-cost laboratory tests and the use of easy-obtainable peripheral blood samples add a very relevant translational value to this study.

Coagulation in Hematological Malignancies

Patients with hematological malignancies, like patients with other types of cancer, are at high risk of thrombo/hemorrhagic complications. The incidence rate of these events is greatly variable and it is influenced by many factors, including the type and stage of disease, the therapeutic regimen, and the presence or not of thromboprophylaxis. A number of clinical risk factors contribute to the increased thrombotic rate in hematological malignancies. Biological properties of the tumor cells also influence the hypercoagulable state of these patients. Of interest, oncogenes responsible for neoplastic transformation might be involved in clotting activation. No recommendations for a routinary thromboprophylaxis in hematologic malignancies are available, with the exception of multiple myeloma. Large prospective randomized clinical trials are needed to establish the best practice for thromboprophylaxis and treatment of venous thromboembolism in these conditions.

Acute promyelocytic leukemia cell adhesion to vascular endothelium is reduced by heparins

Adhesion of acute promyelocytic leukemia (APL) cells to endothelial cells (EC) is among the mechanisms of the APL-associated coagulopathy, responsible for early hemorrhagic deaths in affected patients. We compared the effects of dalteparin and enoxaparin, two low-molecular-weight heparins (LMWH), and unfractionated heparin (UFH), on APL NB4 adhesion to micro- (HMEC-1) and macro-vascular EC (HUVEC), in resting and interleukin-1β (IL-1β)-stimulated conditions. The heparin effect on EC adhesion molecule (ICAM-1, VCAM-1, E-selectin) expression was also assessed. In HMEC-1, dalteparin inhibited IL1β-induced NB4 adhesion by 80%, enoxaparin by 52%, and UFH by 44%. Similar results were obtained in HUVEC. This was associated with a significant decrease of VCAM-1 and ICAM-1 expression. In conclusion, we show that LMWH significantly counteract APL cell adhesion to the vessel wall, by modulating EC adhesion molecule expression. This property of heparins may represent one approach for hampering excess clotting activation and microthrombi deposition in APL.

PO-85 Effects of an anthocyanin (delphinidin-3-glucoside) from wild blueberries on the proangiogenic and prothrombotic properties of endothelial cells

CFPAC1 (Table). The clotting times (CT) mirrored TF expression levels with CFPAC1 giving the fastest time and MIAPaCA-2 the slowest. Blocking of cell-expressed TF with a saturating concentration of a polyclonal antibody (American diagnostica) increased CT in all cases. The highest cell invasion was also observed with CFPAC1, followed by AsPC1 and MIAPaCa-2. Blocking of TF decreased cell invasion to approximately 50% in both CFPAC1 and AsPC1, but showed minimal effect on the already low levels observed with MIAPaCa-2. Conclusions: Clotting time and cell invasion appear to be strongly correlated, with both processes being mediated, at least in part, via TF.