Enhanced cell‐associated plasminogen activator pathway but not coagulation pathway activity contributes to motility in metastatic breast cancer cells

Summary. Background: Activation of tumor cell‐associated coagulation and plasminogen activator pathways occurs in malignant disease processes, including breast cancer, and may promote metastatic activity. Objectives/Methods: To compare the coagulation and plasminogen activator pathways of normal and metastatic cells, we examined two cell lines from the MCF‐10 family of breast cells: near‐normal immortalized MCF‐10A cells, and metastatic MCF‐10CA1 cells. Results: MCF‐10CA1 cell motility was significantly increased as compared with that of MCF‐10A cells. The two cell types supported similar rates of factor Xa generation, plasma thrombin generation, and fibrin formation. MCF‐10A cells produced a stable fibrin network, whereas MCF‐10CA1 cells lysed the surrounding fibrin network within 24 h of network formation. Importantly, fibrin located proximal to (within 10 μm) the MCF‐10CA1 cell surface lysed substantially faster than fibrin located 100 μm from the surface. MCF‐10CA1 cells supported significantly increased plasmin generation rates as compared with MCF‐10A cells, providing a mechanism for the increased fibrinolytic activity of these cells towards the fibrin network. Metastatic MCF‐10CA1 cells had increased expression (mRNA and protein) levels of urokinase plasminogen activator (u‐PA) and decreased levels of plasminogen activator inhibitor‐1 as compared with MCF‐10A cells. Blocking u‐PA activity with the active site‐directed protease inhibitor amiloride substantially decreased MCF‐10CA1 cell motility. Phosphorylated Akt levels were elevated in MCF‐10CA1 cells, which partially explains the increased u‐PA expression. Conclusions: These results suggest that the tumor‐associated plasminogen activator pathway, not the coagulation pathway, is a key distinguishing feature between metastatic MCF10‐CA1 cells and normal MCF‐10A cells.     

The chemotherapy metabolite acrolein upregulates thrombin generation and impairs the protein C anticoagulant pathway in animal‐based and cell‐based models

Summary. Background: Thrombosis is a common complication in cancer patients receiving chemotherapy regimens that include cyclophosphamide. However, the mechanisms by which these agents increase this risk are largely uncharacterized. Objectives: To examine the effects of cyclophosphamide and its metabolite acrolein on procoagulant and anticoagulant pathways in both cell‐based and animal‐based models. Methods: Thrombin and activated protein C (APC) generation were measured in defibrinated plasma exposed to acrolein‐treated endothelial and smooth muscle cells. Tissue factor (TF) activity was measured on acrolein‐treated cells. Cell surface levels of phosphatidylserine, TF, endothelial protein C receptor and thrombomodulin were measured. Healthy BALB/c mice received injections of saline (control), acrolein, or cyclophosphamide; blood was collected, and plasma thrombin–antithrombin (TAT) complex, protein C and APC levels were analyzed. Results: Exposure of acrolein‐treated endothelial and smooth muscle cells to defibrinated plasma increased thrombin generation in the plasma. This was associated with enhanced phosphatidylserine exposure and/or increased TF activity on acrolein‐treated cells. Despite elevated levels of thrombin generation, plasma APC levels were not elevated. In vivo, treatment of mice with cyclophosphamide and acrolein resulted in elevations of plasma TAT complex levels, whereas APC levels remained low. Conclusions: This is the first study to examine thrombin generation and the APC pathway in chemotherapy‐treated mice. Cyclophosphamide and acrolein appear to upregulate procoagulant pathways, while impairing endogenous anticoagulant pathways. This may explain, in part, the increased risk of thrombosis observed in cancer patients receiving cyclophosphamide‐containing chemotherapy.     

The potential use of urine cell free DNA as a marker for cancer

Introduction: Although the role of circulating cell free DNA in cancer has been widely demonstrated, less is known about the role of urine cell free DNA (UcfDNA). UcfDNA can serve as a ‘liquid biopsy’ for urological and non-urological tumors, as it carries information on DNA from cells exfoliated in urine and from circulation.

Areas covered: We review the studies on UcfDNA as a source of biomarkers for cancer, focusing on the new techniques and the differences between urological and non-urological tumors. We searched Pubmed for articles published between 1998 and 2016 with the following key words and phrases: ‘urine’ and ‘cell free DNA’ or ‘liquid biopsy’ or ‘cancer’.

Expert commentary: Despite the few papers published on this topic, UcfDNA is an important component of ‘liquid biopsy’, a useful and non-invasive tool for cancer diagnosis, prognosis and treatment monitoring, containing a wide range of genetic information.     

Citrullinated histone H3, a biomarker of neutrophil extracellular trap formation,predicts the risk of venous thromboembolism in cancer patients

Essentials

• Neutrophil extracellular traps (NETs) might play a role in cancer‐related coagulopathy.
• We determined NET biomarkers and followed cancer patients for venous thromboembolism (VTE).
• We found a constant association with VTE for citrullinated histone H3.
• Biomarkers of NET formation could reflect a novel pathomechanism of cancer‐related VTE.

Summary

Background

Neutrophil extracellular traps (NETs) are decondensed chromatin fibers that might play a role in the prothrombotic state of cancer patients.

Objectives

To investigate whether the levels of citrullinated histone H3 (H3Cit), a biomarker for NET formation, cell‐free DNA (cfDNA) and nucleosomes predict venous thromboembolism (VTE) in cancer patients.

Patients/Methods

Nine‐hundred and forty‐six patients with newly diagnosed cancer or progression after remission were enrolled in this prospective observational cohort study. H3Cit, cfDNA and nucleosome levels were determined at study inclusion, and patients were followed for 2 years. VTE occurred in 89 patients; the cumulative 3‐month, 6‐month, 12‐month and 24‐month incidence rates of VTE were 3.7%, 6.0%, 8.1%, and 10.0%, respectively.

Results

Patients with elevated H3Cit levels (> 75th percentile of its distribution, n = 236) experienced a higher cumulative incidence of VTE (2‐year risk of 14.5%) than patients with levels below this cut‐off (2‐year risk of 8.5%, n = 710). In a competing‐risk regression analysis, a 100 ng mL^?1 increase in H3Cit level was associated with a 13% relative increase in VTE risk (subdistribution hazard ratio [SHR] 1.13, 95% confidence interval [CI] 1.04–1.22). This association remained after adjustment for high VTE risk and very high VTE risk tumor sites, D‐dimer level, and soluble P‐selectin level (SHR 1.13, 95% CI 1.04–1.22). The association of elevated nucleosome and cfDNA levels with VTE risk was time‐dependent, with associations with a higher risk of VTE only during the first 3–6 months.

Conclusion

These data suggest that biomarkers of NET formation are associated with the occurrence of VTE in cancer patients, indicating a role of NETs in the pathogenesis of cancer‐associated thrombosis.

     

Information needs of cancer patients receiving chemotherapy at a day‐case unit in Northern Ireland

? Recent restructuring of cancer services in the United Kingdom and advances in cancer chemotherapy have resulted in the majority of chemotherapy being delivered on an outpatient basis. ? Patients receiving chemotherapy are at risk of developing multiple problems and increased anxiety levels and are now more likely to encounter these problems outside the hospital setting. Therefore, providing all the necessary information at chemotherapy sessions is crucial to self‐care activities and coping. ? A study was carried out to investigate the information needs of patients at various stages of chemotherapy treatment and this article reports these needs at the beginning of treatment. ? Most patients wanted to receive all possible information about their condition and reported satisfaction with the information provided. ? The overwhelming finding was lack of information given to patients regarding family relationships. Whilst almost all patients wanted this information, more than half reported that it had not been given.     

Thrombin‐inhibiting perfluorocarbon nanoparticles provide a novel strategy for the treatment and magnetic resonance imaging of acute thrombosis

Summary. Background: As a regulator of the penultimate step in the coagulation cascade, thrombin represents a principal target of direct and specific anticoagulants. Objective: A potent thrombin inhibitor complexed with a colloidal nanoparticle was devised as a first‐in‐class anticoagulant with prolonged and highly localized therapeutic impact conferred by its multivalent thrombin‐absorbing particle surface. Methods: PPACK (Phe[D]‐Pro‐Arg‐Chloromethylketone) was secured covalently to the surface of perfluorocarbon‐core nanoparticle structures. PPACK and PPACK nanoparticle inhibition of thrombin were assessed in vitro via thrombin activity against a chromogenic substrate. In vivo antithrombotic activity of PPACK, heparin, non‐functionalized nanoparticles and PPACK nanoparticles was assessed through intravenous (i.v.) administration prior to acute photochemical injury of the common carotid artery. Perfluorocarbon particle retention in extracted carotid arteries from injured mice was assessed via ¹⁹F magnetic resonance spectroscopy (MRS) and imaging (MRI) at 11.7 T. Activated partial thromboplastin time (APTT) measurements determined the systemic effects of the PPACK nanoparticles at various times after injection. Results: An optical assay verified that PPACK nanoparticles exceeded PPACK’s intrinsic activity against thrombin. Application of an in vivo acute arterial thrombosis model demonstrated that PPACK nanoparticles outperformed both heparin (P = 0.001) and uncomplexed PPACK (P = 0.0006) in inhibiting thrombosis. ¹⁹F MRS confirmed that PPACK nanoparticles specifically bound to sites of acute thrombotic injury. APTT normalized within 20 min of PPACK nanoparticles injection. Conclusions: PPACK nanoparticles present thrombin‐inhibiting surfaces at sites of acutely forming thrombi that continue to manifest local clot inhibition even as systemic effects rapidly diminish and thus represent a new platform for localized control of acute thrombosis.     

Increased levels of free thyroxine and risk of venous thrombosis in a large population‐based prospective study

Summary. Background: Recent studies have shown that high levels of free thyroxine (FT4), even without leading to hyperthyroidism, are associated with a procoagulant state. Objectives: The aim of our study was to determine whether high levels of thyroid hormones are associated with an increased risk of venous thrombosis. Patients/Methods: From a prospective nested case‐cohort design within the second Nord‐Trøndelag Health Study (HUNT2) cohort (1995–1997; 66 140 subjects), all patients with venous thrombosis during follow‐up (n = 515) and 1476 randomly selected age‐stratified and sex‐stratified controls were included. Relative and absolute risks for venous thrombosis were calculated for different cut‐off levels of thyroid hormones on the basis of percentiles in the controls and different times between blood sampling and thombosis. Results: In subjects with an FT4 level above the 98th percentile (17.3 pmol L^?1), the odds ratio (OR) was 2.5 (95% confidence interval [CI] 1.3–5.0) as compared with subjects with levels below this percentile. For venous thrombosis within 1 year from blood sampling, this relative risk was more pronounced, with an OR of 4.8 (95% CI 1.7–14.0). Within 0.5 years, the association was even stronger, with an OR of 9.9 (95% CI 2.9–34.0, adjusted for age, sex, and body mass index). For thyroid‐stimulating hormone, the relationship was inverse and less pronounced. The absolute risk within 6 months in the population for FT4 levels above the 98th percentile was 6.1 per 1000 person‐years (95% CI 1.7–15.7). Conclusions: Levels of FT4 at the upper end of the normal range are a strong risk factor for venous thrombosis. The risk increased with higher levels of thyroxine and shorter time between blood sampling and thrombosis. Further studies on the effect of clinical hyperthyroidism are warranted.     

The genetics of gastroesophageal adenocarcinoma and the use of circulating cell free DNA for disease detection and monitoring

Introduction: Gastroesophageal adenocarcinoma (GOA) is a frequently occurring cancer worldwide with a poor clinical outcome. Adenocarcinomas of the esophagus and gastroesophageal junction have shown a recent increase in frequency, therefore there is need to increase our understanding of GOA in order to improve our ability to detect, monitor and treat the disease.

Areas covered: The authors discuss the current classification of GOA in the context of recent changes in incidence. The authors also discuss developments in the understanding of disease biology and recent discoveries from whole genome and whole exome sequencing, and studies in immunotherapy. Finally, the authors discuss the recent developments in the use of circulating tumour DNA (ctDNA). PubMed search terms were in English including ‘esophageal/gastric adenocarcinoma’, ‘gastroesophageal junctional tumour’, ‘whole genome/exome sequencing’, ‘immunotherapy’ and ‘circulating tumour DNA’.

Expert commentary: Shared biological and genetic changes in GOA suggest it can be investigated as a single disease entity with different molecular subtypes. A number of genes are recurrently mutated including TP53, SMAD4, PIK3CA and there are frequent somatic copy number alterations and high levels of chromosomal instability. A subset of these genetic alterations have been detected in ctDNA and may provide an important avenue of research for detecting minimal residual disease and response to chemo- and immunotherapies.     

Fondaparinux treatment of acute heparin‐induced thrombocytopenia confirmed by the serotonin‐release assay: a 30‐month, 16‐patient case series

See also Greinacher A. Immunogenic but effective: the HIT‐fondaparinux brain puzzler. This issue, pp 2386–8; Goldfarb MJ, Blostein MD. Fondaparinux in acute heparin‐induced thrombocytopenia: a case series. This issue, pp 2501–3. Summary. Background: Fondaparinux is theoretically an attractive agent for the treatment of immune heparin‐induced thrombocytopenia (HIT), a prothrombotic disorder caused by platelet‐activating anti‐platelet factor 4/heparin antibodies. Although reports of the use of fondaparinux for this indication have thus far been favorable, the diagnosis of HIT in most cases was not based on definitive laboratory confirmation of heparin‐dependent, platelet‐activating antibodies. Objectives: To report thrombotic and major bleeding outcomes with fondaparinux in patients with a high likelihood of having acute HIT based on clinical features and a positive result in the confirmatory platelet serotonin‐release assay (SRA), a sensitive and specific test for platelet‐activating HIT antibodies. Methods/Patients: We reviewed consecutive eligible patients with SRA‐positive HIT (mean peak serotonin release, 91% [normal, < 20%]; mean IgG‐specific PF4/heparin enzyme immunoassay result, 2.53 optical density units [normal, < 0.45 units]) in one medical center over a 30‐month period who received fondaparinux for anticoagulation during acute HIT (platelet count, < 150 × 10⁹ L^?1). Where available, plasma samples were used to measure thrombin–antithrombin (TAT) complex levels. Results: Sixteen patients with SRA‐positive HIT received fondaparinux: 14 surgical (11 after cardiac surgery; three after vascular surgery) and two medical (acute stroke). Fifty‐six per cent of patients had HIT‐associated thrombosis at the time of diagnosis. No patient developed new, recurrent or progressive thrombosis; one patient developed a major bleed (calf hematoma). One patient judged to have irreversible tissue necrosis before receiving fondaparinux therapy ultimately required limb amputation. TAT complex levels were reduced within 24 h of starting fondaparinux, and 13 of 13 patients were successfully switched to warfarin. Conclusion: Fondaparinux shows promise for the treatment of patients with SRA‐positive acute HIT.