PO-25 - FATCAT: an observational cohort study investigating fractal dimension (df) as a biomarker of thrombogenicity in cancer associated thrombosis during chemotherapy for lung cancer.

INTRODUCTION: Venous thromboembolism (VTE), comprising deep vein thrombosis (DVT) and pulmonary embolism (PE) are common complications in patients with cancer, affecting up to 18% of patients. VTE risk is increased by surgery and disease progression, whilst chemotherapy further increases risk up to 7-fold compared to patients without cancer. VTE contributes significantly to morbidity and mortality in patients with cancer, and is the second most common cause of death. Lung cancer is well established to be high risk for VTE, with up to a 22-fold increase in VTE risk associated with this malignancy, and 12% incidence in a recent study of patients with lung cancer undergoing chemotherapy. Furthermore, platinum based chemotherapy agents used in treatment of lung cancer are further associated with increased VTE risk. AIM: Current risk assessment tools have little value in predicting VTE risk, but prophylactic anticoagulation of patients with cancer increases bleeding incidence and no overall survival benefit. There is therefore a need for a pragmatic test with which assesses coagulation in patients with cancer, and potentially predict VTE risk, leading to personalised management and targeted treatment. We have previously demonstrated that fractal dimension (df) is sensitive to changes in clot microstructure in patients with lung cancer, assessing global coagulation in these patients. Furthermore, df is significantly different in patients with extensive disease (stages 3&4), which conventional laboratory markers failed to identify. Given the increased risk of VTE associated with chemotherapy, FATCAT will aim to assess changes in df in a larger cohort of patients with lung cancer undergoing chemotherapy, quantifying changes in df and relating these to clinical outcome. MATERIALS AND METHODS: This is a prospective observational cohort study investigating changes in df in patients with lung cancer undergoing chemotherapy. Patients will have a new diagnosis of cytologically or histologically confirmed lung cancer planned for chemotherapy and no history of previous cancer treatment, any thromboembolic / haemostatic disorders or be on anticoagulation. RESULTS: Following a power calculation, 300 patients will be recruited and followed up for 1 year. df, Doppler ultrasonography and standard coagulation markers will be performed on recruitment, at the mid point, and on completion of chemotherapy in line with standard diagnostic procedures i.e. CT scanning. CONCLUSIONS: The primary endpoint of the study will be VTE diagnosis, whilst secondary outcomes will determine the change in df during and after treatment with chemotherapy.

Effects of exercise intensity on clot microstructure and mechanical properties in healthy individuals.

BACKGROUND: Exercise is well established to lead to exercise-induced hypercoagulability, as demonstrated by kinetic coagulation markers. It remains unclear as to whether exercise-induces changes lead in clot development and increased polymerisation. Fractal dimension (df) has been shown to act as a marker of clot microstructure and mechanical properties, and may provide a more meaningful method of determining the relationship between exercise-induced hypercoagulability and potential clot development. METHODS: df was measured in 24 healthy individuals prior to, after 5min of submaximal exercise, following maximal exercise, 45min of passive recovery and following 60min of recovery. Results were compared with conventional markers of coagulation, fibrinolysis and SEM images. RESULTS: Significantly increased df was observed following exercise, returning to resting values following 60min of recovery. The relationship between df and mature clot microstructure was confirmed by SEM: higher df was associated with dense clots formed of smaller fibrin fibres immediately following exercise compared to at rest. Conventional markers of coagulation confirmed findings of previous studies. CONCLUSION: This study demonstrates that df is a sensitive technique which quantifies the structure and properties of blood clots following exercise. In healthy individuals, the haemostatic balance between coagulation and fibrinolysis is maintained in equilibrium following exercise. In individuals with underlying vascular damage who participate in exercise, this equilibrium may be displaced and lead to enhanced clot formation and a prothrombotic state. df may therefore have the potential to not only quantify hypercoagulability, but may also be useful in screening these individuals.

Application of ROTEM to assess hypercoagulability in patients with lung cancer.

BACKGROUND: Venous thromboembolism (VTE) is common in patients with cancer, contributing significantly to morbidity and mortality Currently, no test reliably identifies patients at increased risk of developing VTE who would therefore benefit from prophylactic intervention. The aim of the current study was to evaluate rotational thromboelastometry (ROTEM) in identifying VTE risk in patients with lung cancer. We also compared parameters of ROTEM in patients with limited and extensive disease. METHODS: Parameters of ROTEM were measured in 67 patients with lung cancer and 72 age-matched healthy controls and compared with conventional markers of haemostasis. Patients were followed up for 12 months and VTE incidence recorded. RESULTS: Lung cancer patients had a reduced clotting time (CT), increased maximum clot firmness (MCF) and increased alpha angle compared with controls. Patients also had significantly higher levels of fibrinogen and PAI-1 than controls and in the former group there was a strong correlation between fibrinogen and both MCF and alpha angle. Six patients developed a VTE during the follow-up period and all had values for MCF at or above the upper limit of normal for EXTEM. CONCLUSIONS: This study demonstrates that several ROTEM parameters are significantly different in lung cancer patients compared to healthy age-matched controls, whereas only one of the parameters measured is significantly different between extensive compared to limited disease. No differences were observed between patients who developed a VTE compared to those who did not, highlighting the limitations of ROTEM use in patients with lung cancer.

Workers' exposure to dust, endotoxin and ß-(1-3) glucan at four large-scale composting facilities.

OBJECTIVES: To characterise compost workers' exposure to dust, endotoxin and ß-(1-3) glucan during various operational practices and investigate whether dust concentrations are a useful indicator of endotoxin exposure in compost workers. METHODS: This study assessed inhalable dust fractions, bacterial endotoxin and ß-(1-3) glucan in 117 personal samples and 88 ambient samples from four large-scale composting facilities. RESULTS: Employees' exposures to inhalable dust, endotoxin and ß-(1-3) glucan exhibited a large range. Inhalable dust was found to be generally low (GM 0.99 mg/m(3), GSD 2.99 mg/m(3)). Analysis of the biological component of the dust showed that employees' exposures to endotoxin were elevated (GM 35.10 EU/m(3), GSD 9.97 EU/m(3)). Employees' exposure to ß-(1-3) glucan was low (GM 0.98 ng/m(3), GSD 13.39 ng/m(3)). Dust levels were elevated during manual sorting and screening of waste and high levels of endotoxin and ß-(1-3) glucan were observed during all practices involving the movement of waste. A significant correlation was observed between the personal dust levels and personal endotoxin concentrations (r=0.783, p<0.05) and that personal inhalable dust concentration may be a valuable predictor for personal endotoxin concentration in the sites studied. CONCLUSIONS: Workers at composting sites are exposed to high levels of bacterial endotoxin consistent with adverse respiratory outcomes even though in most cases, their personal dust exposure is below the suggested regulatory levels. Dose-response data for the biological components present in the dust encountered at composting sites are not well established at this time and site operators should adopt precautionary measures when assessing and managing these potential risks.

Rosiglitazone induces the unfolded protein response, but has no significant effect on cell viability, in monocytic and vascular smooth muscle cells.

Given the safety concerns expressed over negative cardiovascular outcomes resulting from the clinical use of rosiglitazone, and the view that rosiglitazone exerts PPARγ-independent effects alongside its insulin-sensitising PPARγ-dependent effects, we hypothesised that rosiglitazone may trigger Unfolded Protein Responses (UPRs) due to disruptions in [Ca(2+)](i) homeostasis within two cardiovascular cell types: monocytic (MM6) and vascular smooth muscle (A7r5) cells. In microsomal samples derived from both cell types, pre-incubation with rosiglitazone rapidly (30min) brought about concentration-dependent PPARγ-independent inhibition of Ca(2+)ATPase activity (IC(50) ∼2µM). Fluo-3 fluorimetric data demonstrated in intact cells that 1h treatment with 1 or 10µM rosiglitazone caused Ca(2+) ions to leak into the cytoplasm. Gene expression analysis showed that within 4h of rosiglitazone exposure, the UPR transcription factor XBP-1 was activated (likely due to corresponding ER Ca(2+) depletion), and the UPR target genes BiP and SERCA2b were subsequently upregulated within 24-72h. After 72h 1 or 10µM rosiglitazone treatment, microsomal Ca(2+)ATPase activity increased to >2-fold of that seen in control microsomes, while [Ca(2+)](i) returned to basal, indicating that UPR-triggered SERCA2b upregulation was responsible for enhanced enzymatic Ca(2+) sequestration within the ER. This appeared to be sufficient to replenish ER Ca(2+) stores and restore normal cell physiology, as cell viability levels were not decreased due to rosiglitazone treatment throughout a 2-week study. Thus, incubation with 1-10µM rosiglitazone triggers the UPR, but does not prove cytotoxic, in cells of the cardiovascular system. This observation provides an important contribution to the current debate over the use of rosiglitazone in the clinical treatment of Type-2 Diabetes.

DPPC regulates COX-2 expression in monocytes via phosphorylation of CREB.

The major phospholipid in pulmonary surfactant dipalmitoyl phosphatidylcholine (DPPC) has been shown to modulate inflammatory responses. Using human monocytes, this study demonstrates that DPPC significantly increased PGE(2) (P<0.05) production by 2.5-fold when compared to untreated monocyte controls. Mechanistically, this effect was concomitant with an increase in COX-2 expression which was abrogated in the presence of a COX-2 inhibitor. The regulation of COX-2 expression was independent of NF-kappaB activity. Further, DPPC increased the phosphorylation of the cyclic AMP response element binding protein (CREB; an important nuclear transcription factor important in regulating COX-2 expression). In addition, we also show that changing the fatty acid groups of PC (e.g. using l-alpha-phosphatidylcholine beta-arachidonoyl-gamma-palmitoyl (PAPC)) has a profound effect on the regulation of COX-2 expression and CREB activation. This study provides new evidence for the anti-inflammatory activity of DPPC and that this activity is at least in part mediated via CREB activation of COX-2.

Rosiglitazone produces a greater reduction in circulating platelet activity compared with gliclazide in patients with type 2 diabetes mellitus--an effect probably mediated by direct platelet PPARgamma activation.

AIMS: Type 2 diabetes mellitus (T2DM) is associated with enhanced platelet activation. We conducted a randomised double-blind study to compare the effects of combination metformin and rosiglitazone or metformin and gliclazide therapy on platelet function in persons with T2DM. METHODS: Fifty subjects on metformin monotherapy received either rosiglitazone 4 mg or gliclazide 80 mg. HbA1c, HOMA-R, markers of platelet activation, inflammation, endothelial activation and oxidative stress were measured at baseline and after 24 weeks of treatment. Separate in vitro platelet function studies were conducted on platelets pre-incubated with rosiglitazone and gliclazide. RESULTS: A significantly greater reduction in platelet aggregation was observed in the rosiglitazone treated group compared to gliclazide. HbA1c and markers of endothelial activation were reduced to a similar extent in both groups. A significant reduction in HOMA-R, markers of inflammation and oxidative stress was only observed with rosiglitazone. Reduction in platelet aggregation with rosiglitazone correlated with reduction in oxidative stress. In the in vitro study, rosiglitazone produced significantly greater reduction in platelet aggregation compared with gliclazide. CONCLUSION: Greater reduction in platelet activity observed with rosiglitazone may be related to reduced oxidative stress and a possible direct PPARgamma mediated effect on platelet function.

Insulin resistance and inflammation - A further systemic complication of COPD.

Chronic obstructive pulmonary disease (COPD) is associated with a continuous systemic inflammatory response. Furthermore, COPD is associated with an excess risk for cardiovascular disease and type II diabetes. Systemic inflammation in other populations is a factor in atherogenesis and has been associated with insulin resistance. We assessed the association between systemic inflammation and insulin resistance in non-hypoxaemic patients with COPD. Fasting plasma glucose, insulin and inflammatory mediators were measured in 56 patients and 29 healthy subjects. Body mass index (BMI) and height squared fat- and fat-free-mass index were similar between subject groups. Using homeostatic modelling techniques, mean (SD) insulin resistance was greater in the patients, 1.68 (2.58) and 1.13 (2.02) in healthy subjects, p=0.032. Fasting plasma insulin was increased in patients while glucose was similar to that in healthy subjects. Patients had increased circulating inflammatory mediators. Insulin resistance was related to interleukin-6 (IL-6), r=0.276, p=0.039, and tumour necrosis factor alpha soluble receptor I, r=0.351, p=0.008. Both IL-6 and BMI were predictive variables of insulin resistance r(2)=0.288, p<0.05. We demonstrated greater insulin resistance in non-hypoxaemic patients with COPD compared with healthy subjects, which was related to systemic inflammation. This relationship may indicate a contributory factor in the excess risk of cardiovascular disease and type II diabetes in COPD.