Monocyte recruitment in venous pulmonary embolism at time of cancer diagnosis in upper gastrointestinal cancer patients.

Upper gastrointestinal cancer is frequently complicated by venous thromboembolisms (VTE), especially pulmonary embolisms (PE) increase the mortality rate. Monocytes are a part of the innate immune system and up-regulation may indicate an ongoing inflammatory response or infectious disease and has lately been associated with a moderate risk of suffering from VTE. This prospectively study aims to compare the incidence of pulmonary embolism with markers of coagulation and compare it to the absolute monocyte count. A consecutive cohort of 250 patients with biopsy proven upper gastrointestinal cancer (i.e. pancreas, biliary tract, esophagus and gastric cancer) where included at the time of cancer diagnosis and before treatment. All patients underwent bilateral compression ultrasonography for detection of deep vein thrombosis (DVT). Of these 143 had an additionally pulmonary angiografi (CTPA) with the staging computer tomography. 13 of 250 patients (5.2%) had a DVT and 11 of 143 (7.7%) had CTPA proven PE. PE was significantly more common among patients with elevated D-dimer (OR 11.62, 95%CI: 1.13-119, P = 0.039) and elevated absolute monocyte count (OR 7.59, 95%CI: 1.37-41.98, P = 0.020). Only patients with pancreatic cancer had a significantly higher risk of DVT (OR 11.03, 95%CI: 1.25-97.43, P = 0.031). The sensitivity of absolute monocyte count was 63.6 (95%CI: 30.8-89.1) and specificity 80.3 (95%CI: 72.5-86.7), with a negative predictive value of 96.4 (95%CI: 91-99) in PE. An increased absolute monocyte count was detected in patients suffering from PE but not DVT, suggesting a possible interaction with the innate immune system.

Survival after cancer-related venous thrombosis: the Scandinavian Thrombosis and Cancer Study.

Patients with cancer have an increased risk of developing venous thromboembolism (VTE), and this combination is reported to result in poorer survival compared with cancer alone. This study aimed to investigate the impact of VTE on the survival of patients with cancer in a general population. The Scandinavian Thrombosis and Cancer (STAC) cohort, a population-based cohort including 144 952 participants without previous VTE or cancer, was used. During follow-up, cancer and VTE incidences were registered. "Cancer-related VTE" was defined as VTE diagnosed in patients with overt or occult cancer. The survival of participants without cancer and/or VTE ("disease-free") was compared with the survival of participants with cancer and cancer-related VTE. Cox regression models with cancer and VTE as time-varying exposures were performed to calculate hazard ratios for death. Subanalyses were performed across cancer types and stages and VTE type (deep vein thrombosis or pulmonary embolism). During follow-up (mean, 11.7 years), 14 621 participants developed cancer, and 2444 developed VTE, of which 1241 were cancer-related. The mortality rates (per 100 person years) for disease-free participants, VTE only, cancer only, and cancer-related VTE were 0.63, 5.0, 9.2, and 45.3, respectively. Compared with patients with cancer only, the risk of death for patients with cancer-related VTE was increased 3.4-fold. Within all cancer types, the occurrence of VTE increased the mortality risk 2.8- to 14.7-fold. In a general population, patients with cancer with VTE had a 3.4-fold higher mortality risk than patients with cancer without VTE, independent of cancer type.

Association of smoking and cancer with the risk of venous thromboembolism: the Scandinavian Thrombosis and Cancer cohort.

Smoking is a well-established risk factor for cancer, and cancer patients have a high risk of venous thromboembolism (VTE). Conflicting results have been reported on the association between smoking and risk of VTE, and the effect of smoking on VTE-risk in subjects with cancer is scarcely studied. We aimed to investigate the association between smoking and VTE in subjects with and without cancer in a large population-based cohort. The Scandinavian Thrombosis and Cancer (STAC) cohort included 144,952 participants followed from 1993-1997 to 2008-2012. Information on smoking habits was derived from self-administered questionnaires. Active cancer was defined as the first two years following the date of cancer diagnosis. Former smokers (n = 35,890) and those with missing information on smoking status (n = 3680) at baseline were excluded. During a mean follow up of 11 years, 10,181 participants were diagnosed with cancer, and 1611 developed incident VTE, of which 214 were cancer-related. Smoking was associated with a 50% increased risk of VTE (HR 1.49, 95% CI 1.12-1.98) in cancer patients, whereas no association was found in cancer-free subjects (HR 1.07, 95% CI 0.96-1.20). In cancer patients, the risk of VTE among smokers remained unchanged after adjustment for cancer site and metastasis. Stratified analyses showed that smoking was a risk factor for VTE among those with smoking-related and advanced cancers. In conclusion, smoking was associated with increased VTE risk in subjects with active cancer, but not in those without cancer. Our findings imply a biological interaction between cancer and smoking on the risk of VTE.

Prothrombotic genotypes and risk of venous thromboembolism in occult cancer.

BACKGROUND: Studies have reported that the combination of some prothrombotic genotypes and overt cancer yields a synergistic effect on VTE risk. Whether individual prothrombotic genotypes or number of risk alleles in a genetic risk score (GRS) affect VTE risk in occult cancer have not been addressed. The aim of this study was to investigate the joint effect of five prothrombotic genotypes and occult cancer on VTE risk. METHODS: Cases with incident VTE (n = 1566) and a subcohort (n = 14,537) were sampled from the Scandinavian Thrombosis and Cancer Cohort (1993-2012). Five single nucleotide polymorphisms previously reported in a GRS were genotyped: ABO (rs8176719), F5 (rs6025), F2 (rs1799963), FGG (rs2066865) and F11 (rs2036914). Hazard ratios (HRs) for VTE by individual SNPs and GRS were estimated according to non-cancer and occult cancer (one year preceding a cancer diagnosis) exposure. RESULTS: Occult cancer occurred in 1817 subjects, and of these, 93 experienced a VTE. The VTE risk was 4-fold higher (HR 4.05, 95% CI 3.28-5.00) in subjects with occult cancer compared with those without cancer. Among subjects with occult cancer, those with VTE had a higher proportion of prothrombotic and advanced cancers than those without VTE. The VTE risk increased according to individual prothrombotic genotypes and GRS in cancer-free subjects, while no such effect was observed in subjects with occult cancer (HR for ≥4 versus ≤1 risk alleles in GRS: 1.14, 95% CI 0.61-2.11). CONCLUSIONS: Five well-established prothrombotic genotypes, individually or combined, were not associated with increased risk of VTE in individuals with occult cancer.

Venous thromboembolism after lower extremity orthopedic surgery: A population-based nationwide cohort study.

BACKGROUND: Venous thromboembolism (VTE) causes morbidity and mortality in the general population. Several events occur after lower limb orthopedic surgery, but the contribution from various types of lower limb surgery is not well known. OBJECTIVE: To investigate the postoperative incidence of VTE for all types of lower extremity orthopedic surgery compared with the background population. METHODS: Individual-level linkage of Danish nationwide register data for all Danish residents with first-time orthopedic surgery of the lower limb (1996-2017) and, for each of these, four controls from the general population matched on age, sex, and history of VTE. Adjusted hazard ratios (HR) compared the postoperative risk of VTE to the matched controls. RESULTS: In total 7203 of the 1 012 823 patients with a first orthopedic procedure had a VTE within 180 days after surgery, corresponding to a postoperative cumulative incidence of 0.71% (95% confidence interval [CI], 0.70-0.73). The cumulative incidence of VTE among controls was 0.11% (95% CI, 0.11-0.12). The HR of VTE within the first 30 days after surgery below knee level was 20.5 (95% CI, 17.9-23.5) compared with matched controls. The HRs of VTE after minor distal procedures (eg, meniscectomy and arthroscopies) were 2.9 (95% CI, 1.9-4.4) to 7.1 (95% CI, 6.4-8.0). CONCLUSION: All types of lower limb orthopedic surgery including minor distal procedures were associated with higher rates of VTE compared with matched controls, in particular within the first 30 days after surgery.

MicroRNAs and Neutrophil Activation Markers Predict Venous Thrombosis in Pancreatic Ductal Adenocarcinoma and Distal Extrahepatic Cholangiocarcinoma.

Cancer-associated venous thrombosis (VTE) increases mortality and morbidity. However, limited tools are available to identify high risk patients. Upon activation, neutrophils release their content through different mechanisms, thereby prompting thrombosis. We explored plasma microRNAs (miRNAs) and neutrophil activation markers to predict VTE in pancreatic ductal adenocarcinoma (PDAC) and distal extrahepatic cholangiocarcinoma (DECC). Twenty-six PDAC and 6 DECC patients recruited at cancer diagnosis, were examined for deep vein thrombosis and pulmonary embolisms, and were then followed-up with clinical examinations, blood collections, and biCUS. Ten patients developed VTE and were compared with 22 age- and sex-matched controls. miRNA expression levels were measured at diagnosis and right before VTE, and neutrophil activation markers (cell-free DNA, nucleosomes, calprotectin, and myeloperoxidase) were measured in every sample obtained during follow-up. We obtained a profile of 7 miRNAs able to estimate the risk of future VTE at diagnosis (AUC = 0.95; 95% Confidence Interval (CI) (0.987, 1)) with targets involved in the pancreatic cancer and complement and coagulation cascades pathways. Seven miRNAs were up- or down-regulated before VTE compared with diagnosis. We obtained a predictive model of VTE with calprotectin as predictor (AUC = 0.77; 95% CI (0.57, 0.95)). This is the first study that addresses the ability of plasma miRNAs and neutrophil activation markers to predict VTE in PDAC and DECC.

Real-Time Interferometric Refractive Index Change Measurement for the Direct Detection of Enzymatic Reactions and the Determination of Enzyme Kinetics.

Back scatter interferometry (BSI) is a sensitive method for detecting changes in the bulk refractive index of a solution in a microfluidic system. Here we demonstrate that BSI can be used to directly detect enzymatic reactions and, for the first time, derive kinetic parameters. While many methods in biomedical assays rely on detectable biproducts to produce a signal, direct detection is possible if the substrate or the product exert distinct differences in their specific refractive index so that the total refractive index changes during the enzymatic reaction. In this study, both the conversion of glucose to glucose-6-phosphate, catalyzed by hexokinase, and the conversion of adenosine-triphosphate to adenosine di-phosphate and mono-phosphate, catalyzed by apyrase, were monitored by BSI. When adding hexokinase to glucose solutions containing adenosine-triphosphate, the conversion can be directly followed by BSI, which shows the increasing refractive index and a final plateau corresponding to the particular concentration. From the initial reaction velocities, KM was found to be 0.33 mM using Michaelis⁻Menten kinetics. The experiments with apyrase indicate that the refractive index also depends on the presence of various ions that must be taken into account when using this technique. This study clearly demonstrates that measuring changes in the refractive index can be used for the direct determination of substrate concentrations and enzyme kinetics.

Elevated blood plasma levels of tissue factor-bearing extracellular vesicles in patients with atrial fibrillation.

BACKGROUND: The risk of thrombus formation in the left atrial appendage (LAA) in patients with atrial fibrillation (AF) may result from blood stasis, local endocardial changes, and/or changed blood composition. Extracellular vesicles (EVs), especially subtypes exposing tissue factor (TF), have procoagulant capacity. We hypothesized that blood concentrations of TF-bearing EVs and other procoagulant biomarkers are elevated in AF patients, particularly in the LAA lumen. METHODS: From 13 AF patients and 12 controls a venous blood sample was drawn prior to cardiac surgery. Intraoperatively, venous blood and blood directly from the LAA was drawn. Plasma levels of EVs, including TF- and cell type specific antigen-bearing EVs, were measured using a protein microarray platform. Plasma levels of TF, von Willebrand factor (vWF), cell free deoxyribonucleic acid (cf-DNA), procoagulant phospholipids (PPLs), and total submicron particles were also evaluated. RESULTS: Significantly higher EV levels, including a several-fold higher median level of TF-bearing EVs were measured in AF patients compared with controls. Median concentrations of TF and vWF were approximately 40% and 30% higher, respectively, in the AF group than in the control group, while no significant differences in levels of cf-DNA, PPLs, or total submicron particles were observed. No significant differences in levels of any of the measured analytes were observed between intraoperative venous and LAA samples. CONCLUSIONS: Increased plasma concentrations of TF in AF patients are accompanied and probably at least partly explained by increased levels of TF-bearing EVs, which may be mechanistically involved in increased thrombogenicity in AF patients.

Long-Term Incidence of Venous Thromboembolism in Cancer: The Scandinavian Thrombosis and Cancer Cohort.

The risk of venous thromboembolism (VTE) in patients who survive the first years after a cancer diagnosis after the acute effects of disease and treatment in comparison to a similar background population has been sparsely investigated. The aim of the study was to investigate if incidence rates (IRs) of VTE differed in patients who were alive at least 2 years after a cancer diagnosis without VTE compared with cancer-free references in a population-based cohort study. The study entry was 2 years after a first cancer diagnosis. For each cancer-exposed subject, five reference subjects were identified within the cohort. The IRs were calculated as number of VTEs per 1,000 person years (×10 -3 p-y) in total and in distinct cancer types and corresponding reference subjects. Incidence rate ratios (IRRs) were calculated by Poisson's regression. During a mean follow-up of 5.3 years, 110 VTEs occurred among the 7,288 cancer-exposed subjects and 321 VTEs occurred among the 36,297 identified reference subjects. The IR of VTE was higher for cancer-exposed subjects compared with reference subjects, IRs 3.7 × 10 -3 p-y, 95% CI: 3.1 to 4.5 and 1.9 × 10 -3 p-y, 95% CI: 1.7 to 2.2, respectively. IRs of VTE in most solid cancer types declined to almost the same level as in the reference subjects 2 years after cancer diagnosis, but remained higher in hematological cancers, IRR 4.0, 95% CI: 2.0 to 7.8.

Existing data sources in clinical epidemiology: the Scandinavian Thrombosis and Cancer Cohort.

BACKGROUND: Although venous thromboembolism (VTE) is a known common complication in cancer patients, there is limited knowledge on patient-related and cancer-specific risk factors in the general population. The Scandinavian Thrombosis and Cancer (STAC) Cohort was established by merging individual data from three large Scandinavian cohorts (The Tromsø Study, the second Nord-Trøndelag Health Study, and the Danish Diet, Cancer and Health Study). Here, we present the profile of the STAC cohort and provide age-specific incidence rates of VTE and cancer. METHODS: The STAC cohort includes 144,952 subjects aged 19-101 years without previous VTE or cancer. Baseline information collected in 1993-1997 included physical examination, self-administered questionnaires, and blood samples. Validated VTE events and cancer diagnoses were registered up to 2007-2012. RESULTS: There were 2,444 VTE events (1.4 per 1,000 person-years [PY]) during follow-up, and the incidence increased exponentially from 0.3 per 1,000 PY in subjects aged 20-29 years to 6.4 per 1,000 PY in subjects aged 80+. Overall, 51% of the VTE events were provoked, and cancer was the most common provoking factor (19%), followed by immobilization and surgery (both 15%). In total, 19,757 subjects developed cancer during follow-up (9.8 per 1,000 PY), and the 5-year age-specific incidence rates of cancer were coherent with corresponding rates from the Norwegian Cancer Registry. CONCLUSION: The STAC cohort will provide a unique opportunity to explore the epidemiology and impact of genetic and environmental patient-related and cancer-specific risk factors for VTE in the general population.

Impact of incident venous thromboembolism on risk of arterial thrombotic diseases.

BACKGROUND: Growing evidence supports an association between venous thromboembolism (VTE) and arterial thrombotic diseases (ie, myocardial infarction and ischemic stroke). We aimed to study the association between VTE and future arterial events and to determine the population attributable risk of arterial events by VTE in a large prospective cohort recruited from the general population. METHODS AND RESULTS: In 1994 to 1995 and 1993 to 1997, 81 687 subjects were included in the Tromsø Study and in the Diet, Cancer and Health Study and followed up to the date of incident venous and arterial events (myocardial infarction or ischemic stroke), death or migration, or to the end of the study period (2010 and 2008, respectively). There were 1208 cases of VTE and 90 subsequent arterial events during a median follow-up of 12.2 years. An association between VTE and future arterial events was found in all women and men aged <65 years but not in men aged >65 years. Women <65 years old with VTE had 3.3-fold higher risk of arterial disease (adjusted hazard ratio, 3.28; 95% confidence interval, 1.69-6.35) compared with women of the same age without VTE. The corresponding hazard ratio in men aged <65 years was 2.06 (95% confidence interval, 1.32-3.20). Only 0.9% of the arterial events were attributed to VTE, and the VTE explained 63.8% of the risk of arterial events among VTE patients. CONCLUSIONS: Our findings imply that women and young men with VTE have higher risk of arterial thrombotic disease than those without VTE. However, only 1% of the arterial thrombotic events in the population are attributed to VTE.

Extracellular Vesicle (EV) Array: microarray capturing of exosomes and other extracellular vesicles for multiplexed phenotyping.

BACKGROUND: Exosomes are one of the several types of cell-derived vesicles with a diameter of 30-100 nm. These extracellular vesicles are recognized as potential markers of human diseases such as cancer. However, their use in diagnostic tests requires an objective and high-throughput method to define their phenotype and determine their concentration in biological fluids. To identify circulating as well as cell culture-derived vesicles, the current standard is immunoblotting or a flow cytometrical analysis for specific proteins, both of which requires large amounts of purified vesicles. METHODS: Based on the technology of protein microarray, we hereby present a highly sensitive Extracellular Vesicle (EV) Array capable of detecting and phenotyping exosomes and other extracellular vesicles from unpurified starting material in a high-throughput manner. To only detect the exosomes captured on the EV Array, a cocktail of antibodies against the tetraspanins CD9, CD63 and CD81 was used. These antibodies were selected to ensure that all exosomes captured are detected, and concomitantly excluding the detection of other types of microvesicles. RESULTS: The limit of detection (LOD) was determined on exosomes derived from the colon cancer cell line LS180. It clarified that supernatant from only approximately 10(4) cells was needed to obtain signals or that only 2.5×10(4) exosomes were required for each microarray spot (~1 nL). Phenotyping was performed on plasma (1-10 µL) from 7 healthy donors, which were applied to the EV Array with a panel of antibodies against 21 different cellular surface antigens and cancer antigens. For each donor, there was considerable heterogeneity in the expression levels of individual markers. The protein profiles of the exosomes (defined as positive for CD9, CD63 and CD81) revealed that only the expression level of CD9 and CD81 was approximately equal in the 7 donors. This implies questioning the use of CD63 as a standard exosomal marker since the expression level of this tetraspanin was considerably lower.

Increased levels of citrullinated antithrombin in plasma of patients with rheumatoid arthritis and colorectal adenocarcinoma determined by a newly developed ELISA using a specific monoclonal antibody.

Citrullination is a post-translational modification that plays essential roles in both physiological processes and disease. Recent studies have found increased levels of citrullinated antithrombin in patients with rheumatoid arthritis and in different malignant tumours. Antithrombin, the main haemostatic serpin, loses its anticoagulant function via citrullination, which might contribute to the pathogenesis or thrombotic side effects of these disorders. We have developed a specific monoclonal antibody against citrullinated antithrombin. We determined the levels of citrullinated antithrombin and anti-FXa activity in plasma from 66 donors, 17 patients with rheumatoid arthritis and 77 patients with colorectal adenocarcinoma (42 suffering from venous thrombosis). Healthy subjects had negligible amounts of citrullinated antithrombin in plasma (7.9 ± 22.1 ng/ml), while it significantly increased in patients with rheumatoid arthritis or adenocarcinoma (159.7 ± 237.6 ng/ml and 36.8 ± 66.1 ng/ml), levels that, however, did not modify the plasma anticoagulant activity. Moreover, we did not find association between citrullinated antithrombin and the thrombotic risk in patients with adenocarcinoma. In conclusion, we have developed an antibody specific for citrullinated antithrombin that allows its quantification in biological samples, offering a new tool for the analysis of citrullination in different diseases. We confirm increased levels of citrullinated antithrombin in plasma of patients with rheumatoid arthritis and adenocarcinoma. This modification, probably local, could have pathological consequences in both disorders, but only affects a minor fraction of plasma antithrombin, resulting in no significant reduction of global anticoagulant activity. This result explains the absence of association of this marker with an increased risk of thrombosis in patients with colorectal adenocarcinoma.

Genetic susceptibility, smoking, obesity and risk of venous thromboembolism.

The F5 G1691A (Factor V Leiden) and F2 G20210A (prothrombin) mutations are linked to an increase in the incidence rate of venous thromboembolism (VTE), but their effects are highly variable. We investigated whether the effects of smoking and obesity might explain this variability. In a case-cohort study including the participants of the Danish Diet, Cancer and Health study, we computed incidence rates and Cox proportional hazard ratios for VTE in individuals with and without the mutations, categorized by weight and tobacco consumption. The sole effect of heavy smoking was 128 extra VTE events per 100,000 person years in individuals with the F5 G1691A mutation versus 59 in individuals without. The sole effect of obesity was 222 extra VTE events per 100,000 person years in individuals with the F5 G1691A mutation, versus 103 in individuals without this mutation; and 705 extra VTE events per 100,000 person years in individuals with the F2 G20210A mutation versus 107 in individuals without this mutation. The F5 G1691A and F2 G20210A mutations conferred increased susceptibility to the unfavourable effects of smoking and obesity on the risk for VTE. Thus, individuals with genetic risk factors for VTE might benefit from maintaining a healthy lifestyle.

Effects of aspirin and clopidogrel in healthy men measured by platelet aggregation and PFA-100.

There are no generally accepted definitions for low-response (frequently called resistance) to the platelet inhibitors, aspirin and clopidogrel. Low-response may increase the risk of cardiovascular events in atherosclerotic patients. We aimed to define the normal drug responses in healthy men. Platelet function was measured in 20 healthy men during 11 days of aspirin or clopidogrel intake, using light transmission aggregometry (LTA) and the Platelet Function Analyzer 100 (PFA-100). The lower limits for LTA at baseline were 64% and 61%, using arachidonic acid and ADP as agonists, respectively. During aspirin intake the LTA results were stable from day to day, and an upper limit of 9% arachidonic acid stimulated aggregation was found. Clopidogrel intake was best shown by ADP induced aggregation. However, two out of 20 individuals exhibited low-response to clopidogrel. In the remaining 18 volunteers an upper limit of 48% aggregation was found. We found an upper limit for collagen-epinephrine stimulated PFA-100 results of 166 s at baseline. During aspirin intake, these results varied considerably from day to day in nine out of 20 men, resulting in an overlap between the reference ranges at baseline and during therapy. In conclusion, platelet inhibition by aspirin and clopidogrel assessed by aggregometry was stable during 11 days of treatment and reference ranges were established. The PFA-100 results varied greatly and low-response was not precisely defined by this method.

Effect of long-term hormone replacement therapy on plasma homocysteine in postmenopausal women: a randomized controlled study.

OBJECTIVE: The purpose of this study was to investigate the long-term effect of hormone replacement therapy on total homocysteine and to study whether there was any difference in effect between opposed and unopposed hormone replacement therapy or whether the methylenetetrahydrofolate reductase C677T polymorphism was associated with the effect of hormone replacement therapy on total homocysteine. STUDY DESIGN: Two hundred nine healthy postmenopausal women were randomized to hormone replacement therapy (n = 103) or no substitution (n = 106) 5 to 7 years earlier. RESULTS: Women who received hormone replacement therapy had significantly lower total homocysteine concentrations than women in the control group; median total homocysteine values were 8.6 micromol/L and 9.7 micromol/L, respectively, in a per-protocol analysis (P =.02). The effect was comparable in all methylenetetrahydrofolate reductase genotypes, and no difference between unopposed and opposed hormone replacement therapy could be demonstrated. Similar results were obtained when an intention-to-treat analysis was performed. CONCLUSION: Long-term hormone replacement therapy results in lower total homocysteine concentrations in all methylenetetrahydrofolate reductase genotypes without demonstrable difference in effect between unopposed and opposed hormone replacement therapy.