Clinical models and biochemical predictors of VTE in lung cancer.

Venous thromboembolism (VTE) is a frequent complication of lung cancer and its treatment, especially in the advanced stages of disease. The risk of a pro-thrombotic state might increase through the activation of hemostasis, occurring both via the induction of a pro-coagulant activity and with platelet involvement, ultimately leading to the development of metastases. Despite the acknowledgement of an increased thrombophilic condition in cancer patients, and the experimental evidence that heparin compounds may have direct anticancer benefits, there is no univocal consent regarding VTE prevention in cancer outpatients receiving therapy. Thus, many authors highlighted the need for the development of stratification techniques to identify at-risk patients who might benefit from thromboprophylaxis. Clinical risk models were developed and validated, in order to assign high-risk patients to a proper thromboprophylaxis regimen that, however, might not be justified in all clusters. Besides, efforts have been devoted to identify candidate biomarkers that may be used in VTE risk assessment, although none has been recognized, so far, as a predictor for VTE in lung cancer patients. In this review, we will summarize the latest information concerning this very controversial topic, with focus on some of the proposed strategies to select the appropriate patients for prophylaxis.

TNF-α gene promoter polymorphisms and risk of venous thromboembolism in gastrointestinal cancer patients undergoing chemotherapy.

BACKGROUND: TNF-α has been proposed as a predictive factor for venous thromboembolism (VTE). Genetic polymorphisms could regulate TNF-α production. However, the relationship between TNFA gene variants and VTE is not clarified. This study aims to investigate the predictive role of five different TNFA gene promoter SNPs, or their haplotype combination(s), for a first VTE episode in gastrointestinal cancer out-patients treated with chemotherapy. PATIENTS AND METHODS: Serum TNF-α levels and TNFA -863C/A, -857C/T, -376G/A, -308G/A and -238G/A gene promoter polymorphisms were retrospectively evaluated in 314 subjects, including 157 controls and 157 Caucasian patients with histologically diagnosed GI cancers beginning chemotherapy delivery (5-fluorouracil either as monotherapy or in combination with platinum compounds or irinotecan). RESULTS: Haplotype analysis showed that a five-loci haplotype (CTGGG haplotype) has higher frequency in GI cancer patients who developed VTE (n = 15) during chemotherapy [odds ratio = 2.7, 95% confidence interval (CI) 1.04-7.11, P = 0.04]. GI patients who remained VTE-free did not differ in CTGGG haplotype frequency from controls. No association was observed between serum TNF-α levels and TNFA haplotype, but both were independent predictors of VTE. Approximately 20% of GI cancer patients carrying the CTGGG haplotype developed VTE compared with 4% of the remaining 101 patients (hazard ratio = 5.6, 95% CI 1.8-17.6, P = 0.003). CONCLUSION: These results suggest that TNFA might represent a candidate gene contributing to VTE pathogenesis in GI cancer patients and suggest that VTE risk during chemotherapy might be genetically identified. Validation studies are needed for translation into clinical practice.

Impact of statins on the coagulation status of type 2 diabetes patients evaluated by a novel thrombin-generation assay.

PURPOSE: Dyslipidemia is common in type 2 diabetes (T2D) and contributes to cardiovascular disease (CVD) by exacerbating atherosclerosis and hypercoagulability. Statins can stabilize atherosclerotic plaque and reduce prothrombotic status. In the present study we aimed to evaluate the coagulation activity and the effect of statins on procoagulant state of T2D patients using a novel activated protein C (APC)-dependent thrombin-generation assay. METHODS: Procoagulant status (by HemosIL ThromboPath (ThP) assay) and in vivo platelet activation (by plasma soluble (s)CD40L levels) were analyzed in a retrospective, cross-sectional study of 198 patients with long-standing T2D and 198 controls. RESULTS: Procoagulant status of T2D patients was enhanced when compared to control subjects (p < 0.0001). Similarly, sCD40L levels were increased in T2D (p < 0.0001). When testing ThP as the dependent variable in a multivariate regression model, sCD40L (p < 0.0001) and statin treatment (p = 0.019) were independent predictors of the procoagulant state of T2D patients. Subgroup analysis showed a significant improvement of coagulability in T2D patients on statins (p = 0.012). CONCLUSIONS: The use of a standardized, easy-to-run, and commercially available APC-dependent thrombin-generation assay detected the presence of a procoagulant status in a large series of patients with long-standing T2D and demonstrated a significant impact of statins in the coagulation status of patients with T2D.

Contribution of platelet-derived CD40 ligand to inflammation, thrombosis and neoangiogenesis.

CD40-CD40L interactions have been involved in inflammation and thrombosis. Several diseases are characterized by inflammation, hypercoagulability and increased prevalence of thromboembolic events. In the past decade, a series of preclinical and clinical studies has provided more insight into the pathogenetic mechanisms linking inflammatory mediators to the activation and regulation of the haemostatic system. In particular, the study of CD40-CD40L interactions has greatly contributed to understanding the role of platelets in a variety of pathophysiological conditions, including atherothrombosis, immuno-inflammatory diseases and, possibly, cancer. A wide variety of preclinical and clinical studies have generated clinical interest in the use of CD40L as a prognostic marker of thrombotic risk. However, the use of sCD40L in clinical studies requires reliable methods. For the correct interpretation of results, clinical and research laboratories and physicians must be aware of the limitations of immunoassays for this cytokine, which underlines the need for standardization of preanalytic conditions. This review will focus on biochemical evidence of CD40L involvement in platelet activation, contribution of platelet-derived CD40L to inflammation, thrombosis and neoangiogenesis, and possible methodological pitfalls regarding the appropriate specimen and preparation for laboratory evaluation of blood soluble CD40L as a biomarker in various human diseases characterized by underlying inflammation, such as atherothrombosis, cancer and immuno-inflammatory diseases.

Biological and predictive role of ERCC1 polymorphisms in cancer.

Excision repair cross-complementation group 1 (ERCC1) is a key component in DNA repair mechanisms and may influence the tumor DNA-targeting effect of the chemotherapeutic agent oxaliplatin. Germline ERCC1 polymorphisms may alter the protein expression and published data on their predictive and prognostic value have so far been contradictory. In the present article we review available evidence on the clinical role and utility of ERCC1 polymorphisms and, in the absence of a 'perfect' trial, what we call the 'sliding doors' trial, we present the data of ERCC1 genotyping in our local patient population. We found a useful predictive value for oxaliplatin-induced risk of anemia.

Evaluation of the potential role of class II histocompatibility antigen HLA-DR in platelet/tumor cell interaction.

It has been reported that HLA-DR is a potent inducer of thrombin generation. Human colorectal cells (GEO, WiDr, DLD-1, and MIP) that lack the constitutive expression of HLA-DR cause platelet aggregation through a thrombin-dependent mechanism. Treatment with recombinant human gamma-interferon induced the expression of HLA-DR in the GEO, WiDr, and DLD-1 cells, whereas the MIP cell line remained HLA-DR negative. The concurrent analysis of tumor cell/platelet interaction after gamma-interferon treatment showed a decrease in platelet proaggregating activity of either the responsive GEO (highly expressing HLA-DR) or the unresponsive MIP (HLA-DR negative) cells. Furthermore, the DLD-1 (moderately expressing HLA-DR) cells showed an increase of proaggregating activity after gamma-interferon treatment, whereas WiDr (highly expressing HLA-DR) cells did not modify their activity. These results suggest a lack of a role of HLA-DR in the in vitro platelet proaggregating activity of human colorectal tumor cells.

Reactivity to B cell epitopes within hepatitis C virus core protein and hepatocellular carcinoma.

Our aim was to investigate the existence of an association between B cell responsiveness to hepatitis C virus (HCV) core protein and progression of liver disease. In fact, the persistence of HCV infection is permitted by avoidance of viral clearance, despite chronic inflammation in the liver; this process ends with the development of hepatocellular carcinoma in many patients. On the basis of computerized prediction of antigenicity of the genomic sequence of HCV core protein, three 15-mer peptides (named Q15V, R15P, and G15V) were synthesized to be used as antigens in an enzyme immunoassay. Sera from 97 patients (65 males and 32 females) were tested: 43 patients had mild chronic liver disease (steatofibrosis, chronic persistent, or chronic active hepatitis) and 54 had cirrhosis, which was complicated by hepatocellular carcinoma (HCC) in 19. Seventy-six patients were positive to anti-HCV testing by second generation ELISA and 21 were negative. Rates of positivity for synthetic peptides in anti-HCV-positive versus anti-HCV negative patients were as follows: 53 of 76 and 0 of 21 for anti-Q15V; 41 of 76 and 0 of 21 for R15P; and 67 of 76 and 2 of 21 for G15V. Rates of positivity to anti-Q15V and anti-G15V were similar among diagnostic groups (Pearson's chi 2, 1.97, P > 0.10 and 0.45, P > 0.10), whereas anti-R15P antibodies were detected at a significantly lower rate in patients with HCC (2/13) in comparison to mild chronic liver disease (22/35) and cirrhosis (17/28) (Pearson's chi 2, 9.42, P < 0.01). We conclude that anti-R15P antibodies are uncommon in anti-HCV-positive patients with HCC. During the course of chronic HCV infection, anti-R15P testing might help to identify a subgroup at higher risk to develop HCC.

Involvement of GD3 in platelet activation. A novel association with Fcgamma receptor.

Gangliosides are sialic acid-containing glycosphingolipids present in the outer leaflet of the plasma membrane of many cell types where they modulate adhesive processes. The main population of glycolipids in resting platelets is represented by ganglioside M3 (GM3). It has been demonstrated that following platelet activation ganglioside D3 (GD3) is rapidly formed from the GM3 pool. The present study was designed to evaluate the link between platelet activation and GD3 expression and to verify whether this ganglioside might play a role in modulating signal transduction events. Our results suggest that following platelet activation, GD3 is rapidly expressed on the platelet surface and internalised to the cytoskeleton where it transiently associates first with the Src family tyrosine kinase Lyn then with the Fc receptor gamma chain. This sequence of events ultimately leads to an enhanced CD32 (the Fc receptor isoform present in platelets) expression on the platelet membrane. These data drive us to speculate that GD3 might act as second messenger in the activatory cascade, which leads to CD32 expression and triggers platelet adhesion and spreading to the subendothelial matrix.

Association between circulating adiponectin and interleukin-10 levels in android obesity: effects of weight loss.

OBJECTIVE: Adiponectin inhibits vascular inflammation and increases IL-10 mRNA expression in human macrophages. Thus, we investigated the possible relationship between plasma adiponectin and IL-10 levels and the effects of a diet-induced moderate weight loss on both cytokines. PATIENTS AND STUDY DESIGN: Plasma adiponectin and IL-10 levels were analyzed in 64 android [body mass index (BMI), > 28 kg/m2; waist to hip ratio (WHR), > or = 0.86] and 20 gynoid [BMI, > 28 kg/m2; WHR, < 0.86] obese healthy women. Android obese women (49 +/- 14 yr) had a mean BMI of 37.1 +/- 5.3 kg/m2, similar to that of gynoid obese women (49 +/- 11 yr; BMI, 33.4 +/- 2.6 kg/m2). Twenty nonobese control women (46 +/- 11 yr; BMI, 25.2 +/- 2.2 kg/m2) were also studied. In 15 android obese women, measurements were repeated after a 12-wk diet period (1200 kcal/d). RESULTS: Median adiponectin [5.2 (range, 3.3-7.8) vs. 12.1 (9.7-13.9) vs. 15.0 (12.6-18.2) microg/ml; P < 0.0001] and IL-10 [1.8 (1.2-3.3) vs. 3.5 (2.9-4.3) and vs. 4.1 (3.5-4.8) pg/ml; P < 0.0001] levels were lower in android vs. gynoid vs. nonobese women. Among android obese women, low adiponectin levels were independently related (P < 0.0001) to decreased IL-10 levels, independently of BMI, WHR, or insulin resistance. No significant change in either median adiponectin or IL-10 levels was observed after body weight reduction (8 +/- 4 kg; P < 0.01), although percent changes in adiponectin paralleled those in IL-10 (P < 0.05). CONCLUSIONS: Android obesity is associated with a concomitant reduction of IL-10 and adiponectin levels. However, the antiinflammatory status of obesity might require prolonged periods of energy-restricted diets to revert to normal.

A re-evaluation of carcinoembryonic antigen (CEA) as a serum marker for breast cancer: a prospective longitudinal study.

PURPOSE: Carcinoembryonic antigen (CEA) is still a widely used test for monitoring breast cancer, although recent reports discourage its routine use because of low sensitivity. This is a prospective study evaluating the efficacy of CEA and CA 15.3 in monitoring breast cancer. EXPERIMENTAL DESIGN: Serum CEA and CA 15.3 were measured in 2191 patients with either benign (n = 738) or malignant (n = 1453) breast diseases. Five hundred and forty-nine patients were monitored during postsurgical follow-up for either a minimum of 5 years or until time of recurrence. Fifty-three patients with metastases were also monitored during chemotherapy. RESULTS: Elevated CEA and CA 15.3 levels were found in 16.7% and 33.0% of patients, respectively. CEA sensitivity rose to 41.3% and CA 15.3 sensitivity rose to 80.8% in metastatic patients. The adjunct of CEA increased the CA 15.3 sensitivity by 6% in the overall population and by only 2.1% for patients with metastases. During postsurgical follow-up, CEA was elevated in 38.0% and CA 15.3 in 70.2% of patients with recurrence. The combination of CEA and CA 15.3 increased the overall sensitivity by only 1.4%. Longitudinal monitoring of 53 metastatic patients undergoing chemotherapy demonstrated that, when positive, both CEA and CA 15.3 paralleled response to treatment, although CA 15.3 was a significantly more powerful marker for determining response to treatment. The cost effectiveness ratio of CEA was clearly less favorable than that of CA 15.3. CONCLUSIONS: CEA monitoring should be considered an expensive and inefficient method of follow-up evaluation for breast cancer patients, and it provides no additional value when used in combination with CA 15.3.

Biological effects of a software-controlled voltage pulse generator (PhyBack PBK-2C) on the release of vascular endothelial growth factor (VEGF).

BACKGROUND: Electrical stimulation (ES) may induce vascular permeability and physiological angiogenesis. ES of rat muscles significantly increases the microvessel density and vascular endothelial growth factor (VEGF) protein levels. Thus, a pilot study was designed to analyze the effects of low-voltage electric impulses on VEGF levels in patients with dystrophic ulcers. MATERIALS AND METHODS: Circulating VEGF levels were analyzed in 9 patients undergoing an ES session with low voltage software-controlled impulses applied through topical transducers (1-9 micros width, 1-420-Hz frequency and 30-120 V strength-100 microA max). RESULTS: The session was accompanied by a peak of circulating VEGF (3-10 min from start) in all 9 patients, which was preceded by a rise of TNF-alpha (2-min) and was independently associated with soluble E-selectin levels. Nitric oxide generation was significantly improved on the day after treatment. No hemostatic activation or sustained inflammatory reaction were observed. CONCLUSION: ES may represent a safe method for augmenting VEGF-mediated vascular protection, either directly or by induction of NO.

Platelet activation in type 2 diabetes mellitus.

The abnormal metabolic state that accompanies diabetes renders arteries susceptible to atherosclerosis, being capable of altering the functional properties of multiple cell types, including endothelium and platelets. In particular, an altered platelet metabolism and changes in intraplatelet signaling pathways may contribute to the pathogenesis of atherothrombotic complications of diabetes. A variety of mechanisms may be responsible for enhanced platelet aggregation. Among them, hyperglycemia may represent a causal factor for in vivo platelet activation, and may be responsible for nonenzymatic glycation of platelet glycoproteins, causing changes in their structure and conformation, as well as alterations of membrane lipid dynamics. Furthermore, hyperglycemia-induced oxidative stress is responsible for enhanced peroxidation of arachidonic acid to form biologically active isoprostanes, which represents an important biochemical link between impaired glycemic control and persistent platelet activation. Finally, increased oxidative stress is responsible for activation of transcription factors and expression of redox-sensitive genes leading to a phenotypic switch of endothelium toward an adhesive, pro-thrombotic condition, initial platelet activation, adhesion and subsequent platelet aggregate formation. All this evidence is strengthened by the results of clinical trials documenting the beneficial effects of metabolic control on platelet function, and by the finding that aspirin treatment may even be more beneficial in diabetic than in high-risk non-diabetic patients. Attention to appropriate medical management of diabetic patients will have great impact on long-term outcome in this high-risk population.

Platelet proaggregating activity of human colorectal tumour cell lines does not correlate with the degree of differentiation.

Six human colorectal tumour cell lines with various degrees of differentiation were studied. Each of the cell lines studied showed an in vitro platelet proaggregating activity, represented by the induction of typical aggregation waves. This activity was dose-dependent and probably related to a thrombin-dependent mechanism. However, the degree of cell differentiation did not correlate with the proaggregating activity. In fact, significant differences were observed between the two well differentiated cell lines, while a comparison between well and poorly differentiated cell lines did not reveal any difference. These results were confirmed by the ultrastructural observations, indicating that similar platelet-tumour cell interaction may be found in all the cell lines studied. The present results suggest that platelet proaggregating activity of the human colorectal tumour cell lines under investigation is unrelated to their degree of differentiation.

Possible involvement of tumour cell membrane gangliosides in platelet-tumour cell interactions.

The possible correlation(s) between platelet proaggregating activity, and sialic acid content and ganglioside expression of six human colorectal tumour cell lines (CBS, GEO, HT-29, WiDr, MIP and DLD-1) was evaluated. The three cell lines (HT-29, WiDr and DLD-1) capable of inducing remarkable in vitro platelet aggregation, had significantly higher amounts of lipid-bound sialic acid than those cell lines characterised by a lower platelet proaggregating activity (GEO, CBS and MIP). High performance thin-layer chromatography demonstrated the presence of one band comigrating with GM3 in all cell lines, while GD1a and GT1b comigrating gangliosides were present only in HT-29, WiDr and DLD-1 cells. Finally, an increased platelet pro-aggregating activity of GEO and CBS cell lines was observed after the incorporation of exogenous gangliosides. The present data support the hypothesis that lipid-bound sialic acid may be involved in platelet-tumour cell interactions.

Lipoprotein(a) serum levels in patients affected by chronic obstructive pulmonary disease.

A recent study has suggested that symptoms of chronic bronchitis predict the risk of coronary disease independently of the known major cardiovascular risk factors. High serum levels of lipoprotein(a) (Lp(a)) have also been considered as an independent risk factor for coronary heart disease. Therefore, the aim of the present study was to investigate the behaviour of Lp(a) in patients affected by chronic obstructive pulmonary disease (COPD). Serum levels of total-cholesterol (TC), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), triglycerides, apolipoprotein (Apo) B-100, and Lp(a) were measured in 90 COPD patients and in 90 normal subjects matched for age, sex and smoking habit. COPD patients showed lower serum levels of Apo B-100 (P<0.0001) and Lp(a) (P<0.003) compared to controls. Conversely, TC, HDL-C, LDL-C and triglycerides were similar between patients and controls. No significant differences were found in Apo B-100 and Lp(a) levels of patients either undergoing different therapeutic regimens, or with different smoking habits. A significant correlation between Apo B-100 and Lp(a) (rho=0.433, P<0. 0001) was also observed. In conclusion, COPD patients do not show an atherogenetic lipid pattern and their increased risk of coronary disease could be attributable to different factors, such as the ongoing hypercoagulability state often associated with COPD.

A solid-phase enzyme immunoassay (EIA) for detection of HBeAg and anti-HBe.

A sensitive, reproducible and easily performed enzyme immunoassay (EIA) based on a sandwich technique is described for serological detection of HBeAg and anti-HBe. EIA appears to be 600 times more sensitive than immunodiffusion and counterimmunoelectrophoresis and its performance compares well with available radioimmunoassays.

In vivo fate of monoclonal antibody B72.3 in patients with colorectal cancer.

Radiolabeled B72.3 (anti-TAG-72) has been shown to selectively localize metastatic lesions in 70%-80% of the cases. Serum samples from 27 colorectal carcinoma patients who received iodine-131-(131I) B72.3 by i.v. administration were analyzed. Circulating immunoreactive antibody followed a biphasic clearance pattern. High-performance liquid chromatography (HPLC) and SDS-polyacrylamide gel electrophoresis demonstrated that 131I-B72.3 retained its integrity in the patients' sera. HPLC analysis also demonstrated the presence of immune complexes in the sera of 12 patients; this correlated with elevated levels of circulating TAG-72. Several different HAMA response patterns were detected in the 25 patients' sera that were analyzed; some patients developed HAMA as early as 5-7 days post-MAb injection. Higher doses of administered MAb B72.3 correlated with the development of HAMA (p = 0.007). The presence of elevated levels of TAG-72 in the patients' pre-inoculum serum was shown to correlate with the detection of lesions by gamma scanning. Serum TAG-72 may serve as a criteria for patient selection for immunodiagnostic or immunotherapeutic procedures using MAb B72.3.

Hypercoagulability state in patients with chronic obstructive pulmonary disease. Chronic Obstructive Bronchitis and Haemostasis Group.

Since there is some clinical evidence that the clinical course of patients with chronic obstructive pulmonary disease (COPD) may be complicated by thrombosis in the pulmonary vessels, we studied whether a hypercoagulability state (HS) does occur in COPD. Plasma levels of prothrombin F1 + 2 fragment, a marker of thrombin generation, D-dimer, a marker of in vivo thrombin and plasmin activation, and fibrinogen were measured in 37 COPD patients and in 30 controls matched for sex and age. COPD patients had significantly higher values of F1 + 2 (p = 0.0001) and fibrinogen (p = 0.0005) than healthy subjects. The difference persisted after excluding smoking patients. F1 + 2 was not correlated with PaO2 (r = 0.02, p > 0.05) and PaCO2 (p = 0.12, p > 0.05). In six patients with stable COPD and F1 + 2 greater than 1.65 nM (mean + 2 SD of controls) subcutaneous calcium-heparin therapy (5000 IU t.i.d. for 15 days) significantly reduced F1 + 2 (p = 0.03) and PaCO2 (p = 0.01). This study shows that COPD patients have an ongoing prothrombotic state which could potentially account for thrombosis occurring in pulmonary vessels. The effect of calcium-heparin treatment on clotting system activation and blood gas may suggest this treatment as potential candidate for prospective study in COPD patients.

Ganglioside content of human platelets--differences in resting and activated platelets.

Gangliosides may play functional roles in platelet physiology, therefore this study has been designed to evaluate whether changes in ganglioside composition may occur as a consequence of platelet activation. The results obtained indicate that lactosylceramide and GM3 are the major glycosphingolipids of human platelets. The lipid-bound sialic acid (LBSA) content was 1.27 +/- 0.04 micrograms/mg of protein. Resting platelets did not express GD3; GD3 was synthesized upon platelet activation (24 +/- 8 ng/mg of protein). The stimulation of platelets with adenosine diphosphate showed the appearance of GD3 even in the absence of degranulation. Finally, incorporation of pyrene-labeled GM3 into platelet membranes, followed by stimulation with adenosine diphosphate, resulted in the appearance of a fluorescent band comigrating with GD3. The present studies indicate that sialytransferase activation may occur as an early event following platelet stimulation, leading to GD3 synthesis mainly from the GM3 pool.