Inflammation and Thrombophilia in Pregnancy Complications: Implications for Risk Assessment and Clinical Management.

In Italy, each year 500,000 couples refer to specialized centers due to reproductive problems. Among them, recurrent pregnancy loss (RPL) represents a problem of great importance, given that it affects up to 5% of women of childbearing age. Infertility, on the other hand, is a condition that currently covers 10-20% of couples of reproductive age, being idiopathic in 20% of cases. Accumulating evidence support the concept that changes of blood coagulation, generically defined as the presence of a thrombophilic state (congenital or acquired), are the basis of 40-70% of cases of multiple abortions or infertility. Several evidences support the hypothesis that endothelial dysfunction, a hallmark of a condition of low-grade inflammation, is one of the earliest manifestations of thrombotic phenomena. To date, it's believed that, while the antiinflammatory Th2 cytokines (i.e. interleukin-10) can exert a protective role in pregnancy, the pro-inflammatory Th1 ones (i.e. interferon-γ, tumor necrosis factor-α,) have deleterious effects on pregnancy outcome, including fertilization and implantation failure. Moreover, development of many pregnancy complications, first and foremost venous thromboembolism (VTE), recognizes similar mechanism(s). As VTE is the main preventable cause of mortality during pregnancy, thromboprophylaxis is mandatory according to individual VTE risk, influenced by the presence of thrombophilic conditions. In this review, we will analyze the relationship between thrombophilia and pregnancy complications, with particular focus on the role of inflammation. Subsequently, we will consider some issues related to the thromboembolic risk in pregnancy. Finally, the role of thromboprophylaxis in pregnancy will be discussed.

Venous thromboembolism risk prediction in ambulatory cancer patients: clinical significance of neutrophil/lymphocyte ratio and platelet/lymphocyte ratio.

Neutrophil/lymphocyte (NLR) and platelet/lymphocyte (PLR) ratios might represent a yet unrecognized risk factor for venous thromboembolism (VTE) in cancer out-patients receiving chemotherapy. Accordingly, this study was aimed at analyzing the significance of these novel markers in the risk prediction of a first VTE episode in a population representative of a general practice cohort. To this purpose, a mono-institutional cohort study was conducted to retrospectively analyze NLR and PLR in 810 consecutive cancer out-patients with primary or relapsing solid cancer at the start of a new chemotherapy regimen. Over a median follow-up of 9.2 months, VTE occurred in 6.7% of patients. Incidental VTE was diagnosed at time of restaging in 47% of cases. Median pre-chemotherapy NLR (p = 0.015) and PLR (p = 0.040) were significantly higher in patients with intermediate risk class who developed symptomatic VTE with a twofold increased VTE risk for both inflammation-based markers (NLR: p = 0.022; PLR: p = 0.037) and a worst 1-year VTE-free survival for patients with high NLR or PLR. However, only PLR (HR = 2.4, p = 0.027) confirmed to be an independent predictor of future VTE in patients in the intermediate risk class in multivariate analysis, together with ECOG performance status (HR = 3.4, p = 0.0002) and bevacizumab use (HR = 4.7, p = 0.012). We may, thus, conclude that PLR, but to a lesser extent NLR, could represent useful clinical predictors of VTE, especially in selected categories of patients such as those in the intermediate risk class in whom the assessment of PLR could allow a better risk stratification of VTE without additional costs to the national health systems.

Evaluation of mean platelet volume as a predictive marker for cancer-associated venous thromboembolism during chemotherapy.

Mean platelet volume has been proposed as a predictor for venous thromboembolism in cancer. We, therefore, investigated the effects of different anti-cancer drugs on mean platelet volume in order to assess its possible value in the risk prediction of a first thromboembolic episode in cancer outpatients during treatment. Pre-treatment mean platelet volumes were retrospectively evaluated in 589 ambulatory patients at the beginning of a new chemotherapy regimen. Moreover, serial changes were evaluated at baseline and before each chemotherapy cycle on 385 of the 589 patients who consented to have additional blood withdrawals during treatment. Cox proportional hazards survival analysis demonstrated a 2.7 hazard ratio (P=0.01) of developing a first venous thromboembolic episode during chemotherapy for patients with baseline mean platelet volumes below the 10(th) percentile (<7.3 fL). This index significantly declined during the first three months of chemotherapy (-6%; P<0.0001) reverting to baseline at the end of treatment. Multivariate regression analysis showed that normal baseline volumes (P=0.012) and platinum-based regimens (P=0.017) were both independent predictors of mean platelet volume decline during chemotherapy which, in turn, was associated with a 2.4 hazard ratio (P=0.044) of venous thromboembolism. In conclusion, low pre-chemotherapy mean platelet volume might be regarded as a predictor of increased venous thromboembolism risk in cancer patients and chemotherapy further decreases platelet volumes, possibly due to drug-induced platelet activation and destruction. Changes in mean platelet volumes during chemotherapy might provide additional information on thromboembolic risk of patients treated with anti-cancer drugs, particularly platinum compounds.

Plasma plasminogen activator inhibitor-1 (PAI-1) levels in breast cancer - relationship with clinical outcome.

BACKGROUND: Signaling pathways triggered by increased thrombin or plasminogen activator inhibitor-1 (PAI-1) expression drastically alter the tumor microenvironment, contributing to an adverse outcome. This study aimed to evaluate the prognostic value of coagulation/fibrinolytic activities in breast cancer (BC). MATERIALS AND METHODS: Coagulation/fibrinolytic activities were investigated in 187 patients with breast cancer, with respect to possible associations with clinicopathological features and survival outcomes. RESULTS: Levels of plasma PAI-1 (p<0.001), D-dimer (p=0.037) and activated protein C-dependent thrombin generation (p=0.003) were higher in women with breast cancer compared to 187 healthy women. PAI-1 directly correlated with D-dimer levels (p=0.009) and Ki67 expression (p=0.027), which were both predictors of elevated PAI-1 levels at multivariate regression analysis. Cox analysis demonstrated that an elevated plasma PAI-1 level had a negative prognostic impact in terms of relapse-free (hazard ratio=2.5, p=0.021) and overall survival (hazard ratio=2.7, p=0.002). CONCLUSION: Determination of plasma PAI-1 levels might provide important prognostic information in risk stratification and survival outcomes for patients with breast cancer.

Increased risk of chemotherapy-associated venous thromboembolism in elderly patients with cancer.

Data on the relationship between aging, chemotherapy, and risk for venous thromboembolism (VTE) are controversial. We sought to evaluate the risk of chemotherapy-associated VTE in young to middle-aged (YMA) and elderly cancer patients and to analyze the VTE-free survival time in both groups. Patients with histologically confirmed diagnosis of solid malignancy receiving any type of systemic chemotherapy, no clinical diagnosis of VTE before chemotherapy initiation, and Eastern Cooperative Oncology Group (ECOG) performance status (PS) ≤2 were enrolled in this study. Of the 486 consecutive patients included in the study, 380 (78%) were classified as YMA (≤70 years of age) and 106 (22%) as elderly (>70 years of age). At a median follow-up of 1 year, the incidence of VTE events was almost two-fold greater in elderly than in YMA (11% vs. 6%). Age (≤70 years vs. >70 years (hazard ratio [HR], 2.42; 95% confidence interval [CI] 1.15-5.06; p=0.020), ECOG-PS (HR, 6.54; 95% CI 3.10-13.8; p<0.0001), and platinum-based chemotherapy (HR, 2.46; 95% CI 1.06-5.69; p=0.035) were independent risk factors for VTE. In the elderly subset, a trend toward an increased risk of VTE in patients receiving a platinum-based chemotherapy when compared with a non-platinum-containing regimen was observed (15% vs. 9.1%). The Kaplan-Meier analysis showed that elderly patients had a significantly shorter VTE-free survival time compared with younger cancer patients (log-rank test=2.0; p=0.045). Our study reports an increase incidence of VTE in elderly cancer patients treated with chemotherapy compared with the younger group, suggesting that aging is one of the most important risk factors for VTE. On the basis of the results of this study, we believe that a validated predictive model including age, ECOG-PS, and type of chemotherapy (platinum- vs. non-platinum containing regimen) would enable clinicians to target thromboprophylaxis to those patients considered to be at greatest risk.

Association between increased tumor necrosis factor alpha levels and acquired activated protein C resistance in patients with metastatic colorectal cancer.

PURPOSE: The purpose of this study was to investigate the possible association between tumor necrosis factor-α (TNF-α) levels and defects in the activated protein C (APC) system as a determinant of venous thromboembolism (VTE) in metastatic colorectal cancer patients (mCRC) undergoing chemotherapy. METHODS: TNF-α levels (measured by immunoassay) and abnormalities in the APC system [evaluated by an APC-dependent thrombin generation assay (ThromboPath-ThP)] were evaluated in 45 mCRC patients undergoing chemotherapy. VTE events were recorded during follow-up. RESULTS: TNF-α levels were increased (p < 0.01), and APC functionality was decreased (p < 0.0001) in mCRC patients compared to age- and sex-matched controls. An inverse correlation was observed between TNF-α and APC impairment in mCRC (p < 0.0001). TNF-α was confirmed as an independent predictor (p = 0.007) for APC abnormalities at multivariate regression analysis. Nine (20 %) of 45 mCRC patients experienced VTE during chemotherapy. Bayesian analysis of combined ThP/TNF-α showed a positive predictive value of 0.67 in predicting VTE (p = 0.01). Cox proportional hazards survival analysis confirmed the predictive value of combined ThP/TNF-α determination in VTE risk assessment of mCRC patients (either negative vs. both positive: HR = 0.02; p = 0.001), and Kaplan-Meier analysis demonstrated that mCRC patients with either negative TNF-α or ThP values prior to chemotherapy were less likely to experience VTE (13 %) than patients with abnormalities of both markers (67 %, p = 0.002). CONCLUSIONS: These results suggest that the host inflammatory response to cancer cells and/or tumor-derived cytokines could be responsible for an impairment of the APC system and a switch toward a pro-thrombotic state, which might predispose to the occurrence of VTE in mCRC patients undergoing chemotherapy.

Novel high-sensitive D-dimer determination predicts chemotherapy-associated venous thromboembolism in intermediate risk lung cancer patients.

INTRODUCTION: We hypothesized that the use of a novel high sensitivity (HS) assay for D-dimer determination might ameliorate venous thromboembolism (VTE) risk prediction in intermediate risk lung cancer patients in whom chemotherapy could act as a trigger for VTE onset. PATIENTS AND METHODS: Pretreatment HS D-dimer levels were retrospectively evaluated in 108 lung cancer outpatients using a novel automated latex enhanced turbidimetric immunoassay. All patients were at the start of a new platinum-based chemotherapy regimen and were classified as intermediate risk according to Khorana's assessment model. Patients were followed-up for a median period of 6.9 months. RESULTS: Receiver operating characteristic (ROC) curves and corresponding Bayesian analysis showed that the best performance was obtained at a cutoff level of 1500 ng/mL, which resulted in a sensitivity of 81%, a specificity of 69%, a positive predictive value (PPV) of 31%, a negative predictive value (NPV) of 96%, and an accuracy of 70%. Patients with HS D-dimer levels above the cutoff had a worse VTE-free survival (60%) compared with those with levels below the cutoff (95%; P = .0001). Multivariate Cox proportional hazards survival analysis confirmed that pretreatment HS D-dimer levels were able to significantly predict VTE with a hazard ratio of 11 (95% confidence interval, 2.62-46.2; P = .001), independently of classic VTE risk factors. CONCLUSIONS: The use of HS D-dimer determination prior to chemotherapy might allow for VTE risk stratification of intermediate risk cancer patients, helping in identifying those individuals who could benefit from thromboprophylaxis.

Lymphocytes as internal standard in oxidative burst analysis by cytometry: a new data analysis approach.

We propose a new data analysis approach for reactive oxygen species detection using Dihydrorhodamine 123 in blood monocytes and neutrophils. This approach, based on data transformation using lymphocytes as internal standard, allows to appreciate free radical production by monocytes also without Phorbol 12-myristate 13-acetate (PMA) activation. In addition, this method is sensitive to differences in healthy subjects due to sub-pathological conditions, such as hypercholesterolemia.

Factor seven activating protease activity levels in women with recurrent pregnancy loss.

This study was designed to analyze the changes in circulating factor seven activating protease (FSAP) levels in association with the thrombophilic state of 40 women with recurrent pregnancy loss (RPL). All women were trying to conceive and were prospectively followed up until the achievement of spontaneous pregnancy. The results obtained showed that plasma FSAP activity levels were higher in RPL than in fertile women (P < .001) and represented an adverse predictor of pregnancy at multivariate analysis (P = .002). In 7 consenting RPL women, FSAP activity levels increased continuously during pregnancy until the third trimester, remained elevated immediately after delivery, and declined 6-week postpartum, although at levels that were still above the range of control women. These results suggest that FSAP activity levels might provide useful information during pregnancy progression in at-risk women, possibly acting as a predictive factor for adverse pregnancy outcome in RPL even in the absence of other well recognized thrombophilic conditions.

Early changes of a novel APC-dependent thrombin generation assay during chemotherapy independently predict venous thromboembolism in cancer patients--a pilot study.

PURPOSE: Identifying cancer patients who are most at risk for venous thromboembolism (VTE) is essential to improve timely delivery of chemotherapy. Several studies have been performed to identify novel candidate biomarkers, but no agreement has yet been reached. In this light, we sought to analyze whether a dynamic evaluation of early changes of activated protein C (APC) function during chemotherapy could be predictive of a first VTE episode in cancer outpatients, thus improving risk stratification. METHODS: A retrospective single-center pilot study was conducted to investigate the adequacy of a dynamic evaluation of a novel APC-dependent thrombin generation assay (HemosIL ThromboPath (ThP)) in predicting VTE in 208 ambulatory cancer patients, enrolled on the basis of tight inclusion criteria, prior to start and before the second cycle of a new chemotherapy regimen. RESULTS: Retrospective analysis of samples showed the occurrence of an acquired APC resistance during chemotherapy, which was predictive of VTE. Univariate Cox proportional hazards survival analysis showed that early ThP changes predicted VTE (stable vs. decreasing ThP: hazard ratio (HR) 0.21; 95% CI 0.10-0.19; p < 0.0001), which was confirmed in the multivariate model (HR 0.25; CI 0.12-0.52, p < 0.0001). Stratification of patients according to a risk assessment model showed a 0.18 HR for stable vs. decreasing ThP assay results in an intermediate risk group. CONCLUSIONS: We may thus conclude that early changes of ThP assay in patients on active chemotherapy enhance VTE risk stratification, helping in identifying a population of cancer patients who might benefit from thromboprophylaxis.

Functional impairment in video terminal operators is related to low-grade inflammation.

PURPOSE: Progressive functional impairments develop with chronic repetitive tasks possibly involving inflammatory mediators. Aim of this study was to analyze systemic inflammatory changes in relation to the possible occurrence of pain and/or disability in video terminal operators (VTOs) undergoing upper-extremity repetitive stress due to chronic overuse. METHODS: Pain assessments, classification, and grade of impairment relied on self-report questionnaires administered to 21 VTOs and to 21 matched controls. The inflammatory status of the enrolled subjects was analyzed by determination of serum high sensitive C-reactive protein (hs-CRP) as well as systemic levels or monocyte expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). RESULTS: Serum levels of both cytokines were increased in VTOs compared to controls (P = 0.005 for TNF-α and P = 0.004 for IL-6). TNF-α levels correlated to IL-6 (P = 0.019), which, in turn, was associated to increased hs-CRP (P = 0.012). DASH score allowed to categorize VTOs according to disability. VTOs with mild (DASH = 22) or moderate (DASH = 46) disability (n = 10) had higher serum hs-CRP (P = 0.001) and IL-6 (P = 0.035) levels than VTOs without disabilities (DASH < 17) (n = 11). Monocyte stimulatory TNF-α expression was increased in individuals with mild/moderate disability. Monocyte expression of TNF-α was independently associated to that of IL-6, which, in turn, was associated to increased systemic hs-CRP levels together with mild/moderate functional impairment and weekly commitment to the display screen. CONCLUSIONS: The results here reported indicate the occurrence of a low-grade inflammatory condition in VTOs with mild/moderate disability, which might allow the early recognition of arising musculoskeletal disorders induced by repetitive stress.

TNFA gene promoter polymorphisms and susceptibility to recurrent pregnancy loss in Italian women.

The aim of this study was to investigate the relationship between serum tumor necrosis factor alpha (TNF-alpha) levels and single nucleotide polymorphisms (SNPs) of the TNFA gene promoter (-376G/A, -308G/A, and -238G/A) in 100 Italian Caucasian women with reproductive failure and 100 fertile controls. Molecular analysis of TNFA SNPs showed higher frequencies of -238G allele (P = .028) as well as the presence of a 3-loci haplotype (-376G/-308A/-238G; P = .020) in fertile controls compared to women with reproductive failure. Serum TNF-alpha levels were higher in study women compared to controls ( P = .001). Of interest, the TNFA -376G/-308A/-238G haplotype was an independent predictor of low TNF-alpha levels (P = .021) and miscarriage (P = .023) in multivariate analyses. In conclusion, these findings support the concept of an association of TNFA polymorphisms and recurrent pregnancy loss (RPL). In particular, the TNFA -238GG variant and the TNFA -376G/-308A/-238G haplotype might represent protective factors, probably through reduced TNF-alpha production and/or mediated responses.

Increased plasma levels of soluble CD40 ligand correlate with platelet activation markers and underline the need for standardized pre-analytical conditions.

OBJECTIVES: To investigate whether sCD40L dosage might represent a useful tool to explore in vivo platelet function. DESIGN AND METHODS: sCD40L and sP-selectin levels and light transmission aggregometry (LTA) were analyzed in 69 healthy donors. Immunoassays were performed on platelet-depleted citrate plasma samples. The effects of in vitro aspirin treatment on the release of sCD40L were investigated in 15 subjects following platelet stimulation. The effects of a 1-month therapeutic course of low-dose aspirin on sP-selectin and sCD40L levels were also investigated. RESULTS: A significant correlation was observed between sCD40L and sP-selectin (p<0.01). In vitro aspirin treatment remarkably decreased sCD40L levels following platelet activation by exogenous agonists. sCD40L directly correlated with LTA (Rho=0.62, p<0.0001). In vivo aspirin treatment significantly reduced both sP-selectin and sCD40L levels (both p<0.01) in a direct correlation (Rho=0.66, p<0.05). CONCLUSIONS: Citrated plasma samples reflect sCD40L released from platelets, thus yielding the most valid estimates of in vivo circulating levels of this platelet activation markers.

Predictive value of thrombopath determination in women with infertility and pregnancy complications.

BACKGROUND: A condition of maternal thrombophilia, either inherited or acquired, can compromise the success of implantation and foetal survival. METHODS: Malfunctions in protein C pathway were evaluated using a novel assay [HemosIL ThromboPath (ThP)] in a case-control study of 172 women with a history of recurrent miscarriage or infertility and 86 age-matched unrelated fertile women. RESULTS: Thrombophilia was ascertained in 13% of the cases. ThP values were lower in women either with or without thrombophilia compared to controls (both p<0.0001). Abnormal ThP values (below the mean minus 1SD of controls) were found in 62% of cases compared to 12% of controls (p<0.0001). Non-thrombophilic women who achieved spontaneous pregnancy had higher ThP values compared to those who did not (p<0.05). Elevated ThP values were an independent predictor for pregnancy (p<0.01) in women without thrombophilia. A decrease of ThP values was observed during pregnancy progression, which correlated with that of protein S (p<0.05). Miscarriage or major complications occurred in women in whom ThP percent change was approximately 2-fold higher than that observed in women who achieved successful pregnancy (p<0.05). CONCLUSIONS: ThP might represent an efficient assay for screening women with pregnancy complications and might provide prognostic information during pregnancy progression.

Soluble P-selectin as a marker of in vivo platelet activation.

BACKGROUND: Platelets are the major source of circulating sP-selectin. Elevated levels of this protein have been found in many atherothrombotic disorders. Thus, we investigated whether sP-selectin dosage might reflect platelet function in patients with risk factors for or with established cardiovascular diseases and whether its levels can be modulated by aspirin therapy. METHODS: Plasma sP-selectin levels and light transmission platelet aggregometry (LTA) were analyzed in 152 outpatients. The effects of a 6-month aspirin therapeutic course on sP-selectin levels and LTA in 51 consecutive patients have been also investigated. RESULTS: Significant correlations were observed between sP-selectin and Mx% LTA in response to epinephrine (p=0.022) and arachidonic acid (p=0.006), or between sP-selectin and collagen lag-phase (p=0.016). Multiple regression analysis showed that the only predictors of sP-selectin levels were platelet number (p<0.001) and collagen-induced lag-phase (p<0.01). Aspirin-treated patients showed a significant reduction of sP-selectin levels by 13% (p=0.021) which significantly correlated with collagen-induced lag-phase (p=0.005). CONCLUSIONS: sP-selectin dosage could be proposed as a reliable marker of platelet activation in patients with major atherosclerotic risk factors either in the absence of clinically overt disease, and might represent a valid tool to asses in vivo platelet behavior.