Increased coagulation activity and genetic polymorphisms in the F5, F10 and EPCR genes are associated with breast cancer: a case-control study

BackgroundThe procoagulant state in cancer increases the thrombotic risk, but also supports tumor progression. To investigate the molecular mechanisms controlling cancer and hemostasis, we conducted a case-control study of genotypic and phenotypic variables of the tissue factor (TF) pathway of coagulation in breast cancer.Methods366 breast cancer patients and 307 controls were genotyped for SNPs (n = 41) in the F2, F3 (TF), F5, F7, F10, TFPI and EPCR genes, and assayed for plasma coagulation markers (thrombin generation, activated protein C (APC) resistance, D-dimer, antithrombin, protein C, protein S, and TF pathway inhibitor (TFPI)). Associations with breast cancer were evaluated using logistic regression to obtain odds ratios (ORs) and 95% confidence intervals (CIs), or the chi-square test.ResultsFour SNPs in F5 (rs12120605, rs6427202, rs9332542 and rs6427199), one in F10 (rs3093261), and one in EPCR (rs2069948) were associated with breast cancer. EPCR rs2069948 was associated with estrogen receptor (ER) and progesterone receptor (PR) positivity, while the SNPs in F5 appeared to follow hormone receptor negative and triple negative patients. The prothrombotic polymorphisms factor V Leiden (rs6025) and prothrombin G20210A (rs1799963) were not associated with breast cancer. High APC resistance was associated with breast cancer in both factor V Leiden non-carriers (OR 6.5, 95% CI 4.1-10.4) and carriers (OR 38.3, 95% CI 6.2-236.6). The thrombin parameters short lag times (OR 5.8, 95% CI 3.7-9.2), short times to peak thrombin (OR 7.1, 95% CI 4.4-11.3), and high thrombin peak (OR 6.1, 95% CI 3.9-9.5) predicted presence of breast cancer, and high D-dimer also associated with breast cancer (OR 2.0, 95% CI 1.3-3.3). Among the coagulation inhibitors, low levels of antithrombin associated with breast cancer (OR 5.7, 95% CI 3.6-9.0). The increased coagulability was not explained by the breast cancer associated SNPs, and was unaffected by ER, PR and triple negative status.ConclusionsA procoagulant phenotype was found in the breast cancer patients. Novel associations with SNPs in F5, F10 and EPCR to breast cancer susceptibility were demonstrated, and the SNPs in F5 were confined to hormone receptor negative and triple negative patients. The study supports the importance of developing new therapeutic strategies targeting coagulation processes in cancer.

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title: Activated-protein-C resistance in cancer patients.
abstract: Resistance to activated protein C (aPC) is usually linked to factor V Leiden, but may occur in other disorders associated with hypercoagulability. In this study, we investigated the frequency of resistance to aPC in patients with advanced cancer and examined the relationship of aPC resistance to other markers of coagulation activation.



Patients (n = 39) had established diagnosis of advanced cancer; controls (n = 20) were healthy persons. aPC resistance was measured as the ratio of activated partial thromboplastin times with and without aPC (aPC-sensitivity ratio, aPC-SR). The factor V Leiden mutation was detected by a polymerase-chain-reaction based technique. Other assays were performed by standard laboratory methods. Data were analyzed using t tests and the Pearson correlation.



aPC-SR was below 2 SD for 5 of the cancer patients (13%), but none of the controls; only 1 of the 5 had the factor V Leiden mutation. aPC-SR was inversely correlated (p < 0.01) with factor VIII and fibrinogen in patients and with prothrombin activation fragment 1.2 (F1.2) in controls. Patient factor VIII, von Willebrand factor, (vWF), fibrinogen, F1.2 and D dimer were all significantly increased (p < 0.01; antithrombin III, protein C and proteins were similar to controls. Factor VIII correlated with vWF (p < 0.001) and F1.2 with d-dimer (p < 0.001). Other associations (p < 0.05) were observed between factor V and protein C, fibrinogen and protein C, factor V and antithrombin III and protein C and antithrombin III. Four cancer patients had a history of thromboembolism; their aPC-SR was similar to that of patients without thrombosis. Of the several coagulation measures examined, only vWF was higher in the patients with thrombosis (p = 0.01).



Cancer patients have evidence of intravascular coagulation and increases in procoagulants and may have aPC resistance. The aPC resistance is not due to factor V Leiden, but is rather associated with elevated levels of factor VIII and fibrinogen, and in itself does not predict thrombosis.

title: Acquired resistance to activated protein C in breast cancer patients
abstract: In 56 women with a lymph‐node‐positive breast carcinoma and 28 matched healthy control subjects, the sensitivity to activated protein C (APC‐sr) was determined with an APC resistance test that quantifies the effect of APC on thrombin generation initiated via the extrinsic coagulation pathway. Carriers of the Factor V Leiden mutation were excluded from the study. Significant resistance to APC was found in the breast cancer patients: median APC‐sr 2·02 vs 1·03 in the healthy control subjects (P < 0·001). No difference in APC‐sr was found between patients with metastases and without metastases. In patients with metastases, protein S levels were significantly elevated compared with patients without metastases and healthy control subjects: 108·0%vs 96·0% and 94·5% (P = 0·008 and P = 0·007). The APC‐sr correlated with protein S in the control subjects and in patients without metastases but not in patients with metastases. The disturbance of the haemostatic balance probed by the tissue‐factor‐based APC resistance test might contribute to the cancer‐related hypercoagulability.

title: Resistance to Activated Protein C, Factor V Leiden and the Prothrombin G20210A Variant in Patients with Colorectal Cancer
abstract: The aim of our study was to determine the frequency of resistance to activated protein C (APC), factor V Leiden (FVL) and the prothrombin G20210A variant in patients with colorectal cancer.  74 patients with colorectal cancer and 192 colonoscopically selected controls were prospectively investigated for the presence of APC resistance, FVL and the prothrombin G20210A variant. APC resistance was measured as the ratio of activated partial thromboplastin times with and without APC (APC sensitivity ratio, APC-SR). The FVL and prothrombin G20210A variant were detected by a polymerase-chain-reaction-based technique.  FVL was detected in the heterozygous form in 4 of 74 cancer patients (5.4%) and in 7 of 192 controls (3.6%; p > 0.5, odds ratio: 1.51). After excluding patients and controls with FVL, APC-SR was below 2 in 6 of 70 cancer patients (8.5%) and in 1 of 185 controls (0.5%; p < 0.01, odds ratio: 17.25), and the mean value of APC-SR was significantly lower in cancer patients than the respective level of controls (2.8 vs. 3.7, p < 0.001). The G20210A mutation in the prothrombin gene was found in the heterozygous form in 2 of 74 patients with colorectal cancer (2.7%) and in 5 of 192 colonoscopically control subjects (2.6%; p > 0.5, odds ratio: 1.03).  These findings suggest that patients with colorectal cancer have a high frequency of resistance to APC but no significant differences in the frequency of the FVL or G20210A mutation of the prothrombin gene compared to colonoscopically selected controls.

title: The bidirectional relationship of cancer and hemostasis and the potential role of anticoagulant therapy in moderating thrombosis and cancer spread.
abstract: The relationship between hemostasis and malignancy is well recognized, with both elements interacting in a "vicious cycle" where cancers overexpress procoagulants and thrombin, which in turn promote both prothrombotic potential and tumor growth, invasion, and spread. Indeed, venous thromboembolism, particularly idiopathic venous thrombosis, occurs frequently as a paraneoplastic phenomenon, and in turn several components of primary and secondary hemostasis (namely platelets, tissue factor, and thrombin) play an important role in primary tumor growth and metastasization. Despite the many and various mechanisms involved in this multifaceted relationship, anticoagulants might represent an attractive anticancer therapy, in that current research supports the hypothesis that such drugs may offer a better control of cancer progression. The main biological and clinical evidence on the relationship between cancer and hemostasis are briefly summarized in this review, as is the potential benefits of anticoagulant therapy in this setting.

title: Advanced prostate cancer activates coagulation: a controlled study of activation markers of coagulation in ambulatory patients with localized and advanced prostate cancer
abstract: Cancer and increased age are risk factors for coagulation activation. Patients with advanced prostate cancer, which usually presents in the seventh to eighth decade of life, are likely to be at increased risk for thrombosis. We report results of a controlled study of changes in specific and sensitive markers of coagulation activation in patients with prostate cancer. Complete blood count, prothrombin time, partial thromboplastin time, prothrombin fragment 1 + 2 (F1 + 2), thrombin–antithrombin complex (TAT) and quantitative D-dimers (DD) were measured in 30 patients of advanced prostate cancer (androgen ablated), in 30 newly diagnosed localized prostate cancer patients, in 30 healthy age-matched volunteers, and in 20 healthy young volunteers. Plasma F1 + 2 (P < 0.05) and DD (P < 0.05), but not TAT, were significantly elevated in healthy elderly males (mean age, 77 years) when compared with healthy young volunteers (mean age, 35 years). F1 + 2, TAT and DD were significantly elevated in advanced prostate cancer when compared with healthy age-matched controls (P < 0.001). In conclusion, advanced prostate cancer patients have significantly increased levels of sensitive markers of coagulation activation compared with healthy age-matched controls. This data can be used to plan studies to determine the risk of clinically significant coagulopathy and the role of primary prophylaxis in patients with advanced prostate cancer.

title: Plasma fibrin D-dimer levels correlate with tumour volume, progression rate and survival in patients with metastatic breast cancer
abstract: Plasma levels of D-dimer are elevated in cancer patients. Activation of the extrinsic coagulation system and the fibrinolytic cascade within a tumour is thought to be related with growth, invasion and metastasis. We have investigated the relationship between these markers of fibrin metabolism, standard clinicopathological variables and serum levels of angiogenic cytokines in three cohorts: group A (n=30) consisted of 30 healthy female volunteers, group B (n=23) of consecutive patients with operable breast cancer and group C (n=84) of patients with untreated or progressive metastatic breast cancer. Plasma D-dimers, fibrinogen, IL-6, vascular endothelial growth factor and calculated vascular endothelial growth factor load in platelets are clearly increased in patients with breast cancer. D-dimers were increased in nearly 89% of patients with progressive metastatic disease. The level of D-dimers was positively correlated with tumour load (P<0.0001), number of metastatic sites (P=0.002), progression kinetics (P<0.0001) and the cytokines related to angiogenesis: serum vascular endothelial growth factor (P=0.0016, Spearman correlation=0.285), calculated vascular endothelial growth factor load in platelets (P<0.0001, Spearman correlation=0.37) and serum interleukin-6 (P<0.0001, Spearman correlation=0.59). Similarly increased D-dimer levels were positively correlated with increased fibrinogen levels (P<0.0001, Spearman correlation=0.38). The association between markers of fibrin degradation in patients with progressive breast cancer suggests that the D-dimer level is a clinically important marker for progression and points towards a relation between haemostasis and tumour progression. A role of interleukin-6, by influencing both angiogenesis and haemostasis, is suggested by these observations.

title: High D-dimer levels are associated with poor prognosis in cancer patients
abstract: Systemic activation of hemostasis is frequently observed in cancer patients, even in the absence of thrombosis. Moreover, this activation has been implicated in tumor progression, angiogenesis and metastatic spread. Increased levels of D-dimer, which is a degradation product of cross-linked fibrin, indicate a global activation of hemostasis and fibrinolysis.  In a prospective and observational cohort study, we assessed the prognostic value of D-dimer levels for overall survival and mortality risk in 1178 cancer patients included in the Vienna Cancer and Thrombosis Study (CATS). Patients were followed over 2 years at regular intervals until occurrence of symptomatic venous thromboembolism or death. D-dimer levels were measured with a quantitative D-dimer latex agglutination assay  The main solid tumors were malignancies of the lung (n=182), breast (n=157), lower gastrointestinal tract (n=133), pancreas (n=74), stomach (n=50), kidney (n=37), prostate (n=133), and brain (n=148); 201 of the patients had hematologic malignancies; 63 had other tumors. During a median follow-up of 731 days, 460 (39.0%) patients died. The overall survival probabilities for patients with D-dimer levels categorized into four groups based on the 1st, 2nd and 3rd quartiles of the D-dimer distribution in the total study population were 88%, 82%, 66% and 53% after 1 year, and 78%, 66%, 50% and 30% after 2 years, respectively (P<0.001). The univariate hazard ratio of D-dimer (per double increase) for mortality was 1.5 (95% confidence interval: 1.4–1.6, P<0.001) and remained increased in multivariable analysis including tumor subgroups, age, sex and venous thromboembolism.  High D-dimer levels were associated with poor overall survival and increased mortality risk in cancer patients.

title: High plasma levels of extrinsic pathway inhibitor and low levels of other coagulation inhibitors in advanced cancer.
abstract: Serial determinations of plasma coagulation inhibitor levels were performed with chromogenic substrate activity assays in 7 patients with cancer. At time of diagnosis normal median activities of Antithrombin, Protein C, Heparin Cofactor II and Extrinsic Pathway Inhibitor were found. The inhibitor activities changed significantly with the progress of malignant disease; Antithrombin, Protein C and Heparin Cofactor II decreased whereas Extrinsic Pathway Inhibitor increased. Determinations in 13 additional patients in the terminal phase of cancer confirmed this finding. The inhibitor activities were expressed in per cent of a pooled reference plasma. In the total series of 20 patients studied, median activity of Extrinsic Pathway Inhibitor was 183% (range 61-378%) and significantly (p less than 0.005) above age-adjusted normal reference 10 days (range 1-20 days) prior to death. Median activities of Antithrombin was 59% (range 20-109%), of Protein C 54% (range 24-130%) and Heparin Cofactor II 59% (range 33-110%), all significantly below age adjusted normal reference (p less than 0.001). The coagulation inhibitor levels seem related to the stage of disease in patients with cancer.

title: Elevated TFPI in malignant disease: relation to cancer type and hypercoagulation
abstract: We have previously reported high levels of the coagulation inhibitor TFPI in the blood of patients with gastrointestinal cancer. TFPI is not an acute‐phase reactant, but high levels have also been reported in patients with septicaemia and disseminated intravascular coagulation (DIC). To study its relationship with other types of malignancy, TFPI activity was first determined in plasma samples from 214 patients with various malignancies. In a second cohort of 83 patients, total and free TFPI antigen, protein C, antithrombin, fibrin monomer and D‐dimer were also measured. Elevated TFPI activity and antigens were found in about half of the patients with solid tumours. In contrast, elevated TFPI was rare in haematological malignancies (12%). In the 18 patients with acute nonlymphocytic leukaemia (ANLL), elevated free TFPI was found only in patients who also had DIC. No correlation was found between TFPI levels and fibrin monomer or D‐dimer levels. Only four out of 20 patients with solid tumours had normal levels of fibrin monomer and D‐dimer, yet three out of these four had elevated TFPI. In conclusion, elevated TFPI in ANLL is related to the coexistence of DIC. In solid tumour disease increased TFPI may reduce protective fibrin formation, but the pathogenic mechanism is as yet unknown.

title: Hypercoagulable State in Patients with Advanced Gastrointestinal Cancer: Evidence for an Acquired Resistance to Activated Protein C
abstract: Thromboembolic complications are common in patients with cancer and represent the second cause of death in patients with overt malignant disease. The aim of this study was to investigate the activated protein C pathway in cancer.  We studied the coagulation cascade, natural clotting inhibitors, fibrinolytic proteins and resistance to activated protein C in 20 patients with advanced gastrointestinal cancer and 84 volunteers by measuring PT, APTT, fibrinogen, AT III, PC, PS, APC resistance, fibrinolytic system (PLG, ANPL, PAI-1 and t-PA) and activation peptides (D-Dimers, prothrombin 0 fragment 1+2/F1+2).  Laboratory tests confirmed coagulation abnormalities in cancer patients. Fibrinogen, D-Dimers and F1+2 were increased, while t-PA activity was significantly lower than that of controls. APC resistance was higher in cancer patients compared to the control group (55% vs 2%; P < 0.0001). Excess thrombin generation was manifested by increased F1+2 plasma levels in APC-resistant cancer patients. Genetic analyses showed that only one patient with a poor response to APC carried a factor V R506Q mutation in exon 10.  Our findings show a high prevalence of APC resistance in cancer, compatible with an acquired defect in the APC pathway.

title: Mechanisms for acquired activated protein C resistance in cancer patients
abstract: Acquired activated protein C resistance (APCR) is common in cancer patients and may increase the risk of venous thromboembolism (VTE) in these patients. Earlier results published by our group demonstrated the association of acquired APCR with thrombosis in cancer patients . Moreover, acquired APCR was found to be common also in cancer patients without thrombosis . The aim of this study was to investigate potential mechanisms underlying these findings. We prospectively evaluated APC-sensitivity ratio (APC-SR), coagulation factor (F)II, factor (F)V, factor (F)VIII, factor (F)X and protein S levels as well as presence of antiphospholipid antibodies (APLA) in cancer patients compared with controls. The study group included 99 cancer patients (55 females and 44 males, mean age 49 ± 12 years, range 23–65 years) who fulfillled the following criteria: (i) ambulatory performance status (WHO grade 0–3), (ii) no previous history of venous or arterial thrombosis, (iii) no anticoagulant or hormone therapy, (iv) no evidence of active infection, (v) normal serum bilirubin, serum transaminases <2.0 upper limit and creatinine £1.5 mg dL. Their diagnoses included carcinoma (n 1⁄4 62), lymphoma (n 1⁄4 15), sarcoma (n 1⁄4 8), melanoma (n 1⁄4 4) and other solid malignant tumors (n 1⁄4 10). Sixty-six (67%) had received chemotherapy within 1 month before the coagulation evaluation. The patients’ selection criteria used in this study exclude a possible effect of the thrombotic event and/or the anticoagulant or hormone therapy on the coagulation parameters. However, a possible effect of chemotherapy on the study results cannot be excluded. The control group included 79 healthy volunteers (39 females and 40 males, mean age 41 ± 10 years, range 26–62 years) without a clinical history of venous or arterial thromboembolism or malignancy. An additional five cancer patients and two control subjects were found to be heterozygous for FV Leiden and were excluded from the study. The study was approved by the Rambam Medical Center Institutional Ethics Committee. APC-SR was determined by the addition of activated protein C (APC) to activated partial thromboplastin time (APTT) assay without dilution in FV-deficient plasma, using a Coatest APC resistance kit (Chromogenix AB, Molendal, Sweden). Free protein S antigen levels were measured by the Asserachrom free protein S kit (Stago Diagnostics, Asnieres, France). Lupus anticoagulant assays were performed as previously described . Levels of FII, FV, FVIII and FX were determined by one-stage assay using factor-deficient plasma (Instrumentation Laboratories, Barcelona, Spain). Statistical analysis was performed by SPSS software (SPSS Inc., Chicago, IL, USA) using parametric tests, i.e. Fisher’s exact test, T-test and Pearson correlation test. Non-parametric tests included Kendall’s s-b and Mann–Whitney U-tests. APCR (APC-SR <2.0) without FV Leiden was more common in cancer patients. Likewise, levels of coagulation FV and FVIII were significantly higher in cancer patients compared with controls (Table 1). Coagulation factors V and VIII that might be increased due to acute-phase state [4] were inversely correlated with APC-SR in cancer patients but not in healthy controls (for FV, r 1⁄4 ) 0.3, P 1⁄4 0.003 in patients and r 1⁄4 ) 0.2, P 1⁄4 0.17 in controls; for FVIII, r 1⁄4 ) 0.4, P < 0.0001 in patients and r 1⁄4 ) 0.2, P 1⁄4 0.09 in controls). There was no apparent correlation between APC-SR and either age or gender in either patients or controls. FV plays a role in both procoagulant and anticoagulant pathways, i.e. FVa accelerates the activation of prothrombin and is inactivated by APC . A correlation between FV levels and APCR was documented in pregnancy; on the other hand, low FV levels were found to be associated with a reduced APC cofactor activity in the inactivation of FVIII/VIIIa . Mechanisms underlying the effect of FVIII on APC-SR are not clear. It may be related to an imbalance between FVIII and Correspondence: G. Sarig, Thrombosis and Hemostasis Unit, Rambam Medical Center, Haifa 31096,  +97 2 4 854 3052; fax: +97 2 4 854 3636;

title: Genetic link between cancer and thrombosis.
abstract: From the beginning of their lives, cancer cells exert a procoagulant activity in their microenvironment, which can extend systemically and become clinically evident as Trousseau's syndrome, the well-known association between tumor and thrombosis. It is becoming clear that the genetic mechanisms responsible for neoplastic transformation (activation of oncogenes such as RAS or MET, and inactivation of tumor suppressor genes such as p53 or PTEN) directly induce the expression of genes controlling hemostasis. Activation of blood coagulation results in a selective advantage for cancer cells, as fibrin provides a scaffold for anchorage and invasion, and coagulation proteins induce receptor-mediated intracellular signals promoting invasive growth. Targeting the tumor procoagulant activity can fight not only a dangerous tumor adverse effect, but also the core mechanisms of cancer onset and progression.

title: Tissue factor and protease-activated receptor signaling in cancer.
abstract: The activation of the coagulation cascade in the tumor microenvironment is a key feature of advanced malignancies. On tumor cells, tissue factor (TF) plays a central role to initiate cross-talk through the release of procoagulant microparticles or through direct, protease-activated receptor (PAR)-mediated cell signaling that leads to the production of soluble cytokines and angiogenic growth factors. In addition, the hemostatic system in the host compartment sustains crucial circuits that promote metastasis and support tumor growth and angiogenesis. Experimental tumor and genetic models have defined specific pathways that are supported by tumor cell and host TF and have identified potential therapeutic modalities to specifically interrupt TF signaling in tumor biology without impairment of hemostatic functions.

title: The Endothelial Protein C Receptor Supports Tissue Factor Ternary Coagulation Initiation Complex Signaling through Protease-activated Receptors*
abstract: Protease-activated receptor (PAR) signaling is closely linked to the cellular activation of the pro- and anticoagulant pathways. The endothelial protein C receptor (EPCR) is crucial for signaling by activated protein C through PAR1, but EPCR may have additional roles by interacting with the 4-carboxyglutamic acid domains of procoagulant coagulation factors VII (FVII) and X (FX). Here we show that soluble EPCR regulates the interaction of FX with human or mouse tissue factor (TF)-FVIIa complexes. Mutagenesis of the FVIIa 4-carboxyglutamic acid domain and dose titrations with FX showed that EPCR interacted primarily with FX to attenuate FX activation in lipid-free assay systems. In human cell models of TF signaling, antibody inhibition of EPCR selectively blocked PAR activation by the ternary TF-FVIIa-FXa complex but not by the non-coagulant TF-FVIIa binary complex. Heterologous expression of EPCR promoted PAR1 and PAR2 cleavage by FXa in the ternary complex but did not alter PAR2 cleavage by TF-FVIIa. In murine smooth muscle cells that constitutively express EPCR and TF, thrombin and FVIIa/FX but not FVIIa alone induced PAR1-dependent signaling. Although thrombin signaling was unchanged, cells with genetically reduced levels of EPCR no longer showed a signaling response to the ternary complex. These results demonstrate that EPCR interacts with the ternary TF coagulation initiation complex to enable PAR signaling and suggest that EPCR may play a role in regulating the biology of TF-expressing extravascular and vessel wall cells that are exposed to limited concentrations of FVIIa and FX provided by ectopic synthesis or vascular leakage.

title: Endothelial Protein C Receptor Function in Murine and Human Breast Cancer Development
abstract: Several markers identify cancer stem cell-like populations, but little is known about the functional roles of stem cell surface receptors in tumor progression. Here, we show that the endothelial protein C receptor (EPCR), a stem cell marker in hematopoietic, neuronal and epithelial cells, is crucial for breast cancer growth in the orthotopic microenvironment of the mammary gland. Mice with a hypomorphic allele of EPCR show reduced tumor growth in the PyMT-model of spontaneous breast cancer development and deletion of EPCR in established PyMT tumor cells significantly attenuates transplanted tumor take and growth. We find expansion of EPCR+ cancer stem cell-like populations in aggressive, mammary fat pad-enhanced human triple negative breast cancer cells. In this model, EPCR-expressing cells have markedly increased mammosphere- and tumor-cell initiating activity compared to another stable progenitor-like subpopulation present at comparable frequency. We show that receptor blocking antibodies to EPCR specifically attenuate in vivo tumor growth initiated by either EPCR+ cells or the heterogenous mixture of EPCR+ and EPCR- cells. Furthermore, we have identified tumor associated macrophages as a major source for recognized ligands of EPCR, suggesting a novel mechanism by which cancer stem cell-like populations are regulated by innate immune cells in the tumor microenvironment.

title: Involvement of serum retinoids and Leiden mutation in patients with esophageal, gastric, liver, pancreatic, and colorectal cancers in Hungary.
abstract: To analyze the serum levels of retinoids and Leiden mutation in patients with esophageal, gastric, liver, pancreatic, and colorectal cancers.



The changes in serum levels of retinoids (vitamin A, alpha- and beta-carotene, alpha- and beta-cryptoxanthin, zeaxanthin, lutein) and Leiden mutation were measured by high liquid performance chromatography (HPLC) and polymerase chain reaction (PCR) in 107 patients (70 males/37 females) with esophageal (0/8), gastric (16/5), liver (8/7), pancreatic (6/4), and colorectal (30/21 including 9 patients suffering from in situ colon cancer) cancer. Fifty-seven healthy subjects (in matched groups) for controls of serum retinoids and 600 healthy blood donors for Leiden mutation were used.



The serum levels of vitamin A and zeaxanthin were decreased significantly in all groups of patients with gastrointestinal (GI) tumors except for vitamin A in patients with pancreatic cancer. No changes were obtained in the serum levels of alpha- and beta-carotene, alpha- and beta-cryptoxanthin, zeaxanthin, lutein in patients with GI cancer. The prevalence of Leiden mutation significantly increased in all groups of patients with GI cancer.



Retinoids (as environmental factors) are decreased significantly with increased prevalence of Leiden mutation (as a genetic factor) in patients before the clinical manifestation of histologically different (planocellular and hepatocellular carcinoma, and adenocarcinoma) GI cancer.

title: Clotting factor gene polymorphisms and colorectal cancer risk.
abstract: Increased coagulation has been associated with cancer onset and progression. Mainly small studies have addressed the association between clotting factor gene polymorphisms and the onset of colorectal cancer. We examined the association between six well-known clotting factor gene polymorphisms and colorectal cancer risk in a large case-control study.



Factor V Leiden (rs6025), prothrombin G20210A (rs1799963), PAI-1 4G/5G (rs1799889), MTHFR 677C>T (rs1801133), fibrinogen gamma 10034C>T (rs2066865), and factor XIII Val34Leu (rs5985) were genotyped in 1,801 patients with colorectal cancer and 1,853 healthy controls from a large German population-based study. The risk of colorectal cancer associated with gene variants was determined by calculating odds ratios (ORs) and their 95% CIs using logistic regression.



Homozygous carriers of the prothrombotic factor V Leiden polymorphism showed a 5.8-fold increased risk (95% CI, 1.69 to 19.72) for colorectal cancer compared with noncarriers. A 30% reduced risk was found for heterozygous carriers of factor V Leiden (OR = 0.68; 95% CI, 0.52 to 0.90) and prothrombin G20210A (OR = 0.69; 95% CI, 0.49 to 0.96), implying an advantage for slightly increased thrombin generation. Carriers of the antithrombotic factor XIII Val34Leu polymorphism showed a 15% reduced risk of developing colorectal cancer (OR = 0.85; 95% CI, 0.74 to 0.97) compared with noncarriers. Our results did not support an effect of PAI-1 4G/5G, MTHFR 677C>T, and fibrinogen gamma 10034C>T on colorectal cancer risk.



Our results support a role of clotting factor polymorphisms and thereby the coagulation system in the risk of colorectal cancer.

title: Prevalence of factor V Leiden and prothrombin G20210A in patients with gastric cancer.
abstract: To analyze the prevalence of the two commonest thrombophilic mutations, factor V Leiden and prothrombin G20210A, in patients with gastric cancer.



One hundred and twenty-one patients with primary gastric carcinoma and 130 healthy subjects, comparable for age and sex, were investigated. Factor V Leiden was detected by using polymerase chain reaction and restriction enzyme digestion, and prothrombin G20210A gene mutation by allele-specific PCR.



Among the 121 cancer patients, factor V Leiden was found in 4 cases (GA genotype: 3.3%) and prothrombin G20210A in 10 cases (GA genotype: 8.3%). Of the 130 control subjects, factor V Leiden was detected in 6 cases (GA genotype: 4.6%) and prothrombin G20210A in 8 cases (GA genotype: 6.1%). No double heterozygous carriers of both mutations were found in either group. The prevalence of both factor V Leiden and prothrombin G20210A variant was not statistically different between the cancer patients and the healthy subjects.



Our study suggests that, in gastric cancer, the risk factors of thrombophilic cancer state are on acquired rather than on a genetic basis and that prothrombin G20210A does not seem to be a cofactor in gastric cancer pathogenesis.

title: Impact of thrombophilic gene mutations on thrombosis risk in patients with gastrointestinal carcinoma
abstract: Patients with malignancies have an increased risk for thromboembolic events due to the release of tissue factor by the tumor, damage to the vessel wall, and immobilization. Moreover, tumors may improve their growth and metastatic spread by utilizing the coagulation system. To date, no information is available on the additional role of prothrombotic mutations in these patients.

title: The Risk of Cancer Progression in Women With Gynecological Malignancies and Thrombophilic Polymorphisms: A Pilot Case-Control Study
abstract: Cancer produces a hypercoagulable state, which might lead to thrombosis, and on contrary, unprovoked venous thromboembolism might be the manifestation of an occult cancer. In this pilot case-control study, we assessed the risk of gynecological malignant diseases related to the presence of the factor V Leiden and prothrombin G20210A polymorphisms. Fifty-two women underwent an operation for gynecological malignancy and were enrolled in the study. Women who underwent an operation for gynecological nonmalignant disease in the same days of cases were considered as controls. The presence of factor V Leiden and prothrombin G20210A was assessed in case and control groups. In all, 7 out of 52 cases were carriers of the 2 polymorphisms compared with 20 out of 198 controls (odds ratio = 1.3; 95% confidence interval, 0.6-3.0). The results were also similar when the risk was considered separately for the site of cancer. As for advanced and metastatic malignancies, the odds ratios were 2.3 (95% confidence interval, 0.9-6.0) and 3.3 (95% confidence interval, 1.0-11), respectively, compared to noncancer patients. When these 2 groups were compared to nonadvanced cancer group, the odds ratios for carriers of polymorphisms were 2.7 (95%confidence interval, 0.7-11.0) and 3.9 (95%confidence interval, 0.8-18.6) for advanced cancer and metastatic malignancies, respectively. Women with factor V Leiden or prothrombin G20210A polymorphisms who developed gynecological malignancy might present with a higher stage of cancer at the time of surgery. Larger case-control studies in similar cohort of patients are needed to confirm these findings.

title: Are factor V and prothrombin mutations associated with increased risk of oral cancer?
abstract: Methylenetetrahydrofolate reductase is associated with pathogenesis of both thrombosis and oral cancer. Therefore, a search for a similar association of other thrombosis-related factors with oral cancer is justified.



In order to investigate whether the coagulation factor V Leiden and prothrombin G20210A mutations increase the risk of oral cancer, we searched for these mutant alleles by RFLP analysis in DNA samples of 102 patients with oral cancer and 120 healthy controls.



Neither the Leiden nor the G20210A alleles were found in statistically different frequencies in the two groups. In addition, no statistical difference was observed in parameters such as sex, age and positive family history for cancer. Nevertheless, a significant difference was observed for Leiden in patients with a positive history for thrombophilia (p < 0.001).



There seems to be no association of prothrombin, and possibly a minor contribution of factor V, in oncogenesis in the oral region.

title: Association between the −402GA, −401GT, and −323ins10-bp polymorphisms of factor VII gene and breast cancer
abstract: Background and PurposeRecent data have shown that the hemostatic system may play a role in cancer development and progression. To test whether factor VII (FVII) can be a candidate factor for breast cancer, we have evaluated the distribution of FVII gene polymorphisms in breast cancer patients and healthy subjects.MethodsThe nested case–control study consisted of 92 women with breast cancer (group 1) and 80 control subjects (in age-matched women) (group 2). Genotyping of the −323ins10-bp, −401GT, and −402GA polymorphisms of the FVII gene was performed by the method of single-strand conformation polymorphism analysis and sequencing.ResultsA significant difference was observed in the distribution of the −402GA genotype and allele frequencies in breast cancer and control cases (p < 0.05). For other polymorphisms of the FVII gene, the distributions of genotypes and allele frequencies were not significantly different between two groups (p > 0.05). There was also a significant difference between the distributions of the haplotypes in breast cancer patients and control subjects (p < 0.05).ConclusionAlthough the number of cases in this study was small, the preliminary findings revealed a possible contribution of the FVII −402GA polymorphism in the development of breast cancer. However, further case–control studies with larger series are needed to confirm our findings.

title: Molecular classification of breast cancer
abstract: Breast cancer is a complex, multifaceted disease encompassing a great variety of entities that show considerable variation in clinical, morphological and molecular attributes. Traditional classifications including histological assessment and clinical staging are used to guide patient management. In recent years, there has been exponential progress in molecular analysis with profound implications for our understanding of breast cancer biology and, hence, classification. There are now genome-based frameworks for the molecular categorisation of breast cancer including the development of prognostic and predictive signatures that potentially allow individualisation of treatment. Here we review the current state of the molecular classifications of in situ and invasive breast cancer including special subtypes. Future perspectives are also provided.

title: Resistance to activated protein C is a risk factor for pregnancy‐related venous thrombosis in the absence of the F5 rs6025 (factor V Leiden) polymorphism
abstract: Resistance to activated protein C (aPC) is most commonly due to the F5 rs6025 (factor V Leiden) polymorphism, which increases the risk of venous thrombosis. In the present population‐based study of 313 cases and 353 controls, we investigated whether reduced sensitivity to aPC was associated with a history of pregnancy‐related venous thrombosis. Calibrated automated thrombography was used to determine the sensitivity to aPC, and normalized aPC sensitivity ratio (n‐aPC‐sr) was calculated. Pregnant women and women using oral contraceptives and/or anticoagulants were excluded due to the effect on the n‐aPC‐sr. In women without the F5 rs6025 polymorphism, free tissue factor pathway inhibitor (TFPI), free protein S and protein C activity were associated with n‐aPC‐sr. Unadjusted odds ratio for venous thrombosis for women with n‐aPC‐sr in the 4th quartile as compared with n‐aPC‐sr below the 4th quartile was 2·4 (95% confidence interval 1·7–3·6). Adjusting for free protein S, free TFPI and age did not influence the odds ratios. Also in carriers of the F5 rs6025 polymorphism the risk for venous thrombosis was increased for women with higher n‐aPC‐sr. Our findings substantiate the importance of the aPC resistant phenotype as a risk factor for pregnancy‐related venous thrombosis.

title: Calibrated Automated Thrombin Generation Measurement in Clotting Plasma
abstract: Calibrated automated thrombography displays the concentration of thrombin in clotting plasma with or without platelets (platelet-rich plasma/platelet-poor plasma, PRP/PPP) in up to 48 samples by monitoring the splitting of a fluorogenic substrate and comparing it to a constant known thrombin activity in a parallel, non-clotting sample. Thus, the non-linearity of the reaction rate with thrombin concentration is compensated for, and adding an excess of substrate can be avoided. Standard conditions were established at which acceptable experimental variation accompanies sensitivity to pathological changes. The coefficients of variation of the surface under the curve (endogenous thrombin potential) are: within experiment ∼3%; intra-individual: <5% in PPP, <8% in PRP; interindividual 15% in PPP and 19% in PRP. In PPP, calibrated automated thrombography shows all clotting factor deficiencies (except factor XIII) and the effect of all anticoagulants [AVK, heparin(-likes), direct inhibitors]. In PRP, it is diminished in von Willebrand’s disease, but it also shows the effect of platelet inhibitors (e.g. aspirin and abciximab). Addition of activated protein C (APC) or thrombomodulin inhibits thrombin generation and reflects disorders of the APC system (congenital and acquired resistance, deficiencies and lupus antibodies) independent of concomitant inhibition of the procoagulant pathway as for example by anticoagulants.

title: Controlling the false discovery rate: a practical and powerful approach to multiple testing
abstract: The common approach to the multiplicity problem calls for controlling the familywise error rate (FWER). This approach, though, has faults, and we point out a few. A different approach to problems of multiple significance testing is presented. It calls for controlling the expected proportion of falsely rejected hypotheses -the false discovery rate. This error rate is equivalent to the FWER when all hypotheses are true but is smaller otherwise. Therefore, in problems where the control of the false discovery rate rather than that of the FWER is desired, there is potential for a gain in power. A simple sequential Bonferronitype procedure is proved to control the false discovery rate for independent test statistics, and a simulation study shows that the gain in power is substantial. The use of the new procedure and the appropriateness of the criterion are illustrated with examples.

title: SNAP: a web-based tool for identification and annotation of proxy SNPs using HapMap
abstract: The interpretation of genome-wide association results is confounded by linkage disequilibrium between nearby alleles. We have developed a flexible bioinformatics query tool for single-nucleotide polymorphisms (SNPs) to identify and to annotate nearby SNPs in linkage disequilibrium (proxies) based on HapMap. By offering functionality to generate graphical plots for these data, the SNAP server will facilitate interpretation and comparison of genome-wide association study results, and the design of fine-mapping experiments (by delineating genomic regions harboring associated variants and their proxies).


AVAILABILITY
SNAP server is available at http://www.broad.mit.edu/mpg/snap/.

title: Annotation of functional variation in personal genomes using RegulomeDB.
abstract: As the sequencing of healthy and disease genomes becomes more commonplace, detailed annotation provides interpretation for individual variation responsible for normal and disease phenotypes. Current approaches focus on direct changes in protein coding genes, particularly nonsynonymous mutations that directly affect the gene product. However, most individual variation occurs outside of genes and, indeed, most markers generated from genome-wide association studies (GWAS) identify variants outside of coding segments. Identification of potential regulatory changes that perturb these sites will lead to a better localization of truly functional variants and interpretation of their effects. We have developed a novel approach and database, RegulomeDB, which guides interpretation of regulatory variants in the human genome. RegulomeDB includes high-throughput, experimental data sets from ENCODE and other sources, as well as computational predictions and manual annotations to identify putative regulatory potential and identify functional variants. These data sources are combined into a powerful tool that scores variants to help separate functional variants from a large pool and provides a small set of putative sites with testable hypotheses as to their function. We demonstrate the applicability of this tool to the annotation of noncoding variants from 69 full sequenced genomes as well as that of a personal genome, where thousands of functionally associated variants were identified. Moreover, we demonstrate a GWAS where the database is able to quickly identify the known associated functional variant and provide a hypothesis as to its function. Overall, we expect this approach and resource to be valuable for the annotation of human genome sequences.

title: Genetics and Beyond – The Transcriptome of Human Monocytes and Disease Susceptibility
abstract: Variability of gene expression in human may link gene sequence variability and phenotypes; however, non-genetic variations, alone or in combination with genetics, may also influence expression traits and have a critical role in physiological and disease processes.  To get better insight into the overall variability of gene expression, we assessed the transcriptome of circulating monocytes, a key cell involved in immunity-related diseases and atherosclerosis, in 1,490 unrelated individuals and investigated its association with >675,000 SNPs and 10 common cardiovascular risk factors. Out of 12,808 expressed genes, 2,745 expression quantitative trait loci were detected (P<5.78×10−12), most of them (90%) being cis-modulated. Extensive analyses showed that associations identified by genome-wide association studies of lipids, body mass index or blood pressure were rarely compatible with a mediation by monocyte expression level at the locus. At a study-wide level (P<3.9×10−7), 1,662 expression traits (13.0%) were significantly associated with at least one risk factor. Genome-wide interaction analyses suggested that genetic variability and risk factors mostly acted additively on gene expression. Because of the structure of correlation among expression traits, the variability of risk factors could be characterized by a limited set of independent gene expressions which may have biological and clinical relevance. For example expression traits associated with cigarette smoking were more strongly associated with carotid atherosclerosis than smoking itself.  This study demonstrates that the monocyte transcriptome is a potent integrator of genetic and non-genetic influences of relevance for disease pathophysiology and risk assessment.

title: Association of coagulation‐related and inflammation‐related genes and factor VIIc levels with stroke: the Cardiovascular Health Study
abstract: Thrombosis and inflammation are critical in stroke etiology, but associations of coagulation and inflammation gene variants with stroke, and particularly factor VII levels, are inconclusive.  To test the associations between 736 single‐nucleotide polymorphisms (SNPs) between tagging haplotype patterns of 130 coagulation and inflammation genes, and stroke events, in the 5888 participants aged ≥ 65 years of the observational Cardiovascular Health Study cohort.  With 16 years of follow‐up, age‐adjusted and sex‐adjusted Cox models were used to estimate associations of SNPs and FVIIc levels with future stroke.  Eight hundred and fifteen strokes occurred in 5255 genotyped participants without baseline stroke (748 ischemic strokes; 586 among whites). Among whites, six SNPs were associated with stroke, with a nominal P‐value of < 0.01: rs6046 and rs3093261 (F7); rs4918851 and rs3781387 (HABP2); and rs3138055 (NFKB1A) and rs4648004 (NFKB1). Two of these SNPs were associated with FVIIc levels (units of percentage activity): rs6046 (β = − 18.5, P = 2.38 × 10−83) and rs3093261 (β = 2.99, P = 3.93 × 10−6). After adjustment for age, sex, race, and cardiovascular risk factors, the association of FVIIc quintiles (Q) with stroke were as follows (hazard ratio; 95% confidence interval): Q1, reference; Q2, 1.4, 1.1–1.9); Q3, 1.1, 0.8–1.5); Q4, 1.5, 1.1–2.0); and Q5, 1.6, ). Associations between SNPs and stroke were independent of FVIIc levels.  Variations in FVII‐related genes and FVIIc levels were associated with risk of incident ischemic stroke in this elderly cohort, suggesting a potential causal role for FVII in stroke etiology.

title: Effect of thrombosis-related gene polymorphisms upon oral cancer: a regression analysis.
abstract: It is well-known that there is an interplay between hemostasis, thrombosis and cancer. Functional DNA polymorphisms in genes encoding factors related to thrombosis have been associated with increased risk for oral squamous cell carcinoma (OSCC). The present study investigated the possible combinatory effect of 10 such polymorphisms as primary risk predictors for OSCC in a European population. Two groups including 160 patients with OSCC and 168 healthy controls of Greek and German origin were studied. The patient and control groups were comparable regarding ethnicity, age and gender. For all studied individuals, 10 genotypes of functional polymorphisms were investigated: 5,10-methylene tetrahydrofolate reductase (MTHFR) C677T, coagulation factor V (F5) Leiden, coagulation factor II (F2, also known as prothrombin) G20210A, coagulation factor XII (F12) C46T, coagulation factor XIII A1 subunit (F13A1) Val34Leu, serpine1 (SERPINE1, also known as plasminogen activator inhibitor-1) 4G/5G, protein Z (PROZ) -A13G, angiotensin I-converting enzyme (ACE) I/D, angiotensinogen (AGT) Met325Thr, and carboxypeptidase B2 (CPB2, also known as thrombin-activatable fibrinolysis inhibitor) C1040T. Multivariate logistic regression models were used in order to evaluate the relation and contribution of homozygous and heterozygous variant polymorphisms upon overall, early and advanced stages of OSCC. Five out of the studied polymorphisms, influencing the expression of SERPINE1 and ACE genes, as well as the activity of CPB2, F12 and F13 proteins, were recognized as significant predictive factors for OSCC. The 'mode of inheritance' regression model, in particular, revealed the low expression I allele of ACE to be a primary predictor in overall, early and advanced stages of oral cancer. Comparing the present findings with previous knowledge, possible interactions of these factors and their relation to the risk for OSCC development are discussed.

title: More on: high thrombin generation and the risk of recurrent venous thromboembolism
abstract: 11 Masini S, Ticconi C, Gravina P, Tomassini M, Pietropolli A, Forte V, Federici G, Piccione E, Bernardini S. Thrombin-activatable fibrinolysis inhibitor polymorphisms and recurrent pregnancy loss. Fertil Steril 2008, doi: 10.1016/j.fertnstert.2008.07.015. 12 Isermann B, Sood R, Pawlinski R, Zogg M, Kalloway S, Degen JL, Mackman N, Weiler H. The thrombomodulin–protein C system is essential for the maintenance of pregnancy.  2003; 9: 331–7. 13 Preston FE, Rosendaal FR,Walker ID, Briet E, Berntorp E, Conard J, Fontcuberta J, Makris M, Mariani G, Noteboom W, Pabinger I, Legnani C, Scharrer I, Schulman S, van der Meer FJ. Increased fetal loss in women with heritable thrombophilia. Lancet 1996; 348: 913–16. 14 Meinardi JR,Middeldorp S, de Kam PJ, KoopmanMM, van Pampus EC, Hamulyak K, Prins MH, Buller HR, van der Meer J. Increased risk for fetal loss in carriers of the factor V Leiden mutation.  The risk of abortion and stillbirth in antithrombin-, protein C-, and protein S-deficient women.

title: [Factors influencing thrombin generation measured as thrombin-antithrombin complexes levels and using calibrated automated thrombogram in patients with advanced coronary artery disease].
abstract: Haemostatic factors play an important role in atherothrombosis. Thrombin generation is a crucial stage of blood coagulation.



Comparison of different thrombin generation markers: thrombin-antithrombin complex (TAT) generation and calibrated automated thrombogram method (CAT). Identification of factors influencing thrombin generation in patients with stable angina (SA) enrolled to the coronary artery bypass grafting (CABG) surgery. Analysis of traditional (age, gender, hypertension and diabetes) and novel (fibrinogen and C-reactive protein [CRP]) risk factors and the antiplatelet therapy (aspirin 75-150 mg/d) in relation to coagulation.



In 135 SA patients with left main coronary artery stenosis (> 50%) or major epicardial artery stenosis (> 70%), plasma TAT levels, maximal thrombin concentration (C(max)) and endogenous thrombin potential (ETP) were determined. A marker of the platelet activation (beta-thromboglobulin) was also measured.



No correlations among TAT, C(max), ETP, risk factors and beta-thromboglobulin were observed. Linear regression model showed that independent predictors of TAT levels were age (beta = 0.5; p = < 0.0001), male gender and diabetes (beta = 0.36; p = 0.02). CRP independently predicted TAT and ETP (beta = -0.24 and beta = 0.22; p < 0.05, respectively), while fibrinogen predicted C(max) (beta = 0.21; p < 0.05). Independent predictors of beta-thromboglobulin were a male gender and aspirin use cessation (beta = 0.46; p = 0.01). Aspirin treatment had no effect on thrombin generation.



Age, higher fibrinogen, CRP, diabetes and male gender influence thrombin generation and/or coagulation activation in SA patients. Plasma levels of thrombin-antithrombin complexes do not correlate with the parameters obtained using the calibrated automated thrombogram method (C(max), ETP).

title: Coagulation tests show significant differences in patients with breast cancer
abstract: Activated coagulation and fibrinolytic system in cancer patients is associated with tumor stroma formation and metastasis in different cancer types. The aim of this study is to explore the correlation of blood coagulation assays for various clinicopathologic factors in breast cancer patients. A total of 123 female breast cancer patients were enrolled into the study. All the patients were treatment naïve. Pretreatment blood coagulation tests including PT, APTT, PTA, INR, D-dimer, fibrinogen levels, and platelet counts were evaluated. Median age of diagnosis was 51 years old (range 26–82). Twenty-two percent of the group consisted of metastatic breast cancer patients. The plasma level of all coagulation tests revealed statistically significant difference between patient and control group except for PT (p < 0.001 for all variables except for PT; p = 0.08). Elderly age (>50 years) was associated with higher D-dimer levels (p = 0.003). Metastatic patients exhibited significantly higher D-dimer values when compared with early breast cancer patients (p = 0.049). Advanced tumor stage (T3 and T4) was associated with higher INR (p = 0.05) and lower PTA (p = 0.025). In conclusion, coagulation tests show significant differences in patients with breast cancer.

title: Increased acquired activated protein C resistance in unselected patients with hematological malignancies
abstract: We have previously found that activation of coagulation in patients with various hematological malignancies was apparently not initiated by tissue factor (TF). Acquired activated protein C (APC) resistance may be another mechanism responsible for such hypercoagulation, and has been demonstrated in patients with solid tumors, but not in patients with hematological malignancy.  To investigate acquired APC resistance in a hypercoagulable cohort of patients with hematological malignancies.  Blood samples from 93 patients with acute myeloid leukemia (AML), chronic lymphatic leukemia, multiple myeloma, or non‐Hodgkin’s lymphoma, were analyzed before start and after completion of cancer therapy. APC resistance was measured using calibrated automated thrombography. The APC sensitivity ratio (APC‐SR) was calculated as the ratio of the endogenous thrombin potential (ETP) determined in plasma probed with either APC or buffer.  Untreated patients were found to have higher APC‐SR than healthy controls, and patients with AML had higher APC‐SR as compared to the other diagnoses, both findings being consistent with acquired APC resistance. The acquired APC resistance was partly ameliorated with cancer treatment. Decreased levels of protein S and TF pathway inhibitor were inversely correlated to APC resistance.  APC resistance may contribute to the hypercoagulable state in hematological malignancies.

title: Actiated protein C resistance in the absence of factor V Leiden mutation is a common finding in multiple myeloma and is associated with an increased risk of thrombotic complications
abstract: Thromboembolism is not uncommon in multiple myeloma (MM) patients on treatment, but its pathogenesis remains poorly understood. We report the results of a prospective randomized trial of 62 newly diagnosed MM patients tested at baseline for hypercoagulability and treated with intensive chemotherapy with or without thalidomide in a randomized fashion. During the induction phase, 12 patients (19%) developed evidence of deep venous thrombosis (DVT), which was significantly more common in the thalidomide arm (36%) than in the control group (3%) (P = 0.001). Fourteen patients (23%) were found to have a baseline-reduced response to activated protein C (APC) in the absence of factor V Leiden mutation. Using a Kaplan–Meier analysis, a significantly higher proportion of patients with APC resistance developed DVT (5/14 versus 7/38;P = 0.04) irrespective of thalidomide administration. The risk of DVT was highest (50%) in patients with APC resistance on thalidomide. None of the patients with normal APC response and not receiving thalidomide developed DVT. In conclusion, in this series, acquired APC resistance was present in almost one-quarter of newly diagnosed myeloma patients and significantly increased the risk of DVT.

title: Factor V Cambridge: a new mutation (Arg306-->Thr) associated with resistance to activated protein C.
abstract: A new factor V mutation associated with resistance to activated protein C and thrombosis (factor V Cambridge, Arg306-->Thr) was found in one patient from a carefully selected group of 17 patients with venous thrombosis and confirmed APC resistance in the absence of the common Gln506 mutation. The Arg306 mutation was also present in a first degree relative who also had APC resistance. Other potential causes of APC resistance, such as a mutation at the Arg679 site and the factor V HR2 haplotype, were excluded. Subsequent screening of 585 patients with venous thromboembolism and 226 blood donors did not show any other individual with this mutation. Factor VThr306 is the first description of a mutation affecting the Arg306 APC cleavage site and is the only mutation, other than factor V Leiden (Arg506-->Gln), that has been found in association with APC resistance. This finding confirms the physiologic importance of the Arg306 APC-cleavage site in the regulation of the prothrombinase complex. It also supports the concept that APC resistance and venous thrombosis can result from a variety of genetic mutations affecting critical sites in the factor V cofactor.

title: Plasma D-dimer levels in operable breast cancer patients correlate with clinical stage and axillary lymph node status.
abstract: To investigate the relationship between preoperative plasma D-dimer levels and extent of tumor involvement in operable breast cancer patients.



A total of 140 preoperative plasma specimens were obtained from women scheduled to undergo diagnostic breast biopsies. Ninety-five patients in the initial group went on to undergo axillary lymph node dissection. Of the 140 patients from whom plasma samples were obtained, 102 were subsequently diagnosed with invasive breast carcinoma, nine were subsequently diagnosed with ductal carcinoma-in-situ, and 20 were subsequently diagnosed with benign breast disease. Plasma D-dimer levels were quantitated using a commercially available immunoassay kit (DIMERTEST; American Diagnostica, Greenwich, CT). The relationships between plasma D-dimer and other prognostic variables (tumor size, estrogen receptor, progesterone receptor, nuclear grade, histologic grade, lymphovascular invasion, and clinical stage grouping) were then examined using univariate and multivariate linear and logistic regression analyses.



Median plasma D-dimer levels were significantly higher in patients with invasive carcinoma than those patients with either benign breast disease or carcinoma-in-situ (P =.0001). A significant relationship existed between the presence of elevated D-dimer (> 100 ng/mL) and involved axillary lymph nodes (chi(2) test; P =.001). Elevated D-dimer levels predicted positive lymph node involvement in both univariate regression (P =.0035) and multivariate linear regression (P =.012) models. In addition, elevated D-dimer levels predicted the presence of lymphovascular invasion in univariate logistic regression (P =. 0025) and multivariate logistic regression analysis (P =.0053). Quantitative D-dimer levels were highly correlated with clinical stage grouping (analysis of variance test; P =.002).



Plasma D-dimer levels were markers of lymphovascular invasion, clinical stage, and lymph node involvement in operable breast cancer. This correlation suggests that detectable fibrin degradation, as measured by plasma D-dimer, is a clinically important marker for lymphovascular invasion and early tumor metastasis in operable breast cancer.

title: High preoperative plasma D-dimer level is associated with advanced tumor stage and short survival after curative resection in patients with colorectal cancer.
abstract: An elevated plasma D-dimer level indicates activation of coagulation and fibrinolysis. Previous studies demonstrated that the preoperative plasma D-dimer level correlates with tumor stage in patients with colorectal cancer. This study examined the relationship between preoperative plasma D-dimer level and both pathological findings and TNM classification and examined the prognostic significance of preoperative plasma D-dimer level.



Preoperative plasma D-dimer levels were measured in 93 patients who underwent curative resection of colorectal cancer and 40 patients with benign colorectal diseases other than inflammatory bowel disease. The results were analyzed for correlations between preoperative plasma D-dimer levels in patients with colorectal cancer and pathological findings, TNM classification and postoperative survival.



Preoperative plasma D-dimer levels were significantly higher in patients with colorectal cancer than in patients with benign colorectal diseases. Plasma D-dimer levels were higher in patients with tumors that were relatively large, had relatively deep wall penetration and were at a relatively advanced TNM stage. Higher preoperative plasma D-dimer levels were significantly associated with shorter postoperative overall survival. Results of analysis with a multivariate proportional hazard model suggested that preoperative plasma D-dimer level was the third strongest prognostic factor; exceeded in importance only by lymph node status and preoperative carcinoembryonic antigen level.



Elevated plasma D-dimer levels in patients with colorectal cancer are associated with relatively advanced tumor stage and short postoperative survival after curative resection. It appears that measurement of preoperative D-dimer level would be useful in the preoperative diagnosis of tumor stage and prediction of postoperative survival.

title: Evaluating prethrombotic state in lung cancer using molecular markers.
abstract: Clotting abnormalities are well-recognized complications that occur with high frequency in patients suffering from underlying malignant diseases. New and highly sensitive molecular markers of hemostasis, thrombin-antithrombin III complex (TAT III), D-dimer fragments (DD), and plasmin-alpha 2-antiplasmin complex (PIC) were measured in 58 consecutive lung cancer patients. Significant elevation in the blood concentrations of DD, PIC, and TAT was found in lung cancer patients, with either extensive or limited disease compared with values obtained in a healthy control group and in another group of patients with chronic obstructive pulmonary disease. Patients with distant metastasis exhibited significantly higher levels of these parameters as compared to those without metastasis. These data indicated that there was a subclinical activation of blood coagulation and fibrinolysis in lung cancer from the early clinical stages of the disease. In addition, there appeared to be different levels of clotting activation according to histologic type of tumor and response to chemotherapy.

title: Clinical and Prognostic Significance of Coagulation Assays in Gastric Cancer
abstract: PurposeActivation of coagulation and fibrinolysis is frequently found among cancer patients. Such tumors are considered to be associated with a higher risk of invasion, metastases, and eventually, worse outcome. The aim of this study is to explore the clinical and prognostic value of blood coagulation tests in gastric cancer (GC) patients.Materials and MethodsA total of 44 consecutive patients with a pathologically confirmed diagnosis of GC were enrolled into the study. Pretreatment blood coagulation tests including prothrombin time (PT), activated partial thromboplastin time (APTT), international normalized ratio (INR), D-dimer (DD), fibrinogen (F) levels, and platelet (PLT) counts were evaluated. Control group comprised 50 age- and sex-matched individuals without history of malignancy and coagulation disorder.ResultsMedian age at diagnosis was 55 years, range 19–80 years; most had men (n = 32, 73 %) and metastatic disease (n = 31, 70 %). The level of blood coagulation tests showed a statistically significant difference between the patient and the control groups (P < 0.001 for all, but p = 0.07 for PT). DD levels were significantly associated with elevated PLT and LDH levels (p = 0.009 and p = 0.01, respectively). Patients with metastatic disease had higher levels of F (p = 0.001) and INR (p = 0.027) levels. Elevated DD levels tended to be a poor prognostic factor on outcome (p = 0.06).ConclusionChange in almost all coagulation tests are found in GC patients and only DD level seems to be of prognostic value.

title: Elevated D-dimers are also a marker of underlying malignancy and increased mortality in the absence of venous thromboembolism
abstract: D-dimers are used in conjunction with clinical probability scores in the assessment of venous thromboembolism (VTE), and they are elevated in other conditions, including malignancy, infection and arrhythmias. High levels of D-dimers in VTE are associated with adverse outcomes, including increased mortality. Their significance in patients without VTE has not previously been established.  To establish the clinical significance of elevated D-dimer levels in patients without VTE.  This prospective study included 2263 patient episodes of suspected deep vein thrombosis, which were excluded radiologically. Patients were followed up for survival and adverse events for a median of 22 months.  D-dimer levels greater than 4000 ng FEU/ml (4.9% of patients), and greater than 8000 ng FEU/ml (1.8%) were associated with a reduced overall survival. D-dimer levels greater than 8000 ng FEU/ml and age over 60 years were independent poor prognostic factors for overall survival (p<0.001.). D-dimer levels greater than 8000 ng FEU/ml were associated with an increased incidence of malignancy (p=0.003).  This study provides evidence of very high D-dimer levels in patients with cancer who do not have VTE. This suggests that elevated D-dimer levels in patients with VTE and malignancy are not solely due to presence of thrombus. High D-dimer levels in malignancy are likely to reflect the biology of the underlying tumour, with higher levels observed in breast, prostate and bowel cancers.

title: Relation between hemostatic parameters and prognostic/predictive factors in breast cancer.
abstract: In our study, we searched for a relation between various prognostic and predictive factors and hemostatic parameters.



One hundred women with newly diagnosed breast cancer after surgery were included. Patients did not receive systemic therapy or radiotherapy. The control group included 100 healthy, age-matched women. In the patient group, age, menopausal status, tumor size, grade, axillary lymph node status, steroid receptor status, p53, and HER2/neu were evaluated. Plasma levels of factor VIII, factor IX, D-dimer, fibrinogen, protein C, protein S, vWF, and antithrombin III were measured in both groups.



Plasma levels of factor VIII, factor IX, vWF, and CRP in patients with breast cancer were higher than those in controls. Protein S levels in patients were lower than in controls. There was no significant difference in other hemostatic parameters between the groups. In patients with axillary lymph node metastasis, factor VIII levels were significantly higher than in node-negative patients. There was a strong correlation between axillary lymph node status, number of metastatic nodes, and factor VIII levels. There was no correlation between factor VIII levels and CRP. Factor VIII levels were higher in the group having high HER2/neu (3+) than in the group with negativity for HER2/neu.



There was a strong correlation between axillary lymph node involvement, number of metastatic nodules, overexpression of HER2/neu, hemostatic parameters, and factor VIII levels. Our study showed that factor VIII level measurement can provide additional data for evaluation of breast cancer patients' prognosis.

title: Incidence of venous thromboembolism and the impact on survival in breast cancer patients.
abstract: The incidence of venous thromboembolism (VTE) and the risk factors associated with development of VTE have not been reported in a large population-based study of breast cancer patients.



The California Cancer Registry was merged with the Patient Discharge Data Set, and the number of VTE events determined among patients diagnosed between 1993 and 1999.



Among 108,255 patients with breast cancer, the 2-year cumulative VTE incidence was 1.2%, with a rate of 1.2 and 0.6 events/100 patient-years during the first and second half-year, respectively. The 1-year incidence of VTE was significantly increased compared with the general population (standardized incidence ratio of VTE, 4.2; 95% CI, 3.9 to 4.4). In a multivariate model, significant predictors of developing VTE within 2 years were: age (hazard ratio [HR], 2.0 if > 75 years v < 45; 95% CI, 1.6 to 2.6), the number of chronic medical comorbidities (HR, 2.9 if 3 v 0; 95% CI, 2.4 to 3.5), and advancing cancer stage (HR, 6.3; 95% CI, 5.3 to 7.5 for metastatic v local disease). In multivariate models, VTE was a significant predictor of decreased 2-year survival (HR, 2.3; 95% CI, 2.1 to 2.6) and when stratified by initial cancer stage, the effect was highest in patients with localized (HR, 5.1; 95% CI, 3.6 to 7.1) or regional stage (HR, 3.5; 95% CI, 2.5 to 4.8) cancer compared with patients with metastatic disease (HR, 1.9; 95% CI, 1.5 to 2.4).



Approximately 1% of breast cancer patients developed VTE within 2 years, with the highest incidence in the first 6 months after diagnosis. Metastatic disease and comorbidities were the strongest predictors. The diagnosis of VTE was associated with a higher risk of death within 2 years.

title: Increased incidence of neoplasia of the digestive tract in men with persistent activation of the coagulant pathway
abstract: Thrombin promotes angiogenesis and cell proliferation in cancer. Whether thrombin turnover influences cancer incidence is unknown.  To explore the relation between the status of the coagulant pathway and cancer incidence by population survey.  Of 4009 middle‐aged men clinically free of malignancy, 3052 (76.1%) were recruited. Measurements of hemostatic status were made annually for 4 years, and follow‐up for morbidity and mortality was maintained thereafter. Persistent activation of the coagulant pathway was diagnosed when prothrombin fragment 1+2 and fibrinopeptide A concentrations exceeded the upper quartiles of the population distribution in two consecutive annual examinations. Cancer incidence rates in men developing persistent activation (taking the time of onset of activation as baseline) were compared with those in men remaining free of this condition.  Persistent activation of the hemostatic pathway was a distinct entity found in 111 men [43 expected by chance alone (P < 0.001)], and associated with activation throughout the coagulation pathway. Total mortality (/1000 person‐years) was higher in those with persistent activation than in others (17.1 and 9.7, respectively, P = 0.015), owing to a higher mortality from all cancers (11.3 and 5.1, respectively, P = 0.01), due in turn largely to a higher mortality from cancers of the digestive tract (6.3 and 1.9, respectively, P = 0.004). Trends were similar for non‐fatal cancers.  Persistent activation of the coagulant pathway plays a role in the preclinical phase of cancer and is associated with an increased incidence of clinical malignancy, especially of the digestive tract.