Hemostatic biomarkers in cancer progression

Thrombosis Research 164 (2018) S54–S61

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Hemostatic biomarkers in cancer progression ⁎

T

Anna Falanga , Marina Marchetti Division of Immunohematology and Transfusion Medicine, Hospital Papa Giovanni XXIII, Bergamo, Italy

A R T I C L E I N F O

A B S T R A C T

Keywords: Cancer Hypercoagulability Cancer prognosis Biomarkers Predictive factors Fibrinogen D-dimer Thrombin generation NETs Microparticles

Malignant disease is characterized by a hemostatic imbalance, usually shifted towards a procoagulant direction, and a high incidence of thrombotic complications. The mechanisms of hemostasis that are critically involved in thrombosis are also implicated in tumor progression, angiogenesis, and metastatic spread. As there is a close relationship between cancer and the clotting system, circulating biomarkers of activation of various hemostasis compartments (i.e. coagulation, fibrinolysis, platelets, endothelium, and other blood cells) have been extensively studied to predict cancer outcomes along with predicting the thrombotic risk. In this review, we will summarize the results of published studies and will focus on ongoing research and future directions of clotting activation bioproducts as biomarkers of cancer disease and progression.

1. The biological significance of hemostatic biomarkers for cancer progression The National Institute of Health defines a biomarker as a cellular, biochemical, and/or molecular entity that can be objectively measured and serve as an indicator of ongoing normal or pathogenic biological processes, or pharmacological responses to therapeutic interventions. In the context of malignant disease, biomarkers can play a crucial role in aiding the diagnosis of early stage cancers (diagnostic biomarker), estimation of tumor aggressiveness, predict the likelihood of patient survival in the absence of treatment (prognostic biomarker), and predict patient response to antitumor therapy (predictive biomarker). Despite the remarkable advances in tumor biology research and in “omics” technologies, limited tumor biomarkers have been adopted successfully into routine clinical care of oncologic patients over the last 30 years [1, 2]. The requirement for new biomarkers is continuously increasing for personalized medicine, the growing of the therapeutic armamentarium, and the evidence that early cancer detection in otherwise asymptomatic patients does improve both survival and quality of life. Patients with cancer are commonly characterized by abnormalities in laboratory coagulation tests, underlying a subclinical hypercoagulable condition [3]. The results of laboratory tests demonstrate that a process of fibrin formation and removal is very active during the development of malignancy. Histopathological analyses demonstrate the presence of fibrin and platelet aggregates in and around various types of tumors, indicating local blood clotting activation. Different molecular mechanisms can cause the onset of a hypercoagulable state. In cancer, the interaction between ⁎

tumor cells with the vascular endothelium, and constituents of the coagulation cascade is responsible for the conversion of the intravascular milieu into a prothrombotic, proinflammatory, and proadhesive environment (Fig. 1) [4, 5]. Hypercoagulability in cancer increases the risk of thromboembolic complications, which actually occur at a high rate compared to a non-cancer population [6]. Accordingly, a number of hemostatic biomarkers have been evaluated with regard to their capacity to predict primary or recurrent venous thromboembolism (VTE) in these patients, and some of these have been integrated in risk score models for the evaluation of individual VTE risk [7, 8]. However, hypercoagulability also influences the biology of the tumor, favoring its growth and development of metastasis [5]. Activation of blood coagulation results in a selective advantage for cancer cells. Indeed fibrin, the final product of the coagulation cascade, provides a scaffold for tumor cell anchorage and invasion, and protects the tumor cell from immune system recognition and destruction. Further, while activated coagulation proteases (i.e. thrombin, FVIIa, FXa) induce receptor-mediated intracellular signals promoting invasive growth [9] [10]. Furthermore, the cancer-associated hypercoagulable state can be linked to known oncogenetic lesions causal for the onset and progression of malignancy [11]. Due to the relationship between cancer and hemostasis, the biomarkers of hemostatic system activation can be a promising tool in predicting cancer outcomes. Several laboratory abnormalities of hemostasis have been described in cancer, including prolonged and shortened prothrombin time (PT), partial thromboplastin time (PTT), increased and decreased levels of thrombin, FV, FVIII, FIX, FXI, FXII,

Corresponding author at: Division of Immunohematology and Transfusion Medicine, Hospital Papa Giovanni XXIII, Piazza OMS, 1, 24127 Bergamo, Italy. E-mail address: [email protected] (A. Falanga).

https://doi.org/10.1016/j.thromres.2018.01.017 Received 1 December 2017; Received in revised form 8 January 2018; Accepted 10 January 2018 0049-3848/ © 2018 Elsevier Ltd. All rights reserved.

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Fig. 1. Tumor cell associated biomarkers during cancer development and dissemination. PANEL A. Extravascular activation of blood coagulation by tumor cells. Tumor-associated vasculature is characterized by an enhanced permeability induced by tumor cell derived proangiogenic factors. This permeability allows the extravasation of coagulation proteins into the tumor microenvironment, where they interact with tumor-associated TF. The assembly of coagulation proteins on tumor cells causes the local activation of blood clotting cascade, which is potentiated by the TF-bearing MP released by the tumor cells, and the low levels of TFPI, and that culminates in thrombin generation and fibrin formation. The fibrin formed around tumors serves as a temporary matrix for tumor cell migration, which is supported by the proteins of the fibrinolytic system expressed by the tumor tissues (i.e. uPA, tPA, uPAR, and PAI-1). PANEL B. Intravascular activation of blood coagulation by tumor cells. Once in circulation, the tumor cell can activate, as well as blood coagulation, induce the prothrombotic features of endothelium and platelets, and NETosis by neutrophils. Reductions in the levels of natural anticoagulant (Protein C, Antithrombin), the increased thrombin generation, fibrin degradation products (D-dimer, FDP), and in fibrinogen levels are some of the hemostatic biomarkers modifications induced by the tumor cells. These alterations have a significant impact on both tumor metastasis and cancer-associated thrombosis. Abbreviations: MP = microparticles, uPA = urokinase plasminogen activator, t-PA = tissue plasminogen activator, PAI-1, plasminogen activator inhibitor –1, TF = Tissue Factor, TFPI = TF pathway inhibitor, FDP fibrin degradation products, =NETs = neutrophil extracellular traps, EPCR = endothelial protein C receptor, F1 + 2 = prothrombin fragment 1 + 2.

fibrinogen, fibrinogen/fibrin degradation products, the thrombin-antithrombin complex (TAT), and thrombocytosis. These laboratory abnormalities increase with cancer progression, which are consistent with a tight relationship between tumor burden and clotting deregulation [12]. Accordingly, consistent evidence emerging from the literature demonstrates that plasma levels of thrombotic biomarkers are significantly lower in patients with early cancer compared to those with advanced cancer. These observations have pathophysiological and clinical implications. In the last three decades, many studies have been performed with the aim to evaluate the levels of hemostatic markers in patients with advanced, as well as limited, disease in relation to the overall survival (OS), disease specific survival (DSS), disease free survival (DFS), progression free survival (PFS), and in relation to tumor response to therapies (Fig. 2). Many hemostatic biomarkers have been involved in these studies, ranging from standard clotting tests to more sophisticated hemostatic assays, evaluated alone or in combination of two or more. In this review, we will focus on hemostasis biomarkers associated with tumor progression in specific cancer types, from those extensively investigated (i.e. D-Dimer, fibrinogen, plasminogen activator proteins) to those, which are novel with limited evaluation (i.e. plasma microparticles, endogenous thrombin potential, neutrophil extracellular traps or NETs).

these assays in most hospitals, their low cost, and their use in pre-operative routine screening. Overall, data from these studies agree on a significant association between elevated plasma levels of D-dimer and/ or fibrinogen and advanced tumor stage and poor prognosis in patients with different types of cancer, including lung, colorectal, gastric, esophageal, ovarian, renal, pancreatic and breast cancer. Furthermore, the finding of majority of these studies highlights a strong association of these biomarkers with cancer outcomes and patient survival, even independently from VTE. 2.1.1. Lung cancer Elevated D-dimer and fibrinogen plasma levels have been frequently reported in lung cancer patients in association with large tumor burden, clinical progression and poor prognosis [13–16]. In 70 patients with non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) a significant prognostic role of D-dimer for OS was found, independent of tumor stage, histological type, performance status and tumor size [15]. In 2003, the results of the first large retrospective study of 826 consecutive patients with a new diagnosis of lung cancer [14], reported the median survival periods were shorter in patients with abnormally elevated compared to normal values (154 days vs. 308 days; p < .01). The difference was larger in patients with adenocarcinoma and in those with earlier stages of the disease (i.e., Stage T1 tumors). In the subsequent years, new studies reinforce previous evidences. In 100 newly diagnosed lung cancer patients (87% with NSCLC), D-dimer levels predicted OS independently of the clinical stage of disease, histologic tumor type and performance status (HR: 5.1, p = .013) [16]. Similarly, the pre-operative plasma D-dimer level was an important prognostic biomarker for 1-year OS, independently of VTE, in 232 patients with operable NSCLC [13]. Particularly, for patients with high D-dimer

2. Extensively investigated thrombotic biomarkers for cancer progression 2.1. D-DIMER AND FIBRINOGEN Fibrinogen and D-dimer are the most studied hemostatic biomarkers in relation to cancer disease features, likely due to the availability of S55

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Fig. 2. Role of hemostatic biomarkers in cancer progression. Different circulating and tumor cell-associated hemostatic proteins and coagulation gene polymorphisms have been evaluated as biomarkers in cancer disease. Studies have been performed to test their utility, alone or in combination with classical tumor markers, in helping the diagnosis, predicting the prognosis and cancer recurrence, and how the patient will respond to antitumor therapy.

progression than CEA [26]. Subsequently, the prognostic value of plasma high D-dimer levels (> 375 μg/l) was prospectively confirmed in 51 patients with CRC [27]. Otherwise, the clinical utility of preoperative D-Dimer assessments suggested by previous studies was not established by Pedrazzani et al., in 199 patients who underwent resection for CRC, and CEA was confirmed to be the most reliable and valid indicator of prognosis [28]. Similar to D-dimer, the independent prognostic value of high preoperative plasma fibrinogen levels have been investigated in different retrospective cohorts of CRC patients [22, 24, 25, 29]. Additionally, the studies found a significant association of preoperative plasma fibrinogen levels with tumor size and depth of tumor invasion. In 341 patients with CRC [24], using multivariate analysis, preoperative fibrinogen levels, histologic grade, and lymph node involvement were defined as significant independent prognostic factors. In 170 patients who underwent curative surgery for CRC, prolonged prothrombin time (≥11.7 s), high fibrinogen (≥327 mg/dl), D-dimer (≥1.3 μg/ml) and FDP (≥2.7 μg/ml) levels were associated with shorter OS [25]. The prospective study by Hong et al. confirmed the negative prognostic role of high preoperative plasma fibrinogen, but not of D-dimer in non-metastatic CRC treated by curative resection [22]. Particularly, in a multivariate model, hyperfibrinogenemia was a negative prognostic marker (HR 3.39, 95% CI 1.34–8.67) in patients with normal Glasgow Prognostic scores. Otherwise, the retrospective cohort study in 652 Western patients with stage I-IV CRC undergoing surgery, failed to identify a prognostic value of high preoperative fibrinogen for OS and DSS [29]. The predictive value of fibrinogen as a response to therapy was investigated in a retrospective cohort of 947 patients who received preoperative chemo-radiotherapy followed by curative surgery for primary rectal cancer [30]. In this study, multivariate analysis revealed a presurgery CEA level ≤ 5.0 ng/ml and fibrinogen level ≤ 270 mg/dl were predictors of down-staging, primary tumor regression, and complete response. Interestingly, the predictive value of fibrinogen-CEA in combination of tumor response following preoperative chemo-radiotherapy was higher than that of fibrinogen alone. A recent meta-analysis that included 15 eligible observational studies (2283 cases), demonstrated that high pretreatment plasma D-dimer was a predictor of poor survival of CRC patients (HR 2.143, 95% CI 1.922–2.390) [31].

values, the adjusted HR for 1-year mortality and OS was 3.19 (95% CI, 1.18–7.12) and 1.54 (95% CI, 1.11–2.78), respectively. These results were recently confirmed in a retrospective cohort of 393 patients with SCLC [17], in which elevated D-dimer levels before chemotherapy predicted for significantly (p < .001) shorter PFS (6.2 vs. 9.6 months) and OS (15.7 vs. 24.4 months). This study, interestingly, additionally demonstrated the importance of a longitudinal evaluation of a biomarker. Indeed, patients with D-dimer converting from high to normal levels had significantly better PFS and OS than those with D-dimer levels remaining high after two cycles of chemotherapy. Two prospective studies specifically evaluated the clinical significance of plasma fibrinogen and D-dimer in predicting both the response to chemotherapy and prognosis in lung cancer patients [18, 19]. Zhu et al., prospectively measured plasma fibrinogen and D-dimer levels before and during chemotherapy in 74 SCLC patients who received first-line therapy [19]. Using multivariate analysis, high fibrinogen and D-dimer levels following two chemotherapy cycles were independently unfavorable factors for PFS and OS. Similarly, in 78 patients with advanced (stage III and IV) lung cancer (both NSCLC and SCLC), median survival times were 405 days and 207 days (p < .001) for patients with low D-dimer (≤0.65 μg/ml) and high D-dimer level (> 0.65 μg/ml), respectively [18]. A significant association between the plasma levels of D-dimer and the response to chemotherapy was also observed (p < .03). Finally, the results of two meta-analyses endorse the prognostic role of D-dimer in lung cancer [20, 21]. The first, including seven eligible studies (1377 patients), found that the HR for OS of patients with a high pre-treatment D-dimer level was 1.12 times higher than those of patients with low D-dimer level. Similarly, the second meta-analysis that included 11 studies, reported a worse OS (HR 2.06, 95% CI 1.64–2.58) for lung cancer patients with high D-dimer [20]. Additional information provided by this analysis is that the HR for OS was increased in Asian compared to non-Asian patients (HR 2.48 vs. 1.89), and that D-dimer had a greater predictive value when using the ELISA compared to latex method.

2.1.2. Colorectal cancer (CRC) The clinical utility of D-dimer and fibrinogen has been largely investigated in patients with CRC undergoing surgery or receiving chemotherapy [22–26]. Elevated pre-operative D-dimer levels were associated with advanced tumor stage and short survival following curative resection in CRC [23], and by multivariate analysis, D-dimer was the third strongest independent prognostic factor, exceeded in importance only by lymph node status and carcinoembryonic antigen (CEA) level. Similarly, in a Phase II randomized trial comparing bevacizumab plus 5-fluorouracil/leucovorin (5-FU/LV) with 5-FU/LV alone in metastatic CRC patients, D-dimer level was a better predictor of OS and disease

2.1.3. Gastric cancer As observed for lung and CRC, data from literature undoubtedly suggest that high plasma fibrinogen and D-dimer levels are valuable biomarkers to predict peritoneal dissemination and poor survival in gastric cancer patients [32–34]. High preoperative plasma fibrinogen levels were significantly associated with the presence of metastatic lymph nodes and distant metastasis in 354 patients [32], and positively correlated with advanced tumor stage and poor survival in another S56

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cohort of 1196 patients [34]. Pretreatment D-dimer > 1.465 μg/ml predicted a significant shorter OS in 247 gastric cancer patients (22 vs. 48.10 months) (HR 2.28, 95% CI 1.36–3.81) [33].

metastatic BRAFV600-mutated melanoma who were receiving BRAF inhibitors, high pre-treatment D-dimer levels were associated with significantly poorer PFS and OS [49].

2.1.4. Breast cancer Plasma D-dimer levels was largely correlated with clinical stage grouping in breast cancer [35–37]. Blackwell et al., established plasma D-dimer as a clinically important marker for lymphovascular invasion and early tumor metastasis in operable breast cancer [35]. This finding has been reinforced by a recent prospective study demonstrating a significant association between high plasma D-dimer and circulating tumor cells in patients with metastatic breast cancer [38]. Cancer cells in peripheral blood are thought to play a crucial role in the metastatic cascade [39]. Two retrospective studies investigated the prognostic role of plasma fibrinogen in breast cancer patients, according to the estrogen receptor status [36, 37]. In 2073 consecutive breast cancer patients, preoperative fibrinogen levels > 2.83 g/l was an independent predictive factor for OS (HR 1.475, p = .001) [36], and predominantly in stage IIIII, luminal subtypes, and triple-negative breast cancer patients. In 520 consecutive breast cancer patients, an elevated plasma fibrinogen level predicted shorter DSS both in the overall group of patients (HR 1.71, 95% CI: 1.02–2.85; p = .042), and in the subgroup with ER/PR+, HER2- tumors (HR 3.63, 95% CI 1.37–9.64, p = .010) [37]. Hypercoagulable states potentially affect antitumor treatment efficiency, as suggested by the poor treatment response to trastuzumab in advanced breast cancer patients (HER-2 positive) with fibrinogen values > 2.88 g/l [40].

2.2. THE PLASMINOGEN ACTIVATOR SYSTEM PROTEINS The plasminogen activator system includes urokinase-type plasminogen activator (u-PA) and its receptor (u-PAR), tissue PA (t-PA) and type 1 and 2 inhibitors (PAI-1 and PAI-2). Many of these proteins play a role in tumor invasion and metastasis [50], and have been evaluated as potential tumor biomarkers, both in cancer tissue specimens and in plasma. Concerning their role as tissue-associated biomarkers, several retrospective and prospective studies, and multicenter breast cancer therapy trials, have reported that elevated levels of u-PA and PAI-1 in breast tumor tissue are statistically independent and potent predictors of poor patient outcome [51, 52]. Currently, tissue u-PA and PAI-1 are among the best-validated prognostic biomarkers in lymph node negative breast cancer, and high levels of uPA and PAI-1 are also associated with benefit from adjuvant chemotherapy in patients with early breast cancer. uPA/PAI-1 measurement by ELISA has undergone detailed analytical validation. A recent retrospective analysis in lymph nodenegative invasive breast cancer patients, comparing the prognostic impact of HER2 status, uPA, PAI-1, and traditional prognostic factors, found an HR 1.98 (95% CI 0.83–4.76) for death for patients with high uPA and/or PAI-1 compared to patients with low levels of both [53]. Although no significant correlation for uPA, uPAR and PAI-1 between plasma/serum and tumor tissue could be demonstrated, several studies reported that pre-operative plasma levels uPAR and PAI- 1 can predict poor prognosis in patients with colorectal [54–56], ovarian [57], or breast cancers [58]. Particularly, in the study by Riisbro et al. [56], the independent prognostic value of soluble uPAR was retrospectively determined by two different ELISA analyses in pre-operatively collected plasma samples from a Swedish (n = 354) and Danish (n = 255) cohort of rectal cancer patients. The results confirm the preoperative concentration of plasma uPAR as an independent prognostic biomarker in patients with rectal cancer, independently of the two different versions of ELISA utilized to perform the analyses. Plasma levels of PAI-1, D-dimer, and activated PC-dependent thrombin generation were assessed in a cohort of 187 breast cancer patients in relation with clinical outcome [58]. Cox analysis demonstrated that an elevated plasma PAI-1 level had a negative prognostic impact in terms of relapse-free (HR 2.5, p = .021) and OS (HR 2.7, p = .002) in this group of patients.

2.1.5. Gynecological cancers Among gynecological cancers, studies have been performed in patients with ovarian [41, 42] and cervical carcinomas [43]. In these tumors, again elevated fibrinogen and D-dimer levels were associated with advanced tumor stage, older age, large tumor size, and deep stromal invasion. Regarding tumor prognosis, pre-surgical plasma levels of D-dimer, fibrinogen, and platelet, were promising biomarkers to predict disease progression and surgery outcome in patients with epithelial ovarian carcinoma (EOC) [41]. Using a multivariate analysis, high pre-treatment D-Dimer levels were independently associated with poor OS in patients with EOC stages II to IV [42], and in patients with primary serous ovarian cancer [44], while high fibrinogen levels were significantly predictive of worse outcome in patients with early-stage cervical cancer (stage I-IIA) [43]. Two meta-analysis were recently published on the prognostic value of D-dimer levels in gynecological cancer [45, 46]. The first meta-analysis, including 15 eligible studies (1437 cases), demonstrated that D-dimer level paralleled the clinical signs of disease progression in ovarian cancer patients. However, the predictive value of poor prognosis was significantly dependent on the sample sizes of the study, and elevated D-dimer could predict increased risk of mortality when the sample size was > 100 patients (HR 1.80, 95% CI: 1.283–2.523). In the second meta-analysis that included 7 eligible studies (1112 cases), all conducted in East Asia, plasma D-dimer levels were predictive of a shorter OS in ovarian, cervical and endometrial carcinoma (HR 2.09, 95% CI 1.59–2.74) [45].

2.3. TISSUE FACTOR (TF) AND TF PATHWAY INHIBITOR (TFPI) Tissue Factor (TF), the main initiator of the blood coagulation cascade, is the cell-associated receptor of activated factor VII (FVIIa) expressed by vascular cells upon stimulation. Different tumor cells constitutively express TF [59] able to induce the generation of thrombin and fibrin formation [60]. TF is involved in a variety of biologic processes, and numerous in vitro and in vivo studies have clearly established a central role for TF in cancer progression and spread [61, 62]. In addition, TF represents a potential target in the treatment of several malignancies, and different methods of targeting TF have been investigated [63, 64]. Tumor TF expression was an independent prognostic indicator for OS in breast cancer [65] and for DFS in osteosarcoma [66]. Under pathological conditions, TF can be detected at increased concentration in plasma where it circulates alone or associated to cellular derived microparticles (MP) expressing also the procoagulant phosphatidylserine (PS) [67, 68]. TF-containing MP is the main source of TF activity released from human cancer cells [69]. A potential diagnostic value of MP-TF activity has been suggested in women with suspected ovarian cancer [70], whereas in pancreatic cancer, MP-TF activity represent a biomarker for a poorly differentiated

2.1.6. Other malignant diseases Finally, fibrinogen and dimer were evaluated in other tumor types, including gallbladder cancer, renal cell carcinoma and melanoma. For example, pre-operative high fibrinogen values (> 402 mg/dl) were independently associated with worse OS, tumor stage, margin status, and lymphatic metastasis in patients with gallbladder cancer [47]. In patients with renal cell carcinoma, multivariate analyses demonstrated that fibrinogen independently predicted poor DFS (HR 2.52; 95% CI 1.04–6.31; p = .029) and DSS (HR 3.89, 95% CI 1.13–13.40; p = .032), whereas D-dimer had a negative independent prognostic value on OS (HR 4.01, 95% CI 1.54–10.50; p = .005) [48]. In patients with S57

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evaluate the functionality of the PC pathway, and highlights the presence of an acquired APC-resistant phenotype. Patients with solid tumor and hematological malignancies can show an increased TG potential [92–97], and its role as a biomarker of increased risk of VTE in cancer has been explored in large prospective trials [92, 98, 99]. Only one study, to our knowledge, has been published on the prognostic role of TG in cancer [100]. In this case-control study of association of genotypic and phenotypic variables of coagulation with breast cancer, the thrombin generation parameters, as well as high D-dimer, high APC resistance and low antithrombin levels, significantly predicted breast cancer diagnosis.

and invasive pancreatic cancer phenotype and poor survival [71, 72]. Among the soluble forms of TF, alternatively spliced TF in the plasma of patients with pancreatic cancer may predict an aggressive tumor phenotype [73]. TFPI, and its structural homolog TFPI-2, belong to the family of Kunitz-type serine proteinase inhibitor. TFPI inhibits the TF/FVIIa and exhibits antiangiogenic and antimetastatic effects [74]. The expression of TFPI-2 by tumor tissues is inversely related to an increasing degree of malignancy [75]. Low TFPI-2 protein expression in tumor tissues is an independent prognostic factor for poor DFS (p < .001) and OS (p < .001) in pancreatic carcinoma [76], and of cancer progression, recurrence and poor OS following surgery in breast cancer [77]. Considered as a putative tumor suppressor gene, TFPI-2 gene is frequently inactivated by promoter hypermethylation in nasopharyngeal carcinoma [78], glioblastoma, and NSCLC where it predicts poor OS [79]. The measurement of plasma TFPI, together with tenascin and CA 19–9's has been found of clinical utility for early detection of surgically resectable early stage pancreatic cancer, as well as for enhanced survival from this routinely lethal cancer [80].

3.4. COAGULATION GENE POLYMORPHISMS Coagulation gene polymorphisms have been investigated for their role in the risk of cancer, as well as in tumor progression [101]. 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 1801 subjects for CRC risk in a large case-control study. Carriers of the antithrombotic FXIII Val34Leu polymorphism showed a 15% reduced risk of developing CRC (OR = 0.85; 95% CI, 0.74 to 0.97) compared with non-carriers, while no effects of PAI-1 4G/5G, MTHFR 677C > T, and fibrinogen gamma 10034C > T on CRC risk were found [102]. Associations with SNPs in FV, FX and EPCR to breast cancer susceptibility were demonstrated by a case–control study [100]. Regarding tumor progression, FV Leiden, prothrombin G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms were not associated with DFS in breast cancer patients [103]. Conversely, in CRC, the inherited homozygous CC polymorphism of TFPI (−33 T → C) associated with higher TFPI levels, predicted for improved DFS [104].

3. Less investigated thrombotic biomarkers in cancer progression 3.1. ANTITHROMBIN, PROTEIN C (PC) AND ENDOTHELIAL PROTEIN C RECEPTOR (EPCR) Antithrombin is a key inhibitor of the coagulation cascade, but it may also function as an antiinflammatory, antiangiogenic, antiviral and antiapoptotic protein. A novel function of antithrombin is the capability to act as a modulator of tumor cell migration and invasion [81]. There are limited studies investigating the prognostic significance of antithrombin levels on cancer outcomes [82, 83]. Low antithrombin levels and high D-dimer significantly predicted short survival in lung cancer patients [82]. In ovarian cancer, by univariate analysis, reduced antithrombin and elevated von Willebrand factor (vWF) and D-dimer levels were associated with poor OS [83]. PC is a natural coagulation inhibitor involved in hemostasis, inflammation and signal transduction, and has a protective effect on the endothelial barrier. Recently, Wilts et al., associated lower levels of PC with increased mortality in 477 patients with advanced prostate, NSCLC, or pancreatic cancers [84]. EPCR is the cellular receptor of PC that plays a key role in mediating activated PC-induced cytoprotective effects [85] and has been implicated in cancer mechanisms [86]. In aggressive triple-negative breast cancer cells, EPCR expression characterizes a cancer stem cell-like population with tumor initiating properties in vivo [86].

4. Prospective clinical studies of hemostatic biomarkers for cancer progression Except for tumor tissue-associated uPA and PAI-1 levels, which are presently the best validated prognostic biomarkers for breast cancer, all other hemostatic biomarkers still require an extensive analytical and clinical validation. As discussed above, the majority of studies published so far are retrospective in nature, often involving small groups of patients, and not designed ad hoc to evaluate the predictive role of the hemostatic biomarkers in relation to cancer outcome measures. Nevertheless, the available data call for the conduction of large prospective clinical studies to demonstrate the validity or not of hemostatic biomarker utility in cancer management. In this regard, two large prospective studies evaluating hemostatic biomarkers in cancer are ongoing. The Vienna Cancer and Thrombosis Study (CATS) is a prospective and observational cohort study designed to investigate and establish risk factors predictive of VTE occurrence in cancer patients and to improve risk assessment of VTE in patients diagnosed with cancer. In the frame of this study, that included newly diagnosed patients with a broad range of malignancies, the authors found that D-dimer value at enrollment was an independent prognostic factor for OS and mortality [105]. The univariate HR of D-dimer (per double increase) for mortality was 1.5 (95% CI 1.4–1.6, p < .001) and remained significant in multivariable analysis including tumor subgroups, age, sex and VTE. The HYPERCAN (“HYPERcoagulation and CANcer”) is an ongoing prospective, multicenter, observational study specifically designed to evaluate whether hypercoagulation is perhaps an innovative tool for risk assessment, early diagnosis and prognosis in cancer (ClinicalTrials. gov # NCT0262281) [106]. Funded by the “Italian Association for Cancer Research (AIRC)”, it began on January 2012 and includes two main projects, i.e., project 1 and 2. The objective of project 1 is to assess whether thrombotic markers potentially act as a tool for cancer risk

3.2. NEUTROPHIL EXTRACELLULAR TRAPS (NETS) NETs are extracellular structures composed of DNA strands and neutrophil granule proteins that bind pathogens to prevent their spread and ensure increased local concentrations of toxic factors. In the recent years, NETs have emerged as significant contributors to thrombosis and cancer [87, 88]. Tumor cells, through a systemic effect on the host, can induce an increase in peripheral blood neutrophils, which are predisposed to NETs formation [89], but can also directly stimulate NETs release [90, 91]. To date, no studies have evaluated the role of NETs as biomarkers in predicting cancer outcomes. 3.3. THROMBIN GENERATION POTENTIAL Hypercoagulability may be due to increased levels of coagulation factors, decreased activity of anticoagulant proteins or the presence of thrombophilic polymorphisms. The thrombin generation (TG) assay is a global hemostatic test sensitive to several genetic and acquired thrombophilic states. When performed in the presence of exogenous activated PC (APC) or thrombomodulin, the TG assay can globally S58

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prediction in healthy subjects. Project 2 aims to prospectively assess whether the occurrence of hypercoagulability in patients with a newly diagnosed breast, NSCLC, CRC, or gastric cancer may predict for OS, PFS, RFS, response to therapy, and occurrence of VTE. Various thrombotic biomarkers are evaluated, including D-dimer, fibrinogen, TG, TF, prothrombin fragment 1 + 2 (F1 + 2), MP procoagulant activity, PC, PS, coagulation factor polymorphisms, t-PA, PAI-1, FVIII and FXIII. In project 1, a total of 8125 healthy blood donors have been enrolled so far, and the enrollment is ongoing to reach the 10.000 subjects ‘accrual’. In project 2, a total of 3429 cancer patients have been enrolled: 1893 with limited resected tumors, 310 with locally advanced tumors, and 1226 with metastatic disease. Preliminary results in a subgroup of 690 patients with limited-resected breast cancer, demonstrated after 2 years follow-up, a 5.7% early disease recurrence. By multivariate analysis, TG peak ≥ 394 nM at enrollment was observed as an independent risk factor for early disease recurrence, as well as after correcting for gender, age and triple-negative subtype (HR 2.28; 95% CI 1.25–4.16; p < .01). Alternatively, early disease recurrence was not significantly associated with fibrinogen or D-dimer levels. These preliminary data demonstrate, for the first time, that measurements of TG may assist in identifying breast cancer patients at high risk of early disease recurrence. Further studies are required to validate the prognostic role of TG [107].

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5. Conclusions The activation of the clotting-fibrinolytic system in cancer patients is common and represents an unfavorable clinical sign. A hypercoagulable state often is associated with a large tumor burden, clinical progression, low rates of response to chemotherapy, and a poor prognosis. A substantial amount of data suggests that hemostatic biomarkers are perhaps a potential utility in predicting cancer outcomes. As positive remark, the majorities of available publications in this setting are focused on specific cancer types, and report a significant relationship of hemostatic biomarkers with different tumor outcome measures, even after correcting for many potential confounding factors including VTE and surgical interventions. Among limitations, the majorities of these studies are retrospective in nature and not specifically designed to address the role of hemostatic biomarkers in cancer disease. In addition, recruitment is often mono-institutional, and includes small heterogeneous cohorts of patients with different local and systemic treatments. Furthermore, in a majority of the cases the study design includes a single time point measurement of the given biomarker(s), and only rarely involves a longitudinal biomarker assessment. Finally, the cut-off values for each biomarker are derived from specific study populations and therefore, cannot be easily extrapolated and compared from one laboratory setting to another. In conclusion, except from tumor-tissue uPA and PAI-1 that have achieved a high level of validation in breast cancer, all biomarker evaluations are required to be standardized at analytical levels, rely on well-established cut-off values, and clinically validated by means of prospective clinical trials. Details of nature of conflict of interest None. Acknowledgements The HYPERCAN project is funded by AIRC “5xMILLE” n. 12237 grant from the “Italian Association for Cancer Research (AIRC)”. References [1] A. Mordente, E. Meucci, G.E. Martorana, A. Silvestrini, Cancer biomarkers discovery and validation: state of the art, problems and future perspectives, Adv. Exp.

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