Original Articles. Hyuk-Chan Kwon 1, Sung Yong Oh 1, Suee Lee 1, Sung-Hyun Kim 1, Jin Yeong Han 2, Ri Young Koh 2, Min Chan Kim 3 and Hyo-Jin Kim 1

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1 Jpn J Clin Oncol 2008;38(1)2 7 doi: /jjco/hym157 Original Articles Plasma Levels of Prothrombin Fragment F112, D-dimer and Prothrombin Time Correlate with Clinical Stage and Lymph Node Metastasis in Operable Gastric Cancer Patients Hyuk-Chan Kwon 1, Sung Yong Oh 1, Suee Lee 1, Sung-Hyun Kim 1, Jin Yeong Han 2, Ri Young Koh 2, Min Chan Kim 3 and Hyo-Jin Kim 1 Departments of 1 Internal Medicine, 2 Laboratory Medicine and 3 Surgery, Dong-A University College of Medicine, Busan, South Korea Received July 4, 2007; accepted October 23, 2007 Objective: The principal objective of this study was to determine the relationship between preoperative coagulation tests and the extent of tumor involvement in gastric cancer patients. Method: A total of 110 patients with adenocarcinoma of the stomach were studied in order to evaluate this relationship. Platelet count (P), prothrombin time (PT), activated partial thromboplastin time, D-dimer, fibrinogen degradation product, thrombin antithrombin complex and prothrombin fragment F1þ2 (F1þ2) were evaluated. Results: The D-dimer levels were positively correlated with the depth of invasion (P ¼ 0.007). Plasma D-dimer and PT were highly correlated with degree of lymph node involvement (P ¼ 0.006, 0.004, respectively). D-dimer level, PT and plasma F1þ2 level were correlated with clinical stage (P ¼ 0.001, 0.017, 0.031, respectively). PT and F1þ2 levelswere significant in the prediction of the presence of lymph node involvement on the multivariate logistic regression models (odds ratio ( ); P ¼ and odds ratio ( ); P ¼ 0.023, respectively). Conclusion: PT and plasma levels of F1þ2 and D-dimer could be markers of degree or presence of lymph node involvement and clinical stage in patients with operable gastric cancer. Key words: coagulation test gastric cancer INTRODUCTION An increased frequency of thrombosis in patients with gastrointestinal cancer was first documented in 1865 (1). Since that time, great deal of biological data and several experimental studies have confirmed the important relationship between cancer and phenomena associated with blood coagulation (2 5). Approximately 50% of patients with malignant disease and over 90% of those with metastatic lesions show evidence of some abnormality in clotting and/ or fibrinolysis (4). For reprints and all correspondence: Hyo-Jin Kim, Department of Internal Medicine, Dong-A University College of Medicine, 3-1 Dongdaeshin-dong, Seo-gu, Busan , South Korea. kimhj@dau.ac.kr Cancer cells can activate the clotting system directly, thereby generating thrombin, or indirectly, by stimulating mononuclear cells to synthesize and express a variety of procoagulants (5). In fact, tissue factors and cancer procoagulants are expressed in tumor cells, resulting in the activation of clotting factors VII and X (6,7). Cytokines released from tumor cells activate coagulant activity on monocytes, thrombocytes and endothelial cells. Fibrin formation occurs in many types of tumor tissues, and the formation of a fibrin matrix appears to foster tumor growth via the promotion of neoangiogenesis, and by shielding tumor cells against attack from immunocompetent cells (8). Thrombin also functions as a potent promoter of cancer growth and spread via an increase in tumor cell adhesion and by affecting angiogenesis (6,9,10). Furthermore, the tissue factor is considered to # The Authors (2008). Published by Oxford University Press. All rights reserved.

2 Jpn J Clin Oncol 2008;38(1) 3 be the primary cancer-related procoagulant, and has been associated with tumor angiogenesis (7). Many researchers have documented the association between hemostatic abnormalities and gastric cancer (11,12), and various hemostasis markers associated with tumor staging and prognosis have previously been studied (13 18). Plasma fibrinogen levels are correlated with tumor extent, lymphatic metastasis and clinical outcomes (15 17). Elevated tissue factor expression has been associated with advanced stages and poor prognosis, particularly in the case of intestinal-type gastric cancer (18). However, much remains unknown regarding the association between gastric cancer and other markers of hemostasis. The extent of such activation has been correlated with tumor stage and prognosis in some malignancies. A single determination of coagulation markers, particularly those of the thrombin antithrombin complex (TAT), fibrin monomer and D-dimer, is sufficient to accurately predict survival in cancer patients (19). The primary objective of this study was to determine that hemostasis markers are associated with clinical and pathological findings in cases of gastric cancer. PATIENTS AND METHODS PATIENTS From April 2005, a total of 110 ambulatory patients with adenocarcinoma of the stomach, all of whom were candidates for surgical laparotomy, were studied in order to evaluate the presence and extent of hemostasis abnormalities in cases of gastric cancer. Patients with cardiovascular diseases, diabetes, acute or chronic inflammatory diseases, previous malignancy, or previous thromboembolic events were excluded from the present study. In all patients, several hemostasis markers associated with platelet activation, extrinsic, intrinsic and common pathways, as well as fibrinogen activation, were evaluated prior to surgical intervention. This study was approved by the review board of our hospital, and written informed consent was obtained from all patients. METHODS For coagulation and fibrinolysis, venous blood samples were collected in Vacuette tubes (Greiner Holding AG, Kremsmunster, Austria). Platelet-poor plasma was obtained via min of centrifugation at 3500 g. Prothrombin time (PT) and activated partial thromboplastin time (aptt) tests were immediately conducted. Aliquots of plasma were transferred to plastic tubes without delay, and then stored frozen at 2808C until analysis for fibrin/fibrinogen degradation products (FDP), D-dimer, TAT and prothrombin fragment F1þ2 (F1þ2). Complete blood cell counts were also determined with an automatic cell counter, XE-2100 (Sysmex Corp., Kobe, Japan) with EDTA blood. PT and aptt were determined with a CA-1500 coagulometer (Sysmex) using Thromborel S (Dade Behring GmbH, Marburg, Germany) and Dade Actin FS commercial kits (Dade Behring GmbH), respectively. FDP and D-dimer assays were conducted using Iatron commercial kits (Mitsubishi Kagaku Iatron, Inc., Tokyo, Japan) by CA-1500 (Sysmex). TAT and F1þ2 levels were measured via ELISA with Dade Behring commercial kits (Dade Behring GmbH). Each patient underwent gastrectomy, and D2 or more extended lymph node dissection was usually conducted. Tissue specimens were examined for the following characteristics: depth of tumor invasion, presence of lymph mode involvement, macroscopic and histological type, tumor size and lymphovascular invasion. The staging of gastric cancer and the clinicopathological factors utilized in this study were based on the sixth edition of the American Joint Committee on Cancer AJCC Cancer Staging Manual (20). All statistical analyses were conducted using the SPSS 12.0 statistical package for Windows (SPSS, Chicago, Illinois, USA). The continuous data are expressed as the means + standard deviation (SD) and the categorical data are expressed as percentages (%). The significance of differences was assessed via Mann Whitney s U-test for unpaired data. Spearman correlation coefficients were utilized to determine the association between pairs of variables. Multivariate stepwise logistic regression analyses were conducted in order to identify independent variables correlated with lymph node metastasis. A P-value of,0.05 was considered to be statistically significant. RESULTS PATIENT CHARACTERISTICS The characteristics of the study population are provided in Table 1. Sixty-two patients (56.4%) were male and 48 (43.6%) were female. The mean age of the patients was 59 years. All patients underwent curative gastric resection with a D2 or more lymph node dissection. Eighty-one patients (73.6%) were in Stage I, and approximately 65% patients evidenced no lymph node metastasis. HEMOSTASIS MARKERS AND HISTOPATHOLOGIC VARIABLES The mean values of hemostasis markers in a total of 110 gastric cancer patients are shown in Table 2. In this study, we assessed the association of hemostasis markers with the histopathological findings. Table 3 shows the correlation between hemostasis markers and histopathologic findings. Of all the examined hemostasis markers, the plasma D-dimer levels were correlated with tumor size (r s ¼ 0.224, P ¼ 0.009), tumor stage (T stage) (r s ¼ 0.258, P ¼ 0.007), lymph node stage (N stage) (r s ¼ 0.263, P ¼ 0.006) and TNM stage (r s ¼ 0.304, P ¼ 0.001) as shown in Fig. 1. The PT levels were correlated with N stage (r s ¼ 0.272, P ¼ 0.004) and TNM stage (r s ¼ 0.228, P ¼ 0.017). Plasma F1þ2 levels were correlated with tumor size (r s ¼ 0.231, P ¼ 0.015) and TNM stage (r s ¼ 0.206, P ¼ 0.031). Plasma TAT levels were

3 4 Hemostatic alterations in gastric cancer Table 1. Patient characteristics Table 2. Mean value of hemostasis markers (n ¼ 110) correlated with lymphovascular invasion (r s ¼ 0.203, P ¼ 0.034). However, these markers were not associated with age, sex or Lauren classification. Platelet count (P) and aptt and FDP levels were not related to any histological parameters. MULTIVARIATE LOGISTIC REGRESSION MODEL FOR PREDICTION OF LYMPH NODE METASTASIS No. of patients % Total number of patients 110 Sex Male Female Age Mean Range Lymphovascular invasion Negative Positive Lauren classification Intestinal Diffuse Mixed Undetermined Tumor size T stage T T T N stage N N N N TNM stage I II III IV TNM, tumor-node-metastasis. T stage, tumor stage; N stage, lymph node stage. PT and plasma F1þ2 levels were found to be significant in the prediction of the presence of lymph node metastasis in both univariate and multivariate logistic regression models Table 3. Correlation between hemostasis markers and stage (odds ratio ( ); P ¼ and odds ratio ( ); P ¼ 0.023, respectively) (Table 4). Elevated D-dimer levels predicted lymph node metastasis only in univariate regression (P ¼ 0.027). Other markers were not used in the prediction of lymph node metastasis. DISCUSSION T stage N stage TNM stage Correlation a P Correlation a P Correlation a P Platelet PT (INR) aptt TAT F1þ FDP D-dimer a Spearman correlation. Mean value Normal range Platelet count ( 10 3 /ul ) PT (INR) aptt (s) TAT (mg/l) F1þ2 (nm/l) FDP (mg/ml) ,5 D-dimer (mg/ml) ,0.83 PT, prothrombin time; aptt, activated partial thromboplastine time; TAT, thrombin-antithrombin complex; F1þ2, Prothrombin fragment F1þ2; FDP, Fibrinogen degradation product. Coagulation dysfunctions encountered in cancer patients may range from subtle abnormalities observed in laboratory tests to clinically overt thrombosis and disseminated intravascular coagulation (2,3). Many researchers have documented the association of hemostasis abnormalities with gastric cancer (11,12). Recently, it appears that certain components of the hemostasis system may modulate aggressive behaviors of cancer (14 18). Of all hemostasis markers examined, the smallest and unique degradation product of cross-linked fibrin is the D-dimer fragment, which results from the proteolytic actions of plasmin on fibrin. Plasma D-dimer levels were correlated

4 Jpn J Clin Oncol 2008;38(1) 5 Figure 1. (A) Plasma D-dimer levels according to the tumor size. (B) Plasma D-dimer levels according to the depth of tumor. (C) Plasma D-dimer levels according to lymph node metastasis. (D) Plasma D-dimer levels according to tumor-node-metastasis (TNM) stage. T stage, tumor stage; LN stage, lymph node stage. 8, mild outlier (between 1.5 interquartile range (IQR) and 3.0 IQR); *, extreme outlier (more than 3.0 IQR). with lymphovascular invasion, clinical stage and lymph node involvement in cases of operable breast cancer and lung cancer (21 23). Di Micco et al. reported that plasma D-dimer levels were elevated in gastric cancer patients (11), but the possible association between plasma D-dimer levels and clinicopathologic findings has yet to be determined. The results of our study indicated that preoperative plasma D-dimer levels were higher in patients with larger tumors, advanced T, N and TNM stage in patients with gastric cancer. Prothrombin is cleaved into two peptides, the active thrombin and the prothrombin fragment F1þ2. Thrombin

5 6 Hemostatic alterations in gastric cancer Table 4. Regression model predicting positive lymph nodes Univariate P* Multivariate P* Odds ratio 95% Confidence intervals Platelet NS PT (INR) aptt NS TAT NS F1þ FDP NS D-dimer NS *By logistic regression model. may subsequently be inactivated by antithrombin III, thereby forming the TAT. Therefore, both the prothrombin fragment F1þ2 and TAT can be considered markers of in vivo coagulation activation (24). The plasma levels of F1þ2 and TAT are significantly elevated in patients with specific types of malignancies, including gastric cancer (11,25,26). In our study, the plasma levels of F1þ2 were significantly correlated with tumor size and stage, and were statistically insignificantly correlated with the depth of invasion and lymph node involvement. Plasma TAT levels were correlated with lymphovascular invasion. The levels of PT did not differ significantly in cases of gastric cancer as compared with patients without gastric malignancies (11). However, some studies have shown that abnormally prolonged PT was predictive of a grave prognosis (22). We discovered that PT levels are correlated with lymph node involvement and TNM stage. Lymph node metastasis in gastric cancer is important for the confirmation of clinical stage and treatment. Although our results showed that prolonged PT and plasma levels of D-dimer correlated with degrees of lymph node involvement, we thought even only one lymph node metastasis would be of importance. Thus, we have attempted to determine which hemostasis markers best predict lymph node metastasis in cases of gastric cancer, via multivariate logistic regression tests. As a result, prolonged PT and plasma F1þ2 levels were found to be statistically significant markers. However, the plasma D-dimer levels were not significant markers. We propose that an elevated level of D-dimer might be associated with an increased concentration of F1þ2 in gastric cancer patients. This is because an increase in F1þ2 concentration is highly indicative of thrombin generation, which in turn converts fibrinogen to fibrin, so this factor may be excluded. This study started from April 2005, and 75% of patients were in early stage. Owing to the small number of patients, lots of whom were in early stages, and shortness of follow-up duration, it is not possible to get accurate survival and progression free survival data. In our study, the hemostasis markers almost appeared close to normal values. Although they are generally considered to be meaningless markers, they were statistically significantly correlated with disease status in cases of gastric cancer. First of all, we should verify that these markers were collected with appropriate quality. If the laboratory data were collected by fair means, even tiny differences may prove important in the prediction of disease status and clinical outcomes. Therefore, the measurement of plasma hemostasis markers using commercially available kits need to be neither complicated nor overly time-intensive. According to the results of this study, the preoperative laboratory examination of hemostasis in gastric cancer patients, especially D-dimer, F1þ2 and PT might provide supplementary information regarding disease status, including lymph node involvement and tumor stage. Conflict of interest statement None declared. References 1. Trousseau A. Clinique Medicale de L Hotel Dieu de Paris. 2nd edn. Paris, Balliere 1865, Bick RL. Coagulation abnormalities in malignancy: a review. Semin Thromb Hemost 1992;18: Luzzatto G, Schafer Al. The prethrombotic state in cancer. Semin Oncol 1990;17: Editorial. Haemostatic abnormalities and malignant disease. Lancet 1986;8: Hillen HF. 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