Significance of Thrombocytosis in Clinicopathologic Characteristics and Prognosis of Gastric Cancer

Clinical abnormalities of coagulation may be detected as the first symptom of malignancy. One-third of cancer patients have elevation of blood platelet count at the time of diagnosis and before treatment (Naschitz et al. 1996), and the thrombocytosis is reported to be present in 10% to 57% of various neoplasm patients (Sierko and Wojtukiewicz 2004). Previous studies have showed that thrombocytosis is associated with later stage, lymphatic and blood-borne metastasis and higher risk of recurrence in many cancers (Gao et al., 2013; Sasaki et al., 2012; Unal et al., 2013). It was also suggested that patients with thrombocytosis usually inclined to poorer prognoses in ovarian cancer (Lee et al., 2011), renal cell carcinoma (Cho et al., 2011), colorectal cancer and gastric cancer (Hwang et al., 2012). Although, several mechanisms of platelet action in facilitating cancer progression and metastasis have been supposed, exact pathophysiological mechanisms of it have not been completely elaborated. Based on the pivotal role of platelet and thrombocytosis in developing, metastasis and prognosis of cancers, several studies have suggested thrombocytosis as a therapeutic targets in cancer treatments, especially against to the metastatic cancer cells. Several platelet aggregation inhibitors have been reported to retard tumor metastasis in certain animal models and some clinical trials (Choe


Introduction
Clinical abnormalities of coagulation may be detected as the first symptom of malignancy.One-third of cancer patients have elevation of blood platelet count at the time of diagnosis and before treatment (Naschitz et al. 1996), and the thrombocytosis is reported to be present in 10% to 57% of various neoplasm patients (Sierko and Wojtukiewicz 2004).Previous studies have showed that thrombocytosis is associated with later stage, lymphatic and blood-borne metastasis and higher risk of recurrence in many cancers (Gao et al., 2013;Sasaki et al., 2012;Unal et al., 2013).It was also suggested that patients with thrombocytosis usually inclined to poorer prognoses in ovarian cancer (Lee et al., 2011), renal cell carcinoma (Cho et al., 2011), colorectal cancer and gastric cancer (Hwang et al., 2012).Although, several mechanisms of platelet action in facilitating cancer progression and metastasis have been supposed, exact pathophysiological mechanisms of it have not been completely elaborated.Based on the pivotal role of platelet and thrombocytosis in developing, metastasis and prognosis of cancers, several studies have suggested thrombocytosis as a therapeutic targets in cancer treatments, especially against to the metastatic cancer cells.Several platelet aggregation inhibitors have been reported to retard tumor metastasis in certain animal models and some clinical trials (Choe
Gastric cancer is the fourth most common cancer in the world and accounts for nearly 8.6% of all new cancers.It remains be a leading cause of cancer-related death which causes 12% of all cancer-related deaths each year (Jemal et al., 2011;Li et al., 2012).Also in gastric cancer, the higher platelets counts could be detected at initial diagnosis, the incidence of thrombocytosis was ranged from 6.4%-20.4% in gastric cancer (Ikeda et al., 2002).The prognostic significance of thrombocytosis was previously reported by some literatures.However, these studies should be complemented because of their more or less shortcomings.Aliustaoglu et al. (2010) and Ikeda et al. (2002) analyzed a relatively small number of patients while Heras et al. (2010) analyzed only a short-term mortality rate without delineating the prevalence of thrombocytosis in gastric cancer patients.Based on a larger group, Hwang et al. (2012) focused on the association between thrombocytosis and hematogenous recurrence.However, undetailed TNM staging and group division in statistic analysis reduced the study's reference meanings in the thrombocytosis research.In this study, we aimed to study the relationship with thrombocytosis and the clinicopathology characters of gastric cancer based on retrospective analysis of 1596 gastric cancer patients.Furthermore, we can investigate the association between platelet counts and gastric cancer progression upon different TNM stages.

Materials and Methods
The eligibility criteria for this study included: (1) Histologically confirmed gastric carcinoma at the Gastric Cancer Surgery Department, Tianjin Medical University Cancer Institution andHospital, between January 1997 andDecember 2000.(2) Patients who had complete clinical and follow-up data.(3) Patients received the surgery and chemotherapy according to the rules of the Japanese Research Society for Gastric Cancer(JRSGC).
The exclusion criteria included: (1) Patients with other-organ malignancy, severe liver cirrhosis, recent infectious disorder or inflammatory disease.(2) Gastric cancer patients at I-III stage who did not received a curative resection (R0 resection) with lymphadenectomy (dissected lymph nodes were more than 15).

Patients
Based on eligibility and exclusion criteria given above, there were 1596 patients enrolled in our study.In these patients, 1127 patients were males and 469 patients were females.Their ages ranged from 25 to 89 years with a median age of 60 years.Medical records were reviewed to extract the patients' data, including age at the time of diagnosis (<60 or ≥60), sex (male or female), tumor location(upper & diffuse or middle &lower), size of tumor (≤5 cm or >5 cm), histological type (differentiated type or undifferentiated type), depth of tumor invasion (T1, T2, T3, T4a or T4b), lymph node metastasis (N0, N1, N2 or N3).All histopathological data were analyzed and determined according to the Union for international cancer control (UICC) TNM classification (7th edition) (Greene and Sobin 2009).In all patients, 1510 patients received radical resection, meanwhile 1462 patients received chemotherapy.
The current study was conducted in compliance with the Declaration of Helsinki for the use of patients' data and protection of their confidentiality.

Platelet count and thrombocytosis
The pretreatment platelet count was recorded according to the last preoperative complete blood count performed either at admission for surgery or at outpatient clinic within 2 weeks before surgery.Platelet count >400×1000/μL was defined as thrombocytosis (Ikeda et al., 2002).

Follow-up plan
Follow-up were conducted through outpatient department visiting.All of the patients were followed every 3 months for the first year, every 6 months for second year, then every year until death.

Statistic analysis
Independent sample t test was used in comparison of continuous variables.The chi-squared and Fisher exact tests were applied in categorical variable for univariate analysis of potential factors for thrombocytosis.Logistic regression was used for the multivariate analysis of relative factors of thrombocytosis.Overall survival was estimated by the Kaplan-Meier method and the Log-rank test was used to determine univariate significance.The Cox regression was applied in multivariate prognosis analysis.Significance was defined as p<0.05(two sides).The statistical analysis was performed using the statistical analysis program package SPSS 17.0.
The higher platelet count were observed in patients with older age (p=0.001),larger tumor size (p<0.001),deeper tumor invasion (p<0.001),lymph node metastasis (p=0.017),distant metastasis (p=0.010), and advanced TNM stage (p<0.001),shown in Table 1.The association between the maximum tumor diameter, depth of tumor invasion, lymph node metastasis, TNM stage and the platelet count were showed in Figures 1-4.

The association between thrombocytosis and clinicopathological factors
Table 2 showed the results of univariate analysis of the association between thrombocytosis and clinicopathological variables.The significant correlation between the thrombocytosis and clinicopathological features was demonstrated in tumor size, tumor location, depth of tumor invasion, lymph node metastasis, distant metastasis and TNM stage (All P values were <0.05).The thrombocytosis was observed more frequently in patients with larger tumor size (p<0.001),location at middle or lower third stomach (p=0.013),deeper tumor invasion (p=0.012),later N stage (p=0.046),distant metastasis (p=0.048) and advanced TNM stage (p<0.001).
These significant features were enrolled in multivariate logistic regression, we detected that tumor size (p<0.001),depth of tumor invasion (p=0.045),lymph node metastasis (p=0.031) and TNM stage (p=0.023)were independent risk factors for thrombocytosis of gastric cancer patients.

Discussion
Elevation of blood platelet count is commonly observed in malignancy diseases.In our study, the mean level of platelet counts was 253.23±88.47×1000/μL,and 7.5% of gastric cancer patients had thrombocytosis, which was coincident with previous literatures (Aliustaoglu et al., 2010;Heras et al., 2010).Although the mechanism responsible for the elevated levels of serum IL-6 and TPO in cancer patients remains vague, it had been confirmed that the significantly higher frequency of elevated IL-6 levels (Kabir and Daar, 1995) and up-regulation of TPO (McConnell et al., 2007) were observed in patients with malignancies in many reports.Meanwhile, cancer cells can release vascular endothelial growth factor (VEGF), which activates the coagulation cascade, resulting in platelets to be activated (Kuenen et al., 2002;Roselli et al., 2003).Activated platelets also secrete TPO further, which stimulates bone marrow to generate new platelets.Thus, platelets and cancer cells make up a positive feedback cascade in which each stimulates the other, potentiating the effect (Folman et al., 2000).
This study demonstrated the palate count was correlated with clinical features, which was in line with previous study (Aliustaoglu et al., 2010;Aminian et al., 2011).Thrombocytosis was also found more frequently in patients with advanced stage and a higher histological grade, which also can be investigated in colorectal cancer (Sasaki et al., 2012) and nasopharyngeal carcinoma (Gao et al., 2013) etc.
Platelets synthesize and transport several angiogenic factors such as VEGF, platelet derived growth factor (PDGF), basic fibroblastic growth factor (bFGF), epidermal growth factor (EGF), and matrix metalloproteinases (MMP), which are known as stimulators of tumor angiogenesis (Buergy et al., 2012;Wang et al., 2011).Mohle et al showed that platelets adhere to the tumorrelated endothelium and release high concentrations of VEGF.Thus, the formation of the microvascular arrest of tumor cells at distant sites was facilitated (Mohle et al., 1997).Various experimental models have established the crucial steps on different molecular levels that underlie the pro-metastatic function of platelet-tumor cell aggregates within the microenvironment of tumor.
In addition, Labelle et al., recently demonstrated in their experimental lung metastasis model that plateletderived TGF-β/Smad and NF-Κb pathway are critical steps in which circulating tumor cells undergo epithelialmesenchymal transition (EMT) to acquire invasive and metastatic phenotypes, and ultimately colonize distant organs (Labelle et al., 2011).
In a number of cancers, lymphatic metastasis was found to be a common pattern of tumor cells after drop out from the primary tumor.In our study, the higher platelet counts and thrombocytosis was associated with lymph node metastasis.Previous researches suggested that platelets were important in the development of lymphatic vessels (Bertozzi et al., 2010;Liu et al., 2013;Suh et al., 2012).Bertozzi et al found that platelets and podoplanin function in the developmental separation of blood and lymphatic circulation in the embryo.Also, lymph-angiogenic factors such as VEGF, PDGF, and βFGF can be synthesized and transported by platelets into the blood circulation and tissues, these factors were able to improve the development of lymphatic vessels (Bertozzi et al., 2010).
What's more, activated platelet-tumor cell aggregates in the circulation facilitate immune evasion of tumor cells.Platelets facilitate cohesion of tumor cells and leukocytes to form hetero-aggregates, which adhere to vascular endothelial cells and act as physiologically protecting tumor cells against NK cell-mediated lysis and shear stress (Cho et al., 2012;Gupta and Massague, 2004).
Owing to above mechanisms, aberrant activation of platelets and coagulation pathway is associated with poorer prognosis in many tumors (Aminian et al., 2011;Sasaki et al., 2012).In Kawai et al's study, thrombocytosis Fang-Xuan Li et al before pre-operative chemoradiotherapy predicts poor response and shorter local recurrence-free survival in rectal cancer (Kawai et al., 2012).Consistently, in our study, we clearly demonstrated that thrombocytosis is an independent factor of survival.
Due to the presence of thrombocytosis in some solid tumors has been associated with poor prognosis, there is increasing interest in the potential role of antithrombotic agents in the treatment of cancer patients.Several literature have showed that anticoagulants significantly improved survival in cancer patients.Nevertheless, clinical studies have been limited and have reported some controversial conclusions.A meta analysis of 11 studies demonstrated a significant reduction in overall mortality with anticoagulant therapy in cancer patients (Kuderer et al., 2007).Phillips et al. have demonstrated that low molecular weight heparin application significantly increases tumor chemo-responsiveness (Phillips et al., 2011).However, Van Doormaal et al did a multicenter, randomized and open-label trial in patients with advanced malignancy, nadroparin had no effect on time to disease progression (van Doormaal et al., 2011).These studies suggested the complicated and manifold molecular mechanisms in cancer-platelet interactions (Rebuffat et al., 2010).Large scale multi-center prospective clinical trials were called for confirming the mechanisms and effect of anticoagulants treatment in cancer therapy before the antithrombotic treatment applied in clinical therapy of cancer.

Figure 3 .
Figure 3.The Association between Platelet Count and Lymph Node Metastasis

Figure 4 .
Figure 4.The Association between Platelet Count and TNM Stage

Figure
Figure 1.The Association between Platelet Count and Maximum Tumor Diameter