Expression Of TIM-4in NON-small Cell Lung Cancer And Its Significance

Background and purpose:Lung cancer is one of malignant tumors harming human health, which is the leading cause of cancer morbidity and mortality in China. Recently, great progress has been made in diagnosis and therapy of lung cancer. However, the lung cancer mortality rates have risen465%during the past30years in China according to a latest report by ministry of health in2008. The development and progression of lung cancer is an extremely complex process related to many genes. Conventional diagnosis methods, such as chest imaging, bronchofiborscope and sputum cytology, don’t make much sense in the early diagnosis of lung cancers, which is really disadvantageous to the overall survival of patients with lung cancer. The prognosis of patients with lung cancer is related to the tumor size, lymph node metastasis, clinical stages, and tumor differentiation levels. But the non-small cell lung cancer (NSCLC) patients with the same tumor types or the clinical stages don’t have the same5years survival rates. This indicates that there should be some other factors affecting the prognosis of the cancer patients. So it is urgent to find out the new target involved in lung cancer pathogenesis and prognosis, which would benefit to prolong patients survival, decrease metastasis and recurrence.Macrophage is a kind of innate immune cells, and it’s an important part of the inherent immune system. In the process of immune response, macrophages can phagocytose tumor cells directly. On the one hand, macrophages inhibit the proliferation of tumor cells by secreting cytokines, on the other hand, tumor related macrophages are closely related to formation of the blood vessels in tumor tissues. The more macrophages infiltration within tumors, the poorer the prognosis of patients with tumor. Since macrophages play critical roles in the development and progress of tumors, we infer that molecules regulating macrophages would be involved in the process of tumor pathogenesis.Tim (T cell immunoglobulin domain and mucin domain containing molecule) is a new gene family related to many autoimmune diseases, and can regulate the immune response mediated by Thl and Th2cells. Human Tim gene family contains3members:Tim-1, Tim-3, Tim-4. Tim-4is mainly expressed on the surface of antigen presenting cells, such as activated dendritic cells, macrophages. Low expression of Tim-4is also found in the lung, liver and the thymus of healthy people. Tim-4is the natural ligand of Tim-1. Interaction of Tim-4and Tim-1could stimulate T cell proliferation and cytokine secretion. Previous studies in our group showed that Tim-4blockade could promote macrophage activity and transfer macrophages overexpressing Tim-4could attenuate liver injury induced by ConA. In addition, Tim-4is identified as the receptor of phosphatidylserine and mediates phagocytosis of apoptotic cells by macrophages. It is reported Tim-4gene could be detected in stomach cancer and colon cancer. However its role remains unclear. The present study aims to detect the expression of Tim-4in NSCLC tumor tissues and adjacent tissues using immunohistochemical method. The relationship of Tim-4expression level in the lung cancer with the clinical pathological features of lung cancer and their prognosis was analyzed. This study would provide new candidate molecules for early diagnosis, treatment and prognosis evaluation of lung cancer.Methods:A retrospective study was designed with surgical resection specimen from70NSCLC patients who underwent surgery at Shandong Provincial Hospital, Shandong University. All patients did not receive any chemotherapy or radiative therapy before operation. Tissues adjacent to cancer, which are more than5cm far from the tumor, were set as control.57male patients and13female cases were involved in the study.34patients with lung cancer were more than60years old and36patients were less than60years old. There were31patients with adenocarcinoma and39patients with squamous cell carcinoma. Tim-4expression in lung cancer tissues and cancer adjacent tissues was detected by immunohistochemical method according to standard protocols. Positive expression of Tim-4is located in cytoplasma of lung cancer cells. Positve staining cells is shown as faint yellow or claybank. Tim-4staining was reported according to two grade scoring system. Briefly, each sample was scored according to the number of stained cells (0-5%=0;5-25%=1;25-50%=2;50-75%=3;75-100%=4) and staining intensity (weak staining=1; moderate staining=2; strong staining=3). Final immunoreactive scores were determined by adding the staining intensity and the number of stained cells. The data are analysed by SPSS12.0statistical software. Chi-square test was used to compare group differences, and P values <0.05were considered to be significant.A call follow up was made to record survival time of patients after operation. The survival date is from the operation date to the death date or to the end date of our follow-up. Univariate survival analysis using Gehan statistic was performed to compare survival experience, and a multivariate analysis using COX proportional hazards regression model was also performed to evaluate the prognostic significance of Tim-4expression when clinical prognostic factors were adjusted.Results:1. The Tim-4positive staining was detected in the cell plasma of lung cancer cells. Out of70lung cancer tissues,7lung cancer tissues did not expressTim-4, and cancer tissues of63NSCLC patients expressed Tim-4. The rate of Tim-4positive staining in lung cancer tissues was90%. In contrast, the rate of Tim-4postive staining in non-tumor tissues was63%. The Tim-4expression in lung cancer tissues was significantly higher than that of cancer adjacent tissues (x2=3.91, P<0.01). No significant differences of Tim-4expression were found in lung cancer patients with ages, gender, tissue types, tumor sizes, clinical stages and lymph node metastasis. However, Tim-4expression level was significantly correlated to tumor differentiation (χ2=11.278, P<0.01).2. Univariate survival analysis showed that there was no association of ages, gender,:issue types with overall survival rates of lung cancer patients. Tumor sizes, clinical stages, the differentiation degree, lymph node metastasis and Tim-4expression intensity affected the prognosis of tumor patients. The survival rate of the patients whose tumor size was more than3cm was significantly lower than those less than3cm (χ2=18.101, P=0.000). The survival rate of the patients with low tumor differentiation was significantly lower than those with mediate or higher tumor differentiation (x2=65.053, P=0.000). Survival rates of lung cancer patients decreased as the clinical stages were upgraded (χ2=18.511, P=0.000). The survival rate of the patients with lymph node metastasis was lower than those without lymph node metastasis (χ2=4.941, P=0.000). The overall survival rate decreased as the Tim-4expression intensity increased (χ2=18.511,P=0.000). Tumor size (χ2=10.41, P=0.000), tumor differentiation degree (x2=31.483, P=0.000), the Tim-4expression intensity (x2=8.318, P=0.04) belonged to the disadvantageous factors for prognosis of NSCLC patients at Ⅰ stage and Ⅱ stage. While ages, gender, tissue types and lymph node metastasis were not related to the prognosis of NSCLC patients at Ⅰ stage and Ⅱ stage.3. COX proportional hazards regression analysis showed the mortality risk of lung cancer patients with low tumor differentiation degree was17.275times higher that of patients with mediate and high tumor differentiation degree. The mortality risk of patients with tumor size larger than3cm was higher than those smaller than3cm, the death risk was10.417(1/0.096) times. Tumor differentiation degree (P=0.000), and tumor size (P=0.000) were independent prognostic factors of NSCLC patients, while tumor size (P=0.003) was an independent risk factor for prognosis of NSCLC patients at Ⅰ stage and Ⅱ stage.Conclusions:1. Tim-4expression intensity in lung cancer tissues is significantly higher than that of cancer adjavant tissues. Tim-4expression level is related to tumor differentiation degree. Lung cancer tissues with low differentiation degree express more Tim-4than those with mediate and high tumor differentiation degree.2. Tumor sizes, clinical stages, the differentiation degree and the Tim-4expression intensity influence the prognosis of NSCLC patients to some extent. Tim-4expression intensity in lung cancer tissues is a risk factor affecting the survival rate of NSCLC patients at Ⅰ stage and Ⅱ stage, indicating that detection of Tim-4in lung tissues might be helpful to evaluate the prognosis of NSCLC patients at Ⅰ and Ⅱ stage.3. Tumor differentiation degree and tumor size are two independent prognostic factors for NSCLC patients. Tumor size is an independent prognostic factor for NSCLC patients at Ⅰ and Ⅱ stage. The combination of the tumor differentiation degree and Tim-4expression intensity may help assess prognosis of NSCLC patients after operation.