| The lung cancer has become the global serious health problem in 21st century,for the incidence and mortality rate have been increasing fast during the past decades. In China, during 2025, the numbers of lung cancer patients will reach to 100 million,ranking first in the world.So the study about formation, development, treatment and prognosis of lung cancer has become an important issue in the medical sphere.Tiaml, T lymphoma invasion and metastasis inducing factor 1, is one of guanine nucleotide exchange factor (GEF), associated with invasion and metastasis of a variety of tumors, such as breast cancer, colon cancer; But some scholars believe that Tiaml can inhibit invasion and metastasis of tumor.Research has shown that overexpression of Tiam1 was tightly associated with the development, lymph node metastasis and prognosis of non-small cell lung cancer. Expression of Tiaml was an independent predictor for the prognosis of liver cancer, nasopharyngeal cancer and prostate cancer. But the quantitative expression of Tiaml and the quantitative changes in lung cancer, the role of joint detection of Tiaml, Ki67 and p53 protein for prognosis has not been reported in lung cancer.Ki67 is the most widely used marker of cell proliferation, overexpressed in many malignant tumors, plays an important role in assisting diagnosis, analyzing prognosis and guiding treatment. There are many different opinions in term of the connection of Ki67 and the clinicopathological features of lung cancer, the association of Ki67 and the prognosis of lung cancer also has been the research focus. p53 gene is an important tumor suppressor gene, its mutation connected with tumorous development, progression and metastasis.There was p53 mutation in the tumorigenesis process of lung, research has shown that the expression of p53 was correlated with the clinical stage, tumor size, lymph node metastasis of lung cancer,but some showed the opposite results. Based on the problem above, we designed this research.OBJECTIVEThis study was to evaluate the quantitative expression of Tiam1, Ki67 and p53 protein in lung adenocarcinoma cells and lung cancer, from protein levels and quantitative aspect revealed the connection of Tiam1, Ki67 and p53 and lung cancer, and the role of the three joint detection for prognosis of lung cancer.METERLAILS AND METHODS1.The quantitative analysis of Tiaml expression in lung adenocarcinoma cells and lung cancerImmunohistochemistry was performed to detect the expression of Tiam1 in three lung cancer cell lines (A549, GLC-82, H460), lung cancer paraffin tissue microarray (including 102 lung cancer tissues,20 normal adult lung tissues and 20 embryonic lung tissues),205 lung cancer tissues and 37 lymph node metastasis tissues. Through the Zoom Browser Ex photograph system,the positive image was randomly photoed under the Nikon microscope linked Canon digital camera.The expression intensity (in positive unit,PU) of Tiam1 in these tissues was assessed quantitatively using Image-pro Plus image analysis software.2.The quantitative analysis of Ki67 and p53 expression in lung adenocarcinoma cells and lung cancerImmunohistochemistry was performed to detect the expression of Ki67 and p53 in three lung cancer cell lines (A549, GLC-82, H460)and 84 lung cancer tissues Through the Zoom Browser Ex photograph system,the positive image was randomly photoed under the Nikon microscope linked Canon digital camera.The expression intensity (in positive unit,PU) of Tiam1 in these tissues was assessed quantitatively using Image-pro Plus image analysis software.3.Correlation analysis of expression of Tiaml,Ki67 and p53 proteinThe Immunohistochemistry detection and quantitative test of Tiam1, Ki67 and p53 protein of 205 lung cancer tissues(121 cases of Ki67 and p53 protein had completed before) were the same with the former(1,2 above).Semi-quantitative determination is determined according to degree of staining and the percentage of stained cells:> 4 scores is divided into high expression,<3 scores is divided into low expression.4.The survival analysis of Tiaml,Ki67 and p53 in lung cancer prognosisThe case data extracted and follow-up study were used to collected the clinical and follow-up data of the primary lung cancer patients who were under surgical treatment in the nanfang hospital during 2000-2006 year,then survival analysis was carried out combined with the expression of Tiaml, Ki67 and p53 protein.RESULTS1. The quantitative analysis of Tiaml expression in lung adenocarcinoma cells and lung cancer1.The Tiaml PU value of H460 cells was greater than A549, GLC-82 cells, the difference was statistically significant (P=0.032, P=0.000); the PU value of A549 cells was similar with GLC-82 cells (P=0.084).2.The Tiaml PU value of lung squamous cell carcinoma, adenocarcinoma, large cell carcinoma cells were greater than normal adult alveolar typeⅡepithelial cells and embryonic epithelial cells, the difference was statistically significant (P＜0.05); The Tiaml PU value of small cell lung cancer cells was also greater than them,but the difference did not show statistical significance (P=0.254,.P=0.283). Amongst them, the PU value is basically the same (P=0.916). PU of Tiaml was greater in primary lung carcinomas with lymph node metastases than in those without metastases (P=0.000),but the PU value is basically the same between the primary tumor and lymph node metastasis cells(P=0.836). Lung cancers of TNM stageⅢ-Ⅳhad stronger expression than those of stageⅠ-Ⅱ(P=0.008). The intensity of Tiaml expression was not associated with the patients'gender,age, smoking history,the general types and differentiation of lung carcinoma(P>0.05).3.PU of Tiaml in squamouscell cellcarcinoma were less than adenocarcinoma,1a rge cell carcinoma and small cell carcinoma(P=0.000) were diversity.But the three latter have the same PU value (P>0.05). PU of Tiaml in undifferentiated lung cancer were greater than that in the moderately to poorly differentiated cancer (P= 0.011), the well-differentiated carcinoma had the same PU value with them (P=0.813, P=0.133). PU of Tiaml in women patients were greater than that in men (P=0.000) PU of Tiaml was greater in primary lung carcinomas with lymph node metastases than in those without metastases (P=0.004).PU value in lymph node metastasis were greater than that in the primary tumor (P=0.001). Lung cancers of TNM stageⅢ-Ⅳhad stronger expression than those of stageⅠ-Ⅱ(P=0.000).The intensity of Tiaml expression was not associated with the patients' age, smoking history,the general types and distant metastasis of lung carcinoma(P>0.05).2. The quantitative analysis of Ki67 and p53 expression in lung adenocarcinoma cells and lung cancer1.The PU value of Ki67 of H460 cells were greater than A549, GLC-82 cells (P=0.000,P=0.001); the PU value of A549 cells were similar with GLC-82 cells (P=0.901). The PU value of p53 of H460 cells were greater than A549, GLC-82 cells; the PU value of A549 cells were greater than GLC-82 cells (P=0.000, P=0.000,P=0.000)2.PU of Ki67 was greater in primary lung carcinomas with lymph node metastases than in those without metastases (P=0.035), but there were no diffrenrence in p53 PU value (P=0.556). The intensity of Ki67,p53 expression was not associated with the patients' gender,age,smoking history,the general types,differentiation, distant metastasis and TNM stage of lung carcinoma(P>0.05).3. Correlation analysis of expression of Tiaml,Ki67 and p53 protein1.From the view of quantitation and semi-quantitation showed that:There was significant positive correlation between Ki67 expression and p53 expression, but not closely related (Spearman correlation coefficient was 0.209, Pearson correlation coefficient was 0.332). There was positive correlation between p53 expression and tumorous maximum area,but not closely related (Pearson correlation coefficient 0.153). Tiaml and Ki67, Tiaml and p53, Tiaml and Ki67 with the tumorous maximum area had no correlation (P> 0.05).2.Consistency analysis of Ki67 and p53 showed (P=0.003), but consistency is poor(Kappa=0.200).The significant did not exist in Tiaml and Ki67, Tiaml and p53 (P=0.949,P=0.714),so it indicatied that the consistency is poor among the three markers.4. The survival analysis of Tiaml,Ki67 and p53 in lung cancer prognosis(1)The 1-8-year survival rates of lung cancer patients respectively were 80%, 59%,42%,34%,30%,22%,17%,13%.(2) Clinical and pathological features of lung cancer is affect on the prognosis of patients with single-factor analysis showed that:lymph node metastasis, TNM stage, radiotherapy and chemotherapy, Tiaml, Ki67, Ki67and Tiaml,Ki67 and p53,p53 and Tiaml, Ki67 and Tiaml and p53 combined detection of these factors affect the prognosis of survival time, the differences were statistically significant (P＜0.05). Other factors have no significant effect with the prognosis survival time of patients, the differences were not statistically significant (P>0.05).(3) Ki67 and Tiaml combined to detect lung cancer a group(Ki67 high/Tiaml high), b group(Ki67 high/Tiaml low), c group(Ki67 low/Tiaml high), d group (Ki67 low/Tiaml low)changes in survival time of patients:The median survival time of a,b, c,d group respectively were 18,35,34,54 months, the differences were statistically significant (P=0.006). The 1-5-year survival rates of a group respectively were 76%,46%,25%,18%,16%; that of b group respectively were 81%,64%,49%,38%,35%; that of c group respectively were 82%,65%,47%,39%,33%;that of d group respectively were 86%,69%,58%,55%,46%. Among the four groups, the difference of 3,4 and 5-year survival rate were statistically significant(P=0.004,P=0.003,P=0.007); but the difference of land 2-year survival rates did not show statistically significant (P>0.05) (4) Ki67 and p53 combined to detect lung cancer A group(Ki67 high/p53 high), B group(Ki67 high/p53 low), C group(Ki67 low/p53 high), D group (Ki67 low/p53 low)changes in survival time of patients:The median survival time of A,B, C,D group respectively were 30,16,31,43 months, the differences were statistically significant (P=0.004). The 1～5-year survival rates of A group respectively were 86,59%,43%,34%,32%; that of B group respectively were 69%,47%,26%,17%,15%; that of C group respectively were77%,62%,50%,38%,38%;that of D group respectively were 88%,71%,53%,50%,40%. Among the four groups, the difference of 1,3,4 and 5-year survival rate were statistically significant (P=0.041,P=0.018,P=0.003, P=0.026); but the difference of 1-year survival rates did not show statistically significant (P>0.05)(5) p53 and Tiaml combined to detect lung cancer①group(p53 high/Tiam1 high),②group(p53 high/Tiam1 low),③group(p53 low/Tiaml high),④group (p53 low/Tiaml low)changes in survival time of patients:The median survival time of①,②,③,④group respectively were 20,56,25,27 months, the differences were statistically significant(P=0.047). The 1～5-year survival rates of①group respectively were 77%,47%,32%,24%,24%; that of②group respectively were 91%,79%,64%,51%,47%; that of③group respectively were 80%,59%,37%,31%, 25%;that of④group respectively were 77%,59%,45%,40%,34%. Among the four groups, the difference of 2,3,and 4-year survival rate were statistically significant (P=0.038,P=0.025,P=0.050); but the difference of 1and 5-year survival rates did not show statistically significant (P>0.05)(6) Ki67,Tiam1 and p53 combined to detect lung cancer 1 group(Ki67 high/p53 high/Tiam1 high),2 group(Ki67 high/p53 low/Tiam1 high),3 group (Ki67 high/p53 high/Tiam1 low),4 group(Ki67 high/p53 low/Tiam1 low),5 group (Ki67 low/p53 high/Tiam1 high),6 group (Ki67 low/p53 low/Tiam1 high),7 group (Ki67 low/p53 high/Tiam1 low),8 group (Ki67 low/p53 low/Tiam1 low)changes in survival time of patients:The median survival time of 1,2,3,4,5,6,7,8 group respectively were 18,16,56,18,24,34,36,54 months, the differences were statistically significant (P=0.002). The 1～5-year survival rates of 1 group respectively were 78%,42%,28%,22%,22%; that of 2 group respectively were 73%,49%,24%,16%,13%; that of 3 group respectively were96%,81%,65%,53%,49%;that of 4 group respectively were 62%,43%,29%,19%,19%; that of 5 group respectively were 75%,56%,44%,29%,29%; that of 6 group respectively were 86%,69%,49%,43%,36%;that of 7 group respectively were 80%,70%,60%,50%,50%; that of 8 group respectively were 92%,75%,58%,58%,45%.Among the eight groups, the difference of 2,3,4 and 5-year survival rate were statistically significant (P-0.013,P=0.006,P=0.002,P=0.017); but the difference of 1-year survival rates did not show statistically significant (P>0.05)(7) Cox regression analysis about gender,age,smoking history,gross type, histological type,differentiation degree,lymph node metastasis,distant metastasis, TNM stage, tumorous maximum size, radiotherapy and chemotherapy, Ki67 PU value, Tiaml PU value, p53 PU value, independent testing of Ki67,Tiaml and p53, joint detection of Ki67 and Tiaml joint detection of Ki67 and p53, joint detection of p53 and Tiaml, joint detection of Tiaml, Ki67, p53.It is show that joint detection of Ki67 and Tiaml,tumorous maximum size,radiotherapy and chemotherapy,TNM stage was independent prognostic factors which affect survival (P=0.030,P=0.004,P=0.001, P=0.017), suggest that:patients with high expression of Ki67, Tiaml have greater death risk than patients with low expression; patients with radiotherapy and chemotherapy have higher death risk than patients without; the larger tumorous maximum size, the higher TNM stage, the greater the death risk.CONCLUSIONS1. There is significant difference of Tiaml protein expression in normal lung tissue, fetal lung tissue, lung cancer tissue, indicating that the expression of Tiaml was associated with the formation of lung carcinoma;Tiaml protein were highly expressed in lung cancer cells and lung cancer, and the expressive intensity was closely related to histological types, differentiation, lymph node metastasis and distant metastasis, TNM stage of lung cancer, indicating that the expression of Tiaml protein was highly associated with the degree of malignancy and metastasis of lung cancer.2. The expressive intensity of Ki67 protein was related to lymph node metastasis of lung cancer, indicating that the intensity of Ki67 protein was associated with metastasis of lung cancer.3. There were no correlation between Tiam1 and Ki67,Tiam1 and p53 protein in lung cancer, also no consistency, indicating that the expression of Tiam1, Ki67 and p53 protein were independent in lung cancer. There was significant positive correlation between Ki67 expression and p53 expression, but not closely related; so it was important significance to joint detect the expression of the three marker for analyzying comprehensively the biological characteristics of lung cancer.4. The expression of Ki67,the expression of Tiam1, lymph node and distant metastasis, radiotherapy and chemotherapy, TNM stage were independent significant prognostic markers for lung cancer patients. Joint detection of Ki67 and Tiam1 was the best combination in the Ki67, p53, Tiam1 random combinations, the 3-5 year survival rate from high to low were:Ki67 low/Tiam1 low group> Ki67 high/Tiam1 low group, Ki67 low/Tiam1 high group> Ki67 high/ Tiaml high group. Multivariate Cox regression analysis:Ki67 and Tiam1 joint detection, tumorous maximum size, TNM stage, radiotherapy and chemotherapy were the independent predictor for thel prognostis of lung cancer patients; therefore, combined detection of Ki67, Tiaml expression can predict effectively the prognosis of lung cancer patients. |
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