The Mechanism And Relationship Between Downregulative Classical HLA Class I Antigen Expression And Metastatic Lymph Node Lesions In Non-Small Cell Lung Cancer

Objective: To detect expression of classical HLA classⅠantigens in primary lesions and metastatic lymph node in non- small cell lung cancer (NSCLC) by Flow cytometry, elucidate correlation of classical HLA class I antigen expression between primary lesions and metastatic lesions , probe the relationship between loss of TAPK LMP2 mRNA and loss or downregulation of classical HLA classⅠantigen expression in tumor cells, and explore the relationship between onco-gene(K-ras,C-erbB-2) and loss or downregulation of classical HLA classⅠantigen expression in tumor cells.Methods: (1) Expression of classical HLA classⅠantigens in 65 primary lesions in NSCLC and 31 metastatic cancer lesions in lymph node was examined by Flow cytometry (FCM). (2) Expression of TAP1 and LMP 2 mRNA in 31 primary lesions in lung cancer with classical HLA-Ⅰantigens downregulation or loss and 31 metastatic lesions in lymph nodes with HLA-Ⅰantigens downregulation or loss were detected by RT-PCR. (3) The detection of C-erbB-2 and K-ras activation in 65 primary lesions and 31 metastatic lesions were performed by differential PCR and PCR-RFLP, respectively.Different variants were analysed by different statistic methods, for example, t test, Spearman correlation analysis, chi-square test, Fisher's exact test of probability or logistic regression analysis.Results: (1) Compared with expression of classical HLA class I antigens in normal lung tissue, expression of classical HLA class I antigens was detected in 58 out of 65 (89.2%) primary lesions. Expression rate of classical HLA classⅠantigens in 31 lymph node metastatic lesions(15.35±6.24%) was remarkably lower than that in primary lesions(39.68±12.46%) (t=2.06, P<0.05),and positive correlation between downregulation of classical HLA classⅠantigens in primary lesions in lung cancer and metastatic lesions in lymph nodes was discovered (r_s=0.487,P<0.01). (2) The expression of TAP1 and LMP2 mRNA was detected 13 cases(41.9%) and 12 cases(38.7%) in 31 primary lesions ,and 5 cases (16.1%) and 4cases (12.9%) in 31 metastatic lesions, respectively. The difference of the expression of TAP1 and LMP2 mRNA in primary lesions and metastatic lesions was statistically significant(P< 0.05).(3) K-ras gene mutations were detected in 15 cases among the 65 primary lesions (23.1%) and in 13 cases among 31 metastatic lesions in lymph nodes(41.9%).The difference of K-ras gene mutations in primary lesions and metastatic lesions was not statistically significant(x~2 = 3.56,P>0.05). The difference of incidences of K-ras gene mutations between primary lesions with downregulation and loss of classical HLA classⅠantigen was significant(x~2=5.38,P<0.05). C-erbB-2 gene amplifications were detected in 21cases among the 65 primary lesions (32.3%) and in 15 cases among 31 metastatic lesions in lymph nodes(48.4%).The difference of C-erbB-2 gene amplifications in primary lesions and metastatic lesions was not significant(x~2=2.32,P>0.05). The incidences of C-erbB-2 gene amplifications between primary lesions with downregulation of classical HLA classⅠantigen and normal erpression of classical HLA classⅠantigen was significant (P=0.02).Conclusions: (1) Expression of classical HLA classⅠantigens in NSCLC lesions can be accurately detected by Flow cytometry. Expression of classical HLA classⅠantigens in most primary lesions was down-regulation. Expression of classical HLA class I antigens in metastatic lesions was lower than that in primary lesions.There is positive correlation between expression of classical HLA classⅠantigens in primary lesions in lung cancer and metastatic lesions in lymph nodes.Loss or downregulation of classical HLA classⅠantigen expression in primary lesions is one of mechanisms of cancer metastasis to lymph node in NSCLC. (2) Negative expression of TAP1 and LMP2 mRNA is one of mechanisms of calssical HLA-Ⅰ antigens loss or downregulation in primary lesions and metastatic lesions in lung cancer. (3) There is negative correlation between K-ras gene mutations and C-erbB-2 gene amplifications and expression of HLA-Ⅰ antigen in primary lesions and metastatic lesions with non small cell lung cancer PART 1DETECTING CLASSICAL HLA-1 ANTIGENS EXPRESSION IN PATIENTS WIYH NON-SMALL CELL LUNG CANCER BY FLOW CYTOMETRYObjective:To detect expression of classical HLA classⅠantigens in primary lesions and metastatic lymph node in non- small cell lung cancer (NSCLC) by Flow cytometry, elucidate correlation of classical HLA classⅠantigen expression between primary lesions and metastatic lesions ,and explore relationship between downregulation of classical HLA classⅠantigen expression and cancer metastasis to lymph node.Methods: Expression of classical HLA classⅠantigens in 65 primary lesions in NSCLC and 31 metastatic cancer lesions in lymph node was examined by Flow cytometry (FCM). The relationship between expression of classical HLA classⅠantigens in primary lesions and metastatic lesions was analyzed by t test. The correlation of expression of classical HLA classⅠantigens in primary lesions and metastatic lesions was analysed by Spearman correlation analysis.The relationship between loss or downregulation of classical HLA classⅠantigen expression and cancer metastasis to lymph node was analysed by logistic regression analysis.Result: Compared with expression of classical HLA classⅠantigens in normal lung tissue, expression of classical HLA class I antigens was detected in 58 out of 65 (89.2%) primary lesions. Expression rate of classical HLA classⅠantigens in 31 lymph node metastatic lesions(15.35±6.24%) was remarkably lower than that in primary lesions (39.68±12.46%) (t=2.06 , P<0.05),and positive correlation between downregulation of classical HLA class I antigens in primary lesions in lung cancer and metastatic lesions in lymph nodes was discovered (r_s= 0.487, P<0.01). Loss of classical HLA classsⅠantigens was risk factor,which lead to cancer metastasis to lymph node(OR=2.570, P=0.036). Conclusion: Expression of classical HLA classⅠantigens in NSCLC lesions can be accurately detected by Flow cytometry. Expression of classical HLA classⅠantigens in most primary lesions was down-regulation. Loss or downregulation of classical HLA classⅠantigen expression in primary lesions is one of mechanisms of cancer metastasis to lymph node in NSCLC. Expression of classical HLA classⅠantigens in metastatic lesions was lower than that in primary lesions.There is positive correlation between expression of classical HLA classⅠantigens in primary lesions in lung cancer and metastatic lesions in lymph nodes. Moreover, Loss of classical HLA classsⅠantigens significantly increases the risk of lymph node metastasis in NSCLC. PART 2THE RELATIONSHIP BETWEEN LOSS OF TAP1 AND LMP2 mRNA AND EXPRESSION OF CLASSICAL HLA-ⅠANTIGENS IN NSCLCObjective: To explore the relationship between loss of TAP1 and LMP2 mRNA and expression of classical HLA-Ⅰantigens in primary lesions and metastatic lesions in non-small cell lung cancer and metastatic lymph node, and to explore the correlation between loss of TAP1 and LMP2 mRNA and cancer metastasis to lymph node.Methods: Expression of TAP1 and LMP2 mRNA in 31 primary lesions in lung cancer with classical HLA-Ⅰantigens downregulation or loss and 31 metastatic lesions in lymph nodes with classical HLA-Ⅰantigens downregulation or loss were detected by RT-PCR. The results were analysed by chi-square test or Fisher's exact test of probability.The risk factors of cancer metastasis to lymph node were analysed by logistic regression analysis.Results: The expression of TAP1 and LMP2 mRNA was detected 13 cases(41.9%) and 12 cases(38.7%) in 31 primary lesions ,and 5 cases (16.1%) and 4 cases (12.9%) in 31 metastatic lesions, respectively. The difference of the expression of TAP1 and LMP2 mRNA in primary lesions and metastatic lesions was statistically significant(P<0.05). Negative expression of TAP1 mRNA, LMP2 and adeno-carcinoma were risk factors leading to cancer metastasis to lymph node.Conclusions: Negative expression of TAP1 and LMP2 mRNA is one of mechanisms of HLA-Ⅰantigens loss or downregulation in primary lesions and metastatic lesions in lung cancer and,and is risk factor of cancer metastasis to lymph node. PART 3THE RELATIONSHIP BETWEEN ACTIVATED K-RAS AND C-erbB-2 ONCOGENES AND CLASSICAL HLA-ⅠANTIGEN DOWNREGULATION OR LOSS IN NSCLC PRIMARY LESIONS AND METASTATIC LYMPH NODE LESIONSObjective: To probe the relationship between K-ras and C-erbB-2 oncogenes and downregulation or loss of classical HLA class I antigen in primary lesions of non small cell lung cancer and metastatic lesions of lymph nodes.Methods: The detection of C-erbB-2 and K-ras activation in 65 primary lesions and 31 metastatic lesions were performed by differential PCR and PCR-RFLP, respectively. The results were analysed by chi-square test or Fisher's exact test of probabilities.Results: K-ras gene mutations were detected in 15 cases among the 65 primary lesions (23.1%) and in 13 cases among 31 metastatic lesions in lymph nodes(41.9%).The difference of K-ras gene mutations in primary lesions and metastatic lesions was not statistically significant(x~2=3.56,P>0.05). The difference of incidences of K-ras gene mutations between primary lesions with downregulation of classical HLA classⅠantigen and loss of classical HLA classⅠantigen was significant(x~2=5.38,P<0.05). C-erbB-2 gene amplifications were detected in 21cases among the 65 primary lesions (32.3%) and in 15 cases among 31 metastatic lesions in lymph nodes(48.4%).The difference of C-erbB-2 gene amplifications in primary lesions and metastatic lesions was not significant(x~2=2.32,P>0.05). The incidences of C-erbB-2 gene amplifications between primary lesions with downregulation of classical HLA class I antigen and normal erpression of classical HLA classⅠantigen was significant (P=0.02). The difference of the incidences of coexistence of K-ras gene mutations and C-erbB-2 gene amplifications between two groups(in 12 cases among the 65 primary lesions and in 9 cases among 31 metastatic lesions in lymph nodes) was not significant(x~2=1.37,P>0.05). The difference between the incidences of coexistence in primary lesions with loss of classical HLA classⅠantigen (45.5%) and downregulation of classical HLA classⅠantigen (15.6%) was significant(x~2=4.69,P<0.05), The difference between the incidences of coexistence in metastatic lesions with loss of classical HLA classⅠantigen (50%) and downregulation of classical HLA class I antigen (11%) was not significant (P=0.14).Conclusions: There is negative correlation between K-ras gene mutations and C-erbB-2 gene amplifications and expression of classical HLA-Ⅰantigen in primary lesions and metastatic lesions with non small cell lung cancer...