| Objective:Mediastinal (N2) lymph node involvement is heterogenous with huge variation in the extent and grouped together under stageⅢA. However, they showed a different survival even in the same stage. Some investigators have also postulated the possible classification of a subgroup of multiple station N2 patients who have more unfavorable results compared to single station N2 patients with stage IIIA-N2 NSCLC. We detected gene expression profiles of single station and multi-station lymph node metastasis N2 non-small cell lung cancer and explored the molecular mechanisms of further progression from single station mediastinal lymph node metastasis to multiple stations N2. Conventional lymph node (LN) staging does not effectively reflect the wide differences in LN involvement and patient survival, even in patients classified within the same LN stage. We used N2 NSCLC prognosis related genes to re-stage the N2 stage non-small cell lung cancer and further guided on the individualized treatment of N2 lung cancer that can be proposed for different therapeutic strategies.Methods:The single station and multi station tumor tissue that have consistent clinical information ofⅢA stage were detected by Agilent's Human Genome microarray and using bioinformatics software for comparative analysis. of differentially expressed genes according to functional classification, on which the tumor lymphatic invasion and metastasis gene were analyzed. Using RT-PCR, immunohistochemistry and western blot method verified differentially expressed genes from mRNA levels and protein level, respcyively. And we collect 96 patients with N2 lung cancer paraffin specimens, using two-step EnVision immunohistochemical detect the expression of MUC4, IGF2 in N2 lung cancer tissues. We analysis the level of MUC4 and IGF2 expression with clinicopathological parameters, lymph node metastasis, prognosis and correlation with each other and using SPSS 13.0 statistical software for data analysis.Results:1 Compared with the single station, there were 329 tumor tissue differences in gene expression levels of up to 10 times in multiple stations tissue. In these genes, and tumor metastasis-related genes were 88 and 51 unregulated,37 downregulated.2 Using RT-PCR, part of the differentially expressed genes were analyzed. Compared to single station N2 lung cancer, MUC4, ADAM28 were low expression in the multi-station N2 lung cancer, while KRTDAP, IGF2, CLDN1 were highly expressed.3 Using Western blot, relative expression levels of MUC4 protein in a single station and multiple station in the cancer tissue were 0.891±0.144,1.057±0.193. the differences of MUC4 protein expression were significantly different between single and multi station lung cancer (P<0.05).4 Using immunohistochemical, the positive rate of of MUC4 in the single-station N2 lung cancer were 73%, which is significantly higher than that of multi station(x2 =16.466, P=0.000), and he positive rate of IGF2 in the single-station N2 was 46% lower than the multi-station N2 lung cancer(x2=2.089, P=0.048).5 The level of MUC4 expression collated with clinicopathologic parameters, mediastinal lymph node metastasis and clinical prognosis(1) MUC4 stain are mainly located in the cell membrane and (or) cytoplasm, and no expression within the nucleus. In normal lung tissue, the positive rate of MUC4 was 25.4% and 47.9% in N2 lung cancer, the difference was statistically significant difference (P<0.05).(2) In different clinical and pathological parameters, MUC4 expression was no statistical difference significance in different gender, age, smoking status, tumor location, general location, pathology, tumor size and T staging of N2 lung cancer tissues.(3) For the mediastinal lymph node metastasis, MUC4 in different lymph node stations, number of lymph node metastasis, subcarinal lymph node metastasis, regional lymph node metastasis and clinical (pathological) N2 of the cancer, the emergence of differential expression (P<0.05);(4) In the whole group of N2 patients, high expression of MUC4 in patients with a total of 46 cases, including median survival of 43 months, and the 5-year survival rate was 32.6%, in terms of DFS, significantly better than patients with low expression (P =0.000). In MUC4 high expression group, the median DFS was 35 months and 5 years disease-free survival rate reached 25.3%, far better than the low expression group (P=0.000). The high expression of IGF2 total of 53 patients, median survival period of 19 months,5-year survival rate was only 15.1%, significantly lower than the low expression group (P=0.000), in the DFS, too, the same conclusion.(5) In the COX multivariate analysis, the expression of 1GF2 and of MUC4 in N2 non-small cell lung cancer are independent prognostic factor (P=0.000).(6) Spearman rank correlation analysis showed that:in N2 lung cancer tissues, MUC4 and IGF2 expression was negatively correlated (r=-0.226, P= 0.027).6 Combined with MUC4 and IGF2 on the N2 non-small cell lung cancer molecular staging, in which MUC4+ +IGF2- group, the best prognosis, with median survival of 61 months, the median DFS was 55 months,5 year survival rate was 49.7% 5-year disease-free survival rate reached 44.3%, better than the other three groups (P =0.000).Conclusion:1 Advances in N2 disease from single-station to multi stations are involved in multiple genes, multiple signaling pathways, which is a multi-step process. MUC4, IGF2, ADAM28, CLDN1, S100 family and MMP family may play a key role in the progression course of a single station N2 and in further understanding of N2 mediastinal lymph node metastasis of lung cancer causes and molecular mechanisms of progress. We can make molecular treatment for these genes in the future.2 United MUC4 and IGF2 is divided N2 non-small cell lung cancer into different subgroups. MUC4+ +IGF2+ group have the best prognosis, and aggressive surgical treatment is the better choice for them. For subgroups with poor prognosis, surgery is not advocated resection, and the choice of chemoradiotherapy is much better. |
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