Prognostic Stratification Of Patients With Lung, Breast And Liver Cancers By EMT-related Gene-expression Profiling In Tumor-adjacent Tissues

Clinical evidence revealed that approximately25%of stage la primary non-small cell lung cancer (NSCLC) patients may suffer relapse or metastasis within two years after complete surgical resection. We presumed that the sustaining field effect of cancerization in the surgical margins account for cell malignant transformation and cancer relapse. In this study, we identified a set of EMT (Epithelial-to-Mesenchymal Transition)-related genes in tumor-adjacent histologically normal tissues (TAHNTs) from lung squamous cell carcinoma (LSCC) patients, and we validated this gene-expression profile in three independent cohorts of LSCC, breast cancer, and hepatocellular carcinoma (HCC) patients.Based on microarrays, global gene expression analysis was performed on69tumor tissues and60TAHNTs from LSCC cases, as well as15mature lung tissues from pulmonary non-tumor patients. Data analysis revealed that:1) TAHNTs exhibited deregulated gene expression profiles, and shared some common differentially expressed genes with the tumor tissues;2) TAHNTs had less inter-sample heterogeneity in their gene expression compared to tumors;3) extracellular matrix and cell movement related genes in TAHNTs were significantly correlated with lymph node metastasis. An EMT model of the immortalized human bronchial epithelial cell line, M-BE, was induced by recombinant TGF-β1in vitro, leading to the induction of1,490EMT-related genes,33of which overlapped with TAHNT-derived genes. Gene Set Enrichment Analysis (GSEA) revealed that EMT-related genes were significantly enriched in TAHNTs derived from lymph node positive LSCC patients. Integrating gene expression data from TAHNTs in vivo and the EMT model in vitro, we identified four key EMT-related genes. Independent validation revealed that the4-gene signature was activated in TAHNTs from LSCC, breast cancer, and HCC patients, and it was significantly correlated with the prognosis of the three malignancies. For LSCC, up-regulation of the4-gene signature in TAHNTs was associated with poor overall patient survival (N=50,P=0.009), including stage I patients (N=21,,P=0.032). For breast cancer, elevated expression of these four genes in TAHNTs significantly correlated with shorter survival time in ER-positive patients (N=43, P=0.039). For HCC, patients with higher expression of these four genes in TAHNTs showed significantly lower overall survival (N=82, P=0.008). We also measured the protein levels of the four key EMT-related genes in the periphery circulating blood from HCC patients by ELISA, and we found that up regulation of ECM1and FBN1in the plasma was an independent prognostic factor for disease free survival (DFS, P=0.024) and overall survival (OS, P<0.001) of HCC patients.In conclusion, these results suggest that histologically normal tissues exhibited malignance-promoting gene expression alterations that may contribute to relapse or metastasis of the tumor after surgical resection. Assessment of these molecular alterations represents a useful approach for determining the diagnosis or prognosis of cancer patients. Meanwhile, these genes may also be potential therapeutic targets to prevent cancer relapse or metastasis.