Multiple Tumor Markers Combined Analysis Of Prognosis In Patients With Esophageal Carcinoma

Background:Cancer represents one of the most concerns of human health worldwide. Approximately20%global cancer cases and24%cancer mortalities occur in China. Esophageal cancer (EC)is one of most common cancer across the world and is characterized by distinct geographicdistribution, which displays500-fold incidence difference between low and high risk areas forEC. Linzhou and adjacent Huixian, Anyang are one of the highest areas for EC in terms ofincidence and mortality. On the other hand, the prognosis of EC is poor and the5-yearsurvival rate for advanced EC is only about10%. The mean survival time of the majority ofEC patients is less than one year after confirmed diagnosis and the mortality is very high.Although there are considerable advances in therapeutic modalities of EC and it is wellknown that EC takes a multi-factorial, multi-stage and complicated process, the incidence andmortality of EC in high risk areas still remain unchanged in the past decades. Therefore, ECstill represents one of the main cancer-related mortalities in the high risk areas for EC innorthern China. Conceivably, it is merited to elucidate the molecular mechanisms ofmulti-stage evolution of EC pathogenesis, to screen risk-associated molecules, to identifysensitive and specific biomarkers for EC early diagnosis and therapeutic targets, with aim toreduce the incidence and mortality of EC.By using a complete set of proteomic techniques, i.e. two-dimensional electrophoresis(2DE), stable isotope labeling with amino acids in cell culture (SILAC), two-dimensionalchromatography, ESI-MS/MS, MALDI-TOF-TOF-MS, we have identified156differentiallyexpressed proteins implicated in esophageal squamous cell carcinoma (ESCC), such as MIF（Macrophage migration inhibitory factor）、prohibitin、 HSP27（Heat shock protein27）、annexin A2、GRP94(Glucoseregulated protein)、stratifin、CD（Cathepsin D）, etc. HSP27is one of the members of small heat shock protein. Under stress conditions,HSP27expression is induced, which functions to avoid injury of cell, and is involved in cellproliferation and apoptosis as well. Recent studies suggest that HSP is implicated in tumorbiology, such as regulation of cell cycle, cell proliferation, differentiation, and apoptosis.Furthermore, HSP is correlated with tumor immunity and multi-drug resistance. Theup-regulated expression of HSP27is associated with prognosis with distinct biologicalfunctions and clinical relevance in various types of tumor.GRP94, one of the molecular chaperones, belongs to HSP90family and plays functionsin co-regulating protein folding, stretching, assignment, and inhibiting secretion ofmis-folding proteins. In tumor biology, GRP94is correlated with tumor cell differentiation,invasion, proliferation, metastasis and acquiring of multi-drug resistance. Furthermore,GRP94binds to a variety of peptides possessing immunogenicity, which implicates thatGRP94has potential values in cancer therapy and prognosis.Macrophage migration inhibitory factor (MIF) exists in T cells and is a cytokine withpleiotrophic functions, such as activation of immune cells, inhibiting migration ofmacrophage, induction of tumor cell detachment and invasion, etc. MIF expression iscorrelated with prognosis through potentiating the ability of invasion and metastasis of tumorcells.Cathepsin D (CD) is a major lysosomal aspartic endopeptidase and comprisespro-cathepsin D (pCD), intermediate and mature forms. The main function of CD is todegrade proteins in acid milieu of lysosome. In pathologic conditions, however, CD candegrade extracellular matrix and basement membrane, which enhances the invasion andmetastasis of malignant cells. Recent published investigations indicate that over-expression ofCD is correlated with recurrence of cancer and worse prognosis.The present study verified the differential expression of CD, pCD, MIF, GRP94and HSP27between ESCC and adjacent non-tumor tissue samples by Western blot andcharacterized the expressions of HSP27, GRP94and MIF in archival paraffin-embeded ESCCtissue samples with5-year follow-up data. We also digged the clinical significance andprognostic value of the expressions of HSP27, GRP94and MIF, which provide rationale formolecular diagnosis, prognosis evaluation and identification of biomarkers of EC.AimsTo determine the clinical and prognostic values of the multiple biomarkers (CD, pCD,MIF, GRP94and HSP27) in different TNM staging of ESCC and to provide more insights ofmulti-molecule, multi-stage of ESCC evolution, which forms the theoretic basis ofidentification of biomarkers for high-risk population screening, early diagnosis, andprognostic monitoring.Methods1. To detect the differential expressions of CD, pCD, MIF, GRP94and HSP27in resectedESCC and adjacent non-tumor tissue samples by Western blot;2. To characterize the expressions of MIF, GRP94and HSP27in various TNM staging ESCCand analyze the correlation between expressions of above biomarkers with clinicalparameters and prognosis.Results1. Over-expressions of CD, pCD, MIF, GRP94and down-expression of HSP27wereverified in ESCC compared to adjacent non-tumor tissue;2. Over-expressions of MIF and GRP94were aggressively correlated with lymph nodemetastasis (P=0.047, P=0.016), TNM staging (P=0.008, P=0.002) and shorterover-all5-year survival (P=0.01, P=0.001). Over-expression of GRP94was correlatedwith differentiation as well (P=0.002);3. Over-expression of HSP27was correlated inversely with lymph node metastasis (P= 0.004), TNM staging (P=0.008), and shorter over-all5-year survival (P=0.007);4. MIF and GRP94were independent factors in risk predication post-surgery of ESCC bymulti-factorial Cox model.Conclusions1. CD, pCD, MIF, GRP94and HSP27may be crucial molecules implicating multi-stageevolution of ESCC;2. MIF, GRP94and HSP27may play important roles in malignant transformation ofesophageal epithelium and be novel molecular markers for prognostic prediction.