Expression And Clinical Significance Of C-Cbl,Cbl-b And EGFR In Gastric Carcinoma

IntroductionCurrently,gastric carcinoma remains an important public health problem. Worldwide,the incidence of gastric cancer lies in the fourth place of all malignant tumors,meanwhile it is the second largest cause of death.There are 1,000,000 new cases each year all of the world.The incidence of gastric cancer in China is in the second place among all the malignancies.Though surgical treatment is the only method to cure gastric cancer at present,most patients have been diagnosed at the advanced phase of the disease.So chemotherapy is still the main method for advanced gastric cancer.So far there is no standard chemotherapy plan for gastric cancer,and the efficacy of existing chemotherapy is hardly satisfactory.Targeted therapy has arised recent years. With the progress of molecular biology mechanism,targeted therapy will gradually be applied for the gastric cancer clinical practice.EGFR has usually been uesed as the molecular target for therapy,which is widerly applided in the clinical practice.EGFR,a 170 KD membrane receptor protein with tyrosine kinase activity,is a member of the ErbB family,which combines growth factor to transduct cell signals and promote cell proliferation.Meanwhile EGFR itself is internalized and deregulated,and Cbl proteins play a critical role during the degradation of EGFR.c-Cbl and Cbl-b are two main members of CBL(The Casitas B-cell lymphoma)family,which can target a series of proteins to degrade as adaptors and the E3 ubiquitin ligase,including a series of proteins with receptor tyrosine kinase activity,such as the EGF receptor,to maintain the homeostasis of the body.c-Cbl and Cbl-b structurally include several domains:a tyrosine kinase binding domain(TKB), the ring finger(RF),Linker domain,and the C-terminal with ubiquitin ligase activity. At present,the studies of c-Cbl,,Cbl-b and EGFR association with gastric cancer remain rare.The existing studies have shown that the EGFR is an independent prognostic indicator of gastric cancer,but c-Cbl isn't.So far there is none study of Cbl-b in the area.So this study was designed to investigate the expression of c-Cbl, Cbl-b and EGFR as well as their clinical significance in gastric cancer.Materials and Methods1.PatientsA total of 124 gastric adenocarcinoma cases were used.The tumor tissues and corresponding non-neoplastic gastric mucosa were obtained from surgeries at the first hospital of China Medical University(CMU)and Liaoning Cancer Hospital,and none of them underwent radiochemotherapy before operation.For immunohistochemistry, tissues were fixed in 10%buffered-formalin and embedded in paraffin.There were 92 male and 32 female patients with median age of 60 years.We classified the gastric carcinoma according to the P-TNM criteria of the UICC 1997.There were 11 cases of well differentiation(papillary adenocarcinoma and well-differentiated tubular adenocarcinoma)(8.9%),29 moderated differentiation(moderately differentiated tubular adenocarcinoma)(23.4%)and 84 poor differentiation(solid-and non-solid-type poorly differentiated adenocarcinoma,signet-ring-cell carcinoma and mutinous adenocarcinoma)(67.7%).Depth of carcinoma invasion was classified as follows: t1,mocosa and submucosa(3 cases);t2,muscularis propria and subserosa(73 cases); t3,serosa-exposed(se)(40 cases);t4,serosa-infiltrating(si)(8 cases).There were 100 cases with lymph node metastasis(80.6%).There were 17 cases in stageⅠ(13.7%),46 in stageⅡ(37.1%),37 in stageⅢ(29.8%)and 24 in stageⅣ(19.4%).2.MaterialsAntidodies for immunohistochemistry:The monoclonal antibodies against c-Cbl was purchased from BD corporation;The monoclonal antibodies against Cbl-b was purchased from SANTA CRUZ corporation;The monoclonal antibodies against EGFR was purchased from Fuzhou Maixin Biotechnology,Inc.;S-P immunohistochemical kits and DAB substrate solution were purchased from Fuzhou Maixin Biotechnology,Inc.3.MethodsImmunohistochemical staining S-P method was performed on tissue microarray sections made of 124 cases of gastric carcinoma and 24 cases corresponding non-neoplastic gastric mucoma.4.AssessmentOutcome definition of c-Cbl,Cbl-b and EGFR were stained yellow particles in membrane and/or cytosol.Judging reference consided the scores of both proportion and intension of positive stained.(-)was negative stained,(+)was defined as weekly positive stained,(++)～(+++)was defined as strongly positive stained.5.Statistical analysisThe Chi-square test,Mann-Whitney U test and Spearman rank correlation test were performed by SPSS software version 13.0.The statistical difference was considered significant when P＜0.05.Results1.The expression of c-Cbl,Cbl-b and EGFR in patients with gastric carcinomaThe positive expression rate of c-Cbl,Cbl-b and EGFR were 71.0%,82.3%and 56.5%.They were all highly expressed in gastric carcinoma.2.The relationship between expression of c-Cbl,Cbl-b and EGFR and clinicopathologic parameters in patients with gastric carcinomaThe expression of c-Cbl was positively correlated with depth of invasion (r=0.219,P=0.015)and TNM staging(r=0.266,P=0.003);The expression of Cbl-b was positively correlated with metastasis of tumor cells in lymph nodes(r=0.190,P=0.034) and TNM staging(r=0.298,P=0.001);The expression of EGFR was positively correlated with depth of invasion(r=0.286,P=0.001)and TNM staging(r=0.362, P=0.000).3.The relationship between c-Cbl,Cbl-b and EGFRThere was a positive relationship between the expression of c-Cbl and EGFR(r=0.241,P=0.007);there was also a positive relationship between the expression of Cbl-b and EGFR(r=0.183,P=0.042).4.The relationship between synchronous strongly positive expression of the three proteins and clinicopathologic parameters in patients with gastric cancerSynchronous strongly positive expression of c-Cbl,Cbl-b and EGFR was observed in 27 cases.Among 27 cases with simultaneous strongly positive expression of c-Cbl, Cbl-b and EGFR,most were found in poorly differentiation,deep invasion,nodal metastasis and advaced cases.Conclusion1.c-Cbl,Cbl-b and EGFR are highly expressed in gastric carcinoma.The positive expressions of c-Cbl and EGFR are associated with deep invasion and late TNM stage, and Cbl-b is associated with metastasis of tumor cells in lymph nodes and late TNM stage.Meanwhile synchronous strongly positive expression of c-Cbl,Cbl-b and EGFR are mostly found in poorly differentiation,deep invasion,nodal metastasis and advaced cases.Our results suggest that the positive expression of c-Cbl,Cbl-b and EGFR may be a prognosis indicator,especially the synchronous strongly positive expression of the three proteins.2.c-Cbl/Cbl-b in connection with EGFR system may be associated with stomach carcinogenesis,invasion and metastasis,c-Cbl and Cbl-b may serve as a novel molecular marker for aggressive gastric carcinoma.