食管癌组织中Survivin和Ki67的表达及临床意义

　　作者：李秀娟 范 婕 李玉珍 王淑强 作者单位：(河北北方学院医学院病理学教研室，河北 张家口 075000)

　　【摘要】 目的：观察Survivin和Ki67在食管鳞癌中的表达，并探讨Survivin与Ki67的表达关系及其临床意义。方法：应用免疫组织化学法检测70例食管鳞癌组织石蜡切片Survivin和Ki67的表达，分析Survivin表达与临床病理因素及Ki67的关系。结果：Survivin在食管鳞癌组织中的表达率为72.9%(51/70)，Ki67在食管鳞癌组织中的表达率为67.1%(47/70)。Survivin表达和Ki67表达与食管鳞癌分化程度、淋巴结转移、TNM分期明显相关(P<0.05)。Survivin表达和Ki67表达呈正相关(P<0.05)。结论：Survivin和Ki67可作为判断食管癌组织恶性程度和预后的指标之一。

　　【关键词】 食管;鳞状细胞/肿瘤;Survivin;Ki67

　　Esophageal carcinoma is one of the most frequently malignant tumors in China.A method for identifying cancer cell proliferation and spreading is important to commence the effective treatment and to improve survival for ESCC patients.The process of esophageal tumorigenesis at the molecular level is related to disorders of cell amplification,differentiation,senescence and apoptosis.The genetic bases underlying esophageal tumorigenesis have been partly understood in the past few years,including a loss of the antioncogene p53 and overexpression of epidermal growth factor receptor or cMyc[1].However,other molecular mechanisms involved in esophageal tumorigenesis progress are still widely unknown.

　　Survivin is a kind of protein that is highly expressed in a vast number of malignancies,but is minimally expressed in normal tissues.It plays a role as an inhibitor of cell death in cancer cells,thus facilitating the growth of these cells[2].Ki67 is a marker of cell growth.This muclear antigen is expressed during all phases of the cell cycle(G1,S,G2 and mitosis),except phase G0.In recent studies,important correlation was found between ki67 and the growth,invision,metastasis and prognosis[3].In this study,the effects of Survivin and Ki67 in the formation and progression of ESCC were investigated,and the expression of them in ESCC was detected.

　　1 MATERIALS AND METHODS

　　1.1 Clinical Data

　　70 cases with ESCC were selected from the First Affiliated Hospital of Hebei North University from January 2000 to June 2005.There were 23 male and 47 female.The patients were given preoperative examination including biopsy for diagnosis,barium Xray and CT,and no treatment was given before operation.Radical resection was performed in each patient,and all the samples underwent postoperative pathological examination.There were 40 cases with stage Ⅰ～Ⅱ and 30 cases with stage Ⅲ～Ⅳ cancer according to the American Joint Committee on Cancer staging manual(AJCC,2002)[4].With regard to postoperative histological results,33 were welldifferentiated and 37 poorly differentiated.And 10 cases normal esophagus tissues were used as negative control.

　　1.2 Immunohistochemistry

　　A paraffin section of the ESCC sample was deparaffinized and rehydrated in graded alcohol to water.Antigenic enhancement was performed by submerging in citrate buffer(pH6.0)and microwaving.Endogenous peroxide activity was quenched by applying 0.3% hydrogen peroxide for 10 min.The primary antibodies such as rabbit polyclonal antihuman Survivin and rat monoclonal antihuman Ki67(both from Beijing Zhongshan Golden Bridge Biotechnology Ltd)were incubated for 60 min at 37℃.After washing,the tissue sections were incubated in the second antibodies for 30 min at 37℃.The reaction products were visualized by 3,3diaminobenzidine tetrahydrochloride(DAB).

　　Slides were then counterstained with hematoxylin,dehydrated through alcohol of increasing concentration,placed in xylene,coverslipped using Permount,and analyzed under light microscopy.The positive Survivin and Ki67 were stained brown yellow,the former was localized in the cytoplasm of the cells and the later in the nucleus.The percentage of positive tumor cells was recorded as follows:negative(≤5% of tumor cells positive),positive(>5% of tumor cells positive)[5].

　　1.3 Statistical analysis

　　Data analysis was carried out using SPSS statistical software package and verified with χ2test and spearman,s rank.The value with P<0.05 was considered statistically significant.

　　2 RESULTS

　　2.1 The expression of Survivin in ESCC

　　As determined by IHC,51(72.9%)of 70 cases was positive for in ESCC.In 10 cases of normal esophageal mucosal samples,Survivin did not express.The relationship between expression and clinicopathological variables was examined between the positive and negative groups.No statistical difference between two groups was found with respect to gender(P>0.05).However,Survivin expression was correlated to cell differentiation,lymph node metastasis and tumor stage(P<0.05).Survivin protein was highly expressed in ESCC(Table 1).Table 1 The expression of Survivin or Ki67 in ESCC

　　clinicalnSurvivin expressionpositiveχ2 valueP valueKi67 expressionpositiveχ2 valueP valueSex male2317 female47340.019>0.0517300.712>0.05Tissue differentiation well3319 low37327.37<0.0518294.49<0.05Lymph node Metastasis negative2614 positive44377.56<0.0513345.51<0.05TNM Stage Ⅰ、Ⅱ4025 Ⅲ、Ⅳ30265.06<0.0522256.23<0.05

　　2.2 The expression of Ki67 in ESCC

　　As table 1 shows,the expression of Ki67 was significantly higher in poorly differentiated,lymphnode metastasis and stage Ⅲ～Ⅳ group.

　　2.3 The relationship between Survivin and Ki67 in ESCC

　　After spearmans rank correlation analysis,it could be found that in ESCC the expression of Ki67 was correlated with Survivin(r=0.37,P<0.05).

　　Table 2 The relationship between Survivin and Ki67 in ESCC(n)

　　SurvivinKi67+-Total+35 1651-14519Total492170

　　3 DISCUSSION

　　Regulation of apoptosis is critical for normal embryonic development and for homeostasis in adult tissues.Cancellation of this process with increased resistance to cell death is a common feature of malignant cells and represents a significant obstacle to therapy of human cancers[6].Apoptosis resistance in ESCC accounts for its poor response to chemotherapy and enhanced metastasis[7].Inhibitor of apoptosis(IAP)has been proved to be crucial regulators of the molecular mechanisms of apoptosis.Among the IAP members,Survivin is unique in which it is involved in both control of cell division and inhibition of apoptpsis[8].It is expressed during fetal development,but not in nonproliferating adult tissuess.Survivin is also overexpressed in most human cancers,including ESCC.

　　In ESCC,overexpression of Survivin has been demonstrated by many groups.Survivin expression has been reported to correlate with the proliferative activity of cancer cells[9],overall survival of patients[10],the response of patients to chemotherapy[11] and recently the apoptotic cell ratio in esophageal cancer[12].Survivin inhibits apoptosis in cells exposed to diverse apoptotic stimuli by associating with microtubules of the mitotic spindles and inhibiting caspase3 and caspase7 activity.Overexpression of survivin has oncogenic potential because it may overcome the G2/M phase checkpoint to enforce progression of cells through mitosis.The vast majority of cancers express survivin protein,suggesting that reactivation of survivin gene expression occurs commonly in cancers.Correspondingly,previous reports have shown that the presence of survivin is associated with poor survival among patients with colorectal cancer,non smallcell lung cancer,breast cancer and neuroblastoma.

　　In the study on 70 patients with ESCC,it was showed that Survivin protein was expressed in 72.9%(51/70)of these tumours.The positive rate of Survivin in the well differentitation was 57.6%(19/33),while it was 86.5%(32/37)in the low differentitation,the comparison between the two groups had a significant difference(P<0.05).Correspondingly,the expression of Survivin was significantly associated with lymph mode metastasis and TNM stage of ESCC(P<0.05)，while less associated with gender(P>0.05).This means that Survivin plays a vital role in the occurrence process of ESCC.Several studies on ESCC have indicated that there is a relationship between survivin expression and the ultimate behavior of the carcinoma.Since the expression pattern of survivin is selective to cancer cells,it has been described as an “ideal target” for cancer therapy.Currently,several preclinical and clinical trials are ongoing to investigate the effects of interfering with survivin function in cancer cells as a biologic therapy.

　　Ki67 is a maker of cell growth.This nuclear antigen is expressed during all phases of the cell cycle(G1,S,G2 and mitosis),except phase G0 and,in several studies,cells in the growth phase appear to be more radiosensitive than quiescent cells,particularly in esophageal squamous cell cancer.Research showed that overexpression of Ki67 was a independent factor for complete endoscopic response after chemoradiotherapy for esophageal cancer[13].In this research，the expression of Ki67 was significantly associated with tissue differentitation,lymph mode metastasis and TNM stage of ESCC(P<0.05)，while less associated with gender(P>0.05).

　　In the present study,significant correlation was found between Survivin and Ki67 expression with histological differentiation grade，lymphnode metastasis，TNM stage of ESCC(P<0.05).Survivin expression was significantly correlated with Ki67 expression(r=0.37,P<0.05).So the expression of Survivin protein might promote cell proliferation of esophageal cancer.

　　In the case of ESCC,Survivin is a potentially significant protein in the diagnosis,prognosis and treatment of ESCC.Survivin and Ki67 are overexpressed in tumor cells and play role in the carcinogenesis process,further prospective studies are necessary to investigate the impact of these prognostic factors.

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