Association of E-cadherin and β-catenin with metastasis in nasopharyngeal carcinoma

Background This study was designed to detect methylation of E-cadherin gene promoter and gene mutation of β-catenin in exon 3 and their expression of protein and mRNA in primary tumor and lymph node metastatic tumor of nasopharyngeal carcinoma (NPC), and investigate the mechanism of invasion and metastasis of neoplastic cells in NPC. Methods Fourty-two fresh biopsy samples were taken from untreated NPC patients at the Affiliated Hospital of Sun Yat-sen Medical College, Sun Yat-sen University, Guangzhou, China during the period of 1999 -2002. Among them 21 were taken from primary tumors and the other 21 from lymph node metastatic tumors. The gene promoter methylation of E-cadherin was detected by methylation-specific PCR (MSP). The mutation in exon 3 of β-catenin was detected by direct sequencing analysis. RT-PCR, Western blot and immunohistochemical staining were used to detect the mRNA and protein expression patterns in both primary and metastatic tumors of NPC. Results Down-regulated expression of E-cadherin in metastatic tumor was compared with that in primary tumor. Reduced expression of E-cadherin was found to be correlated with lymph node metastatic tumor of NPC ( P = 0. 004) ; but there was no obvious correlation between primary and metastatic tumors in the expression of β-catenin (P = 0. 698). The mRNA expression level of Ecadherin in metastatic tumors decreased significantly compared with that in primary tumors. However, little change was observed in the mRNA level of β-catenin in different tumor tissues. Only 4 samples (19. 1%) displayed gene promoter methylation of E-cadherin in primary tumor and 10 samples (47. 6%) showed methylated form of E-cadherin. The gene promoter methylation of E-cadherin was more common in metastatic tumor than in primary tumor of NPC ( P =0. 024). Only 2 (4. 76% ) of the 42 samples showed mutations in exon 3 of β-catenin at 41 (T41A, ACC→GCC) and codon 47(S47T, AGT→ACT). The cytoplasmic and nuclear expression of β-catenin in tumor was not found in any samples of NPC. Conclusions The results suggest that the downregulation of E-cadherin results from the gene promoter aberrant methylation of E-cadherin and that the methylation of E-cadherin plays an important role in invasion and metastasis of tumor cells in NPC. However, β-catenin mutation is an infrequent event in NPC, and β-catenin is not a critical factor influencing the invasion and metastasis of tumor cells in NPC.     

Expression and Significance of Cox-2 and Livin in Oral Squamous Cell Carcinoma with Tissue Microarray

Objective To detect the expression of Cox-2 and livin in oral squamous cell carcinoma and precancerous lesion with tissue microarray, and discuss their significance and relationship in the occurrence and development of oral squamous cell carcinoma. Methods Immunohistochemical （IHC） staining and tissue microarray technique were used to detect the expression of Cox-2 and livin in noma! oral mucous membrane, precancerous lesion and oral squamous cell carcinomas. Results The expression of Cox-2 was negative in normal oral mucous membrane, and positive in precancerous lesion （81.6%） and squamous carcinoma （85.2%）; while the expression of livin was negative or weakly positive in normal oral mucous membrane, and positive in precancerous lesion （89.8%） and squamous carcinoma （100%）. The positive expression of Cox-2 and livin were both closely related to pathological classifications of oral squamous cell carcinomas. But there was no correlation between them. Conclusion Cox-2 and livin have close relationship with the occurrence and development of oral squamous cell carcinoma, but no correlation with the expression.     

Establishment of cervical lymph node metastasis model of squamous cell carcinoma in the oral cavity in mice

Background Oral squamous cell carcinoma （OSCC） is the most prevalent malignant tumor in the head and neck region, comprising more than 90% of all oral malignancies. A feasible approach for an animal model to study OSCC lymph node metastasis was established and biological behaviors of three oral squamous cell carcinoma cell lines were compared. Methods After implanting three kinds of cell lines （GDC185, Tca8113, Tca83） into three different anatomical sites in nude mice, namely the tongue, floor of the mouth, and axillary fossa, we observed the tumorigenicity and the metastatic capacity, which was confirmed by histopathology under a surgical microscope. Results The animal model injected with GDC185 cells into the floor of the mouth had the highest rate of neck lymph node metastasis （55.6%） and the cell lines had significantly different biological behaviors. Conclusions Nude mice injected with GDC185 cells into the floor of the mouth could be used as a feasible animal model to study neck metastasis of oral squamous cell carcinoma.     

Her2阳性乳腺癌中β-catenin和E-cadherin异常表达及其意义

目的 探讨人乳腺癌中Her2阳性表达与β连环蛋白(β-catenin)及上皮性钙粘索(E-cadherin)异常表达间的关系及意义.方法 应用免疫组织化学方法检测147例乳腺癌中Her2、β-catenin和E-cadherin表达情况,同时检测19例癌旁正常乳腺和17例乳腺良性病变中β-catenin和E-cadherin的表达作为对照.结果 在乳腺癌组织中,Her2阳性表达与腋窝淋巴结转移、较晚的临床分期及ER、PR阴性表达显著相关(P(0.05):β-catenin的异常表达与腋窝淋巴结转移和较高的组织学分级显著相关(P<0.01);E-eadherin的异常表达与腋窝淋巴结转移和较晚的临床分期显著相关(P<0.05);β-catenin的异常表达与E-cadherin的异常表达正相关(P<0.01);Her2的阳性表达与β-catenin的异常表达正相关(P<0.01),并且二者还协同促进了乳腺癌的腋窝淋巴结转移(P<0.01).结论 在人乳腺癌组织中Her2的阳性表达与β-catenin的异常表达呈正相关性,Her2的阳性表达与Wnt/β-eatenin信号通路的异常激活之问可能存在功能上的相互联系.

Abstract：

Objective To investigate the relationship between positive expression of Her2 and abnormal expressions of β-catenin and E-cadherin and its implications. Methods Immunohistochemistry was used to detect the expressions of Her2, β-catenin and E-cadherin in 147 samples of human breast carcinoma. The expressions of β-catenin and E-cadherin were also detected in 19 tissues adjacent to the carcinoma and 17 benign breast lesions as controls. Results In breast carcinoma, positive Her2 expression was associated with lymph node metastasis, advanced clinical stage and negative expression of ER and PR (P<0.05). Abnormal β-catenin expression was associated with positive lymph node status and high histological grade (P<0.01). Abnormality of E-cadherin expression was related to lymph node metastasis and advanced clinical stage (P<0.05). Abnormal β-catenin expression was directly correlated with abnormal E-cadherin expression (P<0.01). Her2 positivity showed a direct correlation to abnormal β-catenin expression (P<0.01), and they cooperated in promoting axillary lymph node metastasis in human breast carcinoma (P<0.01). Conclusion A direct correlation between positive Her2 expression and abnormal β-catenin expression exists in human breast carcinoma, and positive Her2 expression may have functional interactions with abnormal activation of Wnt/β-catenin signaling pathway.     

Expression of β-catenin in renal cell carcinoma

Objective To investigate the expression of β-catenin and its mRNA in renal cell carcinoma.Methods Twenty-six cases with renal cell carcinoma (RCC) were studied by immunohistoche mistry, Western blot and RT-PCR.Results We found the expression of β-catenis is higher in cancer tissues than in norma l kidney tissues and the level of β-catenin is associated with the tumor stage . Its expression in tumor of pT3 and pT4 is obviously higher than pT1 and pT2 ( P&lt;0.01). That is to say, there was an overexpression of β-catenin protei n in RCC and its level was related to the tumor stage, but the expression of β -catenin mRNA had no difference between tumor tissue and normal tissue.Conclusion β-catenin may be related to the occurrence and progress of RCC.     

Expression of p63 and Cyclooxygenase-2 and Their Correlation in Skin Tumors

To study the expression of p63 and cyclooxygenase-2 (cox-2) in skin tumors and evalu- ate the correlation between p63 and cox-2, the expressions of cox-2 and p63 were measured by streptavidin-peroxidase complex immunohistochemical technique in 17 cases of skin squamous cell carcinoma(SCC), 19 cases of Bowen's disease(Bowen), 11 cases of actinic keratosis(AK), 12 cases of seborreic keratosis(SK) and 13 specimens of normal skin. Our results showed that the expression of p63 in skin squamous cell carcinoma, Bowen's disease and actinic keratosis were significantly higher than that in seborreic keratosis, while the expression of p63 in seborreic keratosis was sig- nificantly higher than that in normal skin. The expression of cox-2 in skin squamous cell carcinoma, Bowen's disease and actinic keratosis were significantly higher than that in seborreic keratosis, while no statistical difference was noted in the expression of cox-2 between seborreic keratosis and normal skin. Cox-2 expression was positively correlated with the high p63 expression in malignant skin tu- mors. The increased expression of cox-2 and p63 may play an important role in the development of skin tumors and work synergetically in malignant skin tumors.     

Detailed deletion mapping of loss of heterozygosity on 9p13-23 in laryngeal squamous cell carcinoma by microsatellite analysis

Background This study was designed to investigate the hot spots of microsatellite loss of heterozygosity (LOH) on 9p13-23 in laryngeal squamous cell carcinoma and to find out the correlation between the incidence of microsatellite LOH and the clinicopathological parameters.Methods Tumor tissues were obtained from paraffin embedded sections with microdissection. Genomic DNA was extracted from tumor tissues and peripheral blood lymphocytes with the phenol-chloroform. Polymerase chain reaction (PCR) amplification and denaturing gel electrophoresis were carried out in a set of 42 squamous cell carcinoma (SCC) of larynx and corresponding peripheral blood lymphocytes using 13 highly polymorphic microsatellite markers on 9p13-23. The correlation was analyzed between microsatellite LOH at the high frequency on 9p13-23 and clinicopathological parameters in the patients with squamous cell carcinoma of larynx.Results Of the 42 laryngeal cancers, 41 (97.6%) showed LOH in at least one of the microsatellite markers tested on 9p13-23. The most frequently deleted marker was D9S162 in 17 of the 19 (89.5%) informative samples. The marker D9S171, which is located on 9p21, had LOH detected in 12 of the 15 informative cases (80.0%). LOH at the D9S1748 marker (closest to the p16 gene locus) was detected in 18 of the 36 informative cases (50.0%). Allelic deletion mapping revealed two minimal regions of LOH encompassing markers D9S161-D9S171 on 9p21 and IFNA-D9S162 on 9p22-23. Multiple LOH (≥4) on 9p21-23 was found more frequently in the patients under 60 years, with supraglottic SCC or cervical lymph node metastasis than those over 60 years, with glottic SCC or without cervical lymph node metastasis (P&lt;0.01 or 0.01, 0.05, respectively). On the contrary, there was no correlation between T stages or pathologic classification and the frequency of LOH on 9p21-23 in 42 SCC of Larynx.Conclusions These findings imply the presence of at least two putative tumor suppressor genes on 9p13-23 in laryngeal SCC. Multiple genetic alterations are probably implicated in supraglottic SCC with cervical lymph node metastasis in younger patients.     

Esophagus,cardia of stomach

208100 Pattern of lymph node metastasis in patients with squamous cell carcinoma of the thoracic esophagus and its significance in lymphadenectomy/Li Hecheng(李鹤成,Dept Thorac Surg,Cancer Hosp Fudan Univ,Shanghai 200032)…∥Chin J Thorac Cardiovasc Surg.-2007,23(4).-252～254Objective To explore the pattern of lymph node metastasis in patients with squamous cell carcinoma of the thoracic esophagus and its significance in lymphadenectomy.Methods The clinical data of 230 patients who received radical esophagectomy with three-field lymphadenectomy was analyzed.     

Expression of Heat Shock Protein 70 and 27 in Non-small Cell Lung Cancer and Its Clinical Significance

The heat shock proteins （HSPs） 70 and HSP 27 expression in patients with non-small cell lung cancer （NSCLC） was studied and the relation.ship between HSP 70 and HSP 27 with the clinicopathological features of NSCLC was investigated. The expression of HSP 70 and HSP 27 was detected in tumor tissues from 60 patients with NSCLC by S-P immunohistochemistry. The findings were analyzed in combination with the histological types, histopathological differentiation, lymph node metastasis, patients＇ clinical stages, smoking history and gender. The results showed that of the 60 NSCLC tissue specimens studied, the immunoreactivity of HSP 70 and HSP 27 was detected in 47 （78.3 %） and 43 （71.7 %） specimens, respectively. A positive correlation was found between the overexpression of HSP 70 and HSP 27. The histopathological differentiation, lymph node metastasis, clinical stages and smoking history were correlated to the expression of HSP 70, but not to the expression of HSP 27. No statistical significance was observed in histological types and gender with respect to both HSP 70 and HSP 27 expression. It is suggested that the HSP 70 expression is a powerful and significant prognostic indicator and is related to histopathological differentiation, lymph node metastasis, patients＇ clinical stages, smoking history, whereas HSP 27 expression is not.     

Expressions of cyclin E, cyclin dependent kinase 2 and p57KIP2 in human astric cancer

Objective To investigate the expressions of cyclin E, cyclin dependent kinase 2（CDK-2）a nd cyclin-dependent kinase inhibitor p57 (KIP2) in human gastric cancer, and to evaluate the relationships between protein levels and clinicopathological pa rameters. Methods Western blot was used to measure the expressions of cyclin E, CDK-2 and p57 (KIP2) proteins in the surgically resected gastric carcinoma, adjacent normal mu cosa and metastatic lymph nodes from 36 patients. Results Cyclin E and CDK-2 protein levels were higher in gastric cancer tissues in comp arison with normal tissues ( P &lt;0. 05).Overexpression of cyclin E was correl ated with lymph node involvement, poor histological grade and serosa invasion ( P &lt;0. 05).Overexpression of CDK-2 was correlated with lymph nodes involvem ent ( P &lt;0. 05).No statistically significant difference between cyclin E and CDK-2 expression was found when samples were stratified according to tumor siz e ( P &gt;0. 05).Expression of cyclin E and CDK-2 showed a positive linear cor relation( r =0. 451,P =0. 01).Protein levels of p57 (KIP2) were lower in gastric cancer tissues than in the normal mucosa ( P &lt;0. 05).Decreased expr ession of p57 (KIP2) was correlated with lymph node involvement ( P &lt;0. 05) .No statistically significant difference in p57 (KIP2) expression was found when sample were stratified according to tumor size, histological grade or sero sa invasion ( P &gt;0. 05).In metastatic lymph nodes, expression of cyclin E wa s increased and the expression of p57 (KIP2) decreased. Conclusion Overexpressions of cyclin E, CDK-2 and downregulated expression of p57KIP2 may play important roles in tumorigenesis and metastatic potential of gastric cancer.     

Local injection of liposomal adriamycin to inhibit metastatic cell proliferation in axillary nodes in rabbits bearing breast tumors

Objective： To assess the inhibitory effects of local injection of liposomal adriamycin （LADR） on the proliferation of lymph node metastases in rabbits bearing VX2 carcinoma in the mammary gland. Methods：Thirty female New Zealand white rabbits were divided into 3 groups, with 10 in each. VX2 tumor mass suspensions were injected into the breast tissues of rabbits. Treatment initiated once the axillary lymph node reached 5 mm in the maximum diameter. Group 1 received a sham treatment. Group 2 received a subcutaneous injection of LADR adjacent to tumor. Group 3 received an intravenous injection of free ADR （FADR） at the same dose and concentration to group 2. The breast tumors and axillary lymph nodes were resected after the treatment was repeated 3 times. The tumor and node sizes before and after treatment were measured. PCNA mRNA expressions in breast tumors and axillary nodes were determined using RT-PCR. Results： The mean growth ratios of lymph nodes after treatment were 3. 70, 1.55, and 2.89, respectively, in groups 1, 2, and 3. The slowest node growth was observed in animals of group 2, with significant differences from group 1 （P〈0. 001） and group 3 （P= 0. 002）. The relative values of PCNA mRNA expression in lymph nodes were 0. 541, 0. 329, and 0. 450, respectively, in groups 1, 2, and 3. Group 2 exhibited a significantly reduced PCNA mRNA expression in metastatic lymph node, as compared to group 1 （P〈0. 001） and group 3 （P=0. 004）. Intravenous FADR injection effectively lowered the mRNA expressions of PCNA in breast tumors, which were not apparently altered after local LADR injection. Conclusion： Local injection of LADR holds a strong inhibitory effect on the proliferation of metastatic tumor cells in lymph nodes and appears to be an effective method for the treatment of lymphatic metastases of breast cancer.     

Flow cytometric DNA-ploidy as a prognostic indicator in oral squamous cell carcinoma.

In an attempt to evaluate the significance of DNA-ploidy as an objective prognostic indicator of oral squamous cell carcinoma, a retrospective analysis of 70 paraffin-embedded specimens using flow cytometry was performed. Forty-one patients (58.6%) had aneuploid tumors and 29 patients (41.4%) had diploid DNA distribution patterns. There was a significant difference in 5-year disease-free survival rates between patients with aneuploid and diploid tumors. The difference in cervical metastatic rates between aneuploid and diploid group approximated statistical significance. No obvious correlation was observed between DNA indices and histologic grade, clinical stage or tumor size. It is concluded that patients with aneuploidy primary tumors have a significantly poorer prognosis and higher risk for regional metastases than those with diploid tumors.

     

Gene expression profiles of ERCC1, TYMS, RRM1, TUBB3 and EGFR in tumor tissue from non-small cell lung cancer patients

Background Personalized medicine becomes essential in lung cancer treatment, however lung-cancer-related gene expression profiles in Chinese patients remain unknown. In this study, the correlation of gene expression profiles and clinical characteristics in non-small-cell lung cancer （NSCLC） was investigated. Methods Seventy-six Chinese patients with NSCLC were enrolled in the study to investigate mRNA expression profiles of excision repair cross complement group 1 （ERCC1）, thymidylate synthetase （TYMS）, ribonucleotide reductase （RRM1）, class Ill 13-tubulin （TUBB3）, and epidermal growth factor receptor （EGFR） genes and their correlation with patient clinical characteristics. A novel liquidchip technology was used to detect mRNA expression levels in formalin fixed paraffin embedded tumor pathology samples. The relationships between gene expression and clinical characteristics were assessed using the Mann-Whitney test. Results ERCC1 mRNA levels were higher in tumors from patients with metastatic disease than patients with non- metastatic disease （P=-0.021）, and higher in adenocarcinomas than squamous cell carcinomas （P=0.006）. Increased TUBB3 mRNA expression levels were found in patients with performance status （PS） 1 in comparison with PS 0 （P=0.049）, with poorly differentiated tumors in comparison with tumors that were moderately and well differentiated （P 〈0.000 1）, and with advanced stage in comparison with early stage disease （P 〈0.000 1）. Conclusions ERCC1 mRNA levels were higher in metastatic adenocarcinoma NSCLC; TUBB3 mRNA levels were significantly higher in poorly differentiated tumors and in advanced stage NSCLC, which indicates the poor prognosis.     

Relations of proliferative activities of gastric carcinoma cells to lymphatic involvement, venous invasion and prognosis

Background This study was to evaluate bivariate bromodeoxyuridine(BrdUrd)/DNA flow cytometric analysis in detection of gastric carcinoma and to study the relations of cellular BrdUrd labeling indices (LI), G2/M-phase fraction(G2/MPF) and DNA ploidy pattern to lymphatic involvement, venous invasion and prognosis.Methods Fresh tumor samples from 60 patients with gastric carcinoma were analyzed by bivariate BrdUrd/DNA flow cytometry. The results were correlated with lymphatic vessel invasion, lymphatic node metastasis, the number of matastatic lymphatic nodes, and venous invasion. Propidium iodide (PI) was used as a fluorescent probe for total cellular DNA, and a monoclonal antibody against BrdUrd was used as a probe for BrdUrd incorporated into DNA. Fluorescent-labeled goat anti-mouse antibody was used as a second antibody. S-phase fractions were measured by in vitro BrdUrd labeling, and DNA ploidy and G2/MPF were also measured. Comparison of survival was performed with the log-rank test, the Chi-square test for qualitative data, and Student’s t test for quantu data. Results BrdUrd LI and G2/MPF values were significantly higher in tumors with lymphatic vessel invasion than in those without invasion respectively (P&lt;0.01); the patients who had tumors with lymphatic vessel invasion showed a significantly poor prognosis (P&lt;0.01). Both BrdUrd LI and G2/MPF values were significantly higher in tumors with lymphatic node metastasis than in those without metastasis (P&lt;0.01). A statistical significant difference was noted in the 5-year survival rates between the patients with lymph node metastasis and those without metastasis. Compared with diploid carcinoma, the incidence of lymph node metastasis was significantly higher in aneuploid carcinoma (P&lt;0.05), and the patients with aneuploid carcinoma showed a significantly poor prognosis (P&lt;0.05). BrdUrd LI was significantly higher in patients with more than 5 metastatic lymph nodes than those with 1-4 metastatic lymph nodes (P&lt;0.05) and those without metastasis (P&lt;0.01). G2/MPF values in those patients either with more than 5 metastatic lymph nodes or 1-4 metastatic lymph nodes were higher than those without metastasis (P&lt;0.01 and P&lt;0.05). A statistical significance was seen in the 5-year survival rates among the patients with no metastatic lymph node, 1-4 metastatic nodes and more than 5 metastatic nodes (P&lt;0.01). G2/MPF values were significantly higher in patients with venous invasion than in those without invasion (P&lt;0.01).Conclusions Positive correlations exist between cellular BrdUrd LI, G2/MPF with lymphatic involvement and prognosis, and DNA aneuploid with lymphatic involvement and prognosis. The same was true between G2/MPF value and venous invasion in gastric carcinoma.     

Investigation of tumor necrosis factor receptor-p55 expression in human colorectal cancer

Objective: To investigate the clinical significance of tumor necrosis factor receptor-p55 (TNFR-p55) expression and its relationship with the clinical pathology and Dukes‘ classification of human colorectal cancer. Methods: SABC immunohistochemistry method was used to examine TNFR-p55 expression in 91 specimens of colorectal cancer, 81 surrounding mucosas of the tumors and 13 normal tissues. Results: TNFR-p55 expression in colorectal cancer was significantly higher than that in the surrounding mucosa andnormal tissues, and it was significantly higher in the surrounding mucosa than in normal tissue. TNFR-p55 was inversely correlated to serous membrane invasion and lymph node metastasis of colorectal cancers.TNFR-p55 expression in Dukes‘ A and B stages was significantly higher than that of C stage. Conclusion: TNFR-p55 expression was important to determine the degree of malignancy and assess invasion depth, lymph node metastasis and Dukes‘ classification in colorectal cancer.     

Silencing MTA1 by RNAi Reverses Adhesion,Migration and Invasiveness of Cervical Cancer Cells （SiHa） via Altered Expression of p53, and E-cadherin/β-catenin Complex

It has been reported that metastasis-associated gene 1 (Mta1) is overexpressed in many malignant tumors with high metastatic potential. In addition, some studies indicated that MTA1 participated in invasion, metastasis, and survival of cancer cells by regulating cell migration, adhesion and proliferation. But the role of MTA1 is unclear in vitro in the development of cervical cancer cells. This study investigated whether and how MTA1 mediated cell proliferation, migration, invasion and adhesion in cervical cancer. MTA1 expression level was detected by Western blot in two cervical cancer cell lines of different invasion potentials. The effects of MTA1 expression on SiHa cell apoptosis, cycle, proliferation, migration, invasion and adhesion were tested by flow cytometry, MTT, wound-healing assay, Transwell assay and adhesion assay, respectively. The expression levels of p53, E-cadherin, and β-catenin activity were evaluated in untreated and treated cells. The results showed that MTA1 protein expression was significantly higher in SiHa than in HeLa, which was correlated well with the potential of migration and invasion in both cell lines. Furthermore, the cell invasion, migration and adhesion capabilities were decreased after inhibition of MTA1 expression mediated by Mta1-siRNA transfection in SiHa. However, no significant differences were found in cell apoptosis, cycle, and proliferation. In addition, E-cadherin and p53 protein levels were significantly up-regulated, while β-catenin was significantly down-regulated in SiHa transfected with the siRNA. These results demonstrated that MTA1 played an important role in the migration and invasion of cervical cancer cells. It was speculated that the decreased migration and invasion capability by inhibiting the MTA1 expression in the SiHa cell line may be mediated through the altered expression of p53, and E-cadherin/β-catenin complex. MTA1 could serve as a potential therapeutic target in cervical cancer.     

Inactivation of Smad4 is A Prognostic Factor of Intrahepatic Cholangiocarcinoma

Objective: To study the expression of Smad4 in intrahepatic cholangiocarcinoma and its relationship with the biological behaviors and prognosis of the disease. Methods: Forty-nine paraffin-embedded intrahepatic cholangiocarcinoma specimens and nine normal liver tissues were analyzed by immunohistochemical methods using Smad4 monoclonal antibodies. The expression of Smad4 was compared with the clinical pathological characteristics of the patients. Results: The expression of Smad4 was 100% positive in normal liver tissues, which was higher than that in the ICC (44.9%). Negative labeling of the Smad4 protein was found in 26.1% (6/23) of well differentiated ICC and 61.5% (16/26) of poorly to moderately differentiated ICC. 34.3% (12/35) and 71.4% (10/14) showed negative Smad4 labeling (P=0.018) of ICC at pTNM stageⅠ-Ⅱand stageⅢ-Ⅳ, separately. Furthermore, there were 72% (8/11) lymph node metastatic ICC and 73.3% (11/15) intrahepatic metastatic ICC showed negative labeling of the Smad4 protein. The loss of Smad4 expression in those metastatic ICCs was significant severe compared to non- metastatic ICCs (P=0.000). Conclusion: The expression of Smad4 was associated with the histological grade, clinical stage and metastasis of ICC (P < 0.05). Detection of Smad4 may be helpful in the determination of the malignant degree and the prognosis of ICC.