Gene expression profiling of colorectal adenomas and early invasive carcinomas by cDNA array analysis

It is generally accepted that most colorectal carcinomas arise in pre-existing adenomas. Morphologically, colorectal adenomas can be divided into two groups, protruded type and flat type. The aim of this study was to clarify relevant alterations of gene expression associated with the early stage of colorectal carcinogenesis. Using cDNA array, we analysed the expression profiles of 550 cancer-related genes in 36 colorectal adenomas (18 flat-type and 18 protruded-type adenomas) and 14 early invasive carcinomas. Among the 550 genes, we chose 32 genes the average expression levels of which were at least three-fold up- or downregulated in tumour tissues compared with levels in matched normal tissues. A total of 13 and 19 genes were identified as up- and downregulated genes in tumour tissues, respectively. Among the upregulated genes, the average expression levels of E1AF, bone morphogenic protein (BMP)-4, insulin-like growth factor (IGF)-2, inducible nitric oxide synthase (iNOS), tissue inhibitors of metalloproteinase (TIMP)-1, Smad4, and nm23 in tumour tissues were over five times higher than those in matched normal tissues. Colorectal adenomas and early invasive carcinomas were divided into two major clusters by clustering analysis. Moreover, flat- and protruded-type adenomas were divided into two major clusters by clustering analysis. The expression profiles obtained by the cDNA array clearly indicate that colorectal adenomas and early invasive carcinomas have specific expression profiles. Likewise, the gene expression profiles of flat- and protruded-type adenomas are different. These results indicate that molecular classification of early colorectal tumours by a cDNA array is feasible.     

Ezrin在散发性结直肠癌中的表达及意义

Objective： The membrane-linking protein Ezrin is highly expressed in several types of human cancers. The correlations between its immunoreactivity and histopathological data as well as patient outcome have previously been shown. However, the role played by Ezrin in the carcinogenesis, progression and metastasis of primary sporadic colorectal carcinoma （SCRC） is still under investigation. This study assessed Ezrin protein expression in a series of clinical specimens. Methods： Immunohistochemical analysis was used to characterize patterns of Ezrin expression in 132 cases of SCRC, including 74 metastatic cases and 58 non-metastatic cases and 43 adjacent normal colorectal mucosa. Results： （1） The expression rate of Ezrin in SCRC （79.5%） was significantly higher than in adjacent normal colorectal mucosa （11.6%） （P 〈 0.001）; （2） The total expression rate of Ezrin was 86.5% and 70.7% in metastatic group and non-metastatic group, respectively （P = 0.026）; the membrane expression rate of Ezrin was 31.1% and 6.9% in the two groups, respectively （P 〈 0.01）; （3） There was no relationship between the expression of Ezrin with age, gender, tumor size, location, degree of differentiation and invasive depth; （4） In the cases with followed-up data, univariate analysis demonstrated that Ezrin expression and its membrane translocation was correlated with worse patient＇s disease-free survival （DFS） （Puni 〈 0.05）. Conclusion： Ezrin was expressed in the majority of SCRC and associated with adverse prognostic factors. The increase expression and the switch of Ezrin localization from the cytoplasm to the membrane were closely correlated with metastasis in SCRC. It might be served as an important parameter for determining tumor biological behavior.     

Correlation of c-MET expression with clinical characteristics and the prognosis of colorectal cancer

BackgroundThe proto-oncogene c-MET (mesenchymal-epithelial transition factor gene) plays a critical role in cellular proliferation, survival, migration, and invasion in cancers. The aim of this study is to explore the relationship between c-MET expression and the clinicopathological characteristics of colorectal cancer (CRC) patients.MethodsA total of 337 enrolled patients were collected in present study. Here, the c-MET and EGFR expression were detected by immunohistochemistry (IHC). The mutational statuses of KRAS in exons 2, 3, and 4, NRAS in exons 2, 3, and 4, and BRAF in exon 15 from formalin-fixed sections were detected by direct DNA sequencing.ResultsOur results showed that high c-MET expression was significantly associated with tumor perineural invasion (P=0.007) and gender (P=0.016). High level c-MET expression (c-MET-high) in the primary tumors was observed in 68.2% of patients. In the 337 enrolled patients, 43.2% of patients had KRAS mutations, 3.3% of patients had NRAS mutations, and 4.7% of patients had BRAF mutations. However, KRAS, NRAS, and BRAF gene mutations had no association with c-MET protein levels in primary tumors. Additionally, c-MET protein expression had a strong correlation with EGFR expression (P=0.002). The survival time was not significantly longer for patients with c-MET-high primary tumors than for those with c-MET-low primary tumors.Conclusionsc-MET immunohistochemistry was significantly higher in primary tumors with perineural invasion, female gender, and EGFR high expression. However, c-MET-high in the primary tumors was not significantly associated with longer survival compared with c-MET-low tumors. Further studies are required to investigate c-MET as potential molecular marker of progression and to test the possibility of its incorporation as a new therapeutic target.     

Matrilysin gene expression in sporadic and familial colorectal adenomas

We examined the expression of matrilysin mRNA in sporadic and hereditary colorectal adenomas to clarify the role of matrilysin in tumorigenesis. Matrilysin mRNA was not detected in normal colorectal mucosa from patients with either sporadic or familial adenomas. Matrilysin mRNA expression in sporadic adenomas correlated with the degree of dysplasia and the size of the mass, whereas most of the adenomas in patients with familial adenomatous polyposis coli expressed matrilysin mRNA irrespective of adenoma size or degree of dysplasia. Because matrilysin is more likely to be expressed in adenomas with a potential for malignancy, this enzyme may play a role in the malignant conversion of colorectal adenomas. Mol. Carcinog. 19:225–229, 1997. © 1997 Wiley-Liss, Inc.     

P27kip1在结直肠癌中的表达及临床意义

Objective To investigate the significance of p27 expression in colorectal carcinomas (CRCs). Methods The samples were obtained from 44 cases. P27 expression was detected by immunohistochemistry using the SABC staining method at the three sites: carcinoma tissue, pericarcinoma (1 cm away from CRC) and the incision margin (3 cm away from CRC). Results Expression of p27 was found in 56.82% (25/44) of CRCs, in 88.64% (39/44) of percarcinoma and and in 97.73% (43/44) of the mucosa at the incison margin. P27 expression in CRCs was correlated significantly with Dukes stage (P<0.05), histology grade (P<0.01) and local lymph node involvement (P<0.05), but not with age, gender, and site or size of the CRC. Conclusion The decrease of p27 expression may be involved in the malignant transformation of colorectal epithelial cells to CRC. Expression of p27 in CRC correlates with some clinicopathological characteristics and may be of prognostic significance.     

Ezrin在散发性结直肠癌中的表达及意义

背景与目的：细胞骨架和细胞浆膜的连接蛋白Ezrin（埃兹蛋白）被认为可能参与肿瘤的发生发展，是近年来肿瘤转移的研究热点，但Ezrin与结直肠癌发生发展的关系尚不明确。本研究探讨Ezrin在散发性结直肠癌中的表达情况及其临床病理意义。方法：应用EnVision免疫组织化学的方法检测132例散发性结直肠癌及43例癌旁正常肠粘膜中Ezrin的表达情况，并对其有无转移（转移组74例，未转移组58例）进行分组比较。结果：①Ezrin在结直肠癌中的阳性率（79．5％）显著高于癌旁正常肠粘膜的阳性率（11．6％）（P〈0．001）；②Ezrin总阳性率在转移组（86．5％）高于未转移组（70．7％）（P=0．026），其中在膜的表达率转移组（31．1％）显著高于未转移组（6．9％）（P〈0．001）；③Ezrin在结直肠癌中的表达与年龄、性别、肿瘤大小、发生部位、分化程度、浸润深度等临床病理参数均无关（P〉0．05）。结论：Ezrin的表达增高与结直肠癌转移密切相关，其从胞质表达到胞膜表达在肿瘤细胞转移过程中可能起着至关重要的作用，并可作为临床判断散发性结直肠癌转移及预后等生物学行为的重要参考指标。     

大肠腺瘤和腺癌组织中Survivin的表达及其意义

目的:研究Survivin在大肠腺瘤和腺癌组织中的表达及其意义。方法:SP法检测大肠腺瘤50例(管状腺瘤35例,绒毛状腺廇9例,混合腺廇6例)和大肠腺癌89例组织中Survivin表达情况,正常大肠黏膜组织20例作为对照。结果:在正常大肠黏膜组织、腺瘤和大肠腺癌中,Survivin的阳性表达率分别为10.0%(2/20)、26.0%(13/50)和75.3%(67/89);随腺瘤不典型增生程度增高,Survivin的表达也随之明显增高,P=0.019。Survivin的表达随大肠腺瘤分化程度的降低而升高[高、中和低分化组阳性表达率分别为65.0%(26/40)、86.0%(37/43)和100%(6/6)]。伴有淋巴结转移组大肠腺癌Survivin阳性表达率85.2%(52/61)明显高于无淋巴结转移组53.6%(15/28),P=0.003。在生存时间≥2年的大肠腺癌中,阳性表达率为73.7%(14/19),在<2年组中为64.7%(11/17),两者差异无统计学意义,P=0.41。结论:正常大肠黏膜、大肠腺瘤和大肠腺癌中Survivin的阳性表达逐步增高。随大肠腺瘤不典型增生程度增高,Survivin的表达也随之明显增高。Survivin的表达随大肠腺癌分化程度的降低而升高。伴有淋巴结转移组大肠腺癌Survivin表达的阳性率明显高于与无淋巴结转移组。     

Galectin-3蛋白表达与结直肠腺癌临床病理特征的关系

目的：探讨结直肠腺癌组织中Galectin-3蛋白表达及其与临床病理参数的关系。方法：采用微波-EliVisionTM免疫组化染色方法检测60例结直肠腺癌组织中Galectin-3的表达情况并分析其与结直肠腺癌浸润转移等的关系。结果：Galectin-3阳性表达主要在细胞质。60例结直肠腺癌组织中Galectin-3蛋白阳性表达率为68.3%（41/60）。在结直肠腺癌浆膜浸润、淋巴结转移和TNMⅢ＋Ⅳ期中明显高于无浆膜浸润、无淋巴结转移及TNMⅠ＋Ⅱ期（P〈0.05）,不同分化肿瘤之间也有显著差异（P〈0.05）。结论：Galectin-3蛋白的高表达与结直肠腺癌高侵袭能力、淋巴结转移等有一定相关性,可作为潜在预测大肠癌浸润转移的指标。     

144例结直肠癌肝转移原发灶切除术后临床病理特征和预后分析

目的:分析144例结直肠癌肝转移原发灶切除术后患者的临床病理特征和预后。方法:回顾性分析病理证实的144例结直肠癌肝转移原发灶切除术后患者的临床病理特征和预后,Kaplan-Meier法分析生存率,Log-rank检验比较组间生存差异。结果:144例结直肠癌肝转移组及其中77例结直肠癌同时性肝转移亚组的中位生存期分别为28个月和21个月,所有病例分析显示原发肿瘤分化程度、肝转移发生时间、肝转移灶数目及治疗方法与预后显著相关(P＜0.05);同时性肝转移亚组分析显示原发肿瘤分化程度、性别、TNM分期与预后显著相关(P＜0.05);两组病例分析显示年龄对患者总生存期的影响无统计学差异(P＞0.05)。结论:原发肿瘤低分化、同时性肝转移、多发肝转移及单纯化疗是结直肠癌肝转移原发灶术后患者独立预后不良因素。原发肿瘤低分化、男性及Ⅱ-Ⅲ期是结直肠癌同时性肝转移原发灶术后患者独立预后不良因素。年龄对结直肠癌肝转移原发灶切除术后患者总生存期的影响无统计学意义。     

CD44v6和survivin在大肠癌中的表达及其意义

目的：探讨大肠癌组织中CD44v6和survivin蛋白表达及其与临床病理参数的关系。方法：采用微波-LSAB免疫组化染色方法检测60例大肠癌组织中CD44v6和survivin的表达情况并分析其与大肠癌浸润转移的关系。结果：阳性表达物质CD44v6主要在细胞膜和细胞质,survivin主要在细胞质。60例大肠癌CD44v6和survivin蛋白阳性表达率分别为71.7%和65.0%。与肿瘤临床Dukes分期、浸润程度、淋巴结转移及术后复发密切相关（P〈0.05）。结论：检测CD44v6和survivin蛋白表达对了解大肠癌的生物学行为和判断预后有一定价值。     

The Notch pathway in ovarian carcinomas and adenomas

Elements of the Notch pathway regulate differentiation; we investigated the expression of such elements in epithelial ovarian tumours. A total of 32 ovarian tumour samples (17 adenocarcinomas, three borderline tumours, 12 adenomas), two human ovarian cancer (A2780, OVCAR3), and one ovarian surface (IOSE 144) cell lines were analysed. The expression of Notch pathway elements was assessed by RT-PCR, real-time PCR (Notch 1), and by immunoblots (Notch 1 extracellular domain (EC), HES1). The proliferation and colony formation of A2780 cells were measured after stable transfection with activated Notch 1 (intracellular domain). Jagged 2, Delta-like-1, Manic Fringe, and TSL1 were expressed more frequently in adenocarcinomas whereas Deltex, Mastermind, and Radical Fringe were more frequent in adenomas. Quantitative PCR revealed decreased Notch 1 mRNA in ovarian adenocarcinomas compared with adenomas. The expression of Notch 1-EC protein was similar in benign and malignant tumours. HES1 protein was strongly expressed in 18/19 ovarian cancers and borderline tumours but not in adenomas. Transfecting A2780 cells with active Notch 1-IC resulted in a proliferative and colony formation advantage compared to mock transfected cells. Thus, Notch pathway elements are expressed in ovarian epithelial tumours and some of them are differentially expressed between adenomas and carcinomas. The Notch pathway could be a target for the development of therapies for ovarian cancer.     

Nuclear BAG-1 expression reflects malignant potential in colorectal carcinomas

BAG-1 is a recently identified Bcl-2-interacting anti-apoptotic protein. The aim of our study was to investigate the immunohistochemical staining pattern of BAG-1 protein in patients with colorectal cancer and examine associations of BAG-1 expression with various clinicopathological factors and patient survival. Tumour samples were collected from 86 patients diagnosed with colorectal cancer. There was significant variation in the immunohistochemical staining patterns for BAG-1, including absent staining and staining of either the cytoplasm, nucleus or both. Twenty-one colorectal carcinomas (24.4%) exhibited a nuclear staining pattern whilst 56 (65.1%) exhibited cytoplasmic staining. The percentage of cases exhibiting nuclear BAG-1 positivity was significantly higher in distant metastasis-positive cases (55.6%) than in distant metastasis-negative cases (20.8%; P=0.036). Overall survival was significantly shorter for patients with tumours exhibiting BAG-1 positive nuclei than those with absent nuclear BAG-1-staining (P=0.011). In addition, the multivariate cox proportional hazard models indicated that nuclear BAG-1 expression was the only independent prognostic variable for mortality (P=0.013). These studies demonstrate that nuclear BAG-1 expression is a useful predictive factor for distant metastasis and a poor prognosis in patients with colorectal cancer.     

Tumour-site-dependent expression profile of angiogenic factors in tumour-associated stroma of primary colorectal cancer and metastases

Background:

Tumour-associated stroma has a critical role in tumour proliferation. Our aim was to determine a specific protein expression profile of stromal angiogenic cytokines and matrix metalloproteinases (MMPs) to identify potential biomarkers or new therapy targets.

Methods:

Frozen tissue of primary colorectal cancer (n=25), liver (n=25) and lung metastases (n=23) was laser-microdissected to obtain tumour epithelial cells and adjacent tumour-associated stroma. Protein expression of nine angiogenic cytokines and eight MMPs was analysed using a multiplex-based protein assay.

Results:

We found a differential expression of several MMPs and angiogenic cytokines in tumour cells compared with adjacent tumour stroma. Cluster analysis displayed a tumour-site-dependent stromal expression of MMPs and angiogenic cytokines. Univariate analysis identified stromal MMP-2 and MMP-3 in primary colorectal cancer, stromal MMP-1, -2, -3 and Angiopoietin-2 in lung metastases and stromal MMP-12 and VEGF in liver metastases as prognostic markers (P>0.05, respectively). Furthermore, stroma-derived Angiopoietin-2 proved to be an independent prognostic marker in colorectal lung metastases.

Conclusion:

Expression of MMPs and angiogenic cytokines in tumour cells and adjacent tumour stroma is dependent on the tumour site. Stroma-derived MMPs and angiogenic cytokines may be useful prognostic biomarkers. These data can be helpful to identify new agents for a targeted therapy in patients with colorectal cancer.     

Correlations between cyclooxygenase-2 expression and angiogenic factors in primary tumors and liver metastases in colorectal cancer

BACKGROUND: Angiogenesis is required for growth and metastasis of colorectal cancer (CRC), and several positive regulators of tumor angiogenesis have been identified. Cyclooxygenase-2 (COX-2), known to be elevated in several human cancers, regulates angiogenesis by inducing angiogenic factors. The aim of this study was to clarify the levels and evaluate the relationships of COX-2, vascular endothelial growth factor A and C, thymidine phosphorylase (TP) and microvascular density (MVD) in paired tissue specimens between primary CRC and corresponding metastatic liver cancer. METHODS: Tissue samples from pairs of primary tumors and corresponding metastatic liver tumors from 44 patients with CRC were immunohistochemically evaluated for COX-2, VEGF-A, VEGF-C, TP and MVD. RESULTS: The primary and corresponding metastatic liver tumors tended to show concordant immunoreactivity for COX-2 (P = 0.005, rs = 0.428), VEGF-A (P = 0.039, rs = 0.314), TP (P = 0.005, rs = 0.422) and MVD (P = 0.046, rs = 0.304) by Spearman rank test. The rate of COX-2 immunoreactivity was higher in liver metastases than in primary tumors (P = 0.002), while the rate of VEGF-A was higher in primary tumors than in liver metastases (P = 0.0004). The incidence of TP immunoreactivity and the level of MVD did not differ between primary and metastatic liver tumors (P = 0.247; P = 0.229). Significant correlations were found between COX-2 immunoreactivity and VEGF-A immunoreactivity in metastatic liver tumors (P = 0.033) as well as in primary tumors (P = 0.008). CONCLUSION: The positive correlations between COX-2, VEGF-A, TP and MVD in primary CRC and liver metastasis as demonstrated here will help to predict the angiogenic activity of liver metastasis by analyzing primary tumors, allowing for individualized cancer treatment options.     

Clinical significance of mTOR and p-mTOR protein expression in human colorectal carcinomas

Aim: To investigate the significance of mammalian target of rapamycin (mTOR) and its active form, p-mTOR in colorectal carcinomas. Methods: Immunohistochemistry was used to detect the expression of mTOR and p-mTOR proteins in 108, 40 and 40 tissue samples from colorectal carcinoma, normal colonic mucosa and adenomatous polyps samples, respectively. The correlation of mTOR and p-mTOR expression with clinicopathological characteristics of colorectal carcinoma was analyzed. Results: The positive rates of mTOR and p-mTOR were significantly higher in colorectal carcinoma (61.1% and 61.1%, respectively, p<0.05) than in normal colonic mucosa (7.5% and 2.5%) and adenomatous polyps (27.5% and 20%). Overexpression of total mTOR protein was significantly associated with T1/T2 stage tumors, lymph node metastasis, distal metastasis) and degree of differentiation. p-mTOR overexpression was additionaly linked with degree of differentiation and TNM stage. Conclusion: The overexpression of mTOR and p-mTOR may play important roles in colorectal carcinogenesis with relations to the degree of differentiation, invasiveness and metastasis.