VEGF expression in the colorectal adenoma-carcinoma sequence

Angiogenesis is essential for tumor growth and metastasis. It is controlled by multiple factors, one of the most important being vascular endothelial growth factor (VEGF). VEGF and p53 expression were evaluated in 16 hyperplastic polyps, 35 solitary tubular and tubulovillous adenomas, and 47 cases of sporadic colorectal carcinomas arising on the basis of preexisting adenomas, using immunohistochemistry. In parallel, angiogenesis was assessed by the Chalkley score (CS) method. VEGF positivity was detected in 19/47 carcinoma cases (40%). In the respective adenomatous part of the tumor, VEGF positivity was detected in 11/47 cases (23%). Carcinomas arising from VEGF-positive adenomas were mostly VEGF positive (10/11, 91%), whereas in 28/36 (78%) carcinomas arising from VEGF-negative adenomas VEGF expression was not detected. CS was higher in VEGF-positive compared with VEGF-negative carcinomas (9.1 +/- 1.8 and 7.8 +/- 2.3, respectively, p < 0.05), whereas there was no statistically significant difference between the CS in the VEGF-negative and VEGF-positive adenomatous part of the tumor (3.3 +/- 1.8 and 4.3 +/- 2.3, respectively). Nuclear p53 positivity was detected in 26/47 (55%) cases in the cancerous part and in 14/47 (29%) cases in the adenomatous part of the tissue, and no significant correlation with VEGF expression was observed. We conclude that VEGF associates with angiogenesis in colorectal cancer, and its pattern of expression in adenomas is maintained in the arising carcinomas. Further investigation is warranted to clarify whether these findings could be used as indicators of prognosis in screening programs or in patients with limited stage disease where the usefulness of adjuvant therapies with either cytotoxic drugs or inhibitors of angiogenesis is still unclear.     

结直肠肿瘤微血管计数及血管内皮细胞生长因子表达

目的 探讨血管生成与结直肠肿瘤的发生,发展关系,评估微血管计数（ＭＶＤ值）及血管内皮细胞生长因子（ＶＥＧＦ）表达与结直肠肿瘤预后的相关性。方法 应用免疫组化法回顾性地研究了３２例结直肠肿瘤石蜡包埋的病理组织。结果 正常粘膜,腺瘤,癌组织的ＭＶＤ值递增。不同病理状态下的结直肠癌ＭＶＤ值有差异,ＶＥＧＦ阴性组ＭＶＤ值低于ＶＥＧＦ阳性组,低ＭＶＤ值主ＶＥＧＦ阴性组生存率高于高ＭＶＤ值组及ＶＥＧＦ阳性组。     

Concurrent Expression of Angiogenic Growth Factors and Neovascularization during Tumourigenesis in Colorectal Carcinoma Patients

Details of concurrent expression of angiogenic growth factors (AGFs) and microvessel density (MVD) in human colorectal adenomas and carcinomas remain obscure. Eighty lesions, 20 each from colorectal adenoma, Tis, T1 and T2 cancers were evaluated immunohistochemically for basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), thymidine phosphorylase (dThdPase) and MVD. MVD (p = 0.0001) and bFGF expression (p = 0.0001) increased in the order of adenoma, Tis, T1 and T2 cancers. VEGF expression was same in adenomas and cancers while dThdPase was weak in adenomas but strongly expressed in invasive carcinomas ( > T1). MVD had positive correlation with tumour size in small (< or = 4 cm) colorectal cancers. Concurrent expression of AGFs was noticed in invasive carcinomas. bFGF seems to be the strongest among the three AGFs expressed during colorectal carcinogenesis and had a significant correlation with tumour MVD. Concurrent expression of multiple AGFs is a crucial step in the transition from non-invasive to invasive carcinoma.     

Expression of survivin correlates with apoptosis, proliferation, and angiogenesis during human colorectal tumorigenesis

BACKGROUND: To identify the role of survivin, a novel inhibitor of apoptosis (IAP) in colorectal tumorigenesis, the authors investigated tissue expression of survivin in human colorectal tumors including 43 hyperplastic polyps, 171 adenomas with low dysplasia, 42 adenomas with high dysplasia, and 60 carcinomas in adenoma, and examined whether the expression of survivin correlated with tumor cell apoptosis, proliferation, and angiogenesis, which is known to initiate the imbalance between cell proliferation and apoptosis. METHODS: Immunohistochemical staining for the paraffin sections by using the monoclonal antibodies, survivin, p53, bcl-2, Ki-67, and CD34, was performed by the standard avidin-biotin-peroxidase technique. The apoptotic cells were detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method, using an Apop Tag in situ detection kit. RESULTS: The immunoreactivity of survivin significantly increased in the transition from adenoma with low dysplasia to high dysplasia/carcinoma (P < 0.001). Similar changes in protein expression were observed for p53 but not for bcl-2, which was expressed throughout the colorectal tumorigenesis. This transition was associated with a significant decrease in the apoptotic index (AI) and significant increases in the Ki-67 labeling index (LI) and microvessel density (MVD; P < 0.001 for both). The expression of survivin inversely correlated with AI and was positively correlated with Ki-67 LI and MVD (P < 0.001 for both). CONCLUSIONS: These results suggest that, like p53, survivin plays an important role in transition from adenoma with low dysplasia to high dysplasia during human colorectal tumorigenesis.     

Fhit, Mlh1, P53 and phenotypic expression in the early stage of colorectal neoplasms

There are two different pathways for the development of colorectal carcinoma (CRC), adenoma-carcinoma sequence (ACS) and de novo (DN) carcinogenesis. To clarify the molecular and clinicopathological characteristics in colorectal carcinogenesis, we examined endoscopically resected specimens of 30 adenomas, 30 carcinoma in adenomas (CIAs), and 18 early pure colorectal carcinomas without any adenoma component (EPCs, so called DN carcinoma) and compared the expression of Fhit, Mlh1, Msh2, P53 and cellular phenotype (HGM, MUC2 and CD10). Markedly reduced or absent Fhit expression was noted in 8 (44%) of 18 EPCs, but none of the adenomas or CIAs (p<0.0001). Six (33%) of 18 EPCs showed loss of Mlh1 expression, but rarely in adenomas and CIAs (p=0.008). This altered Fhit expression was significantly higher in submucosal invasive cancers (p=0.001), lymphatic or venous invasive cancers (p=0.0018), and tumors with altered expression of Mlh1 (p=0.01). The incidence of P53 overexpression was significantly higher in EPCs (39%) and CIAs (27%) than in adenomas (3.3%) (p<0.05). There were significant differences in phenotypic expression between the adenomatous and carcinomatous areas. Moreover, in CIAs and EPCs, the rate of P53 overexpression was significantly higher in the CD10-positive cases (53%) than CD10-negative cases (19%) (p=0.04). The present findings suggested that aberrant Fhit and Mlh1 expression could be related to DN carcinogenesis and that P53 overexpression and changes in phenotypic expression could contribute to the malignant transformation of colorectal precursor lesions.     

Prognostic significance of p53 overexpression in gastric and colorectal carcinoma.

p53 expression was examined in 55 gastric and 107 colorectal carcinomas with an immunoperoxidase technique, using the polyclonal antibody CM1 on routinely fixed, paraffin embedded tissue. p53 protein was detected in 47% gastric and in 46% colorectal carcinomas and found to correlate with stage of disease and unfavourable clinical outcome (P less than 0.001). Thus, the proportion of positively reacting neoplasms increased as the stage progressed, tumours which had invaded regional lymph-nodes overexpressed p53 more frequently than localised carcinomas and an elevated level of p53 was associated with early relapse and death. In colorectal carcinoma p53 positivity was also linked with site and macroscopic configuration of the primary tumour and was most frequently expressed in carcinomas from the rectum and in ulcerative tumours. p53 overexpression was irrespective of tumour grade. Uniform negative reactivity with anti-p53 antibody was seen in normal epithelium adjacent to carcinoma, intestinal metaplasia, atrophic gastritis and in colonic adenomas. There was a good correlation between immunohistochemical staining on paraffin and frozen sections. These studies suggest that in gastric and colorectal carcinoma, immunohistochemical detection of p53 protein in routinely fixed tissue can be used along with other established parameters to assess prognostic outcome, especially to identify patients with poor short-term prognosis.     

Correlation of p53 and bcl-2 expression with vascular endothelial growth factor (VEGF), microvessel density (MVD) and clinico-pathological features in colon cancer

This study was designed to elucidate the possible relationship between tumour related genes and angiogenesis in colon cancer. The protein expression of p53, bcl-2, Von Willebrand factor and vascular endothelial growth factor (VEGF) were analysed by immunohistochemistry in 57 paraffin-embedded colon cancer. The results showed that microvessel density (MVD) was lower in VEGF negative tumours than in VEGF positive ones (P<0.0001). MVD and VEGF in p53 negative tumours were significantly lower than in p53 positive tumours (respectively, P=0.003 and P<0.0001). Moreover, positive correlations were recorded between VEGF expression and MVD, and bcl-2 expression (respectively, P<0.0001 and P=0.009). Our data confirm the central role of VEGF in angiogenesis and suggest direct correlations among p53, bcl-2 and VEGF expression in colon cancer.     

p53、血管内皮生长因子在大肠癌组织中的表达与血管生成

目的　探讨p53、血管内皮生长因子 (VEGF)在大肠癌组织中的表达及其与血管生成的关系。方法　利用免疫组化SABC法 ,对 1 0 6例大肠癌组织及 2 0例正常大肠组织中的 p53、VEGF的表达及微血管密度 (MVD)进行研究。 结果　p53、VEGF的表达与肿瘤的分化程度及Dukes分期无明显相关性 (P >0 .0 5 )。p53表达阳性或VEGF表达阳性的大肠癌组织MVD明显高于p53表达阴性 (P <0 .0 1 )或VEGF表达阴性者 (P <0 .0 1 )。p53表达阳性的大肠癌组织中VEGF的表达阳性率显著高于 p53表达阴性者 (P <0 .0 1 )。结论　p53、VEGF在大肠癌的发生和发展中起着重要作用 ,可反映大肠癌的恶性程度和进展情况并作为预后的指标 ,p53作用的发挥是通过上调VEGF的表达水平来实现的     

Intratumoral angiogenesis: a new prognostic indicator for stage I endometrial adenocarcinomas?

The prognostic significance of three recently emerged parameters, namely intratumoral angiogenesis and the antiapoptotic proteins bcl-2 and mutant p53, was investigated in a series of 124 patients with endometrial adenocarcinomas of the endometrioid cell type. All patients were treated with total abdominal hysterectomy and bilateral oophorectomy, without node dissection. When deep myometrial invasion or advanced stage of disease was confirmed, adjuvant radiotherapy was given. Intratumoral angiogenesis was assessed in tissue samples, after immunohistochemical staining, with the anti-CD31 monoclonal antibody. The mean microvessel density (MVD) was 23.2 +/- 14.1 (range 4-60; 95% CI 20-25.8). Microvessel density was high (> 30) in 30% of endometrial adenocarcinomas, medium (15-30) in 33% of the tumors, and low (< 15) in the remaining cases (37%). A strong cytoplasmic and/or perinuclear expression of bcl-2 in more than 10% of the neoplastic cells was considered as being positive, and noted in 35.5% of the endometrial neoplasms; it was more frequent in the less vascularized carcinomas (P = 0.03). Nuclear p53 accumulation in an equal percentage of neoplastic cells (> 10%) was less common (7.2%). In univariate analysis, early stage of disease, absence of lymphatic-vascular space invasion (LVI), and low intratumoral MVD were the parameters associated with an improved survival (P = 0.0001, P = 0.001, and P = 0.009, respectively). In multivariate analysis, however, the only independent variable noted was stage of disease (P < 0.0001). Within stage I endometrial adenocarcinomas, only intratumoral angiogenesis was associated with prognosis (univariate analysis): high MVD cases had a significantly worse prognosis compared to medium MVD (P = 0.02). Low MVD adenocarcinomas, on the other hand, were associated with an intermediate prognosis, indicating that other factors, such as hypoxia and related mechanisms, may also be important. It is suggested that intratumoral angiogenesis may prove useful in selecting a subgroup of cancer patients, among others with stage I endometrial disease, that would benefit from additional treatment.     

Association of Bcl-2 protein expression with gallbladder carcinoma differentiation and progression and its relation to apoptosis

BACKGROUND: Bcl-2 protein is believed to play a role in neoplasia by inhibiting tumor cell apoptosis. To assess its contribution to gallbladder tumorigenesis and cancer progression, an immunohistochemical study was performed. METHODS: Fifteen adenomas and 68 adenocarcinomas were immunohistochemically and histopathologically investigated for the relation of Bcl-2 expression to p53 status, apoptosis (apoptotic index, AI), and proliferation activity (mitotic index, MI; Ki-67 labeling index, Ki-67 LI). RESULTS: The Bcl-2 score, based on intensity and extent, decreased in the order of adenoma, well-differentiated, and moderately to poorly differentiated adenocarcinoma. Early stage carcinomas demonstrated significantly higher Bcl-2 scores than their advanced counterparts (P < 0.05). On the other hand, p53 score, MI, Ki-67 LI, and AI increased in the same order. The Bcl-2 negative adenocarcinomas displayed higher AI and AI-to-MI ratios than the Bcl-2 positive group, especially in the early stage, well-differentiated lesions. A significantly positive correlation between MI(r=0.549) or Ki-67 LI(r = 0.446) and AI was observed. In early stage carcinomas, adenomatous components in the lesions were found more frequently in the polypoid lesions than in the nonpolypoid lesions (P < 0.05). CONCLUSIONS: Expression of Bcl-2 protein in gallbladder tumors appears to be positively associated with tumor cell differentiation and inversely with tumor progression. It may thus play a role in regulating carcinoma growth, especially in the early stage of tumorigenesis. It is believed that the polypoid carcinomas may arise from preexisting adenomas but the nonpolypoid carcinomas may arise as de novo carcinoma.     

In oesophageal squamous cell carcinoma vascular endothelial growth factor is associated with p53 mutation, advanced stage and poor prognosis.

Vascular endothelial growth factor (VEGF) affects malignant tumours by promoting angiogenesis. The tumour-suppressor gene p53 has been thought to regulate VEGF. We investigated the effect of VEGF on oesophageal carcinoma and the connection between VEGF and p53. One hundred and nine resected oesophageal squamous cell carcinomas were examined. VEGF expression was analysed by immunohistochemical staining. Sixty-five tumours (59.6%, 65 out of 109) were classified as VEGF positive. A significant correlation was found between the VEGF expression and both the depth of invasion (P = 0.0001) and lymph node metastasis (P < 0.0001). With regard to p53, we compared the expression of VEGF with the mutation of p53, examined using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct sequencing in tumour samples obtained from 36 patients who we have reported previously. The VEGF expression was significantly correlated to p53 mutation (P = 0.0291). To evaluate the angiogenesis, microvascular density (MVD) was counted, and endothelial cells were stained immunohistochemically using anti-CD34 monoclonal antibody against 29 cases with invasion limited to the submucosal layer. The average MVD had a tendency to correlate to VEGF expression (P = 0.1626). The prognoses of patients with VEGF-positive primary tumours were significantly worse than for those with VEGF-negative primary tumours (P = 0.0077). We have assumed that VEGF contributes to aggressive characteristics in oesophageal carcinomas and that VEGF expression might be affected by p53 status.     

Expression and prognostic significance of angiopoietin in colorectal carcinoma

BACKGROUND AND OBJECTIVES: Growth and metastasis of malignant tumors depend on the angiogenesis. The aim of this study was to elucidate the prognostic significance of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) expression in advanced colorectal carcinoma. METHODS: Totally, 101 patients with surgically resected advanced colorectal carcinomas were enrolled. The tumor expressions of Ang-1 and Ang-2 were evaluated immunohistochemically, and their relationships with clinicopathological factors and prognosis were investigated. Tumor microvessel density (MVD) was also calculated and correlated with angiopoietin expression. RESULTS: Ang-1 and Ang-2 were detected in 26 (25.7%) and 45 (44.6%), respectively, of 101 cancerous lesions. Overexpression of Ang-1 was correlated with high MVD. Overexpression of Ang-2 was correlated with lymph node metastasis, venous invasion, preoperative carcinoembryonic antigen levels, and high MVD (P < or = 0.05). MVD was not significantly upregulated by Ang-1 expression, but was significantly upregulated by Ang-2 expression (P < or = 0.01). However, only patients with Ang-2 overexpression showed a significantly worse prognosis than those without Ang-2 overexpression. Multivariate analysis with logistic regression for 5-year survival revealed that cancerous stage and Ang-2 overexpression were independent prognostic indicators. CONCLUSIONS: The Ang-1 expression correlated with MVD. However, Ang-2 expression was a useful prognostic marker in the management of patients with advanced colorectal carcinoma.     

Immunohistochemical expression of fatty acid synthase, apoptotic-regulating genes, proliferating factors, and ras protein product in colorectal adenomas, carcinomas, and adjacent nonneoplastic mucosa.

The normal mucosa-adenoma-carcinoma sequence in colon pathology provides an attractive model of tumor progression. The role of tumor suppressor genes, oncogenes, and proliferative markers in tumorogenesis has evolved considerably in the last decade. By immunohistochemistry means, we have studied p53, bcl-2, c-myc, p21-ras, ki67, and fatty acid synthase (a fatty-acid-synthesizing enzyme) in normal, dysplastic, and neoplastic mucosa. The results were correlated with clinicopathological features and overall survival (OS). Formalin-fixed, paraffin-embedded archival material from 100 nonconsecutive adenomas and 100 adenocarcinomas (ADCs), including adjacent-to-tumor nonneoplastic mucosa (ANNM), from patients with a 5-year follow-up period were studied. Negative controls were obtained from colon resections for nonneoplastic disease. Fatty acid synthase was associated with ADC (P = 0.0001). p53 protein was associated with high-grade dysplasia adenoma (AHGD), ADC (P = 0.0001), and pT stage (P = 0.003). bcl-2 was associated with adenomas with low-grade dysplasia (P = 0.009); c-myc was associated with ANNM (P = 0.005) and pT stage (P = 0.006). p21-ras was associated with AHGD (P = 0.0001) and ANNM (P = 0.01). Ki67 was associated with AHGD (P = 0.02) and ADC (P = 0.0001). Univariate analysis on neoplastic tissue revealed histological grade, pT stage, pN stage, p21-ras, and p53 to be significant markers of OS; p21-ras, p53, and c-myc were reliable markers when evaluated on ANNM. Multivariate analysis revealed pT stage, pN stage, and p21-ras to be independent prognosticators of OS on ADC; p21-ras and c-myc staining in the ANNM were correlated with worse survival (OS). We suggest that the evaluation in concert of clinicopathological data and immunohistochemical markers on both normal and abnormal colon tissue provides an attractive model of tumor progression; moreover, it may give important messages about the prediction of survival.     

p53 gene mutations occur in combination with 17p allelic deletions as late events in colorectal tumorigenesis

Coordinate loss of one copy of the p53 gene and mutation of the remaining copy occur in colorectal carcinomas and in many other human malignancies. However, the prevalence of p53 gene mutations in carcinomas which maintain both parental copies of p53 has not previously been evaluated. Moreover, it is not known whether p53 gene mutations are limited to malignant tumors or whether they can also occur in benign neoplasms. To answer these questions, a total of 58 colorectal tumors have been examined; in each tumor, allelic losses were assessed using restriction fragment length polymorphisms and p53 gene mutations were assessed by sequencing cloned polymerase chain reaction products. The following conclusions emerged: (a) p53 gene mutations occurred but were relatively rare in adenomas, regardless of size and whether the adenomas were derived from patients with familial adenomatous polyposis; (b) In carcinomas as well as in adenomas, p53 gene mutations were infrequently observed in tumors which contain both copies of chromosome 17p (17% of 30 tumors), while tumors which lost one copy of chromosome 17p usually had a mutation in the remaining p53 allele (86% of 28 tumors); (c) p53 gene mutations were found at similar frequencies in primary tumor samples and in cell lines derived from tumors. These and other data suggest that the rate limiting step in p53 inactivation is point mutation and that once a mutation occurs, loss of the remaining wild-type allele rapidly follows. Both mutations and allelic losses generally occur near the transition from benign to malignant growth, and the p53 gene may play a causal role in this progression.     

大肠腺瘤和腺癌组织中缺氧诱导因子-1α的表达及其与VEGF、微血管密度的关系

背景与目的:缺氧诱导因子-1α(hypoxia-inducible factor 1 alpha,HIF-1α)是肿瘤细胞适应缺氧而产生的一种核转录因子,在促进肿瘤新生血管生成中起重要作用。本研究旨在探讨大肠腺瘤和腺癌组织中HIF-1α的表达及其与血管内皮生长因子(vascular endothelial growth factor,VEGF)、微血管密度(microvessel density,MVD)的关系。方法:采用原位杂交技术检测HIF-1α mRNA,应用免疫组织化学方法检测VEGF蛋白的表达,用CD34单克隆抗体标记血管内皮细胞并计数MVD。结果:大肠腺癌组织HIF-1α mRNA阳性表达率为67.8％(42/62),腺瘤组织为44.4％(8/18)。腺癌组织从Dukes’A期到Dukes’C+D期HIF-lα mRNA表达阳性率不断增加(P<0.05)。HIF-1α mRNA表达平均阳性率为:腺瘤44.4％;腺癌Dukes’A期41.2％,Dukes’B期72.2％,Dukes’C+D期81.5％。腺癌组VEGF阳性表达率高于腺瘤组(59.7％ vs 33.3％,P<0.005).HIF-1α表达与VEGF呈正相关(r_s=0.768,P<0.01),与MVD呈正相关(r_s=0.683,P<0.05)。结论:HIF-1α及其靶基因 VEGF的过度表达与肿瘤新生血管形成呈正相关,这在大肠腺瘤癌变及大肠腺癌发展过程中可能起重要作用。     

胸苷磷酸化酶在癌组织中表达的研究

目的　研究胸苷磷酸化酶 (TP)在不同种类癌组织中的表达 ,探讨TP与癌组织血管生成的关系。方法　采用免疫组织化学方法检测 2 5 1例癌组织和相对应的 92例正常组织TP和微血管密度 (MVD)的表达。癌组织包括 :胃癌 4 8例 ,大肠癌 5 3例 ,乳腺癌 4 7例 ,宫颈癌 5 6例 ,肺癌 4 7例 ;正常组织包括 :胃 2 5例 ,大肠 2 5例 ,宫颈 17例 ,肺 2 5例。分析癌组织和正常组织TP表达差异 ,及癌组织TP表达与癌组织MVD的关系。结果　胃癌、大肠癌、乳腺癌、宫颈癌和肺癌的TP表达阳性率分别为6 4 .6 %、6 7.9%、80 .9%、82 .1%和 6 3.8%。癌组织的TP表达阳性率显著高于正常组织 (P =0 .0 0 0 0 )。癌组织TP表达与胃癌、大肠癌、乳腺癌、宫颈癌的MVD值关系密切 (P <0 .0 0 1)。结论　TP在胃癌、大肠癌、乳腺癌、宫颈癌和肺癌组织中过度表达 ,并对胃癌、大肠癌、乳腺癌和宫颈癌的血管生成有明显促进作用。     

p73 Overexpression and angiogenesis in human colorectal carcinoma

BACKGROUND: Solid tumors requires neovascularization for growth and metastasis. Angiogenesis depends on the local balance between positive and negative effectors, the production of which can be regulated by oncogenes and tumor suppressor genes. The aim of this study was to investigate expression of p73, a gene homologous to the tumor suppressor gene p53, in colorectal cancer and its relationship to angiogenesis. METHODS: p73 expression was examined by immunohistochemistry and western blot analysis on 56 primary colon carcinomas with matched normal mucosas. Vascular endothelial growth factor (VEGF) and microvessels were highlighted using a monoclonal antibody specific to VEGF and von Willebrand factor (vWF). RESULTS: The immunoexpressions of p73 were significantly higher in the primary colorectal carcinomas than that in the matched normal mucosa (P < 0.001). Western blotting showed that 85% patients have a high level of p73 expression (more than double the normal level). A close association between p73 and VEGF expression level was observed (P = 0.016). Colorectal adenocarcinoma that expressed p73 showed significantly greater vascularity than p73-negative tumors (P = 0.012). However, no association between immunoexpression of p73 and tumor stage or differentiation was observed. CONCLUSION: These findings suggest a potential role of p73 in tumor angiogenesis.