Angiogenesis and p53 expression in the colorectal adenoma-carcinoma sequence

Angiogenesis, the formation of new vessels, is essential for tumor growth and metastasis. Mutations of p53 tumor suppressor gene are frequent and play an important role in colorectal oncogenesis. A role of p53 as an angiogenesis inhibitor has also been proposed. We evaluated angiogenesis and p53 expression in 16 hyperplastic polyps, 35 solitary tubular and tubulovillous adenomas, and 47 cases of sporadic colorectal carcinomas arising on the basis of preexisting adenomas, with standard immunohistochemical techniques. The mean microvessel density (MVD) in carcinomas was significantly higher compared with the respective adenomatous part of the same tumor (27.9 vs. 7; P=0.0001). Linear regression analysis of MVD between cancerous and adenomatous areas showed a significant correlation (P = 0.0001, r = 0.56), raising the possibility that carcinomas arising from better vascularized adenomas might show increased vascularity. The MVD was significantly higher in stage C compared with stage A cases (P=0.04). p53 positivity was detected in 26 of 47 cancerous (55%) and in 14 of 47 adenomatous areas (30%; P = 0.0002). All carcinomas arising from p53-positive adenomas were also p53 positive. p53 positivity associated with a higher MVD in adenomas (P = 0.02), but not in carcinomas (P = 0.78). We conclude that angiogenesis and p53 play a critical role in colorectal neoplasia, and the process of malignant transformation in tumors arising from highly angiogenic adenomas, particularly those carrying p53 mutations, is accelerated with rapid tumor progression from stage to stage, indicating a more aggressive tumor phenotype.     

Up-regulation of gelatinases in the colorectal adenoma-carcinoma sequence

Gelatinase activity has been associated with colorectal cancer (CRC) invasion and metastasis. However, it remains unresolved whether these proteases participate in early colorectal carcinogenesis. The activity of metalloproteinases (MMP) 2 and 9 were measured by zymography in 122 colorectal adenomas, 22 CRC samples, 12 hyperplasic polyps and in 114 matched normal mucosal samples from 114 patients undergoing colonoscopy. There was a progressive and significant increase of pro-MMP-9 activity from adenoma to CRC tissue, whereas the activity of the latent and active forms of MMP-2 was exclusively up-regulated in CRC samples. Among neoplastic polyps, pro-MMP-9 activity was significantly higher in advanced versus non-advanced adenomas and in those harbouring high grade dysplasia. In addition, a positive correlation was observed between MMP-9 activity and the size of the adenomas. The present study demonstrates that MMP-9 is markedly up-regulated in the adenomatous tissue and suggests that this gelatinase might be a marker for early colorectal carcinogenesis.     

Effects of expression of hRFI on adenoma formation and tumor progression in colorectal adenoma-carcinoma sequence

For colorectal carcinomas as well as colonic polyps we investigated the expression of a newly discovered gene, hRFI, which is isolated by the yeast two-hybrid screening using hTid as a bait and expressed highly in esophageal carcinomas. Immunohistochemical staining was performed on 48 colorectal carcinomas and 77 colorectal polyps consisting of 70 adenomas and 7 hyperplastic polyps using the antibody of hRFI. We analyzed the expression of hRFI and the correlation between the percentage of staining of each and their clinico-pathological characteristics. Protein coding by hRFI was specifically and diffusely expressed in most of the cancerous regions of the colorectum. Also, in the early stage of colorectal adenomas, staining of hRFI was focal, and the percentage area of diffuse staining increased as the degree of dysplasia progressed. Although all normal colorectal glands and most hyperplastic polyps (71.4%) showed no staining of hRFI, most colorectal adenomas and carcinomas (93.2%) showed a focal or diffuse staining (P<0.001). Furthermore, the percentage of diffuse staining in carcinomas (81.3%) was significantly higher than in adenomas (5.7%) (P<0.001). hRFI is highly expressed in colorectal carcinomas. In the adenoma-carcinoma sequence, hRFI is involved at the initial tumor formation and its diffuse expression is associated with colorectal carcinogenesis. This evidence suggests that hRFI may act as an oncogenic molecule affecting the apoptotic pathway.     

胆囊腺瘤-腺癌中的DNA含量和基因表达

目的 研究胆囊腺瘤从增殖到恶变中腺上皮细胞的增殖变化和基因表达。方法PCR-RFLP法测定APC、ras基因突变,ABC免疫化学组织染色法测定p53蛋白表达。用TJTY-300全自动图像分析仪测定胆囊上皮细胞核异型性和DNA含量。结果 上皮细胞核异型性和DNA含量在胆囊腺瘤-腺癌过程中逐渐增加。胆囊腺瘤中异倍体比例与腺瘤的大小有明显的关系,直径≥1cm异倍体显著增加(P<0.05)。在胆囊腺瘤-腺癌过程中可检测到APC基因和RAS基因突变。P53蛋白在这一过程中不表达。结论 胆囊腺瘤-腺癌的过程是胆囊癌发生中一条重要的途径。当胆囊腺瘤直径≥1cm时具有明显的癌变潜能。其分子改变机制与胆囊上皮不典型增生-原位癌途径不同。     

Active TGF-β1 correlates with myofibroblasts and malignancy in the colorectal adenoma-carcinoma sequence

Transforming growth factor-β1 (TGF-β1), a cytokine involved in various stages of cancer, is produced as a latent complex and requires processing to become active. We have determined total and active TGF-β1 levels in homogenates of colorectal neoplasia. In contrast to total TGF-β levels, showing a stepwise increase in the mucosa-adenoma-carcinoma sequence, active TGF-β1 levels are increased only in carcinomas but not in premalignant adenomas. Furthermore, solely active TGF-β1 levels are associated with the stage of the carcinomas and worse patient prognosis. Active TGF-β1 levels correlated significantly with plasminogen activator inhibitor (PAI)-1, α-smooth muscle actin (SMA) and several matrix-remodeling proteinases. Interestingly, SMA levels are also significantly increased in colorectal carcinomas but not in adenomas, suggesting that despite the enhanced total TGF-β1 levels, myofibroblast accumulation is not (yet) occurring in these premalignant neoplasias. The correlation between active TGF-β1 and SMA expression in tumors indicates that tumor-promoting myofibroblasts might arise as a result of increased TGF-β1 activation. These data underline the significance of the interaction between malignant cells and (myo)-fibroblasts in the tumor microenvironment, modulating the biologic behavior of colorectal cancer. ( Cancer Sci 2009; 100: 663–670)     

p53 gene mutations in early colorectal carcinoma. de novo vs. adenoma-carcinoma sequence

p53 gene mutations in early and advanced colorectal cancer were detected by PCR-SSCP analysis. Early colorectal cancer was classified intode novo cancer and polyp-forming cancer, respectively, according to morphology and presence of adenomatous components. A total of 94 paraffin-embedded tissue specimens from patients with colorectal cancer were analysed.p53 gene mutations were detected in 40.0% (12/30) of de novo cancer, 36.7% (11/30) of polyp-forming cancer, and 44% (15/34) of advanced cancer. p53 mutations were detected at a high frequency in both types of early colorectal cancer as well as in advanced cancer. No correlations were found between p53 mutations and clinicopathological data. Our data suggest that the p53 gene mutations occurred in the early colorectal cancer stage of carcinogenesis regardless of the pathway. © 1995 Wiley-Liss, Inc.     

Allelotype of the adenoma-carcinoma sequence of the stomach.

In order to identify the significant allelic loss involving the gastric adenoma-carcinoma sequence, we used 49 genome-wide microsatellite markers in an allelotype study of 30 cases of stomach resections that harbored both adenomas and carcinomas. Frequent loss of heterozygosity was demonstrated on 12q (53.3%), 2p (50.0%), and 18p (50.0%) in adenomas and on 8q (80.0%), 2p (70.0%), 18p (66.7%), and 17p (61.9%) in carcinomas. Significant difference in the loss of heterozygosity rate between the adenoma and the carcinoma was noted on 17p. Our results suggested that the critical target of loss of heterozygosity in gastric adenomacarcinoma sequences may be the p53 gene on 17p.     

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。     

Investigations on the significance of the adenoma-carcinoma sequence in the small bowel

In view of the rarity of small-bowel epithelial neoplasms as compared with the case for the large bowel, evidence for an adenoma-carcinoma sequence in the small bowel was studied based on a search for data in the medical literature for the years 1927 through 1986. Sufficiently defined data were found for comparison of 185 benign adenomas, 76 adenoma-with-carcinomas, and 1333 carcinomas in patients without familial polyposis disease and for 63, five, and 30, respectively, in patients with disease. For patients without polyposis, it was found that (1) 29.8% of all small-bowel adenomas (33.6% if those at Vater's ampulla are excluded) showed malignancy; (2) the mean and median ages were lower for benign adenoma than for adenoma-with-carcinoma and carcinoma, although the ratios by sex were the same; (3) there is a nearly identical spatial distribution of the three types of epithelial neoplasms within the small bowel; and (4) both the frequency of finding adenomatous residues existing in continuity with carcinoma and the life history of the adenoma-carcinoma sequence are similar in the small bowel as in the large. In comparing these results with those from patients with familial polyposis disease, it was particularly noted that (1) the only difference was that adenomas in familial polyposis occurred earlier and multiply, and (2) the spatial distributions of adenomas and carcinomas for both cases were closely similar. It is therefore postulated that the adenoma-carcinoma sequence is as significant in the small bowels as in the large. A hypothesis regarding the relationship of epithelial neoplasms in people with and without familial polyposis disease is suggested.     

Genetic analysis of multiple synchronous lesions of the colon adenoma-carcinoma sequence

The colorectal adenoma-carcinoma sequence represents a well-known paradigm for the sequential development of cancer driven by the accumulation of genomic defects. Although the colorectal adenoma-carcinoma sequence is well investigated, studies about tumours of different dignity co-existent in the same patient are seldom. In order to address the distribution of genetic alterations in different lesions of the same patient, we coincidently investigated carcinomas, adenomas and aberrant crypt foci in patients with sporadic colon cancer. By utilizing polymerase chain reaction, single-strand conformation polymorphism, heteroduplex-analysis, restriction fragment length polymorphism, protein truncation test and sequencing techniques we looked for mutations and microsatellite instability of APC, H-ras, K-ras, p53, DCC and the DNA repair genes hMLH1/hMSH2. In accordance with the suggested adenoma-carcinoma sequence of the colon, four patients reflected the progressive accumulation of genetic defects in synchronously appearing tumours during carcinogenesis. However, two patients with non-hereditary malignomas presented different genetic instabilities in different but synchronously appearing tumours suggesting non-clonal growth under almost identical conditions of the environment. Thus, sporadically manifesting multiple lesions of the colon were not necessarily driven by similar genetic mechanisms. Premalignant lesions may transform into malignant tumours starting from different types of genetic instability, which indicates independent and simultaneous tumorigenesis within the same organ.     

Association between VEGF expression in tumour-associated macrophages and elevated serum VEGF levels in primary colorectal cancer patients

OBJECTIVE: Angiogenesis is stimulated by angiogenic factors released by tumour cells, though other cells, such as tumour-associated macrophages (TAMs), also contribute towards increasing the angiogenic process in colorectal cancer (CRC). The aim of this study was to determine in CRC patients the contribution of vascular endothelial growth factor (VEGF) expression in TAMs and tumour cells towards circulating VEGF levels, their association with p53 expression and microvascular density (MVD), and their prognostic value. METHODS: Immunohistochemical techniques were used to identify TAMs and p53 protein, and to evaluate the VEGF expression in TAMs, MVD and tumour cells in 110 primary CRC patients. Serum VEGF levels were determined using an enzyme immune assay. RESULTS: There was a greater expression of VEGF in tumours with a positive p53 expression than a negative stain (p<0.01). The macrophage index was not related to tumour VEGF secretion. No significant association was observed between serum VEGF levels and VEGF tumour expression, node status, histological grade, MVD or p53 expression. However, the patients with high values of VEGF expression in TAMs showed significantly higher presurgery serum VEGF levels than those patients with low values of VEGF expression in TAMs (p=0.021). No statistical significant differences in survival were found when we compared patients with high VEGF expression in TAMs vs low or median VEGF expression in TAMs (p=0.093). Serum VEGF levels were increased 6-8 hours after tumour removal (p=0.001). CONCLUSIONS: Our data suggest that in primary CRC, presurgery circulating VEGF levels are related to VEGF produced by TAMs.     

VEGF gene sequence variation defines VEGF gene expression status and angiogenic activity in non-small cell lung cancer

Different vascular endothelial growth factor (VEGF) gene polymorphisms have been shown to result in different VEGF gene responsiveness to various stimuli and different capacity for VEGF protein production. In the present study, we examined four VEGF gene polymorphisms in thirty-six individuals with non-small cell lung cancer (NSCLC). Gene polymorphisms were correlated with the VEGF protein expression in cancer cells and the tumor angiogenic activity. The -2578C/C, -634G/G and -1154A/A and G/A alleles in the VEGF gene were linked with low VEGF expression, while the -2578C/A, the -634 G/C and the -1154G/G alleles were linked with high VEGF expression. Tumors with -2578C/C had a significantly lower vascular density (VD) compared to the -2578C C/A. Similarly, cases with the -634G/G VEGF polymorphism had a singinificanltly lower vascular density compared to the combined C/C and G/C groups. In addition, the -1154A/A polymorphism seemed to relate with poor vaccularization but the difference did not reach significance. It is concluded that inherited VEGF sequence variations, which characterize the tumor genome itself, are strong determinants of the molecular VEGF and VEGF-downstream phenotype of NSCLC. The large variation in angiogenicity between tumors of similar histologic morphology emerges as a consequence of the 'parental' VEGF gene ability to produce VEGF.     

Impact of the coxsackievirus and adenovirus receptor on the adenoma-carcinoma sequence of colon cancer

Background:

Coxsackie and adenovirus receptor (CAR) has been suggested to function as a tumour suppressor. Its impact on the adenoma–carcinoma sequence of the colon, however, is unclear.

Methods:

Coxsackie and adenovirus receptor was analysed in non-cancerous and neoplastic colon samples using immunohistochemistry and quantitative RT–PCR. The function of CAR in colon cancer cell lines was determined following application of CAR siRNA or ectopic expression of a human full-length CAR cDNA.

Results:

Compared with healthy mucosa, increased CAR-mRNA expression was found in adenomas, whereas primary cancers and metastases displayed a marked decline. At the plasma membrane, CAR was present in normal mucosa samples (93%), adenomas, and metastases (100% ea.), whereas in colon cancers, it was found less frequently (49%, P<0.0001). Cytoplasmic CAR immunopositivity increased from normal mucosa (22%), to adenomas (73%, P=0.0006), primary cancers (83%, P<0.0001), and metastases (67%, P=0.0019). In cancer cell lines, CAR inhibition resulted in increased proliferation, whereas enforced ectopic CAR expression led to opposite results. Blocking the extracellular portion of CAR increased cell invasion in vitro. In mice, xenotransplants of colon cancer cells with enforced CAR expression formed significantly smaller tumours, whereas CAR inhibition increased the formation of liver metastases.

Conclusion:

We conclude that CAR facilitates complex effects during colon carcinogenesis, potentially mediated by its stage-dependent subcellular distribution; high CAR expression potentially prevents apoptosis in adenomas, loss of CAR at the plasma membrane promotes growth, and dissemination of primary cancers, and high membranous CAR presence may support the establishment of distant metastases.     

大肠癌组织c-Met和VEGF表达及其与微血管密度关系的初步研究

目的:探讨大肠癌组织c-Met和血管内皮生长因子(VEGF)的表达以及两者与肿瘤血管生成的关系及其临床病理学意义。方法:选取经病理确诊的51例大肠腺癌手术切除组织标本,免疫组化SP法检测c-Met和VEGF的表达及微血管密度(MVD)。结果:大肠癌组织中c-Met和VEGF的阳性表达率分别为72.55%(37/51)和52.94%(27/51);与Dukes分期密切相关,P<0.05。大肠癌组织c-Met、VEGF均为阳性时MVD值为37.94±7.53,单一阳性时分别为30.67±1.45和23.82±7.45,均为阴性时MVD值最小,为13.64±5.33。大肠癌组织中c-Met和VEGF的表达呈显著正相关,rs=0.614,P<0.05。结论:c-Met和VEGF在大肠癌发生、发展和转移过程中起重要作用,并与肿瘤血管生成相关。     

Dissecting karyotypic patterns in colorectal tumors: two distinct but overlapping pathways in the adenoma-carcinoma transition

More than 500 colorectal tumors with clonal chromosomal abnormalities have been reported. Although the pattern of aberrations is nonrandom, no specific primary or secondary karyotypic abnormality has been identified. Also, the chronological order in which the aberrations appear during disease progression is not well known. One reason why our understanding of the cytogenetic evolution is unclear is the high degree of karyotypic complexity seen in these tumors. To overcome some of these difficulties we have previously used several statistical methods that allow identification and interpretation of karyotypic pathways as well as establishment of a temporal order of appearance of the imbalances. These methods were applied on 531 colorectal tumor karyotypes. By using a resampling strategy, 1p-, +7, 7q-, and +12p were identified as early events. Two major and two minor cytogenetic pathways were identified by means of principal component analysis. The two major pathways were initiated with 1p- and +7, and the minor pathways were initiated with +12p and 7q-. The +7/+12p tumors were found to be hyperdiploid, whereas those with 1p-/7q- were pseudodiploid. We also show that the adenoma-carcinoma transition in the 1p- pathway is strongly linked to karyoytypic evolution, whereas the +7 pathway is not, and that the cytogenetic pathways are separated at both early and late stages.