Expression of APO-1 (CD95), a member of the NGF/TNF receptor superfamily,in normal and neoplastic colon epithelium

APO-I is a 48-kDa cell-membrane protein identical to the Fas antigen now designated CD95. It is a member of the NGF/TNF receptor superfamily. Anti-APO-I monoclonal antibody induces apoptosis in a variety of cell types expressing this antigen. We immunohistochemically investigated APO-I expression in normal colon mucosa, 20 adenomas, 258 colon carcinomas and 10 liver metastases and carried out in vitro studies using a panel of colon-carcinoma cell lines. Immunohistochemically, APO-I was regularly expressed at the basolateral membrane of normal colon epithelia. In a minor fraction of colon adenomas and in 39.1% of colon carcinomas APO-I expression was diminished and in 48.1% of carcinomas, predominantly of the non-mucinous type, APO-I expression was completely abrogated. The normal level of APO-I in carcinomas was correlated with the mucinous type. Reduced/lost APO-I expression was more frequent in rectal carcinomas. Complete loss of APO-I was more frequent in tumors that had already metastasized. APO-I expression in liver metastases essentially corresponded to that of the primary tumors. Comparative analysis with data from previous studies revealed that the mode of APO-I expression is correlated with that of HLA-A,B,C./β₂m, HLA-DR, HLA-D-associated invariant chain and of the secretory component. Surface expression of APO-I was heterogeneous in colon-carcinoma cell lines; SW480 expressed considerable amounts of APO-I on all cells, while HT-29 constitutively did less so and only in a minority of cells. Surface density of APO-I and the fraction of positive cells in HT-29 was enhanced by interferon-gamma (IFN-γ) and, additively, by tumor necrosis factor-alpha (TNF-α), whereas in SW480 APO-I expression was not modulated by these cytokines. We conclude that neoplastic transformation of colon epithelium often leads to a loss of the physiologic, high level of surface APO-I by giving rise either to a stable lack of APO-I or to an IFN-γ/TNF-α-sensitive phenotype of inducible APO-I expression. © 1994 Wiley-Liss, Inc.     

Expression of Bcl-2 and c-ErbB-2 in colorectal neoplasia

Several studies have been demonstrated the value of c-ErbB-2 and Bcl-2 in predicting the biological behaviour of tumors. The aim of this study was to investigate Bcl-2 and c-ErbB-2 expression in colorectal carcinomas and the correlation between their presence and other clinicopathologic parameters. Eighty-six colorectal carcinomas and 17 adenomas were stained with Bcl-2 and c-ErbB-2 immunohistochemically. Staining patterns were assessed semi-quantitatively and correlated with tumor size, Duke s classification, tumor differentiation, mucinous characteristic and anatomic locations. We detected Bcl-2 expression in 10 of 17 adenomas (58.8 %) and 31 of 86 carcinomas (36.04 %). Positive staining in normal mucosa was observed only in the compartment of cryptic cells. However neither the difference in the rates of Bcl-2 positivity in adenoma and carcinoma groups, nor the correlation with other mentioned clinicopathological parameters, were found statistically significant. Bcl-2 expression was found to be significantly high in mucinous carcinomas. Expression of c-ErbB-2 was observed in 12 of 86 (13.95 %) carcinomas. It was not detected in adenomas and normal mucosa. Although the incidence of c-ErbB-2 in nonmucinous carcinoma was higher than that of mucinous carcinoma, this was not significant. In addition we were unable to show any significant relation between c-ErbB-2 expression and other clinicopathologic features. Our result suggest that c-ErbB-2 protein expression in colorectal carcinomas, is not very frequent event. There is no correlation between c-ErbB-2 expression and malignant potential of colorectal carcinomas. Higher expressions of Bcl-2 in adenomas than carcinomas suggest us a possible role of Bcl-2 in early carcinogenesis of colon. However since we were unable to find any significant correlation between Bcl-2 expression and other parameters the impact of this gene on biological behavior is still unclear for us.     

Expression of cripto-1 in human colorectal adenomas and carcinomas is related to the degree of dysplasia

The expression and localization of cripto-1 (CR-1) and epidermal growth factor receptor (EGFR) were assessed by immunocytochemistry in 41 human colorectal carcinomas, 57 adenomas, 9 hyperplastic polyps and in 98 noninvolved colonic mucosa samples that were adjacent to adenoma and/or carcinoma. Thirty-two (78.0%) and 19 (46%) carcinomas showed staining for CR-1 and EGFR, respectively, whereas 24 (42.0%) and 25 (43.8%) of the adenoma samples were reactive with the anti-CR-1 and anti-EGER antibodies, respectively. Two (22.2%) and 1 (11.1%) of the hyperplastic polyps demonstrated moderate levels of staining with anti-CR-1 and anti-EGFR antibodies. In contrast, none of the normal, noninvolved colonic mucosa samples reacted with the CR-1 antibody, whereas only 1 (1.0%) reacted with the EGFR antibody. Between EGFR and CR-1 expression, there was no significant association within either adenomas or carcinomas. A significant difference in the incidence for CR-1 expression was observed between adenomas and carcinomas (p<0.001). Within adenomas, the frequency of CR-1 was related to the histological degree of atypia. Immunostaining for p53 was also observed in 10 (24%) of the carcinomas, in 10 (17%) of the adenomas and in none of the hyperplastic polyps nor colonic mucosa samples. No statistically significant difference for p53 staining was observed between the adenomas and carcinomas. However, adenomas with moderate atypia exhibited relatively strong positive staining for p53 (p<0.05) compared to either adenomas with mild or severe atypia. A slight trend (p<0.05) for coexpression of p53 and CR-1 was detected in adenomas but not in carcinomas. These data demonstrate that CR-1 is a tumor marker for colon carcinomas and additionally that the expression of CR-1 may be an important factor in the early stages of colon cancer development during the adenomacarcinoma transition.     

Bcl2 expression and its correlation with neuroendocrine differentiation in colon carcinomas

AIMS AND BACKGROUND: In this study we investigated the association between BCL2 expression and neuroendocrine differentiation in tubular adenomas and colon carcinomas. We also evaluated the prognostic significance of BCL2 expression and neuroendocrine differentiation in the carcinoma group. METHODS: Forty-eight colon carcinomas and twelve tubular adenomas were stained immunohistochemically with BCL2 and chromogranin A (CgA). Staining patterns were scored semiquantitatively and correlated with tumor type, tumor grade, Dukes stage, and survival time. RESULTS: BCL2 expression was detected in 7 of 12 (58.3%) adenomas and 37 of 48 (77.0%) carcinomas. In normal mucosa we observed positive staining only in the basal portions of the crypts. However, diffuse positivity was observed in the adenomas and carcinomas. Carcinomas had significantly higher BCL2 scores than the normal group, but we did not observe any significant differences either between the adenoma and carcinoma group or between the adenoma and normal group. BCL2 scores were higher in stage B than in stage C tumors, and in adenocarcinomas than in mucinous carcinomas. CgA positivity was observed in 24 of 48 (50%) carcinomas. It was not detected in adenomas. We did not find a significant correlation between CgA expression and any of the clinicopathological parameters. CONCLUSION: On consecutive sections BCL2 and CgA positivity closely paralleled each other and a significant positive correlation was observed between CgA and BCL2 expression. These findings suggest a close association between BCL2 expression and neuroendocrine differentiation and indicate that BCL2 may be involved in neuroendocrine differentiation in addition to its role in protecting cells from apoptosis.     

Expression of the nuclear bile acid receptor/farnesoid X receptor is reduced in human colon carcinoma compared to nonneoplastic mucosa independent from site and may be associated with adverse prognosis

The nuclear bile acid receptor/farnesoid X receptor (FXR; NR1H4) is involved in bile acid homeostasis, cell proliferation and apoptosis and has been linked to intestinal carcinogenesis in mice. Aim of this study was to analyze FXR expression in human normal intestinal mucosa and colon carcinoma. We achieved systematic mapping of FXR expression of human intestinal mucosa and analysis of 75 human colon carcinomas using FXR immunohistochemistry on formalin-fixed, paraffin-embedded tissue. FXR expression gradually decreased from terminal ileum to the sigmoid colon with strongest expression in the terminal ileum (p < 0.001). FXR expression in carcinomas was reduced compared to peritumoral nonneoplastic mucosa (p < 0.000). Loss of FXR expression was significantly correlated with grading in tumors of the right colon (p = 0.008). FXR expression in tumor and normal tissue showed an inverse correlation with stage. FXR expression in tumor was inversely correlated with clinical outcome. No association was found with patients' age and sex. In nonneoplastic mucosa FXR expression concurred with low expression of Ki-67. In carcinomas, no association was found between FXR expression and Ki-67 and cyclin D1, respectively. Development of colon carcinoma in humans is associated with reduced FXR expression independent of site and may reflect an impaired defense against potentially carcinogenic bile acids along their intestinal gradient. In contrast to normal colon mucosa, FXR expression in carcinomas is not associated with low proliferation. Colon carcinomas with FXR expression seem to be associated with lower stage and a more favourable outcome compared to FXR negative carcinomas.     

An immunohistochemical analysis of ras oncogene expression in epithelial neoplasms of the colon

Colonic epithelial tumors (101) including villoglandular adenomas, carcinomas in situ, adenocarcinomas, and neuroendocrine (NE) carcinomas were studied immunohistochemically with monoclonal antibodies (MoAb) RAP-5 and RAS-10 recognizing altered and unaltered ras oncogene products. In addition, 20 samples from multiple polyposis including adenomas with and without dysplasia, carcinomas in situ, and invasive carcinomas were studied. Using immunostaining techniques, normal mucosa was weakly stained, whereas the mucosa in the vicinity of tumors or inflammation showed enhanced staining. More tumors stained intensely with MoAb RAP-5 than with MoAb RAS-10. With MoAb RAP-5, most benign and malignant tumors showed enhanced staining. No significant differences in staining were noted in relation to superficial versus deeply invasive carcinomas or clinical staging. Immunostaining was also noted in some metastases. No significant differences in enhanced staining were found in carcinomas. Interestingly, the most extensive and enhanced immunostaining was noted in the villoglandular adenomas, dysplastic adenomas, and carcinomas in situ. The authors conclude that (1) ras protein expression is detectable in most benign, borderline, and malignant epithelial tumors of the colon as determined with MoAb RAP-5 and RAS-10, whereas enhanced expression is more often detected with RAP-5; (2) enhanced ras product expression in colon carcinomas does not seem to correlate with advanced tumor stages or with exocrine, NE, or phenotypically mixed tumors; and (3) the finding of the most intensely enhanced ras products expression in villoglandular polyps and carcinomas in situ suggests a possibly significant role for the oncogene in the early phases of transformation.     

Differential immunohistochemical detection of amphiregulin and cripto in human normal colon and colorectal tumors.

Thirty-six primary human colorectal tumors, 43 noninvolved colon samples that were adjacent to either carcinomas of adenomas, 22 adenomas, and nine normal colon specimens were immunohistochemically examined for the presence and localization of two epidermal growth factor-related peptides, amphiregulin (AR) and cripto. Within the primary tumors, 18 (50%) showed moderate levels of AR expression. Approximately 60% of the tubular and tubulovillous adenomas were positive for AR expression, whereas only 15% of the adjacent, noninvolved colon mucosa expressed AR. A greater proportion of well-differentiated tumors (71%) were positive for AR expression than were poorly differentiated tumors (18%). All of the nine normal colon specimens were positive. Consequently, AR expression appeared to be associated with both normal and malignant epithelial cells that were more differentiated. The distribution of cripto expression was different. Seventy-nine % of the colon tumors expressed cripto with a frequency of expression that was approximately equivalent between well-differentiated and poorly differentiated tumors. Approximately 86% of the tubulovillous adenomas, but only 43% of the tubular adenomas, were positive for cripto expression. In contrast, whereas AR was expressed in normal colon specimens, none of these tissues expressed cripto, and only 12% of the noninvolved normal colon samples adjacent to tumors or adenomas were positive for cripto. Cripto expression therefore appeared related to neoplasia. These data suggest that AR and cripto may be functioning as potential autocrine and/or paracrine growth factors in the colon and that the differential expression of cripto may serve as a potential tumor marker for colonic carcinogenesis.     

Vascular endothelial growth factor-D expression is an independent prognostic marker for survival in colorectal carcinoma

The angiogenic factor vascular endothelial growth factor-D (VEGF-D) isa ligand for VEGF receptor-3 (VEGFR-3/Flt-4) and receptor-2 (VEGFR-2/KDR)and is implicated in the development of lymphatic vessels and promotion of lymphatic metastases. We assessed the expression of VEGF-D and VEGFR-3 in relation to microvessel density (MVD) in colorectal carcinomas (CRC), adenomas, and adjacent normal tissue by immunohistochemistry on consecutive archival sections. VEGF-D was detected in malignant and benign epithelium and in some smooth muscle of the colorectum. High-grade VEGF-D expression was observed frequently (74%) in CRC compared with adenomas (0%) and adjacent normal mucosa (22%). High-grade VEGF-D expression was not correlated with MVD, Dukes' stage (A to C), or tumor differentiation, but was associated with lymphatic involvement and patient survival. By multivariate analysis, VEGF-D expression was found to be an independent prognostic factor for both disease-free and overall survival. VEGFR-3 expression was detected in a subset of vessels, typically thin-walled and devoid of RBCs, in 89% of CRC cases examined. VEGFR-3-positive vessel densities increased progressively from normal mucosa to adenomas and carcinomas and were correlated with MVD, but not with Dukes' stage (A to C), tumor differentiation, or VEGF-D expression. VEGFR-3 expression was spatially associated with macrophage-rich inflammatory infiltrates, which were significantly more frequent among VEGFR-3-positive cases. We conclude that VEGF-D expression, but not that of its receptor VEGFR-3, is an independent prognostic indicator in CRC. VEGF-D expression may be associated with disease outcome through the promotion of lymphatic involvement/metastases.     

Loss of colonic HLA antigens in familial adenomatous polyposis.

The loss of HLA antigens by neoplastic cells is considered important for tumor growth and metastasis, since it may allow tumors to escape immune surveillance. We studied the expression of HLA class I and II antigens in the colons of 10 patients with familial adenomatous polyposis (FAP), a condition which leads inevitably to colorectal cancer. Expression of HLA class antigens was studied by immunohistochemistry in (a) adenomas from patients with FAP, (b) histologically normal mucosa distant from the adenomas, and (c) histologically normal colonic mucosa from normal subjects. The expression of HLA class I and II antigens was decreased in histologically normal mucosa from FAP patients compared to normal controls. Adenomas showed a similar but quantitatively more pronounced reduction (or loss) of HLA antigen expression. The reduction of HLA expression in adenomas was comparable to that observed in sporadic colon carcinomas. This generalized suppression of HLA gene expression in the colon of FAP patients, which precedes the onset of overt histological manifestations of neoplasia, may be an important early event in colon carcinogenesis.     

Expression of cripto and amphiregulin in colon mucosa from high risk colon cancer families

We assessed the expression of the epidermal growth factor (EGF)-related peptides, cripto-I (CR-I) and amphiregulin (AR), in a small panel of human colon adenomas and carcinomas. CR-I immunoreactivity was found in 17/31 (55%) of colon adenomas, and in 33/39 (84%) colon carcinomas. AR immunostaining was observed in 16/26 adenomas (61%) and in 20/26 carcinomas (77%). CR-I and AR staining were also assessed in 29 specimens from 24 individuals that belong to families with high incidence of colorectal carcinoma, and in 5 non-high risk individuals. Expression of CR-I was detected in 18/29 (62%) of high risk colon mucosa specimens, but only in 1/5 (20%) specimens from non-high risk individuals, while AR staining was found in 20/29 (69%) and in 4/5 (80%) of colon mucosa samples from high and low risk individuals, respectively. A majority (21/29; 72%) of the specimens from the high risk individuals had a high proliferative rate, as measured by Ki-67 staining. A statistically significant correlation was found between high proliferative rate, increased expression of CR-I and reduced expression of AR in the mucosa specimens from high risk individuals, suggesting that these might represent early events in colon tumorigenesis.     

Expression of multiple isoforms of protein kinase C in normal human colon mucosa and colon tumors and decreased levels of protein kinase C β and η mRNAs in the tumors

Previous studies have suggested that protein kinase C (PKC) may play an important role in colon carcinogenesis and that human colon tumors have less total PKC enzyme activity than normal tissue. Because PKC is a multigene family that enocodes for at least 11 distinct isofroms, in the study reported here we analyzed the expression of six of these isoforms at the mRNA level by northern blot hybridization in 22 pairs of primary colon tumors (of various stages), and adjacent normal mucosa samples. We found that the normal mucosa samples expressed the mRNA of the following isoforms of PKC, in decreasing order of abundance: PKCδ>PKC>PKCα>PKCβ>PKC?. There was no consistent difference in the levels of PKCα, PKCδ, and PKCη. mRNAs between the normal mucosa and the tumor samples. PKCγ was expressed at a very low level in two of the colon tumors but could not be detected in the remaining tumors of any of the normal mucosa samples. The levels of both PKCβ and PKCη mRNAs were singificantly lower in the tumor samples than in the normal mucosa samples, and this was true of adenomas as well as Dukes' stage A, B, and Cadenocarcinomas. Furthermore, the devrease in PKCη mRNA appeared to be greater in the more poorly differentiated carcinomas. This finding in of interest becouse PKCη is normally expresed in the more differentiated cells of epithelial tissues. The devreased levels of both PKCβ and PKCη mRNAs ocurred early in the multistage process of colon carcinogenesis, as it was also seen in adenomas. The functional significane of these changes remains to be determined.     

High mobility group HMGI(Y) protein expression in human colorectal hyperplastic and neoplastic diseases

HMGI(Y) proteins are overexpressed in experimental and human malignancies, including colon, prostate and thyroid carcinomas. To determine at which step of the carcinogenic process HMGI(Y) induction occurs, we analysed the expression of the HMGI(Y) proteins in hyperplastic, preneoplastic and neoplastic tissues of colorectal origin by immunohistochemistry. All the colorectal carcinomas were HMGI(Y)-positive, whereas no expression was detected in normal colon mucosa tissue. HMGI(Y) expression in adenomas was closely correlated with the degree of cellular atypia. Only 2 of the 18 non-neoplastic polyps tested were HMGI(Y)-positive. These data indicate that HMGI(Y) protein induction is associated with the early stages of neoplastic transformation of colon cells and only rarely with colon cell hyperproliferation.     

Influence of major histocompatibility complex class I and II antigens on survival in colorectal carcinoma

HLA-A,B,C and HLA-D molecules present antigenic peptides to the antigen-specific receptor of autologous T-lymphocytes. T-cell-mediated host-versus-tumor response might therefore depend on the presence of these molecules on tumor cells, although the absence of HLA-A,B,C determinants on a cell has been shown to increase its susceptibility to lysis by natural killer cells. To investigate whether the presence or absence of HLA-A,B,C and/or HLA-DR in colorectal carcinoma influences relapse rate and time of tumor-related death, 152 patients who underwent putatively curative surgical treatment were surveyed for a maximum of 65 months (mean, 48 months). As determined by immunohistochemistry, aberrant reduction or loss of HLA-A,B,C/beta 2-microglobulin molecules was more frequent in tumors of the proximal colon than of the rectosigmoid (P = 0.032) and in mucinous carcinomas than in nonmucinous ones (P = 0.022). An abnormal induction of the HLA-D-associated invariant chain (Ii) was more frequent in Dukes' A and B than in stage C (P = 0.046). Reduction/loss of HLA-A,B,C/beta 2-microglobulin was correlated with the absence of HLA-DR (P = 0.024) and Ii (P = 0.005). In contrast to the prognostic role of tumor stage and grade, the presence versus the absence of HLA-A,B,C/beta 2-microglobulin and HLA-DR/Ii molecules was not correlated with recurrence rate or survival. We conclude that in spite of an increasing amount of experimental data suggesting the contrary, the status of HLA-A,B,C and HLA-DR expression in colorectal carcinoma seems to be irrelevant in vivo, regarding survival and growth of residual tumor cells after putatively curative resection of the initial tumor burden.     

Conserved expression of hepatocyte growth factor activator inhibitor type-2/placental bikunin in human colorectal carcinomas

Hepatocyte growth factor activator inhibitor type 2 (HAI-2) was recently identified as a potent inhibitor of hepatocyte growth factor activator. It was also independently reported as placental bikunin (PB) and as a protein over-expressed in pancreatic cancer. The expression of HAI-2/PB was analyzed in human normal colon mucosa, adenomas, and carcinomas. HAI-2/PB mRNA was consistently expressed in the colorectal mucosa. The expression was conserved in the neoplastic colorectal mucosa, and no relationship was found between HAI-2/PB mRNA levels and tumor stages. Moreover, 13 out of 14 colorectal carcinoma cell lines expressed HAI-2/PB mRNA. Immunohistochemically, HAI-2/PB proteins were predominantly stained beneath the apical surface of normal enterocytes. In tumor tissues, rather disarranged intracytoplasmic granular staining was observed. The HAI-2/PB immunoreactivity was well conserved in the colonic adenoma-carcinoma sequence, and this protein may have important unknown function in the intestinal mucosa.     

Expression of beta-catenin and full-length APC protein in normal and neoplastic colonic tissues

Mutations of the APC gene are thought to be early events in the process of colorectal carcinogenesis. Although the complete function(s) of the APC gene product is not known, it has been shown that the APC protein interacts with beta-catenin in a multi-protein complex to regulate the level of expression of beta-catenin. Loss of normal APC protein function can lead to an accumulation of beta-catenin in the cytosol and the nucleus. Immunohistochemical methods were used to determine the relationship between APC and beta-catenin protein expression in human colonic tissues (150 normal, 9 hyperplastic, 58 adenomas and 83 carcinomas) and 12 paired samples of normal and cancer tissue in mouse colon. In all samples of normal human and mouse colonic mucosa and in human hyperplastic polyps both APC and beta-catenin immunoreactivity were present in colonocytes. APC expression was cytoplasmic, with maximal immunoreactivity in the goblet cells. beta-Catenin expression was predominantly localized to the plasma membrane, with no nuclear immunoreactivity. APC immunoreactivity was absent in all of the mouse adenocarcinomas and 83% of the human colon cancers. All of the human and mouse carcinomas had nuclear and cytoplasmic beta-catenin expression. In contrast, only 29% of the 58 colonic adenomas were completely negative for APC immunoreactivity. Regardless of the presence or absence of APC, all of the adenomas had cytoplasmic and nuclear beta-catenin immunoreactivity. Many colonic adenomas retain expression of full-length APC protein whereas it is usually lost in colorectal cancers. Regardless of the status of APC protein expression, beta-catenin protein was found in the cytoplasm and nucleus of all neoplastic colonic mucosa. The dissociation between loss of expression of APC and accumulation of beta-catenin in the nucleus suggests that inactivation of both alleles of the APC gene may not be required for beta-catenin nuclear accumulation in colonic adenomas.     

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。     

Overexpression of c-met proto-oncogene but not epidermal growth factor receptor or c-erbB-2 in primary human colorectal carcinomas.

The epidermal growth factor receptor (EGFR) and the protein products of c-erbB-2 and c-met proto-oncogenes belong to a family of growth factor receptors with tyrosine kinase activity. In human colonic carcinomas, the expression of the EGFR and c-erbB-2 have been studied at the protein level only, while c-met expression has not been reported. We have examined the mRNA expression of these genes in human normal colorectal mucosa and primary carcinomas. The results demonstrate that the normal mucosa shows highly variable levels of EGFR and c-erbB-2 mRNAs, but expresses consistently low amounts of c-met mRNA. Colorectal carcinomas did not express significantly higher levels of the EGFR and c-erbB-2 mRNAs than the normal mucosa. In contrast, c-met was consistently and significantly overexpressed (mean sixfold) in carcinomas as compared with normal mucosa. Seventy percent of paired normal-tumor specimens showed a tumor to normal c-met mRNA ratio of greater than 4. The expression of c-met mRNA was also enhanced in the adenomas, suggesting that over-expression of this proto-oncogene may have mechanistic significance in the early stages of human colorectal carcinogenesis.