Tumor initiating cells are a small subset of cancer cells responsible for tumor growth and recurrence. The status of tumor initiating cells was measured using the surface markers CD133 (prominin-1) and ESA (epithelial-specific antigen). The aims of this study were to investigate the significance of CD133(+)/ESA(+) cells in mesenteric venous blood (MVB) and tumor mass (TM) for overall survival (OS) and disease-free survival (DFS) in colorectal cancer (CRC) patients undergoing curative resection.

A total of 229 CRC patients undergoing curative resection were prospectively enrolled in the study. Using CD133 and ESA as surface markers, CD133(+)/ESA(+) cells were enumerated from MVB and TM using flow cytometry.

We analyzed the presence of CD133(+)/ESA(+) cells in TM from 158 patients and found no correlation to patient DFS, OS, or clinical stage. In 135 patients, an analysis of CD133(+)/ESA(+) cells in MVB showed an inverse correlation with both DFS and OS (P = 0.014 and P = 0.008, respectively). It exhibited an increase-then-decrease pattern with the peak in stage II patients. A multivariate Cox analysis demonstrated that the status of CD133(+)/ESA(+) cells in MVB, but not the TM, was a significant prognostic factor for DFS and OS (P = 0.003 and P = 0.011, respectively).

The status of CD133(+)/ESA(+) cells in MVB, but not in TM, could be a useful indicator for predicting tumor recurrence and a prognostic marker for CRC patients.

Tumor budding is a histological feature that reflects loss of adhesion of tumor cells and is associated with locoregional metastasis of colorectal carcinoma. Although nuclear localization of beta-catenin is associated with tumor budding, the molecular mechanism remains largely elusive. In this study, we hypothesize that the epithelial cell adhesion molecule (Ep-CAM) is involved in tumor budding. In order to address this question, we performed immunohistochemistry on Ep-CAM using three different antibodies (monoclonal antibodies Ber-ep4 and 311-1K1 and a polyclonal antibody) and a double staining on beta-catenin and Ep-CAM. In addition, Ep-CAM mRNA was monitored with mRNA in situ hybridization. Subsequently, we determined the effect of Ep-CAM staining patterns on tumor spread in rectal cancer. In contrast to the tumor mass, budding cells of colorectal carcinoma displayed lack of membranous but highly increased cytoplasmic Ep-CAM staining and nuclear translocation of beta-catenin. mRNA in situ hybridization suggested no differences in Ep-CAM expression between the invasive front and the tumor mass. Importantly, reduced Ep-CAM staining at the invasive margin of rectal tumor specimens (n=133) correlated significantly with tumor budding, tumor grade and an increased risk of local recurrence (P=0.001, P=0.04 and P=0.03, respectively). These data demonstrate abnormal processing of Ep-CAM at the invasive margin of colorectal carcinomas. Our observations indicate that loss of membranous Ep-CAM is associated with nuclear beta-catenin localization and suggest that this contributes to reduced cell-cell adhesions, increased migratory potential and tumor budding.

The early detection of colorectal cancer is desired because this cancer can be cured surgically if diagnosed early. The purpose of the present study was to determine the feasibility of a new methodology for isolating colonocytes from naturally evacuated feces, followed by cytology or molecular biology of the colonocytes to detect colorectal cancer originating from any part of the colorectum.

Several simulation studies were conducted to establish the optimal methods for retrieving colonocytes from any portion of feces. Colonocytes exfoliated into feces, which had been retrieved from 116 patients with colorectal cancer and 83 healthy volunteers, were analyzed. Part of the exfoliated colonocytes was examined cytologically, whereas the remainder was subjected to DNA analysis. The extracted DNA was examined for mutations of the APC, K-ras, and p53 genes using direct sequence analysis and was also subjected to microsatellite instability (MSI) analysis.

In the DNA analysis, the overall sensitivity and specificity were 71% (82 of 116) of patients with colorectal cancer and 88% (73 of 83) of healthy volunteers. The sensitivity for Dukes A and B was 72% (44 of 61). Furthermore, the sensitivity for cancers on the right side of the colon was 57% (20 of 35). The detection rate for genetic alterations using our methodology was 86% (80 of 93) when the analysis was limited to cases in which genetic alterations were present in the cancer tissue.

We have developed a new methodology for isolating colonocytes from feces. The present study describes a promising procedure for future clinical evaluations and the early detection of colorectal cancers, including right-side colon cancer.

Because of their suggested link with microsatellite instability high colorectal cancers, right sided hyperplastic polyps (HPs) may differ from their distally located counterparts. This is highlighted by the recognition of a variant HP, termed sessile serrated adenoma (SSA), which predominates in the proximal colon. HPs displaying the morphological features now associated with SSAs have been shown to have altered expression of "cancer associated" markers, but no studies have investigated whether this is dependent on anatomical location of the polyps.

To evaluate morphological and functional features in right versus left sided HPs from patients without colorectal cancer with the aim of identifying distinguishing characteristics.

HPs originating in the proximal and distal colorectum were histochemically and immunohistochemically stained to evaluate a panel of markers related to proliferation and differentiation. In addition, a series of morphological features was evaluated for each polyp.

Crypt serration, crypt dilatation, and horizontal crypt growth were more common among HPs from the right side, whereas histochemical factors including mucin changes, global methylation status, and expression of carcinoembryonic antigen were not significantly different. An age disparity was also seen between patients with right versus left sided lesions, with patients with right sided lesions being an average of more than 10 years younger than those with left sided lesions.

These findings suggest that right and left sided HPs differ mainly in terms of growth regulation rather than cellular differentiation, implying that these lesions belong to a continuous spectrum of serrated polyps that differ quantitatively rather than qualitatively.

Cancer vaccination has become an important focus of oncology in recent years. Active immunization with tumor-associated antigens such as colorectal cancer antigen GA733-2 is thought to potentially overcome the reoccurrence of metastasis. As recombinant protein production in bioreactors is costly and subject to growing safety concerns, we tested plants as an alternative for the expression of a potential colorectal cancer vaccine. Comparing colorectal cancer antigen GA733-2 produced in tobacco plants with the same antigen produced in insect cell culture, we found a similar humoral immune response to injection of either of the two antigen preparations into mice. Some minor differences were observed in the cellular response that might be due to impurities. Our studies compare for the first time, immunization with the same antigen expressed in either plants or insect cell culture. This will provide important data for use of plants as production systems of therapeutics.

Cell adhesion receptors (CAMs) are actively involved in regulating various cell processes, including growth, differentiation, and cell death. Therefore, CAMs represent a large group of morphoregulating molecules, mediating cross-talk between cells and of cells with their environment. From this perspective, CAMs do contribute to cells and tissue organization, and in diseased tissue, to the disease development and biological characteristics. Therefore, observed changes in expression patterns of adhesion molecules may contribute to establish a diagnosis. A distinct shift in expression patterns in neoplastic epithelium has been described, for example for cadherins, integrins, and CD44. A relatively novel cell CAM, Ep-CAM, was first reported to be a pan-carcinoma antigen, although it is rather a marker of epithelial lineage. Several antibodies directed to Ep-CAM have been generated, and many epithelial tissues and their neoplastic appendages have been studied. This article outlines the results of these studies. Based on the results of these studies, we conclude that Ep-CAM immunohistochemistry can be a useful tool in the diagnosis of disturbed epithelial tissues.

It has now been demonstrated in both experimental models and recent clinical trials that certain "self" antigens, which are functionally non-immunogenic in the host, can become immunogenic if presented to the immune system in a certain way. Here, we describe recombinant vaccines and vaccine strategies that have been developed to induce and potentiate T-cell responses of the host to such self-antigens. These strategies include: (a) the use of recombinant poxvirus vectors in which the tumor-associated antigen (TAA) is inserted as a transgene. Recombinant vaccinia vaccines and recombinant avipox (replication-defective) vaccines have been employed to break tolerance to a self-antigen; (b) the use of diversified prime and boost strategies using different vaccines; and (c) the insertion of multiple T-cell co-stimulatory molecules into recombinant poxvirus vectors, along with the TAA gene, to enhance T-cell immune responses to the TAA and induce anti-tumor immunity.

Increasingly, data from distinct experimental systems show that immunity can be activated to prevent tumors. The rationale for prevention is strong because, in that setting, one deals with an immune system that is neither impaired by tumor- and treatment-induced suppression nor tolerant to tumor-associated antigens that have been encountered in the absence of correct presentation and costimulatory/danger signals. The use of overexpressed or mutated proteins, or mutated oncogenic growth factor receptors, as tumor-associated antigens yields rational targets for specific immunoprevention. Transgenic mouse models are providing encouraging indications of future usefulness of vaccines that are based on these molecules.

Adenomatous polyps (AP) of the gastrointestinal tract in children are very rare. Because of their potential malignancy, they are of great clinical importance. There is little experience in the management of children with AP. The immunohistochemical expression of the Lewis blood group antigens (BGA) (sialosyl-Le(a), Le(a), Leb, Le(x), and Le(y)) and the number of activated nucleoli with the silver staining method for nucleolar organizer regions (AgNORs) were studied in two children with AP. In a girl with isolated AP of the stomach and colon, it was found that antigens Le(b) and s-Le(a) were expressed extensively in the gastric adenoma, and sialosyl-Le(a) throughout the entire length of the rectal adenoma crypts, but in the AgNORs stain the number of nucleoli ranged from two to four, evidencing changes of a benign character. In the case of familial adenomatous polyposis diagnosed in a 9-year-old boy, in some colonic adenomas the number of activated nucleoli was greater than five, and the Le(b) antigen was expressed in superficial epithelial cells in one of the adenomas. Also, extensive expression of antigens Le(y) and s-Le(a) throughout the entire length of the crypt in another polyp removed was observed. We believe that immunohistochemical study of the intensity and extent of the expression of Lewis BGA in the polyp tissue simultaneously with the determination of the number of activated nucleoli by the AgNORs staining method can be helpful in better analysis of cytological risk factors of a malignant transformation.

Clinical utility of murine mAbs is limited because many elicit Abs to murine Ig constant and variable regions in patients. An Ab humanized by the current procedure of grafting all the complementarity determining regions (CDRs) of a murine Ab onto the human Ab frameworks is likely to be less immunogenic, except that its murine CDRs could still evoke an anti-variable region response. Previous studies with anticarcinoma mAb CC49 showed that light chain LCDR1 and LCDR2 of humanized CC49 could be replaced with the corresponding CDRs of a human Ab with minimal loss of Ag-binding activity. The studies reported in this paper were undertaken to dissect the CC49 Ag-binding site to identify 1) specificity determining residues (SDRs), the residues of the hypervariable region that are most critical in Ag-Ab interaction, and 2) those residues that contribute to the idiotopes that are potential targets of patients' immune responses. A panel of variants generated by genetic manipulation of the murine CC49 hypervariable regions were evaluated for their relative Ag-binding affinity and reactivity to sera from several patients who had been immunized with murine CC49. One variant, designated HuCC49V10, retained only the SDRs of CC49 and does not react with the anti-variable region Abs of the sera from the murine CC49-treated patients. These studies thus demonstrate that the genetic manipulation of Ab variable regions can be accomplished by grafting only the SDRs of a xenogeneic Ab onto human Ab frameworks. This approach may reduce the immunogenicity of Abs to a minimum.

One of the major obstacles in the successful clinical application of monoclonal antibodies has been the development of host immune responses to murine Ig constant and variable regions. While the CDR grafting of MAbs may alleviate many of these problems, the potential remains that one or more murine CDRs on the human Ig backbone of a "humanized" MAb may still be immunogenic. Studies were undertaken employing a MAb of potential clinical utility, CC49, to define those CDRs that are essential for antigen binding and those that may be immunogenic in humans. We previously developed a humanized CC49 (HuCC49) by grafting the MAb CC49 hypervariable regions onto frameworks of human MAbs. To identify those CDRs essential for binding, a panel of variant HuCC49 MAbs was generated here by systematically replacing each of the murine CDRs with their human counterparts. The relative affinity constant of each variant was determined. Serum from a patient who received murine CC49 was used to determine the potential immunogenicity of each CDR in humans. The serum was shown to react with the anti-CC49 variable region. Results showed that patients' anti-idiotypic responses are directed mainly against LCDR3 and moderately against LCDR1 and HCDR2. These studies demonstrate for the first time that variants containing individual CDR substitutions of a humanized MAb can be constructed, and each CDR can be defined for the two most important properties for potential clinical utility: antigen binding and immunogenicity.

Ep-CAM, an epithelium-specific cell-cell adhesion molecule (CAM) not structurally related to the major families of CAMs, contains a cytoplasmic domain of 26 amino acids. The chemical disruption of the actin microfilaments, but not of the microtubuli or intermediate filaments, affected the localization of Ep-CAM at the cell-cell boundaries, suggesting that the molecule interacts with the actin-based cytoskeleton. Mutated forms of Ep-CAM were generated with the cytoplasmic domain truncated at various lengths. All of the mutants were transported to the cell surface in the transfectants; however, the mutant lacking the complete cytoplasmic domain was not able to localize to the cell-cell boundaries, in contrast to mutants with partial deletions. Both the disruption of the actin microfilaments and a complete truncation of the cytoplasmic tail strongly affected the ability of Ep-CAM to mediate aggregation of L cells. The capability of direct aggregation was reduced for the partially truncated mutants but remained cytochalasin D sensitive. The tail truncation did not affect the ability of the transfectants to adhere to solid-phase-adsorbed Ep-CAM, suggesting that the ability to form stable adhesions and not the ligand specificity of the molecule was affected by the truncation. The formation of intercellular adhesions mediated by Ep-CAM induced a redistribution to the cell-cell boundaries of alpha-actinin, but not of vinculin, talin, filamin, spectrin, or catenins. Coprecipitation demonstrated direct association of Ep-CAM with alpha-actinin. Binding of alpha-actinin to purified mutated and wild-type Ep-CAMs and to peptides representing different domains of the cytoplasmic tail of Ep-CAM demonstrates two binding sites for alpha-actinin at positions 289 to 296 and 304 to 314 of the amino acid sequence. The results demonstrate that the cytoplasmic domain of Ep-CAM regulates the adhesion function of the molecule through interaction with the actin cytoskeleton via alpha-actinin.

The contribution of noncadherin-type, Ca2+-independent cell-cell adhesion molecules to the organization of epithelial tissues is, as yet, unclear. A homophilic, epithelial Ca2+-independent adhesion molecule (Ep-CAM) is expressed in most epithelia, benign or malignant proliferative lesions, or during embryogenesis. Here we demonstrate that ectopic Ep-CAM, when expressed in cells interconnected by classic cadherins (E- or N-cadherin), induces segregation of the transfectants from the parental cell type in coaggregation assays and in cultured mixed aggregates, respectively. In the latter assay, Ep-CAM-positive transfectants behave like cells with a decreased strength of cell-cell adhesion as compared to the parental cells. Using transfectants with an inducible Ep-CAM-cDNA construct, we demonstrate that increasing expression of Ep-CAM in cadherin-positive cells leads to the gradual abrogation of adherens junctions. Overexpression of Ep-CAM has no influence on the total amount of cellular cadherin, but affects the interaction of cadherins with the cytoskeleton since a substantial decrease in the detergent-insoluble fraction of cadherin molecules was observed. Similarly, the detergent-insoluble fractions of alpha- and beta-catenins decreased in cells overexpressing Ep-CAM. While the total beta-catenin content remains unchanged, a reduction in total cellular alpha-catenin is observed as Ep-CAM expression increases. As the cadherin-mediated cell-cell adhesions diminish, Ep-CAM-mediated intercellular connections become predominant. An adhesion-defective mutant of Ep-CAM lacking the cytoplasmic domain has no effect on the cadherin-mediated cell-cell adhesions. The ability of Ep-CAM to modulate the cadherin-mediated cell-cell interactions, as demonstrated in the present study, suggests a role for this molecule in development of the proliferative, and probably malignant, phenotype of epithelial cells, since an increase of Ep-CAM expression was observed in vivo in association with hyperplastic and malignant proliferation of epithelial cells.

Tissues and sera from 110 patients diagnosed with colorectal primary carcinoma, 20 patients with benign colorectal diseases and 31 healthy donors were subjected to quantitative CEA analysis. Multiple samples from tumor lesions and autologous histologically normal mucosa (10 cm from the tumor) were obtained at the time of surgery (cancer patients) or endoscopy (benign patients and healthy volunteers). CEA content was measured in protein extracts obtained from these tissues using a quantitative RIA method. A limit of normality for CEA content was established as 300 ng/mg of protein. When this was taken as cut-off, 104 of 110 (94.5%) tumor lesions and 51 of 110 (46.4%) autologous histologically normal colonic mucosa from cancer patients had elevated CEA levels. No correlation with stage of disease was found, while a correlation was observed with degree of tumor differentiation. A statistically significant difference between CEA content in tumor lesions and in histologically normal mucosa from cancer patients was observed (p = -0.001). Moreover, CEA content was statistically higher in the normal mucosa from cancer patients than in that from healthy donors (p = 0.005). CEA content in tissue specimens from benign lesions differed significantly from that in tissue from healthy donors (p = 0.005) and in carcinoma lesions (p < 0.001). The highest CEA content was observed in benign lesions with severe dysplasia. No statistical correlation between CEA content in carcinoma tissues and serum CEA levels (r = 0.195, p = .13) was found. Therefore, in considering diagnosis or therapy with anti-CEA MAbs for colorectal-carcinoma patients, or potential therapies with anti-CEA recombinant vaccines, serum CEA levels should not be taken as indicating CEA expression in tumor lesions.