Changing roles of cadherins and catenins during progression of squamous intraepithelial lesions in the uterine cervix.

Uterine cervix represents a convenient model for the study of the gradual transformation of normal squamous epithelium via low- to high-grade squamous intraepithelial lesions (SILs). Because SIL, on the basis of the cytokeratins expressed, are thought to originate from the reserve cells, we analyzed whether SILs also show a reserve cell phenotype with respect to intercellular interactions. The changes in expression and subcellular localization of the components of the adherens junction and desmosomal complexes were investigated in normal, metaplastic, and premalignant cervical epithelium, as well as in cell cultures derived from these tissues. The results suggest that 1) during progression of SILs, E-cadherin is suppressed, with its role in cell-cell connections diminishing; 2) P-cadherin, in contrast, becomes the predominant cadherin in high-grade SILs; 3) the level of cellular alpha-catenin is dramatically decreased in high-grade SILs; 4) the level of beta-catenin is decreased during progression of SILs, with plakoglobin suggestively becoming the predominant catenin mediating connection of cadherins to the cytoskeleton; 5) the assembly of desmosomes is affected during progression of SILs and is accompanied by a dramatically decreased expression for desmogleins and desmoplakins (I, II); and 6) expression of differentiation markers (involucrin, CK13) in high-grade SILs seems to be controlled by P-cadherin as opposed to E-cadherin in the normal tissue counterpart. We conclude that during development of cervical lesions substantial (both quantitative and qualitative) changes occur in cell-cell junctions, making the interactions of cells in lesions dissimilar from those of reserve cells, basal cells, or cells of immature squamous metaplasia, despite existing morphological similarity between all of these cell types and cells of high-grade lesions.

Expression of Ep-CAM in normal, regenerating, metaplastic, and neoplastic liver.

Ep-CAM is a homophilic, Ca2+-independent cell-cell adhesion molecule that is expressed in many human epithelial tissues. Its increased expression is closely associated with active cell proliferation. Furthermore, in epithelial cell types that in adults lack Ep-CAM (i. e. squamous epithelia), up-regulation of Ep-CAM coincides with the early stages of neoplastic change. This study has analysed the expression of Ep-CAM in liver, in the hepatocytes and cells of the biliary duct system, in relation to proliferative diseases and carcinogenesis. Adult hepatocytes are Ep-CAM negative, with only bile duct epithelium being positive in the liver tissue. However, in the 8-week embryonic liver, the majority of hepatocytes express Ep-CAM. During regeneration and repair of liver tissues associated with focal nodular hyperplasia and (biliary) cirrhosis, activation of Ep-CAM expression was observed, with high expression levels in so-called 'ductular proliferations'-regenerating stem cells. During precursor cell differentiation into mature hepatocytes, several intermediate morphological stages could be observed, all Ep-CAM positive, including cells morphologically close to mature hepatocytes. Full maturation of the precursor resulted in the disappearance of Ep-CAM expression. The results suggest that expression of Ep-CAM is a prerequisite of the proliferative phenotype during differentiation of hepatocyte precursors. In liver neoplasia, Ep-CAM was expressed in almost all cholangiocarcinomas (10/11), whereas the majority of hepatocellular carcinomas (8/10) were negative, suggesting that malignant proliferation of hepatocellular carcinoma cells is not related to expression of Ep-CAM and that hepatocellular carcinoma originates from a highly differentiated precursor. The results indicate that Ep-CAM can be used as an additional immunohistochemical marker to distinguish cholangiocarcinoma from hepatocellular carcinoma due to the differential expression of Ep-CAM in these tumours.

The impact of antigen density and antibody affinity on antibody-dependent cellular cytotoxicity: relevance for immunotherapy of carcinomas.

Antibody-dependent cellular cytotoxicity (ADCC) is considered to be the major mechanism through which tumour cells, upon treatment with anti-tumour MAbs, are eliminated in vivo. However, the relative importance of various parameters that influence the efficacy of ADCC is unclear. Here we present in vitro data on the impact of MAb affinity and antigen density on ADCC, as obtained by comparison of two MAbs against the tumour-associated antigen Ep-CAM. The low-affinity MAb 17-1A (Ka = 5 x 10(7)M(-1)) currently used for therapy, and the high-affinity MAb 323/A3 (Ka = 2 x 10(9) M(-1)), were compared in ADCC experiments against murine and human tumour target cells transfected with the Ep-CAM cDNA under the control of an inducible promoter to enable regulation of the target antigen expression levels. Data obtained from these studies revealed that the high-affinity MAb, in contrast to the low-affinity MAb, could mediate killing of tumour cells with low antigen expression levels. Even at comparable MAb-binding levels, ADCC mediated by the high-affinity MAb was more effective. The kinetics of ADCC was also found to be determined by the level of antigen expression, and by the affinity and the concentration of the MAb used. The efficacy of ADCC with both low- and high-affinity MAbs further depended on adhesive interactions between effector and target cells mediated by CD18. However, at every given MAb concentration these interactions were of less importance for the high-affinity MAb than for the low-affinity MAb. As heterogeneity of a target antigen expression is a common feature of all tumours, and some tumour cells express very low levels of the antigen, the use of high-affinity MAbs in immunotherapy may significantly improve the clinical results obtained to the present date in the treatment of minimal residual disease.

The role of monoclonal antibody affinity in tumor immunotherapy evaluated in in vivo models for minimal residual disease.

To evaluate the role of affinity in monoclonal antibody (mAb)-mediated treatment of carcinomas, we compared the antibodies 17-1A and 323/A3 that bind with different affinities overlapping epitopes on the epithelial adhesion molecule Ep-CAM. This comparison was performed in several models for minimal residual disease in mice grafted with Ep-CAM transfected B16 melanoma cells originating from C57BL/6 mice. These cells were either grafted subcutaneously or injected intravenously into nude BALB/c mice, or grafted subcutaneously in immunocompetent C57BL/6 mice. In the BALB/c subcutaneous model, significant therapeutic results (p < 0.05) compared with the control mAb were obtained with both mAbs 17-1A and 323/A3. However, when treating lung metastases in nude BALB/c mice that had developed after intravenous injection of the B16/Ep-CAM tumor cells, only the high-affinity 323/A3 mAb could significantly (p < 0.05) reduce the number of metastases that appeared. In syngeneic C57BL/6 mice grafted subcutaneously with B16/ Ep-CAM cells, a single 323/A3 or 17-1A mAb injection had no effect, in contrast to that observed for the nude BALB/c mouse model. However, multiple injections of the 323/A3 mAb significantly (p < 0.005) reduced the mean tumor volume, although they did not prevent tumor development. The results show that in vivo antibody-mediated effector cell activation and subsequent tumor cell elimination is determined by mAb affinity and target antigen density. Therefore, treatment of minimal residual disease with high-affinity mAb 323/ A3 is expected to improve the clinical results obtained with mAb 17-1A.

Expression of Ep-CAM in cervical squamous epithelia correlates with an increased proliferation and the disappearance of markers for terminal differentiation.

Ep-CAM, an epithelial adhesion molecule, is absent in normal squamous epithelia but can be detected in some squamous carcinomas. Using a panel of monoclonal antibodies to keratinocyte differentiation and proliferation markers, we investigated the association of EP-CAM expression with differentiation-related and/or neoplastic changes in cervical epithelium. Normal endocervical glandular epithelium (Both columnar and reserve cells) appeared strongly positive for EP-CAM, whereas ectocervical squamous epithelial cells did not express this molecule. Expression of Ep-CAM (in basal cells) was sometimes observed in morphologically normal ectocervical tissue but only in areas bordering cervical intraepithelial neoplasia (CIN) lesions. At the early stages of neoplasia the expression of Ep-CAM was regularly present in squamous epithelium, in general consistent with the areas of atypical, undifferentiated cells. Thus, in CIN grades I and II, the basal/suprabasal layers of the epithelia were positive, whereas in CIN grade III lesions, up to 100% of the cells over the whole thickness of the epithelium sometimes excluding the very upper layers, expressed Ep-CAM. A clear increase, not only in number of positive cells but also in levels of Ep-CAM expression (intensity) was observed during progression from CIN I to CIN III. Expression of Ep-CAM in ectocervical lesions did not coincide with a reappearance of the simple epithelium cytokeratins (CK8 and CK18). On the other hand, expression of Ep-CAM in atypical cells of CIN lesions correlated with the disappearance of CK13, which normally marks cells undergoing squamous differentiation. As was shown with Ki-67, a marker for proliferating cell populations, the areas of Ep-CAM expression were also the areas of enhanced proliferation. Cells expressing Ep-CAM did not express involucrin, a marker for cells committed to terminal differentiation. In the majority of both squamous and adenocarcinomas of the cervix a strong expression of Ep-CAM was observed, although some decrease in the expression (both the intensity and the number of positive cells), as compared with CIN III lesions, was observed in the areas of squamous differentiation. This study demonstrates that the expression of Ep-CAM in cervical squamous epithelium is associated with abnormal proliferation of cell populations that are not committed to terminal differentiation.

Immunotherapy with low and high affinity monoclonal antibodies 17-1A and 323/A3 in a nude mouse xenograft carcinoma model.

The therapeutic effects of the low and high affinity mAbs 17-1A and 323/A3 were investigated in nude mice xenografted with LS 180 human colorectal carcinoma cells. Treatment of mice grafted with dispersed tumor cells, before formation of a tumor nodule, was started 1 day after s.c. injection of tumor cells and consisted of a single i.p. injection of murine 17-1A or 323/A3 mAb. Tumor appearance after a single injection of either 17-1A or 323/A3 was delayed as compared to injection of an irrelevant mAb. Both 17-1A and 323/A3 reduced the tumor growth rate, and both mAbs decreased the total number of mice that eventually developed a tumor. In all experiments, 323/A3 showed consistently better treatment effects on xenografted mice than mAb 17-1A. For treatment of established tumors with mAb 17-1A or 323/A3 therapy was delayed until a tumor nodule was macroscopically detectable. One single i.p. injection of mAb 17-1A had no effect on further tumor growth and mean tumor size as compared to the control group injected with irrelevant mAb. One single i.p. injection with mAb 323/A3 reduced the tumor growth rate in some mice with established tumors and resulted in a significant difference of mean tumor size of this group as compared to the 17-1A treated mice and the control groups. Multiple injections with mAb 17-1A also had no effects on established tumors, in contrast to mAb 323/A3, where serial injections resulted in tumor growth reduction and, eventually, in some mice reduction in tumor size. In summary, we showed that in nude mice mAb 323/A3 (Ka = 2 x 10(9) M-1) is much more potent than mAb 17-1A (Ka = 5 x 10(7) M-1) in eradication of nonestablished tumor cells and treatment of small established tumors. These results suggest that high affinity mAbs like 323/A3 might dramatically improve the clinical results obtained thus far with the low affinity mAb 17-1A in the adjuvant treatment of surgically resected Dukes C colorectal cancer patients with minimal residual disease.