Connie L. Finstad

Serological and immunochemical analysis of Lewis Y (Ley) blood group antigen expression in epithelial ovarian cancer

The expression of Ley blood group antigen in epithelial ovarian cancer tissues and cell lines has been studied using a Ley‐specific monoclonal antibody (MAb 35193). In ovarian cancer specimens, Ley was expressed in 75% of the 140 tumor specimens examined, with strong or moderate expression being observed in 56% of the samples. Seven of the 11 ovarian cancer cell lines studied were Ley‐positive. Using immunochemical approaches, Ley epitopes were found to be expressed on 4 types of carrier molecules: CA125 ovarian cancer antigen, MUC‐I mucins, lower m.w. glycoprotiens and glycolipids. In cell lines, Ley was more commonly expressed on MUC‐I mucin than on CA125, whereas in tumor specimens Ley was commonly found on both CA125 and MUC‐I. The biochemical nature of the smaller Ley glycoproteins was not determined, but it was shown that they were not CEA and LAMP‐I, known Ley carriers in some other tumor types. Glycolipids carrying Ley epitopes were detected in both ovarian cancer cell lines and tumor specimens. The presence of Ley epitopes on a number of different molecular carriers, including 2 major ovarian cancer antigens (CA125 and MUC‐I), explains the high incidence of Ley in ovarian cancer. The high expression of Ley in ovarian cancer and the availability of specific murine and humanized MAbs make Ley an attractive candidate target for clinical studies.

Expression and localization of aminopeptidase A, aminopeptidase N, and dipeptidyl peptidase IV in benign and malignant human prostate tissue.

Cell‐surface peptidases are ectoenzymes which regulate the access of bioactive peptides to their receptors on cell membranes. Abnormalities in their expression and function result in altered peptide activity which contribute to neoplastic transformation and/or progression.

Comparison of MUC-1 mucin expression in epithelial and non-epithelial cancer cell lines and demonstration of a new short variant form (MUC-1/Z).

Mucins, including MUC‐1, are generally considered to be products of epithelial tissues and of their tumors. To examine the possible expression of MUC‐1 in other cell types, a panel of human epithelial and non‐epithelial tumor cell lines was studied by reverse transcriptase polymerase chain reaction (RT‐PCR), Northern blot analysis, immunocytology and radio‐immunoprecipitation. Using the highly sensitive RT‐PCR method, products corresponding to the non‐repetitive 5′ and 3′ MUC‐1 sequences were detected in all the cell lines examined. Amplified products lacking the tandem repeat region of MUC‐1, including a new short form (designated MUC‐1/Z) different from the previously reported MUC‐1/Y protein, were also detected in most cell lines tested. Northern blot analysis, using a probe to the variable number tandem repeat (VNTR) region, confirmed the presence of MUC‐1 mRNA in the astrocytoma, melanoma and neuroblastoma cell lines studied. MUC‐1 protein was detected by immunocytology in these cell lines using monoclonal antibody (MAb) 139H2. Immunoprecipitation analysis with [3H]‐glucosamine‐labeled cell lysates and MAb 139H2 or an antibody to the cytoplasmic domain, CT‐1, detected MUC‐1 protein in 2 epithelial cell lines, an astrocytoma cell line (SK‐MG‐4) but not in the melanoma and neuroblastoma cell lines studied. Northern blot analysis using a probe to the 3′ end of MUC‐1 mRNA, confirmed the presence of MUC‐1 mucin and also identified short products corresponding to the size of the short variant forms. Protein products corresponding to the MUC‐1/Y and MUC‐1/Z variant forms were not ob‐ served using either [3H]‐glucosamine‐labeled OVCAR‐3 cells or [3H]‐amino acid‐labeled MCF‐7 cells and either CT‐1 antibody or MAb 232A1, detecting an epitope to the C‐terminal region. Thus, depending on the sensitivity of the assay used, varying amounts of MUC‐1 mRNA and protein could be detected in non‐epithelial tumor cell lines. Although the amounts of MUC‐1 in these cell lines are much lower than in carcinomas, it is possible that MUC‐1 mucin serves a similar function in non‐epithelial as in epithelial cells. The possible role of MUC‐1/Y and MUC‐1/Z variant forms in these cell lines is not understood. Int. J. Cancer 72:87–94, 1997.

Selection of carbohydrate antigens in human epithelial ovarian cancers as targets for immunotherapy: Serous and mucinous tumors exhibit distinctive patterns of expression

Expression of blood group–related carbohydrate antigens was examined in frozen sections from a series of ovarian carcinomas of different histological types using an indirect immunoperoxidase technique. Antigenic specificities belonging to the O(H) and Lewis blood group families (H‐1, H‐2, Lea, sLea, Lex, sLex, Leb and Ley) or the mucin‐core family (Tn, sTn and TF) were studied. A distinct difference in antigen expression between mucinous and other ovarian carcinomas (serous and endometrioid) was observed. Specifically, mucinous tumors tended to express sTn, Lea and sLea strongly and homogeneously, whereas serous and endometrioid tumors rarely expressed these specificities and, in contrast, expressed Ley and H type 2 antigen strongly. When expressed in serous tumors, sTn was usually distributed in a heterogeneous pattern, whereas sTn expression in mucinous tumors was much more homogeneous. The distribution of Ley in serous tumors was noticeably homogeneous. H‐1, Lex, sLex,, Leb , TF and Tn specificities were rarely expressed in any type of ovarian carcinoma. Our results provide further support for the different histogenesis of mucinous and non‐mucinous tumors and indicate alternative differentiation pathways for the 3 pathological subtypes of ovarian tumor. They also provide the basis for the choice of carbohydrate antigens for active and passive immunotherapy of ovarian carcinomas. Int. J. Cancer 81:193–198, 1999.

Expression of P-glycoprotein in epithelial ovarian cancer: Evaluation as a marker of multidrug resistance

The multidrug-resistance gene, MDR1, encodes a plasma membrane glycoprotein termed P-glycoprotein that mediates active cellular efflux of certain chemotherapeutic agents. P-Glycoprotein expression was evaluated in 98 frozen tumor specimens from 57 patients with epithelial ovarian cancer by the indirect immunoperoxidase technique with monoclonal antibodies C219 and JSB-1 used for detection. Tumor specimens were further characterized antigenically with a panel of monoclonal antibodies representing a variety of epithelial cell antigens. Included were 57 specimens from 33 previously untreated patients; 11 specimens were also available from eight patients in this group after chemotherapy. An additional 30 specimens were studied from 24 other patients after chemotherapy. In only four of the 57 patients with ovarian cancer (7%) did one or more of the specimens express P-glycoprotein. Two of these patients had tumors that were considered clinically drug resistant. No increase in P-glycoprotein expression was noted after exposure to chemotherapy, including the eight individuals for whom specimens were available both before and after treatment. Although drug resistance is a major problem in treatment of ovarian cancer, resistance to the drugs most active against these tumors probably occurs through a mechanism other than expression of the MDR1 gene product.

A longitudinal study of antigen expression in epithelial ovarian cancer

The extent to which the antigenic phenotype of human epithelial ovarian cancer changes during the course of the disease is an issue that must be addressed in order to maximize the potential of antibody-directed imaging and therapy. We have obtained tumor specimens at two separate operations from ten patients with epithelial ovarian cancer and typed these specimens with a panel of 19 monoclonal antibodies to cell surface glycoprotein and carbohydrate antigens including blood group antigens. Antibodies with relatively high specificity for malignant cells as well as those detecting more widely distributed epithelial antigens were used in the study. Mean time between the two operations was 8.5 months. Five patients received intraperitoneal therapy during the interval between the two operations, including platinum-based regimens (four patients) and tumor necrosis factor (one patient). Four patients received intravenous platinum-based chemotherapy; one received no treatment. Frozen sections of specimens were stained with the antibodies by the indirect immunoperoxidase technique. Antigenic phenotypes were found to be unrelated to the patients age, stage, tumor grade, histologic cell type, prior chemotherapy, and interval between operations. Most significantly, little difference was seen in antigenic expression between tumors obtained at the two operations for either the cell surface or blood group markers. Variations in the staining pattern were seen with antibody B72.3 and, to some extent, with the anti-blood group antibodies, which are known for producing heterogeneous staining. The antigenic phenotype of the tumor specimens in a given patient as determined immunohistochemically by our panel of antibodies showed only minor variation between operations, even under the selective pressures of chemotherapy and immunotherapy.

Altered Regulation of Cell Surface Peptidases in Human Cholesteatoma

Cholesteatoma is a destructive process involving an accumulation of desquamated keratin arising from squamous epithelium that pathologically has invaded the middle ear or mastoid process. The clinical hallmarks of cholesteatomas, namely invasion of healthy tissues, migration, unrestrained proliferation, aggressiveness, recidivism, and uncoordinated differentiation predict the existence of defects in the normal biology and biochemistry of the cellular constituents that compose a cholesteatoma, as well as in the cellular interactions between these cells, the surrounding normal tissue, and the host. In the current report, we analyzed 11 cholesteatomas and matched healthy tissue for altered expression in four different cell surface peptidases, aminopeptidase A, aminopeptidase N, dipeptidyl peptidase IV, and neutral endopeptidase. We suggest that peptidases may modulate cell growth and differentiation by inactivating stimulatory signals (or conversely, by activating inhibitory signals).

Reversal of the low-affinity neurotrophin receptor stromal-epithelial expression pattern between benign and malignant human prostate

Reduced expression of the low-affinity p75 neurotrophin receptor (p75(NTR)) occurs in prostate epithelial cells during malignant transformation. Recent studies indicating that the p75(NTR) can transduce signals that induce apoptosis suggest that diminished p75(NTR) in transformed prostate cells may contribute to immortalization. Mutations in the transmembrane domain of the p75(NTR) gene have been associated with decreased p75(NTR) protein expression and may block the ability of the p75(NTR) to induce apoptosis. Therefore, we used Western blot to analyze prostate cancer (PC) cell lines for p75(NTR) protein expression and gene single strand conformation polymorphism (SSCP) analysis and direct DNA sequencing to analyze mutations in the transmembrane domain of the p75(NTR). p75(NTR) Protein was present in all cell lines, and mutations in the p75(NTR) gene were not detected in cDNA derived from any cell line. To define the expression pattern of p75(NTR) in PCs in vivo, we used immunohistochemical techniques to examine tissue specimens from 20 benign, 19 malignant primary, and 14 metastatic prostate specimens. In benign prostate tissues, expression of p75(NTR) was universally detected in basal cells but not in secretory epithelial or stromal cells. In both primary and metastatic PC tissues, p75(NTR) immunoreactivity could not be detected in malignant prostate epithelial cells. However, in contrast to the benign prostate, p75(NTR) protein was expressed in stromal cells surrounding malignant epithelial cells. Stromal p75(NTR) expression was present in 84% (16 of 19) primary and in 86% (12 of 14) metastatic specimens. These data show that in the benign prostate p75(NTR) protein is expressed by basal cells and not stromal cells whereas in malignant prostate p75(NTR) protein is expressed by stromal cells but not prostatic carcinoma cells. Reversal of the p75(NTR) stromal-epithelial pattern of expression between benign and malignant prostate suggests that p75(NTR) may contribute to the development and maintenance of prostate cancer.