Mark G. Federici

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.

High IL‐6 levels in ascitic fluid correlate with reactive thrombocytosis in patients with epithelial ovarian cancer

Summary. Non‐haematopoietic malignancies are commonly associated with thrombocytosis. The aetiology of tumour‐associated thrombocytosis is still unclear but may be related to tumour‐derived thrombopoietin‐like factors. Epithelial ovarian tumour cells have been shown to release IL‐6 in vitro and high IL‐6 levels have been identified in ascites of patients with ovarian cancer. Since IL‐6 is a potent stimulator of megakaryocytopoiesis we examined IL‐6 production at the tumour site and its relationship to serum IL‐6 levels and circulating platelet counts in patients with ovarian cancer. Forty patients undergoing exploratory laparotomy for epithelial ovarian cancer [stage I + II: 6 (15%); stage III: 25 (62.5%); stage IV: 9 (22.5%)] and 24 women with benign ovarian conditions were evaluated. Sera were available from 39 cases with ovarian cancer and from 19 cases with benign ovarian tumours. Ascites was obtained from 35 patients with ovarian cancer. IL‐6 activity in serum and ascitic fluid was determined by the standard B9 proliferation assay (detection level: 1 pg/ml). IL‐6 bioactivity was detectable in 22 (56%) sera from patients with ovarian cancer, but in only five (26%) of the serum samples obtained from benign cases (P < 0.001). Serum IL‐6 levels in patients with ovarian cancer were significantly higher (median 3 pg/ml; range < 1 to 1221 pg/ml) than in patients with benign ovarian conditions (median 0 pg/ml; range < 1 to 4 pg/ml) (P < 0.001). However, much higher concentrations of IL‐6 were measured in malignant ascites specimens (median 22 100 pg/ml; range < 1 to 182 600 pg/ml). IL‐6 bioactivity in serum and ascites samples was completely inhibited by a neutralizing goat antihuman IL‐6 antiserum. Thrombocytosis (platelet counts > 400 × 109/l) occurred in 25 (62.5%) of the 40 patients with ovarian cancer, but in only two (8%) of the 24 cases with benign ovarian tumours. In eight (20%) cases with malignant disease platelet counts ranged between 600 × 109/l and 1060 × 109/l. IL‐6 bioactivity in ascitic fluid correlated significantly with circulating platelet counts (r = 0.5916; P < 0.001). Maximum IL‐6 bioactivity in ascites and highest platelet counts occurred in patients with undifferentiated ovarian adenocarcinoma or advanced disease. In conclusion, these observations strongly suggest a role for IL‐6 in the development of tumour‐associated thrombocytosis.

Male breast cancer in the hereditary nonpolyposis colorectal cancer syndrome

A male member of a large HNPCC kindred, affected by primary malignancies of the breast and colon, was identified. This individual was found to harbor a germline mutation of the MLH1 mismatch repair gene previously shown to segregate with disease in this kindred. The breast tumor exhibited somatic reduction to homozygosity for the MLH1 mutation, and microsatellite instability was evident in the breast tumor. We conclude that hereditary male breast cancer can occur as an integral tumor in the HNPCC syndrome.

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.

Analysis of antigen expression at multiple tumor sites in epithelial ovarian cancer

The question of whether the antigenic phenotype of human epithelial ovarian cancer varies in a given patient between the primary tumor and metastatic sites or among metastatic sites themselves is an important issue in planning potential therapeutic strategies for ovarian cancer. We have obtained tumor specimens from at least two separate sites during operations on 12 patients with epithelial ovarian cancer, and we have typed these specimens with a group of 18 monoclonal antibodies that react with cell-surface glycoprotein and carbohydrate antigens, including blood group antigens. Antibodies with relative specificity for malignant cells as well as those that detect more widely distributed epithelial antigens were used. A total of 31 specimens from 12 patients with advanced adenocarcinoma (8 serous, 3 undifferentiated, 1 endometrioid) of the ovary were studied, including fresh ascites cells in two patients. Frozen sections of tumor specimens were stained with the antibodies by the indirect immunoperoxidase technique and graded semiquantitatively. Little difference was seen in antigenic expression of tumors that were obtained from various sites in the same patient for either the epithelial cell markers or blood group markers. Intratumoral antigenic heterogeneity was seen, but this was generally quite consistent within a given patients specimens. As anticipated, variations in antigen expression were seen among specimens from different patients. The antigenic phenotype of the tumor specimens in a given patient, as determined immunohistochemically by our group of antibodies, showed only minor variation among primary and metastatic sites.

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.

Expression and mutational analysis of P53 in stage IB and IIA cervical cancers

OBJECTIVE This study evaluates overexpression of the p53 protein and point mutation in the P53 gene in a group of patients with stage IB and IIA cervical cancer. STUDY DESIGN We reviewed the medical records of all patients who underwent radical hysterectomy for the treatment of stage IB and IIA cervical cancer between 1980 and 1985 at Memorial Sloan-Kettering Cancer Center. Overexpression of p53 protein was determined with the use of immunohistochemistry on fixed and paraffin-embedded tissue. Two blocks were selected for each tumor, and tissue sections from each block were tested with both monoclonal (Ab-6) and polyclonal (CM-1) anti-p53 antibodies. Molecular analysis for determination of specific P53 gene mutations was performed with single-strand conformation polymorphism analysis. A group of 132 patients was identified for inclusion in the study. RESULTS Fifty-eight of 132 tumors (44%) showed overexpression of the p53 protein and were subjected to molecular analysis. Discrepancy between pairs of blocks (7/132, 5.3%) and between antibodies for the same block (5/264, 1.9%) was uncommon. High-level overexpression was rare (5/132, 3.8%). No difference in survival was found on the basis of overexpression of p53 protein. Only one of the 58 cases (1/58, 1.7%) that showed overexpression of the p53 protein exhibited a point mutation (exon 8) in P53 by single-strand conformation polymorphism. This case had a low level of overexpression of p53 protein on immunohistochemistry. CONCLUSIONS Low levels of overexpression of p53 were frequently seen in early cervical cancers (40/132, 30%). However, mutation of the P53 gene was rarely seen in these tumors. Overexpression of p53 protein as detected by immunohistochemistry is not predictive of a somatic mutation in the P53 gene in cervical cancer. Molecular analysis is required for confirmation of P53 mutations in these tumors.

Synergistic activity of tumor necrosis factor and interferon in a nude mouse model of human ovarian cancer.

In vitro and in vivo studies suggest a synergistic inhibition of tumor growth by the combination of certain interferons and tumor necrosis factors. We have examined the activity of two of these agents in an animal model of human ovarian cancer. The human ovarian cancer cell line SK-OV3 was grown subcutaneously in immunodeficient mice. Mean relative tumor volume was determined weekly by external tumor measurements and a standard formula. Animals were treated by intraperitoneal injection with (1) recombinant human tumor necrosis factor alpha (rHTNF) 5 or 10 micrograms/day; (2) recombinant murine interferon gamma (rMIFN) 5 x 10(4) units per day; or (3) a combination of rHTNF 5 micrograms/day and rMIFN 5 x 10(4) units/day. Simultaneous control groups were run for all experiments. Administration of rHTNF alone in either dose had no effect on rate of tumor growth. Administration of rMIFN at a dose of 5 x 10(4) units/day produced a marginal decrease in rate of tumor growth. Animals receiving both rHTNF 5 micrograms/day and rMIFN 5 x 10(4) units daily showed marked inhibition of tumor growth, with the mean relative tumor volume after 4 weeks of treatment measuring 0.75 +/- 0.91 in the treatment group and 11.06 +/- 2.96 in the control group. rHTNF and rMIFN demonstrate a synergistic inhibition of tumor growth in this animal model of human ovarian cancer.

The APC I1307K Allele and BRCA-Associated Ovarian Cancer Risk

This work was supported by National Institutes of Health grant R01-CA71840. The authors are also grateful to Drs. William J. Hoskins and Patrick I. Borgen for their support of this laboratory.

Characterization of a human endometrial carcinoma cell line producing intraperitoneal tumor growth in immunodeficient mice

Establishment of laboratory models of gynecologic neoplasms provides an important means of studying the biologic characteristics of these tumors. We report a previously uncharacterized human endometrial adenocarcinoma cell line that produces both intraperitoneal and subcutaneous growth in nude mice. The line was derived from a poorly differentiated endometrial cancer and has been carried in continuous tissue culture for greater than 100 passages. Doubling time in culture is approximately 48 hr. Antigenic phenotyping against a panel of murine monoclonal antibodies by rosetting cell surface assay on live cells or peroxidase assay on fixed cells has shown reactivity with a number of determinants, including MH99, MT334, MQ49, and the blood group antigens F3, 118, and 41-83. Cytogenetically, the line displays an aneuploid human karyotype with several chromosomal rearrangements and deletions. When injected intraperitoneally into nude mice, animals develop intraperitoneal nodules and ascites and succumb with wasting in 30-40 days. The intraperitoneal tumor has been passaged multiple times in nude mice by direct transfer of ascites. Subcutaneous injection of tumor cells produces nodules that grow at a reproducible rate. By light and electron microscopy, the nude mouse tumor is a poorly differentiated adenocarcinoma, similar to the original patients tumor. It expresses both estrogen and progesterone receptors. CA 125 is not elevated in the serum of animals with tumor implants. The line appears to be cisplatin sensitive as determined by rates of growth of subcutaneous nodules. This cell line may be useful in studying the in vitro and in vivo properties of human endometrial carcinoma.