Kathy O'Briant

Expression and amplification of the HER-2/ neu (c-erbB-2) protooncogene in epithelial ovarian tumors and cell lines

Amplification of the c-erbB-2 protooncogene has been associated with a poor prognosis in human breast and ovarian cancers. Our study was undertaken to examine whether amplification, rearrangement, or overexpression of c-erbB-2 and other protooncogenes was frequently observed in epithelial ovarian cancers. c-erbB-2 was expressed in 87% of 22 ovarian cancers analyzed, but expression was significantly increased in only one of the 22 tumor specimens. In this case elevated c-erbB-2 expression was associated with dramatic amplification of the gene. In another tumor a 3.8 kb EcoRI fragment was found, in addition to the usual 4.4 and 6.0 kb fragments; this is consistent with a possible gene rearrangement or a restriction fragment length polymorphism. To place these results in perspective, expression of several other protooncogenes has been examined in ovarian carcinomas. The c-fos, c-myc, n-myc, c-fms, and c-Ha-ras protooncogenes were expressed in different fractions of tumors, but expression of l-myc, c-erbB, c-myb, c-sis, and c-mos was not detectable. Aside from c-erbB-2, neither amplification nor rearrangement was observed among the other protooncogenes studied. Expression of c-erbB-2, c-fms, c-myc, n-myc, c-fos, and c-Ha-ras deserves further evaluation as a prognostic factor in ovarian cancer.

Lung cancer and the human gene for ribonucleotide reductase subunit M1 (RRM1)

Abstract. LOH11A is a region of Chromosome (Chr) 11p15.5 where 75% of lung cancers show loss of heterozygosity (LOH). Clinical and cell biological studies suggest that LOH11A contains a tumor/metastasis suppressor gene. We have mapped this region (650 kb) using overlapping genomic P1/PAC/BAC clones, and one of the genes that we have identified is RRM1. This gene encodes the large subunit (M1) of ribonucleotide reductase, the heterodimeric enzyme that catalyzes the rate-limiting step in deoxyribonucleotide synthesis. By comparing our genomic sequences with the previously published cDNA, we have found that the human gene is composed of 19 exons. It is oriented telomere to centromere and is Alu rich. In order to verify that RRM1 maps within the boundaries of LOH11A, we assessed the frequency of LOH at a SacI polymorphism within intron IX of the gene. We observed LOH in 48% (15/31) of informative lung tumor specimens. To determine whether RRM1 was mutated in tumors, SSCP analysis of the 19 RRM1 exons was performed. No mutations were revealed in 12 pairs of normal and tumor DNA samples. Immunoblots on protein extracts from normal/tumor pairs indicated that a protein of the expected size was present in both. Our conclusion is that RRM1 lies within the LOH11A region, but that its exons are not mutated in tumors. The potential for RRM1 to act as a tumor suppressor is discussed.

Relative cytotoxic activity of immunotoxins reactive with different epitopes on the extracellular domain of the c-erbB-2 (HER-2/neu) gene product p185

Different epitopes on the extracellular domain of the HER‐2 receptor can serve as distinct targets for immunotoxins. To determine the optimal epitope target for immunotoxin therapy, 7 anti‐HER‐2 ricin A chain murine monoclonal immunotoxins, each reactive with different epitopes of HER‐2 receptor, were tested for cytotoxic activity. The immunotoxins produced 1.2–4.6 logs of cytotoxicity in limiting dilution clonogenic assays with 2 breast cancer cell lines that overexpressed HER‐2. Cytotoxicity did not correlate with immunoglobulin isotype, binding affinity, relative position of epitopes or internalization of the anti‐HER‐2 immunotoxins. Interestingly, the most and least effective immunotoxins bound to epitopes in very close proximity. Competitive binding assays with unconjugated antibodies have previously indicated that our antibodies recognized epitopes that are arranged in a linear array. To orient this relative epitope map, deletions were prepared in the HER‐2/neu gene and these mutant constructs were expressed in NIH3T3 cells. Epitope expression was determined by antibody binding and radioimmunoassay. Epitopes targeted by the PB3, 454C11 and NB3 antibodies are localized N‐terminal to the epitopes recognized by ID5, BD5, 741F8 and 520C9 antibodies. The 2 non‐conformational epitopes PB3 and NB3 were localized to regions corresponding to amino acides 78–242 of the p185HER‐2 protein. Int. J. Cancer 82:525–531, 1999.

Delineation of the centromeric and telomeric chromosome segment 11p15.5 lung cancer suppressor regions LOH11A and LOH11B

We have reported frequent allele loss for two separate regions identified by the markers D11S12 and HRAS on chromosome 11p15.5. D11S12 allele loss was associated with tumor stage and type and HRAS allele loss with cigarette consumption, sex, and survival. To positionally clone the putative tumor suppressor genes located in these regions, we here report a reduction in the size of these intervals to approximately 250 kb. The markers used, ordered from centromere to telomere, were D11S932 ‐ D11S1331 ‐ D11S1760 ‐ D11S1323 ‐ D11S4891(HBB) ‐ D11S1758 ‐ D11S12 ‐ D11S988 ‐D11S860 ‐ D11S1318 ‐ TH ‐ HRAS ‐D11S1363 ‐ D11S2071. We analyzed 44 tissue pairs from patients with primary lung cancer. The smallest common regions of allele loss were located between D11S1758 and D11S12 in the centromeric region of chromosome segment 11p15.5 (region of LOH on chromosome 11 in lung cancer, LOH11A) and between HRAS and D11S1363 in the telomeric region (region of LOH on chromosome 11 in lung cancer, LOH11B). Loss of heterozygosity was observed in 24/39 (62%) primary lung cancers informative for LOH11A and in 17/34 (50%) informative for LOH11B. The pattern of allele loss strongly suggests that two lung cancer suppressor genes are located on chromosome segment 11p15.5, one between D11S1758 and D11S12 and the other between HRAS and D11S1363. The estimated physical size of each of these regions is 250 kb. Genes Chromosom. Cancer 18:111–114, 1997.

Elimination of clonogenic breast cancer cells from human bone marrow. A comparison of immunotoxin treatment with chemoimmunoseparation using 4‐hydroperoxycyclophosphamide, monoclonal antibodies, and magnetic microspheres

Autologous bone marrow transplantation (ABMT) may aid in the management of breast cancer, but is currently limited to patients without bone marrow metastases. In earlier studies, 5 logs of malignant clonogenic breast cancer cells could be eliminated from human bone marrow using a combination of chemoseparation with 4‐hydroperoxycyclophosphamide (4‐HC) and immunoseparation with monoclonal antibodies and magnetic microspheres. In this report the authors compare chemoimmunoseparation to treatment with immunotoxins for elimination of tumor cells from human bone marrow and for the preservation of normal precursors. Breast cancer cells from each of five cell lines were mixed with a tenfold excess of irradiated human bone marrow cells. Treatment with a combination of five immunotoxins reduced clonogenic tumor cell growth by 1.8 to 5.5 logs depending upon the cell line. With two of the five cell lines, clonogenic tumor cells were eliminated quantitatively. Using the CAMA‐1 breast cancer cell line, treatment with multiple immunotoxins was compared with chemoimmunoseparation with 4‐HC, a panel of five unconjugated monoclonal antibodies and magnetic microspheres. Chemoimmunoseparation eliminated 3.5 to 5.4 logs of malignant cells, while preserving 21% of Colony‐forming unit‐granulocyte‐macrophage (CFU‐GM) and 37% of burst‐forming unit‐erythrocyte (BFU‐E). No clonogenic breast cancer cells could be detected. Immunotoxin treatment eliminated 2.2 to 5.4 logs of clonogenic breast cancer cells, but had no effect on the bone marrow precursors. In seven of ten experiments, however, clonogenic breast cancer cells remained after immunotoxin treatment. Consequently, treatment with 4‐HC, multiple murine monoclonal antibodies and magnetic microspheres provided more consistent elimination of tumor cells than separation with immunotoxins, but was significantly more toxic for marrow precursors.

Alkylating agents and immunotoxins exert synergistic cytotoxic activity against ovarian cancer cells. Mechanism of action.

Alkylating agents can be administered in high dosage to patients with ovarian cancer using autologous bone marrow support, but drug-resistant tumor cells can still persist. Immunotoxins provide reagents that might eliminate drug resistant cells. In the present study, concurrent treatment with alkylators and immunotoxins proved superior to treatment with each agent alone. Toxin immunoconjugates prepared from different monoclonal antibodies and recombinant ricin A chain (rRTA) inhibited clonogenic growth of ovarian cancer cell lines in limiting dilution assays. When alkylating agents and toxin conjugates were used in combination, the addition of the immunotoxins to cisplatin, or to cisplatin and thiotepa, produced synergistic cytotoxic activity against the OVCA 432 and OVCAR III cell lines. Studies performed to clarify the mechanism of action showed that cisplatin and thiotepa had no influence on internalization and binding of the 317G5-rRTA immunotoxin. Intracellular uptake of [195m]Pt-cisplatin was not affected by the immunoconjugate and thiotepa. The combination of the 317G5-rRTA and thiotepa, as well as 317G5-rRTA alone, increased [195m]Pt cisplatin-DNA adduct levels. The immunotoxin alone and in combination with the alkylators decreased intracellular glutathione levels and reduced glutathione-S-transferase activity. Repair of DNA damage induced by the combination of alkylators and 317G5-rRTA was significantly reduced when compared to repair after damage with alkylators alone. These findings suggest that immunotoxins affect levels and activity of enzymes required for the prevention and repair of alkylator damage.

SSA/RO52gene and expressed sequence tags in an 85 kb region of chromosome segment 11p15.5.

Frequent allelic loss in lung cancer has been described in a region on chromosome segment 11p15.5 (LOH11A). The region is approximately 650 kb in size and flanked by the markers D11S988 centromeric and D11S860 telomeric. Clinical and cell biological studies suggest that it contains a gene associated with metastatic tumor spread. One of the genes identified within this region is SSA/Ro52, which has a RING finger domain and may be involved in gene regulation. We studied this gene for mutations using SSCP analysis and for expression using RT‐PCR and Western blotting on lung cancer cell lines and tumor–normal tissue pairs. No mutations and no differences in mRNA or protein expression between tumor tissue and normal tissue pairs were identified. We discovered a novel polymorphic site (SSA44C/T) within exon 1 of this gene. Among 141 primary lung cancers, allelic loss was observed in 16% of informative cases. Our analyses excluded SSA/Ro52 as a tumor‐suppressor gene in lung cancer and newly defined the centromeric border of the LOH11A region from D11S988 previously to SSA44C/T. This reduced the region of the putative suppressor gene to 460 to 485 kb. A significant difference (p = 0.01) in the frequency of alleles for this polymorphism between Caucasians and African‐Americans was observed. The “T” allele frequency was 0.12 in Caucasians and 0.23 in African‐Americans. A genomic EcoRI map over 85 kb surrounding the SSA/Ro52 gene was constructed, and 4 expressed sequence tags were identified by sequencing and studied. Int. J. Cancer 87:61–67, 2000.

Ha-ras polymorphisms in epithelial ovarian cancer

Unusual restriction fragment length polymorphisms (RFLPs) of the Ha-ras locus have been found in DNA from leukocytes and tumor tissue of cancer patients. To determine whether rare alleles would be observed frequently in patients with ovarian cancer, Ha-ras RFLPs were studied in DNA from 42 different ovarian epithelial tumors and from the peripheral blood leukocytes of 76 normal individuals. Four common, seven intermediate, and seven rare alleles were detected overall. Similar fractions of rare alleles were found in DNA from ovarian cancers and from the peripheral blood of normal individuals. Thus, the frequency of unusual Ha-ras RFLPs did not distinguish patients with ovarian cancers from apparently healthy individuals.