Ovarian Carcinoma Specimens Research Articles

Calcifications in Ovary and Endometrium and Their Neoplasms

In this study, we investigated the role of hormones in the pathogenesis of calcifications in ovary and in endometrium and their neoplasms of the gynecologic tract and assessed the anatomic location and incidence of these calcifications. The study consists of three parts designed to investigate the pathogenesis, the location, and the incidence of calcifications in ovary and endometrium and their neoplasms. In the first part, 79 female guinea pigs were divided into 10 groups, and different hormones, given weekly for 12 months, were administered to the guinea pigs by group. A control group of 7 guinea pigs received sterile water. Calcifications developed in 5 of 7 guinea pigs treated with prolactin, 10 of 20 treated with human chorionic gonadotropin, 5 of 11 treated with estradiol, 3 of 7 treated with estrone, 1 of 6 treated with growth hormone, and 1 of 10 treated with testosterone; in 20 of the guinea pigs, the calcifications developed in the stroma of the endometrium, and in 5 guinea pigs, they developed in the ovary. The second part of the study consisted of an evaluation of the specific location of calcifications in 43 consecutive human surgical ovaries and endometria. Calcifications were seen only in the stroma in 100% of the ovarian serous adenofibroma specimens; in ovarian serous borderline neoplasms, the stroma contained 70 to 100% of the calcifications, and the epithelium had 0 to 30% of the calcifications. In ovarian serous carcinoma specimens, the calcifications were seen in the stroma in 50 to 60% of the cases, in the epithelium in 40% of the cases, and in areas of necrosis in 10% of the cases. The third part of the study was directed to determine the frequency of calcifications in ovarian lesions. We found that all cases of endosalpingiosis and ovarian low-grade serous carcinoma had calcifications, whereas 80% of the cases of serous borderline tumor had calcifications, and only 50% of the cases of ovarian high-grade serous carcinoma contained calcifications. The results of this study indicate that the majority of the calcifications in the ovary and the endometrium and their neoplasms are present in the stroma. This is most probably secondary to metabolic changes, which could be related to hormones and not caused by degenerative changes in epithelial cells.

Expression of metastasis-related genes in human epithelial ovarian tumors

We examined the expression level of several genes that regulate distinct steps of metastasis in 55 formalin-fixed, paraffin-embedded, archival specimens of primary human ovarian carcinoma from patients undergoing curative surgery. The expression of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), basic fibroblast growth factor (bFGF), E-cadherin, type IV collagenase, matrix metalloproteinase (MMP-2 and MMP-9), and interleukin 8 (IL-8) was examined by a colorimetric in situ mRNA hybridization technique. The expression level of E-cadherin, MMP-2, MMP-9, VEGF, and IL-8 mRNA correlated with disease stages. The ratio of type IV collagenase expression (mean of the expression of MMP-2 and MMP-9) to E-cadherin expression (MMP:E-cadherin ratio) increased with increasing stage of disease (p<0.0001). Death rates significantly increased with high MMP:E-cadherin ratio (p=0.0005). Multivariate analysis of overall survival showed that the MMP:E-cadherin ratio was a significant independent prognostic factor, even after adjustment for known prognostic factors, such as histology, stage, and age.

Ovarian epithelial carcinoma tyrosine phosphorylation, cell proliferation, and ezrin translocation are stimulated by interleukin 1alpha and epidermal growth factor.

Ezrin is a member of the ezrin, radixin, and moesin family. These proteins are membrane-actin cross-linking proteins. Furthermore, ezrin is an important signal transduction protein that undergoes phosphorylation and translocation on stimulation by growth factors. Ezrin phosphorylation and translocation are thought to be correlated with cell motility, invasion, and carcinoma metastasis. Recently, the authors reported that an ezrin antisense phosphorothionate could significantly inhibit endometrial carcinoma cells' penetration in the Matrigel membrane cancer invasion assay. In the current study, the authors measured ezrin content in clinical ovarian epithelial carcinoma (OVCA) specimens and cell lines and investigated whether interleukin (IL)-1alpha and epidermal growth factor (EGF) induce an invasive phenotype in OVCA cells via ezrin phosphorylation and translocation. Twenty-five normal ovary, 25 primary OVCA, 21 metastatic OVCA tissue (7 in omentum, 16 in ascites), and 3 OVCA cell lines were collected for Western blot detection of ezrin content. The OVCA cell line SKOV3 was treated with IL-1alpha or EGF. Indirect immunofluorescence staining followed by confocal laser scanning and double-staining electron microscopic immunohistochemistry were used to investigate changes in the intracellular distribution of ezrin and cell morphology after IL-1alpha or EGF treatment. The content of ezrin was measured by Western blotting and analyzed by the National Institutes of Health Image computer program. Immunoprecipitation and Western blot techniques were used for ezrin phosphorylation studies. Genistein was used to block tyrosine phosphorylation. (1) Ezrin was overexpressed in OVCA, with the highest values in metastases. (2) Interleukin-1alpha and EGF significantly increased OVCA tyrosine phosphorylation, ezrin translocation, and cell growth. (3) These effects were abolished by treatment with the tyrosine kinase inhibitor, genistein. (4) Treatment with IL-1alpha or EGF induced an invasive phenotype, i.e., membrane ruffling, and process formation. High expression and activation of ezrin appear to be related to OVCA metastatic behavior. Interleukin-1alpha and EGF may regulate OVCA invasive behavior by activating ezrin tyrosine phosphorylation, translocation, and cancer cell proliferation. The authors' results may partially explain why OVCA patients with positive macrophage colony stimulating factor (a chemoattractant of IL-1alpha secreting monocytes) or EGF receptors (c-erb B-2) have a poor prognosis.

Single Mutations of the PTEN Gene in Recurrent Ovarian Carcinomas

To establish the role of phosphate and tensin homologue on chromosome 10 (PTEN) mutations in tumorigenesis of the ovary, we determined the mutational spectrum of the PTENgene in surgical specimens of ovarian carcinomas. The study group consisted of 86 ovarian cancer specimens (18 fluids, 68 solid specimens), including 30 primary ovarian cancer specimens and 56 of relapsed ovarian cancer from women with a median age of 57.9 years and a range of 27-85 years. Each of the nine exons of the PTEN gene was amplified separately by polymerase chain reaction (PCR). Both strands of the PCR products were sequenced directly by standard cycle sequencing procedures and subsequent computer-aided alignments with the wild-type sequence. In ascitic fluids of two women with recurrence of cancer, we observed mutations: one seven-base-pair insertion at codon 52 (GATGATG) and the other a base-pair substitution resulting in an amino acid change (T131I). We found no mutation in the primary ovarian cancers. Our data indicate that PTEN mutations have a subordinate role in tumorigenesis of the ovary.

Genetic Alterations of the WT1 Gene in Papillary Serous Carcinoma of the Peritoneum

Objective. The Wilms' tumor (WT1) gene product is consistently detectable in both normal ovarian germinal epithelium and human mesothelium. Ovarian carcinomas frequently exhibit alterations in WT1 function. Papillary serous carcinoma of the peritoneum (PSCP) is believed to develop de novo from the peritoneal lining (mesothelium) of the pelvis and abdomen. The purpose of this study was to determine if genetic alterations of the WT1 gene are associated with the development of PSCP.Methods. Normal and tumor tissue specimens were retrieved from patients with stage III and IV PSCP (n = 38) and serous epithelial ovarian carcinoma (n = 38). Immunohistochemistry was performed using the anti-WT1 (C-19) antibody. Loss of heterozygosity (LOH) was performed at the WT1 locus. Clinical data were obtained and correlated with molecular findings.Results. Loss of normal WT1 expression was detected in 18 (51%) of 35 PSCP specimens and 18 (53%) of 34 ovarian carcinoma specimens. Six (27%) of 22 PSCP specimens and 3 (13%) of 24 ovarian carcinoma specimens had LOH at the WT1 locus (P = 0.27). Normal WT1 gene expression was maintained in 86% of tumors exhibiting LOH. Genetic alterations of the WT1 gene were not predictive of survival, nor were they associated with other clinical or molecular factors.Conclusions. Genetic alterations of the WT1 gene are associated with the development of PSCP. The loss of normal WT1 gene expression is a common event in both PSCP and advanced ovarian carcinoma, likely resulting from down-regulation by other regulatory factors—not from inactivating gene mutation and subsequent allelic loss.

Antitumor activity of a novel glyco‐nitric oxide conjugate in ovarian carcinoma

BACKGROUND Several studies have shown that nitric oxide (NO)-releasing agents can kill tumor cells. Unfortunately, currently available NO delivery molecules do not target tumor cells preferentially. To exploit the overexpression of glucose transport proteins and the high level of glucose transport characteristics of tumor cells, glucose was conjugated to S-nitroso-N-acetyl-penicillamine (2-gluSNAP) and evaluated for cytotoxicity in human ovarian carcinoma cells. METHODS The cytotoxicity of 2-gluSNAP and SNAP was assessed by clonogenic cell survival assays performed in A2780S (cisplatin sensitive) and A2780cP (cisplatin-resistant) ovarian carcinoma cells in vitro. Immunoblotting and immunohistochemistry were used to assess the expression of Glut-1 hexose transport protein in the cell lines as well as in paraffin blocks from 28 surgical specimens of epithelial ovarian carcinoma. Apoptosis was assessed by an end-labeling assay. RESULTS The ovarian carcinoma cell lines consistently were more sensitive to 2-gluSNAP than SNAP alone. The median effective doses (MEDs) for 2-gluSNAP and SNAP in the A2780s cell line were 0.0042 μM and 20.4 μM, respectively. Therefore, 2-GluSNAP was nearly 5000-fold more potent than the NO-donating moiety (SNAP) alone. In the A2780cP cells, the MED for 2-gluSNAP (0.38 μM) was 250-fold lower than that for SNAP alone (100 μM). Immunoblotting and immunohistochemistry studies showed overexpression of Glut-1 in the cell lines and in 23 of 28 epithelial ovarian carcinoma specimens. CONCLUSIONS The novel glyco-NO conjugate 2-gluSNAP exhibits a much greater cytotoxicity than the parent NO donor without the hexose moiety. These agents have the potential to target tumor cells preferentially, that overexpress Glut-1. This transporter is expressed highly in epithelial ovarian carcinoma. Cancer 2000;88:381–8. © 2000 American Cancer Society.

Antitumor activity of a novel glyco-nitric oxide conjugate in ovarian carcinoma

Several studies have shown that nitric oxide (NO)-releasing agents can kill tumor cells. Unfortunately, currently available NO delivery molecules do not target tumor cells preferentially. To exploit the overexpression of glucose transport proteins and the high level of glucose transport characteristics of tumor cells, glucose was conjugated to S-nitroso-N-acetyl-penicillamine (2-gluSNAP) and evaluated for cytotoxicity in human ovarian carcinoma cells. The cytotoxicity of 2-gluSNAP and SNAP was assessed by clonogenic cell survival assays performed in A2780S (cisplatin sensitive) and A2780cP (cisplatin-resistant) ovarian carcinoma cells in vitro. Immunoblotting and immunohistochemistry were used to assess the expression of Glut-1 hexose transport protein in the cell lines as well as in paraffin blocks from 28 surgical specimens of epithelial ovarian carcinoma. Apoptosis was assessed by an end-labeling assay. The ovarian carcinoma cell lines consistently were more sensitive to 2-gluSNAP than SNAP alone. The median effective doses (MEDs) for 2-gluSNAP and SNAP in the A2780s cell line were 0.0042 microM and 20.4 microM, respectively. Therefore, 2-GluSNAP was nearly 5000-fold more potent than the NO-donating moiety (SNAP) alone. In the A2780cP cells, the MED for 2-gluSNAP (0.38 microM) was 250-fold lower than that for SNAP alone (100 microM). Immunoblotting and immunohistochemistry studies showed overexpression of Glut-1 in the cell lines and in 23 of 28 epithelial ovarian carcinoma specimens. The novel glyco-NO conjugate 2-gluSNAP exhibits a much greater cytotoxicity than the parent NO donor without the hexose moiety. These agents have the potential to target tumor cells preferentially, that overexpress Glut-1. This transporter is expressed highly in epithelial ovarian carcinoma.

Primary Ovarian Cancer Cultures are Resistant to Fas-Mediated Apoptosis

Objectives. Fas, a primary mediator of cellular apoptosis, is expressed by the normal ovarian epithelium. We analyzed the levels of Fas and soluble Fas (sFas) expression in ovarian cancer tissue and determined the susceptibility of primary ovarian cancer cell (CSOC) cultures to Fas-mediated apoptosis.Methods. Fas mRNA levels were detected by RT-PCR, and Fas protein levels were determined by immunohistochemistry and Western blot analysis. Secreted sFas levels were measured by ELISA. Localization of Fas to the cell surface was demonstrated by flow cytometry. The effect of Fas on cell proliferation was measured by MTT assay.Results. Intense Fas staining was detected on the cell surface and in the cytoplasm of ovarian carcinoma specimens. We also found that mean levels of sFas, which can function as a Fas agonist, were significantly increased in 18 sera from cancer patients (0.98 ng/ml) compared to those of 8 healthy individuals (0.61 ng/ml, P = 0.004). Fas mRNA and protein were expressed in all primary ovarian cancer cell cultures. Despite abundant Fas expression, CSOC cultures were significantly less sensitive to Fas-mediated apoptosis (11.3%) than primary cultures of normal ovarian epithelial cells (HOSE) (50.0%) (P = 0.00001). The sFas level in CSOC-conditioned medium was minimal (0.07 ng/ml) and not significantly different from that of HOSE-conditioned medium (0.09 ng/ml). The small amount of sFas secreted by CSOC does not likely account for the observed resistance to Fas-mediated apoptosis.Conclusion. Decreased sensitivity to Fas-mediated apoptosis could contribute to ovarian tumorigenesis through resistance to cytotoxic T lymphocyte-mediated cytotoxicity and may play a role in ovarian tumorigenesis.

High-Affinity Folate Receptor in Human Ovary, Serous Ovarian Adenocarcinoma, and Ascites: Radioligand Binding Mechanism, Molecular Size, Ionic Properties, Hydrophobic Domain, and Immunoreactivity

High-affinity folate receptors are expressed in normal ovaries and ovarian carcinomas. Binding of [3H]folate in human ovary, serous ovarian carcinoma tissue, and ascites is a complex process that has not been well characterized. This study shows changes in binding affinity and mechanism of binding with decreasing receptor concentration, inhibition by folate derivatives, and a slow radioligand dissociation at pH 7.4 becoming rapid and complete at pH 3.5. The receptor seems to be positively charged since it elutes in the front effluent of a DEAE-Sepharose CL-6B ion-exchange column at pH 6.3. The gel filtration profile of Triton X-100-solubilized tissue and ascites contained two peaks of radioligand-bound receptor (25 and 100 kDa). Exposure of ascites to cleavage by phosphatidylinositol-specific phospholipase C resulted in a partial conversion of the 100-kDa peak to a 25-kDa peak. This suggests that the receptor may be anchored to the membrane by a glycosylphosphatidyl residue that inserts into Triton X-100 micelles, resulting in a large molecular size on gel filtration. The receptor in ovarian carcinoma tissue immunoreacts with antibodies against purified human milk folate receptor protein as shown by enzyme-linked immunosorbent assay, immunoprecipitation, sodium dodecyl sulfate–polyacrylamide gel electrophoresis immunoblotting (a single band of 45 kDa), and immunohistochemistry. In only three of seven ovarian carcinomas did expression of radioligand-bound receptors exceed levels found in five normal ovaries. However, only receptors in ovarian carcinoma specimens showed a high degree of immunoreactivity. Hence, even without elevations of the total receptor level, a folate receptor isoform homologous to human milk folate receptor protein seemed to prevail in serous ovarian carcinomas.

In VitroSensitivity of Fresh Ovarian Carcinoma Specimens to CPT-11 (Irinotecan)

Objective.Irinotecan (CPT-11) is a DNA topoisomerase I inhibitor which has shown activityin vitroagainst several cancer cell lines and some promising clinical responses in phase I and II trials in various solid tumors. The cytotoxicity of CPT-11 on human ovarian epithelial malignancies was testedin vitroutilizing the ATP chemosensitivity assay. Flow cytometry was also performed on the fresh carcinoma specimens.Methods.Fresh tumor samples were obtained at laparotomy from 20 patients with primary adenocarcinoma of the ovary and 1 patient with heavily pretreated recurrent ovarian carcinoma. Tumors were plated in anin vitrosystem and treated with varying doses of both CPT-11 and its active metabolite SN-38 (7-ethyl-10-hydroxycamptothecin), in addition to a panel of standard chemotherapeutic agents used in treating ovarian cancer. On the sixth day after incubation, ATP levels in remaining cells were measured using a luminometer as an indication of the amount of cell kill compared to untreated controls.Results.Twelve of the 21 tumors were evaluable for a response to CPT-11. Of the 12 tumors tested, 7 were found to have median effect doses of CPT-11 significantly under the clinically achievable peak plasma concentration (range 1.00 × 10−4to 44.67 μM) and 11 of the 12 tumors showed sensitivity to the active metabolite SN38 with median effect doses ranging from 2.00 × 10−4to 0.147 μM. Some of these tumors showed resistance to the standard agents, such as platinum, based on our assay. The heavily pretreated recurrent tumor tested was very sensitive to both CPT-11 and SN38 by our assay. The results of flow cytometry showed that the tumors were composed of heterogeneous populations of cells with mean S phase fractions varying from 7.0 to 17.3%. The only tumor not sensitive to SN38 showed the highest mean S phase activity of 17.3%.Conclusions.Overall, these results suggest that CPT-11 deserves further investigation as a clinically useful chemotherapeutic agent for ovarian cancer.

Altered expression of transforming growth factor-beta ligands and receptors in primary and recurrent ovarian carcinoma.

Resistance to the potent growth inhibitory effects of transforming growth factor-beta (TGF-beta) is a characteristic of many malignancies. TGF-beta insensitivity has been attributed to alterations in the number and function of the TGF-beta receptors as well as disturbances of downstream signal transduction. Paradoxically, increased levels of TGF-beta ligand have been demonstrated in several types of malignant tumors. TGF-beta also may play a role in ovarian carcinogenesis; however, the nature of this interaction has yet to be defined completely. To explore the potential role of TGF-beta-mediated autocrine and paracrine influences in epithelial ovarian carcinoma, mRNA expression levels of the three TGF-beta ligand isoforms (TGF-beta1, TGF-beta2, and TGF-beta3) and the three TGF-beta receptors (TbetaR-I, T/betaR-II, and TbetaR-III) were examined by Northern blot analysis in both primary and recurrent ovarian carcinoma specimens. Immunohistochemical analysis was performed to localize expression of TbetaR-I and TbetaR-II, whereas the presence of genetic alterations in TbetaR-1 was examined through Southern blot analysis. Compared with normal ovarian tissue, both primary and recurrent ovarian carcinomas demonstrated significant overexpression of the TGF-beta1 and TGF-beta3 mRNA transcripts. TGF-beta2 expression was detectable in 75% of primary and only 53% of recurrent tumor specimens. Alterations also were detected in TbetaR mRNA expression. Expression levels of TbetaR-III were significantly reduced in both primary and recurrent ovarian carcinomas. Furthermore, detectable levels of TbetaR-I and TbetaR-III mRNA transcripts were present in only 47% and 50% of recurrent ovarian tumors, respectively. Immunohistochemical staining demonstrated that TbetaR-I and TbetaR-II expression localized to tumor cells; however, receptor staining in stromal tissue also was detected. Southern blot analysis of TbetaR-I did not reveal any major genetic changes to account for the absence of TbetaR-I expression. Alterations in expression of TGF-beta ligands and receptors consistently were greater in recurrent ovarian carcinomas compared with primary tumors, and may reflect a phenotype that promotes tumor recurrence or chemoresistance. Together, these data suggest that enhanced expression of TGF-betaI and TGF-beta3, as well as the loss of expression of TbetaR-I and TbetaR-III, contribute to ovarian carcinogenesis and/or tumor progression.

Melanin content and downregulation of glutathione S-transferase contribute to the action of l-buthionine- S-sulfoximine on human melanoma

l-buthionine- S, R-sulfoximine ( l- S, R-BSO) was enriched for the active l-buthionine- S-sulfoximine ( l- S-BSO) diastereomer. Comparative analysis was performed to determine if this enriched form possessed an increased capacity to deplete glutathione (GSH), and to inhibit the proliferation of tumor cell lines and fresh human tumor samples. Increased activity was observed for the enriched preparation of l- S-BSO in direct proportion to its increased l- S-diastereomeric percentage. Significant antitumor activity towards melanoma, breast and ovarian carcinoma specimens was noted, with the greatest activity directed against malignant melanoma. The activity of BSO on melanoma specimens was found to be correlated with their melanin content, suggesting that free radicals generated during melanin synthesis may become cytotoxic after GSH-dependent scavenging has been eliminated by BSO treatment. The antimelanoma activity of melphalan and BCNU were found to be significantly enhanced in combination with l- S-BSO. With respect to the mechanism of l- S-BSO synergy with alkylators, l- S-BSO treatment of M14 and ZAZ human melanoma cell lines resulted in decreased GSH levels and glutathione S-transferase (GST) activity. Western and Northern blot analyses indicated that GST- μ was the predominant isozyme downregulated after l- S-BSO treatment. Both M14 and ZAZ cell lines selected for resistance to l- S-BSO also showed decreased levels of GST- μ expression. However, in drug free media GST enzyme activity returned to pre-treatment levels without altering the BSO-resistance status of the cell lines. We conclude that l- S-BSO may be an active agent in the treatment of melanoma, and that it may enhance alkylator activity on melanoma through depletion of GSH and down-regulation of GST expression. Purified l- S-BSO should be explored clinically as an active agent for the treatment of melanoma.

Immunocytochemical localization of Cathepsin D and CA 125 in ovarian cancer

Objective: To study the expression of Cathepsin D (Cath D) and CA 125 antigens and ER and PR receptors on freshly obtained surgical specimens of ovarian carcinomas and their relationship with menopausal status, tumor histology, primary tumor size and lymph node invasion. Method: The tumors obtained from 100 women were measured and cut in half. The cut surface of one half was pressed against glass slides which were air dried and stained using the Avidin-Biotin peroxidase method for Cath D and CA 125 antigens. The slides were viewed under the light microscope for the characteristic brown granules in the cytoplasm or membrane of the malignant cells. The other half of the tumor was subjected to routine histological examination and part used for the demonstration of ER and PR receptors. The results were analyzed using χ 2 analysis. Results: Cath D positivity was as common as CA 125 positivity. Cath D positivity is more frequently associated with serous carcinomas than with others. No relationship was observed between ER/PR positivity and Cath D or CA 125 positivity. Conclusion: The high incidence of Cath D positivity makes it a possible complementary method for following up ovarian carcinoma patients especially those who are CA 125 negative.

Transforming growth factor-alpha promotes tumor markers secretion from human ovarian cancers in vitro.

The regulatory mechanism of tumor markers secretion has not been well clarified. Serum levels of CA 125 and tissue polypeptide antigen (TPA) from 17 patients with Stage III serous cystadenocarcinoma were measured prior to an initial surgical treatment. Epidermal growth factor receptor (EGFR) status was examined by an 125I-EGF binding assay in a human serous cystadenocarcinoma cell (SHIN-3) and in the 17 primary carcinomas. SHIN-3 cell and the EGFR-expressing primary cancer cells (n = 4) were cultured with or without various concentrations of transforming growth factor (TGF-alpha), a ligand for EGFR, and the CA 125 and TPA concentrations in the conditioned media were measured. EGFR was expressed in 12 primary carcinomas and in the SHIN-3 cell, and it was absent in the remaining 5 carcinomas. Pre-therapeutic serum CA 125 and TPA levels were significantly greater (P < 0.05) in patients with EGFR-expressing carcinomas (n = 5). These data suggest a possible involvement of EGFR in regulating these tumor markers secretion. TGF-alpha increased the CA 125 and TPA secretion from SHIN-3 cell. It also promoted the CA 125 secretion in 2 of 4 EGFR-expressing primary ovarian carcinoma specimens. These results suggest that a signal through the EGFR may be involved in regulating the CA 125 and TPA secretion from human ovarian carcinomas.

Oncogene amplification in archival ovarian carcinoma detected by fluorescent differential polymerase chain reaction - a routine analytical approach

For quantitative determination of gene amplification of oncogenes involved in ovarian cancer development we have established a rapid, nonradioactive approach. Determination of c-erbB-2 and c-myc gene amplification in archival ovarian carcinoma specimens was based on differential polymerase chain reaction (dPCR) and fluorescent DNA technique. c-erbB-2 or c-myc sequences were amplified by PCR, in which one of each primer pair was fluorescently labeled, simultaneously with a reference gene (γ-IFN) as internal single copy gene control. PCR products were separated by polyacrylamide gel electrophoresis with an automated DNA sequencer (A.L.F.™, Pharmacia, Freiburg, Germany) and directly quantified after laser activation and emission scanning using appropriate software (Fragment Manager ™, Pharmacia, Freiburg, Germany). This fluorescent differential PCR (fdPCR) method was used for quantitative determination of c-erbB-2 and c-myc gene amplification in 79 formalin-fixed, paraffin-embedded epithelial ovarian carcinoma tissues and 15 benign ovarian tissues. c-erbB-2 gene amplification was found in 18 (22%) and c-myc gene amplification in 13 (17%) of these carcinomas, but could not be detected in benign ovarian tissues. Single oncogene amplification correlated significantly with higher stage and higher grade (P<0.05). Patients with either c-erbB-2 or c-myc gene amplification or both had significantly shorter relapse-free survival and overall survival. The fdPCR assay is a valuable tool for determination of oncogene amplification even in archival tumor tissue. More detailed information about individual tumor biology of the primary tumor and metastasis as well as criteria for additional therapeutic decisions may be acquired by this analytical approach.

Aberrations of the p53 Tumor Suppressor Gene in Human Epithelial Ovarian Carcinoma

Aberrations of the p53 gene in 26 surgical specimens of human epithelial ovarian carcinomas were examined by single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) products. Seven (27%) of the tumors demonstrated a SSCP band shift in exons 4 to 9 of the gene, including 5 in the region encompassing exons 5 and 6, 1 in exon 7, and 1 in the region encompassing exons 8 and 9. Mutations were clustered in exon 5 in highly conserved regions of the p53 gene. All of the abnormal DNA fragments have been further characterized by direct DNA sequencing. These include five missense mutations (five transitions), a one-base-pair deletion introducing, by frameshift, a stop codon further downstream, and a two-base-pair insertion introducing a stop codon downstream by frameshift. Most mutations were base substitutions, and were clustered in exon 5 (71%), especially codons 175 and 179. The aberrations of the p53 gene were only found in tumors of FIGO stages III and IV. Histologic grading was also reviewed with respect to p53 aberrations. The aberrations were absent in well-differentiated carcinomas. The more undifferentiated the primary tumor, the more frequent p53 mutation (P < 0.05). Our results indicated that the aberrations of the p53 gene were common in epithelial ovarian cancers and p53 aberration may occur late during ovarian cancer evolution.

Co-cultivation of ovarian carcinoma cells with dermal fibroblasts induces fibroblast expression of sex steroid receptor transcripts and protein.

We have previously reported that stromal fibroblasts of ovarian carcinoma specimens may express estrogen (ER) and progesterone receptors (PR) when the malignant epithelial cells do not, and even when the specimens have been obtained from such non-Müllerian structures as the omentum whose fibroblasts normally express neither ER nor PR. In an attempt to investigate whether our observations of the expression of ER and PR in fibroblasts surrounding metastatic invasive epithelial ovarian carcinoma cells might result from an interaction involving malignant epithelial cells and stromal fibroblasts, we co-cultivated in vitro BG1 ovarian carcinoma cells with sex steroid receptor-negative dermal fibroblasts to determine whether carcinoma cells might induce the latter to express ER or PR protein and transcripts at levels detectable by standard immunocytochemical (ICC) and in situ hybridization (ISH) techniques. We report the in vitro induction of ER and PR transcripts and protein in previously steroid receptor-negative skin fibroblasts after co-cultivation with BG1 ovarian adenocarcinoma cells. Such observations suggest that a juxtacrine mechanism is responsible for the observed phenomenon, possibly involving ER- and PR-inducing factors (ER-IF and PR-IF).

Comparison of U-73,975 and cisplatin cytotoxicity in fresh cervical and ovarian carcinoma specimens with the ATP-chemosensitivity assay

U-73,975 (U-73), a closely related synthetic analogue of the antitumor agent CC-1065, acts by binding tightly in the minor groove of DNA. A comparison was made between the cytotoxicity of U-73 and cisplatin (DDP) on 11 fresh cervical and 7 fresh ovarian carcinoma specimens. The ATP-chemosensitivity assay as previously described (Sevin et al. Gynecol. Oncol. 31, 191–204, 1988) was used to determine the cytotoxic effect of U-73 and DDP. IC 50s were calculated using regression analysis. The mean IC 50s for U-73 and DDP were 519 pg/ml and 2918 ng/ml, respectively, for the cervical carcinoma specimens and 324 pg/ml and 2649 ng/ml, respectively, for the ovarian carcinoma specimens. Significance comparing U-73 and DDP for cervical and ovarian tissue was demonstrated with P < 0.001. U-73 was 4000 times as cytotoxic per unit of mass as DDP on cervical carcinoma compared to over 8000 times for ovarian carcinoma. Based on these in vitro data, U-73 appears to be a very promising antitumor agent for cervical and ovarian carcinoma.

Development of a Cell Surface Reacting Human Monoclonal Antibody Recognizing Ovarian and Certain Other Malignancies

A human monoclonal antibody designated AC6C3 was developed by fusing regional lymph node lymphocytes from a patient with epithelial ovarian carcinoma with cells of the hybrid myeloma SPAZ 4. This monoclonal antibody recognized a determinant expressed on the cell surface of ovarian tumor cell lines. The AC6C3 hybridoma has been maintained for more than 24 months by repeated cloning and secretes IgM at concentrations of 2-8 micrograms/10(6) cells/24h. The AC6C3 monoclonal antibody reacted with a cell surface component of ovarian tumor cell lines, as determined by cell surface immunofluorescence staining using the fluorescent activated cell sorter (FACS). In contrast, nylon wool nonadherent peripheral blood lymphocytes or red blood cells from normal donors were negative (less than 5% of the cells were stained). Immunoperoxidase staining with the AC6C3 monoclonal antibody of nonpermeabilized cryostat sections of freshly obtained or cryopreserved ovarian carcinoma specimens and human ovarian tumor xenografts demonstrated strong reactivity of these specimens. Most normal tissues including brain, liver, heart, kidney and peritoneum demonstrated negative or weak reactions with AC6C3. Other carcinomas including breast, colon and some malignancies of neuroectodermal origin were strongly reactive with AC6C3. AC6C3 mediated complement-dependent cytotoxicity and identified a 32 Kd band in Western blotting and immunoprecipitation experiments conducted on surface labelled SKOV3 cells. The association constant for AC6C3 was determined at 2.3 x 10(10) M-1.

Clinical significance of oncogenes and growth factors in ovarian carcinomas

The expression of epidermal growth factor receptor (EGF-R), transforming growth factor alpha (TGFα) and the c- myc oncogene was investigated in different specimens of gynecologic carcinomas. EGF specific binding sites were detected in about 50% of adenocarcinomas (ovarian, endometrial, breast) and in over 90% of squamous carcinomas (cervical). There is a positive correlation between the EGF-R binding assay, immunohistochemistry and the relative amounts of mRNA by Northern blotting. TGFα was investigated by immunohistochemistry and Northern blotting. TGFα immunoreactivity was detected exclusively in the epithelial cells of nonmalignant tissues (skin, cervix, endometrium, large bowel, lung) as well as different ovarian carcinomas. The TGFα immunostaining score correlates with the TGFα mRNA amounts. The c- myc expression was analyzed by Northern blotting in the specimens of ovarian carcinomas. Whereas, a positive correlation between the c- myc and TGFα expression was noticed, no correlation existed between EGF-R and c- myc expression. Progressive disease (PD) of ovarian carcinomas after chemotherapy was mainly noticed in the group of EGF-R − tumors and those with high amounts of c- myc mRNA. EGF-R + ovarian carcinomas responded significantly better to chemotherapy. However, similar survival times existed between the EGF-R + and EGF-R − group and the survival times of patients having responded to the treatment was reduced in the EGF-R + group. This indicates that EGF-R + and those carcinomas expressing high amounts of c- myc constitute a more aggressive group of ovarian carcinomas.