Molecular approaches to personalizing management of ovarian cancer.

Cytoreductive surgery and empirical combination chemotherapy have improved 5-year survival for ovarian cancer patients, but have not increased the overall rate of cure. Poor outcomes relate, at least in part, to late diagnosis and to the persistence of dormant ovarian cancer cells that have resisted conventional drugs. Increased understanding of the molecular, cellular and clinical biology of ovarian cancer must be translated into personalized therapy with conventional and targeted agents as well as personalized detection of high-grade cancers in early stages. Different strategies will be required to treat low-grade and high-grade serous cancers as well as other histotypes. Activating mutations of Ras and Raf can be targeted in low-grade cancers. Activation of the PI3K pathway-PI3Kness-and inactivation of BRCA function-BRCAness-can be targeted in high-grade lesions. Inhibition of multiple pathways will be required. Sensitivity of primary cancers to paclitaxel and platinum can be modulated by inhibiting kinases and other molecules that regulate the cell cycle. Dormant ovarian cancer cells may depend upon autophagy, cytokines and growth factors for survival. Early detection can utilize two stage strategies where rising serum biomarker levels prompt imaging in a small fraction of women. Screening can be personalized by taking into account each woman's baseline biomarker levels.

Heterogeneity of the cellular immune response. I. Kinetics of lymphocyte stimulation during sensitization and recovery from tolerance.

Lymph node cells from guinea pigs immunized with HSA in complete Freund's adjuvant were grown in cultures containing different concentrations of specific antigen. Stimulation of thymidine incorporation was induced with progressively lower concentrations of HSA at successive intervals after sensitization. Moreover, the intensity of delayed skin reactions and the magnitude of stimulation in vitro increased over the same interval. These events are considered compatible with an evolution of the cellular immune response resulting from the selection of lymphoid cells by decreasing concentrations of antigen in vivo. Cells from animals rendered tolerant to HSA failed to respond to specific antigen in culture. As tolerance waned, stimulation was achieved at high but not low antigen concentrations. Tolerance, measured by cutaneous reactivity or by lymphocyte stimulation, was less readily induced in animals sensitized with adjuvant containing a reduced concentration of mycobacteria. Lymph nodes from these animals contained a large population of cells reactive at high antigen concentration, presumably less susceptible to the toleragenic effect of intravenous antigen. The dissociation of delayed hypersensitivity and antibody formation observed early in the immune response and upon recovery from tolerance has permitted correlation of lymphocyte stimulation with delayed hypersensitivity and cutaneous basophil hypersensitivity respectively.

Heterogeneity of the cellular immune response. II. The role of adjuvant, lymphocyte stimulation in cutaneous basophil hypersensitivity.

Antigen-mediated stimulation of thymidine incorporation was demonstrated in lymph node cells from guinea pigs immunized with 100 microg human serum albumin in either Freund's incomplete or Freund's complete adjuvant. Animals receiving HSA in IFA exhibited both cutaneous basophil (Jones-Mote) hypersensitivity and lymphocyte stimulation at 1, but not at 6 wk after immunization. Significant stimulation required >/= 10 microg HSA/ml of culture. Sensitization with HSA in CFA produced delayed hypersensitivity and permitted lymphocyte stimulation at both 1 and 6 wk. Stimulation was observed with as little as 0.1 microg HSA/ml at the later interval. Administration of 5 mg HSA intravenously at the time of sensitization with 100 microg HSA in IFA reduced but did not eliminate both CBH and lymphocyte stimulation at 1 wk. Antigen-specific inhibition of macrophage migration could be demonstrated with exudates from animals immunized with HSA in CFA, but not with HSA in IFA at 3 wk after sensitization. HSA was cleared from depots of CFA and IFA at similar rates, but significantly more antigen appeared in the plasma and subsequently in the draining lymph nodes following administration in IFA. Conversely, accumulated antigen disappeared more rapidly following CFA immunization.

Somatic activation of rasK gene in a human ovarian carcinoma.

A tumor isolate from a patient with serous cystadenocarcinoma of the ovary contained an activated rasK gene detected hy transfection of NIH/3T3 cells. In contrast, DNA from normal cells of the same patient lacked transforming activity, indicating that activation of this transforming gene was the consequence of somatic mutation in the neoplastic cells. The transforming gene product displayed an electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels that differed from the mobilities of rasK transforming proteins in other tumors, indicating that a previously undescribed mutation was responsible for activation of rasK in this ovarian carcinoma.

HER2 signaling modulates the equilibrium between pro- and antiangiogenic factors via distinct pathways: implications for HER2-targeted antibody therapy.

We determined the impact of HER2 signaling on two proangiogenic factors, vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), and on an antiangiogenic factor, thrombospondin-1 (TSP-1). Re-expression of HER2 in MCF-7 and T-47D breast cancer cells that endogenously express low levels of HER2 resulted in elevated expression of VEGF and IL-8 and decreased expression of TSP-1. Inhibition of HER2 with a humanized anti-HER2 antibody (trastuzumab, or Herceptin) or a retrovirus-mediated small interfering RNA against HER2 (siHER2) decreased VEGF and IL-8 expression, but increased TSP-1 expression in BT474 breast cancer cells that express high levels of HER2. These in vitro results were further evaluated by treatment of BT474 xenografts in immunosuppressed mice with trastuzumab. Trastuzumab inhibited growth of BT474 xenografts and decreased microvascular density associated with downregulation of VEGF and IL-8 and with upregulation of TSP-1 expression. Inhibiting the PI3K-AKT pathway decreased VEGF and IL-8 expression. AKT1 overexpession increased VEGF and IL-8 expression, but did not increase TSP-1 expression. A p38 kinase inhibitor, SB203580, instead blocked TSP-1 expression and a p38 activator, MKK6, increased TSP-1 expression. Trastuzumab stimulated sustained p38 activation and SB203580 attenuated the TSP-1 upregulation induced by trastuzumab. HER2 signaling therefore influences the equilibrium between pro- and antiangiogenic factors via distinct signaling pathways. Trastuzumab inhibits angiogenesis and tumor growth, at least in part, through activation of the HER2-p38-TSP-1 pathway and inhibition of the HER2-PI3K-AKT-VEGF/IL-8 pathway.

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.

Reactivity of a monoclonal antibody with human ovarian carcinoma.

A murine monoclonal antibody (OC125) has been developed that reacts with each of six epithelial ovarian carcinoma cell lines and with cryopreserved tumor tissue from 12 of 20 ovarian cancer patients. By contrast, the antibody does not bind to a variety of nonmalignant tissues, including adult and fetal ovary. OC125 reacts with only 1 of 14 cell lines derived from nonovarian neoplasms and has failed to react with cryostat sections from 12 nonovarian carcinomas.

Constitutive production of macrophage colony-stimulating factor by human ovarian and breast cancer cell lines.

Many nonhematologic tumors produce growth factors that may influence cellular proliferation either by autocrine or by paracrine mechanisms. In the current study, human tumor cell lines were investigated for the constitutive production of macrophage colony-stimulating factor (M-CSF). Culture supernatants obtained from cell lines were analyzed using a radioimmunoassay and a radioreceptor assay specific for M-CSF. Among the various cell types analyzed, all the ovarian cell lines and a majority of the breast cancer cell lines secreted significant amount of an M-CSF-like factor. Treatment of mouse bone marrow cultures with culture supernatants from ovarian cancer cells stimulated the production of macrophage colonies. Analysis of total cellular RNA obtained from the ovarian cell lines by Northern blot showed multiple sizes of M-CSF transcripts with an abundance of a 4.2-kb message. The relative amount of M-CSF transcripts correlated with the level of immunoreactive material seen in the culture supernatants.

Local antitumor activity of a primary and an anamnestic response to a syngeneic guinea pig hepatoma.

After intradermal (id) injection, the line-10 hepatoma grew progressively in nonimmune guinea pigs, whereas the line-1 hepatoma grew for approximately 2 weeks, developed central necrosis, ulcerated, and regressed. Growth of the line-10 hepatoma was suppressed when line-10 hepatoma cells were mixed with antigenically distinct line-1 hepatoma cells before id injection into syngeneic strain-2 guinea pigs. Mixture of line-10 with irradiated line-1 or viable strain-2 embryo cells did not inhibit tumor growth. Preimmunization of recipients to line-1 cells abrogated the suppression of tumor growth from mixtures of line-1 and line-10.

Antitumor activity of bacterial infection. I. Effect of Listeria monocytogenes on growth of a murine fibrosarcoma.

Growth of a murine fibrosarcoma was suppressed when tumor cells were mixed with viable Listeria monocytogenes (LM) before intradermal injection into nonimmune syngeneic recepients. Immunization of recipients, by intravenous injection of LM 11 days before transplantation of LM-tumor cell mixtures, eliminated the mortality associated with large doses of LM but did not alter the antitumor activity of the microorganisms. Simultaneous injection of LM and tumor cells at separate sites failed to affect tumor growth, which suggested that contact between LM and tumor cells was required for tumor suppression. Tumor-specific immunity was not observed; mice surviving injection of LM and tumor cells did not resist a second tumor-cell challenge. At least 100 times more heat-killed LM was required to produce the antitumor effect of viable organisms. The ability of heat-killed LM to suppress tumor growth was abolished by treatment of recipients with rabbit antiserum to mouse thymocytes, which was consistent with a requirement for a host response to the LM. Regression of established fibrosarcoma transplants was produced by the intratumor injection of viable LM 5 days after injection of tumor cells. Intratumor injection of BCG at this interval was not effective. The incidence of tumor regression was not increased by multiple intratumor injections of LM, by intratumor injection of a combination of LM and BCG, or by preimmunization with LM prior to the intratumor injection of the same organism.

Aryl hydrocarbon (benzo[a]pyrene) hydroxylase in guinea pig lymphoid tissue.

Aryl hydrocarbon hydroxylase (AHH) activity was detected in a variety of guinea pig lymphoid tissues and was increased by intradermal or ip administration of 3-methylcholanthrene AHH levels were generally greater in spleen than in lymph node, bone marrow, or thymus. Similar AHH activities were observed in lymphoid tissues from Sewall-Wright inbred strain 2 and strain 13 guinea pigs. Greater AHH activities were associated with peritoneal and alveolar macrophages than with lymph nodes lymphocytes in the absence of mitogens.

Eradication of microscopic lymph nodes metastases after injection of living BCG adjacent to the primary tumor.

Guinea pigs with established intradermal tumors and microscopic axillary lymph node metastases were treated with a combination of surgery and BCG. The tumors were excised and BCG was given in attempts to eliminate residual malignant disease. Injection of BCG into established intradermal tumors 7 days before local excision successfully eradicated microscopic axillary lymph node metastases and cured significant numbers of animals. Injection of BCG into dermal tumors 20 minutes or 1 day before excision prolonged survival but did not cure a significant number of animals. Injection of BCG into the skin adjacent to the dermal tumor 7 days before local excision eradicated microscopic axillary lymph node metastases. However, such injection 1 day before local excision did not eradicate metastases. BCG administered by intravenous, intra-arterial, or intranodal injection did not eliminate reidual malignant disease. Several factors were evaluated as possible correlates of successful immunotherapy. The development of tuberculin hypersensitivity, the magnitude of regional adenopathy, and the number of BCG organisms in axillary nodes were not useful correlates. Histologically, the presence of tumor cells, multiple focal granuloma, or histiocytosis in axillary nodes faiiled to correlate with results of therapy. The development of tumor-specific transplantation immunity provided the best correlate of successful immunotherapy.

Antitumor activity of bacterial infection. II. effect of Listeria monocytogenes on growth of a guinea pig hepatoma.

Growth of a guinea pig hepatoma was suppressed when tumor cells were mixed with viable Listeria monocytogenes (LM) before intradermal (id) injection into syngeneic recipients. Heat-killed LM were less effective than viable organisms in suppressing tumor growth. A vaccine containing oil droplets and LM cell walls lacked antitumor activity. Intratumor injection of viable LM on the 7th day after id injection of tumor cells prolonged survival of guinea pigs that did not succumb to LM infection. After intratumor injection of 0.6 times 10-8-1.0 times 10-8 LM, 5 of 22 guinea pigs died from acute infection (23 percent). In the 17 survivors, 3 tumors regressed completely (18 percent). Animals surviving injections of LM and tumor cells were immune to a second challenge with tumor cells. Immunization ofguinea pigs with an intravenous injection of LM decreased the mortality from intratumor injection of LM, but the intratumor injection of LM failed to cure a significant fraction of LM-immune animals bearing 7-day hepatoma transplants. BCG was more effective than LM in producing tumor regression. Synergism between LM and BCG was not observed, and simultaneous intratumor injection of BCG and LM was no more effective than intratumor injection of BCG alone in the treatment of 12-day tumor transplants.

Overexpression of the protein tyrosine phosphatase PTP1B in human breast cancer: association with p185c-erbB-2 protein expression.

BACKGROUND: The p185c-erbB-2 growth factor receptor protein tyrosine kinase (PTK) is overexpressed in one third of human breast cancer patients and indicates a poor prognosis in these patients. Protein tyrosine phosphatases (PTPs) may balance PTK activity as part of normal growth-regulation pathways. PTP1B is an intracellular PTP that is involved in linkage between signal transduction pathways and may interface with inappropriate PTK activity in transformed cells. PURPOSE: The aim of this study was to determine if PTP1B is overexpressed in human mammary tumors and to determine if such overexpression is associated with the overexpression of the p185c-erbB-2 receptor PTK. METHODS: Our samples were frozen sections from 29 human mammary tumors (19 pure infiltrating, two pure intraductal, and eight combined intraductal and infiltrating) and nine sections from normal breast tissue. The sections were immunohistochemically stained for PTP1B and p185c-erbB-2, and the results were analyzed statistically for association between overexpression of the two proteins. Northern blot analysis was used to assess if PTP1B overexpression was coincident with increased transcription of the PTP1B gene. RESULTS: Overexpression of the PTP1B protein was observed in 72.4% of the tumor sections compared with normal epithelium, with maximal expression occurring in 37.9% of the tumors. All of the tumor subtypes, including the pure intraductal lesions, overexpressed PTP1B. Statistical analyses demonstrated a significant association between PTP1B overexpression and breast cancer (P < .038) and between the overexpression of PTP1B and the overexpression of p185c-erbB-2 (P < .006). PTP1B messenger RNA steady-state transcription was consistently increased in tumors versus normal tissues. CONCLUSIONS: PTP1B overexpression is a common phenotypic manifestation in human breast cancers and is associated with over-expression of p185c-erbB-2. Steady-state PTP1B transcription is increased in tumor tissue. IMPLICATIONS: Further studies are needed to determine if PTP1B overexpression balances or augments PTK activity. PTP1B overexpression might also be evaluated as a clinical prognostic factor in human breast cancers.

Spectrum of mutation and frequency of allelic deletion of the p53 gene in ovarian cancer.

BACKGROUND: The p53 gene encodes a nuclear phosphoprotein present in low levels in normal human cells. The wild-type form of this protein functions to restrain inappropriate cellular proliferation. Approximately one half of human epithelial ovarian cancers have mutations in the p53 gene and overexpress the mutant protein product. Deletion of one allele of the p53 gene also frequently occurs in these cancers. PURPOSE: We sought to define the spectrum of mutations in the p53 gene in epithelial ovarian cancer with respect to both the specific codons involved and the type of mutations observed. We also examined the frequency of allelic deletion of the p53 gene in cancers containing p53 gene mutations. METHODS: Tissue samples from the epithelial ovarian cancers of 62 patients were obtained during initial laparotomy. Histologic examination was done to ensure that the experimental samples used in this study contained more than 75% cancer cells. Total RNA was extracted from these samples and separately from matched control noncancerous regions of the surgical specimen or white blood cells. The purified RNAs were reverse transcribed to generate cDNA copies of exons 4-10 of the p53 gene. Two rounds of polymerase chain reaction (PCR) were conducted to produce enough template for DNA sequence analysis of the regions of interest within the p53 gene. Dideoxy sequencing of at least two independent productions of each amplified DNA template was done to confirm the validity of the mutations found. Allelic deletions were identified by PCR and gel electrophoretic techniques to examine three polymorphisms within the p53 gene in cancer-normal DNA pairs. RESULTS: We identified 45 mutations in exons 5-8 of the p53 gene, where mutations frequently have been found in other cancer types. An additional mutation was identified in exon 4. Overall, 72% of the mutations were transitions, 24% transversions, and 4% microdeletions. Allelic deletion of the other p53 allele was seen in 67% of ovarian cancers in which a p53 mutation was present. Germ-line p53 mutations were not found in any patients whose cancers had p53 mutations. CONCLUSIONS AND IMPLICATIONS: Like p53 mutations in other types of human cancers, those in epithelial ovarian cancers are diverse and occur frequently in exons 5-8. The predominance of transition mutations suggests that p53 mutations in ovarian cancer arise because of spontaneous errors in DNA synthesis and repair rather than the direct interaction of carcinogens with DNA. These molecular data are consistent with data from epidemiologic studies that have failed to demonstrate a convincing relationship between exposure to environmental carcinogens and the development of ovarian cancer.

Clonal origin of epithelial ovarian carcinoma: analysis by loss of heterozygosity, p53 mutation, and X-chromosome inactivation.

BACKGROUND: It has been suggested that multiple sites of epithelial ovarian carcinoma on the peritoneal surface reflect polyclonal disease arising from multiple primary tumors in the peritoneal mesothelium, rather than monoclonal disease spread by metastases from one primary ovarian cancer. PURPOSE: The purpose of this study was to investigate whether ovarian cancer has a monoclonal or polyclonal origin. METHODS: DNA specimens were obtained from peripheral blood lymphocytes (normal DNA) and from multiple tumor deposits of 17 women with epithelial ovarian carcinoma: primary tumors, metastatic deposits, and ascites. The clonal origin of each tumor was determined by performing (a) analysis to detect loss of heterozygosity at five loci on chromosomes 5, 11, 13, and 17; (b) sequencing of exons 5-8 of the p53 gene; and (c) X-chromosome inactivation analysis of the phosphoglycerate kinase (PGK) gene. RESULTS: In 15 of the 17 cases analyzed, there was clear evidence of monoclonal origin. The probability that the genetic events documented in these 15 cases occurred as independent events in each tumor deposit ranged from 2.5 x 10(-1) to 3.7 x 10(-16). In two cases, the pattern of allelic deletion and p53 gene mutation was compatible with either a monoclonal origin or origin from two primary ovarian tumors. CONCLUSIONS: The results did not support the hypothesis that ovarian cancer is a multifocal, polyclonal disease. Instead, the data suggest that sporadic epithelial ovarian carcinoma has either a monoclonal or a dual primary origin. IMPLICATIONS: These findings have important implications for understanding of the natural history of ovarian cancer and for clinical strategies aimed at prevention and early detection. Further studies will be required to determine the clonal origin of familial hereditary ovarian cancer.

Specificity of heteroantisera developed against purified populations of intact murine ovarian carcinoma cells.

Antisera were raised in New Zealand White rabbits against purified populations of murine ovarian carcinoma (MOT) cells that were freed from contaminating host leukocytes and erythrocytes. In contrast to other antisera raised against this tumor, heteroantisera from rabbits immunized with purified tumor cell suspensions consistently retained antitumor activity after exhaustive absorption with syngeneic (C3HeB/FeJ) adult and fetal tissues. Absorbed antisera inhibited tumor growth in vivo and reacted with MOT cells in vitro as judged by indirect immunofluorescence, binding of staphylococcal protein A, and complement-mediated cytotoxicity. Appropriately absorbed antisera failed to bind to fetal tissues or to adult spleen, ovary, and kidney cells. Antisera with similar specificity could be obtained with the use of populations purified on a fluorescence-activated cell sorter or on discontinuous rabbit serum albumin gradients. Optimal titers against tumor were raised with multiple injections of 5 x 10(5) gradient-purified MOT cells.

Regression of established tumors and induction of tumor immunity by intratumor chemotherapy.

The inoculation of a mixture of drugs and guinea pig hepatoma cells (line-10) induced tumor-specific immunity in about 20% of guinea pigs. When guinea pigs with established intradermal tumors were given various drugs ip, no cures were observed; in contrast, multiple intralesional injections of actinomycin D, 1,3-bis(2-chlorethyl)-1-nitrosourea, adriamycin, mitomycin C, and melphalan were effective in curing animals of their intradermal tumors at a time when there were tumor cells in the draining lymph nodes; dimethyl-triazenoimidazole carboxamide, methotrexate, 5-fluorouracil, and 6-mercaptopurine were not effective. More than 80% of the cured animals were immune to rechallenge with 10(6) line-10 tumor cells.