A feasibility study of low-dose, prolonged oral topotecan in patients with advanced ovarian, fallopian tube, or primary peritoneal serous cancer who have attained a complete clinical response following platinum-based chemotherapy.

To determine the tolerability of oral maintenance topotecan when administered to patients with advanced ovarian, fallopian tube, and primary peritoneal serous cancers who have achieved a complete clinical response after first-line platinum-based therapy. Oral topotecan was given at a starting dose of 0.4 mg/m(2)/dose, twice a day (BID) for 21 consecutive days out of 28 days. The dose was subsequently increased to 0.5 mg/m(2)/dose, twice a day as tolerated. If the patient experienced toxicities during cycle 1 or subsequent cycles, doses were delayed and/or reduced. The lowest dose allowed on protocol was 0.3 mg/m(2)/dose twice daily. Thirteen patients were enrolled in the study, representing a total of fifty-nine cycles of oral topotecan. The starting dose of 0.4 mg/m(2) by mouth (PO) BID for 21 days was generally difficult for patients to tolerate, usually due to progressive anemia and fatigue, and a dose reduction to 0.3 mg/m(2) was necessary in 10/13 patients. A median of six cycles was administered, although 6 of 13 patients could not tolerate the planned 6 cycles due to toxicity. Hematologic toxicity was the most common side effect, although there were no episodes of febrile neutropenia. Diarrhea was the most common nonhematologic side effect, occurring in 8 of 13 patients. Six patients were removed from the study prior to completing the planned six cycles of therapy, after receiving a median number of 2.5 cycles of treatment. This dose and schedule of oral topotecan does not appear to be feasible in this patient population.

Opposite association of two PPARG variants with cancer: overrepresentation of H449H in endometrial carcinoma cases and underrepresentation of P12A in renal cell carcinoma cases.

Peroxisome proliferator activated receptor gamma (PPARgamma) is a nuclear hormone receptor that has been shown to regulate differentiation and cell growth. Studies of the differentiative effects of PPARgamma agonists on several cancer cell lines led to the hypothesis that dysfunction of PPARgamma contributes to tumorigenesis. These functional observations were strengthened by genetic evidence: somatic loss-of-function mutations in PPARG, encoding PPARgamma, in sporadic colorectal carcinomas and somatic translocation of PAX8 and PPARG in follicular thyroid carcinoma. Recently overrepresentation of the H449H variant was found in a cohort of American patients with glioblastoma multiforme. The glioblastoma multiforme data suggest that PPARG contributes common, low-penetrance alleles for cancer susceptibility. To test this hypothesis in a broader range of cancers we examined a series of carcinomas of the cervix, endometrium, ovary, prostate, and kidney for germline sequence variation in PPARG. In addition to the two common sequence variants, P12A and H449H, there were five other sequence variants. P12A alleles were underrepresented in renal cell carcinoma patients compared to country-of-origin race-matched controls (3.75% vs. 12.1%, P<0.04). In contrast, the H449H variant was overrepresented in individuals with endometrial carcinoma compared to controls (14.4% vs. 6.25%, P<0.02). These observations lend genetic evidence consistent with our hypothesis that PPARG serves as a common, low-penetrance susceptibility gene for cancers of several types, especially those epidemiologically associated with obesity and fat intake.

Frequent loss of PTEN expression is linked to elevated phosphorylated Akt levels, but not associated with p27 and cyclin D1 expression, in primary epithelial ovarian carcinomas.

PTEN (MMAC1/TEP1), a tumor suppressor gene on chromosome subband 10q23.3, is variably mutated and/or deleted in a variety of human cancers. Germline mutations in PTEN, which encode a dual-specificity phosphatase, have been implicated in at least two hamartoma tumor syndromes that exhibit some clinical overlap, Cowden syndrome and Bannayan-Riley-Ruvalcaba syndrome. Among several series of ovarian cancers, the frequency of loss of heterozygosity (LOH) of markers flanking and within PTEN, is approximately 30 to 50%, and the somatic intragenic PTEN mutation frequency is <10%. In this study, we screened primary adenocarcinomas of the ovary for LOH of polymorphic markers within and flanking the PTEN gene and for intragenic mutations of the PTEN gene and compared them to PTEN expression using immunohistochemistry. Furthermore, we sought to detect the expression of the presumed downstream targets of PTEN, such as P-Akt, p27, and cyclin D1 by immunohistochemistry. LOH at 10q23 was observed in 29 of 64 (45%) cases. Of the 117 samples, 6 somatic intragenic PTEN mutations, 1 germline mutation, and 1 novel polymorphism were found in 7 (6%) patients. Immunostaining of 49 ovarian cancer samples revealed that 13 (27%) were PTEN immunostain-negative, 25 (51%) had reduced staining, and the rest (22%) were PTEN expression-positive. Among the 44 informative tumors assessed for 10q23 LOH and PTEN immunostaining, there was an association between 10q23 LOH and decreased or absent staining (P = 0.0317). Of note, there were five (11%) tumors with neither mutation nor deletion that exhibited no PTEN expression and 10 (25%) others without mutation or deletion but had decreased PTEN expression. Among the 49 tumors available for immunohistochemistry, 28 (57%) showed P-Akt-positive staining, 24 (49%) had decreased p27 staining, and cyclin D1 was overexpressed in 35 (79%) cases. In general, P-Akt expression was inversely correlated with PTEN expression (P = 0.0083). These data suggest that disruption of PTEN by several mechanisms, allelic loss, intragenic mutation, or epigenetic silencing, all contribute to epithelial ovarian carcinogenesis, and that epigenetic silencing is a significant mechanism. The Akt pathway is prominently involved, but clearly not in all cases. Surprisingly, despite in vitro demonstration that p27 and cyclin D1 lies downstream of PTEN and Akt, there was no correlation between p27 and cyclin D1 expression and PTEN or P-Akt status. Thus, in vivo, although PTEN and Akt play a prominent role in ovarian carcinogenesis, p27 and cyclin D1 might not be the primary downstream targets. Alternatively, these observations could also suggest that pathways involving other than Akt, p27 and cyclin D1 that lie downstream of PTEN play roles in ovarian carcinogenesis.

Phase I trial of carboplatin, paclitaxel, etoposide, and cyclophosphamide with granulocyte colony stimulating factor as first-line therapy for patients with advanced epithelial ovarian cancer.

OBJECTIVES: The goal of this study was to determine the maximally tolerated doses (MTDs) of carboplatin, paclitaxel (Taxol), etoposide, and cyclophosphamide (CTEC) with granulocyte-colony stimulating factor (G-CSF, Filgrastim) support as first-line chemotherapy in women with advanced epithelial ovarian cancer (EOC). METHODS: Newly diagnosed patients with either stage IV EOC, or stage III EOC and any amount of gross residual tumor after surgical debulking were eligible to receive six cycles of CTEC over five different dose levels in this phase I trial (planned 21-day cycle length). Paclitaxel, carboplatin, and cyclophosphamide were administered intravenously on Day 1, and oral etoposide was administered on Days 1, 2, and 3. G-CSF was administered beginning Day 4. RESULTS: Twenty patients received a total of 98 cycles of CTEC over the five dose levels evaluated. Bone marrow suppression was the major toxic effect, with grade 4 neutropenia and thrombocytopenia being observed in 25 and 23% of cycles, respectively. The overall incidence of febrile neutropenia was 10%, and no toxic deaths occurred. No grade IV thrombocytopenia or febrile neutropenia was observed once the carboplatin dose was reduced from AUC of 7 to 5. Nonhematologic toxicity was generally mild (grade 2 or less). Dose-limiting toxicity was not observed at the highest dose level evaluated in this study, preventing assignment of the MTD. The clinical complete response rate was 92%, although 15 of 16 evaluable patients have progressed with a median progression-free interval of 4 months (range, 2-11 months). One patient remains disease-free 9 months from the completion of CTEC. CONCLUSIONS: The CTEC regimen is well tolerated and highly active. Although the MTD was not reached in this study, the short median progression-free interval suggests that this regimen is unlikely to be superior to standard treatment with paclitaxel and carboplatin. Strategies to optimize the development of future combination chemotherapy regimens in the treatment of newly diagnosed ovarian cancer are discussed.

Beta1-integrins partly mediate binding of ovarian cancer cells to peritoneal mesothelium in vitro.

Ovarian cancer cells frequently metastasize by implanting onto the peritoneal mesothelial surface of the abdominal cavity. Although the CD44 molecule expressed by ovarian cancer cells is known to partly mediate this process, the role of other adhesion proteins such as beta1-integrins has been previously difficult to demonstrate using the 4B4 anti-beta1 neutralizing antibody. In view of the widespread expression of beta1-integrins in ovarian cancer, however, we have further examined the potential role of this class of molecules in ovarian cancer cell implantation through the use of an alternative anti-beta1 neutralizing antibody, MAB13. We now report that MAB13 is capable of inhibiting the binding of three separate human ovarian cancer cell lines (36M2, CAOV-3, SKOV-3) to mesothelium (mean 37 +/- 4% inhibition, n = 21, P < 0.001). An additive inhibitory effect was observed when MAB13 was combined with anti-CD44 antibody (clone 515) (mean 63 +/- 3% inhibition, n = 19, P < 0.001), suggesting that binding occurs through two independent pathways involving both beta1-integrins and CD44. Because fibronectin is an extracellular matrix ligand recognized by many types of integrins and is abundantly expressed on mesothelial cells, the inhibitory effects of MAB13 and 4B4 on ovarian cancer cell binding to fibronectin were directly compared. MAB13 inhibited ovarian cancer cell binding to fibronectin to a significantly greater degree than did 4B4, suggesting that the discordant effects of these two antibodies on mesothelial adhesion may be partly related to their differential ability to neutralizing fibronectin-mediated binding. Studies using anti-alpha5 neutralizing antibody demonstrated that the alpha5beta1 fibronectin receptor contributes to approximately 50% of integrin-mediated binding of 36M2 and CAOV-3 cells (which express the alpha5 chain in 54 and 58% of cells, respectively). Since the RGD sequence of fibronectin is a known recognition site for many types of integrins, including alpha5beta1 and alphanubeta3, we performed binding in the continued presence of both anti-alpha5 and RGD peptide in order to exclude other types of fibronectin-integrin interactions. The addition of RGD peptides at concentrations known to be capable of blocking fibronectin binding resulted in no additional inhibitory effect over that observed with anti-alpha5 antibody alone, suggesting that alpha5beta1 was the major receptor responsible for fibronectin-mediated ovarian cancer binding to mesothelium. These data demonstrate that ovarian cancer cell binding to peritoneal mesothelium is mediated by several adhesion pathways and that simultaneous inhibition of both beta1-integrin and CD44 function may be necessary in order to significantly limit the intraabdominal spread of this tumor in vivo.

In vivo cytotoxicity of ovarian cancer cells through tumor-selective expression of the BAX gene.

The BAX proapoptotic protein is capable of inducing cell death either directly, through its effects on mitochondrial function, or indirectly, by lowering the apoptotic threshold in response to certain chemotherapy agents. In this study, we tested the hypothesis that selective expression of BAX in human ovarian cancer through adenoviral gene transfer might represent a novel approach to eradicating tumor cells in vivo. Two constructs were prepared using replication-deficient adenoviral vectors containing either the cDNA for beta-galactosidase (Ad.DF3.betaGAL) or hemagglutinin (HA)-tagged BAX (Ad.DF3.BAX) under the control of the DF3 promoter. The DF3 promoter was used to confer tumor-specific gene expression in view of its restricted pattern of expression in the majority of human ovarian cancers and its limited expression in normal peritoneal mesothelial cells. In vitro infection of up to seven different epithelial cancer cell lines with Ad.DF3.betaGAL or Ad.DF3.BAX resulted in expression of either beta-galactosidase activity or HA-BAX protein, respectively, which was highly correlated with DF3 levels. Furthermore, infection with Ad.DF3.BAX was capable of highly selective cytotoxicity of DF3-positive ovarian cancer clonogenic cells in vitro. The effect of i.p. administration of Ad.DF3.BAX was also assessed in nude mice inoculated with the DF3-positive 36M2 human ovarian cancer cell line. Expression of either beta-galactosidase activity (after Ad.DF3.betaGAL treatment) or HA-BAX transcripts (after Ad.DF3.BAX treatment) was restricted to tumor tissue in vivo. Importantly, administration of Ad.DF3.BAX on days 2 and 3 after tumor inoculation was capable of eradicating >99% of tumor implants. These results demonstrate the feasibility of tumor selective expression of a proapoptotic protein such as BAX through adenoviral gene transfer.

BAD partly reverses paclitaxel resistance in human ovarian cancer cells.

Although paclitaxel is an important chemotherapy agent for the treatment of patients with epithelial ovarian cancer, its utility is significantly limited by the frequent development of drug resistance. Recent evidence suggests that resistance to chemotherapy may be partly related to defects in the apoptotic pathway. In this study we have investigated whether enhancement of apoptotic pathway function through stable expression of the BAD protein is capable of sensitizing human epithelial ovarian cancer cells to the effects of chemotherapy. Expression of HA-BAD in six separate clonal transfectants from two different ovarian cancer cell lines was found to significantly enhance the cytotoxic effects of paclitaxel, vincristine, and, to a lesser extent, etoposide. Enhancement of paclitaxel-induced apoptosis in HA-BAD-expressing clones was demonstrated by trypan blue exclusion, clonogenic cell assay, and flow cytometric evaluation. Importantly, this effect was associated with binding of HA-BAD to BCL-xL and concomitant disruption of BAX:BCL-xL interaction. Taken together, these data suggest that the development of small molecules which mimic the effects of BAD may represent a new class of drugs capable of preventing or reversing resistance to chemotherapy agents such as paclitaxel.

BAX expression is associated with enhanced intracellular accumulation of paclitaxel: a novel role for BAX during chemotherapy-induced cell death.

SW626 cells that overexpress BAX are sensitized to the cytotoxic effects of paclitaxel and vincristine. It has been assumed that BAX mediates these effects through its ability to alter mitochondrial function, specifically by promoting the release of cytochrome c and facilitating the mitochondrial permeability transition. However, we have found that several early paclitaxel-mediated events are enhanced in SW626 transfectants that overexpress BAX, including G2-M-phase arrest, tubulin polymerization, and BCL-2 phosphorylation. We now demonstrate that these seemingly disparate effects are explained by an enhanced accumulation of paclitaxel in BAX-overexpressing cells, an effect due to diminished drug efflux. In contrast, drug efflux is increased in cells that do not overexpress BAX, resulting in low intracellular paclitaxel levels and relative resistance to the effects of this drug. Drug efflux in SW626 cells is mediated by a verapamil-inhibitable, non-MDR-1, non-MRP-1 transporter whose function or expression may be inhibited by BAX. These data suggest that stable transfectants that overexpress BAX may be sensitized to apoptotic cell death through a novel mechanism involving the enhancement of intracellular levels of naturally occurring toxins such as alkaloid derivatives.

BAX protein expression and clinical outcome in epithelial ovarian cancer.

PURPOSE: Expression of the pro-apoptotic protein BAX sensitizes ovarian cancer cell lines to paclitaxel in vitro by enhancing the pathway of programmed cell death. The present study was performed to determine the relationship between BAX expression and clinical outcome in 45 patients with newly diagnosed ovarian cancer. METHODS: BAX protein expression was analyzed by immunohistochemistry, and its relationship with clinical outcome was determined. Assessment of BAX mRNA transcript levels and mutational analysis of the BAX coding region were also performed. RESULTS: BAX protein was expressed at high levels (defined as > or = 50% of tumor cells positive) in tumor tissue from 60% of newly diagnosed patients. All patients whose tumors expressed high levels of BAX achieved a complete response (CR) to first-line chemotherapy that contained paclitaxel plus a platinum analogue, compared with 57% of patients in the low-BAX group (P = .036). After a median follow-up of 1.9 years, the median disease-free survival (DFS) of patients in the high-BAX group has not been reached, compared with a median DFS of 1.1 years for low-BAX expressors (P = .0061). BAX retained independent prognostic significance in multivariate analysis when corrected for stage and histology. BAX mRNA transcripts were easily detected in samples with low BAX protein expression, and no BAX mutations were identified. CONCLUSION: The correlation between high BAX levels and improved clinical outcome suggests that an intact apoptotic pathway is an important determinant of chemoresponsiveness in ovarian cancer patients who receive paclitaxel.

Enrichment for DNA mismatch repair-deficient cells during treatment with cisplatin.

In addition to playing a role in tumorigenesis, loss of DNA mismatch repair results in low-level intrinsic resistance to cisplatin and carboplatin. We used a mismatch repair-deficient (clone B) and -proficient (clone B/rev) pair of Chinese hamster ovary sublines to determine the ability of cisplatin to enrich for repair-deficient cells during growth in vitro and in vivo. Clone B cells were 1.8-fold resistant to cisplatin as measured by a clonogenic assay. These cells were molecularly engineered to express constitutively the green fluorescent protein, and changes in the fraction of these repair-deficient cells were monitored by flow cytometric analysis. A single 1-hr exposure to cisplatin at an IC50 concentration enriched populations initially containing either 5 or 10% clone B cells by 81 and 75%, respectively, when measured at 5 days. Enrichment increased as a function of drug concentration to 158 and 169%, respectively, following an IC90 exposure. When grown as a xenograft, a single LD10 dose of cisplatin enriched the tumors by 48% from 4.6 to 6.8% repair-deficient cells (p = 0.04). To determine whether similar enrichment occurs during the treatment of human ovarian cancer patients, paired tumor samples were obtained from 38 patients before and after treatment with a minimum of 3 cycles of platinum drug-based primary chemotherapy and analyzed immunohistochemically for changes in the fraction of tumor cells expressing hMHL1. Following treatment there was a reduction in hMLH1 staining in 66% of the cases (p = 0.0005). Our results demonstrate that, despite the fact that loss of mismatch repair yields only modest levels of cisplatin resistance, even a single exposure to cisplatin produces quite a marked enrichment for repair-deficient cells in vitro and in vivo. Our results are consistent with the concept that treatment with cisplatin or carboplatin selects for preexisting mismatch repair-deficient cells, and that this contributes to the frequent development of clinical resistance.

Radiation-induced apoptosis is not enhanced by expression of either p53 or BAX in SW626 ovarian cancer cells.

The p53 protein is known to play a central role in mediating G1 arrest or apoptosis in response to ionizing radiation in some cell types. It has been proposed that the link between p53 and induction of apoptosis is provided in part by p53-mediated upregulation of BAX. In this study, we used the human SW626 ovarian cancer cell line, which lacks functional p53, to further investigate the relationship between wildtype p53, BAX, and apoptosis. SW626 cells expressing a temperature sensitive (ts) p53 mutant did not undergo G1 arrest or apoptosis and did not exhibit enhanced sensitivity to radiation at the permissive temperature of 32 degrees C. The tsp53 protein was functional in these cells as evidenced by rapid induction of p21 at 32 degrees C, but not at 37 degrees C. Interestingly, restoration of wildtype p53 function at 32 degrees C was not associated with BAX upregulation. In addition, stable overexpression of BAX in SW626 cells was not capable of enhancing apoptotic cell death in response to radiation. Thus, failure of p53 to upregulate BAX is not the sole reason for its inability to promote radiation-induced apoptosis in SW626 cells. Taken together, our data suggest that neither p53 nor BAX upregulation is sufficient for the induction of apoptosis in response to genotoxic damage in some cell types.

In vivo inhibition of CD44 limits intra-abdominal spread of a human ovarian cancer xenograft in nude mice: a novel role for CD44 in the process of peritoneal implantation.

Ovarian cancer cells frequently metastasize by implanting onto the peritoneal mesothelial lining of the abdominal cavity. Data obtained from in vitro adhesion studies have suggested a possible role for the CD44 molecule in this process. The purpose of the present study was to determine the in vivo role of CD44 in ovarian cancer metastasis by using a nude mouse xenograft model of peritoneal implantation. Three groups of 10 athymic female nude mice each received an i.p. inoculum of 10 x 10(6) cells from a CD44-positive human ovarian cancer cell line (36M2) in the presence of either anti-D144 antibody (Ab; nonreactive IgG1), anti-DF3 Ab (reactive IgG1 Ab that does not inhibit in vitro binding), or neutralizing anti-CD44 Ab (IgG1). The number of peritoneal and diaphragmatic implants at 5 weeks for anti-D144 and anti-DF3-treated groups was 103 +/- 17 and 120 +/- 20, respectively (mean +/- SE; P > 0.2). In contrast, animals treated with anti-CD44 Ab experienced a significant reduction in the number of tumor implants (35 +/- 4; P < 0.002). Anti-CD44 Ab was not inhibitory to the growth of 36M2 cells in vitro and did not inhibit s.c. tumor growth in vivo, suggesting that the observed effect was related to inhibition of peritoneal implantation. These data suggest that the CD44 molecule plays an important in vivo role in ovarian cancer cell implantation and that strategies to inhibit CD44 function may represent a novel approach to limiting the intra-abdominal spread of this highly lethal tumor.

BAX enhances paclitaxel-induced apoptosis through a p53-independent pathway.

To investigate the role of BAX in chemotherapy-induced apoptosis, we transfected the SW626 human ovarian cancer cell line, which lacks functional p53, with a cDNA encoding for murine BAX. Immunoblotting revealed that BAX transfectants expressed a mean of 10-fold increased levels of BAX compared with neo-transfected control clones, with similar levels of BCL-2 and BCL-xL. The cytotoxicity of paclitaxel, vincristine, and doxorubicin was significantly enhanced in BAX transfectants compared with control clones, whereas the cytotoxicity profile of carboplatin, etoposide, and hydroxyurea was unchanged. Increased paclitaxel-induced cytotoxicity of BAX clones was associated with enhanced apoptosis, as assessed by morphologic and flow cytometric criteria. These data suggest that sufficient levels of BAX may bypass the need for upstream molecules such as p53 in the process of chemotherapy-induced apoptosis.

Absence of constitutive EGF receptor activation in ovarian cancer cell lines.

Previous investigators have noted that certain ovarian cancer cell lines secrete and respond to transforming growth factor-alpha (TGF-alpha), suggesting that endogenous activation of the epidermal growth factor (EGF) receptor through autocrine or paracrine mechanisms might contribute to the proliferative response. In order to determine whether autocrine stimulation was partly responsible for the proliferative response in ovarian cancer, we investigated whether the EGF receptor expressed by ovarian cancer cell lines was constitutively activated as assessed by the presence of tyrosine phosphorylation. A specific anti-phosphotyrosine antibody was used in conjunction with an immunoblotting technique in order to detect EGF receptor phosphorylation in ovarian cancer cell lines in the absence and presence of exogenous EGF. The effects of neutralising anti-EGF receptor antibody on the proliferation of ovarian cancer cell lines was also examined. We found no evidence for constitutive tyrosine phosphorylation of the p170 EGF receptor in eight epithelial ovarian cancer cell lines tested, although each line demonstrated inducible phosphorylation in response to exogenous EGF. The absence of constitutive EGF receptor activation was also noted when cells were grown under high density conditions, thus excluding a role for membrane-bound EGF or TGF-alpha in this process. Media conditioned by five ovarian cancer cell lines, as well as malignant ascites obtained from 12 different ovarian cancer patients, were not capable of stimulating EGF receptor phosphorylation. Finally, the proliferation of ovarian cancer cell lines was not significantly inhibited in the presence of neutralising anti-EGF receptor antibody. These data suggest that EGF receptor activation through autocrine pathways is not a major mechanism for the growth of many ovarian cancer cell lines. Other pathways of signal transduction which bypass the requirement for EGF receptor activation may be important in the proliferation for ovarian cancer cells. Such EGF receptor-independent pathways may limit the effectiveness of strategies designed to inhibit ovarian cancer cell growth through disruption of EGF receptor function.