Systemic administration of bevacizumab prolongs survival in an in vivo model of platinum pre-treated ovarian cancer.

Ovarian cancer patients often suffer from malignant ascites and pleural effusion. Apart from worsening the outcome, this condition frequently impairs the quality of life in patients who are already distressed by ovarian cancer. This study investigated whether single intraperitoneal administration of the anti-VEGF antibody bevacizumab is capable of reducing the ascites-related body surface and prolonging survival. The study was performed in an orthotopic murine model of peritoneal disseminated platin-resistant ovarian cancer. Mice were treated with bevacizumab and/or paclitaxel or buffer (control). Reduction of body surface and increased survival rates were assessed as therapeutic success. Survival of mice in all treatment groups was significantly enhanced when compared to the non-treatment control group. The combination of paclitaxel plus bevacizumab significantly improved body surface as well as overall survival in comparison to a treatment with only one of the drugs. Treatment of malignant effusion with a single dose of bevacizumab as an intraperitoneal application, with or without cytostatic co-medication, may be a powerful alternative to systemic treatment.

Combination of a MDR1-targeted replicative adenovirus and chemotherapy for the therapy of pretreated ovarian cancer.

PURPOSE: Targeted oncolytic adenoviruses capable of replication selectively in cancer cells are an appealing approach for the treatment of various cancer types refractory to conventional therapies. The aim of this study was to evaluate the effect of Ad5/3MDR1E1, a multidrug resistance gene 1 (MDR1)-targeted fiber-modified replication-competent adenovirus for the therapy of platinum-pretreated ovarian cancer in combination with cytostatic agents. METHODS: MDR1-specific tumor cell killing of Ad5/3MDR1E1 was systematically evaluated in chemotherapy naïve and pretreated ovarian cancer cells in vitro. Combinations of Ad5/3MDR1E1 and cytostatic agents were studied in vivo and in vitro. An in vivo hepatotoxicity model was used to evaluate liver toxicity. RESULTS: We demonstrate efficient oncolysis of Ad5/3MDR1E1 in chemotherapy-resistant ovarian cancer cells as well as therapeutic efficacy in an orthotopic mouse model. Further, combining Ad5/3MDR1E1 with paclitaxel resulted in greater therapeutic benefit than either agent alone. CONCLUSION: These preclinical data suggest that a fiber-modified adenovirus vector under the control of the MDR1 promoter represents a promising treatment strategy for platinum-pretreated ovarian cancer as a single agent or in combination with conventional anticancer drugs.

Hysteroscopic management of residual trophoblastic tissue is superior to ultrasound-guided curettage.

STUDY OBJECTIVE: The aim of this study was to estimate the rate of intrauterine adhesions and subsequent pregnancy outcome in patients with residual trophoblastic tissue treated with hysteroscopic resection versus ultrasound-guided dilation and evacuation (D&E). DESIGN: Cohort study from 2 centers (Canadian Task Force classification II-2). SETTING: Two surgical teams at the University of Duesseldorf Medical Center and the PAN Clinic in Cologne, Germany. PATIENTS: Women with residual trophoblastic tissue after first- or second-trimester miscarriage or term delivery. INTERVENTION: Two techniques were used for the removal of residual trophoblastic tissue: ultrasound-guided evacuation with a curette (D&E) and hysteroscopic resection of trophoblastic tissue (HR). MEASUREMENTS AND MAIN RESULTS: We evaluated 95 patients who underwent secondary intervention for residual trophoblastic disease. A total of 42 patients underwent dilation of the cervix and ultrasound-guided curettage. In a second series of 53 patients, a resectoscope fitted with a 4-mm cutting loop was used for the removal of residual trophoblastic tissue used without application of current. Three months after the intervention, second-look office hysteroscopy was performed. Differences between both treatment groups were statistically significant. After HR, mild intrauterine adhesions were found in 2 patients (4.2%). After D&E, 12 patients (30.8%) presented with intrauterine adhesions (mild intrauterine adhesions: n = 7 [17.9%]; single dense adhesions: n = 3 [7.7%]; and extensive endometrial fibrosis n = 1 [2.6%]). Eighty-two patients wanted to become pregnant. Conception rate of all patients examined was 68.8% (HR) and 59.9% (D&E) (p < .05). In patients younger than 35 years of age who underwent HR, the pregnancy rate was significantly (p < .05) increased compared with patients who underwent D&E (78.1% vs 66.6%). In addition, patients from the HR group demonstrated a significantly (p < .05) shorter time to conception (11.5 month vs 14.5 month). CONCLUSION: The results of this study indicate that selective HR of residual trophoblastic tissue significantly reduces the incidence of intrauterine adhesions and increases pregnancy rates.

Relaxin expression correlates significantly with serum changes in VEGF in response to antidiabetic treatment in male patients with type 2 diabetes mellitus.

Recent studies indicate that relaxin as well as VEGF possess cardioprotective properties. This study aimed to determine the association of relaxin with VEGF in patients with type 2 diabetes. We therefore analyzed samples from a recent study showing the benefits of anti-diabetic treatment on cardiovascular risk markers independently from glycemic control. VEGF, relaxin and markers of endothelial dysfunction, s-ICAM-1 and s-VCAM-1, were compared after 26 +/- 2 weeks of antidiabetic treatment with pioglitazone or glimepiride with their base line values. A total of 151 data sets (patients age, 62.7 +/- 8.1 years, diabetes duration, 6.8 +/- 6.6 years, 57 women, 94 men) were available for the analysis. Baseline values were in median, relaxin: 27.4 pg/mL 125% quartile 15.8; 75% quartile: 45.21, s-ICAM-1: 294 ng/mL [25% quartile: 260; 75% quartile: 331], s-VCAM-1: 677 ng/mL [25% quartile: 589; 75% quartile 871], VEGF: 350 pg/mL [25% quartile: 251; 75% quartile: 514]. Parameter variation after therapy showed a significant correlation of relaxin expression with VEGF expression (p = 0.02) in the entire study group. The correlation was seen in the subgroup of male patients (p < 0.01) but did not reach significance in the female patients (p = 0.71). No further correlation was observed analyzing the other investigated parameters. Our data suggest that relaxin may exert its cardioprotective action possibly via VEGF increase, particularly in men. In women, other pathways may superimpose this effect. In conclusion, our study supports the hypothesis of different regulating pathways and effects of relaxin in men and women also in patients with type 2 diabetes.

Anticancer drugs induce mdr1 gene expression in recurrent ovarian cancer.

Ovarian cancer is currently the most lethal gynecologic malignancy in Europe and the US. Platin analogues and paclitaxel demonstrate high remission rates, but unfortunately the efficacy of cytostatic agents is limited by the development of multidrug resistance (mdr). Clinical paclitaxel resistance is often associated with mdr1 overexpression. In a recent study, we introduced a highly specific quantitative real-time reverse transcriptase polymerase chain reaction for the quantification of mdr1 transcripts. In the present study, we demonstrate that primary tumor cells from patients with recurrent ovarian cancer overexpress mdr1. To evaluate mdr1 expression, we collected tumor cells from 77 ovarian cancer patients (13 chemotherapy-naive ovarian cancer, 64 recurrent ovarian cancer). Cancer cells were aspirated from 49 solid specimens (63%) and 28 ascitic fluids (37%). Subsequently, cancer cells were exposed in 221 short-term cultures either to blank medium (control) or to a single anticancer drug, cisplatin, doxorubicin or paclitaxel. The drug concentrations applied referred to clinical relevant doses. mdr1 mRNA expression was significantly higher in specimens from recurrent ovarian cancer incubated in paclitaxel than in specimens from chemotherapy-naive ovarian cancer. No significant differences were detectable between the mean value of mdr1 mRNA expression in tumor specimens from recurrent ovarian cancer incubated in cisplatin or doxorubicin. Differences within the untreated patients group were also not statistically significant. The result of this study confirms clinical observations, as well as in-vitro studies based on tumor cell lines, that paclitaxel resistance is correlated with mdr1 overexpression.

A new targeting approach for breast cancer gene therapy using the heparanase promoter.

Gene therapy with adenoviral (Ad) vectors is a promising new approach in the treatment of cancer. Strategies to restrict adenoviral-mediated transgene expression are important to avoid gene transfer into normal cells. Heparanase (HPR) is overexpressed in breast cancer but downregulated in differentiated normal tissue. Expression of the HPR gene was evaluated in breast cancer cells. Biodistribution and liver tropism was evaluated in a mouse model. HPR is highly expressed in breast cancer tissue. The HPR promoter retained its fidelity in an adenovirus context and was activated in breast cancer cells but showed low activity in normal breast cells and the murine liver. We conclude that the HPR pathway is a promising target for the development of breast cancer directed gene therapy strategies.

High apoptotic index correlates to p21 and p27 expression indicating a favorable outcome of primary breast cancer patients, but lacking prognostic significance in multivariate analysis.

This study was performed in order to investigate the role of the apoptotic index (AI) as a prediction parameter for the prognosis of patients with primary breast cancer. AI was determined by DNA fragmentation on 298 primary breast cancer samples and compared to clinically established breast cancer parameters. Additionally, we determined the expression of functional parameters including proliferating cell nuclear antigen, p21waf and p27kip by immunohistochemistry. The mean AI was found to be 11.9% (range, 0-90%). 189 tumors (63.4%) were negative for apoptosis, while 109 tissue samples (36.6%) were apoptotic with >5% positive cells. Using univariate analysis (chi2 test), the AI did not show any significant correlation to one of the established prognostic parameters of primary breast cancer (p > 0.05). In contrast, we found a significant positive correlation to the expression of the cell cycle inhibitors p21waf (p = 0.04) and p27kip (p = 0.024). During the clinical follow-up (median observation time for disease-free survival 87 months), several clinically established prognostic parameters including menopausal status, nodal status, tumor size, tumor grade, and hormone receptor expression could be confirmed and were analyzed with respect to the AI in the tumor. Furthermore, AI displayed a significant positive correlation to disease-free survival using Kaplan-Meier survival analysis (log-rank test, p = 0.04). However, AI lost its prognostic significance in multivariate analysis based on the Cox proportional hazard model (relative risk 0.8, confidence interval 0.52-1.33, p = 0.44). Our data indicate that high apoptotic rates in cancer tissues are indicative of a favorable patient outcome. However, the AI was not an independent factor. The study provides indirect evidence that this process may involve cell cycle inhibitors physiologically.

Hypermethylation of the PTEN gene in ovarian cancer cell lines.

This study was performed to investigate the hypermethylation status of the PTEN gene in ovarian cancer. To this end, we incubated eight ovarian cancer cell lines with the demethylating agent 5-aza-2' deoxycytidine in three different concentrations for 5 days. Subsequently, the PTEN expression was quantified by both real time RT-PCR and quantitative western analyses. PTEN mRNA varied considerably in response to demethylation whereas PTEN protein concentrations remained constant in all cell lines except OAW42 cells (12.5%). The data suggest that PTEN is highly regulated at translational level. However, methylation of the PTEN gene plays a subordinate role in ovarian cancer.

Dysregulation of protein kinase C activity in chemoresistant metastatic breast cancer cells.

This study was performed to evaluate the role of protein kinase C (PKC) activity in the development of chemoresistance in clinical breast cancer cells. To simulate the clinical situation, native tumor cells derived from 10 patients with advanced breast cancer were brought into short-term cultures, and treated with anthracyclines (doxorubicin, mitoxantrone), paclitaxel and combinations, respectively. After 3 days of incubation, we determined total PKC activity relative to each control incubated with blank medium. Furthermore, we determined the chemoresistance against these drugs from each cell population separately. Relative PKC activity ranged from 14 to 249%; 64% (37 of 58) of the breast cancer cell suspensions were considered chemoresistant. There was a non-significant trend to a higher relative PKC activity in resistant cells compared to non-resistant cells (p=0.058), regardless of the antineoplastic agent investigated. The individual variability in both PKC activity and chemoresistance pattern revealed that dysregulated PKC activity mediates resistance to antineoplastics. In order to achieve clinical value, evaluation of more data concerning the PKC signal-transduction pathway is necessary. New protocols of cancer treatment will require this information in order to be successful.

The V109G polymorphism of the p27 gene CDKN1B indicates a worse outcome in node-negative breast cancer patients.

Although p27 plays a central role in cell cycle regulation, its role in breast cancer prognosis is controversial. Furthermore, the p27 gene CDKN1B carries a polymorphism with unknown functional relevance. This study was designed to evaluate p27 expression and p27 genotyping with respect to early breast cancer prognosis. 279 patients with infiltrating metastasis-free breast cancer were included in this study. p27 expression was determined in tumor tissue specimens from 261 patients by immunohistochemistry. From 108 patients, the CDKN1B genotype was examined by PCR and subsequent direct sequencing. 55.2% of the tumors were considered p27 positive. p27 expression did not correlate with any of the established parameters except for nodal involvement but significantly correlated to prolonged disease-free survival. In 35% of the tumors analyzed, the CDKN1B gene showed a polymorphism at codon 109 (V109G). The V109G polymorphism correlated with greater nodal involvement. In the node-negative subgroup, V109G correlated significantly with a shortened disease-free survival. In conclusion, the determination of the CDKN1B genotype might be a powerful tool for the prognosis of patients with early breast cancer.

Expression of the tumor suppressor gene PTEN is not altered in the progression of ovarian carcinomas and does not correlate with p27Kip1 expression.

This study was designed to investigate the role of PTEN in the progression of ovarian cancer. We performed mutation analysis and determined PTEN gene expression in tissue from both primary and relapsed cancers and in the corresponding occult metastases. Furthermore, p27Kip1 staining was conducted in order to explore a putative functional link. The study group comprised 112 tumor tissue specimens from 37 ovarian cancer patients. Expression of both PTEN and p27Kip1 was determined by immunohistochemistry. The PTEN mutational spectrum was determined by PCR-based sequence analysis. Fifty-six per cent of the tumors were positive for PTEN expression and 75% were p27Kip1 positive. For both markers, tumor cells ranged from 0 to 90% positivity. In 55% (20/37) of the cases, PTEN expression in the primary tumor was consistent and in the corresponding advanced cancer tissues, whereas the remainder showed considerable variation. p27Kip1 was consistently expressed in 16 out of 37 cases (43%). No mutations were observed in the coding region of the PTEN gene. No correlation was observed between PTEN and p27Kip1 expression. Our data indicate that expression of PTEN, but not p27Kip1 (one of the major mediators of PTEN function) is unchanged during the progression of ovarian cancer. This study suggests that in ovarian cancer PTEN does not play a major role in disease progression and is not involved in the alteration of p27Kip1 expression.

Hematological side-effect profiles of individualized chemotherapy regimen for recurrent ovarian cancer.

The long-term results for patients with recurrent ovarian cancer (ROC) are poor. There is a need to optimize treatment strategies to improve outcome by avoiding ineffective regimens which are often associated with exacerbated side-effects. Individualized chemotherapy regimens guided by a chemosensitivity assay (ATP-tumor chemosensitivity assay) have already been used successfully to direct chemotherapy. Taking the results of this assay into account, application of drug combinations appears more advisable. Here we present a systematic evaluation of toxicities seen with individualized chemotherapy for ROC. A total of 62 patients who received 314 cycles of antineoplastic therapies were evaluated. Three single agents (topotecan, paclitaxel and gemcitabine) and five combinations (cisplatin/gemcitabine, carbopatin/gemcitabine, gemcitabine/treosulfan, mitoxantrone/paclitaxel and carboplatin/paclitaxel) were examined. With respect to myelotoxicity, most single agents except topotecan revealed favorable results in comparison to drug combinations. However, this observation lacks statistical significance. Generally, severe myelosuppression was rare. The highest incidence of leukopenia was seen in regimens with mitoxantrone/paclitaxel or gemcitabine/treosulfan, respectively. Thrombocytopenia accompanied most commonly a topotecan therapy. In the present study combination regimens tend to be more toxic than monotherapies. When response rates are comparable, empirically chosen treatment combination therapies should only be practiced in carefully planned clinical studies.

HER-2/neu determination in blood plasma of patients with HER-2/neu overexpressing metastasized breast cancer: a longitudinal study.

This retrospective study was designed to evaluate the serological HER-2/neu determination as a predictor of the clinical outcome of patients with metastasized breast cancer receiving a trastuzumab therapy. Twenty trastuzumab patients were included into this study. Plasma samples of each patient were collected from the beginning of a trastuzumab therapy until the first imaging diagnostics. Serological HER-2/neu was quantified automatically using the Bayer Immuno 1 immunoanalyzer. Each assay was performed in duplicate. The values were analyzed in terms of the clinical course of each patient. The observation time was 13 months (median) with a range from 4 to 22 months. The data were analyzed by means of prediction of a therapy response as defined by the time to progression. HER-2/neu determination served as a predictive marker for the clinical course in 16 out of 20 patients (80%). All ten therapy responders displayed a normal HER-2/neu either from the beginning of the trastuzumab administration or a value normalization paralleled the course. Patients with permanent elevated or increasing HER-2/neu levels displayed a poor clinical outcome in six out of ten cases. Our data suggest that HER-2/neu determination in the plasma of patients with metastasized breast cancer could be a powerful tool for prediction of the patient's outcome after a trastuzumab-based therapy.

Association of the vitamin D receptor genotype with bone metastases in breast cancer patients.

This study was designed in order to evaluate specific vitamin D receptor (VDR) genotypes as indicators of the likelihood of developing osseous metastases in breast cancer patients. Therefore, we determined polymorphisms of the VDR gene in a study group comprising 183 breast cancer patients. Specific fragments spanning over intron 8 and exon 9 of the VDR gene were amplified by polymerase chain reaction. The fragments were then incubated with each of the specific endonucleases APAI, BSMI or TAQI, respectively. The VDR gene polymorphisms were detected by the presence or absence of the particular restriction site using agarose gel electrophoresis. Statistical analyses revealed a significant correlation between both the VDR gene polymorphisms indicated as AA (absence of the APAI restriction site in both alleles) or TT (absence of the TAQI restriction site in both alleles), respectively, and the occurrence of bone metastases. Patients with the AA genotype have a 1.7-fold increased risk of developing bone metastases, whereas patients with the TT genotype have a 0.5-fold risk. Neither other genotypes nor allelic combinations displayed any further correlation with the clinical stage. The data suggest that the AA genotype of the VDR gene might be useful to identify breast cancer patients with a high probability of forming occult bone metastases who are considered to benefit from an adjuvant bone-protective therapy.

Cisplatin, doxorubicin and paclitaxel induce mdr1 gene transcription in ovarian cancer cell lines.

The clinical observation of the multidrug resistance (MDR) phenotype is often associated with overexpression of the mdrl gene, in particular with respect to ovarian cancer. However, until now the mdrl-inducing potential of commonly used antineoplastics has been only incompletely explored. We performed short-term cultures of six ovarian cancer cell lines (MZOV4, EF027, SKOV3, OAW42, OTN14, MZOV20) exposed to either blank medium or cisplatin, doxorubicin or paclitaxel at concentrations related to the clinically achievable plasma peak concentration. A highly specific quantitative real-time RT-PCR was used to detect the Mdr1 transcripts. Mdrl mRNA contents were calibrated in relation to coamplified GAPDH mRNA. Mdrl mRNA was detectable in each cell line. In 13 out of 18 assays (72%) the specific anticancer drug being tested induced mdr1 transcription. No decrease in mdr1 mRNA concentration was observed. Our data suggest that mdr1 induction by antineoplastics is one of the reasons for failure of ovarian cancer therapy but may vary individually.

Tumor M2 pyruvate kinase--determination in breast cancer patients receiving trastuzumab therapy.

This study was designed to investigate the value of tumor M2 pyruvate kinase (tumor M2-PK) determination as an early marker for response to trastuzumab therapy in patients with metastasized breast cancer. Plasma samples of 20 trastuzumab patients were collected immediately after standard hematological investigations. The tumor M2-PK level was quantified using an enzyme linked immunosorbent assay (ScheBo Biotech) and CA 15-3 was measured automatically using the Bayer Immuno 1 immunoanalyzer and the corresponding assay. Each assay was performed in duplicate. The values were analyzed for correlation to the clinical course of each patient. Median observation time was 13 months with a range from 4 to 22 months. In 17/20 (85%) patients, tumor M2-PK determination was a marker for the clinical course of their disease. In this 'tumor M2-PK sensitive' group, 49 known clinical events (remission or progression according to UICC criteria) were recorded. The variation in tumor M2-PK level paralleled 63% of the clinical events (31/49). Our data suggest that tumor M2-PK determination in the plasma of patients with metastasized breast cancer could be a helpful tool for monitoring therapeutic success.

Lack of prognostic significance of nm23 expression in human primary breast cancer.

This retrospective study was designed to evaluate the prognostic value of immunohistochemically detected nm23-expression in invasive breast cancer. Cellular expression of nm23 was assessed in 325 primary breast cancer tissues by immunohistochemistry. The data were correlated to established functional factors of prognosis (age, tumor size, nodal status, tumor grade, steroid hormone receptor status), and to clinical follow-up (median observation time, 92 months). 215/325 (66.2%) tumor tissues were considered nm23 positive. nm23 expression did not correlate to any of the investigated markers. Similar results were obtained after subdivision of the patients into node-negative patients (n=153) and node-positive cases (n=172). With respect to clinical outcome, nm23 expression displayed no statistical significance to predict the clinical course. In conclusion, the determination of nm23 expression is an independent factor but lacks prognostic or predictive value in breast cancer patients.

Induction of MDR1-gene expression by antineoplastic agents in ovarian cancer cell lines.

BACKGROUND: Development of resistance to anticancer drugs is a major concern in clinical oncology and might be particularly involved in the secondary treatment failure frequently seen in ovarian cancer. Clinical observation of the multidrug resistance (MDR) phenotype is often associated with overexpression of the mdr1-gene. However, until now the mdr1-inducing potential of commonly used antineoplastics has been only incompletely explored. MATERIALS AND METHODS: We perfomed short-term cultures of 7 established ovarian cancer cell lines exposed to either blank medium or one of three single anticancer drugs (cisplatin, doxorubicin, paclitaxel) at concentrations related to the clinically achievable plasma peak concentration. Mdr1-transcripts were detected using the highly specific quantitative real time RT-PCR. To calibrate each approach, mdr1-mRNA content was calculated in relation to co-amplified GAPDH-mRNA. RESULTS: Mdr1-mRNA was detectable in each cell line. In 13 assays (62%) the specific anticancer drug being tested induced mdr1-transcription. No decrease in mdr1-mRNA concentration was observed. The method described here is easy to perform and could be of striking value in predicting the development of tumor chemoresistance. CONCLUSION: Our data indicate that mdr1-induction by antineoplastics is one of the reasons for failure of ovarian cancer therapy but may vary from one individual to another.