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.

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.

Immunohistochemical analysis reveals a protective effect of H-ras expression mediated via apoptosis in node-negative breast cancer patients.

Recently, we were able to demonstrate the favourable indication of H-ras expression in the clinically very important group of node-negative breast cancer patients. In order to further resolve a conceivable cellular effect mediated by the H-ras signal transduction pathway, we ascertained the apoptotic activity of the tumor cells of this overall 298 patients group. To this end, fragmented genomic DNA of breast cancer tissue was determined by specific DNA-end-labelling and subsequent visualization, thereby indicating early apoptosis induction. Tissues were considered apoptotic by means of the apoptotic index (quotient of apoptotic tumor cells and total tumor cells). One hundred and eighty-nine tumors (63.4%) were negative for apoptosis and 109 tissues (36.6%) were considered apoptotic (mean apoptotic index 11.9%, range, 0 to 90%). H-ras expression correlated significantly (chi2-test, p=0.004) to apoptosis. Furthermore, restricted to the node-negative subgroup, both H-ras expression and apoptosis were indicative of a better outcome (log-rank test p=0.0001, and p=0.012, respectively) during the observation time (median 87 months). These data suggest that H-ras expression effects the particular breast tumor cells by induction of apoptosis in breast cancer patients in an early stage without node involvement. The study indicates a possible mechanism, by which H-ras may act protectively.

Cytokine expression in peripheral blood lymphocytes indicates a switch to T(HELPER) cells in patients with preeclampsia.

We sought to determine whether cytokine expression in peripheral blood mononuclear cells is altered in patients with preeclampsia and in patients with a history of recurrent spontaneous abortion (RSA). Twenty-four patients with preeclampsia and twenty patients with a history of RSA were included into the study. Two control groups consisted of twenty healthy pregnant and twenty healthy non-pregnant women. The intracellular expression of interleukin-2 (IL-2), interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) were determined in peripheral blood mononuclear cells (PBMCs) by flow cytometry as a measure of cytokine production. IL-2 synthesis was significantly elevated in the third trimester in preeclamptic patients in comparison with the control group. Non-pregnant women with RSA showed a significantly lower expression of IFN-gamma compared to the non-pregnant control group. Our data suggest an abnormal immune response in preeclamptic patients characterised by a shift to a predominantly Th1-type immunity.

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.

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.

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.

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.

Comparison of MammaPrint and TargetPrint results with clinical parameters in German patients with early stage breast cancer.

The 70-gene expression profile MammaPrint is a powerful prognostic indicator for disease outcome in breast cancer patients with improved prediction of recurrence risk compared to currently used guidelines. The microarray-based test TargetPrint further provides reliable, quantitative assessment of mRNA expression levels of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). This study was performed as a validation of MammaPrint and TargetPrint in an unselected German breast cancer population and was designed to determine the degree of concordance with currently applied clinical parameters. One hundred and forty cases of breast cancer stage I and II were classified as being low or high risk for distant metastasis using MammaPrint. Results were compared to current clinical risk classifications and adjuvant treatment management. Immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH)/chromogenic in situ hybridization (CISH) assessments of ER, PR and HER2 were further compared with gene expression read-outs using TargetPrint. Thirty-two percent of patients (19/59) with a poor prognosis-signature identified via MammaPrint did not receive adjuvant systemic treatment apart from endocrine therapy and were potentially undertreated; whereas 42% (35/77) of patients with a good prognosis-signature received chemotherapy and were potentially overtreated. Comparison of microarray receptor results with IHC and FISH/CISH were concordant in 97% for ER; 86% for PR; and 94% for HER2. In this German study population, MammaPrint would have resulted in altered treatment advice for adjuvant systemic therapy in 40% of patients. Furthermore, TargetPrint presented high concordance for ER, PR and Her2 with IHC and FISH/CISH analysis.

Does Center Volume Correlate with Survival from Breast Cancer?

With its high incidence and long history of patient advocacy, breast cancer has generated the most concern about the quality of its care and the volume-outcome relationship. In breast cancer surgery, the risk of perioperative morbidity or mortality is low, but surgery is only one single piece in the mosaic of multidisciplinary care that eventually determines survival. Only a limited number of articles is available investigating the relationship between case volume of physicians and hospitals and specialization of surgeons and survival. In summary, there is evidence to support the hypothesis that specialization, research interest, and caseload of physicians and hospitals is positively correlated with providing state-of-the-art care and with survival. However, it is less clear what impact might be attributed to the surgical routine gained with increasing number of procedures compared to the deeper insight into the biology of breast cancer that comes with specialization in oncology and the weight of the multidisciplinary setting that is more easily established and maintained with a higher caseload.

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.

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.

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.