Effects of EpCAM overexpression on human breast cancer cell lines.

BACKGROUND: Recently, EpCAM has attracted major interest as a target for antibody- and vaccine-based cancer immunotherapies. In breast cancer, the EpCAM antigen is overexpressed in 30-40% of all cases and this increased expression correlates with poor prognosis. The use of EpCAM-specific monoclonal antibodies is a promising treatment approach in these patients. METHODS: In order to explore molecular changes following EpCAM overexpression, we investigated changes of the transcriptome upon EpCAM gene expression in commercially available human breast cancer cells lines Hs578T and MDA-MB-231. To assess cell proliferation, a tetrazolium salt based assay was performed. A TCF/LEF Reporter Kit was used to measure the transcriptional activity of the Wnt/ß-catenin pathway. To evaluate the accumulation of ß-catenin in the nucleus, a subcellular fractionation assay was performed. RESULTS: For the first time we could show that expression profiling data of EpCAM transfected cell lines Hs578TEpCAM and MDA-MB-231EpCAM indicate an association of EpCAM overexpression with the downregulation of the Wnt signaling inhibitors SFRP1 and TCF7L2. Confirmation of increased Wnt signaling was provided by a TCF/LEF reporter kit and by the finding of the nuclear accumulation of ß-catenin for MDA-MB-231 EpCAM but not Hs578T EpCAM cells. In Hs578T cells, an increase of proliferation and chemosensitivity to Docetaxel was associated with EpCAM overexpression. CONCLUSIONS: These data show a cell type dependent modification of Wnt signaling components after EpCAM overexpression in breast cancer cell lines, which results in marginal functional changes. Further investigations on the interaction of EpCAM with SFRP1 and TCF7L2 and on additional factors, which may be causal for changes upon EpCAM overexpression, will help to characterize unique molecular properties of EpCAM-positive breast cancer cells.

14-3-3 sigma and p53 expression in gastric cancer and its clinical applications.

14-3-3 sigma (σ) induces G2 arrest enabling the repair of damaged DNA. The function of 14-3-3 σ is frequently lost in tumor cells, indicating a potential tumor suppressor function. The purpose of this study was to evaluate the prognostic value of 14-3-3 σ expression in human gastric cancer. 14-3-3 σ expression was analyzed by immunohistochemistry in 157 tumor samples of patients, who underwent resection for gastric cancer. Since 14-3-3 σ is involved in the p53 network, p53 expression was detected in parallel and correlated with 14-3-3 σ. 14-3-3 σ was found to be overexpressed in 75 (47.8%) of 157 cases, the overexpression rate of p53 protein was 27.4%. 14-3-3 σ overexpression was statistically significantly associated with pT-stage (p=0.041) pN-stage (p=0.015) and UICC-stage (p=0.019) and showed a borderline significance with Lauren classification (p=0.057). Univariate survival calculations revealed a coexistent 14-3-3 σ and p53 overexpression as a significant predictor of disease-free survival. Multivariate analysis did not unfold 14-3-3 as an independent prognostic factor for disease-free survival and overall survival. Concomitant 14-3-3 σ and p53 overexpression in tumor cells of patients with gastric cancer identifies a population of patients with relatively unfavorable prognosis.

Immunohistochemically detectable dickkopf-3 expression in tumor vessels predicts survival in gastric cancer.

Dickkopf-3 (Dkk-3) may act as a tumor suppressor as it is downregulated in various types of cancer. Moreover, a putative role in tumor neovascularization is discussed. Here, we investigated the expression of Dkk-3 protein in gastric cancer and its potential value as a prognostic marker. Dkk-3 expression was analyzed by immunohistochemistry in 136 tumor samples and was correlated with microvessel density (MVD), tumor stage, and grading as well as the clinical outcome of the patients. Dkk-3 expression was detected in endothelial cells of the tumor vessels in 129/136 (94.9%) and in tumor cells in 85/136 (62.5%) samples. MVD was high and low in 57 (42.9%) and 76 (57.1%) specimens respectively. In tumor cells, overexpression of Dkk-3 was found in 41 (30.1%) of all cases and was correlated significantly to pT-stage (p < 0.05) and UICC stage (p < 0.05). Survival analysis regarding Dkk-3 expression in tumor endothelial cells showed that Dkk-3 is an independent predictor of disease-free survival (p < 0.05). Dkk-3 expression in tumor vessels of patients with gastric cancer identifies a population of patients with relatively favorable prognosis.

Circulating cell-free DNA in plasma of locally advanced rectal cancer patients undergoing preoperative chemoradiation: a potential diagnostic tool for therapy monitoring.

Circulating cell-free DNA opens up an interesting field for therapy monitoring, in particular during multimodal therapy protocols. The objective of this proof of principle study was to evaluate whether the amount of circulating plasma DNA has the potential to serve as a marker for therapy monitoring during the treatment course of locally advanced rectal cancer patients. We especially focused on kinetics of circulating DNA to assess whether variances in kinetics have the potential to discriminate between therapy responders and nonresponders. The amount of circulating DNA in plasma of rectal cancer patients undergoing preoperative chemoradiation was determined using real-time PCR before chemoradiation, after the end of chemoradiation and at the end of treatment. The study population was divided into responders (ypT0-T2 stage) and nonresponders (ypT3-T4 stage). Both groups showed comparable median plasma DNA values before and after the end of chemoradiation. At the end of treatment responders showed a further decrease in circulating DNA, whereas in nonresponders the circulating DNA manifestly increased (P = 0.006). This study demonstrates that circulating DNA in plasma of rectal cancer patients undergoing preoperative chemoradiation might serve as a surrogate marker to discriminate between responders and nonresponders. Therefore, we hypothesize that quantification of plasma DNA could be of use as an easily accessible tool for therapy monitoring in these patients.

Dickkopf-3 as a new potential marker for neoangiogenesis in colorectal cancer: expression in cancer tissue and adjacent non-cancerous tissue.

Gene expression of Dickkopf-3 (Dkk-3) has been shown to be upregulated in tumor endothelium of colorectal cancer (CRC). For the first time, we analyzed Dkk-3 protein expression in CRC and its potential as a marker for neoangiogenesis. We used tissue microarrays (TMAs) to investigate Dkk-3 in microvessels of 403 CRC samples, 318 appropriate adjacent non-cancerous samples and 127 normal colorectal samples. Of cancer samples with CD31-positive microvessels, 67.7% were positive for Dkk-3. Dkk-3 staining was demonstrated in endothelial cells of all microvessels in nearly all cases. Dkk-3-positive samples showed a higher mean microvessel count than did Dkk-3-negative samples (P=0.001). Dkk-3 expression increased with rising numbers of microvessels per sample (P<0.0001). In adjacent samples with CD31-positive microvessels, 56% were Dkk-3-positive in all microvessels. Similar to cancer samples, Dkk-3-positive adjacent samples had a higher mean microvessel count than did Dkk-3-negative samples (P<0.0001), and Dkk-3 expression also increased with rising numbers of microvessels (P<0.0001). All microvessels in normal mucosa samples were negative for Dkk-3. Dkk-3 can be considered a putative pro-angiogenic protein in neovascularization and may possibly be a marker for neoangiogenesis in CRC. Further investigations will elucidate whether Dkk-3 is a target structure for novel therapies.

SFRP2 methylation in fecal DNA--a marker for colorectal polyps.

INTRODUCTION: DNA methylation of secreted frizzled-related proteins (SFRPs) can be detected in colorectal cancer (CRC) tissue, in tissue of adenomas, and in aberrant crypt foci, whereas in normal colorectal mucosa tissue, SFRP genes are unmethylated. Recently, our study group was able to demonstrate SFRP2 methylation as the most sensitive single DNA-based marker in stool for identification of CRC. The purpose of this study was to clarify whether SFRP2 methylation in fecal DNA can be found in stool of individuals with hyperplastic and adenomatous colorectal polyps. MATERIALS AND METHODS: Patients who were diagnosed with colorectal polyps or showed negative colonoscopy were included in this study. DNA from stool samples was isolated. SFRP2 methylation was assessed by means of MethyLight. RESULTS: Stool samples from 68 individuals were checked for DNA content; 23% of the samples (6 of 26) from healthy controls, 46% of the samples (6 of 13) from patients with hyperplastic polyps, and 45% of the samples (13 of 29) from patients with adenomas were positive for human DNA. SFRP2 methylation in stool samples was found in none of the healthy controls, in 33% (2 of 6) patients with hyperplastic polyps, and in 46% (6 of 13) patients with adenomas. Statistical analysis revealed that the frequency of SFRP2 methylation increased significantly (P=0.028) from healthy controls to patients with hyperplastic polyps and to patients with adenomas. CONCLUSIONS: In the current study, we report for the first time that SFRP2 methylation in fecal DNA increases significantly from healthy controls to patients with hyperplastic polyps and to patients with adenomas. SFRP2 methylation may serve as a marker for molecular stool-based adenoma and CRC screening.

DNA methylation in colorectal cancer--impact on screening and therapy monitoring modalities?

Colorectal cancer (CRC) is a common malignancy. It arises from benign neoplasms and evolves into adenocarcinomas through a stepwise histological progression sequence, proceeding from either adenomas or hyperplastic polyps/serrated adenomas. Genetic alterations have been associated with specific steps in this adenoma-carcinoma sequence and are believed to drive the histological progression of CRC. Recently, epigenetic alterations (especially DNA methylation) have been shown to occur in colon polyps and CRC. The aberrant methylation of genes appears to act together with genetic alterations to drive the initiation and progression of colon polyps to CRC. DNA methylation changes have been recognized as one of the most common molecular alterations in human tumors, including CRC. Because of the ubiquity of DNA methylation changes and the ability to detect methylated DNA in several body fluids (blood, stool), this specifically altered DNA may serve, on the one hand, as a possible new screening marker for CRC and, on the other hand, as a tool for therapy monitoring in patients having had neoplastic disease of the colorectum. As many CRC patients present with advanced disease, early detection seems to be one of the most important approaches to reduce mortality. Therefore, an effective screening test would have substantial clinical benefits. Furthermore, early detection of progression of disease in patients having had CRC permits immediate commencement of specific treatment regimens (e.g. curative resection of liver and lung metastases) and probably longer survival and better quality of life.

Disseminated tumor cells in peripheral blood: a novel marker for therapy response in locally advanced rectal cancer patients undergoing preoperative chemoradiation.

PURPOSE: This study was designed to examine whether disseminated tumor cells in peripheral blood of locally advanced rectal cancer patients undergoing preoperative chemoradiation have the potential to serve as a marker for therapy response. Studies suggest that patients with advanced rectal cancer who respond to preoperative chemoradiation most likely benefit from this treatment. METHODS: From advanced rectal cancer patients undergoing preoperative chemoradiation, peripheral blood was obtained at defined times: before, during, and after chemoradiation and during surgery. Patients were divided into histopathologic responders (ypT0-T2) and nonresponders (ypT3-T4). Cytokeratin 20 and carcinoembryonic antigen reverse transcriptase-polymerase chain reaction were performed to detect disseminated tumor cells. A blood sample was deemed positive for disseminated tumor cells if both carcinoembryonic antigen and cytokeratin 20 were detected. RESULTS: The overall population (n = 26) showed a positivity rate of 32 percent for disseminated tumor cells before initiation of chemoradiation. Of the responders (n = 8), 63 percent were positive for disseminated tumor cells before chemoradiation, whereas only 18 percent of nonresponders (n = 18) were positive (P = 0.026). From initiation of chemoradiation to the end of surgery, a significant decrease was seen in tumor cell positivity in the blood of responders (P = 0.042). Moreover, the responders represented a trend toward a decrease in tumor cell positivity during chemoradiation (P = 0.079). In contrast, there were no noticeable alterations within the treatment course in nonresponders. CONCLUSIONS: This prospective proof of principle study demonstrates that locally advanced rectal cancer with preoperative chemoradiation shows different biologic behavior in terms of tumor cell dissemination in peripheral blood when therapy responders compared with nonresponders.

Circulating nucleic acids in plasma or serum (CNAPS) as prognostic and predictive markers in patients with solid neoplasias.

It is now widely accepted that there is a need for the development of molecular markers of cancer that can be used for clinical prognostication and monitoring. Approximately a decade ago tumor-derived circulating nucleic acids in the plasma or serum (CNAPS) of cancer patients were introduced as a noninvasive tool for cancer detection. This review focuses on the various types of CNAPS of patients with solid neoplasias (genetic alterations in circulating DNA, microsatellites, methylated DNA, viral DNA, nucleosomes, mitochondrial DNA and cell-free mRNA) and their putative potential as prognostic or predictive parameter or even as a tool for therapy monitoring during follow-up. Additionally, this review aims to point out the difference between a prognostic and a predictive factor in patient bloodstream. However, with rapid technical improvement and well-designed studies we conclude that the next years will see CNAPS analysis integrated in the prognostication and monitoring of cancer patients, thus producing more specific treatment regimens for patients with various stages of neoplastic disease and ultimately longer survival and better quality of life.