The importance of hCG in human endometrial adenocarcinoma and breast cancer.

Human chorionic gonadotropin (hCG) is a peptide hormone which plays an important role during pregnancy. But its impact is not limited to pregnancy; it also influences tumor formation and metastatic outgrowth, especially in endometrial adenocarcinoma and breast cancer. This review summarizes what has been written in the literature about the role of hCG as a tumor marker in these 2 gynecological malignancies and also about the signal transduction pathways in which hCG is involved. HCG can, on the one hand, be a marker for the progression of a malignant disease, and on the other hand, it may be a point for therapeutical intervention, so further research into this molecule would be very much worthwhile.

Triple-negative breast cancer: New therapeutic options via signalling transduction cascades.

Triple-negative breast cancer is a highly aggressive type of mammalian carcinoma. It is defined by a rather weak expression of estrogen-, progesterone- and Her2-receptor, and is thus difficult to treat, resulting in low disease-free and overall survival rates of the affected patients. Hence it is important to find new therapeutic options. To this aim we analysed the incidence of some molecules from different signal transduction cascades by immunohistochemistry, which are known to correlate with triple-negative breast cancer, and correlated the expression of these molecules to different tumour traits, such as size, grading, menopausal stage, histology, lymph node affection, remote metastasis formation, and to the incidence of local and lymph node recurrence and metastasis by statistical analysis. Statistically significant correlations were found for a number of tumour characteristics and signalling molecules: HIF1α is correlated to tumour grading, ß-catenin to the menopausal state of the patient, and for Notch1 a relation to lymph node affection is seen. In terms of different recurrences, a correlation of ß-catenin to metastasis formation and lymph node affection could be shown, as well as coherences between XBP1 and lymph node recurrence, Notch1 and metastasis formation and FOXP3 and the occurrence of local recurrence. The presented results are in accordance with formerly published studies and therefore might comprise opportunities to develop new therapeutical strategies, which could help to handle this aggressive form of breast cancer in a manner, by which side effects would be reduced and therapeutical efficiency is increased.

Influence of VEGFR and LHCGR on endometrial adenocarcinoma.

Endometrial adenocarcinoma is a common gynecological malignancy that is usually treated by surgical resection followed by radiation. However, the frequency of remote metastasis is high. The present study aimed to investigate whether patients with endometrial adenocarcinoma exhibited a positive response to treatment with a gonadotropin-releasing hormone analogue or inhibitors of neoangiogenesis, which are applied for the treatment of other malignancies. Immunohistochemical analyses were performed using 203 paraffin-embedded tissue samples of endometrial adenocarcinomas from patients who had undergone surgery at the Department of Obstetrics and Gynecology of the Ludwig Maximilians University of Munich, Germany. The tissues were incubated with antibodies against luteinizing hormone/choriogonadotropin receptor (LHCGR) and vascular endothelial growth factor receptor 2 (VEGFR2), and evaluated by bright field microscopy. The staining was categorized according to the Immune-Reactive-Score (IRS). The IRS scores were then statistically associated with various tumor traits, including tumor size, lymph node status, metastasis, grade, expression of steroid hormone receptors and patient survival. There was a significant association between VEGFR2 expression and tumor grading and estrogen receptor-α (ERα). For LHCGR, a correlation was observed with ERα and progesterone receptor (PR). No correlations were identified between VEGFR2 or LHCGR expression and the other examined tumor traits or patient survival. The associations between VEGFR2 and ERα, and between LHCGR and ERα or PR, may be explained by the interaction of these signal transduction molecules in the regulation of cellular growth and differentiation. These mechanisms also have an important role in the formation of remote metastases, which is the main cause for tumor-associated mortality. The results of the present study suggested that patients with endometrial adenocarcinoma may benefit from treatment with inhibitors of ERα, PR, VEGFR2 or LHCGR, since it could lead to a better prognosis. However, further studies are required in order to elucidate the roles of these receptors in endometrial adenocarcinoma.

Real-Time qPCR-Based Detection of Circulating Tumor Cells from Blood Samples of Adjuvant Breast Cancer Patients: A Preliminary Study.

BACKGROUND: Circulating tumor cells (CTCs) are cells that detach from a primary tumor, circulate through the blood stream and lymphatic vessels, and are considered to be the main reason for remote metastasis. Due to their origin, tumor cells have different gene expression levels than the surrounding blood cells. Therefore, they might be detectable in blood samples from breast cancer patients by real-time quantitative polymerase chain reaction (RT-qPCR). MATERIALS AND METHODS: Blood samples of healthy donors and adjuvant breast cancer patients were withdrawn and the cell fraction containing white blood cells and tumor cells was enriched by density gradient centrifugation. RNA was isolated and reverse transcribed to cDNA, which was then used in TaqMan real-time PCR against cytokeratin (CK)8, CK18 and CK19. 18S and GAPDH were used as controls. RESULTS: All 3 CKs were, on average, found to be significantly higher expressed in adjuvant breast cancer samples compared to negative controls, probably due to the presence of CTCs. Unfortunately, gene expression levels could not be correlated to tumor characteristics. CONCLUSIONS: RT-qPCR could make up a new approach for the detection of CTCs from blood samples of breast cancer patients, but a correlation of the PCR data to gold standard methods in CTC detection would help to further improve the informative value of the qPCR results.

The Significance of Epithelial-to-Mesenchymal Transition for Circulating Tumor Cells.

Epithelial to mesenchymal transition (EMT) is a process involved in embryonic development, but it also plays a role in remote metastasis formation in tumor diseases. During this process cells lose their epithelial features and adopt characteristics of mesenchymal cells. Thereby single tumor cells, which dissolve from the primary tumor, are enabled to invade the blood vessels and travel throughout the body as so called "circulating tumor cells" (CTCs). After leaving the blood stream the reverse process of EMT, the mesenchymal to epithelial transition (MET) helps the cells to seed in different tissues, thereby generating the bud of metastasis formation. As metastasis is the main reason for tumor-associated death, CTCs and the EMT process are in the focus of research in recent years. This review summarizes what was already found out about the molecular mechanisms driving EMT, the consequences of EMT for tumor cell detection, and suitable markers for the detection of CTCs which underwent EMT. The research work done in this field could open new roads towards combating cancer.

Quantitative PCR marker genes for endometrial adenocarcinoma.

Endometrial adenocarcinoma is a common malignancy in women worldwide, with formation of remote metastasis occurring following oncological treatment. Circulating tumor cells (CTCs) are regarded to be the origin of haematogenous metastasis formation. The present study aimed to identify suitable marker genes using a quantitative polymerase chain reaction (qPCR) approach to detect CTCs from blood samples of patients with endometrial carcinoma. Therefore, RNA was isolated from endometrial adenocarcinoma cell lines and from healthy endometrial tissue and reverse transcribed to cDNA, which was then used in qPCR on a number of marker genes. Cytokeratin 19 and claudin 4 were identified as suitable marker genes for CTCs in endometrial adenocarcinoma, due to their high expression in the majority of the cell lines investigated. The expression values of the genes examined varied widely between the different cell lines, which is similar to the variation in the patient samples. Therefore, the necessity for a set of genes for CTC detection and not one single marker gene is demonstrated. qPCR is a fast, cost­efficient and easy to perform technique, which may be used in the detection of CTCs. Investigation of the occurrence of CTCs in cancer patients would aid in the prevention of metastasis and thereby refine treatment.

Do signal transduction cascades influence survival in triple-negative breast cancer? A preliminary study.

BACKGROUND: Triple-negative breast cancer (TNBC) is a rather aggressive form of breast cancer, comprised by early metastasis formation and reduced overall survival of the affected patients. Steroid hormone receptors and the human epidermal growth factor receptor 2 are not overexpressed, limiting therapeutic options. Therefore, new treatment options have to be investigated. The aim of our preliminary study was to detect coherences between some molecules of intracellular signal transduction pathways and survival of patients with TNBC, in order to obtain some hints for new therapeutical solutions. METHODS: Thirty-one paraffin-embedded tumor tissue samples, which were determined to be negative for steroid hormone receptors as well as human epidermal growth factor receptor 2, were immunohistochemically stained for a number of signal transduction molecules from several signaling pathways. ß-Catenin, HIF1α, MCL, Notch1, LRP6, XBP1, and FOXP3 were stained with specific antibodies, and their staining was correlated with patient survival by Kaplan-Meier analyses. RESULTS: Only two of the investigated molecules have shown correlation with overall survival. Cytoplasmic staining of HIF1α and centro-tumoral lymphocyte FOXP3 staining showed statistically significant correlations with survival. CONCLUSION: The coherence of signal transduction molecules with survival of patients with TNBC is still controversially discussed in the literature. Our study comprises one more mosaic stone in the elucidation of these intracellular processes and their influences on patient outcome. Lots of research still has to be done in this field, but it would be worthwhile as it may offer new therapeutic targets for a group of patients with breast cancer, which is still hard to treat.

Endometrial Adenocarcinoma: Analysis of Circulating Tumour Cells by RT-qPCR.

BACKGROUND: Endometrial adenocarcinoma is a frequently occurring cancer in women, accounting for 42,000 deaths every year. Despite treatment with standard therapy, occurrence of remote metastases and local recurrences is high. Through help of RT-qPCR minimal residual disease could be detected and characterized, facilitating therapeutic decision making. MATERIALS AND METHODS: A number of marker genes were first tested in model systems and genes that performed best, were consequently used for the examination of 13 blood samples from endometrial carcinoma patients. RESULTS: Cytokeratin 19 and MIG7 were chosen for the analysis in patient samples. Both genes were found up-regulated in small tumours and in one large tumour, but no statistical correlations could be revealed between expression levels of these two genes and tumour characteristics. CONCLUSION: There seems to be a coherence between gene expression and the stage of tumorigenesis, but the number of samples is still too small, to be able to obtain statistical significant differences.

New Marker Genes for Real-Time PCR-based Detection of Circulating Tumour Cells from Blood of Breast Cancer Patients.

BACKGROUND: The detection of circulating tumour cells (CTCs) from peripheral blood of cancer patients can be carried out by real-time PCR approaches using different gene expression levels of tumour cells and surrounding blood cells. MATERIALS AND METHODS: Potential marker genes were first analyzed in a model system and then applied to 20 blood samples of adjuvant breast cancer patients and gene expression levels were correlated to tumour characteristics. RESULTS: The mean of gene expression levels was found elevated for the four genes analyzed in the adjuvant breast cancer patient group in comparison to the samples of the group of healthy donors, but no correlation between gene expression and tumour characteristics could be detected as being statistically significant. CONCLUSION: The results demonstrated, that the employed methodology is functional, but has to be refined by certain approaches like simultaneously running a state-of-the-art system of CTC-detection comparing the results, and by an enlargement of patient collective and number of marker genes.

Immunocytochemical Characterization of Disseminated Tumour Cells from Bone Marrow of Breast Cancer Patients.

BACKGROUND: The occurrence of disseminated tumour cells in bone marrow of patients with breast cancer is linked to a worse prognosis. We present a method for DTC detection from bone marrow samples based on immunocytochemistry, using breast cancer-associated glycosylation molecules as markers for detection and characterization. MATERIALS AND METHODS: A double immunofluorescence staining of a pan-cytokeratin (CK) marker and either Tn or O-Acetyl-GD3 was carried out in artificial and patient bone marrow samples. RESULTS: The sample in which most cells stained positive for CK/Tn and CK/O-AC-GD3, was obtained from a patient who certainly had remote metastases. All other bone marrow samples showed heterogenous staining, so no correlation to tumour characteristics could be revealed. CONCLUSION: A certain characterization of tumour cells can be achieved by a double staining of bone marrow samples with CK and a glycosylation marker. For future studies, analysis should be extended to a larger patient collective and further examination of more glycosylation markers should be carried out.

Role of N-acetylgalactosaminyltransferase 6 in early tumorigenesis and formation of metastasis.

Glycosylation is one of the most important posttranslational modifications of proteins and lipids that contributes to the structural diversity of cellular molecules. Enzymes of the glycosyltransferase class are responsible for altering glycosylation patterns by adding carbohydrate chains to the respective acceptor molecules. It is well known that glycosylation is commonly altered in cancerous tissue. Therefore, the present study aimed to determine the incidence of N­acetylgalactosaminyltransferase 6 (GALNT6), a prominent member of the glycosyltransferase class, in breast cancer tissue of different developmental stages by immunohistochemistry. Although no correlation was identified between tumour characteristics and GALNT6 staining intensity, to the best of our knowledge, this is the first study to demonstrate that tissue from carcinoma in situ­tumours and metastases were more heavily stained than late­stage breast cancers. This may indicate an important role of glycosylation aberration in escaping the immune system at early phases of tumour development. The present study also hypothesised that nascent or early metastasizing tumours are normally recognized by the immune system of the patient, but glycosylation pattern changes may facilitate tumor escape from immune recognition. In follow­up studies, our group will aim to confirm and consolidate these results in a larger patient cohort that may give greater insight into breast cancer characterization as well as tumour treatment.

Real-time RT-PCR systems for CTC detection from blood samples of breast cancer and gynaecological tumour patients (Review).

Cells, which detach from a primary epithelial tumour and migrate through lymphatic vessels and blood stream are called 'circulating tumour cells'. These cells are considered to be the main root of remote metastasis and are correlated to a worse prognosis concerning progression-free and overall survival of the patients. Therefore, the detection of the minimal residual disease is of great importance regarding therapeutic decisions. Many different detection strategies are already available, but only one method, the CellSearch® system, reached FDA approval. The present review focusses on the detection of circulating tumour cells by means of real-time PCR, a highly sensitive method based on differences in gene expression between normal and malignant cells. Strategies for an enrichment of tumour cells are mentioned, as well as a large panel of potential marker genes. Drawbacks and advantages of the technique are elucidated, whereas, the greatest advantage might be, that by selection of appropriate marker genes, also tumour cells, which have already undergone epithelial to mesenchymal transition can be detected. Finally, the application of real-time PCR in different gynaecological malignancies is described, with breast cancer being the most studied cancer entity.

The role of TF- and Tn-antigens in breast cancer metastasis.

Almost 40 years ago, researchers found out that the Thomsen-Friedenreich (TF) and the Thomsen nouvelle (Tn) antigens could be detected in carcinoma, but not in healthy tissue. A short time after that it became clear that TF and Tn are precursor molecules of the MN-blood group antigens. In normal tissue TF and Tn are coated by glycosyl structures, thereby forming the glycoproteins which are known to account for the MN-blood group, but in malignant tissue these molecules are uncovered.TF, which has an additional Galectin-residue compared to Tn, is correlated with a more favourable prognosis for patients. On the contrary, patients with Tn-bearing tissues have a worse prognosis for overall and progression-free survival. It is known that TF and Tn are involved in the adhesion of tumour cells to the endothelium via a mechanism recruiting Galectin-3 and MUC-1, which is the first step in metastasis formation. Furthermore, it became clear that this pathway can be blocked by a growing number of molecules, thereby creating ways of therapeutical intervention.

The Role of Glycosylation in Breast Cancer Metastasis and Cancer Control.

Glycosylation and its correlation to the formation of remote metastasis in breast cancer had been an important scientific topic in the last 25 years. With the development of new analytical techniques, new insights were gained on the mechanisms underlying metastasis formation and the role of aberrant glycosylation within. Mucin-1 and Galectin were recognized as key players in glycosylation. Interestingly, aberrant carbohydrate structures seem to support the development of brain metastasis in breast cancer patients, as changes in glycosylation structures facilitate an overcoming of blood-brain barrier. Changes in the gene expression of glycosyltransferases are the leading cause for a modification of carbohydrate chains, so that also altered gene expression plays a role for glycosylation. In consequence, glycosylation and changes within can be useful for cancer diagnosis, determination of tumor stage, and prognosis, but can as well be targets for therapeutic strategies. Thus, further research on this topic would worthwhile for cancer combating.

Glycosyltransferases as Markers for Early Tumorigenesis.

BACKGROUND: Glycosylation is the most frequent posttranslational modification of proteins and lipids influencing inter- and intracellular communication and cell adhesion. Altered glycosylation patterns are characteristically observed in tumour cells. Normal and altered carbohydrate chains are transferred to their acceptor structures via glycosyltransferases. Here, we present the correlation between the presence of three different glycosyltransferases and tumour characteristics. METHODS: 235 breast cancer tissue samples were stained immunohistochemically for the glycosyltransferases N-acetylgalactosaminyltransferase 6 (GALNT6), ß-1,6-N-acetylglucosaminyltransferase 2 (GCNT2), and ST6 (α-N-acetyl-neuraminyl-2,3-ß-galactosyl-1,3)-N-acetylgalactosamine α-2,6-sialyltransferase 1 (ST6GALNac1). Staining was evaluated by light microscopy and was correlated to different tumour characteristics by statistical analysis. RESULTS: We found a statistically significant correlation for the presence of glycosyltransferases and tumour size and grading. Specifically smaller tumours with low grading revealed the highest incidences of glycosyltransferases. Additionally, Her4-expression but not pHer4-expression is correlated with the presence of glycosyltransferases. All other investigated parameters could not uncover any statistically significant reciprocity. CONCLUSION: Here we show, that glycosyltransferases can identify small tumours with well-differentiated cells; hence, glycosylation patterns could be used as a marker for early tumourigenesis. This assumption is supported by the fact that Her4 is also correlated to glycosylation, whereas the activated form of Her4 does not show such a connection with glycosylation.

Glycosyltransferases as marker genes for the quantitative polymerase chain reaction-based detection of circulating tumour cells from blood samples of patients with breast cancer undergoing adjuvant therapy.

Altered glycosylation is a predominant feature of tumour cells; it serves for cell adhesion and detachment, respectively, and facilitates the immune escape of these cells. Therefore changes in the expression of glycosyltransferase genes could help to identify circulating tumour cells (CTCs) in the blood samples of cancer patients using a quantitative polymerase chain reaction (PCR) approach. Blood samples of healthy donors were inoculated with certain numbers of established breast cancer cell line cells, thus creating a model system. These samples were analysed by quantitative PCR for the expression of six different glycosyltransferase genes. The three genes with the best results in the model system were consecutively applied to samples from adjuvant breast cancer patients and of healthy donors. FUT3 and GALNT6 showed the highest increase in relative expression, while GALNT6 and ST3GAL3 were the first to reach statistically significant different ∆CT-values comparing the sample with and without addition of tumour cells. These three genes were applied to patient samples, but did not show any significant results that may suggest the presence of CTCs in the blood. Although the relative expression of some of the glycosyltransferase genes exhibited reasonable results in the model system, their application to breast cancer patient samples will have to be further improved, e.g. by co-analysis of patient blood samples by gold-standard methods.

Glycodelin A - a famous lipocalin and its role in breast cancer.

Lipocalins are a large protein family with only little sequence homology but highly conserved structural similarity. Many lipocalins play crucial roles in the generation of epithelial cancer, influencing pathways which regulate cell motility, cell differentiation and neovascularisation. Thereby they can be used as biomarkers of cancer, in most cases for a rather good prognosis. Glycodelin is a lipocalin existing in three isoforms which differ only by glycosylation, but which have different functions. In breast cancer, glycodelin A is known to contribute to a more differentiated cell morphology and is a biomarker for a favourable prognosis, but also plays a role in angiogenesis. Glycodelin A is a useful prognostic marker as it can be detected in serum samples, but is also a target for therapeutical interventions.

Detection of Circulating Tumour Cells from Blood of Breast Cancer Patients via RT-qPCR.

Breast cancer is still the most frequent cause of cancer-related death in women worldwide. Often death is not caused only by the primary tumour itself, but also by metastatic lesions. Today it is largely accepted, that these remote metastases arise out of cells, which detach from the primary tumour, enter circulation, settle down at secondary sites in the body and are called Circulating Tumour Cells (CTCs). The occurrence of such minimal residual diseases in the blood of breast cancer patients is mostly linked to a worse prognosis for therapy outcome and overall survival. Due to their very low frequency, the detection of CTCs is, still a technical challenge. RT-qPCR as a highly sensitive method could be an approach for CTC-detection from peripheral blood of breast cancer patients. This assumption is based on the fact that CTCs are of epithelial origin and therefore express a different gene panel than surrounding blood cells. For the technical approach it is necessary to identify appropriate marker genes and to correlate their gene expression levels to the number of tumour cells within a sample in an in vitro approach. After that, samples from adjuvant and metastatic patients can be analysed. This approach may lead to new concepts in diagnosis and treatment.

Detection of breast cancer cells in blood samples by immunostaining of the Thomsen-Friedenreich antigen.

AIM: Disseminated tumor cells are found in the bone marrow of patients with epithelial carcinoma and are correlated with a poor prognosis of the disease. Their detection is a technical challenge. This report describes a model system for the detection of cancer cells by co-immunostaining of Thomsen-Friedenreich and Her-2 antigens. METHODS & RESULTS: Small numbers of cancer cells from different cancer cell lines were mixed with blood samples of healthy donors. Cytospins were prepared and double immunostaining against Thomsen-Friedenreich antigen and Her-2 was carried out by fluorochrome-coupled antibodies. Quantification of Thomsen-Friedenreich and/or Her-2-positive cells was performed with an epifluorescence microscope. On average, 83% of cancer cells were recovered by this method. CONCLUSION: Immunostaining is a useful method for the detection of cancer cells in blood samples. Results of this model system will be transferred to bone marrow patient samples to prove the benefits for detection of disseminated tumor cells.

Detection of tumor cell-specific mRNA in the peripheral blood of patients with breast cancer­evaluation of several markers with real-time reverse transcription-PCR.

It is widely known that cells from epithelial tumors, e.g., breast cancer, detach from their primary tissue and enter blood circulation. We show that the presence of circulating tumor cells (CTCs) in samples of patients with primary and metastatic breast cancer can be detected with an array of selected tumor-marker-genes by reverse transcription real-time PCR. The focus of the presented work is on detecting differences in gene expression between healthy individuals and adjuvant and metastatic breast cancer patients, not an accurate quantification of these differences. Therefore, total RNA was isolated from blood samples of healthy donors and patients with primary or metastatic breast cancer after enrichment of mononuclear cells by density gradient centrifugation. After reverse transcription real-time PCR was carried out with a set of marker genes (BCSP, CK8, Her2, MGL, CK18, CK19). B2M and GAPDH were used as reference genes. Blood samples from patients with metastatic disease revealed increased cytokine gene levels in comparison to normal blood samples. Detection of a single gene was not sufficient to detect CTCs by reverse transcription real-time PCR. Markers used here were selected based on a recent study detecting cancer cells on different protein levels. The combination of such a marker array leads to higher and more specific discovery rates, predominantly in metastatic patients. Identification of CTCs by PCR methods may lead to better diagnosis and prognosis and could help to choose an adequate therapy.