Histone H3 lysine 9 acetylation is downregulated in GDM Placentas and Calcitriol supplementation enhanced this effect.

Despite the ever-rising incidence of Gestational Diabetes Mellitus (GDM) and its implications for long-term health of mothers and offspring, the underlying molecular mechanisms remain to be elucidated. To contribute to this, the present study's objectives are to conduct a sex-specific analysis of active histone modifications in placentas affected by GDM and to investigate the effect of calcitriol on trophoblast cell's transcriptional status. The expression of Histone H3 lysine 9 acetylation (H3K9ac) and Histone H3 lysine 4 trimethylation (H3K4me3) was evaluated in 40 control and 40 GDM (20 male and 20 female each) placentas using immunohistochemistry and immunofluorescence. The choriocarcinoma cell line BeWo and primary human villous trophoblast cells were treated with calcitriol (48 h). Thereafter, western blots were used to quantify concentrations of H3K9ac and the transcription factor FOXO1. H3K9ac expression was downregulated in GDM placentas, while H3K4me3 expression was not significantly different. Cell culture experiments showed a slight downregulation of H3K9ac after calcitriol stimulation at the highest concentration. FOXO1 expression showed a dose-dependent increase. Our data supports previous research suggesting that epigenetic dysregulations play a key role in gestational diabetes mellitus. Insufficient transcriptional activity may be part of its pathophysiology and this cannot be rescued by calcitriol.

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 MATERIALS AND METHODS: Blood samples of healthy donors and adjuvant breast cancer patients were withdrawn and the cell fraction containing white blood cells and tumor cells was enriched by density gradient centrifugation.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.

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.

Prototype and Chimera-Type Galectins in Placentas with Spontaneous and Recurrent Miscarriages.

Galectins are galactose binding proteins and, in addition, factors for a wide range of pathologies in pregnancy. We have analyzed the expression of prototype (gal-1, -2, -7, -10) and chimera-type (gal-3) galectins in the placenta in cases of spontaneous abortions (SPA) and recurrent abortions (RA) in the first trimester. Fifteen placental samples from healthy pregnancies were used as a control group. Nine placentas were examined for spontaneous abortions, and 12 placentas for recurrent abortions. For differentiation and evaluation of different cell types of galectin-expression in the decidua, immunofluorescence was used. For all investigated prototype galectins (gal-1, -2, -7, -10) in SPA and RA placenta trophoblast cells the expression is significantly decreased. In the decidua/extravillous trophoblast only gal-2 expression was significantly lowered, which could be connected to its role in angiogenesis. In trophoblasts in first-trimester placentas and in cases of SPA and RA, prototype galectins are altered in the same way. We suspect prototype galectins have a similar function in placental tissue because of their common biochemical structure. Expression of galectin 3 as a chimera type galectin was not found to be significantly altered in abortive placentas.

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.

Unique trophoblast stem cell- and pluripotency marker staining patterns depending on gestational age and placenta-associated pregnancy complications.

Preeclampsia (PE) and intrauterine growth retardation (IUGR) are rare but severe pregnancy complications that are associated with placental insufficiency often resulting in premature birth. The clinical pathologies are related to gross placental pathologies and trophoblastic deficiencies that might derive from inflammatory processes and oxidative stress injury. The mesenchymal core of placental villi has been identified as a possible niche for trophoblast progenitor cells that are called upon to replenish the injured syncytiotrophoblast layer. These progenitor cells are known to express trophoblast stem cell (CDX2) and pluripotency (SOX2, NANOG and OCT4A) markers, however only little data is available characterizing the expression of these transcription factors beyond the blastocyst stage. We aimed to describe the expression of these factors in healthy 1st and 3rd trimester placentae as well as PE, IUGR and combined PE+IUGR placentae. We analyzed 8 respective samples derived from 1st trimester (elective abortions), and 3rd trimester (healthy controls, PE, IUGR and combined PE+IUGR). We accomplished immunoperoxidase staining to detect the stem cell markers: CDX2 (trophectoderm), SOX2, NANOG and OCT4A (embryonal). Immunoreative scoring was used for objective analyses of staining patterns. All markers display clearly elevated signals in 1st trimester villous samples as compared to healthy 3rd trimester counterparts. Especially CDX2 and NANOG were specific to the cytotrophoblast layer and the mesenchymal core. Specific and differential expression patterns were visible in the villous/extravillous compartment of each placenta-associated pregnancy complication (PE: pan elevated expression; IUGR elevated SOX2 in basal plate; combined PE+IUGR pan loss of expression). Reduction of stem cell transcription factor expression in term placentae indicates temporal regulation, and probably a specific function which is yet to be elucidated. The differential expression patterns within placentae complicated with placenta-associated pregnancy complications indicate that PE, IUGR and combined PE+IUGR are separate entities. It is unclear whether the alterations are the cause or the effect of the clinical pathology.

Gal-1 silenced trophoblast tumor cells (BeWo) show decreased syncytium formation and different miRNA production compared to non-target silenced BeWo cells.

Galectin-1 (gal-1), a member of the mammalian ß-galactoside-binding proteins, exerts biological effects by recognition of glycan ligands, including those involved in cell adhesion and growth regulation. In previous studies, we demonstrated that gal-1 induces cell differentiation processes on the membrane of choriocarcinoma cells BeWo, including the receptor tyrosine kinases (RTKs) REarranged during Transfection (RET), Janus Kinase 2 (JAK2) and Vascular endothelial growth factor receptor 3 (VEGFR3). Furthermore, Mitogen-Activated Protein Kinases (MAPK) and serine/threonine kinases were phosphorylated by gal-1. In addition, gal-1 in trophoblast cells in vitro induced syncytium formation especially after concentration dependent stimulation of the cells with this galectin. This is in contrast to MAPK-inhibitor U0126 that reduced syncytium formation of BeWo cells. The aim of this study was to analyze the syncytium formation abilities of BeWo cells that were gal-1 silenced. We found a significantly reduced syncytium formation rate in gal-1 silenced BeWo cells. In addition, these cells show a different miRNA expression profile. In summary, we found that gal-1 is a major trigger for fusion processes in BeWo cells. This function is accompanied by different regulation of miRNA synthesis in the BeWo cell culture model.

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.

GDM Alters Expression of Placental Estrogen Receptor α in a Cell Type and Gender-Specific Manner.

OBJECTIVE: The nuclear receptor estrogen receptor α (ERα) is one of the key players in energy balance, insulin resistance, and trophoblast differentiation. We tested the hypothesis that gestational diabetes mellitus (GDM) alters expression of placental ERα in a cell type-specific manner and that this regulation may involve epigenetic changes. STUDY DESIGN: Expression of ERα was analyzed by immunohistochemistry using the semiquantitative immunoreactive score in 80 placentas (40 GDM/40 controls). Quantitative real-time polymerase chain reaction (PCR) measured ERα messenger RNA (mRNA) in decidual tissue. Methylation-specific PCR was performed to analyze cytosine-phosphatidyl-guanine-island methylation of the ERα promoter. RESULTS: Expression of ERα protein is upregulated (P = .011) in GDM in extravillous trophoblasts but not in syncytiotrophoblast. Gestational diabetes mellitus downregulated ERα in decidual vessels only in pregnancies with male but not female fetuses. Furthermore, mRNA of the ERα encoding gene estrogen receptor gene 1 (ESR1) was increased (+1.77 fold) in GDM decidua when compared to controls (P = .024). In parallel, the promoter of ESR1 was methylated only in decidua of healthy control individuals but not in GDM. CONCLUSION: Gestational diabetes mellitus affects expression of placental ERα in a cell type-dependent way, on epigenetic level. These data link GDM with epigenetic deregulations of ERα expression and open new insights into the intrauterine programming hypothesis of GDM.

Gestational diabetes mellitus upregulates vitamin D receptor in extravillous trophoblasts and fetoplacental endothelial cells.

OBJECTIVE: Gestational diabetes mellitus (GDM) is often accompanied by low maternal vitamin D, that is, calcitriol (1,25[OH]2 vitamin D3), levels. Here, we tested the hypothesis that the placental vitamin D receptor (VDR) is regulated by calcitriol and altered in GDM with distinct changes in different placental cell types. Specifically, we aimed to localize VDR in human term placentas from normal and GDM pregnancies, to quantify its cellular expression and to study in vitro its regulation by its physiological agonist calcitriol. STUDY DESIGN: Placental tissue slides of 80 patients (40 with GDM/40 controls) were double stained for VDR and human leukocyte antigen G to identify extravillous trophoblasts (EVTs). Staining intensity was semiquantified. Quantitative real time-polymerase chain reaction and Western blotting measured VDR messenger RNA (mRNA) and protein in decidual tissue. The trophoblast cell line BeWo was used to study in vitro VDR regulation by calcitriol (0.01, 0.1, and 1 nmol/mL). RESULTS: Vitamin D receptor protein and mRNA levels are upregulated (P < .05) in EVT (1.8-fold) as well as in placental endothelium (5.8-fold) of patients with GDM. Expression of VDR is regulated by calcitriol in a bimodal manner: high doses (0.1 and 1 nmol/mL) caused downregulation, whereas the low dose (0.01 nmol/mL) resulted in VDR upregulation. CONCLUSION: Vitamin D receptor is upregulated in EVT and endothelium of GDM placentas. This could be due to low maternal vitamin D levels in patients with GDM because in vitro low calcitriol doses upregulate VDR in trophoblast cells.

Peroxisome proliferator-activated receptor-gamma (PPARγ) is down regulated in trophoblast cells of gestational diabetes mellitus (GDM) and in trophoblast tumour cells BeWo in vitro after stimulation with PPARγ agonists.

AIMS: Peroxisome proliferator-activated receptor-gamma (PPARγ) plays an important role in insulin metabolism, trophoblast differentiation and anti-inflammatory circuits. The aim of this study was to investigate the expression of PPARγ in the placenta of patients with gestational diabetes mellitus (GDM) and the regulation of PPARγ by its agonists in trophoblast tumour cells BeWo in vitro. METHODS: PPARγ expression in a total of 80 placentas (40 GDM/40 controls) was analysed by immunohistochemistry using the semi-quantitative immunoreactive score. Furthermore, a quantitative reverse transcription-polymerase chain reaction (PCR) was performed to determine the PPARγ mRNA-expression in both groups. We used a fused and a non-fused BeWo cell culture model for the stimulation with arachidonic acid and 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2). Afterwards PPARγ mRNA-expression was analysed by quantitative real-time PCR (RT-PCR) (TaqMan). RESULTS: Using immunohistochemistry we identified a decreased expression of PPARγ in the syncytiotrophoblast and the extravillous trophoblast of GDM placentas compared to normal controls. Furthermore, PPARγ mRNA-expression was reduced in GDM placentas. Stimulation of BeWo cells with arachidonic acid and 15d-PGJ2 caused a downregulation of PPARγ expression. CONCLUSION: As PPARγ is down regulated by arachidonic acid and 15d-PGJ2, the reduced PPARγ expression in GDM placentas may be due to an altered concentration of fatty acid derivates.

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.

Macrophages: are they involved in endometriosis, abortion and preeclampsia and how?

Macrophages hold a key role in both regulating and executing the body's own immune response under various conditions. Hence, although endometriosis, preeclampsia and abortions are clinically different, all three are regarded to involve highly complex immunological processes. The aim of our current work was to evaluate the role of macrophages within these gynaecological disorders. Macrophages have been shown to invade endometriosis lesions and to mediate propagation of endometriotic cyst growth. However this is the first time that significant GPER up-regulation in macrophages is demonstrated. This highlights a potential alternative way through which estrogen may modulate immune response of macrophages in endometriosis. In addition, during spontaneous miscarriages the macrophage population increases significantly. This deregulation may possibly support an inflammatory scheme further triggering abortive procedures. Macrophage-mediated apoptosis of extravillous trophoblasts (EVT) has been associated with preeclampsia. Larger numbers of apoptotic EVT were detected in preeclamptic placentas compared with normal. In preeclamptic placentas, decidual macrophages were found to be Fas ligand (FasL)-positive. Our results highlight a new aspect of macrophage biology in endometriosis and pregnancy physiology and patho-physiology. Further studies with larger samples are needed to verify the current results and evaluate their clinical impact. Our data strongly indicate that macrophages hold key roles in various gynaecological disorders and might be crucial to further elucidate their patho-physiology.

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.

The Role of PPARs in Placental Immunology: A Systematic Review of the Literature.

Pregnancy is a state of immunotolerance, and pregnancy outcome is strongly linked to the correct activation and balancing of the maternal immune system. Besides abortion as possible result of improper early pregnancy development, other pregnancy associated conditions like preeclampsia (PE), intrauterine growth retardation (IUGR), preterm labour, or gestational diabetes mellitus (GDM) are linked to immunologic overactivation and dysregulation. Both the innate and the adaptive immune system, and therefore B and T lymphocytes, natural killer cells (NK), macrophages and dendritic cells (DCs) are all involved in trophoblast invasion, pregnancy maintenance, and development of pregnancy disorders. Peroxisome proliferator activated receptors (PPARs) are nuclear transcription factors with three known isotypes: PPARα, PPAR ß / δ , and PPAR γ . They are expressed in most human organs and their function extends from regulating metabolism, homeostasis, and carcinogenesis to immune response. In the recent years, PPARs have been identified in most reproductive tissues and in all lines of immune cells. Only in few cases, the role of PPARs in reproductive immunology has been elucidated though the role of PPARs in immune answer and immunotolerance is evident. Within this paper we would like to give an update on today's knowledge about PPARs and immune cells in reproduction and highlight interesting interferences in regard of future therapeutic targets.

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.