Cancer cell invasion and EMT marker expression: a three-dimensional study of the human cancer-host interface.

Cancer cell invasion takes place at the cancer-host interface and is a prerequisite for distant metastasis. The relationships between current biological and clinical concepts such as cell migration modes, tumour budding and epithelial-mesenchymal transition (EMT) remains unclear in several aspects, especially for the 'real' situation in human cancer. We developed a novel method that provides exact three-dimensional (3D) information on both microscopic morphology and gene expression, over a virtually unlimited spatial range, by reconstruction from serial immunostained tissue slices. Quantitative 3D assessment of tumour budding at the cancer-host interface in human pancreatic, colorectal, lung and breast adenocarcinoma suggests collective cell migration as the mechanism of cancer cell invasion, while single cancer cell migration seems to be virtually absent. Budding tumour cells display a shift towards spindle-like as well as a rounded morphology. This is associated with decreased E-cadherin staining intensity and a shift from membranous to cytoplasmic staining, as well as increased nuclear ZEB1 expression.

Gastrointestinal stem cells in development and cancer.

An enormous body of knowledge about the biology of stem cells and their role in development, tissue homeostasis and cancer formation has been gained in the last 20 years. This review gives a comprehensive overview on knowledge about localization and regulation of normal gastrointestinal stem cells and links it to our understanding of gastrointestinal tumourigenesis and malignant progression in the light of the cancer stem cell concept. The focus is on intestinal stem cells and newly identified stem cell factors, such as the beta-catenin target gene Lgr5. The basis of intestinal stem cell regulation is a permanent crosstalk between epithelial and underlying mesenchymal cells in the intestinal stem cell niche. This crosstalk is mediated by crucial pathways, including the Wnt, Hedgehog (HH), Notch, PI3K and BMP pathways. Disturbances in this fine-regulated interaction can both initiate intestinal tumours and, in association with additional genetic alterations or environmental activation of embryonic processes such as epithelial-mesenchymal transition (EMT), lead to tumour invasion and metastasis.

[Tumour stem cells and metastasis]. / Tumorstammzellen und Metastasierung.

The underlying mechanisms of cancer development, invasion and metastasis are still only partly unraveled. Cancer stem cells (CSC) and the so-called epithelial-mesenchymal transition (EMT) seem to contribute to these oncological phenomena. Cancer stem cells which, according to our present knowledge, play an important role in tumour development and persistence, are operationally defined. The embryonic programme of EMT is activated aberrantly in cancer cells at the invasive front and seems to contribute to tumour invasion and metastasis. Recent observations suggest that the EMT and CSC traits are closely related. This provides new explanatory models for cancer development and metastasis.

Frizzled-7 dictates three-dimensional organization of colorectal cancer cell carcinoids.

Progression of colorectal cancer (CRC) involves spatial and temporal occurrences of epithelial-mesenchymal transition (EMT), whereby tumour cells acquire a more invasive and metastatic phenotype. Subsequently, the disseminated mesenchymal tumour cells must undergo a reverse transition (mesenchymal-epithelial transition, MET) at the site of metastases, as most metastases recapitulate the pathology of their corresponding primary tumours. Importantly, initiation of tumour growth at the secondary site is the rate-limiting step in metastasis. However, investigation of this dynamic reversible EMT and MET that underpins CRC morphogenesis has been hindered by a lack of suitable in vitro models. To this end, we have established a unique in vitro model of CRC morphogenesis, which we term LIM1863-Mph (morphogenetic). LIM1863-Mph cells spontaneously undergo cyclic transitions between two-dimensional monolayer (migratory, mesenchymal) and three-dimensional sphere (carcinoid, epithelial) states. Using RNAi, we demonstrate that FZD7 is necessary for MET of the monolayer cells as loss of FZD7 results in the persistence of a mesenchymal state (increased SNAI2/decreased E-cadherin). Moreover, FZD7 is also required for migration of the LIM1863-Mph monolayer cells. During development, FZD7 orchestrates either migratory or epithelialization events depending on the context. Our findings strongly implicate similar functional diversity for FZD7 during CRC morphogenesis.

The transcription factor ZEB1 (deltaEF1) promotes tumour cell dedifferentiation by repressing master regulators of epithelial polarity.

Epithelial to mesenchymal transition (EMT) is implicated in the progression of primary tumours towards metastasis and is likely caused by a pathological activation of transcription factors regulating EMT in embryonic development. To analyse EMT-causing pathways in tumourigenesis, we identified transcriptional targets of the E-cadherin repressor ZEB1 in invasive human cancer cells. We show that ZEB1 repressed multiple key determinants of epithelial differentiation and cell-cell adhesion, including the cell polarity genes Crumbs3, HUGL2 and Pals1-associated tight junction protein. ZEB1 associated with their endogenous promoters in vivo, and strongly repressed promotor activities in reporter assays. ZEB1 downregulation in undifferentiated cancer cells by RNA interference was sufficient to upregulate expression of these cell polarity genes on the RNA and protein level, to re-establish epithelial features and to impair cell motility in vitro. In human colorectal cancer, ZEB1 expression was limited to the tumour-host interface and was accompanied by loss of intercellular adhesion and tumour cell invasion. In invasive ductal and lobular breast cancer, upregulation of ZEB1 was stringently coupled to cancer cell dedifferentiation. Our data show that ZEB1 represents a key player in pathologic EMTs associated with tumour progression.

Transforming growth factor beta and cyclosporin A inhibit the inducible activity of the interleukin-2 gene in T cells through a noncanonical octamer-binding site.

Transforming growth factor beta (TGF-beta) has a growth-inhibitory effect on numerous different cell types of the immune system, including T lymphocytes. We show in this study that the inhibitory action of TGF-beta on T lymphocytes is accompanied by a block of interleukin 2 (IL-2) gene expression which is mediated, at least in part, by inhibition of IL-2 promoter/enhancer activity. The functional analysis of cis-regulatory (proto-enhancer) elements of the IL-2 enhancer/promoter region showed that the most TGF-beta-responsive element maps to its so-called upstream promoter site. The proto-enhancer activity of the upstream promoter site element is also inhibited by cyclosporin A. The upstream promoter site DNA harbors two noncanonical, closely linked binding sequences for octamer and AP-1-like factors. Both sites are involved in the establishment of IL-2 enhancer activity. Since the activity of genuine octamer sites but not that of AP-1-binding sites is also impaired by TGF-beta and cyclosporin A in El4 T lymphoma cells, we conclude that both immunosuppressives interfere with the activity but not the DNA binding of octamer factors in T lymphocytes.

A point mutation in the extracellular domain of L1 blocks its capacity to confer metastasis in colon cancer cells via CD10.

The neural L1 transmembrane cell adhesion receptor of the immunoglobulin-like family is a target gene of Wnt-ß-catenin signaling in human colorectal cancer (CRC) cells and is expressed at the invasive edge of the tumor tissue. L1 overexpression in cultured CRC cells confers enhanced proliferation, motility and liver metastasis. We have analyzed the mechanisms of L1-mediated signaling in CRC cells by using various point mutations in the L1 ectodomain that are known to cause severe genetically inherited mental retardation disorders in patients. We found that all such L1 ectodomain mutations abolish the ability of L1 to confer metastatic properties in CRC cells. Using gene array analysis, we identified L1-mutation-specific gene expression signatures for the L1/H210Q and L1/D598N mutations. We identified CD10, a metalloprotease (neprilysin, neutral endopeptidase) and a gene that is specifically induced in CRC cells by L1 in an L1/H210Q mutation-specific manner. CD10 expression was required for the L1-mediated induction of cell proliferation, motility and metastasis, as suppression of CD10 levels in L1-expressing CRC cells abolished the L1 effects on CRC progression. The signaling from L1 to CD10 was mediated through the L1-ezrin-NF-κB pathway. In human CRC tissue L1 and CD10 were localized in partially overlapping regions in the more invasive areas of the tumor tissue. The results suggest that CD10 is a necessary component conferring the L1 effects in CRC cells. The identification of gene expression patterns of L1-domain-specific point mutations may provide novel markers and targets for interfering with L1-mediated CRC progression.

Expression of the invasion factor laminin gamma2 in colorectal carcinomas is regulated by beta-catenin.

The migration-inducing gamma2 chain of laminin-5, one of the best known invasion markers, is strongly overexpressed in disseminating and infiltrating tumor cells at the invasive front of colorectal carcinomas. The same tumor cells show nuclear accumulation of the oncoprotein beta-catenin, which together with T-cell factor-DNA-binding proteins, functions as transcriptional activator of genes involved in tumor progression. Here we show that beta-catenin activates the human laminin-5 gamma2 gene through two T-cell factor-binding elements in a synergistic manner together with hepatocyte growth factor and conclude that laminin-5 gamma2 is another important target gene of nuclear beta-catenin during tumor progression.

Induction of the intestinal stem cell signature gene SMOC-2 is required for L1-mediated colon cancer progression.

Overactivation of Wnt-ß-catenin signaling, including ß-catenin-TCF target gene expression, is a hallmark of colorectal cancer (CRC) development. We identified the immunoglobulin family of cell-adhesion receptors member L1 as a ß-catenin-TCF target gene preferentially expressed at the invasive edge of human CRC tissue. L1 can confer enhanced motility and liver metastasis when expressed in CRC cells. This ability of L1-mediated metastasis is exerted by a mechanism involving ezrin and the activation of NF-κB target genes. In this study, we identified the secreted modular calcium-binding matricellular protein-2 (SMOC-2) as a gene activated by L1-ezrin-NF-κB signaling. SMOC-2 is also known as an intestinal stem cell signature gene in mice expressing Lgr5 in cells at the bottom of intestinal crypts. The induction of SMOC-2 expression in L1-expressing CRC cells was necessary for the increase in cell motility, proliferation under stress and liver metastasis conferred by L1. SMOC-2 expression induced a more mesenchymal like phenotype in CRC cells, a decrease in E-cadherin and an increase in Snail by signaling that involves integrin-linked kinase (ILK). SMOC-2 was localized at the bottom of normal human colonic crypts and at increased levels in CRC tissue with preferential expression in invasive areas of the tumor. We found an increase in Lgr5 levels in CRC cells overexpressing L1, p65 or SMOC-2, suggesting that L1-mediated CRC progression involves the acquisition of a stem cell-like phenotype, and that SMOC-2 elevation is necessary for L1-mediated induction of more aggressive/invasive CRC properties.

Tip60 is a cell-type-specific transcriptional regulator.

Tip60 was originally identified as cellular HIV-Tat interacting protein and has been shown to augment Tat-dependent transcription. It has also been shown to interact with various cellular transcription factors and to belong to the nuclear histone acetyltransferase (HAT) family. To further elucidate the function of Tip60 and its HAT domain in transcription regulation, we compared Tip60 activity in HeLa and Jurkat T lymphoma cells. Here we show that Tip60 augments the HIV-1 Tat activity at the HIV-LTR promoter in HeLa but inhibits it in Jurkat cells. Moreover, we isolated two new variants of the Tip60 protein (Tip60Delta1, Tip60Delta2) from Jurkat cells. The Tip60Delta2 variant lacks the entire HAT domain but modulates HIV-1 Tat activity like full-length Tip60. In addition, Tip60 and the transcriptional repressor ZEB (zinc finger E box binding protein) interact specifically in the yeast two-hybrid system and additively inhibit the CD4 enhancer/promoter activity in Jurkat cells. Thus, Tip60 may function as corepressor of the ZEB protein. In summary, these data show that Tip60 functions as a cell-type-specific transcriptional regulator and that the HAT domain is not required for either transcriptional activation or inhibition. This indicates that Tip60 may function by recruiting additional cell-type-specific cofactors.

Apoptosis in chronic gastritis and its correlation with antigastric autoantibodies.

In the course of time, chronic gastritis may result in gastric atrophy, as in type A gastritis, where autoimmune reactions against parietal cells result in a loss of corpus glands. Two antigastric autoantibodies have been detected in Helicobacter pylori gastritis and are described as anti-luminal and anti-canalicular autoantibodies. The aim of this study was to determine whether increased apoptosis may be responsible for the loss of gastric epithelium and whether this apoptosis is correlated with antigastric autoimmunity. Gastric biopsies from normal mucosa and Helicobacter pylori gastritis were analysed for the presence of apoptosis using the TUNEL method. Helicobacter pylori gastritis was divided into cases (1) without autoantibodies, (2) with anti-luminal, and (3) with anti-canalicular autoantibodies. Apoptotic cells of the foveolar and of the glandular epithelium in the antrum and corpus were counted. The number of apoptotic cells in the gastric mucosa was significantly increased in all cases of gastritis. The highest number of apoptotic cells was observed in the gastric glands of the corpus mucosa in Helicobacter pylori gastritis with anti-canalicular autoantibodies. Apoptosis contributes to the development of gastric atrophy and there are various types of Helicobacter pylori gastritis. The positive correlation between apoptotic cell loss in the glandular zone of the corpus mucosa and the presence of anti-canalicular autoantibodies indicates a possible link between anti-gastric autoimmunity and atrophy in this type of Helicobacter pylori gastritis--similar to that in classic type A gastritis.

Metaplasia, intraepithelial neoplasia and early cancer of the stomach are related to dedifferentiated epithelial cells defined by cytokeratin-7 expression in gastritis.

Cancer presumably arises from stem cells, preserved in an undifferentiated status since fetal development, or from a dedifferentiation of mature cells that return into a fetal phenotype with the potential for proliferation and renewal. Dedifferentiation in this context could represent a transient phase, passed through by cells, before they switch to redifferentiation, metaplasia or neoplasia. Cytokeratin-7 (CK7) is present in fetal, largely absent in normal adult, and transiently neoexpressed in metaplastic and neoplastic epithelial cells of the stomach according to previous observations. CK7 neoexpression in the stomach could, hence, define a fetal-like, dedifferentiated, cellular phenotype during the development of metaplasia and neoplasia. To test this hypothesis, we investigated CK7 expressions in fetal stomachs, non-neoplastic control stomachs, and neoplastic stomachs exhibiting metaplasia, intraepithelial neoplasia, and early cancer. Proliferation and beta-catenin expression of CK7-positive cells were also evaluated. The chronology of CK7 expression was studied during the experimental gastritis-cancer sequence in Mongolian gerbils. Our results show that metaplastic and neoplastic changes in the gastritis-cancer sequence are related to dedifferentiated epithelial cells which are defined by CK7 expression and can phenotypically be linked to fetal cells at the start of gastric pit development. The dedifferentiated cells exhibit a low proliferation and beta-catenin accumulation, similar to stem cells. Thus, the "stem cell" and "dedifferentiation" hypotheses for cancer origin could complement one another, and dedifferentiation-redifferentiation processes might be decisive for carcinogenesis in the stomach.

Mismatch repair deficiency in sporadic synchronous colorectal cancer.

BACKGROUND: Hereditary non-polyposis colorectal cancer (HNPCC) patients frequently develop synchronous colorectal cancer (SCRC) which also occurs sporadically in other patients. Recent studies on microsatellite instability (MSI) in sporadic SCRC diverge completely in their findings (0%-100%). In the present study MSI and mismatch repair (MMR) proteins were evaluated according to standardised criteria (exclusion of a family history, MSI analysed according to NCI recommendations) METHODS: Paraffin embedded sections of SCRC of 30 patients were evaluated for MSI and the loss of protein expression of hMLH1 and hMSH2. RESULTS: 3 out of 30 (10%) patients exhibited MSI-H which 5 out of 30 (17%) showed MSI-L. Loss of protein expression of either hMLH1 or hMSH2 was found in all cases of MSI-H and none of the MSI-L cancers. CONCLUSION: MSI is found in sporadic cases of SCRC to about the same extent as it is mentioned in the literature on sporadic single colorectal cancers. Immunohistochemistry with mismatch repair proteins could be used as a pre-screening for MMR deficiency in sporadic SCRC.

Relationship between microsatellite instability, response and survival in palliative patients with colorectal cancer undergoing first-line chemotherapy.

BACKGROUND AND AIMS: The aim of this work was to investigate the relationship between microsatellite instability (MSI), treatment response and survival in palliative patients with colorectal cancer (CRC) undergoing first-line treatment with weekly 24-hour infusion (24-h inf.) of high-dose 5-fluorouracil (5-FU) and folinic acid (FA). PATIENTS AND METHODS: Tumour material from the colorectal primary carcinomas was analysed for 43 patients. MSI analysis was carried out and immunohistochemistry was performed with hMLH1 and hMSH2. RESULTS: Tumours of 7 patients (16%) were highly instable (MSI-H). These patients had a better response rate (72% vs. 41%; p = 0.072) and a significantly better median survival (33 months, [95% CI 20-46] vs. 19 months, [95% CI 10-28]; p = 0.021) than microsatellite stable (MSS) patients (n = 36). Furthermore, MSI status was shown to be an independent predictive marker for survival (p = 0.037). CONCLUSION: These data provide further support for the hypothesis that MSI-H CRC might have a better response and survival than (MSS) CRC in palliative first-line treatment.

Ki-67 expression and residual tumour (R) classification are associated with disease-free survival in desmoid tumour patients.

BACKGROUND: In order to evaluate prognostic factors in the long-term survival of desmoid tumour patients, analysis of clinicopathological, immunohistochemical and follow-up data was performed. PATIENTS AND METHODS: Between 1969 and 1998, 54 patients underwent resection of aggressive fibromatosis (desmoid) and 33 of them (10 patients with FAP and 23 sporadic) were followed-up with a median time of 130 months (range 10-355 months). Additionally, immunohistochemical analysis of the desmoid tumours using Ki-67 was performed. RESULTS: In univariate analysis, curative resection (R0) (p<0.001) and low proliferation of Ki-67 (p=0.002) were of significant positive prognostic value concerning disease-free survivaL R0 and absence of Ki-67 staining were significantly associated with each other (p=0.004). CONCLUSION: Ki-67 seems to serve as a predictive marker concerning disease-free survival of desmoid tumour patients. In patients presenting with Ki-67 positive desmoids, which are unlikely to be resected in a curative manner, alternative treatment (e.g. sulindac) may be preferable.

Nuclear overexpression of the oncoprotein beta-catenin in colorectal cancer is localized predominantly at the invasion front.

Sixty to eighty percent of all colorectal cancers are characterized by mutations in the APC tumor suppressor gene. Recently, it was shown that these mutations lead to a nuclear overexpression of beta-Catenin by disruption of the wingless/WNT signal pathway. Since nuclear beta-Catenin functions as a transcriptional activator of hitherto unknown tumor genes, this form of beta-Catenin is now considered a major oncoprotein in colorectal cancer. Using immunohistochemistry, we investigated the distribution of overexpressed beta-Catenin within individual colorectal carcinomas. In the majority of the tumors, we found no homogeneous staining, but a strong nuclear expression of beta-Catenin predominantly localized at the invasion front with strongest nuclear staining of isolated, scattered tumor cells. In contrast, cells in the tumor center often showed no nuclear staining, but retained a membranous expression of beta-Catenin, comparable to normal colon epithelium. It is, therefore, likely that in addition to the overexpression of beta-Catenin caused by defects in the APC locus, regulatory events in the tumor itself lead to a different distribution of this oncoprotein. Possibly, surrounding tissue at the invasion front can give signals to the tumor cells, leading to a nuclear translocation of beta-Catenin, where it may play a direct role in tumor invasion processes.

In vivo functions of catenins.

The adhesion of cells to neighbor cells determines cellular and tissue morphogenesis and regulates major cellular processes including motility, growth, survival, and differentiation. Regions of cell-cell adhesion are adherens junctions, desmosomes, and tight junctions. Cadherins are transmembrane molecules whose extracellular domains transmit the direct interaction of two cells. The intracellular cadherin domains bind directly or indirectly to the submembranous catenins, which are linked to the cytoskeleton. Four types of catenins, alpha-catenin, beta-catenin, gamma-catenin, and p120 catenin are known. Three of them, beta-, gamma-, and p120 catenin, are structurally related and possess similar protein interaction domains, the so-called armadillo repeats. These catenins are also parts of signal transduction pathways and play a role in phenotypical changes of cells, e.g., during switches from adherent to migratory cells. The function of catenins in such basic cellular processes also determines a role of catenins in embryogenesis, adult tissue homeostasis, and disease. In particular, beta-catenin is known to be an important oncoprotein in human cancer development.

Global analysis of L1-transcriptomes identified IGFBP-2 as a target of ezrin and NF-κB signaling that promotes colon cancer progression.

L1, a neuronal cell adhesion receptor of the immunoglobulin-like protein family is expressed in invading colorectal cancer (CRC) cells as a target gene of Wnt/ß-catenin signaling. Overexpression of L1 in CRC cells enhances cell motility and proliferation, and confers liver metastasis. We recently identified ezrin and the IκB-NF-κB pathway as essential for the biological properties conferred by L1 in CRC cells. Here, we studied the underlying molecular mechanisms and found that L1 enhances ezrin phosphorylation, via Rho-associated protein kinase (ROCK), and is required for L1-ezrin co-localization at the juxtamembrane domain and for enhancing cell motility. Global transcriptomes from L1-expressing CRC cells were compared with transcriptomes from the same cells expressing small hairpin RNA (shRNA) to ezrin. Among the genes whose expression was elevated by L1 and ezrin we identified insulin-like growth factor-binding protein 2 (IGFBP-2) and showed that its increased expression is mediated by an NF-κB-mediated transactivation of the IGFBP-2 gene promoter. Expression of a constitutively activated mutant ezrin (Ezrin567D) could also increase IGFBP-2 levels in CRC cells. Overexpression of IGFBP-2 in CRC cells lacking L1-enhanced cell proliferation (in the absence of serum), cell motility, tumorigenesis and induced liver metastasis, similar to L1 overexpression. Suppression of endogenous IGFBP-2 in L1-transfected cells inhibited these properties conferred by L1. We detected IGFBP-2 in a unique organization at the bottom of human colonic crypts in normal mucosa and at increased levels throughout human CRC tissue samples co-localizing with the phosphorylated p65 subunit of NF-κB. Finally, we found that IGFBP-2 and L1 can form a molecular complex suggesting that L1-mediated signaling by the L1-ezrin-NF-κB pathway, that induces IGFBP-2 expression, has an important role in CRC progression.

[Liver metastases: pathogenesis and oncogenesis]. / Lebermetastasen: Pathogenese und Onkogenese.

Liver metastases are most often caused by colorectal cancer, followed by pancreatic and breast cancer. Metastasis constitutes the last step of malignant tumor progression. Molecular investigations point towards an important role of the epithelial-mesenchymal transition (EMT) as a mechanism of local invasion and distant metastasis formation. Furthermore, the existence of a subpopulation of cancer stem cells (CSC) could be demonstrated in solid tumors. Recent observations show a dynamic induction of CSC properties by EMT. Therefore, theoretically migrating cancer stem cells (MCSC) can be induced which form the basis for the development of distant metastases.

Detection of disseminated tumour cells in mediastinoscopic lymph node biopsies and endobronchial ultrasonography-guided transbronchial needle aspiration in patients with suspected lung cancer.

PURPOSE: Ultrasound-guided transbronchial needle aspiration of mediastinal lymph nodes (EBUS-TBNA) is apparently more accurate for cancer diagnosis than standard transbronchial needle aspiration (TBNA), but it is less sensitive than mediastinoscopy. The detection of disseminated tumour cells in transbronchial needle aspiration and mediastinoscopic biopsies could improve staging and might be helpful concerning indications for neoadjuvant regimen. The goal of this study was to develop a quantitative method for the detection of disseminated tumour cells (DTCs) in lymph node samples from patients with suspected lung cancer. PATIENTS AND METHODS: We compared in a prospective trail EBUS-TBNA (n=58 patients, 86 samples) and mediastinoscopy (n=22 patients, 37 samples) in two largely independent cohorts of lung cancer patients. Eleven patients, 14 samples were analysed using both methods. Patients without evidence of malignant disease were available as controls for EBUS-TBNA (n=20 patients, 28 samples) and mediastinoscopy (n=6 patients, 8 samples). Real-time quantitative mRNA analysis was performed for the cytokeratin 19 (CK19) and MAGE-A genes (MAGE-A 1-6, MAGE-A12) as markers, using a LightCycler 480 instrument. RESULTS: CK19 mRNA expression in EBUS-TBNA samples was detected in 84/86 (98%) and in 28/28 control samples (100%). After mediastinoscopy 16/37 (43%) samples of lung cancer patients were CK19 mRNA positive while controls showed no CK19 mRNA expression (0/8). MAGE-A expression was detectable in 42/86 (49%) EBUS-TBNA samples and in 13/37 (35%) mediastinoscopy samples. MAGE-A expression was detected in EBUS-TBNA controls in 3/28 (11%) and 1/8 (12%) mediastinoscopy controls. High MAGE-A expression correlated with increased tumour stage. CONCLUSION: Since CK19 expression was detected in all EBUS-TBNA samples from the control patients, but not in mediastinoscopy samples, we conclude that CK19 is not suitable as a marker for disseminated tumour cells in samples attained by EBUS-TBNA. One possible explanation is a contamination with epithelial cells from the bronchial tubes. MAGE-A genes are promising markers for disseminated tumour cells in lymph nodes in patients with suspected lung cancer which merit further investigation.