Showing papers by "Klaus Pantel published in 2005"

Changes in Cytoskeletal Protein Composition Indicative of an Epithelial-Mesenchymal Transition in Human Micrometastatic and Primary Breast Carcinoma Cells

Abstract: Purpose: The bone marrow is a frequent and clinically important homing site for early disseminated breast cancer cells. Here, we aimed to profile the protein expression of these cells using unique cell line models and to evaluate the prognostic relevance of candidate gene expression for breast cancer patients. Experimental Design: To identify expression patterns characteristic for micrometastatic cells, three different cell lines (BC-K1, BC-P1, and BC-S1) established by SV40 immortalization of cancer cells isolated from the bone marrow of patients with breast cancer were compared with MCF-7 breast cancer and SV40 immortalized normal breast ductal cells (MTSV-1.7) using two-dimensional gel electrophoresis followed by MALDI-ToF analysis. The prognostic significance and clinicopathologic associations of selected differentially expressed proteins were evaluated using high-density breast cancer tissue microarrays. Results: In contrast to MCF-7 and MTSV1-7 reference cell lines, all micrometastatic cancer cell lines displayed loss of epithelial cytokeratins (CK8, CK18, and CK19) and ectopic expression of vimentin commonly present in mesenchymal cells. Immunohistochemical analysis of 2,517 samples of breast cancer further showed that loss of cytokeratin and ectopic vimentin expression were significantly associated with a higher tumor grade, high mitotic index, and negative estrogen/progesterone-receptor status. Although in univariate analyses significantly related to clinical outcome, none of the cytokeratins analyzed were independently associated with either overall or cancer-specific survival. Conclusions: Micrometastatic cancer cells exhibit marked changes in the expression pattern of cytoskeletal proteins indicative of an epithelial-mesenchymal transition. This phenotypical change could already be detected in primary tumors and is associated with the aggressive behavior of breast cancer cells in vivo .

Tumor-Cell Homing to Lymph Nodes and Bone Marrow and CXCR4 Expression in Esophageal Cancer

Abstract: BACKGROUND The chemokine and bone marrow-homing receptor CXCR4 has been implicated in metastatic dissemination of various cancers. We investigated CXCR4 expression in esophageal cancer specimens and its association with survival, lymph node microinvolvement, and bone marrow micrometastasis. METHODS We analyzed frozen tumor specimens from 136 patients with completely resected esophageal cancer for CXCR4 expression by immunohistochemistry. Lymph node microinvolvement and bone marrow micrometastasis were assessed by immunohistochemistry with monoclonal antibodies Ber-EP4 (against epithelial cell adhesion molecule) and pancytokeratin A45-B/B3 (against several cytokeratins), respectively. Associations between CXCR4 expression and clinicopathologic features, including tumor stage, histologic grade, lymph node metastasis and microinvolvement, bone marrow micrometastasis, and survival, were investigated with Fisher's test, log-rank test, and Cox multivariable analysis. All statistical tests were two-sided. RESULTS CXCR4 protein was expressed in 75 (55%) of 136 esophageal tumors examined. CXCR4 expression was statistically significantly associated with reduced median overall and disease-specific survival, compared with CXCR4 nonexpression (P < .001; log-rank test). The median overall survival of patients with CXCR4-positive tumors was 20 months and with CXCR4-negative tumors, 76 months (difference = 56 months, 95% confidence interval [CI] = 4 to 108 months; P < .001). The median disease-specific survival of patients with CXCR4-positive tumors was 25 months and with CXCR4-negative tumors was 97 months (difference = 72 months, 95% CI = 34 to 110 months; P < .001). CXCR4 expression was statistically significantly associated with increased lymph node microinvolvement (P < .001) and with increased bone marrow micrometastasis (P < .001). In multivariable analysis, CXCR4 expression, compared with its nonexpression, was identified as the independent variable that was most strongly associated with reduced disease-specific survival (relative risk [RR] of death = 2.03, 95% CI = 1.20 to 3.41; P = .008) and overall survival (RR of death = 2.18, 95% CI = 1.33 to 3.59; P = .002). CONCLUSION CXCR4 expression was associated with poor clinical outcome in esophageal cancer patients. CXCR4 may have a role in early metastatic spread because its expression was associated with micrometastases to both the lymph nodes and bone marrow. Thus, CXCR4 should be explored further as a target for adjuvant therapy for micrometastatic disease.

Key role of myosin light chain (MLC) kinase-mediated MLC2a phosphorylation in the α1-adrenergic positive inotropic effect in human atrium

Abstract: Objective : Mechanisms of the positive inotropic response to α1-adrenergic stimulation in the heart remain poorly understood, but recent evidence in rat papillary muscle suggests an important role of regulatory myosin light chain (MLC2) phosphorylation. This study investigated α1-adrenergic contractile effects and the role of MLC kinase (MLCK)-dependent phosphorylation of MLC2 in human atrial muscle strips. Methods : Force measurement was performed on electrically stimulated atrial muscle strips ( n =140; 20 hearts) in the presence of the beta-blocker nadolol. MLC2a phosphorylation was determined by 2D-polyacrylamide gel electrophoresis and Western blotting of muscle strips that were immediately freeze-clamped following force measurements. Results : The α1-agonist phenylephrine (PE; 0.3–100 μM) exerted a concentration-dependent, monophasic, sustained positive inotropic effect (86% above basal) that was accompanied by an 80% increase in MLC2a phosphorylation. Desinhibition of MLC phosphatase by the Rho kinase inhibitor Y-27632 (10 μM) reduced the effect of PE by 16%. The MLCK inhibitor wortmannin (10 μM) completely abolished both the PE-induced increase in force and MLC2a phosphorylation. The structurally unrelated MLCK inhibitor ML-7 (10 μM) had similar effects. Neither Y-27632 nor wortmannin or ML-7 affected β-adrenergic force stimulation. In contrast to our findings in atrial muscle strips, we observed no increase in MLC2v phosphorylation after PE in human ventricular muscle strips and wortmannin failed to inhibit PE-induced force of contraction. Conclusion : α1-Adrenergic receptors mediate a prominent increase in contractile force in human atria that depends on MLCK activity and is accompanied by an increase in MLC2 phosphorylation.

Influence of immunomagnetic enrichment on gene expression of tumor cells

Abstract: Background Metastasis is the leading cause of cancer-related death. Bone marrow (BM) is a frequent site for the settlement of disseminated tumor cells which occurs years before overt metastases signal incurability.

Disseminated tumor cells (DTC) in bone marrow (BM) and clinical outcome: Final results of pooled analysis on 10-year survival of 4,703 breast cancer patients

Abstract: 502 Background: Statistically powerful prognostic data on long-term outcome of DTC +ve breast cancer patients is needed to enable evidence-based therapeutic strategies designed to improve patient o...

Bone marrow: Homing organ for dormant micrometastatic cancer cells

Abstract: Proc Amer Assoc Cancer Res, Volume 46, 2005 SY34-2 Although many different assays have been developed over the past ten years to detect micrometastatic tumor cells, the two major approaches used involve either immunocytochemical staining or polymerase chain reaction (PCR) analysis. If these assays are sensitive and specific enough, they can detect a single metastatic cell in the background of millions of normal cells. For epithelial tumors, cytokeratins have become the best marker for the immunocytochemical detection of micrometastatic tumor cells[1][1]. Bone marrow (BM), which can be easily collected from the iliac crest, is the most important site for detecting micrometastatic epithelial tumor cells, which are present in BM samples of 20-40% of patients with carcinomas at various primary sites; even in the absence of lymph node metastases (stage N0) or clinical signs of overt distant metastases (stage M0) [2][2]–[4][3]. BM samples can also be monitored for the presence of micrometastatic tumor cells after primary surgical treatment, to detect tumor recurrence[5][4],[6][5]. Interestingly, the presence of disseminated tumor cells in the BM is not only useful in predicting the development of skeletal metastases, but also in predicting the development of metastases in other distant organs, such as lung or liver. This is even true for tumors that rarely show skeletal metastases, such as colon cancer [3][6]. In fact disseminated cancer cells have also been found in the BM of patients with head and neck cancer that did not have lymph node metastases, although the clinical significance of these cells is not clear [7][7]. So BM might be an important reservoir that allows for disseminated epithelial tumor cells to adapt and disseminate into other organs. An alternative explanation is that the presence of occult cancer cells in the BM might only reflect the general propensity of these cells to disseminate and to survive in organs, rather than just in the BM. Several studies have documented the persistence of dormant micrometastatic tumor cells in BM of cancer patients without clinical signs of overt metastases[5][4],[6][5],[8][8]–[12][9]. These findings are direct proof of the concept of dormancy. Moreover, the persistence of these dormant cells is an indicator for subsequent metastatic relapse. Heiss et al. previously showed a close association between post-operative persistence of tumor cells in BM and metastatic relapse in patients with gastric cancer [6][5]. More recent data in breast cancer published by two independent groups demonstrated that micrometastatic tumor cells in BM three years after initial diagnosis in disease-free breast cancer patients predicted unfavourable clinical outcome [11][10],[12][9]. Persistence of tumor cells in BM was an independent prognostic factor. These findings indicate that adjuvant systemic treatment may be insufficient to eliminate micrometastatic cells and that at least some of the surviving micrometastatic cells can escape dormancy. It will be now a major challenge to unravel the mechanisms underlying this capacity. One approach to achieve this task might be the further characterization of micrometastatic cells. Anti-cytokeratin antibodies, which are used to identify disseminated tumor cells in BM, can be used in combination with antibodies against other tumor-associated antigens to profile these cells. Using such immunocytochemical procedures, it has been possible to identify a number of tumor-associated characteristics of the cytokeratin-positive cells found in BM[1][1] (see Fig. 1, in Reference 1). Phenotyping of disseminated tumor cells in BM of early stage patients has also yielded additional prognostic information. In patients with gastric cancer, the presence of micrometastatic cells that express the urokinase-type plasminogen activator receptor (uPAR) correlates with an unfavourable prognosis [6][5]. Similar observations have been made in patients with breast cancer; Her2/neu overexpression by tumor cells that have disseminated to the BM predicts poor clinical outcome [13][11]. So uPAR and Her2/neu might be important for the survival and growth of disseminated tumor cells. Recently, Jonathan Uhr′s group presented evidence that the genotype of persistent circulating tumor cells may change towards a Her2-amplified genotype[14][12], which might be in line with our previous findings on the selection of a Her2-positive phenotype in BM micrometastases[13][11],[15][13]. Recent technical developments have made it possible to examine the genome of disseminated tumor cells. A combination of immunocytochemistry and fluorescence in situ hybridization (FISH) have shown that BM contains proliferating micrometastatic cells with various numeric chromosomal aberrations found of malignant origin[16][14]. The viability and proliferative capacity of these cells has also been monitored in these models, and correlated with clinical outcome[16][14],[17][15]. By developing a new procedure for whole genome amplification and subsequent comparative genomic hybridisation (CGH) of single immunostained cells, it was found that cytokeratin-positive cells in BM of epithelial breast cancer patients without clinical signs of overt metastases (stage M0) are genetically heterogeneous[18][16]. This heterogeneity was strikingly reduced with the emergence of clinically evident metastasis (stage M1). The stage at which individual cells leave the primary tumor is unclear. In patients with early stage invasive breast cancer, the cytokeratin-positive cells isolated from the BM had few features in common with those found in their respective primary tumors[19][17]. A provocative interpretation of this surprising finding is that the disseminated tumor cells separated from the primary lesion at a very early stage. This hypothesis is also supported by the finding that only few disseminated tumor cells in these patients had TP53 mutations, which are associated with the later stages of tumourigenesis[18][16],[20][18]. Disseminated tumor cells might therefore evolve independently into overt metastases, driven by the specific selective pressures of the BM environment. Functional analysis of micrometastatic cells remains a challenge, because even after short-term culture their numbers are still small (on average between 1,000 and 10,000 cells). Permanent cell lines have therefore been established, and show gene expression and genomic characteristics that are typical of epithelial tumor cells in situ [21][19],[22][20]. These could therefore serve as models for functional studies on dormancy of micrometastatic cells. The detection and characterization of micrometastatic tumor cells in cancer patients has provided important new information about the cascade of metastatic events. This information may also have important implications for cancer prognosis and for therapy. [1]: #ref-1 [2]: #ref-2 [3]: #ref-4 [4]: #ref-5 [5]: #ref-6 [6]: #ref-3 [7]: #ref-7 [8]: #ref-8 [9]: #ref-12 [10]: #ref-11 [11]: #ref-13 [12]: #ref-14 [13]: #ref-15 [14]: #ref-16 [15]: #ref-17 [16]: #ref-18 [17]: #ref-19 [18]: #ref-20 [19]: #ref-21 [20]: #ref-22

Homing ösophagealer Karzinomzellen in Lymphknoten und ins Knochenmark — Rolle der CXCR4-Expression

Abstract: Background: The chemokine and bone marrow homing receptor CXCR4 has been implicated in metastatic dissemination in different tumor types. In this study we investigated CXCR4 expression in esophageal cancer and the association with survival, lymph node and bone marrow micrometastasis. Methods: We retrospectively analyzed frozen tumor samples of 136 patients with completely resected esophageal cancer for CXCR4 expression by immunohistochemistry. Lymph node micrometastasis was determined by staining with mAb Ber-EP4, bone marrow micrometastasis by using pancyto-keratin mAb A45-B/B3. CXCR4 expression was correlated with these results and other clinicopathological features. Results: CXCR4 expression was found in 75 out of 136 (55%) esophageal tumors. Patients with positive immunostaining of CXCR4 had a significantly shorter overall and tumor-specific survival than patients without (p = 0.002; log-rank test). Further, CXCR4 expression significantly correlated with lymph node as well as bone marrow micrometastasis. Multivariate analysis identified CXCR4 expression as an independent prognostic factor (p = 0.001). Conclusion: CXCR4 expression is associated with a significantly worse outcome in esophageal cancer patients. Our results implicate a role for CXCR4 in systemic spread since expression correlates with lymph node and bone marrow micrometastasis and it could be a potential target for immunotherapy.