Guenther Gastl

CD4+CD25+ Regulatory T Cells Inhibit Experimental Anti-Glomerular Basement Membrane Glomerulonephritis in Mice

CD4+CD25+ regulatory T cells (Treg) are of critical importance for the maintenance of tolerance. The kidney is frequently involved in autoimmune diseases, such as lupus erythematosus or glomerulonephritis (GN). Therefore, the therapeutic efficacy of Treg in a T cell-dependent murine model of experimental anti-glomerular basement membrane (anti-GBM) GN was tested. Transfer of 1 x 10(6) CD4+CD25+ T cells (day -1) into mice that were previously immunized with rabbit IgG (day -3) and subsequently received an injection of anti-GBM rabbit serum (day 0) significantly attenuated the development of proteinuria when compared with animals that received an injection of 1 x 10(6) CD4+CD25- T cells (control group). Treg injection induced a dramatic decrease of glomerular damage as well as a marked decrease of CD4+ T cell, CD8+ T cell, and macrophage infiltration. Of note, deposition of immune complexes was not prevented by Treg, showing that Treg rather inhibited cell-mediated organ damage than priming of the humoral immune response. Accordingly, a significant reduction of IFN-gamma, TNF-alpha, and TGF-beta1 mRNA in kidneys from animals that received Treg injection was observed. Tracking of enhanced green fluorescence protein-transgenic Treg revealed a predominant migration to secondary lymphoid organs with a significant increase of regulatory T cells (CD4+CD25+CD69-CD45RB(low)) in the lymph nodes. In contrast, enhanced green fluorescence protein-and FoxP3-positive cells by reverse transcription-PCR and CD4+CD25+CD69-CD45RB(low) T cells by flow cytometry in the kidney of nephritic animals were not detected. This report provides first evidence that Treg are potent suppressors of anti-GBM GN. Treg therefore might be of therapeutic value for the treatment of severe GN in humans.

EpCAM expression in primary tumour tissues and metastases: an immunohistochemical analysis.

Aims Epithelial cell adhesion molecule (EpCAM) is a cell surface protein with oncogenic features that is expressed on healthy human epithelia and corresponding malignant tumours. EpCAM expression frequently correlates with more aggressive tumour behaviour and new EpCAM-specific therapeutic agents have recently been approved for clinical use in patients with cancer. However, no consensus exists on how and when to evaluate EpCAM expression in patients with cancer. Material and methods EpCAM expression was assessed by a well-established immunohistochemical staining protocol in 2291 primary tumour tissues and in 108 metastases using the EpCAM-specific antibody clone VU1D9. A total immunostaining score was calculated as the product of a proportion score and an intensity score. Four expression subgroups (no, weak, moderate and intense) were defined. As described previously, the term ‘EpCAM overexpression’ was reserved for tissues showing a total immunostaining score >4. Results EpCAM was highly expressed in most tumours of gastrointestinal origin and in some carcinomas of the genitourinary tract. However, hepatocellular carcinomas, clear cell renal cell cancer, urothelial cancer and squamous cell cancers were frequently EpCAM negative. EpCAM expression in breast cancer depended on the histological subtype; lobular histology usually showed no or weak expression. Most metastases were EpCAM positive and they frequently reflected the expression phenotype of the primary tumour. Conclusion EpCAM expression was detected on adenocarcinomas of various primary sites. If EpCAM-specific antibodies are intended to be used in patients with cancer, we recommend prior immunohistochemical evaluation of EpCAM expression, particularly in patients with renal cell cancer, hepatocellular carcinoma, urothelial carcinoma, breast cancer and squamous cell carcinomas.

Overexpression of Epithelial Cell Adhesion Molecule Antigen in Gallbladder Carcinoma Is an Independent Marker for Poor Survival

Purpose: Gallbladder carcinoma is an aggressive type of cancer that is difficult to cure by conventional procedures. There thus is a need to identify novel molecular markers for the assessment of prognosis and as potential therapeutic targets. This retrospective study was designed to investigate the prognostic significance of epithelial cell adhesion molecule (Ep-CAM) overexpression in human gallbladder carcinoma. Experimental Design: Ep-CAM expression was examined immunohistochemically on paraffin-embedded tissue specimens from 99 patients who underwent surgical treatment for gallbladder carcinoma in the period between August 1988 and May 1999. Results: Ep-CAM overexpression was found in 63 (63.6%) of the tumor samples. Kaplan-Meier curves showed that Ep-CAM overexpression was significantly related to decreased overall survival (P < 0.01). Overall survival gradually worsened with increasing Ep-CAM scores. Notably, in the subgroup of pT1 tumors (n = 17), patients without Ep-CAM overexpression had a 5-year overall survival rate of 100% compared with 38% (P = 0.01) for patients with Ep-CAM-overexpressing tumors. By univariate analysis, no correlation was found with conventional clinicopathological parameters. Multivariate analysis, including Ep-CAM expression, pT stage, tumor grade, and resection margin involvement, showed that Ep-CAM overexpression was an independent prognostic marker in gallbladder carcinoma (P = 0.03; relative risk, 1.8). Conclusions: These results demonstrate for the first time that Ep-CAM overexpression is an independent prognostic marker in gallbladder carcinoma and that its prognostic impact should be validated prospectively. Furthermore, the Ep-CAM antigen represents an attractive target for specific therapies with monoclonal antibodies or specific vaccines in patients with Ep-CAM-overexpressing gallbladder carcinoma.

TROP2: a novel prognostic marker in squamous cell carcinoma of the oral cavity

Squamous cell carcinoma is by far the most common type of cancer of the oral cavity, representing more than 90% of all oral cancers. Despite refinement of surgical techniques and adjuvant therapies, the prognosis for patients with oral squamous cell carcinoma remains poor. Identification of prognostic factors related to tumor biology might improve this assessment. Recently, the human trophoblast cell-surface antigen TROP2 was found to be highly expressed in colorectal cancer, correlating with aggressiveness and poor prognosis. Thus, the aim of this study was to investigate TROP2 expression and its prognostic impact in oral squamous cell carcinoma patients. TROP2 expression was examined by immunohistochemistry in a series of 90 patients on a tissue microarray of paraffin-embedded specimens. Survival was calculated using Kaplan–Meier estimates. Parameters found to be of prognostic significance in univariate analysis were verified in a multivariate Cox regression model. TROP2 overexpression was observed in 52 (58%) of the tumor samples. Kaplan–Meier curves showed that TROP2 overexpression was significantly associated with decreased overall survival (P<0.01). Overall survival gradually worsened with increasing TROP2 scores. By univariate analyses, no correlation with conventional clinicopathological features was found. Multivariate Cox regression analysis revealed TROP2 overexpression to be an independent factor predictive of poor disease outcome (P<0.01). These results demonstrate that TROP2 overexpression is an independent prognostic marker in patients with oral squamous cell carcinoma. TROP2 overexpression was detectable in 58% of the tumor samples, indicating it to be a potential novel therapeutic target in squamous cell carcinoma of the oral cavity.

Low concentrations of STI571 in the cerebrospinal fluid: a case report

Summary. We report a 53‐year‐old man with lymphoid blast crisis of Ph+ chronic myeloid leukaemia who was treated with STI571, a selective inhibitor of the enzymatic activity of BCR–ABL. He responded excellently to STI571 (600 mg/d), obtaining a complete cytogenetic remission after 3 months of therapy. Although remission in the bone marrow was sustained, the patient developed an isolated central nervous system relapse. Subsequent analyses of STI571 concentrations in the cerebrospinal fluid (CSF) revealed 2‐log lower CSF levels of STI571 than corresponding plasma levels. These are the first data demonstrating a low penetration of orally administered STI571 into the CSF in humans.

GRP-78 secreted by tumor cells blocks the antiangiogenic activity of bortezomib.

Antiangiogenic effects of the proteasome inhibitor bortezomib were analyzed on tumor xenografts in vivo. Bortezomib strongly inhibited angiogenesis and vascularization in the chicken chorioallantoic membrane. Bortezomibs inhibitory effects on chorioallantoic membrane vascularization were abrogated in the presence of distinct tumor xenografts, thanks to a soluble factor secreted by tumor cells. Through size-exclusion and ion-exchange chromatography as well as mass spectroscopy, we identified GRP-78, a chaperone protein of the unfolded protein response, as being responsible for bortezomib resistance. Indeed, a variety of bortezomib-resistant solid tumor cell lines (PC-3, HRT-18), but not myeloma cell lines (U266, OPM-2), were able to secrete high amounts of GRP-78. Recombinant GRP-78 conferred bortezomib resistance to endothelial cells and OPM-2 myeloma cells. Knockdown of GRP78 gene expression in tumor cells and immunodepletion of GRP-78 protein from tumor cell supernatants restored bortezomib sensitivity. GRP-78 did not bind or complex bortezomib but induced prosurvival signals by phosphorylation of extracellular signal-related kinase and inhibited p53-mediated expression of proapoptotic Bok and Noxa proteins in endothelial cells. From our data, we conclude that distinct solid tumor cells are able to secrete GRP-78 into the tumor microenvironment, thus demonstrating a hitherto unknown mechanism of resistance to bortezomib.

Ep-CAM expression in pancreatic and ampullary carcinomas: frequency and prognostic relevance

Aims: Pancreatic adenocarcinoma is an aggressive gastrointestinal malignancy with only a few long-term survivors even after radical surgery. Patients with ampullary cancer have a better prognosis but adjuvant therapy needs further improvement. Epithelial cell adhesion molecule (Ep-CAM) is strongly expressed in a variety of epithelial cancers and represents a promising target for immunological tumour therapy. Thus, the aim of this study was to investigate Ep-CAM expression and its potential prognostic impact in pancreatic and ampullary carcinomas. Methods: Ep-CAM expression was investigated retrospectively by immunohistochemistry in paraffin-embedded primary tumour tissue samples from a series of consecutive patients with pancreatic (n = 153) and ampullary cancer (n = 34). Results: Ep-CAM overexpression was observed in 85 of 153 pancreatic cancer specimens (56%) and in 29 of 34 ampullary cancer samples (85%). Overall, Ep-CAM failed to be an independent prognostic marker. However, subgroup analyses showed that Ep-CAM overexpression correlated with shorter overall survival among patients with ampullary cancer and advanced stage pancreatic cancer. In the latter subgroup, survival gradually worsened with increasing Ep-CAM scores. Furthermore, in ampullary cancer, Ep-CAM overexpression was found to correlate with tumour stage. Conclusions: Ep-CAM overexpression was detectable in the majority of cases with pancreatic and ampullary cancer. Therefore, Ep-CAM represents an attractive target for immune-based therapeutic interventions in these tumour entities. However, the prognostic value of Ep-CAM overexpression remains undetermined.

Hodgkin's disease variant of Richter's syndrome in chronic lymphocytic leukaemia patients previously treated with fludarabine

The transformation of chronic lymphocytic leukaemia (CLL) into large‐cell lymphoma (Richters syndrome, RS) is a well‐documented phenomenon. Only rarely does CLL transform into Hodgkins lymphoma (HL). To further analyse the clinico‐pathological and genetic findings in the HL variant of RS, we performed a single‐institution study in four patients, who developed HL within a mean of 107 months after diagnosis of CLL. All were treated with fludarabine. Three cases were Epstein–Barr virus (EBV)‐associated mixed cellularity (MC) HL, the fourth was nodular sclerosis (NS) HL without EBV association. The sites involved by HL included supra‐ and infradiaphragmal lymph nodes and the tonsils; stage IV disease was also documented. All patients presented with CLL treatment‐resistant lymphadenopathies and B‐symptoms. In two of the MC cases, molecular analysis performed on CLL samples and microdissected Hodgkin and Reed–Sternberg cells (HRSC) suggested a clonal relationship, while in NS no indication of a clonal relationship was detected. In summary, HL can occur in CLL patients at any site, up to 17 years after initial diagnosis, especially after treatment with fludarabine. The majority present with B‐symptoms and CLL treatment‐resistant lymphadenopathy, are of the MC type, clonally related to CLL and might be triggered by an EBV infection.

TROP2 expression as prognostic marker for gastric carcinoma

Background: In gastric cancer the recurrence rate is unacceptably high, even after R0 resection and (neo)adjuvant chemotherapy. Therefore, there is an urgent need for identification of predictive and/or prognostic biomarkers to select high-risk patients who might benefit from additional therapies. Expression of TROP2 has been shown to be associated with tumour aggressiveness and poor prognosis in patients with various epithelial cancers. Aims: To investigate TROP2 expression in gastric cancer and its correlation with clinicopathological features and disease outcome. Methods: Expression of TROP2 was investigated by immunohistochemistry of tumour specimens from 104 patients who underwent resection for gastric cancer. Parameters found to be of prognostic significance in univariate analysis were verified in a multivariate Cox regression model. Results: TROP2 was found to be overexpressed in 58 (56%) tumour samples. Significantly higher expression of TROP2 could be detected in intestinal-type carcinomas (p = 0.03). In intestinal-type gastric cancer, TROP2 overexpression was significantly correlated with shorter disease-free survival (DFS) (p = 0.03). Among the total group, TROP2 overexpression was predictive for poor disease-free (p<0.01) and overall (p = 0.03) survival in lymph node positive patients. Multivariate Cox regression analysis revealed TROP2 overexpression to be an independent prognostic marker for poor DFS in the subgroup of patients with intestinal-type gastric cancer irrespective of lymph node involvement. Conclusion: Results show that TROP2 is an independent prognostic marker for disease recurrence in intestinal type gastric cancer. Due to its wide distribution TROP2 may become an attractive therapeutic target in a subgroup of patients with gastric cancer.

Phenotype-dependent effects of EpCAM expression on growth and invasion of human breast cancer cell lines

BackgroundThe epithelial cell adhesion molecule (EpCAM) has been shown to be overexpressed in breast cancer and stem cells and has emerged as an attractive target for immunotherapy of breast cancer patients. This study analyzes the effects of EpCAM on breast cancer cell lines with epithelial or mesenchymal phenotype.MethodsFor this purpose, shRNA-mediated knockdown of EpCAM gene expression was performed in EpCAMhigh breast cancer cell lines with epithelial phenotype (MCF-7, T47D and SkBR3). Moreover, EpCAMlow breast carcinoma cell lines with mesenchymal phenotype (MDA-MB-231, Hs578t) and inducible overexpression of EpCAM were used to study effects on proliferation, migration and in vivo growth.ResultsIn comparison to non-specific silencing controls (n/s-crtl) knockdown of EpCAM (E#2) in EpCAMhigh cell lines resulted in reduced cell proliferation under serum-reduced culture conditions. Moreover, DNA synthesis under 3D culture conditions in collagen was significantly reduced. Xenografts of MCF-7 and T47D cells with knockdown of EpCAM formed smaller tumors that were less invasive. EpCAMlow cell lines with tetracycline-inducible overexpression of EpCAM showed no increased cell proliferation or migration under serum-reduced growth conditions. MDA-MB-231 xenografts with EpCAM overexpression showed reduced invasion into host tissue and more infiltrates of chicken granulocytes.ConclusionsThe role of EpCAM in breast cancer strongly depends on the epithelial or mesenchymal phenotype of tumor cells. Cancer cells with epithelial phenotype need EpCAM as a growth- and invasion-promoting factor, whereas tumor cells with a mesenchymal phenotype are independent of EpCAM in invasion processes and tumor progression. These findings might have clinical implications for EpCAM-based targeting strategies in patients with invasive breast cancer.