Stephanie J. Gros

Frequent homogeneous HER-2 amplification in primary and metastatic adenocarcinoma of the esophagus

HER-2 is the target for antibody based treatment of breast cancer (Herceptin®). In order to evaluate the potential role of such a treatment in esophageal cancers, HER-2 amplification and overexpression was investigated in primary and metastatic cancers of the esophagus. A tissue microarray was constructed from 255 primary esophageal cancers (110 adenocarcinomas and 145 squamous cell carcinomas), 89 nodal and 33 distant metastases. Slides were analyzed by immunohistochemistry (HercepTest™; DAKO) and fluorescence in situ hybridization (FISH; PathVysion™; Vysis-Abbott) for HER-2 amplification and overexpression. Amplification was seen in 16/110 (15%) adenocarcinomas and in 7/145 (5%) squamous cell carcinomas. There was a strong association between HER-2 amplification and overexpression, especially in adenocarcinomas (P<0.0001, log rank). There was a 100% concordance of the HER-2 results in primary tumor and corresponding metastases in 84 analyzed pairs. Amplification was typically high-level with more than 10–15 HER-2 copies per tumor cell. Amplification was unrelated to survival, grading, pT, pN, pM or UICC stage. We conclude that esophageal adenocarcinomas belong to those cancer types with relevant frequency high-level HER-2 gene amplification clinical trials or individual case studies investigating the response of metastatic HER-2-positive esophageal cancers to Herceptin® should be undertaken. The strong concordance of the HER-2 status in primary and metastatic cancers argues for a possible response of metastases from patients with HER-2-positive primary tumors to Herceptin®.

Effective Therapeutic Targeting of the Overexpressed HER-2 Receptor in a Highly Metastatic Orthotopic Model of Esophageal Carcinoma

This study aimed to determine the targeted efficacy of trastuzumab (Herceptin) on human epidermal growth factor receptor 2 (HER-2)-overexpressing metastatic esophageal cancer in an orthotopic mouse model. HER-2 overexpression and amplification of human esophageal primary and metastatic tumors were shown with HER-2–fluorescence in situ hybridization analysis and HER-2 immunostaining. Following orthotopic implantation with the HER-2–overexpressing OE19 human esophageal cancer cell line, mice were treated with trastuzumab. Sequential magnetic resonance imaging was used to monitor primary tumor and metastasis during treatment. After six weeks, a significant inhibition of primary tumor development was imaged in trastuzumab-treated animals in comparison with the control group. Trastuzumab treatment also led to a reduction of lymphatic metastasis. Thus, HER-2 targeted therapy with trastuzumab resulted in a significant primary tumor growth reduction as well as a decrease of lymph node metastases in the orthotopic model of metastatic esophageal carcinoma. The results of the present study suggest the clinical use of trastuzumab for HER-2–overexpressing esophageal cancer, which is a significant fraction of the patient population. Treatment of this highly treatment-resistant disease with trastuzumab in the adjuvant setting to prevent lymph node metastasis after primary tumor resection is suggested by the data in this report. Mol Cancer Ther; 9(7); 2037–45. ©2010 AACR.

Involvement of CXCR4 chemokine receptor in metastastic HER2-positive esophageal cancer.

A functional linkage of the structurally unrelated receptors HER2 and CXCR4 has been suggested for breast cancer but has not been evaluated for esophageal carcinoma. The inhibition of HER2 leads to a reduction of primary tumor growth and metastases in an orthotopic model of esophageal carcinoma. The chemokine receptor CXCR4 has been implicated in metastatic dissemination of various tumors and correlates with poor survival in esophageal carcinoma. The aim of this study was to investigate a correlation between the expression levels of HER2 and CXCR4 and to evaluate the involvemnent of CXCR4-expression in HER2-positive esophageal carcinoma. The effects of HER2-inhibition with trastuzumab and of CXCR4-inhibition with AMD3100 on primary tumor growth, metastatic homing, and receptor expression were evaluated in vitro and in an orthotopic model of metastatic esophageal carcinoma using MRI for imaging. The clinical relevance of HER2- and CXCR4-expression was examined in esophageal carcinoma patients. A significant correlation of HER2- and CXCR4-expression in primary tumor and metastases exists in the orthotopic model. Trastuzumab and AMD3100 treatment led to a significant reduction of primary tumor growth, metastases and micrometastases. HER2-expression was significantly elevated under AMD3100 treatment in the primary tumor and particularly in the metastases. The positive correlation between HER2- and CXCR4-expression was validated in esophageal cancer patients. The correlation of CXCR4- and HER2-expression and the elevation of HER2-expression and reduction of metastases through CXCR4-inhibition suggest a possible functional linkage and a role in tumor dissemination in HER2-positive esophageal carcinoma.

Complementary use of fluorescence and magnetic resonance imaging of metastatic esophageal cancer in a novel orthotopic mouse model

We describe the development of an aggressive orthotopic metastatic model of esophageal cancer, which is visualized in real time with combined magnetic resonance imaging (MRI) and fluorescence imaging. The aim of the study was to describe the development of a novel model of metastatic tumor disease of esophageal carcinoma and use this model to evaluate fluorescence and MRI in early detection of local and metastatic disease. The human esophageal adenocarcinoma cell line PT1590 was stably transfected with green fluorescent protein (GFP). Nude mice were orthotopically implanted with PT1590‐GFP cells. Orthotopic tumor growth as well as metastatic spread was examined by fluorescence imaging and high‐resolution MRI at defined intervals after orthotopic implantation. Highly aggressive novel fluorescent cell lines were isolated from metastatic tissues and put into culture. After implantation of these cells, 100% of the animals developed orthotopic primary tumors. In 83% of animals, metastatic spread to liver, lung and lymph nodes was observed. Primary tumor growth could be visualized with fluorescence imaging and with MRI with high correlation between the 2 methods. Fluorescence imaging allows fast, sensitive, and economical imaging of the primary and metastatic tumor without anesthesia. With MRI, anatomical structures are visualized more precisely and tumors can be more accurately localized to specific organs. This model should prove highly useful to understand esophageal carcinoma and to identify novel therapeutics for this treatment‐resistant disease.

L1 is Highly Expressed in Tumors of the Nervous System: A Study of Over 8000 Human Tissues

BACKGROUND L1 cell adhesion molecule (CD171) has been detected in different malignant tumors and is associated with unfavorable outcome. It thus represents a target for tumor diagnosis and therapy. In this study, we assessed L1 expression in more than 8000 normal human tissues and different types of tumors, both malignant and non-malignant, and neural and non-neural. MATERIALS AND METHODS Tissue micro-arrays, including a multi-tumor-array of 128 different tumor types, up to 50 samples of each type (approximately 5500 different samples), arrays with approximately 3000 different prostate and 600 mesenchymal tumor samples, and a normal human tissue-array were analyzed by immunohistochemistry with a monoclonal antibody using immunoperoxidase staining. RESULTS L1 expression was detected in tumors of neural and neural crest origin and other types of non-neural tumors, but not in those of epithelial origin. In normal human tissues, L1 was detected in skin basal cells and small blood vessels, most notably in the mature placenta and peripheral nerves. CONCLUSION This first comprehensive study of the importance of L1 expression in human demonstrates strong L1 overexpression in tumors of neuroectodermal and neural crest origin and an expression in only very few normal human tissues. L1 thus is a potentially important therapeutic target, particularly with respect to malignant melanoma, gastrointestinal stromal tumor, neuroblastoma, and certain subtypes of non-neural tumors.

ErbB2 signaling activates the Hedgehog pathway via PI3K–Akt in human esophageal adenocarcinoma: Identification of novel targets for concerted therapy concepts

The Hedgehog pathway plays an important role in the pathogenesis of several tumor types, including esophageal cancer. In our study, we show an expression of the ligand Indian hedgehog (Ihh) and its downstream mediator Gli-1 in primary resected adenocarcinoma tissue by immunohistochemistry and quantitative PCR in fifty percent of the cases, while matching healthy esophagus mucosa was negative for both proteins. Moreover, a functionally important regulation of Gli-1 by ErbB2-PI3K-mTORC signaling as well as a Gli-1-dependent regulation of Ihh in the ErbB2 amplified esophageal adenocarcinoma cell line OE19 was observed. Treatment of OE19 cells with the Her2 antibody trastuzumab, the PI3K-mTORC1 inhibitor NVP BEZ235 (BEZ235) or the knockdown of Akt1 resulted in a downregulation of Gli-1 and Ihh as well as in a reduction of viable OE19 cells in vitro. Interestingly, the Hedgehog receptor Smo, which acts upstream of Gli-1, was not expressed in OE19 cells and in the majority of primary human esophageal adenocarcinoma, suggesting a non-canonical upregulation of Gli-1 expression by the ErbB2-PI3K axis. To translate our findings into a therapeutic concept, we targeted ErbB2-PI3K-mTORC1 by trastuzumab and BEZ235, combining both compounds with the Gli-1/2 inhibitor GANT61. The triple combination led to significantly stronger reduction of tumor cell viability than cisplatinum or each biological alone. Therefore, concomitant blockage of the ErbB2-PI3K pathway and the Hedgehog downstream mediator Gli-1 may provide a new therapeutic strategy for esophageal cancer.

Carbonic anhydrase IX correlates with survival and is a potential therapeutic target for neuroblastoma

Abstract Carbonic anhydrase IX (CAIX) is involved in pathological processes including tumorgenicity, metastases and poor survival in solid tumors. Twenty-two neuroblastoma samples of patients who were surgically treated at the University Medical Center Hamburg-Eppendorf were evaluated immunohistochemically for expression of CAIX. Results were correlated with clinical parameters and outcome. Neuroblastoma Kelly and SH-EP-Tet-21/N cells were examined for CAIX expression and inhibited with specific inhibitors, FC5-207A and FC8-325A. 32% of neuroblastoma tumors expressed CAIX. This was significantly associated with poorer survival. Kelly and SH-EP-Tet-21/N cells showed a major increase of CAIX RNA under hypoxic conditions. Proliferation of Kelly cells was significantly decreased by CAIX inhibitors, FC5-207A and FC8-325A, while proliferation of SH-EP-Tet-21/N cells was only significantly affected by FC8-325A. CAIX is a potent biomarker that predicts survival in neuroblastoma patients. CAIX-targeted therapy in neuroblastoma cell lines is highly effective and strengthens the potential of CAIX as a clinical therapeutic target in a selected patient collective.

PGK1 as predictor of CXCR4 expression, bone marrow metastases and survival in neuroblastoma.

Background and Aim A close relationship between phosphoglycerate kinase 1 (PGK1) and the CXCR4/SDF1 axis (chemokine receptor 4/stromal cell derived factor 1) has been shown for several cancers. However, the role of PGK1 has not been investigated for neuroblastoma, and PGK1 might be a therapeutic target for this tumor entity. The aim of the current study was to evaluate the role of PGK1 expression in neuroblastoma patients, to determine the impact of PGK1 expression levels on survival, and to correlate PGK1 expression with CXCR4 expression and bone marrow dissemination. Materials and Methods Samples from 22 patients with neuroblastoma that were surgically treated at the University Medical Center Hamburg-Eppendorf were evaluated for expression of PGK1 and CXCR4 using immunohistochemistry. Results were correlated with clinical parameters, metastases and outcome of patients. Immunocytochemistry, proliferation and expression analysis of CXCR4 and PGK1 were performed in neuroblastoma cell lines. Results PGK1 is expressed in neuroblastoma cells. PGK1 expression is significantly positively correlated with CXCR4 expression and tumor dissemination to the bone marrow. Moreover the expression of PGK1 is significantly associated with a negative impact on survival in patients with neuroblastoma. PGK1 is downregulated by inhibition of CXCR4 in neuroblastoma cells. Conclusion PGK1 appears to play an important role for neuroblastoma, predicting survival and tumor dissemination. Further in vivo studies outstanding, it is a candidate target for novel therapeutic strategies.

Orthotopic models of esophageal carcinoma and their use in drug discovery.

The protocol detailed in this unit is for the establishment of an orthotopic model of human esophageal adenocarcinoma in NMRI/nu mice. The resultant tumor has high metastatic potential, spreading readily to liver, lungs, and lymph nodes. This model is useful for studying primary esophageal carcinoma, tumor biology, pathogenesis, tumor progression, metastatic homing, and the efficacy of therapeutic approaches for treating this condition. The practical use of this preclinical model for drug discovery is illustrated with data from a study on the chemotherapeutic effects of HER2‐targeted therapy. Curr. Protoc. Pharmacol. 54:14.20.1‐14.20.15.

Adhesion molecule L1 is down-regulated in malignant peripheral nerve sheath tumors versus benign neurofibromatosis type 1–associated tumors

Type 1 neurofibromatosis (NF-1), also known as von Recklinghausen disease, is caused by a disorder of a single gene on chromosome 17 that usually restrains cell division. A sequence that is frequently associated with NF-1 is tumor progression from neurofibromas to malignant peripheral nerve sheath tumors (MPNSTs). The aim of this study was to determine the expression of the neural L1 cell adhesion molecule in dermal-diffuse neurofibromas, plexiform neurofibromas, and MPNSTs of NF-1. We retrospectively analyzed surgically resected primary tumors, including 20 dermal neurofibromas, 23 plexiform neurofibromas, and 17 MPNSTs, by immunohistochemistry in paraffin sections of NF-1 tumors with the use of the L1-specific monoclonal antibody UJ127, which does not cross-react with other members of the L1 family. Immunostainings for CD34 and S100 were included to distinguish and allocate L1-expressing Schwann cells and perineural (specialized) fibroblasts. Our data showed that L1 is highly expressed in all benign NF-1 tumors and in some but not all MPNSTs. Furthermore, we demonstrated a correlation between L1 expression and differentiation grade of MPNSTs. There was a significant trend toward lower or nondetectable expression in the poorly differentiated MPNSTs, in contrast to all other tumor entities so far investigated, in which L1 expression correlated positive with malignancy, except for juvenile but not adult-derived neuroblastomas. Future studies are warranted to elucidate the molecular basis of the varying effects of the degree of L1 expression, receptor, and signal transduction mechanisms in different tumors.