Imaging, Diagnosis, Prognosis AxlandGrowthArrest^SpecificGene6AreFrequently Overexpressed in Human Gliomas and Predict Poor Prognosis in Patients with Glioblastoma Multiforme Markus Hutterer,1, 5 Pjotr Knyazev,5 ArianeAbate,1Markus Reschke,5 Hans Maier,2 Nadia Stefanova,1 Tatjana Knyazeva,5 Verena Barbieri,4 Markus Reindl,1Armin Muigg,1Herwig Kostron,3 Guenther Stockhammer,1and Axel Ullrich5,6 Abstract Purpose:The receptor tyrosine kinaseAxl has recently been identified as a critical element in the invasive properties of glioma cell lines. However, the effect of Axl and its ligand growth arrest ^ specific gene 6 (Gas6) in human gliomas is still unknown. Experimental Design: Axl and Gas6 expression was studied in 42 fresh-frozen and 79 paraffin- embedded glioma specimens by means of reverse transcription-PCR and immunohistochemistry. The prognostic value of Axl and Gas6 expression was evaluated using a population-based tissue microarray derived from a cohort of 55 glioblastoma multiforme (GBM) patients. Results: Axl and Gas6 were detectable in gliomas of malignancy gradesWHO 2 to 4. Moderate to highAxlmRNAexpressionwasfoundin61%,Axlproteinin55%,Gas6mRNAin81%,andGas6 proteinin74%of GBMsamples,respectively.GBMpatients withhigh Axlexpressionand Axl/Gas6 coexpression showed a significantly shorter time to tumor progression and an association with poorer overall survival. Comparative immunohistochemical studies showed that Axl staining was most pronounced in glioma cells of pseudopalisades and reactive astrocytes. Additionally, Axl/ Gas6 coexpression was observed in glioma cells and tumor vessels. In contrast, Axl staining was not detectable in nonneoplastic brain tissue and Gas6 was strongly expressed in neurons. Conclusions: In human gliomas, Axl and Gas6 are frequently overexpressed in both glioma and vascular cells and predict poor prognosis in GBM patients. Our results indicate that specific targeting of the Axl/Gas6 signaling pathway may represent a potential new approach for glioma treatment. Glioblastoma multiforme (GBM) is the most common and have a minor effect on the natural course of this tumor with a most aggressive primary brain tumor in adults with an mean overall survival (OS) time of only 9 to 15 months from incidence of 5 to 8 per 100,000 inhabitants per year (1, 2). the time of diagnosis (2, 3). Therefore, there is an urgent need Advances in surgery, radiotherapy, and chemotherapy do only for more effective therapeutic approaches based on a better understanding of the molecular mechanisms of GBM growth, invasiveness, and vascularization. Authors’ Affiliations: 1Clinical Department of Neurology, 2Institute of Pathology, The receptor tyrosine kinase Axl is characterized by an 3Clinical Department of Neurosurgery, and 4Department of Medical Statistics, extracellular domain consisting of two immunoglobulin-like Informatics and Health Economics, Innsbruck Medical University, Innsbruck, Austria; domains in juxtaposition to two fibronectin type III domains 5Department of Molecular Biology, Max Planck Institute of Biochemistry, Munich/Martinsried, Germany; and 6Center of Molecular Medicine, Institute of (4, 5), typical for cell adhesion molecules of the immunoglob- Molecular and Cell Biology, Singapore, Singapore ulin superfamily (6). The growth arrest–specific gene 6 (Gas6) Received 4/11/07; revised 9/5/07; accepted 10/8/07. is the natural ligand of Axl (7, 8). Axl/Gas6 signaling has been Grant support: Medizinischer Forschungsfond Tirol grant 120/2005 (G. shown to regulate survival, proliferation, and migration in a Stockhammer) and Austrian Cancer Society, Krebshilfe Tirol grant 23/2005 (G. variety of cells in vitro, including tumor-derived cell lines of Stockhammer). The costs of publication of this article were defrayed in part by the payment of page epithelial, mesenchymal, and hematopoietic origin (5, 9). charges. This article must therefore be hereby marked advertisement in accordance Recently, Axl has been identified to be overexpressed in the with 18 U.S.C. Section 1734 solely to indicate this fact. majority of glioma cell lines and was associated with invasive Note: M. Hutterer and P. Knyazev contributed equally as first authors. A. Ullrich and properties of glioma cell lines in a spheroid as well as in a G. Stockhammer contributed equally as senior authors. The authors confirm the originality of this study and disclose previous reports or xenograft mouse model (10). However, the biological effect of publications. In part, preliminary results of this study were presented at the 7th Axl and its ligand Gas6 in human gliomas is still unknown. Congress of the European Association for Neurooncology, September 14-17, In this study, we investigated the mRNA and protein 2006,Vienna, Austria (poster presentation). expression of Axl and Gas6 in human gliomas WHO grades 2 Requests for reprints: Markus Hutterer, Clinical Department of Neurology, to 4, determined its staining pattern in comparison with non- Innsbruck Medical University, Anichstrasse 35, A-6020 Innsbruck, Austria. Phone: 43-512-504-24365;Fax: 43-512-504-24266;E-mail:[email protected]. neoplastic brain tissue derived from epilepsy surgery, and F 2008 American Association for Cancer Research. compared the Axl and Gas6 protein expression with the clinical doi:10.1158/1078-0432.CCR-07-0862 course of patients with GBM. Clin Cancer Res 2008;14(1) January 1, 2008 130 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2008 American Association for Cancer Research. Axl and Gas6 Expression in Human Glioma A Materials and Methods the cDNA probe was denatured with 10 L 1 N NaOH, pH neutralized with 5 AL 2N HCl and 5 AL 2mol/L Tris-HCl (pH 7.5), and purified using the QIAquick PCR Purification kit (Qiagen) according to the Fresh-frozen tissue. This study was done according to the ethical manufacturer’s instructions. regulative of the institutional review board. During open neurosurgery, PCR. The PCR method was used to determine mRNA expression tissue samples from brain tumor patients were snap frozen in liquid levels of Axl, Gas6, and tubulin (housekeeping gene) in fresh-frozen nitrogen within 3 min of harvesting and stored at -80jC. mRNA glioma samples. The following primer sequences were applied: Axl, expression levels of Axl, Gas6, and tubulin (housekeeping gene) were 5¶-GGTGGCTGTGAAGACGATGA-3¶ (forward) and 5¶-CTCAGATACTC- investigated using reverse transcription-PCR. CATGCCACT-3¶ (reverse); Gas6, 5¶-ACATCTTGCCGTGCGTGCCCTTCA-3¶ Total RNA isolation and cDNA synthesis. Total RNA was isolated (forward) and 5¶-ATTCCGCGCCAGCTCCTCAACAGA-3¶ (reverse); and from 42fresh-frozen glioma specimens using the acid guanidium tubulin,5¶-AAGTGACAAGACCATTGGGGGAGG-3¶ (forward) and 5¶- thiocyanate-phenol-chloroform extraction method (11). After NaCl GGGCATAGTTATTGGCAGCATC-3¶ (reverse). All primers were synthe- acetate treatment to reduce genomic DNA, total RNA was extracted with sized by MWG Biotech. PCR was done using 1 AL cDNA as template in a phenol-chloroform, precipitated with isopropanol, resuspended with  lysis buffer, reprecipitated with isopropanol, washed with 80% ethanol, mastermix containing 10 PCR buffer without MgCl2, 25 mmol/L A and finally solved in TES/diethyl pyrocarbonate solution. Afterwards, MgCl2, 10 mmol/L deoxynucleotide triphosphates, 10 pmol/ L of each the total RNA concentration was determined by absorbance measure- specific primer, and 0.5 IU Taq DNA polymerase (Fermentas Taq ment (260 and 280 nm) and the quality of total RNA was verified by Polymerase kit, Roche) for each sample and carried out in a DNA a 1% agarose gel electrophoresis. Only samples with 28S/18S ratios thermal cycler (Eppendorf). PCR cycling conditions began with an j >2and with no evidence of DNA contamination and RNA degradation initial DNA denaturation step at 94 C for 2min followed by 32cycles j j j were used for continuative cDNA synthesis. cDNA synthesis was done at 94 C for 30 s, 55 C for 30 s, and 72 C for 30 s. Amplified PCR using 5 Ag of total RNA in an oligo(dT) primer mix (1 pmol, total products were applied to a 2% agarose gel for electrophoresis done at volume 9 AL; Chipco), which was heated at 70jC for 3 min and then 80 V for 30 min. To analyze the mRNA expression frequency, reverse cooled on ice. A mastermix containing 5 reverse transcriptase buffer, transcription-PCR results were evaluated semiquantitatively (no, low, 10 mmol/L deoxynucleotide triphosphates, 100 mmol/L DTT, 1 unit moderate, or high). (0.5 AL) RNase inhibitor, and 50 units (2 AL) avian myeloblastosis virus Whole tissue sections and tissue microarray population. To investigate reverse transcriptase (Molecular Diagnostics, Roche) for each cell line the immunohistochemical staining patterns of Axl and Gas6 protein in and frozen tumor sample was added into a tube and subsequently neoplastic and nonneoplastic tissue samples, we applied representative heated at 42jC for 2h. A stop reaction was done with 80 AL Tris-EDTA whole sections of paraffin-embedded glioma tissue WHO grades 2to 4 10/0.1 followed by heating at 72jC for 7 min. The quality of the (30 samples) and nonneoplastic hippocampal tissue derived from cDNA was verified using a 1.5% agarose gel electrophoresis. Afterwards, epilepsy surgery (10 samples). For clinicopathologic correlation studies, Table 1. Clinical characteristics of 76 patients with gliomas included in the population-based TMA Variable Histologic classification (WHO) Grade 4 Grade 3 Grade 2 No. patients 55 6 15 Age,*y (range) 56.5 (18-78) 56.4 (47-69) 41.8 (26-60) z60 24 1 2 <60 31 5 13 Sex Female 20 1 6 Male 35 5 9 KPS,c mean (range) 81.5 (70-100) 85.0 (80-90) 88.7 (80-100) z80 42 6 15 <80 13 0 0 Tumor location Frontal 20 1 9 Nonfrontal 35 5 6 Extend of resectionb x Total and subtotal 26 48 x Partial 26 27 Chemotherapy +4050 -15115 k Radiation +4558 -1017 TTP, mo (range) 7.3 (1.6-17.0) 9.5 (9.4-9.6) 44.1 (6.0-64.0) OS, mo (range) 12.6 (0.8-27.5) 30.2 (10.8-59.5) 72.0 (30.0-102.5) *Age at first diagnosis.