Abstract LB-A06: Analysis of gene expression and protein expression of the AXL receptor in clinical biospecimen

Abstract

Abstract Introduction It has become obvious that targeted therapy of patients has to be monitored by taking and analyzing biopsies on a regular basis. Therefore, laboratory methods have to be optimized to be able to handle minute amounts of clinical biospecimen, e.g. biopsies. In this study we are presenting the development of a Simple Western Size assay and a quantitative Real-time PCR assay for the detection and analysis of AXL protein and gene expression in biopsies. Background Targeted therapy in personalized medicine is often affected by resistance mechanisms, such as activating mutations of signaling molecules and signaling pathway bypasses. AXL, a tyrosine kinase receptor, is expressed in a variety of cancers and has been revealed as the most highly expressed gene in preclinical models with acquired resistance, and second most common alteration in EGFR (epidermal growth factor receptor) inhibitor-resistant tumors, behind the T790M mutation. Up-regulation and/or activation of AXL are shown to be predictive for lack of response to ErbB family receptor-targeted inhibitors, e.g. to Her2-targeted agents. Methods Protein expression was examined by Simple Western Size technology and gene expression was analyzed using quantitative Real-time PCR. For assay development, high and low AXL expressing breast cancer cell lines were used to establish “fit for purpose” assays. Subsequently, Her2-positive and Her2-negative samples from breast cancer patients were analyzed by screening for AXL gene and protein expression. Furthermore, correlations of gene and protein expression were conducted. Results In this study, we developed and validated an assay for the detection of AXL protein expression using the Simple Western technology. This highly sensitive technology enables high-throughput screening of extremely small sample amounts such as biopsies or laser capture microdissected material. Our results showed a broad linear dynamic range of AXL protein in both, high and low AXL expressing cell lines as well as a high reproducibility between multiple runs. The protein loading range suitable to detect AXL within whole cell lysates showed detection levels of AXL protein down to 10 ng of total protein. AXL protein expression results were highly comparable to AXL gene expression results in analyzed breast cancer cell lines and breast cancer patient samples. This newly developed assay will allow us to analyze and quantify AXL protein expression profiles in breast cancer tissue and subsequently correlate them with e.g. Her-2 expression status. Conclusion The elucidation of networks and mechanisms underlying drug resistance will greatly improve the development of new compounds and promote personalized therapy. In order to gain scientific knowledge regarding gene and protein expression of resistance relevant marker, e.g. AXL, it is mandatory to establish assays that are very robust, sensitive and furthermore, if not most important, applicable to biospecimen being collected in the clinical setting. Citation Format: Kristina Bernoth, Florian T. Unger, Nicole Lange, Hartmut Juhl, Kerstin A. David. Analysis of gene expression and protein expression of the AXL receptor in clinical biospecimen. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A06.