Abstract 3989: Analysis of minute amounts of clinical biospecimen for the AXL receptor expression using real-time PCR and Simple Western size technology
Authors: Unger Florian T. , Bernoth Kristina , Lange Nicole , Juhl Hartmut , David Kerstin A. 
Published: July 2016

Abstract
Targeted therapy in personalized medicine is often affected by resistance mechanisms, such as activating mutations of signaling molecules and signaling pathway bypasses. It has become obvious that targeted therapy of patients has to be monitored by taking biopsies on a regular basis. Therefore, laboratory methods have to be optimized to be able to handle minute amounts of 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 analysis of AXL protein and gene expression in biopsies.
AXL, a tyrosine kinase receptor, is expressed in a variety of cancers and 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 of AXL is shown to be predictive for lack of response to ErbB family receptor-targeted inhibitors, e.g. to Her2-targeted agents. 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 clinical samples from breast cancer patients were analyzed by screening AXL gene and protein expression.
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. Therefore, the assay is well-suited for the monitoring of patients by using biopsies. Our results showed a broad linear dynamic range 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 a lower limit of detection of 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.
The elucidation of networks and mechanisms underlying drug resistance will greatly improve the development of new drugs 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 amounts of biospecimen being collected in the clinical setting.
Citation Format: Florian T. Unger, Kristina Bernoth, Nicole Lange, Hartmut Juhl, Kerstin A. David. Analysis of minute amounts of clinical biospecimen for the AXL receptor expression using real-time PCR and Simple Western size technology. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3989.