Abstract 3389: A multiplexed immunohistochemistry test to screen for protein overexpression of ROS1, TrkA, TrkB and TrkC in multiple tumor tissue types
Authors: Boomer Aaron , Fernandez Diane , Murphy Danielle , Christiansen Jason , Lamoureux Jennifer 
Published: August 2015

Abstract
Introduction: A consequence of precision medicine is the increased challenge to identify small patient populations, while maintaining testing efficiency and manageable costs. One area of focus is identifying gene rearrangements in patients for targeted kinase inhibitor therapies, where the prevalence in a patient population can be ∼1-10%. Diagnostic detection of gene rearrangements is primarily accomplished by fluorescence in situ hybridization (FISH), while next generation sequencing (NGS) or immunohistochemistry (IHC) show promise. FISH does not provide information regarding the gene fusion partner, while NGS is expensive and labor intensive. IHC is both high throughput and cost efficient, but not specific to rearrangements. To enable rapid screening of large tissue populations for prevalence, we sought to develop a method that could be broadly applied to multiple receptor tyrosine kinases simultaneously to streamline screening for multi-targeted therapeutics. To this end, we have developed a multiplexed IHC method that assesses expression of the clinically promising markers: ROS1, TrkA, TrkB, and TrkC in multiple tumor tissues. Specimens selected positive by this pan-receptor IHC assay can then be reflexed to a method with higher sensitivity and specificity for gene rearrangements (e.g. NGS).
Methods: Standard IHC was conducted as follows: Antigen retrieval was performed in EDTA (pH 9) at 98°C for 30 min. The primary antibody cocktail consists of antibodies against ROS1 (D4D6, Cell Signaling) and a pan-Trk antibody (C17F1, Cell Signaling). A signal amplification step was performed before the secondary antibody conjugated to a dextran-HRP polymer (Envision FLEX, Dako) step. Signal was developed using DAB and counterstained with hematoxylin.
Results: We have developed an assay for measuring expression of multiple receptor tyrosine kinases in multiple tissue types using a cocktail of antibodies that is sensitive, accurate, and with minimal background or cross-reactive staining. This has been demonstrated using characterized cell lines and human tissues with results confirmed by testing individual IHC markers and alternative methods (e.g. FISH or NGS). Since IHC measurements are assessing in-frame expression, we can also identify false positive samples such as FISH detected rearrangements which do not lead to active protein product.
Conclusions: We have demonstrated a simple, sensitive, multiplexed IHC assay to screen for molecular alterations (ROS1, TrkA/B/C) that are currently not part of standard tissue annotation. We have shown that the assay is able to identify molecular alterations that have been confirmed by sequencing. The test is expandable and can be adjusted to suit the needs of researchers studying specific protein families.
Citation Format: Aaron Boomer, Diane Fernandez, Danielle Murphy, Jason Christiansen, Jennifer Lamoureux. A multiplexed immunohistochemistry test to screen for protein overexpression of ROS1, TrkA, TrkB and TrkC in multiple tumor tissue types. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3389. doi:10.1158/1538-7445.AM2015-3389