Computational Analysis Of Multiplexed Immunohistochemistry For Understanding Immune Profiles In Clinical Biopsies

Using immunohistochemistry (IHC) to examine immune infiltrates in tissue biopsies to support exploratory investigations for immuno-oncology drug development is critical for understanding the abundance and spatial relationships of different immune cell types and how they may change with drug treatment. Multiplex IHC approaches are utilized to examine these types of pharmacodynamic responses because of the limited tissue available from repeat needle core biopsies and the need to visualize multiple biomarkers in the same tissue section. While several different methods of fluorescent multiplexing approaches exist, the complexity of these fluorescent assays limits the ability to develop and validate bespoke assays to meet the needs of hypothesis-driven research which aims to elucidate predictors of clinical response. Furthermore, these methodologies present known challenges for design control processes and regulatory approval as companion diagnostics, preventing wide use beyond exploratory research settings. In contrast, development and analytical validation of chromogenic IHC assays enables an agile and bespoke approach to IHC assay development which can support all phases drug development, including direct translations of these methods into companion diagnostic building approaches. However, investigating multiple biomarkers using chromogenic assays presents its own challenges, as there is a more limited repertoire of chromagens than fluorophores, which have significant spectral overlap in wavelength absorbance and optical density. As such, a multiplex chromogenic IHC assay requires specialized performance specifications as well as sophisticated interpretation methods to ensure accurate interpretations. While these limitations often create too great a challenge for pathologists to interpret, computational analysis of tissue using Flagship9s cTA™ platform resolves these challenges and enables drug developers to rely on chromogenic IHC approaches to meet the needs of drug and diagnostic development. To demonstrate the application of this method, we developed several chromogenic multiplex IHC assays which capture critical immune profiles for immuno-oncology studies and interpreted the endpoints using Flagship9s cTA™ approach. These assays/interpretations provide quantitative data about: 1) General immune profile (CD8/CD68/FoxP3); 2) T-cell ratio (CD3/CD8); 3)T-cell activation (CD8/Ki67); and 4) T-cell suppression (CD8/FoxP3). Using this approach, we demonstrate how Flagship9s cTA™ platform enables investigation of complex biological questions using chromogenic assays, which cannot be achieved by relying on traditional manual pathology interpretation. Citation Format: Joseph S. Krueger, Brooke Hirsch, Mandy Bell, Karen Ryall, Nick Landis, G David Young, Kile McFadden, Ben Landis. Computational analysis of multiplexed immunohistochemistry for understanding immune profiles in clinical biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1694.