Computational Analysis of Multiplexed Immunohistochemistry for Understanding Immune Profiles in Clinical Biopsy Samples


Using immunohistochemistry (IHC) to examine immune infiltrates in tissue biopsy samples 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 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, the development and analytical validation of chromogenic IHC assays enables an agile and bespoke approach to IHC assay development that can support all phases of drug development, including direct translations of these methods into companion diagnostic building approaches. Investigating multiple biomarkers using chromogenic assays presents its own challenges, however, as there is a more limited repertoire of chromogens than fluorophores, which have more 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 pathologist interpretation, computational analysis of tissue using Flagship’s cTA® platform resolves these challenges and enables drug developers to rely on chromogenic IHC approaches to meet the needs of drug and diagnostic development.


Multiparametric cTA profiles can capture key immune response phenotypes by:

  • Utilizing multiplexed IHC images to profile pharmacodynamic changes of immune content and context in tumor biopsy samples.
  • Combining multiple measurements to identify specific immune phenotypes.
  • Incorporating spatial relationships of different cell types to represent biological behaviors of a patient’s tumor that are key for predicting a response.

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