Multiplexed Immunohistochemistry for Molecular and Immune Profiling in Lung Cancer—Just About Ready for Prime-Time?

Abstract

As targeted molecular therapies and immuno-oncology have become pivotal in the management of patients with lung cancer, the essential requirement for high throughput analyses and clinical validation of biomarkers has become even more intense, with response rates maintained in the 20%–30% range. Moreover, the list of treatment alternatives, including combination therapies, is rapidly evolving. The molecular profiling and specific tumor-associated immune contexture may be predictive of response or resistance to these therapeutic strategies. Multiplexed immunohistochemistry is an effective and proficient approach to simultaneously identify specific proteins or molecular abnormalities, to determine the spatial distribution and activation state of immune cells, as well as the presence of immunoactive molecular expression. This method is highly advantageous for investigating immune evasion mechanisms and discovering potential biomarkers to assess mechanisms of action and to predict response to a given treatment. This review provides views on the current technological status and evidence for clinical applications of multiplexing and how it could be applied to optimize clinical management of patients with lung cancer.

Procedural Requirements and Recommendations for Multiplex Immunofluorescence Tyramide Signal Amplification Assays to Support Translational Oncology Studies

“…In the field of multiplex IF, the use of scanners represents a major technological advance that enables the use of multiple sometimes unstable fluorochromes and thus more than seven different antibodies on the same slide [16]. A Vectra or Vectra Polaris scanner allows extremely precise imaging in brightfield or fluorescence detection with high resolution by combining multiband filter cubes, which increase the flexibility of the multispectral camera [39].…”

“…Areas of disinterest, such as necrosis, tissue artifacts, and staining artifacts, must be excluded with the Detection and phenotypic characterization of cells in ROIs require both the scanner system and image analysis software, such as the inForm software (Akoya/PerkinElmer). Image analysis software allows automatic analysis of parameters that cannot be accurately discerned by the human eye (e.g., cell forms, multiple cell phenotype networks, and vascular network) [16]. InForm in particular includes a user-trainable algorithm for tissue analysis based on morphology as well as specified markers.…”

“…When the training for each marker is complete and saved, we can apply the algorithm of each marker in a set of cases to carefully verify that the algorithms are working properly in our samples. The education of the software may be time-consuming, with a phase of learning or "teaching" [16]. Using this approach, the optical signal from each biomarker in the spectral unmixing image can be isolated, assessed separately, and quantified [42] to integrate the data at the end of the process, using R studio software with the phenoptr program (Akoya/PerkinElmer).…”

“…This technology therefore has an important role in translational oncology research [11][12][13][14][15]. Thus, multiplexed IF can help characterize the topography of immune cells in cancer in detail, including the relative localizations and interactions of marker expression on cancer cells, immune cells, stromal cells, and endothelial cells, furthering our understanding of the disease [8,16]. This approach can reproducibly quantify multiple protein levels and functional activities in intact tissue specimens.…”

The Cancer Immunotherapy Biomarker Testing Landscape

“…144 A detailed overview of the currently available IHC multiplexing methods and technologies is beyond the scope of this article, but can be found in recently published reviews. 145,146 The historic challenge of creating a multiplexed indirect IHC assay that avoids multiple species of primary antibodies and cross-reactivity between same-species primary antibodies has largely been solved by elegant chemical and heat deactivation reactions. 145 Chromogenic multiplexing allows light microscopy visualization, with novel methods such as multiplexed immunohistochemical consecutive staining on a single slide and reagents such as narrow-band tyramineconjugated dyes demonstrating the feasibility of medium-to high-dimensional analysis.…”