Abstract B109: In-depth tissue analysis using multiplexed immunohistochemical consecutive staining on single slide
Authors: Remark Romain , Merghoub Taha , Damotte Diane , Wolchok Jedd D. , Merad Miriam , Gnjatic Sacha 
Published: May 2016

Abstract
Recent successes of cancer immunotherapy, in particular immune checkpoint blockade, have been largely attributed to factors in the tumor microenvironment (TME). In addition, many studies have found that high lymphocyte infiltration in tumors is prognostic of progression-free or overall survival, while in some cases, myeloid cells rather than lymphocytes are associated with favorable clinical outcome. Importantly, assessment of immune markers usually requires not only quantifying their presence in tumors but also assessing their spatial localization.
To assess spatial distribution of tumor-infiltrating immune cells and suppressive molecules, multidimensional immunohistochemical analyses are poised to become the new standard in pathology. A major limitation for such high-dimensional analyses however is tumor tissue availability and new multiplexed imaging approaches are desperately needed.
Here, we will describe the development of a new multiplexed immunohistochemistry (IHC) method independent of proprietary reagents or equipment to assess a large panel of phenotypical markers that could easily be integrated in routine clinical pathology. This new technique named Multiplexed Immunohistochemical Consecutive Staining on Single Slide (MICSSS) can be performed in any laboratory already conducting traditional IHC with limited cost and no additional instrumentation. The MICSSS method is performed on formalin-fixed paraffin-embedded tissue using iterative cycles of staining, revelation, scanning, and bleaching of chromogenic substrate. It should easily be adaptable to most existing staining protocols with primary antibody staining and secondary antibody detection, thus retaining previously established specificity and sensitivity parameters. The MICSSS method can characterize a large panel of parameters, including marker colocalization on cells, using a single slide while preserving tissue antigenicity and architecture. It is not limited by antibody cross-reactivity, photo-bleaching, or autofluorescence, while still allowing prolonged slide storage for future reuse as new markers become available. Here, we will describe the MICSSS method workflow and show potential applications of multiplex immunostaining including its use to characterize the tumor immune environment and identify prognostic factors or predictive biomarkers of response to CTLA-4 blockade.
In conclusion, the MICSSS method will provide new powerful ways to map the tumor microenvironment with precision, in a sample-sparing manner, to monitor the immune response at the tumor site during therapy and discover new prognostic and predictive markers that might be of major interest for the clinical management of patients with various diseases.
Citation Format: Romain Remark, Taha Merghoub, Diane Damotte, Jedd D. Wolchok, Miriam Merad, Sacha Gnjatic. In-depth tissue analysis using multiplexed immunohistochemical consecutive staining on single slide. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B109.