Introduction: Multiplexed analysis of limited tissue samples can improve our understanding of tumor biology and tumor microenvironment. Chromogenic and fluorescent multiplexed immunohistochemistry (IHC) approaches are available and offer great insights while conserving limited tissue however these approaches have their limitations. Multiplexed chromogenic IHC methods can at best accommodate up to 2-3 distinct markers. Fluorescence-based approaches can support higher degree of multiplexing however spectral overlap issues and differences in labeling efficiency/photostability complicate experimental procedures and data interpretation. Imaging mass cytometry system (IMC) has recently emerged as a novel technology for tissue imaging that enables multiplexed analysis protein expression (up to 40 markers) in a single tissue sample while circumventing the limitations of chromogenic and fluorescent IHC techniques. Metal-conjugated antibodies are used to perform qualitative and quantitative analysis of expression of multiple proteins of interest on a single formalin fixed paraffin embedded (FFPE) tissue slide. Here, we compare the performance of the IMC method with conventional, established IHC techniques using a small panel of markers.

Methods: Serial sections of FFPE tonsil and non-small cell lung carcinoma tissues were assessed by monoplex IHC and multiplex IMC for CD3 (Cell Signaling Technology, D7A6E), CD8 (LS Bio, C8/144B), CD68 (Abcam, KP1), PD-L1 (Spring Bio, SP142) and Histone H3 (D1H2). Digital image analysis using Flagship's image analysis software was used to compare performance characteristics of multiplex IMC platform with standard monoplex chromogenic IHC.

Results: The staining patterns of the corresponding biomarkers were similar between IMC and IHC on sequential sections. Digital image analysis demonstrated concordance in the percentage of biomarker positive cells within analyzed matched IHC and IMC areas. It was also demonstrated that cellular image segmentation can be performed on IMC images thus allowing for utilization of various software packages for high dimensional single cell analysis of IMC data.

Conclusion: Comparative digital image analysis indicates that on FFPE tissues multiplexed IMC platform generates data comparable to that obtained from the monoplex chromogenic IHC platform. We believe that an IMC platform is a new tool capable of dramatically enhancing our ability to study biology of cancer using highly multiplexed analysis of limited tissue samples.

Citation Format: Navi Mehra, Carsten Schnatwinkel, Elliott Ergon, Joseph Krueger, Karl Calara-Nielsen, Brad Foulk, Kirti Sharma, Denis Smirnov, Chandra Rao, Tatiana Perova, Rengasamy Boominathan. Comparison of multiplexed imaging mass cytometry in FFPE tissue to monoplex immunohistochemistry [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 3037.