Immune contexture analysis in immuno‐oncology: applications and challenges of multiplex fluorescent immunohistochemistry

Abstract

The tumor microenvironment is an integral player in cancer initiation, tumor progression, response and resistance to anticancer therapy. Understanding the complex interactions of tumor immune architecture (referred to as 'immune contexture') has therefore become increasingly desirable to guide our approach to patient selection, clinical trial design, combination therapies, and patient management. Quantitative image analysis based on multiplexed fluorescence immunohistochemistry and deep learning technologies are rapidly developing to enable researchers to interrogate complex information from the tumor microenvironment and find predictive insights into treatment response. Herein, we discuss current developments in multiplexed fluorescence immunohistochemistry for immune contexture analysis, and their application in immuno-oncology, and discuss challenges to effectively use this technology in clinical settings. We also present a multiplexed image analysis workflow to analyse fluorescence multiplexed stained tumor sections using the Vectra Automated Digital Pathology System together with FCS express flow cytometry software. The benefit of this strategy is that the spectral unmixing accurately generates and analyses complex arrays of multiple biomarkers, which can be helpful for diagnosis, risk stratification, and guiding clinical management of oncology patients.

Next-Generation Digital Histopathology of the Tumor Microenvironment

“…Conventional IHC/IF staining techniques are restricted by the number of markers which can be detected at once within one tissue section. This problem was bypassed by staining consecutive tissue sections, with the main limitation being that high-dimensional co-expression analysis is not possible and very precious information is lost [ 14 ]. However, in recent years the ability of multiplexing, in terms of visualizing a high number of markers at one time within a sample, has evolved and thereby represents a powerful tool for investigating complex molecular/functional processes and interactions within cells as well as in the complex native tissue environment.…”

“…Published IF multiplexing techniques include “TSA Opal multiplex immunohistochemistry” (Opal mIHC, PerkinElmer, Waltham, MA, USA) [ 30 ], “in silico multiplexing workflow” [ 31 ], “tissue-based cyclic immunofluorescence” (t-Cycif), MultiOmyx (MxIF) and “multi-epitope-ligand cartography” (MELC) technology as well as DNA barcoding-based techniques such as “CO detection by InDEXing” (CODEX, Akoya Biosciences ,Marlborough, MA, USA) and GeoMx ® (NanoString, Seattle, WA, USA). IF-based methods are much more effective and faster than the IHC-based methods, given that more than one marker can be stained simultaneously in each staining round [ 14 , 31 , 32 , 33 , 34 , 35 ]. The Opal mIHC technique is based on sequential staining rounds, and the secondary antibodies are tagged with tyramide signal amplification system (TSA)-conjugated fluorescence molecules.…”

“…As the name already indicates, these scanners are able to acquire whole slides instead of only individual captures of fields of view, and thereby provide complete composite digitized images of slides in high resolution. The technology used is image acquisition by either tile scanning or line scanning with a follow-up stitching of the images [ 14 , 40 ]. Depending on the specific next-generation digital pathology platform configuration, these scanners are able to perform whole slide imaging in different imaging modes such as brightfield, widefield fluorescence, confocal, structured illumination, multiplexing, and/or multispectral.…”

“…Converting immunohistochemistry (IHC), immunofluorescence (IF), or RNA in situ hybridization (RNA ISH) stained markers within tissues sections into digitized images is a prerequisite, but for pathology to become really “digital” and automated, further processing and extraction of quantitative data, termed as image cytometry, is required. Several commercial systems are available [ 14 ] that offer specialized software solutions utilizing image cytometry, but are methodically focused on the analysis of histological sections and are thus referred to as tissue cytometry [ 15 , 16 ]. Due to the constant evolvement and the increasing reliability of these systems, two commercially available whole slide imagers are approved by the ‘U.S.…”

Multiplexed Immunohistochemistry and Digital Pathology as the Foundation for Next-Generation Pathology in Melanoma: Methodological Comparison and Future Clinical Applications

“…In addition, to identify all the inflammatory subpopulations that are present in a histological sample, the evaluation of more than 20 markers is needed, ideally on the same tissue section (“high-plexing”). Nowadays, several methods for tissue multiplexing are available ( 69 ) and any technique representing a surrogate to investigate co-expression of markers at single cell level should be replaced by multiplexed IHC. First investigated in the context of colorectal cancer ( 70 ), the implementation of the concept of an ‘Immunoscore’ or immunoprofiling into a renewed cancer staging system incorporating the effects of the host immune response based on the numeration of specific lymphocyte populations alongside with the tumor cell-autonomous characteristics has been proven useful in the context of advanced melanoma as well ( 23 ).…”