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. 2020 May;8(1):e000155.
doi: 10.1136/jitc-2019-000155.

The Society for Immunotherapy of Cancer statement on best practices for multiplex immunohistochemistry (IHC) and immunofluorescence (IF) staining and validation

Janis M Taube  1 , Guray Akturk #  2 , Michael Angelo #  3 , Elizabeth L Engle #  4 , Sacha Gnjatic #  2 , Shirley Greenbaum #  3 , Noah F Greenwald #  3   5 , Cyrus V Hedvat #  6 , Travis J Hollmann #  7 , Jonathan Juco #  8 , Edwin R Parra #  9 , Marlon C Rebelatto #  10 , David L Rimm #  11 , Jaime Rodriguez-Canales #  10 , Kurt A Schalper #  11 , Edward C Stack #  12 , Cláudia S Ferreira  13 , Konstanty Korski  13 , Ana Lako  14   15 , Scott J Rodig  14   15 , Emanuel Schenck  10 , Keith E Steele  10 , Michael J Surace  10 , Michael T Tetzlaff  9   16 , Katharina von Loga  17 , Ignacio I Wistuba  9 , Carlo B Bifulco  18 ; Society for Immunotherapy of Cancer (SITC) Pathology Task Force.
Affiliations

The Society for Immunotherapy of Cancer statement on best practices for multiplex immunohistochemistry (IHC) and immunofluorescence (IF) staining and validation

Janis M Taube et al. J Immunother Cancer. 2020 May.

Erratum in

Abstract

Objectives: The interaction between the immune system and tumor cells is an important feature for the prognosis and treatment of cancer. Multiplex immunohistochemistry (mIHC) and multiplex immunofluorescence (mIF) analyses are emerging technologies that can be used to help quantify immune cell subsets, their functional state, and their spatial arrangement within the tumor microenvironment.

Methods: The Society for Immunotherapy of Cancer (SITC) convened a task force of pathologists and laboratory leaders from academic centers as well as experts from pharmaceutical and diagnostic companies to develop best practice guidelines for the optimization and validation of mIHC/mIF assays across platforms.

Results: Representative outputs and the advantages and disadvantages of mIHC/mIF approaches, such as multiplexed chromogenic IHC, multiplexed immunohistochemical consecutive staining on single slide, mIF (including multispectral approaches), tissue-based mass spectrometry, and digital spatial profiling are discussed.

Conclusions: mIHC/mIF technologies are becoming standard tools for biomarker studies and are likely to enter routine clinical practice in the near future. Careful assay optimization and validation will help ensure outputs are robust and comparable across laboratories as well as potentially across mIHC/mIF platforms. Quantitative image analysis of mIHC/mIF output and data management considerations will be addressed in a complementary manuscript from this task force.

Keywords: image analysis; immunology; oncology; tumors.

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Conflict of interest statement

Competing interests: JMT has served on an advisory board, consulting and received research funding from Bristol-Myers Squibb; is a consultant and an advisory board member for Merck and Co., Inc., Kenilworth, New Jersey, USA, Amgen and AstraZeneca. MA is a founder, consultant and board member of IONpath. SG (Gnjatic) is a co-inventor on an issued patent that is filed through Icahn School of Medicine at Mount Sinai (ISMMS) and non-exclusively licensed to Caprion; reports consultancy/advisory fees from Merck and Co., Inc., Kenilworth, New Jersey, USA, Neon Therapeutics and OncoMed Pharmaceutical; and has received research funding from Agenus, Bristol-Myers Squibb, Genentech, Immune Design, Janssen R&D, Pfizer, Regeneron and Takeda Pharmaceutical. CVH is a stockholder of Bristol-Myers Squibb. TJH has received research support from Bristol-Myers Squibb and General Electric. JJ is an employee of Merck Sharp & Dohme, a subsidiary of Merck & Co., Inc., Kenilworth, New Jersey, USA and stockowner of Merck & Co., Inc., Kenilworth, New Jersey, USA; and a stockholder of Regeneron Pharmaceuticals, Illumina and Google. MCR is an employee and stockholder of AstraZeneca. DLR is a consultant, advisor, or serves on a scientific advisory board for Amgen, AstraZeneca, Biocept, Bristol-Myers Squibb, Cell Signaling Technology, Cepheid, Daiichi Sankyo, GlaxoSmithKline, Invicro Konica Minolta, Merck and Co., Inc., Kenilworth, New Jersey, USA, NanoString, PerkinElmer, Ultivue and Ventana; is a founder and equity holder of PixelGear; has received research support or instrument support from AstraZeneca, Cepheid, Eli Lilly, Navigate BioPharma/Novartis, NextCure, Ultivue, Akoya Biosciences/PerkinElmer, NanoString and Ventana; and has received royalty from Rarecyte. JRC is an employee and unvested stock options holder of AstraZeneca. KAS has received consultant honoraria from Clinica Alemana de Santiago, Celgene, Moderna Therapeutics, Pierre-Fabre Research Institute, Shattuck Labs, AbbVie, AstraZeneca, EMD Serono, Ono Pharmaceuticals, Dynamo Therapeutics and Takeda Pharmaceutical; speaker honoraria from Merck & Co., Inc., Kenilworth, New Jersey, USA, Takeda Pharmaceutical, The PeerView Institute and Fluidigm; and research funding from Genoptix/Navigate BioPharma (Novartis), Tioma Therapeutics (formerly Vasculox), Tesaro, Moderna, Takeda Pharmaceutical, Surface Oncology, Pierre-Fabre Research Institute, Merck & Co., Inc., Kenilworth, New Jersey, USA, Bristol-Myers Squibb, AstraZeneca and Eli Lilly. ECS is an employee of Jounce Therapeutics. CSF is an employee of Roche Diagnostics GmbH. KK is an employee and shareholder of Roche. SJR receives research grant support from Bristol-Myers Squibb, Merck and Co., Inc., Kenilworth, New Jersey, USA, Kite/Gilead and Affimed Inc. ES is an employee of AstraZeneca. KES is an employee and stockholder of AstraZeneca; and a spouse of an employee of Arcellx. MJS is an employee and stockholder of AstraZeneca. MTT has served on an advisory board for Myriad Genetics, Seattle Genetics and Novartis; and has presented as a speaker for NanoString. IIW has served on an advisory board for as well as received research support from Genetech/Roche, Bayer AG, Bristol-Myers Squibb, AstraZeneca, Pfizer, HTG Molecular Diagnostics and Merck and Co., Inc., Kenilworth, New Jersey, USA; has served on an advisory board for Asuragen, GlaxoSmithKline, Guardant Health and Merck Sharp & Dohme, a subsidiary of Merck & Co., Inc., Kenilworth, New Jersey, USA; has received research support from DEPArray/Menarini Silicon Biosystems, Adaptive Biotechnologies, Adaptimmune, EMD Serono, Takeda Pharmaceutical, Amgen, Karus Therapeutics, Johnson & Johnson, Iovance Biotherapeutics, 4D pharma, Novartis, Akoya Biosciences; and has presented as a speaker for Genetech/Roche, Pfizer and Merck Sharp & Dohme, a subsidiary of Merck & Co., Inc., Kenilworth, New Jersey, USA. CBB has served on the scientific advisory boards at Bristol-Myers Squibb, Roche, PrimeVax and Halio DX; is a stockholder of PrimeVax and holds the patent US20180322632A1.

Figures

Figure 1
Figure 1
Light microscopy multiplex chromogenic immunohistochemistry staining. Representative image of triplex FOXP3/CD8/KRT staining with purple (Discovery HRP, Ventana, Roche Tissue Diagnostics), yellow (Discovery AP, Ventana, Roche Tissue Diagnostics) and teal (Discovery HRP, Ventana, Roche Tissue Diagnostics) chromogens and a hematoxylin counterstain.
Figure 2
Figure 2
Light microscopy multiplexed immunohistochemical consecutive staining on single slide (MICSSS). (A) As the name suggests, MICSSS uses iterative cycles of tagging, image scanning, and destaining of chromogenic substrate on a single slide to generate a multiplex image. (B) Representative triple negative breast cancer tissue specimen stained FOXP3, DC-LAMP, CD163, CD20, CD8 and CD3. Upper panels show each individual chromogenic stain. The lower panel shows the resultant composite image that has been pseudo-colored for fluorescence. HIER, Heat-induced epitope retrieval; HRP, horseradish peroxidase; QC, quality control.
Figure 3
Figure 3
Imaging area varies by the multiplex immunohistochemistry (mIHC)/multiplex immunofluorescence (mIF) approach. Representative slide from a malignant melanoma showing the relative area of regions of interest (ROIs) acquired for analysis by each technology. It is possible to acquire adjacent ROIs such that the entire tumor is profiled using mIF, digital spatial profiling (DSP), or mass spectrometry. This latter approach currently requires consideration for acquisition time, data management, and analysis. For example, chromogenic IHC with light microscopy requires approximately 2–4 min to acquire a whole slide image, while each ROI for mass spectrometry requires 15–120 min, depending on the platform used and desired resolution (see online supplementary table 1). MICSSS, multiplexed immunohistochemical consecutive staining on single slide.
Figure 4
Figure 4
Multiplex immunofluorescence (IF) using tyramide signal amplification (TSA)-based detection methods and multispectral imaging. Representative non-small cell lung carcinoma stained with six markers (cytokeratin (CK), programmed death-ligand 1 (PD-L1), programmed cell death protein-1 (PD-1), CD68, CD8, FOXP3). The image acquisition of all markers occurs simultaneously. Individual markers (or select combinations of markers) can then be displayed.
Figure 5
Figure 5
Tissue-based mass spectrometry. Representative images from a 40-marker panel applied to human decidua and acquired using multiplexed ion beam imaging by time of flight. Six-color overlay (top left) and enlarged two-color insets (border) of a representative sampling of the simultaneously acquired markers.
Figure 6
Figure 6
Digital spatial profiling. (A) A multiplex immunofluorescence image is first used to create molecular compartments. (B) The molecular compartments (green=CK, yellow=CD45, blue=CD68) are used to guide the UV laser and subsequent sipping process in this representative spot on a non-small cell lung carcinoma tissue microarray (TMA). (C) The amount of signal for a given marker is then assessed within a given compartment. Shown here is normalized CD8 signal in the CD45 compartment (blue) and the remainder of the tissue, that is, non-CD45 compartment, (red) by tumor tissue spot number on the TMA.
See this image and copyright information in PMC

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