Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

Surace, Michael; Dacosta, Karma; Huntley, Anna; Zhao, Weiguang; Bagnall, Christopher; Brown, Charles O.; Wang, Tao; Roman, Kristin; Cann, Jennifer A.; Lewis, Arthur; Steele, Keith; Rebelatto, Marlon C.; Parra, Edwin R.; Hoyt, Clifford; Rodriguez‐Canales, Jaime

doi:10.3791/58390

Cited by 21 publications

(23 citation statements)

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“…A useful approach to determine antibody/ fluorophore interference or blocking is the drop controls method described by Surace et al, to find which one is causing the interference. 57 Potential corrective actions then include increasing the primary antibody concentration(s), reducing TSA fluorophore concentration(s), and/or changing the order of targets in the panel, among others.…”

Section: Open Accessmentioning

confidence: 99%

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

Taube

Aktürk

Angelo

et al. 2020

J Immunother Cancer

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180

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ObjectivesThe 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.MethodsThe 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.ResultsRepresentative 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.ConclusionsmIHC/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.

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Section: Open Accessmentioning

confidence: 99%

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

Taube

Aktürk

Angelo

et al. 2020

J Immunother Cancer

Self Cite

180

141

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“…This phenomenon is easily detected during the optimization process through comparison with chromogenic IHC or uniplex IF using the same markers and can be corrected by increasing the primary antibody concentrations, reducing TSA fluorophore concentrations, and/or changing the order of targets in the panel. A useful approach when a staining artifact persists is to determine which antibody or fluorophore is causing the interference or blocking using the drop-control method described by Surace and colleagues [9].…”

Section: Staining Interferencesmentioning

confidence: 99%

“…Although various sophisticated multiplex immunohistochemistry (IHC) methods are available for analyzing formalin-fixed, paraffin-embedded (FFPE) material, the current applications have limited scalability and throughput, because even with high levels of multiplexing, the analysis is limited to small regions of interests and/or a limited number of fields of view [8] The immune profiling analysis of tissue samples from FFPE biopsies has become a key tool for understanding the complexity of tumor immunology and discovering novel predictive biomarkers for cancer immunotherapy. Immune profiling analysis of tissues requires the evaluation of combined markers, including inflammatory cell subpopulations and immune checkpoints, in the tumor microenvironment [9].…”

Section: Introductionmentioning

confidence: 99%

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

Parra

Jiang

Solis

et al. 2020

Cancers

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In the development of a multiplex immunofluorescence (IF) platform and the optimization and validation of new multiplex IF panels using a tyramide signal amplification system, several technical requirements are important for high-quality staining, analysis, and results. The aim of this review is to discuss the basic requirements for performing multiplex IF tyramide signal amplification (TSA) in formalin-fixed, paraffin-embedded cancer tissues to support translational oncology research. Our laboratory has stained approximately 4000 formalin-fixed, paraffin-embedded tumor samples using the multiplex IF TSA system for immune profiling of several labeled biomarkers in a single slide to elucidate cancer biology at a protein level and identify therapeutic targets and biomarkers. By analyzing several proteins in thousands of cells on a single slide, this technique provides a systems-level view of various processes in various tumor tissues. Although this technology shows high flexibility in cancer studies, it presents several challenges when applied to study different histology cancers. Our experience shows that adequate antibody validation, staining optimization, analysis strategies, and data generation are important steps for generating quality results. Tissue management, fixation procedures, storage, and cutting can also affect the results of the assay and must be standardized. Overall, this method is reliable for supporting translational research given a precise, step-by-step approach.

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“…Methods utilizing multiplex immunofluorescence to identify and quantify immune cell subsets in tumor specimens are being actively investigated as potential predictors of response to ICB (Surace et al 2019). These methods may preserve the spatial localization of lymphocytes within tumors and allow for multiparameter characterization of protein expression and precise cell counting; they have been characterized by some investigators as flow on a slide.…”

Section: Multiplex Immunofluorescencementioning

confidence: 99%

Biomarkers for Response to Immune Checkpoint Blockade

Ganesan

Mehnert²

2020

Annu. Rev. Cancer Biol.

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Immune checkpoint blockade (ICB) has significant clinical activity in diverse cancer classes and can induce durable remissions in even refractory advanced disease. However, only a minority of cancer patients treated with ICB have long-term benefits, and ICB treatment is associated with significant, potentially life-threatening, autoimmune side effects. There is a great need to develop biomarkers of response to guide patient selection to maximize the chance of benefit and prevent unnecessary toxicity, and current biomarkers do not have optimal positive or negative predictive value. A variety of potential biomarkers are currently being developed, including those based on assessment of checkpoint protein expression, evaluation of tumor-intrinsic features including mutation burden and viral infection, evaluation of features of the tumor immune microenvironment including nature of immune cell infiltration, and features of the host such as composition of the gut microbiome. Better understanding of the underlying fundamental mechanisms of immune response and resistance to ICB, along with the use of complementary assays that interrogate distinct features of the tumor, the tumor microenvironment, and host immune system, will allow more precise use of these therapies to optimize patient outcomes.

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Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

Cited by 21 publications

References 0 publications

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

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

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

Biomarkers for Response to Immune Checkpoint Blockade

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