Abstract
Immune-profiling is becoming an important tool to identify predictive markers for the response to immunotherapy. Our goal was to validate multiplex immunofluorescence (mIF) panels to apply to formalin-fixed and paraffin-embedded tissues using a set of immune marker antibodies, with the Opal™ 7 color Kit (PerkinElmer) in the same tissue section. We validated and we described two panels aiming to characterize the expression of PD-L1, PD-1, and subsets of tumor associated immune cells. Panel 1 included pancytokeratin (AE1/AE3), PD-L1, CD4, CD8, CD3, CD68, and DAPI, and Panel 2 included pancytokeratin, PD-1, CD45RO, granzyme B, CD57, FOXP3, and DAPI. After all primary antibodies were tested in positive and negative controls by immunohistochemistry and uniplex IF, panels were developed and simultaneous marker expressions were quantified using the Vectra 3.0™ multispectral microscopy and image analysis InForm™ 2.2.1 software (PerkinElmer).These two mIF panels demonstrated specific co-localization in different cells that can identify the expression of PD-L1 in malignant cells and macrophages, and different T-cell subpopulations. This mIF methodology can be an invaluable tool for tumor tissue immune-profiling to allow multiple targets in the same tissue section and we provide that is accurate and reproducible method when is performed carefully under pathologist supervision.
Highlights
Novel and effective immunotherapies for patients with various types of cancer are becoming a clinical reality, in part because of the remarkable clinical efficacy observed with immune checkpoint inhibitors such as programmed cell death protein 1 (PD-1, a T-cell co-inhibitory receptor) and one of this protein’s ligands, programmed cell death ligand 1 (PD-L1, known as B7-H1 or CD274)[1,2,3,4,5,6,7,8,9,10,11,12]
Our goal was to validate multiplex immunofluorescence (mIF) panels in the same tissue section to apply to formalin-fixed and paraffin-embedded (FFPE) carcinoma tissues using a set of immune marker antibodies, including those against PD-L1 and Tumor-associated immune cells (TAICs), multispectral microscopy and image analysis software
We validated mIF panels using the Opal workflow in the same tissue section to a set of immune marker antibodies, including those against PD-L1 and TAICs, to apply to FFPE tissues. We applied those panels in carcinoma tissues to compare and quantify the expression of those markers using mIF and conventional chromogenic IHC
Summary
Novel and effective immunotherapies for patients with various types of cancer are becoming a clinical reality, in part because of the remarkable clinical efficacy observed with immune checkpoint inhibitors such as programmed cell death protein 1 (PD-1, a T-cell co-inhibitory receptor) and one of this protein’s ligands, programmed cell death ligand 1 (PD-L1, known as B7-H1 or CD274)[1,2,3,4,5,6,7,8,9,10,11,12]. Characterization of the tumor microenvironment in patients with cancer has become a fundamental step in discovering evidence for the presence of distinct immunologic phenotypes, based on the presence or absence of various immune cells[1,13,14] These observations have generated candidate predictive biomarkers that can respond to immunotherapies and are guiding the identification of new immunotherapeutic interventions[15]. Our goal was to validate mIF panels in the same tissue section to apply to FFPE carcinoma tissues using a set of immune marker antibodies, including those against PD-L1 and TAICs, multispectral microscopy and image analysis software
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