Validation of multiplex immunofluorescence panels using multispectral microscopy for immune-profiling of formalin-fixed and paraffin-embedded human tumor tissues

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.

Figure 1

Microphotographs of representative examples of validation from IHC (left panels), uniplex IF tumor-associated immune cell expression (middle panels), and details of uniplex IF in tonsil tissue (right panels). Immune panel 1: AE1/AE3 (cytokeratin-positive), PD-L1–positive, helper T cell (CD4-positive), cytotoxic T cell (CD8-positive), T-cell lymphocyte (CD3-positive), and macrophages (CD68-positive). ×200 magnification and high-power magnification of the positive cells.

Figure 2

Microphotographs of representative examples of validation from uniplex IHC (left panels), uniplex IF tumor-associated immune cell expression (middle panels), and details of uniplex IF in tonsil tissue (right panels). Immune panel 2: AE1/AE3 (cytokeratin-positive), PD-1–positive, granzyme B–positive, natural killer cell (CD57-positive), memory T cell (CD45RO-positive), and regulatory T cell (FOXP3-positive). ×200 magnification and high-power magnification of the positive cells.

Figure 3

Microphotographs of representative examples of individual IHC and multiplex IF markers in tonsil tissue. Immune panels 1 and 2. ×200 magnification and high-power magnification of the positive cells.

Figure 4

Microphotographs of representative examples of co-localization of the cell markers observed in panel 1.

Figure 5

Microphotographs of representative examples of co-localization of the cell markers observed in panel 2.