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Multiplex chromogenic immunohistochemistry is a valuable tool that enables characterization and enumeration of immune cells in cancer biopsies. When subcellular localization does not completely overlap between the targets (e.g. one is nuclear, the other is cytoplasmic) or expression on cell types is mutually exclusive (e.g. CD68 and CD3), results can be analyzed rapidly across entire tissue biopsies using image analysis of whole slide scans. Whole slide scan image capture times for chromogenic immunohistochemistry are typically 2-4 minutes on an Aperio AT Turbo vs. >60 minutes for whole slide scans of immunofluorescence methods. For the current study, we developed the following multiplex assays: Ki-67+CD8, CD3+CD4+Ki-67, FoxP3+PD-1+CD8, and CD68+PD-L1+CD3 using brown, red, and green chromogens. Evaluating the density of immune cell subtypes and their relative spatial positioning in cancers has become an important tool in understanding response to immune checkpoint inhibitors. The purpose of this study was to investigate protein expression of PD-L1, PD-1, CD3, CD4, CD8, CD68, FoxP3, and Ki-67 with four multiplex immunohistochemical assays (Ki-67+CD8, CD3+CD4+Ki-67, FoxP3+PD-1+CD8, and CD68+PD-L1+CD3) using DAB, red, and green chromogens within formalin-fixed, paraffin embedded non-small cell lung (NSCLC) tissues. The Ki-67+CD8 multiplex was developed to evaluate the density of cytotoxic T cells and the percentage that are proliferating. The CD3+CD4+Ki- 67 multiplex characterizes the frequency of CD4+ T cells and evaluate proliferation in this subset. The FoxP3+PD-1+CD8 multiplex evaluates the regulatory T cell subset and evaluates PD-1 expression. The CD68+PD-L1+CD3 multiplex characterizes PD-L1 expression on macrophages and T cells. Cell densities were evaluated in the center of tumor and invasive margin regions, and image analysis was performed to quantitate each stain as a single agent and co-expression within each multiplex assay. Multiplex Immunohistochemical Staining of PD-L1, PD-1, CD3, CD4, CD8, CD68, FoxP3, and Ki-67 and Image Analysis of Tumor and Invasive Margin in Human FFPE NSCLC Tissue Lisa M Dauffenbach, Gela C. Sia, Jianping Zheng, Natalia Jun, Eric P. Olsen, Christopher A. Kerfoot Mosaic Laboratories, Lake Forest, CA 92630, (949) 472-8080, www.mosaiclabs.com Abstract Materials and Methods Conclusions Results 1669 Ki-67+CD8 multiplex IHC assay (MOS727-DR) FoxP3+PD-1+CD8 multiplex IHC assay (MOS739-RDG) Tonsil Tonsil Presented at the 2017 AACR Meeting, Washington DC, USA; Monday Apr 3, 2017 8:00 AM - 12:00 PM; Poster Section 28 CD68+PD-L1+CD3 multiplex IHC assay (MOS738-RDG) Tonsil Center of Tumor (CT) Invasive Margin (IM) Five 20X Multispectral Images in CT Region Five 20X Multispectral Images in IM Region Whole Slide Scan, IM and CT Regions Delineated Non- overlapping chromogens (or incomplete overlap) Overlapping chromogens Multiplex Parameters: • Percentage of one cell type positive for another analyte • Density and percentage of cells positive for multiple analytes DAB (Brown) Vulcan Red Vina Green Imaging Ki-67+CD8 Ki-67 CD8 N/A Aperio CD3+CD4+Ki-67 CD3 CD4 Ki-67 Nuance FoxP3+PD-1+CD8 PD-1 FoxP3 CD8 Nuance CD68+PD-L1+CD3 PD-L1 CD68 CD3 Nuance Slides imaging and analysis methods were selected based on whether chromogens had complete overlap (e.g. CD4 completely overlaps CD3), Imaging methods and color combinations are listed in the table above. Image analysis of Aperio whole slide scans was performed with Aperio ImageScope software with Indica’s Cytonuclear algorithm. Regions of interest were manually drawn around the invasive margin (IM; +/- 500 microns from the tumor margin), center of tumor (CT; region of tumor up to the inner IM boundary), and the all tumor region (ALL; tumor area up to the margin). The tumor regions included small stromal bands but excluded large stromal regions or regions of necrosis. Image analysis using the Nuance multispectral imaging system was performed by capturing up to five 20X fields (when present) within the IM or CT regions and performing image analysis using inForm software. Results from each of the fields were added together and calculations were performed based on the number of cells present in all fields imaged per region. Sample ID % Ki67+ Cells CD8+ Cells/mm^2 % CD8+ Cells % Ki67+ CD8+ Cells Ki67+ CD8+ Cells/mm^2 % of CD8+ Cells that are Ki67+ Sample 1 1.02 127 1.66 0.18 14 10.66% Sample 2 37.46 38 0.84 0.09 4 11.11% Sample 3 28.32 558 8.31 3.44 231 41.37% Table 1: Results of Ki-67 + CD8 Multiplex IHC in Region = ALL Table 4: Results of CD3+CD4+Ki-67 Multiplex IHC in Regions = IM and CT Sample ID Sample 10 Sample 10 Sample 11 Sample 11 Sample 12 Sample 12 Region CT IM CT IM CT IM % CD3+ 15.83% 7.03% 16.56% 11.53% 45.26% NA % CD4+ 38.63% 13.40% 1.99% 3.59% 32.88% NA % Ki67+ 4.62% 2.19% 13.57% 17.56% 19.64% NA % of CD3+ Cells that are CD4+ 58.00% 55.56% 6.02% 14.38% 42.38% NA % of CD3+ Cells that are Ki67+ 2.96% 3.65% 6.18% 8.52% 9.74% NA Sample ID Sample 7 Sample 7 Sample 8 Sample 8 Sample 9 Sample 9 Region CT IM CT IM CT IM % CD8+ 6.32% 10.27% 11.15% NA 3.73% 11.98% % FoxP3+ 5.29% 3.35% 2.15% NA 2.17% 3.44% % PD-1+ 2.93% 6.28% 1.14% NA 3.66% 7.39% % of CD8+ Cells that are PD-1+ 9.80% 7.47% 6.90% NA 4.62% 4.47% Table 3: Results of FoxP3+PD-1+CD8 Multiplex IHC in Regions = IM and CT Sample ID Sample 4 Sample 4 Sample 5 Sample 5 Sample 6 Sample 6 Region CT IM CT IM CT IM % CD3+ Cells 26.82% NA 19.48% NA 15.96% 17.91% % CD68+ Cells 2.89% NA 4.76% NA 6.65% 5.10% % PD-L1+ Cells 25.89% NA 17.00% NA 71.81% 58.58% % PD-L1+ Low 15.73% NA 8.92% NA 16.69% 10.85% % PD-L1+ Moderate 6.86% NA 4.34% NA 17.83% 8.01% % PD-L1+ Strong 3.30% NA 3.74% NA 37.29% 39.72% % of CD3+ Cells that are PD-L1+ Cells 1.16% NA 6.87% NA 13.45% 8.31% % of CD68+ Cells that are PD-L1+ Cells 45.18% NA 68.16% NA 88.54% 73.38% Table 2: Results of FoxP3+PD-1+CD8 Multiplex IHC in Regions = IM and CT Multiplex chromogenic immunohistochemistry is a tool to evaluate immuno- oncology biomarkers in tumor samples . Multiplex chromogenic IHC assays can be generated for at least three targets without cross-talk even if all three antibodies are from the same species. Imaging and image analysis methods must be evaluated carefully to ensure detection and proper discrimination of overlapping chromogens. Selection of invasive margin and center of tumor regions can be performed either using whole slide scans or spectral imaging. CD3+CD4+Ki-67 multiplex IHC assay (MOS763-DRG) Tonsil NSCLC NSCLC NSCLC NSCLC Parameters: • Percent positive • Cells per mm2 (immune cells) • Staining intensity (cancer markers)
