Abstract 4146: MultiOmyx multiplexed tumor infiltrating lymphocyte panel provides comprehensive immunophenotyping from a single FFPE slide
Authors: Au Qingyan , Nguyen Kathy , Padmanabhan Raghav , Kuller Anne , Moler Eddie , Hoe Nicholas 
Published: July 2016

Abstract
Immune checkpoint therapies target immune regulatory pathways to enhance anti-tumor immune response. These therapies have contributed to important clinical advances, and are a promising approach to combat cancer. Development of effective immune checkpoint therapies requires an understanding of the host immune response within the tumor microenvironment. GE Healthcare, through its affiliate Clarient Diagnostics Service Inc., has developed a multiplexed Tumor Infiltrating Lymphocyte (TIL) Panel* consisting of 12 key cancer immune markers: CD3, CD4, CD8, CD20, CD45RO, CD56, CD68, CTLA4, FOXP3, PD1, PD-L1 and Pan-CK. This MultiOmyx TIL panel identifies individual Thelper (CD3+CD4+), Tcytotoxic (CD3+CD8+), Tregulatory (CD3+CD4+FoxP3), memory T-cells (CD3+CD4+CD45RO), anergic T-cells (PD1+CD8+), natural killer cells (CD56+), macrophages (CD68+), and B-cells (CD20+) within the tumor and the stromal regions.
MultiOmyx multiplexed immunofluorescence technology enables qualitative and quantitative analysis of these 12 biomarkers’ expression and co-localization from a single formalin-fixed, paraffin-embedded (FFPE) slide. MultiOmyx assay utilizes a pair of directly conjugated Cyanine dye-labeled (Cy3, Cy5) antibodies per round of staining. Each Cy-dye conjugated antibody recognizes different target proteins. Each round of staining is imaged and followed by novel dye inactivation chemistry, enabling repeated rounds of staining.
Herein, we report an analysis of immune response in the tumor microenvironment within solid tumors including breast cancer, lung cancer, colorectal cancer, esophageal cancer, prostate cancer, and melanoma utilizing the MultiOmyx TIL Panel. The results revealed two distinct immunologic phenotypes, high TIL tumors and Low TIL tumors. The high TIL tumors showed enhanced T cell population within the tumor and in the peritumoral stroma including CD8+ cytotoxic T cells, CD4+ helper T cells and CD45RO+ memory T cells. Increased expression of immune checkpoints markers such as CTLA4 and PD-1 were also observed. PD-1 was predominantly expressed in CD8+ cytotoxic T cells while CTLA4 was mostly found on CD4+ FOXP3+ regulatory T cells. PD-L1 expression was also induced, mainly on Pan-CK+ tumor cells and CD68+ macrophages. High density of PD-L1+ |CD68+ macrophages was localized in the stroma surrounding the tumor region. Conversely, many of the immune markers were not expressed in low-TIL tumors.
Immunophenotyping analysis offered by the MultiOmyx TIL panel may facilitate the identification of appropriate immunotherapeutic approach. Tumor with high TIL profiling may be effectively treated with single-agent immune checkpoint therapy, while tumor with low TIL profiling may require combination therapy with an agent that enhances endogenous antitumor response.
*MultiOmyx TIL panel is currently research use only tool.
Citation Format: Qingyan Au, Kathy Nguyen, Raghav Padmanabhan, Anne Kuller, Eddie Moler, Nicholas Hoe. MultiOmyx multiplexed tumor infiltrating lymphocyte panel provides comprehensive immunophenotyping from a single FFPE slide. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4146.