A multiplex immunofluorescence assay to assess immune checkpoint inhibitor-targeted CD8 activation and tumor colocalization in FFPE tissues.

癌症研究 免疫系统 间质细胞 CD8型 肿瘤微环境 细胞毒性T细胞 组织微阵列 医学 免疫检查点 T细胞 FOXP3型 免疫荧光 肿瘤浸润淋巴细胞 抗体 免疫疗法 病理 生物 免疫学 免疫组织化学 体外 生物化学
作者
Tony Navas,Kristin Fino,King Leung Fung,Facundo Cutuli,Robert J. Kinders,Aditi Sharma,Geraldine O’Sullivan Coyne,Alice Chen,Toby T. Hecht,James H. Doroshow,Ralph E. Parchment
出处
期刊:Journal of Clinical Oncology [Lippincott Williams & Wilkins]
卷期号:37 (15_suppl): 2629-2629 被引量:6
标识
DOI:10.1200/jco.2019.37.15_suppl.2629
摘要

2629 Background: Immune checkpoint inhibitors promote antitumor immune responses by enhancing T-cell activity. Measuring the pharmacodynamic effects of these drugs is challenging, as it requires assessing both immune cell and cancer cell populations. To evaluate T cell activation in tumor tissue from patient biopsies, we developed a robust multiplexed immunofluorescence assay. Methods: Our assay uses novel oligo-conjugated antibodies (Ultivue) for simultaneous quantitation of TCR activation (phospho-CD3zeta), immune checkpoint signaling via PD-1 (p-SHP1/p-SHP2), and the net stimulation/inhibition resulting from the integration of these two pathways in CD8 cells (p-ZAP70), while also providing the proximity of CD8 cells to tumor tissues, identified by β-catenin. The method was clinically validated using custom tissue microarrays (TMA) containing tumor biopsies of 3 different histologies (CRC, NSCLC, and breast). Results: From a total of 192 tumor core biopsies, 20/64 NSCLC, 9/64 CRC, and 3/65 breast TMA cores were found to have a significant number of CD8+ tumor infiltrating lymphocytes (TILs) at baseline ( > 50 cells in the examined section). In 18 of the 20 NSCLC cores, ≥50% of CD8 cells both inside and outside of the tumor were activated (CD3z-pY142+). In 6/9 CRC cores, ≥50% of CD8+ cells inside tumor tissues were activated, and in 4/9 CRC cores, ≥50% of CD8+ cells in stroma were activated. In 2/3 breast tumor cores, 90% of CD8+ cells inside tumor tissues were activated; in the remaining core, 90% of CD8+ cells in stroma were activated. Interestingly, all 192 cores had minimal to no expression of activated Zap70 (pY493) in CD8+ cells. Conclusions: Depending on tumor histology, baseline biopsy samples may contain variable numbers of activated CD8+ TILs (CD3z-pY142+), which may reside inside or outside of tumor regions and express very low levels of Zap70-pY493. Anti-PD-1 therapy is predicted to enhance T-cell cytotoxic activity, as demonstrated by an increased number of TILs and elevated Zap70-pY493 expression. This assay is being used for pharmacodynamic evaluations in ongoing immunotherapy clinical trials. Funded by NCI Contract No HHSN261200800001E.

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