2019 ASCO Annual Meeting!
Session: Developmental Immunotherapy and Tumor Immunobiology
Type: Poster Session
Time: Saturday June 1, 8:00 AM to 11:00 AM
Location: Hall A
A multiplex immunofluorescence assay to assess immune checkpoint inhibitor-targeted CD8 activation and tumor colocalization in FFPE tissues.
Developmental Immunotherapy and Tumor Immunobiology
2019 ASCO Annual Meeting
Poster Board Number:
Poster Session (Board #273)
J Clin Oncol 37, 2019 (suppl; abstr 2629)
Author(s): Tony Navas, Kristin Fino, King Leung Fung, Facundo Cutuli, Robert J. Kinders, Aditi Sharma, Geraldine O'Sullivan-Coyne, Alice P. Chen, Toby Tucker Hecht, James H. Doroshow, Ralph E. Parchment; Clinical Pharmacodynamics Biomarker Program, Applied/Developmental Directorate, Frederick National Laboratory for Cancer Research, Frederick, MD; Ultivue, Inc, Cambridge, MA; Developmental Therapeutics Clinic/Early Clinical Trials Development Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD; Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD; Division of Cancer Treatment and Diagnosis and Center for Cancer Research, National Cancer Institute, Bethesda, MD
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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