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Updated: Jul 15, 2025
Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells
Published on: February 8, 2018
Digital spatial profiling of melanoma shows CD95 expression in immune cells is associated with resistance to
Sandra Martinez-Morilla1, Myrto Moutafi1, Aileen I Fernandez1
1Department of Pathology, Yale School of Medicine, New Haven, CT, USA.
Abstract:
Although immune checkpoint inhibitor (ICI) therapy has dramatically improved outcome for metastatic melanoma patients, many patients do not benefit. Since adverse events may be severe, biomarkers for resistance would be valuable, especially in the adjuvant setting. We performed high-plex digital spatial profiling (DSP) using the NanoString GeoMx® on 53 pre-treatment specimens from ICI-treated metastatic melanoma cases. We interrogated 77 targets simultaneously in four molecular compartments defined by S100B for tumor, CD68 for macrophages, CD45 for leukocytes, and nonimmune stromal cells defined as regions negative for all three compartment markers but positive for SYTO 13. For DSP validation, we confirmed the results obtained for some immune markers, such as CD8, CD4, CD20, CD68, CD45, and PD-L1, by quantitative immunofluorescence (QIF). In the univariable analysis, 38 variables were associated with outcome, 14 of which remained significant after multivariable adjustment. Among them, CD95 was further validated using multiplex immunofluorescence in the Discovery immunotherapy (ITX) Cohort and an independent validation cohort with similar characteristics, showing an association between high levels of CD95 and shorter progression-free survival. We found that CD95 in stroma was associated with resistance to ICI. With further validation, this biomarker could have value to select patients that will not benefit from immunotherapy.
Insights
Biomarkers identifying resistance to immune checkpoint inhibitors (ICI) in metastatic melanoma are crucial. High CD95 levels in tumor stroma indicate resistance to ICI therapy, potentially guiding patient selection.
Area of Science:
- Oncology
- Immunology
- Biomarker Discovery
Background:
- Immune checkpoint inhibitor (ICI) therapy offers significant benefits for metastatic melanoma patients.
- However, a substantial number of patients do not respond to ICI treatment, and severe adverse events necessitate predictive biomarkers, especially in the adjuvant setting.
Purpose of the Study:
- To identify biomarkers associated with resistance to immune checkpoint inhibitor therapy in metastatic melanoma patients.
- To investigate the role of specific molecular targets within distinct tumor microenvironment compartments.
Main Methods:
- High-plex digital spatial profiling (DSP) was employed on 53 pre-treatment metastatic melanoma specimens.
- Seventy-seven targets were simultaneously analyzed across tumor, macrophage, leukocyte, and nonimmune stromal compartments.
- Validation of key markers, including CD95, was performed using quantitative immunofluorescence (QIF) and multiplex immunofluorescence.
Main Results:
- Thirty-eight variables were associated with patient outcome in univariable analysis, with 14 remaining significant after multivariable adjustment.
- High levels of CD95, particularly within the stromal compartment, were significantly associated with shorter progression-free survival.
- CD95 in stroma emerged as a potential biomarker for resistance to ICI therapy.
Conclusions:
- CD95 in the tumor stroma is a promising biomarker for predicting resistance to immune checkpoint inhibitor therapy in metastatic melanoma.
- Further validation of CD95 could aid in selecting patients who are unlikely to benefit from immunotherapy, optimizing treatment strategies.
