ABSTRACT
BACKGROUND: Vascular endothelial growth factor is associated with reduced immune response and impaired anti-tumor activity. Combining antiangiogenic agents with immune checkpoint inhibition can overcome this immune suppression and enhance treatment efficacy. METHODS: This study investigated the combination of ziv-aflibercept anti-angiogenic therapy with pembrolizumab in patients with advanced melanoma resistant to anti-PD-1 treatment. Baseline and on-treatment plasma and PBMC samples were analyzed by multiplex protein assay and mass cytometry, respectively. RESULTS: In this Phase 1B study (NCT02298959), ten patients with advanced PD-1-resistant melanoma were treated with a combination of ziv-aflibercept (at 2-4 mg/kg) plus pembrolizumab (at 2 mg/kg), administered intravenously every 2 weeks. Two patients (20%) achieved a partial response, and two patients (20%) experienced stable disease (SD) as the best response. The two responders had mucosal melanoma, while both patients with SD had ocular melanoma. The combination therapy demonstrated clinical activity and acceptable safety, despite the occurrence of adverse events. Changes in plasma analytes such as platelet-derived growth factor and PD-L1 were explored, indicating potential alterations in myeloid cell function. Higher levels of circulating CXCL10 in non-responding patients may reflect pro-tumor activity. Specific subsets of γδ T cells were associated with poor clinical outcomes, suggesting impaired γδ T-cell function in non-responding patients. CONCLUSIONS: Although limited by sample size and follow-up, these findings highlight the potential of the combination of ziv-aflibercept antiangiogenic therapy with pembrolizumab in patients with advanced melanoma resistant to anti-PD-1 treatment and the need for further research to improve outcomes in anti-PD-1-resistant melanoma. TRIAL REGISTRATION NUMBER: NCT02298959.
Subject(s)
Antibodies, Monoclonal, Humanized , Melanoma , Receptors, Vascular Endothelial Growth Factor , Recombinant Fusion Proteins , Humans , Melanoma/drug therapy , Leukocytes, Mononuclear , Vascular Endothelial Growth Factor AABSTRACT
We developed a comprehensive method for functional assessment of the changes in immune populations and killing activity of peripheral blood mononuclear cells after cocultures with cancer cells using mass cytometry. In this study, a 43-marker mass cytometry panel was applied to a coculture of immune cells from healthy donors' peripheral blood mononuclear cells with diverse cancer cell lines. DNA content combined with classical CD45 surface staining was used as gating parameters for cocultures of immune cells (CD45high/DNAlow) with hematological (CD45low/DNAhigh) and solid cancer cell lines (CD45neg/DNAhigh). This strategy allows for universal discrimination of cancer cells from immune populations without the need for a specific cancer cell marker and simultaneous assessment of phenotypical changes in both populations. The use of mass cytometry allows for simultaneous detection of changes in natural killer, natural killer T cell, and T cell phenotypes and degranulation of immune populations upon target recognition, analysis of target cells for cytotoxic protein granzyme B content, and cancer cell death. These findings have broad applicability in research and clinical settings with the aim to phenotype and assess functional changes following not only NK-cancer cell interactions but also the effect of those interactions on other immune populations.
Subject(s)
Cytotoxicity, Immunologic , Neoplasms , Leukocytes, Mononuclear , Killer Cells, Natural , T-Lymphocytes , Coculture Techniques , Flow Cytometry , Neoplasms/metabolismABSTRACT
With the increasing use of mass cytometry in clinical research, a simplified and standardized protocol for immunophenotyping human peripheral blood mononuclear cells (PBMCs) in clinical trials is needed. We present a simplified in-plate staining protocol for up to 80 samples, for laboratories of all mass cytometry expertise levels, aimed to generate reproducible datasets for large clinical cohorts. In this protocol, we provide details on the requirements to obtain meaningful results, spanning from sample quality, barcoding, and batch-freezing of stained samples.
Subject(s)
Cryopreservation , Leukocytes, Mononuclear , Cryopreservation/methods , Flow Cytometry/methods , Humans , Immunophenotyping , Staining and LabelingABSTRACT
This protocol details a staining technique optimized for immunophenotyping of human bone marrow immune populations using mass cytometry. The protocol accounts for the limitations of working with human bone marrow, such as reduced viability, low cell counts, and fragile cell pellets, to successfully acquire single viable cells ready for downstream analysis. This assay can be used to characterize the activation, exhaustion, and cytotoxicity of immune populations and ensure comprehensive immunophenotyping of human bone marrow clinical samples.
