Abstract C078: Pembrolizumab response in melanoma patients: an analysis of the tumor microenvironment
Authors: Juncker-Jensen Anna , Wortman Juliana , Beaton Nigel , Beeler Kristina , Treiber Tobias , Levesque Mitchell , Gomez Julia Martinez , Vowinckel Jakob , Nunns Harry 
Published: December 2023

Abstract
Background: Melanoma has been associated with poor prognosis and limited treatment options. Immunotherapy, particularly the use of immune checkpoint inhibitors, has revolutionized the treatment landscape for advanced melanoma. Pembrolizumab, a PD-1 inhibitor, has shown promising results; however, the underlying mechanisms of its efficacy and the influence of the tumor microenvironment (TME) on treatment response remain incompletely understood. This study aims to investigate the response to pembrolizumab in melanoma patients and analyse the TME using a multiplexed immunofluorescence (mIF) spatial tissue analysis to gain insights into potential predictive factors and therapeutic targets.
Methods: A retrospective analysis was conducted on formalin-fixed paraffin-embedded (FFPE) tumour samples from baseline melanoma patient samples, classified as responders (n = 6) or non-responders (n = 4) based on the response at 3 months post pembrolizumab treatment. Analysis was done using the mIF platform MultiOmyx™, a proprietary, immunofluorescence (IF) multiplexing assay that enables visualization and characterization of up to 60 biomarkers on a single FFPE section. Image segmentation, cell phenotyping, and spatial analytics were performed using our AI-powered image analytics software, NeoLYTX™.
Results: Analysis of the 10 FFPE samples from metastatic melanoma patients treated with pembrolizumab revealed significant differences in immune cell populations between non-responders and responders. Patients who responded favorably to pembrolizumab exhibited increased infiltration of cytotoxic T lymphocytes (CTLs) and tumor-associated macrophages within the TME. This finding suggests that an immune-inflamed microenvironment, characterized by high levels of tumor-infiltrating lymphocytes may be indicative of a robust antitumor immune response. Moreover, immune checkpoint molecules PD-1, CTLA-4, LAG3, and TIM3 were also significantly increased in responding patients, suggesting potential therapeutic targets to enhance pembrolizumab response, including combination strategies targeting multiple immune checkpoints. When spatially characterizing T cell activation and exhaustion status in different regions of the TME we found an increase in checkpoint molecules among intra-tumoral CTLs as compared to intra-stromal CTLs. In particular, the fraction of CTLs with quadruple-positive CTLA4+LAG3+PD1+TIM3+ expression was doubled. This suggests an immunosuppressive TME in which CTLs are rapidly exhausted upon reaching the tumor. Work is now ongoing to characterize how this cell population spatially interacts with other immune and tumor cells in the TME.
Conclusions: This study provides valuable insights into the response to pembrolizumab in melanoma patients and highlights the crucial role of the TME in determining treatment outcomes. Identification of immune cell subsets, and immunosuppressive mechanisms associated with pembrolizumab response may guide the development of novel therapeutic approaches to improve patient outcomes in advanced melanoma.
Citation Format: Anna Juncker-Jensen, Juliana Wortman, Nigel Beaton, Kristina Beeler, Tobias Treiber, Mitchell Levesque, Julia Martinez Gomez, Jakob Vowinckel, Harry Nunns. Pembrolizumab response in melanoma patients: an analysis of the tumor microenvironment [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C078.