Abstract 2001: Biomarker discovery in immunotherapy-treated melanoma patients with imaging mass cytometry
Authors: Martinez-Morilla Sandra , Villarroel-Espindola Franz , Wong Pok Fai , Kluger Harriet , Toki Maria , Aung Thazin New , Pelekanou Vasiliki , Bourke-Martin Brian , Rimm David L. 
Published: September 2020

Abstract
Background: Understanding the complexity of the tumor microenvironment could allow identification of new biomarkers that predict response to immunotherapy in cancer patients and anticipate the toxicity associated with the treatment. Since the number of targets measurable by quantitative immunofluorescence (QIF) is limited, we applied Imaging Mass Cytometry (IMC), a metal-conjugated antibody-based high-plex technology, to generate a multidimensional analysis of the microenvironment. We interrogated a 25-antibody panel, including tumor and immune cell markers, in a cohort of melanoma patients treated with immune checkpoint blockers.
Methods: We used a validated, optimized panel of 25 antibodies conjugated to unique metals, including a DNA intercalator conjugated with 191/193Ir, for detection using HyperionTM (Fluidigm). Digital image analysis was performed using a newly designed version of the AQUA software (Navigate BP) to measure marker intensity in molecularly defined masks or compartments as follows: all cells (DNA intercalator Ir191/193), tumor cells (HMB45/S100), stroma (tumor cells subtracted from all cells), T cells (CD3), B cells (CD20) and macrophages (CD68). Results were compared to QIF and Digital Spatial Profiling (DSP), a NanoString technology based on primary antibodies conjugated to indexing DNA oligos. We examined archived formalin-fixed paraffin-embedded (FFPE) samples from a metastatic melanoma cohort treated with immunotherapy (n=60) in tissue microarray format.
Results: Immune markers such as CD3, CD4 and CD8 showed a high correlation between the three multiplexing methods: IMC, DSP and QIF (r=0.48-0.89, p&lt;0.0001). Moreover, high CD3 and CD8 levels assessed by IMC were statistically significantly associated with favorable outcome, confirming results previously published on the same cohort (Toki et al, CCR, 2018). Multivariable analyses revealed significant associations of MHC-I, CSF1R, IRF1, LAG3, PD1, MHC-II and beta2-microglobulin (B2M) in tumor with progression-free survival (PFS) independent of age, sex, mutation, stage, treatment, and prior immune checkpoint blockade treatment. In stroma, high TIM3 and high PD-L2 also predicted response to immunotherapy. Furthermore, we tested for validation by QIF B2M, MHC-I and CSF1R, predictive markers identified by IMC. However, only B2M showed a statistically significant correlation with overall survival.
Conclusion: Using IMC technology and digital image analysis based on pixel colocalization, we were able to evaluate simultaneously 25 markers on FFPE tissue microarray samples. There was a high concordance with QIF and DSP for immune markers such as CD3, CD4 and CD8. A series of potentially predictive biomarkers for immunotherapy in metastatic melanoma were identified.
Citation Format: Sandra Martinez-Morilla, Franz Villarroel-Espindola, Pok Fai Wong, Harriet Kluger, Maria Toki, Thazin New Aung, Vasiliki Pelekanou, Brian Bourke-Martin, David L. Rimm. Biomarker discovery in immunotherapy-treated melanoma patients with imaging mass cytometry [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2001.