Background: Stratifying melanoma patients for adjuvant immunotherapy trials has quickly become an urgent need. Tumor-infiltrating lymphocyte (TIL) analysis is predictive but insufficiently precise for clinical application. A novel pathology method, quantitative multiplex immunofluorescence (qmIF), allows for complex analysis of the tumor microenvironment (TME) for development of new biomarkers. Given that CD3+CD8+ cytotoxic lymphocytes (CTLs) promote antitumor immunity while CD68+ macrophages (Mϕ) impair immunity, we hypothesized that precise quantification and spatial analysis of Mϕ and CTLs would correlate with survival in melanoma.
Methods: We applied qmIF to 104 stage II/III primary melanomas from Columbia University Medical Center (CUMC); known cause of death is available for 64 patients. 5-µm slides were stained using qmIF for DAPI, CD3, CD8, CD68, SOX10, HLA-DR and Ki67. Tumor areas were preselected by a dermatopathologist and visualized using Mantra. Tissue and cell segmentation, multiparameter immune phenotyping, and quantitative spatial analysis (qSA) were performed using inForm. Local spatial analysis was implemented by partitioning images into 100-µm square windows. The interquartile range of cellular ratios among windows was used as the measure of dispersion.
Results: We find high CTL and low Mϕ density in stroma (p=0.0038, p=0.0006) correlate with disease-specific survival (DSS). Correlation was strongest for stromal HLA-DR+ CTLs (p=0.0005). CTL distance to HLA-DR- Mϕ associates with poor DSS (p=0.0016), while distance to Ki67- tumor cells associates inversely with DSS (p=0.0006). Low CTL/ Mϕ ratio in stroma confers a hazard ratio (HR) of 3.719 for death from melanoma and correlates with shortened overall survival (OS) in the complete 104-patient cohort by Cox analysis (p=0.009). HLA-DR- Mϕ are more evenly dispersed throughout the stroma of survivors (n=42) than patients known to have died of melanoma (n=22) (p=0.0036), while conversely, HLA-DR+ CTLs are less evenly dispersed in survivors (p=0.023).
Conclusions: CTL/ Mϕ ratio is prognostically superior to conventional clinical biomarkers in nonmetastatic melanoma. Local spatial features such as leukocyte dispersion and intercellular distances also associate with survival, highlighting the importance of microenvironmental structure. Future efforts will further parse the large output of data from qmIF to generate biomarkers that are increasingly precise, spatially informed, and quantitatively robust.
Citation Format: Robyn D. Gartrell, Andrew Chen, Douglas K. Marks, Thomas D. Hart, Gen Li, Alan Wu, Yan Lu, Camden Esancy, Zoe Blake, Bret Taback, Raul Rabadan, Howard L. Kaufman, Charles G. Drake, Basil Horst, Anthea Monod, Yvonne Saenger. Quantitative compositional and spatial analysis of tumor microenvironment (TME) in primary melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2091.