Quantitative Analysis of Immune Infiltrates in Primary Melanoma

Area of Science:

• Immunology
• Oncology
• Biomarker Discovery

Background:

• Accurate stratification of melanoma patients for immunotherapy requires novel methods to analyze the tumor microenvironment (TME).
• Conventional tumor-infiltrating lymphocyte (TIL) analysis lacks the precision needed for clinical application.
• Quantitative multiplex immunofluorescence (qmIF) offers advanced TME evaluation through multiparameter phenotyping, tissue segmentation, and quantitative spatial analysis (qSA).

Purpose of the Study:

• To investigate the correlation between the quantification and spatial distribution of cytotoxic lymphocytes (CTLs) and macrophages within the TME and clinical outcomes in melanoma patients.
• To determine if spatial analysis of immune cell interactions can serve as a predictive biomarker for adjuvant immunotherapy response.

Main Methods:

• Application of qmIF to analyze 104 primary stage II-III melanoma tumors.
• Quantitative spatial analysis (qSA) to assess the proximity of CTLs (CD3+CD8+) to activated (CD68+HLA-DR+) and nonactivated (CD68+HLA-DR-) macrophages.
• Evaluation of CTL and macrophage proximity to proliferating (SOX10+, Ki67+) and non-proliferating tumor cells.
• Correlation analysis with disease-specific survival (DSS) and overall survival (OS) using Cox regression.

Main Results:

• CTLs were found in closer proximity to activated macrophages than nonactivated ones.
• CTLs were further from proliferating melanoma cells compared to non-proliferating cells.
• High stromal CTL density and low stromal macrophage density correlated with improved DSS.
• A low CTL/macrophage ratio in the stroma was significantly associated with a higher hazard ratio for melanoma-related death and shortened OS.

Conclusions:

• The spatial relationship and density of CTLs and macrophages within the melanoma TME are critical determinants of patient survival.
• A low CTL/macrophage ratio in the stroma emerges as a promising predictive biomarker for melanoma prognosis and response to immunotherapy.
• qmIF and qSA provide a precise method for evaluating the TME and hold potential for clinical application in patient stratification.