Cytotoxic t lymphocyte (CTL) to macrophage ratio to predict survival in stage II-III melanoma: Validation in a second cohort.
Authors: Djavid A. Reza , Fullerton Benjamin T. , Wang Samuel W. , Drastal Meghan S. , Barker Luke W. , Gartrell Robyn D. , Rohr Bethany R. , Ferringer Tammie C. , Saenger Yvonne M. 
Published: June 2021

e21595 Background: Cutaneous melanoma is an aggressive dermatologic malignancy with stage II-III recurrence rates ranging from 27-46%. Analysis of the tumor microenvironment (TME) is a promising mechanism to identify the patients who would benefit most from adjuvant immunotherapy given the risk for potential immune-related adverse events. Previous research by our group established the potential of utilizing CD3+CD8+ cytotoxic lymphocyte (CTL) and CD68+ macrophage densities, as well as the CTL/macrophage ratio in the TME, as prognostic biomarkers for metastatic progression and disease-specific survival (DSS). We sought to test the prognostic accuracy of the CTL/macrophage ratio in a second cohort of stage II-III melanoma patients from the Geisinger Health System. Methods: We utilized quantitative multiplex immunofluorescence (qmIF) to analyze 43 patient samples with stage II-III melanoma to validate the prospective biomarkers established in our previous CUIMC cohort. TME cell phenotype and cell density data were acquired for both the stroma as well as the combined tumor + stroma (“total TME”) via utilization of a trainable machine learning program. Cell densities and ratios were subsequently analyzed to determine associations with clinical outcomes including both disease recurrence and DSS. Results: In patients without distant metastatic recurrence (DMR), the density of CTLs and activated HLA-DR+ CTLs was higher in both the stroma ( p= 0.0003, p= 0.0152, respectively), as well as total TME ( p< 0.0001, p= 0.0165, respectively), when compared to those with DMR. The CTL/macrophage ratio in the total TME was greater in those without recurrence ( p= 0.0031). The ratio of CTLs to MPO+ neutrophils was higher in both the stroma ( p= 0.0015) and the total TME ( p= 0.0016) in those without recurrence. Survival analysis of 38 patients with known cause of death indicated a higher CTL/macrophage ratio in the total TME was associated with improved survival ( p= 0.0005). Improved survival was also associated with high CTL/neutrophil ratios in both the stroma ( p< 0.0001) and the total TME ( p< 0.0001). Cox regression analysis demonstrated a strong association between low CTL/macrophage and CTL/neutrophil ratios in the total TME and an increased risk of death ( p= 0.003, HR: 7.24, CI: 1.99-26.31; p= 0.003, HR: 22.35, CI: 2.87-173.97, respectively). Conclusions: Patients without disease recurrence display an increased infiltration of CTLs and activated HLA-DR+ CTLs, in addition to higher ratios of CTLs to macrophages and neutrophils, in the TME. High CTL/macrophage and CTL/neutrophil ratios in the total TME were also independently associated with improved DSS. Validation is underway with an additional cohort to further explore the use of these prospective biomarkers as clinical decision-making support tools to help guide the determination of patient risk and the use of adjuvant immunotherapy in melanoma.