Evaluation of S100A8/A9 and neutrophils as prognostic markers in metastatic melanoma patients under immune-checkpoint inhibition

Abstract

Immune-checkpoint inhibitors (ICIs) have revolutionized melanoma treatment, yet approximately half of patients do not respond to these therapies. Identifying prognostic biomarkers is crucial for treatment decisions. Our retrospective study assessed liquid biopsies and tumor tissue analyses for two potential biomarkers: danger-associated molecular pattern (DAMP) S100A8/A9 and its source, neutrophils. In 43 metastatic unresected stage III/IV melanoma patients, elevated serum levels of S100A8/A9 and neutrophils before and during ICI treatment correlated with worse outcomes. Furthermore, in 113 melanoma patients, neutrophil expression in the tumor microenvironment (TME) was associated with relapse and reduced survival. Measuring S100A8/A9 and neutrophils could enhance immunotherapy monitoring by predicting impaired clinical outcomes and non-response to ICIs. Serum S100A8/A9 levels and neutrophil counts at baseline (T0) and during treatment (T3) correlated with reduced progression-free survival (PFS). Elevated S100A8/A9 levels at T0 and T3 negatively impacted overall survival (OS). Notably, neutrophil infiltration was more prevalent in primary melanomas than in nevi and metastases, and its presence in primary melanomas was linked to poorer survival. S100A8/A9 serum levels, neutrophil counts, and tumor-associated neutrophil infiltration represent promising biomarkers for predicting treatment response and clinical outcomes in melanoma patients receiving ICIs. SIGNIFICANCE: These findings underscore the critical need for reliable biomarkers in melanoma research, particularly for predicting responses to immune-checkpoint inhibitors (ICIs). Identifying S100A8/A9 levels and neutrophil infiltration as potential indicators of treatment outcomes offers valuable insights for personalized therapy decisions. By enhancing monitoring and prognosis assessment, these biomarkers contribute to refining treatment strategies, ultimately improving patient care and outcomes. This research bridges gaps in understanding melanoma response mechanisms and highlights avenues for further investigation into immune-related markers, fostering advancements in precision medicine for melanoma patients.

Keywords: Biomarker; Immune-checkpoint inhibitors; Inflammation; Melanoma; Neutrophils; S100A8/A9; Tumor progression.

Graphical abstract

Fig. 1

Univariate survival analyses: impact of baseline serum levels and T3 serum levels of S100A8/A9 and neutrophils on progression-free survival (PFS) and overall survival (OS). Kaplan-Meier curves are shown for the melanoma patients in the systemic study. A) For high S100A8/A9 serum levels before therapy initiation, there was a negative effect on PFS of the melanoma patients. In this analysis, the "number at risk" at day 0 for baseline serum S100A8/A9=low was 17 patients and for baseline serum S100A8/A9=high, it was 24 patients. The cut-off value was 2864.35 ng/ml. B) In the analysis at T3, high serum S100A8/A9 levels had a negative effect on PFS. Here, the "number at risk" at day 0 for T3 serum S100A8/A9=low was 27 patients and for T3 serum S100A8/A9=high, it was 14 patients. The cut-off value was 3770.73 ng/ml. C) For high baseline serum S100A8/A9 levels, there was a negative effect on OS of melanoma patients. In this analysis, the "number at risk" at day 0 for baseline serum S100A8/A9 =low was 28 patients and for S100A8/A9 =high, it was 13 patients. The cut-off value was 4499.26 ng/ml. D) Low serum S100A8/A9 levels at T3 had a positive effect on the OS of melanoma patients. In this analysis, the "number at risk" at day 0 for the T3 serum neutrophils=low was 27 patients and for T3 serum neutrophils=high, it was 14 patients. The cut-off value was 3770.73 ng/ml. E) In this analysis regarding PFS, there was no significant difference between patients with high neutrophil count in peripheral blood at baseline and those melanoma patients who had low systemic neutrophil count. The "number at risk" at day 0 for baseline neutrophil count in peripheral blood=low was 31 patients and for baseline count in peripheral blood=high, it was 6 patients. The cut-off value was 6.02/nl. F) For a high count of neutrophils at T3, there was a negative effect on PFS of melanoma patients. The "number at risk" at day 0 was 16 patients for T3 neutrophil count in peripheral blood=low and 14 patients for T3 neutrophil count in peripheral blood=high. The cut-off value was 4.35/nl. G) For high neutrophil count in peripheral blood at baseline, there was a positive effect on OS of the melanoma patients. In this analysis, the "number at risk" at day 0 for baseline neutrophil count in peripheral blood=low was 7 patients and for baseline neutrophil count in peripheral blood=high, it was 30 patients. The cut-off value was 3.84/nl. H) In the analysis with the endpoint OS, high T3 neutrophil count in peripheral blood were associated with no significant impact on survival. In this analysis, the "number at risk" at day 0 for T3 neutrophil count in peripheral blood=low was 5 patients and for T3 neutrophil count in peripheral blood=high, it was 25 patients. The cut-off value was 3.17/nl.

Fig. 2

Immunofluorescence stainings and analyses of an average low neutrophil infiltrated nevus (A) and an average high infiltrated primarius (B) and metastasis (C). Statistical analysis of all cell counts obtained (D). Shown are microscopic overview images of the tissue punches of a nevus (TMA 1, punch L17), a primary melanoma (TMA 2.1, punch A11), and a metastasis (TMA 2.2, punch E8) as representation of the respective tissue types. The tissue samples were stained with an anti-CD15 antibody in conjunction with the secondary antibody Alexa 594 to visualize neutrophil granulocytes in red color. A single neutrophil from each punch is shown magnified. Blue nuclear counterstaining was performed with DAPI. Graphically, the neutrophil counts of the total 44 nevi, 86 primaries, and 162 metastases examined are listed in D. Significance levels: *: p < 0.05; ***: p < 0.001. Abbreviations: MN: melanocytic nevus, PM: primary melanoma, M: metastasis.

Fig. 3

Univariate survival analyses: impact of neutrophil infiltration on progression-free survival (PFS) within melanocytic nevi, primary melanomas and metastases. Kaplan-Meier curves are shown for the melanoma patients in the tissue study. A) No significant difference in PFS was shown for high or low neutrophil infiltration in melanocytic nevi. Patients were stratified by high and low neutrophil infiltration in their melanocytic nevus. The "number at risk" at day 0 for CD15=low was 13 patients; for CD15=high, 4 patients. The cut-off value was 0/mm². B) High neutrophil infiltration in primary melanomas could be associated with shortened PFS. Patients were stratified by high and low neutrophil infiltration in their primary melanoma. The "number at risk" at day 0 for CD15=low was 7 patients; for CD15=high, 19 patients. The cut-off value was 0.99/mm². C) No significant difference in PFS was shown for high or low neutrophil infiltration in metastases. Patients were stratified by high and low neutrophil infiltration in their metastasis. The "number at risk" at day 0 for CD15=low was 13 patients; for CD15=high, 6 patients. The cut-off value was 6.27/mm².