Variation in Immune and Inflammatory Blood Markers in Advanced Melanoma Patients Treated with PD-1 Inhibitors: A Preliminary Exploratory Study

Abstract

Background: Immune checkpoint inhibitors (ICIs) used for the treatment of advanced melanoma have yielded significant results, with long-term responses and improved survival rates, but not for all treated patients. Therefore, predictive biomarkers of response to ICI therapy have been intensively explored. Our study aimed to evaluate the dynamics of peripheral blood lymphocyte variation and their correlation with a set of related inflammatory factors in Nivolumab-treated advanced melanoma patients. Methods: The immunophenotypic assessment of peripheral blood immune cell subpopulations (CD3⁺, CD4⁺, and CD8⁺ T cells; CD19⁺ B cells; CD16⁺CD56⁺ NK cells; and CD4⁺/CD8⁺ ratio) was performed by the flow cytometry technique, concomitantly with a complete blood count; levels of S100, IL-6, and TNF-α proteins were quantified in serum by immunoassays, and lactate dehydrogenase (LDH) by a chemiluminescence assay. Results: Approximately 85% and 79% of patients recorded a trend of increasing levels of CD8⁺ lymphocytes and NK cells, respectively, during therapy. The percentage of NK cells negatively correlated with CD3⁺, CD4⁺, and CD19⁺ cells; the last three cell populations also established negative correlations with the inflammatory neutrophile/lymphocyte ratio (NLR). Furthermore, CD19⁺ cells were negatively correlated with the systemic inflammatory response index (SIRI) and systemic immune-inflammation index (SII). The evaluation of progression biomarkers showed that LDH levels directly correlated with IL-6 and S100 proteins, but no correlation was found with TNFα; IL-6 levels negatively correlated with percentages of CD3⁺, CD4⁺, and CD8⁺ lymphocytes. Conclusions: Variation in lymphocyte subpopulations during immunotherapy of advanced melanoma patients, associated with other cellular and/or molecular inflammatory markers, might provide insights about immune system response, but additional prospective studies are needed.

Keywords: PD-1 inhibitors; advanced melanoma; cytokines; inflammatory markers; peripheral blood immune cells; predictive melanoma biomarkers.

Figure 1

Graphical representation of patients’ categories, divided according to the variation trends (increasing, decreasing, or stationary) of the levels of cellular subpopulations, during treatment with Nivolumab. Percentages of Nivolumab-treated patients’ categories, divided according to the variation trends of (a) total T lymphocytes (CD3⁺), (b) helper/inducer T lymphocytes (CD3⁺CD4⁺), (c) suppressor/cytotoxic T lymphocytes (CD3⁺CD8⁺), (d) the helper/suppressor T cell ratio (CD4⁺/CD8⁺), (e) total B lymphocytes (CD19⁺), and (f) natural killer (NK) cells (CD3⁺/CD16⁺CD56⁺). Color legend: percentages of patients with stationary (grey), increasing (green), or decreasing (blue) status of cellular subsets.

Figure 2

Circulating levels of the cytokines TNF-α (a) and IL-6 (b) during the course of Nivolumab therapy, for the time points T₁, T₂, T₃. T1–T3, sampling times from Nivolumab-treated melanoma patients, taken at 3-month intervals.

Figure 3

Circulating levels of the cytokines TNF-α (a) and IL-6 (b) in patients with advanced stage III or IV melanoma during treatment with Nivolumab. Levels of TNF-α or IL-6 were detected in the entire cohort of peripheral blood samples, taken at 3 time points at 3-month intervals, from the start of therapy.

Figure 4

Correlation analyses between systemic inflammatory markers and several soluble melanoma markers: (a) SII vs. SIRI; (b) SII vs. S100; (c) SIRI vs. S100; (d) SIRI vs. IL-6; (— fitted linear regression curve; --- equality line). SII, systemic immune-inflammation index; SIRI, systemic inflammatory response index; IL-6, interleukin-6.

Figure 5

Correlation analyses between LDH and other soluble melanoma markers: (a) LDH vs. IL-6; (b) LDH vs. S100 (— fitted linear regression curve; --- equality line). LDH, lactate dehydrogenase; IL-6, interleukin 6.

Figure 6

Correlation analyses between soluble cytokine IL-6 levels and the percentages of T lymphocyte subsets ((a) IL-6 vs. CD3⁺ cells; (b) IL-6 vs. CD4⁺ cells; (c) IL-6 vs. CD8⁺ cells) or between the percentages of CD19⁺ and NK cells (d) (— fitted linear regression curve; --- equality line). IL-6, interleukin 6.