Comprehensive Assessment of Selected Immune Cell Subpopulations Changes in Chemotherapy-Naïve Germ Cell Tumor Patients

Abstract

The pattern of immune cell distribution in testicular germ cell tumors (GCT) significantly differs from the immune environment in normal testicular tissues. The present study aimed to evaluate the role of different leukocyte subpopulation in GCTs. A cohort of 84 chemotherapy-naïve GCT patients was analyzed. Immunophenotyping of peripheral blood leukocyte subpopulations was carried out by flow cytometry. In addition, the data assessing the immunophenotypes and the baseline clinicopathological characteristics of the included subjects were statistically evaluated. Their prognostic value for the assessment of progression-free survival (PFS) and overall survival (OS) was estimated. The percentage of different innate/adaptive immune cell subpopulations was significantly associated with poor risk-related clinical features, including the number of metastatic sites, presence of retroperitoneal, mediastinal, lung, brain and non-pulmonary visceral metastases as well as with the S-stage and International Germ Cell Consensus Classification Group (IGCCCG) risk groups. In univariate analysis, the percentages of neutrophils, eosinophils, dendritic cells type 2, lymphocytes and T cytotoxic cells were significantly associated with PFS, while the neutrophil, non-classical monocyte and lymphocyte percentage were associated with OS. However, all these outcome correlations were not independent of IGCCCG in multivariate analysis. The data indicated a link among different innate/adaptive peripheral immune cell subpopulations in GCT patients. In addition, the association between these subpopulations and tumor characteristics was also investigated. The findings of the present study may contribute to a deeper understanding of the interactions between cancer and innate/adaptive immune response in GCT patients.

Keywords: adaptive immune cells; biomarkers; germ cell tumors; innate immune cells; patient outcome; tumor burden.

Figure 1

Flow cytometry gating strategy used for immunophenotyping of selected leukocyte subpopulations in the analyzed cohort of patients with GCT. The cells were initially gated by FSC and SSC following doublet exclusion using forward scatter area (FSC-A)/forward scatter height (FSC-H) (not shown). DCs were identified by CD45+ HLADR+ lin- and subsequently distinguished by CD123+ CD11c-(pDC) and CD11c+ (mDC) expression, as shown on the top of the figure. The total lymphocyte percentage was gated using a CD45/SSC plot and subsequently gating was performed for CD19+ (B cells) vs. CD3+ (T cells), CD4+ (Th cells) vs. CD8+ (Tc cells), CD56 + CD16 + CD3 (NK cells) and CD56+CD16+CD3+ (NKT cells) as well as their subpopulation (CD4+ NKT, CD8+NKT). CD4+ cells were used for the identification of CD25+ CD127-/low (Tregs). GCT, germ cell tumors; FSC, forward scatter; SSC, side scatter; CD, cluster of differentiation; HLA, human leukocyte antigen; DCs, dendritic cells; Th, T helper; Tc; T cytotoxic; NK, natural killer; Tregs, regulatory T cells. CD15 + CD33 + CD62L-HLADR-/low are polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and subpopulations of monocytes- CD14+CD16- (Classical monocytes), CD16+CD14- (Nonclassical monocytes) and CD14+CD16+ (Intermediate monocytes) are shown on the bottom of the figure.

Figure 2

Kaplan-Meier survival curves for the assessment of the neutrophil percentage in chemotherapy-naïve patients with GCT. The log-rank test was used to assess the (A) PFS (p = 0.0084) and the (B) OS (p = 0.0022). GCT, germ cell tumors; PFS, progression-free survival; OS, overall survival.

Figure 3

Kaplan-Meier survival curves for the assessment of the lymphocyte percentage in chemotherapy-naïve patients with GCT. The log-rank test was used to assess the (A) PFS (p = 0.0089) and the (B) OS (p = 0.0033). GCT, germ cell tumor; PFS, progression-free survival; OS, overall survival.