Absolute monocyte count has a diagnostic role in distinguishing tumor marker-negative TGCT from benign testicular tumor via CCL2 regulation

Abstract

Clinically, for testicular tumor patients with negative tumor markers, how to distinguish the malignant from the benign is a difficult problem. This study aimed to assess the clinical significance of the absolute monocyte count (AMC) in differential diagnosis of testicular germ cell tumor with stage S0 (TGCTS0) and benign testicular tumor. In this retrospective single-center study, a total of 90 patients newly diagnosed with benign testicular tumor or TGCTS0 were reviewed. All patients received surgical intervention as the primary treatment method. AMC and other clinicopathological parameters were analyzed. Receiver operating characteristic (ROC) curves were constructed to assess the diagnostic power of investigated parameters, and to determine the optimal cutoff values. Kaplan-Meier curve analysis was used to study the survival of patients with TGCTS0. qRT-PCR and immunohistochemistry (IHC) were performed to examine the expression of C-C motif chemokine ligand 2 (CCL2) mRNA and protein respectively. Differential gene expression and functional enrichment analysis were performed using Gene Expression Omnibus and the Cancer Genome Atlas databases. The mean preoperative AMC in patients with TGCTS0 was significantly higher than that in patients with benign testicular tumor (P = .020). AMC > 0.485*10^9/L was identified to be associated with the presence of TGCTS0 (hazard ratio [HR] = 3.074, P = .026), and patients with higher AMC level had worse progression free survival (PFS) (P = .047). Furthermore, AMC combined with lactate dehydrogenase (LDH) achieved a better diagnostic efficacy for TGCTS0 (area under curve [AUC] = 0.695). Tumor-associated macrophages (TAMs) signature gene CCL2 was highly expressed in TGCT compared with normal testicular tissue. Functional enrichment analysis showed that CCL2 is closely involved in the Extracellular Matrix Organization pathway and positively correlated with the expression of various matrix metalloproteinases (MMPs). Elevated AMC may serve as a predictor of higher risk of TGCTS0, and CCL2 mediated TAMs infiltration and MMPs secretion is essential for the tumorigenesis of TGCT.

Figure 1.

Patients included in this study. CC = choriocarcinoma, EC = Embryonal carcinoma, TE = teratoma, TGCT^S0 = testicular germ cell tumor with stage S0, YST = yolk sac tumor.

Figure 2.

Diagnostic value of AMC in distinguishing TGCT ^S0 from benign tumors. (A) Differences in PLR, NLR, and MLR between TGCT ^S0 and benign tumors. (B) ROC curves for determination of cutoff value of preoperative variables regarding distinguishing TGCT^S0 from benign tumors. The following table shows the data of ROC curves. (C) Univariate logistic regression analysis of preoperative variables on prediction of TGCT^S0. (D) Kaplan–Meier plot showing PFS in patients with TGCT^S0 divided into high AMC and low AMC groups. (E) Kaplan–Meier plot showing PFS in patients with TGCT^S0 divided into high LDH and low LDH groups. AMC = absolute monocyte count, AUC = area under curve, HR = hazard ration, LDH = lactate dehydrogenase, MLR = monocyte to lymphocyte ratio, NLR = neutrophil to lymphocyte ratio, NPV = negative predictive value, PFS = progression free survival, PLR = platelet to lymphocyte ratio, PPV = positive predictive value, ROC = receiver operating characteristic, TGCT^S0 = testicular germ cell tumor with stage S0, YI = Youden index.

Figure 3.

Monocyte/macrophage signature gene enrichment analysis in TGCT. (A) The proportion of different cell types of macrophage in TGCT and normal testis analyzed by GEPIA2021. (B) Top: Representative heat map of the 10 macrophage signature genes using GSE8607 data. Bottom: Bar graph showing the fold difference of 10 monocyte/macrophage signature genes between TGCT and normal tissues. (C) Bar graph showing the fold difference of 11 monocyte/macrophage signature genes between TGCT and normal tissues using GSE3218 data. (D) ROC curves of preoperative variables regarding discriminating between TGCT and normal tissues in TCGA_GTEx database. *P < .05; **P < .01; ***P < .001. CCL2 = C-C motif chemokine ligand 2, GEPIA2021 = Gene Expression Profiling Interactive Analysis 2021, GTEx = The Genotype-Tissue Expression, LogFC = log₂(fold change); ROC = receiver operating characteristic, TAMs = tumor-associated macrophages, TCGA = the Cancer Genome Atlas, TGCT = testicular germ cell tumor.

Figure 4.

Expression of CCL2 in TGCT^S0. (A) The expression of CCL2 mRNA expression in benign testicular tumor (n = 28) and TGCT^S0 (n = 36). (B) The expression of CCL2 protein examined in benign testicular tumor (n = 28) and TGCT^S0 (n = 36) by IHC analysis. Scale bars represent 100 μm and (C) H-scores (right panel) were indicated. (D) Kaplan–Meier plot showing PFS in patients with TGCT^S0 divided into high CCL2 and low CCL2 groups. (E) Correlation between CCL2 expression and clinicopathological features of patients with TGCT^S0. CCL2 = C-C motif chemokine ligand 2, H-score = (percentage of weak intensity × 1) + (percentage of moderate intensity × 2) + (percentage of strong intensity × 3), HR = hazard ration, IHC = immunohistochemistry, PFS = progression free survival, TGCT^S0 = testicular germ cell tumor with stage S0.

Figure 5.

Functional analyses of CCL2 in TGCT. (A) The GO&KEGG analysis and (B) molecular network of CCL2 in TGCT. (C) GSEA analysis on High CCL2 versus Low CCL2 TGCT specimens in TCGA database. GSEA enrichment plot showing for [Reactome] Extracellular Matrix Organization and [Reactome] Activation of Matrix Metalloproteinases gene set. (D) Correlation analyses between the expression of CCL2 and MMPs expression in TGCT. (E) CCL2/MMPs mediated macrophages infiltrating promoting tumorigenesis of TGCT. BP = biological process, CC = cellular component, CCL2 = C-C motif chemokine ligand 2, GSEA = Gene Set Enrichment Analysis, GO = Gene Ontology, KEGG = The Kyoto Encyclopedia of Genes and Genomes, MF = molecular function, MMPs = matrix metalloproteinases, NES = normalized enrichment score, TAMs = tumor-associated macrophages, TCGA = the Cancer Genome Atlas, TGCT = testicular germ cell tumor.