adjuvant chemotherapy) was not associated with recurrence rate, but this analysis is underpowered. death-ligand 1 (PD-L1)) and markers of systemic inflammation could predict progression/relapse and death in the cohort of 180 patients with testicular germ-cell tumors (GCTs). Expression of PD-L1 and VISTA was assessed by immunohistochemistry utilizing tissue microarrays. To estimate systemic inflammation neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR), and platelet-to-lymphocyte ratio (PLR) were calculated. We found high PD-L1 and VISTA expression on tumor-associated immune cells (TAICs) in 89 (49.44%) and 63 (37.22%) of GCTs, respectively, whereas tumor cells besides trophoblastic elements were almost uniformly negative. High PD-L1 was associated with seminomatous histology and lower stage. Relapses in stage I patients occurred predominantly in cases with low numbers of PD-L1 and VISTA-expressing TAICs. In stage II/III disease, the combination of low VISTA-expressing TAICs and high PLR was identified as predictor of shorter event-free survival (HR 4.10; 1.48C11.36, = 0.006) and overall survival (HR 15.56, 95% CI 1.78C135.51, = 0.001) independently of tumor histology and location of metastases. We exhibited that the assessment of immune checkpoint proteins on TAICs may serve as a valuable prognostic factor in patients with high-risk testicular GCTs. Further study is usually warranted to explore the predictive utility of these biomarkers in GCTs. ( 0.05. Differences in BZS EFS between groups were assessed using log-rank test and visualized with KaplanCMeier curves. Statistical analysis was performed with Statistica 13.3 software (TIBCO Software Inc., Palo Alto, CA, USA) and R statistical environment . Boxplots were plotted using the ggplot2 package . KaplanCMeier curves were plotted using the survminer and ggsci packages [33,34]. 3. Results 3.1. Expression of PD-L1 and VISTA in Testicular Germ Cell Tumors High expression of both VISTA and PD-L1 was noted in the choriocarcinoma component. Otherwise, tumor cells were unfavorable for both markers, except for 3 embryonal carcinoma cases with a focal PHA690509 PD-L1 staining. Tumor-associated immune cells with high VISTA expression were observed in 63 cases (37.22%), while those with high PD-L1 expression in 89 cases (49.44%). A complete lack of PD-L1 expression was noted in seven seminomas (7.2% of seminomas), and three cases of MGCT (3.6% of nonseminomas). Only two cases of seminoma showed complete absence of VISTA+ cells (2.1% of seminomas). Pure teratomas and areas of teratoma PHA690509 in mixed tumors were uniformly characterized by low number of TAICs and very weak or absent expression of immune checkpoints (PD-L1 histoscore 5, percentage of VISTA+ cells 5%). As previously described, in seminomas PD-L1-expressing TAICs were distributed mainly along the tumor interface and within the fibrovascular septa  (Physique 2ACC)?, while VISTA-positive TAICs were mainly located within septa with a PHA690509 less prominent interface enhancement (Physique 3A,D,E). In nonseminomatous tumors, VISTA-expressing cells frequently formed small clusters within tumor parenchyma or in peritumoral borders of fibrovascular septa (Physique 3B,C), while the distribution of PD-L1 positive TAICs was more heterogeneous and patchy (Physique 2D). Non-neoplastic Leydig cells and endothelia showed VISTA expression regardless of tumor histology. Open in a separate window Physique 2 Representative examples of PD-L1 staining. (A). Intense staining in TAICs along the interface between fibrovascular septa and tumor cells; (B). Moderately positive to unfavorable TAICs in seminoma; (C). Weakly positive to unfavorable TAICs in seminoma; (D). Embryonal carcinoma with moderate PD-L1 staining in TAICs. Abbreviations: PD-L1: programmed death-ligand 1; TAICs: tumor-associated immune cells. Open in a separate window Physique 3 Representative examples.