Immune checkpoint inhibitors (ICI) have been approved for patients with clear cell renal cell carcinoma (ccRCC), but not all patients benefit from ICI. One reason is the tumor microenvironment (TME) that has substantial influence on patient's prognosis and therapy response. Thus, we comprehensively analyzed the TME of ccRCC regarding prognostic and predictive properties. Tumor-infiltrating CD3-positive T-cells, CD8-positive cytotoxic T-lymphocytes (CTLs), regulatory T-cells, B-cells, plasma cells, macrophages, granulocytes, programmed cell death receptor 1 (PD-1), and its ligand PD-L1 were examined in a large hospital-based series of ccRCC with long-term follow-up information (n = 756) and in another patient collective with information on response to nivolumab therapy (n = 8). Tissue microarray technique and digital image analysis were used. Relationship between immune cell infiltration and tumor characteristics, cancer-specific survival (CSS), or response to ICI was examined. Univariate survival analysis revealed that increased tumor-infiltrating B-cells, T-cells, and PD-1-positive cells were significantly associated with favorable CSS and high levels of intratumoral granulocytes, macrophages, cytotoxic T-cells, and PD-L1 significantly with poor CSS. High CTL or B-cell infiltration and high PD-L1 expression of ccRCC tumor cells qualified as independent prognostic biomarkers for patients' CSS. Significantly higher densities of intratumoral T-cells, CTLs, and PD-1-positive immune cells were observed in ccRCC with response to ICI compared with patients with mixed or no response (CD3: p = 0.003; CD8: p = 0.006; PD-1: p = 0.01). This study shows that subsets of tumor-infiltrating leukocytes in the TME and also PD-1/PD-L1 provide prognostic and predictive information for patients with ccRCC.

Whereas the lack of biomarkers in penile cancer (PeCa) impedes the development of efficacious treatment protocols, preliminary evidence suggests that c-MET and associated signaling elements may be dysregulated in this disorder. In the following study, we investigated whether c-MET and associated key molecular elements may have prognostic and therapeutic utility in PeCa. Formalin-fixed, paraffin-embedded tumor tissue from therapy-naïve patients with invasive PeCa was used for tissue microarray (TMA) analysis. Immunohistochemical staining was performed to determine the expression of the proteins c-MET, PPARg, β-catenin, snail, survivin, and n-MYC. In total, 94 PeCa patients with available tumor tissue were included. The median age was 64.9 years. High-grade tumors were present in 23.4%, and high-risk HPV was detected in 25.5%. The median follow-up was 32.5 months. High expression of snail was associated with HPV-positive tumors. Expression of β-catenin was inversely associated with grading. In both univariate COX regression analysis and the log-rank test, an increased expression of PPARg and c-MET was predictive of inferior disease-specific survival (DSS). Moreover, in multivariate analysis, a higher expression of c-MET was independently associated with worse DSS. Blocking c-MET with cabozantinib and tivantinib induced a significant decrease in viability in the primary PeCa cell line UKF-PeC3 isolated from the tumor tissue as well as in cisplatin- and osimertinib-resistant sublines. Strikingly, a higher sensitivity to tivantinib could be detected in the latter, pointing to the promising option of utilizing this agent in the second-line treatment setting.

The PI3K/mTOR/AKT pathway might represent an intriguing option for treatment of penile cancer (PeCa). We aimed to assess whether members of this pathway might serve as biomarkers and targets for systemic therapy. Tissue of primary cancer from treatment-naïve PeCa patients was used for tissue microarray analysis. Immunohistochemical staining was performed with antibodies against AKT, pAKT, mTOR, pmTOR, pS6, pPRAS, p4EBP1, S6K1 and pp70S6K. Protein expression was correlated with clinicopathological characteristics as well as overall survival (OS), disease-specific survival (DSS), recurrence-free survival (RFS) and metastasis-free survival (MFS). AKT inhibition was tested in two primarily established, treatment-naïve PeCa cell lines by treatment with capivasertib and analysis of cell viability and chemotaxis. A total of 76 patients surgically treated for invasive PeCa were included. Higher expression of AKT was significantly more prevalent in high-grade tumors and predictive of DSS and OS in the Kaplan–Meier analysis, and an independent predictor of worse OS and DSS in the multivariate regression analysis. Treatment with pan-AKT inhibitor capivasertib in PeCa cell lines induced a significant downregulation of both total AKT and pAKT as well as decreased cell viability and chemotaxis. Selected protein candidates of the mTOR/AKT signaling pathway demonstrate association with histological and survival parameters of PeCa patients, whereas AKT appears to be the most promising one.

Background: In the treatment of clear cell renal cell carcinoma (ccRCC), nivolumab is an established component of the first-line therapy with a favorable impact on progression free survival and overall survival. However, treatment-related adverse effects occur and, to date, there is no approved predictive biomarker for patient stratification. Thus, the aim of this study was to establish an ex vivo tissue slice culture model of ccRCC and to elucidate the impact of nivolumab on tumor infiltrating immune cells. Methods: Fresh tumor tissue of ccRCC was treated with the immune checkpoint inhibitor nivolumab using ex vivo tissue slice culture (TSC). After cultivation, tissue slices were formalin-fixed, immunohistochemically stained and analyzed via digital image analysis. Results: The TSC model was shown to be suitable for ex vivo pharmacological experiments on intratumoral immune cells in ccRCC. PD1 expression on tumor infiltrating immune cells was dose-dependently reduced after nivolumab treatment (p < 0.01), whereas density and proliferation of tumor infiltrating T-cells and cytotoxic T-cells were inter-individually altered with a remarkable variability. Tumor cell proliferation was not affected by nivolumab. Conclusions: This study could demonstrate nivolumab-dependent effects on PD1 expression and tumor infiltrating T-cells in TSC of ccRCC. This is in line with results from other scientific studies about changes in immune cell proliferation in peripheral blood in response to nivolumab. Thus, TSC of ccRCC could be a further step to personalized medicine in terms of testing the response of individual patients to nivolumab.

Zinc finger protein 580 (ZNF580) was reported to modulate angiogenesis, endothelial homeostasis and blood pressure control. ZNF580 regulated genes include VEGF-A and IL-8. However, it is unknown if ZNF580 could play a role during inflammation. The aim of this study was to find out if ZNF580 affects the expression of IL-6, if it occurs in monocytic cells and responds to inflammatory mediators. Overexpression of ZNF580 reduced LPS-induced promotor activity of IL-6. Consistently, overexpression of ZNF580 reduced by half the LPS-induced expression of IL-6. ZNF580 was strongly expressed in the nucleus of MonoMac6, a human monocytic cell line. LPS-stimulated IL-6 secretion increased when ZNF580 was suppressed with siRNA. After stimulation of MonoMac6 with LPS for 24 h, ZNF580 negatively correlated with the amount of secreted IL-6. In response to LPS, ZNF580 was increased within the first 8 h, followed by a marked decrease after 16 h. This decrease coincided with sustained IL-6 production. This study demonstrated that ZNF580 inhibits LPS-induced expression of IL-6. ZNF580 was highly expressed in monocytic cells and therefore may contribute to the modulation of its IL-6 production, at least in response to LPS. This suggests cooperation between ZNF580 and NFκB, which could play a role during sepsis.