• Combination of GP88 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Is an Independent Prognostic Factor for Bladder Cancer Patients

      Eckstein, Markus; Lieb, Verena; Jung, Rudolf; Sikic, Danijel; Weigelt, Katrin; Stöhr, Robert; Geppert, Carol; Weyerer, Veronika; Bertz, Simone; Serrero, Ginette; et al. (MDPI AG, 2021-07-15)
      Urothelial bladder cancer (BCa) is the ninth most commonly diagnosed cancer worldwide and accounts for approximately 3% of global cancer diagnoses. We are interested in prognostic markers that may characterize tumor cells (TCs) and immune cells (ICs) and their relationship in BCa. A potential candidate marker that meets these criteria is progranulin (GP88), which is expressed separately in TCs and ICs. We analyzed GP88 expression by immunohistochemistry (IHC) in 196 muscle-invasive BCa samples using a tissue microarray. The immunoreactive score for GP88 staining in TCs and the percentage of GP88-positive ICs was determined. An easy cutoff for the staining status of TCs (positive vs. negative) and ICs (0% vs. >0%) and, more generally, negative vs. positive GP88 staining could be applied. We detected 93 patients (47.4%) and 92 patients (46.9%) with GP88-positive TCs or ICs, respectively. The IHC results were correlated with clinicopathological and survival data. Positive GP88 staining in TCs appeared to be an independent poor prognostic factor for disease-specific survival (DSS) (RR (relative risk) = 1.74; p = 0.009) and recurrence-free survival (RFS) (RR = 1.92; p = 0.002). In contrast, negative GP88 staining in ICs was an independent negative predictor for overall survival (OS) (RR = 2.18; p < 0.001), DSS (RR = 2.84; p < 0.001) and RFS (RR = 2.91; p < 0.001) in multivariate Cox's regression analysis. When combining GP88 staining in TCs and ICs, a specific combination of GP88-positive TCs and GP88-negative ICs was associated with a 2.54-fold increased risk of death, a 4.21-fold increased risk of disease-specific death and a 4.81-fold increased risk of recurrence compared to GP88-negative TCs and GP88-positive ICs. In summary, GP88 positivity in TCs is a negative prognostic factor for DSS and RFS. In addition, GP88 positivity can mark ICs that are associated with a good prognosis (OS, DSS and RFS). The combination of GP88 staining in TCs and ICs appears to be a significant independent prognostic biomarker in muscle-invasive BCa.
    • Endogenous Retroviral-K Envelope Is a Novel Tumor Antigen and Prognostic Indicator of Renal Cell Carcinoma

      Weyerer, Veronika; Strissel, Pamela L; Stöhr, Christine; Eckstein, Markus; Wach, Sven; Taubert, Helge; Brandl, Lisa; Geppert, Carol I; Wullich, Bernd; Cynis, Holger; et al. (Frontiers Media S.A., 2021-04-22)
      Renal cell carcinoma (RCC) is one of the ten most common cancers for men and women with an approximate 75% overall 5-year survival. Sixteen histological tumor subtypes exist and the most common are papillary, chromophobe and clear cell renal cell carcinoma (ccRCC) representing 85% of all RCC. Although epigenetically silenced, endogenous retroviral (ERV) genes become activated in tumors and function to ignite immune responses. Research has intensified to understand ERV protein function and their role as tumor antigens and targets for cancer (immune) therapy. ERV-K env is overexpressed and implicated as a therapeutic target for breast cancer, however studies in RCC are limited. In this investigation a human RCC tissue microarray (TMA) (n=374) predominantly consisting of the most common histological tumor subtypes was hybridized with an ERV-K env antibody and correlated with patient clinical data. TMA results showed the highest amount of ERV-K env protein expression and the strongest significant membrane expression in ccRCC versus other RCC subtypes. High ERV-K env total protein expression of all tumor subtypes significantly correlated with low tumor grading and a longer disease specific survival using multivariable analyses. Cell proliferation and invasion were assayed using the kidney cell lines HEK293 with wild-type p53 and a ccRCC cell line MZ1257RC mutated for p53. Transfecting these cell lines with a codon optimized ERV-K113 env overexpressing CMV vector was performed with or without 5'-Aza-2'-deoxycytidine (Aza) treatment to sustain promoter de-methylation. MZ1257RC showed induction of ERV-K113 expression and significantly increased both proliferation and invasion in the presence or absence of Aza. HEK293 cells demonstrated a restriction of ERV-K113 env expression and invasion with no changes in proliferation in the absence of Aza. However, in the presence of Aza despite increased ERV-K113 env expression, an inhibition of HEK293 proliferation and a further restriction of invasion was found. This study supports ERV-K env as a single prognostic indicator for better survival of RCC, which we propose represents a new tumor antigen. In addition, ERV-K env significantly regulates proliferation and invasion depending on p53 status and Aza treatment.
    • Expression of AR-V7 (Androgen receptor variant 7) protein in granular cytoplasmic structures is an independent prognostic factor in prostate cancer patients

      König, Paul; Eckstein, Markus; Jung, Rudolf; Abdulrahman, Amer; Guzman, Juan; Weigelt, Katrin; Serrero, Ginette; Hayashi, Jun; Geppert, Carol; Stöhr, Robert; et al. (MDPI AG, 2020-09-01)
      Prostate cancer (PCa) is the second most common cancer, causing morbidity and mortality among men world-wide. The expression of the androgen receptor (AR) and its splice variants is a crucial factor of prostate cancer biology that has not been comprehensively studied in PCa tumors. The aim of this study was to characterize the protein expression of the AR and its splice variant, AR-V7, and their subcellular distributions in PCa by immunohistochemistry and to correlate the results to the clinicopathological data and prognosis. Immunohistochemical staining for AR and AR-V7 was performed on a tissue microarray (TMA) with specimens from 410 PCa patients using an immunoreactive score (IRS) or only the percentage of AR-V7 staining in cytoplasmic granules. Nuclear or cytoplasmic AR staining was not associated with prognosis. AR-V7 staining was only occasionally observed in the nucleus. However, AR-V7 staining in the cytoplasm or in cytoplasmic granules was associated with relapse-free survival (RFS). AR-V7 staining of the cytoplasm was associated with a shorter RFS, whereas AR-V7 staining of cytoplasmic granules was associated with a longer RFS. In a multivariate Cox’s regression analysis, only negative (<5%) AR-V7 staining of cytoplasmic granules remained an independent prognostic factor for RFS (HR = 5.3; p = 0.006). In a further subgroup analysis by multivariate Cox’s regression analysis, AR-V7 was an independent prognostic factor in the following groups: age ≤ 65 (HR = 9.7; p = 0.029), negative CK20 staining (HR = 7.0; p = 0.008), and positive perineural invasion (HR = 3.7; p = 0.034). Altogether, AR-V7 protein in granular cytoplasmic structures is an independent prognostic factor for RFS in PCa patients.
    • Integration of spatial PD-L1 expression with the tumor immune microenvironment outperforms standard PD-L1 scoring in outcome prediction of urothelial cancer patients

      Weyerer, Veronika; Strissel, Pamela L.; Strick, Reiner; Sikic, Danijel; Geppert, Carol I.; Bertz, Simone; Lange, Fabienne; Taubert, Helge; Wach, Sven; Breyer, Johannes; et al. (MDPI AG, 2021-05-02)
      Background: Immune therapy has gained significant importance in managing urothelial cancer. The value of PD-L1 remains a matter of controversy, thus requiring an in-depth analysis of its biological and clinical relevance. Methods: A total of 193 tumors of muscle-invasive bladder cancer patients (MIBC) were assessed with four PD-L1 assays. PD-L1 scoring results were correlated with data from a comprehensive digital-spatial immune-profiling panel using descriptive statistics, hierarchical clustering and uni-/multivariable survival analyses. Results: PD-L1 scoring algorithms are heterogeneous (agreements from 63.1% to 87.7%), and stems from different constellations of immune and tumor cells (IC/TC). While Ventana IC5% algorithm identifies tumors with high inflammation and favorable baseline prognosis, CPS10 and the TCarea25%/ICarea25% algorithm identify tumors with TC and IC expression. Spatially organized immune phenotypes, which correlate either with high PD-L1 IC expression and favorable prognosis or constitutive PD-L1 TC expression and poor baseline prognosis, cannot be resolved properly by PD-L1 algorithms. PD-L1 negative tumors with relevant immune infiltration can be detected by sTILs scoring on HE slides and digital CD8+ scoring. Conclusions: Contemporary PD-L1 scoring algorithms are not sufficient to resolve spatially distributed MIBC immune phenotypes and their clinical implications. A more comprehensive view of immune phenotypes along with the integration of spatial PD-L1 expression on IC and TC is necessary in order to stratify patients for ICI. © 2021 by the authors.