Tissue Engineering von zellbesiedelten Polyurethan-Aortenklappenprothesen

In fortgeschrittenen Stadien von Herzklappenerkrankungen stellt der Klappenersatz durch eine Herzklappenprothese eine anerkannte Therapie dar. Derzeit zugelassene und erhältliche Klappenprothesen sind limitiert durch folgende Faktoren: eingeschränkte Verfügbarkeit (z.B. Homograft), begrenzte Haltbarkeit (biologische Klappenprothesen) oder Blutungsrisiko durch erforderliche Antikoagulation (mechanische Klappenprothesen). Tissue engineerte Herzklappenprothesen sind ein vielversprechender Ansatz, um diese Einschränkungen zu überwinden. Das Ziel dieser Arbeit ist es die mechanische Integrität der Zellschichten, inklusive der Ausbildung einer extrazellulären Matrix und Genexpressionsveränderungen auf tissue engineerten Herzklappen zu vergleichen: eine Gruppe nach highflow Perfusion, und eine Gruppe nach highflow Perfusion mit vorangegangener niedrigeren Konditionierungs-Perfusion. Polyurethan Herzklappen wurden in einem speziellen 3D-rotierbarem Bioreaktor dynamisch mit Fibroblasten und Endothelzellen aus humanen Vena saphena magna Segmenten beschichtet. Die besiedelten Polyurethanklappen wurden in zwei Bioreaktoren unterschiedlichen Fluss-Bedingungen ausgesetzt: Gruppe A (n = 6) ausschließlich im highflow Bioreaktor: 3 Tage, je 24 Stunden bei 1 l/min, 1.5 l/min, und anschließend bei 2 l/min; Gruppe B (n = 6): initial 5 Tage im Konditionierungs-Bioreaktor bei 1 l/min, mit anschließender Perfusion im highflow Bioreaktor für weitere 3 Tage, je 24 Stunden bei 1 l/min, 1.5 l/min, und bei 2 l/min. Gewebeproben wurden mittels Immunzytologie, Immunhistochemie, Rasterelektronenmikroskopie und real time Polymerase Kettenreaktion (rt-PCR) analysiert. Die immunhistochemische Färbung ergab eine gleichmäßige Färbung des mehrlagigen Zellüberzuges in beiden Gruppen. Rasterelektronenmikroskopie zeigte ebenfalls eine kunfluente Zellschicht in beiden Gruppen. Rt-PCR verzeichnete höhere Expressionen von IL-6 und MCP-1 in der Konditionierungs-Reaktor Gruppe. Immunhistochemisch konnte man in dieser Gruppe insgesamt eine stärkere Expression extrazellulärer Matrixproteine sowie eine deutlichere Färbung des Zellüberzuges mit dem Hinweis auf höhere Zellschichten beobachten. Wir konnten zeigen, dass highflow Perfusion nicht die Integrität der Zelloberfläche von besiedelten Polyurethanherzklappen kompromittiert. Bioreaktoren unserer Arbeitsgruppe ermöglichen Methoden, um die Reaktion von Zellen auf Scherkräfte in vitro zu erhöhen und letztendlich die mechanischen Eigenschaften von tissue engineerten kardiovaskulären Prothesen zu optimieren

Tissue Engineering von zellbesiedelten Polyurethan-Aortenklappenprothesen

In fortgeschrittenen Stadien von Herzklappenerkrankungen stellt der Klappenersatz durch eine Herzklappenprothese eine anerkannte Therapie dar. Derzeit zugelassene und erhältliche Klappenprothesen sind limitiert durch folgende Faktoren: eingeschränkte Verfügbarkeit (z.B. Homograft), begrenzte Haltbarkeit (biologische Klappenprothesen) oder Blutungsrisiko durch erforderliche Antikoagulation (mechanische Klappenprothesen). Tissue engineerte Herzklappenprothesen sind ein vielversprechender Ansatz, um diese Einschränkungen zu überwinden. Das Ziel dieser Arbeit ist es die mechanische Integrität der Zellschichten, inklusive der Ausbildung einer extrazellulären Matrix und Genexpressionsveränderungen auf tissue engineerten Herzklappen zu vergleichen: eine Gruppe nach highflow Perfusion, und eine Gruppe nach highflow Perfusion mit vorangegangener niedrigeren Konditionierungs-Perfusion. Polyurethan Herzklappen wurden in einem speziellen 3D-rotierbarem Bioreaktor dynamisch mit Fibroblasten und Endothelzellen aus humanen Vena saphena magna Segmenten beschichtet. Die besiedelten Polyurethanklappen wurden in zwei Bioreaktoren unterschiedlichen Fluss-Bedingungen ausgesetzt: Gruppe A (n = 6) ausschließlich im highflow Bioreaktor: 3 Tage, je 24 Stunden bei 1 l/min, 1.5 l/min, und anschließend bei 2 l/min; Gruppe B (n = 6): initial 5 Tage im Konditionierungs-Bioreaktor bei 1 l/min, mit anschließender Perfusion im highflow Bioreaktor für weitere 3 Tage, je 24 Stunden bei 1 l/min, 1.5 l/min, und bei 2 l/min. Gewebeproben wurden mittels Immunzytologie, Immunhistochemie, Rasterelektronenmikroskopie und real time Polymerase Kettenreaktion (rt-PCR) analysiert. Die immunhistochemische Färbung ergab eine gleichmäßige Färbung des mehrlagigen Zellüberzuges in beiden Gruppen. Rasterelektronenmikroskopie zeigte ebenfalls eine kunfluente Zellschicht in beiden Gruppen. Rt-PCR verzeichnete höhere Expressionen von IL-6 und MCP-1 in der Konditionierungs-Reaktor Gruppe. Immunhistochemisch konnte man in dieser Gruppe insgesamt eine stärkere Expression extrazellulärer Matrixproteine sowie eine deutlichere Färbung des Zellüberzuges mit dem Hinweis auf höhere Zellschichten beobachten. Wir konnten zeigen, dass highflow Perfusion nicht die Integrität der Zelloberfläche von besiedelten Polyurethanherzklappen kompromittiert. Bioreaktoren unserer Arbeitsgruppe ermöglichen Methoden, um die Reaktion von Zellen auf Scherkräfte in vitro zu erhöhen und letztendlich die mechanischen Eigenschaften von tissue engineerten kardiovaskulären Prothesen zu optimieren

Performance of the Food and Drug Administration/EMA-approved programmed cell death ligand-1 assays in urothelial carcinoma with emphasis on therapy stratification for first-line use of atezolizumab and pembrolizumab

Background: Recently, the Food and Drug Administration (FDA)/European Medicines Agency (EMA) restricted first-line use of atezolizumab and pembrolizumab in patients with metastasised urothelial carcinoma by defining distinct programmed cell death ligand-1 cut-offs. We analysed the diagnostic performance of all FDA/EMA-approved programmed cell death ligand-1 assays with emphasis on new restrictions for first-line treatment with atezolizumab and pembrolizumab. Patients and methods: Two hundred fifty-one urothelial carcinomas were analysed on tissue microarrays with four cores of each tumour. Stains were performed in certified laboratories on Ventana Benchmark Ultra and Dako Link 48 autostainers. Stains were read on an assay-by-assay basis by two trained pathologists. Overall percentage agreement (OPA) was calculated across the preset cut-offs. Positive percentage agreement (PPA) and negative percentage agreement (NPA) were calculated across different scoring algorithms. Venn diagrams were constructed to illustrate discordance according to the recent FDA/EMA guidelines. Results: The Dako 28-8, 22c3 and the Ventana SP263 assays showed high interassay correlation (r-range 0.83-0.91). Interassay correlation between the Ventana SP142 and the three other assays was moderate (r-range 0.66-0.75). OPA of 93.3% was achieved between the Dako 28-8, 22c3 and Ventana SP263 assays. OPA including the SP142 was 84.1%. Pooled PPA and NPA of different scoring algorithms was 89.4% and 95.3% for the Dako 28-8, 22c3 and the SP263 assays, respectively. With the SP142 assay, pooled PPA was 59.1%. The SP142 assay identifies fewer eligible patients for first-line treatment with atezolizumab/pembrolizumab. Conclusion: Dako 28-8, 22c3 and SP263 assays show interchangeable performance. The SP142 assay shows divergent staining results. Interassay variability leads to different detection rates of eligible patients for first-line treatment with atezolizumab and pembrolizumab. (C) 2018 Elsevier Ltd. All rights reserved

The Tumor Immune Microenvironment Drives a Prognostic Relevance That Correlates with Bladder Cancer Subtypes

Muscle-invasive bladder cancer (MIBC) represents approximately two-thirds of invasive urothelial bladder cancers (UBC) and has high morbidity and mortality. Men are over 3-fold more frequently affected by UBC than women. Despite intensive efforts to improve patient treatment and outcome, two-thirds of patients with UBC will have a recurrence or disease progression within 5 years. We demonstrated that the quantity and spatial distribution of stromal tumor-infiltrating lymphocytes (sTIL) within the tumor immune microenvironment (TIME) predict stages of tumor inflammation, subtypes, and patient survival and correlate with expression of immune checkpoints in an analysis of 542 patients with MIBC. High sTILs indicated an inflamed subtype with an 80% 5-year DSS, and a lack of immune infiltrates identified an uninflamed subtype with a survival rate of less than 25%. A separate immune evading phenotype with upregulated immune checkpoints associated with poor survival. Within the TIME are tertiary lymphoid structures (TLS), which can mediate antitumor activity via immune cells. High TLS amounts and close tumor distance correlated significantly with an inflamed phenotype and favorable survival. The uninflamed and evasion phenotypes showed lowest TLS numbers, farthest tumor distances, and shortest survival. High inflammation also correlated with increased neoantigen load and mutational burden. Patients treated with adjuvant chemotherapy showed a favorable prognosis, which was dependent on high sTILs. Determination of sTILs and tumor subtypes may stratify therapy success and patient survival, and considering sTILs can easily be quantified using simple morphologic parameters, like hematoxylin and eosin, sTILs can be implemented for predicting patient survival in a routine manner

cMET: a prognostic marker in papillary renal cell carcinoma?

The tyrosine-protein kinase c-Met plays a decisive role in numerous cellular processes, as a proto-oncogene that supports aggressive tumor behavior. It is still unknown whether c-Met could be relevant for prognosis of papillary RCC (pRCC). Specimen collection was a collaboration of the PAN-ZAR consortium. Patients' medical history and tumor specimens were collected from 197 and 110 patients with type 1 and 2 pRCC, respectively. Expression of cMET was determined by immunohistochemistry. In total, cMET staining was evaluable in of 97 of 197 type 1 and 63 of 110 type 2 pRCC cases. Five-year overall survival revealed no significant difference in dependence of cMET positivity (cMET(-) vs. cMET(+): pRCC type 1: 84.8% vs. 80.3%, respectively [p = 0.303, log-rank]; type 2: 71.4% vs. 64.4%, respectively [p = 0.239, log-rank]). Interestingly, the subgroup analyses showed a significant difference for cMET expression in T stage and metastases of the pRCC type 2 (p = 0.014, p = 0.022, chi-square). The cMET-positive type 2 collective developed more metastases than the cMET-negative cohort (pRCC type 2 M+: cMET 2 [4.3%] vs. cMET(+): 12 [19%]). cMET expression did not qualify as a prognostic marker in pRCC for overall survival. (C) 2021 Elsevier Inc. All rights reserved

The prognostic impact of Claudin 6 in papillary renal cell carcinoma

Background: Claudins are promising biomarkers for diagnosis and prognosis or targets for treatment. They play a major role in signal transduction and are important in nearly all aspects of tumorigenesis. Claudin 6 is a member of the claudin family and is part of the tight junction molecule. It is reactivated in several cancer types and serves as prognostic marker in, for example, gastric, breast or non small cell lung cancer. The prognostic role of Claudin 6 in renal cell carcinoma (RCC), especially in papillary RCC (pRCC), is still unclear. Patients and Methods: The patients' sample collection was a joint collaboration of the PANZAR consortium. Patients' medical history and tumor specimens were collected from n = 240 and n = 128 patients with type 1 and 2 pRCC, respectively. Expression of Claudin 6 was determined by immunohistochemistry. Results: In total, Claudin 6 staining was positive in 55 of 240 type 1 and 30 of 128 type 2 pRCC cases. Kaplan-Meier analysis disclosed an overall survival of 84% for Claudin 6-compared to 78% for Claudin 6 + in pRCC type 1 tumors (p = 0.449, log-rank) and 68% for Claudin 6-compared to 65.4% for Claudin 6 + in pRCC type 2 tumors (p = 0.364, log-rank). Conclusion: In this study, claudin 6 expression showed no significant association regarding overall survival (OS) and therefore did not qualify as a prognostic marker in pRCC. Future studies will have to determine, whether Claudin 6 plays a prognostic role in other RCC entities. In addition, the function of Claudin 6 as a predictive marker for therapeutic approaches has to be evaluated in future studies

Characterization of PD-1 and PD-L1 Expression in Papillary Renal Cell Carcinoma: Results of a Large Multicenter Study

Understanding the impact of programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) expression becomes increasingly important in renal cell carcinoma (RCC) owing to increasing therapeutic implications. However, little is known in non -clear-cell RCC about the relevance of those immune checkpoint surrogates. Here, we suggest that PD-1/PD-L1 expression in papillary RCC does not have prognostic impact, neither for type 1 nor type 2. However, in advanced disease, further evaluation according to PD-1/PD-L1 is warranted. Background: Programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) play a decisive role as prognostic markers in clear-cell renal cell carcinoma (RCC). To date, the role of PD-1/PD-L1 as a prognostic marker in papillary RCC (pRCC) remains scarce. Patients and Methods: Patients' sample collection was a joint collaboration of the nationwide PANZAR consortium -a multicenter study. Medical history and tumor specimens were collected from 245 and 129 patients with pRCC types 1 and 2, respectively. Expression of PD-1 and PD-L1 was determined by immunohistochemistry in pRCC and tumor-infiltrating mononuclear cells. Results: Of 374 pRCC specimens, 204 type 1 and 97 type 2 were evaluable for PD-1 and PD-L1 expression analysis. In total, PD-1 and PD-L1 expression were found in 8 (4.9%) of 162 and 12 (7.2%) of 166 evaluable pRCC type 1 specimens. Comparably, PD-1 and PD-L1 expression were found in 2 (2.4%) of 83 and 5 (6.2%) of 81 evaluable pRCC type 2 specimens. Hardly any clinically relevant associations between PD-1 and PD-L1 positivity and clinicopathologic or clinical courses were observed, neither in pRCC type 1 nor type 2. Conclusion: The analysis of a large pRCC cohort from a multicenter consortium revealed no impact of PD-1/PD-L1 expression on prognosis in patients with pRCC with predominantly limited disease status, neither for type 1 nor type 2. However, the impact of PD-1 and PD-L1 in more advanced pRCC disease needs further elucidation

Expression of nectin-4 in papillary renal cell carcinoma

Background Nectin-4 contributes to tumor proliferation, lymphangiogenesis and angiogenesis in malignant tumors and is an emerging target in tumor therapy. In renal cell carcinoma (RCC) VEGF-directed tyrosine kinase inhibitors and checkpoint inhibitors are currently treatments of choice. Enfortumab vedotin-ejf (EV) is an antibody drug conjugate that targets Nectin-4. The aim of our study was to investigate the expression of Nectin-4 in a large cohort of papillary RCC specimens. Patients and methods Specimens were derived from the PANZAR consortium (Erlangen, Heidelberg, Herne, Homburg, Mainz, Mannheim, Marburg, Muenster, LMU Munich, TU Munich, and Regensburg). Clinical data and tissue samples from n = 190 and n = 107 patients with type 1 and 2 pRCC, respectively, were available. Expression of Nectin-4 was determined by immunohistochemistry (IHC). Results In total, Nectin-4 staining was moderately or strongly positive in of 92 (48.4%) of type 1 and 39 (36.4%) type 2 of pRCC cases. No associations between Nectin-4 expression and age at diagnosis, gender, grading, and TNM stage was found. 5 year overall survival rate was not statistically different in patients with Nectin-4 negative versus Nectin-4 positive tumors for the overall cohort and the pRCC type 2 subgroup, but higher in patient with Nectin-4 positive pRCC type 1 tumors compared to Nectin-4 negative tumors (81.3% vs. 67.8%, p = 0.042). Conclusion Nectin-4 could not be confirmed as a prognostic marker in pRCC in general. Due to its high abundance on pRCC specimens Nectin-4 is an interesting target for therapeutical approaches e.g. with EV. Clinical trials are warranted to elucidate its role in the pRCC treatment landscape

The Prognostic Impact of PD-L2 in Papillary Renal-Cell Carcinoma

Introduction: Programmed death-1 ligand (PD-L1) has been often studied in different types of renal-cell carcinoma (RCC). For example, in clear-cell renal carcinoma it is well established that programmed death-1 receptor and PD-L1 are important prognostic markers. In contrast, the role of programmed death-2 ligand (PD-L2) as prognostic marker remains unclear. The aim of this study was to evaluate if PD-L2 expression could play a role as a prognostic marker for papillary RCC (pRCC). Methods: The patients' sample collection was a joint collaboration of the PANZAR consortium. Patients' medical history and tumor specimens were collected from n = 240 and n = 128 patients with type 1 and 2 pRCC, respectively. Expression of PD-L2 was determined by immunohistochemistry. In total, PD-L2 staining was evaluable in 185 of 240 type 1 and 99 of 128 type 2 pRCC cases. Results: PD-L2 staining was positive in 67 (36.2%) of type 1 and in 31 (31.3%) of type 2 pRCC specimens. The prevalence of PD-L2+ cells was significantly higher in high-grade type 1 tumors (p = 0.019) and in type 2 patients with metastasis (p = 0.002). Kaplan-Meier analysis disclosed significant differences in 5-year overall survival (OS) for patients with PD-L2- compared to PD-L2+ in pRCC type 1 of 88.4% compared to 73.6% (p = 0.039) and type 2 of 78.8% compared to 39.1% % (p < 0.001). However, multivariate analysis did not identify the presence of PD-L2+ cells neither in type 1 nor type 2 pRCC as an independent predictor of poor OS. Discussion/Conclusion: PD-L2 expression did not qualify as an independent prognostic marker in pRCC. Future studies will have to determine whether anti-PD-L2-targeted treatment may play a role in pRCC and expression can potentially serve as a predictive marker for these therapeutic approaches