Untersuchung der Mediatorkomplex-Untereinheiten MED12 und MED15 in verschiedenen Tumorentitäten

Ziel dieser Arbeit war zu untersuchen, ob den Mediator-Untereinheiten MED12 und MED15 eine Bedeutung in der Tumorentwicklung und –progression zukommt. Zunächst sollten Gewebsanalysen zeigen, ob MED12 und MED15 ein unterschiedliches Expressionsmuster im benignen und Tumorgewebe verschiedener Stadien aufweisen. Die Analyse der immunohistochemischen Färbung zeigt im Vergleich zum Normalgewebe eine verstärkte nukleäre MED12- und MED15-Expression in primären Prostatakarzinomen und Lymphknotenmetastasen. In kastrations-resistenten Tumoren (CRPC) sind MED12 und MED15 am stärksten exprimiert. Der Vergleich der MED12- und MED15-Expression zwischen kastrations-resistenten Karzinomen vor und nach Androgenentzugs-Behandlung zeigt, dass MED15 nach Therapie eine signifikant stärkere Expression aufweist. Während unsere Analysen keine Assoziation zwischen der MED12-Expression und der Überlebensrate der Patienten ergaben, weisen Patienten mit MED15-Überexpression im CRPC ein reduziertes Überleben auf. Im Vergleich zu benignem Plattenepithel weisen primäre Plattenepithelkarzinome, Lymphknotenmetastasen und Rezidivtumore ein stärkeres nukleäres MED15-Expressionslevel auf. Der MED15-Überexpressionsstatus im Primärtumor gleicht dem in korrespondierenden Lymphknotenmetastasen in 74% der Fälle. Das Vorhandensein einer MED15-Überexpression in Tumoren von Patienten, die einen Rezidivtumor entwickelten, geht mit einer höheren Mortalitätsrate einher. Außerdem weisen Tumore aus der Mundhöhle und dem Oropharynx eine signifikant stärkere MED15-Expression auf als Tumore aus dem Hypopharynx oder Larynx, und sind mit einer höheren Mortalitätsrate assoziiert. Im Gewebe primärer Prostatakarzinome und Metastasen konnten wir mittels IHC feststellen, dass die nukleäre MED12-Expression mit der Anzahl der für die Proliferationsmarker Ki67 und pHH3 positiv gefärbter Kerne signifikant korreliert. Im MTT-Assay zeigten MED12-Knockdown-Zellen eine reduzierte Viabilität. Wir stellten keinen Unterschied der Annexin-V-Intensität zwischen Kontroll- und MED12-Knockdown-Zellen fest. Im Gegensatz dazu zeigten Zellen mit MED12-Knockdown einen veränderten Zellzyklus. In Übereinstimmung damit beobachteten wir eine gesteigerte Expression des Zellzyklus-Inhibitors p27 in MED12-Knockdown-Zellen, die wir mittels ICC analysierten. In den Plattenepithelkarzinomzelllienen HSC3 und SCC25 führte ein MED15-Knockdown zu signifikant reduzierter Zellviabilität im MTT-Assay sowie einer verringerten Expression des Proliferationsmarkers Ki67. MED15-Knockdown-Prostatakarzinomzellen zeigten unter Androgenentzug eine um 50% reduzierte Viabilität sowie eine Apoptoserate von 13% im Vergleich zu 1% in Kontrollzellen. In anschließenden Untersuchungen sollte herausgearbeitet werden, in welche Signalwege MED12 und MED15 involviert sind und ob wir im Tumorgewebe sowie in Zelllinien eine Assoziation zwischen der Aktivierung potenziell onkogener Signalwege und der Expression von MED12 und MED15 feststellen können. Im Prostatakarzinomgewebe stellten wir fest, dass die MED12-Überexpression mit einer stärkeren Expression des TGFß Aktivitätsmarkers phospho-SMAD3 assoziiert ist. Wir beobachteten in der immunozytochemischen Färbung und im Western blot eine verminderte Vimentin-Expression in Zellen mit MED12-Knockdown. Mittels IHC stellten wir eine signifikante Korrelation zwischen der MED15-Expression und der Aktivität des PI3K und TGFß Signalweges fest. In Übereinstimmung damit führte eine Behandlung von PC3-Zellen mit rekombinantem TGFß sowie von LNCaP-Zellen mit dem PI3K-Aktivator Epidermal growth factor (EGF) zur verstärkten MED15-Expression, die wir mittels Western blot verifizierten. Umgekehrt zeigten Zellen, die unter Androgenentzug mit dem TGFß-Rezeptor-Inhibitor SB431542 oder dem PI3K-Inhibitor LY294002 behandelten wurden, eine reduzierte MED15-Expression. In Plattenepithelkarzinomzellen des Kopf- und Halsbereiches untersuchten wir ebenfalls die Korrelation zwischen der MED15-Expression und der TGFß-Aktivität. Hierbei zeigte sich wie in Prostatakarzinomzellen, dass MED15 mit stärkerer phospho-SMAD3-Expression assoziert ist und das ein MED15-Knockdown zu verringerter TGFß-Aktivität führt. Zusammenfassend weisen unsere Ergebnisse von Untersuchungen an Prostatakarzinomen und Plattenepithelkarzinomen darauf hin, dass MED12 und MED15 an Regulationsmechanismen beteiligt sind, die das Malignitätspotenzial von Tumorzellen steigern. Basierend auf diesen Ergebnissen vermuten wir, dass Patienten in fortgeschrittenen Tumorstadien von einer zielgerichteten Therapie profitieren, die eine Reduktion der Aktivität von MED12 und MED15 beinhaltet

Early CT and FDG-metabolic tumour volume changes show a significant correlation with survival in stage I-III small cell lung cancer: A hypothesis generating study

BACKGROUND: Many patients with stage I–III small cell lung cancer (SCLC) experience disease progression short after the completion of concurrent chemoradiotherapy (CRT). The purpose of the current study was to evaluate whether CT or FDG metabolic response early after the start of chemotherapy, but before the beginning of chest RT, is predictive for survival in SCLC. METHODS: Fifteen stage I–III SCLC patients treated with concurrent CRT with an FDG-PET and CT scan available before the start of chemotherapy and after or during the first cycle of chemotherapy, but before the start of radiotherapy, were selected. The metabolic volume (MV) was defined both within the primary tumour and in the involved nodal stations using the 40% (MV40) and 50% (MV50) threshold of the maximum SUV. Metabolic and CT response was assessed by the relative change in MV and CT volume, respectively, between both time points. The association between response and overall survival (OS) was analysed by univariate cox regression analysis. The minimum follow-up was 18 months. RESULTS: Reductions in MV40 and MV50 were −36 ± 38% (126.4 to 68.7 cm(3)) and −44 ± 38% (90.2 to 27.8 cm(3)), respectively. The median CT volume reduction was −40 ± 64% (190.6 to 113.8 cm(3)). MV40 and MV50 changes showed a significant association with survival (HR = 1.02, 95% CI: 1.00–1.04 (p = 0.042); HR = 1.02, 95% CI: 1.00–1.04 (p = 0.048), respectively), indicating a 2% increase in survival probability for 1% reduction in metabolic volume. The CT volume change was also significantly correlated with survival (HR = 1.01, 95% CI: 1.00–1.03, p = 0.007). CONCLUSIONS: This hypothesis generating study shows that both the early CT and the MV changes show a significant correlation with survival in SCLC. A prospective study is planned in a larger patient cohort to allow multivariate analysis, with the final aim to select patients early during treatment that could benefit from dose intensification or alternative treatment

Prognostic Value of the New Prostate Cancer International Society of Urological Pathology Grade Groups

Gleason grading is the best independent predictor for prostate cancer (PCa) progression. Recently, a new PCa grading system has been introduced by the International Society of Urological Pathology (ISUP) and is recommended by the World Health Organization (WHO). Following studies observed more accurate and simplified grade stratification of the new system. Aim of this study was to compare the prognostic value of the new grade groups compared to the former Gleason Grading and to determine whether re-definition of Gleason Pattern 4 might reduce upgrading from prostate biopsy to radical prostatectomy (RP) specimen. A cohort of men undergoing RP from 2002 to 2015 at the Hospital of Goeppingen (Goeppingen, Germany) was used for this study. In total, 339 pre-operative prostatic biopsies and corresponding RP specimens, as well as additional 203 RP specimens were re-reviewed for Grade Groups according to the ISUP. Biochemical recurrence-free survival (BFS) after surgery was used as endpoint to analyze prognostic significance. Other clinicopathological data included TNM-stage and pre-operative PSA level. Kaplan–Meier analysis revealed risk stratification of patients based on both former Gleason Grading and ISUP Grade Groups, and was statistically significant using the log-rank test (p < 0.001). Both grading systems significantly correlated with TNM-stage and pre-operative PSA level (p < 0.001). Higher tumor grade in RP specimen compared to corresponding pre-operative biopsy was observed in 44 and 34.5% of cases considering former Gleason Grading and ISUP Grade Groups, respectively. Both, former Gleason Grading and ISUP Grade Groups predict survival when applied on tumors in prostatic biopsies as well as RP specimens. This is the first validation study on a large representative German community-based cohort to compare the former Gleason Grading with the recently introduced ISUP Grade Groups. Our data indicate that the ISUP Grade Groups do not improve predictive value of PCa grading and might be less sensitive in deciphering tumors with 3 + 4 and 4 + 3 pattern on RP specimen. However, the Grade Group system results less frequently in an upgrading from biopsy to the corresponding RP specimens, indicating a lower risk to miss potentially aggressive tumors not represented on biopsies

Immune Cell Infiltration of the Primary Tumor, Not PD-L1 Status, Is Associated With Improved Response to Checkpoint Inhibition in Metastatic Melanoma

Immune checkpoint inhibition has resulted in dramatic improvements in overall and relapse-free survival in patients with metastatic melanoma. The most commonly used immune checkpoint inhibitors are monoclonal antibodies targeting programmed cell death protein 1 and cytotoxic T-lymphocyte-associated protein 4. Unfortunately, a significant subset of patients fail to respond to these therapies, which has resulted in intense research efforts to identify the factors which are associated with treatment response. To this end, we investigated immune cell infiltration in primary melanomas and melanoma metastases, in addition to tumor cell PD-L1 expression, to determine whether these factors are associated with an improved outcome after immune checkpoint inhibition. Indeed, the extent of the immune cell infiltration in the primary melanoma, measured by the Immunoscore, was associated with a significantly improved response to immune checkpoint inhibition in terms of increased overall survival. However, the Immunoscore did not predict which patients would respond to treatment. The Immunoscore was significantly reduced in metastases when compared to primary melanomas. In contrast, PD-L1 expression, exhaustively tested using four commercially available anti-PD-L1 clones, did not differ significantly between primary tumors and melanoma metastases and was not associated treatment response. Whilst replication in larger, prospective studies is required, our data demonstrates the relevance of immune cell infiltration in the primary melanoma as a novel marker of improved overall survival in response to immune checkpoint inhibition

Comprehensive characterization of the prostate tumor microenvironment identifies CXCR4/CXCL12 crosstalk as a novel antiangiogenic therapeutic target in prostate cancer

Background: Crosstalk between neoplastic and stromal cells fosters prostate cancer (PCa) progression and dissemination. Insight in cell-to-cell communication networks provides new therapeutic avenues to mold processes that contribute to PCa tumor microenvironment (TME) alterations. Here we performed a detailed characterization of PCa tumor endothelial cells (TEC) to delineate intercellular crosstalk between TEC and the PCa TME. Methods: TEC isolated from 67 fresh radical prostatectomy (RP) specimens underwent multi-omic ex vivo characterization as well as orthogonal validation of both TEC functions and key markers by immunohistochemistry (IHC) and immunofluorescence (IF). To identify cell-cell interaction targets in TEC, we performed single-cell RNA sequencing (scRNA-seq) in four PCa patients who underwent a RP to catalogue cellular TME composition. Targets were cross-validated using IHC, publicly available datasets, cell culture expriments as well as a PCa xenograft mouse model. Results: Compared to adjacent normal endothelial cells (NEC) bulk RNA-seq analysis revealed upregulation of genes associated with tumor vasculature, collagen modification and extracellular matrix remodeling in TEC. PTGIR, PLAC9, CXCL12 and VDR were identified as TEC markers and confirmed by IF and IHC in an independent patient cohort. By scRNA-seq we identified 27 cell (sub)types, including endothelial cells (EC) with arterial, venous and immature signatures, as well as angiogenic tip EC. A focused molecular analysis revealed that arterial TEC displayed highest CXCL12 mRNA expression levels when compared to all other TME cell (sub)populations and showed a negative prognostic role. Receptor-ligand interaction analysis predicted interactions between arterial TEC derived CXCL12 and its cognate receptor CXCR4 on angiogenic tip EC. CXCL12 was in vitro and in vivo validated as actionable TEC target by highlighting the vessel number- and density- reducing activity of the CXCR4-inhibitor AMD3100 in murine PCa as well as by inhibition of TEC proliferation and migration in vitro. Conclusions: Overall, our comprehensive analysis identified novel PCa TEC targets and highlights CXCR4/CXCL12 interaction as a potential novel target to interfere with tumor angiogenesis in PCa

PD-L1 Dependent Immunogenic Landscape in Hot Lung Adenocarcinomas Identified by Transcriptome Analysis

Background: Lung cancer is the most frequent cause of cancer-related deaths worldwide. The clinical development of immune checkpoint blockade has dramatically changed the treatment paradigm for patients with lung cancer. Yet, an improved understanding of PD-1/PD-L1 checkpoint blockade- responsive biology is warranted. Methods: We aimed to identify the landscape of immune cell infiltration in primary lung adenocarcinoma (LUAD) in the context of tumoral PD-L1 expression and the extent of immune infiltration ("hot" vs. "cold" phenotype). The study comprises LUAD cases (n = 138) with "hot" (≥150 lymphocytes/HPF) and "cold" (<150 lymphocytes/HPF) tumor immune phenotype and positive (>50%) and negative (<1%) tumor PD-L1 expression, respectively. Tumor samples were immunohistochemically analyzed for expression of PD-L1, CD4, and CD8, and further investigated by transcriptome analysis. Results: Gene set enrichment analysis defined complement, IL-JAK-STAT signaling, KRAS signaling, inflammatory response, TNF-alpha signaling, interferon-gamma response, interferon-alpha response, and allograft rejection as significantly upregulated pathways in the PD-L1-positive hot subgroup. Additionally, we demonstrated that STAT1 is upregulated in the PD-L1-positive hot subgroup and KIT in the PD-L1-negative hot subgroup. Conclusion: The presented study illustrates novel aspects of PD-L1 regulation, with potential biological relevance, as well as relevance for immunotherapy response stratification

Expression of Prostate-Specific Membrane Antigen (PSMA) on Biopsies Is an Independent Risk Stratifier of Prostate Cancer Patients at Time of Initial Diagnosis

Background: Stratifying prostate cancer (PCa) patients into risk groups at time of initial diagnosis enabling a risk-adapted disease management is still a major clinical challenge. Existing studies evaluating the prognostic potential of PSMA (prostate-specific membrane antigen) for PCa were performed on radical prostatectomy specimens (RPE), i.e., decision making for disease management was already completed at time of sample analysis. Aim of our study was to assess the prognostic value of PSMA expression for PCa patients on biopsies at time of initial diagnosis.Methods: PSMA expression was assessed by immunohistochemistry on 294 prostate biopsies with corresponding RPE, 621 primary tumor foci from 242 RPE, 43 locally advanced or recurrent tumors, 34 lymph node metastases, 78 distant metastases and 52 benign prostatic samples. PSMA expression was correlated with clinico-pathologic features. Primary endpoint was recurrence free survival. Other clinicopathologic features included WHO/ISUP grade groups, PSA serum level, TNM-stage, and R-status. Chi-square test, ANOVA-analyses, Cox-regression, and log-rank tests were performed for statistical analyses.Results: High PSMA expression on both biopsy and RPE significantly associates with a higher risk of disease recurrence following curative surgery. The 5-year-recurrence free survival rates were 88.2, 74.2, 67.7 and 26.8% for patients exhibiting no, low, medium, or high PSMA expression on biopsy, respectively. High PSMA expression on biopsy was significant in multivariate analysis predicting a 4-fold increased risk of disease recurrence independently from established prognostic markers. PSMA significantly increases during PCa progression.Conclusion: PSMA is an independent prognostic marker on biopsies at time of initial diagnosis and can predict disease recurrence following curative therapy for PCa. Our study proposes the application of the routinely used IHC marker PSMA for outcome prediction and decision making in risk-adapted PCa management on biopsies at time of initial diagnosis

Regulation of the Fruit-Specific PEP Carboxylase SlPPC2 Promoter at Early Stages of Tomato Fruit Development

The SlPPC2 phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) gene from tomato (Solanum lycopersicum) is differentially and specifically expressed in expanding tissues of developing tomato fruit. We recently showed that a 1966 bp DNA fragment located upstream of the ATG codon of the SlPPC2 gene (GenBank AJ313434) confers appropriate fruit-specificity in transgenic tomato. In this study, we further investigated the regulation of the SlPPC2 promoter gene by analysing the SlPPC2 cis-regulating region fused to either the firefly luciferase (LUC) or the β-glucuronidase (GUS) reporter gene, using stable genetic transformation and biolistic transient expression assays in the fruit. Biolistic analyses of 5′ SlPPC2 promoter deletions fused to LUC in fruits at the 8th day after anthesis revealed that positive regulatory regions are mostly located in the distal region of the promoter. In addition, a 5′ UTR leader intron present in the 1966 bp fragment contributes to the proper temporal regulation of LUC activity during fruit development. Interestingly, the SlPPC2 promoter responds to hormones (ethylene) and metabolites (sugars) regulating fruit growth and metabolism. When tested by transient expression assays, the chimeric promoter:LUC fusion constructs allowed gene expression in both fruit and leaf, suggesting that integration into the chromatin is required for fruit-specificity. These results clearly demonstrate that SlPPC2 gene is under tight transcriptional regulation in the developing fruit and that its promoter can be employed to drive transgene expression specifically during the cell expansion stage of tomato fruit. Taken together, the SlPPC2 promoter offers great potential as a candidate for driving transgene expression specifically in developing tomato fruit from various tomato cultivars