Eckhard Klieser

T cell exhaustion: from pathophysiological basics to tumor immunotherapy

The immune system is capable of distinguishing between danger- and non-danger signals, thus inducing either an appropriate immune response against pathogens and cancer or inducing self-tolerance to avoid autoimmunity and immunopathology. One of the mechanisms that have evolved to prevent destruction by the immune system, is to functionally silence effector T cells, termed T cell exhaustion, which is also exploited by viruses and cancers for immune escape In this review, we discuss some of the phenotypic markers associated with T cell exhaustion and we summarize current strategies to reinvigorate exhausted T cells by blocking these surface marker using monoclonal antibodies.

Histone deacetylation meets miRNA: epigenetics and post-transcriptional regulation in cancer and chronic diseases

Introduction: Epigenetic regulation via DNA methylation, histone acetylation, as well as by microRNAs (miRNAs) is currently in the scientific focus due to its role in carcinogenesis and its involvement in initiation, progression and metastasis. While many target genes of DNA methylation, histone acetylation and miRNAs are known, even less information exists as to how these mechanisms cooperate and how they may regulate each other in a specific pathological context. For further development of therapeutic approaches, this review presents the current status of the crosstalk of histone acetylation and miRNAs in human carcinogenesis and chronic diseases. Areas covered: This article reviews information from comprehensive PubMed searches to evaluate relevant literature with a focus on possible association between histone acetylation, miRNAs and their targets. Our analysis identified specific miRNAs which collaborate with histone deacetylases (HDACs) and cooperatively regulate several relevant target genes. Expert opinion: Fourteen miRNAs could be linked to the expression of eight HDACs influencing the α-(1,6)-fucosyltransferase, polycystin-2 and the fibroblast-growth-factor 2 pathways. Focusing on the complex linkage of miRNA and HDAC expression could give deeper insights in new ‘druggable’ targets and might provide possible novel therapeutic approaches in future.

Robust linear regression model of Ki-67 for mitotic rate in gastrointestinal stromal tumors.

Risk stratification of gastrointestinal stromal tumors (GISTs) by tumor size, lymph node and metastasis status is crucially affected by mitotic activity. To date, no studies have quantitatively compared mitotic activity in hematoxylin and eosin (H&E)-stained tissue sections with immunohistochemical markers, such as phosphohistone H3 (PHH3) and Ki-67. According to the TNM guidelines, the mitotic count on H&E sections and immunohistochemical PHH3-stained slides has been assessed per 50 high-power fields of 154 specimens of clinically documented GIST cases. The Ki-67-associated proliferation rate was evaluated on three digitalized hot spots using image analysis. The H&E-based mitotic rate was found to correlate significantly better with Ki-67-assessed proliferation activity than with PHH3-assessed proliferation activity (r=0.780; P<0.01). A linear regression model (analysis of variance; P<0.001) allowed reliable predictions of the H&E-associated mitoses based on the Ki-67 expression alone. Additionally, the Ki-67-associated proliferation revealed a higher and significant impact on the recurrence and metastasis rate of the GIST cases than by the classical H&E-based mitotic rate. The results of the present study indicated that the mitotic rate may be reliably and time-efficiently estimated by immunohistochemistry of Ki-67 using only three hot spots.

Role of histone deacetylases in pancreas: Implications for pathogenesis and therapy

In the last years, our knowledge of the pathogenesis in acute and chronic pancreatitis (AP/CP) as well as in pancreatic cancerogenesis has significantly diversified. Nevertheless, the medicinal therapeutic options are still limited and therapeutic success and patient outcome are poor. Epigenetic deregulation of gene expression is known to contribute to development and progression of AP and CP as well as of pancreatic cancer. Therefore, the selective inhibition of aberrantly active epigenetic regulators can be an effective option for future therapies. Histone deacetylases (HDACs) are enzymes that remove an acetyl group from histone tails, thereby causing chromatin compaction and repression of transcription. In this review we present an overview of the currently available literature addressing the role of HDACs in the pancreas and in pancreatic diseases. In pancreatic cancerogenesis, HDACs play a role in the important process of epithelial-mesenchymal-transition, ubiquitin-proteasome pathway and, hypoxia-inducible-factor-1-angiogenesis. Finally, we focus on HDACs as potential therapeutic targets by summarizing currently available histone deacetylase inhibitors.

GERD—Barrett—Adenocarcinoma: Do We Have Suitable Prognostic and Predictive Molecular Markers?

Due to unfavorable lifestyle habits (unhealthy diet and tobacco abuse) the incidence of gastroesophageal reflux disease (GERD) in western countries is increasing. The GERD-Barrett-Adenocarcinoma sequence currently lacks well-defined diagnostic, progressive, predictive, and prognostic biomarkers (i) providing an appropriate screening method identifying the presence of the disease, (ii) estimating the risk of evolving cancer, that is, the progression from Barretts esophagus (BE) to esophageal adenocarcinoma (EAC), (iii) predicting the response to therapy, and (iv) indicating an overall survival—prognosis for EAC patients. Based on histomorphological findings, detailed screening and therapeutic guidelines have been elaborated, although epidemiological studies could not support the postulated increasing progression rates of GERD to BE and EAC. Additionally, proposed predictive and prognostic markers are rather heterogeneous by nature, lack substantial proofs, and currently do not allow stratification of GERD patients for progression, outcome, and therapeutic effectiveness in clinical practice. The aim of this paper is to discuss the current knowledge regarding the GERD-BE-EAC sequence mainly focusing on the disputable and ambiguous status of proposed biomarkers to identify promising and reliable markers in order to provide more detailed insights into pathophysiological mechanisms and thus to improve prognostic and predictive therapeutic approaches.

Comprehensive immunohistochemical analysis of histone deacetylases in pancreatic neuroendocrine tumors: HDAC5 as a predictor of poor clinical outcome ☆ ☆☆

Epigenetic factors contribute to carcinogenesis, tumor promotion, and chemoresistance. Histone deacetylases (HDACs) are epigenetic regulators that primarily cause chromatin compaction, leading to inaccessibility of promoter regions and eventually gene silencing. Many cancer entities feature overexpression of HDACs. Currently, the role of HDACs in pancreatic neuroendocrine tumors (pNETs) is unclear. We analyzed the expression patterns of all HDAC classes (classes I, IIA, IIB, III, and IV) in 5 human tissue microarrays representing 57 pNETs resected between 1997 and 2013 and corresponding control tissue. All pNET cases were characterized clinically and pathologically according to recent staging guidelines. The investigated cases included 32 (56.1%) female and 25 (43.9%) male pNET patients (total n=57, 47.4% immunohistochemically endocrine positive). Immunohistochemical profiling revealed a significant up-regulation of all HDAC classes in pNET versus control, with different levels of intensity and extensity ranging from 1.5- to >7-fold up-regulation. In addition, expression of several HDACs (HDAC1, HDAC2, HDAC5, HDAC11, and Sirt1) was significantly increased in G3 tumors. Correlation analysis showed a significant association between the protein expression of HDAC classes I, III, and IV and rate of the pHH3/Ki-67-associated mitotic and proliferation index. Furthermore, especially HDAC5 proved as a negative predictor of disease-free and overall survival in pNET patients. Overall, we demonstrate that specific members of all 4 HDAC classes are heterogeneously expressed in pNET. Moreover, expression of HDACs was associated with tumor grading, proliferation markers, and patient survival, therefore representing interesting new targets in pNET treatment.

Relevance of MicroRNA200 Family and MicroRNA205 for Epithelial to Mesenchymal Transition and Clinical Outcome in Biliary Tract Cancer Patients

Extensive stromal interaction is one reason for the dismal outcome of biliary tract cancer (BTC) patients. Epithelial to mesenchymal transition (EMT) is involved in tumor invasion and metastasis and is partly regulated by microRNAs (miRs). This study explores the expression of anti-EMT miR200 family (miR141, −200a/b/c, −429) and miR205 as well as the EMT-related proteins E-cadherin and vimentin in a panel of BTC cell lines and clinical specimens by quantitative real-time polymerase chain reaction, Western blot and immunohistochemistry, respectively. MicroRNA expression was correlated to (i) the expression patterns of E-cadherin and vimentin; (ii) clinicopathological characteristics; and (iii) survival data. MicroRNA-200 family and miR205 were expressed in all BTC cells and clinical specimens. E-cadherin and vimentin showed a mutually exclusive expression pattern in both, in vitro and in vivo. Expression of miR200 family members positively correlated with E-cadherin and negatively with vimentin expression in BTC cells and specimens. High expression of miR200 family members (but not miR205) and E-cadherin was associated with longer survival, while low miR200 family and high vimentin expression was a predictor of unfavorable survival. Overall, the current study demonstrates the relevance of the miR200 family in EMT of BTC tumors and suggests these miRs as predictors for positive outcome.

Differential role of Hedgehog signaling in human pancreatic (patho-) physiology: An up to date review.

Since the discovery of the Hedgehog (Hh) pathway in drosophila melanogaster, our knowledge of the role of Hh in embryonic development, inflammation, and cancerogenesis in humans has dramatically increased over the last decades. This is the case especially concerning the pancreas, however, real therapeutic breakthroughs are missing until now. In general, Hh signaling is essential for pancreatic organogenesis, development, and tissue maturation. In the case of acute pancreatitis, Hh has a protective role, whereas in chronic pancreatitis, Hh interacts with pancreatic stellate cells, leading to destructive parenchym fibrosis and atrophy, as well as to irregular tissue remodeling with potency of initiating cancerogenesis. In vitro and in situ analysis of Hh in pancreatic cancer revealed that the Hh pathway participates in the development of pancreatic precursor lesions and ductal adenocarcinoma including critical interactions with the tumor microenvironment. The application of specific inhibitors of components of the Hh pathway is currently subject of ongoing clinical trials (phases 1 and 2). Furthermore, a combination of Hh pathway inhibitors and established chemotherapeutic drugs could also represent a promising therapeutic approach. In this review, we give a structured survey of the role of the Hh pathway in pancreatic development, pancreatitis, pancreatic carcinogenesis and pancreatic cancer as well as an overview of current clinical trials concerning Hh pathway inhibitors and pancreas cancer.

NCCN-IPI score-independent prognostic potential of pretreatment uric acid levels for clinical outcome of diffuse large B-cell lymphoma patients

Background:Blood-based parameters are gaining increasing interest as potential prognostic biomarkers in patients with diffuse large B-cell lymphoma (DLBCL). The aim of this study was to comprehensively evaluate the prognostic significance of pretreatment plasma uric acid levels in patients with newly diagnosed DLBCL.Methods:The clinical course of 539 DLBCL patients, diagnosed and treated between 2004 and 2013 at two Austrian high-volume centres with rituximab-based immunochemotherapy was evaluated retrospectively. The prognostic influence of uric acid on overall survival (OS) and progression-free survival (PFS) were studied including multi-state modelling, and analysis of conditional survival.Results:Five-year OS and PFS were 50.4% (95% CI: 39.2–60.6) and 44.0% (33.4–54.0) in patients with uric acid levels above the 75th percentile of the uric acid distribution (Q3, cut-off: 6.8 mg dl−1), and 66.2% (60.4–71.5) and 59.6% (53.7–65.0%) in patients with lower levels (log-rank P=0.002 and P=0.0045, respectively). In univariable time-to-event analysis, elevated uric acid levels were associated with a worse PFS (hazard ratio (HR) per 1 log increase in uric acid 1.47, 95% CI: 1.10–1.97, P=0.009) and a worse OS (HR=1.60, 95% CI: 1.16–2.19, P=0.004). These associations prevailed upon multivariable adjustment for the NCCN-IPI score. Uric acid levels significantly improved the predictive performance of the R-IPI and NCCN-IPI scores, and in multi-state analysis, it emerged as a highly significant predictor of an increased risk of death without developing recurrence (transition-HR=4.47, 95% CI: 2.17–9.23, P<0.0001).Conclusions:We demonstrate that elevated uric acid levels predict poor long-term outcomes in DLBCL patients beyond the NCCN-IPI risk index.

Thermographic real-time-monitoring of surgical radiofrequency and microwave ablation in a perfused porcine liver model

Radiofrequency ablation (RFA) and microwave ablation (MWA) are currently the dominant modalities to treat unresectable liver tumors. Monitoring the ablation process with b-mode-sonography is often hampered by artefacts. Furthermore, vessels may cause cooling in the adjacent tumor target (heat-sink-effect) with risk of local recurrence. The present study evaluated infrared-thermography to monitor surgical RFA/MWA and detect heat-sink-effects in real-time. RFA and MWA of perfused porcine livers was conducted at peripheral and central-vessel-adjacent locations, and monitored by real-time thermography. Ablation was measured and evaluated by gross pathology. The mean time for ablation was significantly longer in RFA compared with MWA (8 vs. 2 min). Although mean macroscopic ablation diameter was similar (RFA, 3.17 cm; MWA, 3.38 cm), RFA showed a significant heat-sink-effect compared with MWA. The surface temperature during central RFA near vessels was 1/3 lower compared with peripheral RFA (47.11±8.35°C vs. 68.72±12.70°C; P<0.001). There was no significant difference in MWA (50.52±8.35°C vs. 50.18±10.35°C; P=0.74). In conclusion, thermography is suitable to monitor the correct ablation with MWA and RFA. The results of the current study demonstrated a significant heat-sink-effect for RFA, but not MWA near vessels. MWA reaches consistent surface temperatures much faster than RFA. With further in vivo validation, thermography may be useful to ensure appropriate ablation particularly near vulnerable or vascular structures.