Markus W. Löffler

Phosphoglycerate Kinase 1 a Promoting Enzyme for Peritoneal Dissemination in Gastric Cancer

Peritoneal carcinomatosis is a frequent finding in gastric cancer associated with a poor prognosis. The features that enable gastric tumors to disseminate are poorly understood until now. Previously, we showed elevated mRNA levels of phosphoglycerate kinase 1 (PGK1), an adenosine triphosphate‐generating enzyme in the glycolytic pathway, the chemokine receptor 4 (CXCR4), the corresponding chemokine ligand 12 (CXCL12) and β‐catenin in specimens from gastric cancer patients with peritoneal carcinomatosis. In this study, the influence of PGK1 on CXCR4 and β‐catenin was assessed as well as the invasiveness of PGK1 overexpressing cancer cells. In this current study, we found that PGK1 regulates the expression of CXCR4 and β‐catenin at the mRNA and protein levels. On the other hand, CXCR4 regulates the expression of PGK1. Plasmid‐mediated overexpression of PGK1 dramatically increased the invasiveness of gastric cancer cells. Interestingly, inhibition of CXCR4 in cells overexpressing PGK1 produced only a moderate reduction of invasiveness suggesting that, PGK1 itself has a critical role in tumor invasiveness. Immunohistochemistry in specimens from diffuse gastric cancer patients also revealed an overexpression of PGK1 in patients with development of peritoneal carcinomatosis. Therefore, PGK1 may be a crucial enzyme in peritoneal dissemination. Together these findings suggest that the enhanced expression of PGK1 and its signaling targets CXCR4 and β‐catenin in gastric cancer cells promote peritoneal carcinomatosis. Thus, PGK1 may serve as prognostic marker and/or be a potential therapeutic target to prevent dissemination of gastric carcinoma cells into the peritoneum.

Phosphoglycerate kinase 1 promoting tumor progression and metastasis in gastric cancer - detected in a tumor mouse model using positron emission tomography/magnetic resonance imaging.

Background/Aims: Tumor dissemination is frequent in gastric cancer and implies a poor prognosis. Cure is only achievable provided an accurate staging is performed at primary diagnosis. In previous studies we were able to show a relevant impact of increased phosphoglycerate kinase 1 expression (PGK1; a glycolytic enzyme) on invasive properties of gastric cancer in-vivo and in-vitro. Thus the aim of the present study was to evaluate the effect of enhanced PGK1 expression in gastric cancer employing magnetic resonance (MR)-imaging combined with positron emission tomography (PET), a recently emerging new high resolution imaging technique in a mouse model. Methods: A metastatic nude mouse model simulating human gastric cancer behavior by orthotopic tumor implantation was established. Mice were divided into one control group (n=5) and two experimental groups (n=30) divided by half in animals baring tumors from MKN45-cells and MKN45-cells with plasmid-mediated overexpression of PGK1. In the course of tumor growth MR-imaging and PET/MRI fusion was performed. Successively experimental animals were examined macroscopically and histopathologically regarding growth, metastasis and PGK1 expression. Results: Elevated PGK1 expression increased invasive and metastatic behavior of implanted gastric tumors significantly. MR/PET- imaging results in-vivoand subsequent ex-vivo findings concerning tumor growth and metastasis correlated excellently and could be underlined by concordant immuohistochemical PGK1 staining. Conclusion: Consistent in-vivo findings suggest that PGK1 might be crucially involved in gastric malignancy regarding growth and metastasis, which was also underlined by novel imaging techniques. Thus, PGK1 may be exploited as a prognostic marker and/or be of potential therapeutic value preventing malignant dissemination.

An impedance-based cytotoxicity assay for real-time and label-free assessment of T-cell-mediated killing of adherent cells

The in vitro assessment of T-cell-mediated cytotoxicity plays an important and increasingly relevant role both in preclinical target evaluation and during immunomonitoring to accompany clinical trials employing targeted immunotherapies. For a long time, the gold standard for this purpose has been the chromium release assay (CRA). This end point assay, however, shows several disadvantages including the inevitable use of radioactivity. Based on electrical impedance measurements (using the xCELLigence system), we have established a label-free assay, facilitating the real-time monitoring of T-cell-mediated cytotoxicity. The coculture of peptide-specific T-cell lines with peptide-loaded target cells reproducibly led to a decrease in impedance due to induced apoptosis and detachment of target cells. Comparing our results to the standard CRA assay, we could demonstrate that impedance-based measurements show comparable results after short incubation periods (6h) but outperform the CRA both in reproducibility and sensitivity after prolonged incubation (24h), enabling the detection of target cell lysis with an effector to target ratio as low as 0.05:1. The impedance-based assay represents a valuable and highly sensitive tool for label-free real-time high throughput analysis of T-cell-mediated cytotoxicity.

The immunopeptidomic landscape of ovarian carcinomas

Significance Despite the revolution in cancer therapy initiated by checkpoint inhibitors, durable clinical responses remain sporadic in many types of cancer, including ovarian cancer. Understanding which antigens are essentially presented by tumor cells and further able to be recognized by T cells provides a major step toward novel effective targeted immunotherapies. In this study, we comprehensively analyzed the immunopeptidomic landscape of ovarian carcinoma and compared it to variety of benign sources to identify antigens exclusively presented on tumor cells. With personalized therapies moving into the focus of clinical cancer therapy, we further present insights on how gene-expression analysis and immunohistochemistry can support antigen selection for individualized immunotherapy. Immunotherapies, particularly checkpoint inhibitors, have set off a revolution in cancer therapy by releasing the power of the immune system. However, only little is known about the antigens that are essentially presented on cancer cells, capable of exposing them to immune cells. Large-scale HLA ligandome analysis has enabled us to exhaustively characterize the immunopeptidomic landscape of epithelial ovarian cancers (EOCs). Additional comparative profiling with the immunopeptidome of a variety of benign sources has unveiled a multitude of ovarian cancer antigens (MUC16, MSLN, LGALS1, IDO1, KLK10) to be presented by HLA class I and class II molecules exclusively on ovarian cancer cells. Most strikingly, ligands derived from mucin 16 and mesothelin, a molecular axis of prognostic importance in EOC, are prominent in a majority of patients. Differential gene-expression analysis has allowed us to confirm the relevance of these targets for EOC and further provided important insights into the relationship between gene transcript levels and HLA ligand presentation.

Personalized peptide vaccine-induced immune response associated with long-term survival of a metastatic cholangiocarcinoma patient

Graphical abstract

Superficial Wound Swabbing: A novel method of sampling and processing wound fluid for subsequent immunoassay analysis in diabetic foot ulcerations

OBJECTIVE In diabetic foot ulcers, wound fluid inflammatory mediators have previously been proposed as surrogate markers for nonhealing. However, currently available wound fluid sampling techniques are not suitable for clinical practice due to low levels of exudate and a high logistical effort. The aim of this investigation was to assess 1) the technique of superficial wound swabbing for harvesting wound fluid; and 2) the quality of the collected fluid for immunoassay analysis of inflammatory mediators. RESEARCH DESIGN AND METHODS Both nylon-flocked swabs and film dressings were used to collect wound fluid from foot ulcers of diabetic patients. In randomly selected patients, levels of wound fluid inflammatory mediators and matrix metalloproteases were determined using multiplexed bead-based sandwich immunoassays with respect to both sampling methods. Wound fluid spike-in experiments were performed to evaluate the impact of different sample processing protocols on subsequent immunoassay analysis. RESULTS Using the swabbing technique, a median amount of 40 µL (2–120 µL) wound exudate was collected, which allowed the measurement of several multiplex panels. Comparing both sampling methods, a similar qualitative protein recovery was observed with a trend to analyte enrichment by swabbing. Sample processing using swabs did not affect analyte recovery, with the exception of interleukin (IL)-8, thymus and activation-regulated chemokine, IL-17A, interferon-γ–induced protein 10, and IL-4. CONCLUSIONS The quality of wound fluid collected by superficial swabbing is not inferior to the current standard technique. Combined with subsequent bead-based sandwich immunoassay analysis, this new method offers a noninvasive technique, suitable for daily clinical routines, for assessment of inflammatory activity in diabetic foot ulcers.

A frequent hypofunctional IRAK2 variant is associated with reduced spontaneous hepatitis C virus clearance

Patients carrying very rare loss‐of‐function mutations in interleukin‐1 receptor–associated kinase 4 (IRAK4), a critical signaling mediator in Toll‐like receptor signaling, are severely immunodeficient, highlighting the paramount role of IRAK kinases in innate immunity. We discovered a comparatively frequent coding variant of the enigmatic human IRAK2, L392V (rs3844283), which is found homozygously in ∼15% of Caucasians, to be associated with a reduced ability to induce interferon‐alpha in primary human plasmacytoid dendritic cells in response to hepatitis C virus (HCV). Cytokine production in response to purified Toll‐like receptor agonists was also impaired. Additionally, rs3844283 was epidemiologically associated with a chronic course of HCV infection in two independent HCV cohorts and emerged as an independent predictor of chronic HCV disease. Mechanistically, IRAK2 L392V showed intact binding to, but impaired ubiquitination of, tumor necrosis factor receptor–associated factor 6, a vital step in signal transduction. Conclusion: Our study highlights IRAK2 and its genetic variants as critical factors and potentially novel biomarkers for human antiviral innate immunity. (Hepatology 2015;62:1375–1387)

Rapamycin-Induced Impaired Wound Healing Is Associated with Compromised Tissue Lactate Accumulation and Extracellular Matrix Remodeling

Introduction: Extracellular matrix (ECM) remodeling involving matrix metalloproteinases (MMPs) and wound lactate accumulation are essential elements of tissue repair. The aim of this study was to investigate whether rapamycin-induced impaired healing is associated with compromised wound fluid lactate accumulation and altered ECM remodeling. Methods: Polyvinyl alcohol sponges were subcutaneously implanted in male C57/BL6 mice. Animals were randomized to daily intraperitoneal treatment with either vehicle or 1.5 mg/kg rapamycin. After 7 or 14 days, sponges were harvested to collect wound fluid for subsequent analyses. Wounds were excised for assessment of tensile strength. Results: After 7 days, wound hydroxyproline content was significantly decreased due to rapamycin therapy, whereas the observed difference in tensile strength marginally failed to show statistical significance. In addition, rapamycin reduced wound lactate accumulation and enhanced MMP-2 protein expression, and both MMP-2 and MMP-9 activity. At day 14, wound tensile strength and hydroxyproline content were significantly lower along with an increase in MMP-2 and MMP-9 activity in rapamycin-treated mice. Similarly, wound fluid lactate concentration and MMP-2 protein expression were found to be persistently decreased and increased, respectively. Conclusions: Rapamycin affects tissue repair by interfering with fundamental mechanisms involved in healing, namely lactate accumulation and ECM remodeling.

Wound Fluid in Diabetic Foot Ulceration: More Than Just an Undefined Soup?

Valid and reproducible sampling techniques as well as processing protocols are required for the assessment of biomarkers and mediators contained in wound exudate. Moreover, the ideal technique should be easy to use even in daily clinical routine. This is challenging since wound fluid represents an inhomogeneous mixture of different exogenous and endogenous sources. Analyzing wound fluid, however, may facilitate clinical decision making. Many techniques for obtaining wound fluid have been described. There is very little validation data, and the array of different techniques appears confusing. Structuring and new standards are needed to avoid wound fluid sampling yielding an “undefined soup.” A lot of wound fluid parameters have been analyzed, although none of them have made its way into clinical practice. Nevertheless, basic principles of wound healing have been established from wound fluid analysis. With adequate techniques suitable for daily practice, basic research might foster our clinical understanding of wound healing with implications for new therapies. So far, research has mainly concentrated on analyzing available sample material with respect to either a wide variety of analytes or comparing acute with chronic wound exudate. Clinical endpoints such as healing or wound infection as well as longitudinal data may indeed be more valuable for clinical practice, enabling the discovery of meaningful biomarkers using a suitable technique.

Haploidentical hematopoietic cell transplantation using in vitro T cell depleted grafts as salvage therapy in patients with disease relapse after prior allogeneic transplantation

Disease relapse after one or more allogeneic hematopoietic cell transplantations (HCT) represents a therapeutic challenge with all options bearing a significant morbidity and mortality. Haploidentical HCT may induce more pronounced anti-leukemic effects and was evaluated at our center in 25 consecutive patients with disease relapse after preceding HCT receiving haploidentical grafts after in vitro T cell depletion. Overall survival at 1 and 2xa0years was 32 and 14%, respectively. Of note, patients with complete remission (CR) before haploidentical HCT had a very favorable overall survival of 41.7% at 2xa0years. Cumulative incidence of non-relapse mortality was 36 and 40% at 1 and 2xa0years, respectively. With a cumulative incidence for relapse of 36 and 45.6% at 1 and 2xa0years, disease-free survival (DFS) was 28 and 14.4%, respectively. Here also, patients with CR before haploidentical HCT had a favorable DFS of 42% at 2xa0years. Only very limited acute (11 patients (44%) with a median grade 1) and chronic graft versus host disease (GvHD) (5 patients (11%), limited grade only) was observed. The main complications and causes of death comprised—besides relapse—infections and bleeding complications. Hence, haploidentical HCT can achieve long-term survival comparable to second transplantation with matched or mismatched donors for patients with otherwise deleterious prognosis and should be considered as a treatment option for patients experiencing disease relapse after previous allogeneic HCT.