Stefan Wirths

Immunologic and Clinical Responses after Vaccinations with Peptide-Pulsed Dendritic Cells in Metastatic Renal Cancer Patients

A phase I trial was conducted to evaluate the feasibility, safety, and efficacy of a dendritic cell-based vaccination in patients with metastatic renal cell carcinoma (RCC). Autologous mature dendritic cells derived from peripheral blood monocytes were pulsed with the HLA-A2-binding MUC1 peptides (M1.1 and M1.2). For the activation of CD4(+) T-helper lymphocytes, dendritic cells were further incubated with the PAN-DR-binding peptide PADRE. Dendritic cell vaccinations were done s.c. every 2 weeks for four times and repeated monthly until tumor progression. After five dendritic cell injections, patients additionally received three injections weekly of low-dose interleukin-2 (1 million IE/m(2)). The induction of vaccine-induced T-cell responses was monitored using enzyme-linked immunospot and Cr release assays. Twenty patients were included. The treatment was well tolerated with no severe side effects. In six patients, regression of the metastatic sites was induced after vaccinations with three patients achieving an objective response (one complete response, two partial responses, two mixed responses, and one stable disease). Additional four patients were stable during the treatment for up to 14 months. MUC1 peptide-specific T-cell responses in vivo were detected in the peripheral blood mononuclear cells of the six patients with objective responses. Interestingly, in patients responding to the treatment, T-cell responses to antigens not used for vaccinations, such as adipophilin, telomerase, or oncofetal antigen, could be detected, indicating that epitope spreading might occur. This study shows that MUC1 peptide-pulsed dendritic cells can induce clinical and immunologic responses in patients with metastatic RCC.

Dendritic cells in cancer vaccines.

Dendritic cells (DC) are recognized as the most potent antigen-presenting cells with the ability to stimulate naive resting T cells and to initiate primary immune responses. Encouraging results in vaccination studies in animal models and the development of protocols to generate sufficient numbers of human DC for clinical application have led to attempts to verify the feasibility and efficacy of this approach in patients in the context of Phase I/II vaccination trials. This review aims to present a concise overview of the current knowledge in DC development and biology and describes the recent data of the first published DC-based vaccination studies. These preliminary trials indicate that immunotherapies utilizing DC-presenting tumor-associated antigens can safely be administered to patients with cancer and induce significant immunologic and clinical responses.

Steady-state neutrophil homeostasis is dependent on TLR4/TRIF signaling

UNLABELLED Polymorphonuclear neutrophil granulocytes (neutrophils) are tightly controlled by an incompletely understood homeostatic feedback loop adjusting the marrows supply to peripheral needs. Although it has long been known that marrow cellularity is inversely correlated with G-CSF levels, the mechanism linking peripheral clearance to production remains unknown. Herein, the feedback response to antibody induced neutropenia is characterized to consist of G-CSF–dependent shifts of marrow hematopoietic progenitor populations including expansion of the lin-/Sca-1/c-kit (LSK) and granulocyte macrophage progenitor (GMP) compartments at the expense of thrombopoietic and red cell precursors. Evidence is provided that positive feedback regulation is independent from commensal germs as well as T, B, and NK cells. However, in vivo feedback is impaired in TLR4-/- and TRIF-/-, but not MyD88-/- animals. In conclusion, steady-state neutrophil homeostasis is G-CSF–dependent and regulated through pattern-recognition receptors,thereby directly linking TLR-triggering to granulopoiesis. KEY POINTS Steady-state and emergency granulopoiesis are both dependent on TLR signaling.

Current insights into neutrophil homeostasis

Neutrophil granulocytes represent the first immunologic barrier against invading pathogens, and neutropenia predisposes to infection. However, neutrophils may also cause significant collateral inflammatory damage. Therefore, neutrophil numbers are tightly regulated by an incompletely understood homeostatic feedback loop adjusting the marrows supply to peripheral needs. Granulocyte colony‐stimulating factor (G‐CSF) is accepted to be the major determinant of neutrophil production, and G‐CSF levels have, soon after its discovery, been described to be inversely correlated with neutrophil counts. A neutrophil sensor, or “neutrostat,” has, therefore, been postulated. The prevailing feedback hypothesis was established in adhesion molecule–deficient mice; it includes macrophages and Th17 cells, which determine G‐CSF levels in response to the number of peripherally transmigrated, apoptosing neutrophils. Recent work has deepened our understanding of homeostatic regulation of neutrophil granulopoiesis, but there are still inconsistent findings and unresolved questions when it comes to a plausible hypothesis, similar to the feedback control models of red cell or platelet homeostasis.

RANKL Expression, Function, and Therapeutic Targeting in Multiple Myeloma and Chronic Lymphocytic Leukemia

Bone destruction is a prominent feature of multiple myeloma, but conflicting data exist on the expression and pathophysiologic involvement of the bone remodeling ligand RANKL in this disease and the potential therapeutic benefits of its targeted inhibition. Here, we show that RANKL is expressed by primary multiple myeloma and chronic lymphocytic leukemia (CLL) cells, whereas release of soluble RANKL was observed exclusively with multiple myeloma cells and was strongly influenced by posttranscriptional/posttranslational regulation. Signaling via RANKL into multiple myeloma and CLL cells induced release of cytokines involved in disease pathophysiology. Both the effects of RANKL on osteoclastogenesis and cytokine production by malignant cells could be blocked by disruption of RANK-RANKL interaction with denosumab. As we aimed to combine neutralization of RANKL with induction of antibody-dependent cellular cytotoxicity of natural killer (NK) cells against RANKL-expressing malignant cells and as denosumab does not stimulate NK reactivity, we generated RANK-Fc fusion proteins with modified Fc moieties. The latter displayed similar capacity compared with denosumab to neutralize the effects of RANKL on osteoclastogenesis in vitro, but also potently stimulated NK cell reactivity against primary RANKL-expressing malignant B cells, which was dependent on their engineered affinity to CD16. Our findings introduce Fc-optimized RANK-Ig fusion proteins as attractive tools to neutralize the detrimental function of RANKL while at the same time potently stimulating NK cell antitumor immunity.

Neutrophil homeostasis and its regulation by danger signaling

Hematopoiesis in general is demand driven and adaptive, but in contrast to erythropoiesis or thrombocytopoiesis, our knowledge on how neutrophil production is adapted to individual needs remains incomplete. Recently, neutrophil homeostasis has been shown to depend on danger receptors, macrophages, and even circadian rhythms. Puzzle pieces for a broader view of neutrophil homeostasis accumulate, and we will herein try to put seemingly contradictory evidence in a perspective of neutrophil homeostasis and emergency granulopoiesis determined by innate immunologic signaling.

Shared Cell Surface Marker Expression in Mesenchymal Stem Cells and Adult Sarcomas

Advanced adult soft‐tissue sarcomas (STSs) are rare tumors with a dismal prognosis and limited systemic treatment options. STSs may originate from mesenchymal stem cells (MSCs); the latter have mainly been isolated from adult bone marrow as plastic‐adherent cells with differentiation capacity into mesenchymal tissues. Recently, a panel of antibodies has been established that allows for the prospective isolation of primary MSCs with high selectivity. Similar to cancer stem cells in other malignancies, sarcoma stem cells may bear immunophenotypic similarity with the corresponding precursor, that is, MSCs. We therefore set out to establish the expression pattern of MSC markers in sarcoma cell lines and primary tumor samples by flow cytometry. In addition, fibroblasts from different sources were examined. The results document a significant amount of MSC markers shared by sarcoma cells. The expression pattern includes uniformly expressed markers, as well as MSC markers that only stained subpopulations of sarcoma cells. Expression of W5C5, W8B2 (tissue nonspecific alkaline phosphatase [TNAP]), CD344 (frizzled‐4), and CD271 marked subpopulations displaying increased proliferation potential. Moreover, CD271+ cells displayed in vitro doxorubicin resistance and an increased capacity to form spheres under serum‐free conditions. Interestingly, another set of antigens, including the bona fide progenitor cell markers CD117 and CD133, were not expressed. Comparative expression patterns of novel MSC markers in sarcoma cells, as well as fibroblasts and MSCs, are presented. Our data suggest a hierarchical cytoarchitecture of the most common adult type sarcomas and introduce W5C5, TNAP, CD344, and CD271 as potential sarcoma progenitor cell markers.

Severe paraneoplastic hypereosinophilia in metastatic renal cell carcinoma

BackgroundRenal cell carcinoma can cause various paraneoplastic syndromes including metabolic and hematologic disturbances. Paraneoplastic hypereosinophilia has been reported in a variety of hematologic and solid tumors. We present the first case in the literature of severe paraneoplastic hypereosinophilia in a patient with renal cell carcinoma.Case presentationA 46 year-old patient patient with a history of significant weight loss, reduced general state of health and coughing underwent radical nephrectomy for metastasized renal cell carcinoma. Three weeks after surgery, the patient presented with excessive peripheral hypereosinophilia leading to profound neurological symptoms due to cerebral microinfarction. Systemic treatment with prednisolone, hydroxyurea, vincristine, cytarabine, temsirolimus and sunitinib led to reduction of peripheral eosinophils but could not prevent rapid disease progression of the patient. At time of severe leukocytosis, a considerable increase of cytokines associated with hypereosinophilia was measurable.ConclusionsParaneoplastic hypereosinophilia in patients with renal cell carcinoma might indicate poor prognosis and rapid disease progression. Myelosuppressive therapy is required in symptomatic patients.

Diffuse alveolar hemorrhage in patients with hematological malignancies: HRCT patterns of pulmonary involvement and disease course

OBJECTIVE To analyze high-resolution computed tomography (HRCT) patterns of lung involvement and disease course in patients with hematological malignancies experiencing diffuse alveolar hemorrhage (DAH) after chemotherapy ± allogeneic stem cell transplantation (allo-SCT). MATERIALS AND METHODS Sixteen patients experiencing DAH after chemotherapy ± allo-SCT were enrolled. A total of 74 computed tomography (CT) scans obtained before, during, and after onset of DAH were evaluated retrospectively. RESULTS CT features of DAH are each, by oneself, nonspecific. However, conjoint bilateral, diffuse, and dependent ground glass opacity ± crazy paving, accompanied by airspace bronchograms, should suggest this complication. The HRCT course comprises a wide range of trends that are not predictive for patients outcome, but progression of parenchymal consolidations at follow up was more often detrimental.

NFAT2 is a critical regulator of the anergic phenotype in chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is a clonal disorder of mature B cells. Most patients are characterised by an indolent disease course and an anergic phenotype of their leukaemia cells, which refers to a state of unresponsiveness to B cell receptor stimulation. Up to 10% of CLL patients transform from an indolent subtype to an aggressive form of B cell lymphoma over time (Richter´s syndrome) and show a significantly worse treatment outcome. Here we show that B cell-specific ablation of Nfat2 leads to the loss of the anergic phenotype culminating in a significantly compromised life expectancy and transformation to aggressive disease. We further define a gene expression signature of anergic CLL cells consisting of several NFAT2-dependent genes including Cbl-b, Grail, Egr2 and Lck. In summary, this study identifies NFAT2 as a crucial regulator of the anergic phenotype in CLL.NFAT2 is a transcription factor that has been linked with chronic lymphocytic leukaemia (CLL), but its functions in CLL manifestation are still unclear. Here the authors show, by analysing mouse CLL models and characterising biopsies from CLL patients, that NFAT2 is an important regulator for the anergic phenotype of CLL.