Differential immunomodulatory activity of tumor cell death induced by cancer therapeutic toll-like receptor ligands.

Synthetic toll-like receptor (TLR) ligands stimulate defined immune cell subsets and are currently tested as novel immunotherapeutic agents against cancer with, however, varying clinical efficacy. Recent data showed the expression of TLR receptors also on tumor cells. In this study we investigated immunological events associated with the induction of tumor cell death by poly(I:C) and imiquimod. A human head and neck squamous cell carcinoma (HNSCC) cell line was exposed to poly(I:C) and imiquimod, which were delivered exogenously via culture medium or via electroporation. Cell death and cell biological consequences thereof were analyzed. For in vivo analyses, a human xenograft and a syngeneic immunocompetent mouse model were used. Poly(I:C) induced cell death only if delivered by electroporation into the cytosol. Cell death induced by poly(I:C) resulted in cytokine release and activation of monocytes in vitro. Monocytes activated by the supernatant of cancer cells previously exposed to poly(I:C) recruited significantly more Th1 cells than monocytes exposed to control supernatants. If delivered exogenously, imiquimod also induced tumor cell death and some release of interleukin-6, but cell death was not associated with release of Th1 cytokines, interferons, monocyte activation and Th1 recruitment. Interestingly, intratumoral injection of poly(I:C) triggered tumor cell death in tumor-bearing mice and reduced tumor growth independent of TLR signaling on host cells. Imiquimod did not affect tumor size. Our data suggest that common cancer therapeutic RNA compounds can induce functionally diverse types of cell death in tumor cells with implications for the use of TLR ligands in cancer immunotherapy.

Results from a safety cohort of patients with renal anemia receiving the biosimilar epoetinzeta: The PASCO I study.

BACKGROUND: Epoetin-zeta (epoetin-ζ) (sold as Retacrit™/Silapo™) is a biologic product that was approved by the European Medicines Agency in 2007 after demonstrating biosimilarity to its reference product epoetin-α (Eprex™), based on a comprehensive comparability exercise including extensive biophysical characterization and three double-blind randomized controlled trials. Since 2008, epoetin-ζ has been prescribed by physicians across Europe to treat anemia of renal disease in many thousands of patients. METHODS: Provided here are results of the PASCO I study (post-authorization safety cohort observation of silapo/retacrit (epoetin-ζ) administered intravenously for the treatment of renal anemia). The primary study endpoint was the frequency of adverse events of special interest (AESI) occurring in patients receiving epoetin-ζ over a 1-year study observation period. RESULTS: The safety set included 1,634 patients who received at least 1 dose of epoetin-ζ during the study period. These patients experienced AESI at these frequencies: clotting of artificial kidney 9.8%, lack of efficacy 2.3%, cerebrovascular events (including cerebrovascular accident, cerebral infarction, cerebral hemorrhage, and transient ischemic attack) 1.8%, myocardial infarction 1.7%, acute myocardial infarction 1.2%, clinically relevant hyperkalemia 0.4%, deep vein thrombosis 0.2%, convulsion 0.2%, hypertensive encephalopathy 0.1%, and pulmonary embolism 0.1%. No patients were reported as having anaphylactoid reactions, angioedema, erythropoietinneutralizing antibodies, or pure red cell aplasia. The median weekly follow-up dose of epoetin-ζ was 158.6 IU/kg. Mean hemoglobin concentration ranged between 11.3 and 11.7 g/dL. From the safety set, 228 patients died (14.0%), while 1,135 patients (74.9%; excluding 119 with data missing) continued treatment with epoetin-ζ following the 12-month observation. CONCLUSION: The PASCO I study contributes significantly to current knowledge about the frequency of adverse events associated with the use of epoetin-ζ for the treatment of renal anemia and demonstrates a pattern of adverse events comparable with data for other existing epoetin products in Europe.

HMGB1 is overexpressed in tumor cells and promotes activity of regulatory T cells in patients with head and neck cancer.

HMGB1 has gained a prominent role in cancer development and is implicated in tumor escape phenomena. To date, only few data are available on effects of HMGB1 on regulatory T cells (Treg) in cancer patients. This study evaluates the prevalence of HMGB1 and its effects on Treg in patients with head and neck squamous cell carcinoma (HNSCC). Sixty-seven patients with HNSCC and seventeen healthy donors were included in this study. Tumor tissues of patients were analyzed for expression of HMGB1 employing immunofluorescence and qRT-PCR. HMGB1 serum levels were assessed using ELISA. Tumor-infiltration and Treg from peripheral blood were phenotyped with flow cytometry and immunofluorescence microscopy. Migration and suppressive function of Treg upon HMGB1 stimulation was analyzed in chemotaxis assays and CFSE assays. HMGB1 is overexpressed in tumor cells of HNSCC, and serum levels are significantly elevated. Tumor-infiltrating Treg express HMGB1-recognizing receptors, TLR4 and RAGE. HMGB1 is a chemoattractant for Treg and promotes their suppressive function. Our data provide new aspects how the HMGB1 tumor-derived danger signal augments function of Treg in patients with HNSCC.

Human tumor-induced and naturally occurring Treg cells differentially affect NK cells activated by either IL-2 or target cells.

NK cells play a crucial role in the eradication of tumor cells. Naturally occurring (n) Treg cells and induced (i) Treg cells are two distinct Treg subsets. While the interaction of nTreg cells with NK cells has been investigated in the past, the role of tumor iTreg cells in the modulation of NK-cell function remains unclear. Tumor iTreg cells were generated from CD4(+) CD25(-) T cells in the presence of autologous immature DCs, head and neck cancer cells and IL-2, IL-10, and IL-15. The effect of iTreg cells and nTreg cells on the expression of NKG2D, NKp44, CD107a, and IFN-γ by NK cells, as well as NK tumor-cytolytic activity, were investigated. iTreg cells - similar to recombinant TGF-ß and nTreg cells - inhibited IL-2-induced activation of NK cells in the absence of target cell contact. Surprisingly, and in contrast to nTreg cells, iTreg cells enhanced NK-cell activity elicited by target cell contact. The cytolytic activity of NK cells activated by iTreg cells was mediated via perforin and FasL. We conclude that tumor iTreg cells inhibited IL-2-mediated NK-cell activity in the absence of target cells, whereas the tumoricidal activity of NK cells was enhanced by iTreg cells. Our data suggest a complex, previously not recognized, differential regulation of human NK activity by iTreg cells in the tumor microenvironment.

Toll-like receptor 4 single-nucleotide polymorphisms Asp299Gly and Thr399Ile in head and neck squamous cell carcinomas.

BACKGROUND: Chronic inflammation plays an important role in head and neck squamous cell carcinomas (HNSCC). This study addresses the impact of two single nucleotide polymorphisms (SNP) Asp299Gly and Thr399Ile of the toll-like receptor (TLR) 4 gene on the clinical outcome while accounting for the influence of adjuvant systemic therapy in a large cohort of HNSCC patients. METHODS: Genotype analysis was done using DNA from tissue samples from 188 patients with HNSCC; TLR4 protein expression was assessed immunohistochemically in tissue microarrays. Classical survival models were used for statistical analyses. RESULTS: Ten percent of patients with HNSCC presented with the TLR4 299Gly and 17% with the TLR4 399Ile allele. Patients with the heterozygous genotype TLR4 Asp299Gly had a significantly reduced disease-free and overall survival. Also, patients with the heterozygous genotype TLR4 Thr399Ile had a reduced disease-free survival. Notably, these associations seem to be attributable to relatively poor therapy response as e.g. reflected in a significantly shorter DFS among HNSCC patients carrying the Asp299Gly variant and receiving adjuvant systemic therapy. CONCLUSION: According to this study, TLR4 299Gly und 399Ile alleles may serve as markers for prognosis of head and neck cancer in patients with adjuvant systemic therapy, particularly chemotherapy, and might indicate therapy resistance.