Transcriptomic and proteomic insight into the effects of a defined European mistletoe extract in Ewing sarcoma cells reveals cellular stress responses.

BACKGROUND: The hydrophobic triterpenes, oleanolic and betulinic acid as well as the hydrophilic mistletoe lectins and viscotoxins possess anticancer properties. They do all occur in combination in European mistletoe (Viscum album L.). Commercial Viscum album L. extracts are aqueous, excluding the insoluble triterpenes. We have previously shown that mistletoe lectins and triterpene acids are effective against Ewing sarcoma in vitro, ex vivo and in vivo. METHODS: We recreated a total mistletoe effect (viscumTT) by combining an aqueous extract (viscum) and a triterpene extract (TT) solubilised with cyclodextrins and analysed the effects of viscumTT and the single extracts on TC-71 Ewing sarcoma cells in vitro by transcriptomic and proteomic profiling. RESULTS: Treatment with the extracts strongly impacted Ewing sarcoma cell gene and protein expression. Apoptosis-associated and stress-activated genes were upregulated, proteasomal protein abundance enhanced and ribosomal and spliceosomal proteins downregulated. The mechanism of action of viscum, TT and viscumTT in TC-71 and MHH-ES-1 cells suggests the involvement of the unfolded protein response. While viscum and viscumTT extract treatment indicate response to oxidative stress and activation of stress-mediated MAPK signalling, TT extract treatment suggests the involvement of TLR signalling and autophagy. CONCLUSIONS: Since the combinatory extract viscumTT exerts highly effective pro-apoptotic effects on Ewing sarcoma cells in vitro, this phytopolychemotherapy could be a promising adjuvant therapeutic option for paediatric patients with Ewing sarcoma.

A Cre-conditional MYCN-driven neuroblastoma mouse model as an improved tool for preclinical studies.

Neuroblastoma, a childhood cancer that originates from neural crest-derived cells, is the most common deadly solid tumor of infancy. Amplification of the MYCN oncogene, which occurs in approximately 20-25% of human neuroblastomas, is the most prominent genetic marker of high-stage disease. The availability of valid preclinical in vivo models is a prerequisite to develop novel targeted therapies. We here report on the generation of transgenic mice with Cre-conditional induction of MYCN in dopamine ß-hydroxylase-expressing cells, termed LSL-MYCN;Dbh-iCre. These mice develop neuroblastic tumors with an incidence of >75%, regardless of strain background. Molecular profiling of tumors revealed upregulation of the MYCN-dependent miR-17-92 cluster as well as expression of neuroblastoma marker genes, including tyrosine hydroxylase and the neural cell adhesion molecule 1. Gene set enrichment analyses demonstrated significant correlation with MYC-associated expression patterns. Array comparative genome hybridization showed that chromosomal aberrations in LSL-MYCN;Dbh-iCre tumors were syntenic to those observed in human neuroblastomas. Treatment of a cell line established from a tumor derived from a LSL-MYCN;Dbh-iCre mouse with JQ1 or MLN8237 reduced cell viability and demonstrated oncogene addiction to MYCN. Here we report establishment of the first Cre-conditional human MYCN-driven mouse model for neuroblastoma that closely recapitulates the human disease with respect to tumor localization, histology, marker expression and genomic make up. This mouse model is a valuable tool for further functional studies and to assess the effect of targeted therapies.

[Diagnosis and treatment of retinoblastoma: current strategies for effective tumour control and preservation of vision]. / Diagnose und behandlung des retinoblastoms: aktuelle konzepte zur sicheren tumorkontrolle bei erhalt des sehvermögens.

There are approximately 40 new cases of retinoblastoma in Germany per year. Children in whom the tumour is detected when still intraocular have an excellent overall survival rate (> 95%). However, the prognosis of metastasised retinoblastoma remains poor. About 40% of retinoblastoma patients have tumours in both eyes. For these children in particular it is important to save the eye and visual function as much as possible. There are several options for conservative treatment of localised retinoblastoma including laser coagulation, thermotherapy, cryotherapy, brachytherapy and chemotherapy. In recent years, systemic chemotherapy has become the established standard for primary treatment of intraocular retinoblastoma. In case series, intra-arterial, intravitreal and periocular applications of chemotherapy were also shown to be effective in treating intraocular retinoblastoma. Genetic testing is an integral part of the routine diagnostics of all patients. Mutation analysis of tumour material is invaluable for identification of somatic mutations including mutational mosaicism. Genetic testing also identifies children with heritable retinoblastoma, which represent 50% of cases. These children also have a predisposition for the development of tumours outside of the eye (second primary neoplasm). To adequately address these and other late effects in survivors of retinoblastoma, a multidisciplinary approach is needed that optimises therapy and long-term follow-up. Upcoming multicentre clinical trials will evaluate treatment concepts for localised and metastasised retinoblastoma to improve survival rates and quality of life of children with retinoblastoma. This article was translated and modified and was primarily published in Klin Padiatr 2012; 224: 339-347.

Current concepts for diagnosis and treatment of retinoblastoma in Germany: aiming for safe tumor control and vision preservation.

Retinoblastoma affects approximately 40 children in Germany per year. Most children are diagnosed early with localized intraocular disease, and the overall survival rate exceeds 95%. However, the prognosis of metastasized retinoblastoma remains poor. In 40% of the patients, retinoblastoma occurs bilaterally and, especially for these children, the salvage of the eye and visual function is of major importance. The variety of conservative treatment options for localized retinoblastoma includes laser coagulation, thermotherapy, cryotherapy, brachytherapy and chemotherapy. While systemic chemotherapy has nearly completely replaced external beam radiotherapy in the primary treatment of intraocular retinoblastoma, intra-arterial, intravitreal and periocular application of chemotherapy was also shown to be effective in treating intraocular retinoblastoma in case series. Genetic testing is an integral part of the routine diagnostics of all patients. Available tumor material should be analyzed to detect mutational mosaicism, that affects >10% of children with unilateral retinoblastoma. Genetic testing also identifies children with heritable (50% of patients) retinoblastoma. These children have a genetic predisposition for second malignancies. For this reason, late effects are an increasing concern and the care of patients with retinoblastoma requires a multidisciplinary approach to tailor therapy and long-term follow-up. Multicenter clinical trials are being developed to evaluate evidence-based treatment concepts for localized and metastasized retinoblastoma to improve survival rates and quality of life of children with retinoblastoma.

Detection of neuroblastoma cells during clinical follow up: advanced flow cytometry and rt-PCR for tyrosine hydroxylase using both conventional and real-time PCR.

PURPOSE: Real-time reverse-transcriptase PCR (RT-qPCR) or conventional RT-PCR (RT-cPCR) detection of tyrosine hydroxylase (TH) is increasingly used to detect neuroblastoma (NB) cells in clinical samples. However, TH expression in normal tissues can limit its usefulness and make additional diagnostic strategies necessary. METHODS: We analysed TH in 857 tumour, bone marrow aspirate and peripheral blood stem cell samples from 65 NB patients using RT-cPCR, and compared results from 666 samples analysed by RT-qPCR. TH was investigated in 84 samples from patients with other diagnoses and 354 samples from healthy donors as controls, and 132 samples from the entire collection were evaluated for NB cells using 5-colour flow cytometry (FC). RESULTS: Cohen's kappa coefficient demonstrated a substantial agreement between RT-cPCR and RT-qPCR as well as RT-cPCR and FC and a moderate agreement between RT-qPCR and FC. TH expression was also detected in samples from individual patients with Ewing sarcoma, nephroblastoma and rhabdomyosarcoma, but not from healthy donors. FC panels were an effective complementary strategy, detecting as few as 0.002% NB cells, characterised as CD45negCD9+CD81+CD56+ch14:18+GD2+ cells with occasional CD57+CD138+CD166+ expression. CONCLUSION: TH RT-qPCR alone is limited for detection of NB cells because of "false positives" in samples from patients with other diseases. Advanced FC may serve as a complementary method to detect residual NB, but needs further confirmation in larger patient cohorts.

Microarray-analysis: a new approach to study the molecular mechanisms of thermo-chemotherapy.

BACKGROUND: Experimental studies for the treatment of relapsed neuroblastoma include the use of hyperthermia in combination with chemotherapeutic drugs. Cytotoxic effects of alkylants and platinum compounds on tumor cells can be enhanced by hyperthermia in various in vitro models. However, the underlying molecular mechanisms are still largely unknown. METHOD: In this study, we used microarray-analysis as a new biological approach to gain insight into the pharmacogenomics and possible target genes of thermochemotherapy. As a model, LAN 1 neuroblastoma cells were treated for 1 h with low doses of cisplatin alone, with simultaneous heating to 42 degrees C or with hyperthermia alone. Gene expression was analyzed at five time points 0 to 24 h after treatment using U95Av2 oligonucleotide arrays (Affymetrix Inc). Significant changes of gene expression levels were calculated by similarity metrices and Pearson correlation. RESULTS: Only a few genes (n = 23) demonstrated altered expression following treatment of LAN 1 cells with cisplatin alone. Hyperthermia alone resulted in significant expression changes of 136 genes in comparison to untreated control cells. Combination therapy of cisplatin and hyperthermia resulted in expression changes of 251 genes, interestingly including 131 genes with unchanged expression under treatment with either cisplatin or hyperthermia alone. Significant changes of expression levels could be annotated to genes involved in heat shock response, protein degradation and apoptosis. These results are now being validated on mRNA- and protein levels by RT-PCR and Western Blot analysis. CONCLUSION: Microarray-Analysis is a suitable new approach for the identification of target genes, which might play an important role for the synergistic effect of hyperthermia and chemotherapy in tumor cells.

Calcitriol vs. dithranol in combination with narrow-band ultraviolet B (311 nm) in psoriasis.

BACKGROUND: Narrow-band ultraviolet (UV) B (311 nm) phototherapy is an effective treatment for psoriasis. In order to reduce cumulative UV doses and to enhance clearance of psoriasis plaques, combination therapies with topical agents such as dithranol and calcipotriol have been established. OBJECTIVES: To compare the clinical efficacy, in a half-side manner, of UVB (311 nm) in combination with either calcitriol or dithranol. METHODS: Ten patients with symmetrical stable plaque psoriasis were treated with narrow-band UVB (311 nm) five times a week. In addition, topical calcitriol was applied twice daily to one arm, whereas the other arm and the rest of the body were treated once daily with dithranol. The follow-up period was at least 4 weeks. Efficacy was assessed separately for both arms prior to treatment and once weekly thereafter by a modified Psoriasis Area and Severity Index (PASI) score. The cumulative irradiation dose and the number of treatment sessions required for clearance of psoriasis lesions were determined for each patient. Additionally, all patients completed a quality of life questionnaire. RESULTS: Both treatment modalities notably reduced the PASI score. A clinical comparison of UVB (311 nm) in combination with either calcitriol or dithranol revealed no significant therapeutic differences between the regimens. CONCLUSIONS: Combination of narrow-band UVB (311 nm) therapy with calcitriol is equally effective as the combination with dithranol for the treatment of psoriasis. However, patients preferred calcitriol rather than dithranol when both quality of life and treatment acceptability were assessed.

Dendritic cells break tolerance and induce protective immunity against a melanocyte differentiation antigen in an autologous melanoma model.

Tyrosinase-related protein (TRP) 2 belongs to the melanocyte differentiation antigens and has been implicated as a target for immunotherapy of human as well as murine melanoma. In the current report, we explored the efficacy of nonmutated epitopes with differential binding affinity for MHC class I, derived from mouse TRP2 to induce CTL-mediated, tumor-reactive immunity in vivo within the established B16 melanoma model of C57BL/6 mice. The use of nonmutated TRP2-derived epitopes for vaccination provides a mouse model that closely mimics human melanoma without introduction of xenogeneic or otherwise foreign antigen. The results demonstrate that vaccination with TRP2 peptide-loaded bone marrow-derived dendritic cells (DCs) results in activation of high avidity TRP2-specific CTLs, displaying lytic activity against both B16 melanoma cells and normal melanocytes in vitro. In vivo, protective antitumor immunity against a lethal s.c. B16 challenge was observed upon DC-based vaccination in this fully autologous tumor model. The level of protective immunity positively correlated with the MHC class I binding capacity of the peptides used for vaccination. In contrast, within this autologous model, vaccination with TRP2 peptide in Freund's adjuvant or TRP2-encoding plasmid DNA did not result in protective immunity against B16. Strikingly, despite the observed CTL-mediated melanocyte destruction in vitro, melanocyte destruction in vivo was sporadic and primarily restricted to minor depigmentation of the vaccination site. These results emphasize the potency of DC-based vaccines to induce immunity against autologous tumor-associated antigen and indicate that CTL-mediated antitumor immunity can proceed without development of adverse autoimmunity against normal tissue.