K Seeger

Prognostic value of genetic alterations in children with first bone marrow relapse of childhood B-cell precursor acute lymphoblastic leukemia.

Despite risk-adapted treatment, survival of children with relapse of acute lymphoblastic leukemia (ALL) remains poor compared with that of patients with initial diagnosis of ALL. Leukemia-associated genetic alterations may provide novel prognostic factors to refine present relapse treatment strategies. Therefore, we investigated the clinical relevance of 13 recurrent genetic alterations in 204 children treated uniformly for relapsed B-cell precursor ALL according to the ALL-REZ BFM 2002 protocol. The most common alterations were deletions of CDKN2A/2B, IKZF1, PAX5, ETV6, fusion of ETV6-RUNX1 and deletions and/or mutations of TP53. Multivariate analysis identified IKZF1 deletion and TP53 alteration as independent predictors of inferior outcome (P=0.002 and P=0.001). Next, we investigated how both alterations can improve the established risk stratification in relapsed ALL. Intermediate-risk relapse patients with low minimal residual disease are currently considered to have a good prognosis. In this group, deletion of IKZF1 and alteration of TP53 identify patients with significantly inferior outcome (P<0.001). In high-risk relapse patients, deletion of IKZF1 is strongly predictive of a second relapse after stem cell transplantation (P<0.001). We conclude that IKZF1 and TP53 represent relevant prognostic factors that should be considered in future risk assessment of children with relapsed ALL to indicate treatment intensification or intervention.

Therapeutic use of mistletoe for CD30+ cutaneous lymphoproliferative disorder/lymphomatoid papulosis.

558 JEADV 2007, 21, 536–578

Drug Cocktail Optimization in Chemotherapy of Cancer

Background In general, drug metabolism has to be considered to avoid adverse effects and ineffective therapy. In particular, chemotherapeutic drug cocktails strain drug metabolizing enzymes especially the cytochrome P450 family (CYP). Furthermore, a number of important chemotherapeutic drugs such as cyclophosphamide, ifosfamide, tamoxifen or procarbazine are administered as prodrugs and have to be activated by CYP. Therefore, the genetic variability of these enzymes should be taken into account to design appropriate therapeutic regimens to avoid inadequate drug administration, toxicity and inefficiency. Objective The aim of this work was to find drug interactions and to avoid side effects or ineffective therapy in chemotherapy. Data sources and methods Information on drug administration in the therapy of leukemia and their drug metabolism was collected from scientific literature and various web resources. We carried out an automated textmining approach. Abstracts of PubMed were filtered for relevant articles using specific keywords. Abstracts were automatically screened for antineoplastic drugs and their synonyms in combination with a set of human CYPs in title or abstract. Results We present a comprehensive analysis of over 100 common cancer treatment regimens regarding drug-drug interactions and present alternatives avoiding CYP overload. Typical concomitant medication, e.g. antiemetics or antibiotics is a preferred subject to improvement. A webtool, which allows drug cocktail optimization was developed and is publicly available on http://bioinformatics.charite.de/chemotherapy.

The sequence of application of methotrexate and histone deacetylase inhibitors determines either a synergistic or an antagonistic response in childhood acute lymphoblastic leukemia cells

The sequence of application of methotrexate and histone deacetylase inhibitors determines either a synergistic or an antagonistic response in childhood acute lymphoblastic leukemia cells

FLT3 mutations in childhood acute lymphoblastic leukemia at first relapse

The FMS-like tyrosine kinase 3 (FLT3) plays an important role in the pathogenesis of hematopoietic malignancies. Constitutive activation of FLT3, resulting in cell proliferation and survival, occurs either through coexpression of FLT3 ligand and autocrine signalling or through mutations of the FLT3 gene, which lead to autophosphorylation and activation of the FLT3 receptor. FLT3 activating mutations, internal tandem duplications (ITD) and kinase domain (KD) mutations were initially discovered in acute myeloid leukemias (AML) and are associated with poor prognosis in both adult and pediatric AML. Recently, it was shown that FLT3 mutations are common in two subtypes of childhood acute lymphoblastic leukemia (ALL). In infant ALL with translocations involving the mixed lineage leukemia (MLL) gene and in hyperdiploid ALL, FLT3 mutations are detected in 18.2% and in 21.5%–25%, respectively. While the presence of FLT3 mutations in infant ALL with MLL rearrangements was found to be associated with an inferior prognosis, the presence of the mutations in hyperdiploid ALL did not affect clinical outcome. To substantiate these important findings, we investigated whether the frequency of FLT3 mutations is increased in relapsed childhood ALL, and if FLT3 mutations are of prognostic relevance. We analyzed 134 diagnostic leukemic samples from patients with first relapse of ALL with bone marrow (BM) involvement enrolled within two consecutive years, 2000 and 2001, into the multicenter relapse trials of the Berlin–Frankfurt– Muenster Study group (ALL-REZ BFM). Although leukemic samples were not available from 62 patients within the indicated time period, statistical analysis of patient characteristics of the studied (n1⁄4 134) vs the not available samples (n1⁄4 62) did not disclose any significant differences. Only the percentage of samples with TEL/AML1 fusion transcripts was slightly higher in the studied group (P1⁄4 0.018). Informed consent for treatment and accompanying scientific studies was obtained from the parents or the guardians. FLT3 length mutations in the juxtamembrane domain were identified by Genescan analysis, and RFLP-mediated PCR was used for detection of activation loop mutations in the KD, as published elsewhere. All samples with detectable mutations were sequenced (different control DNA were a gift from S Schnittger, University Hospital Grosshadern, Munich, Germany). We found FLT3 mutations in leukemic cell samples of three patients: one I836M (49% mutant allele), one D835Y (5%) and one ITD of 30 bp (65.6%). The FLT3 length mutation was detected in a patient with MLL rearrangement (MLL/AF4). At initial ALL diagnosis, when the patient was an infant, the leukemic cells were MLL/AF4 positive but FLT-ITD negative, suggesting the occurrence of an FLT-ITD positive subclone. All other included patients with an MLL aberration at relapse (n1⁄4 4) depicted no FLT3 mutations and were older than 1 year at initial diagnosis. Both FLT3 activation loop mutations were neither associated with known cytogenetics nor with hyperdiploidy. While FLT3-D835Y could also not be detected in the corresponding initial ALL, no leukemic sample was available to study the clonal evolution of the FLT3-I836M mutation. All three patients suffered a subsequent relapse or died after achieving complete remission, while in the entire study group 77/134 patients and in the not analyzed group 41/62 had an adverse event (P1⁄4 0.126). As the blast count in only 12/134 analyzed BM samples was below 70% (but above 10%), it is unlikely that mutations were missed. Taken together, our results indicate that FLT3 mutations emerged at ALL relapse (2/3) and occur with a lower frequency than Taketani et al and Armstrong et al found in their analyses regarding ALL at first presentation in patients older than 1 year, 6/112 and 10/71, respectively. As we found no FLT3 mutations in hyperdiploid ALL, our data are in agreement with the observation of Taketani et al that these patients have good clinical outcomes and that the mutations may not affect the growth advantage of hyperdiploid ALL cells. In conclusion, albeit FLT3 mutations do not accumulate in relapsed ALL, recent findings indicate that even wild-type FLT3 remains an interesting therapeutic target in ALL. Constitutively activated wild-type FLT3 was found in some B-cell precursor ALL cell lines, and childhood ALL cells with high levels of FLT3 expression were killed by inhibition of FLT3 signalling.

Pediatric Colorectal Carcinoma is Associated With Excellent Outcome in the Context of Cancer Predisposition Syndromes.

Colorectal carcinoma (CRC) is the second most common adult cancer in Germany, however, it is extremely rare in children and adolescents. In these patients, previous literature describes aggressive behavior and diagnosis at advanced stage.

P01.18. Triterpene acid containing Viscum album L. extracts mediate apoptosis in paediatric solid cancer cells

Purpose Paediatric solid cancers such as osteosarcoma, Ewing s sarcoma, rhabdomyosarcoma and neuroblastoma are the most common cancers in children besides leukemia. These cancers have a poor prognosis, are highly metastatic and often resistant to current therapeutic approaches. Viscum album L. (mistletoe) is one of the most widely used complementary cancer therapies in Germany but little is known about its actual effects on paediatric solid cancers. Approved Viscum album L. extracts (VAE) basically contain water soluble compounds of the plant (lectins, viscotoxins). However, mistletoe also contains triterpene acids (mainly oleanolicand betulinic acid) that are water-insoluble. The antitumorigenic properties of these solubilized triterpene acids are the subject of ongoing research. The aim of this study is to determine the effects of different VAE containing either lectins (viscum), triterpene acids (TT) or a combination thereof (viscum TT) on solid tumor models in vitro and in vivo.

The Serotonin Receptor‐Antagonist Ondansetron Induces Significant Increases in the Expression of Interferon‐Gamma Which Correlate with Antiproliferative Properties in the Acute Lymphoblastic Leukaemia Cell Line REH

To the Editor: Ondansetron is a serotonin (5-hydroxytriptamine, 5HT) receptor 3 (5-HT3) antagonist widely used as antiemetic agent in the prophylactic treatment of chemotherapy-induced nausea and vomiting in children with acute lymphoblastic leukaemia (ALL) [1, 2]. In a recent report from our group [3], it has been described that ondansetron exhibits antiproliferative effects in the B-cell precursor (BCP)-ALL cell line REH [4, 5]. REH cells in the logarithmic growth phase were cultured for 72 h, as previously described [3]. The concentrations of interferon-gamma (IFNc) in supernatants were measured with aid of a commercially available h-IFNc EASIA/ELISA kit (Biosource Europe, Nivelles, Belgium). IFNc levels were expressed in international units of IFNc active protein per ml of supernatant (IU/ml, 1 IU = 100 pg active protein). Ondansetron (Zofran) was from Glaxo Wellcome GmbH, Munich (Germany). All experiments were performed at least three times in double series of triplicates, and results were considered statistically significant for P < 0.05 in the unpaired Student’s t-test. The ‘Pearson’s Correlation Coefficient’ (PCC) was used for correlation analysis. In the presence of 5–50 lM ondansetron, the release of IFNc was found to be significantly increased by more than 200% after 72 h incubation (means 206 and 260%; P = 0.003 and P = 0.004, respectively; Fig. 1A). Significant increases in IFNc expression were also observable in the presence of further 10 lM 5-HT (means for 5 and 50 lM ondansetron: 291 and 276%; with P values of 0.005 and 0.0480, respectively; Fig. 1A). Interestingly, the addition of 10 lM 5-HT significantly increased the release of IFNc by approximately 400% (mean 396%; P = 0.015, Fig. 1A). No significant differences were found between the increases in IFNc release for 5 and 50 lM ondansetron, either in the absence, or in the presence of 10 lM 5-HT (Fig. 1B), so indicating that 5 lM ondansetron was enough to act on all the available receptors in the analysed cells. Furthermore, the detected increases in IFNc production were found to correlate with previously described reductions in cell proliferation [3], showing a PCC of 0.6903 with each other. An inhibition of cell proliferation because of a selective effect of exogenous rec-IFNc has previously been reported in REH cells [6]. Although the acting concentrations of exogenous rec-IFNc differed from those in endogenous release, the reported inhibition of cell proliferation confirms that IFNc is related to antiproliferative features in REH cells [6]. Moreover, it has also been reported that a 5-HT1B antagonist significantly inhibited the production of IFNc in blood cells isolated from healthy donors [7]. Figure 1 Effects of ondansetron on the release of interferon-gamma (IFNc) in the acute lymphoblastic leukaemia cell line REH, after 72 h incubation at 37 °C. The observed IFNc levels are presented as concentrations of IFNc, expressed in international units of active protein per ml of analysed sample (IU/ml, 1 IU = 100 pg active protein, Panel A), or as per cent increases related to the corresponding samples with no ondansetron (%, Panel B). Values are means and standard error of the means (SEM). All experiments were performed at least three times in double series of triplicates. Stars indicate statistical significance when compared with controls with no ondansetron in the unpaired Student’s t-test (one star: P < 0.05; two stars: P < 0.01).

P01.33. A new development of Triterpene Acids- containing extracts from Viscum album L. displays synergistic induction of apoptosis in childhood leukemia

Purpose Aqueous Viscum album L. extracts (VAE) are widely used in complementary cancer therapies. Due to their low solubility, triterpene acids, which are known to possess anti-cancer properties, do not occur in aqueous extracts in significant amounts. Using cyclodextrins it was possible to solubilize mistletoe triterpene acids and to determine the anti-cancer properties in different acute lymphoblastic (ALL) and myeloid leukemia cell lines (AML). Methods The experimental extracts contain either mistletoe lectin-I and viscotoxins (viscum) or solubilized oleanolicand betulinic acids (TT) and more interestingly, a combination thereof (viscumTT). The cytotoxicity of increasing concentrations of VAE preparations was tested in NALM-6, U937 and HL-60 cells in vitro. Apoptosis was determined using mitochondrial membrane potential measurement, Annexin/PI, Western blot analysis and caspase assays. A C.B-17/SCID model of pre-B ALL/NALM-6 was used to test efficacy and mechanisms of treatment with lectin- and triterpenecontaining preparations in vivo. Results All three cell lines have shown distinct apoptosis induction for viscum, TT and viscumTT. However, differences between ALL and AML cell lines toward the lectin and triterpene acids sensitivity were observed. Annexin/PI and mitochondrial membrane potential assays indicated that dose-dependent induction of apoptosis was the main mechanism. The combination (viscumTT) of lectin- (viscum) and triterpene acidscontaining (TT) extracts showed the strongest apoptosis induction. Furthermore, caspase activity demonstrated that these extracts are able to induce apoptosis via caspase-8 and -9 dependent pathways. The in vivo experiment showed that treatment of mice with the viscumTT combination prolonged the mean survival significantly compared control group. Conclusion

P01.16. A root extract of Helleborus niger possess cytotoxic properties in neuroblastoma cells

Purpose Helleborus niger (Ranunculaceae), commonly known as Christmas rose, is used in anthroposophically extended cancer therapy in the adjuvant treatment of different entities and reduction of chemotherapy-associated side effects. Although Helleborus niger is widely used in anthroposophic medicine, there is a lack of scientific clinical and preclinical data and until now it is applied on an empirical basis. Neuroblastoma is one of the most common extracranial solid tumors of childhood, and more than 50 % of these children initially present with nonresectable primary tumors and disseminated metastasis to distant organ sites, predominantly bone marrow. In this study, we determined for the first time the cytotoxic properties of Helleborus niger Root (HNR) extract for neuroblastoma in vitro.