Astrid Voigt

Identification of a Set of Seven Genes for the Monitoring of Minimal Residual Disease in Pediatric Acute Myeloid Leukemia

Background: Monitoring of minimal residual disease (MRD) has become a strong diagnostic tool in acute lymphoblastic leukemia. It is used for risk-adapted therapy and for the recognition of pending relapses. In acute myeloid leukemia (AML), there is still a need for more suitable MRD markers. Experimental Design: A stepwise approach which combined genome-wide expression profiling, TaqMan low density arrays, and a TaqMan real-time PCR-based screening was used to identify new markers for the monitoring of MRD in AML. Leukemic cells from 52 children with AML and 145 follow-up samples from 25 patients were analyzed. Results: Seven genes were identified which are vastly overexpressed in many patients with AML compared with healthy bone marrow: CCL23, GAGED2, MSLN, SPAG6, and ST18 as well as the previously described markers WT1 and PRAME. The expression of all genes decreased to normal levels in patients who achieved a continuous complete remission. Elevated levels of at least one gene were found prior to relapse in 7 out of 10 patients who relapsed. Conclusions: This set of genes should allow a sensitive and specific monitoring of MRD in AML. Notably, some of these markers could also serve as therapeutic targets or might be involved in leukemogenesis. MSLN is already used as a target for immunotherapy in clinical trials in other malignancies.

Expression of the BCRP gene (ABCG2/MXR/ABCP) in childhood acute lymphoblastic leukaemia

Summary.u2002 The breast cancer resistance protein (BCRP), also known as mitoxantrone resistance protein (MXR) or placenta ABC protein (ABC‐P), is the second member of the ABCG subfamily of ABC transport proteins (gene symbol ABCG2). BCRP has been detected in acute myeloid leukaemia and in breast, colon and gastric cancer but there has been no reports regarding BCRP expression in acute lymphoblastic leukaemia (ALL). We report the first results of BCRP expression in childhood ALL. Sixty‐seven children (47 initial stage, 20 relapses) with ALL were analysed for BCRP gene expression by TaqMan real‐time polymerase chain reaction. The expression of BCRP in mononuclear cells obtained from the bone marrow (BM) and peripheral blood (PB) of healthy donors was also investigated. There was no relationship between BCRP expression and age, sex, initial blast cell count, prednisolone response or BM response on du200315 and 33. Patients with T‐lineage ALL showed a lower expression of BCRP (Pu2003=u20030·044). Kaplan–Meier analysis of the relapse‐free interval showed no prognostic significance of BCRP expression when different levels of BCRP expression were used as cut‐off points. No significant difference in expression of BCRP mRNA was measured between initial‐stage and relapsed‐stage ALL or between normal MNC obtained from BM and ALL patients. The results indicate a low expression of BCRP in childhood ALL. Relationships between BCRP and clinical, molecular or in vivo resistance characteristics of the patients were not observed.

Differentiation, proliferation and adhesion of human neuroblastoma cells after treatment with retinoic acid.

Because of the known property of spontaneous regression in stage IVS of neuroblastoma all attempts are made to elucidate whether differentiation inducers possibly could be applied for neuroblastoma therapy. Here we examined the influence of retinoic acid (RA) in vitro on differentiation, proliferation and adhesion of 10 permanent and 4 primary cell lines as well as of several SCID-mouse tumour transplants. In general, after RA treatment morphologically different cell types which are characteristic for neuroblastoma cells have changed. N (neuronal)-type cells prolonged their neuronal processes, whereas S (epithelial, substrate-adherent, Schwann cell-like)-type cells lost their adherence to substratum and became apoptotic. Additionally, the reactions of all neuroblastoma cell lines with monoclonal antibodies against β-tubulin (for neuronal cells) and glial fibrillary acidic protein (for epithelial cells) were determined. The anti-proliferative effect of all-trans-RA as well as 13-eis-RA was more profound in S-type cells (up to 40% in primary cell lines). To elucidate the role of adhesion molecules during neuronal cell differentiation, we have analysed the adhesion of neuroblastoma cells on poly-D-lysin-precoated plates under RA influence. While N-type cells displayed an increased adhesion, all S-type cell lines as well as all primary cell lines exhibited a reduced adhesion (IMR-5 and IMR-32: p < 0.001; JW, SR and PM: p < 0.05). RA treatment increased predominantly the tested antigens (HCAM, ICAM-1, NCAM, PECAM-1, VCAM-1, cadherin, FGF-R, IGF-R, NGF-R, TGF-β/1, NF200, NF160, NF68, NSE, HLA-ABC) in all cell lines independently of their phenotypes (TGFβ/1: p < 0.001; NF68: p < 0.01; PECAM-1 and NGF-R: p < 0.05). In recultured SCID-mouse-passaged tumour cells antigens were down-regulated (FGF-R: p < 0.01), but increased again after RA influence (TGF-β/1: p < 0.05). In summary, the RA differentiation model demonstrates the possibility to interfere in cell adhesion and to diminish growth potential both in N-type as well as S-type neuroblastoma cells.

Messenger RNA Analysis of the Multidrug Resistance Related Protein (MRP1) and the Lung Resistance Protein (LRP) in de novo and Relapsed Childhood Acute Lymphoblastic Leukemia

In this study, 86 children (58 initial ALL and 28 children with relapsed disease) were investigated for lung resistance protein (LRP) and multidrug resistance related protein (MRP1)-mRNA expression by semiquantitative RT-PCR. The majority of investigated cases demonstrated variable LRP and MRP1 mRNA expression, when normalized for β -microglobulin expression. LRP and MRP1 mRNA expression may be coordinately regulated, as expression of both transcripts was found to be significantly correlated (p =0.0001). No differences of LRP and MRP expression were observed between initial and relapsed stage patients (LRP: p =0.89 and for MRP: p =0.09 ). The prognostic value of both resistance mechanisms was subjected to Kaplan-Meier analysis for event-free survival. For this analysis the patients were divided into groups with high or low LRP or MRP1 mRNA expression by utilizing the median value as the cut-off point. Overexpression of both resistance mechanisms had no prognostic significance in our retrospective study (log-rank test for LRP: p =0.12 and for MRP1: p =0.95 ), however, patients who showed high LRP expression exhibited a lower tendency of remaining in continuous first remission.

Expression of CD34 and other haematopoietic antigens on neuroblastoma cells: consequences for autologous bone marrow and peripheral blood stem cell transplantation.

Autologous peripheral blood stem cells, obtained by CD34+ stem cell selection, are being used with increasing frequency for transplantation in patients with neuroblastoma. Here, we examined the surface membrane antigens of neuroblastoma cells with a panel of hematopoietic monoclonal antibodies (mAbs), including anti-CD34 mAbs, by flow cytometric analysis. We found stronger binding of anti-CD34 mAbs to clonogenic, less differentiated, non-adherent neuroblastoma cells than to adherent neuroblastoma cells. Moreover, the majority of neuroblastoma cell lines shared hematopoietic-associated antigens with all blood cells. Because of these cross-reactions, especially found with the anti-CD34 mAbs 12.8 and ICH3, we have demonstrated that there is a potential risk of cell harvest contamination by circulating neuroblastoma cells during CD34+ stem cell selection.

Neuroblastoma cells can express the hematopoietic progenitor cell antigen CD34 as detected at surface protein and mRNA level

Recently, we have shown the expression of the hematopoietic precursor cell antigen CD34 on neuroblastoma cells. Here, we present the CD34 expression on 16 permanent neuroblastoma cell lines and primary cell lines at the mRNA level and the flow cytometric results on neuroblastoma cells grown in the same culture and split for flow cytometric analysis and total mRNA extraction. The flow cytometry was performed using a panel of anti-CD34 antibodies covering the epitope classes I to III. In eight neuroblastoma cell lines, CD34 mRNA expression could be detected and corresponded always with the protein surface expression. Alternatively, when CD34 mRNA expression was not seen, CD34 antigen expression ranged from negative to as high as 78%. Based on these results caution should be taken with transplants obtained by CD34+ stem cell selection from neuroblastoma patients.

Das wachstum der thermophilen pilzstämme Aspergillus fumigatus und Mucor lusitanicus in n-alkan-medium

Summary Growth parameters of the thermophilic hydrocarbon utilizing strains Aspergillus fumigatus and Mucor lusitanicus in n-alkanes (KW)- and glucose (Glc)-medium in standing and fermenter -cultures were investigated. The temperature limits for growth were dependent on the medium composition. The temperature range for sporulation was smaller than for that hyphal growth. Both strains were cultivated on carbon limitation with subsequent determinations of n-alkanes glucose, ammoniumsulfate-nitrogen, phosphate-phosphorus and the trace elements Mn, Cu, Zn and Fe. A method for estimation of hydrocarbon utilization was developed. In a medium containing a mixture of n-alkanes and glucose as C-sources the strains utilized at first glucose. Both the strains excreted monocarbon acids with a low carbon number into the culture liquid. The results were discussed.

Enzymatische Untersuchungen an den thermophilen Kohlenwasserstoff-verwertenden Pilzstämmen Aspergillus fumigatus und Mucor lusitanicus

Summary The activities of catalase, peroxydase, and of the chitinolytic enzymes of the thermophilic hydrocarbon-utilizing fungal strains of Aspergillus fumigatus and Mucor lusitanicus, grown in n-alkane (KW) or glucose (Glc) medium at different temperatures with additions of KCN, NaNO3, 3-amino-1,2,4-triazole (ATA), and CaCl2 and with a shift culture from Glc to KW medium were determined. The enzymatic activities, able to cleave H2O2, were cytochemically shown in the hyphae with 3,3-diamino-benzidine-tetrahydrochloride (DAB) reagent. The colouring of the mycelium was most intense during the linear growth phase in the KW medium. The DAB oxydation could completely be suppressed by pre-incubation of the mycelium with the enzyme inhibitors KCN or ATA and incubation in the standard medium without H2O2. The catalase and peroxydase activities were higher in the KW medium than in the Glc medium, where highest activities occurred at the start of the linear and the stationary growth phase. The pre-incubation of the enzyme solution with the inhibitors KCN, NaNO2 or ATA gave maximum inhibition with ATA, likewise gave the addition of ATA to the medium the highest inhibition of the enzymatic activities, connected with an extension of the initial growth phase. Addition of CaCl2 increased the catalase and peroxydase activities, where catalase at 40 °C and peroxydase at 50 °C showed maximum growth. A shift of the growing mycelium from Glc to KW medium confirmed the correlation of KW utilization and high catalase activity. The highest chitinolytic activities were ascertained at the beginning of the linear growth phase at a temperature of 40 °C. — The results were discussed.