Felix Zintl

Clinical implications of PRAME gene expression in childhood acute myeloid leukemia

The expression of the PRAME gene (preferentially expressed antigen of melanoma) was measured by quantitative reverse transcriptase polymerase chain reaction in 50 children with newly diagnosed acute myeloid leukemia (AML), three samples of CD34(+) stem cells, six bone marrow samples, and 10 peripheral blood samples of healthy donors, as well as three AML cell-lines (KG-1, U937, and HL-60). Eight patients were also analyzed in relapse. Contrary to previous reports, we could show that the PRAME gene is expressed by CD34(+) stem cells. This might constitute a problem in using PRAME for tumor immunotherapy. Overexpression of PRAME was found in 62% (n=31) of our patients. The rates of overall and disease-free survival in this group were higher than in patients with no or low expression (P<0.05). PRAME expression was negatively correlated to the white blood cell count at diagnosis (P<0.05) and significantly higher in patients with t(8;21). The levels of expression at diagnosis corresponded with those at relapse (P<0.001) and increased levels could be found prior to the relapse in one patient who was regularly monitored. Our results suggest that the expression of PRAME is an indicator of favorable prognosis and could be a useful tool for monitoring minimal residual disease in childhood AML.

PRAME gene expression in childhood acute lymphoblastic leukemia

The gene PRAME (preferentially expressed antigen of melanoma) was found to be expressed at high levels in a large fraction of different tumors and adult leukemias. Since PRAME is only expressed at low levels in a few normal tissues and encodes an antigen recognized by autologous cytolytic T lymphocytes, it might be a good candidate for tumor immunotherapy. In this study, quantitative reverse transcriptase polymerase chain reaction was used to measure PRAME gene expression in 50 children with newly diagnosed acute lymphoblastic leukemia (ALL). Nine patients were also analyzed in relapse. Overexpression of PRAME was found in 42% (N = 21) of the patients. In accordance with our findings in acute myeloblastic leukemia (AML) patients, the rate of disease-free survival was higher and white blood cell counts at diagnosis were lower in patients with an overexpression of PRAME. However, in our group of ALL patients these findings were not statistically significant. The levels of expression at diagnosis corresponded well with those at relapse (P = 0.017). Although overexpression of PRAME was less frequent than in children with AML (62%) our results suggest that PRAME could be a useful target for immunotherapy in some children with ALL.

Fatty acid composition of lymphocyte membrane phospholipids in children with acute leukemia

The composition of phospholipid fatty acids (PLFA) of separated mononuclear blood cells (MNC) from patients with leukemia was established by high-resolution gas chromatography. Abnormal fatty acid concentrations are detected in the MNC membrane phospholipids in patients with acute lymphoblastic leukemia (ALL) without a deficiency of essential fatty acids (EFA). Significantly reduced relative levels of linoleic acid (4.35 vs. 7.82%; P<0.001) are found in the MNC-PL in patients with ALL as compared to a healthy control group. Moreover, the Delta6-desaturated fatty acids are increased: gamma-linoleic acid (3.56 vs. 0.17%; P<0.001), arachidonic acid (21.82 vs. 16.27%; P<0.05), docosatetraenoic acid (3.52 vs. 1.56%; P<0.001), docosapentaenoic acid (0.34 vs. 0.04%; P<0.001), octadecatetraenoic acid (0.53 vs. 0.23%; P<0.05), eicosatetraenoic acid (1.83 vs. 0.08%; P<0.001) and docosahexaenoic acid (2.77 vs. 1.54%; P<0.001). A increased Delta(6)-desaturase activity is postulated as the cause for the increased level of desaturate products or the increased Delta6-activity index (Ratio of gamma-linoleic acid+dihomogamma-linolenic acid to linoleic acid) (1.21 vs. 0.27; P<0.001). The Delta6-enzyme activities measured using linoleic acid and alpha-linoleic acid as substrate underscore these findings (Delta6(n-6); 2.49 vs. 0.65 and Delta6(n-3); 2.75 vs. 1.12 nmol x h(-1)/10(8) MNC). In contrast, patients with acute myeloid leukemia (AML) do not show any significant differences in the lymphocyte membrane PLFA and no Delta6-desaturase abnormalities.

Hybridoma cell growth and anti-neuroblastoma monoclonal antibody production in spinner flasks using a protein-free medium with microcarriers.

The main disadvantages of foetal calf serum as the world-wide common serum supplement for cell growth are its content of various proteins of variable concentrations between batches as well as its high cost. The use of serum-free and protein-free media is gradually becoming one of the goals of cell culture especially for standardizing culture conditions or for simple purification of cell products like monoclonal antibodies. The mouse hybridoma cells 14/2/1 were cultivated either in protein-free UltraDOMA medium or in serum-containing RPMI medium with and without microcarriers to generate high quantities of monoclonal antibodies against neuroblastoma tumour cells. Cell growth rate, IgG production, viability, glucose and lactate concentrations, attachment rate and doubling time have been used as investigation criteria. Modifications of culture procedures (static or stirred), inoculum density, and microcarrier concentration caused an improvement of monoclonal antibody production. The kinetics of antibody synthesis was best in spinner culture with 2 ml of microcarriers in protein-free medium. These results of short-term microcarrier culture in stirred spinner flasks indicate that IgG yields in protein-free medium 2.5-fold higher to those in serum-supplemented medium can be achieved.

Are MTT assays the right tool to analyze drug resistance caused by ABC-transporters in patient samples?

Drug resistance can be caused by ATP-binding-cassette (ABC)-transporters which function as outward pumps for chemotherapeutic drugs. The aim of the present study was to analyze the association between eight ABC-transporters (BCRP, MDR1, SMRP, MRP1, MRP2, MRP3, MRP4, and MRP5) and in vitro drug resistance. Leukemic cells from 52 children with previously untreated acute leukemia (ALL: n=37; AML: n=15) were analysed. The expression of the ABC-transporters was measured by TaqMan real-time PCR. In vitro drug resistance to cytarabine, vincristine, tioguanine, daunorubicin, etoposide, dexamethasone, and prednisone was analysed with methyl-thiazol-tetrazolium (MTT) assays.MDR1 was weakly associated with resistance to vincristine (p<0.05) in AML samples. No other correlation between an ABC-transporter and a higher in vitro drug resistance was found. In vitro drug resistance was not associated with the simultaneous expression of a larger number of ABC-transporters.MTT assays are a widely used and validated method to analyse in vitro drug resistance but they may not be a useful tool to detect resistance which is caused by drug efflux in patient samples. If that is the case, MTT assays and the expression of ABC-transporters could provide complementary information on the drug resistance profile of patients with acute leukemia.

Monoclonal antibodies against neuroblastoma. Production and preliminary characterization of their specificity

Neuroblastoma (NB) is a common childhood tumor that originates from neuroblasts of the neural crest. In this paper we will describe the production of murine monoclonal antibodies (mAbs) to human NB cell lines. The hybridomas were selected by ELISA and immunofluorescence for antibody binding to multiple human neuroblastoma cultured cell lines, but not to hematopoietic cells and leukemic cells. The mAbs were characterized in terms of their ability to bind to human cell lines and tissues. The IgG2a and IgG2b mAbs may prove useful in the diagnosis of therapy of neuroblastoma.

Polyunsaturated but not Conjugated Linoleic Acid Supplementation of Leukemic U937 Cells Can Act as an Amplification Factor for Photofrin-mediated Photodynamic Therapy

Abstract Polyunsaturated fatty acids located in leukemia cell membranes are excellent targets for peroxidation. They can significantly enhance the effectiveness of Photofrin-mediated photodynamic therapy (PDT)-induced cell killing. In this study, the peroxidizability of conjugated fatty acid isomers (9c,11t-linoleic acid and 9c,11c-linoleic acid) and polyunsaturated fatty acids (PUFAs; linoleic acid, γ-linolenic acid and arachidonic acid) with 2,2′-azo-bis(2-amidinpropane)dihydrochloride, soybean lipoxygenase and photomediated peroxidation are compared with each other. Peroxidation was determined using different methods: by means of gas chromatography to estimate the fatty acid (FA) consumption, by photometry for the level of FA peroxides or phospholipid peroxides and by definition of the content of malondialdehyde for thiobarbituric acid reactive substances (TBARS). The results suggest that the generation of oxidation products from individual FAs indicate a different formation rate of oxidation products. Radical FA peroxides were produced most by polyunsaturated arachidonic acid, followed by linoleic acid and γ-linolenic acid, whereas conjugated FA isomers did not generate peroxides. Accordingly, the levels of lipid peroxides and TBARS were substantially increased after incorporation and oxidation of polyunsaturated FAs into U937 cells and could significantly enhance the effectiveness of Photofrin-PDT–induced cytotoxicity. The results showed that PUFA, but not conjugated FA supplementation of U937 cells, can act as a PDT amplification factor.

Risk group definition in children with acute myeloid leukemia by calculating individual risk factors on the basis of a multivariate stepwise Cox regression analysis.

To define risk groups in children with acute myeloid leukaemia (AML), we conducted a multivariate stepwise Cox regression analysis of three consecutive multicentre studies in East Germany. The total number of patients was 240, but cytogenetics and remission status on day 15 were routinely investigated only in the most recent study, AML-III/93 (78 patients). We derived an equation to calculate individual risk factors, determined those risk factors for all patients of study AML-III/93 and divided them into three groups with 26 patients in each. The variables in the equation were: WBC, FAB-type, auer rods, cytogenetics and response status on day 15. The event-free survival was 80% in the low risk, 55% in the intermediate risk and 15% in the high risk group. Our results strongly suggest that calculating individual risk factors on the basis of a multivariate stepwise Cox regression analysis is a useful tool in defining risk groups.