TNF-α receptor 1 expression on acute myeloid leukemic blasts predicts differentiation into leukemic dendritic cells

Abstract

TNF-a receptor 1 expression on acute myeloid leukemic blasts predicts differentiation into leukemic dendritic cells Leukemia (2004) the unique ability to differentiate into leukemic DC, thereby conserving their intrinsic leukemia antigen repertoire and obtaining full potential of antigen presentation. We have shown that DC generated from acute myeloid leukemic (AML) blasts are able to initiate autologous cytotoxic T-cell responses, and thus hold promise for vaccination purposes in a minimal residual disease (MRD) status. 1 In a recently performed phase-I study on CML-DC vaccination in advanced stage chronic myeloid leukemia (CML), we were able to detect strong DTH-responses representing autologous CML-specific T-cell responses. 2 The presence of various cytokines such as GM-CSF, SCF, Flt3-L, TNF-a, IL-3 and IL-4 induces differentiation into AML-DC in approximately 2 weeks, also under clinical grade serum-free The PIG3 polymorphic promoter shows LOH in de novo AML. Electropherograms obtained with labeled PCR products containing the PIG3 microsatellite (TGYCC)n. Top: sample obtained at diagnosis displayed a loss of the 17 repeats allelle (arrow). Bottom: sample obtained at remission. 1149Leukemia conditions. 1, 3 We showed previously that it is also possible to generate AML-DC in a 2-day culture period by the incubation with calcium ionophores (CI), thereby bypassing receptor mediated signalling. 1 AML-DC cultured by CI displayed, both phenotypically and functionally, a more mature state than those cultured with a combination of cytokines. However, CI-cultured cells were found to be less viable. 1 Consequently, the CI-based method can only be used if high amounts of AML blasts are available. The common use of the cytokine-based method to develop AML-DC is accompanied by a variable DC differentiation inducibility, irrespective of French-American-British (FAB) classification. For these reasons, we examined the possibility to select a culture method in advance that will result in the most optimal generation of AML-DC for each individual patient. In this study we investigated whether the cytokine receptor profile of AML blasts is predictive for the ability to differentiate into AML-DC. After informed consent, peripheral blood and bone marrow mononuclear cells of AML patients were collected. A total of 33 patients (21 female, 12 male) were included with a median age of 60 years (range: 22-88), with the following FAB classifications: M0 (2), M1 (5), M2 (5), M4 (8), M5 (10), RAEB-t (1), unclassified (2). AML cells were cultured as published previously. 1,3 Briefly, fresh or thawed mononuclear cells were cultured in either RPMI-1640; 20% fetal calf serum; 100 U/ml penicillin and 100 mg/ml streptomycin (n ¼ 6) or Stem Spant SFEM serum-free medium (n ¼ 27); 100 U/ml penicillin and 100 mg/ml streptomycin. Differentiation towards DC was induced by the addition of a combination of cytokines (n ¼ 33): GM-CSF, TNF-a, IL-3, SCF, Flt3-L and IL-4 or CI A23187 and IL-4 (n ¼ 28). 1,3 IL-4 was added to the CI culture to prevent the...

Flt-3 internal tandem duplication hampers differentiation of AML blasts towards leukemic dendritic cells

“…For CD120a, a cutoff value of 15% was used, as described previously. 6 In the multivariate analysis powerful predictors of cytokine-mediated AML-DC culture outcome proved to be Flt-3 ITD (regression coefficient B:-3.41, P ¼ 0.0001), CD14 (B:3.28, P ¼ 0.0001) and CD120a (B:2.82, P ¼ 0.0006). Based upon the three relevant predictors Flt-3 ITD, CD14 and CD120a, cytokine-mediated AML-DC culture outcome can be predicted by the following formula:…”

“…6 Median AML-DC yield in the cytokine-based culture method was 12% (range: 0-70%). Defined by the median AML-DC yield, cultures resulting in X10% AML-DC were considered successful.…”

“…5 Because of the limitations of these assays, a simple and reliable test is needed for screening and genotyping. We developed a microchip diagnostic platform (NMW NanoChip Molecular Biology Workstation, manufactured by Nanogen Inc., San Diego, CA, USA) 6 to identify the JAK-2 mutation in granulo-…”

Employing the immunological synapse in AML: Development of leukemic dendritic cells for active specific immunization

“…Reflecting the heterogeneity of the disease, cultured AML-DC derived from a spectrum of AML patients form a heterogeneous population with a variable expression of, especially, the costimulatory molecules CD80 and CD86. However, the possibility to predict AML-DC culture outcome by the expression of certain surface markers on AML blasts enables selection of culture methods optimal for the generation of mature AML-DC (Houtenbos et al, 2004;Mohty et al, 2002;Re et al, 2002). In a group of 152 patients we succeeded in culturing mature AML-DC in 63% of the patients by the use of either a cytokine-based culture method or a calcium-ionophore-based culture method (Houtenbos et al, in preparation).…”

The characterization and role of leukemia cell-derived dendritic cells in immunotherapy for leukemic diseases

“…Other factors also affect AML-DC formation and functioning. For example, only AML primary cells expressing CD14 and TNF-α-RI can be successfully induced to differentiate into DCs (AML-DCs) by cytokines (35,36). The FLT3-ITD (FLT3 internal tandem duplication) can inhibit AML-DC formation and down-regulate VEGF expression and induce the production of functional AML-DCs (34).…”