Abstract

Abstract 2193

Introduction.

Vaccination with acute myeloid leukemia derived dendritic cells (AML-DC) is a promising immunotherapeutic tool to prevent relapse of AML due to presence of residual AML cells. However, in the clinical setting AML-DC vaccination is hampered in 40% of cases where AML-DC cannot be cultured from diagnosis material or where numbers of AML-cells were too low to obtain sufficient numbers of AML-DC. Here we explore whether expansion prior to differentiation is feasible for those cases where vaccine preparation is hampered due to low numbers of cells.

Material & Methods.

After obtaining informed consent, peripheral blood or bone marrow mononuclear cells from 51 AML patients were isolated by density centrifugation using Ficoll-Paque. AML blasts were resuspended in culture medium supplemented with different cocktails of cytokines with known myeloid growth-promoting effects (i.e. GM-CSF, G-CSF, SCF, FLT3-L, IL-3). Cells were passaged every 3–4 days when expansion exceeded more than 2-fold. After 1–2 months the capacity the expanded cells to differentiate into DC was tested by culture with IL-4, TNFα, GM-CSF or the calcium ionophore A23187 and IL-4. The cells were analyzed for progenitor (CD34, CD14) and DC (CD1a, DC-SIGN, CD40, CD80, CD86) markers by flow cytometry pre- and post differentiation.

Results.

Cells from 51 patients were able to proliferate to varying numbers of passages (mean 16, range 1–66). Of all cases, 76% (39/51) could be kept in culture for at least two months, thus leading to an expansion factor of at least 256. Overall, low expression of TNFαRI and high expression of TNFαRII was associated with an ongoing proliferative capacity. No correlation was found with various FAB subtypes. Expansion was achieved by most of the tested cocktails but we identified a slight advantage for the combination of GM-CSF, FLT3L, IL-3 and SCF. Upon culture and expansion, marker expression changed: CD34 expression decreased in 7 out of 9 evaluated patients, whereas CD14 expression increased significantly (p= 0.04) in 8 out of 13 evaluated patients (from no expression on blasts cells to 51% (range 19–89%)). High expression levels of CD14 correlated with the ability to subsequently differentiate into DC, with the preferred GM-CSF, IL-4 and TNFα.differentiation cocktail. In 7 out of 13 expanded cultures DC could be cultured.

Conclusion.

DC progenitors were successfully expanded from AML blood and bone marrow samples, enabling the generation of sufficient numbers of AML-DC for vaccination purposes. Expanded cells gained CD14 expression, which is a positive predictor for DC-differentiation [Mohty et al. Leukemia 2002; Houtenbos et al. Leukemia 2003]. Thus, more AML patients will become eligible for DC vaccination upon progenitor expansion.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.