Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Sep;90(9):1047-58.
doi: 10.1007/s00277-011-1231-2. Epub 2011 Apr 26.

Elevated Frequencies of Leukemic Myeloid and Plasmacytoid Dendritic Cells in Acute Myeloid Leukemia With the FLT3 Internal Tandem Duplication

Affiliations
Free PMC article

Elevated Frequencies of Leukemic Myeloid and Plasmacytoid Dendritic Cells in Acute Myeloid Leukemia With the FLT3 Internal Tandem Duplication

Mareike Rickmann et al. Ann Hematol. .
Free PMC article

Abstract

Some 30% of acute myeloid leukemia (AML) patients display an internal tandem duplication (ITD) mutation in the FMS-like tyrosine kinase 3 (FLT3) gene. FLT3-ITDs are known to drive hematopoietic stem cells towards FLT3 ligand independent growth, but the effects on dendritic cell (DC) differentiation during leukemogenesis are not clear. We compared the frequency of cells with immunophenotype of myeloid DC (mDC: Lin(-), HLA-DR(+), CD11c(+), CD86(+)) and plasmacytoid DC (pDC: Lin(-), HLA-DR(+), CD123(+), CD86(+)) in diagnostic samples of 47 FLT3-ITD(-) and 40 FLT3-ITD(+) AML patients. The majority of ITD(+) AML samples showed high frequencies of mDCs or pDCs, with significantly decreased HLA-DR expression compared with DCs detectable in ITD(-) AML samples. Interestingly, mDCs and pDCs sorted out from ITD(+) AML samples contained the ITD insert revealing their leukemic origin and, upon ex vivo culture with cytokines, they acquired DC morphology. Notably, mDC/pDCs were detectable concurrently with single lineage mDCs and pDCs in all ITD(+) AML (n = 11) and ITD(-) AML (n = 12) samples analyzed for mixed lineage DCs (Lin(-), HLA-DR(+), CD11c(+), CD123(+)). ITD(+) AML mDCs/pDCs could be only partially activated with CD40L and CpG for production of IFN-α, TNF-α, and IL-1α, which may affect the anti-leukemia immune surveillance in the course of disease progression.

Figures

Fig. 1
Fig. 1
Experimental design and validation of methods. a Schema of the flow cytometry analyses for detection of mDCs and pDCs. b Frequencies of DCs detectable in fresh (black) versus thawed (grey) PBMCs obtained from healthy donors (n = 10) do not differ significantly (p values of mDCs, 0.15; pDCs, 0.44.). c Frequencies of cells with mDC and pDC immunophenotypes as total cells or with additional expression of the activation marker CD86 and maturation marker CD83 (numbers indicate the average and standard deviation for each analyses; n = 10)
Fig. 2
Fig. 2
Flow cytometry analyses of cells with DC immunophenotype in AML diagnostic samples. a Frequencies of mDCs, and b pDCs detected in FLT3-ITD+ (upper graphs) and FLT3-ITD- AML samples (lower graphs). All analyses indicated significantly higher frequencies of mDCs and pDCs in ITD+ and ITD AML samples in comparison with DCs analyses in healthy subjects (p < 0.05). The panels on the right side show representative FACS analyses samples with the gating strategy
Fig. 3
Fig. 3
Flow cytometry analyses of HLA-DR expression in AML cells. a Mean fluorescence intensity (M.F.I.) of HLA-DR expression on mDCs showing significant (asterisk) differences in the expression level comparing healthy (n = 10), ITD (n = 42) and ITD+ (n = 33) PBMC. b M.F.I. of HLA-DR expression on pDCs
Fig. 4
Fig. 4
Sequencing of PCR-ITD mutational insert product from ITD+ patients. In addition to the w.t. FLT3 amplification product, the ITD mutational insert is detectable in the original AML patient samples and in the sorted DCs
Fig. 5
Fig. 5
Morphological analyses of cytospin/Giemsa preparations of FLT3-ITD+ AML diagnostic samples prior and post-sorting of mDCs and pDCs. AML samples obtained from three patients and containing high frequencies of mDCs (a) or pDCs (b) before and after sorting resemble leukemia blasts. c ITD+ AML-mDCs after sorting and culture with GM-CSF/IL-4 and maturation with CD40L show cell enlargement and veils. d ITD+ AML pDCs after sorting and culture with IL-3 and maturation with CD40L show cell enlargement and high granularity
Fig. 6
Fig. 6
Immunophenotypic detection of mDCs/pDCs’ mixed lineages. a Schematic presentation of flow cytometry analyses. b Average frequency of mixed lineage mDCs/pDCs, single mDCs and pDCs obtained for ITD+ and ITD patients
Fig. 7
Fig. 7
Functional analyses of double positive CD11c/CD123 mDCs/pDCs in ITD+ AML samples of three patients. a Gating approach for detection of mDCs/pDCs. b Frequency of mDC/pDCs with detectable intracellular cytokines after stimulation with CD40L or CpG

Similar articles

See all similar articles

Cited by 10 articles

See all "Cited by" articles

References

    1. Rovati B, Mariucci S, Manzoni M, Bencardino K, Danova M. Flow cytometric detection of circulating dendritic cells in healthy subjects. Eur J Histochem. 2008;52(1):45–52. - PubMed
    1. Li L, Piloto O, Kim KT, Ye Z, Nguyen HB, Yu X, Levis M, Cheng L, Small D. FLT3/ITD expression increases expansion, survival and entry into cell cycle of human haematopoietic stem/progenitor cells. Br J Haematol. 2007;137(1):64–75. - PubMed
    1. Naik SH. Demystifying the development of dendritic cell subtypes, a little. Immunol Cell Biol. 2008;86(5):439–452. doi: 10.1038/icb.2008.28. - DOI - PubMed
    1. Karsunky H, Merad M, Cozzio A, Weissman IL, Manz MG. Flt3 ligand regulates dendritic cell development from Flt3+ lymphoid and myeloid-committed progenitors to Flt3+ dendritic cells in vivo. J Exp Med. 2003;198(2):305–313. doi: 10.1084/jem.20030323. - DOI - PMC - PubMed
    1. Kelly LM, Liu Q, Kutok JL, Williams IR, Boulton CL, Gilliland DG. FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myeloproliferative disease in a murine bone marrow transplant model. Blood. 2002;99(1):310–318. doi: 10.1182/blood.V99.1.310. - DOI - PubMed

MeSH terms

Substances

Feedback