Novel Long-Term Co-Culture Approach Identifies Prognostically Important Heterogeneity Of Stem/Progenitor Cell Involvement In Human Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1318-1318
Author(s):  
George S. Laszlo ◽  
Jack M. Lionberger ◽  
Kimberly H. Harrington ◽  
Chelsea J. Gudgeon ◽  
Megan Othus ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Progenitor Cells ◽  
Growth Pattern ◽  
Myeloid Leukemia ◽  
Culture Method ◽  
Molecular Abnormalities ◽  
Human Acute Myeloid Leukemia ◽  
Acute Myeloid

Abstract Background The cellular origin of human acute myeloid leukemia (AML) remains controversial. Limited G6PD-based X chromosome inactivation (XCI) studies suggested heterogeneity of leukemic stem cells (LSCs), with evidence of some AMLs resulting from multipotent CD34+/CD33- stem cells and others emerging from, or predominantly involving, lineage-committed CD34+/CD33+ myeloid precursors. So far, the difficulty of growing stem/progenitor cells from primary AML specimens has provided a major challenge for precise study of human LSCs and clinical implications of their heterogeneity. To overcome this, we developed a novel co-culture method to support long-term in vitro growth of primary AML specimens together with a highly sensitive XCI assay for isolated immature progenitor cells. Methods Diagnostic bone marrow specimens from 50 adult females with newly diagnosed AML were obtained from the SWOG Sample Repository. All patients received standard induction therapies without the CD33-targeting immunoconjugate, gemtuzumab ozogamicin. Human umbilical vein endothelial cells, transduced with a lentiviral construct encoding the open reading frame 1 of the early region 4 of adenovirus to provide growth factor/serum-independence, were co-cultured with aliquots of flow cytometrically-isolated CD34+/CD33- and CD34+/CD33+ cells in medium containing the aryl hydrocarbon receptor antagonist, StemReginin-1. Cultures were replenished with fresh medium weekly and kept in hypoxia for up to 8 weeks, and defined fractions plated after 4, 6, and 8 weeks for colony-forming cell assays. Resulting colony forming units-granulocyte and/or macrophage (CFU-GMs) were harvested and individually assessed for XCI via methylation-sensitive PCR-based analysis of a polymorphic short tandem repeat (STR) in the X-linked human androgen-receptor gene (HUMARA). Specimen growth was considered if it yielded ≥5 CFU-GMs after ≥4 weeks of co-culture. Specimens with >75% disease-specific CFU-GMs were considered clonal. Results Among the 50 specimens, 45 (90%) were informative with regard to HUMARA STR, allowing clonal assessment of isolated cell progeny. Sixteen (35.6%) yielded CFU-GMs derived from CD34+/CD33- cells after long-term (≥4 weeks) co-culture sufficient for clonal analyses, whereas the remaining samples yielded either no growth (n=22) or insufficient CFU-GM growth (n=8) from this starting cell population. Five of the 16 specimens with sufficient CFU-GM growth derived from CD34+/CD33- cells (31.3%) yielded colonies consistent with a polyclonal growth pattern, and 3 samples without sufficient CFU-GM growth derived from CD34+/CD33- cells yielded colonies from unsorted cells that were consistent with a polyclonal growth pattern; these 8 leukemias were cytogenetically classified as favorable (n=3) or intermediate-risk (n=5, including 2 with normal karyotype and NPM1 but not FLT3/ITD mutation). In contrast, 11 specimens yielded CFU-GMs growth derived from CD34+/CD33- with a pattern consistent with clonal growth. The complete remission rate was slightly but statistically non-significantly higher among patients whose specimens yielded polyclonal CFU-GMs derived from CD34+/CD33- cells relative to those with clonal progenitor growth (87.5% vs. 63.6%, P=0.34). More importantly, however, the overall and relapse-free survival of patients whose specimens yielded polyclonal CFU-GMs derived from CD34+/CD33- or unsorted cells was significantly longer than that for patients whose specimens yielded a clonal progenitor growth pattern after long-term culture of CD34+/CD33- cells (P=0.0006 and P=0.0054, respectively; Figure 1); restriction of this analysis to the 16 specimens with sufficient CFU-GM growth from CD34+/CD33- cells did not change this outcome difference (e.g. hazard ratio for overall survival for restricted dataset: 0.13 [95% confidence interval: 0.06-0.72] vs 0.08 [0.04-0.44]). Conclusion Our novel co-culture method allows for the study of progenitor/stem cell involvement in a significant portion of human AMLs. Our data suggest that a subset of adult AMLs emerges from, or predominantly involves, only lineage-committed CD34+/CD33+ myeloid precursors but not the less mature CD34+/CD33- precursors. This subset, which is comprised of AMLs with diverse cytogenetic/molecular abnormalities, is characterized by an excellent outcome with intensive chemotherapy. Disclosures: No relevant conflicts of interest to declare.

5-Azacytidine and Decitabine Induce Demethylation and Re-Expression of FAS (CD95) and Promote Apoptosis in Neoplastic Cells in Acute Myeloid Leukemia (AML),

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3463-3463
Author(s):  
Heidrun Karlic ◽  
Rene Reitermaier ◽  
Viviane Ghanim ◽  
Harald Herrmann ◽  
Roman Thaler ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Neoplastic Cells ◽  
Drug Induced ◽  
Secondary Aml ◽  
Induced Apoptosis ◽  
Acute Myeloid

Abstract Abstract 3463 Epigenetic and apoptosis-regulating mechanisms have been implicated as critical factors contributing to the progression from myelodysplastic syndromes (MDS) to secondary acute myeloid leukemia (AML). However, the exact molecular mechanisms and genes involved in disease evolution have not been identified yet. We screened for epigenetically regulated pro-apoptotic effector molecules in neoplastic cells in patients with MDS (n=50) and AML (n=30). Among a series of potential regulators, we identified FAS (CD95) as an epigenetically regulated critical death regulator in neoplastic cells. As assessed by qPCR, bone marrow cells obtained from patients with low risk MDS were found to display high levels of FAS, whereas FAS mRNA levels were lower or undetectable in patients with advanced MDS (with excess of blasts) or secondary AML. Moreover, we were able to show by multicolor flow cytometry that CD34+/CD38+ progenitor cells and CD34+/CD38- stem cells in MDS and AML display measurable FAS (CD95) on their surface, with slightly higher levels detectable in progenitor cells in low risk MDS compared to high risk MDS and secondary AML. Methylation-specific PCR and qPCR revealed that the FAS-promoter is hypermethylated in primary AML cells as well as in various AML cell lines including KG1 and HL60 thus repressing mRNA-synthesis. In addition, we found that exposure to 5-Azacytidine or Decitabine leads to demethylation of CpG-rich regions closest to the transcription starting sites, and thus to re-expression of FAS in AML cells. In vitro-targeting of AML cells by demethylating drugs was also found to revert epigenetic inactivation of other tumor suppressor genes such as CDKN2B (P15), CDKN2A (P16), ESR1 (estrogen-receptor alpha), with subsequent normalization of mRNA expression levels. Next, we asked whether CD95 acts as a critical death regulator involved in drug-induced apoptosis in neoplastic cells. In these experiments, both demethylating agents, 5-Azacytidine or Decitabine, were found to induce dose-dependent apoptosis and growth inhibition in primary AML cells, primary MDS cells, and in all AML cell lines examined. Drug-induced apoptosis in AML cells was accompanied by activation of caspase 8 and caspase 3 as well as increased expression of proapoptotic FAS/CD95 as determined by qPCR, Western blotting, and flow cytometry. Moreover, both drugs were found to induce expression of the FAS-ligand and DAPK1 in neoplastic cells. We then applied a siRNA against FAS. The siRNA-induced knock-down of FAS was found to block drug-induced FAS expression and FAS-induced apoptosis in KG1 cells and HL60 cells. In conclusion, our data show that FAS is hypermethylated in neoplastic cells in patients with advanced MDS and AML, that demethylating agents lead to re-expression of FAS, and that drug-induced FAS expression mediates apoptosis in leukemic cells. As FAS is also expressed on neoplastic stem cells, these observations may have clinical implications and may explain beneficial effects seen with 5-Azacytidine or Decitabine in patients with advanced MDS. Disclosures: Valent: Novartis: Consultancy, Honoraria, Research Funding.

Most Acute Myeloid Leukemia Progenitor Cells With Long-Term Proliferative Ability In Vitro and In Vivo Have the Phenotype CD34+/CD71−/HLA-DR−

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4325-4335 ◽  
Author(s):  
A. Blair ◽  
D.E. Hogge ◽  
H.J. Sutherland
Keyword(s):  
Acute Myeloid Leukemia ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Scid Mice ◽  
Hla Dr ◽  
Proliferation In Vitro ◽  
Acute Myeloid

Acute myeloid leukemia (AML) occurs as the result of malignant transformation in a hematopoietic progenitor cell, which proliferates to form an accumulation of AML blasts. Only a minority of these AML cells are capable of proliferation in vitro, suggesting that AML cells may be organized in a hierarchy, with only the most primitive of these cells capable of maintaining the leukemic clone. To further investigate this hypothesis, we have evaluated a strategy for purifying these primitive cells based on surface antigen expression. As an in vitro endpoint, we have determined the phenotype of AML progenitor cells which are capable of producing AML colony-forming cells (CFU) for up to 8 weeks in suspension culture (SC) and compared the phenotype with that of cells which reproduce AML in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. AML cells were fluorescence-activated cell sorted (FACS) for coexpression of CD34 and CD71, CD38, and/or HLA-DR and the subfractions were assayed in vitro and in vivo at various cell doses to estimate purification. While the majority of primary AML CFU lacked expression of CD34, most cells capable of producing CFU after 2 to 8 weeks in SC were CD34+/CD71−. HLA-DR expression was heterogeneous on cells producing CFU after 2 to 4 weeks. However, after 6 to 8 weeks in SC, the majority of CFU were derived from CD34+/HLA-DR− cells. Similarly, the majority of cells capable of long-term CFU production from SC were CD34+/CD38−. Most cells that were capable of engrafting NOD/SCID mice were also CD34+/CD71− and CD34+/HLA-DR−. Engraftment was not achieved with CD34+/CD71+ or HLA-DR+subfractions, however, in two patients, both the CD34+and CD34− subfractions were capable of engrafting the NOD/SCID mice. A three-color sorting strategy combining these antigens allowed approximately a 2-log purification of these NOD/SCID leukemia initiating cells, with engraftment achieved using as few as 400 cells in one experiment. Phenotyping studies suggest even higher purification could be achieved by combining lack of CD38 expression with the CD34+/CD71− or CD34+/HLA DR− phenotype. These results suggest that most AML cells capable of long-term proliferation in vitro and in vivo share the CD34+/CD71−/HLA-DR− phenotype with normal stem cells. Our data suggests that in this group of patients the leukemic transformation has occurred in a primitive progenitor, as defined by phenotype, with some degree of subsequent differentiation as defined by functional assays.

scholarly journals Long-term observation reveals high-frequency engraftment of human acute myeloid leukemia in immunodeficient mice

Haematologica ◽  
2017 ◽  
Vol 102 (5) ◽  
pp. 854-864 ◽  
Author(s):  
Anna M. Paczulla ◽  
Stephan Dirnhofer ◽  
Martina Konantz ◽  
Michael Medinger ◽  
Helmut R. Salih ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
High Frequency ◽  
Myeloid Leukemia ◽  
Immunodeficient Mice ◽  
Human Acute Myeloid Leukemia ◽  
Long Term Observation ◽  
Acute Myeloid
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