Identification of Leukemic Stem Cells in Acute Myeloid Leukemia Patients

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5309-5309
Author(s):  
Manal M W Elmasry ◽  
Alaa Elhaddad
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Mononuclear Phagocytes ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Two Samples ◽  
Acute Myeloid

Abstract Background: Acute myeloblastic leukemia (AML) can be viewed as newly formed, abnormal hemopoeitic tissue initiated by few leukemic stem cells (LSCs). Recognizing the LSC and identifying their behavior, plays a pivotal role in the approach of a targeted therapy.Colony-stimulating factor 1 (CSF-1), also known as M-CSF, is a protein ligand that acts on the CSF1R promotes mononuclear phagocytes survival, proliferation and differentiation. Aim of the work: Defining the self-renewing [Thy1-, CD34+, CD38-] LICs in AML cases before and after induction chemotherapy as a predictor for relapse and to determine how CSF1R (Fms) and CD34 markers affect the growth and survival of human leukemic cells in the CD38- Thy1- population. Patients and methods: This study was carried out on 30 samples from the peripheral blood of adult patients with de-novo acute myeloid leukemia. The majority of the patients were monocytic AML Samples were sorted into four populations (Fms+CD34-, Fms+CD34+, Fms-CD34+ and Fms-CD34-) according to the surface markers of the cells. Cells were cultured on mouse stromal cells transfected with a plasmid containing human CSF-1. Samples were cultured using Iscove's modified Dulbecc's medium (IMDM).The cultures were assessed for survival of leukemic cells in days. Results: The mean survival in days of the cells was 13.9 before chemotherapy and 14.1 after chemotherapy. The difference in growth was insignificant (p>0.05). The Fms-CD34+ population in all but two samples tested had the longest survival time in culture. Conclusion: Our results suggest that leukemic stem cells may survive chemotherapy mainly due to their quiescence. Human CSF-1 was shown to increase the number of leukemic cells in co-culture with mouse stroma after 5 weeks. A novel leukemic stem cell (Fms-CD34+) has been identified and is the cell responsible for the growth and maintenance of the leukemic bulk. Disclosures No relevant conflicts of interest to declare.

scholarly journals Evidence for malignant transformation in acute myeloid leukemia at the level of early hematopoietic stem cells by cytogenetic analysis of CD34+ subpopulations

Blood ◽  
1995 ◽  
Vol 86 (8) ◽  
pp. 2906-2912 ◽  
Author(s):  
D Haase ◽  
M Feuring-Buske ◽  
S Konemann ◽  
C Fonatsch ◽  
C Troff ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Hematopoietic Stem Cells ◽  
Malignant Transformation ◽  
Cell Sorting ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Acute myeloid leukemia (AML) is a heterogenous disease according to morphology, immunophenotype, and genetics. The retained capacity of differentiation is the basis for the phenotypic classification of the bulk population of leukemic blasts and the identification of distinct subpopulations. Within the hierarchy of hematopoietic development and differentiation it is still unknown at which stage the malignant transformation occurs. It was our aim to analyze the potential involvement of cells with the immunophenotype of pluripotent stem cells in the leukemic process by the use of cytogenetic and cell sorting techniques. Cytogenetic analyses of bone marrow aspirates were performed in 13 patients with AML (11 de novo and 2 secondary) and showed karyotype abnormalities in 10 cases [2q+, +4, 6p, t(6:9), 7, +8 in 1 patient each and inv(16) in 4 patients each]. Aliquots of the samples were fractionated by fluorescence-activated cell sorting of CD34+ cells. Two subpopulations, CD34+/CD38-(early hematopoietic stem cells) and CD34+/CD38+ (more mature progenitor cells), were screened for karyotype aberations as a marker for leukemic cells. Clonal abnormalities and evaluable metaphases were found in 8 highly purified CD34+/CD38-populations and in 9 of the CD34+/CD38-specimens, respectively. In the majority of cases (CD34+/CD38-, 6 of 8 informative samples; CD34+/CD38+, 5 of 9 informative samples), the highly purified CD34+ specimens also contained cytogenetically normal cells. Secondary, progression-associated chromosomal changes (+8, 12) were identified in the CD34+/CD38-cells of 2 patients. We conclude that clonal karyotypic abnormalities are frequently found in the stem cell-like (CD34+/CD38-) and more mature (CD34+/CD38+) populations of patients with AML, irrespective of the phenotype of the bulk population of leukemic blasts and of the primary or secondary character of the leukemia. Our data suggest that, in AML, malignant transformation as well as disease progression may occur at the level of CD34+/CD38-cells with multilineage potential.

The Target Receptor Siglec-3 (CD33) Is Expressed on AML Stem Cells in a Majority of All Patients with AML.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4324-4324
Author(s):  
Alexander W. Hauswirth ◽  
Stefan FLorian ◽  
Maria-Theresa Krauth ◽  
Gerit-Holger Schernthaner ◽  
Edgar Selzer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Staining Technique ◽  
Scid Mice ◽  
Leukemic Cells ◽  
Cytostatic Drug ◽  
Leukemic Stem Cells ◽  
Targeting Therapy ◽  
Acute Myeloid

Abstract The cell surface antigen Siglec-3 = CD33 is becoming increasingly important as target of therapy in acute myeloid leukemia (AML). In particular, a conjugate consisting of the humanized CD33 antibody P67.6 (gemtuzumab) and the cytostatic drug calicheamicin has been developed for clinical use and was found to work as an effective antileukemic agent (Mylotarg®) in patients with CD33+ AML. In normal myelopoiesis, expression of CD33 is restricted to advanced stages of differentiation, whereas primitive stem cells do not express CD33 (Siglec-3). In line with this notion, CD33-targeting therapy is a non-myeloablative approach. In the present study, we asked whether leukemic stem cells in patients with AML express CD33. For this purpose, a multicolor-staining technique was applied in eleven patients with AML. Leukemic stem cells were defined as CD34+/CD38−/CD123+ cells. In all patients in whom the majority of myeloblasts expressed CD33 (=CD33+ AML, n=8), the AML progenitor cells reacted with the CD33 antibody P67.6. Repopulation experiments utilizing NOD/SCID mice confirmed that the AML stem cells in these patients reside within the CD33+ subpopulation of leukemic cells. Moreover, AML stem cells (CD34+/CD38−/CD123+ cells) highly purified (>98% purity) from patients with (CD33+) AML by cell sorting, were found to express CD33 mRNA in RT-PCR analyses. To demonstrate that AML stem cells can also reside within the CD33-negative fraction of the AML clone, we purified CD33-negative cells in a patient with AML in whom a majority of leukemic stem cells were found to lack CD33. In this particular patient, the CD33-negative cells were found to repopulate NOD/SCID mice with leukemias in the same way as the entire leukemic clone did. The CD33 antigen was neither detectable on CD34+/CD38− cells in the normal bone marrow nor on leukemic stem cells in patients with CD33-negative AML. In summary, our data show that leukemic stem cells in patients with CD33+ AML frequently express the target receptor CD33. This observation is in favor of novel treatment concepts employing CD33-targeting antibodies (Mylotarg®) in acute myeloid leukemia.

Impact of Leukemia Stem Cells Phenotype Expression on Response to Induction Therapy in Acute Myeloid Leukemia Patients

2020 ◽  
Vol 20 (2) ◽  
pp. 145-151 ◽  
Author(s):  
Faez Almohsen ◽  
Subh S. Al-Mudallal
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Complete Remission ◽  
Induction Therapy ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Laboratory Data ◽  
Leukemic Stem Cells ◽  
Color Flow ◽  
Acute Myeloid

Background: Laboratory data suggest that acute myeloid leukemia AML originates from a rare population of cells, termed Leukemic Stem Cells (LSCs) or leukemia-initiating cells, which are capable of self-renewal, proliferation and differentiation into malignant blasts. There is a universal agreement that LSCs lie within the CD34+ compartment of hemopoietic cells and most of leukemic stem cells express the interleukin-3 alpha chain receptor, CD123 and lack CD38. This study aimed to estimate the expression of LSC phenotype in AML patients and to correlate it with response to induction therapy. Methods: A cohort of 41 patients older than 15 years with newly diagnosed de novo AML were enrolled in this study. They were obtained from the National center of hematology in Baghdad and Baghdad teaching hospital between February and July 2013. The expression of CD34, CD38 and CD123 was assessed by multi-color flow cytometry. LSC positive (LSC+) samples must express CD34 and CD123 and lack the expression of CD38 in >1% of cells. French American British (FAB) classification system was used in this study. : After four weeks of induction therapy; three groups were found: those who reached the Complete morphological Remission (CR), those who failed to reach CR and those who died before the assessment of morphological remission. The last two groups were merged for statistical purposes. Results: After the course of induction therapy, 41.46% of patients had complete morphological remission while 58.54% of the studied patients failed to reach complete remission. The Complete Remission (CR) rate was higher (53.33%) in patients who were negative for LSC phenotype than patients who were positive for LSC phenotype (34.61%). Conclusions: LSCs were expressed in 63.41% of AML cases and were in approximate distribution in FAB M3 and non-M3 patients. The expression of LSC phenotype was associated with poor response to induction therapy in AML patients.

scholarly journals Evidence for malignant transformation in acute myeloid leukemia at the level of early hematopoietic stem cells by cytogenetic analysis of CD34+ subpopulations

Blood ◽  
1995 ◽  
Vol 86 (8) ◽  
pp. 2906-2912 ◽  
Author(s):  
D Haase ◽  
M Feuring-Buske ◽  
S Konemann ◽  
C Fonatsch ◽  
C Troff ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Hematopoietic Stem Cells ◽  
Malignant Transformation ◽  
Cell Sorting ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a heterogenous disease according to morphology, immunophenotype, and genetics. The retained capacity of differentiation is the basis for the phenotypic classification of the bulk population of leukemic blasts and the identification of distinct subpopulations. Within the hierarchy of hematopoietic development and differentiation it is still unknown at which stage the malignant transformation occurs. It was our aim to analyze the potential involvement of cells with the immunophenotype of pluripotent stem cells in the leukemic process by the use of cytogenetic and cell sorting techniques. Cytogenetic analyses of bone marrow aspirates were performed in 13 patients with AML (11 de novo and 2 secondary) and showed karyotype abnormalities in 10 cases [2q+, +4, 6p, t(6:9), 7, +8 in 1 patient each and inv(16) in 4 patients each]. Aliquots of the samples were fractionated by fluorescence-activated cell sorting of CD34+ cells. Two subpopulations, CD34+/CD38-(early hematopoietic stem cells) and CD34+/CD38+ (more mature progenitor cells), were screened for karyotype aberations as a marker for leukemic cells. Clonal abnormalities and evaluable metaphases were found in 8 highly purified CD34+/CD38-populations and in 9 of the CD34+/CD38-specimens, respectively. In the majority of cases (CD34+/CD38-, 6 of 8 informative samples; CD34+/CD38+, 5 of 9 informative samples), the highly purified CD34+ specimens also contained cytogenetically normal cells. Secondary, progression-associated chromosomal changes (+8, 12) were identified in the CD34+/CD38-cells of 2 patients. We conclude that clonal karyotypic abnormalities are frequently found in the stem cell-like (CD34+/CD38-) and more mature (CD34+/CD38+) populations of patients with AML, irrespective of the phenotype of the bulk population of leukemic blasts and of the primary or secondary character of the leukemia. Our data suggest that, in AML, malignant transformation as well as disease progression may occur at the level of CD34+/CD38-cells with multilineage potential.

Cytosine Arabinoside Chemotherapy Does Not Enrich For Leukemic Stem Cells In Xenotransplantation Model Of Human Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1651-1651 ◽  
Author(s):  
Fabienne de Toni ◽  
Robin L. Perry ◽  
Estelle Saland ◽  
Mayumi Sugita ◽  
Marion David ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Frequent Relapses ◽  
Cell Cycle Status ◽  
Acute Myeloid

Abstract Despite a high rate of complete remission after treatment with conventional genotoxic agents, the overall survival of patients with acute myeloid leukemia (AML) is poor due to frequent relapses caused by the chemoresistance of rare leukemic stem cells (LSCs, also called Scid-Leukemia Initiating Cells). This unfavorable situation leads to a strong need to characterize those cells in order to target them with new specific therapies. Using a robust immunodeficient mouse model (NOD/LtSz-scid IL2Rγchainnull or NSG), we have previously shown that these LSCs were rare and not restricted to the CD34+CD38- immature compartment. This phenotypical heterogeneity of LSCs suggests that pharmacological targeting of LSC will not work if solely based on their cell surface markers. A better understanding of the mechanisms underlying the in vivo chemoresistance is required for the development of innovative targeted therapies. Aracytine (Ara-C, a pyrimidine analog), the most clinically used chemotherapeutic agents for AML patients, inhibits DNA synthesis and, therefore, targets and kills cycling AML cells in S phase of the cell cycle. Based on this mechanism of action, we hypothesized that Ara-C treatment will spare and enrich quiescent LSCs in vivo. We analyzed the response to Ara-C and residual disease in NSG mice engrafted with primary AML cells from 13 patients in two clinical centers (University of Pennsylvania, Philadelphia, USA and Purpan Hospital, Toulouse, France). A sub-lethal treatment of 60 mg/kg Ara-C given daily for five days induced a 5- to 50- fold reduction of peripheral blood blasts and total tumor burden in spleen and bone marrow in all patients tested. For 5 patients, we observed relapse within 4 to 6 weeks post-chemotherapy. Surprisingly, residual viable cells after Ara-C treatment showed no significant enrichment in quiescent cells and CD34+CD38- cells for the majority of primary samples tested (12 and 10 out of 13 total tested, respectively). Of note, the largest fraction (70-90%) of leukemic cells is in G0/G1 phase (including 0.5-20% in G0) in untreated engrafted mice. Moreover, we observed no significant changes in cell cycle profile of residual leukemic cells during the time course of the disease progression for 3 out of 4 patients. Finally, we assessed the frequency of LSCs in Ara-C-treated and control mice using transplantation and limiting dilution analysis in secondary recipients. Interestingly, we observed that Ara-C treatment did not increase the frequency of SL-ICs in residual cells, suggesting that blasts and LSC were equally sensitive to Ara-C in vivo. Our results show that sub-lethal regimen of Ara-C does not lead to enrichment of LSCs and induces cell death of both leukemic bulk and stem/progenitor cells independently of their cell cycle status probably through another in vivo mechanism such as apoptosis, autophagy or necroptosis. This study also suggests that further characterization of chemoresistant leukemic cells beyond phenotype and cell cycle status must rely on more functional properties in order to better elucidate the molecular basis of resistance in AML. Disclosures: Perry: MERCK: Employment. Carroll:Leukemia and Lymphoma Society: Research Funding. Sarry:AFFICHEM SA: Membership on an entity’s Board of Directors or advisory committees.

Pathway Based Pharmacogenomics of Cytarabine In Pediatric Acute Myeloid Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 294-294
Author(s):  
Jatinder Lamba ◽  
Amit Mitra ◽  
Kristine Crews ◽  
Pounds Stanley ◽  
Xueyuan Cao ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Continuous Infusion ◽  
Myeloid Leukemia ◽  
Childhood Leukemia ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Leukemic Cells ◽  
High Dose ◽  
To Receive ◽  
Acute Myeloid

Abstract Abstract 294 Acute myeloid leukemia (AML) is the second most common form of childhood leukemia and has the worst prognosis of all major childhood cancers. The mainstay of AML chemotherapy is the nucleoside analog cytarabine (ara-C). Numerous studies suggest that the intracellular concentrations of the ara-C active metabolite, ara-CTP, vary widely among patients, and in turn, are associated with variability in clinical response to AML treatment. In the present study, we have taken a pathway directed approach to identify genetic predictors of ara-C response in pediatric patients treated with ara-C based antileukemic chemotherapy in the AML02 (n=187) and AML97 (n=55) clinical trials. The AML02 trial enrolled AML patients <22 years of age excluding APL or Down's syndrome patients, but those with all other subtypes of de novo or secondary AML, as well as patients with mixed-lineage leukemia, were eligible. Patients were randomized to receive induction I therapy containing high-dose cytarabine or low-dose cytarabine plus daunorubicin and etoposide. We genotyped the genomic DNA from patients enrolled in AML02 study for potentially significant single nucleotide polymorphisms (SNPs) in 10 key ara-C pathway genes and screened for association with 3 endpoints in AML02 study: in vitro ara-C LC50 determined in diagnostic leukemic cells, event free survival (EFS) and overall survival (OS). In samples from St. Jude AML97 study, we screened for association of SNPs with 4 endpoints: intracellular ara-CTP levels after start of induction, DNA synthesis relative to baseline, morphological response after induction I, and EFS. In the St Jude AML 97 study patients were randomly assigned to receive ara-C as either a short daily infusion (500 mg/m2/dose intravenously over 2 hrs daily for 5 days) or a continuous infusion (500 mg/m2/day as a continuous infusion over 5 days). Bone marrow was collected at the end of the ara-C infusion on day 1 for patients receiving the short daily infusion (n=27), and at 10 hrs after the start of the infusion for those receiving the continuous infusion (n=28). Ara-CTP levels in leukemia cells were analyzed by HPLC. Intracellular accumulation of ara-CTP was significantly higher when given as short daily infusion, as compared to continuous infusion (p = 0.01). The inter-patient variability for blast ara-CTP concentrations was 40-fold in the short infusion arm and 101-fold in the continuous infusion arm. We found significant correlations between SNPs in ara-C pathway genes (such as DCK, DCTD, CMPK, CTPS, CDA and NT5C2) and various clinical parameters (after adjusting for arm and/or risk group), some of which are listed below. These results suggest that genetic variation in key candidate genes in ara-C metabolic pathway could in?uence and predict the variability observed in cellular sensitivity and treatment response. The pharmacogenomic factors identi?ed in the present study could be potentially used for tailoring medications to better individualize cytarabine based AML therapy. Disclosures: No relevant conflicts of interest to declare.

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