scholarly journals Multicolor Immunophenotyping of Candidate Leukemic Stem Cell Markers in CD34+CD38- Chronic Myeloid Leukemia Stem Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2922-2922
Author(s):  
Martin Culen ◽  
Marianna Romzova ◽  
Dagmar Smitalova ◽  
Tomas Loja ◽  
Jiri Mayer
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
Correlation Coefficient ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Surface Expression ◽  
Specific Marker ◽  
Stem Cell Markers ◽  
Similar Frequency ◽  
Median Difference

Introduction: Detection of leukemic stem cells (LSCs) may represent a new potential prognostic parameter in chronic myeloid leukemia (CML) and a tool for minimal residual disease monitoring in combination with standard qPCR method. To date CD26 is studied as a most specific marker for CML LSC detection. Several other candidate LSC markers have been reported, such as IL1-RAP, CD25 and CD93, however a side-by-side testing of their specificity is lacking. Recently, we have identified CD69 molecule to be overexpressed in CD34+CD38-CD26+ cells, which makes this antigen another candidate marker for LSC. Aim: To compare the surface expression of LSC markers CD69, CD26, CD25, CD56, IL1-RAP, CD56, CD93 in bulk CD34+CD38- population at diagnosis using a multicolor phenotypization assay Methods: In total, 44 patients were analyzed at diagnosis of chronic phase CML before administration of any treatment. Fresh (n=38) or cryopreserved (n=6) leukocytes obtained by erythrolysis were stained with CD45, CD34, CD38, CD25, CD26, CD56, CD69, CD93, IL1-RAP antibodies and 7-AAD for selection of live cells. Analysis was performed on FACSAria Fusion (BD Biosciences). Acquisition of live mononuclear cells ranged from 2×104 to 2.5×106. Results: Expression of candidate LSC markers CD26, CD25, CD56, IL1-RAP, CD56, CD93 was analyzed in BM of 35 patients who carried at least 30 CD34+CD38- cells. Median percentage of marker positive cells was 34% for IL1-RAP, 31% for CD25, 23% for CD26, 16% for CD56 and 2% for CD93, from the parent CD34+CD38- population. Next we analyzed the overlap and combination for the three best markers - IL1-RAP, CD25 and CD26. The 3-combination (defined as IL1-RAP or CD25 or CD26 expression) identified 40% of CD34+CD38- positive cells, which was more than any of the markers alone. Expression of the three markers showed good overlap and ruled out mutually exclusive expression of the markers. This was demonstrated by median difference of 0.4% of CD34+CD38- cells (range: 0-18%) and a correlation coefficient r2=0.9914, when comparing the 3-combination and the best performing marker in each patient. In contrast, in 12/35 (34%) of patients, one of the three markers failed to identify at least half of the cells positive for another marker. In 21/35 patients, we also analyzed the expression of CD69 in the CD34+CD38- compartment. The CD69 showed similar performance as the 3-combination of CD26, CD25, and IL1-RAP, 59% vs 54% positive cells, respectively. We observed excellent overlap between CD69 and 3-combination expression in individual patients with median difference of 0% of CD34+CD38- cells (range 0-15%) and correlation coefficient r2=0.9890. Furthermore, we compared marker positivity in BM vs PB in 19 paired samples. Both, sample types showed similar frequency of CD34+CD38- cells (5×10-3 in BM, and 2×10-3 for PB), but PB carried higher percentage of LSCs identified by the 3-combination - median 76 vs 52% cells. Conclusions: We show an overlap in surface expression of three previously reported CML LSC markers - IL1-RAP, CD25 and CD26. Nevertheless, a combination of these markers can detect more positive cells than any of the markers alone. Moreover, we demonstrate that CD69 identifies the same cells within the CD34+CD38- compartment as the combination of three above mentioned markers, which makes CD69 the best candidate for routine CML LSC quantification. Disclosures Mayer: AOP Orphan Pharmaceuticals AG: Research Funding.

scholarly journals Identification of BCR/ABL-negative primitive hematopoietic progenitor cells within chronic myeloid leukemia marrow

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 801-807 ◽  
Author(s):  
T Leemhuis ◽  
D Leibowitz ◽  
G Cox ◽  
R Silver ◽  
EF Srour ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Polymerase Chain Reaction Analysis ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem

Chronic myeloid leukemia (CML) is a malignant disorder of the hematopoietic stem cell. It has been shown that normal stem cells coexist with malignant stem cells in the bone marrow of patients with chronic-phase CML. To characterize the primitive hematopoietic progenitor cells within CML marrow, CD34+DR- and CD34+DR+ cells were isolated using centrifugal elutriation, monoclonal antibody labeling, and flow cytometric cell sorting. Polymerase chain reaction analysis of RNA samples from these CD34+ subpopulations was used to detect the presence of the BCR/ABL translocation characteristic of CML. The CD34+DR+ subpopulation contained BCR/ABL(+) cells in 11 of 12 marrow samples studied, whereas the CD34+DR- subpopulation contained BCR/ABL(+) cells in 6 of 9 CML marrow specimens. These cell populations were assayed for hematopoietic progenitor cells, and individual hematopoietic colonies were analyzed by PCR for their BCR/ABL status. Results from six patients showed that nearly half of the myeloid colonies cloned from CD34+DR- cells were BCR/ABL(+), although the CD34+DR- subpopulation contained significantly fewer BCR/ABL(+) progenitor cells than either low-density bone marrow (LDBM) or the CD34+DR+ fraction. These CD34+ cells were also used to establish stromal cell-free long-term bone marrow cultures to assess the BCR/ABL status of hematopoietic stem cells within these CML marrow populations. After 28 days in culture, three of five cultures initiated with CD34+DR- cells produced BCR/ABL(-) cells. By contrast, only one of eight cultures initiated with CD34+DR+ cells were BCR/ABL(-) after 28 days. These results indicate that the CD34+DR- subpopulation of CML marrow still contains leukemic progenitor cells, although to a lesser extent than either LDBM or CD34+DR+ cells.

Dipeptidylpeptidase IV (CD26) defines leukemic stem cells (LSC) in chronic myeloid leukemia

Blood ◽  
2014 ◽  
Vol 123 (25) ◽  
pp. 3951-3962 ◽  
Author(s):  
Harald Herrmann ◽  
Irina Sadovnik ◽  
Sabine Cerny-Reiterer ◽  
Thomas Rülicke ◽  
Gabriele Stefanzl ◽  
...  
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Specific Marker ◽  
Leukemic Stem Cells ◽  
Dipeptidylpeptidase Iv ◽  
Key Points

Key Points DPPIV (CD26) is a new specific marker of CML LSC that aids CML diagnostics and the measurement, characterization, and purification of LSC. DPPIV on CML LSC degrades SDF-1 and thereby promotes the niche-escape of LSC, which may contribute to extramedullary myeloproliferation in CML.

scholarly journals Identification of BCR/ABL-negative primitive hematopoietic progenitor cells within chronic myeloid leukemia marrow

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 801-807 ◽  
Author(s):  
T Leemhuis ◽  
D Leibowitz ◽  
G Cox ◽  
R Silver ◽  
EF Srour ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Polymerase Chain Reaction Analysis ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem

Abstract Chronic myeloid leukemia (CML) is a malignant disorder of the hematopoietic stem cell. It has been shown that normal stem cells coexist with malignant stem cells in the bone marrow of patients with chronic-phase CML. To characterize the primitive hematopoietic progenitor cells within CML marrow, CD34+DR- and CD34+DR+ cells were isolated using centrifugal elutriation, monoclonal antibody labeling, and flow cytometric cell sorting. Polymerase chain reaction analysis of RNA samples from these CD34+ subpopulations was used to detect the presence of the BCR/ABL translocation characteristic of CML. The CD34+DR+ subpopulation contained BCR/ABL(+) cells in 11 of 12 marrow samples studied, whereas the CD34+DR- subpopulation contained BCR/ABL(+) cells in 6 of 9 CML marrow specimens. These cell populations were assayed for hematopoietic progenitor cells, and individual hematopoietic colonies were analyzed by PCR for their BCR/ABL status. Results from six patients showed that nearly half of the myeloid colonies cloned from CD34+DR- cells were BCR/ABL(+), although the CD34+DR- subpopulation contained significantly fewer BCR/ABL(+) progenitor cells than either low-density bone marrow (LDBM) or the CD34+DR+ fraction. These CD34+ cells were also used to establish stromal cell-free long-term bone marrow cultures to assess the BCR/ABL status of hematopoietic stem cells within these CML marrow populations. After 28 days in culture, three of five cultures initiated with CD34+DR- cells produced BCR/ABL(-) cells. By contrast, only one of eight cultures initiated with CD34+DR+ cells were BCR/ABL(-) after 28 days. These results indicate that the CD34+DR- subpopulation of CML marrow still contains leukemic progenitor cells, although to a lesser extent than either LDBM or CD34+DR+ cells.

scholarly journals C/EBPβ is a critical mediator of IFN-α–induced exhaustion of chronic myeloid leukemia stem cells

2019 ◽  
Vol 3 (3) ◽  
pp. 476-488 ◽  
Author(s):  
Asumi Yokota ◽  
Hideyo Hirai ◽  
Ryuichi Sato ◽  
Hiroko Adachi ◽  
Fumiko Sato ◽  
...  
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Interferon Α ◽  
Dependent Manner ◽  
Distal Enhancer ◽  
Induced Differentiation ◽  
Ifn Γ

Abstract Even in the era of ABL tyrosine kinase inhibitors, eradication of chronic myeloid leukemia (CML) stem cells is necessary for complete cure of the disease. Interferon-α (IFN-α) has long been used for the treatment of chronic-phase CML, but its mechanisms of action against CML stem cells remain unclear. We found that IFN-α upregulated CCAAT/enhancer binding protein β (C/EBPβ) in BCR-ABL–expressing mouse cells by activating STAT1 and STAT5, which were recruited to a newly identified 3′ distal enhancer of Cebpb that contains tandemly aligned IFN-γ–activated site elements. Suppression or deletion of the IFN-γ–activated site elements abrogated IFN-α–dependent upregulation of C/EBPβ. IFN-α induced differentiation and exhaustion of CML stem cells, both in vitro and in vivo, in a C/EBPβ-dependent manner. In addition, IFN-α upregulated C/EBPβ and induced exhaustion of lineage− CD34+ cells from CML patients. Collectively, these results clearly indicate that C/EBPβ is a critical mediator of IFN-α–induced differentiation and exhaustion of CML stem cells.

PTEN Regulated BCRP/ABCG2 and Side Population Through PI3K/Akt Pathway In Chronic Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5408-5408
Author(s):  
Fang-Fang Huang ◽  
Li Zhang ◽  
Yan-Hui Yu ◽  
Fang-ping Chen ◽  
Hui Zeng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Conflicts Of Interest ◽  
Side Population ◽  
K562 Cells ◽  
Akt Pathway ◽  
Specific Marker ◽  
Pi3k Inhibitor Ly294002

Abstract A small population of cancer stem cells named as “side population (SP)” has been demonstrated to be responsible for many solid tumor maintenance. However, the role of SP in leukemic pathogenesis is still controversial. The resistance of leukemic stem cells in response to targeted therapies such as tyrosine kinase inhibitors (TKIs) results in therapeutic failure or refractory/relapsed disease in chronic myeloid leukemia (CML). The drug pump, ABCG2, is well-known as a specific marker of SP and could be controlled by several pathways including PI3K/Akt. Our data demonstrated that compared to wild type K562 cells, the higher percentage of ABCG2+ cells corresponded to the higher SP fraction in K562/ABCG2 (ABCG2 overexpressing) and K562/IMR (resistance to imatinib) cells, which exhibited enhanced drug resistance along with downregulated PTEN and activated p-Akt. It could be abrogated by both PI3K inhibitor LY294002 and mTOR inhibitor rapamycin. Moreover, in CML patients at accelerated phase/blastic phase (AP/BP), increased SP phenotype rather than ABCG2 expression accompanied with loss of PTEN protein and up-regulation of p-Akt expression was observed. These results suggested that expression of ABCG2 and fraction of SP may be regulated by PTEN through PI3K/Akt pathway, which will be the potentially effective strategy for targeting CML stem cells. Disclosures: No relevant conflicts of interest to declare.

Targeting β-arrestin2 Enhances Survival in a Murine Model of Chronic Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 857-857
Author(s):  
Lindsay A.M. Rein ◽  
Minyong Chen ◽  
Barbara S. Theriot ◽  
James W. Wisler ◽  
Laura M. Wingler ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Male Mice ◽  
Donor Cells ◽  
Wbc Count

Abstract Background Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm of hematopoietic stem cells characterized by presence of a dysregulated BCR-ABL fusion protein which leads to constitutive activation of tyrosine kinase activity. Classically, CML is treated with tyrosine kinase inhibitors (TKIs). However, TKI therapy is non-curative, and persistence of quiescent leukemia stem cells likely accounts for the inability of these agents to cure CML. β-arrestins are multifunctional adapter proteins which regulate G protein-coupled receptor (GPCR) signaling and trafficking and have recently been identified as mediators of distinct cellular signaling cascades independent of G proteins. β-arrestins also play a role in the Smoothened/Hedgehog pathway, as well as in the Wingless/Frizzled (Wnt/Fz) signaling axis, both of which have been associated with the development of CML. We, therefore, hypothesize that β-arrestin2 (βarr2) is necessary for the development and propagation of CML and may function as a therapeutic target. Aim Demonstrate that loss of βarr2, using an inducible conditional knockout mouse model, slows progression of CML. Methods We used a standard murine retroviral transduction system to model chronic phase BCR-ABL positive CML. KLS cells (Lin-, Sca-1+, c-kit+) were harvested from bone marrow of donor C57BL6/J βarr2F/F-CreERT2+/- (Cre positive, CD45.2) and age matched C57BL6/J βarr2F/F-CreERT2-/- (Cre negative, CD54.2) male mice. KLS cells were retrovirally transduced with MSCV-BCR-ABL-IRES-GFP and were subsequently injected retro-orbitally into sublethally irradiated congenic wild type recipient male mice (CD45.1). Donor mice were engineered using global Cre-ER/loxP technology in order to induce site-specific recombination and loss of βarr2 when treated with tamoxifen. Mice who received Cre positive cells lost βarr2 only in hematopoietic cells. Recipient mice were treated with tamoxifen 75mg/kg daily via intraperitoneal injection for 5 days starting day 3 after transplant and were monitored for signs of leukemia development. Weekly blood analysis included WBC count, number of donor cells by flow cytometry, blood film, and qPCR for BCR-ABL expression. Survival was compared between animals receiving Cre positive (βarr2F/F-CreERT2+/-) and Cre negative (βarr2F/F-CreERT2-/-) donor cells as well as both tamoxifen treated and untreated conditions. Results Treatment of donor C57BL6/J βarr2F/F-CreERT2+/- mice with tamoxifen resulted in decreased βarr2 expression within 10 days in multiple tissues including spleen and bone marrow. By day 10, βarr2 expression was 9.5 ± 3.0% in Cre positive mice relative to pretreatment expression levels (Figure 1). In total, 8 mice received Cre positive cells and 11 mice received Cre negative cells. Ten of 11 (90.9%) Cre negative mice developed CML as evidenced by splenomegaly, leukocytosis, increased BCR-ABL expression measured by qPCR and increased number of donor cells detectable by flow cytometry. Median survival was 15 days. Six of 8 (75%) Cre positive mice developed disease with median survival of 27 days (HR 3.2, 95% CI; 1.525-12.2, p=0.013)(Figure 2). At day 11, flow cytometry for donor CD45.2 cells present in peripheral blood of recipient mice was 38 ± 2.05% in Cre negative versus 16.5± 3.9% in Cre positive mice (p<0.0001). Spleen size at death and WBC count at day 11 were not significantly different between groups. Conclusions These data demonstrate that targeting βarr2 prolongs the course of disease in chronic phase CML and raise the possibility that loss of βarr2 after disease onset may lead to disease regression. βarr2 may therefore represent an alternative therapeutic target for CML independent of tyrosine kinase inhibition. Disclosures: No relevant conflicts of interest to declare.

Chronic Myeloid Leukemia Stem Cells

2008 ◽  
Vol 26 (17) ◽  
pp. 2911-2915 ◽  
Author(s):  
Edward Kavalerchik ◽  
Daniel Goff ◽  
Catriona H.M. Jamieson
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Single Agent ◽  
Chronic Phase ◽  
Leukemia Stem Cells ◽  
Molecular Events ◽  
Advanced Phase ◽  
Dividing Cells

Although rare, chronic myeloid leukemia (CML) represents an important paradigm for understanding the molecular events leading to malignant transformation of primitive hematopoietic progenitors. CML was the first cancer to be associated with a defined genetic abnormality, BCR-ABL, that is necessary and sufficient for initiating chronic phase disease as well as the first cancer to be treated with molecular targeted therapy. Malignant progenitors or leukemia stem cells (LSCs) evolve as a result of both epigenetic and genetic events that alter hematopoietic progenitor differentiation, proliferation, survival, and self-renewal. LSCs are rare and divide less frequently, and thus, represent a reservoir for relapse and resistance to a molecularly targeted single agent. On subverting developmental processes normally responsible for maintaining robust life-long hematopoiesis, the LSCs are able to evade the majority of current cancer treatments that target rapidly dividing cells. Enthusiasm for the enormous success of tyrosine kinase inhibitors at controlling the chronic phase disease is tempered somewhat by the persistence of the LSC pool in the majority of the patients. Combined therapies targeting aberrant properties of LSC may obviate therapeutic resistance and relapse in advanced phase and therapeutically recalcitrant CML.

Chronic Myeloid Leukemia Stem Cells

Hematology ◽  
2008 ◽  
Vol 2008 (1) ◽  
pp. 436-442 ◽  
Author(s):  
Catriona H. Jamieson
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Targeted Therapy ◽  
Chronic Myeloid Leukemia ◽  
Disease Progression ◽  
Myeloid Leukemia ◽  
Progenitor Cell ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Self Renewal

Abstract Chronic myeloid leukemia (CML) is typified by robust marrow and extramedullary myeloid cell production. In the absence of therapy or sometimes despite it, CML has a propensity to progress from a relatively well tolerated chronic phase to an almost uniformly fatal blast crisis phase. The discovery of the Philadelphia chromosome followed by identification of its BCR-ABL fusion gene product and the resultant constitutively active P210 BCR-ABL tyrosine kinase, prompted the unraveling of the molecular pathogenesis of CML. Ground-breaking research demonstrating that BCR-ABL was necessary and sufficient to initiate chronic phase CML provided the rationale for targeted therapy. However, regardless of greatly reduced mortality rates with BCR-ABL targeted therapy, most patients harbor quiescent CML stem cells that may be a reservoir for disease progression to blast crisis. While the hematopoietic stem cell (HSC) origin of CML was first suggested over 30 years ago, only recently have the HSC and progenitor cell–specific effects of the molecular mutations that drive CML been investigated. This has provided the impetus for investigating the genetic and epigenetic events governing HSC and progenitor cell resistance to therapy and their role in disease progression. Accumulating evidence suggests that the acquired BCR-ABL mutation initiates chronic phase CML and results in aberrant stem cell differentiation and survival. This eventually leads to the production of an expanded progenitor population that aberrantly acquires self-renewal capacity resulting in leukemia stem cell (LSC) generation and blast crisis transformation. Therapeutic recalcitrance of blast crisis CML provides the rationale for targeting the molecular pathways that drive aberrant progenitor differentiation, survival and self-renewal earlier in disease before LSC predominate.

Primitive, quiescent, Philadelphia-positive stem cells from patients with chronic myeloid leukemia are insensitive to STI571 in vitro

Blood ◽  
2002 ◽  
Vol 99 (1) ◽  
pp. 319-325 ◽  
Author(s):  
Susan M. Graham ◽  
Heather G. Jørgensen ◽  
Elaine Allan ◽  
Charlie Pearson ◽  
Michael J. Alcorn ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Growth Factors ◽  
Chronic Myeloid Leukemia ◽  
Antiproliferative Activity ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Chronic Phase ◽  
Quiescent State

In clinical trials, the tyrosine kinase inhibitor STI571 has proven highly effective in reducing leukemic cell burden in chronic myeloid leukemia (CML). The overall sensitivity of CML CD34+ progenitor cells to STI571 and the degree to which cell death was dependent on cell cycle status were determined. Stem cells (Lin−CD34+) from the peripheral blood of patients with CML in chronic phase and from granulocyte–colony-stimulating factor–mobilized healthy donors were labeled with carboxy-fluorescein diacetate succinimidyl diester dye to enable high-resolution tracking of cell division. Then they were cultured for 3 days with and without growth factors ± STI571. After culture, the cells were separated by fluorescence-activated cell sorting into populations of viable quiescent versus cycling cells for genotyping. For healthy controls, in the presence of growth factors, STI571 affected neither cell cycle kinetics nor recovery of viable cells. In the absence of growth factors, normal cells were unable to divide. For CML samples, in the presence or absence of growth factors, the response to STI571 was variable. In the most sensitive cases, STI571 killed almost all dividing cells; however, a significant population of viable CD34+ cells was recovered in the undivided peak and confirmed to be part of the leukemic clone. STI571 also appeared to exhibit antiproliferative activity on the quiescent population. These studies confirm that CML stem cells remain viable in a quiescent state even in the presence of growth factors and STI571. Despite dramatic short-term responses in vivo, such in vitro insensitivity to STI571, in combination with its demonstrated antiproliferative activity, could translate into disease relapse after prolonged therapy.

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