Leukemic Stem Cell Detection in Bone Marrow for CML Patients With MMR Undergoing TKIs

2019 ◽  
Author(s):  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cell Detection ◽  
Leukemic Stem Cell

PRMT1 Activates Leukemic Stem Cell Program in MLL-Rearranged Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3493-3493
Author(s):  
Wing Chi Lui ◽  
Yuen Fan Chan ◽  
Ray Ng
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Leukemic Cells ◽  
Leukemic Stem Cell ◽  
Self Renewal ◽  
Mll Gene ◽  
Acute Myeloid

Abstract In MLL-rearranged leukemia, the Mixed Lineage Leukemia (MLL) gene undergoes chromosomal translocation that results in the loss of C-terminal histone methyltransferase SET domain, whereas the N-terminal of MLL gene fuses in-frame with one of the 60 identified partner genes. The resultant MLL fusion proteins lead to a characteristic aberrant gene expression pattern in human acute myeloid and lymphoblastic leukemia. Epigenetic dysregulation mediated by MLL fusion proteins has been suggested to be a key event in MLL-rearranged leukemia. It has been demonstrated that MLL-EEN/PRMT1 oncogenic complex induces transformation of primary myeloid progenitors via introduction of aberrant H4R3me2 at target Hoxloci. PRMT1 is the predominant protein arginine methyltransferase in mammals and is responsible for over 85% of arginine methylation activity in mammalian cells. Dysregulation of PRMT1 has been implicated in different cancers such as leukemia, suggesting the expression of PRMT1 is positively correlated with cancer progression and clinical parameters. Nevertheless, the leukemogenic role of PRMT1 in the establishment of leukemic stem cell (LSC) remains unclear. Previously we have demonstrated that a MLL fusion protein, MLL-EEN, can strongly enhance the self-renewal ability of murine primary hematopoietic cells through multiple rounds of replating assays. We have created a conditional Mll-Een invertor mouse model (MllEen/+) in which the expression of fusion protein is restricted to hematopoietic progenitors. Immunophenotypic analysis demonstrated a significant increase in the immature myeloid cell population (c-kit+Mac-1+) in bone marrow of MllEen/+ mice, suggesting that the expression of Mll-Een induces the development of acute myeloid leukemia. We have also established an Mll-Een expressing cell line from the bone marrow of MllEen/+ mouse. These leukemic cells can persistently form colonies and they also demonstrated deregulation of Hox genes, which is frequently observed in human leukemia cases. The leukemogenicity of Mll-Een is closely associated with Prmt1, which was demonstrated through knockdown of Prmt1. Strikingly, we discovered a subpopulation of CD41+Mll-Een expressing cells, which showed enhanced self-renewal ability in the serial colony forming assays. The percentage of CD41+ leukemic cells is reduced once Prmt1 was knocked down, suggesting that Prmt1 is crucial in the maintenance of this subpopulation of cells. In addition, the CD41+ cells showed enhanced expression of genes associated with hematopoietic stem cell (HSC) activities (Bmi-1, Runx1, Tal-1 and Lmo2), implying that part of the HSC transcriptional program has been re-activated in these cells. We therefore speculate that the CD41+ cells may represent a group of MLL leukemic cells that harbors strong stem cell features, and presumably functions as LSCs. The CD41+ leukemic cells will be further characterized by their LSC functions and CD41 can potentially serve as a novel LSC marker in MLL-rearranged leukemia. Taken together, studies on the role of PRMT1 can provide novel insights on the establishment of LSC and the development of effective clinical treatment for MLL-rearranged leukemia. Disclosures No relevant conflicts of interest to declare.

The Novel AML Stem Cell Associated Antigen CLL-1 Discriminates between Normal and Leukemic Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4-4 ◽  
Author(s):  
Anna van Rhenen ◽  
Nicole Feller ◽  
Angèle Kelder ◽  
Guus Westra ◽  
Lex Bakker ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Clinical Outcome ◽  
Stem Cell Marker ◽  
Cell Detection ◽  
The Novel ◽  
Antibody Treatment ◽  
Cell Compartment ◽  
Stem Cell Compartment

Abstract In CD34-positive acute myeloid leukemia (AML), the leukemia-initiating event likely takes place in the CD34+CD38- stem cell compartment. Survival of these cells after chemotherapy hypothetically leads to minimal residual disease (MRD) and relapse. We have previously shown that a high CD34+CD38- frequency correlates with both MRD frequency, especially after the third course of chemotherapy and poor survival (Clin Cancer Res, in press). Furthermore, we have shown that a monoclonal antibody against the novel cell surface marker C-type lectin-like molecule-1 (CLL-1), directed against myeloid cells, stains 92% of diagnosis AML (Bakker et al., Cancer Res.64:8443, 2004). In the present study we investigated whether this antibody can be used to identify AML stem cells in remission bone marrow. Such would offer opportunities for MRD stem cell detection and stem cell-directed therapy. We found that anti-CLL-1 antibody homogeneously stained the CD34+CD38- compartment in 77/89 cases (median expression of 33.3% in all 89 cases, range 0–100%). The median stem cell expression of CLL-1 in control bone marrow was 0% ranging from 0–11% (n=11). Furthermore, CLL-1 expression on AML stem cells is highly stable: no differences between paired diagnosis and relapse samples (p=0.9, n=12). Like most antigens CLL-1 is expressed on part of the CD34+CD38+ compartment, but expression is absent on megakaryocytic precursors, which for therapeutic use would circumvent delayed platelet recovery. For antibody-mediated therapy it is crucial that normal stem cells remain negative throughout treatment of the disease. Therefore we tested bone marrow regenerating after high dose chemotherapy, obtained from either non-AML hematological patients or CD34 negative or CLL-1 negative AML patients. In those patients complete absence of CLL-1 expression was found in CD34+CD38− cells (n=4). Under MRD-conditions CLL-1 staining thus enables to accurately discriminate between normal and malignant CD34+CD38− stem cells. In agreement with this, the different ratios of AML and normal stem cells that were found in a number of patients, paralleled clinical outcome in terms of probability of relapse. For comparison, the stem cell marker CD123 was studied. Although anti-CD123 antibody homogeneously stained CD34+CD38− cells with high intensity in almost all AML samples studied (35/36 cases) with also no differences between diagnosis and relapse (p=0.6, n=6) and with low expression in normal bone marrow (median 14.9%, range 0–18.8%, n=5), a high expression was found in regenerating bone marrow (median 60%, range 53–84%, n=4). The latter suggests that anti-CD123 antibody is not AML stem cell specific under all conditions of disease. In conclusion, our data provide strong evidence that a large CD34+CD38− population at diagnosis reflects a higher percentage of chemotherapy-resistant cells, which, in remission, will lead to the outgrowth of MRD, thereby affecting clinical outcome. The specificity of anti-CLL-1 antibody under all conditions of disease enables both reliable detection and quantification of the stem cell compartment for prognostic use under MRD conditions, as well as characterization. Moreover, it shows that AML stem cell targeting using antibody treatment at different stages of disease has now become an option in the treatment of AML patients.

Gene Expression Profiling In Leukemic Stem Cell-Enriched AML CD34+ Cell Fraction Identifies Target Genes That Predict Prognosis In Normal Karyotype AML

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 952-952
Author(s):  
Carolien M Woolthuis ◽  
Hendrik JM de Jonge ◽  
Annet Z Vos ◽  
Andre B Mulder ◽  
Eva van den Berg ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Stem Cell ◽  
Expression Profiles ◽  
Normal Karyotype ◽  
Cell Fraction ◽  
Normal Bone Marrow ◽  
Leukemic Stem Cell ◽  
Transcript Levels ◽  
Normal Bone

Abstract Abstract 952 Acute myeloid leukemia (AML) is clinically, cytogenetically and molecularly a heterogeneous disease which makes it challenging to classify it properly. In recent years major advances have been achieved in predicting outcome. However, there is still need for more powerful and independent prognostic factors that can guide treatment decisions, especially for the large subgroup of patients presenting with normal karyotype AML. In order to improve the identification of prognostic markers, gene expression studies have been performed. However, most of these studies have analyzed the mononuclear cell fraction. So, very little has been revealed about gene expression programs that drive leukemic transformation in the small population of leukemic stem cells (LSCs). Although considerable heterogeneity appears to exist in the phenotype of LSCs, CD34 is uniformly expressed and LSCs have been found to reside in the CD34+ compartment in the vast majority of leukemias. In the present study AML CD34+-specific gene expression profiles were indentified and used to distinguish prognostically relevant target genes in normal karyotype AML. AML mononuclear cells (n=46) were sorted in CD34+ and CD34- subfractions and genome-wide expression analysis was performed using Illumina BeadChip Arrays. AML CD34+ and CD34- gene expression was compared to a large group of normal CD34+ bone marrow cells (n=31). Unsupervised hierarchical clustering analysis showed that CD34+ AML samples belonged to a distinct cluster compared to normal bone marrow and that in 61% of the cases the AML CD34+ transcriptome did not cluster together with the paired CD34- transcriptome. These data indicate that in the majority of AML cases the leukemic stem cell-enriched CD34+ gene expression profile is quite distinct from the leukemic CD34- compartment. GO analysis revealed that common differences in gene expression between CD34+ and CD34- groups were particularly enriched for genes that were associated with T-cells and erythropoiesis. This association with a more committed phenotype was found in all AML samples and not just in those samples where CD34+ and CD34- transcriptomes did not cluster together. Among the GO-ontologies representing the differentially expressed genes between CD34+ AML versus CD34+ normal bone marrow cells were gene sets related to DNA damage and a number of mitotic and metabolic processes. A top 50 of AML CD34+-specific genes was selected by comparing the AML CD34+ transcriptome with the AML CD34- and CD34+ normal bone marrow transcriptomes. The prognostic relevance of these 50 genes was assessed using univariate cox regression analyses between the continuous transcript levels of these 50 genes and overall survival (OS) in a large series of normal karyotype AML (n=163) (Metzeler et al. Blood 2008). The findings were validated in another independent cohort of 218 normal karyotype AML patients (Valk et al. NEJM 2004). Interestingly, higher transcript levels of three CD34+ AML specific genes, i.e. ankyrin repeat domain 28 (ANKRD28), guanine nucleotide binding protein, alpha 15 (GNA15) and UDP-glucose pyrophosphorylase 2 (UGP2) were associated with a significant poorer OS in both cohorts (p<0.01). For the cohort of 218 normal karyotype AML patients also event free survival (EFS) data were available for further analyses. A significant association between the continuous transcript levels of ANKRD28, GNA15 and UGP2 with poor EFS was evident. Also for the sum of expression of ANKRD28, GNA15 and UGP2 higher transcript levels were strongly associated with poorer OS (p=0.007) and EFS (p=0.006) in the cohort of 218 normal karyotype AML. Similar results were obtained in the cohort of 163 normal karyotype AML patients for OS (p<0.001). Importantly, the prognostic value of the continuous transcript levels of these three genes was independent from the well known risk factors FLT3-ITD, NPM1c+ and CEBPA mutation status as determined by a multivariate analysis in the cohort of 218 normal karyotype AML patients. In conclusion, by microarray analysis of the leukemic stem cell-enriched AML CD34+ cell fraction novel insight was obtained in gene expression programs that are potentially associated with leukemic stem cell self-renewal and transformation. Moreover, the identified new gene expression profiles were shown to have prognostic relevance in normal karyotype AML independent of well known risk factors. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Up-regulation of SPINT2/HAI-2 by Azacytidine in bone marrow mesenchymal stromal cells affects leukemic stem cell survival and adhesion

2018 ◽  
Vol 23 (2) ◽  
pp. 1562-1571 ◽  
Author(s):  
Fernanda Marconi Roversi ◽  
Nathalia Moreno Cury ◽  
Matheus Rodrigues Lopes ◽  
Karla Priscila Ferro ◽  
João Agostinho Machado-Neto ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cell Survival ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Leukemic Stem Cell ◽  
Stem Cell Survival

The CD34+CD38LowCD19+ Candidate Leukemic Stem Cell Phenotype Revisited: Useful for Flow MRD Monitoring?

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2505-2505
Author(s):  
Kerrie Wilson ◽  
Marian Case ◽  
Lynne Minto ◽  
Simon Bailey ◽  
Josef Vormoor ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cancer Stem Cell ◽  
Cell Population ◽  
Stem Cell Population ◽  
Cell Phenotype ◽  
Leukemic Stem Cell ◽  
Childhood All ◽  
Cancer Stem Cell Population ◽  
Minimal Residual

Abstract The assessment of minimal residual disease during therapy is a powerful prognostic marker in childhood acute lymphoblastic leukemia but current techniques, whether molecular analysis of antigen receptor gene rearrangements or flow cytometric analysis of aberrant immunophenotypes, track the blast population(s) that predominate(s) at diagnosis. Recent evidence from diagnostic TEL/AML1 positive cases (Hong et al, 2008) suggests that the candidate leukemic stem cell population may be characterised by the immunophenotype CD34+CD38LowCD19+ and thus the identification and quantitation of this cell population at diagnosis and during therapy may have greater clinical significance. Cytogenetic subgroup Number with candidate population Number without candidate population TEL/AML1 8 1 High Hyperdiploid 6 5 Other 15 16 Therefore, we have sought this proposed leukemic stem cell population in 51 consecutive diagnostic precursor-B acute lymphoblastic leukaemia samples using multi-parametric flow cytometry. Patient blasts were stained with the antibodies CD34PerCP, CD38FITC and CD19APC and 50,000 events acquired on a FACSCalibur. The postulated cancer stem cell population, CD34+CD38LowCD19+, was found in 57% of patients (29 of 51). Correlation with major cytogenetic subgroups showed that 89% (8 of 9) of TEL/AML1 positive cases had evidence of the population, compared to 55% (6 of 11) of high hyperdiploid and 48% (15 of 31) of the remainder (see Table). There was no evidence of the population in ‘normal marrow’ samples which were from children in the latter stages of therapy for ALL i.e. end of treatment bone marrow samples (n=4). We also examined bone marrow samples taken 28 days after the start of treatment in a cohort of patients scoring positive for the candidate population at diagnosis (n=11) and found evidence for persistence of the cells in 4 of them. CD38 under-expression, in relation to normal B cell precursors, is a common feature of ALL blasts and classification of CD38 expression levels revealed that 92% (24/26) of these under-expressers unsurprisingly housed the population because their blasts were also CD34+ and CD19+. Most importantly, since 43% of the diagnostic samples analysed show no evidence of this population (or it exists below the limits of detection of the assay i.e.&lt;0.1% ), it seems unlikely that this represents a definitive cancer stem cell population in childhood ALL per se and would, therefore, not be appropriate as a generic marker of minimal residual leukemic stem cells.

scholarly journals Comparison of human cord blood engraftment between immunocompromised mouse strains

Blood ◽  
2010 ◽  
Vol 116 (2) ◽  
pp. 193-200 ◽  
Author(s):  
Sean P. McDermott ◽  
Kolja Eppert ◽  
Eric R. Lechman ◽  
Monica Doedens ◽  
John E. Dick
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cord Blood ◽  
Hematopoietic Stem Cell ◽  
Cell Activity ◽  
Scid Mice ◽  
Cell Detection ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Stem Cell Activity

AbstractThe nonobese diabetic/severe combined immune deficiency (NOD-scid) xenotransplantation model is the “gold standard” for assaying human hematopoietic stem cell activity. Systematic advancements, such as depletion of natural killer cell activity with anti-CD122 antibody, direct intrafemoral injection, and deletion or truncation of IL2Rγ, have improved human cell engraftment; however, questions remain whether these mouse models are equivalent or, if not, which model is superior for assaying hematopoietic stem cell activity. To address this, we compared overall engraftment and multilineage differentiation of near-limiting doses of lineage-depleted human umbilical cord blood cells by direct intrafemoral injection into NOD/Lt-scid, NOD/Shi-scid, NOD/Lt-scid/IL2Rγnull (NSG), and NOD/Shi-scid/IL2Rγnull mice. Transplantation into NSG mice generated moderately higher human engraftment levels in bone marrow compared with other strains. At limiting doses, NSG mice of both sexes were 3.6-fold more sensitive in detecting SCID-repopulating cells compared with NOD/Lt-scid mice. However, NSG females exhibited higher engraftment at limiting cell doses, resulting in an overall increase in SCID-repopulating cell detection of 9-fold. Both NSG and NOD/Shi-scid/IL2Rγnull support significantly improved engraftment in peripheral tissues compared with NOD/Lt-scid and NOD/Shi-scid mice, whereas NSG mice provide greater human engraftment in bone marrow than all other strains, especially at limiting doses.

Sternal bone marrow is a suitable autologous cell source for cardiac stem cell therapies

2014 ◽  
Vol 62 (S 01) ◽  
Author(s):  
M. Arar ◽  
A. Rotärmel ◽  
A.-K. Knoefel ◽  
H. Baraki ◽  
I. Kutschka ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cardiac Stem Cell ◽  
Stem Cell Therapies ◽  
Cell Therapies ◽  
Autologous Cell ◽  
Cell Source

A pilot study with autologous bone marrow-derived stem cell therapy in patients with severe peripheral arterial disorder

2008 ◽  
Vol 149 (12) ◽  
pp. 531-540 ◽  
Author(s):  
Zoltán Boda ◽  
Miklós Udvardy ◽  
Katalin Farkas ◽  
Judit Tóth ◽  
László Jámbor ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Pilot Study ◽  
Cell Therapy ◽  
Color Doppler ◽  
Autologous Bone Marrow ◽  
Thromboangiitis Obliterans ◽  
Arteriosclerosis Obliterans ◽  
Autologous Bone ◽  
Peripheral Arterial

Súlyos perifériás artériás érbetegségekben a gyógyszeres és/vagy érsebészeti beavatkozások kimerülését követően a tűrhetetlen fájdalom, kiterjedt végtagi fekélyek, gangraenák megszüntetésének egyetlen módja a végtag amputációja. Betegek és módszerek: A szerzők – hazánkban elsőként – 5 előrehaladott perifériás artériás érbetegben (1 arteriosclerosis obliterans és 4 thromboangiitis obliterans) autológ csontvelői eredetű őssejtterápiát végeztek. A csontvelői őssejteket (CD34+ sejtek) crista biopsia végzésével nyerték. Mágneses sejtszeparálással CD34+ sejtszuszpenziót állítottak elő. Meghatározták a CD34+, CD133+ és CD45± sejtek számát és arányát. Az őssejtszuszpenziót intramuscularis injekció formájában a beteg végtagba juttatták vissza. Betegenként 0,37–1,14 × 10 5 /kg őssejt visszaadására került sor. Betegeiket 12 hónapig követték. Vizsgálatok történtek a beavatkozás előtt és után (1, 3, 6, 9 és 12 hónappal). Klinikai vizsgálatok: nyugalmi fájdalom, dysbasiás távolság, ischaemiás fekélyek gyógyhajlama, boka-kar index. Laboratóriumi vizsgálatok: angiográfia (az őssejtterápia előtt és után 1 és 6 hónappal), duplex ultrahang- és lézer-Doppler-scan, transcutan oxigéntenzió mérése, az endothelfunkciók vizsgálata. Eredmények: A nyugalmi fájdalom mind az öt betegük esetében megszűnt. A dysbasiás távolság szignifikánsan nőtt (36/440 m). Három beteg végtagi ischaemiás fekélye begyógyult, egy beteg nagyméretű fekélye lényegesen kisebbé és felületesebbé vált, egy betegben a végtagi fekély nem változott. A kezelt oldalon a boka-kar index szignifikánsan nőtt (0,41/0,83) tizenkét hónappal az őssejtterápiát követően, s nem változott az ellenoldalon. Három betegben tapasztaltak számottevő változást angiográfiával az őssejtterápia után hat hónappal. Csak szerény javulást észleltek color-Doppler- és lézer-Doppler-vizsgálatokkal. Az őssejtterápia előtt és után 1, 6 és 12 hónappal a transcutan oxigéntenzió-értékek a lábháton 18,10/16,78/23,83/37,50 Hgmm-re, míg a lábszáron 36,66/31,25/45,00/37,30 Hgmm-re változtak. A makro- és mikrocirkulációs paraméterek nem mutattak javulást az őssejtterápiát követően 1 hónappal, azonban az őssejtterápia után 3, 6, 9 és 12 hónappal már mérhető javulást tapasztaltak. Szövődményt, mellékhatást nem észleltek. Következtetések: Klinikai eredményeik alapján az autológ csontvelői eredetű őssejtterápiát hatásosnak, tartósnak és biztonságosnak tartják előrehaladott perifériás artériás érbetegségben. Szükség van további klinikai tapasztalatgyűjtésre.

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