Prognosis of Low- or Intermediate-1-Risk MDS with Del(5q) Chromosome Abnormality During Lenalidomide Therapy Depends on the Capacity of the Neoplastic Stem Cell to Differentiate Into Maturing Erythroid Committed Stem Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3819-3819
Author(s):  
Guntram Buesche ◽  
Aristoteles Giagounidis ◽  
Gudrun Göhring ◽  
Brigitte Schlegelberger ◽  
Stephanie Dieck ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Chromosome Abnormality ◽  
Clonal Evolution ◽  
Cytogenetic Response ◽  
Prognostic Information ◽  
Transfusion Independence ◽  
Erythroid Precursors

Abstract Abstract 3819 Transfusion-dependent anemia is a characteristic complication of myelodysplastic syndromes (MDS) with del(5q) chromosome abnormality. Background appears to be a haplo-insufficiency of the RPS-14 gene located within the CDR. Lenalidomide has improved the treatment of MDS with 5q deletion inducing a hematologic (erythroid) and cytogenetic response in the majority of patients. The exact mechanisms of action of lenalidomide explaining its effects on erythropoiesis and their possible prognostic relevance are not clear so far. PATIENTS: We present the first data on in-vivo changes at the level of hematopoietic stem cells (SC), erythroid committed SC (SC-e), maturation of SC-e to erythroblastic islands and normoblasts observed during lenalidomide therapy within the bone marrow from a total of 39 transfusion-dependent patients with low- or intermediate-1-risk MDS with del(5q) chromosome aberration (MDS.5q-) who were recruited and treated with lenalidomide (10 mg / d) at the European site of the MDS-003 study and whose bone marrow samples (aspirates and biopsies) were taken prospectively at 6-month intervals after start of treatment. Mature erythroid precursors were marked by anti-hemoglobin-A-antibody (HbA); immature, HbA- erythroid precursors by anti-glycophorin-C-antibody (GPC), and the SC compartment by anti-CD34 antibody. Due to their low number, the numerical densities of SC-e were estimated indirectly by the numerical density of erythroblastic islands within bone marrow whereas the total number of SC was estimated by a statistical approach assuming that SC give rise to clusters of CD34+ precursors. The results were correlated with lenalidomide dose, RPS-14, SPARC, and glycophorin A (GPA) gene expression, degree of anemia, transfusion dependence, cytogenetic and molecular (FISH) response, clonal evolution, AML-free and overall survival of patients. RESULTS: Lenalidomide therapy resulted in a marked increase of SC-e, differentiation of SC-e to GPC+HbA- erythroid precursors, pronounced production of HbA+ precursors and erythrocytes resulting in transfusion independence in 54 % of patients. Lenalidomide-induced increase of SC-e did not correlate with a rise in the total number of SC. It was more pronounced in patients with a cytogenetic response, but it occurred also in patients without a cytogenetic remission. 17 / 39 patients with a minimal therapy effect on erythropoiesis achieving the 1% percentile of normal healthy marrow and continuing for > 12 months showed an excellent prognosis with a probability of survival > 90 % 60 months after start of treatment and 120 months after diagnosis of disease irrespective of their hematologic response whereas the median survival time of the patients without minimal effect on erythroid precursors (n = 22) was shorter than 20 months after start of treatment (P < 0.0002). In multivariate survival analysis, changes of erythropoiesis occurring during lenalidomide treatment, clonal evolution of disease, and age of patients were the only variables providing independent prognostic information (P < 0.00005). Classification of disease (FAB, WHO, IPSS, WPSS), changes of peripheral blood cell counts, transfusion independence, cytogenetic and molecular response, RPS-13, SPARC, or GPA gene expression did not provide additional independent prognostic information (P > 0.05). CONCLUSIONS: Lenalidomide promotes erythroid differentiation at stem cell level as well as at more mature stages of erythropoiesis affecting both, the neoplastic clone as well as non-neoplastic hematopoiesis. The anti-neoplastic effect of lenalidomide does not seem to be relevant for the prognosis of the patients; (1) genetic instability of neoplastic SC / precursor resulting in clonal evolution on the one and (2) the capacity of neoplastic SC / precursors to respond to lenalidomide therapy with the production of SC-e and more mature erythroid precursors on the other hand appear to be significant. By combining both variables, three groups can be discriminated allowing for a therapy-specific prognostic scoring: one group with excellent prognosis (100 % survival 5 years after start of therapy) comprising ∼ 33 % of patients, another with poor prognosis (0 % survival 5 years after start of therapy) comprising ∼ 28 % of patients, and a third group with intermediate prognosis (53 % dying during this period of observation) comprising ∼ 39 % of patients (P < 0.000001). Disclosures: Buesche: Celgene Corp.: reference pathologist of the MDS-004 and the MDS-Le-Mon-5 studies. Giagounidis:Celgene Corp: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Göhring:Celgene Corp.: reference cytogeneticist of the MDS-004 study. Schlegelberger:Celgene Corp.: reference cytogeneticist of the MDS-004 study. Knight:Celgene Corp.: Employment.

TNFα Overproduction in FANCC-Deficient Cells Is TLR8 Dependent.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 494-494
Author(s):  
Scott Vanderwerf ◽  
Johanna Svahn ◽  
Praveen Anur ◽  
Ricardo Pasquini ◽  
Grover C. Bagby
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Clonal Evolution ◽  
Hematopoietic Cells ◽  
Mononuclear Phagocytes ◽  
C Cells ◽  
Mutant Cells

Abstract Abstract 494 The Fanconi anemia (FA) proteins play a role in regulating genome stability but it is not clear that loss of genoprotection in FA hematopoietic cells accounts for the molecular pathogenesis of bone marrow failure so characteristic of this disease. Other factors are known to influence survival and replication of FA stem cells. For example, not only are FA progenitors and stem cells hypersensitive to the apoptotic effects of TNFα, FA cells over-produce TNFα. Most importantly over-production of and hypersensitivity to TNFα in hematopoietic cells of Fancc-/- mice results in bone marrow hypoplasia 1;2 and long-term ex-vivo exposure of murine Fancc -/- hematopoietic cells to both growth factors and TNFα results in the evolution of cytogenetically marked preleukemic clones.3 Therefore, the hematopoietic phenotype of FA is likely multifactorial and may evolve from the overproduction of precisely the cytokine to which FA stem cells are hypersensitive. Methods: We sought to clarify the molecular basis of aberrant TNFα-production. We conducted gene expression microarray experiments using RNA samples from low density marrow cells obtained from 11 normal volunteers and 22 Fanconi anemia patients with uncomplicated marrow hypoplasia without clonal cytogenetic defects. Because the FA complex is known to enhance ubiquitinylation of FANCD2, we reasoned that the ubiquitinylation state of proteins involved in the TNF pathways might also be influenced by core FA proteins. Therefore, we conducted in vitro ubiquitinylation assays using hexahistidine-tagged ubiquitin and an ATP-recycling system added to lysates of FANCC-deficient lymphoblasts (HSC536) and control cells (isogenic cells complemented with WT FANCC cDNA). Following the ubiquitinylation reaction, ubiquitinylated proteins were affinity purified, digested and analyzed by 2D capillary LC-MS/MS. Mass spectra were obtained and peptide precursor-MS/MS spectrum pairs were analyzed using SEQUEST and support vector machine learning.4 Peptides identified only in one or the other cell line were considered. Results: Initially we anticipated focusing on the set of proteins uniquely ubiquitinated in normal cells. However, the transcriptomal results indicated that genes encoding proteins in the ubiquitin pathway were over-represented in the list of genes that were over-expressed in FA samples. Consequently, we examined both differential ubiquitination lists and found that a major regulator of TNF-gene expression, TLR8, appeared in the ubiquitinylated fraction only in mutant cells. In co-immunoprecipitation studies we confirmed that TLR8 (or a TLR8-associated protein) is ubiquitinylated in mutant FA-C cells, and using RNAi determined that high level TNFα synthesis in mutant cells depended upon TLR8 and its downstream signaling intermediates IRAK-1 and IKK-alpha/beta. FANCC deficient THP1 blue cells were created using lentiviral shRNA targeting FANCC. These cells exhibited the MMC hypersensitive phenotype and over-expressed both TNFα and an NF-kappaB reporter gene (secreted embryonic alkaline phosphatase) in response to TLR8 agonists but not to other TLR agonists. Primary splenic macrophages from Fancc-/- mice were also hypersensitive to the TLR8 agonist R848. TNFα production in FA-C cells was suppressed by inhibitors of TLR8, p38 MAPK, IRAK, and IKK. Engineered point mutants of FANCC were capable of complementing the mitomycin C hypersensitivity phenotype of FANCC mutant cells but did not suppress TNFα overproduction in FANCC mutant cells. In conclusion, TNF over-expression in FANCC-deficient cells reflects the loss of FANCC function as a suppressor of TLR8 activation. In addition, FANCC suppresses TLR8 dependent production of TNFα in normal mononuclear phagocytes at least in part by suppressing either TLR8 ubiquitinylation or by inhibiting its association with an ubiquitinylated protein. Finally, this function of FANCC is independent of its function in protecting the genome from cross-linking agent-induced damage. In light of the role of TNFα in bone marrow failure and clonal evolution in this disease, control of TNF-production by targeting the TLR8 pathway might provide an opportunity to enhance hematopoietic activity and forestall clonal evolution in patients with this disorder. 1. Sejas DP, et al, J Immunol 2007;178:5277-5287. 2. Zhang × et al, J.Cell Sci. 2007;120:1572-1583. 3. Li J, et al, J.Clin.Invest. 2007;117:3283-3295, 4. Anderson DC, et al, J Proteome.Res 2003;2:137-146. Disclosures: No relevant conflicts of interest to declare.

Cord Blood CD34+ Stem Cell Sialyl Lewis X Levels and E-Selectin Binding Are Predictive Of Engraftment In Mice: Functional Separation Of Stemness and Homing To Improve Engraftment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 893-893 ◽  
Author(s):  
Patrick A Zweidler-McKay ◽  
Simon N Robinson ◽  
Michael W Thomas ◽  
JunJun Lu ◽  
Hong Yang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Cord Blood ◽  
Ex Vivo ◽  
Sialyl Lewis X ◽  
Sialyl Lewis ◽  
Cord Blood Cd34 ◽  
Selectin Binding

Abstract Background Cord blood (CB) is an increasingly used alternative to bone marrow and mobilized peripheral blood as a source of hematopoietic tissue for transplantation. However, the relatively low cell dose and significantly delayed engraftment when compared to BM and mPB remain significant hurdles. A deficit in the homing of CB hematopoietic progenitor and stem cells (HPSC) to the hematopoietic microenvironment due to suboptimal expression and/or activity of homing molecules is thought to be responsible in part for the delayed engraftment seen with CB in patients. Sialyl Lewis X (sLeX) bearing cells can bind E-selectin, and we have previously reported that ex vivo fucosylation of CB HPSCs with fucosyltransferase VI or VII enhance the rapidity and magnitude of engraftment in mice. Here we explore the engraftment potential of endogenous sLeX bearing CD34+ CB HPSCs to determine if physiologic levels of E-selectin binding predicts engraftment in murine xenografts. Approach CB cells were sorted with CD34 and HECA-452 (anti-sLex) antibodies. sLeX-bearing CD34+ HPSCs (CD34+HECA+) and CD34+ HPSCs lacking sLeX (CD34+HECA-) were compared phenotypically for stem cell and differentiation markers, by gene expression profiling, E/P/L-selectin binding, colony-forming assays, and for multi-lineage engraftment into NOD-SCID-IL2Rg immune-deficient mice. Results Cord blood CD34+HECA+ cells represent 10-20% of CB MNCs and show no significant phenotypic differences from the CD34+HECA- cells in stem cell (CD133, CD90 (Thy-1), CD117 (c-kit), CD143/BB9) and differentiation (CD38, CD33, CD14, CD3, CD19) markers. In agreement, similar percentages of CD34+CD38- and CD34+CD38+ CB cells were found to be HECA+ (18% and 15% respectively, p=0.38), showing no significant bias toward the more immature CD34+CD38- phenotype. mRNA-seq expression analysis revealed relatively few differences in gene expression patterns, although CD34+HECA+ cells express higher levels of the gamma globin genes HBG1 and HBG2, the components of fetal hemoglobin. As predicted, CD34+HECA+ cells demonstrated significantly increased ability to bind recombinant E-selectin in vitro, with no differences in P- and L-selectin binding. Importantly, colony forming assays revealed a small (30%) disadvantage to the CD34+HECA+ cells revealing that the CD34+HECA+ CB cells do not have enriched stem cells activity by CFU assay. However, CD34+HECA+ cells demonstrated significantly higher rate and magnitude of engraftment when compared to CD34+HECA- cells in three independent NSG experiments (Figure 1). Indeed bone marrow, peripheral blood and splenic levels of human hematopoietic cells were consistently 3-5-fold higher in CD34+HECA+ injected mice than in CD34+HECA- injected controls (Figure 2). Multi-lineage engraftment data reveals higher levels of myeloid (CD33+/CD14+), B-lymphocytes (CD19+/CD20+) and platelets (6-14-fold, CD41a+/CD61+) in CD34+HECA+ cells, but interestingly lower levels of T-lymphocytes (CD3+). Finally, secondary transplants had equal magnitude of engraftment, indicating no bias in short- versus long-term HSPCs. Conclusions Data presented here supports the hypothesis that endogenous sLex levels on CD34+ cells is associated with enhanced engraftment rapidity and magnitude but that this is not reflective of an enriched stem cell fraction. Rather it appears to be an indicator of homing to the bone marrow through E-selectin binding. Functional separation of stemness and homing supports the approach to improve CB transplantation through decoration of CB cells with sLex via ex vivo fucosylation (see clinical trial abstract by Popat and Shpall) Disclosures: No relevant conflicts of interest to declare.

Clonal Evolution Underlying the Progression from Myelodysplastic Syndromes to Ph-Positive Acute Lymphoblastic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5514-5514
Author(s):  
Masataka Taguchi ◽  
Tomoko Kohno ◽  
Hiroyuki Mishima ◽  
Hiroaki Taniguchi ◽  
Takeharu Kato ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Acute Lymphoblastic Leukemia ◽  
Myelodysplastic Syndromes ◽  
Copy Number ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Intron 1

Abstract Introduction: Myelodysplastic syndromes (MDS) are considered as a "stem cell disorders", in which hematopoietic stem cells and lineage-committed progenitor cells acquire genetic and epigenetic alterations and provide aberrant, clonal hematopoiesis, sometimes resulted in the progression to acute myeloid leukemia (Elias HK et al, Oncogene 2014). We previously reported a rare case of which the patient developed Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) 2.5 years after being diagnosed with MDS (Kohno T et al, Br J Haematol 1996). p190 BCR-ABL1 mRNA was detected in the Ph+ALL cells. Metaphase cytogenetics showed the karyotypes: 46, XY, 20q- in MDS phase and 46, XY, t(9;22)(q34;q11), 20q- in ALL phase, indicating that MDS and Ph+ALL in this patient were of the same clonal origin. To uncover the detail of the clonal evolution, we analyzed bone marrow samples of MDS and Ph+ALL in this patient by targeted massively parallel sequencing with a panel of 154 genes including known driver genes of hematologic malignancies. Methods: Genomic DNAs (gDNAs) were extracted from the bone marrow mononuclear cells of MDS and Ph+ALL in this patient. Targeted sequencing was performed on the Illumina HiSeq2500 platform. Single nucleotide variants (SNVs) and small insertions and deletions (INDELs) were called using HaplotypeCaller of Genome Analysis Toolkit (GATK) version 3.4-46. We also attempted to detect the breakpoint of BCR-ABL1 translocation from the targeted sequencing data using the computational method, BreaKmer (Abo RP et al, Nucleic Acids Research 2015). The candidates of the mutations and structural variations were validated by amplicon-based deep sequencing and Sanger sequencing. Copy number variations were analyzed using Affymetrix CytoScan HD Array. Results: The mutations in ASXL1 and U2AF1 genes were identified in the MDS sample with variant allele frequencies (VAFs) of about 45%. At the progression of Ph+ALL, the mutations in SETBP1, SMC1A, and SLC5A8 genes were newly acquired while the ASXL1 and U2AF1 mutations were also identified with the same level of VAFs (about 50%) as the other mutations. VAFs of all of the mutations were decreased to about 20% after the chemotherapy for Ph+ALL, and then increased to about 40% at the recurrence of the disease. Furthermore, we identified the breakpoint of BCR-ABL1 translocation at intron 1 of ABL1 genes and intron 1 of BCR genes, that is the well-known cluster region, m-bcr, only among the samples of Ph+ALL. Copy number analysis confirmed that both MDS and Ph+ALL samples harbored the deletion of chromosome 20q. And the deletion of IKZF1 gene, which is frequently identified in Ph+ALL cases (Mullighan CG et al, Nature 2008), was not identified during the progression from MDS to Ph+ALL. These results demonstrated that the MDS clone harboring 20q- and ASXL1 and U2AF1 mutations acquired the mutations in SETBP1, SMC1A, and SLC5A8 genes and the p190 BCR-ABL1, resulted in the development of Ph+ALL in this patient. Conclusion: The alterations of SETBP1, SMC1A, and SLC5A8 genes are usually reported in myeloid malignancies (Makishima H et al, Nat Genet 2013, Kon A et al, Nat Genet 2013, Whitman SP et al, Blood 2008). Previous study in transgenic mouse demonstrated the distinct role of p190 BCR-ABL1 in the development of an ALL (Voncken JW et al, Blood 1995). Recapitulating this scenario, p190 BCR-ABL1 may play a critical role in the development of Ph+ALL from the MDS stem cells in this patient. This study may provide a new insight into the stem cell origin of MDS and the role of p190 BCR-ABL1 in the development of Ph+ALL. Disclosures No relevant conflicts of interest to declare.

Clonal Evolution of Fanconi Anemia (FA) Stem Cells Results from Clonal Adaptation and Clonal Selection. A Report from the International Fanconi Anemia Transcriptome Consortium.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3416-3416
Author(s):  
Grover Bagby ◽  
Jane Yates ◽  
Shannon McWeeney ◽  
Byung Park ◽  
Keaney Rathbun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Fanconi Anemia ◽  
Mixed Model ◽  
Linear Mixed Model ◽  
Clonal Selection ◽  
Clonal Evolution ◽  
Differentially Expressed

Abstract FA stem cells and progenitor cells are apoptotic in the ground state and hypersensitive to a variety of extracellular apoptotic cues. The relative lifetime risk of AML or MDS in FA is high and clonal cytogenetic defects are universally found in evolved clones, but the mechanisms involved in leukemogenesis are unknown. The combined influences of genetic instability and high level stem cell apoptosis represents a de-facto selective pressure that in our view favors the emergence of more resistant stem cell clones. Predictions of this model are: (1) Non clonal FA progenitor cells (FAwt) are hypersensitive to apoptotic cues but FA progenitors derived from cytogenetically abnormal clones (FAclon) will be resistant, (2) anti-apoptotic events that attend cytogenetic clonal evolution should interdict precisely those apoptotic pathways activated by FA gene mutations, and (3) Key transcripts differentially expressed in the normal (N) vs. FAwt comparison should normalize (ie. should register as "no change") in the N vs. FAclon comparison. Preliminary studies support the first 2 predictions (Lensch et al, Leukemia, 1999 and Haneline et al, Blood, 2003). To further test the adaptive model, we compared transcriptomes of 41 low density bone marrow cell samples: (11 normal volunteers [N], 9 FAclon [marrow replaced [>75%] by cells bearing clonal cytogenetic defects] and 21 FAwt [no cytogenetic abnormalities]). RNA was prepared in the participating institutions and shipped to Portland on dry ice. Complementary DNA and cRNA was prepared, cRNA fragments labeled for use as targets of the probes in the human U133A Affymetrix chip. MAS 5.0 was utilized to process images, quantify signals, adjust background, and to scale the data. A linear mixed model was used for inter-chip normalization. Unsupervised hierarchical clustering and multidimensional scaling (MDS) distinguished N from FAwt samples but FAclon fell within the mathematical space defined by the N RNA. Of 17,044 genes tested, 1,430 were expressed differentially (FDR adjusted p <0.05, fold change ≥2) between N and FAwt samples. Differences did not derive from changes in cell lineages from case-to-case, and did not vary between geographical sites and were not linked to particular complementation groups. 659 transcripts were UP in FAwt (vs. N), but 587 of these were not UP in FAclon (vs N). The two dominant over-represented (p<0.01) ontologies (biological process) of the 659 genes were apoptosis and transcriptional control. 771 transcripts were DN in FAwt (vs. N) but 734 of these were not DN in the FAclon vs. N comparison. Dominant ontologies included cell proliferation, and responses to biotic stimuli. Our state assessment of FA bone marrow cells has defined a transcriptomal difference between and FAwt and normal cells and reveals that only 5-10% of these differentially expressed genes are differentially expressed between normal and FAclon cells. These observations support the notion that cytogenetically marked clones evolve adaptively from initially pro-apoptotic stem cells.

Leukemic Stem Cell Assessment in Remission Bone Marrow of Acute Myeloid Leukemia Patients Is a New Prognostic Parameter.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 399-399 ◽  
Author(s):  
Monique Terwijn ◽  
Angèle Kelder ◽  
Arjo P Rutten ◽  
Alexander N Snel ◽  
Willemijn Scholten ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
Prognostic Information ◽  
Cell Compartment ◽  
Hematopoietic Stem ◽  
Stem Cell Compartment ◽  
Acute Myeloid

Abstract Abstract 399 In acute myeloid leukemia (AML), relapses originate from the outgrowth of therapy surviving leukemic blasts know as minimal residual disease (MRD). Accumulating evidence shows that leukemia initiating cells or leukemic stem cells (LSCs) are responsible for persistence and outgrowth of AML. Monitoring LSCs during and after therapy might thus offer accurate prognostic information. However, as LSCs and hematopoietic stem cells (HSCs) both reside within the immunophenotypically defined CD34+CD38- compartment, accurate discrimination between LSCs and HSCs is required. We previously showed that within the CD34+CD38- stem cell compartment, LSCs can be discriminated from HSC by aberrant expression of markers (leukemia associated phenotype, LAP), including lineage markers like CD7, CD19 and CD56 and the novel LSC marker CLL-1 (van Rhenen, Leukemia 2007, Blood 2007). In addition, we reported that flowcytometer light scatter properties add to even better detection of LSCs, allowing LSCs detection in AML cases lacking LAP (ASH abstract 1353, 2008). Using this gating strategy, we determined LSC frequency in 64 remission bone marrow samples of CD34+ AML patients. A stem cell compartment was defined as a minimum of 5 clustered CD34+CD38- events with a minimal analyzed number of 500,000 white blood cells. After first cycle of chemotherapy, high LSC frequency (&gt;1 × 10-3) clearly predicted adverse relapse free survival (RFS, figure 1a). LSC frequency above cut-off led to a median RFS of 5 months (n=9), while patients with LSC frequency below cut-off (n=22) showed a significantly longer median RFS of &gt;56 months (p=0.00003). In spite of the relatively low number of patients, again a high LSC frequency (&gt;2 × 10-4) after the second cycle and after consolidation therapy predicted worse RFS: after second cycle, median RFS was 6 months (n=9) vs. &gt;43 months for patients with LSC frequency below cut-off (p=0.004). After consolidation, these figures were 6 months (n=7) vs. &gt;32 months (n=6, p=0.03). Although total blast MRD (leukemic blasts as % of WBC) is known to predict survival (N.Feller et al. Leukemia 2004), monitoring LSCs as compared to total blast MRD has two major advantages: the specificity is higher (van Rhenen et al. Leukemia 2007) and well-known LSC makers like CLL-1, CD96 and CD123 can in principle be used for LSC monitoring, but not for total blast MRD detection since these markers are also expressed on normal progenitor cells. On the other hand, LSCs constitute only a small fraction of all leukemic blasts and therefore monitoring total blast MRD may have the advantage of a higher sensitivity. We thus tested the hypothesis that even more accurate prognostic information could be obtained by combining LSC frequency with total blast MRD. Total blast MRD after first cycle was predictive for survival with borderline significance (p=0.08): a cut-off of 0.3% resulted in two patient groups with median RFS of 9 months vs. &gt;56 months. Figure 1b shows the result of the combined data of LSC and MRD frequency after first cycle therapy. We used the terms LSC+ and MRD+ for cell frequencies above cut-off and LSC- and MRD- for those below cut-off. We could clearly identify that apart from LSC+/MRD+ patients, LSC+/MRD- patients too have very poor prognosis, while MRD+/LSC- patients show an adverse prognosis as compared to LSC-/MRD- patients. These results from the first study on the in vivo fate of LSCs during and after therapy, strongly support the hypothesis that in CD34+ AML the leukemia initiating capacity originates from the CD34+CD38- population and is important for tumor survival and outgrowth. These results show that LSC frequency might be superior in predicting prognosis of AML patients in CR as compared to MRD total blast frequency, while the combination of both may offer the most optimal parameter to guide future intervention therapies. This work was supported by Netherlands Cancer Foundation KWF. Disclosures: No relevant conflicts of interest to declare.

Similarities and differences in aging and in malignant transformation of human hematopoietic stem cells

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 6555-6555
Author(s):  
H. Lannert ◽  
T. Able ◽  
T. Franz ◽  
R. Hofmann ◽  
A. Lenze ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Leukemia Cells ◽  
Cell Aging ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Genomics And Proteomics ◽  
Cell Fractions

6555 Background: The tracking of stem cell aging, differentiation and deterioration by gene expression profiling and proteome analysis allows the comparison of different stages. The overall aim of a proteomic study is characterization of the complex network of cell regulation. We focused our investigations on different subsets of highly enriched CD34+ stem cells from different human origins: fetal liver, cord blood, bone marrow (BM), and mobilized stem cells from peripheral blood (PBSC), as well as CD34+ leukemia cells, thus e.g. to identify pathways and new targets for leukemia therapy. Methods: Mononuclear cells were isolated by a standard Ficoll separation method from the different blood sources. An Auto-MACS (Miltenyi) and FACS Vantage SE cell sorter (Becton Dickinson) was used to highly enrich (>99%) CD34+ cells fractions. Sample preparation: Total RNA was isolated from sorted 1 × 10e6 cells by standard methods using RNA isolation kit (Qiagen). For gene expression analysis topic-defined PIQOR stem cell microarrays (936 genes) were performed. Proteomics started with the determination of protein concentrations, 2D-gel-electrophoresis were described in Proteome Works System (BioRad). Sypro ruby and/or coomasie stained gels were used for protein identification by Q-TOF analyses. The subcellular localization of the identified proteins were performed by fluorescence and confocal microscopy of all cell fractions. Results: 1. The microarray gene expression correlation shows many similarities between human healthy stem cells of different sources and ages, otherwise many differences: 125 upregulated genes (kinases: PAK1, ATM1, CDKN2A) and 32 downregulated genes (EBCTF, RAMP1) in malignant cells compared to healthy stem cells. 2. Proteomics analyses of the different cell fractions show a large overlap of the most dominant protein spots, >200 spots were identified by Q-TOF. For example stathmin (oncoprotein Op18) is expressed at very high levels in leukemia cells and in PBSCs but not in BM cells, additionally demonstrated by fluorescence microscopy. Conclusions: Combining genomics and proteomics assays, pathways e.g. Op18 for proliferation and migration of healthy (mobilized) CD34+ cells from bone marrow and malignant leukemia cells could be identified. No significant financial relationships to disclose.

scholarly journals Ph+/VE-cadherin+ identifies a stem cell–like population of acute lymphoblastic leukemia sustained by bone marrow niche cells

Blood ◽  
2007 ◽  
Vol 110 (9) ◽  
pp. 3334-3344 ◽  
Author(s):  
Lin Wang ◽  
Heather O'Leary ◽  
James Fortney ◽  
Laura F. Gibson
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Stromal Cell ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Cell Contact ◽  
Leukemic Stem Cells ◽  
Self Renewal

Abstract Although leukemic stem cells (LSCs) show a symbiotic relationship with bone marrow microenvironmental niches, the mechanism by which the marrow microenvironment contributes to self-renewal and proliferation of LSCs remains elusive. In the present study, we identified a unique subpopulation of Philadelphia chromosome–positive (Ph+) acute lymphoblastic leukemia (ALL) cells coexpressing markers of endothelial cells (including VE-cadherin, PECAM-1, and Flk-1) and committed B-lineage progenitors. After long-term coculture with bone marrow stromal cells, tumor cells formed hematopoietic colonies and cords, expressed early stem- cell markers, and showed endothelial sprouting. Gene expression profiles of LSCs were altered in the presence of stromal cell contact. Stromal cell contact promoted leukemic cell VE-cadherin expression, stabilized β-catenin, and up-regulated Bcr-abl fusion gene expression. Our study indicates that these specific tumor cells are uniquely positioned to respond to microenvironment-derived self-renewing and proliferative cues. Ph+/VE-cadherin+ tumor subpopulation circumvents the requirement of exogenous Wnt signaling for self-renewal through stromal cell support of leukemic cell VE-cadherin expression and up-regulated Bcr-abl tyrosine kinase activity. These data suggest that strategies targeting signals in the marrow microenvironment that amplify the Bcr-abl/VE-cadherin/β-catenin axis may have utility in sensitizing drug-resistant leukemic stem cells.

Circadian Rhythms of Whole Bone Marrow and Lineage Negative Stem Cells of the Mouse.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4188-4188
Author(s):  
Galatia Politopoulou ◽  
Mark S. Dooner ◽  
Gerald A. Colvin ◽  
Deborah Greer ◽  
Delia Demers ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Circadian Rhythm ◽  
Circadian Rhythms ◽  
Stem Cell Factor ◽  
Clock Genes ◽  
Circadian Variation

Abstract Circadian rhythms underlie most biological processes. In mammals circadian control of physiology and behavior is mediated via a central master oscillator, in the supra-schismatic nuclei of the hypothalamus. At the cellular level this oscillator is composed of an auto-regulatory transcription-translation loop of clock genes. The Period2 (Per2) gene is one of the clock genes which plays a key role in controlling the circadian rhythm in mammals. Mice with mutations in Per2 become arrhythmic. Expression of clock genes is also present in many peripheral tissues, including the bone marrow. Stem cell engraftment has been shown to vary with cell cycle transit (Habibian et al, 1998). A diurnal circadian variation in the ability of bone marrow to engraft sub-lethally irradiated mice has been previously shown by our laboratory. An increase in numbers of progenitors in S-phase underlined the engraftment nadirs. The host’s ability to accept incoming cells did not show circadian variation. To further study the interplay of circadian rhythm with cell cycle in bone marrow and populations of engraftable stem cells, we utilized a transgenic mouse model for the Per2 gene. The mouse Period2 (mPer2) real time gene expression reporter of circadian dynamics, constructed by Takahashi et al., was employed for these studies. In this reporter a Luciferin (Luc) gene was fused in frame to the 3′ of the promoter of the endogenous mPer2 gene. This system allows for detection of Per2 gene expression in the presence of luciferase, by recording light given off, during the luciferase catalyzed conversion of Luciferin to Oxyluciferin. We have detected circadian rhythm in whole bone marrow and Lineage negative cells i.e. whole bone marrow mononuclear cells depleted of B220, Ter119, GR1, CD4, CD8 and CD11b, with one peak every 24 hours for up to 14 days, from as few as 500,000 cells. Dissociated lung cells also show a circadian rhythm as do Lineage Negative Sca+ marrow stem cells. The later show an intermittent rhythm, for up to 10 days. The best rhythms were obtained from cells grown on a 12mm dish bathed in 4 mls of media or a 1 ml drop of media, the later covered with mineral oil. The media was DMEM with L Glutamine, low glucose no phenol red, 1% Penicillin, 10,000 U/ml/streptomycin, 4.18mM NaHCO3, 10mM Hepes, 0.019mM D-glucose pH 7.2 supplemented with 1×B27, 5–15% HIFCS and either stem cell factor alone (50ng/ml) or IL3, IL6, IL11 and stem cell factor (steel) (50 ng or units/ml). Feeding of the cells after 7 days increased the amplitude of the rhythm. Absence of cytokines dampened the rhythm, especially for Lineage Negative Sca+ cells. Steel in the presence of HIFCS induces some rhythm, but is not as effective as a cocktail of IL3, IL6, IL11 and steel, together with HIFCS. Given work from our laboratory on synchronized progenitor cells entering and progressing through cell cycle in cytokine cocktails, including IL3, IL6, IL11 and steel, and the correlation of engraftment potential with cell cycle phase and adhesion molecule phenotype, the appearance of the best circadian rhythm in proliferating cytokine cocktails, in this system, is intriguing. The cell cycle kinetics of the marrow cells exhibiting circadian rhythmicity, are being explored further, in this culture system. The links between expression of cell cycle control molecules and adhesion molecules in Lineage Negative Sca+ cells and circadian rhythms for engraftment are also under investigation.

Following Autologous Stem Cell Transplantation (1 Yr) Mesenchymal Stem Cells Are Functionally Impaired with Reduced Hematopoietic Support

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3888-3888
Author(s):  
Catharina Hazenberg ◽  
Fiona A.J. van den Heuvel ◽  
Edo Vellenga ◽  
Annet Z. Brouwers-Vos ◽  
Gerbrig Berger ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Autologous Stem Cell Transplantation ◽  
Population Doubling ◽  
Bone Marrow Biopsies

Abstract Autologous stem cell transplantation (ASCT) is frequently applied in patients with multiple myeloma and malignant lymphoma. Although adequate steady state hematopoiesis with normal peripheral blood counts is attained after ASCT, marked cytopenias may occur in times of stress such as sepsis or re-exposure to chemotherapy. Our group has previously shown impairment of the hematopoietic stem cell (HSC) compartment 1 year post ASCT (pASCT), reflected by reduced HSC frequency and quiescence, and increased ROS production (Haematologica 2013;98:1264). Considering the essential role for mesenchymal stem cells (MSCs) in supporting hematopoiesis, we studied the MSC compartment 1 year post ASCT. Bone marrow biopsies from pASCT patients (n=17) were studied and compared to normal bone marrow from healthy donors (NBM, n=20) by performing immunohistochemistry staining of endothelial cells by CD34 (indicating microvessel density, MVD) and MSCs by nestin, CD146 (Melanoma Cell Adhesion Molecule, MCAM) and CD271 (Nerve Growth Factor Receptor, NGFR). A significant increase in CD271+ MSCs was observed in pASCT bone marrow biopsies compared to NBM (p<0.0001), while the expression of additional markers did not differ between pASCT vs. NBM. MSCs were cultured from the CD34- fraction of bone marrow mononuclear cells, obtained from pASCT patients (n=17) and MSCs derived from NBM (n=20). MSCs were selected by their plastic-adherency and replated to generate MSCs. Although pASCT MSCs and NBM MSCs had similar population doubling times (1.92±0.22 and 3.52±1.02 in passage 4 (P4), pASCT MSCs cultured in vitro demonstrated a change in morphology from the onset of P4. We also observed premature exhaustion of growth in 45% of the studied patients at P5 (vs. 18% in NBM) and increased senescence shown by B-galactosidase staining in P5/P6 (p=0.04). Differentiation assays did not show impairment in differentiation towards osteoblasts or adipocytes of pASCT MSCs. Gene expression analysis on early passage MSCs showed upregulation of pro-inflammatory and cell cycle genes, such as IL6 and p21, in pASCT MSCs compared to NBM MSCs. Co-culture studies with cord blood-derived CD34+ cells on pASCT MSCs showed a significant reduction in output in CFC assays and significant reduction in number of cobblestone-area forming cells in pASCT co-cultures versus NBM (p < 0.05). Given the higher incidence of MDS and AML after ASCT, we questioned whether the observed phenotype of pASCT MSCs resembles MSCs from patients with MDS and AML. Therefore the endothelial and mesenchymal compartments of MDS (n=20) and AML (n=23) patients were studied. An increase in MVD was detected in MDS/AML bone marrow biopsies in contrast to NBM and pASCT (p < 0.05), while the expression of CD146, CD271 and nestin in MDS/AML patients was not significantly increased. 25% of AML MSC cultures showed no growth in the first passage. When MSC growth did occur, the remaining cultures did not show a difference in population doubling time or expansion. However, a change in morphology of MDS/AML MSCs similar to pASCT MSCs was observed. Studies of early passages of MDS/AML MSCs demonstrated a significantly increased gene expression of IL-6 and p21 comparable to pASCT MSCs. In addition PITX2 and Foxc1 expression was increased but no difference was observed in pASCT vs. MDS/AML MSCs. PITX2 has been linked to increased senescence of MDS MSCs while Foxc1 is linked to adipo-osteoprogenitor cell differentiation thereby affecting the HSC compartment. Since none of the pASCT patients did develop MDS, immunohistochemical stainings were also performed on bone marrow biopsies of patients that developed therapy related (t-)MDS/AML following ASCT for lymphoma and myeloma (n=7), after a mean of 117 (MDS) and 50 months (AML). An increase in MVD was observed shortly before or during MDS/AML development, which is probably related to the emergence of malignant cells. No major changes in the phenotype of the MSC compartment were observed before or during the emergence of t-MDS/AML, indicating that t-MDS/AML is preceded by an increase in MVD without distinct changes in the MSC compartment. In summary our results demonstrate that MSCs are affected after ASCT, as shown by expression pattern and functionality. These changes result in a pro-inflammatory phenotype with premature senescence and impaired support of hematopoietic cells, which may account for the reduced bone marrow reserve observed in pASCT patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Comparison of phenotype and differentiation marker gene expression profiles in human dental pulp and bone marrow mesenchymal stem cells

2014 ◽  
Vol 08 (03) ◽  
pp. 307-313 ◽  
Author(s):  
Deepa Ponnaiyan ◽  
Visakan Jegadeesan
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Dental Pulp ◽  
Expression Profiles ◽  
Stem Cell Marker ◽  
Fatty Acid Binding ◽  
Dental Tissues

ABSTRACT Objective: Bone marrow (BM) is the most utilized and well-studied source of stem cells. Stem cells from dental tissues have provided an alternate source of mesenchymal stem cells (MSCs). Dental pulp stem cells (DPSCs) have been shown to share a similar pattern of protein expression with BMMSCs in vitro. However, differences have been noted between DPSCs and BMMSCs. This study focuses on variation in expression of stem cell and differentiation markers between DPSCs and BMMSCs. Materials and Methods: The two stem cells were isolated and compared for clonogenic potential, growth characteristics, multipotency, and stem cell marker expression. Specifically, the fatty acid binding protein 4, perilipin, alkaline phosphatase and osteonectic gene expression was analyzed by real-time polymerase chain reaction to confirm the capacity for adipogenic and osteogenic differentiation. Results: MSCs from these cell sources were similar in their morphology and immune phenotype except for the expression of CD105. Growth curves and colony formation assay revealed proliferation rate of DPSCs was significantly faster than BMMSCs (P < 0.05). DPSCs appeared less able to differentiate into adipogenic lineage, although more able to differentiate into osteogenic lineage. Conclusion: Data from the present study indicate how DPSCs are different from BMMSCs though they are a population of MSCs. DPSCs are a novel population of MSCs as observed by their unique expression of differentiation and lineage specific genes. Further microarray analysis could be used to determine, which genes are differentially regulated in BMMSCs and DPSCs to establish uniqueness of each population of MSCs.

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