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  • Fischer, Johannes C.  (4)
  • 2005-2009  (4)
  • Medicine  (4)
  • 1
    In: Blood, American Society of Hematology, Vol. 110, No. 11 ( 2007-11-16), p. 3170-3170
    Abstract: During the last decade, chronic myeloid leukemia (CML) has been mainly characterized by the reciprocal translocation between chromosomes 9 and 22, resulting in the formation of the protooncogene BCR-ABL. This constitutively active tyrosine kinase is widely considered as the cause of the disease. Even though BCR-ABL transcripts are found in every dividing hematopoietic cell and thus, the disease is likely to originate from a primitive stem cell, the “cell of origin” is still a matter of debate. Despite the active “leukemia stem cell” discussion, very few characteristics of the “cancer stem cell” are established to date. In order to get further molecular insights into CML stem and progenitor cells, we examined CD34+ cell subsets obtained from bone marrow of 7 patients with CML in chronic phase in comparison with 5 healthy volunteers. CD34+ cells were immunomagnetically selected and high-speed cell sorting of lineage-negative, CD34+, CD38−, hematopoietic stem cells and myeloid progenitors was performed. Progenitors were further subdivided by anti-IL-3Ralpha and anti-CD45RA staining. Following RNA extraction, a two-cycle amplification procedure was used to generate cDNA for the hybridization with Affymetrix U133A2.0 arrays. After performing smoothening spline normalization, we applied the perfect match-mismatch difference model algorithm to calculate expression values (dChip). Hierarchical cluster analysis was performed using a correlation based centroid linkage algorithm. Hereby we could discriminate the HSCs, CMPs, and MEP subsets. Corroboration of RNA expression was performed by real-time RT-PCR for selected genes. Comparing the HSC subsets of CML patients with healthy controls we found 98 differentially expressed genes. 87 genes had a lower expression level in CML HSCs whereas 11 genes had a higher one. Among the downregulated genes in CML were transcriptions factors involved in myelogenesis and proliferation and several adhesion molecules associated with homing and migration of the HSCs. On the other hand, the Leptin receptor and BCR-ABL downstream targets were found to be upregulated. Within the common myeloid progenitor (CMP) compartment 37 genes were significantly differentially regulated. Twenty genes had a higher expression level in CML CMPs, 17 genes were downlegulated. Hematopoietic cell-specific cell cycle inhibitor MS4A3 was among the significantly downregulated genes whereas genes of the retinoblastoma and E2F families as well as inhibitors of the Wnt-signaling pathway were upregulated. Looking at megakaryocte-erythrocyte progenitors (MEP) in CML, key mediators of G2-M cell cycle transition were downregulated indicating a lower proliferative capacity of this subset. No transcriptional differences have been observed between granulocyte-macrophage progenitors from CML patients and healthy volunteers. Interestingly, among all other subsets myeloperoxidase (MPO) was downregulated in the CML samples and the Leptin receptor was upregulated. Our results provide novel insights into the biology of CML and potentially provide the basis for the characterization of a candidate CML stem cell.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2007
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  • 2
    In: Blood, American Society of Hematology, Vol. 110, No. 11 ( 2007-11-16), p. 1974-1974
    Abstract: BACKGROUND HLA-C epitopes can be grouped in C1C1, C1C2 or C2C2 ligands and mediate NK cell dependent immune response. Especially in haploidentical allogeneic stem cell transplantation a HLA-C ligand mismatch improves event free survival (EFS) in patients with AML known as Velardi effect. Recently, we could show in 109 CML patients that those with a C1C1 phenotype showed better overall survival (OS) and lower rates of treatment related mortality (TRM) (Fischer JC et al. J Immunology. 2007). But, the role of HLA-C ligands in allogeneic transplantations remains controversial. PATIENTS AND METHODS In this study we retrospectively analyzed a group of 88 patients with AML or CML (n=34), MDS (n=21) or lymphoid malignancies (Non-Hodgkin-Lymphoma or ALL) (n=31) receiving unrelated allogeneic blood stem cell transplantation after myeloablative and non-myeloablative conditioning regimens. HLA-C alleles were determined by DNA-based direct sequencing of all donors and recipients included into this study. RESULTS Looking at the group of 34 patients with AML or CML, the 13 recipients with a C1C1 phenotype showed increased OS compared to those with C1C2 and C2C2 phenotypes (all patients alive with a median follow-up of 154 days, range 90 to 665 days vs. a mean survival of 381 days, respectively; p=0.049). All recipients with a C1C1 phenotype received grafts with matched HLA-C alleles. Within the subgroup of patients with C1C2 or C2C2 phenotypes 6 patients had a HLA-C mismatch which was associated with significantly (p=0.016) increased OS (all patients alive with a median follow-up of 575 days, range 133 to 899) compared to matched HLA-C phenotypes (median survival of 254 days). In recipients with C1C1 phenotype the risk for TRM following HLA-C matched hematopoietic stem cell transplantation was reduced as reflected by an odds ratio of 0.13. In turn, the group receiving HLA-C mismatched grafts had a lower incidence of relapse. This effect was independent from the direction of the mismatch, graft vs. host or host vs. graft. The effects described above were not observed in patients with MDS, ALL or lymphoid malignancy. CONCLUSION The beneficial effects of a C1C1 HLA-C phenotype could be confirmed for patients with CML and AML in our patient cohort. Our data also suggest that patients with myeloid malignancies and an unfavourable C1C2 or C2C2 HLA-C phenotype benefit from a donor with HLA-C ligand mismatch.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    RVK:
    RVK:
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2007
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
    Location Call Number Limitation Availability
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  • 3
    In: Blood, American Society of Hematology, Vol. 110, No. 11 ( 2007-11-16), p. 4920-4920
    Abstract: Mobilized peripheral blood stem and progenitor cells are nowadays widely used for transplantation of hematopoietic stem and progenitor cells (PBSCT). These cells can be mobilized into the peripheral blood with cytotoxic chemotherapy, cytokines or both. Currently, G-CSF is most frequently used due to its high efficacy and lack of serious toxicity. However, a serious patient-to-patient variation in the yield of peripheral blood stem and progenitor cells is a feature common of all mobilizations schemes. Therefore, factors determining the collection efficacy have been identified for G-CSF mobilization. Recently a polyethylenglycole-conjugated G-CSF (Peg-G-CSF) has been introduced which has a 12-fold longer half-life than the original compound and therefore leads to long-lasting G-CSF serum-levels after a single injection. Studies on Peg-G-CSF included only small cohorts and no attempts have been made to identify factors influencing the mobilization of blood stem and progenitor cells. Therefore, we retrospectively analyzed 101 unselected patients (66 with multiple myeloma, 26 with non-Hodgkin-lymphoma, 7 with Hodgkin’s disease, 1 with Ewing sarcoma, 1 with malignant germ cell tumor). 27% of patients had active disease, while all others where at least in partial remission after conventional chemotherapy. Patients were treated with a broad range of chemotherapy regimens. The number of cytotoxic chemotherapy cycles administered prior to the mobilization therapy ranged from 1 to 11 (median 4). Mobilization chemotherapy was followed by 6 mg or 12 mg Peg-G-CSF (median 6 mg). Median peripheral blood CD34+ cell maximum in all patients was 65.3/μl (range 0.2–1084 per μl). 12 mg Peg-G-CSF led to a significantly earlier CD34+ cell maximum in the peripheral blood compared to 6 mg Peg-G-CSF (median 13 days vs 15 days, respectively; p=0.01). Overall, a median yield of 8.5 x 10^6 CD34+ cells/kg bodyweight (range 0.2–72.4 x 10^6) was reached with a single apheresis (median, range 1–4). To search for predictors of hematopoietic stem and progenitor cell mobilization, multiple regression analysis was used and revealed CD34+ cell count/μl peripheral blood at the day of apheresis and the processed blood volume during apheresis as predictors for the CD34+ cell yield per kilogram bodyweight. Age, sex, disease type and status were not significantly related to the CD34+ cell count/μl peripheral blood nor the CD34+ cell yield. Interestingly, the number of previous chemotherapy cycles was correlated with the CD34+ cell maximum (p=0.027) with fewer chemotherapy cycles leading to a higher peripheral blood CD34+ cell count and vice versa. In contrast, radiation therapy prior to CD34+ cell mobilization led to a significantly later occurrence of the CD34+ cell maximum in the peripheral blood. Our results confirm the feasibility and efficacy of PBPC mobilization with single dose Peg-G-CSF after cytotoxic chemotherapy shown in previous clinical trials analyzing the largest patient cohort to date and predictors for successful stem cell mobilization with Peg-G-CSF could be identified.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    RVK:
    RVK:
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2007
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
    Location Call Number Limitation Availability
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  • 4
    In: Blood, American Society of Hematology, Vol. 108, No. 11 ( 2006-11-16), p. 3382-3382
    Abstract: Current regimens for peripheral blood stem cell (PBSC) mobilization in patients with multiple myeloma are based on daily subcutaneous injections of G-CSF starting shortly after cytotoxic therapy. Recently a polyethylenglycole (PEG)-conjugated G-CSF has been introduced which has a substantially longer half-life than the original formula and therefore provides the basis for long-lasting G-CSF serum-levels after a single injection. In this study, we compared gene expression patterns, subset composition and functional properties of CD34+ cells and highly purified HSC mobilized with cyclophosphamide and either Peg-G-CSF or G-CSF. Cells were derived from peripheral blood of patients with multiple myeloma. After the end of chemotherapy, 7 patients got a single injection of Peg-G-CSF whereas 9 patients received daily G-CSF resulting in an equal cumulative dose. Gene expression analysis was performed using Affymetrix HG Focus GeneChips. Key functional genes were verified by RT-PCR. Subset analysis and fluorescence based cell sorting has been conducted to assess the effects of stimulation with either pegylated or unconjugated G-CSF on CD34+ subset composition and to obtain HSCs. Cell cycle and apoptosis assays as well as clonogenic assays were for functional corroboration. The same patients with multiple myeloma who had donated CD34+ cells for the molecular and biological studies were transplantated with Peg-G-CSF- or G-CSF-mobilized PBSC. Peg-G-CSF-mobilized cells showed lower expression of genes characteristic for erythroid and later stages of myeloid differentiation as well as a lower BFU-E/CFU-GM ratio compared to G-CSF-mobilized cells. In turn, we found higher expression levels of genes indicative of early hematopoiesis including HOXA9, MEIS1, MLL and GATA3. Subset analyses revealed a greater number of HSC and CMP (common myeloid progenitors) and a lower number of MEP (megakaryocyte-erthrocyte progenitors) in Peg-G-CSF-mobilized CD34+ cells. Cell cycle-promoting genes including cyclins and kinases were higher expressed in Peg-G-CSF-mobilized cells. On the other hand human HTm4, which causes cell cycle arrest in hematopoietic cells, was lower expressed compared to G-CSF-mobilized cells. This is emphasized by a significant higher proportion of actively cycling CD34+ cells after pegfilgrastim-mobilization. Higher gene expression levels of HOXA9, MEIS1 and GATA3 were also found in sorted Peg-G-CSF-mobilized HSC in comparison to G-CSF-mobilized HSC. Moreover, Peg-G-CSF-mobilized HSC showed a lower apoptosis rate and a greater proportion of cells in S- and G2/M phase of cell cycle. After transplantation of Peg-G-CSF-mobilized stem- and progenitor cells we observed earlier leukocyte recovery compared to G-CSF-mobilized transplants. Our data demonstrate that Peg-G-CSF and G-CSF stimulation differentially affects the expression of key regulatory genes and functional properties of mobilized HSC as well as their progeny, which might be important for their application in stem cell transplantation.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    RVK:
    RVK:
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2006
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
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