Hypomethylation Therapy of Decitabine in Patients with Myelodysplastic Syndromes (MDS) Induces Apoptosis and Reduces Proliferation.

Abstract Background: MDS is characterized by cytopenias believed to be the direct result of increased apoptosis of the hematopoietic cells in the bone marrow despite an increase in proliferation. Aberrant methylation resulting in leukemogenesis is frequent in MDS and is a potential target for pharmacologic therapy. Decitabine (Dacogen™; DAC), which has been shown to be effective in treating patients with MDS (Saba et al, Kantarjian et al), indirectly depletes methylcytosine, resulting in hypomethylation of target genes. However, it is not known whether this correction of methylation leads to reducing the apoptosis, allowing normal cells to grow, or by increasing the apoptosis killing off the tumor cells. Methods: We studied apoptosis and proliferation in patients with MDS treated on a randomized protocol to receive either DAC or best supportive care (SC). Apoptosis as measured by annexin V and mitochondrial potential was studied in various subpopulations of cells using multiparameter flow cytometry. Proliferation was also measured in a similar fashion using BrdU incorporation. Bone marrow (BM) and peripheral blood (PB) samples were collected from patients at baseline and at various times on therapy. Results: At baseline, the DAC group showed no significant difference in apoptosis, proliferation or percent of CD34+ cells from the SC group in BM (n = 39 and 33 patients, respectively) or PB (n = 51 and 50, respectively). After three months on treatment a significant increase in apoptosis (annexin V) in CD34+ cells was noted in the DAC arm but not in the SC arm (Wilcoxon test, P=0.01). Similar results were obtained when disturbance in mitochondrial potential was measured in the blast population (P=0.02). Interestingly, a similar increase in apoptosis was observed in CD8+ cells in the DAC arm (P=0.02). The increase in apoptosis was augmented with additional courses of DAC therapy. A greater reduction in proliferation (BrdU incorporation) in the CD34+ cells in the decitabine arm compared to SC (P=0.01) was also observed. Those DAC-treated patients who had a higher proliferation rate (BrdU incorporation) at diagnosis were more likely to achieve a CR than those with low level of proliferation (P=0.03). Conclusions: Decitabine therapy in patients with MDS leads to hypomethylation, but the net effects include high levels of apoptosis and the death of the neoplastic cells and a complimentary reduction of proliferation of the leukemic cells.

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Genotypic Representation of Myelodysplastic/Myeloproliferative Neoplasms in Nrg, Nrg-3GS and Srg-W41 Mice with Transgenic Expression of Human Cytokines

Blood ◽

10.1182/blood-2018-99-111728 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2038-2038

Author(s):

Hein Than ◽

Naoto Nakamichi ◽

Anthony D. Pomicter ◽

John O'Shea ◽

Orlando Antelope ◽

...

Keyword(s):

Bone Marrow ◽

Myeloid Leukemia ◽

Myeloproliferative Neoplasms ◽

Model Systems ◽

Juvenile Myelomonocytic Leukemia ◽

Functional Screening ◽

Human Cd34 ◽

Cd34 Cells ◽

Significant Difference ◽

Myelomonocytic Leukemia

Abstract Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are complex clonal hematopoietic stem cell malignancies with overlapping dysplastic and proliferative features. Genomic analyses have charted the somatic mutation spectrum of MDS/MPN and revealed a major role for epigenetic dysregulation in their pathogenesis. No disease-modifying therapies are currently available, as progress has been hampered by a lack of genetically faithful in vivo model systems suitable for the preclinical development of new strategies. Yoshimi et al (Blood. 2017;130:397-407) recently showed that patients' chronic myelomonocytic leukemia (CMML) and juvenile myelomonocytic leukemia (JMML) cells transplanted into NOD/SCID-IL2Rγ-/-mice expressing human IL3, GM-CSF and SCF transgenes (NSG-3GS mice) produced xenografts that had mutations characteristic of the input cells. Since we had demonstrated a superior level of chimerism achieved from transplants of normal human CD34+cord blood cells in SirpaNOD/Rag1-/-/IL2rγc-/-/W41/41mice with c-KIT deficiency (with an otherwise mixed NOD-C57Bl/6 background - SRG-W41 mice) compared to conventional NSG or NRG hosts (Miller et al. Exp Hematol. 2017;48:41-49), it was of interest to explore their use as hosts of samples from patients with MDS/MPN: CMML, atypical chronic myeloid leukemia (aCML) and secondary acute myeloid leukemia (sAML) progressed from CMML or aCML. Heparinized blood or bone marrow samples were obtained from patients treated at Huntsman Cancer Institute after informed consent. Diagnoses included CMML (n=5), aCML (n=2), and sAML (n=2). Unseparated cells were shipped by overnight courier to Vancouver and CD34+cells isolated on the same day were injected intravenously into sub-lethally irradiated female NRG mice or SRG-W41 mice, or in some cases the same sex and strains also carrying the human 3GS transgenes (NRG-3GS or SRG-W41-3GS mice) in accordance with British Columbia Cancer Agency institutional guidelines. Occasionally when mice were not immediately available, or large numbers of cells were available, cells were viably cryopreserved and transplanted later after thawing. Mice were observed for up to 36 weeks after xenotransplantation with .05 to 1.1x106 human CD34+cells. Engraftment of human CD45+cells in xenografts was evaluated by immunophenotyping, and a median of 90% human chimerism (range: 1% - 95%) was achieved at the time of bone marrow harvest from xenografts. Variant allele frequencies (VAF) were determined in genomic DNA extracted from both the patient samples (CD34+cells) and matching fluorescence-activated cells (FACS)-sorted human CD45+cells (hCD45+cells) purified from xenografts (1-5 xenografts per patient sample). DNA samples were subjected to PCR amplification with extension primers and analyzed using a MALDI-TOF mass spectrometer (MassArray, Agena Bioscience, San Diego, CA). Each mutation call was assigned by the software based on the molecular weight of the extended primer. Analysis of hCD45+cells from eight xenograft samples so far demonstrated a strong correlation of VAF between the patient samples and hCD45+cells from xenografts, in both SRG-W41-3GS (R2=0.94, p<0.01) and NRG-3GS (R2=0.97, p<0.01) models (Figure 1). This tight correlation of VAF was illustrated in hCD45+cells from xenografts transplanted with CMML, aCML or sAML cells. The majority of mutations detected were those in epigenetic regulator genes, such as ASXL1, EZH2 and TET2. No significant difference in VAF was observed between CD34+and CD34- compartments within the hCD45+cells. Additional samples, including specimens from patients with the related myeloproliferative neoplasm, chronic neutrophilic leukemia (CNL) are being analyzed and will be presented. These findings demonstrate the utility of SRG-W41-3GS as well as NRG-3GS as receptive hosts of primary human MDS/MPN cells with genetic evidence of their growth in these mice closely recapitulating the mutational profiles of the transplanted cells. These new strains may facilitate the development of functional screening and pre-clinical testing of novel therapeutic strategies for a range of human MDS/MPN and related myeloid disorders. Disclosures Deininger: Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint: Consultancy.

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Comparative Analysis on Cellular Components of Bone Marrow Microenvironment in Normal and Aberrant Hematopoiesis

Blood ◽

10.1182/blood.v118.21.4808.4808 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4808-4808

Author(s):

Young-Ho Lee ◽

Young-hee Kwon ◽

Kyoujung Hwang ◽

Hyunju Jun ◽

Byungbae Park ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

Conflicts Of Interest ◽

T Cell Subsets ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Significant Difference ◽

Stromal Cell Derived Factor ◽

And Control ◽

Cellular Components

Abstract Abstract 4808 Background: It is now evident that hematopoietic stem cells (HSCs) reside preferentially at the endosteal region within the bone marrow (BM) where bone-lining osteoblasts are a key cellular component of the HSC niche that directly regulates HSC fate. We investigated the microenvironmental differences including osteoblastic activities and HSC components in myeloproliferative (chronic myeloid leukemia, CML) and hypogenerative disease (aplastic anemia, AA) as well as normal control (NC). Methods: The immunohistochemistry for osteonectin, osteocalcin, stromal cell derived factor (SDF, CXCL12), T cell, T helper/inducer cell, T suppressor/cytotoxic cell, hematopoietic stem/progenitor (CD34, CD117) and megakaryocytes was performed on BM biopsy specimens from 10 AA patients, 10 CML patients and 10 NC (lymphoma without BM involvement). The positive cells for immunohistochemical stainings except osteocalcin on each slide were calculated on 10 high power fields (HPF, ×400), and then corrected by the cellularity. The positive cells for osteocalcin were counted on the peritrabecular line on each slide, and then corrected by the mean length measured. Results: The CD34+ cells (p=0.012) and megakaryocytes (p<0.0001) were significantly lower in AA than in NC, but CD117+ cells was comparable in AA, CML, and control samples. The osteonectin+ cells (p=0.0003) were lower in CML than in AA and NC, however the osteocalcin+ cells showed wide variation (0-903/2035um) and no significant difference. The SDF+ cells (p<0.0001) was significantly higher in AA and very lower in CML, compared with NC. The counts for T cell and T cell subsets were significantly lower in CML than in NC, and higher in AA than in NC (p<0.0001). Conclusions: Cellular components of BM microenvironment in 2 hematologic diseases representative of myeloproliferation (CML) and hyporegeneration (AA) respectively are quite different. Further studies would be required to explore the role of these components for hematopoiesis and the rationale for therapeutic application. Disclosures: No relevant conflicts of interest to declare.

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Thymic Stromal Lymphopoeitin Signaling Contributes To Increased Risk Of Relapse Of Pediatric Acute Lympoblastic Leukemia Through Enhanced Survival Of Leukemic Cells That Overexpress Thymic Stromal Lymphopoeitin Receptor

Blood ◽

10.1182/blood.v122.21.2543.2543 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2543-2543

Author(s):

Christopher D Chien ◽

Matthew Kreitman ◽

Haiying Qin ◽

Terry J Fry

Keyword(s):

Bone Marrow ◽

Annexin V ◽

Thymic Stromal Lymphopoietin ◽

Leukemic Cells ◽

Leukemia Cells ◽

Early Stages ◽

Increased Risk ◽

Significant Difference ◽

Risk Of Relapse

Abstract Pediatric acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Although the cure rate for this disease is greater than 85%, ALL remains the number one cause of cancer-related deaths in children due to relapse of ALL. Therefore, there is a great need to identify new therapies for patients who have recurrent disease. Recently, a subset of pediatric ALL patients whose leukemic cells express high levels of thymic stromal lymphopoietin receptor (TSLPR/CRLF2) have been shown to have an increased risk of relapse and shorter disease free and poorer overall survival. Overexpression of TLSPR occurs in 8% of unscreened pediatric precursor B ALL and occurs by genomic rearrangement of the TSLPR gene, which fuses the unmutated TSLPR gene to altered transcriptional control or by other yet to be described means. The mechanism by which Thymic Stromal Lymphopoietin (TLSP) signaling contributes to increased risk of relapse is unknown. Studies have shown aberrant signaling in high TSLPR expressing ALL patient derived cell lines relying heavily on in vitro experiments. As no pre-clinical model of high TSLPR ALL has been published, we created a model of high TSLPR expressing leukemia to study TSLPR overexpressing leukemia progression. We have created a high TSLPR expressing leukemia cell line through retroviral transduction of a transplantable syngeneic mouse leukemia model in which the leukemic progression can be studied with physiologic levels of TSLP. This high TSLPR leukemia has levels of expression of TSLPR comparable to what is found on human leukemia that overexpress TSLPR. The TSLPR is functional in these cells and we see increased phosphorylation of STAT5 protein in response to IL-7 or TSLP stimulation. When we introduce the leukemia into mice and look at disease progression, we observed an 8 fold difference in the numbers of cells in the bone marrow 5 days after intravenous injection corresponding to an early stage of leukemia progression (Figure 1. high TSLPR 1.61%+/-0.95 vs. low TSLPR 0.20%+/-0.16). Interestingly we find no significant difference in long term survival of mice injected with either low or high TSLPR leukemia lines. The increased numbers of leukemic cells in the bone marrow at early stages of leukemic progression could be due to an increased rate of proliferation or better survival/engraftment. Low and high TSLPR expressing cells show no significant difference in growth rate in vitro or in vivo in dye dilution assays (Figure 2) suggesting that the increase in leukemic cells in the bone marrow is through enhanced survival. To test this, we treated low and high TSLPR leukemia lines with the steroid dexamethasone in the absence or presence of TSLP. We found that the addition of TSLP significantly reduced the Annexin V positive relative to cells not treated with TSLP in the high TSLP expressing leukemia cells, while in low TSLPR expressing cells we observed no decrease in Annexin V positive cells (Figure 3). This suggests that high TSLPR expression sensitizes leukemia cells to TSLP in the leukemia microenvironment. To confirm that this is the case we have found by gene expression analysis that we can detect TSLP in mouse bone marrow. We hypothesize that therapies targeting the TSLP signaling axis in ALL would decrease the risk of relapse. To test this hypothesis we have generated TSLPR-Fc conjugates to block TSLP signaling. We plan on using these reagents to block TSLP signaling to see if we can reverse the increased amounts of leukemia we find in mice at early stages of leukemic progression as well as the eliminate the survival advantage provided by TSLP to high TSLPR expressing leukemic cells in response to chemotherapeutic agents. Disclosures: No relevant conflicts of interest to declare.

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Abnormalities of Bone Marrow Immune Micro-Environment in Patients with Immune Thrombocytopenia

Blood ◽

10.1182/blood.v126.23.3464.3464 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3464-3464

Author(s):

Yang Song ◽

Yu-tong Wang ◽

Xiao-jun Huang ◽

Yuan Kong

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

Endothelial Cells ◽

Th17 Cells ◽

Immune Thrombocytopenia ◽

Perivascular Cells ◽

Platelet Production ◽

Cd34 Cells ◽

Significant Difference

Abstract Background: Immune thrombocytopenia (ITP) is an immune-mediated disease that is characterized by excessive platelet destruction and decreased platelet production. Although antiplatelet antibodies are considered as the primary immunologic defect in ITP, dysfunctional cellular immunity is also important in the pathophysiology of ITP. The current publications have observed excessive activation and proliferation of platelet auto-antigen-reactive CTLs, production abnormal Th cells, abnormal numbers and function of Tregs in peripheral blood of ITP, but no one focus on the bone marrow (BM) micro-environment in ITP patients. Many cell types including osteoblastic, perivascular, endothelial cells, and various mature immune cells contribute to the BM micro-environment. We have recently reported that the impaired BM vascular micro-environment may affect the thrombopoiesis of CD34+ cells by disrupting the interaction between megakaryocytes and BM endothelial cells (BMECs), resulting in the delayed platelet engraftment in allotransplant patients with prolonged isolated thrombocytopenia (Kong Y, et al. Biol Blood Marrow Transplant. 2014; 20:1190-1197). In mice model, the cross-talk between megakaryocytes and BMECs in BM vascular micro-environment regulates the megakaryocyte maturation and thrombopoiesis. Therefore, we hypothesized that the abnormal BM vascular micro-environment and immune micro-environment may operate in the occurrence of ITP. Aims: To investigate whether abnormal BM vascular and immune micro-environment are involved in ITP patients. Methods: The compartments of BM immune micro-environment were analyzed by flow cytometry in 26 untreated ITP patients and 26 healthy donors (HD). The fractions of T cells, including Th1, Tc1,Th2, Tc2 ,Th17 and Treg were identified as CD3+ CD8- IFN-gama+, CD3+ CD8- IFN-gama+, CD3+ CD8+ IL4+, CD3+ CD8+ IL-4+, CD3+ CD8- IL17A+ and CD3+ CD4+ CD25+ Foxp3+, respectively. The BMECs and perivascular cells, acting as key elements of vascular micro-environment, were identified as CD45- CD34+ VEGFR2+ and CD45- CD34- CD146+, respectively. Hematoxylin-eosin (H&E) staining and immunohistochemistry (IHC) using rabbit anti-human CD34 and CD146 primary antibodies were performed on each BM trephine biopsies (BMB) derived from the patients and controls. Results: The proportion of Th1 cells and Tc1 cells among the bone marrow mononuclear cells (BMMNCs) was significantly increased in ITP patients compared to HD (27.7% ± 11.6% vs. 16.3% ± 7.7%, P<0.001; 39.8%±17.7% vs. 24.1%±11.8%, P<0.005), whereas there was no significant difference in the percentages of Th2 and Tc2 cells. In addition, the proportion of Th17 cells in ITP patients was remarkable higher than HD (3.2%±0.51%1.5%vs 1.7%±1.0%, P<0.0001). We also found the significantly decreased percentage of Treg in ITP patients compared to HD (2.5%±2.0% vs 3.7%±2.6%, P<0.001). However, the frequency of CD34+ cells as well as BMECs and perivascular cells were similar in BM between the ITP patients and HD. Consistent with our flow cytometry data, histological analysis of the recipient BMBs in situ showed no significant differences in CD34-positive BMECs and CD146-positive perivascular cells between ITP patients and HD. Summary/Conclusion: The BM CD34+ cells and vascular micro-environment were normal in ITP patients. However, the abnormal BM immune micro-environment, including the excessive polarization of Th1, Tc1 and Th17 cells and a remarkable decrease of Treg cells were observed in ITP patients. Our data indicated that the desregulated T cells responses in BM may abrogate the thrombopoiesis through the impaired megakaryocytes maturation and decreased platelet production, and eventually contributing to the occurrence of ITP. Acknowledgment: Supported by the National Natural Science Foundation of China (grant nos. 81370638&81230013), and the Beijing Municipal Science and Technology Program (grant nos. Z141100000214011& Z151100004015164& Z151100001615020). Disclosures No relevant conflicts of interest to declare.

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Therapeutic Targets in Childhood B-Acute Lymphoblastic Leukemia : What about HER2/Neu?

Blood ◽

10.1182/blood-2020-140273 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 15-16

Author(s):

Margaux Camuset ◽

Alice Huault ◽

Audrey Grain ◽

Beatrice Clemenceau ◽

Fanny Rialland ◽

...

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Age Groups ◽

Therapeutic Targets ◽

Antigen Expression ◽

Multiparameter Flow Cytometry ◽

Significant Difference ◽

Measurable Residual Disease

Relapsed B-lineage acute lymphoblastic leukemia (ALL) in children is associated with poor prognosis. Therefore, it seems essential to stratify patients according to prognostic factors in order to adapt therapies. The evaluation of minimal/measurable residual disease (MRD) during chemotherapy constitutes the most powerful prognostic factor in all age groups, indeed, patients with early low MRD having better outcomes. New immunotherapies based on monoclonal antibodies have recently been developed and are giving promising results in chemoresistant forms with limited toxicities. Indeed, CD20, CD38, CD22 and, less developed, HER2/neu constitute therapeutic targets. However, few studies have reported on the expression level of these markers and compared it to the normal counterpart, precursor B-cells or hematogones. Such investigations would be useful to appreciate the potential efficacy of immunotherapies targeting these markers. Moreover, it could help for the detection of minimal/measurable residual disease during follow-up. A cohort of 125 B-ALL patients (1-25 years old) was retrospectively enrolled in this study, treated between January 2011 and February 2020. Samples collected from bone marrow or peripheral blood were studied in multiparameter flow cytometry (MFC). Cells were analyzed with a Canto II flow cytometer (BD Biosciences), and Diva (BD Biosciences) software was used to assess the expression of the antigenic markers. The same MFC assay had been performed over the whole period, allowing for fluorescence intensities comparison. Additionally, results from 36 normal bone marrow samples were examined, to compare the level of antigen expression by hematogones. CD20, CD38 and CD22 were expressed in respectively 53.6%, 99.2% 98.4% of the cases, rather homogeneously and at intermediate levels. The mean fluorescence intensity of CD38 was much higher on hematogones than blasts, which made it a leukemia-associated immunophenotype (LAIP) for 101 patients (81.4%). HER2/neu, a marker of breast cancer also expressed in a subset of ALL, was present in 16 samples (13.4%), but not detectable on hematogones, and can thus always be considered a LAIP. Interestingly, in this subgroup, patients had a significantly lower 5-year EFS compared to patients without expression of HER2/neu (63% versus 80.5%, p=0.02) (Figure 1). No significant difference in the expression of tested potential therapeutic markers was found between age groups. In conclusion, these 4 antigens have sufficient expression intensities to make them potential therapeutic targets, which could be an interesting alternative for the treatment of refractory/relapsed childhood B-ALL. For example, trastuzumab could be a potential immunotherapy in the HER2/neu expressing group, especially regarding their poorer prognosis. Moreover, CD38 and HER2/neu are good candidates for monitoring MRD. These results are of interest as it has been shown that monitoring MRD early was prognostic on patients' outcome. Figure Disclosures Chevallier: Incyte Corporation: Honoraria.

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Immunoexpression of CD34, CD117, and p53 in Hypocellular Bone Marrow Disorders

Journal of Laboratory Physicians ◽

10.1055/s-0041-1732491 ◽

2021 ◽

Author(s):

Pooja Sharma ◽

Anshu Palta ◽

Anita Tahlan ◽

Manveen Kaur ◽

Ram Singh

Keyword(s):

Bone Marrow ◽

Myeloid Leukemia ◽

The Other ◽

Immunohistochemical Expression ◽

Cd34 Cells ◽

Significant Difference ◽

Blast Count ◽

Bone Marrow Disorders ◽

Acute Myeloid

Abstract Objectives Hypocellular bone marrow (BM) disorders comprise heterogeneous entities associated with peripheral cytopenias and decreased production of hematopoietic cells in BM. This study was undertaken to analyze immunohistochemical expression of CD34, CD117, and p53 in morphologically diagnosed patients of hypocellular BM (aplastic anemia [AA], hypocellular myelodysplastic syndrome [h-MDS], and hypocellular acute myeloid leukemia [h-AML]). Materials and Methods BM specimens were obtained from patients presenting with pancytopenia/bicytopenia. On 30 patients diagnosed as hypocellular BM, immunohistochemistry (IHC) for CD34, CD117, and p53 was performed. Results BM cellularity was < 30% in all (100%) patients. Blast count was increased in h-MDS and h-AML. Features of dysplasia were noted in six (20%) patients. Out of these, three patients were diagnosed as h-MDS having bilineage/trilineage dysplasia, and the other three patients were of AA (11.5% patients) displaying only dyserythropoiesis. On IHC, percentage of BM CD34+ cells was increased in h-MDS+ h-AML (3.87 ± 0.86) as compared with AA (0.19 ± 0.15) and controls (0.81 ± 0.21), p = 0.01. Percentage of BM p53+ cells was also increased in h-MDS+ h-AML (2.9 ± 2.07) as compared with AA and controls, which did not show any p53+ cells, p = 0.0. No statistically significant difference was observed in the expression of CD117 in h-MDS+ h-AML (4.95 ± 3.40) compared with AA (4.49 ± 1.07), p = 0.99. Conclusion The study demonstrates the usefulness of CD34 and p53 immunoexpression as an important ancillary method in distinguishing various hypocellular BM disorders, especially h-MDS and AA. However, the role of CD117 remains unclear and needs to be evaluated further by larger studies.

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Erythropoiesis Is Highly Stimulated in CD34+ Cells in Low-Risk Myelodysplastic Syndromes (MDS) with an Improper Mitochondrial Function.

Blood ◽

10.1182/blood.v104.11.473.473 ◽

2004 ◽

Vol 104 (11) ◽

pp. 473-473

Author(s):

Ramin Tehranchi ◽

Rosangela Invernizzi ◽

Boris Zhivotovsky ◽

Bengt Fadeel ◽

Ann-Mari Forsblom ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Erythroid Differentiation ◽

Low Risk ◽

Refractory Anemia ◽

Continuous Increase ◽

Atp Production ◽

Cd34 Cells ◽

Significant Difference ◽

Cyt C

Abstract The apoptosis of early erythroblasts from patients with low-risk MDS, refractory anemia (RA) and RA with ringed sideroblasts (RARS), is mediated through a constitutive cytochrome c (cyt c) release from mitochondria (Tehranchi etal, 2003). Moreover, mature erythroblasts in RARS, but not in RA, show mitochondrial accumulation of aberrant ferritin (MtF) (Cazzola etal, 2003). This study aimed at further describing the pathophysiology of ineffective hematopoiesis in low-risk MDS, by studying cyt c release and MtF expression during erythroid differentiation and mitochondria ATP production in MDS bone marrow cells. We assessed freshly isolated CD34+ cells and day 4–14 erythroblasts from RARS, RA and normal bone marrow (NBM). CD34+ cells from all individuals were negative for MtF. NBM showed only few positive cells (0–4%, d4–14), and RA erythroblasts a median of 3% (0–8%) MtF+ cells. RARS erythroblasts, on the contrary, showed an early increase in MtF+ cells and a continuous increase during the culture period (d4=10%, d7=17%, d14=19%). There was a positive correlation between MtF expression and cyt c at day 14 ( r2=0.8). There was no significant difference in mitochondria ATP production between RARS, RA and NBM (all complexes or cyt -dependent complex IV). We found a significant over-expression (mRNA) of the pro-apoptotic genes for cyt c, Bid and Bax at day 0. Moreover, genes involved in erythroid differentiation were significantly up-regulated in MDS CD34+ cells: 6-fold for GATA-1 and 23-fold for β-globin; p<,0005 for both. GATA-1 and β-globin expression increased during normal erythroid maturation, but in MDS erythroblasts GATA-1 declined and β-globin showed only a weak increase. Comparing RARS with RA, the former showed both higher expression of the β-globin and GATA-1 genes, and a higher degree of cyt c release and MtF expression. This indicates that the cellular abnormalities leading to erythroid apoptosis as well as efforts to compensate for these defects are present at the stem cell level in RARS. G-CSF that reduces cyt c release in MDS erythroblasts (RARS>RA) showed no effect at all on ATP production or cyt c mRNA. Moreover, G-CSF tended to increase MtF expression in some RARS erythroblast cultures, indicating that it allows survival of pro-apoptotic MDS erythroblasts rather than addressing the cause of apoptosis. In conclusion, the aberrant MtF expression of RARS erythroblasts occurs at a very early stage of erythroid differentiation and is paralleled by an up-regulation of genes involved in erythroid differentiation. Alternative mechanisms may be involved in RA pathogenesis, since these cells show cyt c release but only moderate MtF expression, and less gene up-regulation.

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Recovery of Viable CD34+ Cells from Cryopreserved Haemopoietic Stem Cell Products.

Blood ◽

10.1182/blood.v104.11.5018.5018 ◽

2004 ◽

Vol 104 (11) ◽

pp. 5018-5018

Author(s):

Mary M. Sartor ◽

Frances Garvin ◽

Vicki Antonenas ◽

Melina Webb ◽

Kenneth F. Bradstock

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Cell Count ◽

Adult Stem Cell ◽

Autologous Bone Marrow ◽

Freeze Thaw ◽

Cd34 Cells ◽

Significant Difference ◽

The Mean ◽

Autologous Bone

Abstract The recovery of viable CD34+ cells reinfused into patients at the time of autologous or allogeneic transplantation is clinically an important variable, which can determine graft success or failure. In this study we analyse the recovery of viable CD34+ cells /kg pre and post cryopreservation on a total of 86 autologous stem cell products from adult and paediatric patients as well as 4 cryopreserved stem cell products from allogeneic donors. CD34 enumeration was performed on all samples pre and post cryopreservation using a novel in-house no-lyse CD34 assay (previously described ASH 2003 abstract no.1685). Cells were labelled with CD45, CD34 and 7AAD in TRUCOUNT tubes using a modified single platform ISHAGE protocol. The absolute number of viable CD34 + cells per Kg was determined. For the 77 PBSC harvest samples the mean viable CD34+ cell count was 6.0 x10^6/Kg (range 0.3 – 25.2 x 10^6/Kg) before freezing. For post thaw samples the mean viable CD34+ cell count was 5.5 x 10^6/Kg (range 0.2 – 24.6 x 10^6/Kg). The median recovery was 95% (range 48–124%). This represents a median loss post freeze/thaw of 5%. Further analysis showed a median recovery of 90% for NHL (range 48–119%, n=34), 87% for MM (range 56–115%, n=12), 92.5% for acute leukaemia (range 71–124% n=8) and 97% for non-hematological malignancies (range 50–120% n=21). There was no significant difference in the recovery of viable CD34+ cells within the four groups of malignancies (p>0.17 for all groups tested). Similarly, autologous bone marrow collections (n=9) also showed a good recovery of viable CD34+ cells post thaw. The median viable CD34+ cell count was 8.1x10^6 /Kg (range 0.6–30.3x10^6/kg) pre-cryopreservation, compared to a median viable cell count of 6.5 x10^6/Kg CD34+ cells (range 0.6–26x10^6/Kg) post thaw, this represents a median recovery of 90% viable CD34+cells from autologous bone marrow collections. There was no significant difference in the recovery of viable CD34+ cells from autologous PBSC harvests and autologous bone marrow collections (p=0.169). We also compared the recovery of viable CD34+ cells post thaw between adult and pediatric stem cells collections. The median recovery of viable CD34+ cells from 56 adult stem cell products post thaw was 91% (range 48–120%), compared to a median recovery of 96.5% (range 50–124%) viable CD34+ cells from 30 pediatric stem cell products (p=0.06). Interestingly the greatest loss occurred in allogeneic donors, where viable CD34+ counts on fresh samples averaged 5.7 x 10^6/Kg (range 3.1–11.8 x 10^6/Kg, n=4), whereas post freeze/thaw averaged 2.2 x 10^6/Kg (range 1.2–3.3 x 10^6/Kg). Representing a mean loss of 58% of CD34+ cells. Twenty-nine patients were transplanted with a median number of 3.8x10^6 viable CD34+ cells per Kg (range 1.8–18.4x10^6/Kg), The median time to neutrophil and platelet engraftment was 12 days (range 10–18) and 14 days (range 8–65) respectively. Assaying the viability of CD34+ cells post cryopreservation may identify patients at risk of poor haematological recovery that could benefit from further stem cell collections.

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Ineffective Hematopoiesis in Megaloblastic Anemia Is Associated with Higher Frequency of Apoptosis in Each Lineage Immature and Mature Cells.

Blood ◽

10.1182/blood.v106.11.3601.3601 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3601-3601

Author(s):

Tatsuo Oyake ◽

Shigeki Ito ◽

Shugo Kowata ◽

Kazunori Murai ◽

Yoji Ishida

Keyword(s):

Bone Marrow ◽

Mononuclear Cells ◽

Bone Marrow Cells ◽

Megaloblastic Anemia ◽

Flow Cytometric Analysis ◽

Annexin V ◽

Cell Number ◽

Color Flow ◽

Cd34 Cells ◽

Normal Controls

Abstract A deficiency of Vitamin B12 is a major cause of megaloblastic anemia (MBA). Ineffective hematopoiesis is observed in MBA, characterized by cytopenia, bone marrow cells with dysplastic change and normal to hypercellularity. We reported that excessive apoptosis of each lineage CD34(+) cells was observed in myelodysplastic syndrome (MDS) in the last ASH meeting. In this study, we investigated the hypothesis that excessive apoptosis induced the ineffective hematopoiesis in MBA. We performed the three color flow cytometric analysis of bone marrow mononuclear cells in 12 MBA patients using PE labeled Annexin V, PerCP labeled anti-CD34 antibody and FITC labeled anti-each lineage antibody (anti-glycophorin A (GPA) antibody, anti-CD33 antibody and anti-CD41 antibody). The frequency of apoptosis in subpopulations of immature (CD34(+)) and each lineage (+) cells or those of mature (CD34(−)) and each lineage (+) cells were calculated as the ratio (%) of (cell number with Annexin V(+)) divided by (cell number in the subpopulation). The subpopulations include CD34(+)GPA(+) (immature erythroid), CD34(+)CD33(+) (immature myeloid), CD34(+)CD41(+) (immature megakaryocytic), CD34(−)GPA(+) (mature erythroid), CD34(−)CD33(+) (mature myeloid) and CD34(−)CD41(+) (mature megakaryocytic) cells. Much higher frequency of apoptosis was observed in each lineage CD34(+) cells in MBA (median: 23.8% (range: 10.8–43.6%) in erythroid, 43.5% (12.7–67.3%) in myeloid, 50.1% (21.0–64.1%) in megakaryocytic lineages, P< 0.05, respectively, n=12), compared to those in normal controls (8.5% (1.5–9.9%) in erythroid, 8.5% (2.2–8.8%) in myeloid, 7.7% (4.4–9.3%) in megakaryocytic lineages, respectively, n=10). While, the relatively higher frequency of apoptosis was observed in each lineage CD34(−) cells in MBA patients (median: 15.9% (range: 5.1–20.6%) in erythroid, 16.4% (5.6–23.2%) in myeloid, 16.1% (10.2–24.8%) in megakaryocytic lineages, P< 0.05, respectively, n=12), compared to those in normal controls (4.8% (1.3–6.6%) in erythroid, 2.2% (0.6–4.4%) in myeloid, 3.3% (1.5–7.1%) in megakaryocytic lineages, respectively, n=10). These results suggest that the excessive apoptosis occurs not only in CD34(+) but also in CD34(−) cells, which induces ineffective hematopoiesis in MBA. Figure 1. The frequency of apoptosis in CD34+ BM cells Figure 1. The frequency of apoptosis in CD34+ BM cells Figure 2. The frequency of apoptosis in CD34− BM cells Figure 2. The frequency of apoptosis in CD34− BM cells

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Survivin Is Required for Proliferation, but Not Polyploidization of Megakaryocytes.

Blood ◽

10.1182/blood.v112.11.1328.1328 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1328-1328

Author(s):

Jeremy Q Wen ◽

Cindy Leung ◽

Zan Huang ◽

Sara Small ◽

John Crispino

Keyword(s):

Bone Marrow ◽

Ex Vivo ◽

Survivin Expression ◽

Annexin V ◽

Retroviral Infection ◽

Hematopoietic Stem ◽

Significant Difference ◽

Megakaryocyte Progenitors

Abstract Survivin is a member of chromosome passenger complex, which plays an important role in chromosome alignment, separation and cytokinesis. We recently reported that survivin is necessary for the proliferation and survival of hematopoietic stem and progenitor cells. Furthermore, we previously showed that reduced levels of survivin expression facilitates megakaryocyte development, whereas elevated levels of survivin inhibit their maturation and polyploidization. However, the extent to which survivin is necessary for polyploidization and terminal differentiation of committed megakaryocytes remains unclear. To determine whether survivin is required for megakaryocyte and platelet biogenesis, we mated mice with floxed alleles of survivin (sur fl/fl) to mice that express Cre recombinase under the control of the PF4 promoter. Compound mutant animals appeared grossly normal and harbored normal platelet counts. Furthermore, survivin deleted and control littermates displayed similar expression of CD41 and CD42, as well as similar DNA content within the CD41+ population. The only significant difference detected was an increase in annexin V staining of CD41+ cells within the bone marrow of the mice with survivin deletion. Analysis of DNA extracted from these bone marrows showed no evidence of the survivin deletion, indicating that the surviving cells all escaped excision. These in vivo findings are consistent with a requirement for survivin in the survival or proliferation of megakaryocyte progenitors. Next, to induce megakaryocyte development ex vivo, we cultured bone marrow from surv fl/fl mice in vitro in the presence of TPO. Using this approach, we were able to induce survivin deletion in 75% of the cells as evidenced by PCR. Despite the deletion of survivin, polyploidization of the ex vivo generated megakaryocytes was unaffected. Finally, we induced deletion of survivin by retroviral infection of surv fl/fl progenitors with MSCV-Cre and found that megakaryocyte polyploidization was actually increased in the excised population. Taken together, our results suggest that survivin is not required for polyploidization, but is necessary for proliferation of megakaryocyte progenitors.

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