The Impact of NFAT Inhibition on Neutrophil Antifungal Defense and Myelopoiesis in Cyclosporine A Treated and NFATc1LysM Mice

Abstract Background and Aims: Immunodeficient patients after allogeneic stem cell transplantation (HSCT) are heavily threatened by opportunistic fungal infections like invasive pulmonary aspergillosis (IPA), partly due to immunosuppressive medication e.g. by calcineurin inhibitors like cyclosporine A (CsA) or tacrolimus. It is well known that the nuclear factor of activated T cells (NFAT) is an important transcription factor downstream of calcineurin in the adaptive immune system especially in T cells. Additionally, there is a growing body of evidence that NFAT also plays a substantial role in innate immune response against invasive fungal diseases by polymorphonuclear neutrophils (PMN), as well as in regulation of myelopoiesis and myeloid differentiation, as indicated by recent data in rodent models. Methods: Firstly, we used a murine IPA model (C57BL/6) to clarify the role of NFAT in antifungal innate immune response in vivo. To do so, we treated mice intraperitoneally with CsA (18mg/kg/d) or vehicle for 2 weeks and challenged them with Aspergillus fumigatus (A. f.) conidia intratracheally. 24 hours later, some mice were sacrificed and PMN recruitment to the lungs and pulmonary fungal clearance were examined by analyzing bronchoalveolar lavages (BAL) and peripheral blood (PB) by flow cytometry and murine lungs by fungal culture assays and histopathologic examination. In addition, survival of remaining infected mice was studied with neutropenic animals (by depletion with anti-Gr1) serving as positive controls. Secondly, LysM-specific NFATc1 knockout (NFATc1LysM) mice were bred lacking NFATc1 expression solely in myelomonocytic cells (i.e. PMN and monocytes). Furthermore, these animals were infected with A. f. and analyzed as described above. Secondly, we investigated myelopoiesis and myeloid differentiation under steady state conditions by quantifying bone marrow derived myeloid progenitor cells from CsA treated or NFATc1LysM mice using flow cytometry and simultaneously counting PMN in PB. Results: While the infection was lethal in CsA or vehicle treated neutropenic mice, all CsA or vehicle treated mice survived the infection. CsA treated mice showed enhanced PMN recruitment in BAL by trend (55.2% +/- 12.0 (CsA) vs. 33.7% +/- 8.0 (control), mean +/- SEM, p=0.053), whereas pulmonary inflammation and PMN counts in PB were comparable to controls. In contrast, fungal clearance was clearly impaired in animals after CsA treatment (2.1 x 105 CFU/lung after 48 hours +/- 0.5 (CsA) vs. 1.7 x 105 +/- 0.2 (control), p<0.005). Along with that, NFATc1LysM mice infected with A. f. showed unimpaired survival. However, there were no detectable differences in PMN recruitment or fungal clearance, whereas pulmonary inflammation and PMN counts in PB seemed to be more pronounced in knockout mice (1.0 inflammation points/lung +/- 0.12 (NFATc1LysM) vs. 0.7 +/- 0.07 (control), p=0.057; 1.5 x 103 PMN/µl +/- 0.2 (NFATc1LysM) vs. 0.9 +/- 0.1 (control), p=0.036). Distribution of bone marrow derived murine myeloid progenitor cells was unaffected through NFAT inhibition by CsA but clearly impaired in NFATc1LysM mice especially in megakaryocyte-erythroid progenitor cells (1.2 x 105 cells +/- 0.2 (NFATc1LysM) vs. 2.7 +/- 0.6, p=0.015) whereas PMN blood counts in PB were unchanged. Conclusions: In a mouse model, NFAT inhibition via treatment with CsA does not influence survival after infection with A. f. in vivo but affects PMN recruitment and local fungal clearance. To some extent this may be due to impaired PMN phagocytic and migratory capabilities as indicated by our in vitro and ex vivo studies (data not shown). However, solely NFATc1 downregulation in PMN apparently results in slightly different effects given that infected NFATc1LysM mice displayed enhanced pulmonary inflammation and elevated PMN blood counts compared to controls. Additionally, NFATc1 inhibition in NFATc1LysM mice leads to constrained myelopoiesis under steady state conditions without affecting peripheral PMN blood counts compared to untreated wild type controls. Further studies are needed to clarify underlying mechanisms and clinical relevance in HSCT of our findings. Disclosures No relevant conflicts of interest to declare.

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The Impact of NFAT Inhibition on Neutrophil Effector Functions in Patients after Allogeneic Hematopoietic Stem Cell Transplantation and on Neutrophil Antifungal Defense and Myelopoiesis in Cyclosporin Α Treated and NFATc1LysM Mice

Blood ◽

10.1182/blood.v128.22.3679.3679 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3679-3679

Author(s):

Daniel Teschner ◽

Katharina Plein ◽

Christian Michel ◽

Steve Pruefer ◽

Matthias Bros ◽

...

Keyword(s):

Progenitor Cells ◽

Ex Vivo ◽

Pulmonary Inflammation ◽

Blood Samples ◽

Effector Functions ◽

Myeloid Progenitor ◽

Healthy Donors ◽

Myeloid Progenitor Cells

Abstract Background and Aims: Immunosuppressive medication e.g. by calcineurin inhibitors substantially contributes to the risk for opportunistic fungal infections in patients after allogeneic transplantation (HSCT). It is well known that the nuclear factor of activated T cells (NFAT) is an important transcription factor downstream of calcineurin especially in T cells. Additionally, recent data in rodent models indicate that NFAT also seems to play a relevant role in innate antifungal immune responses by polymorphonuclear neutrophils (PMN), as well as in regulation of myelopoiesis and myeloid differentiation. Methods: Firstly, isolated PMN from healthy donors were analyzed in vitro in absence or presence of CsA regarding their effector functions and activation-induced release of inflammatory mediators. Consecutively, blood samples of CsA-treated patients after allogeneic HSCT (n=17) and healthy donors (n=8) were analyzed ex vivo at two different time points as described above. Secondly, we used a murine IPA model (C57BL/6) and treated mice with CsA (18 mg/kg/d) or vehicle and challenged them with Aspergillus fumigatus (A. f.) conidia intratracheally. PMN recruitment to the lungs and pulmonary fungal clearance were examined by analyzing bronchoalveolar lavages (BAL) and peripheral blood (PB) using flow cytometry and cytometric bead array and murine lungs by fungal culture assays and histopathologic examination. Furthermore, survival was studied with neutropenic animals serving as positive controls. Moreover, LysM-specific NFATc1 knockout (NFATc1LysM) mice were bred lacking NFATc1 expression solely in myelomonocytic cells. These animals were also infected with A. f. and analyzed as further mentioned. In addition, we investigated myelopoiesis and myeloid differentiation by quantifying bone marrow derived myeloid progenitor cells from CsA treated or NFATc1LysMmice using flow cytometry and simultaneously counting PMN in PB under steady state conditions. Results: CsA enhanced phagocytosis of PMN in vitro and ex vivo in patients' blood samples (54.2 % +/- 4.1 (patients) vs. 43.8 +/- 1.5, LPS, p=0.006). Moreover, PMNs migratory capabilities were reduced in vitro, whereas other effector functions or release of IL-8 were rather unaffected. PMNs of CsA-treated patients showed increased activation, degranulation and production of inflammatory mediators, but production of ROS was slightly decreased. In our in vivo model, IPA was lethal in neutropenic mice whereas solely CsA or vehicle treated mice survived the infection. CsA treatment resulted in enhanced PMN recruitment in BAL by trend, while pulmonary inflammation and PMN counts in PB remained stable. Indeed, fungal clearance was clearly constrained in CsA　treated animals (2.1 x 105 CFU/lung +/- 0.5 (CsA) vs. 1.7 x 105 +/- 0.2, p<0.005). In our murine knockout model, NFATc1LysM mice infected with A. f. showed unimpaired survival without displaying detectable differences in PMN recruitment or fungal clearance. However, pulmonary inflammation and PMN counts in PB seemed to be more pronounced in knockout mice. Interestingly, BALs of CsA treated mice showed increased levels of IL-6 by trend (4634 pg/mL +/- 1073 (CsA) vs. 3108 +/- 729, p=0.48) but decreased levels of MCP-1 and TNF-α. In contrast, MCP-1, RANTES and TNF-α were enhanced by trend in BALs of NFATc1LysM mice, while IL-6 was reduced compared to wild type controls (3762 pg/mL +/- 729 vs. 4770 +/- 1613, p=0.81). PMN counts in PB were unaffected in NFATc1LysM mice but distribution of bone marrow derived murine myeloid progenitor cells was clearly impaired especially in megakaryocyte-erythroid progenitor cells (1.2 x 105 cells +/- 0.2 (NFATc1LysM) vs. 2.7 +/- 0.6, p=0.015), whereas solely CsA treatment had no influence. Conclusion: Results from our in vitro and ex vivo studies on patients' blood samples as well as from our murine in vivo IPA model indicate that NFAT regulates not only myelopoiesis, but also PMN functionalities in mice and humans. Nevertheless, these interactions are obviously multidimensional and potentially derive from involvement of different pathways. The underlying molecular mechanisms and clinical relevance of our findings in HSCT remain to be determined. Disclosures No relevant conflicts of interest to declare.

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STAT3 Deletion after Birth Results in Marked Decreases in Absolute Numbers and Cell Cycling Status of Marrow and Spleen Myeloid Progenitor Cells, and STAT3 Is a Critical Molecule in Mediating Synergistically Stimulated Progenitor Cell Proliferation.

Blood ◽

10.1182/blood.v106.11.1346.1346 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1346-1346

Author(s):

Hal E. Broxmeyer ◽

Scott Cooper ◽

Giao Hangoc ◽

Wenjun Zhang ◽

Akira Moh ◽

...

Keyword(s):

Bone Marrow ◽

Growth Factors ◽

Mouse Model ◽

Progenitor Cells ◽

Ample Evidence ◽

Gm Csf ◽

Myeloid Progenitor ◽

Myeloid Progenitor Cells

Abstract STAT3 is an important transcription factor involved in mediating intracellular signals initiated at the cell membrane by cytokines and growth factors. There is ample evidence that STAT3 acts as a positive regulator of cell growth, but most of this information derives from studies done with isolated cells in vitro. Because functional deletion of STAT3 in mice is lethal, it was difficult to evaluate a role for STAT3 in mediating hematopoietic effects in vivo in mice after birth. To address this problem, a unique strain of mice was developed with tissue specific disruption of STAT3 in bone marrow and hematopoietic cells (Welte et.al. PNAS100: 1879, 2003). The availability of this conditional STAT3 −/− mouse model demonstrated a critical role for STAT3 in innate immunity. We have now utilized this conditional STAT3 −/− mouse model to evaluate a role for STAT3 in hematopoiesis after birth, with the hypothesis that STAT3 would be one critical factor involved in the proliferation of myeloid progenitor cells (MPC: CFU-GM, BFU-E, CFU-GEMM) in bone marrow and spleen. STAT3 −/− and their littermate control mice were evaluated at 4 weeks of age. STAT3 −/− mice manifested 40–44% decreases in absolute numbers of nucleated cells in the marrow (femur) and spleen. This was associated with decreased absolute numbers of CFU-GM (70%), BFU-E (70%) and CFU-GEMM (50%) per femur and CFU-GM (50%), BFU-E (30%), and CFU-GEMM (50%) per spleen for these MPC which are responsive in vitro to stimulation of colony formation by the combination of EPO, SCF, TPO and growth factors in PWMSCM. Moreover, MPC from STAT3 −/− mice were in a slow or non cycling state (0–4% MPC in S-phase) in marrow and spleen compared to 50–60% marrow and 32–48% spleen MPC from +/+ mice being in active cell cycle. There were also large decreases per femur in STAT3 −/− mice in terms of GM-CSF-, IL-3-, M-CSF-, GM-CSF plus SCF-, GM-CSF plus Flt3 ligand (FL)-, IL-3 plus SCF-, IL-3 plus FL-, M-CSF plus SCF-, and M-CSF plus FL- responsive CFU-GM. These decreases may in part reflect the finding that CFU-GM from STAT3 −/− mice did not respond to the synergistic proliferation effects of GM-CSF, IL-3, or M-CSF, each in combination with either SCF or FL. At best these cytokine combinations resulted in additive proliferative effects on MPC from marrow of STAT3 −/− mice in contrast to CFU-GM from +/+ mouse marrow where the effects were clearly synergistic. In terms of survival of MPC, there were no apparent differences between the survival of MPC from STAT3 −/− and +/+ mice after withdrawal of growth factors in vitro and their delayed addition to the cell cultures. MPC from STAT3 −/− and +/+ marrow responded similarly to the survival enhancing effects in vitro of SDF-1/CXCL12. Our results demonstrate that after birth STAT3 acts as a positive mediator of the proliferation of MPC in vivo, and STAT3 is a critical mediator of the synergistic proliferation effects of cytokines on MPC.

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Bone marrow lymphoid and myeloid progenitor cells are suppressed in 7,12-dimethylbenz(a)anthracene (DMBA) treated mice

Toxicology ◽

10.1016/j.tox.2010.02.009 ◽

2010 ◽

Vol 271 (1-2) ◽

pp. 27-35 ◽

Cited By ~ 11

Author(s):

A.U. N’jai ◽

M. Larsen ◽

L. Shi ◽

C.R. Jefcoate ◽

C.J. Czuprynski

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Myeloid Progenitor ◽

Myeloid Progenitor Cells

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The influence in vivo of murine colony-stimulating factor-1 on myeloid progenitor cells in mice recovering from sublethal dosages of cyclophosphamide

Blood ◽

10.1182/blood.v69.3.913.913 ◽

1987 ◽

Vol 69 (3) ◽

pp. 913-918 ◽

Cited By ~ 13

Author(s):

HE Broxmeyer ◽

DE Williams ◽

S Cooper ◽

A Waheed ◽

RK Shadduck

Keyword(s):

Progenitor Cells ◽

Colony Formation ◽

Colony Stimulating Factor ◽

Myeloid Progenitor ◽

Accessory Cells ◽

Myeloid Progenitor Cells ◽

In Vivo Administration ◽

Stimulating Factor

Abstract Pure murine colony-stimulating factor-1 (CSF-1) was assessed for its effects in vivo in mice pretreated seven days earlier with a sublethal dosage of cyclophosphamide. The multipotential (CFU-GEMM), erythroid (BFU-E), and granulocyte-macrophage (CFU-GM) progenitor cells in these mice were in a slowly cycling or noncycling state. Intravenous administration of 20,000 units of CSF-1 to these mice stimulated the hematopoietic progenitors into a rapidly cycling state in the marrow and spleen within three hours. Significant increases in absolute numbers of marrow and spleen CFU-GM and spleen BFU-E and CFU-GEMM were also detected. No endotoxin was detected in the CSF-1 preparation by Limulus lysate assay, and treatment of CSF-1 at 100 degrees C for 20 to 30 minutes completely inactivated the in vitro and in vivo stimulating effects. The effects of CSF-1 were not mimicked by the in vivo administration of 0.1 to 10 ng Escherichia coli lipopolysaccharide. These results suggest that the effects of CSF-1 in vivo were not due to contaminating endotoxin or to a nonspecific protein effect. CSF-1 did not enhance colony formation by BFU-E or stimulate colony formation by CFU-GEMM in vitro, thus suggesting that at least some of the effects of CSF-1 noted in vivo are probably indirect and mediated by accessory cells.

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Human bone marrow depleted of CD33-positive cells mediates delayed but durable reconstitution of hematopoiesis: clinical trial of MY9 monoclonal antibody-purged autografts for the treatment of acute myeloid leukemia

Blood ◽

10.1182/blood.v79.9.2229.2229 ◽

1992 ◽

Vol 79 (9) ◽

pp. 2229-2236 ◽

Cited By ~ 69

Author(s):

MJ Robertson ◽

RJ Soiffer ◽

AS Freedman ◽

SL Rabinowe ◽

KC Anderson ◽

...

Keyword(s):

Monoclonal Antibody ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Progenitor Cells ◽

Myeloid Leukemia ◽

Red Blood Cell Transfusion ◽

Hematopoietic Stem ◽

Myeloid Progenitor ◽

Myeloid Progenitor Cells ◽

Acute Myeloid

Abstract The CD33 antigen, identified by murine monoclonal antibody anti-MY9, is expressed by clonogenic leukemic cells from almost all patients with acute myeloid leukemia; it is also expressed by normal myeloid progenitor cells. Twelve consecutive patients with de novo acute myeloid leukemia received myeloablative therapy followed by infusion of autologous marrow previously treated in vitro with anti-MY9 and complement. Anti-MY9 and complement treatment eliminated virtually all committed myeloid progenitors (colony-forming unit granulocyte- macrophage) from the autografts. Nevertheless, in the absence of early relapse of leukemia, all patients showed durable trilineage engraftment. The median interval post bone marrow transplantation (BMT) required to achieve an absolute neutrophil count greater than 500/microL was 43 days (range, 16 to 75), to achieve a platelet count greater than 20,000/microL without transfusion was 92 days (range, 35 to 679), and to achieve red blood cell transfusion independence was 105 days (range, 37 to 670). At the time of BM harvest, 10 patients were in second remission, one patient was in first remission, and one patient was in third remission. Eight patients relapsed 3 to 18 months after BMT. Four patients transplanted in second remission remain disease-free 34+, 37+, 52+, and 57+ months after BMT. There was no treatment-related mortality. Early engraftment was significantly delayed in patients receiving CD33-purged autografts compared with concurrently treated patients receiving CD9/CD10-purged autografts for acute lymphoblastic leukemia or patients receiving CD6-purged allografts from HLA- compatible sibling donors. In contrast, both groups of autograft patients required a significantly longer time to achieve neutrophil counts greater than 500/microL and greater than 1,000/microL than did patients receiving normal allogeneic marrow. CD33(+)-committed myeloid progenitor cells thus appear to play an important role in the early phase of hematopoietic reconstitution after BMT. However, our results also show that human marrow depleted of CD33+ cells can sustain durable engraftment after myeloablative therapy, and provide further evidence that the CD33 antigen is absent from the human pluripotent hematopoietic stem cell.

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Involvement of p21cip-1 and p27kip-1 in the molecular mechanisms of steel factor-induced proliferative synergy in vitro and of p21cip-1 in the maintenance of stem/progenitor cells in vivo

Blood ◽

10.1182/blood.v88.10.3710.bloodjournal88103710 ◽

1996 ◽

Vol 88 (10) ◽

pp. 3710-3719 ◽

Cited By ~ 82

Author(s):

C Mantel ◽

Z Luo ◽

J Canfield ◽

S Braun ◽

C Deng ◽

...

Keyword(s):

Progenitor Cells ◽

Proliferative Response ◽

Myeloid Cell ◽

Cyclin Dependent Kinase ◽

Hematopoietic Progenitor ◽

Gm Csf ◽

Myeloid Progenitor ◽

Myeloid Progenitor Cells

Steel factor (SLF) is a hematopoietic cytokine that synergizes with other growth factors to induce a greatly enhanced proliferative state of hematopoietic progenitor cells and factor-dependent cell lines. Even though the in vivo importance of SLF in the maintenance and responsiveness of stem and progenitor cells is well documented, the molecular mechanism involved in its synergistic effects are mainly unknown. Some factor-dependent myeloid cell lines respond to the synergistic proliferative effects of SLF plus other cytokines in a manner similar to that of normal myeloid progenitor cells from bone marrow and cord blood. We show here that SLF can synergize with granulocyte-macrophage colony-stimulating factor (GM-CSF) to induce an enhanced phosphorylation of the retinoblastoma gene product and a synergistic increase in the total intracellular protein level of the cyclin-dependent kinase inhibitor, p21cip-1, which is correlated with a simultaneous decrease in p27kip-1 in the human factor-dependent myeloid cell line, M07e. Moreover, these cytokines synergize to increase p21cip- 1 binding and decrease p27kip-1 binding to cyclin-dependent kinase-2 (cdk2), an enzyme required for normal cell cycle progression; these inverse events correlated with increased cdk2 kinase activity. It is also shown that exogenous purified p21cip-1 can displace p27kip-1 already bound to cdk2 in vitro. These data implicate increased p21cip-1 and decreased p27kip-1 intracellular concentrations and their stoichiometric interplay in the enhanced proliferative status of cells stimulated by the combination of SLF and GM-CSF. In support of these findings, it is shown that hematopoietic progenitor cells from mice lacking p21cip-1 are defective in SLF synergistic proliferative response in vitro. Moreover, the cycling status of marrow and spleen progenitors and absolute numbers of marrow progenitors were significantly decreased in the p21cip-1 -/-, compared with the +/+ mice. We conclude that the cdk threshold regulators p21cip-1 and p27kip- 1 play a critical role in the normal mitogenic response of M07e cells and murine myeloid progenitor cells to these cytokines and particularly in the SLF synergistic proliferative response that is important to the normal maintenance of the stem/progenitor cell compartment.

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Diversity and Molecular Modeling of Neutrophil Elastase Mutations in Patients with Severe Congenital Neutropenia and Acute Myelogenous Leukemia.

Blood ◽

10.1182/blood.v104.11.1454.1454 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1454-1454

Author(s):

Andrew A.G. Aprikyan ◽

Steve Stein ◽

Nara A. Markosyan ◽

Maxim Totrov ◽

Ruben Abagyan ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Neutrophil Elastase ◽

Apoptotic Cell ◽

Myelogenous Leukemia ◽

Congenital Neutropenia ◽

Deletion Mutations ◽

Myeloid Progenitor ◽

Myeloid Progenitor Cells ◽

Acute Myelogenous

Abstract Severe congenital neutropenia (SCN) is an inheritable hematopoietic disorder that is characterized by extremely low levels of neutrophils in peripheral circulation and maturation arrest of bone marrow myeloid progenitor cells at the promyelocytic stage of differentiation. SCN patients have recurring severe infections and approximately 10% of these patients evolve to develop acute myelogenous leukemia. Recently we reported that an impaired cell survival and cell cycle arrest of bone marrow myeloid progenitor cells was observed in SCN patients compared with controls. We also reported various heterozygous mutations in the neutrophil elastase (NE) gene encoding a serine protease in approximately 80% of SCN patients. We hypothesized that mutations in the NE gene trigger apoptotic cell death of myeloid progenitor cells and subsequent severe neutropenia. Mutational analysis of 15 families with one or more affected family members revealed that mutant NE was present only in affected but not in healthy members of these families suggesting the causative role for mutant NE in pathogenesis of SCN. Sequencing analysis revealed that none of SCN patients negative for NE mutations examined had mutations in the Gfi-1 or WAS gene. Sequencing DNA samples of SCN and SCN/AML patients revealed 40 mutations that are distributed primarily throughout the exons 2 through 5 of the NE gene and result in substitution, deletion, insertion, or truncation mutations. Molecular modeling of the tertiary structure of NE revealed that all these mutations can be grouped into three major categories. The first category includes 19 substitution and insertion mutations that are grouped around the N-glycosylation sites of the neutrophil elastase and may lead to abnormal targeting and subcellular localization of the mutant protease. The second group includes 9 substitution and deletion mutations that alter the side loop of the NE that is necessary for proper oligomerization of neutrophil elastase. The third category includes 12 substitution, truncation, and deletion mutations that either alter or completely eliminate the carboxy-terminus of the mutant protein leading to conformational changes of the binding pocket of the NE, and subsequently to altered substrate specificity and/or an acquired resistance to elastase inhibitors. SCN patients that evolved to develop AML had either substitution, deletion, or truncation mutations from each of the three categories described above. Most mutations are clearly non-conservative, have destabilizing effect on oligomeric structure of mutant protein, and alter dramatically the affinity of mutant NE to various factors participating in its processing and intracellular transport. Flow cytometry analysis of annexin V-labeled cells revealed that expression of representative mutant but not normal NE from each of the three categories of NE mutations in human promyelocytic HL-60 cells triggered apoptotic cell death similar to that observed in bone marrow progenitor cells in SCN patients. These data indicate that impaired cell survival and block of differentiation in SCN is due to heterozygous mutations in the neutrophil elastase gene. Current studies focused on design and screen of specific protease inhibitors capable of blocking the pro-apoptotic effect of mutant neutrophil elastase.

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Peripheral Blood Cytogenetic Studies in Hematological Neoplasms: Predictors of Obtaining Metaphases for Analysis.

Blood ◽

10.1182/blood.v110.11.3504.3504 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3504-3504

Author(s):

Kebede Hussein ◽

Rhett P. Ketterling ◽

Gordon W. Dewald ◽

Rachael L. Hulshizer ◽

Daniel G. Kuffel ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Clinical Diagnosis ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Cytogenetic Study ◽

Lymphocytic Leukemia ◽

Myeloid Progenitor ◽

Cytogenetic Studies ◽

Myeloid Progenitor Cells

Abstract Background: Peripheral blood (PB) is sometimes used in place of bone marrow (BM) for cytogenetic studies during the evaluation of hematologic malignancies. We looked for clinical or laboratory features that predict success in obtaining analyzable metaphases during PB chromosome studies. Methods: The Mayo Clinic cytogenetics database was queried to identify adult cases (age > 18 years) with suspected or established hematologic neoplasm in whom PB cytogenetic studies were performed. Success defined as the acquisition of at least two metaphases, was correlated with clinical and laboratory information corresponding to the time of the PB cytogenetic study. Results: A total of 242 PB cytogenetic studies were performed: clinical diagnosis was a myeloid neoplasm in 169 patients (70%), lymphoid neoplasm in 50 (21%), and unexplained cytopenia or leukocytosis in 23 (9%). The 169 myeloid cases included 59 patients with either primary (n=39) or post-polycythemia vera/essential thrombocythemia (post-PV/ET MF) myelofibrosis (n=20), 42 with acute myeloid leukemia (AML), 15 with chronic myeloid leukemia, 9 with myelodysplastic syndrome (MDS), 8 with ET, 6 with PV, and 30 with other MPDs. The 50 lymphoid cases included 19 with chronic lymphocytic leukemia, 12 with lymphoma, 11 with acute lymphocytic leukemia (ALL), and 8 with plasma cell proliferative disorders. PB cytogenetic studies resulted in at least two analyzable metaphases (median 20, range 2–31) in 142 of the 242 study cases (59%); in univariate analysis, this was predicted by the specific clinical diagnosis (p<0.0001), presence and degree of circulating myeloid progenitor cells (p<0.0001), higher leukocyte count (p<0.001), lower platelet count (p=0.003), lower hemoglobin level (p=0.002), and presence of palpable splenomegaly (p=0.002). In multivariable analysis, only the presence of circulating myeloid progenitor cells sustained its significance and this was consistent with the high yield rates seen in PMF (80%), post-PV/ET MF (85%), AML (76%), and ALL (80%) as opposed to the low rates seen in ET (0%) and PV (2%). In 104 cases, BM cytogenetic studies were performed within one month of the PB cytogenetic studies; an abnormal BM cytogenetic finding was another independent predictor of a successful PB study (p=0.002). Conclusion: PB cytogenetic studies are most appropriate in diseases characterized by presence of circulating myeloid progenitors or blasts (e.g. PMF, AML, ALL); the yield otherwise is too small to be cost-effective. The current study also suggests a higher likelihood of a successful PB cytogenetic study in the presence of an abnormal bone marrow karyotype.

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Post-Induction-BM Status Combining Minimal Residual Disease, Return to MDS and Hematogones in Predicting Prognosis in Adult AML,

Blood ◽

10.1182/blood.v118.21.3532.3532 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3532-3532

Author(s):

Sung-Chao Chu ◽

Tso-Fu Wang ◽

Yu-Chieh Su ◽

Ruey-Ho Kao ◽

Yi-Fung Wu ◽

...

Keyword(s):

Flow Cytometry ◽

Progenitor Cells ◽

Minimal Residual Disease ◽

Residual Disease ◽

Prognostic Impact ◽

Post Induction ◽

Myeloid Progenitor ◽

Flow Cytometric ◽

Myeloid Progenitor Cells ◽

Minimal Residual

Abstract Abstract 3532 Introduction: The study is to analyze the prognostic impact of post-induction-BM status combining minimal residual disease (MRD), neutrophil/monocyte maturation return to MDS and quantitation of hematogones in adult AML achieving first morphologic complete remission (mCR). The hypothesis is that the detection of aberrant myeloid progenitor cells, aberrant neutrophil/monocyte maturation and hematogones in post-induction BM by flow cytometry may predict the outcome of AML in mCR even without diagnostic specimen. The positive MRD and the aberrant neutrophil/monocyte maturation will predict worse prognosis. In contrast, positive hematogones will predict better prognosis. Methods: Multidimensional flow cytometry was performed on bone marrow specimens from 47 consecutive non-M3 AML patients who had achieved mCR after standard 3+7 induction treatment. The in-remission immunophenotypic evaluation of BM was done before first consolidation treatment. The aberrant myeloid progenitor cells and aberrant neutrophil/monocyte maturation were defined by the flow cytometric scoring system (FCSS)1. Two investigators were blinded to corresponding pathologic, clinical or diagnostic flow cytometric data during initial analysis phase. After reaching agreement of the three parameters: aberrant myeloid progenitor cells (MRD ≥ 0.2% or not), aberrant neutrophil/monocyte maturation (FCSS ≥ 2 points or not) and hematogones (stages I / II B lymphoid progenitor cells ≥ 0.02% or not), those prognostic impacts on leukemia free survival (LFS) and overall survival (OS) were analyzed retrospectively. Results: Table 1 summarizes the clinical characteristics of patients according to the status of MRD and hematogones. Nine (19%) patients who had MRD ≥0.2% had a significantly worse median LFS (9.0 months vs not reached; P =.006) and worse OS (24.0 months vs not reached; P =.059). Fourteen (30%) patients who had hematogone levels ≥ 0.02% had a significantly better median LFS (not reached vs 13.5 months; P =.045) and a trend of better OS (not reached vs 24.0 months; P =.070). Six (13%) patients who had FCSS ≥ 2 points had a worse median LFS (8.0 vs 48.0 months; P =.052) but not significantly worse OS (17.0 vs 28.0 months; P =.804). A Spearman coefficient for MRD ≥0.2% and hematogone levels ≥ 0.02% was -0.317 (P <.029), indicating a mildly negative correlation. However, MRD ≥0.2% and hematogones ≥ 0.02% were almost mutually exclusive. For clinical convenience, a MRD/Hematogone score was proposed that patient with MRD ≥0.2% was defined as 1 point and hematogone ≥ 0.02% was defined as -1 point. All patients could be categorized into three subgroups: 14(30%) patients with -1 point, 24(51%) patients with 0 point and 9(19%) with 1 point. Patients were stratified based on MRD/Hematogone score to assess survival using the Kaplan-Meier method and the Log-rank test. The median LFS were not reached, 48 months and 9 months for patients with -1 point, 0 point and 1 point, respectively (P =.015; Fig. 1). The median OS were not reached (-1 point), not reached (0 point) and 24 months (1 point) (P =.088). Multivariate analyses using Cox proportional hazard model demonstrated MRD/Hematogone score was an independent predictor of LFS (HR = 2.5, 95% CI, 1.2–5.1, P =.016; Table 2) after adjusting for MRC cytogenetic classification, WBC count (above or below 50000/uL) and whether undergoing allogenetic hematopoietic stem cell transplant before 1st relapse or not. Conclusion: Even without a diagnostic specimen, flow cytometry can assess and quantify aberrant myeloid progenitor cells, aberrant neutrophil/monocyte maturation and hematogones in post-induction marrow. MRD ≥0.2% or neutrophil/monocyte abnormalities (FCSS ≥ 2) predicts shorter LFS. Hematogones ≥ 0.02% predicts longer LFS and correlates negatively with MRD ≥0.2%. Proposed MRD/Hematogone score for post-induction adult AML in mCR maybe offer a simple and practical risk assessment. And the score is worthy to be validated by prospective and larger scale study. Disclosures: No relevant conflicts of interest to declare.

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Elevated EGR2 Expression Provides a Potential Link Between Cell Cycle Arrest and Induced Differentiation in Myeloid Progenitor Cells

Blood ◽

10.1182/blood.v124.21.2733.2733 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2733-2733

Author(s):

Joshua B. Bland ◽

Jose R. Peralta ◽

William T. Tse

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Progenitor Cells ◽

Myeloid Differentiation ◽

Phenotypic Effect ◽

Induced Differentiation ◽

Hl60 Cells ◽

Cycle Arrest ◽

Myeloid Progenitor ◽

Myeloid Progenitor Cells

Abstract Similar to many immature cell types, myeloid progenitor cells need to exit cell cycle to undergo terminal differentiation, but the mechanism linking the two is still unclear. Elucidating this mechanism could lead to the development of new differentiation therapies to treat myeloid leukemia. Recent studies have suggested that the processes regulating myeloid differentiation and cell cycle progression together constitute a positive feedback loop where each process reciprocally affects the other. To study the relationship between these processes, we examined early cellular and molecular events associated with induced differentiation of the HL60 human promyelocytic leukemia cells. We treated HL60 cells with 3 classical inducers of differentiation (vitamin D3 analog EB1089 (EB), all-trans retinoic acid (ATRA), and dimethyl sulfoxide (DMSO)), along with PD0332991 (PD), a selective cyclin D-dependent kinase 4/6 inhibitor that caused G1-phase-specific cell-cycle arrest. We evaluated differentiation of the treated cells by flow cytometric analysis of CD11b (integrin αM) and CD71 (transferrin receptor) expression. In untreated HL60 cells, a baseline subset of 3-5% of cells exhibits a differentiated, CD11b+CD71- phenotype. Exposure to the various inducers revealed a progressive increase in the percentage of CD11b+CD71- cells with time, such that by day 4 of treatment, it has increased to 50-90% in the treated samples, indicating that all 4 agents tested were effective in inducing myeloid differentiation. To understand how differentiation induced by each agent affects cell cycle progression, the cell cycle status of the induced cells were evaluated by a BrdU-incorporation assay after a 30-minute pulse of BrdU labeling. Uninduced cells exhibited a baseline cell cycle phase distribution of 64%-28%-8% (G1-S-G2/M phases). After 1 day of induction, EB-treated sample showed no changes in the distribution (58%-33%-9%), but ATRA, DMSO and PD-treated samples showed significant changes, with an increase of cell numbers in G1 phase and decrease in S phase (74%-18%-8%, 79%-13%-8%, and 93%-4%-3%, respectively). These results reveal that an early induction of G1 arrest was caused by treatment with ATRA, DMSO and PD, but not EB, and that the cell cycle arrest occurred before major changes in the myeloid phenotype were observed. To determine how the cell cycle perturbation relates to changes in the underlying genetic regulatory network, we examined by quantitative RT-PCR analysis the expression of several transcription factors associated with myeloid differentiation. PU.1 and CEBPA were found to be expressed at high levels but these levels did not change upon treatment with the inducing agents. Similarly, the expression levels of GFI1 and EGR1 did not change significantly with induction. In contrast, the expression level of EGR2 (Early Growth Response 2) was found to be low initially but became elevated upon treatment with 3 of the 4 inducers. EGR2 is a zinc finger transcription factor implicated in the control of a switch between pro- and anti-proliferation pathways. EGR2 has been shown to regulate the transition between differentiation states of Schwann cells, induction of anergic and regulatory T cells, growth and survival of osteoclasts, and proliferation and apoptosis of acute myeloid leukemia blasts. We found that EGR2 expression, after 1 day of treatment with ATRA, DMSO or PD, was increased by 5.2 ± 0.9, 7.6 ± 1.9, 5.8 ± 0.9 folds, respectively, whereas treatment with EB led to no significant change (1.5 ± 0.2 fold). We evaluated whether simultaneous treatment of the cells with 2 inducers would result in an additive effect. Treatment of HL60 cells with a combination of ATRA/DMSO, ATRA/PD, or DMSO/PD increased the percentage of CD11b+CD71- cells to 55%, 70% and 25% after just 1 day of treatment. In line with the enhanced phenotypic effect, the expression level of EGR2 was further elevated to 7.7 ± 1.4, 15.4 ± 3.5, and 11.3 ± 3.4 folds, respectively, when the cells were treated with the above inducer combinations, indicating a tight association between EGR2 expression and the phenotypic effect. In summary, our data suggest that elevated expression of EGR2 is an early event in the induction of myeloid differentiation in HL60 cells. Because of its known role in cell cycle regulation, EGR2 could function as a mechanistic link between cell cycle arrest and induced differentiation in myeloid progenitor cells. Disclosures No relevant conflicts of interest to declare.

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