Bone Marrow Of Healthy Controls Research Articles

Establishing a Pathogenic Role for NK Cells As Drivers of Bone Marrow Failure in the Myelodysplastic Syndromes (MDS)

Background: Myelodysplastic syndromes (MDS) are characterized by aberrant bone-marrow (BM) morphology leading to a clonal impairment of hematopoiesis. Worsening BM failure (BMF) and increasing myeloblasts are observed as the predominant consequences of MDS disease progression from very low risk (VLR) to higher risk (HR) to acute myeloid leukemia (AML). Patients are stratified based on the degree of BMF and risk of AML transformation to help guide treatment decisions. Approximately 30% of MDS patients exhibit various autoimmune diseases that can include autoantibodies targeting neutrophils, erythrocytes, and/or platelets. Autoimmune phenomena play an important role in MDS yet are poorly characterized. We hypothesized that early in LR-MDS, dysregulated interactions between stromal cells and developing Natural Killer (NK)/T-cells promote rapid/unregulated development resulting in autoreactivity ( Fig. 1A). We predict dual effector mechanisms: CD16-mediated antibody-dependent cellular cytotoxicity (ADCC) and loss of HLA class I (HLA-I) expression by developing hematopoietic lineage cells making them targets for “missing-self” reactivity ( Fig. 1A). NK cells are important sentinel cells that recognize tumors and dysfunctional cells that lose expression of HLA class I. Such surveillance is guided by interactions between NK inhibitory receptors and HLA class I ligands that ‘train’ NK cells to distinguish diseased cells, exhibiting perturbed HLA expression, from healthy cells with normal expression. The effects of these interactions combined with expression of activating receptors (e.g. CD16, NKG2D, DNAM1, FasL, TRAIL) allow NK cells to engage a target cell through many activating mechanisms, importantly including antibodies eliciting an antibody-dependent cellular cytotoxicity (ADCC) reaction. Autoantibodies are well-established in MDS, yet no one has profiled whether they may trigger ADCC/complement. Further, no one has ever thoroughly characterized BMMCs from LR-MDS patients before. In our study, we observed a clear link between signatures of bone marrow failure at LR-MDS and a significant increase in BM CD57+NK and CD8 T cells expressing a highly activated and dysregulated phenotype. Method: Ex-vivo profiling of 19 MDS patients (N=10 LR/IR-MDS, N=9 HR-MDS) BM mononuclear cells (BMMCs) was performed by mass cytometry (CyTOF)( Fig.1B). Cells were stained with three different antibody panels each consisting of 44 antibodies focused on: specific markers of hematopoiesis and myeloid or lymphoid cell lineages; specific markers of development, effector function, homing, exhaustion, and T-cell memory and proliferation; expression of HLA class I and activating ligands which are critical regulators of NK inhibition. Results: In our study we observed that a major fraction of MDS patients present with clonal-like expansions of BM-derived CD57+CD8+T-cells and natural killer (NK) cells that strongly correlate with autoimmune-cytopenias and BMF-enriched in LR-MDS (Fig.#). We observed that NK-cells and non-classical T-cells were significantly expanded (>2-fold) in LR MDS compared to HR-MDS and healthy-control BM (p<0.01). These non-classical CD57+CD8+T-cells co-expressed HLA-I-reactive and inhibitory NKG2A/KIR receptors as well as activating CD16, NKG2C, NKG2D, NKp46, TRAIL, FasL. In cancers, these cells use innate-like, TCR-independent mechanisms (traditionally ascribed to NK-cells) to target HLA-I-deficient tumor cells. Strikingly, HLA-I was significantly elevated on myeloblasts in LR-MDS compared to HR-MDS (p=0.0007)( Fig.1B), demonstrating NK-evasion strategies, but strongly reduced on dendritic cells, granulocytes, and CD4+T-cells, suggesting susceptible immune targets for destruction by NK-cells (and innate-like CD8+T-cells) (p< 0.0001) ( Fig.1B). BM-derived CD57+NK and CD8+T-cells all co-express very high levels of CD16 making them particularly ADCC reactive. Conclusion: We identified novel innate-like CD8+CD57+ T-cell and NK-cell populations that may play a role in LR-MDS disease pathogenesis. We predict that a strategy to target these cells could help restore hematopoiesis early in the disease state. This may also reduce pressure on myeloblasts to increase HLA-I, permitting improved immune surveillance to control the blast burden and prevention MDS/AML progression. Further experiments are ongoing to validate our observations.

MicroRNA-25-3p suppresses osteogenic differentiation of BMSCs in patients with osteoporosis by targeting ITGB3

This study was conducted to investigate the impact of the microRNA (miR)-25–3p/ITGB3 axis on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) from patients with osteoporosis (OP). BMSCs isolated from the bone marrow of healthy controls and OP patients were identified by flow cytometry, in which ITGB3 mRNA and miR-25–3p expression was detected by RT-qPCR and ITGB3, Runx2, OPN, ALP, and OSX protein expression by western blot. The binding between ITGB3 and miR-25–3p was assessed by dual-luciferase reporter gene and Ago2-RIP assays. BMSC osteogenic differentiation was observed by alizarin red staining and ALP activity. The differentiation of BMSCs to adipocytes and chondrocytes was measured by oil red O staining and alcian blue staining, respectively. BMSCs were successfully isolated from the bone marrow of healthy controls (normal-BMSCs) and OP patients (OP-BMSCs). ITGB3, Runx2, OPN, ALP, and OSX expression was poorer and miR-25–3p expression was higher in OP-BMSCs than in normal-BMSCs. Mechanistically, ITGB3 was negatively targeted by miR-25–3p. After osteogenic, adipogenic, and chondrogenic differentiation of BMSCs were successfully induced, adipogenic differentiation increased and osteogenic and chondrogenic differentiation decreased in OP-BMSCs compared with normal-BMSCs. Overexpression of ITGB3 facilitated mineralized nodule formation and elevated ALP activity and Runx2, OPN, and ALP expression in OP-BMSCs. miR-25–3p upregulation diminished mineralized nodule formation, ALP activity, and Runx2, OPN, and ALP expression in OP-BMSCs and normal-BMSCs, which was annulled by additional ITGB3 overexpression. miR-25–3p targets ITGB3, thereby suppressing osteogenic differentiation of BMSCs from OP patients.

Chromosomal Abnormalities in MDS Are Linked to Dysregulation of CDC20 and CEP55 Genes

Myelodysplastic syndrome (MDS) is a clonal hematopoietic disorder characterized by cytopenias and an increased risk of progression to acute myeloid leukemia (AML). Its pathogenesis is strictly linked to chromosomal instability, which in turn provides a valuable prognostic marker. Malignant cells develop alternative routes to escape mitosis checkpoints, overcoming the mitotic arrest imposed by Spindle Assembly Checkpoint (SAC), a process dependent on CDC20 inactivation. Abnormal levels of CDC20 can inhibit mitotic arrest, promoting premature exit from mitosis. Overexpression of CEP55 also facilitates the mitotic exit, resulting in polyploidy (an event called Mitotic Slippage). Since chromosomal abnormalities are one of the most important prognostic factors for patients with MDS, this study aimed to analyze the possible link between chromosomal abnormalities and CDC20 and CEP55 mRNA expression in MDS. We evaluated the bone marrow cells from 45 patients diagnosed as MDS according to 2016 WHO-classification (1 MDS-SLD, 15 MDS-RS-MLD, 5 MDS-MLD, 1 t-MDS, and 23 MDS-EB) and 5 bone marrow of healthy controls. Conventional Karyotyping was performed by G-banding of 20 metaphases whenever possible. TaqMan expression assays for CDC20 (Hs00426680_mH) and CEP55 (Hs01070181_m1) were performed in duplicate and the expression ratios were calculated using the 2−ΔCq method. Normality was evaluated by Shapiro-Wilk test. Outliers were removed. The Student's t-test or one-way ANOVA with Tukey/Games Howell post-hoc test was used to analyze the influence of relative expression regarding variables. Patients with MDS showed increased expression of CDC20 and CEP55 compared to healthy individuals (p<0.0001 and p<0.0001). Regarding karyotype, there was the overexpression of CDC20 and CEP55 in patients with altered karyotype and aneuploid karyotype when compared to patients with normal karyotype (p <0.0001 and p =0.001; p = 0.013 and p = 0.022, respectively) (Figure 1A-D). CDC20 and CEP55 have fundamental functions in controlling the progression of metaphase to anaphase and both, when upregulated, induce chromosomal instability. Additionally, patients with del(7q) and complex karyotype showed hyperexpression of CEP55 when compared with patients with normal karyotype (p = 0.005 and p = 0.019) (Figure 1E-F), while patients with deletion (5q) had an increased expression of CDC20 when compared with patients with normal karyotype (p <0.0001). Our group previously demonstrate that high CDC20 protein expression is associated with complex karyotype in MDS patients. Thus, we hypothesized that the deregulation of CDC20 and CEP55 expression induces chromosomal changes, each one in its way. Both can cause disturbances in crucial phases of mitosis (anaphase and cytokinesis, respectively). Finally, we detected a strong correlation between CDC20 and CEP55 (r = 0.646; p <0.0001), suggesting both genes may play a synergistic role during chromosomal abnormalities in MDS, creating possible new targets to be evaluated in MDS. Our data suggest CDC20 and CEP55 as possible new therapeutic targets in MDS. There is a need for further studies, validations and urgent in-depth investigations in cell lines/primary samples or murine models. Disclosures No relevant conflicts of interest to declare.

Detailed immunophenotyping of B-cell precursors in regenerating bone marrow of acute lymphoblastic leukaemia patients: implications for minimal residual disease detection.

Flow cytometric detection of minimal residual disease (MRD) in children with B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) requires immunophenotypic discrimination between residual leukaemic cells and B-cell precursors (BCPs) which regenerate during therapy intervals. In this study, EuroFlow-based 8-colour flow cytometry and innovative analysis tools were used to first characterize the immunophenotypic maturation of normal BCPs in bone marrow (BM) from healthy children, resulting in a continuous multiparametric pathway including transition stages. This pathway was subsequently used as a reference to characterize the immunophenotypic maturation of regenerating BCPs in BM from children treated for BCP-ALL. We identified pre-B-I cells that expressed low or dim CD34 levels, in contrast to the classical CD34high pre-B-I cell immunophenotype. These CD34-dim pre-B-I cells were relatively abundant in regenerating BM (11-85% within pre-B-I subset), while hardly present in healthy control BM (9-13% within pre-B-I subset; P=0·0037). Furthermore, we showed that some of the BCP-ALL diagnosis immunophenotypes (23%) overlapped with CD34-dim pre-B-I cells. Our results indicate that newly identified CD34-dim pre-B-I cells can be mistaken for residual BCP-ALL cells, potentially resulting in false-positive MRD outcomes. Therefore, regenerating BM, in which CD34-dim pre-B-I cells are relatively abundant, should be used as reference frame in flow cytometric MRD measurements.

Monitoring of T-cell acute lymphoblastic leukemia by flow cytometry

Abstract Minimal residual disease (MRD) predicts the outcome of acute lymphoblastic leukemia (ALL). Flow cytometry (FC) is one of the most sensitive and most applicable methods for MRD diagnostics, but there is still no agreement on the “gold standard” of the method. We tried to optimize flow cytometric MRD detection in T-ALL. Fourteen adults and 11 children with T-ALL and 12 normal bone marrow (BM) donors were enrolled in the study. We found that the most common phenotypic aberrations in T-ALL were TdT and CD99 coexpression on T-cells in BM. Therefore for MRD detection we developed a limited four-color marker panel (TdT/CD7/cCD3/CD19 and CD99/CD7/cCD3/CD2) and a standard analysis strategy. This assay was evaluated on BM of healthy controls. Less than 0.01% TdT+ or CD99 bright T-cells were found in normal BM. MRD was detected in 9 adult patients and 1 child at different time-points of treatment. The average TdT and CD99 mean fluorescence intensity (MFI) value of residual blasts fluctuated during therapy, but it still remained higher than MFI of normal T-cells. Our established MRD detection method differentiated leukemic lymphoblasts with sensitivity in the range of 0.01% and did not give any false positive results in normal BM.

Expression of Midkine(MDK) and Sensitivity to Anti-Leukemic Drugs in Pediatric Acute Lymphoblastic Leukemia (ALL).

Midkine (MDK), a heparin-binding growth factor, is highly expressed in pediatric ALL, neuroblastomas, and various solid tumors. Previous gene expression profiling studies conducted in our laboratory on a cohort of 220 children with B precursor ALL treated on Children's Oncology Group (COG) clinical trials have shown that higher levels of MDK expression in leukemic blasts are strongly associated with continuous complete remission (CCR) and that MDK is a powerful prognostic predictor of outcome (Wilcoxon ranksum test, p=0.000001), particularly in patients with central nervous system (CNS) involvement (Wilcoxon ranksum test, p =0.009). To explore the role of MDK in hematopoiesis and leukemogenesis and to further explore MDK as a potential novel target for ALL therapy, we examined MDK mRNA expression in three stages of normal B cell development in healthy controls and compared it to expression levels in leukemic blasts from children with B-ALL. Quantitative RT-PCR assays showed that MDK mRNA levels in CD19+ cells from peripheral blood mononuclear cells (PBMNC) were significantly higher than those in CD34+ or CD19+ cells from bone marrow (BM) (Mann Whitney test, p=0.0025 and 0.0018, respectively). ALL leukemic blasts samples expressed significantly higher levels of MDK than those in CD19+ cell in the BM of healthy controls (Mann Whitney test, p=0.0043). Similar results were obtained by ELISA detection of MDK protein in plasma of ALL patients and healthy controls. We have also investigated the relationship of MDK expression and the chemo-sensitivity of ALL cell lines. Using a lentiviral system, we have successfully over-expressed or knocked down MDK gene expression in the REH cell line containing the t(12;21). Unexpectedly, MTS assays revealed that MDK expression is inversely associated with sensitivity to L-asparaginase and doxorubicin, but not to vincristine and cytarabine, in REH cells. Higher levels of MDK were associated with chemo-resistance. Finally, we tested the effects of MDK expression on proliferation and apoptosis in REH cells and determined that although MDK expression was not associated with REH cell proliferation in vitro, it protected REH cells from doxorubicin-induced apoptotic cell death. Taken together, these results suggest that expression levels of MDK in ALL is inversely associated with in vitro drug sensitivity to specific anti-leukemic agents; consistent with observations in solid tumor models. It has been suggested that 4β1- and 6β1-integrins bind directly to MDK and are involved in MK-dependent cell migration. Further studies of the role of MDK signaling in vivo in the complex marrow/adhesion microenvironment and the effects on ALL survival and chemo-sensitivity and resistance are in progress.

Localized COSY and DQF‐COSY 1H‐MRS sequences for investigating human tibial bone marrow in vivo and initial application to patients with acute leukemia

To develop 2D 1H-MRS measurement sequences for the evaluation of bone marrow lipids, and to assess these measurement sequences in healthy and diseased bone marrow. Single-voxel localized variants of COSY and DQF-COSY 2D 1H-MRS sequences were developed for use at 1.5 T to investigate the biochemical composition of human bone marrow in vivo. The performance of each sequence was initially tested in vitro using lipid phantoms. An unsaturated lipid proton index was developed to interrogate the degree of unsaturation within the triacylglyceride (TAG) acyl chains. Localized 2D 1H-MRS data were obtained from the bone marrow of healthy controls (N = 6), patients presenting with acute leukemia (N = 6) and patients with acute leukemia in remission (N = 4). The COSY and DQF-COSY data recorded from all subject cohorts were similar, and the unsaturated lipid proton index did not reveal significant differences between patient groups. Variations in water content and measured relaxation times showed minor differences between the measurement groups. No significant differences were observed in the spectra obtained from bone marrow using the 2D 1H-MRS sequences. A novel unsaturated lipid proton index was developed.