Chunkang Chang

Overexpression of the EZH2, RING1 and BMI1 genes is common in myelodysplastic syndromes: relation to adverse epigenetic alteration and poor prognostic scoring.

Epigenetics refers to the study of clonally inherited changes in gene expression without accompanying genetic changes. Previous research on the epigenetics of myelodysplastic syndromes (MDS) mainly focused on the inactivation of tumor suppressor genes as a result of DNA methylation. However, the basic molecular pathogenesis of epigenetics in MDS remains poorly understood. Recent studies have revealed that DNA methylation and histone modification may be controlled by Polycomb-group (PcG) proteins, which may give new clues toward understanding the epigenetic mechanism of MDS. In this study, we explored for the first time the expression of PcG genes, including EZH2, EED, SUZ12, RING1, and BMI1, in various MDS subsets and acute myeloid leukemia (AML), as well as the relationship between the expression of PcG genes and epigenetic alteration and prognosis-risk scoring. Patients with MDS/AML showed overexpression of EZH2, RING1, and BMI1 genes compared to their expression levels in patients with non-clonal cytopenia diseases. The MDS patients with DNA methylation had higher EZH2 expression than those without DNA methylation. The patients who received decitabine treatment presented significantly reduced expression of EZH2 and RING1 besides decreased p15INK4B methylation after decitabine treatment. Moreover, overexpression of EZH2, RING1, and BMI1 was always linked to poor prognostic scoring. In conclusion, overexpression of the EZH2, RING1, and BMI1 genes is common in MDS and indicate poor prognosis. The products of these genes might participate in epigenetic regulation of MDS. These studies may also contribute to our understanding of the effective mechanism of decitabine.

Flow cytometric scoring system (FCMSS) assisted diagnosis of myelodysplastic syndromes (MDS) and the biological significance of FCMSS-based immunophenotypes

In the myelodysplastic syndromes (MDS), the haematopoietic cells show various levels of abnormal maturation and differentiation, which can be detected by flow cytometry. Testing the anomalies of stage‐ or lineage‐specific surface antigens in CD34+ blasts can distinguish MDS from non‐clonal cytopenic diseases, and also reflect the pathological characteristics of MDS as a class of clonal diseases for providing new clues to basic research. The present study established a flow cytometric scoring system (FCMSS) based on theproportion and antigenic co‐expression of CD34+ blasts. This FCMSS showed good sensitivity and specificity (77·8% and 100%) in the assisted diagnosis of low‐risk MDS without chromosome anomalies, ringed sideroblasts and excess marrow blasts. Moreover, we explored and reported different modes of abnormal expression of CD34+ blasts antigens in different disease stages and analyzed the biological significance of the immunotypes for the first time. We found expression of mature myeloid antigens and lymphoid antigens gradually decreased, and early functional antigens gradually increased from low‐risk MDS with normal karytype to low‐risk MDS with abnormal karyotype then to high‐risk MDS. The patients with higher FCM scores were generally accompanied with HLA‐DR15 allele or hypocellular marrow. Evolution of clones and immunological factors might have influence on expression of antigens in CD34+ blasts.

Down-regulation of Dicer1 promotes cellular senescence and decreases the differentiation and stem cell-supporting capacities of mesenchymal stromal cells in patients with myelodysplastic syndrome.

Although it has been reported that mesenchymal stromal cells are unable to provide sufficient hematopoietic support in myelodysplastic syndrome, the underlying mechanisms remain elusive. In this study, we found that mesenchymal stromal cells from patients with myelodysplastic syndrome displayed a significant increase in senescence, as evidenced by their decreased proliferative capacity, flattened morphology and increased expression of SA-β-gal and p21. Senescent mesenchymal stromal cells from patients had decreased differentiation potential and decreased stem cell support capacity. Gene knockdown of Dicer1, which was down-regulated in mesenchymal stromal cells from patients, induced senescence. The differentiation and stem cell-supporting capacities were significantly inhibited by Dicer1 knockdown. Overexpression of Dicer1 in mesenchymal stromal cells from patients reversed cellular senescence and enhanced stem cell properties. Furthermore, we identified reduced expression in the microRNA-17 family (miR-17-5p, miR-20a/b, miR-106a/b and miR-93) as a potential factor responsible for increased p21 expression, a key senescence mediator, in Dicer1 knockdown cells. Moreover, we found that miR-93 and miR-20a expression levels were significantly reduced in mesenchymal stromal cells from patients and miR-93/miR-20a gain of function resulted in a decrease of cellular senescence. Collectively, the results of our study show that mesenchymal stromal cells from patients with myelodysplastic syndrome are prone to senescence and that Dicer1 down-regulation promotes cellular senescence and decreases the differentiation and stem cell-supporting capacities of mesenchymal stromal cells. Dicer1 down-regulation seems to contribute to the insufficient hematopoietic support capacities of mesenchymal stromal cells from patients with myelodysplastic syndrome.

Xenogeneic immunosuppression of human umbilical cord mesenchymal stem cells in a major histocompatibility complex-mismatched allogeneic acute graft-versus-host disease murine model.

Mesenchymal stem cells (MSCs) hold great promise for treating immune disorders owing to their immunoregulatory capacity, but the mechanism remains controversial. As we show here, the mechanism of human umbilical cord mesenchymal stem cell (HUCMSC)‐mediated immunosuppression involves TGF‐β and indoleamine 2,3‐dioxygenase (IDO). In this study, we investigated the influence of xenogeneic HUCMSCs on acute graft‐versus‐host disease (aGVHD) in murine allogeneic bone marrow transplantation (BMT). In the HUCMSC‐treated group, lethally irradiated DBA/2(H‐2Kd) mice were adoptively transferred with expanded HUCMSCs, bone marrow (BM), and splenocytes (SCs) from C57BL/6 (H‐2Kb) mice. Recipients in the control group were transferred only BM and SCs. The two groups were compared in survival, weight, histopathologic specimens, and aGVHD scoring. In the HUCMSC‐treated group, 60% of the mice survived past day 30 after BMT, but in the control group, all mice died within 18 d. The mice treated with HUCMSCs exhibited light symptoms of aGVHD after day 30. The results suggest that xenogeneic HUCMSCs could alleviate aGVHD symptoms and prolong survival after allogeneic BMT. Our study suggests that in vitro expanded HUCMSCs might be used to inhibit severe aGVHD effectively in allogeneic hematopoietic cell transplantation clinically.

Effect of low-dose cytarabine, homoharringtonine and granulocyte colony-stimulating factor priming regimen on patients with advanced myelodysplastic syndrome or acute myeloid leukemia transformed from myelodysplastic syndrome.

A total of 32 patients (25 with advanced MDS and 7 with t-AML) were enrolled in this study to evaluate the efficacy and toxicity of the low-dose cytarabine and homoharringtonine in combination with granulocyte colony-stimulating factor (G-CSF) (CHG protocol) in patients with advanced myelodysplastic syndromes (MDS) or MDS-transformed acute myeloid leukemia (t-AML). All the patients were administered the CHG regimen comprising low-dose cytarabine (25 mg/day, intravenous continuous infusion, days 1–14), homoharringtonine (1 mg/day, intravenous continuous infusion, days 1–14), and G-CSF (300 µg/day, subcutaneous injection, days 0–14, interrupted when the peripheral white blood cell count reached >20 × 109/L). The overall response rate was 71.9% after the administration of one course of the CHG regimen. Of the 32 patients, 15 (46.9%) achieved complete remission (CR) and 8 (25%) achieved partial remission (PR). This regimen was followed by a post-remission therapy that included conventional chemotherapy, when CR was achieved. Of the patients with CR who just received post-remission regimens as homoharringtonine and cytarabine (HA) and daunorubicin and cytarabine (DA) 6 relapsed rapidly and just had a mean 6.1 months of CR. Otherwise, the other 8 out of 14 patients with CR alternatively received subsequent chemotherapy, which combined mitoxantrone, idarubicin, pirarubicin, or aclarubicin with cytarabine. The mean CR duration of the 8 patients had reached 10.6 months, and 5 of the 8 still kept a continuous CR. The median overall survival (OS) was 18.2 months. There were no statistically significant differences for CR, PR, and OS when the patients were grouped by age, blasts in bone marrow, and karyotypes, respectively. No treatment-related deaths were observed. Myelosuppression was mild to moderate, and no severe non-hematological toxicity was observed. Thus, a CHG priming regimen as an induction therapy was well tolerated and effective in patients with advanced MDS or t-AML. Stronger and alternative subsequent chemotherapy is necessary for patients with CR to maintain longer CR and better OS.

Senescence of bone marrow mesenchymal stromal cells is accompanied by activation of p53/p21 pathway in myelodysplastic syndromes

The contribution of bone marrow mesenchymal stromal cells (BMMSCs) to the pathogenesis of myelodysplastic syndrome (MDS) has created controversies. In this study, we confirmed that BMMSCs from MDS patients showed prominent features of senescence, which were characterized by increased cell size, decreased proliferation and colony‐forming potential, alteration of cytoskeleton, and increased senescence‐associated β‐galactosidase (SA‐β‐Gal) activity. Interestingly, the apoptosis assay results showed that the percentage of apoptosis cells was very low and the difference was not significant between MDS patients and normal controls. Moreover, the osteogenic differentiation potential of BMMSCs from lower risk but not higher risk MDS was impaired, indicated by cytochemical stainings and reduced expressions of RUNX2. In addition, BMMSCs from MDS patients had impaired hematopoietic supporting function. Furthermore, the expression of p53 and p21 which played an important role in regulating the senescence progress of BMMSCs was significantly increased, whereas levels of p16 and pRb expression were not changed in the BMMSCs from MDS patients. Taken together, our comprehensive analysis shows that BMMSCs from MDS patients exhibited senescent behavior and activation of p53/p21 pathway probably played an important role in the senescence process.

Immunophenotypic analysis of erythroid dysplasia and its diagnostic application in myelodysplastic syndromes.

Background/Aim:  Abnormal immunophenotypes of haematopoietic cells in myelodysplastic syndromes (MDS) have been identified by flow cytometry (FCM) as a typical characteristic of myeloid dysplasia. Considering that most MDS patients show varying degrees of erythroid dysplasia, we analysed the immunophenotypic feature of erythroblasts to evaluate its diagnostic application in MDS.

Impaired osteogenic differentiation of mesenchymal stem cells derived from bone marrow of patients with lower-risk myelodysplastic syndromes

The pathogenesis of myelodysplastic syndromes (MDS) has not been completely understood, and insufficiency of the hematopoietic microenvironment can be an important factor. Mesenchymal stem cells (MSCs) and osteoblasts are key components of the hematopoietic microenvironment. Here, we measured the expression of multiple osteogenic genes in 58 MSCs from MDS patients with different disease stages and subtypes by real-time PCR and compared the osteogenic differentiation of MSCs from 20 MDS patients with those of MSCs from eight normal controls quantitatively and dynamically. The mRNA level of Osterix and RUNX2, two key factors involved in the early differentiation process toward osteoblasts, was significantly reduced in undifferentiated MSCs from lower-risk MDS. After osteogenic induction, lower-risk MDS showed lower alkaline phosphatase activity, less intense alizarin red S staining, and lower gene expression of osteogenic differentiation markers; however, higher-risk MDS was normal. Finally, in bone marrow biopsy, the number of osteoblasts was significantly decreased in lower-risk MDS. These results indicate that MSCs from lower-risk MDS have impaired osteogenic differentiation functions, suggesting their insufficient stromal support in MDS.

Deviation of type I and type II T cells and its negative effect on hematopoiesis in myelodysplastic syndrome

Immunemediated hematopoietic suppression has been considered as one of significant pathophysiological changes in less‐advanced myelodysplastic syndrome (MDS). To explore deviation of T cell subsets and its relationship to marrow cells apoptosis, measurement of helper‐T (Th)/cytotoxic‐T (Tc) subsets as well as the deviation situation within this two subsets (Th1/Th2 and Tc1/Tc2) in marrow was performed by flow cytometry from 39 MDS patients and 13 normal controls. Interferon (INF)‐γ and tumor necrosis factor (TNF)‐α in marrow serum was simultaneously detected by ELISA (enzyme‐linked immunosorbent assay). Furthermore, apoptosis rate of marrow cells was demonstrated by TUNEL (TdT‐mediated dUTP nick end labeling). Results showed that Th and Tc subsets were unevenly activated, both deviating to type I response, which was especially obvious in patients with RCMD (according to WHO classification) and in lower‐risk cases defined by International Prognosis Scoring System (IPSS). Level of INF‐γ/TNF‐α in MDS marrow serum was markedly elevated, and so did the apoptosis rate of marrow cells. Although type I deviation was observed both in Th and Tc subsets, just Th1 cell percentage showed positive correlation with level of INF‐γ/TNF‐α and apoptotic index of nucleated cells. In addition, cytokines level in marrow serum presented positive correlation to apoptosis. We then deduced that the increased Th1 cells in marrow may account for nucleated cells apoptosis in MDS through overproduced proapoptotic cytokines such as INF‐γ and TNF‐α. Our results suggested that type I deviation of T cell subsets may play a role in pantocytopenia in MDS and the deviation pattern may be as a direct and effective parameter to predict response of immunosuppression therapy.

Interleukin-17 enhances the production of interferon-γ and tumour necrosis factor-α by bone marrow T lymphocytes from patients with lower risk myelodysplastic syndromes

Lower risk myelodysplastic syndromes (MDSs) are characterised by increased apoptosis of haematopoietic cells in the bone marrow (BM). The mechanism driving this excessive apoptosis involves multiple immune molecules, including inflammatory cytokines such as interferon‐γ (IFN‐γ), tumour necrosis factor‐α (TNF‐α) and interleukins (ILs). Interleukin‐17 (IL‐17) is the hallmark cytokine produced by CD4+ Th17 cells, and IL‐17 mediates activation of the adaptive T‐cell response inducing an inflammatory cytokine environment. However, little is known about the role of IL‐17 in MDS‐associated immune dysfunction.