| Section ITh22cells as well as Th17cells expand differentially in patients with early-stage and late-stage myelodysplastic syndromeBackground:Myelodysplastic syndrome (MDS) encompasses a heterogeneous group of clonal hematopoietic stem-cell disorders, characterized by ineffective hematopoiesis and an increased probability of developing acute leukemia. Autoimmune-mediated myelosuppression and immune surveillance of malignant clone play recognized crucial roles in the process of MDS. Findings of expanded clonal T lymphocytes and clinical responses to immunoregulatory therapy have led to the speculation that both T helper and cytotoxic T cells are involved in the immunological pathophysiology of MDS.Recently, a separate Th cell subset which secrets the cytokine interleukin(IL)-17and expresses the lineage-specific transcription factor retinoic acid receptor-related orphan receptor C (RORC) was identified and named T-helper cell type17(Th17). Since then, Th17cells have entered the limelight because of their comprehensive involvement in inflammations. IL-17A, a representative Th17cytokine, has been described in various models of immune-mediated tissue injury, such as rheumatoid arthritis, lupus, myeloma and so on. Although one study reported an increased number of peripheral Th17cells in low risk MDS lately, the mechanism of cellular immune abnormalities still remains undiscovered.Th22subset, which has the ability of secreting IL-22and tumor necrosis factor alpha (TNF-a), has become a new subset clearly separated from other Th cells. This subset does not express any of cytokine interferon (IFN)-y, IL-17or their associated transcription factors T-bet and RORC. Aryl hydrocarbon receptor (AHR) activation participates in priming naive CD4+T cells to Th22subset plus the promotion by IL-6and TNF-a. Th22cells have been found regulating epidermal responses within the epidermal layer prominently in inflammatory skin diseases. In addition, a preferential expansion of Th22cells contributing to gastric cancer progression has been described. These data above support that Th22cells are involved in the pathophysiology of inflammatory immune reactions and immune evasion of neoplasms.IL-22belongs to the IL-10cytokine family and is primarily secreted by Th22cells on activation. The expression trend of IL-22in cancers and autoimmune disorders is diverse, with IL-17as siblings but not twins based on the local biological characteristics. The expression level of IL-22was up-regulated in skin inflammatory pathology and anaplastic lymphoma kinase positive anaplastic large cell lymphoma, inducing directional signal linking the immune system to the targeted tissue-resident cells. Meanwhile, it was down-regulated in systemic lupus erythematosus. Within disorders such as inflammatory bowel disease (IBD), once inducted by the naive or memory/effector cells, IL-22has access to inhibit or accelerate the inflammatory reactions regarding the idiographic microenvironment.To date, no report exist with regard to Th22cell subset or their association with Th17or Thl subset among MDS patients. To investigate their possible roles in the immunological pathophysiology of MDS, we measured the percentages of circulating Th22, Th17, Th1and mRNA expression levels of RORC, IL-6, TNF-a and IL-23in peripheral blood mononuclear cells (PBMCs) as well as the cytokine expression levels of IL-22or IL-17in peripheral blood (PB) and bone marrow (BM). To explore the potential relationship between Th polarization and different stages of MDS, the correlations of various Th subsets were evaluated at the same time.Objective:Measure the frequencies of periphery Th22, Th17subsets and determine the mRNA expression of transcription factor RORC, cytokines such as IL-6, TNF-a and IL-23. Explore the regulation effects of transcription factor and cytokines on Th22/Th17polarization in different stages of myelodysplastic syndrome. Attempt to elucidate the relevance between the switched immune status and clinical prognosis of myelodysplastic syndrome as well as search for peripheral blood immunological index to guide the diagnosis of different disease stages.Materials and methods:1. PB was collected from seventeen patients with early-stage MDS (E-MDS), twenty patients with late-stage MDS (L-MDS) and twenty age-matched healthy volunteers. BM was obtained from twenty-five MDS patients and ten hematologically normal surgical patients without any oncological or immunological issues.2. The percentages of circulating Th22, Th17, Thl subset were measured in E-MDS patients, L-MDS patients and healthy controls by flow cytometry.3. The cytokine levels of IL-22and IL-17both in PB and BM plasma were examined by enzyme-linked immunosorbent assay.4. For RNA isolation, PBMCs were isolated from the anticoagulated blood donated by E-MDS patients, L-MDS patients and healthy controls. The mRNA expression levels of RORC, IL-6, TNF-a and IL-23were determined by real-time quantitative polymerase chain reaction.5. Results were expressed as mean±SD or median (range). Comparisons between two groups were assessed by the non-paired t-test or the Wilcoxon rank-sum test to compare parametric and non-parametric data respectively. Statistical significance among E-MDS, L-MDS and healthy control three groups was determined by ANOVA, and difference between any two groups was determined by Newman-Keuls multiple comparison test (q test) unless the data were not normally distributed, in which case Kruskal-Wallis test (H test) and Nemenyi test were used. The Pearson or Spearman correlation test was used for correlation analysis depending on data distribution. All tests were performed by SAS9.1system. P value less than0.05was considered statistically significant.Results:1. The percentage of circulating Th22subset in E-MDS patients, L-MDS patients and healthy controls Compared with healthy controls, the percentage of peripheral Th22cells was significantly higher in total MDS patients (0.71±0.17%vs.1.55±0.74%, P<0.0001). The number of peripheral Th22cells in E-MDS is higher than that in controls (1.27±0.50%vs.0.71±0.17%, P=0.002). Also a significant increase was shown in L-MDS compared with E-MDS patients (1.77±0.84%vs.1.27±0.50%, P=0.03).2. The percentage of circulating Th17subset in E-MDS patients, L-MDS patients and healthy controls The percentage of peripheral Th17cells was significantly elevated in E-MDS (median,1.90%; range,0.58-6.01%) when compared with L-MDS (median,1.16%; range,0.15-1.86%)(P=0.002) or healthy control group (0.97±0.29%; P=0.002).3. The percentage of circulating Th1subset in MDS patients and healthy controls For peripheral Thl cells, no significant difference was observed between MDS patients and healthy controls.4. The cytokine levels of IL-22and IL-17in PB and BM plasma from MDS patients and healthy controls No significant difference of PB IL-22or IL-17level between MDS patients (median,22.64pg/ml; range,16.02-54.66) and healthy controls (median,23.86pg/ml; range,14.05-36.49) was observed, consistent with BM findings.5. mRNA expression levels of RORC, IL-6, TNF-a and IL-23in E-MDS, L-MDS cohort and controls The relative amount of RORC mRNA in E-MDS patients was markedly increased compared with healthy controls and L-MDS patients (P=0.0007; P=0.002). The relative amount of mRNA of IL-6in L-MDS patients was much higher than that in E-MDS patients (P<0.05) and healthy controls(P<0.001). TNF-a mRNA level was also present on higher levels in L-MDS compared with E-MDS and controls (P<0.05; P<0.005). There was no significant difference in IL-23p19mRNA expression levels among E-MDS, L-MDS and healthy controls.6. Correlation between Th22, Thl7, and Thl Cells in MDS Patients In E-MDS patients, there existed a statistically positive correlation between peripheral Th22cells and Thl7cells (r=0.675, P=0.004) while no statistical correlation was shown among L-MDS patients (r=0.138, P=0.610). Peripheral Th22subset showed no significant correlation with peripheral Thl subset (P=0.053).Conclusion:E-MDS cohort demonstrated the dominance of Thl7subset and its specific transcription factor RORC. On the other hand, L-MDS cohort showed an increased frequency of Th22cells in peripheral blood, along with higher mRNA expression levels of IL-6and TNF-a. Our study indicates that Th22cells along with Th17cells or not are involved in regulating the autoimmune reaction of E-MDS and immune evasion of L-MDS. As for the disease stages, peripheral Th22/Th17ratio is important to the stratified diagnosis of E-MDS and L-MDS. section ⅡClinical study of decitabine in the segmented treatment and evaluation system for myelodysplastic syndromeBackground:Epigenetics refers to alteration in gene expression that does not involve changes to the corresponding DNA sequence. The common epigenetic events include DNA methylation, RNA interference, histone modification and so on. Despite that cytogenetic heterogeneity constitutes the principal pathophysiological mechanism of MDS, haploinsufficiency, secondary mutation as well as epigenetic modification also contribute to the disease phenotype. What top the ratings of epigenetic modifications in MDS are DNA methylation and histone modification on genomic level, but in fact these modifications are focally increased around the promoter region of tumor suppressor gene and other mitosis inhibitors. It is noted that there are more hypermethylation sites in high risk MDS compared with low risk MDS. DNA methyltransferase3A (DNMT3A) and TET2mutations in the promoter region have been recognized to be involved in DNA methylation. In the CpG dinucleotides enriched site, DNMT functions in the methylation of cytimidine converting to5-methylcytosine, which is thought to mediate much of the increased methylation process and has therefore become a crucial treatment target in MDS.Researchers have proved that high-dose decitabine has direct cytotoxicity to clonal cells whereas low-dose decitabine induces hypomethylation through inhibiting the activity of DNMTs, which is distinct from immediate cytotoxicity. The normal demethylated state of former silencing tumor suppressor genes such as E-cadherin, p16, hMLHl, VHL, p15and P21shall be restored, followed with reactivation and gene transcription.Decitabine was recently approved by the US Food and Drug Administration (FDA) for treating patients with intermediate risk/high risk/very high risk MDS or nonresponsive MDS. A number of retrospective and prospective studies have revealed that decitabine favors to cytogenetic improvements, decreases the probability of developing acute leukemia and prolongs the overall survival in the majority of patients with higher risk MDS. However, during the clinical practice of decitabine, myelosuppression often occurs, resulting in thrombocytopenia, neutropenia or anemia. What’s worse, higher grade3/4myelosuppression happens more frequently.Given that the current clinical scheme of decitabine often goes wrong due to dose delay or early discontinuance, we designed a novel segmented treatment and evaluation system to improve the long-term efficacy of low-dose decitabine. At present, we describe this novel segmented treatment and evaluation system in detail and make comparison with the original prevailing protocol.Objective:Expound the rationality, feasibility and drug safety of the segmented treatment and evaluation system of decitabine for treating MDS; compare and analyze the clinical effectiveness of decitabine used in the segmented system and conventional system.Methods:A total of57patients with MDS were recruited in this study. Enrollment took place between February2012and December2013in the Department of Hematology of Qilu Hospital, Shandong University.24patients were treated with decitabine in the conventional scheme, i.e.20mg/m2/d, intravenous (IV) infused for5days and repeated every4weeks. The myelogram and hemogram changes were reexamined after the second course to evaluate the marrow remission and hematologic remission.33MDS patients were incorporated into the segmented treatment and evaluation system with the same dose of decitabine as the conventional scheme. However, before starting the segmented treatment, the baseline characteristics of the subjects should be assessed. Antibiotics, antivirals and antifungals were preventively applied. Best supportive care was supposed to be aggressive. After the second course, only platelet count was monitored whereas after the third course, complete blood count and blasts in bone marrow were tested. Intensification therapy was carried on after the forth course and maintainance therapy after the sixth course. The incidence of grades3to4hematological side effects and infections were analyzed in the conventional remedy system and segmented remedy system after the first course, as well as the incidence of dose delay and early discontinuance. The platelet counts were highlightly concerned in the second course and were dynamicly monitored until achieving platelet hematologic improvement (HI-P). The myelogram and hemogram changes were reexamined after the third course in the segmented remedy system whereas these had been reexamined after the second course in the conventional remedy system. The overall response rate (ORR), transfusion-dependence and median course in these two different systems were compared using chi-square test and non-parametric test via SPSS17.0software.Results:1. Infections in the conventional and segmented remedy systems after the first decitabine courseThe infection rate in the conventional treatment group after the first decitabine course was66.7%versus39.4%in the segmented treatment and evaluation group (P=0.042<0.05). The infection rate in the segmented treatment and evaluation group was significantly lower compared with that in the conventional treatment group.2. Grades3to4neutropenia in the conventional and segmented remedy systems after the first decitabine courseCompared with the conventional remedy group, the incidence rate of Grades3to4neutropenia was significantly lower in the segmented remedy group (9.1%vs.33.3%, P=0.039<0.05).3. Grades3to4thrombocytopenia in the conventional and segmented remedy systems after the first decitabine courseThe incidence rate of Grades3to4thrombocytopenia in the segmented remedy group was obviously reduced when compared with the conventional remedy group (24.2%vs.50.0%, P=0.044<0.05).4. Grades3to4anemia in the conventional and segmented remedy systems after the first decitabine courseThe incidence rate of Grades3to4anemia in the segmented remedy group was markedly reduced compared with that in the conventional remedy group (6.1%vs.29.2%, P=0.027<0.05).5. Dose delay or early discontinuance in the conventional and segmented remedy systems after the first decitabine courseSignificant difference in the incidence of dose delay between the segmented remedy group and the conventional remedy group was observed (36.4%vs.66.7%, P=0.024<0.05). No difference in the incidence of early discontinuance after the first course was found between the two groups.6. Comparision of responses to decitabine in the conventional and segmented remedy systemsSignificant increment of ORR (CR%plus PR%) was obtained in the segmented remedy group when compared with the index in the conventional remedy group (36.4%vs.12.5%, P=0.043<0.05).The incidence rate of platelet hematologic improvement (HI-P) in the conventional and segmented remedy groups was41.7%and69.7%respectively (P=0.034), showing a much better platelet response in the latter group.The platelet counts of MDS patients who finally achieved HI-P in the segmented group usually began to rebound during the second to third course whereas it was the third to forth course during which platelet counts began to rebound in the conventional group.In the conventional group,13cases were transfusion-dependent at baseline,4cases (30.8%) became transfusion-independent during the trial. In the segmented group,24cases were transfusion-dependent at baseline,16cases (66.7%) became transfusion-independent during the trial, P=0.036, indicating that the segmented treatment and evaluation system can alleviate transfusion-dependence.7. Comparision of the median courses of decitabine in the conventional and segmented remedy systemsThe number of medication courses in the conventional scheme reached to5as the upper limit with a median of4while the courses in the segmented scheme reached to8at most, with a median of7. The therapeutic courses were noteworthyly increased in the segmented remedy group so as to guarantee the consecutive demethylation therapy.Conclusion:Different from the therapeutic strategy in the conventional system, prophylactic antibiotics, antivirals and antifungals were simultaneously applied in the segmented system, resulting in reduced infection; patients incorporated in the segmented system were given best supportive care such as hematopoietic growth factors or blood-component transfusion in time to promoting the hematological recovery, leading to diminished dose delay or dose reduction; the tolerance and recovery situation in peripheral blood and bone marrow were detected in different time periods in the segmented system. The segmented system of decitabine turned out to have more access to guaranteeing the consecutive and sufficient medication courses, reducing the opportunities for hematologic toxicity and secondary infection, dose delay or blood transfusion, and ultimately improving the overall response rate of MDS patients who were newly diagnosed or nonresponsive to previous therapies. |
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