Myelodysplastic Syndrome Refractory Anemia Research Articles

Glutathione promotes the synergistic effects of venetoclax and azacytidine against myelodysplastic syndrome‑refractory anemia by regulating the cell cycle.

Azacitidine is a DNA methyltransferase inhibitor that has been used as a singular agent for the treatment of myelodysplastic syndrome-refractory anemia with excess blast-1 and -2 (MDS-RAEB I/II). However, recurrence and overall response rates following this treatment remain unsatisfactory. The combination of azacitidine and venetoclax has been used for the clinical treatment of a variety of hematological diseases due to the synergistic killing effect of the two drugs. Venetoclax is a BCL-2 inhibitor that can inhibit mitochondrial metabolism. In addition, azacitidine has been shown to reduce the levels of myeloid cell leukemia 1 (MCL-1) in acute myeloid leukemia cells. MCL-1 is an anti-apoptotic protein and a potential source of resistance to venetoclax. However, the mechanism underlying the effects of combined venetoclax and azacitidine treatment remains to be fully elucidated. In the present study, the molecular mechanism underlying the impact of venetoclax on the efficacy of azacitidine was investigated by examining its effects on cell cycle progression. SKM-1 cell lines were treated in vitro with 0-2 µM venetoclax and 0-4 µM azacytidine. After 24, 48 and 72 h of treatment, the impact of the drugs on the cell cycle was assessed by flow cytometry. Following drug treatment, changes in cellular glutamine metabolism pathways was analyzed using western blotting (ATF4, CHOP, ASCT2, IDH2 and RB), quantitative PCR (ASCT2 and IDH2), liquid chromatography-mass spectrometry (α-KG, succinate and glutathione) and ELISA (glutamine and glutaminase). Venetoclax was found to inhibit mitochondrial activity though the alanine-serine-cysteine transporter 2 (ASCT2) pathway, which decreased glutamine uptake. Furthermore, venetoclax partially antagonized the action of azacitidine through this ASCT2 pathway, which was reversed by glutathione (GSH) treatment. These results suggest that GSH treatment can potentiate the synergistic therapeutic effects of venetoclax and azacitidine combined treatment on a myelodysplastic syndrome-refractory anemia cell line at lower concentrations.

The effect of granulocyte-colony stimulating factor, decitabine, and busulfan–cyclophosphamide versus busulfan–cyclophosphamide conditioning on relapse in patients with myelodysplastic syndrome or secondary acute myeloid leukaemia evolving from myelodysplastic syndrome undergoing allogeneic haematopoietic stem-cell transplantation: an open-label, multicentre, randomised, phase 3 trial

Relapse remains high in patients with myelodysplastic syndrome-refractory anaemia with excess blasts (RAEB) or secondary acute myeloid leukaemia evolving from myelodysplastic syndrome undergoing allogeneic haematopoietic stem-cell transplantation (HSCT). We aimed to investigate whether granulocyte-colony stimulating factor (G-CSF) and decitabine plus busulfan-cyclophosphamide conditioning reduced relapse compared with busulfan-cyclophosphamide in this population. We did an open-label, randomised, phase 3 trial at six hospitals in China. Eligible patients (aged 14-65 years) had myelodysplastic syndrome-RAEB or secondary acute myeloid leukaemia evolving from myelodysplastic syndrome, and an Eastern Cooperative Oncology Group performance status of 0-2 and HSCT comorbidity index of 0-2. Patients were randomly assigned (1:1) to receive G-CSF, decitabine, and busulfan-cyclophosphamide conditioning or busulfan-cyclophosphamide conditioning. Randomisation was done with permuted blocks (block size four) with no stratification and was implemented through an interactive web-based response system, which was independent of study site staff and investigators. G-CSF, decitabine, and busulfan-cyclophosphamide conditioning comprised G-CSF 5 μg/kg daily subcutaneously (days -17 to -10), decitabine 20 mg/m2 daily intravenously (days -14 to -10), busulfan 3·2 mg/kg daily intravenously (days -7 to -4), and cyclophosphamide 60 mg/kg daily intravenously (days -3 and -2). Busulfan-cyclophosphamide conditioning comprised the same dose and duration of busulfan and cyclophosphamide. The primary endpoint was 2 year cumulative incidence of relapse. All efficacy and safety endpoints were assessed in the intention-to-treat population. This trial is registered with ClinicalTrials.gov, NCT02744742; the trial is complete. Between April 18, 2016, and Sept 30, 2019, 297 patients were screened for eligibility, 202 of whom were randomly assigned to G-CSF, decitabine, and busulfan-cyclophosphamide (n=101) or busulfan-cyclophosphamide (n=101) conditioning. 123 (61%) participants were male and 79 (31%) were female. Median follow-up was 32·4 months (IQR 10·0-43·0). The 2-year cumulative incidence of relapse was 10·9% (95% CI 5·8-17·9) in the G-CSF, decitabine, and busulfan-cyclophosphamide group and 24·8% (16·8-33·5) in the busulfan-cyclophosphamide group (hazard ratio 0·39 [95% CI 0·19-0·79]; p=0·011). Within 100 days after transplantation, the most common grade 3-4 adverse events in the G-CSF, decitabine, and busulfan-cyclophosphamide group and the busulfan-cyclophosphamide group were infections (34 [34%] and 32 [32%]), acute graft-versus-host disease (30 [30%] and 30 [30%]), and gastrointestinal toxicity (28 [28%] and 29 [29%]). 11 (11%) patients in the G-CSF, decitabine, and busulfan-cyclophosphamide group and 13 (13%) in the busulfan-cyclophosphamide group died of adverse events. There were no treatment related deaths. Our results suggest that G-CSF, decitabine, and busulfan-cyclophosphamide conditioning is a better choice than busulfan-cyclophosphamide conditioning for patients with myelodysplastic syndrome-RAEB or secondary acute myeloid leukaemia evolving from myelodysplastic syndrome undergoing allogeneic HSCT. This conditioning could be a suitable therapuetic option for this patient population. None. For the Chinese translation of the abstract see Supplementary Materials section.

Treatment Patterns and Characteristics Among Patients with Myelodysplastic Syndromes Initiating Oral Decitabine and Cedazuridine or Intravenous/Subcutaneous Hypomethylating Agents in a Real-World Setting

Introduction: Hypomethylating agents (HMAs) are recommended as standard of care treatment for patients with higher-risk myelodysplastic syndromes (MDS); however, intravenous (IV) and subcutaneous (SC) administration of HMA therapy has been associated with additional patient burden. In July 2020, the HMA oral decitabine and cedazuridine (DEC-C) was approved by the US Food and Drug Administration (FDA) for the treatment of MDS, as an alternative to IV or SC HMAs. This study reports initial results evaluating DEC-C and IV/SC HMA treatment patterns and population characteristics among MDS patients in a real-world setting. Methods: This retrospective observational study utilized the IQVIA PharMetrics® Plus database which comprises adjudicated claims for more than 190 million unique patients across the US. Adults with ≥1 claim for HMAs between July 1, 2020 and November 30, 2021, and continuous enrollment for 6-months prior to and ≥1-month following the index date were included. Patients newly initiating HMA therapy were identified, where index was the date of the first claim for DEC-C (DEC-C-new cohort) or IV/SC HMA therapy (IV/SC-new cohort), and patients had no claims for HMA therapy in the prior 6 months; a third cohort including all patients initiating DEC-C as either new HMA therapy or switching from IV/SC HMAs was also identified, where index was the date of first claim for DEC-C (DEC-C-all cohort). Patients in the IV/SC-new cohort also had to have ≥1 diagnosis codes for high-grade MDS lesions and/or refractory anemia with excess blasts. Patients had a variable follow-up period of up to 1-year post-index and were followed through December 31, 2021. Demographic/clinical characteristics and treatment patterns were evaluated across the 3 cohorts according to HMA therapy. Results: Of 208 patients meeting inclusion criteria, 90 initiated DEC-C (DEC-C-all) and 118 initiated IV/SC HMAs (IV/SC-new) during the study period; 59 patients newly initiated HMA therapy with DEC-C (DEC-C-new). Demographic and clinical characteristics were similar across the 3 cohorts (Table), with no statistically significant differences observed between the DEC-C and IV/SC cohorts newly initiating HMA therapy. Overall, the proportion of males was higher (vs females) across the DEC-C-all, DEC-C-new, and IV/SC-new cohorts (61.1%, 62.7%, and 53.4%, respectively), and most patients were aged ≥55 years (93.4%, 91.5%, and 80.4%; median age 65, 67, and 64 years, respectively). All 3 groups had high levels of comorbidity, with mean Charlson Comorbidity Index (CCI) scores of 3.4, 3.3, and 3.1, respectively, and 27.8%, 27.1%, and 24.6% having CCI scores ≥5. Across the DEC-C-all, DEC-C-new, and IV/SC-new groups, common comorbidities included malignancy (58.9%, 55.9%, and 46.6%, respectively), chronic pulmonary disease (25.6%, 27.1%, and 22.9%) and congestive heart failure (20.0%, 15.3%, and 17.8%). In all groups, some patients had a diagnosis of acute myeloid leukemia (AML) during the pre-index period (20.0%, 10.2%, and 21.2%, respectively), and a small proportion received hematopoietic stem cell transplantation (HSCT) pre-index (6.7%, 1.7%, and 1.7%). Red blood cell transfusions were reported in 57.8%, 54.2%, and 53.4%, and platelet transfusions in 28.9%, 25.4%, and 22.9%, for the DEC-C-all, DEC-C-new, and IV/SC-new groups, respectively. Among patients with ≥6-months continuous enrollment post-index, mean number of HMA cycles of therapy (based on number of claims) over the 6-months post-index were similar (4.1, 4.1, and 4.2 cycles in the DEC-C-all, DEC-C-new, and IV/SC-new cohorts, respectively). Conclusions: To our knowledge, this study provides the first real-world data on treatment patterns and characteristics among MDS patients initiating oral DEC-C. Characteristics were similar among patients initiating DEC-C and IV/SC HMAs; data also suggest ongoing off-label use of HMAs post-HSCT transplant and in AML. The similar number of HMA claims between groups suggests comparable compliance with oral therapy at home vs IV/SC treatment in the clinical setting, with a possible advantage of DEC-C in reducing the treatment burden associated with existing IV/SC HMA therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MDS-516 A Rare Case of X-Linked Recessive Sideroblastic Anemia (XLSA) Masquerading as Myelodysplastic Syndrome-Refractory Anemia With Ringed Sideroblasts (MDS - RARS)

XLSA may be confused clinically with MDS-RARS, which can have patient management implications. Sideroblastic anemia is categorized as a rare type of anemia by National Organization for Rare Disorders. Fewer than 200 cases with less than 100 unrelated probands have been described for X-linked recessive sideroblastic anemia (XLSA). XLSA is the most common type of non-syndromic congenital sideroblastic anemias (CSA), with almost 40% of all CSA cases involving mutations in the ALAS2 (δ- aminolaevulinic acid synthase) gene. Male patients (two-thirds of known cases) usually present in childhood or adolescence with symptoms of anemia, while female patients present in middle age. We present a case of XLSA in a male patient with symptom-onset in middle age. He initially presented with symptoms of anemia and received multiple blood transfusions over a period of 10 years. Subsequent bone marrow biopsy showed a hypercellular bone marrow with erythroid hyperplasia, increased iron and ringed sideroblasts consistent with refractory anemia with ringed sideroblasts, no abnormal karyotype in cytogenetics, and normal flow cytometry results. A liver biopsy showed hemosiderotic changes, and he was initially considered to have myelodysplastic syndrome-refractory anemia with ringed sideroblasts (MDS-RARS). Genetic testing, including SF3B1 gene mutation, revealed no findings suggestive of MDS. The patient was then started on iron chelating agents and pyridoxine with improvement in anemia and did not require any further transfusions with significant improvement in his symptoms. A hereditary anemia NGS gene sequencing and deletion/duplication panel was then done which showed pathogenic X-linked recessive hemizygous mutation involving ALAS2 gene-OMIM 301300. Response to the pyridoxine treatment, absence of SF3B1 gene mutation, and presence of ALAS2 gene mutation helped confirm the diagnosis of XLSA in our patient. This case report highlights the necessity for extensive workup in patients with sideroblastic anemia, given the rarity of XLSA and similarity in its clinical symptoms to MDS-RARS, and the need for its early diagnosis. In our patient, iron overload and subsequent liver cirrhosis developed due to multiple transfusions for refractory sideroblastic anemia which was treated as MDS-RARS and could have been avoided if XLSA had been suspected earlier in the course of the disease.

Intestinal Beh\xe7et\u2019s disease complicated by myelodysplastic syndrome and secondary pulmonary alveolar proteinosis: a case report

BackgroundGastrointestinal lesions, which sometimes develop in Behçet’s disease (BD), are referred to as intestinal BD. Although rare, intestinal BD can be accompanied by myelodysplastic syndrome (MDS) with abnormal karyotype trisomy 8, which is refractory to immunosuppressive therapy. Pulmonary alveolar proteinosis is a rare lung complication of BD and MDS. Herein, we present an extremely rare case of intestinal BD presenting with MDS and several chromosomal abnormalities, followed by secondary pulmonary proteinosis.Case presentationA 58-year-old Japanese woman with a 3-year history of genital ulcers and oral aphthae was admitted to our hospital. The patient developed abdominal pain and persistent diarrhea. Colonoscopy revealed multiple, round, punched-out ulcers from the terminal ileum to the descending colon. Intestinal BD was diagnosed and the patient was treated with colchicine, prednisolone, and adalimumab. However, her symptoms were unstable. Bone marrow examination to investigate the persistent macrocytic anemia revealed the presence of trisomy 8, trisomy 9, and X chromosome abnormalities (48, + 8, + 9, X, i(X) (q10) in 12 out of the examined 20 cells). Based on her hypoplastic bone marrow, the patient was diagnosed with low-risk MDS (refractory anemia). At the age of 61, the patient developed pneumonia with fever and diffuse ground-glass opacities on the lung computed tomography (CT). Chest high-resolution CT and histopathology via transbronchial lung biopsy revealed the presence of pulmonary alveolar proteinosis (PAP). These findings combined with the underlying disease led to the diagnosis of secondary PAP.ConclusionsSecondary pulmonary proteinosis may accompany intestinal BD with MDS and several chromosomal abnormalities. Physicians should pay attention to lung complications, such as PAP, in patients with intestinal BD complicated by MDS. Genetic abnormalities may be associated with the development of such diseases.

Outcomes of Transplant-Eligible Patients with Myelodysplastic Syndrome-Refractory Anemia with Excess Blasts Registered in a Prospective Observational Study: The JALSG-CS11-MDS-SCT

Outcomes of Transplant-Eligible Patients with Myelodysplastic Syndrome-Refractory Anemia with Excess Blasts Registered in a Prospective Observational Study: The JALSG-CS11-MDS-SCT

The cell body space occupied by the nucleus during the cell differentiation in human lymphocytic, granulocytic and erythroid cell lineages.

The present nuclear and cell body diameter measurements demonstrated size differences of the approximate cell space estimate occupied by the cell nucleus during the cell differentiation in lymphocytic, granulocytic and erythroid cell lineages. These lineages were used as convenient models because all differentiation steps were easily identified and accessible in diagnostic peripheral blood or bone marrow smears of blood donors (BDs), patients suffering from chronic lymphocytic leukemia (CLL), patients with chronic myeloid leukemia (CML) and refractory anemia (RA) of the myelodysplastic syndrome (MDS). The cell space occupied by the nucleus was constant and did not change during the cell differentiation in the lymphocytic cell lineages of BDs and CLL patients despite the decreased cell size. In contrary, the cell space occupied by the nucleus markedly decreased in differentiating cells of granulocytic and erythroid lineages of patients suffering from CML. In the erythroid cell lineage in patients with RA of MDS the small reduction of the cell space occupied by the nucleus during the differentiation was not significant. The measurements also indicated that in progenitor cells of all studied cell lineages nuclei occupied more than 70 % of the cell space. Thus, the nucleus-cytoplasmic morphological and functional equilibrium appeared to be characteristic for each differentiation step and each specific cell lineage.

A New Recurrent Translocation, t(5;11)(q35;p15.5), Associated With del(5q) in Childhood Acute Myeloid Leukemia

AbstractPartial deletion of the long arm of chromosome 5, del(5q), is the cytogenetic hallmark of the 5q-syndrome, a distinct subtype of myelodysplastic syndrome-refractory anemia (MDS-RA). Deletions of 5q also occur in the full spectrum of other de novo and therapy-related MDS and acute myeloid leukemia (AML) types, most often in association with other chromosome abnormalities. However, the loss of genetic material from 5q is believed to be of primary importance in the pathogenesis of all del(5q) disorders. In the present study, we performed fluorescence in situ hybridization (FISH) studies using a chromosome 5-specific whole chromosome painting probe and a 5q subtelomeric probe to determine the incidence of cryptic translocations. We studied archival fixed chromosome suspensions from 36 patients with myeloid disorders (predominantly MDS and AML) and del(5q) as the sole abnormality. In 3 AML patients studied, this identified a translocation of 5q subtelomeric sequences from the del(5q) to the short arm of an apparently normal chromosome 11. FISH with chromosome 11-specific subtelomeric probes confirmed the presence of 11p on the shortened 5q. Further FISH mapping confirmed that the 5q and 11p translocation breakpoints were the same in all 3 cases, between the nucleophosmin (NPM1) and fms-related tyrosine kinase 4 (FLT4) genes on 5q35 and the Harveyras-1–related gene complex (HRC) and the radixin pseudogene (RDPX1) on 11p15.5. Importantly, all 3 patients with the cryptic t(5;11) were children: a total of 3 of 4 AML children studied. Two were classified as AML-M2 and the third was classified as M4. All 3 responded poorly to treatment and had short survival times, ranging from 10 to 18 months. Although del(5q) is rare in childhood AML, this study indicates that, within this subgroup, the incidence of cryptic t(5;11) may be high. It is significant that none of the 24 MDS patients studied, including 11 confirmed as having 5q-syndrome, had the translocation. Therefore, this appears to be a new nonrandom chromosomal translocation, specifically associated with childhood AML with a differentiated blast cell phenotype and the presence of a del(5q).

Clinical Efficacy of Low-dose Decitabine Combined with CAG Regimen in Patients with Myelodysplastic Syndrome-refractory Anemia with Excess Blasts(MDS-RAEB)

To investigate the clinical efficacy of low-dose decitabine combined with CAG regimen in patients with myelodysplastic syndrome-refractory anemia with excess blasts (MDS-RAEB) through retrospective analysis. Thirty-six patients with MDS-RAEB who ever received low-dose decitabine combined with CAG regimen were enrolled into decitabine + CAG group and 40 patients with MDS-RAEB treated by decitabine alone in our center were enolled into the control group. The clinical characteristics, efficacy and adverse reactions (AE) were compared between the 2 groups. Compared with the control group, the overall response rate (ORR) [complete remission (CR)+partial remission (PR) + hematologic improvement (HI) rate] of the decitabine+CAG group was higher (83.3% vs 62.5%)(P=0.043), and the AEs were not significantly increased. Cytopenia grade ≥3 and infection after treatment were the most prevalent AEs, which occurred in the early stage (within the first 2 cycles) and gradually decreased later. Other non-hematologic AEs were infrequent. Low-dose decitabine combined with CAG regimen has better clinical efficacy for patients with MDS-RAEB than that of decitabine alone. It is worthy to be applied in clinic, especially for these patients who are not ineligible for hematopoietic stem cell transplantation.

"C1ORF150", a Novel Mediator of EPO/EPOR/JAK2 Dependent Human Erythroid Progenitor Cell Formation

An understanding of cellular events that are propagated within erythroid progenitor cells upon HGF-R / JAK complex activation is of basic importance for generating new insight into regulated red cell formation, anemia and myeloproliferative disease. Using the EPO/EPOR/JAK2 system as a paradigm, our group is successfully applying post-translational modification-based proteomics to uncover important new mediators of EPO-dependent human erythropoiesis (certain of which may also relate to EPO's untoward effects on hypertension and cancer progression). Here, we report on the discovery of a novel ORF, "C1ORF150", that is strongly tyrosine phosphorylated in response to EPO, possesses several unique features, and modulates EPO- dependent erythroid progenitor cell formation.In human erythroid progenitor UT7epo cells, EPOR ligation leads to C1ORF150 phosphorylation at tandem tyrosine p-Y69, p-Y89 and p-Y110, p-Y129 sites (up to 10-fold within 15 minutes). For each PTM site, EPOR/JAK2 mediated- phosphorylation was validated in independent LC-MS/MS experiments using Hematide as an EPOR agonist. p-Y69 and p-Y89 are predicted SFK sites, while p-Y110 and p-Y129 are predicted RTK sites (including KIT). Notably, C1ORF150 is conserved in H. sapiens and primates, but is not represented in mouse, rat or lower vertebrates. In addition, C1ORF150 has no obvious orthologues, but within EPO-regulated pY regions exhibits sequence homology with HGAL, an important factor for B cell receptor signaling.To assess C1ORF150’s functional effects, we used a lentiviral shRNA loss-of-function approach (80% knockdown efficiency). At physiological EPO levels, the knockdown of C1ORF150 substantially compromised UT7epo erythroid progenitor cell (EPC) survival, including 200% increases in apoptosis observed relative to control sh-NT transduced EPCs (p < 0.01). The ectopic expression of C1ORF150, in contrast, heightened baseline JAK2 activation, and potentiated STAT5 activation following EPO challenge. C1ORF150’s subcellular localization proved to be predominantly membrane associated.With regards to expression profiles, C1ORF150 levels were markedly elevated in bone marrow (among 30 human tissues), and during erythroid development were maximal at a CFUe stage. Furthermore, transcriptome profiles of myelodysplastic syndrome (MDS) CD34pos hematopoietic progenitor cells revealed elevated C1ORF150 expression in MDS refractory anemia (p=0.005) and refractory anemia-blast patients (p=0.05) compared to normal controls. In summary, via PTM-proteomics we have discovered "C1ORF150" as a major new pY- regulated EPOR/JAK2 target and membrane associated phosphoprotein that is proposed to have evolved in human erythroid progenitor cells to support EPO's cytoprotective effects, and red cell formation, in part by reinforcing JAK2 and STAT5 activation. In anemia and pre-leukemic contexts, attention also is brought to possible roles for C1ORF150 in the onset and progression of MDS. DisclosuresNo relevant conflicts of interest to declare.

Analysis of the impact of decitabine treatment cycles on efficacy and safety in patients of myelodysplastic syndrome-refractory anemia with excess blasts

目的评估地西他滨治疗骨髓增生异常综合征(MDS)难治性贫血伴有原始细胞过多(RAEB)患者的疗效和不良反应与治疗疗程数的关系。方法2008年12月至2016年3月中国医学科学院血液病医院MDS诊疗中心共有56例RAEB患者接受了地西他滨20 mg·m−2·d−1×5 d方案治疗，回顾性分析其临床特征、疗效、疗效影响因素及不良反应。结果RAEB-1 25例，RAEB-2 31例。中位治疗3(1~15)个疗程。总有效[完全缓解(CR)＋骨髓完全缓解(mCR)+血液学改善(HI)]率为67.9%(38/56)，其中10例(17.8%)CR, 8例(14.3%)mCR但无HI，17例(30.4%)mCR同时有HI, 3例(5.4%)单纯HI。中位随访7.9(1.0~56.3)个月，中位生存期为21.1(95%CI 16.0~26.1)个月。RAEB-1患者的疗效(总有效率为88.0%)显著优于RAEB-2患者(总有效率为51.6%)(P=0.004)，多因素分析证实其为影响疗效的独立预后因素(OR=6.03，95%CI 1.39~26.09，P=0.016)。地西他滨治疗有效的38例患者中，37例(97.4%)在1~2个疗程起效，25例(65.8%)在2个疗程内达到最佳疗效，37例(97.4%)在4个疗程内达最佳疗效。主要不良反应为3级以上血细胞减少及感染，在治疗早期(第1~2疗程)中较常见，治疗有效后随疗程增加，发生率逐渐下降，其他非血液学不良反应发生率低。结论地西他滨标准剂量5天方案治疗RAEB疗效肯定。治疗有效患者常在第1~2个疗程起效，4个疗程内达最佳疗效。主要不良反应为血细胞减少及感染，常见于前2个疗程，起效后后续疗程不良反应发生率显著降低。

Detection of erythroblast antibodies in mitogen-stimulated bone marrow cultures from patients with myelodysplastic syndromes.

Low-risk myelodysplastic syndromes (MDS) show several immunologic abnormalities, including increased frequency of autoimmune manifestations and/or overt autoimmune diseases, whose prognostic significance still remains controversial. We studied the presence of erythroblast antibodies in mitogen-stimulated bone marrow (BM) cultures of 70 patients with early-stage MDS (refractory anemia and refractory anemia with ringed sideroblasts). Sixty-six percent of patients showed positive erythroblast antibodies, along with BM erythroid hyperplasia and a hemolytic picture in the peripheral blood. Supernatants from positive cultures induced an increase of overall cellularity, the appearance of erythroblastic clustering, and dyserythropoietic signs in normal BM. We identified CD45(dim) Gly-A(dim) CD71(bright) cells (red blood cell precursors at different maturation stage) as the target of the antibodies. Erythropoietin (EPO) levels were reduced and EPO receptors (EPO-R) increased in BM culture supernatants from positive patients. However, flow cytometric analysis showed that neither EPO nor EPO-R was involved in an abnormal stimulation driven by these autoantibodies. Values of the proapoptotic protein Bax were increased in positive patients and Bcl-2 levels were decreased, although not significantly. MDS patients with anti-erythroblast autoimmunity showed increased BM apoptosis, suggesting that the autoimmune reaction may contribute to an unfavorable BM microenvironment for optimal erythropoiesis.

Survey of experts on therapeutic policies and proposals for the optimal timing for allogeneic peripheral blood stem cell transplantation in transfusion-dependent patients with myelodysplastic syndrome-refractory anemia.

BackgroundMost hypomethylating agent (HMA) responders with myelodysplastic syndrome (MDS) eventually need allogeneic stem cell transplantation (SCT) because they often acquire resistance to HMAs within two years of treatment. Considering the nature of MDS and the poor outcomes of SCT when performed after confirming the progression of MDS to acute myeloid leukemia (AML), allogeneic SCT should be performed with caution in patients with low-risk MDS.MethodsTo address low-risk MDS, the Korean AML/MDS working party group designed a survey for 34 MDS experts in Korea on therapeutic HMA and allogeneic SCT policies for low-risk MDS. The level of consensus was defined as the percentage of agreement among the experts.ResultsWith regard to the optimal time for allogeneic SCT for HMA responders with MDS-RA, 76% experts agreed that allogeneic SCT should be performed when a patient has a low platelet count. With regard to the relapse pattern that was most commonly found during HMA treatment in responding patients with MDS-RA, 54% experts agreed that the most common pattern that indicated HMA failure was the gradual worsening of cytopenia.ConclusionThe optimal time to perform allogeneic SCT in RA patients who achieved hematologic complete remission during HMA treatment is when the platelet count decreases. However, these suggestions need to be evaluated in larger future studies. Therefore, careful decisions should be taken at each step of allogeneic SCT to maximize the outcomes for patients with MDS-RA and iron overload.

Granulocytic and Monocytic Myeloid-Derived Suppressor Cells May Have Different Role on Allogeneic Stem Cell Transplant Outcomes

Background: Myeloid-derived suppressor cells (MDSC), a heterogeneous group of myeloid cells, have emerged as immune regulators, having a high potential to suppress T cell responses. Although uniform characterization of human MDSC needs to be elucidated, they can be divided into the categories of granulocytic (G-MDSC) and monocytic (M-MDSC). Recent studies have reported that MDSC, generated in vitro or in vivo, alleviated the severity of graft-versus-host disease (GVHD) in murine allogeneic transplant models and in human delayed M-MDSC reconstitution was associated with the occurrence of acute GVHD. However, whether G-MDSC and M-MDSC may have different role on the outcomes after allogeneic stem cell transplantation (SCT) remains obscure.Methods: This prospective study was aimed to identify the clinical implications of early G-MDSC and M-MDSC expansion as a predictor for the occurrence of acute GVHD (aGVHD), infections, CMV reactivation, and survival outcomes after allogeneic SCT. The peripheral blood samples from 130 patients with acute myeloid leukemia and myelodysplastic syndrome-refractory anemia with excess blasts, who underwent allogeneic SCT between Jan. 2013 through Oct. 2014 were taken at engraftment and analyzed by flow cytometry.Results: Seventy-eight men and 52 women were enrolled in this study. The median age was 45.5 years (range, 17-68). To compare the predictive role of MDSC for various transplants, the patients were grouped according to the median values of the frequency of G-MDSC and M-MDSC. High G-MDSC at engraftment was a potential factor promoting the occurrence of ≥ grade 2 aGVHD at 100 days (30.8% vs. 47.7%, P = 0.023), whereas high M-MDSC group had no difference in the occurrence of ≥ grade 2 GVHD compared that of low M-MDSC group. There was no difference in CMV reactivation, infection rate, and TRM according to G-MDSC recovery. In contrast, patients in the high M-MDSC group had a higher cumulative incidence of infection at 100 days (25.1% vs. 48.2%, P = 0.002), and TRM (6.4% vs. 22.6%, P = 0.018), compared with the patients in the low group. Ultimately, multivariate analyses reveal that high G-MDSC had a trend for the occurrence of ≥ grade 2 GVHD at 100 days (RR 1.72, 95%CI (0.95-3.11), P = 0.071) and high M-MDSC could predict a higher infection rate (RR 2.30, 95%CI (1.30-4.07), P = 0.004) and higher transplant related mortality (TRM) (RR 3.30, 95%CI (1.10-9.90), P = 0.033). In addition, high M-MDSC was associated lower event-free survival (P = 0.008).Conclusion: Our data demonstrated that the high G-MDSC in the peripheral blood at engraftment was associated with a trend toward higher incidence of aGVHD and high M-MDSC was an independent factor for infection and TRM. Discrepancy of the role of G-MDSC and M-MDSC after allogeneic SCT suggests that difference of MDSC reconstitution into the more differentiated subset may predict transplant outcomes, including aGVHD, infections, and TRM. DisclosuresNo relevant conflicts of interest to declare.

To investigate the relationship between P15INK4B gene promoter methylation degree and different types of MDS and prognosis

Objective To determine the methylation level of P15INK4B gene promoter in different types of myelodysplastic syndromes (MDS) and its correlation with its prognosis. Methods Methylation frequency of the P15INK4B gene promoter in 44 cases of MDS were determined by methylation-specific polymerase chain reaction (PCR) and pyrosequencing, and its correlation with clinical classification and characteristics of MDS were statistically analyzed. Results Frequency of P15INK4B gene promoter methylation in myelodysplastic syndromes-refractory anemia with excess blasts Ⅱ (MDS-RAEB Ⅱ) patients was (46.89±15.41)%, significantly higher than that in other types of MDS (P 0.05); frequency of P15INK4B gene promoter methylation was found to be correlated with decline in platelet upon diagnosis (t=9.02, P 0.05). As for the correlation between P15INK4B gene promoter methylation and MDS risk stratification, no significant difference was detected between the low-risk and very low-risk groups (P> 0.05), but significant differences were detected among the medium-risk, high-risk, and very high-risk groups (P< 0.05). In addition, frequency of P15INK4B gene promoter methylation was (49.21±8.78)% in MDS patients that developed leukemia in the following two year, significantly higher than that in MDS patients who didn't (19.64±6.24)% (P<0.05). Conclusions P15INK4B gene promoter methylation frequency is a valuable indicator of prognosis of MDS patients. Key words: Tumor suppressor proteins/GE; Methylation; Myelodysplastic syndromes/GE; Prognosis

Effects of the Order of Administration of Total Body Irradiation and Cyclophosphamide on the Outcome of Allogeneic Hematopoietic Cell Transplantation

▪Background and objectives: Total body irradiation (TBI) combined with cyclophosphamide (CY) is one of the most common myeloablative conditioning regimens used in allogeneic hematopoietic stem-cell transplantation (HSCT) for treating hematological malignancies. However, it remains unclear whether the order of administrating TBI and CY has an effect on the outcome in clinical transplantation. The aim of this study is to clarify the effects of the order of TBI and CY administration on the outcome of allogeneic HSCT.Patients and Methods: We retrospectively investigated the clinical outcome of 504 adult patients with acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), and myelodysplastic syndrome who received allogeneic HSCT with myeloablative conditioning regimens consisting of TBI and CY at the transplant centers participating in the Kanto Study Group on Cell Therapy between January 2001 and August 2012. Patients were divided into two groups based on the order in which TBI and CY were administered. The outcome of HSCT and incidences of acute and chronic GVHD, sinusoidal obstruction syndrome / veno-occlusive disease, and idiopathic pneumonia were compared between the two groups.Patients who underwent HSCT during the first or second remission of acute leukemia or refractory anemia of myelodysplastic syndrome were considered as having standard-risk disease. All other conditions were considered as high-risk disease.Results: A total of 218 patients received CY before TBI (CY-TBI) and 286 received CY after TBI (TBI-CY). AML was more common in the CY-TBI group (62.8%) compared with the TBI-CY group (51.0%), and ALL was less common in the CY-TBI group (25.7%) compared with the TBI-CY group (37.8%; P = 0.013). High-risk disease was more frequent in the CY-TBI group (38.5%) compared with the TBI-CY group (23.4%; P < 0.001). The proportion of unrelated bone marrow (54.6% vs. 43.4%) and cord blood transplantation (22.9% vs. 17.8) were higher among patients in the CY-TBI group than in the TBI-CY group (P = 0.0014). TBI was administered at a dose of 12 Gy in 212 patients (97.2%) in the CY-TBI group and 266 patients (93.0%) in the TBI-CY group (P = 0.013). More patients received TBI administered in six fractions in the CY-TBI group (72.9%) than in the TBI-CY group (53.1%; P < 0.001). Female to female transplantation was lower in the CY-TBI (13.3%) group compared with the TBI-CY group (24.1%; P = 0.023). Age, gender, GVHD prophylaxis, and blood mismatch were not significantly different between the two groups.The order in which TBI and CY was administered did not affect the incidence of grades II–IV acute GVHD (45.3% vs. 49.3% at day 100; P = 0.28) and chronic GVHD (36.0% vs. 43.8% at 2 years; P = 0.10), overall survival (52.4% vs. 53.4% at 5 years; P = 0.44), disease-free survival (50.5% vs. 51.5% at 5 years; P = 0.58), relapse rate (30.2% vs. 31.8% at 5 years; P = 0.96) and non-relapse mortality (19.3% vs. 16.7% at 5 years; P = 0.52) in the two groups (CY-TBI and TBI-CY, respectively) by univariate analysis. Moreover, the cumulative incidences of sinusoidal obstruction syndrome / veno-occlusive disease (4.1% vs. 3.8%; P = 0.81) and idiopathic pneumonia were comparable (3.1% vs. 3.4%; P = 0.87) between the two groups (CY-TBI and TBI-CY, respectively).Conclusions: This study demonstrates that the order of administration of TBI and CY does not have an effect on the outcome of allogeneic HSCT. Further studies are warranted to confirm this result. DisclosuresNo relevant conflicts of interest to declare.

Comparison of DNA Methyltransferase Inhibitors in Patients with Myelodysplastic Syndrome

The aim of this retrospective study was to compare the response rates (RR), overall survival (OS), time–to–acute myeloid leukemia–transformation (TTAMLT), hematologic toxicity and transfusion requirement of azacitidine and decitabine in patients with Myelodysplastic Syndrome Refractory Anemia with Excess Blasts (MDS RAEB) types 1 and 2. A total of 51 patients (35 male, 16 female, median age (years): 67) diagnosed with MDS RAEB-1 and RAEB-2 treated with azacitidine (n=31) or decitabine (n=20) in three Turkish institutes were evaluated . Response to treatment was classified according to International Working Group response criterias. Competing risk analysis was used to compare TTAMLT. There was no significant difference between the two groups in terms of age, gender, performance score, diagnosis, etiology, treatment cycles, bone marrow cellularity and blast percentage. Most patients received at least 4 cycles of azacitidine or decitabine. Patients who received azacitidine or decitabine had comparable response rates (58.1% vs 66.7%, respectively, p= 0.55). There was no difference in terms of red blood cell and platelet transfusion requirement and febrile neutropenia episodes (p= 0.46, p= 0.27, p= 0.19, respectively). Median follow–up was 31 months for azacitidine and 20 months for decitabine. Median OS was 18.4 months for azacitidine and 13.4 months for decitabine (p= 0.16). Only predictor of OS was at least 4-cycle treatment with any DNA methyltransferase inhibitors. 1–year risk of AML transformation was comparable across the groups (26% vs 33%, p= 0.55). In conclusion, both azacitidine and decitabine have similar efficacy and toxicity profiles in the treatment of MDS RAEB-1 and RAEB-2. Until a head–to–head comparison in prospective, randomized studies are conducted, the current source of available data will derive from meta–analyses that consist of the indirect comparison of treatment arms, or retrospective analyses with inevitable biases. DisclosuresNo relevant conflicts of interest to declare.

A Novel Hemizygous I418S Mutation in the ALAS2 Gene in a Young Korean Man with X-Linked Sideroblastic Anemia

A Novel Hemizygous I418S Mutation in the ALAS2 Gene in a Young Korean Man with X-Linked Sideroblastic Anemia

Salvaged Cord Blood Transplantation For 19 Patients With Progressive Hematologic Malignancies

Few clinical studies have investigated the role of salvaged unrelated cord blood transplantation (CBT) for progressive hematologic malignancies. The aim of this report is to identify the potential benefits of unrelated CBT in progressive hematologic malignancies.19 consecutive patients with progressive myeloid and lymphoid malignancies who received salvaged CBT following myeloablative conditioning regimens (12 TBI/CY/Ara-C, 6 Bu / CY / Ara-C, and 1 Bu /CY) from July 2005 to December 2012 were analyzed retrospectively. Of the 19 patients, 6 suffered from acute myeloid leukemia(AML), 5 acute lymphoid leukemia(ALL),：1 acute mixed lineage leukemia (AMLL), 1 myelodysplastic syndrome-refractory anemia with excess blasts(MDS-RAEB), 2 acute myeloid leukemia transformed from myelodysplastic syndrome, and 4 lymphoma, all of them in non-remission (NR) before transplantation. Median age of them were 13（range 6-32）years and median body weight were 45 (range 18–73 ) kg. All patients received 1 CBT unit ≤2 loci HLA-mismatched with the recipient. Infused TNC was 4.07（range 2.76-6.02）×107/kg and CD34+ stem cell 2.08（range 0.99-8.65）×105/kg. All patients were engrafted with neutrophil exceeded 0.5×109/L on median day +17（range 14-37d）and plt counts of＞20×109/L on median day +35 (range 17-70d). 10 patients (52.6%) experienced pre-engraftment syndrome (PES) and 3 (15.8%) patients progressed to acute GVHD. The incidence of Ⅱ-Ⅳ aGVHD and cGVHD were 10.5% and 21.1%. With a median follow up of 10(range 1-58) months，10 cases survived and 2 relapsed．The estimated 2 year overall survival (OS), disease-free survival (DFS) and non-relapse mortality (NRM) rate was 45.6%, 34.2% and 39.8%. Salvaged CBT might be a promising modality for treatment of progressive hematologic malignancies, even with high leukemia burden. Disclosures:No relevant conflicts of interest to declare.

Impairment of HuR-Mediated FOS mRNA Stabilization in Granulocytes From Myelodysplastic Syndrome Patients.

Abstract 2805Infection is a major cause of death in patients with myelodysplastic syndromes (MDS). Although qualitative and quantitative granulocyte defects have been described, the underlying molecular basis of granulocyte dysfunction in MDS is largely unknown. Recently, we found that FOS mRNA elevation following translation inhibition was significantly smaller in MDS granulocytes than in healthy granulocytes (Shikama et al, Br J Haematol 154, 525–527, 2011). FOS is an immediate early gene that is rapidly expressed in response to various stimuli and cellular stresses, and possesses an AU-rich element (ARE) in its mRNA 3’UTR, to which mRNA stability-regulating proteins, such as HuR, bind. The aim of this study was to clarify the cause of impaired FOS induction following translation inhibition in MDS.We first confirmed impaired FOS induction in MDS by comparing 17 patients with MDS (refractory anemia) with 17 age-matched healthy controls. Granulocyte fractions, enriched to >90% neutrophils, were cultured with 200 ng/mL of the translation inhibitor emetine at 37 °C for 30 min. FOS mRNA increased 6.2±1.1-fold in controls, vs. 3.5±0.8-fold in MDS (p<0.01). In contrast, when 5 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) or 100 ng/mL lipopolysaccharide (LPS) was added instead of emetine, there were no significant differences in the FOS mRNA elevation between controls and MDS (GM-CSF: 2.4±0.5-fold vs. 2.3±1.3-fold, LPS: 2.2±0.9-fold vs. 2.4±1.1-fold), suggesting that FOS mRNA induction by a translation inhibitor was specifically impaired in MDS granulocytes.To study the mechanisms of FOS mRNA elevation by emetine, we next examined the involvement of (1) MAPK signaling pathways, (2) FOS transcription and mRNA stabilization, and (3) ARE-binding protein HuR, using healthy granulocytes. Inhibition of MAPK p38, which completely blocked the FOS-upregulating effect of LPS, reduced FOS elevation by emetine to nearly 50%. Neither inhibition of JNK nor ERK altered the effects of emetine. Thus, emetine increases FOS mRNA via MAPK p38-dependent and MAPK-independent mechanisms. Emetine treatment for 30 min increased nascent FOS mRNA 3.5±0.8-fold, which was reduced to 1.1±0.8-fold by MAPK p38 inhibition (p<0.01). When transcription was inhibited, FOS mRNA decayed to 19.2±9.1% of the initial level in 45 min. In the presence of emetine, 78.1±23.5% of FOS mRNA remained at 45 min, and was not affected by p38 inhibition. These data indicate that FOS mRNA elevation by emetine results from both upregulation of transcription via MAPK p38 and p38-independent mRNA stabilization. RNA-protein complex immunoprecipitation showed that 2.5±1.2-fold more FOS mRNA 3’UTR coprecipitated with HuR in emetine-treated cells than in untreated cells (p<0.05). GAPDH mRNA 3’UTR without ARE was not detected regardless of emetine treatment. Although HL60 cells treated with HuR and control siRNA showed similar FOS mRNA decay rates in the absence of emetine, emetine treatment led to a significantly lower FOS mRNA at 45 min in HuR-reduced cells (48.0±12.8%) than in control cells (77.4±13.9%, p<0.05). Thus, emetine stabilizes FOS mRNA via upregulated interaction of HuR with FOS mRNA.Finally, we investigated which process was impaired in MDS, p38-mediated FOS transcription, or HuR-mediated mRNA stabilization. The increase of FOS transcription in MDS (2.0±0.3-fold) did not significantly differ from that in controls (2.4±0.3-fold). When FOS transcription was blocked by a MAPK p38 inhibitor, FOS mRNA increase by emetine was significantly smaller in MDS (2.9±0.7-fold) than in controls (4.4±1.5-fold, p<0.05), suggesting that the aberrant FOS induction in MDS was not due to impaired FOS transcription. FOS mRNA, which decayed similarly in MDS and control cells in the absence of emetine, remained 41.9±24.6% and 61.0±19.9% in MDS and control granulocytes, respectively, at 45 min in the presence of emetine (p<0.05).In conclusion, the aberrant FOS mRNA induction by emetine in MDS results from the impairment of HuR-mediated FOS mRNA stabilization. Since nearly 15% of the transcriptome in mammalian cells is targeted by HuR, these results may provide new insights into the functional defects in MDS. Disclosures:No relevant conflicts of interest to declare.