Non-severe Aplastic Anemia Research Articles

Plasma Concentration of Eltrombopag Correlated with the Efficacy in the Treatment of Refractory Aplastic Anemia: A Single-Center Study in China

Background and purpose: Eltrombopag (ELT) was effective in the treatment of relapse/refractory aplastic anemia (AA) patients. Response and adverse drug reaction (ADR) differed greatly among individuals even at the same dosage of ELT. Methods: Patients diagnosed with non-severe aplastic anemia (NSAA) between January 2018 to January 2019 in Peking Union Medical Colleague Hospital who were refractory to immunosuppressive therapy were treated with ELT and followed up for at least 6 months. Plasma concentrations of ELT were detected by the high-performance liquid chromatography-mass spectrometry after at least two months of ELT treatment and staying for that dosage for at least two weeks. Dose-concentration, concentration-response and concentration- ADR relation were evaluated. Results: Among the 72 patients treated with ELT during that period, 44 patients with complete data were enrolled. 6 (13.6%) were males and 38 were females (86.4%) with a median age of 54 years (IQR: 38.5-63). By the time of plasma concentration detected, the median dosage of ELT was 75(IQR 50-100) mg/d and median time for total ELT exposure was 3 (IQR 2.0-6.0) months, and 37 (70.5%) patients responded to ELT. The median concentration of ELT was 10.4μg/ml (IQR 3.7-24.4μg/ml). The concentration of ELT was positively correlated with the daily dose of ELT (r = 0.68, p < 0.001). Multivariate logistic regression analysis showed that the risk of inefficacy of ELT with a concentration between 11.2-15.2 μg/ml was 0.028-fold (95% CI: 0.001-0.864; P=0.041) to those with a concentration between 3.2-7.2μg/ml. The cutoff value of concentration for ELT efficacy was 12.50μg/ml by receiver operation characteristic curve. A higher risk of ADR was related to a longer total exposure to ELT (P=0.012). Although not significant, odd ratio (OR) increased with ELT concentration, suggesting a possible elevated risk of ADR correlated with blood concentration. Conclusions: ELT is effective for the treatment of NSAA with accepTable side effects. Plasma concentration of ELT correlated with the dose and the effects of ELT. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Eltrombopag (ELT) has been proven to induce trilinear hematopoietic responses in relapse/refractory AA patients and has been approval as a monotherapy in relapsed/refractory SAA in the USA and Europe. However, the use of ELT is only approved for idiopathic thrombocytopenic purpura (ITP) in China by the National Medical Products Administration (NMPA), for AA it is still "off-label".

The change of quality of life in 52 patients with non-severe aplastic anemia after cyclosporine A therapy

目的研究非重型再生障碍性贫血（NSAA）患者接受环孢素A（CsA）治疗前后生活质量的变化，并分析生活质量改善的影响因素。方法收集2014年1月至2016年1月在北京协和医院门诊初次治疗、仅使用CsA的NSAA患者，在治疗前和治疗2年后分别填写SF-36量表，与正常中国人模型（常模）比较；并调查评估患者的年龄、性别、文化程度、家庭年收入、付费方式、依从性、治疗前一般健康状况及治疗2年后的疗效，分析其与生活质量改善的关系。结果共52例患者符合入组条件，其中男性27例（51.9％），女性25例（48.1％），中位年龄48（21～85）岁。CsA治疗2年，完全治疗反应（CR）15例（28.8％），部分治疗反应（PR）25例（48.1％），无治疗反应（NR）12例（23.1％），治疗总有效率（CR+PR）76.9％。患者治疗前SF-36量表躯体和心理健康得分差于常模（P值均<0.05）。治疗后，躯体方面及总体生活质量虽仍然低于常模（P值均<0.05），但心理健康与常模比较差异无统计学意义（P值均>0.05），精力（VT）、精神健康（MH）得分高于常模［VT：（58.8±20.1）分对（52.3±20.9）分，P＝0.023；MH：（65.9±17.6）分对（59.7±22.9）分，P＝0.014］。不同社会背景与生活质量改善均无显著相关性。起病时体能状况评分（ECOG评分）较低者生活质量改善更大。CsA治疗有效者生活质量均得到显著改善。结论NSAA患者生活质量低于常人，CsA治疗可以改善患者的生活质量，尤其是心理健康情况。不同社会背景的患者均可从治疗中获益，而起病较重者获益更大。

Efficacy of Rabbit Antithymocyte Globulin as a First-line Therapy in Children With Aplastic Anemia.

The efficacy, safety, and outcome of rabbit antihuman thymocyte globulin (rATG) as initial therapy for children aplastic anemia (AA) were evaluated. Sixty-one children with AA were retrospectively analyzed, including 43 patients with severe AA and 18 patients with transfusion-dependent nonsevere AA. All patients received rATG in combination with cyclosporine A between September 2005 and January 2015. The overall response rates were 55.7%, 68.9%, and 68.9% at 6, 12, and 18 months, respectively. Surprisingly, the overall complete response rate kept increasing from 9.8% at 12 months to 39.3% at 18 months, indicating a delayed response for rATG. Overall survival at 5 and 10 years was 72.1% and 67.2%, respectively. The overall survival of patients who responded between 3 and 12 months was significantly higher than that of nonresponders (71.4% vs. 47.4%).Antithymocyte globulin-related adverse reactions were significantly higher in severe AA (83.7%) than in nonsevere AA (55.6%) and these reactions were controllable and not life threatening with comprehensive measures. This retrospective study shows an encouraging response and survival results in children with AA treated with rATG. Prolonged assessments were needed to evaluate the delayed responses to rATG. rATG could be used as an alternative in the first-line treatment of childhood AA.

Eltrombopag is effective in patients with relapse/refractory aplastic anemia-report from a single center in China.

Eltrombopag has shown a promising effect on patients with refractory/relapsed aplastic anemia (AA). However, data in Asian patients are limited due to the short launching time. Data from relapsed/refractory AA patients treated with eltrombopag in Peking Union Medical College Hospital from December 2017 to May 2019 were analyzed retrospectively. Totally 41 patients including 37 non-severe AA and 4 severe AA were analyzed with a median age of 47 (13~81) years old. The dosage of eltrombopag varies from 25 mg every other day (qod) to 100 mg every day (qd) (median 75 mg qd) with the duration of 13 (3~23) months. The overall response rates were 51.2%, 58.5%, and 55.2% at 3-month, 6-month, and 1-year follow-up. Seventy-five percent of patients achieved the best response below the dosage of 75 mg qd at 3 (1-6) months. Two patients stopped eltrombopag after achieving complete remission and remained stable. Two patients relapsed and evolved to myelodysplastic syndrome. Adverse events included gastrointestinal discomfort, hepatic toxicity, and skin reactions, which were mild and controllable. Eltrombopag is effective for Chinese relapsed/refractory AA patients with a relatively lower dose and acceptable side effects.

Evaluation of the efficacy of cyclosporin A combined with recombined human thrombopoietin for treating patients with non-severe aplastic anemia

目的比较环孢素A（CsA）联合重组人血小板生成素（rhTPO）和单用CsA治疗非重型再生障碍性贫血（NSAA）的疗效。方法回顾2014年8月至2019年2月83例初治NSAA患者临床资料，男35例，女48例，中位年龄45（14~85）岁。其中57例使用CsA+rhTPO治疗（TPO治疗组），TPO 15 000 U，皮下注射，每日1次，共7 d，28 d为1个疗程，最多治疗3个疗程；26例为同期基线相匹配的患者，仅单用CsA治疗（对照组）。入选患者CsA使用至少6个月，并随访至少1年。比较两组的疗效及转归。结果TPO治疗组患者男23例，女34例；中位年龄46（14~85）岁；CsA中位应用时间为17（8~28）个月；中位随访时间为27（12~45）个月。对照组26例，男12例，女14例；中位年龄40（20~64）岁；CsA中位应用时间为19（9~30）个月；中位随访时间为29（16~66）个月。两组基线水平差异均无统计学意义（P值均>0.05）。两组1、3、6、12和24个月的CR率及OR率差异均无统计学意义（P值均>0.05）。TPO治疗组1个月[8（−12~86）×109/L对3（16~57）×109/L，P=0.029]、3个月[24（−6~102）×109/L对7（−9~76）×109/L，P=0.006]和6个月[33.5（−4~123）×109/L对12.5（−14~109）×109/L，P=0.048]血小板升高水平均显著高于对照组，但12个月及24个月与对照组差异无统计学意义（P值均>0.05）。两组6个月时骨髓巨核细胞变化水平差异无统计学意义[3（0~4）个对2（0~5）个，z=−0.868，P=0.385]。TPO治疗组10.5%（6/57）出现注射部位疼痛，除此之外，两组不良反应无明显差异。随访期间，两组患者各有1例阵发性睡眠性血红蛋白尿（PNH）克隆>10%；TPO治疗组1例进展为SAA；均无死亡、无血栓事件发生，未见网状纤维及胶原纤维增生，未见骨髓增生异常综合征和急性髓系白血病转化。结论CsA联合TPO治疗相比CsA单药治疗可较快提升NSAA患者血小板水平，而不增加明显的不良反应。

Abnormal miR-214/A20 expression might play a role in T cell activation in patients with aplastic anemia.

Aberrant T cell activation is a major cause of aplastic anemia (AA) pathogenesis. Recent studies have shown that miRNAs regulate T cell activation and are involved in AA. A previous study found that miR-214 was significantly up-regulated upon T cell activation in a CD28-dependent fashion by targeting PTEN. However, the expression characteristics of miR-214 and its target genes in AA have not been defined. In this study, target genes for miR-214 were predicted and confirmed by bioinformatics and luciferase reporter assays. The expression levels of miR-214 and target genes were detected in 36 healthy individuals and 35 patients with AA in peripheral blood mononuclear cells by real-time quantitative reverse transcriptase-polymerase chain reaction. Bioinformatics and luciferase reporter assays identified that miR-214 could bind to the A20 3′ untranslated regions. Significantly increased miR-214 and the decreased A20 expression level were detected in the AA patients compared with the healthy group. In addition, significantly increased miR-214 was found in non-severe aplastic anemia compared with severe aplastic anemia patients. These results suggested that the A20 gene was a potential target of miR-214, and elevated miR-214 might medicate T cell activation at least in part by regulating A20 expression in AA. We firstly confirmed that miR-214 regulated A20 expression, and aberrant miR-214/A20 expression might contribute to immunopathology in AA. The miR-214 expression might be used as a potential biomarker that assisted in diagnosing AA severity.

MiR-335-5p-Mediated Dysfunction of T Lymphocytes in Patients with Acquired Aplastic Anemia

To explore the effect of miR-335-5p/ADCY3 interaction on the lymphocyte function in the patients with aplastic anemia (AA). Blood samples were collected from 22 healthy volunteers (HC) and 50 AA patients including 38 severe AA (SAA) and 12 non-severe AA (NSAA). Peripheral blood mononuclear cells (PBMNC) were isolated. The expression of miR-335-5p and ADCY3 mRNA was detected by using RT-PCR. Negative control miR-335-5p (NC group) and miR-335-5p mimic (mimic group) were transfected to AA-PBMNC by using RNAimax reagent, respectively. The proliferative ability, activation and cytokines of CD4+ T and CD8+ T cells were measured by flow cytometry. Dual-luciferase reporter assay was used to verify the targeted relationship between miR-335-5p and target gene. The expression of miR-335-5p was significantly downregulated in SAA-PBMNC and NSAA-PBMNC compared with HC-PBMNC (0.08±0.01 vs 0.74±0.10, P＜0.01; 0.17±0.02 vs 0.74±0.10, P＜0.01). Meanwhile, the expression of miR-335-5p in SAA-PBMNC was very statistically significantly lower than that in NSAA-PBMNC (P＜0.01). Compared with NC group, upregulation of miR-335-5p in vitro could significantly inhibited the proliferation of CD4+ T and CD8+ T cells in AA-PBMNC (P＜0.05 and P＜0.05, respectively). And, upregulating miR-335-5p in AA-PBMNC could significantly inhibited the activation of CD4+ and CD8+ T cells (P＜0.01 and P＜0.01, respectively). The ratio of CD4+TNFα+ T, CD8+IFNγ++T and CD8+TNFα+ T cell by up-regulating the expression of miR-335-5p from AA-PBMNC in vitro was also significantly lower (P＜0.01, P＜0.05 and P＜0.05, respectively). In addition, the expression of ADCY3 was higher in AA-PBMNC than that in HC-PBMNC (1.70±0.15 vs 0.76±0.12, P＜0.01). Furthermore, by means of dual-luciferase reporter assay, the luciferase activity of ADCY3'UTR wildtype could be inhibited by miR-335-5p. The expression of miR-335-5p was significantly downregulated in AA, and that correlates with disease severity. Up-regulating miR-335-5p can correct the hyperimmune status in AA patients by targeting ADCY3. These changes may relates with the strengthen of inhibition for targeted gene ADCY3.

Flowcytometric Cytokine Profiling of T- Lymphocytes in Pediatric Aplastic Anemia

Background: Aplastic anemia is a result of complex immune mediated marrow injury. As activated T cells play a pivotal role in immune response, study of cytokine profile in patients of aplastic anaemia (AA) will be of use in understanding the initiation and propagation of the disease. Methods: Thirty children with AA between ages 6 months to 18 years were included along with age & sex matched controls. Complete blood count (CBC) and bone marrow examination was done. They were grouped as Very severe aplastic anemia (VSAA), Severe aplastic anemia (SAA) and Non severe aplastic anemia (NSAA) as per standard guidelines. Intracytoplasmic expression of Type 1 cytokine (IFNγ) and Type 2 cytokine (IL4) was determined by Flow cytometry (FCM). CD8 and CD4 cell counts were also determined by FCM. Result: CD8 cell count was higher than the CD4 counts with reversal of CD4:CD8 ratio in all patients. Mean intracytoplasmic IFNγ expression in CD8 and CD4 cells was higher in cases than in controls (p<0.0001) and showed graded positive correlation with the severity of the disease (p<0.0001). Type 2 cytokine (IL4) did not show any such correlation. Conclusion: IFN gamma expression in T cells can be useful in predicting severity of the disease and also response to immunosuppression.

Prognostic factors of cyclosporine A combined with androgen in the treatment of transfusion dependent non-severe aplastic anemia

目的调查影响环孢素A（CsA）联合雄激素方案治疗输血依赖非重型再生障碍性贫血(TD-NSAA)获得血液学反应的因素。方法回顾性分析2010–2013年连续收治的77例TD-NSAA患者临床资料，单因素和多因素分析影响CsA联合雄激素治疗方案获得血液学反应患者的基线临床和血液学特征。结果77例TD-NSAA患者治疗后6个月和12个月获得血液学反应分别为43例（55.8％）和53例（68.8％），单因素分析基线血小板计数［19（6～61）×109/L对13.5（5～45）×109/L，P＝0.001］是影响6个月获得血液学反应的唯一因素；基线血小板计数［18（6～61）×109/L对10.5（5～45）×109/L，P<0.001］、网织红细胞绝对值［0.03（0.01～0.06）×1012/L对0.03（0.02～0.06）×1012/L，P＝0.043］、血小板输注依赖（P＝0.007）和红细胞及血小板输注依赖（P＝0.012）为治疗后12个月能否获得血液学反应相关因素。多因素分析显示基线血小板水平为获得血液学反应独立影响因素（P值分别为0.010和0.009）。受试者工作特征曲线（ROC曲线）方法显示基线PLT界值为15.5×109/L。结论TD-NSAA患者初诊时较高的血小板基线水平、网织红细胞基线水平和不伴血小板输注依赖均提示预后较好，血小板水平≥15.5×109/L时可以考虑采用CsA联合雄激素治疗。

Th17 Cell Subset levels in Peripheral Blood of Patients with Aplastic Anemia and Their Clinical Significance

To study the level and clinical value of Th17 cell subset in the peripheral blood of the patients with aplastic anemia (AA). Eighty-five patients with aplastic anemia in our hospital from May 2017 to February 2018 were selected. Among them, 51 patients with poor curative effect were enrolled in group A. and the 34 patients with good curative effect were enrolled in group B, the 35 healthy persons were selected as controls. The levels of serum IL-17, IL-6, IL-4 and IFN-β were analyzed by ELISA; the levels of peripheral blood Th17 cell subset were analyzed by flow cytometry; the expression levels of RORγt and STAT3 mRNA in lymphocytes were analyzed by RT-PCR; the expression levels of RORγt and STAT3 protein in lymphocytes were analyzed by Western blot. There was no significant difference in sex, age, WBC count and complications between group A and group B (P＞0.05). Non-severe aplastic anemia (NSAA) in group B accounted for 23 cases, and the NSAA ratio (23/34) was significantly higher than that in group A (20/51) (P＜0.05). The Hb level (86.25±7.9 g/L) and Plt count (54.7 ± 6.3×109/L) in group B were significantly higher than those in group A (P＜0.05). ELISA showed that the levels of IL-17 and IL-6 in group A were significantly higher than those in control group (P＜0.05); the levels of IL-17 and IL-6 in group B were significantly lower than those in group A (P＜0.05); the levels of IL-4 and IFN-β in group A were significantly lower than those in control group (P＜0.05); the levels of IL-4 and IFN-βin group B were significantly higher than those in group A (P＜0.05). Flow cytometry showed that the proportion of Th17 cells in peripheral blood of group A was higher than that of group B (P＜0.05). RT-PCR showed that the levels of RORγt and STAT3 mRNA in group A were significantly higher than those in control group (P＜0.05), and the mRNA levels of RORγt and STAT3 in group B were significantly lower than those in group A (P＜0.05). Western blot showed that the protein levels of RORγt and STAT3 in group A were significantly higher than those in control group (P＜0.05), and the protein levels of RORγt and STAT3 in group B were significantly lower than those in group A (P＜0.05). The Th17 cells in peripheral blood of AA patients increase, and the inflammatory reaction in the patients are enhanced. It may be related with the rise of RORγt and STAT3 gene expression, which provides a research direction for clinical treatment of AA.

Alternative donor HSCT for 109 children with acquired severe aplastic anemia: a single center retrospective analysis

目的了解替代供者（AD）移植一线治疗儿童再生障碍性贫血（AA）的疗效及安全性。方法回顾性分析2010年4月1日至2016年12月31日在上海儿童医学中心一线接受AD移植治疗的AA患儿临床资料，统计分析总生存（OS）率、植入成功率、移植物抗宿主病（GVHD）发生率等指标。结果共纳入109例患者，极重型AA（VSAA）32例，重型AA（SAA）64例，非重型AA（NSAA）伴输血依赖13例，中位年龄6（0.8~18）岁，其中44例患者接受全相合无关供者（MUD）移植，44例接受8–9/10位点不全相合无关供者（MMUD）移植，21例接受不全相合亲缘供者（MMRD）移植，所有患者均接受以外周血干细胞（PBSC）为主的移植，≥3个位点不合的单倍型移植加第三方脐血（UCB）一份。所有患者移植前均未接受过抗胸腺细胞球蛋白（ATG）治疗，并排除活动性感染。106例（97.2%）获造血重建，中性粒细胞中位重建时间为13（9~19）d，血小板中位重建时间为16（10~81）d。死亡13例，5年OS率为88.1%（95%CI 81.1%~91.4%），MUD、MMUD及MMRD三组患者OS率差异无统计学意义（P=0.361）。总体急性GVHD（aGVHD）及Ⅱ~Ⅳ度aGVHD发生率分别为74.3%和39.4%，总体慢性GVHD（cGVHD）和中度cGVHD发生率分别为30.7%和9.9%，无一例患者发生重度cGVHD。结论对于无同胞全相合供者的SAA/VSAA患儿，早期一线接受AD移植可能是一个选择，但需要进一步探索更有效的预防及治疗GVHD的措施。

Th17 Cell Subset levels in Peripheral Blood of Patients with Aplastic Anemia and Their Clinical Significance

To study the level and clinical value of Th17 cell subset in the peripheral blood of the patients with aplastic anemia (AA). Eighty-five patients with aplastic anemia in our hospital from May 2017 to February 2018 were selected. Among them, 51 patients with poor curative effect were enrolled in group A. and the 34 patients with good curative effect were enrolled in group B, the 35 healthy persons were selected as controls. The levels of serum IL-17, IL-6, IL-4 and IFN-β were analyzed by ELISA; the levels of peripheral blood Th17 cell subset were analyzed by flow cytometry; the expression levels of RORγt and STAT3 mRNA in lymphocytes were analyzed by RT-PCR; the expression levels of RORγt and STAT3 protein in lymphocytes were analyzed by Western blot. There was no significant difference in sex, age, WBC count and complications between group A and group B (P＞0.05). Non-severe aplastic anemia (NSAA) in group B accounted for 23 cases, and the NSAA ratio (23/34) was significantly higher than that in group A (20/51) (P＜0.05). The Hb level (86.25±7.9 g/L) and Plt count (54.7 ± 6.3×109/L) in group B were significantly higher than those in group A (P＜0.05). ELISA showed that the levels of IL-17 and IL-6 in group A were significantly higher than those in control group (P＜0.05); the levels of IL-17 and IL-6 in group B were significantly lower than those in group A (P＜0.05); the levels of IL-4 and IFN-β in group A were significantly lower than those in control group (P＜0.05); the levels of IL-4 and IFN-βin group B were significantly higher than those in group A (P＜0.05). Flow cytometry showed that the proportion of Th17 cells in peripheral blood of group A was higher than that of group B (P＜0.05). RT-PCR showed that the levels of RORγt and STAT3 mRNA in group A were significantly higher than those in control group (P＜0.05), and the mRNA levels of RORγt and STAT3 in group B were significantly lower than those in group A (P＜0.05). Western blot showed that the protein levels of RORγt and STAT3 in group A were significantly higher than those in control group (P＜0.05), and the protein levels of RORγt and STAT3 in group B were significantly lower than those in group A (P＜0.05). The Th17 cells in peripheral blood of AA patients increase, and the inflammatory reaction in the patients are enhanced. It may be related with the rise of RORγt and STAT3 gene expression, which provides a research direction for clinical treatment of AA.

A Common HLA Allelic Mutation of exon1 in Leukocytes Defines Class I Alleles Responsible for Autoantigen Presentation of Acquired Aplastic Anemia

[Background] Acquired aplastic anemia (AA) is thought to be caused by cytotoxic T lymphocyte (CTL) attack specific to antigens presented by class I HLA alleles on hematopoietic stem cells (HSCs) although the target antigens on HSCs are still unknown. HLA alleles that are responsible for the auto-antigen presentation, which can be inferred from the presence of leukocytes that lack particular HLA class I alleles (HLA-lacking leukocytes [HLA-LLs]), may provide useful information on the identification of autoantigens in AA. We previously reported that deep sequencing of HLA class I genes of HLA-LLs revealed various loss-of-function mutations in alleles, including HLA-A*02:06 and B*40:02, suggesting that limited HLA-class I alleles are involved in the autoantigen presentation of AA (ASH 2018). Intriguingly, 60% of patients possessing HLA-LLs due to 6pLOH or several inactivating mutations in different HLA-A or HLA-B genes shared a nonsense mutation in exon 1 (Exon1mut) of which allelic frequency was very low (range 1.0-37.3%, median 4.5%). We hypothesized that the nonsense mutation, which efficiently lacks the corresponding HLA-allelic expression, might have been overlooked due to its low VAFs, and if we could establish a highly sensitive assay for detecting Exon1mut and determine the HLA alleles that undergo the mutation for a large number of AA patients, we might be able to define all HLA alleles that are involved in autoantigen presentation of AA. [Objectives/Methods] To test this hypothesis, we developed a highly sensitive droplet digital PCR (ddPCR) assay for precisely detecting Exon1mut in the peripheral blood (PB) of AA patient. In brief, the exon 1 regions of HLA-A and HLA-B alleles were amplified using two different sets of primer pairs that are complementary to the consensus sequences of the HLA-A and HLA-B alleles. The amplicons were subjected to a ddPCR assay using TaqMan probes complementary to wild-type (WT) and mutant-specific (MT) sequences, which were labeled with different fluorochromes (6-FAM for MT and HEX for WT). Peripheral blood leukocytes from 363 patients with AA (mean 64 [range, 11-93]) years of age, 134 with severe AA and 229 with non-severe AA; 173 males and 190 females; 84 6pLOH[+] and 279 6pLOH[-]) were subjected to the ddPCR assay. All blood samples were analyzed for 6pLOH by SNP array-based methods or a ddPCR assay as previously described. The HLA allele that underwent Exon1mut was determined by targeted deep sequencing using a unique molecular identifier (UMI), which enabled us to detect variant calling at a VAF as low as 0.1%, or deduced from the alleles contained in the lost haplotype, which are known to be the frequently lost alleles due to 6pLOH. [Results] Using 2 different ddPCR mixtures for HLA-A and HLA-B, the presence of Exon1mut was evaluable in all 363 AA patients. The sensitivity of the ddPCR assay for detecting Exon1mutwas 0.07%. 6pLOH was detected in 84 (23.1%) of the 363 AA patients. Ninety-nine (27.3%) of the 363 patients with (55 [65.5%] of 84) or without (44 [15.8%] of 279) 6pLOH were found to be positive for Exon1mut. The median allele frequency of Exon1mut in DNA from the Exon1mut(+) patients was 0.6% (range, 0.074% to 21.3%). In 17 patients whose blood samples were serially available, Exon1mutwas persistently detected in 13 and disappeared in 4 patients for 10-77 months (Figure 1). Among 43 different HLA-A and HLA-B alleles carried by the Exon1mut(+) patients, those with Exon1mutcould be identified by targeted deep sequencing in 54 patients. In 13 of the remaining 42 patients with Exon1mut, the Exon1mut-involved HLA alleleswere deduced from the alleles contained in the lost haplotype due to 6pLOH. These were 12 alleles and included A*02:06 (n=11), A*31:01 (n=3), B*13:01 (n=2), B*40:01 (n=3), B*40:02 (n=26), B*40:03 (n=1), B*54:01 (n=6), A*02:01 (n=2), A*02:07 (n=1), B*44:03 (n=1), B*55:02 (n=2) and B*56:01 (n=1) (Figure 2). The last five infrequent alleles were newly identified as "risk alleles" using the Exon1mutdetection. Two-hundred and twenty (92%) of 239 patients with PNH-type cells possessed at least 1 of the 12 alleles, while 103 (85%) of 121 patients without PNH-type cells did (P=0.045). [Conclusions] The Exon1mutdetection assay identified 12 HLA-alleles that are closely and exclusively involved in the autoantigen presentation of AA in Japanese patients. Similarity analyses of their antigen-presentation motifs may help to identify autoantigen peptides in AA. Disclosures Nakao: Takeda Pharmaceutical Company Limited: Honoraria; SynBio Pharmaceuticals: Consultancy; Ono Pharmaceutical: Honoraria; Novartis Pharma K.K: Honoraria; Bristol-Myers Squibb: Honoraria; Kyowa Kirin: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Celgene: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria.

DDX41 mutation in Patients with Idiopathic Cytopenia of Undetermined Significance, Myelodysplastic Syndrome, and Acute Myeloid Leukemia

Background Following the advances in genetic tests, including next-generation sequencing, there have been new insights into hereditary hematopoietic malignancies. The germline mutation in DDX41 was included in a new category, myeloid neoplasms with germline predisposition, of the updated 2016 WHO classification. Based on the reported data to date, there seem to be racial differences in the mutation variants of DDX41 gene, which were found in patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Idiopathic cytopenia of undetermined significance (ICUS) is known to be a precursor lesion of MDS, but the DDX41 mutations have not been evaluated in patients with ICUS. In this study, we aimed to reveal the incidence, genetic characteristics, and clinical features of the DDX41 mutations in patients with ICUS, MDS, and AML. Methods We performed targeted deep sequencing of 141 genes with a MiSeqDx sequencer (Illumina) using bone marrow (BM) samples obtained from the patients with ICUS (n=77), MDS (n=175), and AML (n=148) between May 2009 and June 2019. ICUS was defined by the proposed criteria of 2007 Consensus Group. The cut-off level of variant allele frequency (VAF) was set to 2.0% of mutant allele reads. We divided ICUS into clonal cytopenia of undetermined significance (CCUS), which was defined as ICUS with ≥ 2% VAF of somatic mutations of myeloid malignancy-associated genes and non-CCUS. Results Overall, DDX41 mutations were detected in 6 (7.8%) of 77 ICUS, 19 (10.9%) of 175 MDS, and 8 (5.4%) of 148 AML patients. Thirty-eight (49.4%) of 77 ICUS patients had CCUS. Of 6 DDX41 mutated patients with CCUS, 5 showed biallelic mutations with the median VAF of 44.7% (range, 29.3−50.0) and 10.2% (range, 3.3−25.4), indicating that one germline and one somatic mutation exists. Of 175 MDS patients, 78 were categorized into lower-risk MDS (revised international prognostic scoring system [IPSS-R] &lt; 3.5) and 97 into higher-risk MDS (IPSS-R ≥ 3.5), and DDX41 mutations were identified in 6 (7.7%) of 78 lower-risk MDS and 13 (13.4%) of 97 higher-risk MDS patients. Interestingly, biallelic mutations were found in 16 of 18 DDX41-mutated MDS patients with the median VAF of 47.75% (range, 43.4−55.6) and 13.8% (range, 2.7−35.8). In contrast, only one of 8 DDX41-mutated AML patients had biallelic mutation. Patients with DDX41 mutations typically showed hypocellular marrow (median BM cellularity, 30%; range, 5−95) with significant neutropenia (median neutrophil counts, 607/μL; range, 142−1675), male predominance (29/33, 87.9%), and relatively older age (median age, 64 years; range, 41−79) at diagnosis. In addition, we found novel mutation locations, which were different between presumed germline and somatic variants: V152G in germline, and T227M in somatic (Table 2). During a median follow-up duration of 2.9 years, 1 of 6 ICUS patients progressed to MDS-EB-1 after 17.3 months and 1 to non-severe aplastic anemia after 51.3 months. Conclusion Our data show that a significant proportion of ICUS, MDS, and AML patients had DDX41 mutations, many of which are presumably germline. These findings suggest that careful consideration of the predisposing germline mutation is important when selecting a familial donor for allogeneic HCT. We also found novel mutation locations of DDX41 gene which were different between somatic and germline variants. Further studies are warranted to define the clinical and molecular characteristics of DDX41 mutations and therapeutic implications in myeloid neoplasms. Disclosures No relevant conflicts of interest to declare.

Real-World Effectiveness and Safety of Eltrombopag in Patients with Aplastic Anemia: A Chinese Nationwide Survey

Immunosuppressive therapy (IST) based on antithymoglobin (ATG) and cyclosporin (CsA) is the first-line treatment for severe aplastic anemia (SAA) patients who are unfit for transplantation,and the overall response rate (ORR) is about 70%.The utility of eltrombopag (EPAG),a TPO receptor agonist, achieved robust hematologic response in refractory and treatment-naïve SAA patients in clinical trials and some other studies. However, only a few data came from Asia countries where higher incidence of AA has been reported. A retrospective study on the use of EPAG in AA was conducted in mainland China. Aplastic anemia (transfusion dependent non-severe, severe, and very severe) patients who started eltrombopag before Feb 2019 and continued for at least 3 months either as first-line treatment or as rescue treatment, were enrolled. The maximum daily dosage of EPAG used continuously for at least 2 weeks is defined as the stable dosage. Response criteria were referred to that used in previous reports (Townsley DM, NEJM 2017; BCSH, BJH 2016). Fifty-six patients from eleven centers were enrolled in this study, including 26 males and 30 females at the median age of 39 (7-80) years. All patients were transfusion-dependent by the time of EPAG administration, and there were 14 VSAA, 24 SAA and 18 transfusion dependent non-severe aplastic anemia (TD-NSAA). Nineteen treatment-naïve patients received EPAG and IST (ATG+CsA, n=10; CsA/CsA+androgen, n=9) as first-line treatment. Thirty-seven patients were refractory to IST. Eltrombopag was administered at a median dose of 75 (25-150) mg per day for 7 (3-31) month. The median follow-up time was 9 (3-40) months. The overall response rate in patients receiving EPAG as first-line therapy was 78.9% (15/19), and most patients achieved complete response (CR) (10/15). Among the 10 patients receiving ATG+CsA, 6 patients achieved hematologic response (HR) at 3 months post-treatment, including 3 CR. Six patients were diagnosed as VSAA and three achieved HR. For the 9 patients treated with CsA/CsA+androgen, 8 achieved HR (88.9%) and 4 were CR (44.4%) at 3 months. By last follow-up, the cumulative HR rate was 70% in ATG+CsA group and 89% in CsA/CsA+ androgen group. Among the 14 responders, 11 patients receiving EPAG at a stable dosage ≤75mg/d and achieved HR at 3 months. The overall response rate in IST-refractory patients was 46% (17/37), with trilineage response in 27% patients at 3 months. For the 18 ATG+CsA refractory SAA patients,trilineage HR occurred in 4 patients (22.2%, 4/18), bi-lineage HR in one patient and single lineage HR in one patient. Thus, the total HR was 33.3% (6/18) at 3 months and increased to 44% (8/18) by last follow-up. Among the 19 CsA/CsA+ androgen refractory patients, 6 (31.5%, 6/19) achieved trilineage HR, one achieved bi-lineage HR and 4 achieved single lineage response. Total HR rate was 57.9% (11/19) at 3 months after EPAG initiation and 68% (13/19) by last follow-up, including 9 patients with trilineage HR. Among 17 responders, 13 received a stable EPAG dose of≤75mg/d. Most patients tolerated EPAG well. Adverse events occurred in 29 patients (52%) and most were mild to moderate, including gastrointestinal symptom (n=15, e.g. abdominal pain, nausea), impaired liver function (n=5), skin changes (n=7, e.g. skin pruritus and rash) and musculoskeletal pain (n=6), and venous thrombus (n=2). Eltrombopag dosage was reduced in 2 patients due to severe digestive symptoms at 100 mg/d and discontinued in one patient who suffered from upper limb venous thrombus. In conclusion, EPAG is effective and safe in treating Chinese AA patients at a daily dose of 75mg and less. The real-world result of EPAG in Chinese patients is similar to those reported in Western countries. Disclosures No relevant conflicts of interest to declare.

Characteristic overview of aplastic anemia at Sanglah Hospital, Bali-Indonesia in 2017-2018

Background: Aplastic anemia is a rare disease of anemia which characterized by pancytopenia due to spinal cord hypoplasia. This can occur in people of any age, race, and gender with different degree of severity, with incidence rate is two until three times higher in Asia than the West. This study aims to determine the characteristic of aplastic anemia based on age, gender, laboratory examination, and severity in Sanglah Hospital.Methods: This research is a cross sectional descriptive study with sources of data were medical records of aplastic anemia patients at Sanglah Hospital in the last 2 years, 2017-2018. Distribution of research variables is gender, age, laboratory examination, and severity.Results:The result showed that 11 data were 6 (55%) male and 5 female (45%), with the mean age was 48 ± 14 years. The highest incidence occurred in the age range of 26-60 years old with 7 patients (64%). In this study the average hemoglobin level obtained was 9.94 ± 1.46 g/dL with range between 6.65 to 12.49 g/dL. The average number of leukocytes was 11.91 ± 24.52 x 109 L with range of leukocytes is 1.99-84.58 x 109/L. The average number of absolute Neutrophils and percent was 1.53 ± 1.52 x 109/L and 25.99 ± 25.79% with range between 0.06 – 4.53 x 109/L and 2.3 - 70.83%. The average platelet count was 24.77 ± 11.35 x 109/L with range between 4.25 – 42.58 x 109/L. The highest cases were nonsevere aplastic anemia (NSAA) with 7 people (64%).Conclusion: The value of laboratory parameters for complete blood examination (leukocytes, platelets, hemoglobin, and neutrophils) in patients with aplastic anemia at Sanglah General Hospital is still very low, so it is a challenge for clinicians to carry out treatment of aplastic anemia and management related complications.

18F-FLT PET/MRI for bone marrow failure syndrome-initial experience

BackgroundBone marrow failure syndrome (BMFS) is a heterogeneous group of disorders associated with single- or multiple-lineage cytopenia and failure of normal hematopoiesis. We assessed the feasibility of integrated PET/MRI with 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) to assess the pathophysiology of whole-body bone marrow for the diagnosis and monitoring of BMFS. Twenty-five consecutive patients with BMFS underwent a pre-treatment 18F-FLT PET/MRI scan. They included 7 patients with aplastic anemia (AA), 16 with myelodysplastic syndrome (MDS), and 2 with myeloproliferative neoplasms (MPNs), primary myelofibrosis (MF), and secondary [post-essential thrombocythemia (post-ET)] MF. Two of the seven AA patients underwent a post-treatment scan. Eight of the 16 MDS patients who exhibited decreased 18F-FLT uptake in the pelvis were considered to have hypoplastic MDS (hypo-MDS). 18F-FLT PET and diffusion-weighted imaging (DWI) were visually and quantitatively evaluated.ResultsThe 18F-FLT uptake in the ilium was strongly correlated with bone marrow cellularity based on biopsy samples (ρ = 0.85). AA patients exhibited heterogeneously decreased uptake of 18F-FLT according to disease severity. Multiple 18F-FLT foci were observed in the proximal extremities, and they were in the central skeleton in severe AA patients. Post-treatment 18F-FLT PET scans of severe AA patients reflected the response of hematopoietic activity to treatment. MDS patients had marked 18F-FLT uptake in the central skeleton and proximal extremities, whereas hypo-MDS patients had heterogeneously decreased uptake, similar to that of non-severe AA patients. 18F-FLT PET and DWI were unable to predict the progression to leukemia for both MDS and hypo-MDS patients. A primary MF patient had slightly decreased 18F-FLT uptake in the central skeleton, but marked expansion of bone marrow activity to the distal extremities and high uptake of tracer in the extremely enlarged spleen (extramedullary hematopoiesis). In contrast, a secondary (post-ET) MF patient demonstrated marked bone marrow uptake, reflecting the hypercellular marrow with fibrosis. DWI revealed diffusely high signal intensities in both the primary and secondary MF patients.Conclusion18F-FLT PET can be used to noninvasively assess whole-body bone marrow proliferative activity and DWI may reflect the different aspects of bone marrow pathophysiology from 18F-FLT PET. 18F-FLT PET/MRI is useful for the diagnosis and monitoring of BMFS, except for the differentiation between non-severe AA and hypo-MDS, and the prediction of progression to leukemia.

Contribution of autophagy-related gene 5 variants to acquired aplastic anemia in Han-Chinese population.

Immune-mediated quantitative and qualitative defects of hematopoietic stem/progenitor cells (HSPCs) play a vital role in the pathophysiology of acquired aplastic anemia (AA). Autophagy is closely related to T cell pathophysiology and the destiny of HSPCs, in which autophagy-related gene 5 (ATG5) is indispensably involved. We hypothesized that genetic variants of ATG5 might contribute to AA. We studied six ATG5 polymorphisms in a Chinese cohort of 176 patients with AA to compare with 157 healthy controls. A markedly decreased risk of AA in the recessive models of rs510432 and rs803360 polymorphisms (adjusted odds ratio [OR], 95% confidence interval [CI] = 0.467 [0.236-0.924], P = 0.029 for ATG5 rs510432; adjusted OR [95% CI] = 0.499 [0.255-0.975], P = 0.042 for ATG5 rs803360) was observed. Furthermore, the decreased risk was even more pronounced among nonsevere AA compared with healthy controls under recessive models (adjusted OR [95% CI] = 0.356 [0.141-0.901], P = 0.029 for ATG5 rs510432; adjusted OR [95% CI] = 0.348 [0.138-0.878], P = 0.025 for ATG5 rs803360; adjusted OR [95% CI] = 0.352 [0.139-0.891], P = 0.027 for ATG5 rs473543). Above all, rs573775 can strongly predict the occurrence of newly onset hematological event in patients with AA. Our results indicate that genetic ATG5 variants contributed to AA, which may facilitate further clarifying the underlying mechanisms of AA and making a patient-tailored medical decision.

Abnormalities of quantities and functions of CD56bright natural killer cells in non-severe aplastic Anemia

ABSTRACTObjectivesThe mechanism of non-severe aplastic anemia (NSAA) is not clear. It may be different from severe aplastic anemia (SAA). CD56bright NK cells (regulatory NK cells) is a subgroup of NK cells that produce immunoregulatory cytokines and express high-affinity IL-2 receptor. To investigate CD56bright NK cells quantities and function in patients with NSAA and to explore how CD56bright NK cells participate in the progress of this disease.MethodsIn this study, we analyzed the quantitative and functional changes of CD56bright NK cells in peripheral blood of patients with NSAA by using Flow Cytometry (FCM) before and after immunosuppressive therapy (IST). The expressions of activating receptor (NKG2D, NKp46, NKp44), inhibitory receptor (NKG2A, CD158a, CD158b) and perforin and granzyme B were detected by FCM. IL-2 and IL-18 levels in serum were detected by ELISA. The correlation between these parameters and clinical indicators of patients were evaluated.ResultsWe found that the percentage of CD56bright NK cells in newly diagnosed NSAA patients was higher than that in normal controls (p = .011, p < .05). The median expression of NKG2D in patients with NSAA was higher compared to that in normal controls (p = .021, p < .05), and the expression of CD158a was lower (p = .047, p < .05). The concentrations of IL-2 and IL-18 in the serum of patients with NSAA were higher than those in normal group.ConclusionThese findings suggest that increased and activated CD56bright NK cells might play a protective role in the pathogenesis of NSAA.

Wilms’ tumor 1 mRNA expression: a good tool for differentiating between myelodysplastic syndrome and aplastic anemia in children?

ABSTRACTObjectives: To evaluate the value of Wilms’ tumor 1 mRNA (WT1) expression in the differential diagnosis of childhood myelodysplastic syndrome (MDS) and aplastic anemia (AA).Methods: This study compared WT1 expression levels in children of MDS and AA to evaluate its value in differential diagnosis.Results: WT1 overexpression rate and mean WT1 expression level were significantly higher in MDS compared to AA (P = 0.000 and P = 0.013, respectively). Patients with RCC and normal cytogenetics exhibited significantly greater portion of patients exposing WT1 overexpression, compared to all AA subtypes (P = 0.001, P = 0.000 and P = 0.001, respectively). ROC curve analysis revealed that WT1 expression could differentiate between RCC with normal cytogenetics and non-severe AA. Based on a cut-off value of 1.45%, WT1 expression provided a sensitivity of 23.2% and a specificity of 100%.Discussion: In the present study, WT1 overexpression rate was gradually decreased in RAEB group, RCC group and AA subtypes, and the mean WT1 expression level of the MDS patients was significantly higher than that of the AA group. It is very difficult to differentiate between RCC with normal cytogenetics and NSAA in children. Our results showed significant differences in WT1 overexpression rate between these two groups. When we set the cut-off value as 1.45%, WT1 expression levels could be used to differentiate between cases of RCC with normal cytogenetics and NSAA in children.Conclusion: WT1 expression might be useful for distinguishing between myelodysplastic syndrome and aplastic anemia in children.