Non-severe Aplastic Anemia Patients Research Articles

Red blood cell distribution width as a prognostic factor in patients with aplastic anemia treated with cyclosporin A plus androgen or cyclosporine A alone: a retrospective study

ABSTRACTObjective:To explore the prognostic value of red blood cell distribution width (RDW) in newly diagnosed aplastic anemia (AA) patients treated with cyclosporine A (CsA) plus androgen or CsA alone.Methods:We retrospectively analyzed the clinical outcome of 220 patients with AA. According to the baseline level of RDW before treatment, the patients were divided into the high-RDW group (RDW ≥ 15%) and the normal-RDW group (RDW < 15%).Results:The median RDW of non-severe AA (NSAA) and severe AA (SAA) patients was 15.65% and 15.35%, respectively; this were significantly higher than that of very severe AA (VSAA) patients (13.35%). With median follow-up being 46 months, AA patients in the high-RDW group showed better 5-year OS and PFS than the normal-RDW group (93%: 75.3%; 74.3%: 61%). There was a higher ORR in the high-RDW group than the normal-RDW group (68.7%: 52.3%). The ORR of NSAA patients in the high-RDW group was better than that in the normal RDW group (75.8%: 60%). The 5-year OS of SAA/VSAA patients in the high-RDW group was significantly higher than the normal-RDW group (81.8%: 50.8%).Conclusion:This is the first documentation on the prognostic value of RDW in AA patients receiving CsA treatment with long-term follow-up, which had shown that high RDW at diagnosis was a better prognostic factor.

Analysis of Mean Corpuscular Volume and Red Cell Distribution Width in Patients with Aplastic Anemia

To explore the characteristics of hemogram in patients with aplastic anemia (AA), especially mean corpuscular volume (MCV) and red cell distribution width (RDW). We examined the blood routine of 180 new-onset AA patients and used 166 patients with myelodysplastic syndrome (MDS) as controls. Among the 180 AA patients, 105 (58.3%) were diagnosed with severe AA (SAA), while 75 (41.7%) were diagnosed with non-severe AA (NSAA). Compared to MDS, patients with SAA generally had unfavorable hemogram, including significantly lower white blood cell (WBC), absolute neutrophil count (ANC), hemoglobin (Hb), platelet (PLT) and reticulocyte counts (RET). However, WBC, ANC and lymphocyte counts were higher in the NSAA group than in the MDS group; Hb and Ret were comparable between the two groups. 8.5% of SAA patients and 58.1% of NSAA patients presented with macrocytic anemia, whereas 25.7% of SAA and 64.0% of NSAA had a high RDW. In the MDS group, 54.7% of patients presented with macrocytic anemia, and 84.7% had increased RDW. WBC, ANC, PLT, and Ret in a high-RDW group (25.7% of SAA) were significantly higher than in a normal-RDW group (74.3% of SAA). Overall, most SAA patients exhibited normocytic–normochromic anemia, and their hemograms decreased more significantly; more than half of NSAA patients showed macrocytic-heterogeneous anemia, and their hemograms were similar to those of MDS. Patients with elevated RDW may have better residual bone marrow hematopoietic function than those with normal RDW but with more severe anemia.

Serum level of interleukin: 8 in correlation with aplastic anemia severity

Background: Aplastic anemia (AA) is a disease in which the bone marrow (BM) and the hematopoietic stem cells are damaged causing pancytopenia. It may be hereditary or acquired and it can range from mild to severe. Interleukin-8 (IL-8) is a chemokine that is critical for tissue damage in acute inflammation and has been shown to be potent inhibitors of myelopoiesis. Interleukin promotes macrophages to produce IL-8 in AA. The aim of the present work was to evaluate the level of IL-8 in patients with AA and its correlation with severity of the disease.Methods: The present study was conducted on 40 individuals, 30 newly diagnosed AA patients and 10 healthy subjects served as control. They are classified to group I (8 patients with very severe AA 20%), group II (16 patients with severe AA 40%), group III (6 patients with non-severe AA 15%) and group IV (10 apparently healthy individuals as control). All groups were subjected to complete blood count (CBC), BM aspiration and biopsy for patients only, erythrocyte sedimentation rate (ESR) and lactate dehydrogenase (LDH), measuring level of serum IL-8 of AA patients by enzyme-linked immunosorbent assay (ELISA).Results: Serum levels of IL-8 were significantly higher in AA patients at diagnosis when compared to the levels in normal controls. A significant negative correlation was found between serum level of IL-8 and Hb level, platelets count, neutrophil count, reticulocyte count and BM cellularity, also a significant positive correlation was found between serum level of IL-8 and ESR.Conclusion: Level of serum IL-8 is higher in very severe AA patients than severe AA patients than non-severe AA patients, that suggested it may be involved in the mechanisms of AA hence it correlates with the disease progression.

Use of Cyclosporine A and danazol in treatment of aplastic anemia: A real-world data from a teaching hospital in South India

Background: Due to financial constraints, there are limited treatment options for Aplastic anemia (AA) in developing countries. Aims and Objectives: In the present observational retrospective study, utility of Cyclosporine A (CsA) along with Danazol (an anabolic steroid) in AA was assessed. Materials and Methods: The study included patients of AA treated in our hospital from 2017 to 2020. Results: The present study included total of 95 patients. Out of these, 69 patients (73%) were lost to follow-up. It was observed that, out of 26 patients who could be followed up, 18 (69%) patients responded to the treatment (transfusion independent) at the end of 6 months. Out of 18 patients who had responded, two were very severe AA, 12 were severe AA, and four were non-severe AA patients. Eleven out of 18 (61.1%) females responded, whereas seven out of eight males (87.5%) responded to the treatment. In four patients who received Eltrombopag in addition to CsA-Danazol, three patients responded to the therapy. Conclusion: Treatment with CsA and Danazol may represent a promising therapy for patients with AA, especially in resource limited settings. Further, large-scale collaborative studies are necessary to evaluate such approaches in management of AA.

Interferon-gamma and perforin-positive T cells in acquired aplastic anemia: implication in therapeutic response.

Acquired aplastic anemia (aAA) is an autoimmune disease, characterized by infiltration of T lymphocytes in the bone marrow with destruction of hematopoietic stem cells by the effector cells. Interferon-gamma (IFN-γ) and perforin are important mediators of cell destruction. In this flow cytometry-based study, we have investigated the percentage of intracellular IFN-γ+ and perforin+ CD5+ T cells in peripheral blood of newly diagnosed aAA patients before and after immunosuppressive therapy (IST). Patients were categorized as per standard disease severity and response to IST. The median percentage of IFN-γ+ and perforin+ CD5+ T cells was higher in untreated patients compared to healthy controls. The percentage of these cells was also increased in untreated severe and very severe aplastic anemia when compared with non-severe aplastic anemia patients. In patients before and after IST the median percentage of T cells producing IFN-γ and perforin was elevated in non-responders as compared to partial plus complete responders. The higher percentage of IFN-γ+ and perforin+ CD5+ T cells may be useful as an early diagnostic marker for aberrant activation of immune system and predict poor response to IST in aAA patients, who will benefit from alternative therapy.

Bone Marrow Lymphoid Infiltrates and Aplasia after CD19-Directed CAR T Cell Therapy in Pediatric B-Lymphoblastic Leukemia

IntroductionChimeric Antigen Receptor (CAR) T cell therapy is used to treated relapsed/refractory B-Acute Lymphoblastic Leukemia (B-ALL) patients. Long term side effects include B cell aplasia and cytopenia that are treated with supportive therapy. Prolonged cytopenia is seen in 16% of CAR T-cell treated B-ALL patients. The etiology of cytopenia is not clear, and could be attributed to the impact of chemotherapy, CAR T cells and disease or patient-specific factors. The impact of CAR T cell infusion on bone marrow cellularity, lymphocyte compartment and its correlation with cytopenia has not been investigated.MethodsWe analyzed pre- and post-CAR bone marrow biopsies in 178 B-ALL patients who received a CD19-directed CAR T-cell product between 2012-2017 and followed for at least 12 months. Responses were categorized into sustained responders, non-responders, CD19-positive relapses and CD19-negative relapses as previously described. Bone Marrow biopsy (BMB) overall cellularity, complete blood counts (CBC) in a 12-month post infusion period were analyzed. BMB were considered mildly, moderately and severely hypocellular based on cellularity of 50, 25 and 5% respectively. Hypocellularity was further stratified by CBC per aplastic anemia (AA) classification guidelines: Non-Severe Aplastic Anemia (NSAA) and Very Severe Aplastic Anemia (VSAA). Immunohistochemistry (IHC) for CD3, CD4, CD8, CD163, granzyme and perforin were performed on pre- and post-CAR BMB. CD3 IHC was digitally scanned and analyzed quantitatively (Leica Aperio ImageScope) and qualitatively. Targeted RNA sequencing-based gene expression profiling (EdgeSeq Immuno-Oncology Panel, HTG Diagnostics) was performed on pre-and post-treatment biopsies, with differential expression assessed by DESeq2 within HTG Reveal software. RNAseq gene expression was deconvoluted to impute relative expression of immune cell subsets.Results31% of patients were mildly hypocellular but none were severely hypocellular at baseline pre-CAR timepoints. The highest proportion of hypocellularity was at the 1-month time point. 81% of patients were mildly hypocellular (≤50%), 42% were markedly hypocellular (≤25%), and 13% were severely hypocellular (≤5%) at the 1-month time point. By month 12, the proportion of mildly hypocellular patients was 74%, markedly hypocellular patients was 26%, and severely hypocellular patients was 7% (Figure 1). The proportion of NSAA and VSAA was 57% and 10% in moderately hypocellular BMB. The proportion of NSAA and VSAA was 64% and 18% in severely hypocellular BMB. Severely hypocellular patients had a higher average day minus 1 disease burden (33% involvement) compared to their moderate (24%) and non-hypocellular patients (16%). VSAA patients had a lower baseline BMB cellularity (47%) compared to NSAA patients (71%) and those without AA (60%)Increased CD3+ T cells were noted in the post-CAR BMB compared to the pre-CAR BMB (Figure 1B). Lymphocyte were singly scattered or formed loose aggregates and tight lymphohistiocytic clusters. IHC and RNAseq analysis showed increased CD8+ granzyme+ cells in the post-CAR BMB compared to pre-CAR BMB. Sustained responders showed higher T cell infiltration compared to other categories of patients (Figure 1C and D). Lymphoid infiltrates did not correlate with hypocellularity or cytopenia.ConclusionWe describe for the first time the changes in bone marrow cellularity and lymphocyte compartment after CD19-directed CAR T-cell infusion in B-ALL. A subset of patients were moderately or severely hypocellular and met criteria for aplastic anemia. However, most of them recovered bone marrow cellularity and CBC. Hypocellularity and AA were correlated with pre-CAR disease burden and baseline cellularity rather than post-CAR lymphocyte infiltration. We also show for the first time that sustained responders to CD19-CAR showed increased CD3+ CD8+ T cell infiltrates compared to CD19-positive relapses and non-responders.Figure 1. A. Bone marrow hypocellularity post-CAR T-cell infusion. Proportion of patients who were mildly hypocellular, markedly hypocellular, and severely hypocellular in the 12month follow up period. B. Increased CD3+ T cells and aggregates after CAR T cell infusion. C and D. Sustained responders show significantly greater CD3+ T cells after CAR T cell infusion compared to other categories. *P<0.05. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Effective Tacrolimus Treatment for Patients with Non-Severe Aplastic Anemia That is Refractory/Intolerant to Cyclosporine A: A Retrospective Study

BackgroundFor symptomatic non-severe aplastic anemia (NSAA) patients who cannot afford anti-thymocyte globulin (ATG) or allogeneic hematopoietic stem cell transplantation (HSCT), tacrolimus (FK) may be an option if these patients do not respond or become tolerant to cyclosporine A (CsA).MethodsWe enrolled 101 NSAA patients who were refractory or intolerant to CsA with no chance of HSCT or ATG treatment and treated these patients with tacrolimus for at least 6 months, with follow-up for at least one year.ResultsThe overall response rate (ORR) was 38.6% (complete response: 9.9%; partial response: 28.7%), and the median time to optimal response was 6 (3～10) months. Thirty-two (31.7%) cases had elevated creatinine levels. Eight (7.9%) cases had elevations in AST/ALT. A total of 25.6% (10/39) of patients relapsed at the end of follow-up. Age (P=0.0005), FK concentration (4.0~12 ng/mL, P=0.0005) and intolerance to CsA (P=0.012) were the independent risk factors for ORR. Treg cell levels pre-FK treatment were much lower than those of healthy controls (3.7±0.6% vs 6.8±0.7%, P=0.0004) but increased significantly after FK treatment (3.7±0.6% vs 7.1±0.8%, P=0.0039).ConclusionOur data suggest that tacrolimus is a salvage treatment for patients with NSAA that is refractory or intolerant to CsA.

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.

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.

Evaluation of Bone Marrow Microvessel Density in Patients with Aplastic Anemia.

Bone marrow microenvironment plays a crucial role in the growth of hemopoietic cells and bone marrow function, which in turn depends on an intact microvasculature. Our study assesses the microvessel density (MVD) in the bone marrow of aplastic anemia (AA) patients, compares with MVD of controls and MVD among the different types of AA. Bone marrow specimens from 60 patients with AA and 17 controls were studied. There were 33 patients with non severe AA (NSAA), 12 patients with severe AA (SAA) and 15 patients with very severe AA (VSAA). MVD was calculated on sections stained immunohistochemically for CD34. The mean bone marrow MVD in AA group was 1.28±0.36, being significantly lower than that in control group (6.80±1.59, p<0.001). MVD of SAA and NSAA patients were 1.16±0.35 and 1.49±0.27, respectively, being significantly different (p=0.003). MVD of VSAA was 0.93±0.25 and the difference with NSAA is significant, however there was no significant difference between SAA and VSAA. Bone marrow MVD is low in AA patients and is likely to have a role in pathophysiology of bone marrow failure. Proangiogenic agents together with specific therapy might accelerate the recovery of hematopoiesis in AA patients.

Effect of Blood Concentration of Cyclosporine A on Cyclosporine A Combined with Male Hormone and Compound Ferrous Sulfate in Treatment of Aplastic Anemia

Objective To study effect of blood concentration of cyclosporine A on cyclosporine A combined with male hormone and compound ferrous sulfate in treatment of aplastic anemia (AA),providing reference for rational use of cyclosporine A.Methods From January 2010 to December 2013,a total of 126 cases with AA were selected into this study,all of whom were given cyclosporine A combined with male hormone and compound ferrous sulfate therapy.Treatment effect was observed at two months,three months and six months of treatment.And blood concentration of cyclosporine A was compared between effective and ineffective group.Influences of different blood concentration of cyclosporine A on treatment outcome were compared.The study protocol was approved by the Ethical Review Board of Investigation in Human Being of Canglang Hospital of Suzhou.Informed consent was obtained from all participants.There were no signifcant differences of patients' general clinical features between severe AA patients and nonsevere AA patients (P＞0.05).Results At six months of immunotherapy with cyclosporine A combined with male hormone and compound ferrous sulfate,the effective rate (78.86 ％) was significantly higher than those at 2 months and 3 months (P＜0.05).The effective rate of non-severe AA patients at two months,three months and six months of treatment were 83.10％,88.72％ and 95.71％,respectively,and all of them were significantly higher than that of severe AA patients (P＜0.05).At 3 months of treatment,blood trough concentration and blood peak concentration of cyclosporine A in effective group were (198.87 ±68.45) μg/L and (742.15±188.95) μg/L,respectively,and both of them were significantly higher than those in ineffective group (P＜0.05).The effective rate of blood trough concentration ≥200 μg/L group (94.83 ％) was significantly higher than that of blood trough concentration ＜200 μg/L group (P＜0.05).The effective rate of blood peak concentration ≥700 μg/L group (92.31％) was significantly higher than that of blood peak concentration ＜700 μg/L group.Conclusions Blood concentration of cyclosporine A is associated with the treatment outcome of cyclosporine A combined with male hormone and compound ferrous sulfate in AA patients.Blood trough concentration ≥200 μg/L and blood peak concentration ≥700 μg/L can be a good therapeutic window. Key words: Anemia, aplastic; Cyclosporine; Compound ferrous sulfate; Plasma concentration; Therapeutic window

Busuishengxue ranules mediate their effects upon non-severe aplastic anemia via mitogen-activated protein kinase/extracellular signal-regulated kinase pathway

ObjectiveTo observe the clinical efficacy of Busuishengxue granules on non-severe aplastic anemia (NSAA) and investigate its effect on the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. MethodsSixty NSAA patients were divided equally into two groups. Subjects in the experimental group were treated with Busuishengxue granules, and the control group with Zaizaoshengxue tablets. The treatment course was 6 months and curative efficacy was compared between the two groups as well as with 10 healthy individuals. Flow cytometry (FCM) was used to detect the intracellular concentration of Ca2 + ([Ca2 +]i). Western blotting was employed to detect the expression of enzymes in the MAPK/ERK pathway. ResultsThe efficacy of Busuishengxue granules was significantly better than that of Zaizaoshengxue tablets (P<0.05). Before treatment, expression of JNK, phospho-ERK 1/2 and p-JNK was higher, and [Ca2 +]i higher, than that of the control group (P< 0.05). After treatment with Busuishengxue granules, expression of all enzymes related to signal transduction pathways in the blood cells of NSSA patients were altered to different degrees. ConclusionBusuishengxue granules had a better effect with regard to improving symptom scores, increasing the number of blood leukocytes, and increasing hemoglobin levels than Zaizaoshengxue tablets, and they differed slightly in terms of increasing the number of platelets.

Change and significance of Th22 cells in patients with aplastic anemia

To explore the proportion of Th22 cells in peripheral blood of patients with aplastic anemia (AA) and evaluate its significance. From January 2011 to June 2012, a total of 47 AA patients were recruited and divided into 4 groups: severe aplastic anemia (SAA) pre-therapy (group A, n = 11), non-severe aplastic anemia (NSAA) pre-therapy (group B, n = 12), SAA post-therapy (group C, n = 12), NSAA post-therapy (group D, n = 12) and healthy donor controls (n = 12). The proportion of Th22 cells in peripheral blood of each group was evaluated by flow cytometry. The cytokines interleukin-22 (IL-22), transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured by ELISA. And the level of IL-22 mRNA was examined by reverse transcription-PCR (RT-PCR). The percentage of Th22 cells and the level of IL-22, TNF-α, IL-6 and IL-22 mRNA in group A (4.3% ± 1.4%, (57 ± 17) ng/L, (497 ± 123) ng/L, (323 ± 88) ng/L, 1.65 ± 0.51) and group C (2.6% ± 0.6%, (34 ± 10) ng/L, (314 ± 79) ng/L, (187 ± 45) ng/L, 0.92 ± 0.28) were significantly higher than that in control group (1.2% ± 0.3%, (19 ± 6) ng/L, (228 ± 50) ng/L, (134 ± 26) ng/L, 0.47 ± 0.09,all P < 0.05). The percentage of Th22 cells and the level of IL-22, TNF-α , IL-6 and IL-22 mRNA in group A were higher than those in group C (all P < 0.05). NSAA patients had similar results. The percentage of Th22 cells and the level of IL-22, TNF-α , IL-6 and IL-22 mRNA in group A were higher than those in group B (all P < 0.05). But the level of TGF-β in groups A and C were significantly lower than that in control group ((3.4 ± 1.1) and (5.8 ± 1.7) vs (9.7 ± 2.8) ng/L, P < 0.05). And the level of TGF-β in group A was lower than that of group B (P < 0.05). The number of Th22 cells is elevated in AA patients. Th22 cells may be positively correlated with the development of AA. And a higher level of TNF-α, IL-6 and a lower level of TGF-β promote the differentiation of Th22 cells.

The progression risk factors of children with transfusion-independent non-severe aplastic anemia

Non-severe aplastic anemia (NSAA) in children is a rare, idiopathic condition of bone marrow insufficiency that can resolve spontaneously, persist for months or years, or progress to severe aplastic anemia (SAA). We reviewed clinical and laboratory data of patients diagnosed with transfusion-independent non-severe aplastic anemia (NSAA) from 1996 to 2009 at the Institute of Hematology and Blood Diseases Hospital, Peking Union Medical College, and analyzed the clinical course and outcomes in these patients. NSAA was defined as bone marrow cellularity <50 % and two or three cytopenias that persisted for 6 weeks or more: absolute neutrophil count (ANC) <1.5 × 10(9)/L, absolute reticulocyte count (ARC) <40 × 10(9)/L, platelet count <100 × 10(9)/L, without meeting criteria for SAA (bone marrow cellularity <30 % and two or three cytopenias: ANC <0.5 × 10(9)/L, ARC <20 × 10(9)/L, platelet count <20 × 10(9)/L). All patients were treated with reasonable supportive care, cyclosporine A, and stanozolol (0.1 mg/kg/day). Of a total of 284 patients, 117 (41.2 %) were female, and 167 (58.8 %) were male. With a median follow-up of 43 months (range 2-196 months), 38 patients (13.4 %) progressed to transfusion-dependent NSAA and among them 26 patients (9.2 %) progressed to SAA. One hundred and ninety-eight patients (69.7 %) had persistent NSAA. Forty-eight patients (16.9 %) showed the complete resolution of NSAA. The Kaplan-Meier estimates of all patients for progression-free survival were 86 ± 2.7 % and 66 ± 7.3 % at 60 and 120 months after diagnosis, respectively. Patients with ANC <1.0 × 10(9)/L or female had a higher probability of progression to transfusion-dependent NSAA (18.5 vs. 5.4 %, respectively; p = 0.002, and 17.1 vs. 10.8 %, respectively; p = 0.022). The patients with ARC <60 × 10(9)/L or with ANC <1.0 × 10(9)/L had a higher probability of progression to SAA (11.5 vs. 3.6 %, respectively; p = 0.035, and 12.7 vs. 3.6 %, respectively; p = 0.011). A categorical risk factor analysis showed that patients with ANC <1 × 10(9)/L had a higher probability of progression to SAA (p = 0.03) and had a higher probability of progression to transfusion-dependent AA (p = 0.007). NSAA patients may be benefited from early intervention with cyclosporine A and stanozolol.

Clinical Features of Chinese Refractory Cytopenia of Childhood: Lower Overall Survival and Higher Possibillity of Clonal Evolution Than Nsaa Patients

AbstractAbstract 4947 Objective:To analyze the clinical features and survival time in patients with refractory cytopenia of childhood (RCC), and to develop a new diagnostic scoring system for RCC. Methods:The clinical information, laboratory findings at primary diagnosis and survival time in 97 non-severe aplastic anemia (NSAA) children diagnosed in August, 2000 to June, 2006 were analyzed retrospectively. According to the criteria for RCC proposed in the 2008 edition of the World Health Organization classification of hematopoietic and lymphoid tissues, diagnosis of these NSAA patients was reassessed. Results:37/97 cases (38. 1%) were reassessed as RCC, 60/97 cases (61. 9%) were still diagnosed as NSAA. There was no significant differences in distribution of age, gender and chromosomal abnormalities, absolute neutrophil count, PLT, MCV, absolute reticulocyte count, the number of cytopenias and duration from onset to starting treatmeat between RCC and NSAA. All patients received the treatment of CsA and androgen. There was no significant differences in distribution of treatment response. The overall survival (OS) of RCC patients was significantly lower than NSAA patients (P=0. 041) in long-term follow-up. The former had 91. 5% of 6-year prospective survival (PS) and the latter had 96. 5% of 6-year PS. At a median follow-up of 93 months (range 6–138), 2 patients reassessed as RCC progressed into AML-M5 and PNH respectively, while no NSAA patient progressed into clonal diseases. There was significant differences in the distribution of HGB<11g/dl at onset and lymphocyte count<3×109/L, dysplastic megakarycytes≥10% (CD41 monoclonal antibogy immunohistochemical staining) and periodic acid schiff (PAS) staining (+) in bone marrow aspirate between RCC and NSAA (P=0. 034, 0. 025, 0. 032, 0. 029). Compared with NSAA, there was significant differences in the distribution of bone marrow cellularity down to 10% of the normal age matched value, dysplastic erythroblastic island, number of erythroblastic mitoses and micromegakaryocytes in bone marrow biopsy of RCC(P<0. 0001, 0. 0001, 0. 0001, 0. 0001). Using Logistic regression, a new diagnostic scoring model including increased dysplastic erythroblastic island, having micromegakaryocytes in bone marrow biopsy, and lymphocyte count<3×109/L in peripheral blood was developed. By ROC curve analysis, score≥2 points was identified as a cut-off value with sensitivity of 70. 3% and specificity of 95%. Conclusion:RCC patients had similar clinical features with NSAA patients but had lower OS and higher possibillity of clonal evolution. This new diagnostic scoring model had significance to differentiate diagnose RCC and NSAA. Disclosures:No relevant conflicts of interest to declare.

Clinical Implications of Clonal Cytogenetic Abnormalities of Aquired Aplastic Anemia.

Abstract 4217 BackgroundCytogenetic abnormalities (CA) have been reported infrequently in patients with otherwise typical aplastic anemia (AA). The relevance of CA in AA to the prognosis of AA and the evolution to the hematologic malignancies is controversial. Design and MethodsOne hundred and twenty-nine adult AA patients from four centers located in Busan, South Korea, who had successful cytogenetics at initial diagnosis were retrospectively analyzed. Results.The median follow-up duration of the overall patients was 46.8months. The ratio of severe AA to non-severe AA was 59:41. The patients were classified into 5 groups according to the CA and progression to the hematologic malignancies. Among the patients with normal cytogenetics at initial diagnosis, 117 remained AA with normal cytogenetics (Group 1). Six patients (4.7%) had CA at initial diagnosis (Group 2). The CA showed trisomy 8 in two cases and trisomy 11, deletion of Y chromosome, t(2;9), and t(22;?) in each case. One with trisomy 11 later developed monosomy 1. None of the Group 2 evolved to acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Two patients with normal cytogenetics at initial diagnosis later developed monosomy 4, and monosomy 7, respectively, with persistent AA (Group 3). Group 3 patients were treated only with intermittent transfusion but spontaneously recovered from cytopenia and are still alive without transfusion requirement. Among the AA with normal cytogenetics at initial diagnosis, four patients (3.2%) progressed to AML or MDS; two remained normal cytogenetics (Group 4), and two patients obtained structural CA (Group 5) at follow-up, respectively. ConclusionThe majority of the AA patients had normal cytogenetics at initial diagnosis. Non-severe AA patients may have CA. AA patients with CA at initial diagnosis or at follow-up are not at greater risk of evolution to the hematologic malignancies, and have no significant difference in survival. Prospective studies and more patients are needed to establish the clinical relevance of CA. Disclosures:No relevant conflicts of interest to declare.

Characteristics and Clinical Outcomes of Adult Aplastic Anemia with Abnormal Cytogenetics at Diagnosis.

Background and methods Occasionally, patients with acquired aplastic anemia (AA) present with abnormal cytogenetics in bone marrow cells at diagnosis. The diagnosis and treatment of such patients have not been established and have been center-dependent. We have treated adult AA patients with abnormal cytogenetics in a same way as those with normal cytogenetics. Presently, the characteristics and clinical outcomes of 600 adult AA patients who had successful cytogenetics at diagnosis were retrospectively evaluated. Our aim was to determine the characteristics and clinical courses of AA patients with cytogenetic abnormalities at diagnosis who were treated as those with normal cytogenetics.ResultsCharacteristics: Of the evaluable patients, 572 (95.3 %) had normal cytogenetics and 28 (4.7 %) had abnormal cytogenetics at diagnosis. The most frequent abnormality was trisomy 8 (n=15) followed by monosomy 7/deletion 7q (n=5) and deletion 1q (n=5). Other chromosome abnormalities were isochromosome 17q (n=1), trisomy 15 (n=1) and monosomy 21 (n=1). There was no significant statistical difference in gender (P=0.562), Hepatitis B or C infection (P=0.402), paroxysmal nocturnal hemoglobinuria (P=0.709) and severity of AA (P=0.325) between patients displaying normal cytogenetics and abnormal cytogenetics. The age of abnormal cytogenetics patients was significantly lower than normal cytogenetics patients (P<0.001).Immunosuppressive therapy: A total of 334 patients received immunosuppressive therapy using antithymocyte globulin and cyclosporine. Six months after commencement of therapy, 165 (50.9 %) patients responded partially or completely. Multivariate analysis revealed abnormal cytogenetics (HR=0.250; 95% CI=0.077–0.808; P=0.021), absence of paroxysmal nocturnal hemoglobinuria and age (≥ 67) as independent predictors for the poor response to immunosuppressive therapy.Leukemic transformation: Kaplan-Meier modeling revealed that abnormal cytogenetics was also associated with higher cumulative leukemic transformation rate (P<0.001) and lower leukemia-free survival (P=0.021). Of note, the cause of all deaths in non-severe AA patients with abnormal cytogenetics was leukemic transformation.Conclusion: Patients with abnormal cytogenetics at diagnosis of AA tend to respond poorly to immunosuppressive therapy and present with a high leukemic transformation risk, which suggests that cytogenetic abnormalities should be emphasized more than morphological features in diagnosis and treatment decision in AA.

Association of human leukocyte antigen-DRB1 alleles with disease susceptibility and severity of aplastic anemia in Korean patients

Although association of human leukocyte antigen (HLA)-DR2 (DRB1*1501) with susceptibility to aplastic anemia (AA) has been well documented in several different ethnic groups, little is known about the protective role of HLA in this disease. HLA-DRB1 alleles were analyzed in 109 Korean AA patients (26 nonsevere and 83 severe) and 800 healthy controls. The frequency of DRB1*1501 was significantly higher in AA patients compared with controls [33.0% vs 15.3%, p=0.000004, p(c)=0.0001, odds ratio (OR)=2.74]. Nonsevere AA (30.8%, OR=2.47) and severe AA patients (33.7%, OR=2.83) showed similar changes, and DRB1*1501 was considered a susceptibility factor to AA in both forms of the disease. The frequency of DRB1*1302 in total AA patients was not different from controls (12.8% vs 17.9%), but it was significantly lower in severe AA compared with nonsevere AA patients (6.0% vs 34.6%, p=0.0006, p(c)=0.02, OR=0.12). DRB1*1302 was considered a protective factor against severe AA. In Koreans, DRB1*1501 was associated with disease susceptibility to AA and DRB1*1302 with protection against the severe form of the disease.

The Clinical Significance of Some Specific HLA Class I Alleles in Japanese Patients with Bone Marrow Failure (BMF).

Autoreactive T lymphocytes are implicated in the immune mechanisms involved in the bone marrow failure (BMF) syndrome, including aplastic anemia (AA), paroxysmal nocturnal hemoglobinuria (PNH), and myelodysplastic syndrome (MDS). However, the significance of the HLA class I alleles remains unknown in the BMF syndrome. Nevertheless, from many clinical and basic studies, it is certain that CD8+ T lymphocytes are implicated in some of the immune mechanisms involved in the occurrence of AA. To clarify some clinical significance of the HLA class I alleles in the BMF syndrome, we investigated the alleles using a high-resolution method of genotyping in 78 Japanese patients with BMF, including 32 AA, 24 PNH, and 22 MDS patients. Subsequently, we compared various clinical findings, including age, sex, white blood cell counts, absolute neutrophil counts, hemoglobin concentrations, reticulocyte counts, platelet counts, values of lactate dehydrogenase, durations of illness, chromosomal findings, and proportions of CD55− and CD59− erythrocytes, between the groups with and without some alleles. The diagnosis and grading of the severity of AA were based on the criteria of the International Agranulocytosis and Aplastic Anemia Study Group (Blood 1987; 70: 1718–21) and that of Frickhofen et al (N Engl J Med 1991; 324: 1297–304), respectively. A patient with a CD55− and CD59− population of more than 1% was judged to have PNH erythrocytes (Blood 1996; 87: 5332–40). The diagnosis of MDS was determined according to the FAB criteria (Br J Haematol 1982; 51: 189–99). The frequencies of the HLA-B* 4002 allele in AA patients (21.9%) and of the HLA-A* 0206 allele in PNH patients (22.9%) were significantly different from those in controls (n=371; 8.6%, p<0.002 and 7.7%, p<0.001, respectively), while we found no specific HLA class I alelles in MDS patients. The frequency of the HLA-DRB1*1501 allele in PNH patients (31.3%) was significantly higher than that in controls (6.1%, p<0.0001), while we could not find the high frequencies of the HLA-DRB1*1501 (10.9%) and *1502 (10.9%) alleles in AA patients. The proportion of severe or very severe AA patients with the HLA-B* 4002 allele (10/17, 58.8%) was significantly higher than that of non-severe AA patients with the allele (3/15, 20%; p<0.05). In contrast, the proportion of severe or very severe AA patients (4/17; 23.5%) with the HLA-DRB1* 1501 allele was not different from that of non-severe AA patients (3/15; 20%) with the allele. Subsequently, the reticulocyte counts (138 ± 73 x 10 9/L) and values of lactate dehydrogenase (2399 ± 235 IU/L) at the time of examination in PNH patients (n=10) with the HLA-A* 0206 allele were significantly higher than those in PNH patients (n=14) without the allele (78 ± 34 x 109/L, p<0.02 and 972 ± 770 IU/L, p<0.05, respectively). In addition, the frequency of PNH patients with over 30% of complement-sensitive erythrocytes, consisting of intermediate and negative populations of CD55 and CD59 expressions on erythrocytes by flow cytometry, was significantly higher in PNH patients with the HLA-A* 0206 than in those without the allele (80% versus 28.6%, p<0.05). In conclusions, our results suggest that the HLA-B* 4002 allele in AA or the HLA-A* 0206 allele in PNH is related to grading of the severity of AA or grading of hemolysis of PNH, respectively.

Prospective Multicenter Trial Comparing Repeated Immunosuppressive Therapy (IST) with Stem Cell Transplantation (SCT) from an Alternative Donor as a Second-Line Treatment for Children with Acquired Aplastic Anemia (AA).

Immunosuppressive therapy (IST) has been shown to be an effective therapy for aplastic anemia (AA) patients without HLA-matched family donors. However, a significant proportion of patients are refractory to IST. Although the efficacy of repeated IST and stem cell transplantation (SCT) from an alternative donor has been confirmed, a direct comparison between these two procedures has not yet been conducted. In this study, we prospectively compared the efficacy of these two treatments. Between Oct. 1997 and Apr. 2003, 231 patients (125 male, 106 female; median age, 9 y; range, 0 to 17 y) were enrolled in the AA-97 study. Analysis was performed in April 2004. A total of 103 very severe AA and 74 severe AA patients received first-line IST consisting of antithymocyte globulin (ATG; Lymphoglobulin), Cyclosporin (CyA) and methylprednisolone (Mepred), while 54 non-severe AA patients received IST consisting of ATG and Mepred with or without CyA. G-CSF was administered only to patients with very severe AA. Sixty patients (26%) were non-responders to first-line IST and were eligible for 2 nd -line therapies. Of these, 29 patients lacking suitable donors received 2 nd-line IST with ATG (Lymphoglobulin), CyA and Mepred. Four developed severe anaphylactoid reactions and could not complete the 2 nd-line treatment. Twenty-nine patients received SCT as follows: BMT from an unrelated donor (n=22), BMT from an HLA-mismatched family donor (n=4) and cord blood SCT from an unrelated donor (n=3). Two patients did not receive any 2 nd-line treatment. Median interval between 1 st -line IST and 2 nd-line treatment was 8 mo for the SCT group (range: 4–20 mo) and 7 mo for the IST group (range: 5–14 mo). One patient receiving 2 nd-line IST and 5 patients receiving SCT died of infections. The overall probability of survival at 5 y from the start of 2 nd-line treatment was 82 ± 10% in the SCT group and 97 ± 5% in the IST group (p:0.08 ). However, trilineage response to a 2 nd course of IST was seen in only 3 of 25 evaluable patients (12%), and 10 non-responders to 2 nd-line IST later received BMT from an unrelated donor as a 3 rd -line therapy. Blood counts normalized in all patients following SCT. Failure-free survival (FFS) was defined as survival with treatment response. The estimated FFS at 5 y was significantly better (p <0.01) in the SCT group (81 ± 15%) than in the IST group (13 ± 12%). The probability of survival at 6 y from the start of 1 st treatments was 90 ± 6% in the 103 very severe AA patients, 97 ± 3% in the 74 severe AA patients and 95±4% in the non-severe 54 AA patients.Conclusion: SCT from alternative donors offers a better chance of FFS than 2 nd-line IST in non-responders to 1 st -line IST. However, overall survival rate is not different between two approaches.