Very Severe Aplastic Anemia Research Articles

Comparison of efficacy of eltrombopag combined with immunosuppression in the treatment of severe aplastic anemia and very severe aplastic anemia: real-world data and evidence.

Eltrombopag combined with immunosuppressive therapy (IST) was superior to IST alone for severe aplastic anemia (SAA) in the previous studies. But in China, horse antithymocyte globulin (hATG) is not available, instead, we use rabbit ATG (rATG). Here, we compared the efficacy and safety of IST (rATG combined with cyclosporine) combined with or without eltrombopag for the first-line treatment of SAA and very severe aplastic anemia (VSAA). A total of 371 patients in ten institutions in China from April 1, 2017 to December 1, 2022 were enrolled. The overall response (OR) rate at 3 months (54.2% vs. 41%; P = 0.046), the complete response (CR) (31.3% vs. 19.4%; P = 0.041) and OR (78.3% vs. 51.1%; P < 0.0001) rates at 6 months were significantly higher with IST combined with eltrombopag than with IST alone in SAA patients. While in VSAA patients, the addition of eltrombopag to IST only increased the CR rate at 6 months (29.8% vs. 9.43%; P = 0.010). Liver injury increased significantly in groups treated with IST combined with eltrombopag (P < 0.05). Serious treatment-related toxicities were similar (P > 0.05). In patients with SAA, 3-year failure-free survival (FFS) of eltrombopag combined with IST group was significantly higher than that of IST group (70.7 ± 5.3% vs. 50.3 ± 3.9%; P = 0.007). In patients with VSAA, the addition of eltrombopag significantly improved 3-year overall survival (OS) (82.2 ± 5.7% vs. 57.3 ± 7.2%; P = 0.020). Our findings suggested that IST combined with eltrombopag could improve the hematological recovery of newly diagnosed SAA without increasing severe toxicities. But in VSAA, the addition of eltrombopag seemed to show no other improvement to efficacy except the CR rate at 6 months.

The Efficacy and Influencing Factors of Cyclosporine Alone in the Treatment of Children with Acquired Aplastic Anemia

To analyze the efficacy and influencing factors of cyclosporine (CsA) alone in the treatment of children with acquired aplastic anemia (AA). The clinical data of children diagnosed with AA and treated with CsA alone from January 1, 2016 to December 31, 2020 in the Children's Hospital of Chongqing Medical University were collected, and the efficacy and influencing factors of CsA treatment were evaluated. Among the 119 patients, there were 62 male and 57 female, with a median age of 7 years and 1 month. There were 45 cases of very severe AA (VSAA), 47 cases of severe AA (SAA), and 27 cases of non-severe AA (NSAA). At 6 months after treatment, the efficacy of VSAA was lower than that of SAA and NSAA, and there was a statistical difference (P < 0.01). 6 cases died early, 16 cases relapsed, 2 cases progressed to AML and ALL. The results of univariate analysis showed that the high proportion of lymphocyte in the bone marrow at 6 months was an adverse factor for the efficacy of CsA, while high PLT count was a protective factor (P =0.008, P =0.002). The ROC curve showed that the cut-off values of PLT count and the proportion of bone marrow lymphocyte at 6 months were 16.5×109 /L, 68.5%, respectively. Multivariate analysis showed that the high proportion of lymphocyte in bone marrow at 6 months was an independent adverse factor for IST (P =0.020, OR =0.062), and high PLT count was a protective factor (P =0.044, OR =1.038). At 3 months of treatment, CsA response and NSAA were the risk factor for recurrence (P =0.001, 0.031). The efficacy of NSAA was higher than that of SAA and VSAA after 6 months of treatment with CsA alone. A high PLT count at the initial diagnosis was a good factor for the effectiveness of CsA, and a high proportion of bone marrow lymphocyte was an unfavorable factor. CsA response at 3 months and NSAA were risk factors for recurrence.

Efficacy, Safety, and Population Pharmacokinetics of Eltrombopag in Children with Different Severities of Aplastic Anemia.

Eltrombopag was approved as a first-line treatment for patients older than 2 years old with severe aplastic anemia (SAA). However, data on eltrombopag in children with different types of aplastic anemia (AA), especially non-severe AA (NSAA), are limited. We performed a prospective, single-arm, and observational study to investigate eltrombopag's efficacy, safety, and pharmacokinetics in children with NSAA, SAA, and very severe AA (VSAA). The efficacy and safety were assessed every 3 months. The population pharmacokinetic (PPK) model was used to depict the pharmacokinetic profile of eltrombopag. Twenty-three AA children with an average age of 7.9 (range of 3.0-14.0) years were enrolled. The response (complete and partial response) rate was 12.5%, 50.0%, and 100.0% after 3, 6, and 12 months in patients with NSAA. For patients with SAA and VSAA, these response rates were 46.7%, 61.5%, and 87.5%. Hepatotoxicity occurred in one patient. Fifty-three blood samples were used to build the PPK model. Body weight was the only covariate for apparent clearance (CL/F) and volume of distribution. The allele-T carrier of adenosine triphosphate-binding cassette transporter G2 was found to increase eltrombopag's clearance. However, when normalized by weight, the clearance between the wild-type and variant showed no statistical difference. In patients with response, children with NSAA exhibited lower area under the curve from time zero to infinity, higher CL/F, and higher weight-adjusted CL/F than those with SAA or VSAA. However, the differences were not statistically significant. The results may support further individualized treatment of eltrombopag in children with AA.

Clinical characteristics of hepatitis-associated aplastic anemia in children.

To understand the current situation of hepatitis-related aplastic anemia (HAAA) in children, we analyzed the patients with HAAA admitted to our hospital in the past 5 years to understand the disease characteristics and prognosis.The clinical data of patients with HAAA admitted to our hospital from February 2017 to May 2022 were retrospectively analyzed.A total of 81 patients with HAAA, 56 males and 25 females. The median onset age was 5.9 years. The median time from hepatitis to occurrence of hemocytopenia was 30 days, and the median follow-up time was 2.77 years. There were 23 cases (28.5%) of severe aplastic anemia (SAA), 50 cases of very severe aplastic anemia (VSAA), and 8 cases of non-severe aplastic anemia (NSAA). At the beginning of the disease, cytotoxic T lymphocyte (CTL) was higher than normal in 60% of patients, and the median CD4/CD8 ratio was 0.2. As of follow-up, 72 children survived, 4 were lost, and 5 died. Thirty-four cases were treated with immunosuppressive therapy (IST), with a median follow-up time of 0.97 years. The total reaction rate was 73.5% (25/34), the complete reaction rate was 67.6% (23/34), and the nonreaction rate was 26.5% (9/34). Multivariate analysis suggested that co-infection was an independent risk factor affecting the efficacy of IST at 6 months, with an OR value of 16.76, 95% CI (1.23, 227.95), P=0.034. No independent influencing factors were found at the end of follow-up.The proportion of CTL cells in peripheral blood of children with HAAA is relatively increased, and IST is effective in 73.5% of children. Co-infection may prolongs the time to response to IST.

Efficacy, Safety, and Population Pharmacokinetics of Eltrombopag in Children with Different Severities of Aplastic Anemia

OBJECTIVES: Eltrombopag (EPAG) was initially approved by the U.S. Food and Drug Administration (FDA) as first-line treatment, combined with standard immunosuppressive therapy, for patients older than two years old with severe aplastic anemia (SAA). However, the use of EPAG in children with different types of aplastic anemia (AA), especially non-severe AA (NSAA) has been limited. Therefore, we decided to investigate the efficacy, safety, and pharmacokinetics of EPAG in children with NSAA, SAA, and very severe AA (VSAA). METHODS: A prospective, open-label, observational study was conducted at the Institute of Hematology and Blood Diseases Hospital, Tianjin, China. The efficacy including complete response (CR), partial response (PR), and no response (NR), as well as safety was assessed every three months after the start of treatment. A non-linear mixed-effects population pharmacokinetic (PPK) model was used to depict the pharmacokinetic profile of EPAG. We explored the exposure-response relationship of EPAG in patients with different types of AA. This study was registered at ClinicalTrials.gov (NCT03844360). RESULTS: A total of 23 AA children with an average age of 7.9 (range of 3.0 - 14.0) years were enrolled in this study for efficacy, safety, and PPK analysis. Fifteen patients had a treatment response (5 had CR and 10 had PR). The response rate was 12.5% after 3 months, 50.0% after 6 months, and 100% after 12 months of treatment for patients with NSAA. For patients with SAA and VSAA, these response rates were 46.7%, 61.5%, and 87.5%. Hepatotoxicity occurred in one patient. Fifty-three blood samples were collected and analyzed to build the PPK model using allometric scaling with fixed exponents. Body weight significantly affected EPAG's apparent clearance and volume of distribution. The mean (range) of the area under the concentration-time curve from time zero to infinity (AUC ∞) was 316 (131 - 809) μg×h /mL. Twenty patients with genotypes were included in the genotype analysis. The apparent clearance of EPAG was significantly higher in allele-T carriers of adenosine triphosphate-binding cassette G2 (ABCG2) (rs2231142, G&amp;gt;T) ( p=0.02). In patients with clinical response, children with NSAA exhibited lower AUC ∞, higher apparent clearance, and higher weight-adjusted apparent clearance than those with SAA or VSAA, although the differences were not significant. CONCLUSIONS: This study enriched the efficacy and safety of EPAG in children with different types of AA. Body weight and ABCG2 genotype significantly influence EPAG clearance. The results may support further individualized treatment of EPAG in children with AA.

Efficacy and Safety of Recombinant Thrombopoietin Combined with Immunosuppressive Therapy for Primary Treatment of Severe Aplastic Anemia: A Single-Center, Open-Label, Randomized, Clinical Trial

Standard immunosuppressive therapy (IST) combined with eltrombopag is the first-line treatment regimen for severe aplastic anemia patients who are ineligible for allo-hematopoietic stem cell transplantation (allo-HSCT). However, there was no second-generation TPO receptor agonist available in China before 2019, and only a first-generation TPO preparation was available for clinical treatment. Although rhTPO and eltrombopag act on different receptor binding sites, the differentiation pathways are similar, so rhTPO can theoretically also stimulate hematopoietic stem cells and promote hematopoietic recovery in AA. In 2015, we used rhTPO combined with IST as the first-line treatment for SAA. The results showed that the OR and quality of response in the rhTPO group were significantly higher than those in the control group. Multivariate analysis showed that the application of rhTPO was an independent prognostic factor affecting the efficacy of IST. Based on these results, we performed a single-center, randomized, open-label, parallel-controlled clinical trial to assess the hematologic response rate and long-term safety 3, 6, and 12 months after treatment. The study was conducted between 2015 and 2019 with 188 patients with primary SAA who were randomly assigned to the control or rhTPO group (95 with IST alone and 93 with rhTPO). For rhTPO, 15000 IU was administered subcutaneously starting on day 14 after p-ATG administration and then once every other day for a total of 14 injections. The median age of the 93 patients in the rhTPO group was 33 years (range 16-67). These included 58 patients with SAA and 35 patients with very severe aplastic anemia (VSAA), which were comparable to the control group. There were no significant differences in baseline clinical parameters between groups. No significant differences were observed in the overall hematologic response rates at 3, 6, and 12 months between the rhTPO and control groups (3 months: 40.9% vs 41.1%, P = 0.979; 6 months: 52.7% vs 52.6%, P = 0.994; 12 months: 65.6% vs 63.2%, P = 0.782, respectively). The median time of platelet transfusion independent was 49 (0-376) days in the rh-TPO group and 47 (0-195) days in the control group, which was not different between the two groups ( P = 0.757). The median time of RBC transfusion independence was 47 (5-404) days and 49 (0-295) days, respectively ( P = 0.592). The median follow-up time was 64 (1.3-86.2) months. The 5-year overall survival was 86.4% (95% confidence interval [CI], 77.2-92.0%) in the rh-TPO group and 84.1% (95% CI, 74.5-90.3%, P = 0.898) in the control group. The 5-year event-free survival was 51.7% (95% CI, 41.0-61.5%) and 54.1% (95% CI, 43.5-63.6%, P = 0.947) in the two groups, respectively. No significant differences were observed in the relapse rate and clonal evolution rate between the two groups (11.5% vs. 11.7%, 4.3% vs.3.2%, repectively). Subcutaneous rhTPO injection was well tolerated by patients, with no significant adverse effects other than mild localized pain at the injection site. The patients in the rhTPO group did not have concomitant thrombotic or embolic complications; moreover, reticulo-fibrillary or collagen fibrillary hyperplasia in bone marrow was not detected in any patients at three and six months after IST. Ten patients had anti-TPO antibodies at the end of TPO treatment, with no positive anti-TPO antibodies. In conclusion, our study is the only prospective randomized controlled clinical trial in China of rhTPO combined with IST for the treatment of SAA, with no difference in the hematologic response rate of the 28-day rhTPO combination regimen compared to standard IST; however, we did confirm the safety of rhTPO at this dose. Further clinical studies are required to determine the most effective dosage and duration of rhTPO treatment.

Haploidentical Stem Cell Transplantation Using Total Body Irradiation (600 cGy) and Fludarabine with ATG in Upfront and Salvage Setting in Adult Patients with Severe Aplastic Anemia

Background: Haploidentical stem cell transplantation from a related mismatched donor (Haplo-SCT) has extended into adult patients with severe aplastic anemia (SAA) with advances in controlling graft failure and GVHD. However, because of concerns for morbidity and mortality, Haplo-SCT has been used as salvage treatment after the failure of immunosuppressive therapy (IST). In Haplo-SCT for SAA patients, the achievement of successful engraftment without GVHD is fundamental for better long-term survival with good quality of life. We optimized the dose of rabbit ATG and fractionated TBI (fTBI) with fixed dose of fludarabine (Flu, 150mg/m 2/day) using step-by-step dose de-escalation (Lee et al, Am J Hematol, 2018) and reached fTBI 600 cGy/Flu/intermediate-dose ATG (5 mg/kg). This protocol showed an overall survival rate of 91.0% after a median follow-up of 32.3 months in a study of 47 patients (Lee et al, BBMT, 2020). Here, we evaluated the feasibility of this protocol in both upfront and salvage setting for Haplo-SCT in adult patients with SAA. Methods: We analyzed 68 consecutive patients who underwent Haplo-SCT between Oct. 2014 and Jan. 2023. Fifty-six (82%) patients had salvage Haplo-SCT after the failure of standard IST (ATG + CsA) (n=36) and CsA monotherapy (n=20). Twelve (18%) patients underwent upfront Haplo-SCT. All patients received a conditioning regimen of 600 cGy fTBI (200 cGy, 3 times) and Flu (30 mg/m 2/day) for 5 days. GVHD prophylaxis consisted of ATG/tacrolimus/methotrexate (MTX). ATG (Thymoglobulin ®,2.5 mg/kg/day) was administered on days -2 and -3. All patients received PBSC. Results: The median age was 34.0 years (17-61) and 30 (44%) patients had very SAA (VSAA) at transplantation. Haploidentical donors are composed of mother (n=10), father (n =8), sibling (n=30), offspring (n=17), and others (n=3). All patients achieved primary engraftment. The cumulative incidence of acute GVHD (grade ≥2) and chronic GVHD (≥ moderate) was 26.5% at 100 days and 9.3% at 4 years, respectively. With a median follow-up of 54 months, the 4-year overall survival (OS) and failure-free survival (FFS) was 93.9% and 92.4%, respectively. 4-year OS of upfront and salvage Haplo-SCT was 100 and 94.7%, respectively (P = 0.352). The 4-year GFFS was 78.9%, and there was no significant difference between upfront and salvage Haplo-SCT (83.3% vs 78.1%, P = 0.709). In the subgroup analysis, 4-year GFFS of patients who failed the standard IST (ATG+CsA) excluding CsA monotherapy also showed no statistical difference compared to the others (85.8% vs 71.3%, P = 0.143). Furthermore, after adjusting for the potential factors affecting GFFS (patient's age, donor type, and comorbidity index), no different GFFS was shown between upfront and salvage Haplo-SCT. Conclusions: Our results suggest that Haplo-SCT with fTBI 600 cGy/Flu/ATG-5 can be an effective alternative option in both upfront and salvage settings, when fully matched donors are not available.

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.

Efficacy and safety of allogeneic hematopoietic stem cell transplantation in the treatment of 28 patients with hepatitis-related aplastic anemia

Objective: To evaluate the efficacy and safety of HLA-haploidentical hematopoietic stem cell transplantation (allo-HSCT) for hepatitis-related aplastic anemia (HRAA) patients. Methods: Retrospective analysis was performed on hepatitis-associated aplastic anemia patients who received haplo-HSCT at our center between January 2012 and June 2022. October 30, 2022 was the final date of follow-up. Results: This study included 28 HRAA patients receiving allo-HSCT, including 18 males (64.3% ) and 10 females (35.7% ), with a median age of 25.5 (9-44) years. About 17 cases of severe aplastic anemia (SAA), 10 cases of very severe aplastic anemia (VSAA), and 1 case of transfusion-dependent aplastic anemia (TD-NSAA) were identified. Among 28 patients, 15 patients received haplo-HSCT, and 13 received MSD-HSCT. The 2-year overall survival (OS) rate, the 2-year failure-free survival (FFS) rate, the 2-year transplant-related mortality (TRM) rate, the 100-day grade Ⅱ-Ⅳ acute graft-versus-host disease (aGVHD) cumulative incidence rate, and the 2-year chronic graft-versus-host disease (cGVHD) cumulative incidence rate were 81.4%, 81.4% (95% CI 10.5% -20.6% ), 14.6% (95% CI 5.7% -34.3% ), 25.0% (95% CI 12.8% -45.4% ), and 4.2% (95% CI 0.6% -25.4% ), respectively. After transplantation, all patients had no significant liver function damage. Compared with the MSD-HSCT group, only the incidence of cytomegaloviremia was significantly higher in the haplo-HSCT group [60.0% (95% CI 35.2% -84.8% ) vs 7.7% (95% CI 0-22.2% ), P=0.004]. No statistically significant difference in the Epstein-Barr virus was found in the 2-year OS, 2-year FFS, 2-year TRM, and 100-day grade Ⅱ-Ⅳ aGVHD cumulative incidence rates and 2-year cGVHD cumulative incidence rate. Conclusion: Allo-HSCT is safe and effective for HRAA, and haplo-HSCT can be used as a safe and effective alternative for newly diagnosed HRAA patients who cannot obtain HLA-matched sibling donors.

Development and validation of early death risk score model for emergency status prediction in very severe aplastic anemia.

This study developed and validated the Early Death Risk Score Model for early identification of emergency patients with very severe aplastic anemia (VSAA). All 377 patients with VSAA receiving first-line immunosuppressive therapy (IST) were categorized into training (n=252) and validation (n=125) cohorts. In the training cohort, age >24 years, absolute neutrophil count ≤0.015×109/L, serum ferritin >900ng/mL and times of fever before IST >1 time were significantly associated with early death. Covariates were assigned scores and categorized as: low (score 0-4), medium (score 5-7) and high (score ≥8) risk. Early death rate was significantly different between risk groups and the validation cohort results were consistent with those of the training cohort. The area under the receiver operating characteristic curve for the model was 0.835 (0.734,0.936) in the training cohort and 0.862 (0.730,0.994) in the validation cohort. The calibration plots showed high agreement, and decision curve analysis showed good benefit in clinical applications. The VSAA Early Death Risk Score Model can help with early identification of emergency VSAA and optimize treatment strategies. Emergency VSAA with high risk is associated with high early death rate, and alternative donor hematopoietic stem cell transplantation could be a better treatment than IST even without HLA-matching.

Association between human leukocyte antigen and immunosuppressive treatment outcomes in Chinese patients with aplastic anemia.

Activated cytotoxic T cells (CTLs) recognize the auto-antigens presented on hematopoietic stem/progenitor cells (HSPCs) through class I human leukocyte antigen (HLA) molecules and play an important role in the immune pathogenesis of aplastic anemia (AA). Previous reports demonstrated that HLA was related to the disease susceptibility and response to immunosuppressive therapy (IST) in AA patients. Recent studies have indicated that specific HLA allele deletions, which helped AA patients to evade CTL-driven autoimmune responses and escape from immune surveillance, may lead to high-risk clonal evolution. Therefore, HLA genotyping has a particular predictive value for the response to IST and the risk of clonal evolution. However, there are limited studies on this topic in the Chinese population. To explore the value of HLA genotyping in Chinese patients with AA, 95 AA patients treated with IST were retrospectively investigated. The alleles HLA-B*15:18 and HLA-C*04:01 were associated with a superior long-term response to IST (P = 0.025; P = 0.027, respectively), while the allele HLA-B*40:01 indicated an inferior result (P = 0.02). The allele HLA-A*01:01 and HLA-B*54:01 were associated with high-risk clonal evolution (P = 0.032; P = 0.01, respectively), and the former had a higher frequency in very severe AA (VSAA) patients than that in severe AA (SAA) patients (12.7% vs 0%, P = 0.02). The HLA-DQ*03:03 and HLA-DR*09:01 alleles were associated with high-risk clonal evolution and poor long-term survival in patients aged ≥40 years. Such patients may be recommended for early allogeneic hematopoietic stem cell transplantation rather than the routine IST treatment. HLA genotype has crucial value in predicting the outcome of IST and long-term survival in AA patients, and thus may assist an individualized treatment strategy.

Interleukin-6 and interleukin-8 levels in children with aplastic anemia and its correlation with disease severity and response to immunosuppressive therapy.

Aplastic anemia (AA) is an uncommon condition characterized by pancytopenia and hypocellular bone marrow. Interleukin (IL)-6 and IL-8 have been shown to inhibit myelopoiesis and are major mediators of tissue damage. The primary goal of this study was to determine the IL-6 and IL-8 levels in children with AA, as well as their relationship to illness severity and immunosuppressive medication response. The IL-6 and IL-8 levels were tested in 50 children aged 3-18 years who had AA. As controls, 50 healthy age and sex matched individuals were used. A sandwich enzyme-linked immunosorbent assay kit (solid-phase) was used to measure IL-6 and IL-8 levels quantitatively. The concentrations of IL-6 and IL-8 in pg/mL were used to represent the results. Immunosuppressive medication was given to the patients in accordance with the British Committee for Standards in Haematology Guidelines 2009. The patients' average age was 11.3 ± 3.7 years. Patients with AA had significantly higher IL-6 and IL-8 levels than controls (278.88 ± 216.03 vs. 4.51 ± 3.26; P < 0.001) and (120.28 ± 94.98 vs. 1.79 ± 0.78; P < 0.001), respectively. The IL-6 and IL-8 levels were also investigated with respect to AA severity, with statistically significant differences (P < 0.01) between different grading strata. Patients with very severe AA (VSAA) had the highest IL-6 levels (499.52 ± 66.19), followed by severe AA (SAA) (201.28 ± 157.77) and non-SAA (NSAA) (22.62 ± 14.63). For IL-8 levels, a similar trend (P < 0.01) was detected, with values of 209.81 ± 38.85, 92.12 ± 78.0, and 9.29 ± 10.68 for VSAA, SAA, and NSAA, respectively. After 6 months of immunosuppressive treatment (IST), mean levels of IL-6 and IL-8 in responders and nonresponders were again assessed. The mean IL-6 level in the responders' group (46.50 ± 45.41) was significantly lower, when compared to the nonresponders' group (145.76 ± 116.32) (P < 0.001). Similarly, the mean IL-8 level in the responder's group (33.57 ± 27.14) was significantly lower, compared to the nonresponder's group (97.49 ± 69.00) (P < 0.001). Children with AA had higher IL-6 and IL-8 levels than normal age- and sex-matched controls. Increased levels were linked to the severity of the condition, suggesting that IL may have a role in AA. IL levels can be monitored in AA patients during IST, which can assist in predicting response to IST.

Apolipoprotein-a Is a Potential Prognosis Biomarker for Severe Aplastic Anemia Patients Treated with ATG-Based Immunosuppressive Therapy: A Single-Center Retrospective Study

Aplastic anemia (AA) is a bone marrow failure disorder characterized by pancytopenia with a risk of hemorrhage and infection. AA can be further divided into severe AA (SAA) and non-severe AA (NSAA) based on the severity of the disease. Anti-thymoglobulin (ATG)-based immunosuppressive therapy (IST) is the standard first-line management for SAA and very severe AA (VSAA) patients not eligible for hematopoietic stem cell transplantation (HSCT). Several studies identified the influencing factors on response and survival, including patient age, disease severity, absolute reticulocyte count and the interval between diagnosis and treatment. However, the prognosis cannot be improved if the patient exhibits poor response indicators when ATG-based IST is the only appropriate method. Adipocytes occupy the bone marrow of SAA, but the function of these cells is not yet clarified. Herein, we conducted this study aimed to explore the serum lipid changes during the ATG-based IST and identify the putative indicators that may predict the prognosis, thereby implying them as potential therapeutic target. A total of 61 newly diagnosed SAA/VSAA patients aged between 10 and 60 years who received ATG-based IST were enrolled, and divided into IST-response (IST-R, n=40) and IST-non-response (IST-NR, n=21) groups based on the 9-month response effect. Differences were observed in blood lipids level, immunoglobulins and complements among groups after ATG-based IST. Significantly higher serum total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), apolipoprotein A (Apo-A) levels were detected in the IST-NR group than the IST-R group pretreatment (all P<0.05). After ATG-treatment, the Apo-A levels increased in the IST-R group while decreased drastically in the IST-NR group (Fig. A-F). Both groups showed decreased IgA after 3 months after IST, and the level in the IST-R group was much lower than that in the IST-NR group (P<0.05) (Fig. G-K). In order to predict the 9-month response early, we conducted a correlation analysis and found that the 9-month IST response had a moderate negative correlation with IgA level at 3 months (Spearman's r=-0.516, P=0.006) and a negative correlation to pretreatment Apo-A (Spearman's r=-0.380, P=0.004). Considering the dramatic changes between the IST-R and IST-NR groups, enrolled patients were respectively divided into two subgroup based on the cutoff value of Apo-A and IgA by receiver operating characteristic (ROC), and found that patients with lower Apo-A (<1.205 g/L) level pretreatment had a better event-free survival (EFS) (P=0.008)，but no differences were observed on OS (Fig. L-M). The patients with IgA level ≥1.72 g/L showed poorer EFS compared to those with IgA <1.72 g/L, although no statistically significant difference was noted (Fig. N-O). In conclusion, the current study found that serum Apo-A is a potential prognosis biomarker for the newly diagnosed <60-year-old SAA/VSAA patients who received ATG-based IST, and the level of 3-month IgA can also be an indicator for 9-month IST response. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cytokines help suggest aplastic anemia with pulmonary bacterial or co-fungal infection

Although aplastic anemia (AA) does not come under the category of blood malignant diseases, the infection that frequently occurs in this bone marrow failure can make it worse. Pulmonary infection is the most prevalent but limiting clinical diagnosis. To find biomarkers predicting bacterial or bacterial-combined fungal infections in the lungs, we reviewed 287 AA medical records including 151 without any infection, 87 with pure pulmonary bacterial infection, and 49 with bacterial and fungal infection were reviewed. There were substantial changes in IL-17F, IL-17A, IFN-γ, IL-6, IL-8, and IL-10 levels between the non-infected and lung bacterial infection groups (P < 0.05). Further, a significant variation in IL-17A, TNF-β, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-22, and IL-12p70, between the uninfected group and the pulmonary bacterial and fungal infection group (P < 0.05) was observed. The results further revealed significant differences in TNF-β, IL-12p70, IL-6, IL-8, and IL-10 between the pulmonary bacterial infection group and the fungal infection group (P < 0.05). Moreover, by calculating ROC and cut-off values, we determined that IL-6 (AUC = 0.98, Cut-off = 14.28 pg/ml, P = 0.0000) had a significant advantage than other cytokines, body temperature (AUC = 0.61, P = 0.0050), PCT (AUC = 0.57, P = 0.0592), and CRP (AUC = 0.60, P = 0.0147) in the detection of lungs bacterial infections. In addition, IL-6 (AUC = 1.00, Cut-off = 51.50 pg/ml, P = 0.000) and IL-8 (AUC = 0.87, Cut-off = 60.53 pg/ml, P = 0.0000) showed stronger advantages than other cytokines, body temperature (AUC = 0.60, P = 0.0324), PCT (AUC = 0.72, Cut-off = 0.63 ng/ml, P = 0.0000) and CRP (AUC = 0.79, Cut-off = 5.79 mg/l, P = 0.0000) in distinguishing bacteria from fungi. This may suggest that IL-8 may play a role in differentiating co-infected bacteria and fungi. Such advantages are repeated in severe aplastic anemia (SAA) and very severe aplastic anemia (VSAA).In conclusion, aberrant IL-6 elevations in AA patients may predict the likelihood of bacterial lung infection. The concurrent increase of IL-6 and IL-8, on the other hand, should signal bacterial and fungal infections in patients.These findings may help to suggest bacterial or fungal co-infection in patients with AA (Focus on VSAA and SAA).

Frontline Therapy Options for Adults With Newly Diagnosed Severe Aplastic Anemia: Intensive immunosuppressive Therapy Plus Eltrombopag or Matched Sibling Donor Hematopoietic Stem Cell Transplantation?

Severe aplastic anemia (SAA) is a life-threatening bone marrow failure disease. Allogeneic hematopoietic stem cell transplantation from a matched sibling donor (MSD-HSCT) and intensive immunosuppressive therapy (IST) are 2 major comparable treatments for SAA. As the addition of eltrombopag (EPAG) to standard IST therapy has greatly improved the survival prognosis of SAA, whether MSD-HSCT or IST/EPAG is the better choice has become a matter of debate. A study was performed involving 99 patients with newly diagnosed acquired SAA from 5 medical centers, including 48 MSD-HSCT cases and 51 IST/EPAG cases, which consisted of rabbit antithymocyte globulin or porcine-antilymphocyte globulin, cyclosporine plus eltrombopag. The results suggested that patients treated with MSD-HSCT or IST/EPAG had similar overall survival (OS) rates exceeding 95% (P = .97). However, the event-free survival rate (EFS) of IST/EPAG (71.0%) was significantly lower than that of MSD-HSCT (89.6%), P = .04. Subgroup analysis indicated that the OS of the MSD-HSCT group was superior to that of the IST/EPAG group (100% versus 85.7%, P = .04) among those with very severe aplastic anemia (VSAA). Both the complete response rate (CR) and overall response rate (OR) with MSD-HSCT were significantly higher than those with IST/EPAG (CR: 79.2% versus 15.7%, P < .001; OR: 97.9% versus 72.6%, P = .001). In conclusion, IST/EPAG or MSD-HSCT treatment achieves an equally high OS in SAA, but MSD-HSCT leads to a better OS in patients with VSAA and shows advantages in improving EFS and accelerating hematopoietic reconstruction in patients with SAA.

A New Immunosuppressive Therapy for Very Severe Aplastic Anemia in Children with Autoantibodies.

At present, a number of very severe aplastic anemia (VSAA) patients cannot receive hematopoietic stem cell transplantation (HSCT) or standard immunosuppressive therapy (IST) due to the high cost of therapy, shortage of sibling donors, and lack of resources to support the HSCT. In addition, some VSAA patients with autoantibodies have no life-threatening infections or bleeding at the time of initial diagnosis. Considering the disease condition, economics and other factors, the present study designed a new and relatively mild treatment strategy: cyclosporine A plus pulsed high-dose prednisone (CsA+HDP). The present study retrospectively analyzed 11 VSAA patients, who were treated with CsA+HDP in our hospital from August 2017 to August 2019. The median follow-up time for these patients was 24.9 months. The overall response rate was 54.5% (6/11) at six months after the initiation of IST and 81.8% (9/11) at deadline. Five patients achieved complete remission and four patients met the criteria for partial response at the last follow-up. The median time to response for responders was 110 days. Three patients underwent HSCT due to the poor effect of CsA+HDP or to find a suitable transplant donor. Recurrence and clonal evolution were not found in any of these patients. The estimated 3-year overall survival rate and 3-year failure-free survival rate were 100.0% and 72.7%, respectively. In addition, the results revealed that the cyclosporine-prednisone-associated toxicity was mild and well-tolerated by most patients. The novel CsA+HDP regimen has good therapeutic effect and safety for VSAA patients with autoantibodies, who have no serious life-threatening infections or bleeding at the time of initial diagnosis.

Nutritional assessment of patients with aplastic anemia: comparison of four nutritional screening tools.

Introduction: nutritional status can affect the treatment of hospitalized patients, and malnutrition can even lead to death. However, the nutritional status of patients with aplastic anemia (AA) is unclear. Objective: to assess the nutritional status of aplastic anemia patients with body mass index (BMI) ≥ 24 kg/m2 (high BMI group) and BMI < 24 kg/m2 (low BMI group), and to compare the consistency between different nutritional screening tools. Methods: patients with aplastic anemia hospitalized from January 2016 to December 2020 were collected. We used the combined index generated by Nutritional Risk Index (NRI), Prognostic Nutritional Index (PNI), Control Nutritional Status (CONUT) and Instant Nutritional Assessment (INA) to assess nutritional status of patients with aplastic anemia. Kappa index was used to measure the consistency between different nutritional screening tools. Receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of different nutritional screening tools. Results: one hundred and ninety-five patients with aplastic anemia were enrolled. The overall prevalence of malnutrition calculated by the combined index in patients with aplastic anemia was 51.3 %. The malnutrition rates of patients in the low BMI group and high BMI group were 60.9 % and 38.8 %, respectively. The malnutrition rates of very severe aplastic anemia (VSAA) patients, severe aplastic anemia (SAA) patients and ordinary patients were 76 %, 63.8 % and 45.1 %, respectively. Compared with the combined index, NRI had the highest consistency and area under the curve. Conclusions: the nutritional status of patients with aplastic anemia was very poor; the more serious the disease, the worse the nutritional status. Although the malnutrition rate in the low-BMI group was higher than in the high BMI group, the nutritional status of overweight or obese patients can not be ignored. NRI is the best tool for assessing the nutritional status of patients with aplastic anemia.

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.

Multipotent Mesenchymal Stromal Cells from the Bone Marrow of Untreated Aplastic Anemia Patients Preserve Their Ability to Support Hematopoietic Precursors However Display the Pronounced Changes in Gene Expression

Aplastic anemia (AA) is believed to be an autoimmune disorder characterized by the pancytopenia due to the depletion of hematopoietic stem and progenitor cells in the bone marrow. There are three forms of AA depending on the severity of pancytopenia: moderate, or non-severe AA (NAA), severe AA (SAA), and very severe AA (VSAA). Clones of cells typical for paroxysmal nocturnal hemoglobinuria (PNH-clones) are frequently present in AA patients in various proportions.We aimed to study stromal microenvironment of untreated AA patients depending on the AA severity, presence or absence of PNH-clone, and on the response to the therapy after 3 and 6 months of treatment. We analyzed the bone marrow (BM) multipotent mesenchymal stromal cells (MMSCs) in their ability to maintain hematopoietic precursors and examined relative expression levels (REL) of selected genes.The study included 17 patients with NAA (53% females, 47% males, 33.8±2.2 years old), 12 patients with SAA (33% females, 67% males, 29.3±3.8 years old). Among NAA patients 7 had PNH-clone, and among SAA - 6 patients. Control group consisted of 19 donors (42% females, 58% males, 30.4±3.1 years old). The ability to support hematopoietic precursors by MMSCs from the BM of AA patients was measured by cobble stone area forming cells (CAFC) assay, where BM cells from one healthy donor were seeded on different MMSCs; REL of selected genes was analyzed with TaqMan RT-PCR. Only the genes with statistically significant differences are presented. The data are presented as mean ± standard error of measures, the differences were statistically significant when p<0.05 when Student's unpaired t-test or Mann-Whitney test was applied.MMSCs from AA patients preserve their ability to maintain hematopoietic precursors. CAFC 7 frequency reflects the number of late hematopoietic precursors. CAFC 7 frequency was slightly higher on MMSCs from NAA patients (9.92±2.73 per 10 6 healthy BM cells) then on MMSCs from healthy donors (5.56±1.14 per 10 6 healthy BM cells), although the difference was not statistically significant. MMSCs from SAA patients maintained CAFC 7 as well as donors' MMSCs (6.75±1.96 per 10 6 healthy BM cells). The frequency of CAFC 28, reflecting the number of early hematopoietic precursor, displayed similar but more pronounced dynamics. CAFC 28 frequency on NAA patients' MMSCs was significantly higher than on donors' ones (2.17±0.34 versus 1.11±0.31 per 10 6 healthy BM cells, p=0.03), while on SAA patients' MMSCs it was also high (1.92±0.57 per 10 6 healthy BM cells) but the difference was insignificant (Table 1). The presence of PNH-clone does not affect the ability of stromal cells to maintain hematopoiesis. MMSCs from the patients that had responded to the therapy in 90 or 180 days did not differ in their ability to maintain hematopoietic precursors from the MMSCs of treatment resistant the patients. Therefore, we can assume that physiological function of stromal microenvironment is not affected deeply in the debut of AA.Gene expression analysis revealed statistically significant upregulation of FGFR1, PDGFRA, VEGFA and downregulation of ANG1 (in MMSCs from both NAA and SAA patients), and upregulation of FGFR2 and CFH (only in NAA patients' MMSCs) (Table 2). In MMSCs of AA patients (both NAA and SAA) without PNH-clone the upregulation of CFH gene was detected (Table 3). CFH is one of the players in the complement system which is disrupted in PNH. This fact needs to be further scrutinized. In addition, IL1R, SDF1 and VEGFA were statistically significantly downregulated in MMSCs from AA patients with PNH-clone compared with MMSCs from patients without PNH-clone. It seems that the presence of PNH-clone corresponds with the changes in stromal microenvironment. Gene expression of analyzed genes was the same in MMSCs of the patients that had responded or not responded to the treatment in 90 or 180 days since the therapy begun.Thus, MMSCs from the BM of untreated AA patients preserve their ability to support hematopoietic precursors however display the pronounced changes in gene expression.The work is supported by the RFBR, project 19-015-00280. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Adult aplastic anemia in Thailand: incidence and treatment outcome from a prospective nationwide population-based study

The incidence and outcomes of aplastic anemia (AA) in Asia remain limited. This study aimed to explore the incidence and outcomes of patients with adult AA across the country of Thailand. This is a prospective multi-center nationwide population-based observational study of AA patients aged at least 15 years old, diagnosed from August 2014 to July 2016, with a longitudinal follow-up period over 2 years. There were 348 newly diagnosed adult AA patients during the enrollment period, giving an annual incidence of 4.6 per million. The incidence of severe (SAA) and very severe aplastic anemia (VSAA) (3.8 per million) was higher than non-severe AA (NSAA, 0.8 per million). The peak incidence was observed in the patients aged from 80 to 89 years old (14.4 per million). The 2-year overall survival (OS) in NSAA, SAA, and VSAA were 65.5%, 49.3%, and 20.1%, respectively (P < 0.001). With regard to the response to immunosuppressive therapy, the overall response rate (ORR) in SAA/VSAA treated with rabbit anti-thymocyte globulin with/without cyclosporin A (rATG ± CsA) were significantly superior to those treated with CsA alone, or anabolic steroids (44.4% vs 36.4% and 31.2%, respectively, P < 0.001). The 2-year OS in SAA/VSAA treated with rATG ± CsA, CsA, and anabolic steroids were 54.8%, 54.5%, and 37.6% (P = 0.037), respectively. The incidence of adult AA in Thailand is higher than those in Western countries, and the peak incidence is in the elderly. rATG ± CsA provided a better response than anabolic steroids, translating to the superior survival in SAA/VSAA treated with rATG ± CsA.