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The bone marrow (BM) is responsible for generating and maintaining lifelong output of blood and immune cells. In addition to its key hematopoietic function, the BM acts as an important lymphoid organ, hosting a large variety of mature lymphocyte populations, including B cells, T cells, natural killer T cells, and innate lymphoid cells. Many of these cell types are thought to visit the BM only transiently, but for others, like plasma cells and memory T cells, the BM provides supportive niches that promote their long-term survival. Interestingly, accumulating evidence points toward an important role for mature lymphocytes in the regulation of hematopoietic stem cells (HSCs) and hematopoiesis in health and disease. In this review, we describe the diversity, migration, localization, and function of mature lymphocyte populations in murine and human BM, focusing on their role in immunity and hematopoiesis. We also address how various BM lymphocyte subsets contribute to the development of aplastic anemia and immune thrombocytopenia, illustrating the complexity of these BM disorders and the underlying similarities and differences in their disease pathophysiology. Finally, we summarize the interactions between mature lymphocytes and BM resident cells in HSC transplantation and graft-versus-host disease. A better understanding of the mechanisms by which mature lymphocyte populations regulate BM function will likely improve future therapies for patients with benign and malignant hematologic disorders.

Platelet transfusion refractoriness results in adverse outcomes and increased health care costs. Managing refractoriness resulting from HLA alloimmunization necessitates the use of HLA antigen–matched platelets but requires a large platelet donor pool and does not guarantee full matching. We report the first randomized, double-blind, noninferiority, crossover trial comparing HLA epitope–matched (HEM) platelets with HLA standard antigen–matched (HSM) platelet transfusions. Alloimmunized, platelet-refractory, thrombocytopenic patients with aplastic anemia, myelodysplastic syndrome, or acute myeloid leukemia were eligible. HEM platelets were selected using HLAMatchMaker epitope (specifically eplet) matching. Patients received up to 8 prophylactic HEM and HSM transfusions provided in random order. The primary outcome was 1-hour posttransfusion platelet count increment (PCI). Forty-nine patients were randomized at 14 UK hospitals. For intention to treat, numbers of evaluable transfusions were 107 and 112 for HEM and HSM methods, respectively. Unadjusted mean PCIs for HEM and HSM methods were 23.9 (standard deviation [SD], 15) and 23.5 (SD, 14.1), respectively (adjusted mean difference, −0.1; 95% confidence interval [CI], −2.9 to 2.8). Because the lower limit of the 95% CI was not greater than the predefined noninferiority limit, the HEM approach was declared noninferior to the HSM approach. There were no differences in secondary outcomes of platelet counts, transfusion requirements, and bleeding events. Adequate 1-hour PCI was more frequently observed, with a mean number of 3.2 epitope mismatches, compared with 5.5 epitope mismatches for inadequate 1-hour increments. For every additional epitope mismatch, the likelihood of an adequate PCI decreased by 15%. Epitope-matched platelets should be considered to support HLA alloimmunized patients. This trial was registered at www.isrctn.com as #ISRCTN23996532.

Introduction: β-thalassemia major (TM) is one of the most prevalent inherited hemoglobinopathies in Pakistan. It has one of the highest prevalence of transfusion-dependent TM patients globally, with an estimated greater than 100,000 active cases. Each year, an estimated 5000-9000 new cases of TM are being diagnosed in the country. Blood transfusions (BT) are essential in the management of severe TM; it is critical to have a safe BT to reduce the risk of transfusion transmissible infections (TTIs). Frequent blood transfusions in these patients increase their risk of acquiring TTIs compared to the general population. In this systematic review, we aimed to identify the prevalence of TTIs in transfusion-dependent β -thalassemia major patients in Pakistan. Methods & Material: We performed a systematic literature search to identify studies related to the TTIs and transfusion-related infections in Pakistan from January 1, 2010, to January 31, 2020. The search was conducted using PubMed and PakMediNet (Largest medical database of Pakistan), with initial search retrieved 981 studies. Among these, 166 studies met the inclusion criteria. After further screening by reviewing the articles for relevance and availability of full-length articles, only 14 studies met the final criteria for qualitative synthesis. Results Analysis of 14 studies (n=3786) showed that the seroprevalence of Hepatitis B virus (HBV) of 3.13% (0.66 % to 7.4%) and Hepatitis C virus (HCV) of 26 % (5.56% to 68.2%). There were only two studies reported HIV seroprevalence of 0 % & 0.5% (n=6). The rate of seropositivity for HBV and HCV was directly related to the number of transfusions, higher ferritin levels, and older age groups. There was an increase in the HCV rate with the increasing age of patients. Thalassemia patients who were older than ten years of age had a greater HCV compared to those who were less than ten years of age, i.e., 22% vs. 8.4%, p:0.005, respectively. The mean age was higher in HCV reactive children than non-reactive children. A comparison of HCV in healthy donors vs. thalassemia patients showed a rate of 1.9% vs. 13.1% for T.M. patients. There was HCV infection rate of 74% in the group with greater than 100 BTs compared to 33 % in a group with fewer than 35 BTs. The rate of HCV increased to 75% for the patients who had more than 100 BTs. The majority of the patients were males (51% to 88%). The seroprevalence of TTIs was higher in males than in females (73.4% vs. 26.6%). On average, a single TM patient is exposed to at least 17 different donors annually, requiring 1-2 transfusions every month. The free BT is accessible only in 1 out of 4 thalassemia centers. The majority of patients either need to bring their donors or are dependent on an external source of financial aid as they could not afford the cost of BT treatment. More than half of thalassemia patients (57.2%) need to contact multiple BT centers to search for required blood products. About 42.1% of parents of TM patients did not know about TTIs, whereas 31.6% of them did not know about the bloodborne transmission of HBV and HCV. The majority of parents of TM children had a low income, with 75% of them having income less than 10,000 Pakistani rupees (PKR) per month. The prevalence of TTIs in TM patients was significantly higher (96% vs. 4%) compared to the patients requiring multiple transfusions due to other causes such as leukemia, aplastic anemia, and thrombocytopenia. Conclusion: Our data highlights that the prevalence of transfusion-transmitted infections, especially HCV, is alarmingly higher (26%) in the TM population than in the general population. This is because of a lack of resources, inadequate safety measures, and a fragmented blood transfusion system. These findings warrant the urgent need for better public health measures, safe blood transfusion practices, voluntary remunerated blood-based transfusions, and universal quality-assured donor screening. Without these positive interventions, the current transfusion system can lead to a further worsening of the situation. Large prospective multi-centered clinical trials are required to understand better the high prevalence of TTIs in patients with TM. Disclosures Anwer: Incyte, Seattle Genetics, Acetylon Pharmaceuticals, AbbVie Pharma, Astellas Pharma, Celegene, Millennium Pharmaceuticals.:Honoraria, Research Funding, Speakers Bureau.

Myelodysplastic syndromes (MDS) are clonal diseases defined by clinical, morphologic, and genetic features often shared by related myeloid disorders. The diagnostic boundaries between these diseases can be arbitrary and not necessarily reflective of underlying disease biology or outcomes. In practice, measures that distinguish MDS from related disorders may be difficult to quantify and can vary as disease progression occurs. Patients may harbor findings that are not consistent with a single diagnostic category. Several overlap disorders have been formally described, such as the myelodysplastic/myeloproliferative neoplasms (MDS/MPNs). These disorders are characterized by hematopoietic dysplasia with increased proliferation of monocytes, neutrophils, or platelets. They may have mutational profiles that distinguish them from the disorders they resemble and reflect important differences in pathophysiology. MDS also shares diagnostic borders with other diseases. For example, aplastic anemia and hypoplastic MDS can be difficult to distinguish in patients with pancytopenia and bone marrow hypocellularity. Genetic features may help in this regard, because they can identify differences in prognosis and risk of progression. The boundary between MDS and secondary acute myeloid leukemia (sAML) is arbitrarily defined and has been redefined over the years. Genetic studies have demonstrated that sAML clones can precede clinical progression from MDS by many months, suggesting that MDS with excess blasts could be viewed as an overlap between a dysplastic bone marrow failure syndrome and an oligoblastic leukemia. This review will describe the diagnostic boundaries between MDS, MDS/MPNs, sAML, clonal hematopoiesis of indeterminate potential, clonal cytopenia of undetermined significance, and aplastic anemia and how genetic approaches may help to better define them.

Interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) have been implicated historically in the immune pathophysiology of aplastic anemia (AA) and other bone marrow (BM) failure syndromes. We recently defined the essential roles of IFN-γ produced by donor T cells and the IFN-γ receptor in the host in murine immune-mediated BM failure models. TNF-α has been assumed to function similarly to IFN-γ. We used our murine models and mice genetically deficient in TNF-α or TNF-α receptors (TNF-αRs) to establish an analogous mechanism. Unexpectedly, infusion of TNF-α−/− donor lymph node (LN) cells into CByB6F1 recipients or injection of FVB LN cells into TNF-αR−/− recipients both induced BM failure, with concurrent marked increases in plasma IFN-γ and TNF-α levels. Surprisingly, in TNF-α−/− recipients, BM damage was attenuated, suggesting that TNF-α of host origin was essential for immune destruction of hematopoiesis. Depletion of host macrophages before LN injection reduced T-cell IFN-γ levels and reduced BM damage, whereas injection of recombinant TNF-α into FVB-LN cell-infused TNF-α−/− recipients increased T-cell IFN-γ expression and accelerated BM damage. Furthermore, infusion of TNF-αR−/− donor LN cells into CByB6F1 recipients reduced BM T-cell infiltration, suppressed T-cell IFN-γ production, and alleviated BM destruction. Thus, TNF-α from host macrophages and TNF-αR expressed on donor effector T cells were critical in the pathogenesis of murine immune-mediated BM failure, acting by modulation of IFN-γ secretion. In AA patients, TNF-α–producing macrophages in the BM were more frequent than in healthy controls, suggesting the involvement of this cytokine and these cells in human disease.

Idiopathic aplastic anemia (AA) has 2 key characteristics: an autoimmune response against hematopoietic stem/progenitor cells and regulatory T-cells (Tregs) deficiency. We have previously demonstrated reduction in a specific subpopulation of Treg in AA, which predicts response to immunosuppression. The aims of the present study were to define mechanisms of Treg subpopulation imbalance and identify potential for therapeutic intervention. We have identified 2 mechanisms that lead to skewed Treg composition in AA: first, FasL-mediated apoptosis on ligand interaction; and, second, relative interleukin-2 (IL-2) deprivation. We have shown that IL-2 augmentation can overcome these mechanisms. Interestingly, when high concentrations of IL-2 were used for in vitro Treg expansion cultures, AA Tregs were able to expand. The expanded populations expressed a high level of p-BCL-2, which makes them resistant to apoptosis. Using a xenograft mouse model, the function and stability of expanded AA Tregs were tested. We have shown that these Tregs were able to suppress the macroscopic clinical features and tissue manifestations of T-cell–mediated graft-versus-host disease. These Tregs maintained their suppressive properties as well as their phenotype in a highly inflammatory environment. Our findings provide an insight into the mechanisms of Treg reduction in AA. We have identified novel targets with potential for therapeutic interventions. Supplementation of ex vivo expansion cultures of Tregs with high concentrations of IL-2 or delivery of IL-2 directly to patients could improve clinical outcomes in addition to standard immunosuppressive therapy.

Introduction: Aplastic anemia (AA) is a life-threatening disorder characterized by pancytopenia and a hypocellular bone marrow. Pure red cell aplasia (PRCA) is a similar disorder with primary reduction in the red blood cell population and virtual absence of erythroid precursors in the bone marrow. While the etiology of immune mediated marrow failure is multifactorial, preceding viral infections have been associated with the disease; these include parvovirus B19, cytomegalovirus, and Epstein-Barr virus. We present four cases of immune mediated marrow failure with either preceding or simultaneous SARS-CoV-2 infection. Methods: The medical records of patients treated for AA or PRCA at the University of Texas Southwestern Medical Center, Parkland Hospital, and the National Institutes of Health (NIH) were reviewed for SARS-CoV-2 infection. Four patients without prior hematological diseases were identified who had SARS-CoV-2 infection prior to or with simultaneous the diagnosis of AA or PRCA. Results: Patient #1 was a 22-year-old white female who was diagnosed with asymptomatic COVID-19 10 days prior to her pancytopenia and AA diagnosis was confirmed by bone marrow biopsy (5% cellularity; Table 1). Her extensive work-up including HIV, hepatitis panel, immunoglobulins, B12 and folate was negative, and she underwent HLA-matched family donor hematopoietic stem cell transplant. Patient #2 was a 69-year-old Asian female who presented to her primary care physician with symptoms of fatigue and was found to be pancytopenic. CBC from a few months prior was completely normal. Further work-up was positive for COVID-19 and negative for HIV, nutritional deficiency, or hemolysis. She did not have respiratory symptoms, was eventually diagnosed with pRBC and platelet transfusion-dependent severe AA (5-10% cellularity on bone marrow), and underwent treatment with cyclosporine, equine antithymocyte globulin, and eltrombopag. She has had a partial response to this therapy. Both patients had bone marrow specimens stained for SARS-CoV-2 by immunohistochemistry that were negative. Patient #3 was a 76-year-old white male who was diagnosed with COVID-19 4 months prior to presenting with a non-ST segment myocardial infarction and found to be profoundly anemic, requiring pRBC transfusion. He re-presented with chest pain one week later and was found to be anemic again, and required transfusion. A trial of darbepoetin alfa was unsuccessful. Extensive work-up for malignancy, infection, and autoimmune etiologies were negative. He was diagnosed with PRCA based on the bone marrow biopsy and initiated treatment with cyclosporine. Patient # 4 was diagnosed with severe AA (presenting as pancytopenia) and COVID-19 infection. He had fatigue for one month and fever, chills and sore throat one-week prior seeking medical care. Testing for hepatitis, HIV, EBV, and CMV was negative. He was treated on a clinical trial (NCT04304820) at NIH with cyclosporine and eltrombopag until SARS-CoV-2 PCR was negative then received equine anti-thymocyte globulin. He has achieved a complete hematologic response at 6 months and remains well at last follow-up. Conclusion: The four patients described had minimal respiratory COVID-19 symptoms, but they presented with cytopenia and were eventually diagnosed with bone marrow failure. It is possible that this is co-incidental due to the high prevalence of SARS-CoV-2. However, there is emerging evidence that COVID-19 pneumonia is a hyperinflammatory and immune dysregulated state improved by dexamethasone therapy. Other immune mediated hematologic conditions, such as autoimmune hemolytic anemia and immune thrombocytopenia, have been reported. The onset from infection to cytopenia appears rapid, although patients often presented with symptoms for many days prior to diagnosis and thus testing may have been delayed from the onset of infection. This case series does not provide a mechanistic link between SARS-CoV-2 infection and bone marrow failure, but it raises the possibility that SARS-CoV-2 may mediate an immunologic response that contributes to marrow failure. Patients appear to respond well to standard immunosuppressive treatment. Further cases and studies are needed to determine if this is directly linked to SARS-CoV-2 and whether the natural history and response to standard therapy is different than idiopathic cases. Figure 1 Figure 1. Disclosures Young: Novartis: Research Funding.

Backgrou n d : Patients with low-risk myelodysplastic syndrome (MDS) and aplastic anemia (AA) often need transfusions, which may accelerate the iron overload. The aim of this study was to evaluate the efficacy, safety and dose-effect relation of deferasirox (DFX) for patients with low-risk MDS and AA who were refractory to regular treatment in the real-world setting. M ethods: This was a retrospective study. Patients with low-risk MDS or AA who failed to standard treatments and were transfusion-dependent were enrolled. DFX was given as the only treatment apart from transfusion. Patients were recorded for their medical history, laboratory tests, nuclear magnetic resonance (MRI), echocardiography and calculated for their overall survival (OS). Dose-effect relations of DFX were evaluated after the first 6 months. Total annual exposure of DFX was calculated after 12 months, expressed as accumulated exposure time at dose of 20mg/kg/d. R esults: Of the 112 patients finally enrolled, 61 (54.5%) were low-risk MDS and 51 (45.5%) were AA. The median age was 56 (10, 89) years and 52.7% patients were males. The minimum dose of DFX for significant SF decrease was 20 mg/kg/d at 6 months; and the minimum accumulation of DFX had to reach 9 months at 20 mg/kg/d to maintain the efficacy at the time of 12 months for patients with low-risk MDS (p＜0.050). Different from MDS, the minimum dose for significant SF decrease was 10 mg/kg/d at 6 months; and the minimum accumulation had to reach 3 months at 20 mg/kg/d to maintain the efficacy at the time of 12 months for patients with AA (p＜0.050). Meanwhile, with same dose of exposure, significant improvements in hematological parameters were also observed in AA, but no dose-effect relations were found in MDS. 62.3% MDS and 51.0% AA patients stopped transfusion in the next 6 months. Erythroid responders had lower SF than non-responders after 12 months of DFX, both for MDS and AA (p＜0.05). Lower alanine aminotransferase (ALT) and aspartate aminotransferase (AST) compared with baseline after 12 months of DFX were observed and longer exposure time correlated with lower ALT (p＜0.050). No significant changes in cardiac function, however. Similar side effects were found in MDS and AA, with gastrointestinal disorders and elevated serum creatinine the most common. Higher dose and longer exposure time of DFX correlated with longer overall survival, both for patients with MDS and AA (p＜0.050). Conclusion: Although dose of DFX varies greatly in different individuals, a significant decrease in SF and an improvement of hematologic parameters, organ functions, or even overall survival can be achieved if the accumulate dose reaches a certain level. In that case, patients with low-risk MDS need higher dose than those with AA. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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 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. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Despite therapeutic successes, AA patients (pts) exhibit a much higher risk of leukemic evolution than the general population. Secondary myeloid neoplasia (sMN) remains the most serious AA complication with major therapeutic and prognostic implications. Historically, multiple theories have been proposed as to the origin of leukemic evolution. For instance, sMN may be the consequence of a relentless autoimmune attack producing a maladaptive response to immunosuppression (IST). Alternatively, occurrence of leukemogenic drivers may be an event setting in motion initially successful and overshooting tumor surveillance reactions. Finally, sMN pathogenesis may be related to either CHIP evolution or acceleration of its progression. Each of these theories is supported by clinical-molecular features (e.g. HLA mutations, secondary PNH evolution, somatic mutations at presentation etc.). Here, we took advantage of a multicentric cohort of pts with AA (n=1010; to our knowledge the largest yet explored) and primary MN (n=3599) to define immunogenetic, iatrogenic and molecular determinants of MN progression. Among AA pts (M:F ratio 0.98; median age 34 years, IQR 20-54, median follow-up 89 months), the 5 and 10 years cumulative incidences of sMN were 6% and 11% respectively, with a median time to progression of 56 months (IQR 23-96). Analysis of available data showed that younger pts (3.5) were observed in 59% (vs 43% in pMDS, p=.02) due to the enrichment in poor/very poor cytogenetic risk groups. In particular, chr.7 abnormalities were the most frequent (54%, of which 88% were del7). By comparison, del7/7q was present in 8% of pMN cases (p At the time of AA onset only 18% of pts harbored somatic myeloid mutations with their presence/absence not influencing evolution, whereas mutations were found in 81% of sMN (1.7 mutations/patient, n=86/101). No difference in mutational burden was observed according to presence/absence of del7/7q, which constituted the founder lesion in 60% of cases, when the analysis was possible. ASXL1 (24% vs 14%, p=.01), RUNX1 (21% vs 11%, p=.008), SETBP1 (14% vs 3%, p AA malignant evolution is characterized by an orchestra of molecular events with an invariant genomic signature (e.g. CUX1, SETBP1, ASXL1). Immunogenetic and immune escape mechanisms may also play a role in shaping the fate of individual patients' trajectories towards PNH vs sMN progression, which may be considered a maladaptive escape event resulting from a bottlenecked hematopoiesis. Disclosures Sebert: BMS: Consultancy; Abbvie: Consultancy. Patel: Apellis: Consultancy, Other: educational talks, Speakers Bureau; Alexion: Consultancy, Other: educational talks, Speakers Bureau. Voso: Celgene: Consultancy, Research Funding, Speakers Bureau; Novartis: Speakers Bureau. Calado: Novartis Brasil: Honoraria; Alexion Brasil: Consultancy; AA&MDS International Foundation: Research Funding; Agios: Membership on an entity's Board of Directors or advisory committees; Instituto Butantan: Consultancy; Team Telomere, Inc.: Membership on an entity's Board of Directors or advisory committees. Peffault De Latour: Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Amgen: Research Funding; Alexion Pharmaceuticals: Consultancy, Honoraria, Research Funding; Apellis Pharmaceuticals Inc: Consultancy, Honoraria; Swedish Orphan Biovitrum AB: Consultancy, Honoraria. Maciejewski: Regeneron: Consultancy; Alexion: Consultancy; Novartis: Consultancy; Bristol Myers Squibb/Celgene: Consultancy.

Introduction: Temozolomide-induced aplastic anemia (TIAA) is a very rare and highly challenging complication of temozolomide (TMZ) chemotherapy. TMZ is a mainstay of therapy in patients with central nervous system (CNS) malignancies. Single-patient case reports and small case series have described variable morbidity and mortality associated with TIAA. However, quantitative evidence describing prognosis, clinical characteristics, and treatment of this entity is absent or very limited. Methods: We performed a 5-center, 22-year observational cohort study of patients with CNS malignancies treated with temozolomide who developed TIAA, retrospectively analyzing prognosis, complications, and recovery. We compared patients who achieved meaningful hematologic recovery [partial hematologic recovery (PHR) or complete hematologic recovery (CHR)] to those who did not recover [refractory disease (RD)]. We used descriptive statistics, T-tests, and chi-square tests to evaluate patient characteristics, outcomes, and laboratory values. Overall time to hematologic recovery and overall survival of patients with PHR, CHR, and RD were estimated using the Kaplan-Meier method and compared using the log-rank test, and a multivariable logistic model evaluated potential predictors of achieving CHR. TIAA was defined using adapted evidence-based severe aplastic anemia criteria incorporating profound cytopenias and a minimum duration (4 weeks) without hematologic recovery (TABLE 1). Results: Study Population and TIAA Prevalence. Of 3,821 patients with CNS malignancies receiving TMZ, 34 patients (0.89%) met criteria for TIAA (FIGURE 1). Onset was rapid, with 29 patients (85.3%) developing TIAA before completing a second TMZ cycle (TABLE 2). Complications and Outcomes of Hematologic Recovery. 23 patients (67.6%) ultimately achieved a hematologic recovery and the remaining 11 patients (32.4%) had refractory disease. Figure 2A illustrates the time from TIAA diagnosis to PHR and CHR for the cohort. In Kaplan-Meier survival analysis, the median time from TIAA diagnosis to PHR was 84 days and the median time to CHR was 134 days. Patients without recovery were more likely to develop serious complications (TABLE 3), including febrile neutropenia (72.7% vs. 30.4%, P=0.03), major infection (45.5% vs. 8.7%, P=0.02), hospitalization (81.8% vs. 43.5%, P=0.04), and death (100.0% vs. 60.9%, P=0.02). This increased risk of complications in patients not achieving hematologic recovery was observed despite a similar median time from TMZ initiation to TIAA diagnosis in the two groups (45 days vs. 40 days). Figure 2B illustrates the overall survival in the entire cohort and by each hematologic recovery subgroup. In Kaplan-Meier survival analysis, median overall survival from the time of TIAA diagnosis was as follows: entire cohort, 355 days; achieved PHR, 752 days; achieved CHR, 1414 days; refractory disease, 28 days (P Treatment of TIAA and Outcomes of Therapy. 29 patients (85.3%) received hematopoietic growth factors (TABLE 3); no patients were treated with immunosuppression or hematopoietic stem cell transplant. Hematologic recovery rates were numerically higher in patients receiving thrombopoietin receptor agonists vs. those who did not (81.8% vs. 54.2%, P=0.15), but were not higher in patients receiving granulocyte colony stimulating factors vs. those who did not (68.0% vs. 66.7%, P=0.99). Conclusions: TIAA occurs in less than 1% of patients receiving temozolomide for CNS malignancies, occurring rapidly after TMZ initiation, but is highly morbid and often fatal when it occurs, precluding further use of cytotoxic therapy in the vast majority of patients. In the one-third who do not recover, risks of febrile neutropenia, infection, and hospitalization are increased, and overall survival greatly diminished. Hematologic recovery is potentially aided by the use of thrombopoietin receptor agonists, as has been demonstrated in classical autoimmune aplastic anemia. Further study of TIAA treatment is needed for this serious complication of TMZ therapy. Figure 1 Figure 1. Disclosures Forst: Eli Lilly: Current holder of individual stocks in a privately-held company. Al-Samkari: Rigel: Consultancy; Argenx: Consultancy; Moderna: Consultancy; Novartis: Consultancy; Amgen: Research Funding; Dova/Sobi: Consultancy, Research Funding; Agios: Consultancy, Research Funding.

Introduction Aplastic anemia (AA) is characterized by a hypoplastic marrow and bone marrow failure (BMF), leading to peripheral pancytopenia. The treatment for AA currently consists of either hematopoietic stem cell transplant (HSCT) or immunosuppressive therapy (IST) with anti-thymocyte globulin and cyclosporine. We have previously reported poor responses to IST in Indian children with AA while adults (>15 years) show responses of 60%. Therefore, we wanted to study if we could identify constitutional variants in children and young adults with AA. Methods We included 102 young patients (≤40 years of age) diagnosed to have AA between year Jan 2006 to April 2021. Genomic DNA was extracted from peripheral blood and relative telomere length (rTL) was measured using quantitative real-time PCR (qPCR). The DNA was used to perform targeted gene capture using a custom capture kit which covered 2196 genes associated with various hematological disorders. Among the 2196 genes, the analysis was restricted to 96 genes associated with AA/IBMFS. Bioinformatics analysis was carried out using Genome Analysis ToolKit (GATK) best practices pipeline. Based on the American College of Medical Genetics and Genomics (ACMG) guidelines, the variants were labelled as pathogenic/likely pathogenic/variant of uncertain significance (VUS)/likely benign/benign. The candidate variants were validated by Sanger sequencing. Results The median age of patients was 9 (0-40) years, including 56 males (55%) and 46 females (45%). There were 15 patients with non-severe AA (NSAA) (15%), 63 with severe AA (SAA) (62%) and 24 with very severe AA (VSAA) (23%). The median relative telomere length of the entire cohort was 0.85 (0.21-3.10). The characteristics of patients age-wise are mentioned in Table 1. Genetic variants were identified in 34 patients (33.3%). This included germline pathological (PAT) genetic variants in 12.7%, variants of limited significance in 9.8% and variants of uncertain significance (VUS) in 10.7% patients. Of the 13 patients who had PAT variants, 6 patients had variants in telomere associated genes. This includes 2 patients with splice site and missense mutation in TINF2 (c.1221+5G>A & R282H); 2 patients with missense mutation in TERT (C828W & S947C); and 2 patients with frameshift and nonsense mutation in RTEL1 (L962S & R1010X). Homozygous MPL mutations [L79Q, R522T & P530L(n=3)] were observed in 5 patients. Two patients had PAT variants in DNAJC21 and NLRP12. Eleven VUS variants were present in the following genes, ATM (n=3), TINF2 (n=1), WRAP53 (n=1), BRCA2 (n=1), AK2 (n=1), FANCA (n=1), FANCN (n=1), ERCC6L2 (n=1) & PRF1 (n=1). The incidence of mutations in the age group ≤5, 6-10, 11-15 and 16-30 years were 51.8%, 25.6%, 27.8% & 33.4% respectively. There was significant difference in median rTL between patients with variants and no variants in the age-group 11-15 years (p=0.043). Discussion Genetic analyses studies in aplastic anemia patients were confined to single genes or limited gene sets. Keel et al., reported 5.1% of AA patients carried pathogenic variants in inherited BMF/MDS genes. We observed a high frequency of causative genetic variants (37%) in children ( Figure 1 Figure 1. Disclosures Mathews: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor.

Introduction Aplastic anemia is a challenging disease to treat due to its rarity and severity. The current standard of care for severe and very severe aplastic anemia includes consideration of frontline allogeneic stem cell transplant (ASCT) in patients who are under 40 years of age and have an HLA-matched sibling. For patients who do not meet this criteria, immunosuppressive therapy (IST) consisting typically of horse antithymocyte-globulin (ATG) and cyclosporine (CsA) is considered, with recent data supporting the addition of eltrombopag. The main risks of IST are infusion reactions, serum sickness, kidney injury, and infection. While it is generally well tolerated in younger patients, older patients have historically been treated with transfusion support alone due to concerns for higher complications. Unfortunately, data to support clinical decisions regarding these concerns is limited, with small studies showing that reduced dosing or attenuated doses of ATG with or without CsA is safe and can be effective. At the University of Michigan, we have been treating older patients with attenuated doses of ATG +/- CsA since the late-2000s and have recently added eltrombopag to our standard of care. Herein, we present the results of our experience using IST to treat aplastic anemia, specifically presenting our outcomes in patients over 60 years old at the time of initiation of IST. Methods We used the Electronic Medical Record Search Engine (EMERSE) to query patients with aplastic anemia who were treated at the University of Michigan between 1995 and 2021. Patients included in our analysis were diagnosed with idiopathic aplastic anemia or hepatitis-associated aplastic anemia, were 18 years or older at the date of initiating IST, were treated with IST first-line, had adequate physician follow up at the University of Michigan, and were reported as being complaint with IST. Patients diagnosed or with high concern for hypoplastic myelodysplastic syndrome (MDS) were excluded in addition to those who received treatment for paroxysmal nocturnal hemoglobinuria (PNH). Statistical analysis was performed using SPSS Statistics v.27 (IBM, 2020). Results We identified 62 patients aged 18 - 60 and 54 patients over 60 years old who met inclusion criteria. Descriptive statistics of the two cohorts are available in Table 1. All patients in our study received horse ATG in addition to cyclosporine. 63% of patients over 60 received full dose horse ATG (20mg/kg for 5 days) and 37% received attenuated horse ATG (20mg/kg for 5 days), while 46% received eltrombopag as part of their IST. OS was superior in patients 18-60 receiving IST with median OS NR vs. 2760 days (Figure 1; p=0.009). In patients over 60, no difference in OS was observed in those who received attenuated (3646 days) vs. full dose IST (2760 days) (Figure 2, p=0.83). Lastly, no difference in median OS was seen in patients > 60 years old who received eltrombopag compared to those who did not (2821 vs 2760 days, Figure 3, p=0.795). Using a Chi-squared test, no statistically significant differences in rates of PR at 12 months, CR at 12 months, primary refractory disease, febrile neutropenia within 6 months of IST, bacterial infections within 6 months IST, and viral infections within 6 months of IST were noted between those who received dose attenuated ATG and those who received full dose ATG in patients > 60. PR in older patients was 48% (12) and 36% (9) at 3 and 12 months with eltrombopag, compared to 40% (10) and 36% (9) without. CR in older patients was 12% (3) and 16% (4) with eltrombopag at 3 and 12 months, compared to 12% (3) and 12% (3) without. In patients > age 60 who received dose attenuated IST, the addition of eltrombopag did not affect OS (Figure 4). A trend toward better OS was seen in patients 18-60 who received eltrombopag, but this was not statistically significant (Figure 5). Discussion In conclusion, we present our experience using IST to treat aplastic anemia at the University of Michigan. Dose attenuated IST has been proposed as a potential option for elderly patients, but we found no significant difference in OS, rates of response, or rates of infection between patients who received full dose and those who did not. There was also a trend toward improved survival with the addition of eltrombopag in younger patients, but additional follow up time is needed. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background：The lipid metabolomic profile has been well defined in the pathogenesis and differential diagnosis in patients with different myeloid diseases. However, the role of plasma lipidome was rarely explored, especially in aplastic anemia (AA), a disease which has close connection with lipid metabolism. Methods: Peripheral fasting serum levels of patients newly diagnosed with AA from March 2019 to December 2019 were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the serum lipid profiles. Meanwhile, the lipidomes for patients with hypocellular myelodysplastic syndrome (h-MDS) and age and sex matched healthy volunteers were taken as controls. The lipid profiles were tested again 6 months after standard cyclosporin A（CsA）treatment in patients with AA. For all the patients, those with a history of hyperlipidemia, diabetes, obesity (body mass index > 28 kg/m 2) or complications with other malignant diseases at diagnosis were excluded. Results: The study enrolled 15 patients with AA, 11 patients with h-MDS and 20 healthy controls. All the enrolled AA patients were non-severe and transfusion dependent, and were treated with CsA 3-5mg/kg/d for at least 6 months. For h-MDS patients, five were MDS with single lineage dysplasia, three were MDS with multilineage dysplasia, and three were MDS-Excess Blasts 1 (MDS-RAEB1). Metabolites in arachidonic acid pathway and retinol metabolism were significantly decreased in the AA patients compared with the healthy controls (P Conclusion: The lipid profiles showed significant difference not only between patients with AA and healthy controls, but also between AA and h-MDS. Meanwhile, some of baseline value and the change in lipid molecules may predict the CsA response at 6 months. Disclosures No relevant conflicts of interest to declare.

Hepatitis-associated aplastic anemia (HAAA) refers to aplastic anemia (AA) in which pancytopenia appears within 6 months after an acute attack of hepatitis. The incidence of HAAA in the Far East and West accounts for 4-10% and 2-5%, respectively, in all AA cases. HAAA frequently occurs in young men, with a median age of 19 (6-56) years, and the two-month mortality after the onset of HAAA can reach 78-88% if the disease was leaved untreated. To date, the underlying pathogenesis is not yet clarified, and no specific correlation has been established between HAAA and chemical toxicants, neither radiation. Based on available studies, the hyper-activation of T lymphocytes and abnormal humoral immunity were confirmed in HAAA, and a large number of infiltrated lymphocytes are also noted in liver at the early stage of the disease, which resulting in liver dysfunction. Some viruses, including hepatitis A, hepatitis B, hepatitis C, parvovirus B19, human herpesvirus (HHV), and Epstein-Barr virus were correlated to the development of HAAA. However, the clinical results of the serological tests for hepatitis-associated viruses of most patients are negative, i.e., serologically negative HAAA. The frontline treatments are agreed to be allogeneic hematopoietic stem cell transplantation (allo-HSCT) or immunosuppressive therapy (IST). Because of the relatively scattered clinical data, compared to other acquired AA, the basis of treatment decision-making is insufficient. Some studies speculated that both IST and HSCT are effective, while others believe that transplantation should be the first choice if suitable donors are available. Therefore, we conducted a retrospective study and meta-analysis for better understanding and choice of the treatment. Our retrospective cohort study enrolled 18 HAAA patients, including 12 males and 6 females. A total of 12 (66.7%) patients received IST treatment, in which 4(33.33%) achieved completed remission (CR), 1(8.33%) partial remission (PR), and 7(58.33%) no response (NR), with a total effective rate of 41.7%. 6 patients (33.3%) received HSCT treatment, in which, 2 achieved CR, 1 PR, and 3 NR, with an effective rate of 50% (Table 1). The one-year overall survival (OS) rate of the IST group was significantly higher than that of the HSCT group (P16-years-old was 0.86 (95% CI: 0.50-1.46)(Figure E). The effective rate of ATG/ALG combined with CSA was 51.35%, and that of CSA alone was 46.7%. Compared to the HSCT group, the efficacy of ATG/ALG + CSA group (RR=0.73, 95% CI: 0.51-1.04) was better than that of CSA alone (RR=0.59, 95% CI: 0.32-1.09) (Figure F). The result of comparing ATG/ALG + CSA with CSA showed that the pooled RR was 1.35(95% CI: 0.70, 2.59) (Figure G), and the difference was not significant. This phenomenon could be attributed to the small number of articles included and insufficient sample size after subgroup analysis according to the IST treatment. We speculated that ATG/ALG combined with CSA has advantages in the treatment of HAAA compared to CSA alone. In summary, HSCT is more effective than IST in the treatment of HAAA, especially for patients 16-years-old, there is no significant difference in the efficacy between the HSCT and ATG/ALG + CsA strategies. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background Aplastic anemia (AA) is a life-threatening disease characterized by bone marrow (BM) failure and pancytopenia. Immunosuppressive therapy can rescue most patients with AA. However, the pathogenesis of AA is still not well elucidated and the new strategies need to be developed for AA patients. Increasing evidences suggested the dysfunctional BM microenvironment may be involved in the pathogenesis of AA. As important components of the BM microenvironment, endothelial cells (ECs) play a crucial role in supporting hematopoiesis and regulating immune. However, whether BM ECs are involved in the occurrence of AA and whether repairing BM ECs could improve the hematopoietic and immune status of AA patients remain to be elucidated. Aims To evaluate the quantity and function of BM ECs from AA patients. Moreover, to determine whether the dysfunctional BM ECs are involved in the occurrence of AA by affecting hematopoiesis and regulating immunity in vitro and in vivo. Finally, to uncover the therapeutic potential of repairing dysfunctional BM ECs to alter the hematopoietic and immunological status in AA patients. Methods This study enrolled 30 patients with AA and 30 healthy donors(HD). Flow cytometry and BM in situ immunofluorescence staining were used to analyze the proportion of ECs in BM of the two groups. The level of intracellular reactive oxygen species(ROS) and the proportion of apoptosis were detected by flow cytometry. The functions of BM ECs were evaluated by double-positive staining, migration and tube formation assays. To determine the effect of BM ECs on hematopoiesis and immunity, primary human BM ECs were separately cocultured with CD34 + and CD3 + cells. To further validate the role of BM ECs in the occurrence of AA, a classical AA mice model and VE-cadherin blocking antibody that could antagonize the function of BM ECs were used. Moreover, to explore potential approach of targeting the dysfunctional BM ECs, the exogenous EC infusion or N-acetyl-L-cysteine (NAC, a ROS scavenger) for repairing BM ECs were administrated to the AA mice. To further explore the repairing effect of NAC on BM ECs, the primary BM ECs from AA patients were treated by NAC in vitroand then the functions of BM ECs were evaluated. Results Compared with HD, BM ECs in AA patients were decreased and dysfunctional, which characterized by higher levels of ROS and apoptosis, impaired abilities of migration and angiogenesis. Furthermore, dysfunctional BM ECs from AA patients not only impaired their hematopoiesis-supporting ability but also promoted co-cultured T cells to polarize towards pro-inflammatory T cells in vitro, which resulted in an unbalanced T cell subsets. Consistently, AA mice demonstrated decreased BM ECs with increased level of intracellular ROS. Moreover, hematopoietic failure and immune imbalance in AA mice became more severe when the function of BM ECs was antagonized, whereas the administration of NAC or infusion of exogenous EC improved the hematopoietic and immunological status of AA mice via repairing BM ECs in vivo. In addition, we found the NAC treatment also restored the hematopoiesis-supporting ability and immunity-regulating ability of the primary ECs derived from AA patients in vitro. Summary/Conclusion Our study demonstrates for the first time that dysfunctional BM ECs with impaired hematopoiesis-supporting ability and abnormal immunomodulatory ability are involved in the pathogenesis of AA. Although further validation is required, restoring dysfunctional BM ECs via EC infusion or administration of ROS scavenger NAC might be a potential therapeutic approach for AA patients. Disclosures No relevant conflicts of interest to declare.

Introduction: Platelet transfusion have saved lives of patients with thrombocytopenia through preventing or treating bleeding complications. Currently, platelet products are provided from blood banks which collect blood from healthy donors. However, our ageing society bears the risk of supply in the future. Furthermore, although the rate is decreasing, alloimmune platelet transfusion refractoriness (allo-PTR) is still found in 5% of platelet transfusion patients. Gestation and previous platelet transfusion cause sensitization to produce alloantibodies mostly against class I human leukocyte antigens (HLA-I) and less frequently against human platelet antigens (HPA), resulting in allo-PTR. In these cases, platelets from compatible donors are transfused, but for patients with rare HLA or HPA, donors are difficult to find. As a possible solution, we proposed the application of platelets from induced pluripotent stem cells (iPSC-platelets), which we have succeeded in the ex vivo production at clinical scale. iPSC-platelets are produced from expandable megakaryocyte cell lines (imMKCLs) as master cells and using a "turbulent flow" bioreactor and various new drugs. imMKCLs are established from iPSCs during megakaryocytic differentiation by overexpression of c-MYC, BMI1 and BCL-XL under the doxycycline control promoter. Switching off these transgenes leads to the maturation of imMKCLs. Turbulent flow was found to be a crucial factor in efficient ex vivo production of healthy iPSC-platelets from imMKCLs. We also developed TA-316, a thrombopoietin mimetic compound, KP-547, an ADAM17 inhibitor that inhibits CD42b shedding and stores platelet function, and also found the combination of AhR antagonist and ROCK inhibitor enables feeder-free liquid culture in imMKCL maturation. Aim: To evaluate the safety of autologous iPS-platelets administered to an aplastic anemia patient with allo-PTR due to anti-HPA alloantibody, who experienced systemic post-transfusion purpura-like complication and have no compatible donor in Japan. Methods and Results: Preclinical studies showed that iPSC-platelets were competent in in vitro assays and mouse and rabbit models for circulation and hemostasis, and pathogen and tumorigenicity free. The clinical study was approved by the Certified Special Committee for Regenerative Medicine of the Kyoto University and by the Health Sciences Council of the Japan Ministry of Health, Labour and Welfare as meeting the Act on the Safety of Regenerative Medicine. The clinical study started in March 2019. Using imMKCL master cells derived from the patient, iPS-platelets were manufactured at the Facility for iPS Cell Therapy (FiT) in Center for iPS Cell Research and Application (CiRA), Kyoto University according to the GMP standard. Three doses of 1x10^10, 3x10^10 and 1x10^11 were administered in a dose escalation single-center open-label uncontrolled study at Kyoto University Hospital. The primary endpoint was safety as measured by frequency and extent of adverse events, which were evaluated by the Efficacy Safety Assessment Committee for each dose cohort. Three doses of the administration of autologous iPS-platelets have been completed. No significant adverse event was observed during the full observation period of one year after the last dose. Conclusion: The iPLAT1 study completed the administration of iPSC-platelets for the first time and confirmed the safety in an allo-PTR patient who would otherwise have no HPA-compatible platelet product. The insights gained from the current study should further contribute to development of allogeneic iPSC-platelet products that can be readily administered to wide range of patients. (Japan Registry of Clinical Trials number jRCTa050190117) Figure 1 Figure 1. Disclosures Sugimoto: Astellas Pharma Inc.: Honoraria; Ebara Corporation: Honoraria; Kyowa Kirin Co., Ltd.: Honoraria; Megakaryon Co: Consultancy, Honoraria; Novartis Pharma K.K.: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria. Kanda: Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; SymBio Pharmaceuticals, Ltd.: Membership on an entity's Board of Directors or advisory committees; Sanofi K.K.: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Ono Pharma Inc.: Honoraria; Novartis Pharma K.K.: Honoraria; NextGeM Inc: Patents & Royalties; Megakaryon Co: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kyowa Kirin Co., Ltd.: Honoraria; Janssen Pharmaceutical K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Eisai: Research Funding; DAIICHI SANKYO Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees; CHUGAI PHARMACEUTICAL Co., Ltd.: Honoraria; Bristol-Myers Squibb Co: Honoraria; Astellas Pharma Inc.: Consultancy, Honoraria; Amgen Astellas BioPharma: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria, Membership on an entity's Board of Directors or advisory committees; TEIJIN PHARMA LIMITED.: Honoraria. Kondo: Asahi Kasei Pharmaceutical: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Chugai Pharma: Honoraria; MSD Pharmaceutical: Honoraria; Sumitomo Dainippon Pharma: Honoraria. Shimizu: Megakaryon co: Consultancy. Hirai: Kyowa Kirin: Patents & Royalties, Research Funding; Bristol-Myers K.K.: Research Funding; CSL Behring: Research Funding; Mitsubishi Tanabe Pharma: Research Funding; Sumitomo Dainippon Pharma: Research Funding; Novartis Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding. Takaori-Kondo: Bristol-Myers K.K.: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Research Funding; Celgene: Research Funding. Eto: Megakaryon co: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Kyowa Kirin Co., Ltd.: Honoraria; Takeda Pharmaceutical Co Ltd: Honoraria; Kowa Co Ltd: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; TEIJIN PHARMA LIMITED.: Honoraria; Kyoto Manufacturing Co., Ltd.: Research Funding.

To the editor: The course of aplastic anemia (AA) is often complicated by the development of clonal disorders such as paroxysmal nocturnal hemoglobinuria (PNH) and secondary myelodysplastic syndromes (sMDS).[1][1][⇓][2][⇓][3][⇓][4]-[5][5] Identification of patients at risk for development of