Destruction Of Hematopoietic Stem Cells Research Articles

Aplastic Anemia Following COVID-19 Vaccination: A Systematic Review of Case Reports and Case Series.

Background: Aplastic anemia (AA) is the prototypical bone marrow failure syndrome due to the destruction of hematopoietic stem cells by cytotoxic T cells. According to case reports, vaccines could lead to the development of AA. We conducted the present systematic review to evaluate cases of AA following vaccination against coronavirus disease (COVID-19). Materials and Methods: We searched the following databases: PubMed, Scopus, and EMBASE in English, Portuguese, and Spanish languages until April 24, 2023. Published reports and case series on AA following vaccination against COVID-19 were included. The Joanna Brigs Institute (JBI) was used to assess study quality and risk of bias. Results: Six studies were selected from 102 research studies, and data were extracted according to the inclusion criteria. All case reports and case series reported the occurrence of AA following COVID-19 vaccination. AA events were mainly observed in vaccines with messenger ribonucleic acid technology (Moderna; Pfizer-BioNTech). AA was diagnosed by bone marrow biopsy, and severity was determined by Camitta criteria. Conclusion: All cases of AA were properly diagnosed. The sample size was small; therefore, further investigations are required to demonstrate and elucidate the complete pathophysiological mechanisms of AA development after receiving COVID-19 vaccination.

Haploidentical Hematopoietic Cell Transplant with Post-Transplant Cyclophosphamide Expedites Donor-Derived Immune Reconstitution in a Pediatric Severe Aplastic Anemia Cohort

Severe aplastic anemia (SAA) is a life-threatening, idiopathic process involving the immune-mediated destruction of hematopoietic stem cells. Treatment involves immunosuppressive therapy (IST) and/or allogeneic hematopoietic cell transplantation (HCT). The latter has recently been optimized with use of a post-transplant cyclophosphamide (PTCy) based graft-versus-host disease (GvHD) platform, permitting successful HCT with only half-HLA-matched haploidentical donors. In pediatrics, this platform results in >90% overall survival with low rates of GvHD and graft failure. However, reports on donor chimerism and immune reconstitution are limited. We hypothesized that a PTCy platform would expedite CD3 donor engraftment and CD4 T cell recovery. In a phase III clinical trial, we treated three cohorts of pediatric (age </=25 years) patients with SAA requiring alloHCT: MUD, MSD, and HAPLO. The MUD cohort included n=15 recipients of 7-8/8 HLA-matched unrelated donor grafts (n=5 7/8, n=10 8/8) with median age 14 years (range 2-17 years), following cyclophosphamide (Cy) 50 mg/kg, fludarabine (Flu) 200 mg/m 2, rabbit anti-thymocyte globulin (rATG) 9 mg/kg total dose rabbit and low dose total body irradiation (TBI) at 200 cGy. The MSD cohort included n=9 recipients of 8/8 HLA-matched sibling donor grafts with median age 15 years (range 12-24 years), following Cy 200 mg/kg and 9 mg/kg rATG. The HAPLO cohort included n=4 recipients of 4/8 HLA-matched related donor grafts with median age 8.5 years (range 2-21 years) following Cy 29 mg/kg, Flu 150 mg/m 2, 9 mg/kg rATG, and low dose TBI at 200 cGy. GvHD prophylaxis consisted of 30 days of mycophenolate mofetil, and 100 days of a calcineurin inhibitor (followed by an 8-10 week taper), with the HAPLO cohort additionally receiving post-transplant Cy (PTCy) 50 mg/kg/day on days +3 and +4. Peripheral blood lymphocyte counts and peripheral blood and bone marrow donor chimerism were evaluated on days +100, +180 and +365. Recipient characteristics were largely equivalent between cohorts, with 43% receiving IST prior to HCT. Clonality was overrepresented in the HAPLO cohort (2 of 4, 1 with monosomy 7 myelodysplastic syndrome and 1 with a large PNH clone), with only 2 of 15 in the MUD and none in the MSD cohorts. Active controlled infections at the time of HCT were additionally overrepresented in the HAPLO cohort, with disseminated pseudomonas in 1. The MUD cohort had 1 with Rhizopus invasive sinusitis (none in the MSD cohort). Achievement of neutrophil engraftment was statistically significantly delayed in the HAPLO cohort at a median of 17 days compared to 12 for MSD and 14 for MUD, p=0.004), however there were no episodes of primary or secondary graft failure. With only a single late death from intracranial hemorrhage in the MUD cohort, OS was equivalent at 2 years (100% in MSD and HAPLO cohorts, 93.3% in MUD). GvHD rates were low with only 1 (MUD) experiencing grade II acute GvHD, and 1 (MUD) experiencing mild chronic GvHD. Immune reconstitution varied by cohort with statistically significantly greater CD3 peripheral blood donor chimerism (p<0.001) as well as CD4 (p=0.04) and CD8 (p=0.03) T cell counts over time for the HAPLO recipients (CD3 chimerism in Fig 1, CD4 count in Fig 2; 2-way ANOVA fixed effects model). Considering a CD4 >200 cell/uL as clinically meaningful, we assessed the proportion of each cohort achieving this milestone at each timepoint. While each cohort had an increase in proportion achieving CD4>200 over time, the HAPLO cohort reached 50% with CD4 >200 at day +100, compared to 11% MUD and 0% MSD (p=0.09). At day +180, the proportion with CD4 >200 were 36% MUD, 25% MSD and 75% HAPLO (1 yr: 90% MUD, 100% MSD and HAPLO). Donor availability remains a challenge to successful transplant for patients with SAA, and haploidentical matches overcome donor limited accessibility. PTCy as GvHD prophylaxis is associated with low incidences of graft failure and severe GvHD. While PTCy might be anticipated to inhibit donor T cells and immune reconstitution, our data, albeit limited by small cohorts, demonstrates superior and more efficient CD3 donor engraftment and CD4 T cell count recovery following HAPLO HCT with PTCy compared to non-PTCy based MUD and MSD cohorts.

Hypoplastic form of myelodysplastic neoplasm.

Hypoplastic myelodysplastic neoplasm (MDS-h) is a rare hematopoietic disorder characterized by peripheral cytopenia, hypoplasia (cellularity ≤ 25%) and dysplastic changes in the bone marrow. Compared to normo- /hypercellular MDS, in addition to hypocellularity, MDS-h patients have more profound neutropenia and thrombocytopenia, a lower percentage of blasts, and less frequent abnormal karyotype. It is difficult to distinguish MDS-h from aplastic anemia in differential diagnosis. Abnormal karyotype is found in 15-50% of MDS-h patients and the most common chromosomal aberrations include -5/del (5q), -7/del (7q), +8, 17pLOH, del (20q), UPD at 4q, 11q, 13q, and 14q. Approximately 35% of MDS-h patients harbour somatic mutations that are most often detected in PIGA, TET2, DNMT3A, RUNX1, NPM1, ASXL1, STAG2, and APC genes. An autoimmune destruction of hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) mediated by abnormally activated T cells plays a key role in the pathophysiology of MDS-h. Expanded T cells overproduce proinflammatory cytokines (IFN- g and TNF-a), which inhibit proliferation and induce apoptosis of HSC/HPCs. The antigens that trigger the immune response are not known, but potential candidates have been suggested such as WT1 protein and HLA class I molecules. MDS-h does not represent a phenotypically homogeneous subtype of MDS, but rather it is a mixed entity comprising both patients showing features similar to myelodysplastic neoplasm and patients with features of non-malignant bone marrow failure. Determining the prevailing phenotype in MDS-h is important for choosing the optimal treatment and prognosis prediction. The aim of this article is to point out an interesting hypoplastic MDS, the diagnosis of which is difficult, and to provide an overview of its main clinical-pathological features, genetic background, and mechanisms of aberrant immune response.

Bone marrow expression of CD68/CD163 macrophages, IL-17 and FOXP3 cells in aplastic anemia and their relation to prognosis.

The primary mechanism for bone marrow failure in aplastic anemia (AA) is autoimmune hematopoietic stem cell destruction. AA can be cured with antithymocyte globulin (ATG) treatment, and some smaller studies have indicated that the number of regulatory T cells (Tregs) may be predictive of response. Additionally, AA patients appear to have elevated numbers of Th17 cells and bone marrow macrophages, but outcome data are missing. We performed immunohistochemistry on bone marrow biopsies from 121 ATG-treated AA patients and 14 healthy controls, using antibodies against FOXP3 (for Tregs), IL-17 (for Th17), CD68 (for pan-macrophages) and CD163 (for M2 type macrophages) to study their possible relation to ATG response and AA prognosis. AA patients had significantly fewer Tregs and Th17 cells but significantly more macrophages compared with controls. Treg, Th17 and pan-macrophage cell numbers were not associated with ATG response or differences in survival. Patients with higher levels of M2 macrophages had improved 5-year overall survival: 79.6% versus 57.4% (p=.017), and this benefit was primarily seen in AA patients with non-severe disease. We found that Treg and Th17 cell numbers did not predict ATG response or survival, whereas M2 macrophages may be associated with improved survival.

Interleukin-10 promoter variability is associated with the susceptibility, severity, and clinical outcomes of aplasitc anemia in Han-Chinese population.

Acquired aplastic anemia (AA), a heterogeneous bone marrow (BM) failure disease, is mainly mediated by the immune destruction of hematopoietic stem cells (HSCs). Given the predominant role of immunosuppressive therapy (IST) in AA, it is sensible to theorize that variants of cytokine genes might affect the outcome of IST. In this study, we analyzed three single nucleotide polymorphisms (SNPs) of interleukin (IL)-10 gene in promoter region to clarify their relationship with susceptibility, clinical efficacy and prognosis of AA. We observed that CT genotype of IL-10 rs1800896 was associated with a decreased risk of AA (adjusted OR=0.541 [95% CI 0.295-0.993], p=.047). Besides, the disease severity differed considerably by IL-10 gene promoter genotypes and alleles. Furthermore, IL-10 SNPs influenced efficacy of IST, with unfavorable response exhibited by rs1800871 and rs1800872 in dominant models (GG + AG vs. AA, adjusted OR=0.409 [95% CI 0.178-0.943, p=.036] for rs1800871 and GG + GT vs. TT, adjusted OR=0.396 [95% CI 0.173-0.909, p=.028] for rs1800872, respectively). The polymorphisms of IL-10 promoter region were informatively genetic risk factors which might be conducive to the insights into the mechanisms of AA and the design of individual regimens.

Efficacy and Safety of Long-Term Romiplostim Use for Refractory Aplastic Anemia

Introduction: Aplastic anemia (AA) is a life-threatening bone marrow failure syndrome characterized by pancytopenia and hypocellular bone marrow caused by the destruction of hematopoietic stem cells by lymphocytes. About one-third of patients with severe AA who cannot undergo allogeneic hematopoietic stem cell transplantation from a matched sibling donor are refractory to single or repeated immunosuppressive therapy courses. Romiplostim (ROMI), a thrombopoietin receptor agonist, signals and activates intracellular transcriptional pathways via the thrombopoietin receptor, resulting in stimulation of hematopoietic stem and progenitor cells. Our previous research, a phase II/III, multicenter, open-label interventional study, showed that ROMI induced the recovery of at least one lineage of blood cells in >80% of patients with refractory AA. However, data on the long-term efficacy and safety of ROMI in refractory AA beyond 52 weeks are scarce. Additionally, the pretreatment baseline factors of patients who achieve hematological response during treatment and maintain response after discontinuing ROMI are unclear. To address these issues, we report the results at the end of the extension phase (June 2020) of the aforementioned phase II/III study. Methods: We conducted a long-term follow-up analysis of the efficacy and safety of ROMI 10 μg/kg administered subcutaneously once weekly for 4 weeks (Weeks 1-4), followed by 1-step titration to 5, 10, 15, and 20 μg/kg once weekly up to 52 weeks (Weeks 5-52) depending on platelet response. The extension study was started from Week 56 after a 4-week suspension period. The patients continued to receive the same dose of ROMI they received on Week 52 until its commercially available date for the treatment of AA in the study country or the end of June 2020 (end of the extension phase), whichever occurred first. Results: Of the 31 patients enrolled in the phase II/III study, 27 completed the 53-week assessment. Twenty-five hematological responders (any cell lineage) and 2 non-responders entered the extension phase. ROMI was administered for 2 years in 23 patients, 3 years in 7 patients, and 3.5 years in 6 patients in this extension study. The median dose of ROMI was 20 μg/kg at Week 53, which was reduced to 15 μg/kg at 2 years, and 7.5 μg/kg at 3.5 years (Figure 1). Of the 2 non-responders, 1 patient showed platelet response at 2 years, which was sustained thereafter, while the other failed to respond by the end of the extension study at 103 weeks. Of the patients who failed to achieve the platelet (n=7) and erythrocyte (n=6) responses at 1 year, 3 (43%) and 1 (17%) showed responses at 2 years; the mean platelet count increased from 38.3 to 76.7 ×109/L in the 3 platelet responders, and the hemoglobin level increased from 8.0 to 9.2 g/dL in the erythrocyte responder (Figure 2). Of the 23 patients who needed blood transfusions at baseline, 4 and 2 of them still required erythrocyte and platelet transfusions at 1 year, and 4 and 5 of them did so at 2 years, respectively. Five patients remained in response after discontinuing ROMI lasting 29-111 weeks even after discontinuing treatment. These 5 patients had relatively high baseline reticulocyte counts (45.8-93.8 ×109/L, median 57.5 ×109/L) compared with the remaining 22 patients (2.7-114.7 ×109/L, median 46.5 ×109/L). The most common adverse drug reactions included headache (3 cases, 9.7%) and muscle spasms (2 cases, 6.5%); cytogenetic abnormality (-7) led to discontinuation of the study drug in 1 case. Two patients presented with chromosomal abnormalities of unknown significance, including 46,XX,t(11;17) at Week 108 and 46,XX,add(10) at Week 134. No transformation to acute myeloid leukemia or myelodysplastic syndromes occurred. Conclusions: The up to 3.5-year follow-up study of ROMI administration in adult patients with refractory AA demonstrated its safety and efficacy during the extension period. The dose of ROMI was on a downward trend during the long-term follow-up. Nearly half the patients who did not achieve platelet or erythrocyte responses to ROMI at Week 52 eventually achieved responses by continuing the administration until the end of the study, demonstrating the efficacy of prolonged ROMI administration. High reticulocyte counts at ROMI initiation may predict achievement of response after discontinuing ROMI. Further studies with larger sample sizes should confirm these findings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Alemtuzumab and Eltrombopag Combination for Patients with Refractory or Relapsed Severe Aplastic Anemia

Background: Immune aplastic anemia (AA) results from cytotoxic T-lymphocyte-mediated destruction of hematopoietic stem cells. Although upfront immunosuppressive therapy (IST) is effective, patients, with relapsed and refractory disease need further treatment with a second round of IST or bone marrow transplantation (BMT). In this retrospective study, we assess the safety and efficacy of alemtuzumab combined with eltrombopag (EPAG) as second-line IST. Methods: Severe AA patients who relapsed after or were refractory to IST (horse anti-thymocyte globulin, cyclosporine and EPAG) were treated with alemtuzumab and EPAG in combination at the Clinical Center between 2017-2022. Alemtuzumab was given as previously reported in SAA: 10 mg/dose/day for 10 days as an intravenous infusion after a 1 mg test dose in adults and in children < 50Kg, 0.2mg/kg/day for 10 days (not to exceed 10 mg/day). EPAG was administered at 150 mg daily in adults and weight based for children as previously reported. Periodic blood counts and bone marrow evaluations were performed to assess for response and progression to myeloid malignancy. Toxicity was assessed based on the previous profile of the two drugs. Results: Six patients were included with a median age of 16 years (range 13- 39 years); half were children. Four of six had very severe disease at SAA presentation and the majority were refractory to initial IST. Two patients with relapsed SAA had achieved a response to IST at 6-month evaluation but then had declining blood counts at 60 and 49 days, respectively. All patients received alemtuzumab as second-line IST (Table 1). EPAG was subsequently added at a median of 60 days from alemtuzumab administration (range 26 days - 199 days) when there was no signs of response or when the counts declined further, .was performed at 3-months and 6-months from EPAG addition. Both patients with relapsed disease achieved a response at 6-month evaluationUPN3 showed early partial response (PR) at 3-months and then ultimately achieved a complete response (CR); at last follow-up, EPAG was being down-titrated with stable blood counts and no signs of evolution to myeloid malignancy. UPN4 achieved a PR at 6 months and at last follow-up maintained stable counts despite discontinuation of EPAG. Of the four refractory patients, only one (UPN1), responded to the addition of EPAG and continued treatment for 2 years. While the EPAG was reduced in dosage, evaluation for declining blood counts with a bone marrow biopsy revealed progression to acute myeloid leukemia, with monosomy 7 and an ASXL1 clone at 24% variant allele frequency. The patient achieved normal blood counts post BMT. Two of the non-responders (UPN2 and UPN5) died from SAA complications; UPN2 died shortly after BMT from refractory infection. UPN6 also had refractory disease; she is 5 months from alemtuzumab and 3 months from EPAG treatment without a response undergoing evaluation for BMT. Patients that responded (UPN1, UPN3, and UPN4) had a median hemoglobin baseline of 7 g/DL (range 6-7.6 g/DL) and increased to a median of 9.3 g/DL (range 8.4-11.6 g/DL), 6 months after EPAG. Responders had a median ANC baseline of 0.69 k/mcL (range 0.46-1.58 k/mcL) and increased to a median of 1.5 k/mcL(range 1.5-2.11). At baseline they also presented with a median platelet of 17 k/mcL at baseline (range 5-18 k/mcL) and increased to 23 k/mcL (range 23-37 k/mcL), 6 month,s after EPAG. None of the patients had toxicity, specifically liver function abnormalities, with the addition of EPAG. The median AST and ALT were 36 U/L (range 20-66 U/L) and 93 U/L (range 13-170 U/L) 6 months after EPAG, respectively. Patients had a median total bilirubin of 0.4 mg/DL (range 0.3-0.9 mg/DL). Conclusion: In absence of BMT, second-line IST is administered for relapsed and refractory SAA patients. The addition of EPAG to alemtuzumab after 1-3 months resulted in responses in relapsed patients but not in refractory cases. The combination was safe without increased toxicities, but prolonged use of EPAG in a single refractory patient associated with development of myeloid malignancy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Anti-COX-2 autoantibody is a novel biomarker of immune aplastic anemia

In immune aplastic anemia (IAA), severe pancytopenia results from the immune-mediated destruction of hematopoietic stem cells. Several autoantibodies have been reported, but no clinically applicable autoantibody tests are available for IAA. We screened autoantibodies using a microarray containing >9000 proteins and validated the findings in a large international cohort of IAA patients (n = 405) and controls (n = 815). We identified a novel autoantibody that binds to the C-terminal end of cyclooxygenase 2 (COX-2, aCOX-2 Ab). In total, 37% of all adult IAA patients tested positive for aCOX-2 Ab, while only 1.7% of the controls were aCOX-2 Ab positive. Sporadic non-IAA aCOX-2 Ab positive cases were observed among patients with related bone marrow failure diseases, multiple sclerosis, and type I diabetes, whereas no aCOX-2 Ab seropositivity was detected in the healthy controls, in patients with non-autoinflammatory diseases or rheumatoid arthritis. In IAA, anti-COX-2 Ab positivity correlated with age and the HLA-DRB1*15:01 genotype. 83% of the >40 years old IAA patients with HLA-DRB1*15:01 were anti-COX-2 Ab positive, indicating an excellent sensitivity in this group. aCOX-2 Ab positive IAA patients also presented lower platelet counts. Our results suggest that aCOX-2 Ab defines a distinct subgroup of IAA and may serve as a valuable disease biomarker.

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

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

Expression Levels of the TERT Gene Can be Used to Risk Stratify Aplastic Anemia Patients: Study in an Indian Cohort

Introduction: Acquired Aplastic anemia (AA) is a bone marrow failure syndrome characterized by pancytopenia and marrow hypoplasia, and is mediated by immune destruction of hematopoietic stem cells. Mutations in several genes including telomerase, a ribonucleoprotein enzyme complex, consisting of a reverse transcriptase enzyme (TERT), an RNA template (TERC), and several stabilizing proteins, and the associated shelterin complex have been found in cases of both congenital and idiopathic AA. In particular, several TERT and TER C mutations reduce telomerase activity in vitro and accelerate telomere attrition in vivo . Shortened telomeres have been observed in a third of idiopathic AA patients, but only 10% of these patients have mutations in genes of the telomerase complex. We have previously demonstrated that in addition to keeping telomeres from shortening, telomerase directly regulates transcriptional programs of developmentally relevant genes (Ghosh et al, Nat Cell Biol, 2012). We postulate that changes in expression of telomerase associated genes, specifically TERT, contribute to the etiology of aplastic anemia.Patient cohort and Methods: In an effort to better understand the molecular and clinical correlates of this disease, 24 idiopathic AA patient blood samples were collected at a tertiary medical center in Bangalore, India. After obtaining informed consent, high throughput exome panel sequencing on an Illumina HiSeq 2500 platform of the 24 samples was performed, followed by variant calling, annotation and filtration of the generated data to reveal rare pathogenic variants. Additionally, RT-PCR analysis of harvested RNA from each patient was carried out and expression of levels of TERT measured in comparison to samples from normal individuals.Results: Mutational profiling of 24 AA patients by exome sequencing revealed a total number of 2837 non- synonymous mutations (MAF < 0.01%). These included nonsense, frame shift, splice site, missense and in-frame mutations. Recurrent gene mutations were identified in 129 genes. However, pathway analysis of these gene mutations was uninformative with respect to disease etiology. Since DNA mutational data from the AA patients was unrevealing, and given that AA is a telomeropathy, we next chose to measure RNA expression levels of the TERT gene, an integral component of the Telomerase complex. RT-PCR of TERT expression compared to that of normal samples (n=6) revealed an 8 fold reduction in TERT expression 17/24 patients, while 7/24 patients maintained normal TERT expression.Examination of the average number of gene mutations in the TERT-normal versus the TERT-Low patients revealed that the mutational burden was significantly higher in TERT-normal (166 mutations) compared to TERT-low (112 mutations) expressers (p=0.04). High throughput sequencing data also revealed that while TERT-normal patients had no mutations in genes associated with the telomerase/shelterin complex, TERT-low patients had mutations in TERT, TEP1, TINF2, NBN, TPP1, HSP90A and POT1 genes: all associated with the telomerase complex, most of which were predicted to be pathogenic variants. TERT-low patients also showed more evidence of clonal evolution (multiple patients with mutations in TET2, and in BCORL1, FLT-3, MLP, BRAF genes) compared to TERT-normal patients.Clinically TERT-low patients were younger in age (average age 29) compared with the TERT-normal patients (average age 40). TERT-low patients were more likely to have severe aplastic anemia (SAA) leading to higher mortality and poorer response to therapy, with 6/17 patients dying and 4/17 not responding to ATG therapy.Summary and Conclusions: Our observations suggest that expression of TERT can serve as a risk stratification marker in patients with AA. In spite of the higher mutational burden in TERT-normal patients (probably due to their comparative advanced age) TERT-low patients had poorer clinical outcomes which correlated with mutations in the TERT/Shelterin complex and with mutations in genes associated with clonal evolution. While these findings need to be validated in larger cohorts of patients, we suggest that evaluation of TERT expression levels in newly diagnosed AA patients could serve to risk stratify these patients and influence the choice of therapy administered. DisclosuresNo relevant conflicts of interest to declare.

Acquired Somatic Mutations in T Cells in Patients with Aplastic Anemia and Hypoplastic Myelodysplastic Syndromes

Background: Vigorous cell division increases the probability of acquiring mutations during DNA replication. Such may occur in T cells when they recognize their cognate antigen and proliferate. Conventionally, acquired somatic mutations have been associated with the development of malignancy. In T cells, somatic mutations may also lead to autoimmunity, as in large granular lymphocyte (LGL) leukemia with STAT3 mutations. Somatic STAT3 mutations have also been detected in patients with immune-mediated bone marrow failure (BMF) disorders without concomitant LGL lymphoproliferation. These BMF disorders, aplastic anemia (AA) and hypoplastic myelodysplastic syndromes (hMDS), are responsive to immunosuppressive therapy, indicating autoimmune nature of diseases. In AA and hMDS, cytotoxic T cell receptor (TCR) repertoire is skewed and clonal autoreactive cytotoxic T cells are thought to cause hematopoietic stem cell destruction. Because previous findings suggest that these immune-mediated BMF disorders and LGL leukemia share common features, we investigated whether also in AA and hMDS cytotoxic T cells accumulate acquired somatic mutations in genes that are important for cell survival and immune regulation.Methods: We sorted CD8+ and CD4+ T cell fractions from blood and BM samples of patients diagnosed with AA (n=24, median age 60) or hMDS (n=13, median age 64). TCR repertoire was analyzed with TCRB CDR3 deep sequencing or TCR Vb-antibody based flow cytometry, and all selected patients had at least one cytotoxic CD8+ T cell clone >7%. CD4+ and CD8+ T cells were then sequenced with our custom gene panel covering 2000 essential genes of innate and adaptive immunity with mean sequencing depth of 270x. Results were compared to healthy controls (n=9, median age 48). For each subject, somatic mutations in CD8+ cells were called with VarScan2 using CD4+ cells as a “germline” control and vice versa. To reduce possible false positives, all detected mutations were evaluated visually in Integrative Genomics Viewers by two independent researchers in a blinded manner, with agreed and precise criteria to distinguish real and false mutations. Concordant results were validated with amplicon sequencing. The enrichment of cell specific somatic mutations in biological pathways among AA and MDS patients was compared to healthy controls.Results: Somatic mutations in CD4+ and CD8+ T cells were detected both in patients (29/35, 83%) and controls (7/9, 78%). The number of found somatic mutations was associated with the TCR clonality index in cytotoxic CD8+ T cells (r=0.76, p<0.0001). Although single mutations were frequently found in healthy controls' T cells, the number of mutations per subject was significantly higher in patients' CD8+ T cells compared to controls (mean[range] of found somatic mutations per subject for each group: AA CD4 0.9[0-6] and CD8 3.5[0-10]; MDS CD4 0.7[0-2] and CD8 2.3[0-8]; controls CD4 1.1[0-3] and CD8 0.9[0-3]). The number of mutations did not correlate with age. In cases from which both blood and BM samples were available, mutations were detected in T cells derived from both compartments. Most of the genes that had somatic mutations in healthy controls had also mutations in AA and MDS patients (e.g. DNMT3A, TLR2). Although mutations were mostly distinct in individual patients, they accumulated in important immune regulatory pathways. When compared to healthy controls, AA and hMDS patients had more mutations in JAK-STAT signaling (SOCS7, STAT3, PTPN2), MAPK signaling (KRAS, PTPN5, TP53), other cancer-related pathways and in pathways related to viral carcinogenesis and HTLV-I infection. Notably, most of the BMF patients' T-cell mutations were in other genes than STAT3 or genes related to clonal hematopoiesis.Conclusions: Our findings confirm that a subset of immune-mediated BMF disorders may have similar pathogenesis to LGL leukemia highlighting the importance of JAK-STAT signaling pathway in immunoregulation and autoimmunity. Acquired somatic mutations in inflammatory cells leading to dysregulation of immune system may be the common denominator in the pathogenesis of autoimmune disorders and hematologic malignancies. Deep molecular depth analysis and functional studies on found somatic mutations can further illuminate the pathogenesis of immune-mediated BMF syndromes and other autoimmune disorders. DisclosuresMaciejewski:Apellis Pharmaceuticals: Consultancy; Ra Pharma: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Speaker Fees. Mustjoki:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Ariad: Research Funding; Celgene: Honoraria.

Impact of HLA Evolutionary Divergence on Clinical Features of Patients with Aplastic Anemia and Paroxysmal Nocturnal Hemoglobinuria

HLA evolutionary divergence (HED) is a continuous metric that quantifies the breadth of the immunopeptidome that can be presented by an individual's HLA allotype. High HED scores have been shown to be associated with better outcome after immune check point inhibitor therapy in solid tumors.1 However, the role of HED has never been assessed in autoimmune disorders, and may have an impact on disease susceptibility and outcomes. Here we explored the impact of HED on clinical features of aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH). The immunological landscape of those diseases is characterized by an HLA-restricted CD8+ and CD4+ mediated autoimmunity which leads to hematopoietic stem cell destruction, with a crucial role played by immune-dominant PNH clones and immune escape mechanisms likely associated with the down-regulation of the HLA-antigen presentation. Since higher HED scores reflect a wider antigenic spectrum and potentially more diverse peptide presentation capabilities, we hypothesize that this context not only may elicit T-cellular responses by auto-antigen presentation but also that HLA structural diversity can impact on disease phenotype and have a role within the definition of an immune-dominant (higher HED) or immune-escape (lower HED) environment. Using an NGS-based platform targeting the entire HLA locus, we have sequenced DNA samples of a cohort of 98 well-annotated AA, PNH, or AA/PNH patients. We then calculated HED at the classical HLA class I and II loci (A, B, C, DRB1, DQA1, DQB1, DPB1, DPA1), and investigated its impact on disease course and outcomes (Fig. A, B). Homozygosity for at least 1 out of 8 loci was frequently found (70%), especially for class II, however only 2% of patients had &amp;gt;1 homozygous locus). HLA-B and HLA-DQA1 genotypes demonstrated the highest HED among HLA-I and II loci, respectively (Fig. C). While for class I loci no difference was detected across diagnostic subgroups, mean HED for class II loci was significantly lower for PNH patients as compared to AA (6.031 vs 7.623 respectively p=0.047). Analysis of HED metrics across several binary outcomes within logistic regression models demonstrated that higher HED at class I and class II loci was generally associated with unfavorable disease characteristics and response outcomes. Specifically, high mean HED in class I and particularly in B and C loci tracked with disease severity (severe/very severe AA according to modified Camitta criteria: OR= 1.36 [95%CI 1.12, 1.70], p=0.003),2,3 whereas higher mean HED in class II was associated with refractoriness to immunosuppression [OR=1.18 (95%CI: 1.01-1.42), p=0.041]. In particular, a strong relationship was found for DQB1 locus [p=0.032, OR=1.12 (95%CI: 1.01-1.25)] in severe AA patients. Moreover, high HED in DPB1 was associated with early disease presentation [OR= 1.32 (95%CI 1.02, 1.80), p=0.047]. No correlation was found with overall survival. In summary, our results indicate that higher HED scores correlate with severity, early disease onset and refractoriness to immune-suppression in AA patients. High HED may increase the probability of presenting self-peptides that elicit dominant autoimmune T cell responses. Consistent with this line of reasoning, it is not surprising that lower HED (for class II HLA) is found in PNH subjects and that disruptive HLA somatic mutations in AA are preferably found in loci with increased structural diversity, consistent with the idea that less diversity may produce more immune-escape (see Abstract #: 142501). Our results suggest that HED may be useful for risk stratification of patients with immune-mediated bone marrow failure disorders, and more generally, that the ability to present a wider antigenic peptide spectrum may produce a more pervasive autoimmune response. Disclosures Patel: Alexion: Other: educational speaker. Saunthararajah:EpiDestiny: Consultancy, Current equity holder in private company, Patents &amp; Royalties: University of Illinois at Chicago.

Landscape of bone marrow T cells in acquired aplastic anaemia revealed by single-cell sequencing

Abstract Objective Acquired aplastic anaemia (AA) is a T-cell-mediated autoimmune disease characterized by hematopoietic stem cell destruction in the bone marrow (BM). The aim of our study is to explore the cellular heterogeneity, lineage and functional states of T cells involved in the pathogenesis of AA. Methods CD3+CD4+ and CD3+CD8+ mature T cells, isolated from BM of five newly diagnosed severe AA patients and two healthy controls by FACS, were subjected to single-cell RNA sequencing (ScRNA-seq) and T cell receptor (TCR) sequences using 10X Genomics Chromium Single Cell 5′ and Single-Cell V(D)J protocols. Libraries were sequenced on Illumina NovaSeq platforms. Results We obtained transcriptome data for 47,508 cells from severe AA patients and 20,758 cells from controls. Bioinformatic analysis of the transcriptional profiles and TCR sequences revealed their molecular and functional properties and developmental trajectory. All clusters are not library specific, but consisted of cells from 7 different donors. We identified all major cell types and signature genes for each subset. We discovered a marked decrease of three heat shock protein family A (Hsp70) members (HSPA6, HSPA1A and HSPA1B) as a signature of AA patients. Interestingly, this phenomenon occurred for all T cell subpopulations. GO annotations related to these genes included response to temperature stimulus, unfolded protein binding and protein folding. We suspected these Hsp70 members were closely related to the pathogenesis of AA. Conclusions Our results establish a reference map of human BM T cells in health and AA patients. and demonstrate that T cells have unique characteristics in AA patients, which may represent a pathogenesis of this autoimmune disease.

PB1863 APLASTIC ANEMIA ‐ THE CASUISTRY OF A CENTRAL HOSPITAL

Background:Aplastic Anemia (AA) is a rare haematological disease associated to significant morbidity and mortality, with the autoimmune destruction of hematopoietic stem cells having a central role in its pathogenesis. The diagnostic criteria for AA includes the presence of bone marrow hypoplasia, with no evidence of fibrosis, associated with haemoglobin (Hb) &lt;10 g / dL, platelets &lt;50G / L or neutrophils (ANC) &lt;1.5G / L. The therapeutic algorithm is based mainly on fitness and age, immunosuppression and transfusion support being the standard treatment and allogeneic transplantation of hematopoietic progenitors (ASCT), the only curative treatment.Aims:The purpose of this paper is to describe the AA casuistry of the Haematology Service of a Central Hospital.Methods:A retrospective study of all patients diagnosed with AA was carried out over a period of 10 years (2008–2018), from the Hematology Service of the Centro Hospitalar e Universitário de Coimbra, in Portugal.Results:In the selected period, 34 patients were diagnosed, 71% being female, and the average age at diagnosis was 60 years (18‐85 years). Anemia (44%), haemorrhagic dyskinesia (38%) and infection (3%) were the most frequent reasons for consultation; 15% of the patients were asymptomatic. The lowest values observed were Hb 3.8 g / dL, 0.02G / L ANC and 2G / L platelets. According to Camitta criteria, 17% of patients had non‐severe AAA, 43% severe and 26% very severe.Of the patients considered fit and considered eligible for ASCH (7 patients ‐ 21%), only 1 had a related donor for which he underwent allogeneic transplantation. Of the remaining patients, 22 received immunosuppressive therapy [9 cyclosporin (CsA) and 13 anti‐thymocyte globulin (ATG) + CsA] and 5 of them received supportive therapy alone. From the statistical analysis performed, TRG showed a favorable trend to CsA + ATG (64%), when compared to CsA and TS (40%) (p = NS), being lower in patients aged ≥ 65 years (27% vs 68%; p = 0.036).The time to partial response (PR) was higher in the group that did CsA compared to CsA + ATG (15.6 vs 7.8 M; p = 0.016).Complete response (CR) was achieved in 18% and 36% of patients treated with CsA and ATG + CSA (p = NS), respectively. The average overall survival (OS) was 87 months, being lower in the elderly (patients ≥ 65 years, p = 0.024). Despite the absence of significant difference (p = NS), the CsA and CsA + ATG groups showed a tendency to higher OS at 3 M (76% vs 86%, respectively) and 2A (55% vs 64%, respectively), when compared to the group of supportive therapy.In the 15 patients who died (48.4%), infection was the main cause of death (60%), followed by hemorrhage (20%) and heart disease (20%). The infection was higher in the CsA + ATG group (75% of the causes of death), and was associated with a higher risk of death (p = 0.021).Summary/Conclusion:AA is a disease of complex pathophysiology, presenting diagnostic and therapeutic gaps. In the last decade, significant progress has been made to understand its pathophysiology and to find appropriate therapeutic strategies for these patients. In the cohort of patients presented, the analysed variables, namely the rates of response to the therapies and OS, are overlapping with those published in previous studies.The enlargement of the sample may contribute to a greater robustness of these data, helping in the selection of an individualized therapy.

Population Pharmacokinetics of Vancomycin in Patients Undergoing Allogeneic Hematopoietic Stem-Cell Transplantation.

Vancomycin is a commonly used antimicrobial agent for patients undergoing allogeneic hematopoietic stem-cell transplantation (allo-HSCT). Vancomycin has large inter- and intraindividual pharmacokinetic variability, which is mainly described by renal function; various studies have indicated that vancomycin pharmacokinetics are altered in special populations. However, little is known regarding vancomycin pharmacokinetics in patients undergoing allo-HSCT. Therefore, we aimed to develop a population pharmacokinetic (PopPK) model of vancomycin in patients undergoing allo-HSCT for effective and safe antimicrobial therapy and to develop a vancomycin dosing nomogram for a vancomycin optimal-dosing strategy. In total, 285 observations from 95 patients undergoing allo-HSCT were available. The final PopPK parameter estimates were central volume of distribution (V1, L),39.2; clearance (L/h),4.25; peripheral volume of distribution (V2, L), 56.1; and intercompartmental clearance (L/h),1.95. The developed vancomycin model revealed an increase in V1 and V2 compared with those in the general population that consisted of patients with methicillin-resistant Staphylococcus aureus. Moreover, serum creatinine was reduced because of an increase in the plasma fraction because of destruction of hematopoietic stem cells accompanying allo-HSCT pretreatment, suggesting that the Cockcroft-Gault equation-based creatinine clearance value was overestimated. To our knowledge, this is the first PopPK study to develop a dosing nomogram for vancomycin in patients undergoing allo-HSCT and was proven to be useful in optimizing the dosage and dosing interval of vancomycin in these patients. This strategy will provide more useful information for vancomycin therapy with an evidence-based dose adjustment.

SWI/SNF subunit expression heterogeneity in human aplastic anemia stem/progenitors

Acquired aplastic anemia (AA) is a bone marrow (BM) failure associated with autoimmune destruction of hematopoietic stem cells (HSCs). Although somatic mutations have been identified in AA patients, mutations alone do not explain AA pathophysiology. SWI/SNF is an evolutionarily conserved, multi-subunit, ATP-dependent chromatin-remodeling protein complex that plays an important role in mammalian hematopoiesis. Herein, gene expression analysis identified a significant loss of the SWI/SNF core component SMARCC1, along with ARID1B, ACTL6A, and SMARCD1, in human AA BM CD34+ HSCs and hematopoietic stem and progenitor cells (HSPCs) compared with normal HSPCs. However, expression of SMARCA4, SMARCB1, SMARCD3, and DPF2 remained intact in our AA cohort. PBRM1, BRD7, and SMARCA2 expression were significantly upregulated in both untreated and follow-up AA patients. Clonal hematopoiesis in AA is associated with evolution to late clonal disorders, including myelodysplastic syndromes (MDS). Apart from SMARCD1 loss, we did not observe significant alteration of SWI/SNF expression in MDS HSPCs, indicating SWI/SNF differential expression in AA and MDS. In addition, except for ACTL6A, SWI/SNF expression was unaltered in aged HSPCs. Importantly, our results provide evidence for loss of SWI/SNF in AA, and may implicate AA HSPC-autonomous defective SWI/SNF regulation as an integral component of BM failure, in addition to autoimmune destruction of AA HSCs. These findings illustrate for the first time SWI/SNF subunit expression heterogeneity in human AA HSPCs and require prognostic validation in a larger cohort.

Autoimmune Processes as an Important Parameter for the Pathogenesis of Aplastic Anemia and Paroxysmal Nocturnal Hemoglobinuria

Aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH) are two rare and often concomitant hematologic diseases. Their development is - despite lack of decisive evidence - attributed autoimmune mechanisms. AA is characterized by cytopenias combined with a hypocellular bone marrow. The pathophysiology is believed to be immune-mediated with destruction of hematopoietic stem cells by autoreactive lymphocytes, a hypothesis supported by its response to immunosuppressive therapy. A large proportion of patients with AA also have PNH. In PNH, somatic mutations in the PIGA gene in hematopoietic stem cells blocks synthesis of the glycosylphosphatidylinositol (GPI) anchor. As a result, blood cells deriving from the GPI-deficient clone lack GPI-anchored proteins, most notably the complement inhibiting factors CD55 and CD59. The clinical manifestations primarily arise because of uncontrolled complement activation on erythrocytes and thrombocytes. This review summarizes current knowledge and theories about of the pathophysiology of both diseases with focus on immune mechanisms attributing to the two diseases.

Microbe-Induced Inflammatory Signals Triggering Acquired Bone Marrow Failure Syndromes

Acquired bone marrow failure syndromes encompass a unique set of disorders characterized by a reduction in the effective production of mature cells by the bone marrow (BM). In the majority of cases, these syndromes are the result of the immune-mediated destruction of hematopoietic stem cells or their progenitors at various stages of differentiation. Microbial infection has also been associated with hematopoietic stem cell injury and may lead to associated transient or persistent BM failure, and recent evidence has highlighted the potential impact of commensal microbes and their metabolites on hematopoiesis. We summarize the interactions between microorganisms and the host immune system and emphasize how they may impact the development of acquired BM failure.

Mutations in the Telomerase Complex and Expression Levels of the TERT Gene Determine Severity and Outcome of Disease in Aplastic Anemia Patients

Acquired Aplastic anemia (AA) is a bone marrow failure syndrome characterized by pancytopenia and marrow hypoplasia, and is mediated by immune destruction of hematopoietic stem cells. Mutations in several genes including telomerase, a ribonucleoprotein enzyme complex, consisting of a reverse transcriptase enzyme (TERT), an RNA template (TERC), and several stabilizing proteins, and the associated shelterin complexes have been found in both congenital and idiopathic AA. In particular, several TERT and TERC mutations reduce telomerase activity in vitro and accelerate telomere attrition in vivo. Shortened telomeres have been observed in a third of idiopathic AA patients, but only 10% of these patients have mutations in genes of the telomerase complex. We have recently demonstrated that in addition to keeping telomeres from shortening, telomerase directly regulates transcriptional programs of developmentally relevant genes (Ghosh et al, Nat Cell Biol, 2012, 14, 1270). We postulate that changes in expression of telomerase associated genes, specifically TERT, contribute to the etiology of aplastic anemia.In an effort to better understand the molecular and clinical correlates of this disease, 24 idiopathic AA patient samples were collected at a tertiary medical center in Bangalore, India. Following informed consent, we performed RT-PCR analysis on harvested RNA from each patient and measured levels of TERT expression compared to that of normal controls (n=6). An 8 fold reduction in TERT expression was observed in 17/24 patients, while 7/24 patients maintained normal TERT expression. In general, TERT-low patients were younger in age (mean age 29y) compared with the TERT-normal patients (mean age 40y). TERT-low patients were more likely to have severe aplastic anemia (SAA) leading to higher mortality and poorer response to therapy, with 6/17 patients dying and 4/17 not responding to ATG therapy. Targeted panel sequencing of the 24 samples on an Illumina platform revealed that while TERT-normal patients had no mutations in genes associated with the telomerase/shelterin complex, TERT-low patients carried predicted pathogenic variants in TERT, TEP1, TINF2, NBN, TPP1, HSP90A and POT1 genes, all associated with the telomerase complex. Somatic gene variants were also identified in other AA associated genes, PRF1 and CDAN1, in the TERT-low cohort. In addition, novel predicted pathogenic mutations associated with the shelterin complex were found in two TERT-low patients in the TNKS gene. We also detected mutations in TET2, BCORL1, FLT-3, MLP and BRAF genes in TERT-low patients. Mutations in these genes are associated with clonal evolution, disease progression and poor prognosis. Our observations were further illustrated in a single patient where normal TERT expression was noted at initial clinical presentation. ATG therapy led to CR, but the patient returned within a year and succumbed to E.coli related sepsis. At that stage he had low TERT expression, suggesting that TERT expression can change as the disease progresses. Taken together, our data support the hypothesis that loss of TERT expression correlates with disease severity and poor prognosis. Our observations further suggest that preliminary and periodic evaluation of TERT expression levels in AA patients is likely to serve as a predictor of disease severity and influence the choice of therapy. DisclosuresNo relevant conflicts of interest to declare.

Molecular Characterization of Cytotoxic T Cell Repertoire in Aplastic Anemia and Myelodysplastic Syndromes

Background: Aplastic anemia (AA) and some subclasses of myelodysplastic syndromes (MDS) are immune-mediated bone marrow failure disorders. Previous studies indicate that cytotoxic T lymphocyte (CTL) clones play a role in the pathophysiology of these diseases by targeting hematopoietic stem cells. Current high throughput sequencing methods provide an unprecedented view to the characteristics of the T cell receptor (TCR) repertoire at the in-depth molecular level and opportunity to compare TCR repertoire in these diseases.Methods: We investigated the TCR repertoire in patients with AA (n=12, mean age 58) and hypoplastic MDS (RA, RCMD) (n=20, mean age 61). Sorted CD8+ T cell fractions were analyzed with a multiplexed PCR assay that targets the variable CDR3 region of the rearranged TCRβ locus. Next-generation sequencing was performed and data was analyzed with the ImmunoSEQ, VDJtools (Shugay M et al. 2015) and VDJdb-standalone. The found rearrangements of T cell locus were compared to an age-matched control group (n=7, mean age 58) and previously published data from healthy adults (n=587), as well as to known immunodominant clones found in patients with large granular lymphocyte (LGL) leukemia. Lastly, VDJdb, a curated database of TCR sequences of known antigen specificity (https://vdjdb.cdr3.net) was used to investigate putative antigen-specific T cells.Results: The overall T cell clonality of CD8+ T cells was significantly higher in patients with AA when compared to age matched controls (clonality index mean 0.39 vs. 0.28, p=0.03). However, in patients with hypoplastic MDS no significant difference in clonality was observed (0.30 vs. 0.28), although some patients had large expansions (clonality index >0.5). When all patients were analyzed as a group, age correlated both with the productive clonality (r=0.52, p=0.02) and the frequency of biggest productive rearrangement (r=0.53, p=0.01).Several TCR rearrangements that were expressed in high frequency (>10% cumulative productive frequency) were shared among the AA and MDS patients, but were virtually absent in healthy individuals. Because LGL leukemia is closely connected to AA and hypoplastic MDS, and mono/oligoclonal LGL cells mediate autoimmune manifestations, we also compared our results to previously published TCR repertoire data of LGL leukemia patients. The immunodominant rearrangements found in AA and MDS patients (CTL rearrangements larger that 10%) were not shared with LGL leukemia cases. Similarly, the previously reported immunodominant rearrangements in LGL leukemia did not correspond to any major (>10%) clones in our patients. Only minor shared sequences were found which have also been discovered with a similar frequency in healthy controls.Annotation of AA and MDS sequences to TCR database revealed that the reported antigen-specific sequences were not found in the immunodominant clones of our patients. The most frequent rearrangements with known antigen-specificity (up to 6.3% cumulative frequency) in our patients were specific for cytomegalovirus (CMV) and Epstein-Barr-virus. However, reported CMV-specific rearrangements were also found in a CMV-IgG negative patient, but with a low frequency (<10^-4 cumulative productive frequency).From two patients we had both peripheral blood and bone marrow samples that were taken simultaneously. By comparing individual TCR sequences between 2 sample types we could observe that all T cell clones >0.1% were present in both samples with similar frequencies and there were only minor differences between the overall repertoires (r=0.97, r=0.96). We also compared T cell repertoires of patients before and after immunosuppressive treatment. A marked change in T cell repertoire was observed after successful treatment with cyclosporine A and anti-thymocyte globulin.Conclusions: Next-generation TCRB sequencing of patients with AA and hypoplastic MDS confirmed that large cytotoxic T cell clones are found in these diseases. The molecular TCR structure of the clones was typical to AA and hypoplastic MDS and was not found in healthy individuals or patients with LGL leukemia. Furthermore, the rearrangements were not found among reported virus-specific sequences. Our results suggest that there is a common specific T cell target in AA and MDS. Deep molecular analysis of TCRs can further illuminate the mechanism of hematopoietic stem cell destruction in these diseases. DisclosuresMustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding.