ALBERT

Description: Clonal hematopoiesis associated with loss of HLA class I alleles due to somatic mutations and/or 6p loss of heterozygosity (LOH) is frequent in immune aplastic anemia (AA). HLA-B*40:02 is more likely to be involved in HLA loss in Japanese AA patients, suggesting a role for this allele in immune pathophysiology (Zaimoku Y et al, Blood 2017). Mutations in non-B*40:02 HLA class I alleles have been reported in a limited number of patients from the United States (Babushok D et al, Blood Adv 2017) and Japan (Mizumaki H et al, 60th ASH meeting), but their prevalence and clinical significance are not well characterized. We investigated somatic mutations of HLA class I alleles, HLA allele frequencies, and their correlations with outcomes of therapy in a total of 532 AA patients, aged 2 years or older, treated on various Hematology Branch protocols (clinicaltrials.gov NCTs 00001964, 00061360, 00195624, 00260689, 00944749, 01193283, and 01623167). HLA allele-lacking (HLA-) monocytes from cryopreserved peripheral blood mononuclear cells were screened by flow cytometry after staining with allele-specific monoclonal antibodies for HLA-A and/or HLA-B (HLA-flow) in 172 AA patients. HLA- monocytes accounting for 0.5% to 100% (median 9.5%) of total monocytes were detected in 49 (28%) of the 172 patients and in 59 (15%) of 382 alleles analyzed (Figure 1). Loss of cell surface expression was frequent for HLA-B14 (46%), B27 (33%), B49 (33%), A68 (26%), A2 (23%), B40 (21%), and B8 (21%). One percent to 60% (median, 8.9%) of glycosylphosphatidylinositol-linked protein-negative (GPI-) monocytes were also present in 43% (21 of 49) of the patients with HLA- monocytes, but GPI- clones had normal HLA cell surface expression. Deep sequencing of HLA-A, HLA-B and HLA-C on sorted HLA- and HLA+GPI+ monocytes was performed in 42 of the 48 patients from whom adequate cells were available. Somatic mutations and/or LOH corresponding to the lacking alleles were detected in all 42 cases (Figure 1): 9 had both somatic mutations and LOH, 20 had somatic mutations only, and 13 had LOH only. Among the 13 patients who showed only LOH in the absent allele, 6 had somatic mutations in other alleles of HLA+ monocytes that was not analyzable of HLA expression, and 2 had a breakpoint of LOH between HLA-A and HLA-C, leading to loss of a single HLA-A allele. Somatic mutations or LOH involving only one allele were present in 37 patients among 6 HLA-A alleles (in 02:01 [7 patients], 02:05 [1], 02:06 [3], 02:11 [1], 68:01 [2], 68:02 [2]) and 10 HLA-B alleles (07:02 [1], 08:01 [4], 14:01 [1], 14:02 [7], 27:05 [1], 35:02 [1], 35:05 [1], 40:01 [1], 40:02 [3], 45:01 [1]), but were not found in HLA-C alleles. HLA allele frequencies in AA patients, including 271 white Americans, 120 African-Americans, and 99 Hispanics and Latinos, were compared with ethnicity-matched individuals in bone marrow donor datasets of the National Marrow Donor Program, and underwent random-effects meta-analyses. HLA-B*07, B*14, and B*40 were overrepresented in AA, while A*02, A*68, and B*08 frequencies were similar to those of healthy donors (Figure 2). In 164 severe AA patients who were initially treated with horse antithymocyte globulin (hATG), cyclosporine, and eltrombopag between 2012 and 2018, 36 and 79 were positive and negative for HLA- monocytes, respectively, and 49 were not tested by HLA-flow. There was no significant difference in overall and complete response rates at six months among the three groups (Figure 3). Clonal evolution, defined as acquisition of abnormal bone marrow cytogenetics or morphology, especially high-risk evolution to chromosome 7 abnormalities, complex cytogenetics, or morphological MDS/AML, tended to be more frequent in patients with HLA- monocytes, compared to the other two groups, but the difference did not reach statistical significance. Clinical outcomes were also assessed according to the presence of specific HLA alleles in 400 severe AA patients who were treated with hATG-based initial immunosuppressive therapy from 2000 to 2018: there was no significant differences in probabilities of response and clonal evolution according to the alleles associated with somatic mutations. Our study revealed that somatic mutations in HLA genes in AA are broadly distributed, but some alleles are preferentially affected. Inconsistent with previous studies, we found that outcomes of therapy did not significantly correlate with HLA gene mutations or with distinct HLA alleles. Disclosures No relevant conflicts of interest to declare.

Description: Background: Immune aplastic anemia (SAA) disproportionally affects children and young adults. Immunosuppression (IST) remains the treatment of choice for patients less than 40 years of age without a fully human leukocyte antigen (HLA) matched sibling. In the pediatric population (aged

Description: Background: Immune aplastic anemia (AA) is caused by cytotoxic T cells (CTLs) that destroy hematopoietic stem and progenitor cells. Regulatory T cells (Tregs) are reduced in AA and increase in response to immunosuppressive therapy (IST; Solomou E et al, Blood 2007). Recent studies suggested an immune regulatory role of regulatory B cells (Bregs). Human CD19+CD24hiCD38hi Bregs suppress Th1 response of CD4+ T cells as well as IFN-γ production by CD8+ CTLs (Mauri C, Menon M, J Clin Invest 2017). The quantity and/or function of Bregs are impaired in autoimmune diseases, malignancies, chronic graft-versus-host disease, and during rejection of transplanted organs. Methods: We investigated B cell phenotypes including CD24hiCD38hi Bregs in previously untreated severe AA (SAA) and very severe AA (VSAA) patients, and healthy individuals aged 18 years and older, and tested their correlation with severity and response to IST. Absolute numbers of lymphocyte subsets, including CD19+ B cells, CD8+ T cells, CD4+ T cells, and NK cell (TBNK), were quantified in fresh blood. Percentages of B cell subsets among total CD19+ B cells, including CD24hiCD38hi Bregs, CD24loCD38lo mature naïve B cells, CD24hiCD38lo memory B cells and CD24loCD38hi plasma cells/plasmablasts, were analyzed using cryopreserved peripheral blood mononuclear cells (PBMCs). Blood samples were obtained from patients close to time of diagnosis and before institution of definitive therapy. All patients were treated with horse anti-thymocyte globulin, cyclosporine, and eltrombopag between 2012 and 2018 at the Hematology Branch, NHLBI (clinicaltrials.gov NCT01623167). Results: TBNK analysis revealed no significant difference in total B cell counts in 104 AA patients compared to 40 healthy individuals (median, 137/μl [IQR, 73-212] vs 163/μl [106-242], P=.11); NK cells were significantly decreased in patients with AA, as previously reported (Gascon P et al, Blood 1986). Total B cell count did not correlate with severity of AA (P=.89) nor with overall response at six months (P=.93). CD8+ T cells and NK cells were lower in VSAA patients compared to SAA patients. None of the TBNK subsets was predictive of overall response in six months after IST. When we assessed the phenotype of B cells among 60 AA patients whose cryopreserved PBMCs were available, CD24hiCD38hi Bregs were markedly decreased as compared to 29 healthy individuals (0.31% [0.14-0.85%] vs 1.9% [1.3-3.6%], P=3×10-7; Figure, Table), while there was no significant difference in other B cell phenotypes. Among these 60 patients, the percentage of CD24hiCD38hi Bregs was especially decreased in VSAA patients compared to SAA (0.18% [0.11-0.34%] vs 0.50% [0.17-1.4%], P=.017). In contrast, CD24loCD38lo mature naïve B cells were higher in VSAA than in SAA (69% [58-86%] vs 60% [42-70%], P=.024). CD24hiCD38hi Breg frequency was positively associated with neutrophil and reticulocyte counts (correlation coefficients [r], 0.34 and 0.26, respectively), while the frequency of CD24loCD38lo mature naïve B cells was negatively correlated (r, -0.34 and -0.40). CD24loCD38lo mature naïve B cells before IST were significantly lower in 47 patients who achieved overall responses at six months compared to 13 non-responders (64% [42-71%), vs 73% [58-88%], P=.014), but CD24hiCD38hi Breg frequency was not correlated with IST responses. At six months after IST, CD24hiCD38hi Bregs in AA patients had recovered to levels present in healthy individuals (2.3% [0.98-4.8%]), in both 34 responders and five non-responders; non-responders showed non-significant increased CD24loCD38lo mature naïve B cells at six months (P=.068). Discussion: A deficit of circulating CD24hiCD38hi Bregs in immune AA with recovery after IST, as occurs with Tregs, suggests Bregs may contribute to the immune pathophysiology in AA. We unexpectedly observed a higher percentage of CD24loCD38lo mature naïve B cells to be associated with more severe disease and a lower probability of responses to IST. B cell phenotype analysis may be beneficial for monitoring of AA and predicting outcomes of therapy. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Identifying the causes of adult-onset rheumatic diseases remains a challenge, and limits diagnosis, prognosis, and targeted treatment. We hypothesized that mutations in genes regulating the post-translational modification ubiquitin, previously implicated in two autoinflammatory diseases, may define new rheumatic disorders. Methods: We analyzed peripheral blood exome sequence data from 2,560 individuals with inflammation-related diagnoses for deleterious mutations in 〉800 ubiquitin-related genes. After discovering three patients with novel UBA1 mutations, we identified additional cases based on clinical similarities through screening multiple independent cohorts. Clinical evaluation of all patients combined with Sanger sequencing, digital droplet PCR, immunoblotting, immunohistochemistry, flow cytometry, and transcriptome/cytokine profiling were performed. CRISPR/Cas9 edited zebrafish provided an in vivo model to assess UBA1 gene function. Results: Twenty-eight adult males were identified with somatic mutations at methionine 41 in UBA1, an X-linked gene, encoding the major E1 enzyme that initiates ubiquitylation. Methionine 41 is highly conserved in UBA1, and these mutations were not observed in exome sequences from over 80,000 healthy controls. Among affected individuals, mutations were found in more than half of hematopoietic stem cells, exclusively in peripheral blood myeloid cells, and not in lymphocytes or fibroblasts. The variant allele fraction of UBA1 p.Met41 mutations in peripheral blood ranged from 20-95%. Patients developed an often fatal, treatment-refractory inflammatory syndrome in late adulthood, with fever, neutrophilic cutaneous and pulmonary inflammation, chondritis, and vasculitis and some individuals met clinical criteria for relapsing polychondritis, Sweet syndrome, polyarteritis nodosa, or giant cell arteritis. In addition, the majority of subjects developed myelodysplastic bone marrow with cytopenias, characteristic vacuoles in myeloid and erythroid precursors cells, progressive bone marrow failure and thromboembolic disease, and some fulfilled clinical criteria for myelodysplastic syndrome or plasma cell dyscrasia. Transformation into MDS with excess blasts or acute myeloid leukemia did not occur in any case. Mutations at p.Met41, the initiation start site of the canonical cytoplasmic isoform, caused loss of this protein and expression of a novel, catalytically impaired isoform initiated at p.Met67. Mutant peripheral blood cells exhibited decreased ubiquitylation and activated innate immune pathways. Knockout of the zebrafish cytoplasmic UBA1 isoform homologue, but not the nuclear isoform, caused systemic inflammation. These results identify somatic mutations in UBA1 as the cause, and not the consequence, of this inflammatory disease. Conclusions: We have defined a novel disorder, VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic), which connects seemingly unrelated adult-onset inflammatory and hematologic diseases. Our work also reveals somatic mutations in a hematopoietic stem cell as a cause of adult-onset rheumatic syndromes that overlap with hematologic conditions. Identification of UBA1 mutations as a cause of these diseases has important implications for classification, prognosis, and treatment of patients, and for the role of somatic mutations of hematopoietic origin in multi-organ pathophysiology. Figure Disclosures Young: Novartis: Research Funding.

Description: Cell-free DNA (cfDNA) extracted from peripheral blood plasma has been increasingly used as a non-invasive approach for diagnosis and surveillance of solid and hematologic malignancies. Somatic variants associated with clonal hematopoiesis of indeterminate potential (CHIP) are commonly detected in such liquid biopsies, suggesting that cfDNA may be useful for their detection. CHIP has emerged as a predictor of progression to hematological malignancies; however, clones are still largely detected using peripheral blood (PB) and bone marrow (BM) cells. In this study, we investigated the performance of cfDNA for detection and quantification of CHIP compared to matched PB/BM cells. cfDNA initially was collected from a healthy cohort (n=106), part of the Baltimore Longitudinal Study of Aging/NIH; later we expanded to screen patients with aplastic anemia (AA; n=53) and myelodysplastic syndrome (MDS; n=27) monitored at the National Heart, Lung, and Blood Institute. Samples were screened for somatic mutations in myeloid neoplasm-related genes using a commercial panel of 177 genes. In HC (median age:72 [range:24-96]), 78/106 subjects (73%) were found to have one (28%) or two and more (45%) cfDNA variants in CHIP-related genes, most frequently in DNMT3A,TET2, TP53, and ASXL1 (Figure 1A). CHIP was observed in more than 60% of individuals older than 40 years, higher than typically reported. In contrast, only 17/53 of AA patients (32%; median age:51 [range:13-82]) were found to have one (n=10) or two or more (n=7) cfDNA variants, most commonly DNMT3A and SF3B1 (Figure 1B). In MDS, 17/29 (58%; median age:63 [range:35-85]) had one (n=10) or two or more (n=7) variants, TET2 and SF3B1 being most frequent (Figure 1C). Median VAF of cfDNA variants was significantly different among cohorts (HC:2.5% [95CI%:2-4] vs AA:18% [95CI%:6-32] vs MDS:38.6% [95CI%:27-42]; t-test,p

Description: Eltrombopag (EPAG) is a non-peptide, thrombopoietin receptor agonist approved for several forms of thrombocytopenia, as well as severe aplastic anemia in first line (combined with immunosuppressive therapy) and refractory (single agent) settings. It is effective in moderate aplastic anemia and under investigation for myelodysplastic syndrome (MDS) and other marrow failure conditions. Although aplastic anemia (AA) patients often respond to EPAG and become drug-independent, this may take months to years, and some patients remain either continuously or intermittently EPAG-dependent. Previously, we and others have reported that EPAG chelates and mobilizes iron in a cohort of AA patients. We present a larger cohort of AA/MDS patients from 4 prospective clinical trials of EPAG, and report declining iron stores on extended EPAG therapy, including some patients developing frank iron deficiency anemia (IDA) responsive to oral or parenteral supplementation, and others normalizing prior severe iron overload during extended EPAG therapy. We report the kinetics of this effect and analyze its impact, if any, upon response and relapse. 317 patients were treated with EPAG for unilineage cytopenia, AA or MDS. 206 AA patients treated on non-EPAG protocols served as historical controls. Average follow-up was 42.9 months (2.2-206.7) for EPAG and 69.6 months (6.1-196) for controls. Average time on EPAG was 9.0 months (0.4-81.1); 73 patients (23.0%) were treated for ≥ 7 months. Average baseline serum ferritin (SF) was 1757 ng/mL (29-18977) for EPAG; 1628 ng/mL (14-8438) for controls (N.S.), with iron overload (SF ≥ 1000 ng/mL) in 56.5% of EPAG patients; 53.9% of controls (N.S.). On EPAG, serum iron (2x over baseline, P 〈 1.3 x 10-39) and transferrin saturation (85.1% of patients have 100% saturation) are elevated due to binding by EPAG, while serum ferritin (SF) does not correlate with iron or transferrin saturation. These effects resolve following EPAG therapy: iron is 15% lower than baseline (P = 5.1 x 10-5), median saturation is 40%, and SF and iron correlate. No such dissociation is noted for historical controls. This implies that SF is the most reliable measure of iron status while on EPAG due to its chelating properties. EPAG duration correlates with SF fold-reduction (R2 = 0.19, P 〈 4 x 10-14). SF levels during EPAG treatment follow first order (exponential) kinetics (R2 = 0.35, P 〈 2 x 10-20), with a clearance half-life of 15.3 months, independent of baseline SF. There is minimal correlation of treatment response with SF kinetics (P = 0.04). In comparison, historical responders demonstrate a significantly slower SF clearance (P 〈 8 x 10-10) with a half-life of 47.5 months. SF half-life on EPAG is comparable to that of chelators such as deferoxamine or deferasirox used for transfusion-related iron overload in AA/MDS. This similarity in kinetics and the fact that this effect is independent of treatment response (and thus transfusion burden) support a role for EPAG in actively depleting total body iron. Of 305 evaluable patients, 62 (9.8%) had iron-depletion as measured by ferritin. 30 (7.5%) were during EPAG treatment with a median time of 55.0 months. 11 (3.6%) of patients experienced falling hemoglobin or other signs of anemia. 9 started iron supplementation or discontinued EPAG, with improvement in the 5 patients who have had follow-up. Patients with EPAG-induced IDA follow the same kinetics (15.5-month half-life) as the general cohort. Logistic models do not predict response based upon either baseline ferritin or kinetics. Furthermore, neither logistic nor Kaplan-Meier models identify any timepoint ferritin, baseline iron overload, or kinetics as predictors of relapse risk. While iron and ferritin may be biomarkers of disease and transfusion burden, they do not appear to drive outcomes, and do not support the recent hypothesis that response of marrow failure to EPAG is based on iron chelation and reversal the toxic impact of intracellular iron on hematopoietic stem cells. Our data suggest a potential role for EPAG or its derivatives as iron chelators. More importantly, they demonstrate that EPAG can deplete iron stores, paradoxically requiring supplementation in previously overloaded patients. In addition to relapse, IDA should also be considered in responders on long term EPAG with declining hemoglobin. We recommend maintaining SF greater than 100 ng/mL so that these patients may continue this life-saving therapy. Figure 1 Disclosures Young: Novartis: Research Funding. Dunbar:Novartis: Research Funding.

Description: Somatic mutations in UBA1 in hematopoietic stem cells and myeloid cells have recently been described and are associated with adult-onset severe autoinflammatory diseases including relapsing polychondritis, Sweet syndrome, polyarteritis nodosa, and giant cell arteritis. This newly defined syndrome is named VEXAS (vacuoles, E1, X-linked, autoinflammatory, somatic). We performed clinicopathologic, cytogenetic, flow cytometric and molecular bone marrow assessment of 15 patients diagnosed with VEXAS and peripheral cytopenias. All patients were male, with a median age of 64y (IQR 45-80) and all had somatic missense mutations affecting the p.Met41 residue in UBA1 with variant allele frequencies (VAF) over 20% from peripheral blood or bone marrow samples and with lineage restriction to mature myeloid cells. Germline mutations at this residue are not reported in any public databases. Peripheral blood findings included macrocytic anemia in all patients (100%), thrombocytopenia in 9/15, and neutropenia in 1/15. All bone marrow aspirates (15/15; 100%) showed prominent vacuolization of both myeloid and erythroid precursors with few vacuoles noted in mature cells. Of the 9 patients tested for serum copper levels all were normal or borderline low. Marrow was hypercellular with granulocytic and megakaryocytic hyperplasia in 12/15 (80%). Definitive WHO criteria for MDS was met in 5/15 (40%) with 3 cases of MDS-MLD, and 2 of MDS-SLD. The remaining cases were suspicious for MDS with low or borderline levels of dyspoiesis that did not exceed greater than 10% of cells in respective lineages meeting criteria for clonal cytopenia of undetermined significance (CCUS). Of the MDS cases, dysplasia was seen in megakaryocytes (5/5), myeloid (2/5) and erythroid (1/5) precursors. Cytogenetic abnormalities were detected in 2/5 MDS patients involving t(3;12)(q21;q13) in one case, and del (5q)/del (13q) in another case. Next generation sequencing analyses were performed in 9/15 patients and showed mutations in DNMT3A in 2/5 MDS cases (VAF 43% and 44%), CSF1R in 1/5 MDS (VAF 3.1%), GNA11 in 1/5 MDS (VAF3.3%) and EZH2 mutation (VAF 22%) in one of the patients without overt MDS. Clonal plasma cell or B-cell populations were diagnosed in 6/15 patients with 3 plasma cell dyscrasias, and 2 cases of monoclonal B-cell lymphocytosis. The most common abnormalities detected by bone marrow flow cytometry analysis were severely reduced or absent B-cell precursors, inverted CD4:CD8 ratios and abnormal expression of CD56 on monocytes. In summary, hematologic disorders were identified in 10/15 VEXAS patients, including both myeloid and lymphoid clonal disease comprising MDS, plasma cell dyscrasias, and monoclonal B cell lymphocytosis. Based on the presence of UBA1 somatic mutations in hematopoietic cells of all VEXAS patients, the patients without evidence of overt neoplasia in this study met criteria for CCUS. Importantly, all patients had characteristic vacuoles in myeloid and erythroid precursors in the marrow without evidence of copper deficiency. These findings suggest that vacuolization of hematopoietic precursors in marrow of cytopenic patients, particularly in male patients and in the setting of systemic inflammation and normal copper levels, should prompt evaluation for somatic UBA1 mutations. In addition to severe autoinflammatory disease manifestations, VEXAS patients appear to confer increased risk for development of both myeloid and lymphoid/plasma cell neoplasia and require surveillance for disease progression. Figure Disclosures Young: Novartis: Research Funding.