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Immune aplastic anemia (AA) is a severe blood disease characterized by T-lymphocyte− mediated stem cell destruction. Hematopoietic stem cell transplantation and immunosuppression are effective, but they entail costs and risks, and are not always successful. The Janus kinase (JAK) 1/2 inhibitor ruxolitinib (RUX) suppresses cytotoxic T-cell activation and inhibits cytokine production in models of graft-versus-host disease. We tested RUX in murine immune AA for potential therapeutic benefit. After infusion of lymph node (LN) cells mismatched at the major histocompatibility complex [C67BL/6 (B6)⇒CByB6F1], RUX, administered as a food additive (Rux-chow), attenuated bone marrow hypoplasia, ameliorated peripheral blood pancytopenia, preserved hematopoietic progenitors, and prevented mortality, when used either prophylactically or therapeutically. RUX suppressed the infiltration, proliferation, and activation of effector T cells in the bone marrow and mitigated Fas-mediated apoptotic destruction of target hematopoietic cells. Similar effects were obtained when Rux-chow was fed to C.B10 mice in a minor histocompatibility antigen mismatched (B6⇒C.B10) AA model. RUX only modestly suppressed lymphoid and erythroid hematopoiesis in normal and irradiated CByB6F1 mice. Our data support clinical trials of JAK/STAT inhibitors in human AA and other immune bone marrow failure syndromes.

VEXAS is caused by somatic mutations in UBA1 (UBA1mut) and characterized by heterogenous systemic auto-inflammation and progressive hematologic manifestations, meeting criteria for myelodysplastic syndrome (MDS) and plasma cell dyscrasias. The landscape of myeloid-related gene mutations leading to typical clonal hematopoiesis (CH) in these patients is unknown. Retrospectively, we screened 80 VEXAS patients for CH in their peripheral blood (PB) and correlated findings with clinical outcomes in 77. UBA1mutwere most common at hotspot p.M41 (median variant allele frequency/VAF = 75%). Typical CH mutations co-occurred with UBA1mut in 60% of patients, mostly in DNMT3A and TET2, and were not associated with inflammatory or hematologic manifestations. In prospective single-cell proteogenomic sequencing (scDNA), UBA1mutwas the dominant clone, present mostly in branched clonal trajectories. Based on integrated bulk and scDNA analyses, clonality in VEXAS followed two major patterns: with either typical CH preceding UBA1mutselection in a clone (Pattern 1), or occurring as an UBA1mutsubclone or in independent clones (Pattern 2). VAF in PB differed markedly between DNMT3A and TET2 clones (median VAF of 25% vs 1%). DNMT3A and TET2 clones associated with hierarchies representing patterns 1 and 2, respectively. Overall survival for all patients was 60% at 10 years. Transfusion-dependent anemia, moderate thrombocytopenia, and typical CH mutations, each correlated with poor outcome. In VEXAS, UBA1mut cells are the primary cause of systemic inflammation and marrow failure, being a new molecularly defined somatic entity associated with MDS. VEXAS-associated MDS is distinct from classical MDS in its presentation and clinical course.

Severe aplastic anemia, a disease characterized by pancytopenia and a hypocellular marrow, is treatable by either immunosuppressive therapy (IST) or hematopoietic stem cell transplant. Much is understood about the immune-mediated pathophysiology of AA now, but the inciting factor remains elusive. Many groups around the globe contributed to understanding the disease pathophysiology and optimizing the IST regimen. Horse antithymocyte globulin and cyclosporine, the initial IST regimen, achieved a hematologic response rate in about 60% to 65% of treated patients, with less than 10% achieving a complete count recovery. However, adding a thrombopoietin receptor agonist, eltrombopag (EPAG), to IST improved these response rates to nearly 80% and an unprecedented increase in complete response to almost 40%. The latest report indicates that a high-risk clonal evolution to myeloid malignancies is not increased with hematopoietic stem cell stimulation by adding EPAG in the front line setting. Despite the great success of IST and EPAG in improving early outcomes, relapse remains a problem. Further optimization of upfront therapy and treatment protocol is needed to prevent relapses and decrease clonal evolution rates for even better long-term results.

The choice to postpone treatment while awaiting genetic testing can result in significant delay in definitive therapies in severely pancytopenic patients. Conversely, inherited bone marrow failure (BMF) misdiagnosis can expose patients to ineffectual and expensive therapies, toxic transplant conditioning regimens, and inappropriate use of an affected family member as a stem cell donor. To predict the likelihood of patients having acquired or inherited BMF, we developed a two-step data-driven machine-learning model using 25 clinical and laboratory variables typically recorded at the initial clinical encounter. For model's development, patients were labeled as having acquired or inherited BMF depending on their genomic data. Datasets were unbiasedly clustered and an ensemble model was trained with cases from the largest cluster of the training cohort (n=359) and validated with an independent cohort (n=127). Cluster A, the largest group, was mostly immune or inherited aplastic anemia, whereas Cluster B was composed of underrepresented BMF phenotypes, and not included in the next step of data modeling due to small sample size. The ensemble model Cluster A-specific was accurate (89%) to predict BMF etiology, correctly predicting inherited and likely immune BMF in 79% and 92% of cases, respectively. Our model represents a practical guide for BMF diagnosis and highlights the importance of clinical and laboratory variables in the initial evaluation, particularly telomere length. Our tool can be potentially used by general hematologists and health care providers not specialized in BMF, and in under-resourced centers, to prioritize patients for genetic testing or for expeditious treatment.

VEXAS syndrome has an unmet need for therapeutic interventions. Even if few data exist regarding the treatment of this newly described syndrome, different options can be proposed given the unique pathophysiological consequences of the clonal dominance of UBA1 mutated hematopoietic stem cells. To date, allogeneic transplantation is the only curative option, but many questions remain regarding the selection of eligible patients, the conditioning regimen or management of toxicities that may be unique to VEXAS patients. Alternatively, drugs used in myelodysplastic syndrome such as hypomethylating agents or lenalidomide are interesting candidates, which could theoretically have also an effect on the clone. Another strategy is to target the inflammatory cascade, by inhibiting proinflammatory cytokines (such as TNFα, IL1, IL6) or effector cells, for example with JAK inhibitors. Whatever the choice of treatment for VEXAS patients, supportive care is always needed to be considered to manage frequent complications such as cytopenia, thrombosis and infections. Finally, we discuss the challenges of the design of clinical trials for VEXAS patients, from inclusion criteria to clinical and biological endpoints of activity.

Objective Somatic mutations in UBA1 cause a newly defined syndrome known as VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic syndrome). More than 50% of patients currently identified as having VEXAS met diagnostic criteria for relapsing polychondritis (RP), but clinical features that characterize VEXAS within a cohort of patients with RP have not been defined. We undertook this study to define the prevalence of somatic mutations in UBA1 in patients with RP and to create an algorithm to identify patients with genetically confirmed VEXAS among those with RP. Methods Exome and targeted sequencing of UBA1 was performed in a prospective observational cohort of patients with RP. Clinical and immunologic characteristics of patients with RP were compared based on the presence or absence of UBA1 mutations. The random forest method was used to derive a clinical algorithm to identify patients with UBA1 mutations. Results Seven of 92 patients with RP (7.6%) had UBA1 mutations (referred to here as VEXAS-RP). Patients with VEXAS-RP were all male, were on average ≥45 years of age at disease onset, and commonly had fever, ear chondritis, skin involvement, deep vein thrombosis, and pulmonary infiltrates. No patient with VEXAS-RP had chondritis of the airways or costochondritis. Mortality was greater in VEXAS-RP than in RP (23% versus 4%; P = 0.029). Elevated acute-phase reactants and hematologic abnormalities (e.g., macrocytic anemia, thrombocytopenia, lymphopenia, multiple myeloma, myelodysplastic syndrome) were prevalent in VEXAS-RP. A decision tree algorithm based on male sex, a mean corpuscular volume >100 fl, and a platelet count

Somatic mutations in UBA1 involving hematopoietic stem and myeloid cells have been reported in patients with the newly defined VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome. Here, we report clinical hematologic manifestations and unique bone marrow (BM) features in 16 patients with VEXAS. All patients were male and had a history of severe autoinflammatory and rheumatologic manifestations and a somatic UBA1 mutation (p.Met41). Ten patients had hematologic disorders: myelodysplastic syndrome (MDS; 6 of 16), multiple myeloma (2 of 16), monoclonal gammopathy of undetermined significance (2 of 16), and monoclonal B-cell lymphocytosis (2 of 16), and a few of those patients had 2 co-existing clonal processes. Although macrocytic anemia (100%) and lymphopenia (80%) were prevalent in all patients with VEXAS, thrombocytopenia and neutropenia were more common in patients with progression to MDS. All BMs in VEXAS patients had prominent cytoplasmic vacuoles in myeloid and erythroid precursors. In addition, most BMs were hypercellular with myeloid hyperplasia, erythroid hypoplasia, and varying degrees of dysplasia. All patients diagnosed with MDS were lower risk (low blast count, very good to intermediate cytogenetics) according to standard prognostic scoring with no known progression to leukemia. In addition, 10 of 16 patients had thrombotic events, including venous thromboembolism and arterial stroke. Although VEXAS presents symptomatically as a rheumatologic disease, morbidity and mortality are associated with progression to hematologic disease. Given the increased risk of developing MDS and multiple myeloma, surveillance for disease progression is important.

Patients with severe aplastic anaemia (SAA) are often not vaccinated against viruses due to concerns of ineffective protective antibody response and potential for pathogenic global immune system activation, leading to relapse. We evaluated the impact of COVID-19 vaccination on haematological indices and disease status and characterized the humoural and cellular responses to vaccination in 50 SAA patients, who were previously treated with immunosuppressive therapy (IST). There was no significant difference in haemoglobin (p = 0.52), platelet count (p = 0.67), absolute lymphocyte (p = 0.42) and neutrophil (p = 0.98) counts prior to and after completion of vaccination series. Relapse after vaccination, defined as a progressive decline in counts requiring treatment, occurred in three patients (6%). Humoural response was detectable in 90% (28/31) of cases by reduction in an in-vitro Angiotensin II Converting Enzyme (ACE2) binding and neutralization assay, even in patients receiving ciclosporin (10/11, 90.1%). Comparison of spike-specific T-cell responses in 27 SAA patients and 10 control subjects revealed qualitatively similar CD4

Predictors, genetic characteristics, and long-term outcomes of patients with SAA who clonally evolved after immunosuppressive therapy (IST) were assessed. SAA patients were treated with IST from 1989–2020. Clonal evolution was categorized as “high-risk” (overt myeloid neoplasm [meeting WHO criteria for dysplasia, MPN or acute leukemia] or isolated chromosome-7 abnormality/complex karyotype without dysplasia or overt myeloid neoplasia) or “low-risk” (non-7 or non-complex chromosome abnormalities without morphological evidence of dysplasia or myeloid neoplasia). Univariate and multivariable analysis using Fine-Gray competing risk regression model determined predictors. Long-term outcomes included relapse, overall survival (OS) and hematopoietic stem cell transplant (HSCT). Somatic mutations in myeloid cancer genes were assessed in evolvers and in 407 patients 6 months after IST. Of 663 SAA patients, 95 developed clonal evolution. Pre-treatment age >48 years and ANC >0.87×10(9)/L were strong predictors of high-risk evolution. OS was 37% in high-risk clonal evolution by 5 years compared to 94% in low-risk. High-risk patients who underwent HSCT had improved OS. Eltrombopag did not increase high-risk evolution. Splicing factors and RUNX1 somatic variants were detected exclusively at high-risk evolution; DNMT3A, BCOR/L1 and ASXL1 were present in both. RUNX1, splicing factors and ASXL1 mutations detected at 6 months after IST predicted high-risk evolution.

Somatic mutations in UBA1 cause vacuoles, E1 ubiquitin-activating enzyme, X-linked, autoinflammatory somatic (VEXAS) syndrome, an adult-onset inflammatory disease with an overlap of hematologic manifestations. VEXAS syndrome is characterized by a high mortality rate and significant clinical heterogeneity. We sought to determine independent predictors of survival in VEXAS and to understand the mechanistic basis for these factors. We analyzed 83 patients with somatic pathogenic variants in UBA1 at p.Met41 (p.Met41Leu/Thr/Val), the start codon for translation of the cytoplasmic isoform of UBA1 (UBA1b). Patients with the p.Met41Val genotype were most likely to have an undifferentiated inflammatory syndrome. Multivariate analysis showed ear chondritis was associated with increased survival, whereas transfusion dependence and the p.Met41Val variant were independently associated with decreased survival. Using in vitro models and patient-derived cells, we demonstrate that p.Met41Val variant supports less UBA1b translation than either p.Met41Leu or p.Met41Thr, providing a molecular rationale for decreased survival. In addition, we show that these 3 canonical VEXAS variants produce more UBA1b than any of the 6 other possible single-nucleotide variants within this codon. Finally, we report a patient, clinically diagnosed with VEXAS syndrome, with 2 novel mutations in UBA1 occurring in cis on the same allele. One mutation (c.121 A>T; p.Met41Leu) caused severely reduced translation of UBA1b in a reporter assay, but coexpression with the second mutation (c.119 G>C; p.Gly40Ala) rescued UBA1b levels to those of canonical mutations. We conclude that regulation of residual UBA1b translation is fundamental to the pathogenesis of VEXAS syndrome and contributes to disease prognosis.

Somatic mutations have been increasingly identified as etiologic for many hematologic and autoinflammatory disorders. VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome and Erdheim-Chester disease (ECD), a type of histiocytosis, can be classified as inflammatory myeloid diseases, characterized by systemic inflammation and multi-organ disease with predisposition to myeloid malignancies. VEXAS is a novel disease caused by UBA1 mutations that was first discovered using a genotype-driven approach (genotype was used to identify patients with undiagnosed inflammatory diseases). Since the initial description, many VEXAS cases have been reported and disease phenotype is expanding rapidly. In contrast, ECD was first characterized in the 1930s based on patients' phenotype, and only recently found to be caused by recurrent somatic mutations in the MAPK pathway (traditional phenotype-driven approach). The discovery of these mutations and development of target therapies have revolutionized the treatment of patients with histiocytosis, particularly ECD. Here we discuss the impact of causal and associated somatic mutations in VEXAS and ECD at both clinical and molecular levels.

Background Adult-onset inflammatory syndromes often manifest with overlapping clinical features. Variants in ubiquitin-related genes, previously implicated in autoinflammatory disease, may...

VEXAS syndrome (vacuoles in myeloid progenitors, E1 ubiquitin activating enzyme, X-linked, autoinflammatory manifestations and somatic) is an autoinflammatory condition caused by somatically acquired UBA1 mutations. Heiblig et al report on an international retrospective analysis of 30 patients with VEXAS syndrome treated with different Janus kinase (JAK) inhibitors, finding encouraging evidence supporting the use of the JAK1/2 inhibitor ruxolitinib with clinical remissions and reductions in steroid use seen in the majority of patients.

Patients with severe aplastic anemia (SAA) are either treated with bone marrow transplant (BMT) or immunosuppression (IST) depending on their age, comorbidities, and available donors. In 2017, our phase 2 trial reported improved hematologic responses with the addition of eltrombopag (EPAG) to standard IST for SAA when compared with a historical cohort treated with IST alone. However, the rates and characteristics of long-term complications, relapse, and clonal evolution, previously described in patients treated with IST alone, are not yet known with this new regimen, IST and EPAG. Patients were accrued from 2012 to 2020, with a total of 178 subjects included in this secondary endpoint analysis. With double the sample size and a much longer median follow-up (4 years) since the original publication in 2017, we report a cumulative relapse rate of 39% in responding patients who received cyclosporine (CSA) maintenance and clonal evolution of 15% in all treated patients at 4 years. Relapse occurred at distinct timepoints: after CSA dose reduction and EPAG discontinuation at 6 months, and after 2 years when CSA was discontinued. Most relapsed patients were retreated with therapeutic doses of CSA +/− EPAG, and two-thirds responded. Clonal evolution to a myeloid malignancy or chromosome 7 abnormality (high-risk) was noted in 5.7% of patients and conferred a poorer overall survival. Neither relapse nor high-risk evolution occurred at a higher rate than was observed in a historical comparator cohort, but the median time to both events was earlier in IST and EPAG treated patients. This trial was registered at www.clinicaltrials.gov as #NCT01623167.

Myelodysplastic syndromes (MDS) are a group of clonal myeloid disorders characterized by low blood counts and a propensity to develop acute myeloid leukemia. The management of lowerrisk (LR) MDS with persistent cytopenias remains suboptimal. Eltrombopag, a thrombopoietin-receptor agonist, can improve platelet counts in LR-MDS and trilineage hematopoiesis in aplastic anemia. We conducted a phase II dose modification study to investigate the safety and efficacy of eltrombopag in LR-MDS. The eltrombopag dose was escalated from 50 mg/day to a maximum of 150 mg/day over a period of 16 weeks. The primary efficacy endpoint was hematologic response at 16-20 weeks. Eleven of 25 (44%) patients responded; five and six patients had uni- or bi-lineage hematologic responses, respectively. The predictors of response were presence of a paroxysmal nocturnal hemoglobinuria clone, marrow hypocellularity, thrombocytopenia, and elevated plasma thrombopoietin levels at study entry. The safety profile was consistent with that found in previous eltrombopag studies in aplastic anemia; no patients discontinued the drug due to adverse events. Three patients developed reversible grade 3 liver toxicity and one patient had increased reticulin fibrosis. Ten patients discontinued eltrombopag after achieving a robust response (median time 16 months); four of them reinitiated eltrombopag because of declining blood counts, and all attained a second robust response. Six patients had disease progression not associated with expansion of mutated clones and no patient progressed to develop acute myeloid leukemia on study. In conclusion, eltrombopag was well-tolerated and effective in restoring hematopoiesis in some patients with low or intermediate-1 risk MDS. This study was registered at clinicaltrials.gov as #NCT00932156.

Acquired severe aplastic anaemia (SAA) has an immune pathogenesis, and immunosuppressive therapy (IST) with anti-thymocyte globulin and cyclosporine is effective therapy. Eltrombopag (EPAG) added to standard IST was associated with higher overall and complete response rates in patients with treatment-naive SAA compared to a historical IST cohort. We performed a paediatric subgroup analysis of this trial including all patients aged

Venetoclax is a BCL2 inhibitor approved for 17p-deleted relapsed/refractory chronic lymphocytic leukemia with activity following kinase inhibitors. We conducted a multicenter retrospective cohort analysis of patients with chronic lymphocytic leukemia treated with venetoclax to describe outcomes, toxicities, and treatment selection following venetoclax discontinuation. A total of 141 chronic lymphocytic leukemia patients were included (98% relapsed/refractory). Median age at venetoclax initiation was 67 years (range 37-91), median prior therapies was 3 (0-11), 81% unmutated IGHV, 45% del(17p), and 26.8% complex karyotype (≥ 3 abnormalities). Prior to venetoclax initiation, 89% received a B-cell receptor antagonist. For tumor lysis syndrome prophylaxis, 93% received allopurinol, 92% normal saline, and 45% rasburicase. Dose escalation to the maximum recommended dose of 400 mg daily was achieved in 85% of patients. Adverse events of interest included neutropenia in 47.4%, thrombocytopenia in 36%, tumor lysis syndrome in 13.4%, neutropenic fever in 11.6%, and diarrhea in 7.3%. The overall response rate to venetoclax was 72% (19.4% complete remission). With a median follow up of 7 months, median progression free survival and overall survival for the entire cohort have not been reached. To date, 41 venetoclax treated patients have discontinued therapy and 24 have received a subsequent therapy, most commonly ibrutinib. In the largest clinical experience of venetoclax-treated chronic lymphocytic leukemia patients, the majority successfully completed and maintained a maximum recommended dose. Response rates and duration of response appear comparable to clinical trial data. Venetoclax was active in patients with mutations known to confer ibrutinib resistance. Optimal sequencing of newer chronic lymphocytic leukemia therapies requires further study.

Kidney stone disease is an increasing disorder of humans. It affects about 12% of the world population. Epidemiological data have shown that calcium oxalate is the predominant mineral in a majority of kidney stones. [1] It has been associated with an increased risk of end-stage renal failure. Kidney stones result from a succession of several physicochemical events including super saturation, nucleation, growth, aggregation, and retention within the kidneys. Kidney stones may cause extreme pain and blockage of urine flow .The average life time risk of stone formation has been reported in the range of 5-10 %.Recurrent stone formation is a common part of the medical care of patients with stone disease.[2] Kidney stone disease is usually treated with medications that may cause a number of side-effects. Even improved and besides the high cost that imposes, compelling data now suggest that exposure to shock waves in therapeutic doses may cause acute renal injury, decrease in renal function and an increase in stone recurrence. Data from in vitro, in vivo and clinical trials reveal that phytotherapeutic agents could be useful as either an alternative therapy in the management of urolithiasis. The present review therefore critically explains the potential usefulness of herbal medicines in the management of urolithiasis. Keywords: Kidney stones, Calcium oxalate, Herbal plant extracts, Alternative medicine

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.

Introduction: Immune aplastic anemia (AA) is effectively treated with either immunosuppressive treatment (IST) or allogeneic hematopoietic stem cell transplant (HSCT). Clonal evolution remains the most feared long-term complication after IST. We investigated predictor factors, genetic characteristics, and long-term outcomes of patients who developed either secondary myeloid neoplasia or isolated chromosomal abnormalities without morphologic dysplasia after immunosuppression. Methods: All patients with severe AA treated at the NIH Clinical Center with IST from 1989-2020 who underwent clonal evolution were categorized as "high-risk" (overt myeloid neoplasia, or isolated chromosome 7 abnormality / complex cytogenetics) or "low-risk" (isolated chromosome abnormalities without overt myeloid neoplasia or dysplasia; isolated chromosome 7 abnormality or complex cytogenetics were characterized as high-risk). Univariable analysis was performed using the Fine-Gray competing risk regression model using death as a competing risk to determine predictors of clonal evolution. Classification and regression tree analysis of time to clonal evolution was performed on continuous baseline variables to partition the data based on the best categorical cutoff. Long term outcomes assessed included overall survival (OS) and HSCT. Error corrected next-generation sequencing (ECS) was used to assess for pathogenic somatic variants in known myeloid cancer genes in clonal evolvers both at time of evolution and in serial samples prior when available. Results: Of 659 patients with severe AA included in this study, 95 developed clonal evolution: 59 high-risk and 36 low-risk. Age >48 years at diagnosis and pre-treatment ANC >0.87x10 9/L were strong predictors of high-risk clonal evolution. High-risk clonal evolution was increased in patients aged >48 years, with cumulative incidence (CI) of 13.9% by 5 years compared to patients aged 0.87 x10 9/L (independent of age) predicted an even higher risk of evolution; CI for high-risk evolution was 17% by 5 years (p48 years) were prognostic of the greatest risk of high-risk evolution, with a hazard ratio (HR) of 5.51; conversely, ANC RUNX1 (13 variants in 8 [35%] patients) and ASXL1 (13 variants in 10 [43%] patients) were the most frequent mutated genes at time of clonal evolution in high-risk patients, and BCOR/L1 (14 variants in 8 [32%] patients) was the most frequently mutated in the low-risk group. Longitudinal data were available in five high-risk and eight low-risk patients. Three of five high-risk patients had acquisition or expansion of RUNX1 clones at evolution. Small RUNX1 variants were present in two patients as early as three years prior to high-risk evolution. Splicing factor genes and RUNX1 somatic variants were detected exclusively in the high-risk group; DNMT3A, BCOR/L1 and ASXL1 gene mutations were present in both groups. Conclusion: Age and pre-treatment ANC strongly predict high-risk clonal evolution in AA patients after IST and may be used determine at-risk patients for long term follow-up. Outcomes in patients with low-risk evolution are favorable but poor in high-risk evolution without HSCT. The clonal landscape differs between high-risk and low-risk evolution; MDS-associated genetic mutations are enriched in high-risk evolution, in particular RUNX1. Further study of the role of RUNX1 in high-risk clonal evolvers may give insight into leukemogenesis in AA. Figure 1: Cumulative incidence (CI) of clonal evolution since immunosuppression with death treated as competing risk. (A) CI for development of all clonal evolution in patients >37 years (B) and high-risk clonal evolution in patients >48 years (C) CI for development of all clonal evolution when baseline ANC >0.87x10 9/L and (D) high-risk clonal evolution when baseline ANC >0.87x10 9/L. Figure 1 Figure 1. Disclosures Young: Novartis: Research Funding.

Introduction: Telomere Biology Disorders (TBD) are due to germline variants in telomere maintenance and repair genes. Clinical manifestations include bone marrow failure (BMF), liver and lung fibrosis, and risk of cancer, especially myeloid neoplasia. We characterized clinical phenotype, pathology, and clonal landscape of patients with TBD and myelodysplastic syndrome (MDS) or acute leukemia (AL). We assessed if somatic variants in myeloid cancer genes are associated with MDS/AL or overall survival (OS). Methods: Patients (n=109) from the National Institutes of Health and University of São Paulo (NIH/USP) (n=91) or MD Anderson (MDA) (n=18) met TBD clinical criteria or had a pathogenic/likely pathogenic germline variant in a telomere maintenance/repair gene. All patients gave informed consent. Clinical characteristics and details of hematologic malignancy were collected. MDS was defined using WHO 2016 criteria. In the NIH/USP cohort, somatic variants in myeloid cancer genes were assessed in peripheral blood using error-corrected DNA sequencing (ECS; minimum variant allele frequency [VAF] of 0.5%) and in the MDA cohort patients were screened by targeted amplicon-based next-generation sequencing (minimum VAF of 2%). Serial samples were available in 31 (41%) patients. Results: Eighteen out of 109 (17%) patients from both the NIH/USP and MDA cohorts had a diagnosis of MDS/AL, developing in patients with TERC (n=8/31; 25%), TERT (n=8/48; 16%), RTEL1 (n=1/5; 20%), and DKC1 (n=1/2; 50%). Karyotypes were normal diploid (n=6), chromosome 1 abnormality (n=5), trisomy 8 (n=2), deletion 20q (n=1), complex (n=2), monosomy 7 (n=1) and deletion 7q (n=1). Six patients had >5% blasts. Four patients had concurrent liver fibrosis and 9 had pulmonary fibrosis. Somatic mutation data at time of MDS/AL was available for 16/18 patients; splicing factor genes (n=14) were most frequent, seen in 11 (69%) patients, with U2AF1 predominating (n=7; 44% of patients) (Figure 1A). More than 1 mutation was present in 12/16 (75%) of patients. One patient developed AML 6 years after MDS diagnosis . U2AF1 variants were also present in 8 patients without MDS/AL; 5 had no sequential samples and 3 had stable mutations over 2 (n=2) and 4 (n=1) years. Recurrent variants in U2AF1 were present at p.S34 (n=12) and p.Q157 (n=2). Median U2AF1 variant allele fraction (measured in NIH/USP cohort only) was 23% in patients with MDS/AL compared to 7% in those without. Variant association analysis was confined to NIH/USP cohort due to DNA sequencing assay differences; data were available in 75 patients. Patients were divided into groups defined by specific variants at any time of sampling; "Clonal-hematopoiesis of indeterminate potential / aplastic anemia (CHIP/AA) associated" (DNMT3A, TET2, ASXLI, or BCOR/L1), "MDS-associated" (containing splicing factor genes, RUNX1, SETBP1, ETV1, KRAS, STAG2, GATA2/1, TP53), "PPM1D" (containing PPM1D), and a subgroup, "Splicing factors" (U2AF1, ZRSR2, SF3B1, SRSF2). Patients with MDS-associated variants (Figure 1A; p=0.02), particularly splicing factors (Figure 1B; p=0.007) more likely had or later developed MDS/AL, compared to patients without somatic variants or in other variant groups. MDS/AL was not associated with PPM1D or CHIP/AA-associated variants. OS from time of first detected somatic variant was lower in the MDS-associated variant group (Figure 1C p=0.013) compared to those without variants at time of first assessment; this was not the case for CHIP/AA-associated variants or PPM1D. Patients with somatic variants (median ages 36, 41, and 42 for PPM1D, CHIP/AA-associated, and MDS-associated respectively) were significantly older than patients without variants (median age 24) though ages were similar within variant groups. Conclusion: Splicing factor gene variants predominate the clonal landscape of TBD-associated hematologic malignancy; U2AF1 p.S34 is recurrent and can occur in the absence of neoplasia. Splicing factor gene variants such as U2AF1 in the peripheral blood may be potential biomarkers for hematologic malignancy in TBD patients. PPM1D, previously associated with therapy-related MDS, is commonly mutated in TBD but does not associate with MDS/AL. OS was decreased in patients with MDS-associated variants but not with PPM1D or CHIP/AA-associated variants. Further study of U2AF1 variants in TBD patients may give insight to the underlying drivers of MDS/AL. Figure 1 Figure 1. Disclosures DiNardo: Agios/Servier: Consultancy, Honoraria, Research Funding; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; ImmuneOnc: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; AbbVie: Consultancy, Research Funding; Forma: Honoraria, Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Takeda: Honoraria; Foghorn: Honoraria, Research Funding; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding. Calado: Novartis Brasil: Honoraria; Instituto Butantan: Consultancy; AA&MDS International Foundation: Research Funding; Alexion Brasil: Consultancy; Agios: Membership on an entity's Board of Directors or advisory committees; Team Telomere, Inc.: Membership on an entity's Board of Directors or advisory committees. Young: Novartis: Research Funding.

Introduction: Telomere biology (TBD) disorders are caused by pathogenic germline variants in genes related to telomere maintenance. In TBD, clonal hematopoiesis (CH) has been hypothesized to compensate for restricted cell fitness and to lead to development of myelodysplastic syndromes and acute myeloid leukemia (MDS/AML). We sought to characterize the clonal landscape and dynamics by deep sequencing of a large cohort of TBD patients with a broad spectrum of phenotypes and ages. Methods: We screened 120 TBD patients (median age=29) from the National Institutes of Health and the University of Sao Paulo for somatic mutations in genes related to myeloid malignancies and telomere diseases using an error-correcting DNA sequencing panel (minimum allele frequency [VAF] of 0.5%). Patients had either a pathogenic germline variant in telomere-related genes or short telomeres in blood and a strong clinical suspicion for TBD. Relatives were included if they harbored the proband's germline mutation. Single-cell DNA sequencing was performed in marrow samples from two TBD patients with MDS (TBD-MDS) to elucidate clonal trajectories Results: Fifty-eight TBD patients (48%) had somatic mutations in peripheral blood (median age and range, 42 years; 9-57), most frequently in PPM1D (all exon 6 truncated; n=18) , TERTp (-57, -124, and -146; n=14), POT1 (n=12), U2AF1 (n=12), and other MDS-associated genes. Clinically, these patients had dyskeratosis congenita (DC; n=12/27), aplastic anemia (AA; n=11/27), isolated cytopenias (n=7/10), MDS/AML (n=7/8), pulmonary or liver fibrosis (n=4/8), and multi-organ disease (n=19/26). In this series, no relatives had somatic mutations (n=14). CH frequency increased with age and was significantly more frequently observed than in healthy controls, regardless of age (p Patients' clonal profiles correlated with the underlying germline defect. Somatic P OT1 mutations strongly associated with TINF2 germline variants, and consequently DC: 5/9 TINF2 patients had one (n=2) or >2 POT1 clones (n=3). In contrast, both TERTp and PPM1D clones were mostly detected in TERT/TERC patients with multi-organ disease, especially pulmonary fibrosis and marrow failure. No telomere elongation or improved blood counts were seen in serial samples. TINF2 patients with somatically mutated POT1 clones were older despite their DC diagnosis (median age=19 vs 5 years in POT1 mutated and wild type, respectively). A single patient with a germline TINF2 R282C and somatically mutated POT1 clone at VAF=29%, which was stable for 5 years, had MAA. The median ages (range) of TERT/TERC patients with TERTp and PPM1D mutations were 41 (25-64) and 43 (12-72), respectively, whereas TERT/TERC patients without TERTp and PPM1D mutations were at a median age of 27 (8-58). Most clones were stable regardless of clinical phenotype, even after danazol treatment. PPM1D clones were stable for 2-9 years of follow-up. TERTp and POT1 clones' size decreased while on androgens but consistently increased after the drug was discontinued. In single-cell DNA analysis of two TBD-MDS patients, the U2AF1 S34F and Q157R were driver mutations and occurred with mutations in RUNX1, ETV6, ASXL1; these clones were stable for 3-6 years. In the first case, the U2AF1 clone subsequently acquired a RUNX1 mutation; this clone was coincident with an independent clone containing PPM1D and POT1 mutations. In the second patient, a U2AF1 clone acquired successive mutations in SETBP1 and AXSL1; a second clone with U2AF1 and additional mutations in GATA2 and KRAS arose at evolution to AML. Conclusion: In TBD, the somatic landscape differed from age-related CH, with recurrent TERTp, POT1, and truncated PPM1D mutations. Mutations' frequency increased with age but was related to the underlying germline mutation. It is uncertain whether clonal selection is a probabilistic consequence of older age or the cause of mild phenotypes and prolonged lifespan. Despite the association of POT1 and PPM1D with malignancies, no patients in our cohort had POT1-related cancers or had received chemotherapy. POT1 and PPM1D like TERTp mutations may arise to compensate for cell fitness. Clinically, this distinct clonal landscape, not found in immune BMF, could serve as a molecular marker of underlying TBD. Disclosures Calado: Instituto Butantan: Consultancy; AA&MDS International Foundation: Research Funding; Agios: Membership on an entity's Board of Directors or advisory committees; Novartis Brasil: Honoraria; Alexion Brasil: Consultancy; Team Telomere, Inc.: Membership on an entity's Board of Directors or advisory committees. Young: Novartis: Research Funding.

Background VEXAS syndrome (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) is due to a somatically acquired mutation of the E1-ubiquitin ligase UBA1, leading to the expression of a catalytically impaired isoform in myeloid cells. VEXAS syndrome combines severe auto-inflammatory manifestations and is frequently associated with myeloid neoplasia (MN). The outcome of VEXAS is poor, and most patients require high dose corticosteroids to reduce inflammation (Bourbon et al. Blood 2021). Therapeutic options besides steroids are currently limited in those patients. In this multicenter retrospective study, we report some clinical efficacy of JAK inhibitors (JAKi) in VEXAS patients. Patients We analyzed retrospectively 24 UBA1 mutated patients (Met41 or previously reported alternative splicing site) treated with JAKi (11 with ruxolitinib (RUXO), 11 with tofacitinib (TOFA), 1 with baricitinib, 1 with upadicitinib) in 7 French, 1 Portugese and 2 US centers. Complete clinical (CCR) and complete biological response (CBR) were defined as complete resolution of clinical symptoms and normalization of inflammation markers (C reactive protein, CRP) respectively. Partial clinical (PCR) and biological (PBR) response were defined by reduction of at least 50% of clinical or inflammation markers, respectively. Results All 24 patients were males with a median age at VEXAS diagnosis of 72 years [range 54-89]. Thirteen had documented myeloid neoplasia (MN) (1 CMML-0, 1 other MDS/MPN, 10 MDS). Clinical manifestations at VEXAS diagnosis include skin involvement (87.5%), arthritis or arthralgia (83.3%), vasculitis (37.5%), fever (75%), ocular manifestations (29.2%) and pulmonary infiltrates (41.6%). IPSS-R was very low/low/intermediate in 8/3/2 cases respectively. Median time between first VEXAS related clinical manifestations and JAKi onset was 2.45 years [0.15-5.45]. Prior to JAKi onset, patients had received a median of 2.5 immunosuppressive/immunomodulatory treatments [range 0-9]. After 1 month, 12/24 (50%) patients had achieved clinical and/or biological response. CCR and CBR was achieved in 7/11 (64%) and 6/11 (54%) patients treated with RUXO, and in 3/13 (23%) and 2/13 (15%) patients treated with other JAKi (figure A). After 3 months, CCR and CBR was 100% and 80% (10 evaluable patients) in the RUXO group as compared to 25% and 25 % in patients treated with other JAKi (8 evaluable patients) (p=0.0036 et 0.0055 respectively, figure B). RUXO efficacy was similar in patients with (n=9) or without (n=2) associated MN. In RUXO treated patients, median CRP and steroid dose reduction was 72.5% [range 21.5- 99.5] and 66.25% [range 0-75] respectively at 3 months. With a median follow-up of 4 months [range 1.4-12], only 1 RUXO treated patient had lost response, whereas median time to next of treatment was 3.4 months with other JAKi (figure C). Of the 13 patients with MN, 7 were RBC transfusion dependent at JAKi onset (6 with RUXO, 1 with other JAKi). Four of 6 patients treated with RUXO achieved RBC transfusion independence at 3 months, but not the patient treated with other JAKi. Regarding safety, severe adverse events were reported in 6 patients: 3 deep vein thrombosis, (2 with TOFA/1 with RUXO), 1 pneumonia (RUXO), 1 enterohemorrhagic E. Coli infection (RUXO), and 1 lethal legionellosis (TOFA)). Conclusion Ruxolitinib (and less often other JAK inhibitors used in this study) provides rapid response in most VEXAS patients, allowing in two third of the cases corticosteroid dose reduction/withdrawal and RBC transfusion independence in 4/6 patients with MN who were initially transfusion dependent. Those retrospective preliminary results, with limited follow up, must be interpreted with caution and will be updated at the meeting. The effect of RUXO on VEXAS patients with concomitant MN will soon be studied prospectively in a Groupe Francophone des Myélodysplasies (GFM) clinical study. Figure 1 Figure 1. Disclosures Galicier: Novartis Pharma Sas, Sanofi Aventis France: Consultancy; Lilly France, Baxalta France, Sanofi Aventis France Sas: Other: Payments as Speaker for Educational Program; Shire France SA, Janssen-Cilag, Pfizer Sas: Other: Invitation to Congress. Hirsch: Novartis Pharma: Consultancy; Daiichi Sankyo Oncology: Consultancy. Warrington: Eli Lilly: Research Funding; Kiniksa: Research Funding. Fenaux: Novartis: Honoraria, Research Funding; JAZZ: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Celgene/BMS: Honoraria, Research Funding; Syros Pharmaceuticals: Honoraria.

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 cfDNA results were validated against matched PB/BM cells collected from HC (n=25), AA (n=56), and MDS patients (n=54). cfDNA variants were classified as true, and false positives or negatives according to their presence in PB/BM (Figure 1E). In HC, cfDNA sensitivity was moderate (58%), but specificity and PPV were low compared to PB. In AA and MDS, sensitivity, specificity and PPV values were high compared with BM (Table 1). In all cohorts, the median VAF of discordant variants was significantly lower than VAF of true-positives (9.5% [95CI%:8-12] vs. 36% [95CI%:31-37]; t-test,p40yo; Figure 1A). Discordant pairs were found most often in ASXL1, KIT, TET2, and ZRSR2 (Figure 1E). Although linear regression showed high correlation between the VAF of paired samples (R2>0.7), agreement of VAF by Bland-Altman analysis was poor; this approach calculates the bias, the mean difference between the VAF of matched samples, and its standard deviation (SD) to more accurately evaluate agreement between paired values. Here, the bias and SD were both as high as 9% in HC and patients. These large variations translated to a wide range of upper and lower limits of agreement (mean ± 2*SD), which represent the limits of acceptable differences. In summary, the landscape of somatic cfDNA variants depended on variant VAF. Technical factors were an important source of assay discordance, and although cfDNA variants were reliably detected at higher VAFs, their quantification was not comparable to VAF detected in blood cells. A single cfDNA clone varied up to 9% in size and SD was unacceptably high in all cohorts. cfDNA and PB/BM may not be interchangeable, as cfDNA may either over- or under-estimate clone size, regardless of disease status. Since small changes in clone size and dynamics may influence clinical evaluation and decisions, the use of cfDNA for CHIP detection requires robust sequencing coverage and validation of variants at VAF Disclosures Young: Novartis: Research Funding.

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.

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.