Search

There is no standard or widely effective treatment of patients with moderate aplastic anemia (MAA) or hypo-productive uni-lineage cytopenias (UC). Eltrombopag (EPAG), a small molecule thrombopoietin mimetic, has previously been shown to result in durable multi-lineage hematologic responses with low toxicity in patients with refractory severe aplastic anemia (SAA). Its safety and efficacy in MAA are unknown. This prospective phase 2 study enrolled previously untreated and treated MAA and UC patients with clinically relevant cytopenias. EPAG was administered at doses escalating from 50 to 300 mg/d. Hematologic responses were assessed at 16 to 20 weeks. Responding patients were continued on EPAG until reaching defined robust or stable blood counts. EPAG was reinstituted for relapse. Thirty-four patients were enrolled between 2012 and 2017, including 31 with MAA and 3 with UC. Seventeen patients responded in at least 1 eligible lineage by the primary end point. A striking improvement in anemia was observed in a patient with Diamond-Blackfan anemia. EPAG was well tolerated, and it was discontinued for robust or stable blood counts in 12 of 17 patients after a median of 8 months. A majority required re-initiation of EPAG for declining counts, and all regained response. Two of 34 patients developed non–chromosome 7 bone marrow cytogenetic abnormalities while taking EPAG, without dysplasia or increased blasts. Somatic mutation allele frequencies in cancer genes did not increase overall on EPAG. EPAG is a well-tolerated oral treatment of cytopenias in patients with MAA/UC. This trial was registered at www.clinicaltrials.gov as #NCT01328587.

BACKGROUND: Loss-of-function mutations in genes that encode for components of the telomere repair complex cause accelerated telomere shortening. Hepatic involvement has been recognized as a cause of morbidity in telomere diseases, but very few studies have characterized the nature and extent of liver involvement in affected patients. We report the prevalence and characteristics of liver involvement in a large cohort of patients with telomere disease evaluated serially at the National Institutes of Health. METHODS: One hundred twenty-one patients with known or suspected telomere disease were screened; 40 patients with liver involvement were included in the current study. Median follow up was 2.4 years. Data were collected regarding their demographic information, laboratory analysis, imaging and histopathology. RESULTS: Forty patients (40% of the cohort) with a median age of 42 years were found to have liver involvement. Liver enzyme elevation was cholestatic in pattern; 8(21%) had drug related enzyme elevations. The most common imaging finding was increased hepatic echogenicity on ultrasound in 39% (9) patients, followed by hepatomegaly in 26% (6) subjects. Biopsies were infrequent due to risk associated with thrombocytopenia, but in six patients there were varying findings: nodular regenerative hyperplasia, steatohepatitis, hemosiderosis, cholestasis, and cirrhosis with hepatic steatosis. Almost half the cohort had pulmonary diffusion abnormalities, and 25% died during the follow up period. CONCLUSION: In patients with telomere disease, hepatic involvement is common and can present in diverse ways, including elevated liver enzymes, histopathologic as well as imaging abnormalities. Liver disease has important implications for morbidity and mortality in patients with telomere disease.

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.

The anemia of MDS often results in decreased quality of life, which is invoked to justify red cell transfusions; however, there are sparse data regarding the minimum hemoglobin (Hb) at which it is safe to forgo transfusions for patients with no evidence of end-organ damage. This issue is even more important in the COVID-19 era, where decreases in blood donations have stressed the blood supply. In March 2018, using a modified Delphi method, we convened a panel of 13 expert MDS clinicians for three iterative rounds to discuss a minimum safe Hb for this population. While the panel was unable to reach the pre-set consensus of 75% for a specific Hb threshold, there was 100% consensus that it be no greater than 7.5 g/dL. Our data suggest that, given no end-organ effects of anemia, patients with MDS can safely forgo transfusions with a Hb of 7.5 g/dL or higher.

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

Purpose: Immune checkpoint blockade has demonstrated clinical benefits across multiple solid tumor types; however, resistance and relapse often occur. New immunomodulatory targets, which are highly expressed in activated immune cells, are needed. MEDI0562, an agonistic humanized mAb, specifically binds to the costimulatory molecule OX40. This first-in-human study evaluated MEDI0562 in adults with advanced solid tumors. Patients and Methods: In this phase I, multicenter, open-label, single-arm, dose-escalation (3+3 design) study, patients received 0.03, 0.1, 0.3, 1.0, 3.0, or 10 mg/kg MEDI0562 through intravenous infusion every 2 weeks, until confirmed disease progression or unacceptable toxicity. The primary objective evaluated safety and tolerability. Secondary endpoints included antitumor activity, pharmacokinetics, immunogenicity, and pharmacodynamics. Results: In total, 55 patients received ≥1 dose of MEDI0562 and were included in the analysis. The most common tumor type was squamous cell carcinoma of the head and neck (47%). Median duration of treatment was 10 weeks (range, 2–48 weeks). Treatment-related adverse events (TRAEs) occurred in 67% of patients, most commonly fatigue (31%) and infusion-related reactions (14%). Grade 3 TRAEs occurred in 14% of patients with no apparent dose relationship; no TRAEs resulted in death. Two patients had immune-related partial responses per protocol and 44% had stable disease. MEDI0562 induced increased Ki67+ CD4+ and CD8+ memory T-cell proliferation in the periphery and decreased intratumoral OX40+ FOXP3+ cells. Conclusions: MEDI0562 was safely administered at doses up to 10 mg/kg in heavily pretreated patients. On-target pharmacodynamic effects were suggested in this setting. Further evaluation with immune checkpoint inhibitors is ongoing.

GATA2 deficiency syndrome is caused by autosomal dominant, heterozygous germline mutations with widespread effects on immune, pulmonary and vascular systems. Patients commonly develop hematological abnormalities including bone marrow failure, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). We present a patient with GATA2 mutation and MDS who progressed to AML over four months. Whole exome and targeted deep sequencing identified a new p.Q61K NRAS mutation in the bone marrow at the time of AML development. Rapid development of AML is possible in the setting of germline GATA2 mutation despite stable MDS, supporting close monitoring and consideration of early allogeneic transplantation.

Clinical utility of gene panel-based testing for hereditary myelodysplastic syndrome/acute leukemia predisposition syndromes

Background. Aplastic anemia (AA) is an acquired bone marrow (BM) disease characterized by marrow hypoplasia, a paucity of hematopoietic stem and progenitor cells, and pancytopenia of the peripheral blood, due to immune attack on the BM. Although the immune pathophysiology of AA is well characterized, there are no biomarkers that would allow a better understanding of the immunological status of an individual AA patient. Extracellular miRNAs are cell-free circulating molecules residing in microvesicles, exosomes, and microparticles. Recent studies have reported circulating miRNAs as biomarkers to monitor T cell mediated autoimmune diseases. Our work aimed to analyze the circulating miRNA profile in the plasma of AA patients and to assess whether specific miRNAs could serve as new biomarkers for AA. Method. Human blood samples were obtained from patients (n = 129) and healthy controls (HC) (n = 54) who were enrolled on clinical protocols between 2006 and 2015 at NIH. All AA patients were diagnosed as severe AA and received immunosuppressive therapy (IST) on a clinical research protocol (#NCT00260689). Plasma samples from myelodysplastic syndrome (MDS) patients and HC were used for comparison (NCT00217594 and NCT00961064). RNA was isolated from EDTA plasma using the miRCURY RNA Isolation Kit - Biofluids (Exiqon). A total of 179 miRNAs were analyzed in 35 plasma samples from 13 AA, 11 MDS, and 11 HC (a discovery set) using the Serum/Plasma Focus microRNA PCR Panel (Exiqon). Subsequently, 19 miRNAs from the discovery set were investigated in the 108 plasma samples from 41 AA, 24 MDS, and 43 HC (a validation set) for validation using a custom PCR panel. To assess the effect of IST, 40 out of 41 AA patients with plasma samples available both before and after 6 months of IST were further analyzed. Results and Discussion . We first analyzed 35 discovery-set plasma samples. Between AA and HC, 14 miRNAs displayed more than 1.5 fold change (FC) (P < 0.05): seven miRNAs were upregulated (miR-501-3p, miR-146b-5p, miR-150-5p, let-7b-5p, miR-200a-3p, miR-1260a, and miR-424-5p) and seven miRNAs were downregulated (miR-1, miR-29b-3p, miR-30e-5p, miR-143-3p, let-7e-5p, let-7c-5p, and let-7f-5p) in AA, compared to HC. In the validation set, group comparison revealed distinct expression profiles (> 1.5 FC, P < 0.05): upregulated miR-150-5p and miR-146b-5p and downregulated miR-1 in AA, compared to HC; upregulated miR-146b-5p and miR-22-3p in MDS, compared to HC; and downregulated miR-1, miR-22-3p, and miR-424-5p in AA, compared to MDS. By multivariate analysis, miR-1 [odds ratio (OR) = 0.34, adjusted P = 0.005], miR-146b-5p (OR = 3.82, adjusted P = 0.03), and miR-150-5p (OR = 2.19, adjusted P = 0.04), independently associated with the AA diagnosis. A receiver operating characteristic curve analysis indicated strong association of miR-146b-5p [95% confidence interval (CI), 0.65-0.86, P = 0.0001], miR-150-5p (95% CI, 0.63-0.85, P = 0.0002), and miR-1 (95% CI, 0.62-0.85, P = 0.0004) with AA [area under the curve (AUC) of 0.76, 0.74, and 0.73, respectively). Further, logistic regression demonstrated that a linear combination of expression levels of miR-150-5p, miR-146b-5p, and miR-1 produced the best model. A miRNA biomarker panel composed of miR-150-5p, miR-146b-5p, and miR-1 provided significantly increased AUC of 0.86 (95% CI, 0.78-0.94, P < 0.00001), compared with use of each miRNAs alone. At onset of AA, miR-150-5p and miR-146b-5p showed modest but significant negative correlations with platelet counts and absolute reticulocyte counts, respectively. Significant changes after IST were detected in all three miRNA (miR-150-5p, miR-146b-5p, and miR-1) levels, suggesting restoration of dysregurated miRNA expressions after therapy (P = 0.0001 for miR-150-5p, P = 0.0263 for miR-146b-5p, and P= 0.0003 for miR-1). Specifically, miR-150-5p expression was significantly reduced after successful IST but did not change in non-responders. Conclusion. We demonstrate that expression levels of three dysregulated miRNAs in AA plasma associated with clinical parameters and normalized after IST, suggesting them as potential biomarkers in AA. We developed a diagnostic logistic model using combined miRNA panels. Additional studies in larger patient cohorts are required to validate miRNAs as disease biomarkers for diagnostic and therapeutic purposes in BM failure. Disclosures Hosokawa: Aplastic Anemia and MDS International Foundation: Research Funding. Desierto:GSK/Novartis: Research Funding. Rios:GSK/Novartis: Research Funding. Weinstein:GSK/Novartis: Research Funding. Townsley:GSK/Novartis: Research Funding.

Prevalence of somatic mutations in patients with aplastic anemia using peripheral blood cfDNA as compared with BM

Background: Oncolytic viruses selectively infect tumors and activate antitumor immune responses via innate and adaptive pathways, which may potentiate the efficacy of immune checkpoint inhibitors. MEDI5395 is a recombinant Newcastle disease virus incorporating a granulocyte macrophage colony-stimulating factor (GM-CSF) transgene that exhibits tumor cell-specific viral replication, stimulates PD-L1 expression, and induces tumor regression in murine and patient-derived xenograft models. Increased T cell recruitment and expression of IFNγ-inducible genes observed in vitro suggest immune activation in the tumor microenvironment (TME). Antitumor activity is enhanced by concurrent PD-L1 blockade. This clinical study is evaluating IV MEDI5395 in combination with the anti-PD-L1 antibody durvalumab in patients with advanced solid tumors. Methods: This is a Phase 1, first-in-human, multicenter, open-label trial (NCT03889275) enrolling patients with breast cancer, colorectal cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, renal cell carcinoma, non-small-cell lung cancer or melanoma. Eligibility criteria include relapsed/refractory disease or intolerance to standard therapy after ≥1 prior line of treatment for recurrent/metastatic disease, and ≥1 tumor measurable by RECIST v1.1. Exclusion criteria include rapidly progressing disease precluding a break of ≥8 weeks from systemic therapy, life expectancy ≥12 weeks by GRIm Score, uncontrolled metastatic central nervous system disease, and concomitant immunosuppressive therapy. The initial dose escalation phase will enroll up to 84 patients across all eligible tumor types. Three sequentially ascending dose levels of MEDI5395 will be evaluated. Durvalumab is administered either sequentially or concurrently with MEDI5395, for up to 2 years or until radiologically confirmed disease progression, clinical deterioration, withdrawal of consent, or unacceptable toxicity. The subsequent dose expansion phase will include 2 cohorts of ~40 patients, each enrolling a single tumor type. The tumor types, MEDI5395 dose level and schedule will be selected based on dose escalation data. On-treatment biopsies of the primary tumor or a metastatic site are required in the dose escalation phase and optional in the dose expansion phase. Primary objectives are evaluating the safety, tolerability and incidence of dose-limiting toxicities of MEDI5395, and identifying the optimal dose/schedule in combination with durvalumab. Secondary objectives include assessing MEDI5395 pharmacokinetics (viremia and GM-CSF transgene expression), pharmacodynamics in the TME, immunogenicity, and initial efficacy in combination with durvalumab. The trial is actively accruing. Citation Format: Grace K. Dy, Diwakar Davar, Evanthia Galanis, Danielle Townsley, Djuro Karanovic, Maria Schwaederle, Beth Kelly, Dmitriy Zamarin, Mitesh Borad, Kevin Harrington. A phase 1 study of IV MEDI5395, an oncolytic virus, in combination with durvalumab in patients with advanced solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT244.

On behalf of the Clinical Care Consortium for Telomere-associated Ailments (CCCTAA)., Vascular complications such as bleeding due to gastrointestinal telangiectatic anomalies, pulmonary arteriovenous malformations, hepatopulmonary syndrome, and retinal vessel abnormalities are being reported in patients with telomere biology disorders (TBDs) more frequently than previously described. The international clinical care consortium of telomere-associated ailments and family support group Dyskeratosis Congenita Outreach, Inc. held a workshop on vascular abnormalities in the TBDs at the National Cancer Institute in October 2017. Clinicians and basic scientists reviewed current data on vascular complications, hypotheses for the underlying biology and developed new collaborations to address the etiology and clinical management of vascular complications in TBDs.

Germline mutation in GATA2 can lead to GATA2 deficiency characterized by a complex multi-system disorder that can present with many manifestations including variable cytopenias, bone marrow failure, myelodysplastic syndrome/acute myeloid leukemia (MDS/AML), and severe immunodeficiency. Penetrance and expressivity within families is often variable. There is a spectrum of bone marrow disease in symptomatic cytopenic patients ranging from hypocellular marrows without overt dysplasia to those with definitive MDS, AML, or chronic myelomonocytic leukemia. Relatives of probands with the same mutations may demonstrate minimal disease manifestations and normal marrows. A comprehensive clinical, hematological and genetic assessment of 25 patients with germline GATA2 mutation was performed. MDS-associated mutations were identified in symptomatic GATA2 patients both with overt MDS and in those with hypocellular/aplastic bone marrows without definitive dysplasia. Healthy relatives of probands harboring the same germline GATA2 mutations had essentially normal marrows that were overall devoid of MDS-associated mutations. The findings suggest that abnormal clonal hematopoiesis is a common event in symptomatic germline mutated GATA2 patients with MDS and also in those with hypocellular marrows without overt morphologic evidence of dysplasia, possibly indicating a pre-MDS stage warranting close monitoring for disease progression.

Eltrombopag (EPAG) received approval from the US Food and Drug Administration for the treatment of refractory severe aplastic anemia (rSAA) based on treatment of 43 patients with doses escalating from 50 to 150 mg daily for 12 weeks. Response kinetics suggested that more prolonged administration of EPAG at a dose of 150 mg could speed and improve response rates. We enrolled 40 patients with rSAA in a study of EPAG 150 mg daily, with a primary end point of response at 24 weeks. Twenty (50%) of 40 patients responded at 24 weeks; 5 (25%) of 20 would have been deemed nonresponders at 12 weeks, the end point of the previous study. Fifteen of the 19 responding patients continuing on EPAG had drug discontinued for robust response; 5 of the 15 required EPAG re-initiation for relapse, with all recovering response. To analyze risk of clonal progression, we combined long-term data from the 83 patients with rSAA enrolled in both studies. Evolution to an abnormal karyotype occurred in 16 (19%), most within 6 months of EPAG initiation. Targeted deep sequencing/whole-exome sequencing was performed pre-EPAG and at primary response end point and/or time of clonal evolution or longest follow-up. Cytogenetic evolution did not correlate with mutational status, and overall mutated allele fractions of myeloid cancer genes did not increase on EPAG. In summary, extended administration of EPAG at a dose of 150 mg for 24 weeks rescued responses in some patients with rSAA not responding at 12 weeks. The temporal relationship between clonal evolution and drug exposure suggests that EPAG may promote expansion of dormant preexisting clones with an aberrant karyotype. The studies were registered at www.clinicaltrials.gov as #NCT00922883 and #NCT01891994.

TO THE EDITOR: The thrombopoietin receptor agonist eltrombopag (EPAG) has been shown to restore hematopoiesis in patients with severe aplastic anemia (SAA) refractory to immunosuppressive therapy and to significantly improve response rates when used in combination with immunosuppressive therapy in

Memory stem T cells (TSCMs) constitute a long-lived, self-renewing lymphocyte population essential for the maintenance of functional immunity. Hallmarks of autoimmune disease pathogenesis are abnormal CD4+ and CD8+ T cell activation. We investigated the TSCM subset in 55, 34, 43, and 5 patients with acquired aplastic anemia (AA), autoimmune uveitis, systemic lupus erythematosus, and sickle cell disease, respectively, as well as in 41 age-matched healthy controls. CD8+ TSCM frequency was significantly increased in AA compared with healthy controls. An increased CD8+ TSCM frequency at diagnosis was associated with responsiveness to immunosuppressive therapy, and an elevated CD8+ TSCM population after immunosuppressive therapy correlated with treatment failure or relapse in AA patients. IFN-γ and IL-2 production was significantly increased in various CD8+ and CD4+ T cell subsets in AA patients, including CD8+ and CD4+ TSCMs. CD8+ TSCM frequency was also increased in patients with autoimmune uveitis or sickle cell disease. A positive correlation between CD4+ and CD8+ TSCM frequencies was found in AA, autoimmune uveitis, and systemic lupus erythematosus. Evaluation of PD-1, CD160, and CD244 expression revealed that TSCMs were less exhausted compared with other types of memory T cells. Our results suggest that the CD8+ TSCM subset is a novel biomarker and a potential therapeutic target for AA.

Summary Background T-cell large granular lymphocytic leukaemia (T-LGL) is a lymphoproliferative disease that presents with immune-mediated cytopenias and is characterised by clonal expansion of cytotoxic CD3+ CD8+ lymphocytes. Use of methotrexate, ciclosporin, or cyclophosphamide as first therapy improves cytopenias in 50% of patients, but long-term use of these can lead to toxicity. We aimed to explore the activity and safety of alemtuzumab, an anti-CD52 monoclonal antibody, in patients with T-LGL. Methods We did this single-arm, phase 2 trial in consecutively enrolled adults with T-LGL referred to the National Institutes of Health in Bethesda, MD, USA. Alemtuzumab was given intravenously at 10 mg per day for 10 days. The primary endpoint was haematological response at 3 months after infusion. A complete response was defined as normalisation of all affected lineages, and a partial response was defined in neutropenic patients as 100% increase in the absolute neutrophil count to more than 5 × 10 8 cells per L, and in those with anaemia, as any increase in haemoglobin of 20 g/L or higher observed in at least two serial measurements 1 week apart and sustained for 1 month or longer without exogenous growth factors support or transfusions. Analysis was by intention to treat. We report results from the first stage of this Simon two-stage design trial; enrolment into the second stage is continuing. This study is registered with ClinicalTrials.gov, number NCT00345345. Findings From Oct 1, 2006, to March 1, 2015, we enrolled 25 patients with T-LGL. 14 patients (56%; 95% CI 35–76) had a haematological response at 3 months. Four patients with associated myelodysplastic syndrome and two who had received haemopoietic stem cell transplantation had either no response or were not evaluable, meaning 14 (74% [49–91]) of the 19 patients with classic T-LGL responded. All patients had an infusion reaction (24 [96%] patients grade 1–2, one [4%] patient grade 3), which improved with symptomatic therapy. All patients developed lymphopenia, with 22 (88%) patients having grade 3 or 4 lymphopenia. The other most common grade 3 and 4 adverse events were leukopenia (eight [32%]) and neutropenic infections (five [20%]). Seven patients died; all were non-responders. Interpretation This is the largest and only prospective study of alemtuzumab in patients with T-LGL. The activity reported with a single course of a lymphocytotoxic drug in patients with mainly relapsed and refractory disease suggests that haematological response can be achieved without continued use of oral immunosuppression. Funding National Heart, Lung, and Blood Institute.

Emerging evidence indicates that microRNA control and modulate immunity. MicroRNA have not been investigated in acquired aplastic anemia, a T-cell-mediated immune disease. Analysis of 84 microRNA expression levels in CD4(+) and CD8(+) T cells of patients with aplastic anemia revealed concurrent down-regulation of miR-126-3p, miR-145-5p, miR-223-3p, and miR-199a-5p (3-fold change, P0.05) in both T-cell populations, which were unique in aplastic anemia compared to other hematologic disorders. MiR-126-3p and miR-223-3p were down-regulated in CD4(+) T effector memory cells, and miR-126-3p, miR-145-5p, and miR-223-3p were down-regulated in CD8(+) T effector memory and terminal effector cells. Successful immunosuppressive therapy was associated with restoration to normal expression levels of miR-126-3p, miR-145-5p, and miR-223-3p (2-fold change, P0.05). In CD4(+) and CD8(+) T cells in aplastic anemia patients, MYC and PIK3R2 were up-regulated and proved to be targets of miR-145-5p and miR-126-3p, respectively. MiR-126-3p and miR-145-5p knockdown promoted proliferation and increased interferon-γ and granzyme B production in both CD4(+) and CD8(+) T cells. Our work describes previously unknown regulatory roles of microRNA in T-cell activation in aplastic anemia, which may open a new perspective for development of effective therapy. Clinicaltrials.gov identifier: NCT 01623167.

Exosomal microRNAs modulate cancer cell metabolism and the immune response. Specific exosomal microRNAs have been reported to be reliable biomarkers of several solid and hematologic malignancies. We examined the possible diagnostic and prognostic values of exosomal microRNAs in two human bone marrow failure diseases: aplastic anemia and myelodysplastic syndromes. After screening 372 microRNAs in a discovery set (n=42) of plasma exosome samples, we constructed a customized PCR plate, including 42 microRNAs, for validation in a larger cohort (n=99). We identified 25 differentially expressed exosomal microRNAs uniquely or frequently present in aplastic anemia and/or myelodysplastic syndromes. These microRNAs could be related to intracellular functions, such as metabolism, cell survival, and proliferation. Clinical parameters and progression-free survival were correlated to microRNA expression levels in aplastic anemia and myelodysplastic syndrome patients before and after six months of immunosuppressive therapy. One microRNA, mir-126-5p, was negatively correlated with a response to therapy in aplastic anemia: patients with higher relative expression of miR-126-5p at diagnosis had the shortest progression-free survival compared to those with lower or normal levels. Our findings suggest utility of exosomal microRNAs in the differential diagnosis of bone marrow failure syndromes. (Registered at clinicaltrials.gov identifiers: 00260689, 00604201, 00378534, 01623167, 00001620, 00001397, 00217594).

Single-stranded oligonucleotides containing deoxyuridine are aptamers (SOMAmers) that can bind proteins with high specificity and affinity and slow dissociation rates. SOMAscan, an aptamer-based proteomic technology, allows measurement of more than 1,300 proteins simultaneously for the identification of new disease biomarkers. The aim of the present study was to identify new serum and plasma protein markers for diagnosis of acquired aplastic anemia (AA) and response to immunosuppressive therapies (IST). SOMAscan was used to screen 1,141 serum proteins in 28 AA patients before and after therapy and 1,317 plasma proteins in seven SAA patients treated with standard IST and a thrombopoietin receptor agonist. From our analysis, 19 serum and 28 plasma proteins were identified as possible candidate diagnostic and prognostic markers. A custom immunobead-based multiplex assay with five selected serum proteins (BMP-10, CCL17, DKK1, HGF, and SELL) was used for validation in a verification set (n = 65) of samples obtained before and after IST and in a blinded validation cohort at baseline (n = 16). After technical validation, four biomarkers were employed to predict diagnosis (accuracy, 88%) and long-term response to IST (accuracy, 79%). In conclusion, SOMAscan is a powerful tool for the identification of new biomarkers. We propose further larger studies to validate new candidate serum and plasma diagnostic and prognostic markers of AA. Published by Elsevier Inc. on behalf of ISEH – Society for Hematology and Stem Cells.

The treatment of aplastic anemia is currently with immunosuppressive therapy (IST) with anti-thymocyte globulin (ATG) and cyclosporine, to which two thirds of patients respond. However, a significant proportion of these responders relapse and many have persistent cytopenias. The management of these patients is challenging. Modifications to this standard approach using alternative immunosuppressive agents or adding hematopoietic cytokines such as granulocyte colony-stimulating factor (G-CSF) and erythropoietin (EPO) have not improved outcome. A recent trial has shown that eltrombopag, a thrombopoeitin mimetic, is efficacious in the treatment of patients with severe aplastic anemia (SAA) refractory to IST. There is evidence that this drug works by directly stimulating marrow stem and progenitor cells thereby promoting hematopoietic recovery in patients with bone marrow failure. Several trials are ongoing in our institution using this very promising drug in combination therapy in the upfront treatment of SAA, in IST-refractory SAA and in moderate disease.

Although hematopoietic growth factors are at high levels in aplastic anemia (AA) patients, little is known about their dynamic change over time post treatment. We examined plasma concentrations of hematopoietic growth factors sequentially in 55 severe AA patients (45 treatment-naïve patients who had received immunosuppressive therapy (IST) or IST and eltrombopag; 10 IST-refractory patients who had received eltrombopag only), focusing on thrombopoietin (TPO). TPO concentrations were much higher than normal range in patients before treatment, then decreased in responders but not in non-responders. We followed up a cohort of 9 patients who obtained stable complete remission for up to 7 years post immunosuppressive therapy, and found that TPO levels declined gradually by 3 months post treatment accompanying an increase in platelet counts, but stabilized at the levels higher than normal range. An inverse correlation was noted between TPO levels and platelet counts. The increased plasma TPO levels could be required to maintain normal platelet counts in remission, as well as attributed to the reduced consumption by circulating platelets.

Acquired aplastic anemia results from immune-mediated destruction of bone marrow. Immunosuppressive therapies are effective, but reduced numbers of residual stem cells may limit their efficacy. In patients with aplastic anemia that was refractory to immunosuppression, eltrombopag, a synthetic thrombopoietin-receptor agonist, led to clinically significant increases in blood counts in almost half the patients. We combined standard immunosuppressive therapy with eltrombopag in previously untreated patients with severe aplastic anemia.We enrolled 92 consecutive patients in a prospective phase 1-2 study of immunosuppressive therapy plus eltrombopag. The three consecutively enrolled cohorts differed with regard to the timing of initiation and the duration of the eltrombopag regimen (cohort 1 received eltrombopag from day 14 to 6 months, cohort 2 from day 14 to 3 months, and cohort 3 from day 1 to 6 months). The cohorts were analyzed separately. The primary outcome was complete hematologic response at 6 months. Secondary end points included overall response, survival, relapse, and clonal evolution to myeloid cancer.The rate of complete response at 6 months was 33% in cohort 1, 26% in cohort 2, and 58% in cohort 3. The overall response rates at 6 months were 80%, 87%, and 94%, respectively. The complete and overall response rates in the combined cohorts were higher than in our historical cohort, in which the rate of complete response was 10% and the overall response rate was 66%. At a median follow-up of 2 years, the survival rate was 97%; one patient died during the study from a nonhematologic cause. Marked increases in bone marrow cellularity, CD34+ cell number, and frequency of early hematopoietic progenitors were noted. Rates of relapse and clonal evolution were similar to our historical experience. Severe rashes occurred in two patients, resulting in the early discontinuation of eltrombopag.The addition of eltrombopag to immunosuppressive therapy was associated with markedly higher rates of hematologic response among patients with severe aplastic anemia than in a historical cohort. (Funded by the National Heart, Lung, and Blood Institute; ClinicalTrials.gov number, NCT01623167 .).

We recently reported that rabbit antithymocyte globulin was markedly inferior to horse antithymocyte globulin as a primary treatment for severe aplastic anemia. Here we expand on our findings in this unique cohort of patients. Rabbit antithymocyte globulin was detectable in plasma for longer periods than horse antithymocyte globulin; rabbit antithymocyte globulin in plasma retained functional capacity to bind to lymphocytes for up to 1 month, horse antithymocyte globulin for only about 2 weeks. In the first week after treatment there were much lower numbers of neutrophils in patients treated with rabbit antithymocyte globulin than in patients receiving horse antithymocyte globulin. Both antithymocyte globulins induced a “cytokine storm” in the first 2 days after administration. Compared with horse antithymocyte globulin, rabbit antithymocyte globulin was associated with higher levels of chemokine (C-C motif) ligand 4 during the first 3 weeks. Besides a much lower absolute number and a lower relative frequency of CD4+ T cells, rabbit antithymocyte globulin induced higher frequencies of CD4+CD38+, CD3+CD4−CD8− T cells, and B cells than did horse antithymocyte globulin. Serum sickness occurred around 2 weeks after infusion of both types of antithymocyte globulin. Human anti-antithymocyte globulin antibodies, especially of the IgM subtype, correlated with serum sickness, which appeared concurrently with clearance of antithymocyte globulin in blood and with the production of cytokines. In conclusion, rabbit and horse antithymocyte globulins have very different pharmacokinetics and effects on neutrophils, lymphocyte subsets, and cytokine release. These differences may be related to their efficacy in suppressing the immune system and restoring hematopoiesis in bone marrow failure. Clinicaltrials.gov identifier: NCT00260689.

About a quarter of patients with severe aplastic anemia remain pancytopenic despite immunosuppressive therapy. We have previously demonstrated that eltrombopag has efficacy in this setting with 44% (11/25) of patients having clinically significant hematologic responses. We now report safety and efficacy data on a further 18 patients and long-term follow-up on the entire cohort of 43 patients. The overall response rate was 17 of 43 patients (40%) at 3 to 4 months, including tri- and bilineage responses. The majority of patients who remained on eltrombopag in an extension study (14/17) continued to show improvement, and 7 eventually had significant increases in neutrophil, red cell, and platelet lineages. Five patients with robust near-normalization of blood counts had drug discontinued at a median of 28.5 months after entry (range, 9-37 months), and all maintained stable counts a median of 13 months (range, 1-15 months) off eltrombopag. Eight patients, including 6 nonresponders and 2 responders, developed new cytogenetic abnormalities on eltrombopag, including 5 with chromosome 7 loss or partial deletion. None evolved to acute myeloid leukemia to date. Eltrombopag is efficacious in a subset of patients with aplastic anemia refractory to immunosuppressive therapy, with frequent multilineage responses and maintenance of normalized hematopoiesis off treatment. This study is registered at www.clinicaltrials.gov as #NCT00922883.

Nontransplant therapeutic options for acquired and constitutional aplastic anemia have significantly expanded during the last 5 years. In the future, transplant may be required less frequently. That trilineage hematologic responses could be achieved with the single agent eltrombopag in refractory aplastic anemia promotes new interest in growth factors after years of failed trials using other growth factor agents. Preliminary results adding eltrombopag to immunosuppressive therapy are promising, but long-term follow-up data evaluating clonal evolution rates are required before promoting its standard use in treatment-naive disease. Danazol, which is traditionally less preferred for treating cytopenias, is capable of preventing telomere attrition associated with hematologic responses in constitutional bone marrow failure resulting from telomere disease.

Pulmonary arteriovenous malformations are under-recognized in telomere biology disorders and present diagnostic and therapeutic challenges.

The effectiveness of salvage therapy for aplastic anemia patients unresponsive to initial rabbit antithymocyte globulin (r-ATG) or cyclophosphamide is not known. We investigated the administration of standard horse ATG (h-ATG) plus cyclosporine (CsA) in patients who were refractory to initial r-ATG/CsA (n = 19) or cyclophosphamide/CsA (n = 6) (registered at clinicaltrials.gov as NCT00944749). The primary endpoint was hematologic response at 3 months and was defined as no longer meeting the criteria for severe aplastic anemia. Of the 19 patients who received r-ATG as initial therapy, 4 (21%) achieved a hematologic response by 3 months, and of the 6 patients who received cyclophosphamide, only 1 (17%) responded by 6 months. Among the responders there were no cases of relapse, and in nonresponders 2 patients evolved to monosomy 7. The overall survival for the cohort at 3 years was 68% (95% CI, 50-91%). These results suggest that only a minority can be successfully salvaged after receiving as first therapy either r-ATG or cyclophosphamide. Although h-ATG may be utilized in the salvage setting, the overall response rate probably will be lower than when h-ATG is used as initial treatment.

Eltrombopag (EPAG), a thrombopoietin receptor agonist, has been shown to improve hematopoiesis in patients with aplastic anemia (AA), but in MDS patients the effect of thrombopoietin mimetics in bone marrow function is still unclear. In this phase-2 dose escalation study, we investigated the safety and effectiveness of EPAG treatment in low to intermediate-2 risk MDS patients (NCT 00961064). Thirty patients were enrolled from March 2011 to July 2017. Preceding enrollment the majority of patients were either diagnosed with AA (n=13) or hypoplastic MDS (n=5). EPAG was started at 50 mg/day, up to a maximal dose of 150 mg/day, increasing the dose by 25mg every 2 weeks. The primary endpoint was hematologic response at 16 or 20 weeks, defined as either: (1) an increase in platelet counts ≥20.000/uL or transfusion independence for a minimum of 8 weeks; (2) hemoglobin (Hb) increase of ≥1.5g/dL from baseline, or a reduction in red blood cells (RBC) transfusion of at least 50%; or (3) an increase in absolute neutrophil counts (ANC) of ≥0.5x109/L or by at least 100% in patients with a baseline ANC The primary endpoint of hematological response was met in 14/30 patients (47%). All responders continued EPAG on the extension arm. In 3 patients, peripheral blood cell counts declined on EPAG after the initial response. One patient withdrew from the study. Ten of the 14 responding patients achieved a robust response (RR) after a median treatment duration of 15 months (range 7-27 months). Robust response was defined as stable hematopoiesis with at least a hemoglobin >10g/dl, and thrombocytes >50.000/L, and ANC>1000/L. However, peripheral blood cell counts significantly declined in 5/10 RR and EPAG was restarted per protocol. In 4 of these patients peripheral blood cell counts recovered. One patient did not achieve a second response. Based on International Prognostic Score System (IPSS), 4/30 (13%) patients progressed on study, including 3 non-responders and 1 responder, at a median follow-up of 4 months (3-35 months). The responding patient was diagnosed with increased bone marrow myeloblast 7 months after discontinuation of EPAG for robust response and 35 months after enrolling in the study. New cytogenetic abnormalities determined progression in non-responding patients (Figure). Novel dose limiting toxicities were not observed. Three patients developed CTCAE grade III hepatic toxicities. One of them discontinued EPAG at 3 months. Elevated transaminases returned to baseline after EPAG discontinuation in 2 patients. In both cases EPAG was resumed either at the same (150mg/day) or reduced dose (50mg/day) level. There were no treatment-related death cases. One patient died on study before the primary endpoint from acute respiratory distress syndrome. Sequential acquisition of genomic aberrations has been associated with malignant transformation. Targeting next-generation sequencing for somatic variants in genes previously associated with myeloid malignancies (Myeloid cancer genes, MCG) was performed in 29/30 patients with sufficient material (bone marrow mononuclear cells) available from baseline, primary endpoint, and at time of progression. At baseline, 22/29 (76%) patients were found with at least one mutation:TET2 (14.5%), ASXL1 (12.5%), SF3B1 (8.3%), SETBP1 (8.3%), ATM (8.3%), and ZRSR2 (8.3%). After EPAG, additional somatic variants in different genes were detected in 4/14 responders and 7/16 non-responders. Variants present at baseline were no longer detected in post EPAG samples from 4 responding and 6 non-responding patients. The VAF of variants detected at both time points were similar, indicating no selective expansion of clones with EPAG in neither responder, non-responder nor patients with progression based on IPSS. In conclusion, our results suggest that EPAG is well-tolerated and effective in restoring hematopoiesis in patients with low to intermediate-2 risk MDS, particular with a prior history of hypoplastic bone marrow failure syndromes. EPAG was discontinued for robust response in the majority of responders but declining blood cell counts were observed in about 50% of them. Variants in MCG were more common at study entry compared to patients with aplastic anemia (Yoshizato, NEJM, 2015). However, EPAG appears not to selectively promote expansion of clones harboring MCGs in this patient population. Disclosures Townsley: National Institute of Health: Research Funding. Scheinberg:Pfizer: Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Janssen: Honoraria, Research Funding. Dunbar:National Institute of Health: Research Funding. Young:GlaxoSmithKline: Research Funding; CRADA with Novartis: Research Funding; National Institute of Health: Research Funding. Winkler:National Institute of Health: Research Funding.

Eltrombopag (EPAG) received FDA approval for treatment of refractory severe aplastic anemia (rSAA) in 2014, based on our phase I/II dose escalation trial of single agent EPAG for patients failing one or more treatment cycles with ATG/cyclosporine (Olnes NEJM 2012; Desmond Blood 2014). There is no standard treatment for patients with moderate aplastic anemia (MAA) or hypo-productive uni-lineage cytopenias (MAA/UC), conditions that can also impact on morbidity, mortality and quality of life. To explore the safety and effectiveness of EPAG in MAA/UC, we conducted a phase II study of EPAG given at escalating doses from 50-300mg/day (25-150mg/day for East Asians) through a primary hematologic response endpoint at 16-20 weeks (NCT 01328587). 34 patients enrolled between February 2012 and March 2017. 27 had never been treated with ATG/CSA IST. Responding patients could continue EPAG treatment on an extension arm. The drug was well-tolerated in 33/34 patients, with 1 patient coming off study at 10 weeks for nausea and vomiting. 25 patients reached the maximal dose. The median duration of follow-up in all patients was 16 months, and 27 months in responding patients. 17 of 34 (50%) of patients met criteria for response at the primary endpoint in at least one initially protocol-qualifying lineage (Hb 1.5 gr/dL increase in Hb or > 50% reduction in transfusions), including a patient with RSP19-mutated Diamond-Blackfan anemia. 7/24 with severe thrombocytopenia had a platelet response (>20,000/ul or transfusion-independence). 2/13 with both severe anemia and thrombocytopenia had bi-lineage responses at the primary endpoint, and an additional 5 went on to bi-lineage responses during the extension period. 3 patients without a response to EPAG were later treated with ATG/CSA and responded. EPAG was discontinued in 11/17 (65%) responding patients upon achievement of robust blood counts (Hb > 10mg/dL, platelets > 50,000/uL, and ANC > 1000) or stable blood counts for 6 months in 1 patient (6%) after a median duration of drug administration of 8 months (2-14 months) (Fig 1). In contrast to our prior experience in rSAA, the majority of patients still being followed on study after drug discontinuation (8/10) needed to have EPAG re-initiated for declining counts at a median of 6 months (2-38) later. All 8 responded again, with 4/8 able to discontinue EPAG after a second robust or stable response. 2/34 patients (6%) developed marrow cytogenetic abnormalities while on drug in contrast to 16/83 (16%) rSAA patients treated with EPAG in our prior studies. Both MAA patients were responders and neither had dysplastic changes or increased blasts. Patient # 5 had +8 in 7/20 metaphases at 22m, went off drug and relapsed, and EPAG was restarted off protocol. Repeat cytogenetics on EPAG were normal. Patient #20 had del13q in 5/20 metaphases at 9.5m, EPAG was stopped, and repeat cytogenetics off drug were normal. Of note, patient #12 had a robust response and had been off drug for 25m when counts declined and BM revealed dysplastic changes with no increase in blasts and normal cytogenetics. The acquisition and selection of somatic mutations, particularly in myeloid candidate genes (MCG) recurrently mutated in MDS/AMLhas been proposed to be an initiating step in clonal evolution. We performed targeted next generation exome sequencing (NGS) of 66 MCG and additional genes previously found to be somatically-mutated in SAA (Yoshizato, NEJM, 2015) pre-EPAG treatment and at the primary response endpoint of 16-20 weeks. Only 5 patients showed somatic mutations in these genes at baseline (SETBP1, CBL, SF3B1, PPMID, EP300). There were no significant changes in VAF. 2 mutations became detectable on EPAG (BCOR, and DNMT3A transiently), and 1 disappeared (SF3B1). In summary, administration of EPAG at escalating doses up to of 300mg/day was well-tolerated and 50% of patients with MAA/UC had clinically-meaningful responses, including those not previously treated with IST. The responses were durable, often robust, although frequently required ongoing EPAG treatment, in contrast to the experience in rSAA. Clonal cytogenetic evolution was rare, with no instances of chromosome 7 abnormalities, and there was no consistent expansion of MCG somatically-mutated clone size in patients during EPAG treatment. Figure. Figure. Disclosures Winkler: National Institute of Health: Research Funding. Young:CRADA with Novartis: Research Funding; GlaxoSmithKline: Research Funding; National Institute of Health: Research Funding. Dunbar:National Institute of Health: Research Funding.

Inherited and acquired bone marrow failure syndromes (BMF) may be difficult to distinguish due to heterogeneity and overlap of clinical phenotypes. Genomic screening has been increasingly used to identify mutations in BMF-related genes that are known to be etiologic in inherited BMF. However, genomic testing is expensive, results may not return for several seeks, and findings can be difficult to interpret as some reported variants are of unclear clinical significance. To guide the decision-making for genetic testing and results interpretation, we aimed to identify clinical and molecular parameters associated with a higher probability of patients having an inherited disease. We screened 323 BMF patients from two independent cohorts for germline mutations in BMF-related genes using a targeted next-generation sequencing (NGS) assay, and correlated the results with patients' prior diagnosis, family history, telomere length (TL), karyotype, and the presence of a paroxysmal nocturnal hemoglobinuria (PNH) clones. Patients were followed at the Hematology Branch of NHLBI (NHLBI, n=179) and the Ribeirão Preto Medical School, University of São Paulo (USP, n=144). Diagnoses included were severe (SAA) and moderate aplastic anemia (MAA), isolated cytopenias, myelodysplastic syndrome (MDS), hypocellular MDS (HypoMDS), dyskeratosis congenita (DC), and Diamond-Blackfan anemia (DBA). Patients were classified as suspected to have inherited BMF (phenotype suggestive for constitutional disease, short or very short telomeres, family history of hematologic, pulmonary, or liver disease, and idiopathic cytopenias), or acquired BMF (normal TL and no signs of constitutional disease) (Figure 1A). Pathogenicity of novel and rare variants was assessed using the ACMG criteria. We identified a pathogenic (or likely pathogenic) germline variant in 21 (18%) and 44 (47%) inherited BMF patients from NHLBI and USP cohorts, respectively (Figure 1B). Altogether, mutated genes were associated with telomeropathies (mostly DC and MAA), congenital cytopenias, DBA, cryptic Fanconi anemia, and myeloid malignancies (Figure 1C). In both cohorts, inherited BMF patients with DC, DBA, MAA, and isolated cytopenias were more likely to have a pathogenic variant. BMF patients suspected to have an acquired disease were rarely found with a pathogenic variant; one patient from each cohort (NHLBI, 1.5% and USP, 2%), carried the R166A RUNX1 and A202T TERT variants, respectively. Overall, patients with SAA were highly unlikely to have a pathogenic variant, regardless of the clinical suspicion for constitutional disease (Figure 1B). The presence of PNH clone and chromosomal abnormalities were poorly associated with variants' pathogenicity; only one patient from the USP cohort had a PNH clone of 6% and the pathogenic TERT D718E variant, and three patients had an abnormal karyotype (indicated by asterisks in Figure 3C). In both cohorts, we additionally screened 101 acquired BMF and 140 inherited BMF patients for somatic clones in myeloid-driver genes. These results recapitulated the clonal landscape previously observed in AA by our group; the frequency of variants in ASXL1, DNMT3A, TET2, and JAK2, but not in BCOR and BCORL1, increased with aging. In the current study, TP53, RUNX1, and Ras genes were more frequently mutated in the patients suspected to have inherited BMF (Fisher's exact test, 14% vs. 4.4%; p In summary, inherited BMF patients were more likely to have a pathogenic variant compared to acquired BMF (18% vs. 1.5%, p Figure. Figure. Disclosures Dunbar: National Institute of Health: Research Funding. Young:CRADA with Novartis: Research Funding; GlaxoSmithKline: Research Funding; National Institute of Health: Research Funding.

International audience; Background - Genetic defects in telomere maintenance and repair cause bone marrow failure, liver cirrhosis, and pulmonary fibrosis, and they increase susceptibility to cancer. Historically, androgens have been useful as treatment for marrow failure syndromes. In tissue culture and animal models, sex hormones regulate expression of the telomerase gene. Methods - In a phase 1-2 prospective study involving patients with telomere diseases, we administered the synthetic sex hormone danazol orally at a dose of 800 mg per day for a total of 24 months. The goal of treatment was the attenuation of accelerated telomere attrition, and the primary efficacy end point was a 20% reduction in the annual rate of telomere attrition measured at 24 months. The occurrence of toxic effects of treatment was the primary safety end point. Hematologic response to treatment at various time points was the secondary efficacy end point. Results - After 27 patients were enrolled, the study was halted early, because telomere attrition was reduced in all 12 patients who could be evaluated for the primary end point; in the intention-to-treat analysis, 12 of 27 patients (44%; 95% confidence interval [CI], 26 to 64) met the primary efficacy end point. Unexpectedly, almost all the patients (11 of 12, 92%) had a gain in telomere length at 24 months as compared with baseline (mean increase, 386 bp [95% CI, 178 to 593]); in exploratory analyses, similar increases were observed at 6 months (16 of 21 patients; mean increase, 175 bp [95% CI, 79 to 271]) and 12 months (16 of 18 patients; mean increase, 360 bp [95% CI, 209 to 512]). Hematologic responses occurred in 19 of 24 patients (79%) who could be evaluated at 3 months and in 10 of 12 patients (83%) who could be evaluated at 24 months. Known adverse effects of danazol--elevated liver-enzyme levels and muscle cramps--of grade 2 or less occurred in 41% and 33% of the patients, respectively. Conclusions - In our study, treatment with danazol led to telomere elongation in patients with telomere diseases. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT01441037.).

[BACKGROUND]In patients with acquired aplastic anemia, destruction of hematopoietic cells by the immune system leads to pancytopenia. Patients have a response to immunosuppressive therapy, but myelodysplastic syndromes and acute myeloid leukemia develop in about 15% of the patients, usually many months to years after the diagnosis of aplastic anemia. [METHODS]We performed next-generation sequencing and array-based karyotyping using 668 blood samples obtained from 439 patients with aplastic anemia. We analyzed serial samples obtained from 82 patients. [RESULTS]Somatic mutations in myeloid cancer candidate genes were present in one third of the patients, in a limited number of genes and at low initial variant allele frequency. Clonal hematopoiesis was detected in 47% of the patients, most frequently as acquired mutations. The prevalence of the mutations increased with age, and mutations had an age-related signature. DNMT3A-mutated and ASXL1-mutated clones tended to increase in size over time; the size of BCOR- andBCORL1-mutated and PIGA-mutated clones decreased or remained stable. Mutations in PIGA and BCOR and BCORL1 correlated with a better response to immunosuppressive therapy and longer and a higher rate of overall and progression-free survival; mutations in a subgroup of genes that included DNMT3A and ASXL1 were associated with worse outcomes. However, clonal dynamics were highly variable and might not necessarily have predicted the response to therapy and long-term survival among individual patients. [CONCLUSIONS]Clonal hematopoiesis was prevalent in aplastic anemia. Some mutations were related to clinical outcomes. A highly biased set of mutations is evidence of Darwinian selection in the failed bone marrow environment. The pattern of somatic clones in individual patients over time was variable and frequently unpredictable. (Funded by Grant-in-Aid for Scientific Research and others.), 再生不良性貧血における遺伝子変異の解明 -白血病発症にいたる過程を初めて解明-. 京都大学プレスリリース. 2015-07-09.

Germ-line GATA2 gene mutations, leading to haploinsufficiency, have been identified in patients with familial myelodysplastic syndrome/acute myeloid leukemia, monocytopenia and mycobacterial infections, Emberger syndrome, and dendritic cell, monocyte, B-, and NK-cell deficiency. GATA2 mutations have also been reported in a minority of patients with congenital neutropenia and aplastic anemia (AA). The bone marrow (BM) from patients with GATA2 deficiency is typically hypocellular, with varying degrees of dysplasia. Distinguishing GATA2 patients from those with AA is critical for selecting appropriate therapy. We compared the BM flow cytometric, morphologic, and cytogenetic features of 28 GATA2 patients with those of 32 patients being evaluated for idiopathic AA. The marrow of GATA2 patients had severely reduced monocytes, B cells, and NK cells; absent hematogones; and inverted CD4:CD8 ratios. Atypical megakaryocytes and abnormal cytogenetics were more common in GATA2 marrows. CD34(+) cells were comparably reduced in GATA2 and AA. Using these criteria, we prospectively identified 4 of 32 patients with suspected AA who had features suspicious for GATA2 mutations, later confirmed by DNA sequencing. Our results show that routine BM flow cytometry, morphology, and cytogenetics in patients who present with cytopenia(s) can identify patients for whom GATA2 sequencing is indicated.

Eltrombopag (EPAG) received FDA approval for treatment of refractory severe aplastic anemia (rSAA) in 2014, based on our phase I/II dose escalation trial of single agent EPAG at 50-150 mg daily over a period of 12 weeks (Olnes NEJM 2012; Desmond Blood 2014). Two observations warranted further investigation of EPAG in this unique patient population. First, cell count kinetics and lack of acute toxicities suggested that extended administration of EPAG at a fixed dose of 150mg could speed and improve response rates. Second, 19% of patients developed new cytogenetic abnormalities on EPAG, raising concerns that EPAG might promote progression to MDS/AML. We conducted a subsequent phase II study of EPAG given at a fixed daily dose of 150mg for 6 months in patients with rSAA (NCT01891994). Thirty-nine patients enrolled between July 2013 and April 2017. Primary endpoint was hematologic response at 6 months. Responding participants could continue EPAG treatment. Secondary endpoints included response at 3 months and the rate of clonal cytogenetic evolution. Nineteen of 39 (49%) patients met criteria for hematologic response at 6 months. Of these, 5/19 (26%) patients would have been deemed non-responders at 3 months of treatment. EPAG was continued in 18 patients on the extension arm. EPAG was discontinued for robust response in 13/18 (72%) after a median duration of drug administration of 12 months (6-27.5 months). EPAG was re-initiated for relapse in 3/13 patients, and all 3 recovered response. At median follow up of 6 months (range 2 - 39 m), 6/39 patients (15%) developed marrow cytogenetic abnormalities, a rate comparable to our previous cohort. Given the similar rates of response and clonal evolution in our two consecutive studies, we analyzed the relationship between outcomes and cytogenetic progression for all patients (n=83) at up to 8 years of follow-up. Sixteen of 83 (18%) patients clonally evolved (Table 1). Clonal evolution was an early event after EPAG initiation. Evolution occurred within 6 months in 13/16 evolvers (81%), and in 6/6 evolvers with high risk chromosome 7 abnormalities (5/6 within 3 months). The frequency of high risk clonal evolution 24 months post intervention is comparable to historic controls with rSAA. However, direct temporal comparisons of evolution events are limited by differences in the sequence of cytogenetic tests. Non-chromosome 7 cytogenetic abnormalities were often transient, and not associated with dysplasia. Two evolvers continued EPAG off the rSAA protocol and cytogenetics normalized (UPN 71,14). The acquisition and selection of somatic mutations, particularly of myeloid candidate genes recurrently mutated in MDS/AML, has been proposed to be an initiating step in clonal evolution. We performed whole exome sequencing (WES) on samples obtained pre-EPAG treatment and at the primary response endpoint and/or time of clonal evolution in 21 responding patients and in 11 patients with cytogenetic evolution. Candidate gene mutations were detected in patients who responded (6/21) and in those with cytogenetic evolution (4/11). Clonal hematopoiesis without an identifiable driver mutation was common. In post-EPAG samples, additional myeloid candidate gene somatic mutations were detected in 2 cytogenetic evolvers and in 4 responding patients, all at low variant allele frequencies (VAF) that were close to the 2.5% detection threshold. There was no significant change in VAF in either candidate or in non-candidate genes in responders and in cytogenetic evolvers. Only one early cytogenetic evolver (UPN 01) showed an expansion of mutated clones (SETBP1 and RUNX1), from VAF of 5% to 40%, when 80% of bone marrow metaphases showed chromosome 7q deletion. In summary, extended administration of EPAG at a fixed dose of 150 mg daily for 6 instead of 3 months induces additional responses in a subgroup of patients with rSAA. After EPAG was discontinued, most patients maintained durable robust responses. The temporal relationship between clonal evolution and drug exposure suggests that in a subgroup of patients, EPAG may promote expansion of dormant pre-existing clones with an aberrant karyotype. No clinical or laboratory findings prior to therapy, including WES, predicted response or risk of clonal evolution. Careful monitoring of refSAA patients treated with EPAG is indicated, particularly in the first 6 months of treatment. Table 1 Table 1. Disclosures Winkler: Novartis/GSK to institute: Research Funding. Cooper: Novartis/GSK to institute: Research Funding. Townsley: Novartis/GSK to institute: Research Funding. Grasmeder: Novartis/GSK to institute: Research Funding. Young: Novartis/GSK to institute: Research Funding. Dunbar: Novartis/GSK to institute: Research Funding.

First-line therapy of severe aplastic anemia (SAA) with high-dose cyclophosphamide causes toxicity and increased short-term mortality. We investigated cyclophosphamide at a lower, more moderate dose in combination with aggressive supportive care to determine whether severe infections might be avoided and hematologic outcomes defined for this regimen. From 2010 to 2012, 22 patients received cyclophosphamide at 120 mg/kg plus cyclosporine and antibacterial, antiviral, and antifungal prophylaxis. Toxicity was considerable, mainly due to prolonged absolute neutropenia, which occurred regardless of pretherapy blood counts, and persisted an average of 2 months. Granulocyte transfusions for uncontrolled infection were required in 5 patients, confirmed fungal infections were documented in 6, and 9 patients died. Nine patients (41%) responded at 6 months. After a median follow-up of 2.2 years, relapse occurred in 2 patients, and cytogenetic abnormalities (including monosomy 7) were observed in 4 patients. Although cyclophosphamide has activity in SAA, its toxicity is not justified when far less dangerous alternatives are available. This trial was registered at www.clinicaltrials.gov as #NCT01193283.

A 24-year-old medical editor presented to her primary care physician with fevers, epigastric pain, nausea, and vomiting upon returning from a business trip in Brazil in 2001. Symptoms resolved, but a few weeks later, she developed episodic but persistent severe central epigastric pain, accompanied

Pregnancy induces a number of physiologic changes that affect the hematologic indices, either directly or indirectly. Recognizing and treating hematologic disorders that occur during pregnancy is difficult owing to the paucity of evidence available to guide consultants. This review discusses specifically the diagnosis and management of benign hematologic disorders occurring during pregnancy. Anemia secondary to iron deficiency is the most frequent hematologic complication and is easily treated with oral iron formulations; however, care must be taken not to miss other causes of anemia, such as sickle cell disease. Thrombocytopenia is also a common reason for consulting the hematologist, and distinguishing gestational thrombocytopenia from immune thrombocytopenia (ITP), preeclampsia, HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets), or thrombotic thrombocytopenic purpura (TTP) is essential since the treatment differs widely. Occasionally the management of mother and infant involves the expeditious recognition of neonatal alloimmune thrombocytopenia (NAIT), a condition that is responsible for severe life-threatening bleeding of the newborn. Additionally, inherited and acquired bleeding disorders affect pregnant women disproportionately and often require careful monitoring of coagulation parameters to prevent bleeding in the puerperium. Finally, venous thromboembolism (VTE) during pregnancy is still largely responsible for mortality during pregnancy, and the diagnosis, treatment options and guidelines for prevention of VTE during pregnancy are explored.

Aplastic anemia is a bone marrow failure syndrome that causes pancytopenia and can lead to life-threatening complications. Bone marrow transplantation remains the standard of care for younger patients and those with a good performance status but many patients may not have a suitable donor. Immunosuppressive therapy is able to resolve cytopenias in a majority of patients with aplastic anemia but relapses are not uncommon and some patients remain refractory to this approach. Patients may require frequent blood and platelet transfusion support which is expensive and inconvenient. Life-threatening bleeding complications still occur despite prophylactic platelet transfusion. Thrombopoietin (TPO) mimetics, such as romiplostim and eltrombopag, were developed to treat patients with refractory immune thrombocytopenia but are now being investigated for the treatment of bone marrow failure syndromes. TPO is the main regulator for platelet production and its receptor (c-Mpl) is present on megakaryocytes and hematopoietic stem cells. Trilineage hematopoietic responses were observed in a recent clinical trial using eltrombopag in patients with severe aplastic anemia refractory to immunosuppression suggesting that these agents can provide a new therapeutic option for enhancing blood production. In this review, we discuss these recent results and ongoing investigation of TPO mimetics for aplastic anemia and other bone marrow failure states like myelodysplastic syndromes. Clonal evolution or progression to acute myeloid leukemia remains a concern when using these drugs in bone marrow failure and patients should only be treated in the setting of a clinical trial.

The major complication of severe aplastic anemia is clonal evolution, defined as any new cytogenetic abnormality or progression to MDS/AML, which occurs in about 15% of SAA patients, usually many months to years after the diagnosis. Eltrombopag, a thrombopoietin receptor agonist, appears capable of stimulating hematopoietic stem cell proliferation in patients with bone marrow failure. Addition of eltrombopag to standard immunosuppressive treatment (IST) with horse antithymocyte globulin (hATG) and cyclosporine (CsA) markedly increases hematologic response rates in treatment-naive SAA, with overall response rates up to 90% and complete response rates approaching 60% (Townsley DM et al, ASH 2015, clinicaltrials.gov NCT01623167). In comparison, IST alone achieves 60% overall response rates, of which 10% are complete (Scheinberg Blood 2012). Somatic mutations in myeloid cancer candidate genes are present in one-third of patients after IST alone (Yoshizato T et al, NEJM 2015). Specific subsets of mutations were associated with clinical outcomes: a group including ASXL1 and DNMT3A with a poor response to IST, inferior survival, and clonal evolution, while BCOR and PIGA were associated with good response and favorable outcomes. Monosomy 7, the most prevalent cytogenetic abnormality defining clonal evolution, can develop in the absence of gene mutations, underscoring the non-determinative and complex role mutations play in clonal evolution (Dumitriu B et al, Blood 2015). Of patients with disease refractory to IST who were subsequently treated with single-agent eltrombopag, 19% (8/43) developed cytogenetic abnormalities, usually within the first year of treatment, but only rarely with morphologic changes consistent with MDS/AML (Desmond R et al, Blood 2014). The frequency of somatic mutations following treatment with eltrombopag added to IST in treatment-naïve SAA patients is unknown. We used amplicon-based next-generation sequencing to assess mutations in 54 candidate genes recurrently mutated in myeloid neoplasms. Bone marrow cells of 90 subjects who had been treated with IST/eltrombopag were obtained at 6 months following treatment initiation, or at the time of clonal evolution. At least one detectable mutation was identified in 21 (23%) subjects. All 21 patients had exhibited a hematologic response to treatment by 6 months; of those patients with somatic mutations, 14 of 19 (74%) had a complete hematologic response. In comparison, of the 69 patients who lacked mutations, 20 (29%) had a complete response. Nine different genes were mutated in total, with the most frequent genes being ASXL1 and BCOR. BCOR was associated with more robust responses (6 of 7 had a complete response) and younger age (range 12 - 49 years; Table). One subject with a longstanding history of JAK2-positive essential thrombocytosis and myelofibrosis at baseline had two additional mutations detectable following treatment, TET2 and ASXL1. With a median follow up of 21 months, clonal cytogenetic evolution occurred in 7/90 (8%) subjects. Three of seven patients also had a mutation in a myeloid cancer gene (two with DNMT3A and one with ASXL1/RUNX1); in the other four, no somatic mutation was detected, either at the 6-month time point or at time of cytogenetic evolution. Four patients had monosomy or partial deletion of chromosome 7: one patient had complex (t(3;3)(q21;q26), -7), one patient had deletion 13q that later disappeared, and one patient had trisomy 6 and trisomy 15 in 2 metaphases. The patient with complex cytogenetics did not have somatic mutations detected at evolution, and she later died due to relapsed AML following transplant. The rates of somatic mutations in myeloid cancer genes and of cytogenetic evolution in patients treated with IST/eltrombopag do not appear to be higher than we and others have reported in aplastic anemia treated with standard IST, without eltrombopag (Kulasekararaj AG et al, Blood 2014). The distribution of genes mutated and the allelic frequency of these mutations also were similar to patients treated with standard IST. These results suggest that the benefits of a higher response rate and quality of response associated with the addition of eltrombopag to IST for the initial treatment of SAA are not associated with a higher risk of clonal progression. Disclosures Townsley: GSK/Novartis: Research Funding. Cooper:GSK/Novartis: Research Funding. Winkler:GSK/Novartis: Research Funding. Scheinberg:Novartis: Consultancy, Speakers Bureau. Rios:GSK/Novartis: Research Funding. Weinstein:GSK/Novartis: Research Funding. Desierto:GSK/Novartis: Research Funding. Fernandez Ibanez:GSK/Novartis: Research Funding. Dunbar:GSK/Novartis: Research Funding. Ma:Neogenomics Laboratories: Employment. Albitar:Neogenomics Laboratories: Employment, Equity Ownership. Young:GSK/Novartis: Research Funding.

DBA is a rare bone marrow failure syndrome clinically defined by onset of hypoproliferative anemia in early childhood accompanied by a grossly normocellular bone marrow. With age, a significant fraction of patients progress to pancytopenia and a hypocellular bone marrow, with some increased risk of progression to leukemia. Autosomal dominant or sporadic heterozygous mutations in genes encoding for ribosomal subunit proteins have been etiologically linked to DBA. How loss of function mutations in ribosomal proteins results in selective loss of erythroid progenitors with variable severity is incompletely understood. Chronic transfusion therapy and corticosteroid treatment can alleviate anemia, but both cause severe long term toxicities. Hematopoietic stem cell transplantation, when available, is the only definitive treatment modality for the hematological manifestations, and the only known treatment for DBA patients once pancytopenia develops. Here we report on a 28 year old female patient diagnosed with DBA at age of one month based on clinical criteria. Recent mutational analysis identified a novel mutation in intron 4(c.356+3>C) of the RPS19 gene affecting a known splice donor site that has previously reported in DBA patients. She did not respond to corticosteroids and was maintained on transfusions. She failed various experimental therapies including danazol, hematopoietic growth factors including IL-3 and GM-CSF, and anti-thymocyte globulin/cyclosporine without sustained clinical response. After treatment with daclizumab in 2006 her transfusion requirements decreased for about 4 years. In 2012 she presented with progressive pancytopenia and hypocellular bone marrow without overt dysplasia, and normal cytogenetic analysis. She required red cell transfusions. The patient was enrolled in a clinical research protocol investigating the efficiency and safety of the thrombopoietin receptor agonist eltrombopag (EPAG) in patients with moderate aplastic anemia or those with bone marrow failure with unilineage cytopenia (ClinicalTrials.gov: NCT01328587). The study is designed as a non-randomized, phase II, dose modification study with the primary endpoint hematological response at 16 weeks. EPAG was administered at 50mg daily and escalated every 2 weeks by 25 mg to the maximal dose of 300mg daily. At response assessment the patient's hemoglobin (Hgb) improved from 7.0g/dl to 8.9g/dl and platelets from 112K/ul to 171k/ul. She required no further transfusions, and was deemed a responder. EPAG was subsequently continued at the same dose level for an additional 6 months when the peripheral cells counts peaked at Hgb 11.9g/dl, platelets 251K/ul, ANC 3.19 K/ul, and EPAG was discontinued for protocol-defined robust response. After discontinuation of EPAG the patient's hemoglobin steadily declined over a period of 8 months. EPAG was reinitiated at 300mg per protocol. Peripheral blood cell counts rapidly improved and the EPAG dose was slowly tapered. The patient is currently on EPAG 50mg daily with normal blood cell counts. We have not observed any EPAG related toxicities in our patient, and repeated cytogenetic studies on bone marrow documented a normal karyotype. To the best of our knowledge this is the first report documenting EPAG efficacy in a DBA patient. Trautman et al. (2012) previously reported a DBA patient that failed treatment with low dose EPAG after six months. Our observation warrants further systematic investigation of EPAG treatment for DBA patients within a clinical research study setting. Figure Figure. Disclosures Winkler: GSK/Novartis: Research Funding. Townsley:Novartis: Research Funding. Desmond:Novartis: Honoraria. Dumitriu:GSK/Novartis: Research Funding. Grasmeder:GSK/Novartis: Research Funding. Young:GSK/Novartis: Research Funding. Dunbar:GSK/Novartis: Research Funding.

Background:We have reported that peripheral blood cell-free DNA (cfDNA) is reliable for detecting bone marrow molecular abnormalities in patients with hematologic neoplasms. However, not clear is whether cfDNA is sufficient to detect mutations present at low variant allele frequency (VAF). Since patients with aplastic anemia (AA usually have relatively small clones in blood and bone marrow (BM), we compared mutations detected in BM cells with those detected in peripheral blood cfDNA from patientswith this disease. Methods: A total of 120 paired BM aspirate and PB plasma samples were tested by the commercially available TruSight Myeloid Sequencing Panel (Illumina; San Diego, CA). We extracted DNA from bone marrow aspirate using the QIAamp DNA Mini Kit. We used NucliSenS EasyMAG automated platform for extracting total nucleic acid from PB plasma collected in EDTA. All paired BM and plasma samples were tested by the commercially available TruSight Myeloid Sequencing Panel (Illumina; San Diego, CA), which covers hot spot mutations in 54 genes. The average depth of sequencing was 10,000X. Results: One hundred twenty paired BM and cfDNA samples from 96 patients with aplastic anemia were tested. Of the 96 patients, 33 (34%; equivalent to 48 samples, 40%) had one or more mutations. We identified 54 different somatic mutations in these patients, of which 45 were unique. There was no significant difference (P=0.71, Sign test) in allele frequency between cfDNA and BM. The median mutant allele frequency was 10.9% in cfDNA and 12.6% in BM cells, and 40 of the 54 mutations had allele frequency ≤20% in BM cells, while 45 samples had allele frequency ≤20 in cfDNA. Six of the 33 patients with somatic mutations (18%) showed mutations in plasma cfDNA but not in BM. In contrast, 2 patients (6%) showed mutations in BM cells and not in cfDNA. One of these two patients had a mutation in ASXL1 gene detected in BM cells but not in cfDNA and a subsequent sample showed the same ASXL1mutation in BM cells and not in cfDNA, and a second clone with a different ASXL1 mutation detected in both BM cells and cfDNA. Overall concordance between BM cells and cfDNA in the 120 samples was 92% and there was no statistically significant difference between the two sample types (P=0.6). Summary and Conclusions: Seven samples (from 7 patients) of the 120 tested samples showed mutations in cfDNA and not in BM cells while 3 samples (from 2 patients) showed mutations in BM and not in cfDNA. VAF of mutations in cfDNA were similar to those in BM cells. Therefore, peripheral blood cfDNA should be tested in addition to BM cells for detecting mutations in patients with AA. Peripheral blood cfDNA can be used as a reliable means for monitoring patients with AA. cfDNA testing can be used as an alternative testing to bone marrow even when mutant allele frequency in bone marrow is Disclosures Albitar: Neogenomics Laboratories: Employment. Townsley:Novartis: Research Funding. Ma:Neogenomics Laboratories: Employment. De Dios:Neogenomics Laboratories: Employment. Funari:Neogenomics Laboratories: Employment. Young:GSK/Novartis: Research Funding. Albitar:Neogenomics Laboratories: Employment, Equity Ownership.

Background. Aplastic anemia (AA), the prototypical bone marrow (BM) failure syndrome, is caused by immune-mediated destruction of hematopoietic stem/progenitor cells (HSPCs). CD8+ cytotoxic T cells with restricted TCR diversity (oligoclonal T cells) are expanded in AA, leading to production of proinflammatory cytokines, such as IFN-γ, which induce apoptosis of HSPCs. Recent studies have identified a new subset of memory T cells with stem cell-like properties, TSCM, which are the least differentiated cells of all distinct memory populations. Functionally, TSCM possess an enhanced capacity for self-renewal and can generate multiple memory T cell populations, and they likely have an important role in controlling immunity. In autoimmune diseases, there is abnormal CD4+ and CD8+ T cell activation. We evaluated TSCM frequency in AA and its association with severity, treatment response, relapse, and changes after immunosuppressive therapy (IST). Further, to evaluate the TSCM in other autoimmune diseases, we examined CD4+ and CD8+ TSCM frequencies in uveitis, systemic lupus erythematosus (SLE), and sickle cell disease (SCD), as compared with healthy controls. Method. We retrospectively analyzed CD4+ and CD8+ TSCM populations by flow cytometry. PB specimens were collected from 55 AA samples and 41 age-matched healthy donor samples. Among 55 AA samples, 21 samples were analyzed at diagnosis and 34 after IST. For comparison, blood samples were obtained from 34 uveitis patients (27 inactive or 7 active cases), 43 SLE patients who met the American College of rheumatology (ACR) criteria for the disease [19 inactive SLE (SLE disease activity index-2K (SLEDAI-2K) score < 3; and 24 active SLE (SLEDAI-2K score > 3)], and 5 SCD patients who were receiving frequent transfusions. TSCM was defined as CD3+ CD4 (CD8)+ CD45RO- CD45RA+ CCR7+ CD27+ CD95+ population. Results and Discussion. In healthy controls, TSCM represented a relatively small percentage of circulating CD4+ or CD8+ T cells (median 2.4% CD4+ TSCM and 2.1% CD8+ TSCM, Fig. 1A). A significantly higher CD8+ TSCM frequency was detected in AA patients (4.2% vs. 2.1%, p < 0.05) while there was no difference in the CD4+ TSCM frequency (p > 0.05), compared to controls (Fig. 1B-C). In AA, high CD8+ TSCM frequency at diagnosis correlated with complete (CR) or partial response (PR) to IST [5.0 % in CR and PR vs 2.8 % in non-responders (NR), p < 0.05). In AA patients prior to IST (n=21), CD8+ TSCM frequency was not correlated with age, sex, absolute neutrophil count, platelet count, time from diagnosis to therapy, and serum ferritin levels. CD8+ TSCM were significantly increased in the two AA cohorts (with or without IST), relative to controls (p < 0.05, respectively). Higher CD8+ TSCM frequency after IST associated with treatment-failure (3.5 % in responders vs 5.5 % in NR or relapse, p < 0.05). Stimulation with anti-CD3/CD28 beads successfully induced cytokine production in CD4+ and CD8+ T cells from AA and healthy controls. Elevated IFN- γ and IL-2 levels were seen in CD4+ and CD8+ TSCM in AA compared to healthy controls. We next compared CD4+ or CD8+ TSCM frequency between each patient group (AA, uveitis, SLE, or SCD) and a healthy control group. Among the four patient groups, the uveitis group alone displayed a reduction in CD4+ TSCM frequency (1.8%) relative to the healthy controls (2.4 %; p < 0.05). An elevated CD8+ TSCM frequency was observed in AA (4.2 %), uveitis (3.6 %), and SCD (4.3 %), but not in SLE, compared to controls (2.1%; p < 0.05) (Fig. 2A). Positive correlation between CD4+ and CD8+ TSCM frequencies was found in AA, autoimmune uveitis, and SLE (Fig. 2B). Evaluation of PD-1 expression revealed that TSCM were the least exhausted T cell compartment, as compared to other types of memory T cells. Immune therapies appeared to have negative effects on the TSCM population both in uveitis and SLE patient cohorts, as well as in AA. Conclusion. We provide evidence for increased circulating CD8+ TSCM in AA, underscoring the importance of this novel subset in regulation of immune responses and pathogenesis of autoimmunity. Our work described previously unknown potential roles of TSCM in AA, such as cytokine secretion correlated with effector functions. Understanding the CD8+ TSCM population may offer new therapeutic strategies and novel mechanistic insight into the various autoimmune diseases. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Townsley: Novartis: Research Funding; GSK: Research Funding. Dumitriu:Novartis: Research Funding; GSK: Research Funding. Young:Novartis: Research Funding; GSK: Research Funding.

In bone marrow failure, levels of circulating hematopoietic growth factors (HGFs) are extremely elevated, in compensation for low blood counts. In general, administration of recombinant HGFs is not effective in aplastic anemia (AA) and related syndromes. We have established the utility of eltrombopag, a thrombopoietin (Tpo) mimetic, in severe AA (SAA) refractory to immunosuppression (IST) (Olnes, N Engl J Med, 2012; Desmond, Blood, 2014). Eltrombopag in combination with standard IST also appears to increase the rate and speed of recovery in treatment-naive SAA (Townsley, EHA. 2015). These clinical results are surprising, as plasma Tpo is markedly increased in SAA (Feng, Haematologica, 2011). In investigating possible mechanisms of action of eltrombopag, we studied HGF dynamics over time in SAA patients undergoing various treatment regimens and manifesting a range of hematologic responses. In a cohort of 37 treatment-naive SAA patients who were treated with either standard IST (horse antithymocyte globulin/cyclosporine A:ATG/CsA, n = 10) alone or with the addition of eltrombopag (n = 27), among whom 8 were non-responders and 29 responders at 6 months, we measured plasma Tpo, granulocyte-colony stimulating factor (G-CSF) and erythropoietin (Epo) using magnetic multiplex assays. Concentrations of these HGFs were greatly elevated before treatment. In a majority of patients, G-CSF declined rapidly to the normal range by 6 months, with no difference between responders and non-responders, and no correlation between G-CSF and absolute neutrophil counts. Epo concentrations decreased to the normal range at 6, 12, and 24 months post treatment in responders, to a significantly greater degree than in non-responders (p = 0.001, 0.0012, 0.038, respectively). There was a negative correlation between Epo and hemoglobin (r2 = 0.3126, p Disclosures Townsley: Novartis: Research Funding; GSK: Research Funding. Winkler:Novartis: Research Funding; GSK: Research Funding. Dumitriu:Novartis: Research Funding; GSK: Research Funding. Young:Novartis: Research Funding; GSK: Research Funding.

Immunosuppressive treatment (IST) with horse antithymocyte globulin (hATG) and cyclosporine (CsA) for severe aplastic anemia (SAA) has a 60-65% rate of overall hematologic response (OR) and approximately 10% complete response (CR). These rates have remained stable over the last three decades despite efforts to improve upon this regimen (Scheinberg Blood 2012). Eltrombopag (EPAG), an oral thrombopoietin receptor agonist has activity in refractory SAA producing hematologic responses, the majority multilineage, in about 40% of patients when administered as a single agent (Olnes NEJM 2012; Desmond Blood 2014). We conducted an investigator-initiated, phase II, single-center trial to test the efficacy of adding EPAG to h-ATG/CsA (clinicaltrials.gov, NCT01623167), using a triple cohort design to optimize the regimen. Eighty-eight patients with treatment-naive SAA have been enrolled from July 2012 to October 2015. All received standard h-ATG and CsA. EPAG was administered at 150 mg daily to three consecutive cohorts: in the first (n=30) and second (n=31) cohorts, EPAG was introduced after hATG, from day 14 until 6 months (cohort 1) or 3 months (cohort 2), due to initial concern of hepatotoxicity. In the third cohort, EPAG was concurrent with hATG on day 1 and continued for 6 months (n=27). As analysis of our previous IST trials suggested that CR correlates with better survival and less evolution to MDS/AML, the primary endpoint in the current protocol was CR at 6 months. Baseline enrollment characteristics were similar across cohorts: median age was 31 years (range 3 – 82; 20% were 1%. EPAG was well tolerated when combined with CsA and hATG; 2 patients discontinued EPAG for severe cutaneous reactions, and 6 patients withdrew before 6 months due to refractoriness (n=4) or evolution to MDS (n=2). OR and CR were higher at 3 months and 6 months for all cohorts compared to historic rates (Table): OR at 6 months was 80%, 87% and 92%, respectively for the three cohorts; CR at 6 months was 33%, 26%, and 54%, respectively. For all patients evaluable to date, OR at 3 and 6 months of 80% and 85% and CR rates of 28 and 34% were higher when compared to our historical rates (p Improvements in blood counts among partial responders were robust by 3 months in all cohorts: median ANC 1790/ul; hemoglobin 10 gm/dL; and platelets 60,500/ul. For patients with very severe neutropenia (ANC500/uL was 47 days for all cohorts (n=23), and 35 days for cohort 3 (n=7). Among responders, median time to transfusion-independence for platelets was 32 days and for red cells 42 days. Serial BM biopsies showed improved cellularity in 80% without increased fibrosis. Median increase from baseline in BM CD34+ cells, as measured by flow cytometry, was 17-fold and 4-fold at 3 months for cohorts 1 and 2 respectively (p At median follow up 15 months (range 1 – 42 m), cytogenetic abnormalities have occurred in 7 patients: 4 monosomy or partial deletion of chromosome 7, 1 complex (t(3;3)(q21;q26), -7), 1 deletion 13q that later normalized, and 1 subject with trisomy 6 and trisomy 15 in 2 metaphases. We sequenced 54 candidate genes, recurrently mutated in MDS/AML: somatic mutations were not identified in 4 of 7 subjects prior to treatment or at the time of cytogenetic evolution. Clonal evolution to MDS occurred at a similar frequency compared to our historic experience with standard IST. Addition of EPAG to IST markedly increases overall and complete hematologic response rates in treatment-naive SAA. These results suggest that immediate institution of EPAG with IST may salvage and expand residual hematopoietic stem cells in aplastic anemia, accelerating the rate and quality of hematopoietic recovery. Rapid blood count improvement and reduced transfusion burden support early use of EPAG for patients with newly diagnosed severe aplastic anemia. Disclosures: Townsley: Novartis: Research Funding ; GSK: Research Funding. Off Label Use: Eltrombopag (Promacta) is not FDA approved for treatment naive aplastic anemia. Dumitriu:Novartis: Research Funding ; GSK: Research Funding. Winkler:GSK: Research Funding ; Novartis: Research Funding. Larochelle:Novartis: Research Funding ; GSK: Research Funding. Dunbar:Novartis: Research Funding ; GSK: Research Funding. Young:Novartis: Research Funding ; GSK: Research Funding.

Telomeres, the terminal complex of hexameric repeats and shelterin protein of linear chromosomes, shorten with every mitosis. Telomere attrition is accelerated in patients with mutations in telomerase complex genes (Calado and Young, NEJM 2009) and with replicative stress, as in chronic bone marrow failure. Historically, male hormones were effective in some patients with aplastic anemia (AA), and case reports and retrospective observations have suggested hematologic improvement in patients with telomeropathies treated with male hormones. Exposure of normal lymphocytes and CD34+ cells to androgens increased telomerase activity in vitro, and in cells from individuals carrying loss-of-function TERT mutations to normal levels (Calado et al. Blood 2009). We have conducted a phase I/II single-center trial (www.clinicaltrials.gov NCT01441037) assessing the safety and the effect of male hormones on telomere attrition in patients with telomere disease. Entry criteria included age-adjusted mean telomere content ≤1%ile, ± identified mutations in telomerase complex genes, and low blood counts (hemoglobin 1.5 g/dL or platelets >20,000/uL or neutrophils >500/uL). Twenty seven patients were enrolled, accrual commencing August 2011. Most patients had moderate (n=20) or severe (n=4) AA, one had myelodysplasia, and two pulmonary fibrosis. Median age was 41 years (range 17-66); 15 patients were females. There was only one severe adverse event possibly related to drug. Frequent reported symptoms were muscle cramping with dehydration and exacerbation of headaches. Changes in serum lipid profiles were observed in all patients, with increased serum LDL and decreased HDL. Severe elevation in liver enzymes was not observed. One death occurred on study, not treatment related (pneumonia in a pulmonary fibrosis case). Mean telomere content of leukocytes at enrollment was compared with mean telomere content at 6, 12, and 24 months on drug as well as available samples before starting danazol. Telomere attrition prior to protocol entry, determined by q-pcr, was estimated at loss of 227 bp/year (95% CI, 58-368bp; p=0.009). Androgen administration appeared to elongate telomeres: the average increase in telomere length at 6 months was 205 bp (95% CI, 82-329 bp; p=0.002), at 12 months 441 bp (95% CI, 263-620 bp; p=0.0001), and at 24 months 347 bp (95% CI, 87-607 bp; p=0.01). A similar trend of increase in mean telomere content with danazol was confirmed in flow-sorted lymphocytes. Hematologic response rate, as defined by protocol, was 67% at 3 months and 60% at 6 months. Nine of eleven patients who required RBCs became transfusion-independent; two of them also became platelet transfusion independent. Liver cirrhosis was present in 6 patients at enrollment; worsening of liver disease in one occurred with continued alcohol abuse. To date 8 patients have completed two years of danazol, all of them responders; 10 patients remain on danazol, and 9 patients stopped drug prior to 2 years. Blood counts in all patients have been stable with drug discontinuation, with median follow up of 258 days (range 31-335). In conclusion, male hormones are safe and efficacious in patients with inherited bone marrow failure associated with telomeropathies, producing clinically meaningful hematologic improvement. Increased mean telomere content in patients, suggests that in vitro demonstration of up-regulation by sex hormones of TERT and of telomerase enzymatic activity is the mechanism of action of androgens in vivo. To our knowledge, this is the first successful prospective effort to favorably modulate telomere length by pharmacologic intervention in humans. Sex hormones may be useful in other conditions of accelerated telomere attrition, as for example after chemotherapy, and other drugs and small molecules may be usefully screened for their effects on telomerase in vitro. Disclosures Off Label Use: we want to determine if treatment with male hormone danazol is safe and improves hematologic response as first-line treatment in patients with AA and telomere disease(www.clinicaltrials.gov NCT01441037)..

Introduction: The risk of graft-rejection after allogeneic hematopoietic cell transplantation using bone marrow and conventional cyclophosphamide-based conditioning is increased in HLA allo-immunized and heavily transfused patients with bone marrow failure syndromes. Recently, we showed that fludarabine-based peripheral blood stem cells transplantation (PBSCT) overcomes the risk of graft-failure in patients with SAA who have failed immunosuppressive therapy (IST). However, this approach was complicated by a high incidence of acute and chronic GVHD. Multivariate analysis showed rapid donor T-cell engraftment (defined as >= 95% donor T-cell chimerism by post-transplant day 30) significantly increased the risk of cGVHD. Based on these data, we developed a novel transplant approach in which a G-CSF mobilized PBSC allograft that was T-cell depleted and CD34+ selected was co-infused with a bone marrow transplant (BMT)-equivalent dose of non-mobilized donor T-cells to facilitate donor engraftment and reduce GVHD by delaying the speed of donor T-cell engraftment. Method: Patients with transfusion-dependent SAA, refractory to conventional IST, underwent allogeneic PBSCT following cyclophosphamide (60 mg/kg/d IV x 2 days), equine antithymocyte globulin (hATG; 40 mg/kg/d IV x 4 days), and fludarabine (25 mg/m2/d IV x 5 days) conditioning. On day 0, patients received a G-CSF mobilized PBSC allograft from an HLA identical sibling, containing CD34+ selected cells (MiltenyiCliniMACS system: target CD34+ cell dose 8 x 106 cells/kg and target T-cell dose < 3 x 105 cells/kg) combined with 2 x 107CD3+ T-cells/kg that had been collected by apheresis and cryopreserved from the same donor prior to G-CSF mobilization. CSA and mini-dose MTX (5 mg/m2 IV on days 1, 3, 6) were used as GVHD prophylaxis. Transplant outcomes were compared to our historical cohort of patients (n=56) with SAA and other bone marrow failure syndromes who received a T-cell replete PBSC from an HLA matched donor following the identical conditioning and GVHD prophylaxis regimens. Results: 11 patients with SAA were transplanted. Patients were heavily transfused and highly allo-immunized; the pre-transplant serum ferritin level was markedly elevated at a median 3003 µg/L (range 286 to 13928 µg/L) and 7 patients (64%) were HLA allo-immunized with a median 21% (HLA class I) and 31% (HLA class II) panel-reactive antibodies. All 11 patients (100%) engrafted. The median time to neutrophil and platelet recovery was 14 (range 12 to 23) and 18 (range 14 to 321) days respectively. All patients achieved full and sustained donor T-cell chimerism and myeloid chimerism, which occurred at a median 45 and 15 days post-transplant, respectively. Among those at risk, 6/7 (86%) developed CMV reactivation. EBV reactivation occurred in all cases, including 5 who received preemptive treatment with rituximab. At a median follow-up of 2 years, only 1 patient (9%) has developed acute and another (9%) developed cGVHD (limited, skin). Neither corticosteroid-refractory aGVHD nor extensive cGVHD occurred. Long-term survival was excellent; 10 of 11 patients (91%) survived to day 200; at a median follow-up of 2.7 years, 9/11 (80%) survive. One patient died on day 46 from Klebsiella Pneumoniae carbapenemase bacteremia, which predated the transplant, and another died 18 months post-transplant from bacterial pneumonia. Compared to our historical cohort of marrow failure patients who received a T-cell replete PBSC allograft, patients receiving CD34+ selected cells combined with non-mobilized T-cells (partially T cell depleted PBSC) had similar survival (80% vs 87%;p=0.5), a delay in the time to achieving full donor T-cell chimerism (45 days vs 30 days; p=0.046) and dramatic reductions in both acute grade II-IV GVHD (9% vs 52%; p=0.017) and cGVHD (1 year incidence 9% vs 63%; p=0.002). Conclusion: In SAA, transplantation of a PBSC allograft containing high numbers of CD34+ selected cells co-infused with a BMT-equivalent dose of non-mobilized T-cells results in excellent engraftment and reduces acute and chronic GVHD by delaying the speed of donor T-cell engraftment. Figure: Figure:. Disclosures Townsley: GSK: Research Funding.

Donor-cell derived leukemia (DCL) is a rare but serious complication of hematopoietic stem cell transplantation (HSCT), reported in 5% of all relapses following allogeneic HSCT. Only 12 cases of DCL after umbilical cord blood (UCB) HSCT are reported in the literature. Multiple mechanisms have been proposed for DCL, including occult donor leukemia, impaired immunity, drug toxicity, or a bone marrow “leukemogenic milieu”. We propose a novel mechanism of leukemogenesis, mediated by very short telomeres of donor cells with subsequent severe telomere attrition in vivo, genomic instability, and progression to complex cytogenetics acute myeloid leukemia (AML), based on study of a patient who underwent UCB HSCT for myelodysplasia (MDS). The patient was a 44-year-old woman, subsequently shown to have a T354M mutation in GATA2, who presented at age 19 years with multiple infections and MDS. At age 41, due to progression of MDS to AML, she received induction chemotherapy and underwent a single 4/6 HLA-matched UCB HSCT. She had delayed engraftment. achieving an absolute neutrophil count of 500/ul more than 100 days post-transplant. Chimerism studies demonstrated 100% donor cell at all time-points post-transplant. Two years and eight months after HSCT she presented with pancytopenia, circulating myeloblasts, and 50% myeloblasts in bone marrow, indicating recurrence of with AML. However, cytogenetics revealed complex abnormalities, t(2p;3q) and an interstitial deletion of 5q, male cells, indicating donor origin of AML. Telomere length of the transplanted cord blood cells measured by Q-PCR showed a severe decrease in length to 7.7 kb compared with an average length of 13 kb in control UCB (n=12). Moreover the telomere length decreased precipitously to 6.6 kb 2 years after transplantation and 5.6 kb at the time of diagnosis of AML. Single telomere length assay (STELA) was used to assess chromosome-specific telomere length. Very short telomeres ( Telomere attrition occurs in the first year after HSCT, presumably due to proliferative stress on the stem cell compartment. In comparison to bone marrow, UCB have longer telomeres, but HSC number may be lower than in a BM inoculum, especially for adult recipients. In our case, initial telomere length of UCB was extremely short, for unclear reasons but possibly related to low cord blood cell viability. The long time that was needed for engraftment generated additional telomere attrition with accumulation of very short telomere and genomic instability leading to development of leukemia. Screening UCB for telomere length prior to HSCT might prove useful in identifying situations at risk for graft failure or malignant transformation. Disclosures: No relevant conflicts of interest to declare.

Abstract 3488 Germline heterozygous mutations in GATA2 have been reported to cause familial myelodysplasia–acute myeloid leukemia (MDS/AML), monocytopenia and mycobacterial infection (monoMAC syndrome), dendritic cell, myeloid and NK cell lymphopenia (DCML), and Emberger syndrome (lymphedema and MDS). GATA2 is a zinc finger transcription factor that plays a crucial role in regulating growth of hematopoietic progenitors. In some pedigrees, patients or family members have manifestations of bone marrow failure. We hypothesized that patients with aplastic anemia (AA) may harbor mutations in GATA2. The coding regions and regulatory regions of GATA2 were sequenced in 99 patients with confirmed AA. Sequences from 100 normal individuals as well as published human genomes from unaffected individuals (dbSNP build 135 and 1000 Genomes Project) were used as controls. Genetic variants were confirmed in hematopoietic and somatic tissues. We identified 4 heterozygous mutations in regulatory regions of GATA2 in 5 patients. In two patients, a mutation at nucleotide 59T>G in exon 1 of isoform 2 was identified; both had severe AA in early adulthood refractory to immunosuppressive therapy. We noted this 59T>G mutation in two unrelated individuals with severe disseminated mycobacterial disease. We identified a mutation at nucleotide 20G>A in exon 2 of isoform 1, in a 3 year-old male with hepatitis-associated severe AA whose disease was refractory to multiple rounds of immunosuppressive therapy. Another mutation was present in 38G>A in exon 2 of isoform 1 in a 32 year old male with moderate AA and paroxysmal nocturnal hemoglobinuria (PNH). We also identified the exon 2 38G>A mutation in a patient with disseminated mycobacterial disease where reduced transcription of the mutant 38G>A allele was noted on RT-PCR. Finally, an intron 5, c.512+573 G>A variant was identified in an 18 year old male with severe AA who progressed after immunosuppressive therapy to MDS/AML. This variant, which causes a disruption of the FLI1 binding site, has also been found to be pathogenic in monoMAC syndrome. In summary, a subset of patients with AA were found to have mutations in GATA2 suggesting a role for the gene in the pathogenesis of bone marrow failure. It also may identify patients at higher risk of infectious complications, those who may have less advantageous responses to immune suppression, and command earlier bone marrow transplantation. Disclosures: No relevant conflicts of interest to declare.