Moderate Aplastic Anemia Research Articles

A Napaac Survey to Understand Practices in Diagnosis and Treatment of Children with Moderate Aplastic Anemia

Among patients with acquired aplastic anemia, approximately 20% do not meet current classification as severe based on established criteria that reflect blood counts and marrow cellularity. For patients with moderate aplastic anemia (MAA), there is no set of defined diagnostic criteria, or clear standard-of-care for management. Little is known about the natural history of spontaneous recovery versus progression to severe bone marrow failure (BMF), response to conventional immunosuppressive treatment (IST), or evolution to myelodysplasia (MDS). Thus, institutional and individual practice can range from observation to immunosuppression or hematopoietic stem cell transplantation (HSCT). Recent studies of this patient population have begun to identify individuals harboring pathogenic mutations in genes known to cause BMF. To better understand population characteristics and management practices for patients with MAA, a survey was conducted through the North American Pediatric Aplastic Anemia Consortium (NAPAAC), consisting of 52 centers with expertise in BMF across North America. A total of 65 surveys were completed by pediatric hematology-oncology providers (55 attending physicians, 5 nurse practitioners and 5 fellows). Across all groups, there was marked variability of the MAA definition. Cytopenias were the defining criteria for diagnosis, including ANC<1000 cells/ul (79%), platelets <100K cells/ul (43%), and anemia for age (47%). In contrast, bone marrow hypocellularity (<50% cellularity) was considered by less than half (48%) of the respondents to be necessary for diagnosis. The combination of hypocellular bone marrow plus two cytopenias was considered the most common diagnostic definition of MAA (60% of participants). Although genetic testing has begun to transform diagnosis for many hematological diseases and can alter treatment decisions for pediatric BMF disorders, only 51% currently consider next generation sequencing (NGS) and 4% consider whole exome/whole genome sequencing as part of the initial work-up for MAA. Conventional cytogenetics and FISH are used more frequently in the initial work-up (86% and 73%, respectively). The two main triggers for treatment were dependence on red blood cell- (92%) or platelet (87%) transfusions, followed by persistent cytopenias (54%) and cytogenetic abnormalities (53%). Where available, acquired somatic variants triggered treatment in 30% of responses. Interestingly, infections were a less frequent trigger for treatment (35%). The most common management approaches were observation with transfusions (79%), single agent thrombopoietin receptor agonists (71%) or ATG+Cyclosporine based immune suppressive therapy (IST, 61%). Progression to severe aplastic anemia (SAA, 97%), myelodysplastic syndrome (MDS, 97%), or refractoriness to IST (61%) were the most common indications for HSCT.Availability of a matched sibling donor (MSD, 12%) was an infrequent indication for HSCT. A growing body of work, albeit largely in adult patients, suggests that patients with MAA represent a heterogenous group of individuals, some with genetic predisposition to BMF and inherent risk of myelodysplastic evolution. We identified that half of the responding NAPAAC practitioners use NGS panels routinely, suggesting a need for increased awareness to consider this work-up at initial diagnosis. Future studies are important to determine the yield of standard NGS testing, as the diagnosis of inherited BMF syndromes alter the strategy for long term surveillance of patients and genetic counseling for the family, as well as the use of related donor HSCT. We observed variability regarding the diagnostic criteria and management of children with MAA by providers who are members of NAPAAC. The heterogeneous responses to our survey suggest the need to establish management guidelines for uniform work-up and treatment, and to facilitate research of this understudied population. PC, LO: contributed equally as first authors. PK, JD: contributed equally as senior authors

Optimized Rabbit Antithymocyte Globulin Protocol Plus Sequential Cyclosporine and Levamisole Regimen for Newly Diagnosed Severe Aplastic Anemia: A Single-Arm, Single-Centre, Prospective, Phase 2 Trial

Background The inferior effect of rATG is due to the early fatal blow caused by the more potent immunosuppression. In order to retain its sustainable immunosuppression while weakening the early fatal blow. We explored a modifying schedule of administration of rATG by prolonging the course of rATG administration based on the equality of total dose, and then reported the better response and survival in severe aplastic anemia (SAA). And previously, we reported the encouraging effectiveness of cyclosporine alternately combined with levamisole (CSA and LMS regimen) in moderate AA, refractory or relapsed SAA, and SAA. We aim to establish whether the optimized rATG protocol plus sequential CSA and LMS regimen could improve SAA outcomes. Methods We conducted a single-arm, prospective, phase 2 trial at The Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Science & Peking Union Medical College (CAMS & PUMC). Patients aged 6 years or older with confirmed, newly diagnosed SAA were eligible. Patients received optimized rATG administration (1.97 mg/kg/d for 9 days) based on the equality of total does with standard protocol (3.55 mg/kg/d for 5 days). And then CSA and LMS were alternatively administrated every other day from day 14. The initial dose of CSA was 3mg/kg/day in adults and 5mg/kg/day in children (6-18 years old). Oral LMS was alternatively administrated at a dose of 150 mg per day in adults and 2.5 mg/kg per day in children (< 40 kg) in three divided doses. The primary endpoint was overall response rate at 6 months. The trial is registered at www.clinicaltrials.gov as NCT02203396. Results: Between Aug 1, 2014 and Sep 30, 2017, 40 patients with newly diagnosed SAA were treated (median age 24 years [range 7-57 years]; 19 [47.5%] male; 18 [45%] VSAA; 1[2.5%] post-hepatitis). All of these patients were transfusion dependent. Demographic data and clinical characteristics of these patients are outlined in Table 1. The 3-month overall response rates (ORRs) was 40.0%, including 2.5% CR and 37.5% PR. The ORRs increased to 60% (15% CR and 45% PR) at 6 months and 72.5% (42.5% CR and 30% PR) at 12 months. With a median follow-up of 77 months (range 0.1-97 months), estimated 8-year OS rate and event-free survival rate were 84.9% (95% CI, 69.35-92.9%) and 66.5 (95%CI, 49.1-79.1%), respectively. No patient discontinued therapy because of treatment-related toxicity. There were two deaths-one due to infection and another due to cerebral hemorrhage. In this cohort, 4/40 patients (5.7%) evolved to clonal disorders. Of the 4 patients, 1 patients progressed to overt PNH, 2 MDS and 1 acute myeloid leukemia (AML). Cytogenetic abnormalities involved chromosome 7 occurred in one cases with AML. A total of 2 (7.1%) patients relapsed at 33 and 79 months after initial response. Both of the 2 relapsed patients acquired response again with the administration of CSA. Conclusions: Front-line sequential optimized rATG protocol with CSA and LMS regimen resulted in encouraging long-term survival. Future randomized studies should evaluate the optimized IST regimen in the treatment of patients with SAA.

Atgam Efficacy and Safety in Moderate-to-Very Severe Acquired Aplastic Anemia: Outcome of a Large Multicenter Cohort of 634 Children and Adults from the French Authorization for Temporary Use Surveillance Program

Introduction Acquired aplastic anemia (AA) is a rare immunological disease leading to bone marrow failure. First-line treatment in patients aged >40 years or without a matched related donor is immunosuppressive therapy (IST) based on anti-human T lymphocyte immunoglobulin (ATG) plus cyclosporine. This ATGAM Temporary Use Authorization program was a retrospective, multicenter study to report safety and efficacy surveillance data on ATGAM use in patients with AA. Methods This study collected surveillance data from the ATGAM Named Patients Program for French authorities ahead of ATGAM registration. Patients were treated with ATGAM 40 mg/kg for 4 days in addition to cyclosporine. Safety and efficacy data were collected from patients treated from September 2011 to August 2022 and reported to the French authority every 6 months. Patients were classified as severe AA if they had 2 of the following criteria: neutrophil count <0.5x10 9/L, platelet count <20x10 9/L, or reticulocyte count <60x10 9/L. Very severe AA: same as severe except neutrophil count <0.2x10 9/L. Patients not meeting criteria for very severe/severe AA were classified as moderate. Patient response was evaluated using the RACE study criteria (Peffault de Latour, et al [2022]), for severe AA: complete response was defined as: hemoglobin >100g/L, neutrophil count >1.0x10 9/L, and platelet count >100x10 9/L; partial response: no longer meeting criteria for severe disease; no response: still severe AA and/or transfusion dependent. For moderate AA (Marsh, et al [1999]), complete response was defined as: neutrophil count >2.0x10 9/L and platelet count >100x10 9/L; partial response: neutrophil count >1.0x10 9/L and platelet count >30x10 9/L; no response: transfusion dependent. Patients were classified as receiving first-line treatment (never received ATG), refractory (failed to respond to previous IST with ATG within 12 months before initial ATGAM), or relapse (recurrence of AA after a positive response to IST with ATG), regardless of AA severity. Results In total, 634 patients with moderate-to-very severe AA were treated with ATGAM (n=537 first-line; n=68 refractory/relapse; n=29 not classified) in addition to cyclosporine (40 first-line patients also received eltrombopag) from January 2012 to August 2022. By severity, n=124 were moderate, n=317 severe, n=133 very severe, and n=60 severity unknown. Patient demographics are shown in Table 1. Overall response (partial+complete) in patients on first-line therapy at 3, 6, and 12 months was 22.5% (n=78), 50.6% (n=156), and 79.2% (n=164), respectively. By severity, overall response rates in first-line therapy for moderate and severe/very severe cohorts, respectively, were: 25.0% (n=20) and 21.7% (n=58) at 3 months; 56.8% (n=42) and 48.7% (n=114) at 6 months; and 74.1% (n=40) and 81.0% (n=124) at 12 months. Overall response at 6 and 12 months by age and AA severity in patients on first-line treatment is shown in Table 2. Median duration of follow-up was 12.4 months. Overall survival (95% confidence interval) at 12 months for all patients was 91.5% (88.8-93.6). The treatment was well tolerated, and no new safety signals were observed with ATGAM. Cumulatively, 1,087 adverse events were reported in 364 patients over the entire program period, the majority of which were disease related. Conclusion This real-world, retrospective study utilizing surveillance data showed response rates in line with the recent RACE study (Peffault de Latour, et al [2022]) for patients with first-line severe AA treated with a combination of ATGAM and cyclosporine. No new safety risks were identified in this large cohort of patients. Treatment with ATGAM remains of benefit in patients with moderate-to-very severe AA.

Telomere Length Testing of Lymphocytes Alone By Flow-FISH Is a Highly Sensitive and Specific Test to Screen for a Telomere Biology Disorder in a Cohort of Children and Very Young Adults with Bone Marrow Failure

Background Uncovering an underlying telomere biology disorder (TBD) in patients with bone marrow failure (BMF) has crucial implications for treatment and outcomes, including hematopoietic cell transplantation and malignancy risk. Telomere length (TL) testing of peripheral blood leukocytes by clinical Flow-FISH has become an important test in diagnosing TBDs. In seminal studies, Alter et al. compared age-adjusted TL lengths (< or ≥1 st percentile) in lymphocytes (lymphs), granulocytes (grans), and four lymph subsets in individuals with a known TBD and their unaffected relatives. They found that very low (VL) TL (<1 st percentile) in lymphs had a sensitivity (Sens) of 97%, specificity (Spec) of 91%, and positive predictive value (PPV) of 85% for a TBD diagnosis. VL TL in 3 or 4 lymph subsets had slightly better test performance with a Sens of 98%, Spec of 94%, and PPV of 89%. We hypothesized that test performance characteristics of flow FISH would be different if applied to a ‘real-world’ cohort, which could inform the use of TL in lymphs alone versus in the four lymph subsets for different clinical indications. Methods We performed a search of the electronic medical record to identify all individuals who had Flow-FISH performed at Texas Children's Hospital from 2007 - 2022 (Figure 1). We excluded those who had the 2-panel test (lymphs and grans) or family TL cascade testing. We collected the indication for and results of Flow-FISH, demographic and clinical characteristics, and genetic testing information via chart review. A composite score (Comp Score) was created to define TBD (Comp Score of 1 or 2) vs. Not TBD (Comp Score <1). Performance of lymph TL alone and TL in 3 or 4 lymph subsets in predicting TBD was evaluated using Sens, Spec, PPV, NPV, area under ROC curve (AUC), and Cohen's Kappa statistic (k). Results Our final cohort included 433 individuals who received the 6-panel test (lymphs, grans, and four lymph subsets) with a median age of 9.7 years, range 0.06-22.6. Top indications for Flow-FISH were severe aplastic anemia (SAA) (n=106), mild aplastic anemia (AA) (i.e., not fulfilling criteria for MAA or SAA) (103), cytopenias without known or documented bone marrow hypoplasia or dysplasia (70), moderate aplastic anemia (MAA) (37), non-therapy-related myelodysplastic syndrome (26), and an incidental TBD gene variant identified through comprehensive sequencing (17). Twenty-two subjects (5.1%) had a TBD, 17 (3.9%) of which had a pathogenic/likely pathogenic variant (P/LPV) in a known TBD gene (Figure 1). Seven of those with SAA (6.6%) had VL TL in lymphs, 5 (4.7%) of which also had VL TL in 3 or 4 of the lymph subsets, and only 1 (0.9%) had a TBD based on Comp Score. Importantly, none had VL TL in 3 or 4 of the lymph subsets without VL TL in lymphs. Five of those with MAA (13.5%) had VL TL in lymphs, all of which had VL TL in 3 or 4 lymph subsets. Three had a TBD based on Comp Score, and each had a PV in a TBD gene; a TBD variant was not identified in two despite exome sequencing and chromosomal microarray testing. Eighteen of those with mild AA (17.5%) had VL TL in lymphs, 15 (14.6%) also had VL TL in 3 or 4 lymph subsets, 9 (8.7%) had a TBD based on Comp Score, and 7 (6.8%) had a P/LPV in a TBD gene. The Sens of TL in lymphs < vs. ≥ 1 st percentile and TL < 1 st percentile in 3 or 4 vs. < 3 lymph subsets against TBD based on Comp Score in those with AA regardless of severity was 100%, and the Spec ranged from 90.4% (lymph TL testing alone in mild AA) to 96.2% (lymph subsets testing in SAA) (Table 1). The PPV for both test approaches was low (≤20%) in SAA due to the low prevalence of TBD in this population. PPV was between 50-60% in the other AA groups. The AUC for detecting a TBD in the various AA groups was comparably high for both test approaches (0.95-0.98). The two tests had a substantial to almost perfect agreement (k=0.82, p<0.001). Conclusions This single institutional cohort demonstrates that the prevalence of TBD in young patients presenting with SAA is remarkably low, and the Sens and Spec of TL testing in lymphs alone are sufficiently high to detect the rare TBD patient and prompt additional testing (e.g., genetics) in few patients due to false positive testing. The prevalence of TBD is higher in those with MAA or mild AA, yet both test approaches detect all TBD patients. Spec level in mild AA is marginally higher with lymph subset TL testing but, at ~94%, underscores the need for other clinical parameters (e.g., genetics) to establish a TBD diagnosis in a small percentage of patients.

Aberrant myelomonocytic CD56 expression predicts response to cyclosporine therapy in pediatric patients with moderate aplastic anemia.

This study aimed to investigate the expression patterns and clinical significance of neural cell adhesion molecule-positive (CD56+) myelomonocytes in pediatric patients with moderate aplastic anemia (mAA). Fifty-six pediatric patients with mAA were enrolled in this study. The patients' clinical characteristics, laboratory data, and response to cyclosporine therapy were obtained. CD56 expression on bone marrow myelomonocytic cells was investigated using flow cytometry. The association between aberrant CD56 expression and cyclosporine response was evaluated by a multivariate analysis. CD56+ myelomonocytes were detected in 43% of the mAA cases. Aberrant CD56 expression was frequent on immature CD45dimCD16dim granulocytes and mature CD45brightCD14bright monocytes. Compared with patients with CD56- myelomonocytes (CD56- patients), patients with CD56+ myelomonocytes (CD56+ patients) were in moderate hematological condition and had a distinct bone marrow cellular composition profile, which included an increased proportion of myeloid cells and CD56bright natural killer cells and a reduced proportion of CD4+ T cells, CD8+ T cells, and B cells. The multivariate analysis determined that CD56+ myelomonocytes were a favorable factor for achieving response at 6 months after cyclosporine therapy. There was a trend towards a lower 3-year rate of evolution to severe aplastic anemia or relapse among the CD56+ patients (8%) than the CD56- patients (22%). CD56+ patients had an increased myeloid compartment and better prognosis compared with CD56- patients. The findings demonstrated the favorable role of CD56+ myelomonocytes in aplastic anemia progression.

32295 Looking beyond scalp hair loss: Factors influencing dermatologists’ assessment of alopecia areata severity

Background: Severity criteria for AA currently rely on extent of scalp hair loss (HL) without formally assessing other relevant factors that may influence disease severity. Objective: Identify factors beyond extent (percent) of scalp HL that are associated with dermatologist assessed AA severity. Methods: Real-world data from a 2019 cross-sectional survey of US dermatologists and their adult AA patients were analyzed. Dermatologists provided assessments of patients’ clinical characteristics. Two methodologies were used to evaluate the predictive power of risk factors including percent scalp HL: classification and regression tree (CART) and multinomial logistic regression (MLR) models. Seven domains of dermatologist-reported risk factors were sequentially tested: HL percent, patient impairments, disease/treatment history, demographics, dermatologist treatment satisfaction, comorbidities, and dermatologist characteristics. Results: Patients’ (n = 442) mean (SD) age was 39.3 (13.8) years, time since diagnosis was 4.5 (7.5) years, 51% female. Patient’s AA severity as assessed by the dermatologist was 20% mild, 53% moderate and 27% severe. CART identified percent scalp HL as the primary differentiator between Mild (0-10%), Moderate (11-40%) and Severe (≥41%) AA; treatment success was an additional predictor that differentiated between mild and moderate severity. In MLR, scalp HL was the most important risk factor (generalized R-sq = 60% out of 70% in the final model). Additional differentiating factors included fear of recurrence and day-to-day activity impairment. Conclusions: Although dermatologists’ assessment of AA severity primarily relies on extent of scalp HL, it is also influenced by a combination of physician-related (treatment success) and patient-related (fear of recurrence and activity impairment) factors.

Spectrum of Aplastic Anaemia; presentation, etiology and overall survival: ASpectrum of Aplastic Anaemia: Presentation, Etiology and Overall Survival-A Tertiary Care Hospital Experience Tertiary Care Hospital Experience

Aplastic anemia (AA) is a hematological disorder with immune pathophysiology. There is a decrease in bone marrow precursors and cellularity which lead to a peripheral decline in at least two cell lines. The incidence of AA is higher in the Asian population as compared to that in the west. Objective: To highlight the disease spectrum of aplastic anemia patients from the clinical presentation and etiological factors to the disease outcome and overall survival. Methods: It was a descriptive cross-sectional study, conducted at Hematology Department King Edward Medical University. All the patients of both genders, diagnosed with aplastic anemia on peripheral smear and bone marrow trephine biopsy findings were included. The patients were followed up, the results were recorded and data were analyzed using SPSS V22. Results: The study included total 91 patients of aplastic anemia out of which 46(50.5%) were males, 45(49.5%) were females The patients were divided into three groups according to the severity of which moderate aplastic anemia was present in 19 (20.9%) patients, severe aplastic anemia was seen in 42 (46.2%) patients and very severe aplastic anemia was seen in 30 (33%) patients. The etiological factors seen included idiopathic 61.5%, hakim medication 14.3%, infections 12% drugs 9.8% and radiation exposure 2.1%. It was observed that the patients with severe aplastic anemia presented with high ECOG score. Conclusion: Aplastic anemia has life detrimental outcome with high mortality rate if left untreated can be provided to the patients since it is one of the high incidences hematological disorders in Asian population.

PSIII-18 Effect of Increasing L-Lysine-HCl and Amino Acid Ratios on Performance of Finishing Pigs from 109 to 129 Kg

Abstract A total of 1,789 pigs (PIC; 337×1050; initial BW 108.9 ± 1.14 kg) were used to determine the effects of increasing L-Lys-HCl and AA ratios on performance of late finishing pigs fed corn-soybean meal diets without DDGS. The study used 2 groups of pigs and each study lasted 18 and 27 d, respectively. Pigs were housed in mixed gender pens with 20 to 25 pigs/pen and 19 replications/treatment (10 and 9 replications/group, respectively). Pens of pigs were blocked by BW and randomly allotted to 1 of 4 dietary treatments. Treatment diets were formulated to 0.70% digestible Lys and consisted of low, medium, or high levels of feed-grade AA and moderate or high AA ratios relative to Lys. Low, medium and high feed-grade AA treatments had increased L-Lys-HCl (0.15, 0.30 and 0.39%) replacing soybean meal to achieve CP levels of 13.6, 12.0, and 11.0. Minimum AA:Lys ratios were 53% Ile, 128% Leu, 58% Met&Cys, 66% Thr, 18.5% Trp, 68% Val, and 34% His in Moderate AA ratio diets and 60% Ile, 128% Leu, 60% Met&Cys, 70% Thr, 21.2% Trp, 72% Val, and 33% His for the high AA ratio treatment. Overall, there was a marginally significant increase in ADFI (quadratic, P = 0.097) observed in pigs fed medium feed-grade AA and moderate AA ratios. Treatment diets had no effect on ADG or G:F. At the end of group 2, carcass data was collected with no differences (P > 0.10) observed for HCW, carcass yield, backfat depth, loin depth or percentage lean. In summary, differing levels of feed-grade AA and AA ratios used in this study did not impact growth performance or carcass characteristics of late finishing pigs with the exception of a marginally significant increase in ADFI.

Immunogenetic, Molecular and Clinical Determinants of Clonal Evolution in Aplastic Anemia and Paroxysmal Nocturnal Hemoglobinuria

Immunogenetic, Molecular and Clinical Determinants of Clonal Evolution in Aplastic Anemia and Paroxysmal Nocturnal Hemoglobinuria

Tacrolimus Is Effective Immunosuppressive Therapy in Aplastic Anemia

Introduction:For patients with aplastic anemia (AA) who are ineligible or unsuited for hematopoietic stem cell transplantation (HSCT), immunosuppressive therapy (IST) with cyclosporine A (CsA) and horse antithymocyte globulin (ATG) remains the standard therapeutic regimen of choice. However, CsA has notable side effects including hirsutism and gingival hyperplasia that may limit the overall duration and effective dosage of this therapy. In both renal and heart transplant patients, tacrolimus has shown an improved toxicity profile when compared to CsA, however the use of tacrolimus as an alternative to CsA in AA has not been systematically examined. We report our experience using tacrolimus plus horse ATG as first- and second-line IST in a diverse ethnic cohort of patients with acquired AA at two urban hospitals.Methods:We conducted a retrospective chart review of patients with AA treated at the University of Southern California (USC) Norris Cancer Hospital from 2008 to 2017 and the LAC+USC Medical Center from 2015 to 2017. Out of a cumulative 197 AA patients meeting inclusion criteria, 23 patients were identified who had received tacrolimus-based IST in either the first- or second-line setting. In these patients, tacrolimus was combined with horse ATG, glucocorticoids and more recently, eltrombopag. Demographic data as well as 3, 6, and 12 month overall response rate (ORR), time to last transfusion, time to transplant and toxicity were assessed. The Camitta criteria were used to categorize the severity of AA based on bone marrow cellularity and peripheral cell count. Criteria for complete response (CR) were ANC>1000, HGB>10 g/dL, and PLT >100,000. Partial response (PR) was defined as no longer meeting criteria for severe AA, or if moderate AA, transfusion independence or an increase in ANC by 500, HGB by 3 g/dL, and/or PLTs by 20,000.Results:Among the 23 patients in our cohort treated with tacrolimus IST, mean age at diagnosis was 40, 65% were female, 47.8% were Latino and 34.7% were Caucasian. 12 patients (52%) received tacrolimus as front-line immunosuppression. Of these patients, 4 (33%) had moderate AA and the rest (66%) had severe or very severe AA. The ORR was 75% at 3 months (3 CR + 6 PR), 80% at 6 months (1 CR + 7 PR) and 100% in those evaluable at 12 months (4 PR). Among those with severe disease, ORR were 75%, 71% and 27% at 3, 6 and 12 months respectively, with 2 receiving subsequent HSCT. 11 patients received tacrolimus in the second-line setting due to cyclosporine toxicity or incomplete response to cyclosporine IST. Prior to starting tacrolimus, 73% had moderate AA and 18% had severe or very severe AA. ORR was 55% (1CR + 4 PR) at 3 months, 36% (2 CR + 2 PR) at 6 months and 45% (4 CR + 1 PR) at 12 months. All responses were in patients with moderate AA.Conclusion:Front-line tacrolimus IST with horse ATG demonstrated in the treatment of AA with an overall favorable safety profile. For patients who are transplant ineligible, tacrolimus should be considered a suitable alternative to CsA as part of an IST front-line regimen. More strikingly, tacrolimus in the second-line IST setting can achieve significant responses in patients that continue to have moderate AA despite initial cyclosporine-based IST. DisclosuresWeitz:Alexion Pharmaceuticals: Speakers Bureau.

Identification of T Cell Receptors Specific to Antigens Presented By HLA-B5401 on IPS Cell-Derived Hematopoietic Stem Cells in a Patient with Acquired Aplastic Anemia Carrying B5401-Lacking Leukocytes

[Background] Cytotoxic T lymphocytes (CTLs) specific to hematopoietic stem cells (HSCs) are thought to play an important role in the development of acquired aplastic anemia (AA). The presence of HLA class I allele-lacking leukocytes as a result of 6pLOH or allelic mutations in about 25% of AA patients strongly suggests that CTLs specific to antigens presented by the specific HLA alleles are involved in the destruction of normal HSCs as well as in the escape of HLA-lacking HSCs. Establishing CTLs from these patients by culturing the patients' CD8+ cells with targeted HSCs may be useful for identifying autoantigens of AA. However, it is generally impossible to obtain sufficient numbers of HSCs from the patients for functional assays and in vitro stimulation of patient's T cells with HSCs may induce clonal expansion of CTLs irrelevant to pathogenesis of AA. To solve these problems, we generated iPS cells (iPSCs) from an untreated AA patient who possessed HLA-B5401-lacking leukocytes for inducing HSCs and attempted to identify cDNAs of T-cell receptor (TCR) α and β chains of predominant T cells in the patient's bone marrow (BM) using a single T-cell analysis, which may specifically recognize autologous HSCs. [Methods] iPSCs were generated from the peripheral blood mononuclear cells of an AA patient (56-year-old man), whose moderate AA later responded to cyclosporine therapy, using Yamanaka's method. The patient's monocytes accounted for 20% wild-type, 20% 6pLOH(+) and 60% B5401-lacking cells at the time of sampling. CD34+ cells were induced from these iPSCs and injected into the BM of C57BL/6.Rag2 mice harboring NOD-Sirpa (BRGS). CD8+ T cells in the BM of the patient were subjected to a single cell sorting followed by cDNA amplification of TCRVβ chains. TCRα chains of five T cells that showed the five most recurrent TCRVβs were determined, and the TCRβ cDNAs were retrovirally transfected to Jurkat cells that lacked TCRβ chain and were transfected with IFN-γ cDNA. Specific responses of each TCR transfectant to iPSC-derived CD34+ cells were determined using ELISA for detecting IFN-γ. [Results] Five iPSC clones, including wild type (n=2), 6pLOH(+) (n=1), and B5401-lacking (B5401[-]) (n=2) clones, that had different mutations in B*54:01 were generated. Three-week culture of the iPSCs in the presence of various growth factors produced hematopoietic cells consisting of 50% CD34+ cells of each phenotype, which were able to reconstitute of BRGS mice with 3.5% to 8.9% human CD45+ cells in the peripheral blood at 9-12 weeks after injection. When the four TCR transfectants were tested for their response to wild-type and B5401-lacking CD34+ cells, all four transfectants secreted higher amounts of IFN-γ in response to wild-type cells than to B5401(-) CD34+ cells. [Conclusions] HSCs with original genotypes capable of reconstituting hematopoiesis in immunodeficient mice were successfully induced from iPSCs that had been generated from an AA patient's monocytes. The TCR transfectants showing a specific response to B5401(+) wild-type CD34+ cells will be useful for identifying the ligand peptides presented by B5401. DisclosuresNo relevant conflicts of interest to declare.

Germline Mutations in CTLA4 and Spectrum of Bone Marrow Features Overlapping with Aplastic Anemia and Large Granular Lymphocyte Lymphocytosis

Introduction: CTLA4 haploinsufficiency due to germline heterozygous mutations in the cytotoxic T-lymphocyte antigen-4 gene (CTLA4) results in severe systemic immune dysregulation; infiltrative lymphocytic lesions of lungs (e.g. lymphocytic pneumonitis), GI tract (e.g.lymphocytic colitis), and CNS; and a spectrum of autoimmunity, immunodeficiency and peripheral blood cytopenias. We describe the hematologic, bone marrow (BM), and flow cytometry (FC) features of CTLA4 haploinsufficiency of a cohort of 14 patients, which show overlapping morphologic features with acquired aplastic anemia (acAA), and large granular lymphocyte lymphocytosis (LGL) in a subset of patients. We compared the findings to those in a separate cohort of 20 age matched patients diagnosed with acAA. There were significant differences between acAA and CTLA4 deficiency based on analysis of BM lymphocyte subsets and marrow lymphocyte infiltration patterns.Materials and Methods: Peripheral blood (PB), BM and FC analysis of patients with germline mutations in CTLA4, and acAA was performed along with review of clinical, molecular and laboratory data. Statistical comparisons were made using Mann Whitney test, with p < 0.05 for significance.Results: Median age at initial evaluation for patients with CTLA4 haploinsufficiency was 23.5 years (range 11-71 years), with a male-to-female ratio of 11:3. 11 out of 14 patients had cytopenias. Of those with cytopenias 2/11 had pancytopenia, 4/11 had bicytopenia and 5/11 had unilineage cytopenia. Hypogammaglobulinemia was common (13/14). In some cases cytopenias were recognized as autoimmune in etiology with immune thrombocytopenic purpura and autoimmune hemolytic anemia, among the most common hematologic manifestations. Three patients met criteria for a diagnosis of moderate AA, 1 for severe AA, and 1 for LGL. Overall bone marrow biopsies were hypocellular for age (10/14) with a subset (29% [4/14]) showing severe trilineage hypoplasia with ≤20% cellularity. Atypical interstitial CD8-positive T-cell infiltrates with prominent non-paratrabecular CD4-positive T-cell aggregates were evident by immunohistochemistry in most cases (10/14), accompanied by a near absence of B-cells. T-cell clonality was assessed by T-cell receptor (TCR) gamma PCR and showed abnormal oligoclonal T-cell populations in 3/8, and one case with a clonal T-cell receptor gene rearrangement. B-cell IgH PCR reactions were polyclonal. In all cases, cytogenetic studies were either negative or showed only minor abnormalities (i.e. loss of Y chromosome). Given the morphologic overlap of a subset of CTLA4 cases with acAA, we compared bone marrow flow cytometry analysis of 8 CTLA4 cases with 20 acAA cases. CTLA4 patients showed >5 fold reduction in the median percentage of B-cells in comparison to acAA (p=0.0005). While acAA cases demonstrated relative preservation the B-cell compartment with intact B-cell maturation sequence; in contrast CTLA4 cases showed abnormal B-cell maturation patterns in half of cases characterized by few detectable early B-cell precursors, and progressive loss of intermediate and mature forms. T-cell populations in CTLA4 showed inverted CD4:CD8 ratios with relatively increased CD57+ T-cells, and atypical dim expression of CD20 on T-cells. Morphologically, when the markedly hypocellular CTLA4 cases were compared to acAA on core biopsies, little difference in marrow cellularity was seen; however, the CTLA4 cases showed multiple large atypical T-cell aggregates and near total absence of B-cells, which was not seen in acAA; acAA cases showed scattered background T-cells and B-cells.Conclusions: CTLA-4 deficiency presents with cytopenias, autoimmunity, and immunodeficiency with a subset of patients that have features overlapping with bone marrow failure, specifically AA and LGL. Herein we demonstrate a combination of abnormal BM findings, previously unreported, comprising hypocellularity, CD8-positive interstitial T-cell infiltrates with atypical CD4-positive T-cell aggregates, marked B-cell lymphopenia, increased LGLs and abnormal T-cell clonality patterns by PCR. These features may help identify patients harboring CTLA4 mutations for early detection and optimal therapeutic management. DisclosuresYoung:Novartis/GSK to institute: Research Funding. Townsley:Novartis/GSK to institute: Research Funding.

A Diamond-Blackfan Anemia Patient's Response to Eltrombopag and Genomic Analysis in Different Lineages

Diamond-Blackfan Anemia (DBA) is a rare, inherited bone marrow (BM) failure syndrome characterized by pure red cell aplasia generally presenting within the first year of life. The majority of cases are due to heterozygous mutations in ribosomal genes, either autosomal dominant inherited or sporadicde novoevents. Despite the recognition of the importance of ribosomal biology, the underlying mechanisms are still poorly understood. The only curative treatment is hematopoietic stem cell transplantation, otherwise steroids or transfusions will cause significant long-term morbidity. There is an unmet need for new therapies. We describe the unique clinical course and molecular analyses of a 32-year-old Hispanic woman, provide insights into disease pathophysiology as well as hope for an effective new treatment. She was diagnosed with DBA at the age of one month with isolated severe normochromic anemia. She failed various therapies, including corticosteroids, danazol, growth factors, and additional immunosuppression and developed significant iron overload despite desferal chelation. She was enrolled in a phase II clinical trial investigating the safety and efficiency of the thrombopoietin receptor agonist eltrombopag (EPAG) in patients with moderate aplastic anemia or unilineage cytopenias (NCT01328587). EPAG was administered in an escalating manner reaching 300mg daily. She achieved transfusion-independence by 16 weeks. This dose was continued for an additional 6 months and discontinued for protocol-defined robust response, ie Hb &amp;gt;10g/dL. After discontinuation, her hemoglobin steadily declined over a period of 8 months. EPAG was reinitiated, her Hb again rapidly improved and the EPAG dose was slowly tapered. Seven years since enrollment, the patient has remained on drug, currently at a dose of 25mg daily with normal blood cell counts and no EPAG related toxicity. BM cytogenetics and morphology have remained normal throughout follow up, as has a myeloid somatic mutation panel. Next generation sequencing has revealed a novel, pathogenic mutation in intron 4(c.356+3&amp;gt;C) of theRPS19gene affecting a splice donor site previously implicated in DBA. Testing of both parents was negative, documenting this to be ade novomutation. With digital droplet PCR (ddPCR), we have monitored the variant allele frequency (VAF) in fibroblasts (germline) and different hematopoietic populations (Figure 1). Fibroblasts showed a VAF 50% (heterozygous), while her whole BM and whole blood show a lower VAF between 25% and 37%, both before and on EPAG treatment. Across lineages were analyzed when she was on EPAG, lymphocytes showed the highest VAF (42% to 45%), followed by HSPCs and granulocytes at 24% to 32%. Of note, the erythrocytes progenitors (CD45-CD71+) had the lowest VAF of 8% to 14%. Sequencing of myeloid and erythroid colony forming units (CFU) with both pre and on EPAG BM samples confirmed only heterozygous mutations or wild-type (WT) CFUs in percentages comparable to their respective VAF detected by bulk ddPCR. These results suggest the emergence of WT clones within the BM either through mosaicism or a reversion event. The discrepancy in VAF between peripheral and BM samples is consistent with a proliferative advantage for WT clones, especially in the erythroid and myeloid lineages. Yet despite the presence of WT HSPCs, profound transfusion-dependent anemia persisted in this patient long-term. An imbalance between globin chain and heme production has been documented to result from ribosomal dysfunction in DBA, leading to increased iron-mediated cellular stress and death of erythroid progenitors. We hypothesize the residual mutant cells suppress the growth of WT cells, given the close proximity of large numbers of erythroid cells aggregated by macrophages in erythroid islands, potentially via passage of free iron. These observations have implications for the development of gene addition or gene correction therapies for DBA, suggesting that correcting a fraction of HSPCs will not revert the phenotype. Given the potent intracellular chelation activity of EPAG, and the lack of the EPAG target MPL receptor on erythroid progenitors, we hypothesize that iron chelation is a potential mechanism for improvement of red blood cell production by EPAG in DBA. The results in our patient have encouraged the opening of a prospective clinical trial (NCT04269889) to further explore the potential of EPAG for DBA patients. Figure Disclosures Young: Pharos I&amp;BT Co., Ltd.:Research Funding.Pakbaz:Amgen:Membership on an entity's Board of Directors or advisory committees;Dova:Membership on an entity's Board of Directors or advisory committees;Novartis:Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Terumo BCT:Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Global Blood Therapeutics:Speakers Bureau.Dunbar:Novartis:Research Funding.

Long-Term Eltrombopag for Bone Marrow Failure Depletes Total Body Iron

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

The Genomic Landscape of Myeloid Neoplasms Evolved from AA/PNH

The Genomic Landscape of Myeloid Neoplasms Evolved from AA/PNH

A study on epidemiological features of alopecia areata in children: At tertiary care center, Telangana

Aim & Objective: The objective of this study to assess the epidemiology of alopecia areata, including its incidence, prevalence, and distribution by sex and age. Methodology: A cross sectional study was carried out at the Department of Dermatology 2017 January-2019 December. During the study period all patients presenting to the dermatology outpatient with a possible diagnosis of alopecia areata were evaluated. Results: Out of 50 children, 33 (66%) were males, 17 (34%) were females. five (10%) were in 0-5years age group, 31 (62%) were in 6-10 years age group, 14 (28 %) were in 11-15 years age group. Mean age of onset was 8.63 years. In present analysis duration of the illness, out of 50 patients, 24 (48%) had duration less than 3 months, 17 (35%) had duration 4-6 months, five (10%) patients had duration 7 months - 1 year and four (8%) patients had AA of more than one year duration. In the present investigation, out of 50 children, 15(30%) had a positive family history of alopecia areata. Out of 15 patients with family history of AA, twelve were males (24%) and three (6%) were females. Out of 33 males with AA, 15 (45.45%) had a family history of AA. Out of 17 females with AA, only three (17.64%) had a positive family history of AA. scalp was the initial site of involvement in 48 (96%) children whereas the face was the initial site involved in two (4%) child. Out of 48 patients with scalp as initial site of onset, Occipital region was the most common presenting initial site for 22 (45.83%) cases followed by vertex in 13 (27.08%) cases, temporal and parietal in five (10.41%) cases each. In the present study, out of 50 children, 23 (46%) had only single lesion, while 27 (54 %) had multiple lesions. Out of 50 children, patchy AA was found in 28(58 %) children, combined patchy and ophiasis was found in 9 (17 %), ophiasis was found in 7 (16%), combined patchy and diffuse AA was found in 1(2.08%) child, and alopecia universalis, subtotal AA, reticular AA were found in 1(2.08%) child each. Severity of AA in 48 patients with scalp involvement was assessed using the SALT (SEVERITY OF ALOPECIA TOOL) scoring system. 72.91% (35) of the children were having mild AA, 22.91% (11) were having moderate AA and only 4.16% (2) had severe AA. Out of 35 children with mild AA, 20 (57.14%) were males and 15 (42.86%) were females. Out of 11 children with moderate AA, 8 (72.72%) were males and 3 (27.27%) were females. two children with severe alopecia was male. Conclusion: AA is the most prevalent autoimmune disorder and the second most prevalent hair loss disorder after androgeneticalopecia, and the lifetime risk in the global population is approximately 2%. AA is associated with psychiatric and medical co-morbidities including depression, anxiety, and several autoimmune disorders, and an increased global burden of disease.

Eltrombopag for patients with moderate aplastic anemia or uni-lineage cytopenias

AbstractThere 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.

Correlation between serum ferritin and severity of alopecia areata

Correlation between serum ferritin and severity of alopecia areata - IJCED- Print ISSN No: - 2581-4710 Online ISSN No:- 2581-4729 Article DOI No:- 10.18231/j.ijced.2019.070, IP Indian Journal of Clinical and Experimental Dermatology-IP Indian J Clin Exp Dermatol

Evolution of Moderate Aplastic Anemia into Subclinical PNH/AA and Successful Treatment with Reduced Intensity Haplo-HSCT: A Pediatric Case Report

Introduction: Aplastic Anemia (AA) is diagnosis made after the exclusion of other acquired or inherited disease that can present with pancytopenia. In pediatric AA, there is often overlap with many other diseases including Paroxysmal Nocturnal Hemoglobinuria (PNH) and hypocellular myelodysplastic syndrome (MDS). The diagnosis is often difficult and can evolve. There are new tests and methodologies to guide diagnosis and treatment decision making. Case report: The patient is a previously healthy, 18-year-old Caucasian female referred for pancytopenia. Patient reported progressive fatigue and easy bruising for a month prior to evaluation. Initial white blood count was 2.4 x109/L, hemoglobin 9.7g/dl, platelets 39 x109/L, absolute neutrophil count (ANC) of 590, and absolute retic count 68 x 109/L. A bone marrow exam revealed a hypocellular marrow with 30% cellularity with no dysplasia or blasts on flow cytometry. Viral studies, folate and vitamin B12 levels were normal. Chromosomal analysis, Fluorescent In situ Hybridization (FISH) study for MDS, and telomere length assay were normal. A bone marrow failure (BMF) panel for sixty targeted genes showed no mutations associated with BMF. PNH studies by flow cytometry showed 2.6% erythroid, 2% granulocytes, 2.7% monocytes failed to express GPI associated anchoring proteins (GPI-APs) representing a small PNH clone, not unusual in patients with aplastic anemia (AA). Studies were c/w with moderate AA (mAA). Standard management of mAA is careful observation and treatment if the patient requires transfusions or ANC falls below 500. However, since our patient was soon starting college out of state, we decided to treat with Immunosuppressive Therapy (IST), consisting of equine Anti-thymocyte globulin (eATG) and Cyclosporine. At 24 weeks post IST, no response was observed and the patient was requiring more frequent platelet and red blood cell transfusions. Repeat PNH testing showed 2% erythroid, 5% monocytes and 4% granulocytes that were deficient in GPI-Aps (Table1). BM exam showed 60% cellularity that was consistent with a recovering marrow but there was relative erythroid hyperplasia, without evidence of dysplasia or blasts. At this time, with increasing marrow cellularity but persistent pancytopenia we began to strongly suspect MDS. However, repeat chromosomal analysis and FISH studies were normal and a Foundation One myeloid panel was negative for mutations associated with MDS. At 28 weeks post-IST, repeat BM exam revealed 70% cellularity, again with relative erythroid hyperplasia. At the same time PNH testing, showed that 1.3% erythrocytes, 5.6% granulocytes and 5.4% monocytes were negative for GPI-APs. We concluded that our patient evolved had from mAA to subclinical PNH/AA which accounted for the normocellular marrow. High Disease Activity (HDA) is defined as lactate dehydrogenase (LDH) ≥ 1.5 times upper limit of normal and is associated with large PNH clones. HDA can guide the use of eculizumab as pre-transplant therapy. (DeZern et al, 2012) Throughout the course of the disease, her LDH was normal. She did not have HDA and therefore, was not treated with eculizumab. Because of the normocellular marrow she was not eligible for CTN 1502-CHAMP study. However, the patient underwent reduced intensity conditioning (RIC) and a haplo paternal BMT, followed by post-transplant cyclophosphamide, as per CTN 1502- CHAMP. She tolerated her BMT well without major complications. She is now five months post-transplant, alive and well, fully engrafted with 100% donor chimerism and no signs of GVHD. Post-transplant PNH testing revealed all PNH clones were eliminated. Conclusion: This case illustrates the diagnostic challenges of patients presenting with pancytopenia and the overlap and possible evolution of disease classifications; new diagnostic testing including gene mutation screens to aid in diagnosis; new concepts such as HDA in PNH to help guide treatment decision making; and the successful use of RIC and haplo BMT in a patient with PNH/AA and a normocellular BM Disclosures No relevant conflicts of interest to declare.

Telomere Elongation and Clinical Improvement in Telomeropathy Patients: A Prospective Clinical Trial of Nandrolone in Telomeropathies

Telomeres are a complex of hexameric repetitive DNA sequences at the end of linear chromosomes. Telomeres erode during mitosis, and this process is accelerated in patients carrying germline pathogenic mutations in telomere-biology genes (Calado and Young, NEJM 2009). In vitro, the exposition to androgens stimulates TERT transcription and telomerase activity of lymphocytes and bone marrow CD34+ cells (Calado et al. Blood 2009). In a phase 1-2 prospective trial, the use of danazol led to telomere elongation in patients with telomeropathies (Townsley et al. NEJM 2016). The hematological response was observed in 79% of evaluable patients. Here, we describe a phase 1-2 single-center prospective study assessing the safety and the effect of the male hormone nandrolone decanoate on telomere erosion in patients with telomere diseases (ClinicalTrials.gov Identifier: NCT02055456). Entry criteria comprised patients older than two-year-old with age-adjusted mean telomere length below the 1st percentile ± identified mutations in telomerase complex genes, associated with at least one cytopenia and/or diagnosis of idiopathic pulmonary fibrosis (IPF). Patients were treated with nandrolone decanoate intramuscular at a dose of 5 mg/kg every 14 days for 24 months. The primary efficacy end point was a 20% decrease in the annual rate of telomere erosion, and the incidence of toxic effects was the primary safety end point. Secondary endpoints were hematologic response and pulmonary response. Flow-FISH was used to determine the telomere length of peripheral blood leukocytes. From May 2014 to October 2017, 20 consecutive patients were eligible for participation, and 17 were enrolled. The median age was 36 years (range, 4 - 59 years), and five patients were female. In all but one patient germline pathogenic mutations were identified (TERT, 7; RTEL1, 4; TERC, 2; WRAP53, 1; TINF2, 1; and SAMD9, 1). Eleven patients were diagnosed with bone marrow failure (9 with moderate and one with severe AA, and one with myelodysplasia). Four patients were diagnosed with IPF, and 2 patients had both moderate AA and IPF. One patient diagnosed with IPF had received an allogeneic bone marrow transplant before enrollment. Four patients had additional hepatic involvement, and 5 displayed cutaneous features of dyskeratosis congenita. The most common adverse events associated with drug were elevations in liver enzyme levels in 88%, acne in 59%, and virilization in 59%. Severe adverse events possibly related to drug occurred in six instances. Dose reductions were necessary in 5 patients due to severe or moderate adverse events. Seven patients withdrew from the study before 2 years: two patients discontinued therapy due to grade 3 adverse events (depression and acne); one patient sought alternative therapies; and four patients died during the study period, two due progressive IPF and two due intracranial hemorrhage. Out the 17 patients enrolled, a total of 15 reached 12 months of treatment and 13 were evaluable (one patient was transplanted and telomere length not evaluated; and for one patient the telomere sample was missing). Ten patients met the primary efficacy end point, showing telomere elongation. As of July 2019, nine patients reached 24 months of treatment and telomere length measured for seven patients at this time-point; all of them met end point criteria and showed telomere elongation at two years. The average increase in telomere length at 12 months was 1,119 bp (95% CI, 180-2,059 bp; P&lt;0.04), and 1,257 bp (95% CI, 675-1,839 bp; P&lt;0.02) at 24 months. The hematologic response rate was 60% at 3 months and 80% at 6 months. Only one patient relapsed during nandrolone use. Five of 8 transfusion-dependent patients became transfusion-independent, and one patient showed a reduction in transfusion requirements &gt;50%. The pulmonary response rate was observed in 2 of 6 patients who had a pulmonary disease at baseline, and one patient eventually became oxygen-independent. In our study, nandrolone decanoate treatment was safe and led to telomere elongation in patients with telomeropathies. Nandrolone also improved hematologic function in patients with marrow failure. Pulmonary function remained stable or ameliorated in two-thirds of patients with pulmonary involvement. Known nandrolone side effects were common, especially liver toxicity and virilization. OffLabel Disclosure: We want to determine if treatment with male hormone nandrolone decante is safe and improves hematologic response as first-line treatment in patients with telomere disease.