ALBERT

Description: Haploinsufficiency of GATA2 caused by heterozygous loss-of-function mutations is associated with cytopenias and predisposition to myelodysplasia and AML with other variable extrahematopoietic manifestions, including lymphedema, pulmonary alveolar proteinosis, and hearing loss. The authors report on 2 siblings with the disorder whose father was asymptomatic because of an acquired missense mutation in the affected allele that was restricted to hematopoietic cells; surprisingly, he also had no extrahematopoietic complications.

Description: Introduction: Burkitt lymphoma (BL) has become an AIDS-defining disease by Centers for Disease Control (CDC) definition since 1993, emphasizing the strong relationship between this subtype of non-Hodgkin Lymphoma (NHL) and the Human Immunodeficiency Virus (HIV) infection. Although it is widely recognized that the HIV-associated BL represents a distinct entity and a more difficult-to-treat disease, reported results from developed countries seem to corroborate that these regimens do not need to be tailored to the HIV-positive population. There is no available data on outcomes of this population in developing countries. In this study, we report a real-world cohort of adult HIV-associated BL patients in Brazil. Methods: This is a retrospective multicenter cohort encompassing 4 academic hospitals in Brazil. Ethics approval for the study was obtained in all centers. Patients were treated according to their local protocol, which included Hyper-CVAD, CODOX-M IVAC or CHOP-like regimens, on a clinical judgement basis. Further reductions in the dose could be done at discretion of physician in case of excessive toxicity. For all patients who were not using cART, HIV-directed therapy was promptly started and administered along with chemotherapy. A cytoreductive regimen could be administered prior to the actual regimen to minimize the risk of tumor lysis. Survival analysis was performed using the Kaplan-Meier method and log-rank test for comparison. Cumulative incidence of relapse (CIR) was calculated using death as a competitor. Results: A total of 54 patients with HIV-associated BL were included in this analysis. Median age was 39 years (range, 15 - 64) and the vast majority were male (78%). 41% of patients were using cART prior to the BL diagnosis. Advanced disease was found in 86% of patients. CNS disease was observed in 31%. Regarding the immune status at the presentation, 52% had CD4+ T-cells count lower than 200 cells/mm3 and 19% had undetectable levels of HIV viral load. Other features are summarized in Table 1. Five patients died before starting any regimen due to sepsis. Among the 49 patients, most were treated with a modified Hyper-CVAD (26/49, 53%), CODOX-M IVAC (9/49, 18%) and a CHOP-like regimen (8/49). Only 8/49 (16%) patients received rituximab in upfront therapy. Radiotherapy was used in 4/49 patients (2 cranial irradiation, 1 in initial bulky site and 1 in testicles). A treatment-related mortality of 38.7% (95% confidence interval [CI] 25.5 - 53.7) was found and the complete response (CR) rate was 44.9% (95% CI 30.9 - 59.6). Primary refractory disease was found in 14%. Regarding the patients who achieved a CR at the end of treatment, 3-year CIR was 6.2% (95% CI 1.6 - 15.7%). There was no recommended regimen for salvage, and most patients received an alternate classic regimen for BL if feasible. Only one relapsed patient proceeded to autologous stem-cell transplantation, but eventually died after a second relapse. Median follow-up time was 4.4 years. A 4-year OS of 36.1% (95% CI 25.2 - 51.8) for total cohort and 39.8% (95% CI 28.1 - 56.5) for those patients who received any regimen was calculated (Figure 1). A univariate analysis for CR and OS did not show any statistically significant difference regarding sex, age, staging, CD4+, viral load, prior cART, extranodal infiltration, blood counts, lactate dehydrogenase (LDH), employed regimen or use of rituximab. All relapsed and primary refractory patients eventually died in our cohort. Aside from those patients who died from disease progression per se, remaining patients died from infections (24/34), despite intensive antimicrobial prophylaxis and associated cART. Conclusion: Outcomes of HIV-positive BL population are scarcely reported worldwide. While HIV patients seem to have a higher relapse rate in other studies, our early mortality and overall survival were far worse than those reported in developed countries and with more selected cohorts. In comparison with literature, our patients experienced higher toxicity with classic regimens and higher refractoriness rate. Therefore, these findings warrant further cooperative multicenter studies in developing countries, aiming at improving supportive care and optimizing regimens locally feasible. Disclosures Delamain: Novartis: Honoraria.

Description: Telomerase reactivation by acquisition of mutations in the TERT promoter (TERTp) region is a mechanism of tumorigenesis. The most common TERTp mutations are located in positions -146, -124, and -57 upstream the initiation codon. In non-malignant diseases, TERTp mutations only have been reported in patients with idiopathic pulmonary fibrosis (IPF) caused by germline mutations in telomere biology genes, that are also etiologic in a broader spectrum of diseases collectively named telomeropathies (such as IPF, aplastic anemia [AA], dyskeratosis congenita [DC], and cirrhosis). We screened blood from 136 patients with telomeropathies (median age=29 years; range, 1-76), 52 relatives (median age=40 years; range, 8-72), and 195 controls using a customized low-cost amplicon-based next-generation sequencing (NGS) assay for identification and quantification TERTp mutations. Patients had DC (n=21), AA (n=86), IPF with or without another telomeropathy-related phenotype (n=18), or other phenotypes (n=11). Inclusion criteria were telomere length (TL) below the 10th percentile of age-matched controls or a germline mutation in a telomere-related gene classified as pathogenic/likely pathogenic or of uncertain significance by the ACMG criteria. Patients' relatives were only studied if they carried the same germline mutation as the proband or had short telomeres, regardless of symptoms or evidence of disease. Patients with acquired AA (n=70), IPF (n=12), other inherited bone marrow failure (n=7), and acute myeloid leukemia (AML; n=106) were controls. All TERTp mutations identified by NGS were confirmed and tracked over time by droplet digital PCR. We identified the -124 or -146 mutations in leukocytes from 12 unrelated patients diagnosed with IPF (n=6), DC (n=2), or moderate AA (n=4). Five relatives also had the -146 (n=1), and -124 (n=4) mutations, all carriers of a germline mutation in telomere biology gene. The frequency of TERTp mutations was much higher in IPF patients compared to AA cases (33% vs. 4.6%; Fisher's exact test, P=0.0016). However, no difference in frequency of TERTp mutations among patients with IPF vs. marrow failure was observed (41% vs. 58%; Fisher's exact test, P〉0.05), suggesting TERTp mutations occurred in both clinical presentations. MutTERTp clones positively correlated with age, as they were only present in individuals older than 18 years old and more frequent in those 60 to 80 years old. Also, TERTp mutations more frequently co-ocurred with germline TERT mutations (n=13) compared to mutations in TERC (n=2), RTEL1 (n=1), or DKC1 (n=2) (76% vs. 23%; Fisher's exact test, P=0.002). All germline variants were pathogenic or had some evidence of pathogenicity. MutTERTp clones size varied from 1.2% to 50% in total leukocytes and was at higher allele frequencies (VAF) in the granulocytic fraction from four patients. In serial samples (available for five patients), the mutTERTp clone size expanded over time, suggesting a selective growth advantage in comparison to unmutated hematopoietic cells. Despite that, mutTERTp clones did not associate with blood counts or telomere elongation; most subjects carrying a TERTp mutation, which is known to upregulate TERT expression, nevertheless had short or very short telomeres (15 out of 17 individuals). Six patients with mutTERTp clones (VAF ranging from 3-33% in myeloid cells) were treated with danazol for two years; four were responders and two were off-study after 3-6 months. In serial samples (available for two patients), the mutTERTp clone sizes decreased during danazol treatment while blood counts improved. After treatment, mutTERTp clones VAF increased. TERTp mutations were found in telomeropathy patients who had a germline variant in telomere biology genes but not in controls or patients with very short telomeres without germline variant in telomere biology genes. We have expanded the spectrum of non-malignant diseases associated with somatic TERTp mutations to DC, AA, and cirrhosis. Our data indicate that mutTERTp clones are specifically and randomly selected with aging in a marrow environment deficient in telomerase function, and mutTERTp selection did not associate with patients' peripheral blood counts, TL, and response to danazol treatment. TERTp emergence may be a useful clonal indicator for telomere dysfunction and may help to assess the pathogenicity of unclear constitutional variants in telomeropathies. Disclosures Young: CRADA with Novartis: Research Funding; National Institute of Health: Research Funding; GlaxoSmithKline: Research Funding.

Description: 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

Description: In patients with severe acquired aplastic anemia (AA) not eligible for hematopoietic stem cell transplant (HSCT), immunosuppression with horse antithymocyte globulin (h-ATG) and cyclosporine (CsA) is standard and is associated with a high response rate of about 60-70% at 6 months (Scheinberg et al. N Engl J Med 365: 430, 2011). Patients who are unresponsive to initial h-ATG can be rescued with rabbit ATG (r-ATG) in a third of cases (Scheinberg et al. Br J Haematol 133: 622, 2006). Since 2007, h-ATG is no longer available in most Latin American, Asian and European countries, with rabbit ATG (r-ATG) the only ATG formulation available in these markets. However, the outcome with r-ATG as first therapy in SAA is significantly inferior to that of h-ATG with worst response rate and overall survival (Scheinberg et al. N Engl J Med 365: 430, 2011). The salvage rate for patients who failed initial r-ATG is low with alemtuzumab and horse ATG [Scheinberg et al. Blood 119: 345, 2012; Am J Hematol 89: 467, 2014]. However, the salvage rate of a repeat course of r-ATG after initial r-ATG is unknown. Thus, we conducted a retrospective analysis in marrow failure referral Brazilian and Argentinian centers to address this question. The primary endpoint was hematologic response at 3 and 6 months, which was defined as transfusional independence and no longer meeting criteria for severe AA. Secondary endpoints included relapse, clonal evolution, and overall survival. Since 2005, 37 patients (32 refractory and 5 relapsed; 57% males, median age, 17 years; range 3-63 years) were re-treated with r-ATG (Thymoglobuline¨, Genzyme, Cambridge, MA, USA) with median dose of 3.5 mg/kg/d (range 1.67-5.0) for 5 days and oral cyclosporine adjusted to maintain blood levels between 150 and 400 ng/mL for 6 months. Corticosteroids, usually methylprednisolone, were given for at least 2 weeks to prevent serum sickness and trimethoprim–sulfamethoxazole was administered as Pneumocystis jiroveci prophylaxis. Only patients that completed the 5 day r-ATG course were included in the analysis. Second treatment was administered at a median of 283 days from first r-ATG/CsA (range 118-2379 days). After a median follow-up of 726 days (range 7-2320 days), the overall response rates at 3 and 6 months for initial r-ATG refractory patients was 5/32 (16%) and 7/32 (22%), respectively, and for those who relapsed 60% (3/5) (Table). Among all responders, 2 (20%) relapsed at 170 and 897 days after second treatment. In total, clonal evolutions were observed in 6 patients; 5 in non-responders (4 to monosomy 7; 1 trisomy 8 and 21), and 1 in a responder (myelodysplastic syndrome with normal karyotype). Three non-responders underwent matched-unrelated donor allogeneic HSCT. Twelve patients died, all who were non-responders to repeat r-ATG. Overall survival at 4.1 years (censored at the time of HSCT) was 58% (95% CI, 36-75%) (Figure). Our findings indicate that only a minority of r-ATG refractory AA patients may be successfully rescued with a second course of the same immunosuppression. Similar low salvage rates have been reported with alemtuzumab and h-ATG for those refractory to initial r-ATG. In the aggregate, these data show that: 1) other therapies should be considered for those refractory to initial r-ATG such as alternative donor HSCT, thrombopoietin agonists, or other experimental therapies; 2) the high success rate of initial h-ATG therapies cannot be recapitulated when r-ATG is administered first given the low salvage rate with alemtuzumab, h-ATG and now (current data) with r-ATG; 3) for patients that relapse after first r-ATG treatment, second course r-ATG may be a reasonable option. Table: Hematologic response at 3 and 6 months to second course of rabbit ATG plus cyclosporine Refractory to first r-ATG/CsA(n=32) 95% CI Relapsed to first r-ATG/CsA(n=5) 95% CI At 3 months no. (%) 5 (16) 3-28 3 (60) 17-100 At 6 months no. (%) 7 (22) 8-36 3 (60) 17-100 Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: 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