ALBERT

Description: 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 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.

Description: Excessive telomere erosion is the molecular etiology of a group of disorders (dyskeratosis congenita, aplastic anemia, idiopathic pulmonary fibrosis) collectively called telomeropathies. Telomere length measurement is an essential diagnostic test for these diseases. The most commonly used methods are terminal restriction fragment (TRF) analysis by Southern blotting (the gold-standard method), flow cytometry combined with fluorescence in situ hybridization (flow-FISH), and quantitative PCR (qPCR). Although the clinical use of these methods has been reported, their utility and characteristics have not been widely compared. Measurement techniques and coefficients of variations often differ among diagnostic services. Here, we directly compared the accuracy, reproducibility, sensitivity, and specificity of flow-FISH and qPCR in comparison to TRF to measure peripheral blood leukocyte’s telomere length in healthy individuals and patients with telomeropathies. TRF analyses and flow-FISH showed good correlation in the analysis of samples from healthy subjects (R2=0.60; p

Description: 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.

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

Description: 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: Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome known as the prototype of telomere diseases. In addition to the clinical triad (nail dystrophy, hyperpigmentation, and leukoplakia), very short telomeres (below the 1st percentile) is a marker for the diagnosis of DC (Calado & Young, NEJM 2009). Telomere dysfunction was associated with DC after discovery of DKC1 mutations in patients presenting the X-linked form of the syndrome. Novel mutations in telomere biology genes (TERC, TERT, NOP10, NHP2, TINF2, TCAB1, CTC1, RTEL1, ACD, and PARN) have been described in patients with DC. Variations in all these genes can affect telomere protection and maintenance, leading to telomere shortening and development of telomeropathies. In this study, we mapped the TERT, TERC, DKC1, and TINF2 genes for mutations in 15 patients (median age = 10 years; M/F = 11/4) with DC and very short telomeres, in order to classify these cases in a molecular level and determine the frequency of these mutations in our cohort. Survival for twelve patients who underwent allogeneic hematopoietic stem cell transplant (HSCT) was assessed and correlated with mutational status and telomere length. Diagnosis of DC was made according to the definition of Calado & Young (2009). Telomere length was measured in nucleated blood cells by flow-FISH and mutational screening was performed on genomic DNA extracted from peripheral blood cells by direct sequencing. Seven non-synonymous mutations were identified in TINF2 (40%), two in DKC1 (13%), one in TERT (6%), and one in TERC (6%). The TINF2 variants R282H and R282C had been already described as pathogenic, as well T66A and A353V DKC1 variants (Knight et al, 1999; Savage et al, 2008; Walne et al, 2008). The heterozygous variant R282H (c. 845 G〉A) in TINF2 were found in 4 unrelated patients. One of them also harbor the variants Q120R and Q157H in the same gene. The heterozygous mutation in TINF2 R282C (c. 844 C〉T) was found in one patient, that also presented the common polymorphism A279T in TERT. The pathogenic variants T66A (c.196A〉G) and A353V (c.1058C〉T) in DKC1 were found in two different male patients. Moreover, three novel mutations were identified in our cohort, r.94 C〉T in TERC, F290C in TINF2, and R696Cin TERT. The heterozygous mutation r.94 (C〉T) found in TERC was located at the pseudoknot P2b region of the gene and the patient who carries that presented a severe aplastic anemia and all DC clinical triad. The novel heterozygous F290C (c.859 T〉G) variant is located at the "hot spot" in exon 6 of TINF2 andwas found in one patient that presented a severe phenotype of DC. In silico analysis with SIFT and Polyphen-2 predicted that this variant is not tolerated and probably damaging, which is consistent with the pathogenicity of the mutation. The homozygous mutation R696C (c.2086 C〉T) in TERT was found in one patient and also in his two brothers. All of them presented reduced blood cell count, clinical features of DC, and severe aplastic anemia. The family screening identified the father and sister as heterozygous for the same mutation, but both asymptomatic. DNA sample from the mother were not available for this study. In silico analysis by SIFT and Polyphen 2.0, predicted that the R696C mutation is not tolerant and possible damaging to telomerase activity, respectively. To validate in silico analysis, TRAP assay with cell lysates obtained from telomerase-negative VA13 cell line transfected with wild type or R696C mutated TERT vector and TERC vector is under evaluation. Consistently with previously studies, telomere length in patients with TINF2 mutations were the shortest compared with the other telomeres genes mapped in this study. Although, the phenotype and severity of the disease does not appear to change according to the mutated gene. Also, the mutational status (p=0.28) or telomere length (p=0.21) did not influence the survival rates of patients after HSCT. Flow-FISH was able to identify patients with very short telomeres and validated telomere length measurement as a diagnostic tool for DC. Direct sequencing of the most commonly mutated genes in DC in a cohort of patients with telomeres below 1st percentile was able to characterize the genetic cause of this disease in more than 70% of the cases. The identification of genetic defect in DC can manage clinical decisions and is essential to genetic counseling prior to bone marrow transplantation. Disclosures No relevant conflicts of interest to declare.

Description: The pathophysiology of bone marrow failure (BMF) can be immune, as in acquired aplastic anemia (AA), or constitutional, due to germline mutations in genes critical for DNA repair and telomere maintenance. Variability in penetrance and phenotype can complicate diagnosis, as patients with underlying genetic defects may present in adulthood and without characteristic physical anomalies. RTEL1 encodes a helicase crucial for telomere maintenance and DNA repair. The gene has two main transcripts in human cells: the 1300 amino acid isoform 3 and the 1219 amino acid isoform 1. RTEL1 isoform 3 contains a conserved C4C4-RING domain responsible for resolving the t-loop required for telomere replication. Using next-generation sequencing (NGS), RTEL1 germline variants with unknown clinical significance have been found in AA patients. Functional tests may elucidate RTEL1 variants' pathogenic role in telomere biology. Here, we describe RTEL1 heterozygous germline mutations in patients with BMF and investigate their impact in telomere maintenance. We screened 63 patients with a suggestive familial phenotype for germline mutations in peripheral blood cells using a targeted, 49 gene NGS panel. To investigate variants' impact in telomere functions, telomere length (TL) was measured by Southern blot (SB), t-circles were quantified by telomere circle assay, and single-stranded overhang was measured by non-denaturing SB. Eight patients carried novel heterozygous non-synonymous RTEL1 variants: four nucleotide changes were located in the RAD3 domain, six in the harmonin-like domain, and one in the RING domain. Clinical features and TL were heterogeneous (Table 1). The only RTEL1 variant predicted as pathogenic in silico was F1262L (c.3786 C〉G) in patient 2; this mutation affects a highly conserved amino acid residue located in the RING domain, which is responsible for RTEL1 interaction with TRF2 at telomeres and t-loop unwinding. Patient 2 had very short telomeres, abnormal accumulation of t-circles, and eroded single-stranded telomeric overhangs in leukocytes, indicating a disrupted RTEL1 RING domain. To confirm observations made in clinical samples, 293T cells transfected with a plasmid carrying wild-type RTEL1-FLAG isoform 3 or its F1262L mutated version were assessed for TRF2 and FLAG co-localization in the nucleus. By confocal microscopy, wild-type RTEL1, but not mutant RTEL1 co-localized with TRF2. These findings strongly implicate RTEL1-F1262L as pathogenic, and thus the first autosomal dominant mutation in the RING domain in an AA patient. In patient 1, D743N variant in silico prediction was indeterminate, but telomeres were very short and there was a family history of typical telomeropathy (AA, liver cirrhosis, and pulmonary fibrosis) without any other suspicious germline mutations. The D743N variant is located close to the V745M variant that has been reported in a patient with dyskeratosis congenita. Increased amounts of t-circles and telomeric overhang attrition were observed in three other patients (#4, 5, and 7). While not specific for RTEL1 function, these results suggest telomere dysfunction, despite TLs in the normal range for patient 4 and 5. The RTEL1 P82L variant also appeared related to clonal evolution and leukemic progression observed in patient 5. For patients 3, 4, 6, 7, and 8, several mutations were observed in other genes concomitant to RTEL1, and a more complex genomic architecture may be the cause of patients' phenotype. A previously reported TERC variant, and a TERT variant of undetermined in silico prediction, could be pathogenic in patients 7 and 6, respectively. In these cases, RTEL1 variants may modulate disease, or represent only coincidental abnormalities. To our knowledge, this is the first report of heterozygous RTEL1 mutations in AA. We also describe a TL-independent association between RTEL1 haploinsufficiency and telomere dysfunction in humans. Haploinsufficiency of RTEL1 may disrupt DNA repair, destabilize the genome, and promote leukemogenesis by a mechanism different than typical accelerated telomere attrition associated with very short telomeres. T-circle quantification and overhang measurement may be better measures of telomere dysfunction in patients with RTEL1 variants than simple TL assessment. The combination of different functional tests was useful to the assessment of novel variants impact in telomere maintenance and DNA repair. Disclosures Fernandez Ibanez: GSK/Novartis: Research Funding. Desierto:GSK/Novartis: Research Funding. Townsley:GSK/Novartis: Research Funding. Young:GSK/Novartis: Research Funding.