Mutations in the Telomerase Complex and Expression Levels of the TERT Gene Determine Severity and Outcome of Disease in Aplastic Anemia Patients

Abstract Acquired Aplastic anemia (AA) is a bone marrow failure syndrome characterized by pancytopenia and marrow hypoplasia, and is mediated by immune destruction of hematopoietic stem cells. Mutations in several genes including telomerase, a ribonucleoprotein enzyme complex, consisting of a reverse transcriptase enzyme (TERT), an RNA template (TERC), and several stabilizing proteins, and the associated shelterin complexes have been found in both congenital and idiopathic AA. In particular, several TERT and TERC mutations reduce telomerase activity in vitro and accelerate telomere attrition in vivo. Shortened telomeres have been observed in a third of idiopathic AA patients, but only 10% of these patients have mutations in genes of the telomerase complex. We have recently demonstrated that in addition to keeping telomeres from shortening, telomerase directly regulates transcriptional programs of developmentally relevant genes (Ghosh et al, Nat Cell Biol, 2012, 14, 1270). We postulate that changes in expression of telomerase associated genes, specifically TERT, contribute to the etiology of aplastic anemia. In an effort to better understand the molecular and clinical correlates of this disease, 24 idiopathic AA patient samples were collected at a tertiary medical center in Bangalore, India. Following informed consent, we performed RT-PCR analysis on harvested RNA from each patient and measured levels of TERT expression compared to that of normal controls (n=6). An 8 fold reduction in TERT expression was observed in 17/24 patients, while 7/24 patients maintained normal TERT expression. In general, TERT-low patients were younger in age (mean age 29y) compared with the TERT-normal patients (mean age 40y). TERT-low patients were more likely to have severe aplastic anemia (SAA) leading to higher mortality and poorer response to therapy, with 6/17 patients dying and 4/17 not responding to ATG therapy. Targeted panel sequencing of the 24 samples on an Illumina platform revealed that while TERT-normal patients had no mutations in genes associated with the telomerase/shelterin complex, TERT-low patients carried predicted pathogenic variants in TERT, TEP1, TINF2, NBN, TPP1, HSP90A and POT1 genes, all associated with the telomerase complex. Somatic gene variants were also identified in other AA associated genes, PRF1 and CDAN1, in the TERT-low cohort. In addition, novel predicted pathogenic mutations associated with the shelterin complex were found in two TERT-low patients in the TNKS gene. We also detected mutations in TET2, BCORL1, FLT-3, MLP and BRAF genes in TERT-low patients. Mutations in these genes are associated with clonal evolution, disease progression and poor prognosis. Our observations were further illustrated in a single patient where normal TERT expression was noted at initial clinical presentation. ATG therapy led to CR, but the patient returned within a year and succumbed to E.coli related sepsis. At that stage he had low TERT expression, suggesting that TERT expression can change as the disease progresses. Taken together, our data support the hypothesis that loss of TERT expression correlates with disease severity and poor prognosis. Our observations further suggest that preliminary and periodic evaluation of TERT expression levels in AA patients is likely to serve as a predictor of disease severity and influence the choice of therapy. Disclosures No relevant conflicts of interest to declare.

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Paroxysmal Nocturnal Hemoglobinuria Clones Are Infrequent in Hepatitis-Associated Aplastic Anemia at the Time of Diagnosis

Blood ◽

10.1182/blood-2019-125620 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5016-5016

Author(s):

Wenrui Yang ◽

Xin Zhao ◽

Guangxin Peng ◽

Li Zhang ◽

Liping Jing ◽

...

Keyword(s):

Aplastic Anemia ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Clonal Evolution ◽

Extrinsic Factors ◽

Clone Size ◽

Hematopoietic Stem ◽

Over Time ◽

Pnh Clone

Aplastic anemia (AA) is an immune-mediated bone marrow failure, resulting in reduced number of hematopoietic stem and progenitor cells and pancytopenia. The presence of paroxysmal nocturnal hemoglobinuria (PNH) clone in AA usually suggests an immunopathogenesis in patients. However, when and how PNH clone emerge in AA is still unclear. Hepatitis associated aplastic anemia (HAAA) is a special variant of AA with a clear disease course and relatively explicit immune pathogenesis, thus serves as a good model to explore the emergence and expansion of PNH clone. To evaluate the frequency and clonal evolution of PNH clones in AA, we retrospectively analyzed the clinical data of 90 HAAA patients that were consecutively diagnosed between August 2006 and March 2018 in Blood Diseases Hospital, and we included 403 idiopathic AA (IAA) patients as control. PNH clones were detected in 8 HAAA patients (8.9%,8/90) at the time of diagnosis, compared to 18.1% (73/403) in IAA. Eight HAAA patients had PNH clone in granulocytes with a median clone size of 3.90% (1.09-12.33%), and 3 patients had PNH clone in erythrocytes (median 4.29%, range 2.99-10.8%). Only one HAAA patients (1/8, 12.5%) had a PNH clone larger than 10%, while 24 out of 73 IAA patients (32.9%) had larger PNH clones. Taken together, we observed a less frequent PNH clone with smaller clone size in HAAA patients, compared to that in IAAs. We next attempted to find out factors that associated with PNH clones. We first split patients with HAAA into two groups based on the length of disease history (≥3 mo and < 3mo). There were more patients carried PNH clone in HAAA with longer history (21.4%, 3/14) than patients with shorter history (6.6%, 5/76), in line with higher incidence of PNH clone in IAA patients who had longer disease history. Then we compared the PNH clone incidence between HAAA patients with higher absolute neutrophil counts (ANC, ≥0.2*109/L) and lower ANC (< 0.2*109/L). Interestingly, very few VSAA patients developed PNH clone (5%, 3/60), while 16.7% (5/30) of non-VSAA patients had PNH clone at diagnosis. We monitored the evolution of PNH clones after immunosuppressive therapy, and found increased incidence of PNH clone over time. The overall frequency of PNH clone in HAAA was 20.8% (15/72), which was comparable to that in IAA (27.8%, 112/403). Two thirds of those new PNH clones occurred in non-responders in HAAA. In conclusion, PNH clones are infrequent in HAAA compared to IAA at the time of diagnosis, but the overall frequency over time are comparable between the two groups of patients. In SAA/VSAA patients who are under the activated abnormal immunity, longer clinical course and relatively adequate residual hematopoietic cells serve as two important extrinsic factors for HSCs with PIGA-mutation to escape from immune attack and to expand. Disclosures No relevant conflicts of interest to declare.

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23 Years of Management: A Retrospective Review of Treatment for Aplastic Anemia.

Blood ◽

10.1182/blood.v114.22.1091.1091 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1091-1091

Author(s):

Connie M Piccone ◽

Marie Boorman Martin ◽

Zung Vu Tran ◽

Kim Smith-Whitley

Keyword(s):

Bone Marrow ◽

Aplastic Anemia ◽

Retrospective Review ◽

Pediatric Patients ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Complete Response ◽

Primary Objective ◽

Hematopoietic Stem ◽

Transfusion Independence

Abstract Abstract 1091 Poster Board I-113 Introduction Aplastic anemia (AA) is a syndrome of bone marrow failure characterized by peripheral pancytopenia and marrow hypoplasia. In the past, AA was considered to be a fatal disease; however, current therapies, including bone marrow transplantation or immunosuppressive therapy (IST) with antithymocyte globulin (ATG) and cyclosporine (CSA), are curative in the majority of patients. IST is effective at restoring hematopoietic stem cell production, but relapse and evolution to myelodysplastic syndromes remain clinical challenges. Additionally, there is no real consensus regarding optimal CSA levels, duration of CSA treatment, or the optimal use of growth factors and their relationship to the development of clonal disease. Objectives The primary objective was to review treatment management for severe AA in pediatric patients in order to elucidate treatment differences and review morbidity and mortality as they relate to treatment variation. Study Design/Methods A retrospective review of pediatric patients treated at the Children's Hospital of Philadelphia for AA (both severe and moderate) over a 23 year period was performed. Results A total of 70 patients with AA were treated at our institution from 1985 to July 2008. Exclusions included: 6 patients who received some type of initial treatment at outside institutions, 4 patients who had missing records, and 2 patients who had a diagnosis of moderate AA. Thus, a total of 58 patient records were included in the analysis. Of the total patients reviewed, 60% were male and 40% were female. 34.5% of patients were African-American, and 57% were diagnosed in 2000 or later. The mean age at diagnosis was 9.5±5.8 years. 52% fell into the category of very severe AA based on published diagnostic criteria, 45% had severe AA, and 2 patients (3%) had moderate AA. 15.5% of patients developed AA in the setting of acute hepatitis. More than half of the patients treated with IST had a complete response (CR). The average time to CR was 15±15 months. Average duration of CSA treatment was 15±13 months and 8.6±10.7 months for growth factor. Two patients (3.5%) died, one from complications unrelated to AA and one from infectious complications post-BMT after initial IST failure. Average time to transfusion independence for all patients was 8±11 months (with a range of 0-54 months). Not surprisingly, the time to transfusion independence was significantly associated with IST failure (p=0.010). Patients who failed treatment had an average time to transfusion independence of 17±16 months as compared to those who were complete responders who had an average time to transfusion independence of 3±3 months. Additionally, there was a significant association between IST failure and CSA levels (p=0.014). Patients who had nontherapeutic CSA levels overall had an increased rate of treatment failure. Of those patients who were nontherapeutic, 56% were noncompliant with CSA administration. There was no significant association between IST failure and bone marrow cellularity (p=0.251). PNH was diagnosed in 5% of patients; there were no patients with evidence of myelodysplastic syndrome (MDS). Two of the 3 patients with PNH failed initial IST. Another 2 patients had evidence of a cytogenetic abnormality (16q deletion), but never progressed to MDS. (Note: averages presented as mean±SD) Conclusions/Methods With current IST regimens, AA is curative in the majority of pediatric patients. IST failure was associated with nonadherence to CSA treatment. For patients with confirmed clonal disease, it is possible that IST failure and the ultimate development of clonal disease are related. Disclosures No relevant conflicts of interest to declare.

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Genetics in Inherited Bone Marrow Failure Disorders

Blood ◽

10.1182/blood.v126.23.sci-1.sci-1 ◽

2015 ◽

Vol 126 (23) ◽

pp. SCI-1-SCI-1

Author(s):

Sioban Keel

Keyword(s):

Bone Marrow ◽

Aplastic Anemia ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Accurate Diagnosis ◽

Cell Transplant ◽

Medical Monitoring ◽

Hematopoietic Stem ◽

New Genes ◽

Increased Risk

The classical Inherited Bone Marrow Failure Syndromes (IBMFS) such as Fanconi anemia, Dyskeratosis Congenita, Shwachman-Diamond syndrome, and Diamond-Blackfan anemia are a heterogeneous group of disorders, all of which are characterized by impaired hematopoiesis, varying degrees of peripheral cytopenias and marrow hypoplasia and dysplasia. Many of these are associated with an increased risk of clonal dominance and evolution to myelodyplastic syndrome (MDS) and acute myeloid leukemia (AML). For the purposes of this talk, the familial MDS and acute leukemia predisposition syndromes are also included in the broad term IBMFS. The genes responsible for a subset of IBMFS have been identified and will be reviewed. However, the causative mutations in many patients presenting with seemingly inherited marrow failure remain unknown. Gene discovery in IBMFS has been difficult in large part due to the phenotypic heterogeneity of these syndromes. Some patients with IBMFS display a distinct clinical phenotype with associated syndromic abnormalities, others are variable and overlap with one another or with acquired MDS or idiopathic acquired aplastic anemia, and additional cases are more obscure and have evaded classification altogether. Accurate diagnosis of IBMFS inform patient care as it allows appropriate screening of siblings to avoid choosing an affected donor if marrow transplant is indicated and the selection of an appropriate transplant conditioning regiment to avoid undue toxicity. Additionally, accurate diagnosis allows appropriate medical monitoring and early intervention to successfully treat disease-specific non-hematologic medical complications. The application of next generation sequencing approaches for comprehensive genetic screening of IBMFS, including these cryptic or atypical presentations will be reviewed. In addition to providing accurate diagnoses in a subset of patients, genetic characterization in small family kindreds or even in single individuals presents unique opportunities to discover new genes and pathways contributing to dysfunctional hematopoiesis and clonal progression. The frequency of inherited mutations in known IBMFS genes among seemingly idiopathic acquired aplastic anemia patients or pediatric and younger adults with MDS referred for hematopoietic stem cell transplant will be reviewed. Future genetic studies are needed to characterize the secondary genetic events that lead to disease progression in IBMFS. Disclosures No relevant conflicts of interest to declare.

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Chronic Infection Drives Hematopoietic Stem Cell Exhaustion through Differentiation and a Lowered Threshold for Apoptosis

Blood ◽

10.1182/blood.v126.23.2406.2406 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2406-2406

Author(s):

Katie A Matatall ◽

Mira Jeong ◽

Sun Deqiang ◽

Claudine Salire ◽

Katherine Y. King

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Aplastic Anemia ◽

Chronic Infection ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Terminal Differentiation ◽

Self Renewal ◽

Hematopoietic Stem ◽

Risk Of Death

Abstract Background: While inflammation is necessary to fight infection and repair damaged tissue, excessive inflammation can cause bone marrow suppression and promote cancer. In an extreme example, high levels of the inflammatory cytokine interferon gamma (IFNg) deplete hematopoietic stem cells (HSCs), resulting in aplastic anemia. Patients with this dangerous disease are pancytopenic and therefore at high risk of death from infection. Pancytopenia also occurs to a lesser extent in other inflammatory conditions such as chronic infections (tuberculosis, HIV), and autoimmune diseases (hemophagocytic histiocytosis). However, the mechanism by which HSCs are damaged by IFNg remains poorly understood. We used a mouse model of Mycobacterium avium infection to study the effects of sustained IFNg exposure on primitive hematopoiesis. In prior work, we found, surprisingly, that IFNg promotes division of quiescent HSCs. We hypothesized that cell division might lead to loss of HSCs through terminal differentiation, displacement, or activation of p53-dependent apoptosis pathways. Objective: We sought to determine whether prolonged IFNg stimulation would lead experimentally to exhaustion of the HSC compartment, and to determine the mechanism of inflammation-mediated HSC loss. Methods: We conducted repeated monthly infection of C57Bl/6 WT mice with 2 x 106 cfu M. avium, thereby generating a sustained chronic IFNg response. We characterized the blood and bone marrow of treated mice by histology, flow cytometry, colony forming assays, and bone marrow transplant. Results: Mice infected with M. avium became anemic and leukopenic after 6 months of repeated infection. High IFNg levels were sustained in the mice, with evidence of IFNg production by T cells and NK cells in the bone marrow. The number of committed hematopoietic progenitors gradually decreased and HSCs were depleted in the bone marrow by four months following initial infection, without evidence of extensive myelofibrosis. The marrow was hypercellular with a significant increase in granulocytes. Meanwhile, the myeloid differentiation capacity of the marrow was reduced, consistent with terminal differentiation of myeloid-biased HSCs, as we have previously described. Despite an overall reduction in HSC number, the HSCs that remained in chronically infected animals mostly retained their self-renewal potential, with subtle self-renewal defects evident only after two rounds of transplantation. Homing of HSCs from infected animals was not impaired, but ex vivo culture and apoptosis assays indicated that HSCs from chronically infected animals had reduced colony forming ability and were more prone to cell death upon secondary stress. These findings were recapitulated by introduction of recombinant IFNg alone. RNAseq profiling of HSCs from infected and control animals reflected increased proliferation and differentiation during infection, consistent with the above findings. Conclusions: We have established a novel mouse model of bone marrow failure related to chronic IFNg stimulation. We demonstrate that chronic infection can deplete the HSC pool by promoting HSC differentiation and lowering the threshold for apoptosis. These mechanisms may drive marrow suppression in patients with aplastic anemia, hemophagocytic histiocytosis (also associated with high IFNg levels), and patients with marrow failure associated with chronic infection. Furthermore, since a reduction in HSC number results in depletion of clonal heterogeneity, our findings have significant implications regarding the mechanism by which chronic inflammation can contribute to the emergence of clonal hematopoiesis and hematologic malignancies with age. Disclosures No relevant conflicts of interest to declare.

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Aplastic Anemia in Thailand: Incidence and Treatment Outcome from a Prospective Nationwide Population-Based Study

Blood ◽

10.1182/blood-2020-137047 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 8-9

Author(s):

Lalita Norasetthada ◽

Somchai Wongkhantee ◽

Jindaratn Chaipokam ◽

Kanyaporn Charoenprasert ◽

Suporn Chuncharunee ◽

...

Keyword(s):

Aplastic Anemia ◽

Anabolic Steroid ◽

Real World ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Population Based ◽

Annual Incidence ◽

Severe Aplastic Anemia ◽

Treatment Modalities ◽

Population Based Study

Background: Incidence of Aplastic Anemia (AA) in Asia tends to be higher than in western countries, but contemporary real-world incidence and outcomes of AA in Asia remain limited. This study aimed to explore the incidence across the country regions and to evaluate the patient outcomes according to age, the severity of disease, and treatment modalities. Method: This is a prospective multicenter nationwide population-based observational study of patients with AA aged over 15 years old, diagnosed between August 1st, 2014 to July 31st, 2016, with a longitudinal follow-up period over 2 years, from 30 medical centers. Patients with suspected hypocellular MDS and congenital bone marrow failure syndrome were excluded. Results: During the study period of 2 years, there were 348 newly diagnosed patients with aplastic anemia, giving the annual incidence of 4.6 per million inhabitants. There was a higher annual incidence of severe (SAA) and very severe aplastic anemia (VSAA) (3.8 per million) than non-severe aplastic anemia (NSAA) (0.8 per million). The incidence was greater among older patients with a peak incidence in patients aged 60-89 years old. (Figure 1) There was a high variation in the geographic incidences across country regions, ranging from 2.6 to 6.6 per million per year. (Figure 2) The 2-year overall survival (OS) for NSAA, SAA, and VSAA were 65.5%, 49.3%, and 20.1%, respectively (P < 0.001). Patients aged older than 60 years had the worst OS (42.6% as compared with 47.7% for the age 41-60 years and 64.5% for the age 15-40 years, P = 0.002). Among patients with SAA and VSAA (n = 280), the overall response rate (ORR) among patients treated with rabbit anti-thymocyte globulin and/or cyclosporin A (rATG±CsA) was significantly superior than those treated with CsA-based therapy and those treated with anabolic steroid (44.4% vs. 36.4% and 31.2%, respectively, P < 0.001). Among evaluable patients, ORR after the 1st treatment with rATG±CsA at 3, 6, 12 and 24 months were 23.9%, 43.8%, 68.4% and 89.2%, respectively. The 2-year OS among SAA/VSAA patients treated with rATG±CsA, CsA-based therapy, and anabolic steroid were 54.8%, 54.5%, and 37.6% (P = 0.037), respectively (Figure 3). From multivariate analysis, age > 60 years (HR 1.63, 95%CI, 1.14-2.33, P = 0.007), VSAA (HR 2.24, 95% CI, 1.45-3.46, P < 0.001) and not receiving immunosuppressive therapy or anabolic steroid (HR 4.96, 95%CI, 2.88-8.54, P <0.001), were independently associated with inferior OS among patients with SAA/VSAA. Conclusion: The incidence rate of AA in Thailand from this contemporary nationwide population-based study is high, especially in the elderly. Patients treated with rATG±CsA had superior survival than those receiving anabolic steroid. The real-world outcome of patients with SAA/VSAA, especially in those aged over 60 years, is substantially poor. Figure 1 Disclosures No relevant conflicts of interest to declare.

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Secondary CNL after SAA reveals insights in leukemic transformation of bone marrow failure syndromes

Blood Advances ◽

10.1182/bloodadvances.2020001541 ◽

2020 ◽

Vol 4 (21) ◽

pp. 5540-5546

Author(s):

Laurent Schmied ◽

Patricia A. Olofsen ◽

Pontus Lundberg ◽

Alexandar Tzankov ◽

Martina Kleber ◽

...

Keyword(s):

Bone Marrow ◽

Aplastic Anemia ◽

Myeloid Leukemia ◽

Bone Marrow Failure ◽

Driver Mutations ◽

Leukemic Transformation ◽

Hematopoietic Stem ◽

Bone Marrow Failure Syndromes ◽

Proinflammatory Responses ◽

Stem And Progenitor Cells

Abstract Acquired aplastic anemia and severe congenital neutropenia (SCN) are bone marrow (BM) failure syndromes of different origin, however, they share a common risk for secondary leukemic transformation. Here, we present a patient with severe aplastic anemia (SAA) evolving to secondary chronic neutrophilic leukemia (CNL; SAA-CNL). We show that SAA-CNL shares multiple somatic driver mutations in CSF3R, RUNX1, and EZH2/SUZ12 with cases of SCN that transformed to myelodysplastic syndrome or acute myeloid leukemia (AML). This molecular connection between SAA-CNL and SCN progressing to AML (SCN-AML) prompted us to perform a comparative transcriptome analysis on nonleukemic CD34high hematopoietic stem and progenitor cells, which showed transcriptional profiles that resemble indicative of interferon-driven proinflammatory responses. These findings provide further insights in the mechanisms underlying leukemic transformation in BM failure syndromes.

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A Complex Karyotype but Not Monosomy 7 Is an Independent Prognostic Factor in Advanced Childhood MDS.

Blood ◽

10.1182/blood.v110.11.2452.2452 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2452-2452

Author(s):

Gudrun Gohring ◽

Kyra Michalova ◽

Berna Beverloo ◽

David Betts ◽

Jochen Harbott ◽

...

Keyword(s):

Chromosome Aberrations ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Prognostic Significance ◽

Complex Karyotype ◽

Similar Frequency ◽

Hematopoietic Stem ◽

Monosomy 7 ◽

Significant Difference ◽

Secondary Mds

Abstract Disclosure: No relevant conflicts of interest to declare. To study the clinical significance of recurrent chromosome aberrations in childhood MDS, cytogenetic data of 394 consecutive children with refractory cytopenia (RC) (N=215), RAEB (N=141) and RAEB-T (N=38) analyzed in the regional cytogenetic reference centers and registered in the prospective study EWOG-MDS 98 between 1998 and 2005 were evaluated. At diagnosis, a karyotype could be defined in 279/394 patients (pts) (71%). No karyotype was obtained in 16% of pts with RC compared to 8% pts with RAEB and RAEB-t (p<0.001). Clonal chromosome aberrations were more common in pts with advanced MDS (RAEB and RAEB-T, 61%) compared to RC (29%), and in pts with secondary (69%) compared to primary MDS (36%) (p<0.001). Monosomy 7 was the most frequent aberration occurring with similar frequency in RC (47% of abnormal karyotypes) compared to advanced MDS (49%) and in primary (53%) compared to secondary (41%) MDS. In addition, aberrations typical for de novo AML such as aberrations involving 11q23 or 3q, t(6;9) and del(9q) were noted in morphologically and clinically unequivocal MDS cases. Recurrent aberrations of adult MDS like isolated del(5q), del(20q) and -Y were very uncommon indicating a different pathogenesis of these cases. In pts with advanced MDS, there was no significant difference in overall survival (OS) of pts with normal karyotype (44% ± 18) compared to pts with monosomy 7 (58% ± 19) and patients with other karyotypes (61% ± 22). However, pts with advanced MDS and a complex karyotype (defined by ≥ 3 chromosome aberrations, presence of structural aberrations and excluding clonal evolution of monosomy 7) had a shorter OS (16% ±15, p<0.01). OS and event-free survival after hematopoietic stem cell transplantation (HSCT) in pts with complex karyotypes was inferior compared to that of pts with other cytogenetic aberrations (p=0.012 and 0.039, respectively). Within the group of pts with secondary MDS, complex karyotypes were found in MDS evolving from inherited bone marrow failure disorders or after radio-/ chemotherapy, but absent in familial MDS and cases evolving from acquired aplastic anemia. As shown in a multivariate Cox analysis, advanced MDS, secondary MDS, the presence of a complex karyotype and HSCT were identified as independent prognostic factors for OS. Thus, this study demonstrates the prognostic significance of cytogenetic findings in advanced childhood MDS independent of HSCT.

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Long-Term Clinical Outcome of Children with Acquired Aplastic Anemia in Brazil.

Blood ◽

10.1182/blood.v114.22.4216.4216 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4216-4216

Author(s):

Marlene Pereira Garanito ◽

Vicente Odone Filho ◽

Marcela Vieira dos Santos ◽

Elvira Velloso ◽

Frederico L. Dulley ◽

...

Keyword(s):

Stem Cell ◽

Stem Cell Transplantation ◽

Aplastic Anemia ◽

Immunosuppressive Therapy ◽

Cell Transplantation ◽

Survival Probability ◽

Conflicts Of Interest ◽

Time History ◽

Hematopoietic Stem ◽

Significant Difference

Abstract Abstract 4216 Introdution/ Backgound Acquired Aplastic Anemia (AAA) is a rare hematologic disorder characterized by pancytopenia and hypocelular bone marrow. The pathophysiology is immune mediated in most cases. Environmental exposures to drugs, viruses and toxins, are thought to trigger the aberrant immune response in some patients. However, 50 to 74 percent of cases are classified as idiopathic. The highest frequency occurs in young population (15 to 25 years) with a second peak at age of 65 to 69 years. Immunosuppressive therapy is the best treatment in children with AAA who do not have a suitable donor for allogeneic stem cell transplantation. Materials and methods We reviewed the medical records of patients diagnosed with severe (SAA) and very severe acquired aplastic anemia (vSAA) at the Department of Pediatrics, Instituto da Criança – Hospital das Clínicas, University of Sào Paulo, Brazil from December, 1992 to December, 2007. We analyzed the clinical characteristics of the patients at diagnosis and the response to immunosuppressive therapy (IST) and hematopoietic stem cell transplantation (HSCT). Results In this study, 47 patients (27 boys and 20 girls), younger than 16 years, were diagnosed with vSAA (n= 21) or SAA (n=26). The median age was 7,71 years, ranging from 0.5 to 16 years and the average time history (beginning of signs and symptoms related to the disease and diagnosis) of the disease was 4,82 months, ranging from 0,25 to 48 months. Of the 47 patients, 45 had idiopathic AAA and 2 had hepatitis-associated. The median follow-up was 6,91 years for the patients treated with IST and 3,10 years for the patients who underwent to HSCT. One patient died before any treatment. For the eight patients who underwent to allogenic HLA-matched HSCT the 5-years-survival probability was 50%. For the 38 patients treated with IST, ten of them received cyclosporine and a short course of corticosteroids (CsA/CE) and 28 received antithymocyte globulin plus cyclosporine (ATG/CSA). The 5 years survival probability was 40% and 55%, respectively (p:0,0054). According to the severity of AAA, we did not show a significant difference in survival (p:0,32). Eight patients received second treatment after 1 year and 6 months (6 ATG from different species and CsA, 1 CsA and 1 thalidomide) and the probably of survival at 5 years was 60%. Among the 18 patients who responded to IST, four relapsed (22%). Two patients developed acute myeloid leukemia at 5 and 12 years after diagnosis. Conclusion Our results both for patients undergoing HSCT, as well as patients undergoing IST are lower in comparison to other hematological centers. Probably, this discrepancy is related to the prolonged time of disease when patients are admitted to our service. Unfortunately, the difficulty of access to specialized centers for diagnosis and early treatment in our country is a reality and this fact contributes to the delay to the beginning of treatment. Disclosures: No relevant conflicts of interest to declare.

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Akt-Mediated Phosphorylation of Bmi1 Regulates Its Chromatin Association and Growth Promoting Properties.

Blood ◽

10.1182/blood.v114.22.3605.3605 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3605-3605

Author(s):

Yan Liu ◽

Fan Liu ◽

Xinyang Zhao ◽

Goro Sashida ◽

Anthony Deblasio ◽

...

Keyword(s):

Stem Cell ◽

Signaling Pathway ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Akt Signaling ◽

Polycomb Group ◽

Akt Pathway ◽

Akt Signaling Pathway ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Abstract 3605 Poster Board III-541 The Polycomb group (PcG) protein Bmi1 maintains silencing of the Ink4a-Arf locus and plays a key role in stem cell self-renewal and oncogenesis. The phosphoinositide 3-kinase-Akt (PI3K-Akt) signaling pathway regulates cell survival, growth, metabolism, migration and angiogenesis. In response to acute Pten loss (which results in Akt activation), mouse embryonic fibroblasts (mefs) accumulate p16Ink4a and p19Arf and undergo senescence. Similarly, Bmi1 −/− mefs undergo premature senescence and accumulate p16Ink4a and p19Arf. PTEN and Bmi1 have similar effects on hematopoiesis; Pten deletion promotes hematopoietic stem cell (HSC) proliferation, resulting in HSC depletion, whereas loss of Bmi1 impairs HSC self-renewal capability, also leading to bone marrow failure. These similarities led us to examine whether the PI3K/Akt pathway functions upstream of Bmi1 to negatively regulate its function and indeed we found that PKB/Akt phosphorylates Bmi1 in vivo, which results in its dissociation from chromatin and in de-repression of the Ink4a-Arf locus. Furthermore, activation of the PI3K/Akt pathway suppresses the ability of Bmi1 to promote cell growth and tumourigenesis and decreases the global level of histone H2A ubiquitination. PI3K/Akt signaling is not active in hematopoietic stem cells, but it is active in more committed progenitor cells. Thus, phosphorylation and inactivation of Bmi1 by Akt may limit HSC self-renewal. Our study also provides a mechanism for the upregulation of p16Ink4a and p19Arf seen in cancer cells that have activation of the PI3K/Akt signaling pathway, and identifies important crosstalk between phosphorylation and chromatin structure. Disclosures: No relevant conflicts of interest to declare.

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Inherited Failure Syndromes.

Blood ◽

10.1182/blood.v114.22.sci-10.sci-10 ◽

2009 ◽

Vol 114 (22) ◽

pp. SCI-10-SCI-10

Author(s):

Grover C. Bagby

Keyword(s):

Relative Risk ◽

Diagnostic Tests ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Genetic Diagnosis ◽

Hematopoietic Cells ◽

Dyskeratosis Congenita ◽

Inherited Disorders ◽

Hematopoietic Stem ◽

Diagnostic Certainty

Abstract Abstract SCI-10 Global and lineage-restricted bone marrow failure syndromes can be acquired or inherited. Each of the known inherited disorders was initially described by observant clinicians before the field had access to modern tools of molecular biology and genetics. Consequently until recently, diagnosis had depended entirely on a clinical context drawn from the medical and family history, careful physical examinations and a few laboratory tests, none of which were pathognomonic. Today, many of the mutated genes responsible for these phenotypes have been identified and diagnostic tests of good reliability are emerging. This presentation will review the advantages and pitfalls associated with the application of newer diagnostic tests for many of these diseases. The molecular genetic insights have provided additional clinically relevant lessons. For example, the diseases are not limited to patients with the “classic” phenotype and can be diagnosed initially in adulthood. A substantial fraction of patients with Fanconi anemia (FA), for example, does not exhibit the cutaneous or skeletal manifestations of the disease. Some FA patients have entirely normal hematopoiesis, including the responses of hematopoietic cells to mitomycin C or diepoxybutane (abnormalities of which are considered to be the diagnostic standard for this disease). Such patients are “mosaics” in which a single hematopoietic stem cell has corrected the defect on one mutant FA allele and gives it and its progeny such a competitive advantage that they repopulate the entire marrow. Establishing the diagnosis in such cases is essential because the patients remain at high risk for squamous cell carcinoma and because their non-hematopoietic cells remain hypersensitive to cross-linking agents. Between these syndromes there are some shared genetic dysfunctions. For example, cells from patients with dyskeratosis congenita, Shwachman-Diamond syndrome, and Diamond-Blackfan anemia have genetic lesions that directly perturb ribosome biogenesis. Few of these disorders are caused by the inactivation of only one gene. There are at least 13 FA genes, more than 6 dyskeratosis congenita genes, more than one Shwachman-Diamond gene, more than 6 Diamond-Blackfan genes, and at least 7 genes for congenital neutropenia. Not surprisingly, some of these genes encode mutually interacting proteins (the most widely studied of these form a nuclear Fanconi interactome). While the identification of involved genes has advanced our levels of diagnostic certainty, the discoveries have posed many challenges and the list of unanswered questions is growing longer. The most cost-effective approaches to diagnosis are not perfectly defined and although mutation analysis is of profound research importance and required for certain management strategies (e.g. in vitro fertilization and pre-implantation genetic diagnosis) the clinical value of assigning patients to specific complementation groups or mutations has not yet been clearly demonstrated. Leading research questions for hematologists focusing on these disorders include: How do these disparate genetic lesions influence the function of hematopoietic stem cells so profoundly? What environmental influences play a role in marrow failure progression and do the mutant gene products interact biochemically with signals evolving from environmental cues? What accounts for the high relative-risk of myelodysplasia and acute leukemia in all of these disorders? Will effective gene therapy reduce the relative risk of MDS and AML? Hypotheses centering on each of these points are now being tested in a number of laboratories and some of them will be summarized in this presentation. Disclosures No relevant conflicts of interest to declare.

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