Genetics in Inherited Bone Marrow Failure Disorders

Blood ◽  
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.

KIT Blockade Is Sufficient to Sustain Donor Hematopoietic Stem Cell Engraftment in Fanconi Anemia Mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1206-1206
Author(s):  
Shanmuganathan Chandrakasan ◽  
Rajeswari Jayavaradhan ◽  
Ernst John ◽  
Archana Shrestha ◽  
Phillip Dexheimer ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Fanconi Anemia ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Conditioning Regimen ◽  
Cell Transplant ◽  
Hematopoietic Stem ◽  
Increased Risk

Abstract Background: Fanconi anemia (FA) is the most common cause of inherited bone marrow failure (BMF). Currently, the only curative option for the BMF in FA is an allogenic hematopoietic stem cell transplant (HSCT). However, due to the underlying DNA repair defect, FA patients poorly tolerate alkylating chemotherapy or irradiation based conditioning, which is necessary for donor engraftment. However, this results in significant short and long term morbidity/mortality and augments the inherent increased risk of malignancies in FA patients. To overcome the adverse effects associated with alkylating conditioning agents, alternate experimental approaches exploiting the inherent hematopoietic stem cell (HSC) defect in FA are of utmost clinical necessity. Objective: To develop a safe KIT blocking antibody (KIT-Ab) based HSCT conditioning regimen for FA that does not involve chemotherapy or irradiation. Method: High purity KIT-Ab was made from the ACK2 hybridoma and its specificity to KIT binding was validated using mast cell assay. Baseline peripheral blood cells and the bone marrow hematopoietic stem and progenitor cell (HSPC) compartment (Lin-Kit+Sca+ and Lin-Kit+Sca+CD150+CD48- cells) of FANCA-/- and FANCD2-/- murine models were analyzed. Mechanistic studies using sorted FA bone marrow HSPC were performed ex vivo. This was followed by definitive primary and secondary transplants experiments following injection of KIT-Ab. Results: Several features of FA hematopoietic stem/progenitor cells (HSPC) suggested their susceptibility to KIT-Ab blockade-mediated killing: (a) Expression of KIT was significantly lower in FANCA-/- HSPC, while expression of its ligand was higher in bone marrow stroma; (b) Moreover, genes associated with apoptosis/senescence, stress and inflammatory signaling that were upregulated in WT-HSPC following KIT-Ab blockade, were upregulated in FANCA-/- HSPC at baseline; (c) Furthermore, FANCA-/- HSPC demonstrated increased susceptibility to KIT-Ab mediated apoptosis and had a reduced proliferative capacity. In-vivo studies following ACK2 injection showed a marked reduction of colony-forming units (CFU-C) from both FANCA-/- and FANCD2-/- mice one week following injection, when compared to WT mice (48% and 76% decrease in CFU-C, respectively). Based on these findings, we evaluated the role of ACK2 as a sole HSCT conditioning regimen in FANCA-/- and FANCD2-/- mice. Indeed, definitive HSCT in both FANCA-/- and FANCD2-/- mice using KIT-Ab based conditioning resulted in donor HSC engraftment with multi-lineage chimerism, which progressively increased to 22-24% by 4-months, and was sustained in secondary transplants. Overall, we show that KIT-blockade alone is an adequate non-genotoxic HSPC-targeted conditioning in FA mice, and its clinical translation could circumvent the extensive transplant-related morbidity/mortality in this disease. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽  
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.

Excelent Option Therapy of BONE Marrow Failure in Fanconi Anemia Patients Withouth Full Match Donnor

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5075-5075 ◽  
Author(s):  
Lisandro L Ribeiro ◽  
Samantha Nichele ◽  
marco Antonio Bitencourt ◽  
Ricardo Petterle ◽  
Gisele Loth ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Hematological Parameters ◽  
Severe Neutropenia ◽  
Cell Transplant ◽  
Variable Degree ◽  
Duration Of Treatment ◽  
Hematopoietic Stem ◽  
Hematological Response

Abstract The main cause of morbidity and mortality of FA pts is bone marrow failure (BMF), which usually arises in the first decade of life and progresses to transfusion dependence and severe neutropenia. Androgen treatment has been recommended for FA pts with BMF for whom there is no acceptable hematopoietic stem cell transplant donor. Oxymetholone and Danazol are frequently used in these pts. We retrospectively analyzed data on 67 FA pts who received oxymetholone or danazol for the treatment of their BMF. The starting dose was approximately 1mg/kg for oxy and 2-4mg/kg for danazol. The hematological parameters at the initiation of treatment were hemoglobin (Hb) < 8 g/dL and/or thrombocytes < 30.000/μl. Patients were diagnosed between 01.2005 and 01.2016. The median age was 10.5 ys (2.9 - 40ys). Gender: 39M/27F. The median duration of treatment was 18m (3m - 95m). Fifty-three patients (79%) showed hematological response and became transfusion independence at a median of 3 months after beginning oxymetholone (2-9m) and 5 months after danazol (4-7m). Two adult pts treated with danazol achieved total hematological response with 2.5mg/kg. Seven pts are stable after tapering and stopping androgen with a median follow up of 4 ys (6m-8.5ys). Fourteen pts did not respond to treatment (21%). Eleven pts received an HSCT and seven are alive and well. Three pts were not transplanted and two are alive but transfusion dependent and one pt died from CNS bleeding. All patients developed variable degree of virilization but it was more evident with oxymetholone therapy. Older age at starting therapy was related to less virilization. Conclusion: This study shows the largest number of FA pts treated with androgen up till now. Androgen is an effective and well-tolerated treatment option for FA pts who develop BMF with 79% of them showing transfusion free after 3-5 months. This response may give us time to search for better donors. Disclosures No relevant conflicts of interest to declare.

Acute Lymphoblastic Leukemia in a Patient with Monomac Syndrome/GATA2 Haploinsufficiency

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3729-3729
Author(s):  
Ashley Koegel ◽  
Venee N. Tubman ◽  
Inga Hofmann
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Lymphoblastic Leukemia ◽  
Killer Cell ◽  
Cell Transplant ◽  
Hematopoietic Stem ◽  
Monosomy 7 ◽  
Definitive Therapy

Abstract Background: Heterozygous germline mutations in GATA2 have been described in three distinct conditions: 1) familial myelodysplastic syndrome (MDS)/ acute myeloid leukemia (AML), 2) Emberger syndrome which is characterized by lymphedema, warts and predisposition to MDS/AML, 3) MonoMac syndrome which is comprised of atypical nontuberculous mycobacterial infection, monocyte, and B and natural killer cell lymphoid deficiency. It is now recognized that these conditions represent a spectrum of hematopoietic, lymphatic and immune system disorders due to GATA2 haplosinsufficiency. MDS/AML due to GATA2 mutation shows a unique histopathology with characteristic dysplasia and is often associated with monosomy 7. Although many patients with GATA2 haploinsufficiency are initially asymptomatic the majority of patients will ultimately experience a significant complication such as severe infections due to immunodeficiency, pulmonary alveolar proteinosis (PAP), thrombotic events, bone marrow failure, MDS and progression to AML. Allogenic hematopoietic stem cell transplant (HSCT) is the only curative treatment for patients with GATA2 haploinsufficiency and those who develop MDS/AML. Here we report a unique patient who presented with with acute lymphoblastic leukemia (ALL) and was later found to have classical features of MonoMAC syndrome and GATA2 haploinsufficiency. Case Summary: A previously healthy 11 year-old girl presented with fever, cellulitis, and pancytopenia. Bone marrow biopsy and aspirate were diagnostic for B-precursor acute lymphoblastic leukemia (ALL) with associated monosomy 7 and the following karyotype: 45,XX,-7,del(9)(p13),del(10)(q24). She was treated on Dana Farber Cancer Institute (DFCI) Consortium ALL Protocol 05-001, achieving a morphological and cytogenetic remission. During induction, she developed necrotizing aspergillus pneumonia and molluscum contagiousum. Her planned course of therapy was abbreviated due to the development of restrictive lung disease associated with PAP and disseminated Mycobacterium kansasii infection. Serial off therapy bone marrow studies were obtained given poor count recovery and revealed significant morphologic dysplasia, most prominent in the megakaryocytes. These findings were reminiscent of those characteristically seen in patients with GATA2 haploinsufficiency. Her infectious complications, profound monocytopenia, PAP and bone marrow dysplasia raised concern for MonoMAC Syndrome. Sanger Sequencing of GATA2 revealed a point mutation in the regulatory enhancer region of intron 5 (c.1017+572C>T) confirming the diagnosis. More than 3 years following remission of ALL, she developed a bone marrow relapse with her initial clone. Given her diagnosis of GATA2 haploinsufficiency, HSCT was selected as consolidation therapy in second remission. She succumbed to complications of HSCT 4 months after transplantation. Conclusion: Patients with GATA2 haploinsufficiency show a heterogeneous clinical presentation and are at high risk for MDS/AML often associated with monosomy 7. The development of ALL in association with GATA2 haploinsufficiency has not been described in the literature. Hematologist and oncologists should be aware that ALL may be associated with GATA2 haploinsufficiency and should be attuned to the clinical, laboratory and histopathologic features of the MonoMAC syndrome that would prompt additional testing and potentially alter treatment regimens. As allogenic HSCT is the only definitive therapy for patients with GATA2 mutation, consideration of immediate HSCT following induction of remission should be considered in patients with ALL and GATA2 haploinsufficiency. Further, as patients with GATA2 mutations can be asymptomatic, it is imperative to screen family members for GATA2 mutations and offer genetic counselling prior to consideration as potential bone marrow donors. Disclosures No relevant conflicts of interest to declare.

scholarly journals Clinicohematological Characteristics and Survival Analysis of Aplastic Anemia in Pakistan; A Single Centre Experience

2021 ◽  
Vol 5 (4) ◽  
pp. 157-161
Author(s):  
Nida Anwar ◽  
◽  
Aisha Arshad ◽  
Naveena Fatima ◽  
Sumaira Shaheen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Aplastic Anemia ◽  
Clinical Characteristics ◽  
Standard Treatment ◽  
Bone Marrow Failure ◽  
Treatment Options ◽  
Total Leucocyte Count ◽  
Cell Transplant ◽  
Hematopoietic Stem ◽  
Male Predominance

Abstract: Objective: Aplastic anemia (AA) is characterized by pancytopenia and hypocellular bone marrow. Several factors like infections, toxins, chemotherapeutics and radiations are known causes for the suppression of hematopoietic cells which results in bone marrow failure but the exact etiology is unknown. The current study was conducted to assess the baseline demographics, laboratory and clinical characteristics of patients presenting with aplastic anemia and evaluate their cytogenetic profile. Materials and Methods: A retrospective cohort study conducted at National Institute of Blood Diseases and Bone Marrow Transplantation after approval by Institutional Ethics Committee. In this study, AA patients were enrolled from January 2010 to December 2018. Data collection included demographic, laboratory and clinical characteristics including age, gender, symptoms, treatment, and blood counts. Cytogenetic analysis was performed on bone marrow samples. Data analysis was done by using SPSS version 23. Results: Based on camitta classification, a total of 362 aplastic anemia patients were enrolled in the study. The frequency of severe aplastic anemia was most common 199(55%). Median and interquartile range (IQR) age of overall patients was 17(11-26) years, for children and adult population it was 12(9-14) years and 28 (21-43) years respectively. Male predominance was observed i.e.251(69%). The most common presenting complaint was fever 202(55.8%). The median and IQR of hemoglobin (Hb) was 7.8(5.8-9.4)g/dl, MCV 90(83-91)fl, total leucocyte count (TLC) 2.6 (1.9-3.6) × 109/l, absolute neutrophil count (ANC) 0.64 (0.27-1.2)×109/l and platelet count 13 (5-27) ×109/l. Bone marrow cytogenetics was done and 76 (67%) patients were found to have normal karyotype. CMV was positive in 24(6.6%). Majority of patients were treated with blood transfusion and supportive care only 230(64%) and the survival rate was 84%. Conclusion: In conclusion, the study represents a large cohort of aplastic anemia in the country. Majority of cases were acquired aplastic anemia predominantly being severe aplastic. Limited numbers of patients opted for standard treatment options probably due to financial reservations to afford standard treatments like immune suppression therapy and hematopoietic stem cell transplant. Further, local studies with larger number of sample size and provision of standard treatment options are needed to explore the treatment response, etiological factors, prognosis and outcomes. Keywords: Aplastic anemia, Cytogenetics, Clinicohematological characteristics, Laboratory analysis, Survival outcome, Pakistan.

scholarly journals Rpl22 Deficiency Predisposes Hematopoietic Stem and Progenitor Cells to Leukemogenesis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 899-899 ◽  
Author(s):  
Bryan Harris ◽  
Jaqueline Perrigoue ◽  
Rachel M. Kessel ◽  
Shawn Fahl ◽  
Stephen Matthew Sykes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Stem Cell ◽  
Ribosomal Protein ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Ectopic Expression ◽  
Hematopoietic Stem ◽  
Increased Risk

Abstract Mutations and deletions in ribosomal proteins are associated with a group of diseases termed ribosomopathies. Collectively, these diseases are characterized by ineffective hematopoiesis, bone marrow failure, and an increased risk of developing myelodysplastic syndrome (MDS) and subsequently acute myeloid leukemia (AML). This observation highlights the role of dysregulation of this class of proteins in the development and progression of myeloid neoplasms. Analysis of gene expression in CD34+ hematopoietic stem cells (HSC) from 183 MDS patients demonstrated that ribosomal protein L22 (Rpl22) was the most significantly reduced ribosomal protein gene in MDS. Interestingly, we observed that AML patients with lower expression of Rpl22 had a significant reduction in their survival (TCGA cohort, N=200, Log Rank P value <0.05). To assess the mechanism of reduced expression, we developed a FISH probe complementary to the RPL22 locus and assessed for deletion of this locus in an independent set of 104 MDS/AML bone marrow samples. Strikingly, we found that RPL22 deletion was enriched in high-risk MDS and secondary AML cases. We, therefore, sought to investigate whether reduced Rpl22 expression played a causal in leukemogenesis. Using Rpl22-/- mice, we found that Rpl22-deficiency resulted in a constellation of phenotypes resembling MDS. Indeed, Rpl22-deficiency causes a macrocytic reduction in red blood cells, dysplasia in the bone marrow, and an expansion of the early hematopoietic stem and progenitor compartment (HSPC). Since MDS has been described as a disease originating from the stem cell compartment, we next sought to determine if the hematopoietic defects were cell autonomous and resident in Rpl22-/- HSC. Competitive transplantation revealed that Rpl22-/- HSC exhibited pre-leukemic characteristics including effective engraftment, but a failure to give rise to downstream mature blood cell lineages. Importantly, there was a strong myeloid bias in those downstream progeny derived form Rpl22-/- HSC. Because human MDS frequently progresses to AML, we examined the potential for Rpl22-deficient HSC to be transformed upon ectopic expression of the MLL-AF9 oncogenic fusion. Indeed, Rpl22-deficient HSPC exhibited an increased predisposition to transformation both in vitro and in vivo, in MLL-AF9 knockin mice. To determine how Rpl22-deficiency increased the transformation potential of HSC, we performed whole transcriptome analysis on Rpl22-/- HSC. Interestingly, four expression signatures were observed that were consistent with the altered behavior exhibited by Rpl22-/- HSC. Rpl22-deficient HSC exhibited increased expression of: 1) genes associated with stem cell function, consistent with the basal expansion and effective engraftment of Rpl22-/- HSC upon adoptive transfer; 2) markers of the myeloid lineage, providing a potential explanation for the myeloid bias exhibited by Rpl22-/- HSC; 3) cell cycle regulators, consistent with the increased proliferation exhibited by Rpl22-/- HSC; and 4) components of the mitochondrial respiratory chain, a metabolic program on which leukemic stem cell function depends. Together, these data suggest that Rpl22 controls a program of gene expression that regulates the predisposition of HSPC to myeloid transformation. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽  
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.

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

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1502-1502 ◽  
Author(s):  
Arati Khanna-Gupta ◽  
Durga Sarvepalli ◽  
Snigdha Majumder ◽  
Coral Karunakaran ◽  
Malini Manoharan ◽  
...  
Keyword(s):  
Aplastic Anemia ◽  
Disease Severity ◽  
Poor Prognosis ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Response To Therapy ◽  
Hematopoietic Stem ◽  
Expression Levels ◽  
Shelterin Complex ◽  
Tert Expression

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.

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

Blood ◽  
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.

scholarly journals The Platelet Millionaire: A Rare Cause of Thrombocytosis in an Adolescent

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5391-5391
Author(s):  
Ritika Walia ◽  
Theresa Sepulveda ◽  
Sharon Wretzel ◽  
Philip H Brandt
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Biopsy ◽  
Primary Care Physicians ◽  
Conflicts Of Interest ◽  
Abdominal Ultrasound ◽  
Cell Transplant ◽  
Peripheral Smear ◽  
Hematopoietic Stem ◽  
Marrow Fibrosis

Objectives: Primary myelofibrosis is rare in pediatrics, often manifesting as persistent idiopathic thrombocytosis.Transitions from pediatric to adult medical care can be complicated by workup requiring invasive procedures. J.M., an 18-year-old healthy male, presented for excessive gingival bleeding after wisdom tooth extraction. Workup revealed persistent thrombocytosis to 1,165K, prompting a referral to hematology-oncology. A peripheral smear was notable for many platelets but normal RBC morphology. He had splenomegaly on abdominal ultrasound and a decreased von-Willebrand's activity to antigen ratio, suggesting acquired vWD. A bone marrow biopsy was advised; however, J.M. became lost to follow up for over 9 months owing to self-reported anxiety about the procedure. He remained asymptomatic in this interim until he re-presented to clinic for easy bruising, with no other evidence of bleeding at the time. The biopsy was pursued, revealing hypercellular marrow for age with left shifted granulocytic and erythroid maturation, abnormal megakaryocytes, and 3% blasts. This was consistent with primary early myelofibrosis (PMF), positive for MF-1, CALR, and TP53 mutations and negative for JAK2 and BCR-ABL. He was transitioned to adult hematology, maintained on baby aspirin, and referred for potential allogeneic hematopoietic stem cell transplant (HSCT). PMF is characterized by marrow fibrosis due to secretion of fibroblast growth factor by clonally proliferative megakaryocytes. It is a disease of adulthood, with 67 years being the median age at diagnosis. Only 100 cases have been reported in children, most of which are secondary to AML, ALL or other malignancies.1 Most patients present with complications of extramedullary hematopoiesis or bleeding.2 Diagnosis is suggested by a leukoerythroblastic picture on peripheral smear and confirmed with a bone marrow biopsy "dry tap" revealing marrow fibrosis.3 Prognosis in pediatric PMF is difficult to predict but outcomes tend to be worse;4 TP53 mutation is rare and based on limited adult studies may portend a poorer prognosis.5 Our young patient with this rare mutation was therefore referred for HSCT evaluation. Further complicating this case was J.M.'s anxiety, which delayed definitive diagnosis by biopsy. He only agreed to it when, at the med-peds clinic, the concept of local pain management was discussed. Anticipation of upcoming procedures by primary care physicians and close follow-up is especially important for patients transitioning from pediatric to adult providers. Disclosures No relevant conflicts of interest to declare.

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