Published on September 26, 2018
1. Bone Marrow Failure Syndromes Idiopathic Aplastic Anemia Paroxysmal Nocturnal Hemoglobinuria (PNH) Myelodysplastic Syndrome (MDS) Constitutional (Congenital/ Inherited) Aplastic Anemia Abdul-Kader Souid Peripheral blood showing pancytopenia Normal peripheral blood Fatty bone marrow in aplastic anemia Normal cellular bone marrow http://www.healthsystem.virginia.edu/internet/hematology/HessImages/Aplastic-Anemia-Pancytopenia-and-macrocytes-40x-website.jpg moon.ouhsc.edu/kfung/JTY1/Com05/Com509-1-Diss.htm
2. • Idiopathic aplastic anemia as a result of autoreactive T-cells that destroy the hematopoietic cells (anhematopiesis). • Paroxysmal nocturnal hemoglobinuria (PNH) as a result of complement-mediated intravascular hemolysis (clonal disease). • Myelodysplastic Syndrome (MDS) = Cytopenia + oligoblastic leukemia (clonal disease). • Constitutional/ congenital/ inherited: Diamond-Blackfan anemia (DBA, ribosomopathy); Fanconi anemia (FA, defect in DNA repair); dyskeratosis congenita (DC, short telomere). • Viruses: Epstein-Barr virus, HIV, seronegative hepatitis. • Toxic exposure: Radiation, benzene, pesticides (organophosphates), chlorinated solvents (e.g., chloroform), anticancer drugs (cyclophosphamide, etoposide). • Idiosyncratic (peculiar): Chloramphenicol, NSAID, sulfonamides, anti-epileptic drugs, psychotropics. Bone Marrow Failure Syndromes: Etiology
3. Bone Marrow Failure Syndromes: Definition • These disorders are characterized by “near absence of hematopoietic cells”. The marrow is replaced by fat cells (“fatty marrow”). • Clinical manifestations include various severities of anemia, neutropenia, lymphopenia, and thrombocytopenia. • In most cases, the disease occurs without a known precipitating cause (termed “idiopathic aplastic anemia”), resulting from “autoreactive T-cells” that destroy the hematopoietic cells. • Severe aplastic anemia is defined as “bone marrow cellularity <25% with 2 of the following cytopenias: neutrophil count <0.5 x109/L, platelet counts <20 x109/L, and reticulocyte count <40 x109/L. • All patients need bone marrow biopsy, cytogenetic studies for clonal abnormalities, chromosome breakage for Fanconi anemia by diepoxybutane [DEB] test, flow cytometry for CD55/59 for paroxysmal nocturnal hemoglobinuria (PNH), and telomere length study for dyskeratosis congenita (DC).
4. • Mutations in telomere repair genes, e.g., TERT (OMIM#187270), the gene for telomerase reverse transcriptase (a ribonucleoprotein polymerase that maintains telomere ends by adding the telomere repeat TTAGGG); NEJM 2005;352:1413-1424. (OMIM#614742) • Inheritance of such mutations results in short telomeres in the hematopoietic cells, which predispose to apoptosis. Genetic Susceptibility to Marrow Failure A telomere is a region of repetitive nucleotide sequences (TTAGGG) at each end of a chromatid, which protects the end of the chromosome from deterioration. The telomere is short in dyskeratosis congenita (DC; OMIM#613989). NaildystrophyinDC
5. Case Presentation • A 9-year-old boy presents with pallor and bruises. He has increased fatigue and bleeding from his gum. His examination reveals fever (38.6oC), tonsillitis, pallor, and ecchymoses. The liver, spleen, and lymph nodes are not palpable. – WBC count 0.8 x109/L; neutrophil 0.3 x109/L; lymphocyte 0.5 x109/L. – Hemoglobin 73 g/L; RBC count 2.1 x1012/L; reticulocyte count 7 x109/L; MCV 110 fL, RDW 14%. – Platelet count 13 x109/L. – Blood film shows pancytopenia without abnormal cells. 5 Problem list = Leukopenia + Neutropenia + Lymphopenia + Macrocytic anemia + Reticulocytopenia + Thrombocytopenia
6. Idiopathic Aplastic Anemia: Treatment • Supportive care include blood and platelet transfusions, antibiotics, and growth factors [granulocyte colony-stimulating factor (G-CSF), thrombopoietin, erythropoietin]. • The disease is curable with hematopoietic stem-cell transplantation from allogeneic HLA-matched (usually sibling) donor. • About 80% of patients with no HLA-matched donor show hematopoietic recovery after transient T-cell depletion by anti- thymocyte immunoglobulins (ATG, cytolytic antibodies) + cyclosporine (an immunosuppressant that reduces T-cell function); relapse usually responds to repetitive treatment. 9/26/2018 6
7. Complement-mediated Hemolysis CD59 (terminal complement inhibitor) is tethered to the membrane by GPI anchor (glycosylphosphatidylinositol). PNH (paroxysmal nocturnal hemoglobinuria) cells lack GPI and CD59. Consequently, complement activation creates numerous pores in the membrane. J Cell Biochem 1986;30:133-170 7 Paroxysmal Nocturnal Hemoglobinuria (PNH) (OMIM#300818; intravascular hemolysis + cytopenia + venous thrombosis)
8. • PNH is a clonal hematopoietic stem cell disorder caused by somatic de novo (neither transmitted nor parent possessed) mutation in the phosphatidylinositol glycan class A (PIGA) gene (OMIM#311770) on the X chromosome. • PIGA protein is required for formation of the phosphatidylinositol (GPI) anchor. Its absence results in missing many membrane proteins, including inhibitors of the complement cascade. Red cells are especially sensitive to the hemolytic effect of complements. • The disease usually evolves as an abnormal clone in the milieu of a bone marrow failure syndrome. 8 Paroxysmal Nocturnal Hemoglobinuria (PNH) (OMIM#300818; intravascular hemolysis + cytopenia + venous thrombosis)
9. • The clinical presentation includes chronic intravascular hemolytic anemia, venous and arterial thrombosis (commonly in abdominal and cerebral vessels; leading cause of death), abdominal pain and esophageal spasm. – Depletion of nitric oxide (NO) from increased free hemoglobin leads to smooth muscle dystonias (abdominal pain, dysphagia, pulmonary hypertension, erectile dysfunction). – The most common severe complication is thrombosis. Testing for PNH when patients present with unprovoked thrombus, particularly if they have hemolysis or cytopenia. • Diagnosis is by flow cytometry using anti-CD59 (granulocytes missing the terminal complement inhibitor, CD59). • Treatment is supportive with transfusion, folate/B12/iron, and anticipatory guidance about ↑bacterial infection and ↑thrombosis. – Therapeutic options also include stem-cell transplantation and eculizumab (Soliris™, monoclonal antibodies to the complement protein C5). Eculizumab blocks cleavage of C5, thus, halting complement-mediated cell destruction. It deceases the hemolysis, transfusion requirement, and debilitating fatigue. Paroxysmal Nocturnal Hemoglobinuria (PNH) (OMIM#300818; intravascular hemolysis + cytopenia + venous thrombosis)
10. 9/26/2018 Brodsky RA. Paroxysmal nocturnal hemoglobinuria. Blood 2014;124:2804-11. doi: 10.1182/blood-2014-02-522128.
11. • A 17-year-old male presents with fatigue, pallor (anemia), abdominal pain, dysphagia, and red urine (hematuria). He is icteric (jaundice = hemolysis). • Hemoglobin is 84 g/L, reticulocytes 322 x109/L (hemolysis), platelets 96 x109/L (bone marrow disease). • Lactate dehydrogenase (↑LDH = hemolysis) is 2,950 units/L (normal, <200). • Urinalysis reveals hemoglobinuria (+hemoglobin with no red cells = free hemoglobin in the blood = intravascular hemolysis). • Flow cytometry using anti-CD59 reveals granulocytes missing the glycosylphosphatidylinositol–(GPI) anchored protein. • He is being treated with weekly eculizumab. Problem list = Anemia + Intravascular Hemolysis + Thrombocytopenia (bone marrow disease) + GI complaints Case Presentation
12. Myelodysplastic Syndrome (OMIM#614286): “Clonal Cytopenia + Oligoblastic Leukemia” • MDS (myelodysplasia) is ineffective hematopoiesis (cellular bone marrow + pancytopenia) resulting from an evolving clone of genetically injured hematopoietic stem cells. – Cytogenetic abnormalities exist in most patients; most commonly involving chromosomes 5, 7 and 8. • Cases could be sporadic (de novo) or result from stem cell injuries, e.g., – Cyclophosphamide (AML-associated with monosomy 5 or 7; (del)5q or (del)7q) – Etoposide (AML-associated with rearrangements involving the mixed lineage leukemia, MLL [MIM#602409], gene on chromosome 11q23) • Patients present with uni-lineage, bi-lineage or tri-lineage (pancytopenia), progressing to acute myelogenous leukemia in many of the cases.
13. Myelodysplastic Syndrome: “Clonal Cytopenias and Oligoblastic Leukemia” A 16-y-old girl is treated for Hodgkin lymphoma at 10 y of age. She received standard chemotherapy and radiation. She now presents with fatigue and pallor. CBC reveals pancytopenia. Bone marrow examination shows myelodysplasia with 30% myeloblasts possessing a deletion of chromosome 7. Which of the following is the most likely cause of her disease? A. Bleomycin B. Cyclophosphamide C. Etoposide D. Vincristine E. Radiation
14. • Refractory anemia • Refractory anemia with ringed sideroblasts • Refractory anemia with multi-lineage dysplasia (cytopenias) • Refractory anemia with excess myeloblasts (oligoblastic leukemia) • 5q- syndrome Myelodysplastic Syndrome: WHO Classification 9/26/2018 14 pathy.med.nagoya-u.ac.jp/ atlas/doc/node71.htm Karyotype showing 5q- www.scielo.br/img/revistas/ rbhh/v26n3/3a19f02.jpg Blasts and a mono- lobed neutrophil Myeloblast with auer rods Ringed sideroblast
15. • It primarily affects older females. It presents with anemia and dysmorphic hematopoiesis (lobulated erythroblast nuclei and hypolobulated micromegakaryocytes). The platelet count is normal or high. The disease is indolent and has low propensity to evolve into AML. • It requires supportive care (erythropoietin, granulocyte–colony stimulating factor, transfusions and antibiotics). • Allogeneic stem cell transplantation is curative. Chromosome 5q Deletion Syndrome (OMIM#153550) [Macrocytic anemia, refractory, due to 5q deletion, somatic] Hypolobulated micromegakaryocytes PEIR Digital Library (Pathology image database Lobulated erythroblast nuclei pathologyoutlines.com/images/marrow/048.jpg 15
16. • DBS is characterized by isolated anemia (↑MCV, ↑erythrocyte adenosine deaminase [eADA], and ↑hemoglobin F) with severe reticulocytopenia and absence of marrow erythroid precursors. The neutrophil, lymphocyte and platelet counts are normal. – It appears in early life and may improve with glucocorticoids. – Congenital malformations occur in 50% of the patients (e.g., cleft palate, thumb defect). • Patients have mutations in RPS19 [ribosomal protein S19; OMIM#603474; autosomal dominant], RPL11 [ribosomal protein L11; OMIM#604175], or GATA1 [OMIM#305371; encodes a zinc finger DNA-binding transcription factor that is critical for the development of hematopoiesis). Acquired red cell aplasia: − Parvovirus infection (cytotoxic to erythroid progenitors) − “Transient erythrocytopenia of childhood” (TEC, a short-lived suppression of erythropoiesis due to viral infection). Diamond-Blackfan Anemia (DBA, OMIM#105650) - Ribosomopathy DBA in a 3-y-old boy from Pakistan Dr. Diamond
17. Fanconi Anemia (OMIM#227650) - A defect in DNA repair • This entity was first reported in 1927 by Guido Fanconi, who described 3 siblings with progressive pancytopenia, physical anomalies and predisposition to malignancy (particularly acute myelogenous leukemia). • The disease is autosomal recessive involving one of the 13 FA genes (OMIM#607139; FANC-A, B, C, D1, D2, E, F, G, I, J, L, M and N), which regulate DNA repair and cell cycle. • The disorder is heterogeneous, characterized by hypersensitivity to chromosome-breaking agents (diepoxybutane, DEB). • Heterozygotes for FA genes (e.g., FANCD1/BRCA2) have an increased risk of breast and other cancers. 9/26/2018 17
18. Fanconi Anemia: Natural History and Treatment [A defect in DNA repair] • Bone marrow failure occurs in childhood with petechiae, bruising and hemorrhage from thrombocytopenia; pallor and fatigue from anemia; and infection from neutropenia. • The major cause of death is bone marrow failure, followed by leukemia (AML) and solid tumors (most commonly liver adenomas and hepatomas in patients treated with oral androgens). The projected median survival is ~20 years. • Treatment: Supportive care (transfusions, antibiotics, growth factors), androgen therapy and stem-cell transplantation. [Blood 2003;101:1249-1256] 9/26/2018 18
19. Fanconi Anemia: Birth Defects • Skeletal anomalies (short stature, abnormal thumbs, abnormal thumbs and radi, microcephaly). • Altered skin pigmentation (café au lait spots, hyperpigmentation, hypopigmentation). • Other congenital malformations (abnormal gonads, eye anomalies, renal defects, low birth weight, developmental delay, abnormal ears or hearing). Café au lait spot Bifurcated thumb Absent thumb & radius
20. Fanconi Anemia: Chromosome Breaks Arleen D. Auerbach: Diagnosis of Fanconi Anemia by Diepoxybutane Analysis. Rockefeller University, New York. Current Protocols in Human Genetics , UNIT 8.7, 10.1002/0471142905.hg0807s37 , April, 2003. untreated treated with 0.1 mg/mL DEB 9/26/2018 20 Increased chromosome breaks with diepoxybutane (DEB). Molecular diagnosis has improved the diagnosis of Fanconi anemia.
21. 1. Exposure to alkylating agents 2. Complement-mediated hemolysis 3. Autoreactive T-cells destroying bone marrow 4. Thumb terminal defects 5. Blood cells lacking CD59 6. Treatment with anti-thymocyte globulins 7. Hypersensitivity to diepoxybutane (DEB) 8. Chromosome 5q Deletion Syndrome 9. Parvovirus 10. Mutation in phosphatidylinositol glycan gene 11. Treatment with eculizumab 12. Ringed sideroblasts 13. Hemoglobinuria 14. Predisposition to solid tumors 15. Defect in DNA repair 16. Short telomere 17. Ribosomopathy 18. Café au lait spots 19. Abnormal thumbs 20. Cyclophosphamide 21. Anti-thymocyte immunoglobulins (ATG)9/26/2018 A. Idiopathic aplastic anemia B. Paroxysmal nocturnal hemoglobinuria C. Myelodysplastic syndrome D. Diamond-Blackfan anemia E. Fanconi anemia F. Acquired red cell aplasia G. Dyskeratosis congenita “Must Know Pearls”
22. Required Reading • Scheinberg P, Wu CO, Nunez O, Young NS. Long-term outcome of pediatric patients with severe aplastic anemia treated with antithymocyte globulin and cyclosporine. J Pediatr 2008;153:814. • Young NS, Bacigalupo A, Marsh JC. Aplastic anemia: pathophysiology and treatment. Biol Blood Marrow Transplant 2010;16:S119. • Korthof ET, Békássy AN, Hussein AA. Management of acquired aplastic anemia in children. Bone Marrow Transplant 2013;48:191. 9/26/2018 22