1. Aplastic anemia Deepa Jeyakumar, MD Assistant Clinical Professor of Medicine 10/15/14

2. ST  Pt is a 43 year old woman who was in her usual state of health until 03/16/11 when she presented to outside ER with left flank pain and dark urine for two days.  Found to have hct of 26 with platelet count of 26k.  No hemolysis on labs.  Peripheral smear reveals no atypical cells with few large platelets. Initially thought to have ITP- given IVIG x 1. Did not response to IVIG.  Required daily platelet transfusions.

3. Bone marrow biopsy  markedly hypocellular marrow with 80% fat. Cellularity is composed of entirely maturing erythroid elements. Myeloid elements are markedly decreased. Occasional segmented forms noted. Megakaryocytes rare. Stainable iron increased. No ringed sideroblasts. Reticulin focally increased. No features of parvovirus seen. Several blast forms seen.

4. Diagnosis of Aplastic Anemia  Marrow is profoundly hypocellular with decrease in all elements.  Residual hematopoietic cells are morphologically normal.  Malignant infiltrates and fibrosis is absent.  Hematopoiesis is non-megaloblastic.

5. Severity  Moderate aplastic anemia Marrow cellularity <30% Absence of severe pancytopenia Depression of at least two of three blood elements below normal.  Severe o Bone marrow cellularity <25% or marrow showing <50% normal with two of three peripheral blood count criteria: o ANC <500  Plt <20k  Retic count <40k  Very Severe All of above plus ANC less than 200.

6. Classification  Inherited Fanconi’s anemia, dyskeratosis congenita, Shwachman-Diamond Syndrome, Reticular dysgenesis, Amegakaryocytic thrombocytopenia, familial aplastic anemia, preleukemia (monosomy 7) and nonhematologic disease (Down, Dubowitz, Seckel)  Acquired Irradiation drugs and chemicals: cytotoxic agents, benzene, idiosyncratic reaction, chloramphenicol, NSAIDS, antiepileptics, Gold viruses: EBV, Hepatitis virus (non-A,non-B, non-C, non-G), Parvovirus (transient aplastic crisis or pure red cell aplasia), HIV Immune diseases: eosinophilic fasciitis, hyperimmunoglobulinemia, thymoma and thymic carcinoma, GvHD in immunodeficiency PNH Pregnancy Idiopathic

8. Differential Diagnosis  Pancytopenia with hypocellular bone marrow Acquired aplastic anemia- Inherited aplastic anemia Hypoplastic MDS- Hypoplastic AML  Pancytopenia with cellular bone marrow Primary bone marrow diseases-MDS PNH- Myelofibrosis Myelophthisis- Bone marrow lymphoma Hairy cell leukemia- SLE, Sjogren’s disease Hypersplenism- Vitamin B12 and folate deficiency Overwhelming infection- Alcoholism Brucellosis- Ehrlichiosis Sarcoidosis- tuberculosis  Hypocellular bone marrow with or without cytopenia Q fever- Legionaires disease Mycobacteria- Tuberculosis Hypothyroidism- Anorexia nervosa

10. Hypocellular AML & hypocellular MDS

11. Epidemiology  International Aplastic Anemia and Agranulocytosis Study (IAAAS) found 2 confirmed cases per one million people (two PNH units)  Thailand- 4 cases per million

12. Cumulative survival has increased over the past few decades

13. Etiology and Pathogenesis  Genetic predisposition found in HLA- DR2.  This correlates to response to immunosuppressants.  Similar results found in hypoplastic MDS.

14. Pathogenesis  Immune-mediated T-cell destruction of marrow Young demonstrated that removal of lymphocytes from aplastic bone marrow improved colony number in tissue culture and addition of lymphocytes to normal marrow inhibited hematopoiesis in vitro.

16. Immune Destruction of Hematopoiesis

19. Telomere shortening  Originally thought to be due to stem-cell exhaustion.  Telomere shortening also found in X-linked form of dyskeratosis congenita due to mutations in DKCI.  Telomere shortening also found in mutations in TERC found in AD patients with constitutional  Subsequent analysis of patients with acquired aplastic anemia found mutations in TERC and TERT.  Interestingly, family members of patients who share these mutations can have normal blood counts but hypocellular marrows, reduced CD34 counts and poor hematopoietic colony formations and short telomeres.  Therefore, 1/3 to ½ of patients with aplastic anemia have short telomeres but mutations are only found in 10% of patients.

20. Treatment

21.  ATG: Lymphocyte numbers decreased within the first few days of therapy and then return to pretreatment levels within a week or so. Appears to be immunomodulatory as well as lymphocytotoxic- producing a state of tolerance by preferential depletion of activated T cells. Rabbit appears to be more potent that the horse formulation.  Cyclosporine: its selective effects on T-cell function is due to direct inhibition on the expression of nuclear regulatory proteins, resulting in decreased T-cell proliferation and activation.

22. Intensive Immunosuppression

23. Clinical Endpoints  Response defined as transfusion independence. About 50% response rate with horse ATG.  Relapse defined as requirement of additional immunosuppresants. Happens in 30-40% of patients.  Clonal evolution occurs in 15% of cases. Into MDS, AML, PNH

24. Improving on ATG & cyclosporine for first line management of AA?  Addition of high dose steroids did not improve outcomes and just added to toxicity.  Addition of G-CSF and GM-CSF did not improve outcomes  Addition of mycophenolate did not improve response rates or outcomes.  Sirolimus was equally ineffective.  Cyclophosphamide was associated with a higher death rate due to prolonged neutropenia.

25. Relapsed/Refractory Aplastic Anemia  Rabbit ATG- if patient has not seen it before and had a decent response to initial treatment.  Alemtuzumab has been shown in the relapsed setting to be effective.  Cyclophosphamide has a 50% response rate in relapsed setting.

26. Moderate Aplastic Anemia  Role for duclizumab, humanized monoclonal antibody to IL-2 receptor  Role for androgen therapy

27. HLA-matched sibling allotransplantation

28. Risk factors for graft failure  Heavily transfused  Long time between diagnosis and transplantation

29. Alternative Donor Transplant

31. A 24-year-old man undergoes follow-up evaluation for treatment of aplastic anemia. Two of his siblings are HLA-identical matches. Hemoglobin 8.3 g/dL (83 g/L) (following transfusion of 1 unit of irradiated packed erythrocytes last week) Leukocyte count 500/µL (0.5 × 10 9 /L) with 23% neutrophils, 3% band forms, and 71% lymphocytes Platelet count26,000/µL (26 × 10 9 /L) Reticulocyte count0.2%

32.  Review of the bone marrow biopsy done 2 weeks ago confirms the diagnosis of aplastic anemia, demonstrating an aplastic bone marrow with normal cytogenetics.  Which of the following is the most appropriate treatment? A: Allogeneic hematopoietic stem cell transplantation B: Antithymocyte globulin, corticosteroids, and cyclosporine C: Autologous hematopoietic stem cell transplantation D: Corticosteroids E: Granulocyte colony-stimulating factor

33. Thank You!