1. Aplastic Anemia Tissue Conference 1/19/00 Brad Kahl, MD

2. Pancytopenia Reduction of counts in all three cell lines Differential Diagnosis –aplastic anemia –myelodysplasia –marrow replacement leukemia, lymphoma, carcinoma, myelofibrosis –B12, folate –chemotherapy induced

3. Pancytopenia Differential Diagnosis continued –splenomegaly (any cause) –PNH –SLE –Congenital Fanconi’s, Schwamann-Diamond, Folate uptake def

4. Pancytopenia Presentation varies with degree of cytopenia –anemiafatigue –thrombocytopeniabruising/bleeding –neutropeniainfection Approach –history constitutional symptoms, pain, early satiety, etc... diet, EtOH, exposures, occupation

5. Pancytopenia Approach –PE nodes, spleen, sensory, portal htn –Labs B12, folate, LFT’s, PNH, ANA view smear (macrocytosis, megaloblastosis, tear drops, nuc RBC’s, malignant cells) abdominal imaging bone marrow evaluation

6. Aplastic Anemia Bone Marrow Failure –WHY?????????? Stem cell defect (seed) Stromal cell defect (soil) Growth Factor defect (fertilizer) –Evidence suggests that majority of cases of idiopathic AA are due to immune suppression of the hematopoietic stem cell

7. Aplastic Anemia Classification Direct Toxicity –Iatrogenic (radiation, chemotherapy) –Benzene –Drug metabolites Immune Mediated –Drug metabolites –transfusion associated –hepatitis associated –idiopathic

8. Aplastic Anemia Pathophysiology Evidence for an immunological basis arose from observations after BMT –unexpected improvement of pancytopenia in some patients after allogeneic graft failure –successful BMT of identical twins generally requires some sort of immunosuppressive conditioning regimen

9. Aplastic Anemia Pathophysiology Evidence for stem cells (seed) as targets –in vitro colony forming assays are used to define the stem cell compartment –two papers in 1996 showed profound deficits in the stem cell population in patients with AA –at the time of clinical presentation the absolute number of stem cells is < 1% of normal

10. Aplastic Anemia Pathophysiology What about the stroma (soil) and growth factors (fertilizer)? –successful BMT implies intact stroma since it is not replaced in the transplant –laboratory studies have shown the stroma of AA patients is able to support normal stem cell growth –stromal cells of AA patients tend to make increased levels of several growth factors (EPO, TPO, G-CSF) –clinical studies using factor replacement haven’t worked

11. Aplastic Anemia Pathophysiology Laboratory Evidence for Immune Destruction of Hematopoietic Stem Cells –mononuclear cells from blood and marrow of AA patients suppress hematopoietic colony formation by normal marrow stem cells –if selectively remove T cells from the sample, generally improve in vitro colony formation

12. Aplastic Anemia Pathophysiology What are the T cells doing? –Direct cellular cytotoxicity blood and marrow of AA patients contain increased numbers of activated cytotoxic lymphocytes the number and activity of these cells decreases after successful treatment with ATG

13. Aplastic Anemia Pathophysiology Cytokines –T cells of AA patients overproduce both IFN-gamma and TNF-alpha –both of these cytokines inhibit colony formation in vitro IFN-gamma induces nitric oxide synthase (NOS) and production of nitric oxide (NO) both induce expression of Fas receptor on CD34+ cells and activation of this receptor by its ligand induces apoptosis –both appear to inhibit mitosis IFN-gamma increases IFN regulatory factor 1 which inhibits transcription of cellular genes and entry into the cell cycle

14. Aplastic Anemia Pathophysiology

15. Inciting Events –much less clear, most cases--no clue –a few cases clearly associated with a non-A, non-B, non-C, non-G hepatitis severe pancytopenia 1-2 months after an apparent viral hepatitis patients tend to have a marked activation of cytotoxic lymphocytes and tend to respond favorably to immunosuppressive therapy

16. Aplastic Anemia Pathophysiology Drugs –implicated in 15-25% cases (difficult to study) –no animal model –some cases may be a direct toxic effect –some cases appear immune mediated –in general patients have similar characteristics as idiopathic AA and respond similarly to immunosuppression

17. Aplastic Anemia Treatment Options –BMT from donor vs. immunosuppression with ATG, CSA, or ATG/CSA combination –steroids, androgens generally ineffective Trend towards separating severe AA and non-severe AA in current clinical trials

18. Aplastic Anemia Treatment Severe Aplastic Anemia Criteria –blood: neutrophils < 500/mm 3 platelets < 20k retics < 1% (corrected) –marrow severe hypocellularity moderate hypocellularity with hematopoietic cells representing < 30% of residual cells need 2/3 blood and one marrow criteria

19. Aplastic Anemia Treatment Non-severe AA (Blood, April 99) –patients randomized to CSA vs. ATG/CSA –Overall Response Rate at 6 months CSA 46%ATG/CSA 74%P=.02 –Similar early toxicity/infections

20. Aplastic Anemia Treatment Severe AA (Ann Int Med 1997) Allo BMT vs. Immunosuppression ORR15 Yr OS allogeneic BMT89%69% Immunosuppression44%38% –40% BMT patients clinically extensive chronic GVHD –1/227 receiving immunosuppression got ATG/CSA –50/227 received ATG + mismatched bone marrow

21. Aplastic Anemia Treatment Severe Aplastic Anemia –NEJM 1991 ORR ATG/Pred31% ATG/Pred/CSA65% –Blood 1992 ATG/LDM/oxymethalone36% ATG/HDM/oxymetholone48% –Blood 1995 ATG/CSA78%

22. Aplastic Anemia Treatment Future –High Dose Cyclophosphamide vs. ATG –Addition of MMF to ATG/CSA combinations –? allo BMT vs optimal immunosuppression?

23. Aplastic Anemia Summary –idiopathic AA appears to be an AI disorder directed against hematopoietic stem cells –mediated by cytotoxic T cells and cytokines –allo BMT is the gold standard treatment –intensive immunosuppressive therapy has improved the outlook for patients ineligible for BMT due to age or lack of a suitable donor –expect further refinements in therapy as the pathophysiology is further elucidated