1. Ramon V. Tiu, MD Cleveland Clinic Taussig Cancer Institute Dept. of Translational Hematology and Oncology Research Cleveland, Ohio USA New Directions in Aplastic Anemia Treatment: What’s on the Horizon? AA & MDS International Foundation Living with Aplastic Anemia, MDS, or PNH Patient and Family Conferences in 2014 April 5, 2014

2. Objectives To provide new information on emerging therapies in aplastic anemia (AA) To provide updates in the development of biomarkers that impact survival, clonal evolution and relapse in AA To provide new developments on molecular genetics in AA

3. Emerging Therapies (Iron Chelators) Deferasirox Rationale: –Too much iron can lead to suppressive effects on immature RBC precursors –Evaluation of Patient’s Chelation with Exjade (EPIC) Trial. Phase IIIb, one-year, open-label, single-arm study in patients diagnosed with transfusion-dependent iron overload How does the drug work? JW Lee et al. Haematologica. April 2013 Hartmann J et al. Leuk Res. Mar 2013

4. Emerging Therapies (Iron Chelators) Definition of Response JW Lee et al. Haematologica. April 2013 † TI- at least, a one 8 week period without transfusions

5. Emerging Therapies (Iron Chelators) Clinical Responses JW Lee et al. Haematologica. April 2013 Total AA Patient in the study: 116 No Complete Responders Median time to response: 85 days (range 1-277) One patient had a platelet response

6. Emerging Therapies (Iron Chelators) Deferasirox JW Lee et al. Haematologica. April 2013 Baseline Ferritin (ng/ml) Change in ferritin level post- Tx (ng/ml) % Change from baseline P value Partial Responder N=11 6693 ± 7014 - 3948 ± 4998- 45.7 %.0029 No Response N=13 4365 ± 3063 - 2021 ± 3242- 27.6%.0171

7. Emerging Therapies (Novel T cell Therapies) Alefacept Rationale: –Novel immunosuppressive therapies with less toxicities are needed for AA patients How does the drug work? Alefacept Humanized recombinant dimeric fusion protein composed of LFA3 and Fc portion of human IgG FDA approved for the treatment of chronic plaque psoriasis Have been found to be of use in GVHD RV Tiu. AA and MDS International YIA Grant Report. 2012

8. Mechanism of Action of Alefacept

9. Emerging Therapies (Novel T cell Therapies) Clinical Response PatientDoseResponseType of Hematologic Response † Side effects 17.5 mg/ week for 12 weeks PRNcough, sore throat, and nasal congestion 27.5 mg/ week for 12 weeks NR-Mild muscle aches 37.5 mg/ week for 12 weeks PRH/ N/ PNone 410 mg/ week for 12 weeks PRH/ N/ PNone †: H- Hemoglobin; N: Neutrophils; P: Platelets RV Tiu. AA and MDS International YIA Grant Report. 2012

10. Emerging Therapies (Novel T cell Therapies) Patient #3 Complete blood cell count 1 2 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 Pre (mos) Post (mos) During (week) Abs Retic (10 8 /uL) ANC (10 4 /uL) Hgb (g/dL) Platelets (10 3 /uL) Treatment 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 25 27.5 30 32.5 35 RV Tiu. AA and MDS International YIA Grant Report. 2012

11. Emerging Therapies (Novel T cell Therapies) 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 25 27.5 30 32.5 35 1 2 3 4 1 2 3 4 5 6 7 8 9 101112 1 2 3 4 Abs Retic (10 8 /uL) ANC (10 4 /uL) Hgb (g/dL) Platelets (10 3 /uL) Pre (mos) Post (mos) During (week) Treatment Complete blood cell count Patient #4 Unfortunately: In December 2011, Astellas Pharma US announced that the company has voluntarily discontinued the promotion, manufacturing, distribution and sales of alefacept because of business needs but not related to safety issues. RV Tiu. AA and MDS International YIA Grant Report. 2012

12. Emerging Therapies (Combination Therapies) Cyclosporine plus Levamisole Rationale: –Developing novel therapies for patients with moderate AA Patient Cohort –118 patients with mAA o 42 newly diagnosed o 76 chronic Regimen –CsA 3 mg/kg per day in adults plus Levamisole 150 mg per day in adults –CsA 5 mg/kg per day in children plus or 2.5 mg/kg per day in children, –Either regimen will be continued for 12 more months after achieving maximal hematologic response, followed by a slow taper X Li et al. Ann Hematol. April 2013

13. Emerging Therapies (Combination Therapies) Clinical Response X Li et al. Ann Hematol. April 2013 Newly dx mAAChronic mAAP value Overall Response Rate 100 %86.8 % 24 month PFS 95.2 % (95% CI: 85.9- 100) 93.6 % (95% CI: 86.9- 100 %) 0.50 2 year EFS86.8 (95% CI: 70.4- 100) 57.0 % (95% CI: 43.5- 70.4 %).001

14. Biomarkers Predictive/ Prognostic Biomarkers (ARC and ALC) Scheinberg P et al. BJH. 2008 NIH Study of 316 SAA patients (1989-2005) Defining important predictors of response to hATG +CsA therapy at 6 mos of therapy Factors looked at includes age, PNH clone, hematological factors like ANC, platelet count, Hgb, Absolute reticulocyte count (ARC), absolute lymphocyte count Presence of PNH clone is defined as presence of positive Ham test for samples until yr 2000 and subsequently GPI-(neg) Neutrophils or red cells >1% ARC and ALC are predictive

15. Biomarkers Predictive/ Prognostic Biomarkers (ARC/ ALC) Scheinberg P et al. BJH. 2008

16. Biomarkers Predictive/ Prognostic Biomarkers (ANC and ARC) Chang M et al. EJH. 2009 Korean Study of 62 SAA patients (1994- 2007) Defining important predictive/ prognostic markers Patient got rATG + CsA or hATG + CsA Factors looked at includes age, type of ATG clone, baseline hematological factors like ANC, platelet count, Absolute reticulocyte count (ARC), absolute lymphocyte count (ALC), total WBC, sveerity of disease, etiology Only predictive factor is ANC >0.3 x10 9 /L Prognostic Factors: ARC >10.9 x10 9 /L and response status

17. Importance of Telomeres in Aplastic Anemia Telomeres. Young N S Hematology 2010;2010:30-35

18. Importance of Telomeres in Aplastic Anemia Telomerase Complex. Young N S Hematology 2010;2010:30-35

19. Importance of Telomeres in Aplastic Anemia Telomere Length and Disease Relapse. Scheinberg P et al. JAMA. 2010

20. Importance of Telomeres in Aplastic Anemia Telomeres and Clonal Evolution. Scheinberg P et al. JAMA. 2010

21. Importance of Telomeres in Aplastic Anemia Telomere Length as a predictor for clonal evolution in AA. Calado RT et al. Leukemia. April 2012

22. Importance of Telomeres in Aplastic Anemia More Monosomy 7 and chromosomal Instability in patients with short telomeres. Calado RT et al. Leukemia. April 2012

23. Importance of Telomeres in Aplastic Anemia Telomeres and Survival. Scheinberg P et al. JAMA. 2010

24. Differentiating AA vs hypocellular MDS using SNP-A Karyotyping SNP-Array Karyotyping #57 whole genome Chromosome 7Chromosome X Total Copy number Allele specific copy number Ideogram Heterozygous SNP call Ideogram Smoothed copy number Raw copy number Allele call AA AB BB BM 250K UPD6p12.1-pterChr7 monosomy Pat#96 B. A. 2N 1N 3N 2N 1N 3N Afable M et al. Blood. 2011

25. Differentiating AA vs hypocellular MDS using SNP-A Karyotyping Afable M et al. Blood. 2011 More chromosomal defects detected by SNP-Array Karyotyping compared to metaphase cytogenetics in AA and hypocellualr MDS

26. SNP-A Karyotyping Afable M et al. Blood. 2011 Early Detection of Clonal Evolution by SNP-Array Karyotyping

27. Genetic Causes of Inherited AA Presence of MPL mutations in Inherited cases of AA. Walne A J et al. Haematologica 2012;97:524-528

28. Cytogenetic Defects and Molecular Mutations in Fanconi Anemia Rare NRAS, RUNX1, Flt-3 and MLL mutations in FA that transformed to AML. Quentin S et al. Blood 2011;117:e161-e170

29. STAT3 mutations in Aplastic Anemia And MDS. Epling-Burnette PK et al. ASH Website.

30. STAT3 mutations in Aplastic Anemia And MDS. Jerez A et al. Blood 2013;122:2453-2459 Frequency of STAT3 mutations in AA and MDS

31. STAT3 mutations in Aplastic Anemia And MDS. Jerez A et al. Blood 2013;122:2453-2459 Impact of STAT3 mutations on survival outcomes in AA and MDS

32. Conclusions Some patients with AA treated with iron chelator, deferasirox achieved transfusion independence but the results will need to be clarified in bigger studies Targeting the CD2-LFA3 pathway is a viable treatment option in AA ARC and ALC are important in predicting immunosuppressive response in AA Shorter Telomeres have adverse impact on survival, clonal evolution and relapse in AA SNP-A karyotyping can improve cytogenetic detection in AA Molecular genetics can unravel new information on the pathophysiology of AA.