1. Non Transplant Options for Aplastic Anemia Jong Wook Lee, MD, PhD. Department of Hematology, Seoul St. Mary’s Hospital The Catholic University of Korea, Seoul, Korea

3. Cytotoxic T cell Haemopoietic cell Interleukin-2 TNF-β INF-γ Fas ligand INF-γ TNF-β INF-γ receptor TNF-β receptor Fas receptor Expansion of T-cell clones IRF-1 ↑ ICE apoptosis Jak 1 Stat 1 Stat 3 i NOS NO Toxicity for other cells dsRNA-PKR RNaseL elF-2 ↓protein synthesis ↓Cell cycling Immune-mediated aplastic anemia Anemia Leukopenia (neutropenia) Thrombocytopenia CD34 + cell↓ CFU-GM, BFU-E ↓ LTC-IC ↓ Young N, Maciejewski J. N Engl J Med. 1997;336:1365-72. Young N, et al. Blood 2006;108:2509-19.

4. Genetic aplastic anemia Telomere shortening 1–3 ●Abnormal telomere shorting in AA: relapse after IST, clonal evolution, survival in SAA 1. Ball SE, et al. Blood. 1998;91:3582-92. 2. Brummendorf TH, et al. Blood 2001;97:895-900. 3. Scheinberg P, et al. J Am Med Assoc. 2010;304:1358-64. 4. Yamaguchi H, et al. N Engl J Med. 2005;352:1413-24. 5. Fogarty PF, et al. Lancet. 2003;362:1628-30. 6. Yamaguchi H, et al. Blood. 2003;102:916-8. Mutation in both TERT and TERC genes 4–6 ●Mutation of TERC: 3/210 of AA, PNH, and DKC ●Mutation of TERC in 2 families ●Mutation of TERT: 7/200 AA vs 0/528 control –heterozygous mutations in the TERT gene impair telomerase activity and may be risk factors for bone marrow failure Embryonic stem cells Adult stem cells Telomeres Chromosome Telomere is a repeating DNA sequence Telomerase inactive or absent Telomere short Telomere long Telomerase active DKC = dominant dyskeratosis congenita; IST = immunosuppressive therapy; PNH = paroxysmal nocturnal haemoglobinuria; SAA = severe AA.

5. Auto-antibodies (Ab) against distinct proteins Kinectin - a 1300 amino acid protein expressed on human hematopoietic cell, liver, ovary, testis & brain - IgG Anti-kinectin Ab found in 7/18 (39%) of US AA vs. in 3/30 (10%) of Japanese AA (Hirano, Blood, 2003) Anti-PMS1 (postmeiotic segregation increased 1) Ab - Japanese AA, 3/10 (30%) vs. US AA, 0% (0/18) (Hirano, BJH, 2004) Diazepam-binding related protein-1 - anti-DRS-1 in 38% (27/71) of PNH+AA, 6.3% (2/32) of PNH-AA, 38.5% (5/13) of PNH+MDS. - response to IST in AA patients with DRS-1 Ab (100% vs. 55%) (Feng, Blood, 2004) Moesin: - a membrane cytoskeleton linker protein - antimoesin Abs in 25/67 (37%) of AA - marker for response to IST (Takamatsu, Blood, 2007)

6. Treatment options of aplastic anemia 1.Allogeneic BMT –age < 50 years –HLA-matched donor (+) –severe or very severe AA 2.Immunosuppressive therapy (IST) –hematological recovery in 60~70% of patients 1,2 3.Supportive care –blood transfusions, management of infections, iron chelation therapy 1. Rosenfeld SJ, et al. Blood. 1995;85:3058-65. 2. Bacigalupo A, et al. Blood. 1995;85:1348-53. BMT = bone marrow transplantation.

7. Supportive care 1. Transfusion Red cell (PRC) : Hb 7.0 g/dl ↓ PLT : 10,000/mm 3 ↓ * Irradiation (2,500 rad) * Leukocyte-depleted filter * Iron chelation therapy 2. Infection control

8. Algorithm for treatment Maciejewski JP, Risitano AM. Hematology Am Soc Hematol Educ Program. 2005:110-7. BMT uBMT; androgens, 2nd IST Adequate blood count Observation Blood counts Acquired AA SevereNon-severe Older adults No family donor ATG+CsA R Follow for relapse, late clonal ds Allo-BMT; androgens, 2nd IST Clinical status ATG+CsA R CsA; androgen, 2nd IST ATG+CsA Clinical status Tx- dependent Children, young adults Age Sibling match HLA typing Blood count improvement at 3–6 mo Non-R ATG = antithrombocyte globulin; CsA = ciclosporin; ds = disease; R = response; uBMT = unrelated bone marrow transplantations.

9. ATG CyclophosphamideCyclosporin Inhibition of T cell function

10. Proposed mechanisms through which ATG can interfere with the immune response 1)T-cell depletion in blood and lymphoid tissues through complement-dependent lysis and T-cell apoptosis 2) induction of B-cell apoptosis 3)modulation of cell-surface molecules (adhesion and chemokine receptors) that mediate leukocyte/endothelium interactions 4)Interference with DC functional properties (maturation and migration) 5) Induction of Treg and NK-T cells. Morthy M. Leukemia 21:1387–1394, 2007

11. Comparative study of IST in AA Treatment (mg/kg/day) No. of patients (SAA) RR (CR+PR)Survival (F/U duration) German study ATG+CsA (12) ATG 43 (31) 41 (29) 70% 46% p <0.05 64% (41 mon) 58% p =0.16 NIH ATG+CsA (12) ATG 51 (51) 70 (70) 67% 29% p =0.001 62% (3 yrs) _ EBMTATG+CsA (5) CsA 54 (0) 61 (0) 74% 46% p =0.02 90% (6 mon)* 67% p =0.001 Korean study ATG+CsA (5) ATG 53 (39) 40 (27) 41% 18% p =0.01 98% (6 mon) 90% p =0.16

12. Factors for predicting response of IST ●Young age ●Presence of PNH clone ●Higher reticulocyte count (>25x10 9 /L) ●Higher lymphocyte counts (>1x10 9 /L) ●Early treatment within 21 days after diagnosis ●CsA therapeutic level/late tapering ●HLA-DR15

13. Predictors for response to IST (n=324) Kim SY et al, GENES, CHROMOSOMES & CANCER 49:844–850, 2010

14. Factors to predict relapse after IST ●Shorter telomeres ●Rapid tapering of CsA : General recommendation is to taper CsA by 10% every month, starting at least 1 year from IST (Sarraco et al, Br J Haematol 2008, Marsh et al, Br J Haematol 2009) ●Suboptimal therapeutic range of CsA

15. Improving on ATG+CSA ●Addition of G-CSF - improved neutrophil counts but similar response or survival ● Addition of other immunosuppressive drugs + High dose of methylprednisolone: toxicities ↑ + MMF: no benefit (Scheinberg, 2006) + Rapamycin/sirolimus: not superior (Scheinberg, 2009)

16. Alternative immunosuppressive regimens ●Cyclophosphamide (CY) 1) John’s Hopkins (Brodsky et al, 1996 & 2010) - CY 50 mg/kg/d for 4d in 33 untreated SAA - CR at 50 months; 65%, OS 86% 2) NIH trial (Tisdale et al, 2000 & 2002) - CY (50 mg/kg/d for 4d) + CsA vs. ATG + CSA - Response rate: 6/13 (46%) vs. 9/12 (75%) - Higher fungal infection & death in CY arm ● Alemtuzumab 1) EBMT study : OR 58% (Risitano et al, Br J Haematol 2010) 2) NIH study : 10mg/d, IV for 10 days (Scheinberg et al, Blood 2012) Refractory SAA (n=27) : OR 37% Relapsed disease (n=25) : OR 56% Treatment-naïve (n=16) : OR 19%

17. Moderate AA ●IST –Better survival with ATG + CyA than CyA alone (Marsh, 1999) ● Daclizumab (humanized monoclonal Ab of IL-2 receptor) - 16 AA : response 6/16 (38%) with little toxicity (Maciejewski, 2003) ● Androgen - stimulate TERT expression via estrogen receptor  increase telomerase activity in lymphocytes and CD34+ cells (Calado, 2005)

18. Various ATG preparations ●Horse ATG - ATGAM ® (40 mg/kg per day for 4 d) - Lymphoglobuline ® (15 mg/kg per day for 5 d) ●Rabbit ATG - Thymoglobuline ® (2.5~3.75mg/kg per day for 5 D) - ATG-Fresenius ® (5mg/kg/d for 5 D) : inferior response to hATG (53% vs 78%, p=0.03), 5 yr-OS (66% vs 80%, p=0.002) (Zheng et al, Exp Hematol 34:826, 2006)

19. Phase II prospective study from Cleveland SAA treated with rabbit ATG (n=21) versus matched historical control treated with horse ATG (n=67). Rabbit ATG : 3.5 mg/kg/day for 5 days Horse ATG: 40mg/kg/day for 4 days Cyclosporine – maintain until response or developing renal complications. Afable et al, Haematologica 96:1269, 2011 Type of ATG nEarly mortality Response (%)Relapse (%) 3 months6 months12 months Rabbit ATG20240 (CR: 0)45 (CR: 0)50 (CR: 0)5 Horse ATG67655 (CR: 5)58 (CR: 6)58 (CR: 8)16 p value1.00.430.440.610.28

20. Atta et al, Annals of hematology. 89:851, 2010 Horse ATGRabbit ATGp value N4229 Partial response at 6 months20 (47.6%)8 (27.6%)0.14 Complete response at 6 months5 (11.9%)2 (6.9%)0.69 Overall response at 6 months25 (59.5%)10 (34.5%)0.05 Interval between ATG and response (days)99 (39-166)88.5 (39-161)0.98 Overall survival at 2 years78.4%55.4%0.03 Relapse9 (36%)3 (30%)1 A retrospective comparative study from Brazil Rabbit ATG : 2.5 mg/kg (range, 1.5-3 mg/kg), Horse ATG : 15 mg/kg (range, 10-21.4 mg/kg)

21. Randomized prospective study in NIH (n=120) Rabbit ATG (3.5mg/kg for 5 days) vs Horse ATG (40mg/kg for 4 days) Cyclosporine – continued for at least for 6 months Scheinberg et al, NEJM 365:430, 2011 ResponseHorse ATG Rabbit ATG p 3 months37 (62)20 (33%)0.002 6 months41 (68%)22 (37%)<0.001

22. Comparison of response between horse ATG (n=53) and rabbit ATG group (n=46) in SAA Horse ATG (OR) Rabbit ATG (OR) Horse ATG (CR) Rabbit ATG (CR) p valueOR0.3190.8340.9250.757 CR0.1230.0480.3870.905 Shin, et al 2011 ASH #3430

23. Similar OS between rATG and hATG P=0.46 Shin, et al 2011 ASH #3430

24. Lymphocytes depletion after ATG treatment Scheinberg et al, NEJM 365:430, 2011

25. Lymphocytes depletion after ATG treatment Scheinberg et al, NEJM 365:430, 2011

26. Comparison between horse and rabbit ATG 2.5mg/kg/d Х 5 days 3.5mg/kg/d Х 5 days 2.5mg/kg/d Х 5 days n=22 n=67 n=29 n=42n=60 n=33 n=29n=53 n=46 (1) Atta E, et al. Ann Hematol. 2010 89(9):851-9.(2) Sheinberg, et al. N Engl J Med. 2011 365(5):430-8. (3) Chang M, et al. Eur J Haematol. 2010 84(2):154-9.(4) Afable M, et al. Haematologica. 2011 96(9):1269-75 (5) Shin, et al 2011 ASH #3430.

27. International Clinical Trial ●A prospective randomized multicenter study comparing different dosages of rabbit antithymocyte globulin (Thymoglobuline  ) in patients with severe aplastic anemia ●A total of 256 SAA patients will be enrolled, with 128 patients randomized to arm A (2.5mg/kg/d) and 128 patients to arm B (3.5mg/kg/d). ●Korea, Japan, China

28. Conclusions 1. For the last 20 years, the standard immunosuppressive regimen for AA patients has been ATG plus CsA, which results in response rates ranging between 50% and 60%, and even higher overall survival. 2. Response rates of rabbit ATG seem to be similar to horse ATG. 3. Thymoglobuline might need more time to reach the best response after IST in comparison to horse ATG. 4. CsA should be continued for at least 12 months after achieving maximal hematological response, followed by a very slow tapering, to reduce the risk of relapse. 5. Optimal dose of rabbit ATG to be determined.