New Directions in Aplastic Anemia Treatment Whats on

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 Ramon V. Tiu, MD Cleveland Clinic Taussig Cancer Institute Dept. of Translational Hematology and Oncology Research Cleveland, Ohio USA

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

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

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

Emerging Therapies (Iron Chelators) Clinical Responses • 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 JW Lee et al. Haematologica. April 2013

Emerging Therapies (Iron Chelators) Deferasirox Baseline Change in ferritin 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 JW Lee et al. Haematologica. April 2013

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 LFA 3 and Fc portion of human Ig. G • 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

I A l N ce T e iv v e III m PC A II A m A ll ve l i Mechanism of Action of Alefacept K ce a el N N c ll D C 2 γ Fc T -3 FA e iv ev m L D C e c tic T o d pt te o a p A ctiv A 2 A D C 2 ve l i a l N ce T -3 PC A A LF D C 2 2 D C at iv l t c l A ce T ve l i a l N ce T C em St c i et i o ed t a iv l t c l A ce T op t a em H l el

Emerging Therapies (Novel T cell Therapies) Clinical Response Patient Dose Response Type of Hematologic Response† Side effects 1 7. 5 mg/ week PR for 12 weeks N cough, sore throat, and nasal congestion 2 7. 5 mg/ week for 12 weeks NR - Mild muscle aches 3 7. 5 mg/ week for 12 weeks PR H/ N/ P None 4 10 mg/ week for 12 weeks PR H/ N/ P None †: H- Hemoglobin; N: Neutrophils; P: Platelets RV Tiu. AA and MDS International YIA Grant Report. 2012

Emerging Therapies (Novel T cell Therapies) Complete blood cell count Patient #3 35 32. 5 30 27. 5 25 22. 5 20 17. 5 15 12. 5 10 7. 5 5 2. 5 0 Abs Retic (108/u. L) ANC (104/u. L) Hgb (g/d. L) Platelets (103/u. L) 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 Pre (mos) During (week) Post (mos) Treatment RV Tiu. AA and MDS International YIA Grant Report. 2012

Emerging Therapies (Novel T cell Therapies) Patient #4 Complete blood cell count Abs Retic (108/u. L) ANC (104/u. L) 35 32. 5 30 27. 5 25 22. 5 20 17. 5 15 12. 5 10 7. 5 5 2. 5 0 Hgb (g/d. L) Platelets (103/u. L) 1 2 3 4 1 2 3 4 5 6 7 8 9 101112 1 2 3 4 Pre (mos) During (week) Post (mos) Treatment Unfortunately: RV Tiu. AA and MDS International YIA Grant Report. 2012 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.

Emerging Therapies (Combination Therapies) Cyclosporine plus Levamisole • Rationale: – Developing novel therapies for patients with moderate AA • Patient Cohort – 118 patients with m. AA o 42 newly diagnosed o 76 chronic • Regimen – Cs. A 3 mg/kg per day in adults plus Levamisole 150 mg per day in adults – Cs. A 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

Emerging Therapies (Combination Therapies) Clinical Response Newly dx m. AA Chronic m. AA P value Overall Response Rate 100 % 86. 8 % 24 month PFS 95. 2 % (95% CI: 85. 9100) 93. 6 % (95% CI: 86. 9100 %) 0. 50 2 year EFS 86. 8 (95% CI: 70. 4100) 57. 0 % (95% CI: 43. 570. 4 %) . 001 X Li et al. Ann Hematol. April 2013

Biomarkers Predictive/ Prognostic Biomarkers (ARC and ALC) • NIH Study of 316 SAA patients (1989 -2005) • Defining important predictors of response to h. ATG +Cs. A 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 Scheinberg P et al. BJH. 2008

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

Biomarkers Predictive/ Prognostic Biomarkers (ANC and ARC) • Korean Study of 62 SAA patients (19942007) • Defining important predictive/ prognostic markers • Patient got r. ATG + Cs. A or h. ATG + Cs. A • 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 x 109/L • Prognostic Factors: ARC >10. 9 x 109/L and response status Chang M et al. EJH. 2009

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

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

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

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

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

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

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

Differentiating AA vs hypocellular MDS using SNP-A Karyotyping A. SNP-Array Karyotyping 3 N 2 N 1 N #57 whole genome 3 N 2 N 1 N Total Copy number Ideogram Heterozygous SNP call Allele specific copy number Allele call Chromosome 7 Chromosome X AA AB BB Raw copy number Smoothed copy number Ideogram B. Pat#96 UPD 6 p 12. 1 -pter Chr 7 monosomy BM 250 K Afable M et al. Blood. 2011

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

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

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

Cytogenetic Defects and Molecular Mutations in Fanconi Anemia Rare NRAS, RUNX 1, Flt-3 and MLL mutations in FA that transformed to AML . Quentin S et al. Blood 2011; 117: e 161 -e 170

STAT 3 mutations in Aplastic Anemia And MDS . Epling-Burnette PK et al. ASH Website.

STAT 3 mutations in Aplastic Anemia And MDS Frequency of STAT 3 mutations in AA and MDS . Jerez A et al. Blood 2013; 122: 2453 -2459

STAT 3 mutations in Aplastic Anemia And MDS Impact of STAT 3 mutations on survival outcomes in AA and MDS . Jerez A et al. Blood 2013; 122: 2453 -2459

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 CD 2 -LFA 3 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.