Bone Marrow Failure

Robert A. Brodsky, MD Johns Hopkins Family Professor of Medicine and Oncology Director, Division of Hematology Johns Hopkins University Disclosures

• Dr. Brodsky serves as a Scientific Advisory Board member to Alexion Pharmaceuticals.

• Alexion Pharmaceuticals – grant funding • Apellis: Scientific Advisor • Achillion: Scientific Advisor Aplastic Anemia Diagnosis And Nomenclature • SAA – Bone marrow (< 25% cellular) – Peripheral cytopenias (at least 2 of 3) • ANC < 500 per µl 2 year • Platelets < 20,000 per µl mortality • Absolute retic < 60,000 or corrected retic < 1% > 70% • VSAA: as above, but ANC < 200

• Moderate AA or (NSAA) – Hypocellular marrow but does not meet criteria for SAA APLASTIC ANEMIA Etiology • Acquired AA (most cases idiopathic) – Drugs (5 – 10%) – Infection (hepatitis –usually seronegative) – Toxic (benzene, radiation)

• Inherited – Fanconi anemia – Shwachman-Diamond – Dyskeratosis congenita Aplastic Anemia: Differential Dx

• Rule out inherited forms – Fanconi: all patients < 40yo (DEB,MMC) – Others: careful history

• PNH - flow cytometry

• Hypoplastic MDS – morphology, cytogenetics, CD34 count

• LGL - flow cytometry 156 patients with marrow failure, 20 of whom have IBMFS. None of the IBMSF patients had (PNH) clones. Aplastic Anemia: Pathophysiology

• Autoimmune - mediated bone marrow failure – cytotoxic T cells attack hematopoietic progenitors

• CD34 pool is markedly reduced – Usually < 0.05 % , (normal 0.5% to 1.0%) APLASTIC ANEMIA: Pathophysiology

High quality HSC Low quality HSC Progenitors

T cells

SAA Normal CD34 Pool CD34 Pool Autoimmune attack is a set-up for clonal hematopoiesis

6pLOH ~ 10-15%; PIGA mutations ~ 50%

NKG2D receptor GPI-AP+ NK HSC Normal cell Cell Strong Cell kill ULBPs GPI-anchored

MICA/B

NK GPI-AP- Cell HSC PNH cell Weak Cell kill

Katagiri T et al. Blood 2011;118:6601-6609 ©2011 by American Society of Hematology Hanaoka et al, Blood 2006 Relationship among bone Marrow Failure Diseases CHIP + PIGA

PIGA AA PNH CLASSICAL PNH

6pLOH 10~12% DNMT3A ASLX1 Acquired AA Monosomy 7 MDS

PIGA clonal escape AA/PNH Take Home: Autoimmune attack on stem cells Predisposes to clonal escape/malignancy Initial Management - Essentials

• CBC, diff & retic, biochem profile • Bone marrow aspirate, bx, FISH and cytogenetics, CD34 Count • Peripheral blood for PNH and Fanconi

• Transfuse CMV-neg., Irradiated blood products (avoid family members) • Consider prompt referral to specialized center Severe Aplastic Anemia

Definitive management • Allogeneic bone marrow transplantation – HLA-identical – unrelated – mismatched

• Immunosuppressive therapy – ATG/CSA

• High-dose cyclophosphamide Probability of Survival after Allogeneic Transplants for SAA, Probability of Survival after Allogeneic Transplants for SAA, 2000-2009 - By Donor Type and Age - 2000-2009 100 - By Donor Type and Age - 100 90 ≤ 20y, Sibling Donor (N=1,191) 90

80 80 > 20y, Sibling Donor (N=1,256) 70 70 ≤ 20y, Unrelated Donor (N=574) 60 60

50 50 > 20y, Unrelated Donor (N=550) 40 40

30 30 Probability of Survival, % 20 20

10 10 P < 0.0001 0 0 0 1 2 3 4 5 6 CIBMTR Years Horse ATG is superior to rabbit ATG

Response to ATG/CSA is roughly 60%

Mortality Decreasing But response fixed

Scheinberg et al, NEJM 2011,;365:430-38 ATG/CSA: Late complications Rosenfeld et. al, Jama 2003;289: 1130-35

Risk of relapse > 40% in Risk of clonal evolution responders 25-35% failure-free survival ELTROMBOPAG ADDED TO STANDARD IMMUNOSUPPRESSION AS FIRST TREATMENT IN APLASTIC ANEMIA Hematologic response

h-ATG Day 1 to 4 3 mo 6 mo

CSA x 6 months

Cohort

EPAG 150 mg  Day 14 to 6 months

5 year follow-up

EPAG 150 mg  Day 14 to 3 mos

EPAG 150 mg Day 1 to 6 months

Supplemental methods: The protocol was amended starting with subject # 46 on cohort 2, so that cyclosporine was continued at a 6 fixed daily dose, 2mg/kg/day, for an additional 18 months in order to prevent relapse. Townsley, DM et al, NEJM 2017; 376:1540-50. Supplemental Figure 4 Median follow-up 23 months Refractory SAA

• Repeat IST – low response rate

• Supportive care

• Eltrombopag (Olnes et al, NEJM 2012)

• BMT – usually from a MUD or alternative donor Post Transplant High Dose Cy

• Mitigates GVHD

• Allows for greater use of alternative donors (haplo BMT) – No difference for engraftment or GVHD btw matched sibs and HLA-haplo identical donors

• Average person in US has 4.5 HLA haplo- identical donors Conditioning for HLA Haplo- identical BMT

Flu Flu Flu Flu Flu GVHD Prophylaxis: Tacrolimus, MMF, & Cy Cy Post-HSCT Cy rATG rATG rATG

TBI Cy Cy

-9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4

Flu = Fludarabine 30 mg/m2 IV daily Cy = Cyclophosphamide 14.5 mg/kg IV daily TBI = 200 cGy Cy = Cyclophosphamide 50 mg/kg IV daily rATG = Thymoglobulin 0.5 mg/kg (day-9) & 2 mg/kg (day -8,-7)

20 Mini Haplo BMT with PTCy for Refractory SAA

• 21 patients with refractory SAA (median f/u 24 mos (range 3-72) − Median age: 29 years (range 5-69) − 15/21 had evidence of clonality (PNH and/or cytogenetic abnormality) − 18 haplo 1 mm URD(9/10) 2 URD (10/10)

• Rapid and consistent engraftment − Day 60 chimerism 100% in 20/21 patients • One primary graft failure (engrafted with 2nd BMT from different donor) − ANC 15 days Reds 25 days Platelets 28 days

• Excellent Disease Free Survival − All 21 alive, transfusion-independent, without clonality (KPS 100) − Acute GVHD grade II-IV 2/21 (9.5%) − Extensive chronic GVHD 0/21 − All off IST – median f/u 30 months

Dezern et al, Biol Blood Marrow Transplant 2017, 23:498-504 Conclusions

• SAA: IST vs BMT – Exciting clinical trials of IST and BMT – BMT advancing faster; solves problem of relapse and clonality – Upfront mini-haplo BMT: requires increased TBI to 400: 6/6 engrafted.

• Clonality underlies most of acquired SAA

• Post-transplant Cy is safely expanding the donor pool in SAA – Adopted by BMT-CTN – Standard of care for refractory SAA – Haplo BMT front-line? Paroxysmal Nocturnal Hemoglobinuria Biology

• Acquired Clonal Hematopoietic Stem Cell Disease

• PIG-A mutation – X(p22.1) • PIG-A gene product necessary for 1st step in the biosynthesis of GPI anchors • PNH cells have deficiency or absence of all GPI anchored proteins GPI-AP Biosynthesis: Involves >10 steps and > 25 genes

plasma membrane raft

N N N

PIG-A

PI

GlcNAc-PI endoplasmic reticulum PNH Pathogenesis of hemolytic anemia

• CD59 – Membrane inhibitor of reactive lysis – Prevents incorporation of C9 into C5b-8; thus, MAC does not form

• CD55 – Decay accelerating factor – Block C3 convertase

• Protect cells from complement-mediated destruction Effects of Terminal Complement on RBCs and Clinical Consequences in PNH

esophageal spasm, abdominal pain, male ED Complement Activation Free hemoglobin is a nitric oxide scavenger Fatigue

Intact RBC Thrombosis ? Free hemoglobinAnemia in the blood from destroyed PNH RBC Renal Failure

Anemia/Transfusions Flow Cytometric Diagnosis of PNH

PNH Aplastic anemia

Brodsky RA, Blood 2009; 113(26):6522-7. PNH: Therapy

• Eculizumab: – FDA approved monoclonal antibody for PNH

• Allogeneic BMT - only curative therapy – Indications: severe cytopenias, poor response to eculizumab – Non-myeloablative conditioning acceptable.

• Supportive care – Fe, folic acid, transfusions

Brodsky RA, Blood 2009, 113:6522-7 Lectin Pathway Classical Pathway Alternate Pathway

MBL, MASP, C1q, C1r, C1s C3 C4 + C2 C4 + C2 Factor B + D

C3 convertases CD55 C4b2a, C3bBb

C3b

C5 convertases C4b2a3b, C3bBb3b

C5a C 5 CD59 C6,C7,C8,C9

Eculizumab C5b

Membrane attack complex (MAC) Dosing schedule: Intravenous infusion over 30 min

– Induction period • 600 mg every 7±2 days for 4 doses

– Maintenance period • 900 mg 1 week later, followed by… • 900 mg every 14±2 days for a total of 26 weeks

Hillmen et al, NEJM 2006, 355:1233-43 Brodsky et al, Blood 2008, 111:1840-7 Effect of Eculizumab on Hemolysis – lactate dehydrogenase (LDH)

3000 Placebo 2500

2000

1500 P < 0.00000000001

1000 Eculizumab 500 Normal Lactate Dehydrogenase (IU/L) 0 Screening-4 -2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 Study Week Response to Eculizumab

Hematocrit Retic count LDH

Pre eculizumab 19.2 7.6 1650 post eculizumab 33.2 5.5 301

Patients continue to have compensated extravascular hemolysis Intravascular and Extravascular Hemolysis in PNH

Absence of CD55 & CD59 MAC forms  intravascular hemolysis PNHpatient

= C3dg

MAC

Absence of CD55 & CD59

Extravascular hemolysis PNH patienton eculizumab Long‐term safety and efficacy of sustained eculizumab treatment in patients with paroxysmal nocturnal haemoglobinuria

British Journal of Haematology Volume 162, Issue 1, pages 62-73, 25 APR 2013 DOI: 10.1111/bjh.12347 Lessons from Eculizumab Trials (TRIUMPH, SHEPHERD, EXTENSION)

• Safe – Mild side-effects – 1000-2000 fold increased risk for Neisserial infections (~0.5% per year) – Strongly consider microbial prophylaxis – PCN – Macrolide if Pen allergic

• Effective – Decreases intravascular hemolysis – Decreases (>90%) or eliminates (>75%) need for PRBC – Improves quality of life – Reduces the risk for thrombosis by >90% Lessons from Eculizumab Trials – cont. (TRIUMPH, SHEPHERD, EXTENSION)

• Drawbacks – Lifelong therapy intravenous therapy – Cost (> 350K a year) – Some patients have a lot of extravascular hemolysis

• Indications – Severe anemia – thrombosis – Disabling fatigue or other severe symptoms – Labs: large clone, high retic, LDH > 2x ULN – Bone marrow: normal to hypercellular • Not as effective in patients with AA/PNH – Does not treat bone marrow failure How to monitor PNH pt of eculizumab

• CBC with retic, LDH, comp once a month – Draw labs before giving drug

• PNH clone every 12 months

• Direct coombs if persistent anemia – IgG neg – C3 pos Future Complement Inhibitors

APL-2

Eculizumab ALXN1210 ALNCC5 Conversin Near Future Drugs for PNH

•Ravulizumab •C5 Inhibitor (non-inferior to ecu in randomized phase III trial) •Intravenous every 8 weeks •May be approved in 2019 •APL2 •C3 inhibitor •Add on to eculizumab •Daily subcutaneous •ACH4471 •Factor D inhibitor •Add on to ecu •TID oral Pure Red Cell Aplasia Diagnostic Criteria

• Severe anemia

• Reticulocytes less than 1.0 %

• Paucity (< 0.5%) of erythroblasts in the marrow – Other lineages usually unaffected Pure Red Cell Aplasia pathophysiology

• Congenital - (Diamond-Blackfan anemia) • Acquired – immunologic • Antibody-mediated - TEC, ABO in BMT, EPO • Neoplasia: thymoma, B cell malignancies • LGL T cell or NK cell • Drugs – Pregnancy – MDS – Parvovirus B-19 Parvovirus Induced Red Cell Aplasia

• Globoside (p-antigen) – Viral target on erythroid progenitors • Susceptible hosts – immunosuppressed – Chronic hemolytic anemia • Diagnosis – DNA based assays – Antibodies – Giant pronormoblast • Treatment - IVIG PRCA: Clinical Approach

• CBC, retic, bone marrow to establish diagnosis

• Look for LGL (PBS), flow cytometry if indicated

• Consider other associated diseases (SLE, MDS, thymoma, RA)

• Marrow culture – BFU-E maturation in vitro helps distinguish from MDS and predicts response to immunosuppression BFU-E ASSAY for PRCA

BFUE growth by Diagnosis 150

p=0.0001 100 Systemic Inflammation

LGL 50 Normals

PRCA (no LGL) BFUE 10E5(bursts/ nucleated cells)

MDS 0

DeZern et al, Exp Heme 2013;41:808-16 Large Granular Lymphocytes

Neutropenia

Anemia

Thrombocytopenia

Pancytopenia

- may be T cells or NK cells Immunologic Classification T-CELL LGL

• Immunophenotype: CD3+, CD8+, CD57+

• Clonal disorder: TCR (gamma chain) gene rearrangement assessed by PCR (Sensitivity is 90%). Pure Red Cell Aplasia: Therapy

• Stop unnecessary drugs and establish etiology

• LGL mediated PRCA – First line drugs: CSA, Cy, MTX, steroids – Second line: ATG/CSA; CAMPATH, HiCy