Bone Marrow Failure

Robert A. Brodsky, MDJohns 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 AlexionPharmaceuticals.

• Alexion Pharmaceuticals – grant funding• Apellis: Scientific Advisor• Achillion: Scientific Advisor

Aplastic AnemiaDiagnosis And Nomenclature

• SAA – Bone marrow (< 25% cellular)– Peripheral cytopenias (at least 2 of 3)

• ANC < 500 per µl• Platelets < 20,000 per µl• Absolute retic < 60,000 or corrected retic < 1%

• VSAA: as above, but ANC < 200

• Moderate AA or (NSAA)– Hypocellular marrow but does not meet criteria for

SAA

2 yearmortality> 70%

APLASTIC ANEMIAEtiology

• 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%)

SAACD34 Pool

High quality HSCLow quality HSCProgenitors

T cells

NormalCD34 Pool

APLASTIC ANEMIA: Pathophysiology

Autoimmune attack is a set-up for clonal hematopoiesis

Katagiri T et al. Blood 2011;118:6601-6609©2011 by American Society of Hematology Hanaoka et al, Blood 2006

6pLOH ~ 10-15%;

NKCell

NKG2D receptor

GPI-AP+

HSC

NKCell

GPI-AP-

HSC

Strong Cell kill

Weak Cell kill

ULBPs

MICA/B

Normal cell

PNH cell

GPI-anchored

PIGA mutations ~ 50%

Relationship among bone Marrow Failure Diseases

AA PNH

MDS

10~12%

PIGA

6pLOH

DNMT3AASLX1

Monosomy 7

Take Home: Autoimmune attack on stem cellsPredisposes to clonal escape/malignancy

CHIP + PIGA

CLASSICAL PNH

Acquired AA

PIGA clonal escapeAA/PNH

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

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

• Immunosuppressive therapy– ATG/CSA

• High-dose cyclophosphamide

Definitive management

Years

0 2 61 3 4 5

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- By Donor Type and Age -

0

20

40

60

80

100

10

30

50

70

90

0

20

40

60

80

100

10

30

50

70

90

Prob

abili

ty o

f Sur

viva

l, %

P < 0.0001

≤ 20y, Sibling Donor (N=1,191)

> 20y, Sibling Donor (N=1,256)

≤ 20y, Unrelated Donor (N=574)

> 20y, Unrelated Donor (N=550)

CIBMTR

Horse ATG is superior to rabbit ATG

Scheinberg et al, NEJM 2011,;365:430-38

Response to ATG/CSAis roughly 60%

Mortality DecreasingBut response fixed

ATG/CSA: Late complications

Risk of relapse > 40% in responders

Risk of clonal evolution

Rosenfeld et. al, Jama 2003;289: 1130-35

25-35% failure-free survival

ELTROMBOPAG ADDED TO STANDARD IMMUNOSUPPRESSION AS FIRST TREATMENT

IN APLASTIC ANEMIAh-ATG

Day 1 to 4

EPAG 150 mg Day 14 to 6 months

Hematologic response

EPAG 150 mg Day 14 to 3 mos

CSA x 6 months

Cohort

5 yearfollow-up

EPAG 150 mg Day 1 to 6 months

3 mo 6 mo

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 preventrelapse. Townsley, DM et al, NEJM 2017; 376:1540-50.

Supplemental Figure 4Median 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

20

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

Flu FluFluFlu

CyCy

Flu

Cy TBI

= Fludarabine 30 mg/m2 IV daily

= Cyclophosphamide 14.5 mg/kg IV daily = 200 cGy

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

TBI

Cy = Cyclophosphamide 50 mg/kg IV daily

Cy Cy

Flu

rATG rATGrATG

rATG = Thymoglobulin 0.5 mg/kg (day-9) & 2 mg/kg (day -8,-7)

Mini Haplo BMT with PTCy forRefractory 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

• 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

Biology

GPI-AP Biosynthesis:Involves >10 steps and > 25 genes

N NN

PIG-A

endoplasmic reticulum

plasma membrane

raft

GlcNAc-PI

PI

PNH

• 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

Pathogenesis of hemolytic anemia

Intact RBC

ComplementActivation

Effects of Terminal Complement on RBCs and Clinical Consequences in PNH

Anemia

Anemia/Transfusions

Free hemoglobin in the blood from destroyed

PNH RBC

Free hemoglobin isa nitric oxidescavenger

esophageal spasm, abdominal pain,

male ED

Fatigue

Thrombosis ?

Renal Failure

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, C4 + C2

C1q, C1r, C1sC4 + C2

C3Factor B + D

C3 convertasesC4b2a, C3bBb

C3b

C5 convertasesC4b2a3b, C3bBb3b

C 5C5a

C6,C7,C8,C9

Membrane attack complex (MAC)

EculizumabC5b

CD55

CD59

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, 355:1233-43Brodsky et al, Blood 2008, 111:1840-7

Effect of Eculizumab on Hemolysis –lactate dehydrogenase (LDH)

0

500

1000

1500

2000

2500

3000

-4 -2 0 2 4 6 8 10 12 14 16 18 20 22 24 26

Study Week

Placebo

Eculizumab

Lac

tate

Deh

ydro

gena

se (I

U/L

)

P < 0.00000000001

Normal

Screening

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

PNH p

atient

PNH p

atient

on

eculizum

ab

Absence of CD55 & CD59

= C3dg

Extravascularhemolysis

MAC

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

British Journal of HaematologyVolume 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

EculizumabALXN1210ALNCC5Conversin

APL-2

Future Complement Inhibitors

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

• Severe anemia

• Reticulocytes less than 1.0 %

• Paucity (< 0.5%) of erythroblasts in the marrow– Other lineages usually unaffected

Diagnostic Criteria

Pure Red Cell Aplasiapathophysiology

• 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 InducedRed 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

Systemic Inflammation

LGL

Normals

PRCA (no LGL)

MDS

p=0.0001

050

100

150

BFU

E (b

urst

s/ 1

0E5

nucl

eate

d ce

lls)

BFUE growth by Diagnosis

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

Large Granular Lymphocytes

- may be T cells or NK cells

Neutropenia

Anemia

Thrombocytopenia

Pancytopenia

Immunologic ClassificationT-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