Thalassemia Care and Research Elliott Vichinsky, M.D. January 18, 2014 Thalassemia Patient and Family Conference Northern California Comprehensive Thalassemia Center Children's Hospital Oakland Global distribution and description of the thalassemia disorders, treatment and complications.

1. Elliott Vichinsky, MD
Thalassemia Patient and Family Conference
January 18, 2014
Thalassemia Care and Research

2. Thalassemia: Global Problem
Next 20 yrs > 900,000 annual births of E/β, β0 thal
 400,000 S.China
 400,000 Thai, India, Sri Lanka, Malaysia
 100,000 Middle East
 10% of health budget in ~ SEA
 Blood safety/availability[ 1.5 mil RBC units/yr in Indonesia
alone]
-- Chelation availability/cost
In the last decade 75% immigrants to NA came from these
regions (20% annual births)

3. Due to the continual migration of populations from one area to
another, there is virtually no country of the world now in which
thalassaemia does not affect some percentage of the inhabitants
Thalassemia—Global Distribution
Cappellini N, et al, eds. Thalassaemia International Federation 2000, with permission.

4. Alpha,BetaThalassemiaandHemoglobinSwitchingInUtero
 

 
Gene ArrangementChromosome 16

B
  
Gene ArrangementChromosome 11

Hemoglobin A
Hemoglobin F



Hemoglobin A2
Adult HemoglobinsEmbryonic Hemoglobins


 
8 weeks 34 weeks Birth 24 weeks 48 weeks
Hemoglobin A


Hemoglobin H
Bart’s Hemoglobin5 to 30% at birth 
 

 
Gene Arrangement Chromosome 16

B
  
Gene Arrangement Chromosome 11


Fetal Hemoglobins

cs
cs

cs
G  A G  A 
FAH BART’S

5. The Thalassemia Disorders
Alpha Thalassemia
 Hemoglobin H
 Hemoglobin H Constant Spring
 Homozygous Alpha Thal
Beta Thalassemia
 Beta Thal Intermedia
 EB Thal
 Beta Thal Major

6. Transfusion dependant
Severe anemia
Diagnosed in early childhood
Mild
Generally
asymptomatic
ThalassemiaClinicalSeveritySpectrum
Non Transfusion dependent
Intermediate severity
Moderate anemia
Diagnosed usually in late childhood
α-thalassemia silent carrier/trait
β-thalassemia minor/trait
Hemoglobin Constant Spring
α-thalassemia intermedia-HbH
β-thalassemia intermedia
Hemoglobin E β-thalassemia
α-thalassemia major/Hb Barts
β-thalassemia major
Severe Hb E β-thalassemia

7. GDF15 = growth differentiation factor 15;
HIF = hypoxia-inducible transcription factor;
LIC = liver iron concentration;
RES = reticulo-endothelial system.
Iron absorption in response to anemia
7

8. ComplicationsofIronOverload
Excess iron promotes the
generation of free hydroxyl
radicals, propagators of
oxygen-related tissue
damage
Liver cirrhosis/
fibrosis/cancer
Insoluble iron complexes are
deposited in body tissues
and end-organ toxicity
occurs
Diabetes
mellitus
Cardiac failure Infertility Growth Failure
Non-transferring-bound
iron NTBI in the plasma
Capacity of serum transferrin
bind iron is exceeded

9. ChelatableIronPoolsPreventionof AccumulationMore
EfficientthanRemovalofStoredIron
100%
30%
Normal: No
NTBI produced
Subsequent
formation of
NTBI in plasma
Fe
Fe
Fe
Fe
Fe
Fe
Fe
Iron overload
Transferrin saturation
occurs due to frequent
blood transfusions
Uncontrolled iron
loading of organs,
such as:
Chelators may prevent iron uptake into these tissuesChelation of storage iron is slow and inefficient
Courtesy of Dr. J. Porter.

10. Transferrin-bound andNTBIironuptake by
myocardial cells
0
10
20
30
40
0 4 8 12 16 20 24
time (h)
Fe-transferrin
NTBI
Link et al: J Lab Clin Med 1985

11. Reprinted from Porter JB, et al. Blood. 1996;88:705, with permission from the American Society of
Hematology.
544842363024181260-6
-1
0
1
2
3
4
5
6
7
Time (hours)
NTBIorDFO(µM)
DFO–ControlofPlasmaNTBILevels
DFO (µM)
NTBI (µM)
Intravenous continuous infusion
DFO = desferrioxamine, NTBI = non–transferrin-bound iron.

12. Chronic hypoxia
Compensatory reactions
Pulmonary hypertension
poor
transfusions
hyper
transfusions
TI pathway TM pathway
Iron overload
LV dysfunction
ThalassemiaHeartDisease

13. Pulmonary Vessel PHT

14. Thalassemia Intermedia complications increase
withage
6.7
3.3 3.3 3.3
0
13.3
3.3
0 0
6.7
0
13.3
6.7
13.3
6.7
3.3
10.0
6.7
0
3.3
16.7 16.716.7 16.7
20.0
10.0
6.7
20.0
10.0
3.3
10.0
23.3 23.3
40.0
26.7 26.7
20.0
10.0
33.3
13.3 13.3
16.7
30.0
20.0
0
5
10
15
20
25
30
35
40
45
1 2 3 4 5 6 7 8 9 10 11
Frequency(%)
Series1 Series2 Series3 Series4
*
* *
*
*
*
* = statistically significant trend.
< 10 years 11–20 years 21–32 years > 32 years
Complications in 120 treatment-naive patients with TI
ALF = abnormal liver function; DM = diabetes mellitus;
HF = heart failure; PHT = pulmonary hypertension. Taher A, et al. Br J Haematol. 2010;150:486-9.

15. Morbidity
absent
Morbidity
present
LIC(mgFe/gdrywt)
Musallam KM, et al. Haematologica 2011.
p = 0.027p = 0.490 p = 0.245 p = 0.682p = 0.002 p < 0.001 p < 0.001 p = 0.040 p < 0.001 p < 0.001
0
3
6
9
12
15
18
21
Thalassemia Intermedia complications and LIC

16. Thalassemia
major
NTDT HH
Study Study 0107 Study 2209 Loreal 2007
Mean age in years
(SD)
17.2 (9.7) 32.2 (12.1) 44.5 (12.7)
Mean LIC in mg Fe/g
dw (SD)
13.7 (9.7) 14.8 (9.1) 16.4 (8.1)
Mean SF in μg/L
(SD)
2682 (1867) 1207 (846) 1661 (1741)
Non-transfused patients develop comparable iron burden to
thalassemia major patients at an older age
LIC and serum ferritin in Thalassemia Major, Non-transfusion-
dependent Thalassemia, and Hereditary Hemochromatosis

17. 17
SacralMass

18. Thal andCNSDisease
T2- and PD-weighted images T1- and T2-weighted images

19. LiverLiver
LiverFepredictsbodyiron andcardiacironinnonchelated
patientsbutmaynotpredictCardiacFechelatedpatients
Anderson LJ. Eur Heart J 2001; 22: 2171-9

20. IronQuantitationPituitaryGland:
Relationshipbetweenpituitaryironandhypogonadism?
Control: R2 = 10.8 s-1
TM: R2 = 20.6 s-1
Pituitary iron an
early indicator of
hypogonadism?
Normal (n=13, 2-19y)
-thal maj with hypogonadism (n=4, 20-24 y)
-thal maj no hypogonadism (n=18, 14-24 y)
from Christoforidis et al, Eur J Radiol 2007;
62:138-42.MRI-R2 is the adequate method for pituitary iron measurement due to
the pituitary-air interface.

21. Anterior Pituitary Volume and R2

22. Hematopoietic stem cells as vehicles
for therapeutic gene delivery
Allogeneic stem cell
transplantation
Autologous stem cell gene
transfer
–Transplantation using autologous stem
cells which have been corrected by
transfer of a normal or therapeutic gene
•Retroviral vectors
•Phase 1 trials open one e thal pt out 7 yrs
–Transplantation using
allogeneic stem cells from a
normal donor
•HLA-matched ,
sibling

23. Payen E , and Leboulch P Hematology 2012
©2012 by American Society of Hematology
GeneTherapy InducedTransfusion
IndependenceinE-Beta-0

24. low dose TBI, campath, sirolimus conditioning =mixed hematopoietic
chimerism sufficient to prevent sickling
Low toxicity allows rx adults with severe disease
 11 patients off immunosuppression with stable mixed chimerism
Longer follow-up and further accrual necessary
Alternative stem cell sources need exploration to

25. HLA-haploidentical bone marrow transplantation
with post transplant cyclophosphamide
•14 haploidentical SCD recipients
15-33 years old
•Conditioning with Flu/Cy/ATG/low
dose TBI and PTCy with Tacro or
Siro/MMF
•OS 100%
•DFS ~50%
•Rejection ~50%
•Expands access to potentially
curative transplantation to nearly
all patients

26. CuringSiblingGeneticDisease:
PGD and HLATyping
3:4 unaffected / carrier embryo
1:4 embryo HLA matches sibling
= 3:16 chance of both matching
Couple with serious genetic risk
ICSI to produce embryos in vitro
• Embryos screened for genetic defect
and HLA match to affected sibling
• Embryo (s) transferred to produce child
who can be a bone marrow match for
affected sibling - 20 SCD HLA matched
pregnancies 50 % success
Centre for Preimplantation Genetic Diagnosis

27. Hb F Modulators

28. Prospective Targets for Fetal Hemoglobin
Reactivation
Jiang Blood 2006; 108:1077-8; Bianchi Blood; 116: e99-110; Xu Gene Dev 2010; 24: 783-8; van Dijk Blood; 116: 4349-52; Sripichai Blood 2009; 114: 2299-
306; Azzouzi,et al ASH 2010 Abstr 3220; Sankaran et al PNAS 2011; 108: 1519-24
Bauer et al Blood 2012
• Hypomethylation agents ,HDAC inhibitors
• Reducing BCL11A activity
• Reducing KLF1 levels
• Altering MYB activity

29. Hypomethylating Agents
5-azacytidine
 Increased Hb F in baboons, humans with SCD, β thal
 No further trials due to concern re. carcinogenesis (in rat model)
Decitabine
 Analog of 5-azacytidine, IV or SQ
 Decreased incidence of tumors in animal models renewed interest
 Previous clinical trials (3)
 Increased γ-globin synthesis, Hb F, F-cells, total Hb (in all pts)
 DLT: reversible neutropenia, thrombocytosis
Open Trials
 Phase 1 RCT -- PO decitabine + THU (inhibits intestinal
metabolism of decitabine, extends absorption time and S-phase
depletion of DNMT)
 Phase 2 – high-risk adult patients

30. HQK-1001 (Dimethylbutyrate)
 Oral, short chain fatty acid derivative
 HDAC inhibitor- activates ϒ-globin gene promoter by displacing
HDAC-3, BCL11A and other repressors
 Phase 1 /2 RCT- Hb F, Hb response with higher dose
HbF response (day 97, +/- HU) Hb Response (day 97, +/- HU)
 Phase 2 trial using higher dose, longer duration
Kutlar A et al (Hemaquest)

31. HDAC Inhibitors: Panobinostat, Vorinostat
 Small molecule screen of bioactive compounds (inc 5’aza, butyrate)
 Identified HDAC 1, HDAC2 as targets for Hb F induction
 10-fold increase in γ/β globin ratio
 Panobinostat >1000X more potent inhibitor of HAD than butyrate, valpraote
 Panobinostat increased Hb F in lymphoma patients
 rationale for trial in SCD
 Vorinostat (SAHA), Panobinostat (LBH-589) in phase 1/ 2 trials
M Okam, NIH; Novartis (A. Kutlar, PI)
HbF(%)
Bradner JE et al. 2010 PNAS

32. ARG/NO Agents

33.  The nitrite anion is an intravascular and tissue storage form of nitric
oxide (NO): NO prodrug
 NO is produced from nitrite, using hemoglobin and myoglobin as nitrite
reductase
 This reaction is favored in tissues with low oxygen tension and low pH,
likely to occur at the leg ulcer site
 NO has vasodilating, anti-bacterial, angiogenetic and anti-platelet
activity and stimulates extracellular matrix production
Why use sodium nitrite in leg
ulcers?
Huang Z, J Clin Invest 2005, Fang FC J Clin Invest 1997, Soneja,A.
Pharmacol.Rep 2005, Alexis W, Chem Soc Rev 2012

35. Improving Anemia+Transfusion
Therapy

36. Number Mean
on tx
Mean on Tx and
HU
P
Hemoglobin (g/dL) 14 88 96 0.017
Brousse V 2013 BJH
Are Transfusion Therapy and
Hydroxyurea Synergistic?

37. ProspectiveComparisonofSpermparametersinSCD
thalassemaAdultPatientsbeforeandaftera6Months
Treatmentwith Hydroxyurea(HYDREPstudy)
D. Bachir, F. Lionnet, J.Gellen-Dautremer, K.Stankovic,
A.Habibi , P.Bartolucci, C. Chalas, I.Berthaut,
F. Eustache, C.Ravel , J.M.Kuntsman ,
R. Girot, J.Mandelbaum, F. Galactéros
Sickle Cell Referral Centre Tenon Paris, H. Mondor Créteil .
CECOS Tenon, Cochin, Jean Verdier

38. New approach to iron overload
Chelators
Intracellular ferritin access using peptides
Hepicidins
Jak2 inhibitors
Transferrin infusions

39. From:ASHabstracts2012,HamiltonJ,etal.
Hyperbranched polyglycerol (HPG) with DFO
Compari-
son of
urinary iron
excretion
from iron
overloaded
mice for
DFO and an
HPG-DFO
conjugate
(50 kDa, 80
molecules
of DFO

40. Peptidealtersferritinironbyopeningferritiniron
chelationratesinsolutionpools(Theil)
Time to formation of the iron chelator + Fe2 complex

41. ModificationofTGF-Bcorrectsanemiaand
ineffectiveerythropoiesisinthalassemia
Normal adults
ACE-536 recombinant fusion
protein is a ligand trap for TGF-B
super family. This improves rythroid
differentiation by inhibiting specific
TGF beta ligands.
In animal models, it improved
thalassemia mice (decreased
ineffective erythropoiesis) and
decreased iron overload
In normal individuals, it increases
hemoglobin through a non-epo
mechanism (no change in Hb F)
Phase I thal trials have been initiated

42. Normal adults
ACE-536 recombinant fusion
protein is a ligand trap for TGF-B
super family. This improves eythroid
differentiation by inhibiting specific
TGF beta ligands.
In animal models, it improved
thalassemia mice (decreased
ineffective erythropoiesis) and
decreased iron overload
In normal individuals, it increases
hemoglobin through a non-epo
mechanism
Phase I thal trials have been initiated
Thalassemia disorders
Modification ofTGF-B improves anemiaand
ineffective erythropoiesis

43. 0
10
20
30
40
50
60
1 2 3
GatedPopulation(%)
Differentiation Profiling
Series1 Series2
Series3 Series4
***
***
** ***
**
0
5
10
15
20
25
30
1 2 3
%Differencefrom
vehicle
Complete Blood Count
Series1
Series2
Series3***
*** ***
Additive Effect of ACE-536 and EPO
Complete RBC Count
Spleen Differentiation Profile
-Wild type mice
Mice at 72 hours post 10 mg/kg RAP-536, EPO 1800units/kg, N=4 * P≤0.05; ** P≤0.01 *** P≤0.001; vs EPO

44. ACE-536 Improves RBC Morphology in β-Thal Mice
Hbb-/- β-thal Mice
Hbb-/- β-Thal Mice Blood Smears
TBS RAP-536

45. Wild type mice Hbb-/- β-Thal Mice
TBS RAP-536
Reduces Iron Deposition in Organs
RAP-536DecreasesOrganIronOverloadin
-thalassemicMice-Hbb-/- Model
Kidney
Liver

46. Treatment with RAP-011 increases hemoglobin
levels and restores RBC morphology

47. Potential effect of JAK2 inhibitors
on ineffective erythropoiesis
ß-thalassemia
Red cell
JAK2 inhibitor
: pJAK2Ineffective erythropoiesis
Libani et al, Blood 2008; Melchiori L, et al. Adv Hematol. 2010;.

48. Use of Jak2 in ß-thalassemia intermedia (NTDT) and
TM
Time
Iron overload
Reduced iron absorption &
iron overload
+ Jak2iNTDT: Non Transfusion Dependent thalassemia
Erythroid
progenitors
RBC

49. Development and Use of Minihepcidins
Hepcidin: 25 AA
The key residues were identified  Minihepcidins: 7-9 AA
Several minihepcidins showed greater molar activity in vitro and in vivo
than natural hepcidin and had substantial activity when given by gavage.

50. Potential effects of Hepcidin agonists or activators on
iron absorption in ß-thalassemia intermedia
Normal Conditions
Hepcidin
activity
Iron absorption
Normal organ iron
concentrations
ß-Thalassemia
Iron absorption
Iron overload
ß-Thalassemia
Correct activity
Iron absorption
Iron overload
Amelioration
of erythropoiesis
a-chain/heme
aggregates

51. Transferrin adm ameliorates disease in ß-thal mice Erythroid
precursors: Reduced hemichrome formation
and proliferation