1. Bone Disease in haemoglobin disorders
Ersi Voskaridou1, MD, PhD, Evangelos Terpos2, MD, PhD
1Thalassaemia Reference Center, Laikon General Hospital
2Department of Clinical Therapeutics, University of Athens School of Medicine
Athens, Greece

2. Bone Disease in Patients With Haemoglobinopathies
Abnormalities in children with undermanaged thalassemia include
Enlarged cranial and facial bones
Spinal deformities
Nerve compression
Spontaneous fractures
All thalassemia and SCD patients: effects of bone marrow expansion and other imbalances
Localized changes in bone (loss and formation)
Generalized decrease in bone mineral density (BMD), osteppenia/osteoporosis
Mikroinfarction of vertebral bone marrow (fish mouth)

3. Clinical Factors in the Pathogenesis of Thalassemia and SCD-Induced Osteoporosis
Bone marrow expansion
Mechanical interruption of bone formation
Cortical thinning
 Fragility of bones
 haemoglobin levels
 chronic haemolytic anaemia
Imbalances in multiple endocrine pathways can alter bone metabolism in thalassemia patients
Hypogonadism
Iron deposition in pituitary gonadotropic cells
Iron deposition in testes and ovaries
Defects in growth hormone/insulin-like growth factor I signaling

4. Acquired Factors in the Pathogenesis of Thalassemia and SCD-Induced Osteoporosis
Iron overload and chelation therapies
Iron deposition in bone
Impaired maturation of bone cells (osteoids)
 Local mineralisation
High-dose iron chelation therapy with desferoxamine
 Differentiation and proliferation of bone-forming cells (osteoblasts)
 Collagen (bone matrix) formation
 Osteoblast programmed cell death (apoptosis)
Deficiencies in vitamins and minerals
Vitamin D deficiency
Impaired regulation of bone metabolism
Zinc deficiency
Prevalent in hemoglobinopathies and associated with low bone mineral density
Role of zinc is not well defined, but might involve activation of bone-specific alkaline phosphatase (bALP) and inhibition of osteoclast activity1,2
1. King JC. Am J Clin Nutr. 1996;64(3): ; 2. Reviewed in Voskaridou E, et al. Br J Haematol. 2004;127(2):2004;

5. Genetic predisposition to reduced BMD
Polymorphisms in genes that play key roles in bone maintenance and remodeling:
- Collagen type I
- Vitamin D receptor
- Transforming growth factor β 1
- Calcitonin receptor
- Oestrogen receptor
- Interleukin 6
To date, although some genetic traits correlate with thalassaemia-induced osteoporosis, their roles in its development are unclear
Reviewed in Voskaridou E, et al. Br J Haematol. 2004;127(2):

6. Haemoglobinopathies-Induced osteoporosis
Most of these factors act through the imbalance in bone remodelling; they inhibit osteoblast activation and/or increase osteoclast function leading to bone loss and osteoporosis.
The necessity of understanding the underlying mechanisms for bone destruction in these patients seems to be compulsory
Determination of optimal therapy

7. Bone Metabolism: A Balance Between Osteoblasts and Osteoclasts
Reproduced from Seeman E, et al. N Engl J Med. 2006;354(21):

8. Results From an Imbalance in Bone Remodeling
Decreased bone mineral density (BMD)
Micro-architectural deterioration
Decreased bone strength
Increased fracture risk
Osteoporotic Bone
Normal Bone
Eastell R. N Engl J Med. 1998;
Boyle JB, et al. Nature. 2003;423:
Photos reproduced from Dempster DW, et al. J Bone Miner Res. 1986;1:15-21 with permission of the American Society for Bone and Mineral Research.

9. Osteoporosis
Osteoporosis is a disease characterized by low bone mass and microarchitectural deterioration of bone issue, leading to enhanced bone fragility and a consequential increase in fracture risk
Osteoporosis is the silent disease that makes bones prone to fracture and is a major public health

10. Osteoporosis T-score Definition Z-score
Number of standard deviations (SD) that a patient’s bone mass is above or below the mean peak bone mass for a 30-year-old healthy woman
T-score –1 to +1 = normal BMD
1 SD decrease in T-score = 10% to 15% decrease in BMD
Z-score
Number of SDs that a patient’s bone mass is above or below the mean bone mass for age- and sex-matched controls
Definition
Osteoporosis is defined as BMD T-score < – 2.5 leading to higher risk of fracture
Osteopenia: T-score –1.0 to –2.5
Normal BMD: T-score > –1.0
BMD = BMD = Bone mineral density.
Consensus Development Conference. Am J Med. 1993;94(6):
National Osteoporosis Foundation
BMD = Bone mineral density.
Consensus Development Conference. Am J Med. 1993;94(6):
National Osteoporosis Foundation

11. Dual-Energy X-ray Absorptiometry (DEXA) for Determination of BMD
Typical DEXA equipment1
Sample DEXA scans of lumbar spine (top) and hip (bottom)2
BMD = Bone mineral density.

12. Relationship Between BMD and Fracture Risk
BMD = Bone mineral density. Adapted from Faulkner KG. J Bone Miner Res. 2000;15(2):
Osteoporosis
Osteopenia
Normal

13. Osteoporosis: A Severe Problem in Adult Patients With Thalassemia and SCD
Osteoporosis is prevalent in adult thalassemia and SCD patients
Incidence: 40%-60%1 and 70-80%2
Mean annual change in lumbar-spine BMD: –1.36%1
Lumbar spine 89%1
Hip %1
Distal radius %1
1. Dresner Pollack R, et al. Br J Haematol. 2000;111(3): Baldanzi G, et al. Clinics (Sao Paulo) 2011; 66:

14. Biochemical Markers of Bone Metabolism
Enzymes, protein fragments, or other molecules released into blood as a result of bone turnover
In order to be clinically useful, markers should be
Highly specific to bone
Detectable in body fluids (blood or urine) using standard assay methods (chemical, enzymatic or immunologic)

15. Biochemical Markers of Bone Metabolism Resorption Formation Calcium
TRAcP-5b
BSP
OH-proline OH-lysine glycosides
Pyridinium crosslinks
Collagen type I telopeptides (eg, NTX, CTX)
Collagen type I propeptides
BALP
Osteocalcin
Osteoblasts
Bone matrix
Osteoclasts
Formation
Resorption
BALP = Bone-specific alkaline phosphatase; TRAcP-5b = Tartrate-resistant acid phosphatase type 5b; BSP = Bone sialoprotein; NTX = N-telopeptide of type I collagen; CTX = C-telopeptide of type I collagen. Adapted by permission from Fohr B, et al. J Clin Endocrinol Metab. 2003;88(11):

16. Bone Metabolism Unit
Voskaridou & Terpos. BJH 2004;127:127-39

17. RANKL: An Essential Mediator of Osteoclasts
CFU-M
RANK
RANK
Prefusion
osteoclast
Growth factors Hormones Cytokines
Multinucleated
osteoclast
Mature osteoclast
Osteoblast
lineage
Bone
RARANK = Receptor activator of nuclear factor kappa B; RANKL = RANK ligand; CFU-M = Colony-forming unit macrophage. Adapted by permission of Macmillan Publishers Ltd [NATURE] Boyle WJ, et al. Nature. 2003;423(6937):

18. Mouse lacking the RANKL gene
Increased Bone Density Associated With Absence of RANKL in Preclinical Experiments
Normal mouse
Normal mouse
Mouse lacking the RANKL gene
RANKL = Receptor activator of nuclear factor kappa B ligand. Adapted from Li J, et al. PNAS. 2000;97(4):

19. Osteoprotegerin (OPG): The Decoy Receptor of RANKL
Osteoclast formation, function, and survival inhibited by OPG
Bone
Osteoblast
lineage
Inactive
osteoclast
CFU-M
Prefusion
Multinucleated
Growth factors Hormones Cytokines
RANK
RANKL
OPG
OPG = Osteoprotegerin; RANK = Receptor activator of nuclear factor kappa B; RANKL = RANK ligand; CFU-M = Colony-forming unit macrophage. Adapted by permission of Macmillan Publishers Ltd [NATURE] Boyle WJ, et al. Nature. 2003;423(6937):

20. Reduced Bone Density Associated With Absence of OPG
BV % TV 50
+/+
–/– 40 30 * * 20
Normal mice
When OPG is lacking, you see, (in the right image) and the osteoporosi of the OPG deficient mice.
OPG-deficient mice
Normal mice
OPG-deficient mice 10 * *
OPG = Osteoprotegerin; BV % TV = Trabecular bone density as a percentage of total tissue density. *P < .001 versus wild type. Adapted from Bucay N, et al. Genes Dev. 1998;12(9):
–10
OPG +/+
OPG +/–
OPG –/–

21. RANKL/OPG Balance Drives Osteoclast Activity
Alterations of the RANKL / OPG ratio are critical in the pathogenesis of bone diseases that result in increased bone resorption1-3
RANKL
OPG
Prevents OC activaion
Promotes OC activation
OC Activity
RANKL = Receptor activator of nuclear factor kappa B ligand; OPG = Osteoprotegerin; OC = Osteoclast.
1. Hofbauer LC, et al. JAMA. 2004;292(4): ; 2. Lacey DL, et al. Cell. 1998;93(2): ; 3. Boyle WJ, et al. Nature. 2003;423(6937):

22. OPG/sRANKL ratio in thalassaemia
2.0
Parameter
Patients
(N=26)
(m±SD)
Controls
(N=30)
(μέση±SD)
p-value
OPG (pmol/L)
sRANKL (pmol/L)
OPG/sRANKL
2,6±1,6
5,6±3,8
0,4±0,4
4,0±0,4
4,6±1,4
0,8±0,2
0,002
0,09
0,006
1.5
1.0 .5
p<0.01
Log sRANKL/OPG
0.0
Parameter
Patients
(N=54)
(m±SD)
Controls
(M±SD)
p-value
OPG (pmol/L)
sRANKL (pmol/L)
OPG/sRANKL
3,0±1,3
8,1±2,8
0,4±0,2
3,6±1,4
4,5±1,2
0,8±0,2 NS
<0,0001
-.5
-1.0
-1.5
N = 30 26
L1-L4 and femoral neck BMD strongly correlated with
OPG/sRANKL
Controls
Thalassaemia patients
Voskaridou et al. Br J Haematol 2003;123:730-7
Morabito et al. J Bone Miner Res 2004;19:722-7

23. Markers of Bone Remodeling in SCD patients
Nouraie M, et al. Haematologica. 2011; 96(8):1092-8

24. HbS/b-thalassemia induced osteoporosis
Ersi Voskaridou et al. Haematologica 2006; 91:

25. A model that intergrates Hh and Wint signaling in osteoblast development
Hu et al. Development 2005;132:49

26. Regulation of Osteoblast Function Proliferation and Differentiation
Marie PJ. Arch Biochem Biophys. 2008;473(2):

27. Insulin-Like Growth Factor-1 (IGF-1) and Bone Loss in Thalassemia Major (TM)
250
200
150
P < .0001
IGF-1, ng/mL
100
50.0
25.0
0.00
Controls
TM patients (n = 54)
Morabito N, et al. J Bone Miner Res. 2004;19(5):

28. Dickkopf-1 and Bone Metabolism
Reduced Dkk-1 levels correlate with high bone mass:
d/– versus +/+ mice:
75%  in Dkk-1 expression
and
18 to 25%  in cortical thickness at the femur diaphysis; P < .01
Dkk-1 = Dickkopf-1 (protein). Adapted from MacDonald B, et al, Bone 2007;41(3):
© 2007, with permission from Elsevier.

29. Dkk-1: Dramatic Increase in Thalassemia Patients With Osteoporosis
Dkk-1, ng/mL 60 40 20
Patients (n=66)
Controls (n=30)
P <
Dkk-1: Dickkopf-1 (protein). Voskaridou E, et al. Haematologica 2009;94(5):

30. Correlation Between Baseline Dkk-1 and Bone Turnover
TRAcP-5b
6.0
5.0
4.0
3.0
2.0
1.0
0.0
Dkk-1, ng/mL 60 40 20
r = 0.310, P = .011
bALP
120.0
100.0
80.0
60.0
40.0
20.0
r = –0.289, P = .018
Bone resorption
Bone formation
Dkk-1: Dickkopf-1 (protein); TRAcP-5b = Tartrate-resistant acid phosphatase type-5b; BALP = Bone-specific alkaline phosphatase. Voskaridou E, et al. Haematologica 2009;94(5):

31. Correlations Between Baseline Dkk-1 and BMD60
r = –0.415, P = .001 60 40 40
Dkk-1, ng/mL
Dkk-1, ng/mL 20 20
r = –0.290, P = .022
0.5
0.6
0.7
0.8
0.9
1.0
1.1
0.2
0.4
0.6
0.8
1.0
1.2
Wrist BMD
L1-L4 BMD
Dkk-1: Dickkopf-1 (protein); BMD = Bone mineral density. Voskaridou E, et al. Haematologica 2009;94(5):

32. Treatment Options for Thalassemia-Induced Osteoporosis
Prevention and general principles
Hormonal replacement
Calcitonin
Bisphosphonates
Denosumab (?)

33. Prevention and Treatment of Early Bone Loss Consists the Best Policy
Annual checking of BMD starting in adolescence is considered indispensable
Physical activity must always be encouraged
Smoking should be discouraged
Adequate calcium and zinc intake during skeleton development can increase bone mass in adult life
Low doses of vitamin D
Early diagnosis and treatment of diabetes mellitus
Adequate iron chelation may prevent iron toxicity in the bone and sufficient blood transfusions may inhibit uncontrolled bone marrow expansion

34. Hormonal replacement
Prevention of hypogonadism seems to be the most effective way for preventing osteoporosis and other bone deformities in TM patients
Therapeutic agent
Dosage
No of patients
Authors
Males: long-acting testosterone esterase
Females: equine oestrogen
+ metroxyprogesterone
for months
250mg, im, every 4weeks
0.625mg/d
5mg/d for 10 days monthly 67
Anapliotou et al, 1995
(Clin Endocrinol 42:279-87)

35. Hormonal replacement
Hormonal replacement do not seem to be the most effective treatment for preventing osteoporosis in TM patients and it is not the only cause of bone disease
in β-TM
TM patients with hypogonadism and osteoporosis HRT
POF patients HRT
Both groups had significant improvement but the POF group had normalization of spinal T scores following HRT in contrast to the β-TM patients
Chatterjee R, et al, Hemoglobin 2011;35(5-6):653-8.

36. Bisphosphonates Evaluated in Thalassemia-Induced Osteoporosis
The increased bone resorption observed in patients with thalassaemia-induced osteoporosis has led to the use of bisphosphonates (inhibitors of osteoclastic bone resorption) for the management of bone loss in these patients

37. Bisphosphonates Evaluated in Thalassemia-Induced Osteoporosis
Alendronate (first generation)
Clodronate (first generation)
Pamidronate (second generation)
Zoledronic acid (third generation)
Neridronate (third generation)

38. First-Generation Bisphosphonates for Treatment of Thalassemia-Induced Osteoporosis
N = 25 patients with thalassemia major and osteoporosis
Bisphosphonate treatment for 2 years1
Alendronate (10 mg PO qd)
Clodronate (100 mg IM qd for 10d/month)
At 2 years
BMD  at all measured sites in placebo arm
BMD constant in clodronate arm
BMD  in alendronate arm
Lack of efficacy of clodronate confirmed in a second study2
PO = Oral; IM = Intramuscular; BMD = Bone mineral density.
1. Morabito N, et al. Osteoporosis Int. 2002;13(8): ; 2. Pennisi P, et al. J Bone Miner Metab. 2003;21(6):

39. Pamidronate in Thalassemia-Induced Osteoporosis
Methods
26 patients with beta thalassaemia
Randomised to 30 mg or 60 mg monthly pamidronate for 1 year
Results
Pamidronate improved lumbar-spine BMD
For regular transfusion + 30 mg pamidronate arm (n = 13), BMD Z-scores were –3.0 at baseline and –2.5 at 12 mo (P = .007)
For regular transfusion + 60 mg pamidronate arm (n = 5), BMD Z-scores were –2.4 at baseline and –2.0 at 12 mo (P = .007)
30 mg/month was sufficient
BMD = Bone mineral density.
Voskaridou E, et al. Br J Haematol. 2003;123(4):

40. Pamidronate in Thalassemia-Induced Osteoporosis
Pamidronate every month IV (1 mg/kg body weight) for 3 years.
Non randomised: 1. N=12 adult TM and 5 TI received Pamidronate +HRT
2. N=17 pts received only HRT
Methods
- DEXA
- Markers of bone turnover (PICP and ICTP)
- Histomorphometric measurements (trabecular bone volume, osteoid
volume, osteoid surface (formation indices) and resorption surface
(bone resorption)
Results
Pamidronate improved BMD of lumbar spine and total hip
 plasma PICP and plasma ICTP levels
 bone formation and resorption in TM
 bone formation and resorption in TI
BMD = Bone mineral density.
Ratna Chatterjee et al. BJH 2012

41. Pamidronate in Thalassemia-Induced Osteoporosis
Ratna Chatterjee et al. BJH 2012

42. Pamidronate in Thalassemia-Induced Osteoporosis Histomorphometry
Ratna Chatterjee et al. BJH 2012

43. Long-term Follow-up of ZOL-Treated Patients
Baseline
36 months
Patients
(N = 66)
Group A (n = 23)
ZOL q 6 mo for 12 mo
Group B (n = 21)
ZOL q 3 mo for 12 mo
Group C (n = 22)
Placebo for 12 mo
Follow-up
ZOL 4 mg q 3 mo
Treat 12 mo
Follow-up 12 mo
Follow-up 24 mo 2 –2 –4 –6
L1-L4 12 mo
L1-L4 36 mo
Femoral neck Baseline
Femoral neck 12 mo
Femoral neck 36 mo C
P < .01
P = .01
ZOL = Zoledronic acid.
Voskaridou E, et al. Haematologica. 2008;93(10):

44. ZOL Decreases Bone Turnover Marker Levels in Patients With Thalassaemia
Control
ZOL 4 mg q3m
ZOL = Zoledronic acid; CTX = C-telopeptide of type I collagen; TRAcP-5b = Tartrate-resistant acid phosphatase type 5b; BALP = Bone-specific alkaline phosphatase. Voskaridou E, et al. Haematologica. 2006;91(9):
Bone resorption
Pain Score
Voskaridou E, et al. Haematologica. 2006;91(9):

45. Safety of ZOL in Patients With Thalassemia-Induced Osteoporosis
ZOL (4 mg q 3 or 6 mo) was generally well tolerated
No alteration in serum hemoglobin or ferritin levels
Adverse events
Diffuse bone pain (in 9% of patients)
Fever (in 20% of patients)
Noted after first dose of ZOL
Duration < 24 hours
Symptoms relieved by antipyretics (paracetamol/acetaminophen)
No bone pain or fever after subsequent infusions of ZOL
No cases of
Renal abnormalities
Hypocalcemia
Osteonecrosis of the jaw (ONJ)
ZOL = zoledronic acid.
Voskaridou E, et al. Haematologica. 2006;91(9):

46. Neridronate a third generation bisphosphonate
Randomized: a. 54 pts with neridronate (100mg X 3m),
calcium, and vitanin D x 3 ys
b. 64 pts with calcium and vitamin D x 3 ys
Forni GL et al. Br J Haematol. 2012;158(2):274-82

47. Reduction of Dkk-1 in ZOL Groups80
P = .004
P = .08 60
Dkk-1, ng/mL 40
12-mo placebo
Baseline
12-mo ZOL
Baseline 20
Groups A + B (ZOL)
Group C (Placebo)
ZOL = Zoledronic acid; Dkk-1 = Dickkopf-1 (protein) Voskaridou E, et al. Haematologica 2009;94(5):

48. Sclerostin is a Wnt signaling inhibitor Sclerostin in TM vs. Controls
p<0.001
Voskaridou E et al. Sclerostin in Thalassemia Osteoporosis Horm Metab Res 2012; 44: 1–5

49. Sclerostin Correlations
with BMD in TM
L1-L4 BMD (g/cm2)
and correlated with their BMD.
The high circulating sclerostin & Dkk-1 levels and their association with BMD support the notion of a disrupted Wnt signaling in patients with thalassemia and osteoporosis which leads, in turn, to osteoblast deregulation
These findings give the rationale for the use of novel drugs targeting sclerostin and Dkk-1 in patients with thalassemia-induced osteoporosis
Sclerostin (pg/ml)
Voskaridou E et al. Sclerostin in Thalassemia Osteoporosis Horm Metab Res 2012; 44: 1–5

50. Novel drugs?
These findings give the rationale for the use of novel drugs targeting sclerostin and Dkk-1 in patients with thalassemia-induced osteoporosis

51. Conclusions and Future Perspectives I
Thalassemia-induced osteoporosis is multifactorial and, therefore, very difficult to manage
Monitoring BMD with DXA is especially important in adolescents and adults patients with hemoglobinopathies
Preventive measures are essential
Bisphosphonates effectively restores BMD and reduces pain in hemoglobinopathies patients with osteoporosis
Generally well tolerated
Benefits continue for ≥ 2 years after cessation of ZOL or NER
Outstanding questions regarding the use of bisphosphonates in this setting include
Most suitable agent
Optimal dose, frequency, and duration of therapy
Combination therapies using bisphosphonates and other agents

52. Conclusions and Future Perspectives II
New data suggest that the reduced osteoblastic activity, which is believed to be the basic mechanism of bone loss in thalassemia, is accompanied by a comparable or even greater increase in bone resorption through the RANK/RANKL/OPG pathway
Wingless-type (Wnt) signaling is an important pathway for osteoblast differentiation
The high circulating sclerostin & Dkk-1 levels and their association with BMD support the notion of a disrupted Wnt signaling in patients with thalassemia and osteoporosis which leads to osteoblast deregulation
Use of novel antiresorptive agents (denosumab/anti-RANKL) or novel drugs targeting sclerostin and Dkk-1, may
be effective in patients with hemoglobinopathies

53. Thanks all of you