Iron Overload in NTDT

3rd Pan-European Conference on Haemoglobinopathies & Rare Anaemias

Limassol, 24 – 26 October 2012

Khaled M. Musallam, MD, PhDAmerican University of Beirut, Beirut, Lebanon

University of Milan, Milan, Italy

● Primary: Increased intestinal absorption

● Secondary: Blood transfusions

Excess Iron in NTDT

Musallam KM et al. Blood Rev 2012;26:16-9.

● Occasional

• Infection

• Pregnancy

• Surgery

● More regular

• Poor growth and development

• Specific complications (advanced age)

Leg ulcer, pulmonary hypertension, extramedullary hematopoietic psuedotumors, thrombotic disease

Transfusions in NTDT

Musallam KM et al. Cold Spring Harb Perspect Med 2012;2:a013482.

Primary Iron Overload

Musallam KM et al. Blood Rev 2012;26:16-9.Ginzburg Y and Rivella S. Blood 2011;118:4321-30.

Ineffective erythropoiesis, Anemia, Hypoxia

↓ Hepcidin↑ Erythropoietin

↓ Ferroportin↑ Intestinal absorption

↑ Release of recycled iron from reticuloendothelial system

↑ Liver iron concentration↓ Than expected serum ferritin level

GDF-15TWGF-1

HIFsTmprss6

? Other erythroid regulators

Primary Iron Overload (2)

r=0.63, P=0.0141 r=0.36, P=0.0022

1. Musallam KM et al. Blood Cells Mol Dis 2011;47:232-4.2. Taher AT et al. Br J Haematol 2009;146:569-72.

● Cumulative process

• Positive correlations between iron overload indices and advancing age1-5

● Slower than transfusional siderosis

• 3-4 mg/day or as much as 1,000 mg/year6

• Annual increase in liver iron concentration of 0.38 ± 0.49 mg Fe/g dry7

Iron Overload in NTDT

1. Taher A et al. Br J Haematol 2009;146:569-72.2. Taher AT et al. Br J Haematol 2010;150:486-9.3. Taher A et al. Haematologica 2008;93:1584-6.4. Lal A et al. N Engl J Med 2011;364:710-8.5. Chen FE et al. N Eng J Med 2000;343:544-50.6. Musallam KM et al. Blood Rev 2012;26:16-9.7. Taher AT et al. Blood 2012;120:970-7.

β-thalassemia intermedia2

Hemoglobin H disease5

r=0.65P<0.001

Considerable iron overload warranting concern?

● Iron overload as early as 5 years1

● Iron-related morbidities beyond 10 years2

● Mean LIC values at cross-sectional assessment of NTDT cohorts with a mean age in early-mid adulthood range between 7 and 15 mg Fe/g dw3-7

1. Cossu P et al. Eur J Pediatr 1981;137:267-71.2. Taher AT et al. Br J Haematol 2010;150:486-9.3. Origa R et al. Haematologica 2007;92:583-8.4. Musallam KM et al. Haematologica 2011;96:1605-12.5. Taher AT et al. Blood 2012;120:970-7.6. Lal A et al. N Engl J Med 2011;364:710-8.7. Pakbaz Z et al. Pediatr Blood Cancer 2007;49:329-32.

The Heart

20

25

30

35

40

45

50

55

60

65

0 500 1000 1500 2000 2500 3000 3500

Serum ferritin (ng/mL)

Ca

rdia

c T

2*

(ms

)

Normal LIC (< 3 mg Fe/g dry wt)

Mild LIC (3–7 mg Fe/g dry wt)

Moderate LIC (7–15 mg Fe/g dry wt)

Severe LIC (> 15 mg Fe/g dry wt)

Normal cardiac R2*

0

5

10

15

20

25

30

0 20 40 60 80 100

LIC

(m

g F

e/g

dry

wt)

Cardiac R2* (Hz)

1. Origa R et al. Haematologica 2008;93:1095-6.2. Roghi A et al. Ann Hematol 2010;89:585-9.3. Taher AT et al. Am J Hematol 2010;85:288-90.4. Mavrogeni S et al. Int J Cardiovasc Imaging 2008;24:849-54.

n=20 n=49

No evidence of cardiac siderosis even in NTDT patients with considerable iron overload1-4

The Liver

● Several case reports and case series suggest an association between iron overload and hepatocellular carcinoma in hepatitis C negative patients with NTDT1-4

1. Macaron J et al. Ann Hepatol 2012. In Press.2. Restivo Pantalone G et al. Br J Haematol 2010;150:245-7.3. Borgna-Pignatti C et al. Br J Haematol 2004;124:114-7.4. Mancuso A. World J Hepatol 2010;2:171-4.

The Liver (2)

Musallam KM et al. Blood Cells Mol Dis 2012;49:136-9.

ChelatedNon-chelated

● β-TI (n=42)

● Median age 38 yrs, 50% men

● Hepatitis C negative

● 28 non-chelated, 14 chelated

● Two consecutive Transient Elastography (FibroScan®) measurements (median 2 yrs, range 1-3 yrs)

R2: 0.836P<0.001

The Liver (3)

Musallam KM et al. Blood Cells Mol Dis 2012;49:136-9.

● ∆ TE in non-chelated patients:

4.4 to 5.7 kPa, P<0.001

● ∆ TE in chelated patients:

7.0 to 4.7 kPa, P=0.005

● ∆ TE/yr non-chelated vs. chelated:

+0.3 vs. -0.9 kPa/year, P<0.001

S <3 (n=11)

S 3 (n=1)

S 4 (n=1)

S 5 (n=1)

S <3 (n=11)

S 3 (n=3)

S <3 (n=28) S <3 (n=26)

S 3 (n=2)

First measurement

Lastmeasurement

Chelated

Non-chelated

Transient elastography values corresponding to fibrosis stages are: ≤7.9 kPa for S <3; >7.9 to 10.3 for S 3; >10.3 to 12.0 for S 4; and >12.0 for S 5.

Other Morbidities

Musallam KM et al. Haematologica 2011;96:1605-12.

Morbidity absent

Morbidity present

p = 0.027

p = 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

168 non-chelated β-TI, mean age 35.2 yrs, mean LIC 8.4 mg Fe/g dw

On multivariate analysis, a 1 mg Fe/g dw increase in LIC was significantly associated with higher odds of thrombosis, pulmonary hypertension,

hypothyroidism, osteoporosis, and hypogonadism

Adjusted for age, gender, splenectomy status, transfusion history, total hemoglobin level, fetal hemoglobin level, platelet count, NRBC count, and

serum ferritin level

On multivariate analysis, a 1 mg Fe/g dw increase in LIC was significantly associated with higher odds of thrombosis, pulmonary hypertension,

hypothyroidism, osteoporosis, and hypogonadism

Adjusted for age, gender, splenectomy status, transfusion history, total hemoglobin level, fetal hemoglobin level, platelet count, NRBC count, and

serum ferritin level

Other Morbidities (2)

Musallam KM et al. Haematologica 2011;96:1605-12.

MorbidityLIC cut-off(mg Fe/g dry wt)

AUC 95% CI p value Sensitivity Specificity AOR (95% CI)a

Thrombosis ≥ 7 0.669 ± 0.049 0.573–0.765 0.001 70.5% 61.3% 2.86 (1.22–5.91)

Pulmonary hypertension

≥ 6 0.684 ± 0.042 0.601–0.767 < 0.001 75% 58% 3.30 (1.54–7.08)

Vascularb ≥ 7 0.723 ± 0.039 0.647–0.800 < 0.001 66.3% 71.8% 3.76 (1.81–7.81)

Hypothyroidism ≥ 6 0.630 ± 0.056 0.521–0.739 0.025 76.7% 52.2% 2.65 (1.03–6.77)

Osteoporosis ≥ 9 0.796 ± 0.041 0.624–0.787 < 0.001 58.4% 81.3% 5.13 (2.46–10.71)

Hypogonadism ≥ 6 0.689 ± 0.053 0.585–0.793 0.002 78.6% 52.1% 3.35 (1.2–9.26)

Endocrinec ≥ 6 0.724 ± 0.039 0.647–0.801 < 0.001 71.3% 70.3% 4.05 (1.96–8.35)

aAdjusted for age, gender, splenectomy status, transfusion history, total hemoglobin level, fetal hemoglobin level, platelet count, NRBC count, and serum ferritin level. Model was built using forward-stepwise selection. p ≤ 0.1 was used as the criterion for inclusion. Multi-colinearity was absent in the model as evident from a variation inflation factor ≤ 3 (acceptable limit up to 10).bPatients having PHT or thrombosis.cPatients having hypothyroidism, osteoporosis, or hypogonadism.

Other Morbidities (3)

Musallam KM et al. Eur J Haematol 2011;87:539-46.

>0.8<0.7<0.5<0.3<0.1

Probability

Total Hemoglobin (g/l)

NTBI (µmol/l)

1.0

0.8

0.6

0.4

0.2

40 50 60 70 80 90 100 110 120 130 140

12

10

8

6

4

2

0

● 29 β-TI

● Splenectomized

● Mean age 32 yrs

● Significant association between the occurrence of large-vessel cerebrovascular disease (MRA) and high NTBI levels

Other Morbidities (4)

Musallam KM et al. Ann Hematol 2012;91:235-41.

● 30 β-TI

● Splenectomized

● Mean age 32 yrs

● Significant association between decreased neuronal function (PET-CT) and high LIC

1.0

LIC (mg Fe/g dry wt)

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0 5 10 15 20 25 30 35 I I I I I I

II

II

II

II

I

0

Association with vascular disease truly causal?

Splenectomyn (%)

Transfusion n (%)

LIC (mg Fe/g dw) n (%)

Vascular morbidity n (%)

+

P<0.001

P=0.016

P=0.001

N=168

No47 (28)

Yes121 (72)

Yes103 (85.1)

No18 (14.9)

Yes21 (44.7)

No26 (55.3)

<723 (88.5)

≥73 (11.5)

<727 (69.2)

≥712 (30.8)

<739 (37.9)

≥764 (62.1)

47 (73.4)

16 (41)

7 (58.3)

9 (33.3)

1 (33)

3 (13)

P=0.020

P<0.001

Mild phenotype (neither splenectomized nor transfused)

Moderate phenotype (either splenectomized or transfused)Severe phenotype (both splenectomized and transfused)

Musallam KM et al. Haematologica 2011;96:1605-12.

*p < 0.05; **p < 0.01; ***p < 0.001.

1.0

0.8

0.6

0.4

0.2

0

Pro

ba

bil

ity

of

va

sc

ula

r m

orb

idit

y

0 10 20 30 40 6050 70

Age (years)

LIC < 7 mg Fe/g dry wtLIC ≥ 7 mg Fe/g dry wt

*

**

Mild phenotype (neither splenectomized nor transfused)

Moderate phenotype (either splenectomized or transfused)

Severe phenotype (both splenectomized and transfused)

1.0

0.8

0.6

0.4

0.2

0

Pro

ba

bil

ity

of

va

sc

ula

r m

orb

idit

y

0 10 20 30 40 6050 70

Age (years)

LIC < 7 mg Fe/g dry wtLIC ≥ 7 mg Fe/g dry wt

**

Association with vascular disease truly causal? (2)

Musallam KM et al. Haematologica 2011;96:1605-12.

Musallam KM et al. Thromb Res 2012;130:695-702.

Hemoglobin

Denaturation Degradation

Excess α-chains

Hemichromes

Inclusion bodies

Band 3 clustering

Oxidation

ROS

Fe++

Spectrin & Band 3 abnormalities

PS exposure

PS

PSThrombin

FVa, FXa, FII

↓ Protein C & S

↑ RBC adhesion & aggregation

↑ Platelet activation& adhesion

↑ WBC activation

Fibrin

Endothelial damage/activation

Tissue factor, ELAM-1, ICAM-1, VCAM-1, VWF

Thrombus formation

RBC

Assessment of Iron Overload

● Serum ferritin

● Liver iron concentration● SQUID

● MRI

● Biopsy

● Cardiac MRI?

● Other markers (NTBI, Transferrin Sat)?

Musallam KM et al. Blood Rev 2012;26:16-9.

Spot Serum Ferritin Measurement

● Caution with interpreting spot serum ferritin values to tailor iron chelation therapy in NTDT

● The 1000 and 2500 ng/ml thresholds are used in thalassemia major patients as they predict survival and cardiac outcomes -> less relevant in NTDT and no similar predictive assessment exists

● Although serum ferritin correlates with LIC in NTDT1-4, the ratio of serum ferritin to LIC is lower relative to patients with β-thalassemia major1,4-6

1. Taher A et al. Haematologica 2008;93:1584-6.2. Lal A et al. N Engl J Med 2011;364:710-8.3. Taher AT et al. Blood 2012;120:970-7.4. Pakbaz et al. Pediatr Blood Cancer 2007;49:329-32.5. Origa R et al. Haematologica 2007;92:583-8.6. Taher AT et al. Am J Hematol 2010;85:288-90.

Serum Ferritin vs. LIC in TI and TM

1,0002,000

3,0004,0005,0006,0007,000

8,0009,000

10,000

0 5 10 15 20 25 30 35 40 45 50LIC (mg Fe/g dry wt)

Ser

um

fer

riti

n l

evel

(μ

g/L

)

TI TMLinear (TI) Linear (TM)

0

Taher A et al. Haematologica 2008;93:1584-6.

Conclusions

● NTDT patients show considerable iron overload despite their transfusion-independence

● Ineffective erythropoiesis leading to hepcidin suppression and increased intestinal iron absorption is the primary implicated mechanism

● Iron overload in this patient population is associated with morbidities involving several organs and organ systems

● Timely detection is warranted, and caution regarding serum ferritin level interpretation is essential