1. Iron Overload in NTDT 3rd Pan-European Conference on Haemoglobinopathies & Rare Anaemias Limassol, 24 – 26 October 2012 Khaled M. Musallam, MD, PhD American University of Beirut, Beirut, Lebanon University of Milan, Milan, Italy

2. ● Primary: Increased intestinal absorption ● Secondary: Blood transfusions Excess Iron in NTDT Musallam KM et al. Blood Rev 2012;26:16-9.

3. ● 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.

4. 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-15 TWGF-1 HIFs Tmprss6 ? Other erythroid regulators

5. Primary Iron Overload (2) r=0.63, P=0.014 1 r=0.36, P=0.002 2 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.

6. ● Cumulative process Positive correlations between iron overload indices and advancing age 1-5 ● Slower than transfusional siderosis 3-4 mg/day or as much as 1,000 mg/year 6 Annual increase in liver iron concentration of 0.38 ± 0.49 mg Fe/g dry 7 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 intermedia 2 Hemoglobin H disease 5 r=0.65 P<0.001

7. Considerable iron overload warranting concern? ● Iron overload as early as 5 years 1 ● Iron-related morbidities beyond 10 years 2 ● 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 dw 3-7 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.

8. The Heart 20 25 30 35 40 45 50 55 60 65 0500100015002000250030003500 Serum ferritin (ng/mL) Cardiac T2* (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 020406080100 LIC (mg Fe/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 overload 1-4

9. The Liver ● Several case reports and case series suggest an association between iron overload and hepatocellular carcinoma in hepatitis C negative patients with NTDT 1-4 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.

10. The Liver (2) Musallam KM et al. Blood Cells Mol Dis 2012;49:136-9. Chelated Non-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) R 2 : 0.836 P<0.001

11. 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 Last measurement Chelated Non-chelated Transient elastography values corresponding to fibrosis stages are: ≤7.9 kPa for S 7.9 to 10.3 for S 3; >10.3 to 12.0 for S 4; and >12.0 for S 5.

12. Other Morbidities Musallam KM et al. Haematologica 2011;96:1605-12. Morbidity absent Morbidity present LIC (mg Fe/g dry wt) Leg ulcers p = 0.027 EMH Heart failure Diabetes mellitus p = 0.490p = 0.245p = 0.682 Thrombosis PHT ALF Hypothyroidism Osteoporosis Hypogonadism p = 0.002 p < 0.001 p = 0.040p < 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

13. Other Morbidities (2) Musallam KM et al. Haematologica 2011;96:1605-12. Morbidity LIC cut-off (mg Fe/g dry wt) AUC95% CI p valueSensitivitySpecificityAOR (95% CI) a Thrombosis≥ 70.669 ± 0.0490.573–0.7650.00170.5%61.3%2.86 (1.22–5.91) Pulmonary hypertension ≥ 60.684 ± 0.0420.601–0.767< 0.00175%58%3.30 (1.54–7.08) Vascular b ≥ 70.723 ± 0.0390.647–0.800< 0.00166.3%71.8%3.76 (1.81–7.81) Hypothyroidism≥ 60.630 ± 0.0560.521–0.7390.02576.7%52.2%2.65 (1.03–6.77) Osteoporosis≥ 90.796 ± 0.0410.624–0.787< 0.00158.4%81.3%5.13 (2.46–10.71) Hypogonadism≥ 60.689 ± 0.0530.585–0.7930.00278.6%52.1%3.35 (1.2–9.26) Endocrine c ≥ 60.724 ± 0.0390.647–0.801< 0.00171.3%70.3%4.05 (1.96–8.35) a Adjusted 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). b Patients having PHT or thrombosis. c Patients having hypothyroidism, osteoporosis, or hypogonadism.

14. 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 Probability of Stenosis on MRA 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

15. 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 II IIII I I I I I I I I I Probability of abnormality on PET-CT 0

16. Association with vascular disease truly causal? Splenectomy n (%) Transfusion n (%) LIC (mg Fe/g dw) n (%) Vascular morbidity n (%) + P<0.001 P=0.016 P=0.001 N=168 No 47 (28) Yes 121 (72) Yes 103 (85.1) No 18 (14.9) Yes 21 (44.7) No 26 (55.3) <7 23 (88.5) ≥7 3 (11.5) <7 27 (69.2) ≥7 12 (30.8) <7 39 (37.9) ≥7 64 (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.

17. *p < 0.05; **p < 0.01; ***p < 0.001. 1.0 0.8 0.6 0.4 0.2 0 Probability of vascular morbidity 010203040605070 Age (years) LIC < 7 mg Fe/g dry wt LIC ≥ 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 Probability of vascular morbidity 010203040605070 Age (years) LIC < 7 mg Fe/g dry wt LIC ≥ 7 mg Fe/g dry wt ** Association with vascular disease truly causal? (2) Musallam KM et al. Haematologica 2011;96:1605-12.

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

19. 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.

20. 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 NTDT 1-4, the ratio of serum ferritin to LIC is lower relative to patients with β-thalassemia major 1,4-6 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.

21. Serum Ferritin vs. LIC in TI and TM 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000 9,000 10,000 05101520253035404550 LIC (mg Fe/g dry wt) Serum ferritin level (μg/L) TITM Linear (TI) Linear (TM) 0 Taher A et al. Haematologica 2008;93:1584-6.

22. 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