Slide 1 of 50 Clinical Case: Hereditary Haemochromatosis Pierre Brissot, MD Professor of Medicine Liver Disease Department University Hospital Pontchaillou Rennes, France

Slide 2 of 50 Patient Presentation Male, Age 55 Years Background Hypertension (treated) Increased weight (BMI 26.6) High cholesterol (untreated) Hyperglycaemia (1.25 g/L) No alcoholism No tobacco History Fatigue (1 year) Arthralgias of 2nd and 3rd metacarpo- phalangeal joints (8 months) Lab Values Ferritin 2500 µg/L Transferrin saturation 95%

Slide 3 of 50 Diagnosis? Polymetabolic Syndrome Hypertension Increased weight High cholesterol Hyperglycaemia Could explain Fatigue Hyperferritinaemia Could not explain This type of arthropathy Such high ferritin level Elevated transferrin saturation

Slide 4 of 50 Diagnosis? Polymetabolic Syndrome + Hereditary Haemochromatosis Could explain 1,2 This type of arthropathy Elevated transferrin saturation High ferritin level 1. Brissot P, et al. Blood Rev. 2008;22: Aguilar-Martinez P, et al. Am J Gastroenterol. 2005;100:

Slide 5 of 50 Diagnostic Grid

Slide 6 of 50 Diagnostic Grid

Slide 7 of 50 Diagnosis? Polymetabolic Syndrome + Hereditary Haemochromatosis HFE is Key Test C 282 Y/C 282 Y = type 1 haemochromatosis Brissot P, et al. Blood Rev. 2008;22:

Slide 8 of 50 Search for Visceral Complications Degree of Body Iron Excess: Beware Ferritin Interpretation Abbreviations: ALT, alanine aminotransaminase; AST, aspartate aminotransaminase; DW, dry weight; ECG, electrocardiogram; LIC, liver iron concentration. LIC (MRI): 9 mg/g dw (N <2mg/g dw) ALT/AST: normal Ultrasound: hyperechoic Liver biopsy? Liver Test/FindingOrgan

Slide 9 of 50 Diagnostic Grid

Slide 10 of 50 Search for Visceral Complications Degree of Body Iron Excess: Beware Ferritin Interpretation ECG, ultrasound: normalHeart Testosterone: normalGonads X-ray: arthropathy Bone mineral density: normal Joints/bones HyperglycaemiaPancreas Abbreviations: ALT, alanine aminotransaminase; AST, aspartate aminotransaminase; DW, dry weight; ECG, electrocardiogram; LIC, liver iron concentration. LIC (MRI): 9 mg/g dw (N <2mg/g dw) ALT/AST: normal Ultrasound: hyperechoic Liver biopsy? Liver Test/FindingOrgan

Slide 11 of 50 Haemochromatosis Grade Brissot P, et al. Hematology. 2006;1: Type 1 haemochromatosis, grade 3 a T Sat, transferrin saturation >45%; b Ferritin, >300 µg/L in men and >200 µg/L in women; c symptoms such as fatigue, impotence, arthropathies; d conditions of vital risk, such as cirrhosis or cardiomyopathy Ferritin b Quality of life c T Sat a Ferritin b Quality of life c Life d T Sat a

Slide 12 of 50 ferritin overexpression as compared with liver iron concentration Accounting for: Contributing to: hyperglycaemia Final Diagnosis Polymetabolic Syndrome + Type 1 Haemochromatosis, Grade 3

Slide 13 of 50 Beware ferritinaemia interpretation! Treatment Metabolic syndrome Diet Exercise Haemochromatosis Phlebotomies (7 mL/kg/wk) 50 µg/L: not an appropriate goal in this case

Slide 14 of 50 Diagnostic Grid

Slide 15 of 50 Sister, age 52 y T Sat: 30% Ferritin: 40 µg/L no C 282 Y Patient Family Study Brother, age 48 y T Sat: 37% Ferritin: 95 µg/L C 282 Y heterozygote Son, age 28 y T Sat: 75% Ferritin: 450 µg/L C 282 Y homozygote Spouse, age 53 y T Sat: 29% Ferritin: 50 µg/L C 282 Y heterozygote Daughter, age 31 y T Sat: 32% Ferritin: 45 µg/L C 282 Y heterozygote

Slide 16 of 50 Conclusions Increased plasma transferrin saturation is a key diagnostic parameter Beware confounding associated condition, which can increase ferritinaemia (eg, polymetabolic syndrome) In presence of confounding associated conditions –Quantify hepatic iron load by MRI –Carefully monitor Hb levels when treating iron overload It is essential to perform a family study In type 1 haemochromatosis

Slide 17 of 50 Clinical Case: Myelodysplastic Syndromes (MDS) Aristoteles A. N. Giagounidis, MD Medizinische Klinik II St. Johannes Hospital Duisburg, Germany

Slide 18 of 50 The Diagnostic Challenge of MDS Megaloblastic anaemia Myelo- dysplastic syndromes AA CDA Immune cytopaenias (AIHA/ITP) TTP HUS Anaemia of chronic disease Hypersplenism AML Bone marrow infiltration (NHL, solid tumors) AIDS Nutritive and pharmacologic toxins Abbreviations: AA, aplastic anaemia; AIDS, acquired immune deficiency syndrome; AIHA, autoimmune haemolytic anaemia; AML, acute myelocytic leukaemia; CDA, congenital dyserythropoietic anaemia; HUS, haemolytic uremic syndrome; ITP, immune thrombocytopaenic purpura; PNH, paroxysmal nocturnal haemoglobinuria; TTP, thrombotic thrombocytopaenic purpura. Graphic courtesy of Dr. A.A.N. Giagounidis.

Slide 19 of 50 Cumulative Survival of 1806 Untreated Patients with Primary MDS (Düsseldorf MDS Registry, 1970–2003) Years Courtesy of Dr. U. Germing Cumulative Survival

Slide 20 of 50 SubtypeBloodMarrow Refractory cytopaenia Anaemia, thrombocytopaenia, neutropaenia Anaemia Blasts ≤1% Dyserythropoiesis only Blasts <5% Ring sideroblasts <15% Refractory anaemia with ring sideroblasts Anaemia Blasts ≤1% Dyserythropoiesis only Blasts <5% Ring sideroblasts >15% Refractory cytopaenia with multilineage dysplasia with or without ring sideroblasts Cytopaenia Blasts ≤1% No Auer rods Monocytes <1000/mL Dyserythropoiesis in >10% of other cell lines Blasts <5% No Auer rods MDS with isolated del(5q)Anaemia Platelets, normal or elevated Blasts <5% No Auer rods Megakaryocytes with hypolobulated nuclei MDS, unclassifiableCytopaeniaDysplasia of noneyrthroid line 2008 WHO Proposals for the Classification of MDS Swerdlow SH, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. Geneva, Switzerland: World Health Organization; Table courtesy of Dr. A.A.N. Giagounidis. Abbreviation: WHO, World Health Organization.

Slide 21 of 50 SubtypeBloodMarrow Refractory anaemia with excess blasts I Cytopaenia Blasts <5% No Auer rods Monocytes <2000/mL Unilineage or multilineage Dysplasia Blasts 5%–9% No Auer rods Refractory anaemia with excess blasts II Cytopaenia Blasts <19% Auer rods possible Unilineage or multilineage Dysplasia Blasts 10%–19% Auer rods possible 2008 WHO Proposals for the Classification of MDS Swerdlow SH, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. Geneva, Switzerland: World Health Organization; Courtesy of Dr. A.A.N. Giagounidis.

Slide 22 of 50 Patient Case Presentation Symptoms Exertional dyspnoea Fatigue No changes in bowel habits No melaena No overt blood loss or haematuria No hepatosplenomegaly or lymph node enlargement Laboratory values Haemoglobin 8.9 g/dL Platelets 278,000 cells/µL (normal range: 150,000–350,000/µL) White blood cell count 4300 cells/µL (normal range: 4000–10,000 cells/µL) Absolute neutrophil count 2600 cells/µL (normal range: 1800–7000 cells/µL) Mean corpuscular volume 92 fL (normal range: 80–95 fL) 77-year-old woman, admitted to hospital in January 2007

Slide 23 of 50 Patient History Interventions had been unsuccessful in correcting anaemia –Administration of iron, folic acid, vitamin B 12 and B 6 –Transfusions with 2 units of packed red blood cells every 4 weeks during the last 4 months Imaging studies had not revealed any pathology –Gastroscopy –Colonoscopy –CT scans of thorax and abdomen Abbreviation: CT, computed tomography.

Slide 24 of 50 Anaemia Testing Ferritin 1140 ng/mL (normal range: 15–350 ng/mL) Transferrin 172 mg/dL (normal range: 200–400 mg/dL) Transferrin saturation 87% Vitamin B 12 and folic acid within normal range Lactate dehydrogenase 225 U/L (normal range: <240 U/L) Erythropoietin 149 U/L Direct and indirect bilirubin not elevated Coombs test negative Haptoglobin within normal limits

Slide 25 of 50 Bone Marrow Aspirate and Core Biopsy Trilineage dysplastic changes Blast count 3% Cytogenetics 46,XX,del(20q) Diagnosis: refractory cytopaenia with multilineage dysplasia

Slide 26 of 50 Treatment Options in Myelodysplastic Syndromes Low risk Intermediate 1 Intermediate 2 High risk Risk stratification according to IPSS a Abbreviation: IPSS, International Prognostic Scoring System. a Greenberg P, et al. Blood. 1997;89:

Slide 27 of 50 Decision-Making Process Low-risk myelodysplastic syndrome Relatively long life expectancy Good quality of life Isolated anaemia with transfusion dependency What is the best treatment for this patient?

Slide 28 of 50 EPO ± G-CSF Lenalidomide Therapeutic Options for Myelodysplastic Syndromes ATG/CSA Iron chelation Transfusions Low risk Intermediate 1 Risk stratification according to IPSS Abbreviations: ATG, antithymocyte globulin; CSA, cyclosporin A; EPO, erythropoietin; G-CSF, granulocyte colony-stimulating factor.

Slide 29 of 50 Trial with erythropoietin resulted in 12 months of transfusion independence Relapse occurred in January 2008 Regular blood transfusions (2 units/4 weeks) When to start iron chelation? Therapy

Slide 30 of 50 Ferritin 1630 ng/mL Transferrin saturation 89% Substantial iron overload in liver MRI Low cardiac iron deposits –Cardiac T2* MRI 28 ms Decision: commencement of iron chelation to prevent damage to liver and heart (ie, to maintain good quality of life) Iron Chelation Diagnostics

Slide 31 of 50 Diagnostic Grid

Slide 32 of 50 Conclusions MDS are heterogeneous and have a broad differential diagnosis –Morphologic expertise needed for correct classification Low-risk MDS have a relatively favourable overall survival –Supportive care is a sensible treatment option Iron overload is a continuous threat to patients with MDS who are transfusion-dependent Evaluating iron overload includes laboratory testing of ferritin, transferrin, and transferrin saturation levels –To assess organ-specific iron deposits, MRI techniques are valuable

Slide 33 of 50 Clinical Case: Thalassaemia Major Ali T. Taher, MD Professor Department of Internal Medicine American University of Beirut Medical Center Beirut, Lebanon

Slide 34 of 50 Patient Presentation 12-year-old boy of Mediterranean origin Previously diagnosed with –β-thalassaemia major at age 6 months –Hepatitis C virus infection at age 4 years Splenectomized at the age of 6 years Received ~45 packed red blood cell transfusions in his childhood Never received iron chelation therapy Presenting for assessment of iron overload

Slide 35 of 50 Relevant Laboratory Value Serum ferritin level = 7200 ng/mL How reliable is serum ferritin for the assessment of iron overload in this case?

Slide 36 of 50 Diagnostic Grid

Slide 37 of 50 Measuring and Interpreting Serum Ferritin 1-3 AdvantagesDisadvantages Easy to evaluate Inexpensive Serial measures to monitor chelation therapy Positively correlates with morbidity and mortality Allows longitudinal follow-up of patients Indirectly measures iron burden Fluctuates in response to inflammation, abnormal liver function, ascorbate deficiencies Individual measures may not accurately reflect iron levels and response to chelation therapy Serial measurement of serum ferritin is a simple, reliable, indirect measure of total body iron 1. Taher A, et al. Semin Hematol. 2007;44(suppl 3):S2-S6. 2. TIF. Guidelines for the clinical management of thalassaemia. 2nd ed. Nicosia, Cyprus; Brittenham GM, et al. Blood. 2003;101:15-19.

Slide 38 of 50 Serum Ferritin Underestimates Iron Burden in Thalassaemia Intermedia Patients , Liver Iron Concentration (LIC) (mg Fe/g dry weight) Serum Ferritin Level (ng/mL) Thalassaemia intermedia (TI) Thalassaemia major (TM) Linear (TI) Linear (TM) With permission from Taher A, et al. Haematologica. 2008;93: Serum ferritin correlates with LIC in patients with TM and TI. However, for the same LIC, patients with TI had lower ferritin levels than corresponding patients with TM.

Slide 39 of 50 Case Continues—Liver Biopsy A liver biopsy was recommended –To determine the liver iron concentration –To evaluate histopathologic changes secondary to hepatitis C infection Patient’s mother refused due to concerns about the associated risks of invasive intervention

Slide 40 of 50 Measuring LIC by Liver Biopsy 1,2 AdvantagesDisadvantages Directly measures LIC (quantitative, specific, sensitive) Validated reference standard Measures nonheme storage iron Evaluates liver histology/pathology Positively correlates with morbidity and mortality Invasive, painful, and potentially serious complications (eg, bleeding) Sampling error risk, especially in patients with cirrhosis Inadequate standardization between laboratories Difficult to follow up 1. Taher A, et al. Semin Hematol. 2007;44(suppl 3):S2-S6. 2. TIF. Guidelines for the clinical management of thalassaemia. 2nd ed. Nicosia, Cyprus; 2008.

Slide 41 of 50 Case Continues—Assessing Liver Iron Patient underwent R2 MRI of the liver = 16 mg/g dry weight How well does liver R2 MRI correlate with liver biopsy?

Slide 42 of 50 Correlation Between R2 MRI and Liver Biopsy With permission from St. Pierre TG, et al. Blood. 2005;105: Biopsy Iron Concentration (mg/g -1 dry weight) R2 MRI is a validated and standardized technique approved by the Australia Therapeutic Goods Administration, FDA, and European Medicines Agency Hereditary haemochromatosis Hepatitis β-thalassaemia β-thalassaemia/ haemoglobin E Mean Transverse Relaxation Rate (s -1 )

Slide 43 of 50 Measuring LIC with MRI 1,2 AdvantagesDisadvantages Estimates iron content throughout the liver Increasingly available worldwide Status of liver and heart can be assessed in parallel Validated relationship with LIC Allows longitudinal patient follow- up Indirectly measures LIC Requires MRI imager with dedicated imaging method Children younger than age 7 years require a general anaesthetic 1. Taher A, et al. Semin Hematol. 2007;44(suppl 3):S2-S6. 2. TIF. Guidelines for the clinical management of thalassaemia. 2nd ed. Nicosia, Cyprus; 2008.

Slide 44 of 50 Diagnostic Grid

Slide 45 of 50 Case Continues—Assessing Cardiac Iron The patient underwent myocardial T2* MRI = 16 ms Can cardiac dysfunction be predicted on the basis of this value alone?

Slide 46 of 50 T2* MRI—Emerging New Standard for Cardiac Iron Assessment in TM Patients Left Ventricular Ejection Fraction (LVEF) (%) Heart T2* (ms) Cardiac T2* value of 37 in a normal heart Cardiac T2* value of 4 in a significantly iron overloaded heart With permission from Anderson LJ, et al. Eur Heart J. 2001;22: Photos courtesy of Maria D. Cappellini, MD. Myocardial T2* values <20 ms are associated with progressive and significant decline in LVEF

Slide 47 of 50 T2* and Left Ventricular Ejection Fraction (LVEF) A shortening of myocardial T2* to <20 ms (ie, increased myocardial iron) is associated with an increased chance of decreased LVEF T2* Value (ms) Chance of Decreased LVEF >20Low chance 10–2010% 8–1018% 638% 470% TIF. Guidelines for the clinical management of thalassaemia. 2nd ed. Nicosia, Cyprus; 2008.

Slide 48 of 50 Measuring Cardiac Iron with MRI 1,2 AdvantagesDisadvantages Rapidly assesses iron content in the septum of heart Relative iron burden can be estimated reproducibly Functional parameters can be examined concurrently Iron status of liver and heart can be assessed in parallel Allows longitudinal follow-up Indirectly measures cardiac iron Requires MRI imager with dedicated imaging method MRI is a nonvalidated method to rapidly and effectively assess cardiac iron 1. Taher A, et al. Semin Hematol. 2007;44(suppl 3):S2-S6. 2. TIF. Guidelines for the clinical management of thalassaemia. 2nd ed. Nicosia, Cyprus; 2008.

Slide 49 of 50 Thresholds for Parameters Used to Evaluate Iron Overload Parameter Normal Range Iron Overload State MildModerateSevere LIC (mg Fe/g dw) 1 <1.23–7>7>15 Serum ferritin (ng/mL) 2,3 <300, male <200, female >1000 to <2500>2500 Cardiac T2* (ms) 4 >2014–208–14<8 1. Wood JC, et al. Blood. 2005;106: Taher A, et al. Semin Hematol. 2007;44 (suppl 3):S2-S6. 3. Brissot P, et al. Blood Rev. 2008;22: Anderson LJ, et al. Eur Heart J. 2001;22:

Slide 50 of 50 Conclusions Assessment of iron overload is essential in the clinical management of patients with thalassaemia because it guides chelation therapy Historically, serum ferritin and liver biopsy have been the diagnostic methods of choice –However, limitations to the reliability of the first and invasiveness of the latter call for novel noninvasive approaches Liver R2 MRI and cardiac MRI T2* are becoming highly sought methods for the diagnosis of iron overload and monitoring of chelation therapy